Differential lymphoid-myeloid determination of leukocytes in whole blood

• US 4,286,963 A
• Filed: 11/23/1979
• Issued: 09/01/1981
• Est. Priority Date: 11/23/1979
• Status: Expired due to Term

• Current Assignee: Coulter Electronics, Inc.
• Original Assignee: Coulter Electronics, Inc.
• Inventors: Crews, Harold R., Ledis, Stephen L., Chastain, David L. Jr.
• Primary Examiner(s): Turk, Arnold
• Application Number:
  US06/096,697
• Time in Patent Office: 648 Days
• Field of Search: 252/408, 23/230 B, 23/913
• US Class Current: 436/63
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/721 Haemoglobin
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

This invention relates to a lytic diluent and a method for rapid lysing of red blood cells in whole blood for a differential determination of lymphoid and myeloid populations of leukocytes, and measurement of hemoglobin, particularly for use in automatic particle counting systems.

The diluent is a mixture of at least one quaternary ammonium salt having surface active properties, and an additive in suitable proportions, buffered to an acid pH of 3.5 to 5.0. The additive includes 2-phenoxyethanol and other short chain alkanols substituted by one phenyl or phenoxy, as well as certain polyhydroxy compounds.

When the reagent is employed in lower concentrations, an alkaline reagent comprising a quaternary ammonium salt and an alkali metal cyanide, buffered to an alkaline pH, may be added to complete the chromagen formation.

Claims

1. A lytic diluent comprising a mixture of ingredients (A) and (B) where

(A) is an aqueous saline solution of at least one quaternary ammonium salt having surface active properties, and

(B) is at least one additive selected from the group consisting of

(1) a short chain alkanol substituted by phenyl or phenoxy;

and

(2) a polyhydroxy compound;

said lytic diluent being buffered to an acid pH within the range of 3.5 to 5.0, and each of said ingredients (A) and (B) being present in an amount sufficient to bring about rapid and complete lysing of the red cells in whole blood for making a differential determination of lymphoid and myeloid populations of leukocytes, and also for measuring hemoglobin by chromagen formation.

2. The lytic diluent of claim 1 wherein said quaternary ammonium salt is selected from the group of compounds having the formula ##STR2## where one of the radicals R₁, R₂, R₃ and R₄ is a long chain alkyl radical having from 12 to 18 carbon atoms, and the remaining radicals are short chain alkyl radicals having from 1 to 6 carbon atoms, and compounds where two of the radicals together with the nitrogen to which they are attached form a heterocyclic ring;

and X^- is Cl, Br, I, PO₄, HSO₄, CH₃ SO₄.

3. The lytic diluent of claim 1 wherein said quaternary salt is tetradecyltrimethyl ammonium bromide.

4. The lytic dilutent of claim 1 wherein said quaternary salt is hexadecyltrimethyl ammonium bromide.

5. The lytic diluent of claim 1 wherein said quaternary ammonium salt is a mixture of hexadecyltrimethyl ammonium bromide and tetradecyltrimethyl ammonium bromide.

6. The lytic diluent of claim 1 wherein said additive is sorbitol.

7. The lytic diluent of claim 1 wherein said additive is 2-phenoxyethanol.

8. The lytic diluent of claim 7 wherein said 2-phenoxyethanol is present in a concentration of about 0.2% to 1.0%.

9. The lytic diluent of claim 1 wherein said quaternary ammonium salt is present in a concentration in the range of about 200 to 500 mg/liter by weight of said solution.

10. A lytic diluent comprising a mixture of ingredients (A) and (B) where

(A) is an aqueous saline solution of at least one quaternary ammonium salt having surface active properties, and

(B) is at least one additive selected from the group consisting of

(1) a short chain alkanol substituted by phenyl or phenoxy;

and

(2) a polyhydroxy compound;

said lytic diluent being buffered to an acid pH within the range of 3.5 to 5.0, and each of said ingredients (A) and (B) being present in an amount sufficient to bring about rapid, but incomplete, lysing of the red cells in whole blood for making a differential determination of lymphoid and myeloid populations of leukocytes, and also for measuring hemoglobin for chromagen formation, wherein said chromagen formation is completed by the addition of an alkaline reagent comprising an aqueous solution of a quaternary ammonium salt and an alkali metal cyanide, the mixture being buffered with a basic buffer in sufficient concentration to allow rapid neutralization of said lytic diluent which is buffered to an acid pH.

11. The lytic dilutent of claim 10 wherein said quaternary ammonium salt is present in a concentration of about 80 to 200 mg/liter by weight of said solution, and said chromagen formation is completed by subsequent addition of an alkaline reagent comprising an aqueous solution of said quaternary ammonium salt and alkali metal cyanide, buffered with a basic buffer in sufficient concentration to allow rapid neutralization of said acid lytic diluent.

12. The lytic diluent of claim 10 wherein said alkali metal cyanide is present in a concentration in the range of 200 to 400 mg/liter.

13. The lytic diluent of claim 10 wherein said basic buffer is selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate.

14. A method for the differential determination of lymphoid and myeloid populations of leukocytes and also for measuring hemoglobin by chromagen formation which comprises employing a diluent which is a mixture of ingredients (A) and (B) where

(A) is an aqueous saline solution of at least one quaternary ammonium salt having surface active properties, and

(B) is at least one additive selected from the group consisting of

(1) a short chain alkanol substituted by phenyl or phenoxy;

and

(2) a polyhydroxy compound;

said lytic diluent being buffered to an acid pH within the range of 3.5 to 5.0; and

each of said ingredients (A) and (B) being present in an amount sufficient to bring about rapid and complete lysing of red cells in whole blood for making a differential determination of lymphoid and myeloid populations of leukocytes, and also for measuring hemoglobin by chromagen formation.

15. The method of claim 14 wherein said quaternary ammonium salt is a mixture of hexadecyltrimethyl ammonium bromide and tetradecyltrimethyl ammonium bromide, which is present in the range of about 200 mg to 500 mg/liter by weight of said solution.

16. A method for the differential determination of lymphoid and myeloid populations of leukocytes and also for measuring hemoglobin by chromagen formation which comprises employing a diluent which is a mixture of ingredients (A) and (B) where

(A) is an aqueous saline solution of at least one quaternary ammonium salt having surface active properties, and

(B) is at least one additive selected from the group consisting of

(1) a short chain alkanol substituted by phenyl or phenoxy;

and

(2) a polyhydroxy compound;

said lytic diluent being buffered to an acid pH within the range of 3.5 to 5.0, and each of said ingredients (A) and (B) being present in an amount sufficient to bring about rapid, but incomplete, lysing of red cells in whole blood for making a differential determination of lymphoid and myeloid populations of leukocytes, and also for measuring hemoglobin by chromagen formation, wherein said chromagen formation is completed by the addition of an alkaline reagent comprising an aqueous solution of a quaternary ammonium salt and an alkali metal cyanide, the mixture being buffered with a basic buffer in sufficient concentration to allow rapid neutralization of said lytic diluent which is buffered to an acid pH.

17. The method of claim 16 wherein said quaternary ammonium salt in said diluent is hexadecyltrimethyl ammonium bromide which is present in a concentration of about 80 to 200 mg/liter of said solution, and said chromagen formation is completed using as quaternary ammonium salt tetradecyltrimethyl ammonium bromide in a concentration of about 30 to 50 mg/liter.