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

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

Severe thrombocytopenia and intracranial hemorrhage in a newborn with Noonan syndrome and neonatal alloimmune thrombocytopenia.

Noonan syndrome (NS) is a genetic disorder with distinctive physical features and often multiple organ involvement. Bleeding disorders are reported in over half of patients with NS, including thrombocytopenia and platelet dysfunction. Neonatal alloimmune thrombocytopenia (NAIT) is an alloantigenic thrombocytopenia that can present with severe bleeding. Here, we present a case of intracranial hemorrhage and severe thrombocytopenia in a neonate found to have both NAIT and a de novo heterozygous pathogenic variant in PTPN11, consistent with Noonan syndrome.

Type 2B von Willebrand Disease: Early Manifestation as Neonatal Thrombocytopenia.

Here, we report about a preterm female newborn with a prolonged course of severe thrombocytopenia and hematomas. The family history was positive for von Willebrand disease type 2B (VWD 2B). Diagnosis of VWD 2B was identified analyzing von Willebrand factor (VWF) parameters (VWF:antigen, VWF:activity, VWF multimer analyses) and performing light transmission aggregometry (with half concentration of ristocetin). In addition, the diagnosis was confirmed by molecular genetic analysis: identification of a disease-causing missense mutation (Val1316Met) in the VWF gene associated with a severe course of VWD 2B, which had been previously reported. Treatment with a VWF-containing plasma concentrate was initiated. Because the combination of prematurity and very low platelet count is often associated with intracranial bleeding, at the beginning platelet concentrates were transfused. Fortunately, the patient did not develop serious bleeding episodes. Interestingly, the patient had a mutation in the VWF gene, which had been described to be associated with aggravation of thrombocytopenia especially in stressful situations. Therefore, we replaced venous blood withdrawals by capillary blood samplings when possible and, consequently, we observed an increase of the platelet count after this change in management. At the age of 2 months, the patient was discharged after stabilization of the platelet count without any bleeding signs and without a need of long-term medication.

Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin ß3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Platelet CD36 deficiency is present in 2.6% of Arabian individuals and can cause NAIT and platelet refractoriness.

BACKGROUND: CD36 isoantibodies are capable of inducing neonatal alloimmune thrombocytopenia (NAIT) and platelet refractoriness. As to now the CD36 type I deficiency has been reported in East Asian and African individuals. However, it is virtually unknown in Caucasians. The aim of this study was to display the prevalence of the CD36 deficiency within parts of the Arabian population in Germany. Secondly, we are presenting the case of a newborn suffering from NAIT which was induced by CD36 antibody. METHODS: Platelet (p) CD36 was determined by flow cytometry on 1328 samples mainly from individuals of Arabian origin and a family with a neonate affected by NAIT. DNA sequencing was performed on all pCD36-negative samples. RESULTS: Thirty-five (2.64%) of all donor samples were pCD36 negative, 19 (1.43%) had a weak expression. Including only individuals from the Arabian peninsula, frequencies were 3.39% and 1.75%, respectively. CD36 type I deficiency on both platelets and monocytes combined with a CD36 isoantibody were detected in the mother of the NAIT baby. The baby was successfully transfused with two HPA-unselected platelet concentrates. In case of need, two platelet units with a weak pCD36 expression were on hand. A total of 45 different CD36 mutations were detected within pCD36-negative individuals, some being homozygous, most of them only present on one allele. CONCLUSION: The CD36-negative phenotype is present in a significant number of individuals of Arabian origin and enables CD36 isoimmunization in NAIT or refractoriness. Blood transfusion services should be aware of such cases.

A nait-associated and previously unreported mutation in the ITGB3 gene with a low frequency in the local population.

BACKGROUND: Neonatal alloimmune thrombocytopenia is a rare but potentially severe postnatal complication caused by maternal allo-antibodies against platelet antigens of the newborn. In relatively few cases, immunisation against low-frequency antigens has been reported. METHODS: Platelet antigens of a newborn with severe thrombocytopenia and his family members were investigated by serological and molecular biological methods. A real-time PCR assay was developed to reliably detect this mutation in pools of DNA from up to seven individuals. RESULTS: Serological testing showed positive reactions of maternal plasma with paternal platelets but not with conventional platelet donor panels. Sequencing of the ITGB3 gene revealed a G > A polymorphism in position c.1915 of exon 12 for the father, the newborn and three of four paternal relatives. Screening of samples from a local population of 1575 Caucasian blood donors identified only a single individual with this mutation. CONCLUSION: This finding of a previously unreported private platelet antigen demonstrates that the identification of the target glycoprotein by MAIPA assay followed by sequencing of the affected gene can be combined with an efficient population screening by real-time PCR with pooling of DNA samples.

Congenital thrombocytopenia associated with GNE mutations in twin sisters: a case report and literature review.

BACKGROUND: Neonatal thrombocytopenia is common in preterm and term neonates admitted to neonatal intensive care units. The etiology behind neonatal thrombocytopenia is complex. Inherited thrombocytopenia is rare and usually results from genetic mutations. CASE PRESENTATION: Here we report a case of twins with severe inherited thrombocytopenia presented in the neonatal period who were shown to be compound heterozygotes for 2 UDP-N-acetylglucosamine 2-epimerase (GNE) gene mutations, c.1351C > T and c.1330G > T, of which c.1330G > T is a novel mutation. CONCLUSION: These two GNE mutations may help in the diagnosis and management of thrombocytopenia diagnosed in neonates.

Neonatal alloimmune thrombocytopenia due to anti-HPA 5a in a HPA-5a homozygous neonate.

The most frequently involved antigen in severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the human platelet antigen 1a. Cases of FNAIT caused by HPA-5a antigen are extremely rare, and usually not severe. We report a case of FNAIT caused by anti-HPA antibodies directed to the HPA-5a antigen. The thrombocytopenia was moderate with a minimal platelet count of 36 × 109/L by day 3, and spontaneously resolved by day 10. The pregnancy had been obtained by in vitro fertilization using embryo donation, creating a complete genetic disparity between the HPA 5b5b mother and the HPA 5a5a homozygous neonate. The use of ART with gamete donation can increase the risk and the severity of alloimmune thrombocytopenia and must be considered in new and subsequent pregnancies.

The prevalence of HPA-1a alloimmunization and the potential risk of FNAIT depend on both the DRB3*01:01 allele and associated DR-DQ haplotypes.

Alloimmunization against human platelet antigen (HPA)-1a during pregnancy can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) and severe bleeding in the foetus or newborn and likely depends on several factors. HPA-1a alloimmunization is associated with DRB3*01:01, which is associated with several DR-DQ haplotypes. However, it is not known to what extent these haplotypes contribute to the prevalence of HPA-1a alloimmunization. HPA-1a-alloimmunized women, identified in a prospective study, and random donors were typed for selected DRB3, DRB4, DRB1, DQA1 and DQB1 alleles to determine allele and DR-DQ haplotype frequencies. DRB3*01:01 was carried by 94% HPA-1a-immunized women compared to 27% in the general population. In the first population, the DR3-DQ2 haplotype was overrepresented (P < .003). The prevalence of HPA-1a alloimmunization was estimated to be about twice as frequent with DR3-DQ2 compared to DR13-DQ6, together accounting for about 90% of DRB3*01:01-positive individuals. Further, we examined DQB1*02 and DRB4*01:01 alleles for their reported association with HPA-1a alloimmunization, in the context of DR-DQ haplotypes. Since ~ 80% of DQB1*02 alleles are linked to the DR3-DQ2 haplotype, the association might be coincidental. However, the DQB1*02:02-associated DR7-DQ2 haplotype was also overrepresented in alloimmunized women, suggesting a role for this allele or haplotype in HPA-1a alloimmunization. As DRB4*01:01 is predominantly associated with the DR7-DQ2 haplotype in HPA-1a-alloimmunized individuals, the reported association with FNAIT may be coincidental. Typing for DR-DQ haplotypes revealed important genetic associations with HPA-1a alloimmunization not evident from typing individual alleles, and the presence of different DRB3-associated DR-DQ haplotypes showed different prevalence of HPA-1a alloimmunization.

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

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

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

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

Prenatal Management of Pregnancies at Risk of Fetal Neonatal Alloimmune Thrombocytopenia (FNAIT): Scientific Impact Paper No. 61.

WHAT IS IT?: Fetal neonatal alloimmune thrombocytopenia (FNAIT), also known as neonatal alloimmune thrombocytopenia (NAIT) or fetomaternal alloimmune thrombocytopenia (FMAIT), is a rare condition which affects a baby's platelets. This can put them at risk of problems with bleeding, particularly into the brain. One baby per week in the UK may be seriously affected and milder forms can affect one in every 1000 births. HOW IS IT CAUSED?: Platelets are blood cells that are very important in helping blood to clot. All platelets have natural proteins on their surface called human platelet antigens (HPAs). In babies, half of these antigens are inherited from the mother and half from the father. During pregnancy, some of the baby's platelets can cross into the mother's bloodstream. In most cases, this does not cause a problem. But in cases of FNAIT, the mother's immune system does not recognise the baby's HPAs that were inherited from the father and develops antibodies, which can cross the placenta and attack the baby's platelets. These antibodies are called anti-HPAs, and the commonest antibody implicated is anti-HPA-1a, but there are other rarer antibody types. If this happens, the baby's platelets may be destroyed causing their platelet count to fall dangerously low. If the platelet count is very low there is a risk to the baby of bleeding into their brain before they are born. This is very rare but if it happens it can have serious effects on the baby's health. HOW IS IT INHERITED?: A baby inherits half of their HPAs from its mother and half from its father. Consequently, a baby may have different HPAs from its mother. As the condition is very rare, and even if the baby is at risk of the condition we have no way of knowing how severely they will be affected, routine screening is not currently recommended. WHAT CAN BE DONE?: FNAIT is usually diagnosed if a previous baby has had a low platelet count. The parents are offered blood tests and the condition can be confirmed or ruled out. There are many other causes of low platelets in babies, which may also need to be tested for. As the condition is so rare, expertise is limited to specialist centres and normally a haematologist and fetal medicine doctor will perform and interpret the tests together. Fortunately, there is an effective treatment for the vast majority of cases called immunoglobulin, or IVIg. This 'blood product' is given intravenously through a drip every week to women at risk of the condition. It may be started from as early as 16 weeks in the next pregnancy, until birth, which would be offered at around 36-37 weeks. Less common treatments that may be considered depending on individual circumstances include steroid tablets or injections, or giving platelet transfusions to the baby. WHAT DOES THIS PAPER TELL YOU?: This paper considers the latest evidence in relation to treatment options in the management of pregnancies at risk of FNAIT. Specifically, we discuss the role of screening, when IVIg should be started, what dose should be used, and what evidence there is for maternal steroids. We also consider in very rare selected cases, the use of fetal blood sampling and giving platelet transfusions to the baby before birth. Finally, we consider the approaches to blood testing mothers to tell if babies are at risk, which is offered in some countries, and development of new treatments to reduce the risk of FNAIT.

[Polymorphism of human platelet antigens in Tunisian population: Clinical and anthropological interests]. / Polymorphisme des antigènes plaquettaires humains dans la population tunisienne : intérêt clinique et anthropologique.

OBJECTIVES: Human Platelet Antigens (HPA) are of considerable interest in obstetric transfusion medicine and anthropological genetics. This study aims to provide clinicians with a detailed database of HPA antigenic variants, which allows them to estimate the probability of allo-immunisation of each antigen. In addition, it aims to make an interethnic comparison of the Tunisian population with other populations. METHODS: The target population consists of 324 healthy and unrelated Tunisian blood donors recruited from the National Blood Transfusion Center in Tunis. DNA extraction was performed by the Salting Out method and molecular genotyping was performed by the PCR-SSP technique. The statistical analysis was performed using two approaches: manual calculation and computerized calculation. Phylogenetic trees were constructed through the use of Standard Genetic Distances that were calculated from allelic frequencies. RESULTS: With the exception of the HPA-4 system, statistical analysis showed that all HPA systems are polymorphic especially the two systems HPA-3 and HPA-15. The inter-ethnic analysis showed that Tunisians are closer to North Africans and Caucasians than Sub-Saharan and Asian populations, which shows genetic mixing between Tunisians, Arabs, Europeans and Africans. CONCLUSION: The results of this study could be exploited to prepare a ready-to-use genotyping plate dedicated to HPA antigens, with the aim of ensuring better management, especially for polytransfused patients.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

BACKGROUND: Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS: The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS: NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). CONCLUSION: The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

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

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

Noninvasive prenatal HPA-1 typing in HPA-1a negative pregnancies selected in the Polish PREVFNAIT screening program.

BACKGROUND: Anti-HPA-1a alloantibodies in HPA-1a negative mothers can lead to fetal/neonatal alloimmune thrombocytopenia (FNAIT). Noninvasive prenatal testing (NIPT) of HPA-1a determines fetuses at risk and the course of maternal antenatal treatment. STUDY DESIGN AND METHODS: The aim was to develop and validate HPA-1a NIPT by real-time polymerase chain reaction (PCR) or next-generation sequencing (NGS) for a high-throughput screening setting. DNA from 328 plasma samples of 299 HPA-1a negative pregnant women was examined for HPA-1a by real-time PCR and in two cases also by NGS (Ion Torrent). The results were compared with neonatal HPA-1a genotyping in 281 cases. RESULTS: HPA-1a NIPT was negative in 44 of 51 HPA-1a negative fetuses, inconclusive in five, and false positive in two. In 228 of 229 HPA-1a positive fetuses, the NIPT results were positive (mean threshold cycle 36.0 ± 1.7) and inconclusive in one. In 22 cases with HPA-1a positive fetuses analyzed twice, the sensitivity of HPA-1a detection was significantly higher at 28 weeks compared with 16 to 20 weeks. NGS efficiently detected the ITGB3 coding HPA-1a/b (1% and 5% fetal HPA-1a reads). CONCLUSION: Real-time PCR is reliable to predict the fetal HPA-1a positive genotype in a screening study, but false-positive results are reported in 4%, with unnecessary prenatal treatment if anti-HPA-1a is detected.