Fetal And Neonatal Alloimmune Thrombocytopenia Research Articles

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

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

Does anti-HPA-1a affect birthweight in fetal and neonatal alloimmune thrombocytopenia?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) ensues from parental incompatibility for platelet alloantigens with maternal sensitization. HPA-1a/1b incompatibility is the most common cause of FNAIT in Caucasians. Placental villitis and lower birthweight in FNAIT suggest anti-HPA-1a may have effects beyond inducing thrombocytopenia. Does FNAIT secondary to anti-HPA-1a result in smaller newborns and, the corollary, does antenatal management of FNAIT increase birthweight? Birthweights of 270 FNAIT-affected newborns from a randomized clinical trial and a NAITbabies.org survey (135 paired siblings) were compared with those of published controls and treated to untreated FNAIT-affected siblings. Birthweights were converted to percentiles to account for gestational age, sex, and role of birth order in birth weight. Body weights of FNAIT-affected and -unaffected pups in a mouse FNAIT model were analyzed. Untreated siblings in both the clinical trial and NAITbabies.org cohorts were not small, compared with normal controls. However, treated siblings in both cohorts had significantly higher birthweight percentiles compared with their previous untreated affected sibling. After accounting for gestational age, sex, and birth order, increased birthweight percentile in treated compared with the untreated siblings remained significant in both cohorts. FNAIT-affected neonatal mice had lower bodyweights than FNAIT-unaffected pups. Untreated FNAIT-affected newborns were not small; however, treatment of FNAIT-affected pregnancies increased newborn birthweights despite corrections to account for other factors that might have influenced the results. High dose IVIG is believed to "block" FcRn and lower maternal anti-HPA-1a levels, and thus increase birthweights by reducing levels of maternal anti-HPA-1a and reducing placental villitis.

Natural history of human platelet antigen 1a-alloimmunised pregnancies: a prospective observational cohort study

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare disease that untreated can lead to intracranial haemorrhage or death. The natural history of FNAIT is still unclear; therefore, the benefits of screening cannot be estimated and no routine screening is yet in place. We aimed to assess the incidence of clinically detectable FNAIT among pregnancies in human platelet antigen-1a (HPA-1a)-immunised women. We did a prospective observational cohort study of pregnant women negative for rhesus D (RhD) and rhesus c (Rhc) antigens, without age limits, who underwent routine antenatal screening for red cell antibodies at 27 weeks' gestation and were typed for HPA-1a between March 1, 2017, and May 1, 2020. HPA-1a-negative women were tested for HPA alloantibodies. Health-care professionals were masked to all test results. The main outcome was the proportion of neonates with severe, clinically detectable FNAIT, defined as having an intracranial bleed, organ bleed, or bleeding-related death observed during pregnancy or within the first week of life. Cases of clinically detectable FNAIT not categorised as severe were categorised as mild. This study is registered with ClinicalTrials.gov (NCT04067375). Of 153 106 women typed for HPA-1a, 3722 (2·4%) were negative for HPA-1a. 913 HPA-1a-negative women gave informed consent, underwent HPA-1a antibody screening, and were included in the study. Anti-HPA-1a antibodies were detected in 85 HPA-1a-negative participants, among whom three with HPA-1a-negative fetuses and one with a previous child with FNAIT were excluded. As controls, 820 HPA-1a-negative, non-immunised pregnancies and 2704 randomly selected pregnancies of women negative for RhD and Rhc who were typed HPA-1a positive were included. Of 81 fetuses included, one (1·2%) was diagnosed with severe HPA-1a-mediated intracranial haemorrhage and three (3·7%) had mild FNAIT. Gravidity and parity did not seem to be risk factors for HPA-1a immunisation. 73 (90·1%) of 81 HPA-1a-immunised women were positive for HLA-DRB3*01:01. Our data suggest that, without intervention, the incidence of major clinically detectable bleeding in FNAIT is estimated as 11 (95% CI 0-32) per 10 000 HPA-1a-negative pregnancies. These findings imply that severe bleeding is a rare event that potentially could be prevented by a screening programme. Landsteiner Foundation for Blood Transfusion Research and Sanquin.

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

Pregnant Women at Low Risk of Having a Child with Fetal and Neonatal Alloimmune Thrombocytopenia Do Not Require Treatment with Intravenous Immunoglobulin.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition in which maternal alloantibodies to fetal platelets cause fetal thrombocytopenia that may lead to intracranial hemorrhage (ICH). Off-label intravenous immunoglobulin (IVIg) has for 30 years been the standard of care for pregnant women who previously have had a child with FNAIT. The efficacy of this treatment has never been tested in a placebo-controlled clinical trial. Although IVIg treatment may improve the neonatal outcome in women who previously have had a child with FNAIT-associated ICH, the question is whether IVIg is necessary for all immunized pregnant women at risk of having a child with FNAIT. The results from some recent publications suggest that antenatal IVIg treatment is not necessary for women who are (1) HPA-1a-immunized and HLA-DRB3*01:01-negative, (2) HPA-1a-immunized with a previous child with FNAIT but without ICH or (3) HPA-5b-immunized. If IVIg is not used for these categories of pregnant women, the amount of IVIg used in pregnant women with platelet antibodies would be reduced to less than ¼ of today's use. This is important because IVIg is a scarce resource, and the collection of plasma for the treatment of one pregnant woman is not only extremely expensive but also requires tremendous donor efforts.

HLA antibodies in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by antibodies against human platelet antigens (HPA). However, in many cases that meet clinical criteria for the condition, maternal sera do not have HPA antibodies. In studies examining whether human leukocyte antigen (HLA) antibodies cause FNAIT, the results are limited and inconclusive. This study sought to examine whether clinically suspected FNAIT cases with absent maternal HPA antibodies had different HLA antibody strength and specificity compared to controls. A retrospective case-control study assessed class I HLA antibody strength and specificity in cases submitted for testing to Versiti, Wisconsin. There were 813 cases that met initial screening criteria, but written consent could only be obtained for 50. After review of medical records and expert panel review, 31 cases with clinical criteria of FNAIT and maternal HLA but not HPA antibodies were included. Each case was matched for maternal age, gestational age at delivery, parity, and race/ethnicity to two controls from unaffected pregnancies that had maternal serum HLA antibodies. FNAIT cases were found to have both significantly higher HLA antibody strength, measured by mean fluorescence index (MFI), and broader HLA antibody specificity at antigen epitope level, compared to matched controls (p < .001). p-values remained significant after controlling for parity and gestational age at delivery. Additional studies are needed to further examine whether the strong HLA antibodies identified in HPA-antibody-negative cases directly cause neonatal thrombocytopenia and whether prenatal treatment may be warranted in select cases to prevent recurrence.

In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA).

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.

Clinical and laboratory predictors of fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the most common cause of intracranial hemorrhage (ICH) in thrombocytopenic term infants. We investigated clinical and laboratory predictors of severe FNAIT in a tertiary care referral center. Retrospective cohort study over a 30-year period. We defined FNAIT as recurrence of neonatal thrombocytopenia in a subsequent pregnancy; and severe outcomes as any of: (1) a birth platelet count below 20 × 109 /L; (2) ICH or (3) fetal death. We used a generalized estimating equations analysis and classification tree analysis to identify risk factors for severe FNAIT in a subsequent pregnancy. During index pregnancies (n=135 in 131 mothers), 71 infants (52.6%) had severe outcomes including a platelet count <20 × 109 /L (n=45), fetal or neonatal ICH (n=32), or fetal death (n=4). During subsequent pregnancies (n=72), 15 infants (20.8%) had severe outcomes including birth platelets <20 × 109 /L (n=10), ICH (n=2), or death (n=3). Forty-two women (58.3%) received antenatal intravenous immune globulin (IVIG) during subsequent pregnancies. Eight mothers (n=9 infants) had severe FNAIT outcomes despite receiving antenatal IVIG. Maternal antibodies to human platelet antigens (HPA) was the only independent predictor of severe FNAIT in a subsequent pregnancy (OR=25.3, p=.004). Nevertheless, one of 43 infants from antibody-negative mothers had a severe outcome. The presence of anti-HPA is highly indicative of the diagnosis of severe FNAIT; however, we observed one infant who had severe FNAIT recurrence, defined using strict clinical criteria, without a maternal antibody. Improved diagnostic and therapeutic strategies are needed to prevent severe FNAIT in high-risk mothers.

Severe course of COVID-19 in pregnant woman and newborn's alloimmune thrombocytopenia - case study and review of the literature.

So far, little is known about the impact of the coronavirus disease 2019 (COVID-19) on pregnancy and data is often inconsistent. Even less information has been published on the management of severe courses of COVID-19 in pregnant women. By writing this article, we aim to share our experience in the treatment of pregnant woman critically ill with COVID-19 and newborn's condition, FNAIT (Fetal and neonatal alloimmune thrombocytopenia), as well as literature review of this disease. After admission,the woman's respiratory status rapidly worsened, requiring administration of oxygen and in the end ECMO therapy. At the 9th day of ECMO support, and 28 weeks 3 days of gestation, due to mother's prognosis and increased feto-placental vascular resistance a decision of Caesarean section was taken. The neonate required intensive care not only due to extreme prematurity but coagulation disorder, alloimmune thrombocytopenia, which we diagnosed a few weeks after delivery.

Postnatal treatment for children with fetal and neonatal alloimmune thrombocytopenia: a multicentre, retrospective, cohort study

Children affected by fetal and neonatal alloimmune thrombocytopenia (FNAIT) are at risk of severe intracranial haemorrhage. Management in the postnatal period is based on sparse evidence. We aimed to describe the contemporary management and outcomes of patients with FNAIT in high-income countries. In this multicentre, retrospective, cohort study, we set up a web-based registry for the collection of deidentified data on the management and course of neonates with FNAIT. Eight centres from seven countries (Australia, Norway, Slovenia, Spain, Sweden, the Netherlands, and the USA) participated. Eligibility criteria comprised neonates with FNAIT being liveborn between Jan 1, 2010, and Jan 1, 2020; anti-human platelet antigen (HPA) alloantibodies in maternal serum; confirmed maternal and fetal HPA incompatibility; and bleeding detected at antenatal ultrasound, neonatal thrombocytopenia (<150 × 109 platelets per L), or both in the current or previous pregnancy. Clinical data were retrieved from local medical records of the first neonatal admission and entered in the registry. The key outcome was the type of postnatal treatment given to neonates with FNAIT. Other outcomes were daily median platelet counts in the first week of life, median platelet count increment after first unmatched versus first matched transfusions, and the proportion of neonates with mild or severe bleeding. 408 liveborn neonates with FNAIT were entered into the FNAIT registry, of whom 389 from Australia (n=74), Norway (n=56), Slovenia (n=19), Spain (n=55), Sweden (n=31), the Netherlands (n=138), and the USA (n=16) were included in our analyses. The median follow-up was 5 days (IQR 2-9). More neonates were male (241 [64%] of 379) than female (138 [36%]). Severe thrombocytopenia (platelet count <50 × 109 platelets per L) was reported in 283 (74%) of 380 neonates, and extreme thrombocytopenia (<10 × 109 platelets per L) was reported in 92 (24%) neonates. Postnatal platelet count nadir was higher in the no-treatment group than in all other groups. 163 (42%) of 389 neonates with FNAIT received no postnatal treatment. 207 (53%) neonates received platelet transfusions, which were either HPA-unmatched (88 [43%] of 207), HPA-matched (84 [41%]), or a combination of both (35 [17%]). The proportion of neonates who received HPA-matched platelet transfusions varied between countries, ranging from 0% (Slovenia) to 63% (35 of 56 neonates; Norway). Postnatal intravenous immunoglobulin treatment was given to 110 (28%) of 389 neonates (alone [n=19] or in combination with platelet transfusions [n=91]), with the proportion receiving it ranging from 12% (17 of 138 neonates; the Netherlands) to 63% (ten of 16 neonates; the USA) across countries. The median platelet increment was 59 × 109 platelets per L (IQR 35-94) after HPA-unmatched platelet transfusions and 98 × 109 platelets per L (67-134) after HPA-matched platelet transfusions (p<0·0001). Severe bleeding was diagnosed in 23 (6%) of 389 liveborn neonates, with one having a severe pulmonary haemorrhage and 22 having severe intracranial haemorrhages. Mild bleeding was diagnosed in 186 (48%) neonates. Postnatal management of FNAIT varies greatly between international centres, highlighting the absence of consensus on optimal treatments. Our data suggest that HPA-matched transfusions lead to a larger median platelet count increment than HPA-unmatched transfusions, but whether HPA matching is also associated with a reduced risk of bleeding remains unknown. Sanquin.

Neonatal and fetal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the result of maternal alloantibodies interaction with human platelets antigen. Maternal alloantibodies are generated following the contact of maternal immune system with paternally acquired antigens on the fetal human platelets (PLTs). HPA-1a alloantibodies are responsible for more than 80% of cases of FNAIT. Low prevalence of FNAIT that occurs in less than 1 case out of 1000 births, and even a lower risk for intracranial hemorrhage makes a controversial discussion whether a screening in pregnant patients would bring benefits. Concerning pathogenesis of FNAIT, it has been shown that fetal platelets caring HPA-1a antigen enters maternal circulation and produce an immune response, that leads to production of HPA-1a maternal antibodies that afterwards can pass the placenta causing fetal thrombocytopenia. The majority of mothers who gave birth to a newborn with FNAIT usually are asymptomatic, although they might have a previous affected pregnancy. Although management of pregnancies complicated by FNAIT is still in discuss and controversial, a systemic review studies in 2017 showed that best guidance and delivery for pregnancies that are at risk for FNAIT is a weekly maternal administration of intravenous immunoglobulin, adding glucocorticoids depending on severity of FNAIT.

Noninvasive Prenatal Testing in Immunohematology-Clinical, Technical and Ethical Considerations.

Hemolytic disease of the fetus and newborn (HDFN), as well as fetal and neonatal alloimmune thrombocytopenia (FNAIT), represent two important disease entities that are caused by maternal IgG antibodies directed against nonmaternally inherited antigens on the fetal blood cells. These antibodies are most frequently directed against the RhD antigen on red blood cells (RBCs) or the human platelet antigen 1a (HPA-1a) on platelets. For optimal management of pregnancies where HDFN or FNAIT is suspected, it is essential to determine the RhD or the HPA-1a type of the fetus. Noninvasive fetal RhD typing is also relevant for identifying which RhD-negative pregnant women should receive antenatal RhD prophylaxis. In this review, we will give an overview of the clinical indications and technical challenges related to the noninvasive analysis of fetal RBCs or platelet types. In addition, we will discuss the ethical implications associated with the routine administration of antenatal RhD to all pregnant RhD-negative women and likewise the ethical challenges related to making clinical decisions concerning the mother that have been based on samples collected from the (presumptive) father, which is a common practice when determining the risk of FNAIT.

Antenatally Diagnosed Fetal Subdural Hematoma: A Case Report

Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) is a disease that affects babies. In FNAIT, the platelet count is decreased because the mother’s immune system attacks her fetus platelets. A low platelet count increases the risk of bleeding in the fetus and newborn. FNAIT is the leading cause of severe thrombocytopenia in the fetus and neonate. The most severe complication of FNAIT is intracranial hemorrhage, which occurs in 10%-20% of symptomatic infants. If the bleeding occurs in the brain, there may be long-term effects. The Pathophysiology of atraumatic fetal Subdural Hematoma (SDH) remains unknown. It is extrapolated from the SDH of a shaken baby syndrome in infants. A detailed prenatal investigation is crucial to elucidate the underlying etiology, which may be associated with a better prognosis. We present a case of antenatally diagnosed SDH secondary to FNAIT that was normal up to one month after follow-up.

Generation of induced pluripotent stem cell line derived from FNAIT patient with CD36 deficiency mutations

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by anti-CD36 isoantibodies is a common disease and the frequency of type I CD36 deficiency is relatively high in eastern Asian populations.Currently, patient-specific induced pluripotent stem cells (hiPSC) are believed to be useful tools for studying anti-CD36 mediated FNAIT and finding new therapeutic approaches to the disease.We generated an iPSC line from peripheral blood mononuclear cells of a patient carrying a 329-330delAC of the CD36 gene.The iPSC expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had a normal karyotype, and retained the patient-specific mutation.

Cerebral vasculature exhibits dose-dependent sensitivity to thrombocytopenia that is limited to fetal/neonatal life

AbstractWhether increasing platelet counts in fetal and neonatal alloimmune thrombocytopenia (FNAIT) is effective at preventing intracerebral hemorrhage (ICH) has been a subject of debate. The crux of the matter has been whether thrombocytopenia is the major driver of ICH in diseases such as FNAIT. We recently demonstrated in mice that severe thrombocytopenia was sufficient to drive ICH in utero and in early neonatal life. It remains unclear what degree of thrombocytopenia is required to drive ICH and for how long after birth thrombocytopenia can cause ICH. By inducing a thrombocytopenic range, we demonstrate that there is a large buffer zone of mild thrombocytopenia that does not result in ICH, that ICH becomes probabilistic at 40% of the normal platelet number, and that ICH becomes fully penetrant below 10% of the normal platelet number. We also demonstrate that although the neonatal mouse is susceptible to thrombocytopenia-induced ICH, this sensitivity is rapidly lost between postnatal days 7 and 14. These findings provide important insights into the risk of in utero ICH with varying degrees of thrombocytopenia and into defining the developmental high-risk period for thrombocytopenia-driven ICH in a mouse model of FNAIT.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody.

Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.4) with modified Fc region or altered N-glycan structures. The mAb 26.4 wild-type (WT) variants all showed efficient platelet clearance capacity and ability to mediate phagocytosis independent of their N-glycan structure, compared with an effector silent variant (26.4.AAAG), although the modified N-glycan variants showed differential binding to FcγRs measured in vitro. In an in vivo model, female mice were transfused with platelets from transgenic mice harboring an engineered integrin β3 containing the HPA-1a epitope. When these preimmunized mice were bred with transgenic males, Abs against the introduced epitope induced thrombocytopenia in the offspring, mimicking FNAIT. Prophylactic administration of the mAb 26.4.WT, and to some extent the mAb 26.4.AAAG, prior to platelet transfusion resulted in reduced alloimmunization in challenged mice and normal platelet counts in neonates. The notion that the effector silent variant hampered alloimmunization demonstrates that rapid platelet clearance, as seen with mAb 26.4.WT, is not the sole mechanism in action. Our data thus successfully demonstrate efficient Ab-mediated immunosuppression and prevention of FNAIT by anti-HPA-1a monoclonal variants, providing support for potential use in humans.

[Management of high-risk pregnancies in a context of maternal antiplatelet alloimmunization: Expert opinion of the French-speaking working group].

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating disease, seen in 1/800-1000 neonates. FNAIT is the most common cause of early-onset isolated severe neonatal thrombocytopenia in maternity wards. A working group on fetomaternal platelet alloimmunization was created in 2017, under the auspices on the French Group of Thrombosis and Hemostasis (GFHT). The objective was to survey clinical practices for management of high-risk pregnancies in a context of suspected or confirmed FNAIT. Recommendations published by the ICTMG were translated in French, and discussed (Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach. British J of Haematology, 2019, 185, 549-562). The study involved centers from France, Switzerland and Belgium: Angers, Besançon, Bordeaux, Brest, Créteil/Clamart, Genève, Grenoble, Liège, Lille, Lyon, Marseille, Nantes, Nîmes, Paris (hôpitaux Necker, Robert Debré et Trousseau), Poitiers, Rennes, Saint-Etienne, Strasbourg, Toulouse, Tours. Expert opinion was validated on September 23, 2020 (consensus≥90%).

Are HPA-5b antibodies a significant cause of FNAIT and associated bleeding or merely an incidental finding?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal IgG antibodies produced against incompatible human platelet antigens (HPA) on fetal platelets. The IgG antibodies cross the placenta and can cause clearance of fetal platelets, thrombocytopenia and bleeding, sometimes severe. Most cases are identified in first pregnancies when bleeding, bruising and thrombocytopenia are recognized after delivery of the infant. Laboratory tests of the parent’s blood that show HPA-specific antibodies in maternal serum and incompatibility between the parents for the cognate HPA confirm a diagnosis of FNAIT. In white populations, HPA-1a antibodies are the most frequently implicated (>80%), followed by HPA-5b antibodies (˜15%). It is well-established that infants affected by FNAIT involving HPA-1a antibodies frequently have severe thrombocytopenia (platelets ≤25 000/µl) associated with organ bleeding and intracranial haemorrhage (ICH). However, our understanding of the clinical significance of HPA-5 antibodies is less clear. Case reports of infants born with FNAIT due to HPA-5a or HPA-5b antibodies generally have higher platelet counts than infants born with FNAIT due to HPA-1a antibodies, and bleeding is often less severe. One possible reason for this difference is that platelets express significantly more (˜80 000 molecules/platelet) of the glycoprotein complex (GP)IIb/IIIa that carries HPA-1a on GPIIIa (CD61) than they do of GPIa/IIa (3,000–5,000 copies per platelet) that carries HPA-5 on GPIa (CD49b). However, there are exceptional cases of FNAIT involving HPA-5 antibodies, describing infants affected by severe thrombocytopenia with or without ICH.1, 2 In their paper, Vos et al report results from a large, retrospective cohort study of suspected FNAIT cases with a focus on clinical outcomes in cases with maternal HPA-5b antibodies, in an effort to determine the clinical significance of HPA-5b antibodies in FNAIT.3 Data were collected over an 18-year period from 1,864 cases of suspected FNAIT that were submitted for laboratory evaluation to the reference laboratory Sanquin Diagnostic Laboratories, (Amsterdam, the Netherlands). HPA antibodies were detected in maternal sera from 262 (14%) cases: 161 (61%) HPA-1a antibodies and 60 (23%) HPA-5b antibodies. Complete clinical histories could be obtained for 129 HPA-1a and 40 HPA-5b cases. Severe bleeding, infant postnatal and nadir platelet counts, mortality and postnatal treatment were compared between the two groups. HPA-1a cases showed more bleeding than HPA-5b cases (76% vs. 31%, respectively), however, severe bleeding (mostly ICH) was similar (11% vs. 10%). The median platelet count after birth was lower for HPA-1a cases, but the nadir platelet counts were not significantly different between groups. There were six (5%) HPA-1a and one (3%) HPA-5b cases that resulted in perinatal death. Postnatal treatments consisting of platelet transfusions, IVIgG or both, were administered more frequently in HPA-1a cases (69%) compared to HPA-5b cases (22%). Antenatal IVIgG treatment was given in five HPA-1a and 11 HPA-5b cases. Interestingly, physicians were aware of a FNAIT diagnosis because of a previously affected infant in only one case in each group. All other cases were suspected when cerebral abnormalities were detected during routine ultrasound screens. Four of the treated HPA-1a pregnancies were either terminated or resulted in fetal death, and the infants’ platelet counts for two of the antenatally detected cases were both <30 000/µl. However, infants in all 11 of the HPA-5b IVIgG-treated cases were born with platelets >120 000/µl. Overall, these findings confirm what previous studies have indicated, that HPA-1a FNAIT is associated with more severe clinical outcomes and that HPA-5b FNAIT is often, but not always, less severe. When the clinical data were stratified by the presence of other risk factors for neonatal alloimmune thrombocytopenia there were no major differences observed in the results. Two major strengths of the study are the comprehensive clinical case histories presented and the thorough platelet antibody and genotyping workups performed on parental blood, that when combined, significantly increase the accuracy and clinical significance of the findings. There are a few other notable results that deserve discussion. As mentioned,4, 5 ≥80% of FNAIT cases are reported to be associated with antibodies against HPA-1a, so why did de Vos et al only detect HPA-1a antibodies in 61% of cases? This was probably because many hospital laboratories can detect HPA-1a antibodies using commercial platelet antibody detection kits and, therefore, reference laboratories like Sanquin receive fewer HPA-1a cases and more difficult FNAIT cases for testing. In contrast, the authors detected a higher percentage (23%) of anti-HPA-5b than has been reported in previous studies (˜10–15%).4-6 This could be due to the authors’ practice of performing repeat postpartum testing 6 weeks later to ensure that HPA-1a or HPA-5b antibodies were not missed; however, the authors did not indicate how many HPA-5b antibodies were only detected by testing later maternal samples. Last, the authors detected 10 FNAIT cases with maternal anti-HPA-5b in which there was no fetal-maternal incompatibility for HPA-5b. This is not an uncommon finding, including in our own platelet reference laboratory here at Versiti, Milwaukee, WI. As the authors discovered, this finding is usually a result of a previous pregnancy with a different father, maternal blood transfusions or in rare cases, non-paternity. Why it occurs more often for HPA-5b is not known. However, the authors questioned whether HPA-5b antibodies might be an incidental finding. So, they calculated the expected frequencies of incompatibility between mother and fetus for either HPA-1a or HPA-5b and for the actual frequencies in their study cases. They found that the study frequencies were higher than expected for both HPA-1a (86% expected and 100% study) and HPA-5b (52% expected and 79% study), in support of HPA-5b antibodies not being incidentally present. Therefore, how can physicians determine which HPA-5b FNAIT cases will result in a mild or more severely affected infant? It helps to remember that in FNAIT cases associated with antibodies against any HPA, even HPA-1a, outcomes vary from modest to severe thrombocytopenia and no bleeding, to minor signs of bleeding, and no ICH to severe ICH. As a result, I agree with the authors that for pregnancies where maternal serum has antibodies against any HPA and there is documented fetal-maternal incompatibility for that HPA, antenatal IVIgG protocols should be discussed with the patients and implemented as required. A large, prospective clinical study of FNAIT cases associated with maternal HPA-5 antibodies would be required to begin to understand their true clinical significance, but such a study of a rare disorder (<1 in 1,000 births) is near impossible to conduct. However, de Vos et al announce that they have such a study underway! I look forward to the results.

Maternal Anti-αIIb Antibodies Target Fetal Hematopoietic Stem Cells and May Lead to Pancytopenia and Miscarriage in Fnait

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a devastating disorder characterized by an aberrant maternal immune response against paternally inherited polymorphisms on fetal platelet antigens. Affected fetuses can experience severe bleeding (e.g. intracranial hemorrhage; ICH), neurological sequelae and death. The targeted platelet antigens are located predominantly on platelet surface receptor αIIbβ3 integrin. It is estimated that 0.5-1.5/1000 live neonates are born with FNAIT although this incidence may largely underestimate the prevalence of the disease due to miscarriage. We have previously demonstrated that anti-β3 alloantibodies can target not only fetal platelets but also the β3 subunit on endothelial cells leading to ICH and placental vascular pathologies (J Clin Invest. 2015; Nat Commun. 2017). Although the number of polymorphisms in the αIIb subunit is similar to the β3 subunit, reported incidences of anti-β3 mediated FNAIT are far greater. It is currently unknown whether this due to miscarriage in anti-αIIb mediated FNAIT. αIIb was previously considered to be exclusively expressed on platelets, however hematopoietic stem cells (HSCs) and their early progenitors can express αIIbβ3 during embryonic development and αIIb is one of the earliest markers of hematopoietic commitment. Throughout gestation, HSCs migrate from yolk sac to the aorta-gonadal-mesonephros, placenta, fetal liver and finally bone marrow. We hypothesize that miscarriage is prevalent in anti-αIIb mediated FNAIT and that maternal anti-αIIb antibodies may target HSCs resulting in decreased blood cell counts, increased bleeding and fetal death.Methods: We established an animal model of anti-αIIb mediated FNAIT by immunizing αIIb-/- mice with 108 wild-type (WT, αIIb+/+) platelets. Immunized αIIb-/- mice were then bred with WT males. We used non-immunized (naïve) αIIb-/- x WT breeding pairs as a control. Sera of immunized and naïve mice were collected prior to breeding and ultrasound was used to detect fetal death. Mice were sacrificed at E14.5 and fetuses, placentas and fetal livers were weighed. Cell suspensions were prepared from these tissues as well as fetal blood and analyzed with flow cytometry. Fluorescent markers were used to identify HSCs (CD34+/CD117+/Lin- or CD34+/CD45+) and blood cell counts for T cells (CD3+,CD4+,CD8+), B cells (CD19+), myeloid lineage cells (CD11b+) and megakaryocytes (GPIbα+).Results: Sera of immunized mice showed a significant anti-αIIb immune response. Pups of immunized mice had low platelet counts, reduced body weight and bleeding diathesis. Ultrasound revealed miscarriage occurred mainly at E14.5 of immunized αIIb+/- fetuses. The miscarriage rate in our anti-αIIb mediated FNAIT model appeared significantly higher than the anti-β3 model. At E14.5, our results showed live fetuses of immunized mice had significantly lower body and liver weight although no significant difference in placental weight was observed. We found fetal liver HSCs have increased maternal antibody binding when incubated with sera from immunized αIIb-/- mice versus naïve αIIb-/- mice in vitro, suggesting that maternal anti-αIIb antibodies target fetal HSCs in vivo . Preliminary data also demonstrated that fetuses in the immunized group had reduced overall HSCs as well as αIIb+ HSC subsets in the placenta, liver and yolk sac. Fetal blood analysis showed reduced hematopoietic lineage cells in the immunized group, particularly T cells (CD3+), B cells and megakaryocytes.Conclusions: We established an anti-αIIb mouse model of FNAIT and revealed a high rate of miscarriage, which may explain the paucity of reported human cases. Our results indicate maternal anti-αIIbantibodies may bind and target fetal HSCs during embryonic development, which contributes significantly to impaired placental and fetal liver development, and fetal death. Destruction of early αIIb+ HSC progenitors may explain the decrease in HSCs (both αIIb+ and αIIb-) and account for the pancytopenia in FNAIT fetuses. Further experiments are in progress to elucidate the mechanism by which destruction of αIIb+ HSCs contributes to fetal pathology.*J.S. and B.E.O contributed equally to this work DisclosuresNo relevant conflicts of interest to declare.

Placental Complement Activation in Fetal and Neonatal Alloimmune Thrombocytopenia: An Observational Study.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.