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In pregnancy, D- pregnant women may be at risk of becoming immunized against D when carrying a D+ fetus, which may eventually lead to hemolytic disease of the fetus and newborn. Administrating antenatal and postnatal anti-D immunoglobulin prophylaxis decreases the risk of immunization substantially. Noninvasive fetal RHD genotyping, based on testing cell-free DNA extracted from maternal plasma, offers a reliable tool to predict the fetal RhD phenotype during pregnancy. Used as a screening program, antenatal RHD screening can guide the administration of antenatal prophylaxis in non-immunized D- pregnant women so that unnecessary prophylaxis is avoided in those women who carry a D- fetus. In Europe, antenatal RHD screening programs have been running since 2009, demonstrating high test accuracies and program feasibility. In this review, an overview is provided of current state-of-the-art antenatal RHD screening, which includes discussions on the rationale for its implementation, methodology, detection strategies, and test performance. The performance of antenatal RHD screening in a routine setting is characterized by high accuracy, with a high diagnostic sensitivity of ≥99.9 percent. The result of using antenatal RHD screening is that 97-99 percent of the women who carry a D- fetus avoid unnecessary prophylaxis. As such, this activity contributes to avoiding unnecessary treatment and saves valuable anti-D immunoglobulin, which has a shortage worldwide. The main challenges for a reliable noninvasive fetal RHD genotyping assay are low cell-free DNA levels, the genetics of the Rh blood group system, and choosing an appropriate detection strategy for an admixed population. In many parts of the world, however, the main challenge is to improve the basic care for D- pregnant women., (© 2024 Frederik B. Clausen, published by Sciendo.)

Purpose of Review: The aim of this review is to explore a possible link between cell-free DNA (cfDNA) and cardiovascular disease (CVD), which may hold valuable potential for future diagnostics., Recent Findings: cfDNA has become topic of high interest across several medical fields. cfDNA is used as a diagnostic biomarker in cancer, prenatal care, and transplantation. In addition, cfDNA may play an unrecognized role in biological processes that are involved in or underlying various disease states, for example, inflammation. Elevated levels of cfDNA are associated with various elements of CVD, cardio-metabolic risk factors, and autoimmune diseases. Mitochondrial cfDNA and neutrophil extracellular traps may play distinct roles. Total circulating cfDNA may reflect the unspecific accumulation of stressors and the organism's susceptibility and resilience to such stressors. As such, cfDNA, in a stressful situation, may provide predictive value for future development of CVD. We suggest exploring such possibility through a large-scale prospective cohort study of pregnant women., Summary: There is no doubt that cfDNA is a valuable biomarker. For CVD, its potential is indicated but less explored. New studies may identify cfDNA as a valuable circulating cardiovascular risk marker to help improve risk stratification., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Noninvasive fetal blood group antigen genotyping serves as a diagnostic tool to predict the risk of hemolytic disease of the fetus and newborn in pregnancies of immunized women. In addition, fetal RHD genotyping is used as an antenatal screening to guide targeted use of immunoglobulin prophylaxis in non-immunized RhD negative, pregnant women. Based on testing of cell-free DNA extracted from maternal plasma, these noninvasive assays demonstrate high performance accuracies. Consequently, noninvasive fetal blood group antigen genotyping has become standard care in transfusion medicine.

Background and Objectives: For 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in the following., Materials and Methods: Genotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application., Results: In total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner-Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population. ABO genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of ABO*A2, was validated against a series of 1287 samples with Dolichos biflorus lectin determination of the A1 phenotype., Conclusion: We report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology., (© 2023 International Society of Blood Transfusion.)

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rieneck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

Background: Susceptibility to severe acute respiratory syndrome coronavirus 2 shows individual variability in un-vaccinated and previously un-exposed individuals. We investigated the impact of ABO blood group, titers of anti-A and anti-B, other blood group antigens, and the extracellular deposition of ABH antigens as controlled by secretor fucosyltransferase 2 (FUT2) status., Study Design and Methods: We studied incidents in three different hospitals between April to September 2020, where un-diagnosed coronavirus disease 2019 (COVID-19) patients were cared for by health care workers without use of personal protection and with close contact while delivering therapy. We recruited 108 exposed staff, of whom 34 were diagnosed with COVID-19. ABO blood type, titer of anti-A and -B, blood group specific alleles, and secretor status were determined., Results: Blood group O was associated with lower risk of COVID-19 (OR 0.39, 95 %CI (0.16-0.92), p = 0.03) compared to non-O, i.e., blood groups A, B and AB. High titer anti-A immunoglobulin G (IgG) compared to low titer was associated with lower risk of COVID-19 (OR 0.24 95 %CI (0.07-0.78), p = 0.017). High titer of anti-B immunoglobulin M (IgM) compared to no anti-B (IgM) was associated with lower risk of COVID-19 (OR 0.16, 95 %CI (0.039-0.608), p = 0.006) and the same applies to low titer anti-B (IgM) compared to no titer (OR 0.23, 95 %CI (0.07-0.72), p = 0.012). The 33Pro variant in Integrin beta-3, that is part of human platelet antigen 1b (HPA-1b), was associated with lower risk of COVID-19 (OR 0.23, 95 %CI (0.034-0.86), p = 0.028)., Conclusion: Our data showed that blood group O, anti-A (IgG) titer, anti-B (IgM) titer as well as HPA-1b are associated with lower risk for COVID-19., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background and Objectives: Identification of antibody characteristics and genetics underlying the development of maternal anti-A/B linked to inducing haemolytic disease of the foetus and newborn could contribute to the development of screening methods predicting pregnancies at risk with high diagnostic accuracy., Materials and Methods: We examined 73 samples from mothers to 37 newborns with haemolysis (cases) and 36 without (controls). The secretor status was determined by genotyping a single nucleotide polymorphism in FUT2, rs601338 (c.428G>A)., Results: We found a significant association between secretor mothers and newborns developing haemolysis (p = 0.028). However, stratifying by the newborn's blood group, the association was found only in secretor mothers to blood group B newborns (p = 0.032). In fact, only secretor mothers were found in this group. By including antibody data from a previous study, we found higher median semi-quantitative levels of IgG1 and IgG3 among secretor mothers than non-secretor mothers to newborns with and without haemolysis., Conclusion: We found that the maternal secretor status is associated with the production of anti-A/B, pathogenic to ABO-incompatible newborns. We suggest that secretors experience hyper-immunizing events more frequently than non-secretors, leading to the production of pathogenic ABO antibodies, especially anti-B., (© 2023 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

In solid organ transplantation, donor-derived cell-free DNA (dd-cfDNA) is a promising universal noninvasive biomarker for allograft health, where high levels of dd-cfDNA indicate organ damage. Using Droplet Digital PCR (ddPCR), we aimed to develop an assay setup for monitoring organ health. We aimed to identify the least distinguishable percentage-point increase in the fraction of minute amounts of cfDNA in a large cfDNA background by using assays targeting single nucleotide polymorphisms (SNPs). We mimicked a clinical sample from a recipient in a number of spike-in experiments, where cfDNA from healthy volunteers were mixed. A total of 40 assays were tested and approved by qPCR and ddPCR. Limit of detection (LOD) was demonstrated to be approximately 3 copies per reaction, observed at a fraction of 0.002%, and which would equal 6 copies per mL plasma. Limit of quantification (LOQ) was 35 copies per reaction, estimated to 0.038%. The lowest detectable increase in percentage point of dd-cfDNA was approximately 0.04%. Our results demonstrated that ddPCR has great sensitivity, high precision, and exceptional ability to quantify low levels of cfDNA. The ability to distinguish small differences in mimicking dd-cfDNA was far beyond the desired capability. While these methodological data are promising, further prospective studies are needed to determine the clinical utility of the proposed method., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Clausen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Previous studies have shown that intracorporeal anastomosis (ICA) in minimally invasive right colectomy may improve postoperative recovery compared with extracorporeal anastomosis (ECA). It has been hypothesized that creating the anastomosis extracorporeally may cause mesenteric traction and compromised intestinal perfusion. The purpose of this study was to investigate the effect of either ICA or ECA on intestinal perfusion., Method: This was a substudy to a multicenter, triple-blind randomized clinical trial comparing ICA with ECA in patients undergoing robotic right colectomy for colonic cancer. Videos from intraoperative Indocyanine Green (ICG) fluorescence imaging were analyzed with quantitative ICG perfusion assessment (q-ICG). q-ICG was performed by extracting perfusion metrics from a time-intensity curve generated from an image analysis software: F MAX : maximal fluorescence intensity, T MAX : time until maximal fluorescent signal, T 1/2MAX : time until half-maximal fluorescent signal, time ratio (T 1/2MAX /T MAX ) and slope., Results: A total of 68 patients (33 ICA and 35 ECA) were available for analysis. Demographics were similar between the groups, except for mean arterial blood pressure at the time of ICG infusion, which was significantly lower in the ICA group. We found a significantly steeper slope in the ICA group compared to the ECA group (6.3 vs. 4.7 AU/sec, P = .048). There were no significant differences in F MAX , T MAX , T 1/2MAX , and time ratio., Conclusion: We found evidence of an improved intestinal perfusion following ICA compared with ECA. This finding may be related to patient outcomes and should be explored further in the future., Clinicaltrials: gov NCT03130166., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Objectives: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021., Materials and Methods: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented., Results: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030)., Conclusion: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/)., (© 2022 International Society of Blood Transfusion.)

Objective: To determine if minimally invasive right colectomy with intra-corporeal anastomosis improves postoperative recovery compared to extra-corporeal anastomosis., Background: Previous trials have shown that intracorporeal anastomosis improves postoperative recovery; however, it has not yet been evaluated in a setting with optimized perioperative care or with patient-related outcome measures., Methods: This was a multicenter, triple-blind, randomized clinical trial at two high-volume colorectal centers with strict adherence to optimized perioperative care pathways. The patients underwent robotic right colectomy with either intracorporeal or extracorporeal anastomosis. The primary outcome was patient-reported postoperative recovery measured using the "Quality of Recovery-15" questionnaire. ClinicalTrials.gov NCT03130166., Results: A total of 89 patients were randomized and analyzed according to the "Intention-to-treat"-principle. We found no statistically significant differences in patient-reported recovery between the groups. Postoperative pain, nausea, time to ambulation, time to first passage of flatus/stool, length of hospital stay, and pathophysiological tests showed no differences either. The duration of time to create the anastomosis was significantly longer with intracorporeal anastomosis (17 vs 13 min, P = 0.003), while all other intraoperative, postoperative, and pathology variables showed no difference., Conclusion: There were no significant differences in postoperative recovery between the two groups., Competing Interests: Conflicts of Interest and Sources of Funding: ND received unrestricted funding from the Louis-Hansen Foundation, Jacob & Olga Madsen’s foundation, Trigon Foundation, Toyota Foundation, Dagmar Marshalls Foundation, Vissing Foundation, the Kjaer Foundation, and the Nyegaard Foundation. The funding sources had no role in the design and conduct of the study; data collection, analysis, and interpretation of data; the preparation, review or approval of the manuscript or the decision to submit for publication. For the remaining authors none were declared. The study did not receive funding from National Institutes of Health (NIH); Wellcome Trust; or Howard Hughes Medical Institute (HHMI). The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The standard operation for mid- and low rectal cancer total mesorectal excision (TME) is routinely performed as minimally invasive surgery. TME is associated with temporary or permanent functional impairment of pelvic organs, causing reduced quality of life (QoL). Concerns have been raised that the newest minimally invasive approach, transanal TME (TaTME), may further reduce urogenital and anorectal functions., Objective: To determine if functional outcomes affecting QoL are altered after TaTME. Primary end-point is the impact of TaTME on QoL and functional outcomes. Secondary end-point is assessing differences in QoL and functional outcomes after TME surgery from below (TaTME) or above (transabdominal TME)., Design, Setting, and Participants: Observational study consisting of prospectively registered self-reported questionnaire data collected at baseline and follow-ups after TaTME. All patients who underwent TaTME during the Danish national implementation phase were included. Central surveillance of the implementation included questionnaires concerning QoL and functional outcomes. Analyses of functional results from the Danish cohort of the ROLARR trial (Jayne et al. in JAMA 318:1569-1580, (2017) are reported separately for perspective, representing the transabdominal approach to TME, i.e., laparoscopic- or robotic-assisted TME (LaTME/RoTME). Applied questionnaires include EORTC QLQ-C30, SF-36, LARS, ICIQ-MLUTS, ICIQ-FLUTS, IPSS, IIEF, SVQ, and FSFI., Results: A total of 115 TaTME procedures were registered August 2016 to April 2019. LaTME/RoTME patients (n = 92) were operated on January 2011 to September 2014. A temporary postoperative decrease of QoL (global health status and functional scales) was observed, yet long-term results were unaffected by surgery in both groups. In TaTME patients, the anorectal dysfunction increased significantly (p < 0.001) from preoperative baseline to 13.5 months follow-up, where 67.5% (n = 52) reported major LARS symptoms. Urinary function was not significantly impaired after TME regardless of technique. The paucity of responses concerning sexual function precludes conclusions., Conclusions: Although an initial reduction in QoL after TME occurs, it normalizes within the first year postoperatively. In concurrence with international results, we found that significant anorectal dysfunction is common after TaTME. No data on anorectal function was available for LaTME/RoTME patients for comparison. We found no indications that transanal TME is inferior to transabdominal TME surgery concerning urogenital functions or health-related QoL., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Objectives: Prediction of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti-A/-B enables timely therapy, thereby preventing the development of kernicterus spectrum disorder. However, previous efforts to establish accurate prediction methods have been only modestly successful., Materials and Methods: In a case-control study, we examined 76 samples from mothers and 76 samples from their newborns; 38 with and 38 without haemolysis. The IgG subclass profile of maternal anti-A and anti-B was determined by flow cytometry. Samples from newborns were genetically analysed for the A 2 subgroup, secretor and FcγRIIa receptor alleles., Results: Surprisingly, we found a correlation between the newborn secretor allele and haemolysis (p = 0.034). No correlation was found for FcγRIIa alleles. The A 2 subgroup was found only in newborns without haemolysis. Unexpectedly, different reaction patterns were found for maternal anti-A and anti-B; consequently, the results were treated separately. For the prediction of haemolysis in A-newborns, the maternal IgG1 subclass determination resulted in an accuracy of 83% at birth. For B-newborns, an accuracy of 91% was achieved by the maternal IgG2 subclass determination., Conclusion: We improved the prediction of ABO-HDFN by characterizing maternal anti-A and anti-B by flow cytometry and we presented genetic traits in newborns with correlation to haemolysis. We propose a new understanding of A- and B-substances as immunogens that enhance the maternal immune response and protect the newborn, and we suggest that the development of ABO-HDFN is different when caused by maternal anti-A compared to maternal anti-B., (© 2021 International Society of Blood Transfusion.)

Background and Objectives: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations., Materials and Methods: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors., Results: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed., Conclusion: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays., (© 2021 International Society of Blood Transfusion.)

Introduction: In the Kell blood group system, the K and k antigens are the clinically most important ones. Maternal anti-K IgG antibodies can lead to the demise of a K-positive fetus in early pregnancy. Intervention can save the fetus. Prenatal K status prediction of the fetus in early pregnancy is desirable and gives a good basis for pregnancy risk management. We present the results from 7 years of clinical experience in predicting fetal K status as well as some theoretical considerations relevant for design of the assay and evaluation of results., Methods: Blood was collected from 43 women, all immunized against K, at a mean gestational age of 18 weeks (range 10-38). A total of 56 consecutive samples were tested. The KEL * 01.01 / KEL * 02 single nucleotide variant that determines K status was amplified from maternal plasma DNA by PCR without allele specificity. The PCR product was sequenced by NGS technology, and the number of sequenced KEL * 01.01 and KEL * 02 reads were counted. Prediction of the fetal K status was based on this count and was compared with the serologically determined K status of the newborns., Results: All fetal K predictions were in accordance with postnatal serology where available ( n = 34), using our current data analysis., Conclusion: We have developed an NGS-based method for the non-invasive prediction of fetal K status. This approach requires special considerations in terms of primer design, stringent preanalytical sample handling, and careful analytical procedures. We analyzed samples starting at GA 10 weeks and demonstrated the correct prediction of fetal K status. This assay enables timely clinical intervention in pregnancies at risk of hemolytic disease of the fetus and newborn caused by maternal anti-K IgG antibodies., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Background: The Rh blood group system has considerable clinical importance. The C, c, and E antigens are targets of alloantibodies. Anti-C, anti-c or anti-E alloreactive antibodies produced in pregnant women can cause anemia of a fetus carrying the corresponding antigens., Aims: Based on NGS technology, we have developed a noninvasive diagnostic assay to predict the fetal blood group of C, c or E antigens by sequencing cell-free DNA (cfDNA) during pregnancy., Materials and Methods: The SNVs underlying either the C, c or E antigens were PCR amplified and sequenced using NGS on a MiSeq instrument. The DNA sequences encoding the C, c or E antigen were counted, as were the number of total sequences. Based on the percentage of fetally derived target SNVs inherited from the father, the fetal blood group could be predicted., Results: The results of 55 consecutive RHCE prenatal analyses with postnatal serological blood group determination of 30 newborns showed no discordant results. A threshold discerning positive from negative samples was set at 0.05% specific reads., Discussion: Noninvasive, prenatal prediction of fetal blood groups by sequencing cfDNA for the detection of low-level RHCE*C, RHCE*c and RHCE*E sequences was established as an accurate and robust assay applicable for use in clinical settings., (© 2021 John Wiley & Sons Ltd.)

Background: Laboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark., Summary: All pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by S. Karger AG, Basel.)

Background: Hemolysis in fetus/newborns is often caused by maternal antibodies. There are currently no established screening procedures for maternal ABO antibodies harmful to fetus/newborn. We investigated the clinical significance, and predictive value of maternal anti-A/B titer for hyperbilirubinemia in ABO-incompatible newborns., Methods: We conducted a case-control study of blood group O mothers and their ABO-compatible (O) vs. -incompatible (A/B) newborns receiving phototherapy, and of ABO-incompatible newborns receiving phototherapy vs. no phototherapy. Newborn data and treatment modalities were recorded, and total serum bilirubin and hemoglobin were measured. Maternal anti-A/B immunoglobulin-γ (IgG) titers were measured prenatally and perinatally, and negative and positive predictive values (NPV, PPV) were calculated to assess the risk of developing hyperbilirubinemia requiring phototherapy., Results: We found a significantly higher maternal IgG antibody titer in the case group (p < 0.001). Maternal anti-A/B titers at first trimester had modest predictive values: NPV = 0.82 and PPV = 0.65 for neonatal hyperbilirubinemia; titers at birth improved the predictive values: NPV = 0.93 and PPV = 0.73. Newborn hemoglobin was significantly lower in incompatibles compared to compatibles (p = 0.034). Furthermore, increased anti-A/B IgG production during pregnancy was associated with hyperbilirubinemia and hemolysis in incompatible newborns., Conclusions: There was a significant association between maternal anti-A/B IgG titer and hyperbilirubinemia requiring treatment., Impact: Maternal anti-A/B IgG titer in the first trimester and at birth is predictive of hemolytic disease of the ABO-incompatible newborn. Increased IgG anti-A/B production throughout pregnancy in mothers to ABO-incompatible newborns developing hyperbilirubinemia contrasts a constant or reduced production in mothers to newborns not developing hyperbilirubinemia. Screening tools available in most immunohematology laboratories can identify clinically important IgG anti-A/B. Use of maternal samples taken at birth yielded NPV = 0.93 and PPV = 0.73., (© 2020. International Pediatric Research Foundation, Inc.)

Purpose: Patients with a defunctioning ileostomy after rectal resection experience substantial ileostomy-related morbidity and decreased quality of life. Early reversal of the defunctioning ileostomy has been proposed as a method of mitigating these problems. We aimed to evaluate the safety of early ileostomy closure within 6 weeks., Method: Randomized controlled trials investigating the safety of early ileostomy closure were identified through a systematic search and review of the current literature. Meta-analysis of the extracted outcome data was performed, and the methodological quality of the individual studies was assessed., Results: The search identified six eligible studies yielding a total of 528 patients, with 269 in the early closure (EC) group and 259 in the standard closure (SC) group. Major complications in the EC group was 5.2% compared with 3.6% in the SC group (RR = 1.12, 95% CI 0.33-3.79). Anastomotic leakage in the EC group was 3.3% compared with 3.5% in the SC group (RR = 0.89, 95% CI 0.29-2.75). The meta-analysis resulted in no statistically significant differences between the groups in any of the primary or secondary outcomes., Conclusion: This review was not able to discern a statistically significant difference in postoperative complications when comparing early and standard ileostomy closure. The current literature indicates that early ileostomy closure is not associated with higher complication rates in patients with an uncomplicated postoperative course and radiologically verified intact distal anastomosis after index surgery.

Background and Objective: Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR., Materials and Methods: Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6 years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at -25°C., Results: We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA., Conclusion: Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes., (© 2020 International Society of Blood Transfusion.)

Background and Objectives: Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories., Materials and Methods: Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly., Results: Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory., Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting., (© 2020 International Society of Blood Transfusion.)

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information., Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group., Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples., Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women ( n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO * O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity., Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO * O.01/ABO * O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 by S. Karger AG, Basel.)

Background: In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible information. Here we designed and validated a high-throughput extended genotyping setup., Study Design and Methods: We developed 35 competitive allele-specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes., Results: We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen-negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction., Conclusion: We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping-only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost-efficient than previous technologies., (© 2019 AABB.)

Introduction: The aim of this clinical pilot study was to examine the accuracy of noninvasive fetal RHD genotyping in early pregnancy (8 +0  to 11 +6  weeks) and to clarify whether targeted administration of Rhesus immunoglobulin (RhIg) is possible for women undergoing an induced abortion such that unnecessary injections can be avoided. The study examines the correlation between gestational age and the amount of cell-free fetal DNA in maternal plasma, the fetal fraction of DNA and whether transportation time or body mass index affects these parameters., Material and Methods: Fifty-two RhD-negative women undergoing a surgically induced abortion were included. A maternal blood sample was collected prior to the abortion and a tissue sample was collected from the placental part of the abortion material after the intervention. Fetal RhD type was determined by PCR analysis of cell-free fetal DNA extracted from maternal plasma and on DNA from the tissue sample, with the latter providing a reference standard. Copies of RHD/mL were determined on RHD-positive samples and the fetal fraction of DNA was calculated., Results: We demonstrated complete concordance between results from plasma and tissue, with 31 RhD-positive and 21 RhD-negative samples, corresponding to 40% being RhD-negative, specificity 100% [95% confidence interval (CI) 88.8-100] and sensitivity 100% (95% CI 83.9-100). We found no significant correlation between gestational age and the amount or the fraction of cell-free fetal DNA in maternal plasma, nor did we find that transportation time or BMI significantly affected these factors in this setup., Conclusions: Fetal RHD genotyping can be accurately performed from the 8th week of gestation and unnecessary injections of RhIg can be avoided for women undergoing an induced abortion. A larger study is needed to determine a more accurate sensitivity for the analysis early in pregnancy., (© 2019 Nordic Federation of Societies of Obstetrics and Gynecology.)

Background and Objectives: Fetal RHD genotyping of cell-free fetal DNA from RhD-negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 28 laboratories., Materials and Methods: Aliquots of pooled maternal plasma were sent to each laboratory. One sample was positive, and the second sample was negative for fetal RHD, verified by pre-workshop testing using quantitative real-time PCR (qPCR) analysis of RHD exons 4, 5, 7 and 10. Plasma samples were shipped at room temperature. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. Different methodological approaches were used, all employing qPCR with a total of eight different combinations of RHD exon targets. The samples were tested blindly., Results: Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD-positive sample, and four inconclusive results were reported for the RHD-negative sample. All clinical conclusions were satisfactory., Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting., (© 2019 International Society of Blood Transfusion.)

RhD negative pregnant women who carry an RhD positive fetus are at risk of immunization against the D antigen, which may result in hemolytic disease of the fetus and the newborn. Predicting the fetal RhD status by noninvasive antenatal screening for the fetal RhD gene (RHD) can guide targeted use of antenatal anti-D prophylaxis.Cell-free fetal DNA is extracted from maternal plasma from RhD negative pregnant women at a gestational age of 25 weeks. A real-time PCR-based detection of two RHD exons enables reliable prediction of the fetal RhD status to determine the administration of antenatal prophylaxis, as well as postnatal prophylaxis.

Background and Objectives: The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session., Methods: As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented., Results and Conclusions: Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies., (© 2018 International Society of Blood Transfusion.)

Background: Circulating cell-free fetal DNA (cffDNA) provides the opportunity for noninvasive prenatal diagnosis. Early knowledge of the fetal sex is essential in cases with a risk of a sex-linked genetic disease. A reliable and highly sensitive sex determination test is required for first trimester testing because of the low amounts of cffDNA., Methods: First trimester blood samples from 326 pregnant women were analyzed by real-time quantitative polymerase chain reaction (qPCR) for the presence of Y chromosome repetitive sequence (YRS). Blood samples were collected from gestational weeks 4-12. Fetal sex was predicted on the basis of results from the YRS assay of cffDNA extracted from maternal plasma. The predicted sex was compared with the phenotypic sex of the newborn baby (n = 294)., Results: There was high concordance between the test results from the YRS assay and the actual sex at birth. There were no false-positive results, indicating agreement between male YRS results and male sex at birth. Two results were false negative (from gestational weeks 4 and 6) predicting female fetuses, when the actual sex at birth was male. Overall, the sensitivity of the YRS assay was 98.6% (95% CI, 95.1%-99.8%), specificity was 100% (95% CI, 97.5%-100%), and accuracy was 99.3% (95% CI, 97.5%-99.9%). From 7 weeks of gestation, sensitivity, specificity, and accuracy were 100%., Conclusions: This study shows that qPCR can be used to detect and quantify repetitive DNA sequences from 0.3 genome equivalents per milliliter of plasma. Prenatal sex determination by qPCR of YRS in cffDNA from maternal plasma was reliable and robust with cffDNA extracted from 1 mL of nonhemolyzed plasma, with a plasma equivalent per PCR of 167 μL. The YRS assay can be used for early noninvasive prenatal sex determination from a gestational age of 7 weeks., (© 2018 American Association for Clinical Chemistry.)

Introduction: In the fight against hemolytic disease of the fetus and newborn, pregnant RhD negative women are offered antenatal and postnatal anti-D immunoglobulin prophylaxis to prevent the development of antibodies against the fetal D antigen. Antenatal prophylaxis has traditionally been provided to all D negative pregnant women, as the fetal RhD type remains unknown until birth. With noninvasive prenatal testing of cell-free DNA, predicting the fetal RhD type has become highly feasible based on analysis of the fetal RHD gene. Fetal RHD screening can guide targeted antenatal prophylaxis, treating only women who carry an RhD positive fetus, thereby avoiding the unnecessary treatment of approximately 40% of the RhD negative women. Areas covered: Areas covered are the current clinical practice, performance, and challenges of fetal RHD screening, and relevant issues for its implementation. Expert commentary: Fetal RHD screening is highly accurate, with sensitivities of 99.9%, as reported from clinical programs. From gestational week 10, sensitivities are approximately 99%. Despite challenges in assay design, low levels of cell-free fetal DNA in the maternal plasma, and concerns regarding implementation and medical necessity, the clinical experience with fetal RHD screening and targeted prophylaxis has generated encouraging results, and its future widespread use is expected.

Purpose of Review: In this review, we analyzed the current literature on noninvasive fetal RHD typing to answer the question whether the administration of RhIg to prevent D-alloimmunization during pregnancy can be safely guided by fetal RHD typing., Recent Findings: Recently the first centers that implemented large-scale nationwide fetal RHD typing in the second trimester for targeted RhIg administration have published their studies evaluating the diagnostic accuracy of their screening programs. These data show that fetal RHD typing in a routine setting is, at least in a population of European descent, accurate enough to guide both antenatal and postnatal immunoprophylaxis., Summary: Depending on the ethnic background and the organization of pregnancy care the decisions regarding RhIg can be safely and cost-effectively based on fetal RHD typing by a duplex real-time PCR. As a result, the unnecessary administration of 40% of antenatal RhIg can be prevented, and cord blood serology can be omitted.

Hemolytic disease of the fetus and newborn (HDFN) is a condition characterized by a decreased lifespan of fetal red blood cells caused by maternally produced allospecific antibodies transferred to the fetus during pregnancy. The antibodies bind to the corresponding blood group antigens on fetal red blood cells and induce hemolysis. Cell-free DNA derived from the conceptus circulates in maternal blood. Using next-generation sequencing (NGS), it can be determined if this cell-free fetal DNA encodes the corresponding blood group antigen that is the target of the maternal allospecific antibodies. This determination carries no risk to the fetus. It is important to determine if the fetus is at risk of hemolysis to enable timely intervention. Many tests for blood groups are based solely on the presence or absence of a single nucleotide polymorphism (SNP). Antenatal determination of fetal blood group by NGS analysis holds advantages over polymerase chain reaction (PCR) determination based on allele specific amplification., (Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.)

High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

The KEL1 antigen can give rise to immunization of KEL2 mothers. Maternal antibodies can be transferred to the fetus and destroy fetal red blood cells and their stem cell precursors and give rise to serious fetal disease. It is important to be able to predict the fetal KEL status in order to intervene in those pregnancies where the fetus is at risk, and to ascertain when the fetus is not at risk. Technically it can be demanding to predict KEL1 status from a maternal blood sample. The KEL1 allele is based on a single SNP present in about 1-10 % of cell-free maternal DNA after gestation week 10. Here we describe our protocol for antenatal prediction of fetal KEL1 status by NGS analysis of maternal DNA on a MiSeq instrument.

Objective: Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening., Methods: Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies., Results: The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%., Conclusion: The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance., (© 2014 John Wiley & Sons, Ltd.)

Incompatibility of red blood cell blood group antigens between a pregnant woman and her fetus can cause maternal immunization and, consequently, hemolytic disease of the fetus and newborn. Noninvasive prenatal testing of cell-free fetal DNA can be used to assess the risk of hemolytic disease of the fetus and newborn to fetuses of immunized women. Prediction of the fetal RhD type has been very successful and is now integrated into clinical practice to assist in the management of the pregnancies of RhD immunized women. In addition, noninvasive prediction of the fetal RhD type can be applied to guide targeted prenatal prophylaxis, thus avoiding unnecessary exposure to anti-D in pregnant women. The analytical aspect of noninvasive fetal RHD typing is very robust and accurate, and its routine utilization has demonstrated high sensitivities for fetal RHD detection. A high compliance with administering anti-D is essential for obtaining a clinical effect. Noninvasive fetal typing of RHC/c, RHE/e, and KEL may become more widely used in the future., (© 2014 John Wiley & Sons, Ltd.)

Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10-11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next-generation sequencing (NGS) technology., Study Design and Methods: The KEL1/2 single-nucleotide polymorphism was polymerase chain reaction (PCR) amplified with one adjoining base, and the PCR product was sequenced using a genome analyzer (GAIIx, Illumina); several millions of PCR sequences were analyzed., Results: The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell-free DNA purified from maternal plasma., Conclusion: This method requires only one primer pair, and the large amount of sequence information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA-based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity, various conditions of chimerism could also be examined using this approach. To our knowledge, this is the first report focused on antenatal blood group determination using NGS., (© 2013 Department of Clinical Immunology, University Hospital Copenhagen. Transfusion © 2013 American Association of Blood Banks.)

Background: Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening., Methods: Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104)., Results: The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317)., Conclusion: The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.

Background: The objective was to investigate whether women who develop preeclampsia can be identified in a routine analysis when determining fetal RHD status at 25 weeks' gestation in combination with PAPP-A levels at the first-trimester combined risk assessment for Trisomy 21., Study Design and Methods: D- women participating in the routine antenatal RHD screening program in the capital region of Denmark were retrospectively studied. We used a standard dilution curve to quantify the amounts of cell-free fetal DNA (cffDNA) and divided women into groups according to cffDNA levels. PAPP-A was measured at 11 to 14 weeks. Information about pregnancy outcome and complications was obtained from the National Fetal Medicine Database, medical charts, and discharge letters., Results: The odds ratio (OR) of developing severe preeclampsia given a cffDNA level above the 90th percentile compared to cffDNA below the 90th percentile was 8.1 (95% confidence interval [CI], 2.6-25.5). The OR of developing mild preeclampsia given a cffDNA level below the 5th percentile compared to cffDNA levels above the 5th percentile was 3.6 (95% CI, 1.1-11.7). PAPP-A levels below the 5th percentile were associated with mild preeclampsia, but adding it to the analysis did not increase the detection rate (DR)., Conclusion: Women with cffDNA levels below the 5th percentile and above the 90th percentile quantified at 25 weeks' gestation are at increased risk of developing preeclampsia. Adding PAPP-A levels to the analysis did not increase the DR of preeclampsia., (© 2013 American Association of Blood Banks.)

Objective: To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation., Study Design: Cohort study including RhD negative women participating in a routine RHD screening programme. A standard dilution curve was used to quantify the amounts of cffDNA. Values above the 95(th) centile for the study population defined high levels of cffDNA., Results: We found a highly significant association between preterm delivery and cffDNA levels above the 95(th) centile (p = 0.002). Using logistic regression, women with high levels of cffDNA had an odds ratio of 6.3 (95% confidence interval: 1.9-20.9) for preterm delivery before 37 weeks and an odds ratio for delivery before 34 weeks of 16.6 (95% confidence interval: 3.2-84.7) when adjusting for gestational age at sampling, body mass index and previous miscarriages/terminations of pregnancy., Conclusion: High levels of cffDNA at 25 weeks are associated with increased risk of spontaneous preterm delivery., (© 2012 John Wiley & Sons, Ltd.)

Background and Objectives: A targeted routine antenatal anti-D prophylaxis programme was implemented in Denmark where anti-D immunoglobulin is given based on the result from noninvasive antenatal screening for fetal RHD. Our objective was to evaluate compliance with this new programme right after its initiation., Materials and Methods: We examined the treatment outcome of 239 D-negative pregnant women who gave birth at our hospital between June and September 2010., Results: The majority of these women (90%) underwent antenatal RHD screening, 86% of the women who were recommended antenatal prophylaxis received anti-D, and 99% of the women who delivered RhD-positive infants received postnatal anti-D., Conclusion: These compliance results are acceptable as they were obtained only a few months after the initiation of the new prophylaxis regime. However, suggestions to further improve compliance are presented., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Background: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D- women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening., Study Design and Methods: In each of the five Danish health care regions, blood samples were drawn from D- women in Gestational Week 25. DNA was extracted from the maternal plasma and analyzed for the presence of the RHD gene by real-time polymerase chain reaction targeting two RHD exons. Prediction of the fetal RhD type was compared with serologic typing of the newborn in 2312 pregnancies, which represented the first 6 months of routine analysis., Results: For the detection of fetal RHD, the sensitivity was 99.9%. The accuracy was 96.5%. The recommendation for unnecessary antenatal RhD prophylaxis for women carrying a D- fetus was correctly avoided in 862 cases (37.3%), while 39 women (1.7%) were recommended for antenatal RhD prophylaxis unnecessarily. Two RHD+ fetuses (0.087%) were not detected, and antenatal RhIG was not given., Conclusion: These data represent the first demonstration of the reliability of routine antenatal fetal RHD screening in D-, pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Our findings should encourage the implementation of such screening programs worldwide, to reduce the unnecessary use of RhIG., (© 2011 American Association of Blood Banks.)