One-tube method for complete HPA-1 genotyping by PCR using sequence-specific primers

Human platelet antigens (HPA) can be targets for antibody responses that cause life-threatening thrombocytopenia following platelet transfusions or pregnancy. As an aid to diagnosis and prevention, serologic and DNA-based methods have been developed to type HPA. Of the DNA-based strategies, those using the polymerase chain reaction (PCR) are very sensitive, but often require processing of amplification products. Sequence-specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, current methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA-1 in a single PCR. In addition, because the absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genotype.     

Maternal antibodies against paternal class I human leukocyte antigens are not associated with foetal and neonatal alloimmune thrombocytopenia

The causative role of maternal, anti-human leukocyte antigen (anti-HLA) class I antibodies in foetal and neonatal alloimmune thrombocytopenia (FNAIT) remains controversial. Furthermore, in FNAIT cases caused by anti-human platelet antigen-1a (anti-HPA-1a) antibodies, the possible additive effect of maternal anti-HLA class I antibodies on outcomes is unclear. Among 817 mother–father–neonate trios of suspected FNAIT, we assessed the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, and the incidence of FNAIT caused by anti-HPA-1a antibodies. In 144 FNAIT cases caused by anti-HPA-1a antibodies, we investigated the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, birth weight, and the incidence of intracranial haemorrhage (n = 16). Maternal anti-HLA class I antibodies were not associated with neonatal platelet count in suspected cases of FNAIT. There was no significant interaction between the presence of anti-HLA class I antibodies and anti-HPA-1a antibodies. In FNAIT cases caused by anti-HPA-1a antibodies, there was no association between the presence of anti-HLA class I antibodies and neonatal platelet count, birth weight, or occurrence of intracranial haemorrhage. This study’s findings do not support the concept that maternal anti-HLA class I antibodies represent a risk factor of FNAIT or disease severity.     

Marked thrombocytopenia in a neonate is associated with anti‐HPA‐5b,anti‐HLA‐A31, and anti‐HLA‐B55 antibodies

Maternal antibodies against human platelet antigen (HPA) and/or human leukocyte antigen (HLA) cause fetal and neonatal alloimmune thrombocytopenia (FNAIT) in 0.09‐0.15% of live births. Severe cases account for 5‐31% and the frequency of multiple kinds of alloantibodies is 6.9‐9% of FNAIT. We present a case of severe FNAIT associated with anti‐HPA‐5b, anti‐HLA‐A31, and anti‐HLA‐B55 antibodies, successfully treated with immunoglobulin and platelet transfusion. The anti‐HLA‐B55 antibody was detected in the newborn's serum, but disappeared on the 20th day, which was followed by an increase of the platelet count. These findings suggested the potential involvement of an anti‐HLA antibody in the pathogenesis of FNAIT.     

Pregnancy IFN-gamma responses to foetal alloantigens are altered by maternal allergy and gravidity status

Background: During pregnancy, variations in maternal–foetal cellular interactions may influence immune programming. This study was carried out to determine if maternal responses to foetal alloantigens are altered by maternal allergic disease and/or previous pregnancies. Methods: For this cohort study, peripheral blood was collected from allergic (n = 69) and nonallergic (n = 63) pregnant women at 20, 30, 36‐week gestation and 6‐week postpartum (pp). Cord blood was collected at delivery. Mixed lymphocyte reactions were used to measure maternal cytokine responses [interleukin‐6 (IL‐6), IL‐10, IL‐13 and (interferon‐γ) IFN‐γ] at each time point towards foetal mononuclear cells. Results: Maternal cytokine responses during pregnancy (20, 30 and 36 weeks) were suppressed compared to the responses at 6‐week pp. The ratio of maternal IFN‐γ/IL‐13 and IFN‐γ/IL‐10 responses were lower during pregnancy. Allergic mothers had lower IFN‐γ responses at each time‐point during pregnancy with the greatest difference in responses observed at 36‐week gestation. When allergic and nonallergic women were further stratified by gravidity group, IFN‐γ responses of allergic multigravid mothers were significantly lower than nonallergic multigravid mothers during pregnancy. Conclusions: During normal pregnancy, peripheral T‐cell cytokine responses to foetal alloantigens may be altered by both allergic status of the mother and previous pregnancies. These factors could influence the cytokine milieu experienced by the foetus and will be further explored in the development of allergic disease during early life.     

Review of neonatal alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT), with an incidence of one in 1000 live births, is the most common cause of severe thrombocytopenia and intra-cerebral haemorrhage in term neonates. NAIT results from trans-placental passage of maternal antibodies against a paternally derived fetal platelet alloantigen. Clinical presentation varies from unexpected thrombocytopenia on a blood film in a well newborn to intracranial haemorrhage (ICH). In contrast to haemolytic disease of the newborn, NAIT can present in a first pregnancy, and subsequent pregnancies are usually more severely affected. The role of antenatal screening for maternal alloantibodies instead of fetal blood sampling to identify at-risk fetuses remains uncertain, but there is a trend towards less invasive maternally directed treatment for at-risk pregnancies. Neonatal management is aimed at preventing or limiting thrombocytopenic bleeding with transfusion of antigen-matched platelets.     

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT‐1

We have recently described the immunosuppressive properties of AR‐C117977 and AR‐C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT‐1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR‐C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft‐versus‐host (GvH) response in the rat, and in high‐ and low‐responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT‐1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT‐1.     

European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia

The aims of this study were to determine whether the severity of fetomaternal alloimmune thrombocytopenia (FMAIT) in the current pregnancy could be predicted from the history of FMAIT in previous pregnancies, and to assess the effects of different types of antenatal intervention. Fifty-six fetuses were studied that all had a sibling affected by FMAIT due to human platelet antigen 1a (HPA-1a) alloimmunization. Cases with a sibling history of antenatal intracranial haemorrhage (ICH) or severe thrombocytopenia (platelet counts of < 20 x 109/l) had significantly lower pretreatment platelet counts than cases whose siblings had less severe thrombocytopenia or postnatal ICH. Maternal therapy resulted in a platelet count exceeding 50 x 109/l in 67% of cases. None of the fetuses managed by serial platelet intrauterine transfusions (IUT) suffered ICH following treatment. However, several serious complications arose with fetal blood sampling (FBS). Overall, intervention improved outcome, as three study cases suffered from antenatal ICH and three others died whereas 15 study cases had a sibling with an ICH, eight of whom died. The results of this study suggest that the start of therapy can be stratified on the basis of the sibling history of FMAIT, and support the use of maternal therapy as first-line treatment.     

In vitro culture of colony forming unit-megakaryocyte (CFU-MK) in fetal alloimmune thrombocytopenia

Summary . Perinatal alloimmune thrombocytopenia (PAITP) causes intracranial haemorrhage in the fetus and neonate. However, the severity of the thromobocytopenia correlates poorly with maternal anti-platelet antibody titres. To test the hypothesis that reduced platelet production contributes to fetal thrombocytopenia in PAITP, maternal sera from three HPA-la-negative mothers whose pregnancies were complicated by anti-HPA-la (two severe cases, one mild case) were added to colony forming unit-megakaryocyte (CFU-MK) cultures from HPA-la positive and negative individuals. Sera from the two severely affected pregnancies containing anti-HPA-la caused 66–100% inhibition of HPA-la-positive fetal and neonatal CFU-MK, whereas CFU-MK from two HPA-la-negative mothers were not inhibited by the anti-HPA-la-containing sera. Maternal serum from the case of mild PAITP caused only mild inhibition of HPA-la-positive cord and adult CFU-MK and did not inhibit HPA-la-positive fetal CFU-MK. Taken together, these findings suggest that reduced megakaryocyte production contributes to fetal thrombocytopenia due to maternal anti-HPA-la antibodies and also that the degree of CFU-MK inhibition correlates with severity of fetal thrombocytopenia.     

Feto-maternal alloimmune thrombocytopenia: antenatal therapy with IvIgG and steroids — more questions than answers

The optimal antenatal therapy for fetal thrombocytopenia has not been determined. We analysed 37 cases managed by maternal therapy and observed a successful outcome of maternal treatment in 26% of IvIgG cases and in 10% of steroid-treated cases. The significance of a plateau of the fetal platelet counts during pregnancy, 41% of IvIgG cases and 20% of cases treated with steroids, is uncertain. It may indicate a stabilization of thrombocytopenia, hence a beneficial effect of therapy, or the natural course of the platelet count in a low-risk pregnancy. Overall outcome was unpredictable, but amongst the therapy failures there were proportionally more severely affected siblings. Further multicentre studies are necessary to establish the optimal antenatal management of high-risk pregnancies.