Approach to Neonatal Alloimmune Thrombocytopenia: The Perspective from a Transfusion Medicine Service

KEY POINTS

1. In fetal/NAIT, the mother forms antibodies against paternal antigens expressed on the surface of platelets of her fetus/infant. These antibodies cross the placenta and damage the fetal/neonatal platelets.

2. Neonatal alloimmune thrombocytopenia (NAIT) is a major cause of severe, isolated thrombocytopenia in term neonates. The incidence may be as high as 1 in 1,000 live births.

3. Although the term NAIT emphasizes the disease manifestations after birth, the condition can commence in utero with serious consequences including intrauterine death or intracerebral hemorrhage during the 20–24 weeks’ period of pregnancy.

4. Nearly in 85% of all Caucasian mothers develop some alloimmunization against HPA-1a.

5. We have limited information on the immunogenicity of various platelet antigens in terms of the alloantibody production, the efficacy of various antibodies in terms of transplacental transfer, and the impact of different alloantibodies on platelet function or on the incidence of bleeding complications. Our population data on the distribution of different platelet antigens in various ethnic groups is also limited. Consequently, the development of screening programs for NAIT has been difficult.

INTRODUCTION

Thrombocytopenia is a frequently seen hematological abnormality in neonates.^1,2 Platelet counts reach levels of around 150 × 10⁹/L by the late second trimester in fetuses and then plateau at these levels until term gestation.³ Platelets counts between 100–150 × 10⁹/L have been defined as mild thrombocytopenia, 50–100 × 10⁹/L as moderate, and counts <50 × 10⁹/L as severe thrombocytopenia.⁴ Mild thrombocytopenia may be seen in up to 25–30% of term infants and is usually self-limiting and of short duration. Moderate/severe thrombocytopenia occurs less frequently and is seen in 5–10% infants.^5–8

Neonatal thrombocytopenia with platelet counts less than 30–50 × 10⁶/L has been associated with an increased risk of serious hemorrhages into vital organs.^9,10 There are important associations with intrauterine infections, low Apgar scores, sepsis, and an overall higher acuity of illness even when the etiology is unclear. In premature infants, thrombocytopenia is a stronger predictor of intracranial hemorrhage (ICH) than their birth weight or gestational age.¹¹

In this review, we summarized the current definitions of neonatal thrombocytopenia and then focused on NAIT. It is noted that NAIT is an important cause of severe thrombocytopenia in neonates; we present the current evidence on its pathogenesis, clinical manifestations, evaluation, treatment, outcomes, and the future directions. This article combines peer-reviewed evidence from our own studies with an extensive literature search in the databases PubMed, EMBASE, and Scopus.

NORMAL PLATELET COUNTS IN NEONATES

Existing studies show that 98% of term neonates have platelet counts at or above 150 × 10⁹/L, and thrombocytopenia is usually defined as a number of circulating platelets below these levels. Some extremely premature infants born at 22–24 weeks’ gestation may have lower platelet counts at less than 100 × 10⁹/L in the first few days after birth, and most of them are asymptomatic.³ The timing of presentation of neonatal thrombocytopenia can also be used in diagnostic evaluation. Early-onset thrombocytopenia is noted within the first 72 hours after birth, and it may be caused by intrauterine infections, immune-mediated causes, perinatal asphyxia, and infections. Late-onset thrombocytopenia may be related to more diverse causes including bacterial and viral infections, systemic inflammation, hepatitis, necrotizing enterocolitis, and sometimes, may be iatrogenic due to thrombi in central lines or may develop as adverse drugs of certain drugs.^12–26 Genetic disorders with bone marrow dysfunction are less frequent, but can appear at any age.^27–29

Baer et al.²³ examined 11281 NICU admissions and identified severe thrombocytopenia in 273 (2.4%). Nearly 30% presented in the first 3 days after birth. Half presented by day 10, 75% by day 27, and 95% by day 100. The prevalence was inversely related to birth weight. Cutaneous bleeding was more common in patients with platelet counts of less than 20 × 10⁹/L; however, there was no statistically significant correlation between platelet counts and pulmonary, gastrointestinal, or intraventricular bleeding. The most common explanations for severe thrombocytopenia were acquired varieties of consumptive thrombocytopenia. Platelet transfusions (median, 5; range, 0–76) were administered to 86% of the patients. No deaths were ascribed to exsanguination. The mortality rates did not correlate with the lowest platelet counts but were proportionate to the number of platelet transfusions.

Wiedmeier et al.²⁴ examined platelet counts in neonates between the first and the ninetieth day after birth, from 47, 291 neonates delivered at 22–42 weeks gestation. The platelet counts obtained in the first 3 days of life, increased over the range of 22–42 weeks gestation. In those born in less than or 32 weeks gestation, the lower reference range (fifth percentile) was less than 104 × 10⁹/L, but it was less than 123 × 10⁹/L in late-preterm and late-term neonates. Advancing postnatal age affected platelet counts; during the first 9 weeks, the counts showed a sinusoidal pattern with two peaks; one at 2–3 weeks and a second at 6–7 weeks. The upper limit of expected counts (95th percentile) during these peaks were as high as less than 750 × 10⁹/L.

Christensen et al.²⁵ examined blood counts from extremely-low-birth-weight (ELBW) infants. Multiple platelet counts were obtained in 284, and 208 (73%) had one or more platelet counts less than 150 × 10⁹/L. Most were detected during the first days of life; 80% were detected before postnatal day 7. Thrombocytopenia was seen frequently in the smallest infants; 85% incidence among those born with weights less than or 800 gm, 60% among those 801–900 gm, and 53% among those 901–1000 gm. In 48% of cases, the cause of the thrombocytopenia went undiagnosed. The most common explanations were being small-for-gestational-age (SGA) or delivery to a hypertensive mother, disseminated intravascular coagulation, bacterial infection, fungal infection, and necrotizing enterocolitis, respectively.

The same group of scientists²⁶ studied a large cohort of SGA infants. A total of 31% (905 of 2,891) showed first-week thrombocytopenia compared to the 10% of matched non-SGA controls (p <0.0001). Of the 905, 102 had a recognized cause of thrombocytopenia (disseminated intravascular coagulation, early-onset sepsis, or extracorporeal membrane oxygenation). The remaining 803 did not have an obvious cause for their thrombocytopenia and were grouped as having “thrombocytopenia of SGA.” These infants had a mean nadir count on postnatal day 4 of 93 × 10⁹/L (standard deviation 51.8 × 10⁹/L, tenth percentile 50 × 10⁹/L, ninetieth percentile 175 × 10⁹/L). By postnatal day 14, platelet counts were more than or 150 × 10⁹/L in more than half of the patients. Severely SGA neonates (less than first percentile) had lower counts and longer duration of thrombocytopenia (p <0.001). Thrombocytopenia was more associated with SGA status than with the diagnosis of maternal preeclampsia.

NEONATAL ALLOIMMUNE THROMBOCYTOPENIA

Neonatal alloimmune thrombocytopenia is a condition in which maternal antibodies are formed against the paternal alloantigen expressed on fetal platelets.³⁰ The pathogenesis is analogous in some ways to that of the hemolytic disease of the newborn, which affects red blood cells. The fetal platelets carrying paternal antigens cross into the maternal circulation during normal low-grade transplacental cellular exchange or during larger-scale fetal-maternal hemorrhages/transfusions, which may occur during miscarriage or delivery. Antigen-presenting cells in maternal lymph nodes and spleen recognize these fetal antigens and stimulate the production of alloantibodies. The antiplatelet immunoglobulin G (IgG) antibodies are then actively transferred into the fetus and promote phago-immune destruction (Fig. 1).

The fetal platelets express specific human platelet antigens (HPAs) from sixteenth week onward.³¹ Platelets carrying HPA epitopes such as HPA-1a present on the glycoprotein (GP) IIIa binding to the syncytiotrophoblasts-derived microparticles (STMPs) increases the likelihood of alloimmunization (Fig. 2). Trophoblasts normally escape allorecognition because of low expression of human leukocyte antigen (HLA) class I and II molecules. There is some expression of HLA-G, which is a non-classical HLA-I molecule and promotes alloantigen tolerance.³²

Several antigen systems can be seen on the surface of human platelets, including the HPAs, the ABO antigens, and the HLA class I.^33,34 So far, 29 HPA systems have been identified on six platelet membrane GPs (GPIa, GPIbα, GPIbβ, GPIIb, GPIIIa, and CD109); 12 are grouped into 6 biallelic systems (HPA-1, -2, -3, -4, -5, and -15). All but one of these HPAs represents single nucleotide polymorphisms (SNPs) that result in single amino acid substitutions.

Most HPAs are located on the GPIIb/IIIa although the distribution of various HPAs may show some ethnic/geographic variation.³⁵ Anti-HPA-1a alloantibodies are the major cause of immune mediated thrombocytopenia in Caucasian, whereas the HPA-4 and Naka (anti-CD36) antibodies are the predominant cause in Asian population, especially in the Japanese.

Most infants with NAIT develop mild-moderate thrombocytopenia, although these reductions can add to the morbidity and mortality if these infants become critically-ill.³⁶ The destruction of platelets by maternal antibodies can increase the risk of bleeding, particularly that of ICH. Alloimmunization has been best studied with the HPA-1a antigen expressed on the β3 integrin (platelet GPIIIa; CD61).³⁷ This integrin may be intrinsically expressed on placental STMPs or may be acquired from circulating platelets.³⁸ The syncytiotrophoblasts-derived microparticles show these antigens complexed with CD51, which evokes an immune response. Platelets also express various surface molecules such as the integrin β2, β3, αIIb, CD109, and the complex GPIbα that may carry various HPAs.³⁹ The syncytiotrophoblasts-derived microparticles can induce variable immune responses, which include fetal alloantigen tolerance or induce immune responses that cause antibody-mediated platelet destruction.⁴⁰

EPIDEMIOLOGY OF NAIT

The incompatibility between fetal and maternal platelet antigens evokes the synthesis of maternal IgG antibodies, which then cross the placenta to induce fetal platelet destruction and cause NAIT.⁴¹ Similar to red cell alloimmunization such as in Rh antigen-mediated hemolysis, most cases of NAIT follow immune sensitization against platelets at the time of delivery in a previous pregnancy. However, many cases are seen in the very first pregnancy.⁴²

Human platelet antigens-1a is the best-studied trigger for the production of antiplatelet antibodies and causation of NAIT.⁴³ In one study, the incidence of thrombocytopenia in incompatible HPA-1a positive infants was 1:1000–2000.⁴⁴ The HPA-1bb phenotype in Caucasian population was about 2.5% and out of these one-third expressed the HLA-DR antigen B3*0101.⁴⁵ One-third of infants in this subset developed antibodies against HPA-1a and with moderate-to-severe thrombocytopenia.³

PLATELET ANTIGENS

ANTENATAL SCREENING

Neonatal alloimmune thrombocytopenia can be associated with intracranial hemorrhages in fetuses in utero. About 40 in 100,000 pregnancies can present with fetal-onset NAIT, with severe bleeding episodes in about three to four of these cases.⁵⁸ Most of these bleeds seem to before 36 weeks of gestation. Hence, antenatal screening is justified in pregnancies following one with documented NAIT.

To design and implement an appropriate screening program for NAIT, resources are needed to identify women at a risk for fetal-onset NAIT.⁵⁹ We need both experienced personnel and access to cost-effective, continuously-available laboratory protocols. These antenatal screening programs need to include both HLA typing and HPA detection in at-risk pregnancies.⁶⁰

LABORATORY DIAGNOSIS OF NAIT

When thrombocytopenia is detected in a newborn, a CBC should be obtained to ascertain whether thrombocytopenia is isolated or is a part of pancytopenia syndrome. Maternal blood counts should be obtained to refute the possibility of autoimmune thrombocytopenia. These should be followed by platelet serological tests on parental blood to confirm NAIT. The diagnostic testing for NAIT has the following two objectives: (a) To determine the incompatibility between the maternal and fetal platelet antigenic profile and (b) The detection of alloantibodies in the mother’s serum. Based on the results, the risk to the neonate can be projected.²⁴

The assays for detecting antigen are performed on parents’ blood, and if an incompatibility is detected, serum samples from the mother are tested to identify antibodies against any antigen(s) that may be detectable on the father’s platelets. If there are differences in parental genotypes and there are specific antibodies in the mother’s serum against the putative antigen, the diagnosis of NAIT needs consideration. The certainty of NAIT as a diagnosis is higher when an alloantibody against specific paternal antigen(s) identified on neonatal platelets is detectable in the maternal serum.^34,43 The antiplatelet antibodies in maternal serum can be detected by a variety of tests including the platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilization of platelet antigens (MAIPA), radioimmunoprecipitation (RIA), and flow cytometry-based assays. These tests are briefly described below:

Platelet Suspension Immunofluorescence Test (PSIFT)

The intact platelets are incubated with the patient’s or the control serum and allowed to bind to the antigenic epitopes. Then, fluorescence-labeled anti-human IgG/IgM are added as the secondary antibody and allowed to bind to the antibody bound to the antigenic epitope. The fluorescence-labeled platelets are then analyzed by fluorescence microscopy or by flow-cytometry.⁶¹

Flow-cytometry is highly sensitive to detect antibodies against most HPAs except for those against HPA-5 and HPA-15.⁶² These two antigens are expressed in lower densities on the platelet surface; only about 3,000–5,000 HPA-5 antigenic sites and only 1,000 HPA-15 sites are expressed on platelets. The binding assays can be confounded due to the simultaneous presence of multiple antibodies, particularly those against the HPA and HLA. To remove reactivity against anti-HLA antibodies, platelets can be pre-treated with chloroquine or acid to destroy the platelet surface β2 microglobulin. However, it might be difficult to completely eliminate this cross-reactivity if the anti-HLA antibodies are present in high titers. To reduce the confounding effect of anti-A and anti-B antibodies, we use blood group O platelets for these assays.

Antigen Capture Assays

There are three types of antigen capture assay [antigen capture Elisa (ACE)]; the ACE, the modified ACE (MACE), and the MAIPA.⁶³ These methods differ in the way the GP antigens are captured. The MAIPA is widely used in Europe and other countries, whereas MACE is preferred in the USA.

Radioimmunoprecipitation (RIP)

Radioimmunoprecipitation is more sensitive than MAIPA.⁶⁶ It utilizes unbound radioisotopes such as Iodine¹²⁵ for tagging surface GPs on platelets. These immunoprecipitated GPs are captured on a solid phase such as protein agarose, where these are recognized by maternal alloantibodies. The immunoprecipitated GPs are first eluted, and then identified using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by autoradiography. These proteins are identified based on characteristics such as molecular weight. More recently, several sensitive modifications of the RIP using non-fluorescent labeling have also been developed (Fig. 3).⁶⁷

In 2019, Vrbensky et al.⁶⁸ evaluated direct and indirect antiplatelet antibody tests for the diagnosis of immune thrombocytopenia (ITP). They concluded that the overall sensitivity of antiplatelet antibody testing was low (53%), but its specificity was high (>90%).

Newer Laboratory Tests

TREATMENT OPTIONS AND TRANSFUSION PRACTICES FOR NAIT

The treatment of choices for full-term neonates with suspected NAIT, with and without bleeding includes intravenous immunoglobulins (IVIG), corticosteroids, and antigen-negative or irradiated maternal platelets as emergency supportive measures.^30,85,86

Clinicians usually do not have continuous 24-hour access to maternal HPA-1a negative platelets.⁸⁷ Therefore, most physicians choose IVIG at a dose of 1 gm/kg weight for 2 consecutive days to the neonates who had no signs of bleeding but with platelet counts below a pre-decided threshold.⁸⁸ Small doses of corticosteroids may help by improving capillary fragility.³⁰ We will soon describe the current norms and preferences for treatment of NAIT in another review.

SUMMARY

Neonatal alloimmune thrombocytopenia is an important cause of severe neonatal thrombocytopenia. The clinical presentation may range from incidental, isolated abnormalities in laboratory tests to major clinical hemorrhages with life-threatening sequalae. If the platelet counts are less than 150 × 10⁹/L with no obvious cause to explain the thrombocytopenia, NAIT should be considered in the differential diagnosis. Although a large number of antigen systems have been clearly associated with NAIT, there are still many infants who have with suggestive clinical/laboratory profiles but unclear molecular diagnosis. These infants may have still-unidentified low-frequency and rare antigens.

ORCID

Akhil Maheshwari https://orcid.org/0000-0003-3613-4054

REFERENCES

1. Donato H. Neonatal thrombocytopenia: A review. II. Non-immune thrombocytopenia; platelet transfusion. Arch Argent Pediatr Aug 2021;119(4):e303–e314. DOI: 10.5546/aap.2021.eng.e303.

2. Caserta S, Zaccuri AM, Innao V, et al. Immune thrombocytopenia: options and new perspectives. Blood Coagul Fibrinolysis 2021;32(7):427–433. DOI: 10.1097/MBC.0000000000001058.

3. Williamson LM. Screening programmes for foetomaternal alloimmune thrombocytopenia. Vox Sang 1998;74(Suppl. 2):385–389. DOI: 10.1111/j.1423-0410.1998.tb05446.x.

4. Roberts I, Murray NA. Neonatal thrombocytopenia: causes and management. Arch Dis Child Fetal Neonatal Ed 2003;88(5):F359–F364. DOI: 10.1136/fn.88.5.f359.

5. Ulusoy E, Tufekci O, Duman N, et al. Thrombocytopenia in neonates: causes and outcomes. Ann Hematol 2013;92(7):961–967. DOI: 10.1007/s00277-013-1726-0.

6. Stanworth SJ. Thrombocytopenia, bleeding, and use of platelet transfusions in sick neonates. Hematology Am Soc Hematol Educ Program 2012;2012:512–516. DOI: 10.1182/asheducation-2012.1.512.

7. Beiner ME, Simchen MJ, Sivan E, et al. Risk factors for neonatal thrombocytopenia in preterm infants. Am J Perinatol 2003;20(1):49–54. DOI: 10.1055/s-2003-37948.

8. Sola–Visner M, Saxonhouse MA, Brown RE. Neonatal thrombocytopenia: what we do and don’t know. Early Hum Dev 2008;84(8):499–506. DOI: 10.1016/j.earlhumdev.2008.06.004.

9. von Lindern JS, van den Bruele T, Lopriore E, et al. Thrombocytopenia in neonates and the risk of intraventricular hemorrhage: a retrospective cohort study. BMC Pediatr 2011;11:16. DOI: 10.1186/1471-2431-11-16.

10. Fustolo-Gunnink SF, Fijnvandraat K, Putter H, et al. Dynamic prediction of bleeding risk in thrombocytopenic preterm neonates. Haematologica 2019;104(11):2300–2306. DOI: 10.3324/haematol.2018.208595.

11. Morrone K. Thrombocytopenia in the Newborn. Neoreviews 2018;19(1):e34–e41. DOI: 10.1542/NEO.19-1-E34.

12. Reed MAB, Rikabi N, Krugh D, et al. Thrombocytopenia in a neonate. Lab Med 2003;34(12):833–835. https://doi.org/10.1309/A6VH2BBEHYPNFBCN.

13. Kasivajjula H, Maheshwari A. Pathophysiology and current management of necrotizing enterocolitis. Indian J Pediatr 2014;81(5):489–497. DOI: 10.1007/s12098-014-1388-5.

14. Maheshwari A. Immunologic and hematological abnormalities in necrotizing enterocolitis. Clin Perinatol 2015;42(3):567–585. DOI: 10.1016/j.clp.2015.04.014.

15. MohanKumar K, Namachivayam K, Cheng F, et al. Trinitrobenzene sulfonic acid-induced intestinal injury in neonatal mice activates transcriptional networks similar to those seen in human necrotizing enterocolitis. Pediatr Res 2016;81(1):99–112. DOI: 10.1038/pr.2016.189.

16. MohanKumar K, Namachivayam K, Song T, et al. A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions. Nat Commun 2019; 10(1):3494. DOI: 10.1038/s41467-019-11199-5.

17. Namachivayam K, MohanKumar K, Garg L, et al. Neonatal mice with necrotizing enterocolitis-like injury develop thrombocytopenia despite increased megakaryopoiesis. Pediatr Res 2017;81(5):817–824. DOI: 10.1038/pr.2017.7.

18. Kennedy J, Holt CL, Ricketts RR. The significance of portal vein gas in necrotizing enterocolitis. Am Surg 1987;53(4):231–234. PMID: 3579031.

19. Kenton AB, Hegemier S, Smith EO, et al. Platelet transfusions in infants with necrotizing enterocolitis do not lower mortality but may increase morbidity. J Perinatol 2005;25(3):173–177. DOI: 10.1038/sj.jp.7211237.

20. Khalak R, Chess PR. Fulminant necrotizing enterocolitis in a premature neonate treated for supraventricular tachycardia. J Perinatol 1998;18(4):306–307. PMID: 9730203.

21. Kilic N, Buyukunal C, Dervisoglu S, et al. Maternal cocaine abuse resulting in necrotizing enterocolitis. An experimental study in a rat model. II. Results of perfusion studies. Pediatr Surg Int 2000;16(3):176–178. DOI: 10.1007/s003830050717.

22. Kim WY, Kim WS, Kim IO, et al. Sonographic evaluation of neonates with early-stage necrotizing enterocolitis. Pediatr Radiol 2005;35(11):1056–1061. DOI: 10.1007/s00247-005-1533-4.

23. Baer VL, Lambert DK, Henry E, et al. Severe thrombocytopenia in the NICU. Pediatrics 2009;124(6):e1095–e1100. DOI: 10.1542/peds.2009-0582.

24. Wiedmeier SE, Henry E, Sola–Visner MC, et al. Platelet reference ranges for neonates, defined using data from over 47,000 patients in a multihospital healthcare system. J Perinatol 2009;29(2):130–136. DOI: 10.1038/jp.2008.141.

25. Christensen RD, Henry E, Wiedmeier SE, et al. Thrombocytopenia among extremely low birth weight neonates: data from a multihospital healthcare system. J Perinatol 2006;26(6):348–53. DOI: 10.1038/sj.jp.7211509.

26. Christensen RD, Baer VL, Henry E, et al. Thrombocytopenia in small-for-gestational-age infants. Pediatrics 2015;136(2):e361–e370. DOI: 10.1542/peds.2014-4182.

27. Li X, Li Y, Lei M, et al. Congenital thrombocytopenia associated with GNE mutations in twin sisters: a case report and literature review. BMC Med Genet 2020;21(1):224. DOI: 10.1186/s12881-020-01163-2.

28. Sillers L, Van Slambrouck C, Lapping–Carr G. Neonatal thrombocytopenia: etiology and diagnosis. Pediatr Ann 2015;44(7):e175–e180. DOI: 10.3928/00904481-20150710-11.

29. Christensen RD, Wiedmeier SE, Yaish HM. A neonate with congenital amegakaryocytic thrombocytopenia associated with a chromosomal microdeletion at 21q22.11 including the gene RUNX1. J Perinatol 2013;33(3):242–244. DOI: 10.1038/jp.2012.53.

30. Bussel JB, Vander Haar EL, Berkowitz RL. New developments in fetal and neonatal alloimmune thrombocytopenia. Am J Obstet Gynecol 2021;225(2):120–127. DOI: 10.1016/j.ajog.2021.04.211.

31. O’Toole TE, Loftus JC, Plow EF, et al. Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Blood 1989;74(1):14–18. PMID: 2752106.

32. Maslanka K, Yassai M, Gorski J. Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia. J Clin Invest 1996;98(8):1802–1808. DOI: 10.1172/JCI118980.

33. Curtis BR, McFarland JG. Human platelet antigens–2013. Vox Sang 2014;106(2):93–102. DOI: 10.1111/vox.12085.

34. Davoren A, Curtis BR, Aster RH, et al. Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia. Transfusion 2004;44(8):1220–1225. DOI: 10.1111/j.1537-2995.2004.04026.x.

35. Hayashi T, Hirayama F. Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping. Blood Transfus 2015;13(3):380–390. DOI: 10.2450/2015.0275-14.

36. Van den Hof MC, Nicolaides KH. Platelet count in normal, small, and anemic fetuses. Am J Obstet Gynecol 1990;162(3):735–739. DOI: 10.1016/0002-9378(90)90997-l.

37. Bayat B, Traum A, Berghofer H, et al. Current anti-HPA-1a standard antibodies react with the beta3 integrin subunit but not with alphaIIbbeta3 and alphavbeta3 complexes. Thromb Haemost 2019;119(11):1807–1815. DOI: 10.1055/s-0039-1696716.

38. Kumpel BM, Sibley K, Jackson DJ, et al. Ultrastructural localization of glycoprotein IIIa (GPIIIa, beta 3 integrin) on placental syncytiotrophoblast microvilli: implications for platelet alloimmunization during pregnancy. Transfusion 2008;48(10):2077–2086. DOI: 10.1111/j.1537-2995.2008.01832.x.

39. Huang J, Li X, Shi X, et al. Platelet integrin alphaIIbbeta3: signal transduction, regulation, and its therapeutic targeting. J Hematol Oncol 2019;12(1):26. DOI: 10.1186/s13045-019-0709-6.

40. Chen ZY, Oswald BE, Sullivan JA, et al. Platelet physiology and immunology: pathogenesis and treatment of classical and non-classical fetal and neonatal alloimmune thrombocytopenia. Ann Blood 2019;4:29. DOI: 10.21037/aob.2019.12.04.

41. Arnold DM, Smith JW, Kelton JG. Diagnosis and management of neonatal alloimmune thrombocytopenia. Transfus Med Rev 2008;22(4):255–267. DOI: 10.1016/j.tmrv.2008.05.003.

42. Stuge TB, Skogen B, Ahlen MT, et al. The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia. Transfus Apher Sci 2011;45(1):53–59. DOI: 10.1016/j.transci.2011.06.003.

43. Peterson JA, McFarland JG, Curtis BR, et al. Neonatal alloimmune thrombocytopenia: pathogenesis, diagnosis and management. Br J Haematol 2013;161(1):3–14. DOI: 10.1111/bjh.12235.

44. Kjeldsen–Kragh J, Killie MK, Tomter G, et al. A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia. Blood 2007;110(3):833–839. DOI: 10.1182/blood-2006-08-040121.

45. Kjeldsen–Kragh J, Titze TL, Lie BA, et al. HLA-DRB3*01:01 exhibits a dose-dependent impact on HPA-1a antibody levels in HPA-1a-immunized women. Blood Adv 2019;3(7):945–951. DOI: 10.1182/bloodadvances.2019032227.

46. Metcalfe P, Ouwehand WH, Sands D, et al. Collaborative studies to establish the first WHO reference reagent for detection of human antibody against human platelet antigen-5b. Vox Sang 2003;84(3):237–240. DOI: 10.1046/j.1423-0410.2003.00281.x.

47. Newman PJ, Derbes RS, Aster RH. The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. J Clin Invest 1989;83(5):1778–1781. DOI: 10.1172/JCI114082.

48. Clemetson KJ. Platelets and primary haemostasis. Thromb Res 2012;129(3):220–224. DOI: 10.1016/j.thromres.2011.11.036.

49. Newman PJ, Aster R, Boylan B. Human platelets circulating in mice: applications for interrogating platelet function and survival, the efficacy of antiplatelet therapeutics, and the molecular basis of platelet immunological disorders. J Thromb Haemost 2007;5(Suppl. 1):305–309. DOI: 10.1111/j.1538-7836.2007.02466.x.

50. Srzentic SJ, Lilic M, Vavic N, et al. Genotyping of eight human platelet antigen systems in Serbian blood donors: Foundation for Platelet Apheresis Registry. Transfus Med Hemother 2021;48(4):228–233. DOI: 10.1159/000514487.

51. Peterson JA, Balthazor SM, Curtis BR, et al. Maternal alloimmunization against the rare platelet-specific antigen HPA-9b (Max a) is an important cause of neonatal alloimmune thrombocytopenia. Transfusion 2005;45(9):1487–1495. DOI: 10.1111/j.1537-2995.2005.00561.x.

52. Ghevaert C, Wilcox DA, Fang J, et al. Developing recombinant HPA-1a-specific antibodies with abrogated Fcgamma receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia. J Clin Invest 2008;118(8):2929–2938. DOI: 10.1172/JCI34708.

53. Ogasawara K, Ueki J, Takenaka M, et al. Study on the expression of ABH antigens on platelets. Blood 1993;82(3):993–999. PMID: 8338959.

54. Kankirawatana S, Kupatawintu P, Juji T, et al. Neonatal alloimmune thrombocytopenia due to anti-Nak(a). Transfusion 2001;41(3):375–377. DOI: 10.1046/j.1537-2995.2001.41030375.x.

55. King KE, Kao KJ, Bray PF, et al. The role of HLA antibodies in neonatal thrombocytopenia: a prospective study. Tissue Antigens 1996;47(3):206–211. DOI: 10.1111/j.1399-0039.1996.tb02542.x.

56. Thude H, Schorner U, Helfricht C, et al. Neonatal alloimmune thrombocytopenia caused by human leucocyte antigen-B27 antibody. Transfus Med 2006;16(2):143–149. DOI: 10.1111/j.1365-3148.2006.00634.x.

57. Sasaki M, Yagihashi A, Kobayashi D, et al. Neonatal alloimmune thrombocytopenia due to anti-human leukocyte antigen antibody: a case report. Pediatr Hematol Oncol 2001;18(8):519–524. DOI: 10.1080/088800101753328484.

58. Espinoza JP, Caradeux J, Norwitz ER, et al. Fetal and neonatal alloimmune thrombocytopenia. Rev Obstet Gynecol 2013;6(1):e15–e21. PMCID: PMC3651544.

59. Tiller H, Killie MK, Skogen B, et al. Neonatal alloimmune thrombocytopenia in Norway: poor detection rate with nonscreening versus a general screening programme. BJOG 2009;116(4):594–598. DOI: 10.1111/j.1471-0528.2008.02068.x.

60. Kamphuis MM, Paridaans N, Porcelijn L, et al. Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review. BJOG 2010;117(11):1335–1343. DOI: 10.1111/j.1471-0528.2010.02657.x.

61. von dem Borne AE, Verheugt FW, Oosterhof F, et al. A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol 1978;39(2):195–207. DOI: 10.1111/j.1365-2141.1978.tb01089.x.

62. Peterson JA, Gitter M, Bougie DW, et al. Low-frequency human platelet antigens as triggers for neonatal alloimmune thrombocytopenia. Transfusion 2014;54(5):1286–1293. DOI: 10.1111/trf.12450.

63. Kiefel V. The MAIPA assay and its applications in immunohaematology. Transfus Med 1992;2(3):181–188. DOI: 10.1111/j.1365-3148.1992.tb00153.x.

64. Sarkar RS, Philip J, Jain N. Detection and identification of platelet-associated alloantibodies by a solid-phase modified antigen capture Elisa (MACE) technique and its correlation to platelet refractoriness in multi platelet concentrate transfused patients. Indian J Hematol Blood Transfus 2015;31(1):77–84. DOI: 10.1007/s12288-014-0374-4.

65. Brighton TA, Evans S, Castaldi PA, et al. Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias. Blood 1996;88(1):194–201. PMID: 8704174.

66. Dreyfus M, Kaplan C, Verdy E, et al. Frequency of immune thrombocytopenia in newborns: a prospective study. Immune Thrombocytopenia Working Group. Blood 1997;89(12):4402–4406. PMID: 9192764.

67. Smith JW, Hayward CP, Warkentin TE, et al. Investigation of human platelet alloantigens and glycoproteins using non-radioactive immunoprecipitation. J Immunol Methods 1993;158(1):77–85. DOI: 10.1016/0022-1759(93)90260-e.

68. Vrbensky JR, Moore JE, Arnold DM, et al. The sensitivity and specificity of platelet autoantibody testing in immune thrombocytopenia: a systematic review and meta-analysis of a diagnostic test. J Thromb Haemost 2019;17(5):787–794. DOI: 10.1111/jth.14419.

69. Metzner K, Bauer J, Ponzi H, et al. Detection and identification of platelet antibodies using a sensitive multiplex assay system–platelet antibody bead array. Transfusion 2017;57(7):1724–1733. DOI: 10.1111/trf.14122.

70. Bertrand G, Jallu V, Gouet M, et al. Quantification of human platelet antigen-1a antibodies with the monoclonal antibody immobilization of platelet antigens procedure. Transfusion 2005;45(8):1319–1323. DOI: 10.1111/j.1537-2995.2005.00195.x.

71. Hamidpour M, Khalili G, Tajic N, et al. Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP. Am J Blood Res 2014;4(2):86–92. PMID: 25755908.

72. Winkelhorst D, Porcelijn L, Muizelaar E, et al. Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing. Transfusion 2019;59(9):2989–2996. DOI: 10.1111/trf.15454.

73. Hurd CM, Cavanagh G, Schuh A, et al. Genotyping for platelet-specific antigens: techniques for the detection of single nucleotide polymorphisms. Vox Sang 2002;83(1):1–12. DOI: 10.1046/j.1423-0410.2002.00187.x.

74. Kengkate M, Butthep P, Kupatawintu P, et al. Comparison of a simple-probe real-time PCR and multiplex PCR techniques for HPA-1 to HPA-6 and HPA-15 genotyping. J Clin Lab Anal 2015;29(2):94–99. DOI: 10.1002/jcla.21734.

75. Wu GG, Kaplan C, Curtis BR, et al. Report on the 14th International Society of Blood Transfusion Platelet Immunology Workshop. Vox Sang 2010;99(4):375–381. DOI: 10.1111/j.1423-0410.2010.01348.x.

76. Curtis BR, Edwards JT, Hessner MJ, et al. Blood group A and B antigens are strongly expressed on platelets of some individuals. Blood 2000;96(4):1574–1581. PMID: 10942408.

77. Curtis BR. Genotyping for human platelet alloantigen polymorphisms: applications in the diagnosis of alloimmune platelet disorders. Semin Thromb Hemost 2008;34(6):539–548. DOI: 10.1055/s-0028-1103365.

78. Hashmi G, Shariff T, Seul M, et al. A flexible array format for large-scale, rapid blood group DNA typing. Transfusion 2005;45(5):680–688. DOI: 10.1111/j.1537-2995.2005.04362.x.

79. Dunbar SA. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin Chim Acta 2006;363(1–2):71–82. DOI: 10.1016/j.cccn.2005.06.023.

80. Shehata N, Denomme GA, Hannach B, et al. Mass-scale high-throughput multiplex polymerase chain reaction for human platelet antigen single-nucleotide polymorphisms screening of apheresis platelet donors. Transfusion 2011;51(9):2028–2033. DOI: 10.1111/j.1537-2995.2011.03082.x.

81. Montpetit A, Phillips MS, Mongrain I, et al. High-throughput molecular profiling of blood donors for minor red blood cell and platelet antigens. Transfusion 2006;46(5):841–848. DOI: 10.1111/j.1537-2995.2006.00805.x.

82. Arinsburg SA, Shaz BH, Westhoff C, et al. Determination of human platelet antigen typing by molecular methods: importance in diagnosis and early treatment of neonatal alloimmune thrombocytopenia. Am J Hematol 2012;87(5):525–528. DOI: 10.1002/ajh.23111.

83. Morel–Kopp MC, Clemenceau S, Aurousseau MH, et al. Human platelet alloantigen typing: PCR analysis is not a substitute for serological methods. Transfus Med 1994;4(1):9–14. DOI: 10.1111/j.1365-3148.1994.tb00237.x.

84. Watkins NA, Schaffner–Reckinger E, Allen DL, et al. HPA-1a phenotype–genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope. Blood 2002;99(5):1833–1839. DOI: 10.1182/blood.v99.5.1833.

85. Wyckoff SL, Hudson KE. Targeting the neonatal Fc receptor (FcRn) to treat autoimmune diseases and maternal–fetal immune cytopenias. Transfusion 2021;61(5):1350–1354. DOI: 10.1111/trf.16341.

86. Ohto H. Neonatal alloimmune thrombocytopenia. Nihon Rinsho 1997;55(9):2310–2234. PMID: 9301295.

87. Kjeldsen–Kragh J, Bengtsson J. Fetal and neonatal alloimmune thrombocytopenia: new prospects for fetal risk assessment of HPA-1a-negative pregnant women. Transfus Med Rev 2020;34(4):270–276. DOI: 10.1016/j.tmrv.2020.09.004.

88. Wagner N, Kagan K, Maden Z, et al. Neonatal alloimmune thrombocytopenia. Geburtshilfe Frauenheilkd 2008;68(4):406–408. DOI: 10.1055/s-2008-1038593.