Note: Descriptions are shown in the official language in which they were submitted.
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CO2dPOSITIONB AND MBTHODS FOR THE RAPID
ANALYBIS OF RBTICIILOCYTBS
The present invention relates to a method and stable
aqueous reagent compositions for the rapid analysis of
reticulocytes. More particularly the present invention relates
to automatable methods and stable aqueous reagent compositions
for the rapid analysis of r,eticulocytes utilizing light scatter
and fluorescence flow cytometric techniques. The methods and
stable aqueous reagent compositions enable the rapid analysis
of reticulocytes, reportable by count and maturity, on high
throughput multi-parameter hematology instruments. The method
also permits the real-time analysis of reticulocytes and
complete blood cell counts ("CBC"), including nucleated red
cell counts ("NRBC"), without a separate incubation step.
BACKGRntTND
Red blood cells ("RBCu) normally enter the blood stream as
reticulocytes. Erythropoiesis begins with the erythroblast and
proceeds through about five generations of intermediate,
nucleated cells in the bone marrow, and ending with the
reticulocyte. The reticulocyte is an immature red blood cell
which still contains reticular material (ribosomal and
messenger RNA) even though at this stage of it's development
the cell -has expelled the nucleus.
Under anemic or hypoxic conditions this process may be
shortened. Reticulocytes of an earlier stage than normal may
enter the blood stream under these conditions. These early
reticulocytes are recognized by the extra quantity of RNA they
contain, as well as their larger size, lower content of
hemoglobin, and by the greater length of time they persist as
reticulocytes in the blood stream.
In the early 19301s, L. Heilmeyer (Ztschr. Klin. Ned.
121:361-379, 1932) differentiated reticulocytes into four age-
groups according to the quantity of reticulum (vitally
stainable substance contained within reticulocytes) they
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contain. Traditionally, Group I contains 30 or more reticulum,
Group II contains 15-30, Group III contains 3-15, and Group IV
contains 1-2 reticulum. The normal reticulocyte count is from
about 0.5 to 2.5% of the RBC count for adults, and from about
E. 2.0 to 6.0% of the RBC count for newborn infants. In terms of
age groups, a normal adult will have the majority of
reticulocytes in group IV, and none i.n group I.
The reticulocyte count is a measure of red cell
production. It is useful, therefore, in the diagnosis and
treatment of anemia. Historically, reticulocyte counts have
been very closely associated with the etiology and
classification of the anemias. The reticulocyte count is
increased in hemolytic anemias, pyruvate kinase deficiency,
sickle cell anemia, thalassemias, and decreased in
megaloblastic anemia, aplastic anemia, general bone marrow
dysfunction. Recently, the reticulocyte count has also been
used to assess the toxic effects of chemotherapeutic agents on
the marrow. Therefore, hematologists have unanimously promoted
the reticulocyte count and reticulocyte maturity index as very
valuable information.
The most: commonly used method for counting reticulocytes
is the manual microscopic procedure. Brilliant Cresol blue was
predominantly used until New Methylene Blue ("NMB") was
introduced in 1949 by G. Brecher (Am. J. Clin. Pathol. 19:895-
896, 1949). The most recent National Committee for Clinical
Laboratory Standards ("NCCLS") publication (NCCLS DOC. H44-P march 1993)
endorses the NMB stain in which an equal volume of blood is
mixed with NMB stain and incubated for about fifteen minutes to
allow the RNA to precipitate. Blood smears are then made, and
the stained reticulocytes are counted microscopically. A
Miller ocular disc is inserted into an eyepiece. The area of
the smaller square viewed within the eyepiece is 1/9 that of
the larger square. The RBC are enumerated in the smaller
square, while the reticulocytes are enumerated in the larger
square. Twenty successive fields are counted and the % of
reticulocytes is calculated by dividing total reticulocytes in
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large squares by total red blood cells in small squares after
multiplying by 9. The draw back of the manual method is that
it is labor intensive, imprecise, time-consuming and
subjective.
Many attempts have been made to correct these shortcomings
by means of flow cytometric technology. In the 1980's pyronin
Y("PY") and Acridin Orange ("AO") were used to stain and count
reticulocytes on flow cytometers. Several semi-automated, flow
cytometric methods are now available to enumerate reticulocytes
lo from a whole blood sample. In each of the existing methods, a
diluent containing an organic cationic dye, such as Auramine 0
("AuO"), or Thiazole Orange ("TO') is used to stain the RNA
within the reticulocytes. During the incubation period, the
dye slowly penetrates the cell membrane and binds to the RNA
within each reticulocyte. The amount of signal generated by
the stained reticulocytes as the sample passes through the
detection zone is roughly proportional to the RNA content
within each reticulocyte. A flow cytometer equipped with the
proper excitation light source and emission detection system
can, therefore, be=used to determine the percentage of
reticulocytes in a whole blood sample.
However, there are many sources of problems complicating
the automation of reticulocyte methodologies, especially when
the instrument is a multi-parameter hematology system. The
most important of which is the necessity to prepare a stained
sample "off-line". This sample preparation step requires a
lengthy incubation period of several minutes or longer before
the dye has penetrated the cell to sufficiently stain the
reticulocyte's RNA to enable the sample to be processed through
a flow cytometer. 3n addition, the majority of the dyes
currently in use to stain RNA in the reticulocytes are not
stable in aqueous solutions and bind not only to RNA and DNA
but can also bind to the instrument's plastic tubing, glass
surfaces, including the flow cell. This requires the long and
arduous task of cleaning the complete system after running the
test.
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U.S. Patent 3,684,377 to Kaminsky, et al., U.S. Patent
3,883,247 to Adams and U.S. Patent 4,336,029 to Natale, all
disclose the use of AO for staining reticulocytes. Although AO
has excellent properties for staining reticulocytes (it binds
to RNA and generates red fluorescence), it also binds to plasma
and generates high background sample stream fluorescence,
making it very difficult to obtain a good signal to noise
("S/N") ratio. Even more problematical, using AO on a flow
cytometer is AO's propensity to bind to plastic tubing and flow
cell surfaces, adding to the detection problems. In addition,
the AO methods require 5 to 7 minutes to incubate which is inuch
too long a time period to be incorporated onto the high
throughput multi-channel hematology analyzers of today.
U.S. Patent 4,707,451 to Sage, Jr. discloses a reagent
composition comprised of thioflavin T or chtysaniline. The dye
uptake by reticulocytes takes about 7 minutes in this method
and the background fluorescence is too high to obtain a good
S/N ratio in Group IV reticulocyte detection.
U.S. Patent 4,883,867 to Lee et al. discloses a dye for
staining RNA and DNA. TO is their preferred dye for
reticulocyte analysis and the method requires a minimum
incubation time of 30 minutes. Although TO has a high nucleic
acid binding affinity and quantum yield, the rate of membrane
permeation by TO is very slow (30 minutes) and thus, is
unsuitable for use on a high throughput multi-parameter
clinical hematology instrument.
U.S. Patent 4,971,917 to Kuroda teaches a reagent
composition which contains AuO and a carbonate salt to reduce
the non-specific staining of the mature erythrocytes. U.S.
Patent 4,985,176 dis'cioses another reticulocyte staining
reagent for flow cytometric use in which AuO is included as a
preferred dye and the sample incubation time for staining is
anywhere between 30 seconds to 20 minutes. The disadvantages
of using AuO on a flow cytometer is that the dye, like AO, has
:5 an affinity not only to DNA and RNA but also to various types
of plastic and glass surfaces, making it extremely difficult to
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incorporate the methods into a multi-parameter hematology
instrument. TOA Medical Electronics Co., Ltd. has managed to
incorporated an AuO method onto it's Sysmex R-1000 and R-3000
automated, stand-alone reticulocyte analyzers, but has been
unable to incorporate the method onto their multi-parameter
hematology instruments such as the NE8000 or SE9000
instruments.
U.S. Patent 5,284,771 to Colella et al. discloses a method
and a reagent composition which comprises treating a whole
blood sample with a single reagent solution containing a
cationic dye (Oxazine 750) to stain RNA in reticulocytes and a
zwitterionic sphering agent to eliminate the orientational
noise of the flat red blood cell when the stained subjected to
flow cytometric light measurement system.
Recently, Miles Inc. has incorporated the above method on
their latest H*3 hematology instrument. This instrument is
equipped with a helium/neon ("HeNe") electro-optical detection
system and Oxazine 750 nucleic acid dye is used to stain
reticulocytes while a zwitterionic surfactant is used to sphere
the red blood cells and reticulocytes in preparation for
volumetric measurements. The H*3 reticulocyte method is only
a semi-automated method which requires manual mixing of the
blood with the reticulocyte reagent followed by a 15 minute
off-line incubation. The sensitivity of the method in
detectinq Group IV reticulocytes is poor.
Coulter Corporation has also incorporated a semi-automated
reticulocyte method on their STKS and Maxine hematology
analyzers. Coulter calls their technology: VCS (Volume,
Conductivity and Scatter). In practicing VCS, a blood sample
is first mixed with Coulter's retic dye (which contains NMB).
The mixture is then incubated at room temperature for 5
minutes. Then, a small volume of the stained blood sample is
diluted with a clearing solution which contains sulfuric acid.
The accuracy of the method suffers greatly from poor
sensitivity and a poor resolution of the red blood cells. This
is due to reticulocyte stroma (lysed by the clearing solution),
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other cellular debris and platelets creating noise, making it
very difficult to differentiate the proper signals. Only
clumped platelets will generate signals that are larger and
which, therefore, will not interfere with the signals generated
by RBC and reticulocyte stroma.
U. S. Patent 4,981,803 to Kuroda discloses a reagent
containing AuO for reticulocyte counting by flow cytometric
methods which comprises two solutions, a stock solution for
staining, in which a dye (AuO) is dissolved in a non-aqueous
3.0 solvent, and a buffer solution which satisfies the optimum
staining conditions for RNA in reticulocytes. Kuroda claims
that by combining these two solutions immediately before
staining, a stable final staining solution for reticulocyte
counting can always be obtained. The problems of this method
is are many. Most importantly, the group of non-aqueous solvents
that they selected (ethylene glycol, triethylene glycol,
diethylene glycol) have a high, refractive index. Such indices
of refraction make them unsuitable for use on a multi-parameter
hematology analyzer. This is due to the aqueous sheath
20 solution which is necessary (such as phosphate buffered saline)
and which has a refractive index of about 1.334. As described
in the Kuroda patent, other solvents, such as methanol, ethanol
or propanol (which have refractive index close to saline
sheath) are highly volatile and thus, are also unsuitable.
25 Additionally, it is technically very difficult to mix such
small amounts of stock solutions with large volumes of aqueous
buffer on-line, as is required by this methodology, to provide
a stable reticulocyte reagent.
Therefore, a long felt need has existed for a rapid and
30 accurate method for reticulocyte analysis which can be
incorporated on a flow cytometer or a high throughput multi-
parameter hematology analyzer while'utilizing a reagent that is
stable in an aqueous environment.
'SH~
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Summary of the Invention
A method for the simultaneous enumeration of CBCs and
reticulocytes in a whole blood sample, without the need for a
separate incubation step for the reticulocyte enumeration, is
provided.
One embodiment provides for an aqueous, nucleic acid
staining reagent which comprises a reticulocyte staining
amount of an unsymmetrical cyanine dye, from about 20 mM to
about 60 mM of a buffer solution selected from the group
consisting of imidazole, Hepes, Bis-Tris and Tris buffers, and
a dye stabilizing amount of a non-ionic surfactant selected
from the group consisting of N,N-bis[3-D-Glucon-
amidopropyl]cholamide, n-Dodecyl-D-Maltoside, a
polyoxypropylene-polyoxyethylene block copolymer, n-
Tetradecyl-D-Maltoside, Decanoyl-N-methyl-glucamide, n-
Dodecyl-D-glucopyranoside and n-Decyl-D-glucopyranoside. The
reagent has a pH from about 6.0 to about 8.0 and an osmolarity
adjusted to about 230 to about 340 mOsm/1 with mono-, or di-,
valent alkali salts which do not interfere with the cyanine
dye or precipitate in the aqueous reagent solution.
In another embodiment a method of enumerating
reticulocytes is provided. First an aliquot of a whole blood
sample is mixed with the aqueous, nucleic acid staining
reagent. This sample/reagent aliquot is then transported to
an optical flow cell for analysis without separately
incubating the aliquot. While passing the cells through an
illuminated optical flow cell, essentially one cell at a time,
the light scatter and the fluorescent characteristics are
detected and the enumeration of reticulocytes in the sample is
determined therefrom.
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More particularly, in one aspect there is provided a
method of enumerating reticulocytes from a whole blood sample
while simultaneously differentiating a separate aliquot of the
sample to obtain a complete blood cell ("CBC") analysis,
wherein said method comprises: a) directing one or more
aliquots of the sample to various positions within an
automated analyzer for analysis and differentiation; b)
combining a reticulocyte aliquot of the sample with a reagent
of the invention, while directing the combined
reagent/reticulocyte aliquot to an optical sensing zone of an
automated analyzer; c) causing the reagent/reticulocyte
aliquot to pass through the sensing zone essentially one cell
at a time; d) illuminating the stained sample in said optical
sensing zone with an incident light beam to cause fluorescence
events; e) measuring the fluorescence events for the
reticulocytes in said sample solution as the stained
reticulocytes pass through said optical sensing zone; and f)
determining the number of reticulocytes present in said sample
by counting the measured fluorescence events of the stained
reticulocytes.
In another particular aspect there is provided a method
of enumerating reticulocytes from a whole blood sample,
wherein said method comprises: a) combining an aliquot of the
sample with a reagent of the invention, while directing the
combined reagent/reticulocyte aliquot to an optical sensing
zone of an automated analyzer; b) causing the
reagent/reticulocyte aliquot to pass through the sensing zone
essentially one cell at a time; c) illuminating the stained
sample in said optical sensing zone with an incident light
beam to cause fluorescence events; d)measuring the
fluorescence events for the reticulocytes in said sample
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solution as the stained reticulocytes pass through said
optical sensing zone; and e) determining the number of
reticulocytes present in said sample by counting the measured
fluorescence events of the stained reticulocytes.
A further embodiment provides an aqueous, nucleic acid
staining reagent which comprises from about 0.1 g/ml to about
0.3 g/ml of a dye selected from the group consisting of 2-
butyl-4-(2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide, 2-diethylamino-4-
(2, 3-
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dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, 2-methylthio-4-(2,3-dihydro-3-methyl-
(benzo-2,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium
iodide and 4-(2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium tosylate (all obtainable from
Molecular Probes, Eugene, OR), from about 40 mM to about 60 mM
of imidazole buffer, and a dye stabilizing amount of a non-
ionic surfactant selected from the group consisting of N,N-
bis[3-D-Glucon-amidopropyl]cholamide and a polyoxypropylene-
polyoxyethylene block copolymer wherein the reagent has a pH
from about 6.8, to about 7.2 and an osmolarity adjusted ~o about
280 to about 310 mOsm/1 with a mono-, or di-, valent alkali
salt selected from the group consisting of NaCl, KC1, LiCl,
CaC12, MgC12 and 2nC12.
i5 In yet another embodiment a method for the simultaneous
enumeration of reticulocytes and a complete blood cell count is
provided. While an aliquot of a whole blood sample is being
analyzed for a CBC determination ri second aliquot of the sample
is being mixed with the aqueous, nucleic acid staining reagent
and the reticulocyte component of the sample is analyzed as
described above.
Rrief Descrintion of the Drawinc4
Figure la is a two dimensional FACScan display
(cytogram) of forward Scatter ("FSC") vs green fluorescence
("FLI") signals of a clinical sample with 5.63 % reticulocytes.
Figure lb is an FLl histogram of the gated red blood cell
("RBC") population of Figure la.
Figure lc is 4 FACScan. cytogram of FSC vs FL1 signals of
a clinical sample with 2.63 % reticulocytes and elevated white
blood cell ("WBC") population.
Figure id is an FL1 histogram of the gated RBC population
of Figure ic.
Figures 2a and 2b are graphical depictions of the results
of a time study performed on a FACScan instrument.
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Figure 3a is a FACScan cytogram of FSC vs FL1 signals of
a clinical sample with elevated reticulocytes
Figure 3b is the FL1 histogram of the gated RBC population
the blood sample of Figure 3a.
Figure 4a is an Abbott Laboratories hematology analyzer
cytogram of a sample of normal blood showing the four decade
log Intermediate Angle Light Scatter ("IAS") vs the four decade
log FLl.
Figure 4b is a FL1 histogram of the gated RBC population
and reticulocyte population from Figure 4a. The lx scale
display of the histogram shows the RBC Peak and the 5x scale
displays of the histoQram exhibits the reticulocyte population.
Figure 5a is an Abbott Laboratories hematology analyzer
cytogram of IAS vs FLl of a clinical blood sample with very low
reticulocytes but elevated WBC.
Figure 5b is a FLI histogram of the gated RBC and
reticulocyte population from Figure 5a. Both lx and 5x scale
displays of the histogram are shown.
Figure 6a is an Abbott Laboratories hematology analyzer
cytogram of IAS vs FL1 of an anti-coagulated clinical blood
sample with elevated reticulocyte (20.5 %) and WBC (96.4k/L)
levels.
= Figure 6b is a FLl histogram of the gated RBC and
reticulocyte population from Figure 6a. Both 2x and 5x scale
displays-ot the histogram are shown.
Figure 7 is a graphical depiction of the comparison of
reticulocyte results obtained by practicing the National
Committee for Clinical Laboratory Standards (NNCCLS") method vs
an automated method of the present invention.
3o Figure 8 is a. graphical depiction showing a comparison of
linear regression data for reticulocyte results obtained by
practicing a thiazole orange staining method vs an automated
method of the present invention for detecting reticulocytes.
The same clinical samples presented in Figure 7 were used for
the analysis.
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Figure 9 is a graphical depiction of the linearity of the
~ reticulocyte measured by practicing an automated method of
the present invention.
Figure 10 is a shelf-life study emission spectra of the
RNA bound proprietary cyanine dye, 2-butyl-4-(2,3-dihydro-3-
methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquinoliniuin iodide (Molecular Probes, Eugene, OR), in the
presence and absence of various non-ionic surfactants. The
reagents were kept at ambient temperature for three and one-
half months.
Figure 11 is a shelf-life study emission spectra of the
RNA bound cyanine proprietary dye, 2-butyl-4-(2,3-dihydro-3-
methyl-(benzo-l,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, demonstrating four month stability
is under refrigeration.
Figure 12 is a shelf-life study emission spectra of the
RNA bound proprietary cyanine dye, 2-butyl-4-(2,3-dihydro-3-
methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, demonstrating a three week elevated
temperature stability.
Figure 13 is a time-study emission spectra of the RNA
bound proprietary cyanine dye, 2-methylthio-4-(2,3-dihydro-3-
methyl-(benzo-l,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide (Molecular Probes, Eugene, OR), in
Bis-Trisbufferwith and without 1.0 $ Polyoxypropylene-
polyoxyethylene block copolymer ("Pluronic(D F1271) and stored
at ambient temperature for six and one-half months.
Figure 14 is a shelf-life study emission spectra of the
RNA bound proprietary cyanine dye, 2-butyl-4-(2,3-dihydro-3-
methyl- (benzo-1, 3-t-hiazol-2-yl) -rnethylidene) -1-
phenylquinolinium iodide, in different buffers with 0.2 ~
Pluronic F127. All reagents were stored at room temperature
for six weeks.
Figure 15a is a FACScan cytometer two dimensional
display of FSC vs FL1 signals.
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Figure 15b is the FLl histogram of the gated red cell
population of Figure 15a.
Figure 15c is a time study performed on the FACScan
flow cytometer expressed as reticulocyte % from 15 seconds up
to 4.0 minutes.
D taiied DescriDtion of tre Tnv nion
Embodiments of the present invention comprise methods and
stable aqueous reagent systems for the rapid and accurate
analysis of a blood eample for reticulocytes by utilizing flow
cytometric methodologies. Generally, these embodiments relate
to the rapid and simultaneous analysis of reticulocytes and
complete blood cell counts ("CBC"), including nucleated red
blood cells ("NRBC ). Embodiments of the present invention can
comprise the use of an analytical instrument and a method for
analyzing blood. Generally, one such analytical instrument
includes an automated impedance analyzer. In one such
automated instrument an automated light scatter analyzer is
integrated with the automatedimpedance analyzer, and an
automated fluorescence analyzer is integrated with the
automated light scatter analyzer.
For the sake of this disclosure, an automated analyzer is
distinguished in that an operator does not need to intervene in
analysis-of the sample, viz, after the operator presents the
sample to the automated analyzer, no further operator
intervention is required. Additionally, a hematology analyzer
quantifies and classifies cells in substantially absolute
terms. Such an analyzer uses at least one of electrical
impedance and optical scattering properties of the cells to
classify cells by size and shape.
Implementations of the invention can generally utilize an
automated hematology analyzer with a controller which monitors
and controls the various analyzers, collects data from the
analyzers and reports a result. Illustrating by example,
integration of the analyzers with a controller allows an
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operator to input data about a whole blood sample into the
controller. The operator selects a bank of tests to be
performed on the whole blood sample with the aid of the
controller. The operator presents the whole blood sample to
the integrated analyzers at a centralized sample processing
area. The controller activates the analyzers, allowing the
analyzers to automatedly perform analyses on the whole blood
sample under the direction of the controller. The controller
utilizes data obtained from the analyzers to formulate a
result. The controller reports the result to the operator. It
is to be noted that no operator action is needed after the
whole blood sample is presented to the integrated analyzers.
Because the whole blood sample preparation is entirely
automated, in a preferred embodiment, conventional hematology
zs tests are done first with the incubated sample tests to follow.
Secause the analyzers are integrated with the controller, the
controller obtains data from both the hematology analyzer and
the flow cytometry analyzer. Thus,- the controller is able to
report a combined patient blood analysis to the operator.
While specific embodiments of the invention will be
discussed in detail to clarify understanding, it is to be
remembered that other embodiments are also possible. Any
desirable combination of elements of the described embodiments
is also possible.
In one aspect of the invention a stable, aqueous reagent
composition is provided. This reagent comprises: an
unsymmetrical cyanine dye capable of staining reticulocytes,
from abollt (i0 liLLl ~1..o abo11t: C7V 11LL1 Vf a Lllffter seie\~..ted from
tiae
group consisting of Imidazole buffer, 4-(2-Hydroxyethyl)-1-
peperazineethane-sulfonic acid ("Hepes") buffer, Bis (2-
Hydroxyethyl)-1-piperazineethane*-sulfonic acid ("Bis-Tris")
buffer and Tris Hydroxyanethyl Aminomethane ("Tris") buffer; a
pH from about 6.0 to about 8.0; an osmolarity adjusted to about
230 to about 340 mOsm/L with a mono, or di, valent alkali salt;
and a non-ionic surfactant (from about 5 mg/dl to about 1.0
g/dl depending on the surfactant) which facilitates the
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membrane permeation and stabilizes the cyanine dyes in an
aqueous isotonic solution. Preferably the dyes are cyclic
substituted and exhibit enhanced fluorescence upon binding with
DNA or RNA. Even more preferably, the reagent comprises from
S about 0.1 ug/ml to about 0.3 ug/ml of a cyclic substituted,
unsymmetrical cyanine dye.
Another aspect involves methods for the rapid and
continuous detection and enumeration of reticulocytes and CBC
differentials, utilizing the present inventive reagent system.
Such methods are distinct due to 'the particular absence of the
need to provide for a separate incubation step. The incubation
minimal period required, from about 10 to 60 seconds is all
that is necessazy.
One such embodiment is a method of enumerating
is reticulocytes from a whole blood sample while simultaneously
differentiating a separate aliquot of the sample to obtain a
complete blood cell ("CBC") analysis. This method comprises,
directing one or more aliquots of the sample to various
positions within an automated analyzer for analysis and
differentiation, while a reticulocyte aliquot of the sample is
combined with a staining reagent. This reagent comprises, a
reticulocyte staining amount of an unsymmetrical cyanine dye,
from about 20 mM to about 60 mM of a buffer solution, selected
from the group consisting of imidazole, Hepes, Bis-Tris and
Tris bufi-ers, and a dye stabilizing amount of a non-ionic
surfactant selected from the group consisting of N,N-bis[3-D-
Glucon-amidopropyl] cholamide, n-Dodecyl-D-Maltoside, a
polyoxypropylene-polyoxyethylene block copolymer, n-Tetradecyl-
D-Maltoside, Decanoyl-N-methyl-glucamide, n-Dodecyl-D-
glucopyranoside and n-Decyl-D-glucopyranoside, wherein said
reagent has a pH from about 6.0 to about 8.0 and an osmolarity
adjusted to about 230 to about 340 mOsm/1 with a mono-, or di-,
valent alkali salt which does not interfere with the cyanine
dye or precipitate in the aqueous reagent solution such as
NaCZ, KC1, LiCl, CaC12, MgC12 and ZnC12. The combined
reagent/reticulocyte aliquot is then directed to an optical
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sensing zone of an automated analyzer. Thereafter the
reagent/reticulocyte aliquot is passed through an illuminated
sensing zone essentially one cell at a time to cause
fluorescence and scattered light events. These events are
S detected and the number of reticulocytes present in said sample
are determined therefrom.
The unsymmetrical dyes usable with the reagent system of
the present invention generally have the following
characteristics;
1. Absorption Maxima : 488 +20 nm
2. High nucleic acid binding affinity
~:.
3. High quantum yield . 0.1
4. Molar Extinction Coefficient: 10,000
5. Fluorescence Enhancement upon binding to RNA
or DNA: 20
6. Membrane Permeation Rate: <2 minutes
Typically, the dyes utilized in this inventive aqueous
reagent and reticulocyte enumerating methods are highly
unstable in aqueous environments. The stability data for
several dyes tested in this class are shown in Figures-10
through 14.
One embodiment of the present reagent system comprises
from about 0.05 ug/ml to about 0.5 w/ml of 2-butyl-4-(2,3-
dihydro-3-methyi-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, a proprietary dye sold by Molecular
Probes, Inc. (Eugene, OR), from about 20 mM to about 50 mM
Imidazole buffer, and from about 5 mg/dl to about 20 mg/dl of
N,N-bis[3-D-Glucon-amidopropyl]cholamide ("BIGCHAP"), from
about 0.02 % to about 0.05 5t Proclin 300 (5-chloro-2-methyl-
4-isothiazoline-3-one + 2-methyl-4-isothiazoline-3-one). The
pH is adjusted to from about 6.8 to about 7.2 with 1N HC1 and
the Osmolarity adjusted with NaCi from about 270 to about 310
mOsm/L.
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A main ingredient of' the reagent system is the dye.
One such class of dyes are unsymmetrical cyanine dyes
such as those disclosed in W094/24213, "CYCLIC-
SUBSTITUTED UNSYMMETRIC DYES. Additionally, the dyes
utilized in this invention exhibit enhanced fluorescence upon
binding with DNA or RNA. Such useful dyes must also have high
binding affinity to RNA and DNA and a high quantum yield. It
is anticipated that. a variety of unsymrnetrical cyanine dyes
which exhibit the characteristics described and claimed herein
can be used. Some of the examples of such dyes include, but
are not limited to 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-l,3-
thiazol-2-yl)-methylidene)-l-phenylquinolinium iodide, 2-
diethylamino-4-(2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide, 2-methylthio-4-(2,3-
dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, also obtained from Molecular Probes,
Inc. (Eugene, OR) ("MPI"). Other unsyrranetrical cyanine dyes
such as 4-(2,3.-dihydro-3-;:nethyl-(benzo-l,3-thiazol-2-yl)-
methylidene)-1--phenylquinolinium tosylate, also sourced from
MPI, are also useful in practicing the present invention. 4-
(2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium tosylate is believed to be a neutral,
unsymmetrical cyanine dye comprising a substituted benzazolium
ring system linked to a methine bridge to a pyridinic or
quinoline ring system.
A further ingredient of the reagerit system is a buffer
whose pKa is from about 6.0 to about 8.0 and is capable of
maintaining the required (for staining RNA or DNA)
concentration of the cyanine dye in an aqueous solution iri an
extended period of time. Such buffers should not react with
the cyanine dyes or the non-ionic surfactants used in the
practice of this invention to stabilize the dye. Exemplary
buffers include Imidazole, Hepes, Bis-Tris, and Tris.
Another ingredient of the reagent system is a non-ionic
surfactant. Depending upon the surfactant, or combination of
non-ionic surfactants, that are use, the concentration should
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be from about 5 mg/dl to about 1 g/dl_ The surfactant(s)
appear to enhance the rate of the cyanine dye permeation
through the cell membrane (within 30 seconds). In addition,
the solubility and the stability of the cyanine dyes in an
isotonic aqueous solution are enhanced by the surfactant. Such
surfactant(s) should not, however, precipitate or react with
the cyanine dyes or lyse RBCs, even at the low concentrations.,
Examples of such surfactants are, but are not limited to,
BIGCHAP, n-Dodecyl-D-Maltoside, Polyoxypropylene-
polyoxyethylene block copolymer ('PluronicM F1270), n-
Tetradecyl-D-Maltoside, Decanoyl-N-methyl-glucamide, n-Dodecyl-
D-glucopyranoside and n-Decyl-D-glucopyranoside.
Yet another ingredient of the reagent system is a mono-,
or di-, valent alkali salt to adjust the osmolarity of the
reagent from about 230 mOsm/L to about 340 mOsm/L to prevent
the lysis of red cells, including the reticulocytes, or the
white cells. Such salts should not react with the either the
cyanine dyes or precipitate in solution. Examples of such
salts include NaCI, KC1, LiCl, CaClz, MgC12, ZnC12 and others.
An optional ingredient, is a preservative to prevent
microbial growth in the reagent. Such a preservative should
not change the light scattering or fluorescent emission
properties of the cells, or stained cells. Examples of such
preservatives include Proclin 300, Proclin 150 (5-chloro-2-
rnethyl-4-isothiazolin-3-one (1.05-1.25%) and 2-nmethyl-4-isothiazolin-3-one
(0.25-0.45%)), sodium azide and others.
Utilizing the above described reagent system the rapid
analysis of reticulocytes on a flow cytometer, or other
automated hematology analyzer, is.possible. In one embodiment,
5 l of a whole bload sample is mixed with 1.0 ml of the
reagent system disclosed and claimed herein, and the
sample/reagent mixture is run on a commercially available flow
cytometer within 30 to 60 seconds of mixing, and as little as
ten (10) seconds. Figures la through ld show the results of
such a method using a normal blood sample prepared as described
in Example 1.
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A further embodiment of the present invention allows
fo the rapid and quantitative analysis of reticulocytes
on an automated high throughput multi-parameter hemato-
logy analyzer. U.S. Patent 5,656,499 discloses
an automated hematology analyzer which utilizes light scatter
signals axial light loss ("ALL") and int.ermediate angle scatter
---("IAS"),-among others, as-well as detecting various fluorescent
.i0 _signals to enable the instru=ent to automatically differentiate
cells and subclasses cF cells-from a whole---blood samp~e:
Generally, however, a method for practicing the present
invention comprises the mixing of a whole blood sample with a
reagent to stain the RNA of any reti.culocytes present, flowing
15 the mixture, essentially one cell at a time, through an
illuminated optical flow cell, detecting the light scattered
and fluorescence emitted therefrom and determining the amount
of reticulocytes present in the sample without subjecting the
sample/reagent mixture to a separate incubation step or period.
20 One reagent of this invention comprises from about 0.1 ug/ml to
about 0.3 ug/ml of an unsymmetrical cyanine dye; from about 20
mM to about 60 mM of a buffer selected from the group
consisting of Imidazole, Hepes, Bis-Tris, and Tris buffers;
from about 5 mg/dl to about 1.0 g/dl oi: a non-ionic surfactant,
25 depending on the surfactant or combination of surfactants, and
the reagent has a pH from about 6.0 to about 8Ø; and an
osmolarity adjusted to about 230 to about 340 mOsm/L with a
mono-, or di-, valent alkali salt.
In order to ana-lyze a whole blood sample for the
30 percentage as well as t'r,e absolute counts of reticulocytes on
the multi-parameter hematology analyzer described above, about
18.75 ul of a whole blood sample is deposited by means of a
sample aspiration probe into an RBC cup which contains about
7856 ul of a diluent/sheath solution (an isotonic saline) and
35 the fluids are mixed. The diluted sample is then transported
to a sheathed impedance aperture to electronically determine
CA 02218728 2002-01-09
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the absolute RBC counts of the sample. (See details
of instrument calibration for RBC analysis in U.S.
Patent 5,656,499). In the mean
time, about 200 ul of the diluted sample is transferred into
another receptacle ("reti.culocyte receiving cup"), which
contains 600u1 of the reagent of the present invention, where
it is mixed. The prepared (mixed) sample is then transported
to the sheathed optical flow cell for detection. The
measurement process begins as the cell stream passes through
the flow cell essentially one cell at a time, in a laminar
flowing sample stream surrounded by a diluent/sheath solution.
The volume is illuminated. by a beam of light and is bounded in
the two dimensions normal to the flow axis by the
hydrodynamically focused cell stream, and in the dimension
parallel to the flow axis by the vertical beam waist of the
laser beam which is about 17 microns. When doing this test,
the sample flow rate is about 2.0 ul. per second, and the
corresponding illuminated sensing volume of the RBC and
reticulocyte sample stream approximates an elliptical cylinder
with dimension of about 80 x 5 x 17 microns. The 17 micron
dimension is measured along the axis of the cylinder.
At this point, and as shown in the two dimensional feature
space of IAS and FL1 of the cytogram of Figure 4a, the presence
of a cell is detected by an intermediate angle scatter photo-
diode which detects light in a 3 to 100 cone, and a
photomultiplier tube ("PMT") which detects green fluorescence,
FL1. When cells pass through the aforementioned illuminated
volume, pulses are generated and measured by these detectors.
The amplitudes of these pulses are then filtered, amplified,
digitized, and stored in list mode in the corresponding two
dimensional feature space of IAS and FL1. The cells are
counted for 8 seconds. At: the flow rate and the dilution ratio
described above, with a normal subject RBC counts of 5 millions
per microliter of blood volume, the resulting event count rate
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would be 5950 per second. Algorithms are then applied to the
list mode data of the aforementioned feature space of IAS and
FL1 and the following parameters are measured within 20 seconds
of computational time:
1. RBC gate: WBCs and platelets are excluded by gating
the RBC population, including reticulocytes, but excluding WBCs
and platelets.
2. The percent of reticulocytes: The gated RBC
population is re-analyzed according to the size of their FL1
signals. A log fit is applied to the FL1 histogram to define
the region which belongs to mature RBCs, and the cells whose
FL1 signals fall above the region are labeled as reticulocytes.
Reticulocyte % is computed by dividing the counts of
reticulocytes by the total RBC counts.
3. The absolute reticulocyte counts: Obtained by
multiplying the percent of reticulocytes by absolute RBC counts
of the sample from the CBC mode.
4. Reticulocyte Maturity Index ("RMI"): RMI is expressed
as the percent of reticulocytes whose FL1 signals'are more than
one (1) standard deviation ("S.D.") above the mean fluorescence
of a normal reticulocyte population.
Such a description is merely for convenience and by no
means is the expression of RMI of the present invention limited
to only the algorithms discussed herein.
The f-ollowing examples set forth reagent compositions and
methods incorporating the same for the rapid analysis of
reticulocytes using light-scatter and fluorescence flow
cytometric techniques. It will be understood that the
following examples are for illustrative purposes only and are
not intended to limit the scope o.f this invention in any
manner.
Ex=p le 1
Five (5) microliters of a clinical sample was mixed with
1.0 ml of the reagent comprising 50 mM imidazole buffer, pH
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adjusted with iN HC1 to 7.0 + 0.1, 6.4 g/L NaCl, 0.1 micrograms
per ml of 2-diethylamino-4-(2,3-dihydro-3-methyl-(benzo-1,3-
thiazol -2 -yl) -methylidene) -1 -phenylquinolinium iodide
(Molecular Probes, Inc., Eugene, OR), 0.2 $ Pluronic F127, and
0.03 % Proclin 300. The sample/reagent mixture is then run
within 30-60 seconds on a FACScan flow cytometer (Becton
Dickinson & Co.) to determine the rate of inembrane-permeation
by the dye in the reagent composition. Figure la is a two
dimensional display of forward Scatter (FSC) vs green
fluorescence (FL1) signals;, lb is the FL1 histogram of the
gated red cell population revealing the stained reticulocyte
population to the right of the mature RBC population. Figures
ic and 2d show the results of a second clinical sample
displayed a similar manner as Figures la and lb, respectively.
Exam3 e 2
Figure 2a is a time study performed on the FACScan flow
cytometer expressed as reticulocyte % over an extended time
period (from 30 seconds to 30 minutes) and 2b is expressed as
reticulocyte mean FL1. As can be seen in the Figures, the % of
reticulocyte reaches the steady state within 30 seconds and the
staining intensity reaches about 80% of the peak within 30
seconds. Such rapid staining by means of the present invention
enables.the incorporation of the method and reagents disclosed
herein, onto automated hematology analyzers, as we21 as flow
cytometers, without the need for separate incubation equipment,
or methods.
-- ~~~mD1 e
Five (5) microliters of a clinical samples with about 15 t
reticulocytes were mixed with 1.0 ml of the reagent of the
present invention comprised of 50 mM imidazole buffer, pH
adjusted with iN HC1 to 7.0 + 0.1, 6.4 g/L NaCl, 0.2
micrograms/mi of 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-l,3-
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thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide
(Molecular Probes, Inc., Eugene, OR), 0.2 % Pluronic F127,
and 0.03 % Proclin 300. The sample/reagent mixtures were then
run on a FACScan flow cytometer within 30 seconds to
determine the % of reticulocytes in the samples. Figure 3a is
a two dimensional display of FSC vs FL1 signals and Fig 3b is
the FL1 histogram of the gated red cell population. As the
data show, WBCs and platelets are well separated from the RBC
and reticulocyte population, the FL1 intensity of the stained
reticulocytes is such that defining the region of
reticulocytes in the FL1 histogram of red cells is easy for
quantitative analysis of reticulocyte concentration in a blood
sample.
Example 4
For this example, an automated, high-throughput multi-
parameter hematology analyzer (U.S. Patent 5,656,499) was
used. 18.75 ul of an EDTA-anti coagulated whole blood sample
from a normal subject was deposited by means of a sample
aspiration probe into the RBC cup which contains about 7856 ul
of a diluent/sheath solution (an isotonic phosphate buffered
saline) and mixed. The diluted sample was then transported to
a sheathed impedance aperture to determine the absolute RBC
counts of the sample. At the same time, about 200 microliters
of the diluted whole blood sample was transferred into the
reticulocyte cup which contains 600 microliters of the reagent
of the present invention. This reagent contained of 50 mM
imidazole buffer, pH adjusted with 1N HC1 to 7.0 +0.1, 6.4 g/L
of NaCl, 0.2 ug/ml of 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-
1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide, 10
mgs per ml BIGCHAP, 0.03 % Proclin 300. The sample/reagent
mixture was then transported to the sheathed optical flow cell
for scatter and fluorescence detection. The system detection
process has previously been described above, and in the
CA 02218728 2002-01-09
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contents of U.S. Patent 5,656,499. The dwell time
for the transportation of the sample/reagent mixture
can be as little as ten (10) seconds.
''he RBC population including reticulocytes is gated on the IAS
vs FL1 cytogram (Figure 4a; and the FL1 histogram of the gated
RBC population (Figure 4b) is used for reticulocyte counts and
RMI measurements. The lx display of the FL1 histogram is
scaled to show the RBC poptilation peak and the 5x display shows
t.he reticulocyte population. These results show that it is
possible to analyze a sample for reticulocytes while
simultaneously determining a CBC analysis, and without the need
for additional sample incubation equipment or methods, or off-
l.ine sample preparation steps.
Example 5
A clinical sample wi-h low concentration of reticulocytes
and elevated WBC, as determined by reference methods, is
d.eposited by means of a sanlple aspiration probe into the RBC
cup of the instrument described in U.S. Patent
5,656,499, which contains about 7856 l of a
diluent/sheath solution (arL isotonic saline) and mixed. The
diluted sample is then transported to the sheathed impedance
aperture to determine the absolute RBC counts of the sample.
In the interim, about 200 niicroliters of the diluted sample is
transferred into the reticulocyte cup which contains 600
microliters of the reagent of the presen' invention comprised
of 50 mM imidazole buffer, pH adjusted with 1N HC1 to 7.0 +
0.1, 6.4 g/L of NaCl, 0.2 micrograms per 1.0 ml of 2-butyl-4-
(2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide, 0.2% Pluronic F127 and 2.5 mg/dl n-
Dodecyl-D-Maltoside, 0.03% Proclin 300 and mixed. The
sample/reagent mixture was then transported.to the sheathed
optical flow cell 100 for detection. The system detection
CA 02218728 2002-01-09
- 23-
process has already been described above. The RBC population
including reticulocytes is gated on the IAS vs FL1 cytogram
(Figure 5a) and the FL1 histogram of the gated RBC population
(Figure 5b) is used for reticulocyte counts and RMI
measurements (Retic counts were too low to calculate RMI for
this sample). The Figure 5b FL1 histograms are scaled to show
both the RBC population peak (lx display) and the reticulocyte
population (5x display). No detectable reticulocyte population
is observable in this sample.
Examnle 6
For this example, a clinical sample with elevated
reticulocytes (20 % as determined by a reference method) and
elevated WBC (96 k/L) was used. The sample was processed as
described in Example 4. 7he results are presented in Figures
6a and 6b. As the data show, the present invention accurately
excludes WBCs and platelets and able to identify and count both
dim and bright reticulocytes. The system produced an elevated
RMI value of 69.2 % on this sample using the algorithm
descrified above.
Exam-ole 7
Seventy seven (77) clinical samples were analyzed on a
high throughput multi-parameter hematology analyzer as
described in Example 4 and also on a FACScan flow cytometer as
described in Example 1 but using a thiazole orange (TO) method
which requires 30 minutes of incubation at room temperature
(U.S. Patent 4,883,867). The NCCLS reference method
NCCLS DOC. H44P, March 1993 was also performed on each
sample. Then the results of the present, no incubation,
:_nventive method were compared to that of the TO and NCCLS
riethods. The linear regression plots are presented in Figures
7 and 8. As the data show, the results of the present
invention correlate well with that of both NCCLS microscopic
CA 02218728 2002-01-09
- 24-
method (R = 0.982, slope = 1.05, Y-intercept = 0.002) and the
TO method (R =: 0.986, slope = 0.964, Y-intercept = 0.005).
Example 8
Two EDTA-anti-coagulated rabbit blood samples were
obtained, one from a normal rabbit (sample #1) and another from
a rabbit with reticulocytosis (sample #2). Sample #1 contained
1.9 % reticulocytes and sample #2 contained over 90 %
reticulocytes. The linearity samples were prepared according
to the following protocol:
1. RBC concentrat,_on of both samples was adjusted to 3.5
+ 0.05 M/L.
2. Six (6) levels of linearity samples were prepared by
mixing the two samples as shown in the Table below:
Tube No. Sample #1 Samnle #2
1 0.00 ml 1.50 ml
2 0.30 ml 1.20 ml
3 0.60 ml 0.90 ml
4 0.90 ml 0.60 ml
5 1.20 ml 0.30 ml
6 1.35 ml 0.15 ml
3. The samples were analyzed on the hematology analyzer
of U.S. Patent 5,656,499:
4. The theoretical values for reticulocyte % and
absolute count per ul of the whole blood sample were calculated
according to the fol-lowing equation:
Theoretical Retic % of the sample =[(n/1.5) x A%] +
[(m/1.5) x B%] x 100 where:
n= volume of sample #1 in the linearity sample
m = volume of sample #2 in the linearity sample
1.5 = total volume of the linearity sample
A% = Retic % in the undiluted sample #1
Bo = Retic % in --he undiluted sample #2
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Theoretical Retic Absolute# ~[(n x A#) +(m x S#)]/2.5 x
Theoretical Retic % of the sample where:
n = volume of sample #1 in the linearity sample
m= volume of sample #2 in the linearity sample
1.5 = total volume of the linearity sample
A# = Retic # in the undiluted sample #1
B# = Retic # in the undiluted sample #2
S. The linearity curve was plotted using the abscissa
for the theoretical values and the ordinate for the values
obtained from the multi-parameter hematology instrument.
The results'are presented in Figure,9. As the data show,
the method and reagent of the present invention produce a
linear response up to reticulocyte concentration of 90%.
F2amnl e 9
The stability of several reagents prepared according to
the present invention, in which various surfactants are
evaluated is shown in Figures 10-14. This is accomplished by
adding RNA to the reagents and scanning the emission spectra of
the cyanine nucleic acid stains bound to RNA on a HITACHI
F4500 instrument (Excitation 488 nm) according to the
following-protocol:
1. RNA (Calf Liver RNA, Sigma Cat. No. R7250) solutions
(1 mg/ml) were prepared in deionized water.
2. 100 ul of either RNA stock solution was added to 1.6
ml of a test reagent of the present invention and mixed.
3. The emission spectrum of the cyanine nucleic acid
stain bound to RNA was then scanned from 45.0 nm to 650 nm on an
HITACHI F4500 instrument (Excitation 488 nm). The full scale
for RNA-bound cyanine dye is set at 2,000 for all test
reagents.
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ExamDle 10
Figure 10 is the emission spectra of the RNA bound 2-
butyl-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
s methylidene)-1-phenylquinoliniunt iodide dye (Molecular Probes,
Inc., Eugene, OR) in 50 mM imidazole buffer prepared as
described in Exampie 4 except that 5 different surfactants were
evaluated for their effectiveness in stabilizing the cyanine
dye, 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
methylidene) -1-phenylquinolinium iodide, in an aqueous
solution. All reagents were stored at room temperature for a
period of 3.5 months. Starting from the highest peak:
A = BIGCHAP (10 mg%);
B = Maltoside (5 mg%);
C = Pluronic F127 (0.2%);
D= Cholic Acid, Na Salt (10 mg%);
E = No surfactant added;
F = Caprilic Acid, Na Salt (10 mg%).
As the data show, BIGCHAP, Maltoside and Pluronic F127
are very effective in keeping the dye in an aqueous buffered
solution while Cho,lic Acid, Na salt and Caprilic Acid, Na Salt
were not. As a matter of fact, adding Caprilic Acid to the
reagent shortened the shelf-life of the dye.
Examti21 e 1-1
Figure 11 is the emission spectra of the RNA bound cyanine
dye, 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide, prepared as described
in Example 4 with BIGCHAP or Pluronic F127 surfactants and
stored at 4 C for 4 months. Starting from the highest peak:
A = Freshly prepared control reagent;
B = BIGCHAP (10 mg%);
C = Pluronic F127 (0.24);
D = Pluronic F127 (1.0%);
E = no surfactant.
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As can be seen in the figure, there is no significant
deterioration of the dye in the present invention during the 4
months test period.
Examp le 12
Figure 12 is the emission spectra of the RNA bound cyanine
dye, 2-butyl-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide, demonstrating a three
lo (3) week elevated temperature stability in the reagent of
present invention with 5 mgs % BIGCHAP. The test reagents were
prepared as described in Example 4 and RNA addition was
prepared according to Example 9 protocol. Starting from the
highest peak:
A = 4 C;
B = 25 C;
C = 37 C;
D = 45 C.
As the data demonstrates, the addition of the nonionic
surfactant=, BIGCHAP, to an appropriate aqueous buffered
solution makes the cyanine dye less sensitive to elevated
temperatures, and therefore, more stable.
Ex mn 1 ~ ~
-
Figure 13 is the emission spectra of the RNA bound 2-
methylthio-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide in Bis-Tris buffer with
and without 1.0 % Pluronic F127. The reagents were prepared
as described in Example 4 except that Imidazole buffer was
replaced with Bis-Tris buffer and 2-butyl-4-(2,3-dihydro-3-
methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquino2inium iodide was replaced with 2-methylthio-4-(2,3-
dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-
phenylquinolinium iodide dye (Molecular Probes, Inc., Eugene,
OR) and stored at ambient temperature for 6.5 months.
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Starting from the highest peak:
A = 2-methylthio-4-(2,3-dihydro-3-methyl-(benzo-1,3-
thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide reagent
with 1.0 % Pluronic F127;
B = 2-methylthio-4-(2,3-dihydro-3-methyl-(benzo-1,3-
thiazol-2-yl)-methylidene)-1-phenyiquinolinium iodide in Bis-
Tris buffer without any surfactant;
C = 2-methylthio-4-(2,_-dihydro-3-methyl-(benzo-l,3-
thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide in
Imidazole buffer without any surfactant;
D = 2-methylthio-4-(2,3-dihydro-3-methyl-(benzo-1,3-
thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide in
Glycyl-glycine buffer with 1.0% Pluronic F127.
The results demonstrate that the stabilization of this
cyanine dye requires an appropriate combination of a buffer and
a surfactant.
xa e- 1A
Figure 14 is the emission spectra of the RNA bound 2-
butyl-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
methylidene)-1-phenylquinolinium iodide cyanine dye in
different buffers along with 0.2 % Pluronic F127. All
reagents were stored at room temperature for six weeks.
Starting from the highest peak:
A = Tris buffer;
B = Bis-Tris buffer;
C = Zmidazole;
D = Hepes buffer;
E = Glycyl-glycine buffer;
F = Phosphate buffer:
As this data show, Tris, Bis-Tris and Imidazole buffers
preserve the dye well while Glycyl-glycine and Phosphate
buffers do not.
F=~mp 1e 1S
CA 02218728 1997-10-20 ~MMM SHMT
}
JUN 30 '97 04:23PM ABEOTT LEGAL DEPT(70E)93E2623 P.30i37
PCTjl1S96/Q5520
1PEA/US 3 0 J U N 1997
- 29-
Five (5) microliters of a normal sample with about 2.3 ~
reticulocytes were mixed with 1.0 ml of the reagent of the
present invention coznprised of 50 mM Tris buffer, pH adjusted
with iN HC1 to 7.0 + 0.1, osmolarity adjusted to 290 rnOsm/L
with NaCl, 0.2 ug/ml of 4-(2,3-dihydro-3-methyl-(benzo-1,3-
thiazol-2-yl)-methylidene)-1-phenylquinolinium tosylate
(Molecular Probes, Inc., Eugene, OR), 0.2 % Pluronic F127,
and 0.03 % Proclin 300. The sample/reagent mixtures were
then run on a FACScan flow cytometer within 15 seconds to
deternline the % of reticulocytes in the samples. Figure 15a
shows the FACScan* cytometer two dimensional display of FSC
vs FLl signals; and Figure 15b shows the FL1 histogram of the
gated red cell population. Figure 15c is a time study
i5 performed on the FACScan flow cytometer expressed as
reticulocyte % from 15 seconds up to 4.0 minutes. As can be
seen in the Figures the RBC+ reticulocyte gate cleanly
separates reticulocytes from platelets and WBCs and the
reticulocyte staining is so rapid that the % reticulocyte
reaches the steady state within 15 seconds. Such rapid
staining enables the incorporation of the detection onto
automated hematology analyzers, as well as flow cytometers.
CA 02218728 1997-10-20 aMEMM &4ET
