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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 (I²  =  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 (I²  =  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.

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.

This brief report summarizes the use of surface plasmon resonance technology (SPRT) in probing HPA-1a antigen-antibody interactions, based on a poster presented at the 60th meeting of the American Association of Blood Banks. It was concluded that the GP purification method could affect the performance of antigen in SPRT. It also highlighted that chips immobilised with Monoclonal antibody (Mab)-purified GP-IIb/IIIa work satisfactorily with both monoclonal and recombinant Abs with the appropriate concentration and binding affinity, while determination of the avidity and concentration of maternal polyclonal antibodies in respect to clinical severity on NAIT warrants further development.

A 65-year-old woman, blood group A RhD positive, who had completed her first course of induction chemotherapy for acute myeloid leukaemia was transfused with apheresis platelets over a number of days. On three occasions she received group O RhD positive units, which had been screened and found not to contain high-titre anti-A,B isoagglutinins. Following the third unit, she developed a haemolytic transfusion reaction and died soon thereafter. This has led to change in policy of the supplying centre in testing for high-titre anti-A,B isoagglutinins. Blood group O apheresis platelets and fresh-frozen plasma units are now labelled as high titre with a cut-off of 1/50 as compared to the previous cut-off of 1/100 for anti-A,B isoagglutinins. A universal approach to testing donations for high-titre anti-A,B isoagglutinins, better compliance of guidelines and monitoring of patients is necessary.

The RhD protein is expressed only on human red blood cells (RBC), and is one of the most immunogenic of the blood groups. It is of clinical importance since the alloantibody (anti-D) can hemolyse D positive RBC after blood transfusion, or cause hemolytic disease of the newborn. The immunogenicity of D is better understood with the knowledge of the genetic basis of the protein(s) involved, the molecular orientation in the RBC membrane, and the nature of the cellular immune response to proteins. The adaptive humoral response consists of antigen presenting cells, T-cells and B-cells, which interact cooperatively to result in antibody against the antigen in question. The anti-D that B-cells produce is targeted against surface membrane determinants (B-cell epitopes) and are conformational i.e. non-contiguous amino acids. The antigen specific T-cells recognize short linear peptides in the context of MHC class II, and these T-cell epitopes can reside anywhere in the protein. Since the RhD protein in D-positive individuals differs by some 35 amino acids from the RhCcEe protein in D-negative individuals, the opportunity for generating immunogenic T-cell epitopes is much greater than that for alleles characterized by a single amino acid difference e.g. E and e. Multiple conformational B-cell epitopes are also created by the presence of several D-specific amino acids in extracellular loops on the red cells surface, which may stimulate several B-cell clones and develop a strong polyclonal antibody response. With greater understanding comes the possibility of manipulating the immune response to D in clinical situations.

Using an in vitro perfusion model, therapeutic intravenous immunoglobulin (IVIgG) and IgG anti-D have been shown to cross the placenta from the maternal circuit to the fetal circuit. The transfer of all IgG species was linear with respect to time, and the amount of IgG transferred was proportional to the concentration of IgG in the maternal circuit ([IgG]m), but reached saturation at upper limits. With total [IgG]m at 6.5 g/l, 11.1 g/l or 26.2 g/l the increase in the fetal concentration of total IgG was 4.6 mg/l/h. 8.9 mg/l/h and 9.9 mg/l/h respectively. The rate of transfer of specific anti-D antibody to the fetal circuit was 0.026 IU/ml/h at a concentration of 38 IU/ml in the maternal circuit ([anti-D]m). High-dose therapeutic IVIgG added to the maternal circuit (total [IgG]m 29.2 g/l) significantly inhibited (P < 0.001) anti-D transfer to 0.004 IU/ml/n. Addition of the same IVIgG at a lower concentration (total [IgG]m 11.1 g/l) also reduced anti-D transfer, but only to 0.015 IU/l/h. The inhibitory effect of IVIgG does not appear to be mediated by anti-idiotypic or non-specific complexing with the anti-D, but may be the result of competition with IgG anti-D for placental Fc gamma receptors involved in the endocytotic uptake of IgG. The efficacy of IVIgG in this model suggests that it may be clinically useful in preventing HDN and other immune cytopenias, provided a sufficiently high dose is given.

An ADCC system has been developed using anti-D and papainized group O rhesus (D) positive red cells as the targets. Monocyte depleted mononuclear cell suspensions were effective in lysing appropriately sensitized red cells and papainization considerably enhanced the degree of specific lysis. Variation in culture volume and incubation in tubes or microplates were not critical to the degree of specific lysis obtained provided that the number of effector cells and target cells per culture was constant and the anti-D not diluted below the optimal concentration. Cytolytic activity was seen down to levels of 3 ng anti-D per culture. Specificity for lysis resided with the anti-D and not the effector cells. Several sources of anti-D were effective in inducing lysis of D positive red cells although individual variation was noted. Anti-c and anti-E were also shown to be effective in inducing specific lysis of red cells with the appropriate antigens.

The mechanism of lysis of anti-D coated human erythrocytes by human mononuclear K-cells was investigated. Red cell lysis was measurable after 30 min incubation and reached a maximum by 18--20 h. Cell-to-cell contact was necessary for lysis, phagocytosis was not a prerequisite, and intact microfilament function was required. The divalent cations Ca2+ and Mg2+ were both required for lysis to occur. Studies with metabolic inhibitors indicate that some RNA and protein synthesis is required for maximum expression of ADCC and intact microtubule function is essential. In the present system lysis was mediated by IgG1 anti-D antibodies and was significantly inhibited by IgG1 and IgG3 subclasses with some inhibition by IgG2 but not by IgG4, IgA or IgM. This suggests that the K-cell receptor is specific for IgG but that there is major cross-reactivity between IgG1 and IgG3. The inhibiting effect of hydrocortisone suggests that ADCC inhibition may be one mode of action of corticosteroids in ameliorating autoimmune haemolytic anaemia.

Conventional manual (enzyme and antiglobulin titres) and AutoAnalyser 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. AutoAnalyser (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 IgG1/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 IgG1 titres were greater than or equal to IgG3 titres and ADCC activity was very low.

An in vitro homologous system using human Rhesus alloantibodies and target erythrocytes labelled with 51Cr has been used to demonstrate the ability of normal peripheral blood lymphoid cells to lyse antibody-coated human red cells. The results obtained suggest that this type of mechanism may be relevant to certain haemolytic diseases in man.