Platelet Antigens Research Articles

Informing pregnancy dose via target-mediated drug disposition modeling and simulations for a recombinant human monoclonal antibody.

RLYB212 is a human monoclonal anti-human platelet antigen (HPA)-1a immunoglobulin gamma 1 in clinical development as a subcutaneous injection for the prevention of maternal alloimmunization to fetal HPA-1a leading to fetal and neonatal alloimmune thrombocytopenia (FNAIT). This analysis developed a target-mediated drug disposition (TMDD) model to simultaneously characterize RLYB212 pharmacokinetics (PK) and HPA-1a-positive platelet dynamics in HPA-1b/b (HPA-1a-negative) volunteers. The model was then used to perform simulations to inform a dosing regimen in a phase II clinical study in pregnant women, where simulations accounted for physiological changes throughout pregnancy. Allometric scaling (0.75) for clearance and intercompartment transfer rate and volume (1) was included in the base model to account for variations in body weight. A 0.06 mg RLYB212 dose with a loading dose of 0.12 mg was identified as the optimal dosing regimen of RLYB212, which maintained exposures below the target upper boundary of ~10 ng/mL throughout pregnancy. This work presents an application of the TMDD model that advances the quantitative clinical pharmacology toolkit to understand monoclonal antibody PK in pregnancy.

Association of HPA Antigens with Immune Thrombocytopenia: A Case-Control Study by PCR-SSP Method

Background: Human platelet antigens (HPAs) play a clinically significant role in alloimmunization and the development of immune-mediated disorders such as immune thrombocytopenia (ITP), fetal and neonatal alloimmune thrombocytopenia (FNAIT), and post-transfusion purpura (PTP). Understanding the genetic profiles of HPAs is critical for preventing and treating these conditions. Given the limitations of serological methods in determining HPA genotypes, this study aims to investigate the association between the genotypes of HPA1, HPA2, HPA3, HPA4, and HPA15 antigens and autoimmune thrombocytopenia in Lorestan Province, utilizing the PCR-SSP method. Materials and Methods: This case-control study involved 80 individuals diagnosed with ITP and 120 healthy controls. DNA samples were extracted using a commercial DNA extraction kit, with concentrations quantified via a Nanodrop spectrophotometer. Genotyping was performed using the PCR-SSP method with specific primers for each HPA gene. The genotype data were verified using previously established sample sets. The frequencies of each HPA genotype were recorded, and a comparative analysis was conducted between the patient and control groups to evaluate the study hypothesis. Results: The results revealed that individuals carrying the HPA2b allele had a 5.31-fold increased risk of developing ITP, a statistically significant finding (P < 0.05, OR = 5.31). Similarly, the presence of the HPA15b allele was associated with a 6.54-fold increased risk (P < 0.05, OR = 6.54). Conclusion: These findings, in conjunction with previous studies, suggest the need for larger-scale investigations across different populations. Such research could aid in the early diagnosis and prediction of thrombocytopenia severity, inform treatment strategies, and facilitate the removal of pathogenic antibodies from circulation.

Two Concurrent Cases of NAIT in Pregnant Sisters

Abstract Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal antibodies directed against fetal platelet antigens inherited from the father. Approximately 80% of cases of NAIT are attributed to antibodies against human platelet antigen (HPA)-1a and less commonly to antibodies against HPA-5b. We present two cases of possible NAIT attributed to anti-HPA-5b that were identified in sisters requiring concurrent management. Patient #1 is a 32-year-old G9P4044 woman with one pregnancy complicated by intrauterine growth retardation and partial placental abruption, another pregnancy complicated by fetal anomaly (intracerebral hemorrhage) status post termination of pregnancy at 23 weeks’ gestation and three miscarriages (one with chromosomal abnormality). The patient is homozygous for HPA-1a and HPA-5a, and the partner is homozygous for HPA-5b. HPA-5b antibodies and HLA antibodies were identified in the patient’s serum. Deemed at high risk for subsequent NAIT, she was started on intravenous immunoglobulin twice weekly and steroids during her second trimester until delivery. The patient underwent weekly fetal sonograms during pregnancy which were normal. The patient’s platelet counts were stable. The patient refused caesarean delivery. At 38 weeks’ gestation, the patient gave birth to a healthy female via normal spontaneous vaginal delivery. HPA-5b negative platelets were procured from our blood supplier, but neither the mother nor neonate required transfusion. The neonate showed no signs of bleeding and had normal platelet counts. Ultrasound imaging of the head was unremarkable. The patient and neonate were discharged home. Patient #2 is a 33-year-old G13P6156 woman with a prior pregnancy complicated by stillbirth at 32 weeks’ gestation. Considering her personal and family history, HPA testing was performed and HPA-5b antibodies were identified in the patient’s serum. The patient’s partner is heterozygous for HPA-5b. Crossmatched platelets were procured from our blood supplier. The patient refused caesarean delivery. The patient gave birth at 37 weeks’ gestation to a healthy male via normal spontaneous vaginal delivery. The fetus had mild thrombocytopenia (107 x 103/uL), with no bleeding or bruising identified. Neither the mother nor neonate required transfusion. The patient and neonate were discharged home. Ultrasound imaging of the head was unremarkable, and his thrombocytopenia resolved. While some studies postulate that anti-HPA-5b identification may be coincidental rather contributory to the disease process, these cases re-emphasize the importance of obtaining pregnancy, neonatal and family history to best risk stratify patients and determine management from both the clinical and transfusion medicine perspectives. If transfusion is needed, transfusion options include HPA negative platelets, crossmatched platelets and random donor platelets depending on clinical urgency and platelet availability.

A novel approach to simultaneous genotyping of human platelet antigen systems and human leucocyte antigen class I loci using PacBio long-read sequencing.

Accurate human leucocyte antigen (HLA) and human platelet antigen (HPA) typing is essential for establishing a blood platelet donor bank to deal with refractoriness in patients undergoing multiple platelet transfusions. Current methods, such as Sanger and next-generation sequencing, encounter difficulties in haplotyping. Herein, the aim of this study was to establish a method for HLA and HPA typing based on the long read sequencing. The HPA and HLA class I genotypes of 268 platelet donors from the Taiyuan Blood Center, China were identified using long-read sequencing on the PacBio platform. Allele frequencies for HPA systems and HLA class I genes were calculated, and genetic variability within HPA system genes was analysed. Polymorphisms were identified in 8 of the 35 HPA systems (HPA-1 to HPA-6w, HPA-15 and HPA-21w), with the frequencies of the 'b' allele at 0.0187, 0.0709, 0.4086, 0.0075, 0.0149, 0.0317, 0.4310 and 0.0019, respectively. The alleles with the highest frequencies at the HLA-A, HLA-B and HLA-C loci are HLA-A02:01, B51:01, B46:01 and C06:02, respectively. Additionally, several genetic patterns in HPA systems were identified, including the c.166-1029C>T variant, which was found exclusively in samples carrying the HPA-1b allele. This study developed a targeted long-read sequencing method characterized by high throughput and simultaneity, capable of resolving allele ambiguities for effective HLA class I genotyping in establishing a platelet donor bank.

The effects of complement-independent, autoantibody-induced apoptosis of platelets in immune thrombocytopenia (ITP).

Autoantibodies that cause platelet apoptosis may play a role in the development of immune thrombocytopenia (ITP), specifically antibodies that target GPIIbIIIa and GPIbα. Our research aims to compare the impact of the antigen specificity of antiplatelet antibodies on normal platelets under conditions that do not rely on complement. Using a modified monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay, we detected the levels of autoantibodies against specific platelet membrane glycoproteins (GPIIb/IIIa, GPIb/IX) in the plasma of 36 patients diagnosed with chronic ITP. IgG was isolated and purified using a protein A agarose affinity chromatography column, and their concentrations were measured using spectrophotometry. We obtained normal platelets and treated them with the purified IgG anti-GPIIb/IIIa and/or anti-GPIb/IX antibodies, as well as an IgG-free buffer and healthy control IgG. Flow cytometry was used to analyze markers of apoptosis, including phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), and platelet particle formation. Our results indicate that ITP patients with GPIb/IX-specific autoantibodies can induce platelet apoptosis and platelet particle formation through complement-independent pathways, which are not associated with platelet activation, while GPIIb/IIIa-specific autoantibodies did not have this effect. This suggests that specific autoantibodies may serve as a valuable predictive tool to identify patients who could potentially benefit from complement-inhibiting therapy in the future.

A Phase 1b PK/PD Study to Demonstrate Antigen Elimination with RLYB212, a Monoclonal Anti-HPA-1a Antibody for FNAIT Prevention.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare bleeding disorder of the fetus/newborn caused by development of maternal alloantibodies against fetal human platelet antigens (HPAs), predominantly HPA-1a. Currently there are no treatments available to prevent maternal alloimmunization to HPAs or FNAIT. This proof-of-concept study (EudraCT Number: 2021-005380-49) was designed to assess the ability of subcutaneous (SC) RLYB212, a monoclonal anti-HPA-1a antibody, to eliminate HPA-1a-positive platelets in an antigen challenge model of a 30 mL fetal-maternal hemorrhage. Subjects were randomized to receive a single SC dose of RLYB212 or placebo on day 1 in a single-blinded manner, followed by transfusion of 10 × 109 HPA-1a-positive platelets on day 8. Four subjects received 0.09 mg SC RLYB212, five received 0.29 mg SC RLYB212, and two received placebo. RLYB212 achieved rapid elimination of HPA-1a-positive platelets in a concentration-dependent manner, with concentrations as low as 3.57 ng/mL meeting the prespecified proof-of-concept criterion of ≥90% reduction in platelet elimination half-life versus placebo. Following HPA-1a-positive platelet transfusion, a rapid decline was observed in the concentration of RLYB212 over a period of 2 to 24 hours, corresponding to the time needed for RLYB212 to bind to ∼10% of HPA-1a on cell surfaces. RLYB212 was well tolerated with no reports of drug-related adverse events. The data from this study are consistent with preclinical efficacy data and support the potential use of RLYB212 as a prophylactic treatment for FNAIT that prevents maternal HPA-1a alloimmunization during at-risk pregnancies.

Platelet antibody detection assays: a single-laboratory comparison of MAIPA, PIFT, and microsphere-based multiplex assays Pak-Lx

Background and ObjectivesThe identification of platelet antibodies is essential for diagnosing and managing conditions such as fetal and neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and immune platelet refractoriness. Monoclonal antibody immobilization of platelet antigens (MAIPA) is the standard method for detecting anti-human platelet antigen (HPA) antibodies, while the detection of anti-HLA antibodies once relied on the complement-dependent cytotoxicity method, however advanced technologies such as enzyme-linked immunosorbent assay and Luminex have significantly improved sensitivity and accuracy in identifying these antibodies. Flow cytometry-based techniques (platelet immunofluorescence test - PIFT) and Luminex platform-driven microsphere-based multiplex assays (Pak-Lx) are widely employed in platelet immunology laboratories owing to their remarkable flexibility and versatility. The present study compared the sensitivity, specificity, and concordance of these different serological techniques used in platelet antibody identification. Material and MethodsOne hundred serum samples from patients suspected of immune-mediated platelet disorders were examined. Initially, the samples underwent testing using the MAIPA method. Subsequently, the results were compared with three alternative methods: PIFT and microsphere-based multiplex assays for both HLA and HPA antibodies. ResultsPak-Lx demonstrated a 94 % agreement with MAIPA, while PIFT had 88 % agreement for HPA antibodies. For HLA antibody detection, Pak-Lx versus DLX had 75 % concordance, MAIPA versus DLX showed 77 %, and PIFT versus DLX displayed an 81 % concordance rate. Remarkably, there were no significant differences in concordance levels between Pak-Lx and PIFT compared to MAIPA and DLX for anti-HPA and HLA antibodies, respectively. ConclusionThis study found no significant differences in concordance among the tested assays for detecting anti-HPA and anti-HLA antibodies. These data suggest that no single method can detect all clinically important antibodies. Therefore, it is advisable that each laboratory develops customized protocols based on their expertise and employs complementary methods for comprehensive patient assessments.

A Novel Ultrasound-Based Radiomics Model for the Preoperative Prediction of Lymph Node Metastasis in Cervical Cancer

ObjectiveThe purpose of this retrospective study was to establish a combined model based on ultrasound (US)-radiomics and clinical factors to predict preoperative lymph node metastasis (LNM) in cervical cancer (CC) patients non-invasively. MethodsA total of 131 CC patients who had cervical lesions found by transvaginal sonography (TVS) from the First Affiliated Hospital of Anhui Medical University (Hefei, China) were retrospectively analyzed. The clinical independent predictors were selected using univariate and multivariate logistic regression analysis. US-radiomics features were extracted from US images; after selecting the most significant features by univariate analysis, Spearman's correlation analysis, and the least absolute shrinkage and selection operator (LASSO) algorithm; four machine-learning classification algorithms were used to build the US-radiomics model. Fivefold cross-validation was then used to test the performance of the model and compare the ability of the clinical, US-radiomics and combined models to predict LNM in CC patients. ResultsRed blood cell, platelet and squamous cell carcinoma-associated antigen were independent clinical predictors of LNM (+) in CC patients. eXtreme Gradient Boosting performed the best among the four machine-learning classification algorithms. Fivefold cross-validation confirmed that eXtreme Gradient Boosting indeed performs the best, with average area under the curve values in the training and validation sets of 0.897 and 0.898. In the three prediction models, both the US-radiomics model and the combined model showed good predictive efficacy, with average area under the curve values in the training and validation sets of 0.897, 0.898 and 0.912, 0.905, respectively. ConclusionUS-radiomics features combined with clinical factors can preoperatively predict LNM in CC patients non-invasively.

A rare case of hemolytic disease-associated thrombocytopenia mimicking alloimmune thrombocytopenia: Salvaging a precious baby

Abstract: Thrombocytopenia associated with hemolytic disease of fetus and newborn (HDFN) is usually mild-to-moderate severity. Neonatal alloimmune thrombocytopenia (NAIT) is one of the leading causes of severe thrombocytopenia in neonates. We describe a rare case of very severe thrombocytopenia (5 g/L) with bleeding manifestations associated with HDFN mimicking NAIT. The baby was a preterm female, born to a 34-year-old multiparous mother with a bad obstetric history and multiple alloantibodies (anti-D, anti-C, anti-E, and anti-Jka). She was managed with phototherapy, exchange transfusion with phenotype-matched packed red blood cells, random donor platelets and intravenous immunoglobulin as platelet crossmatching could not provide compatible platelets. NAIT was ruled out with human platelet antigen (HPA) genotyping of the baby, the mother, and the father. It was concluded that the very severe thrombocytopenia might be due to HDFN after ruling out the other causes. HDFN can rarely cause very severe thrombocytopenia. However, when a newborn presents with severe/very severe thrombocytopenia, NAIT is to be ruled out with HPA genotyping, even in resource-limited settings, to plan out subsequent pregnancies.

In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Plain Language SummaryThe identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies.

Analysis of immunogenetics interlaboratory comparisons' success rates. External quality assurance system of the Spanish Society for Immunology GECLID-SEI.

For many years, transplantation outcomes were uncertain and not hopeful, until histocompatibility testing spread. Common criteria for histocompatibility assays and communications' improvement allowed an efficient organ sharing system. The possibility of organ exchanges is closely linked to the importance of interlaboratory comparisons for histocompatibility and immunogenetics methods. The external proficiency testing (EPT) systems are the most powerful quality assurance tools. They help achieve harmonization of analyses, set a standard of performance, and a common interpretation. The external quality assurance program for diagnostic immunology laboratories (Garantía Externa de Calidad para Laboratorios de Inmunología Diagnóstica, GECLID) program nowadays runs 13 external quality assurance (EQA) histocompatibility and immunogenetics schemes, with the first of them from 2011 to date: serological and molecular: low- and high-resolution human leukocyte antigen (HLA), human platelet antigen (HPA), and killer inhibitory receptor (KIR) typing(HLA-B*27, HLA-B*57:01, and coeliac disease-related HLA), cell-dependent cytotoxicity (CDC) and flow cytometry (FC) crossmatches, anti-HLA and anti-HPA antibodies, and chimerism. A total of 85 laboratories participated in this subprogram in the last 12years reporting over 1.69M results: 1.46M for anti-HLA and anti-HPA antibodies, 203.810 molecular typing data (HLA, HPA, and KIR genes), 2.372 for chimerism analyses, and 39.352 for crossmatches. Based on the European Federation for Immunogenetics (EFI) standards for EPT providers, the mean success rates ranged from 99.2% for molecular typing schemes and antibodies and 94.8% for chimerism, was 96.7% regarding crossmatches, and was 98.9% in serological typing. In 2022, 61.3% of the participating laboratories successfully passed every HLA EQA scheme, although 87.9% annual reports were satisfactory. Most penalties were due to nomenclature errors or misreporting of the risk associated to HLA and disease. This EQA confirms the reliability of HLA and immunogenetics assays in routine care. There is little heterogeneity of results of different assays used by participating laboratories, even when in-house assays are used. Reliability of test results is reasonably granted.

Should HLA and HPA-matched platelet transfusions for patients with Glanzmann Thrombasthenia or Bernard-Soulier syndrome be standardized care? A Dutch survey and recommendations.

Glanzmann thrombasthenia (GT) and Bernard-Soulier syndrome (BSS) patients require frequent platelet transfusions and hence have an increased risk for alloimmunization against donor Human Leukocyte Antigens (HLA) when no HLA-matching is performed. Knowing that Human Platelet Antigens (HPA) are located on the platelet glycoproteins that can be absent in these patients, preventive HPA-matching may also be considered. Uniform recommendations on this topic lack in transfusion guidelines making standard practice unclear, therefore, we aimed to provide a framework for matched platelet transfusions. We conducted a targeted literature search and a national survey of Dutch (pediatric) hematologists from July to September 2021. We found 20 articles describing platelet transfusion policies in 483 GT-patients and 29 BSS-patients, both adults and children. Twenty surveys were returned for full analysis. All responders treated patients with platelet disorders, including GT (n = 36 reported) and BSS (n = 29 reported). Of respondents, 75% estimated the risk of antibody formation as "likely" for HLA and 65% for HPA. Formation of HLA antibodies was reported in 5 GT and in 5 BSS-patients, including one child. Fifteen respondents gave preventive HLA-matched platelets in elective setting (75%). Three respondents additionally matched for HPA in GT-patients (15%). Main argument for matched platelet transfusions was preventing alloimmunization to safeguard the effectivity of 'random' donor-platelets in acute settings. Elective HLA-matching for GT and BSS-patients is already conducted by most Dutch (pediatric) hematologists. HPA-matching is mainly applied when HPA-antibodies are formed. Based on the current literature and the survey, recommendations are proposed.

Place of HPA1a Antigen in Neonatal Thrombocytopenia in Gyneco-Obstetric and Pediatric Hospital of Douala and Laquintinie Hospital of Douala

Background: Platelet antigens called human platelet antigens (HPA) present on the surface of platelets are involved in immunological conflicts, sometimes leading to severe thrombocytopenia related to anti-HPA1a antibodies. The aim of this study was to detect platelet antibodies in newborns and the associated risk factors. Methods: We conducted a cross-sectional analytical study from 05 January to 30 June 2017 at the Laquintinie Hospital of Douala and the Gyneco-Obstetric and Pediatric Hospital of Douala among newborns with thrombocytopenia and mothers in compliance with ethical considerations. Socio-demographic and clinical data were collected. Blood was collected on EDTA tubes for newborns and on tubes without anticoagulants for mothers in order to determine the presence of anti-HPA1a antibodies. Statistical analysis was performed using SPSS version 22 software. Results: A total of 35 newborns and 35 mothers were recruited in this study. The mean age of the newborns was 8.5 ± 7.2 days, with a sex ratio of 1.7% in favor of boys. The prevalence of neonatal thrombocytopenia was 4.1%, and the prevalence of HPA1a antibody was 17.1%. Most of the newborns were born to primiparous mothers (57.1%), and 80.0% had prematurity and neonatal jaundice as reasons for hospitalization. Male newborns and those whose mothers had been transfused at least once during or before pregnancy had respectively 4 (OR = 3.53; P-value < 0.0001) and 14 (OR = 14; P-value = 0.0483) times more risk of having the anti-HPA1a antibody. Conclusion: Our study has shown that the anti-HPA1a antibody is a risk factor for neonatal thrombocytopenia and is associated with maternal transfusion.

Platelets retain function and can be stored following disruption of human leucocyte antigens.

Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

PLT antigen discrepancy pattern among couples with recurrent abortion.

Recurrent abortion refers to a condition of two or more consecutive pregnancies without known etiology affected by miscarriage before the completion of the 20th week of gestational age. However, several hypotheses have been proposed, but not much data are available concerning the relationship between human platelet antigens (HPAs) polymorphisms and recurrent abortion. This study was conducted to evaluate the genetic differences between HPA-1, -2, -3, -5, and - 15 in Iranian couples with a history of recurrent abortion. In this cross-sectional study, a total of 74 couples with at least 2 recurrent abortions without any known specified reasons enrolled in the study. HPA polymorphisms genotyping was performed by single-specific primer PCR. Genotype frequency was calculated using the Hardy-Weinberg equation. A total of 39 couples (52.7%) had HPA genotyping partial mismatches. The most common partial mismatch pairs were found concomitantly on both HPA-15a and HPA-15b in three couples (4%), followed by two (2.7%) on HPA-3a and one (1.3%) in each HPA-2b and HPA-5b. There was a deviation from the Hardy-Weinberg equilibrium in the HPA-2 and -5 systems. The present study declared that partial mismatches of HPA-3 and -15 genotypes were common among Iranian couples due to the history of recurrent abortion and approximately half of the couples carried at least one HPA gene that was absent in their partners. Further studies might be helpful to clarify the association between HPA polymorphisms and recurrent abortion, such as an investigation into the alloantibodies against HPAs.

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.

Analysis of transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness

Objective: To analyze the transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness (PTR). Methods: A total of 94 patients with PTR received by Taiyuan Blood Center from January to December 2021 were retrospectively analyzed, including 26 males and 68 females, aged 53(34,66) years. Platelet antibody screening was performed by enzyme-linked immunosorbent assay (ELISA). For patients with positive human leukocyte antigen (HLA) class Ⅰ antibodies, Luminex platform liquid chip assay was used to identify the specificity of antibodies, and platelets with missing allelic expression antigen corresponding to their specific antibodies were found in the platelet donor gene database established in our laboratory. For patients with negative class HLA-Ⅰ antibody screening, medium and high-resolution HLA-A and B alleles were genotyped by polymerase chain reaction restriction sequence specific oligonucleotide (PCR-SSO), and the compatible platelets were searched from the platelet donor gene database by HLA cross-reactive group genotype matching scheme or directly selected by serological cross-matching. The PCI compliance rate and total transfusion effective rate of different mismatch site groups and different matching scheme groups were statistically analyzed. Results: Platelet antibody was detected in 39 of 94 PTR patients with a positive rate of 41.5%, and all of them were HLA-Ⅰ antibodies, and 1 case was accompanied by human platelet antigen (HPA) antibody. A total of 134 times of compatible platelets were supplied to 39 patients with HLA-Ⅰ antibody positive by using antibody avoidance matching method. And the total effective rate of transfusion was 97.8% (131/134); The PCI compliance rates of HLA-A antigen mismatch, HLA-B antigen mismatch and HLA-A and B antigen mismatch groups were 81.6% (31/38), 86.5% (32/37) and 78.6% (22/28), respectively. The total effective rate of transfusion was 97.4% (37/38), 94.6% (35/37) and 100% (28/28), respectively, with no statistical significance (all P>0.05). A total of 118 times of compatible platelets were provided by HLA antigen cross-reaction group genotype matching and serological cross-matching, 90 transfusion effects were collected during follow-up, and the total effective rate was 76.7% (69/90). Conclusion: The combination of different platelet matching schemes can improve the PCI compliance rate and the total effective rate of transfusion in PTR patients.

Detection of human platelets antigen polymorphism (HPA-1 and HPA-3) and human factor XIII mutation in Sudanese women with recurrent pregnancy loss

BackgroundRecurrent pregnancy Loss (RPL) is common problem affecting many couples. A certain genetic variants link to increase the danger of this condition particularly HPA-1, HPA-3 and Human Factor XIII Val34Leu Mutation. The present study aims to find an association between RPL and the Factor XIII Val34Leu polymorphism, as well as HPA-1 and HPA-3 in Sudanese women with RPL.MethodsThis case-control study conducted between June 2022 and December 2022 included 216 women, with 103 cases having minimum three abortions in the past, and 113 healthy controls with at least two full-term births and no abortion history. DNA was isolated from whole blood and the status of three genetic polymorphisms (HPA-1, HPA-3, and factor XIII) was done using a polymerase chain reaction (PCR). Data was analysed using the SPSS version 24 software.ResultsThe A/A genotype was found to be more prevalent in cases (79.6%) and controls (96.5%) regarding HPA-1. A significant difference was observed in overall allele frequency for B allele (97.0%) and expected frequency of A allele was (81.1%) using the Hardy-Weinberg distribution (p < 0.001). The genotype A/A was most common in these patients (90.3%) and controls (100%), while B/B genotype was only (9.7%) in patients regarding HPA-3. Furthermore, the frequency of Val/Val genotype was higher in cases (88.3%) as compared with controls (90.3%). The risk of RPL in patients was nearly the same in Val/Leu individuals and controls group but all these differences were not statistically significant (p > 0.05).ConclusionOur results indicate a link between Human Platelet Antigen-1 (HPA-1), Human Platelet Antigen-3 (HPA-3) and Factor XIII gene polymorphism with RPL.

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

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. 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. 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. 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.

Evidence that CD36 is expressed on red blood cells and constitutes a novel blood group system of clinical importance.

Polymorphic molecules expressed on the surface of certain blood cells are traditionally categorized as blood groups and human platelet or neutrophil antigens. CD36 is widely considered a platelet antigen (Naka) and anti-CD36 can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) in CD36-negative pregnant women. CD36 is used as a marker of differentiation in early erythroid culture. During the experimental culture of CD34+ cells from random blood donors, we observed that one individual lacked CD36. We sought to investigate this observation further and determine if CD36 fulfils the International Society of Blood Transfusion criteria for becoming a blood group. Surface markers were monitored by flow cytometry on developing cells during the erythroid culture of CD34+ cells. Genetic and flow cytometric analyses on peripheral blood cells were performed. Proteomic datasets were analysed, and clinical case reports involving anti-CD36 and foetal anaemia were scrutinized. Sequencing of CD36-cDNA identified homozygosity for c.1133G>T/p.Gly378Val in the CD36-negative donor. The minor allele frequency of rs146027667:T is 0.1% globally and results in abolished CD36 expression. CD36 has been considered absent from mature red blood cells (RBCs); however, we detected CD36 expression on RBCs and reticulocytes from 20 blood donors. By mining reticulocyte and RBC datasets, we found evidence for CD36-derived peptides enriched in the membrane fractions. Finally, our literature review revealed severe cases of foetal anaemia attributed to anti-CD36. Based on these findings, we conclude that CD36 fulfils the criteria for becoming a new blood group system and that anti-CD36 is implicated not only in FNAIT but also foetal anaemia.