Ariadni Mavrou

Single nucleated red blood cells (NRBCs) isolated from maternal circulation were used for prenatal diagnosis of β‐thalassaemia. The study included 22 pregnant women in the first trimester, 6 carriers at risk for β‐thalassaemia and 16 noncarriers. Methodology involved enrichment of NRBCs by magnetic cell sorting (MACS) and microdissection of single NRBCs with a laser micromanipulation system. Single‐cell genotyping based on nested real‐time PCR for genotyping β‐globin gene mutations was performed followed by a multiplexed minifingerprinting to confirm the origin of the isolated cells and possible contamination. Two polymorphic markers (D13S314 and GABRB3) facilitated the identification of fetal NRBCs through comparison of allele sizes found in the respective parents. In this study, 224 single NRBCs were detached and transferred into individual PCR tubes. Allele amplification in at least one microsatellite marker was achieved in 128/224 cells. Minifingerprinting analysis showed that 22 cells were fetal, 26 maternal and 80 were noninformative due to ADO or homozygosity. In 6 NRBCs the β‐globin gene was amplified and in 2, coming from the same pregnancy, only the paternal mutation was detected. The low PCR success when genotyping isolated NRBCs was possibly due to the poor quality of fetal NRBCs and the relatively large size of the β‐globin gene product. Copyright © 2007 John Wiley & Sons, Ltd.

Background: Identification of fetal nucleated red blood cells (NRBCs) in maternal circulation can facilitate non‐invasive prenatal diagnosis, but technical difficulties still exist. An increase in the number of circulating NRBCs, however, could indicate fetal aneuploidies or pregnancy complications.Materials and Methods: The number of NRBCs was determined from 20 mL peripheral blood in 351 women in the second trimester of pregnancy after isolation by magnetic cell sorting (MACS) with anti‐CD71 antibody and identification with May‐Grunwald/Giemsa staining.Results: An average of eight NRBCs (range 1–12) were identified among 282 women with chromosomally normal fetuses. In cases known to carry aneuploid fetuses the mean number was 35 (range 7–113), but when the fetus had trisomy 21 (n = 17) an average of 71 NRBCs were identified. Among 26 carriers of β‐thalassemia, 42 NRBCs (range 22–158) were isolated. In pregnancies with abnormal Doppler findings in both uterine arteries (n = 20), 15 NRBCs (range 2–75) were isolated.Conclusion: Determining the number of NRBCs in maternal circulation could represent an additional screening step for fetal aneuploidies, as long as the anemic status of the mother is taken into consideration. However, more cases with abnormal Doppler results must be investigated before this test is used for in the prediction of pregnancy complications. Copyright © 2006 John Wiley & Sons, Ltd.

This study aimed to determine whether the number of nucleated red blood cells (NRBCs) in maternal circulation during the first trimester of pregnancy could identify pregnancies that will have an anomalous Doppler in the second trimester. A total of 85 blood samples were obtained at 11–14 weeks of gestation with mean uterine arterial perfusion index >1.6, as noted by Doppler ultrasonography. NRBCs were enriched by magnetic automated cell sorting using anti-CD71 and were stained with May/Grunwald/Giemsa. A total of 4.8 NRBCs (range 1–75) were identified in 68 cases. Follow-up scans at 22–24 weeks were available in 46 cases. In 39 women, blood flow in the uterine arteries normalized, whereas in seven, high resistance was noted. One woman in the high-resistance group developed preeclampsia (PET; four NRBCs) and another delivered an intrauterine growth restriction (IUGR) baby (75 NRBCs). The number of NRBCs in women whose Doppler indices later normalized and in those who continued to have increased impedance was similar. The study indicates that NRBC number in maternal circulation during the first trimester cannot be used to screen pregnancies at high risk for developing preeclampsia (PET)/IUGR. High-impedance blood flow in the uterine arteries in the first trimester may be due to an unfinished process of trophoblastic invasion, most likely to be completed successfully by 22–24 weeks.

Aim: To investigate the use of anti-hemoglobin-Ε antibody in order to identify fetal cells in the maternal circulation during pregnancy. Materials and Methods: 48 blood samples were obtained from pregnant women, 26 in the 1st trimester and 22 in the 2nd trimester. Magnetic activated cell sorting was used for fetal cell enrichment followed by immunophenotyping with a monoclonal antibody against hemoglobin-Ε. FISH with X, Y and 21 chromosome-specific probes was performed in 29 cases. Results: The mean number of Ε-positive cells was 9.2 (range 2–23) in the 1st trimester, 4.8 (range 3–13) in the 2nd trimester and 22 (range 15–28) in pregnancies with Down syndrome. No significant difference was noted in the number of Ε-positive nucleated red blood cells (NRBCs) isolated from carriers and noncarriers of β-thalassemia. FISH analysis was successful in 24 cases. In 4 cases with known male fetuses, an average of 4.7 Ε-positive cells showed a Y signal. In 4 cases with Down syndrome, all Ε-positive cells showed 3 signals for chromosome 21. Conclusion: Anti-hemoglobin-Ε antibody has increased specificity for fetal NRBCs and should be preferentially used to improve noninvasive prenatal diagnosis of chromosome abnormalities from fetal cells in maternal blood.

Fetal nucleated red blood cells (NRBCs) entering maternal circulation during pregnancy constitute a potential source of material for safe and reliable noninvasive prenatal diagnosis. The increased prevalence of beta-thalassemia mutations in countries like Greece may create a problem, making it difficult to distinguish between NRBCs of fetal or maternal origin. Use of Ab against embryonic hemoglobin epsilon may increase specificity for fetal NRBC detection. In the present study, Ab against embryonic hemoglobin epsilon was used in the first and second trimesters of pregnancy in order to determine if specificity for fetal NRBC detection could be increased.

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.