Factors affecting the differential counting of human lymphocyte subpopulations in blood smears

We have previously used the antibody-mediated binding of bacteria to identify Ig-bearing cells and the natural binding of bacteria to identify several lymphocyte subpopulations. By using bacteria this identification can be carried out in conventional blood smears since bacteria are small and easily distinguished from all blood elements. To develop a standardized method for identification of lymphocyte subpopulations that may become useful in clinical laboratories, we investigated here several parameters that might affect the safety and accuracy of the test, and that might simplify the procedure. We found that: (1) the rosettes formed between bacteria and lymphocytes cannot be disrupted by vigorous handling; (2) the ability fo form rosettes is a stable property of the bacterial strains; (3) for optimal results the blood sample must not be stored for more than 4 hr at 25 degrees C and the entire procedure must be performed in medium supplemented with 6% bovine serum albumin; (4) a large excess of anti-Ig antibody is required for the optimal coating of bacteria to detect Ig-bearing cells; (5) both the antibody-coated bacteria and formaldehyde-fixed bacteria can be stored at 4 degrees C or at -20 degrees C for at least 6 months; (6) the buffy coat from the blood sample can be used instead of the whole blood to reduce the time required for reading the smears; and (7) some of the pathogenic bacteria can be killed by autoclaving without modifying their binding properties. A complete and simple method which uses the bacterial adherence for the identification of lymphocyte subpopulations in blood smears is presented.