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Blood samples from 24 Rh(D) immunized women were analyzed for antibody titers and quantification of anti-D. The HLA-DR and -DQ polymorphisms were identified as RFLP. In 11 women with titers 16-256 the HLA-DQB1 allele *0201 was found in 18%, i e as in a reference population. In 13 women with titers > or = 512 the HLA-DQB1 allele *0201 was found in 85% indicating a correlation between severe Rh(D) immunization with high titers/quantification values and the DQB1 allele *0201. In this group the fetus was severely affected by the immunization and treatment during pregnancy was frequently needed. HLA phenotyping of women known to have anti-D antibodies early in pregnancy seems to be an effective way to assess the probability of severe hemolytic disease of the newborn.

The ability of the chemiluminescence test (CLT) to predict the severity of haemolytic disease of the newborn (HDN) was determined in 80 alloimmunized pregnant women who delivered antigen-positive babies. In 54 cases of alloimmunization to D, results from the CLT showed better correlation with fetal outcome than anti-D concentration measured by AutoAnalyzer (r = 0.70 and 0.36 respectively). Results from the CLT permitted a threshold level of antibody activity (30%) below which 15/20 babies were unaffected or had mild disease, and only one required transfusion therapy in utero. CLT results above 30% were associated with moderate or severe disease in all cases. Results from the AutoAnalyzer proved a less reliable predictor of disease severity; three women with anti-D levels > 20 iu/ml delivered unaffected babies, and two women with anti-D levels < 10 iu/ml delivered babies who had required transfusion in utero. The clinical usefulness of the CLT derives from the possibility of avoiding invasive monitoring procedures in women with high levels of anti-D which is relatively non-functional.

All rhesus-negative women who completed a pregnancy between January and August 1992 in two Scottish regions were studied to assess whether the administration of the rhesus prevention programme was complete: 671 rhesus positive (or rhesus type unknown) pregnancies were completed in 1120 Rh D negative women. For eight pregnancies no record of Rh D administration at birth was available. For recorded antenatal events that should have resulted in its administration anti-D was given in 195/280 (69.6%). Kleihauer testing was carried out in 9 of 98 instances of antepartum haemorrhage occurring after 20 weeks gestation. No cases of antenatal immunisation were identified. The study identified a need to increase awareness of the necessity for anti-D administration after potentially immunising events during pregnancy, and for increasing compliance with administration of postnatal anti-D in one of the study regions. Re-evaluation is also required of the recommendation that Kleihauer testing should be done when antenatal anti-D is given following an obstetric event after 20 weeks.

Classification of subjects with a partial D antigen is traditionally performed with immune anti-D sera. The development of monoclonal antibodies enables a fine analysis to be made of the specificity of the epitopes that are present or missing in these cases. A systematic search in a Caucasian donor population of 17,500 revealed 8 unrelated male individuals (frequency 0.05%) with a red cell phenotype characteristic of partial D category VI, but without anti-D in their serum. The relation to the 'classic' partial D category VI was investigated and is discussed, as is the observed serological heterogeneity of the partial D category VI group. Clinical consequences for the prevention of immunization of these subjects are mentioned.

The pre- and postpartum maternal serum anti-D concentrations of 28 women with severe Rh(D) immunization who received high-dose intravenous immunoglobulin treatment has been determined. In all cases, including 1 in which the newborn was D negative, a sharp increment in the anti-D titer was observed after delivery. The specific immunoglobulin concentration rose to levels ranging from 4.7 to 204.0 micrograms/ml and, in 20% of the patients, increments of fifty times or greater were observed. Human monoclonal antibodies (hmAb) have been produced from Epstein-Barr virus-transformed lymphoblastoid B cell lines derived from 1 of these naturally hyperimmunized patients whose serum contained an anti-D-category DVI antibody. Four anti-D-secreting cell lines (97.E3.39.214, 44.E4.R1.257, E7.R1.126.83.115 and E11V.117.63; hereafter referred to as 214, 257, 115 and 63) have been established and maintained in continuous culture for periods ranging from several months to 3 years, without loss of antibody production capacity. Antibodies 115 and 214 recognize all Du samples tested at the same level as the polyclonal positive control. Antibodies 63 and 257 show a significantly lower reaction strength with some of the Du samples. Studies with D category cells showed that the DVI category was recognized only by hmAb 214. The reactivity pattern of this antibody is that of an anti-epD4, although the reaction strength varied greatly with different DIVa cell samples. Results obtained with hmAb 257 and 115 using papain-treated D category cells suggest that booth react as anti-epD6/7.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Recent insights into the structure-function relationship of IgG, the nature of Fc receptors and their interactions with antibodies, and the cellular mechanisms involved in the immune destruction of IgG-sensitized cells have all contributed to a fuller understanding of the role of Rh antibodies in HDN. As this understanding has increased, so different diagnostic and therapeutic approaches have been developed and evaluated in order either to predict or ameliorate disease severity. The role of Rh antibodies in HDN can be considered in three contexts: maternal anti-D, monoclonal anti-D and prophylactic anti-D. Anti-D formed after maternal alloimmunization may be transported across the placenta, resulting in destruction of sensitized red cells by mononuclear phagocytes in the fetus and infant. The use of monoclonal anti-D has given an insight into the cellular and molecular mechanisms involved in red cell destruction, and has facilitated the development and evaluation of assays which use maternal anti-D to predict the severity of HDN. Polyclonal anti-D, given prophylactically, can prevent maternal alloimmunization to D-positive fetal red cells. Future developments are likely in several areas. Prophylactic polyclonal anti-D may be replaced, wholly or partially, with monoclonal anti-D. The development and introduction of cellular assays as non-invasive tests for predicting disease severity is likely to continue as preliminary results are encouraging. Finally, new strategies for ameliorating disease severity may be assessed including the role of ivIgG and Fc gamma R-blocking antibodies.