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.