Macedo A, Orfao A, Ciudad J, Gonzalez M, Vidriales B, Lopez-Berges M C, Martínez A, Landolfi C, Cañizo C, San Miguel J F
Servicio de Hematología, Hospital Clínico Universitario, Salamanca, Spain.
Leukemia. 1995 Nov;9(11):1896-901.
In the past, studies on CD34+ cells have been based on the use of monoclonal antibodies conjugated with different fluorochromes that show different fluorescence intensity and yield variable results. Moreover, most of these studies have neither specifically focused on adult human BM samples nor have they used combinations to explore specifically the phenotype of myeloid committed CD34+ cells. The aim of the present study has been to characterize the normal human CD34+ precursor cells from adult BM in order to identify missing or extremely rare phenotypes that can be used for detecting minimal residual disease (MRD) in patients with AML. For this purpose we have utilized the fluorochrome conjugates that provide the most sensitive signals for identifying low antigenic expression, and the technique has been adapted to the characterization of cells present at very low frequencies. Normal human BM samples from 13 adult healthy volunteers have been analyzed using triple stainings at flow cytometry. The mean percentage of CD34+ cells detected was 0.72 +/- 0.33%; these cells displayed an heterogeneous light-scatter distribution. Most CD34+ cells coexpressed CD38 (96.7 +/- 5.7%), HLADR (81.6 +/- 14.0%), CD33 (84.7 +/- 18.3%), CD13 (84.6 +/- 16.2%) and CD71 antigens (65.5 +/- 9.1%). In addition, almost half of CD34+ cells were CD117+ (60 +/- 26.8%). Only a small proportion of CD34+ cells coexpressed CD4 (15.5 +/- 11.7%, CD36 (31.7 +/- 6.2%), CD61 (16.3 +/- 12.9%), CD41 (6.5 +/- 5.5%) or the lymphoid associated markers CD10 (18.6 +/- 11.8%) and CD19 (12.3 +/- 13.2%). Reactivity for the CD15 antigen was observed in a small population of CD34+HLADR+ cells (11.6 +/- 11.2%) although its intensity of expression was lower than that of the more mature granulocytic cells. No CD34+ cells displayed CD14, CD65, CD20, strong CD22, CD3 and CD56 antigens. Accordingly, most adult bone marrow CD34+ cells appeared to be committed to the myeloid lineage (CD13+/CD33+) and displayed an intermediate-to-large FSC/SSC while the lymphoid-committed CD34+ cells (CD19+, CD10+) were in a minority with low FSC/SSC values. By triple marker stainings several phenotypes of CD34+ precursor cells were found to be either undetectable or present at very low frequencies (< 1 x 10(-3)) in the normal human adult bone marrow. These data may be of great value for defining leukemia 'associated' phenotypes used to detect minimal residual disease in adult acute leukemia patients.
过去，对CD34+细胞的研究基于使用与不同荧光染料偶联的单克隆抗体，这些抗体显示出不同的荧光强度并产生可变的结果。此外，这些研究大多既未专门聚焦于成人骨髓样本，也未使用组合方法来专门探索髓系定向CD34+细胞的表型。本研究的目的是对来自成人骨髓的正常人类CD34+前体细胞进行表征，以识别可用于检测急性髓系白血病（AML）患者微小残留病（MRD）的缺失或极其罕见的表型。为此，我们利用了能为识别低抗原表达提供最灵敏信号的荧光染料偶联物，并且该技术已适用于对极低频率存在的细胞进行表征。使用流式细胞术的三重染色分析了来自13名成年健康志愿者的正常人类骨髓样本。检测到的CD34+细胞的平均百分比为0.72±0.33%；这些细胞呈现出异质性的光散射分布。大多数CD34+细胞共表达CD38（96.7±5.7%）、HLA-DR（81.6±14.0%）、CD33（84.7±18.3%）、CD13（84.6±16.2%）和CD71抗原（65.5±9.1%）。此外，几乎一半的CD34+细胞为CD117+（60±26.8%）。只有一小部分CD34+细胞共表达CD4（15.5±11.7%）、CD36（31.7±6.2%）、CD61（16.3±12.9%）、CD41（6.5±5.5%）或淋巴系相关标志物CD10（18.6±11.8%）和CD19（12.3±13.2%）。在一小部分CD34+HLA-DR+细胞（11.6±11.2%）中观察到对CD15抗原的反应性，尽管其表达强度低于更成熟的粒细胞。没有CD34+细胞显示出CD14、CD65、CD20、强CD22、CD3和CD56抗原。因此，大多数成人骨髓CD34+细胞似乎定向于髓系谱系（CD13+/CD33+），并呈现中等至大的前向散射光/侧向散射光，而淋巴系定向的CD34+细胞（CD19+、CD10+）占少数，前向散射光/侧向散射光值较低。通过三重标记染色发现，几种CD34+前体细胞表型在正常成人骨髓中要么无法检测到，要么以极低频率（<1×10⁻³）存在。这些数据对于定义用于检测成人急性白血病患者微小残留病的白血病“相关”表型可能具有重要价值。
