THE RAPID DETECTION OF THE QUANTITY (GENOTYPE) AND CELL LINEAGE (IMMUNOPHENOTYPE) OF CONTAMINATING MATERNAL WHITE CELLS IN CORD-BLOOD SAMPLES BY FLUORESCENCE IN-SITU HYBRIDIZATION COMBINED WITH CONFOCAL LASER-SCANNING MICROSCOPY

The role of maternal blood cell contamination in cord blood as hematop oietic stem cell transplant could be an important factor driving the g raft vs. host reactivity. Therefore, the efficiacy of fluorescence-lab eled chromosome probes for the X and Y chromosomes to study contaminat ing maternal cells (XX(+), Y-) in nucleated cells of male cord blood s pecimen (X(+), Y+) by fluorescence in situ hybridization (FISH) was ev aluated here. Unfortunately, the FISH technique evaluated by standard indirect immunofluorescence was not sufficiently sensitive to safely d etect maternal contamination below 2-3%. Thus, the analysis of FISH ha d to be adapted with confocal laser scanning microscopy, which then ga ve no false-negative reading values. A Bio-Rad MRC-600 laser scanning microscope with a very high sensitivity for low-intensity and hidden s ignals was used for the observation of cord blood cells. Sixteen sampl es from the whole cord blood (CB), 10 analyses of mononuclear cells, 6 of the granulocytic fraction, 6 of picked erythroid burst-forming uni t (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) colo nies and 5 of enriched CD34(+) cells were performed. Only in 1 of 16 c ases was a contamination by maternal cells detected: 10% of all nuclea ted cells from the whole CB, 5% of the CD3+ T-cell fraction, 15% of th e myelomonocytic cells from mononuclear cells (MNCs), and 15% of picke d BFU-E and CFU-GM colonies were of maternal origin. In addition, to d etermine the hematopoietic lineage of contaminating maternal cells, mo nonuclear cells, the granulocytic fraction, CD34(+)-enriched cells as well as picked BFU-E and CFU-GM colonies were analyzed by the simultan eous immunophenotyping (monoclonal antibodies CD13, CD3, CD14, glycoph orin A, CD34) and genotyping (for Y and X) analysis. This approach per mits simultaneous visualization of both the immunophenotype (Mabs, APA AP, red fluorescence) and the genotype (chromosomes, fluorescein isoth iocyanate, green fluorescence) within the same cell.