Vox Sanguinis (2015) 109, 99–113

© 2015 International Society of Blood Transfusion

REVIEW DOI: 10.1111/vox.12265

Haemolytic disease of the fetus and newborn

M. de Haas,1,2 F. F. Thurik,2 J.M. Koelewijn2,3 & C.E. van der Schoot2
1
Department of Immunohaematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, the Netherlands
2
Department of Experimental Immunohaematology, Sanquin Research Amsterdam and Landsteiner laboratory, Academic Medical Centre, University
of Amsterdam, Amsterdam, the Netherlands
3
Department of General Practice, University Medical Centre, Groningen, the Netherlands

Haemolytic Disease of the Fetus and Newborn (HDFN) is caused by maternal

Received: 24 April 2013,
revised 11 January 2015,
accepted 2 February 2015,
published online 20 April 2015
alloimmunization against red blood cell antigens. In severe cases, HDFN may
lead to fetal anaemia with a risk for fetal death and to severe forms of neonatal
hyperbilirubinaemia with a risk for kernicterus. Most severe cases are caused by
anti-D, despite the introduction of antental and postnatal anti-D immunoglobulin
prophylaxis. In general, red blood cell antibody screening programmes are aimed
to detect maternal alloimmunization early in pregnancy to facilitate the identifi-
cation of high-risk cases to timely start antenatal and postnatal treatment. In
this review, an overview of the clinical relevance of red cell alloantibodies in
relation to occurrence of HDFN and recent views on prevention, screening and
treatment options of HDFN are provided.
Key words: haemolytic disease of the fetus and newborn, RBC antigens and anti-
bodies, alloimmunisation in pregnancy, anti-D prophylaxis, fetal genotyping.

induce HDFN is about 1 in 500 pregnancies, albeit there is

Introduction
Haemolytic disease of the fetus and newborn (HDFN) is a
disease which – if untreated – can cause perinatal mortal-
ity and morbidity with a substantial risk for long-term
sequelae [1–5]. HDFN is caused by maternal red cell al-
loantibodies of the IgG class that are actively transported
across the placenta and destroy fetal erythroid cells carry-
ing the involved antigen. In most severe cases of HDFN,
alloimmunization against the RhD antigen (in this review,
we will further refer to the RhD antigen or phenotype as
D) is involved. Introduction of anti-D immunoglobulin (Ig)
prophylaxis has drastically decreased the prevalence of D
antibodies and of anti-D-mediated HDFN. Despite ade-
quate antenatal and postnatal anti-D Ig prophylaxis 1 to 3
in 1000 D-negative women still develop anti-D [6]; overall
in the whole pregnant population, the prevalence of anti-
D sensitized pregnancies is about 1 in 1000, as determined
in the Netherlands (data not shown). The prevalence of red
cell antibodies other than anti-D with the potency to
Correspondence: Masja de Haas, Department of Immunohaematology
Diagnostics, Sanquin Diagnostic Services, Plesmanlaan 125, 1066 CX
Amsterdam, the Netherlands
E-mail: m.dehaas@sanquin.nl
De Haas and Thurik contributed equally to the paper.
a lower risk to develop severe disease [1–4, 7]. In the past
decade, non-invasive monitoring of high-risk cases by
laboratory testing, including fetal antigen typing with
cell-free fetal DNA from maternal plasma, followed, if
necessary, by ultrasound-based techniques to judge the
presence of fetal anaemia, replaced invasive procedures
for monitoring fetal haemolysis and anaemia [8]. Since
the potency of red cell antibodies to induce HDFN differs,
it is questionable whether repeated antibody screening
and laboratory monitoring is necessary in all pregnancies
complicated by the presence of red cell alloantibodies.
Furthermore, introduction of antenatal anti-D prophylaxis
in a screening programme, in combination with the cur-
rent possibilities of non-invasive fetal RHD typing with
cell-free fetal DNA from maternal plasma, has been the
topic of several cost-benefit studies. In this review, we
provide an overview of the clinical relevance of red cell
alloantibodies in relation to development of severe HDFN
and summarize recent views on prevention, screening, fol-
low-up and treatment options of cases with HDFN.
Literature search
A literature search was conducted in Pubmed using the
following search terms (free-text terms): hemolytic dis-

99
100 M. de Haas et al.
ease of the newborn OR haemolytic disease of the new-
born OR hemolytic disease of the newborn and fetus OR
haemolytic disease of the newborn and fetus OR fetal
erythroblastosis OR maternal alloimmunisation OR preg-
nancy immunisation OR red-cell alloimmunisation OR
irregular antibodies OR maternal alloimmunization OR
pregnancy immunization OR red-cell alloimmunization
OR alloimmunised pregnant women OR alloimmunized
pregnant women. Limits were set for time (i.e. published
in the last 10 years), species (i.e. humans), type of article
(i.e. exclusion of letters and editorials) and language (i.e.
English and Dutch). Title and abstract were screened of
1293 articles independently by two of the authors (FT
and MH). In addition, the snowball method was applied
and books written by experts on this specific topic were
consulted in order to avoid overlooking relevant litera-
ture. The search query was last updated in June 2014.
Pathophysiology of haemolytic disease of the
fetus and newborn
Maternal alloimmunization can be triggered by prior
incompatible blood transfusions or by fetomaternal haem-
orrhage (FMH) in a previous or the current pregnancy.
Only antibodies of the IgG class are actively transported
across the placenta. Red cell alloantibody-mediated
destruction of fetal red cells in the fetal spleen may result
in anaemia (see Fig. 1). If the child is positive for the
involved red blood cell antigen, the maternal alloantibod-
ies will bind. Fetal anaemia will induce compensatory
erythropoiesis, nonetheless this may be insufficient. Red
cell alloantibodies directed against antigens of the Kell
system [9, 10] and also against the MNSs system (e.g.
Maternal red cell antibodies
Mother IgM IgG
placenta
Antigen-positive
Fetus fetal blood cells
Extravascular Destruction
haemolysis red cell progenitors
Anaemia with erythroblastosis
Bilirubin Extramedullary Hydrops
erythropoiesis fetalis
(ultrasound)
Enlargement
liver and spleen Heart failure
(ultrasound)
Fetal death
Newborn Icterus
Kernicterus
anti-GPMur [11]) induce destruction of fetal red cells as
well as destruction of erythroid progenitor cells, resulting
in early anaemia without erythroblastosis. Compensation
for the anaemia results in a hyperdynamic circulation in
the fetus causing cardiomegaly and eventually fetal hy-
drops, a condition consisting of oedema in the fetal skin
and serous cavities. Haemolysis of the fetal red cells
results in raised bilirubin levels. Since bilirubin passes the
placenta, excess of bilirubin is cleared via the maternal
circulation during pregnancy. After birth, the haemolytic
process continues, but the relatively immature liver of the
neonate cannot sufficiently conjugate the excess of biliru-
bin. This may result in severe hyperbilirubinaemia and,
when untreated, even in irreversible damage to the cen-
tral nervous system, a condition known as ‘kernicterus’.
This condition is characterized by bilirubin deposition in
the basal ganglia and brain stem nuclei and is correlated
with long-term morbidity that can consist of a severe
form of athetoid cerebral palsy, hearing problems and
psychomotor handicaps. When the presence or develop-
ment of red cell alloantibodies is not detected during
pregnancy, the only, nonspecific and unpredictable clini-
cal features of HDFN during pregnancy are a decrease of
fetal movements or sudden fetal death, while after birth
(early), neonatal jaundice can occur. When the presence
of maternal alloantibodies is detected by screening, the
pregnancy can be monitored by repeated laboratory tests
and – if necessary – by clinical investigations. In those
cases, prenatal anaemia can be corrected by intrauterine
blood transfusions to prevent development of hydrops
and asphyxia at birth and development of kernicterus can
be prevented by timely starting treatment with photother-
apy or, if necessary, with exchange transfusions to lower
Kell and GP antibodies
Child movements
Blood flow rate
(Doppler
ultrasound)
Fig. 1 Illustrates the pathogenesis of
haemolytic disease of the fetus and newborn in
the mother, fetus and newborn.
© 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109, 99–113
 Haemolytic disease of fetus and newborn 101

Maternal
alloimmunisation Fetus antigen- No HDFN Normal
negative*
Antibody with development
risk to cause Fetus antigen- Postnatal moderate
severe HDFN positive* hyperbilirubinaemia
and/or anaemia
No kernicterus
Postnatal severe
hyperbilirubinaemia Neonatal
Fetal death Antenatal anaemia +/– Kernicterus
severe death
anaemia
Postnatal severe
(hydrops)
anaemia
hyperbilirubinaemia +/–
Long-term
Non-invasive Laboratory monitoring: sequelae
fetal antigen typing Repeated titer/bio assay
possible for: If above critical titers:
RhD, C, c, E, K* IUT Phototherapy Exchange RBC
Clinical monitoring with: transfusion transfusion
Doppler ultrasonography

Fig. 2 Disease model maternal alloimmunization leading to haemolytic disease of the fetus and newborn and laboratory testing and eventual clinical
testing to discriminate high-risk cases. *If non-invasive fetal antigen typing is not available, pregnancies should be monitored as if the fetus is antigen
positive when the biological father is homozygous or heterozygous positive for the respective antigen. Pregnancies can be monitored as if the fetus is
antigen negative upon a negative non-invasive fetal antigen typing result, or when the biological father is typed with certainty as homozygous negative
for the respective antigen.

bilirubin levels postnatally (Fig. 2). Thus, a screening pro-
gramme aims to identify those pregnancies in which
intrauterine treatment of the fetus is needed and/or deliv-
ery should be induced to lower the risk of development
of severe haemolytic disease (Fig. 2). Without a screening
programme, treatment may be delayed. In this review,
‘very severe HDFN’ is used for cases of intrauterine death
and cases treated by intrauterine transfusions (IUTs) and/
or exchange transfusion (ET) after birth, ‘severe HDFN’
also includes cases treated with top-up transfusions and
cases in which preterm induction of labour was needed.
A ‘mild course’ is defined as cases without a need of pre-
term induction of labour and in whom treatment with
phototherapy suffices.
Clinical relevance of different red cell
alloantibody specificities
The risk of developing severe HDFN depends on several
factors, including Ig class, specificity of the red cell alloan-
tibodies and level of expression of the involved blood
group antigen on the fetal red cells and other tissues. In
Table 1, an overview is given of red cell antibody specifici-
ties in regard to their risk to induce HDFN (Table 1) [1–4, 7,
12]. Anti-D is correlated with the highest risk of fetal mor-
tality and morbidity [4, 13]. The risk to develop severe
HDFN is much lower in pregnancies complicated by other
maternal red cell alloantibodies, with the exception of
anti-K (Table 1). In the Netherlands, we performed a pro-
spective index-cohort study based on 298 000 screened
pregnant women to study the development of HDFN,
caused by non-D antibodies. K antibodies caused severe
HDFN in 26% of the pregnancies at risk (antigen-positive
fetus), c antibodies in 10%, E antibodies in 2% and anti-
bodies directed against another Rh antigen in 5% of the
pregnancies [7]. Phototherapy was necessary in 42% of the
pregnancies complicated by K antibodies (antigen-positive
fetus), in 33% of the pregnancies complicated by c anti-
bodies, in 19% when E antibodies were present and in 20%
of the pregnancies complicated by antibodies directed
against other antigens of the Rh blood group system [7].
This is comparable to other published single-centre studies
[14, 15]. Antibodies against Duffy antigens (e.g. Fya) have
been reported to induce severe HDFN that requires intra-
uterine blood transfusion. This, however, rarely occurs [7,
12, 16]. In our study, we observed no cases of severe HDFN
in 42 antigen-positive fetuses but phototherapy was
needed in 14% of the cases, compared to 4% in controls
(no antibodies present or antigen-negative fetus) [7]. In the
literature, a wide variety of red cell alloantibodies is
reported as case reports to cause severe HDFN. In popula-
tion studies, however, these antibodies are seldom reported
as cause of severe HDFN (Table 2). However, this may
depend on the ethnic background of the population stud-
ied. For example, in Asian population anti-GPMur may
cause severe HDFN [11] and recently some case reports

© 2015 International Society of Blood Transfusion

Vox Sanguinis (2015) 109, 99–113
102 M. de Haas et al.

Table 1 Correlation of red cell alloantibody specificities with occurrence Given the high rate of ABO incompatibility between the
of haemolytic disease of the fetus and newborn [1–4, 7, 12] mother and her fetus, the prevalence of clinically signifi-
cant haemolysis in neonates because of anti-A or anti-B is
Risk to develop HDFN in antigen-positive children
relatively low. And, if occurring, it shows a mild clinical
and clinical course of disease
course. A and B antigens are expressed by placental tissue
ABO Lowb risk for disease, in general milda, and may bind maternal alloantibodies to some extent.
incidentally severea Furthermore, the expression of A and B blood group anti-
Rh gens on fetal red cells is not fully developed. Anti-A and
D Highb risk for disease, often (very) severe, anti-B are predominantly of the IgM class, whereas anti-
otherwise mild A,B in group O women is often of IgG class; during preg-
c High risk for disease, (very) severe or mild
nancy, the formation of anti-A, anti-B and anti-A,B titres
E Mediumb risk for disease, sometimes severe,
of the IgG class can be strongly increased. There is a strik-
but mostly mild
Other Rh Medium risk for disease, incidentally severe,
ing, and yet not understood, difference in the incidence of
antigens but mostly mild ABO-mediated HDFN between populations. The incidence
Kell is around 0
3–0
8% in the Caucasian population, versus 3–
K High risk for disease, (very) severe or mild 5% in Black or Asian populations, also with a more severe
Other Kell Medium risk mild to severe disease clinical course [18–21].
antigens Expression of some blood group antigens is very low
Duffy on fetal red cells (e.g. Lub, Yta), and therefore, significant
Fya/Fyb Medium risk for disease, mostly mild haemolysis is not induced (Table 1). Finally, in some
Kidd cases, for example with Lutheran antibodies, another rea-
Jka/Jkb Low riskb for disease, only mild
son for absence of HDFN could be the presence of antigen
MNS
on placental cells, preventing transfer of antibody to the
M, N, S, s Low riskb for disease, mostly mild disease,
very rarely severe
fetus.
Other antigens Low riskb for disease, mostly mild disease,
very rarely severe Laboratory monitoring of alloimmunized
I, Le, P1, Lu, Yt No risk, because of very low expression of these
cases
antigens by fetal cells
Other antigen Very low risk, very rarely severe disease can develop Since the last decade, non-invasive fetal blood group typ-
systems ing with cell-free fetal DNA from maternal plasma is a
a
clinical reality and is offered by many laboratories for
Very severe disease: Need for intrauterine treatment and/or exchange
pregnancies complicated by antibodies that can cause
transfusion after birth. Severe disease: Need for intrauterine treatment
and/or preterm induction of labour and/or blood transfusions after birth.
severe HDFN (Fig. 2) [22–28]. Because cell-free fetal DNA
Mild disease: Only treatment with phototherapy is needed. represents only a fraction of the total cell-free DNA in
b
High risk: >50%, medium risk >10–50%, low risk 1–10%, very low/inci- the maternal plasma and is present in extremely low con-
dentally <1%. centrations, highly efficient DNA extraction and sensitive
detection technology, currently with quantitative PCR, is
required. In the literature, different assays have been
report on the occurrence of anti-Ge3 in Hispanic families described for reliable genotyping of D, C, c, E and K in
responsible for HDFN with severe late-onset haemolysis the first trimester of pregnancy and thus in time before
and hyperbilirubinaemia [17]. treatment should start [22–25, 27, 28]. In these assays,
Anti-M is very often found in pregnant women as a the fetal antigen-negative phenotype is not detected
so-called naturally occurring antibody (formed without a directly, but concluded based on a negative result of the
previous immunization event) of the IgM class. Anti-M of antigen-specific PCR. Therefore, confirming the presence
the IgG class can, however, cause severe HDFN, like other of fetal DNA with other paternally inherited DNA
antibodies directed against antigens of the MNS blood sequences (e.g. SRY) or a universal fetal DNA marker (e.g.
group system [11]. We observed in 69 women with methylated RASSF1a) is used by some laboratories to
anti-M of the IgM class early in pregnancy that a class report conclusive results in one single assay [22, 29]. Or
switch to IgG had not occurred as analysed in week 24, the assay is repeated later in pregnancy to assure the
30 and 36 of pregnancy [7]. Therefore, if anti-M of the presence of sufficient amounts of fetal DNA. One can
IgM class is detected early in pregnancy, it is not likely conclude that non-invasive fetal genotyping can reliably
that this antibody will switch to IgG and induce severe be used to target laboratory and clinical monitoring to
HDFN; therefore, repeated testing is not necessary. cases at risk for the development of severe HDFN.

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Vox Sanguinis (2015) 109, 99–113
 Table 2 Studies on the prevalence of maternal alloimmunization for other specificities than anti-D and subsequent occurrence of haemolytic disease of the fetus and newborn

Prevalence of non-D alloimmunization and subsequent

very severe HDFNa

Detected upon 1st Detected after 1st

screening screeningb
Screening Antibodies causing
Study Period Study Population/setting moment(s) Immunization HDFN Immunization HDFN severe HDFN Remarks

Hardy, Wales, 1981 [75] 1948–1978 Retrospective D+ women, regional Unclear 0
19% 0
003% NRc Denominator

Vox Sanguinis (2015) 109, 99–113

733/380 790 11/380 790 women instead
of pregnancies
Bowell, UK, 1986 [76] 1983–1984 Retrospective All pregnant women, Intake 0
45% NR 0
29% NR Denominator:
regional week 28 315/70 000 201/70 000 estimation
after week 28

© 2015 International Society of Blood Transfusion

Belfrage, Sweden, 1992 [77] 1983–1989 Retrospective Referral university ‘Initially’ ND 0
01% Unclear
hospital 17/147 068 (E, c, K, etc.
Cw+Fya)
Heddle, Canada, 1993 [78] 1980–1990 Retrospective Community hospital + 1st trimester 0
33% NR 0
24% NR
1987–1989 secondary care hospital 2nd screening 58/17 568 42/17 510
moment
unclear
Gottvall, Sweden, 1993 [79] 1983–1989 Prospective All pregnant women, Week 25 0
24% 0
01% Moment of NR Only pregnancies
regional Week 35 188/78 300 8/78 300 detection unclear with an antigen-
Filbey, Sweden, 1995 [80] 1980–1991 Retrospective All pregnant women, Week 10 0
15% 0
005% Moment of c, E, cE, K, Fya positive partner
regional Week 35 171/111 939 6/111 939 detection unclear were included
Wong, Hong Kong, 1997 [81] 1995–1996 Prospective Pregnant women, Intake 0
25% 0 ND
regional 5/1997 0/1997
Howard, UK, 1998 [82] 1993–1994 Retrospective All women 7 maternity Intake 0
65% 0
018% c, ce, E
units 144/22 264 4/22 264
Rothenberg, USA, 1999 [83] 1988–1997 Retrospective D+ women, tertiary 1st trimester 0
37% 0
011% 0
06% 0 Fya
care referral 3rd trimester 35/9348 1/9348 6/9313 0/9313
centre + public
hospital
Andersen, Denmark, 1996 Prospective D+ women, regional 1st trimester 1
1% NR 0
43% 0 1st trimester:
2002 [84] 3rd trimester 34/3046 13/3012 0/3012 including Lewis
antibodies
Lee, Hong Kong, 2003 [85] 1997–2001 Retrospective National screening Intake 0
27% 0
005% NR NR E
laboratory, ethnic 57/21 327 1/21 327
Chinese women
Haemolytic disease of fetus and newborn 103
 Table 2 (Continued)

Prevalence of non-D alloimmunization and subsequent

very severe HDFNa

Detected upon 1st Detected after 1st

screening screeningb
Screening Antibodies causing
104 M. de Haas et al.

Study Period Study Population/setting moment(s) Immunization HDFN Immunization HDFN severe HDFN Remarks

Jovanovic, Yugoslavia, 1995–2000 Retrospective National screening Unclear 0
58% ND Unclear whether
2003 [86] laboratory 124/21 370 number of ab’s
or number of
pregnancies is
reported; unclear
whether non-D
antibodies are
combined with
D antibodies
Lurie, Israel, 2003 [88] 1999–2002 Retrospective Women’s Health 1st trimester 0
16% 0
Centre 1/631 0/631
Ameen, Kuwait, 2005 [87] 1992–2001 Retrospective National Blood Bank Unclear 6
2% ND
182/2932
Adeniji, UK, 2007 [89] 2002–2004 Retrospective D+ women, regional 1st trimester ND 0
24% 0
>week 28 34/14 143 0/14 143
Gottvall, Sweden, 2008 [90] 1992–2005 Retrospective All pregnant women, Week 10 0
25% 0
01% Moment of c, E, c+E, Fya Only cases with
regional Week 35 196/78 145 8/78 145 detection antigen-positive
unclear father included
Koelewijn, Netherlands, 2002–2004 Prospective All pregnant women, Intake 0
33% 0
007% 0
002% c, E, K, C+Jka, Mur Denominators
2008 [7] national 1002/305 700 21/305 700 8/393 500 estimated
Dajak, Crotia, 2011 [12] 1993–2008 Retrospective All pregnant women, Intake 0
16% 0
04% 0
01% c, C, E, Rh17, Hospital-based
regional 143/84 000 30/84 000 11/84 000 Cw, K, Fya, S study. Inclusion
from population
with putative
higher risk for
alloimmunization

a
Very severe HDFN: need for intrauterine transfusion and/or exchange transfusion, perinatal death or fetal hydrops because of maternal RBC antibodies.
b
Incidence of antibodies, newly detected later in pregnancy, after a negative first trimester screening.
c
NR Not-reported.

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© 2015 International Society of Blood Transfusion

In many countries, cases with an increased risk for
severe HDFN (further: ‘high-risk cases’) are discriminated
based on red cell alloantibodies titres, as determined by
the indirect antiglobulin test performed in saline (Fig. 2)
[3, 30–32]. The blood sample is retested at regular inter-
vals until a threshold or cut-off value (‘critical titre’) is
exceeded, which implies an increased risk for severe
HDFN and referral to specialized care is indicated (see
‘clinical assessment of high-risk cases’). Efficient prese-
lection of high-risk cases reduces the number of admis-
sions for specialized clinical diagnostics. In some
countries, only pregnant women with anti-D, anti-c or
anti-K are regularly retested and pregnant women with
other types of red cell antibodies are only tested again
in week 28 to detect an infrequent high-risk case with
high antibody titres in this group. Moise concludes that
in most laboratories for anti-D, a critical titre between 8
and 32 is used [3]. In the UK, a threshold value of 15
international units anti-D/ml has been recommended as
critical titre [30]. In general, for non-D alloantibodies,
except for anti-K, a cut-off of 32 is used [3, 32]. Hack-
ney et al. [14] reported for anti-c induced HDFN,
defined as need for intrauterine transfusion or haemo-
globin levels at birth of <11 g/dl, that a titre >32 pre-
dicted all severe cases. Similarly, Joy et al. concluded
for anti-E and McKenna et al. for anti-K, a cut-off of
32 [15, 33]. However, Leggat et al. and Van Wamelen
et al. reported that severe HDFN can already occur at
very low anti-K antibody titres [34, 35]. Therefore, both
the British and American guidelines state that anti-K ti-
tres <32 may already be relevant; a titre of 8 has been
proposed by Moise [3, 30–32].
In the past, several laboratories performed biological
assays for the prediction of the antibody activity [36, 37].
The antibody-dependent cell-mediated cytotoxicity
(ADCC), a monocyte-driven biological assay, is used in
the Netherlands to assess the risk of developing severe
disease. Oepkes et al. [38] showed that the use of this
assay improves the diagnostic accuracy of selection of
high-risk cases, because of the higher specificity of the
test than antibody titres alone. The higher positive predic-
tive value of the ADCC was also confirmed for non-D
antibodies [39]. From these studies, it was concluded that
a non-D alloantibody titre of 16 or higher or an ADCC
test result of 30% or higher indicate high-risk cases for
which clinical monitoring is recommended [7]. In case of
anti-D antibodies, the advice to the clinician is mainly
based on the obtained ADCC test value [38].
Clinical assessment of high-risk cases
The standard test to assess the severity of fetal anaemia
used to be the quantification of bilirubin levels in
© 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109, 99–113
Haemolytic disease of fetus and newborn 105
amniotic fluid upon amniocentesis. Amniocentesis is an
invasive procedure with complications including
spontaneous miscarriage and amniotic fluid leakage
[40]. Moreover, each invasive procedure carries an
increased risk for FMH, which can trigger an antibody
response [41].
Advances in Doppler ultrasonography enabled non-
invasive detection of early fetal anaemia. Mari et al. [42]
reported that peak systolic velocity measurement of the
middle cerebral artery (MCA-PSV) can be used for
detection of fetal anaemia. A velocity of more than 1
5
multiples of the median (MoM) for gestational age pre-
dicted all 70 cases of moderate to severe anaemia correct
with a sensitivity of 100% (95% CI, 86–100). The false-
positive rate was 12%.
In a multi-centre international study, the diagnostic
accuracy of MCA-PSV-Doppler ultrasonography for pre-
diction of severe fetal anaemia in alloimmunized cases
showed a sensitivity of 88% (74 cases), a specificity of
82% and an accuracy of 85% (95% CI, 78–93%, 73–89%
and 79–90%, respectively). In comparison with amniocen-
tesis, Doppler ultrasonography was more accurate by 9
percentage points (95% CI, 1
1–15
9) [43]. Therefore,
Doppler ultrasonography is nowadays used predominantly
to identify cases with fetal anaemia among high-risk
pregnancies [44].
Antenatal treatment
In severe fetal anaemia, it is essential to treat HDFN ante-
natally to prevent hydrops fetalis and fetal death. IUT
should be considered when clinical assessment indicates
fetal anaemia, for example MCA-PSV >1
5 MoM [5]. Most
studies reporting on series of IUT-treated cases report
perinatal survival rates around 90% [45, 46]. Furthermore,
a long-term follow-up study on neurodevelopmental out-
come shows a normal development in more than 95% of
291 included children [47].
In a cohort of 537 IUT-treated fetuses from the refer-
ence centre for fetal treatment in the Netherlands, the
vast majority of IUTs were given because of alloimmuni-
zation against D (81%), compared to K in 13%, c in 5%
and antibodies against other antigens in 2% (D. Oepkes,
Leiden University Medical Centre, personal communica-
tion).
Intrauterine transfusion may lead to further alloimmu-
nization in 25% of the mothers, despite preventive
matching for Rh and K antigens [41].
Additional treatment of alloimmunized women with
intravenous immunoglobulin (IVIg) with or without plas-
mapheresis has only been described in three small case
series, but the efficacy of these experimental treatments
has not yet been proven [48–50].
106 M. de Haas et al.
Postnatal treatment
Phototherapy
Phototherapy has been proven to be effective by drasti-
cally decreasing the necessity of exchange transfusion in
the treatment of hyperbilirubinemia [51]. Guidelines for
treatment of neonatal icterus have been published by the
American Academy of Pediatrics (AAP) in 2004 and state
that factors other than bilirubin level should be taken into
consideration in clinical assessment, such as gestational
age, birth weight and cause of hyperbilirubinemia [52].
In children treated with IUT, a shortened period of
phototherapy was needed compared to neonates suffering
from HDFN who did not receive IUT (3
8 and 5
1 days,
respectively, P-value 0
01) [53]. Rath et al. [54] found
that less phototherapy was also required in neonates
affected by anti-K-mediated haemolytic disease. Since
anti-K also destroys erythroid progenitor cells, in these
cases anaemia manifests earlier and is more pronounced
relative to bilirubin levels.
Exchange and top-up transfusions
Exchange transfusions (ET) have the advantage that both
bilirubin and maternal alloantibodies are removed from
the circulation, and anaemia is corrected [55]. Up to 24%
of neonates treated with ET suffer from a complication,
such as electrolyte imbalance, cardiac arrhythmias, embo-
lism, necrotizing enterocolitis, infection and sepsis. There
is a risk of almost 0
3% of ET-associated mortality in
term neonates [56, 57]. The guidelines published by the
AAP do not recommend early ET, such as within the first
12 h of birth, and bilirubin thresholds at which time ET
should be started differ depending on the gestational age
at which the baby is born [52]. Rath et al. [58] showed
that more restrictive ET criteria reduced the number of
exchange transfusions but resulted in an increase of the
number of top-up transfusions.
In contrast to the effect of IUT on phototherapy, De Boer
et al. [53] found no difference in the number of exchange
transfusions required in neonates treated with or without
IUT. Since maternal alloantibodies can be active for
months in the neonate [59] and recovery of erythropoiesis
may be delayed as an effect of transfusions, additional
transfusions may be necessary in the first months of life
[53]. De Boer et al. [53] found that neonates treated with
IUT and suffering from anti-D-mediated HDFN needed
more top-up transfusions compared to neonates who did
not receive an IUT (77% and 26
5%, respectively, with a P-
value < 0
01). Compared to neonates suffering from anti-
D-mediated HDFN, neonates suffering from anti-K-medi-
ated HDFN require less ET and less phototherapy, but a
similar number of top-up transfusions. In particular, neo-
nates suffering from severe HDFN due to anti-c also
require top-up transfusions in the first months of life, with
a positive correlation between the titre of anti-c measured
in cord blood and the need for postnatal transfusion [60].
Intravenous immunoglobulin
In the literature, neonatal treatment with intravenous
immunoglobulin (IVIg) has been discussed as an alterna-
tive therapy for exchange transfusion. Initially, studies
were published that favoured this approach [61–63], but
two later placebo-controlled randomized control trials did
not show a beneficial effect of IVIg treatment [64, 65].
And recently Santos et al. [66] showed that IVIg does not
prevent the need for exchange transfusion in neonates
with anti-D-mediated HDFN. Hence, efficacy of IVIg
treatment has not been proven.
Erythropoietin
Anaemia presented in the first weeks of life (‘late anae-
mia’) occurs in 71–83% of neonates with HDFN, and it is
characterized by depressed erythropoiesis [57]. Although
administration of EPO may prevent late anaemia and
reduce the need for top-up transfusion of red blood cells,
there is no sufficient evidence to support this [57, 59].
Screening programmes
In most countries, pregnant women are typed for ABO and
D early in pregnancy and screened for the presence of red
cell antibodies [1, 4, 31, 44, 67–69]. Nowadays, screening
tests are very sensitive early in pregnancy. The level of
antibodies developed earlier upon incompatible pregnan-
cies or transfusions, however, may be too low to detect.
During pregnancy, small FMHs may initiate a secondary
antibody response leading to sufficient high antibody levels
in the fetus to induce disease. To ascertain that all clinically
relevant alloimmunizations are detected, screening pro-
grammes in many western countries comprise two or even
three screening moments: in the first trimester, the second
and/or third trimester. A repeated screen is meant to detect
primary responses which may occur during pregnancy and
secondary responses in women already previously immu-
nized. For example, Dajak and co-workers showed that
severe anti-D-mediated HDFN was not detected in 12% (8/
68) of the cases upon first trimester screening, but only
upon screening at week 28 or 34 of pregnancy (in women
who did not receive anti-D Ig prophylaxis) [12]. We
observed in a study in D-negative women who delivered
previously a D-positive child that administration of both
antenatal and postnatal anti-D prophylaxis did not reduce
© 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109, 99–113

the incidence of ‘late’ development of anti-D as detected at
week 30 of pregnancy [6]. But in the group of women who
received both antenatal and postnatal prophylaxis and in
whom immunization was detected in week 30 of preg-
nancy, the incidence of severe HDFN was reduced to 3% (1
out of 29) versus 28% (6 out of 21
5) if only postnatal anti-
D Ig prophylaxis was given in the first pregnancy [6]. The
working mechanism of anti-D Ig prophylaxis is not yet elu-
cidated, and an immunomodulatory effect of anti-D Ig pro-
phylaxis has been proposed [70–72].
Although antenatal screening for the presence of red
cell antibodies is established practice in several countries,
there is discussion about necessity and cost-benefit ratio
of the screening itself and of repetition of red cell anti-
body screening in pregnancies from D-positive women. A
positive red cell antibody screening test does not only
identify red cell antibodies correlated with an increased
risk of development of HDFN, but also a broad range of
irrelevant antibodies (e.g. cold agglutinins) and antibodies
which only rarely cause HDFN (Table 1). Antibody identi-
fication and subsequent laboratory monitoring and possi-
bly also clinical monitoring result in substantial costs for
the healthcare system. In order to evaluate (novel) screen-
ing programmes, the WHO criteria designed by Wilson
and J€ungner can be used [73]. One of the criteria is that
the test should be acceptable to the population. In the
Netherlands, a survey among 233 screened women
showed a positive attitude towards the red cell antibody
screening programme, with a positive balance between
perceived utility and burden of the screening programme,
independent of the screening test results [74]. Table 2 lists
a number of population studies looking into the benefits
of a first trimester red cell antibody screening test in D-
positive women [7, 12, 75–90]. From this overview, it is
apparent that the prevalence of maternal alloimmuniza-
tion shows a wide variation from 0
15% to as high as
6
2% and that of severe HDFN from 0 to 18/100
000
births (Table 2). This variation can be explained by the
heterogeneity of the screening protocols, by the absence
of an unequivocal definition of ‘the presence of clinically
relevant alloantibodies’ and, most importantly, by the use
of preselected high-risk study populations with an
unknown relation to the general population of pregnant
women. Population data are available from three regional
studies in Sweden, one in Croatia and our studies in the
Netherlands [7, 12, 80, 90]. These latter studies show a
prevalence of 0
15–0
25% of alloimmunized pregnancies
with a risk for HDFN (partner antigen positive) caused by
a red cell antibody other than anti-D (non-D mediated).
To assess the validity of repeating the screening in preg-
nancies for D-positive women, a study should be of a suffi-
cient sample size to investigate the effectiveness of a
screening programme. In the studies listed in Table 2, both
© 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109, 99–113
Haemolytic disease of fetus and newborn 107
the observed incidence of early and that of late detected
non-D antibodies are low (ranging from 0
06% to 0
4%)
and because the presence of these red cell antibodies is cor-
related with a lower risk for development of severe HDFN,
most studies lack sufficient power to determine the clinical
relevance of (repeated) red cell alloantibody screening. In a
retrospective two-year nationwide study, representing
about 393 500 screened pregnant women, we identified
seven cases of non-D-mediated severe HDFN with a nega-
tive screening result early in pregnancy, anti-c: n = 3,
anti-c and anti-E: n = 2 and anti-E: n = 2. It was calcu-
lated that a repeated screening test in c-negative (majority
CCDee) pregnant women late in pregnancy would increase
the sensitivity of the screening programme with 22%. Simi-
larly, Dajak et al. [12] showed that 27
5% (11/40) of cases
with severe HDFN were undetected by a first trimester
screening and detected at week 28 of pregnancy. It con-
cerned cases with, respectively, anti-c (n = 7, 6 received an
ET, one only a top-up transfusion), anti-E (n = 1, ET) anti-
C (n = 2, only top-up transfusion) and one case of anti-
Rh17 (perinatal death). The involved pregnant woman in
the latter case and in one of the cases with anti-C received
an incompatible blood transfusion during pregnancy. Both
Koelewijn and Dajak observed that parity is a strong risk
factor for the presence of clinically relevant antibodies dur-
ing a subsequent pregnancy [12, 91, 92].
Thus to increase the cost-benefit ratio of repeated
screening in pregnancy, one might consider to limit this
to all D-negative women and to multiparous, or earlier
transfused, D-positive women (see Fig. 3).
Prevention of alloimmunization in pregnant
women due to blood transfusion
We demonstrated that RBC transfusion is the most impor-
tant risk factor for non-D immunization, even after
adjustment for parity [92]. Over 50% of women with clin-
ically relevant red blood antibodies had a history of prior
blood transfusion. This percentage was even higher in
women with anti-K antibodies (>80%).
In blood transfusion guidelines, it is already included
for a long time that premenopausal D-negative women
should receive D-negative blood. Since anti-c, anti-K and
albeit to a lesser extent anti-E, can cause severe HDFN,
one should consider to transfuse girls and premenopausal
women with RhcDE and K-compatible blood.
Anti-D prophylaxis
Routine postnatal administration of anti-D – introduced in
the 1960s – has been shown to substantially decrease
maternal alloimmunization detected in a subsequent preg-
nancy from 15% to 1
6% [93]. In the more recent years,
108 M. de Haas et al.

Actions Actions
OBSTRETIC CARE GIVER/ LABORATORY
PAEDIATRICIAN

D typing and red cell antibody screening

Blood sampling Consider c and E typing (1)
Booking visit If D-negative mother: consider fetal D typing early in pregnancy to guide antenatal anti-D prophylaxis(5)

Anti-D Ig if indicated,
to women without Red cell antibody Red cell antibody screen positive
anti-D (consider fetal screen negative antibody specificity
RHD typing (5))

No clinical relevant Anti-D, anti-K or other Other

antibodies anti-Rh antibodies specificities

Blood sampling Antigen typing father

father Consider non-invasive fetal antigen typing D, c, C, E, K
(2)

Fetus or father Fetus or father

antigen negative antigen positive

Laboratory monitoring
Consider differential policy (3)
Consider selected second screen in D-positive -Anti-Rh or anti-K repeated testing
D- women for example c-negative women or risk until critical thresholds are met
Blood sampling -Other specificities: one test in last
Week 27–30 negative factor based (4)
trimester to judge risk for HDFN

Consider (5): Antibody.screening

Above critical thresholds
Fetal RHD typing Antibody.specifity
Clinical monitoring by
Doppler ultrasonography

Fetus D-positive Fetus D-negative Anti-D, -K, - other

–Rh antibodies

Between
week 28–34,
consider Anti-D Ig
week 30 Consider single dose (6)

If mother is Consider: no cord D typing, but use RHD typing child

D-negative: fetal RHD typing result (7)
Cord blood
at birth
Anti-D Ig D-positive D-negative

Check HDFN
- Clinical
- Laboratory work-up,
PAEDIATRICIAN only if necessary

- Follow-up of children
with severe HDFN

Fig. 3 Red cell antibody screening and red cell antigen typing for timely detection of haemolytic disease of the fetus and newborn and to prevent D
alloimmunization. White background: actions taken by health care professionals (gynaecologist, midwife, paediatrician). Gray background: tests
performed by laboratories. Box: action or test; box with rounded angles: test result followed by action or test.

© 2015 International Society of Blood Transfusion

Vox Sanguinis (2015) 109, 99–113

routine antenatal and postnatal anti-D prophylaxis, in
which antenatal anti-D is administered between week 28
and 34 of gestation, has become the standard care for
D-negative women in many developed countries [94]. Koe-
lewijn et al. [6] showed in 2008 in a nationwide observa-
tional study, involving over 41 000 women, that antenatal
administration of anti-D Ig prophylaxis halves the risk of
anti-D immunization, detected early in the next pregnancy
(RR 0
46; 95% CI 0
09–0
84), and of subsequent HDFN (RR
0
45; 95% CI 0–1
08). Crowther et al. [95] reported in a
systematic review, including two randomized controlled
trials (RCT), involving over 4500 women, that the risk of
immunization during the current pregnancy (RR 0
42; 95%
CI 0
15–1
17) until 12 months after birth (RR 0
30; 95% CI
0
10–1
62) was not significantly reduced by antenatal anti-
D Ig prophylaxis in the current pregnancy, but that the risk
to have a positive Kleihauer test both during pregnancy
(RR 0
60; 95% CI 0
41–0
88) and within 12 h after birth
(RR 0
60; 95% CI 0
46–0
79) was significantly lower after
antenatal administration of anti-D Ig prophylaxis. Also
Turner et al. [96] demonstrated the effect of antenatal anti-
D Ig prophylaxis, based on a meta-analysis of these two
aforementioned RCTs and eight observational studies,
adjusted for differences in study design and quality.
Despite appropriate prophylaxis D alloimmunization still
occurs [91]. The dosages and timing of antenatal anti-D
prophylaxis differ between countries. In theory, a higher
dosage or two dosages given at different time points in
pregnancy will lead to higher anti-D levels in pregnant
woman. However, compliance to a prevention programme
with one single injection of anti-D prophylaxis may be
higher. In the Netherlands, one injection of 1000 IU
(250 lg) of anti-D is administered in week 30 of preg-
nancy. We analysed in D-alloimmunized women, who were
pregnant with their second child, potential risk factors for
immunization despite correctly administration of antenatal
and postnatal anti-D prophylaxis in the first pregnancy.
Post-term pregnancy (≥42 weeks), non-spontaneous deliv-
ery, a blood transfusion around delivery, which may be an
indicator of a prolonged third stage of labour, and maternal
age (OR 0
89/year) at first delivery were all found to be
independent risk factors of D immunization. In post-term
pregnancy, levels of anti-D prophylaxis may drop below
protective levels, other risk factors point to (unexpected)
large FMH or (higher) activity of the immune system
(maternal age) [91].
Several large-scale feasibility studies showed that non-
invasive fetal RHD typing can be used to restrict antenatal
anti-D administration to D-negative women carrying a D-
positive child (60%) [97–100]. In Denmark and the Nether-
lands, fetal RHD typing to target antenatal anti-D prophy-
laxis has already been introduced in 2010 and 2011,
respectively [97, 101]. Clausen et al. [97] showed that only
© 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109, 99–113
Haemolytic disease of fetus and newborn 109
in 2/2312 (0
09%) of false-negative test results were
obtained upon fetal RHD typing at 26 weeks of gestation
and that in 1
7% of cases, anti-D was unnecessarily admin-
istered. The latter was also calculated by Finning et al.
[98], who calculated that in about 2
1% of cases, anti-D
prophylaxis would be given unnecessarily because of either
false-positive (0
07%; 14/1869) or inconclusive (0
14%;
25/1869) test results.
Calculations on cost efficiency of the combined intro-
duction of fetal RHD genotyping and antenatal anti-D
prophylaxis have been published [102–104]. In Denmark
and the Netherlands, fetal RHD genotyping is now rou-
tinely performed to target anti-D Ig prophylaxis. Cost-
benefit ratios are influenced by costs of tests, which are
largely influenced by economy of scale, and costs of
anti-D Ig prophylaxis, the latter differs between countries.
The earlier in pregnancy fetal RHD genotyping can be
performed reliably, the more one will benefit, since
administration of anti-D given upon possible sensitizing
events can also be targeted [105–107]. In the Netherlands,
the reliability of the fetal RHD genotyping assay has been
regarded to be sufficient to discontinue D cord blood typ-
ing. Since January 2013, both antenatal and postnatal
anti-D Ig administration policy is based on the result of
the fetal RHD genotyping assay. Only in case of twin
pregnancies and in case of an inconclusive fetal typing
result, cord blood D typing will be performed.
Summary
Figure 3 summarizes the considerations one nowadays
can make in the design of a screening programme. It has
been shown that an antenatal screening test for red blood
cell antibodies results in timely detection of fetuses at risk
for a disease that can be fatal or can result in life-long
sequelae. Anti-D, anti-c, anti-K and anti-E, the latter to a
lesser extent, are responsible for the majority of cases
with severe HDFN that need treatment with intrauterine
transfusions or exchange transfusions after birth. Other
antibody specificities may be related to an increased risk
of neonatal icterus demanding phototherapy (e.g. anti-
Fya) but cause very rarely life threatening or disabling
disease. Therefore, intensive laboratory monitoring should
be performed for red cell antibodies directed against Rh
antigens or antigens of the Kell blood group system and
can be less frequent in case of other antibody specifici-
ties. Laboratory monitoring should be aimed to detect
high-risk cases, that is cases at high risk for severe HDFN,
for example those with high antibody titres or relevant
biological activity as measured in a functional assay such
as the ADCC (Fig. 3, consideration 2 and 3). Since red cell
antibodies causing severe HDFN may not be detected
early in pregnancy, a repeated screening test, early in the
110 M. de Haas et al.

third trimester of pregnancy, for example around week
30, should be aimed to detect anti-D, anti-c and anti-E.
We produced some evidence that a repeated screening test
would suffice solely in those women with an increased
risk to develop maternal alloantibodies, such as D- and c-
negative pregnant women (Fig. 3, consideration 1 and 4).
The last decades, invasive procedures to monitor
fetuses at risk have been abandoned. Fetal blood group
typing can be performed with cell-free fetal DNA present
in maternal plasma during pregnancy (Fig. 3, consider-
ation 2). Fetal anaemia can be assessed by Doppler ultra-
sound to identify fetuses in need of intrauterine
transfusions. Finally, intensive phototherapy is a safe
intervention to sufficiently lower bilirubin levels in order
to prevent kernicterus. Affected children may need one or
more transfusions in the first months of life, since the
maternal antibodies stay active during this period. In this
decade, cost-benefit calculations regarding scenario’s to
introduce non-invasive fetal RHD typing to target admin-
istration of anti-D Ig in pregnancy and to make cord
blood D typing presumably obsolete will be made in
many centres (Fig. 3, consideration 5, 6 and 7).

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