Blood Reviews (2000) 14, 4461

2000 Harcourt Publishers Ltd

DOI: 10.1054/ blre.1999.0123, available online at http://www.idealibrary.com on

State of the art

RhD haemolytic disease of the fetus and the

newborn

S. J. Urbaniak, M. A. Greiss

When an RhD negative mother is exposed to the RhD positive red cells (usually as transplacental
haemorrhage), she develops allo-anti-D which crosses the placenta and then results in the destruction
of fetal red cells. Clinical manifestations of RhD haemolytic disease (HDN) range from
asymptomatic mild anaemia to hydrops fetalis or stillbirth associated with severe anaemia and
jaundice. HDN was a significant cause of fetal mortality and morbidity until the introduction of
amniocentesis, intrauterine transfusion, controlled early delivery and exchange transfusion in the
management of severely alloimmunised women and their fetuses. The objective of monitoring
alloimmunised women is to identify fetal anaemia and prevent the development of life-threatening
hydrops. Evaluation involves assessing the history of previous pregnancies; serial estimation of
maternal anti-D levels; serial ultrasound measurements; serial amniocentesis; fetal blood sampling,
and intrauterine transfusion when indicated. Diagnostic genotyping by DNA-based methods can
identify at-risk RhD positive fetuses early in gestation. Identification of transplacental haemorrhage
(TPH) as the stimulus for anti-D antibody production led to the development of anti-D
immunoglobulin prophylaxis for at-risk RhD negative women who are not already alloimmunised.
Prevention includes administration of anti-D immunoglobulin for any event associated with TPH
during pregnancy, and at delivery of an RhD positive infant. Prophylactic routine administration of
anti-D immunoglobulin at 28 (and 34) weeks gestation, in addition to the above, has reduced
alloimmunisation to <1% of RhD negative women carrying an RhD positive fetus. 2000 Harcourt
Publishers Ltd

HISTORICAL BACKGROUND haemolytic anaemia (AIHA), haemolytic transfusion

In vivo haemolytic disease of the newborn (HDN) is
the most complex of the three common forms of IgG-
mediated red cell destruction, i.e. HDN, autoimmune
Stanislaw J. Urbaniak PhD, FRCP, FRCPE, FRCPath Professor
of Transfusion Medicine, and Director of Academic Transfusion
Medicine Unit, Department of Medicine and Therapeutics,
Aberdeen University, UK. Michel A Greiss MBBCh, PhD Clinical
Lecturer, Academic Transfusion Medicine Unit and Associate
Specialist, NE Scotland Regional Transfusion Centre, Foresterhill,
Aberdeen, UK
Correspondence to: S.J. Urbaniak
reaction (HTR), since it involves the production of
antibody(ies) in one individual (maternal) and cell
destruction in another (fetus).
Allegedly, the first description of a clinical condi-
tion called hydrops fetalis was in 1609,1 when a
French midwife called Louise Bourgeois described
the birth of twins: the first was oedematous and died
immediately after birth, the second became deeply
jaundiced (icterus gravis) and died a few days later.
Over the centuries, this clinical picture was recognised
and reported as two separate conditions (Table 1). It
was not until 1932 that Diamond et al.2 realised that
congenital anaemia, icterus gravis and hyrdrops

44

Table 1 Summary of historical events
16091 Louise Bourgeois (midwife) first described hydrops fetalis
19322 Diamond et al. realised that congenital anaemia, icterus gravis and hydrops fetails were manifestations of the same disease,
which they named erythroblastosis
19343 Hawskley & Lightwood recognised that hydrops fetalis, haemolytic anaemia and icterus gravis are variants of the above
19444 Gilmour noted that erythroblastosis fetalis is equivalent to HDN
19385 Darrow noted haemolysis was due to transplacental passage of a maternal antibodies to the fetal circulation
19396 Levine & Stetson found an antibody that caused HDN
19407 Landsteiner & Weiner described the Rhesus blood group
19548 Chown noted that mothers were sensitised following an FMH
19669 A combined study on preventing HDN by anti-D prophylaxis from centres in England & Baltimore
196810 Pollack et al. Results of clinical trails of RhoGAM in women
197111 WHO Prevention of Rh sensitization
199812 Consensus conference on anti-D prophylaxis
fetalis were manifestations of the same disease, which
they named erythroblastosis fetalis. It was also only
comparatively recently confirmed that hydrops fetalis,
haemolytic anaemia of the newborn and icterus gravis
were all recognised to be variants of the same disease
(Hawksley & Lightwood3 and Gilmour4) now univer-
sally referred to as haemolytic disease of the newborn
(HDN). The term erythroblastosis fetalis, which was
at one time widely used to describe the whole clinical
syndrome, more correctly refers to the finding of
extramedullary haemopoiesis and erythroblastaemia.
In 1938, Darrow5 postulated that the haemolysis was
due to transplacental passage of a maternal antibody
into the fetal circulation. One year later (1939), an
antibody was discovered by Levine & Stetson6 after an
unusual transfusion reaction; there was an immediate
reaction in a blood group O woman who had received
her husbands group O blood, shortly after delivery of
a stillborn fetus with erythroblastosis. The events sug-
gested that the infant had inherited a red cell agglu-
tinogen from the father which was foreign to the
mother. In 1940, Landsteiner & Weiner,7 using rhesus
monkeys, discovered the Rh antigen. They observed
that red blood cells from 85% of human subjects
agglutinated in the presence of rhesus monkey red cell
antiserum, whereas the 15% of subjects who lacked
the antigen on their red cells did not. Nevertheless, it
was not until 1954 that Chown8 reported that mothers
were sensitised by a fetomaternal haemorrhage
(FMH), and the association was made between fetal
RBCs and the stimulation of maternal antibodies.
The FMH or transplacental haemorrhage (TPH) had
been shown to occur mainly at delivery, and two
groups in the UK and the USA were able to demon-
strate in clinical trials that immunisation in RhD
negative women could be suppressed, when concen-
trated anti-D IgG was given intramuscularly (i.m.)
soon after delivery.9,10 Because of the difficulty in
measuring the TPH volumes with a degree of accu-
racy, and to err on the safe side, it is recommended
RhD haemolytic disease 45
that 25 g (125 iu) of anti-D Ig should be given i.m. to
cover a TPH of 1 ml RhD positive packed red cells or
2 ml of whole blood.11 Most recently, this was reinforced
by the joint working group of BBTS and RCOG.12
When RhD positive cells from the fetus pass into
the maternal circulation of an RhD negative mother,
this results in the production of antibodies, which
cross the placenta to the fetus circulation and cause a
variety of effects in the fetus ranging from very mild
to severe haemolytic anaemia and, on occasion,
intra-uterine death (IUD).
The most effective treatment is prevention of initial
RhD alloimmunization by prophylactic administra-
tion of passive anti-D immunoglobulin. The mecha-
nism of the antibody-mediated suppression is
incompletely understood (see later) but the most likely
mechanism is a negative modulation of the primary
immune response. Since antigenantibody complexes
are bound to cells bearing Fc receptors in the lymph
nodes and spleen, these cells presumably stimulate
suppressor T cell responses, which prevent antigen-
induced B cell proliferation and antibody formation.
Alternatively, the splenic macrophages remove anti-D
coated red cells prior to contact with the dendritic
antigen-presenting cell (APC), which have low FcR
expression, and the immune response is inhibited by
antigen deviation.1,1315
Once sensitisation has occurred the following
therapeutic options are open to the pregnant woman:
controlled early delivery, intrauterine transfusion
(IUT)/direct intravascular transfusion (IVT), plasma
exchange (PE), and/or intravenous immunoglobulin
(IV IgG). The IUT procedure is not without risk, and
with the reduced incidence of HDN is likely to
become more hazardous as those skilled in the proce-
dure become fewer. With the recent advances in fetal
medicine, introduction of direct intravascular transfu-
sion and improved neo-natology in special centres, the
survival of affected fetuses with HDN is now greater
than 90%.13,1621
46 Blood Reviews
Table 2 Frequency of possible Rh alleleic combinations
Nomenclature
FisherRace Weiner
Rh Positive CDe R1
cDE R2
cDe Ro
CDE Rz
Rh Negative cde r 0.37
Cde r 0.02
cdE r 0.01
CdE ry
SCIENTIFIC BASIS
Rh antigens: structure and inheritance
The Rh blood group antigens are associated with
nonglycosylated human erythrocyte membrane pro-
teins of molecular mass 30 KDa (the Rh poly-
peptides) and a glycoprotein of 40100 KDa (the
Rh-associated glycoprotein). The precise function of
these Rh antigens are unknown. The Rh polypeptides
are embedded in the lipid phase of the red blood cell
membrane and may interact with the membrane
ATPase, functioning as part of a cation or proton
pump to control fluid and electrolyte fluxes across the
cell membrane.13,18,22 However, rare individuals with-
out any Rh antigen (Rhnull) have defective red cell
membranes associated with a degree of haemolytic
anaemia.13,15,18,2325
The human Rh locus is composed of two highly
related genes RHD and RHCE (RhCcEe proteins),
encoding the D, Cc and Ee blood group antigens
respectively. The expression of D indicates an RhD
positive person and the absence of D denotes an Rh
negative person. The genes which determine the anti-
gen are inherited as 2 haplotypes consisting of three
alleles, one haplotype being inherited from each par-
ent, CDe (phenotype designation R1) 4042%, cde
(r) 3537%, and cDE (R2) 1416% being the most
common in Caucasians (Table 2). Of the approxi-
mately 45 Rh antigens, D is the major cause of Rh
incompatibility. It is estimated that 1517% of whites,
57% of blacks and about 2% IndoEurasians do not
express the D antigen and so are called Rh-negative;
RhD negative individuals (as determined serologi-
cally) are < 1% amongst indigenous Chinese.13,15,18
Recent molecular analysis shows that serologically
defined RhD-negative individuals of some ethnic
groups, e.g. South African blacks (and their descen-
dants) carry an RHD pseudogene which does not
express RhD antigen.26
Ethnic group
Whites Blacks Asian
0.42 0.17 0.70
0.14 0.11 0.03
0.04 0.44 0.03
0.00 0.00 0.01
0.26 0.03
0.02 0.02
<0.01 <0.01
<0.01 <0.01 <0.01
A number of variants exist for each of the common
Rh antigens, e.g. Du, Cw, Cx, Ew. The most clinically
significant antigen D is a mosaic composed of at
least 30 determinants as identified by monoclonal
anti-Ds.27 The lack of expression of certain of these D
epitopes at the surface of variant red blood cells deter-
mines the so-called D category phenotypes which
arise from genomic rearrangements of the D and CE
genes.15,28,29 The Du or weak D phenotype is of par-
ticular importance and is common in black popula-
tions. In most individuals weak D differs from normal
D only in having fewer antigenic sites per red cell, and
these cells react weakly/variably with different anti-D
sera.30 Molecular analysis of weak D phenotype
shows that the majority have altered RhD proteins
resulting in diminished expression of the D antigen.31
Some weak D phenotypes are in fact D category vari-
ants and these individuals can be immunised to form
anti-D and should be identified as D variant at a
reference laboratory.
If a structural D variant is excluded, it is not neces-
sary to treat such weak D individuals as RhD negative
for transfusion purposes. Similarly, weak D individu-
als are not considered candidates for either antenatal
or postnatal prophylaxis with passively administered
anti-D immunoglobulin. However, if there are no
facilities for excluding structural D variants then it is
safer to treat all weak D individuals as being RhD
negative.15,18,29,32
Immunogenic factors
The factors which influence the immune response to
the RhD positive cells are:
1. Concurrent ABO incompatibility can protect the
mother against immunization, presumably because
leaked fetal red cells are promptly coated by
circulating isohaemagglutinins (IgM) and
(probably) complement, and then removed from
 RhD haemolytic disease 47

Fig. 1 Mechanism of Rh sensitisation and the effect of ABO incompatibility.

the circulation by the mononuclear phagocyte
system (MPS), mainly in the liver, which is less
immunoresponsive than the spleen and therefore is
less likely to stimulate antibody production.1,13,18
(Fig. 1)
2. The critical factor determining the magnitude of
antibody response is the dose of immunizing
antigen, i.e. the volume of RhD positive RBC.
Haemolytic disease develops in neonates only
when an RhD negative mother has experienced a
48 Blood Reviews
significant transplacental haemorrhage (TPH).
Although the average TPH occurring at delivery is
less than 1 ml of whole blood, approximately 50%
of all women with ABO-compatible pregnancies
have detectable circulating fetal red cells: only 19%
of those with ABO-incompatible pregnancies have
detectable circulating fetal red cells.1,9,14,18
3. The antigen expression on the RBC influences
immunogenicity, e.g. R2r infants are more effective
in sensitizing their mothers to RhD than are
infants of other phenotypes, since the R2
phenotype expresses most D antigen.13,15,18
4. The immune responsiveness of the recipient, e.g.
some women produce potent anti-D in a first
pregnancy sufficient to cause severe hemolytic
disease.
The D antigen is a high-incidence and strongly
immunogenic antigen, 50 times more so than the
other Rh antigens. The incidence of primary RhD
immunization also depends on the dose of RhD posi-
tive red cells: 15% after 1 ml and 7090% after 250 ml
given intravenously.33,34 On the other hand secondary
immune response may occur after exposure to much
smaller amounts (as little as 0.03 ml of RhD positive
red cells).1,13,18,35
However, the immune system is not fully developed
at birth. Infants less than four months of age appear
to be incapable of producing antibodies in response to
multiple transfusions,36,37 and this may be due to the
immaturity of several lymphocyte and monocyte
functions.
Nevertheless there is considerable variation among
adults in immune responses to alloantigens presented
during pregnancy, and the ability to produce antibod-
ies, once developed, does not regress with age. These
variations may be due to: (1) variable antigenicity of
epitopes; (2) the role of the placenta, i.e. the route of
exposure to antigen; and (3) variable maternal
immune responsiveness.
Immune responsiveness is determined, at least in
part, by products of the major histocompatibility
complex (MHC) genes particularly HLA-DR genes
(class II). Several studies have sought to associate
HLA phenotype and immune response to various
antigens, e.g. the HPA-1 platelet antigen has been
strongly associated with the HLA-B8, DR3 haplo-
type.13,18 On the other hand, the production of anti-D
has not been shown to correlate significantly with
HLA type. Perhaps this inability to demonstrate a
close relationship between HLA and blood group
alloimmunisation is because the immune response is
also influenced by genes independent of HLA, such
as T-cell receptor genes, and the T cell repertoire of
the individual might contribute to the variability of
anti-D responses.13,18,22 However, recent evidence indi-
cates that T-cell responses to RhD peptides is
restricted by MHC class II.38
Sensitisation and development of allo anti-D
Between 1940 and 1970 dramatic progress was made
in reducing the mortality from HDN, from 50% to
59% of all pre-natal deaths. Contributory events
were the introduction of Coombs antiglobulin tech-
nique, exchange transfusions, amniocentesis, con-
trolled premature delivery, and intrauterine
transfusion. By 1970 it was not possible to reduce
further the number of babies which were affected by
RhD hemolytic disease. Although since then the
introduction of prophylactic anti-D has led to dra-
matic reductions in the incidence of cases, Rh HDN
does still occur mainly as a result of: (1) already
immunised women subsequently becoming pregnant;
(2) failure to administer an adequate amount of anti-
D immunoglobulin after a sensitizing episode (e.g.
abortion, amniocentesis, normal delivery); and/or (3)
failure of the anti-D immunoglobulin to protect even
when given correctly.1,13,16,18 In Rh, and other forms of
HDN, the antibody is produced as a result of sensiti-
sation by pregnancy or transfusion. However, expo-
sure to an antigen does not necessarily cause
production of an antibody, and a series of small
immunizing doses (immunological challenges) is more
likely to initiate antibody production than a single
dose. Sensitisation through pregnancy occurs partly
due to a small fetal bleed (fetal maternal haemor-
rhage [FMH]) into the mothers circulation via the
placenta (TPH) and a larger volume at delivery when
the placenta separates from the uterus.1,13,39
In the fetus the RhD antigen is well developed by
3040 days gestation, and FMH can occur antenatally
from as early as six weeks.40 Women immunised after a
relatively small TPH or following an abortion (spon-
taneous or therapeutic) are referred to as good
responders, and any RhD positive fetuses in subse-
quent pregnancies may be severely affected. Evidently
the risk of TPH is increased as the pregnancy pro-
gresses (3% in the first trimester, 43% in the second
trimester, 64% in the third trimester).1,16,39 For unex-
plained reasons, a non-immunised unprotected RhD
negative mother has only a 16% chance of becoming
immunised against her RhD positive fetus in any
given pregnancy. Among these are RhD negative
mothers with RhD positive ABO compatible and
incompatible babies (approximately 8% and 2%
respectively) who will produce anti-D following
delivery of their first baby; and another 8% will have
an anamnestic (secondary) response in their second
RhD positive pregnancy.13,18,22 The primary response
 RhD haemolytic disease 49

Table 3 Events following which anti-D Ig must be given to all RhD-negative women with no RhD
antibody and/or with antibodies other than anti-D

l delivery of an RhD positive infant* l antepartum haemorrhage (APH)

l abortion (see Table 4) l external version of the fetus

l invasive prenatal diagnosis l closed abdominal injury

amniocentesis
chorionic villus sampling (CVS) l ectopic pregnancy
fetal blood sampling (FBS)

l other intrauterine procedures l intrauterine death (IUD)

insertion of shunts
embryo reduction l stillbirth
*also if the RhD type of the infant has not been determined or is in doubt, and
the mother is to be discharged
Dose: before 20 weeks gestation 250 IU (50 g)
after 20 weeks gestation 500 IU (100 g)
In conjunction with a Kleihauer or equivalent test to assess the size of any TPH

Table 4 Prophylactic anti-D following abortions to all RhD negative women with no RhD antibody
and/or with antibodies other than anti-D

l therapeutic termination of pregnancy

l spontaneous abortion followed by instrumentation
l spontaneous complete or incomplete abortion after 12 weeks gestation
l 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
l threatened abortion after 12 weeks all women are eligible; in addition:
when bleeding continues intermittently after 12 weeks gestation, anti-D Ig should be given at
approx. 6 weekly intervals, and assess the size of TPH

is usually weak and often IgM antibodies, which do
not cross the placenta, are produced; thereafter the
majority will convert to IgG antibodies. There is
currently no practical way of preventing a pregnant or
transfused woman from becoming immunised to red
cell antigens other than prophylaxis with administra-
tion of anti-D immunoglobulin. It is not always
remembered that potentially sensitizing episodes other
than delivery are indications for administration of
intra-partum anti-D immunoglobulin (e.g. abortion,
ante-partum haemorrhage, amniocentesis, external
version and adventitious trauma see Tables 34.).
Anti-D IgG associated with HDN is produced
only as subclasses IgG1 and/or IgG3. Cases of
HDN caused by IgG1 anti-D reputedly involve a
more serious anaemia than those caused by IgG3
anti-D presumably due to the longer exposure
of fetal red cells in utero to IgG1 than to IgG3
anti-D. Since the infants post-delivery bilirubin
level in HDN caused by IgG3 anti-D is often greater
than that in IgG1-induced HDN it is conceivable
that, once it reaches the infants circulation, IgG3
anti-D causes greater red cell destruction than does
IgG1.13,18

Role of IgG subclasses Mechanisms of red cell destruction

The concentration of all four IgG subclasses has been
found to be slightly higher in cord than in maternal
serum. IgG1 crosses the placenta early in pregnancy
and by 20 weeks gestation maternal IgG1 is detectable
in cord serum at levels which equal, or exceed, those in
the maternal serum. In contrast, the level of IgG3 in
cord serum does not reach the maternal concentration
until 2832 weeks gestation, and may never rise higher
than this during the entire pregnancy.13,18,41
The mechanisms of immune red cell lysis are basically
the same whether mediated by autoantibodies, i.e.
AIHA, allo-antibodies destroying transfused donors
RBCs i.e. HTR, or transplacental transfer of allo-
antibodies, i.e. HDN (Table 5). Binding of red cell
antibodies to their antigenic determinants on the cell
membrane does not directly damage red cells
Although IgG Rh antibodies are almost exclusively
IgG1 and IgG3, they very seldom bind complement,
50 Blood Reviews
Table 5 Antibody-mediated RBC destruction
RBC + (IgM) antibody + C1C9 complement activation intravascular lysis
RBC + (IgG or IgM) antibody + complement activation to C3 removal by mononuclear phagocytic system (mainly liver)
RBC + (IgG) antibody directly removed by mononuclear phagocytic system (mainly spleen)
RBC + (IgG) antibody antibody-dependent cell-mediated lysis
possibly because of steric hindrance due to the loca-
tion of the Rh antigens within the RBC membrane. It
has been suggested that Rh antigens are mobile within
the membrane and may cluster during antigen-
antibody reactions.14,15 It is possible that this ability of
the antigens to cluster may be important in immune
red cell destruction as it may alter the presentation of
membrane-bound IgG to the macrophage (e.g. there
is a greater likelihood of IgG forming doublets).
Mechanism of extravascular haemolysis
The interaction of macrophages with RBCs coated
with IgG occurs through receptors specific for the Fc
portion of the IgG (especially IgG1 and IgG3) dou-
blet.1315,42 The subclass of the IgG antibodies is also
important for binding to macrophage Fc receptors
(IgG3 > IgG1). The pathogenicity of red cell anti-
bodies depends on additional factors:
1. The amount of the antibody
2. Avidity for the erythrocyte antigen
3. Antigen distribution and density
3. The optimum temperature for binding, i.e. 37C
4. The degree of complement binding (not in the
case of Rh)
In the case of HDN, additional factors are the
efficiency of placental transfer of antibody, the func-
tional maturity of the fetal spleen, and the presence of
HLA-related blocking antibodies. Each of these fac-
tors probably contributes independently and cumula-
tively to erythrocyte destruction.
Immune haemolysis in vivo begins with opsonisa-
tion of red cells by antibodies. Opsonised red blood
cells are recognised and cleared from the circulation
by macrophages primarily in the spleen, and to a
lesser extent, the liver. The splenic environment is
especially conducive to immune clearance. A red
blood cell lightly coated with IgG (and therefore inca-
pable of activating the complement cascade) is prefer-
entially removed in the sluggish circulation of the
spleen. IgG-coated red cells are efficiently trapped
from the haemo-concentrated circulation within the
spleen. The relatively low plasma concentration in
splenic sinusoids tends to alleviate competition
between plasma IgG and IgG-coated RBCs for Fc
receptors, thus providing optimal interactions
between opsonised erythrocytes and macrophages.
The sensitised red cell is either wholly phagocy-
tosed, or it loses part of its membrane (scission) as a
result of attack by macrophages and returns to the
circulation as a microspherocyte. Spherocytes are less
easily deformed than normal discoid red cells and
tend to be trapped in the spleen, thus shortening their
life span.13,15,18 In addition to Fc receptor-mediated
phagocytosis it is now thought that antibody-depen-
dent cell-mediated cytotoxicity (ADCC) may also
contribute to cell damage during the phase of close
contact with splenic macrophages.13,15,18,4144
CLINICAL FEATURES
The clinical manifestations of HDN range from pal-
lor in the mildly affected infant to intense jaundice,
widespread oedema, and signs of neurological
involvement in the severely affected fetus a
syndrome called hydrops fetalis.16,45
The anatomical findings in erythroblastosis fetalis
vary with the severity of the haemolytic process.
Infants may be stillborn, die within the first few days,
or recover completely. In its mildest form the anaemia
may be only slight and the child may survive without
further complication. Plasma protein levels sometimes
drop as low as 2025 g/l because of reduced hepatic
synthesis. Hydrothorax can compromise neo-natal
respiratory status after birth.14,16,45
Pathogenesis of HDN
The process of HDN is initiated in utero where fetal
RBCs are coated with maternal antibody and are
removed from the fetal circulation by its MPS system
and destroyed. The anaemia resulting from the RBC
destruction is associated with reduction in the oxygen
carrying capacity of the blood which causes the fetal
haemopoietic tissue to produce more RBCs and to
release them prematurely. This results in an increased
number of reticulocytes and nucleated red blood cells.
In the gravely ill infant with hydrops fetalis there is
intense jaundice, widespread oedema, signs of neuro-
logical involvement, fatty degeneration, hemosiderin
deposition and engorgement of hepatic canaliculi.

Cardiac enlargement, pulmonary haemorrhage and
pleural and pericardial effusions may occur in addi-
tion to massive ascites and subcutaneous oedema
which results in severe dystocia.45 Historically, before
the disease was well understood it was identified by its
more obvious symptoms and due to the increase in the
number of nucleated RBCs it was frequently called
erythroblastosis fetalis and cases exhibiting severe
jaundice (as a result of increased bilirubin) icterus
gravis neonatorum.
Compensatory mechanisms exist within affected
fetuses, e.g. anaemia and hyperbilirubinaemia may
not occur because the marrow hyperactivity compen-
sates for the red cell destruction and the hepatic
excretor system (including feto-maternal transfer of
bilirubin) may be capable of handling the increased
load of bilirubin. When the compensatory capacity of
the marrow is exceeded, extramedullary haemopoiesis
in the liver and spleen may suffice to maintain normal
levels of red cells in the blood. Reduced synthesis of
albumin by the liver leads to hypoalbuminaemia, a fall
in plasma oncotic pressure, and the formation of
hydrops.16 When the haemolytic reaction is severe and
the serum bilirubin level exceeds 20 mg/dl (approxi-
mately 350 mol/L) the anaemia is associated with
jaundice in the newborn. This unconjugated bilirubin
is water insoluble and has an affinity for lipids and in
the infant with a poorly developed bloodbrain
barrier the bilirubin may bind to the lipids in the brain
and cause severe damage. The severity of all these
changes varies quite considerably in each affected
infant.13,18,45
Haemolytic findings in Rh haemolytic disease of the
newborn
Haemoglobin (Hb)
Cord blood Hb is often abnormally low, but does not
always correlate with severity. Mollison46 reported
that approximately half of the infants affected with
HDN had a Hb exceeding 145 gm/L. In 16 of 141
cases the Hb was > 175 g/L; on the other hand, in six
cases it was < g/L.
Erythrocytes
The erythrocyte count of cord blood reflects the Hb
content, i.e. in infants without anaemia the counts are
as high as 5.5 1012/L and at the end of the scale,
counts < 2.0 1012/L are seen in infants born mori-
bund. There is marked macrocytosis and the MCV are
greater in mature normal newborns.47
Stained blood films showed anisocytosis with many
macrocytes and a small degree of poikilocytosis. The
reticulocyte count can be as high as 3040% in infants
RhD haemolytic disease 51
who did not receive an IUT.48 However, neonates who
receive a direct IVT in particular shortly before
delivery, may present with absent reticulocytes.
Furthermore, neonates who receive exchange transfu-
sion can develop reticulocytopenia,49 which is charac-
teristic of hyporegenerative anaemia observed in
infants with the later anaemia of haemolytic dis-
ease.5052 In the presence of a high concentration of
nucleated RBCs, the automated leukocyte count may
be falsely elevated and require correction by a factor
proportionate to the fraction of nucleated red blood
cells.53 Spherocytes are more commonly seen in cases
of ABO incompatibility and in certain RBC
membrane disorders.54
Accelerated red cell destruction leads to various
degrees of haemolysis and fetal anaemia. Prolonged
haemolysis results in erythroid hyperplasia of the
bone marrow, extramedullary haemopoiesis in the
fetal spleen and liver, and hydrops which is charac-
terised by various degrees of hepatomegaly,
splenomegaly and placental oedema.55,56 This
haemopoietic activity is sufficiently striking to
account for increased numbers of reticulocytes,
normoblasts and erythroblasts in the circulating
blood.48 Evidence of subcutaneous and visceral
oedema is present in the hydrops syndrome, along
with fluid in the peritoneal, pleural and pericardial
cavities.13,45,57
Jaundice
The most serious threat in erythroblastosis is central
nervous system damage known as kernicterus. In
jaundiced infants unconjugated bilirubin appears to
be particularly toxic to the brain tissue. The brain is
enlarged and oedematous, and when sectioned is
found to have a bright yellow pigmentation (ker-
nicterus), particularly in the basal ganglia, thalamus,
cerebellum, cerebral grey matter, and spinal cord.58
This pigmentation is transient and fades within 24
hours despite prompt fixation. It is therefore neces-
sary to section the brain immediately in suspected
cases in order to establish the diagnosis. It is worth
noting that such brain pigmentation is not limited
exclusively to erythroblastosis fetalis, as it has also
been described in milder form in infants with severe
physiological jaundice of the newborn.45
Bile pigmentation of the brain is of considerable
theoretical interest, because in the adult a
bloodbrain barrier blocks the passage of bilirubin
into the spinal fluid and substance of the brain, even
when there is severe jaundice. The precise bilirubin
level that will induce kernicterus is not predictable, but
neural damage rarely occurs if the serum bilirubin
concentration is below 20 mg/dl.45,58 However,
52 Blood Reviews
premature infants are at risk from lower serum albu-
min levels. The mechanism by which free (unconju-
gated) bilirubin enters the brain in the newborn is not
clearly understood, but the level of serum albumin
(which binds bilirubin), pH of the blood and hypoxia
are all important factors in allowing passage of
bilirubin into the brain.16,53,5860
TREATMENT
The availability of tests in vitro to monitor the sever-
ity of the haemolytic reaction has opened a variety of
avenues for the treatment of haemolytic disease of the
newborn. When analysis of amniotic fluid discloses
critical levels of bilirubin, premature delivery may be
induced if the fetus is judged to be viable with regard
to size and maturity. Earlier, fetal transfusion may be
attempted. Post-natally, a variety of supportive mea-
sures may be employed including phototherapy
(visual light oxidises toxic unconjugated bilirubin to
harmless, readily excreted water-soluble dipyrroles)
and, in severe cases, total exchange transfusion of the
infant.19,20
Intrauterine fetal transfusion
The first intravascular transfusions (IVT) were per-
formed fetoscopically in the early 1980s. The method
used most widely is ultrasound-guided blood sam-
pling from the umbilical vein at the placental cord
insertion.13,1720 Intravascular transfusion has become
the mainstay of management of very severe alloim-
munization in pregnancy, although intraperitoneal
transfusion still has a place in early gestation between
14 and 18 weeks or, for example, when the cord root is
inaccessible or in order to top-up an incomplete IVT.
Despite considerable experience with IVT, it has been
shown that there is still a need to continue familiarity
with the intraperitoneal approach.13,1921 Intravascular
transfusions carry significantly increased risks for
both mother and fetus when compared to cordocente-
sis alone, and this technique should be offered only
within a fetal medicine unit. It has been recommended
that about 2030 cordocenteses and 1015 transfu-
sions need to be performed annually to maintain com-
petence.13,19
With accurate determination of fetal genotype by
PCR on amniocytes (see below) there is a tendency to
delay the first IUT until after 20 weeks, or signs of
hydrops are developing. If severe hydrops is suspected
very early, the preference is for intraperitoneal trans-
fusion at 1415 weeks followed by FBS/Intravascular
transfusion. With greater understanding of fetal
responses to transfusion, there is a trend to transfuse
to 2.5 standard deviations above the expected Hb for
the gestational age (using nomograms) and to
lengthen the time between transfusions to 23 weeks.
The effect of the first transfusion is shortest because
of the continued presence of antibody coated fetal red
cells; thereafter fetal haemopoiesis is suppressed and
transfused red cells fall by 1% per day. The last fetal
transfusion is given at about 35 weeks with delivery at
37/38 weeks gestation, in preference to early delivery
and exchange transfusion because the fetus tolerates
larger transfusion volumes than the neonate due to
the large capacity of the placenta. With advances in
fetal medicine, introduction of direct intravascular
transfusion (IVT) and improved neonatalogy in
specialised centres, survival of affected fetuses with
HDN is now greater than 90%.13
Plasma exchange (PE)
Removal of as much as 45 litres anti-D containing
plasma, and replacement with albumin/crystalloid
solutions, is possible using automated apheresis
devices. However, the efficacy of this procedure in the
absence of immunosuppression to prevent rebound
synthesis of IgG is debatable, and needs to be
repeated two or three times a week to maintain
reduced levels of anti-D. Results of PE have been vari-
able,1618 and this procedure is not to be undertaken
lightly as the mother is subjected to an arduous proce-
dure. It may be indicated if there is a previous history
of fetal loss or a severely affected infant, a strong pos-
sibility that the father is homozygous for RhD, and to
maintain pregnancy until cordocentesis or IVT sam-
pling and transfusion can be carried out safely. If
decided upon, PE should be started early in preg-
nancy, and potentially immunising factors (e.g.
amniocentesis) should be avoided until 2628
weeks.1921
Intravenous immunoglobulin (IV IgG)
There have been recent reports of the benefits of high
dose intravenous immunoglobulin (HDIV IgG)
administration in severely alloimmunized pregnant
women.13,18,61 Doses of 2 g/kg maternal body weight
every two weeks can reduce circulating allo-
antibody levels by up to 50%, and this is thought to be
mainly due to the negative feedback produced by total
circulating maternal IgG levels of 2530 g/l.13,61,62
Further benefits of IV IgG therapy may be
interference with trans-placental transfer of maternal
antibodies by trophoblastic Fc receptor saturation,61,62
and (similarly) reduction of IgG-coated fetal red cell
haemolysis by fetal mononuclear phagocytic Fc recep-
tor saturation. This is in agreement with the outcome

of a controlled study63 which showed that the
frequency of transfusional therapy was reduced
when HDIV IgG was combined with conventional
phototherapy.
Combined PE/IV IgG therapy may be considered
in the same circumstances as when intensive plasma
exchange would be considered, and only from 1012
weeks gestation. 13,18,53
BLOOD GROUP SEROLOGY MONITORING
The mothers first prenatal visit booking, includes a
history of pregnancies and blood transfusions, ABO
and RhD grouping, and antibody screening. If the
RhD group is negative, a test for weak D should be
performed. Conventional serology is directed towards
the identification of IgG antibodies which react at
37C by using indirect antiglobulin test (IAT) with
untreated red cells. Further testing of all women with-
out antibodies is advised at least once between 28 and
32 weeks gestation18,64,65 (prior to administration of
any antepartum anti-D, immunoglobulin), and at the
time of delivery, which should be accompanied by a
cord blood sample for ABO/RhD grouping and direct
antiglobulin test, and an uncoagulated maternal
sample for estimation of FMH6668 to decide on
the administration/dose of prophylactic anti-D
immunoglobulin (Fig. 2). Some authorities recom-
mend screening twice during pregnancy, at 2832
and 3436 weeks gestation.18,65 Although enzyme-
enhanced techniques may identify antibody at a lower
level compared to the IAT, the vast majority of these
additional antibodies are of no clinical significance as
regards HDN.69
Where alloantibodies are detected which are clini-
cally significant, i.e. known to be associated with
HDN, IAT titre is performed, and followed up by
assessment of the level of antibody and the monitor-
ing of any change during the course of pregnancy. In
the case of anti-D, Auto Analyser quantification is
also done and reported in iu/ml (5 iu = 1 g) in com-
parison with the national reference preparation.18
Samples should be stored frozen and tested in parallel
with the current sample using the same techniques.70
The partner is also phenotyped for the relevant anti-
gen(s). The absolute level of the antibody is not as
important as the trend, with a rising level requiring
more frequent monitoring especially with a homozy-
gous partner, and if there is a history of previous
HDN.18,19,65,70 The likelihood of significant HDN due
to anti-D below 4 iu/ml is very remote, whereas above
15 iu/ml all cases should be treated as at risk of severe
HDN until proven otherwise by the clinical investiga-
tions i.e. testing monthly from booking to 30 weeks,
RhD haemolytic disease 53
Sample required for antibody screen
Sample required when the antibody(ies) are associated with HDN other than D, c, Kell, e.g.
Duffy (Fy), Kidd (Jk).
For ABO, Rhd group; weak D test if apparently negative;29 antibody screen
Confirmation of blood group/antibody; estimation of FMH
For ABO, Rhd group; weak D test if apparently negative; direct antiglobulin test, antibody
elution and identification if positive; and red cell phenotyping as required
* All patients except those with antiD, antic or Kellrelated antibodies.
** Antibody associated with severe HDN e.g. D, c, and Kell related antibodies.
Delivery in Specialized Hospital is advised when there is a possibility or risk of transfusion
problem and/or HDN.
Fig. 2 Suggested frequency of ante-natal testing
fortnightly from 3036 weeks and weekly from 36
weeks onwards (Fig. 2).65,69,70 Where quantification of
anti-D is not routine, laboratories establish critical
titres below which hydrops fetalis is not anticipated,
usually below 1/161/32, with close monitoring if
> 1/8.71
Serological assays measure only the capacity of
antibodies for agglutination whereas in vivo destruc-
tion involves interaction of antibody coated red cells
with Fc receptors of the mononuclear phagocyte
system. Considerable interest has therefore been
shown in developing bioassays as predictors of HDN,
including monocyte monolayer assays, which measure
adherence/phagocytosis, monocyte chemilumines-
cence (CL) which measures the oxidative respiratory
burst associated with adherence/phagocytosis and
antibody dependent cytotoxicity (ADCC) assays
which measure extracellular haemolysis.14,15,18,42,72,73
MONITORING AND EVALUATION OF
ALLOIMMUNIZED WOMEN
Anti-D levels
For mild disease (maternal anti-D levels <2.5 iu/ml,
with good obstetric history or IAT <1/8), monthly
anti-D quantification and Ultrasonography should be
adequate. When the anti-D concentration remains
<5 iu/ml (<1 g/ml), fewer than 5% of affected fetuses
will require neonatal exchange transfusion.
Amniocentesis may be the preferred method of inves-
tigation when anti-D levels are from 4 to 15 iu/ml
(IAT > 1/32),71 as moderate fetal anaemia may occur.
Fetal blood sampling (FBS) may be performed early
(at 18 weeks) when anti-D levels are >20 iu/ml,13,18,19
54 Blood Reviews
or at a titre established locally as being critical. These
levels are only guidelines, and do not precisely predict
the degree of fetal anaemia.
Ultrasonography
Ultrasonography is useful to monitor Rh-alloimmu-
nized pregnancies, and in experienced hands early
hydrops can be reliably detected.13,18 Fetal ascites or
hydrops can be observed with placental thickening,
loss of architecture and increased homogeneity and
hydramnios. The most common progression is from
hydramnios to placental thickening, to enlargement of
the fetal liver, to hydrops. However, placental thick-
ness, extra and intrahepatic umbilical vein diameters,
abdominal circumference and head/abdominal cir-
cumference ratios in one study were not found to be
reliable markers of severe anaemia.13,19 Further work
needs to be performed to assess the value of ultra-
sonic markers of early fetal ascites, such as double
outlining of small bowel loops and abdominal fluid
rim. However, a recent prospective study suggests that
pregnancies with a mild or no history of fetal anaemia
may be successfully monitored without invasive tech-
niques by a combination of serial anti-D quantifica-
tion and Doppler monitoring of umbilical vein
maximal flow velocity (UVVmax); liver length and
spleen perimeter measurements were not as helpful.74
A similar multicentre prospective study showed that
Doppler estimation of peak systemic blood velocity
in the middle cerebral artery accurately predicted
moderate or severe anaemia in non-hydropic fetuses.75
Amniocentesis
Amniocentesis allows spectrophotometric measure-
ment of the deviation in optical density at 450 nm
( OD 450) due to bilirubin in amniotic fluid, which
reflects fetal red blood cell haemolysis. It does not,
however, provide information on fetal erythropoiesis,
and is unreliable prior to 27 weeks gestation.9,13
Different degrees of change are measured/interpreted
differently, because the normal level of bilirubin
varies with gestational age.16,18,22 Liley (1961)76 was the
first to develop a method to measure this deviation
(Liley Graph- Fig. 3); various modifications have been
made in an attempt to extrapolate to the first
trimester, e.g. Queenans modification,77 or Whitfield
action line78 but none are entirely satisfactory in the
first trimester. If all the OD 450 values remain in the
lower zone this should indicate either no disease or
only a mildly affected fetus, but 10% may require
exchange transfusion. Values in the mid zone indicate
a moderately affected fetus which is a candidate for an
early delivery and possible exchange transfusion.
1.00
Immediate Delivery
Intrauterine
Transfusion Upper Zone
0.50
(severe)
0.20
OD450(nm)
0.10
0.05
Mid Zone
(moderate)
0.02
Lower Zone
(mild)
0.01
24 28 32 36 40
Weeks of Gestation
76
Fig. 3 Liley Graph.
When readings are approaching the upper zone, the
fetus requires immediate attention as severe disease is
predicted. Accurate prediction of the severity of
HDN by using amniotic fluid requires serial OD 450
measurements, and amniocentesis is also more accu-
rate in the third trimester than in the second trimester.
Final values falling in the upper and lower zones have
an accuracy of prediction of 95%. On the other hand,
final readings remaining in the mid zone have accu-
racy of prediction of approximately 90%.13,16,18 It
should be borne in mind that amniocentesis is not
without risk, with up to 1% fetal mortality directly
associated with the procedure, and boosting of anti-D
levels following leak of fetal cells into the maternal
circulation.
Fetal blood sampling (FBS)
The current approach of using ultrasound needle
guidance was first introduced for prenatal diagnosis in
the early 1980s.13,16,20 The most accurate method of
assessing fetal anaemia is to measure the haematocrit
of fetal blood directly by cordocentesis, and is ideally
performed when the fetus is estimated to be
significantly anaemic, but before decompensation and
hydrops has developed. Also, the Rh group of the
fetus may be determined with accuracy, particularly in
cases where there is a poor obstetric history, the father
is heterozygous for RhD, and high levels of anti-D
may be sustained from a previous pregnancy. It is not
technically safe to undertake FBS earlier than 1820
weeks gestation and procedural losses have been
estimated at 12% in non-hydropic cases. Procedural
mortality in hydropic cases is approximately 15%

before 20 weeks, and 5% thereafter. Complicat-
ions include fetal bradycardia, haematoma formation,
fetal haemorrhage and boosting of anti-D levels, pre-
term rupture of membranes and premature labour,
and infection. The technique of fetal blood sampling
requires a lengthy learning curve and should be
performed only in specialist referral centres with
facilities to perform immediate intravascular fetal
transfusion if necessary. There is no agreement yet on
a critical haematocrit level which indicates the need
for transfusion. Transfusion has been advocated when
the fetal haematocrit is below 25% before 26 weeks
gestation, and below 30% after that.13,20
Polymerase Chain Reaction (PCR) RhD genotyping
The single most important advance in recent years in
the antenatal management of alloimmunised women
has been the introduction of molecular methods of
blood grouping from genomic DNA. Techniques such
as PCR28,7984 and sequence-specific oligonucleotide
primers (SSP) to detect fetal RHD and RHCE, KEL,
FY and JK genes82,83 are in routine use and it is now
possible to determine the fetal blood group genotype
using DNA extracted from amniotic fluid cells. Such
tests are of value in cases with history of severe HDN
where the partner is heterozygous for the correspond-
ing antigen (or unknown) and allow more accu-
rate, early prediction of the relevant blood group
genotype of the fetus. Diagnostic amniocentesis to
obtain cells of fetal origin (as for detecting fetal
genetic disorders) can be done as early as 1214
weeks. This allows early and intensive monitoring of
fetuses at risk of death in utero, with early interven-
tion when necessary. Selective termination of an
RhD-positive pregnancy may be considered.
Conversely, when the fetus is RhD negative, further
invasive techniques may be avoided.
There are certain caveats to be considered. In
principle, any test which distinguishes between the
presence or absence of the RHD gene is suitable, but
primers for the PCR reactions need to be chosen with
care to minimise false positives and false negatives,
due to gene conversions between RHD and RHCE,
point mutations and dysfunctional RhD genes.30,85
Recent studies have shown that some ethnic groups,
such as South African blacks [and their descen-
dants],26,30 Japanese,86 and Chinese have a high
incidence of partially intact but functionally inert
RHD genes, and will type as RHD positive with the
usual primers devised for Caucasians. Currently, no
single method is sufficient for diagnostic use, and two
or more PCR reactions for different exons of the
RHD gene are required, with due regard for the
ethnicity of the samples.8587 Multiplex assays capable
RhD haemolytic disease 55
of detecting all common variants have been
reported.8890 It is prudent practice to test maternal
samples in parallel, and to include paternal samples
where D variants, or non-caucasians are being
genotyped.
Summary of evaluation
Prediction of the outcome of Rh HDN is currently
based on a number of factors (Box 1).
Box 1
1. The outcome of previous pregnancies gives some
indication of expected severity, especially when the father
is homozygous for RhD.
2. Serial estimation of maternal anti-D during pregnancy
(single antibody titres are not always helpful). RhD
positive babies will be affected by HDN if the maternal
anti-D level is greater than 4 iu/ml, (as follows) although
exceptions can occur:
anti-D < 4 iu/ml HDN unlikely
anti-D 415 iu/ml moderate risk of HDN
anti-D > 15 iu/ml high risk of hydrops fetalis
3. Serial ultrasound measurements allows detection of early
ascites, and the planning of invasive techniques, such as
FBS and IUT.
4. Diagnostic amniocentesis may be undertaken in at-risk
cases for early PCR RhD genotyping.
5. Serial amniocentesis followed by optical density
measurement ( OD 450 mm) of amniotic fluid for
bilirubin content gives an indication of the degree of
haemolysis, and impending hydrops.
5. Fetal blood sampling allows direct assessment of fetal
anaemia and the Rh group of the fetus.
6. Functional assays, such as monocyte binding, CL and
ADCC have been used recently in the prediction of the
outcome of RhD HDN by measuring the potential lytic
activity of the maternal anti-D.
Combined estimates based on previous history, anti-
D levels and amniocentesis can enable accurate
prediction of the outcome in approximately 95% of
cases.1,13 Nevertheless, unexpectedly mild (or severe)
cases can still occur.1,17,18 When a high level of anti-D
justified amniocentesis but the fetus is shown to be
only mildly affected, there is a risk of further anti-D
production as a result of accidental fetal-maternal
haemorrhage. Conversely, a low level of anti-D might
allay suspicion until it becomes apparent that the fetus
is already severely affected. It is in these situations that
functional assays such as ADCC or CL may provide
additional useful information.14,42,72,73
56 Blood Reviews
PREVENTION
Prophylactic anti-D immunoglobulin and mechanisms
of action
Development of RhD alloimmunization prophylaxis
in the 1960s was based on the well-recognised phe-
nomenon of antibody-mediated immune suppression
(AMIS), in which passively administered specific anti-
body was known to prevent active immunization by
the corresponding antigen. Even today the mecha-
nism of the AMIS response is not completely under-
stood.1,13,22 Three general theories have been
proposed: (1) antigen deviation or diversion; (2) com-
petitive inhibition by antigen blocking; and (3) central
inhibition, i.e. negative modulation of the primary
immune response.
There is some evidence for the antigen
deviation/diversion theory in the observation that
RhD immunization is less common in offspring when
the father is ABO incompatible with the mother.1,13,22
It has been estimated that (in Whites) groups A and B
incompatibility between infant and mother gave 90%
and 55% protection respectively against RhD sensiti-
zation. If the fetal red cells which carry the RhD anti-
gen are ABO compatible with the mother they
circulate freely (intact) until removed by the mononu-
clear phagocytes (MPS) of the spleen, which is also a
major site of antibody production. Concurrent ABO
incompatibility can protect the mother against immu-
nization, presumably because leaked fetal red cells are
promptly coated by circulating isohaemagglutinins
(IgM) and (probably) complement, and then removed
from the circulation by the MPS, mainly in the liver
(Fig. 1). The liver is less immunoresponsive than the
spleen and therefore is less likely to stimulate antibody
production. Experimental evidence of a non-specific
suppressive effect of passive IgG antibody to human
RBC was obtained when red cells possessing two anti-
genic determinants (D+ve and K+ve) were injected
into D-ve, K-ve persons together with anti-K18,91 This
antibody suppressed the response to both K and D.
The mechanism of destruction of red cells due to anti-
K is similar to anti-D, i.e. initiates red cell destruction
predominately in the spleen. On the other hand,
studies of Cr-labelled RhD positive red cells infused
into RhD negative volunteers18 have shown that RhD
immunoglobulin increases clearance of RhD positive
cells; the antigen itself is not destroyed. Rather, the
intact antibody-coated-cells (deformed/tagged) are
removed to the spleen or lymph nodes, where an
immune response would normally be initiated. It is
possible that the splenic macrophages, which have
high FcR expression, efficiently remove the anti body
coated cells, and destroy the RhD antigen before it is
seen by the dendritic antigen-presenting cells
(APC).13,18,22
Antigen blocking cannot explain AMIS because:
(1) less than 20% of available antigen is bound by
RhD-immune globulin; and (2) in murine models
of immunization by sheep erythrocytes, F(ab)2
fragments, which bind avidly to sheep red blood cells,
do not suppress the murine immune response, whereas
whole antibody with an intact Fc region does.14,18,22
However, recent evidence has challenged this
hypothesis.92
Central inhibition may represent a plausible expla-
nation for AMIS.1,22 Fetal red cells coated with anti-D
are filtered out of the maternal circulation by the
spleen and lymph nodes, where the increase in local
concentration of complexes of anti-D bound to RhD
antigen may suppress the primary immune response
by interrupting helper T cell-mediated clonal expan-
sion of RhD-specific B cells. Furthermore, the Fc
region appears to be required for AMIS; neither F(ab)
nor F(ab)2 fragments are effective. This model is also
consistent with the observation that neither AMIS
nor prophylactic anti-D inhibits secondary immune
responses.
In animal models,13,22 the antibody and the effector
T cells remaining in an individual which has already
been immunised also prevent activation of naive B
and T cells by the same antigen. This can be shown by
passively transferring antibody or effector T cells to
naive recipients; when the recipient is then immu-
nised, naive lymphocytes do not respond to the origi-
nal antigen while responses to other antigens are
unaffected. While the precise mechanism of suppres-
sion is unclear, it is known that cross-linking of the
antigen receptor on B cells to Fc receptor II (FcRII)
on the B-cell surface inhibits the activation of naive B
cells, which may explain this kind of suppression.13,22,29
For some reason, memory B-cell responses are not
inhibited by antibody to the antigen, so for this treat-
ment to be useful the RhD negative mothers at risk
must be identified before a response has occurred.
This also shows that memory B cells can be activated
to produce antibody even when they are exposed to
pre-existing antibody, allowing secondary antibody
responses to occur in immunised individuals.
Could RhD haemolytic disease of the newborn be
eliminated by administration of prophylactic anti-D
immunoglobulin during pregnancy? It has been
shown in clinical trials that antenatal administration
of anti-D in the third trimester can suppress immuni-
sation to approximately 0.2%.1,13 It is possible that as
the D antigen is processed, free anti-D molecules
(from the Rh immune globulin) compete successfully
for the processed D antigen and prevent it from bind-
ing to the D antigen receptor of the IgM monomers of
 RhD haemolytic disease 57

appropriate B cells. While it is appreciated that the in Ante-partum anti-D immunoprophylaxis

vivo life of the passively administered anti-D
The administration of anti-D immunoglobulin to
molecules will be considerably shorter than that
RhD negative (previously non-RhD-sensitised)
expected of ABO compatible RhD positive fetal cells,
women within 72 hours of each delivery of an RhD
the presence of anti-D may result in the faster (but not
positive fetus prevents Rh immunization in the vast
immediate) sequestration and removal of the fetal
majority of cases.1,32,9397 Although immunoprophy-
cells. It is also possible that in the presence of free
laxis is highly successful in preventing RhD sensitiza-
anti-D molecules, B cells with receptors for D in their
tion, it is not yet perfect.9597 Approximately 2% of
IgM monomers, do not bind processed D antigen
women at risk develop anti-D in the last trimester or
because they interpret the presence of anti-D as indi-
within 72 hours after labour, and thus fail to benefit
cating lack of necessity to make anti-D. Perhaps most
from Rh immunoprophylaxis.19 Some of these cases
likely, the presence of free anti-D may prevent or
may result from unrecognised previous RhD positive
block the signal from the appropriate T-helper cells
abortions, but as many as 40% of all new cases are
that would normally initiate production of anti-D by
considered to be due to significant TPH during the
B cells.1,13,18
early part of the third trimester.1,18,32,65,96,97 In principle
it should be possible to prevent sensitization in such
Dose and administration of anti-D Ig28 women, and thereby increase the success of Rh
immunoprophylaxis if anti-D immunoglobulin is
The clinical situations in which anti-D immuno-
administered ante-partum in the third trimester as
globulin is recommended are detailed in Tables 3 & 4.
well as post-partum.1,12,18,32,37,94107 In North America,
The general principle is that 100 iu (20 g) of anti-D
RhD negative women receive 1500 iu (300 g) of anti-
will provide protection against 1 ml RhD positive
D between 28 and 32 weeks gestation in all pregnan-
fetal red cells (i.e. not whole blood)33,65 when given
cies.1,13,29 If the infant is RhD positive a similar dose is
intramuscularly, and this may be increased to 125 iu
given after delivery. In most European countries, a
(25 g) per ml fetal red cells to give a margin of
smaller dose (10001250 iu) is given in divided doses
safety.11 Different vial sizes are conventionally used in
at 28 and 34 weeks. In the UK,101 (Table 6) a con-
different countries for historical reasons. A dose of
trolled trial of 500 iu at 28 and 34 weeks reduced
300 g (1500 iu) is commonly used in North America,
alloimmunization to 0.2%, as efficacious as the larger
100 g (500 iu) in the UK, and 200250 g
doses used elsewhere. Theoretically, the divided doses
(10001250 iu) in Europe and elsewhere. Prescribing
should be more effective in practice than a single large
details are given in the manufacturers product leaflets,
dose since a higher level of maternal anti-D will be
but the principles are as above. In the UK the recom-
maintained at a time when larger FMH are more
mended dose of anti-D in the event of any potential
common.
TPH depends on the period of gestation (i.e. 250 iu
Antenatal prophylaxis is not universally applied, due
for an event which may precipitate a FMH at less than
to a variety of factors including the incidence of RhD
20 weeks, and 500 iu if the gestation is more than 20
negative women, the birth rate, the availability and
weeks) the larger standard dose will suffice provided
cost of anti-D, and the failure rate with standard post-
that the FMH is less than 4 ml. A Kleihauer acid elu-
natal prophylaxis;108 a cost-benefit analysis would be
tion test, or equivalent,6668 to demonstrate fetal cells
required for each country to determine the utility of
should be performed on the maternal blood (Table 3),
prophylaxis.109,110
and if the FMH is more than 4 ml a larger dose of
anti-D immunoglobulin given (i.e. 125 iu/1 ml of fetal
red cells).32 The 1500 iu dose will protect against 15 ml SAFETY OF ANTI-D IMMUNOGLOBULIN
of fetal RhD positive red cells, and this is the thresh-
old TPH for further anti-D to be given with the larger Because anti-D immunoglobulin is manufactured
dose.1,16,29 Once instituted it is important to repeat from large numbers of plasma donations from indi-
therapy at 6-week intervals in the event of intermit- viduals exposed to human red cells, considerable
tent bleeding as very low levels of passively-adminis- effort is expended in validating and accrediting both
tered anti-D may enhance antibody formation in the the red cell and the plasma donors.34 The potential
presence of an antigenic stimulus.18 If an RhD-posi- implications of transfusion transmitted infection are
tive fetus is delivered, a Kleihauer, or alternative test considerable, since two individuals are at risk the
should be performed to assess any large FMH, and mother, and her unborn child. Fortunately, the safety
then anti-D should be given in the usual way, even if record of intramuscular injections of immune globu-
residual activity from antenatal prophylactic anti-D lins produced by cold-ethanol Cohn fractionation is
can be demonstrated in the maternal serum.28,85,86 excellent since the process itself reduces viral load
58 Blood Reviews

Table 6 Chronology of antepartum prophylactic anti-D trials

1978a99 Bowman JM, Pollock JM. Rh isoimmunisation during pregnancy: antenatal prophylaxis
1978b100 Bowman JM, Pollock JM. Antenatal prophylaxis of Rh isoimmunisation: 28 week gestation service program
1983101 Tovey LAD et al. The Yorkshire antenatal anti-D immunoglobulin trial in primigravidae
1984102 Herman M, Kjellman H, Ljunggren C. Antenatal prophylaxis of Rh immunisation with 250 mcg of anti-D immunoglobulin
1987103 Bowman JM, Pollock JM. Failures of intravenous Rh immune globulin prophylaxis: an analysis of reasons for such failures
1987104 Huchet J et al. The antepartum use of anti-D immunoglobulin in rhesus negative women
1989105 Trolle B. Prenatal Rh-immune globulin prophylaxis with 300 mcg immune globulin anti-D in the 28 week of pregnancy
1989106 Thornton JG et al. Efficacy and long term effects of antenatal prophylaxis with anti-D immunoglobulin
1995107 Lee D, Rawlinson V. Multicentre trial of antepartum low dose anti-D immunoglobulin

(even prior to screening) by several logs and this prod-
uct is safe from viral transmission (reviewed in ref.
111). The few recorded incidents of transmission of
HCV by anti-D immunoglobulin relate to products
manufactured by anion exchange chromatography
when not subject to virus-inactivation.112
The position with respect to vCJD is different,
since there are no screening methods, the extent of the
infection in the donor population is unknown, and the
transmissibility by blood products is not known
(reviewed in ref. 113). For these reasons, the manufac-
ture of immune globulins from UK plasma has
stopped, and material is imported from countries not
known to have clusters of vCJD infection. Similarly,
some countries (including the USA and Canada)
do not accept blood from donors who have been
resident in the UK for a cumulative period of 6
months or more between 1980 and 1996, to minimise
the theoretical risk of transmission of vCJD.
FUTURE PROSPECTS AND RESEARCH
Adequate and timely administration of anti-D
immunoprophylaxis must remain a priority, since
administrative failures are still a significant cause of
Rh immunisation. More data from continued research
may provide firmer criteria for the timing of invasive
transplacental procedures, and to provide better
non-invasive alternatives such as Doppler ultrasound,
and other biophysical measurements of the fetal
condition.74,75
Recently, the introduction of molecular DNA tech-
nology has permitted determination of fetal blood
group status from first trimester chorionic villus sam-
pling, or from amniotic fluid cells.28,80,81 These tests are
invasive, with risks of fetal loss and boosting of anti-
body levels, and current research is focused on identi-
fying fetal genotype from maternal blood samples,
isolating fetal nucleated red cells, or plasma as a
source of fetal DNA/RNA.114 Accurate fetal genotyp-
ing on maternal plasma samples115 would allow
automation, and the possibility of restricting antena-
tal anti-D immunoglobulin to mothers carrying a
RhD positive fetus.
Anti-D produced by biotechnology may be made
available in the future by means of monoclonal anti-
body production.116 Material derived from
EpsteinBarr virus-transformed lymphocytes is being
evaluated in volunteer studies, and if successful, new
preparations of anti-D will become available to be
tested in large clinical trials.
Finally, prospects are emerging for the manufac-
ture of synthetic peptide vaccines to regulate the
immune response to the RhD antigen with the identi-
fication of T-cell epitopes on the Rh polypeptide38
In principle, active suppression of both primary
and secondary responses to RhD could be achieved in
all RhD negative females prior to puberty leading
to eventual eradication of severe RhD haemolytic
disease.
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