Cassens U, Gutensohn K, Garritsen H, Kelsch R, Kuehnl P, Sibrowski W
Department of Transfusion Medicine, University Hospital Muenster, Germany.
Transfus Med. 1998 Jun;8(2):111-8. doi: 10.1046/j.1365-3148.1998.00135.x.
Since the correct determination of CD34+ cells is of great clinical importance for successful transplantation with haematopoietic progenitor cells (HPCs) from cord blood, we investigated the influence of different erythrocyte lysing techniques on the quantification of CD34+ cells in umbilical cord blood. Flow cytometric determinations of CD34+ cells were performed from 20 cord blood samples, using three different erythrocyte lysing procedures and two monoclonal CD34 antibodies (n = 360). Flow cytometric analysis showed characteristic patterns of the forward (FSC) and side (SSC) scatter light properties for the leucocyte subsets for each of the investigated erythrocyte lysing procedures, indicating that these reagents cause different morphological changes on leucocytes. Furthermore, significant differences of CD34+ cell counts were obtained for identical samples using different lysing techniques (P = 0.001 and P = 0.002). In some cases, a more than 100% difference was found comparing different erythrocyte lysing procedures. In contrast, the determination of CD34+ cells by two CD34 antibodies showed a good reproducibility without significant differences between both antibodies for each of the erythrocyte lysing techniques. We conclude that the erythrocyte lysing procedure represents a very critical and important step for accurate determination of CD34+ cells in whole blood samples. Especially for the quantification of HPCs in cord blood transplants, this influence may be of high clinical relevance.
由于正确测定CD34+细胞对于成功进行脐血造血祖细胞（HPC）移植具有重要的临床意义，我们研究了不同红细胞裂解技术对脐血中CD34+细胞定量的影响。使用三种不同的红细胞裂解程序和两种单克隆CD34抗体，对20份脐血样本进行了CD34+细胞的流式细胞术测定（n = 360）。流式细胞术分析显示，对于每种研究的红细胞裂解程序，白细胞亚群的前向（FSC）和侧向（SSC）散射光特性具有特征性模式，表明这些试剂会导致白细胞发生不同的形态变化。此外，使用不同的裂解技术对相同样本进行CD34+细胞计数时存在显著差异（P = 0.001和P = 0.002）。在某些情况下，比较不同的红细胞裂解程序时发现差异超过100%。相比之下，使用两种CD34抗体测定CD34+细胞显示出良好的重复性，对于每种红细胞裂解技术，两种抗体之间没有显著差异。我们得出结论，红细胞裂解程序是准确测定全血样本中CD34+细胞的一个非常关键和重要的步骤。特别是对于脐血移植中HPC的定量，这种影响可能具有高度的临床相关性。
