临床HSPCs移植参考的CD34+细胞检测方法的依据探讨-修改

临床HSPCs移植参考的CD34+细胞检测方法的依据探讨一、欧洲临床细胞分析事务委员会1998年推荐的方法 (检测实验室采用)以下是其发表文章的摘要译文：对造血干/祖细胞(HSPCs)移植可行性的可靠评价指征的需要，推动了以CD34特征分子为基础的流式细胞术定量测定HSPCs方法的发展。虽然流式细胞术测定低含量细胞偏差较大，但近来技术的进展促进了测定的准确性和精度的提高。在此，回顾了现已报道的文献，经过比较我们推荐如下的CD34+HSPCs检测方法的原则：1）用强荧光标记/型的CD34膜单抗；2）在数据采集时剔除碎片或小细胞成分，如血小板、未裂解红细胞以及细胞碎片等；3）进行CD45膜标记复染，界定HSPCs的范围；4）在流式结果分析时，对低表达CD45+（即弱阳性）和侧向角(SSC)低光值细胞群(也就是采集数据图中SSC low-CD45dim部分)，通过圈门从其他干扰细胞中区分出CD34+HPC的比例。5）统计的CD34+细胞计数应包含CD34弱阳性和强阳性细胞；6）结果应是扣除了阴性对照值(非特异性结合的荧光值)；7）对于白细胞制品，计数时采集细胞应满足不少于100个CD34+细胞，以保证10%的精度；现存有待解决的技术问题包括：1）用单平台检测方案取代传统的双平台检测模式，如何用单独流式细胞仪检测CD34+细胞来取代目前的流式细胞仪和血液分析仪联合应用的模式（采用绝对计数管，下文中提到）。2）单平台测定方法的校准问题，3）样本制备的优化问题。有关优化临床应用的问题将提上日程，那就是如何确定满足长期或短期康复治疗所需HSPCs的精确表型及所需的细胞数量。原文：Cytometry. 1998 Jun 15;34(3):128-42. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis.Gratama JW, Orfao A, Barnett D, Brando B, Huber A, Janossy G, Johnsen HE, Keeney M, Marti GE, Preijers F, Rothe G, Serke S, Sutherland DR, Van der Schoot CE, Schmitz GDepartment of Clinical and Tumor Immunology, Daniel den Hoed Kliniek, Rotterdam, The Netherlands. gratamaimmh.azr.nlAbstractThe need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.二、现行应用的三种CD34+流式细胞计数方法的比较报道临床移植用白细胞制品或骨髓中CD34+造血干/祖细胞的三种流式细胞计数方法的比较研究（综合检测实验室现行CD34+检测方法满足ISHAGE认可的双平台分析方案要求）ISHAGE：International Society of Hematotherapy and Graft Engineering血液病治疗及移植工程协会HSPCs中CD34+的流式细胞计数广泛用于评价外周血和骨髓干细胞移植的可行性。在当前的研究中，我们综述并比较了3种主要的干祖细胞的计数方法。这些方法是：Milan/Mullhouse (M/M)方案，ISHAGE双平台分析方法以及单平台分析方法。M/M方法是通过CD34抗体染色和圈门方法来确定HSPCs。ISHAGE建议的检测CD34+细胞是基于4参数流式细胞术的方法（CD34-PE/CD45-PerCP染色，侧向角和前向角散射光检测技术），并应用了多参数圈门策略。双平台分析法是指联合血细胞分析仪进行白细胞计数后再进行CD34绝对计数；而单平台方案是直接利用已知的荧光珠绝对计数管测定CD34数(仅需要流式细胞仪)。CD34计数的样本来源于动员后外周血分离的白细胞(LKP)以及42位恶性血液病患者的骨髓。测定的CD34+细胞平均值比较M/M方法和ISHAGE案方差异不大，而且对白细胞制品，M/M与双平台方案最大均值差异为2.5%，与单平台方案比较均值最大相差2.6%；对骨髓CD34计数均值相应差异为4.8%个4.9%。研究结果显示了三种CD34检测方法的高度相关性。既然三种方法是相互兼容，那么在临床检测CD34时可依据成本、操作的难易性以及临床准确度要求做合理选择。以下是 三种检测方法的流式方案图。图示1 M/M圈门方案检测白细胞制品(LKP)中CD34流式结果图示2 双平台流式检测成分白细胞中CD34数量的方案（目前多采用的方案）图3 单平台方案采用绝对计数管检测成分白细胞中的CD34流式结果图该方案是在选定CD45+细胞中直接通过CD34+绝对计数管 来测定CD34+细胞。报道原文：Folia Histochem Cytobiol. 2006;44(1):53-60.Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods. Gajkowska A, Oldak T, Jastrzewska M, Machaj EK, Walewski J, Kraszewska E, Pojda Z.Department of Experimental Hematology, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland. agnigajkcoi.waw.plAbstractFlow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)-2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-plat