head of red cell immunohaematology, 2 Geoff Daniels, head of molecular diagnostics 1 ABSTRACT Objectives To assess the feasibility of applying a high throughput method, with an automatic robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. Design Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. Setting Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. Participants Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. Main outcome measures Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. Results Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. Conclusions High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of antiRhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

head of red cell immunohaematology, 2 Geoff Daniels, head of molecular diagnostics 1 ABSTRACT Objectives To assess the feasibility of applying a high throughput method, with an automatic robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. Design Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. Setting Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. Participants Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. Main outcome measures Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. Results Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. Conclusions High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of antiRhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocyte-mediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells. Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.

Eight monoclonal IgG antibodies to the Rh antigen D, produced by Epstein-Barr virus transformed B-lymphoblastoid cell lines from two individuals, have been assessed for their suitability as blood grouping reagents. All showed similar specificity and agglutinated all red cells with partial D antigens tested except category DVI cells. They gave strong reactions with Du red cells in indirect antiglobulin tests, and they all gave good reactions with R1R1, R2R2 and R0r cells in manual tests using antiglobulin, enzyme or albumin methods. Initial studies showed that some of the monoclonal antibodies worked well at high dilutions on a Technicon Autogrouper 16C when used for routine D-typing of blood donors. Antibody production by the cell lines was stable for many months in continuous culture. These monoclonal antibodies may be useful diagnostic reagents.

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