Pathology (February 2015) 47(2), pp.
151–155
HAEMATOLOGY
Prevalence of maternal red cell alloimmunisation: a population study
from Queensland, Australia
MANIKA PAL1 AND BRONWYN WILLIAMS2
1Pathology Queensland, Princess Alexandra Hospital, and 2Pathology Queensland, Royal Brisbane and
Women’s Hospital, Qld, Australia
Summary
The aim of this study was to determine the current prevalence
of red cell antigen alloimmunisation in Australia. Blood group
(ABO and RhD) and red cell antibody screen results of
pregnant women who presented at public hospitals in
Queensland between the period of January 2011 and June
2013 were evaluated retrospectively. Antibody prevalence in
pregnancy was compared to other published studies. A total
of 482 positive antibody screens from 66,354 samples
(0.73%) were identified. The prevalence of antibodies was:
anti-E 27.6%; anti-D 10.4%; anti-Kell 9.5%; anti-c 8.7%; anti-
Duffy 3.1%, including Fya and Fyb; anti-MNS 7.9%, including
M, N, S and s; anti-Lewis 6%, including Lea and Leb; and
multiple antibodies (16%, including anti-D). Compared to
other studies, including one from Australia in 1977, the
anti-D alloimmunisation rate had dropped significantly, with
little change in anti-c and some increase in anti-E and anti-Kell
cases. Continued vigilance is required to ensure eligible RhD
negative women receive prophylaxis according to the current
RhD immunoprophylaxis guidelines, especially those who
have a fetomaternal haemorrhage (FMH). RhD positive
women that are at risk of developing an antibody during
pregnancy should have their pregnancy monitored according
to published guidelines. Once antibodies are identified, con-
sideration should be given to paternal antigen status in an
attempt to identify the pregnancy that will be at risk of
alloimmunisation.
Key words: Alloimmunisation, antenatal antibody screen, haemolytic disease
of newborn.
Received 20 March, revised 17 October, accepted 26 October 2014
INTRODUCTION
Maternal alloimmunisation occurs when the mother’s immune
system is sensitised to foreign red cell surface antigens, stimulat-
ing the production of immunoglobulin G (IgG) antibodies. These
IgG antibodies are capable of crossing the placenta and causing
haemolytic disease of the fetus and the newborn (HDFN).1,2 The
most frequent antigen to cause severe incompatibility is the RhD
antigen due to its high prevalence and immunogenicity as
compared to most other red cell surface antigens.3 Since the
widespread use, at least in the developed world, of antenatal and
postnatal RhD immunoprophylaxis, there has been not only a
significant drop in cases of anti-D alloimmunisation but also a
shift towards non-anti-D alloimmunised cases.4 Anti-c and anti-
Kell are the other main antibodies known to be associated with
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2015 Royal College of Pathologists of Australasia
DOI: 10.1097/PAT.0000000000000225
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
clinically significant HDFN, however there are other antigens
that have also been found to be associated with HDFN.5,6 The
antibody prevalence varies between countries reflecting geo-
graphic variations in gene frequencies, transfusion practices and
blood banking techniques amongst others.
The aims of antibody testing in the antenatal and perinatal
setting are to identify RhD negative women who qualify for RhD
immunoprophylaxis, and those women with clinically significant
alloantibodies to assist in management of HDFN. There are over
400 red cell surface antigens, of which more than 43 have been
reported to be associated with HDFN.7 These have been grouped
according to their likelihood of causing HDFN in the Australian
and New Zealand Society of Blood Transfusion (ANZSBT) Ltd
Guidelines for Blood Grouping and Antibody Screening in the
Antenatal and Perinatal Setting (Table 1).8 Group 1 and 2
antibodies have been reported to be most commonly associated
with clinical HDFN with anti-D, anti-c and anti-K most associ-
ated with moderate to severe HDFN.
Regardless of which antibody is implicated, apart from Kell
antibody, the pathogenesis of cell mediated haemolysis is the
same.9,10 Once the antibodies enter the fetal circulation, they can
coat the fetal red cells (if they are positive for the corresponding
antigen), and thereby initiate the destruction and early removal of
the red cells from the circulation by macrophages in the fetal
spleen. In Kell alloimmunisation, however, the mechanism of
action involves suppression of erythropoiesis at the level of the
progenitor cell.11–13 The clinical consequences of any alloim-
munisation can be devastating. Anaemia and hyperbilirubinae-
mia vary from mild to severe, with risk of serious consequences
including hydrops fetalis and kernicterus if timely recognition
and treatment are not implemented. In affected pregnancies, fetal
monitoring by Doppler ultrasound of the middle cerebral artery
peak systolic velocity (MCA PSV) and intrauterine transfusion
(IUT) for treatment of clinically significant fetal anaemia has
become standard antenatal care. Antibody titres are usually done
to assist the clinician in deciding when to start monitoring a
pregnancy more closely, with a lower threshold for anti-Kell
alloimmunised pregnancies because of the unique pathogenesis.
MATERIALS AND METHODS
In this study, the blood group (ABO and RhD) and red cell antibody screen
results of pregnant women tested between the period of 1 January 2011 and 30
June 2013 were retrospectively evaluated. Testing was performed by Pathology
Queensland, which provides pathology services to all Queensland Health public
hospitals and is composed of a hierarchical networked system of 33 laboratories.
These laboratories consist of district laboratories in rural hospitals, group
laboratories in large regional hospitals and unit base laboratories providing
tertiary referral services in the metropolitan teaching hospitals.
 152 PAL and WILLIAMS Pathology (2015), 47(2), February

Table 1 Antibodies grouped according to their likelihood of causing HDFN8

Group 1 Group 2 Group 3

D Cw P1
16%
c Fyb N
E Jka H
e Jkb Lea 2%
C JK3 Leb
K S Leaþb
k s Lua 6%
Fya M Lub
Gea Sda 50%
HLA
8%

Testing was carried out according to the ANZSBT Guidelines for Blood 5%
Grouping and Antibody Screening in the Antenatal and Perinatal Setting.8 As
per the guidelines all RhD negative females underwent testing at first presen- 3%
tation and then again at 28 weeks gestation. RhD positive females underwent 10%
testing at presentation and then again if clinically indicated. Any woman with a
clinically significant antibody, that was likely to result in HDFN, underwent
extra testing including antibody titres and antibody quantitation (available for
anti-D and anti-c only) to assist with antenatal and perinatal management.
Antenatal women with antibodies that were not clinically significant (e.g.,
reacting only at room temperature) were not reported and extra antibody Rhesus
screening was not performed.
Column agglutination technology was used for blood group (BioRad Dia- Kell
Clon ABO/Dþ Reverse grouping for patients) and antibody screen (BioRad Duffy
Coombs Anti IgG) testing. All antibody screens were analysed and individual
patient results were recorded, taking into account possibility of multiple screens Kidd
during the same pregnancy. Any woman with multiple antibody screens during MNS
the same pregnancy was only included as a single entry, thus avoiding
erroneously higher subject numbers. Also women with positive antibody screens Lewis
who were subsequently identified as having passive anti-D were excluded from Other
the study.
2 or more antibodies
Fig. 1 Antibody prevalence in 482 positive antibody screens.
RESULTS
Results from 66,354 pregnant women were evaluated. The
majority of non-anti-D antibodies were found in RhD positive
overall antibody prevalence was 0.73% with the prevalence
women, in particular anti-E. Only a small number of RhD
of anti-D 0.08% and non-anti-D 0.65% (Table 2). The most
negative women had non-anti-D antibodies.
common antibodies were reactive with Rhesus antigens,
accounting for almost half of all antibodies (Fig. 1). Anti-E
(27.6%) was most frequent followed by anti-D (10.4%), anti- DISCUSSION
Kell (9.5%) and anti-c (8.7%). The antibodies and the sub- Recent studies show the overall incidence of maternal alloim-
groups that were detected in the positive screens are listed in munisation has declined significantly since the introduction of
Table 3. Approximately 16% of the positive screens had two or RhD immunoprophylaxis, with lower rates reported with pro-
more antibodies, the majority of which involved clinically tocols that include antenatal dosing.14 This study found an
significant antibodies as described in Table 1. Of the 482 overall prevalence of antibody formation, which is similar to
positive screens, 21% occurred in RhD negative women and that reported in the literature from countries that follow a
79% in RhD positive women (Fig. 2). These positive screens similar antenatal and perinatal RhD immunoprophylaxis pro-
represent 0.9% of all RhD negative women and 0.7% of all RhD gram to Australia. The type and frequency of reported anti-
positive women, respectively. Table 4 illustrates the differences bodies appears to vary according to the population studied and
in antibody prevalence between the RhD negative and RhD time period studied in relation to implementation of immuno-
positive women in the study group. The data showed that the prophylaxis5,15–19 (Table 5).

Table 2 Antenatal antibody screens performed in Queensland between 1 January 2011 and 30 June 2013

RhD negative women RhD positive women

Total antibody screens 66 354 10 890 (16.4%)* 55 464 (83.6%)*

Positive screens (overall antibody prevalence) 482 (0.73%)* 100 382
Anti-D antibody prevalence 50 (0.08%)*
Non-anti-D antibody prevalence 432 (0.65%)*

*
Percentage of total antibody screens

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 MATERNAL RED CELL ALLOIMMUNISATION: A POPULATION STUDY 153
Table 4 Antibody prevalence in RhD negative and positive women

System and antibody RhD negative (n ¼ 100) RhD positive (n ¼ 382)

RhD negative women Rh 58 (58%)* 184 (48.2%)*

21% D 50 0
C 4 4
c 0 42
E 4 129
e 0 6
Cw 0 3
Kell 8 (8%)* 38 (9.9%)*
K (Kell) 7 37
k (Cellano) 1 1
Duffy 0 15 (3.9%)*
Fya 0 13
RhD positive women Fyb 0 2
79% Kidd 1 (1%)* 24 (6.3%)*
Jka 1 20
Jkb 0 3
JK3 0 1
MNS 1 (1%)* 37 (9.6%)*
S 0 7
s 0 2
M 1 28
N 0 0
Lewis 0 29 (7.6%)*
Fig. 2 Positive antibody screens (n ¼ 482). Antibodies represent 0.9% of all Lea 0 28
RhD negative women ((100/10890)
100) and 0.7% of all RhD positive women Leb 0 1
((382/55464)
100). Leaþb 0 0
Lutheran 0 4 (1.1%)*
Lua 0 4
Lub 0 0
Gerbich Gea 0 0
Compared to Australian historical data,17 the incidence of P P1 1 (1%)* 2 (0.5%)*
RhD alloimmunisation (Table 6) has dropped from 13.3 per High frequency LAN 0 1 (0.3%)*
1000 samples in 1977 to 0.8 per 1000 samples in 2013, which is 2 or more antibodies 31 (31%)* 48 (12.6%)*
similar to that reported in other countries after the successful
*
Percentage of total antibodies (n) in RhD negative and positive women.

Table 3 Antibody prevalence in 482 positive antibody screens

System and antibody Number Percentage

implementation of RhD immunoprophylaxis. Even with these
immunoprophylaxis protocols, anti-D alloimmunisation may still
Rh 242 50.2%
occur as a result of chronic transplacental haemorrhage or failure to
D 50 10.4% administer RhD immunoglobulin at times of risk or at a sufficient
C 8 1.7% dose. RhD alloimmunisation is a significant ongoing concern in
c 42 8.7% low socio-economic groups and in the developing countries20–22
E 133 27.6%
e 6 1.2% where immunoprophylaxis is not widely available.
Cw 3 0.6% Whilst HDFN related to anti-D remains a significant clinical
Kell 46 9.5% issue, non-anti-D antibodies may also cause severe disease.
K (Kell) 44 9.1%
k (Cellano) 2 0.4%
Non-anti-D antibodies were more frequent in our population
Duffy 15 3.1% than anti-D, with a prevalence similar to that reported by others
Fya 13 2.7% (Table 5). Our data showed a much higher detection rate of anti-E
Fyb 2 0.4% as compared to the other studies. This may reflect the sensitivity
Kidd 25 5.2%
Jka 21 4.4% of the testing methodology used in various countries to the
Jkb 3 0.6% naturally occurring IgM anti-E. The method used by Queensland
JK3 1 0.2% Pathology is known to be sensitive to IgM anti-E and, hence, the
MNS 38 7.9%
S 7 1.5% actual numbers of women with IgG anti-E may be lower.
s 2 0.4% However testing to differentiate between IgG and IgM anti-E
M 29 6.0% is not currently routinely carried out. Our study reported lower
N 0 0.0%
Lewis 29 6.0%
rates of Kell sensitisation than others (Table 6), which may
Lea 28 5.8% reflect the population screened and/or the selection of Kell
Leb 1 0.2% negative blood for females of childbearing age, as is practised
Leaþb 0 0.0% in many transfusion units across Australia. Women with non-
Lutheran 4 0.8%
Lua 4 0.8% anti-D antibodies should be readily detected if appropriate
Lub 0 0.0% screening is performed in the first trimester. In the absence of
Gerbich Gea 0 0.0% a positive antibody screen at this initial screening, routine follow-
P P1 3 0.6%
High frequency LAN 1 0.2%
up testing is not recommended as it is not cost effective and
Two or more antibodies 79 16.4% clinically significant late onset alloimmunisation is rare.23 This is
in contrast to the recommendations for RhD negative women.8

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 154 PAL and WILLIAMS Pathology (2015), 47(2), February

Table 5 Antibodies in other countries as reported in literature

Koelewijin Geifman-Holtzman Filbey Pepperell Queenan Polesky

et al. (2008)15 et al. (1997)5 et al. (1995)16 et al. (1977)17 et al. (1969)18 (1967)19

Place The Netherlands New York, United States Sweden Australia New York, United States Minnesota, United States
Time period 2003–05 (2yr) 1993–95 (2.5yr) 1980–91 (12yr) 1965–75 (10yr) 1960–67 (8yr) 1960–66 (7yr)
Study size 403,500 37,506 110,765 72,138 18,378 43,000
Total antibodies 4971 550 836 1467 630 2956
Overall prevalence 1.23% 1.47% 0.75% 2.03% 3.43% 6.87%
Anti-D prevalence 0.08% 0.26% 0.14% 1.32% 1.65% 4.33%
Non-anti-D prevalence 1.15% 1.21% 0.61% 0.71% 1.78% 2.54%

Antibody (% of total antibodies)

D 6.74% 18.4% 19.0% 65.3% 48.3% 63.1%
C 0.42% 4.7% 4.3% 0.6% 5.3% 15.2%
c 1.87% 5.8% 4.5% 4.0% 1.9% 2.3%
E 5.43% 14.0% 6.1% 4.7% 5.3% 2.7%
e 0.18% – 0.1% 0.04% 0.4% 0.07%
Cw 1.65% 0.2% 1.2% – – 0.14%
Kell 4.35% 22.0% 5.7% 2.3% 4.7% 3.1%
Duffy 1.39% 5.6% 3.1% 0.5% 1.9% 0.6%
MNS 1.07% 4.7% 4.2% 1.2% 3.1% 1.5%
Kidd 0.87% 1.5% 1.2% 0.14% 1.1% 0.2%
Lewis – 20.5% 28.8% 11.9% 8.1% 3.2%
P – 0.2% 5.7% 8.8% 2.3% 0.9%
Others 2.94% 0.2% 14.4% 0.07% 14.3% 6.6%

Table 6 Antibody frequency compared with that in reported literature (per 1000 samples)

Pal et al. Koelewijin Geifman-Holtzman Filbey Pepperell Queenan Polesky

(2013) et al. (2008)15 et al. (1997)5 et al. (1995)16 et al. (1977)17 et al. (1969)18 (1967)19

Antibody
D 0.8 0.8 2.6 1.4 13.3 16.5 43.3
E 2.0 1.0 2.0 0.5 1.0 1.9 1.9
c 0.6 0.5 0.7 0.3 0.8 0.7 1.6
Kell 0.7 0.7 3.2 0.4 0.5 1.6 2.2

CONCLUSION Address for correspondence: Dr Manika Pal, Pathology Queensland, Princess

This study shows a reduction in anti-D alloimmunisation,
consistent with successful implementation of RhD immuno-
prophylaxis in Australia. Anti-D alloimmunisation remains a
clinically significant issue with affected pregnancies requiring
significant resource allocation. Ongoing efforts should be
made to ensure that immunoprophylaxis is administered
according to the guidelines. Sensitising events should be
assessed thoroughly and additional RhD immunoglobulin
administered if required.
An increasing prevalence of non-anti-D alloimmunisation was
found and currently there are no preventative strategies for this.
In contrast to RhD alloimmunisation, the main risk factor for
non-anti-D alloimmunisation is previous transfusion history.14
Thus, it is important to minimise exposure of women to incom-
patible red cell antigens through unnecessary transfusions where
possible. It is important that all RhD positive women have
antibody screening in the first trimester to detect those pregnan-
cies with clinically significant antibodies so appropriate follow
up testing and clinical management can occur.
Conflicts of interest and sources of funding: The authors state
that there are no conflicts of interest to disclose.
Alexandra Hospital, Wooloongabba, Qld 4102, Australia. E-mail: manika.pal
@health.sa.gov.au
References
1. Riley JZ, Ness PM, Taddie SJ, Barrasso C, Baldwin ML. Detection and
quantitation of fetal maternal hemorrhage utilizing an enzyme-linked
antiglobulin test. Transfusion 1982; 22: 472–4.
2. Salama A, David M, Wittmann G, Stelzer A, Dudenhausen JW. Use of the
gel agglutination technique for determination of fetomaternal hemorrhage.
Transfusion 1998; 38: 177–80.
3. Moise KJ. Non-anti-D antibodies in red-cell alloimmunization. Eur J
Obstet Gynecol Reprod Biol 2000; 92: 75–81.
4. Moise KJJ. Changing trends in the management of red blood cell alloim-
munization in pregnancy. Arch Pathol Lab Med 1994; 118: 421–8.
5. Geifman-Holtzman O, Wojtowycz M, Kosmas E, Artal R. Female alloim-
munization with antibodies known to cause hemolytic disease. Obstet
Gynecol 1997; 89: 272–5.
6. Smith BD, Haber JM, Queenan JT. Irregular antibodies in pregnant women.
Obstet Gynecol 1967; 29: 118–24.
7. Weinstein L. Irregular antibodies causing hemolytic disease of the new-
born: a continuing problem. Clin Obstet Gynecol 1982; 25: 321–32.
8. Australian and New Zealand Society of Blood Transfusion (ANZSBT) Ltd
and The Royal Australian and New Zealand College of Obstetricians and
Gynaecologists (RANZCOG). Guidelines for Blood Grouping and Anti-
body Screening in the Antenatal and Perinatal Setting. 3rd ed, March 2007.
Sydney: ANZSBT, 2007. http://www.anzsbt.org.au/publications/docu-
ments/antenatal_guidelines_mar07.pdf.

Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
 MATERNAL RED CELL ALLOIMMUNISATION: A POPULATION STUDY
9. Kumpel BM. On the immunologic basis of Rh immune globulin (anti-D)
prophylaxis. Transfusion 2006; 46: 1652–6.
10. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000; 95:
375–87.
11. Vaughan JI, Warwick R, Letsky E, Nicolini U, Rodeck CH, Fisk NM.
Erythropoietic suppression in fetal anemia because of Kell alloimmuniza-
tion. Am J Obstet Gynecol 1994; 171: 247–52.
12. Weiner CP, Widness JA. Decreased fetal erythropoiesis and hemolysis in
Kell hemolytic anemia. Am J Obstet Gynecol 1996; 174: 547–51.
13. Daniels G, Hadley A, Green CA. Causes of fetal anemia in hemolytic
disease due to anti-K. Transfusion 2003; 43: 115–6.
14. Koelewijn JM, Vrijkotte TGM, de Haas M, van der Schoot CE, Bonsel GJ.
Risk factors for the presence of non-rhesus D red blood cell antibodies in
pregnancy. BJOG 2009; 116: 655–64.
15. Koelewijn JM, Vrijkotte TGM, van der Schoot CE, Bonsel GJ, de Haas M.
Effect of screening for red cell antibodies, other than anti-D, to detect
hemolytic disease of the fetus and newborn: a population study in the
Netherlands. Transfusion 2008; 48: 941–52.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
155
16. Filbey D, Hanson U, Wesström G. The prevalence of red cell antibodies in
pregnancy correlated to the outcome of the newborn: a 12 year study in
central Sweden. Acta Obstet Gynecol Scand 1995; 74: 687–92.
17. Pepperell RJ, Barrie JU, Fliegner JR. Significance of red-cell irregular
antibodies in the obstetric patient. Med J Aust 1977; 2: 453–6.
18. Queenan JT, Smith BD, Haber JM, Jeffrey J, Gadow HC. Irregular anti-
bodies in the obstetric patient. Obstet Gynecol 1969; 34: 767–71.
19. Polesky HF. Blood group antibodies in prenatal sera. Results of screening
43,000 individuals. Minn Med 1967; 50: 601–3.
20. Basu S, Kaur R, Kaur G. Hemolytic disease of the fetus and newborn:
Current trends and perspectives. Asian J Transfus Sci 2011; 5: 3–7.
21. Zipursky A, Paul VK. The global burden of Rh disease. Arch Dis Child
Fetal Neonatal Ed 2011; 96: F84–5.
22. Gündüz E, Meltem Akay O, Teke HÜ Gülbaş Z. Incidence of red-cell
alloimmunization due to non-anti-D antibodies during pregnancy: an
experience from Turkey. Transfus Apher Sci 2010; 43: 261–3.
23. Judd WJ. When should tests for unexpected antibodies be done during
pregnancy? Transfusion 2011; 51: 1366–8.