Diagnosis and Management of Neonatal Alloimmune Thrombocytopenia

TRANSFUSION MEDICINE REVIEWS Vol 22, No 4

October 2008

Diagnosis and Management of Neonatal Alloimmune Thrombocytopenia Donald M. Arnold, James W. Smith, and John G. Kelton Neonatal alloimmune thrombocytopenia (NAT) is a lifethreatening bleeding disorder caused by maternal platelet antibodies produced in response to fetal platelet antigens inherited from the father. Antiplatelet antibodies cross the placenta and cause destruction of fetal platelets, leading to severe thrombocytopenia, and potentially bleeding, including fatal intracerebral hemorrhage. Incompatibilities between maternal and fetal platelets for the human platelet antigen 1a (previously called PLA1) account for most of the patients with NAT, but other antigens are commonly implicated. Diagnostic testing for NAT involves genotyping of maternal, paternal, and sometimes

fetal DNA; platelet antigen phenotyping; and maternal platelet antibody investigations using specialized platelet glycoprotein specific assays. The management of women and infants at risk for NAT remains largely empiric; and mounting evidence points to prohibitive risks of invasive procedures such as fetal blood sampling and intrauterine platelet transfusions, except in rare circumstances. Improvements in our understanding of the pathophysiology of NAT, and of clinical and laboratory predictors of severity, may help develop better treatments and improve our ability to identify mothers at risk. C 2008 Published by Elsevier Inc.

EONATAL alloimmune thrombocytopenia (NAT) is a devastating bleeding disorder. It often presents unexpectedly and ranges in severity from mild thrombocytopenia at birth to intracerebral hemorrhage (ICH) in utero, at delivery, or in the first days of life. Over the last few decades, clinical and scientific advances have improved our understanding of the pathophysiology of NAT and refined our diagnostic capabilities. However, the management of women at risk for NAT remains largely empiric; and the lack of an adequate screening test limits our ability to prevent NAT in first pregnancies. In this overview, the scientific presentation, pathophysiology, and scientific evidence for management of NAT are reviewed. A diagnostic algorithm for NAT testing in use at our institution is presented, and areas requiring further research are discussed.

result in long-term cognitive or neurologic impairment. Neonatal alloimmune thrombocytopenia is caused by an incompatibility between fetal and maternal platelet antigens stimulating maternal immunoglobulin G (IgG) antibodies that cross the placenta and cause fetal thrombocytopenia.

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CLINICAL PRESENTATION OF NAT

Neonatal alloimmune thrombocytopenia is a syndrome characterized by thrombocytopenia and bleeding in an otherwise healthy infant. Intracerebral hemorrhage occurs in approximately 20% of affected infants at birth or antenatally, and may be fatal or

Epidemiology Neonatal alloimmune thrombocytopenia affects approximately 1 in 2000 pregnancies. This estimate derives from prospective studies of consecutive pregnancies or newborns as reviewed recently by

From the The McMaster Platelet Immunology Diagnostic Laboratory, McMaster University, Hamilton, Ontario, Canada; and Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada. D.M. Arnold holds a New Investigator Award from the Canadian Institutes of Health Research. J.G. Kelton is a Canada Research Chair. Address reprint requests to Donald M. Arnold, MD, 1200 Main St W, Room 3V-48, Hamilton, Ontario, Canada L8N 3Z5. E-mail: [email protected] 0887-7963/08/$ - see front matter n 2008 Published by Elsevier Inc. doi: 10.1016/j.tmrv.2008.05.003

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Kjeldsen-Kragh et al.1 In the largest study in which human platelet antigen 1a (HPA-1a)–negative women with detectable antibodies were identified from among 100 448 consecutive pregnancies, the incidence of NAT was 1 in 1700 live births.1 In 2 other large screening studies of approximately 26 000 pregnancies each, the incidence of NAT was 1 in 25002 and 1 in 5000.3 In the 2 largest prospective studies of consecutive newborns (N = 24 1014 and N = 15 9325), the incidence of NAT was 1 in 5000 and 1 in 1700, respectively. Some of the variability in the reported frequency of NAT may reflect differences in testing methods or diagnostic criteria, ascertainment bias as a result of fetal blood sampling (FBS), or ethnic diversity. Although NAT is relatively rare, neonatal thrombocytopenia is common. Approximately 1 in 100 infants will have a platelet count less than 150 × 109/L at birth, and 1 in 400 will have a platelet count less than 50 × 109/L.5,6 The most common causes of neonatal thrombocytopenia are infection and immune and congenital thrombocytopenia 5,7,8 (Table 1). Severe thrombocytopenia (birth platelet count <20 × 109/L) is strongly supportive of NAT.5 The diagnosis of NAT is frequently missed.3,9 To help develop diagnostic criteria, Bussel and colleaTable 1. Differential Diagnosis of Thrombocytopenia (<150 × 109/L) in a Newborn Infant Infections Toxoplasmosis Rubella Cytomegalovirus Sepsis DIC Maternal ITP Maternal drug exposure Congenital heart disease Kasabach-Merritt syndrome Hereditary thrombocytopenia MYH-9 macrothrombocytopenia May-Hegglin anomaly Sebastian syndrome Fechner syndrome TAR Amegakarocytic thrombocytopenia Wiskott-Aldrich syndrome Fanconi anemia Bone marrow infiltration Congenital leukemia Reticuloendotheliosis Abbreviations: DIC, disseminated intravascular coagulation; ITP, immune thrombocytopenic purpura; TAR, thrombocytopenia with absent radii.

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gues10 analyzed 110 thrombocytopenic infants with NAT due to HPA-1a incompatibility and compared them with 56 non-NAT thrombocytopenic infants. Factors associated with NAT were a platelet count less than 50 × 109/L, ICH in an infant with high Apgar scores and normal birth weight, ICH in utero, petechiae or ecchymosis, and the absence of other medical illnesses. Comparison Between NAT and Hemolytic Disease of the Newborn Hemolytic disease of the newborn (HDN) is the red blood cell counterpart of NAT with some important differences (Table 2). In both disorders, maternal sensitization by fetal antigens stimulates maternal IgG antibodies that target fetal cells. In HDN, red blood cells are destroyed, most commonly as a result of incompatibility for the rhesus D antigen, resulting in fetal anemia, cardiac failure, and erythroblastosis fetalis. In NAT, platelets are rapidly cleared, most commonly because of HPA1a incompatibility, resulting in fetal thrombocytopenia and bleeding. In HDN, first pregnancies are generally unaffected because maternal sensitization occurs only at the time of delivery or after a fetalmaternal hemorrhage, whereas first pregnancies are affected in NAT because of the passage of fetal platelets or platelet antigens into maternal circulation early in the pregnancy. Universal screening programs and prevention with rhesus immunoglobulin have significantly reduced the incidence of HDN; currently, no screening program is available for NAT because of the lack of a well-defined preclinical phase, insufficient specificity of current diagnostic tests, and the high risk and high cost of treatments. Thus, as opposed to a population-based approach for HDN prevention, an individualized patient-based approach is required for NAT management. Bleeding Complications of NAT Infants with NAT are at high risk of bleeding. Generalized petechiae and mucocutaneous purpura are common, but ICH is the most feared bleeding complication, occurring in approximately 20% of affected infants with anti–HPA-1a antibodies.11,12 One third of ICH events are fatal12; and nonfatal cases are often associated with serious neurologic sequelae including mental retardation, cerebral palsy, cortical blindness, and seizures.13 Up to 80% of ICH occur in utero,11,12,14 as early as 16

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Table 2. Comparison of the Etiology and Clinical Features and NAT and HDN

Affected cells Most common antigen Affected pregnancy Timing of maternal sensitization Clinical presentation (infant) Clinical presentation (fetus) Risk factor Treatment Prevention Efficacy of prevention

NAT

HDN

Platelets HPA-1a 1st (and subsequent) 16 weeks' gestation Thrombocytopenia, hemorrhage Intracranial hemorrhage Previously affected infant Supportive IVIg during subsequent pregnancy Unknown

Red blood cells Rh-D 2nd (and subsequent) At birth or following fetal-maternal hemorrhage Hemolytic anemia, jaundice, kernicterus Hydrops fetalis Rh-negative mothers Supportive Rh-immune globulin ~99%

weeks' gestation,15-17 which may lead to structural and/or metabolic central nervous system abnormalities including hydrocephaly, porencephalic cysts, neuronal migrational disorders, and superficial siderosis.15,18,19 Bleeding at other anatomical sites is unusual.20 Predictors of Severity of NAT A history of NAT in a sibling is the most important predictor of NAT.21 Severity of the disease tends to worsen with subsequent pregnancies14 and possibly with increasing gestational age22; however, the severity of NAT is often difficult to predict. In a study of 107 fetuses with NAT, only a history of ICH in a previously affected sibling was a significant predictor of NAT severity.14 Conversely, in 1 study, up to 7% of affected infants developed ICH despite no previous history of ICH in a sibling.23 Therefore, the management of all subsequent pregnancies requires a high level of vigilance. The implicated antigen is also associated with disease severity. Severe thrombocytopenia and bleeding have been associated with HPA-1a (PL A1 ) alloimmunization (which accounts for most of the NAT in the white population) and HPA-3a incompatibility (a rare cause of NAT), whereas HPA-5a/5b (Zav-b/a) and HPA-15a/15b (Gov-b/a) incompatibilities are common but rarely cause severe disease. Antigen density on the platelet surface, immune response modifiers including HLA genotype, timing and titer of antibody production during pregnancy, and antigen expression on other tissues (including endothelial cells)24 may explain the variability in phenotype attributable to different platelet antigens. The titer of maternal anti–HPA-1a antibody has been shown to predict neonatal thrombocytopenia in some screening studies, 2,25-27 but not in others.3,28,29 Discrepant results may be due to

technical differences in the method of antibody detection, timing of antibody screening, and threshold values of antibody concentrations. Standardized anti–HPA-1a serum should help with interlaboratory comparisons in the future.30 A new rise in antibody titer is likely to be an important predictor, especially during the first pregnancy, although antibody titers must be interpreted with caution because NAT can occur in the absence of detectable antibodies and because treatments (such as intravenous immune globulin [IVIg]) can reduce antibody levels.11,31 PATHOPHYSIOLOGY OF NAT

Neonatal alloimmune thrombocytopenia is caused by maternal IgG alloantibodies stimulated by fetal platelet antigens inherited from the father. Fetal platelet antigens are expressed as early as 16 weeks' gestation17 and, soon thereafter, enter the maternal circulation by mechanisms that remain poorly understood.32 Alloimmunization can occur during the first pregnancy, although in a recent screening study, many women with HPA1a antibodies had previously been pregnant.3 The IgG alloantibodies cross the placenta and target fetal platelets, causing rapid Fc-mediated clearance by the fetal reticulendothelial system. The passage of blood through the lungs immediately after birth increases the exposure of IgG-sensitized platelets to reticulendothelial cells and may explain the postnatal drop in platelet count that is often observed.33 Antiplatelet antibodies may persist in maternal circulation for many years and can be further stimulated by additional exposures through subsequent pregnancies or transfusions.34 Several mechanisms have been proposed to explain the frequency of bleeding in NAT, which is higher than other thrombocytopenic disorders such as immune thrombocytopenic purpura. Alloantibodies that cause NAT have been shown

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Fig 1. Pyramid model of NAT. Each tier shows the number of affected pregnancies and infants expected out of a sample of 10 000 white women. Each tier represents a proportion of the tier below, shown in parentheses along the right side of the pyramid.

to induce a platelet function defect characterized by impaired fibrinogen binding, clot retraction, and platelet aggregation35-37; and antibodies to HPA-1a expressed on the endothelial cell surface may result in a loss of endothelial integrity. In addition, the period of thrombocytopenia may last for many weeks38,39 as a result of antibody-mediated destruction of megakaryocytes, thus compounding the risk of bleeding. Conceptual Pyramid Model of NAT Neonatal alloimmune thrombocytopenia can be conceptualized as a pyramid model, using HPA-1a incompatibility as an example (Fig 1). The base of the pyramid includes all HPA-1a– negative mothers, representing all women at risk (approximately 2% of all women). The next tier represents the proportion of HPA-1a–negative women who have detectable antibodies (10% of the women at risk), followed by the proportion of those women with a thrombocytopenic infant (30% of HPA-1a–negative, antibody-positive mothers).1-3 The top tier of the pyramid represents the proportion of thrombocytopenic infants with ICH (approximately 20% of affected infants). This model does not account for affected infants whose mothers have no detectable antibody.11

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NAT remains uncertain.40,41 The ABO antibodies are often found in maternal serum during investigation of NAT42,43,44 ; and although A and B antigens are usually expressed weakly on platelets, maternal immunization to AB antigens may play a role in NAT in those infants who express the type II high-expressor phenotype.45 Investigation of specific platelet antibodies requires appropriate target platelets and sensitive glycoprotein-specific methods capable of identifying specificities to all known platelet alloantigens, including the rare antigens (eg, HPA-6 through HPA-14) and those on CD109 (HPA-15) (Table 3). Nomenclature of the HPA System Initially, platelet alloantigens were identified based on the binding of alloantibodies from immunized individuals to target platelets. Before the introduction of glycoprotein-specific assays, platelet antigens were named according to the first few letters of the proband surname in whom they were detected and alleles were denoted with the superscript a or b according to the chronological order in which they were discovered (eg, Koa Kob, Table 3. Platelet Antigens Implicated in NAT by Frequency New Nomenclature

Old Nomenclature

HPA-1a HPA-5a HPA-5b HPA-15a HPA-15b

PLA1 Br-b, Zav-b Br-a, Zav-a Gov-b Gov-a

HPA-3a HPA-2a HPA-2b

Bak-a Ko-b Ko-a

HPA-6b HPA-7b HPA-8b HPA-9b HPA-10b HPA-11b HPA-12b HPA-13b HPA-14b HPA-16b

Ca-a, Tu-a Mo-a Sr-a Max-a La-a Gro-a Iy-a Sit-a Oe-a Duv-a

Platelet antigens rarely associated with NAT

HPA-1b HPA-3b

PLA2 Bak-b

Platelet antigens commonly implicated in NAT in the Asian population

HPA-4a

Pen-a

Major platelet antigens (high frequency of NAT)

Platelet antigens accounting for up to 2% of NAT collectively

Platelet antigens implicated in NAT but found on very few individuals in the general population

Platelet Antigens There are 3 major antigen systems found on the surface of platelets: ABH blood group antigens, HLA class I antigens, and specific platelet antigens (HPAs). The HLA and ABO antibodies can interfere with diagnostic testing for specific platelet antibodies, but their causative role in

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Fig 2. Schematic representation of the genetic mutations associated with HPAs, including HPA-1a (from Webert et al. In: Wintrobe's Clinical Hematology, 12th Edition, with permission).

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Baka Bakb); however, a rapid increase in the detection of new antigens and the recognition that previously identified antigens had been given new names by different investigators caused confusion. This prompted the establishment of the HPA system as a method of standardizing platelet antigen nomenclature.46 The HPA system assigns a name to platelet antigens based on their recognition by specific antisera in the chronological order in which they were identified; and high- and low-frequency alleles are designated a and b, respectively. Thus, the PLA1 /PLA2 antigens became HPA-1a/HPA-1b; and the Baka/Bakb antigens became HPA-3a/HPA3b. For a few loci, alloantibodies to the lowfrequency antigen had been reported first; and thus, their a/b allele assignment was reversed in the new system. For example, Zava/Zavb became HPA-5b/ HPA-5a and Gova/Govb became HPA-15b/HPA15a. In all, 16 unique polymorphisms encoding HPAs have now been described.47 Human platelet antigens are epitopes on platelet glycoproteins. Most polymorphisms, including most of the low-frequency or private antigens, are expressed on GPIIIa. The genetic polymorphisms of HPAs are defined either by single nucleotide polymorphisms or by in-frame deletion of a codon. For example, HPA-1b results from a Leu to Pro change at residue 33 on GPIIIa, which is encoded by a T to C substitution at position 196 of GPIIIa complementary DNA48; and a single amino acid deletion on GPIIIa induces the expression of the HPA-14b antigen49 (Fig 2). As such, these polymorphisms can be readily detected using standard polymerase chain reaction (PCR) techniques. HPA-1a and Other Platelet Antigens Implicated in NAT In large studies, HPA-1a has been implicated in about 80% of serologically confirmed NAT.50 While HPA-1a is the leading cause of NAT in the white population, it is rare in the Japanese population because of a much lower gene frequency for HPA-1b (0.01 vs 0.15). Conversely, HPA-4a (Pena ) alloimmunization is far more common among the Japanese, in part because of the genetic distribution of the HPA-4a/4b alleles.51 After HPA-1a, HPA-5b and HPA-15a are implicated in up to 20% of patients with NAT.52 All other specificities, including HPA3a and -2a/-2b, account for most of the remaining 2% of patients.

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Genetic incompatibilities to most platelet antigens (except HPA-1a) are common because of the relatively high expression of both alleles in the general population. For example, among whites, approximately 16%, 17%, and 13% of random couples would be incompatible for HPA-5b, -15a, and -3a, respectively. Genetic incompatibility to platelet antigens is necessary, but not sufficient for NAT, as the observed frequency of the clinical syndrome is far lower than what would be expected based on genetic frequency alone. Overall, 2% of the North American population is HPA-1a negative (1 in 50), whereas the observed frequency of NAT due to HPA-1a is only 0.05% (1 in 2000), representing roughly 2% to 3% of mothers at risk. Thus, other factors including immune response modifiers must be important in the pathophysiology; for example, the HLA DRB3⁎0101 (DRw52a) haplotype has been associated with NAT due to HPA-1a incompatibility.53,54 A large number of antibodies to low-frequency or private antigens have also been implicated in NAT (HPA-6b through HPA-14b, and HPA-16b). These antigens are expressed in very few individuals in the general population; therefore, antibodies to these antigens (eg, HPA-8b) will recognize the glycoprotein target on paternal platelets, but not on most random donor target platelets. The implication for diagnostic testing is that the platelets from the father are optimal platelet targets for the detection of NAT antibodies are platelets from the father. One of the private antigens implicated in NAT, HPA-9b (Maxa), may be more common than previously thought. 55,56 Human platelet antigen 9b results from a Val837Met polymorphism on α-IIb and is expressed only in a subset of individuals with a corresponding serine at position 843 that defines the HPA-3b antigen. Therefore, all individuals positive for HPA-9b also express HPA-3b, indicating that the Val837Met polymorphism occurred chronologically after the polymorphism defining HPA-3a/3b. LABORATORY TESTING FOR NAT

In the 20 years since the first description of platelet alloantibodies in 1959, only 3 additional alloantibody specificities were described using the available assays (platelet suspension immunofluorescence test and direct binding enzyme immunoassay). These tests were limited by low specificity, as they were unable to distinguish between specific platelet

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antibodies, nonspecific platelet-associated IgG (PAIgG), anti-ABH, and anti-HLA antibodies. The use of flow cytometry allowed for the detection of antibodies at the individual platelet level26; however, like platelet suspension immunofluorescence, flow cytometry also detects nonspecific antibodies and reactivity to HLA. Thus, a panel of typed platelet targets is required for the flow cytometric investigations of NAT. The emergence of glycoproteinspecific assays such as the monoclonal antibody immobilization of platelet antigen (MAIPA) and the radioimmunoprecipitation (RIP) assay allowed for the detection of alloantibodies with specificity for platelet antigens even at low levels of expression (eg, HPA-15a/15b on CD109).57,58 Glycoprotein-Specific Assays for the Detection of NAT Antibodies The MAIPA assay. The MAIPA is an enzyme immunoassay that uses glycoprotein-specific monoclonal antibodies to identify the antigenic target of maternal alloantibodies. Only those antibodies with specificity to the glycoprotein will be detected, which are readily distinguishable from anti-HLA and platelet-associated IgG. The MAIPA and other similar monoclonal-based assays (eg, antigen capture assay) require specific monoclonal antibodies. It was because of this limitation that anti–HPA-15 (Gov) antibodies, which react with an epitope on CD109, were initially not detected using MAIPA techniques with capture monoclonal antibodies to GPIa/IIa, Ib/IX, and IIb/IIIa. Radioimmunoprecipitation. The RIP assay is more sensitive, albeit more complicated, than MAIPA. It requires the use of unbound radioisotopes (eg, iodine 125) to tag surface glycoproteins on target platelets. Maternal alloantibodies that recognize epitopes on these glycoproteins are immunoprecipitated by capture on a solid phase (eg, protein A–agarose). The immunoprecipitated proteins are eluted and identified by molecular weight using sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by autoradiography. The RIP assay has the capability of identifying all platelet glycoproteins, including previously uncharacterized (or novel) antigen targets. Thus, it is a useful screening test for NAT. Limitations include the cost, the need for specialized expertise, and the use of radioisotopes. As opposed to MAIPA, the RIP assay can readily identify anti–HPA-15 reactivity, which is how this antigen was first discovered.57

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Diagnostic Testing for NAT The goals of NAT investigations are (1) to determine if a maternal-fetal platelet antigen incompatibility is present and (2) to detect (and sometimes quantitate) platelet alloantibodies in maternal serum. Based on those results, the risk to the fetus can be determined. An investigative algorithm for NAT used in our reference laboratory is described herein (Figure 3). Whole blood samples from the mother and father are used for genotyping and platelet phenotyping, and maternal serum is used for antibody investigations. The determination of maternal and paternal genotype is performed using simple PCR techniques.59 We use MAIPA or RIP to determine the platelet antigen phenotype with reference typing sera for all relevant alloantigens.60-63 Enzymelinked immunosorbent assay kits for the common platelet antigens (eg, HPA-1a)64 are available; however, they are limited by their inability to detect new antigen targets and by their high cost. Antibody investigations are done using MAIPA or RIP, with paternal platelets where possible as the target to allow for the detection of antibodies against a low-frequency (eg, HPA-5b, Zava) or new antigen. When indicated, fetal platelet genotyping can be performed on amniocytes procured by amniocentesis. Amniocentesis is a useful tool for confirming fetal-maternal antigen incompatibilities when the father is heterozygous or when paternal status is uncertain. Amniocytes are grown in culture to ensure that an adequate amount of DNA is available for PCR analysis. Differentiating maternal from fetal DNA by variable number of tandem repeat analysis is sometimes required.65 MANAGEMENT OF NAT

The care of women at risk for NAT and their affected infants requires a multidisciplinary treatment team that includes hematologists, obstetricians, and neonatologists, with support from transfusion medicine services and a specialized platelet testing laboratory. Antenatal Management For women at risk for NAT (usually identified because of a previously affected infant), antenatal treatment should begin by 24 weeks' gestation. Serial fetal ultrasounds should be done to monitor

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Fig 3. Diagnostic testing algorithm for NAT investigations and treatment recommendations based on the results (currently in use at the McMaster Platelet Immunology Diagnostic Laboratory).

for ICH or signs of fetal distress, which would be an indication for treatment escalation. Specific treatment options described subsequently derive mostly from studies of HPA-1a–negative women with anti–HPA-1a antibodies, but the same principals can be applied to mothers with other platelet antigen incompatibilities. Intravenous immune globulin. The clinical efficacy of antenatal IVIg administered to pregnant mothers at risk of NAT was first reported in 1988 by Bussel et al.66 In that report, 7 pregnant women were treated with weekly IVIg (1 g/kg) with or without oral dexamethasone. Fetal platelet count increased with treatment such that all 7 infants were born with a birth platelet count greater than 30 × 109/L, and none had ICH. In contrast, 3 untreated

older siblings had ICH; and all had lower birth platelet counts. Since then, IVIg has become the mainstay of antenatal treatment. The favorable effect of IVIg on fetal platelet count and on the reduction of ICH events compared with historical controls has since been confirmed in larger clinical studies.67,68 The dose of IVIg has been tested in a recent randomized controlled trial (RCT) comparing high-dose weekly IVIg (2 g/kg) and standarddose IVIg (1 g/kg) plus prednisone (0.5 mg/kg) in pregnant women with NAT.69 Similar outcomes were observed in both groups. In general, IVIg is safe and well tolerated, although serious adverse events have been described. In a large prospective study of patients with idiopathic thrombocytopenic purpura treated with

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IVIg (N = 97), half of the patients developed headache and 10% had fever.70 Rare adverse effects include chest pain, blood pressure fluctuations, bronchospasm, laryngeal edema, hemolysis, acute renal failure, and aseptic meningitis.71 Thrombotic complications including myocardial infarction, stroke, and deep venous thrombosis have also been described.72 Dexamethasone. In early studies, dexamethasone (3-5 mg/d) appeared to augment the effect of IVIg in some patients73; however, it was also associated with oligohydramnios and fetal growth retardation. Subsequently, the addition of a lower dose of dexamethasone (1.5 mg/d) to antenatal IVIg was evaluated in an RCT of 55 pregnant women in whom NAT (mostly due to HPA-1a) was confirmed serologically, and the fetal platelet count was less than 100 × 109/L.68 Even at these doses, oligohydramnios was observed in 2 infants born to mothers on dexamethasone, and platelet count responses were no different compared with IVIg alone. Prednisone. In a retrospective cohort study, 27 mothers with affected infants due to HPA-1a incompatibility who received antenatal IVIg (1 g/ kg per week) were compared with 10 mothers who received prednisone (0.5 mg/kg per day). 74 A successful or stable response was achieved in 18 (67%) mothers in the IVIg group, compared with 3 (30%) mothers in the prednisone group. In another study, antenatal prednisone (60 mg/d) in addition to IVIg was used to treat fetuses with an inadequate response to either IVIg alone or IVIg plus dexamethasone.68 Of the 10 mothers augmented with prednisone in that study, 5 achieved a response. More recently, a risk-stratified approach to antenatal treatment of mothers at high and moderate risk of NAT was evaluated.69 High-risk mothers (n = 40) (those with a previous infant with ICH or a current fetal platelet count less than 20 × 109/L) were randomized to weekly IVIg (1 g/ kg) or IVIg plus prednisone (1 mg/kg per day), and moderate-risk mothers (n = 39) (those with a previously affected child who did not have ICH and a current fetal platelet count greater than 20 × 10 9 /L) were randomized to IVIg alone or prednisone alone. In high-risk mothers, IVIg plus prednisone resulted in higher fetal platelet counts compared with IVIg alone; and 1 ICH event occurred in the IVIg group. In moderate-risk mothers, no difference in fetal platelet count

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response was observed with IVIg or prednisone monotherapy; however, 2 ICHs were observed in this cohort. The study was underpowered to detect a difference in bleeding outcomes, and the complex design makes it difficult to interpret. In summary, prednisone given in combination with IVIg may be effective in raising fetal platelets and should be considered for high-risk mothers or when treatment escalation is required. Adverse effects of daily prednisone during pregnancy include gestational diabetes, fluid retention, and mood alterations.69 FBS and intrauterine platelet transfusions. Some treatment algorithms for NAT include FBS to diagnose thrombocytopenia and to monitor the fetal platelet count response. Advantages of this approach are that treatment can be directed to women with thrombocytopenic fetuses and treatment failures can be identified early; however, the risks of fetal and maternal complications of FBS is concerning. In 1995, Paidas et al75 reported 5 fetal deaths due to exsanguination after FBS. Subsequently, intrauterine platelet transfusions were used at the time of FBS to reduce the risk of bleeding.74 However, the rate of fetal loss with FBS and platelet transfusions is estimated at 1.3% per procedure and 5.5% per pregnancy.21 In a retrospective cohort of 30 high-risk pregnancies, all 3 pregnancies treated with FBS and weekly intrauterine transfusions resulted in emergency cesarean deliveries and 2 of 3 infants died.76 Other complications of FBS and intrauterine transfusions include induction of labor77 and worsening of maternal alloimmunization.68 In a recent RCT, 11 (6%) serious complications were observed following 176 FBS procedures, including 1 fetal death.67 Overall, these data suggest that the risks of FBS outweigh the benefits and cannot be justified for the routine management of NAT. Noninvasive treatment strategies. Concerns over the risks of FBS have led to the development of empiric management strategies. In a retrospective study, van den Akker et al 78 compared the outcomes of 52 mothers (including 5 with a previously affected infant with ICH) treated noninvasively with IVIg alone and 46 mothers (including 11 with a previously affected infant with ICH) treated with an FBS-based strategy that included intrauterine platelet transfusions with or without IVIg. Birth platelet counts

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were lower in the noninvasive group, but none of the neonates had ICH, whereas 3 FBS procedures were complicated by emergency cesarean deliveries and 1 fetal death. In a retrospective study of 48 women (with 56 pregnancies), empiric IVIg treatment was as safe as FBS-directed management with or without intrauterine platelet transfusions and IVIg.79 Similarly, in a prospective study by Yinon et al,80 all 30 pregnancies managed empirically with weekly IVIg treatment had good outcomes. Women at highest risk for NAT (those with a previously affected infant with ICH) were excluded from those studies. Finally, in an RCT (N = 73) of high- vs standard-dose IVIg using a minimally invasive approach (using only 1 FBS at 32 weeks to determine treatment failures), fetal outcomes and birth platelet counts were similar in both groups.69 Of the 79 FBS procedures performed in that study, 4 resulted in emergency cesarean deliveries. Overall, IVIg without FBS appears to be safe and effective, even though birth platelet counts may be lower using this approach. Mode of delivery. Cesarean delivery is often used as the mode of delivery in NAT, although vaginal delivery has been recommended if the fetal platelet count is greater than 50 × 10 9 /L. 81 However, evidence in support of this practice is lacking; and the use of a fetal platelet count threshold is impractical. Therefore, van den Akker et al82 reviewed 32 pregnancies managed noninvasively, of which 23 were delivered vaginally and 9 underwent cesarean deliveries for obstetric indications. None of the infants had ICH, including 3 infants born vaginally with a platelet count less than 50 × 109/L. Further study of the mode of delivery in NAT is warranted. One important advantage of cesarean delivery is that the procedure can be timed so that personnel and resources are available without delay, including antigen-compatible platelet transfusions. In summary, weekly IVIg (1 g/kg) starting at 24 weeks' gestation is the mainstay of antenatal treatment of NAT.83 The benefit of adjunctive corticosteroids even for the highest-risk mothers has not been proven, but should be considered in women who have failed prior treatments. Data from clinical trials are limited because of complex trial designs, small sample sizes, and the use of FBS-based protocols, but what is apparent across clinical management studies is

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that the risks of FBS and intrauterine transfusions are prohibitive. We, like others,79,80,84 favor a noninvasive approach for the antenatal management of NAT. Screening for NAT Screening consecutive pregnant women for platelet antigen incompatibilities and antiplatelet antibodies would be required to prevent NAT in the first pregnancy. One such program screened 100 448 pregnant women for HPA-1a immunization who were then offered early cesarean delivery with compatible platelet transfusions available at delivery.1 With this program, 161 affected infants were identified, of whom 3 (6%) died or had ICH, compared with 10 (20%) of 51 infants born to mothers who were not screened. The screening program has also been shown to be cost effective.85 Although these results are encouraging, refinements in screening programs are needed before they can be widely applied. Postnatal Treatment of Infants With NAT Once NAT is suspected in an infant, treatment must be instituted promptly, even before the results of diagnostic tests are available. The IVIg (2 gm/kg in divided doses) is effective in approximately 75% of affected infants,86 and platelet transfusions are indicated for severe thrombocytopenia and/or bleeding. Antigen-negative or maternal platelet transfusions, specifically HPA-1a– or HPA-5b–negative platelets, are the platelet product of choice because the platelet count response is higher and lasts longer than random donor platelets87; nevertheless, random donor platelets should be used if matched platelets are not immediately available.88 The platelet transfusion threshold for babies with NAT is 30 × 109/L in most studies, although data from randomized trials are lacking.89 Postpartum Counseling of Women With an Affected Infant All women with a history of NAT must be counseled on the risks of NAT with subsequent pregnancies. For antigen-incompatible couples where a maternal antibody is detected, all future pregnancies should be closely monitored; and sisters of HPA-1a–negative women should also be tested in their reproductive years. In theory, women who lack the HPA-1a antigen are also at risk for

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posttransfusion purpura, a syndrome characterized by severe thrombocytopenia and bleeding after blood transfusion; these women should be counseled appropriately. CONCLUSIONS

The pathophysiology of alloantibody-mediated thrombocytopenia and bleeding in NAT is being clarified, and diagnostic testing for platelet antigens and maternal antibodies continues to improve. Several fundamental features of NAT remain poorly understood and require further research, including

the mechanism of placental passage of platelets or platelet particles, the propensity for ICH as opposed to other bleeding phenotypes, and the identification of reliable clinical and laboratory predictors of disease severity. Better and more specific therapies are needed to improve the outcome of this devastating disorder, and further strategies for screening first pregnancies are needed. ACKNOWLEDGMENT

We thank Genie LeBlanc and Emmy Arnold for their administrative assistance with the manuscript.

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