Cases Of Neonatal Alloimmune Thrombocytopenia Research Articles

O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope

AbstractSialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope—perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

Case Of NAIT Causing Severe Thrombocytopenia due to Anti-HLA Class I

Neonatal alloimmune thrombocytopenia (NAIT) is the leading cause of thrombocytopenia in otherwise healthy new-born. (1,2) Maternal antibodies raised against paternally inherited alloantigen carried on fetal platelet causing NAIT. Maternal IgG antibodies passed through to the fetal via the placenta, attack and cause the destruction of the fetal platelet. (3) We present a case of NAIT without any complications in a premature baby (35 weeks) with VACREL association, G6PD deficiency, left calcified cephalohaematoma, cardiomegaly and hypospadias with severe thrombocytopenia (platelet counts is 23 109/L) at day two of life and received twice platelet transfusion. Platelet count initially 123 109/L at birth but significantly drop and persistently less than 50 109/L until day 10 of life before it normalized. Maternal serum antibody screening was negative, but platelet immunology test detected maternal platelet-reactive antibody Anti-HLA Class I and correlates with incompatible parental crossmatch indicating that parent had “platelet-antigen incompatibility”. The goal of obstetric management is to identify pregnancies at risk and prevent intracranial haemorrhage. (4) There is no evidence to support routine screening for pregnancies as per current practice. (2, 5) The latest treatments include maternal administration of intravenous immunoglobulin to suppress maternal antibody production and or to reduce placental transfer of antibodies; with or without steroids during antepartum period besides planning of mode, timing and method of delivery. (2, 5, 6, 7) This is a rare and unique case of NAIT secondary to Anti-HLA Class I antibody and hence clinician should be au fait with the diagnosis and management as it is infrequent among Malaysian.International Journal of Human and Health Sciences Supplementary Issue-2: 2021 Page: S21

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

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

P017 The first case of neonatal alloimmune thrombocytopenia due to HLA antibodies in Hong Kong

We report the first case of neonatal alloimmune thrombocytopenia (NAIT) secondary to anti-HLA antibody in Hong Kong. The baby girl was born at 39 weeks gestation by spontaneous delivery, with a birth weight at 4.165 kg. She was kept in hospital for investigations of large for gestational age. A full blood count incidentally showed: moderate thrombocytopenia (Platelets, 64 × 10 9 /L), but normal leukocyte count and haemoglobin. No red cell fragments or spherocytes nor platelet clumps were noted. Over the next two days, her platelet count dropped to 8.0 × 10 9 /L on Day 2, but there were no major bleeding. Her and her mother’s blood groups were both B + and direct antiglobulin test was negative. Antibody screen was negative on the maternal serum. Her mother did not have thrombocytopenia. In view of her marked thrombocytopenia, she was transfused one unit of random donor group B + platelet concentrate and IVIG 4.2 g, and the platelet count rose to 98 × 10 9 /L on Day 3. The mother had no history of blood transfusion. No anti-HPA antibodies were detected by antigen-captured ELISA. Maternal serum was crossmatch incompatible with father’s platelets by PIFT. Luminex SAB test demonstrated broad spectrum anti-HLA IgG antibodies with 2,000–5,000 MFI against several HLA-A, -B antigens in the maternal serum, and NAIT caused by HLA antibodies was suspected. Anti-A203 and anti-B55 IgG antibodies were found against the paternal inherited HLA antigens. These specific antibodies with 1,500–2,000 MFI were also detected in her pre-treatment serum. Although the initial platelet response was satisfactory, the platelet count gradually dropped again from 82 × 10 9 /L on Day 4 to 49 × 10 9 /L on Day 7. She was then given further IVIG 4.2 g with the platelet count rose to 79 × 10 9 /L on Day 8. The platelet count then stabilized up to Day 10. Although the mother was a primipara, anti-HLA antibodies could still occur since even non-transfused men may have ‘naturally occurring’ anti-HLA antibodies. Based on the anti-HLA antibodies in the maternal serum, the mother’s and baby’s HLA typing results and the PIFT results, we believe that there is evidence this is a rare and unique case of NAIT secondary to anti-HLA antibodies.

Um Caso de Trombocitopenia Neonatal com Neutropenia

A trombocitopenia neonatal aloimune é a causa mais comum de trombocitopenia grave neonatal. Resulta da incompatibilidade feto-materna de antigénios plaquetares, levando à produção de anticorpos maternos e destruição das plaquetas fetais durante a gravidez. A correlação entre aloanticorpos anti-HLA e neutropenia neonatal é extremamente rara, em parte pela reduzida expressão do sistema HLA leucócitos no recém-nascido. Uma vez que plaquetas e neutrófilos possuem sistema HLA, é possível que anticorpos HLA causem ambas neutropenia e trombocitopenia, embora sejam raras as citopenias combinadas. Reportamos um caso clínico de trombocitopenia neonatal aloimune associada a neutropenia aloimune.
 Recebido: 30/09/2017 - Aceite: 18/10/2017

A Case of Neonatal Alloimmune Thrombocytopenia Due to Anti-HLA A29 Antibody

A Case of Neonatal Alloimmune Thrombocytopenia Due to Anti-HLA A29 Antibody

Anti-HLA Antibodies in Neonatal Alloimmune Thrombocytopenia - Is There Any Clinical Significance?

Background: Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies raised against paternally inherited alloantigens carried on fetal platelets. Platelets express both HLA class I and specific human platelet antigens (HPA). Although anti-HLA class I antibodies are often detectable in pregnant women, NAIT is considered to be mainly associated with antibodies against HPA. Cases where NAIT has been caused by antibodies against HLA class I are relatively rare and the role of these antibodies in NAIT remains debatable.We hereby describe a sample case of NAIT proved to be caused solely by anti-HLA antibodies and discuss laboratory measures aimed at identification of pregnancies at risk of NAIT related to anti-HLA class I antibodies based on a series of similar cases.Methods: This sample case presents laboratory work-up on a young mother who delivered her first son with a platelet count of 20x109/L, minor petechiae and normal WBC count. Thrombocytopenia in the newborn resolved spontaneously two weeks after birth. Laboratory investigation included platelet immunofluorescence test (PIFT), monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, genotyping of both parents and the newborn for platelet antigens, including rare antigens, and HLA antibody identification using the panel reactive antibodies (PRA) assay (Luminex, USA). A serum sample of this mother, drawn during her second pregnancy, and those of ten other women referred to our laboratory with a similar obstetric history of neonatal thrombocytopenia, were evaluated for the anti-HLA antibody titer using the MAIPA assay.Results: The Rambam Platelet & Neutrophil Immunology Laboratory, as well as 32 other laboratories worldwide, that participated in the 2014 International Workshop organized by the ISBT Platelet Immunobiology Working Party failed to detect anti-HPA antibodies in the mother's serum during her second pregnancy, despite using the most sensitive serological analysis and molecular methods. Only strong anti-HLA antibodies with no single specificity were found in the analyzed samples by all the laboratories. Her second child was born by caesarean section with a platelet count of 50x109/L and maternal anti-HLA antibodies were found in his serum and on his platelets. The anti-HLA antibody titer of the mother, determined by the MAIPA assay, was greater than 1:1024, with antibodies being multi-specific, as demonstrated by PRA. The anti-HLA antibody titer ≥1:16 was found to correlate with low platelet counts in the additional ten cases tested, as opposed to the titer of ≤1:4 in cases with mild and not clinically significant neonatal thrombocytopenia.Conclusions: The presence of anti-HLA class I antibodies should be considered as a potential cause of NAIT, especially in cases with a very high titer of antibodies. The mechanism underlying the effect of these antibodies on fetal platelets needs to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous Occurrence of Neonatal Alloimmune Thrombocytopenia and Neutropenia Associated with Multiple Maternal Antibodies

Background: Neonatal alloimmune thrombocytopenia (NAIT) results from feto-maternal platelet antigen incompatibility leading to the production of maternal antibodies and destruction of fetal platelets during pregnancy. Similarly neonatal alloimmune neutropenia (NAN) is caused by maternal alloimmunization to incompatible fetal neutrophil antigens followed by transplacental transfer of the maternal anti-neutrophil antibody causing fetal or neonatal neutropenia. Although there are many reports in the literature describing these rare disorders, the simultaneous occurrence of NAIT and NAN has not been systematically evaluated. In this study we report a case of NAIT associated with NAN.Material and Methods: A healthy 46-year-old primiparous woman gave birth to triplets in the 30.9th gestational week, by C-section, after a fertility treatment using an egg donation banking. The first neonate, a 1,600g female newborn with a first/fifth minute Apgar score of 8/9, healthy at physical examination, showed severe thrombocytopenia (platelet count = 30x109/L) and a neutrophil count of 1.6X109/L at birth. The second neonate, a 1,275g male newborn had a first/fifth minute Apgar score of 7/7, was healthy at physical examination and showed thrombocytopenia (platelet count = 82x109/L) and neutropenia (neutrophil count = 0.66x109/L). Intracranial bleeding, perinatal infections, genetic and hematologic disorders were ruled out and the neonates showed a spontaneously normalization of the platelets and neutrophils counts. Unfortunately, blood sample from the third neonate, a 1,525g female newborn with first/fifth minute Apgar score of 9/9, was not available. Mother and neonates were genotyped for platelets antigens (HPA-1-11,-15) and neutrophil antigens (HNA-1,-3). HPA genotyping was performed by PCR-SSP, PCR-RFPL and xMAP technology (IDHPAXT, Progenika-Grifols, Spain). HNA genotyping was performed by PCR-SSP and PCR-RFPL. Platelets antibodies were investigated in maternal serum by platelet enzyme-linked immunosorbent assay (PaK12G, Immucor GTI Diagnostic Inc, USA); neutrophil antibodies were investigated by granulocyte agglutination test (GAT) using a panel of donors previously genotyped for HNA-1,-3 systems, while anti-HLA antibodies were investigated by ELISA (LAT Mixed, One Lambda Inc., USA and PaK12G, Immucor GTI Diagnostic Inc, USA).Results: Genotyping studies revealed mismatches for HPA-1,-5 and HNA-1,-3 systems between mother and neonates (Table1). Serologic antibody screening tests showed presence of anti-HPA-5b, anti-HNA-3b and anti-HLA in maternal serum. TABLE 1GENOTYPING RESULTSHPA-1HPA-5HNA-1HNA-3MOTHERHPA-1a1aHPA-5a5aHNA-1a1aHNA-3a3aNEONATE 1HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3bNEONATE 2HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3aConclusion: We described the occurrence of NAIT associated with NAN in preterm neonates with important thrombocytopenia and neutropenia due a multiple maternal sensitization to incompatible fetal antigens. As these syndromes pose a risk to the neonate’s life, the early and correct diagnosis is very important in order to apply appropriate treatment to the neonates and allow an effective prenatal therapy for future pregnancies. DisclosuresNo relevant conflicts of interest to declare.

Role Of Maternal Immunization Against Low Frequency Human Platelet Antigens (LFHPA) In Neonatal Alloimmune Thrombocytopenia (NAIT)

Neonatal alloimmune thrombocytopenia (NAIT) is largely caused by maternal immunization against Human Platelet Antigens (HPA) inherited by a fetus from its father. In about 70% of apparent NAIT cases, however, maternal HPA antibodies are not detected by standard serologic testing. At least 24 low frequency HPAs (LFHPA) have now been implicated in NAIT. Fathers are not ordinarily typed for these antigens and the corresponding antibodies can be difficult to detect. We studied paternal and maternal blood samples from suspected NAIT cases to investigate the extent to which maternal immunization against LFHPAs causes “antibody-negative” NAIT.In Phase 1, we studied paternal DNA in 244 NAIT cases not resolved by standard laboratory testing in which there was compelling clinical and/or serologic evidence for NAIT. LFHPAs were identified by full length sequencing of relevant exons encoding platelet glycoproteins (GP). Sixteen LFHPAs (6.6% of cases) were identified. HPA-4b, 6bw, 11bw, 12bw, 13bw, 19bw, 20bw, 21bw, 22bw, and 23bw were found in single cases and HPA-9bw in 6 cases. In 15 of the 16 cases, a maternal antibody reactive with paternal platelets and/or a platelet GP was identified.Cases studied in Phase 1 were highly selected. To obtain an unbiased estimate of the extent to which LFHPAs trigger NAIT, we determined the prevalence of 22 recognized LFHPAs in fathers of 1067 consecutive “antibody-negative” NAIT cases using high throughput screening methods (Phase 2). Mothers of 1338 cases were similarly typed to assess the normal prevalence of LFHPAs in a population racially/ethnically similar to the fathers. Of the 1067 fathers, 28 (2.6%) were positive for a LFHPA. LFHPAs identified were HPA-4b (1 case), 6bw (2), 8bw (1), 9bw (17), 12bw (4) and 21bw (3). Of the 1338 mothers, 27 (2.0%) were positive for a LFHPA. Maternal LFPAs identified were 4b (2 cases), 6bw (5), 9bw (12), 12bw (4), 13bw (3) and 21bw (1). Of the LFHPAs identified, only HPA-9bw was significantly more common in fathers than in mothers (P=0.02). Serologic evaluations done in the Phase 2 cases provided evidence for maternal antibodies against paternal GPIIb/IIIa in only 2 of 23 instances in which the paternal low frequency antigen was located on this GP complex.Findings made in Phase 1, show that efforts to identify a paternal LFHPA in unresolved NAIT cases can be rewarding when maternal serum reacts with paternal platelets and the clinical picture strongly suggests NAIT. The Phase 2 studies of consecutive unresolved NAIT cases show that, with the exception of HPA-9b, the prevalence of LFHPAs is about the same in mothers and fathers of apparent NAIT cases not attributable to maternal immunization against “common” HPAs. It is clear that maternal immunization against an LFHPA can account for only a small fraction of the apparent “diagnostic gap” in serologic diagnosis of NAIT. HPA-9bw is by far the most important immunogen among the recognized LFHPAs and consideration should be given to typing fathers of suspected NAIT cases routinely for this antigen. Recent studies suggest that “low avidity” HPA antibodies not detected using conventional serology may account for an unknown fraction of the “antibody-negative” NAIT cases. Whether HLA antibodies cause NAIT more often than has been thought also deserves re-examination. Disclosures:No relevant conflicts of interest to declare.

Low‐frequency human platelet antigens as triggers for neonatal alloimmune thrombocytopenia

Twenty-four low-frequency human platelet antigens (LFHPAs) have been implicated as immunogens in neonatal alloimmune thrombocytopenia (NAIT). We performed studies to define more fully how often these antigens trigger maternal immunization leading to NAIT. In a Phase 1 study, fathers of selected NAIT cases not resolved by serologic testing but thought to have a high likelihood of NAIT on clinical and serologic grounds were typed for LFHPAs by DNA sequencing. In a Phase 2 study, high-throughput methods were used to type fathers of 1067 consecutive unresolved NAIT cases for LFHPAs. Mothers of 1338 unresolved cases were also typed to assess the prevalence of LFHPAs in a population racially/ethnically similar to the fathers. In Phase 1, LFHPAs were identified in 16 of 244 fathers (6.55%). In Phase 2, LFPAs were found in only 28 of 1067 fathers (2.62%). LFHPAs were identified in 27 of 1338 maternal samples (2.01%). HPA-9bw was by far the most common LFHPA identified in the populations studied and was the only LFHPA that was significantly more common in fathers than in mothers of affected infants (p = 0.02). Maternal immunization against recognized LFHPAs accounts for only a small fraction of the cases of apparent NAIT not resolved by standard serologic testing. Typing of the fathers of such cases for LFHPAs is likely to be rewarding only when a maternal antibody specific for a paternal platelet glycoprotein is demonstrated and/or there is compelling clinical evidence for NAIT.

Neonatal Alloimmune Thrombocytopenia Associated with Massive Chronic Intervillositis: A Case Report and Review of the Literature

Neonatal alloimmune thrombocytopenia (NAIT) presents as isolated thrombocytopenia in a normal neonate as a result of destruction of fetal platelets by maternal antibodies against paternally derived human platelet antigens. Neonatal alloimmune thrombocytopenia affects 0.1% of births, with maternal antibodies crossing the placenta as early as 14 weeks' gestation. Few reports describe placental histopathological changes occurring in NAIT cases. We present a case of NAIT associated with massive chronic intervillositis, a rare entity occurring in 0.06% to 0.8% of reviewed 2nd- and 3rd-trimester placentas; to our knowledge, this is the 1st report of such an association.

Prevalence and clinical significance of low‐avidity HPA‐1a antibodies in women exposed to HPA‐1a during pregnancy

Recent studies suggest that HPA-1a-specific, low-avidity maternal antibodies not detectable by conventional methods can cause neonatal alloimmune thrombocytopenia (NAIT). We performed studies to further define the incidence and clinical significance of this type of antibody. Surface plasmon resonance analysis was used to detect low-avidity antibodies in HPA-1a-negative, "antibody-negative" mothers of suspected NAIT cases. The ability of antibodies detected to promote immune destruction of human platelets (PLTs) was examined in a newly developed NOD/SCID mouse model. Among 3478 suspected cases of NAIT, 677 HPA-1a-negative mothers were identified. HPA-1a-specific antibodies were detected by conventional antibody testing in 616 cases (91%). Low-avidity HPA-1a-specific antibodies were identified in 18 of the remaining 61 cases (9%). Clinical follow-up on 13 cases showed that eight were referred because of suspected NAIT and five because the mother's sister had previously had an infant with NAIT. Only six infants born to the 13 sensitized mothers had clinically significant thrombocytopenia at birth. Three of four low-avidity antibodies tested in the mouse caused accelerated clearance of HPA-1a/a but not HPA-1b/b PLTs. Only 3 of 12 mothers with low-avidity HPA-1a antibodies were positive for HLA-DRB3*0101. The findings confirm previous reports that low-avidity HPA-1a antibodies can cause NAIT but show that the presence of such an antibody does not predict that an infant will be affected. The low incidence of HLA-DRB3*0101 in this cohort (p < 0.0001) suggests that women negative for DRB3*0101 may be predisposed to produce low-avidity HPA-1a antibodies.

The human platelet antigen‐21bw is relatively common among Asians and is a potential trigger for neonatal alloimmune thrombocytopenia

We and others have identified more than 20 low-frequency human platelet (PLT) antigens (HPAs) that can induce antibodies capable of causing neonatal alloimmune thrombocytopenia (NAIT).1–4 Like HPA-1a and HPA-4b, the antigens that most often trigger NAIT in Caucasian and Asian populations, respectively, the incidence of low-frequency antigens is likely to vary significantly in different ethnic and racial populations. We recently described HPA-21bw, a low-frequency antigen, responsible for a single case of severe NAIT in a presumed Caucasian family.1 The HPA-21bw antigen results from a G>A1960 mutation (RS70940817), in ITGB3, encoding PLT glycoprotein (GP) β3 subunit (GPIIIa) and creates a Glu>Lys substitution at Residue 628 in mature β3. No examples of HPA-21bw were identified in 100 unrelated Caucasian individuals.1 We recently encountered a second case of NAIT apparently caused by HPA-21bw in an Asian family (unpublished). The mutation encoding HPA-21bw was also identified in two of 38 Asian individuals tested in a hypertension and insulin resistance study according to the human SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP; ss95216694). These observations suggested that HPA-21bw, like HPA-4b, might be more prevalent in Asian than in Caucasian populations and therefore a more common trigger for NAIT. To examine this possibility, we determined the gene frequency of the mutation encoding HPA-21bw in anonymized DNA samples identified by age, sex, and self-declared race/ethnicity obtained from the Retrovirus Epidemiology Donor Study (REDS) General Serum Repository of the National Heart, Lung, and Blood Institute (NHLBI) made available by the NHLBI Biologic Specimen and Data Repository guardian (BioLINCC). These specimens were collected from blood donors at five locations in the United States (Baltimore, Detroit, Los Angeles, Oklahoma City, and San Francisco) between December 1994 and December 1995. Anonymized samples were also obtained from Caucasian blood donors at BloodCenter of Wisconsin. Genotyping for HPA-21bw was performed with an allelic discrimination assay using PerfeCTa qPCR SuperMix, UNG, Low ROX (Quanta Biosciences, Gaithersburg, MD).2 Results are summarized in Table 1. No examples of HPA-21bw were found in 1110 unrelated Caucasians, 611 African Americans, or 252 Hispanic blood donors. However, 6 of 531 Asians donors were HPA-21a/bw (prevalence, 1.1%; gene frequency, 0.0056). TABLE 1 Frequency analysis of HPA-21bw in various populations We used the exact binomial methods for statistical inference: Clopper-Pearson confidence intervals (CIs) and Fisher’s exact test. The prevalence of HPA-21bw among Asians, estimated as 1.1% (95% CI, 0.4%–2.4%) is significantly greater than in Caucasians (p = 0.001) and African Americans (p = 0.01). The comparison with Hispanics is not significant (p = 0.18); however, the sample size is too small for this comparison. Based on one-sided CIs we are 95% confident that the prevalence of HPA-21bw among Caucasians is less than 0.3%, among African Americans less than 0.5%, and among Hispanics less than 1.2%. To date the HPA-4b (Penb) polymorphism in GPIIIa is the most common trigger for antibodies that cause NAIT in the Asian population. Having an allelic frequency of 0.0045, HPA-4b is expressed in approximately 1% of the Asian population.5 In contrast HPA-6bw in GPIIIa, though found in 2.7% of the Asian population,5 is not a common trigger of NAIT. Taking into consideration that HPA-21bw is known to initiate severe NAIT,1 has already been implicated as the trigger for NAIT in one Asian family (unpublished), and has an allelic frequency of 0.2% to 2.2% (95% confidence limits) in the Asian population, routine typing for this antigen in cases of suspected NAIT involving Asian families appears indicated.

A complicated case of neonatal allo-immune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies directed against fetal paternally derived platelet-specific antigens destroying fetal platelets. We present a patient (HPA-1a negative platelets with genotype 1b/1b) with...

The first two cases of neonatal alloimmune thrombocytopenia associated with the low‐frequency platelet antigen HPA‐21bw (Nos) in Japan

Neonatal alloimmune thrombocytopenia (NAIT) is a disorder characterized by maternal alloimmunization against paternal fetal platelet antigens. Two healthy, unrelated Japanese women each gave birth to a child with severe NAIT. To elucidate the maternal causes of NAIT, we conducted serologic and genetic studies in these two NAIT infants. The serologic experiments localized the antigens to the glycoprotein (GP) IIIa subunit of the GPIIb/IIIa complex. Sequence-based typing studies subsequently identified a G>A mutation at Nucleotide 1960 (a glutamic acid > lysine substitution at Position 628) in the 11th exon of the GPIIIa gene. This mutation was recently identified in a report as HPA-21bw. Next, it was determined that the cause of NAIT in both cases was the HPA-21bw antigen, as shown by the mothers' antibodies reacting with the mutated GPIIIa-transfected cells, but not with transfectants expressing wild-type GPIIIa. A molecular genetic screening for the HPA-21bw allele among Japanese donors showed that its genetic frequency in the population was 0.53% (10/1888), indicating that HPA-21bw occurs at a low but appreciable frequency in the population. Furthermore, in a retrospective study of 50 previous NAIT cases of unknown causes, we found one NAIT case associated with the HPA-21bw antibody. The two NAIT cases in this study represent the first ones to be associated with HPA-21bw in Japan. We identified the HPA-21bw allele from two unrelated Japanese infants with severe NAIT. We identified 10 individuals (1.06%) positive for the HPA-21bw allele from a genetic screening of 944 Japanese blood donors.

Human platelet antigen 1a epitopes are dependent on the cation-regulated conformation of integrin αIIbβ3 (GPIIb/IIIa)

BackgroundThe HPA-1a (Leu33) polymorphism of platelet integrin αIIbβ3 is the target of alloantibodies in 70–80% cases of neonatal alloimmune thrombocytopenia (NAIT) in Caucasians and reliable detection of these antibodies is essential for appropriate clinical management. However, the ability to detect such antibodies is highly variable between laboratories and, in a number of clinical cases where there is a HPA-1 genotype mismatch between mother and neonate, HPA-1a antibodies are undetectable. Furthermore, some studies have not shown a consistent relationship between maternal anti-HPA-1a level and neonatal platelet count. Since the integrity and conformation of the αIIbβ3 complex are dependent on divalent cations, we investigated whether HPA-1a epitope integrity and/or conformation might be affected by the presence of the cation chelator EDTA in patient samples or in assay buffers, thus providing a possible explanation for the variable sensitivity of current assays. Principle findingsExposure of the αIIbβ3 complex to EDTA resulted in reduced reactivity of three anti-HPA-1a mAbs (B2, 19–7 and 23–15). More significantly, cation chelation adversely affected detection of polyclonal anti-HPA-1a, not only in the platelet immunofluorescence assay, where alloantibody binding was reduced compared to control platelets (mean MFI reduction 44.5%, range 17.3–69.7%, n=4), but also in the commonly used monoclonal antibody specific immobilisation of platelet antigens assay (MAIPA) where both alloantibody and monoclonal capture antibody binding were reduced (mean OD reduction 82.8%, range 68.3–96.6%, n=9). ConclusionsThese data show that HPA-1a antibodies recognise epitopes on αIIbβ3 that are sensitive to EDTA treatment and that cation chelation grossly reduces the sensitivity of the MAIPA assay by diminishing not only HPA-1a alloantibody binding but also ‘capture’ monoclonal antibody binding. These findings may, in part, explain the current variability in antibody measurement and will guide the development of more sensitive tests for anti-integrin antibodies in NAIT and other conditions.

Low‐avidity anti‐HPA‐1a alloantibodies are capable of antigen‐positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

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. 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. 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. 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.

New K103 β3 allele identified in a context of severe neonatal thrombocytopenia

A new β3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10(9) /L). Diagnosis was done by use of monoclonal antibody-specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)-sequence-specific primers (SSP) and PCR-restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbβ3 expressed in HEK-293 cells. Serologic analysis revealed in the maternal serum an anti-human PLT alloantigen (HPA)-1a alloantibody associated to an anti-α2β1. Anti-HPA-1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA-16b assignation between PCR-RFLP and PCR-SSP. Sequencing revealed a new c.385C>A mutation in the β3 coding sequence resulting in a false assignation of the HPA-16b allele by PCR-RFLP. This mutation leads to a Q103K substitution in mature β3. The K103-β3 form of the complex was expressed in HEK-293 cells but did not react with the maternal serum. We have characterized a new rare allele (frequency < 1%) of β3 that yields false HPA-16b genotyping in PCR-RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.

Neonatal Alloimmune Thrombocytopenia

Maternal antibody against fetal platelet antigens inherited from the father is a cause of severe thrombocytopenia in the neonate. Most cases of neonatal alloimmune thrombocytopenia (NAIT) are associated with antibodies against the human platelet antigen 1a (HPA-1a). Treatment is usually focused on suppressing the maternal antibodies, but the optimal agents and doses …

A third allele of the HPA‐5 (Br) platelet alloantigen system identified in investigating a case of neonatal thrombocytopenia

HPA-5a/b (Brb/a) was identified in the late 1980s as an antigen system capable of causing maternal immunization leading to neonatal alloimmune thrombocytopenia (NAIT).1 Subsequent studies showed that the HPA-5a/b antigens result from a single-nucleotide polymorphism (G>A1600; “A” of the ATG start codon is here designated Nucleotide 1) in the gene encoding platelet (PLT) glycoprotein (GP) Ia (α2 integrin), leading to a Glu (HPA-5a)>Lys (HPA-5b) amino acid substitution at Residue 505 in GPIa.2 Further studies showed that maternal-fetal incompatibility for HPA-5 antigens is second only to incompatibility for HPA-1a as a cause of NAIT.3 HPA-5a and -5b is thought to be a diallelic system with gene frequencies approximately 0.89 and 0.11, respectively. Here, we describe a third allele of HPA-5 identified in a Caucasian individual while studying paternal DNA in a suspected case of NAIT. A full-term male infant born to a primiparous woman had a normal blood count except for a PLT level of 38 × 109/L. PLTs were 39 × 109/L on Day 4, but increased to normal on Day 8. The infant had physical features of Down’s syndrome and Trisomy 21 was confirmed in genetic studies. No maternal antibody reactive with paternal PLTs was detected using flow cytometry and immunoprecipitation assays. Using polymerase chain reaction (PCR) with sequence-specific priming method, no incompatibility between father and mother was identified for HPA-1, –3, and –5 alloantigen systems. When this study was done, melting point analysis4 was being evaluated to type DNA for HPA antigens. In this technique, fluorescence is produced when PCR products containing the nucleotide sequence being typed anneal to sequence-specific “reporter probes.” As the reaction mixture is heated, the DNA complexes melt (with resulting loss of fluorescence) at temperatures that are allele-specific (Fig. 1). The mother’s DNA produced a single peak at 50°C, indicating that her genotype was HPA-5a/a. The father’s DNA produced two peaks, one corresponding to HPA-5a and the other intermediate between the HPA-5a peak and the one expected for HPA-5b (Fig. 1). Because of this atypical finding, the father’s DNA encoding the HPA-5 mutation was sequenced using standard methods. It was found that father’s DNA contained both the expected G at Position 1600 and an unexpected T substitution at Position 1601 (Table 1), providing an explanation for the atypical melting curves obtained when his DNA was typed for HPA-5 (Fig. 1). The subsitution of A at Nucleotide 1601 to T leads to amino acid substitution at Position 505 from glutamic acid into valine creating a third allele of HPA-5. “T” at position 1601 was identified in one individual in the course of screening 20 DNA samples from NAIT cases when the melting point technique was being evaluated. However, no similar examples of T1601 were identified in typing 128 normal Caucasian subjects for HPA-5 by direct sequencing. Additional normal subjects are being typed to obtain a better estimate of the frequency of this allele. Fig. 1 Melting curve analysis of HPA-5 alleles with LightCycler (Roche Diagnostic, Indianapolis, IN). The first negative derivative of the fluorescence versus temperature (−dF/dT) graph shows the melting temperature (Tm) of HPA-5a probe/DNA complex (50°C) ... TABLE 1 Alleles of HPA-5 It was not possible to obtain DNA from the infant to determine whether he inherited T1601. However, since no maternal antibody reactive with PLTs or GPIa/IIa from the father was identified and transient thrombocytopenia is common in newborns with Down’s syndrome,5 it seems unlikely that the low PLT counts were the result of maternal immunization against GPIa containing Val at Position 505. Knowledge of the GPIa mutation identified in this case will be helpful if other examples are identified in future evaluations of NAIT cases. It is possible, however, that T1601 will not be detected in assays that utilize sequence-specific primers to type for HPA-5a/b antigens.