2. 9
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
because of small transplacental haemorrhages during
pregnancy17,18
.
One year later, a Canadian study by Bowman et al.
showed, in 1,357 Rh(D)-negative primagravida, that the
incidence of Rh(D) alloimmunisation could be reduced
to 0.1% by prophylaxis with antenatal anti-D IgG, in
addition to post-partum prophylaxis19
.There is currently
sufficient evidence demonstrating that antenatal anti-D
prophylaxisalsoreducestheriskofRh(D)immunisation
in the next pregnancy to below the level of 0.4%.
Forty years after Zipursky and Israels first
proposed the use of anti-D IgG to reduce the incidence
of Rh alloimmunisation in pregnancy14
,
immunoprophylaxis has drastically reduced the cases
of Rh-induced HDN; nevertheless, this pathology
continues to be relevant in 0.4 of 1,000 births
(0.04%)20
, for various reasons21
: i) the possible
occurrence of anti-D immunisation during the
pregnancy (which occurs in about 1% of Rh(D)-
negative women carrying a Rh(D)-positive foetus22
);
ii) the lack of efficacy of immunoprophylaxis because
of the administration of an insufficient dose of anti-D
IgG that is not congruent with the volume of the foetal-
maternal haemorrhage; iii) immunoprophylaxis not
administered; iv) possible errors in typing the pregnant
or puerperal woman or the neonate; v) possible errors
in transfusion therapy in women of child-bearing age.
Antenatal prophylaxis
Rationale
Systematic anti-D prophylaxis was proposed in the
1970s with the aim of reducing the percentage of
Rh(D)-negative women who could be sensitised during
pregnancy, despite the use of post-partum prophylaxis.
This percentage ranged from 0.9-2.2% in a series of
studies carried out between 1977 and 198917-19,23-28
, and
from 0.8-1.5% in subsequent studies carried out
between 1995 and 199929-32
; this latter modest reduction
is probably due to intercurrent changes in obstetric care
and the wider use of anti-D prophylaxis following
sensitising events during pregnancy33
.
The D antigen has been demonstrated to be present
on foetal red blood cells from the 7th
week of
pregnancy onwards34
. There is a direct, proportional
relationship between the volume of Rh(D)-positive
red blood cells to which the Rh(D)-negative subject
is exposed and the incidence of anti-Rh immunisation,
although it has been estimated that 0.1 mL is already
sufficient to induce the formation of antibodies14,33
.
During the first trimester of a normal pregnancy,
foetal red blood cells can be found in the maternal
circulation of about 3% of women, albeit in amounts
less than 0.1 mL35
. As the pregnancy proceeds, the
frequency and volume of foetal red blood cells in the
maternal circulation increase, such that during the
second and third trimesters these cells can be detected
in 12% and 45% of women, respectively. At the time
of delivering an ABO-compatible neonate, foetal red
blood cells can be found in the maternal circulation
in as many as 50% of cases12
. However, the main
sensitising event for Rh(D)-negative women, in whom
prevention is based on post-natal prophylaxis, occurs
at the end of the pregnancy, with detachment of the
placenta during delivery33
.
Other sensitising events, in the form of occult or
silent transplacental haemorrhages, are probably the
principal cause of the approximately 1% residual rate
of immunisation. These events provide the rationale
for the strategy, adopted in many countries, of
systemic antenatal anti-D prophylaxis for all non-
immunised Rh(D)-negative pregnant women, as a
complementary measure to post-partum prophylaxis36
.
Numerous studies have demonstrated that the
antenatal administration of anti-D IgG does not have
adverseeffectsonthefoetus,eventhoughsmallamounts
of anti-D can cross the placenta and, by binding to foetal
D-positive red blood cells, can cause weak positivity at
birth in the direct Coombs' test16,23,37-40
.
Antenatal anti-D prophylaxis is generally
recommended from the 28th
week of pregnancy
onwards, because transplacental haemorrhages large
enough to cause sensitisation do not occur until the
third trimester and, therefore, anti-D antibodies usually
develop after the 28th
week of gestation17,41,42
; studies
showing that 92% of Rh(D)-negative women who
develop anti-D do so after the 28th
week of gestation
lend support to this recommendation16,37
.
Anti-D prophylaxis has a strong
immunosuppressive effect, such that subsequent
exposure to the D antigen does not cause a secondary
immune response, but rather a primary response, as if
the immune system has never encountered the D
antigen43
. Numerous mechanisms of action have been
proposed to explain the efficacy of this strategy,
including : (i) accelerated clearance of D-positive cells,
(ii) epitope masking, (iii) inhibition due to antibodies
Antenatal anti-D immunoprophylaxis
3. 10
Liumbruno GM et al.
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
against FcγRIIB (the specific activating receptor for
the Fc fragment of IgG) or anti-idiotype44
, (iv) inhibition
of immature dendritic cells, and (v) inhibition of B-
cell clones specific for the D antigen43
; this last, recently
proposed, mechanism was suggested by the finding of
an increase in the levels of transforming growth factor-
β and prostaglandin E2
, found in a cohort of pregnant
women who were given antenatal prophylaxis.
However, the accelerated destruction of D-positive red
blood cells remains, according to some authors, the
main mechanism of action45
.
Dose
A dose of 20 µ g (100 UI) of anti-D IgG protects
against 1 mL of D-positive red blood cells or 2 mL of
whole blood10,20
. However, the World Health
Organisation (WHO) and the British Medical Research
Council consider that 25 µ g (125 UI) are needed to
protect against the same volume of D-positive red blood
cells46
; according to Bowman and Pollock, this is
equivalent to having a concentration of anti-D IgG in
the maternal circulation of 2.4 ng/mL26
.
There are essentially two approaches to antenatal
prophylaxis: i) a single injection of 300 µ g (1,500
UI) in the 28th
week of pregnancy19,28,47
, or ii) two
injections of 100-125 µ g (500-625 UI), one in the
28th
week, the other in the 34th
week24,32,40
.
In pharmacokinetic studies, the half-life of the IgG
was estimated to be, on average, 17-22 days (range:
11-29)48,49
;arecentlypublishedpharmacokineticstudy46
reported that both protocols are effective and able to
ensure, 12 weeks after administration, the minimum
residual concentration of 25 µ g (2.4 ng/mL) of anti-D
IgG indicated by the WHO as a protective level. This
is probably an overcautious estimate because it is
extremely unlikely that the volume of a transplacental
haemorrhage in the antenatal period exceeds 1 mL of
foetal red blood, which is equivalent to the volume of
foetal-maternal haemorrhage occurring at the time of
delivery in 98% of women46,50
.
A recent systematic review of the literature on the
clinical efficacy of anti-D prophylaxis during
pregnancy33
found 11 studies that compared the
outcome of Rh(D)-negative women (and their
babies)19,23-32
not immunised to D antigen who
underwent antenatal prophylaxis in the 28th
week of
pregnancy or subsequently, with the outcome of
Rh(D)-negative controls (and their babies) who did
not receive prophylaxis. Table I reports the
characteristics of the studies analysed in the review,
which showed that the percentage of sensitised women
decreased from 1.9-2.2% to 0-0.2% with the use of
antenatal prophylaxis; the data also showed that the
main clinical benefit that a Rh(D)-negative woman
can have from antenatal prophylaxis is the possibility
of avoiding HDN in subsequent pregnancies with
Rh(D)-positive foetuses.
Ameta-analysis, carried out on two of the 11 studies
included in the review, demonstrated a notable reduction
in relative risk of sensitisation in women treated with the
antenatal prophylaxis; indeed, the odds ratio (OR) was
0.37 (95% CI: 0.2-0.65), while the absolute reduction in
risk of sensitisation in mothers at risk (carrying a Rh(D)-
positive foetus) was 0.6%; the number of women who
had to be treated to avoid one case of sensitisation (i.e.
the NNT, number needed to treat) was 278.
A meta-analysis by the Cochrane Collaboration in
200042
also included only two studies27,29
, for a total
of more than 4,500 women treated with
immunoprophylaxis in the 28th
and 34th
weeks of
pregnancy; the doses of anti-D IgG used were 100 µ g
(500 UI) x 2in the French trial27
, and 50 µ g (250 UI)
x 2 in the British trial29
. Three outcomes were
evaluated and for each of these the reduction in the
relative risk was statistically significant. As far as
concerns positivity for the Kleihauer test, which
detects foetal red blood cells in the maternal
circulation, the OR was 0.6 (95% CI: 0.41-0.88)
during pregnancy and 0.6 (95% CI: 0,46-0,79) also
after the delivery of a Rh(D)-positive baby. The OR
for the incidence of anti-D alloimmunisation was 0.42
(95% CI: 0.15-1.17) both during pregnancy and after
the birth of a Rh(D)-positive baby; the OR became
0.41 (95% CI: 0.16-1.04) within 12 months of the
birth, while the OR for women at a first pregnancy,
within 12 months after the delivery, was 0.11 (95%
CI: 0.01-2.04). The reduction in the relative risk of
neonatal jaundice was considerably greater, with an
OR of 0.26 (95% CI: 0.03-2.3).
According to the Cochrane reviewers, as a result of
antenatal prophylaxis [100 µ g (500 UI) of anti-D IgG
in the 28th
to 34th
weeks], the risk of alloimmunisation
of Rh(D)-negative pregnant women drops from about
1% to 0.2% and the probability of immunisation in
subsequent pregnancies is also reduced; the NNT to
avoid one case of sensitisation is 213.
4. 11
International recommendations
Various international recommendations (table II)
suggest antenatal anti-D prophylaxis (i.m. or i.v.) in
unsensitised Rh(D)-negative women as a complement
to post-partum prophylaxis.
1) The Society of Obstetricians and Gynaecologists
of Canada (SOGC) suggests 300 µ g in the 28th
week (grade of recommendation:A) or two doses
of 100-120 µ g in the 28th
and 34th
weeks of
pregnancy20
.
2) The Italian Society of Transfusion Medicine and
Immunohaematology (SIMTI), together with the
Table I – Summary of the results of the clinical efficacy of antenatal anti-D prophylaxis
(adapted from: Jones ML et al.33
).
Study Dosage Patient selection Anti-D prophylaxis group Control group
[Study desing] % Sensitised or % Sensitised or
sensibilised sensibilised
(95% CI) (95% CI)
Bowman et al. (1978) 2 x 1500 IU (28 and 34 weeks) Primigravidae 1/1357 0.1 (0.1 to 0.2) 45/2768 1.6 (1.6 to 2.1)
[NRCT]19
Bowman and Pollock (1978) 1 x 1500 IU (28 weeks) Unselected 11/1805 0.6 (0.3 to 1.0) 62/3533 1.8 (1.3 to 2.2)
[NRCT]23
Tovey et al. (1983) 2 x 500 IU (28 and 34 weeks) Primigravidae 5/1238 0.4 (0.1 to 0.8) 30/2000 1.5 (1.0 to 2.0)
[NRCT]24
Hermann et al. (1984) 1 x 1250 IU (34 weeks) Primigravid primiparae 4/236 1.7 (0.0 to 3.3) 5/286 1.7 (0.2 to 3.3)
[NRCT]25
Multigravid primiparae 1/332 0.3 (0.3 to 0.9) 7/359 1.9 (0.2 to 2.6)
Unselected primiparae 5/568 0.9 (0.1 to 1.6) 12/645 1.9 (0.8 to 2.9)
Bowman and Pollock (1987) 1 x 1500 IU (28 weeks) Unselected 30/9295 0.3 (0.2 to 0.4) 62/3533 1.8 (1.3 to 2.2)
[NRCT]26
Huchet et al. (1987) 2 x 500 IU (28 and 34 weeks) Primiparae 0/461 0.0 (0.0 to 0.0) 4/454 0.9 (0.0 to 1.7)
[Quasi RCT]27
Multiparae 1/138 0.7 (0.7 to 2.1) 3/136 2.2 (0.3 to 4.7)
Unselected 1/599 0.2 (0.2 to 0.5) 7/590 1.2 (0.3 to 2.1)
Trolle (1989) 1 x 1500 IU (28 weeks) Unselected 0/291 0.0 (0.0 to 0.0) 6/322 1.9 (0.4 to3.3)
[NRCT]28
Lee and Rawlinson (1995) 2 x 250 IU (28 and 34 weeks) Primigravidae 5/513 1.0 (0.1 to 1.8) 9/595 1.5 (0.5 to 2.5)
[RCT]29
Mayne et al. (1997) 2 x 500 IU (28 and 34 weeks) Primiparae 4/1425 0.3 (0.0 to 0.6) 16/1426 1.1 (0.6 to 1.7)
[Before and after study]30
Parsons et al. (1998) Not stated* (28 weeks) Not stated 72/9684 0.7 (0.6 to 0.9) No data 0.8 (not given)
[NRCT]31
Mackenzie et al. (1999) 2 x 500 IU (28 and 34 weeks) Primiparae 12/3320 0.4 (0.2 to 0.6) 26/3146 0.8 (0.5 to 1.1)
[Controlled before and
after study]32
NRCT = non randomised controlled trial.; RCT = randomised controlled trial; n = number of deliveries of RhD-positive babies to RhD-negative women;
r = number of sensitised RhD-negative women in the trial group. * Most probably the standard Canadian dose of 1500 IU.
Italian Society of Gynaecology and Obstetrics
(SIGO)recommends250-300µ ginthe28th
week21
.
3) The American Society of Clinical Pathologists
(ASCP) suggests 300 µ g in the 28th
-30th
week16
.
4) The American College of Obstetrician and
Gynaecologists (ACOG) recommends
prophylaxis51
, around the 28th
week, but does not
specify the dose (grade of recommendation:A).
5) The U.S. Preventive Services Task Force
(USPSTF) (USA) recommends 300 µ g in the
24th
-28th
week52
.
6) The National Institute for Clinical Excellence
r/n r/n
Antenatal anti-D immunoprophylaxis
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
5. 12
Liumbruno GM et al.
Council (NHMRC) (Australia) suggests 125 µ g
(625 UI) in the 28th
and 34th
weeks56
.
9) The RoyalAustralian and New Zealand College
of Obstetricians and Gynaecologists
(RANZCOG) confirms the above
recommendation57
.
10) The French recommendations are for a dose of
300 µ g in the 28th
(± 1) week of pregnancy58
.
11) The Dutch authorities recommend a single dose
of 200 µ g in the 30th
week59
.
12) The Spanish Society of Blood Transfusion
(SETS) and the Spanish Society of Obstetrics
and Gynaecology indicate a single dose of 300
µ g in the 28th
week60
.
Prophylaxis should not be given to women with a
"weak" D phenotype, in that such women are actually
D-positive21
; however, the use of anti-D IgG in these
women does not have significant side effects.
Conclusions
HDN has become a rare disorder since the
introduction of anti-D prophylaxis. The foetal Rh(D)
antigen is already well developed after 30-40 days of
gestation and the risk of antenatal foeto-maternal
haemorrhage, already possible from the 6th
week61
,
increases exponentially as a pregnancy progresses22,35
.
Although the risk of foetal blood entering the maternal
circulation is high, the average volume of is very small,
being 0.07 mL in the first trimester, 0.08 mL in the
second and 0.13 mL in the third62
. A Rh(D)-negative
woman not immunised and who does not receive
prophylaxis has, in every pregnancy, a 16% risk of
becoming immunised to a Rh(D)-positive foetus63
.
However, the global success rate of post-natal Rh
immunoprophylaxis has now reached 98.4 - 99%1,3
.
Given that anti-D prophylaxis is effective for about
12 weeks, as indicated by pharmacokinetic studies
on anti-D IgG46
, and given the relatively low risk of
significant foeto-maternal haemorrhage before the 28th
week of gestation, antenatal prophylaxis is carried out
from the 28th
week onwards41
.
The currently available evidence demonstrates that
antenatal anti-D prophylaxis, besides being a cost-
effective strategy36,64
, is able to further reduce the
incidence of sensitisation to D antigen down to about
0.2%33,42
; a variety of prophylactic regimens are used
in different countries, although 300 µ g of anti-D IgG
Table II – Recommended antenatal anti-D prophylaxis in
the main international recommendations
Recommended dose Week
Canada (SOGC)20
300 (or 100-120) 28 (or 28 and 34)
Italy (SIMTI-SIGO)21
250-300 28
USA (ASCP)16
300 28-30
USA (ACOG)51
Not specified 28
USA (USPSTF)52
300 24-28
UK (NICE)53
100 28 and 34
UK (BCSH)54
100 28 and 34
Australia (NHMRC)56
125 28 and 34
Australia (RANZCOG)57
125 28 and 34
France (CNGOF)58
300 28
The Netherlands (CHI)59
200 30
Spain (SETS-SEOG)60
300 28
SOGC: Society of Obstetricians and Gynaecologists of Canada
SIMTI: Società Italiana di Medicina Trasfusionale e Immunoematologia
[Italian Society of Transfusion Medicine and Immunohaematology]
SIGO: Società Italiana di Ginecologia e Ostetricia [Italian Society of
Obstetrics and Gynaecology]
ASCP: American Society of Clinical Pathologists
ACOG: The American College of Obstetricians and Gynecologists
USPSTF: U.S. Preventive Services Task Force
NICE: National Institute of Clinical Excellence
BCSH: British Committee for Standards in Haematology
NHMRC: National Health and Medical Research Council
RANZCOG: Royal Australian and New Zealand College of Obstetricians
and Gynaecologists
CNGOF: Collège National des Gynècologues et Obstétriciens Francais
[French College of Obstetricians and Gynaecologists]
CHI: Council of Health Insurances
SETS: Sociedad Española de Transfusión Sanguínea[Spanish Society of
Blood Transfusion]
SEOG: Sociedad Española de Obstetricia y Ginecología [Spanish Society
of Obstetrics and Gynaecology]
(NICE) (UK), in guidelines published in 2002
and revised in 2005, indicates 100 µ g (500 UI)
in the 28th
and 34th
weeks53
; the NICE states that
this scheme is equivalent to a single dose of 300
µ g (1,500 UI) in the 34th
week.
7) The same indications are included in the recent
guidelines from the British Committee for
Standards in Haematology (BCSH)54
, which
replaced the preceding recommendations by Lee
et al. in 199955
.
8) The National Health and Medical Research
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
6. 13
in the 28th
week is the dose most commonly indicated
in international recommendations16,20,52,58
.
It is not, however, possible to completely eliminate
the risk of sensitisation because, despite increased
adherence to antenatal immunoprophylaxis65
, and
although most foeto-maternal haemorrhages able to
cause immunisation occur in the last trimester of
pregnancy, sensitisation does occur before the 28th
week in a small percentage of women; alternatively,
there may be cases in which intramuscular
administration of the anti-D IgG is insufficient to
provide passive prophylaxis.
Furthermore, the risk of intrauterine foetal death
of Rh(D)-positive foetuses of Rh(D)-negative mothers
appears to be increased despite antenatal
immunoprophylaxis, as indicated by a recent,
retrospective observational study carried out in Israel
on more than 140,000 deliveries occurring between
1988 and 200366
; although the subject undoubtedly
needs further investigation, this retrospective study
seems to suggest a possible pathogenic scenario not
limited to maternal-foetal serological
alloimmunisation but also involving the maternal-
foetal interface (i.e. the placenta), which could
undergo immune-mediated damage leading to
intrauterine foetal death.
The IgG subclasses with anti-D activity are IgG1
and IgG3; the anti-D IgG are produced by industrial
fractionation from pools of donor plasma containing
a high titre of anti-D antibodies; chromatographic
procedures, which are well suited to processing small
quantities of plasma and guarantee an excellent yield,
are used for the fractionation67
.
Antenatal prophylaxis with anti-D IgG does not
have side effects on the foetus and can be considered
complementary to post-partum prophylaxis, but the
decision on whether to undertake a programme of
prophylaxis during pregnancy can be influenced by
the commercial availability of anti-D IgG. The raw
material is a limited resource because the
hyperimmune plasma comes exclusively from the
USA, the only country that still carries out active
immunisation of donors which, until about 10 years
ago, was also performed in Switzerland and Slovenia
(Italian National Blood Centre, unpublished data).
An economic analysis carried out in the United
Figure 1 - Anti-D prophylaxis in Rh(D) negative pregnant women: possible scenario following the introduction of
foetal genotyping
Antenatal anti-D immunoprophylaxis
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
Foetal genotyping
(first trimester)
Rh(D) positive
(∼ 60%)
Rh(D) negative
(∼ 40%)
•
No sensitising
events
Sensitising
events
Abortion (spontaneous or
therapeutic)
• Ectopic pregnancy
Anti‐D prophylaxis
within 72 hours of
the event
Antenatal anti‐D
prophylaxis in
28th
week
Post‐partum
anti‐D
prophylaxis
• Antenatal haemorrhage
• Villocentesis
• Abdominal trauma
• Amniocentesis
• Cordocentesis
Anti‐D prophylaxis
within 72 hours of
the event
No prophylaxis
7. 14
Liumbruno GM et al.
Kingdom36,64
demonstrated that the cost per year of
life gained from using antenatal anti-D prophylaxis
for women in their first pregnancy was low compared
to that of other interventions normally offered by the
health care service in that country; the same
prophylaxis, given to all pregnant women, although
costing more, maintained a relatively low cost per year
of life gained, thus confirming that antenatal anti-D
prophylaxis is a decidedly cost-effective strategy.
Methods of foetal genotyping on maternal
plasma68-74
are able to detect foetal DNA sequences in
the maternal plasma; used on a large scale, these
methods could enable elimination of unnecessary
antenatal prophylaxis with anti-D IgG in Rh(D)-
negative mothers of Rh(D)-negative foetuses, who
represent about 40% of all cases, thus sparing this
blood derivative and reserving its appropriate use
exclusively to those women at risk of maternal-foetal
alloimmunisation (Figure 1). In addition to this
benefit, a further advantage of foetal genotyping
would be that of knowing whether, in a women already
immunised in an early stage of a pregnancy, the foetus
of the current pregnancy is Rh(D)-negative or positive,
providing important information for the management
of the pregnancy.
The possible use of monoclonal or recombinant
anti-D antibodies in clinical practice remains very
uncertain75
. These antibodies could, in the future, be
an alternative to the prophylactic use of plasma-
derived polyclonal anti-D IgG76-78
; unfortunately,
however, none of the products developed by industry
so far has been demonstrated to be effective in the
numerous phase I clinical trials that have been carried
out79,80
. During the last 20 years, 19 monoclonal or
recombinant antibodies, produced by different cell
lines, have been tested in humans75
, but the in vivo
clinical efficacy and side effects were very
heterogeneous. Most of the side effects are probably
related to the type of glycosylation to which the IgG
molecule is subjected in the various different cell
systems used for its production75,80
.
The drafting and adoption of national
recommendations on the systematic use of antenatal
immunoprophylaxis must, however, take into
consideration both clinical and organisational
implications81
, and the potential financial impact of
using this blood derivative which, already in the 3
years from 2002 to 2005, led to a 56% increase in
costs, for a 15% rise in sales (Italian National Blood
Centre, unpublished data), caused presumably also
by the increase in the sale price of the product.
Key words: antenatal immunoprophylaxis,
maternal-foetal alloimmunisation, haemolytic disease
of the newborn, Rh(D).
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Received: 12 May 2009 - Revision accepted: 6 August 2009
Correspondence: Dr. Giancarlo Maria Liumbruno,
Viale Italia, 19
57126 Livorno, Italy.
e-mail: iancarlo@liumbruno.it; ricerca.cns@iss.it
Liumbruno GM et al.
Blood Transfus 2010; 8:8-16 DOI 10.2450/2009.0108-09
