Compositions and methods for leukocyte differential counting

Abstract

The present invention provide compositions and methods for classifying leukocytes in a leukocyte population using fluorescence detection. The methods include contacting a leukocyte population in a sample having one or more leukocyte types with a diagnostic composition, exciting the leukocyte population with a light source; and measuring emitted light from each of the one or more leukocyte types to classify the leukocyte population.

1 . A diagnostic composition, said diagnostic composition comprising:
 a first dye having a cationic charge;   a second dye having less of a cationic charge compared to said first dye; and   a third dye being reactive to a protein, having an affinity to a protein or having an anionic charge.   
     
     
         2 . The diagnostic composition of  claim 1 , wherein said first dye has at least two cationic charges. 
     
     
         3 . The diagnostic composition of  claim 1 , wherein said second dye has one cationic charge. 
     
     
         4 . The diagnostic composition of  claim 2 , wherein said first dye is propidium iodide. 
     
     
         5 . The diagnostic composition of  claim 3 , wherein said second dye is Basic Orange 21. 
     
     
         6 . The diagnostic composition of  claim 1 , wherein said third dye is fluorescein isothiocyanate. 
     
     
         7 . A method for classifying leukocytes in a leukocyte population with fluorescence detection, said method comprising:
 contacting a leukocyte population in a sample having one or more leukocyte types with a diagnostic composition, said diagnostic composition comprising:   a first dye having a cationic charge;   a second dye having less cationic charge compared to said first dye; and   a third dye being reactive to a protein, having an affinity to a protein or having an anionic charge to stain the leukocyte population to form a stained leukocyte population;   exciting the stained leukocyte population with a light source; and   measuring emitted light from each of the one or more leukocyte types to classify the leukocyte population.   
     
     
         8 . The method for classifying of  claim 7 , wherein the number of leukocyte types in the leukocyte population is a member selected from the group consisting of 2, 3, 4 or 5 leukocyte types. 
     
     
         9 . The method for classifying of  claim 8 , wherein the fluorescence emitted from each of the leukocyte types is distinguishable from each of the other types in the population. 
     
     
         10 . The method for classifying of  claim 7 , wherein the leukocytes in the population comprise a lymphocyte, a monocyte, a neutrophil, an eosinophil and a basophil. 
     
     
         11 . The method for classifying of  claim 7 , wherein the first dye primarily stains the nucleus of the one or more leukocyte types. 
     
     
         12 . The method for classifying of  claim 7 , wherein the second dye primarily stains the cytoplasm of the one or more leukocyte types. 
     
     
         13 . The method for classifying of  claim 7 , wherein the third dye primarily stains the protein content of the one or more leukocyte types. 
     
     
         14 . The method for classifying of  claim 7 , wherein the excitation light is emitted from a source selected from the group consisting of a diode laser, a light emitting diode (LED), an ion laser, a dye laser, and a lamp. 
     
     
         15 . The method for classifying of  claim 14 , wherein the emitted light is measured at a first wavelength and at a second wavelength to achieve two color fluorescence measurements. 
     
     
         16 . The method for classifying of  claim 15 , wherein the first wavelength is at about 510 nm to about 560 nm. 
     
     
         17 . The method for classifying of  claim 15 , wherein the second excitation light is emitted at about 590 nm to about 650 nm. 
     
     
         18 . The method for classifying of  claim 15 , wherein the emitted light is measured at the first and the second wavelength. 
     
     
         19 . The method for classifying of  claim 18 , wherein the emitted light is measured simultaneously by two photomultiplier tubes. 
     
     
         20 . The method for classifying of  claim 7 , wherein the sample is selected from the group consisting of whole blood, plasma, and serum. 
     
     
         21 - 31 . (canceled)