A simple, simultaneous micro-method for enumeration of T- and B-cell populations with double-rosette assay using immunobeads and sheep red blood cells (SRBC)

Abstract

As an application of Immunobeads, which are the synthetic polyacrylamide particles coated with anti-human immunoglobulin antibodies, the authors have improved Lee's method to a new micro technique to identify and enumerate human T and B lymphocytes simultaneously. Only 1 ml of heparinized blood is required as a specimen.The principle is based on the reaction of lymphocytes attached onto a plastic microplate with SRBC and immunobeads. The key point of this technique is the order of the addition of the two reagents, at first Immunobeads and then SRBC, otherwise the number of B cells will be much affected. The optimum conditions for this technique were established including the concetration and the volume of lymphocytes and reagents, reaction time and temperature.The improved, micro and simultaneous method showed a good coincidence with single rosette assay with SRBC, anti-Ig immunofluorescence and Immunobeads assay on 13 healthy normal lymphocytes. It is simple, easy to perform and clear-cut, resulting very few dublerosette cells.