Artefactual variations in the B and T subpopulations of rabbit blood lymphocytes depending on method of isolation, and blocking of C3 receptors due to in vitro activation of complement.

Marked differences were found in the proportions of lymphocyte subpopulations in rabbit peripheral blood depending on the techniques used for the purification of lymphocytes. Rosette-forming reactions were used to find the numbers of T lymphocytes, Ig-bearing cells and cells with receptors for C3 or IgG-Fc. Some of the methods used for lymphocyte separation altered the relative numbers of T and B lymphocytes, through a disproportionate loss of T cells. Other changes were due to in vitro activation of complement detectable by the presence of C3 on the lymphocyte cell-membrane and causing partial blocking of C3 receptors. Highest yields of lymphocytes were obtained from defibrinated blood treated with carbonyl iron to remove phagocytes and methyl cellulose to sediment erythrocytes. This procedure was accompanied by in vitro activation of complement, with the consequences mentioned. Complement activation was inhibited by taking the blood into either EDTA or citrate. As EDTA was cytotoxic for rabbit T lymphocytes, citrate was considered best although the resulting lymphocyte suspensions were contaminated with up to 25% granulocytes and monocytes owing to the inhibition of carbonyl iron uptake by the prior exposure to citrate.