HOW SHOULD CD34 PLUS CELLS BE ANALYZED - A STUDY OF 3 CLASSES OF ANTIBODY AND 5 LEUKOCYTE PREPARATION PROCEDURES

For patients undergoing stem cell transplantation after intensive marr ow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maxim ize the number of cells collected by leucophoresis for subsequent haem atopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, alt hough there is no consensus as to the best method. In this study, we h ave examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whale blood, bone marrow and leucophoresis samples were analysed either directly a fter labelling with a vital nuclear dye (LDS-751) and fluorochrome-con jugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FAGS Lysing Solution). By comparison with the resul ts obtained from viable leucocytes in unmanipulated samples, it was fo und that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associ ated with a vital nuclear dye (in this instance LDS-751) and the CD34 cells are detected with fluorescein isothiocyanate- or phycoerythrin- conjugated antibodies.