Improved Specificity of NRBC Detection in Chorionic Villus Sample Supernatant Fluids Using Anti-Zeta and Anti-Epsilon Monoclonal Antibodies

Abstract

Objective: Fetal erythrocytes leak from fetal capillaries at the time of chorionic villus sampling (CVS). It has been reported that in approximately 60% of CVS cases fetal nucleated red blood cells (NRBC) can be isolated from the supernatant fluid by immunophenotyping with monoclonal antibody (Ab) against the γ-chain of fetal hemoglobin and used as an additional source for confirmation of the fetal karyotype. However, the increased prevalence of β-thalassemia mutations in countries such as Greece results in many pregnant women who produce γ-positive cells. This makes it difficult to distinguish between the fetal and maternal origin of the NRBC. Use of Abs against embryonic hemoglobin chains ζ and ε may increase specificity for fetal NRBC detection. Methods: Mouse monoclonal Abs against Hb-ζ and Hb-ε were used in order to examine if specificity for fetal NRBC detection in CVS supernatant fluids could be improved. 41 samples were studied using anti-ζ and 20 using anti-ε monoclonal Abs. Results: Anti-ζ or anti-ε positive erythrocytes were, respectively, identified in 52 of 61 CVS samples and anti-ζ or anti-ε positive NRBC were present in all cases. The mean number of Hb-positive erythrocytes identified with the anti-ζ Ab was 58 and the mean number of NRBC 29. The mean number of anti-ε positive erythrocytes was 30 and of NRBC 23. FISH with X and Y chromosome specific probes was performed in 26 cases and the results were concordant with the CVS karyotype. Statistical analysis using the correlation test showed that anti-ζ and anti-ε were more specific for the detection of embryonic NRBCs. Conclusions: Since embryonic monoclonal Abs show increased specificity, they should be preferentially used for NRBC detection in CVS supernatant fluids. Furthermore, the increased specificity of anti-ζ and anti-ε Abs may considerably improve prenatal diagnosis from fetal cells isolated from maternal circulation.

Studies on the Isolation and Identification of Fetal Nucleated Red Blood Cells in the Circulation of Pregnant Women before and after Chorion Villus Sampling

“…In agreement with results of previous studies [11][12][13][14][15], we found that gamma hemoglobin positive NRBCs were present in all and that epsilon hemoglobin positive NRBCs were present in almost all fetal blood samples from gestation weeks 12-20. It is noteworthy that epsilon hemoglobin positive cells were present in a sample as late as 20 weeks.…”

“…Secondly, the gamma marker is not fully fetus specific, a finding that is in accordance with our previous finding using the mRNA for gamma hemoglobin as marker, where we found a few percent of the gamma hemoglobin positive cells to be maternal [to be published]. Others have made similar findings [12][13][14].…”

“…In 18 women (table 1) fetal cord blood was obtained just before or during termination (cases 1-13) or immediately after delivery (cases [14][15][16][17][18]. The fetal blood samples (weeks [12][13][14][15][16][17][18][19][20] were obtained either by vaginal or transabdominal ultrasound-guided puncture of the umbilical cord or by fetal cardiac puncture. These pregnancies were terminated either because of chromosomal abnormalities or structural abnormalities following genetic counseling.…”

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

“…There may be a case for using ␥-globin for sorting, favoring yield over purity, but it is not nearly specific enough for accurate fetal erythroblast identification because of increased maternal fetal hemoglobin production in pregnancy 5 and ␤ thalassemia. 6,7 In contrast, ⑀-or -globin chains appear specific for fetal cells in chorionic villus supernatants 7 but have not been tested in maternal blood. Whereas -globin is occasionally produced in adults with ␣ thalassemia, 8 ⑀-globin has not been found in adult peripheral blood, 9 making it the preferred fetal cell identifier.…”

Identification of Fetal Nucleated Red Blood Cells in the Maternal Circulation during Pregnancy Using Anti-Hemoglobin-ε Antibody

“…In a previous study we demonstrated that anti-hemoglobin-Â antibody is more specific than anti-hemoglobin-Á in detecting fetal NRBCs in chorionic villus sampling (CVS) washings [10]. Choolani et al [11] confirmed the specificity of anti-hemoglobin-Â for fetal NRBCs in mixtures of male fetal erythroblasts with female adult bone marrow erythroblasts.…”