Collaborative study to recalibrate the International Reference Preparation of Anti-D Immunoglobulin.

A collaborative study involving nine independent assays by eight laboratories has recalibrated the anti-D concentration of the International Reference Preparation of Anti-D Immunoglobulin (68/417) in terms of the International Standard for Anti-Rho (anti-D) Incomplete Blood Typing Serum (64/16). This study was carried out when it was found that 68/419 had been calibrated not against 64/16, as originally intended, but inadvertently against another preparation. Based on the results, a revised rounded off value of 300 IU anti-D per ampoule of 68/419 was assigned by the Expert Committee on Biological Standardisation of WHO at its 30th meeting. Many of the known variables in anti-D quantitation using the AutoAnalyzer were considered in the preparation of the protocol for this study. The remarkably close agreement of the results indicated that the format can be used as an acceptable model for interlaboratory studies in the future.

Anti-A haemagglutinins in factor VIII concentrates.

Experimental evidence has been obtained that cryoprecipitation concentrates anti-A in mixtures of group O and group A plasma by a mechanism that does not operate in group O plasma alone. It has been concluded that the anti-A/A polysaccharide complex is less soluble during cryoprecipitation than anti-A immunoglobulin, and this complex dissociates to give free anti-A when the cryoprecipitate is redissolved. From a practical point of view, factor VIII concentrates prepared from cryoprecipitate obtained from single donations of plasma unselected for ABO group contain significantly less anti-A than those prepared from mixing pools of plasma in which partial neutralisation of anti-A has occurred before cryoprecipitation.

Evaluation of an AutoAnalyzer method for quantitating anti-A and anti-B haemagglutinins in factor VIII preparations.

The continuous flow principle employed in the Technicon AutoAnalyzer has been adapted for the assay of anti-A and anti-B. The method has an acceptable degree of reproducibility and has been used, principally, for the quantitation of anti-A and anti-B in factor VIII concentrates of intermediate activity. It is, however, a method that can be applied to the assay of these antibodies in serum samples.

Prenatal determination of fetal rhesus D status by DNA amplification of peripheral blood of rhesus-negative mothers.

We have developed a sensitive PCR-based assay for the RhD gene and used it to detect circulating fetal cells from RhD-positive fetuses from peripheral blood of RhD-negative mothers. With further improvement in diagnostic accuracy, this assay may have implications in the management of RhD-sensitized pregnancies in women whose partners are heterozygous for the RhD gene. Further studies are required to determine the relationship between maternal anti-D levels and circulating fetal cell numbers.

A comparison of PVP and methyl cellulose as rouleaux inducing reagents in the automated anti-D quantification method.

The anti-D potency of 102 samples was determined using the automated anti-D quantification method. On the whole the values obtained when using PVP as the rouleaux inducer were no different to those obtained using methyl cellulose.

Auto analyzer determination of red cell Kell phenotypes in patients with chronic granulomatous disease (CGD) and heterozygous carriers.

One of six boys chronic granulomatous disease was shown to have the rare Kell phenotype, McLeod, by both manual and Auto Analyzer techniques. Using the Auto Analyzer, the red cells of this boy's mother were clearly shown to have weaker expression of k and Kpb antigens than those of the controls, whereas the Kell groups on the father's red cells appeared normal.

Amniotic fluid anti-D and the maternal serum: liquor ratio in rhesus haemolytic disease of the newborn.

Eighty-seven pairs of samples of maternal serum and amniotic fluid were analysed for anti-D levels at varying stages of gestation. There was a marked positive correlation between the maternal serum level and that in the amniotic fluid. The ratio of maternal serum anti-D to liquor anti-D was calculated for 61 pairs of data and was found to increase as gestation advanced. However, it was neither of clinical value in predicting the severity of haemolytic disease of the newborn nor the Rh(D) group of the fetus.

'Partial D' women with anti-D alloimmunization in pregnancy.

We report five women with Rh 'partial D' who were found to have anti-D during pregnancy. Two patients were category IVa, their red cells typing as Go(a+); one was category VI; and two had a 'partial Du' which could not be categorized. The maternal anti-D concentration increased during four of the eight pregnancies studied, but none reached significant levels. Three of six D-positive cord blood samples had a positive direct antiglobulin test (DAGT). Although one baby became jaundiced, no treatment was required. The policy in the Oxford Region for the management of patients with a weak expression of D is outlined.

Blood group antibody screening tests during pregnancy.

In a 2-year period 667 sera from approximately 70,000 (0.95%) antenatal patients were found to contain 726 atypical red blood cell antibodies. Overall, 66% of the immunized mothers were rhesus (D) positive. Apart from four antibody specificities to rhesus system antigens, knowledge of the rhesus (D) group gave no guide to the ability of the patients to form any of the remaining 21 specificities encountered. Of the 726 antibodies 221 (30%) were not detected in the initial sample tested and 50 of the 92 patients who produced antibodies during pregnancy had not developed detectable antibody when they were sampled at 28 weeks. The significance of these late onset antibodies is discussed both in relation to the risk of haemolytic disease in the newborn and transfusion reactions in the mother. An optimum protocol for testing is defined which takes account of antibody production during the pregnancy and use of this protocol constitutes an attempt to combine maximum clinical safety with minimal consumption of resources.

The significance of anti-C sensitization in pregnancy.

A study of maternal blood samples from 280,000 pregnancies in an 8-year period has shown 38 examples of anti-C (without anti-D) sensitization. This frequency (0.14/1000 pregnancies) was lower than that previously found for anti-c (0.63/1000 pregnancies) and for anti-D (2.55/1000 pregnancies). Although most of the 38 babies born to mothers with anti-C were unaffected by haemolytic disease of the newborn and none was anaemic at birth, two required exchange transfusion for hyperbilirubinaemia and one a top-up transfusion. Five cord blood samples had a positive direct antiglobulin test.

Fetal blood group studies during mid-trimester pregnancy and the management of severe isoimmunization.

Fetal blood samples collected at 14-22 weeks' gestation by fetoscopy have been analysed for 25 different red cell antigens and anti-A antibodies. The results obtained demonstrate that the technique can be used to determine fetal blood groups during mid-pregnancy and also to provide the opportunity to study the physiological and pathological developments of blood groups and their antibodies and to manage more rationally those pregnancies threatened by erythroblastosis fetalis.

The significance of anti-Kell sensitization in pregnancy.

In a 10-year period, 407 of 350,000 pregnancies showed maternal anti-Kell allo-immunization, i.e., an incidence of 1.16 per 1000 pregnancies. About 88% of Kell immunized women gave a history of previous transfusion. There were 51 pregnancies with Kell positive partners (all Kk) resulting in 10 Kell positive babies, of whom six had a positive direct antiglobulin test (DAGT). There were two stillbirths due to haemolytic disease of the newborn, when the maternal anti-Kell titres were 1/256. One baby was severely anaemic and given a top-up transfusion, and two babies were jaundiced and given phototherapy. A policy for management of Kell sensitized pregnancies is outlined, based upon maternal anti-Kell titre and where appropriate fetal blood sampling.

Detection of fetal RhD sequence from peripheral blood of sensitized RhD-negative pregnant women.

A sensitive PCR-based assay was developed to amplify fetal-derived rhesus D (RhD) sequence from peripheral blood of RhD-negative pregnant women with circulating anti-D. RhD-PCR positivity was detected in 7/22 samples from women bearing RhD-positive fetuses, despite the presence of varying levels of anti-D. Evidence is presented which suggests that rising maternal anti-D levels might reduce circulating fetal cell numbers. Further development of this assay may have implications in the clinical management of RhD-sensitized pregnancies and aid the understanding of the physiology of feto-maternal cell trafficking.

Production of additional atypical alloantibodies in Rh(D)-sensitized pregnancies managed by intrauterine investigation methods.

The production of additional atypical alloantibodies by previously Rh(D)-alloimmunized antenatal patients can complicate the clinical management of both mother and fetus. The relative risks of stimulating additional antibodies following the use of intrauterine investigation methods currently used for the management of haemolytic disease of the newborn have been assessed. A significantly (P less than 0.05, X2) greater number of additional antibodies were detected during pregnancy in the group of pregnancies managed by intrauterine transfusion (IUT) (3/29) than in those managed without recourse to intrauterine investigation (0/50), although one of the patients in the IUT group produced a further antibody following a fetal blood sample but before an IUT had been carried out. The number of additional antibodies detected post-delivery was significantly greater (P less than 0.02) in pregnancies managed by intrauterine transfusion (2/10) than in those managed by fetal haematocrit determination alone (0/28).

Serial fetal blood sampling for the management of pregnancies complicated by severe rhesus (D) isoimmunization.

Fifty-one pregnancies complicated by rhesus (D) isoimmunization have been managed by serial fetal blood sampling between 17 and 36 weeks gestation as an alternative to amniocentesis for delta OD453 measurements. In 36 pregnancies where the fetus was shown to be rhesus (D) positive and both measurements were made before any intrauterine fetal transfusions, the delta OD453 value gave misleading predictions on 13 of 63 occasions (21%). Fetal haematocrit estimations provided a direct assessment of the haemopoietic compensation occurring, but fetal bilirubin and albumin concentrations did not correlate directly with disease severity. It is proposed that pregnancies complicated by severe isoimmunization can be more precisely managed by serial fetal blood sampling for haematocrit estimation than amniocentesis for delta OD453 measurement thus avoiding unnecessary intervention or delayed treatment.

Antenatal fetal blood sampling for the management of alloimmunized pregnancies: effect upon maternal anti-D potency levels.

Increase in maternal anti-D concentrations after intrauterine investigation has been studied retrospectively in 95 rhesus (D) alloimmunized pregnancies; 48 were managed by fetal blood sampling (FBS) procedures (using fetoscopy or ultrasound-guided needle sampling) and 47 using amniocentesis. In those pregnancies where the fetus was rhesus (D) positive, the frequency of procedure-related increases (greater than 50%) in maternal anti-D potency was estimated following single procedures and found to be similar for the two methods of FBS employed (28%) and for amniocentesis (31%). The proportion of pregnancies showing an increase in anti-D potency was higher after ultrasound-guided needle sampling (75%) than after fetoscopic FBS (40%) and after amniocentesis (44%).

In-utero intravascular transfusion of the fetus for the management of severe Rhesus isoimmunization--a reappraisal.

Ten fetuses, severely affected by Rhesus (D) haemolytic disease, received one to three intravascular blood transfusions at between 18 and 30 weeks gestation, with the use of fetoscopically guided needles into one of the umbilical cord vessels. Although the technique was successfully accomplished in all cases, the fetal response to the procedure was varied. Only two fetuses survived beyond the neonatal period, and one child subsequently died principally because of the problems resulting from premature delivery. The reason for the low rate of survival has been explored and the continued use of the method described is now questioned.

The significance of anti-c alloimmunization in pregnancy.

In an 8-year period, 177 of 280,000 pregnancies were complicated by maternal anti-c alloimmunization. Although there was one neonatal death associated with anti-c haemolytic disease of the newborn, only two infants were severely anaemic at birth. A total of 11 babies required exchange transfusion, but nine of these developed hyperbilirubinaemia alone. The remaining c positive infants were either unaffected or only mildly affected by erythroblastosis fetalis. A strategy for management of these pregnancies is outlined, and proposed methods of prevention and serological control are discussed.