Generation of human antibodies targeting human platelet antigen (HPA)-1a.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS: This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS: Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION: This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

Case series on clinical applications of liquid biopsy in pediatric solid tumors: towards improved diagnostics and disease monitoring.

Background and aims: Solid tumors account for about 30% of all pediatric cancers. The diagnosis is typically based on histological and molecular analysis of a primary tumor biopsy. Liquid biopsies carry several advantages over conventional tissue biopsy. However, their use for genomic analysis and response monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. In this case series we discuss six clinical cases of children with a solid tumor for whom a liquid biopsy assay was performed and demonstrate the potential of liquid biopsy for future clinical decision making. Methods: We performed quantitative real-time PCR (RT-qPCR), droplet digital PCR (ddPCR) or reduced representation bisulphite sequencing of cell-free DNA (cfRRBS) on liquid biopsies collected from six pediatric patients with a solid tumor treated between 2017 and 2023 at the Princess Máxima Center for Pediatric Oncology in the Netherlands. Results were used to aid in clinical decision making by contribution to establish a diagnosis, by prognostication and response to therapy monitoring. Results: In three patients cfRRBS helped to establish the diagnosis of a rhabdomyosarcoma, an Ewing sarcoma and a neuroblastoma (case 1-3). In two patients, liquid biopsies were used for prognostication, by MYCN ddPCR in a patient with neuroblastoma and by RT-qPCR testing rhabdomyosarcoma-specific mRNA in bone marrow of a patient with a rhabdomyosarcoma (case 4 and 5). In case 6, mRNA testing demonstrated disease progression and assisted clinical decision making. Conclusion: This case series illustrates the value of liquid biopsy. We further demonstrate and recommend the use of liquid biopsies to be used in conjunction with conventional methods for the determination of metastatic status, prognostication and monitoring of treatment response in patients with pediatric solid tumors.

Fetal and neonatal alloimmune thrombocytopenia: Current pathophysiological insights and perspectives for future diagnostics and treatment.

FNAIT is a pregnancy-associated condition caused by maternal alloantibodies against paternally-inherited platelet antigens, most frequently HPA-1a on integrin ß3. The clinical effects range from no symptoms to fatal intracranial hemorrhage, but underlying pathophysiological determinants are poorly understood. Accumulating evidence suggests that differential antibody-Fc-glycosylation, activation of complement/effector cells, and integrin function-blocking effects contribute to clinical outcome. Furthermore, some antibodies preferentially bind platelet integrin αIIbß3, but others bind αvß3 on endothelial cells and trophoblasts. Defects in endothelial cells and angiogenesis may therefore contribute to severe anti-HPA-1a associated FNAIT. Moreover, anti-HPA-1a antibodies may cause placental damage, leading to intrauterine growth restriction. We discuss current insights into diversity and actions of HPA-1a antibodies, gathered from clinical studies, in vitro studies, and mouse models. Assessment of all factors determining severity and progression of anti-HPA-1a-associated FNAIT may importantly improve risk stratification and potentially reveal novel treatment strategies, both for FNAIT and other immunohematological disorders.

Natural history of human platelet antigen 1a-alloimmunised pregnancies: a prospective observational cohort study.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare disease that untreated can lead to intracranial haemorrhage or death. The natural history of FNAIT is still unclear; therefore, the benefits of screening cannot be estimated and no routine screening is yet in place. We aimed to assess the incidence of clinically detectable FNAIT among pregnancies in human platelet antigen-1a (HPA-1a)-immunised women. METHODS: We did a prospective observational cohort study of pregnant women negative for rhesus D (RhD) and rhesus c (Rhc) antigens, without age limits, who underwent routine antenatal screening for red cell antibodies at 27 weeks' gestation and were typed for HPA-1a between March 1, 2017, and May 1, 2020. HPA-1a-negative women were tested for HPA alloantibodies. Health-care professionals were masked to all test results. The main outcome was the proportion of neonates with severe, clinically detectable FNAIT, defined as having an intracranial bleed, organ bleed, or bleeding-related death observed during pregnancy or within the first week of life. Cases of clinically detectable FNAIT not categorised as severe were categorised as mild. This study is registered with ClinicalTrials.gov (NCT04067375). FINDINGS: Of 153 106 women typed for HPA-1a, 3722 (2·4%) were negative for HPA-1a. 913 HPA-1a-negative women gave informed consent, underwent HPA-1a antibody screening, and were included in the study. Anti-HPA-1a antibodies were detected in 85 HPA-1a-negative participants, among whom three with HPA-1a-negative fetuses and one with a previous child with FNAIT were excluded. As controls, 820 HPA-1a-negative, non-immunised pregnancies and 2704 randomly selected pregnancies of women negative for RhD and Rhc who were typed HPA-1a positive were included. Of 81 fetuses included, one (1·2%) was diagnosed with severe HPA-1a-mediated intracranial haemorrhage and three (3·7%) had mild FNAIT. Gravidity and parity did not seem to be risk factors for HPA-1a immunisation. 73 (90·1%) of 81 HPA-1a-immunised women were positive for HLA-DRB3*01:01. INTERPRETATION: Our data suggest that, without intervention, the incidence of major clinically detectable bleeding in FNAIT is estimated as 11 (95% CI 0-32) per 10 000 HPA-1a-negative pregnancies. These findings imply that severe bleeding is a rare event that potentially could be prevented by a screening programme. FUNDING: Landsteiner Foundation for Blood Transfusion Research and Sanquin.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

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/).

Measurement of post-transfusion red blood cell survival kinetics in sickle cell disease and ß-Thalassemia: A biotin label approach.

BACKGROUND: Red blood cell (RBC) transfusions are an important treatment modality for patients with sickle cell disease (SCD) and ß-thalassemia. A subgroup of these patients relies on a chronic RBC transfusion regimen. Little is known about RBC survival (RCS) of the transfused allogeneic RBCs. In this study, we aimed to study the RCS kinetics of transfused RBCs in SCD and ß-thalassemia and to investigate factors that determine RCS. METHODS AND MATERIALS: We performed a prospective cohort study on fourteen adults with SCD and ß-thalassemia disease receiving a chronic transfusion regimen. RCS and the influence of donor and patient characteristics on RCS were assessed by simultaneous transfusion of two allogeneic RBCs using RBC biotinylation. Phenotyping of well-known RBC markers over time was performed using flow cytometry. RESULTS: RCS of the two transfused RBC units was similar in most patients. Although intra-individual variation was small, inter-individual variation in RCS kinetics was observed. Most patients demonstrated a non-linear trend in RCS that was different from the observed linear RCS kinetics in healthy volunteers. After an initial slight increase in the proportion of biotinylated RBCs during the first 24 h, a rapid decrease within the first 10-12 days was followed by a slower clearance rate. CONCLUSION: These are the first data to demonstrate that patient-related factors largely determine post-transfusion RCS behavior of donor RBC in SCD and ß-thalassemia, while donor factors exert a negligible effect. Further assessment and modeling of RCS kinetics and its determinants in SCD and ß-thalassemia patients may ultimately improve transfusion therapy.

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients.

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when <20% of recruitment target was achieved. A Bayesian-adaptive individual patient data meta-analysis was implemented. Outpatients aged ≥50 years and symptomatic for ≤7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with ≤5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution. TRIAL REGISTRATION: Clinicaltrials.gov NCT04621123 and NCT04589949. REGISTRATION: NCT04621123 and NCT04589949 on https://www. CLINICALTRIALS: gov.

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.

Extended phenotyping does not preclude the occurrence of delayed haemolytic transfusion reactions in sickle cell disease.

Delayed haemolytic transfusion reaction (DHTR) is a potentially life-threatening complication of red blood cell (RBC) transfusions in sickle cell disease (SCD) and is classically induced by reactivation of previously formed antibodies. Improved antigenic matching has reduced alloimmunization and may reduce DHTR risk. We conducted a retrospective cohort study to investigate the incidence rate of DHTR in SCD patients receiving extended matched units (ABO/RhDCcEe/K/Fya /Jkb /S). Occasional transfusion episodes (OTE) between 2011 and 2020 were reviewed for occurrence of DHTR symptoms using four screening criteria: decreased Hb, increased lactate dehydrogenase (LDH), pain, and dark urine. We included 205 patients who received a cumulative number of 580 transfusion episodes of 1866 RBC units. During follow-up, 10 DHTR events were observed. The incidence rate of DHTR was 13·8/1000 OTEs [95% confidence interval (CI): 7·37-22·2], with a cumulative incidence of 15·2% (95% CI: 8·4-24·0%) after 25 patients having received RBC units. One DHTR event was fatal (10%). Symptoms were misdiagnosed in four DHTR events (40%) as other acute SCD complications. Despite a lower incidence rate compared to most other studies, the incidence rate of DHTR in SCD remains high, in spite of extended matching of donor RBCs. Increased awareness of DHTR is of utmost importance to facilitate early diagnosis and, consequently, improve outcome.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

OBJECTIVES: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. METHODS: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. RESULTS: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. CONCLUSION: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.

Genetic determinants of ferritin, haemoglobin levels and haemoglobin trajectories: results from Donor InSight.

BACKGROUND AND OBJECTIVES: Blood donors might develop iron deficiency as approximately 250 mg of iron is lost with every donation. Susceptibility to iron deficiency and low haemoglobin levels differs between individuals, which might be due to genetic variation. Therefore, the aim of this study was to investigate associations between single nucleotide polymorphisms (SNPs) and haemoglobin trajectories, haemoglobin levels and ferritin levels in blood donors. MATERIALS AND METHODS: In 2655 donors participating in the observational cohort study Donor InSight-III (2015-2017), haemoglobin and ferritin levels were measured in venous EDTA whole blood and plasma samples, respectively. Haemoglobin trajectories (stable/declining) were determined by fitting growth-mixture models on repeated pre-donation capillary haemoglobin measurements. Genotyping was done using the UK Biobank - version 2 Axiom Array. Single SNP analyses adopting an additive genetic model on imputed genetic variants were performed for haemoglobin trajectories, haemoglobin levels and ferritin levels. Conditional analyses identified independent SNPs. RESULTS: Twelve, twenty and twenty-four independent SNPs were associated with haemoglobin trajectories, haemoglobin levels and ferritin levels respectively (P < 1 x 10-5 ). Rs112016443 reached genome-wide significance for ferritin levels, which influences WDSUB1 expression. CONCLUSION: Rs112016443 was genome-wide significantly associated with ferritin levels in Dutch donors. Further validation studies are needed, as well as studies towards underlying mechanisms and predicting iron deficiency using SNPs.

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

Dietary intake of heme iron is associated with ferritin and hemoglobin levels in Dutch blood donors: results from Donor InSight.

Whole blood donors, especially frequently donating donors, have a risk of iron deficiency and low hemoglobin levels, which may affect their health and eligibility to donate. Lifestyle behaviors, such as dietary iron intake and physical activity, may influence iron stores and thereby hemoglobin levels. We aimed to investigate whether dietary iron intake and questionnaire-based moderate-to-vigorous physical activity were associated with hemoglobin levels, and whether ferritin levels mediated these associations. In Donor InSight-III, a Dutch cohort study of blood and plasma donors, data on heme and non-heme iron intake (mg/day), moderate-to-vigorous physical activity (10 minutes/day), hemoglobin levels (mmol/L) and ferritin levels (µg/L) were available in 2,323 donors (1,074 male). Donors with higher heme iron intakes (regression coefficients (ß) in men and women: 0.160 and 0.065 mmol/L higher hemoglobin per 1 mg of heme iron, respectively) and lower non-heme iron intakes (ß: -0.014 and -0.017, respectively) had higher hemoglobin levels, adjusted for relevant confounders. Ferritin levels mediated these associations (indirect effect (95% confidence interval) in men and women respectively: 0.074 (0.045; 0.111) and 0.061 (0.030; 0.096) for heme and -0.003 (-0.008;0.001) and -0.008 (-0.013;-0.003) for non-heme). Moderate-to-vigorous physical activity was negatively associated with hemoglobin levels in men only (ß: -0.005), but not mediated by ferritin levels. In conclusion, higher heme and lower non-heme iron intake were associated with higher hemoglobin levels in donors, via higher ferritin levels. This indicates that donors with high heme iron intake may be more capable of maintaining iron stores to recover hemoglobin levels after blood donation.

Identification of genetic biomarkers for alloimmunization in sickle cell disease.

Most sickle cell disease (SCD) patients rely on blood transfusion as their main treatment strategy. However, frequent blood transfusion poses the risk of alloimmunization. On average, 30% of SCD patients will alloimmunize while other patient groups form antibodies less frequently. Identification of genetic markers may help to predict which patients are at risk to form alloantibodies. The aim of this study was to evaluate whether genetic variations in the Toll-like receptor pathway or in genes previously associated with antibody-mediated conditions are associated with red blood cell (RBC) alloimmunization in a cohort of SCD patients. In this case-control study, cases had a documented history of alloimmunization while controls had received ≥20 RBC units without alloantibody formation. We used a customized single nucleotide polymorphism (SNP) panel to genotype 690 SNPs in 275 (130 controls, 145 cases) patients. Frequencies were compared using multiple logistic regression analysis. In our primary analysis, no SNPs were found to be significantly associated with alloimmunization after correction for multiple testing. However, in a secondary analysis with a less stringent threshold for significance we found 19 moderately associated SNPs. Among others, SNPs in TLR1/TANK and MALT1 were associated with a higher alloimmunization risk, while SNPs in STAM/IFNAR1 and STAT4 conferred a lower alloimmunization risk.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology: Report of the Dubai, Copenhagen and Toronto meetings.

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.

Performance evaluation study of ID RHD XT, a new genotyping assay for the detection of high-prevalence RhD negative and weak D types.

BACKGROUND AND OBJECTIVES: Routine serologic D typing does not distinguish between weak D subtypes and partial D phenotypes. The goal of this study was to validate the performance of the ID RHD XT genotyping assay. MATERIAL AND METHODS: Previously serotyped samples for D antigen (n = 1000; 16% weak D serotyped donors) were analysed. The reference methods used for comparison were licensed serology tests for D antigen phenotype, and bidirectional sequencing (BDS) for weak D type confirmation and HPA-1 phenotype prediction. Discrepancies were solved with BDS and BLOODchip® Reference. RESULTS: There were no system failure, a 100% call rate and no inconclusive results. ID RHD XT correctly called all (88/88) weak D types 1, 2 and 3. Review of other 87 apparent discrepancies identified a small number of serology errors and showed that ID RHD XT correctly signalled the presence of other RHD variants which were further confirmed by BDS and BLOODchip® Reference. The predicted HPA-1 phenotype by ID RHD XT was 100% concordant with BDS. CONCLUSION: ID RHD XT genotype predictions for high-prevalence RhD negative and weak D types 1, 2 and 3 as well as for HPA-1a/HPA-1b antigens were accurate, which is of clinical significance in guiding transfusion needs.

Performance evaluation study of ID CORE XT, a high throughput blood group genotyping platform.

BACKGROUND: Traditionally, red blood cell antigens have been identified using serological methods, but recent advances in molecular biology have made the implementation of methods for genetic testing of most blood group antigens possible. The goal of this study was to validate the performance of the ID CORE XT blood group typing assay. MATERIALS AND METHODS: One thousand independent samples from donors, patients and neonates were collected from three research institutes in Spain and the Netherlands. DNA was extracted from EDTA-anticoagulated blood. The data were processed with the ID CORE XT to obtain the genotypes and the predicted blood group phenotypes, and results were compared to those obtained with well-established serological and molecular methods. All 1,000 samples were typed for major blood group antigens (C, c, E, e, K) and 371-830 samples were typed for other antigens depending on the rarity and availability of serology comparators. RESULTS: The incorrect call rate was 0%. Four "no calls" (rate: 0.014%) were resolved after repetition. The sensitivity of ID CORE XT for all phenotypes was 100% regarding serology. There was one discrepancy in E- antigen and 33 discrepancies in Fyb- antigen. After bidirectional sequencing, all discrepancies were resolved in favour of ID CORE XT (100% specificity). ID CORE XT detected infrequent antigens of Caucasians in the sample as well as rare allelic variants. DISCUSSION: In this evaluation performed in an extensive sample following the European Directive, the ID CORE XT blood genotyping assay performed as a reliable and accurate method for correctly predicting the genotype and phenotype of clinically relevant blood group antigens.