Sutherland D R, Keating A, Nayar R, Anania S, Stewart A K
Oncology Research Laboratories, Toronto Hospital, Ontario, Canada.
Exp Hematol. 1994 Sep;22(10):1003-10.
Peripheral blood stem cell autografts are increasingly used to reconstitute hematopoiesis after intensive, potentially marrow-ablative therapy. Assessment of autograft adequacy by enumeration of hematopoietic progenitors in colony-forming assays is handicapped by lack of reproducibility and prolonged assay time. Alternative approaches of graft assessment by flow-cytometric enumeration of stem/progenitor cells bearing the CD34 antigen can be hampered by low specificity and sensitivity. Here, we report a rapid and reliable multiparameter flow-cytometric approach to accurately enumerate CD34+ cells in peripheral blood (PB) mononuclear cells (MNCs). Total nucleated white blood cells (WBCs) are quantified by staining with fluorescein isothiocyanate (FITC)-conjugated CD45 antibody. Simultaneous staining by phycoerythrin (PE)-conjugated CD34 antibody defines an approximate number for the CD34+ progenitor/stem cell subfraction. When starting CD34+ cell numbers are low (0.01-0.5%), other nonspecifically stained leukocytes make accurate enumeration impossible. However, when the CD34+ fraction is analyzed for CD45 expression vs. side scatter (granularity), true CD34+ blast cells form a discrete cluster exhibiting low-density CD45 expression and low side-scatter characteristics. Cells within this "blast region" can be readily distinguished from lymphocytes, monocytes, granulocytes, and other events that can contaminate the CD34+ population. Here, we used this sensitive procedure to enumerate CD34+ cells in steady-state PB samples (0.03-0.09%), normal bone marrow (BM) aspirates, and umbilical cord blood collections (0.33-1.98%). This approach thus provides a means to analyze CD34+ cells in specimens from patients who have been extensively treated with chemotherapy and those undergoing PB stem cell mobilization with cytokines. Additionally, it is useful for assessment of CD34+ cells in a variety of clinical samples exhibiting perturbations of the hematopoietic progenitor/stem cell compartments.
外周血干细胞自体移植越来越多地用于在强化的、可能导致骨髓清除的治疗后重建造血功能。通过集落形成试验中造血祖细胞的计数来评估自体移植的充分性，因缺乏可重复性和检测时间长而受到阻碍。通过流式细胞术对携带CD34抗原的干/祖细胞进行计数来评估移植物的替代方法，可能会因特异性和敏感性低而受到阻碍。在此，我们报告一种快速可靠的多参数流式细胞术方法，用于准确计数外周血（PB）单个核细胞（MNC）中的CD34+细胞。通过用异硫氰酸荧光素（FITC）偶联的CD45抗体染色来定量总核白细胞（WBC）。用藻红蛋白（PE）偶联的CD34抗体同时染色可确定CD34+祖细胞/干细胞亚群的大致数量。当起始CD34+细胞数量较低（0.01 - 0.5%）时，其他非特异性染色的白细胞会使准确计数变得不可能。然而，当分析CD34+部分的CD45表达与侧向散射（颗粒度）时，真正的CD34+原始细胞形成一个离散的簇，表现出低密度CD45表达和低侧向散射特征。这个“原始细胞区域”内的细胞可以很容易地与淋巴细胞、单核细胞、粒细胞以及其他可能污染CD34+群体的事件区分开来。在此，我们使用这种灵敏的方法来计数稳态PB样本（0.03 - 0.09%）、正常骨髓（BM）抽吸物和脐带血样本（0.33 - 1.98%）中的CD34+细胞。因此，这种方法为分析来自接受过广泛化疗的患者以及接受细胞因子动员PB干细胞的患者的标本中的CD34+细胞提供了一种手段。此外，它对于评估造血祖细胞/干细胞区室出现扰动的各种临床样本中的CD34+细胞很有用。
