APPENDIX I Details of clinical trials of antenatal Anti-D
A) 2 × 300 μg at 28 and 34 weeks12
The first studies involved two doses of the standard Canadian/North American dose of 300 μg at 28 and 34 weeks. There were sound theoretical reasons for using two doses, because the circulating half life of IgC anti-D is 2 I days and the better protection against larger bleeds in the third trimester with the second dose. This schedule was clearly shown to be effective in a relatively small number of primigravidae. The authors quote a 94% reduction to <1/1000 at risk pregnancies (1 ×6% to 0.1%). If one excludes logistic failures, no cases of alloimmunisalion were seen in the treatment group.
Trial details: open study: nonrandomised concurrent controls; two urban hospitals in antenatal group; other three rural hospitals in control group; both groups given standard postpartum prophylaxis of 300μg at delivery.
Treatment group (1968-1976): (300μg at 28 and 34 weeks) 0/1204 primigravidac alloimmunised at delivery; 0/905 developed immune anti-D at 6months post delivery (one immunised before 28 weeks); operational failure rate in primigravidae = 1/1204 = 0.08%.
Control group (1967-1974): 45/2768 primigravidae alloimmunised at delivery; operational failure rate in primigravidae = 1.6%.
Conclusion: alloimmunisation can be reduccd from I .6%i to 0.1% in primigravidae (946 reduction) (x2= 16.14: P<0.001).
B1) 1 × 300 μg at 28 weeks2
Primarily because of anti-D shomges, a single dose at 28 weeks was evaluated early in the Canadian Rh programme as an alternative. In a small number of primigravidae, a single dose of 300μg at 28 weeks reduced alloimmunisation from 1.6% (45/2768) to 0.18% (2/1088). If logistic failures an: excluded, none of the treatment group was alloimmunised. For multigravidae, the observed failures were 0.846, but excluding women estimated to be sensitised in previous pregnancies, the ‘true'failure rate was estimated 3% 0.42%, compared to 2.2% in the control multigravid group.
Trial details: open study with historical controls: bolh groups given standard postpartum prophylaxis of 300 pg at delivery. Treatment group examined at 6 months for anti-D.
Treatment group (1975-1977): (300μg at 28 weeks).
Group I 0/1084 prirnigravidae alloimmunised at time of delivery (expected 14/1084): 0/512 developed immune anti-D after 6months: (two alloimmunised before 28 weeks at 12.5 and 6.5 weeks); operational failure rate in primigravidae is 2/1088 =0-18%.
Group 2 6/719 rnultigravidae alloiinmunised at time of delivery; 3/716 estimated to have a ‘secondary response’ from an earlier pregnancy: 3l7 16 estimatcd ‘true failures’ (expected 127/16); ‘true failures’ in multigravidae 3/716 = 0.42%. operational failure rate in multigravidae is 6/716 = 0.84%.
Control group (1967-1974): 45/2768 primigravidae alloimmunised at delivery; opcralionnl failure rate in prirnigravidae = 1.6%. Of the 45 primigravidae it was noted that 3/45 (6.67%) were alloimmunised before 28 weeks (3/2768 = 0.108%) and 42/45 (93.33%) were alloimmunised after 28 weeks (42/2768 = 1.52%). 17/765 multigravidae alloimmunised at delivery; operational failure rate in multigravidae = 2.2%.
Conclusion: excluding other causes, antenatal alloimmunisation would reduce from 1 6 8 to 0.18 in primigravidae, and from 2.2% to 0.42% in multigravidac (x2= 12.29: P<0.001). Including all patients in treatment and control arms, for primigravidae. alloimmunisation is reduced from 1.6% to 0.18% (88.75% reduction) for multigravidae, from 2.2% to 0.84% (X2= 3 81: P =0.051)
B1a) 1 × 300 μg at 28 weeks9
This paper is an update on the earlier trial with additional numbers, over 8.5 years to February 1986. All cases reported in the earlier trials are included. There is no distinction made between primigravidae and parous women. although it is estimated that 78%: were primigravidae. The dose also changes from an estimated 24Opg for the first 5 years, with a subsequent change to 300μg as standard. It is also not clear at what point the standard postpartum dose changed to l20μg i.v. Nevertheless, the study has value as the largest number of women evaluated under routine conditions, as opposed to clinical trial conditions. It was estimated that the treatment failures (i.e. excluding alloimmunisation before 28 weeks and other causes during pregnancy) were 0.18% (I7/9295). The observed alloimmunisation rate in the treatment groups fell from an estimated (historical) 1.8% (62/3533) to 0.32% (30/9295) in routine practice. Excluding other causes, and including all women regardless or parity, the authors conclude that antenatal alloimmunisation can be reduced from 1.4 % to 0.278 (25/9295) but this method of calculation overestimates the control failures, and underestimates the treatment failures, and gives a more favourable outcome to antenatal prophylaxis. Nevertheless, the reduction in alloimmunisation as a result of the programme is significant, and evidence of the efficacy of prophylactic antenatal anti-D in routine practice.
Trial details: open study with historical controls in one province in Canada. Both groups given standard postpartum anti-D it is not clear if the dose reduced to 120μg i.v. from 300μg, and at what point.
Treatment group (1977–1986): (1977–83 = 240μg = 1200IU at 28 weeks). (1983–86 = 300μg = 1500IU at 28 weeks). 25/9295 alloimmunised (not all primigravidae). failure rate = 0.27%. 8/25 (32%) alloimmunised < 28 weeks: 17/25 (68%) treatment failures. treatment failure rate (17/9295) = 0.18% NB: actual total failures over the time was 30/9295 = 0.32%.
Control group (1967–74 data — same as in earlier studies): 62/3533 total (i.e. not all primigravidae) [note: this also includes anti-D detected up to 3 days after delivery, which gives a higher figure than reported by othcr groups]; operational failure rate = 1.8%. Of the 45 primigravidae in this group it was noted that 3/45 (6.67%) were alloimmunised before 28 weeks (3/2768 = 0.1088) and 42/45 (93.33%:) were alloimmunised alter 28 weeks (42/2768 = 1.52%).
Conclusions: excluding other causes. and including all women regardless of parity. antenatal alloimmunisation can be reduced from 1.8% to 0.274 = 854, reduction in alloimmunisation (x2= 81.72: P < 0.001). Under routine conditions, with no exclusions, anti-D alloimmunisation can be reduced from 1.8% to 0.32% (78% primigravidae in the group) = 82% reduction in alloimmunisation (x2= 78.1: P < 0.001).
B2) 1 × 300 μg at 28 weeks18
In this small study using the same dose as the Canadian trials, alloimniunisation was reduced to less than 0.32% in the treatment group (0/291) compared to historical controls with 146% alloimmunisation.(6/322). The study includes women of all parities, and the data are comparable in practice to the larger Canadian study. confirming the efficacy of a single dose of anti-D i n reducing alloimmunisation. It was noted that 9% of women had anti-D at delivery in the treatment group. but no positive DAT. jaundice or anaemia was observed in the babies. Kleihauer estimation of FMH at delivery was also carried out in both groups. The number of observed FMH was the same in both groups — 63 in treatment group versus 72 in control group. but the number of l a r p FMH (> I ml) was significantly different 5/63 in treatment group versus 28/72 i n the control group (x2: P < 0.001).
Trial details: open study at one hospital in Denmark. with historical controls. Both groups given standard postpartum prophylaxis of 200μg Both groups stated to be comparable as regards first pregnancies, risk events, instrumentation and Caesarian Section rate. Postpartum 10-month follow-up sample obtained in both groups.
Treatment group (1980–85): (300μg at 28 weeks) 609 women; 346 with RhD-positive baby: 0/291 alloimmunised at 10months = <0.32 % (assuming 1/29?).
Control group (historical controls 1972–77): 354 with RhD-positive baby: 6/32? alloimmunised at 10months = 1.86% (or during subsequent pregnancy). Treatment versus controls t-test; P < 0.45.
Conclusion: alloimmunisation can be reduced from 1.86% to <0.32%(83% reduction) (x2= 3.72: P= 0.054).
C) 1 × 250 pg at 32 weeks17
In this study, the European dose of 250μg (rather than 300μg) was given later in pregnancy. at 32–34 weeks. These doses are not signilicantly different, given the limitations of the anti-D assays. Although a reduction in alloimmunisation was observed. from 1.6%) (10/645) to 0.38% (2/529) in the treatment group regardless of parity, including those women immunised during pregnancy before 32 weeks raised the failure rate to 0.95%. In practice. therefore, it is demonstrated that a single dose at 32–34 weeks is too late to provide sufficient protection against intrapartum sensitisation. as might be expected from the natural history of detectable FMH during pregnancy.
Trial details: open study at one hospital in Sweden with historical controls. Both groups given standard postpartum prophylaxis of 250μg anti-D with 8-month follow-up sample for anti-D.
Treatment group (1973–77): (250μg at 32–34 weeks) 830 women 529 with RhD-positive baby: 2/529 alloimmunised = 0.38%. 1/236 primigravidae = 0.42%. 1/293 multigravidae = 0.34%. 0/59 alloimmunised in second pregnancy. NB: three primigravidae excluded because anti-D before 32 weeks; if included, operational failures = 5/529 = 0.95%.
Control group (historical control dates not given): 10/645 alloiminunised = 1.6%. 5/286 primigravidae = 1.75%. 5/359 multigravidae = 1.39% (1.6 versus 0.38 =P < 0.05). The authors note that if weak anti-D is considered to be due to prophylaxis, then 0/529 in trial group, and 9/645 could be considered alloimmunised =P < 0.005.
Conclusions: excluding logistic failures, alloimmunisation can be reduced from 1.6% to 0.38% (x2=2.87: P= 0.09). When including immunisation before prophylactic anti-D at 32–34 weeks. the reduction is 0.95% (x2: P=0.511). In primigravidae. the reduction is from 1.75% to 0.42%. with small numbers in each group (x2= 1.0: P= 0.317). Giving anti-D at 32–34 weeks is therefore probably too late to be most effective.
D1) 2 × 100 μg at 28 and 24 weekss
This was the first study using a divided dose of anti-D calculated t o give the same (or more) anti-D in the maternal circulation at term 300 μg dose at 28 weeks. and specifically addressed first pregnancies. The impetus for such an approach in the UK was primarily hased o n economic use of anti-D, with emphasis in first pregnancie greater benefit with limited amounts of anti-D available. First pregnancies (2069) were treated, in comparison with 2000 historical control primigravidae. This initial report showed a reduction from 0.9% in controls to 0.16% in the treatment group, a similar figure to that demonstratcd by Bowman et al with a single 300μg dose (0.18%).
Trial details: multicentre open study in first pregnancies, with historical controls. Both groups given standard postpartum prophylaxis of 100 μg i.m. Six-monthly testing for anti-D was undertaken in some women.
Treatment group (1980–81): (100μg at 28 and 34 weeks) 2/1238 primigravidae alloimmunised at delivery and 6 months = 0.16% (1059 tested at 6 months).
Control group (historical 1978–79): 18/2000 primigravidae alloimmunised = 0.9%.
Conclusion: Including all Failures, alloimmunisation can be reduced in primigravidae from 0.9% to 0.16% (82% reduction), (x2=5.64: P= 0.018).
D1a) 2 × 100 μg at 28 and 34 weeks4
In a follow-up report to the original study, the treatment cohort had been followed longer. and a more detailed breakdown of the control data was given. If only failures after the first pregnancy are considered, the control alloimmunisation rate is 0.95% (19/2000); in the treatment group, it is only 0.32% (4/1238), giving a 66% reduction in alloimmunisation. It is not possible t o identify the comparative immunisation rate in both groups in subsequent pregnancies, as the original antenatal prophylaxis group were only given standard postpartum anti-D. However, there was a suggestion of protection persisting into subsequent (up to four) pregnancies in the original trial group, with a total of six failures in 2041 RhD-positive pregnancies (0.29%). compared to 32/2985 (1.07%) in RhD-positive pregnancies in the original historical cohort.
Treatment group (1980–81): 4/1238 primigravidae alloimmunised first pregnancy = 0.32%; 1/604 in second (not given antenatal prophylaxis)= 0.17%. 6/2041 all pregnancies including third and fourth = 0.29%.
Control group (historical 1978–79): 19/2OO0 primigravidae alloimmunised during first pregnancy = 0.95% (9/751 women alloimmunised in second = 1.2%; 32/2985 all pregnancies = 1.07%).
Conclusion: in first pregnancies, alloimmunisation can be reduced from 0.95 to 0.32% (66% reduction), (x2=3.42: P= 0.064).
D2) 2 × 100 μg at 28 and 34 weeks3
A second trial also using 100μg in two doses of anti-D was reported from France, this being the only randomised trial reported with a protective dose of antenatal anti-D administration. Considering first pregnancy only. the reduction in alloimmunisation was from 1.5% (7/468) to 0.21% (1/472). which is a similar reduction to that observed by Thornton et al. (1989) with first pregnancies receiving 2 × l00μg anti-D. A signficiant reduction in primigravidae was also observed. from 1.11% (4/360) to <0.28% (0/360). Kleihauer tests to identify FMH were also carried out in both groups. There was no difference observed with the frequency of larger FMH (>1/10000 maternal RBC) at 32–34 weeks — 31 versus 32 in the control group. On the other hand, fewer FMH in total were seen in the treatment group — 4.2% (39/927) versus 7% (67/957) in the control group, indicating that the 28-week anti-D was effective in eliminating small FMH. Likewise at delivery of a RhD-positive baby, 12.2% (73/599) in the treatment group had a positive Kleihauer test compared to 20.2%; (119/590) in the control group.
Trial details: open controlled study involving several centres in Paris. allocated to treatment or control arms by even/uneven date of birth. Both groups received standard postpartum anti-D of 100μg i.v. (the standard French practice). Serum was examined for anti-D at birth and at 2–1 2 months postpartum.
Treatment group: (100μg anti-D i.m. at 28 and 34 weeks) 927 women — 599 with RhD-positive baby — 461 primigravidae/l38 multipara (472 followed for 2–12months); 0/599 alloimmunised at delivery, 1/599 followed 2–12months (at 5 months) = 0.17%, 1/472 primiparae followed 2–1 2 months (at 5 months) = 0.21, 0/362 primigravidae followed 2–12 months = < 0.28%.
Control group: 955 women — 590 with RhD-positive baby — 454 primigravidae/l36 multipara (468 followed for 2–12 months); 6/590 alloinimunised at delivery = 1.02%, 7/468 primiparae followed 2–12 months = 1.92, 4/360 primigravidae followed 2–12 months = 1.11%.
Conclusion: considering primigravidae only, the reduction in alloimmunisation is from 1.1l% to <0.28% (x2=2.28: P= 0.131). Considering primipara only, the reduction in alloimmunisation is 1.58 to 0.21% (86% reduction) (x2=3.22: P= 0.073). The reduction in alloimmunisation observed. considering all groups, is from 1.19% (7/590) to 0.178 (1/599) (x2 3.22 : P= 0.073).
E) 2 × 50 μg at 28 and 34 weeks6
A prospective study to evaluate the efficacy of a lower dose of 50μg at 28 and 34 weeks was undertaken in the UK, but was stopped due to a combination of low recruitment, and evidence that protection against alloimmunisation was incomplete. Although reduction was substantial, 1.5% (9/595) in controls to 0.78% (4/513) in the treatment group, this was less than that observed in the UK trial of 2 × 100μg (Thornton et al. 1989) and it was concluded that the lower dose could not be recommended.
Trial details: open controlled multicentre study in primigravidae with randomisation to treatment or control groups by sealed envelope. Both groups received standard postpartum prophylaxis of 100μg i.m. Both groups examined for alloimmunisation at 6months. Women with preexisting anti-D at 28 weeks excluded.
Treatment group: (50μg i.m. at 28 and 32 weeks) 4/5 13 alloimmunised at delivery = 0.78%; one extra alloimmunised, but antenatal event not given anti-D therefore excluded.
Control group: 7/595 alloimmunised at delivery = 1.2%; 9/595 alloimnunised at 6months follow-up = 1.5%.
Conclusion: although a reduction in alloimmunisation was observed from 1.5% to 0.788, this was less than observed with 2 × 100μg, and 2 × 50μg is insufficient to give full protection (x2=0.72: P=0.395).