WO2011143044A2

WO2011143044A2 - A lytic reagent system for flow cytometric analysis of leukocytes based on morphology or immunophenotypes

Info

Publication number: WO2011143044A2
Application number: PCT/US2011/035346
Authority: WO
Inventors: Juiming Lin; Chiranjit Deka
Original assignee: Idexx Laboratories, Inc.
Priority date: 2010-05-10
Filing date: 2011-05-05
Publication date: 2011-11-17
Also published as: WO2011143044A3

Abstract

A lytic reagent composition and methods for differentiation and enumeration of leukocytes are disclosed. The lytic reagent is useful for rapid and automated sample preparation methods in an automated hematology analyzer, and also for manual and slow sample preparation methods required for immunophenotyping cells in a conventional fluorescence flow cytometer. The reagent system includes a nonionic surfactant in a hypotonic solution, an inorganic buffer to maintain the pH in a range of from about 6 to about 10, and optionally a leukocyte stabilizer. The reagent system is used to lyse red blood cells and stabilize the leukocytes so that antibody binding sites on the leukocytes remain unaffected by the process and the blood samples may be labeled with antibodies either before or after the lysis of the red blood cells.

Description

A LYTIC REAGENT SYSTEM FOR FLOW CYTOMETRIC ANALYSIS OF LEUKOCYTES BASED ON MORPHOLOGY OR IMMUNOPHENOTYPES

BACKGROUND

Technical Field

[0001] The present disclosure relates to a reagent system for preparing whole blood samples for enumerating different subpopulations of leukocytes. Specifically, the present disclosure relates to a reagent system which may be utilized for manual sample preparation methods suitable for flow cytometers and for automated sample preparation methods suitable for hematology analyzers.

Background of Related Art

[0002] Leukocytes are any of several types of blood cells that help with defending the body from infection. The different mature forms of leukocytes include granulocytes, including neutrophils, basophils, and eosinophils; monocytes, including macrophages; and lymphocytes. These mature forms have different functions, including ingesting bacteria, protozoans, and infected or dead body cells; producing antibodies; and regulating the action of other leukocytes. The leukocytes operate mostly in the tissues. Blood normally contains about 5,000 to about 20,000 leukocytes per cubic millimeter, depending on the species.

[0003] Analysis of leukocyte subpopulation in a blood sample is an important step in clinical pathology. It provides information for diagnosis of pathological infection and disease, and is useful in monitoring progress in recovery of patients following treatment. The subtype analysis may be based on morphological differences or it may be based on functional differences, often manifested by the expression of various types of specific protein markers on the surface of the cell. [0004] Traditional methods for blood analysis involve staining a blood sample with vital stains and counting individual cells on a slide under a microscope to determine the absolute number and percentage of various sub populations within a whole blood sample. For accuracy and reliability, this approach is dependent on the skill and experience of the technologist making the slides and counting the cells. In addition, the method is time consuming and often lacks statistical robustness as only a few hundred cells may be counted per sample. Automated hematology analyzers, based on flow cytometry technology, offer an improvement over the limitations of the manual method by counting thousands of cells within seconds.

[0005] A pre-requisite for automated leukocyte analysis is the lysis of red blood cells (RBC) prior to measurement and stabilization of the white blood cells (WBC) during the measurement of each sample. In addition, in order for the different sub populations of WBC to be analyzed individually, the morphological differences between these populations must be maintained and/or enhanced for effective response by the detector system used for the analysis. For measurement systems based on electrical impedance (i.e., Coulter Principle), size differences are of utmost importance in distinguishing one population from another. For light scatter based measurements, resolution between individual leukocyte sub populations depends on a complex combination of size, internal structure, and relative refractive indices of the cellular material. As a result, whether or not a reagent system can enable accurate identification and analysis of individual leukocyte sub populations after removal of the RBC by lysis can only be determined by experimentation.

[0006] While counting of the main subpopulations of WBC based on morphological differences offers significant clinical information, it does not provide direct information on specific infections afflicting a patient; additional immunological information is necessary for that purpose. Immunological responses are measured by further conjugating individual WBC cell types with specific antibodies that recognize only specific cell types. If the antibodies are labeled appropriately with fluorescent markers (i.e., fluorochromes, quantum dots, etc.), then each cell type specific to a particular antibody can be measured in a flow cytometer.

However, in order for such measurements to be accurate, it is important that there is minimal degradation of the target WBC and that the antibody receptor sites on those cells are not negatively affected by any of the reagents to which the sample comes in contact during sample preparation.

[0007] In automated hematology analyzers, the lytic process is designed to work rapidly on the RBCs while maintaining integrity of the WBC for a short period of time (i.e. under about 1 minute) sufficient to count tens of thousands of cells. There is no requirement to retain cellular integrity after this counting period is over. However, when antibodies are also required to be labeled on the WBC in a blood sample manually, as is the case for

conventional flow cytometers, the incubation time of the antibody reagent with the WBC can be much longer than 1 minute (typically about 5 minutes to about 1 hour). Therefore, in such applications the integrity of the WBC has to be maintained for a longer period than is necessary for automated hematology analyzers that are not designed for immunophenotyping.

[0008] Thus, improved reagent systems for blood analysis remain desirable suitable for automated sample preparation such as in a hematology analyzer and manual sample preparation for immunophenotyping such as in a conventional flow cytometer.

SUMMARY

[0009] The reagent system of the present disclosure may be useful for differential analysis of leukocytes, in embodiments, using optical measurements in a flow cytometer or an automated hematology analyzer based on flow cytometry principles. [0010] The reagent system includes a nonionic surfactant, an alkaline metal salt used to adjust osmolality of from about 15 to about 1.50 milliosmoles (mOsm), and a buffer that maintains a pH of from about 6 to about 10.

[0011] The present disclosure also provides a hypertonic solution for discontinuing a lytic reaction which includes an alkaline chloride salt possessing an alkaline ion such as sodium, potassium and/or lithium.

[0012] The present disclosure also provides methods for resolving white blood cell types based on morphology or immunophenotypes in a whole blood sample. The methods include contacting a sample of whole blood with the lytic reagent of the present disclosure to lyse RBCs and then optionally contacting the sample with the hypertonic solution of the present disclosure. The sample is contacted and incubated with fluorescent labeled antibodies for a pre-determined period before or after the addition of the lytic reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Various embodiments of the present disclosure will be described herein below with reference to the figures wherein:

[0014] FIG. 1 illustrates dot plot results of EXT v. RAS depicting the components of a dog blood sample (FIG. 1(a)) and cat blood sample (FIG. 1(b)) utilizing a commercial reagent as described in Comparative Example 1 ;

[0015] FIG. 2 shows dot plot results of EXT v. RAS depicting the components of a dog blood sample (FIG. 2(a)) and a cat blood sample (FIG. 2(b)) utilizing the reagent system of the present disclosure as described in Examples 1-2;

[0016] FIG. 3 illustrates dot plot results of EXT v. RAS depicting the components of a dog blood sample (FIG. 3(a)) and a dog blood sample with additional buffer (FIG. 3(b)) utilizing the reagent system of the present disclosure as described in Example 3; [0017] FIG. 4 depicts dot plot results of forward versus side scatter measurements of a fluorescently labeled aliquot of dog blood using a lytic reagent of the present disclosure (Fig. 4a) and the differentiation of the CD4+ immunophenotype of the same samples by fluorescence (Fig. 4b); and

[0018] FIG. 5 illustrates dot plot results of forward versus side scatter measurements of a fluorescently labeled aliquot of dog blood using a commercially available lytic reagent PharmLyse (Fig. 5a) and the differentiation of the CD4+ immunophenotype of the same samples by fluorescence (Fig. 5b).

DETAILED DESCRIPTION

[0019] In general, the present disclosure provides a lytic reagent system for differential analysis of leukocytes. In embodiments, the lytic reagent system may be utilized in automated sample preparation systems using optical measurements, including flow cytometric hematology analyzers, and manual sample preparation systems for

immunophenotyping such as in a conventional flow cytometer. Such a system may allow rapid lysis of the RBC without degrading or damaging WBC and stabilize the WBC for antibody conjugation. The reagent system maintains the integrity of the cell surface receptors and fluorescently labeled antibodies to such receptors such that specific cells can be analyzed for immunophenotyping.

[0020] The reagent system includes a nonionic surfactant that serves the dual function of lysing red blood cells (RBCs) and solubilizing debris, an alkaline metal salt used to adjust osmolality, and an inorganic buffer to maintain the pH of the reagent system from about 6 to about 10.

[0021] Suitable nonionic surfactants of the lytic reagent system of the present disclosure include fatty acid esters of polyalcohols such as sorbitan, sucrose, and glycerin. In embodiments, the surfactant may be selected from polyoxyethylene (20) sorbitan monolaurate (i.e., Tween® 20), polyoxyethylene (20) sorbitan monooleate (i.e., Tween® 80), octyl phenol ethoxylate (i.e., Triton® X-100, commercially available from Sigma- Aldrich Co.), and combinations thereof.

[0022] The surfactants may be present at a concentration of from about 0.005% to about 0.015%) (w/v) in a hypotonic aqueous solution of an alkaline salt such as NaCl, KCl and LiCl, maintaining the osmolality of from about 15 to about 150 mOsm, in embodiments from about 25 to about 80 mOsm.

[0023] As noted above, the reagent system of the present disclosure also includes an alkaline metal salt to adjust osmolality of the reagent system. Suitable alkaline metal salts include, for example, alkaline halides, including chlorides, bromides, iodides, and the like. In embodiments, the alkaline metal salts may include alkaline chlorides. Suitable alkaline chlorides include, but are not limited to, sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl), and/or combinations thereof. A suitable salt concentration or osmolality may be important for proper lysis of the RBC and resolution of WBC populations, particularly the eosinophils from neutrophils. The salt concentration in the lytic reagent may be from about 3 to about 50 mM, in embodiments from about 6 to about 35 mM.

[0024] As noted above, the reagent system of the present disclosure also contains a buffer to maintain a desired pH. Suitable buffers include any physiologic buffers such as, for example, phosphate. The concentration of the buffer may be from about 2 to about 10 mM, in embodiments from about 3 to about 7 mM, in some embodiments about 5 mM. Utilizing these buffers, the pH of the reagent system may be maintained from about 6 to about 10, in embodiments from about 7 to about 9, and in other embodiments from about 7.2 to about 8. This near physiological pH is suitable for stabilizing white blood cells in a near natural condition. [0025] In embodiments, it may also be desirable to include a stabilizer, sometimes referred to herein as a leukocyte stabilizing agent, in the reagent system of the present disclosure during the lysis of RBC. Suitable stabilizers include, for example, bovine serum albumin (BSA), and the like. Other stabilizers that may be utilized to bind molecules include polysaccharides. The stabilizer may be added to provide additional leukoprotective function at a concentration of from about 0.01 to about 0.2%, in embodiments of from about 0.08% to about 0.13% in deionized water.

[0026] In embodiments, the present reagent system may include an isotonic sheath. The isotonic sheath is a fluid specifically formulated for use in flow cytometers and is intended for use as the delivery medium of the sample to the optics component of a flow cytometer. The isotonic sheath fluid is carefully manufactured for low particle and fluorescence backgrounds to ensure superior signal to noise ratio measurements and to enhance particle identification.

[0027] The reagent may optionally include a hypertonic, stop reagent to permit analysis of the WBC over a longer period of time in their near native state where no structural damage to the outside membrane or internal structures of the cell are observable. The stop reagent composition may include an aqueous buffer solution with alkali metal salts. Examples of alkali metal salts may include but are not limited to NaCl, LiCl, KC1, and/or combinations thereof. The concentration of salts may vary and depends on the volume added into the lysed samples. In embodiments, the hypertonic stop reagent/solution may have alkaline salts present at a concentration of from about 150 mM to about 250 mM, in embodiments from about 175 mM to about 225 mM.

[0028] The stop reagent may be added subsequent to the lysis of red blood cells to stop further lytic activity and prevent lysis of white blood cells. This is done so that the lytic reagent system can be applied to temperatures of up to about 35°C, in embodiments from about 20°C to about 33°C. In one embodiment, the stop reagent is added to the sample after it has been incubated with the lytic reagent of the present disclosure for a period of about 5 to about 60 seconds, in embodiments from about 8 to about 45 seconds, and in other embodiments of from about 10 to about 30 seconds.

[0029] The addition of the stop reagent may provide a stabilizing effect on the leukocytes, further providing sufficient time for analysis, in embodiments of from about 60 seconds to about 15 minutes, in other embodiments of from about 2 minutes to about 10 minutes.

[0030] In a flow cytometer, a fluid that contains a known amount of particles per unit volume passes by a sensor. When external energy such as light from a laser, or

electromagnetic radiation from an electromagnet, is directed into such a flowing fluid, the particles will scatter, absorb or reemit such energy dependent on characteristics peculiar to such particles. Scattered, absorbed or reemitted energy can be measured by a sensor. The exact amount of such energy received by a sensor per unit time gives a direct indication of the quantity of particles that have passed by in the stream. By knowing the number of such particles per unit volume, the amount of volume per unit time that has passed by can be calculated with an automated instrument (the flow rate). In embodiments, suitable flow cytometer detection systems include, for example, the optical detection system of Accuri C6 Cytometer (commercially available from Accuri Cytometer Inc.), LASERCYTE®

hematology analyzer, (commercially available from IDEXX Laboratories Inc.), and flow cytometers described in U.S. Patent Application Publication Nos. 2004/0246480 and

2006/0203226, the entire disclosures of which are incorporated by reference herein. These flow cytometers may utilize forward light scatter (FS), right angle scatter or side scatter (SS), and one or more fluorescence measurements, measuring the signals from the WBC in a flow cell as the cells pass through a sensing region. Additionally, it may include other

measurement channels, including extinction (EXT) or axial light loss (ALL).

[0031] The reagent system may be maintained in near-physiologic pH conditions from about 6 to about 10, in embodiments from about 7 to about 9, in other embodiments from about 7.2 to about 8, to maintain leukocytes in their native states while incubating the whole blood sample for lysis of RBC. The WBC may then be contacted with the hypertonic solution to provide measurements from two optical detectors capable of measuring axial light loss, side scatter, and time of flight, for example, in the flow cytometer as the cells pass through a sensing region, the first optical detector providing a measure of axial light loss and time of flight and the second optical detector providing a measure of side scatter. In another embodiment, the present reagent system and method may optionally be used with more than two light scatter detectors.

[0032] The lytic reagent system of the present disclosure may be utilized in conducting leukocyte differential on whole blood samples from multiple animal species including, but not limited to, canine, feline, equine, bovine, murine, ferret, mouse, rat and human.

[0033] In embodiments, the lytic reagent system includes an optional additive such as bioactive agents. Suitable bioactive agents include, for example, biocide agents, antibiotics, antimicrobial agents, medicaments, growth factors, anti-clotting agents, analgesics, anesthetics, anti-inflammatory agents, and combinations thereof.

[0034] By applying the lytic reagent system of the present disclosure into whole blood samples, leukocytes may be easily differentiated into at least three subpopulations:

lymphocytes, monocytes, granulocytes. The reagent system may be utilized with any suitable blood analysis system, including flow cytometry systems.

[0035] The following Examples are being submitted to illustrate embodiments of the present disclosure. These Examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. Also, parts and percentages are by weight unless otherwise indicated.

EXAMPLES

Comparative Example 1

[0036] A known hypotonic lytic reagent was used that served both as a lytic agent and as sheath fluid. The reagent utilized was commercially available from IDEXX Laboratories, Inc. in relation to its LaserCyte® instrument. The osmolality of the lytic reagent was about 23 mOsm. The lytic reagent was used separately with two different animal blood samples (derived from dog and cat), using the standard automated method by which LaserCyte operates. FIG. 1(a) and 1(b) shows dot plot results of EXT vs. RAS for these control samples. As can be seen in FIG. 1, a significant number of degraded white cells remained overlapped with the rest of the leukocyte population. In particular, the region marked as R8 overlapped with the lymphocyte population and thus had the potential to inaccurately report the lymphocyte count in the sample.

Example 1

[0037] A lytic reagent system of the present disclosure was prepared using a surfactant, an alkaline metal salt, and a buffer. The lytic reagent system was formulated as follows:

TABLE 1

NaHC03 1.07 g/L 12.74mM

HEPES-Na 1.34 g/L 5.15mM

KCI 0.1 g/L 1.34mM

NaCi 0.78 g/L 13.35mM

K₂HP0₄ 0.047 g/L 0.27mM

KH₂P0₄ 0.86 g/L 6.32mM

Polyoxyethylene (20) sorbitan

monolaurate (Tween 20) 0.013% 0.11 mM proclin 300 0.01 g/L * [0038] A sheath reagent was used to hydrodynamically focus the sample stream in the flow cytometer. About 2310 μί, of lytic reagent was added into about 80 μΐ, of a dog blood sample, followed by mixing and incubation for about 80 seconds. The pH of the lytic reagent system was about 7.5 and the osmolality was about 75 mOsm.

[0039] The treated sample was immediately analyzed by a modified LASERCYTE® Hematology analyzer (commercially available from IDEXX Laboratories Inc.). The resultant dot plot of EXT versus RAS is depicted in FIG. 2(a).

[0040] Compared with the results of Comparative Example 1 , there was significantly less debris and ghost cells present after utilizing the lytic reagent system of the present disclosure (as depicted in FIG. 2(a)) compared with the region marked R8 in FIG. 1(a). Moreover, the absence of degraded cell populations R7 and R8 in FIG. 2(a) is apparent (compare FIG. 1(a)), with only intact white blood cells separated out in distinct sub-populations after utilizing the lytic reagent system of the present disclosure.

Example 2

[0041] A lytic reagent system of the present disclosure was prepared using a surfactant, an alkaline metal salt, and a buffer as described in Table 1.

[0042] A known sheath reagent was used to hydrodynamically focus the sample stream in the flow cytometer.

[0043] About 2310 μΕ of lytic reagent was added into about 80 of a cat blood sample, followed by mixing and incubation for about 80 seconds. The pH of the lytic reagent system was about 7.5 and the osmolality was about 75 mOsm.

[0044] The treated sample was immediately analyzed by a modified LASERCYTE® Hematology analyzer. The resultant dot plot of EXT versus RAS is depicted in FIG. 2(b).

[0045] Compared with the results of Comparative Example 1, there was significantly less debris and ghost cells present after utilizing the lytic reagent system of the present disclosure (as depicted in FIG. 2(b)) compared with the region marked R8 in FIG. 1(b). Moreover, the absence of degraded cell populations R7 and R8 in FIG. 2(b) is apparent (compared to FIG. 1 (b)), with only intact white blood cells separated out in distinct sub-populations after utilizing the lytic reagent system of the present disclosure. In addition, FIG. 2(b) illustrates a clearer separation of the eosinophil population from the neutrophils.

Example 3

[0046] A lytic reagent system of the present disclosure was prepared using a surfactant, an alkaline metal salt, and a buffer as described in Table 1.

[0047] A known sheath reagent was used to hydrodynamically focus the sample stream in the flow cytometer.

[0048] About 2 mL of lytic reagent was added into about 60 of a dog blood sample, followed by mixing and incubation for about 10 to about 30 seconds. The pH of the lytic reagent system was about 7.5 and the osmolality was about 75 mOsm. About 120 μΕ of a hypertonic stop solution was then added to the resultant mixture.

[0049] An aliquot of the treated sample was analyzed after about 30 seconds by a LASERCYTE® Hematology Analyzer. The resultant dot plot of EXT versus RAS is depicted in FIG. 3(a). Fig. 3a indicates a standard differential pattern on the same sample measured by the same optical detectors.

[0050] A second aliquot of the treated sample with about 120 μΐ, of additional lOx phosphate buffered saline (PBS) was analyzed after 3 minutes by a LASERCYTE®

Hematology analyzer. The resultant dot plot of EXT versus RAS is depicted in FIG. 3(b). The dot plot results show that even after about 3 minutes the lymphocyte and neutrophil populations are still well preserved and therefore available for further immuphenotype analysis.

Example 4

[0051 ] A lytic reagent system of the present disclosure was prepared using a surfactant, an alkaline metal salt, and a buffer as described in Table 1.

[0052] About 5 μΐ_^ of anti canine CD4 antibody was added into about 50 μϊ_^ of a dog blood sample, followed by mixing and incubation for about 30 minutes. About 2 mL of lytic reagent of the present disclosure was then added to the above mixture and incubated for about 10 to about 30 seconds. The pH of the lytic reagent system was about 7.5 and the osmolality was about 80 mOsm.

[0053] An aliquot of the treated sample was analyzed in an Accuri® C6 flow cytometer (commercially available from Accuri Inc.). Distilled water was utilized as the sheath reagent to hydrodynamically focus the sample stream in the flow cytometer.

[0054] The resultant dot plot of forward scatter (FSC) versus side scatter (SSC) is depicted in FIG. 4(a). FIG. 4(a) illustrates the preservation of the lymphocyte and neutrophil populations after the RBC were lysed using the lytic reagent of the present disclosure. The differentiation of the CD4+ immunophenotype of the same samples by fluorescence was illustrated in the resultant histogram of fluorescence (FL2) in FIG. 4(b) for the gated lymphocyte population.

Example 5

[0055] Comparative example using a commercial lytic reagent designed for

immunophenotyping applications only.

[0056] A lytic reagent system of the present disclosure was prepared using a surfactant, an alkaline metal salt, and a buffer as described in Table 1. [0057] A known sheath reagent was used to hydrodynamically focus the sample stream in the flow cytometer.

[0058] About 5 of anti canine CD4 antibody was added into about 50 μΐ, of a dog blood sample, followed by mixing and incubation for about 30 minutes. Afterwards, about 2 mL of a commercial lytic reagent Pharmlyse® (commercially available from Becton

Dickinson) was added to the above mixture and incubated for about 15 minutes. Then this mixture was centrifuged at 200 x g (centrifugal force, times gravity) for about 5 minutes and washed once with about 2 ml of lx PBS/1%FBS. The cell pellet was added to about 400 μΐ of lx PBS/1%FBS to resuspend the sample.

[0059] An aliquot of the treated sample was analyzed by an Accuri® C6 flow cytometer. The resultant dot plot of forward scatter (FSC) versus side scatter (SSC) is depicted in FIG. 5(a). FIG. 5(a) illustrates the preservation of the lymphocyte and neutrophil populations after the RBC were lysed using the Pharmlyse lytic reagent. The differentiation of the CD4+ immunophenotype of the same samples by fluorescence was illustrated in the resultant histogram of fluorescence (FL2) in FIG. 5(b) for the gated lymphocyte population.

[0060] It will be appreciated that several of the above-disclosed and other features and functions, or alternatives thereof, may be desirable combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Unless specifically recited in a claim, steps or components of claims should not be implied or imported from the specification or any other claims as to any particular order, number, position, size, shape, angle, color, or material.

Claims

CLAIMS What is claimed is:

1. A lytic reagent system for use in the analysis of leukocytes in whole blood

comprising:

a nonionic surfactant having a concentration of from about 0.005% wt% (w/v) to about 0.02 wt% (w/v);

an alkaline metal salt to adjust osmolality of the system of from about 15 mOsm to about 150 mOsm selected from the group consisting of alkaline halides, alkaline chlorides, alkaline bromides, alkaline iodides, and combinations thereof; and

a buffer to maintain a pH of the system of from about 6 to about 10, wherein the lytic reagent system is applied to temperatures of up to about 35° C.

2. The lytic reagent system according to claim 1, wherein the alkaline metal salt is used to adjust the osmolality of the system of from about 25 mOsm to about 100 mOsm.

3. The lytic reagent system according to claim 1, wherein the buffer maintains the pH of the system of from about 7 to about 9.

4. The lytic reagent system according to claim 1 , wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate,

polyoxyethylene (20) sorbitan monooleate, octyl phenol ethoxylate, and combinations thereof.

5. The lytic reagent system according to claim 1, wherein the alkaline metal salt includes an alkaline ion selected from the group consisting of lithium, sodium and potassium.

6. The lytic reagent system according to claim 1, wherein the buffer is selected from the group consisting of phosphate buffers having a combined concentration of from about 2 mM to about 10 mM.

7. The lytic reagent system according to claim 1, further comprising a white blood cell stabilizer.

8. The lytic reagent system according to claim 7, wherein the white blood cell stabilizer includes bovine serum albumin having a concentration of from about 0.01% to about 0.2%.

9. The lytic reagent system according to claim 1, wherein the whole blood is derived from an animal selected from the group consisting of bovine, canine, feline, equine, ferret and human.

10. A method for resolving white blood cell sub-populations in a whole blood sample, comprising:

(i) incubating the whole blood sample with the lytic reagent of claim 1 to lyse blood cells;

(ii) measuring a response in a flow cytometer which includes at least two optical detectors as the blood cells pass through a flow cell intersecting a laser beam, wherein at least one detector measures light scatter; and

(iii) differentiating white blood cells in the whole blood sample.

11. A method according to claim 10, further comprising the step of: (iv) adding a hypertonic solution for discontinuing the lytic reaction after step (i) of claim 10.

12. The method according to claim 11 , wherein the hypertonic solution includes an alkaline metal salt with an alkaline ion selected from the group consisting of sodium, potassium, and lithium.

13. The method according to claim 10, wherein the whole blood sample is derived from an animal selected from the group consisting of bovine, canine, feline, equine, ferret and human.

14. A method for resolving immunophenotypes of white blood cell sub-populations in a whole blood sample, comprising:

(i) adding at least one antibody specific to receptors on or in at least one white blood cell sub-population, and incubating the mixture for a predetermined period of time;

(ii) adding the lytic reagent of claim 1 to lyse the red blood cells;

(iii) adding a hypertonic solution for discontinuing the lytic reaction;

(iv) measuring a response from at least two optical detectors as the white blood cells pass through a sensing region in a flow cell of an optical detection system, wherein at least one detector measures light scatter; and

(v) differentiating the white blood cells in the whole blood sample.

15. The method according to claim 14, wherein the hypertonic solution includes an alkaline metal salt having an alkaline ion selected from the group consisting of sodium, potassium, and lithium.

16. The method according to claim 14, wherein the whole blood sample is derived from an animal.

17. The method according to claim 14, wherein the whole blood sample is derived from an animal selected from the group consisting of bovine, canine, feline, equine, ferret and human.

18. The method according to claim 14, wherein the antibodies are further conjugated with fluorochromes.