A rapid combined immunocytochemical and fluorescence in situ hybridisation method for the identification of human fetal nucleated red blood cells

Development of a preparation and staining method for fetal erythroblasts in maternal blood: Simultaneous immunocytochemical staining and FISH analysis

“…In comparison with methods described by others (13,24,36), the introduction of two-step cytocentrifugation, with addition of 5% BSA to the supernatant before the second step, led to dramatic improvement of cell morphology without concurrent cell loss. The rationale is that adherence to coated slides is better at low protein concentrations, whereas high protein concentrations are needed to protect the cells against salt crystals that arise upon drying.…”

“…Two conflicting interests (cytoplasm preservation and nuclear accessibility) had to be met. Starting from standard fixation methods and modifications as described by others (11,24), we eventually chose a three-step fixation protocol using methanol, acetone, and formaldehyde. When this was combined with the specific immunocytochemical staining protocol and the omission of enzyme treatment in the FISH procedure, it allowed the simultaneous detection of HbF and FISH analysis for the X-and Y-chromosomes, on the same cell.…”

“…This necessitates optimal use of detected cells by combining confirmation of fetal origin (recognition) with simultaneous or successive genetic diagnosis on the same cell. Recently, modifications of a combined genotype and immunophenotype analysis (25) were described for use in NRBC detection (11,24). We describe here a protocol that enables the simultaneous analysis of morphology, NRBC phenotyping (HbF), and FISH signals for both the X-and Y-chromosomes.…”

Liver from bone marrow in humans

“…This effect of residual DAB on fluorescence has been reported previously. 27,28 Results of the cell counts for Y-chromosome-positive hepatocytes, cholangiocytes, and ductular reactions are summarized in Table 2. Y-positive hepatocytes and cholangiocytes were consistently identified in the male control tissue, whereas none were identified in the female control tissue.…”

“…Background fluorescence of precipitated DAB, still adherent to the slide after FISH protease digestion, could be identified in all tissues examined as it is protease resistant. 27,28 Fields were matched to corresponding photomicrographs of the CAM5.2 immunostained slides, relying on location of the tissue on the slide, identity of nuclear positioning, DAB stain, and typical hepatocyte autofluorescence, thereby identifying specific CAM5.2-labeled hepatocytes and cholangiocytes. Hepatocytes and cholangiocytes could be distinguished from each other by morphology, location (parenchymal vs. intraportal), and intensity/density of DAB staining (cholangiocytes greater than hepatocytes).…”