Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2010 Mar; 154(1):38.

M. Lubusky

PREVENTION OF RhD ALLOIMMUNIZATION IN RhD NEGATIVE WOMEN

Marek Lubuskya,b*
a

Department of Obstetrics and Gynecology, Palacky University, University Hospital, Olomouc, Czech Republic
Department of Medical Genetics and Fetal Medicine, Palacky University, University Hospital, Olomouc, Czech Republic
E-mail: [email protected]
b

Received: October 12, 2009; Accepted (with revision): March 1, 2010

Key words: Anti-D immunoglobulin/RhD alloimmunization/Fetomaternal hemorrhage
Background. Despite the introduction of anti-D prophylaxis into clinical practice, RhD alloimmunization still
presents a problem to date. The actual incidence of RhD alloimmunization in pregnant women remains unknown in
most countries. Anti-D immunoglobulin is administered to RhD negative women at a fixed dose and in much greater
amounts than is actually necessary. On the other hand, it is not possible to diagnose cases where greater doses are
needed. To optimize the prevention of RhD alloimmunization in RhD negative women, it is important to diagnose
conditions that lead to fetomaternal hemorrhage (FMH), precisely determine the volume and subsequently administer
the appropriate dose of anti-D immunoglobulin. The possibility to accurately detect FMH and precisely determine its
volume would enable more effective and less costly prevention of RhD alloimmunization. Anti-D immunoglobulin could
be administered only in indicated cases and only in doses essentially necessary for prevention of RhD alloimmunization.
Methods and results. The Cochrane and UpToDate databases of systematic reviews, as well as national guidelines,
were reviewed.
Conclusions. Due to the medical significance and indispensable economic costs associated with prevention of RhD
alloimmunization, it would be appropriate to establish exact methodical guidelines. The text itself should be limited to
a list of potentially sensitising events during which anti-D immunoglobulin should be administered to RhD negative
women if anti-D antibodies are not already present. Following each potentially sensitising event, the minimal dose of
anti-D immunoglobulin necessary for prevention of RhD alloimmunization should be determined. After 20 weeks of
gestation, the volume of FMH should also be determined to specify the necessary dose of anti-D immunoglobulin.

MATERNAL RhD ALLOIMMUNIZATION

Each person who lacks the red blood cell antigen and
is exposed to it will create an antibody. During penetration of RhD positive fetal erythrocytes into the circulation
of an RhD negative mother, the immune system may be
stimulated and trigger the creation of antibodies by alloimmunization. The same immune reaction may also
occur during transfusion of antigen-incompatible erythrocytes.
Anti-D antibodies may cause a severe form of
haemolytic disease of the fetus and newborn (HDFN
Haemolytic disease of the fetus and newborn). Because
the RhD antigen is very potent, even parenteral administration of only 0.1 ml of RhD positive erythrocytes to
RhD negative individuals will stimulate the production of
antibodies. The most common cause of RhD alloimmunization is hemorrhage, during which fetal erythrocytes
enter the mothers circulation.
ABBREVIATIONS
RhD
RHD
FMH
HDFN

rhesus D phenotype (RhD phenotype)

rhesus D genotype (RHD genotype)
fetomaternal hemorrhage
haemolytic disease of the fetus and newborn

Most cases of RhD alloimmunization however may

theoretically be avoided by prophylactic administration of
anti-D immunoglobulin in the necessary dose after every
potentially sensitising event (Table 1).

THE INCIDENCE OF RhD

INCOMPATIBLE PREGNANCIES
The incidence of RhD incompatibility varies according to race and ethnic background. Approximately 15% of
the Caucasian population is RhD negative. In most other
populations however, the incidence of an RhD negative
phenotype is significantly lower, in African-Americans the
incidence is 58%, in Asians and Native Americans 12%.
In the Caucasian population, an RhD negative woman has
an 85% probability that her partner will be RhD positive,
in 60% heterozygous and in 40% homozygous at locus
RHD. In approximately 10% of all pregnancies, the situation arises where an RhD negative mother will have
an RhD positive child and approximately 60% of RhD
negative women will have an RhD positive child in their
first pregnancy.
In the Czech Republic, if prevention of RhD alloimmunization was not performed in RhD negative women
following potentially sensitising events, approximately

4
2000 women would annually become alloimmunized.
Although during the last three decades the introduction
of anti-D prophylaxis has led to a decrease in incidence,
it remains a problem even today. In the USA in the year
2001, the incidence of RhD alloimmunization was 6.7 per
1000 liveborn infants1.
Such data are not available in the Czech Republic.
However, if similar results are assumed, there are about
670 RhD alloimmunized pregnant women annually. If
two-thirds of these women will have an RhD positive fetus, we may assume that there will be about 447 at-risk
fetuses annually.

PREVENTION OF RhD ALLOIMMUNIZATION

IN RhD NEGATIVE WOMEN
At the beginning of every pregnancy, the womans AB0
+ RhD blood types are determined and screening for irregular anti-erythrocyte antibodies (from herein referred
to only as anti-erythrocyte antibodies) is performed.
In the absence of anti-erythrocyte antibodies at the beginning of pregnancy, a follow-up antibody screening is
performed at 28 weeks of gestation in all pregnant women (RhD negative and RhD positive). In RhD negative
women it is performed before antenatal prophylaxis of
RhD alloimmunization, and in RhD positive women it
is performed due to the possible development of other
than anti-D antibodies causing severe haemolytic disease
of the newborn.
If the womans blood type is RhDw positive "weak rhesus
positive" (approx. 1% of those RhD positive), prevention
of RhD alloimmunization is generally not indicated.
In cases of weak RhD phenotype "weak D" (formerly
labeled Du), there is quantitative weakening of D antigen expression. All D epitopes are weakly expressed, but
individuals do not form anti-D antibodies during contact
with erythrocytes with normal D expression.
In contrast, partial RhD phenotypes partial D, variant
D are RhD positive phenotypes in which some epitopes
of the RhD antigen are not expressed. During contact
with RhD positive erythrocytes, individuals with partial
D phenotype may form antibodies against D epitopes,
which are lacking on their erythrocyte surface.
It is necessary to distinguish quantitative weakening of
the RhD antigen where it is unnecessary to perform prevention of RhD alloimmunization and qualitative variants of the RhD antigen where prevention is indicated.
Individual consultation with the laboratory is necessary!
The effectiveness of screening for anti-erythrocyte antibodies at 28 weeks of gestation has not been confirmed
but is performed in a number of countries (Europe, USA,
Canada, Australia).
The incidence of antepartal RhD alloimmunization is
12%; however, in 90% of cases it occurs after 28 weeks
of gestation910. The incidence of RhD alloimmunization
before 28 weeks is therefore 0.10.2%. Follow-up examination of anti-erythrocyte antibodies in all RhD negative women at 28 weeks of gestation could then enable

M. Lubusky
Table 1. Events following which anti-D immunoglobulin
should be given to all RhD negative women
with no anti-D antibodies27,1923. Dosage and timing
of anti-D immunoglobulin administration67,2223.
Delivery of an RhD positive infant*
Abortion
therapeutic termination of pregnancy
spontaneous abortion followed by instrumentation
spontaneous complete or incomplete abortion after
12 weeks gestation
threatened abortion before 12 weeks
when bleeding is heavy or repeated or is associated with abdominal pain; in particular, if these
events occur as gestation approaches 12 weeks
threatened abortion after 12 weeks
when bleeding continues intermittently, anti-D
immunoglobulin should be given at approximately 6-week intervals, and the volume of fetomaternal hemorrhage should be assessed
Invasive prenatal diagnosis
chorionic villus sampling
amniocentesis
cordocentesis
Other intrauterine procedures
evacuation of the uterus because of mola hydatiforma
multifetal reduction
fetal therapy (insertion of shunts etc.)
Antepartum hemorrhage
when bleeding continues intermittently, anti-D immunoglobulin should be given at approximately
6-week intervals, and the volume of fetomaternal
hemorrhage should be assessed
External version of the fetus
Abdominal trauma
Ectopic pregnancy
Intrauterine fetal death
Stillbirth
Dose:
before 20 weeks gestation
after 20 weeks gestation**

50 g (250 IU)
100 g (500 IU)

Timing:
as soon as possible, but no later than 72 hours after
the event.
* also if the RhD type of the infant has not been determined or is in doubt
** in conjunction with a test to assess the volume of any
fetomaternal hemorrhage

Prevention of RhD alloimmunization in RhD negative women

diagnosis of 1020 cases of RhD alloimmunization per
100 000 deliveries annually. The developed alloimmunization of the mother however does not place the fetus
at risk of severe haemolytic disease in the current pregnancy. In such cases, it would therefore not be necessary
to administer anti-D immunoglobulin.
Before screening for anti-erythrocyte antibodies in the
mothers serum, it is always necessary to specifically inquire whether the woman had already been administered
immunoglobulin anti-D (from herein referred to only as
IgG anti-D) during this pregnancy. If so, it is necessary
to include this information on the examination requisition slip, because persisting levels of IgG anti-D could
falsely lead to suspicion of maternal RhD alloimmunization. Similarly, if during the follow-up screening presence
of anti-D antibodies is confirmed, it is best to once more
inquire whether IgG anti-D was administered during this
pregnancy before a diagnosis of RhD alloimmunization
of the mother is established.
The half-life of the administered IgG anti-D is approximately 24 days. In 1520% of patients who were administered IgG anti-D at 28 weeks of gestation, it is
nonetheless still possible to detect a low titre of anti-D
(usually 2 or 4) at the time of labour2.
RhD negative women where the presence of anti-D antibodies in the serum was not confirmed, are administered
a dose of 125 g IgG anti-D intramuscularly at 28 and
34 weeks of gestation. IgG anti-D may also be administered as a single dose of 250 g at 28 weeks of gestation.
This procedure may lead to an 80% decrease in the incidence of antepartal RhD alloimmunization (from 1%
to 0,2%)7.
In the 1st trimester, women who are RhD negative are
administered 50 g IgG anti-D after spontaneous miscarriage followed by evacuation of the uterus, induced
abortion (therapeutic termination of pregnancy), evacuation of the uterus because of mola hydatiforma, chorionic
villus sampling or after operation for ectopic pregnancy.
Alloimmunization by the RhD antigen may already be
detected at 6 weeks of gestation4.
The risk of RhD alloimmunization after spontaneous
miscarriage is 1.52%, after induced abortion 45%
(ref.2), after chorionic villus sampling 14% (ref.11).
The total volume of fetal blood at 12 weeks of gestation =
3ml (FMH 1.5ml), sufficient dose of IgG anti-D = 30 g.
Therefore up until 12 weeks of gestation, a sufficient dose
of IgG anti-D for prevention of D alloimmunization is
50 g.
In the 2nd and 3rd trimester, after induced abortion (therapeutic termination of pregnancy), amniocentesis, cordocentesis, or after other invasive prenatal diagnostic or
therapeutic procedures, after antepartum hemorrhaging,
intrauterine fetal death, attempt at external cephalic version of a breech presentation, after abdominal trauma or
in situations where there is a potential risk of sensitization of the mother by RhD antigens of the fetus, a dose

5
of 50 g of IgG anti-D is administered to RhD negative
mothers until 20 weeks of gestation, after 20 weeks of
gestation 100 g of IgG anti-D are administered. After
20 weeks of gestation, the volume of fetomaternal hemorrhage (FMH) should also be determined to specify dosing.
The risk of RhD alloimmunization after amniocentesis
is 25% (ref.3).
In cases of continued or repeated hemorrhaging after 12
weeks of gestation, 100 g IgG anti-D are repeatedly administered in 6-week intervals, and during each episode
of hemorrhaging the volume of fetomaternal hemorrhage
(FMH) should be determined to specify the dose of IgG
anti-D necessary for prevention of RhD alloimmunization
of the mother25.
For RhD negative women, after delivery of an RhD positive child, if the presence of anti-D antibodies was not
detected in the serum, a 50100 g dose of IgG anti-D
must be applied intramuscularly and the volume of FMH
should be determined to specify the dose of IgG anti-D necessary to prevent RhD alloimmunization of the mother.
If the FMH volume is not determined, a dose of 200300
g is administered. The effectiveness of administering a
standard dose of over 100 g to all women has not been
demonstrated6,22. This dose should be administered immediately following delivery, no later than 72 hours after
delivery.
A 10 g dose of IgG anti-D administered intramuscularly
should cover 0.5 ml of fetal RhD positive erythrocytes or
1ml of whole blood. This means that 125 g (250 g)
of IgG anti-D should prevent alloimmunization after
fetomaternal hemorrhage of 12.5 ml (25ml) of whole
fetal blood.
In approximately 1.5% of deliveries is fetomaternal hemorrhage more than 5 ml, in only 1% of deliveries does
fetomaternal hemorrhage surpass 12.5 ml and only 0.5%
of deliveries have fetomaternal hemorrhage greater than
25 ml1215. However, in nearly 50% of all cases no risk
factor is present1416.
A number of countries therefore recommend assessing
the volume of FMH after delivery to specify the dose of
IgG anti-D necessary to prevent RhD alloimmunization
of the mother (Australia, Canada, USA, Great Britain,
France, Ireland).
There is a greater risk of fetal erythrocytes entering
maternal circulation in deliveries by caesarean section17, stillborn deliveries15, traumatic vaginal deliveries,
deliveries with multiple births, deliveries with signs of
premature separation of the placenta, deliveries with
pathology in the third stage of labour, etc.
The goal of further studies should be establishing optimal doses of IgG anti-D. The effectiveness of immediate
administration of lower doses of IgG anti-D in combination with screening of the volume of fetomaternal hemorrhage and subsequent supplementation of IgG anti-D in
case of necessity should be compared with the effectiveness of administering a single larger dose of IgG anti-D
to everyone.

6
If the amount of fetal erythrocytes which entered the maternal circulation is quantitatively determined, administration of 10 g IgG anti-D per 0.5 ml of fetal erythrocytes
or 1 ml of whole blood is indicated67.
In cases where prevention of RhD alloimmunization is
not performed within 72 hours of a potentially sensitising event, it is still sensible to administer IgG anti-D
within 13 days, and in special cases, administration is
still recommended up to a maximum interval of 28 days
postpartum8.
It is necessary to issue a confirming document to women
who received IgG anti-D, which precisely describes the
amount and form of administration. IgG anti-D should
not be administered to women where the presence of
anti-D antibodies was confirmed in their blood serum.
Exceptions are cases of persisting levels of antenatally
administered IgG anti-D. If there is not absolute certainty
regarding the origin of anti-D antibodies in the mothers
serum, prevention of RhD alloimmunization should be
performed. IgG anti-D should also be administered in
cases where the RhD status of the child is unknown.

DETERMINING THE VOLUME

OF FETOMATERNAL HEMORRHAGE (FMH)
The amount of fetal erythrocytes leaked into the
maternal circulation (FMH) is accurately assessed using
flow cytometry. After labour, a blood sample from the
mother may be drawn no sooner than 1 hour after labour
and a sample of 0.51.0 ml of venous blood is collected
into a test tube containing an anticoagulative substance
(EDTA, Heparin).
It is common practise to always examine the RhD
status of a child born to an RhD negative woman after
labour. Assessing the volume of fetomaternal hemorrhage
(FMH) is then indicated in cases, where the child is RhD
positive as it allows specification of the dose of IgG anti-D
needed for the prevention of RhD alloimmunization of
the mother. IgG anti-D is however always administered
in a minimally 100 g dose immediately after detecting a
positive RhD status of the child.
Optimal and economically most effective would be
to apply a 250 g dose of anti-D antibody in the 28th
week of gestation to RhD-negative women if no anti-D
antibodies were detected in their serum. After the birth
of an RhD positive child, the volume of fetal erythrocytes
which entered maternal circulation should be assessed
and only in indicated cases should a dose of IgG anti-D
be administered postpartum (Australia)4.
The volume of fetomaternal hemorrhage (FMH)
should also be determined in all RhD negative women
where the presence of anti-D antibodies was not determined, in cases of potentially sensitising events (Table 1)
after 20 weeks of gestation57,2023.
The Kleihauer-Betke acid-elution test (UK) or the
Rosette test (USA) may be used to roughly determine the
volume of fetomaternal hemorrhage.

M. Lubusky
ESTABLISHING RHD GENOTYPE OF THE FETUS
At the beginning of pregnancy of an RhD negative
woman, it is possible to establish the RHD genotype of the
fetus from free fetal DNA circulating in maternal peripheral blood. If the fetus is RhD negative, it is unnecessary
to administer IgG anti-D at 28 weeks gestation or perform
RhD alloimmunization prevention in cases of potentially
sensitising events (Table 1)2427.

ACKNOWLEDGEMENTS
Supported by the grant from the Ministry of Health of the
Czech Republic IGA NS 103113/2009 Incidence, volume
and risk factors of fetomaternal hemorrhage during labour.

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