Fritsch G, Buchinger P, Printz D, Fink F M, Mann G, Peters C, Wagner T, Adler A, Gadner H
Children's Cancer Research Institute, Vienna, Austria.
Blood. 1993 May 1;81(9):2301-9.
Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45-RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU-GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst-forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.
通过密度离心法从脐带血、健康骨髓以及化疗后接受粒细胞-巨噬细胞集落刺激因子（GM-CSF）或粒细胞集落刺激因子（G-CSF）治疗的患者外周血（PB）中分离出的单核细胞（MNC），分别用抗CD34单克隆抗体（MoAb）（8G12）与抗CD45、CD45-RB、CD45-RO和CD45-RA进行双重染色，并通过流式细胞术进行分析。在所有标本中，CD34+ MNC低水平共表达CD45，且CD45-RB的表达相似或略高。大多数CD34+ MNC的CD45-RO呈阴性，仅在一些骨髓（BM）和血液样本中可见微弱的共表达。相比之下，CD45-RA可将CD34+群体细分为这些亚群的阴性、弱阳性（+）和正常阳性（++）部分，并且观察到不同造血细胞来源的典型染色模式：在BM中，大多数CD34+ MNC为RA++。在PB中，使用G-CSF后它们的大多数为RA++，而使用GM-CSF后为RA+或RA-。在脐带血中，造血祖细胞主要为RA-/RO-。对分选的CD34+ MNC进行半固体培养显示，簇状和分散的（晚期）集落形成单位-粒细胞巨噬细胞（CFU-GM）源自34+/RA++细胞，而34+/RA- MNC形成紧密的多中心白色和红色集落，源自早期祖细胞。每毫升含有34+/RA-脐带血MNC的培养基中添加20 ng干细胞因子会导致许多爆式红系集落形成单位（BFU-E）转变为混合集落形成单位（CFU-mix），而在血液和BM中至少在这种程度上未观察到这种情况。我们得出结论，早期髓系CD34+细胞为45+/RA-。由于该群体排除了34+/19+ B细胞和33+髓系细胞，这两者均为RA++，因此使用CD34和CD45-RA进行双色流式细胞术分析有助于早期髓系祖细胞的表征和定量。
