One step method for detection and enumeration of absolute counts of one more cell populations in a sample

• US 5,627,037 A
• Filed: 08/09/1994
• Issued: 05/06/1997
• Est. Priority Date: 08/07/1990
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Recktenwald, Diether J., Mercolino, Thomas J., Ward, Anthony J.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/287,759
• Time in Patent Office: 1,001 Days
• Field of Search: 435/7.21, 530/380, 250/459.1, 250/461.2, 436/10, 436/43, 436/63, 436/172, 436/548, 436/800, 436/805
• US Class Current: 435/7.21
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/11 Automated chemical analysis

Abstract

This invention comprises a one step method for the detection and enumeration of absolute counts of one or more cell populations in a blood sample. The method employs a reagent comprising a mixture of one or more cell markers, a fluorescent microparticle and a fixative. The reagent may be combined with unlysed whole blood and analyzed by means of flow cytometry.

Claims

1. A method for determining the absolute counts of CD4⁺ lymphocytes within a whole blood sample by means of flow cytometry comprising the steps of:

(a) adding the sample to a tube containing a diluent comprising an anti-CD4 monoclonal antibody bound to phycoerythrin, paraformaldehyde and a known concentration of a 2 μ

m autofluorescent coumarin microparticle;

(b) setting a fluorescence trigger in the second fluorescence channel to include at least 99% of all microparticles and CD4⁺ lymphocytes;

(c) setting fluorescence gates in the first and second fluorescence channels to distinguish between the phycoerythrin fluorescence associated with the labelled CD4⁺ lymphocytes and the autofluorescence of the microparticle;

(d) counting the number of CD4⁺ lymphocytes and microparticles from step (c); and

(e) calculating the number of CD4⁺ lymphocytes per microparticle from step (d) and multiplying by the concentration of microparticles resulting in the absolute count of CD4⁺ lymphocytes per unit volume.

2. A method of staining cells in a sample containing said cells with one or more immunofluorescence markers, said immunofluorescence markers comprising a monoclonal antibody bound to a fluorochrome, wherein the sample is added directly to a tube containing a diluent, said diluent comprising said one or more immunofluorescence markers and a fixative.

3. The method of claim 2 wherein the fluorochromes are selected from the group consisting of phycobiliproteins, fluorescein derivatives, rhodamine, pthalocyanine derivatives, peridinin chlorophyll complex and coumarin derivatives.

4. The method of claim 3 wherein the fluorochrome is a phycobiliprotein.

5. The method of claim 4 wherein the phycobiliprotein is phycoerythrin.

6. The method of claim 2 wherein the fixative comprises the group consisting of paraformaldehyde and formaldehyde.

7. The method of claim 2 the concentration of the immunofluorescence marker is between 0.02 μ

g/ml and 20 μ

g/ml.

8. The method of claim 2 wherein the diluent further comprises a fluorescent microparticle.

9. A solution for staining cells comprising a diluent, said diluent comprising one or more immunofluorescence markers and a fixative.

10. The solution of claim 9 wherein the diluent further comprises a fluorescent microparticle.

11. The solution of claim 9 wherein the fixative is paraformaldehyde.

12. A solution for staining unlysed whole blood comprising a diluent, said diluent comprising a fluorescently labelled monoclonal antibody, a fixative and a fluorescent microparticle.

13. The solution of claim 12 wherein the antibody is an anti-CD4 monoclonal antibody.

14. The solution of claim 12 wherein the fluorescent label is phycoerythrin.

15. The solution of claim 12 wherein the fixative is paraformaldehyde.

16. The solution of claim 12 wherein the microparticle is an autofluorescent 2 μ

m coumarin bead.

17. A solution for staining CD4⁺ lymphocytes in an unlysed whole blood sample wherein the solution comprises an anti-CD4 monoclonal antibody bound to phycoerythrin, paraformaldehyde and an autofluorescent 2 μ

m coumarin bead.