Changes in L-selectin expression on CD34-positive cells upon cryopreservation of peripheral blood stem cell transplants

Several studies have pointed out that L-selectin on CD34-positive cells pla ys a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin ex pression in lymphocytes and granulocytes can be induced by a variety of str ess situations, we have investigated in this study whether the freeze-thawi ng procedure, used in PBSC transplantation, would affect L-selectin express ion on CD34(+) stem cells. Flow cytometry was performed by labelling the ce lls with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresi s procedure itself caused a slight decrease of L-selectin expression on CD3 4 cells in 11 out of 12 cases (mean decrease of the percentage of positive cells 11.9; range 6-23). A much larger decrease was found upon freeze-thawi ng: a mean of 39% (range 4-78% in 27 cases) compared to fresh material. To determine if L-selectin expression might be up-regulated after cryopreserva tion, thawed transplant samples (n = 11) were incubated at 37 degrees C in RPMI with 10% FCS at 5% CO2. Already early in the course of incubation two CD34-positive populations appeared in the blast region, characterized by ei ther a low or high forward scatter. Simultaneous viability staining with th e DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that th e population with low forward scatter was apoptotic while the population wi th the high forward scatter was non-apoptotic. The latter population is con sidered to be most relevant for transplantation. In this population an incr ease of L-selectin expression after overnight incubation was observed in 8/ 11 samples up to values of 46-120% of the values of the fresh nonfrozen sam ples. In addition, the mean fluorescence intensity was significantly increa sed in 10/11 cases. Kinetic experiments with shorter incubation times revea led that only part of the leucapheresis samples (two out of 8) showed an in crease of L-selectin expression within 4 h. In addition, a decrease of L-se lectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, 11 = 5). In contrast to frozen samples, L-selectin reappearance was seen already wit hin 4 h of incubation in all samples. Both the loss of L-selectin expressio n on CD34 cells occurring upon freeze-thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells var ies largely between individual leucapheresis samples, and therefore it is c oncluded that such processes should be considered when correlations with cl inical outcome after transplantation are made.