Discussion
Main findings
The retrospective data analysis of more than 4000 type-and-screen tests at delivery in RhD negative women with a fetus typed RHDpositive, revealed that 20.5 per cent in the cohort had non-detectable anti-D levels after RAADP at gestational week 28-29. In the prospective interventional study including 39 women the proportion was higher. Forty-four per cent had anti-D below limit of detection (10 IU/L) at screening, and eighteen per cent had non-detectable levels (<1 IU/L) with the sensitive quantification assay, at 38 weeks of gestation, after RAADP 1500 IU at 28-29 weeks of gestation. In previous reports, 44-78 per cent of cases had non-detectable levels of anti-D at delivery after one dose RAADP in gestational week 28 and 15-39 per cent after two doses in gestational weeks 28 and 34.7,8 In pregnancies post 40 weeks, 78 per cent is lacking protection.8Small amounts of fetal blood pass into the maternal circulation, with increasing risk during the length of pregnancy and greatest risk at delivery.18 The volumes are usually low. In an analysis of published studies of more than 20 000 pregnancies, it was found that 74% of the women had less than 0.5 mL, 96% less than 1 mL and 98% had less than 2 mL fetal blood in the circulation.19 In another study of more than 3000 pregnancies, 99% had FMH less than 2.5 mL and 99,8% had FMH less than 5 mL blood, after vaginal delivery. After caesarian section 99,7% had FMH less than 2.5 mL and 99.5% had FMH less than 5 mL.20
In an early recommendation from the World Health Organization21 supported by the British Medical research Council,22 25 µg anti D in the maternal circulation is estimated to protect 2 mL fetal blood. The current product information for anti-D Rhophylac® (CSL Behring), says that 10 µg (50 IU) covers one mL fetal blood.23 Using an estimated maternal plasma volume of 3750 mL and an extracellular volume of 4500 mL at term,11 it corresponds to a concentration anti-D of 2,4 µg/L (12 IU/L) to protect from FMH of 2 mL whole blood. A dose of 300 µg (1500 IU) giving a theoretical concentration of 36 µg/L (180 IU/L) protects from FMH of 30 mL. A concentration of 15 IU/L (3 µg/L) protects a FMH of less than 2,5 mL occurring in 99% of pregnancies.22 In our prospective study 2/39 women had levels below 15 IU/L, 14,4 and 14,5 IU/L respectively. No cases of fetomaternal hemorrhage were found, but the sample size was small. Those who were followed postpartum showed stable protective levels of anti-D concentration up to 30 days postdelivery, 61+ 28 IU/L.
With current anti-D prophylaxis regimens including RAADP in gestational week 28-30 the incidence of anti-D immunisation is low, 0.2-0.4%. Despite this low incidence, anti-D immunisation is still the most common cause of severe fetal anemia requiring intrauterine blood transfusions to the fetus with its inherent risk of complications and perinatal mortality and morbidity. 2,5 Fetal RHDscreening is now a routine in many countries, resulting in that the fetal RHD status is known with high accuracy prenatally, with a sensitivity of the analysis above 99,9%.6 To add anti-D prophylaxis in gestational week 38, to ensure a protective concentration of anti-D at term and post term in RhD negative women at risk of immunisation, seems relevant and logistically possible in most programs. Anti-D prophylaxis in gestational week 38 may replace postpartum prophylaxis, and thus make the strategy cost effective.
Strength and limitations
The prospective intervention design, with the possibility to follow each individual, as well as the inter variability of anti-D concentrations strengthen the validity of the present study. On the other hand, the study population of the intervention study was small. There was an obvious difficulty to include subjects with repeated measurements close to term and postdelivery. There is a limitation by the relatively high incidence of protocol violations regarding the analysis of anti-D concentrations postpartum. Only 25 % was monitored weekly postdelivery, but in all of these, stable concentrations were maintained up to 30 days postdelivery. In addition, analyses of FMH at delivery was missing in 14/39 (36 %) of the cases. FMH above the detection level of 1 mL fetal blood is reported to occur in 4% of pregnancies.20
A considerable number of women will not have measurable levels of anti-D at term. The reported number varies between studies, 20-80%.7,8,13 In the present study, with a retrospective data analysis cohort, with over 4000 RhD negative women, with an RHD positive fetus, where a type-and-screen test was performed at delivery, 20% had a negative antibody screening at delivery. In the prospective part with 39 pregnancies included, the proportion with a negative antibody screen was higher, 44%. The different result between the two cohorts is unclear, the same method for antibody screening was used during the whole time period, BioVue Cassetttes with anti-IgG (Ortho Clinical Diagnostics) and LOD was estimated to 10 IU/L. Though, the retrospective cohort was from 2010-2012 and the prospective group from 2016-2018 and different anti-D preparations were used, Rhesonativ®, (Octapharma AG),1250 IU, in the first period and Rhophylac®, (CSL Behring) 1500 IU, in the latter period. Minor changes in the screening reagents used cannot be excluded.
Interpretation
Several studies have shown that one single dose anti-D in gestational week 28 is non- detectable at delivery in a considerable proportion of women, thus not protecting them from immunisation at the end of the third trimester. This is confirmed in the present study, with a range of 20-44% of women with undetectable levels at term. The two-dose strategy with RAADP at gestational week 28 and 34, gives a higher rate of detectable anti-D, 86%8 at term but is associated with higher costs and lower compliance of receiving the two doses.14 The risk of immunisation after the introduction of RAADP in the third trimester is decreased to an incidence rate of 0.2-0.4% and seems to be the same regardless of one-dose or two-dose strategy.5 Only a small proportion, approximately 1 %, of women with undetectable anti-D levels at delivery are calculated to be immunised,18 but they still represent the most common severe immunisations.
The study showed a large inter variation of anti-D concentration at delivery. The variation of anti-D levels may depend on individual IgG clearance from plasma and consumption of anti-D, giving a variability in residual anti-D levels and in half-life. Uptake from muscular compartments and fat tissue may vary as well. As expected, anti-D levels correlated to BMI.23 The following weeks after delivery the concentration anti-D remained stable.
The results indicate that an alternative strategy with administration of two RAADP doses (1500 IU), at gestational week 28 and 38, may be efficient to protect against immunisations at term, post term as well as postpartum. It has previously been concluded that adding a dose of anti-D in the third trimester cannot be justified due to high costs,19 but if the postdelivery anti-D dose could be excluded, this strategy may be cost effective. The potential of high compliance at 38 gestational weeks, which is crucial, must be assessed in different antenatal programs, but most women have a visit planned in gestational week 37-38. On the other hand, anti-D prophylaxis may be administered within 72 hours postdelivery for women non-compliant to RAADP at gestational week 38 or delivered preterm. If this strategy will lower the incidence of D-immunisation further is unanswered and has to be addressed in a large prospective trial. Ten out of the 39 women in our small study have been tested for D-immunisation in a subsequent pregnancy, all with negative screening test.
Conclusion A large proportion of RhD negative women have non-detectable levels of protective anti-D at term and post term. In addition to RAADP in gestational week 28 a second dose of RAADP in gestational week 38 to women at risk of anti-D immunisation was evaluated in this study. The gestational week 38 anti-D dose gives protective anti-D concentrations up to 30 days postpartum. If the second dose replaces the postpartum dose, the strategy may be cost-effective and possibly further reduce the risk of immunisation.