Method of using a multi-purpose beagent for subclassification of nucleated blood cells

A multipurpose reagent system for rapid analysis of a whole blood sample allowing the determination of at least five classes of peripheral white blood cells, nucleated red blood cells, and lymphocyte immunophenotyping on automated hematology instrumentation. The multipurpose reagent system lyses red cells rapidly, while it concurrently fixes white cells and preserves surface antigens on lymphocytes. The multipurpose reagent system comprises from about 3 to 7 grams per liter of a non-quaternary ammonium salt, from about 0.04 to about 0.10 percent by volume of an aliphatic aldehyde with one to four carbons, from about 10 mM to about 20 mM of a non-phosphate buffer which is inert to the aliphatic aldehyde, and a sufficient amount of water to give a pH between 5.5 and 7.5 and an osmolality of between about 160 to about 310 mOsm per liter.

BACKGROUND 
This invention relates to a multipurpose reagent system and a method for a 
rapid analysis of whole blood samples. More particularly, the present 
invention relates to a multipurpose reagent system capable of rapidly 
lysing red cells and concurrently fixing white cells, useful for 
performing white cell differential analyses and quantitative analyses of 
nucleated red blood cells or lymphocyte subclassification using 
immunophenotyping techniques on an automated clinical hematology analyzer 
or flow cytometer. 
The peripheral blood of a normal subject contains red blood cells, also 
known as erythrocytes, and five major classes of mature white cells, also 
known as leukocytes. There are at least five classes of leukocytes, known 
as neutrophils, eosinophils, monocytes, lymphocytes and basophils. Each 
type of mature blood cell performs specialized functions necessary in 
maintaining the homeostasis of the host. The concentration of each class 
of peripheral blood cells is tightly regulated and monitored by a dynamic 
process involving a variety of factors present in the microenvironment of 
the bone marrow. Under certain disease conditions, the bone marrow may 
release either an increased or decreased number of certain classes of 
white cells. In other conditions, all regulation of the number of 
peripheral blood cells released from the bone marrow is perturbed and an 
uncontrolled number of immature white or red cells are released to the 
peripheral blood. 
Therefore, monitoring the concentration of the five normal classes of 
leukocytes and identifying the presence of immature erythrocytes and 
leukocytes in the peripheral blood is an important diagnostic tool for 
physicians. Typically, these functions have been performed by doing white 
cell differential counts, whereby the relative proportions of the five 
normal classes of leukocytes and any abnormal cells are determined 
microscopically. The manual procedure is very time consuming, subjective 
and labor intensive. 
Recently, automated processes and automated flow system apparatuses have 
been developed to ease the burden of white cell differential analysis. 
Several of these systems are described in U.S. Pat. Nos. 4,099,917; 
4,617,275; 4,521,518; and 4,801,549. Some of these systems are based on 
cytochemical procedures to specifically identify individual cell types; 
some of these systems differentiate three leukocyte types by electronic 
impedance measurements of cell volume; and other procedures utilize a 
combination of optical and electronic impedance measurements to 
differentiate the five classes of peripheral white blood cells. 
Recent advances in cellular immunology and flow cytometry are being 
utilized to identify and quantify lymphocyte subclasses such as helper T 
cells. Lymphocyte subclassification has become an important diagnostic 
tool, particularly in view of the growing AIDS epidemic. Conventional 
lymphocyte subclassification involves the following steps: (1) The 
separation of lymphocytes from other peripheral blood cells by density 
gradient centrifugation; (2) the reaction of the lymphocytes with 
fluorochrome-labeled monoclonal antibodies directed to specific lymphocyte 
surface antigens; and (3) the analysis of lymphocyte-antibody reaction 
products using flow cytometry. Currently, a great deal of effort is being 
directed towards the development of whole blood methods that bypass the 
need for density gradient centrifugation. Recently developed whole blood 
methods for lymphocyte subclassification comprises lysing the red cells, 
removing red cell ghosts and cell debris by centrifugation, and preserving 
the morphology of the remaining white cells by suspending the white cells 
in an isotonic saline solution containing appropriate fixatives. Although 
these methodologies avoid the need for density gradient centrifugation, 
they are still incompatible with available automated clinical hematologic 
analyzers since they still require a centrifugation step. 
Generally speaking, the reagent systems available for use during the 
analysis of nucleated red blood cells (NRBC) are as yet unable to allow 
for the differentiation and counting of NRBC signals from red cell stroma 
or large platelets and only allow the instrument to flag possible NRBC 
signals. 
It is imperative in leukocyte analyses that all of the red blood cells be 
completely lysed. Since red cells outnumber white cells by about 700 to 1, 
even one percent of unlysed red cells may distort white cell counts. Some 
reagents used to lyse red cells require too lengthy an incubation period 
to be practical in an automated clinical analyzer. For example, the Tris 
buffered ammonium chloride solution recommended by K. A. Murihead in 
Clinical Cytometry, Ann. N.Y. Acd. Sci., vol. 468, pp. 113-127 (1986) 
takes 5 to 10 minutes to lyse red cells, which is too impractical for 
automation. 
Furthermore incomplete hemolysis with certain lytic reagents can result in 
red cell stroma that retain sufficient hemoglobin to generate high 
background counts in automated clinical electro-optical systems. 
Therefore, the white cells to be analyzed must first be removed from the 
red cell stroma by centrifugation, a procedure that is a limiting factor 
when adapting a reagent system for automation. 
Other reagent systems, such as those described in U.S. Pat. Nos. 4,902,613 
and 4,654,312, that are used to lyse red cells, contain high refractive 
index solvents. A cell suspending medium which has a high refractive index 
has two disadvantages: (1) The refractive index may be too high for a 
common flow cell saline sheath; and (2) the high refractive index of the 
suspending medium may mask signals from small cellular components such as 
small lymphocytes and cytoplasm-lysed nucleated red cells. Thus, before 
the cells can be analyzed in a flow cell, the cells must be removed from 
the high refractive index medium by centrifugation and resuspended in an 
isotonic solution. Such manual procedures are not desirable or adaptable 
for use on a fully automated clinical analyzer. 
In addition, lytic reagents, such as those described in U.S. Pat. No. 
5,155,044, are too hypotonic and/or acidic. Such lysing reagents require 
the rapid "follow-up" addition of a high salt solution and/or alkaline 
salt solution to preserve the white cell morphology for analysis. 
Similarly, lytic reagents, such as those described in U.S. Pat. No. 
4,751,179, will not only lyse red cells but will also lyse white cells, 
unless a separate fixative is added at the appropriate time and 
concentration to prevent white cell lysis. These reagents introduce the 
potential of white cell damage, particularly in abnormal blood samples 
containing fragile white cells (such as in blood samples from patients 
with chronic lymphocytic leukemia [CCL]). 
Furthermore, reagent systems, such as those described in U.S. Pat. Nos. 
4,099,917, 4,801,549, and 4,978,624, require incubations at high 
temperatures, e.g. over 50.degree. C., to completely lyse the red cells. 
Temperatures over 45.degree. C. will, generally, begin to denature most 
cell surface antigens and cause hemoglobin clumping in the process. 
Although these systems may be used to perform differential analyses of 
white cells, they destroy the means for differentiating subpopulations of 
lymphocytes and cannot be used for immunophenotypic lymphocyte 
classification. 
Many of the currently used reagent systems require the cytochemical 
staining of fixed white cells before they are subjected to differential 
analysis. These systems require the timed addition of multiple reagents 
and incubation periods and are generally not adaptable for the 
quantitation of nucleated red cells or for immunophenotypic lymphocyte 
classification. Furthermore, each step of reagent addition or other 
manipulation of a blood sample decreases the precision of the final counts 
obtained from that sample. 
Based on the foregoing, a need has arisen for a multipurpose reagent system 
which can lyse red cells rapidly and completely, while concurrently 
preserving white cell morphology and lymphocyte cell surface antigens. 
SUMMARY 
The problems discussed above have been solved in the present invention. 
Accordingly, an object of the present invention is to provide a 
multipurpose reagent system, or blood diluent, that will lyse red cells 
rapidly and completely, while concurrently preserving white cell 
morphology and lymphocyte cell surface antigens for the automated 
electro-optical analyses of peripheral whole blood cells. 
Another object of the present invention is to provide a multipurpose 
reagent system that permits the rapid differentiation of white cells on an 
automated clinical hematologic analyzer. 
Still another object of the present invention is to provide a multipurpose 
reagent system which permits the identification and quantitation of 
nucleated red blood cells (NRBCs) on an automated clinical hematologic 
analyzer. 
Yet another object of the present invention is to provide a multipurpose 
reagent system that eliminates the necessity of centrifuging 
lymphocyte-antibody reaction products prior to the enumeration of 
fluorochrome conjugated antibody bound lymphocyte subclasses on an 
automated clinical flow cytometer. 
The multipurpose reagent system of the present invention is comprised of a 
non-quaternary ammonium salt, an aliphatic aldehyde having one to four 
carbons, a non-phosphate buffer substantially inert to the aliphatic 
aldehyde, and water to give an effective pH of between about 5.5 and about 
7.5 and an osmolarity of between about 160 to about 310 mOsm/L 
(milliosmols per liter). Various optional reagents for the present 
invention include a surface active agent such as saponin, an 
anticoagulant, an alkali salt of bicarbonate, a nuclear stain, or an 
antibody directed against specific cell surface antigens. 
One method of the present invention comprises preparing a multipurpose 
reagent system, mixing the multipurpose reagent system with a whole blood 
sample, incubating the reagent system-blood mixture for at least 10 
seconds, and analyzing the blood sample on an automated hematology 
analyzer. 
One feature and technical advantage of the present invention is that the 
disclosed multipurpose reagent system can rapidly and completely lyse red 
blood cells while concurrently preserving white cell morphology, while, 
eliminating the need for the addition of a second reagent or fixative. The 
disclosed process of red cell lysis can take place in less than 20 
seconds. 
Another feature and technical advantage of the present invention is that 
the disclosed multipurpose reagent system fixes white cells adequately and 
will not lyse fragile lymphocytes such as CLL lymphocytes. Further, the 
multipurpose reagent system has been shown to stabilize white cells 
exposed to the reagent over extended periods of time. 
Still another feature and technical advantage of the present invention is 
that the disclosed multipurpose reagent system has a refractive index 
similar to that of isotonic saline, used in other hematologic 
measurements. 
Another feature and technical advantage of the present invention is that 
the lysing power of the disclosed multipurpose reagent system is potent 
enough to completely and rapidly lyse red cells in as low as a 16-fold 
diluted whole blood sample, thus retaining sufficient white cell density 
to allow accurate and rapid cell analysis. This allows for automated 
analysis in a multi-parameter clinical instrument. 
Still another feature and technical advantage of the present invention is 
that the disclosed multipurpose reagent system preserves lymphocyte cell 
surface antigens, for example, CD3, CD4, CD8, and CD19. 
Yet another feature and technical advantage of the present invention is 
that the disclosed method lyses red blood cells so thoroughly that signals 
from red cell ghosts are sufficiently small to be clearly separated from 
those of lymphocytes without washing or otherwise removing the red cell 
stroma while still providing improved subpopulation separation. 
Yet another feature and technical advantage of the present invention is 
that the disclosed method of peripheral blood analysis bypasses the need 
for either conventional or density gradient centrifugation steps. 
Still yet another feature and technical advantage of the present invention 
is that the disclosed method permits the quantification of nucleated red 
blood cells on a clinical flow cytometer. 
A further feature and technical advantage of the present invention is that 
the disclosed multipurpose reagent system enables a rapid, one-reagent, 
one-tube, automated differential analysis of peripheral white blood cells. 
An additional feature and technical advantage of the present invention is 
that method allows for a rapid differential analysis of lymphocyte 
subclasses on an automated flow cytometer. 
These and further features and advantages of the invention will be apparent 
from the following description of the preferred embodiments thereof.

DETAILED DESCRIPTION 
Broadly, the present invention relates to a multipurpose reagent system, or 
blood diluent, suitable for the rapid analysis of nucleated peripheral 
blood cells. The multipurpose reagent system can completely and rapidly 
lyse red blood cells, while concurrently preserving white cell morphology 
and the antigenicity of lymphocyte surface antigens. 
One aspect of the present invention is the multipurpose reagent system, 
comprising of from about 3 to about 7 grams per liter of a non-quaternary 
ammonium salt, from about 0.04 to about 0.1% by weight volume (i.e., grams 
per 100 ml) of an aliphatic aldehyde with one to four carbons, from about 
10 to about 20 mM of a non-phosphate buffer which is substantially inert 
to the aliphatic aldehyde, and water. The pH of the reagent system is 
within a pH range of about 5.5 to about 7.5 and the osmolality of the 
reagent system is between about 160 to 310 mOsm/L. The refractive index of 
the reagent system can be similar to that of saline and would be within 
the range of about 1.333 to about 1.336. The non-phosphate buffer which 
does not contain any primary amino group is inert to the aliphatic 
aldehyde. Thus, generally, the non-phosphate buffer should not contain a 
primary amino group. 
A preferred embodiment of the present invention utilizes a multipurpose 
reagent system comprised of about 95 mM ammonium chloride (5 g/l), about 
0.075% by volume of formaldehyde, from about 10 mM to about 20 mM acetate 
buffer, about 10 mM potassium bicarbonate, and about 0.01% by weight 
volume (i.e., grams per 100 ml) of saponin. The pH of the reagent system 
is adjusted to a range of from about 6.2 to about 6.5 and the osmolality 
of the reagent system is from about 215 to about 270 mOsm/L. 
Osmolality is defined as the number of dissolved particles in a unit volume 
of an aqueous solution. Osmolarity is defined as the number of dissolved 
particles in a unit weight of water solution. As a practical matter, 
osmolality and osmolarity have numerical values which are very close in 
the ranges involved in the present invention. A solution that has 1/1000 
of an osmol dissolved per kilogram has a concentration of 1 milliosmos 
("mOs") per kilogram. An osmol is the number of particles in 1 gram 
molecular weight of undissociated solute. Tonicity is a measure of the 
osmotic pressure of a solution relative to the osmotic pressure of the 
blood fluids. A hypotonic solution is a solution of lower osmotic pressure 
of tonicity than that of blood. The osmolality of a hypotonic solution is 
usually in the range of about 80-250 mOs/l. An isotonic solution has the 
same tonicity as blood. Here, the osmolality usually ranges from about 280 
to about 310 mOs/l. A hypertonic solution is a solution of greater 
tonicity than blood which normally has an osmolality range of about 
310-440 mOs/l. Water has the osmolality of about 10-20 mOs/l. 
The present invention also pertains to the use of the multipurpose reagent 
system in the automated determination of differential white cell counts, 
nucleated red blood cells, and lymphocyte immunophenotyping. The method 
for the rapid analysis of nucleated peripheral whole blood cells includes 
the following steps: mixing the multipurpose reagent system of the present 
invention with an anticoagulated whole blood sample (whereby the blood is 
diluted 16 to 100 fold), incubating the diluent-blood mixture at 
temperatures from about 25.degree. C. to 46.degree. C. for at least 10 
seconds, and analyzing the nucleated peripheral blood cells with automated 
hematology instrumentation. 
The method using the multipurpose reagent system of the present invention 
in the differential analysis of peripheral white blood cells is a rapid, 
one-reagent method of concurrently lysing red blood cells and fixing white 
blood cells, wherein the white cells maintain their light scattering 
characteristics. Example 1 illustrates the application of a preferred 
embodiment of the disclosed multipurpose reagent system in a rapid process 
for white cell differential analysis. FIG. 1 shows the differential 
analysis of white cells in a normal blood sample (processed as described 
in Example 1) by light scattering. In general, the cells flow through an 
optical view chamber where a photoelectric measuring process records the 
light absorbed or type of light scattered by each cell at selected angles. 
Electronic signals, from scattered light collected at different angles, 
are plotted as two dimensional dot plots as illustrated in FIG. 1. 
Granulocytes are identified first on the cytogram, 10 deg vs 90 deg 
scatter plot, by drawing the threshold between the granulocytes and the 
rest of the white cell population as shown in FIG. 1c. Eosinophils are 
identified next on the ORTHOGONAL vs DEPOL cytogram as shown in FIG. 1b. 
Then, monocytes and lymphocytes are identified on the SIZE vs COMPLEXITY 
cytogram (FIG. 1a) along the Y axis because monocytes are larger than 
lymphocytes. The signals that fall between lymphocytes and granulocytes 
along the X axis (COMPLEXITY) and which are lower than that of monocytes 
along the Y axis that do not belong to any of the populations already 
identified (neutrophils and eosinophils) are basophils, as labeled (FIG. 
1a). 
A first ingredient of the multipurpose reagent system is a non-quaternary 
ammonium salt. Preferably, neither di- nor tri-ammonium salts should be 
used. A variety of mono-ammonium salts, particularly the halogenated 
salts, can be used from about three to about seven grams per liter, and 
preferably at 5 grams per liter. Examples of such non-quaternary ammonium 
salts include NH.sub.4 X, where X is a halogen. Preferably, such a 
non-quaternary ammonium salt is NH.sub.4 Cl. 
A second ingredient of the multipurpose reagent system is a short-chain 
aliphatic aldehyde. Preferably, such aliphatic aldehydes have from one to 
four carbons. Exemplary aldehydes include formaldehyde and the polymer, 
paraformaldehyde. In proper ratios and concentrations, the aldehyde, in 
conjunction with the non-quaternary mono-ammonium salt, and the buffer, 
will rapidly and completely lyse the red blood cells. In addition, the 
aldehyde will fix white blood cells and preserve their membrane integrity. 
Formaldehyde, or comparable aldehyde, will be present in the present 
invention in amounts from about 0.04% to about 0.10% by volume, and 
preferably from about 0.08% to about 0.1% by volume. 
A third ingredient of the multipurpose reagent system is a non-phosphate 
buffer that is substantially inert to the aldehyde component of the 
reagent system. Thus, the buffer must not contain a primary amino group. 
The buffer should also have an effective buffering capacity between pH of 
about 5.5 and about 7.5. Examples of effective organic buffers are acetate 
buffer, succinate buffer, maleate buffer, and citrate buffer. Examples of 
effective biologic buffers are 2-(N-morpholine)ethane sulfonic acid (MES) 
buffer, 3-(N-morpholine)propane sulfonic acid (MOPS) buffer, and 
N-(2-hydroxyethyl)piperazine-N'-(2-ethane sulfonic acid) (HEPES) buffer. 
An acetate, or other suitable buffer, will be present in the present 
invention in amounts from about 10 mM to about 20 mM concentrations, and 
preferably at about 20 mM concentration. Embodiments of the present 
invention using MES buffer, MOPS buffer, and HEPES buffer are described in 
Examples 6, 7, and 8, respectively. 
An optional component of the multipurpose blood diluent is a surface active 
reagent. The preferred surface active agent is saponin, a plant extract 
that is available in a commercial grade powder isolated from quillaja tree 
bark as well as other sources. Although the chemical purity of commercial 
saponin varies from lot to lot, it is more selective towards red cells 
than are the quaternary ammonium salts. Saponin, or other surface active 
reagent, is present in the present invention in amounts from about 10 to 
about 200 mg/L, and preferably at about 100 mg/L. Saponin, in concert with 
the other ingredients of the multipurpose reagent system completely lyses 
the red blood cells present in whole blood. The erythrocyte fraction (i.e. 
red blood cells) of normal blood samples will be lysed within about 20 
seconds at ambient temperatures. However, hard-to-lyse blood samples (such 
as blood samples from babies, kidney dialysis patients, multiple myloma 
patients, diabetics, or patients with uremia) require incubating the blood 
with the reagent system at temperatures of about 38.degree. C. to about 
43.degree. C. for up to 20 seconds for complete erythrocyte lysis. 
Incubation of blood samples with the multipurpose reagent system, even at 
these slightly elevated temperatures, effectively preserves white cell 
membrane integrity and retains the antigenicity of lymphocyte surface 
antigens. In contrast, if saponin is used by itself to lyse the red cells, 
it must be used at a concentration 10 to 20 times higher than those used 
in the present invention. Such concentrations are extremely damaging to 
the integrity of the white cells and require a rapid quenching of the 
lytic activity of the reagent to preserve white cell morphology. An 
advantage of the present invention is that the combined constituents of 
the multipurpose reagent system serve to gently fix the white cells at the 
same time that the red cells are being lysed. Therefore, white cell 
integrity is preserved even at relatively long incubation periods. In fact 
even fragile white cells, such as those seen in chronic lymphocytic 
leukemia, are stabilized in the multipurpose reagent system of the present 
invention for incubation periods of up to 20 minutes. 
FIG. 2 shows the distribution of white cells in a normal blood sample 
processed as described in Example 12 and run on the CD3500.TM. analyzer 
(Abbott Diagnostic, Mountain View, Calif.) system directly through its 
optical system but bypassing the system hydraulics. Granulocytes are 
identified first from the rest of the white cell populations, as labeled, 
on the 10 deg vs 90 deg scatter plot, by setting the threshold as shown in 
FIG. 2c. Eosinophils are identified next on the ORTHOGONAL vs DEPOL 
scatter plot (FIG. 2b), as labeled, by setting the threshold between 
eosinophis and neutrophils as shown in FIG. 1b. Then, moHOCytes and 
lymphocytes are identified on the COMPLEXITY vs SIZE scatter plot, as 
labeled (FIG. 2a). The signals that fall between lymphocytes and 
granulocytes along the X axis (COMPLEXITY) and which are lower than that 
of monocytes along the Y axis that do not belong to any of the populations 
already identified (neutrophils and eosinophils) are basophils, as labeled 
(FIG. 2a). 
A preferred but optional ingredient of the multipurpose reagent system 
according to the present invention is an alkali salt, preferably a 
monovalent alkali salt of bicarbonate. Although a monovalent alkali salt 
of bicarbonate is not an essential component of the diluent, it may be 
added to the diluent to raise its osmolality without reducing the red cell 
lysability of the reagent system. Many other compounds, such as sodium 
chloride, potassium chloride or phosphate buffer, will diminish the 
lysability of the reagent system if they are used to increase the 
osmolality of the reagent system. Exemplary monovalent alkali salts of 
bicarbonate are potassium bicarbonate, sodium bicarbonate, or lithium 
bicarbonate. Potassium bicarbonate, or other alkali bicarbonate salt, can 
be present in the present invention in amounts from about 0.005% to about 
0.015% weight/volume (i.e. milligrams per 100 ml), and preferably at about 
0.01% weight/volume. 
Another optional ingredient of the multipurpose reagent system according to 
the present invention is a platelet anti-clumping agent. For example, an 
ethylenediaminetetraacetate (EDTA) salt can be added to the reagent system 
to prevent platelet aggregation in the sample/reagent mixture. Tetrasodium 
EDTA, of other EDTA salts, will be present in the present invention in 
amounts from about 20 to about 200 mgs per liter, and preferably at 100 
mgs per liter. 
Another embodiment of the present invention allows for the quantitative 
analysis of nucleated red cells on automated hematology analyzers. In 
order to analyze the percentage of nucleated red cells present in a whole 
blood sample, a nuclear stain, e.g., ethidium homodimer, is added to the 
multipurpose reagent system before it is added to the blood sample In this 
embodiment, the nuclear stain is added to the reagent system in an amount 
from between about 0.05 mg % to about 0.15 mg % weight/volume (i.e., 
milligrams per 100 ml), and preferably at 0.1 mg % weight/volume. The 
reagent system completely lyses the red cells while simultaneously 
preserving the integrity of white cell membranes. In the multipurpose 
reagent system, the added nuclear stain reacts with the exposed nuclei of 
immature red cells, yet it is impenetrable to intact white cells. Since 
the only nuclear material available to interact with the nuclear stain is 
that from the nucleated red blood cells, the stained nuclear material is 
proportional to the nucleated erythrocyte fraction of the blood sample and 
can be quantitated on an automated electro-optical analyzer. This 
one-reagent process of the present invention allows one to rapidly 
distinguish the different leukocyte populations from nucleated 
erythrocytes, and is particularly useful for certain veterinary 
applications. 
FIG. 3 shows a FACScan.TM. display of a normal blood sample with chicken 
erythrocyte nuclei (CEN processed as described in Example 5. The sample 
shown in FIG. 3a was processed with a nuclear stain but without CEN and 
the sample shown in FIG. 3b was processed in the presence of both a 
nuclear stain and CEN. The two dimensional dot plots on the left have 
plotted side scatter (SSC) versus forward scatter (FSC). The two 
dimensional dot plots on the right have SSC signals plotted versus red 
fluorescence (FL3) from all the cells in the sample. Note the appearance 
of a FL3 stained CEN population in FIG. 3b at the upper left corner. 
A further embodiment of the present invention allows for the quantitative 
analysis of lymphocyte subpopulations. Lymphocyte subclassification is 
achieved by mixing fluorochrome-conjugated monoclonal antibodies, directed 
to specific lymphocyte surface antigens, with whole blood samples before 
adding the multipurpose reagent system, or blood diluent. The 
concentration of labeled antibody fractions added to a blood sample will 
depend upon the individual antibody preparation, but is commonly about 
one-half to one-tenth of the volume of the blood for commercial antibody 
preparations. After the reagent system is added and the red cells are 
lysed, the lymphocyte-antibody reaction products can be analyzed on an 
automated flow cytometric system. The disclosed reagent system does not 
"quench" fluorescent markers, such as fluorescein isothiocyanate (FITC) or 
phycoerytherin (PE), which are frequently used to fluorochrome-label 
antibodies. Lymphocyte subclassification has become increasingly important 
as a diagnostic tool with the advent of the AIDS epidemic. The ability to 
identify surface markers on blood cell populations is likely to become 
increasingly important over the years as scientists increase their 
knowledge of surface components and characteristics of subpopulations of 
lymphocytes and other white cell fractions such as monocytes and 
neutrophils. 
FIGS. 4a through 4d show a FACScan.TM. displays of a normal blood sample 
processed as described in Examples 2, 3 and 4. FIG. 4a was processed 
without the addition of any antibody as a negative control of the donor 
sample; FIG. 4b was processed as described in Example 4 to identify pan B 
cells (CD19+lymphocytes) and pan T cells (CD3+lymphocytes). The lymphocyte 
population was gated first on the Forward Scatter (FSC) vs Side Scatter 
(SSC) plot and reanalyzed in the Green Fluorescence (FL1) vs Orange 
Fluorescence (FL2) channels. As can be seen in FIGS. 4a through 4d 
unlabeled lymphocytes were all in the lower left quadrant, while the 
CD3-FITC antibody labeled Pan T cells moved out to the lower right 
quadrant and the CD19-PE labeled Pan B cells moved up to the upper left 
quadrant. FIG. 4c sample was processed as described in Example 2 to 
identify Helper T cells (CD4+lymphocytes). Helper T cells are a 
subpopulation of T lymphocytes and have both CD3 and CD4 antigens on the 
cell surface and therefore they moved out to the right because of the FITC 
label on the anti-CD3 antibody and moved up to the upper right quadrant 
because of the PE label on the anti-CD4 antibody. FIG. 4d sample was 
processed as described in Example 3 to identify suppressor T cells 
(CD8+lymphocytes). Suppressor T cells are also a subpopulation of T 
lymphocytes and have both CD3 and CD8 antigens. Therefore the cells were 
labeled with both antibody and fell into the upper right quadrant. 
FIGS. 5b, 5d, 5f and 5h represent FACScan.TM. display printouts of a normal 
blood sample processed as described in Examples 2, 3 and 4. FIGS. 5a, 5c, 
5e and 5g represent the same sample processed as described in the same 
examples above, except that the red cells were lysed with a commercial 
lysing solution, Becton Dickinson's FacsLyse.TM. as described in Example 
11. Columns 1 and 3 are FSC vs SSC cytograms and columns 2 and 4 are FL1 
vs FL2 two dimensional dot plots of the gated lymphocytes. The same FSC, 
SSC, FL1 and FL2 gains were used for the analysis of both samples for 
comparison. 
As can be seen in the FSC vs SSC cytograms, the right column cytograms show 
well defined clusters of neutrophils, eosinophils, monocytes and 
lymphocytes, which are all well separated from noise (the signals mostly 
from red cell stroma), indicating that the white cells were well preserved 
in the multi-purpose blood diluent. This allows more accurate lymphocyte 
gating. In comparison, the cell clusters of the left column cytograms are 
less well defined. The resolution of each cell cluster is less clear and 
the signals of the granulocytes are much lower than that of the right 
column, suggesting an alteration in the refractive index of these cells 
which may have resulted from the leakage of some protein components. The 
quality of the FL1 vs FL2 two dimensional dot plots of the gated 
lymphocytes of the last column is essentially equivalent to that of the 
corresponding dot plot of the second column whose red cells were lysed 
with FacsLyse.TM.. 
FIG. 5a is a negative control of a normal blood, processed as described in 
Example 2 but not reacted with any antibody, lysed with FacsLyse.TM.; FIG. 
5b is also a negative control of the same sample but red cells were lysed 
with the multipurpose diluent of one embodiment of the present invention. 
FIGS. 5c, e and g represent the same sample processed as described in 
Examples 2, 3 and 4 but red cells were lysed with FacsLyse.TM. as 
described in Example 1. FIGS. 5d, f and h are the same sample processed as 
described Examples 2, 3 and 4 in which red cells were lysed with the 
multipurpose diluent of one embodiment of the present invention. 
The invention is further defined by reference to the following examples, 
which are intended to be illustrative and not limiting. 
EXAMPLE 1 
WHITE BLOOD CELL DIFFERENTIAL ANALYSIS 
Fifty microliters of an EDTA-anti-coagulated normal blood sample was mixed 
with 1 ml of the multipurpose reagent system prewarmed at 40.degree. C., 
mixed and incubated at room temperature for 16 seconds. The reagent system 
contained 0.5% weight/volume ammonium chloride, 0.08% weight/volume of 
formaldehyde, 0.01% weight/volume of saponin, 0.1% weight/volume of 
potassium bicarbonate, and 20 mM of acetate buffer. The reagent system had 
a pH of about 6.2 and an osmolality of 267 mOsm/L. This mixture was 
incubated at 38.+-.2.degree. C. for 16 seconds and run on the CD3500.TM. 
system directly through the optical system bypassing the system 
hydraulics. The cytograms of the sample are presented in FIG. 1. 
EXAMPLE 2 
LYMPHOCYTE IMMUNOPHENOTYPING 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 10 
microliters of monoclonal antibody solution containing anti-CD3-FITC and 
anti-CD4-PE in a test tube. 
The mixture was incubated at room temperature for 15 minutes before adding 
1.0 milliliter of the multipurpose reagent system of the present invention 
containing 0.5% weight/volume of ammonium chloride, 0.02% of weight/volume 
of tetra sodium EDTA, 0.1% of volume of formaldehyde, 0.0075% 
weight/volume of saponin, 0.01% weight/volume of potassium bicarbonate, 
and 20 mM acetate buffer. The reagent system had a pH of about 6.2 and an 
osmolality of 270 mOsm per liter, while the reagent system-blood solution 
had a pH around 7.0. 
The reagent system-blood solution was incubated from 20 seconds to 10 
minutes at room temperature. This variation in acceptable incubation time 
allowed for the analysis of multiple samples. 
The percent of CD3+ and CD4+ lymphocyte subpopulations was determined on 
the FACScan.TM. flow cytometer as illustrated in FIG. 4a. 
EXAMPLE 3 
LYMPHOCYTE IMMUNOPHENOTYPING 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 10 
microliters of monoclonal antibody solution containing anti-CD3-FITC and 
anti-CD8-PE in a test tube. 
The mixture was incubated at room temperature for 15 minutes before adding 
1.0 milliliter of the multipurpose reagent system of the present invention 
containing 0.5% weight/volume of ammonium chloride, 0.02% weight/volume of 
tetra sodium EDTA, 0.1% of volume of formaldehyde, 0.0075% weight/volume 
of saponin, 0.01% weight/volume of potassium bicarbonate, and 20 mM 
acetate buffer. The reagent system, described in EXAMPLE 1, had a pH of 
about 6.2 and an osmolality of 270 mOsm per liter. 
The whole blood-reagent system solution could be incubated anywhere from 20 
seconds to 10 minutes at room temperature, which allowed for the analysis 
of multiple samples. 
The percent of CD3+ and CDS+ lymphocyte subpopulations were determined 
using the FACScan.TM. flow cytometer. 
EXAMPLE 4 
LYMPHOCYTE IMMUNOPHENOTYPING 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 10 
microliters of monoclonal antibody solution containing anti-CD3-FITC and 
anti-CD19-PE in a test tube. 
The mixture was incubated at room temperature for 15 minutes before adding 
1.0 milliliter of a multipurpose reagent system of the present invention 
containing 0.5% weight/volume of ammonium chloride, 0.02% weight/volume of 
tetra sodium EDTA, 0.1% volume of formaldehyde, 0.0075% weight/volume of 
saponin, 0.01% weight/volume of potassium bicarbonate, and 20 mM acetate 
buffer. The multipurpose reagent system, as described in EXAMPLE 1, had a 
pH of about 6.2, and an osmolality of 270 mOsm per liter. 
The whole blood-reagent system solution could be incubated from 20 seconds 
to 10 minutes at room temperature. This variation in incubation time 
permits the analysis of multiple samples. 
The percent of CD3+ and CD19+ lymphocyte subpopulations were determined 
using a FACScan.TM. flow cytometer as illustrated in FIG. 4b. 
EXAMPLE 5 
NUCLEATED RED BLOOD CELL DETERMINATION 
Fifty microliters of an EDTA-anti-coagulated whole blood samples with and 
without chicken nuclei were mixed with 950 microliters of the multipurpose 
reagent system of the present invention containing 0.1 mg % weight/volume 
of a nuclear stain, 0.5% weight/volume of ammonium chloride, 0.075% of 
volume of formaldehyde, 0.01% weight/volume of saponin, 0.01% 
weight/volume of potassium bicarbonate, and 20 mM acetate buffer. The 
multipurpose reagent system had a pH of about 6.0 and an osmolality of 270 
mOsm per liter. 
The whole blood-reagent system solution was incubated at 38.+-.2.degree. C. 
for 20 seconds. 
The percentage of nucleated red blood cells in the whole blood sample was 
determined on a FACScan.TM. flow cytometer as illustrated in FIG. 3. 
EXAMPLE 6 
WHITE BLOOD CELL DIFFERENTIAL ANALYSIS 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 950 
microliters of the multipurpose reagent system of the present invention 
containing 20 mM MES buffer, 0.5% weight/volume of ammonium fluoride, 
0.08% of volume of formaldehyde, and 0.01% weight/volume of saponin. The 
reagent system had a pH of about 6.2 and an osmolality of 280 mOsm per 
liter. 
The whole blood-reagent system solution was incubated at 40.degree. C. for 
20 seconds. 
A differential analysis of the white blood cells was performed on an 
experimental clinical flow cytometer. 
EXAMPLE 7 
WHITE BLOOD CELL DIFFERENTIAL ANALYSIS 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 1.0 
milliliter of the multipurpose reagent system of the present invention 
containing 20 mM MOPS buffer, 0.5% weight/volume of ammonium chloride, 
0.1% of volume of formaldehyde, 0.012% weight/volume of saponin and 0.01% 
weight/volume of tetrasodium EDTA. The multipurpose reagent system had a 
pH of about 7.0 and an osmolality of 280 mOsm per liter. 
The whole blood-reagent system solution was incubated at 42.degree. C. for 
20 seconds. 
A differential analysis of the white blood cells was performed on an 
experimental clinical flow cytometer. 
EXAMPLE 8 
WHITE BLOOD CELL DIFFERENTIAL ANALYSIS 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 1.0 
milliliter of the multipurpose reagent system of the present invention 
containing 20 mM HEPES buffer, 0.4% weight/volume of ammonium fluoride, 
0.08% of volume of formaldehyde, 0.01% weight/volume of saponin, and 0.1% 
weight/volume of potassium bicarbonate. The reagent system had a pH of 
about 7.0 and an osmolality of 270 mOsm per liter. 
The whole blood-reagent system solution was mixed at 40.degree. C. for 
about 20 seconds. 
A differential analysis of the white blood cells was performed on an 
experimental clinical flow cytometer. 
EXAMPLE 9 
DIFFERENTIAL INCUBATION TIMES FOR WHITE BLOOD CELL DETERMINATIONS 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 950 
microliters of the multipurpose reagent system of the present invention at 
38.+-.2.degree. C. The reagent system contained 0.5% weight/volume of 
ammonium chloride, 0.08% of volume of formaldehyde, 0.01% weight/volume of 
saponin, 0.01% weight/volume of potassium bicarbonate, and 20 mM acetate 
buffer. The reagent system had a pH of about 6.2 and an osmolality of 267 
mOsm per liter. 
The mixture was incubated at 38.+-.2.degree. C. and serial aliquots of the 
mixture were removed at 14 seconds, 2 minutes, 4 minutes, 6 minutes, 8 
minutes, and 10 minutes. 
A five-part differential analysis of the white blood cells was performed on 
each aliquot on an experimental clinical flow cytometer equipped with an 
argon-ion laser. 
EXAMPLE 10 
VARIATIONS IN INCUBATION TIME AND TEMPERATURE FOR WHITE BLOOD CELL 
DETERMINATIONS 
Fifty microliters of EDTA-anti-coagulated whole blood was mixed with 950 
microliters of the multipurpose reagent system of the present invention 
containing 0.5% weight/volume of ammonium chloride, 0.08% of volume of 
formaldehyde, 0.01% weight/volume of saponin, 0.01% weight/volume of 
potassium bicarbonate, and 20 mM acetate buffer. The reagent system had a 
pH of about 6.2 and an osmolality of 267 mOsm/L. 
Aliquots of the resultant mixture were incubated at 36.degree. C., 
38.degree. C., 40.degree. C., 42.degree. C., 45.degree. C., and 46.degree. 
C. respectively for various time intervals up to 10 minutes. 
A five-part white cell differential analysis was determined on samples of 
each aliquot using an automated electrical optical system. 
EXAMPLE 11 
COMATIVE STUDIES OF A COMMERCIAL LYSING SOLUTION AND THE MULTIPURPOSE 
REAGENT SYSTEM OF THE PRESENT INVENTION 
A commercial lysing solution from Becton Dickinson (FacsLyse.TM.) was 
compared with one embodiment of the multipurpose reagent system of the 
present invention (the "Multipurpose Diluent") in an immuno-phenotyping 
experiment. The samples were processed as described in Examples 2, 3, and 
4 and the results are presented in TABLE 1. 
In the case the commercial FacsLyse.TM., the mixture of the test sample and 
the FacsLyse.TM. was first incubated in the dark at room temperature for 
10 minutes. Afterward, the resulting mixture was centrifuged for 5 minutes 
at 3000 g. The supernatant was separated and the cell button was then 
washed with 1 ml of phosphate buffered saline. The cell suspension was 
again centrifuged for 5 minutes at 3000 g. Afterward, the cell button was 
resuspended in a phosphate buffered saline containing 1% by weight of 
paraformaldehyde. The assay was then performed on a FACScan.TM.. 
In contrast to the elaborate and lengthy red cell lysing procedure as 
described above, in the case of one embodiment of the multipurpose reagent 
system of the present invention (the "Multipurpose Diluent"), the entire 
assay procedure was completed in about 20 seconds. No washing step was 
required. 
The comparative data are compiled in TABLE 1. As can be seen from this 
TABLE, the results obtained from the procedure using a commercial lysing 
solution and those obtained from the procedure using the multipurpose 
reagent system of the present invention are essentially equivalent. 
EXAMPLE 12 
WHITE BLOOD CELL DIFFERENTIAL ANALYSIS 
Fifty microliters of an EDTA-anti-coagulated normal blood sample was mixed 
with 1 ml of the multipurpose reagent system prewarmed at 40.degree. C., 
mixed and incubated at room temperature for 16 seconds. The reagent system 
contained 0.5% weight/volume of anunonium chloride, 0.08% volume of 
formaldehyde, 0.01% weight/volume of saponin, 0.1% weight/volume of 
potassium bicarbonate, and 10 mM of acetate buffer. The reagent system had 
a pH of about 6.2 and an osmolarity of 225 mOsm/L. This mixture was 
incubated at 38.+-.2.degree. C. for 16 seconds and run on the CD3500.TM. 
analyzer system directly through the optical system but bypassing the 
system hydraulics. The cytograms of the sample are presented in FIG. 2. 
Although the present invention and its advantages have been described in 
detail, it should be appreciated by those skilled in the art that the 
conception and the specific embodiments disclosed may be readily utilized 
as a basis for modifying or designing other systems or reagents for 
carrying out the same purposes of the present invention. It should also be 
realized by those skilled in the art that such equivalent constructions do 
not depart from the spirit and scope of the invention as set forth in the 
appended claims. 
TABLE 1 
______________________________________ 
COMISON OF LYMPHOCYTE SUB-TYPING RESULTS 
FACS LYSE .TM. vs MULTIPURPOSE BLOOD DILUENT DISCLOSED 
Multi- 
purpose 
Type of Mab. used for 
Donor Labled FacsLyse 
Diluent 
Immunophenotyping 
No. Population % labeled 
% labeled 
______________________________________ 
CD3FITC/CD4PE 
No. 1 CD3 - CD4 + 
1.36 1.75 
CD3 + CD4 + 
32.66 31.84 
CD3 + CD4 - 
35.14 37.43 
CD3 - CD3 + 
30.84 28.98 
CD3FITC/CD8PE 
No. 1 CD3 - CD8 + 
3.94 3.53 
CD3 + CD8 + 
23.54 27.26 
CD3 - CD8 - 
34.19 37.40 
CD3 + CD8 - 
34.61 35.52 
CD3FITC/CD19PE 
No. 1 CD3 - CD19 + 
24.77 26.41 
CD3 + CD19 + 
1.16 0.20 
CD3 - CD19 - 
12.53 10.36 
CD3 + CD19 - 
61.54 63.03 
CD3FITC/CD4PE 
No. 2 CD3 - CD4 + 
2.59 3.45 
CD3 + CD4 + 
46.66 45.86 
CD3 - CD4 - 
24.90 25.73 
CD3 + CD4 - 
25.02 25.80 
CD3FITC/CD8PE 
No 2 CD3 - CD8 + 
7.99 7.80 
CD3 + CD8 + 
24.52 24.95 
CD3 - CD8 - 
24.17 23.40 
CD3 + CD8 + 
43.32 43.85 
CD3FITC/CD19PE 
No. 2 CD3 - CD19 + 
9.83 9.85 
CD3 + CD19 + 
0.35 0.00 
CD3 - CD19 - 
20.94 18.61 
CD3 + CD19 - 
68.88 71.54 
______________________________________