Method for counting leukocytes and leukocyte counter

Abstract

A method for counting leukocytes which comprises liberating elastase from granulocytes contained in a specimen, adding an anti-granulocyte elastase antibody to the thus liberated elastase, measuring the antibody bonded to the elastase to thereby determine the concentration of the elastase, and then calculating therefrom the number of leukocytes contained in the specimen with the use of the ratio of the leukocyte count to the elastase concentration. This method makes it possible to conveniently and less expensively count leukocytes at a high accuracy comparable to the one established by using a conventional automatic blood cell counter, as the figure shows.

1 . A method for counting leukocytes comprising: liberating elastase from neutrophils, eosionphils and basophils (hereafter, these three blood cells are referred to as “granulocytes”) in leukocytes contained in a specimen; adding an antigranulocyte elastase antibody to the thus liberated elastase; measuring the antigranulocyte elastase antibody bonded to the elastase to thereby determine the concentration of the elastase; and calculating the number of leukocytes contained in the specimen from the thus determined concentration of elastase by using a known ratio of the leukocyte count, the granulocyte count or the neutrophil count to the concentration of elastase.  
     
     
         2 . The method according to  claim 1 , comprising: bonding the same protease inhibitor as a protease inhibitor contained in the specimen to the liberated elastase, and then adding an antigranulocyte elastase antibody to the elastase.  
     
     
         3 . The method according to  claim 2 , wherein the specimen is whole blood and the protease inhibitor is α1-antitrypsin.  
     
     
         4 . The method according to  claim 2  or  3 , wherein the protease inhibitor is added in the range from 5 to 10 times weight as the granulocyte elastase.  
     
     
         5 . The method according to any one of  claims 1  to  3 , comprising causing an antigen-antibody reaction between an antibody particle in which the antigranulocyte elastase antibody is bonded to a latex particle and the elastase liberated from granulocytes, and determining the degree of agglutination of the latex particle to thereby measure the antigranulocyte elastase antibody bonded to the elastase.  
     
     
         6 . The method according to  claim 5 , wherein the antibody particle in which the antigranulocyte elastase antibody is bonded to the latex particle is added at the rate of 0.1 to 5 mg with respect to 1 ml of the specimen.  
     
     
         7 . The method according to any one of  claims 1  to  3 , comprising bonding the elastase liberated from granulocytes to the antigranulocyte elastase antibody bonded to a solid phase; adding a labeled antigranulocyte elastase antibody therein in this state; and measuring the labeled antigranulocyte elastase antibody bonded to the elastase.  
     
     
         8 . The method according to  claim 7 , wherein a label of the labeled antigranulocyte elastase antibody is at least one label selected from the group consisting of an enzyme label, a radioactive label and a fluorescent label.  
     
     
         9 . The method according to  claim 7 , wherein the label of the labeled antigranulocyte elastase antibody is an enzyme and a detectable product is produced by a reaction between the enzyme and its substrate.  
     
     
         10 . The method according to  claim 9 , wherein the enzyme is peroxidase and its substrate is orthophenylene diamine and hydrogen peroxide.  
     
     
         11 . The method according to any one of  claims 1  to  3 , wherein the known ratio is a statistical ratio of the leukocyte count or the granulocyte count to the elastase concentration.  
     
     
         12 . The method according to any one of  claims 1  to  3 , wherein the known ratio is the elastase concentration with respect to one granulocyte cell.  
     
     
         13 . The method according  claim 12 , wherein the elastase concentration with respect to one granulocyte cell is in the range from 1 to 5 ng/ml.  
     
     
         14 . The method according to  claim 12 , wherein the number of whole leukocytes is calculated from the number of granulocytes by using a known ratio of a granulocyte count to the number of whole leukocytes.  
     
     
         15 . A leukocyte counter used for performing the method for counting leukocytes according to  claim 1 , comprising a means for calculating the number of leukocytes in a specimen from the concentration of elastase liberated from granulocytes in the specimen by using a known ratio of a leukocyte count, a granulocyte count or a neutrophil count to the concentration of elastase.  
     
     
         16 . The leukocyte counter according to  claim 15 , comprising a means for liberating elastase from granulocytes in the specimen, a means for adding an antigranulocyte elastase antibody to the thus liberated elastase, a means for measuring the antigranulocyte elastase antibody bonded to the elastase to thereby determine the concentration of elastase; and a means for calculating the number of leukocytes contained in the specimen from the determined concentration of elastase by using a known ratio of a leukocyte count, a granulocyte count or a neutrophil count to the concentration of elastase.  
     
     
         17 . A reagent kit used for the method for counting leukocytes according to  claim 1 , including an antigranulocyte elastase antibody.  
     
     
         18 . A reagent kit used for the method for counting leukocytes according to  claim 5 , including an antibody particle in which an antigranulocyte elastase antibody is bonded to a latex particle.  
     
     
         19 . A reagent kit used for the method for counting leukocytes according to  claim 7 , including a labeled antigranulocyte elastase antibody and an antigranulocyte elastase antibody bonded to a solid phase.  
     
     
         20 . A reagent kit according to any one of  claims 17  to  19 , including the same protease inhibitor as the protease inhibitor contained in a specimen to be determined.  
     
     
         21 . The reagent kit according to  claim 20 , wherein the specimen to be determined is whole blood and the protease inhibitor is α1-antitrypsin.  
     
     
         22 . The reagent kit according to any one of  claims 17  to  19 , further including a lysing material.