Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+cells
Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+cells
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DOI：
10.1046/j.1537-2995.1999.39080864.x
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发表时间：
1999-08-01
期刊：
影响因子：
2.9
通讯作者：
García, J
中科院分区：
文献类型：
--
作者：
Cabezudo, E;Querol, S;García, J
背景技术背景：本研究评估了在常规实验室工作中，一种新的容量细胞术系统用于单采成分、外周血和脐带血样本中CD 34+细胞计数的可行性。该系统与以下流式细胞术方案进行比较：米兰，ISHAGE，ISHAGE与7-AAD，和流式计数fluorospheres.Study设计和方法：相关性，线性，和再现性研究进行了各种方法。克隆形成培养，作为外部控制，以评估每μ L的CD 34+细胞的数量和每μ L的集落形成单位的数量之间的相关性。结果：线性回归分析表明，5种方法是可比的（R-2范围从0.86到0.96，斜率接近1）。CD 34+测定和流式细胞计数法显示，当CD 34+细胞计数低于10个细胞/μ l时，线性较差（R-2 = 0.46和0.47）。对于10个细胞/μ L的CD 34+计数的再现性试验，米兰和CD 34+试验方法的CV分别为12%和25%。46份评价样品的所有5种方法的平均CV为20%。结论：CD 34+细胞计数法可用于脐带血、白细胞分离物和外周血中CD 34+细胞的计数，其结果与Milan法、ISHAGE法、ISHAGE +7-AAD法和流式细胞计数法相当。然而，对于CD 34绝对计数较低（低于10个细胞/μ L）的外周血样本，应采用其他流式细胞术方案进行分析。即使同一操作员在单个实验室进行研究，高方法间CV表明样品制备和设门策略的差异是增加变异性的因素。中间步骤较少的方案或全自动方案（如CD 34+检测）预计将降低实验室内和实验室间的变异性。
BACKGROUND: This study assesses the feasibility of a new volumetric cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres.STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per mu L and the number of colony-forming units per mu L.RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R-2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34+ cell counts below 10 cells per mu l (R-2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per mu L showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per mu L and colony-forming units per mu L in cord blood and apheresis samples (r = 0.71-0.81).CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/mu L) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.