Improved method for direct counting of basophil leucocytes.

An improved method for the direct counting of basophil leucocytes from venous or capillary blood is described. The method is based upon the use of cetylpyridinium chloride to lyse the erythrocytes and render the muco-polysaccharides of the granule insoluble. Aluminium sulphate is used to mordant toluidine blue to the granules. Our knowledge of the role of the basophil leucocytes in disease has been greatly hampered by the lack of a satisfactory method for their direct enumeration in capillary or venous blood. The essential difficulty lies in their relative paucity, so that in order to get sufficient numbers to count, concentrated blood solutions have to be used. Most methods for counting basophils are based on the metachromatic staining of mucopolysaccharide in the basophil granules by aqueous solutions of toluidine blue. Saponin is used to lyse the erythrocytes and potassium or ammonium sulphate mixtures are sometimes added to keep the diluted blood in a fluid state (Boseila, 1959; Gurr, 1962). In our hands these methods have been unsatisfactory from two points of view. The staining of the basophils is often diffuse because of the water solubility of the poly-saccharides, making identification difficult and the count time consuming. Clotting and aggregation of platelets makes counting inaccurate and in addition the aggrega-tion is often associated with the degranulation of the basophils in that area thus giving lower counts than the true value. The following method has been devised to overcome these difficulties. SOLUTIONS Two solutions are required (1) 0-1 % E.D.T.A. in saline (disodium salt) and (2) 25 ml. of 0 5 % cetylpyridinium chloride, 25 ml. of distilled water, 20 ml. of 0-8 % toluidine blue in 5 % aluminium sulphate, filtered before use. METHOD Deliver to the bottom of a 75 x 10 mm. tube 0-08 ml. of solution 1. Add 0-02 ml. of blood taken from a free flowing thumb prick after wiping away the first drop that exudes and mix by gentle shaking. Add 0-1 ml. of stain solution 2, mix and stopper. Fill two Fuchs-Rosenthal chambers using a Pasteur pipette, leave the chambers to stand in a moist chamber for five minutes to allow the cells to settle. Basophils are seen as purple/red metachromatically stained cells. Other leucocytes, platelets, and erythrocytes are unstained. The use of E.D.T.A. as an anticoagulant eliminates the clotting of platelets. The cetylpyridinium chloride fulfils two functions: it lyses the erythrocytes and also complexes with …