Heuer Anabel, Löwhagen Svea, Uhlig Stefanie, Hetjens Svetlana, Büttner Sylvia, Pflästerer Britta, Diehlmann Anke, Klein Stefan, Klüter Harald, Bieback Karen, Wuchter Patrick
Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service Baden-Württemberg - Hessen, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
FlowCore, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
Transfus Med Hemother. 2023 Sep 15;50(5):417-427. doi: 10.1159/000533624. eCollection 2023 Oct.
Autologous stem cell transplantation is a successful routine procedure with only a small number of non-engraftment cases, although the time to hematopoietic recovery may vary considerably across patients. While CD34 has been the decisive marker for enumerating hematopoietic stem and progenitor cells (HSPCs) for more than 30 years, the impact of CD34-positive cellular subpopulations in autologous HSPC grafts on hematopoietic reconstitution remains unclear.
The two-color ISHAGE protocol represents the current gold standard for CD34 cell enumeration but includes only the number of viable CD45/CD34 cells relative to the body weight of the recipient. We adapted a multicolor flow cytometry marker panel for advanced characterization of CD34 subpopulations in retained samples of autologous peripheral blood stem cell products ( = 49), which had been cryostored for a wide range from 4 to 15 years. The flow cytometric analysis included CD10, CD34, CD38, CD45, CD45RA, CD133, and viability staining with 7AAD. The findings were correlated with clinical engraftment data, including reconstitution of leukocytes, neutrophils, and platelets after transplantation (TPL).
We demonstrated that the identification of autologous HSPC subpopulations by flow cytometry after cryopreservation is feasible. Regarding the distribution of HSPC subpopulations, a markedly different pattern was observed in comparison to previously published data obtained using fresh autologous material. Our data revealed the largest ratio of lympho-myeloid progenitors (LMPPs) after freezing and thawing, followed by multipotent progenitors and erythroid-myeloid progenitors. A high ratio of LMPPs, representing an immature stage of differentiation, correlated significantly with early neutrophilic granulocyte and leukocyte engraftment ( = 0.025 and = 0.003). Conversely, a large ratio of differentiated cells correlated with late engraftment of neutrophilic granulocytes ( = 0.024). Overall, successful engraftment was documented for all patients.
We established an advanced flow cytometry panel to assess the differentiation ability of cryostored autologous peripheral blood stem cell grafts and correlated it with timely hematopoietic reconstitution. This approach represents a novel and comprehensive way to identify hematopoietic stem and progenitor subpopulations. It is a feasible way to indicate the engraftment capacity of stem cell products.
自体干细胞移植是一种成功的常规手术，仅有少数未植入病例，尽管患者造血恢复时间差异可能很大。30多年来，CD34一直是计数造血干细胞和祖细胞（HSPCs）的决定性标志物，但自体HSPC移植物中CD34阳性细胞亚群对造血重建的影响仍不清楚。
双色ISHAGE方案是目前CD34细胞计数的金标准，但仅包括相对于受者体重的存活CD45/CD34细胞数量。我们采用了一种多色流式细胞术标记物组合，用于对自体外周血干细胞产品（n = 49）的留存样本中的CD34亚群进行高级表征，这些样本已冷冻保存4至15年不等。流式细胞术分析包括CD10、CD34、CD38、CD45、CD45RA、CD133以及用7AAD进行活力染色。研究结果与临床植入数据相关，包括移植后白细胞、中性粒细胞和血小板的重建（TPL）。
我们证明了冷冻保存后通过流式细胞术鉴定自体HSPC亚群是可行的。关于HSPC亚群的分布，与先前使用新鲜自体材料获得的数据相比，观察到明显不同的模式。我们的数据显示，冻融后淋巴-髓系祖细胞（LMPPs）的比例最大，其次是多能祖细胞和红系-髓系祖细胞。高比例的LMPPs代表分化的不成熟阶段，与早期中性粒细胞和白细胞植入显著相关（P = 0.025和P = 0.003）。相反，高比例的分化细胞与中性粒细胞的晚期植入相关（P = 0.024）。总体而言，所有患者均记录到成功植入。
我们建立了一种先进的流式细胞术方法来评估冷冻保存的自体外周血干细胞移植物的分化能力，并将其与及时的造血重建相关联。这种方法代表了一种识别造血干细胞和祖细胞亚群的新颖且全面的方式。它是一种指示干细胞产品植入能力的可行方法。
