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Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14 ++ CD16 - ), intermediate (CD14 ++ CD16 + ), and nonclassical (CD14 + CD16 ++ ) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14 + CD16 - ), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin-semaphoring-integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ames, Baal, Speckmann, Michel, Ratke, Klesser, Cooper, Takahashi, Bayat, Bein and Santoso.)

Most cases of fetal and neonatal thrombocytopenia (FNAIT) are caused by maternal anti-human platelet antigen-1a antibodies (anti-HPA-1a). Anti-HPA-5b antibodies are the second most common antibodies in suspected FNAIT cases. Given the high prevalence of anti-HPA-5b antibodies in pregnant women delivering healthy newborns, the association with FNAIT may be coincidental. This review of the literature related to FNAIT using the MEDLINE database was conducted according to PRISMA guidelines. A retrospective analysis of a single-centre cohort of 817 suspected FNAIT cases was conducted. The pooled prevalence of anti-HPA-5b antibodies in unselected pregnant women of European descent was 1.96% (n = 3113), compared with 3.4% (n = 5003) in women with suspected FNAIT. We found weak evidence that a small proportion of pregnant women presenting with anti-HPA-5b antibodies will give birth to a newborn with mild thrombocytopenia. The neonatal platelet counts were not different between suspected FNAIT cases (n = 817) with and without maternal anti-HPA-5b antibodies. The prevalence of maternal anti-HPA-5b antibodies was not different between neonates with intracranial haemorrhage and healthy controls. The current experimental and epidemiological evidence does not support the hypothesis that anti-HPA-5b antibodies cause severe thrombocytopenia or bleeding complications in the fetus or newborn., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Recent studies have shown that maternal anti-CD36 antibodies represent a frequent cause of fetal/neonatal alloimmune thrombocytopenia (FNAIT) in Asian and African populations. However, little is known about the pathomechanism and antenatal treatment of anti-CD36-mediated FNAIT. Here, we established a novel animal model to examine the clinical features of pups from immunized Cd36-/- female mice after breeding with wild-type male mice. Mild thrombocytopenia was observed, but high pup mortality was also documented (40.26%). Administration of intravenous immunoglobulin (IVIG) (1 g/kg) on days 7, 12, and 17 to immunized Cd36-/- mothers after breeding reduced fetal death (12.70%). However, delaying the IVIG administration series on days 10, 15, and 20 did not reduce fetal death (40.00%). In contrast, injection of deglycosylated anti-CD36 (deg-anti-CD36) polyclonal antibodies (5 mg/kg) on days 10, 15, and 20 significantly reduced fetal death (5.26%). Subsequently, monoclonal antibodies (mAbs) against mouse CD36 were developed, and one clone producing high-affinity anti-CD36 (termed 32-106) effectively inhibited maternal antibody binding and was therefore selected. Using the same approach of deg-anti-CD36, the administration of deg-32-106 significantly reduced fetal death (2.17%). Furthermore, immunized Cd36-/- mothers exhibited placental deficiency. Accordingly, maternal anti-CD36 antibodies inhibited angiogenesis of placenta endothelial cells, which could be restored by deg-32-106. In summary, maternal anti-CD36 antibodies caused a high frequency of fetal death in our animal model, associated with placental dysfunction. This deleterious effect could be diminished by the antenatal administration of IVIG and deg-mAb 32-106. Interestingly, treatment with deg-32-106 seems more beneficial considering the lower dose, later start of treatment, and therapy success., (© 2021 by The American Society of Hematology.)

Objectives: In the present study, we asked whether anti-CD36 antibodies impair the maturation of erythropoietic stem cells to mature red blood cells (RBCs), leading to anaemia and hydrops fetalis (HF)., Background: Recent studies have shown the importance of anti-CD36 antibodies in the development of Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT). In comparison to other types of antibody-mediated FNAIT, anti-CD36 antibodies are frequently associated with anaemia and HF. As mature RBCs do not express CD36, the reason for this phenomenon is currently not fully understood., Material and Methods: A case of FNAIT with signs of HF was characterised in this study. Maternal anti-CD36 antibodies were isolated by an absorption/elution approach. We cultured haematopoietic stem cells (HSCs) with purified anti-CD36 antibodies, and the formation of burst-forming unit-erythroid and colony-forming unit-erythroid (CFU-E/BFU-E) cells was analysed. Apoptosis of HSCs was also investigated., Results: Analysis of the mother showed type-1 CD36 deficiency. Anti-CD36 antibodies were found in maternal serum, as well as on fetal platelets, by ELISA, and the specificity of these antibodies was further substantiated by flow cytometry. In comparison to control IgG, incubation of HSCs with purified anti-CD36 antibodies led to a significant reduction in CFU-E/BFU-E cell formation, and this result was associated with an increased number of apoptotic CD34+ erythroid/myeloid precursor cells. Administration of intra-uterine transfusion with washed RBCs was effective in improving fetal anaemia., Conclusions: Anti-CD36 antibodies may cause anaemia and trigger HF through apoptosis of CD34+ erythroid/myeloid precursor cells. However, the contribution of other cells must also be taken into account., (© 2020 British Blood Transfusion Society.)

The causative role of maternal, anti-human leukocyte antigen (anti-HLA) class I antibodies in foetal and neonatal alloimmune thrombocytopenia (FNAIT) remains controversial. Furthermore, in FNAIT cases caused by anti-human platelet antigen-1a (anti-HPA-1a) antibodies, the possible additive effect of maternal anti-HLA class I antibodies on outcomes is unclear. Among 817 mother-father-neonate trios of suspected FNAIT, we assessed the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, and the incidence of FNAIT caused by anti-HPA-1a antibodies. In 144 FNAIT cases caused by anti-HPA-1a antibodies, we investigated the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, birth weight, and the incidence of intracranial haemorrhage (n = 16). Maternal anti-HLA class I antibodies were not associated with neonatal platelet count in suspected cases of FNAIT. There was no significant interaction between the presence of anti-HLA class I antibodies and anti-HPA-1a antibodies. In FNAIT cases caused by anti-HPA-1a antibodies, there was no association between the presence of anti-HLA class I antibodies and neonatal platelet count, birth weight, or occurrence of intracranial haemorrhage. This study's findings do not support the concept that maternal anti-HLA class I antibodies represent a risk factor of FNAIT or disease severity., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background:  Fetal/neonatal alloimmune thrombocytopenia (FNAIT) results from maternal alloantibodies (abs) reacting with fetal platelets expressing paternal human platelet antigens (HPAs), mostly HPA-1a. Anti-HPA-1a abs, are the most frequent cause of severe thrombocytopenia and intracranial hemorrhage (ICH)., Objectives:  Titration of anti-HPA-1a in maternal serum using standard National Institute for Biological Standards and Control (NIBSC) 03/152 is one diagnostic approach to predict the severity of FNAIT. Recently, we found three anti-HPA-1a subtypes reacting with the β3 subunit independently or dependently from complexes with αIIb and αv. Endothelial cell-reactive anti-αvβ3 abs were found predominantly in cases with ICH. Our aim was to assess whether available standard material represents all anti-HPA-1a subtypes., Materials and Methods:  In this study, anti-HPA-1a sera (NIBSC 03/152) and human monoclonal antibodies (moabs) against HPA-1a (moabs 26.4 and 813) were evaluated using transfected cell lines expressing αIIbβ3, αvβ3 or monomeric cβ3., Results:  Flow cytometry analyses with well-characterized murine moabs recognizing αIIbβ3, αvβ3, or β3 alone demonstrated that AP3 reacts compound-independently, whereas compound-dependent moabs Gi5 and 23C6 reacted only with complexes. NIBSC 03/152, moabs 26.4, and 813 against HPA-1a reacted like AP3, same results were obtained with monomeric cβ3 in immunoblotting. Antigen capture assay targeting endothelial cells showed anti-HPA-1a reactivity disappearance after cβ3 beads adsorption. Furthermore, in contrast to anti-HPA-1a abs from ICH cases, none of NIBSC 03/152, 26.4, and 813 inhibited tube formation., Conclusion:  These results suggest that current anti-HPA-1a standard material contains only the anti-β3 subtype. The absence of anti-αvβ3 makes NIBSC 03/152 less suitable as standard to predict the severity of FNAIT., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Cases of CD36 deficiency are not rare in Asian populations, foetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by anti-CD36 isoantibodies appears more frequent than other HPA alloantibodies. However, little is known about the treatment of anti-CD36 mediated FNAIT in this region. A Chinese male foetus, whose mother had a history of multiple intrauterine foetal demise and/or hydrops, was diagnosed with severe FNAIT at 27 weeks of gestational age. Immunological analysis revealed total absence of CD36 on platelets and monocytes from mother, caused by a 329-330delAC mutation of the CD36 gene. Anti-CD36 and anti-HLA class I antibodies were detected in the maternal serum, whereas only anti-CD36 isoantibodies were detectable in the foetal blood sample. Serial intrauterine transfusions with red blood cells (RBC) and platelets from a CD36null donor were performed to improve the severe anaemia and thrombocytopenia. The baby (2250 g; Apgar scores 10) was delivered vaginally at 32 weeks of gestation with normal haemoglobin (186 g/L) but low platelet count (48 × 10 9 /L). After 2 days the platelet count rose to 121 × 10 9 /L. This report suggests that intrauterine transfusions with compatible RBC and CD36null platelets are useful in preventing the deleterious clinical effects of anti-CD36-mediated severe FNAIT.

Background: Isoantibodies against CD36 (platelet glycoprotein 4), developed in Type I CD36-deficient mothers are frequently reported as the cause of fetal/neonatal alloimmune thrombocytopenia in the Asian population. Therefore, further detailed characterization of anti-CD36-mediated fetal/neonatal alloimmune thrombocytopenia is warranted. Here, we report the characterization of a patient with fetal/neonatal alloimmune thrombocytopenia in a Taiwanese family caused by anti-CD36 isoantibodies using a novel antigen-capture method., Study Design and Methods: Platelets and monocytes were analyzed for CD36 expression by flow cytometry. Sequencing analysis of the CD36 gene was performed to identify the mutation underlying the CD36 deficiency. Stable transfected human embryonic kidney HEK293 cells expressing recombinant CD36 were established. These cells were used for the characterization of anti-CD36 isoantibodies by flow cytometry, immunoprecipitation, and antigen-capture assay., Results: Flow cytometry analysis revealed a total absence of CD36 on both platelets and monocytes of the mother (Type I CD36-deficient) caused by heterozygous deletions of the CD36 gene (332&#95;333delCA and c.1254 + 6&#95;1254 + 11delTATTTG). Analysis of maternal serum with CD36-transfected HEK293 cells by flow cytometry, immunoprecipitation, and antigen-capture assay demonstrated the presence of anti-CD36 isoantibodies in maternal serum. Interestingly, this antibody could not be detected by the monoclonal antibody immobilization of platelet antigens assay when anti-CD36 monoclonal antibody (clone FA6-152) was used as the capture antibody., Conclusion: This case reemphasizes the role of anti-CD36 isoantibodies on the pathomechanism of fetal/neonatal alloimmune thrombocytopenia. The fact that the monoclonal antibody immobilization of platelet antigens assay does not seem to be reliable for the identification of all anti-CD36 antibodies indicates that screening of anti-CD36 isoantibodies by a monoclonal antibody-independent method, as presented here, should be considered., (© 2017 AABB.)

Background: Most cases of fetal and neonatal alloimmune thrombocytopenia (FNAIT) are caused by maternal alloantibodies against human platelet antigen-1a (HPA-1a). Alloimmunization mainly occurs in HPA-1a-negative mothers who are carriers of the HLA-DRB3*01:01 allele. Recently, it has been reported that the combined presence of HLA-DRB3*01:01 and HLA-DRB4*01:01P was associated with severity of FNAIT. We tested this hypothesis by analyzing a large cohort of cases and controls., Study Design and Methods: In total, 101 mothers with a history of FNAIT caused by anti-HPA-1a were investigated. HLA-DRB1, -DRB3, -DRB4, and -DRB5 genotypes were determined by Luminex technology. Haplotype frequencies were compared between cases and 100 controls. The platelet (PLT) counts of neonates and the incidence of intracranial hemorrhage (ICH) were compared between subgroups defined by genotype., Results: Of the HPA-1a-immunized mothers, 98% (99/101) carried at least one copy of HLA-DRB3*01:01. Carriage of HLA-DRB3*01:01 was significantly associated with immune response to HPA-1a (odds ratio, 92.3; 95% confidence interval, 26.9-317.1; p = 1.34 × 10 -12 ). No association between HLA-DRB3*01:01 and HLA-DRB4*01:01P alone or in combination with the PLT count of the newborns or the incidence of ICH was detected., Conclusion: In contrast to HLA-DRB4*01:01P, the inheritance of HLA-DRB3*01:01 is strongly associated with the propensity for mounting a humoral immune response against fetal HPA-1a antigen. Neither a homozygous nor a compound heterozygous gene dose predicts the severity of the disease. Testing for the presence of HLA-DRB3*01:01 might be very useful in counseling women at risk of FNAIT due to anti-HPA-1a., (© 2016 AABB.)

Objective: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups., Approach and Results: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvβ3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvβ3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvβ3 subtype, but not the anti-β3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvβ3 subtype, but not the anti-β3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation., Conclusions: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvβ3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia., (© 2016 American Heart Association, Inc.)

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets (PLTs) in the fetus or newborn by maternal PLT antibodies that crossed the placenta during pregnancy., Study Design and Methods: In this study, we aim to elucidate the properties of a new PLT alloantigen (Lap(a)) that is associated with a severe case of FNAIT. Analysis of maternal serum with phenotyped PLTs by monoclonal antibody-specific immobilization of platelet antigens showed positive reaction against PLT glycoprotein (GP)IIb/IIIa and HLA Class I expressed on paternal PLTs., Results: In contrast to GPIIIa-reactive anti-HPA-1a, anti-Lap(a) alloantibodies precipitated predominantly GPIIb. Indeed, a point mutation G>C at Position 2511 located in Exon 25 of the ITGA2B gene was found in Lap(a)-positive donors. This mutation causes an amino exchange Gln>His at Position 806 located in the calf-2 domain of GPIIb. Lap(a)-positive individuals were not found in 300 random blood donors. Our expression study showed that anti-Lap(a) alloantibodies reacted with stable transfected HEK293 cells expressing the mutated GPIIb isoform (His806). CHO cells carrying this isoform, however, failed to react with anti-Lap(a) alloantibodies, indicating that Lap(a) epitopes depend on the Gln806 His mutation and the carbohydrate composition of the GPIIb. This mutation did not hamper the binding of anti-HPA-3a, which recognizes a point mutation (Ile843 Ser) located in calf-2 domain. Finally, we found that Lap(a) and some HPA-3a epitopes are sensitive to O-glycanase., Conclusions: This study not only underlines the relevance of rare HPAs on the pathomechanism of FNAIT, but also helps to understand the pitfalls of serologic assays to detect anti-GPIIb alloantibodies., (© 2015 AABB.)

Background: Fetal human platelet antigen (HPA) genotyping is required to determine whether the fetus is at risk and whether prenatal interventions to prevent fetal bleeding are required in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Methods for noninvasive genotyping of HPA alleles with the use of maternal plasma cell-free DNA were published recently but do lack internal controls to exclude false-negative results., Study Design and Methods: Cell-free DNA was isolated from plasma of four pregnant women with a history of FNAIT caused by anti-HPA-1a and controls. A primer panel was designed to target sequences flanking single-nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA-1), ITGA2B (HPA-3), ITGA2 (HPA-5), CD109 (HPA-15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and eight anonymous SNPs were massively parallel sequenced by semiconductor technology., Results: The mean (±SD) number of reads for targeted SNPs was 5255 (±2838). Fetal DNA was detected at a median of 4.5 (range, 2-8) polymorphic loci. The mean fractional fetal DNA concentration in cell-free maternal plasma was 8.36% (range, 4.79%-15.9%). For HPA-1, nonmaternal ITGB3 sequences (c.176T, HPA-1a) were detected in all HPA-1ab fetuses. One HPA-1bb fetus was unequivocally identified, showing the pregnancy was not at risk of FNAIT., Conclusion: We have successfully established massively parallel sequencing as a novel reliable method for noninvasive genotyping of fetal HPA-1a alleles. This technique may also allow the safe detection of other fetal blood group polymorphisms frequently involved in FNAIT and hemolytic disease of the newborn., (© 2015 AABB.)

CD36 (also known as GPIV) deficiency is known to be responsible for the production of anti-Nak(a) antibodies in different clinical settings such as fetal/neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion refractoriness (PTR) and post-transfusion purpura (PTP). However, no data regarding the relevance of CD36 immunisation is currently available for China. In this study, healthy blood donors were typed for CD36 deficiency using flow cytometry. Nucleotide sequencing was performed to identify the molecular basis underlying the CD36 deficiency. Anti-Nak(a) antibodies in CD36-deficient individuals were analysed by ELISA and flow cytometry. By analysis of 998 healthy blood donors, 18 individuals failed to express CD36 on their platelets. In 5/12 individuals no CD36 expression was detected both on platelets and monocytes. This result suggested that the frequencies of type I CD36 deficiency (platelets and monocytes) and type II CD36 deficiency (platelets only) are approximately 0.5 and 1.3%, respectively. Nucleotide sequencing analysis of type I CD36 deficient individuals revealed eight different mutations; four of them were not described so far. However, 1228-1239del ATTGTGCCTATT and 329-330delAC appear to be the most common mutations related to type I CD36 deficiency in South Chinese population. Further analysis showed that 1/5 type I CD36 deficient individuals developed anti-Nak(a) antibodies. In addition, anti-Nak(a) antibodies could be identified in two cases of thrombocytopenia associated with FNAIT and PTR. In conclusion, more than 0.5% of CD36 type I-deficient individuals are at risk to be immunised through blood transfusion or pregnancy in China. Testing of anti-Nak(a) antibodies should be considered in FNAIT and PTR suspected cases. A registry of CD36-deficient donors should be established to allow treatment of immune-mediated bleeding disorders caused by anti-Nak(a) antibodies.

Background: Maternal anti-HPA-1a alloantibodies are responsible for most cases of severe fetal and neonatal alloimmune thrombocytopenia (FNAIT). The presence of HPA-1a alloantibodies in maternal blood alone does not predict the fetal platelet (PLT) count, and the predictivity of antibody titers determined by enzyme immunoassays (EIAs) is debated. In contrast to EIA, surface plasmon resonance (SPR) provides information on antibody-binding properties., Study Design and Methods: Sequential sera from pregnant women with expected FNAIT were assessed for HPA-1a alloantibodies using SPR. Group I (n = 6) was treated with intravenous immunoglobulin (IVIG) and steroids beginning at 19 weeks of gestation (w.g.), and Group II (n = 4) received intrauterine PLT transfusions (IUT) beginning at 22 w.g. Maternal alloantibodies were quantified using an HPA-1a monoclonal antibody (MoAb) as a standard. Antibody avidity was determined as the ratio of B700 (end of the dissociation phase) to B350 (end of the association phase); the area under the curve (AUC) was calculated to determine overall antibody binding., Results: After 22 w.g., alloantibody characteristics remained stable in both groups, while there was a steep decrease in B700 and B350 values between 16 and 22 w.g. (assessed only in Group I), indicating a decrease in anti-HPA-1a alloantibody concentrations. Interestingly, the AUCs of the last maternal sample before elective delivery appeared to be correlated with fetal and neonatal PLT counts (p = 0.014 and 0.017, respectively)., Conclusion: SPR provides quantitative information on HPA-1a alloantibody characteristics in addition to monoclonal antibody-specific immobilization of platelet antigens. SPR results can be calibrated using a MoAb standard and should be further assessed for a potential correlation with fetal PLT count., (© 2012 American Association of Blood Banks.)

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is often caused by maternal alloantibodies against the human platelet antigen (HPA)-1a, which opsonizes fetal platelets (PLTs). Subsequent PLT destruction is mediated via the Fc part of the alloantibodies. The monoclonal antibody (mAb) SZ21 binds to the HPA-1a epitope and inhibits the binding of maternal alloantibodies. However, it also promotes complement activation and phagocytosis. Deglycosylation of antibodies abrogates the Fc-related effector functions. We modified the N-glycan of SZ21 by endoglycosidase F. The in vivo transplacental transport of N-glycan-modified (NGM)-SZ21 was not impaired. When injected into pregnant mice, both native-SZ21 and NGM-SZ21 were transported equally into fetal circulation (8.9% vs 8.7%, respectively, P = .58). Neither the binding properties of NGM-SZ21 to HPA-1a in surface plasmon resonance, nor the inhibition of anti-HPA-1a-induced PLT phagocytosis, were affected by N-glycan modification. NGM-SZ21 prevented PLT destruction induced by maternal anti-HPA-1a antibodies in vivo in a mouse model (PLT clearance after 5 hours; 18% vs 62%, in the presence or absence of NGM-SZ21, respectively, P = .013). Deglycosylation of SZ21 abrogates Fc-effector functions without interfering with placental transport or the ability to block anti-HPA-1a binding. Humanized, deglycosylated anti-HPA-1a mAbs may represent a novel treatment strategy to prevent anti-HPA-1a-mediated PLT destruction in FNAIT.

Background: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA-1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti-HPA-1a could be overlooked by standard methods due to low avidity. Low-avidity HPA-1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low-avidity anti-HPA-1a., Study Design and Methods: A retrospective cohort consisting of 82 HPA-1bb mothers of HPA-1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low-avidity antibodies in SPR using purified GPIIb/IIIa (HPA-1a or -1b). The capability of HPA-1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia., Results: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA-1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA-negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA-1a (mean, 31 ± 5 RU), but not to HPA-1b flow cell (mean, 5 ± 2 RU). In vivo, low-avidity antibodies were capable of clearing HPA-1ab PLTs but not HPA-1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA-positive antibodies., Conclusions: Low-avidity HPA-1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo., (© 2011 American Association of Blood Banks.)

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by transplacental passage of maternal antibodies to fetuses whose platelets (PLTs) express the corresponding human PLT antigen (HPA)., Study Designs and Methods: We observed a fetus with FNAIT who died from a severe intracranial hemorrhage. Analysis of maternal serum in antigen capture assay with paternal PLTs showed reactivity with PLT glycoprotein (GP)IIb/IIIa (α(IIb) β(3) ) and GPIa/IIa (α(2) β(1) integrin), indicating the presence of anti-HPA-1a and an additional alloantibody against GPIa (termed anti-Swi(a) )., Results: By immunochemical studies, the localization of the Swi(a) antigen on GPIa/IIa could be confirmed. Analysis of paternal GPIa full-length cDNA showed a single-nucleotide substitution C(3347) T in Exon 28 resulting in a Thr(1087) Met amino acid substitution. Testing of family members by polymerase chain reaction-restriction fragment length polymorphism using MslI endonuclease showed perfect correlation with phenotyping. Extended family and population studies showed that 4 of 10 members of the paternal family but none of 500 unrelated blood donors were Swi(a) carriers. Expression studies on allele-specific transfected Chinese hamster ovary (CHO) cells confirmed that the single-amino-acid substitution Thr(1087) Met was responsible for the formation of the Swi(a) epitope. Adhesion of CHO cells expressing the Swi(a) alloantigen to immobilized collagens was not impaired compared to the wild-type control and was not inhibited by anti-Swi(a) alloantibodies., Conclusion: In this study we defined a new PLT alloantigen Swi(a) that was involved in a case of additional immunization against HPA-1a. Our observations demonstrate that combinations of PLT-specific alloantibodies may comprise low-frequency alloantigens., (© 2011 American Association of Blood Banks.)

Background: The involvement of the human platelet antigen (HPA)-15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA-15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA-15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan., Study Design and Methods: We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti-HPA-15 alloantibodies., Results: Anti-HPA-15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole-coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA-15 genotype between the mother and the baby (both HPA-15a/a), but incompatibility with the paternal one (HPA-15a/b). The hydatid mole's tissue was genotyped as HPA-15b positive. Besides anti-HPA-15b, maternal sera contain strong HLA Class I antibody, Conclusions: Here we reported the first case of anti-HPA-15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA-15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti-HLA Class I being the possible cause.

Background: Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA-1 to -3, -5, and -15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low-frequency HPAs (HPA-4 and -6bw to -17bw) might in part explain the remaining 80% of cases., Study Design and Methods: A total of 1054 paternal DNA samples from referred FMAIT cases (among which 223 cases where antibodies against a common HPA were found) were genotyped for 11 low-frequency HPAs as well as a recently discovered polymorphism (ITGA2B-C2320T). The initial genotyping was carried out by TaqMan and potential heterozygotes were confirmed by DNA sequencing. Clinical and serologic data were collected for each case with a heterozygote father., Results: In total, eight heterozygous fathers were identified: four for HPA-6w, one each for HPA-10w and -11w, and two for HPA-12w. Maternal antibodies against the corresponding antigen were identified in four of the eight cases. In two of these cases, antibodies against HPA-1a and HPA-1b were also found., Conclusion: It was concluded that the minor alleles of HPA-4 and -6bw to -17bw are exceptionally rare in the Caucasian population and therefore do not explain the large number of FMAIT referrals which test negative for the common HPA antibodies.

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies., Study Design and Methods: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time., Results: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (approximately 490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA., Conclusions: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.

Background: Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by transplacental passage of maternal human platelet-specific alloantigen (HPA)-1a antibodies that bind to fetal platelets (PLTs) and mediate their clearance. SZ21, a monoclonal antibody (MoAb) directed against PLT glycoprotein IIIa, competitively inhibits the binding of anti-HPA-1a alloantibodies to PLTs in vitro. The purpose of this investigation was to determine whether SZ21 F(ab')(2) fragments might be therapeutically effective in inhibiting or displacing maternal HPA-1a antibodies from the fetal PLT surface and preventing their clearance from circulation., Study Design and Methods: Resting human PLTs from HPA-1ab heterozygous donors were injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purified F(ab')(2) fragments of SZ21 or control immunoglobulin G (IgG) were injected intraperitoneally 30 minutes before introduction of HPA-1a antibodies. Blood samples were taken periodically and analyzed by flow cytometry to determine the percentage of circulating human PLTs., Results: Anti-HPA-1a IgG from NAIT cases were able to efficiently clear HPA-1a-positive PLTs from murine circulation. Administration of SZ21 F(ab')(2) fragments not only inhibited binding of HPA-1a antibodies to circulating human PLTs, preventing their clearance, but also displaced bound HPA-1a antibodies from the PLT surface., Conclusion: F(ab')(2) fragments of HPA-1a-selective MoAb SZ21 effectively inhibit anti-HPA-1a-mediated clearance of human PLT circulating in an in vivo NOD/SCID mouse model. These results suggest that agents that inhibit binding of anti-HPA-1a to PLTs may have therapeutic potential in the treatment of NAIT.

Background: Immunization against the human platelet alloantigen (HPA)-3a residing on alphaIIbbeta3 integrin accounts for approximately 2 percent of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Anti-HPA-3a alloantibodies are sometimes difficult to detect and can be overlooked by standard antigen capture assays., Study Design and Methods: The reactivity of 12 anti-HPA-3a and 2 anti-HPA-3b alloantibodies from patients with FNAIT and posttransfusion purpura was analyzed by serologic (monoclonal antibody-specific immobilization of platelet antigens [MAIPA] assay, flow cytometry) and immunochemical (immunoprecipitation, immunoblotting) techniques. The influence of platelet (PLT) age, storage conditions, recombinant antigens from Chinese hamster ovary (CHO) cells, and sialic acids (treatment with neuraminidase) were analyzed., Results: The most sensitive anti-HPA-3 alloantibody detection in MAIPA assay could be achieved with fresh homozygous PLTs. During a PLT storage period of 14 days before use, three types of anti-HPA-3 alloantibodies were found: 1) complete loss of reactivity (n = 6), 2) considerably weakened reaction (> or =50% reduction; n = 3), and 3) minor reduction of reactivity (< or =40% decrease; n = 5). When cryopreserved PLTs were used, 10 of 12 anti-HPA-3a and all anti-HPA-3b alloantibodies reacted positive. Only 6 of 10 serum samples reacted with recombinant HPA-3a on CHO cells. Neuraminidase treatment of PLTs showed that some anti-HPA-3a alloantibodies require the presence of sialic acids. The storage lesion seems to be related to cleavage of sialic acids. Immunochemical analysis revealed evidence that most anti-HPA-3a alloantibodies require an intact three-dimensional alphaIIbbeta3 integrin structure., Conclusions: Anti-HPA-3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the alphaIIbbeta3 integrin and protection from enzymatic degradation seem to be important during PLT storage.