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Post-transfusion purpura is a life-threatening adverse reaction associated with a reduction in platelet counts below 10 x 109/L and bleeding. It usually occurs in women who, due to their pregnancies, have developed antibodies directed against human platelet antigen (HPA), most often anti-HPA 1a. The case described by us concerned an 83-year-old woman who received two units of red blood cells (RBC) due to severe secondary anemia. Seven days after the transfusion, she developed skin symptoms of thrombocytopenic diathesis, gastrointestinal bleeding, and a reduced number of platelets (3 x 109/L), and the patient’s serum (genotype HPA-1b/1b) was positive for anti-platelet antibodies with HPA-1a specificity and directed against human leukocyte antigen (HLA) class I and class II antibodies, unresponsive to the lymphocytotoxic test. Prompt diagnosis by our transfusion medicine consultant and the initiation of treatment with intravenous immunoglobulins certainly shortened the duration of thrombocytopenia and bleeding, and possibly also saved the patient’s life.

Intracranial hemorrhage (ICH) associated with fetal/neonatal alloimmune thrombocytopenia (FNAIT) is attributed mainly to endothelial damage caused by binding of maternal anti-HPA-1a antibodies to the αvβ3 integrin on endothelial cells (ECs). We examined the effect of anti-HPA-1a antibodies on EC function using 2 EC lines from different vascular beds, HMVEC of dermal origin and hCMEC/D3 of cerebral origin. Anti-HPA-1a sera significantly increased apoptosis in both HMVEC and hCMEC/D3 cells and permeability in hCMEC/D3 cells only. This increase in both apoptosis and permeability was significantly inhibited by a monoclonal anti-β3 antibody (SZ21) binding to the HPA-1a epitope. Our results indicate that (1) maternal anti-HPA-1a antibodies impair EC function by increasing apoptosis and permeability and (2) ECs from different vascular beds vary in their susceptibility to pathological effects elicited by maternal anti-HPA-1a antibodies on EC permeability. Examination of maternal anti-HPA-1a antibodies for their effect on EC permeability may predict potential ICH associated with FNAIT.

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans. Disclosures Skogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.

International audience; : Abstract Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

Thrombocytopenia is detected in around one percent of newborns. In otherwise healthy term newborns, thrombocytopenia is most often caused by alloantibodies transferred from the mother to the foetus. In the Caucasian populations human platelet antigen (HPA)-1a is the most immunogenic HPA. In Japan and China antibodies in the HPA-4 system are the most frequent cause of FNAIT. The immune response against the HPA must be understood in order to avoid immunization or prevent induction of FNAIT where mothers are already immunized.

Post-transfusion purpura (PTP) is a rare transfusion reaction almost exclusively observed in female patients. Affected patients develop severe immune-mediated thrombocytopenia in the course of a strong anamnestic alloimmune reaction against a platelet-specific antigen. The pathophysiology of thrombocytopenia has remained elusive. Immunological analysis in the HPA-1a-alloimmunized patient described in this report revealed an antibody with features considered typical of PTP: not only was anti-HPA-1a detectable in plasma, but it could also be eluted from the patients' (alloantigen negative) platelets, and anti-HPA-1a could be detected in eluates from both antigen positive and negative test platelets, which had been incubated in the patient's serum. This is in contrast to two sera with HPA-1a alloantibodies obtained from mothers of children with neonatal alloimmmune thrombocytopenia which were strictly HPA-1a specific. It is proposed that alloantibodies with HPA-1a-like specificity explain the patient's immune thrombocytopenia. The technique described in this report is proposed for further investigation, as it might be useful for discrimination of alloantibodes in PTP and alloantibodies of transfused thrombocytopenic patients.

In red cell immunology, it has long been known that no one technique will detect all clinically significant antibodies. The same appears to be true for platelet immunology, and we highlight this fact by showing four examples of anti-human platelet antigen-1a that were not detected by the monoclonal antibody-specific immobilization of platelet antigen test, the most commonly used technique. Each antibody was found in a case of fetomaternal alloimmune thrombocytopenia in which the fetus or neonate was severely affected.

Results from a study of more than 100 000 pregnant women show that the pathophysiology of neonatal alloimmune thrombocytopenia (NAIT) and haemolytic disease of the newborn (HDN) has many equalities [1]. It has been reported that NAIT is more than three times more frequent compared with HDN. This makes it tempting to consider a design for screening for HPA la negativity and follow up in/after the pregnancy based on the same principles as for RhD-negative pregnant women. Even without available vaccination, it is strongly indicated that screening and Caesarean section 2-4 weeks prior to term in mothers with the HPA-1a-negative platelet type, reduce neonatal morbidity and mortality due to NAIT.

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti-HPA-1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.

Introduction In a screening setting, maternal anti-HPA 1a antibody level has been found to be a good prognostic tool to identify newborns at risk for severe NAIT. Aim Identify the optimal MAIPA protocol for quantitation of anti-HPA 1a antibodies. Materials and methods Plasma were analysed for anti-HPA 1a antibodies using different monoclonal antibodies, lyophilized or fresh platelets and MAIPA protocols. Results The anti-HPA 1a antibody level varied significantly when different monoclonal antibodies were used. However, there was a strong correlation between maternal anti-HPA 1a antibody level and platelet count in the newborn. The sensitivity of the assay depended on the adopted MAIPA protocol. Conclusion Consistent tests results are of importance for the clinical impact of the test.

The issue of whether or not antibody quantity during pregnancy is related to severity of neonatal alloimmune thrombocytopenia remains unresolved. In this article we cite studies in support of both sides of the argument and highlight some of the reasons that may lie behind the observed differences amongst those studies. It may well be that some of the reasons for the discrepant results could be due to the type of study carried out (eg retrospective versus prospective), the sample size, the timing of antibody sampling, and possibly the type or protocol of assay used. Another major reason is the absence, until recently, of an international anti-HPA-1a standard.

A large screening and intervention study, aimed at reducing morbidity and mortality associated with severe anti-HPA 1a antibody induced neonatal alloimmune thrombocytopenia (NAIT), has recently been carried out in Norway.[1][1] Recently, it was shown that the anti-HPA 1a levels surprisingly

Background Around 80% of severe neonatal alloimmune thrombocytopenia (NAIT) cases in the Caucasian population are due to anti-HPA-1a antibodies. However, the relationship between anti-HPA-1a antibody quantity and severity of NAIT has been subject to debate, possibly due to discrepant results between studies incorporating different assays (such as ELISA and MAIPA) and the number of samples. The aim of this study was to compare the level of maternal anti-HPA-1a antibodies in the same samples, using two different methods; quantitative ELISA and quantitative MAIPA. At the same time, the relationship between the maternal anti-HPA-1a antibody quantity and the newborn platelet count was examined. Materials and methods Plasma samples were obtained from HPA-1a negative mothers giving birth to children with different platelet counts (i.e. NAIT vs normal platelet count). The antibody levels were quantified blindly in the respective assays using standards calibrated against the international standard NIBSC 03/152. Results A linear correlation was observed between quantitative MAIPA and quantitative ELISA results. However, there were many individual variations giving a mean ratio of disagreement between the quantitative ELISA and quantitative MAIPA of 2.84. Furthermore, regression analysis revealed a significant relation between anti-HPA-1a antibody quantity measured by MAIPA, but not ELISA, and the newborn platelet count. Conclusion The results indicate that differences observed between various studies with regard to NAIT severity and anti-HPA-1a quantity could partly be due to different assays used.

Background and Objectives This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. Materials and Methods Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. Results An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. Conclusions Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).

Eleven thousand one hundred pregnant women were genotyped for human platelet antigen HPA 1, and 198 HPA 1bb women were followed in the pregnancy with quantitative assay for anti-HPA 1a antibodies. Antibodies were detected in 24 women, and nine children were born with severe thrombocytopenia (< 50×109/L). All mothers with high levels of antibodies were delivered of children with severe thrombocytopenia. None of the newborn infants had clinical signs of intra-cranial haemorrhage. The level of maternal anti-HPA 1a antibodies is predictive for fetal thrombocytopenia and may be used in decisions related to time and mode of delivery.

Most cases of neonatal alloimmune thrombocytopenia (NAIT) are diagnosed after birth and infants with severe NAIT remain at risk for intracranial haemorrhage [1]. In suspected severe NAIT cases, treatment must be instituted prior to confirmation of the diagnosis. HPA-1a alloimmunisation is the most common cause of NAIT. The most effective forms of treatment are provision of compatible antigen-negative platelets or provision of maternal platelets [2].