Method for classifying/counting leukocytes, reagent kit for classifying leukocytes, and reagent for classifying leukocytes

Abstract

The present invention provides a method for classifying and counting leukocytes which allows classification and count of normal leukocytes as well as discrimination between blast cells and atypical lymphocytes. The present invention also provides a regent kit and reagent for classifying leukocyte which are used for classifying and counting leukocytes in biological samples.

What is claimed is: 
     
         1 . A method for classifying and counting leukocytes comprising the steps of:
 preparing a measurement sample by mixing a biological sample; a first reagent containing a fluorescent dye capable of staining nucleic acid; and a second reagent containing cationic and nonionic surfactants for lysing erythrocytes and damaging cell membranes of leukocytes so as to be permeable to the fluorescent dye and an aromatic organic acid at a concentration of not less than 20 mM and not more than 50 mM;   applying light to the prepared measurement sample and obtaining scattered light information and fluorescence information generated thereby; and   based on the obtained scattered light information and fluorescence information, classifying the leukocytes in the biological sample and detecting blast cells and atypical lymphocytes by differentiating therebetween;   wherein when the second reagent contains the aromatic organic acid at a concentration of not less than 20 mM and less than 30 mM, the second reagent has pH of not lower than 5.5 and not higher than 6.4, and when the second reagent contains the aromatic organic acid at a concentration of not less than 30 mM and not more than 50 mM, the second reagent has pH of not lower than 5.5 and not higher than 7.0.   
     
     
         2 . The method according to  claim 1 , wherein the aromatic organic acid is at least one selected from the group consisting of an aromatic carboxylic acid, an aromatic sulphonic acid and a salt thereof. 
     
     
         3 . The method according to  claim 1 , wherein the second reagent contains the aromatic organic acid at a concentration of not less than 40 mM and not more than 50 mM. 
     
     
         4 . The method according to  claim 1 , wherein the second reagent has pH of not lower than 5.5 and not higher than 6.2. 
     
     
         5 . The method according to  claim 1 , wherein the scattered light information is side scattered light information. 
     
     
         6 . The method according to  claim 1 , wherein the cationic surfactant is a quaternary ammonium salt surfactant or a pyridinium salt surfactant. 
     
     
         7 . The method according to  claim 1 , wherein the nonionic surfactant is a polyoxyethylene nonionic surfactant represented by the following general formula (VI):
   R 1 —R 2 —(CH 2 CH 2 O) n —H  (VI)
   wherein R 1  is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms; R 2  is an oxygen atom, —COO— or   
       
         
           
           
               
               
           
         
       
       and
 n is an integer of 10 to 50. 
 
     
     
         8 . A reagent kit for classifying leukocytes comprising
 a first reagent containing a fluorescent dye capable of staining nucleic acid; and   a second reagent containing cationic and nonionic surfactants for lysing erythrocytes and damaging cell membranes of leukocytes so as to be permeable to the fluorescent dye and an aromatic organic acid at a concentration of not less than 20 mM and not more than 50 mM,   wherein when the second reagent contains the aromatic organic acid at a concentration of not less than 20 mM and less than 30 mM, the second reagent has pH of not lower than 5.5 and not higher than 6.4, and when the second reagent contains the aromatic organic acid at a concentration of not less than 30 mM and not more than 50 mM, the second reagent has pH of not lower than 5.5 and not higher than 7.0.   
     
     
         9 . The reagent kit according to  claim 8 , wherein the aromatic organic acid is at least one selected from the group consisting of an aromatic carboxylic acid, an aromatic sulphonic acid and a salt thereof. 
     
     
         10 . The reagent kit according to  claim 8 , wherein the second reagent contains the aromatic organic acid at a concentration of not less than 40 mM and not more than 50 mM. 
     
     
         11 . The reagent kit according to  claim 8 , wherein the second reagent has pH of not lower than 5.5 and not higher than 6.2. 
     
     
         12 . The reagent kit according to  claim 8 , wherein the cationic surfactant is a quaternary ammonium salt surfactant or a pyridinium salt surfactant. 
     
     
         13 . The reagent kit according to  claim 8 , wherein the nonionic surfactant is a polyoxyethylene nonionic surfactant represented by the following general formula (VI):
   R 1 —R 2 —(CH 2 CH 2 O) n —H  (VI)
   wherein R 1  is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms; R 2  is an oxygen atom, —COO— or   
       
         
           
           
               
               
           
         
       
       and 
       n is an integer of 10 to 50. 
     
     
         14 . A reagent for classifying leukocytes comprising a fluorescent dye capable of staining nucleic acid, a cationic surfactant, a nonionic surfactant and an aromatic organic acid,
 wherein the reagent contains the aromatic organic acid at a concentration of not less than 20 mM and not more than 50 mM, and   wherein when the reagent contains the aromatic organic acid at a concentration of not less than 20 mM and less than 30 mM, the reagent has pH of not lower than 5.5 and not higher than 6.4, and when the reagent contains the aromatic organic acid at a concentration of not less than 30 mM and not more than 50 mM, the reagent has pH of not lower than 5.5 and not higher than 7.0.   
     
     
         15 . The reagent according to  claim 14 , wherein the aromatic organic acid is at least one selected from the group consisting of an aromatic carboxylic acid, an aromatic sulphonic acid and a salt thereof. 
     
     
         16 . The reagent according to  claim 14 , wherein the second reagent contains the aromatic organic acid at a concentration of not less than 40 mM and not more than 50 mM. 
     
     
         17 . The reagent according to  claim 14 , wherein the second reagent has pH of not lower than 5.5 and not higher than 6.2. 
     
     
         18 . The reagent according to  claim 14 , wherein the cationic surfactant is a quaternary ammonium salt surfactant or a pyridinium salt surfactant. 
     
     
         19 . The reagent according to  claim 14 , wherein the nonionic surfactant is a polyoxyethylene nonionic surfactant represented by the following general formula (VI):
   R 1 —R 2 —(CH 2 CH 2 O) n —H  (VI)
   wherein R 1  is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms; R 2  is an oxygen atom, —COO— or   
       
         
           
           
               
               
           
         
       
       and
 n is an integer of 10 to 50.