Improved fetal nucleated erythrocyte sorting purity using intracellular antifetal hemoglobin and Hoechst 33342.

Fetal nucleated erythrocytes (FNRBC) flow sorted from maternal peripheral blood, using monoclonal antibodies (mAb) that bind fetal cell surface antigens, are a noninvasive source of fetal DNA for prenatal diagnosis. These mAbs, however, also bind antigens shared by maternal cells. In sorted populations, this results in maternal cell contamination and low fetal cell purities, which complicates genetic analysis by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Fetal hemoglobin, (alpha 2 gamma 2), has been proposed as a useful fetal marker. To improve fetal cell enrichment from maternal blood, we developed an intracellular staining protocol that combines anti-gamma mAb with Hoechst 33342 to identify and flow sort FNRBC. Artificial mixtures of male umbilical cord cells (as a source of fetal hemoglobin) and female adult, non-pregnant peripheral blood mononuclear cells were stained and flow sorted using this protocol. FISH analysis was performed using chromosome X and Y specific probes. Fetal cell purities were calculated by microscope confirmation of anti-gamma staining and counting the number of X and Y signals present after FISH. Results from microscope analyses showed a fetal cell yield of 39-100% and fetal cell purities of 59-73%. These purities are significantly higher than the .001-4.8% previously reported by us in maternal samples using cell surface staining protocols. FISH results demonstrated that 83-100% (mean = 98%) of anti-gamma positive cells were male, whereas 82-100% (mean = 92.5%) of anti-gamma negative cells were female. These results confirmed that the anti-gamma mAb is highly fetal specific. When applied to maternal blood samples, this protocol should lead to increased sensitivity for prenatal diagnosis.

Development of a model system to compare cell separation methods for the isolation of fetal cells from maternal blood.

Three major methods have been described for the isolation of fetal cells from maternal blood: fluorescence-activated cell sorting (FACS), immunomagnetic beads, and magnetic-activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe our system using a "model' male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y- and 7-specific sequences. Fetal cell recovery was investigated by selection of CD71+ cells or depletion of CD45+ cells. Our data demonstrated variation in fetal cell recovery for all methods tested, although CD71+ selection by FACS gave the best and most consistent results.

Fetal RhD genotyping in fetal cells flow sorted from maternal blood.

OBJECTIVE: The aim of this study was to determine the accuracy of noninvasive fetal RhD genotyping by fetal cell isolation from maternal blood. STUDY DESIGN: Candidate fetal cells from 18 pregnant women (one twin gestation) were flow-sorted. Polymerase chain reaction amplification of a 261 bp fragment of the RhD gene was performed on sorted fetal cells. The presence of amplified product was considered predictive of the Rhd-positive genotype in the fetus. RESULTS: Sixteen of the 19 fetal RhD genotypes were correctly predicted in fetal cells isolated from maternal blood (10 were Rh positive, 6 were Rh negative). In 3 cases no amplification products were detected in RhD-positive fetuses. The association between presence of the fragment and RhD-positive genotype was significant (p=0.003, Fisher's exact test). CONCLUSIONS: Noninvasive prenatal diagnosis of the fetal RhD genotype is feasible. Absence of amplification products in the reaction requires confirmation that fetal material is present. Improvements in fetal cell purity and yield should increase diagnostic accuracy, although the current protocol has a positive predictive value of 100% and a negative predictive value of 67%.

Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: preliminary observations on maternal samples.

Monoclonal antibody to fetal haemoglobin (alpha 2 gamma 2) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-gamma with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-gamma-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.

Detection of fetal HLA-DQa sequences in maternal blood: a gender-independent technique of fetal cell identification.

The objective of this study was to detect fetal HLA-DQa gene sequences in maternal blood. HLA-DQa genotypes of 70 pregnant women and their partners were determined for type A1. We specifically sought couples where the father, but not the mother, had genotype A1. In 12 women, maternal blood samples were flow-sorted. Candidate fetal cells were isolated and amplified by using PCR primers specific for a paternal HLA-DQa A1 allele. Fetal HLA-DQa A1 genotype was predicted from sorted cells; amniocytes or cheek swabs were used for confirmation. Six of twelve sorted samples had amplification products indicating the presence of the HLA-DQa A1 allele; 6/12 did not. Prediction of the fetal genotype was 100 per cent correct, as determined by subsequent amplification of amniocytes or cheek swabs. We conclude that paternally inherited uniquely fetal HLA-DQa gene sequences can be identified in maternal blood. This system permits the identification of fetal cells independent of fetal gender, and has the potential for non-invasive prenatal diagnosis of paternally inherited conditions.