The prevention of Rh haemolytic disease of the fetus and newborn — general background
Haemolytic disease of the newborn (HDN) is a condition in which the lifespan of the fetal or newborn infant's red cells is shortened by the action of maternal antibodies against antigens present on the infant's red cells. Only maternal IgG antibodies can cross the placenta and enter the fetal circulation; the passage is an active mechanism that involves interaction between the Fc fragment of IgG and Fc receptors on the placenta. Red cell destruction begins in intrauterine life and may lead to severe anaemia and even hydrops fetalis and death in utero. In liveborn infants, red cell destruction is most severe at birth, but anaemia and jaundice will worsen in the first days of life. If jaundice is severe and not treated by exchange transfusion, the high bilirubin levels may impregnate the basal ganglia leading to kernicterus, a syndrome characterised by lethargy, spasticity and opistotonos with eventually death in 70% of affected infants. Those infants who survive have permanent brain damage.
After ABO antibodies which are naturally occurring, anti-RhD (anti-D) is the IgG red cell alloantibody found most frequently in patients, pregnant women and blood donors. Anti-D is by far the commonest cause of moderate and severe HDN, followed by the significantly less frequent disease due to anti-c and anti-K. This is due to the high immunogenicity of the RhD antigen compared with all the other (over 250) red cell antigens. For these reasons, although there are numerous Rh-antigens, RhD is the most important and subjects are classified as Rh-positive or Rh-negative if they are RhD-positive or RhD-negative respectively, without taking into consideration any of the other Rh antigens.
In the UK and most European Whites, the frequency of RhD-negatives is approximately 16% whereas it is about 5% in West Africans and practically nonexistent amongst Chinese’. RhD-negative subjects can make anti-D if they come into contact with RhD-positive red cells either by transfusion or pregnancy. About 90% of RhD-negative subjects transfused with a unit of RhD-positive blood will make anti-D (i.e. they will become Rh sensitised or Rh immunised). The rate of immunisation in RhD-negative women is not as high after pregnancy, firstly because the infant's red cells might not be carrying the RhD antigen and secondly, because the volume of red cells that crosses the placenta from the fetus to the mother (transplacental haemorrhage) is often too small to induce Rh immunisation. Hence, only 17% of Rh-negative women will become immunised to RhD after a pregnancy with an ABO compatible, Rh-positive infant, but often this primary immunisation is inapparent, i.e. often the antibodies will not be serologically detectable in all immunised women until after the second Rh-positive pregnancy. Postnatal prophylaxis with anti-D immunoglobulin has reduced the immunisation rate by a factor of 10 and antenatal prophylaxis has reduced this frequency by an even further factor of 101. In addition, immunisation by transfusion has practically disappeared due to the practice of transfusing Rh-negative patients and especially girls and women of childbearing age with Rh-negative red cells.
The volume of transplacental haemorrhage (TPH) or the passage of red cells from the fetus into the maternal circulation can be assessed by: i) the acid elution technique of Kleihauer–Betke which measures the number of fetal red cells as a proportion of adult cells in a sample of maternal blood; ii) immunofluorescence which measures, by flow-cytometry, the proportion of D-positive cells in a predominantly D-negative sample of maternal blood; iii) the rosetting technique which measures the same as above but under light microscopy; iv) other techniques such as solid-phase immunofluorescence. None of these techniques is well standardised and estimates on the same sample vary widely between different laboratories. The frequency and magnitude of TPH depends largely on gestational age; it is low and small in the first two trimesters unless there is trauma, a therapeutic abortion, or other form of obstetric manipulation. TPH is much more frequent in the third trimester and greatest at delivery; 0.94% of women were found to have TPHs greater than 2.5 ml of red cells at 30–39 weeks’ and 50% of women show detectable fetal red cells in their circulation after delivery. About 1% of these recently delivered women have TPHs of 3 ml or more and 0.3% have l0ml or more1. The obstetric manipulations that can increase the risk of TPH are amniocentesis, chorionic villus sampling, Caesarean section, manual removal of the placenta, external versions and other intrapartum manipulations. Placenta praevia, placental chorioangioma and choriocarcinoma are often associated with large TPHs.
Immediately after a first pregnancy in untransfused RhD-negative women who deliver an ABO compatible D-positive infant, anti-D is found in approximately 1%; in about half of these, anti-D is detectable between 34 and 40 weeks’ gestation1. The incidence of detectable anti-D in these women increases to 4–9% if samples are tested at 6 months post-delivery; the chance of immunisation is related to the magnitude of TPH1. As stated above, the frequency with which anti-D is detected after a second RhD-positive ABO compatible pregnancy is about 17%, indicating the real rate of immunisation after one pregnancy with an RhD-positive infant, i.e. primary immunisation occurred in most of these women during the first pregnancy.
SUPPRESSION OF Rh IMMUNISATION
Although it has been known for some time, from studies in experimental animals, that the passive administration of specific antibody can suppress immunisation to an antigen given at the same time, evidence of suppression of RhD immunisation by anti-D only started to become available in the early 1960s. In 1961, Stern3 administered RhD-positive red cells coated with anti-D to 16 RhD-negative volunteers and none developed anti-D. Subsequently, 10 of the subjects were given RhD-positive red cells without anti-D and five became immunised to the D antigen, thus proving that passive anti-D can suppress primary immunisation to the RhD antigen.
In the 1960s it was known that ABO incompatibility protected partially against RhD immunisation, i.e. RhD-negative mothers who delivered an RhD-positive infant were less likely to develop anti-D if they had ABO antibodies against their infant (e.g. mother group O, infant group A). It was therefore assumed by workers in Liverpool and in the USA that if IgM anti-D, which cannot cross the placenta, is given to mothers before delivery, the RhD-positive fetal red cells that cross the placental barrier during delivery would be destroyed by the passive antibodies in the mother and primary immunisation to the RhD-positive red cells would be suppressed4. However, IgM anti-D did not prevent RhD immunisation and it was soon shown by the two groups in the UK and the USA that IgG anti-D, when given intramuscularly (i.m.) could suppress immunisation to the RhD antigen5–7. As it had been shown that TPH occurs mainly at delivery, the two groups were able to demonstrate in clinical trials on Rh-negative mothers that concentrates of anti-D IgG would suppress RhD immunisation if given soon after delivery8,9.
The mechanism by which the administration of passive IgG anti-D (anti-D Ig) suppresses primary immunisation to the RhD-positive red cells is not known. Suppression may be simply related to the clearance of the red cells from the circulation before they reach immunocompetent sites or it may be due to more complex mechanisms involving recognition of foreign antigen and antigen presentation by the appropriate cells at the appropriate sites in the presence or absence of antibody.
In general, and from the scarce evidence available, it is accepted that 20 μg (100 IU) of anti-D will suppress immunisation to the injection or transplacental passage of 1 ml of packed RhD-positive red cells or 2ml of blood10. It is possible that 100IU might be excessive, but to be on the safe side, because not all preparations of anti-D Ig are totally comparable, and because it is difficult to measure the volume of TPH with any degree of accuracy, it is recommended that 25 μg (125 IU) anti-D should be given i.m. to cover a TPH of I ml RhD-positive packed red cells1,11. Although anti-D Ig is given i.m. in most countries, it is given intravenously in a few countries such as Canada. It is possible that a lower dose of anti-D Ig will suppress immunisation to a given volume of RhD-positive red cells when given intravenously (i.v.) than when given i.m. because it takes about 2 days for anti-D to reach its maximum level in plasma after i.m. administration, thus delaying the clearance of red cells from the circulation. The suppressive effect of anti-D Ig should be maximal if the Ig is given immediately before or at the time the Rh-positive red cells enter the maternal circulation; for this reason it is recommended that anti-D Ig is given within 72h of delivery. However, a proportion of responders can be suppressed if anti-D Ig is given as late as 13 days after a small volume of RhD-positive red cells enters the circulation1.
Passive anti-D Ig will only suppress primary RhD immunisation and it has no effect in women who have already developed anti-D, however weak this may be. Once the immune response has been switched on, it cannot be reversed12,13.
The standard dose of anti-D Ig given to Rh-negative women after delivery of an Rh-positive infant varies in different countries. In the UK this dose is 100 μg (5001U) and is sufficient to cover a TPH of at least 4ml of fetal red cells, i.e. 99% of all TPHs. This means that a screening test, such as the acid elution test of Kleihauer-Betke is mandatory to screen for TPHs >4ml of red cells; this should be measured in order to assess the need for additional doses of anti-D Ig. In the USA and in many other countries, doses of 250—300μg anti-D Ig are used postnatally with the objective of covering TPHs >12 ml, i.e. approximately 99.8% of all TPHs making screening tests for TPH unnecessary. Although most European countries using the large dose of anti-D Ig do not screen for large TPHs, in the USA it is still mandatory to screen all unimmunised Rh-negative women postpartum for TPHs in order to estimate whether any additional anti-D Ig is needed for effective prophylaxis14.
Other potentially sensitising episodes during pregnancy or at termination of pregnancy should always be covered with prophylactic anti-D Ig. In the UK, a smaller dose of anti-D Ig of 50mg (250IU) is available to treat such episodes in the early part of pregnancy when the size of TPH should be smaller than 2 ml of red cells. It is thought that the cut off stage of pregnancy below which the lower dose should be used should be 20 weeks but there is no firm evidence to support this recommendation and some prefer to be cautious and use the lower dose for potentially sensitising episodes and terminations only up to 12–13 weeks’ gestation.
Several series from different countries have shown that, when anti-D is administered only postnatally, 0.1–0.5% of Rh-negative women will have demonstrable anti-D within the 6 months following delivery. At the end of the second pregnancy with an RhD-positive, ABO compatible child, anti-D is found in at least 1.5% of Rh-negative women, i.e. 0.7% immunisation rate due to sensitising TPHs during each pregnancy and approximately 0.2% due to a TPH after the first delivery which was too large to be covered by postnatal prophylaxis1. It is therefore obvious that postnatal prophylaxis decreases the incidence of Rh immunisation to 10% of the original 17% rate found in the pre-prophylaxis era (i.e. from 17% to 1.5–2%). However, if we are to affect significantly the residual rate of RhD immunisation, there is no option but to introduce antenatal prophylaxis and aim to lower the rate of immunisation by a further 10-fold.
Despite initial concerns about the safety to the fetus and the absence of randomised, properly controlled trials of antenatal prophylaxis, this form of treatment is now accepted as safe for the fetus in several countries because, with the doses given (100–300 μg), the concentration in the fetus after crossing the placenta would never be hi h enough to cause significant red cell destruction15,16.
Reports strongly suggesting that antenatal prophylaxis is effective in preventing most of the residual rate of RhD immunisation in pregnancy come from Canada and the UK. The largest series is from Bowman17 in Canada with nearly 10,000 Rh-negative women delivering RhD-positive fetuses and who were given either 300μg of anti-D Ig i.m. or 240—300μg i.v. at 28 weeks’ gestation. Less than 0.1 % of women developed anti-D at delivery whereas historical controls, given only postnatal anti-D Ig showed an immunisation rate of 1.8% (i.e. considerably higher than the 0.7% reported above). In the English series, reported by Tovey18, a total of 1328 Rh-negative primiparae carrying D-positive fetuses were given two doses of 100 μg of anti-D at 28 and 34 weeks; only 0 pg 16% had detectable anti-D at the time of delivery compared with 0 pg 9% Rh-immunisation rate in historical controls given only postnatal prophylaxis. The real rate of RhD immunisation following antenatal and postnatal prophylaxis is slightly higher than quoted in the two series above because anti-D developed in a few more women during a second pregnancy with a D-positive child. Nevertheless, the difference between the rates of immunisation with and without antenatal prophylaxis remained significant1 and argues strongly in favour of antenatal prophylaxis.
There are two modalities for the administration of antenatal prophylaxis: i) a single dose of 300pg of anti-D Ig at 28 weeks; ii) two doses of 100μg anti-D given at 28 and 34 weeks.
With the first regime, it has been shown that some women have no detectable anti-D at delivery2 and Mollison1 has expressed concern that concentrations of passive anti-D at term might be too low to be capable of suppressing Rh immunisation1. On the other hand, with the two lower dose regimes proposed by Tovey18, the maternal concentrations of anti-D achieved at term will be higher than when the 300μg dose is given at 28 weeks. Therefore, there seems to be a clear economic advantage with a firm scientific basis for recommending a split antenatal smaller dose or for postponing the administration of the 300μg dose by a couple of weeks from the 28th to the 30th week.
On the whole, anti-D prophylaxis is an example of a significant triumph of modern preventive medicine. In England and Wales, registered deaths due to RhHDN fell from 1 in 2180 births in 1953 to 1 in 5400 in 1977 and to 1 in 62,500 in 199019–21. The frequency of Rh immunisation has also decreased considerably since the introduction of RhD prophylaxis. For example in a hospital in Michigan, USA, where postnatal prophylaxis was introduced in the 1960s and antenatal prophylaxis in 1985, the frequency of detection of anti-D in pregnant women was 1 in 238 in 1974, 1 in 963 and 1988 and 1 in 1663 in 199222–24
In industrialised countries, good obstetric and neonatal care, coupled with routine anti-D prophylaxis, have contributed to make haemolytic disease of the newborn a very rare occurrence in hospital practice.