Abstract
Background
The aim of this study was to examine which pregnancies are associated with RhD immunisation and haemolytic disease of foetus and newborn (HDFN) when postnatal RhD prophylaxis is applied.
Material and methods
This retrospective cohort study included pregnancies with RhD immunisation; each of the pregnant women received anti-D immunoglobulin after delivery, miscarriage or invasive antenatal diagnostic procedures. For each pregnancy we analysed the order of pregnancy that caused immunisation as well as the order of the monitored pregnancy and whether the anti-D antibodies caused HDFN.
Results
Anti-D antibody was detected in 1.2% of RhD-negative pregnancies. Out of 89 monitored pregnancies, 56 (63%) were immunised by the first pregnancy, 21 (24%) by the second one, and 12 (13%) by subsequent pregnancies. HDFN occurred in 28 cases; 25 of them were the consequence of the immunisation in the first pregnancy. The most severe cases of HDFN, perinatal death (n=2) and intrauterine transfusion (n=7) were consequence of immunisation during the first pregnancy. Significantly more cases of HDFN were caused by immunisation in the first pregnancy than by immunisation in subsequent pregnancies (χ2=12, p<0.01).
Conclusion
RhD immunisation could be reduced in more than half cases by administering anti-D immunoglobulin at the beginning of the third trimester of pregnancy, especially the first pregnancy.
Keywords: anti-D immunoglobulin, haemolytic disease of newborn, pregnancy immunisation, prevention, RhD
Introduction
Haemolytic disease of the foetus and newborn (HDFN) has long been an important cause of perinatal death and morbidity. In most cases it is caused by anti-D antibodies. In the case of RhD immunisation, the common sequence is a prior pregnancy with a RhD-positive foetus, which induces foeto-maternal haemorrhage (FMH)-related immunisation and a subsequent pregnancy with another RhD-positive foetus, which triggers manifest disease1,2.
Considering that FMH most usually occurs during delivery, administering anti-D immunoglobulin (Ig) to RhD-negative women after the birth of a RhD-positive child is of the utmost importance. Additional recommendations are to administer anti-D Ig in conditions prone to FMH, such as miscarriage, termination of pregnancy, invasive antenatal diagnostic procedures, external version and Caesarean section3–7. The combined strategy of routine postnatal administration and of additional anti-D Ig in high-risk conditions during pregnancy and delivery has substantially decreased the rate of RhD immunisation from 13–19% down to 0.9–1.8%4–6.
FMH can occur after the 28th week of pregnancy in the absence of especially high-risk conditions; in some some developed countries anti-D Ig is, therefore, administered at the beginning of the third trimester of pregnancy8,9. The combined strategy of routine postnatal and antenatal prophylaxis and additional anti-D Ig in high-risk conditions during pregnancy and delivery has substantially further decreased RhD immunisation to 0.1–0.3%8–12.
According to the results of an international survey carried out in 2003, anti-D Ig prophylaxis is practiced nationwide in North America, the United Kingdom and the Netherlands while in Spain, Poland, and Austria it is practiced only in parts of the country13. It is not current practice in many other countries to provide D-negative women with antenatal anti-D Ig prophylaxis, although there are tendencies to do so14,15. The programme of antenatal prophylaxis in some countries is applied to all RhD-negative women while in other countries the administration of anti-D Ig to RhD-negative women is restricted to those without a living child, because of the scarcity of anti-D Ig13.
In this study we wanted to examine which pregnancies cause RhD immunisation and link this with HDFN. This research may be useful for the rational planning of antenatal administration of anti-D Ig.
Materials and methods
The study was performed at the Department of Transfusion Medicine of Split University Hospital Centre, Croatia and included pregnant women with anti-D antibodies who delivered between 1 January 2002 and 31 December 2011, and their infants. Split Hospital is a regional institution responsible for overall pregnancy care, monitoring of red blood cell (RBC) alloimmunised women, and the management of deliveries.
Protocol for testing RhD-negative pregnant women
The Croatian programme for preventing and detecting RhD immunisation in pregnancy is free of charge. All RhD-negative women who deliver a RhD-positive child receive 300 g (1,500 IU) of anti-D Ig within 72 hours of delivery. An additional dose of anti-D Ig 300 g (1,500 IU) on top of routine postnatal anti-D prophylaxis is advised after a miscarriage, termination of pregnancy or invasive procedures during pregnancy16. Antibody screening in RhD-negative pregnancies is performed at week 12 of gestation, and the test is repeated at weeks 28 and 3417. Antenatal maternal serum screening for unexpected RBC antibodies was performed using polyspecific microcards (BioVue System, Ortho Clinical Diagnostics, Raritan, NJ, USA) with commercially prepared RBC tests (Selectogen I and II, Ortho Clinical Diagnostics) at 37 °C and an indirect antiglobulin test, according to the manufacturer’s instructions. If the screening test was positive, the antibody specificity was determined. The frequency of testing pregnancies with anti-D alloantibodies is dependent on antibody titre and the duration of the pregnancy.
Pregnancies with anti-D alloantibody
For the follow-up of pregnancies with anti-D alloantibodies, we used Immunisation Tracking Forms, filled out after anti-D antibodies have been detected in a pregnant woman or after a case of HDFN had been identified. For each RhD-negative woman with anti-D antibodies the form for tracking immunisation was created if she denied receiving Rh prophylaxis during pregnancy. The first set of data included the order of the pregnancy and the order of screening in the ongoing pregnancy. Anti-D antibody titres were determined by the tube method and values ≥16 were considered to indicate a very high risk of HDFN. Pregnancies with anti-D antibody were also closely monitored by experienced obstetricians. The main method was to measure the peak velocity of blood flow through the central cerebral artery of the foetus at weekly intervals. The values obtained were then compared with the standard18. In the case of pathological values the woman was referred to the national referral centre for further treatment and supervision.
The second set of data comprised the laboratory tests of the neonate, including serological analysis of cord blood for RhD antigen and the direct antiglobulin test (DAT). Infants who had a positive DAT, based on the sample of venous blood, were tested for ABO and RhD antigen and DAT.
The third set of data included the information on intrauterine transfusion (IUT) therapy of the foetus and exchange transfusions or simple blood transfusions in the neonate. In our hospital, a neonatologist makes a decision whether to perform exchange transfusions or simple blood transfusions or phototherapy based on the current recommendations19.
IUT treatment in Croatia can be performed only at Zagreb University Hospital Centre and the “Holy Ghost” Hospital in Zagreb.
Study design
The number of RhD-negative pregnancies to be tested was acquired from the electronic database of the Department of Transfusion Medicine.
The information on anti-D antibody pregnancies was gathered from tracking forms compiled in our Department. For each pregnancy we analysed the order of the monitored pregnancy regardless of whether it resulted in abortion or delivery.
A pregnancy was considered a cause of immunisation if the screening test was negative at the beginning of a pregnancy (provided that the screening was done prior to week 18) and anti-D antibody was detected at repeat testing, at delivery or at the beginning of the following pregnancy (prior to week 18). If anti-D antibody was detected for the first time at repeated screening at week 28 or 34, or at delivery, the immunisation was considered to be primary.
Pregnant women with RhD immunisation were not included in this study if: (i) the screening test was not done prior to week 18 of pregnancy; (ii) anti-D Ig were not administered after delivery, miscarriage, termination of pregnancy or invasive procedures; (iii) the women were administered anti-D Ig during pregnancy (i.e. antenatal administration).
Forms were also used to analyse the neonates’ laboratory results (DAT and RBC antigens) and the frequency of therapeutic interventions to the foetus and newborn, including IUT, exchange transfusion, and simple transfusion. Severe HDFN was considered to have occurred if the DAT and specific RBC antigen test results were positive and transfusion therapies (IUT and/or exchange transfusion and/or blood transfusion) were treatment options and in cases of perinatal deaths caused directly by HDFN.
Post-mortem records from the hospital’s Pathology Department were used as a source of data in the case of miscarriage, stillbirth or neonatal death.
The study was approved by the Ethics Committee of the University Hospital Centre in Split.
Statistical analysis
Data were analysed using Statistica 7.0. (StatSoft, Inc., Tulsa, OK, USA). The number and proportion of cases of RhD immunisation and HDFN were analysed given the ordinal number of pregnancy. Differences between groups were compared using the χ2 test. P-values less than 0.01 were considered statistically significant.
Results
Correlation of RhD immunisation with order of pregnancy
During a 10-year period from 2002–2011 a total of 8,400 RhD-negative pregnancies were tested. Anti-D antibodies were detected in 102 pregnancies (1.2%). Thirteen pregnancies were excluded from the study (screening test was not done prior to week 18 of pregnancy n=11; anti-D Ig was not administered after miscarriage n=2). Further research was carried out on the 89 pregnancies with anti-D antibodies that satisfied the study inclusion criteria. These pregnancies occurred in 69 immunised women. Of these 89 pregnancies, immunisation occurred in the first pregnancy in 56 cases (63%), in 21 cases (24%) in the second one pregnancy, and in 12 cases (13%) in subsequent pregnancies (Table I).
Table I.
The number of women and anti-D antibody pregnancies linked to the pregnancy that caused immunisation.
| Pregnancy that caused RhD immunisation | N. of women | N. of pregnancies (%) | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| Total* (%) | Repeat screening | First screening in following pregnancy | ||||
|
| ||||||
| week 28 | week 34 | delivery | ||||
| First | 38 (55) | 2 | 5 | 12 | 19 | 56** (63) |
| Second | 19 (28) | 2 | 2 | 8 | 7 | 21*** (24) |
| Third and following | 12 (17) | 3 | 4 | 4 | 1 | 12 (13) |
|
| ||||||
| Total | 69 (100) | 7 | 11 | 24 | 27 | 89 (100) |
First screening in this pregnancy was negative and anti-D antibodies were detected at repeat screenings in this pregnancy or at first screening in the following pregnancy.
Out of 56 pregnancies monitored, 19 were first pregnancies, 22 were second pregnancies and 15 were third or subsequent pregnancies.
Out of 21 pregnancies monitored, 13 were second pregnancies and 8 were third or subsequent pregnancies.
The first pregnancy caused RhD immunisation in 38 women. The anti-D antibodies were first detected during the first pregnancy screenings in 19 women, and in the remaining 19 cases they were detected at the beginning of the second pregnancy. RhD immunisation was associated with the second pregnancy in 19 women, with 12 of these cases being detected for the first time at the second pregnancy screening, and in 7 women at the beginning of the third pregnancy. In subsequent pregnancies (third or more) RhD immunisation was mostly detected at repeat screenings during pregnancy (11 of 12 cases) (Table I). Antibodies detected in the first trimester of the following pregnancy were usually the result of FMH before delivery. In some cases, due to extensive FMH at birth, the amount of postnatal prophylaxis was insufficient. During this study, 20 pregnant women with RhD immunisation had more than one pregnancy.
Correlation of severe cases of haemolytic disease of foetus and newborn with order of pregnancy
Out of 89 monitored pregnancies three ended up in miscarriage and three in perinatal death (HDFN was the direct cause of two perinatal deaths). Transfusion treatment (IUT and/or blood exchange and/or blood transfusion) was required in 26 patients with HDFN (Table II). Comparing the cases of severe HDFN after primary immunisation or reactivation we found more severe cases (perinatal deaths, IUT, more than one exchange transfusion) in the latter situation. There was two cases of HDFN after primary immunisation and both occurred in the first pregnancy (Table III).
Table II.
Severe HDFN cases caused by anti-D antibodies in correlation to the pregnancy that caused RhD immunisation.
| Pregnancy that caused RhD immunisation | RhD pos | HDFN | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| Total (%) | Perinatal death | IUT | Blood exchange | Blood transfusion | ||
| First | 51 | 25 (89) | 2 | 7 | 13 | 3 |
| Second | 20 | 3 (11) | 0 | 0 | 2 | 1 |
| Third and following | 11 | 0 (0) | 0 | 0 | 0 | 0 |
|
| ||||||
| Total | 82 | 28 (100) | 2 | 7 | 15 | 4 |
HDFN: haemolytic disease of foetus and newborn; IUT: intrauterine transfusion.
Table III.
Cases of HDFN in RhD positive children born after primary immunisation or reactivation.
| RhD immunisation | Primary immunisation* N=42 |
Secondary immunisation N=47 |
Total N=89 |
|---|---|---|---|
| RhD pos child | 42 (100%) | 40 (100%) | 82 (100%) |
| No severe HDFN | 40 (95%) | 14 (35%) | 54 (54%) |
| Severe HDFN | 2 (5%) | 26 (65%) | 28 (46%) |
| Blood transfusion | 1 | 3 | 4 |
| Blood exchange | 1 | 14 | 15 |
| IUT | 0 | 7 | 7 |
| Perinatal death | 0 | 2 | 2 |
First screening in the pregnancy was negative and anti-D antibodies were detected at repeat screenings in this pregnancy.
HDFN: haemolytic disease of foetus and newborn; IUT: intrauterine transfusion.
In 25 cases severe HDFN was the result of the first pregnancy and the most severe cases of HDFN cases (perinatal death, n=2; IUT treatment, n=7) occurred only in this group (Table II). Among women immunised during their first pregnancy (n=56), HDFN occurred in 45% of cases, whereas among those immunised during a second or following pregnancy (n=30), the rate of HDFN was 9%, which is five times less. We proved that a significantly greater number of severe HDFN cases were caused by a first pregnancy with RhD immunisation than by subsequent pregnancies (χ2=12, p<0.01).
In pregnant women immunised by the first pregnancy, the risk of HDFN increased with every subsequent pregnancy. In this group, 11% of RhD-positive children were affected by HDFN in the first pregnancy, 65% in the second and 77% in the subsequent ones (Table IV).
Table IV.
HDFN cases in monitored pregnancies in which the first pregnancy was the cause of RhD immunisation.
| Monitored pregnancy | N. of pregnancies | RHD positive children | HDFN | |
|---|---|---|---|---|
|
|
||||
| Number | % | |||
| First | 19 | 19 | 2 | 11 |
| Second | 22 | 20 | 13 | 65 |
| Third and following | 15 | 13 | 10 | 77 |
|
| ||||
| Total | 56 | 52 | 25 | 48 |
HDFN: haemolytic disease of foetus and newborn.
Discussion
Our study showed that, in women administered postnatal prophylaxis, in more than 50% of cases RhD immunisation was caused by the first pregnancy and that almost 90% of cases of severe HDFN were the effect of RhD immunisation during the first pregnancy.
In our study 38 out of 69 cases of RhD immunisation were caused by FMH during the first pregnancy. These findings are in accordance with other studies which reported that in more than 50% of RhD immunised women, the immunisation had occurred with their first-born child20,21. One possible reason why more than half of the RhD immunisations occurred during the first pregnancy in our study is the fact that during the period monitored almost 50% of women were in their first pregnancy22. Another reason may be the strong immune response of some women, who will immunise at the very first exposure to RhD antigen or due to minor FMH while carrying the first RhD positive baby.
According to our research 40% of RhD immunisations caused by the first pregnancy were detected early in the second one. We assume that in most of these cases women were immunised already at labour. In rare cases women were immunised during labour due to extensive FMH (>30 mL of foetal blood) for which the Croatian standard amount of 1,500 IU RhD IgG was not sufficient. Such extensive FMH happens in 1 out of 400 pregnancies23. Some national guidelines advise quantifying FMH by the Kleihaur-Betke test after Caesarean section, abdominal trauma during pregnancy, a twin delivery, or surgical removal of the placenta10. In our hospital, routine testing for post-partum FMH to guide the doses of anti-D Ig was not performed. However, until now there had been poor evidence regarding routine testing for post-partum FMH. Several studies showed a correlation between assisted delivery and the presence of FMH, but none of these studies provided data about subsequent immunisation24,25.
We found that seven of 69 cases of RhD immunisation were due to FMH before 28 weeks. These cases of RhD immunisation cannot be prevented by antenatal RhD prophylaxis at week 28, but could be prevented by additional anti-D Ig in high-risk conditions during pregnancy8–12.
In our study, first pregnancy RhD immunisation caused 89% of the cases of severe HDFN. This can be explained by the fact that the women in this group had mostly had more than one pregnancy, the ratio of the number of pregnancies and the number of women was highest, and so the possibility of HDFN was the greatest. In terms of developing HDFN, the risk was much greater in subsequent pregnancies in women immunised by their first pregnancy. These findings are consistent with those of other studies, in which it appears that if a particular woman already had a baby suffering from HDFN, having more babies carried even greater risks1. One study found that RhD immunised women gave birth to fewer children than non-immunised RhD-negative women. The most likely explanation for this is that some immunised women refrain from becoming pregnant again24. This is more expected in women who already had two or more children, and were immunised in the second or subsequent pregnancies.
In this study RhD immunisation caused two cases of severe HDFN in first pregnancies. In a previous study Dajak et al. already noted that severe HDFN cases are possible in the first pregnancy. In these cases anti-D antibodies were detected at the repeat screening (at week 28) of the pregnancy, apparently caused by FMH prior to week 2826. Other studies also detected cases of HDFN treated with transfusion or exchange transfusion in babies born after primary RhD immunisation10,24,25.
We compared our results of prevalence of RhD immunization with that in studies in which the administration of RhD IgG was only postnatal, as is the case in our country. According to the results of a meta-analysis, which included prospective studies of the incidence of RhD immunisation for certain forms of RhD prophylaxis, the incidence of RhD immunisation in a group of women with only postnatal RhD protection was 1.6% (0.9 to 1.9%)12. Other studies suggest a much higher incidence of RhD immunisation with postnatal protection, with reported rates of more than 3%27,28. We can conclude that the incidence of RhD immunisation in our study population confirms that achieved with regular administration of postnatal RhD prophylaxis. Antenatal prophylaxis is needed to lower the prevalence of RhD immunisation further.
Finally, antenatal administration of anti-D Ig prophylaxis is necessary in all RhD-negative women. Taking into account economic constraints, antenatal prophylaxis will achieve the greatest impact if administered to RhD-negative women in their first pregnancy or in those without a living child. This form of antenatal prophylaxis would reduce the use of anti-D Ig by approximately 50% and significantly prevent the development of HDFN in second and subsequent pregnancies.
Acknowledgments
The Authors are grateful to Jela Mratinovic Mikulandra, Hania Dadic Jadric, Anuska Trlaja, Branka Skelin, Javorka Dodig, Dejana Bogdanic, Eleonora Gjakun, Jelena Varenac Lukacevic and Ivica Bradaric for collecting data.
Footnotes
The Authors declare no conflicts of interest.
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