A machine-learning method for biobank-scale genetic prediction of blood group antigens.

A key element for successful blood transfusion is compatibility of the patient and donor red blood cell (RBC) antigens. Precise antigen matching reduces the risk for immunization and other adverse transfusion outcomes. RBC antigens are encoded by specific genes, which allows developing computational methods for determining antigens from genomic data. We describe here a classification method for determining RBC antigens from genotyping array data. Random forest models for 39 RBC antigens in 14 blood group systems and for human platelet antigen (HPA)-1 were trained and tested using genotype and RBC antigen and HPA-1 typing data available for 1,192 blood donors in the Finnish Blood Service Biobank. The algorithm and models were further evaluated using a validation cohort of 111,667 Danish blood donors. In the Finnish test data set, the median (interquartile range [IQR]) balanced accuracy for 39 models was 99.9 (98.9-100)%. We were able to replicate 34 out of 39 Finnish models in the Danish cohort and the median (IQR) balanced accuracy for classifications was 97.1 (90.1-99.4)%. When applying models trained with the Danish cohort, the median (IQR) balanced accuracy for the 40 Danish models in the Danish test data set was 99.3 (95.1-99.8)%. The RBC antigen and HPA-1 prediction models demonstrated high overall accuracies suitable for probabilistic determination of blood groups and HPA-1 at biobank-scale. Furthermore, population-specific training cohort increased the accuracies of the models. This stand-alone and freely available method is applicable for research and screening for antigen-negative blood donors.

Severe neonatal thrombocytopenia due to anti-HPA-4b alloantibodies: Third report described in a Caucasian mother.

BACKGROUND: Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) results from maternal platelet alloimmunization against paternal antigens inherited by the fetus, most often due to the Human Platelet Antigen (HPA)-1 system in Caucasians. We investigated in 2023, a 30-year-old Caucasian woman Gravida 2 Para 1 who gave birth at 35 weeks of gestation to a male (body weight 2210 g) without signs of bleeding. A severe thrombocytopenia (platelet count at 3 G/L) was discovered incidentally a few hours after delivery in the context of the management of a respiratory distress. The newborn recovered after one platelet concentrate transfusion and normalized his platelet count at Day 5. STUDY DESIGN AND METHODS: FNAIT investigation was performed according to guideline recommendations. Platelet genotyping was carried out by multiplex PCR. Maternal serological investigation included Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA) and Luminex technology. RESULTS: Parental and newborn genotyping pointed out an HPA-4 incompatibility between the mother and the newborn and the father. Serological investigation revealed an anti-HPA-4b alloantibody confirming the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: We described the third case of anti-HPA-4b alloantibody discovered in a Caucasian mother. This case strengthens the need for reference laboratory to genotype a panel of HPA alleles reflecting local genetic population diversity and for crossmatch of maternal serum with fresh paternal platelets in clinical suspected cases of neonatal alloimmune thrombocytopenia.

Genetic landscape of human platelet antigen variants in the Indian population analysed from 1029 whole genomes.

Genetic variants in human platelet antigens (HPAs) considered allo- or auto antigens are associated with various disorders, including neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and post-transfusion purpura. Although global differences in genotype frequencies were observed, the distributions of HPA variants in the Indian population are largely unknown. This study aims to explore the landscape of HPA variants in India to provide a basis for risk assessment and management of related complications. Population-specific frequencies of genetic variants associated with the 35 classes of HPAs (HPA-1 to HPA-35) were estimated by systematically analysing genomic variations of 1029 healthy Indian individuals as well as from global population genome datasets. Allele frequencies of the most clinically relevant HPA systems in the Indian population were found as follows, HPA-1a - 0.884, HPA-1b - 0.117, HPA-2a - 0.941, HPA-2b - 0.059, HPA-3a - 0.653, HPA-3b - 0.347, HPA-4a - 0.999, HPA-4b - 0.0010, HPA-5a - 0.923, HPA-5b - 0.077, HPA-6a - 0.998, HPA-6b - 0.002, HPA-15a - 0.582 and HPA-15b - 0.418. This study provides the first comprehensive analysis of HPA allele and genotype frequencies using large scale representative whole genome sequencing data of the Indian population.

[Identification of a rare platelet-specific antigen HPA-10bw allele among ethnic Han Chinese population in Shandong].

OBJECTIVE: To study the polymorphism of human platelet antigen (HPA) system 10 among ethnic Han Chinese from Shandong, China so as to supplement the data of platelet donor bank in the region. METHODS: Peripheral blood samples of platelet donors from the region were genotyped for HPA-10 alleles by PCR-sequence specific primer (PCR-SSP) and direct sequencing. RESULTS: Among 1401 donors, a rare heterozygote carrier of HPA-10w (a+b+) was identified, which gave an allelic frequency of approximately 0.035%. CONCLUSION: The detection of rare HPA-10bw antigen allele among ethnic Han Chinese from Shandong is useful for the diagnosis and prevention of neonatal alloimmune thrombocytopenia and post-transfusion purpura in the region.

Bioengineered iPSC-derived megakaryocytes for the detection of platelet-specific patient alloantibodies.

Human platelet membrane glycoprotein polymorphisms can be immunogenic in man and are frequently the cause of clinically important immune reactions responsible for disorders such as neonatal alloimmune thrombocytopenia. Platelets from individuals carrying rare polymorphisms are often difficult to obtain, making diagnostic testing and transfusion of matched platelets challenging. In addition, class I HLA antibodies frequently present in maternal sera interfere with the detection of platelet-reactive alloantibodies. Detection of alloantibodies to human platelet antigen 3 (HPA-3) and HPA-9 is especially challenging, in part because of the presence of cell type-specific glycans situated near the polymorphic amino acid that together form the alloepitope. To overcome these limitations, we generated a series of HLA class I-negative blood group O induced pluripotent stem cell (iPSC) lines that were gene edited to sequentially convert their endogenous HPA-3a alloantigenic epitope to HPA-3b, and HPA-9a to HPA-9b. Subjecting these cell lines, upon differentiation into CD41+/CD42b+ human megakaryocytes (MKs), to flow cytometric detection of suspected anti-HPA-3 and HPA-9 alloantisera revealed that the HPA-3a-positive MKs specifically reacted with HPA-3a patient sera, whereas the HPA-3b MKs lost reactivity with HPA-3a patient sera while acquiring reactivity to HPA-3b patient sera. Importantly, HPA-9b-expressing MKs specifically reacted with anti-HPA-9b-suspected patient samples that had been undetectable using conventional techniques. The provision of specialized iPSC-derived human MKs expressing intact homozygous glycoprotein alloantigens on the cell surface that carry the appropriate endogenous carbohydrate moieties should greatly enhance detection of clinically important and rare HPA-specific alloantibodies that, to date, have resisted detection using current methods.

New developments in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia, the platelet equivalent of hemolytic disease of the fetus and newborn, can have devastating effects on both the fetus and neonate. Current management of fetal and neonatal alloimmune thrombocytopenia in a subsequent affected pregnancy involves antenatal administration of intravenous immune globulin and prednisone to the pregnant woman to prevent the development of severe fetal thrombocytopenia and secondary intracranial hemorrhage in utero. That therapy has proven to be highly effective but is associated with maternal side effects and is expensive. This commentary describes 4 advances that could substantially change the current approach to detecting and managing fetal and neonatal alloimmune thrombocytopenia in the near future. The first would be an introduction of a program to screen all antepartum patients in this country for pregnancies at risk of developing fetal and neonatal alloimmune thrombocytopenia. Strategies to implement this complex process have been described. A second advance is testing of cell-free fetal DNA obtained from maternal blood to noninvasively determine the fetal human platelet antigen 1 genotype. A third, in preliminary development, is creation of a prophylactic product that would be the platelet equivalent of Rh immune globulin (RhoGAM). Finally, a fourth major potential advance is the development of neonatal Fc receptor inhibitors to replace the current medical therapy administered to pregnant women with an affected fetus. Neonatal Fc receptor recycles plasma immunoglobulin G to increase its half-life and is the means by which immunoglobulin G crosses the placenta from the maternal to the fetal circulation. Blocking the neonatal Fc receptor is an ideal way to prevent maternal immunoglobulin G antibody from causing fetal and neonatal alloimmune thrombocytopenia in a fetus at risk of developing that disorder. The pertinent pathophysiology and rationale for each of these developments will be presented in addition to our thoughts relating to steps that must be taken and difficulties that each approach would face for them to be successfully implemented.

Association of human platelet alloantigens encoding gene polymorphisms with the risk of Coronary artery disease in Iranian patients.

BACKGROUND: Coronary artery disease (CAD) is characterized by narrowing/ blockade of coronary arteries that is mainly caused by atherosclerotic plaques. Considering the involvement of platelet abnormalities, such as defective aggregation and adhesion, in the cardiovascular-related disorders, genetic variations in human platelet alloantigens (HPA) have been implicated in the CAD susceptibility. Herein, we intended to determine the association of HPA-1 to -6, -9, and -15 biallelic polymorphisms with CAD in an Iranian population. METHODS: In this retrospective case-control study, 200 CAD subjects and 100 matched healthy individuals were enrolled. DNA samples were isolated from peripheral blood samples and genotyping of HPA polymorphisms was accomplished using polymerase chain reaction-sequence-specific primers. RESULTS: The alleles and genotypes of studied HPA polymorphisms were equally distributed among cases and controls and therefore no statistically significant differences were detected. Univariate analysis identified no association of combined haplotypes with CAD risk. However, multivariate analysis showed a positive association of the| HPA1b/2a/3b haplotype with CAD after adjustment for some covariates (including BMI, TG, LDL, FBS and blood pressure) that conferred a CAD susceptibility haplotype (P = 0.015; OR = 2.792; 95% CI 1.45-8.59). CONCLUSIONS: Although alleles, genotypes, and haplotypes of HPA polymorphisms were not associated with CAD risk, HPA1b/2a/3b haplotype was found to be a dependent disease risk haplotype in Iranian population after correcting for confounding factors.

A new efficient tool for non-invasive diagnosis of fetomaternal platelet antigen incompatibility.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the consequence of platelet destruction by maternal alloantibodies against fetal human platelet antigens (HPA). This may result in intracranial haemorrhages (ICH) or even fetal death. Currently, fetal HPA genotyping is performed using invasive procedures. Here, we carried out a proof-of-concept study for non-invasive prenatal diagnosis of fetal platelet genotyping in four HPA systems (HPA-1, -3, -5 and-15) by droplet digital polymerase chain reaction (ddPCR) using cell-free DNA extracts from the plasma of 47 pregnant women with suspected, or history of, FNAIT. Results showed that 74% (35/47) of pregnant women presented incompatibility in at least one HPA system, and 38% (18/47) of cases presented HPA-1 incompatibility, including nine women with multiple incompatibilities. ICH occurred in one case of profound fetal thrombocytopenia with HPA-15 incompatibility, confirming the need for non-invasive prenatal genotyping in systems other than HPA-1. Fetal HPA genotypes predicted by ddPCR were confirmed in all FNAIT cases after amniocentesis or delivery. Fetal HPA genotyping on maternal plasma based on ddPCR is a fast, safe and reliable non-invasive method. This technique will be useful for the early identification of pregnancies at high risk of FNAIT requiring antenatal management to minimize the risk of fetal/neonatal haemorrhage.

The IPD Project: a centralised resource for the study of polymorphism in genes of the immune system.

The Immuno Polymorphism Database (IPD), https://www.ebi.ac.uk/ipd/, is a set of specialist databases that enable the study of polymorphic genes which function as part of the vertebrate immune system. The major focus is on the hyperpolymorphic major histocompatibility complex (MHC) genes and the killer-cell immunoglobulin-like receptor (KIR) genes, by providing the official repository and primary source of sequence data. Databases are centred around humans as well as animals important for food security, for companionship and as disease models. The IPD project works with specialist groups or nomenclature committees who provide and manually curate individual sections before they are submitted for online publication. To reflect the recent advance of allele sequencing technologies and the increasing demands of novel tools for the analysis of genomic variation, the IPD project is undergoing a progressive redesign and reorganisation. In this review, recent updates and future developments are discussed, with a focus on the core concepts to better future-proof the project.

Human platelet antigens polymorphisms; association to the development of liver fibrosis in patients with chronic hepatitis C.

Recently, human platelet antigens (HPAs) polymorphisms are found to play a role in susceptibility to hepatitis C virus (HCV) infection and fibrosis progression. The aim of the current study was to evaluate the possible association between the HPAs polymorphisms with liver fibrosis progression in HCV patients. HPAs polymorphisms genotyping was performed in HCV patients (n = 71) by Sequence-specific primers-polymerase chain reaction. Fibrosis progression was evaluated using the Metavir scoring system and liver biopsy, and the patients were assigned to two groups, namely, G1 (n = 35) that included patients with F1 (portal fibrosis without septa) or F2 (few septa) and G2 (n = 36) that comprised patients with F3 (numerous septa) or F4 (cirrhosis). The data analyses were performed using Pearson's χ2 test. The genotype frequency of HPA-3ab was significantly higher in G1 patients than in G2 patients (P = 0.015). No statistically significant differences were found between the patient groups (G1 and G2) regarding the distributions of the allelic and genotypic frequencies of the HPA-1, -2, -4, -5, and -15 systems. Multivariate logistic regression showed an independent association between the genotype HPA-3aa/BB and severe fibrosis (F3-F4), when compared with genotype HPA-3ab, independent of the viral genotype, high alanine transaminase, sex, age, time of infection, diabetes, and high cholesterol as risk factors. The present study suggested that the HPA-3ab genotype could be noticed as a potential protecting factor against hepatic fibrosis. Therefore, the antigenic variation of integrins might be considered as a part of the coordinated inflammatory process involved in the progression of liver fibrosis.

Targeted exome sequencing designed for blood group, platelet, and neutrophil antigen investigations: Proof-of-principle study for a customized single-test system.

BACKGROUND: Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad-range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen-associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. STUDY DESIGN AND METHODS: An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. RESULTS: Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA-2. Optimal throughput was 12 to 14 samples per run. CONCLUSION: This single-test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.

Frequency of allotype "b" in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high-resolution melt analysis.

BACKGROUND: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibody-mediated thrombocytopenia. STUDY DESIGN AND METHODS: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to high-resolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. RESULTS: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. CONCLUSION: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

A multi-centre study on the performance of the molecular genotyping platform ID RHD XT for resolving serological weak RhD phenotype in routine clinical practice.

BACKGROUND AND OBJECTIVES: There is concern regarding the lack of prevention of unnecessary transfusion of RhD negative red cells and unnecessary administration of Rh immunoglobulin (RhIG) to pregnant women. In this study, performance of ID RHD XT, a genotyping assay for identification of six RHD allelic variants and human platelet antigens HPA-1a/1b was assessed. MATERIALS AND METHODS: Whole blood samples presenting weak, discrepant or inconclusive D phenotype results were genotyped with ID RHD XT and compared to reference molecular tests. Candidacy for RhIG prophylaxis was determined by analysing samples from pregnant women. Hands-on time to complete the procedures was measured. RESULTS: Overall, 167 samples were tested (55 donors, 56 patients, 52 pregnant women and four newborns). Agreement between ID RHD XT and the reference method was 100% (51% weak D type 1, 2 or 3; 35·5% weak D Types 1, 2 or 3 not detected; 4% RHD deletion; 1% RHD*Pseudogene; 1% RHD*DIIIa-CE(3-7)-D; and 4% no amplification variant detected for RHD genotype; and 64% HPA-1a/a; 30% HPA-1a/b; and 3% HPA-1b/b for HPA-1 genotype). Call rate was 98·2%. ID RHD XT identified 40% of the pregnant women that would not have required RhIG prophylaxis. Overall hands-on time was 25-45 min to process a batch of 24 samples, and four hours for total assay time. CONCLUSION: ID RHD XT yielded reproducible results for RHD typing in serologically weak D phenotype individuals. ID RHD XT was proven useful for the correct management of patients with RhD serological discrepancies and the rational use of RhIG in pregnancy.

Human platelet antigen polymorphisms and the risk of chronic Chagas disease cardiomyopathy.

Human platelet antigen (HPA) polymorphisms are considered to be a risk factor for cardiac and vascular diseases, but the role of HPA in chronic Chagas disease cardiomyopathy (CCC) is not available. Therefore, the aim of this study was to investigate the association of HPA polymorphisms, HPA-1, HPA-2, HPA-3, HPA-5 and HPA-15, in the severity of left ventricular systolic dysfunction (LVSD) in CCC patients. For this, 229 CCC patients were separated into three groups: without LVSD, mild/moderate LVSD and severe LVSD. PCR-SSP was performed for HPA genotyping and the risk was assessed using SNPStats software. HPA-1 allele and genotype frequencies were lower in mild/moderate LVSD patients compared to other groups, without statistical significance. After stratified analyzes, the HPA-3a/3b genotype frequency was lower in women with severe LVSD compared to those without LVSD (OR:0.29; 95% CI: 0.10-0.84). In conclusion, HPA-3 variant could be a protection factor for CCC in the female patients.

Role of platelet gene polymorphisms in ischemic pediatric stroke subtypes: a case-control study.

AIM: To assess the role of human platelet antigens (HPA), P-selectin gene (SELP) polymorphisms, and HPA and SELP haplotypes with factor V (FV) R506Q in ischemic pediatric stroke (IPS) subtypes: cerebral sinovenous thrombosis (CSVT), perinatal (PAIS), and childhood (CAIS) arterial ischemic stroke. METHODS: This case-control study enrolled 150 children with confirmed IPS and 150 age- and sex-matched controls. FV R506Q and HPA-1 were genotyped with CVD StripAssay®, HPA-2 and HPA-3 with real-time polymerase chain reaction, SELP S290N, V599L, and T715P with high resolution melting analysis, and SELP N562D with sequence-specific polymerase chain reaction. RESULTS: HPA-1b allele (odds ratio [OR] 2.75, 95% confidence interval [CI] 1.02-7.42, P=0.048) and HPA-1a2a3b (OR 5.46, 95% CI 1.51-19.76, P=0.011), HPA-1b2a3a (OR 7.00, 95% CI 1.25-39.13, P=0.028), and HPA-1b2b3a (OR 11.39, 95% CI 1.39-92.95, P=0.024) haplotypes increased the risk for CSVT. HPA-3b allele was significantly associated with 2-fold lower risk for PAIS (OR 0.49, 95% CI 0.26-0.89, P=0.020) and CAIS (OR 0.47, 95% CI 0.26-0.86, P=0.014) and non-significantly associated with increased risk for CSVT (OR 6.43, 95% CI 0.83-50.00, P=0.022). HPA-1a2b3a haplotype was significantly associated with CAIS (OR 6.76, 95% CI 2.13-21.44, P=0.001). The inclusion of FV R506Q in SELP haplotype analysis increased the risk for PAIS 4-fold in QNDVT carriers (OR 8.14, 95% CI 0.93-71.33, P=0.060) compared with NDVT haplotype (OR 2.45, 95% CI 0.98-6.18, P=0.058), but the result was not significant. CONCLUSION: Individual HPAs, and particularly HPA haplotypes, are involved in IPS subtypes pathogenesis. A possible risk-inducing synergistic effect of SELP haplotypes with FV R506Q is restricted to PAIS only.

The human platelet antigen-1b (Pro33) variant of αIIbß3 allosterically shifts the dynamic conformational equilibrium of this integrin toward the active state.

Integrins are heterodimeric cell-adhesion receptors comprising α and ß subunits that transmit signals allosterically in both directions across the membrane by binding to intra- and extracellular components. The human platelet antigen-1 (HPA-1) polymorphism in αIIbß3 arises from a Leu → Pro exchange at residue 33 in the genu of the ß3 subunit, resulting in Leu33 (HPA-1a) or Pro33 (HPA-1b) isoforms. Although clinical investigations have provided conflicting results, some studies have suggested that Pro33 platelets exhibit increased thrombogenicity. Under flow-dynamic conditions, the Pro33 variant displays prothrombotic properties, characterized by increased platelet adhesion, aggregate/thrombus formation, and outside-in signaling. However, the molecular events underlying this prothrombotic phenotype have remained elusive. As residue 33 is located >80 Å away from extracellular binding sites or transmembrane domains, we hypothesized that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbß3 toward an active state. Multiple microsecond-long, all-atom molecular dynamics simulations of the ectodomain of the Leu33 and Pro33 isoforms provided evidence that the Leu → Pro exchange weakens interdomain interactions at the genu and alters the structural dynamics of the integrin to a more unbent and splayed state. Using FRET analysis of fluorescent proteins fused with αIIbß3 in transfected HEK293 cells, we found that the Pro33 variant in its resting state displays a lower energy transfer than the Leu33 isoform. This finding indicated a larger spatial separation of the cytoplasmic tails in the Pro33 variant. Together, our results indicate that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbß3 to a structural state closer to the active one, promoting the fully active state and fostering the prothrombotic phenotype of Pro33 platelets.

Automated typing of red blood cell and platelet antigens from whole exome sequences.

BACKGROUND: Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES). STUDY DESIGN AND METHODS: Whole exome sequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples. RESULTS: Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fyb and one N serologic typing errors and one undetected SNP encoding a Jknull phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCR-restriction fragment length polymorphism for all samples. CONCLUSION: The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts.

Blood group genotyping of blood donors: validation of a highly accurate routine method.

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.

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. STUDY DESIGN AND METHODS: The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. RESULTS: Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. CONCLUSION: This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.