Simultaneous genotypic and immunophenotypic analysis of interphase cells for the detection of contaminating maternal cells in cord blood and their respective CFU-GM and BFU-E.

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.