Process for discriminating and counting erythroblasts

Abstract

A method for discriminating and counting erythroblasts comprises the steps of: (i) staining leukocytes in a hematologic sample by adding a fluorescent labeled antibody capable of binding specifically with leukocytes to the hematologic sample; (ii) raising the permeability only of cell membranes of erythroblasts in the hematologic sample to a nucleotide fluorescent dye which does not permeate a cell membrane usually, the nucleotide fluorescent dye having a fluorescent spectrum capable of being distinguished from that of a fluorescent labeling compound of the fluorescent labeled antibody in step (i); (iii) staining nuclei of the erythroblasts in the hematologic sample with the nucleotide fluorescent dye; (iv) subjecting the hematologic sample to flowcytometry to detect at least two fluorescent signals from each cell; and (v) discriminating and counting the erythroblasts from difference in intensity between the at least two fluorescent signals.

1 - 13 . (canceled)  
     
     
         14 . A method for discriminating and counting erythroblasts comprising steps of: 
 (i) staining leukocytes in a hematological sample by adding an antibody labeled by a fluorescent labeling compound, the antibody capable of binding specifically with leukocytes in the hematological sample;    (ii) staining nuclei of erythroblasts in the hematological sample by mixing the hematological sample with a nucleotide fluorescent dye, the nucleotide fluorescent dye having a fluorescent spectrum that is distinguishable from that of the fluorescent labeling compound;    (iii) subjecting the hematological sample using to flow cytometry to detect a first fluorescent signal based on the nucleotide fluorescent dye and a second fluorescent signal based on the fluorescent labeling compound;    (iv) discriminating the erythroblasts based on a difference in intensity between the first fluorescent signal and the second fluorescent signal; and    (v) counting the discriminated erythroblasts.    
     
     
         15 . The method according to claim  1 , wherein the steps (i) to (v) are performed in the order recited.  
     
     
         16 . The method according to claim  1 , wherein the antibody recognizes an antigen present on the leukocytes surface and binds with the antigen.  
     
     
         17 . The method according to claim  1 , wherein the antibody comprises at least one compound selected from the group consisting of phycoerythrin, fluorescein isothiocyanate, allophycocyanin, Texas Red, arylsulfonate, cyanine fluorescent dye CY5, peridinin chlorophyll complex, and a combination thereof.  
     
     
         18 . The method according to claim  2 , wherein the nucleotide fluorescent dye comprises at least one compound selected from the group consisting of propidium iodide, N-methyl-4-(1-pyrene)-vinyl-propidium iodide, ethidium bromide, TOTO-1, TOTO-3, YOYO-1, YOYO-3, BOBO-1, BOBO-3, ethidium homodimer-1, ethidium homodimer-2, POPO-1, POPO-3, BO-PRO-1, YO-PRO-1, and TO-PRO-1.  
     
     
         19 . The method according to claim  1 , wherein a first fluorescent intensity based on the first fluorescent signal and a second fluorescent intensity based on the second fluorescent signal are plotted in two coordinate axes to obtain a two-dimensional distribution chart.  
     
     
         20 . The method according to claim  6 , wherein an area in which the erythroblasts appear is defined on the two-dimensional distribution chart and the number of erythroblasts in the area is counted.  
     
     
         21 . The method according to claim  7 , wherein a second area in which the leukocytes appear is defined on the two-dimensional distribution chart and the number of leukocytes in the second area is counted.  
     
     
         22 . A method for discriminating and counting erythroblasts comprising steps of: 
 (i) staining leukocytes in a hematological sample by adding an antibody labeled by a fluorescent labeling compound, the antibody capable of binding specifically with leukocytes in the hematological sample;    (ii) raising the permeability only of cell membranes of erythroblasts in the hematological sample to a nucleotide fluorescent dye which does not permeate a cell membrane usually, the nucleotide fluorescent dye having a fluorescent spectrum that is distinguishable from that of the fluorescent labeling compound;    (iii) staining nuclei of the erythroblasts in the hematological sample by mixing the hematological sample with the nucleotide fluorescent dye;    (iv) subjecting the hematological sample using to flow cytometry to detect a first fluorescent signal based on the nucleotide fluorescent dye and a second fluorescent signal based on the fluorescent labeling compound;    (v) discriminating the erythroblasts based on a difference in intensity between the first fluorescent signal and the second fluorescent signal; and    (vi) counting the discriminated erythroblasts.    
     
     
         23 . The method according to claim  9 , wherein the antibody recognizes an antigen present on the leukocytes surface and binds with the antigen.  
     
     
         24 . The method according to claim  9 , wherein the antibody comprises at least one compound selected from the group consisting of phycoerythrin, fluorescein isothiocyanate, allophycocyanin, Texas Red, arylsulfonate, cyanine fluorescent dye CY5, peridinin chlorophyll complex, and a combination thereof.  
     
     
         25 . The method according, to claim  9 , wherein the nucleotide fluorescent dye comprises at least one compound selected from the group consisting of propidium iodide, N-methyl-4-(1-pyrene)-vinyl-propidium iodide, ethidium bromide, TOTO-1, TOTO-3, YOYO-1, YOYO-3, BOBO-1, BOBO-3, ethidium homodimer-1, ethidium homodimer-2, POPO-1, POPO-3, BO-PRO-1, YO-PRO-1, and TO-PRO-1.  
     
     
         26 . The method according to claim  9 , wherein a first fluorescent intensity based on the first fluorescent signal and a second fluorescent intensity based on the second fluorescent signal are plotted in two coordinate axes to obtain a two-dimensional distribution chart.  
     
     
         27 . The method according to claim  13 , wherein an area in which the erythroblasts appear is defined on the two-dimensional distribution chart and the number of erythroblasts in the area is counted.  
     
     
         28 . The method according to  claim 14 , wherein a second area in which the leukocytes appear is defined on the two-dimensional distribution chart and the number of leukocytes in the second area is counted.  
     
     
         29 . A method for discriminating and counting erythroblasts comprising steps of: 
 (i) staining leukocytes in a hematological sample by adding an antibody labeled by a fluorescent labeling compound, the antibody capable of binding specifically with leukocytes in the hematological sample;    (ii) staining nuclei of erythroblasts in the hematological sample by mixing the hematological sample with a nucleotide fluorescent dye so as to stain the erythroblasts according to degree of maturity, the nucleotide fluorescent dye having a fluorescent spectrum that is distinguishable from that of the fluorescent labeling compound;    (iii) subjecting the hematological sample using to flow cytometry to detect a first fluorescent signal based on the nucleotide fluorescent dye and a second fluorescent signal based on the fluorescent labeling compound; and    (iv) discriminating and counting the erythroblasts based on a difference in intensity between the first fluorescent signal and the second fluorescent signal so as to classify the erythroblasts into at least two groups, each group of erythroblasts differing in degree of maturity.    
     
     
         30 . The method according to  claim 16 , wherein a first fluorescent intensity based on the first fluorescent signal and a second fluorescent intensity based on the second fluorescent signal are plotted in two coordinate axes to obtain a two-dimensional distribution chart.  
     
     
         31 . The method according to  claim 17 , wherein an area in which the erythroblasts appear is defined on the two-dimensional distribution chart and the number of erythroblasts in the area is counted.  
     
     
         32 . The method according to  claim 18 , wherein a second area in which the leukocytes appear is defined on the two-dimensional distribution chart and the number of leukocytes in the second area is counted.  
     
     
         33 . The method according to  claim 16 , wherein the nucleotide fluorescent dye is used at a concentration within the range of 0.003 mg/L to 10 mg/L.