Kögler G, Göbel U, Somville T, Enczmann J, Arkesteijn G, Wernet P
Transplantation Immunology and Bone Marrow Center, Heinrich Heine-University, Düsseldorf, Germany.
J Hematother. 1993 Summer;2(2):235-9. doi: 10.1089/scd.1.1993.2.235.
Contamination of cord blood (CB) specimens by maternal blood provides a source of cells that may be capable of graft-versus-host reactivity. To confirm the genetic purity of collected CB samples (six samples, volume 110 +/- 21 ml, total nucleated cells 1.22 +/- 0.36 x 10(9)) the HLA-DR beta exon 2 for the noninherited material allele was examined by polymerase chain reaction amplification. No maternal cell contamination was detected in samples of 1 x 10(5) cells. In the case where the mother was homozygous for DR and DQ, the purity of the sample could not be tested by PCR and therefore in situ hybridization on interphase cells with a fluorescein labeled Y- and X-probe in male CB specimens was performed. This sensitive method also revealed no contamination of the CB by maternal white cells. In addition, picked CB-CFU-GM and BFU-E colonies (cultured in hu-SLF, GM-CSF, and Epo) were analyzed by simultaneous genotypic (for Y and X) and immunophenotypic analysis (monoclonal antibodies [MAbs] CD13, CD14, CD2, CD8, CD4, and glycophorin A). This approach permits simultaneous visualization of both the immunophenotype (MAbs, APAAP, red fluorescence) and the genotype (chromosomes, fluorescein isothiocyanate, green fluorescence) within the same cell. In contrast to PCR and restriction fragment length polymorphism, this method has the advantage that the donor-recipient origin of each lymphohematopoietic lineage (i.e., BFU-E, CFU-GM, T cells, B cells) can be determined in sex-mismatched transplantations. Thus confocal scanning laser microscopy is most suitable not only for the detection of contaminating maternal cells, but also for the detection of mixed hematopoietic chimerism after transplantation.
母血污染脐血（CB）标本会产生可能具有移植物抗宿主反应性的细胞来源。为确认所采集CB样本（6个样本，体积110±21 ml，总核细胞1.22±0.36×10⁹）的基因纯度，通过聚合酶链反应扩增检测非遗传物质等位基因的HLA - DRβ外显子2。在1×10⁵个细胞的样本中未检测到母细胞污染。当母亲的DR和DQ为纯合子时，无法通过PCR检测样本纯度，因此对男性CB标本的间期细胞进行了荧光素标记的Y和X探针原位杂交。这种灵敏的方法也未显示CB被母白细胞污染。此外，对挑选出的CB - CFU - GM和BFU - E集落（在人干细胞因子、粒细胞 - 巨噬细胞集落刺激因子和促红细胞生成素中培养）进行了同时的基因型（针对Y和X）和免疫表型分析（单克隆抗体[MAb]CD13、CD14、CD2、CD8、CD4和血型糖蛋白A）。这种方法允许在同一细胞内同时观察免疫表型（单克隆抗体、碱性磷酸酶抗碱性磷酸酶复合物、红色荧光）和基因型（染色体、异硫氰酸荧光素、绿色荧光）。与PCR和限制性片段长度多态性不同，该方法的优点是在性别不匹配的移植中可以确定每个淋巴造血谱系（即BFU - E、CFU - GM、T细胞、B细胞）的供体 - 受体来源。因此，共聚焦扫描激光显微镜不仅最适合检测污染的母细胞，也适合检测移植后的混合造血嵌合体。
