Method and reagents for differential determination of four populations of leukocytes in blood
First Claim
1. A method for stromatolysing the red blood cells in a sample of whole blood and for classification of leukocytes into four categories, one of said categories being eosinophils, which comprises the steps of:
- I. treating a sample of whole blood with aqueous solutions of the following components;
(A) a lysing reagent comprising(1) a single lytic diluent containing saponin, or(2) two solutions to be added separately to said sample of whole blood, consisting of(a) a prediluent and(b) a lytic reagent containing saponin; and
then with(B) a fixing reagent containing a cross-linking compound;
each of said components (A) and (B) being maintained at a predetermined concentration, pH and osmolality;
II. measuring by instrumentation at least two of the primary parameters of DC volume, RF size, fluorescence, and light scatter, to produce cytograms; and
III. observing four populations of leukocytes, one of said categories being eosinophils.
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Abstract
This invention describes a reagent system, including saponin in a lysing reagent and a rapidly active cross-linking agent such as glutaraldehyde as a fixing reagent, which reproducably affects whole blood to cause the red blood cells to stromatolyze and modifies the leukocytes to generate data to define four distinct clusters for detection and classification by flow analysis instrumentation. The clusters represent the four major leukocyte types found in blood: lymphocytes, monocytes, neutrophils and eosinophils, thus providing a method of leukocyte differential analysis. The parameters used for the leukocyte classification include combinations of two or more of DC (Coulter) volume, high frequency (RF) size, opacity (RF size/DC volume), light scatter at various angular ranges, and fluorescence at various wavelengths of illumination.
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8 Claims
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1. A method for stromatolysing the red blood cells in a sample of whole blood and for classification of leukocytes into four categories, one of said categories being eosinophils, which comprises the steps of:
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I. treating a sample of whole blood with aqueous solutions of the following components; (A) a lysing reagent comprising (1) a single lytic diluent containing saponin, or (2) two solutions to be added separately to said sample of whole blood, consisting of (a) a prediluent and (b) a lytic reagent containing saponin; and
then with(B) a fixing reagent containing a cross-linking compound;
each of said components (A) and (B) being maintained at a predetermined concentration, pH and osmolality;II. measuring by instrumentation at least two of the primary parameters of DC volume, RF size, fluorescence, and light scatter, to produce cytograms; and III. observing four populations of leukocytes, one of said categories being eosinophils. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification