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 nonselective, 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 (AFT), 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°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.