Confirmed non-invasive prenatal testing for foetal Rh blood group genotyping along with bi-allelic short insertion/deletion polymorphisms as a positive internal control.

BACKGROUND: Determination of foetus rhesus blood group at risk of hemolytic disease has potential application for early non-invasive prenatal testing (NIPT). There are several challenges in developing NIPT rhesus blood group genotyping assays by using cell-free foetal DNA (cff-DNA) in plasma of RhD-negative pregnant women. So, the aim of this study was optimization of Real-time PCR assay for NIPT rhesus genotyping and development of Bi-allelic short insertion/deletion polymorphisms (INDELs) as internal control to optimise and validate rhesus genotyping based on Real-time PCR to avoid false or negative results. MATERIAL AND METHODS: NIPT Rhesus genotyping including RHD (exon 7), RHCc, and RHEe genes were performed by TaqMan Real-time PCR on 104 maternal samples at different gestation ages (12 to ≥40 weeks) from 51 alloimmunized pregnant women. The sensitivity protocol was confirmed with standard DNA samples. Eight selected INDELs were designed and used to detectable cff-DNA in maternal plasma. INDELs frequency and inheritance were determined on 6 family and 61 unrelated individuals. Finally, multiplex Real-time PCR was performed for each sample with INDELs pairs and Rh probes. RESULTS: The results showed 100% accuracy rhesus typing for RHD, RHC and RHE assays and 95.7% accuracy for RHc. Also, eight selected INDELs as internal control for NIPT were 100% concordance for typed samples. CONCLUSION: The Real-time PCR assay is a suitable method with high sensitivity and specificity for rhesus typing as NIPT for prediction of hemolytic disease in foetuses. The INDELs described here are suitable internal control for confirmation of NIPT on cff-DNA.

The highest Duffy (FYX) allele frequency ever reported for Scottish population: A cross-sectional study.

Background and Aim: The Duffy (FY) blood group system has six known antigens among which the Fya and Fyb are known as major antigens. Fyx phenotype forms as a result of two point mutations in the FYB allele leading to instability of Duffy protein and so reduction of Duffy antigen expression in the cells. This study aimed to investigate the FYX allele frequency in the Scottish population. Methods: The Duffy blood group system was serologically and molecularly investigated in 222 samples collected from donors of Aberdeen Regional Blood Transfusion Center (BTC). The haemagglutination and BeadChip microarray chemistry methods were used for phenotyping and genotyping. Confirmatory tests were also used to check the discrepant results. Results: In this study, the frequency of Duffy blood group phenotypes including Fya+, Fya+b+, and Fyb+ were 17.57%, 42.79%, and 39.64%, respectively. Furthermore, the frequency of FYA/FYA, FYA/FYB, and FYB/FYB genotypes was estimated to be 14.41%, 45.95%, and 39.64%, respectively, using the Bioarray method. In the present study, based on Duffy DNA sequencing results, 12 samples (5.41%) had just one FYX allele. Conclusion: The frequency of the FYX allele in this study was estimated to be 0.0270% which is more than the results reported so far.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.

Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer.

Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus.

The development of a quantitative ELISA for antibodies against human platelet antigen type 1a.

BACKGROUND: Severe neonatal alloimmune thrombocytopenia is often due to antibodies against human platelet antigen type 1a (HPA-1a). The aim of this study was to develop a quantitative ELISA for the measurement of antibodies against HPA-1a. STUDY DESIGN AND METHODS: HPA-1a glycoprotein (GP) IIb-IIIa was immobilized and mixed with recalcified anti-HPA-1a-positive plasma overnight at 4 degrees C. The beads were washed, the antibodies against HPA-1a were eluted, and the eluate pH level was promptly adjusted. The purified antibodies were dialyzed and used for the development of an ELISA incorporating HPA-1a-coated plates. RESULTS: Serial doubling dilutions of the purified antibodies resulted in consistent sigmoid standard curves with a sensitivity of 0.5 microg per mL. To determine the reproducibility of the ELISA, antibodies against HPA-1a in five plasma samples (Samples A-E) were measured at serial doubling dilutions in four separate assays. Three of the samples (Samples A-C) contained antibodies against HPA-1a. The mean amounts in microg per mL (+/- SD, percentage of CV) obtained in the four assays were as follows: Sample A, 133 (9.4, 7.1%); Sample B, 16.5 (1.7, 10%); and Sample C, 8 (0.8, 10%). The amounts in the two antibody-negative controls (Samples D and E) were consistently less than 0.2 microg per mL. CONCLUSION: Using immobilized HPA-1a1a, antibodies against HPA-1a has been purified, and a quick and simple quantitative assay has been developed.