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.

Abstract

The role of maternal blood cell contamination in cord blood as hematopoietic stem cell transplant could be an important factor driving the graft vs. host reactivity. Therefore, the efficiacy of fluorescence-labeled chromosome probes for the X and Y chromosomes to study contaminating maternal cells (XX+, Y-) in nucleated cells of male cord blood specimen (X+, Y+) by fluorescence in situ hybridization (FISH) was evaluated here. Unfortunately, the FISH technique evaluated by standard indirect immunofluorescence was not sufficiently sensitive to safely detect maternal contamination below 2-3%. Thus, the analysis of FISH had to be adapted with confocal laser scanning microscopy, which then gave no false-negative reading values. A Bio-Rad MRC-600 laser scanning microscope with a very high sensitivity for low-intensity and hidden signals was used for the observation of cord blood cells. Sixteen samples from the whole cord blood (CB), 10 analyses of mononuclear cells, 6 of the granulocytic fraction, 6 of picked erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) colonies and 5 of enriched CD34+ cells were performed. Only in 1 of 16 cases was a contamination by maternal cells detected: 10% of all nucleated cells from the whole CB, 5% of the CD3+ T-cell fraction, 15% of the myelomonocytic cells from mononuclear cells (MNCs), and 15% of picked BFU-E and CFU-GM colonies were of maternal origin.(ABSTRACT TRUNCATED AT 250 WORDS)