Stanislaw J. Urbaniak

Prediction of the Outcome of Rhesus Haemolytic Disease of the Newborn: Additional Information Using an ADCC Assay

An in vitro test system is described which measures the ability of anti‐D sera to lyse Rh(D)‐positive red cells. This test was applied to anti‐D sera from 11 cases of HDN selected in Glasgow and tested ‘blind’ in Edinburgh. Evidence is presented to support the view that the ADCC (antibody‐dependent cell‐mediated cytotoxity) assay can correctly identify those cases where there is a satisfactory clinical outcome despite a high level of anti‐D suggesting otherwise. Amniocentesis might therefore be avoided in this group.

ADCC (K‐Cell) Lysis of Human Erythrocytes Sensitized with Rhesus Alloantibodies III. COMPARISON OF IgG ANTI‐D AGGLUTINATING AND LYTIC (ADCC) ACTIVITY AND THE ROLE OF IgG SUBCLASSES

Conventional manual (enzyme and antiglobulin titres) and Auto‐Analyser quantitation of anti‐D sera were compared with the ability of the same sera to mediate lysis of Rh(D) positive red cells in an ADCC assay. Auto Analyser (AA) quantitation correlated significantly with manual titration methods, although there were wide discrepancies between individual sera. The ADCC activity of the anti‐D sera did not correlate with any of the conventional assays (AA, enzyme, antiglobulin). There were significant differences between anti‐D sera obtained from females immunized during pregnancy and male volunteers immunized by deliberate injection. The female sera contained significantly less anti‐D when assessed by AA, yet were significantly more active in ADCC activity at equivalent concentrations. These functional differences in anti‐D activity could not be attributed to the absence of IgG subclasses (IgG1 and IgG3) known to induce ADCC. However, there was a relationship between ADCC and IgGI/IgG3 titres in that high ADCC activity was seen where the IgG3 titre was higher than the IgG1 titre. In the male anti‐D sera IgGl titres were ≥ IgG3 titres and ADCC activity was very low.

Management of alloimmune thrombocytopenia

Fetal alloimmune thrombocytopenia is caused by maternal sensitization to paternally-derived antigens on fetal platelets, most commonly HPA-1a.1 It occurs in approximately 1 in 1000 live births and is the commonest cause of severe fetal and neonatal thrombocytopenia, and of intracranial hemorrhage in neonates born at term.2 Since there is currently no routine screening, first-time cases of fetal alloimmune thrombocytopenia are generally identified following the birth of a markedly thrombocytopenic neonate. Antenatal management is thus only possible in subsequent pregnancies. Intracranial hemorrhage is the most devastating complication of fetal alloimmune thrombocytopenia and often occurs antenatally. Assessment of projected clinical severity is thus based on the development of intracranial hemorrhage in a previous sibling. If there is such a history of intracranial hemorrhage, the chance of this complication occurring again in the next pregnancy is extremely high in an untreated, antigen-positive sibling.3 Administration of intravenous immunoglobulin (IVIG) to the mother, initially given in conjunction with dexamethasone, was first used to prevent recurrence of antenatal intracranial hemorrhage in 1988.4 This approach of providing IVIG-based medical therapy administered to the mother to increase the fetal platelet count has since been extensively investigated in hundreds of maternal-fetal pairs.5 The efficacy of IVIG-based therapy has been supported by numerous studies6–16 (Table 1A) but not by others17–19 (Table 1B). The studies presented in Tables 1A and 1B surprisingly report virtually identical percentages of cases of intracranial hemorrhage: 2.7% versus 2.9%, respectively. However, overall mean birth platelet counts differed markedly between the two groups. While platelet counts are considered to be surrogate markers of intracranial hemorrhage, fortunately, the likelihood of fetal and neonatal intracranial hemorrhage, in the absence of this complication having occurred in a previous sibling, is relatively low.

Characterization of the alloreactive helper T-cell response to the platelet membrane glycoprotein IIIa (integrin-β3) in human platelet antigen-1a alloimmunized human platelet antigen-1b1b women

BACKGROUND: The aims were to characterize the helper T‐cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)‐1a, to identify immunodominant epitopes and to examine the HLA Class II associations.

Limited efficacy of universal leucodepletion in reducing the incidence of febrile nonhaemolytic reactions in red cell transfusions

Summary  This article demonstrates a 62% reduction in the number of febrile nonhaemolytic transfusion reactions (FNHTRs) and 50% reduction in febrile reaction rate associated with red cell transfusions following graded introduction of universal leucodepletion. Though this is a statistically significant reduction (P = 0·009), it shows limited efficacy in abrogating this complication. We also found a reduction in the proportion of cases of FNHTRs with lymphocytotoxic antibodies over the period studied from 54% in 1998, 28% in 1999 to 23% in 2000. This corresponds to a relative increase in the number of febrile reactions without human leucocyte antigen (HLA) antibodies following full implementation of universal leucodepletion, as the total number of reported reactions actually fell considerably during the period. The increase in the number of cases without HLA antibodies was directly proportional to the increase in the number of leucodepleted units used.

Fetomaternal alloimmune thrombocytopenia

Abstract Thrombocytopenia is the second commonest haematological abnormality in the neonatal period after anaemia due to iatrogenic blood letting. One to four percent of all newborn babies have a platelet count 9 /l at birth and approximately 20–40% of neonates in intensive care units are affected by neonatal thrombocytopenia. The most common cause of severe neonatal thrombocytopenia is fetomaternal platelet incompatibility and subsequent alloimmunisation. During the last decade recent advances in molecular techniques have led to rapid and efficient methods for diagnosis. Progress in fetal medicine has enabled accurate determination of fetal status, allowing improvements in fetal diagnosis and therapy. Human platelet antigen (HPA)-1a is by far the most frequently involved platelet antigen system in Caucasians accounting for 90% of cases, followed at a much lower frequency by HPA-5b (5–15%) and HPA-3a. The incidence is estimated to be 1 per 2000 to 1 per 5000 live births, but this is low in comparison to the incidence of fetomaternal platelet antigen incompatibility especially for the HPA-1 alloantigen system in the Caucasian population in whom the estimated frequency of HPA-1b1b individuals is 2%. Retrospective and prospective studies have reported that the immunogenetic background is important, and the chance of HPA-1a alloimmunisation is strongly associated with maternal HLA class II DRB3∗0101 (DR52a) type. A significant association ( p =0.004) between severe thrombocytopenia and a third trimester antiHPA titre >1:32 has been observed. It is now possible to genotype the fetus or neonate and the parents, which provides confirmation as to which HPA systems are incompatible between the mother and father. Simultaneous genotyping of HPA-1, 2, 3 and 5 can be carried out using the polymerase chain reaction–sequence specific primers (PCR–SSP) protocol, which has been widely used for HLA class II determination. The platelet count may continue to fall during the first 48 h after birth and the risk of intracranial bleeding is highest during this period. The best option is transfusion of specially selected antigen negative compatible donor platelets or if unavailable, maternal washed platelets. Antenatal screening for the most common form of fetomaternal alloimmune thrombocytopenia (FMAIT), due to antiHPA-1a is under consideration, but there is no established method at present. The Scottish National Blood Transfusion Service started a study in August 1999 on 25,000 pregnancies to carry out a cost benefit analysis of routine antenatal screening. The aims of the study are to determine the frequency of HPA-1b homozygosity; monitor antibody titres during pregnancy and confirm correlation of antibody emergence with HLA-DRB3∗0101, and finally to access cost effectiveness of routine screening across Scotland. Of 26,509 women screened in three Scottish regions 501 (1.9%) are HPA-1b homozygous and about 9% of the consented women are antibody positive.

Routine antenatal anti-D prophylaxis in women who are Rh(D) negative: meta-analyses adjusted for differences in study design and quality.

Background To estimate the effectiveness of routine antenatal anti-D prophylaxis for preventing sensitisation in pregnant Rhesus negative women, and to explore whether this depends on the treatment regimen adopted. Methods Ten studies identified in a previous systematic literature search were included. Potential sources of bias were systematically identified using bias checklists, and their impact and uncertainty were quantified using expert opinion. Study results were adjusted for biases and combined, first in a random-effects meta-analysis and then in a random-effects meta-regression analysis. Results In a conventional meta-analysis, the pooled odds ratio for sensitisation was estimated as 0.25 (95% CI 0.18, 0.36), comparing routine antenatal anti-D prophylaxis to control, with some heterogeneity (I 2 = 19%). However, this naïve analysis ignores substantial differences in study quality and design. After adjusting for these, the pooled odds ratio for sensitisation was estimated as 0.31 (95% CI 0.17, 0.56), with no evidence of heterogeneity (I 2 = 0%). A meta-regression analysis was performed, which used the data available from the ten anti-D prophylaxis studies to inform us about the relative effectiveness of three licensed treatments. This gave an 83% probability that a dose of 1250 IU at 28 and 34 weeks is most effective and a 76% probability that a single dose of 1500 IU at 28–30 weeks is least effective. Conclusion There is strong evidence for the effectiveness of routine antenatal anti-D prophylaxis for prevention of sensitisation, in support of the policy of offering routine prophylaxis to all non-sensitised pregnant Rhesus negative women. All three licensed dose regimens are expected to be effective.

V(D)J germline gene repertoire analysis of monoclonal D antibodies and the implications for D epitope specificity

BACKGROUND: The D antigen is a highly immunogenic human RBC antigen. Alloimmunization against the D antigen produces high‐affinity antibodies that cause hemolytic transfusion reactions and HDN.

The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibodies that cross the placenta in connection with pregnancy and destroy fetal platelets. Recently, maternal T cell responses associated with FNAIT have been studied at the clonal level. These T cell clones recognize an integrin β3 epitope, which is anchored to the HLA-DRB3∗0101-encoded MHC molecule DR52a. The same MHC allele is strongly associated with FNAIT. As the production of pathological antibodies reactive with fetal platelets is likely dependent on these T cell responses, there exists a potential for preventing FNAIT by targeting these T cells.

The relationship of anti-HPA-1a amount to severity of neonatal alloimmune thrombocytopenia – Where does it stand?

The issue of whether or not antibody quantity during pregnancy is related to severity of neonatal alloimmune thrombocytopenia remains unresolved. In this article we cite studies in support of both sides of the argument and highlight some of the reasons that may lie behind the observed differences amongst those studies. It may well be that some of the reasons for the discrepant results could be due to the type of study carried out (eg retrospective versus prospective), the sample size, the timing of antibody sampling, and possibly the type or protocol of assay used. Another major reason is the absence, until recently, of an international anti-HPA-1a standard.