Method and apparatus for measuring white blood cell count

Abstract

A method for quantitatively measuring white blood cell count involves capture of white blood cells from a fluid sample by a retainer, removal of the red blood cells and other interfering substances by a wash solution, and reading the result of a color reaction in which an ester which is present on the white blood cells cleaves a chromogenic substrate which produces a water insoluble dye. The apparatus for use in the present method includes a retainer for white blood cells that optionally has a dye substrate immobilized therein and an absorption layer that wicks and takes up all excess washing solution flowing past the sample.

1 . A method for counting white blood cells in a sample comprising: 
 a. capturing white blood cells from the sample on a retainer;    b. removing interfering substances form the white blood cells captured on the retainer by washing the retainer with a washing solution;    c. contacting the sample with a chromogenic substrate cleaved by an enzyme present on the white blood cells which produces a water insoluble dye, wherein said substrate is either immobilized on the membrane or comprises part of the washing solution; and    d. reading the color change resulting from the enzyme present in the white blood cells.    
   
   
       2 . The method according to  claim 1  wherein the enzyme is esterase.  
   
   
       3 . The method according to  claim 1  wherein the sample is selected from the group consisting of blood, milk, urine, saliva, and perspiration.  
   
   
       4 . The method according to  claim 1  wherein the wash solution contains a buffer, an optional reaction accelerator, and an optional substrate.  
   
   
       5 . The method according to  claim 4  wherein the buffer has a pH range of from about 8 to about 11.  
   
   
       6 . The method according to  claim 5  wherein the buffer has a pH range of from about 9 to about 10.5.  
   
   
       7 . The method according to  claim 4  wherein the wash solution contains both water and a polar organic solvent.  
   
   
       8 . The method according to  claim 4  wherein the wash solution contains a nonionic surfactant.  
   
   
       9 . The method according to  claim 1  wherein the white blood cells contain CD4 component.  
   
   
       10 . A method for detecting mastitis comprising counting white blood cells in a sample of milk comprising: 
 a. capturing white blood cells from the sample on a retainer;    b. removing interfering substances form the white blood cells captured on the retainer by washing the retainer with a washing solution;    c. contacting the sample with a chromogenic substrate cleaved by an enzyme present on the white blood cells which produces a water insoluble dye, wherein said substrate is either immobilized on the membrane or comprises part of the washing solution; and    d. reading the color change resulting from the enzyme present in the white blood cells.    
   
   
       11 . The method according to  claim 10  wherein the enzyme is esterase.  
   
   
       12 . A device for counting white blood cells comprising: 
 a. a white blood cell capture retainer on which is optionally immobilized a dye substrate and    b. an absorption layer that wicks and takes up excess washing solution flowing past a sample.    
   
   
       13 . The device according to  claim 12  wherein the retainer has a pore size ranging from about 3 to about 15 microns and a negative positive charge.  
   
   
       14 . The device according to  claim 13  wherein the retainer is a porous membrane.  
   
   
       15 . The device according to  claim 12  further including a plastic cover having an opening for application of sample and wash solution.