An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.     

Circulating trophoblast in maternal blood

This review describes the status of circulating trophoblast, but is considered in the perspective that only a specific subset of trophoblast cells circulates in the maternal blood. The consequences for isolation, identification and clinical potential are described. Copyright © 2003 John Wiley & Sons, Ltd.     

Rare cell isolation using antibodies covalently linked to slides: application to fetal cells in maternal blood

We describe here a new type of method for isolation of rare cell populations in biological fluids. The method is based on the anthraquinone technology for covalent binding of molecules to a polymer surface. An anthraquinone molecule conjugated via a linker to an electrophilic group (AQ Immobilizer™ reagent, Exiqon A/S) is covalently bound to a polymer surface by UV irradiation. The electrophilic group of this AQ reagent can covalently bind a specific antibody directed against a specific cell marker. Applying a cell sample to the functional surface, the cells having the specific cell marker on the cell surface will bind to the antibody on the functional surface. Using this technique, even extremely small cell populations may be isolated. We succeeded in isolating fetal cells from maternal blood samples in the first trimester for chromosome defects genetic diagnosis. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal β^E-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 µL of plasma from pregnant women whose husbands carried Hb E. The β^E-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal β^E-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal β^E-globin gene should prove useful in a prevention and control program of Hb E/β-thalassemia in countries where the β^E-globin gene is prevalent. Copyright © 2003 John Wiley & Sons, Ltd.     

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

Monoclonal antibody to fetal haemoglobin (a₂γy₂) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-γ with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-γ-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.     

Isolating fetal cells in maternal circulation for prenatal diagnosis

Fetal cells unequivocally exist in and can be isolated from maternal blood. Erythroblasts, trophoblasts, granulocytes and lymphocytes have all been isolated by various density gradient and flow sorting techniques. Chromosomal abnormalities detected on isolated fetal cells include trisomy 21, trisomy 18, Klinefelter syndrome (47,XXY) and 47,XYY. Polymerase chain reaction (PCR) technology has enabled the detection of fetal sex, Mendelian disorders (e.g. β-globin mutations), HLA polymorphisms, and fetal Rhesus (D) blood type. The fetal cell type that has generated the most success is the nucleated erythrocyte; however, trophoblasts, lymphocytes and granulocytes are also considered to be present in maternal blood. Fetal cells circulate in maternal blood during the first and second trimesters, and their detection is probably not affected by Rh or ABO maternal-fetal incompatibilities. Emphasis is now directed toward determining the most practical and efficacious manner for this technique to be applied to prenatal genetic diagnosis. Only upon completion of clinical evaluations could it be considered appropriate to offer this technology as an alternative to conventional invasive and non-invasive methods of prenatal cytogenetic diagnosis.     

New technique using galactose-specific lectin for isolation of fetal cells from maternal blood

To isolate fetal cells from maternal blood, we developed a new method based on galactose-bearing conjugation. Nucleated red blood cells (NRBCs), which highly express galactose on their surface, were selectively attached to a substrate coated with a galactose-containing polymer via soybean agglutinin (SBA), a galactose-specific lectin. Cord blood samples were used to evaluate enrichment efficacy of NRBCs by this method. Blood samples were obtained from 131 pregnant women between 6 and 27 gestational weeks. After preliminary condensation of fetal cells by Ficoll gradient centrifugation, NRBCs were enriched using galactose-positive selection by adjusting SBA concentration. We isolated one to severalhundred NRBCs (mean±SD, 7.8±8.5) in 2.3 ml of peripheral blood samples from 96% of pregnantwomen. The isolated NRBCs were analyzed by a Y-chromosome FISH probe in eight cases carrying male fetuses. Y-signals were detected in all eight cases and more than half of the NRBCs wereoffetal origin. The study demonstrates that our new method using galactose-specific lectin provides effective enrichment of fetal NRBCs allowing non-invasive prenatal diagnosis. Copyright © 2002 John Wiley & Sons, Ltd.