Method and reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples

• US 5,731,206 A
• Filed: 03/29/1996
• Issued: 03/24/1998
• Est. Priority Date: 03/13/1987
• Status: Expired due to Fees

• Current Assignee: Coulter International Corp.
• Original Assignee: Coulter Electronics, Inc.
• Inventors: Fischer, Timothy J., Ledis, Stephen L., Sena, Ted, Crews, Harold R.
• Primary Examiner(s): Allen, Marriane P.
• Application Number:
  US08/630,868
• Time in Patent Office: 725 Days
• Field of Search: 436/8, 436/10, 436/17, 436/18, 436/63
• US Class Current: 436/17
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

A lytic reagent composition and a kit of a lytic reagent system are disclosed for the rapid isolation, identification and/or analysis of at least five subpopulations of leukocytes from a whole blood sample. The composition and system have application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. The lytic reagent composition of this invention comprises saponin and a carboxylic acid of the formula RCOOH wherein R is H or a C_1-3 aliphatic hydrocarbon radial optionally substituted by one or more carbonyl and/or hydroxy groups. The kit of a lytic reagent system comprises the lytic reagent composition and a quench reagent.

Claims

1. A lytic reagent composition for selective chemical treatment of a whole blood sample, said lytic reagent composition comprising saponin and a lytic reagent, wherein said lytic reagent is a carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl; and

wherein the relative concentration of the lytic reagent composition is in an effective amount to effect partitioning of a whole blood sample into a lysed red cell fraction and an essentially intact leukocyte fraction in such a state as to allow differential analysis of at least five sub-populations of such leukocytes.

2. The lytic reagent composition of claim 1, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

3. The lytic reagent composition of claim 2, wherein the concentration of the lytic reagent is from about 0.05 to about 0.5 percent by volume.

4. The lytic reagent composition of claim 1, wherein the saponin is in an amount effective to reduce the size of red blood cell fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

5. The lytic reagent composition of claim 1, wherein the saponin is in an amount from about 0.05 to about 0.20% by volume.

6. A lytic reagent composition for selective chemical treatment of a whole blood sample, said lytic reagent composition comprising saponin and a lytic reagent, wherein said lytic reagent is a carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl; and

wherein the relative concentration of the lytic reagent composition is in an effective amount to effect partitioning of a whole blood sample into a lysed red cell fraction and a leukocyte fraction by (i) causing rapid and essentially complete hemolysis of red blood cells in the blood sample and, (ii) inducing changes in leukocytes in said blood sample to enhance the ability of instrumentation to perform differential analysis and identification of at least five (5) sub-populations of leukocytes.

7. The lytic reagent composition of claim 6, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

8. The lytic reagent composition of claim 7, wherein the concentration of the lytic reagent is from about 0.05 to about 0.5 percent by volume.

9. The lytic reagent composition of claim 6, wherein the saponin is in an amount effective to reduce size of the red blood cell fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

10. The lytic reagent composition of claim 6, wherein the saponin is in an amount from about 0.05 to about 0.20% by volume.

11. A lytic reagent composition for selective chemical treatment of a whole blood sample, said lytic reagent composition comprising a differentiation effective amount of a lytic reagent and saponin, wherein said lytic reagent is a carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl; and

wherein the differentiation effective amount of said lytic reagent composition, when added to a whole blood sample, effecting (i) a decrease in the pH of the sample from its physiological level to a pH in the range of from about 2.6 to about 4.0 while maintaining the osmolality of the fluid at less than about 100 mOs, (ii) rapid and essentially complete hemolysis of the red blood cell fraction in the blood sample, and (iii) changes in the leukocyte cell fraction of the blood sample to enhance the ability of instrumentation to perform differential analysis and identification of at least five (5) sub-populations of leukocytes.

12. The lytic reagent composition of claim 11, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

13. The lytic reagent composition of claim 12, wherein the concentration of the lytic reagent is from about 0.05 to about 0.5 percent by volume.

14. The lytic reagent composition of claim 11, wherein the saponin is in an amount effective to reduce the size of red blood cell fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

15. The lytic reagent composition of claim 11, wherein the saponin is in an amount from about 0.05 to about 0.20% by volume.

16. A kit of a lytic reagent system for white blood cell differential analysis comprising:

(a) a lytic reagent composition for selective chemical treatment of a whole blood sample, said composition comprising saponin and a lytic reagent, wherein said lytic is a reagent carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl;

wherein the relative concentration of the lytic reagent composition is in an effective amount to effect partitioning of a whole blood sample into a lysed red cell fraction and an essentially intact leukocyte fraction in such a state as to allow differential analysis of at least five populations of such leukocytes; and

(b) a quench reagent, said quench reagent being present in sufficient concentration to retard the lytic activity of the lytic reagent composition and restore the physiological environment of the leukocytes within the sample.

17. The kit of claim 16, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

18. The kit of claim 16, wherein said quench reagent comprises an alkaline aqueous salt solution containing at least two ingredients selected from the group consisting of sodium chloride, sodium sulfate, sodium carbonate, and sodium bicarbonate.

19. The kit of claim 16, wherein said quench reagent is in an amount effective to stabilize the blood sample at a pH in the range of from about 6 to about 7.5.

20. The kit of claim 19, wherein said saponin is in an amount effective to reduce the size of red blood fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

21. The kit of claim 20, wherein said saponin is in an amount from about 0.05 to about 0.20% by volume.

22. A kit of a lytic reagent system for selective chemical treatment of a whole blood sample comprising:

a lytic reagent composition comprising;

(a) a lytic reagent composition comprising saponin and a lytic reagent, wherein said lytic reagent is a carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl;

wherein the relative concentration of the lytic reagent composition is in an effective amount to effect partitioning of a whole blood sample into a lysed red cell fraction and a leukocyte fraction by (i) causing rapid and essentially complete hemolysis of red blood cells in the blood sample and, (ii) inducing changes in leukocytes in said blood sample to enhance the ability of instrumentation to perform differential analysis and identification of at least five sub-populations of leukocytes, and

(b) a quench reagent, said quench reagent being present in sufficient concentration to retard the lytic activity of the lytic reagent composition and restore the physiological environment of the leukocytes within the sample.

23. The kit of claim 22, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

24. The kit of claim 22, wherein said quench reagent comprises an alkaline aqueous salt solution containing at least two ingredients selected from the group consisting of sodium chloride, sodium sulfate, sodium carbonate, and sodium bicarbonate.

25. The kit of claim 24, wherein said quench reagent is in an effective amount to stabilize the blood sample at a pH in the range of from about 6 to about 7.5.

26. The kit of claim 25, wherein the saponin is in an amount effective to reduce the size of red blood cell fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

27. The kit of claim 26, wherein the saponin is in an amount from about 0.05 to about 0.20% by volume.

28. A kit of a lytic reagent system for selective chemical treatment of a whole blood sample comprising:

(a) a differentiation effective amount of a lytic reagent and saponin, wherein said lytic reagent is a carboxylic acid of the formula RCOOH or a sulphonic acid of the formula R'"'"'SO₃ H;

wherein R is H or a C_1-3 aliphatic hydrocarbon radical optionally substituted by one or more carbonyl and/or hydroxy groups, and R'"'"' is OH, a C_1-3 aliphatic hydrocarbon radical or aryl; and

wherein the differentiation effective amount of said lytic reagent, when added to a whole blood sample, effecting (i) a decrease in the pH of the sample from its physiological level to a pH in the range of from about 2.6 to about 4.0 while maintaining the osmolality of the fluid at less than about 100 mOs, (ii) rapid and essentially complete hemolysis of the red blood cell fraction in the blood sample, and (iii) changes in the leukocyte cell fraction of the blood sample to enhance the ability of instrumentation to perform differential analysis and identification of at least five sub-populations of leukocytes; and

(b) a quench reagent, said quench reagent being present in sufficient concentration to retard the lytic activity of the lytic reagent composition and restore the physiological environment of the leukocytes within the sample.

29. The kit of claim 28, wherein the concentration of the lytic reagent is from about 0.01 to about 1.0 percent by volume.

30. The kit of claim 28, wherein said quench reagent comprises an alkaline aqueous salt solution containing at least two ingredients selected from the group consisting of sodium chloride, sodium sulfate, sodium carbonate, and sodium bicarbonate.

31. The kit of claim 30, wherein said quench reagent is in an effective amount to stabilize the blood sample at a pH in the range of from about 6 to about 7.5.

32. The kit of claim 31, wherein the saponin is in an amount effective to reduce the size of red blood cell fragments so that they will not cause a derivation of an output electrical signal indicative of a species of leukocyte.

33. The kit of claim 32, wherein the saponin is in an amount from about 0.05 to about 0.20% by volume.