WO1996034283A1

WO1996034283A1 - Lytic system utilizing propionic acid for leukocytes differentiation

Info

Publication number: WO1996034283A1
Application number: PCT/US1996/005700
Authority: WO
Inventors: James D. Lapicola; Janine D. Becker; Franklin J. Carver
Original assignee: Hematronix, Inc.
Priority date: 1995-04-27
Filing date: 1996-04-24
Publication date: 1996-10-31

Abstract

A lytic reagent system for use in the differentiation of leukocyte sub-populations is provided. The lytic reagent system includes a diluent, a lytic reagent consisting essentially of propionic acid at a concentration sufficient to cause lysis of the erythrocyte fraction of the blood sample but leave substantially intact the leukocyte fraction for subsequent differentiation, a detergent at a concentration sufficient to promote clarification and proper sizing of the leukocyte sub-populations, and a quench comprising a mixture of salt solution for neutralizing the system.

Description

LYTIC SYSTEM UTILIZING PROPIONIC ACID FOR LEUKOCYTE DIFFERENTIATION

Background of the Invention

This invention relates to hematology and, more particularly, to a lytic reagent system utilizing propionic acid for the differentiation of leukocytes within a blood sample.

Leukocytes, or white blood cells, are key components in the body's immune system. There are five major sub-populations of leukocytes: neutrophils, eosinophils, basophils, monocytes and lymphocytes.

Leukocytes are formed in the bone marrow or lymph tissue and may be transported by the circulatory system to different parts of the body to accomplish their immunological function. Typically, the immunological function of leukocytes is to seek out and destroy foreign invading organisms.

Differential quantitative and qualitative analyses of leukocyte sub-populations are important to the diagnosis and treatment of illness and disease. Indeed, such analyses have become almost essential to patient treatment in doctors' offices and hospitals around the world. Abnormal concentrations of certain sub-populations may indicate the presence of a foreign organism within the body, such as a bacterial or viral infection, or may be symptomatic of disease, such as leukemia.

There are several automated means available for the differentiation and quantification of leukocytes.

Perhaps most prevalently employed among these devices are Coulter hematology instruments. Coulter devices utilize impedance, conductance and laser technology to analyze and evaluate the physical properties of blood particles, including leukocytes. The Coulter instruments are capable of discriminating leukocyte sub-populations in terms of percentage composition and absolute concentrations. Coulter instruments, such as the Coulter STKS™ and Coulter MAXM™, can visually display differential data by a scattergram diagram plotting light scatter against impedance volume to illustrate the major sub-population groupings.

The Coulter scattergram plots the identified sub- populations in quadrants, each quadrant enclosing a particular sub-population of leukocytes and related cellular fractions, as in Fig. 1. Thus, a particularly beneficial capability of the Coulter instruments is their ability to produce a graphic summary of data which is easily analyzed by the medical professional or clinician to assist in rapid diagnosis or assessment. To analyze the leukocyte fraction of a whole blood sample with a Coulter instrument, it is first necessary to eliminate the erythrocyte fraction. The elimination of the erythrocyte fraction is commonly accomplished with a lysing reagent, such as that disclosed in U.S. Patent No. 5,155,044 to Ledis et al. ("Ledis"). The Ledis reagent was specifically developed for use with the Coulter instruments and includes an acidic solution of formic and/or acetic acid, a quantity of saponin, and a quench solution for neutralizing the reagent. The Ledis reagent is commercially offered for use with the Coulter instruments under the trade name Coulter Scatter Pak™ by Coulter Electronics, Inc. , of Hialeah, Florida. While the Ledis reagent has proven to work satisfactorily under certain circumstances, its inadequacies have been found to limit the accuracy and reliability of the Coulter devices. Perhaps most troublesome among these inadequacies is the inability of the Ledis reagent to perform satisfactorily on aged blood. Aged blood can be defined as blood that has been extracted from the body for about eight hours or more. When the Coulter instruments are used to analyze the leukocyte fraction of an aged blood sample that has been treated with the Ledis reagent, the scattergram results have been found to be inaccurate and unreliable. Moreover, when the Ledis reagent is used on aged blood there has been found to exist increased incidents of false positive error flags. The Ledis reagent yields particularly poor scattergram results on aged blood that has been in a refrigerated environment, which is often the case when blood has been extracted for a significant period of time.

The inability to conduct differential leukocyte analyses using the Ledis reagent has proven to be a substantial obstacle to medical professionals and clinicians located in remote or rural regions who must often ship ' blood samples to distant laboratories for analysis. These samples are usually transported in a refrigerated environment to preserve the properties of the sample. The centralized clinical laboratories which receive blood samples from off-site and oftentimes distant locations have been significantly hampered by their inability to reliably employ Coulter instruments for leukocyte differentiation. Accordingly, many such laboratories have been forced to resort to less convenient, more costly and often less accurate means to conduct leukocyte differential analyses.

The Ledis reagent has also been unable to provide reliable results on blood samples from pregnant women and on blood samples having elevated triglyceride levels. As with aged blood, such Coulter analyses using the Ledis reagent produce vague and nondistinct scattergrams having significantly increased incidents of false positive error flags. However, as opposed to aged blood samples where the clinician may know of the sample's precondition before the analysis, the clinician may be unaware of elevated triglyceride levels present in a blood sample or of early pregnancy blood samples. The inability of clinicians to easily pre-screen such individuals erodes confidence in the performance of the Ledis reagent and, hence, the Coulter instruments.

Summary of the Invention

It is, therefore, a primary object of the present invention to provide a lytic reagent system which allows accurate leukocyte sub-population differentiation on all blood samples, including aged blood samples, blood samples from pregnant women, and elevated triglyceride blood samples. More particularly, it is an object of the present invention to provide a lytic reagent system for use with Coulter instruments which provides accurate leukocyte sub- population differentiation and quantification results for all blood samples, including aged blood samples, blood samples from pregnant women, and elevated triglyceride blood samples.

It is another object of the present invention to provide a lytic reagent system for use with Coulter instruments which permits leukocyte differentiation on all blood samples without necessity for pre-screening individuals for blood irregularities or pre-existing medical conditions.

It is a further object of the present invention to provide a lytic reagent system which can be produced easily and inexpensively and which may be used under a variety of circumstances, including those impractical for use of existing reagents, such as the Ledis reagent.

To accomplish these and other related objects, the present invention relates to a lytic reagent system for use in the differentiation of leukocyte sub-populations. The lytic reagent system of the present invention includes the following components: a diluent, such as deionized water; a lytic reagent, consisting essentially of propionic acid at a concentration sufficient to cause lysis of the erythrocyte fraction of the blood sample but leave substantially intact the leukocyte fraction for subsequent differentiation; a detergent, consisting essentially of saponin at a concentration sufficient to promote clarification and proper sizing of the leukocyte sub- populations; and a quench, comprising a salt solution for neutralizing the system.

Brief Description of the Drawings Fig. 1 is a schematic diagram of a Coulter scattergram having its quadrants identified by sub- population;

Fig. 2.1 is a scattergram of a blood sample one hour post-phlebotomy maintained at room temperature and produced by a Coulter instrument using the lytic system of the present invention;

Fig. 2.2 is a scattergram of the blood sample in Fig. 2.1 but produced by the Coulter instrument using the Ledis reagent; Fig. 3.1 is a scattergram of a blood sample eight hours post-phlebotomy maintained at room temperature and produced by a Coulter instrument using the lytic system of the present invention;

Fig. 3.2 is a scattergram of the blood sample in Fig. 3.1 but produced by the Coulter instrument using the Ledis reagent;

Fig. 4.1 is a scattergram of a blood sample 24 hours post-phlebotomy maintained at room temperature and produced by a Coulter instrument using the lytic system of the present invention;

Fig. 4.2 is a scattergram of the blood sample in Fig. 4.1 but produced by the Coulter instrument using the Ledis reagent; Fig. 5.1 is a scattergram of a blood sample 24 hours post-phlebotomy maintained at approximately 4 degrees Celsius and produced by a Coulter instrument using the lytic system of the present invention; Fig. 5.2 is a scattergram of the blood sample in

Fig. 5.1 but produced by the Coulter instrument using the Ledis reagent;

Fig 6.1 is a scattergram of a blood sample having an elevated triglyceride level produced by a Coulter instrument using the lytic system of the present invention;

Fig. 6.2 is a scattergram of the blood sample in

Fig. 6.1 but produced by the Coulter instrument using the

Ledis reagent;

Fig. 7.1 is a scattergram of a blood sample taken from a 32 week pregnant woman produced by a Coulter instrument using the lytic system of the present invention; and

Fig. 7.2 is a scattergram of the blood sample in Fig. 7.1 but produced by the Coulter instrument using the Ledis reagent.

Description of the Preferred Embodiment The preferred embodiment of the lytic reagent system of the present invention generally includes two components: a lytic solution and a quench solution. The lytic solution contains a diluent, a lytic reagent and a detergent. The quench solution contains a diluent and one or more salts. The preferred diluent of the lytic solution is deionized water. The diluent may be present in the system at varying concentrations, depending on the system formulation, and serves generally as a solvent for the lytic solution components. Generally, the diluent will comprise at least about 98% of the lytic solution by volume.

The lytic reagent is the primary functional component of the system. The lytic reagent partitions the blood sample analyte by lysing the erythrocyte fraction of the sample but leaving substantially intact the leukocyte fraction. The lysing reagent also affects slight alterations in the sub-populations of the leukocyte fraction to aide in subsequent differential analysis. The lytic reagent of the system is propionic acid. Propionic acid may be present in the lytic solution at concentrations from about 2 mL/L to about 12 mL/L. It is preferable, however, that propionic acid be present in the solution at a concentration from about 4 mL/L to about 8 mL/*p+2XIt has been found that the optimum concentration of propionic in the system is about 6 mL/L.

The detergent of the lytic solution serves a threefold purpose: First, and primarily, the detergent functions to dissolve cell debris from the sample, which could interfere with the impedance, conductance and light scatter technology of the analyzing instrument. Second, the detergent serves to "size" the leukocyte sub- populations to improve the accuracy of the differential analysis. Third, the detergent acts as a secondary lysing agent for eliminating the erythrocyte fraction of the sample.

The detergent of the lytic solution may be any detergent capable of achieving the above purposes, such as trimethyl dodecyl ammonium chloride, sodium lauryl sulfate, nonaethylene glycol octylphenol ether, and their equivalents. The preferred detergent is saponin. Saponin may be present in the lytic solution at a concentration from about 0.5 g/L to about 3.0 g/L. Preferably, saponin is present in the solution at a concentration of about 1.0 g/L.

The lytic solution may have an overall pH from about 2.30 to about 3.70. It is preferable that the pH of the lytic solution be from about 2.70 to about 3.30. The optimum pH for the lytic solution is about 3.0.

The quench solution of the lytic reagent system of the present invention may contain one or more salt solutions capable of neutralizing the lytic solution to a pH of about 5.2 to about 8.6, thereby inhibiting the lysing activity of the lytic solution to preserve the leukocyte fraction. It is preferable that the quenching solution neutralize the lytic solution to a pH of about 7.0.

The preferred formulation of the quench solution of the present system includes a mixture of sodium carbonate, sodium chloride and sodium sulfate. In the preferred mixture, sodium carbonate is present at a concentration of about 15 g/L, sodium chloride is present at a concentration of about 7 g/L, and sodium sulfate is present at a concentration of about 32 g/L. The mixture may have a pH from about 9 to about 13, but preferably the mixture should have a pH of about 11. The osmolarity of the mixture can be from about 900 to about 1100, but is preferably about 950. In use, the lytic solution and quench solution are first prepared in accordance with the preferred embodiment. The solutions may, of course, be prepared in advanced and stored until use. The temperature of the solutions should be maintained at about 68 to about 86 degrees Fahrenheit.

After the solutions of the system have been prepared, they are introduced into the Coulter instrument by selecting appropriate software options of the device. A tri-potassium EDTA treated whole blood sample is allowed to set for approximately one-half hour prior to analysis. Using appropriate software options, a predetermined amount of the blood sample is aspirated into the instrument. The aspirated portion of the blood sample is diluted with a predetermined quantity of the lytic solution for approximately five seconds. During this lytic phase, the lytic solution partitions the sample and prepares the leukocyte fraction for analysis. After the lytic phase, the instrument automatically adds a predetermined quantity of quench solution to the mixture and the final solution is allowed to set for approximately ten seconds. During the quench phase, the quench solution normalizes the salt concentration and hydrogen ion concentration of the final solution, thereby inhibiting excess lysis in the sample. The quenched final solution is then subjected to analysis by the Coulter particle detection system. The results are displayed in a scattergram and by numerical differential data.

EXAMPLES The following examples are illustrative of the superior results obtained by the Coulter STKS™ and MAXM™ hematology instruments using the lytic system of the present invention. Each of the following examples shows at least one scattergram from the Coulter instrument using the lytic system of the present invention. For comparative purposes, the examples also each include at least one scattergram from the instrument on the same sample and under the same conditions, but using the Ledis reagent.

The lytic system of the present invention used in the examples was as prepared in accordance with above preferred embodiment. The formulation of the lytic solution included 6.0 mL/L of propionic acid, 1.0 g/L saponin and diluent. The pH of the lytic solution was about 3. The quench formulation of the lytic system included 15 g/L sodium carbonate, 7 g/L sodium chloride, and 32 g/L sodium sulfate.

The Ledis reagent used in the examples was the commercial formulation of the reagent's preferred embodiment, sold under the trade name Coulter Scatter Pak™ by Coulter Electronics, Inc.

The analyses for both reagent systems in the examples were conducted by first collecting a fresh blood using potassium-EDTA anticoagulant. The anti-coagulated blood then was conditioned at room temperature for at least one-half hour. The blood sample was then assayed with a selected Coulter instrument alternatingly using the Ledis reagent and the reagent system of the present invention, under the procedure described above.

EXAMPLE I

The aged blood comparative analyses were performed with a Coulter MAXM™ instrument on blood portions taken from a single blood sample. The fresh blood sample was collected to capacity in two 7 mL industry standard Becton-Dickinson purple K3-EDTA Vacutainers™. A first tube was maintained at room temperature and a second tube was stored uncapped at a temperature of 4 degrees Celsius.

Referring initially to Figs. 2.1 and 2.2, scattergram results are shown for analyses performed approximately one hour post-phlebotomy on blood samples from the first tube at room temperature. The scattergram of Fig. 2.1 was produced by the Coulter instrument using the lytic system of the present invention. The scattergram of Fig. 2.2 was produced by the Coulter instrument using the Ledis reagent.

At one hour post-phlebotomy, both scattergrams appear relatively normal and produce similar quantitative percentage data for the sub-populations. At this point, the instrument displays no abnormal signals, which would indicate the existence of abnormal cells.

Referring now to Figs. 3.1 and 3.2, comparative scattergram results are shown for analyses conducted approximately 8 hours post-phlebotomy on blood samples taken from the first tube at room temperature. The scattergram of Fig. 3.1 was produced by the lytic system of the present invention. The scattergram of Fig. 3.2 was produced by the Ledis reagent.

Note in Fig. 3.2 the beginning stages of quadrant crossover by the sub-populations treated with the Ledis reagent. Specifically, the monocyte and lymphocyte populations have conspicuously shifted to the right and now abut the primary vertical boundary. The scattergram of Fig. 3.1, by contrast, has substantially retained its quadrant distinction and respective sub-population positions.

Turning now to Figs. 4.1 and 4.2, comparative scattergram results are shown for analyses conducted approximately 24 hours post-phlebotomy on blood samples taken from the first tube at room temperature. The scattergram of Fig. 4.1 was produced by the lytic system of the present invention, and the scattergram of Fig. 4.2 was produced using the Ledis reagent.

At twenty-four hours, the Ledis reagent shows little differentiation of sub-populations and demonstrates significant quadrant cross-over. Notably, the lymphocyte and monocyte populations have encroached upon the granulocyte regions making reasonably precise differentiation more difficult. The Coulter instrument also gives a false positive error flag for the sample treated with the Ledis system, identifying abnormal leukocyte populations when, in fact, none exist. By contrast, the sample treated with the lytic system of the present invention, graphed in Fig. 4.1, still plots relatively distinct quadrants and, more importantly, signals no false positive error flag. The advantageous performance of the present lytic system is particularly clear upon examination of the refrigerated sample results. Referring to Figs. 5.1 and 5.2, scattergram results are shown from analyses conducted approximately twenty-four hours post-phlebotomy on blood samples from the second tube maintained at 4 degrees Celsius. Fig. 5.1 was produced by the lytic system of the present invention, and Fig. 5.2 was produced by using the Ledis reagent.

As evident in Fig. 5.1, the scattergram produced by the present lytic system has distinct boundaries and tight sub-population groupings. There are no false positive error flags in this scattergram and all indications from the Coulter analysis are that the blood sample is normal. The scattergram produced by the Ledis reagent, however, is quite different. The scattergram of Fig. 5.2 has virtually no discernable quadrant groupings and substantially no differentiation by sub-population. In fact, there is so little differentiation that there appears to be only one sub-population in the sample. As with the sample of Fig. 4.2, the Coulter instrument here signals the sample contains abnormal leukocytes. EXAMPLE II

A comparative analysis of an elevated triglyceride blood sample was also performed. A single fresh blood sample was collected to capacity in a 7 mL industry standard Becton-Dickinson purple K3-EDTA Vacutainer™. Two analyses were performed on the sample using a Coulter STKS instrument. The first analysis used the lytic system of the present invention and the second analysis used the Ledis reagent. The scattergram produced by the first analysis using the lytic system of the present invention is shown in Fig. 6.1. The scattergram produced by the second analysis using the Ledis reagent is shown in Fig. 6.2.

Referring initially to Fig. 6.1, the scattergram produced by the Coulter device using the lytic system of the present invention can be seen to be vivid and well- defined. Sub-populations are readily apparent in all quadrants, thereby indicating a very strong reading by the instrument. There is tight grouping of the sub-populations which are distinctly separated by the quadrant lines. The Coulter instrument also indicates the sample a normal leukocyte fraction.

The Coulter scattergram using the Ledis reagent, though, is quite faint. As shown in Fig. 6.2, the data points on the graph are dispersed with little or no grouping. There are no discernable patterns in the scattergram. The scattergram further erroneously signifies an abnormal leukocyte fraction. This scattergram would be considered a false positive error flag and would necessitate significant additional experimentation to verify the scattergram results.

EXAMPLE III A blood sample taken from a 32-week pregnant woman was subjected to similar comparative analyses using the Coulter MAXM™ instrument. As with the prior examples, this blood sample was collected to capacity in a 7 mL industry standard Becton-Dickinson purple K3-EDTA Vacutainer™. Two analyses were performed on the sample. The first analysis used the lytic system of the present invention and the second analysis used the Ledis reagent. The scattergram produced by the first analysis using the lytic system of the present invention is shown in Fig. 7.1, and the scattergram produced by the second analysis using the Ledis reagent is shown in Fig. 7.2.

Fig 7.1 illustrates a well-defined scattergram having clear sub-population groupings well within clearly demarcated quadrants. There is shown to be practically no boundary crossover by the sub-populations, nor is there appreciable fading within the primary groups. This sample could easily be qualitatively analyzed on the basis of the scattergram alone.

Fig. 7.2, however, contains dispersed data patterns. Specifically, the monocyte grouping in Fig. 7.2 is shifted to the right, as compared to the same sub- population in Fig. 7.1. The Coulter instrument has also designated this sample as having abnormal leukocytes, an indication clearly rebutted by Fig. 7.1. EXAMPLE IV

As seen from the foregoing examples, Coulter instruments using the Ledis reagent have a marked tendency to signal false positive error flags of abnormal leukocyte sub-populations when used to analyze certain types of blood samples. A clinical trial was conducted at the University of California, Davis, to more accurately assess the incidence of false positive error flags for both the Ledis reagent and the lytic system of the present invention. The trial sought to compare the relative performance of the two systems on a group of unknown randomly selected blood samples using a Coulter model STKS™ instrument.

A total of 59 randomly selected blood samples were drawn. Each of the 59 samples was assayed twice to produce 118 total samples. Duplicate analyses were conducted on each sample using the STKS instrument and, alternatingly, the preferred formulation of the lytic system of the present invention and the Coulter Scatter Pak™ system. Each sample was analyzed in duplicate reverse order at room temperature with both lytic systems at Day zero, approximately one to eight hours post-phlebotomy. The error profile for the analysis of each blood sample was recorded. The samples were refrigerated and then analyzed in duplicate reverse order with both systems at Day one, approximately thirty-one hours post-phlebotomy. The error profile for each blood sample at this time interval was also recorded. For each blood sample analysis with each lytic system, the Day zero error profile was compared to the Day one error profile to determine whether the blood sample showed a changed error profile between the time interval analyses. The results of each comparison were recorded. Of the 118 samples assayed with the Ledis reagent, 86 samples exhibited a changed error profile at Day one analysis as compared to the Day zero analysis. In other words, the Ledis reagent failed to produce consistent error profiles over time on approximately 74 percent of the samples tested.

Of the 118 samples assayed with the lytic system of the present invention, however, only 12 samples displayed a changed error profile at the Day one analysis as compared to the Day zero analysis. Thus, the present lytic system produced inconsistent error profile over time on only approximately ten percent of the samples. The clinical trial results show the lytic system of the present invention effects greater than a sevenfold decrease in erroneous test results over time compared to the Ledis reagent.

The above examples clearly establish the lytic system of the present invention produces clearly superior results as compared to the Ledis reagent on aged samples. The scattergrams produced by a Coulter instrument using the present lytic system are consistently more well-defined and more accurate than scattergrams yielded by the Ledis reagent on the same samples. The superior performance of the present lytic system is clear when used in Coulter analyses on aged blood, elevated triglyceride blood, and blood from pregnant women. The improved results are even more profound when refrigerated samples are assayed.

Perhaps more importantly, the clinical trial data shows the present lytic system effects approximately a sevenfold decrease in changed flagging over the Ledis reagent on aged blood. Thus, the lytic system of the present invention not only promotes confidence in the performance of Coulter instruments by medical professionals and clinicians who use the devices, but it also ensures peace of mind in patients who can rely upon diagnoses based upon Coulter differential data.

From the foregoing it is apparent this invention is well-adapted to obtain all the ends and objectives set forth herein along with other advantages which are obvious to the invention.

It is to be understood that certain features and subcombinations are useful and may be employed without reference to other features and subcombinations. This is contemplated by the disclosure and is within the scope of the claims.

Because many possible embodiments may be made of the present invention without departing from its scope, it is understood that all matters set forth herein and shown in the accompanying drawings are to be interpreted as illustrative only, and not in a limiting sense.

Claims

The following is claimed:

1. A lytic reagent system for the differentiation of leukocyte sub-populations in a blood sample containing a leukocyte fraction and an erythrocyte fraction, said system comprising: a diluent; a lytic reagent consisting essentially of propionic acid at a concentration sufficient to lyse said erythrocyte fraction but leave substantially intact said leukocyte fraction for subsequent differential analysis of said sub-populations therein; and a detergent at a concentration to effect sufficient clarification and sizing of said leukocyte sub- populations to allow differentiation.

2. The system of claim 1 wherein said lytic reagent is present in said system at a concentration from about 2.0 mL/L to about 12.0 mL/L.

3. The system of claim 2 wherein said detergent is present in said system at a concentration from about 0.5 g/L to about 3.0 g/L.

4. The system of claim 3 wherein said lytic reagent is present in said system at a concentration of about 6.0 mL/L.

5. The system of claim 4 wherein said detergent is present in said system at a concentration of about 1.0 g/L.

6. The system of claim 5 wherein said detergent consists essentially of saponin.

7. The system of claim 6 wherein said diluent, lytic reagent and detergent are present in said system at relative concentrations resulting in a pH for said system from about 2.50 to about 3.50.

8. The system of claim 7 wherein said diluent, lytic reagent and detergent are present in said system at relative concentrations resulting in a pH for said system of about 3.0.

9. A lytic reagent system for the differentiation of leukocyte sub-populations in a blood sample containing a leukocyte fraction and an erythrocyte fraction, said system consisting essentially of the following components: a diluent; a lytic reagent consisting essentially of propionic acid at a concentration sufficient to lyse said erythrocyte fraction but leave substantially intact said leukocyte fraction for subsequent differential analysis of said sub-populations therein; and a detergent at a concentration to effect sufficient clarification and sizing of said leukocyte sub-populations to allow differentiation.

10. The system of claim 9 wherein said lytic reagent is present in said system at a concentration from about 2.0 mL/L to about 12.0 mL/L.

11. The system of claim 10 wherein said detergent is present in said system at a concentration from about 0.5 g/L to about 3.0 g/L.

12. The system of claim 11 wherein said lytic reagent is present in said system at a concentration of about 6.0 mL/L.

13. The system of claim 12 wherein said detergent is present in said system at a concentration of about 1.0 g/L.

14. The system of claim 13 wherein said detergent consists essentially of saponin.

15. The system of claim 14 wherein said diluent, lytic reagent and detergent are present in said system at relative concentrations resulting in a pH for said system from about 2.50 to about 3.50.

16. The system of claim 15 wherein said diluent, lytic reagent and detergent are present in said system at relative concentrations resulting in a pH for said system of about 3.0.

17. A lytic reagent system for the differentiation of leukocyte sub-populations in a blood sample containing a leukocyte fraction and an erythrocyte fraction, said system comprising: a diluent; a lytic reagent consisting essentially of propionic acid at a concentration sufficient to lyse said erythrocyte fraction but leave substantially intact said leukocyte fraction for subsequent differential analysis of said sub-populations therein; a detergent at a concentration to effect sufficient clarification and sizing of said leukocyte sub- populations to allow differentiation; and a quench comprising a salt solution effective to neutralize said system.

18. The system of claim 17 wherein said lytic reagent is present in said system at a concentration from about 2.0 mL/L to about 12.0 mL/L.

19. The system of claim 18 wherein said detergent is present in said system at a concentration from about 0.5 g/L to about 3.0 g/L.

20. The system of claim 19 wherein said lytic reagent is present in said system at a concentration of about 6.0 mL/L.

21. The system of claim 20 wherein said detergent is present in said system at a concentration of about 1.0 g/L.

22. The system of claim 21 wherein said detergent consists essentially of saponin.

23. The system of claim 22 wherein said quench comprises a mixture of sodium carbonate, sodium chloride and sodium sulfate.

24. The system of claim 23 wherein said quench has a pH of about 11.

25. The system of claim 24 wherein said quench is present in said system at a concentration to neutralize said system to a pH from about 6 to about 8.

26. The system of claim 25 wherein said quench is present in said system at a concentration to neutralize said system to a pH of about 7.

27. A lytic reagent system for the differen¬ tiation of leukocyte sub-populations in a blood sample containing a leukocyte fraction and an erythrocyte fraction, said system consisting essentially of the following components: a diluent; a lytic reagent consisting essentially of propionic acid at a concentration sufficient to lyse said erythrocyte fraction but leave substantially intact said leukocyte fraction for subsequent differential analysis of said sub-populations therein; a detergent at a concentration to effect sufficient clarification and sizing of said leukocyte sub-populations to allow differentiation; and a quench comprising a salt solution effective to neutralize said system.

28. The system of claim 27 wherein said lytic reagent is present in said system at a concentration from about 2.0 mL/L to about 12.0 mL/L.

29. The system of claim 28 wherein said detergent is present in said system at a concentration from about 0.5 g/L to about 3.0 g/L.

30. The system of claim 29 wherein said lytic reagent is present in said system at a concentration of about 6.0 mL/L.

31. The system of claim 30 wherein said detergent is present in said system at a concentration of about 1.0 g/L.

32. The system of claim 31 wherein said detergent consists essentially of saponin.

33. The system of claim 32 wherein said quench consists essentially of a mixture of sodium carbonate, sodium chloride and sodium sulfate.

34. The system of claim 33 wherein said quench has a pH of about 11.

35. The system of claim 34 wherein said quench is present in said system at a concentration to neutralize said system to a pH from about 6 to about 8.

36. The system of claim 35 wherein said quench is present in said system at a concentration to neutralize said system to a pH of about 7.

37. A lytic reagent system for use in the differentiation of leukocyte sub-populations in a blood sample containing a leukocyte fraction and an erythrocyte fraction, said system consisting essentially of the following components: a diluent comprising deionized water; a lytic reagent consisting essentially of propionic acid at a concentration of about 6.0 mL/L, said reagent being effective to lyse said erythrocyte fraction but leave substantially intact said leukocyte fraction for subsequent differential analysis of said sub-populations therein; a detergent consisting essentially of saponin at a concentration of about 1.0 g/L for effecting sufficient clarification and sizing of said leukocyte sub-populations to allow differentiation; and a quench comprising a mixture of sodium carbonate, sodium chloride and sodium sulfate, said quench being effective to neutralize said system to a pH of about 7.