Clavarino Giovanna, Delouche Noémie, Vettier Claire, Laurin David, Pernollet Martine, Raskovalova Tatiana, Cesbron Jean-Yves, Dumestre-Pérard Chantal, Jacob Marie-Christine
Laboratoire d'Immunologie, Département d'Hématologie, Oncogénétique et Immunologie, Pôle de Biologie, Grenoble University Hospital, Grenoble, France.
BNI, TIMC-IMAG, UMR 5525 CNRS, Grenoble, France.
PLoS One. 2016 Sep 22;11(9):e0162209. doi: 10.1371/journal.pone.0162209. eCollection 2016.
A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.
精确鉴定和表型特征化人类B细胞亚群在基础研究和医学中都至关重要。在文献中，用于B淋巴细胞表型特征化的流式细胞术研究主要集中在描述特定细胞阶段，或在单一类型样本中观察到的特定细胞阶段。在本研究中，我们提出了一种由6种抗体（CD38、CD27、CD10、CD19、CD5和CD45）组成的核心组合以及一种有效的设门策略，以便在单个分析管中鉴定大量B细胞亚群，涵盖从祖细胞到记忆细胞和浆细胞的整个B细胞分化过程。此外，通过在8色组合中添加两种抗体，我们的方法能够分析沿着B细胞分化过程中任何感兴趣的细胞表面标志物的调节情况。因此，我们开发了一组七种8色抗体组合，用于对骨髓、外周血、淋巴结和脐带血样本中的B细胞亚群进行表型特征化。除了双参数表示法提供的定性信息外，我们还对每个鉴定出的B细胞亚群上的抗原表达进行了定量，并提出了一系列信息丰富的曲线，展示了十七种细胞表面标志物沿着B细胞分化过程的调节情况。我们的流式细胞术方法提供了一种有效的工具，通过相对易于操作的技术获得关于B细胞表面标志物表达的定量数据，该技术可应用于常规检测。
