Canadian Patents Database / Patent 2752247 Summary

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(12) Patent: (11) CA 2752247
(54) English Title: APPARATUS, METHODS AND PROCESSES FOR SORTING PARTICLES AND FOR PROVIDING SEX-SORTED ANIMAL SPERM
(54) French Title: DISPOSITIFS, PROCEDES ET PROCESSUS PERMETTANT DE TRIER DES PARTICULES ET D'OBTENIR DU SPERME ANIMAL PRODUISANT UNE PROGENITURE DE SEXE DONNE
(51) International Patent Classification (IPC):
  • C12M 3/00 (2006.01)
  • C12N 5/076 (2010.01)
  • C12M 1/34 (2006.01)
  • G01N 15/14 (2006.01)
  • C12Q 1/68 (2018.01)
(72) Inventors :
  • DURACK, GARY (United States of America)
  • HATCHER, JEREMY T. (United States of America)
  • WESTFALL, LON A. (United States of America)
  • WALLACE, JEFFREY D. (United States of America)
  • VANDRE, GARY P. (United States of America)
  • NAYAK, NIRAJ V. (United States of America)
(73) Owners :
  • INGURAN, LLC (Not Available)
(71) Applicants :
  • INGURAN, LLC (United States of America)
(74) Agent: OSLER, HOSKIN & HARCOURT LLP
(74) Associate agent:
(45) Issued: 2014-06-17
(22) Filed Date: 2004-03-29
(41) Open to Public Inspection: 2004-10-14
Examination requested: 2011-09-07
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
60/458,607 United States of America 2003-03-28
60/458,731 United States of America 2003-03-28

English Abstract

A flow cytometry apparatus nozzle to sort sperm cells in a fluid stream by chromosome content comprising: a. a nozzle body having a fluid stream entry portion and a fluid stream exit portion and an interior surface separating both portions and along which a fluid stream is adapted to flow; b. the interior surface of the nozzle including a first, a second and a third axially tapered region for progressively accelerating the speed of a fluid stream in a downstream direction toward the exit portion, at least two of the regions having generally elliptical cross sectional shapes oriented in different directions for applying torsional forces to a fluid stream passing therethrough, tending to bring sperm cells in the stream into a desired orientation.


French Abstract

Une buse pour appareil de cytométrie à flux pour trier les cellules spermatiques dans un écoulement fluidique par teneur en chromosomes comprenant : a. un corps de buse ayant une partie d'entrée d'écoulement fluidique et une partie de sortie d'écoulement fluidique et une surface intérieure séparant les deux parties et le long de laquelle un écoulement fluidique est adapté pour s'écouler; b. la surface intérieure de la buse comportant une première, une deuxième et une troisième région axialement coniques pour accélérer progressivement la vitesse d'un écoulement fluidique dans une direction descendante vers la partie de sortie, au moins deux des régions ayant des formes transversales elliptiques orientées dans différentes directions pour exercer des forces de torsion sur un écoulement fluidique qui y circule, tendant à amener les cellules spermatiques dans l'écoulement dans un sens désiré.


Note: Claims are shown in the official language in which they were submitted.

264
The embodiments of the present invention for which an exclusive property or
privilege is
claimed are defined as follows:
1. A flow cytometry apparatus nozzle to sort sperm cells in a fluid stream
by chromosome
content comprising:
a. a nozzle body having a fluid stream entry portion and a fluid stream
exit portion
and an interior surface separating both portions and along which a fluid
stream flows when
operating to sort sperm cells;
b. the interior surface of the nozzle comprising: a first axially tapered
region having
a first angle of taper; a second axially tapered region having a second angle
of taper, the
second axially tapered region adjacent to and downstream of the first axially
tapered region; and
a third axially tapered region having a third angle of taper, the third
axially tapered region
adjacent to and downstream of the second axially tapered region, wherein the
angle of taper of
the second axially tapered region is different than the angle of taper of the
first and third axially
tapered regions, and wherein at least two of the axially tapered regions have
generally elliptical
cross sectional shapes oriented in different directions for applying torsional
forces to the fluid
stream passing there through, the torsional forces tending to bring sperm
cells in the stream into
a desired orientation.
2. The nozzle as claimed in claim 1, wherein the generally elliptical cross
sectional shape
of one of the said at least two regions is oriented at about 90 degrees
relative to the generally
elliptical cross sectional shape of the other of the said at least two
regions.
3. The nozzle as claimed in claim 1, wherein the generally elliptical cross
sectional shapes
having major axises of the first and second regions are oriented in
substantially the same
direction to define a first torsional zone, and the generally elliptical cross
sectional shape of the
third region, constitutes a second torsional zone, which is oriented at about
90 degrees relative
to the generally elliptical cross sectional shapes of the first and second
regions.

265
4. The nozzle as claimed in claim 3, wherein the first torsional zone has
an axial length of
3.0-4.5 mm and the second torsional zone has an axial length of 3.5-5.0 mm.
5. The nozzle as claimed in claim 1, wherein the third axially tapered
region tapers at an
angle of 42-48 degrees.
6. The nozzle as claimed in claim 1, wherein all of the regions have
generally elliptical
cross sectional shapes.
7. The nozzle as claimed in any claim 1, wherein the nozzle body has an
exterior surface
with a non- reflective coating thereon.
8. The nozzle as claimed in claim 1, further comprising a mount for
mounting the nozzle in
a position pointing upward in its normal attitude of operation to direct the
fluid stream along an
upward trajectory.
9. A nozzle as claimed in claim 8, wherein the nozzle body is rotatable on
the mount on a
longitudinal axis of the body.
10. The nozzle as claimed in claim 8 wherein the angle of the taper in the
second region is
greater than that of the first region but less than that of the third region.
11. The nozzle as claimed claim 1, wherein interior surface of the nozzle
includes a fourth
non tapered region coaxial with and downstream from the first, second and
third axially tapered
regions, the fourth tapered region being adjacent to the third axially tapered
region.

266
12. A method of orienting animal sperm cells in a flow cytometry apparatus,
the method
comprising the steps of:
introducing a fluid containing sperm cells into a fluid stream;
directing the fluid stream containing the sperm cells under a pressure in the
range of 20
to 40 psi through a nozzle having three or more adjacent axially tapered
regions and an exit
orifice with a diameter in the range of 50-70 microns;
accelerating the speed of the fluid stream in a downstream direction toward
the orifice
through a first axially tapered region having a first angle of taper, a second
axially tapered
region having a second angle of taper and a third axially tapered region
having a third angle of
taper, wherein the second angle of taper is different than the first angle of
taper and the third
angle of taper; and
applying a torsional force to the fluid stream tending to bring the cells into
a desired
orientation as they pass through the orifice at a rate in the range of 30,000
to 50,000 sperm
cells per second with a generally elliptical cross section shape in at least
one of the axially
tapered regions.
13. The method as claimed in claim 12, wherein the nozzle has two torsional
zones defined
by interior surfaces of the nozzle in three or more axially tapered regions.
14. The method as claimed in claim 12, wherein the nozzle has a flow axis
through the
orifice, and wherein at least some of the interior nozzle surfaces have
generally elliptical shapes
which are rotated relative to one another about the axis.
15. The method as claimed in claim 12, wherein the method further includes
directing the
stream along an upward non-vertical trajectory.

267
16. The
method as claimed in claim 12, wherein the method further includes rotating
the
nozzle body on a central longitudinal axis of the nozzle while directing the
stream.

Note: Descriptions are shown in the official language in which they were submitted.

CA 02752247 2011-09-07
1
APPARATUS, METHODS AND PROCESSES FOR SORTING PARTICLES
AND FOR PROVIDING SEX-SORTED ANIMAL SPERM
This is a division of Canadian 2,518,882 filed March 29, 2004.
Backciround of the Invention
This Invention relates generally to apparatus and methods for animal semen
collection,
and more particularly to apparatus and 1-nett-lock using various techniques,
including flow
cytornetry, to yield sperm populations that ere enriched with sperm cells
having one or more
desired characteristics, such as viable populations of sperm cells sorted
according to DNA
characteristics for use by the animal production industry to preselect the sex
of animal offspring.
The fertilization of animals by artificial Insemination (Al) and embryo
transplant following
in vitro fertillz_ation Is an established practice. In the livestock
production industry, the ability to
influence the reproductive outcome toward offspring having one or rnore
desired characteristics
has obvious advantages. By way of example, there would be an economic. benefit
In the dairy
industry to preselect offspring In favor of the female sex to ensure the
production of dairy cows.
E-Iforts have been made toward achieving this goal by using flow cytometry to
sort X and Y
sperm cells, as evidenced by the disclosures in US Patents Nos. 6,357,307
(Buchanan, et al.),
5,985,216 (Rens, et al.), and 5,135,759 (Johnson), However, none of these
efforts has resulted
In the introduction of a commercially successful high-throughput system
capable of producing
production volumes of relatively pure sexed sperrn cells having a motility
sufficient for effective
fertilization.
Accordingly, there is a current need in the animal production industry for a
viable high-
speed system for efficiently Isolating sperm cells based on a specified DNA
characteristic (or
other characteristics) to produce quantities of such cells, which can be used
on a commercial
scale. Also needed Is a spear: handling system that preserves the viability of
such isolated
sperm as It Is processed by the isolating system and that allows for
preservation of such Isolated
sperm until such time that it Is ready for use. The present invention
addresses these needs.
Thls invention also has application to improvements in the field or flow
cylernetry on a
more general basis. Flow cytornetry may broadly be defined as ineasuring
characteristics of
individual particles as they pnss generally single file In a fluid stream
through a measuring device
which, typically, provides information for classifying the particles according
to selected
characteristics. Optionally, the particles may then be sepal-a.ted into
populations using any
number of techniques, Including droplet sorting, droplet Interference sorting,
and fluid switching.
Another option Is to selectively destroy csiwanted particles, for example by
photo ablation.
In an optic:ally-based flow cytometry system, optics are used to direct and
focus a beam
of light. (e.g., visible light or UV light) on the Stream containing the
particles, and to collect
emissions 'MITI the particles, Including scattered light arid/or fluorescence
emissions from the
particles. In one common optic system, for example, a beam of light (e.g., a
laser beam) is
focused on the stream and elTlISSi011S are collected by a pair of collection
units, one positioned
forward of the laser for collecting scattered light emissions and another
positioned orthoganally to
both stream and the laser for collecting fluorescence emissions. Each
collection .unit includes a
separate pholocietector, which Increases the cost of the system. rurther, in
traditional optic

CA 02752247 2011-09-07
2
systems the photodetectors translate the collected emissions into electrical
signals, which are
analyzed using analog systems to classify the particles according to selected
characteristics of
the particles. Analog systems are relatively inexpensive, but only limited
Information can be
derived from the signals.
Others have tried to develop technology that can be used to process sperm
cells to
obtain populations of sperm cells that are enriched with sperm that have a
desired sex
chromosome. However, the existing technology falls short of the inventive
technologies
described herein.
For example, Johnson et al. (U.S. Patent No. 5,135,759) describe the
separation of
Intact X and Y chromosome-bearing sperm populations according to DNA content
using a flow
cytometer/cell sorter Into X and Y chromosome-bearing sperm enriched
populations. As
described, the sperm Is combined with a DNA selective dye at a temperature of
30 to 39 C for a
period of 1 hour (39 C) to 1.5 hours (30 C). A flow cytometer is then used to
measure the
amount of fluorescent light emitted as the sperm passes through a laser beam
that excites the
dye, Because the X chromosome-bearing sperm contains more DNA than the Y
chromosome-
bearing sperm, with most species of mammal having about 3 to 5% difference,
the X
chromosome-bearing sperm emits more fluorescent light than the Y chromosome-
bearing sperm.
in order to account for the fact that the fluorescence measurement may vary
depending on the
rotational orientation of the sperm cells, two photo detectors are used. The
first determines
whether the sperm cells are properly oriented, while the second takes a
measurement that is
used to classify the sperm as having an X or Y chromosome. An oscillator is
used to cause the =
stream containing the sperm to break into droplets downstream of the place
where the sperm
pass through the laser beam. Droplets containing single sperm of a
predetermined fluorescent
intensity are given a charge and electrostatically deflected into collection
vessels. The collected,
gender enriched sperm population, is then used for microinjection, in vitro
fertilization, or artificial
insemination.
Seidel et at. (WO 02/43574) also describe separation of sperm into gender
enriched
populations of X and Y chromosome-bearing cells using flow cytometry. Seidel
et al. describe
staining the cells at a temperature between 30 C and 40 C.
United States Patent Application Pub. No. 2003/0157475 A1 (Schenk, August 21,
2003)
describes a method of cryopreserving sperm cells that have been sorted
according to X or Y
chromosome content. As noted therein, it is desirable to add a cryoprotectant
to sperm cells
before they are cryopreserved to protect the sperm cells during the
'cryopreservation process.
For example, glycerol is one cryoprotectant that is commonly added to bovine
sperm cells prior
to cryopreservation. However, in order to obtain better protection from the
cryoprotectant, it Is
desirable to wait for the cryoprotectant to equilibrate with the sperm cells
before subjecting the
sperm cells to temperatures below 0 C. During the equilibration period, the
cryoprotectant
penetrates the cell membrane to provide intra-cellular protection in addition
to any extra-cellular
protection provided by the cryoprotectant. Thus, the cryopreservation methods
described in
United States Patent Application Pub. No, 2003/0157476 A1 specify that an
extender containing

CA 02752247 2011-09-07
3
glycerol Is added to the sperm cells after they have been cooled to about 6 C.
Then the sperm
cells and glycerol are allowed to equilibrate at 6 C for anywhere between 1
and 18 hours before
the sperm cells are subjected to lower temperatures. The disclosure recommends
an
equilibration period of between three and six hours in order to obtain the
best results.
Unfortunately, the time and expense involved in a 3 to 6 hour equilibration
period will
have a negative impact on profitability of a commercial sperm sorting process.
Furthermore, in
the context of a commercial sperm sorting process, it is believed that the
health of the sperm is
generally Improved by reducing the time between collection of the sperm and
cryopreservation
(other factors being equal). From this standpoint as well, it would be
desirable to have access to
cryopreservation technology that does not require a long equilibration period
to obtain the optimal
benefits of a cryoprotectant. Moreover, the known cryopreservation technology
is reported to
have a detrimental impact on sperm motility, which is indicative of decreased
sperm fertility.
Thus, there is a need for cryopreservation techniques that preserves sperm
health compared to
conventional techniques.
SUmmary of the Invention
This invention is directed to an improved system (methods and apparatus) for
analyzing,
classifying.and sorting particles based on one or more desired
characteristics; the provision of
such a system which, In one embodiment, uses flow cytometry to accurately
isolate and sort cells
by DNA content; the provision of such a system which, in certain embodiments,
incorporates
sorting protocols which enable the output of the system to be controlled as a
function of one or
more factors, Including the purity of the desired sorted population of
particles, the rate at which
the desired particle population is collected, the loss of desired particles
not sorted into the
desired population, and other factors; the provision of such a system which,
in one embodiment,
operates at high-speed to provide sex sorted sperm for commercial use by the
animal production
industry; the provision of such a system which can be used to sort cells
without significant
detrimental effect on the cells, Including the motility of sperm cells; the
provision of a system that
can be used to preserve sorted sperm cells until they are needed with minimal
detrimental effect
on the cells, including, the motility of the cells, the provision of such a
system which, as it relates
to the production of sexed sperm, incorporates techniques which increase the
speed and
accuracy of the classification and sorting of the sperm cells; the provision
of a flow cytometry
system which uses epi-illumination optics to detect various characteristics of
particles to be
analyzed and, optionally, sorted; the provision of such an epi-illumination
flow cytometry system
which Is economical to manufacture; the provision of a system which, In one
embodiment,
Incorporates multiple flow cytometry units which share an integrated platform
for classifying and
(optionally) sorting particles, such as cells in general and sperm cells in
particular, at high rates
of production; the provision of such a multi-channel system which share common
components
and systems to reduce variations between the channels for more efficient
operation; and the
provision of such a sorting system which, in one embodiment, incorporates
protocols which

CA 02752247 2013-11-21
4
enable a sample to be quickly tested to determine the quality of the sample so
that the
profitability of further sorting can be evaluated. In addition, this invention
is directed to an
improved system (methods and apparatus) for digitally processing signals
representing
fluorescence; the provision for such a digital system, in one embodiment, for
detecting analog to
digital converted-pulses as a function of background characteristics; the
provision for such a
digital system, in one embodiment, for initializing discrimination=parameters;
the provision for
such a digital system, in one embodiment, for detecting digital information
corresponding to
waveform pulses; the provision for such a digital system, in one embodiment,
for digital
information analysis including feature extraction; the provision for such a
digital system, in one
embodiment, for classifying pulses and defining decisions boundaries; the
provision for such a
digital system, in one embodiment, employing a droplet break-off sensor to
control transducer
amplitude; and the provision for using such a digital system, in one
embodiment, to distribute and
collect cells for commercial distribution. Further, this invention is directed
an improved
comprehensive system (apparatus and methods) for commercial processing of
animal semen
from the time a semen sample is collected from a male animal through
cryopreservation of a
sperm sample containing a greater percentage of a sperm having a desired
chromosome
characteristic than exists in the collected semen; the provision of such a
system, in one
embodiment, that allows efficient processing of commercial quantities of
viable gender enriched
sperm; the provision of such a system that allows, in one embodiment,
adjustment of the system
to counter day-to-day and animal-to-animal variations in the semen
characteristics; the provision
of such a system that, in one embodiment, allows production of about
18,000,000 gender
enriched sperm per hour by a single flow cytometry unit at 85% purity; and the
provision of such
=
a system that allows, in one embodiment, complete processing of a batch of
semen (e.g., the
amount of semen collected from a male animal) to yield viable sperm samples
having a desired
gender characteristic at 86% purity with less than 10% loss of collected sperm
having the desired
gender characteristic in about 1 hour of processing time.

CA 02752247 2013-11-21
4A
In accordance with one embodiment of the present invention, there is provided
a flow cytometry
apparatus nozzle to sort sperm cells in a fluid stream by chromosome content
comprising: a. a nozzle
body having a fluid stream entry portion and a fluid stream exit portion and
an interior surface separating
both portions and along which a fluid stream flows when operating to sort
sperm cells; b. the interior
surface of the nozzle comprising: a first axially tapered region having a
first angle of taper; a second
axially tapered region having a second angle of taper, the second axially
tapered region adjacent to and
downstream of the first axially tapered region; and a third axially tapered
region having a third angle of
taper, the third axially tapered region adjacent to and downstream of the
second axially tapered region,
wherein the angle of taper of the second axially tapered region is different
than the angle of taper of the
first and third axially tapered regions, and wherein at least two of the
axially tapered regions have
generally elliptical cross sectional shapes oriented in different directions
for applying torsional forces to
the fluid stream passing there through, the torsional forces tending to bring
sperm cells in the stream into
a desired orientation.
Another embodiment of the present invention provides a method of orienting
animal sperm cells
in a flow cytometry apparatus, the method comprising the steps of: introducing
a fluid containing sperm
cells into a fluid stream; directing the fluid stream containing the sperm
cells under a pressure in the
range of 20 to 40 psi through a nozzle having three or more adjacent axially
tapered regions and an exit
orifice with a diameter in the range of 50-70 microns; accelerating the speed
of the fluid stream in a
downstream direction toward nozzle orifice through a first axially tapered
region having a first angle of
taper, a second axially tapered region having a second angle of taper and a
third axially tapered region
having a third angle of taper, wherein the second angle of taper is different
than the first angle of taper
and the third angle of taper; and applying a torsional force to the fluid
stream tending to bring the cells
into a desired orientation as they pass through the orifice at a rate in the
range of 30,000 to 50,000 sperm
cells per second with a generally elliptical cross section shape in at least
one of the axially tapered
regions.

CA 02752247 2013-11-21
4B
Brief Description of the Drawings
Fig. 1 is a work flow diagram for an exemplary sperm sorting process of the
present invention;
Fig. 2 is a schematic diagram of one embodiment of a flow cytometry droplet
sorting system of
the present invention;
Fig. 3 is a side view of a portion of one embodiment of a flow cytometry
apparatus of the present
invention for droplet sorting showing an epi-illumination optic assembly
focusing an excitation beam on an
upward moving fluid stream generated by a nozzle system;
Fig. 4 is an end view of one embodiment of a nozzle and nozzle holder of the
present invention;
Fig. 5 is a sectional view of the nozzle and nozzle holder of Fig. 4 taken
through cutting plant 5 - -
5 on Fig. 4;
25

CA 02752247 2011-09-07
Fig. 6 is a schematic diagram of a sperm cell entrained in a fluid stream
being
interrogated by an elliptically shaped beam spot according to one embodiment
of the present
invention;
Fig. 7 is a schematic diagram showing the angular envelope for the desired
orientation of
5 a sperm cell in which the light beam from the optics system-will strike a
wide face of the cell
generally broadside;
Fig. 8 is a cross sectional view of one embodiment of a nozzle body of the
present
invention;
Fig. 9 is a side view of the nozzle body shown in Fig. 8 showing a series of
cutting
planes (9A-A through to 9K-K) through the nozzle body;
Figs. 9A through to 9K are sectional views of the nozzle body as shown in Fig.
9 along
the corresponding respective cutting planes 9A-A through to 9K-K shown in Fig.
9;
Fig. 10 is a perspective view of a cross section of one embodiment of a nozzle
system
having an orienting baffle in the nozzle;
Fig. 11 is a cross sectional view of the nozzle system shown in Fig. 10;
Fig. 12 is an enlarged partial cross sectional view of a portion of the nozzle
system
shown in Figs. 10 and 11;
Fig. 13 is an enlarged partial cross sectional view similar to the view shown
in Fig. 12,
but taken from a direction that is perpendicular to the viewing direction in
Fig. 12;
Fig. 14 is a side view of one embodiment of baffle holder holding a baffle
plate;
Fig. 15 is a top view of the baffle holder and baffle plate shown in Fig. 14;
Fig. 16 is a top view of one embodiment of a baffle holder rotationally
oriented in a
nozzle so that the legs of the baffle plate intersect in a line that is
parallel to the major axis of
ellipse D in the nozzle;
Fig. 17 is a top view of one embodiment of a baffle holder rotationally
oriented in a
nozzle so that the legs of the baffle plate intersect in a line that is
perpendicular to the major axis
of the ellipse D in the nozzle;
Fig. 18 is a sectional view of one embodiment of a nozzle system including a
baffle
showing a series of cutting planes (18A-A through to 18E-E) through the nozzle
and baffle;
Figs. 18A-18E show the cross sectional flow areas at various points in the
nozzle system
shown in Fig. 18 along the corresponding respective cutting planes 18A-A
through to 18E-E
shown in Fig. 18;
Fig. 19 is a cross sectional view similar to Fig. 12 taken through a nozzle
having a baffle
plate that is perpendicular to the longitudinal axis of the nozzle;
Fig. 20 is a cross sectional view of the nozzle shown in Fig. 19 taken through
the cutting
plane 20-20 shown on Fig. 19;
Fig. 21 is a cross sectional view similar to the cross sectional view of Fig.
18 showing a
nozzle system having a sample introduction conduit at an offset location;
Fig. 22 is a perspective view of one embodiment of a nozzle system mounted on
a
nozzle mount of the present invention;

CA 02752247 2011-09-07
6
Fig. 23 is schematic diagram of a plurality of aligned sperm cells being
rotationally
oriented as they pass through an orifice member of the present invention
toward the interrogation
location;
Fig. 24 is a schematic diagram showing the droplet break-off location
downstream from
the nozzle according to one embodiment of the present invention;
Fig. 25 is a schematic diagram of one embodiment of a break-off sensor system
of the
present invention;
Fig. 26 is a front elevation of one flow cytometry system of the present
invention;
Fig. 27 Is an enlarged perspective view of a portion of the system shown in
Fig. 26 with
'10 parts of the system removed for clarity;
Fig. 28 Is a schematic diagram of one embodiment of an epi-illumination optics
system of
the present invention;
Fig. 29 is a perspective view of one embodiment of an epi-illumination optics
system of
the present invention;
Fig. 30 is a side view of the epi-illurnination optics system shown in Fig:
29;
Fig. 31 is a top view of the epi-illumination optics system shown in Figs. 29
and 30;
Fig. 52 is a sectional view of the epi-illumination optics system along the
cutting plane
32--32 of Fig. 30;
Fig. 33 is a sectional view of a portion of the epi-illumination optics system
along the
cutting plane 33'--33' of Fig. 31;
Fig. 34 is a perspective view showing only elements of the optical filtering
system that
are rearward of the dichroic filter of the epi-illumination optics system
shown in Fig. 29;
Fig. 35 is a perspective view of another epi-illumination optics system of the
present
invention including translational adjustment of the cylindrical lens;
Fig. 36 is a schematic diagram of an interrogation location of one embodiment
of the
present invention showing a laser beam focused on a fluid stream downstream of
the nozzle at a
skewed angle of incidence;
Fig. 37 is a schematic diagrarn of one embodiment of a sort calibration system
of the
present invention;
Fig. 38 is a schematic diagram of one embodiment of an epi-illumination sensor
for use
with the sort calibration shown in Fig. 37;
Fig. 39 is a block diagram of one embodiment of a digital cell analyzer (DCA)
and
processor controller according to the invention.
Fig. 40 is a schematic diagram of one embodiment of a multi-channel sorter of
the
present invention showing two channels;
Fig. 41 is a work flow diagram of one embodiment of a multi-channel sorter of
the
present invention showing four channels;
Fig. 42 is block diagram of one embodiment of an analog cell analyzer (ACA)
according
to the invention;

CA 02752247 2011-09-07
7
Fig. 43 is a graph illustrating a stream of waveform pulses from a
photodetector output
detecting fluorescent pulses from cells streaming at an average rate of 10,000
cells/second;
Fig. 44 is an exploded view of Fig. 43 illustrating the stream from a
photodetector output
detecting three fluorescent pulses from three cells streaming at an average
rate of 10,000
cells/second; a square wave of a 100MHz droplet clock has been superimposed on
the
illustration to show the synchronization between the three pulses and the
square wave pulses of
the droplet clock;
Figs. 45 illustrates movement of a sperm cell relative to a laser beam spot
having a
narrow width;
Fig. 46 illustrates similar movement of the same sperm cell in Fig. 45 but at
a different
point in time as the sperm cell traverses the laser shaped bearn spot;
Fig. 47 illustrates similar movement of the same sperm cell in Figs. 45 and 46
but again
at a different point in time as the sperm cell traverses the laser shaped beam
spot;
Fig. 48 Illustrates similar movement of the same sperm cell in Figs. 45, 46,
and 47 but
once again at a different point in time as the sperm cell traverses the laser
shaped beam spot;
Fig. 49 is an exemplary illustration of the digital information corresponding
to a time-
varying analog output from a photodetector detecting a single fluorescence
pulse based on 122
samples at a 105MHz continuous sampling rate;
Fig. 50 is a schematic diagram illustrating the timing relationship between
laser pulses,
fluorescence emissions from a cell resulting from the laser pulses and the
digital samples of the
photodetector output in one embodiment of the invention;
Fig. 51 is a schematic diagram illustrating how the digital samples shown in
Fig. 50 form
a pulse waveform;
Fig. 52 is a schematic diagram of a pulse waveform from and X sperm cell
synchronized
with the pulse waveform of a Y sperm cell showing higher peak intensity in the
pulse waveform
for the X sperm cell;
Fig. 53 is a schematic diagram of a pulse waveform showing a threshold and
integration
window that can be used for pulse analysis;
Fig. 54 is a histogram of a sample containing X and Y sperm cells showing the
high
resolution attainable with slit scanning techniques;
Fig. 55 is histogram of a sample containing X and Y sperm cells showing the
relatively
poor resolution attained with standard illumination;
Figs. 56-59 show fluorescence histograms and scatter plots of peak vs, area
for sperm
nuclei and live.sperm cells;
Figs. 60-61 illustrate a four-component model,of a fluorescence Intensity
histogram for
sperm cells ¨ Fig. 60 shows raw data and Fig. 61 shows model curves generated
by one
embodiment of an iterative algorithm of the present invention based on the
data shown in Fig. 60;
Figs. 62-63 illustrate a three-component model of a fluorescence intensity
histogram for
sperm cells ¨ Fig. 62 shows raw data and Fig. 63 shows model curves generated
by one
embodiment of an iterative algorithm of the present invention based on the
data shown in Fig. 62;

CA 02752247 2011-09-07
8
Fig. 64 illustrates the non-linear nature of the CSD feature; the top panel
shows average
M plots for X-bearing and Y-bearing sperm cells; the middle panel shows a
graph of the first
derivatives of these average M plots (i.e. M') for signal amplitude values
less than the peak
height of the average Y-bearing fluorescence emission pulse; and the bottom
panel shows the
difference between the first derivatives (M'x ¨ M'y) as a function of signal
amplitude;
Fig. 65 illustrates one embodiment in which the CSD feature is the computed
slope of a
line that passes through two points on the fluorescence emission pulse;
Figs. 66-69 illustrate improved discrimination achieved by use of CSD feature
extraction;
Fig. 70 illustrates a bi-variate sort region set on a scatter plot of CSD vs.
pulse area
scatter;
Fig. 71 illustrates one embodiment of flow cytometry re-analyses for a test in
which the
left panel corresponds to the high recovery/coincident accept sort strategy
(no coincidence abort
strategy) and the right panel corresponds to the high purity/coincident reject
sort strategy
(coincident abort strategy);
Fig. 72 is a work flow diagram of one embodiment of digital signal processing
of the
present invention;
Fig. 73 is an example of a k-Means clustering strategy that may be employed
according
to one embodiment of the present invention;
Fig. 74 is a conceptual illustration and graphical representation of
application of a Bayes
Minimum Error decision rule to pulse feature data as may be employed according
to one
embodiment of the present invention;
Fig. 75 is graphical representation of results obtained using a Bayes Minimum
Error
decision rule and Mahalonobis distance thresh holding as may be employed
according to one
embodiment of the present invention;
Fig. 76 Is a conceptual illustration of moving window statistics to provide
"forgetting" as
may be employed according to one embodiment of the present invention;
Fig. 77 is a graphical representation drift compensation as may be employed
according
to one embodiment of the present invention;
Fig. 78 illustrates a fluid stream containing an exemplary distribution of
particles;
Fig. 79 is a graph showing purity as a function of fluid delivery rate with a
coincident
accept sort strategy;
Fig. 80 Is a graph showing the percentage of desired particles successfully
sorted into
the usable population as a function of fluid delivery rate with a coincident
reject sort strategy;
Fig. 81 is a graph showing the inverse relationship between the percentage of
coincident
droplets accepted for sorting into a population of desired particles compared
to the percentage of
coincident droplets rejected for sorting into that population;
Fig. 82 is a decision flow diagram showing the overall operation of one
embodiment of a
sorting apparatus of the present Invention;
Fig. 83 is a side elevation of a cytometer oriented to produce a stream of
droplets having
a horizontal velocity component and a collection system to collect the
droplets;

CA 02752247 2011-09-07
9
Fig. 84 is an enlarged perspective view of the collection system shown in Fig.
83 shown
relative to the nozzle system and deflector plates;
Fig. 85 is a schematic diagram of one embodiment of a collection system of the
present
invention;
Fig. 86 is a front elevation of an intercepting device of the collection
system shown in
Fig. 83;
Fig. 87 is a side elevation of an intercepting device of the collection system
shown in Fig.
83;
Figs. 88-95 show graphical results of several sperm centrifugation
experiments;
Fig. 96-98 are schematic diagrams illustrating the steps in one embodiment of
a filtration
method of the present invention;
Flg. 99 is a schematic diagram of one embodiment of a filtration system used
to filter
sperm cells;
Fig. 100 is a schematic diagram of another filtration system used to filter
sperm cells;
Figs. 101 and 102 show graphical results of sperm cell filtration experiments;
Fig. 103 is a work flow diagram for one embodiment of a cryopreservation
method of the
present invention;
Fig. 104 shows graphical results for a sperm cell cryopreservation experiment;
Fig. 105 is a work flow diagram for one embodiment of a method of processing
sperm
cells according to the present invention;
Fig. 106 is a perspective view of one embodiment of a multi-channel particle
sorter of the
present invention with parts broken away to show internal features of the
sorter;
Fig. 107 is a perspective view of a manifold system that may be used for fluid
delivery in
the multi-channel particle sorter of Fig. 106;
Fig. 108 is a perspective view of the manifold system of Fig. 107 showing
internal fluid
connections of the manifold system;
Fig. 109 is a perspective view of the particle sorter shown in Fig. 106 with
additional
elements removed or partially removed to better show internal features of the
sorter;
Fig. 110 is a front elevation of the particle sorter shown in Fig. 106;
Fig. 111 is a side elevation of the particle sorter shown in Fig. 106 with the
side wail of
the housing removed to show internal features of the sorter;
Fig. 112 is a side elevation of the particle sorter shown in Figs. 106 (taken
from the side
opposite the side from which Fig. 107 was taken) with the side wall removed to
show internal
features of the sorter;
Fig. 113 is a perspective view of the particle sorter shown in Figs. 106 taken
from an
angle behind the sorter and with the back cover removed to show internal
features of the sorter;
Fig. 114 is a perspective view of a portion of the particle sorter shown in
Fig. 106
showing the mounting of multiple nozzle systems to a cross bar;
Fig. 115 is a perspective view of a portion of the particle sorter shown in
Figs. 106
showing the relative positions of the collection system and other parts of the
particle sorter;

CA 02752247 2011-09-07
lo
Fig. 116 is a schematic diagram of one embodiment of a fluid delivery system
for a multi-
channel sorter of the present invention;
Figs. 117 and 118 are schematic diagrams of two different laser beamsplitting
systems;
Figs. 119 and 120 are perspective views of another multi-channel system of the
present
invention;
Figs. 121-134 show graphical results of various experiments;
Fig. 135 is a schematic diagram of one alternative embodiment for a nozzle
system of
the present invention wherein the nozzle directs the fluid stream through a
capillary tube;
Fig. 136 is a schematic diagram of one embodiment of a photo damage sorting
system
of the present invention;
Fig. 137 is a schematic diagram of an alternative sorting system based on
fluidic switch-
ing that may be used In an apparatus employing the technology of the present
invention; and
Fig. 138 is a schematic diagram of an alternative sorting system based on a
high-speed
droplet interference stream that diverts selected discrete segments of the
fluid stream carrying
the analyzed particles.
Corresponding parts are designated by corresponding reference numbers
throughout the
drawings. A parts list with associated reference numerals for each part
follows. The parts list is
provided with section headings generally corresponding to section headings in
the specification
to facilitate use of the parts list. Generally, each section of the parts list
provides a reference
numeral for the parts that are Introduced for the first time In the
corresponding section of the
Detailed Description.
Parts List with Associated Reference Numerals for Each Part
General Overview
39 Semen Collection
41 Label Semen
41A Add Buffer
43 Quality Control
47 Washing
47A Selection of staining protocol
48 Staining Fluid
49 Staining
51 Incubation
53 Load into Sample Introduction Device of Flow Cytometer
54 Add Sheath Fluid Through Flow Cytometry
55 Sorting
57 Collecting Sorted Sperm
58A Add Collection Fluid
586 Concentrate Sperm Cells
58C Add Cryoextender
59 Load Sorted Sperm into Straws
61 Cryopreservation
63 Packing in Liquid Nitrogen
65 Distribution
67 Sales
69 Storage
71 Artificial Insemination

CA 02752247 2011-09-07
=
11
Flow CytometrV
1 System (Overall)
3 Supply of Carrier Fluid
7 Supply of Sheath Fluid
9 Flow Cytometry Apparatus Having Sorting Capabilities
Fluid Delivery System
17 Carrier Fluid
19 Sheath Fluid
21 Stream of Fluid
10 23 Stream of Particles
Beam of Electromagnetic Radiation
31 Electromagnetic Radiation Emission from Particles
33 Droplets
Particles Contained in Droplets
15 Flow Cytometry Apparatus (Single Channel)
101 Nozzle System
5--5 Cutting plane
103 Nozzle Orifice
105 Transducer
20 107 Droplet Break-off
109 Optics System
115 Interrogation Location
117 Photodetector
119 Sorting System
25 123 First Different Group or Population of Droplets
125 Second Different Group or Population of Droplets
2201 Collection System
131 Processor
Nozzle System
30 133 Cylindrical Flow Body
135 Central Longitudinal Bore
137 Nozzle
139 Funnel-shaped Nozzle Body
141 Passage Through Nozzle Body
35 145 Internally Threaded Counterbore
149 Threaded Projection or Stud
155 0-ring Seal
157 Conduit (Tubular Needle)
167 Annular Space (Gap)
173 Radial Bore in Flow Body (Sheath Fluid)
183 Second Radial Bore (Additional Sheath Fluid)
189 Central Core of Carrier Fluid
191 Outer Co-axial Sheath of Fluid
Cell Orientation
201 Bovine Sperm Cell
205 Paddle-shaped Head
207 Flat Wide Opposite Faces
209 Narrow Edges
211 Sperm Equator
213 Nucleus
215 Tail
217 Nucleus Length
219 Head Length
221 Head Width
223 Overall Length
225 Localized Region Within Nucleus
227 Direction of Stream Flow
229 Angular Envelope in Which Light Beam Strikes Wide Face
R1 Angular Range
P Plane

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12
Nozzle Design
231 Interior of Nozzle Body
233 Interior Surface of Nozzle Body
235 First Axially Tapered Region
237 Second Axially Tapered Region
239 Third Axially Tapered Region
247 Longitudinal Axis of Nozzle
249 Fourth Region Interior of Nozzle
251 Axial Length of Fourth Region
255 Orifice Member
257 Counterbore at Front End of Nozzle
259 First Torsional Zone
261 Second Torsional Zone
271 Torsional Forces
273 Axial Length of First Torsional Zone
275 Axial Length of First Tapered Region
277 Axial Length of Second Tapered Region
279 Axial Length of Second Torsional Zone
=
309 Conical Upstream Surface of Orifice Member
315Cylindrical Downstream Surface of Orifice Member
317 Axial Length of Conical Upstream Surface
327 Axial Length of Downstream Surface
Orienting Baffle
AA A First Angle
BB A Second Angle
20-20 Cutting Plane
2001 Orienting Baffle
2003 Baffle Plate
2005 Baffle Holder
2007 Upstream Leg
2009 Downstream Leg
2015 Line of Intersection
2017 Central Axis of Nozzle Body
2019 Curved Edge of Upstream Leg
2025 Distance Lower Leg Extends Downstream
2027 Overall Length of Baffle Holder
2029 Exterior Diameter of Baffle Holder
2031 Interior Diameter of Baffle Holder
2033 Distance Between Line of Intersection and Center of Nozzle
2035 Upstream End of Baffle
2037 Inclined Surface of Baffle Holder
2039 Side Edges of Downstream Leg
2041 Downstream Edge of Downstream Leg
2049 Gap Between Baffle Plate and Baffle Holder
2051 Inside surface of Baffle Holder
2053 Volume Behind Baffle Plate
2055 Interior Volume of Nozzle
2057 Longitudinal Axis of Cylindrical Baffle Holder
2059 Line Through Major Axis of Ellipse D
2061 Distance Between Injection Needle and Baffle
2067 Downstream End of Baffle Holder
2069 Contact Points Between Baffle Holder and Nozzle
2071 0-Rings
2077 Downstream End of Nozzle Holder (Boss)
2079 Interior Diameter of Boss
2081 Portion of Sheath Fluid Between Core Stream and Nozzle Surface
2087 Cross Section Upstream (A)
2089 Cross Section at Baffle (B)
2091 Cross Section at Baffle (C)
2093 Cross Section at Baffle (D)

CA 02752247 2011-09-07
13
2094 Cross Section Downstream of Baffle (E)
2097 Perpendicular Baffle System
2095 Air Bubble
2099 Perpendicular Baffle Plate
2101 Curved Edge of Perpendicular Baffle Plate
2103 Straight Edge of Perpendicular Baffle Plate
2105 0-ring
2107 Annular Shoulder (Shelf) in Nozzle
2109 Outer Diameter of Sample Injection Needle (Conduit)
2161 Nozzle System Having an Offset Sample Introduction Conduit
Nozzle Mounting and Adjustment
331 Nozzle Mount
333 First Linear Stage
337 Second Linear Stage
339 X Axis
341 Y Axis
343 Third Rotational Stage
345 Z Axis
349 Frame for First Fixed Stage Member
355 Movable First Stage Member
357 Actuator (Micrometer) for First Stage
359 Fixed Second Stage Member
361 Movable Second Stage Member
363 Actuator (Micrometer) for Second Stage
365 Fixed Third Stage Member
371 Movable Third Stage Member
373 Actuator (Micrometer) for Third Stage
375 Generally Upward Direction of Stream Containing Cells
377 Angle of Upward Direction
Transducer and Droplet Formation
379 Collar
383 Terminals
D Diameter of Stream
Break-off Sensor
389 Break-off Sensor
391 Microprocessor
393 Light Source
395 Linear Photoarray (Photodiodes)
401 Lens for Droplet Break-off Sensor
405 Current to Voltage Op-amp Circuits
407 Track/hold Amplifiers
409 Sinevvave Generator (Track/hold Signal)
411 A/D Converter
412 Camera System
413 Strobe
414A Mask
414B Slit-Shaped Opening in Mask
Epi-illumination Optics System
32--32 Cutting Plane
33'--33' Cutting Plane
415 Epi-illumination System
417 Epi-illumination Instrument
419 Longitudinal Optical Axis
425 Beam Spot
427 Axis of Focused Illumination Beam
429 Rectangular Base
431 Reflecting Filter
435 Laser or Arc-lamp
437 Conditioning Lens Assembly

CA 02752247 2011-09-07
14
439 Opening in
441 Side Well of a Dichroic Chamber
443 Dichroic Chamber
445 Retaining Ring
447 Neutral Density Filter
449 Cylindrical Lens
455 Lens Holder
457 Jam Nut
459 Elliptical Cross Section of Beam Spot
461 Clips for Reflecting Filter
463 Filter Holder
465 Angular Face of Filter Holder
467 Openings in Filter Holder
469 Linear Stage for Filter Holder
471 X Axis
473 = Outrigger
475 Actuator for Linear Stage
477 Dichroic Filter
479 Clips for Dichroic Filter
485 Frame for Dichroic Filter
487 Forward Direction
489 Longitudinal Optical Axis of the Optical Instrument
491 Focusing Lens Assembly
497 Fluorescent Pulse Waveform or Signal Emitted by Cell
498 Excitation Spatial Function
501 Microscope Adapter
503 Opening in Front Wall of Dichroic Chamber
505 Front Wall of Dichroic Chamber
507 Focusing Barrel
509 Lens Mount Barrels
511 Focusing Lens
513 Rearward Direction
515 Telescoping Focus Adjustment
517 Collimated Emitted Light
519 Filtering System
521 Emission Filter
523 Emission Filter Holder
525 Opening in Back Wall of Dichroic Chamber
527 Back Wall of Dichroic Chamber
529 Alignment Pellicle Assembly
531 Slider of Alignment Pellicle
533 Rail for Filter Assembly Components
535 Filter Holder for Alignment Pellicle
539 Pellicle Filter Element
541 Clips for Securing Filter Element to Filter Holder
543 Angle for Alignment Pellicle Relative to Optical Axis
545 Fasteners for Securing Slider to Base
547 Parallel Slots in Base
549 Aspheric Lens
551 Holder for Aspheric Lens
553 Frame for Aspheric Lens
559 Spatial Filter
561 Aperture Plates
563 Frame for Spatial Filter Plates
567 Vertical Slit
571 Horizontal Slit
573 Aperture
575 Vertical Dimension
577 Horizontal Dimension
579 Collection Volume

CA 02752247 2011-09-07
583 Plate Holder
587 Fasteners for Plate Holder
589 Backing Member for Aperture Plates
449A Adjustable Mounting Assembly
5 449B Slots
4490 Slots
450 Epi-illumlnation that Reflects Fluorescence Emissions
451 Dichroic Filter
10 Photodetector
591 Mounting Plate for Photodetector
595 Fasteners for Photodetector
Angle of Beam Incidence
15 605 Distance Between Interrogation Location and Nozzle Orifice
609 Beam Axis
A Angle of Incidence
Focused Beam Spot
L1 Length along Major Axis
W1 Width along Minor Axis
Sorting System
627 Charging Device
629 Charged Deflector Plates
631 Charging Element
633 Opening In Charging Element
635 Power Supply for Deflector Plates
5001 Adjustable Mounting Assembly
5003 Mounting Assembly Adjustment Board
5005 Mounting Assembly Backing
5007 Fasteners
5009 Slots
5011 Translation Axis
5013 Translation Axis
5015 Mounting Assembly Adjustment Board
5017 Fasteners
5019 Slots
5021 Fixed Support
5023 Fasteners
5025 Spring
Automat Sort Calibration
4201 Calibration System
4203 Epi-Illumination Sensor
4205 Fiber Optic Cable
4207 Dichroic Filter
4209 Lens System
4211 Fluorescent Emission from Particle in Droplet
4213 Photodetector
4215 Beam Stop
4221 Electrically Insulated Support
4223 Apertures in Electrically Insulated Support
4225 A First Sorted Droplet Stream
4227 A Second Sorted Droplet Stream
4229 A Third Sorted Droplet Stream
Sort System Fault Correction
5047 Debris Removal System for Charging Element
5049 Debris Removal System for Deflector Plates

CA 02752247 2011-09-07
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5051 Support for Charging Element
5053 Vacuum Passage
5055 Vacuum Line
5057 Opening Adjacent Charging Element
5058 Fitting
5059 Compressed Gas Line
5061 Manifold
5063 Air Passages
5064 Openings
5065 Fitting
5066 Side of Deflector Plate
5067 Portions of the Air Passages 5063
Protection of Sorted Sample
4031 Contamination prevention mechanism
4033 Collection Vessel
4035 A Waste Container
4041 Contamination Prevention Mechanism
4043 Pneumatic Actuator
4045 Swing Arm
4047 End of Swing Arm
4051 A Sample Station
4053 A Two-part Pressure Container
4055 A Sample Tube
4059 Upper section of the pressure container
4071 A Spring-biased Swing Arm
4073 Port
4075 A Cam Plate
Fluid Delivery System
645 Syringe Pump
647 Flow Line from Pump to Carrier Supply
649 Vessel for Containing Supply of Carrier Fluid
651 Line from Pump to Injection Needle
657 Supply Line from Syringe Pump to Needle
661 Second Vessel - for Supply of Sheath Fluid
667 Supply Line for Connecting Sheath Fluid to Radial Bore in Nozzle
669 Control Valve in Supply Line
675 Source of Pressurized Gas
679 Air Line for Pressurized Gas
681 Regulator for Controlling Pressure Supplied to Sheath Fluid Tank
683 Two-way Valve in Air Line
Control
689 A/D Converter
Distance Between Nozzle and Droplet Break-off Location
Signal Processing
701 Output Signal From Photodetector
703 Droplet Generation Clock Signals
705 Digital Signal Processing (Digital Cell Analyzer)
707 Digital Signal from ND
715 Input to Control Operation of the Channels by the Microprocessor 131
725 A Rectangular Region
735 PC/Computer Terminal
739 Data Acquisition (HI-11')
741 Initializing Detection Parameters (HH1)
745 Initializing Discrimination Parameters (HH2)
747 Digital Pulse Detection (HH3)
749 Digital Pulse Analysis - Feature Extraction (HH4)
=

CA 02752247 2011-09-07
17
753 Pulse Area (1-11-15)
755 Pulse Peak (HH6)
757 Pulse Discrimination (HH7)
759 Sorting (HH8)
761 Drift Analysis (HH9)
763 Decision Boundary for Bayes Rules
769 Initialize
771 System Check
773 User Interaction
775 Retry (Up to Three Times)
777 Flush
779 Bead Quality Control
781 Aspirate Sample
783 Sample Quality Control
785 Start Sample
787 Sort On
789 Sample Complete
791 Continue Sample
793 Sort Off
795 X/Y Discrimination Optimum
797 Set X/Y Discrimination
799 Discrimination OK
801 Rate Optimum
803 Set Syringe Rate
805 Rate OK
807 System Check
809 System Reset
811 System OK
813 Exemplary Overall Operational Flow
825 Integrator
827 Width/Area Comparator
829 Dynamic Threshold Calculator
831 Pulse Discrimination
* 833 JTAG Port I/0
837 Window Comparator (Area)
839 Pulse Width and Trigger Logic
841 Sort Decision
843 I/0 Controllers
845 Slave Controllers
847 Sort Controller Board
849 USB
851 DSP Board SDRAM
853 Sort Signal
854 Low-Pass Filter
855 I/0 Board SDRAM
857 Processor I/0
859 Peripheral I/0 Bus
861 Sort Pulse Generator
863 Data Management Processor
865 Pulse Detection Processor
867 Feature Extraction Processor
873 Sort Processor
875 OSP Board RAM
OL Inverse Relationship Between Coincident Droplets in Usable Population
Compared to
Coincident Droplets in Unusable Population
Point on Line OL Corresponding to 85% Purity
LL Point on Line OL Corresponding to 60% Collection of Desired Particles
OR Operating Range (Segment of OL Between P and LL)
6000 Raw Data
6001 1st Population of Non-aligned Cells
=

CA 02752247 2011-09-07
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6003 2nd Population of Non-aligned Cells
6005 Aligned Y Population
6007 Aligned X Population
6010 Raw Data
6011 Population of Non-aligned Cells
6015 Aligned Y Population
6017 Aligned X Population
Multi-channel System
1001 Multi-channel System
1003 Flow Cytometry Units
1007 Common Source of Electromagnetic Radiation
1009 Common Housing
1011 Common Input for Control
1019 Common Output
1021 Commoh Fluid Delivery System
1023 Common Temperature Control System
1027 Common Waste Recovery System
1029 Common Deflector Plate System
1031 Common Cleaning System
Common Housing =
1069 Base
1071 Two Side Walls
1073 Lower Pair of Shoulders
1075 Lower Cover Panel
1077 Front of Housing
1081 Upper Pair of Shoulders
1083 Upper Cover Panel
1085 Rear of Housing
1087 Framework for Mounting Multiple Cytometry Units
1089 Cross Bar Affixed to Side Walls of Housing (For Attaching Nozzle Mounts)
1093 Angled Mounting Plate Extending Between Side Walls
Common Fluid SupolY
1105 Pump for Carrier Fluid
1107 Common Supply of Carrier Fluid
1115 Gas Pressure System for Sheath Fluid
1117 Common Supply of Sheath Fluid
1121 Manifold System
1123 Vessel Containing Common Supply of Carrier Fluid
1125 Holder for Vessel
1133 Holding Block
1135 Cavity for Receiving Vessel
1137 Second Cavity for Buffer Material
1139 Vessel for Buffer Material
1141 Syringe Pump
1147 Supply Line from Syringe Pump to Manifold
1149 Three-way Valve Controlling Carrier and Buffer Fluid
1155 Vessel for Common Supply of Sheath Fluid
1157 Supply Line from Sheath Fluid Vessel to Manifold
1161 Source of Pressurized Gas
1163 Gas Line
1165 Regulator in Gas Line
1167 Two-way Valve for Gas Line Between Gas Source and Sheath Fluid Tank
1169 Gas Line for Pressurizing a Supply of Cleaning Solution
1173 Tank for Cleaning Solution
1175 Two-way Valve for Gas Line for Cleaning Solution
1177 Manifold
1179 Laminated Block

CA 02752247 2011-09-07
19
1181 Passages
1185 Fluid Flow Circuit
1189 Inlets Connected to Syringe Pump
1191 Inlets Connected to Supply of Sheath Fluid
1193 Outlets for Carrier Fluid and Sheath Fluid
V1-V6 Valves for Controlling Flow Through Manifold Passages
1203 Frame Member (For Attaching Manifold Block)
1205 Fittings Threaded into Block
1207 Sample Reservoir
V1A-V1D Two-way Valves (For Controlling Flow of Sample Fluid to Nozzles)
1217 Needle of Sample Reservoir
1221 Waste System
1223 Waste Tank (Receptacle)
1225 Mechanism Such as Vacuum Pump (For Generating Vacuum)
1227 Waste Lines (Connecting Valves VIA-V1D to Waste Tank)
1233 Hydrophobic Filter (In Line Connecting Waste Tank and Vacuum Pump)
1235 Fluid Circuit for Sheath Fluid
V2A-V2D Two-Way Valves (For Controlling Flow of Sheath Fluid to
Nozzles)
1241 Sheath Supply Line
1247 Waste Lines Connecting Sheath Fluid Flow Circuitry to Waste Tank
Common Power Supply and Controls
1251 Common Power Delivery Systems
Common Temperature Control
1257 Temperature Control System
1259 Fluid Flow Circuit (For Temperature Control)
1263 Fluid Passages (For Temperature Control in Holding Block)
1265 Control Unit
1269 Fluid Passages (For Temperature Control in Manifold)
V6 = Shut off Valve
Common Light Beam and Beam Splitting System
1270 Beamsplitter
1270A First Beam from Beamsplitter
1270B Second Beam from Beamsplitter
1271 Second Beamsplitter
1271A First Beam from Second Beamsplitter
1271B Second Beam from Second Beamsplitter
1272 Third Beamsplitter
=
1272A First Beam from Third BeamSplitter
1272B Second Beam from Third Beamsplitter
1273 Beam Guidance System
1279 Lower Filter Assembly
1281 Upper Mirror Assembly
1285 Base (For Lower Filter Assembly)
1289 Stage (For Lower Filter Assembly)
1291 Mechanism for Moving Stage (Micrometer)
1293 Tiltable Platform on the Stage
1295 Mirror (On Platform)
1297 Base (For Upper Mirror Assembly)
1299 Stage (For Upper Mirror Assembly)
1301 Tiltable Platform (For Upper Mirror Assembly)
1303 Mirror (For Upper Mirror Assembly)
1305 Mechanism for Moving Upper Stage
1309 Target Plates (Affixed to Side Wall of Housing)
1311 Vertically Aligned Holes (In the Target Plates)
1315 1st Reflecting Filter
1317 2nd Reflecting Filter
1319 3rd Reflecting Filter

CA 02752247 2011-09-07
1321 4th Reflecting Filter
Common Deflector Plates
1331 Two Common Deflector Plates
5 1333 Frame (For Mounting Common Deflector Plates on Housing)
Modular Multi-Channel System
4001 Multi-Channel System
4009 Modular Cytometry Unit
10 4011 Housing for Modular Unit
4013 Laser
4015 Beam Splitting and Guidance System
4019 Hole
4021 Common Housing
Capillary Tube Nozzle System
1335 Capillary Tube Nozzle System
1337 Capillary Tube
1341 Chamber Filled with Light-transmitting Medium
1343 Focusing Lens
Alternative Sorting Systems
1355 Collection Receptacle
1357 Fluid Switching System
1359 Fluid Switching Device
1361 Capillary Branch to First Collection Vessel
1365 Capillary Branch to Second Collection Vessel
1367 Transducer (For Creating Pressure Waves for Selectively Controlling
Direction of Fluid
Flow)
1369 Capillary Tube on End of Nozzle
1371 Droplet Interference Stream Sorting System
1375 Droplet Generation System for High-Speed Droplet Stream
1377 High-Speed Nozzle System
1379 High-Speed Fluid Stream
1381 Transducer for Droplet Interference Stream Generation
1383 High-Speed Droplets
1387 Electric Deflection Plate for High-Speed Droplet Deflection
1389 Uncharged Droplets
1.391 Charged Droplets
1397 Diverted Segment of Fluid Stream
1399 Intersection of High-Speed Droplet Stream with Coaxial Fluid Stream
1403 Collection Capillaries
Collection System
2201 Collection System
2203 Intercepting Device
2205 Impact Surface
2207 Collection Vessel
2211 Droplet Entryway
2213 Bulb of Pipette
2215 Pipette
2217 Inside Wall of Pipette
2225 Guide Tube
2227 Collection System Frame
2229 Circular Holder
2231 Set Screw for Intercepting Device Height
2233 Mounting Plate
2235 Set Screws for Lateral Adjustment

CA 02752247 2011-09-07
21
2243 Tray for Holding Collection Vessels
2245 Exit Window
2247 First Intercepting Device
, 2249 Second Intercepting Device
2265 Stray Droplets
Collection Fluid
2301 Collection Fluid
Filtration
2401 Filter
2403 Collection Vessel for Filtration
2405 Concentrated Slurry Containing Sperm Cells
2409 Syringe Mechanism
2411 Cannula Filter
2413 Resuspension fluid
2415 A Filter
2417 A Low Level Vacuum
2419 Second Container
2421 Syringe for Filtration Experiment
2423 Sample for Filtration Experiment
2425 Filter for Filtration Experiment
2427 Vacuum Pump for Filtration Experiment
2431 Syringe for Filtration Experiment 11
2433 Sample for Filtration Experiment 11
2435 Filter for Filtration Experiment II
2437 Filter Holder for Filtration Experiment!!
Cryopreservation
2501 Adjust Concentration
2503 Add Cryoprotectant
2505 Add Protein Source
2507 Load in Straws
2509 Cool to Holding Temperature
2511 Maintain at Holding Temperature
2513 Cool to Temperature Approaching Critical Zone
2515 Cool Through Range of Ice Crystal Formation
2517 Immerse in Liquid Nitrogen
Method of Processing Sperm Cells
2601 Semen Samples Collected at about 37 C
2603 Semen Samples Transported to Sperm Sorting Facility in a Controlled
Environment
2605 Semen Evaluation at 20 C - 37 C
2607 Stain at >40 C
2609 Water Bath
2611 Flow Cytometer
2613 Concentration
2615 Addition of Extenders, Protein Source Cryo Protectant, etc at Room
Temperature
2617 Load Into Straws
2619 Cryopreserve
Common Collection System
2801 Common Collection System
2803 Common Frame for Intercepting Devices
2805 Waste Trough
2807 Tray for Collection Vessels
Pulsed Laser System
3003 Laser Pulse Sensor

CA 02752247 2011-09-07
22
Detailed Description of Embodiments
The embodiments described below relate to collection and processing of animal
semen,
particularly to processing semen from a domestic animal to sort the sperm
cells according to a
specified DNA characteristic (e.g., X/Y chromosome content to preselect the
gender of offspring).
A number of inventive technologies are combined to achieve the results
described below.
However, It will be understood that the inventive technologies described
herein may be applied to
other applications without deviating from the scope of this Invention.
General Overview
Figure 1 is a work flow diagram providing an overview of the steps in one
exemplary
process of the present invention. The process starts with collection of neat
semen samples from
one or more male animals (e.g., bulls) at step 39. The semen samples are
labeled for
identification at step 41 , contacted with a buffer, at step 41A and
transported to a processing
facility. In addition to the buffer, additives may also be added at step 41A,
including, for example,
an energy source, a protein source, an antibiotic, and/or a composition which
regulates
oxidation/reduction reactions intracellularly and/or extracellularly. An
optional quality control test
may be performed at step 43 to insure that the quality of each sample (e.g.,
sperm motility) is
sufficient to indicate that the final product is likely to meet minimal
quality criteria. An optional
washing step may be performed at step 47. At step 47A the staining protocol
that will be used
for processing Is selected by using various staining protocols to stain
aliquots of the sample and
then analyzing the sortability of each aliquot to identify a desired staining
protocol for that
particular sample. Staining according to the selected staining protocol is
performed at step 49 by
adding a staining fluid 48 containing a chemical dye (e.g., a DNA selective
fluorescent dye) to
each sample. In addition to the staining fluid, additives may also be added at
step 48, including,
for example, an energy source, a protein source, an antibiotic, and/or a
composition which
regulates oxidation/reduction reactions intracellularly and/or
extracellularly_ The samples are
incubated at step 51 to allow for uptake of the dye by the sperm. Then a
sample is loaded into
the sample introduction device of a flow cytometer at step 53. The sample
fluid Is introduced into
the flow cytometer along with a sheath fluid at step 54. In addition to the
sheath fluid, additives
may also be added at step 54, including, for example, an energy source, a
protein source, an
antibiotic, and/or a composition which regulates oxidation/reduction reactions
intracellularly
and/or extracellularly. At step 55 the flow cytometer sorts the sperm cells
according to a
specified DNA characteristic, as will be described below. As the sorted sperm
cells are collected
by the collection system of the flow cytometer at step 57, they are added to a
collection vessel
that contains a collection fluid or cryoextender at step 58A. In addition to
the collection fluid,
additives may also be added at step 58A, Including, for example, an energy
source, a protein
source, an antibiotic, and/or a composition which regulates
oxidation/reduction reactions
intracellularly and/or extracellularly. By this time the sperm cells are in a
solution that has been
diluted by the various fluids added throughout the process. Accordingly, the
population of sperm
cells having the desired DNA characteristic are concentrated at step 58B for
use in commercial

CA 02752247 2011-09-07
23
artificial insemination. A cryoextender is added to the concentrated sorted
sperm cells at step
68C. In addition to the cryoextender, additives may also be added at step MC,
including, for
example, an energy source, a protein source, an antibiotic, and/or a
composition which regulates
oxidation/reduction reactions intracellularly and/or extracellularly. The
sperm cells are then
packed in tubular containers (referred to in the breeding Industry as
"straws") at step 59 and
cryopreserved at step 61. The cryopreserved sperm are packed for storage in
liquid nitrogen at
step 63. The cryopreserved sperm are then distributed through a commercial
distribution system
at step 65 and sold to animal breeders at step 67. The animal breeders may
store the
cryopreserved sperm at step 69 until they are ready to use the sperm to
artificially Inseminate a
female animal (e.g., cow) at step 71. As will be discussed below, one
embodiment of the present
invention involves temperature control through substantially the entire
process. Likewise,
completion of the various steps within defined time limits is one aspect of
another embodiment of
the present invention. This overall process is only one example of how the
present invention can
be used, and it will be understood that some of the aforementioned steps can
be deleted and/or
others added. The sorted sperm cells can also be used for microinjection or
other In vitro
fertilization, followed by embryo transplant Into a recipient female anirnal.
The steps of the overall process incorporating advances of the present
invention are
described in detail below. While a particular process described is in the
context of sorting animal
sperm (e.g., bovine sperm), it will be understood that the various aspects of
this invention are
more generally applicable to any type of sperm (equine, porcine, and others),
even more
generally to any type of cells, and even more generally to any type of
particles, organic and
inorganic, including latex particles, magnetic particles, chromosomes, sub-
cellular elements,
protoplasts, and starch particles. These particles generally fall within a
size range of 0.5 to 200
microns, but the technology of thls invention is not limited to this range.
Sample Collection and Dilution
Sampie Collection
The sperm sample to be sorted may be a freshly collected sample from a source
animal,
such as bovine, equine, porcine, or other mammalian source, or a thawed,
previously
cryopreserved sample. Moreover, the sample may be a single ejaculate, multiple
pooled
ejaculates from the same mammal, or multiple pooled ejaculates from two or
more animals.
Various collection methods are known and Include the gloved-hand method, use
of an
artificial vagina, and electro-ejaculation.
The sperm are preferably collected or quickly
transferred into an Insulated container to avoid a rapid temperature change
from physiological
temperatures (typically about 35 C to about 39 C). The ejaculate typically
contains about 0.5 to
15 billion sperm per milliliter, depending upon the species and particular
animal.
Regardless of the method of collection, an aliquot may be drawn from the sperm
sample
and evaluated for various characteristics, such as for example, sperm
concentration, sperm
motility, sperm progressive motility, sample pH, sperm membrane integrity, and
sperm
morphology. This data may be obtained by examination of the sperm using, for
example, the
Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known
procedures (see,

CA 02752247 2011-09-07
24
for example, Farrell et al. Theriogenology (1998) 49(4): 871-9; and U.S.
Patent Nos. 4,896,966
and 4,896,967).
Dilution
The sperm sample may be combined with a buffer (in the form of a solid or
solution) to
form a sperm suspension. Among other things, the buffer may enhance sperm
viability by
buffering the suspension against significant changes in pH or osmotic
pressure. Generally, a
buffer Is non-toxic to the cells and is compatible with the dye used to stain
the cells. Exemplary
buffers include phosphates, diphosphates, citrates, acetates, lactates, and
combinations thereof.
Presently preferred buffers include TCA, TEST, sodium citrate, HEPES, TL, TES,
citric acid
monohydrate, HEPEST (Grad(pore, St. Louis, MO), PBS (Johnson et al., Gamete
Research,
17:203-212 (1987)), and Dulbecco's PBS (Invitrogen Corp., Carlsbad, CA).
One or more buffers may be combined together or with additives as discussed
below to
form a buffered solution, and the buffered solution combined with the sperm
sample to form a
sperm suspension. A buffered solution may also contain one or more additives,
as described in
greater detail below. Exemplary buffered solutions are described in Table I.
Preferred buffered
solutions include a solution comprising 3% TRIS base, 2% citric acid
monohydrate, and 1%
fructose (w/v) in water at a pH of about 7.0, a solution designated as TCA #1
In Table I, and a
solution designated as TCA #2 in Table I.
=
[rest of page blank]

CA 02752247 2011-09-07
Table I. Buffered Solutions
COMPONENTS TCA#1 TCA#2 -TEST Na Citrate
!ES TL
Sodium chloride (NaCI)
7.6g 5.8'
Potassium chloride (KCI)
0.3g 0.2:
Sodium bicarbonate (NaHCO3) 2.1
Sodium phosphate monobasic
(NaH2PO4-H20)
(-9-2-hydroxyproprionic acid (Na Lactate) 3.68
Magnesium chloride (MgC12)
0.1g L. 0.0/
N-(2-hydroxyethyl)plperazine-N'-(2-
ethansulfonlc acid) (HEPES)
.2.38g , 2.31
trls(hydroxymethyl) amimonethane (TRIS
base) _ 30.3g 32.02g 10.28g
Citric Acid Monohydrate
16.75g 18.68g
Na Citrate Dihydrate
29g
2-[(2-hydroxy-1,1-bis[hydroxymethyl]
ethyl) aminoethanesulfonlc acid (TES) 43.25g
Fructose 12.5g 2.67g 10g
2.52g
D-Glucose
2g _
-Steptamycin 0.25g
Penicillin-G 0.15g
Water 1 liter 'I liter 1 liter 1
liter 1 liter 1 lit,
Target pH 7.35 7.35 7.35 7.35
7.35 7.3
Target osmolality (milliosmols/kg H20) -314 -300 -302 -
316 -298
Alternatively, the sperm may be combined with a metabolic inhibitor to form an
inhibited
sperm suspension. Metabolic inhibitors cause the sperm cells to emulate sperm
cells of the
5 epididymis of a mammal, such as for example a bull, by simulating the
fluid environment of the
epididyrnis or epididymal tract of the mammal. Such an Inhibitor would reduce
or inhibit the
motility and metabolic activity of the sperm. Exemplary inhibitors of this
class include carbonate
based inhibitors, such as for example those disclosed in Salisbury & Graves,
J. Reprod. Fertil.,
6:351-359 (1963). A preferred Inhibitor of this type comprises NaHCO3, KHCO3,
and
10 C81-1807.H20. A more preferred inhibitor of this type comprises 0.204g
NaHCO3, 0.433g KHCO3,
and 0.473g C6H307.1-120 per 25mL of purified water (0.097 moles/L of NaHCO3,
0.173 moles/L of
KHCO3, 0.090 moles/L C81-1307-H20 in water).
In addition to a buffer, the sperm suspension may also contain a range of
additives to
enhance sperm viability or motility. Exemplary additives include energy
sources, protein
15 sources, antibiotics, and compositions which regulate
oxidation/reduction reactions intracellularly
and/or extracellularly. One or more of these additives may be introduced Into
the buffer or
buffered solution before the formation of the sperm suspension or,
alternatively, may be
separately introduced into the sperm suspension.

CA 02752247 2011-09-07
26
One or more energy sources may be added to minimize or inhibit the sperm cells
from
oxidizing intracellular phospholipids and other cellular components. Exemplary
energy sources
Include monosaccharides, such as fructose, glucose, galactose and mannose, and

disaccharides, such as sucrose, lactose, maltose, and trehalose, as well as
other
polysaccharides. For example, the resulting sperm suspension may Include about
1% (w/v) to
about 4% (w/v) of the energy source(s). If included, the energy source is
preferably fructose and
the sperm suspension contains about 2.5% (w/v).
To minimize dilution shock, provide support to the cells, or disperse the
cells throughout
the suspension, a protein source may also be included In the buffer, buffered
solution, or sperm
suspension. Exemplary protein sources include egg yolk, egg yolk extract, milk
(including heat
homogenized and skim), milk extract, soy protein, soy protein extract, serum
albumin, bovine
serum albumin, human serum substitute supplement, and combinations thereof.
Albumin, and
more particularly bovine serum albumin (BSA), is a preferred protein source.
For example, if
included, BSA may be present in the sperm suspension in an amount of less than
about
5.0% (w/v), preferably less than about 2% (w/v), more preferably less than
about 1% (w/v), and
most preferably in an amount of about 0.1% (w/v).
The use of a protein source, such BSA, alone may initiate the process of
capacitation in
a percentage of the sperm cells in the suspension. It Is preferred that this
process take place in
the female reproductive tract. Therefore, in order to inhibit the initiation
of capacitation during
dilution, as well as during the subsequent staining and sorting, an
alternative protein source or a
protein substitute may be included in the sperm suspension. The alternative
protein source or
protein substitute possess the advantageous effects of a typical protein
source, such as BSA, in
addition to the ability to inhibit the initiation of capacitation in a larger
percentage of the cells in
the sperm suspension. Examples of alternative protein sources include human
serum substitute
supplement (SSS) (Irvine Scientific, Santa Ana, CA) and cholesterol enhancer
BSA, while an
example of a protein substitute includes a polyvinyl alcohol, such as for
example, a low to
medium viscosity polyvinyl alcohol generally of a molecular weight of about
30,000 to about
60,000. Generally, if included, these compositions will be present in the same
amounts as
disclosed above with respect to BSA, with the total albumin content of the
buffer or buffered
solution generally not exceeding about 5.0% (w/v).
An antibiotic may be added to the sperm suspension in order to inhibit
bacterial growth.
Exemplary antibiotics include, for example, tylosin, gentamicin, lincomycln,
spectinomycin, Linco-
Spectin (lincomycln hydrochloride-spectInomycin), penicillin, streptomycin,
tIcarcillin, or any
combination thereof. The antibiotics may be present in a concentration of
about 5014 to about =
80014 per ml of semen, regardless of whether the semen is neat, buffered, or
contains
additional substances, such as for example, any of the additives mentioned
herein. The Certified
Seinen Services (CSS) and National Association of Animal Breeders (NAAB) have
promulgated
guidelines regarding the use of antibiotics with respect to sperm collection
and use.
A composition which regulates oxidation/reduction reactions intracellularly
and/or
extracellularly may also be included in the sperm suspension. Such a
composition may provide

CA 02752247 2011-09-07
27
a protective effect to the sperm cells, such as for example by maintaining
sperm viability or
progressive motility. Examples of such a composition include, for example,
pyruvate, vitamin K,
Ilpoic acid, glutathione, flavIns, quinones, superoxide dismutase (SOD), and
SOD mimics. If
included in the sperm suspension, such a composition may be present In a
concentration
sufficient to effect the protective effect without detrimentally affecting
sperm health. Exemplary
concentration ranges include from about 101iM to about 60mM depending upon
such factors as
the particular composition being used or the concentration of sperm in the
suspension. For
example, pyruvate may be present in the sperm suspension in a concentration
from about 1mM
to about 60mM, preferably from about 2.5mM to about 40mM, more preferably from
about 5mM
to 25mM, even more preferably from about 10mM to 15mM, still more preferably
about 15mM,
and most preferably about 10mM, Vitamin K may be present in the sperm
suspension in a
concentration from about 10M to about 100E1M, preferably from about 100M to
about 1000M,
and more preferably about 1000M. Lipoic acid may be present in the sperm
suspension in a
concentration from about 0.1mM to about 1mM, preferably from about 0.5mM to
about 1mM, and
more preferably about 1mM.
Staining of the Cells to be Sorted
Generally, sperm cells may be stained by forming a staining mixture comprising
sperm
cells, a buffer, and a dye. The sperm cells may be derived from a freshly
obtained semen
sample, as discussed above with respect to sample collection and dilution, or
from a thawed
cryopreserved semen sample.
If the semen sample is a thawed, previously cryopreserved sample, the sperm
are
preferably thawed immediately prior to staining. Generally, a straw or other
cryopreservation
vessel containing the frozen sperm may be placed in a water bath, the
temperature of which is
preferably in excess of the glass transition temperature of the sperm cell
membrane (i.e., about
17 C), but not so great as to adversely impact sperm health. For example,
frozen sperm may be
thawed by immersing the cryopreservation vessel in a water bath maintained at
a temperature of
about 17 C to about 40 C for a period of about 30 seconds to about 90 seconds.
Once obtained, the sperm cells may be introduced into the staining mixture in
the form of
neat semen or in the form of a suspension derived therefrom, e.g., a sperm
suspension as
discussed above with respect to sample collection and dilution.
The dye may be in the form of a neat solid or a liquid composition. The dye
may also be
dissolved or dispersed In an unbuffered liquid to form a dye solution.
Alternatively, the dye may
be In the form of a dye sUspenslon comprising a dye and a buffer or buffered
solution that is
biologically compatible with sperm cells. A range exemplary buffers and
buffered solutions are
discussed above with respect to sample collection and dilution. For example,
among the buffers
which may be used is a TCA buffer solution comprising 3% TRIS base, 2% citric
acid
monohydrate, and 1% fructose in water at a pH of about 7.0, or a carbonate-
based inhibitor
solution comprising 0.204g NaHCO3, 0.4339 KHCO3, and 0.473g Ce1-1807=H20 per
25mL of
purified water (0.097 moles/L of NaHCO3, 0.173 moles/L of KFIC03, 0.090
moles/L C6H807.H20

CA 02752247 2011-09-07
28
in water). Thus, for example, a staining mixture may be formed by combining
neat semen with a
dye. Alternatively, the staining mixture may be formed by combining neat semen
with a buffer or
buffered solution and a dye. Additionally, the staining mixture may be formed
by combining a
sperm suspension with a dye.
The staining mixture may be formed by using one or more UV or visible light
excitable,
DNA selective dyes as previously described in U,S. Patent No. 5,135,759 and WO
02/41906.
Exemplary UV light excitable, selective dyes include Hoechst 33342 and Hoechst
33258, each of
which is commercially available from Sigma-Aldrich (St. Louis, MO). Exemplary
visible light
excitable dyes include SYBR-14, commercially available from Molecular Probes,
Inc. (Eugene,
OR) and bisbenzimide-BODIPY" conjugate 6-([3-((2Z)-2-([1-(difluorobory1)-3,5-
dimethyl-1H-
pyrrol-2-yllmethylenel-2H-pyrrol-5-y1)propanoyljamino)-N-E3-(methyli3-[({446-
(4-methylpiperazin-
1-y1)-1H,3'H-2,5'-bibenzimidazol-2'-
yllphenoxy)acetyl)aminolpropyllamino)propyljhexanamide
("BBC") described in WO 02/41906. Each of these dyes may be used alone or in
combination;
alternatively, other cell permeant UV and visible light excitable dyes may be
used, alone or in
combination with the aforementioned dyes, provided the dye does not
detrimentally affect the
viability of the sperm cells to an unacceptable degree when used in
concentrations which enable
sorting as described elsewhere.
The preferred concentration of the DNA selective dye in the staining mixture
is a function
of a range of variables which Include the permeability of the cells to the
selected dye, the
temperature of the staining mixture, the amount of time allowed for staining
to occur, and the
degree of enrichment desired in the subsequent sorting step. In general, the
dye concentration
Is preferably sufficient to achieve the desired degree of staining in a
reasonably short period of
time without substantially detrimentally affecting sperm viability. For
example, the concentration
of Hoechst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture will
generally be
between about 0.1,uM and about 1.0M, preferably from about 0.1pM to about
700pM, and more
preferably from about 100,uM to about 200pM. Accordingly, under one set of
staining conditions,
the concentration of Hoechst 33342 is preferably about 100,uM. Under another
set of staining
conditions, the concentration of Hoechst 33342 is about 150,uM. Under still
another set of
staining conditions the concentration is preferably about 200pM.
In addition to buffer, other additives may be included in the staining mixture
to enhance
the viability or motility of the sperm; these additives may be provided as
part of the sperm source,
the dye source, or separately to the staining mixture. Such additives include
energy sources,
antibiotics, compositions which regulate oxidation/reduction reactions
intracellularly and/or
extracellularly, and seminal plasma, the first three of which are discussed
above with respect to
sample collection and dilution, and the last of which is discussed below with
respect to collection
fluids. Such additives may be added during the staining techniques In
accordance therewith.
In particular, it has been observed that the inclusion of a composition which
regulates
oxidation/reduction reactions intracellularly and/or extracellularly in the
staining mixture may help
to maintain sperm viabiiity at elevated staining temperatures, at elevated dye
concentrations, at
increased staining 'periods, or any combination thereof. Examples of these
compositions and the

CA 02752247 2011-09-07
29
use of the same are discussed above with respect to buffers and diluents. Such
compositions
may be added during the staining techniques in accordance therewith.
The staining mixture may be maintained at any of a range of temperatures;
typically, this
will be within a range of about 4 C to about 50 C. For example, the staining
mixture may be
maintained at a "relatively low" temperature, i.e., a temperature of about 4 C
to about 30 C; in
this embodiment, the temperature is preferably from about 20 C to about 30 C,
more preferably
from about 25 C to about 30 C, and most preferable at about 28 C.
Alternatively, the staining
mixture may be maintained within an "intermediate" temperature range, i.e., a
temperature of
about 30 C to about 39 C; in this embodiment, the temperature is preferably at
about 34 C to
about 39 C, and more preferably about 37 C. In addition, the staining mixture
may be
maintained within a "relatively high" temperature range, i.e., a temperature
of about 40 C to
about 50 C; in thls embodiment, the temperature is preferably from about 40 C
to about 45 C,
more preferably from about 40 C to about 43 C, and most preferably at about 41
C. Selection of
a preferred temperature generally depends upon a range of variables, Including
for example, the
permeability of the cells to the dye(s) being used, the concentration of the
dye(s) in the staining
Mixture, the amount of time the cells will be maintained In the staining
mixture, and the degree of
enrichment desired in the sorting step.
Uptake of dye by the sperm cells In the staining mixture is allowed to
continue for a
period of time sufficient to obtain the desired degree of DNA staining. That
period is typically a
period sufficient for the dye to bind to the DNA of the sperm cells such that
X and Y
chromosome-bearing sperm cells may be sorted based upon the differing and
measurable
fluorescence intensity between the two. Generally, this will be no more than
about 160 minutes,
preferably no rnore than about 90 minutes, still more preferably no more than
about 60 minutes,
and most preferably from about 5 minutes to about 40 minutes.
Accordingly, in one embodiment, a staining mixture is formed comprising sperm
cells
and a dye in a concentration from about 100pM to about 200pM, and the staining
mixture is held
for a period of time at a temperature of about 41 C. In another embodiment,
the staining mixture
further comprises pyruvate in a concentration of about 10mM, vitamin K In a
concentration of
about 100,uM, or lipoic acid in a concentration of about 1mM.
In still another embodiment, a staining mixture Is forrned comprising sperm
cells and a
dye In a concentration from about 100pM to about 200pM, and the staining
mixture is held for a
period of time at a temperature of about 28 C. In another embodiment, the
staining mixture
comprises pyruvate in a concentration of about 10mM, vitamin K in a
concentration of about
100pM, or lipoic acid in a concentration of about 1mM.
In yet another example, a staining mixture is formed comprising sperm cells, a
metabolic
Inhibitor comprising 0204g NaHCO3, 0.433g KHCO3, and 0.473g Col-1807+120 per
25mL of
purified water (0.097 moles/L of NaFIC03, 0.173 moles/L of KHCO3, 0.090
moles/L C31-1307.H20
In water), and a dye in a concentration from about 100pM to about 200pM, and
the staining
mixture is held for a period of time at a temperature of about 28 C. In
another embodiment, the
staining mixture is held for a period of time at a temperature of about 41 C.

CA 02752247 2011-09-07
Sheath Fluid
To sort the sperms cells, the stained cells are introduced as a sample fluid
into the
nozzle of a flow cytometer as described below. As part of the process, the
sample fluid is
5 typically surrounded by a sheath fluid. The sheath fluid permits the
sperm cells in the sample
fluid to be drawn out into a single file line as discussed below. The sheath
fluid Is collected along
with the sperm cells by the collection system of the flow cytometer and
therefore forms part of the
post-sort environment for the sperm cells. Thus, it is desirable that the
sheath fluid provides a
protective effect to the cells upon contact of cells by the sheath fluid.
10 The sheath fluid generally comprises a buffer or buffered solution.
Examples of buffers
and buffered solutions and illustrative concentrations of the same that may be
used in the sheath
fluid are disclosed above with respect to sample collection and dilution. In a
particular
embodiment, the sheath fluid comprises 0.96% Dulbecco's phosphate buffered
saline (w/v),
0.1% BSA (w/v), in water at a pH of about 7Ø
15 Optionally, the sheath fluid may also contain a range of additives that
are beneficial to
sperm viability or motility. Such additives include, for example, an energy
source, a protein
source, an antibiotic, a composition which regulates oxidation/reduction
reactions intracellularly
and/or extracellularly, an alternative protein source, and polyvinyl alcohol.
Each of these
additives, and examples of the same, Is discussed above with respect to sample
collection and
20 dilution. Such additives may be added to the sheath fluid in accordance
therewith.
The sheath fluid may optionally be filtered prior to the sorting step.
Contaminants that
may be present in the sheath fluid, such as non-soluble particulates, may
interfere with sorting.
Therefore, the sheath fluid may be filtered prior to its introduction Into a
flow cytometer. Such
filters and methods of using the same are well known in the art. Generally,
the filter is a
25 membrane of about 0.1 microns to about 0.5 microns, preferably about 0.2
microns to about 0.3
microns, and more preferably about 0.2 microns.
The stained cells may be introduced into the sheath fluid at any time
subsequent to
staining. Typically, a stream of the stained cells in the sample fluid is
Injected into a stream of
sheath fluid within the nozzle of the flow cytometer. Initially, there is
substantially no contacting
30 of the sample fluid and the sheath fluid due to laminar flow of the
fluids as discussed in more
detail below. It is desirable that the sample fluid and the sheath fluid
remain as substantially
discrete flowing streams until after the particles (e.g., the stained sperm
cells) in the sample fluid
have been analyzed. At some point, however, the sheath fluid and the cells of
the sample fluid
come In contact with one another. For instance in a droplet sorting flow
cytometer (discussed
below) the sheath fluid and sample fluid begin contacting one another as
droplets are being
formed downstream of the interrogation location.
At the time of the introduction of the stained cells and the sheath fluid,
both the stained
cells and the sheath fluid may be at a temperature from about 4 C to about 50
C. The sheath
fluid and the stained cells may be at the same or at different temperatures,
with either being at a
higher temperature than the other. Accordingly, In one embodiment, at the time
of the

CA 02752247 2011-09-07
31
introduction of the stained cells and the sheath fluid, both the cells and the
sheath fluid are at the
same temperature; for example, at a "relatively low" temperature, such as for
example at about
C to about 8 C; at an "intermediate" temperature, such as for example at about
25 C to about
30 C; or at a "relatively high" temperature, such as for example at about 40 C
to about 43 C. In
5 another embodiment, the stained cells are at a higher temperature than
the sheath fluid, such as
for example, the cells being at about 40 C to about 43 C and the sheath fluid
being at about
room temperature or at about 5 C. In yet another embodiment, the stained cells
are at a lower
temperature than the sheath fluid.
FLOW CYTOMETRY
One embodiment of the present Invention employs inventive technologies in flow

cytometry to analyze and sort the sperm cells. Referring now to Figs. 2 and 3,
one embodiment
of a flow cytometry system of the present Invention is designated in its
entirety by the reference
numeral 1. As will appear, the flow cytometry system 1 is useful for
classifying and sorting
particles, such as sperm cells, according to selected characteristics. In
general, the system 1
comprises a supply 3 of carrier fluid 17 containing particles to be sorted, a
supply 7 of sheath
fluid 19, flow cytometry apparatus having sorting capabilities, generally
designated 9, and a fluid
delivery system 15 for delivering the carrier 17 and sheath fluids 19 from
respective supplies 3, 7
under pressure to the flow cytometry apparatus 9. The flow cytometry apparatus
9 is adapted for
receiving the carrier 17 and sheath 19 fluids, for combining the fluids 17, 19
to create a stream of
pressurized fluid 21, for directing the stream 21 carrying the particles
through a focused beam of
electromagnetic radiation 25 (e.g., UV laser light), and for analyzing the
electrornagnetic radiation
31 (e.g., fluorescent light) emitted by particles passing through the focused
beam 25. The
apparatus 9 also functions to break the stream 21 up into droplets 33
containing particles to be
evaluated, and to sort the droplets 33 based on the aforesaid measurements
according to one or
more characteristics of the particles contained in the droplets 33. While this
invention may be
used to analyze and preferably sort any type of particle, it has particular
application to sorting
cells according to one or more desired characteristics of the cells (e.g.,
size, DNA content,
shape, density, gene sequence, etc.). This invention is especially suited for
sorting animal sperm
cells for commercial use by the animal production industry for in vivo or in
vitro artificial
insemination, as discussed in more detail below.
SINGLE-CHANNEL SORTING APPARATUS AND METHOD
Flow Cytometry Apparatus
The flow cytometry apparatus 9 shown in Fig. 3 comprises a nozzle system,
generally
designated 101, for delivering a fluid stream 21 containing particles (e.g.,
stained sperm cells)
through a nozzle orifice 103 under pressure with the cells substantially in
single file and, In the
case of sperm cells, with asymmetric heads of the sperm cells substantially in
a desired
orientation which will be described. As In conventional flow cytometry droplet
sorting systems, a

CA 02752247 2011-09-07
32
transducer 105 is provided opposite the nozzle orifice 103 for introducing
acoustical energy into
the fluid stream 21 which causes the stream 21 to break into droplets 33
containing individual
cells at a "droplet break-off" location 107 spaced from the nozzle orifice
103. The system 1 also
includes an optics system, generally designated 109, for focusing a beam of
electromagnetic
radiation 25 (e.g., 350-700 nm UV or visible laser light) on the fluid stream
21 at an
"interrogation" location 115 which, in the described embodiment, is between
the nozzle orifice
103 and the droplet break-off location 107. Thus, the described embodiment Is
a jet-in-air
system. In other embodiments, the interrogation location 107 could be Inside
the nozzle orifice
103 or upstream from the orifice 103. In any event, the cells are adapted to
pass through the
beam of light 25 at the interrogation location 107, resulting in excitation of
a chemical stain (or
other reporting medium) In the cells to cause fluorescence emissions 31 having
a wavelength
different from that of the beam 25 (e.g., if the illumination light 25 has a
wavelength of about 350
to 37011M, the fluorescent emissions 31 may have a wavelength of about 460
nm). A
photodetector 117 Is operable to detect these emissions 31 and to convert them
into electrical
signals which are processed and used to classify the cells according to
selected characteristics,
such as the X/Y chromosome content of sperm cells. The flow cytornetry
apparatus 9 further
comprises a sorting system, generally designated 119, for sorting the droplets
33 Into different
groups or populations (e.g., two populations 123, 125) according to the
classification of the cells
contained In the droplets 33 and a collection system, generally designated
2201 (Fig. 2), for
collecting the droplets 33 and maintaining the segregation of the different
populations 123, 125.
Operation of the system 1 is controlled by a processor 131, such as
microprocessor or
other digital or analog control and/or processor, or combinations thereof,
which controls the
various functions of the components of the system 1 in a manner to be
described. Significantly,
the processor 131 is also responsive to particle analysis information to
control the output of the
system 1 based on selected control and sorting strategies involving different
parameters,
including the desired purity of one of the sorted populations of particles,
the acceptable quantity
(or percentage) of desired particles one of the populations as compared to the
quantity (or
percentage) of desired particles in one or more of the other populations, and
other parameters,
as will be discussed later.
The various components of the system 1 are described in detail below.
Nozzle System
Referring to Figs. 4 and 5, the nozzle system 101 comprises, in one exemplary
embodiment, a generally cylindrical flow body 133 having a central
longitudinal bore 135 through
it, and a nozzle 137 on the flow body 133 having a funnel-shaped nozzle body
139. A passage
141 extends through the nozzle body 139 co-axial with the bore 136 in the flow
body '133 and
terminates in the aforementioned nozzle orifice 103 at the forward end of the
nozzle 137. The
nozzle body 139 has an internally threaded counterbore 145 at its rearward end
for threadably
receiving a threaded projection or stud 149 at the forward end of the flow
body 133 to removably
connect the nozzle '137 to the flow body 133, the connection being sealed by
an 0-ring seal 165.

CA 02752247 2011-09-07
33
it will be understood that the nozzle can be removably connected to the flow
body in other ways
or, alternatively, the parts could be integrally formed as one piece.
Particles are delivered to the nozzle 137 by means of a conduit 157 positioned
co-axially
in the bore 135 of the flow body 133. The outside diameter of the conduit 167
is less than the
inside diameter of the bore 135 so that an annular space 167 is formed around
the conduit 157.
In one particular embodiment, the conduit 157 is a tubular needle (e.g., a 16-
ga. needle having
an inside diameter of 0.01 in.) having a front end which extends into the
counterbore 145 at the
back of the nozzle 137. The back end of the conduit 157 is connected to the
fluid delivery
system 15 for delivery of carrier fluid 15 (e.g., a staining mixture
containing sperm cells) to the
conduit 157. The annular space 167 surrounding the conduit 157 is connected by
means of a
radial bore 173 in the flow body 133 to the fluid delivery system 15 for
delivery of sheath fluid 19
into the annular space 167. As shown in Figs. 3 and 5, an optional second
radial bore 183 may
be provided in the flow body 133 connecting the annular space 167 to another
line (not shown)
for supply of additional sheath fluid 19 to the nozzle 137.
As in conventional flow cytometry systems, sheath fluid 19 Is introduced into
the annular
space 167 surrounding the conduit 157. The velocity of the sheath fluid 19 as
it flows past the tip
of the conduit 157 is much higher that the velocity of the carrier fluid 17
exiting the conduit 157,
so that the carrier fluid 17 and cells (e.g., sperm cells) contained therein
are accelerated by the
sheath fluid 19 toward the orifice 103 of the nozzle 137. This acceleration
functions to space the
cells out generally in a single file arrangement for separate analysis by the
optics system 109.
The sheath fluid 19 surrounds the carrier fluid 17, resulting In the fluid
stream 21 having a central
core 189 of carrier fluid 17 and an outer co-axial sheath 191 of sheath fluid
19 surrounding the
central core 189 (see Fig. 6). As will be understood by those skilled in flow
cytometry, the
laminar flow and hydrodynamic focusing of the central core 189 tends to
confine the particles to
the core 189, with little mixing of the sheath 19 and carrier fluids 17 in the
nozzle 137. Further,
the central core 189 remains essentially intact within the sheath 191 as the
stream 21 moves
through the nozzle system 101, until such time as droplets 33 are formed at
the break-off location
107. This type of co-axial flow is particularly suited for flow cytometry,
because the particles to
be analyzed are confined within the relatively narrow core 189 of the stream.
As a result, a beam
of light 25 focused on the center or core 189 of the stream 21 will illuminate
the particles so that
they may be analyzed substantially one at a time. By confining the core 189
within a sufficiently
narrow diameter, one can obtain more uniform illumination of the particles in
the core fluid 189.
For good analytical results, the diameter of the core containing the particles
should desirably be
within a range of 7 to 20 microns, and more desirably within a range of 7 to
14 microns. The
diameter of the core stream 189 can be increased or decreased by adjusting the
rate of delivery
of the carrier fluid 17 relative to the rate of delivery of the sheath fluid
19.
Cell Orientation
For optimizing analytical results, it is desirable that particles having
asymmetric shapes
be in a desired orientation when they pass through the light beam from the
optics system. As is

CA 02752247 2011-09-07
34
known to those skilled in the art, fluorescence emissions from asymmetric
particles tend to
anisotropic (i.e., the intensity of the emissions are not uniform in all
directions). As used herein,
the term "desired orientation" means an orientation which allows the
processing system to
discriminate between cells having different characteristics with an accuracy
in a range of 70% to
100%, more desirably in a range of 80% to 100%, still more desirably in a
range of 90% to 100%,
and most desirably 95% or greater.
To Illustrate the point, a bovine sperm cell 201 is illustrated in Figs. 6 and
7. Typically,
the cell has a paddle-shaped head 205 with relatively flat wide opposite faces
207 and narrow
edges 209, a nucleus 213 In the head 205 containing the chromatic DNA mass of
the cell, and a
tail 215 extending from the head 205 providlng the motility necessary for
effective fertilization.
The average bovine sperm cell 201 has a head length 219 of about 8 /iM, a head
width 221 of
about 4 pm, and an overall length 223 from the front of the head to the end of
the tail of about
100 pm. In the average bovine sperm cell 201, the nucleus 213 occupies most of
the head
volume and is only slightly smaller than the sperm head 205. Thus, the nucleus
length 217 is
almost equal to the head length 219, again being about 8 pm in length. It has
been observed
that in the bovine the X/Y chromosomes of the sperm cells 201 are localized in
a region of the
nucleus 225 (Fig. 6) below and immediately adjacent the longitudinal midline
or equator 211 or
center of the head 205. More specifically, this sub-equatorial region 225
extends no more than
about 20% of the nucleus length 217 on the lower half (toward the tail 215) of
the nucleus 213,
even more speclfically no More than about 10-15% of the nucleus length 217 on
the lower half of
the nucleus 213, and still more specifically no more than about 1.0-1.5 pm
below the equator 211
of the nucleus 213.
When sperm cells pass through the excitation beam 25, It is desirable that the
cells be
substantially in single file and that the head 205 of the each cell 201 be
substantially similarly
oriented to reduce orientation variability from cell to cell and thus provide
for a more uniform
measurement of the cells. It Is also desired that the cells have an
orientation which will enable
accurate discrimination between X and Y cells. Desirably, this orientation is
one where the
length of the sperm cell 201 is generally aligned with the direction of stream
flow 227 (eit)ier head
leading (shown Fig. 6) or head trailing) and where the head 205 of the sperm
cell 201 is rotated
on its longitudinal axis so that the head 205 falls within an angular envelope
229 in whlch the
light beam 25 from the optics system 109 will strike a wide face 207 of the
cell 201 generally
broadside, as shown schematically in Fig. 7, rather than a narrow edge 209 of
the cell.
Preferably, the envelope 229 defining the desired orientation is generated by
rotation of a sperm
cell 201 through an angular range of R1 relative to a plane P which is
generally perpendicular to
the incoming light beam 25, as viewed in a cross section taken transversely
through the stream
21. The range R1 is preferably 0 to 90 degrees, more preferably 0 to 60
degrees, and even
more preferably 0 to 30 degrees. The nozzle of the present invention is
configured to achieve
this desired orientation with an accuracy of up to 90% or more.
The tolerance for sperm orientation (i.e., the size of the envelope 229
defined by angular
range R1) is related to the numerical aperture of the lens used to collect
fluorescence emissions

CA 02752247 2011-09-07
33
31 from the sperm cells. In the embodiment shown Fig. 7, for example, the
optics system 109
has a fluorescence emission 31 detection volume 579 defined by a solid angle
of 55 degrees.
When the rotational orientation of a sperm head 205 Is outside the envelope
229 defined by R1
as the sperm moves through the beam 25, a relatively stronger fluorescence
emission 31 from
an edge 209 of the sperm head 205 will be collected by the optic system 109,
preventing the
processor 131 from correlating the intensity of the fluorescence emission 31
with the X/Y
chromosome content of the sperm cell 201. However, the optics system 109 does
riot collect the
relatively stronger fluorescence emissions 31 from the narrow edge 209 of the
sperm heads 205
as long as the rotational orientation of a sperm head 205 is within the
envelope 229 as it passes
through the interrogation location 115. Thus, in the embodiment shown Fig. 7,
the orientation of
the sperm cell does not result in collection of the relatively stronger edge-
wise fluorescence
emissions as long as the narrow edges 209 of the sperm head 205 are confined
within angle R1.
The solid angle of the collection volume 579 can be decreased by using a lens
with a smaller
numerical aperture, thereby increasing angle R1 and the tolerance for poorly
oriented sperm.
However, this also decreases the number of photons that can be collected by
the optics system
109, which can impact the measurement of fluorescence emissions 31 by reducing
the intensity
of the emissions 31 detected by the photodetector. Likewise, if the optics
system 109 collects
fluorescence emissions 31 with a high numerical aperture lens to obtain a
stronger intensity of
the fluorescence emissions detected by the photodetector, then the tolerance
for sperm
orientation decreases. Thus, in designing a system of the present invention,
one needs to strike
a balance between the tolerance for sperm orientation and the numerical lens
aperture. The
optimal balance will depend on the orienting capabilities and optical
sensitivity of the system. In
one desirable embodiment, for example, a lens having a numerical aperture 0.65
is used.
Nozzle Design
in one embodiment, as shown In Figs. 8 and 9, the interior 231 of the nozzle
body 139
downstream from the counterbore 145 has an interior surface 233 comprising
first, second and
third axially tapered regions 235, 237, 239 for progressively accelerating the
speed of the fluid
stream 21 in a downstream direction toward the nozzle orifice 103. As noted
previously, this
acceleration functions to space the particles (e.g., cells) in the stream 21
so they assume a
generally single file formation so they can be analyzed substantially one
particle at a time. At
least two of these regions, and preferably all three 235, 237, 239, have
generally elliptical (oval)
shapes in cross sections taken at right angles to the longitudinal axis 247 of
the nozzle 137, as is
shown in Figs. 9A-9H and Figs. 9J-9K. The Interior surface 233 of the nozzle
body 139 also has
a fourth region 249, not tapered, downstream from the first three regions 235,
237, 239 and
immediately upstream of the nozzle orifice '103 which, in one embodiment, is
formed in a
separate orifice member 255 secured In a counterbore 257 at the front of the
nozzle body 139.
In one embodiment, the generally elliptical cross sectional shapes of the
first 235 and second
237 regions are oriented In substantially the same direction to define a first
torsional zone 259,
and the generally elliptical cross sectional shape of the third region 239,
constituting a second

CA 02752247 2011-09-07
36
torsional zone 261, is oriented at an angle (e.g., about 90 degrees) relative
to the generally
elliptical cross sectional shapes of the first 235 and second 237 regions. The
orientation is such
that the interior surface 233 of the nozzle body 139 applies torsional forces
to the fluid stream 21
and thereby tends to orient the sperm cells 201 In the aforestated desired
orientation as they
pass through the nozzle orifice 103. Preferably, the first torsional zone 259
has an axial length
273 of 3.0-4.5 mm, preferably about 3.6 mm, and the first 235 and second 237
tapered regions
making up the zone 259 have approximately equal axial lengths 275, 277 (e.g.,
about 1.8 mm).
The second torsional zone 261 has an axial length 279 of 3.5-5.0 mm,
preferably about 4.45 mm.
The fourth region 249 is preferably generally cylindrical in shape. Each
generally cross-sectional
elliptical shape A-D (Fig. 8) at the boundaries of the first 235, second 237
and third 239 regions
has a major axis diameter and a minor axis diameter, exemplary dimensions of
which are shown
in Flg. 8 and Table 1 below.
Table 1
Ellipse Major Axis Minor Axis Ratio
Diameter Diameter
(mm) (mm)
A 7.0 6.0 1.2
13 6.1 5.3 1.15
2.1 2.1 1
0.9 0.2 1.45
It will be understood that the above dimensions are exemplary, and that other
dimensions and shapes may also be suitable. Functionally, the changes in the
ratios between
the major and minor diameters, and the different orientations of the
elliptical shapes of the
regions, create side forces which act on each cell 201 and apply a torsional
force 271 tending to
rotate the cell 201 on Its longitudinal axis so that its wide faces 207 align
with the minor axis in
the first torsional zone 259 and as the cell is gently twisted (e.g., 90
degrees) to align with the
minor axis of the second torsional zone 261. Each of the tapered surfaces 235,
237, 239 also
serves to accelerate the stream 21 (and cells) flowing through the nozzle 101.
In one
embodiment, the acceleration increases more gradually in the first 235 and
third 239 regions and
more rapidly in the second region 237. Again by way of example, the taper of
the first region 235
may range from about 11-14 degrees; the taper in the second region 237 may
range from about
42-48 degrees; and the taper in the third region 239 may vary from about 8-12
degrees. The
nozzle body 139 is formed from a suitable material such as molded plastic
(ABS) or metal.
The orifice member 255 (Fig. 8) is preferably formed from a hard, wear
resistant
material, such as sapphire, which is capable of being machined or otherwise
formed with precise
dimensions. The orifice member 255 itself has, in one embodiment, a conical
upstream surface
309 of generally circular cross section which decreases in diameter from about
0.92 mm to about
0.060 mm and has an axial length 317 of about 0.54 mm and a taper angle of
about 39 degrees.

CA 02752247 2011-09-07
37
The orifice member 255 also has a generally cylindrical downstream surface 315
with a diameter
of about 0.060 mm and an axial length 327 of about 0.36 mm. These dimensions
are exemplary
only, and it will be understood that the orifice member 255 may have other
sizes and shapes.
For example, the shape of the upstream surface 309 may be generally elliptical
(oval) in cross
section, and the diameter of the orifice 103 at the downstream end of the
nozzle 137 may range
from 40 to 100 microns or more. It Is desirable that the size of the orifice
103 be such that the
cells exiting the nozzle 101 are substantially In single file formation within
the core 189 of the
stream 21 and substantially in the desired orientation, as described
previously. For example, in
the case of sperm cells an orifice 103 having a diameter of about 60-62
microns at the
downstream end has been found to be suitable. Preferably, the nozzle orifice
103 serves to
further accelerate the stream 21 and to shape and size the stream 21 for
optimum cell spacing,
cell orientation and droplet 33 formation, as will be described.
The velocity of the cells as they exit the nozzle 137 will depend on various
factors,
including the pressure at which sheath fluid 19 is Introduced Into the nozzle
system 101. At a
pressure of 20 psi, the cells will exit the nozzle orifice 103 of the above
embodiment at a velocity
of about 16.6 m/s as a generally cylindrical stream 21 containing cells which
are substantially
similarly oriented at the core 189 of the stream 21. At a sheath pressure of
30 psi, the cell
velocity will be about 20.3 m/s. At different sheath fluid 19 pressures, the
velocity of the stream
21 will vary.
introduction of Core Stream to Torsional Zone
Improved orientation of particles may be obtained by altering the flow of the
fluid stream
21 through an orienting nozzle so that the core stream 189 containing the
particles to be oriented
(e.g., sperm cells) is directed along a flow path, at least a portion of which
Is offset from the
center of the nozzle so that the particles are subjected to the hydrodynamic
orienting forces
generated by a nozzle while they are at a location that is offset from the
center of the nozzle.
Directing the core stream 189 along an offset flow path may also improve
orientation of particles
in a traditional nozzle (i.e., one that does not have any torsional zones). In
many nozzles, one
can determine that a given position is offset from the center of the nozzle
because it is displaced
from a longitudinal axis of the nozzle. One can also recognize that a
particular position is offset
from the center of a nozzle because it is displaced from the geometric center
of a cross sectional
area of the nozzle through which the fluid stream flows.
A number of techniques may be used to direct the core stream 189 along a flow
path that
is offset from the center of the nozzle. For example, an orienting baffle may
be positioned in the
nozzle to deflect the core stream to one side of the nozzle. Similarly, the
conduit '157 for
introducing the core stream 189 containing the sample particles may be
relocated from the
traditional center of the nozzle to an offset location. Furthermore, it is
contemplated that an
offset sample introduction conduit '157 may be used in combination with an
orienting baffle.
Exemplary embodiments of use of an orienting baffle and use of an offset
sample introduction
conduit are discussed below.

CA 02752247 2011-09-07
= 38
The improved orientation of particles (e.g., sperm cells) achieved by use of
an orienting
baffle and/or offset sample introduction conduit 157 may be due to a number of
factors. One
factor Is that the deflection of the core stream 189 and/or a change in the
size and shape of the
cross sectional flow area results In application of hydrodynamic forces that
tend to orient
asymmetric particles. (Kachel, et al., Histochemistry and Cytochemistry,
25(7): 774-80 (1977)).
Another factor is that It has been found that asymmetric particles (in
particular sperm cells) tend
to orient as they flow in a fluid stream in close proximity to a solid
surface. Thus, by directing the
core stream 189 so that it is in close proximity to the Interior surface of a
nozzle or a baffle
surface one can obtain improved orientation of the particles. Furthermore, a
baffle and/or offset
sample introduction conduit can be used in conjunction with an orienting
nozzle which applies
additional orienting forces (e.g., torsional forces) to the asymmetric
particles. In that case, the
baffle can operate to direct the fluid stream so that the core stream
containing the particles to be
oriented flows along a path that is offset from the center of the nozzle while
the particles are
subjected to the torsional forces generated by one or more of the torsional
zones.
Orienting Baffle
Figs. 10-13 show one exemplary orienting baffle, generally designated 2001,
positioned
in the orienting nozzle 137 described above. However, the baffle 2001 could be
used In
conjunction with a different nozzle, including a non-orienting nozzle, without
departing from the
scope of this invention. The baffle 2001 Is positioned in the nozzle upstream
from the orifice 103
and downstream from the sample injection needle 157. Referring to Figs. 14 and
15, the baffle
comprises a baffle plate 2003 that is held in place by a baffle holder 2005.
In the embodiment
shown, the baffle plate 2003 is generally L-shaped and constructed of a
substantially rigid,
durable and corrosion-resistant material (e.g., stainless steel), The L-shaped
plate 2003 has an
upstream leg 2007 and a downstream leg 2009, which are desirably substantially
perpendicular
to each other (e.g., within about 5 degrees of being perpendicular). In the
exemplary
embodiment shown in the drawings, the two legs 2007, 2009 of the L-shaped
plate 2003
intersect at a line 2015 that is perpendicular to the longitudinal axis 2017
of the nozzle 137 (Fig.
11). As shown in Fig. 14, the line of intersection 2015 is also spaced a short
distance 2033 (e.g.,
about 0.3 mm) away from the longitudinal axis 2057 of the baffle holder 2005.
The upstream leg
2007 of the L-shaped plate 2003 extends from the line of intersection 2015
away from the
longitudinal axis 2017 of the nozzle 137 all the way to the edge of the baffle
holder 2005, as
shown in Fig. 15. Thus, the upstream leg 2007 is formed with a curved edge
2019 that closely
matches the shape of the baffle holder 2005. As shown In Fig. 14, the upstream
leg 2007 Is
inclined at an angle AA of about 15-25 degrees from perpendicular to the
longitudinal axis 2057
of the baffle holder 2005. The downstream leg 2009 of the L-shaped plate 2007
extends
downstream from the line of intersection 2015 of the two legs 2007, 2009 a
distance 2025 of
about 2.0-2.5 mm at an angle BB that is in the range of about 60-80 degrees
from perpendicular
to the longitudinal axis 2057 of the baffle holder 2005.

CA 02752247 2011-09-07
39
The baffle holder 2005 is sized and shaped to fit inside the nozzle 137, as
shown in Figs.
10-13. The baffle holder 2005 is preferably made of a moldable material (e.g.,
polypropylene)
although the baffle holder 2005 may be constructed from other materials
without departing from
the scope of the present invention. The baffle holder 2005 used in the
exemplary embodiment,
shown in Figs. 14 and 15, is generally shaped as a hollow cylindrical shell
about 4.0-4.5 mm in
overall length 2027. The baffle holder 2005 has an exterior diameter 2029 of
about 5-6 mm and
an Interior diameter 2031 of about 2.5-3.5 mm. If the baffle holder 2005 is to
be molded, a minor
draft (not shown) can be provided on the surfaces of the holder 2005 (e.g., to
allow the baffle
holder to be easlly removed from an injection molding machine). The upstream
end 2035 of the
exemplary baffle holder 2005 has an inclined surface 2037 which is inclined at
the same angle
AA as the upstream leg 2007 of the L-shaped plate 2003. The upstream leg 2007
of the L-
shaped plate 2003 abuts against and is supported by the inclined surface 2037
of the baffle
holder 2005. The side edges 2039 (Fig. 15) of the downstream leg 2009 of the L-
shaped plate
2003 are partially embedded (e.g., received In slots) In the baffle holder
2005 to hold the baffle
plate 2003 in a position In which the downstream leg 2009 spans generally from
one side of the
baffle holder 2005 to the other. The downstream edge 2041 of the downstream
leg 2009 in the
exemplary embodiment forms a straight line which is generally perpendicular to
the longitudinal
axis 2057 of the baffle holder 2057. There is a gap 2049 (Fig. 14) between the
downstream
edge 2041 of the downstream leg 2009 and the interior cylindrical surface 2051
of the baffle
holder 2005. The gap 2049 provides fluid communication between a volume 2053
defined by the
legs 2007, 2009 of the L-shaped plate 2003 and the interior cylindrical
surface 2051 of the baffle
holder 2003 and the rest of the Interior volume 2055 of the nozzle '137.
The baffle holder 2005 is desirably positioned inside the nozzle with the
longitudinal axis
2057 of the baffle holder 2005 generally aligned with the longitudinal axls
2017 of the nozzle 137
so that it holds the L-shaped plate 2003 in the position described above.
Desirably, the
exemplary baffle plate 2003 is rotationally oriented so that the line of
intersection 2015 of the two
legs 2007, 2009 of the plate 2003 is parallel to a line 2059 runnlng through
the major axis of
ellipse D, as shown in Fig. 16. However, the exemplary baffle 2001 also
performs well when the
intersection 2015 of the two legs 2007, 2009 of the L-shaped plate 2003 is
perpendicular to the
line 2059 running through the major axis of ellipse D, as shown in Fig. 17.
Furthermore, the
baffle may have any rotational orientation without departing from the scope of
this invention. As
shown In Fig. 12, the sample injection needle 157 in the exemplary embodiment
is desirably a
distance 2061 of about 0.25 - 1.0 mm upstream from the most upstream portion
2035 of the
baffle 2001. More desirably, the sample injection needle 157 is about 0.55 -
0.65 mm upstream
from the most upstream portion 2035 of the baffle 2001.
The baffle holder 2005 may be held in a desired position relative to the
nozzle in any
number of ways. Referring to Fig. 14, the downstream end 2067 of the baffle
holder 2005 is
stepped so that It fits farther downstream in the nozzle 137. The stepped
downstream end 2067
of the holder 2005 is circular in shape and abuts against the elliptically
shaped interior surface
233 of the nozzle 137. Thus, the contact between the interior surface 233 of
the nozzle 137 and

CA 02752247 2011-09-07
the baffle holder 2005 is generally limited to two points 2069, as shown in
Fig. 13. A pair of 0-
rings 2071 are positioned around the baffle holder 2005 between the nozzle 137
and the
threaded projection 149 of the flow body 133 (Figs. 11-13) and seal the nozzle
system 101
against leakage. The 0-rings 2071 may be made of Viton0, or any other similar
materials. The
5 two 0-rings 2071 are compressed as the nozzle 137 is screwed onto the
threaded projection 149
to provide a fluid-tight seal. Two 0-rings 2071 are used In the exemplary
embodiment because a
single 0-ring cannot be compressed within the space between the nozzle 137 and
the flow body
133 due to the length 2027 of the baffle holder 2005. Any number of 0-rings or
a different type
of seal could be used without departing from the scope of the present
Invention, provided that the
10 number of 0-rings or other type of seal is selected so that there will
be a fluid-tight seal when the
nozzle 137 is screwed onto the flow body 133. This will depend on a number of
factors, including
the size and shape of the nozzle 137, flow body 133, baffle holder 2005, and 0-
rings 2071 as
well as the type of seal. The 0-rings 2071 also help hold the baffle holder
2005 In the desired
position. The 0-rings 2071 occupy the space around the baffle holder 2005,
thereby restricting
15 side-to-side movement of the baffle holder 2005 inside the nozzle 137.
Frictional forces between
the 0-rings 2071 and the baffle holder 2005 also resist rotational movement of
the baffle holder
2005.
When the nozzle 137 is tightened on the flow body 133 as shown in Fig. 12, the

downstream end 2077 of the threaded projection 149 from the flow body 133, In
the form of a
20 boss in this embodiment, Is approximately even with the most upstream
portion 2035 of the baffle
2001. As a result, the baffle holder 2005 Is held axially captive between the
flow body 133 (at
the upstream end 2035 of the baffle holder 2005) and the interior surface 233
of the nozzle 137
(at the downstream end 2067 of the baffle holder 2005). Other retaining
mechanisms may be
= used. In the embodiment shown in the drawings, the interior diameter of
the boss 2079 (Fig. 12)
25 at the downstream end of the threaded projection 149 is roughly equal to
the internal diameter
2031 of the baffle holder 2005.
Those skilled in the art will recognize that the flow through the nozzle
system 101
remains laminar notwithstanding the baffle 2001 because the small cross
sectional area through
which the fluids must flow results in a low Reynolds number for the flow. As
is shown in Fig. 11,
30 the baffle deflects the core stream 189 and sheath stream 191 away from
the central longitudinal
axis 2017 of the nozzle 137 and toward an interior surface 233 of the nozzle
137. In one
embodiment, the core stream 189 also flows very close to the interior surface
233 of the nozzle
137 as the core stream 189 passes between the transition between the first 269
and second 261
torsional zones. However, a portion 2081 of the sheath fluid stream 191
remains between the
35 core stream 189 and the interior surface 233 of the nozzle 137 so the
particles in the core stream
189 do not actually Impact or contact the interior surface 233 of the nozzle
137. Farther
downstream In the nozzle 137, the hydrodynamic forces push the core stream 189
back toward
the center of the nozzle 137 (e.g., in alignment with the longitudinal axis
2017 of the nozzle 137).
Referring to Figs. 18A-18E, the baffle 2001 changes the shape and reduces the
size of
40 the cross sectional flow area in the nozzle 137. (For the sake of
clarity, Figs. 18A-18E do not

CA 02752247 2011-09-07
41
show any nozzle structure downstream from the baffle. The flow area in each of
the Figs. 18A-
18E Is outlined in bold for clarity.) Upstream from the baffle 2001 (Fig.
18A), the cross sectional
flow area 2087 is generally clrcular or elliptical. At the upstream end 2036
of the baffle 2001, the
flow area begins to change from a circular shape to a generally semi-circular
shape 2089 at the
Intersection 2015 of the legs 2007, 2009 of the baffle plate 2003 (Flg. 18B),
although other
shapes may be sultable. There the cross sectional flow area 2089 is smaller
than the flow area
2087 upstream from the baffle. Fig. 18C illustrates the flow area 2091 as
fluid flows through a
part of the baffle holder 2005, and Flg. 18D illustrates the flow area 2093
farther downstream at
the downstream end 2041 of the downstream leg 2009 of the baffle plate 2003.
It will be
observed that flow area 2093 is somewhat larger than flow area 2091 due to the
angular
orientation of the downstream leg 2009 of the baffle plate 2003. Downstream
from the baffle
plate 2003 (Fig. 18E) the flow area 2094 through the baffle corresponds the
shape of the interior
surface 205'1 of the baffle holder 2005, which is circular in the illustrated
embodiment. (Other
shapes may be suitable.) Downstream from the baffle holder 2005 the torsional
zones 259, 261
of the nozzle 137 desirably provide torsional forces as discussed above.
As shown In Fig. 11, It has been observed that one or more air bubbles 2095
may
become trapped In the volume 2053 between the downstream leg 2009 of the L-
shaped plate
2003 and the baffle holder 2005. Furthermore, a portion of a bubble 2095 may
extend through
the gap 2049 between the edge 2041 of the downstream leg 2009 and the baffle
holder 2005.
Thus, the air bubble(s) 2095 can occupy a portion of the cross sectional flow
area downstream of
the downstream leg 2009 of the L-shaped plate 2003, perhaps affecting the flow
of fluid through
the nozzle '137. The exemplary baffle 2001 has been found to work well both
with and without
the air bubble(s) 2095. Thus, a baffle can be used to orient sperm cells
without involvement of
any bubbles without departing from the scope of the present invention.
Another exemplary orienting baffle, generally designated 2097, is shown in
Figs. 19 and
20. The baffle 2097 comprises a flat generally seml-circular baffle plate 2099
in the orienting
nozzle 137 discussed above. The baffle plate 2099 is positioned in the nozzle
137 downstream
of the sample introduction conduit 157 ancl generally perpendicular to the
longitudinal axis 2017
of the nozzle 137. The baffle plate 2099 has a curved edge 2101 that generally
matches the
curvature of the interior surface 233 of the nozzle 137 so that there are no
large gaps between
the curved edge 2101 of the baffle plate 2099 and the Interior surface 233 of
the nozzle 137.
The baffle plate 2099 also has a stralght edge 2103 that extends a short
distance past
longitudinal axis 20'17 of the nozzle 137 so that it is approximately aligned
with the outer
diameter 2109 of the sample introduction conduit 157. The baffle plate 2099 is
held in position
by friction resulting from compression of the baffle plate 2099 between an o-
ring seal 2105,
which is similar to the o-ring seals 2071 descrlbed in connection with the L-
shaped baffle 2001
above, and an annular shoulder or shelf 2107 formed on the interior of the
nozzle 137. As shown
In Fig. 19 the orienting baffle 2099 operates by deflecting the fluid stream
so that the core stream
189 containing the particles to be analyzed is offset from the central
longltudinal axis 2017 of the
nozzle 137 along a portion of Its flow path. For example, the core stream 189
may be directed

CA 02752247 2011-09-07
= 42
along a flow path that is offset frotn the longitudinal axis 2017 of the
nozzle 137 as it flows
through the first torsional zone 259, as well as at least a portion of the
second torsional zone
261. Consequently, the particles (e.g., sperm cells) are subjected to the
torsional =forces
generated by the torsional zones 259, 261 while they are in a position that is
offset from the
central longitudinal axis 2017 of the nozzle 137.
Those skilled in the art will recognize that substantial changes may be made
to the
exemplary baffles 2001, 2097 described above without departing from the scope
of the present
invention. All that is required Is that the baffle be configured to deflect
the core stream 189 and
sheath stream 191 toward an interior surface of the nozzle or to cause the
core '189 and sheath
stream 191 to flow through a cross sectional area that changes in size and/or
shape. Further, it
is understood that the orienting baffle structure may be Integrally formed
with the nozzle or
integrally formed with the nozzle and flow body without departing from the
scope of the present
invention.
Offset Sample Introduction Conduit
The core stream 189 may be directed along a flow path that Is offset from the
central
longitudinal aids 2017 of the nozzle '137 by repositioning the sample
introduction conduit '157
from its traditional position at the center of the nozzle 137 to an offset
position. For example, Fig.
21 shows an exemplary offset sample introducing nozzle system 2151 having an
offset sample
introduction conduit 157. Except as noted, the nozzle system 2151 Is
substantially the same as
the nozzle system 101 shown in Figs. 4 and 5. The significant difference Is
that the sample
Introduction conduit 167 has been moved away from the center of the nozzle 137
so that it is no
longer aligned with the nozzle's longitudinal axis 2017. Thus, the core stream
189 is directed
into the torsional zones 259, 261 of the orienting nozzle 137 along a flow
path that is offset from
the longitudinal axis 2017. Although the exemplary nozzle system 2161 shown in
Fig. 21 uses
the exemplary orienting nozzle 137 describe above, It is contemplated that
offset sample
introduction conduit 157 could be used with a different orienting nozzle or a
non-orienting nozzle
to orient particles in the core stream 189.
Nozzle Mounting and Adiustment
The flow body 133 and nozzle 137 are mounted In a selected orientation and
position by
means of a nozzle mount, generally designated 331. In one embodiment (Fig.
22), the mount
331 comprises a plurality of stages, including first and second linear stages
333, 337 providing
linear adjustment of the flow body 133 and nozzle 137 along X and Y axes 339,
341,
respectively, and a third rotational stage 343 providing rotational adjustment
about a Z axis 345
corresponding to the longitudinal axis 2017 of the flow body 133 and nozzle
137. These stages
333, 337, 343 may be conventional in design, suitable stages being
commercially available, for
example, from Newport Corporation of Irvine CA. In particular, the first
linear motion stage 333
comprises a fixed first stage member (not shown) mounted on a frame 349, a
movable first stage
member 355 slidable on the fixed first stage member along the X axis 339, and
an actuator 357,

CA 02752247 2011-09-07
43
e.g., a micrometer, for precisely moving the movable first stage 355 member to
a selected X-axis
position. The second linear motion stage 337 comprises a fixed second stage
member 359
mounted on the movable first stage member 355, a movable second stage member
361 slidable
on the fixed second stage member 359 along the Y axis 341, and an actuator
363, e.g., a
micrometer, for precisely moving the movable second stage member 361 to a
selected Y-axis
position. The rotational (third) stage 343 comprises a fixed third stage
member 365 mounted on
the movable second stage member 316, a movable third stage member 371
rotatably mounted
on the fixed third stage member 365 for rotation about the Z-axis 345, and an
actuator 373, e.g.,
a micrometer, for precisely rotating the movable third stage member 371 to a
selected angular
position relative to the Z-axis 345. The three-axis adjustment provided by
these stages 333, 337
343 allows the nozzle 137 and the fluid stream 21 exiting the nozzle orifice
103 to be precisely
positioned relative to the optics system 109. Rotation of the nozzle 137 about
the Z-axis 345 is
particularly helpful because it enables the stream 21 exiting the nozzle 137
to be rotated to bring
the cells (e.g., sperm cells) oriented by the nozzle 137 into a position in
which the light beam 26
from the optics system 109 will fall on the desired surfaces of the cells
(e.g., the flat faces 207 of
sperm heads 205), as Illustrated schematically in Fig. 23. Other nozzle mounts
may be suitable.
For example, a 4-axis nozzle mounting system can also be used, providing
linear adjustment
along X, Y and Z axes and rotational adjustment along the Z axis. Further, it
may be desirable to
use one or more stages having an automated alignment feature, such as a servo
or stepper
motor controlled microtranslation stage (e.gõ part number M-110.2DG from
Polytech PI, Inc. of
Auburn, Michigan).
In one embodiment shown schematically in Fig. 36, for example, the nozzle 137
is
oriented to direct a stream 21 containing cells to be analyzed in a generally
upward direction.
The angle 377 between the direction of the fluld stream 21 and horizontal is
preferably in the
range of 5 to 85 degrees, more preferably in the range of 15 to 75 degrees,
even more preferably
about 30 to 65 degrees, still more preferably about 45 to 60 degrees, and most
preferably about
50 to 55 degrees. Thls orientation is advantageous in that any air trapped =in
the nozzle system
101 is readily removed. Also, the velocity of the fluid stream 21 decreases
gradually under the
force of gravity prior to collection of the droplets 33. A more gradual
deceleration of the droplets
33 is believed to be less stressful to the cells being analyzed which, in the
case of sperm cells,
can result in higher motility of the sorted sperm after collection. Of course,
in other embodiments
of the present invention, the nozzle 101 is positioned so that the fluid
stream 21 has a
substantially downward velocity when it exits the orifice 103 as is
conventional for jet-in-air
cytometers.
Optionally, components of the nozzle system 101 such as the flow body 133 and
nozzle
'137 are coated with a non-reflective, non-emissive material (e.g., a dull
dark paint or epoxy which
does not emit light when subjected to UV laser light) to reduce any reflected
and/or emitted light
off these elements 133, 137 which might otherwise cause signal noise or have
other adverse
effects on the optics system 109.

CA 02752247 2011-09-07
44
Transducer and Droplet Formation
The transducer 105 for introducing energy into the fluid stream 21 comprises,
in one
embodiment, a collar 379 containing a piezoelectric element (not shown)
secured around the
flow body 133 of the nozzle system 101 (Figs. 3 - 5). The transducer is of
conventional design,
such as is available from Beckman Coulter, Inc. as part No. 6858368. The
transducer has
terminals 383 for connection to a suitable source of acoustical energy so that
energy can be
delivered to the fluid stream 21 at a frequency which will cause it to break
into droplets 33 at the
droplet break-off location 107 downstream from the nozzle 137 a distance d
(Fig. 24). As will
understood by those skilled in flow cytornetry, the characteristics of the
droplet formation are
governed by the following Equation 1:
(V = fA) Equation 1
where V is the velocity of the stream 21; f is the frequency applied to the
fluid stream 21 through
the nozzle 137; and A is the "wave length" or distance between the droplets
33. It is a known
principle of flow cytometry that droplets 33 will form in a regular pattern
with the distance
between droplets 33 being 4.54 times the diameter of the stream 21. Since the
diameter D of
the stream 21 close to the nozzle 137 generally corresponds to the diameter of
the nozzle orifice
103 at its downstream end, the frequency at which the stream 21 (and nozzle
137) must be
vibrated to form the droplets 33 can be easily calculated using the following
Equation 2:
(f = V/4.54D') Equation 2
The transducer 105 may be operated to generate in the range of 30,000 -
100,000 droplets 33
per second. For example, the transducer 105 may generate 50,000 - 55,000
droplets per
second. Assuming the frequency is 55,000 cycles per second (55 kHz), and
further assuming
that the concentration of cells in the stream 21 is such that cells exit the
nozzle 137 at a
substantially matching rate of 55,000 cells per second, then there will be, on
average, one cell
per droplet 33. (In reality, some droplets 33 will contain no cells, some will
contain one cell, and
some will contain more than one cell.) Of course, any of various factors can
be changed to vary
this average, including a change In frequency (f), stream 21 (orifice 103)
size (D') and stream 21
velocity (V). Ideally, these factors should be such as to reduce the amount of
stress Imparted to
the cells during the course of the process, especially in the case of sperm
cells where the
preservation of motility is important.
Break-off Sensor
Referring to Fig. 2, a break-off sensor 389 may be employed to determine the
location
(e.g., break-off location 107) at which the stream 21 begins to form free
droplets 33. The break-
off location 107 will vary depending on several factors including stream 21
viscosity, surface
tension of the fluid and the amplitude of vibration of the transducer 105. By
monitoring the break-
.

CA 02752247 2011-09-07
off location 107, the amplitude of the transducer 105 may be varied to
maintain the break-off
location 107 within a given. range so that the time at which each droplet 33
breaks off can be
more accurately predicted by the microprocessor 131. This allows the
microprocessor 131 to
accurately control the electrical charge of the droplet 33 which is
accomplished by selectively
5 controlling the charge of the stream 21. Since the charge of the droplet
33 will be the same as
the charge of the stream 21 immediately before droplet 33 formation, the
microprocessor 131
controls the sorting of the droplets 33 by selectively charging the stream 21,
as noted below.
In general, a break-off sensor is for use with any continuous stream of fluid
which is
breaking into droplets at a break-off location. (In the embodiment of Fig. 2,
the break-off sensor
10 389 is located downstream from the nozzle 137 and interrogation location
115.) One exemplary
break-off sensor 389 is shown schematically in Fig. 25. A light source 393 is
positioned on one
side of the stream 21 to illuminate the stream 21 within the given range at
which the break-off
location 107 will be maintained. A linear photoarray 395 positioned on the
other side of the
stream 21 is adapted to be oriented along an axis substantially parallel to
the stream 21. As a
15 result, the photoarray 395 detects light from the light source 393 which
passes through the
droplets 33 and provides output signals corresponding to the detected light.
The output signals are processed to determine the position of the break-off
location 107.
For example, the output signals may be digitized and provided to the processor
131 for
processing. Alternatively, as shown in Fig. 25, the light source 393 may be an
LED or other
20 source which generates a near-infrared portion of the visible spectrum.
The light passing
between the droplets 33 is magnified by a lens 401 and directed toward an 8 by
1 linear array of
photodiodes 395. Each photodiode generates a current that is proportional to
the light intensity
impinging thereon. This current is fed into 8 current to voltage =op-amp
circuits 405. The output
voltage from the op-amps is AC coupled into 8 track/hold amplifiers 407. The
track/hold signal
25 409 used by the amplifiers is taken from the transducer 105. The output
from the track/hoid
amplifier is fed Into the A/D converter 411 of a microprocessor unit (MPU)
391. The digital
values computed by the MPU 391 will be provided to the system control
microprocessor 131. A
lookup table and/or algorithm may be used by the system control microprocessor
131 to convert
between break-off location 107 drift and voltage adjustment to the transducer
105. Alternatively,
30 the output from the MPU 391 may be an analog signal such as a DC voltage
having an amplitude
corresponding to a change in the amplitude of vibration of the transducer 105.
The dc voltage
can be applied to the high voltage amplifier Input driving the droplet
transducer 105 to vary the
amplitude of vibration. Thus, such a processor 391 would constitute a control
for receiving the
output signal from the photoarray 396 and providing a location signal
corresponding to a location
35 of the break-off location 107. Such a processor 391 would also
constitute a control for receiving
the output signal indicative of the position of the break-off location 107 of
the droplets 33 and
varying operation of the transducer 105 as a function of the position of the
location 107.
Alternatively, as is well known to those skilled in the art, a video camera
and strobe light
may be used to monitor and control the droplet break-off location. Thus, as
shown in Figs. 26-
40 27, a video camera system 412 and strobe 413 may be provided to monitor
the break-off location

CA 02752247 2011-09-07
46
107. It is desirable to place the strobe 413 behind a mask 414A (e.g., a cover
with a small slit;
shaped opening 414B) to limit the amount of light produced by the strobe 413
that enters the
optics system 109 (Fig, 27).
Epi-Illumination Optics System
The optics system 109 is adapted for focusing a beam of electromagnetic
radiation 25
(e.g., a laser beam) on the fluid stream 21 as a beam spot, so that the cells
to be analyzed pass
through the spot. The beam 25 may be laser light in the visible or ultraviolet
portion of the
spectrum, for example, having a wavelength of about 350-700 nm, although other
wavelengths
may be used. The wavelength of the laser light may be selected so that it is
capable of exciting a
particular fluorochrome used to analyze particles. If the optics system 109 Is
used to analyze
sperm cells stained with Hoechst 33342, for instance, the wavelength may be
selected to be in
the range of about 350-370 nm. The power output of the laser may vary between
50 and 300
mW. Sperm cells may be analyzed using a 200 mW laser, for example. Referring
to Figs. 28-
34, the system '109 Is an epi-Illumination system 415 comprising an
instrument, generally
designated 417, having a longitudinal optical axls 419. As used herein, the
term "epi-
Illumination" means an optics system where at least some of the fluorescence
emissions from
cells passing through the beam spot are directed back through the optical
instrument along the
same axis as the focused beam 25, but in the opposite direction. This type of
system is
advantageous in that only one set of optics is required, including only one
photodetector 117,
unlike conventional systems which detect forward and side fluorescence and
which use two or
more photodetectors. However, it will be understood that while an epi-
illumination system is
preferred, many of the aspects of this invention can be applied regardless of
the type of optics
system used.
In one embodiment, the epi-illumination instrument 417 comprises a rectangular
base
429 supporting a plurality of optical elements. These optical elements are
described below, with
specific examples of relevant dimensions, focal lengths, and part numbers. As
will be
understood by those skilled in the art, this information is exemplary only,
and alternative optical
elements can be used without departing from the scope of this invention.
Referring to Figs. 28-34, the optical elements include a reflecting filter 431
which reflects
a collimated beam 25 of light from a laser or arc lamp 435, for example,
through a conditioning
lens assembly 437 mounted in an opening 439 in a side wall 441 of a dichroic
chamber 4143
extending up from the base 429. In this particular embodiment, the
conditioning lens assembly
437 comprises a retaining ring 445, neutral density filter 447, cylindrical
lens 449, lens holder 455
and jam nut 457. The cylindrical lens 449 introduces a one-dimensional
divergence into the
beam 225 and directs it toward optical elements (described below) which shape
the beam to
have a desired cross sectional shape 459, preferably generally elliptical. By
way of example, the
cylindrical lens 449 may be a piano-convex lens having a focal length of 16
mm. A beam

CA 02752247 2011-09-07
47
expander (not shown) can optionally be installed in the instrument 417 to
allow adjustments to be
made to the shape of the elliptical beam spot 459.
The reflecting filter 431 is mounted by clips 461 on the angular face 465 of a
filter holder
463 which has openings 467 in it to permit the beam 25 to reflect off the
filter 431 toward the
optics of the Instrument 417. The holder 463 is fastened to a linear stage 469
movable along an
X-axis 471 relative to an outrigger 473 secured to the base 429 and dichroic
chamber 443, the
stage 469 being movable by suitable means 475 (e.g., a micrometer) to
precisely locate the
holder 463 and reflecting filter 431 to reflect the beam 25 into the
instrument 417 at the proper
location. A dichroic filter 477 Is held by clips 479 on a frame 485 mounted in
the dichroic
chamber 443 and functions to reflect the shaped beam 25 in a forward direction
487 along an
axis 489 which, in this particular embodiment, corresponds to the longitudinal
optical axis 419 of
the instrument. The beam 25 passes through a focusing lens assembly 491 which
focuses the
beam 25 on the fluid stream 21 as a beam spot having the aforementioned
generally elliptical
shape 459 (Fig. 6) with the major axis of the ellipse extending generally
perpendicular to the
direction of flow 227 of the stream 21. As each cell passes through the beam
spot 459, the
fluorescing dye (or other reporting agent) in the cell Is activated to emit
fluorescent light 31 (Fig.
23). In the case of sperm cells stained with a DNA selective fluorescing dye,
X cells have more
DNA than Y cells, include more fluorescing dye, and emit a stronger signal
than Y cells (e,g.,
3.8%), which provides a basis for discriminating and sorting cells, as will be
described. The
focusing lens assembly 491 includes, in one embodiment, a microscope adapter
501 mounted in
an opening 503 in a front wall 505 of the dichroic chamber 443, a focusing
barrel 507, a pair of
lens mount barrels 509, and the lens 511 itself, which may be a 12.5 mm
diameter, piano-convex
lens with a focal length of 16 mm, available from Oriel Corporation as part
number 41209, and is
anti-reflective coated for light having a wavelength in the range of 340-550
nm. The lens 511
may be made of fused silica. Other focusing lenses may also be suitable, such
as an infinity-
corrected fluorescence microscope objective. The focusing lens assembly 491
has a
conventional telescoping focus adjustment 515 to focus the elliptically-shaped
beam spot 459 on
the core 189 of the stream 21.
The outgoing fluorescent light 31 emitted by the cells as they pass through
the beam
spot 459 is of a different (longer, due to the Stoke's shift principle)
wavelength than the incoming
laser light 25. Some of the fluorescence emissions 31 are transmitted in a
rearward direction
513 along the incoming beam axis back through the focusing lens 511 which
collects and
collimates the fluorescence emission 31. The collimated fluorescence emissions
517 pass in a
rearward direction from the lens 511 to the dichroic filter 477, which
transmits the fluorescence
emission 517. By way of example, the dIchroic filter 477 may be a filter
available from Omega
Optical as part number XF2001, 400DCLP.
The optics system 415 includes a filtering system 519 positioned rearward of
the dichroic
filter 477 along the optical axis 419 of the instrument 417. In one
embodiment, the filtering
system 519 Includes an emission filter 521 in a holder 523 mounted in an
opening 525 in a back
wall 527 of the dichroic chamber 443. The emission filter 521 attenuates any
laser light scatter

CA 02752247 2011-09-07
48
or other undesired electromagnetic radiation that is transmitted through the
dichroic filter 477. By
way of example and not limitation, the emission filter 521 can be a thin film,
long-pass filter
adapted to transmit more than 90% of light having a wavelength greater than
408 nm, as is
available from Omega Optical as part number XF3097. An alignment pellicle
assembly 529 is
spaced rearwardly along the optical axis 419 from the emission filter. This
assembly includes a
slider 531 movable on a rail 533 extending longitudinally of the base 429
parallel to the
longitudinal optical axls 419 of the instrument 417, a filter holder 535
secured to the slider 531, a
pellicle filter element 539, and clips 541 for securing the pellicle filter
element 539 to the filter
holder 535 at an angle 543 relative to the optical axis 419 of the instrument
417, The pellicle
filter element 539 has the same thickness as the dichroic filter 477 and
functions to translate the
collimated fluorescence emission 517 back onto the optical axis 419 of the
instrument 417.
Fasteners 545 extending up through parallel slots 547 in the base 429 on
opposite sides of the
rail 533 secure the slider 531 to the base 429 in the desired position along
the optical axis 419.
Spaced to the rear of the alignment pellicle assembly 529 is an aspheric lens
549 held by a
holder 651 mounted in a frame 553 which is also slidable on the rail 533 and
secured in selected
position by suitable fasteners. The aspheric lens 549 focuses the collimated
fluorescence
emission 517 onto a spatial filter, generally designated 559, which filters
out reflection or
emission from sources other than the cells to be analyzed. The aspheric lens
549 may be, for
example, an 12,5 mm diameter aspheric lens having a focal length of 15 mm, as
is available from
Oriel Corporation. The lens 549 is preferably anti-reflective coated for
visible emission
wavelengths but made of a material (e.g., flint glass) which further
attenuates transmission of
laser light scatter.
As shown in Fig. 34, the spatial filter 559 comprises, in one embodiment, a
pair of
aperture plates 561 releasably held by a frame 563 mounted on the base 429 of
the instrument
417. Each of the plates 561 has a slit 567, 571 therein, one slit 567
preferably being generally
vertical and the other 571 preferably generally horizontal, the arrangement
being such that the
silts 567, 571 intersect to form an aperture 573. In one embodiment, the
aperture 573 is
generally rectangular in shape and has a vertical dimension 575 of 100 microns
and a horizontal
dimension 577 of 500 microns. The size and shape of the aperture 573 may vary
(or even be
adjusted by changing aperture plates), so long as it functions to remove
reflections and light from
any source other than the collection volume 579. The frame 563 holding the
aperture plates 561
preferably has two parts, namely, a plate holder 583 slidable on the rail 533
of the base 429 and
secured in selected position by fasteners 587, and a backing member 589 for
securing the
aperture plates 461 in position on the plate holder 583.
In one embodiment, the smaller (vertical) dimension 575 of the aperture 573 in
the
spatial filter 559 is sized (or adjusted) to enable use of a "slit scanning"
technique to evaluate the
cell. This technique is described in more detail in the "Focused Beam Spot"
section of this
specification.
Another embodiment of an epi-illumination optics system, generally designated
450, is
Shown in Fig. 35. This embodiment is substantially the same as the embodiment
shown in Figs.

CA 02752247 2011-09-07
49
28-34, except as noted. One significant difference is that the dichroic filter
477 has been
replaced with a different dichrolc filter 451 that transmits (rather than
reflects) the illumination
beam 25 and reflects (rather than transmits) the fluorescent emissions 31.
Also, because the
fluorescence emissions 31 are reflected by the dichroic filter 451 rather than
transmitted, there Is
no need for en alignment pellicle 539 in this embodiment of an epl-
illumination optics system
450. Thus, the epi-illumination system 450 is Just one example of how the
optics system can be
reconfigured if desired without departing from the scope of this invention.
Further, the cylindrical lens 449 is mounted on an adjustable mounting
assembly 449A.
The mounting assembly 449A allows two-axis translational movement of the
cylindrical lens 449
in a plane perpendicular to the illumination beam 25. Releasable fasteners
(e.g., screws (not
shown)) extend through slot-shaped holes 449B (only one of which is visible on
Fig. 35).
Release of the fasteners allows translational movement of the lens 449 in a
first direction
perpendicular to the beam 25. Similar fasteners (not shown) extend through
slot-shaped holes
449C, allowing translational movement of the lens 449 in a second direction
perpendicular to the
first direction. This allows minor adjustment of the relative positions of the
cylindrical lens 449
and beam 25 so that the intersection of the beam 25 and lens 449 can be moved
across the
surface of the lens 449, thereby causing slight changes to the focusing
provided by the cylindrical
lens 449. Once the lens 449 is In the desired position, the fasteners can be
tightened to hold it
there.
Photodetector
The emitted fluorescence passing though the spatial filter 559 falls upon a
photodetector
117 fastened to a mounting plate 591 slidable on the rail 633 of the base 429
at the rear of the
evil-Illumination instrument 417 and securable in fixed position by fasteners
595 (Fig. 32). The
photodetector 117 detects the fluorescent emissions 31 and converts them Into
electrical signals
which can be processed to analyze the desired characteristics of the cells, as
will be described in
more detail later. The photodetector 117 may be a conventional device, such as
a photodetector
available from Hamnnamtsu. The photodetector -117 preferably includes a
preamplifier and PMT
gain which is optimized for emission Intensity produced by the epi-
illumination system for the
particular stained cells being analyzed.
In general, the PMT gain is optimized when between about 200 and 2000 volts
are
applied to the vacuum tube. In the case of detecting fluorescent emissions
from Hoechst 33342,
for Instance, the PMT gain Is optimized when between about 400-800 volts are
applied to the
vacuum tube. One particularly desirable photodetector includes a PMT having a
spectral range
of 185 ¨ 830 nm (530 nm peak), a 0.01 mA maximum average anode current, a
cathode radiant
sensitivity of 70 m/-VVV typical, a cathode luminous sensitivity of 140 auNlm,
anode luminous
sensitivity of 300 A/Im, max anode dark current of 1 nA (0.1 nA typical), and
a 1.4 nanosecond
risetime. The PMT is DC coupled amplifier demonstrating a flat gain to > 37
MHz, having a 1 V
peak output into a 50 0 load and a recovery time of less than 400 nanoseconds.
It Is also

CA 02752247 2011-09-07
desirable for the amplifier to allow high voltage adjustment for compensation
of PMT efficiency
variations without decreasing the signal-to-noise ratio to less than 800 dB.
Angle of Beam Incidence
5 Fig. 36 schematically illustrates one desirable orientation of the
intersection of the light
beam and the fluid stream. Several points are of note. As shown, the light
beam 25 is focused
on the stream 21 at a location 115 that is only a short distance 605 from the
exit orifice 103 of the
nozzle 137, preferably less than 1.0 mm, or even inside the nozzle 137, so
that the cells pass
through the spot 459 while they are still substantially in desired
orientation, as previously
10 described. This is particularly important for cells which are mobile in
the fluid stream 21,
Including sperm cells.
Another point of note is that the beam 25 of this embodiment may be directed
toward the
fluid stream 21 along a beam axis 609 which intersects the fluid stream 21 at
an angle of
incidence A which is skewed (off 90 degrees) relative to a longitudinal axis
of the fluid stream 21,
15 as viewed from a side of the stream 21 (see Fig. 36). When sorting
certain particles, it has been
found that better discrimination of the different types of particles may be
obtained by illuminating
the stream 21 at an angle of incidence other than 0 . Sperm nuclei, for
instance, are desirably
Illuminated at an angle of Incidence A that is In the range of 5 to 45
degrees, more preferably in
the range of 15 to 30 degrees, and even more preferably in the range of '18 to
24 degrees. Other
20 particles (e.g., live sperm cells) are easier to interrogate when the
light beam 25 is generally
perpendicular to the fluid stream 21 (i.e., when angle A is about 0'). Thus,
it is contemplated that
angle A may be any angle without departing from the scope of this invention.
The proper selection of angle A results in improved signal to noise
discrimination in
certain particles and thus more accurate discrimination based on different
characteristics of those
25 particles (e.g., sperm nuclei with X and Y chromosomes sperm cells).
This improvement may be
clue to a number of factors, including reduced laser light scatter entering
the focusing lens 511.
Because the focused beam spot 459 is preferably wider than the stream 21, a
diffraction pattern
is created at the intersection 115 of the beam 25 and the stream 21. When
angle A is greater
than about 12 degrees, the reflected diffraction pattern does not fall on the
lens 511. Another
30 factor may be that the skewed angle A allows the beam 25 to be focused
very close to the nozzle
orifice 103, so that the nozzle body 139 does not interfere with the lens 511.
Relatedly, the cells
are more uniformly aligned closer to the nozzle 137, so that focusing the beam
spot 459 closer to
the nozzle 137 results In an improved signal. Further, the more "head on"
profile of the cell
presented to the lens 511 (beam 25) at the skewed angle A reduces the
variation of total
35 fluorescence Intensity caused by any misalignment of the cells. In this
regard, in the case of
sperm cells it is preferable that the beam 25 fall on one of the wide faces
207 of each sperm cell
201, as discussed above, and that the nozzle 101 and optics system 109 be
positioned to
achieve this result.
While a skewed angle of incidence A Is believed to be beneficial in sorting
some
40 particles, It is contemplated that the angle of intersection between the
beam axis and the stream

CA 02752247 2011-09-07
51
may be 90 degrees or any skewed angle without departing from the scope of this
invention. It is
also expected that the optimal angle of incidence may vary widely depending on
the properties of
the particular particles being analyzed.
Referring to Fig. 6, the focused beam spot of one embodiment is shown as
having a
generally elliptical (oval) shape 459 with a length L1 along a major axis
extending generally at
right angles to the direction of fluid stream flow 227 and a width W1 along a
minor axis extending
generally parallel to the direction of fluid stream flow 227. In one
embodiment, the width W1 is
25 As mentioned previously, slit scanning can also be carried out by sizing
the aperture 573
of the spatial filter 559 to have a vertical dimension 575 such that only a
portion of the light
emitted from a cell, corresponding to a fraction of the cell length in the
direction of stream flow,
passes through the aperture to the photodetector 117. Further, signal
resolution can be
optimized by adjusting the width of the beam and/or the size of the aperture
of the spatial filter to
One way to adjust the shape of the beam spot 459 is by changing to a different

cylindrical lens and/or by making an adjustment to a beam expander in the
optics system 109.
Further any method of shaping the beam 26 to form an elliptically shaped beam
spot 459 is
contemplated as being within the scope of the present invention. Beam spots of
other shapes
Sorting System
Fig. 2 illustrates an exemplary embodiment of the sorting system 119. The
sorting
system 119 comprises an electrostatic charging device 627 for charging and/or
not charging the

CA 02752247 2011-09-07
52
X/Y chromosome content of sperm cells), and a pair of electrostatic charged
deflector plates 629
for sorting the droplets 33 into different groups 123, 125, according to their
charge. It is desirable
to coat the deflector plates 629 with a dull, low-emissive coating (e.g.,
epoxy or paint) to limit light
reflected or emitted by the deflector plates 629. The deflector plates 629 may
be charged by any
suitable power supply 635. it is generally desirable for the electrical
potential between the two
fully charged deflector plates 629 to be In the range of 2000 - 4000 volts.
However, the electrical
potential between the deflector plates 629 may be anywhere between about 1000
and 6000
volts.
The charging device 627 comprises a charging element 631 having an opening 633
therein through which the stream 21 passes at a location near the droplet
break-off location 107
(e.g., within five droplet lengths or closer). It is desirable to mount the
charging element 631 with
a mechanism that facilitates adjustment of the position of the charging
element 631 with respect
to the droplet break-off location 107. As shown in Figs. 26 and 27, for
example, the charging
element 631 arid deflector plates 629 may be attached to an adjustable
mounting assembly 5001
that allows three-axis translation and tilt adjustment of the charging element
631 and deflector
plates 629 with respect to the nozzle system 101. For translation along an
axis 5011 parallel to
the stream 21, the mounting assembly 5001 Includes a board 5003 fastened to a
backing 5005
by releasable fasteners 5007 passing through slots 5009 in the board 5003, the
slots 5009 being
oriented generally parallel to axis 5011. For translation in an axis 5013
perpendicular to the
stream 21, a second adjustment board 5015 is fastened to the first board 5003
by releasable
fasteners 5017 passing through slots 5019 in the second adjustment board 5015,
the slots 5019
being oriented generally parallel to axis 5013. The charging element 631 and
deflector plates 629
are secured to the second adjustment board 5015. Thus, by releasing the
fasteners 5007 and/or
5017, one can adjust the position of the charging element 631 and deflector
plates relative to the
nozzle system 101 in a plane parallel to the fluid stream 21 and then tighten
the fasteners 5007
and/or 5017 to secure the mounting assembly 5001.
For translation along a third axis perpendicular to the first two axes 5011,
5013, the
backing 5005 is fastened to a fixed support 5021 by adjustable fasteners 6023
(e.g., threaded
bolts screwed into tapped holes in the fixed support 5021). In one embodiment,
each adjustable
fastener 5023 passes through a spring 5025 positioned between the backing 5005
and the fixed
support 5021. The amount of compression of any spring 5025 can be adjusted by
tightening or
loosening the respective fastener 5023. Adjusting the compression of all
springs 5025 in the
same amount results in translation along the third axis. The mounting assembly
5001 can be
tilted In virtually any direction by changing the relative compression of one
or more of the springs
5025 with respect to one or more other springs 5025.
In this exemplary embodiment, the relative positions of the charging element
631 and
deflector plates 629 remain fixed with respect to one another because they are
all fastened to the
same adjustment board 5015. This prevents adjustment of the mounting assembly
5001 from
affecting alignment of the changing element 631 with respect to the deflector
plates 629.

CA 02752247 2013-11-21
53
The charging element 631 is connected to a suitable electrical circuit (e.g.,
a 90 volt
selectively charging circuit) under the control of the processor 131 and
coupled to a power supply
for applying an electrical charge to the charging element 631.. The circuit is
used to charge or not
charge the stream 21 immediately prior to the formation of a droplet 33 at the
break-off location
107 depending on whether the droplet 33 contains a particle having the desired
characteristics
(e.g., at least one live X-chromosome sperm cell). The charging element 631 is
positioned
electrostatically near the stream 21 or near the droplets 33 formed from the
stream 21 for
providing an electrical reference with respect to the electrostatic polarity
of the stream 21. The
droplets 33 carry the same charge as the stream 21 at the instant the droplet
33 breaks from the
stream 21. The charged or uncharged droplets 33 then pass between the
deflector plates 629
and are sorted by charge into collection vessels 2207 of the collection system
2201. While
sorting produces two groups or populations of droplets 123, 125 in Fig. 2, the
particles may be
separated into any number of populations from 1 to N sorted by placing
different charges on the
droplets 33 in respective groups, any by supplying the appropriate number of
collection vessels,
each being positioned to collect a different population of droplets.
Automated Drop Delay Calibration
In the sorting system 119 described above, the processor 131 must estimate the
time It
= takes for a particle to move from the interrogation location 115 to the
droplet break-off location
107 so that the charge (or lack of charge) to be applied to the droplet 33
containing that particle
is applied when the particle is in the iast attached droplet 33 at the break-
off location 107. If the
delay setting used by the processor 131 is wrong, the droplets 33 will not be
sorted according to
their contents. Similarly, if the application of electrical charges to the
droplets 33 is even slightly
out of phase with droplet 33 formation this can degrade sorting because none
of the droplets 33
will be fully charged and droplets 33 that are supposed to have neutral charge
will carry a small
positive or negative electrical charge. This will alter the paths of the
droplets 33 through the
electric field between the deflection plates 629.
The best way to verify that the processor 131 is using the appropriate delay
setting or to
adjust the drop delay setting (i.e., calibrate the system's 9 drop delay
setting), is to sort a number
of droplets 33 and examine the results. By incrementally varying the delay
setting and
monitoring the restilts, one can select the optimal delay setting.
Traditionally, this sort calibration
Is performed manually. Recently, automated calibration systems have been
designed to sample
or examine the contents of the droplets in the sorted droplet streams and
automatically adjust the
delay setting without human Intervention. For example U.S. Patent Nos.
6,372,506 (Norton) and
= 35 5,643,796 (van den Engh) both disclose automated sort
calibration systems. The purported
advantages of these systems are that they are less labor intensive and are
capable of verifying the
delay setting throughout the sorting process rather than just during initial
set up. The drawbacks
are that they are cumbersome and take up valuable space unnecessarily.

CA 02752247 2011-09-07
54
(i) Epi-illumination Sensors
Referring to Fig. 37, an automated continuous calibration system 4201 of the
present
invention for a fluorescence activated droplet sorting cytometry system
comprises one or more
epl-illumination sensors 4203 positioned to sense the contents of droplets 33
to verify the delay
setting for droplet charging. Referring to Fig, 38, each epi-illumination
sensor includes a light
source (not shown), a fiber optic cable 4205, a dichroic filter 4207, a lens
system 4209, a
photodetector 4213, and a control system. In one exemplary embodiment, the
processor 131
serves as the control system, but other processors or controls could be used
instead.
The light source may be a low-power solid state laser dedicated solely to the
automated
calibration system 4201. Alternatively, a beamsplitter (not shown) may be used
to divert a
portion (e.g., about 6%) of the energy in the beam 25 used for interrogation
of particles in the
fluid stream 21 to one or more epl-illumination sensors 4203. Similarly, the
fiber optic cable 4209
can be positioned in a beam stop 4215 (Fig. 26) to gather light from beam 25
after It passes
through the interrogation location 115. The light from the light source must
include light having a
wavelength capable of exciting fluorescent molecules in the particles being
sorted, thereby
causing fluorescence emissions 4211 from the particles. If the particles are
stained with Hoechst
33342, for instance, the light soUrce can provide light having a wavelength of
about 350 nm,
about 407 nm or any other wavelength capable of exciting the Hoechst 33342
molecules.
The fiber optic cable 4205 extends from the light source to a location
downstream of the
Interrogation location 115. For example, in the exemplary embodiment the fiber
optic cable 4205
leads to a location adjacent the trajectory of one of the droplet streams as
it moves through the
electric field between the deflector plates 629. The dichroic filter 4207 is
positioned in front of the
end of the fiber optic cable 4205. The dichrolc filter 4207 transmits light
having the spectral
characteristics of the light conducted by fiber optic cable 4205, but reflects
light having the
spectral characteristics of the fluorescence emissions 4211. Thus, the
dichroic filter 4207 may
have the same specifications as the dichrolc filter 477 describe above in
connection with the epl-
illumination optics instrument 417. The focal length of the lens system 4209
Is selected based on
the expected distance of the sensor 4203 from the droplets 33 so that the
Illumination/detection
volume of each sensor 4203 is about equal to the volume of the droplets 33.
Referring to the exemplary embodiment shown in Fig. 37, an epi-illumination
sensor
4203 is positioned adjacent the trajectory of each of the three sorted droplet
streams 4225, 4227,
4229 to sense the contents of droplets 33 in a respective stream. The
cytometer system 9
Includes an electrically Insulated support 4221 for mounting the two
deflection plates 629. The
support has three holes 4223, one adjacent the trajectory of each sorted
droplet stream 4225,
4227, 4229. An epi-Illumlnation sensor 4203 is positioned at each hole 4223 to
observe droplets
33 in one of the droplet streams 4225, 4227, 4229 through the respective hole
4223. This
compact configuration takes up relatively little space and keeps components of
the calibration
system 4201 out of the way, providing better access to other parts of the
cytometer 9.
If a droplet containing a fluorescent particle passes through the
illumination/detection
volume of the sensor 4203, this will result in a flash of fluorescence
emissions 4211, some of

CA 02752247 2011-09-07
which will be collected by the lens system 4209 and reflected off from the
dichroic filter 4207 to
the photodetector 4213. Signals from the photodetector 4213 are provided to
the processor 131.
Based on the signals received from the photodetectors 4213, the processor 131
can determine
the contents of the droplets 33 in each of the sorted droplet streams 4226,
4227, 4229.
5 If a sensor 4203 fails to detect a flash of fluorescence emission 4211
when the processor
131 expects a droplet 33 containing a fluorescent particle to pass by that
sensor 4203, the
processor 131 can use that information to adjust the delay setting or adjust
the location of the
droplet break-off location 107. Likewise, the processor 131 can make an
adjustment If a sensor
4203 detects a fluorescent emission 4211 when the processor 131 does not
expect a droplet 33
10 containing a particle to be passing by the sensor 4203. Furthermore, the
processor can compare
the relative frequency of fluorescent emissions 4211 from the sorted streams
4226, 4227, 4229
to see If the frequency of detected fluorescent emissions 4211 matches the
expected frequency.
The processor 131 can also adjust the amplitude of the charge applied to the
charging element
631 to increase or decrease the amount by which a sorted stream 4225, 4229 is
deflected to
15 maximize the intensity of the detected fluorescence emissions 4211. This
will maintain the
alignment of the trajectory of the deflected droplet streams 4225, 4229 so the
droplets pass
directly through the collection volume of the epi-illumination sensor. Because
the sensors 4203
are positioned to observe the streams 4225, 4227, 4225 as they move through
the electrical field
between the deflector plates 629, the calibration system has a shorter
response time than it
20 would If it observed the streams 4225, 4227, 4229 in the freefall area
downstream of the
deflection plates.
(11) Empty Droplet Test Stream
One sensitive indication of the quality of the calibration can be arranged by
creating =and
25 monitoring a calibration test stream that contains substantially only
empty droplets 33. Referring
to the sort calibration system 4201 shown Fig. 37, droplets 33 containing
desired particles are
sorted Into stream 4225 and droplets 33 containing any other particles and
most of the empty
droplets 33 are sorted into stream 4229 (i.e., the waste stream). The test
stream 4227 is created
by applying a neutral charge to at least a fraction (e.g., 1 out of every 10)
of the empty droplets
30 33. Many droplets 33 that are considered "empty" for traditional sorting
purposes are actually
droplets 33 for which there Is a low probability that the droplet 33 contains
a particle, based on
the arrival time of particles at the interrogation location 115 and estimated
droplet formation
boundaries in the fluid stream 21. These "empty" droplets should not be sorted
into the test
stream 4227 because this would inevitably result in detection of some
particles in the test stream
35 4227.
Instead, for the test stream 4227 the processor 131 should select only
droplets 33 that
the processor 131 believes have substantially zero probability of containing a
particle in order to
create a substantially particle-free test stream 4227. The probability that
any randomly selected
droplet 33 contains a cell is known and Is approximately the average cell
analysis rate divided by
40 the droplet generation rate. This means that by monitoring the rate of
mis-sorts in the test

CA 02752247 2011-09-07
56
stream 4227 it is possible to estimate fractional adjustment of the phase
relationship of droplet
charging needed to match the phase of droplet 33 formation. For example the
processor 13'1
may select droplets that it estimates have about 16% or lower probability of
containing a particle,
about 10% or lower probability of containing a particle, about 6% or lower
probability of
containing a particle, about 1% or lower probability of containing a particle,
about 0.1% or lower
probability of containing a particle, about 0.01% or lower probability of
containing a particle,
about 0.001% or lower probability of containing a particle, or about 0.0001%
or lower probability
of containing a particle. The probabilistic cutoff for substantially zero
probability may be selected
based on sort-speed, tolerance for Impurity, or other sort parameters, with
the cutoff including
higher probabilities that a droplet will include a particle for high-speed
sorting or when there is
more tolerance for impurity.
Failure of the processor 131 to create a substantially particle-free test
stream 4227 (i.e.,
a test Stream 4227 In which the ratio of droplets 33 containing particles to
the total number of
droplets 33 agrees with the probabilistic cutoff used to select droplets 33
for the test stream
4227), as Indicated by detection of more than a threshold number of droplets
33 containing
particles in the test stream 4227, is a definitive indication of sub-optimal
sorting and prompts the
processor 131 to adjust the drop delay setting. The threshold level is
determined in relation to
the probabilistic cutoff used to select droplets 33 for the test stream 4227
and the total number of
droplets 33 selected for the test stream 4227. Ideally, some droplets 33 can
be selected for the
test stream 4227 even though one or more particles In the fluid stream 21 are
relatively close to
an estimated drop formation boundary for the respective droplet 33 to make the
system 4201
more sensitive to slightly sub-optimal drop delay settings.
Of course, the sort calibration system could apply a non-neutral charge to and
deflect
droplets selected for the test stream, without departing from the scope of
this invention. The
relative order of the streams 4225, 4227, 4229 could also be rearranged
without departing from
the scope of this invention, although Interposing the test stream 4227 between
the waste stream
4225 and the stream of desired particles 4229 (as shown in the exemplary
embodiment) reduces
the risk of crossover contamination of the sorted sample by the waste stream.
Further, if the
particles do not emit fluorescent light, different sensors can be used to
detect any scattered light
caused by particles in the test stream without departing from the scope of
this invention.
(iii) Impact of Sort Calibration System
In one embodiment of the invention, the automated calibration system 4201 Is
operable
to automatically determine and set the phase relationship between droplet
formation and droplet
charging to within about 6% of the optimal phase (I.e., within +/- about 18
degrees. In another
embodiment the system 4201 is operable to automatically determine and set the
phase
relationship to within about 1% of the optimal phase (i.eõ within +/- about
3.6 degrees)). In
another embodiment, the calibration system 4201 is operable to continuously
monitor a high-
speed droplet sorting system and automatically maintain the phase relationship
within about 10%
of the optimal phase (i.e., within +/- about 36 degrees). In still another
embodiment, the system

CA 02752247 2011-09-07
57
4201 is operable to continuously monitor a high-speed droplet sorting system
and automatically
maintain the phase relationship without about 3% of the optimal phase (i.e.,
within +/- 10.8
degrees).
Sort System Fault Correction
From time to time, a droplet 33 will stray from its normal trajectory and hit
the charging
element 631 or the deflector plates 629. If one or more droplets 33 hit the
charging element 631,
the charging element 631 may not be able to charge droplets 33 properly.
Further, the normal
droplet 33 trajectory through the charging element 631 can become obstructed
causing even
more droplets 33 to accumulate on the charging element 631. Also, if stray
droplets 33 strike a
deflector plate 629, they can distort or otherwise disrupt electrical field
lines between the
deflector plates 629, thereby changing the trajectory of the sorted droplet
steams 123, 125.
Thus, it is desirable to have a debris removal system to remove debris from
the charging
element 631 and/or the deflector plates 629. In one exemplary embodiment,
shown Figs. 26 and
27, the system 9 includes a debris removal system 5047 for the charging
element 631 and a
debris removal system 5049 for the deflector plates 629.
Referring to Fig. 27, the charging element 631 is held in position by a
support 5051
secured to board 5015 of the adjustable mounting assembly 5001. A vacuum
passage 5053
(shown in phantom) extends through the support 5051 to an opening 5057
adjacent the charging
element 631. The vacuum passage 5053 is connected to a suitable vacuum source
(not shown)
by a vacuum line 5055 attached to a fitting 5058 on the support 5051. Suitable
controls are
provided for selectively applying a vacuum in the passage 5053 to vacuum any
undesired
material (e.g., stray droplets 33) off the charging element 631 and restore
proper function of the
charging element 631.
Relatedly, as shown in Fig. 27, a manifold 5061 fastened to the mounting
assembly 5001
has a network of air passages 5063 therein (shown In phantom) connected via an
air line 5059
and fitting 5065 to a source of compressed air or other gas (not shown). The
passages 5063
have openings 5064 positioned along a side 5066 of each deflector plate 629
and the portions
5067 of the passages 5063 leading to the openings 5065 are oriented so
compressed air blown
through the manifold 5061 will clear any stray droplets 33 or other debris off
the deflector plates
629. Any material blown off the deflector plates 629 will hit a cover panel
(not shown) and drain
into a suitable waste collection device (not shown).
In one embodiment, if the processor or other sensor determines that stray
droplets 33
have hit the charging element 631 or deflector plates 629, as Indicated by the
sort calibration
. system described above for example, the processor can automatically initiate
a fault correction
procedure ,or mode, which can include applying a vacuum to passage 5053 to
vacuum material
from the charging element 631 and/or sending compressed gas through passages
5067 to blow
material off the deflector plates 629.

CA 02752247 2011-09-07
58
Protection of Sorted Sample Durino Fault Mode
One embodiment of the system 9 also includes a contamination prevention
mechanism
4041 (Fig. 26), which can be activated by the processor 131 to limit or
prevent contamination of
the sorted sample any time the sorting system is in the fault correction mode.
The contamination
prevention mechanism includes a pneumatic actuator 4043 operable to
selectively move a swing
arm 4045 between a shielding position (shown Fig. 26) and a non-shielding
position (not shown).
In the shielding position, the end 4047 of the swing arm 4045 covers the
opening of the collection
vessel 4033, thereby preventing collection of droplets 33 by the coilection
vessel 4033. In the
non-shielding position, the collection vessel 4033 Is uncovered. Normally, the
swing arm 4045 is
in the non-shielding position, but the processor 131 causes the actuator 4043
to move the swing
arm 4045 into the shielding position any time the processor 131 determines
that there is a risk of
contamination (e.g., the nozzle system 101 becomes clogged, the droplet break-
off location 107
becomes unstable, or stray droplets 33 have hit the charging element 631 or
deflector plates
629). The end 4047 of the swing arm 4045 Is trough-shaped to drain any fluid
collected by the
swing arm 4045 into the waste container 4035.
Fluid Delivery System
The system 1 described above is capable of effectively producing quantities of
particles
(e.g., X-sperm cells) sorted by selected characteristics. The rate of
production can be increased
or decreased by varying the rates at which the fluid delivery system 15 (Fig.
2) delivers carrier
fluid 17 and sheath fluid 19 to the nozzle 137. In one embodiment, the fluid
delivery system
includes a syringe pump 645, one example of such a pump being MICROLAB Model
PSD/3
available from Hamilton Company. The pump 645 is operable to deliver carrier
fluid 17 to the
nozzle 137 at a rate of about 20 ,u1/min. In general, the pump 645 should be
operable to deliver
sample fluid 17 to the nozzle 137 at a rate in the range of 10 - 50 pl/min.
The pump 645 is
connected by a flow line 647 to the supply 3 of carrier fluid 17, which may be
a suitable vessel
649 containing a volume of material to be analyzed and sorted. Where the
temperature of the
particles being analyzed is a factor, as in the case of sperm cells, for
example, the temperature
of the vessel 649 may be controlled by a suitable temperature control system,
such as
heating/cooling bath (not shown). The syringe pump 645 is movable through an
intake stroke to
aspirate carrier fluid from the supply vessel and through a discharge stroke
to dispense carrier
fluid 17 through a supply line 651 to the injection needle 157 of the nozzle
system 101. The
pump 645 Is preferably driven by a variable speed motor (not shown) under the
control of the
processor 131. By way of example, the pump 645 may be driven by a stepper
motor which
operates at selectively variable rates to pump carrier fluid 17 to the needle
159 at rates
necessary to obtain the desired throughput. Other types of fluid delivery
devices can be used
instead of a syringe pump. To provide just one example, the vessel 649 can be
pressurized by a
pressurized gas source without departing from the scope of the invention.
Furthermore, it Is
desirable to keep the lines 647, 651 as short as Is practically possible
because the line

CA 02752247 2011-09-07
59
environment is not conducive to the health of sensitive cells (e.g., sperm
cells) that may be in the
carrier fluid 17.
The supply 7 of sheath fluid 19 comprises a second vessel 661, e.g., a tank in
Fig. 2,
holding an appropriate volume of sheath fluid 19 connected to the radial bore
173 in the flow
body 133 of the nozzle system 101 by a supply line 667 having a control valve
669 therein. In
the embodiment of Fig. 1, the sheath fluid vessel 661 Is pressurized by a gas
pressure system
comprising a source 675 of pressurized gas (e.g., air or other gas, such as
nitrogen)
communicating with the tank 661 via an air line 679 having a regulator 681 In
it for controlling the
pressure supplied to the tank 661. A two-way valve 683 in the air line 679 Is
movable between a
1 0 first position establishing communication between the tank 661 and the
gas source 675 and a
second position venting the tank 661. The gas pressure regulator 681 is a
conventional regulator
preferably under the control of the processor 131. By controlling the tank 661
pressure, the
pressure at which sheath fluid 19 Is delivered to the flow body 133 may also
be controlled. This
pressure may range from 16 to 100 psi, more preferably from 10 to 50 psi, even
more preferably
15 to 40 psi, and even more preferably from about 20 to 30 psi. The pressure
at which the
sheath fluid 19 is supplied to the flow body 133 can be controlled in other
ways without departing
from the scope of the invention.
In one embodiment, shown Fig. 26 the fluid delivery system 15, includes a
sheath fluid
tank (not shown) and a sample station 4051. The sample station includes a two-
part pressure
container 4053 adapted to hold a sample tube 4055. The bottom section 4057 of
the pressure
container is moveable up and down relative to the upper section 4059 of the
pressure container
4053 between an open position (shown Fig. 26), In which the sample tube 4055
may be loaded
or unloaded, and a closed position (not shown) in which the two parts 4057,
4059 of the pressure
container 4053 come together to form a seal to contain pressurized gas used to
pump carrier
fluid 17 from the sample tube 4055 to the nozzle system 101.
When the pressure container is open a spring-biased swing arm 4071 moves to a
position beneath the line 651 that delivers carrier fluid 17 to the nozzle
system 101 (See also Fig.
119 #4071'). The swing arm 4071 is trough-shaped and adapted to collect fluid
backflushed
through the line 651 and to drain the backflushed fluid to the waste container
through port 4073.
As the pressure container 4053 moves from its open position to its closed
position, a cam plate
4075 attached to the bottom section 4057 of the pressure container 4053 moves
the swing arm
4071 against its spring bias to clear the area between the two sections 4067,
4059 and allow the
pressure container 4053 to close.
Control
Referring again to Fig. 2, the microprocessor 131 (or other digital or analog
control
and/or processor, or combinations thereof) controls the operation of the
system 1. As noted
below with regard to Fig. 39, the microprocessor may be implemented as a
system control
processor and four processors for handling signal processing. Alternatively,
some or all
functions may be integrated Into one or more processors. For example, the
system control

CA 02752247 2011-09-07
microprocessor (see Fig. 36) may be implemented by using one of the four
signal processing
processors. In addition, as noted below, the signal processing may be
implemented by an
analog circuit (e.g., an analog cell analyzer as shown in Fig. 39) or a
combination of analog and
digital circuitry.
5 The microprocessor 131 provides output signals to control the fluid
delivery system 15
(noted below) in response to input signals received from the epi-illumination
system 415,
provides output signals to control the transducers 105 in response to input
signals received from
the break-off sensors 389, and provides output signals to control the sorting
system 119 (noted
below) in response to input signals received from the epi-illumination system
415. The
10 microprocessor 131 may provide output signals to other parts of the
cytometry system 9 as noted
elsewhere herein. Further, the microprocessor 131 may be adapted to process
Information and
provide output signals in real time. Broadly speaking, the term "real time"
refers to operations in
which the operation of the processor 131 matches the human perception of time
or those in
which the rate of the operation of the processor 131 matches the rate of
relevant physical or
15 external processes. In one context, the term "real time" can Indicate
that the system reacts to
events before the events become obsolete.
In general, electrical signals from the epl-illumination system 415 are
converted to digital
information by an A/D converter 689 which supplies the corresponding digital
information to the
microprocessor 131. In response to the Information, the microprocessor 131
controls a sorting
20 system 119 and a fluid delivery system 15, both described above.
The electrical signals output from the photodetector 117 of the epi-
illumination system
415 are time-varying analog voltage signals indicative of the Amplitude of the
emitted
fluorescence 31 at any instant in time generated by each cell as it is
illuminated by the laser
beam 25. Thus, the analog signals (also referred to as analog output) are in
the shape of time-
25 varying waveform pulses 497 as illustrated schematically in Figs. 52 and
53. In general a
waveform pulse 497 is defined as a waveform or a portion of a waveform
containing one or more
pulses or some portion of a pulse. Thus, the amplitude of each waveform pulse
497 at any
instant in time represents the relative rate of photon emission 31 of each
cell at that instant in
time as the cell passes through the laser beam 25. X chromosome bovine sperm
cells have a
30 higher DNA content than Y chromosome bovine sperm cells (e.g., about
3.8%). As a result, live
X cells labeled with a fluorescent stain as noted above will produce a
different waveform pulse
497 than pulses from any other labeled cells. By analyzing the pulses 497 as
noted below (see
Signal Processing, Slit Scanning, and Critical Slope Difference), each cell
can be Identified as an
X cell or not identified as an X cell (¨X). In general, as used herein, X
cells refers to live X cells,
35 Y cells refers to live Y cells and ¨X cells refers to the combination of
live Y cells and cells which
otherwise produce a detectable fluorescence emission 31 but which cannot be
Identified with a
reasonable probability as being live X cells.
The timing of each waveform pulse 497 indicates the position of each cell in
the stream
21. Since the rate at which the sheath fluid 19 is being delivered through the
nozzle 137 remains
40 constant, and since the distance d (in Fig. 25) between the nozzle 137
and the droplet break-off

CA 02752247 2011-09-07
61
location 107 Is known, the position of each droplet 33 is known and the cells,
if any, within each
droplet 33 are known. Thus, the microprocessor 131 can calculate the instant
at which each
forming droplet passes through the charging collar 631 and can control the
polarity of the collar
631 and thus control whether a droplet 33 is charged for deflection by the
charging elements 631
of the sorting system 119. Since the microprocessor 131 knows the droplet
formation rate and
identifies the cells within a droplet as X or -X, the microprocessor 131 knows
the cell content of
each droplet 33 and keeps track of (or enumerates) the number of cells in each
population 123,
125. Depending on the sort strategy, see below, the microprocessor 131
determines which
droplets 33 are charged for deflection and which droplets 33 are not charged
so that they are not
Signal Processing
A. Digital Sampling Introduction
As previously described, the interaction between the laser beam 25 and the
particle

CA 02752247 2011-09-07
62
determine the number and classification of each particle In a droplet 33
allows for accurate,
efficient sorting.
Digital signal processing 705 as illustrated in Fig. 72 may be employed to
analyze
detection of fluorescence pulses 31 as indicated by synchronously sampled
output signals 701
from the photodetector 117. This processing would be implemented in pulse
analysis software
employing instructions and/or algorithms, as noted herein. The time-varying
analog output signal
701 of the photodetector 117 Is provided to an A/D (analog/digital) converter
689 which
synchronously samples it. Synchronously sampling means sampling to produce
digital
information corresponding to the analog output. Synchronously sampling is also
referred to as
continuously sampling or streaming acquisition. As noted below, the sampling
rate depends on
the frequency spectrum of the analog output.
Converter 689 provides an output including digital information 707 which is
provided to
the microprocessor 131 or other digital analysis device which executes the
pulse analysis
software to analyze the digital information 707. In general, the pulse
analysis software would
include digital pulse detection HH3, pulse feature extraction HH4 and pulse
discrimination HH7.
=
B. Sampling Frequency & Signal Frequency Spectrum
The signal output 701 from the PMT 117 is captured by a high speed analog to
digital
converter 689 (ADC) that samples the output 701 continuously at a frequency of
105 MHz. It Is
well understood that when sampling a time varying signal it Is necessary for
the sampling
frequency to be at least twice the maximum frequency contained in the signal
being sampled.
This is known as the Nyquist sampling theorem. For this reason the output
signal 701 from the
PMT 117 is first sent through a 40 MHz low-pass filter 854 (see Fig. 39) to
ensure that the
maximum frequency contained in the signal 701 is under the 52.5 MHz limit
imposed by the
sampling rate. It is important to note that the optical 109, fluidic 15 and
detection systems of the
apparatus 1 have been tuned to produce a pulse waveform 497 having optimum
frequency
characteristics for sampling at the 105 MHz rate. The sampling rate may be
varied between
about 25 and 200 MHz without departing from the scope of the present
invention.
C. Pulse Processing
Pulse processing takes place In four (4) TigerSharc DSP processors that share
memory
and are connected to one another by high-speed parallei ports. As illustrated
in Fig. 39, the four
processors are: 1) a data management processor 863 which receives data from a
high-speed
ADC 689 which digitizes the output signals 701 from the photodetector 117; 2)
a pulse detection
processor 865 which detects the waveform pulses 497 represented by the digital
information; 3)
a feature extraction and discrimination processor 867 which extracts features
from the detected
pulses 497 and discriminates the pulses 497 based on the extracted features;
and 4) a sort
processor 873 which determines a sort classification for each pulse 497 based
on the extracted
features and the discrimination, which determines sort decisions for the
corresponding cells and
droplets 33 and which Is synchronized with droplet formation 105. In general a
processor 863,

CA 02752247 2011-09-07
63
865, 867, 873 completes a task and sets a "flag" so that companion processors
know there is
data available to process.
Each processor 863, 865, 867, 873 runs independently of the others, maximizing
the
overall throughput because they do not interrupt each other. Thus, any
processor 863, 865, 867,
All four processors 863, 865, 867, 873 are linked to a DSP board SDRAM 851 for
The processor I/0 857 receives the output 707 from the analog/digital
converter (ADC)
689, e.g., Bitware Corp. 105MHz/2-channel, 14 bit capable of 105MHz/1-channel
sustained. The
ADC 689 is connected to the photodetector 117 output for converting its time
varying analog
output signals 701 into digital information 707 and Is also connected to an
1/0 board SDRAM 855
In general, the analog output signals 701 from the photodetector 117 are
indicative of
characteristic A or characteristic B (e.g., X or -X). The A/D converter 689
converts the analog
output signals 701 from the photodetector 117 of the flow cytometry system 1
into corresponding
digital information 707. The processors 863, 865, 867, 873 analyze and
classify the digital
D. Data Acquisition
As previously stated, the signal output 701 from the photodetector 117 is
captured by a

CA 02752247 2011-09-07
64
"real time" or the actual time that the cell passed through the laser beam 25.
The ADC 689 ping-
pongs back and forth between two inputs, continuously sampling the time
varying analog output
signals 701 including the waveform pulses 497 and converting them Into digital
information 707
which is provided in blocks 855 to the I/0 Board SDRAM under the control of
the data
management processor 863. Processor 863 assembles the information 707 into a
continuous
stream.
E. Initial!zinc' Detection Parameters
In order effectively distinguish over background noise, the digital pulse
detection
software 747 should be provided with information indicating signal background
second order
statistics, i.e. knowledge of the behavior of the output voltage signal 701
from the photodetector
117 when there is no fluorescence pulse 497. These statistics can be learned
by software for
initializing detection parameters 741 in an unsupervised manner during the
initialization period
immediately following startup of the system 1. In general, a pulse may be
defined as 2 or 3
standard deviations from the background level.
Due to the possibility that introduction of the carrier fluid 17 into the
sheath fluid stream
191 may cause a change In background fluorescence emission, the carrier fluid
17 should be
present for the Initialization of the detection parameters. Simple computation
of the second order
statistics of a time sequence of output voltage signal values may overestimate
the standard
deviation of the background (due to the possible presence of fluorescence
pulses 497 in the
sequence). An Iterative procedure is therefore preferred to gradually
eliminate this effect. The
pulse detection software 747 accomplishes this by computing the statistics of
the total signal 701
(background + pulses), using these values to apply pulse detection logic, re-
computing the signal
= statistics without samples detected to be within pulses, and repeating
this procedure until the
background statistic estimates converge (or a fixed maximum number of
iterations occurs). By
evaluating the background with cells present, a more accurate indication of
the expected correct
pulse 497 amplitude can be determined. Table II summarizes the detection
initialization
procedure for determining detection parameters for use by the pulse detection
software.
trest of page blank]

CA 02752247 2011-09-07
_Algorithm: Initializing detection parameters
Input: vector of floats PMTvolts; float statWindowSize, integer
maxiterations
Output: float bckgmdMean; float bckgrndSTD
Procedure:
i. Initialize background vector bckgmd to last statWindowSize
samples of
PMTvolts vector and numlteraf Ions, lastSampleMean, and lastSampleSTD to
zero:
bckgrnd PMTvolts[1to statWindowSize]
lastSamplelidean = 0
lastSampleSTD = 0
numIterations = 0
2. Compute sample mean and sample standard deviation of bckgrnd and
increment iteration counter:
sutn(belcgmd)
sampleMean
statWindowSize
sampleSTD
sum(bckgrnd - sampleMean)2)
=
statWindowSize
numiterations = numiterations + 1
3. Check for convergence or exceeding maximum number of iterations:
exitFlag = ((sanzpleMean - lastSanzpleMean) < eps A
(sanzpleStd - lastSanzpleStd < eps)) v
(nuntherations > maxiterations)
If exitFlag is true, go to step 6 (else continue with step 4).
4. Apply pulse detection algorithm, obtaining vectors of pulse samples and
new
estimate of background samples:
[pulse,bckgrnd] = pulse_detect(bckgrnd, sanzpleMean, sanzpleSTD)
5. Record statistics estimates from this Iteration and repeat
lastSampleMean = sampleMean
lastSampleSTD sanzpleSTD
Go to step 2.
6. Set background statistics estimates to sample statistics and exit:
bckgrndillean = sanzpleMean
belcgrndSTD = samleSTD
Table 11 Initialization,of pulse detection algorithm parameters.
In general, the A/D converter 689 converts the analog output signals 701 from
the
photodetector 117 into corresponding digital information 707 indicative of
characteristic A or
5 characteristic B (e.g., X or --X). The digital signal processor 865
determines background
characteristics of the time-varying output signals 701 from the digital
information 707
corresponding thereto, detects waveform pulses 497 from the digital
Information 707 as a

CA 02752247 2011-09-07
66
function of the determined background characteristics, and provides a sorting
signal 853 to the
sorting system 119 as a function of the detected pulses 497.
F. Initial Discrimination Parameters
Similar to the detection parameters (and subsequent to their initialization as
shown in
Table 11), parameters for use in a discrimination algorithm may be initialized
in an unsupervised
fashion. Unlike the detection algorithm parameters, however, an Iterative
procedure is not
necessary. In this case, software for initializing the discrimination
parameters 745 detects a
preset number (e.g., 100,000) of fluorescence pulses 497, computes the
features to be used for
discrimination for each detected pulse 497, and uses a clustering procedure
(see Table 11 for a
summary of candidate clustering procedures) to assign these pulses 497 to
populations of
interest (e.g. X, ¨X).
Algorithm Name Algorithm Approach
k-Means Iterative (local) minimization of sum of
squared distance
(Euclidean or Mahalanobls) between points within each
population [1]
Fuzzy k-Means Expectation-Maximization of (Gaussian) mixture
model [2]
Agglomerative Hierarchical Merging of "nearest" clusters (starting with
each data point as
its own cluster) until desired number of clusters is reached.
Various measures for determination of "nearest" clusters
include distance between closest points, distance between
furthest points, distance between cluster means, and average
distance between points. [1]
Table 2. Summary of clustering approaches being considered for use in
discrimination algorithm
parameter Initialization.
Fig. 73 contains an example of the results of application of a k-means
clustering
procedure to define population 1 and population 2 based on statistics of
distribution. The second
order statistics of these populations are then used to set the parameters
necessary for
discrimination (the coefficients of a 1 st or 2nd order polynomial decision
function). Table IV
summarizes the discrimination initialization procedure.
[rest of page blank].

CA 02752247 2011-09-07
67
Algorithm: Initializing discrimination parameters
Input: Matrix of floats detectedPuiseData, vector of floats
popPriorProbabilities
Output: For each class population /: matrix of floats 14th vector of
floats wlI float wio
Procedure:
1. Compute feature values from detected pulses (n values per pulse, where n
is
dimensionality of feature space):
feature Values = feature_extract(detectedPulseData)
2. Cluster feature values In feature space to obtain population memberships

populations = cluster( featureValues)
3. Compute 2nd order statistics of populations:
(for 1= 1 to m, where m is number of populations/classes)
(for j= 1 to n, where n is dimensionality of feature space)
popMeani[j]=sum(featureValues[populations I, j])
# of samples in populations
(for k = 1 to n, where n is dimensionality of feature space)
tinpVal[ j ,k]= (featureValues[populations , j] - populationMean,[j]) =
(featureValues[populations I, lc] - populationlideani[k])
sum(tinpVal[j,k])
popCovariance,[j ,
# of samples in populations,
4. Compute polynomial discriminant function coefficients:
(for i= 1 to m, where m is number of populations/ciasses)
¨1/2 = popCovarinace,-1
--1
= popCovarinace, = popMean,
= ¨1/2 - ln(lpopCovariatcei ()-
1/2 = popMeat( = pop Covariaicei-1 = popMeati +
in(popPriorPtobabilities ,)
Table IV. Initialization of discrimination algorithm parameters.
In general, the A/D converter 689 converts the analog output signals 701 from
the
photodetector 117 into corresponding digital information 707 indicative of
characteristic A or
characteristic B (e.g., X or -X). The digital signal processor 867 generates
initial discrimination
parameters corresponding to the digital information 707, discriminates the
digital Information as a
function of the initial discrimination parameters, and provides a sorting
signal 853 to the sorting
system 119 as a function of the discriminated digital information.
G. Digital Pulse Detection
The first processing step Is pulse detection performed by pulse detection
processor 865
to determine whether a particular waveform is a waveform pulse 497
corresponding to a
fluorescence emission 31 of a cell. The processor 865 executes a pulse
detection algorithm
which identifies sample sets that are likely to represent either particles
targeted for sorting Into a

CA 02752247 2011-09-07
68
population or particles targeted to be avoided because they are potential
contaminants to a
population. In the case of bovine sperm sorting, a dye is added to quench the
emission 31 of
non-viable cells, causing their associated pulse intensities to be -1/3 the
intensity of a live cell.
Nonviable cells are not considered as sorting targets or potential
contamination. They are not
considered detected pulses 497. Pulses 497 from live cells are detected by
monitoring the
Intensity of samples for a successive number of samples that rise above the
background levels.
Once this level crosses a statistically determined threshold the processor 865
jumps to a later
time that is approximately 75% of the expected pulse 497 width for a live
cell. If the level is still
above the threshold, the series of samples are considered to be a pulse 497.
Samples from
detected pulses 497 are moved to a block of memory used by the feature
extraction processor
867.
A statistical anomaly detection approach Is one embodiment which may be
employed by
digital pulse detection software 747 although it is contemplated that other
approaches for
identifying and/or isolating digitized pulses 497 may be used. Essentially,
digital samples 707 of
the output voltage signals 701 from the photodetector 117 detecting
fluorescence which are
statistically anomalous from the background are considered to part of a pulse
497. For additional
robustness (to minimize noise detections), additional temporal criteria may be
included.
Pulse detection proceeds as follows. When the voltage output signal 701 from
the
photodetector 117 is not a pulse, the Mahalanobis distance from the background
of incoming
samples 707 of the signal 701 is computed and compared with a preset
threshold. If the
distance of a given sample exceeds the threshold, it is considered to be the
potential start of a
pulse 497, and the pulse detection software begins to buffer the incoming
samples. If the next
predetermined number of samples (e.g., 25) also exceed the threshold, a pulse
497 is
considered to have started and buffering continues until the pulse end
criteria are met; otherwise,
the buffer is reset and checking for the start ot a pulse resumes. While in a
pulse 497, if a
sample is below the threshold, then it is considered to be the potential end
of a pulse and the
buffer location is recorded (but sample buffering continues). If the next
predetermined number of
samples (e.g., 25) are also below threshold, the pulse 497 is considered to
have ended and the
pulse 497 consists of the buffered samples up to the recorded location. Table
V summarizes the
pulse detection algorithm, and Fig. 49 provides an illustration of the results
of pulse detection on
a digitally acquired fluorescence pulse 497.
[rest of page blank]

CA 02752247 2011-09-07
69
Algorithm: Digital fluorescence pulse detection
Input: vector of floats digSamples, float bkgrndMean, float
bkgrndSigma,.float
putseStartThresh, float pulseEndThresh, integer numStartSamples, integer
numEndSamples
Output: vector of floats puiseBuffer
Procedure:
1. Initialize inPulseFlag 0, pulseStadCount = 0, pul,seEndCount = 0
2. For each sample in digSamples, compute Mahalanobis distance from
background:
mhDist[i],(digSamplEN-bkgrndMea4
bkgrndSignn
3. If inPulseFlag Is not set, go to step 4, else go to step 6.
4. If mhDist > pulseStadThresh, place sample in pulseBuffer, increment
pulseStartCount, and go to step 5; else set pulseStartCoun1=0, go to step 2.
5. If pulseStartCount > numStattSamples, set inPutseFlag and go to step 2.
6. If mhDist < pulseEndThresh, place sample in pulseBuffer, set
lastPulseSample to current buffer position, increment pulseEndCount, and go
to step 7; else set pulseEndCount to zero and go to step 2.
7. If pulseEndCount is greater than numEndSamples, return
pulseBufferM to lastPutseSamplei and exit.
Table V. Summary of digital fluorescence pulse detection.
In general, the AID converter 689 converts the analog output signals 701 from
the
photodetector 117 into corresponding digital information 707 indicative of
characteristic A or
characteristic B (e.g., X or -X). The digital signal processor 865 analyzes
the digital information
and processor 873 provides a sorting signal 853 to the sorting system 119 as a
function of the
detected digital information.
H. Feature Extraction and Discrimination
The next processing step is feature extraction performed by the feature
extraction and
discrimination processor 867. This processor responds to flags set by the
pulse detection
processor 865. Samples from detected pulses are placed in memory shared with
the feature
extraction processor 867. Features such as area, pulse width, pulse height,
Gaussian correlation
coefficient and/or other features are determined for each pulse 497. In some
cases pulses 497
are determined to be "doublets" or Invalid and features are not extracted. For
the case of bovine
sperm 201 features are only extracted for pulses 497 that have the general
amplitude and width
of a live X or Y cell. Typically, the pulse amplitude for a live sperm cell is
In the range of about
700-900 mV, although this range may be as wide as 500-1000 mV. Once the
features are
extracted they are compared to the feature spaces defined for the
population(s) selected for
sorting. If the features match the feature spaces identified for sorting, then
processor 867 sets a
flag indicating a positive sort command to the sort processor 873. In general,
the classification of

CA 02752247 2011-09-07
a particular cell Is made by the discrimination processor 867 and the sort
decision is made by the
sort processor 873.
Digital information 707 representing fluorescence emissions 31 (and thus the
characteristics of corresponding cells which created them) are discriminated
by software 757
based on specific features or characteristics which exhibit distinguishably
different statistical
behavior in feature space (the n-dimensional orthogonal space formed by n
features as the axes)
for the different populations of Interest. Therefore, the first step in
analyzing digital information
707 for the purposes of discrimination is computation of these features, a
process called feature
extraction performed by pulse analysis software 749 executed by the processor
867. Table VI
lists the several candidate features which software 749 may use for this
application. One or
more of these features will be selected to form the feature space for
classification. It should be
noted that there are additional features providing enhanced separation so that
this list is
exemplary, not comprehensive. For example, the software 749 may employ a
subroutine 753 to
determine pulse 497 area and/or may employ a subroutine 755 to determine pulse
497 peak.
Feature Name Feature Description
Pulse Area Approximated by sum (or average) of pulse
samples
Pulse Peak Maximum value of pulse samples
Pulse "Inner" Area= Sum (or average) of Inner TBD samples of pulse
(centered on
pulse mean)
Pulse Width Number of samples in pulse.
Pulse "GaUssianity" MSE or correlation coefficient of pulse with a
Gaussian shape
with the same 2"d order statistics.
Pulse "Lagging Peak" Pulse value at TBD samples past peak (or mean)
Critical Slope Difference (CSD) Slope of pulse at a point along the pulse
at which the
difference between the first derivative of a pulse produced by
particles having characteristic A and the first derivative of a
pulse produced by particles having characteristic B is at or
near a maximum
Table VI. Summary of candidate featUres currently being considered for use in
digital
pulse analysis relating to feature extraction.
I. Slit Scanning
In general, the elliptical spot 459 provided by the illumination system 109
measures the
relative DNA content differences in cells. Resolution can be improved further
by analyzing the
fraction of the pulse 497 of the fluorescence emission 31 detected by the
photodetector 117
more likely to contain characteristics which are being evaluated. A biological
phenomenon of
certain cells (e.g., bovine sperm cells) is the localization of the VY
chromosomes in a sub-
equatorial region 225 which is immediately adjacent the longitudinal midllne
or equator or center
of the nucleus 213 of the cell 201 and which has a length of about 1 pm. (See
Fig. 6). In fact, the
X/Y chromosomes are not necessarily centered in the nucleus 213. Thus,
resolution can be
improved by converting the time-varying analog output 701 of the photodetector
117 into digital
information 707 and analyzing a portion of the digital Information
corresponding to the fraction of

CA 02752247 2011-09-07
71
the pulse 497 of the fluorescence emission 31, e.g., corresponding to the
light emitted from the
circumequatorial region 225 such as such as 20-60% and particularly 20-30% of
the waveform
pulse centered around the pulse 497 peak.
As noted above, slit scanning can be employed to obtain the fluorescence
measurement
from a portion of each cell's chromatin rather than from the chromatin as a
whole. The elliptical
spot 459 provided by the epi-illumination system 415 noted above measures the
relative DNA
content differences In cells from specific sections of the chromatin, so that
the resolution of X
cells and ¨X cells relative to one another is improved. As noted above, the
slit scanning
measurement technique is a fluorescence measurement approach that focuses the
excitation
beam 25 so that a dimension of the focused spot size 459 is much less than a
cell diameter as
shown in Fig. 6. In this way, the cell 201 is scanned by the laser beam 25 as
the cell passes
through the elliptically-shaped beam spot 459. The resulting waveform pulse
497 produced by
the photodetector 117 output 701 detecting the fluorescence emission 31
resulting from slit scan
illumination contains information about the localization of fluorescence along
the length of the cell
201. As shown in Figs. 45-48, as the cell 201 traverses the elliptically-
shaped beam spot 459,
the time-varying waveform pulses 497 are the convolution of the relative beam
intensity and the
relative emitted pulse Intensity (which corresponds to the fluorescence
emissions from stain
excited by the elliptical spot as the cell traverses the beam and which varies
because the
fluorescence distribution along the axis of the cell varies).
By illuminating only a fraction of the cell's chromatin at one time, the
resulting time-
varying analog output 701 from the photodetector 117 contains information
specific to the
localization of fluorescence within the chromatin along the longitudinal axis
of the cell 201.
Although the detected fluorescence emission 31 from slit scanning is less than
the detected
emission 31 from scanning by a beam 25 having a spot width comparable to the
cell diameter,
resulting in waveform pulses 497 from slit scanning having a lower pulse
amplitude, the majority
of difference between the X-chromosome bearing cells and the Y-chromosome
bearing cells
appears in the center 20-30% to 20-60% of the waveform pulse 497. If only the
rectangular area
725 In Fig. 53 is considered for discriminating X-Y sperm cells, then a larger
relative difference
can be measured between the localized variation In DNA content within the
section of chromatin
that corresponds to the rectangular region 725 due to the presence of the X
and Y chromosomes
within that region as compared to the total DNA content of the cells. For
example, bovine X-Y
sperm cells have a difference in total DNA content of about 3.8%. The
fluorescence emission 31
from the X and Y chromosomes will be contained in the rectangular region 725.
If this
rectangular region 725 accounts for 20% of the total waveform pulse 497
corresponding to a
fluorescence emission 31, then a 14% difference in relative DNA content within
the region will
exist. By measuring the relative DNA content differences from specific
sections of the chromatin,
the resolution of X-Y sperm cell differentiation is improved (e.g., from 3.8%
to 14%). Fig. 54
illustrates the resolution attainable using slit scanning illumination and
processing the areas from
only the center 20% of the pulse 497 (i.e., the rectangular region 725 of Fig.
53). The histogram
of Fig. 54 allows a very high percentage (e.g., 98%) of the X chromosome
bearing sperm and Y

CA 02752247 2011-09-07
72
chromosome bearing sperm to be Identified with a high degree of confidence
(e.g., 95%). In
comparison, the histogram of Fig. 55, which illustrates the resolution
obtainable when using
standard Illumination techniques, shows that slit scanning offers a
significant improvement over
the results obtained using standard illumination techniques.
Two approaches which can be employed to obtain the area 725 of the center
portion of
the waveform pulse 497 as illustrated In Fig. 53 are digital signal processing
(DSP) of digitized
photodetector 117 time-varying analog output 701, as discussed in this
section, or analog
integration using an analog threshold trigger, as noted below. As noted
herein, DSP processing
involves continuously sampling the time-varying analog output 701 from the
photodetector 117 to
obtain digital information 707 corresponding to the output 701 and applying
DSP algorithms to
the digital information 707 to extract features, such as area size, from the
digital information
corresponding to the center portion 725 of the waveform pulse 497 which
corresponds to the
difference In DNA content due to the presence of an X or Y chromosome In
different cells 201.
As a simple example, the center 20% of the total area of each waveform pulse
497 would be
determined by analyzing the digital Information 707 corresponding thereto. The
analysis would
be used to generate a histogram such as illustrated in Fig. 53.
J. Pulsed Laser Scanning
In one embodiment, it Is contemplated that the system 1 Include a pulsed laser
to
illuminate the cells. In this embodiment, slit scanning (as described above)
may or may not be
employed. For example, a mode-locked solid-state laser can be used to emit a
train of
electromagnetic pulses having a pulse width (duration) of 1-100 picoseconds at
a pulse
frequency of about 50-150 MHz and at an average power output of about 100-500
milliwatts.
One suitable laser is a Vanguard 350 mode-locked solid-state laser (available
from Spectra-
Physics, Mountain View, CA 94039), which Is operable to emit a series of
pulses about 12
picoseconds in width (duration) at a frequency of about 85 million pulses per
second and at an
average power of about 360 milliwatts. Because the 350 mW of power is
delivered over
extremely short bursts of only 12 picoseconds, the peak power output of such a
laser is several
hundred times (e.g., about 800 times) greater than the average power.
The output of such a laser can be described as quasi continuous wave (quasi-
cw)
because, for many applications, the pulse repetition rate is fast enough to
approximate a
continuous wave (cw) output. Indeed It is possible to operate the system as
described above
with a quasi-cw laser in much the same manner as one would operate with a cw
laser. This
provides certain advantages because solid-state lasers typically operate more
efficiently, require
less extensive cooling systems, and require less maintenance than most other
lasers.
A quasi-cw pulsed solid-state laser can also result in significantly improved
signal-to-
noise ratios using digital signal processing techniques. A timing circuit may
be included and Is
operable to produce a timing signal indicative of the arrival of laser pulses
at the interrogation
location 11 5 (i.e., the area where the laser beam 25 Illuminates the stream
21). For example, the
timing circuit may be a laser pulse sensor 3003 as shown in rig. 40 for
detecting light

CA 02752247 2011-09-07
73
corresponding to the laser pulse including scattered light generated by the
Interaction of each
laser pulse with the fluid stream 21 and/or Including light from the laser
pulses. Alternatively, for
lasers which may be triggered, a triggering signal may be provided to the
microprocessor 131
and/or the AID converter 689 to synchronize either or both to the laser
pulses, as noted below
with regard to Fig. 50. In either embodiment, the laser pulse timing would
provide a clock signal
for the system.
Referring to Fig. 50, a timing diagram illustrates the timing relationship
between the laser
pulses LP, the fluorescence emissions FE from a cell as a result of repeated
excitation by the
laser pulses LP as the cell passes through the beam spot 459 and the digital
samples DS of the
photodetector output 701. As shown in FIGS. 45-49, as a cell passes through
the laser beam
spot 459 the fluorescence emission 31 varies depending upon the amount of
illumination of the
portion of the cell which generates the fluorescence emission 31. Fig. 50
Illustrates twenty (20)
laser pulses LP1-LP20 which impinge upon a cell as the cell passes through the
interrogation
zone 115 of a flow cytometer 1. Each laser pulse LP1-LP20 corresponds to a
fluorescence
emission FE1-FE20, respectively, which exponentially decays after
substantially instantaneous
excitation by the laser pulse.
In one embodiment, the microprocessor 131 controls the ND converter 689 (see
Fig. 40)
so that the converter 689 samples the output signal 701 of the photodetector
117 at or near peak
of each fluorescence emission FEl-FE20, as indicated by digital samples DS1-
DS20,
respectively. In other words, the timing circuit synchronizes the sampling
rate of the AID
converter 689 with the fluorescence emissions FEI-FE20. The resulting digital
signal produced
by transit of a particle through the interrogation zone 115 is the functional
equivalent of the digital
signal that would have been produced by the digitization of a pulse waveform
497 from a
continuous wave laser. As shown in Fig. 51, for example, by considering only
the fluorescence
intensity during the digital samples DS1-DS20 and disregarding fluorescence
intensity drop-off
between laser pulses LP1-LP20, the fluorescence intensity as a function of
time is a pulse
waveform 497. This permits feature extraction by the microprocessor 131 from
the digital signal
707 generated by the pulsed laser in order to analyze the cell providing the
fluorescence
emissions FE1-FE20. In one embodiment, a more sensitive photodetector having
relatively fast
response time of about 2 nanoseconds or less may be used to more accurately
detect the
fluorescence emissions.
Thus, the pulsed laser provides advantages in a flow cytometry system 1 in
that it Is
possible to use a lower power pulsed laser to obtain substantially the same
analysis that would
be obtained with a cw laser operating at an average power much higher than the
average power
of the pulsed laser. Further, the high peak power from a pulsed laser tends to
saturate the
fluorophores so that the fluorescence emissions are maximized thereby reducing
the signal-to-
noise ratio of the output signals of the photodetector. In other words, by
using a laser pulse that
contains much more energy than is required to saturate the fluorophore,
variations in the output
of the laser do not result in variations in the fluorescent emissions 31.

CA 02752247 2011-09-07
74
Those skilled In the art will recognize that there are many ways to cause a
laser to emit a
series of pulses. It is understood that other pulsed lasers, Including other
mode-locked lasers, Q-
switched lasers, and cavity dumping lasers, could be used in place of the mode-
locked laser
discussed above without departing from the scope of this Invention. Similarly,
many other ways
K. Estimation of Population Characteristics
As noted above, flow cytometry can be used to discriminate X-bearing bovine
sperm
25 These characteristics are illustrated on the example pulse that appears
in Figs. 52 and
53. Figures 52 and 53 are examples of pulses 497 from X-bearing and Y-bearing
sperm cells.
The pulses 497 were generated from a computer model that assumed excitation
illumination with
a laser beam 25 having an elliptically-shaped beam spot 459 having a 2,um
Gaussian beam
waist W1 (Fig. 6) and that the DNA content difference was distributed
uniformly across the center
Integration of the pulses 497 results in a 3.8% average difference between the
pulse 497 area for
an X cell and the pulse 497 area for a Y cell.
=
It is possible to generate histogram and scatter plots of the pulse 497 peak
and area

CA 02752247 2011-09-07
cells (i.e., cells that generate relatively weaker fluorescence emissions due
to slight deviations
from the optimal alignment, but that are not so far out alignment to cause the
relatively brighter
fluorescence emission 31 from the narrow edge 209 of the sperm head 205 to be
collected by
the optics system 109). Only about half of the X cells can be easily
identified in the live cell area
5 histogram. The rest overlap with the Y cell population and the non-
aligned cell populations.
Even when peak pulse height is added, as shown in the scatter plots in Figs.
56-59, X-bearing
cell classification may be significantly limited.
Figs. 60-61 illustrate the overlap of the X and Y population distributions. In
Figs. 60-61,
a four-component computer model has been applied to raw data 6000(Fig. 60) to
estimate
10 population statistics for two populations of non-aligned cells (6001,
6003), aligned live Y cells
(6005) and aligned live X cells (6007) (Fig. 61). It Is desirable to
discriminate the X and Y
populations as a function of the coefficient of variation (CV) of the X and Y
populations. For
example, It Is desirable to minimize the coefficient of variation (CV) of the
X and Y populations in
order to Improve discrimination. In particular, when a population of sorted X
cells Is desired, it is
15 desirable for the CV of the X cell population to be less than 1.5%, more
desirably about 1.3%,
and even more desirably less than 1.3%. Traditionally, the CV of a
distribution of fluorescence
intensity of sperm cells has been considered with respect to the distribution
functions for only two
populations (X and Y). Quality control with respect to CV has been limited to
crude subjective
estimation of the CV of the X and Y populations to decide whether continued
analysis or sorting
20 Is worthwhile.
According to one embodiment of the present invention, one function of the
microprocessor 131 is to provide an automated estimation of the CV of the X
population using
the four-component model illustrated in Figs. 60-61. In order to estimate the
CVs of the
populations present in a feature (e.g. pulse area) distribution, it is
necessary to estimate the 2"d
25 order statistics of the population distributions. This may be achieved
by applying a model of a
known form and finding the best fit of that model to the observed data.
Given the expectation of normally-distributed data, an approach consisting of
Parzen
Window based non-parametric density estimation (utilizing a Gaussian kernel
function) followed
by application of a Gaussian mixture parametric model has been chosen.
Specifically, the four-
30 component model illustrated in Figs. 60-61 consists of a sum (or
mixture) of four uni-variate
Gaussian distributions, with these four components being the feature
distributions corresponding
to aligned X cells, aligned Y cells, and a two-component unaligned cell
population. The
parameters characterizing the model then are the population means (averages)
(4), population
standard deviations/variances (4), and prior probabilities (expected % of the
overall distribution)
35 (4). These 12 parameters can then be varied to achieve a best fit of the
model to the observed
data histogram. With the model component parameters thus estimated, an
estimate of the CV of
a population of interest (in particular, X cells) may be determined from the
estimated population
standard deviation and mean:

CA 02752247 2011-09-07
76
standard deviation
CV = ___________________ =1.00%
mean
In order to reduce computational complexity, constraints have been placed on
the model
to reduce the dimensionality of the parameter space. In particular, the
standard deviations of the
model components corresponding to the aligned X and aligned Y populations have
been
constrained to be the same. Also, the aligned X and aligned Y components have
been
constrained to make up the same percentage of the overall mixture ¨ thus the
non-aligned
populations are assumed 50% X cells and 50% Y cells.
Non-parametric density estimation is applied prior to model fitting to obtain
an improved
estimate of the total density function (being the sum of the component
densities) underlying the
raw histogram data. The specific technique applied is known as "Parzen
Windows" (Duda, Hart,
and Stork, 2001), here utilizing a Gaussian kernel or window function due to
the assumed sum-
of-Gaussian nature of the underlying density. The standard deviation of the
Gaussian kernel is
chosen to be 15/0 of the number of populated histogram bins; this value has
been empirically
observed to provide adequate but not excessive smoothing of the histogram.
Each data point in
the histogram then contributes a kernel function centered on the histogram bin
containing the
data point. The density estimate is then obtained as the sum of the kernel
functions.
The methodology chosen for variation of the model parameters to achieve the
best fit to
the data is known as Expectation Maximization (See Duda R.O., Hart, P.E., and
Stork, D.G.,
2001, Pattern Classification 2"d Ed., John Wiley & Sons; and Moore, A., "Very
Fast EM-based
Mixture Model Clustering using Multiresolution kd-trees," in M. Kearns and D.
Cohn, Eds.,
Advances in Neural Information Processing Systems, pages 543-549, 1999, Morgan
Kaufman).
The specific algorithmic implementation utilized is as follows:
1) Initial conditions for the model parameters are set. The top two local
maxima in the
Parzen density estimate are used as the initial Y and X mean locations (the
initial
amplitude or the maxima for both X and Y populations being estimated as the
amplitude
of the right peak). The initial X and Y population variance is estimated as
the variance of
the data to the right of the local minimum occurring between the left and
right peaks
relative to the right peak location. Also, the initial X and Y population
prior probabilities
are set as the percentage of all points falling to the right of this local
minimum. The initial
X and Y population Gaussian density estimates are then computed using these
parameters and subtracted from the total Parzen density estimate. The mean and

variance of the remaining data points are then computed and used to initialize
the two-
component unaligned population model as follows. The two population means are
assumed (arbitrarily) to be 5% apart (2.5% above and below the overall
unaligned
mean). Given an (initial) assumption of equal priors and equal variances,
then, the
component variances are given by:

CA 02752247 2011-09-07
77
=
py,2 cr,2 (
'1,2 'tot 4 kt"2
where a2ls the variance and p is the mean of the respective population.
2) Updated estimates of component population statistics (means, standard
deviations, and
priors) are computed using the Parzen density estimate. Each histogram bin
location is
weighted in the statistical computations by the Parzen density estimate in
that bin.
Additionally, each data point contributes to all component population
statistic
computations weighted by the degree to which that point is believed to belong
to a given
population, based on the current component population parameters. This degree
of
membership is determined as the ratio of a given component population
(Gaussian)
probability density value to the sum of all component population probability
density
values at the data point. Thus we have (for all data points x in the
histogram,
populations cpe{c,,cy,cõ}, and population parameter vector Oe[pp,crp,Pp])
population
component memberships used in the computation of updated parameter estimates
given
by:
1
P(c 1 x,9,) = P = exp __________
P
a -µ127r 2a2
13
P(c I x,0 )
membership(c x,0 ) P P = ParzenDensityEstimate(x)
P P P(cõ I x,On)
110p
Updated means and variances are then computed using the Parzen density
estimate
values weighted by these membership values, with updated priors given by the
average
membership for each component population over all data points.
3) Parameter updating procedure continues until all parameters reach steady-
state (i.e.,
stop changing significantly from one update iteration to the next (or a
maximum allowed
number of iterations occurs)).
As previously mentioned the aligned X and Y populations are constrained in
this
procedure to have the same variance and prior probability. This constraint is
achieved by using
the average of the X a'nd Y variance and prior values computed via the above
procedure at each
Iteration.
Alternatively, a similar modeling approach can be applied to a three-component
model
(Figs. 62-63) in which the cells comprising the two unaligned populations
6001, 6003 in the four-
component model are treated as a single Gaussian distribution 6011 rather than
two distinct
subpopulatlons. The non-aligned cells can be modeled as third Gaussian
distribution (shown

CA 02752247 2011-09-07
78
Fig. 63) having a mean and standard deviation determined by a best fit of the
left shoulder and
left major peak of the raw data 6010 (shown Fig 62). The three-population
model also has
estimated statistics for the aligned Y Population 6015 and aligned X
Population 6017. One
advantage of the three-component model is that it requires only 9-dimensional
parameter space
(compared to 12-dimensional parameter space required for the four-component
model).
However, it has been found that the four-component model typically results in
an estimated
density function that more closely matches the raw data.
Those skilled In the art will recognize that a wide variety of statistical
techniques can be
used to estimate the characteristics of the aligned X and aligned Y
populations. Thus, the four-
component model, the three-component model, or other models may be implemented
by any
parametric or non-parametric computer algorithms to estimate the
characteristics of the aligned X
cell and/or aligned Y cell populations without departing from the scope of
thls invention.
L. CV-Based Selection of Staining Conditions
Several factors affect the efficiency of sorting stained cells within a
population into
enriched subpopulations of cells. Among these factors is the amount of
differential fluorescence
between the various subpopulations of cells within a stained population.
Differential fluorescence
is affected by dye uptake, which varies based upon staining factors, such as
for example, the
concentration of the stain, the length of the staining period, the temperature
at which staining
occurs, and the number and concentration of any additives that may be included
with the stain or
added to the staining mixture. Accordingly, adjustments to any or all of these
factors may be
made to increase the sorting efficiency (the rate at which cells may be Sorted
into at least one
enriched subpopulation of cells with certain degree of purity and/or a minimal
loss of desired
cells) of the population of stained cells. Further, one can increase
efficiency of a multi-sample
sorting system by adjusting one or more of these factors for each sample,
thereby countering any
sample-to-sample variations. In the context of bovine sperm sorting, for
example, sorting
efficiency can be improved by adjusting one or more of the foregoing staining
factors from one
semen sample to the next to counter bull-to-bull variations or sample-to-
sample variations within
the same bull.
A determination of the coefficient of variatioh ("CV") for a given
fluorescence emission
characteristic of a population of cells to be sorted is one manner in which to
determine if
adjustments to the staining conditions could be made to achieve a desired
sorting efficiency. For
example, one may adjust the staining conditions as a function of the CV of any
feature extracted
from the pulse waveform generated by movement of a cell through the
interrogation location,
such as any feature indicative of total fluorescence intensity or peak
fluorescence intensity
(including total fluorescence intensity and peak fluorescence intensity). As
previously discussed
in greater detail, CV is an indicator of the homogeneity or consistency of a
distribution of a
measurable property or characteristic of a population, such as for example a
fluorescence
emission characteristic of a particular subpopulation of a given population.
CV may be
determined by dividing the standard deviation of the nieasured characteristic
of a sample by the

CA 02752247 2011-09-07
79
sample mean. CV can also be determined automatically by the flow cytometry
system 9, such as
by implementation of the Iterative CV estimation algorithm discussed in detail
above. The lower
the CV, the greater the homogeneity or consistency of the distribution of the
measured
characteristic.
As applied to the staining and separation of sperm cells, the CV of a
particular
fluorescence emission characteristic for a sample of X and Y chromosome
bearing sperm cells
may be affected by the staining conditions. The concentration of the stain,
the length of the
staining period, the temperature of the staining mixture, and/or the number
and concentration of
additives affect the CV of a given fluorescence emission characteristic.
Increasing the
concentration of the stain, the length of the staining period, and the
temperature of the staining
mixture and/or decreasing the number and concentration of additives will
generally lower the CV.
Such conditions may be altered individually or in combination. In addition, if
any one of these
factors Is such that it would tend to increase the CV of a fluorescence
emission characteristic,
such as for example, by shortening the staining time, then any one or more of
the other
conditions may be adjusted such that it counteracts the effect of the first,
such as for example, by
increasing the dye concentration, with the overall result being a decrease in
the CV of the
fluorescence emission characteristic to a level sufficient to achieve a
desired sorting efficiency.
Accordingly, by manipulating any one or any combination of these factors In
this manner, the CV
of a fluorescence emission characteristic of the X and Y chromosome bearing
populations may
be decreased to a value that enables sorting of the sperm sample Into a
subpopulation of gender
enriched semen comprising a desired percent purity of X chromosome bearing
cells.
Unfortunately, changes that tend to lower the CV of the X bearing sperm may
have
hegative consequences such as increased cost or decreased sperm motility or
fertility. For
example, other things being equal it is desirable to use lower stain
concentrations and shorter
staining periods to minimize any harmful impact of the staining process on the
sperm. With this
In mind, one may predetermine a CV at which an acceptable sorting efficiency
will be achieved.
Thereafter, a fraction of the cell sample to be sorted is stained and
subjected to flow cytometric
analysis. A fluorescence emission characteristic of the fraction is
determined, and the fraction is
classified into subpopulations based upon the characteristic. The CV of the
fluorescence
characteristic is determined with respect to the cells of one of the
subpopulations (an enriched
subpopulation). If the CV of the fluorescence emission characteristic of the
cells of the enriched
subpopulation is equal to or less than the predetermined CV at which sorting
Is to occur, then the
remainder of the cell sample is stained according to the conditions under
which the fraction was
stained. The sample is thereafter sorted, for example, according to the
methods disclosed
herein. If the CV of the particular fluorescence emission characteristic of
the cells of the enriched
subpopulation is greater than the predetermined CV at which sorting is to
occur, then another
fraction of the same sample is analyzed in a similar manner, but under
staining conditions
believed to achieve a yet lower CV. In such a situation, the CV may be lowered
by, for example,
increasing the length of the staining period, increasing the concentration of
the dye, increasing
the temperature at which the fraction Is stained, or any combination thereof.
This series of steps

CA 02752247 2011-09-07
(Le., removal of a fraction from the sample to ba- sorted, adjustment of the
staining conditions,
and a determination of the CV) Is repeated until the CV of the particular
fluorescence emission
characteristic of the cells of the enriched subpopulation is determined to be
equal to or lesser
than the predetermined CV. Thereafter, the remainder of the sample Is stained
accordingly and
5 may be sorted, for example, according to the methods disclosed herein. In
a particular
embodiment of the invention, the cell sample comprises a semen sample, and the
cells of the
enriched subpopulation comprise X chromosome bearing sperm cells.
Accordingly, one embodiment of the Invention is a process for evaluating a set
of
conditions for staining a population of cells for sorting, the population
comprising a first type and
10 a second type of cell. The process comprises (a) staining a fraction of
the population of cells
with a fluorescent dye under a set of staining conditions; (b) exposing the
stained cells to
electromagnetic radiation as the stained cells are passed through an
interrogation location of a
flow cytometer at a rate, R; (c) determining a fluorescence emission
characteristic of the exposed
cells; (d) using the determined fluorescence characteristic to classify the
cells into two or more
15 sub-populations, one of the subpopulations being an enriched
subpopulation of the first cell type;
(e) determining a coefficient of variation for the fluorescence emission
characteristic of the cells
of the enriched subpopulation; and (f) determining whether to modify any
staining condition under
which the cells are to be stained or the rate, R, at which the stained cells
are passed through the
interrogation location of the flow cytometer. In another embodiment, another
fraction of the
20 population of cells is stained under a dfferent set of staining
conditions and steps (b) through (e)
are repeated with that fraction. This process may be performed on two, three,
four or any
number of additional fractions. In another embodiment, multiple fractions of
cells are drawn from
the sample at the same time. Each fraction may be stained simultaneously, or
each may be
stained subsequent to the previous fraction being passed through the flow
cytometer. In the
25 former case, each fraction may be stained with Its own unique set of
staining conditions and step
(f) may comprise using the respective CVs to determine a set of staining
conditions to be used to
stain additional ceils. In the later instance, the staining conditions of the
subsequently stained
fractions may be altered according to the determination of step (f) with
respect to a previously
analyzed fraction. In one embodiment the process is repeated until the CV is
determined to be
30 about equal to or less than a specified CV (e.g., 1.3%).
Alternatively, once one has predetermined a CV at which an acceptable sorting
efficiency will be achieved, the entire cell sample may be stained. A fraction
of the cell sample is
removed and subjected to flow cytometry analysis. A fluorescence emission
characteristic of the
fraction is determined and used to classify the cells into two or more sub-
populations. The CV of
35 the fluorescence characteristic is determined with respect to the cells
of an enriched
subpopulation. if the CV of the fluorescence emission characteristic of the
cells of the enriched
subpopulation is equal to or less than the predetermined CV at which sorting
is to occur, then the
remainder of the cell sample is thereafter sorted. If the CV of the particular
fluorescence
emission characteristic of the cells of the enriched subpopulation is greater
than the
40 predetermined CV at which sorting is to occur, then a second fraction
from the same sample is

CA 02752247 2011-09-07
81
analyzed in a similar manner and the CV of the same fluorescence
characteristic is determined.
The CV of the second fraction may be lowered by, for example, increasing the
length of the
staining period, increasing the concentration of the dye, or any combination
thereof. This series
of steps (i.e., removal of a fraction from the sample to be sorted and a
determination of the CV) is
repeated until the CV of the particular fluorescence emission characteristic
of the cells of the
enriched subpopulation is determined to be equal to or lesser than the
predetermined CV.
Thereafter, the remained of the sample may be sorted, for example, according
to the methods
disclosed herein. In a particular embodiment of the invention, the cell sample
comprises a
semen sample, and the cells of the enriched subpopuiation comprise X
chromosome bearing
cells.
Accordingly, another embodiment of the invention is a process for evaluating a
set of
conditions for staining a population of cells for sorting, the population
comprising a first type and
a second type of cell. The process comprises (a) staining a fraction of the
population of cells
with a fluorescent dye under a set of staining conditions; (b) exposing the
stained cells to
electromagnetic radiation as the stained cells are passed through an
interrogation location of a
flow cytometer at a rate, R; (c) determining a fluorescence emission
characteristic of the exposed
cells; (d) using the determined fluorescence einission characteristic to
classify the cells Into two
or more subpopulatlons, one of the subpopulations being an enriched
subpopulation of the first
cell type; (e) determining a coefficient of variation for the fluorescence
emission characteristic of
the cells of the enriched subpopulation; (f) determining whether to modify any
staining condition
under which the fraction of cells are to be stained or the rate, R, at which
the stained cells are
passed through the interrogation location of the flow cytometer; and (g)
applying the modified
staining condition to the remainder of the population of cells. In another
embodiment, steps (a)
through (f) are repeated at least once with at least one other fraction of the
population of cells.
Steps (a) through (f) may be repeated once, twice, three times, four times or
a greater number of
times. In another embodiment, multiple fractions of cells are drawn from the
sample at the same
time. Each sample may be stained simultaneously, or each may be stained
subsequent to the
previous fraction being passed through the flow cytometer. In the later
instance, the subsequent
staining of the fractions may be altered according to the determination of
step (f) with respect to a
previously analyzed. In still another embodiment, the process further
comprises prior to step (g),
selecting the modified staining condition that results in the lowest
coefficient of variation for the
fluorescence emission characteristic. In yet another embodiment, the process
comprises the .
repetition steps (a) through (e) until the coefficient of variation for the
fluorescence emission
characteristic of at least one of the fractions is about 1.3% or less. In
another embodiment of the
Invention, the process further comprises prior to step (g), selecting the
modified staining
condition that results in a coefficient of variation of about 1.3 or less.
In addition to performing such an analysis before sorting the entire sample as
detailed
above, a similar analysis may be performed while the staining and sorting of
the sample is
occurring in an effort to ensure that sorting efficiency is maintained.
Accordingly, in another
embodiment, the CV of a fluorescence emission characteristic of the cells of
an enriched

CA 02752247 2011-09-07
82
subpopulation of a fraction of a sample that has been previously stained, a
portion of said sample
which is in the process of being sorted, is determined as described above.
Adjustments to the
staining conditions under which these sample was stained are made according to
the methods
discussed above with respect to the presort adjustments.
The selection of a predetermined CV at which an acceptable sorting efficiency
will be
achieved Is based upon several factors, including for example, the type of
cell being sorted, the
rate of sorting, and the degree of purity desired with respect to sorting of
the population into
enriched subpopulations. Generally, a CV Is selected that will allow for
sorting to the desired
percent purity of the enriched subpopulation while minimizing the amount of
time necessary to
achieve the same, such as for example, by achieving an 85% degree purity of
the enriched
subpopulation while minimizing the length of the staining period. With these
factors in mind, the
CV of the fluorescence emission characteristic of the cells of an enriched
subpopulatIon Is
generally between about 2.0% and about 1.0%, preferably between about 1.5% and
about 1.0%,
more preferably about 1.4%, and still more preferably about 1.3%.
M. Critical Slope Difference Feature Extraction
The microprocessor 131' with digital signal processing (DSP) illustrated in
Fig. 40
employed as part of a cell sorter makes it possible to extract features of the
time resolved
fluorescence emission pulse, particularly features that cannot be easily or
inexpensively obtained
using analog technology. In particular, a pulse feature which exhibits non-
linear properties and
which significantly improves the separation and thus the resolution of
particles A and B (e.g.,
Improves the discrimination of live, aligned X sperm cells) is a feature
referred to as critical slope
difference (CSD). CSD is a quantification of the slope of the fluorescence
emission pulse at a
signal amplitude where the difference between the first derivative of a pulse
produced by particle
A (e.g., a X-bearing cell) and the first derivative of a pulse produced by
particle B (e.g., a Y-
bearing cell) approaches a maximum.
Functionethat describe fluorescence emission pulses may be expressed in terms
of
signal amplitude as a function of time: y x(t). Within the context of
detecting CSD features, a
function may be defined that describes the fluorescence emission pulses in
terms of pulse
duration time as a function of signal amplitude. This function may be referred
to as an M function.
The M function is obtained by transposing the fluorescence emission pulse
function as shown
below.
Fluorescence Emission Pulse function: y=x(t)
M Function: t = M(y)
t= pulse duration
y= signal amplitude
Comparison of the M functions for typical X and Y bovine sperm cells
illustrates the
discriminating power of the CSD feature. The top panel of Fig. 64 shows
average M plots for X-

CA 02752247 2011-09-07
83
bearing and Y-bearIng sperm cells. The middle panel in Fig. 64 shows a graph
of the first
derivatives of these average M plots (i.e. M') for signal amplitude values
less than the peak
height of the average Y-bearing fluorescence emission pulse. It can be seen in
this plot that as
signal amplitude approaches the average peak height of the Y-bearing pulse,
the difference
between the first derivatives (Wry and M'x) increases significantly. Plotted
in the bottom panel of
Fig. 64 is the difference between the first derivatives (M'x ¨ M'y) as a
function of signal amplitude.
The CSD feature quantifies the slope of M (M') for an individual pulse near
the region where the
maximum difference In first derivatives occurs (or the slope at a
corresponding point on the
fluorescence emission pulse function). For the purpose of discriminating X and
Y bearing sperm
cells, CSD Is determined for each pulse at the point where the leading edge of
the pulse
intersects the CSD threshold, as shown In Figs. 62-63. In some embodiments,
CSD may depend
upon the characteristics of the illuminating beam such as beam width whether
the beam is
continuous or pulsed. An algorithm for determining CSD Is discussed below with
regard to Fig.
65.
Figure 64 illustrates that In some cases the CSD feature has a non-linear
nature, such as
in the case of sorting X-Y sperm cell populations. The difference between the
derivatives (Mix ¨
M'y) increases as the CSD threshold approaches the peak of the Y pulse. The
nonlinear
characteristic of this difference places the mean value of the nonaligned
cells and the aligned Y
cells 45% lower than the mean value of the aligned X cells in the CSD feature
space. The
standard deviation in the CSD feature space,of the aligned X cells is largely
unaffected (i.e.
similar to that seen in the peak or area feature spaces). It is this
nonlinear, high gain nature of
the CSD feature that increases the number of aligned X cells that can be
accurately
discriminated.
One computationally efficient method for determining the CSD value for a given
pulse is
Illustrated in Fig. 65. A CSD threshold may be determined as a function of a
peak height of the
fluorescence emission pulses. In particular, it may be determined based on the
average peak
height of the fluorescence emission pulses. The CSD threshold is maintained at
a point where
about 25% of the pulse peaks from live, aligned cells fall at or below the
threshold. Therefore,
the CSD threshold is adjusted dynamically during the sort based on a running
peak height
distribution (i.e., relative to an average peak height). For example, the
threshold may be based
on a weighted running average of peak height (with more recent measurements
being given
more weight than older measurements). The CSD value is the slope of a line
that passes
through two points on the pulse. These points are the CSD pulse threshold and
the pulse peak.
Thus, in this embodiment the CSD value Is only an approximation of the slope
of the pulse
waveform 497 at the intersection of the leading edge of the pulse and the CSD
threshold.
However, other methods of computing the CSD value for a given pulse are
readily apparent,
some of which can provide more precise CSD values if desired.
In another embodiment, the CSD threshold Is dynamically adjusted as a function
of the
CV of the CSD feature extraction for a subpopulation of particles. In the case
of sorting sperm
cells for example, by increasing the CSD threshold from a relatively low level
(e.g., the pulse

CA 02752247 2011-09-07
84
detection threshold) the CSD threshold will reach a level that results in a
substantial increase in
the CV of the CSD of the Y cells but is still low enough that the Increase In
the CV of the CSD for
the X cells is significantly lower In comparison to the CV Increase in the Y
cells. This effect can
be observed In the CSD distribution as a fanning out of one subpopulation in
the overall CSD
distribution. Good discrimination from the CSD feature can be achieved by
maintaining the CSD
threshold at this level.
it should be noted that the discriminating power of the CSD feature Is
enhanced by use
of slit scanning approach to flow cytometry. The shape of the beam spot 459
can influence the
shape of the pulse waveform 497. For example, by using a beam spot 459 having
a relatively
small width W1, a localized fluorescence difference in a sample particle
(e.g., the localized
fluorescent intensity difference resulting from localization of the X or Y
chromosome in the central
region 225 of a sperm nucleus 213) has a greater influence on the first order
derivative of the
pulse waveform. Accordingly, one embodiment of the present invention includes
using the slit
scanning techniques in combination with CSD feature extraction. Conversely,
using a laser
having a beam waist that is too large (e.g., equal to or greater than the
diameter of the particles)
may prevent effective use of the CSD feature to discriminate particles. The
acceptable range for
the width of the beam waist of the focused illumination beam will depend on a
number of factors
including the size and shape of the particles, the distribution of dye within
the particles being
analyzed, and the amount of difference between the typical waveform pulses for
the particles to
be discriminated. In the case of sperm cells, CSD feature extraction from
waveform pulses 497
generated by excitation of bovine sperm cells 201 with a laser having a beam
waist of less than 3
pm has worked well as indicated below. Of course, CSD feature extraction with
any form of slit
scanning discussed in the slit scanning section is considered to be within the
scope of this aspect
of invention.
Use of the CSD feature substantially increases the yield of the system,
particularly in the
case of sorting X-Y sperm cell populations because it allows collection of
many more aligned X
cells. Due to the overlap in the populations defined in peak vs. area or rise-
time vs. area feature
spaces, no more than about 70% of the aligned X cells can be discriminated
with a certainty
about or greater than 85%. When the CSD feature is used, 95% or more of
aligned X cells can
be discriminated, which significantly increases the percentage of live X cells
that can be collected
without reducing the purity of the population of collected X cells below a
desired level of purity.
This is seen graphically in the live cell data shown in Figs. 66-69. The non-
linear nature
of the CSD feature allows X cells to be isolated for sorting. The gross
selection on CSD applied
in the scatter plot shown Fig. 68 results In a 70% pure X area population.
When bl-variate sort
discrimination is applied in the area and CSD feature spaces (Fig. 68), > 95%
of the aligned X
cells can be discriminated for sorting. The data In Figs. 66-69 were collected
at a total cell
throughput of about 22,000 live cells per second on one channel of a four-
channel cytometry
system (see multi-channel system discussion below). Even with coincidence
detection enabled
(high purity), over 6,000 X cells per second were collected at a purity level
of at least 85% purity.

CA 02752247 2011-09-07
Figures 66-69 illustrate one advantage of the CSD feature when used to
discriminate X-
bearing and Y-bearing sperm cells. Figures 66 and 67 are histograms of the
area feature of the
fluorescence emission pulses for the feature space defined in the scatter
plots shown Figs. 68
and 69. In Fig. 68, the CSD feature has moved most of the non-aligned and
aligned Y cells
5 completely out of the display of the scatter plot. This leaves a 70% pure
X population in the
frame of the scatter plot, which Is what Is shown In the pulse area histogram
in Fig. 66. Non-
CSD discrimination is shown in the pulse area/ rise time scatter plot shown In
Fig. 69. Aligned X
cells make up about 30% of the corresponding area histogram (Fig. 67). More
than 95% of the
aligned X cells can be collected at > 85% purity using the CSD feature for
discrimination. By
10 comparison, no more than 70% of the aligned X cells can be discriminated
using the traditional
feature space on the right.
Several live cell sorts have been completed using the CSD vs. pulse area, bl-
variate
discrimination technique. Figure 70 is an example of how a sort region set in
this two dimensional
feature space can be used to exclude non-aligned cells and Y cells. Figure 70
Illustrates a IA-
. 15 varlets sort region set on a scatter plot of CSD vs. pulse area
scatter. Notice how the sort region
drops lower on the area feature for high values of CSD (CSD increase from left
to right and area
increases from bottom to top) and moves higher on the area feature as CSD
drops to lower
values. The bi-varlate sort region set on the above CSD vs. pulse area scatter
plot was used to
sort X cells at a sort decision rate > 6000 X cells per second with a input
live cell rate of 26,000
20 cells per second. Purity based on flow cytometry re-analysis was 87%.
The CSD feature makes possible a high yield, no-coincidence abort (I.e.,
coincident
accept or high recovery) sorting strategy. In some embodiments, a pulse
feature could provide
nearly baseline separation and thus 100% accurate classification of live X and
Y sperm cells.
This condition would make it possible to sort cells at reasonably high rates
without aborting
25 droplets that contain both a cell classified as X and non-X (either
unknown or Y). This sorting
strategy is referred to as the high recovery or coincidence accept strategy.
An experiment was
performed to test this using the CSD feature. Coincidence accept sorts were
performed with an
input rate of 12,000 live X cells per second on one channel of a four-channel
flow cytometer.
77% of the X cells were properly aligned, making 4,600 X cells per second
potentially avaliable
30 for sorting. Under these conditions, 4,300 cells per second were sorted
into the population of X
cells. Subsequent purity analysis indicated a purity from this sort of > 87%
without correction for
dead cells and 89% with correction for dead cells. A high purity, coincidence
reject detection sort
was performed Immediately after this sort under the same conditions. A
collection rate of 3200-
3500 cells per second was observed. Purity analysis Indicated a purity of 92%
without correction
35 for dead cells and a purity of 94% with dead cell correction.
The results of the above experiment indicate that at 12,000 live cells per
second input, >
92% of aligned X cells can be collected at a purity > 85%. This is an
indication that the CSD
feature provides 95% accurate classification of all aligned X cells. Under
these circumstances,
yield from the cell sorter is limited primarily by correct cell alignment,

CA 02752247 2011-09-07
87 =
in Table 3. Fig. 74 shows a graphical example of classification by this
procedure. The illustration
on the left Is a schematic illustration of the two populations 1 and 2 and the
decision boundary
763 separating the populations. The histogram on the right shows two
concentric sets of ellipses
defining the X and Y regions, with the decision boundary 763 being a line
defined by the
Algorithm: Bayes Minimum Error fluorescence pulse classification
(discrimination)
Input: vector of floats pulseFeatures, for each class population i:
matrix of floats WI,
vector of floats wi, float wro
Output: integer classLabel
Procedure:
1. For each class/population I, compute value of discriminant function g/:
gi = pulseFeaturest = Wi = pulseFeatures + w: = pulseFeatures + wio
2. For each class/population compute value of risk function risk:
Initialize riski=0, then for each class/population j:
riski = risk; + costu * g
3. Find j s.t. riskj= min(riski) V I. Return classLabel j and exit.
Table VII. Summary of digital fluorescence pulse classification
(discrimination) by Bayes
Minimum Error decision rule.
For additional robustness, an additional step is taken in the classification
of digital
fluorescence pulses. The Mahalanobis distance of a pulse in feature space from
the population
assigned via Bayes Minimum Error is computed, and if greater than a threshold,
the pulse is
labeled as "not classified" or some other appropriate indication that it is
not likely a member of
any known population. Fig. 75 illustrates the effect of this additional step,
again using features
In general, the AID converter 689 converts the analog output signals 701 from
the
photodetector 117 into corresponding digital Information 707 indicative of
characteristic A or
characteristic B (e.g., X or ¨X). The digital signal processor 865 extracts
features from the digital
information and processor 873 provides a sorting signal 853 to the sorting
system as a function
O. Sort Classification and Droplet Synchronization
The fourth, sort processor 873 manages droplet classification, implements
sorting
strategy and delivers a sort trigger pulse 853 that is synchronized with the
droplet selected for

CA 02752247 2011-09-07
88
particle arrives at the last attached droplet. It will produce a properly
timed output pulse 853 of
appropriate polarity and amplitude for each droplet selected for sorting.
In general, the A/D converter 689 converts the analog output signals 701 from
the
photodetector 117 into corresponding digital information 707 indicative of
characteristic A or
characteristic B (e.g., X or ¨X). The digital signal processor 867
discriminates the digital
Information 707 as indicative of characteristic A or as indicative of
characteristic B (e.g., X or ¨X)
and provides a sorting signal 853 to the sorting system 119 as a function of
the discriminated
digital information.
In general, the digital signal processors 863, 865, 867, 873 include
instructions for
detecting waveform pulses represented by the digital information, instructions
for extracting
features In the detected pulses and instructions for discriminating the
detected pulses as a
function of their extracted features. In addition, the processors Include
instructions for defining a
decision boundary 763 discriminating between the extracted features
representing characteristics
A and the extracted features representing characteristic B. Further, the
processors 863, 865,
867, 873 may optionally adjust the relative location of the decision boundary
763 with respect to
the extracted features representing characteristic A and with respect to the
extracted features
representing characteristic B as a function of at least one of the following:
(1) the purity of the at
least one population with respect to either characteristic A particles or
characteristic B particles,
and (2) the quantity of characteristic A particles or characteristic B
particles In the at least one
population relative to the total quantity of charcicteristic A particles or
characteristic B particles in
the stream. For example, the processor may move the decision boundary 763 to
include less of
population 1 and more of population 2, or visa versa, based on the output of a
particular sample
or based on the desired output (e.g., as noted above with respect to the Bayes
Minimum Risk
decision rule to adjust the decision boundary for differing costs).
P. Drift Compensation
Given that over time the waveform pulses corresponding to the fluorescence
emissions
may vary or exhibit drift over time (due to staining variations, temperature
change, sample age
and/or other factors, for example), the system may optionally employ drift
analysis software 761
(Fig. 72) defining dynamic thresholds which vary to compensate for any effects
of drift. In
particular, the pulse detection thresholds employed by software 747 may be
adjusted for any
slow variations in the signal background characteristics, arid the
discrimination algorithm of
software 757 may adjust the decision boundary 763 (Fig. 74) to account for any
drift in the
populations in feature space.
In the case of the algorithm(s) employed by pulse detection software 747, the
drift
compensation software 761 accomplishes drift compensation by updating the
background mean
and standard deviation estimates based on sample statistics estimates computed
within a
moving window of a given length of samples (e.g., 10-100 samples) ending with
the current
sample. Given the (assumed) slow drift rate relative to the data acquisition
frequency, the
background statistics need not be updated every sample; rather, background
statistic updates

CA 02752247 2011-09-07
89
may occur periodically (e.g., every 6 seconds; see reference character 795 and
Fig. 82).
Additionally, the window may contain less than unity weighting to allow a
"forgetting" rate to de-
weight older samples relative to newer samples in the statistics computations.
Fig. 76 illustrates
the concept of statistic (mean) computation within a moving window without and
with a
"forgetting" rate.
Similar to the detection algorithm drift compensation, the discrimination
algorithm(s)
employed by pulse discrimination software 757 achieve drift compensation by
periodic updates of
the 2' order statistics of the populations in feature space. In this case,
however, only those
feature values from pulses assigned to a given population are used to update
the statistics of that
population. Again, non-unity weighting may be used to include a "forgetting"
rate. Fig. 77 shows
a conceptual illustration of the effects of applying this technique to
populations in feature space.
Fig. 77 illustrates an example of drift compensation for population statistics
In feature space.
Yellow denotes population 1 (X), green popUlation 2 (Y), diamonds the class
mean estimates
(with an exaggerated illustration of drift), and block arrows changes in the
population covariance
estimates reflected in deformation of the constant-sigma ellipses.
in general, the digital signal processor 863 employs a detection threshold for
analyzing
the digital information, which threshold is a function of a background mean
estimate and a
standard deviation of the sampled time-varying output signals computed within
a moving window
of samples ending with the current sample.
Q. Advantage of All Digital
Techniques Over Analog Techniques
One of the main advantages for using an all digital system for sorting is that
there is no
-- "dead time" associated with the detection and analysis of a pulse. With
analog systems there is
always a finite "switching time" required for electronics to reset after the
occurrence and
detection of a pulse. This time is usually on the order of at least one
microsecond. Since the
digital system captures a continuous stream It really has no dead time.
Another advantage of a digital system is the ability to look forward and
backward in time
around a pulse classified for sorting. In general, the digital signal
processing requires about five
(5) droplet periods for analysis. Preferably, the time delay between droplet
illumination 115 and
droplet formation 107 is about seven (7) droplet periods. This allows the
system to classify a
particular particle based on the probability that it will contaminate the
usable population as
indicated by the features of the particular particle and based on the
proximity of the particular
particle to another classified particle. As an example, the sort processor 873
may reject a
particle viewed as having a 50% probability of being a live X cell whereas the
sort processor 873
may accept a particle viewed as having a 50% probability of being a live X
cell when the particle
is coincident with a second particle viewed as having a 95% probability of
being a live X cell.

CA 02752247 2011-09-07
R. Analog Cell Analysis
It is also contemplated that the time-varying output signals from the
photodetector may
be processed by analog circuitry 819, such as a field programmable gate array,
which may be
less expensive than a digital cell analyzer. Fig. 42 Is a block diagram of one
embodiment of an
5 analog cell analyzer which may be employed as part of the system
according to the invention.
Fig. 53 graphically illustrates the analog analysis. A threshold is set to
produce a trigger based
on pulse height. The threshold opens an integration window which gates an
analog integrator to
accumulate charge. The window remains open either for a fixed period or until
the pulse
amplitude fall below the trigger threshold. In this way, only the area of the
portion of the pulse
10 within the integration window is used for relative fluorescence
measurements.
Referring to Fig. 42, the output 701 of the photodetector 117 Is supplied to
an integrator
825 which Integrates the output signal 701 In synchronization with the droplet
clock 703. The
integrated signal is provided to a width/area comparator 827 for comparing the
level of the
integrated signal to a threshold level defining a pulse (e.g., a pulse may be
defined as an
15 integrated signal with 40% of certain a certain threshold.). A dynamic
threshold calculator 829
functions similarly to the drift compensation noted above in that monitors the
integrated signal
level and it varies the threshold level which the width/ area comparator uses
as a function of
variations in the average integrated signal level.
The pulse discriminated signal is provided to a window comparator 837 to
confirm that
20 the pulse area Is within an acceptable range. The pulse discriminated
signal is also provided to a
pulse width and trigger logic circuit 839 to confirm that the pulse width Is
within an acceptable
range. If the area and width are acceptable, the logic provides a trigger
signal to an I/0 controller
843 which indicates the sort decision 841. Thus, the window comparator 837 and
the pulse
width and trigger logic 839 make the decision as to whether a cell should be
classified as an X
25 cell or a ¨X cell.
The I/0 controller 843 provides the sort decision 841 to the sort controller
board 847 In
the form of a X or ¨X signal. The I/0 controller 843 also includes a Universal
Serial Bus (USB)
interface 849 for connecting to the PC 735 and may have I/0 port for
connecting to slave
controllers 845 of the other channels. The analog cell analyzer also includes
a Joint Test Access
30 Group (JTAG) port 833 for programming the width/area, comparator, the
window comparator and
the pulse width and trigger logic.
It is also contemplated that the analog cell analyzer may be employed
simultaneously
with the digital cell analyzer 706. For example, the analog analyzer may be
used to adjust
voltage thresholds used by the digital analyzer. On the other hand, the
digital analyzer may be
35 used to Identify various features of the pulse and this feature
information may be used to
configure the analog cell analyzer, particularly if It Is implemented with a
gate array.
Control Strategies
In general, the microprocessor 131 is programmed to implement control and
sorting
40 strategies which are intended to optimize the efficiency of the system 1
in terms of throughput

CA 02752247 2011-09-07
91
and/or loss of desirable particles to meet any cost requirements of the sorted
product. This may
involve, for example, balancing the need for high purity of at least one
collected population and
the need to recover at least a minimum percentage of desirable particles from
the sample being
sorted. Achieving such a balance Is important, particularly in the context of
commercial
applications where cost and profitability are Important considerations.
To this end, the microprocessor 131 implements a control strategy which Is a
series of
instructions and/or algorithms that control system variables such as fluid
delivery rate and/or sort
parameters. The microprocessor also implements a sorting strategy which
defines the decision
process for determining how each particle or group of particles Is sorted.
Each particular control
strategy may employ one or more sort strategies. Various sorting strategies
may be used
depending on such factors as the selected control strategy, the particle
detection system and/or
information relating to the particle distribution in the fluid stream.
Regarding particle distribution, Fig. 78 Illustrates a fluid stream containing
an exemplary.
distribution of particles. In this particular example, the stream Is formed by
a nozzle similar to the
nozzle described above and contains a mixture of particles having different
characteristics A and
B, e.g., X and Y sperm cells, As shown, the= cells follow generally one after
another in a series
which can be viewed as comprising sequential sets of particles. These sets
include first particle
sets each coMprisIng one or more particles having a characteristic A (e.g.,
indicating a live X
sperm cell), second particle sets each comprising one or more particles having
a characteristic B
(e.g., indicating a Y sperm cell or, more generally, a sperm cell which is not
a live X cell (¨X),
such as a Y cell, or a dead X or Y cell), and third particle sets each
comprising two or more
closely spaced particles at least one of which has characteristic A and at
least one of which has
characteristic B (e.g., one more X sperm cells and one or more ¨X sperm
cells). Third particle
sets are also hereinafter referred to as "coincident" particle sets.
Whether a particular particle is considered as constituting a set by itself or
part of
another set will depend primarily on its spatial=position and/or separation
relative to adjacent
particles. For example, in a droplet sorting system, the various particle sets
will be defined by
the particles in the droplets. In a photo-damage sorting system where a laser
is used to ablate
(kill or otherwise damage) selected particle sets to provide a collected
population having a
desired content, as discussed below in the "Photo-Damage Sorting" section, the
various particle
sets will be defined by the spatial proximity of the particles, i.e., whether
the spatial separation
between particles is sufficient to enable accurate classification of the
particles and/or the ablation
of one or more undesired particles by the laser without also ablating one or
more desired
particles. Similarly, in a fluid-switching sorting system where portions of
the stream containing
selected particles are diverted to provide a collected population having a
desired content, as is
discussed below in the "Fluid Switching Sorting" system, the various particle
sets will be defined
by the spatial proximity of the particles, i.e., whether the spatial
separation between particles is
sufficient to enable accurate classification of the particles and/or diversion
of selected particles.
It will be observed from the foregoing that sort decision applied to the
different particle
sets may be varied, depending on the desired result or throughput of the
system. For example,

CA 02752247 2011-09-07
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In a droplet sorting system, the sorting strategy used may depend on the
treatment of
"coincident" droplets, i.e., droplets containing third particle sets. In the
handling of bovine sperm
cells in a flow cytometry droplet sorting system and method as described
herein, for example, to
enhance the number of X sperm cells in at least one collected population, it
may be desirable to
use a strategy where each coincident droplet containing an X sperm cell is
accepted and sorted
as if It contained only X sperm cells, even though the droplet may also
contain an -X sperm cell
(coincident accept strategy). On the other hand, to enhance the purity of X
sperm cells
collected In the stated population, It may be desirable to reject each
coincident droplet containing
a -X sperm cell even though the same droplet may also contain an X sperm cell
(coincident
reject strategy). In general, as will be pointed out below, there are many
control strategies
which may be employed to maximize particle throughput and there are many
sorting strategies
that may by employed with each particular control strategy. The strategies can
be applied to
various sorting techniques using flow cytometry, such as droplet sorting,
photo-damage sorting,
and fluid-switching sorting. Further, the above strategies can be used to sort
any type of particle
according to any desired characteristic or characteristics of the particle.
According to one embodiment, the microprocessor controls the rate at which the
fluid
delivery system delivers the fluid containing the particles as a function of
other variables of the
system. For example, the microprocessor can the control the fluid delivery
rate as a function of a
desired output result. Since the microprocessor determines the identity of
each particle and
determines whether such is directed to at least one collected population, the
microprocessor can
determine and control the output result by varying the control strategy and/or
by varying the
sorting strategy. A desired output result may generally be defined as at least
one of the
following: (1) the purity of at least one collected population with respect to
characteristic A
particles or characteristic B particles ("high recovery"), and (2) the
quantity of characteristic A
particles in the stated population relative to the total quantity of
characteristic A particles in the
stream, or the quantity of characteristic B particles in the stated population
relative to the total
quantity of characteristic B particles in the stream ("high purity"). As
another example, the
system may employ a substantially constant fluid delivery rate and the
microprocessor can the
control the sort parameters as a function of a desired output result. In this
latter example, the
desired output result may generally be defined as a combination of (1) the
purity of the particles
in at least one collected population and (2) the quantity of desired particles
available in the
stream but not included in the stated population ("constant flow rate").
In general, it my be assumed that when sorting two populations an identified
cell could
have a 50/50 probability of being part of one population or the other.
However, it is also
contemplated that an unidentified cell may in fact have some other probability
other than a 50/50
probability of being part of one population or the other. This other
probability may be determined
by empirical analysis or from other characteristics regarding the sample being
sorted.
Several different control strategies are discussed in more detail below.

CA 02752247 2011-09-07
93
A. High Recovery Control Strategy
One type of control strategy may be referred to as a "high recovery" control
strategy.
The objective of this strategy is to maximize the number of desired particles
sorted into the
population of desired particles as long as the purity of that population is at
or above an
acceptable purity.
Pursuant to this strategy, the first particle sets described above are sorted
Into the
population of desired particles because each of these sets contains one or
more particles having
a desired characteristic A. The third particle sets are also sorted into the
population of desired
particles (coincident accept) because each of these sets also contains one or
more particles
having a desired characteristic A, albeit accompanied by one more particles
having characteristic
B. On the other hand, the second particle sets are rejected (i.e., not sorted
Into the population of
desired particles) because they do not contain a particle having the desired
characteristic. To
optimize throughput using this strategy, the microprocessor increases the
fluid delivery rate as
long as the purity of the collected population Is at or above an acceptable
level. Stated In the
converse, the fluid delivery rate is increased as long as the probable level
of contamination of the
population of desired particles is at or below an acceptable level.
As an example, consider the use of a high recovery control strategy for
sorting X and Y
sperm cells in the fluid stream of Fig. 78. The desired result may be to sort
all of the X cells into
a population of X cells so long as the population remains at or above an
acceptable purity, e.g.,
so long as X/(X+Y) Is greater than 85% or some other acceptable level. To
obtain this result, the
first particles sets are sorted into a population of X cells because they
contain only one or more X
cells. The third particle sets are also sorted Into the population of X cells
because they also
contain one or more X cells, even though they may also contain a Y (or ¨X)
cell. The second
particle sets are sorted into some other population because they do not
contain one or more X
cells. In this example, the rate at which the fluid delivery system delivers
the fluid containing the
cells to the nozzle would continue to be increased as long as the purity of
the population of X
cells Is greater than 85%. Conversely, if the purity of the population of X
cells falls below 85%,
the fluid delivery rate is decreased.
In the context of a droplet sorting system, it is known from Poisson's
equation that for
any given droplet generation rate, the number of multiple-cell droplets will
increase as the cell
delivery rate increases. In other words, increasing the delivery rate of fluid
containing the cells
will increase the number of multiple-cell droplets. Therefore, If the
coincident accept sorting
strategy is used and coincident droplets containing third particle sets are
sorted into the
population of desired particles, increasing the fluid delivery rate will
result in a decrease In the
purity of the collected population because at higher flUid delivery rates more
coincident droplets
are being generated and collected. Figure 79 Illustrates this result for a two
(2) particle fluid so
that 100c/0 of the desired particles are captured. As shown, at very low fluid
delivery rates (FDR
along x axis), the purity (y axis) of the resulting collected population is
very high because very
few coincident droplets containing third particle sets are being generated and
collected. As the
fluid delivery rate increases (FDR increases to the right along the x axls),
the percentage of

CA 02752247 2011-09-07
94
coincident droplets generated increases resulting in more coincident droplets
being collected and
reducing purity of the usable population (along the y axis). In the specific
example illustrated, the
fluid delivery rate Is 30K particles/second at about 80% purity.
The results of using a high recovery strategy can be dramatic, as illustrated
by a simple
example where X and Y sperm cells are sorted using a droplet sorting process.
Assume, for
example that droplets are generated at a rate of 60K/sec, and that sperm cells
are delivered to
the interrogation location at a rate of 30K/sec. According to Poisson's
equation, if all droplets
containing X cells are sorted into the population of X cells, including
coincident droplets
containing X and Y cells, about 15,000 X cells will be collected every second.
The collected
population will Include about 2,600 Y cells, reducing the purity of the
population with respect to X
cells to about 85.2%. However, the number of collected X cells (15,000)
represents a substantial
increase relative to a strategy where coincident droplets are not collected,
as in the high purity
strategy or mode discussed below. In the high purity mode, operating at a
droplet frequency of
40K/sec and cell delivery rate of 40K/sec (10K cells/sec more than in the high
recovery mode
example above), only about 11,800 X cells are collected every second, or about
3,800 X cells
less than in the high recovery strategy. Further, when the high purity
strategy is used, about
9,200 X cells are lost or wasted because coincident droplets are not sorted
into the population of
X cells. Therefore, if less than 100% purity is acceptable, It may be
desirable to use the high
recovery mode to increase the number of X cells collected or, stated
conversely, to decrease the
number of X cells lost.
in summary, in the high recovery control strategy using the coincident accept
sorting
strategy, the particle delivery rate is Inversely related to the purity of the
collected population of
desired particles (sometimes referred to as the "usable" population).
B. High Purity Control Strategy
A second type of control strategy may be referred to as a "high purity"
control strategy.
The objective of this strategy is to maintain the purity of the collected
population with respect to
particles having a desired characteristic at high level, so long as the
quantity of desired particles
in the collected population relative to the total number of desired particles
available in the stream
is at or above an acceptable quantity (i.e., so long as the quantity of
desired particles in the
stream which are not collected remains below an acceptable quantity). Pursuant
to this strategy,
the first particle sets described above are sorted into the population of
desired particles because
each of these sets contains one or more particles having a desired
characteristic A, and because
these sets contain no contaminating particles. On the other hand, the second
and third particle
sets are sorted into one or more "unusuable" populations (coincident reject)
because they
contain contaminating particles (I.e., characteristic 13 particles). To
optimize throughput using this
"high purity" strategy, the microprocessor increases the fluid delivery rate
as long as the quantity
of desired particles that are sorted into the usable population relative to
the total number of
desired particles available in the stream remains at or above an acceptable
quantity.
As an example, consider the use of a high purity control strategy for sorting
X and Y
sperm cells In the fluid stream of Fig. 78. The desired result may be to sort
all of the X cells in a

CA 02752247 2011-09-07
into a population of X cells so long as the quantity of X cells collected from
the stream remains at
or above an acceptable quantity, e.g., at least 60%. To obtain this result,
the first particles sets
are sorted into the usable population because they contain only one or more X
cells. On the
other hand, the second and third particle sets are sorted into one or more
unusable populations
5 because they contain one or more contaminating (¨X) cells. In this
example, the rate at which
the fluid delivery system delivers the fluid containing the cells to the
nozzle would continue to be
increased as long as the quantity of X cells collected in the usable
population as a percentage of
the total available quantity of X cells that have been sorted remains at or
above 60%.
Conversely, if the quantity of X cells not collected in the usable population
rlses above 40% of
10 the total number of available X cells that have been sorted, the fluid
delivery rate is decreased.
As noted above in the context of a droplet sorting system, it is known that
increasing the
fluid delivery rate will increase the number of multiple-cell droplets, and
thus the number of
coincident droplets containing third particle sets. Since coincident droplets
are not sorted into the
population of collected X cells when using a coincident reject sorting
strategy, this means that
15 increasing the fluid delivery rate will result in a increase in the
quantity of live X cells lost to the
unusable population.
Figure 80 illustrates thls result for a two (2) particle fluid so that the
usable population
has 100% purity of desired particles. As shown, at very low fluid delivery
rates (FDR along x
axis), the percentage of desired particles In the usable population is very
high because very few
20 coincident droplets are being generated and rejected. As the fluid
delivery rate increases (FDR
increases to the right along the x axis), the percentage of coincident
droplets containing third
particle sets increases and more such sets are rejected. This reduces the
quantity of desired
particles that are sorted into the usable population relative to the total
quantity of desired
particles available in the stream (i.e., the percentage of desired particles
from the stream which
25 are collected in the usable population). In the specific example
illustrated, the fluid delivery rate
is about 40K particles/second and about 75% of the desired particles are
sorted into the usable
population.
In summary, in the high purity control strategy implementing the coincident
reject sorting
strategy, the particle delivery rate is inversely related to the percentage of
desired particles in the
30 collected population (i.e., high purity of desired particles in the
usable population).
C. Constant Flow Rate Control Strategy
A third type of control strategy may be referred to as a constant flow rate
control
strategy. In this strategy, the microprocessor maintalns the fluid delivery
rate constant (or within
35 a constant range) and varies the percentage of collected (or rejected)
coincident droplets as long
as the purity of at least one collected population is at or above an
acceptable level and as long
as the quantity of desired particles in that population is at or above an
acceptable quantity
relative to a total quantity of desired particles that have been processed.
Stated in the converse,
the fluid delivery rate is constant and the percentage of accepted (or
rejected) coincident droplets
40 varies as long as the probable level of contamination of the usable
population is at or below an

CA 02752247 2011-09-07
96
acceptable level of purity and as long as the probable quantity of desired
particles that is lost to a
population other than the stated (usable) population is at or below an
acceptable quantity.
As an example, consider the use of a constant flow rate control strategy for
sorting the
fluid stream shown in Fig. 78. The desired result may be to sort X sperm cells
into a usable
population having a purity of at least 85% and to collect at least 60% of the
X cells in the stream
so that no more than 40% of the X cells are sorted into the unusable
population. In this example,
the rate at which the fluid delivery system delivers the particles would be
maintained constant
and the percent of collected or rejected third particle sets (coincident sets)
would be varied as
long as the purity of the usable population with respect to X cells Is equal
to or greater than 85%,
and as long as the percentage of X cells sorted into the unusable population
Is less than 40% so
that the percentage of desired particles sorted into the usable population is
equal to or greater
than 60% (variable coincident accept sorting strategy). As the percentage of
accepted third
particle sets increases, the purity of the usable population decreases, but
the quantity of desired
particles (e.g., X cells) sorted Into the unusable population decreases.
Conversely, as the
percentage of accepted third particle sets decreases, the purity of the usable
population
increases, but the quantity of desired particles (e.g., X cells) that are
sorted In the unusable
population increases.
As noted above, it is known from Poisson's equation that the number of
multiple-cell
droplets (and thus the number of coincident droplets containing third particle
sets) is constant for
= 20 a constant fluid (cell) delivery rate. Since the number of
coincident droplets is constant in this
control strategy, the percentage of coincident droplets sorted into the usable
population will
impact both the purity of the usable population and the quantity of X cells
that are wasted by
being sorted Into an unusable population. This Is because the percent of
unwanted Y (or --X)
cells in coincident droplets which are accepted and sorted into the collected
unusable population
is inversely related to the percent of X cells in coincident droplets which
are rejected and thus not
sorted into the collected usable population.
Figure 81 illustrates the constant fluid delivery rate control strategy in a
flow cytometry
droplet sorting system and method as described herein Implementing a variable
coincident reject
sorting strategy for a two (2) particle fluid. As shown, line OL illustrates
the inverse relationship
between the percentage of rejected coincident droplets (x axis) compared to
the percentage of
accepted coincident droplets (y axis). When there is a very low percentage of
accepted
, coincident droplets, there is a very high percentage of rejected
coincident droplets. Conversely,
when there is a very high percentage of accepted coincident droplets, there is
a very low
percentage of rejected coincident droplets. Line OL Illustrates this inverse
relationship and
represents the operating line of the variable coincident accept sorting
strategy at a given
constant particle flow rate. At point P in Fig. 81 along operating line OL,
the purity of the usable
population is a given percentage depending on particle flow rate, e.g., 85%
purity. As the
percentage of accepted coincident droplets Increases (to the left and upward)
along operating
line OL, the number of undesired particles that are sorted into the usable
population increases
and the purity drops below 85%, which may be unacceptable. As the percentage
of accepted

CA 02752247 2011-09-07
97
coincident droplets decreases (to the right and downward) along operating line
OL, the purity
goes above 85% and is acceptable
At point LL along operating line OL, 75% of the coincident droplets are
rejected (i.e.,
sorted into the unusable population) so that the percentage of desired
particles that are wasted
by being sorted into the unusable population is a given percentage based on
the particle delivery
rate, e.g., 40%. As the percentage of rejected coincident droplets increases
(to the right and
downward) along operating line OL, the percentage of desired particles that
are sorted into in the
usable population decreases (e.g., to <60%), which may be unacceptable. As the
percentage of
rejected coincident droplets (to the left and upward) along operating line OL,
the percentage of
.10 desired particles sorted into the usable population increases (e.g., to
>60%) and is acceptable.
Thus, according to this aspect of the invention for a constant flow rate
control strategy
implementing a variable coincident accept sorting strategy, the microprocessor
may operate the
system so the percentage of accepted and rejected coincident droplets varies
in an operating
range between point P and LL as indicated by arrow OR. Note that operating
range OR may
I 5 encompass more or less of the operating line, depending on the level of
tolerance for impurity
and loss of desired particles to the unusable population.
In summary, in the constant flow rate control strategy using the variable
coincident
accept sorting strategy, the percentage of third particle sets which are
accepted is inversely
related to the purity of the usable population and inversely related to the
quantity of desired
20 particles wasted by being sorted to a unusable population.
D. Summary of Control Strategies
The following Table summarizes the control strategies noted above.
[rest of page blank)
= =

CA 02752247 2011-09-07
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CONTROL HIGH RECOVERY HIGH PURITY CONSTANT FLOW RATE
STRATEGY =
Controlled parameter Fluid delivery rate Fluid delivery rate Sort
parameters
Controlling Purity of population Quantity of desired Purity of
population AND
parameter: particles in population Quantity of
desired particles in
population
Sorting strategy Coincident accept Coincident reject Variable
coincident accept
X/Y Sorting strategy Collect X droplets and Collect X
droplets; Collect X droplets; vary
X4--X droplets; reject ¨X reject X+¨X droplets percentage of
collected X+¨X
droplets and ¨X droplets droplets; reject ¨X
droplets
Definition The fluid delivery rate is The fluid delivery rate The
percentage of coincident
Increased as long as the is Increased as long droplets in
the population is
purity of the population as the quantity of increased as
long as the purity
with respect to X particles desired particles in of the population
with respect
is at or above an the usable population to X particles
Is at or above an
acceptable level relative to the total acceptable
level; to continue
quantity of X particles operation, the quantity of
in the stream is at or desired particles
in the usable
above an acceptable population relative
to the total
quantity quantity of X
particles in the
stream must be at or above an
acceptable quantity
Converse Definition The fluid delivery rate is The fluid delivery rate
The fluid delivery rate is
increased as long as the is increased as long increased as
long as the
probability of as the probability of probability
of contamination of
contamination of the loss of the quantity of the usable
population is at or
usable population Is at or desired particles in below an acceptable
level of
below an acceptable level an unusable purity AND as long
as the
of purity population Is at or probability of
loss of the
below an acceptable quantity of desired
particles in
quantity the unusable
population Is at or
_________________________________________________________ below an acceptable
quantity
Desired result > minimum acceptable >minimum > minimum
acceptable purity
purity; e.g., >85% purity acceptable quantity; and >minimum
acceptable
e.g., >60% of desired quantity; e.g., >85% purity and
particles captured >60% of desired
particles
(<40% of desired captured (<40% of
desired
particles lost) particles lost)
Relatedly, a sorted sample obtained using one of the above control strategies
can be
combined with a second sample to obtain a final (e.g., commercial) sample
having the desired
characteristics. For example, a sample sorted according to the high purity
strategy to produce a
100% pure population cah be combined with a population of the same volume
sorted to 80%
purity to produce a final sample having a purity of 90%. Or in the case of
animal sperm sorted to
a high purity, an aliquot amount of the sorted sperm can be combined with an
aliquot amount of
unsorted sperm to produce a final sample of desired purity at lower cost than
sorting the entire
amount of sperm using any of the above sorting methods.
The above description of the control strategies assumes accurate
identification and
sorting of each droplet Including each coincident droplet. In practice, 100%
accuracy is not
possible for any number of reasons. in order to minimize contamination,
therefore, it may be
desirable to reject particles which cannot be classified with certainty as
belonging to the desired
population. On the other hand, If certain particles can be identified and
classified as being in the
desired population within a certain selected probability (e.g., greater than
50% in the case of

CA 02752247 2011-09-07
99
sperm cells), it may be desirable to classify the particles as belonging to
the desired population
so that they are not lost to the unusable population. Thus, as discussed
earlier, particles such as
sperm cells may be accepted or rejected for sorting into a population of
desired cells based on
the probability that such particles belong in the usable population.
The terms "usable" and "unusable" as used in the above table and this
application are
used for convenience only and are not intended to be limiting in any way.
Thus, a "usable"
population includes any "first" population, regardless of how or whether it is
used, and an
"unusable" population includes any "second" population different from the
usable population,
regardless of how or whether it is used. Similarly, a "desired" population
means any population
which is sorted according to selected particle characteristics.
The microprocessor and its signal processing software constitutes a system for

processing the electrical signals from the photodetector to classify particles
(e.g., particles in
general and sperm particles in particular) according to characteristics of the
particles and to
obtain Information relating to the distribution of the particles as described
above with respect to
Fig. 78. Furthermore, the microprocessor constitutes a control system
responsive to =the signal
processing software for varying the rate at which the fluid delivery system
delivers particles to the
nozzle system as a function of the obtained information relating to the
distribution of the particles.
Furthermore, the microprocessor constitutes a control system responsive to the
signal
processing software for varying the sorting strategy as a function of the
obtained information
relating to the distribution of the particles.
In general, the microprocessor constitutes a control system responsive to
information
received from the flow cytometry apparatus for controlling the sorting system
to vary its sorting
strategy or for controlling the fluid delivery system. In other words, the
microprocessor is capable
= of operating in a first mode to vary the sorting strategy, is capable of
operating in a second mode
for controlling the fluid delivery system, is capable of operating in a third
mode to vary the sorting
strategy and for controlling the fluid delivery system, and may be capable of
operating in other
modes. When operating in the first or third mode, the microprocessor is
capable of varying the
rate at which fluid is delivered as a function of at least one of the
following: (1) the purity of the at
least one population with respect to either characteristic A particles or
characteristic B particles,
and (2) the quantity of characteristic A particles or characteristic B
particles in the at least one
population relative to the total quantity of characteristic A particles or
characteristic B particles in
the stream.

CA 02752247 2011-09-07
100
Collection System
A collection system is needed to collect the droplets after they pass between
the
deflector plates. The collection system for a conventional cytometer may be no
more than
collection vessels disposed to catch the droplets in the various droplet
streams after they pass
between the deflection plates. Similar conventional collection systems can be
used in some
embodiments of the present invention.
However, it may be difficult to use a conventional collection system In
embodiments of
the present invention in which the nozzle is oriented to direct the fluid
stream at an upward angle,
thereby giving the droplets a horizontal velocity component. One issue is that
the droplets would
travel some horizontal distance along their arched trajectories before they
begin downward
movement that would be suitable for landing In a collection vessel. For
example, if the nozzle is
pointed upward at a range of 45 to 60 and the droplets exit at a velocity
between 15 m/s and 20
m/s, the droplets will be a horizontal distance of several meters away from
the nozzle before they
reach the apex of their trajectories and begin downward movement. Thus, a good
deal of lab
space would be occupied by the droplet streams. Furthermore, at a range of
several meters it
could also be difficult to make sure the droplets land in the proper
collection vessels. The
trajectories of the droplet streams can change whenever one or more operating
conditions for the
cytometer change (e.g., adjustment to the fluid delivery rate resulting in a
change in the fluid
velocity at the nozzle orifice). Changes In the trajectories of the droplet
streams will be magnified
by the distance that the droplets travel. Thus, changes In the trajectories
that do not result in an
appreciable change in droplet location at a point relatively near the nozzle
could result in a
significant change In location of the droplets at a location that Is farther
away from the nozzle. As
discussed above, some embodiments of the present invention employ feedback to
the droplet
formation and/or sample fluid delivery systems that could result in droplet
streams that constantly.
alter their trajectories. One may also want to vary the pressure at which the
sheath fluid is
delivered to the nozzle. Air currents, temperature variations, and humidity
variations could also
alter the trajectories of the droplet streams. Any factor that could change
the trajectory of the
droplet streams could also require the collection vessels to be repositioned
so the droplets land
in the appropriate collection vessels. In contrast, the trajectories of
droplets streams in a
conventional cytometer having a downward pointing nozzle are less susceptible
to variation. For
example, the fact that the droplet streams have a substantially downward
initial velocity means
that variation in fluid velocity at the orifice does not result in any
significant variation in the
trajectories. Furthermore, the collection vessels are relatively close to the
nozzle which makes
the collection system more tolerant to trajectory variations in the droplet
streams.
Figs. 83-85 show one embodiment of a collection system, generally designated
2201,
that may be used to collect sorted droplets in a system of the present
invention. The collection
system Is particularly suited for collection of droplets 33 when the cytometer
nozzle system 101
Is oriented to direct the fluld stream 21 at an upward angle or any other
angle having a horizontal
component. As the droplets pass between the deflector plates 629, they are
sorted into multiple
droplet streams 123,125 (e.g., two) having different arched trajectories. As
shown in Figs. 84

CA 02752247 2011-09-07
lot
and 85, the trajectory of each droplet stream leads to one of two droplet
intercepting devices
2203. If the droplets are being sorted into more than two droplets streams, a
separate
intercepting device would need to be provided for each additional droplet
stream. Thus, the
number of Intercepting devices in a collection system of the present invention
will depend on the
number of streams into which the droplets are being sorted.
Each Intercepting device 2203 in the exemplary collection system has an impact
surface 2205
positioned to span the trajectory of one of the droplet streams to divert
droplets moving along
that trajectory to a collection vessel 2207 positioned beneath each
intercepting device. The
impact surfaces are preferably made of a pliable material. Without being bound
by a particular
theory, it is believed that pliable materials cushion the impact of droplets
striking the surface of
the intercepting device, thereby reducing damage to the particles (e.g., sperm
cells) In the
droplets. For example, the intercepting devices may be constructed of
polypropylene,
polyethylene, or other similar polymers. Referring to Figs. 86 and 87, the
intercepting devices
2203 have been constructed by cutting a droplet entryway 2211 (e.g.,
rectangular window) in one
side of the bulb 2213 of a pipette 2215. Thus, a portion of the inside wall
2217 of the bulb
opposite the droplet entryway forms a curved impact surface 2205 which spans
the trajectory of
the respective droplet stream. Conveniently, the tube of the pipette serves as
a guide 2225 for
directing fluid from the Impact surface to the collection vessel.
Referring to Fig. 84, the intercepting devices are fastened to a collection
system frame
2227, which holds them in place. In order to account for variability in the
trajectories of the
droplet streams, It is desirable to allow adjustment of the positions of the
intercepting devices.
For example, the vertical height of each intercepting device may be adjusted
by sliding the guide
tube up and down in a circular bore through a holder 2229. When the
intercepting device is at
the desired height, a set screw 2231 may be tightened to hold it at that
height. The holder may
be attached to a mounting plate 2233 which is attached to the collection
system frame, for
example by set screws 2235 (e.g., two set screws). The set screws 2235 pass
through a
horizontal slot 2241 in the mounting plate to allow lateral adjustment of the
intercepting device.
After adjustment, the set screws may be tightened to hold the circular holder
in the desired
location. Those skilled in the art will recognize that a variety of other
fastening devices could be
used to adjust the position of the intercepting device without departing from
the scope of the
present invention. The collection vessels 2207 are held beneath the
intercepting devices in slots
2241 in a tray 2243 for holding the collection vessels. Thus, each collection
vessel may be
moved within a respective slot as necessary for it to remain In position under
the respective
Intercepting device. Also, a water bath (not shown) may be provided around the
collection vessel
if desired to control the temperature of the contents of the collection
vessel.
Referring to Fig. 85, in one embodiment of the present invention an exit
window 2245
(e.g., a rectangular window) has been cut in the back of one of the
intercepting devices 2247 to
allow one or more droplet streams to pass through the Intercepting device. A
second
intercepting device 2249 is positioned behind the exit window to intercept the
droplets that pass
through this window. An exemplary entry window for the second intercepting
device may be

CA 02752247 2011-09-07
102
approximately the same size as the exemplary exit window for the first
intercepting device. For
reasons that will be apparent, it is desirable for the exit window to be
significantly smaller than
the entry window for the first intercepting device. For instance, an exemplary
entry window for
the first intercepting device is about 5/8 of an inch high and about 3/8 of an
Inch wide. In
contrast, an exemplary exit window may be 1/8 of an inch high and 5/16 of an
Inch wide.
During operation of the cytometer, the collection system operates to intercept
the
droplets in the sorted streams. The intercepted droplets then drain down
through the guide tubes
2225 of the intercepting devices 2203 and into the collection vessels 2207. In
a case in which a
cytometer has an upward pointing cytometer nozzle that directs droplet streams
along arched
trajectories, for example, the intercepting devices allow the droplets to be
intercepted at a point
on their trajectory that is significantly closer to the nozzle in comparison
to the point at which the
droplets would be collected by a conventional collection system (I.e., a
collection system without
intercepting devices).
Intercepting the droplet streams relatively early along their arched
trajectories (e.g., while
they are still moving upward) reduces the amount of variation In the location
of the droplets at the
time the droplets first encounter the collection system. Accordingly, the
collection system can
tolerate more variation in the trajectories of the droplet streams than a
convention collection
system could tolerate. Similarly, the droplets are less likely to be buffeted
by air currents
because 'of their shorter paths to the collection system.
A balance must be struck between moving the Intercepting devices 2203 closer
to the
nozzle 101 to increase tolerance for trajectory variations and moving the
Intercepting devices
farther away from the nozzle orifice to reduce or minimize the force of Impact
when droplets
impact the Intercepting devices, as by positioning the Intercepting devices so
they intercept the
droplet streams substantially at the apex of their trajectories. Accordingly,
the best location for
the intercepting devices will depend on the durability of the particles (e.g.
sperm cells) being
sorted, the droplet velocities, and the expected magnitude of variation in the
droplet stream
trajectories. In the case of droplets containing bovine sperm cells having a
velocity at the nozzle
orifice of about 16 to 20 m/s, for example, the intercepting devices may be
positioned In the
range of 4 - 6 inches from the nozzle orifice. In the embodiment In which a
first intercepting
device has a exit window and a second intercepting device is positioned behind
the first
intercepting device, for example, the first intercepting device may be in the
range of about 4 and
5 inches from the nozzle. More desirably, the first intercepting device is
about 4.5 inches from
the nozzle. The second intercepting device may be in the range of about 5 to 6
inches from the
nozzle. More desirably, the second intercepting device is about 5.5 inches
from the nozzle.
The configuration in which one intercepting device 2203 is positioned to
intercept the
droplets that pass through an exit window of another intercepting device is
particularly
advantageous when one is not concerned about the purity of one of the sorted
populations (e.g.,
Y chromosome-bearing sperm in the case of sperm sorted for use in breeding
dairy cattle).
Those skilled in the art will know that a number of stray droplets 2265 (e.g.,
a mist of stray
droplets) having unknown contents may be produced by the cytometer In addition
to the droplets

CA 02752247 2011-09-07
103
in the sorted streams as shown in Fig. 85. The first intercepting device
should be positioned so
that the stream of droplets that are being sorted into the population for
which there is the greatest
tolerance for impurities hit the impact surface 2205 of the first intercepting
device and the stream
for which purity Is most critical passes through the exit window 2245 to hit
the Impact surface of
the second intercepting device. This way the majority of the stray droplets
are collected into the
collection vessel 2207 containing the population for which there is less
concern about purity, as
shown in Fig. 85, and will not contaminate the population for which purity is
critical. Also by
intercepting and collecting the stray droplets, one avoids the need to clean
as often as if the stray
droplets escape the collection system. In contrast to the first intercepting
device, the second
intercepting device only diverts droplets that pass through the smaller exit
window. This
facilitates maintenance of the purity of the population collected by the
second intercepting device.
Those skilled In the art will recognize that the exemplary collection system
could readily
be modified in a number of ways without departing from the scope of the
present invention. For
example, it would be possible to construct a droplet Intercepting device
having an integrally
formed (or otherwise attached) collection vessel beneath it, without departing
from the scope of
this invention. Similarly, although the Intercepting devices in the embodiment
shown in Flgs. 83-
87 are modified pipettes, It is understood that the intercepting devices 2203
can be any of a
variety of shapes without departing from the scope of this invention. For
example, each
Intercepting device may comprise a flat or curved sheet, a spoon, a bowl, or
other similar shape.
The only requirement is that the intercepting device is operable to intercept
droplets moving
along a respective trajectory of a droplet stream and to divert the
intercepted droplets into a
collection vessel. However, one advantage to constructing the intercepting
devices out of a
readily available and relatively inexpensive product, such as a pipette, is
that it may be more
economical to replace and dispose of the used intercepting devices after each
sample run rather
than clean the intercepting devices between sample runs. This could help
reduce costs of
operating the collection system.
Collection Fluid
The sorted sperm are collected in a vessel that contains a collection fluid
2301 (Figs. 56
and 57). Generally, the purpose of the collection fluid includes cushioning
the impact of the
sperm cells with the collection vessel or providing a fluid support for the
cells. Consistent with
these considerations, the collection fluid may comprise a buffer or buffered
solution and a protein
source.
If included, examples of buffers or buffered solutions that may be used in the
collection
fluid are disclosed above with respect to sample collection and dilution.
Typically, these buffers
or buffer solutions will be in a concentration of about 0.001M to about 1.0M
and have a pH of
about 4.5 to about 8.5, preferably of about 7Ø In one embodiment, the
collection fluid contains
buffer comprising 0.96% Dulbecco's PBS (w/v) at a pH of about 7.0 In another
embodiment, the
collection fluid contains a metabolic inhibitor comprising 0.204g NaHCO3,
0.433g KHCO3, and

CA 02752247 2011-09-07
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0.473g C6H807.1-120 per 25mL of purified water (0.097 moles/L of NaHCO3, 0.173
moles/L of
KHCO3, 0.090 moles/L C3H807-1-120 in water).
If included, the protein source may be any protein source that does not
interfere with the
viability of the sperm cells and Is compatible with the particular buffer or
buffered solution being
used. Examples of common protein sources include milk (including heat
homogenized and
sift), milk extract, egg yolk, egg yolk extract, soy protein and soy protein
extract. Such proteins
may be used in a concentration from about 1% (v/v) to about 30% (v/v),
preferably from about
10% (v/v) to about 20% (v/v), and more preferably about 10% (v/v). While milk
may be used In
combination with a buffer or buffered solution, generally milk is used in the
absence of the same,
as milk is a solution itself that may serve the same purpose of a buffer or
buffered solution. In
such instances, the collection fluid may contain about 80% (v/v) to about 90%
(v/v) Milk.
In addition to or in lieu of the protein source, the collection fluid may also
comprise
seminal plasma. Seminal plasma serves the dual benefits of improving sperm
viability and
motility and of stabilizing the sperm membrane (thereby preventing
capacitation during the
collection and storage of the sperm). Maxwell et al., Reprod. Pert. Dev.
(1998) 10: 433-440. The
seminal plasma may be from the same mammal from which the semen sarnple was
obtained,
from a different mammal of the same species, or from a mammal of a different
species. If
included in the collection fluid, typically the percentage of seminal plasma
will be in the range of
about 0.5% (v/v) to about 10% (v/v). If used in combination with a protein
source, such as for
example egg yolk or milk, the total percentage of seminal plasma and protein
source will range
from about 1% (v/v) to about 30% (v/v). In such instances, the percentage of
seminal plasma will
be inversely proportional to the percentage of the protein source.
Accordingly, in one
embodiment, the collection fluid comprises serninal plasma. In another
embodiment, the
collection fluid contains seminal plasma In an amount of about 0.5% (v/v) to
about 10% (v/v),
preferably in an amount of about 4% (v/v) to about 6% (v/v), and more
preferably in an amount of
about 5% (v/v). In another embodiment, the collection fluid contains a protein
source and
seminal plasma. In yet another embodiment, the collection fluid comprises
seminal plasma and
egg yolk, the percentage of both totaling between about 1% (v/v) and about 30%
(v/v).
Optionally, the collection fluid may also contain a range of additives that
are beneficial to
sperm viability or motility. Examples of such additives include an energy
source, an antibiotic,
and a composition which regulates oxidation/reduction reactions
intracellularly and/or
extracellularly, each of which Is discussed above with respect to sample
collection and dilution.
Such additives may be added to the collection fluid In accordance therewith.
Accordingly, In a certain embodiment, the collection fluid comprises 0.96%
Dulbecco's
PBS (My), 1% (w/v) fructose, 10% (v/v) egg yolk in water, at a pH of about
7Ø In yet another
embodiment, the collection fluid further comprises lOrnM pyruvate, 1000M
vitamin K, or 1mM of
lipoic acid.
Alternatively, and In lieu of the use of a collection fluid, the sorted cells
may be collected
into a vessel containing or coated with a cryoextender used in the subsequent
cryopreservation
steps and further described below. Accordingly, in one particular embodiment,
the sorted cells

CA 02752247 2011-09-07
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are collected Into a cryoextender. In another embodiment, the collected cells
are sorted into a
cryoextender comprising water, Triladyle (Mlnitube, Verona, WI, comprising
glycerol, tris, citric
acid, fructose, 5mg/100mItylosin, 25mg/100m1gentamycin,
30mg/100m1Spectinomycin, and
15mg/100mILIncomycin), egg yolk, and pyruvIc acid. In yet another embodiment,
the collection
fluid Is the cryoextender comprising 25g Trilady10, 25g egg yolk, and 10mM
pyruvic acid in 75mL
of water.
It is to be understood that the percent concentrations of protein in the
collection fluid
disclosed herein are those prior to the addition of the flow sorted cells. The
addition of the flow
sorted cells will dilute the final concentration of the collection fluid to
about 1/20 that of what it
was prior to the addition of the flow sorted cells. Therefore, for example,
the collection fluid may
Initially contain about 10% (v/v) egg yolk. After the flow sorted cells are
collected in the collection
vessel containing the collection fluid, the final concentration of egg yolk
will be reduced to about
0.5% (v/v).
Pre-Treatment of Intercepting Devices and/or Collection Vessels
In order to minimize possible damage to particles (e.g., sperm cells) that may
be sorted
according to the present invention, the intercepting devices 2203 and/or
collection vessels 2207
(Figs. 56-60) may be treated prior to use. Such pre-treatment may comprise,
for example,
contacting or soaking the intercepting devices and collection vessels In a
bath containing a
composition that will serve to minimize the impact between the particle and
the intercepting
device. Upon removal of the intercepting devices and collection vessels from
the bath, a certain
amount of the composition will remain on the intercepting devices and
collection vessels and
serve as a cushioning agent for the particles in the droplets. The
composition, therefore, should
have characteristics suitable for providing the desired cushioning effect. In
addition, the
composition should also be compatible with the particle or cell being sorted,
the sheath fluid, and
the collection fluid. Consistent with these considerations, the composition
used to treat the
intercepting devices and collection vessels may comprise a buffer or buffered
solution, a sheath
fluid, a collection fluid, a cryoextender, any components contained in the
buffered solution,
sheath fluid, collection fluid, or cryoextender, or any combination thereof.
I3uffers, buffered
solutions, sheath fluids, and collection fluids used for the staining and
separation of sperm cells
according to the methods of the present invention are described above.
Accordingly, in one
embodiment, the intercepting devices and collection vessels are contacted with
(e.g., soaked in
or brushed with) sheath fluid. In another embodiment, the intercepting devices
and collection
vessels are contacted with collection fluid. In yet another embodiment, the
intercepting devices
and collection vessels are contacted with a cryoextender described below.
The contacting or soaking of the intercepting devices and collection vessels
with the
composition preferably occurs for a period of time sufficient to allow the
composition to adhere to
the surfaces of the intercepting devices and collection vessels. Such a period
of time is generally
less than about 90 minutes, preferably less than about 60 minutes, more
preferably about 30 to
about 60 minutes, and most preferably about 60 minutes. In still another
embodiment, the

CA 02752247 2011-09-07
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intercepting devices and collection vessels are merely contacted with the
composition prior to
In lieu of or in combination with the contacting of the intercepting devices
and collection
vessels with the above-described composition, the Intercepting devices and
collection vessels
may also be contacted with specific components contained in the sheath fluid,
the collection fluid,
and/or the cryoextender, such as for example, BSA, SSS, egg yolk, egg yolk
extract, milk
(Including heat homogenized and skim), milk extract, soy protein, and soy
protein extract.
Accordingly, in one embodiment, the intercepting devices and collection
vessels are contacted
with sheath fluid and subsequently contacted with 0.1% (v/v) bovine serum
albumin. In another
embodiment, the intercepting devices and collection vessels are contacted with
sheath fluid and
subsequently contacted with 10% (v/v) egg yolk. In another embodiment, the
intercepting
devices and collection vessels are soaked in collection fluid and subsequently
contacted with
0.1% (v/v) bovine serum albumin. In another embodiment, the intercepting
devices and
collection vessels are soaked in collection fluid and subsequently contacted
with 10% (v/v) egg
yolk.
Although the intercepting devices and collection vessels receive the same pre-
treatment
in each embodiment described above, It is possible to use different pre-
treatment protocols for
the intercepting devices and the collection vessels without departing from the
scope of this
invention. Likewise, some of the intercepting devices or collection vessels
could receive one pre-
treatment and others of the Intercepting devices or collection vessels could
receive a different
pre-treatment without departing from the scope of this invention. Certain
advantages of the pre-
treatment can also be obtained by pre-treating only the intercepting devices
or only the collection
vessels, again without departing from the scope of this invention.
Concentration
As noted above, the sorted sperm collected by the flow cytometer have been
diluted by
the addition of various buffers and extenders, the staining fluid, the sheath
fluid, and the
collection fluid. Typically, the concentration of sperm cells after sorting by
flow cytometry as
described above is in the range of about 0.7 - 1.4 x 106 sperm cells/ml.
Therefore, it is important
to concentrate the sorted sperm cells to minimize the dilution shock to the
sperm and to attain
the proper concentration of sperm for cryopreservation and artificial
insemination. Standard
practice in the animal breeding industry, for example, is to perform
artificial insemination with
sperm at a concentration of either about 20 x 106 or about 40 x 106 sperm
cells/mi. One way to
concentrate the sperm cells is through centrifugation of the fluid collected
by the cytometer.
Another way to concentrate the sperm is to pass the fluid collected by the
cytometer through a
filtration system. These methods are discussed in more detail below.
A. Centrifugation
Any conventional centrifuge can be used to concentrate sperm. However in a
commercial operation it Is preferable to use a centrifuge having the capacity
to centrifuge a large

CA 02752247 2011-09-07
107
batch of sperm cells at once. During centrifugation a majority of the sperm
cells will collect In a
pellet at the bottom of the centrifuge tube due to the centrifugal force
acting on the sperm cells.
The magnitude of the centrifugal force is conventionally stated as the number
of times the
centrifugal force exceeds the gravitational force. Because the centrifugal
force is the critical
parameter and because the magnitude of the centrifugal force at any given
speed (angular
velocity) will vary depending of the length of the radius of curvature, the
speed of centrifugation Is
typically specified by stating the magnitude of the centrifugal force. For
example, a 600g force
means the angular velocity of the centrifuge Is selected so the resulting
centrifugal force will be
600 times the force of gravity. The majority of the fluids and any sperm cells
that escape being
centrifuged into the pellet will be in the supernatant. Generally, the
supernatant is removed and
the sperm cells in the pellet are resuspended for further processing as
described below. It is
important to maximize the percentage of sperm that are concentrated in the
pellet, while at the
same time minimizing damage to the sperm cells.
According to one method of the present invention, a centrifuge tube containing
about 10
x 10 sorted sperm cells is placed In a centrifuge. To facilitate
concentration, centrifuge tubes
may be used as the collection vessels in the collection system of the
cytometer. This avoids the
need to transfer the sorted sperm cells to a centrifuge tube before
centrifugation. The tube is
centrifuged at a speed and for a duration that is sufficient to cause a pellet
of concentrated sperm
cells to form in the bottom of the tube. The speed and duration of
the.centrifugation is desirably
selected in consideration of several factors, including: the fact that sperm
cells are fragile and
can be damaged by centrifugation at an excessive speed; the size of the
centrifuge tube will
affect the time required for sperm cells to move to the bottom of the tube;
and the sperm cells are
more likely to be damaged by centrifugation at a given speed the longer the
centrifugation
continues. Thus, in one embodiment of the present Invention the centrifuge
tube is centrifuged at
550-800g for a period of about 6 - 10 minutes. According to another embodiment
of the present
invention, the centrifuge tube is centrifuged at 650-750g for a period of
about 6 - 10 minutes. In
still another embodiment, the centrifuge tube is centrifuged at 700g for a
period of about 6 - 10
minutes. In yet another embodiment, the centrifuge tube is centrifuged at 700g
for a period of
about 7 minutes.
As demonstrated in the following experiments, the speed of the centrifuge and
the
duration of centrifugation may affect the percentage of sperm cells recovered
and the motility of
the recovered sperm cells. The experiments were conducted without actually
sorting the sperm
cells. Instead, various fluids including buffers, extenders, sheath fluids and
a staining fluid were
added to semen samples to simulate the sorting process. The samples were then
centrifuged in
an attempt to concentrate the sperm cells.
Centrifuge Example I
In centrifuge example I bovine semen was collected and evaluated as described
above.
The semen sample was diluted with a quantity of Tris-citric acid ("TCA")
having a pH of 7.3 to
attain a concentration of 150 x 106 sperm cells/ml. Spermatozoa were stained
with Hoechst

CA 02752247 2011-09-07
los
33342 (100 ,uM) at 41' C for twenty minutes. Two 15 ml tubes were prepared
with buffers for the
simulation. Tube 1 was partially filled with 750 pi of phosphate buffered
saline ("PBS") with 10%
egg yolk and 14.25 ml PBS with 0.1 % bovine serum albumin ("BSA"). Tube 2 was
partially filled
with 750 ul TCA with 10% egg yolk and 14.25 ml PBS with 0.1% BSA. Each of the
two tubes
received 100 Ul of the solution containing the stained spermatozoa, which were
then incubated at
room temperature for 20 rninutes. The two tubes were then divided into two
aliquots of 7 ml
each. One aliquot from each tube was centrifuged at 2250 rpm (about 540g) for
7 minutes in a
fixed bucket centrifuge. The other aliquot from each of the two tubes was
centrifuged at 2500
rpM (about 660 g) for 7 minutes. Immediately after centrifugation, 10 ml
pipettes were used to
remove and save the supernatant from each aliquot. The pellets were
resuspended in 200 ul of
TCA with 10% egg yolk (pH 7.0). Pre- and post- centrifuge sperm motility was
observed under a
phase contrast microscope. Fifty ul of a fixative (0.1% glutarldehyde In 3.4%
Na citrate) was
added to each pellet and supernatant to immobilize the sperm for concentration
determination
with a hemacytometer. Total numbers of spermatozoa were calculated on the
basis of volume
used/recovered multiplied by the corresponding sperm concentration as
determined by the
hemacytometer. The recovery rate was calculated as the total number of sperm
in the pellet
divided by the sum of the total number of sperm in the pellet and the total
number of sperm in the
supernatant.
The results, as shown in Figs. 88 and 89 show there is little difference in
sperm cell
motility caused by varying the centrifuge speed. The results also show that
motility was slightly
better using TCA compared to PBS.
Centrifuge Example
In centrifuge example II semen samples from three bulls were collected and
evaluated
as described above. One of the samples was disqualified for failure to meet
initial quality control
standards. The other two semen samples were diluted with a quantity of TCA
having a pH of 7.3
In order to obtain a sperm concentration of 150 x 106 sperm/ml. The
spermatozoa were stained
with a 10 pM Hoechst 33342 solution at 41"c for twenty minutes. A simulated
buffer containing
1500 pi PBS with 10% egg yolk and 28.3 rnl PBS with 0.1% BSA was added to each
of two
tubes. Two hundred pl of the stained spermatozoa (30 x 106 sperm cells) were
added to each
tube and Incubated at room temperature for twenty minutes. Three 9 ml aliquots
of semen
mixture were taken from each of the two tubes for centrifugation. One aliquot
from each of the
two samples was centrifuged for seven minutes in a 15 ml centrifuge tube at
each of the
following speeds: 550g; 650g; and 750g. The temperature during centrifugation
was 22 C.
Immediately after centrifugation, supernatant was removed with a 10 ml
pipette, leaving about
200-300 pl supernatant in the pellet. The pellets were resuspended with 200 pi
of TCA having
10% (v/v) egg yolk having a pH of 7Ø Pre- and post- sort sperm motility was
observed under a
phase contrast microscope. Severe sperm agglutination was noted in the post-
centrifuge
samples from one of the two bulls. Fifty pt of a fixative (0.1% glutardehyde
in 3.4% Na citrate)
was added to each supernatant and pellet to immobilize the sperm for
concentration

CA 02752247 2011-09-07
109
determination. Recovery rate was detem-iined according to the formula set
forth in centrifuge
experiment I.
The results are shown in Fig. 90. The results show improved recovery rate of
sperm
cells at 650g compared to 550g. However, there was little difference in
recovery rate between
650g and 750g. There was no significant difference in sperm cell motility
caused by varying the
speed of the centrifuge.
Centrifuge Example III
For centrifuge example III, the procedure of centrifuge example II was
substantially
repeated with the same three bulls on a different day. The results are shown
in Fig. 91. The
results confirm that there is little difference in the recovery rate at 650g
compared to 750g.
Centrifuge Example IV
Semen was collected from two different bulls on two different days. Semen was
transported and evaluated in the manner described above. Based on sperm
concentration of
raw semen, spermatozoa were diluted with Tris-citric acid (TCA, pH 7.3) plus
10mM pyruvate, to
a concentration of 150x106sperm/ml. The spermatozoa were stained with 101.IM
Hoechst 33342
at 41 C for 20 min. After staining, 267 Ill of the solution containing the
stained spermatozoa
were diluted to a concentration of 1x1Oesperm/m1 by addition of the following
simulated buffers:
2 ml PBS with 10% (v/v) egg yolk; and 37.733 ml PBS with 0.1% (w/v) bovine
serum albumin
(BSA). The stained spermatozoa and simulated buffers were incubated at room
temperature for
at least 20 minutes. Four 9 ml aliquots were taken from the stained
spermatozoa and simulated
buffer mixture obtained from each bull. The four aliquots from the first bull
were centrifuged at
varying combinations of centrifuge speed and duration in the following
sequence:
(1) 700g for 7 minutes for the first aliquot;
(2) 700g for 10 minutes for the second aliquot;
(3) 650g for 10 minutes for the third aliquot; and
(4) 650g for 7 minutes for the fourth aliquot.
The four aliquots from the second bull were centrifuged at varying
combinations of centrifuge
speed and duration in the following sequence:
(1) 700g for 10 minutes for the first aliquot;
(2) 700g for 7 minutes for the second aliquot;
(3) 650g for 10 minutes for the third aliquot; and
(4) 650g for 7 minutes for the fourth aliquot.
All centrifugation was performed in 15 ml centrifuge tubes in a swing head
centrifuge (Allegra 6R,
Beckman Coulter Inc. Fullerton, CA) at 22 C. The time interval between semen
collection at

CA 02752247 2011-09-07
110
farm and centrifugation in lab was 4-5 hours. Immediately after
centrifugation, supernatant was
removed with 1 0 ml pipettes, leaving ¨250 pi supernatant with each pellet.
The pellets were
resuspended in 250 rti of Deibecco's PBS (pH 7.0). Sperm motility and
progressive motility were
observed using a Hamilton-Thorn Motility Analyzer (two slides per sample; two
chambers per
slide) after staining but before centrifugation and again after
centrifugation. Sperm concentration
was determined by hemacytorneter measurement of a 100 pi aliquot of the pre-
centrifuge stained
spermatozoa and simulated buffer mixture that had been placed in the freezer
and a 10 pi aliquot
of the resuspended pellet mixed with 90 gi fixative (0.1% glutaraidehyde in
3.4% Na citrate).
Recovery rate was determined as in Centrifuge Example I. The results are shown
in Figs. 92
and 93.
The data indicate that > 85% of the spermatozoa can be recovered after
centrifugation at
650g or 700g, for 7 or 10 minutes (Fig. 92). However, recovery rate was
slightly better (95%) at
700g. The decline in motility after centrifugation (compared to before
centrifugation) in all
treatments could be due to the presence of dead/abnormal/fragile spermatozoa
which could not
withstand the stress of centrifugal force. Sperm motility declined by 10-14%
(Fig. 93) in all
treatments. The higher decline in sperm motility (14%) at 660g for 7 min might
be due to the
longer exposure of sperm to simulated buffer as centrifugation at 650g was
conducted after
700g. Centrifugation did not show any adverse effect on progressive motility
of spermatozoa,
rather there was improvement by 2-3%.
Centrifuge Example V
Semen was collected from one bull on two different days. Semen was evaluated,
diluted
and stained with Hoechst 33342, and further diluted in simulated buffers as
described In
Centrifuge Example IV. Four 9 mi aliquots of the stained spermatozoa and
simulated buffer
mixture were obtained for each of the two semen samples. The aliquots from the
first sample
were centrifuged at one of the following combinations of centrifuge speed and
duration in the
following sequence:
(1) 750g for 10 Minutes for the first aliquot;
(2) 750g for 7 minutes for the second aliquot;
(3) 700g for 10 minutes for the third aliquot; and
(4) 700g for 7 minutes for the fourth aliquot.
For the aliquots obtained from the second sampler the combinations of
centrifuge speed and
duration were the same, but the sequence was modified as follows:
(1) 750g for 7 minutes for the first aliquot;
(2) 750g for 10 minutes for the second aliquot;
(3) 700g for 7 minutes for the third aliquot; and
(4) 700g for 10 minutes for the fourth aliquot.

CA 02752247 2011-09-07
111
Centrifugation was conducted in a 15 ml centrifuge tube in a swing head
centrifuge (Allegra 6R,
Beckman Coulter Inc. Fullerton, CA) at 22 C. The Interval between semen
collection at farm and
centrifugation in laboratory Was about 6.5 hours for the first sample, and
about 4 hours for the
second sample. Post centrifugation processing, i.e. removal of supernatant,
resuspension of
pellet, determination of sperm concentration, and motility estimation via
Hamilton-Thorn Motility
Analyzer, were conducted following the same procedure as described in Example
IV. The
results are shown In Flgs. 94 and 95.
The results show that > 85% of the sperm population In highly diluted
suspension can be
5%).
B. Secondary Centrifuaation
In order to recover sperm that might otherwise be lost in the supernatant, it
is possible to
centrifuge the supernatant after it has been separated from the pellet.
Without being bound by a
particular theory, applicants believe the peliet/supernatant interphase
impedes movement of
C. Filtration
An alternative concentration method that may be used to avoid loss of sperm
cells in the
supernatant is filtration. As shown in Fig. 96, according to one exemplary
embodiment a filter
2415 is incorporated in a collection vessel 2403. The size of the pores in the
filter are desirably
in the range of about 0.2 - 1 microns. It is also desirable that the filter is
not a depth filter (e.g., a

CA 02752247 2011-09-07
112
inches of mercury) to avoid inflicting damage to the sperm cells. In one
embodiment the vacuum
is low enough that the fluid removal rate is about 1.0 m1/15 seconds.'
According to another
embodiment of the present Invention, the vacuum is applied intermittently to
allow the sperm
cells a chance to recover. In still another embodiment, the filter 2415 is
constructed of a material
that is coMpatible with sperm cells, yet has no binding affinity for them. At
the completion of the
sort, about 80-90% of the fluids will have been removed through the filter.
However, enough fluid
remains (about 10-20%) that the sperm cells are in a concentrated slurry 2405,
thereby
preventing the sperm cells from forming a filter cake. The concentrated
suspension may be
transferred to another container 2419, as shown in Fig. 97 for example. A
syringe mechanism
2409 with a cannula-tip filter 2411 can be used to remove some of the
remaining liquid from this
container 2419. However, enough fluids are left in the container to prevent
the sperm cells from
caking on the filter 2411. The same considerations apply to the cannula tip
filter 2411 as the
filter 2415 in the collection vessel. Thus, the cannula filter 2411 pore size
is desirably in the
range of about 0.2 ¨ 1.0 microns and the cannula filter is relatively thin to
avoid having sperm
tails getting caught In tortuous passages In the filter. For example, a
DynaGard hollow
polypropylene fiber syringe tip filter, which is commercially available from
Spectrum Laboratories,
Inc. 6f Rancho Dominguez, CA may be used for the cannula tip filter. As shown
in Fig. 98, a
resuspension fluid 2413 is flushed through the cannula-tip filter to,wash
cells that may be sticking
to the filter surface back into the slurry. The resuspension fluid may include
a quantity of the
filtered fluid and/or a suitable extender. After a quantity of resuspension
fluid sufficient to remove
sperm cells from the filter has been back flushed through the filter,
additional resuspension fluid
may be added if desired. The total quantity of resuspension fluid is selected
to bring the
concentration to a desired concentration (e.g., about 20 x 106 sperm
cells/m1). Thus, the filtration
process of this embodiment Is a three-step process involving the use of a
filter in the collection
vessel, filtration using a cannula filter, and resuspension to obtain the
desired concentration.
In an alternative two-step filtration process, the first and second steps of
the three-step
process described above are combined so that removal of all fluid Is through a
cannula filter. In
this process the sorted sperm cells are directed to a collection vessel that
does not have a filter.
The fluids are removed by low vacuum and/or Intermittent vacuum as described
above that is
applied through the cannula-tip filter 2411. When the sperm cells are in a
concentrated slurry, a
resuspension fluid, such as for example, an extender, is flushed back through
the cannula filter to
obtain the desired concentration of sperm cells.
Filtration Example I
Filtration example I shows the recovery rate and motility of sperm cells after
concentration by a three-step filtration process of the present invention.
Semen samples were
collected from three bulls and evaluated as provided in the sample preparation
section above.
One of the three semen samples was disqualified for failing to meet minimum
initial quality
criteria. Two remaining samples were diluted with a quantity of TCA (pH 7.3)
necessary to attain
a concentration of 150 x 106 sperm cells/ml. Five hundred ul PBS with 10% egg
yolk and 9.5 ml

CA 02752247 2011-09-07
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PBS with 0.1% BSA was added to each of two 16 ml test tubes. Sixty-seven ul of
semen sample
(about 10 x 106 sperm cells) was added to each test tube and incubated for
twenty minutes at
room temperature. Referring to Fig. 99, a vacuum pump 2427 was used to apply
negative
pressure to draw a four ml aliquot of the diluted semen 2423 through a filter
2425. The filtrate
2429 was collected in a syringe 2421. After filtration sperm cells on the
filter were flushed back
with 1 ml TCA buffer in a 15 ml tube. Sperm motility was assessed visually.
Pre- and post-
filtration samples were mixed with a fixative (0.1 % glutardehyde In 3.4 Na
citrate) to immobilize
the sperm cells. Sperm concentration was determined using a hemacytometer.
Total number of
sperm cells was calculated on the basis of volume multiplied by the
concentration of sperm cells.
The recovery rate was calculated as the total number of sperm cells in the
flushed back portion
divided by the total number of sperm cells In the aliquot prior to filtration.
The process was
repeated with a different filter. The experiment tested both of the following
filters: (1) a 1.0 pm
PTFE (not FTPE) membrane disc (syringe) filter (which is available from Pall
Corporation, Life
Science Group, Ann Arbor, MI, Cat # PN4226T or VWR, Batavia, IL, Cat. # 28143-
928); and (2)
0.8 SFCA (surfactant free cellulose acetate) membrane disc (syringe) filter
(Corning, Inc.,
Corning, NY, Cat. # 431221; VWR Batavia, IL, Cat. # 28200-028). The results
are shown in Fig.
101. More spermatozoa were recovered with cellulose acetate filters as
compared to PTFE filter,
i.e. 67 vs 33% due to low protein binding affinity of cellulose acetate.
Visual motility of
spermatozoa recovered ranged from 63% (PTFE) to 68% (Cellulose acetate).
Filtration Example //
Filtration example 11 shows the recovery rate and motility of sperm cells
after
concentration by at two-step filtration process of the present Invention.
Semen samples were
collected from three bulls and evaluated as provided in the sample preparation
section above.
The three samples were diluted with a quantity of TCA (pH 7.3) necessary to
attain a
concentration of 150 x 106 sperm cells/mi. One and one half ml of PBS with 10%
egg yolk and
28.3 ml PBS with 0.1% BSA was added to each of 50 test tubes. Two hundred pi
of semen
sample (about 30 x 106 sperm cells) was added to each test tube and incubated
for twenty
minutes at room temperature. Referring to Fig. 100, a syringe 2431 was used to
apply negative
pressure to draw a 6 ml aliquot of the diluted semen 2433 from each test tube
through a filter
2435. The filter was placed in a filter holder 2437 (a Swinnex filter holder
from Millipore
Corporation, Billerica, MA Cat # SX0002500,). After filtration, the filtration
holder 2437 was
disconnected from the syringe and the tubing, keeping the filter holder
intact. Spermatozoa on
the filter were collected by turning the filter assembly upside down and back
flushing 1 with ml of
TCA buffer using a 3 mi syringe having a small piece of tubing at the tip In a
15 ml test tube.
Sperm motility was assessed visually. Pre- and post- filtration samples were
mixed with a
fixative (0.1 % glutardehyde in 3.4 Na citrate) to immobilize the sperm cells.
Sperm
concentration was determined using a hemacytometer. Total number of sperm
cells and the
recovery rate were calculated as specified in Filtration example 1, The
process was repeated
twice to test different filters. The experiment tested both of the following
filters: (1) a 0.2 pm

CA 02752247 2011-09-07
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Teflon membrane filter (which is available from X-Partek, P.J Cobert
Associates Inc. St. Louis
Cat. # 944106; and (2) a 0.8 cellulose acetate membrane filter (Millipore
Corporation, Billerica,
MA Cat. #AAWP 02500). The results are shown in Fig. 102. In both filters, the
recovery rate of
spermatozoa was low(-25%). It was low in Teflon filter as in example I.
However, low recovery
rate and visUal motility of flushed back spermatozoa in cellulose acetate
filter might be due to the
material used by different vendor and/or ability of spermatozoa to attach with
filter
holder/assembly.
D. Dense Medium Concentration
Another alternative method of concentrating the collected sperm relies on
flotation of
sperm cells in a high-density medium. According to this method, a high-density
medium is added
to the collected sperm cells to raise the specific gravity of the suspension
above about 1.3. For
example, a colloidal silica suspension such as is available under the PercoII
and Isolate
tradenames may be used to increase the specific gravity of the suspension. The
sperm cells will
float to the top of the suspension, where they can be skimmed or otherwise
collected, because of
the increased specific gravity of the suspension. A resuspension fluid Is
added to the cells that
have been. collected from the surface to bring the final concentration to
about 20 x 106 sperm
cells/ml. Some of the suspension fluid may be removed by one of the filtration
methods
described above prior to addition of the high density medium to reduce the
quantity of high
density medium required to attain the desired specific gravity.
Cryoextension
A. Cryoprotection
Once the sperm have been sorted and collected in the collection vessels, they
may be
used for inseminating female mammals. This can occur almost Immediately,
requiring little
additional treatment of the sperm. Likewise, the sperm may also be cooled or
frozen for use at a
later date. In such instances, the sperm may benefit from additional treatment
to minimize the
impact upon viability or post-thaw motility as a result of cooling and
freezing.
Generally, a cryoextender comprises a buffer or buffered solution, a protein
source, and
a cryoprotectant. Examples of buffers and buffered solutions that may be used
in the
cryoextender are disclosed above with respect to sample collection and
extension. Typically,
these buffers will be in a concentration of about 0.001M to about 1.0M and
have a pH of about
4.5 to about 8.5, preferably of about 7Ø
If included, a protein source may be added to provide support to the cells and
to cushion
the contact of the cells with the collection vessel. The protein source may be
any protein source
that does not interfere with the viability of the sperm cells and is
compatible with the particular
buffer or buffered solution being used. Examples of common protein sources
include milk
(including heat homogenized and skim), milk extract, egg yolk, egg yolk
extract, soy protein and
soy protein extract. Such proteins may be found in a concentration from about
10% (v/v) to
about 30% (v/v), preferably from about 'lb% (v/v) to about 20% (v/v), and more
preferably about

CA 02752247 2011-09-07
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20% (v/v). While milk may be used In combination with a buffer or buffered
solution, generally
milk is used in the absence of the same, as mill( is a solution itself that
may serve the same
purpose of a buffer or buffered solution. In such instances, the cryoextender
would contain about
80% (v/v) to about 90% (v/v) milk.
A cryoprotectant is preferably included in the cryoextender to lessen or
prevent cold
shock or to maintain fertility of the sperm. Numerous cryoprotectants are
known in the art.
Selection of a cryoprotectant suitable for use with a given extender may vary,
and depends upon
the species from which the sperm to be frozen were obtained. Examples of
suitable
cryoprotectants include, for example, glycerol, dimethyl sulfoxide, ethylene
glycol, propylene
glycol, trehalose, Triladyl and combinations thereof. If included, generally,
these
cryoprotectants are present in the cryoextender in an amount of about 1% (v/v)
to about
15% (v/v), preferably in an amount of about 5% (v/v) to about 10% (v/v), more
preferably in an
amount of about 7% (v/v), and most preferably In an amount of about 6% (v/v).
In one particular embodiment, the cryoextender comprises water, Triladyl , egg
yolk, and
pyruvic acid. In yet another embodiment, the cryoextender comprises 25g
Trilady , 25g egg
yolk, and 10mM pyruvic acid in 75mL of water.
Optionally, the cryoextender may also contain a range of additives that are
beneficial to
sperm viability or motility and that prevent or lessen the detrimental side
effects of
cryopreservation. Such additives may include, for example, an energy source,
an antibiotic, or a
composition which regulates oxidation/reduction reactions intracellularly
and/or extracellularly,
each of which is discussed above with respect to sample collection and
dilution. Such additives
may be added to the cryoextender in accordance therewith.
B. Cryopreservation of Sorted Sperm Cells
In most cases, it will not be possible to use the sperm cells that have been
sorted as
described above for Immediate artificial insemination. Particularly in the
case of a commercial
sperm sorting operation, the sorted sperm cells must be stored and/or
transported before they
can be used for artificial insemination. This will usually require
cryopreservation of the sperm
cells. The sorted sperm may be loaded into elongate cylinders (known as
"straws" in the
breeding industry) and cryopreserved to preserve the sperm during
transportation and storage.
Cryopreserved sperm cells can be stored for long periods of time in liquid
nitrogen. To use the
cryopreserved sperm, the straw may be immersed in a heated water bath to thaw
the sperm.
Then the straw is loaded into an artificial insemination gun which is used to
inseminate a female
animal Several precautions must be taken to protect the sperm cells during
cryopreservation.
Otherwise the sperm cells will be so damaged (as indicated by a low post-thaw
motility rate of 5-
10%) that they are not suitable for use in artificial Insemination. .
Conventional cryopreservatlon methods involve sequentially adding a protein
source
(e.g., egg yolk), cooling the sperm to a temperature of about 4 - 5 C, adding
a cryoprotectant
(e.g., glycerol), maintaining the sperm and cryoprotectant at a steady
temperature in the range of
about 4 - 5 C for a period of time sufficient to allow the sperm cells to
equilibrate with the

CA 02752247 2013-11-21
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cryoprotectant, and then supercooling the sperm, as by Immersing the sperm
cells in liquid
nitrogen at -196 C for storage. Those skilled in the art will recognize that
the purpose of the
protein source is to protect sperm from damage as they cool from about 14 C to
about 8 C,
which is the temperature at which sperm cells are most susceptible to cold
shock. In contrast,
the cryoprotectant protects the sperm cells from damage at temperatures below
0 C. Even
though the temperatures involved in cryopreservation are well below freezing
and the term
"freezing" Is sometimes used to describe cryopreservation, those skilled in
the art will also know
that cryopreserved sperm are not actually frozen. To be precise, the
cryopreserved sperm are in
a supercooled state. The conventional period during which sperm cells and
cryoprotectant are
maintained at a steady temperature can last anywhere from 60 minutes to many
hours. The
overall time to complete cryopreservation using conventional methods generally
exceeds four
hours. Furthermore, it is believed that up to 50% of the sperm cells are
killed in conventional
cryopreservation processes. Though sperm are cryopreserved using conventional
methods
according to some embodiments of the present invention, other embodiments Of
the present
invention employ improved cryopreservation methods to reduce the time required
for
cryopreservation and/or to improve the health of the cryopreserved sperm.
Fig. 103 shows a work flow diagram outlining the steps of one exemplary
embodiment of
an improved method of cryopreserving sperm according to the present invention.
At step 2501,
the concentration of a solution containing sorted sperm cells is adjusted to
be in the range of
about 1 million - 40 million sperm/ml, depending on the standard used by the
targeted consumer
(e.g., breeding association). For example, the sperm concentration may be
adjusted to be in the
range of about 20 million to 24 million sperm/ml. AdJUstment of the sperm
concentration may
include addition of resuspension fluid, buffers and/or extenders to
concentrated sperm as
described above. At step 2503, a cryoprotectant (e.g., glycerol) is added
before the sperm are
cooled. The sperm cells begin equilibrating with the cryoprotectant as soon as
they come into
contact with the cryoprotectant. At step 2505, a protein source (e.g., egg
yolk) is also added to
the solution containing the sperm cells as described above.
The sperm cell solution, protein source, and cryoprotectant are loaded into
conventional
0.5 or 0.25 ml artificial insemination straws using a conventional loading
machine at step 2507.
Those skilled in the art will be familiar with a number of conventional
apparatus and techniques
that may be used to load semen into straws. For example, United States Patent
No. 5,249,610,
issued October 5, 1993 to CassoU, et al. provides
instruction about the filling of straws with bovine semen using a disposable
injector nozzle.
Moreover, equipment for filling straws is commercially available from Minitube
of America,
located In Verona WI. Any of these or similar conventional loading methods and
apparatus can
be used to load the sorted sperm cells into straws.
After loading, the sperm cells are cooled to a holding temperature at step
2609. in
general, the holding temperature should be selected with the following
considerations in mind:
holding sperm cells at a temperature.that is too high (e.g., 10 C) may cause
unnecessary
damage from cold shock; equilibration of sperm cells with a cryoprotectant
(e.g., glycerol) is

CA 02752247 2011-09-07
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believed to be most active at temperatures in the range of 4-5 C; and holding
sperm cells at
temperatures that are too low (e.g., < 0 ) is believed to be damaging to the
sperm cells. Thus,
according to one embodiment, the holding temperature is in the range of 0-8 C,
More desirably,
the holding temperature is in the range of 2-6 C. Even more desirably, the
holding temperature
Is in the range of 4-6 C. In another embodiment, the cooling rate used for
this step 2509 is
selected to minimize damage to the sperm cells. For example, the cooling rate
may be
controlled (e.g., substantially constant) to provide homogenous cooling and to
prevent the sperm
from suffering temperature shock. The cooling rate should also cool the sperm
quickly enough to
reduce their metabolism before they incur membrane damage, but slowly enough
that they do
not suffer from temperature shock. One can control the cooling rate by placing
the straws
containing the sperm cells in a programmable freezer (e.g., an IceCube 1810CD
freezer which is
available commercially from Minitube of America, located in Verona, WI) to
cool them. According
to one embodiment, the programmable freezer cools the sperm from about room
temperature
(typically In the range of about 22 and 24 C) at a constant cooling rate of
0.1 and 0.3 C/ minute.
More desirably, the cooling rate Is In a range of about 0.16 and 0.25 C/min.
Even more
desirably, the cooling rate is about 0.2 C/min. In another embodiment, the
cooling rate is
selected so the sperm are cooled from their initial temperature to the holding
temperature in
about 90 minutes. In still another embodiment, the cooling rate is selected to
cool the sperm
from their initial temperature to the holding temperature at a constant
cooling rate in about 90
minutes. The cooling rates referred to above actually refer to the rate of the
cooling of the
chamber of the programmable freezer, but because of the thin walls and long,
thin shape of the
straw (e.g., about 5.25 inches long, less than 3 mm in diameter, and about
0.16 mm in wail
thickness) and the conductive properties of the straw, the temperature
difference between the
sperm cells and the cooling chamber is not significant.
After the sperm cells have been cooled to the holding temperature, at step
2511 they are
kept at or near that temperature for a period to allow substantial completion
of their equilibration
with the cryoprotectant. For example, the programmable freezer described above
can be
programmed to hold the sperm cells at a steady temperature during the period.
According to
another embodiment of the present invention, the sperm cells are held at the
holding temperature
for a period that is shortened compared to conventional methods because the
sperm have
already been equilibrating with the cryoprotectant during the cooling process.
For example, the
period may be in the range of about 10 and 60 minutes. More desirably, the
period is In the
range of about 20 and 40 minutes. Even more desirably, the period is about 30
minutes. in
another embodiment the period is less than 60 minutes, In yet another
embodiment, the period
Is less than 40 minutes. The relatively short holding period offers a number
of advantages in a
commercial sperm sorting process. First, it reduces the time required to
process sorted sperm
which can translate to cost savings. Also, the sperm cells still perform
metabolic processes at
temperatures In the range of 0-8 C so reducing the time for which sperm need
to held at this
temperature can improve the health of the sperm cells, which will increase the
value of the sperm
cells to animal breeders who are concerned about artificial insemination
success rates.

CA 02752247 2011-09-07
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After the sperm cells have been held at the holding temperature for a period
described
above, the sperm cells are cooled at step 2513 to a temperature that
approaches the critical
temperature zone for sperm cryopreservation. Those skilled In the art will
know that the critical
temperature zone Is the zone at which ice crystal formation and changes in
osmotic pressure
damage the sperm cells. This temperature may vary depending on the solution in
which the
sperm cells are cryopreserved, but the critical temperature zone is generally
in the range of ¨18
and ¨35 C. Sometimes this critical temperature zone is reported to be in the
range of about ¨18
and -30 C. Thus, according to yet another embodiment of the present invention,
the cooling rate
used to cool the sperm cells from the holding temperature to a temperature
that approaches -
'l 8 C (e.g., -15 C) is selected to protect the health of the sperm. Relevant
factors to consider
include that fact that the sperm cells are still equilibrating with the
cryoprotectant during this
period, the fact that sperm are still performing some metabolic functions, and
the fact that the
sperm are still somewhat sensitive to rapid temperature change. Again, it Is
desirable that the
cooling rate be a controlled rate, such as a rate that may be programmed into
the programmable
freezer described above. More desirably, the cooling rate used to cool the
sperm from the
holding temperature to a temperature that approaches about -18 C is a constant
cooling rate.
Thus, according to another embodiment of the present invention, the sperm
cells are cooled from
the holding temperature to about -15 C at a cooling rate in the range of about
1.0 - 5.0 C/ min.
More desirably, the cooling rate is In the range of about 2.0 ¨ 4.0 C/min.
Even more desirably,
the cooling rate is about 3.0 C/min.
Step 2515 involves rapidly cooling the sperm cells through the critical
temperature zone
to limit the time sperm cells dwell therein. Thus, according to one embodiment
of the present
invention, the cooling rate through the critical temperature zone about (e.g.,
-18 C to about -
C) is selected to be much faster than the cooling rate used to cool sperm
cells to the holding
25 temperature and the cooling rate used to cool sperm cells to the
temperature approaching the
critical temperature zone. Thus, the steeper cooling rate is desirably in the
range of from about 8
- 40 C per minute. More desirably, the steeper cooling rate is in the range of
from about 8 - 12 C
per minute. most desirably, the steeper cooling rate is about 100C per minute.
The temperature
range over which the steeper cooling rate is used may extend beyond the
critical temperature
30 zone. Thus, in yet another embodiment of the present invention, the
sperm cells are cooled at
one of the steeper cooling rates described above from about -15 C to about -40
C. In still
another embodiment, the sperm cells are cooled at one of the steeper cooling
rates described
above from about -15 C to about -80 C. The step of cooling the sperm through
the critical
temperature zone at a steeper rate may be accomplished in the programmable
freezer described
above.
After the sperm cells have been cooled below the critical temperature zone
(e.g., to -
80 C), the straws containing the sorted sperm are immersed in liquid nitrogen
(-196 C) at step
2517 to provide maximum useful life of the sorted sperm cells. The use of
liquid nitrogen to store
cryopreserved sperm is widespread in the animal breeding industry In the
context of unsorted
sperm. Thus, those skilled in the art will be familiar with technologies
involving the transportation

CA 02752247 2011-09-07
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and storage of sperm in liquid nitrogen, which need not be discussed in great
detail herein. It is
sufficient to note that conventional containers are available to provide for
long term storage of
bulk quantities of artificial insemination straws in liquid nitrogen and that
smaller and more
portable containers are also available for providing storage of artificial
insemination straws In
liquid nitrogen for transport to customers and/or for transport to a farm
having one or more
female animals to be inseminated with cryopreserved sperm.
One advantage of the cryopreservation methods described herein is that the
cryopreservation can be completed In less time than is required according to
conventional
methods. Perhaps relatedly, the decline in motility due to cryopreservation
according to the
present invention is only about 5-11%, as indicated by the example discussed
below. Thus,
cryopreservation according to the present invention preserves the health of
the sperm cells as
indicated by tests showing that sperm cells cryopreserved according to the
present invention
have greater than 50% (e.g., about 60%) motility after they are thawed in a 37
C water bath for
about 50 seconds. As discussed above, sperm motility may be analyzed by an
automatic
machine (e.g., the IVOS sperm analyzer from Hamilton Thorn Research) or by
visual
examination.
It should be noted that the cryopreservation methods described above are
contemplated
as being used in a commercial scale sperm sorting process. Thus, according to
one
embodiment of the present invention, the steps of the inventive methods
described herein are
performed simultaneously on a batch of sorted sperm cells to quickly
cryopreserve the entire
batch of sperm cells in a manner that preserves their health. For example, by
using the multi-
channel flow cytometry apparatus described below, it Is possible to obtain
about 840 x 106 sorted
X chromosome-bearing sperm cells in the collection system of the apparatus in
about 20
minutes. This is enough sperm cells to fill several dozen straws. Moreover, a
batch can include
the combined sperm cells by two or more different sorting cytometers. After
being concentrated
as described above, the sperm cells can be loaded into any number of straws
and cryopreserved
as a batch. For example, according to ohe embodiment of the invention, It
takes about 5 minutes
to add an extender (Including both a protein source and a cryoprotectant) to a
batch of sperm
cells, and about 15 minutes to load the sperm cells into artificial
insemination straws using an
automatic loading machine. All the straws in the batch are cooled
simultaneously in a
programmable freezer. Furthermore, the capacity of some programmable freezers
allows
simultaneous cryopreservation of thousands of artificial insemination straws.
For example, the
IceCube 1810CD freezer referred to above has the capacity to cryopreserve
simultaneously over
2,500 0.5 ml straws or over 3,800 0.25 mi straws. Thus, one could wait to
start the cooling step
Until multiple batches have been obtained. Alternatively, multiple batches
could be obtained
substantially at the same time by running multiple multi-channel flow
cytometry machines (see
below) in parallel and simultaneously cooling multiple batches obtained
therefrom together In a
programmable freezer. In one embodiment of the present invention, it takes a
period of less than
220 minutes to cool the sperm cells from room temperature to a supercooled
state and immerse

CA 02752247 2011-09-07
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them in liquid nitrogen (-196 C). In another embodiment, the supercooling
period is less than
190 minutes. In still another embodiment, the supercooling period is less than
150 minutes,
Those skilled in the art will recognize that substantial modifications may be
made to the
foregoing exemplary methods without departing from the scope of the present
invention. For
example, the sperm cells may be cryopreserved in a container other than an
artificial
Insemination straw. Likewise, the steps In the method that involve changing or
maintaining
temperature may be performed by any suitable means, including water baths,
liquid nitrogen
vapors, and conventional programmable or non-programmable freezers, for
example.
Furthermore, a wide variety of substances or combinations of substances could
be used as the
protein source and/or the cryoprotectant without departing from the scope of
the present
invention. These substances include substances and concentrations of
substances listed above
in connection with the discussions regarding buffers, extenders,
cryoprotectants, sheath fluids,
and collection fluids. Moreover, the order of some steps in the method may be
varied without
departing from the scope of thls invention. Although Fig. 95 indicates that
the cryoprotectant is
added after the concentration of the sorted sperm is adjusted, it is also
contemplated that a
cryoprotectant can be added before the concentration is adjusted without
departing from the
scope of the present invention. For example, the cryoprotectant may be
provided in the
collection fluid or in the sheath fluid used in connection with a flow
cytometer. Some of the
benefits of the present invention may also be obtained by partially cooling
the sperm cells and
then adding the cryoprotectant. Likewise, the order in which the protein
source is added may be
varied as long as the protein source Is effective to protect the sperm cells
from cold shock as
they pass through the temperature range of about 14 to 8 C.
Cryopreservation Example l
Bovine semen was collected, transported, and evaluated as described above. Two
test
tubes containing 5 ml each of TCA buffer (pH 7.3) were placed in one of two
water baths for at
least five minutes. One water bath was at a temperature of 35 C and the other
water bath was at
41 C. Spermatozoa at 24 C were added to each tube so that the final
concentration in each
tube was 150 x 106 sperm/mi. The two tubes were each divided into two aliquots
which were
kept in respective water baths. After the sperm had equilibrated with the TCA
buffer for five
minutes, 80 pM Hoechst 33342 was added to one of 35 C aliquots and one of the
41 C aliquots.
After addition of the Hoechst 33342, all four aliquots were incubated for 20
minutes in their
respective water bath. After incubation, the test tubes were removed from the
water baths and
left at room temperature (about 25 C) for five minutes. Then the contents of
each test tube were
diluted with a TCA extender containing 20% egg yolk and 6% glycerol (v/v) (pH
7.0) to a final
concentration of 20 x 106 sperm/mi. The contents of each test tube were then
used to fill a 0.5
mi artificial insemination straw. Each of the four straws was placed in a
programmable freezer
(an IceCube 1810CD freezer from Minitube of America, WI). The following
cooling sequence
was programmed into the programmable freezer: (1) 22 C to 4 C @ -0.2 C/min;
(2) hold at 4 C
for 30 min; (3) 4 C to -15 C @ -3.0 C/mln; and (4) -15 C to -80 C @ -10.0
C/min. After

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reaching -80 C, the straws were immersed in liquid nitrogen (-196 C) for 45
minutes. Then the
straws were immersed in a 37 C water bath for 50 seconds to thaw. Sperm
motility was checked
under a phase contrast microscope both before and after cryopreservation. The
results are
shown in Fig. 104. The post-thaw motility was generally on the order of 60%.
This represents a
decline In motility of only about 5-11% compared to before cryopreservation.
Analysis of
variance revealed no significant effect of either Hoechst 33342 or Incubation
at 41 C on post-
thaw sperm motility.
Operation of the System
The overall operation 813 of the flow cytometry system 9 will now be described
with
reference to Fig. 82 and in the specific context of sperm cells (e.g., bovine
sperm cells), but it will
be understood that the description is exemplary only, and that the system can
be used to
process other types of particles.
The first series of steps leading up to the six second repeat loop involve
calibration of the
system. After initializing 769, a system check 771 is performed to confirm,
among other things,
that the processor 131 or processors are operational. If an error is detected
after three failed
system checks 775, user interaction 773 is requested. If the system check is
positive, the
microprocessor directs the system to flush 777 the nozzle system with a
suitable fluid, and then a
quality control material 779, such as beads or bovine nuclei, are run through
the system to
initialize the detection parameters (see 739 in Fig. 72) and confirm that the
system is operating
within an acceptable quality control. This involves an evaluation of the
control material to test the
sensitivity and precision of the system to confirm that the system can
adequately discriminate a
sample. If the quality control is not confirmed after three attempts 775, user
intervention 773 Is
requested.
If the quality control material indicates an acceptable level of quality
control, 8 sample
781 Is aspirated and a portion or aliquot of the sample to be sorted is
checked for quality 783.
Sample quality may be determined by a calculation of a quality factor (Q-
factor) of the sample.
For example, the type of cells may be detected in a first aliquot of the
sample. During this
detection, the initialized detection parameters (741) are rechecked and the
initial discrimination
parameters (745) are generated. if the type of cells detected in the aliquot
indicates that the
sample meets or exceeds a preset standard (e.g., that the sample can be
discriminated to yield a
certain purity or motility and, in particular, that there are sufficient live
X cells available for
processing), then the system continues operation. If sample quality falls
three times 775, user
interaction is requested.
Continued operation Involves sorting 785 of the remainder of the sample
employing a six
second repeated loop. At the beginning of the loop, the microprocessor
confirms that sorting of
the sample Is not complete 789. If the sorting of the sample is complete 789,
the microprocessor
proceeds to aspirate the next sample 781 if it is available or to turn off the
sorting operation 793 if
additional sample is not available. If the sample Is not complete 789, the
microprocessor Initially
checks the X/Y discrimination 795 of the sample to confirm that it is within
an optimum range. In

CA 02752247 2011-09-07
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other words, drift analysis as noted above (761 in Fig. 72) is conducted. If
any changes should
be made, such changes are implemented and the discrimination 795 is again
checked. If the
discrimination is still unacceptable at this point, the sort is turned off 793
and user interaction is
requested.
Otherwise, the system proceeds to determine whether the fluid delivery system
is
delivering fluid and cells at a rate which is within an optimum range 801.
This determination
depends on the type of control strategy used. For the high recovery control
strategy, the
optimum rate would be determined by evaluating purity or looking at x/x+¨X of
the collected,
population. If the determined purity is higher than a required purity level,
the feed input rate of
the cells is increased by increasing a rate control signal provided to the
syringe pump 803. This
would tend to increase coincident cells and decrease purity because more
coincident cells
including ¨X cells would be collected with the X cells. If the determined
purity is lower than the
required purity, the feed input rate of the cells is decreased by decreasing a
rate control signal
provided to the syringe pump to reduce the frequency of coincident cells 803.
Thus, the cell
Input rate is a function of the determined purity of the collected population
as compared to a
desired purity level, e.g., a function of the identified ¨X sperm cells
collected.
For the high purity control strategy, the optimum rate would be determined by
calculating
lost X cells, e.g., discarded X/ discarded X + collected X. If the quantity or
percentage of lost X
cells are less than an acceptable level, the input rate of the cells is
increased by increasing a rate
control signal provided to the syringe pump 803. This would tend to increase
coincident cells
and increase the number of discarded X cells because more cells including X
cells would be
discarded with the Y cells. If the quantity or percentage of lost X cells is
higher than the
acceptable level, the input rate of the cells is decreased by decreasing a
rate control signal
provided to the syringe pump 803 to decrease coincident cells. Thus, the cell
input rate is a
function of the determined lost X cells of the discarded population as
compared to number of X
cells in the collected population, e.g., a function of the number of X sperm
cells not collected.
If this modified rate is acceptable 805, the system proceeds to another system
check
807. If the system check is acceptable 807, the sort continues in the six
second loop. If not, the
system is reset 809. If after reset the system is not acceptable or if the
revised feed rate is not
acceptable 811, the sort is turned off 793 and user intervention is requested
773.
The sorted droplet streams are collected by the collection system 2201.
Droplets that
are sorted into the population of X cells pass through the exit window 2245 in
the first
intercepting device 2247 to be intercepted by the second intercepting device
2249. From there,
the droplets containing the X cells flow into a collection vessel 2207. Other
droplets are
Intercepted by the first intercepting device 2247 and directed to the waste
trough 2805. Of
course droplets Intercepted by the first intercepting device could also be
saved, as noted above.
When a suitable amount of X-bearing sperm cells have been collected In the
collection vessel,
sorting may be interrupted to allow concentration of sperm cells in the
collection vessel 2207. A
new collection vessel may be placed under the first intercepting device 2247
or the collected fluid
may be poured into a different container and the collection vessel replaced.
Then sorting may

CA 02752247 2011-09-07
123 =
resume. The sperm cells in the collected fluid are concentrated, loaded in
straws, and frozen as
described above.
Temperature Control During Operation
Temperature control throughout the process may be used to improve the results
of the
process. As has already been discussed above, the temperature of the sperm may
be controlled
during various steps in the process (e.g., staining and cryopreservation). In
several
embodiments of this Invention, the temperatures of the sperm cells throughout
the various steps
of the method are controlled to achieve improved results.
For example, Fig. 105 is a work flow diagram of one embodiment of a method of
temperature control according to the present Invention. The temperature of
semen samples at
the time they are collected will be determined by the body temperature of the
animal from which
they are collected. For example, at step 2601 bovine semen samples are
collected at about
37 C. An insulated container is used for transportation of the semen samples
to the lab from the
collection site at step 2603. The insulated container retards cooling of the
sperm.
During sample evaluation at step 2605, the temperature is maintained below the

collection temperature, but in excess of a temperature corresponding to a
glass transition
temperature below which the sperm cells suffer meMbrane damage. For example
the
temperature may be maintained in the range of about 18 - 37 C. In another
embodiment, the
temperature may be maintained In the range of about 24 - 37 C during sample
evaluation. In a
particular embodiment, the sperm cells are placed In an environment having a
temperature in the
range of about 22 - 25 C during sample evaluation. Depending on the
temperature of the sperm
upon arrival at the lab, the effect of placing them In an environment having a
temperature in the
range of about 22 - 25 C may be to continue slow cooling of the sperm, to
maintain the
temperature of the sperm, or to slightly raise the temperature of the sperm.
In one embodiment,
the temperature may be elevated (e.g., to 40 C or higher) for staining at step
2607 as discussed
in the staining section. In another embodiment, the temperature of the sperm
cells during the
staining step may be in the range of about 20-40 C, as is also discussed
above.
At step 2609, the stained semen mixture is held In a water bath until such
time that the
mixture Is introduced into a flow cytometer. The temperature of the water bath
may be similar to
the temperature used for the staining step. In one embodiment the temperature
of the water bath
is in the range of about 40 - 47 C. In another embodiment, the temperature of
the water bath Is
in the range of about 20 - 37 C. In still another embodiment, the temperature
of the water bath is
in the range of about 20 - 25 C. After being held in the water bath for any
time between one
minute and two hours, the stained sperms cells are sorted by flow cytometry as
discussed above
at step 2611. At step 2613, the collected sperm cells are concentrated.
Concentration may be
performed in an environment that has a temperature that will not significantly
change the
temperature of the sperm cells. For example, in one embodiment, concentration
may be
performed in an environment having a temperature in the range of about 20 and
25 C. An
extender, protein source, and cryoprotectant are added to the concentrated
sperm at step 2615.

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Then, at step 2617 the sperm cells are loaded into artificial insemination
straws. In one
embodiment, the loading step is performed In an environment having a
temperature that will not
significantly change the temperature of the sperm cells. 'Finally, at step
2619 the temperature of
the sperm Is controlled during cryopreservation as discussed above.
In another embodiment, sperm cells may be stained at still lower temperatures
without
departing from the scope of the present invention. For example, it may be
desired to sort the
sperm cells in a flow cytometer at a relatively low temperature (e.g., about 0
C to about 8 C).
This may require modification of the overall temperature control. First, when
cooling the sperm
cells prior to introduction into a flow cytometer, egg yolk and other common
protein sources that
protect the sperm cells from cold shock at temperatures below the glass
transition temperature
generally may not be used as such protein-containing substances tend to foul
and/or clog the
fluidics of the flow cytometer. Thus, it is desirable to cool the sperm cells
before performing the
staining step in order to take advantage of natural cold shock protectants
found in neat semen,
such as for example, the seminal fluid. Any attempt to stain the sperm cells
prior to cooling
would require addition of buffers to protect the sperm which would dilute the
neat semen and
reduce the natural protection against cold shock.
Accordingly, one embodiment of the present invention for sorting the sperm
cells at a
temperature in the range of about 0 C to about 8 C includes placing the sperm
cells in an
environment having a temperature less than about 8 C to cool the sperm cells
to a temperature
in the range of about 0 C to about 8 C prior to staining. Any method may be
used to cool the
sperm cells, but it is desirable to use a method that protects against rapid
temperature
fluctuations of the sperm cells during the cooling process. For example, in
one embodiment, a
container holding the sperm cells is placed in a room temperature water bath,
which in turn is
placed In an environment having a temperature less than about 8 C. In another
embodiment, the
temperature of the sperm cells is monitored and ice is added to the water bath
to further cool the
sperm cells. The staining step may be performed as described above except that
the staining
mixture is subjected to a temperature In the range of about 0 C to about 8 C.
Due to the lower
temperature, the Incubation period required to stain the cells is considerably
longer. Once the
sperm cells have been cooled to 8 C or below, it is desirable to avoid warming
them. Thus,
another embodiment of the present invention is to operate the flow cytometer
in an environment
having a temperature in the range of about 0 C to about 8 C. Similarly,
another embodiment of
the present invention is to collect the sorted sperm cells in a collection
vessel that is surrounded
by an environment having a temperature in the range of about 0 C to about 8 C.
Still another
embodiment of the present invention Is to acid any extenders, cryoprotectants,
buffers, protein
sources, antibiotics, antioxidants, or other additives at a temperature In the
range of about 0 C to
about 8 C. With respect to addition of the cryoprotectant, it may be desirable
to add slightly
more of the cryoprotectant than would be added absent sorting the sperm cells
at a temperature
in the range of about 0 C to about 8 C. Thus, in one particular embodiment, a
cryoprotectant
containing 7% glycerol (v/v) Is added to sperm cells after the sperm cells
have been sorted at a
temperature in the range of about 0 C to about 8 C.

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Supercooling of the sperm cells from the temperature in the range of about 0 C
to about
8 C proceeds generally as described in the cryopreservation section above.
However, the sperm
cells will need to be held at a temperature In the range of about 0 C to about
8 C for a period of
time after addition of the cryoprotectant before supercooling to allow time
for the sperm cells to
equilibrate with the cryoprotectant. Thus, according to one embodiment, the
sperm cells are
allowed to equilibrate with the cryoprotectant for a period In the range of
about 30 minutes to
about 3 hours. In another embodiment, the sperm cells are allowed to
equilibrate with the
cryoprotectant for a period in the range of 1 ¨ 2 hours. In another particular
embodiment, the
sperm cells are allowed to equilibrate with the cryoprotectant for a period of
about 90 minutes.
Conventional temperatUre control apparatus and Methods (e.g., water baths,
incubators,
coolers, and freezers) may be used to heat or cool the sample to attain or
maintain the specified
temperatures In the foregoing embodiments of the invention. It is understood
that placing a
sample In an environment having a different temperature than the sample, will
cause the
temperature of the sample to change over time. There may even be temperature
variations
within the sample. As has been mentioned, it Is desirable to change the
temperature of the
sample gradually to help maintain the health of the sperm. Gradual temperature
change also
serves to reduce the temperature variation within the sample. As is well known
by those skilled
in the art, the rate of temperature change of the sample will be influenced by
many factors,
including the volume of the sample, the size and shape of the sample
container, and the
magnitude of the temperature difference between the sample and the
environment. However,
those skilled in the art will readily be able to select an appropriate method
and apparatus to
achieve the desired temperature control after considering all the relevant
factors.
Those skilled in the art will recognize that there is room for substantial
variation in the
exemplary temperature control without departing from the scope of the
invention. in general,
once the sperm cells have been chilled, it is desirable to avoid warming them.
Furthermore,
temperature variations discussed above in connection with sample collection,
staining, sorting,
droplet collection, concentration, and cryopreservation can be incorporated
Into the overall
temperature control without departing from the scope of the present invention.
Moreover, the
time at which sperm cells remain at any temperature can also impact the health
of the sperm.
Thus, processing according to the embodiment in which temperature Is
controlled throughout the
process is desirably completed within a timeline as discussed below.
Timeline for Operation
Generally, it is desirable to complete the sperm sorting process in the least
amount of
time possible to reduce the damage to the sperm. As discussed above, the
present invention
may Include staining at an elevated temperature to reduce the time needed to
stain the sperm
cells. For example, certain embodiments of the improved staining method
described reduce the
time require for staining to about 10 minutes. Likewise, the novel cytometer
described above
may be used to sort sperm cells in less time than would be required by a
conventional cytometer.
For example, a flow cytometer using the technology discussed above can collect
between 2,000

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and 10,000 sperm cells having a desired DNA characteristic per second
Furthermore, the
cryopreservation process may be used to reduce the time needed to complete
cryopreservation
of the processed sperm cells compared to conventional cryopreservation
methods. Accordingly,
one embodiment of the present invention involves processing sperm pursuant to
an overall
method to take advantage of one or more of the timesaving innovations to
reduce the time
required to complete the entire process.
For example, according to one embodiment of the present invention, a batch of
sperm cells (e.g., an ejaculate) is collected from a male mammal (e.g., bull),
evaluated for quality
control, stained, sorted according to a specified DNA characteristic, loaded
into one or more
containers (e.g., straws), and cryopreserved within a period of about 12 hours
from the time of
collection. In another embodiment, the period is less than about 8 hours. =In
another
embodiment, the period is less than about 6 hours. In still another
embodiment, the period is
less than about 3 hours. In yet another embodiment, the period of time is less
than about 2
hours. In another embodiment, the period of time is less than about 1 hour.
MULTI-CHANNEL SORTING APPARATUS AND METHOD
In order to sort more sperm in less time, it is possible to use more than one
cytometry
unit in parallel to sort that same sperm sample. One way to do this is to
simply divide the stained
sperm cells into multiple aliquots and run each aliquot through a different
cytometer. However,
as will be discussed below, certain advantages may be obtained by designing an
apparatus that
comprises multiple cytometry units in a single integrated multi-channel
cytometry unit.
Multi-Channel System Sharing Integrated Platform
Figs. 106-116 show one embodiment of the invention comprising a multi-channel
cytometry system, generally designated 1001, where multiple single-channel
flow cytometry
units, designated 1003, are ganged together as an integrated system to produce
sorted product.
Four such units are illustrated in this particular embodiment, but this number
can vary. The units
may be integrated in various ways, as by sharing an integrated platform
comprising one or more
of the following elements (1) a common supply of particles; (2) a common
source of
electromagnetic radiation 1007; (3) a common housing 1009; (4) a common input
for controlling
operation of the units 1011; (5) a common output 1019 allowing evaluation of
the operation of
one unit relative to another unit; (6) a common fluid delivery system 1021;
(7) a common
temperature control system 1023; (7) a common power source; (8) a common waste
recovery
system 1027; (9) a common deflector plate system 1029; and (9) a common
cleaning system
1031. In one embodiment, the system includes all of these elements, but it
will be understood
that a multi-channel system of this invention can include any combination of
elements. The use
of common elements is beneficial because it allows the system to be run more
efficiently and
profitably, achieves more consistent results among channels by reducing the
number of
variables, facilitates any trouble-shooting that may be needed, and is
economical.

CA 02752247 2011-09-07
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The multi-channel approach also makes the sorting system more amenable to
scale up or scale-
down.
Each of the cytometry units 1003 has components similar to certain components
of the
flow cytometry apparatus 9 of the previous embodiment and, for convenience,
corresponding
parts are designated by the same reference numbers with the addition of a
prime ('). In general,
each unit comprises a nozzle system 101', a mount for mounting the nozzle
system 331', a
transducer 105', and an epl-illumination optics instrument 417' for focusing a
beam of light 25' on
the fluid stream 21' exiting the nozzle orifice 103', all as previously
described. Each unit further
comprises a photodetector 117' operable as in the first embodiment to detect
fluorescence
emissions 31' from the particles in the stream 21' and to convert the
emissions 31' to electrical
signals 701' which are processed to classify the particles by a specified DNA
characteristic.
Each unit 1003 is also equipped for sorting the droplets 33' into different
groups or populations
123', 126' according to the classification of particles contained in the
droplets 35'. The
populations of droplets sorted by the units are collected by the collection
system 2201.
A. Common Housing and Modularity
The flow cytometry units are mounted in a modular arrangement in a common
housing
1009. In the embodiment shown in Figs. 106 and 109-113, the housing has a base
1069 and
two side wails 1071 extending up from the base. The side walls have a lower
pair of shoulders
1073 for supporting a lower cover panel 1075 at the front of the housing 1077,
and an upper pair
of shoulders 1081. A lower cover panel 1075 at the front of the housing 1077
is mounted
between the lower shoulders 1073. The upper shoulders 1081 support an upper
cover panel
1083 at the rear of the housing 1085. The front and rear of the housing 1077,
1085 are
substantially open to provide access to the equipment inside. It will be
understood that the
housing 1009 may have other configurations without departing from the scope of
the invention.
Further, it will be understood that the various units could be installed In
separate housings.
The flow cytometry units 1003 are mounted side-by-sIde as modules on an
appropriate
framework 1087 in the housing 1009. Specifically, the nozzle mounts 331' for
positioning the
nozzles 101' are releasably attached to a cross bar 1089 (Fig. 106) affixed to
the side walls 1071
of the housing, and the bases 429' of the epi-illumination instruments 417'
are releasably
fastened to an angled moUnting plate 1093 extending between the side walls
1071 of the housing
toward the rear of the housing 1085 (Flg. 109), the arrangement being such
that a particular unit
can be installed or removed as a module. This modularity facilitates
installation, removal for
maintenance and/or replacement, and enables any number of flow cytometry units
1003 to be
readily added as needed or desired to increase the throughput capacity of the
system.
B. Common Fluid Supply and Delivery Systems
The fluid delivery system 1021 of this embodiment is equipped to provide
appropriate
fluids to each of the cytometry units 1003. As Illustrated schematically in
Flg. 108, the system
generally comprises a pump 1105 for conveying carrier fluid 17' from a common
supply of carrier

CA 02752247 2011-09-07
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fluid 1107 under pressure, a gas pressure system 1115 for conveying fluid from
a common
supply 1117 of sheath fluid 19' under pressure, and a manifold system 1121 for
receiving the
fluids from respective supplies and delivering the fluids under pressure to
the various cytometry
units 1003, as needed. In the specific embodiment of Flg. 116, the supply of
carrier fluid
comprises a vessel 1123 containing a suitable volume of such fluid (e.g., 5
ml.). The vessel is
held by a holder 1125, which may be a block 1133 having a cavity 1135 sized to
receive the
vessel 1123. The block also has a second cavity 1137 for holding a vessel 1139
containing a
suitable buffer material for conditioning the system during use, as will be
described later.
The pump 1105 for delivering carrier fluid from the vessel is desirably (but
not
necessarily) a syringe pump 1141 as previously described. The plunger of the
pump is movable
through an intake stroke to aspirate a selected volume of carrier fluid 17'
from the vessel 1139
and through a discharge stroke to dispense carrier fluid through a supply line
1147 to the
manifold 1177 and from there to the various nozzles 101' of the system. The
syringe pump Is
also operabie to 6spirate fluid from the vessel 1139 containing buffer and to
pump the buffer
through the system in a manner to be described. A three-way valve 1149
controls the flow of
carrier and buffer fluids to and from the pump 1141. The pump is driven by a
variable speed
motor under the control of the processor 131'. By way of example, the pump may
be driven by a
stepper motor which operates at selectively variable speeds to pump carrier
fluid to the manifold
system 1121 at rates necessary to obtain the desired throughput from the units
1003. Multiple
syringe pumps or other types of fluid delivery devices can be used instead of
a single syringe
pump.
In one embodiment the supply 1117 of sheath fluid comprises a vessel 1155,
e.g., a tank
connected to the manifold 1177 by means of a supply line 1157. The gas
pressure system 1115
is operable to pressurize the tank and comprises a source of pressurized gas
1161 (e.g., alr or
nitrogen) communicating with the tank via a gas line 1163 having a regulator
1165 In it for
controlling the pressure supplied to the tank, and a two-way valve 1167 which,
in a first position,
establishes communication between the tank and the gas source, and In a second
position, is
operable to vent the tank. The gas pressure regulator 1165 is a conventional
regulator
adjustable to control the pressure supplied from the air source. The gas
pressure system 1115
also includes a gas line 1169 for pressurizing a supply 1173 of cleaning
solution (e.g., de-ionized
water in a tank) which can be used to flush the fluid circuitry in a manner to
be described
hereinafter. Flow through the gas line is controlled by a two-way valve 1167
operable In the
same manner as valve 1167.
In one embodiment, the manifold 1177 comprises a laminated block 1179 (Fig.
112)oI
material having passages 1181 formed In it to define a fluid flow circuit 1185
such as that shown
diagrammatically in Fig. 116, (The passages may be formed by machining grooves
in faces of
the laminations prior to assembly of the laminations to form the block.) The
fluid circuit Includes
inlets 1189, 1191 connected to the syringe pump 1141 and to the supply 1117 of
sheath fluid,
and sets of outlets 1193, for providing such fluids to the flow cytometry
units 1003, each such set
including a carrier fluid outlet and a sheath fluid outlet. Flow through the
various flow passages

CA 02752247 2011-09-07
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1181 is controlled by valves V1-V6 which, in one embodiment, are solenoid-
operated valves in
housings attached to the manifold block 1179. The block is desirably of
substantially transparent
material (e.g., acrylic plastic) to facilitate monitoring of the system 1121
and trouble-shooting. In
the embodiment shown, the manifold 1177 is attached to a frame member 1203
extending
between the side walls 1071 of the housing 1009 adjacent the bottom of the
housing below the
nozzle systems 101'. The Inlets and outlets 1193, of the manifold 1177 may
comprise fittings
1205 threaded into the block, such as flangeless nut and ferrule fittings
available from Upchurch
Scientific, a division of Sclvex. It will be understood that the design and
construction of the fluid
circuit 1186 In general and the manifold 1177 in particular may vary without
departing from the
scope of the present invention.
Referring to Fig. 115, the manifold fluid circuit 1185 for the carrier fluid
17' includes a
sample reservoir 1207 for holding a limited supply of carrier fluid (e.g., 1.0
ml). If the carrier fluid
contains sperm cells, for example, providing such a reservoir close to the
nozzles 101' Is
beneficial to the viability and motility of the sperm cells, since the storage
of such cells, even for
short periods of time, In small spaces may be detrimental to motility. Flow of
carrier fluid from the
sample reservoir 1207 to the nozzles 101' is controlled by a series of two-way
valves V1A-V1D,
one for each nozzle. Each valve V1A-V1D has a first position establishing
fluid communication
between the needle 1217 of the sample reservoir and the needle 167' of a
respective nozzle for
delivery of carrier fluid 17' to the needle under pressure generated by the
syringe pump 1141,
and a second position establishing fluid communication between the needle 1217
and a waste
system, generally designated 1221, which is common to all of the flow
cytometry units 1003. In
the embodiment shown, the waste system 1221 comprises a waste tank 1223 for
holding waste
material, a mechanism 1225 such as a vacuum pump for generating a vacuum in
the waste tank,
and waste lines 1227 connecting the valves V1A-V1D and the waste tank. A valve
V3 is
provided in the waste line upstream from the waste tank for opening and
closing the waste line,
as needed. A suitable hydrophobic filter 1233 is provided In the line
connecting the waste tank
1223 and the vacuum pump 1225.
The manifold fluid circuit 1185 for the sheath fluid 19' includes a plurality
of valves V2A-
V2D. Each valve has a first position establishing fluid communication with the
supply 1117 of
sheath fluid In the tank for delivery of sheath fluid 19' to a respective flow
body 133' via a sheath
supply line 1241, and a second position establishing fluid communication
between the flow body
and the waste tank via a waste line 1247. The pressure at which the sheath
fluid is delivered to
the flow bodies 133' will depend on the sheath tank pressure (as controlled by
the regulator
1155) which may range from 1 to 100 psi, more desirably from 10 to 50 psi,
even more desirably
15 to 40 psl, and even more desirably from about 20 to 30 psi.
While the use of a common supply for all of the units has various advantages,
it is
contemplated that at least some of the flow cytometry units could be supplied
with sample
material from separate sources.

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C. Common Power Supply and input and Output Controls
The flow cytometry units 1003 also share a common power supply, common power
delivery systems, a common input (GUI) 715' for controlling operation of the
channels oy tne
microprocessor 131', and a common output provided to the microprocessor
allowing evaluation
of the operation of one channel relative to another channel. For example, the
common output
includes providing the digitized signals from each epi-illumination system to
the microprocessor
for an indication of the fluorescence Intensity measured by each channel, for
an Indication of the
rate at which each channel is separating particles, for an indication of the
staining variations
(which may be indicated by the intensity difference of fluorescence pulses
from X and Y cells)
and for an indication of the decision boundaries 763 used by each channel for
discriminating
between particles. As another example, the common output includes providing
the output
signals from the break-off sensors 389' to the microprocessor for an
indication of the droplet
break-off location 107' of each channel.
D. Common Temperature Control
Optionally, a temperature control system, generally designated 1257, is
provided to
regulate the temperature of the contents of the vessels 1123 in the holding
block 1133 and the
temperature of the manifold 1177. Such temperature control reduces the
variability of the
system, thus providing more consistent measurements between channels and, for
certain types
of cells (e.g., sperm cells), helping to maintain the viability of the cells.
In one embodiment, the temperature control system 1257 comprises a fluid flow
circuit
1259 comprising fluid passages 1263 in the holding block 1133 and fluid
passages 1269 in the
manifold block 1179, and a control unit 1265 for circulating a thermal fluid
(e.g., water) through
the circuit at a selected temperature. The temperature is desirably such as to
maintain the fluid,
especially the carrier fluid, at an optimal temperature to maximize cell
viability and, if sperm cells
are involved, sperm motility. A valve shut-off V6 is positioned in the circuit
for controlling flow
through the circuit. The temperature control unit may be used to maintain the
sperm cells at the
desired temperature prior to sorting as discussed above.
All of the valves in the fluid delivery system 1021 are operated by
conventional means,
such as solenoids, under control of an operator or suitable programming. The
various fluid flow
lines connecting the components of the system outside the manifold block 1179
are desirably of
substantially transparent plastic tubing for observing any blockages. For
example, the tubing
may be 0.0625 in. OD tubing of FEP polymer. The flow lines of the temperature
control system
1257 are desirably somewhat larger (e.g., 0.125 in. OD) to provide greater
flow capacity.
E. Common Light Beam and Beam Splitting System
As previously noted, the multi-channel system shares a common source of
electromagnetic radiation or beam light 1007. By way of example (and not
limitation), the source
may be a laser beam from a UV multiline laser primarily having wavelengths of
351.1 nm and
363.8 nm. Alternatively, 11 may be desirable to use a pulsed laser (e.g., a
mode-locked laser),

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particularly to.synchronize digital sampling with a pulsed laser (as discussed
in the pulsed laser
section) in order to increase the effective power delivered to each cytometry
unit, thereby
increasing the numberof cytometry units that can be operated with a single
laser.
The power required to generate the laser beam will vary depending on the
requirements
of each flow cytometry unit and the number of units. For example, if there are
N Units and each
unit requires a light beam having an effective power of W watts, then it will
be necessary to
generate a laser beam having a power of (W x N) L, where L equals the system
power loss
among the optical elements of the system. Using a single laser to supply all
of the flow
cytometry units is economical compared to a system Using multiple lasers. It
is also efficient and
provides for more consistent measurements from one channel to the next,
because there is no
variability on account of different beam characteristics (e.g., beam
intensity, light polarity, beam
divergence) or electrical noise resulting from the use of multiple lasers.
According to one embodiment of the present invention, a beam splitting and
guidance
system is used to split a single laser beam into three or more separate beams.
As shown in Fig.
117, for example, a 50/50 beamsplitter 1270 (i.e., a beamsplitter that is
operable to divide a
single beam into two separate beams having approximately equal intensity) can
be used to split
a single beam 25' into two beams 1270A, 1270B. By using a second 50/50
beamsplitter 1271 to
split one of the two beams 1270B into two additional beams 1271A, 1271B, one
can generate a
total of three beams 1270A, 1271A, 1271B from a single beam 25'. Each beam can
be directed
into the optics system of a flow cytometer, for example an epi-illuminatton
optics system 415' as
shown in Fig. 117. One could also use additional 60/50 beamsplitters to split
the single laser
beam into any number of additional separate beams. As shown schematically in
Fig. 118, for
example, a third beamsplitter 1272 can be added to the 3-way beamsplitting
system (Fig. 117) so
that the three 50/50 beamsplitters 1270, 1271, 1272 can be used to split a
single 25' beam into
four separate beams 1271A, 1271B, 1272A, 1272B. From this one can readily
appreciate that
the single beam can be split into any number of separate beams. Other beam
splitting
arrangements may be used to split the incoming source beam into multiple light
beams for the
various units.
One desirable embodiment of a bearnsplitting system is shown in Figs. 106 and
109. A
beam guidance system 1273 is provided for guiding the common beam 1007 to the
optics
instruments 417' of the various flow cytometry units 1003. In the embodiment
illustrated in Figs.
106 and 111, the guidance system 1273 includes a lower mirror assembly 1279
mounted on one
side wall 1071 of the housing 1009, an upper mirror assembly 1281 mounted on
the side wall
1071 above the lower mirror assembly, and a series of reflecting filters, one
associated with each
optics instrument 417'. The lower mirror assembly is operable to reflect a
beam 1007 from a
suitable source upwardly to the upper mirror assembly, and the upper mirror
assembly is
operable to reflect the beam through an opening in the side wall 1071 to the
reflecting filters 431'
of the various instruments 417'.
In one embodiment, the lower mirror assembly includes a base 1285 fastened to
the side
wall 1071 of the housing 1009, a stage 1289 movable vertically on the base by
a suitable

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mechanism 1291, such as a micrometer, a tiltable platform 1293 on the stage
(e.g., a kinematic
optical mount Model P100-P available from Newport), and a mirror 1295 on the
platform, the
position of the mirror being adjustable by moving the stage and the mirror
platform to the
appropriate locations. The upper mirror assembly is similar to the lower
assembly, comprising a
base 1297, a vertically movable stage 1299, a tillable platform on the stage
1301, and a mirror
1303 on the platform. A pair of target plates 1309 are affixed to the side
wall of the housing 1009
between the upper and lower mirror assemblies. The target plates 1309 have
vertically aligned
holes 1311 therein to facilitate adjustment of the upper and lower mirrors so
that an Incoming
beam 1007 is precisely reflected toward the reflecting filters 431' of the
instruments 417', all of
which filters are aligned with the Incoming beam.
Each of the first three reflecting filters 1315, 1317, 1319 functions as a
beam splitter, i.e.,
it functions to reflect a specified percentage of the beam and to pass the
remaining percentage of
the beam. For example, in the case of four epi-illumination instruments, the
reflecting filters 431'
of the first three instruments each reflect a percentage of the laser light
1007, so that each of the
first three units of the series receives 25% of the electromagnetic radiation
of the original beam
1007. For example, the reflecting filters of the first, second and third units
may reflect 25%, 33%
and 50% of the incident light, respectively. The last reflecting filter 1321
of the series desirably
reflects all of the remaining light (about 25% of the original beam) to the
last instrument of the
series. As a result, each of the four Instruments should receive the same
intensity of radiation
(light) to interrogate the cells in respective streams.
Depending on the beam splitting devices used in the above system 1273, it may
be
desirable that the laser beam have a particular polarization. The tranmittance-
to-refiectance ratio
of dielectric filters can vary depending on the polarization of the light.
Further, when dealing with
linearly polarized light, dielectric filters (which are manufactured for use
at a specified angle of
incidence) can be too sensitive to variations in the angle of incidence.
Circularly or elliptically
polarized light alleviates this problem to some extent because the
polarization vector of the light
is in a variety of different orientations with respect to the optical axis of
a dielectric filter as the
light interacts with the filter. Thus, elliptically or circUlarly polarized
light simulates randomly
polarized light, which provides more tolerance for variations in the angle of
Incidence on a
dielectric filter. Accordingly, if the laser described above generates a beam
of light having a
vertical polarization, for example, it may be advantageous to convert the
light to circularly
polarized light before it is split. As will be understood by those skilled in
the art, this can be
accomplished by passing the beam through a 1/4-wave retardation plate (filter)
of polarizing
material having its optical axis rotated 45 degrees relative to the plane of
the laser polarization.
The beam thus transmitted by the waveplate will have approximately circular
polarization, and it
can be more easily split to provide multiple beams to the optics systems of
respective flow
cytometer units.
Moreover, by rotating the wave retardation plate to alter the angle between
the laser
polarization and the optical axis of the material used to make the waveplate,
eccentricity can be
introduced into the approximately circular polarization of the beam (i.e., the
polarization can be

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made more elliptical). Changing the eccentricity of the elliptical
polarization of the beam can
change the transmittance-to-reflectance ratio of the dielectric filters by
causing the polarization
vector for a greater percentage of the light to have a particular angle with
repspect to the optical
axis of the dielectric filter. Accordingly, if the balance of light among the
multiple cytometry units
is outside the desired range, one can rotate the wavepiate to increase or
decrease the
eccentricity of the elliptically polarized light, thereby altering the
transmittance-to-reflectance
ratios of the various filters until a better balance is achieved. Similarly,
if the wavepiate is
transmitting elliptically polarized light, one can influence the transmittance-
to-reflectance ratio of
one of the filters by rotating that filter.
Regardless of the method used to split the single beam into multiple separate
beams.
Balance of the power delivered to each cytometry unit can be achieved by
selectively blocking a
percentage of the light to bring all the cytometry units down to the same
level of power. For
example, the neutral density filter 447' of each epi-illumination system 415'
can be selected to
block more or less of the light to balance the illuminating power delivered by
the beam splitting
and guidance system to each individual cytometry unit. If one channel of a
multi-channel unit
receives significantly more illumination from the beam splitting and guidance
system, a neutral
density filter 467' that transmits less light can be used in the epi-
illumlnation system 415' of that
channel to bring the Illumination power for that channel more In line with the
other channels. It is
desirable, though not essential, that channel-to-channel variations in the
illuminating power be
less than about 10%. It Is even more desirable that the channel-to-channel
variations be less
than about 5%.
It will also be appreciated that pulsed laser scanning, as described above,
may be
desirable for multi-channel flow cytometry. For example, the UV multiline
laser can be replaced
with a mode-locked pulsed laser operating at about 85 MHz to allow more flow
cytometry
channels to be powered by a single laser. For example, the peak power provided
in each pulse
of a mode-locked laser emitting pulses having a width (duration) of about 12
picoseconds at a
frequency of about 85 MHz is approximately 800 times the average power output
of the laser.
Thus, a mode-locked laser (e.g., a Vanguard 350 from Spectra-Physics) can
provide enough
Illumination energy to operate a few dozen cytometry units (e.g., 32 cytometry
units) while
operating at only about 350 milliwatts.
The use of fiber optics for supplying light to the units Is also contemplated.
In this
ernbodiment, fibers are used to direct light from the laser to respective
units, thus eliminating the
need for the guidance system described above.
F. Common Deflector Plates
in the embodiment shown in Figs. 106 and 108-116, the sorting system 119' of
each flow
cytometry unit 1003 Is substantially identical to the sorting system 119
described in the first
embodiment, except that the units desirably share two common deflector plates
1331 extending
across the width of the housing 1009 at the front of the housing. There are
advantages to using
a common set of deflector plates, including a consistent charge from one
channel to the next, the

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use of a common power supply, a larger plate area providing a more stable
electric field and
more uniform droplet deflection, and a consistent angle of deflection for
collection of sorted
samples. The deflector plates 1331 are mounted on a frame 1333 fastened to the
housing 1009.
Alternatively, separate plates could be provided for each unit.
G. Common Collection System
In the embodiment shown In Figs. 107 and 116, a common collection system 2801
Includes two Intercepting devices for each cytometry unit as described above
in connection with
the collection system 2201 for the single unit. However, a common frame 2803
is provided to
hold all eight of the intercepting devices. Also, one of the two Intercepting
devices for each
cytometry unit directs fluid Into a common waste trough 2805 rather than a
collection vessel. The
waste trough makes It easier to discard sorted droplets that contain particles
of little value (e.g.,
Y-chromosome bearing sperm cells for breeding dairy cows). If it is desirable
to retain all the
sorted droplets, the waste trough can be removed and collection vessels can be
placed under
each intercepting device. The four collection vessels In the embodiment shown
in Figs. 107 and
116 rest In openings in the surface of a collection tray 2807. A common water
bath (not shown)
may be provided under the surface of the collection tray to control the
temperature of the
contents of the collection vessels.
H. Multi-Channel Control
The various flow cytometry units are controlled by the microprocessor 131',
(or other
suitable processing system) which desirably has a common input and a common
output as
discussed above.
Desirably, the operational parameters of each flow cytometry unit 1003 can be
set
independently of the other units so that such parameters can be varied as
between units. These
parameters may include, for example, the frequency of droplet formation, the
control and sorting
strategies utilized by a particular unit, the criteria used by each unit to
classify and sort particles
in the fluid supplied to the unit, and other parameters. For example, in
certain situations it may
be desirable to supply one or more units with carrier- fluid 17' at a first
flow rate and other units a
second (different) flow rate. Similarly, it may be desirable to use one
control sorting strategy
(e.g., a "high efficiency" strategy) for one or more units while using a
different strategy (e.g., a
"low loss" strategy) for other units. By controlled variation of these
parameters among the units,
based on historical data and data collected on a real-time basis, the
throughput of the units can
be managed and the results of the system optimized. The capability of
independent operation
also allows selected units to be operated in the event fewer than all of the
units are needed or
available.
Operation of MuItl-Channel System
The operation of the multi-channel system of this embodiment is similar to
that described
previously, except that the multiple flow cytometry units are adapted to
conduct flow cytometry

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operations in parallel (i.e., during the same time period or overlapping time
periods) for higher
throughput.
Prior to the start of a run, the fluid delivery system 1021 is flushed, If
necessary, with
cleaning solution from the tank 1173 by moving the valve V5 to its cleaning
position. The system
is then conditioned by with buffer fluid using the syringe pump 1141. During
this procedure, the
valves VIA-V1D and V2A-V2D are moved to establish communication with the waste
receptacle
1223 which is under vacuum. As a result, the cleaning solution and/or buffer
fluid flows through
the system to waste. This process cleans the system 1021, primes the syringe
pump 1141 and
removes air bubbles from the system.
With the three-way valve 1149 suitably positioned, the syringe pump 1141 is
operated
through an intake stroke to aspirate a quantity of carrier fluid 17'
containing particles, e.g., sperm
cells, following which the valve 1149 is moved to establish communication with
the manifold 1177
and the syringe pump moves through a discharge stroke to pump a volume of
carrier fluid into
the sample reservoir 1207 to fill it. The temperature of the carrier fluid 17'
is controlled by the
temperature control system 1257 to maintain the cells In the carrier fluid at
the desired
temperature. With the valves V1A-V1D positioned to establish communication
with the sample
reservoir 1207, further operation of the syringe pump 1141 forces carrier
fluid through the lines to
the needles of respective nozzle assemblies for flow through the nozzles 101',
as previously
described. At the same time, and with the valves V2A-V2O positioned to
establish
communication with the sheath fluid tank 1155, sheath fluid 19' is forced
through the supply lines
to respective flow bodies and through the nozzles, also as previously
described. This process
continues for an appropriate length of time sufficient to pump a suitable
volume of fluid through
the system 1001. The duration of a particular run will vary depending on the
quantity of carrier
fluid in the supply vessel, the rate at which the carrier fluid is pumped
through the system, and
the number of channels in the system. For example, a run may continue for only
a limited period
of time (e.g., 15 minutes during which about one mi. of carrier fluid is
delivered to each nozzle) or
it may continue indefinitely, with the supply of fluid being replenished as
needed.
In the event a needle 157' becomes clogged, the appropriate valve V1 is moved
to
establish communication with the waste receptacle 1223. Sheath fluid 19'
entering the flow body
133' will then flow under the force of the vacuum 1225 back through the needle
157' to waste,
thus fiushing and clearing the needle. If there is a need to shut off the flow
to a particular nozzle,
the valves V1 and V2 are simply switched to their waste positions.
Although the system described herein with respect to both the single channel
and multi-
channel configurations has been described with regard to particle separation,
such as the
separation of X and Y cells, it Is contemplated that such particles include
any particles having
different characteristics which may be arbitrarily noted as characteristic A
and characteristic 13.
Further, it will be understood that in some embodiments, the sorting function
can be eliminated
entirely, so that the flow cytometry apparatus (single-channel or multi-
channel) operates only to
classify the particles and not to sort them.

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While the multl-channel system is described above in the context of operating
the flow
cytometry units in parallel, It will be understood that the units could also
be operated in series.
For example, it is contemplated that particles in one stream could be sorted
by one unit Into
multiple populations, and that one or more of such sorted populations could
then be passed
through one or more other units in series to perform additional sorting
operations to sort different
particles using the same or different sorting strategies.
J. Upright Multi-Channel Embodiment
Figs. 119 and 120 show another exemplary multi-channel flow cytometry system.
This
system, generally designated 4001 comprises four cytometry units 4009 ganged
together. The
nozzle system 101', epi-Illumination optics system 450', deflector plates
629', sample station
4051, contamination prevention mechanism 4031 and other components of each
unit 4009 are
mounted on a shared vertical mounting board 4011. Referring to Fig. 120, a
single laser 4013
and a beam splitting and guidance system 4015, which is substantially similar
to the beam
splitting and guidance system 1273 described above, provide illumination for
each epi-
Illumination system 450'. The laser 4013 passes through a hole 4019 (Fig. 119)
in a common
housing 4021 containing the beam splitting and guidance system 4115. The beam
splitting and
guidance system 4115 and epl-illumination systems 450' are on one side of the
board 4011. The
focusing lens assembly 491' of each epi-iliumination system 450' extends
through the board
4011 to the other side (similarly to the configuration show in the single
channel system shown
Figs. 26 & 27), on which the remainder of the components for the units 4009
are mounted.
The units 4009 are all oriented so that their nozzle systems 101' direct the
fluid streams
21' downward. Each unit 4009 also has a collection system 4031, which includes
a collection
vessel 4033 for collecting droplets 33 containing a desired population of
particles and a waste
container 4035 for collecting other droplets 33. A water bath (not shown) or
other temperature
control may be used to control the temperature of the collection vessel 4033.
The multiple flow cytometry units 4009 can also share a common power supply
(not
shown), a common input for controlling operation of the units (not shown), and
a common output
(not shown) allowing comparative evaluation of the operation of the units 4009
relative to one
another. As demonstrated by comparison of the two exemplary multl-channel
embodiments 1001, 4001, the nature of the integrated platform and the sharing
of features
between or among multiple flow cytometry units in a multi-channel system can
be varied
extensively without departing from the scope of the present invention.
Impact of Multi-Channel Processing on Overall Process
The overall process described above can be performed with multi-channel sperm
sorting
to decrease the time required to sort the sperm cells. With few exceptions,
the method does not
change. One minor change is that sorted sperm cells will be collected in
multiple collection
vessels. The contents of the multiple collection vessels can be combined for
concentration If
desired. Alternatively, the contents of each collection vessel can be
concentrated separately. It

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will be appreciated that the time required to sort a batch of sperm cells
(e.g., an ejaculate) from
collection to completion of the cryopreservation step can be significantly
reduced by using
multiple cytometry units to process the batch. For example, if four cytometry
units operate in
parallel to process the batch of sperm cells, the time required to complete
sorting is reduced to
approximately one quarter of the time required to sort the batch using a
single cytometry unit.
Thus, by substituting the step of sorting sperm with four cytometry units
operating in parallel with
the step of sorting sperm with a single cytometry unit, the exemplary timeline
for completion of
the method from collection to completion of the freezing step can be reduced.
The time can be
reduced even further by increasing the number of cytometers operating in
parallel to sort the
sperm cells in the sample, subject to the practical limitations involved In
operating a parallel
system having more than four such units. Thus, according to one embodiment of
the present
invention, the sorting step in the overall process described above is
performed by sorting the
sperm cells according to a specified DNA characteristic in a multi-channel
flow cytometry
apparatus. In yet another embodiment, a sperm processing method comprises the
step of
sorting sperm cells according to a specified DNA characteristic In a multi-
channel flow cytometry
apparatus In which each channel collects in the range of about 2,000 - 10,000
sperm cells having
a desired DNA characteristic per second.
Mufti-Channel Sorting Example!
Bull semen was collected from a sexually mature bull using an artificial
vagina and the
sample transported to a nearby staining facility in a temperature-controlled
container at 37 C.
Upon receipt, the semen was analyzed for concentration, visual motility,
motility and progressive
motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard
and well known
procedures (Farrell et al. Theriogenology, 49(4): 871-9 (Mar 1998)).
Six tubes of 1 mt.. of 150 X 106 spermimL sperm suspension were prepared by
suspending an aliquot of semen in 41 C TCA #2 buffer containing 10mM pyruvate
bringing the
overall pH to 7.36. Then varying amounts of 10 mM Hoechst 33342 solution in
water were
added to the sperm samples to yield final dye concentrations of 200, 300, 400,
500, 600, & 700
M Hoechst 33342. Each of the six samples was incubated at 41 C for
approximately 30
minutes. The samples were analyzed by flow cytometry and the %CV of the X cell
population
was estimated by iterative computer algorithm for the 200, 300, and 400 .(.M
Hoechst 33342
samples. The %CV for the 300 and 200 ;.i.M Hoechst 33342 were both ascertained
to be within
the acceptable range near 1.3 %CV. Accordingly, it was determined that a
concentration of 250
M Hoechst 33342 would be used to stain a batch of sperm cells for further
processing.
Two tubes containing 2mL each of 150 X 106 sperm/mt.. sperm suspension were
prepared by suspending an aliquot of semen in 41 C TCA #2 buffer containing
10mM pyruvate
(again bringing the overall pH to 7.35). Then 10 mM Hoechst 33342 solution in
water was added
to each of the two sperm suspensions to yield a final dye concentration of 250
M Hoechst
33342. The sperm suspensions were maintained in a 41 C water bath for 30 min.
After 30
=

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minutes, the sperm suspensions were removed from the 41 C water bath and 4 L
of 25mg/mL
FD&C #40 was added to one of the suspensions. The other was stored at ambient
temperature
to provide comparison samples for the assessment assays.
The stained and quenched sperm suspension was loaded onto the sample port of
one
channel of a four channel droplet sorting flow cytometer. Delbecco's PBS was
used as the
sheath fluid. The cytometer was equipped with a orienting nozzle as described
above and
having a 60 micron orifice. An semicircular baffle plate was installed
perpendicular to the
longitudinal axis of the nozzle as described above. The transducer was
operated at 54 KHz and
the droplet break-off location was controlled manually. An epi-illumination
optics system as
described above was used to direct approximately 25% of the beam of a
continuous wave laser
to intersect the fluid stream at a perpendicular angle. The focusing and
collection lens had a
0.65 numerical aperture. The beam was focused to a spot having a width less
than 3 pm for slit
scanning the sperm cells. Digital signal processing was used to extract the
critical slope
difference and pulse area for each detected pulse waveform. Classification
parameters for
classification of X cells, Y cells, and undetermined cells in the two-
dimensional CSD and pulse
area feature space were manually entered Into the processing system for
classifying sperm cells
according to chromosome content.
Sperm were sorting according to X and Y chromosome content using a coincidence

accept sort strategy for collection of X cells, assigning a 50/50 probability
that each unclassified
sperm was an X cell or Y cell. The sample fluid rate was manually adjusted to
maintain purity of
collected X cell population (as indicated by the GUI) at 85% or better and to
maintain the rate of -
X cell collection above a minimum rate. After approximately fifteen million X
sperm had been
collected in a tube that had been soaked in sheath fluid for at least one hour
and then coated
with 0.5mL of 10% egg yolk in TCA #2 buffer at pH 7.0, the tube was removed
and replaced with
an additional tube that has been similarly prepared.
Immediately after removing a collection tube from the flow cytometer, a
comparison
sample from the stained, but not sorted, sperm suspension was prepared. The
sorted and
comparison samples were centrifuged for 7 min @ 750g in a 15 mL tube. The
supernatants
were removed using a transfer pipette to yield a concentration of
approximately 40 million
sperm/mL. TCA #2 buffer pH 7.0 was added to the sperm suspensions to yield a
final
concentration of approximately 20 million sperm/mL. This process continued
until the flow
cytometer had produced four collection tubes (A2-A5). The sorted samples and
'non-sorted'
comparison samples were assessed by IVOS. Sorted sample A3 and its non-sorted
comparison
sample were tested for % Intact acrosomes by differential interference
contrast microscopy. All
the sorted samples were counted by hemacytometer to determine the output rate
of sorted
sperm per hour. The % X chromosome bearing sperm was confirmed by flow
cytometer
reanalysis. Results of the IVOS assessment for the sorted and 'non-sorted'
comparison samples
are provided in Figs. 121 (motility) and 122 (progressive motility). The total
number of sperm
sorted Into each collection tube is shown in Fig. 123. The rate of sperm
sorted per hour for each
collection period Is shown in Fig. 124. Percentage of X chromosome bearing
sperm for each

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sorted sample Is listed In Fig. 125. Results of the assessment of the acrosome
Integrity were
72% intact acrosomes for the sorted sample and 78% for the non-sorted
comparison sample.
The results demonstrate the technical ability to yield more than 6,000 sorted
X cells per
second at greater than 85% purity per channel of multi-channel flow cytometry
system for
sustained periods, The results also show the technical ability to yield more
than 7,000 X cells
per second at greater than 85% purity for sustained periods under ideal
conditions. Further, the
results indicate that samples of sorted sperm cells obtained by such high-
speed flow cytometric
sorting will suffer only slight declines in motility, indicating that the
sorted sperm will have good
fertility.
Multi-channel Sorting Example
Bull semen was collected from a sexually mature bull using an artificial
vagina. The
ejaculate was split into two aliquots. The first aliquot of 250pL of semen was
suspended in 5mL
of 37 C Triladyl . The second aliquot, which comprised the remained of the
ejaculate, was
suspended in two parts 37 C carbonate buffer (pH 6.1-6.2). Both aliquots were
transported at
37 C in a temperature-controlled container to a processing facility. At the
processing facility, the
first aliquot was floated in ¨120mL of 37 C water in a 200mL beaker and placed
In a cold room to
slowly cool to 5 C. The second aliquot was analyzed for concentration,
motility and progressive
motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard
and well known
procedures (Ferrell et al. Theriogenology, 49(4): 871-9 (Mar 1998)).
Three 1 mL tubes of 150 X 10e sperm/mL sperm suspension were prepared by
transferring sub-aliquots containing 150 million sperm from the second aliquot
to empty tubes,
centrifuging at 500g for 5 Min, removing the supernatants, and re-suspending
the sperm pellets
in 1mL of 28 C TCA ##2 buffer containing 10mM pyruvate pH 7.35. Ten mM Hoechst
33342
soiution in water was added to each of the three tubes in various amounts to
yield final dye
concentrations of 100, 150, & 200 uM Hoechst 33342. Each of the three tubes
was held at 28 C
for approximately 60 minutes. Sperm from each of the three tubes was analyzed
by flow
cytometry and the CV of total fluorescence intensity of the X population was
determined for the
100, 150, and 200 LM Hoechst 33342 staining conditions using an interactive
computer
algorithm. The CVs for the 150 and 200 uM Hoechst 33342 were both within the
acceptable
range near 1.3%. Thus, it was determined to use staining conditions including
150 1.I.M Hoechst
33342 concentration for sorting.
One tube containing 5mL of 150 X 106 sperm/mL sperm suspension was prepared by
transferring a sub-aliquot containing 750 million sperm from the second
aliquot, centrifuging at
500g for 5 min, removing the supernatant, and re-suspending the sperm pellet
In 28 C TCA #2
buffer containing 10mM pyruvate (pH 7.35). Ten mM Hoechst 33342 solution in
water was
added to the tube in an amount yielding a final dye concentration of 150uM
Hoechst 33342. The
tube was maintained in a 28 C water bath for 60 min. After 60 minutes, the
tube was removed
from the 28 C water bath and 10 L of 25mg/rnL FD&C #40 was added.

CA 02752247 2011-09-07
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The now stained and quenched sperm suspension was loaded onto the sample port
of
one channel of a multi-channel droplet sorting flow cytometer system. The
sperm suspension
was maintained at 28 C. Using substantially the same instrument settings as
set forth In Mull/-
channel Example I, X & Y chromosome bearing sperm were separated by the flow
cytometry
After the sorted samples reached 5 C, the three tubes of sorted sperm were
combined
into one tube. The pooled sample was analyzed by IVOS to determine the %
motility,
% progressive motility, and concentration. Additional Triladyl cryo-
preservation media
The pooled sorted sample and the first aliquot were loaded into standard
0.25cc straws
35 Thawed straws were analyzed by IVOS for % motility and % progressive
motility after
incubation at 37 C for 30 and 120 minutes. Results from a set of two sorted
and unsorted straws
are summarized in Fig. 127 and Fig. 128.

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Multi-channel Sorting Example III
Bull semen was collected from a sexually mature bull using an artificial
vagina and the
ejaculate split Into two aliquots. A first aliquot of 250,uL of semen was
suspended in 5mL of 37 C
Trilady10. A second aliquot, which comprised the remainder of the ejaculate,
was suspended In
two parts 37 C carbonate buffer (two parts 0.097 moles/L of NaHCO3, 0.173
moles/L of KHCO3,
0.090 moles/L C311807+120 in water) (pH 6.1-6.2). Both aliquots were
transported at 37 C in a
temperature-controlled container to the processing facility. At the processing
facility, the first
aliquot was floated in ¨120mL of 37 C water in a 200mL beaker and placed In a
cold ronm to
slowly cool to 5 C. The second aliquot was analyzed for concentration,
motility and progressive
motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard
and well known
procedures (Farrell et al. Theriogenology, 49(4): 871-9 (Mar 1998)).
Two tubes of 150 X 106 sperm/mL sperm suspension were prepared by transferring
into
each of two empty tubes a fraction containing 900 million sperm from the
second aliquot,
centrifuging each tube at 500 X g for 5 minutes, removing the supernatant from
each tube, and
re-suspending each sperm pellet in 6mL of 28 C TCA #2 buffer containing 10mM
pyruvate (pH
7.35). 10mM Hoechst 33342 solution in water was added to each of the two tubes
to yield final
dye concentrations of 2001.LM Hoechst 33342 in one tube and 400 M Hoechst
33342 In the other
tube. Each of the two tubes was held at 28 C for approximately 120 minutes.
Sperm from each
of the tubes was analyzed by flow cytometry and the CV of total fluorescence
intensity of the X
population was determined for the 200t.EM and 400RM Hoechst 33342 staining
conditions using
an interactive computer algorithm. The CVs for the 200 M and 400p.M Hoechst
33342 were both
within the acceptable range of about 1.3%. The sperm suspension stained with a
concentration
of 200p,M Hoechst 33342 was chosen for sorting. 104 of 25mg/mL FD&C #40 was
added to
this tube of stained sperm suspension just prior to sorting.
The stained sperm suspension was loaded onto the sample port of one channel of
a
multi-channel droplet sorting flow cytometer system. The sperm suspension was
maintained at
28 C. Using substantially the same instrument settings as set forth in Multi-
channel Example l, X
& Y chromosome bearing sperm were separated by the flow cytometry system using
a
coincidence abort sort strategy for a period of time necessary to place an
enriched X
chromosome bearing cell population of approximately eighteen million sperm
into a collection
tube that had been prepared by soaking with sheath buffer for at least one
hour and then adding
0.5mL of Triladyl cryo-preservation media (pH 6.6). The sperm cells were
introduced into the
flow cytometry system at a rate of between about 25,000 and 30,000
cells/second. An enriched
population of X chromosome bearing cells was collected at a rate varying from
4,500 per second
to 6,000 per second. When approximately eighteen million sperm had been sorted
into a
collection tube, the tube was removed and replaced with another tube that had
been similarly
prepared. Immediately after removal of a collection tube from the flow
cytometer, the sorted
sperm suspension was centrifuged for 7 min @ 700 X g. The supernatant was
removed using a
transfer pipette to yield a concentration of approximately 100 million
sperm/mL. Triladyl cryo-
.

CA 02752247 2011-09-07
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preservation media (pH 6.6) was added to the sperm suspensions to yield a
final concentration of
approximately 50 million sperm/mL. This process continued until the flow
cytometer had
produced two collection tubes (C1-C3). Approximately 35 million sperm were
sorted in 193
minutes yielding an overall collection rate of 11 million enriched X
chromosome bearing cells per
hour of sorting. The re-suspended sorted sample tubes were floated in ¨120mL
of 28 C water In
a 200mL beaker and placed in a 5 C cold room to slowly cool.
After the sorted samples reached 5 C, the three tubes of sorted sperm were
combined
Into one tube. The pooled sample was analyzed by IVOS to determine the %
motility, %
progressive motility and concentration. Additional Triladyl cryo-preservation
media (pH 6.6)
was added to the sample to yield a final concentration of approximately 50
million sperm per mL.
The % X-chromosome bearing sperm in the sorted pooled sample was 88% as
determined by
flow cytometer re-analysis. A summary of the 1VOS assessment compared to the
non-sorted
sample of the same ejaculate is Illustrated in rig. 129.
The pooled sorted sample and unsorted sample (Le., the first aliquot from
above) were
loaded Into standard 0.25cc straws In the 5 C cold room. The loaded straws
were transferred to
a programmable freezer and frozen by the following program: 5 min @ 5 C, cool
from 5 C to -
12 C @ 4 C/min, cool from -12 C to -100 C @ 40 C/min, cool from -100 C to -140
C @
C/min, hold at -140 C. After the straws had reached -140 C, they were quickly
removed from
the freezer and plunged into liquid nitrogen.
20 Thawed straws were analyzed by IVOS for % motility and % progressive
motility after
Incubation at 37 C for 30 and 120 minutes. Results from a set of sorted and
unsorted straws are
summarized in Fig. 130 and Fig. 131.
Multi-channel Sorting Example IV
Bull semen was collected from a sexually mature bull using an artificial
vagina and the
ejaculate split into two aliquots. The first aliquot of 250,uL of semen was
suspended in 5 mL of
37 C Triladyl". The second aliquot, which comprised the remained of the
ejaculate, was
suspended In two parts 37 C carbonate buffer (two parts 0.097 moles& of
NaHCO3, 0.173
moles/L of KHCO3, 0.090 moles/L CoH807.H20 in water)(pH 6.1-6.2) and held
under CO2. Both
aliquots were transported at 37 C in a temperature-controlled container to the
processing facility.
At the processing facility, the first aliquot was floated In ¨120mL of 37 C
water In a 200mL
beaker and placed in the cold room to slowly cool to 5 C. The second aliquot
was analyzed for
concentration, motility and progressive motility by the Hamilton-Thorn
Motility Analyzer (IVOS),
according to standard and well known procedures (Farrell et al.
Therlogenology, 49(4): 871-9
(Mar 1998)).
A 5 mL tube of 150 X 10 sperm/mL sperm suspension was prepared by
transferring a
fraction containing 750 million sperm from the second aliquot (pH 6.1-6.2) to
an empty tube and
adding 28 C carbonate buffer (pH 7.35) to a final volume of 5 ml, To this
sperm suspension, 10
mM Hoechst 33342 solution in water was added to yield a final dye
concentration 1501.tM
Hoechst 33342. The suspension was held at 41 C under CO2 for approximately 40
minutes and

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then placed at 28 C for sorting. Ten j.tL of 25mg/ml_ FD&C #40 was added to
the tube of stained
sperm suspension just prior to sorting.
The stained sperm suspension was loaded onto the sample port of one channel of
a
multi-channel droplet sorting flow cytometer system. The sperm suspension was
maintained at
28 C. X & Y chromosome bearing sperm were separated by the flow cytometry
using a
coincidence abort sort strategy for a time period necessary to place an
enriched X chromosome
bearing cell population of approximately eighteen rnillion sperm Into a
collection tube that had
been prepared by soaking with sheath buffer for at least one hour and then
adding 0.5mL of
Triladyl cryo-preservation media (pH 6.6). The sperm cells were introduced
into the flow
cytometry system at a rate of between about 25,000 and 30,000 cells/second. An
enriched
population of X chromosome bearing cells was collected at a rate varying from
4,500 per second
to 6,000 per second. When approximately eighteen million sperm had been sorted
into a
collection tube, the tube was removed and replaced with another tube that has
been similarly
prepared. Immediately after removal of a collection tube from the flow
cytometer, the sorted
sperm suspension was centrifuged for 7 min @ 700 X g. The supernatant was
removed using a
transfer .pipette to yield a concentration of approximately 100 million
sperm/mL. Triladyl cryo-
preservation media and pyruvate (pH 6.6) was added to the sperm suspensions to
yield a final
concentration of approximately 50 million sperm/mL. This process continued
until the flow
cytometer had produced two collection tubes (C2-C3). The re-suspended sorted
sample tubes
were floated in ¨120mL of 28 C water in a 200mL beaker and placed in a 5 C
cold room to
slowly cool.
After the sorted samples reached 5 C, the two tubes of sorted sperm were
combined into
one tube. The pooled sample was analyzed by IVOS to determine the % motility,
% progressive
motility and concentration. Additional Triladyl cryo-preservation media and
pyruvate (pH 6.6)
was added to the sample to yield a final concentration of approximately 50
million sperm per mL.
A summary of the IVOS assessment compared to the non-sorted sample of the same
ejaculate is
illustrated in Fig. 132.
The pooled sorted sample and unsorted sample (Le., the first aliquot from
above) were
loaded into standard 0.25cc straws in the 5 C cold room. The loaded straws
were transferred to
a programmable freezer and frozen by the following program: 5 min @ 5 C, cool
from 5 C to -
12 C @ 4 C/min, cool from -12 C to -100 C @ 40 C/min, cool from -100 C to -140
C
20 C/min, hold at -140 C. After the straws had reached -140 C, they were
quickly removed from
the freezer and plunged into liquid nitrogen.
Thawed straws were analyzed by IVOS for % motility and % progressive motility
immediately after thawing and after incubation at 37 C for 30 minutes. Results
from a set of
sorted and unsorted straws are summarized in Fig. 133 and Fig. 134.

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CAPILLARY TUBE NOZZLE SYSTEM
Fig. 135 illustrates an alternative nozzle system, generally designated 1335,
similar to
that described above except that a capillary tube 1337(of quartz or fused
silica, for example) is
connected to the nozzle 137 so that Mild exiting the nozzle orifice 103 is
directed into and
through the tube. The optics system 109 of the flow cytometer is optically
coupled to the side of
the tube in a suitable manner, as by a chamber 1341 filled with a light-
transmitting medium such
as oil or gel having a known index of refraction. The use of a capillary tube,
compared to the
jetting of the fluid stream through open space, has the benefit of reducing
the lensing of the
stream 21 due to the acoustical energy supplied by the transducer 105, and
enabling the
focusing lens 1343 to be positioned immediately adjacent the fluid stream for
increasing
resolution of the emission signals.
After the particles have been interrogated and classified, they may be sorted
using any
conventional techniques known to those skilled in the art, as by use of a
fluid switching device
shown in Fig. 137 or other suitable devices such as photo-damage systems or
droplet sorting
systems.
SORTING TECHNIQUES OTHER THAN DROPLET SORTING
Photo-damage Sorting
The flow cytometry improvements of this invention are applicable not only to
droplet cell
sorting as described above, but also to other sorting techniques, such as
sorting by photo-
damage (laser ablation). Photodamaue sorting is discussed in United States
Patent No.
4,395,397. Fig 136 schematically
illustrates one embodiment of a single-channel flow cytometry photo-damage
system, generally
designated by the reference number
As shown in Fig. 136, the photo-damage sorting system is similar to the
droplet sorting
system of Fig. 2, and corresponding parts are designated by corresponding
reference numbers
with the addition of a double prime ("). In general, the system comprises the
same components
as the system of Fig. 2, except that the droplet sorting components are
eliminated (e.g., the
transducer 105, the charging device 627, the deflector plates 629, and
associated power sources
635). Instead these components are replaced by a laser or similar device which
is responsive to
instructions received from the microprocessor 131" to ablate undesired
particles In the fluid
stream 21". As a result, the stream collected in a collection receptacle 1365
contains a desired
population of particles. For example, if the particles being analyzed are
sperm cells and the
intended result is to collect sperm cells having a characteristic A (e.g., a
desired chromosome
content), then the microprocessor receives signals from the epi-illumination
system 415" which
identifies cells not having characteristic A and selectively activates the
laser to ablate such cells
or otherwise render them ineffective.
Different control sorting strategies can be employed in a photo-damage system,

including the "high recovery" and "high purity" sorting strategies discussed
above in the context
of a droplet sorter. In a photo-damage system, particles contained in the
fluid stream are spaced
=

CA 02752247 2013-11-21
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at various intervals along the stream and generally follow one after another
in single file. The
particles have different characteristics, some having a characteristic A, for
example, and others
having a characteristic 13. The sequence of particles is random, so viewed as
a continuous
procession, the particles can be divided into different particle series, one
following another,
including a first particle series consisting only of one or more particles
having characteristic A, a
second particle series consisting only of one or more particles having
characteristic B and a third
particle series consisting of two or more closely spaced particles at least
one of which has
characteristic A and at least one of which has characteristic B. The latter
(third) group generally
corresponds to the closely spaced particles in a "coincident" droplet
discussed previously, at
least for sorting strategy purposes. Thus, the two or more particles in the
third group may be
closely spaced in the sense that the spatial separation between the particles
Is insufficient to
allow accurate discrimination/classification of the particles, or because such
separation Is
insufficient to permit one particle in the series to be ablated by the laser
without damaging the
=
other particle(s) In the same series. In any event, the closely spaced
particles in each (or at least
some) of the third series of particles can be ablated or not ablated,
depending on the sorting
strategy employed. It should be noted that multiple particles in a first
series or multiple particles
in a second series could be "closely spaced", but since the particles in any
such series have the
same characteristic (A or B), they are treated as a single-particle series, at
least for sorting
strategy purposes.
The photo-damage system can be a single-channel system or a multi-channel
system,
as described above.
Fluid Switching Sorting
It is contemplated that the principles of this invention can also be applied
to flow
cytometry systems using fluid switching techniques, as disclosed, for example,
in U.S. Patents
Nos, 6,432,246 (Adair), 4,756,427 (GOhde, et al.), and 3,791, 517 (Friedman).
Fig. 137 is a partial view showing such a =
system, generally designated 1357. It is substantially identical to the system
shown in Fig. 2
except that the nozzle system 101" Includes a capillary tube 1369 (e.g., see
Fig_ 135), and the
sorting system comprises a fluid-switching device 1359 coupled to the
capillary tube 1369
downstream from the interrogation location 115'. The construction and
operation of the fluid-
switching device can incorporate any conventional fluid switching technology
such as disclosed
in the above-referenced patents. In general, the device functions to sort
desired particles from
undesired particles in response to instructions received from the processor by
intermittently
diverting portions of the fluid stream containing the desired/undesired
particles along separate
flow paths 1361, 1365 for collection in vessels or the like. The switching is
commonly achieved
by selectively actuating a transducer 1367 in one of the flow paths.
The various sorting strategies described above in regard to droplet sorting
and photo-
damage sorting can also be employed in a fluid-switching system. In the fluid-
switching system,
particles contained in the fluid stream are also spaced at various intervals
along the stream and

CA 02752247 2011-09-07
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generally follow one after another in single file. The particles have
different characteristics, some
having a characteristic A, for example, and others having a characteristic B,
and the sequence of
particles is random. Therefore, as discussed above in regard to the photo-
damage system, the
procession of particles can be divided into different particle series, one
following another,
including a first particle series comprising one or more particles having
characteristic A, a second
particle series comprising one or more particles having characteristic B and a
third particle series
comprising two or more closely spaced particles at least one of which has
characteristic A and at
least one of which has characteristic B. The latter (third) group generally
corresponds to the
closely spaced particles in a "coincident" droplet discussed previously, at
least for sorting
strategy purposes. Thus, the two or more particles in the third group may be
closely spaced in
the sense that the spatial separation between the particles is insufficient to
allow accurate
discrimination/classification of the particles, or because such separation is
insufficient to permit
one particle in the series to be diverted by the fluid-switching device
separate from the another
particle in the same series. In any event, the closely spaced particles in
each (or at least some)
of the third series of particles can be diverted to one collection location or
another, depending on
the sorting strategy employed. As explained above in connection with photo-
damage sorting,
multiple particles in a first series or multiple particles in a second series
could be "closely
spaced", but since the particles in any such series have the same
characteristic (A or B), they
are treated as a single-particle series for the purpose of sorting strategy.
The fluid switching system can be a single-channel system or a multi-channel
system, as
described above.
Droplet Interference Sorting
it is also contemplated that the technology of this invention can be used in
conjunction
with a droplet interference fluidic switching technique. For example, a high-
speed droplet
interference sorting system 1371, shown schematically in Fig. 138, may be used
to sort particles
by diverting selected segments of the coaxial carrier and sheath fluid stream.
In contrast to some other sorting techniques, the droplet interference sorting
technique
does not require the coaxial carrier and sheath stream to be formed into
droplets. Thus, there is
no need to couple the nozzle system 101- used for delivery of the carrier and
sheath fluids with
a droplet generation system. By way of example only, passing the carrier and
sheath fluids
through a nozzle system at 60 psi to create a 50 micron diameter stream is one
suitable
arrangement for formation of a laminar coaxiai fluid stream for delivery of
particles to the droplet
interference sorting system. Particles in the coaxial fluid stream are
analyzed and classified by
the optics system 109' and processor 131" as they move through the
interrogation location
115-, as has been described above for the other sorting systems. Sorting
occurs downstream
from the interrogation location, at a location where the coaxial fluid stream
intersects a high-
speed droplet interference stream.
The droplet interference stream is generated by a droplet generation system
1375 similar
to the droplet generation system used for droplet sorting. A high-speed fluid
stream 1379 passes
. .

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through a high-speed nozzle system 1377 that is coupled to a piezoelectric
transducer 1361 or
other source of acoustical energy for causing the high-speed fluid stream to
break into droplets
1383 downstream from the high-speed nozzle. For exaMple, a particle-free fluid
at 1500 psi may
be passed through the high-speed nozzle to form a 70 micron diameter high-
speed fluid jet. The
high-speed nozzle may be oscillated at 400 KHz to form high-speed droplets.
The high-speed
droplets 1383 pass through an electric field generated by one or more electric
deflection plates
1387 so that the path of the high-speed droplets may be controlled by
selectively applying an
electric charge to the droplets, as was done to control the path of droplets
in the droplet sorting
system. The high-speed droplet interference stream is directed so some high-
speed droplets
intersect the coaxial fluid stream at a point 1399 downstream from the
interrogation location. For
example, uncharged droplets 1389 may be directed to collide with the fluid
stream while charged
droplets 1391 are deflected away from the coaxial fluid stream. When a high-
speed droplet
collides with the coaxial fluid stream, a segment 1397 of the fluid stream and
any particles
contained therein are diverted from the path they would have otherwise taken.
The application of
a charge or no charge to a high-speed droplet may be timed so the arrival of
that droplet at the
intersection 1399 with the coaxial fluid stream coincides with the arrival of
a particular segment of
the coaxial fluid stream. Thus, by selectively charging high-speed droplets
depending on the
classification of particles contained within the coaxial stream segments, one
can sort particles by
diverting all coaxial fluid stream segments that contain one or more selected
particles and not
diverting other coaxial stream segments or vice-versa. Collection capillaries
1403 having a slight
vacuum may be used to collect both the diverted 1397 and undiverted coaxial
stream segments.
The droplet interference sorting system may be set up so the high-speed
droplets merge with
diverted coaxial stream segments or so the high-speed droplets remain separate
from the
diverted stream segments after collision with the coaxial stream segments.
Because there are no particles or cells in the high-speed droplet interference
stream, it is
possible to use very high pressures and very high droplet frequencies without
damaging the
particles or cells to be sorted. This allows sorting of stream segments each
having less volume
(e.g., four times less volume) than the volume of a droplet in the droplet
sorting system. This
greatly increases the maximum throughput of the system while also reducing the
dilution factor of
the sorted particles. Moreover, because finely filtered liquid with no cells
or particles is used to
form the droplet interference stream, more consistent droplet formation is
possible because the
droplet formation nozzle is less likely to become clogged or suffer from
protein buildup than the
nozzle system used in the droplet sorting system. Another advantage is that
the distance
between particle analysis at the interrogation location and the sorting point
1399 can be reduced
(e.g., by a factor of four), allowing more accurate prediction of the time of
arrival of a particular
particle at the sorting point. Furthermore, the droplet interference system
allows more flexibility
in adjustment of nozzle size or pressure for the coaxial fluid stream. If
desired, the droplet
interference sorting system can be combined with the capillary tube nozzle
system. A multi-
channel droplet interference sorting system may use a high-pressure fluidic
pump to supply
multiple droplet interference stream generating nozzles with fluid from a
common fluid supply.

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When introducing elements of the present invention or the embodiment(s)
thereof, the
articles "a," "an," "the," and "said" are intended to mean that there are one
or more of the
elements. The terms "comprising," "including," and "having" are intended to be
inclusive and
mean that there may be additional elements other than the listed elements. The
term "or" is
intended to include "and/or" and is intended to mean "one or another or both."
Thus, an
Indication of "ABC or DEF" means (1) ABC, or (2) DEF, or (3) both ABC and DEF.
The term
"and/or" is intended to have the same meaning as "or" as defined above. Thus,
the term "and/or"
Is intended to include "or" and is intended to mean "one or another or both."
For example, an
Indication of "ABC and/or DEF" means (1) ABC, or (2) DEF, or (3) both ABC and
DEF.
In view of the above, it will be seen that the several objects of the
invention are achieved
and other advantageous results attained.
As various changes could be made in the above constructions, products, and
methods it is
intended that all matter contained in the above description and shown in the
accompanying
drawings shall be interpreted as illustrative of preferred embodiments.
Comments on Inventive Features
Those skilled in the art will recognize that the Invention described above
includes many
inventive aspects, including at least the following:
A. Multi-Channel Sorting Apparatus
A1. A Multi-channel system for sorting particles according to one or
more
characteristics of the particles, said system comprising:
multiple flow cytometry units each of which is operable to sort a desired
population of
particles in a mixture of particles by interrogating a stream of fluid
containing said particles using
a beam of electromagnetic radiation,
said units sharing an integrated platform comprising at least one of the
following
elements: (1) a common supply of particles; (2) a common source of
electromagnetic radiation;
(3) a common housing; (4) a common input for controlling operation of the
units; (5) a common
processor for receiving and processing information from the units; and (6) a
common fluid
delivery system for delivering fluid containing said particles to said flow
cytometry units.
A2, The system of A1 wherein said particles are cells.
A3, The system of A1 wherein said particles are sperm cells.

CA 02752247 2011-09-07
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A4. The system of A1 wherein said system comprises at least element (2), and
wherein
one or more of said multiple flow cytometry units comprises a jet-in-air
droplet sorting flow
cytometry unit.
A5. The system of A1 wherein said integrated platform comprises at least
elements (2)
and (3).
A6. The system of A1 wherein said integrated platform comprises at least
elements (4)
and (5).
A7. The system of A1 wherein said integrated platform comprises at least
element (2),
said common source comprising a single laser beam.
A8. The system of A7 further comprising a beam splitting system for splitting
the single
laser beam into multiple beams and directing the multiple beams into optics
systems of
respective flow cytometry units.
A9. The system of A8 wherein said single laser beam comprises a plurality of
pulses,
each pulse having a peak power that is greater than the average power output
of the laser.
A10. The system of A1 wherein said integrated platform comprises at least
element (3),
said flow cytometry units comprising interchangeable modules removably mounted
in the
housing.
A11. The system of A1 wherein each flow cytometry unit comprises an epi-
illumination
optics system for interrogating a respective fluid stream.
Al2. The system of A1 further comprising a collection system for collecting
said desired
population of particles from each unit.
A13. The system of A1 wherein said integrated platform comprises at least
element (5),
and wherein said common output comprises an Indication of the fluorescence
intensity measured
by each Unit.
A14. The system of A1 wherein said integrated platform comprises at least
element (5),
and wherein said common output comprises an indication of the rate at which
each unit is
separating particles.
A15. The system of A1 wherein said integrated platform comprises at least
element (5),
and wherein said common output comprises an indication of particle staining
variations.

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A16. The system of A1 wherein said integrated platform comprises at least
elernent (6),
and wherein said common output comprises an indication of a decision boundary
used by each
unit for discriminating between particles.
A17. The system of A1 wherein each of said flow cytometry units comprises a
droplet
sorting system.
=
A18. The system of A1 wherein said integrated platform comprises at least
element (5),
and wherein said common output comprises an indication of a droplet break-off
location of each
unit.
A19. The system of A1 wherein at least one of said flow cytometry units
comprises a
photo-damage system.
A20. The system of A1 wherein at least one of said flow cytometry units
comprises a
fluid-switching sorting system.
A21. The system of A1 wherein said flow cytometry units are adapted to operate
in
parallel.
A22. The system of A1 wherein the integrated platform comprises at least a
shared
laser operable to emit a plurality of electromagnetic radiation pulses,
wherein each pulse has a
peak power exceeding the average power of the laser, and wherein one or more
of said flow
cytometry units comprises:
a flow channel for directing a fluid stream containing sample particles
through a particle
interrogation location;
a beam guidance system operable to direct a portion of the electromagnetic
radiation in
a pulse to the Interrogation location;
a timing circuit operable to produce a timing signal indicative of the arrival
of
electromagnetic radiation at the interrogation location;
a detector adapted to detect electromagnetic radiation from the interrogation
location and
operable to output a time-varying analog signal indicative of the intensity of
the detected
electromagnetic radiation;

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an analog to digital converter adapted to receive the time-varying analog
signal as input
and to sample the analog signal to produce a digitized output; and
an electronic processor operable to analyze the digitized output from the
analog to digital
converter as a function of the timing signal.
A23. The system of A1 wherein the multiple flow cytometry units comprise three
or more
flow cytometry units.
A24. The system of A1 wherein the multiple flow cytometry units comprise at
least
twelve flow cytometry units.
A25. The system of claim A1, wherein the integrated platform comprises at
least
element (6), and wherein the common processor performs at least one of: (1)
receiving and
processing said Information in real time; and (2) receiving and processing
said information to
permit evaluation of the operation of one unit relative to another unit.
A26. The system of A1 wherein each flow cytometry unit comprises a sensor
operable
to generate a time-varying output signal indicative of at least one
characteristic of the particles,
wherein said integrated platform comprises at least element (5) and said
information received by
the common processor comprises the output signals from the respective sensors,
and wherein
the processor Is operable to receive the output signals as a substantially
continuous stream and
to substantially continously process the output signals in real time.
A27. The system of Al wherein said Integrated platform comprises a common
processor
operable to send control signals to the flow cytometry units in real time
during a sorting process
to adjust their operation as a function of said information received by the
common processor, and
wherein the flow cytometry units are responsive to the control signals.
B. Multi-Channel Sorting Method
Bl. A multi-channel method of sorting particles according to one or more
characteristics
of the particles, said method comprising:
providing a plurality of flow cytornetry units;
operating said flow cytometry units to conduct a plurality of flow cytometry
operations,
said operations comprising forming separate fluid streams each containing a
mixture of particles,
and sorting desired populations of particles In said mixtures of particles by
interrogating the
streams using beams of electromagnetic radiation; and

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sharing at least one of the following elements while conducting said
operations: (1) a
common supply of particles for said streams; (2) a common source of
electromagnetic radiation
for saki beams; (3) a common operations control input; (4) a common processor
for receiving
and processing information from the units; (5) a common system for delivering
fluid to said
streams; and (6) a common housing for said flow cytometry units.
B2. The method of B1 wherein said particles are cells.
B3. The method of B1 wherein said particles are sperm cells.
B4. The method of B1 wherein at least one of said multiple flow cytometry
units
comprises a jet-In-air droplet sorting flow cytometry unit.
B5. The method of B1 further comprising at least sharing said common source of
electromagnetic radiation in the form of a single laser beam, said method
further comprising
splitting the single laser beam Into multiple beams and directing the multiple
beams into optics
systems of respective flow cytometry units.
B6. The method of B5 further comprising reflecting a percentage of beam light
of the
single beam toward the optics system of one of said flow cytometry units, and
passing a
percentage of beam light of the single beam for transmission to the optics
system of another of
said flow cytometry units.
B7. The method of B6 further comprising using a solid state laser to form said
single
laser beam.
B8. The method of B7 further comprising mode-locking the solid state laser so
that the
single laser beam comprises a plurality of pulses, wherein each pulse has a
peak power that is
B9. The method of B1 further comprising sharing at least element (6), and
wherein said
method further cornprises removably mounting said flow cytometry units in the
common housing.
B10. The method of B1 further comprising at least sharing said common source
of
electromagnetic radiation in the form of a shared laser; the method further
comprising the steps
of:
emitting a plurality of electromagnetic radiation (EMR) pulses from a laser,
wherein the

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directing each pulse into a beam splitting and guidance system to
intermittently illuminate
each fluid stream and the particles contained therein by directing a portion
of the energy in the
pulses along a beam path from the laser to each interrogation location;
detecting EMR from at least one interrogation location;
generating a time-varying analog signal indicative of the Intensity of the
detected EMR
from said interrogation location;
generating a timing signal Indicative of the arrival of a pulse at said
interrogation location;
converting the time-varying analog signal into a digital signal; and
analyzing the=digital signal to determine characteristics of the particles in
the fluid stream
flowing through the respective Interrogation location.
B11. The method of B1 further comprising using a first sorting strategy in a
first
operation of said operations and a second sorting strategy different from the
first sorting strategy
in a second operation of said operations.
B12. The method of B1 further comprising collecting a population of desired
particles
sorted by each flow cytometry unit, and combining the population collected
from one unit with a
population collected from a different unit to produce a blended population of
desired particles.
B13. The method of B1 further comprising varying the rate at which fluid is
delivered to
one or more of the flow cytometry units as a function of at least one of the
following: (1) the purity
of a first sorted population; and (2) the quantity of desired particles In a
second population.
1314. The method of B1 further comprising conducting said flow cytometry
operations in
parallel.
B15. The method of B1 wherein the sharing step comprises sharing at least
element (4),
the method further comprising using the common processor to do at least one of
the following:
(1) receive and process said information In real time; and (2) receive and
process said
information to permit evaluation of the operation of one unit relative to
another unit.
B16. The method of 131 wherein the sharing step comprises sharing at least
element (4),
the method further comprising using a sensor for each respective cytometry
unit to generate a
time-varying output signal Indicative of at least one characteristic of the
particles and using the

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common processor to receive the respective output signals as a substantially
continous stream
and to process the output signals in real time.
817. The method of B1 wherein the sharing step comprises sharing at least
element (4),
the method further comprising sending a control signal to one or more of the
flow cytometry units
in real time during a sorting process to adjust the unit's operation as a
function of the information
received by the common processor.
C. [Reserved]
D. Multi-channel Analyzer
D1. A Multi-channel system for classifying particles according to one or more
characteristics of the particles, said system comprising:
a plurality of flow cytometry units each of which is operable to classify
particles in a
mixture of particles by interrogating a stream of fluid containing said
particles using a beam of
electromagnetic radiation,
said units sharing an integrated platform comprising at least one of the
following elements: (1) a
common supply of particles; (2) a common housing; (3) a common input for
controlling operation
of the units; (4) a common processor for receiving and processing information
from the units; and
(5) a common fluid delivery system for delivering fluid containing said
particles to said flow
cytometry units.
D2. The system of D1 wherein said integrated platform further comprises a
common
source of electromagnetic radiation.
D3. The system of D1 wherein said particles are cells.
D4. The system of p,1 wherein said particles are sperm cells.
D5. The system of D1 wherein said integrated platform comprises at least
elements (3)
and (4).
D6. The system of D5 wherein said integrated platform further comprises a
common
source of electromagnetic radiation.

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D7. The system of D1 wherein said integrated platform further comprises a
common
source of electromagnetic radiation, said common source comprising a single
laser beam.
D8. The system of D7 further comprising a beam splitting system for splitting
the single
laser beam into multiple beams and directing the multiple beams into optics
systems of
respective flow cytometry units.
09. The system of D1 wherein said integrated platform comprises at least
element (2),
said flow cytometry units comprising interchangeable modules removably mounted
in the
housing.
D10. The system of D1 wherein each flow cytometry unit comprises an epi-
Illumination
optics system for interrogating a respective fluid stream.
D11. The system of D1 wherein said integrated platform comprises at least
element (4),
and wherein said processor is operable to output an Indication of the
fluorescence intensity
measured by each unit.
012. The system of D1 wherein said integrated platform comprises at least
element (4),
and wherein said processor is operable to output an indication of the rate at
which each unit is
separating particles.
D13. The system of D1 wherein said integrated platform comprises at least
element (4),
and wherein said processor is operable to output an indication of particle
staining variations.
D14, The system of D1 wherein said integrated platform comprises at least
element (4),
and wherein said processor is operable to output an indication of a decision
boundary used by
each unit for discriminating between particles.
015. The system of D1 wherein said flow cytometry units are adapted to operate
in
parallel.
D16. The system of D1 wherein said plurality of flow cytometry units are
operable to sort
the particles.
D17. The system of D16 wherein the integrated platform further comprises a
common
source of electromagnetic radiation, and wherein said plurality of flow
cytometry units comprises
a jet-in¨air droplet sorting flow cytometry unit.

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018. The system of D1 wherein said integrated platform comprises at least
element (4),
and wherein the common processor is operable to perform at least one of the
following: (1)
receive and process said information In real time; and (2) receive and process
said information to
permit evaluation of the operation of one unit relative to another unit.
D19. The system of claim D1 wherein each flow cytometry unit comprises a
sensor
operable to generate a time-varying output signal indicative of at least one
characteristic of the
particles, wherein said integrated platform comprises at least element (4) and
said information
received by the common processor comprises outpt the signals from the
respective sensors, and
wherein the processor Is operable to receive the output signals as a
substantially continous
stream and to process the output signals in real time.
020. The system of 01 wherein said integrated platform comprises a common
processor operable to send control signals to the flow cytornetry units In
real time during a sorting
process to adjust their operation as a function of said information received
by the common
processor, and wherein the flow cytometry units are responsive to the control
signals.
E. Multl-Channel Analyzing Method
E1. A multi-channel method of classifying particles according to one or more
characteristics of the particles, said method comprising:
providing a plurality of flow cytometry units;
operating said flow cytometry units to conduct a plurality of flow cytometry
operations,
said operations comprising forming separate fluid streams each containing a
mixture of particles,
and classifying particles in said mixtures of particles by interrogating the
streams using beams of
electromagnetic radiation; and
sharing at least one of the following elements to conduct said operations: (1)
a common
supply of particles for said streams; (2) a common operations control input;
(3) a common
processor for receiving and processing information from the units; (4) a
common system for
delivering fluid to said streams; and (5) a common housing for said flow
cytometry units.
E2. The method of E1 further comprising sharing a common source of
electromagnetic
radiation for said beams.
E3. The method of E2 wherein said plurality of flow cytometry units comprises
a jet-In-air
droplet sorting flow cytometry unit.

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E4. The method of El further comprising operating said plurality of flow
cytometers to
sort said mixture of particles based on their classification.
E5. The method of El wherein said particles are cells.
E6. The method of El wherein said particles are sperm cells.
E7. The method of El further comprising sharing a common source of
electromagnetic
radiation for said beams in the form of a single laser beam, splitting the
single laser beam into
multiple beams, and directing the multiple beams into optics systems of
respective flow
cytometry units.
E8. The method of E7 wherein the sharing step comprises at least element (5),
the
method further comprising guiding said single laser beam into said common
housing prior to
splitting the beam.
E9. The method of E7 further comprising reflecting a percentage of beam light
of the
single beam toward the optics system of one of said flow cytometry units, and
passing a
percentage of beam light of the single beam for transmission to the optics
system of another of
said flow cytometry units.
E1O. The method of El wherein the sharing step comprises at least element (5),
the
method further comprising removably mounting said flow cytornetry units in the
common housing.
El 1. The method of El comprising operating each flow cytometry unit to
interrogate a
respective fluid stream using an epl-illumination optics system.
E12. The method of El further comprising operating said flow cytometry units
In parallel.
E13. The method of El wherein said plurality of flow cytometry units comprise
twelve or
more flow cytometry units.
E14. The method of El wherein the sharing step comprises sharing at least
element (4),
the method further comprising using the common processor to perform at least
one of the
following: (1) receive and process said information in real time; and (2)
receive and process said
information to permit evaluation of the operation of one unit relative to
another unit.
El 5. The method of El wherein the sharing step comprises sharing at least
element (4),
the method further comprising using a sensor for each respective cytometry
unit to generate a
time-varying output signal indicative of at least one characteristic of the
particles and using the

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common processor to receive the respective output signals as a substantially
continous stream
and to process the output signals in real time.
E16. The method of El wherein the sharing step comprises sharing at least
element (4),
the method further comprising sending a control signal to one or more of the
flow cytometry units
in real time during a sorting process to adjust the unit's operation as a
function of the information
received by the common processor.
F. [Reserved]
G. Method of Splitting Single Laser for Multi-Channel Cytometry
G1. A method of sorting particles using a system comprising a three or more
flow
cytometry units each of which Is operable to sort a desired population of
particles from a mixture
of particles by Interrogating a stream of fluid containing said particles
using a beam of light, said
method comprising:
generating a single laser beam;
splitting the single beam into three or more light beams and directing the
light beams into
optics systems of the flow cytometry units; and
operating the flow cytometry units to sort particles.
G2. The method of G1 wherein each cytornetry unit interrogates the fluid
stream with a
light beam of about the same Intensity.
G3. The method of G1 wherein each unit requires a light beam having a power of
W
watts, and wherein said method further comprises generating said single laser
beam having a
power of (W x N) L, where N equals the number of flow cytometry units and L
equals power
loss in the system.
04. The method of 01 further comprising balancing the amount of beam light
used by
the cytometry units to interrogate respeCtive fluid streams by using one or
more filters to
attenuate the intensity of at least one of said three or more light beams.
05. The method of G4 further comprising adjusting the intensity of beam light
entering
respective units so that each unit receives the same amount of beam light
within a tolerance of
10%.

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G6. The method of G1 wherein at least one of said flow cytometry units uses a
droplet
sorting process to sort said particles.
07. The method of G1 wherein at least one of said flow cytometry units uses a
photo-
damage process to sort said particles.
G8. The method of G1 wherein at least one of said flow cytometry units uses a
fluid-
switching sorting process to sort said particles.
G9. The method of G1 wherein said flow cytometry units are mounted in a common
housing, the method further comprising guiding said single laser beam into
said common housing
prior to splitting the beam.
G10. The method of G9 further comprising reflecting a percentage of beam light
of the
single laser beam toward the optics system of one of said flow cytometry units
and passing a
percentage of beam light of the single laser beam for transmission to the
optics system of
another of said flow cytometry units.
G11. The method of G1 wherein the step of splitting a single beam comprises
splitting a
single beam Into four separate beams.
G12. The method of G11 wherein the splitting step comprises using a 50/60
beamsplitter to split the single beam into two beams, using a second 50/50
beamsplitter to split
one of the two beams Into two additional beams, and using a third 50/50
beamsplitter to split the
other of the two beams into two more additional beams.
H. Apparatus for Sorting Using Sort Strategy
H1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic 13, said
system comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
a control responsive to Information received from the flow cytometry apparatus
for
controlling the sorting system to vary Its sorting strategy or for controlling
the fluid delivery
system to vary the rate at which fluid is delivered as a function of at least
one of the following: (1)
the purity of said at least one population with respect to either
characteristic A particles or

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characteristic B particles; and (2) the quantity of characteristic A particles
or characteristic B
particles in said at least one population relative to the total quantity of
characteristic A particles or
characteristic B particles in said stream.
H2. The system of H1 wherein the particles are cells and characteristics A and
B relate
to physical characteristics of the cells.
H3. The system of H1 wherein the particles are sperm cells, and wherein
characteristic
A is indicative of a live X sperm cell.
H4. The system of H3 wherein characteristic B Is indicative of other than a
live X cell
(¨X).
H5. The system of H4 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
H6. The system of H1 wherein the control increases the rate of fluid delivery
when said
purity is greater than a desired purity and decreases the rate of fluid
delivery when said purity Is
less than said desired purity.
H7. The system of H1 wherein the control determines the purity of said at
least one
population based on output signals from the flow cytometry apparatus, said
system further
comprising an operator input to the control for indicating a desired purity,
and wherein the control
varies the rate of fluid delivery so that the purity corresponds to the
desired purity.
H8. The system of H7 wherein the control increases the rate of fluid delivery
when the
quantity of characteristic A particles in said at least one population is
greater than an acceptable
quantity of characteristic A particles in the at least one population, and
wherein the control
decreases the rate of fluid delivery when the quantity of characteristic A
particles in said at least
one population is less than said acceptable quantity.
H9. The system of H8 wherein the control determines the quantity of
characteristic A
particles in said at least one population based on output signals from the
flow cytometry
apparatus, said system further comprising an operator input to the control for
indicating an
acceptable quantity of characteristic A particles in said at least one
population, and wherein the
control varies the fluid delivery rate to obtain said acceptable quantity in
said at least one
population.
H10. The system of H1 wherein said stream is formed to contain particles
following
generally one after another in a series which comprises sequential sets of
particles, including first

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particle sets each comprising one or more particles having characteristic A,
second particle sets
each comprising one or more particles having characteristic B, and third
particle sets each
comprising two or more closely spaced particles at least one of which has
characteristic A and at
least one of which has characteristic B.
H11. The system of H10 wherein said sorting system is operable to use a
sorting
strategy in which said first particle sets are selected for collection in said
at least one population
and said second and third particle sets are not selected for collection In
said at least one
population.
H12. The system of FI10 wherein said sorting system is operable to use a
sorting
strategy in which said first and third particle sets are selected for
collection in said at least one
popUlation and said second particle set is not selected for collection In said
at least one
population.
H13. The system of H1 wherein said sorting system comprises a droplet sorting
system.
H14. The system of H1 wherein said sorting system comprises a photo-damage
sorting
system.
H15. The system of Fli wherein said sorting system comprises a fluid-switching
sorting
system.
l. Method for Sorting Particles Using Sorting StrategV
11. A method of using a flow cytometry system to sort a mixture of particles
including
particles having a characteristic A and particles having a characteristic B,
said method
comprising:
delivering a fluid containing said particles;
forming the fluid into a stream and using flow cytometry to classify the
particles in the
stream according to said characteristics;
sorting the particles in the stream according to said classification and
according to a
sorting strategy thereby to provide at least one population containing desired
particles; and
varying the sorting strategy or varying the rate at which fluid is delivered
as a function of
at least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A

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particles or characteristic B particles in said at least one population
relative to the total quantity of
characteristic A particles or characteristic B particles in said stream.
12. The method of II wherein the particles are cells and characteristics A and
B relate to
physical characteristics of the cells.
13. The method of 11 wherein said particles are sperm cells, and wherein
characteristic A
Is indicative of a live X sperm cell.
14, The method of 13 wherein characteristic B is indicative of other than a
live X cell (-X).
15. The method of 13 further comprising maintaining said purity at more than
85% but
less than 95%.
16. The method of 11 further comprising increasing the rate of fluid delivery
when said
purity is greater than a desired purity and decreasing the rate of fluid
delivery when said purity is
less than the desired purity.
17. The method of 11 further comprising Increasing the rate of fluid delivery
when the=
quantity of characteristic A particles In said at least one population is
greater than an acceptable
quantity of characteristic A particles in the at least one population, and
decreasing the rate of
fluid delivery when the quantity of characteristic A particles in said at
least one population is less
than said acceptable quantity.
18. The method of 11 further comprising forming the stream to contain
particles following
generally one after another in a series which comprises sequential sets of
particles, including first
particle sets each comprising one or more particles having characteristic A,
second particle sets
each comprising one or more particles having characteristic B, and third
particle sets each
comprising two or more closely spaced particles at least one of which has
characteristic A and at
least one of which has characteristic B.
19. The method of 18 further comprising sorting said particles according to a
sorting
strategy in which only first particles sets are selected for collection in
said at least one population.
110. The method of 18 further comprising sorting said particles according to a
sorting
strategy in which first and third particle sets are selected for collection in
said at least one
population.
11 I. The method of 11 wherein said sorting comprises using a droplet sorting
process.

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112. The method of 11 wherein said sorting comprises using a photo-damage
sorting
process.
113. The method of 11 wherein said sorting comprises using a fluid-switching
sorting
process.
J. Droplet Sorter Including Sort Strategy
J1. A system for sorting a mixture of particles including particles having a
characteristic
A and particles having a characteristic 13, said system comprising:
a fluid delivery system for delivering a fluid stream containing said
particles;
a flow cytometry apparatus for receiving said stream, forming droplets
containing said
particles, and sorting said droplets into different populations according to a
sorting strategy, said
droplets including first droplets each containing one or more particles having
characteristic A,
second droplets each containing one or more particles having characteristic B
and third droplets
each containing one or more particles having characteristic A and one or more
particles having
characteristic B; and
a control responsive to information received from the flow cytometry apparatus
for
controlling the flow cytometry apparatus to vary the sorting strategy or for
controlling the fluid
delivery system to vary the rate at which fluid is delivered as a function of
at least one of the
following: (1) the purity of at least one droplet population with respect to
either characteristic A
particles or characteristic B particles; and (2) the quantity of
characteristic A particles or
characteristic B particles in at least one droplet population relative to the
total quantity of
characteristic A particles or characteristic B particles in said stream.
J2. The systern of J1 wherein the control controls the fluid delivery system
to maintain
the rate at which fluid is delivered as substantially constant, and wherein
the control varies the
sort strategy.
J3. The system of J2 wherein the control varies the sort strategy in order to
vary the
percentage of third droplets in one of the droplet populations.
J4. The system of The system of J1 wherein the control controls the fluid
delivery
system to vary the rate at which fluid is delivered as a function of the
purity of said at least one
droplet population.
J5. The system of J4 wherein said purity is at least 85% and not more than
95%.

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J6. The system of J1 wherein the control controls the fluid delivery system to
vary the
rate at which fluid is delivered as a function of the quantity of
characteristic A particles in said at
least one droplet population relative to the total quantity of characteristic
A particles in said
stream.
J7. The system of J6 wherein the rate at which the fluid is deliVered is
varied so that the
quantity of characteristic A particles in said at least one droplet population
represents at least
about 60% of the total quantity of characteristic A particles in said stream.
J8. The system of J1 wherein said particles are sperm cells, and wherein
characteristic
A Is Indicative of a live X sperm cell.
J9. The system of J1 wherein the ratio of characteristic A particles to
characteristic B
particles in said mixture is a known ratio, and wherein said control is
operable classify some of
the particles as having characteristic A or characteristic B and to vary the
fluid delivery rate as a
function of the ratio of classified particles to the known ratio.
J10. The system of J1 wherein the control determines a purity of the sorted
particles
based on output signals from the flow cytometry apparatus, said system further
comprising an
operator input to the control for Indicating a desired purity, and wherein the
control varies the rate
so that the purity of said at least one droplet population generally
corresponds to the desired
purity.
K. Droplet Sorting Method Including Sort Strategy
K1. A method of sorting a mixture of particles including particles having a
characteristic
, A and particles having a characteristic B, said system comprising:
delivering a fluid stream containing said particles;
forming droplets containing said particles;
sorting said droplets into different populations according to a sorting
strategy, said
droplets including first droplets each containing one or more particles having
characteristic A,
second droplets each containing one or more particles having characteristic B
and third droplets
each containing one or more particles having characteristic A and one or more
particles having
characteristic B; and
varying the sorting strategy or varying the rate at which fluid is delivered
as a function of
at least one of the following: (1) the purity of at least one droplet
population with respect to either

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characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A
particles or characteristic 8 particles in at least one droplet population
relative to the total quantity
of characteristic A particles or characteristic B particles in said stream.
K2. The method of K1 further comprising maintaining the rate at which fluid is
delivered
as substantially constant, and varying the sorting strategy.
K3. The method of K2 further comprising varying the sorting strategy in order
to vary the
percentage of third droplets in one of the droplet populations.
K4. The method of K1 further comprising varying the rate at which fluid is
delivered as a
function of the purity of at least one of the droplet populations.
K5. The method of K4 further comprising maintaining said purity in the range
of 85% to
95%.
K6. The method of K1 further comprising varying the rate at which fluid is
delivered as a
function of the quantity of characteristic A particles in said at least one
droplet population relative
to the total quantity of characteristic A particles in said stream.
K7. The method of K6 wherein the rate at which the fluid is delivered is
varied so that
the quantity of characteristic A particles In said at least one of the droplet
populations represents
at least about 60% of the total quantity of characteristic A particles in said
stream.
K8. The method of K1 wherein said particles are sperm cells, and wherein
characteristic
A is indicative of a live X sperm cell.
K9. The method of K1 further comprising increasing the rate when the purity of
said at
least one droplet population Is greater than the desired purity and decreasing
the rate when the
purity of said at least one droplet population is less than the desired
purity.
L. Variable Flow Rate Droplet Sorter Having Feedback LOOP
1.1. A system for sorting a mixture of particles including particles having a
characteristic
A and particles having a characteristic 13, said system comprising:
a variable rate fluid delivery system for delivering a fluid stream containing
said particles;
a flow cytometry apparatus for receiving said stream, forming droplets
containing said
particles, and sorting said droplets into different populations according to a
sorting strategy, said

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droplets including first droplets each containing one or more particles having
characteristic A,
second droplets each containing one or more particles having characteristic B
and third droplets
each containing one or more particles having characteristic A and one or more
particles having
characteristic B; and
a control responsive to information received from the flow cytometry apparatus
for
controlling the fluid delivery system to vary the rate at which fluid is
delivered from the fluid
delivery system as a function of at least one of the following: (1) the purity
of at least one of the
droplet populations with respect to either characteristic A particles or
characteristic B particles;
and (2) the quantity of characteristic A particles or characteristic B
particles in at least one droplet
population relative to the total quantity of characteristic A particles or
characteristic B particles In
said stream.
L2. The system of Li wherein the control controls the fluid delivery system to
vary the
rate at which fluid Is delivered as a function of the purity of at least one
of the droplet populations.
L3. The system of L2 wherein the purity is at least 85% and not more than 95%.
L4. The system of Li wherein the control controls the fluid delivery system to
vary the
rate at which fluid is delivered as a function of the quantity or percentage
of characteristic A
particles in at least one droplet population relative to the total quantity of
characteristic A particles
in said stream.
L5. The system of L4 wherein the rate at which the fluid is delivered is
varied so that the
quantity of characteristic A particles in said at least one of the droplet
populations represents at
least about 60% of the total quantity of characteristic A particles in said
stream.
L6. The system of Li wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
L7. The system of Li wherein said cells are sperm cells, and wherein
characteristic A Is
indicative of a live X sperm cell.
L8. The system of L7 wherein at least some of the droplets In said at least
one droplet
population contain at least one live X cell and at least one Y cell in the
same droplet, and wherein
said purity of said at least one droplet population is measured by X/(X+Y).
L9. The system of L8 wherein said control is operable to vary the rate of
fluid delivery to
maintain said purity at more than 85% but less than 95%.

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L10. The system of L7 wherein characteristic B is indicative of cells which
are not live X
cells.
L11. The system of L7 wherein said control Is operable to vary the rate of
fluid delivery
so that percentage of live X cells in said at least one droplet population is
at least 60% of the total
number of live X cells In said first, second and third pluralities of
droplets.
L12. The system of L11 wherein the purity of said at least one droplet
population is
maintained at less than 95%.
L13. The system of L1 wherein the ratio of characteristic A particles to
characteristic B
particles in said mixture is a known ratio, and wherein said control is
operable to classify some of
the particles has having characteristic A or characteristic B and vary the
fluid delivery rate as a
function of the ratio of the classified particles to the known ratio.
L14. The system of L1 wherein the particles having characteristic A are live X
sperm
cells and the particles having characteristic B are sperm cells which are not
live X sperm cells,
and wherein said control is operable to vary the fluid delivery rate as a
function of the ratio of
classified sperm cells to 50%.
L15. The system of L1 wherein the control increases the rate when the purity
of said at
least one droplet population is greater than a desired purity and decreases
the rate when the
purity of said at least one droplet population Is less than the desired
purity.
L16. The system of L1 wherein the control determines a purity of the sorted
particles
based on output signals from the flow cytometry apparatus, said system further
comprising an
operator input to the control for indicating a desired purity, and wherein the
control varies the rate
so that the purity of said at least one droplet population generally
corresponds to the desired
purity.
NI. Droplet Sorting Method Using Variable Flow Rate and Feedback
M1. A method of sorting a mixture of particles including particles having a
characteristic
A and particles having a characteristic B, said system comprising:
delivering a fluid stream containing said particles;
forming droplets containing said particles;

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sorting said droplets into different populations according to a sorting
strategy, said
droplets including first droplets each containing one or more particles having
characteristic A,
second droplets each containing one or more particles having characteristic B
and third droplets
each containing one or rnore particles having characteristic A and particles
having characteristic
B; and
varying the rate at which fluid is delivered as a function of at least one of
the following:
(1) the purity of at least one of the droplet populations with respect to
either characteristic A
particles or characteristic B particles; arid (2) the quantity of
characteristic A particles or
characteristic B particles in at least one droplet population relative to the
total quantity of
=
characteristic A particles or characteristic B particles in said stream.
M2. The method of M1 further comprising varying the rate at which fluid is
delivered as a
function of the purity of at least one of the droplet populations.
M3. The method of M2 wherein said purity is at least 85% and not more than
95%.
M4. The method of M1 further comprising varying the rate at which fluid is
delivered as a
function of the quantity of characteristic A particles in at least one droplet
population relative to
the total quantity of characteristic A particles In said stream.
M5. The method of M4 further comprising varying the fluid delivery rate so
that the
quantity of characteristic A particles in said at least one of the droplet
populations represents at
least about 60% of the total quantity of characteristic A particles in said
stream.
M6. The method of M1 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
M7. The method of M1 wherein said cells are sperm cells, and wherein
characteristic A
Is indicative of a live X sperm cell.
M8. The method of M7 wherein at least some of said droplets in said at least
one of the
droplet populations contain at least one live X cell and at least one Y cell
In the same droplet,
and wherein said purity of said at least one droplet population Is measured by
X/(X+Y).
M9. The method of M8 further comprising varying the rate of fluid delivery to
maintain
said purity at more than 85% but less than 95%.
M10. The method of M7 wherein characteristic B is indicative of cells which
are not live
X cells.

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M11. The method of M7 further comprising varying the rate of fluid delivery so
that
percentage of live X cells In said at least one droplet population is at least
60% of the total
number of live X cells in said first, second and third pluralities of
droplets.
M12. The method of M11 further comprising maintaining the purity of said at
least one
droplet population at less than 95%,
M13. The method of M1 wherein the ratio of characteristic A particles to
characteristic B
particles in said mixture is a known ratio, and said method further comprises
varying the fluid
delivery rate as a function of the ratio of classified particles to the known
ratio.
M14. The method of M1 wherein the particles having characteristic A are live X
sperm
cells and particles having characteristic B are not live X sperm cells, said
method further
comprising varying the fluid delivery rate as a function of the ratio of
classified sperm cells to
50%.
M15. The method of M1 further comprising increasing the rate when the purity
of said at
least one droplet population is greater than the desired purity and decreasing
the rate when the
purity of said at least one droplet population is less than the desired
purity.
N. Sperm Sorting System for High Purity Sort Strategy
= N1, A system for sorting X and Y sperm cells, said system comprising:
a variable rate fluid delivery system for delivering a fluid stream containing
X and Y
sperm cells;
a flow cytometry apparatus for (1) receiving said stream and forming droplets
containing
said particles, said droplets comprising first droplets each containing one or
more X sperm cells,
second droplets each containing one or more Y sperm cells, and third plurality
of droplets each
containing one or more X sperms cells and one or more Y sperm cells, (2)
sorting said first
droplets from said second and third droplets, (3) collecting said first
droplets to provide at least
one population of X sperm cells, and (4) identifying a quantity of X sperm
cells In the at least one
population; and
a control responsive to instructions received from the flow cytometer
apparatus for
varying the rate at which the fluid is delivered to the flow cytometer
apparatus as a function of the
quantity of identified X sperm cells In said at least one population relative
to the total number of X
cells in said first, second and third droplets.

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M11. The method of M7 further comprising varying the rate of fluid delivery so
that
percentage of live X cells in said at least one droplet population is at least
60% of the total
number of live X cells in said first, second and third pluralities of
droplets.
M12. The method of M11 further comprising maintaining the purity of said at
least one
droplet population at less than 95%.
M13. The method of M1 wherein the ratio of characteristic A particles to
characteristic B
particles in said mixture is a known ratio, and said method further comprises
varying the fluid
delivery rate as a function of the ratio of classified particles to the known
ratio.
M14. The method of M1 wherein the particles having characteristic A are live X
sperm
cells and particles having characteristic B are not live X sperm cells, said
method further
comprising varying the fluid delivery rate as a function of the ratio of
classified sperm cells to
50%.
M15. The method of M1 further comprising increasing the rate when the purity
of said at
least one droplet population is greater than the desired purity and decreasing
the rate when the
purity of said at least one droplet population is less than the desired
purity.
N. Sperm Sorting System for High Purity Sort Strategy
= N1. A system for sorting X and Y sperm cells, said system comprising:
a variable rate fluid delivery system for delivering a fluid stream containing
X and Y
sperm cells;
a flow cytometry apparatus for (1) receiving said stream and forming droplets
containing
said particles, said droplets comprising first droplets each containing one or
more X sperm cells,
second droplets each containing one or more Y sperm cells, and third plurality
of droplets each
containing one or more X sperms cells and one or more Y sperm cells, (2)
sorting said first
droplets from said second and third droplets, (3) collecting said first
droplets to provide at least
one population of X sperm cells, and (4) identifying a quantity of X sperm
cells in the at least one
population; and
a control responsive to instructions received from the flow cytometer
apparatus for
varying the rate at which the fluid is delivered to the flow cytometer
apparatus as a function of the
quantity of identified X sperm cells in said at least one population relative
to the total number of X
cells in said first, second and third droplets.

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N2. The system of N1 wherein said flow cytometry apparatus is operable to
identify the
number of X cells collected In said at least one population, and wherein the
control varies the
rate at which fluid Is delivered to the flow cytometer apparatus to maintain
the quantity of X cells
collected in said at least one population at or above an acceptable quantity
relative to the total
number of X cells in said first, second and third droplets.
N3. The system of N2 wherein said acceptable quantity Is at least 60% of said
total
number of X cells.
N4. The system of N3 wherein said X cells are live X cells.
N5. The system of N2 wherein the control is operable to increase the rate of
fluid
delivery when the quantity of X cells in said at least one population is above
said acceptable
quantity and to decrease the rate of fluid delivery when the quantity of X
cells ih said at least one
population is below said acceptable quantity.
O. Sperm Sorting Method Including High-Purity Sort Strategy
01. A method of sorting X and Y sperm cells, said method comprising:
delivering a fluid stream containing X and Y sperm cells to a first location
and causing
said stream to break into droplets at a second location, said droplets
comprising first droplets
each containing one or more X sperm cells, second droplets each containing one
or more Y
sperm cells, and third plurality of droplets each containing one or more X
sperms cells and one or
more Y sperm cells;
sorting said first droplets from said second and third droplets;
collecting said first droplets to provide at least one population of X sperm
cells;
identifying a quantity of X sperm cells collected in said at least one
population; and
varying the rate at which fluid is delivered to said first location as a
function of the
quantity of identified,X sperm cells collected in said at least one population
relative to the total
number of X cells in said first, second and third droplets.
02. The method of 01 further comprising varying the rate at which fluid is
delivered to
said first location to maintain the quantity of X cells collected in said at
least one population at or
above an acceptable quantity relative to the total number of X cells in said
first, second and third
droplets.
=

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03. The method of 02 wherein said acceptable quantity is at least 60% of said
total
number of X cells.
04. The method of 03 wherein said X cells are live X cells.
05. The method of 01 further comprising increasing the rate of fluid delivery
when the
quantity or percentage of X cells collected in said at least one population is
above said
acceptable quantity and decreasing the rate of fluid delivery when the
quantity or percentage of X
cells collected in said at least one population is below said acceptable
quantity.
P. Sperm Sorter for High-Recovery Sort Strategy
P1. System for sorting X and Y sperm cells, said system comprising:
a variable rate fluid delivery system for delivering a fluid stream containing
X and Y
sperm cells;
a flow cytometer apparatus for (1) receiving said stream and forming droplets
containing
said particles, said droplets comprising first droplets each containing one or
more X sperm cells,
second droplets each containing one or more Y sperm cells, and third plurality
of droplets each
containing one or more X sperms cells and one or more Y sperm cells, (2)
sorting said first and
third droplets from said second droplets, (3) collecting said first and third
droplets to provide at
least one population of X sperm cells, and (4) identifying a quantity of Y
sperm cells in the at
least one population; and
a control responsive to instructions received from the flow cytometer
apparatus for
varying the rate at which the fluid is delivered to the flow cytometer
apparatus as a function of the
quantity of identified Y sperm cells in said at least one population.
P2. The system of P1 wherein said control varies the rate at which fluid is
delivered to
the flow cytometer system to maintain the purity of said at least one
population at or above a
desired purity.
P3. The system of P2 wherein said control is operable to increase the fluid
delivery rate
when the purity of said at least one population is greater than said desired
purity and to decrease
the fluid delivery rate when the purity is less than said desired purity.
P4. The system of P2 wherein said desired purity is no greater than 95%.
P5. The system of P2 wherein said X sperm cells are live cells.

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Q. Sperm Sorting Method Including High-Recovery Sort Strategy
Q1. A method of sorting X and Y sperm cells, said method comprising:
= delivering a fluid stream containing X and Y sperm cells to a first
location and causing
said stream to break into droplets at a second location, said droplets
comprising first droplets
each containing one or more X sperm cells, second droplets each containing one
or more Y
sperm cells, and third plurality of droplets each containing one or more X
sperms cells and one or
more Y sperm cells;
sorting said first and third droplets from said second droplets;
collecting said first and third droplets to provide at least one population of
X sperm cells;
identifying a quantity of Y sperm cells collected in said at least one
population; and
varying the rate at which fluid is delivered to said first location as a
function of the
quantity of identified Y sperm cells collected in said at least one
population.
Q2. The method of Q1 further comprising varying the rate at which fluid is
delivered to
maintain the purity of said at least one population with respect to X cells at
or above a desired
purity,
Q3. The method of Q2 further comprising increasing the fluid delivery rate
when the
purity of said at least one population is greater than said desired purity and
decreasing the fluid
delivery rate when the purity is less than said desired purity.
04. The method of Q2 wherein said desired purity is no greater than 95%.
Qb. The method of Q2 wherein said X sperm cells are live cells.
Q6. A method of sorting X and Y sperm cells using flow cytometry, said method
comprising:
delivering a fluid stream containing X and Y sperm cells to a first location
and causing
said stream to break into droplets at a second location, said droplets
comprising first droplets
each containing one or more X sperm cells, second droplets each containing one
or more Y
sperm cells, and third plurality of droplets each containing one or more X
sperms cells and one or
more Y sperm cells;
sorting said first and third droplets from said second droplets; and

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collecting said first and third droplets to provide at least one population of
X sperm cells.
07. The method of Q6 wherein said X sperm cells are live cells and not dead X
cells.
Q8. A method of sorting X arid Y sperm cells using flow cytometry, said method

comprising:
delivering a fluid stream containing X and Y sperm cells and causing said
stream to
break into droplets;
interrogating the fluid stream before it breaks into droplets to identify
which sperm cells
will reside in which droplets;
sorting droplets containing X sperm cells from droplets not containing X sperm
cells; and
collecting said droplets containing X sperm cells, including droplets
containing at least
one X sperm cell and at least one Y sperm cell.
Q9. The method of Q8 wherein said X sperm cells are live X cells and not dead
X cells.
R. Photodamage Sorting_ Having Sort StrateoV
R1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system comprising a laser for
ablating selected
particles in the stream according to said classification and according to a
sorting strategy to
provide at least one population containing desired particles, the Improvement
comprising:
a control responsive to information received from the flow cytometry apparatus
for
controlling the laser to vary its sorting strategy or for controlling the
fluid delivery to vary the rate
at which fluid is delivered as a function of at least one of the following:
(1) the purity of said at
least one population with respect to either characteristic A particles or
characteristic B particles;
and (2) the quantity of unablated characteristic A particles or unablated
characteristic B particles
in said at least one population relative to the total quantity of
characteristic A particles or
characteristic B particles in said stream.

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R2. The system of R1 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
R3. The system of R1 wherein saki cells are sperm cells, and wherein
characteristic A is
indicative of a live X sperm cell.
R4. The system of R3 wherein characteristic B Is indicative of other than a
live X cell
(¨X).
R5. The system of R1 wherein the control increases the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreases the rate of
fluid delivery when the purity is less than the desired purity.
R6. The system of R1 wherein the control determines said purity based on
output
signals from the flow cytometry apparatus, said system further comprising an
operator input to
the control for indicating a desired purity, and wherein the control varies
the rate of fluid delivery
to obtain the desired purity.
R7. The system of R1 wherein said stream is formed into a stream containing
particles
following generally one after another In a series which comprises sequential
sets of particles,
Including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets
each comprising two or more closely spaced particles at least one of which has
characteristic A
and at least one of which has characteristic B.
R8. The system of R7 wherein said laser ablates only the second particle sets.
R9. The system of R8 wherein said control maintains the purity of said at
least one
population at more than 85% but less than 95%.
R10. The system of R'7 wherein characteristic B is indicative of other than a
live X cell
(¨X) and said laser is operable to ablate the second particle sets and the
third particle sets in the
stream.
R11. The system of R10 wherein the control increases the rate of fluid
delivery when the
quantity of unablated characteristic A particles in said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles in the at least
one population, and
wherein the control decreases the rate of fluid delivery when the quantity of
unablated
characteristic A particles in said at least one population is less than said
acceptable quantity.

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R12. The system of R10 wherein the control determines the quantity of
unablated
characteristic A particles in said at least one population based on output
signals from the flow
cytometry apparatus, said system further comprising an operator input to the
control for
indicating an acceptable quantity of unablated characteristic A particles in
said at least one
population, and wherein the control varies the fluid delivery rate to obtain
the acceptable quantity
of unablated characteristic A particles In said at least one population.
S. Photodamage Sorting Method Including Sort Strategy
si. A method of using a flow cytometry system to sort a mixture of particles
including
particles having a characteristic A and particles having a characteristic B,
said method
comprising:
delivering a fluid containing said particles;
forming the fluid into a stream and using flow cytometry to classify the
particles In the
stream according to said characteristics;
sorting the particles In the stream by ablating selected particles according
to said
classification and according to a sorting strategy thereby to provide at least
one population
containing desired particles; and
varying the sorting strategy or varying the rate at which fluid is delivered
as a function of
at least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of unablated
characteristic A particles or unablated characteristic B particles in said at
least one population
relative to the total quantity of characteristic A particles or characteristic
B particles in said
stream.
S2. The method of 31 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
S3. The method of 31 wherein said cells are sperm cells, and wherein
characteristic A is
Indicative of a live X sperm cell.
S4. The method of S1 further comprising increasing the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreasing the rate of
fluld delivery when the purity is less than the desired purity.

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S6. The method of S1 further comprising forming said fluid into a stream
containing
particles following generally one after another in a series which comprises
sequential sets of
particles, including first particle sets each comprising one or more particles
having characteristic
A, second particle sets each comprising one or more particles having
characteristic B, and third
particle sets each comprising two or more closely spaced particles at least
one of which has
characteristic A and at least one of which has characteristic B.
S6. The method of S5 further comprising ablating the second particles sets and
not the
first and third particle sets.
S7. The method of S6 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
S8. The method of S5 further comprising ablating the second and third particle
sets and
not the first particle sets.
S9. The method of S8 further comprising increasing the rate of fluid delivery
when the
quantity of unablated characteristic A particles in said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles in the at least
one population, and
decreasing the rate of fluid delivery when the quantity of unablated
characteristic A particles in
said at least one population Is less than said acceptable quantity.
=
T. Photodamage Sorter Having Variable Flow Rate and Feedback
Tl. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a
variable rate fluid delivery system for delivering a fluid containing said
particles, a flow cytometry
apparatus for receiving said fluid, forming it into a stream and using flow
cytometry to classify the
particles according to said characteristics, and a sorting system comprising a
laser for ablating
selected particles in the stream according to said classification and
according to a sorting
strategy to provide at least one population containing desired particles, the
improvement
comprising:
a control responsive to information received from the flow cytometry apparatus
for
controlling the laser to vary the rate at which fluid Is delivered as a
function of at least one of the
following: (1) the purity of said at least one population with respect to
either characteristic A
particles or characteristic B particles; and (2) the quantity of unablated
characteristic A particles
or unablated characteristic B particles in said at least one population
relative to the total quantity
of characteristic A particles or characteristic B particles in said stream.

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T2. The system of T1 wherein the particles are cells and characteristics A and
B relate
to physical characteristics of the cells.
T3. The system of T2 wherein said cells are sperm cells and characteristic A
is
indicative of a live X sperm cell.
T4. The system of T3 wherein characteristic B is indicative of other than a
live X cell
(¨X) and said laser ablates only the second particle sets.
T5. The system of T4 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
T6. The system of T2 wherein characteristic B is indicative of other than a
live X cell
(¨X), and wherein said laser ablates the second and third particles sets.
T7. The system of T1 wherein the control increases the rate of fluid delivery
when the
purity of the at least one population is greater than the desired purity and
decreases the rate of
fluid delivery when the purity is less than the desired purity.
T8. The system of T1 wherein the control determines a purity of the at least
one
population based on output signals from the flow cytometry apparatus, said
system further
1. comprising an operator input to the control for indicating a desired
purity, and wherein the control
varies the rate of fluid delivery to obtain the desired purity.
T9. The system of T8 wherein the control increases the rate of fluid delivery
when the
quantity of characteristic A particles in said at least one population is
greater than an acceptable
quantity of characteristic A particles in said at least one population, and
wherein the control
decreases the rate of fluid delivery when the quantity of characteristic A
particles in the at least
one population is less than an acceptable quantity of characteristic A
particles in the at least one
population.
T10. The system of T8 wherein the control determines the total number of
characteristic
A particles In said at least one population based on output signals from the
flow cytometry
apparatus, said system further comprising an operator input to the control for
indicating an
acceptable quantity of characteristic A particles in the at least one
population, and wherein the
control varies the rate so that the quantity of characteristic A particles in
the at least one
population to obtain said acceptable quantity or percentage.
Ti 1. The system of T1 wherein said stream is formed into a stream containing
particles
following generally one after another in a series which comprises sequential
sets of particles,

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including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets
each comprising two or more closely spaced particles at least one of which has
characteristic A
and at least one of which has characteristic B.
T12. The system of T11 wherein characteristic A is indicative of a live X
sperm cell (X),
wherein characteristic B is indicative of other than a live X cell (¨X),
wherein the laser ablates the
second particles sets and third particle sets and not the first particle set,
and wherein the control
varies the rate at which the fluid is delivered to the flow cytometer
apparatus as a function of the
number of live X sperm cells in the at least one population.
T13. The system of T11 wherein characteristic A Is indicative of a live X
sperm cell (X),
wherein characteristic B is indicative of other than a live X cell (¨X),
wherein the laser ablates the
second particle sets and not the first and third particle sets, and wherein
the control varies the
rate at which the fluid is delivered to the flow cytometer system as a
function of the number of ¨X
sperm cells in said at least one population.
U. Photodamade Sorting Method Including Variable Flow Rate Sort
Strategy
U1.. A method of using a flow cytometry system to sort a mixture of particles
including =
particles having a characteristic A and particles having a characteristic B,
said method
comprising:
delivering a fluid containing said particles;
forming the fluid into a stream and using flow cytometry to classify the
particles in the
stream according to said characteristics;
sorting the particles in the stream by ablating selected particles according
to said
classification and according to a sorting strategy thereby to provide at least
one population
containing desired particles; anti
varying the rate at which fluid Is delivered as a function of at least one of
the following:
(1) the purity of said at least one population with respect to either
characteristic A particles or
characteristic B particles; and (2) the quantity of unablated characteristic A
particles or unablated
characteristic B particles in said at least one population relative to the
total quantity of
characteristic A particles or characteristic B particles in said stream.
U2. The method of Ul wherein the particles are cells and characteristics A and
B relate
to physical characteristics of the cells.

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U3. The method of U2 wherein said cells are sperm cells and characteristic A
is
indicative of a live X sperm cell.
U4. The method of U3 wherein characteristic B is indicative of other than a
live X cell
(¨X) and said laser ablates only the second particle sets.
U5. The method of U4 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
U6. The method of U2 wherein characteristic B is indicative of other than a
live X cell
(¨X), and wherein said laser ablates the second and third particles sets.
U7. The method of U1 wherein the control increases the rate of fluid delivery
when the
purity of the at least one population is greater than the desired purity and
decreases the rate of
fluid delivery when the purity Is less than the desired purity.
U8. The system of U1 wherein the control determines a purity of the at least
one
population based on output signals from the flow cytometry apparatus, said
system further
comprising an operator input to the control for indicating a desired purity,
and wherein the control
varies the rate of fluid delivery to obtain the desired purity.
U9. The system of U8 wherein the control increases the rate of fluid delivery
when the
quantity of characteristic A particles in said at least one population is
greater than an acceptable
quantity of characteristic A particles in said at least one population, and
wherein the control
decreases the rate of fluid delivery when the quantity of characteristic A
particles in the at least
one population is less than an acceptable quantity of characteristic A
particles in the at least one
population.
U10. The system of U8 wherein the control determines the total number of
characteristic
A particles in said at least one population based on output signals from the
flow cytometry
apparatus, said system further comprising an operator input to the control for
indicating an
acceptable quantity of characteristic A particles In the at least one
population, and wherein the
control varies the rate so that the quantity of characteristic A particles in
the at least one
population to obtain said acceptable quantity or percentage.
U11. The system of U1 wherein said stream is formed into a stream containing
particles
following generally one after another in a series which comprises sequential
sets of particles,
including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets

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each comprising two or more closely spaced particles at least one of which has
characteristic A
arid at least one of which has characteristic 13.
U12. The system of U11 wherein characteristic A is Indicative of a live X
sperm cell (X),
wherein characteristic B is indicative of other than a live X cell (-X),
wherein the laser ablates the
second particles sets and third particle sets and not the first particle set,
and wherein the control
varies the rate at which the fluid is delivered to the flow cytometer
apparatus as a function of the
number of live X sperm cells in the at least one population.
U13. The system of U11 wherein characteristic A is indicative of a live X
sperm cell (X),
wherein characteristic B is indicative of other than a live X cell (-X),
wherein the laser ablates the
second particle sets and not the first and third particle sets, and wherein
the control varies the
rate at which the fluid is delivered to the flow cytometer system as a
function of the number of -X
sperm cells in said at least one population.
V. Fluid Switching Particle Sorter Including Sort StrategV
V1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming It Into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a fluid switching sorting system for
sorting selected
particles in the stream according to said classification and according to a
sorting strategy to
provide at least one population containing desired particles, the improvement
comprising:
a control responsive to information received from the flow cytometry apparatus
for
controlling the fluid switching sorting system to vary its sorting strategy or
for controlling the fluid
delivery system to vary the rate at which fluid is delivered as a function of
at least one of the
following: (1) the purity of said at least one population with respect to
either characteristic A
particles or characteristic B particles; and (2) the quantity of
characteristic A particles or
characteristic 13 particles in said at least one population relative to the
total quantity of
characteristic A particles or characteristic B particles in said stream.
V2. The system of V1 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
V3. The system of V1 wherein said cells are sperm cells, and wherein
characteristic A is
indicative of a live X sperm cell.
V4. The system of V3 wherein characteristic B is indicative of other than a
live X cell
(-X).

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V5. The system of V1 wherein the control increases the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreases the rate of
fluid delivery when the purity is less than the desired purity.
V6. The system of V1 wherein the control determines said purity based on
output
signals from the flow cytometry apparatus, said system further comprising an
operator Input to
the control for Indicating a desired purity, and wherein the control varies
the rate of fluid delivery
to obtain the desired purity.
V7. The system of V1 wherein said stream is formed into a stream containing
particles
following generally one after another In a series which comprises sequential
sets of particles,
including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets
each comprising two or more closely spaced particles at least one of which has
characteristic A
and at least one of which has characteristic B.
V8. The system of V7 wherein said laser ablates only the second particle sets.
V9. The system of V8 wherein said control maintains the purity of said at
least one
population at more than 85% but less than 95%.
V10. The system of V7 wherein characteristic B is indicative of other than a
live X cell
(¨X) and said laser is operable to ablate the second particle sets and the
third particle sets in the
stream.
V11. The system of V10 wherein the control increases the rate of fluid
delivery when the
quantity of unablated characteristic A particles In said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles in the at least
one population, and
wherein the control decreases the rate of fluid delivery when the quantity of
unablated
characteristic A particles in said at least one population is less than said
acceptable quantity.
=
V12. The system of V10 wherein the control determines the quantity of
unablated
characteristic A particles in said at least one population based on output
signals from the flow
cytometry apparatus, said system further comprising an operator input to the
control for
indicating an acceptable quantity of unablated characteristic A particles in
said at least one
population, and wherein the control varies the fluid delivery rate to obtain
the acceptable quantity
of unablated characteristic A particles in said at least one population.

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W. Method of Fluid Switching Sorting Including Sort StrategV
VV1. A method of using a flow cytometry system to sort a mixture of particles
including
particles having a characteristic A and particles having a characteristic B,
said method
comprising;
delivering a fluid containing said particles;
forming the fluid into a stream and using flow cYtometry to classify the
particles in the
stream according to said characteristics;
sorting the particles in the stream by diverting selected particles in the
stream according
to said classification and according to a sorting strategy thereby to provide
at least one
population containing desired particles; and
varying the sorting strategy or varying the rate at which fluid is delivered
as a function of
at least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A
particles or characteristic B particles in said at least one population
relative to the total quantity of
characteristic A particles or characteristic B particles in said stream.
W2. The method of W1 wherein the particles are cells, and wherein
characteristics A
and B relate to physical characteristics of the cells.
W3. The method of W1 wherein said cells are sperm cells, and wherein
characteristic A
is indicative of a live X sperm cell.
W4. The method of W1 further comprising increasing the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreasing the rate of
fluid delivery when the purity is less than the desired purity.
W5. The method of W1 further comprising forming said fluid into a stream
containing
particles following generally one after another in a series which comprises
sequential sets of
particles, including first particle sets each comprising one or more particles
having characteristic
A, second particle sets each comprising one or more particles having
characteristic B, and third
particle sets each comprising two or more closely spaced particles at least
one of which has
characteristic A and at least one of which has characteristic B.
W6. The method of W5 further comprising ablating the second particles sets and
not the
first and third particle sets.

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W7. The method of W6 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
W8. The method of W5 further comprising ablating the second and third particle
sets
and not the first particle sets.
W9. The method of W8 further comprising Increasing the rate of fluid delivery
when the
quantity of unablated characteristic A particles in said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles in the at least
one population, and
decreasing the rate of fluid delivery when the quantity of unablated
characteristic A particles in
said at least one population is less than said acceptable quantity.
X. Fluld Switching Sorter Including Variable Flow Rate Sort Stratecw
X1. in a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a fluid switching sorting system for
sorting selected
particles in the stream according to said classification to provide at least
one population
containing desired particles, the improvement comprising:
a control responsive to information received from the flow cytometry apparatus
for
controlling the fluid delivery system to vary the rate at which fluid is
delivered as a function of at
least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A
particles or characteristic B particles in said at least one population
relative to the total quantity of
characteristic A particles or characteristic B particles in said stream.
X2. The system of X1 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
X3. The system of X1 wherein said cells are sperm cells, and wherein
characteristic A is
indicative of a live X sperm cell.
X4. The system of X3 wherein characteristic 13 is Indicative of other than a
live X cell
(¨X).
X5. The system of X1 wherein the control increases the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreases the rate of
fluid delivery when the purity is less than the desired purity.

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X6. The system of X1 wherein the control determines said purity based on
output
signals from the flow cytometry apparatus, said system further comprising an
operator input to
the control for indicating a desired purity, and wherein the control varies
the rate of fluid delivery
to obtain the desired purity.
X7. The system of X1 wherein said stream is formed into a stream containing
particles
following generally one after another In a series which comprises sequential
sets of particles,
including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets
each comprising two or more closely spaced particles at least one of which has
characteristic A
and at least one of which has characteristic 13.
X8. The system of X7 wherein said laser ablates only the second particle sets.
X9. The system of X8 wherein said control maintains the purity of said at
least one
population at more than 85% but less than 95%.
=
X10. The system of X7 wherein characteristic B is indicative of other than a
live X cell
(¨X) and said laser Is operable to ablate the second particle sets and the
third particle sets in the
stream.
X11. The system of X10 wherein the control increases the rate of fluid
delivery when the
quantity of unablated characteristic A particles in said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles In the at least
one population, and
wherein the control decreases the rate of fluid delivery when the quantity of
unablated
characteristic A particles in said at least one population is less than said
acceptable quantity.
X12. The system of X10 wherein the control determines the quantity of
unablated
characteristic A particles in said at least one population based on output
signals from the flow
cytometry apparatus, said system further comprising an operator input to the
control for
indicating an acceptable quantity of unablated characteristic A particles in
said at least one
population, and wherein the control varies the fluid delivery rate to obtain
the acceptable quantity
of unablated characteristic A particles In said at least one population.
Y. Fluid Switching Sorting Method Including Variable Flow Rate and
Feedback
Y1. A method of using a flow cytometry system to sort a mixture of particles
Including
particles having a characteristic A and particles having a characteristic B,
said method
comprising:

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delivering a fluid containing said particles;
forming the fluid into a stream and using flow cytometry to classify the
particles in the
sorting the particles in the stream by diverting selected particles in the
stream according
to said classification thereby to provide at least one population containing
desired particles; and
varying the rate at which fluid is delivered as a function of at least one of
the following:
(1) the purity of said at least one population with respect to either
characteristic A particles or
characteristic B particles; and (2) the quantity of characteristic A particles
or characteristic B
particles in said at least one population relative to the total quantity of
characteristic A particles or
characteristic B particles In said stream.
Y2. The method of Y1 wherein the particles are cells, and wherein
characteristics A and
B relate to physical characteristics of the cells.
Y3. The method of Y1 wherein said cells are sperm cells, and wherein
characteristic A Is
Y4. The method of Y1 further comprising increasing the rate of fluid delivery
when the
purity of said at least one population is greater than a desired purity and
decreasing the rate of
fluid delivery when the purity is less than the desired purity.
Y5. The method of Y1 further comprising forming said fluid into a stream
containing
particles following generally one after another in a series which comprises
sequential sets of
particles, Including first particle sets each comprising one or more particles
having characteristic
A, second particle sets each comprising one or more particles having
characteristic B, and third
Y6. The method of Y5 further comprising ablating the second particles sets and
not the
first and third particle sets.
Y7. The method of Y6 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
YB. The method of Y5 further comprising ablating the second and third particle
sets and

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Y9. The method of Y8 further comprising increasing the rate of fluid delivery
when the
quantity of unablated characteristic A particles in said at least one
population is greater than an
acceptable quantity of unablated characteristic A particles in the at least
one popaiation, and
decreasing the rate of fluid delivery when the quantity of unablated
characteristic A particles in
said at least one population is less than said acceptable quantity.
Z. [Reserved]
AA. AID converter for PMT output signals and DSP analyzing and
classifying
AA1. In a flow cytometry system for sorting a mixture of particles Including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the Improvement comprising:
an analog to digital converter synchronously sampling a time-varying analog
output from
said flow cytometry apparatus and providing an output including digital
information corresponding
to said time-varying analog output wherein said time-varying analog output and
the
corresponding digital information are indicative of characteristic A or
characteristic B;
a digital signal processor analyzing and classifying the digital information
and providing a
sorting signal to said sorting system as a function of the analyzed and
classified digital
Information.
AA1A. The system of AA1 wherein the time-varying analog output comprises a
series of
waveform pulses, each of which is representative of characteristic of a
particle, wherein the
digital signal processor detects portions of the digital information
corresponding to the waveform
pulses and classifies said detected portions, and wherein the digital signal
processor provides
said sorting signal as a function of said detected and classified portions.
AA113. The system of AA1 further comprising a control responsive to
information
received from the flow cytometry apparatus for controlling the sorting system
to vary its sorting
strategy or for controlling the fluid delivery system to vary the rate at
which fluid is delivered as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of

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characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.
AA2. The system of AA1B wherein the particles are cells and characteristics A
and B
relate to physical characteristics of the cells.
AA3. The system of AA1B wherein the particles are sperm cells, and wherein
characteristic A is indicative of a live X sperm cell.
AA4. The system of AA3 wherein characteristic B is Indicative of other than a
live X cell
(¨X).
AA5. The system of AA4 further comprising maintaining the purity of said at
least one
population at more than 85% but less than 95%.
AA6. The system of AA1 wherein the control increases the rate of fluid
delivery when
said pUrIty Is greater than a desired purity and decreases the rate of fluid
delivery when said
purity Is less than said desired purity.
AA7. The system of AA1 wherein the control determines the purity of said at
least one
population based on output signals from the flow cytornetry apparatus, said
system further
comprising an operator input to the control for indicating a desired purity,
and wherein the control
varies the rate of fluid delivery so that the purity corresponds to the
desired purity.
AM. The system of AA7 wherein the control increases the rate of fluid delivery
when
the quantity of characteristic A particles in said at least one population is
greater than an
acceptable quantity of characteristic A particles in the at least one
population, and wherein the
control decreases the rate of fluid delivery when the quantity of
characteristic A particles In sald
at least one population is less than said acceptable quantity.
AA9. The system of AA8 wherein the control determines the quantity of
characteristic A
particles in said at least one population based on output signals from the
flow cytometry
apparatus, said system further comprising an operator Input to the control for
indicating an
acceptable quantity of characteristic A particles in said at least one
population, and wherein the
control varies the fluid delivery rate to obtain said acceptable quantity in
said at least one
population.
AA10. The system of AA1 wherein said stream is formed to contain particles
following
generally one after another In a series which comprises sequential sets of
particles, including first
particle sets each comprising one or more particles having characteristic A,
second particle sets

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each comprising one or more particles having characteristic B, and third
particle sets each
comprising two or more closely spaced particles at least one of which has
characteristic A and at
least one of which has characteristic B.
AA11. The system of AA10 wherein said sorting system is operable to use a
sorting
strategy in which said first particle sets are selected for collection in said
at least one population
and said second and third particle sets are not selected for collection in
said at least one
population.
AA12. The system of AA10 wherein said sorting system is operable to use a
sorting
strategy in which said first and third particle sets are selected for
collection In said at least one
population and said second particle set is not selected for collection in said
at least one
population.
AA13. The system of AA1 wherein said sorting system comprises a droplet
sorting
system.
AA14. The system of AA1 wherein said sorting system comprises a photo-damage
sorting system.
AA15. The system of AA1 wherein said sorting system comprises a fluid-
switching
sorting system,
AA16. The system of AM wherein said digital signal processor includes
instructions for
detecting pulses corresponding to the digital information, instructions for
extracting features In
the detected pulses, and instructions for discriminating the detected pulses
as a function of their
extracted features.
AA17. The system of AA16 wherein said digital signal processor includes
instructions for
defining a decision boundary discriminating between the extracted features
representing
characteristics A and the extracted features representing characteristic B.
AA18. The system of AA17 wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.

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AA1 9. The system of AA1 wherein said digital signal processor comprises a
data
management processor for assembling the digital information into a continuous
stream.
AA20. The system of AA1 wherein sald digital signal processor comprises a
pulse
detection processor for detecting waveform pulses represented by the digital
Information, and
wherein said digital signal processor classifies the digital information as a
function of the
detected waveform pulses.
AA21. The system of AA20 further comprising a filter for filtering the analog
output at a
frequency equal to or less than one half the continuous sampling rate of the
converter, wherein
the converter converts the filtered analog output into corresponding digital
information, and
wherein said digital signal processor classifies the digital information as a
function of a
discrimination boundary.
AA22. The system of AA21 wherein the continuous sampling rate is about 105 MHz
or
higher.
AA23. The system of AA1 wherein said digital signal processor comprises a
feature
extraction and discrimination processor for defining a decision boundary
discriminating between
extracted features representing characteristics A and extracted features
representing
characteristic 13, and wherein the feature extraction and discrimination
processor extracts
features represented by said digit6I information and classifies the features
as a function of the
decision boundary.
AA24. The system of AA1 wherein said digital signal processor comprises a sort
processor responsive to the classifying for providing the sort signals to said
sorting system.
AA25. The system of AA1 wherein the processor innumerates the number of
classified
particles having characteristic A or having characteristic B.
AA26. The system of AA1 wherein said digital signal processor classifies the
digital
information as a function of reducing a coefficient of variation of a
population of the particles
having characteristic A to be substantially equal to or less than a preset
amount or as a function
of minimizing a coefficient of variation of a population of the particles
having characteristic B to
be substantially equal to or less than a preset amount.
AA27. The system of AA26 wherein the preset amount Is about 1.5% or less.
AA27a. The system of AA26 wherein the preset amount is about 1.3% or less.

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AA28. The system of AA1 wherein said digital signal processor classifies the
digital
information such that a population of the particles having characteristic A
and a population of the
particles having characteristic B correspond to a computer model of three
populations including a
first model population of particles having characteristic A, a second model
population of particles
having characteristic B and a third model population of unaligned particles,
said model estimating
population statistics for each of the first, second and third model
populations.
AA29. The system of AA28 wherein the third model population comprises two
populations of unaligned particles, and wherein the model estimates the
population statistics for
the two populations.
AA30. The system of AA1 wherein said digital signal processor comprises a
pulse
detection processor for detecting waveform pulses represented by the digital
Information, and
wherein said digital signal processor classifies the digital information as a
function of a coefficient
of variation of a population of the particles having characteristic A or as a
function of a coefficient
of variation of a population of the particles having characteristic B.
AA30a. The system of AA30 further comprising a filter for filtering the analog
output at a
frequency equal to or less than one half the continuous sampling rate of the
converter, wherein
the converter converts the filtered analog output into corresponding digital
Information, and
wherein said digital signal processor classifies the digital information as a
function of a coefficient
of variation of a population of the particles having characteristic A or as a
function of a coefficient
of variation of a population of the particles having characteristic B.
AA30b. The system of AA1 wherein said digital signal processor comprises a
feature
extraction and discrimination processor for defining a decision boundary
discriminating between
extracted features representing characteristics A and extracted features
representing
characteristic B, and wherein the feature extraction and discrimination
processor extracts
features represented by said digital information and classifies the features
as a function of a
coefficient of variation of a population of the particles having
characteristic A or as a function of a
coefficient of variation of a population of the particles having
characteristic B.
AA3.1. A method of using a flow cytometry system to sort a mixture of
particles Including
particles having a characteristic A and particles having a characteristic B,
said method
comprising:
delivering a fluid containing said particles;
forming the fluid into a stream and using flow cytometry to detect the
particles in the
stream according to said characteristics;

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sorting the particles In the stream and according to a sorting strategy
thereby to provide
at least one population containing desired particles;
converting an analog output frorn said flow cytometry into corresponding
digital
Information wherein the analog output is indicative of characteristic A or
characteristic B; and
analyzing and classifying the digital Information and sorting the particles as
a function of
the analyzed and classified digital information.
AA31A. The method of AA31 further comprising varying the sorting strategy or
varying
the rate at which fluid is delivered as a function of at least one of the
following: (1) the purity of
said at !east one population with respect to either characteristic A particles
or characteristic B
particles; and (2) the quantity of characteristic A particles or
characteristic B particles in said at
least one population relative to the total quantity of characteristic A
particles or characteristic B
particles in said stream.
AA32. The method of AA31 wherein the particles are cells and characteristics A
and B
relate to physical characteristics of the cells.
AA33. The method of AA31 wherein the particles are sperm cells, and wherein
characteristic A is indicative of a live X sperm cell.
AA311. The method of AA33 wherein characteristic B is indicative of other than
a live X
cell (¨X).
AA35. The method of AA33 further comprising maintaining said purity at more
than 85%
but less than 95%.
=
AA36. The method of AA31 further comprising increasing the rate of fluid
delivery when
said purity is greater than a desired purity and decreasing the rate of fluid
delivery when said
purity Is less than the desired purity.
AA37. The method of AA31 further comprising Increasing the rate of fluid
delivery when
the quantity of characteristic A particles in said at least one population Is
greater than an
acceptable quantity of characteristic A particles In the at least one
population, and decreasing the
rate of fluid delivery when the quantity of characteristic A particles in said
at least one population
is less than said acceptable quantity.

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AA38. The rnethod of AA31 further comprising forming the stream to contain
particles
following generally one after another In a series which comprises sequential
sets of particles,
including first particle sets each comprising one or more particles having
characteristic A, second
particle sets each comprising one or more particles having characteristic B,
and third particle sets
each comprising two or more closely spaced particles at least one of which has
characteristic A
and at least one of which has characteristic B.
AA39. The method of AA38 further comprising sorting said particles according
to a
sorting strategy in which only first particles sets are selected for
collection in said at least one
population,
AA40. The method of AA38 further comprising sorting said particles according
to a
sorting strategy in which first and third particle sets are selected for
collection in said at least one
population.
AA41. The method of AA31 wherein said sorting comprises using a droplet
sorting
process.
AA42. The method of AA31 wherein said sorting comprises using a photo-damage
sorting process.
AA43. The method of AA31 wherein said sorting comprises using a fluid-
switching
sorting process.
AA44. The method of AA31 further comprising detecting waveform pulses
represented
by the digital information, extracting features of the waveform pulses from
the digital information,
and discriminating the detected waveform pulses as a function of their
extracted features.
AA45. The method of AA44 further comprising defining a decision boundary
discriminating between the extracted features representing characteristics A
and the extracted
features representing characteristic B.
AA46. The method of AA46 further comprising adjusting the relative location of
the
decision boundary with respect to the extracted features representing
characteristic A and with
respect to the extracted features representing characteristic B as a function
of at least one of the
following: (1) the purity of said at least one population with respect to
either characteristic A
particles or characteristic B particles; and (2) the quantity of
characteristic A particles or
characteristic B particles in said at least one population relative to the
total quantity of
characteristic A particles or characteristic B particles in said stream.

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AA47. The method of AA31 wherein said converting comprises synchronously
sampling
the analog output.
AA48. The method of AA31 wherein classifying the digital information comprises
classifying the digital information as a function of minimizing a coefficient
of variation of a
population of the particles having characteristic A to be substantially equal
to or less than a
preset amount or as a function of minimizing a coefficient of variation of a
population of the
particles having characteristic B to be substantially equal to or less than a
preset amount.
AA49. The method of AA48 wherein the preset amount is about 1.5% or less.
AA50. The method of AA31 wherein classifying the digital information comprises

classifying the digital information such that a population of the particles
having characteristic A
and a population of the particles having characteristic B correspond to a
computer model of three
populations including a first model population of particles having
characteristic A, a second
model population of particles having characteristic B and a third model
population of unaligned
particles, said model estimating population statistics for each of the first,
second and third model
populations.
AA51. The method of AA31 wherein classifying the digital information comprises
classifying the digital information as a function of a coefficient of
variation of a population of the
particles having characteristic A or as a function of a coefficient of
variation of a population of the
particles having characteristic B.
BB. Determining initiat detection parameters
BB1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
an analog to digital converter synchronously sampling a time-varying analog
output from
said flow cytometry apparatus and providing an output including digital
Information
corresponding to said time-varying analog output wherein said time-varying
analog output and
the corresponding digital information are indicative of characteristic A or
characteristic B;

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a digital signal processor determining background characteristics of said time-
varying
analog output from said digital information;
detecting waveform pulses represented by said digital information as a
function of said
determined background characteristics; and
providing a sorting signal to said sorting system as a function of the
detected waveform
pulses.
BB2. The system of claim BB1 wherein said digital signal processor employs an
iterative
procedure for determining a pulse detection threshold for defining the
background
characteristics, said iterative procedure including:
computing background statistic estimates from the digital information;
using the computed estimates to apply a pulse detection logic to said digital
information
in order to identify pulses indicative of characteristic A or characteristic
B;
recomputing the estimates without using the digital information corresponding
to the
identified pulses; and
repeating the above procedure until the background statistic estimates
converge or a
fixed maximum number of iterations occurs.
BB3. The system of BB1 wherein said digital signal processor includes
instructions for
detecting waveform pulses represented by the digital information, instructions
for extracting
features in the detected pulses and instructions for discriminating the
detected pulses as a
function of their extracted features.
BB4. The system of BB3 wherein said digital signal processor includes
instructions for
defining a decision boundary discriminating between the extracted features
representing
characteristics A and the extracted features representing characteristic B.
BB5. The system of B134 wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.

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BB6. The system of BB1 further comprising a control responsive to information
received
from the flow cytometry apparatus for controlling the sorting system to vary
its sorting strategy or
for controlling the fluid delivery system to vary the rate at which fluid is
delivered as a function of
at least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A
particles or characteristic B particles in said at least one population
relative to the total quantity of
characteristic A particles or characteristic B particles in said stream.
BB7. The system of B1 wherein said digital signal processor comprises a pulse
detection processor for detecting waveform pulses represented by the digital
information, and
wherein said digital signal processor classifies the digital information as a
function of the
detected waveform pulses.
BB8. The system of BB7 further comprising a filter for filtering the analog
output at a
=frequency equal to or less than one half a sampling rate of the converter,
and wherein the
converter converts the filtered analog output into corresponding digital
information.
CC. Generating initial discrimination parameters
CCI. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A arid particles having a characteristic B, said
system comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
an analog to digital converter synchronously sampling a time-varying analog
output from
said flow Cytometry apparatus and providing an output including digital
Information corresponding
to sald time-varying analog output wherein said time-varying analog output and
the
corresponding digital information are indicative of characteristic A or
characteristic B; and
a digital signal processor generating initial discrimination parameters from
the digital
information,
discriminating the digital information as a function of the initial
discrimination parameters, and
providing a sorting signal to said sorting system as a function of the
discriminated digital
Information.

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CC2. The system of claim CC1 wherein the digital signal processor employs at
least one
of the following algorithms to generate the initial discrimination parameters:
k-Means, Fuzzy k-
Means and Agglomerative Hierarchical.
CC3. The system of CC1 wherein said digital signal processor includes
instructions for
detecting waveform pulses represented by the digital information, instructions
for extracting
features in the detected waveform pulses and instructions for discriminating
the detected
Waveform pulses as a function of their extracted features.
CC4. The system of CC3 wherein said digital signal processor includes
instructions for
defining a decision boundary discriminating between the extracted features
representing
characteristics A and the extracted features representing characteristic B.
CC5. The system of CC4 wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.
CC6. The system of CC1 further comprising a control responsive to information
received
from the flow cytometry apparatus for controlling the sorting system to vary
Its sorting strategy or
for controlling the fluid delivery system to vary the rate at which fluid is
delivered as a function of
at least one of the following: (1) the purity of said at least one population
with respect to either
characteristic A particles or characteristic B particles, and (2) the quantity
of characteristic A
particles or characteristic B particles in said at least one population
relative to the total quantity of
characteristic A particles or characteristic B particles in said stream.
DD. Synchronously sampling waveform pulses to Classify
DD1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising:
a fluid delivery system for delivering a fluid containing said particles, and
a flow
cytometry apparatus for receiving said fluid, forming it into a stream and
using flow cytometry to
classify the particles according to said characteristics;
an analog to digital converter synchronously sampling a time-varying analog
output
comprising a series of waveform pulses from said flow cytometry apparatus and
providing an

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output including digital information corresponding to said waveform pulses
wherein said
waveform pulses and the corresponding digital information are indicative of
characteristic A or
characteristic 13; and
a digital signal processor analyzing the digital information and classifying
the particle as
a function of the analyzed digital 'information corresponding thereto.
DD2. The system of claim DD1 wherein said digital processor employs a
detection
threshold for defining the waveform the pulses, and wherein said detection
threshold is a function
DD3. The system of claim DD1 wherein the digital control employs a statistical
anomaly
detection analysis, and wherein the digital information statistically
anomalous from background
DD4. The system of DD1 wherein said digital signal processor includes
instructions for
detecting pulses corresponding to the digital information, instructions for
extracting features in
the detected pulses and instructions for discriminating the detected pulses as
a function of their
DD5. The system of DD4 wherein said digital signal processor Includes
instructions for
defining a decision boundary discriminating between the extracted features
representing
characteristics A and the extracted features representing characteristic B.
DP6. The system of DIM wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of at least one
population with respect to
DD7. The system of DD1 further comprising a filter for filtering the analog
output at a
DD8. The system of DD1 further comprising a control responsive to information
received
from the flow cytometry apparatus for controlling the sorting system to vary
its sorting strategy or

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at least one of the following; (1) the purity of at least one population with
respect to either
characteristic A particles or characteristic B particles; and (2) the quantity
of characteristic A
particles or characteristic B particles in at least one population relative to
the total quantity of
characteristic A particles or characteristic B particles in said stream.
DD9. The system of DD1 wherein the processor inntimerates the number of
classified
particles having characteristic A or having characteristic B.
DD10. The system of DD1 further comprising a pulsed illumination device in
synchronization with synchronous sampling for illuminating the particles to
produce the
corresponding waveform pulses.
DD11. The system of DD10 wherein said digital signal processor comprises a
pulse
detection processor for detecting waveform pulses represented by the digital
information, and
wherein said digital signal processor classifies the digital information as a
function of a coefficient
of variation of a population of the particles having characteristic A or as a
function of a coefficient
of variation of a population of the particles having characteristic B.
EE. Feature Extraction
EEl. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
an analog to digital converter synchronously sampling a time-varying analog
output from
said flow cytometry apparatus and providing an output Including digital
information corresponding
to said time-varying analog output wherein said time-varying analog output and
the
corresponding digital information are indicative of characteristic A or
characteristic B;
a digital signal processor extracting features from the digital information
and providing a
sorting signal to said sorting system as a function of the extracted features.
EE2. The apparatus of claim EE1 wherein the extracted feature corresponds to
one or
more of the following features: pulse area, pulse peak, pulse inner area,
pulse width, pulse
gaussianity, pulse lagging peak or pulse slope.

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EE3. The apparatus of claim EE2 wherein the extracted feature comprises an
approximation of a derivative of the pulse or slope of the pulse at a point of
the pulse relative to
an average peak height of the pulse.
EE4. The apparatus of claim EE3 wherein said point along the pulse corresponds
to a
point at which there is a difference between a first derivative of a pulse
produced by particles
having characteristic A and a first derivative of a pulse produced by
particles having
characteristic B.
EE5. The apparatus of claim EE3 wherein the time-varying analog output
corresponds to a
fluorescence emission pulse from the particles, and wherein said point along
the pulse
corresponds to a point at which a difference between a first derivative of a
pulse produced by
particles having characteristic A and a first derivative of a pulse produced
by particles having
characteristic B is at or near a maximum.
EE. The apparatus of claim EE3 wherein the time-varying analog output
corresponds to
a fluorescence emission pulse from the particles, and wherein said point along
the pulse is a
function of a peak height of the fluorescence emission pulses.
EE7. The apparatus of EE1 wherein said digital signal processor includes
instructions
for detecting waveform pulses represented by the digital information,
instructions for extracting
features in the detected waveform pulses and instructions for discriminating
the detected
waveform pulses as a function of their extracted features.
EE8. The apparatus of EE7 wherein said digital signal processor includes
instructions
for defining a decision boundary discriminating between the extracted features
representing
characteristies A and the extracted features representing characteristic B.
EE9. The apparatus of EE8 wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of said at least one
population With respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.
EE10. The apparatus of EE1 further comprising a control responsive to
information
received from the flow cytometry apparatus for controlling the sorting system
to vary its sorting
strategy or for controlling the fluid delivery system to vary the rate at
which fluid is delivered as a
function of at least one of the following: (1) the purity of said at least one
population with respect

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to either characteristic A particles or characteristic 13 particles; arid (2)
the quantity of
characteristic A particles or characteristic 13 particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles In
said stream.
EE11. The apparatus of EE1 wherein said digital signal processor comprises a
pulse
detection processor for detecting waveform pulses represented by the digital
information and for
classifying the detected waveform pulses.
EEl2. The apparatus of EE11 further comprising a filter for filtering the
analog output at
a frequency equal to or less than one half a sampling rate of the converter,
and wherein the
converter converts the filtered analog output into corresponding digital
Information.
FF. Discriminating waveform guises
FF1. In a flow cytometry system for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it Into a stream and using flow cytometry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
an analog to digital converter synchronously sampling a time-varying analog
output
comprising waveform pulses from said flow cytometry apparatus and providing an
output
Including digital Information corresponding to said waveform pulses wherein
said waveform
pulses and the corresponding digital information are Indicative of
characteristic A or characteristic
B;
a digital signal processor discriminating the digital information as
indicative of
characteristic A or as indicative of characteristic B and providing a sorting
signal to said sorting
system as a function of the discriminated digital information.
FF2. The apparatus of FF1 wherein said digital signal processor includes
Instructions for
detecting waveform pulses represented by the digital information, instructions
for extracting
features In the detected waveform pulses, and instructions for discriminating
the detected
waveform pulses as a function of their extracted features.
FF3. The apparatus of FF2 wherein said digital signal processor includes
instructions for
defining a decision boundary discriminating between the extracted features
representing
characteristics A and the extracted features representing characteristic B.

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FF4. The apparatus of FF3 wherein said digital signal processor adjusts the
relative
location of the decision boundary with respect to the extracted features
representing
characteristic A and with respect to the extracted features representing
characteristic B as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles In
said stream.
FF6. The apparatus of FF1 wherein said digital signal processor comprises a
pulse
detection processor for detecting waveform pulses corresponding to the digital
information, and
wherein said digital signal processor classifies the digital information as a
function of the
detected waveforn-i pulses.
FF6. The apparatus of FF5 further comprising a filter for filtering the analog
output at a
frequency equal to or less than one half a sampling rate of the converter, and
wherein the
converter converts the filtered analog output into corresponding digital
Information.
FF7. The apparatus of FF1 further comprising a control responsive to
information
received from the flow cytometry apparatus for controlling the sorting system
to vary its sorting
strategy or for controlling the fluid delivery system to vary the rate at
which fluid is delivered as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic B particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.
FF8. The apparatus of FF1 further comprising a pulsed illumination device in
synchronization with synchronous sampling for Illuminating the particles to
produce the
corresponding waveform pulses.
FF9. The apparatus of FF8 wherein said digital signal processor comprises a
pulse
detection processor for detecting waveform pulses represented by the digital
information, and
wherein said digital signal processor classifies the digital information as a
function of a coefficient
of variation of a population of the particles having characteristic A or as a
function of a coefficient
of variation of a population of the particles having characteristic 13.
GG. LED array break-off sensor
GG1. An apparatus for use with a continuous stream of fluid which is breaking
into
droplets at a break-off location, comprising:

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a light source positioned on one side of the stream to Illuminate the stream;
a linear photoarray positioned on the other side of the stream adapted to be
oriented
along an axis substantially parallel to the stream, said photoarray adapted to
detect light fronn the
light source which passes through the stream and said photoarray adapted to
provide an output
signal corresponding to the detected light;
a control for receiving the output signal and providing a location signal
corresponding to
a location of the break-off location.
GG2. The apparatus of GG1 further comprising a display for providing a display

indicating the location of the break-off location.
GG3. The apparatus of GG1 further comprising a transducer applying a force to
stream
for varying the location of the break-off location, said transducer varying an
amplitude of the force
as a function of an input signal, and wherein the location signal from the
detector is provided to
the transducer as the input signal.
GG4. The apparatus of GG3 further comprising a look up table for specifying
variations
of the amplitude of the force applied to the stream as a function of the
location of the break-off
location.
HH. Flow Cytometry Break-off Sensor
HH1. In a flow cytometry System for sorting a mixture of particles including
particles
having a characteristic A and particles having a characteristic B, said system
comprising a fluid
delivery system for delivering a fluid containing said particles, a flow
cytometry apparatus for
receiving said fluid, forming it into a stream and using flow cytornetry to
classify the particles
according to said characteristics, and a sorting system for sorting the
particles according to said
classification and according to a sorting strategy to provide at least one
population containing
desired particles, the improvement comprising:
a light source positioned at the second location on one side of the stream to
illuminate
the stream;
a linear photoarray positioned at the second location on the other side of the
stream
adapted to be oriented along an axis substantially parallel to the stream,
said photoarray adapted
to detect light from the light source which passes through the stream and said
photoarray
adapted to provide an output signal corresponding to the detected light;

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a control for receiving the output signal indicative of a position of the
second location,
said control varying operation of said transducer as a function of the output
signal.
HH2. The apparatus of HH1 further comprising a control responsive to
Information
received from the flow cytometry apparatus for controlling the sorting system
to vary its sorting
strategy or for controlling the fluid delivery system to vary the rate at
which fluid is delivered as a
function of at least one of the following: (1) the purity of said at least one
population with respect
to either characteristic A particles or characteristic 13 particles; and (2)
the quantity of
characteristic A particles or characteristic B particles in said at least one
population relative to the
total quantity of characteristic A particles or characteristic B particles in
said stream.
HH3. The apparatus of HI-11 further comprising a display for providing a
display
indicating the location of the break-off location.
HH4. The apparatus of HH1 further comprising a transducer applying a force to
stream
for varying the location of the break-off location, said transducer varying an
amplitude of the force
as a function of an input signal, and wherein the location signal from the
detector is provided to
the transducer as the input signal.
HH5. The apparatus of HH4 further comprising a look up table for specifying
variations
of the amplitude of the force applied to the stream as a function of the
location of the break-off
location.
11. Droplet Sorter Having Epi-illumination Optics
Ili. Apparatus for sorting particles contained in a fluid stream according to
one more
characteristics of the particles, said system comprising:
flow cytometry apparatus for delivering a fluid stream containing said
particles to a first
location and for causing the stream to break into droplets at a second
location, said flow
cytometry apparatus being operable using flow cytometry to classify the
particles according to
said characteristics and to sort said droplets according to the classification
of particles contained
in the droplets;
said flow cytometry apparatus comprising an epi-illumination optics system
including a
focusing lens, said optics system being operable to direct a laser beam
through said focusing
lens in a forward direction along a beam axis intersecting the fluid stream at
said first location so
that said cells pass through the beam, resulting in emissions of
electromagnetic radiation from
the cells directed along said beam axis in a rearward direction.

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112. The apparatus of 111 wherein said particles are cells.
113. The apparatus of 111 wherein said particles are sperrn cells.
114. The system of 113 wherein said beam is focused by said optics system as a
spot on
the fluid stream at said first location, said spot having a generally
elliptical shape with a length
along a major axis extending generally at right angles to the direction of
fluid stream flow and a
width along a minor axis extending generally parallel to the direction of
fluid stream flow, said
width being less than the length of the head of a sperm cell passing through
the spot.
115. The apparatus of 113 wherein said optics system includes a single
photodetector to
the rear of said focusing lens for detecting said emissions.
116. The apparatus of 111 wherein said flow cytometry apparatus further
comprises a
nozzle having an interior surface configured to exert a force on the particles
tending to bring
them into a desired orientation prior to passing through said beam.
117. The apparatus of 114 wherein said nozzle Is rotatable about a
longitudinal axis of the
nozzle to adjust said desired particle orientation relative to the beam axis.
118. The apparatus of 111 wherein said flow cytometry apparatus comprises a
nozzle =
oriented to direct the fluid stream in an upward, non-vertical direction.
119. The apparatus of 111 wherein said nozzle has an exterior surface coated
with a non-
reflective, non-emissive coating.
1110. The apparatus of 111 wherein said optics system includes a single
photodetector to
the rear of said focusing lens for detecting said emissions.
111 1. The apparatus of 111 wherein said flow cytornetry apparatus comprises a
nozzle
having a nozzle orifice, and a capillary tube extending from the orifice
through which said fluid
stream is delivered to said first location.
1112. The apparatus of 119 wherein said first location is Inside said
capillary tube.
1113. The system of 111 wherein said flow cytometry apparatus comprises a
plurality of
flow cytometry units operable to analyze multiple fluid streams
simultaneously, each flow
cytometry unit comprising said epi-illumination optics system.

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JJ. Method of Sorting Particles Using Epi-illurnination Optics
JJ1. A method of sorting particles contained in a fluid stream according to
one more
characteristics of the particles, said method comprising:
delivering a fluid stream containing said particles to a first location and
causing the
stream to break into droplets at a second location; and
using a flow cytometry process to classify the particles according to said
characteristics
and to sort said droplets according to the classification of particles
contained in the droplets,
said flow cytometry process comprising directing a laser beam through a
focusing lens In
a forward direction along a beam axis intersecting the fluid stream at said
first location so that
said cells pass through the beam, resulting In emissions of electromagnetic
radiation from the
cells directed along said beam axis in a rearward direction.
JJ2. The method of JJ1 wherein said particles are cells.
JJ3. The method of JJ1 wherein said particles are sperm cells.
JJ4. The method of JJ3 wherein the step of delivering a fluid stream comprises
directing
said stream through a nozzle orifice having a diameter of 50 to 70 microns at
a pressure of 20-40
psi and at a rate of 30,000 to 50,000 sperm cells per second.
JJ5. The method of JJ3 further comprising focusing said beam on said fluid
stream as a
spot of generally elliptical shape having a length along a major axis
extending generally at right
angles to the direction of fluid stream flow and a width along a minor axis
extending generally
parallel to the direction of fluid stream flow, said width being less than the
length of the head of a
sperm cell passing through the beam spot.
JJ6. The method of JJ5 wherein the head of said sperm cell includes a DNA
region
having a length in the direction of stream flow, and wherein said beam spot
width is less than the
length of said DNA region.
JJ7. The method of 113 further comprising generating a plurality of separate
fluid
streams each containing sperm cells and, for each such stream, carrying out
steps (a) (b).
JJ8. The method of 117 further comprising using a common laser beam to
illuminate
said fluid streams.

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JJ9. The method of JJ1 further comprising exerting a force on the particles
tending to
bring them into a desired orientation prior to passing through said beam.
JJ10. The method of JJ4 further comprising flowing the fluid stream through a
nozzle
configured for exerting said force, and rotating the nozzle about a
longitudinal axis of the nozzle
to adjust said desired particle orientation relative to the beam axis.
JJI 1. The method of JJ1 wherein said flow cytometry process further comprises
using
only one photodetector to detect the emission of electromagnetic radiation.
KK. Photodamage Sorter Having SO-Illumination 0_ptics
KK1. Apparatus for sorting particles contained in a fluid stream according to
one more
characteristics of the particles, said system comprising:
a flow cytometry apparatus for delivering a fluid stream containing said
particles to a first
location, classifying the particles according to said characteristics, and
sorting said particles
according to said classification into at least one population containing
desired particles; and
a laser for ablating particles in said stream,
said flow cytometry apparatus comprising an epi-illumination optics system
including a focusing
lens, said optics system being operable to direct a laser beam through said
focusing lens in a
forward direction along a beam axis intersecting the fluid stream at said
first location so that said
cells pass through the beam, resulting in emissions of electromagnetic
radiation from the cells
directed along said beam axis in a rearward direction.
KK2. The apparatus of KK1 wherein said particles are cells.
KK3. The apparatus of KK2 wherein said cells are sperm cells.
KK4. The apparatus of KK1 wherein said flow cytometry apparatus further
comprises a
nozzle having an interior surface configured to exert a force on the particles
tending to bring
them Into a desired orientation prior to passing through said beam.
KK5. The apparatus of KK4 wherein said nozzle is rotatable about a
longitudinal axis of
the nozzle to adjust said desired particle orientation relative to the beam
axis.
KK6. KK1 (copy claims AA2 etc.)

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LL. Method of Sorting_ Particles Using Epi-illumination Optics and
Photodamage
LL1. A method of sorting particles contained in a fluid stream according to
one more
characteristics of the particles, said method comprising:
using a flow cytometry process to classify particles in a fluid stream
according to said
characteristics and to sort said particles according to said classification of
particles; and
ablating particles in said fluid stream,
said flow cytometry process comprising directing a laser beam through a
focusing lens in a
forward direction along a beam axis intersecting the fluid stream so that said
cells pass through
the beam, resulting in emissions of electromagnetic radiation from the cells
directed along said
beam axis in a rearward direction.
MM. Sperm Sorter Having Epi-illumination Optics
MM1. A system for sorting sperm cells according to chromosomal DNA
characteristics,
comprising:
flow cytometry apparatus for delivering a fluid stream containing said cells
to a first
location and for causing the stream to break into droplets at a second
location, said flow
cytometry apparatus being operable to classify the cells according to said DNA
characteristics
and to sort said droplets according to the classification of cells contained
in the droplets,
said flow cytometry apparatus comprising an epi-illuminatIon optics system
including a
focusing lens, said optics system being operable to direct a laser beam
through said focusing
lens in a forward direction along a beam axis intersecting the fluid stream at
said first location so
that said cells pass through the beam, resulting In emissions of
electromagnetic radiation from
the cells directed along said beam axis in a rearward direction.
MM2. The system of MM1 wherein said optics system further comprises a
photodetector
on said beam axis to the rear of said focusing lens operable to detect and
convert at least some
of said emissions into electrical signals indicative of said DNA
characteristics. .
MM3. The system of MM1 wherein said droplets are sorted into first droplets
each of
which contains at least one live X sperm cell and second droplets each of
which contains at least
one Y cell.

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MM4. The system of MM3 wherein at least some of the first droplets contain a
live X cell
and a live Y cell.
MM5. The system of MM1 wherein said flow cytometry apparatus comprises a
nozzle
having an Interior surface configured to exert a force on the cells tending to
bring them into a
desired orientation prior to passing through said beam.
MM6. The system of MM5 wherein said nozzle is rotatable about a longitudinal
axis of
the nozzle to adjust said desired cell orientation relative to the beam axis.
MM7. The system of MM1 wherein said beam axis intersects the fluid stream at
an
angle of incidence which is skewed relative to a longitudinal axis of the
stream at said first
location.
MM8. The system of MM1 wherein said beam is focused by said optics system as a
spot on the fluid stream at said first location, said spot having a generally
elliptical shape with a
length along a major axis extending generally at right angles to the direction
of fluid stream flow
and A width along a minor axis extending generally parallel to the direction
of fluid stream flow,
said width being less than the length of the head of a sperm cell passing
through the beam spot.
MM9. The system of MM8 wherein the head of said sperm cell includes a DNA
region
having a length in the direction of stream flow, and wherein said beam spot
width Is less than the
length of said DNA region.
MM10. The system of MM1 wherein said flow cytometry apparatus comprises a
plurality
of flow cytometry units operable to analyze multiple fluid streams
simultaneously, each flow
cytometry unit comprising said epi-illumination optics system.
MM11. The systern of MM1 wherein said flow cytometry apparatus comprises a
nozzle
oriented to direct the fluid stream in an upward, non-vertical direction.
MM12. The system of MM1 wherein said optics system includes only one
photodetector
for detecting said emissions.
MM13. The system of MM1 further comprising a collector for collecting at least
said first
droplets.
MM14. The system of MM1 wherein said flow cytometry apparatus comprises a
nozzle
having a nozzle orifice, and a capillary tube extending from the orifice
through which said fluid
stream is delivered to said first location.

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MM15. The system of MM14 wherein said first location is inside said capillary
tube.
MM16. The system of MM15 wherein said optics system is optically coupled to
said
capillary tube for transmission of the bearn into the stream flowing through
the tube.
NN. Method of Sorting Sperm Using EpHilumination Optics
NN1. A method of sorting animal sperm cells according to their chromosomal DNA
characteristics, comprising the steps of:
(a) delivering a fluid stream containing said cells to a first location and
causing the
stream to break into droplets at a second location;
(b) directing a laser beam through a focusing lens in a forward direction
along a beam
axis intersecting the fluid stream at said first location so that said cells
pass through the beam,
resulting in emissions of electromagnetic radiation from the cells directed
along said beam axis in
a rearward direction;
(c) detecting and converting at least some of said emissions into electrical
signals
indicative of said DNA characteristics;
(d) processing said electrical signals and classifying the cells according to
said DNA
characteristics; and
(e) sorting said droplets according to the classification of cells contained
in the droplets.
NN2. The method of NN1 wherein said droplets are sorted into first droplets
each of
which contains at least one live X sperm cell and second droplets each of
which contains at least
one Y cell.
NN3. The method of NN2 wherein at least some of said first droplets contain a
live X cell
and a live Y cell.
NN4. The method of NN1 wherein step (a) comprises directing said stream
through a
nozzle orifice having a diameter of 60 to 70 microns at a pressure of 20-40
psi and at a rate of
30,000 to 50,000 sperm cells per second.
NN5. The method of NN1 further comprising exerting a force on the cells
tending to
bring them into a desired orientation prior to passing through said beam.

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NN6. The method of NN5 wherein said sperm cells have heads with wide faces and

narrow edges, and wherein said desired orientation is one where the beam
strikes said wide
faces.
NN7. The Method of NN6 wherein said fluid delivery step comprises directing
said
stream through a nozzle, and wherein said method further comprises rotating
the nozzle about a
longitudinal axis of the nozzle to adjust said desired cell orientation
relative to the beam axis.
NN8. The method of NN1 further comprising focusing said beam on said fluid
stream as
a spot of generally elliptical shape having a length along'a major axis
extending generally at right
angles to the direction of fluid stream flow and a width along a minor axis
extending generally
parallel to the direction of fluid stream flow, said width being less than the
length of the head of a
sperm cell passing through the beam spot.
NN9. The method of NN8 wherein the head of said sperm cell includes a DNA
region
having a length in the direction of stream flow, and wherein said beam spot
width is less than the
length of said DNA region.
NN1O. The method of The method of NN5 wherein step (d) comprises deflecting at
least
said first droplets.
NN11. The method of NN1 further comprising directing at least some of said
emissions
through a filtering system including a spatial filter having an aperture with
length and width
dimensions, at least one of said dimensions being adjustable to vary the size
of the aperture.
NN12. The method of NN1 further comprising directing said fluid stream along
an
upward, non-vertical trajectory.
NN13. The method of NN1 further comprising generating a plurality of separate
fluid
streams each containing sperm cells and, for each such stream, carrying out
steps (a), (b), (c)
and (d).
NN13A. The method of NN13 further comprising using a common laser beam to
illuminate said fluid streams.
NN14. The method of NN13 wherein said fluid streams contain fluid from a
common
supply of fluid.

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NN15. The method of NN1 wherein said fluid stream is delivered to said first
location
through a capillary tube.
00. Sperm Analyzer Having Epl-illumination Optics
001. Apparatus for classifying anirnal sperm cells by chromosomal DNA
characteristics, comprising:
a nozzle system for delivering a fluid stream containing sperm cells to a
first location and
for exerting a force on the sperm cells tending to bring them Into a desired
orientation before the
stream reaches said first location;
an epi-illumination optics system for directing a beam of electromagnetic
radiation in a
forward direction along a beam axis which intersects said fluid stream at said
first location at an
angle of incidence other than 0 degrees stream so that said cells pass through
the beam,
resulting In emissions of electromagnetic radiation from the cells directed
along sald beam axis in
a rearward direction;
=
a photodetector operable to detect and convert at least some of said emissions
into
electrical signals indicative of said DNA characteristics; and
a processor for processing said electrical signals and classifying the cells
according to
said DNA characteristics.
002. The apparatus of 001 wherein said nozzle system is part of a droplet cell
sorting
system.
003. The apparatus of 001 wherein said nozzle system is part of a photo-damage
cell
sorting system.
004. The apparatus of 001 wherein said nozzle system Is part of a fluid-
switching cell
sorting system.
005. The apparatus of 001 wherein said optics system further comprises a
photodetector on said beam axis to the rear of said focusing lens operable to
detect and convert
at least some of said emissions into electrical signals indicative of said DNA
characteristics
006. The apparatus of 001 wherein said flow cytometry apparatus comprises a
nozzle
having an interior surface configured to exert a force on the cells tending to
bring them into a
desired orientation prior to passing through said beam.

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007. The apparatus of 006 wherein said nozzle is rotatable about a
longitudinal axis of
the nozzle to adjust said desired cell orientation relative to the beam axis.
008. The apparatus of 001 wherein said beam axis intersects the fluid stream
at an
angle of Incidence which is skewed relative to a longitudinal axis of the
strearn at said first
location.
009. The apparatus of 001 wherein said beam is focused by said optics system
as a
spot on the fluid stream at said first location, said spot having a generally
elliptical shape with a
length along a major axis extending generally at right angles to the direction
of fluid stream flow
and a width along a minor axis extending generally parallel to the direction
of fluid stream flow,
said width being less than the length of the head of a sperm cell passing
through the beam spot.
0010. The apparatus of 009 wherein the head of said sperm cell includes a DNA
region having a length in the direction of stream flow, and wherein said beam
spot width is less
than the length of said DNA region.
0011. The apparatus of 001 wherein said flow cytometry apparatus comprises a
plurality of flow cytometry units operable in parallel to analyze multiple
fluid streams
simultaneously, each flow cytometry unit comprising said epi-illumination
optics system.
0012. The apparatus of 001 wherein said nozzle system comprises a nozzle
oriented
to direct the fluid stream in an upward, non-vertical direction.
0013. The apparatus of 001 wherein said optics system includes only one
photodetector for detecting said emissions.
0014. The apparatus of 001 wherein said flow cytometry apparatus comprises a
nozzle having a nozzle orifice, and a capillary tube extending from the
orifice through which said
fluid stream is delivered to said first location.
0015. The apparatus of 0014 wherein said first location is inside said
capillary tube.
0015. The apparatus of 0015 wherein said optics system is optically coupled to
said
capillary tube for transmission of the beam into the stream flowing through
theotube.

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PP. Method of Analyzing Sperm Using Epi-illumination Optics
PP1. A method of classifying animal sperm cells by chromosomal DNA
characteristics,
comprising the steps of:
(a) delivering a fluid stream containing sperm cells to a first location and
exerting a force
on the sperm cells tending to bring them into a desired orientation before the
stream reaches
said first location;
(b) directing a beam of electromagnetic radiation In a forward direction along
a beam
axis which Intersects said fluid stream at said first location at an angle of
incidence other than 0
degrees stream so that said cells pass through the beam, resulting In
emissions of
electromagnetic radiation from the cells directed along said beam axis In a
rearward direction;
(c) detecting and converting at least some of said emissions into electrical
signals
indicative of said DNA characteristics; and
(d) processing said electrical signals and classifying the cells according to
said DNA
characteristics.
PP2. The method of PP1 further comprising sorting said cells according to said
DNA
characteristics.
PP3. The method of PP1 further comprising using a photo-damage sorting process
to
sort said cells.
PP4. The method of PP1 further comprising using a fluid-switching sorting
process to
sort said cells.
PP5. The method of PP1 further comprising using a droplet sorting process to
sort said
cells.
PPS. The method of PP1 wherein step (a) comprises directing said stream
through a
nozzle orifice having a diameter of 50 to 70 microns at a pressure of 20-40
psi and at a rate of
30,000 to 50,000 sperm cells per second.
PP7. The method of PP1 further comprising exerting a force on the sperm cells
tending
to bring them into a desired orientation prior to passing through said beam.

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PP8. The method of PP7 wherein said sperm cells have heads with wide faces and
narrow edges, and wherein said desired orientation is one where the beam
strikes said wide
faces.
PP9. The method of PP8 wherein said fluid delivery step comprises directing
said
stream through a nozzle, and wherein said method further comprises rotating
the nozzle about a
longitudinal axis of the nozzle to adjust said desired cell orientation
relative to the beam axis.
PP10. The method of PP1 further comprising focusing said beam on said fluid
stream as
a spot of generally elliptical shape having a length along a major axis
extending generally at right
angles to the direction of fluid stream flow and a width along a minor axis
extending generally
parallel to the direction of fluid stream flow, said width being less than the
length of the head of a
sperm cell passing through the beam spot.
PP11. The method of PP10 wherein the head of said sperm cell includes a DNA
region
having a length In the direction of stream flow, and wherein said beam spot
width is less than the
length of said DNA region.
PP12. The method of PP1 further comprising directing at least some of said
emissions
through a filtering system including a spatial filter having an aperture with
length and width
dimensions, at least one of said dimensions being adjustable to vary the size
of the aperture.
PP1 3. The method of PP1 further comprising directing said fluid stream along
an
upward, non-vertical trajectory.
PP14. The method of PP1 further comprising generating a plurality of separate
fluid
streams each containing sperm cells and, for each such stream, carrying out
steps (a), (b), (c)
and (d).
PPM The method of PP14 further comprising using a common laser beam to
Illuminate
said fluid streams.
PP16. The method of PP14 wherein said fluid streams contain fluid from a
common
supply of fluid.
PP17. The method of PP1 wherein said fluid stream is delivered to said first
location
through a capillary tube.

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QQ. Particle Analyzer Having Skewed Angle of Incidence Epi-illumination
Optics
QQ1. Apparatus for analyzing particles contained in a fluid stream,
comprising:
an epi-Illumination optics system for directing a beam of electromagnetic
radiation in a
forward direction along a beam axis which intersects said fluid stream at an
angle of Incidence
which Is skewed relative to a longitudinal axis of the fluid stream;
said epi-illumination system including a focusing lens on said beam axis path
for
focusing said beam of electromagnetic radiation on the fluid stream as a spot,
said particles
being adapted to pass through said spot resulting in emissions of
electromagnetic radiation from
the particles directed along said beam axis in a rearward direction; and
a photodetector to the rear of the focusing lens for detecting and converting
at least
some of said emissions into electrical signals to be processed to obtain
information regarding
said particles.
QQ2. The apparatus of QQ1 wherein said angle of incidence Is in the range of 5
to 45
degrees.
QQ3. The apparatus of QQ1 wherein said angle of incidence is in the range of
16 to 30
degrees.
QQ4. The apparatus of QQ1 wherein said particles are cells, and wherein said
information relates to DNA characteristics of the cells.
005. The apparatus of QQ1 wherein said particles are sperm cells, and wherein
said
information relates to the X/Y chromosomal DNA characteristics of the cells.
QQ6. The apparatus of QQ5 further comprising a nozzle system for directing
said fluid
stream and for exerting a force on the sperm cells tending to bring them into
a desired orientation
before the stream passes through said beam.
0Q7. The apparatus of QQ1 wherein said nozzle system comprises a nozzle, and
wherein said beam strikes said fluid stream at a location less than 1.0 mm
from the nozzle.
QQ8. The apparatus of QQ1 further comprising a droplet cell sorting system for
sorting
said particles according to said information.
QQ9. The apparatus of QQ1 further comprising a photo-damage cell sorting
system for
sorting said particles according to said information.

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QQ10. The apparatus of QQI further comprising a fluid-switching cell sorting
system for
sorting said particles according to said information.
RR. Method of analyzing Particles Using Skewed Epi-illumination Optics
RRI. A method of analyzing particles contained in a fluid stream, comprising:
directing a beam of electromagnetic radiation In a forward direction along a
beam axis
which intersects said fluid stream at an angle of incidence which is skewed
relative to a
longitudinal axis of the fluid stream;
focusing said beam of electromagnetic radiation on the fluid stream as a spot,
said
particles being adapted to pass through said spot resulting in emissions of
electromagnetic
radiation from the particles directed along said beam axis In a rearward
direction;
detecting and converting at least some of said emissions into electrical
signals indicative
of characteristics of the particles; and
processing said electrical signals to obtain information regarding said
characteristics.
RR2. RRI (add claims similar to QQ2-etc.)
SS. Cell Analyzer Haying Epi-illumination Optics and Elliptical Spot FOCUS
SSi. Apparatus for analyzing DNA characteristics of cells in a fluid stream,
each cell
comprising a DNA region having a length in the direction of stream flow, said
apparatus
comprising:
an epi-illumination optics system for directing a beam of electromagnetic
radiation In a
forward direction along a beam axis so that the beam intersects said fluid
stream;
said optics system including a focusing lens on said beam axis for focusing
the beam on
the fluid stream as a generally elliptical spot having a length along a major
axis extending
generally at right angles to the direction of fluid stream flow and a width
along a minor axis
extending generally parallel to the direction of fluid stream flow, the width
of the beam spot being
less than the length of said DNA region, said cells being adapted to pass
through said spot
resulting in an emissions of electromagnetic radiation from the cells directed
along said beam
axis in a rearward direction, Including emissions from said DNA regions
indicative of said DNA
characteristics;

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a photodetector to the rear of the focusing lens for detecting and converting
at least
some of said emissions from said DNA regions into electrical signals
indicative of said DNA
characteristics; and
a system for processing said electrical signals, identifying said DNA
characteristics, and
classifying the cells according to said DNA characteristics.
SS2. The apparatus of SS1 wherein said spot width is less than about 3.0 pm.
SS3. The apparatus of S52 wherein said spot length is greater than about
80,um.
SS4. The apparatus of SS1 wherein said cells are sperm cells and said DNA
characteristics are indicative of the sex of the sperm cells.
SS5. The apparatus of SS1 further comprising a nozzle system for exerting a
force on
the cells in the stream tending to bring them into a desired orientation
before the stream passes
through said beam.
5S6. The apparatus of SS1 further comprising a droplet cell sorting system for
sorting
said particles according to said DNA characteristics.
SS7. The apparatus of SS1 further comprising a photo-damage cell sorting
system for
sorting said particles according to said DNA characteristics.
SS8. The apparatus of SS1 further comprising a fluid-switching cell sorting
system for
sorting said particles according to said DNA characteristics.
SS9. Apparatus for analyzing chromosomal DNA characteristics of animal sperm
cells
in a fluid stream having a direction of flow, each sperm cell having a head
with a nucleus
comprising a localized chromosome region containing at least one chromosome,
said nucleus
having a length in the direction of stream flow, said apparatus comprising:
an optics system for focusing a beam of electromagnetic radiation on the fluid
stream as
a generally elliptical spot having a length along a major axis extending
generally at right angles to
the direction of stream flow and a width along a minor axis extending
generally parallel to the
direction of stream flow, the width of the beam spot being less than the
length of said nucleus,
said sperm cells being adapted to pass through said spot resulting in a
emissions of
electromagnetic radiation from the sperm cells, including emissions from said
chromosome
regions Indicative of chromosomal characteristics of the regions;

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a photodetector for detecting and converting at least some of said emissions
from the
chromosome regions Into electrical signals indicative of said chromosomal
characteristics; and
a processor for processing said electrical signals, identifying said
chromosomal
characteristics, and classifying the sperrn cells according to said
chromosomal characteristics.
SS10. The apparatus of claim SS9 wherein said chromosomal characteristics are
indicative of the sex of the sperm cells.
SS11. The apparatus of claim SS10 wherein said sperm cells are bovine sperm
cells.
SS12. The apparatus of claim SS10 wherein said chromosome region is localized
within
an area of the nucleus extending no more than about 20% of the nucleus length
on either side of
a longitudinal center of the nucleus.
SS=13. The apparatus of claim SS10 wherein said chromosome region is localized
within
an area of the nucleus extending no more than about 10%-15% of the nucleus
length on either
side of a longitudinal center of the nucleus.
SS14. The apparatus of claim SS9 further comprising an analog to digital
converter
synchronously sampling a time-varying analog output from said photodetector
and providing an
output including digital information corresponding to said time-varying analog
output wherein said
time-varying analog output and the corresponding digital information are
indicative of said
chromosomal characteristics; and wherein said processor comprises a digital
signal processor
analyzing and classifying the digital Information.
SS15. The apparatus of claim SS9 wherein said spot width is less than about
3.0,um.
S316. The apparatus of claim SS15 wherein said spot length is about 80pm.
SS17. The apparatus of claim SS9 further comprising a nozzle system for
exerting a
force on the sperm cells in the stream tending to bring them into a desired
orientation before the
stream passes through said beam.
SS18. The apparatus of claim SS9 further comprising a droplet cell sorting
system for
sorting said sperm cells according to said chromosomal DNA characteristics.
3319. The apparatus of claim SS9 further comprising a photo-damage sperm cell
sorting system for sorting said sperm cells according to said chromosomal DNA
characteristics.

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SSW. The apparatus of claim SS9 further comprising a fluid-switching sperm
cell
sorting system for sorting said sperm cells according to said chromosomal DNA
characteristics.
TT. Method of analyzing cells Using Epi-illumlnation Optics and Elliptical
Spot Focus
Tr1. A method of analyzing DNA characteristics of cells in a fluid stream,
each cell
comprising a DNA region having a length In the direction of stream flow, said
method comprising:
directing a beam of electromagnetic radiation in a forward direction along a
beam axis so
that the beam intersects said fluid stream;
focusing said beam of electromagnetic radiation on the fluld stream as a
generally
elliptical spot having a length along a rnajor axis extending generally at
right angles to the
direction of fluid stream flow and a width along a minor axis extending
generally parallel to the
direction of fluid stream flow, the width of the beam spot being less than the
length of said DNA
region, said cells being adapted to pass through said spot resulting in an
emissions of
electromagnetic radiation from the cells directed along said beam axis In a
rearward direction;
detecting and converting said emissions into electrical signals indicative of
said DNA
characteristics; and
processing said electrical signals, including differentiating between
electrical signals from
emissions from the DNA region of a sperm head and electrical signals from
emissions from other
regions of the sperm cell; and
classifying the cells according to said DNA characteristics.
TT2. The method of TT1 wherein said cells are sperrn cells.
TT3. The method of TT1 further comprising exerting a force on the cells in the
stream
tending to bring them into a desired orientation before the stream passes
through said beam.
TT4. The method of 1T1 further comprising droplet sorting said cells according
to said
DNA characteristics.
TT5. The method of TT1 further comprising photo-damage sorting said particles
according to said DNA characteristics.
TT6. The method of TT1 further comprising fluld-switching sorting said
particles
according to said DNA characteristics.

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TT'7. A method of analyzing chromosomal DNA characteristics of animal sperm
cells in
a fluid stream having a direction of flow, each sperm cell having a head with
a nucleus
comprising a localized chromosome region containing at least one chromosome,
said nucleus
having a length in the direction of stream flow, said method comprising:
focusing a beam of electromagnetic radiation on the fluid stream as a
generally elliptical
spot having a length along a major axls extending generally at right angles to
the direction of
stream flow and a width along a minor axis extending generally parallel to the
direction of stream
flow, the width of the beam spot being less than the length of said nucleus,
said sperm cells
being adapted to pass through said spot resulting in a emissions of
electromagnetic radiation
from the sperm cells, including emissions from said chromosome regions
indicative of
chromosomal characteristics of the regions;
detecting and converting at least some of said emissions from the chromosome
regions
into electrical signals indicative of said chromosomal characteristics;
processing said electrical signals, including identifying said chromosomal
characteristics;
and
classifying the sperm cells according to said chromosomal characteristics.
TT8. The method of claim T17 wherein said chromosomal characteristics are
indicative
of the sex of the sperm cells.
TT9. The method of claim TT8 wherein said sperm cells are bovine sperm cells.
TT10. The method of claim TT8 wherein said chromosome region Is localized
within an
area of the nucleus extending no more than about 20% of the nucleus length on
either side of a
longitudinal center of the nucleus.
TT11. The method of claim T110 wherein said chromosome region is localized
within an
area of the nucleus extending no more than about 10%-15% of the nucleus length
on either side
of a longitudinal center of the nucleus.
TT12. The method of claim TT7 wherein said electrical signals comprise a time-
varying
analog output, and further comprising synchronously sampling the time-varying
analog output
and providing an output including digital information corresponding to said
time-varying analog
output wherein said time-varying analog output and the corresponding digital
information are
indicative of said chromosomal characteristics; wherein said processing
comprises analyzing the

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digital information; and wherein said classifying comprises classifying
analyzed digital
inforniation.
TF13. The method of claim TT7 wherein said spot width is less than about
3.011M.
Tria. The method of claim TT13 wherein said spot length is greater than about
180 pm.
1716. The method of claim TT7 further comprising exerting a force on the sperm
cells in
the stream tending to bring them into a desired orientation before the stream
passes through said
beam.
TT16. The method of claim TT7 further comprising droplet sorting droplet said
sperm
cells according to said chromosomal DNA characteristics.
TT17. The method of claim TT7 further comprising a photo-damage sorting said
sperm
cells according to said chromosomal DNA characteristics.
TT18. The method of claim TT7 further comprising fluid-switching sorting sperm
cells
according to said chromosomal DNA characteristics.
UU. Sperm Sorter Haying Only One Photodetector
UU'l. Apparatus for classifying and sorting sperm cells according to
chromosomal DNA
characteristics of the cells, said apparatus comprising:
a nozzle system for delivering a fluid stream containing said cells to a first
location and
for causing the stream to break into droplets at a second location, said
nozzle system comprising
a nozzle having an interior surface configured to exert a force on the cells
tending to bring them
into a desired orientation before reaching said first location;
an optics system for directing a beam of electromagnetic radiation to
intersect the fluid
stream at said first location so that cells in the stream pass through said
beam resulting in
emissions of electromagnetic radiation from the cells;
only one photodetector for detecting and converting at least some of said
emissions into
electrical signals; and
a system. for classifying the cells according to said DNA characteristics and
for sorting
said droplets according to the classification of cells contained in the
droplets.

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UU2. The apparatus of UU1 wherein said optics system is an epi-illumination
system.
VV. Orienting Nozzle
VV1. A nozzle for use in flow cytometry apparatus for sorting animal sperm
cells in a
fluid stream by chromosome content, said nozzle comprising:
a nozzle body having an Interior surface and an orifice through which said
fluid stream is
adapted to flow;
said interior surface of the nozzle comprising first, second and third axially
tapered
regions for progressively accelerating the speed of said fluid stream in a
downstream direction
toward said nozzle orifice, at least two of said regions having generally
elliptical cross sectional
shapes oriented in different directions for applying torsional forces to said
fluid stream tending to
bring the sperm cells Into a desired orientation.
VV2. The nozzle of VV1 wherein said generally elliptical cross sectional shape
of one of
said at least two regions is oriented at about 90 degrees relative to the
generally elliptical cross
sectional shape of the other of said at least two regions.
W3. The nozzle of VV2 wherein sald generally elliptical cross sectional shapes
of the
first and second regions are oriented In substantially the same direction to
define a first torsional
zone, and wherein the generally elliptical cross sectional shape of the third
region, constituting a
second torsional zone, is oriented at about 90 degrees relative to the
generally elliptical cross
sectional shapes of the first and second regions.
VV4. The nozzle of VV3 wherein the first torsional zone has an axial length of
3.0-4.5
mm and the second torsional zone has an axial length of 3.5-5.0 mm.
VV5. The nozzle of VV1 wherein the third region tapers at an angle of 42-48
degrees.
VV6. The nozzle of VV1 wherein all of said regions have generally elliptical
cross
sectional shapes.
VV7. The nozzle of VV1 wherein said nozzle body has an exterior surface with a
non-
reflective coating thereon.
VV8. The nozzle of VV1 further comprising a mount for mounting said nozzle in
a
position pointing upward to direct said fluid stream along an upward
trajectory.

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VV9. The nozzle of VV8 wherein said nozzle body is rotatable on said mount on
a
longitudinal axis of the body.
WW. Method of Orienting Sperm Cells
WW1. A method of orienting animal sperm cells in flow cytometry apparatus,
said
method comprising:
introducing a fluid containing sperm cells into a fluid stream;
directing the fluid stream containing said sperm cells under a pressure in the
range of 20
to 40 psi through a nozzle having one or more tapered regions for accelerating
the speed of said
fluid stream in a downstream direction toward a nozzle orifice having a
diameter in the range of
50-70 microns, one or more of said tapered regions having a generally
elliptical cross sectional
shape for applying a torsional force to said fluid stream tending to bring the
cells into a desired
orientation as they pass through said orifice at a rate in the range of 30,000
to 50,000 sperm
cells per second.
WW2. The method of WW1 wherein said nozzle has three or more tapered regions
for
progressively accelerating the fluid stream toward the nozzle orifice.
WW3. The method of WW2 wherein said nozzle has two torsional zones defined by
interior surfaces of the nozzle in said three or more tapered regions.
WW4. The method of WW3 wherein said nozzle has a flow axis through said
orifice, and
wherein at least some of said interior nozzle surfaces have generally
elliptical shapes which are
rotated relative to one another about said axis.
WW5. The method of WW1 further comprising directing said stream along an
upward
non-vertical trajectory.
WW6. The method of WWI further comprising rotating the nozzle body on a
central
longitudinal axis of the body while directing said stream.
XX. Particle Sorter Having Upward Pointing Nozzle
XXI. Apparatus for sorting particles using flow cytometry, comprising:

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a nozzle system for delivering a fluid stream containing said particles
through a nozzle
orifice along an upward, non-vertical trajectory;
an optics system for directing a beam of electromagnetic radiation to
intersect said fluid
stream at said first location resulting In emissions of electromagnetic
radiation from the particles;
a photodetector operable to detect and convert at least some of said emissions
into
electrical signals Indicative of said particle characteristics;
a processor for processing said electrical signals and classifying the
particles according
to said characteristics; and
a sorting system for sorting said particles according to the classification of
the particles.
= 15 XX2. The apparatus of XX1 wherein said sorting system comprises
a droplet sorting
system.
XX3. The apparatus of XX1 wherein said sorting system comprises a photo-damage

sorting system.
XX4. The apparatus of XX1 wherein said sorting system comprises a fluid-
switching
sorting system.
XX5. The apparatus of XX1 wherein said nozzle system comprises a nozzle having
a
non-reflective coating thereon.
XX6. The apparatus of XX1 wherein said upward trajectory at the nozzle orifice
Is at an
angle in the range of 5-85 degrees off horizontal.
XX7. The apparatus of XX1 wherein said upward trajectory at the nozzle orifice
is at an
angle in the range of 15-75 degrees off horizontal.
XX8. The apparatus of XX1 wherein said upward trajectory at the nozzle orifice
is at an
angle in the range of 30-65 degrees ofr horizontal.
XX9. The apparatus of XX1 wherein said upward trajectory at the nozzle orifice
is at an
angle in the range of 45-60 degrees off horizontal.

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YY. Method of Sorting Particles Using Upward Pointing Nozzle
YY1. Method of sorting particles using flow cytometry, comprising:
delivering a fluid stream containing said particles through a nozzle orifice
along an
upward, non-vertical trajectory to a first location;
directing a beam of electromagnetic radiation to intersect said fluid stream
at said first
location resulting in emissions of electromagnetic radiation from the
particles;
detecting and converting at least some of sald emissions into electrical
signals indicative
of said particle characteristics;
processing said electrical signals and classifying the particles according to
said
characteristics; and
sorting said particles according to the classification of the particles.
YY2. The method of YY1 wherein said sorting step comprises sorting by using a
droplet
sorting process.
YY3. The method of YY1 wherein said sorting step comprises sorting by using a
photo-
damage sorting process.
YY4. The method of YY1 wherein said sorting step comprises sorting by using a
fluid-
switching sorting process.
ZZ. Process Parameters
ZZ1. A method of separating a desired cell population from a mixture of cells
using flow
cytometry, said population having a light detectable characteristic, said
method comprising:
directing a stream of fluid containing said cells through a nozzle orifice
having a diameter
of from about 50 to 70 pm at a pressure in the range of about 20 to 40 psi and
at a cell rate of
about 30,000 to 50,000 cells per second;
I causing the fluid stream to break into droplets at a frequency of
about 20 to 100 KHz;
and
sorting the droplets using flow cytometry to separate said desired cell
population from
said mixture of cells.

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ZZ2. The method of ZZ1 wherein said cells are sperm cells.
ZZ3. The method of ZZ2 wherein said desired cell population contains live X
cells.
AAA. Business Method
AAA1. A method of doing business including the handling of semen having cells
therein,
said method comprising:
obtaining a supply the semen;
using a programmable machine to conduct a plurality of integrated flow
cytometry
operations, said operations comprising: (a) receiving said supply of semen;
(b) forming multiple
streams containing said cells; and (c) sorting said cells into a first
population of cells having
characteristic A and a second population of cells having characteristic B; and
distributing the first or the second population for commercial use.
AAA2. The method of AAA1 wherein said cells are sperm cells, and wherein
characteristic A is indicative of a live X sperm cell.
AAA3. The method of AAA2 wherein the semen is bovine semen, and wherein the
first
population is sold for use in artificially inseminating cows.
AAA4. The method of AAA3 wherein said programmable machine Is operable to
conduct said operations in parallel.
BBB. Business Method
BBB1. A method of doing business Including the handling of a sample of semen
having
cells therein, said method comprising:
obtaining a supply of semen;
using a programmable machine to conduct a plurality of integrated flow
cytometry
operations, said operations comprising: (a) receiving said supply of semen;
and (b) sorting said
cells from an initial portion of said supply into different populations,
including a first population
containing cells having characteristic A and a second population containing
cells having
characteristic B; and

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sorting said cells from the remaining portion of said supply into different
groups, including
a first group containing cells having characteristic A and a second group
containing cells having
characteristic B, only If the cells initially sorted from the initial portion
meet or exceed a preset
standard.
BBB2. The method of BBB1 further comprising distributing the first or the
second
population for commercial use.
BBB3. The method of BBB1 wherein the preset standard is a minimum recovery
rate of
cells having characteristic A or characteristic B or a minimum purity for at
least one of the
populations.
BBB4. The method of BBB1 further comprising conducting said plurality of
integrated
flow cytometry operations in parallel.
CCC. Business Method
CCC1. A method of doing business including the handling of a sample of semen
having
cells therein, said method comprising:
obtaining a supply of semen;
using a machine to conduct a piurality of integrated flow cytometry
operations, said
operations comprising: (a) receiving said supply of semen; and (b) sorting
said cells from a first
portion of said supply into different populations, including a first
population containing cells
having characteristic A and a second population containing cells having
characteristic B, and
sorting said cells from a second portion of said supply into different
populations, including a first
population containing cells having characteristic A and a second population
containing cells
having characteristic B; and
blending one of the populations of the first portion with one of the
populations of the
second portion to obtain a blended population.
CCC2. The method of CCC1 wherein the first population of the first portion has
an
unacceptable purity, wherein the first population of the second portion has an
acceptable purity,
and wherein the blended population has an acceptable purity.

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DDD. Droplet Interference Sorting W/ Epi-illumination
DDDl. A droplet interference system for sorting sperm cells according to
chromosomal
DNA characteristics, comprising:
flow cytometry apparatus for delivering a fluid stream containing said cells
to a first
location and for causing selected stream segments of the fluid stream to be
hit by a droplet from
a droplet interference fluid stream at a second location, thereby separating
the selected
segments and cells contained therein from the fluid stream, said flow
cytometry apparatus being
operable to classify the cells according to said DNA characteristics and to
sort the stream
segments according to the classification of cells contained in the stream
segments,
said flow cytometry apparatus comprising an epi-illumination optics system
including a
focusing lens, said optics system being operable to direct a laser beam
through said focusing
lens in a forward direction along a beam axis intersecting the fluid stream at
said first location so
that said cells pass through the beam, resulting in emissions of
electromagnetic radiation from
the cells directed along said beam axis in a rearward direction.
DDD2. The system of DDD1 wherein said optics system further comprises a
photodetector on said beam axis to the rear of said focusing lens operable to
detect and convert
at least some of said emissions into electrical signals indicative of said DNA
characteristics.
DDD3. The system of DDD1 wherein the cells are sperm cells and the DNA
characteristics comprises sex chromosome content of the sperm cells.
DDD4. The system of DDD3 wherein said beam is focused by said optics system as
a
spot on the fluid stream at said first location, said spot having a generally
elliptical shape with a
length along a major axis extending generally at right angles to the direction
of fluid stream flow
and a width along a minor axis extending generally parallel to the direction
of fluid stream flow,
said width being less than the length of the head of a sperm cell passing
through the beam spot.
DDD6. The system of DDD4 wherein the head of said sperm cell Includes a DNA
region
having a length in the direction of stream flow, and wherein said beam spot
width is less than the
length of said DNA region.
DDD6. The system of DDD1 wherein said flow cytometry apparatus comprises a
nozzle
having an interior surface configured to exert a force on the cells tending to
bring them into a
desired orientation prior to passing through said beam.
DDD7. The system of DDD6 wherein said nozzle is rotatable about a longitudinal
axis of
the nozzle to adjust said desired cell orientation relative to the beam axis.

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DIDD8. The system of DDD1 wherein said flow cytometry apparatus comprises a
plurality of flow cytometry units operable to analyze multiple fluid streams
simultaneously, each
flow cytometry unit comprising said el-A-illumination optics system.
DDD9. The system of DDD1 wherein said optics system includes only one
photodetector for detecting said emissions.
DDD10. The system of DDD1 wherein said flow cytometry apparatus comprises a
nozzle having a nozzle orifice, and a capillary tube extending from the
=orifice through which said
fluid stream is delivered to said first location.
DDD11. The system of DDD10 wherein said first location is inside said
capillary tube.
DDD12. The system of DDD11 wherein said optics system is optically coupled to
said
capillary tube for transmission of the beam into the stream flowing through
the tube.
A'. Cryopreservation
a. (add cryoprotectant then cool)
A1'. A method of cryopreserving sperm cells comprising the steps of:
adding a cryoprotectant to a quantity of sperm cells;
cooling said quantity of sperm cells and said cryoprotectant to a holding
temperature in a
range of about 0 - 8 C;
maintaining said sperm cells and said cryoprotectant substantially at said
holding
temperature for a period of less than 60 minutes; and
supercooling said quantity of sperm cells to a temperature of -40 C.
A2'. The method of claim A1' wherein said holding temperature is in a range of
about 2 -
6 C.
A3'. The method of claim A1' wherein said holding temperature is in a range of
about 4
5 C.
A4'. The method of claim A1 wherein the step of adding a cryoprotectant
comprises
adding glycerol.

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A6'. The method of claim A1 wherein the step of adding a cryoprotectant
comprises
adding about 6% glycerol (v/v).
A6'. The method of claim A1' wherein said holding period Is less than about 40
minutes.
A7'. The method of claim Al wherein said holding period is about 30 minutes.
A8'. The method of claim A1' wherein the cooling step comprises cooling said
quantity of
sperm at a substantially constant cooling rate.
A9'. The method of claim A8' wherein the cooling rate is selected so that said
quantity of
sperm cells is cooled from a temperature above a glass transition temperature
below which
sperm cells are subject damage from cold shock to the holding temperature in
about 90 minutes.
A10'. The method of claim A8' wherein the substantially constant cooling rate
is In the
range of about 0.1 - 0.3 C per minute.
A11'. The method of claim A8' wherein the substantially constant cooling rate
is in the
=
range of about 0.15 - 0.25 C per minute.
Al2'. The method of claim A11' wherein said holding period is less than about
40
minutes in length.
A13'. The method of claim A1' wherein the cooling step is performed by using a
programmable freezer to cool the sperm cells at a programmed rate.
A14'. The method of claim A13' wherein said holding period is less than 40
minutes in
length.
A15'. The method of claim A14' wherein the programmed cooling rate comprises a

constant cooling rate of about 0.2 C per minute.
A16'. The method of claim A1' wherein the step of supercooling said quantity
of sperm
comprises cooling the sperm cells at a first cooling rate to a temperature
that approaches a
critical temperature zone at which ice crystal formation and changes in
osmotic pressure damage
sperm cells and cooling the sperm at a second cooling rate faster than said
first cooling rate to a
temperature that Is less than about -30 C.

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A17'. The method of claim A16' wherein said first cooling rate Is In the range
of about 1-
C per minute.
A18'. The method of claim A16' wherein said first cooling rate is in the range
of about 2-
5 4 C per minute.
A19'. The method of claim A16' wherein said first cooling rate is about 3 C
per minute.
A20. The method of claim A16' wherein said second cooling rate Is in the range
of
about 8-12 C per minute.
A21'. The method of claim A16' wherein said second cooling rate is about 10 C
per
minute.
A22'. The method of claim A16' wherein the sperm cells are cooled to a
temperature of
about -15 C at said first cooling rate.
A23'. The method of claim A22' wherein the sperm cells are cooled from about
¨15 C to
a temperature of about ¨80 C at said second cooling rate.
A24'. The method of claim A16' wherein the sperm cells are cooled to a
temperature of
about -18 C at said first cooling rate.
A25'. The method of claim A24' wherein the sperm cells are cooled from about
¨18 C to
a temperature of about ¨80 C at said second cooling rate.
A26'. The method of claim A16' wherein the sperm cells are cooled at said
first rate and
said second rate in a programmable freezer.
A27'. The method of claim A1' wherein the step of adding a cryoprotectant to a
quantity
of sperm cells comprises adding a cryoprotectant to a sheath fluid and using
said sheath fluid in
a flow cytometer that analyzes sperm cells.
A28'. The method of claim A27' further comprising the step of using said flow
cytometer
to sort said sperm cells into a population of sperm cells having a desired
characteristic to obtain
said quantity of sperm cells.
A29'. The method of claim Al' wherein the steps of cooling said quantity of
sperm,
maintaining said sperm cells at the holding temperature, and supercooling said
quantity of sperm
cells are all completed in less than 220 minutes.

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A30'. The method of claim A1' wherein the steps of cooling said quantity of
sperm,
maintaining said sperm cells at the holding temperature, and supercooling said
quantity of sperm
cells are all completed in less than about 190 minutes.
A31'. The method of claim A1' wherein the steps of cooling said quantity of
sperm,
maintaining said sperm cells at the holding temperature, and supercooling said
quantity of sperm
cells are all completed in less than about 150 minutes.
A32'. The method of claim A1' further comprising the step of loading said
quantity of
sperm cells into an artificial insemination straw after the oryoprotectant has
been added to said
quantity of sperm cells.
A33'. The method of claim A1' wherein said quantity of sperm cells constitutes
a first
quantity of sperm cells, the method further comprising the steps of obtaining
a sorted population
of sperm cells comprising more than 500 x 106sperm cells from a flow cytometer
to thereby
obtain a plurality of quantities of sperm cells including said first quantity
of sperm cells, loading
each of said plurality of quantities of sperm cells into an artificial
insemination straw, and
performing the steps of cooling, maintaining, and supercooling on each of said
plurality of
. quantities of sperm cells in a batch process to obtain a batch of artificial
insemination straws
containing cryopreserved sperm cells.
A34'. The method of claim A33' wherein said sorted population of sperm cells
comprises
more than 800 x 106 sperm cells.
A35'. The method of claim A33' wherein the steps of the method are completed
in less
than about 240 minutes.
A36'. The method of claim A33' wherein the steps of the method are completed
in less
than about 210 minutes.
A37'. The method of claim A33' wherein the steps of the method are completed
In less
than about 170 minutes.
A38'. The method of claim A1' further comprising the step of staining said
quantity of
sperm cells with a DNA selective fluorescent dye before the cooling step.
A39'. The method of claim A38' wherein said DNA selective dye comprises
Hoechst
33342.

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A40'. The method of claim A39' wherein said step of staining said quantity of
sperm
comprises the step of incubating said quantity of sperm cells for a period of
time in a solution
comprising Hoechst 33342 at a temperature that exceeds 40 C.
b. cold temperature sorting
A41'. A method of cryopreserving sperm cell comprising the steps of:
cooling a quantity of sperm cells to a holding temperature in the range of
about 0 ¨ 8 C;
adding a cryoprotectant to said quantity of sperrn cells; and
maintaining said sperm cells and said cryoprotectant substantially at said
holding
temperature for a period of less than 60 minutes.
A42'. The method of claim A41' wherein said period is in the range of about 30
minutes to less than 60 minutes.
A43'. The method of claim A41' wherein the step of adding a cryoprotectant
comprises
adding glycerol.
A44'. The method of claim A41' wherein the step of adding a cryoprotectant
comprises
adding about 7% glycerol (v/v/).
B'. Nozzle with Baffle
B1'. A nozzle for use in flow cytometry apparatus for analyzing particles in a
fluid
stream, said fluid stream comprising a sheath stream surrounding a core stream
containing said
particles, said nozzle comprising:
a nozzle having an interior surface defining a flow path for said fluid
stream, and an
orifice for exit of the fluid stream from the nozzle; and
a baffle in the nozzle positioned In said flow path upstream from said orifice
for deflecting
the fluid stream as it moves along said flow path,
the baffle and Interior surface of the nozzle being configured to orient the
particles In a desired
orientation as they exit the orifice.

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62'. The nozzle of claim 131' wherein said baffle Is configured for deflecting
said core
stream away from a central longitudinal axis of the nozzle and toward said
interior surface of the
nozzle.
133'. The nozzle of claim 131' wherein said nozzle has a first cross sectional
flow area
upstream of said baffle and a second cross sectional flow area different from
said first cross
sectional flow area at the baffle.
134'. The nozzle of claim 133' wherein said second cross sectional flow area
is smaller
than said first cross sectional flow area.
136'. The nozzle of claim B3' wherein said first and second cross sectional
flow areas
have different shapes.
Be'. The nozzle of claim B5' wherein said second cross sectional flow area is
generally
semi-cylindrical.
B7'. The nozzle of claim B1' wherein said interior surface of the nozzle Is
shaped to
define at least two axially tapered regions downstream from said baffle for
progressively
accelerating the speed of the fluid stream in a downstream direction toward
the nozzle orifice.
B8'. The nozzle of claim B7' wherein said baffle is configured for deflecting
said core
stream toward a portion of said interior surface of the nozzle defining at
least one of said at least
two axially tapered regions.
B9'. The nozzle of claim 138' wherein said at least two axially tapered
regions have
generally elliptical cross sectional shapes oriented In different directions
for applying torsional
forces to said fluid stream that tend to bring the particles into said desired
orientation.
B10'. The nozzle of claim B9' wherein said generally elliptical cross
sectional shape of
one of said at least two regions is oriented at about 90 degrees relative to
the generally elliptical
cross sectional shape of the other of said at least two regions.
1311'. The nozzle of clain-i B1' wherein the nozzle has an exterior surface
with a non-
reflective coating thereon.
B12'. The nozzle of claim B1 further comprising a mount for mounting said
nozzle in a
position pointing upward to direct said fluid stream along an upward
trajectory.

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B13'. The nozzle of claim B13 wherein the nozzle is rotatable on said mount
about a
longitudinal axis of the nozzle.
B14'. The nozzle of claim B1' wherein said baffle comprises a baffle member
for
deflecting said fluld stream and a holder for holding sald baffle member in
fixed position in said
nozzle.
B15'. The nozzle 6f claim B14' wherein said baffle member comprises a baffle
plate.
B16'. The nozzle of claim B15' wherein said baffle plate has a first leg
extending
generally transversely relative to said fluid stream and a second leg
extending generally in the
direction of said fluid stream.
B17'. The nozzle of claim 815' wherein said holder comprises a generally
cylindrical
shell holding said baffle plate and positioned in said nozzle.
B18'. The nozzle of claim B16' wherein said interior surface of the nozzle has
a region of
generally elliptical cross section downstream from said baffle, and wherein
said first and second
legs of the baffle member intersect along a line which Is ubstantially
parallel with a major axis of
said generally elliptical cross section.
B19'. The nozzle of claim B16' wherein sald interior surface of the nozzle has
a region of
generally elliptical cross section downstream from said baffle, and wherein
said first and second
legs of the baffle member intersect along a line which is substantially
perpendicular to a major
axis of said generally elliptical cross section.
B20'. The nozzle of claim 815' wherein the baffle plate is generally
perpendicular to a
longitudinal axis of the nozzle.
B21'. The nozzle of claim B20' wherein the baffle plate has a semi-circular
shape.
822'. The nozzle of claim B20' wherein the baffle plate has a semi-elliptical
shape.
B23'. The nozzle of claim B20' wherein the interior surface of the nozzle is
shaped to
define a shoulder and the holder comprises an o-ring seal capable of pressing
the baffle plate
against the shoulder to hold the baffle plate in a desired position.
B24'. The method of claim B20' whereln the baffle holder holds the baffle
plate in a
position in which the baffle plate intersects the longitudinal axis of the
nozzle.

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B25'. The nozzle of claim 131' wherein sald particles comprise sperm cells.
C. Nozzle with Baffle (Method)
C1'. A method of orienting particles in a flow cytometry apparatus, said
method
comprising:
causing a fluid stream haying a core stream of sample fluid containing said
particles and
a sheath stream of sheath fluid surrounding the core stream to flow through a
nozzle having an
Interior surface that generally tapers from an upstream portion of the nozzle
to an orifice at the
downstream end of the nozzle; and
deflecting the core stream as It flows through the nozzle to subject the
particles in the
core stream to hydrodynamic forces that tend to cause the particles to assume
a desired
orientation.
C2'. The method of claim C1' wherein the nozzle has a longitudinal axis and
the step of
deflecting the core stream comprises deflecting the core stream away from the
longitudinal axis
toward said interior surface.
03'. The method of claim C1' wherein the nozzle has a longitudinal axis and
the step of
deflecting the path of the core stream comprises deflecting the core stream
from a path that
generally coincides with the longitudinal axis to a deflected path, at least a
portion of the
deflected path being offset from the longitudinal axis of the nozzle.
04'. The method of claim C1' wherein the nozzle has a longitudinal axis, the
method
further comprising introducing the core stream into the sheath stream at a
location that is offset
from the longitudinal axis.
05'. The method of claim C1' wherein the deflecting step comprises using a
baffle to
deflect the core stream.
C6'. The method of clairn C1' wherein the deflecting step comprises using a
baffle plate
to deflect the core stream and using a baffle holder to hold the baffle plate
in position in the
nozzle.
C7'. The method of claim C6' wherein the baffle plate intersects the
longitudinal axis of
the nozzle.

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C8'. The method of claim C1' wherein the nozzle has an interior surface that
is shaped
to define at least one torsional zone for subjecting the particles to
hydrodynamic orienting forces.
C9'. The method of claim C8' wherein that at least one torsional zone
comprises a
tapered region of the nozzle having a generally elliptical cross section.
C10'. The method of claim 08' wherein said interior surface is shaped to
define multiple
torsional zones.
C11'. The method of claim C10' wherein each of said multiple torsional zones
comprises
a tapered region of the nozzle having a generally elliptical cross section.
C12'. The method of claim 011' wherein the generally elliptical cross section
for a first of
the multiple torsional zones is oriented in a different direction that the
generally elliptical cross
section for a second of the multiple torsional zones.
C13'. The method of claim C1' wherein the step of deflecting the core stream
comprises
using a baffle to deflect the core stream, the method further comprising the
step of causing the
fluid stream to flow through a first cross sectional flow area and then a
second cross sectional
flow area downstream from the first cross sectional flow area, wherein the
first and second cross
sectional flow areas have different shapes.
C14'. The method of claim C15' wherein the second cross sectional flow area is
smaller
than the first cross sectional flow area.
D'. Nozzle vvIth Asymmetric Injection Needle (Apparatus)
D1'. A nozzle system for use in a flow cytometer for analyzing particles In a
fluid stream,
said fluid stream comprising a sheath stream surrounding a core stream
containing said
particles, said nozzle system comprising:
a nozzle having a longitudinal axls, an interior surface defining a flow path
for said fluid
stream, and an orifice at the downstream end of the nozzle for exit of the
fluid stream from the
nozzle, said Interior surface being shaped to define at least one torsional
zone comprising an
axially tapered region having a generally elliptical cross section for
orienting said particles in a
desired orientation as the fluid stream flows through the torsional zone
toward the orifice; and
a conduit from a particle source to the nozzle, said conduit being positioned
to introduce
said core stream Into the nozzle at a location that Is offset with respect to
the longitudinal axis of
the nozzle.

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D2'. The nozzle system of claim D1' wherein said location is upstream from
said at least
one torsional zone.
03'. The nozzle system of claim DI wherein said at least one torsional zone
constitutes
a first torsional, said nozzle further comprising a second torsional zone.
D4'. The nozzle system of claim D3' wherein said location is upstream from the
first
torsional zone and at least a portion of the second torsional zone.
1.0
D5'. The nozzle system of claim D3' wherein the second torsional zone
comprises an
axially tapered region of the nozzle having a generally elliptical cross
section.
D6'. The nozzle system of claim D5' wherein the major axis of the generally
elliptical
cross section of the first torsional zone is oriented in a different direction
than the major axis of
the generally elliptical cross section of the second torsional zone.
D7'. The nozzle system of claim D6' wherein the generally elliptical cross
section of the
first torsional zone is oriented at an angle of about 90 degrees with respect
to the generally
elliptical cross section of the second torsional zone.
D8'. The nozzle system of claim D7' wherein said location is upstream from the
first,
torsional zone and at least a portion of the second torsional zone.
E'. Asymmetric Sample Introduction (Method)
El'. A method of orienting particles in a flow cytometer comprising:
causing a sheath fluid to flow through a nozzle having a longitudinal axis and
at least one
torsional zone comprising an axially tapered region of the nozzle having a
generally elliptical
cross section; and
introducing a core fluid stream containing particles Into the sheath fluid
stream at a
location that is offset from said longitudinal axis for flow of the sheath
fluid stream and core fluid
stream through the at least one torsional zone.
E2'. The method of claim El wherein the step of causing the fluid stream
through at
least one torsional zone comprises causing the core stream to flow along a
flow path, a portion of
said flow path being offset from the longitudinal axis of the nozzle, and
subjecting said particles

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to hydrodynamic orientation forces generated by said torsional zone while said
particles are
moving along said offset portion of the flow path.
E3'. The method of claim E1 wherein said at least one torsional zone
constitutes a first
torsional zone, the method further comprising the step of causing the sheath
fluid stream and
core fluid stream to flow through a second torsional zone for orienting the
particles in a desired
orientation.
E4'. The method of claim E3 wherein said second torsional zones comprises a
tapered
region in the nozzle having generally elliptical cross sectional area.
E5'. The method of claim E4 wherein the major axis of the generally elliptical
cross
sectional area of the first torsional zone is oriented in a different
direction than the major axis of
the generally elliptical cross sectional area of the second torsional zone.
E6'. The method of claim E4 wherein the major axis of the generally elliptical
cross
sectional area of the first torsional zone is perpendicular to the major axis
of the generally
elliptical cross sectional area of the second torsional zone.
F'. Concentration of Sorted Sperm by Secondary Centrifugation
F1'. A method of processing animal sperm cells comprising the steps of:
collecting sperm cells from a male animal;
sorting the sperm cells into one of multiple populations of sperm cells on the
basis of
one or more specified DNA characteristics;
obtaining a quantity of sperm cells having a desired DNA characteristic from
one of said
multiple populations of sperm cells, said quantity of sperm cells being
contained in a volume of
collection fluid;
subjecting said volume of collection fluid to a first centrifugation process
to form a first
pellet of sperm cells and a supernatant overlying the first pellet;
separating the first pellet from the supernatant;
subjecting the supernatant to additional centrifugation to form a second
pellet of sperm
cells that remained In the supernatant after the first centrifugation; and

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adding a volume of resuspension fluid to the first arid second pellets to
obtain a
suspension of sperm cells having the desired DNA characteristic, the amount of
said volume of
resuspension fluid being selected to result in a desired concentration of
sperm cells in the
suspension.
F2'. The method of claim F1' wherein the first centrifugation process
comprises
centrifuging at a speed sufficient to generate a g-force in the range of 550-
800g.
F3'. The method of claim F2' wherein the first centrifugation process
comprises
centrifuging said volume of collection fluid at said speed for a period in the
range of 7 - 10
minutes.
F4'. The method of claim Fl ' wherein said one or more specified DNA
characteristics
comprises whether the sperm cell contains an X or a Y sex chromosome.
F5'. The method of claim F1' further comprising obtaining a plurality of
quantities of
sperm cells having a desired DNA characteristic and distributing said
plurality of quantities of
sperm cells to animal breeders through a commercial distribution system.
G'. Filtration with low pressure
G1'. A method of processing animal sperm cells comprising the steps of:
collecting sperm cells from a male animal;
sorting the sperm cells to obtain a quantity of sperm cells having a desired
characteristic,
said quantity of sorted sperm cells being contained in a first volume of fluid
having a first
concentration of sperm cells therein; arid
subjecting the sperm cells to a concentration step in which the concentration
of said
sperm cells is increased to a second concentration greater than said first
concentration,
wherein said concentration step comprises flowing at least a portion of the
first volume of fluid
through a first filter at a pressure differential across the first filter of
less than about 20 in.
mercury, said filter having filter pores sufficiently small to inhibit passage
of said sperm cells
therethrough, and retaining a second volume of unfiltered fluid containing
said sperm cells.
G2'. The method of claim G1' wherein the pressure differential across the
filter during
said flowing is substantially constant.

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G3'. The method of claim G1' further comprising the step of intermittently
reducing the
pressure differential across the filter for a period and then substantially
restoring the pressure
differential.
G4'. The Method of claim G3' wherein the step of reducing the pressure
differential
comprises the step of reducing the pressure differential to about zero.
G5'. The method of claim G1' wherein the filter pores have a size of in the
range of
about 0.2 - 1.0 microns.
G6'. The method of claim G1' wherein about 80%-90% of said first volume of
fluid flows
through said first filter.
G7'. The method of claim G1' wherein said second volume of unfiltered fluid is
sufficient
to prevent caking of the sperm cells on said first filter.
G8'. The method of claim G1' further comprising flowing at least a portion of
said second
volume of unfiltered fluid through a second filter having filter pores
sufficiently small to inhibit,
passage of said sperm cells therethrough, retaining a third volume of
unfiltered fluid containing
said sperm cells, and flushing said second filter with a resuspension fluid to
remove sperm cells
from the second filter for addition to said third volume to arrive at said
second concentration.
G9'. The method of claim G8' wherein about 80% of the second volume of
unfiltered
fluid flows through said second filter.
G10'. The method of claim G8' wherein said third volume of unfiltered fluid is
sufficient to
prevent caking of the sperm cells on said second filter.
G11'. The method of claim G8 wherein said second filter is a thin filter
having a
thickness in the range of about 50 - 500 microns.
G12'. The method of claim G8' wherein said second filter is a thin filter
having a
thickness in the range of about 75 - 250 microns. =
G13'. The method of claim G8' wherein said second filter is a thin filter
having a
thickness in the range of about '100 - 150 microns.
G14'. The method of claim G1' wherein said first filter is a thin filter
having a thickness in
the range of about 50 - 500 microns.

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015'. The method of claim G1' wherein said first filter is a thin filter
having a thickness in
the range of about 75 - 250 microns.
G16'. The method of claim G1' wherein said first filter is a thin filter
having a thickness in
the range of about 100 - 150 microns.
H'. Overall Temperature Control (High Temperature Staining)
H1'. A method of processing sperm cells comprising the steps of:
collecting a semen sample containing sperm cells from a male animal;
transporting the semen sample to a sperm processing facility;
conducting an initial quality control check on the semen;
staining the sperm cells in said semen sample with a DNA selective fluorescent
dye that
selectively binds to DNA in the sperm cells by exposing said sperm cells to a
DNA selective
fluorescent dye to form a staining mixture, and subjecting the staining
mixture to a temperature of
at least about 40 C;
using a flow cytometer to sort the sperm cells on the basis of a specified DNA

characteristic;
obtaining a quantity of sperm cells having a desired DNA characteristic
suspended in a
volume of fluid;
adjusting the concentration of sperm cells in said suspension by performing a
concentration process to achieve a desired concentration; and
supercooling said quantity of sperm cells.
H2'. The method of claim H1' further comprising maintaining the temperature of
the
sperm cells from the time they are collected until the beginning of the
staining step at
temperatures in the range of about 20 - 37 C and insulating the sperm cells
from temperature
fluctuations during that time.
H3'. The method of claim H2' wherein the step of insulating the sperm cells
comprises
keeping the semen sample in an insulated container during transportation to
the processing
facility.

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1-14'. The method of claim H1' further comprising the step of placing the
stained sperm
cells in an environment having a temperature in the range of about 18 - 25 C
before they are
introduced into said flow cytometer to cool the sperm cells from the
temperature they attained
during the staining step before beginning the sorting step.
1-15'. The method of claim H1' wherein the supercooling step comprises cooling
the
sperm cells from a temperature that exceeds 20 C to a temperature in the range
of about 0 -
8 C, and adding a protein source and cryoprotectant to said sperm cells before
the temperature
of the sperm cells has been cooled below 20 C.
H6'. The method of claim H5' further comprising using a programmable freezer
to: (a)
cool the sperm cells to a holding temperature in the range of about 0 - B C;
(13) maintain the
sperm at said holding temperature for a period of less than about 60 minutes
to allow the sperm
cells to substantially equilibrate with the cryoprotectant; (c) cool the sperm
at a first cooling rate
to a temperature that approaches a critical temperature zone in which ice
crystal formation and
changes in osmotic pressure damage sperm cells; and (d) cool the sperm cells
through said
critical temperature zone at a second cooling rate faster than said first
cooling rate.
H7'. The method of claim H6' further comprising cooling the sperm cells from
said
holding temperature to about -15 C at said first cooling rate.
H8'. The method of claim H7' further comprising cooling the sperm cells from
about -
18 C to at least about - 30 C at said second cooling rate.
H9'. The method of claim H6' further comprising cooling the sperm cells from
said
holding temperature to about -18 C at said first cooling rate.
H10'. The method of claim H6' further comprising using the programmable
freezer to
cool the sperm cells to said holding temperature at a cooling rate in the
range of about 0.1 - 0.3
C per minute.
1-111'. The method of claim H10' further comprising using the programmable
freezer to
cool the sperm cells from said holding temperature to a temperature of about -
15 C at said first
rate of about 1 - 5 C per minute.
H12'. The method of claim H6' further comprising using the programmable
freezer to cool
the sperm cells from a temperature of about -18 C to at least about - 30 C at
said second cooling
rate of about 8 - 12 C per minute.

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H13'. The method of claim H12' further comprising using thekprogrammable
freezer to
cool the sperm cells from said holding temperature to a temperature of at
least about -15 C at
said first cooling rate of about 1 - 5 C per minute.
H14'. The method of claim H6' wherein the sperm cells are maintained at said
holding
temperature for a period of about 30 minutes.
H15'. The method of any one of claims H1'-H14' wherein the staining mixture
further
comprises an antioxidant.
H16'. The method of claim H15' wherein the antioxidant is selected from the
group
consisting of pyruvate, vitamin K, lipoic acid, and a combination thereof.
H17'. The method of any one of claims H1' or H5'-H16' further comprising
placing the
stained sperm cells in an environment having a temperature of at least about
37 C until they are
introduced into a flow cytometer.
H18'. The method of any one of claims H1' or H5'-H16' further comprising
placing the
stained sperm cells in an environment having a temperature of at least about
40 C until they are
introduced into a flow cytometer.
H19'. The method of claim H1' wherein the step of obtaining a quantity of
sperm cells
comprises obtaining a population of live sperm cells having said desired DNA
characteristic at a
rate of at least 5,000 sperm cells per second.
H20'. The method of claim H19' wherein the purity of said population is at
least 85%.
J'. Overall Temperature Control (without High-Temp staining)
J1'. A method of processing sperm cells comprising the steps of:
collecting a semen sample containing sperm cells from a male animal;
transporting the semen sample to a sperm processing facility;
performing an initial quality control check on the semen sample;
staining sperm cells in said semen sample with DNA selective fluorescent dye;

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sorting the sperm cells with a flow cytometer to obtain one or more
populations of sperm
cells having a desired DNA characteristic;
obtaining a quantity of sperm cells having the desired DNA characteristic
suspended in a
fluid;
adjusting the concentration of said quantity of sperm cells to attain a
desired
concentration of sperm cells having the desired DNA characteristic In a fluid
suspension;
adding a cryoprotectant to a quantity of sperm cells having said desired
characteristic
while said quantity of sperm cells has a temperature In excess of a glass
transition temperature
below which sperm cells are subject to damage from cold shock;
cooling said quantity of sperm cells and cryoprotectant said from said glass
transition
temperature to a holding temperature in the range of about 0 - 8 C;
maintaining said quantity of sperm cells and cryoprotectant In a temperature
range of
about 0 - 8 C for period of less than 60 minutes; and
supercooling said quantity of sperm to a temperature below -40 C.
J2'. The method of claim J1' wherein said cryoprotectant is added while said
quantity of
sperm cells has a temperature in excess of about 20 C.
J3'. The method of claim J1' wherein the stained sperm cells are placed in an
environment having a temperature in the range of about 20 - 25 C until =they
are sorted in a flow
cytometer.
J4'. The method of claim J1' further comprising using a programmable freezer
to cool
said quantity of sperm at a first cooling rate from said holding temperature
to a temperature that
approaches a critical temperature zone in which Ice crystal formation and
changes in osmotic
pressure damage sperm cells, and then to cool said quantity of sperm cells
through said critical
temperature zone at a second cooling rate that Is faster than said first
cooling rate.
J5'. The method of claim J1' wherein the step of maintaining said quantity
of sperm
cells comprises holding said quantity of sperm cells at said holding
temperature for a period of
less than about 40 minutes.

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J6'. The method of claim J1' wherein the step of maintaining said quantity of
sperm cells
comprises holding said quantity of sperm cells at said holding temperature for
a period of about
30 minutes.
J7'. The method of any one of claims J1'-J6' wherein the staining step further
comprises
adding an antioxidant to the sperm cells.
J8'. The method of claim J7' wherein the antioxidant is selected from the
group
consisting of pyruvate, vitamin K, Ilpoic acid, and a combination thereof.
J9'. The method of claim J1' wherein the step of obtaining a quantity of sperm
cells
having a desired DNA characteristic comprises obtaining a population of live
sperm cells having
said desired DNA characteristic at a rate of at least 5,000 sperrn cells per
second.
J10'. The method of claim J9' wherein the purity of said population of sperm
cells Is at
least 85%.
Kt. Overall Temperature Control (cold staining)
K1'. A method of processing sperm cells comprising the steps of:
collecting a semen sample containing sperm cells from a male animal;
transporting the semen sample to a sperm processing facility;
performing an initial quality control check on the semen sample;
cooling the sperm cells to a temperature in the range of about 0 - 8 C before
any
substantial dilution of the sperm cells Is performed;
forming a mixture comprising a solution containing the sperm cells and a DNA
selective
dye and subjecting the mixture to a temperature in the range of 0 - 8 C to
stain the sperm cells;
sorting the sperm cells with a flow cytometer to obtain one or more
populations of sperm
cells having a desired DNA characteristic;
obtaining a quantity of sperm cells having the desired DNA characteristic
suspended in a
fluid;

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adjusting the concentration of said quantity of sperm cells to attain a
desired
concentration of sperm cells having the desired DNA characteristic in a fluid
suspension;
1 .
adding a cryoprotectant to said quantity of sperm cells;
.
holding said quantity of sperm cells and said cryoprotectant at a holding
temperate in
the range of about 0 ¨ 8 C for a period In the range of about 30 minutes to 3
hours; and
supercooling said quantity of sperm cells to a temperature below 40 .
K2'. The method of claim K1' further comprising the step of substantially
avolg
warming the cells at any time during the process. ,
K3'. The rnethod of claim K1' wherein the step of cooling the sperm cells
to a
temperature in the range of 0 - 8 comprises placing a container holding the
sperm cells o a
water bath that is substantially the same temperature as the pre-cooled sperm
cells and cIng
the water bath in an environment having a temperature less than about 8 C.
K4'. The method of claim K3' wherein the cooling step further comprises m
bring
the temperature of the sperm cells a's they cool and adding ice to the water
bath after t perm
cells have cooled to a temperature less than about 10 C.
K5'. The method of claim K1' wherein said holding period is in the range o
out 1 ¨
2 hours.
1
K6'. The
method of claim K1' wherein said holding period is in the range bout 90
minutes.
K7'. The method of claim K1' wherein said holding period is in the rangeli
bout 30
minutes to less than 60 minutes.
K8'. The method of claim K1' wherein the step of supercooling said qu/ of
sperm
cells comprises using a programmable freezer to cool said quantity of sperm at
a fi ooling
rate from said holding temperature to a temperature that approaches a critical
tem ture zone
in which ice crystal forn-iation and changes In osmotic pressure damage sperm
ce nd then to
cool said quantity of sperm cells through said critical temperature zone at a
seconoling rate
that is faster than said first cooling rate.
K9'. The method of claim K1' wherein the step of adding a
cryoprotecicomprises
adding glycerol.

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K10'. The method of claim K1' wherein the step of adding a cryoprotectant
comprises
adding 7% glycerol (v/v).
K11'. The method of claim K1' where in the step of obtaining a quantity of
sperm cells
having a desired DNA characteristic comprises obtaining a population of live
sperm cells having
said desired DNA characteristic at a rate of at least 5,000 sperm cells per
second.
K12'. The method of claim K11' wherein the purity of said population of sperm
cells is
at least 85%.
Collection System (Apparatus)
L1'. A collection system for collecting a stream of droplets sorted by a
droplet sorting
flow cytorneter that produces a stream of droplets moving along a trajectory
that includes a
horizontal component, the system comprising an Intercepting device for
intercepting droplets in
said stream as they move along said trajectory, and a collection vessel
disposed to collect said
Intercepted droplets.
L2'. The collection system of claim L1' wherein the intercepting device
comprises an
impact surface disposed across said trajectory against which said droplets are
adapted to
impact.
L3'. The collection system of claim L2' wherein the irnpact surface is
disposed across
said trajectory at a location at which said droplets have an upward velocity
component.
L4'. The collection system of claim L2' wherein at least some of the droplets
contain
particles and the impact surface is coated with a substance that reduces
damage to said
particles upon impact with the impact surface.
L5'. The collection system of claim L4' wherein said substance comprises one
or more
substances selected from the group consisting of egg yolk, bovine serum
albumin, and
phosphate buffered saline.
L6'. The collection system of claim L4' wherein said particles are sperm
cells.
L7'. The collection system of claim L2' wherein said intercepting device is
configured for
guiding said Intercepted droplets to the collection vessel.

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L8'. The collection system of claim L7' wherein the Intercepting device
comprises a
guide below said impact surface for guiding said droplets down to said
collection vessel.
L9'. The collection system of claim L7' wherein the intercepting device
comprises a
hollow enclosure defining said impact surface and having an entry window for
entry of droplets
moving along said trajectory into said enclosure for impact against said
impact surface.
L10'. The collection system of claim L1' wherein said stream of droplets
constitutes a
first stream of droplets, said intercepting device constitutes a first
intercepting device, said
collection vessel constitutes a first collection vessel, and said cytometer
also produces a second
stream of droplets moving along a second trajectory that Is different from
said first trajectory, said
collection system further comprising a second intercepting device for
intercepting droplets
moving along said second trajectory and a second collection vessel for
collecting droplets
intercepted by the second intercepting device.
L11'. The collection system of claim L10' wherein said first intercepting
device has a
window therein through which said second stream is adapted to pass, and
wherein said second
intercepting device is positioned behind said first intercepting device for
intercepting droplets in
the second stream passing through said window.
L12'. The collection system of claim L10' wherein said second intercepting
device is
configured for guiding said intercepted droplets in the second stream to the
second collection
vessel.
L13'. The collection system of claim L1' wherein said droplets contain
particles, the
collection system further comprising a substance coating the impact surfaces
of the Intercepting
devices for reducing damage to said particles upon impact.
L14'. The collection system of claim L13' wherein said particles comprise live
sperm
cells.
L15'. The collection system of claim L13' wherein said substance comprises a
substance selected from the group consisting of: egg yolk, bovine serum
albumin, and
phosphate buffered saline.
L16'. The collection system of claim L1' in combination with a flow cytometer
positioned
to direct a droplet stream into the Intercepting device.

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M. Collection System (Method)
M1'. A method of collecting droplets sorted by a droplet sorting flow
cytometer that
produces a stream of droplets moving along a trajectory that includes a
horizontal component,
said method comprising the steps of:
intercepting said droplets as they move along said trajectory; and
=
collecting said intercepted droplets in a first collection vessel.
M2'. The method of claim M1' wherein said collecting step comprises guiding
said
Intercepted droplets into said first collection vessel.
M3'. The method of claim M2' wherein said intercepting step
comprises positioning
= an impact surface across said trajectory at a location at which said
droplets have an upward
velocity component.
M4'. The method of claim M3' further comprising adjusting the
position of said impact
surface to accommodate different droplet trajectories.
M5'. The method of claim M1' wherein said flow cytometer produces first and
second
streams of droplets moving along respective first and second trajectories,
each trajectory having
a horizontal component, said method further comprising intercepting said
droplets in said second
stream as they move along said second trajectory, and collecting said
intercepted droplets of the
second stream In a second collection vessel.
M6'. The method of claim M5' wherein said first and second trajectories lie

substantially in one plane.
M7'. The method of claim M5' wherein droplets in said first stream are
intercepted by
positioning a first impact surface across said first trajectory at a location
at which said droplets
have an upward velocity component, and wherein droplets in said second stream
are intercepted
by positioning a second impact surface across said second trajectory at a
location at which said
droplets have an upward velocity component.
M8'. The method of claim M7' wherein said second impact surface is
positioned at a
different elevation than said first impact surface.
M9'. The method of claim M1' wherein said droplets contain particles and the
step of
intercepting the said droplets comprises using an intercepting device to
intercept said droplets,

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the method further comprising contacting said intercepting device with a
composition to reduce
damage to said particles.
M10'. The method of claim M9' wherein said particles comprise live sperm
cells.
M11'. The method of claim M10' wherein said composition comprises a substance
selected from the group consisting of: egg yolk, bovine serum albumin, and
phosphate buffered
saline.
M12'. The method of claim M10' wherein the step of contacting said
intercepting device
with a composition comprises soaking said intercepting device In said
composition for a period
that Is sufficient for the composition be absorbed by and/or adhere to the
Intercepting device.
M13'. The method of claim M12' wherein said period Is in the range of about 30
- 90
minutes.
M14'. The method of claim M13' wherein said period Is In the range of about 30
- 60
minutes.
M15'. The method of claim M14' wherein said period is about 60 minutes.
M16'. The method of any of claims M10'-M15' wherein the step of collecting
said
droplets comprises collecting said droplets in a collection vessel, the method
further comprising
the step of contacting the collection vessel with a composition to reduce
damage to said
particles.
M17'. The method of claim M16' further wherein the step of contacting the
collection
vessel with a composition to reduce damage to the particles comprises
contacting the collection
vessel with the same composition used to contact the intercepting device.
M18'. The method of claim M16' wherein the step of contacting the collection
vessel with
a composition to reduce damage to said particles comprises soaking the
collection vessel in said
composition for a period that is sufficient for the composition to be absorbed
by and/or adhere to
the collection vessel.
M19'. The method of claim M18' wherein said period is In the range of about 30
- 90
minutes.
M20'. The method of claim M18' wherein said period Is about 60 minutes.

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N'. Synergistic Combinations
a. Multi-channel FCM (with sort strategy)
Nal'. A method of processing animal sperm cells comprising the steps of:
sorting the sperm cells into different populations of sperm cells on the basis
of one or
more specified DNA characteristics using a flow cytometry process comprising
delivering a
sarnple fluid containing the sperm cells to a plurality of flow cytometry
units each of which is
operable to sort the sperm cells on the basis of said one or more DNA
characteristics, and
operating said units while sharing an Integrated platform comprising at least
one of the following
elements: (1) a common supply of sperm cells; (2) a common source of
electromagnetic
radiation; (3) a common housing; (4) a cornmon input for controlling operation
of the units; (5) a
common processor for receiving and processing Information from the units to
permit evaluation of
the operation of one unit relative to another unit; and (6) a common fluid
delivery system for
delivering fluid containing said sperm cells to said flow cytometry units; and
varying the rate at which sample fluid is delivered to one or more of the flow
cytometry
units as a function of at least one of the following: (1) the purity of a
first sorted population of
sperm cells having a desired DNA characteristic; and (2) the quantity of sperm
cells having said
desired DNA characteristic in a second sorted population.
Na2'. The method of claim NO' further comprising operating said flow cytometry
units in
parallel.
Na3'. The method of claim Nal' wherein said one or more specified DNA
characteristics
comprises whether a sperm cell contains an X or a Y sex chromosome.
b. Muitl-channel FCM with Digital Processing
Nbl'. A method of processing animal sperm cells comprising the steps of:
sorting the sperm cells into different populations of sperm cells on the basis
of one or
more specified DNA characteristics using a flow cytometry process cornprising
delivering a
sample fluid containing the sperrn cells to a plurality of flow cytometry
units each of which Is
operable to sort the sperm cells on the basis of said one or more DNA
characteristics, and
operating said units while sharing an integrated platform comprising at least
one of the following
elements: (:1) a common supply of sperm cells; (2) a common source of
electromagnetic
radiation; (3) a common housing; (4) a common input for controlling operation
of the units; (5) a
common processor for receiving and processing information from the units to
permit evaluation of

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the operation of one unit relative to another unit; and (6) a common fluid
delivery system for
delivering fluid containing said sperm cells to said flow cytometry units; and
using one or more analog to digital converters to synchronously sample a time-
varying
output from each fiow cytometry unit and provide an output including digital
information
corresponding to said time-varying analog output wherein said time-varying
analog output and
the corresponding digital information are indicative of said specified DNA
characteristic.
Nb2'. The method of claim Nbl' wherein said one or more specified DNA
characteristics
comprises whether a sperm cell has an X or a Y sex chromosome.
c. Multi-channel FCM (w/ high-temperature staining)
Nei'. A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C; and
sorting the sperm cells into different populations of sperm cells on the basis
of one or
more specified DNA characteristics using a flow cytometry process comprising
delivering a
sample fluid containing the sperm cells to a plurality of flow cytometry units
each of which is
operable to sort the sperm cells on the basis of said one or more DNA
characteristics, and
operating said units while sharing an Integrated platform comprising at least
one of the following
elements: (1) a common supply of sperm cells; (2) a common source of
electromagnetic
radiation; (3) a common housing; (4) a common input for controlling operation
of the units; (5) a
common processor for receiving and processing information from the units to
permit evaluation of
the operation of one unit relative to another unit; and (6) a common fluid
delivery system for
delivering fluid containing said sperm cells to said flow cytometry units.
Nc2'. The method of claim Nc1' wherein the dye is a UV excitable or a visible
light
excitable dye.
Nc3'. The method of claim Nc2' wherein the dye is selected from the group
consisting of
a bisbenzirnide, SYBR-14, and a conjugate, an analog, or a derivative thereof.
Nc4'. The method of any one of claims Nc1.-Nc3' wherein the staining mixture
Is
subjected to the temperature for a period of time sufficient to allow the dye
to bind the DNA such
that X and Y bearing sperm cells can be differentially sorted based upon
fluorescence.
Nc5'. The method of claim Nc4' wherein the period of time Is in the range of
about 1 -
160 minutes,

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Nc6'. The method of any one of claims Nc11-Nc5'wherein the staining mixture
further
comprises an antioxidant.
Nc7'. The method of clair n NM' wherein said one or more characteristics
comprises
whether a sperm cell contains an X or a Y sex chromosome:
d. Multi-channel (with rapid cryopreservation)
Ndl'. A method of processing animal sperm cells comprising the steps of:
sorting the sperm cells into dfferent populations of sperm cells on the basis
of one or
more specified DNA characteristics using a flow cytometry process comprising
delivering a
sample-fluid containing the sperm cells to a plurality of flow cytometry units
each of which Is
operable to sort the sperm cells on the basis of said one or more DNA
characteristics, and
operating said units while sharing an integrated platform comprising at least
one of the following
elements: (1) a common supply of sperm cells; (2) a common source of
electromagnetic
radiation; (3) a common housing; (4) a common input for controlling operation
of the units; (5) a
common processor for receiving and processing Information from the units to
permit evaluation of
the operation of one unit relative to another unit; and (6) a common fluid
delivery system for
delivering fluid containing said sperm cells to said flow cytometry units;
obtaining a quantity of sperm cells having a desired DNA characteristic from
one or more
of the sorted populations of sperm cells;
adding a cryoprotectant to said quantity of sperm cells;
cooling said quantity of sperm cells and the cryoprotectant to a holding
temperature of
about 0-8 C;
maintaining said sperm cells and said cryoprotectant substantially at said
holding
temperature for a period of less than 60 minutes; and
supercooling said quantity of sperm cells to a temperature of -40 C.
Nd2'. The method of claim Ndl' wherein the step of adding a cryoprotectant
comprises
adding glycerol.
Nd3'. The method of claim Ndl' wherein said one or more specified DNA
characteristics
comprises whether a sperm cell has an X or a Y sex chromosome.

CA 02752247 2011-09-07
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Nd4'. The method of claim Nd1' wherein the step of cooling said quantity of
sperm cells
to said holding temperature comprises using a cooling rate that is selected so
that said quantity
of sperm cells ls cooled from a temperature above a glass transition
temperature below which
sperm cells are subject to damage from cold shock to said holding temperature
in about 90
minutes.
Nd6'. The method of claim Nd4' wherein the cooling rate is a substantially
constant
cooling rate in the range of about 0.1 -0.3 C per minute.
Nd6'. The method of claim Ndl' wherein the step of supercooling said quantity
of sperm
comprises cooling the sperm cells at a first cooling rate to a temperature
that approaches a
critical temperature zone at which ice crystal formation and changes in
osmotic pressure damage
sperm cells and cooling the sperm at a second cooling rate faster than said
first cooling rate to a
temperature that is less than about -30 C.
Nd7'. The method of Nd1' further comprising:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C.
e. Sort Strategy (with high-temperature staining)
Ne1'. A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
delivering a fluid stream of said staining mixture containing the sperm cells
to a first
location and causing said stream to break into droplets at a second location,
said droplets
comprising first droplets each containing one or more sperm cells having a
desired sex
chromosome, second droplets each containing one or more sperm cells having an
undesired sex
chromosome, and third droplets each containing one or more sperm cells having
the desired sex
chromosome and one more sperm cells having the undesired sex chromosome;
sorting said first droplets from said second and third droplets;
collecting said first droplets to provide at least one population of sperm
cells having a
desired sex chromosome;

CA 02752247 2011-09-07
256
identifying a quantity of sperm cells having the desired sex chromosome in
said at least
one population; and
varying the rate at which fluid is delivered to said first location as a
function of the
quantity of sperm cells identified as having the desired sex chromosome in
said at least one
population relative to the total number of sperm cells having the desired sex
chromosome in said
first, second, and third droplets.
Ne2'. A method of processing animal sperm cells comprising the steps of:
=
' forming a staining mixture containing the sperm cells and a DNA
selective fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
delivering a fluid stream of said staining mixture containing the sperm cells
to a first
location and causing said stream to break into droplets at a second location,
said droplets
comprising first droplets each containing one or more sperm cells having a
desired sex
chromosome, second droplets each containing one or more sperm cells having an
undesired sex
chromosome, and third droplets each containing one or more sperm cells having
the desired sex
chromosome and one more sperm cells having the undesired sex chromosome;
sorting said first droplets and third droplets from said second droplets;
collecting said first and third droplets to provide at least one population of
sperm cells
having a desired sex chromosome;
identifying a quantity of sperm cells having the undesired sex chromosome in
said at
least one population; and
varying the rate at which fluid is delivered to said first location as a
function of the
quantity of sperm cells identified as having the undesired sex chromosome in
said at least one
population.
f. High-temp staining (w/ rapid cryopreservation)
Nfl'. A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
sorting said sperm cells in said staining mixture on the basis of a specified
DNA
characteristic;

CA 02752247 2011-09-07
257
obtaining a quantity of sperm cells having a desired DNA characteristic;
adding a cryoprotectant to said quantity of sperm cells;
cooling said quantity of sperm cells and the cryoprotectant to a holding
temperature of
about 0-8 C;
maintaining said sperm cells and said cryoprotectant substantially at the
holding
temperature for a period of less than 60 minutes; and
supercooling said quantity of sperm cells to a temperature of -40 C.
Nf2'. The method of claim NH wherein the step of adding a cryoprotectant
comprises
adding glycerol.
Nf3'. The method of claim Nf1 wherein said specified DNA characteristic is
whether a
sperm cell contains an X or a Y sex chromosome,
Nf4'. The method of claini'Nf1 wherein the step of cooling said quantity of
sperm cell
comprises using a cooling rate that is selected so that said quantity of sperm
cells is cooled to
about 0-8 C in about 90 minutes.
NM'. The method of claim Nd4 wherein the cooling rate is a substantially
constant
cooling rate in the range of about 0.1 -0.3 C per minute.
Nf6'. The method of claim Nf1 wherein the step of supercooling said quantity
of sperm
comprises cooling the sperm cells at a first cooling rate to a temperature
that approaches a
critical temperature zone at which ice crystal formation and changes in
osmotic pressure damage
sperm cells and cooling the sperm at a second cooling rate faster than said
first cooling rate to a
temperature that is less than about -30 C.
g. Multiple Combinations
Ng1'. (high-temperature staining/Multi-channel sorting/ rapid
cryopreservation)
A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the stalning mixture to a temperature of at least about 40
C;

CA 02752247 2011-09-07
258
sorting the sperm cells in said staining mixture into different populations of
sperm cells
on the basis of one or more specified DNA characteristics using a flow
cytometry process
comprising delivering a sample fluid to a plurality of flow cytometry units
each of which is
operable to sort the sperm cells on the basis of said one or more DNA
characteristics, and
operating said units while sharing an Integrated platform comprising at least
one of the following
elements: (1) a common supply of sperm cells; (2) a cornmon source of
electromagnetic
radiation; (3) a common housing; (4) a common input for controlling operation
of the units; (5) a
common processor for receiving and processing information from the units to
permit evaluation of
the operation of one unit relative to another unit; and (6) a common fluid
delivery system for
delivering fluid containing said sperm cells to said flow cytometry units;
obtaining a quantity of sperm cells having a desired DNA characteristic;
adding a cryoprotectant to said quantity of sperm cells;
cooling said quantity of sperm cells and the cryoprotectant to a holding
temperature of
about 0-8 C;
maintaining said sperm cells and said cryoprotectant substantially at the
holding
temperature for a period of less than 60 minutes; and
supercooling said quantity of sperm cells to a temperature of -40 C.
Ng2'. (High temperature staining/multi-channel sorting/high purity sort
strategy)
A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
delivering a fluid stream of said staining mixture containing the sperm cells
to each of a
plurality of flow cytometry units and using the flow cytometry units to break
each fluid stream into
droplets, said droplets comprising first droplets each containing one or more
sperm cells having a
desired sex chromosome, second droplets each containing one or more sperm
cells having an
undesired sex chromosome, and third droplets each containing.one or more sperm
cells having
the desired sex chromosome and one more sperm cells having the undesired sex
chromosome;
sorting said first droplets from said second and third droplets using a
droplet sorting flow
cytometry process comprising operating said plurality of flow cytometry units
while sharing an
Integrated platform comprising at least one of the following elements: (1) a
common supply of
sperm cells; (2) a common source of electromagnetic radiation; (3) a common
housing; (4) a

CA 02752247 2011-09-07
259
common input for controlling operation of the units; (5) a common processor
for receiving and
processing information from the units to permit evaluation of the operation of
one unit relative to
another unit; and (6) a common fluid delivery system for delivering fluid
containing said sperm
cells to said flow cytometry units;
collecting said first droplets to provide at least one population of sperm
cells having a
desired sex chromosome;
identifying a quantity of sperm cells having the desired sex chromosome in
said at least
one population; and
varying the rate at which fluid is delivered to one or more of the flow
cytometry units as a
function of the quantity of sperm cells identified as having the desired sex
chromosome in said at
least one population relative to the total number of sperm cells having the
desired sex
chromosome in said first, second, and third and third droplets.
Ng3'. (High temperature staining/multi-channel sorting/high recovery sort
strategy)
A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
delivering a fluid stream of said staining mixture containing sperm cells to
each of a
plurality of flow cytometry units and using the flow cytometry units to break
each fluid stream into
droplets, said droplets comprising first droplets each containing one or more
sperm cells having a
desired sex chromosome, second droplets each containing one or more sperm
cells having an
undesired sex chromosome, and third droplets each containing one or more sperm
cells having
a desired sex chromosome and one more sperm cells having an undesired sex
chromosome;
sorting said first droplets and third droplets from said second droplets using
a droplet
sorting flow cytometry process comprising operating said plurality of flow
cytometry units while
sharing an integrated platform comprising at least one of the following
elements: (1) a common
supply of sperm cells; (2) a common source of electromagnetic radiation; (3) a
common housing;
(4) a common input for controlling operation of the units; (5) a common
processor for receiving
and processing information from the Units to permit evaluation of the
operation of one unit
relative to another unit; and (6) a common fluid delivery system for
delivering fluid containing
said sperm cells to said flow cytometry units;
collecting said first and third droplets to provide at least one population of
sperm cells
having a desired sex chromosome;

CA 02752247 2011-09-07
260
Identifying a quantity of sperm cells having the undesired sex chromosome in
said at
least one population; and
varying the rate at which fluid is delivered to one or more of the flow
cytometry units as a
function of the quantity of sperm cells identified as having the undesired sex
chromosome in said
at least one population.
NO'. (High temperature staining/multi-channel sorting/sort strategy/ rapid
cryopreservation)
A method of processing animal sperm cells comprising the steps of:
forming a staining mixture containing the sperm cells and a DNA selective
fluorescent
dye, and subjecting the staining mixture to a temperature of at least about 40
C;
delivering a fluid stream of said staining mixture containing sperm cells to
each of a
plurality of flow cytometry units, using the flow cytometry units to break
each fluid stream Into
droplets, and sorting said droplets according to one or more specified DNA
characteristics of the
sperm cells contained in the droplets to obtain one or more populations of
sperm cells having a
desired DNA characteristic by using a droplet sorting flow cytometry process
comprising
operating said plurality of flow cytometry units while sharing an integrated
platform comprising at
least one of the following elements: (1) a common supply of sperm cells; (2) a
common source
of electromagnetic radiation; (3) a common housing; (4) a common input for
controlling operation
of the units; (5) a common processor for receiving and processing information
from the units to
permit evaluation of the operation of one unit relative to another unit; and
(6) a comrnon fluid
delivery system for delivering fluid containing said sperm cells to said flow
cytometry units;
varying the rate at which fluid is delivered to one or more of the flow
cytometry units as a
function of at least one of the following: (1) the purity of said one or more
populations of sperm
cells having a desired DNA characteristic; and (2) the quantity of sperm cells
having the desired
DNA characteristic in said one or more populations of sperm cells relative to
the total number of
sperm cells having the desired DNA characteristic in said droplets;
adding a cryoprotectant to protect a quantity of sperm cells obtained from
said one or
more populations of sperm cells;
cooling said quantity of sperm cells and said cryoprotectant to a holding
temperature of
about 0-8 C;

CA 02752247 2011-09-07
261
maintaining said sperm cells and said cryoprotectant substantially at said
holding
temperature for a period of less than 60 minutes; and
supercooling said quantity of sperm cells to a temperature of -40 C.
0'. Overall Time Line
01'. A method of processing animal sperm cells comprisIng the steps of:
collecting sperm cells from a male animal;
staining the sperm cells with a DNA selective fluorescent dye;
Illuminating the sperm cells with electromagnetic radiation to cause the DNA
selective
fluorescent dye to emit fluorescent light;
detecting the fluorescent light emitted by the sperm cells;
analyzing the fluorescent light emitted by the sperm cells to determine one or
more
specified DNA characteristics of the sperm cell;
sorting the sperm cells into multiple populations of sperm cells on the basis
of said one
or more specified DNA characteristics;
obtaining a quantity of sperm cells having a desired DNA characteristic from
one of said
multiple populations of sperm cells; and
supercooling said quantity of sperm cells;
whereln the step of supercooling is completed less than about 12 hours after
the sperm cells are
collected.
02'. The method of claim 01' wherein the step of supercooling said quantity of
sperm
cells is completed less than about 8 hours after the sperm cells are
collected.
03'. The method of claim 01' wherein the step of supercooling said sperm cells
is
completed less than about 6 hours after the sperm cells are collected.
04'. The method of cialm 01' wherein the step of supercooling said quantity of
sperm
cells is completed less than about 3 hours after the sperm cells are
collected.

CA 02752247 2011-09-07
262
05. The method of claim 01' wherein the step of supercooling said quantity of
sperm
cells Is completed less than about 2 hours after the sperm cells are
collected.
06'. The method of claim 01' wherein the step of supercooling said quantity of
sperm
cells is completed less than about 1 hour after the sperm cells are collected.
07'. The method of claim 01' wherein the sperm cells comprise live
sperm cells.
= 08'. The method of claim OT wherein said one or more DNA
characteristics
comprises whether the sperm cell contains an X or a Y sex chromosome.
09'. The method of claim 01' further comprising the steps of thawing said
quantity of
sperm cells after the supercooling step and then fertilizing an egg cell with
a sperm cell from said
quantity of sperm cells by artificial insemination.
010'. The method of claim 01' further comprising producing a plurality of
quantities of
sperm cells having a desired characteristic and distributing said plurality of
quantities of sperm
cells to animal breeders through a commercial distribution system.
P'. Sort Speeds
P1'. A method of sorting liVe sperm cells according to chromosome
content
comprising:
(a) adding a DNA selective fluorescent dye to a supply of live sperm cells;
(b) causing a sample fluid containing said sperm cells and a sheath fluid to
flow through
an orienting nozzle that tends to rotate the sperm cells into a desired
orientation, said sample
fluid and sheath fluid exiting the nozzle as a fluid stream comprising a core
stream formed by the
sample fluid and sperm cells and a sheath fluid stream surrounding the core
stream;
(c) applying acoustical energy to the nozzle to cause the fluid stream to
break into a
stream of droplets at a droplet break-off location downstream from the nozzle;
(d) directing a beam of electromagnetic radiation to Intersect the fluid
stream at an
interrogation location upstream from the droplet break-off location to excite
the fluorescent dye in
the sperm cells, thereby causing fluorescent emissions from the sperm cells;

CA 02752247 2011-09-07
263
(e) detecting the fluorescent emissions with a photodetector that outputs a
time-varying
analog signal indicative of the intensity of the detected fluorescent
emissions;
(f) electronically processing said time-varying analog signal to determine the
chromosomal content of a plurality of the sperm cells;
(g) selectively applying an electrostatic charge to the fluid stream to
selectively apply or
not apply an electrical charge to the droplets according to the chromosomal
content of sperm
cells contained in the droplets;
(h) using an electric field to deflect the droplets according to their charge,
thereby
separating sperm cells contained in droplets having the same charge from sperm
cells contained
in droplets having a different charge; and
(i) collecting at least one population of live sperm cells at a rate of at
least 5,000 sperm
cells per second.
P2'. P1' wherein the at least one population collected in the collecting step
has a purity
of at least 85%.
P3'. P1' wherein said collecting step comprises collecting the at least one
population of
live sperm cells at a rate of at least 6,000 sperm cells per second.
P4'. P1' wherein said collecting step comprises collecting the at least one
population of
live sperm cells at a rate of at least 7,000 sperm cells per second.
P5'. P1' further comprising operating two or more cytometry units in parallel
so that each
performs steps (b) through (i) while they share an integrated platform, said
integrated platform
comprising one or more of the following: (1) a common supply of particles; (2)
a common source
of electromagnetic radiation; (3) a common housing; (4) a common input for
controlling operation
of the units; (5) a common processor for receiving and processing information
from the units to
permit evaluation of the operation of one unit relative to another unit; and
(6) a common fluid
delivery system for delivering fluid containing sald particles to said flow
cytometry units.
P6'. P5' wherein the step of operating said two or more flow cytometry units
while they
share an integrated platform comprises operating a mode-locked solid state
laser to produce a
single beam comprising a plurality of electromagnetic pulses, the peak power
of each pulse
being greater than the average power output of the laser, splitting the single
beam into two or
more beams, and directing each of said two or more beams to one of the flow
cytometry units.

A single figure which represents the drawing illustrating the invention.

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Admin Status

Title Date
Forecasted Issue Date 2014-06-17
(22) Filed 2004-03-29
(41) Open to Public Inspection 2004-10-14
Examination Requested 2011-09-07
(45) Issued 2014-06-17

Abandonment History

There is no abandonment history.

Maintenance Fee

Description Date Amount
Last Payment 2020-04-01 $450.00
Next Payment if small entity fee 2021-03-29 $225.00
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Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Request for Examination $800.00 2011-09-07
Filing $400.00 2011-09-07
Maintenance Fee - Application - New Act 2 2006-03-29 $100.00 2011-09-07
Maintenance Fee - Application - New Act 3 2007-03-29 $100.00 2011-09-07
Maintenance Fee - Application - New Act 4 2008-03-31 $100.00 2011-09-07
Maintenance Fee - Application - New Act 5 2009-03-30 $200.00 2011-09-07
Maintenance Fee - Application - New Act 6 2010-03-29 $200.00 2011-09-07
Maintenance Fee - Application - New Act 7 2011-03-29 $200.00 2011-09-07
Maintenance Fee - Application - New Act 8 2012-03-29 $200.00 2012-03-15
Maintenance Fee - Application - New Act 9 2013-04-02 $200.00 2013-03-06
Final Fee $2,118.00 2014-01-31
Maintenance Fee - Application - New Act 10 2014-03-31 $250.00 2014-03-28
Maintenance Fee - Patent - New Act 11 2015-03-30 $250.00 2015-03-23
Section 8 Correction $200.00 2015-08-10
Maintenance Fee - Patent - New Act 12 2016-03-29 $250.00 2016-03-29
Maintenance Fee - Patent - New Act 13 2017-03-29 $250.00 2017-03-27
Maintenance Fee - Patent - New Act 14 2018-03-29 $250.00 2018-03-26
Maintenance Fee - Patent - New Act 15 2019-03-29 $450.00 2019-03-22
Maintenance Fee - Patent - New Act 16 2020-03-30 $450.00 2020-04-01
Current owners on record shown in alphabetical order.
Current Owners on Record
INGURAN, LLC
Past owners on record shown in alphabetical order.
Past Owners on Record
None
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Prosecution-Amendment 2013-05-23 5 310
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