Note: Descriptions are shown in the official language in which they were submitted.
CA 02192196 2004-02-05
1
0 0 o G o
BACKGROUND OF TFiE INVENTION
The present invention relates generally to devices
and methods for analyzing biological fluids. In particular,
it relates to the design and use of improved centrifugal
rotors having siphons which allow delivery of a~precise volume
of liquid to a chamber in the rotor.
is ~ Biological tests of blood plasma and other
biological fluids frequently require that fluids be quickly
divided into predetermined volumes for analysis in a variety
of optical tests or assays. It is also frequently desirable
to separate potentially interfering cellular components of the
material from the other fluid grior to testing. Such'
measurement and separation steps have previously been
typically performed by centrifugation to separate, for
instance, blood plasma from the cellular components, followed
by manual or automated pipetting of predetermined volumes of
the blood plasma into separate test wells. Such procedures
are labor intensive and time-consuming. As a result, various
automated systems and methods have been proposed for providing
multiple aliquots of plasma suitable for testing in a more
efficient manner.
A major advance in the analysis of biological fluids
has been the use of centrifugal rotors. These rotors are
designed to measure volumes of a biological fluid, such as
blood, remove cellular components, and mix the fluid with an
appropriate diluent for analysis, for example by optical
testing. Typically, the rotors provide a plurality of
discrete volumes of sample in separate cuvettes in which the
sample is optically analyzed.
CA 02192196 2004-02-05
2
To ensure accurate and consistent results, such
rotors require the delivery of precisely measured volumes of
liquid to various chambers in the rotor. This must often be
accomplished in circumstances in which the rotor quickly
accelerates and decelerates or is otherwise perturbed during
operation. This perturbation can often lead to.delivery of
inaccurately measured volumes. The present invention
addresses these and other needs.
Description of the Background Art
U.S. Patent Nos. 4,894,204, and 5,160,?02 disclose
siphons for transferring fluids between chambers in a rotor.
U.S. Patent No. 4,244,916 discloses a rotor comprising a
plurality of cuvettes positioned radially outward of a central
receptacle. Each cuvette is connected to the central
receptacle by a duct and comprises a separate air escape
orifice. U.S. Patent No. 4,314,968 relates to rotors having
cells positioned on the periphery of the rotor. Each cell
includes a peripheral orifice for removing fluid introduced
into the cell. U.S. Patent No. 4,902,4?9 discloses a multi-
cuvette rotor comprising elongated, radially extending
cuvettes. Each elongated cuvette comprises a first chamber
for receiving a first constituent and a second chamber for
receiving a second constituent. A divider structure between
the first and second chambers prevents mixing of the
constituents before a predetenained time. Mixing occurs as
the rotor is spun at a sufficient speed. U.S. Patent No.
4,963,498 discloses devices which rely upon capillaries,
chambers, and orifices to pump and mix fluids for optical
analysis. U.S. Patent No. 5,0??,013 discloses rotors
comprising peripheral cuvettes connected to holding chambers
positioned radially inward from the cuvettes.
SUMMARY OF THE INVENTION
The present invention provides centrifugal rotors
comprising siphons for delivering a premeasured volume of
liquid, typically a biological sample such as plasma, between
a first and a second chamber in the rotor.
CA 02192196 2004-02-05
2A
Accordingly, the present invention provides a
centrifugal rotor comprising:
a rotor body comprising a liquid-dispensing chamber, a
liquid-receiving chamber, and a siphon;
the siphon being connected to the liquid-dispensing
chamber through a siphon inlet and connected to the liquid-
receiving chamber through a siphon outlet, the siphon inlet
being radially outward of the siphon outlet, said siphon
traveling radially inward to a point radially inward of said
siphon inlet, and then radially outward to said siphon
outlet; the rotor further comprising
a cuvette, wherein said cuvette is radially outward of
said liquid-dispensing chamber and said liquid-receiving
chamber, and;
a distribution ring which permits flow of a liquid to
said cuvette from an output siphon connected to the liquid-
receiving chamber.
The present invention also provides a centrifugal
rotor comprising:
a rotor body comprising a liquid-dispensing chamber, a
liquid-receiving chamber, and a siphon;
the siphon being connected to the liquid-dispensing
chamber through a siphon inlet and connected to the liguid-
receiving chamber through a siphon outlet, the siphon inlet
being radially outward of the siphon outlet, said siphon
traveling radially inward to a point radially inward of said
siphon inlet, and then radially outward to said siphon
outlet; the rotor further comprising
a distribution ring positioned radially outward of the
liquid-receiving chamber; and
a delivery channel connecting the distribution ring to
CA 02192196 2004-02-05
2B
the liquid-receiving chamber, said distribution ring being
connected to a cuvette through an inlet channel.
The present invention also provides a centrifugal
rotor comprising:
a sample chamber containing a liquid;
an unvented receiving chamber positioned radially
outward from the sample chamber;
a delivery channel connected to the sample chamber for
removing the liquid from the sample chamber under
centrifugal force;
a distribution channel directly connected to the
delivery channel for removing the liquid from the delivery
channel under centrifugal force;
an inlet channel connected to the unvented receiving
chamber for receiving the liquid from the sample chamber
through the delivery channel and the distribution channel;
wherein, the resistance to flow of the liquid in the
delivery channel is greater than the resistance to flow of
the liquid in the inlet and distribution channels such that
the cross-sectional area of the liquid flowing through the
inlet and distribution channels is less than the cross-
sectional area of said inlet and distribution channels,
whereby spinning the rotor effects the flow of the liquid
from the sample chamber through the delivery channel, the
distribution channel and the inlet channel to the receiving
chamber such that air escapes from the receiving chamber
through the inlet channel as the liquid enters the receiving
chamber.
In a further aspect, the present invention
provides a method of delivering a premeasured volume of
CA 02192196 2004-02-05
2C
liquid from a first chamber to a second chamber in a rotor,
the method comprising:
providing a rotor comprising a first chamber with a
first volume, a second chamber, and a siphon connected to
the first chamber through a siphon inlet and connected to
the second chamber through a siphon outlet, the siphon inlet
being radially outward of the siphon outlet;
spinning the rotor, thereby introducing an unmeasured
volume of liquid into the first chamber;
stopping the rotation of the rotor, thereby priming the
siphon connecting the first chamber to the second chamber;
and
spinning the rotor, thereby initiating the operation of
the siphon and delivering the premeasured volume of the
liquid from the first chamber to the second chamber, the
premeasured volume being determined by the radial position
of the siphon outlet and the first volume of the first
chamber.
The siphons of the invention have an elbow that i.s
radially inward of the
W O 95133986 ' , r '_r ~~rt7S95107145
3
radially most inward point of the fluid in the first chamber.
As the rotor is spinning the fluid does not flow past the
elbow. After the rotor stops, capillary forces "prime" the
siphon by pulling fluid just around the elbow. When the rotor
' 5 is restarted, centrifugal force draws the remaining fluid out
of the metering chamber into the receiving chamber until the
' level of the fluid in the metering chamber is at the same
radial distance as the outlet of the siphon. The siphons of
the invention are designed such that the inlet of the siphon
on the first chamber is radially outward of the siphon outlet
on the second chamber.
The positioning of the inlets and outlets of the
siphons of the invention provide a number of advantages. For
example, the inlet of the siphon is always positioned radially
outward of the final position of the meniscus of the fluid in
the first chamber, after fluid has been transferred to the
second chamber. Thus, inaccuracy in measurement associated
with different shaped menisci in different fluids is minimized
since the meniscus is minimized. In addition, one of skill
will recognize that all siphons are semi-stable because the
train of fluid in a siphon is stable but easily broken if the
rotor is perturbed. When the train of fluid is broken, under
centrifugal force, the fluid contained in the siphon will flow
to the radially most outward point. In prior art siphons this
point is the siphon outlet. Thus, the potential exists for
the delivery of unmetered volumes of fluid to the receiving
chamber. In the siphons of the present invention, the
radially most outward point in the siphon is the siphon inlet.
In this design, the problem of delivering unmetered volumes of
fluid is avoided because the fluid flows back into the first
chamber when the train of fluid is broken.
The chambers connected by the siphons of the
invention are used to perform any of a number of functions,
such as metering liquids, separating solid components from a
sample, mixing diluent with the sample, and the like. In the
' preferred embodiments, the siphons connect a plasma metering
chamber to a mixing chamber for mixing the premeasured volume
of plasma with diluent.
WO 95/33986 2 ~ 9 219 6. : ~' PC'1'/US95107145
4
In addition, the rotors of the invention comprise
unmodified inlet chanrie~s connecting a distribution ring to
cuvettes comprising reagents for optical analysis of a
biological sample. The inlet channels are sized such that, as
the rotor spins, gas escapes from the cuvette through the
inlet channel as the liquid enters the cuvette through the
inlet channel. An °'unmodified inlet channel" as used herein
refers to a simple inlet channel, typically having a
rectangular cross section, which is not modified (e.g., by
altering the cross-sectional shape, surface texture, and the
like) to provide a pathway for gas to escape from a cuvette
that is not otherwise vented.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. lA-1F are top plan views of a rotor of the
invention showing the flow of fluids through the chambers and
channels of the rotor as the rotor is spun.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides methods and devices
for the delivery of liquids to chambers in an analytical
rotor. The rotors of the invention comprise siphons which
ensure precise delivery of metered volumes of liquid to a
desired chamber in the rotor.
The rotors of the invention are suitable for the
analysis of any liquid, typically a biological sample such as
whole blood or plasma. It is also useful with numerous other
biological fluids, such as urine, sputum, semen, saliva,
ocular lens fluid, cerebral fluid, spinal fluid, amniotic
fluid. Other fluids that can be tested include tissue culture
media, food and industrial chemicals, environmental samples
and the like.
The rotors typically provide chambers which can
separate cellular components from the biological sample (e. g.
whole blood), measure a precise volume of liquid sample (e. g.
plasma), mix the sample with an appropriate diluent and
deliver the diluted sample to cuvettes for optical analysis.
The fluid delivered to the cuvettes, undergoes reactions)
within the cuvettes, e.g., reaction with a reagent which forms
CA 02192196 2004-02-05
part of an analytical procedure to detect one or more analytas
within the fluid. The sample may further be optically
analyzed while present in the rotor, either with or without
prior reaction.
5 The apparatus of the present invention comprises an
analytical rotor having a rotor body which is capable of being
mounted on a conventional laboratory centrifuge of the type
which is commercially available from suppliers, such a Beckman
Instruments, Inc., Spinco Division, Fullerton, California;
Fisher Scientific, Pittsburgh, Pennsylvania; VWR Scientific,
San Francisco, California, and the like. Generally, the
centrifugal rotor will include a receptacle or other coupling
device suitable for mounting on a vertical drive shaft
provided by the centrifuge. The particular design of_the
receptacle or coupling device will depend on the nature of the
centrifuge, and it will be appreciated that the centrifugal
rotor of the present invention may be adapted for use with all
or most types of centrifuges which are now available or which
may become available in the future.
The rotor body comprises a structure which maintains
a desired geometric pattern or relationship between a
plurality of chambers, interconnecting passages, and vents, as
described in more detail below. Various specialized chambers
and channels suitable for use in the rotors of the invention
25' aze disclosed in U.S. Patent Nos. 5,061,381; 5,122,284;
5,186,844 and 5,242,606.
Usually, the body will be a substantially solid
plate or disk with the chambers and passages formed as spaces
- or voids in the otherwise solid matrix. Conveniently, such
solid plate structures may be formed by laminating a plurality
of separately-formed layers together into a composite
structure where the chambers and horizontal passages are
generally formed between adjacent layers. The vertical
passages may be formed through the layers. The individual
layers may be formed by injection molding, machining, or
combinations thereof, and will usually be joined together,
typically using a suitable adhesive or by ultrasonic welding.
CA 02192196 2004-02-05
6
The final enclosed volumes are formed when the layers are
brought together.
Of course, the centrifugal rotor could also be
formed as a plurality of discrete components, such as tubes,
vessels, chambers, etc., arranged in a suitable framework.
Such assemblies of discrete components, however, are generally
more difficult to manufacture and are therefore less desirable
than those formed within a substantially solid plate.
The rotor body may be formed from a wide variety of
to materials and may optionally include two or more materials.
Usually, the materials) will be transparent so that the
presence and distribution of the biological fluid, cellular
components, and reagents, may be observed within the various
internal chambers and passages. Optionally, to the extent
analytical chambers, e.g., cuvettes, or other test wells are
formed within the rotor, it is desirable to have suitable
optical paths formed within the rotor so that the contents of
the cuvettes may be observed spectrophotometrically,
fluorometrically, or by other optical assessment instruments.
The construction of suitable cuvettes having particular
optical paths formed therethrough is disclosed in U.S. Patent
No. 5,173,193.
In the preferred embodiment, the rotor is
formed with an acrylic resin having suitable optical
properties, at least in those areas which define an optical
path.
The apparatus and method of the present invention
are suitable for performing a wide variety of analytic
procedures and assays which are beneficially or necessarily
3o performed on blood plasma and other samples. The analytic
procedures may require that the sample be combined with one or
more reagents so that some detectable change occurs which may
be related to the presence and/or amount of a particular
component (analyte) or characteristic of the sample. For
instance, the sample may undergo a reaction or other change
which results in a change in color, fluorescence,
luminescence, or the like, which may be measured by
conventional spectrophotometers, fluorometers, light
CA 02192196 2004-02-05
7
detectors, and the like. In some cases, immunoassays and
other specific binding assays may be performed within the
cell-free fluid collection chamber or within cuvettes which
are connected to the collection chamber. Generally, such
assay procedures should be homogeneous and not require a
separation step. In other cases, however, it may be possible
to accommodate heterogeneous assay systems by providing a
means to separate the sample (e.g., blood plasma) from the
collection chamber or another test well or cuvette after the
immunological reaction step has occurred. One of skill will
recognize that the means of analyzing the sample is not an
important aspect of the invention. Any of a number of
analytical methods can be adapted for use in the rotors of the
invention, depending upon the particular sample being analyzed
and component being detected.
In the case of blood analyses, conventional blood
assays are typically preformed. Examples of assays which may
be performed include those designed to detect glucose, lactate
dehydrogenase, serum glutamic-oxaloacetic transaminase (SGOT),
serum glutamic-pyruvic transaminase (SGPT), blood urea
nitrogen (HUN), total protein, alkalinity, phosphatase,
bilirubin, calcium, chloride, sodium, potassium, magnesium,
and the like. This list is not exhaustive and is intended
merely as being exemplary of the assays which may be performed
using the apparatus and method of the present invention.
Usually, these tests will require that the blood and plasma be
combined with one or more reagents which result in an
optically detectable, usually photometrically detectable,
change in the plasma. The reagents Which are required are
well known and amply described in the patent and scientific
literature.
The reagents are preferably provided in lyophilized
form to increase stability. Ideally, they are provided in the
form of lyophilized reagent spheres as described in U.S. Patent
No. 5,413,732.
Referring now to Figures lA-F, an analytical rotor
comprising the chambers and channels of the present invention
can be seen. Figure lA shows the position of a blood sample
CA 02192196 2004-02-05
8
102 in the blood application chamber 104 after the sample has
been loaded in the rotor body 100. A diluent container in
chamber 106 is opened upon mounting of the rotor on the
spindle of the centrifuge as described in copending and
commonly assigned application, U.S. patent No. 5,275,016.
Fig. 18 shows the position of the diluant 108 and
blood sample 102 after the rotor is spun at 4,000 rpm. The
blood sample 102 begins to exit the blood application chamber
104 and enters the plasma metering chamber 110. At the same
time, diluent 108 empties from the diluent container into the
holding chamber 112. The diluent immediately begins to enter
the diluent metering chamber 114 through channel 116.
Fig, iC shows the position of the liquids as the
rotor 100 continues to spin. Here, the blood sample 102 has
emptied the blood application chamber 104 and overflows the
plasma metering chamber 110 into the overflow chamber 118
where it flows to the hemoglobin cuvette 120 and the excess
blood dump 122. Meanwhile, diluent 108 fills the dilusnt
metering chamber 114 and excess flows through channel 124 to
diluent-only cuvettes 126 and excess diluent dump 127.
Fig. iD shows the position of the liquids at the
conclusion of the first spin. The blood sample 102 has
separated into cells 128 and plasma 130. The diluent-only
cuvettes 126 are filled and a predetermined amount of diluent
remains in the diluent metering chamber 114. The rotor 100 is
then stopped and the siphon 132 from the diluent metering
chamber 114, as well as the siphon 134 from the plasma
metering chamber 110, are allowed to prime, as described
above. Siphon 134 is a siphon of the present invention. It
is connected to the plasma metering chamber 110 at inlet 138.
The inlet 138 is position radially outward of the siphon
outlet 139, through which the siphon 134 empties into the
mixing chamber 136.
Fig. lE shows the position of the liquids during the
second spin of the rotor. The diluent metering chamber 114
empties into the mixing chamber 136 through siphon 132. A
predetermined amount of plasma 130 is metered into the mixing
R'O 95133986 , ~ . ~ PCT/US95107145
9
chamber 136 and the two fluids are mixed, thereby forming
diluted plasma 131. The amount of plasma 130 delivered to the
mixing chamber 136 is determined by the position of the outlet
139 on the siphon 134. As can be seen in this figure, the
final level of the plasma 133 in the plasma metering chamber
110 is at the same radial position as the outlet 139. Thus,
the volume of plasma delievered to the mixing chamber 136 is
determined by the volume of the plasma metering chamber 110
between the exit to the overflow chamber 129 and the final
level of plasma 133. After the plasma and diluent are mixed
in the mixing chamber 136, the rotor is stopped again and the
output siphon 140 is primed.
Fig. iF shows the position of the diluted plasma 131
as the rotor is spun during the third spin. This figure
illustrates the movement of the diluted plasma 131 through the
distribution ring 142 and inlet channels 144 to the cuvettes
146 and excess plasma dump 147. The resistance to flow in the
output siphon 140 is selected to be higher than the resistance
to flow in the distribution ring 142 and the inlet channels
144 so that air present in the cuvettes 146 can escape as the
cuvettes are filled. Specifically, siphon 140 is dimensioned
such that the ratio of the cross sectional area of the inlet
channels 144 to the cross sectional area of the liquid in them
is greater than 2:1, preferably greater than about 4:1. The
cross sectional area of the inlet channels 144 is typically
the same as or slightly smaller than that of the distribution
channel 142 so that gas in the unvented cuvettes escapes
through the inlet channels 144 and distribution 142. If the
sample is plasma or diluted plasma and the channels are
rectangular in cross-section, their dimensions are typically
as follows: siphon: 0.150 mm depth, 0.200 mm width;
distribution channel 0.300 mm depth, 0.50omm width; inlet
channels: 0.150 depth, 0.500 width.
After the cuvettes have been filled, reagents
present in the cuvettes are mixed with the solution and the
necessary photometric analyses are made on the sample. Such
analyses are carried out as described above according to
methods known to those of skill in the art.
WO 95133986 ~ ~ ~ ~ PCTIUS95107145
Although the foregoSing invention has been described
in detail for purposes of.clarity of understanding, it will be
obvious that certain modifications may be practiced within the
scope of the appended claims.
