Neonatal Platelet Research Articles

The first case of foetal neonatal alloimmune thrombocytopenia caused by anti‐HPA‐1a antibodies in Asian population

Background and ObjectivesThe foetal neonatal platelet alloimmune thrombocytopenia (FNAIT) is one of the most frequent clinical implications of immunization against human platelet antigen (HPA). Maternal IgG platelet alloantibodies induce the destruction of foetal platelets leading to thrombocytopenia. In Caucasian, alloantibodies against HPA‐1a are responsible for the majority of severe FNAIT. As the frequency of HPA‐1bb homozygous individuals is very low in Asia, the clinical relevance of anti‐HPA‐1a alloantibodies is doubtful in this population.Study design and methodsRecently, we studied retrospectively sera from mothers with suspected FNAIT in Malaysia, a multi‐ethnic country. Surprisingly, we found in one maternal serum antibodies which may react with HPA‐1a. In this study, this FNAIT case, suspected to be caused by the anti‐HPA‐1a alloantibody, was investigated in more details.ResultsAntibody screening analysis using HPA‐phenotyped platelets by the antigen capture assay, MAIPA, showed specific reaction with the platelets GPIIb/IIIa carrying HPA‐1a antigenic determinants. In the control experiment, this antibody did not react with GPIIb/IIIa from HPA‐1bb individuals. In addition, no reaction was observed with other HPAs expressed on GPIIb/IIIa, GPIb/IX, GPIa/IIa, CD 109 and HLA Class I, both by screening tests and by cross‐match analysis (maternal serum against paternal platelets), in MAIPA. This result could be supported by the genotyping analysis of the family members (mother HPA‐1bb, father and the index child HPA‐1a positive).ConclusionsAlthough considered rare, this case shows for the first time the clinical relevance of anti‐HPA‐1a antibody in the pathogenesis of FNAIT among Asian population.

Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping.

Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.

True risk of fetal/neonatal alloimmune thrombocytopenia in subsequent pregnancies: a prospective observational follow‐up study

To assess neonatal platelet counts by comparing alloimmunised pregnancies from a Norwegian screening and intervention study with subsequent pregnancies from the same women. Prospective observational follow-up study. A university hospital. HPA-1a immunised women from a large Norwegian screening study that gave birth to one or more children after the screening study ended (2004-2012). Follow-up of maternal anti-HPA-1a antibody levels and neonatal platelet counts from the screening pregnancies were compared with subsequent pregnancies. None of the women received antenatal intravenous immunoglobulin (IVIG) treatment and neonatal platelet counts were therefore comparable. Change in neonatal platelet counts from one HPA-1a incompatible pregnancy to the next. Maternal anti-HPA-a1 antibody levels from one HPA-1a incompatible pregnancy to the next. Forty-five incompatible subsequent pregnancies were identified. Overall, the neonatal platelet count in the subsequent pregnancy was improved (18%), unchanged (52%), or worse (30%), compared with the corresponding screening pregnancy. There was one case of fetal intracranial haemorrhage (ICH) identified in the screening (intrauterine fetal death detected at 30weeks of gestation) and no ICH cases recorded for the subsequent pregnancies. In cases where the platelet count was lower in the subsequent pregnancy, the maternal anti-HPA-1a antibody level was higher compared with the screening pregnancy. In comparison, the maternal antibody level was lower in subsequent pregnancies where the platelet count improved. In contrast to what is often stated, we found that the neonatal platelet count was increased or unchanged in the majority of subsequent pregnancies of HPA-1a-immunised women.

Platelets in neonates: central mediators in haemostasis, antimicrobial defence and inflammation.

Platelets are not only centrally involved in haemostasis, but also in antimicrobial defence and inflammation. Since evaluation of platelet physiology in the particular patient group of preterm and term neonatal infants is highly restricted for ethical reasons, there are hardly any data available in healthy and much less in extremely immature or ill neonates. By summarising current knowledge and addressing both platelet researchers and neonatologists, we describe neonatal platelet count and morphology, report on previous analyses of neonatal platelet function in primary haemostasis and provide insights into recent advances in platelet immunology that considerably impacts our clinical view on the critically ill neonatal infant. We conclude that neonatal platelets, originating from liver megakaryocytes, substantially differ from adult platelets and may play a pivotal role in the pathophysiology of neonatal sepsis or intraventricular haemorrhage, both complications which seriously augment perinatal morbidity and mortality.

In Newborn Mice, Tpo Signaling Is Required for Normal Megakaryocyte Proliferation, Maturation, Platelet Formation, and Platelet Survival

Congenital amegakaryocytic thrombocytopenia (CAMT) is a disorder caused by c-mpl mutations that block or severely reduce thrombopoietin (Tpo) signaling, leading to amegakaryocytic thrombocytopenia and ultimately aplastic anemia. In neonates and infants, CAMT can have different clinical manifestations than in adults, and cases have been described of newborn infants who presented with severe thrombocytopenia but normal numbers of immature-appearing megakaryocytes (MKs) in the bone marrow. To elucidate the potential mechanisms underlying the developmental differences in the manifestations of Tpo deficiency, we evaluated MKs and platelets in newborn c-mpl-/- mice on postnatal day 1 (P1). Since the liver is the main site of megakaryopoiesis at that developmental stage (Liu et al., Blood 2014), liver MKs in P1 mice were immunohistochemically stained for VWF, quantified, measured, and ultrastructurally evaluated using transmission electron microscopy (TEM), as previously described. Histological analysis showed that MKs in newborn c-mpl-/- mice were present at a concentration of 30% and were significantly smaller compared to WT pups, a finding previously reported in adult mice. Unlike in adult c-mpl-/- mice, however, which have ultrastructurally normal MKs, TEM analysis of neonatal c-mpl-/- liver MKs revealed a significant shift toward immature cells and maturational abnormalities in 68% of MKs, including a disorganized demarcation system, reduced granules, and an abnormally wide peripheral zone. Ultrastructural analysis of platelets from c-mpl-/- P5 mice also revealed a larger open canalicular system and more glycosomes compared to age-matched WT platelets. These findings were not associated with increased levels of baseline P-selectin expression, suggesting that they were not the result of activation during processing. Next, we serially evaluated platelet counts in WT and c-mpl-/- mice during the transition from fetal/neonatal to adult megakaryopoiesis. At P2, c-mpl-/- platelet counts were 170±54x103/µl, representing 26% of those in P2 WT mice (648±137x103/µl). Over the first two weeks of life, WT platelet counts doubled and reached nearly adult levels, as previously described. In contrast, c-mpl-/- platelet counts failed to increase, and remained stably low at P14. Since we previously showed that the rise in platelet counts during this period was associated with a one-day prolongation of the neonatal platelet lifespan (Liu et al., Blood 2014), we evaluated platelet survival in c-mpl-/- compared to WT mice, using in vivo biotinylation. These studies suggested that the platelet lifespan was significantly shorter in c-mpl-/- compared to WT newborn (P2) mice. In contrast, we found no differences in platelet lifespan between c-mpl-/- and WT adult mice. Taken together, our findings suggest that, in fetal and neonatal life, Tpo signaling is required for normal MK proliferation, maturation, platelet formation, and platelet survival. This is in contrast to adult life, when Tpo deficiency leads only to reduced MK and platelet counts. We hypothesize that these differences reflect the lack of Tpo-independent pathways of MK maturation in the environment of the fetal/neonatal liver, and have implications in the developmental stage-specific clinical manifestations of CAMT. DisclosuresNo relevant conflicts of interest to declare.

Neonatal platelets: mediators of primary hemostasis in the developing hemostatic system.

The human hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis in neonates and infants. Although gestational age-related differences in coagulation factor levels occur, the existence of a unique neonatal platelet phenotype remains controversial. Complicated by difficulties in obtaining adequate neonatal blood volumes with which to perform functional assays, ambiguity surrounds the characterization of neonatal platelets. Thus, much of the current knowledge of neonatal platelet function has been based on studies from cord blood samples. Studies suggest that cord blood-derived platelets, as a surrogate for neonatal platelets, are hypofunctional when compared with adult platelets. This relative platelet dysfunction, combined with a propensity toward thrombocytopenia in the neonatal intensive care unit population, creates a clinical conundrum regarding the appropriate administration of platelet transfusions. This review provides an appraisal of the distinct functional phenotype of neonatal platelets. Neonatal platelet transfusion practices and the impact of the relatively hypofunctional neonatal platelet on those practices will be considered.

PMM.25 Outcomes in women with thrombocytopenia in pregnancy

BackgroundAntenatal thrombocytopenia occurs in 5% of pregnancies and is predominantly due to gestational thrombocytopenia (GTP), or more rarely is immunologically-mediated (ITP). The risk of significant neonatal thrombocytopenia (<50 × 109/L)...

Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan

AbstractThe fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets.

Platelets for Neonatal Transfusion - Study 2: A Randomised Controlled Trial to Compare Two Different Platelet Count Thresholds for Prophylactic Platelet Transfusion to Preterm Neonates

Introduction: Neonatal thrombocytopenia is a common and important clinical problem in preterm neonates. A trial assessing clinically relevant outcomes in relation to the different platelet count thresholds used to trigger transfusion has never been undertaken in preterm neonates with severe thrombocytopenia. Objectives: Platelets for Neonatal Transfusion - Study 2 (PlaNeT-2) aims to assess whether a higher prophylactic platelet transfusion threshold is superior to the lower thresholds in current standard practice in reducing the proportion of patients who have a major bleed or die up to study day 28. Methods: PlaNeT-2 is a two-stage, randomised, parallel-group, superiority trial. PlaNet-2 compares clinical outcomes in preterm neonates (<34 weeks' gestation at birth) randomised to receive prophylactic platelet transfusions to maintain platelet counts at or above either 25 × 10⁹/l or 50 × 10⁹/l. The primary outcome measure is the proportion of patients who either die or experience a major bleed up to and including study day 28. A total of 660 infants will be randomised. Results and Conclusions: This trial will help define optimal platelet transfusion support for severely thrombocytopenic preterm neonates by evaluating the risks and benefits of two different prophylactic neonatal platelet transfusion thresholds.

C0405: Role of Syntaxin-11 in Granule Release of Neonatal Platelets

C0405: Role of Syntaxin-11 in Granule Release of Neonatal Platelets

Factors predictive of neonatal thrombocytopenia in pregnant women with immune thrombocytopenia

To determine predictive factors for neonatal thrombocytopenia in deliveries with immune thrombocytopenia (ITP), we conducted a retrospective study at a tertiary hospital between 1997 and 2013. During this period, 30 women with ITP delivered 44 children. Neonatal thrombocytopenia (<100 × 10(9)/L) at birth was observed in seven neonates; four of these cases were severe (<50 × 10(9)/L). No cases were complicated by intracranial hemorrhage, and there was no neonatal mortality. Platelet counts at birth of neonates born to mothers, who had first been diagnosed with ITP during pregnancy were significantly higher than those born to mothers diagnosed with ITP before pregnancy. There were significant correlations between neonatal platelet counts in the first and second siblings at birth (P = 0.015) and at nadir (P = 0.035). Platelet counts of neonates born vaginally were significantly more likely to decline after birth than those delivered by cesarean section (13/16 vs. 10/23, P = 0.024). In conclusion, diagnosis of ITP before pregnancy was significantly associated with neonatal thrombocytopenia, and the platelet count of an older sibling is a strong predictor for that of the next baby. The delivery mode may be an indicator of the timing of platelet count nadir after birth.

Can we predict neonatal thrombocytopenia in offspring of women with idiopathic thrombocytopenic purpura?

BackgroundWe aimed to investigate which factors in the clinical profile of mothers with idiopathic thrombocytopenic purpura (ITP) can predict neonatal risk of thrombocytopenia.MethodsData was retrospectively collected from all pregnant women with ITP who presented to our institution between 2001 and 2013. Neonatal offspring of these women were classified into 2 groups based on the presence or absence of neonatal thrombocytopenia (platelet count <100×109/L). Several parameters were compared between the 2 groups, including maternal age, maternal platelet count, maternal treatment history, and thrombocytopenia in siblings. We further examined the correlation between maternal platelet count at the time of delivery and neonatal platelet count at birth; we also examined the correlation between the minimum platelet counts of other children born to multiparous women.ResultsSixty-six neonates from 49 mothers were enrolled in the study. Thrombocytopenia was observed in 13 (19.7%) neonates. Maternal treatment for ITP such as splenectomy did not correlate with a risk of neonatal thrombocytopenia. Sibling thrombocytopenia was more frequently observed in neonates with thrombocytopenia than in those without (7/13 vs. 4/53, P<0.01). No association was observed between maternal and neonatal platelet counts. However, the nadir neonatal platelet counts of first- and second-born siblings were highly correlated (r=0.87).ConclusionThrombocytopenia in neonates of women with ITP cannot be predicted by maternal treatment history or platelet count. However, the presence of an older sibling with neonatal thrombocytopenia is a reliable risk factor for neonatal thrombocytopenia in subsequent pregnancies.

Prevention and Treatment of Thrombosis in Pediatric and Congenital Heart Disease

Thrombosis has long been recognized as a potentially life-threatening complication in children with congenital heart disease (CHD), children with acquired heart disease, and in adults with CHD. High-risk groups include patients with shunt- dependent single ventricles (shunt thrombosis, 8%–12%; 4%

Development Of a Novel Method To Assess Neonatal Platelet Function

Background The hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis. Age-dependent values of pro- and anti-coagulant proteins have been determined. However, the task of defining age-dependent normal values of neonatal platelet function has been met with challenges owing to difficulties in obtaining adequate blood volumes for functional assays and inconsistent results amongst varying testing methods. In order to overcome many of these challenges, cord blood is often used as a source of neonatal platelets. Platelet aggregometry comparing adult and cord blood derived platelets has demonstrated a near lack of platelet response to epinephrine, collagen, and thromboxane in cord blood samples. In contrast, other studies of platelet function, such as flow cytometry, have failed to demonstrate this phenotypic difference. Assays of primary hemostasis reveal that neonatal blood mediates primary hemostasis as effectively as adult blood. In order to overcome the challenges associated with studying neonatal platelets, we have developed a novel platelet function assay employing small volumes of blood obtained directly from the neonate in order to assess platelet adhesion, activation, and aggregation simultaneously. Methods Eight-well slide chambers were coated with either fibrillar collagen or fibrinogen and allowed to adsorb at room temperature for one hour. Blood was obtained from healthy adult controls via venipuncture and neonatal samples via heelstick into sodium citrate. The blood was separated into two 200 µl aliquots, and TRAP (Thrombin Receptor Activating Peptide: 30 mM) was added to one aliquot. 100 µl of plain whole blood was added to both a collagen and a fibrinogen coated well and 100 µ of whole blood plus TRAP was added to a fibrinogen coated well. The samples were then incubated at 37°C for 30 minutes. Non-adherent cells were washed three times with modified HEPES-Tyrode buffer. FITC-P-selectin was then added (10 µg/ml), and the samples were incubated at 37 oC for 10 minutes and subsequently washed. Samples were imaged with differential interference contrast (DIC) and fluorescence microscopy on a Zeiss Axiovert 200 M microscope. Results Platelet adhesion, activation, and aggregation were assessed for 3 neonatal samples and 3 adult control samples. Both adult and neonatal platelets adhered to fibrinogen and collagen equally. Exposure to collagen and fibrinogen (+/- TRAP) resulted in alpha granule release and P-selectin expression in both neonatal and adult platelets. In addition, both adult and neonatal platelets were observed to undergo the characteristic cytoskeletal changes that result in platelet spreading on fibrinogen (+/- TRAP) and collagen surfaces. Both neonatal and adult platelets were observed to form platelet aggregates on both surfaces under static conditions. (Figure 1) Conclusions We have successfully developed a novel platelet function assay using small volumes of whole blood to assess three key platelet functions: adhesion, activation, and aggregation. This is the first study to demonstrate that neonatal platelets spread on adhesive and extracellular matrix proteins and suggests that neonatal platelets contain the cytoskeletal machinery necessary to undergo this change in platelet formation. This assay fills a critical need in clinical pediatric hematology where efforts to diagnose and treat neonatal platelet dysfunction are often met with technical challenges related to conventional platelet function assays. Disclosures: No relevant conflicts of interest to declare.

Small molecule inhibitors of phagocytosis for treatment of immune cytopenias

Immune cytopenias are caused by endogenous production of auto- or alloantibodies to blood cells resulting in the destruction of autologous, transfused or neonatal red cells, platelets or neutrophils. These conditions arise primarily due to extravascular destruction of the blood cells by mononuclear phagocytes present in the liver and/or spleen through specific Fc receptor (FcR) recognition of the Fc portion of the antibody opsonizing the blood cells. Although there are a number of treatments available, they require invasive splenectomy or immunoglobulin therapy; or drugs such as rituximab and thrombopoietin receptor agonists; or corticosteroids that take hours to be effective.

Distinct differences in platelet production and function between neonates and adults: implications for platelet transfusion practice

Thrombocytopenia is a common problem among sick neonates admitted to the neonatal intensive care unit. Among neonates, preterm infants are the subgroup at highest risk for thrombocytopenia and hemorrhage, which is frequently intracranial. Although there is no evidence of a relationship between platelet (PLT) count and occurrence of major hemorrhage, preterm infants are commonly transfused prophylactically when PLT counts fall below an arbitrary limit, and this threshold is usually higher than for older infants or adults. This liberal practice has been influenced by the observation that, in vitro, neonatal PLTs are hyporeactive in response to multiple agonists. However, full-term infants exhibit normal to increased primary hemostasis due to factors in neonatal blood that enhance the PLT-vessel wall interaction. Additionally, cardiorespiratory problems are considered the main etiologic factors in the development of neonatal intraventricular hemorrhage. In this review, we will discuss the developmental differences that exist in regard to PLT production and function, as well as in primary hemostasis in preterm and term neonates, and the implications of these developmental differences to transfusion medicine. PLT transfusions are not exempt of risk, and a better understanding of the PLT function and the hemostatic profile of premature infants and their changes over time and in response to illness is the starting point to design randomized controlled trials to define optimal use of PLT transfusions in premature neonates. Without these future trials, the marked disparities in PLT transfusion practice in neonates between hospitals and countries will remain over time.

PM.34 Immune Thrombocytopenia in Pregnancy

Immune thrombocytopenia (ITP) is the most common cause of thrombocytopenia in the first half of pregnancy and occurs in one or two per thousand pregnancies. The management of these pregnancies...

Do we know when to treat neonatal thrombocytopaenia?

Thrombocytopaenia is highly prevalent in neonatology affecting around 25% (22–35%) of all neonates admitted to neonatal intensive care units.1 Clinical signs of bleeding are also commonly documented in preterm neonates....

Usefulness of maternal anti‐HPA‐1a antibody quantitation in predicting severity of foetomaternal alloimmune thrombocytopenia

To study the clinical usefulness of maternal anti-HPA-1a antibody levels in predicting severe foetomaternal alloimmune thrombocytopenia (FMAIT). Recent studies using an international anti-HPA-1a standard have shown a correlation between maternal antibody levels and neonatal thrombocytopenia. Cut-off values for identifying high-risk pregnancies have also been suggested. In 1986-2010, HPA-1a alloimmunisation was confirmed in 84 women with 129 pregnancies. Maternal samples were obtained at delivery and during subsequent pregnancies. Anti-HPA-1a was quantified using a MAIPA assay with a detection limit of 0·8 IU mL(-1) (WHO reference serum 03/152). Antibody levels were compared with the severity of neonatal disease in the index and in the subsequent pregnancies. In the index cases, the correlation between an anti-HPA-1a level and neonatal platelet count did not reach statistical significance (n = 77, P = 0·074). However, the platelet counts and antibody levels in cases with cutaneous (n = 45) or intracranial haemorrhage (n = 7) were significantly different from cases with no evidence of bleeding (n = 20). In the subsequent pregnancies, there was a stronger association between the second trimester anti-HPA-1a level and the foetal platelet count (n = 16, P = 0·046). The positive predictive value of the maternal antibody level for a foetal platelet count <20 × 10(9) L(-1) was 90%, but the negative predictive value only 31%. Although a higher anti-HPA-1a level correlated with a more severe neonatal disease, barely detectable antibody levels were also observed in severely affected pregnancies. Cut-off values with sufficient sensitivity and specificity to identify these foetuses could not be found. A previous obstetric history still remains the most useful predictive parameter for severe FMAIT in clinical practice.

Factors predicting the hematopoietic stem cells content of the umbilical cord blood

Umbilical cord blood (UCB) has been demonstrated to be alternative source of hematopoietic stem cells (HSCs). Unfortunately, the wide use of UCB Transplantation is limited due to the low number of HSCs. The aim of this study was to determine factors that affect the number of HSCs collected from UCB. 200 eligible donors were included for HSCs testing, including total nucleated cells (TNCs) and CD34+ cell number, by using univariate and multivariate analysis. In univariate analysis, factors positively associated with higher number of TNCs were maternal weight (P=0.002), preeclampsia (P=0.03), neonatal weight (P<0.001), neonatal platelet count (P=0.02), neonatal Rh (P=0.03), gestational age (P=0.04) and delivery type (P<0.001). Factors positively associated with higher number of CD34+ cells were maternal weight (P<0.007), preeclampsia (P=0.02), maternal hypertension (P=0.02) neonatal weight (P<0.001), neonatal Rh type (P=0.02) and delivery type (P=0.04). In multivariate analysis, factors significantly influence TNCs were neonatal weight (P<0.001), preeclampsia (P=0.008), neonatal Rh type (P=0.02) and delivery type (P<0.001). While factors significantly influence number of CD34+ cells were maternal weight (P=0.025), neonatal weight (P=0.005), neonatal Rh (P=0.006), nuchal cord (P=0.026) and delivery type (P=0.009). Conclusions factors significantly influence TNCs content of UCB were neonatal weight, preeclampsia, neonatal Rh and delivery type. While factors significantly influence number of CD34+ cells were maternal weight, neonatal weight, neonatal Rh, nuchal cord and delivery type.