Two-colour immunocytochemical staining of gamma (gamma) and epsilon (epsilon) type haemoglobin in fetal red cells.

We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well as chorionic villus samples (10-13 weeks) were stained for gamma and epsilon chains using CY3 and FITC labelled antibodies. Morphometric analysis was applied to determine cell size. Samples from organs involved in early embryonic development contained relatively large erythroblasts expressing the epsilon globin chain (megaloblasts); later in gestation the gamma chain was co-expressed by the same cells which ultimately became smaller and contained HbF (alpha 2 gamma 2) only. This phenomenon was confirmed in CVS samples in which all cell types were abundantly present. Since fetal erythroblasts are considered candidate cells for non-invasive prenatal diagnosis using FISH, we studied the phenotype of erythroblasts circulating in the maternal blood. The majority of erythroblasts in maternal blood appeared to be of the relatively small gamma globin-containing cell type. However, careful screening of the same maternal blood samples also revealed erythroblasts expressing epsilon or epsilon and gamma globins simultaneously, although at low frequency. Control specimens from non-pregnant women did not show nucleated red cells expressing either of the haemoglobin types. These observations may contribute to the better recognition of fetal cells in the maternal blood for prenatal diagnosis.

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment.

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.

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

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3-diaminobenzidin (DAB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X- and Y-chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis.

Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis.

A protocol to detect fetal nucleated red blood cells (NRBCs) was tested in 217 pregnant women and in 19 nonpregnant controls. All the pregnant women were sampled after chorionic villus sampling (CVS); 20 were also sampled pre-CVS. NRBC recognition was based upon morphology by using staining of hemoglobin with 3,3-diaminobenzidin (DAB) or by immunocytochemical staining for fetal hemoglobin (HbF). This was combined with FISH analysis for both the X- and Y-chromosomes on the same cells. Progressive refinement of the methods increased the number of cases where NRBCs were detected from 53% (DAB) to 75% and 78% for DAB and HbF staining, respectively, with on average 43 NRBCs (range, 1-220). DAB gave a slightly higher yield than HbF in the lower cell count range (<25). In 6 out of 18 controls, NRBCs were detected with DAB, vs. 1 out of 19 (5%) with HbF. FISH analysis in 41 cases resulted in correct sex prediction in 80% (DAB) and 89% (HbF), respectively. Our data demonstrated an increase of cases with NRBCs (30% to 75%), as well as a rise of the mean number of NRBCs (6 to 29 cells), after CVS. We conclude that DAB staining is a straightforward way to screen for the presence of NRBCs in maternal blood, but is not specific for NRBCs of fetal origin. HbF immunophenotyping is a reliable marker for fetal NRBCs, which detected slightly fewer NRBCs than DAB-staining, but improved sex prediction and significantly reduced false-positive results. CVS at 10-13 weeks of gestation causes a significant increase of NRBCs in maternal blood. These data indicate that further refinement of NRBC detection is needed before application of noninvasive prenatal diagnosis using maternal blood is feasible.

Simultaneous detection of X and Y chromosomes by two-colour fluorescence in situ hybridization in combination with immunophenotyping of single cells to document chimaerism after sex-mismatched bone marrow transplantation.

A powerful approach to documenting engraftment after allogeneic BMT is the quantification of the degree of chimaerism in distinct haematopoietic cell lineages. This cannot be achieved by the recently developed, quantitative, modifications of PCR amplification of highly polymorphic DNA markers, unless this technique is applied to separated cell populations. Here, we report the development of a new method, in which cells are simultaneously characterized by enzymatic immunophenotyping and identified for their origin by two-colour fluorescence in situ hybridization with X and Y chromosome-specific DNA probes (XY-FISH/immunostaining). The method enables the rapid, reliable and quantitative analysis of chimaerism within distinct cell lineages after sex-mismatched BMT, without the requirement for cell separation techniques. This is illustrated by investigation of the pattern of chimaerism in patients receiving a sex-mismatched BMT for the treatment of primary immunodeficiencies. The results obtained with the quantitative XY-FISH/immuno staining method show a good correlation with the data generated by the semi-quantitative analysis of PCR amplified minisatellites in FACS-sorted cell fractions. In addition, XY-FISH/immunostaining was successfully applied to detect materno-fetal engraftment of T cells in a SCID patient.

Detection of 'rare event' fetal erythroblasts in maternal blood using automated microscopy.

This paper describes the use of automated microscopy to detect fetal erythroblasts in maternal blood. The technology is based on the following approach: (1) the use of centrifugal cytology for the preparation of monolayers; (2) simultaneous staining of fetal hemoglobin (immunoalkaline phosphatase) and chromosome sequences (FISH); (3) multi-mode microscopy to detect rare events; (4) visual evaluation of image memories containing detected objects. Model systems show that fetal cells in frequencies as low as 1 in a million cells can be detected easily (manually or by automated microscopy). Algorithms for automated cell selection were developed for a test set of 6 patients. Optimization of hardware and software routines will make analysis of several million cells in approximately 1 h feasible.

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.