De Boer F, Dräger A M, Van der Wall E, Pinedo H M, Schuurhuis G J
Department of Haematology, University Hospital Vrije Universiteit Amsterdam, The Netherlands.
Bone Marrow Transplant. 1998 Dec;22(11):1103-10. doi: 10.1038/sj.bmt.1701495.
Several studies have pointed out that L-selectin on CD34-positive cells plays a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin expression in lymphocytes and granulocytes can be induced by a variety of stress situations, we have investigated in this study whether the freeze-thawing procedure, used in PBSC transplantation, would affect L-selectin expression on CD34+ stem cells. Flow cytometry was performed by labelling the cells with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresis procedure itself caused a slight decrease of L-selectin expression on CD34 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-thawing: 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 cryopreservation, 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 either a low or high forward scatter. Simultaneous viability staining with the DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that the population with low forward scatter was apoptotic while the population with the high forward scatter was non-apoptotic. The latter population is considered to be most relevant for transplantation. In this population an increase 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 samples. In addition, the mean fluorescence intensity was significantly increased in 10/11 cases. Kinetic experiments with shorter incubation times revealed that only part of the leucapheresis samples (two out of 8) showed an increase of L-selectin expression within 4 h. In addition, a decrease of L-selectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, n = 5). In contrast to frozen samples, L-selectin reappearance was seen already within 4 h of incubation in all samples. Both the loss of L-selectin expression on CD34 cells occurring upon freeze-thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells varies largely between individual leucapheresis samples, and therefore it is concluded that such processes should be considered when correlations with clinical outcome after transplantation are made.
多项研究指出，CD34阳性细胞上的L-选择素在外周血干细胞（PBSC）移植后的造血重建中发挥作用。由于已知多种应激情况可诱导淋巴细胞和粒细胞中L-选择素表达降低，我们在本研究中调查了PBSC移植中使用的冻融程序是否会影响CD34 +干细胞上L-选择素的表达。通过用抗CD34（HPCA2 PE）和抗CD62L（FMC46 FITC）标记细胞进行流式细胞术检测。白细胞分离程序本身导致12例中的11例CD34细胞上L-选择素表达略有下降（阳性细胞百分比平均下降11.9；范围6 - 23）。冻融后发现下降幅度大得多：与新鲜材料相比，平均下降39％（27例中范围为4 - 78％）。为了确定冷冻保存后L-选择素表达是否可能上调，将解冻的移植样本（n = 11）在含有10％胎牛血清的RPMI中于37℃、5％二氧化碳条件下孵育。在孵育过程早期，原始区域出现了两个CD34阳性群体，其特征在于前向散射低或高。用DNA染料7-氨基放线菌素D和DNA/RNA染料Syto16同时进行活力染色显示，前向散射低的群体是凋亡的，而前向散射高的群体是非凋亡的。后一个群体被认为与移植最相关。在这个群体中，8/11的样本在过夜孵育后观察到L-选择素表达增加，达到新鲜未冷冻样本值的46 - 120％。此外，10/11的病例中平均荧光强度显著增加。较短孵育时间的动力学实验表明，只有部分白细胞分离样本（8例中的2例）在4小时内显示L-选择素表达增加。此外，通过磁性分离从新鲜白细胞分离材料中纯化CD3时发现L-选择素表达下降（下降范围为59％至92％，n = 5）。与冷冻样本相反，在所有样本孵育4小时内就观察到L-选择素重新出现。冻融时CD34细胞上L-选择素表达的丧失、凋亡的出现以及非凋亡细胞上L-选择素表达的恢复在各个白细胞分离样本之间差异很大，因此得出结论，在与移植后的临床结果进行相关性分析时应考虑这些过程。
