Technical aspects of rosette tests for the demonstration of lymphocyte subpopulations.

Lymphocytes from heparinized or defibrinated blood were separated on Lymphoprep and washed in phosphate-buffered saline (PBS) or Hanks' balanced salt solution (HBSS). Defibrination caused a decreased yield of lymphocytes compared to heparin treatment. The cell loss was probably non-selective, as only minor differences in lymphocyte subpopulations were found. However, lymphocytes from defibrinated blood, washed in PBS gave a lower percentage of rosette-forming cells (RFC) in most tests, and higher number of latex-phagocytizing cells. For the stabilization of sheep erythrocyte (E)-RFC, treatment of E with 2-amino-ethylisothiouronium bromide hydrobromide (AET), with addition of fetal calf serum (FCS), and E-RFC without FCS fixed with glutaraldehyde gave similar results, and higher percentages of RFC than the RFC test performed with FCS. Storage of whole blood or separated lymphocytes for 24 h at 4 degrees C generally resulted in a reduction in the percentages of E-RFC, particularly active E-RFC, but not of EA- or EAC-RFC. The ranges of the results were usually wider after storage.