Various methods of lymphocyte separation and their relevance with the formation of non-immune rosettes

Lymphocytes (leukocytes mainly containing mononuclear cells) are separated by six different methods and these lymphocytes are used for non-immune rosette formation. It is found that the percentage of non-immune rosettes varies according to the method of separation of lymphocytes. Buffy coat on Dextran-70 and Gravity Sedimentation method give the best rosette yields as compared with lymphocytes separated by centrifugation of heparinized blood (unsupported centrifugation) P less than 0.001) or on Hypaque-Ficoll or Diaginol-Ficoll gradients (supported centrifugation) P less than 0.05). Furthermore, the buffy coat separated on Dextran-70, layered on Ficoll-gradients (supported centrifugation) yields lower values of rosettes (P less than 0.05). It is possible that in certain methods which yield lower values of rosettes there is loss of selected subpopulations of lymphocytes. This loss of subpopulation appears to be due to the centrifugation of the blood and the loss is appreciably more on 'unsupported' than 'supported' centrifugation.