I M M U N O H E M AT O L O G Y
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
J.M. Koelewijn, T.G.M. Vrijkotte, C.E. van der Schoot, G.J. Bonsel, and M. de Haas
BACKGROUND: Hemolytic disease of the fetus and
newborn (HDFN) is a severe disease, resulting from
maternal red cell (RBC) alloantibodies directed against
fetal RBCs. The effect of a first-trimester antibody
screening program on the timely detection of HDFN
caused by antibodies other than anti-D was evaluated.
STUDY DESIGN AND METHODS: Nationwide, all
women (1,002 in 305,000 consecutive pregnancies
during 18 months) with alloantibodies other than anti-D,
detected by a first-trimester antibody screen, were
included in a prospective index-cohort study. In a
parallel-coverage validation study, patients with HDFN
caused by antibodies other than anti-D, that were
missed by the screening program, were retrospectively
identified.
RESULTS: The prevalence of positive antibody screens
at first-trimester screening was 1,232 in 100,000; the
prevalence of alloantibodies other than anti-D was 328 in
100,000, of which 191 of 100,000 implied a risk for
occurrence of HDFN because the father carried the
antigen. Overall, severe HDFN, requiring intrauterine or
postnatal (exchange) transfusions, occurred in
3.7 percent of fetuses at risk: for anti-K in 11.6 percent;
anti-c in 8.5 percent; anti-E in 1.1 percent; Rh antibodies
other than anti-c, anti-D, or anti-E in 3.8 percent; and for
antibodies other than Rh antibodies or anti-K, in none of
the fetuses at risk. All affected children, where antibodies
were detected, were promptly treated and healthy at the
age of 1 year. The coverage validation study showed a
sensitivity of the screening program of 75 percent. Five
of 8 missed cases were caused by anti-c, with delay-
induced permanent damage in at least 1.
CONCLUSION: First-trimester screening enables timely
treatment of HDFN caused by antibodies other than
anti-D, however, with a sensitivity of only 75 percent.
A second screening at Week 30 of c– women will
enhance the screening program. Severe HDFN, caused
by antibodies other than anti-D, is associated with anti-K,
anti-c, and to a lesser extent with other
Rh-alloantibodies.
H
emolytic disease of the fetus and newborn
(HDFN) has, for a long time, been a major
cause of perinatal morbidity and mortality.1
In 1941, Levine and colleagues2 suggested
that destruction of red cells (RBCs) in the fetus and
newborn may be due to maternal alloimmunization
against blood group antigens of the unborn child. Immu-
nization is triggered by previous incompatible blood
transfusion or by fetomaternal hemorrhage, mostly
during a previous pregnancy from a blood group antigen–
mismatched fetus. In most cases of severe HDFN, alloim-
munization against the D antigen is involved.3,4 Several
strategies have been developed to prevent D immuniza-
tion, leading to a substantial decrease of D immunization
in developed countries.5 Consequently, alloantibodies
other than anti-D emerged as an important cause of
ABBREVIATIONS: HDFN = hemolytic disease of the fetus and
newborn; IUT(s) = intrauterine transfusion(s).
From Sanquin Research, Amsterdam, and Landsteiner Labora-
tory, Academic Medical Center, University of Amsterdam,
Amsterdam; and the Institute of Health Policy and Manage-
ment, Erasmus Medical Center, Rotterdam, the Netherlands.
Address reprint requests to: M. de Haas, Sanquin Research,
Amsterdam, and Landsteiner Laboratory, Academic Medical
Center, University of Amsterdam, Plesmanlaan 125, 1066 CX
Amsterdam, the Netherlands; e-mail: m.dehaas@sanquin.nl.
The study was supported by the Dutch Council of Health
Insurances.
Source of the study: OPZI (detection and prevention of
pregnancy immunization)-project: the nationwide evaluation of
pregnancy screening for RBC antibodies other than anti-D, and
of antenatal anti-D-prophylaxis in the Netherlands, performed
by Sanquin Research and the Academic Medical Center of the
University of Amsterdam, financed by the Council of Health
Insurances.
Received for publication August 28, 2007; revision received
October 16, 2007, and accepted October 31, 2007.
doi: 10.1111/j.1537-2995.2007.01625.x
TRANSFUSION 2008;48:941-952.
Volume 48, May 2008 TRANSFUSION 941
KOELEWIJN ET AL.
severe HDFN, in particular anti-K and anti-c.6-10 Severe
HDFN caused by antibodies other than anti-D can be
treated with intrauterine transfusions (IUTs) during preg-
nancy and with exchange transfusions after birth. Screen-
ing for RBC alloantibodies in all pregnant women has
been implemented in most developed countries, to detect
possible HDFN caused by anti-D and other antibodies.
Moreover, screening during pregnancy facilitates quick
identification of the specificity of detected antibodies, if a
blood transfusion to the mother is necessary during deliv-
ery. Evidence for the effectiveness of this universal screen-
ing approach, however, is lacking.
In the Netherlands, universal screening for RBC anti-
bodies in the first trimester (200,000 pregnancies/year) has
been implemented since July 1, 1998.11 Our prospective
nationwide controlled study evaluated this program along
the criteria of Wilson and Jüngner12 for screening pro-
grams. This set of 10 criteria is widely accepted for evalu-
ating a new screening program. Our research questions,
reported in this article, elaborate the first (“Is the health
problem important?”), the fifth (“Is an appropriate screen-
ing test available?”), and the seventh criteria (“Is the
natural course of the disease understood?”), respectively.
To evaluate the health problem of HDFN, the following
three main questions were consequently addressed in
general, and for each alloantibody specificity, other than
anti-D, separately: 1)What is the prevalence of pregnancies
with a fetus at risk for HDFN caused by antibodies other
than anti-D; 2) in what proportion of these pregnancies
does the fetus develop HDFN; and 3) what is the diagnostic
performance in terms of sensitivity, specificity, and predic-
tive value of a first-trimester RBC antibody screening for
detecting the fetuses at risk for severe HDFN due to RBC
antibodies other than anti-D? To evaluate the benefit of
screening with regard to blood transfusion around delivery
to the mother, the following secondary questions were
addressed: 4) what is the prevalence of blood transfusion
during pregnancy and delivery to the mother; 5) what is the
diagnostic value of a single first-trimester RBC antibody
screening in detecting all antibodies that may be relevant
for transfusions during delivery; and 6) are, in case of blood
transfusion, all antibodies detected by RBC antibody
screening known at the transfusion laboratory? Empirical
answers should support if not improve the current expert
opinion-based programs.
MATERIALS AND METHODS
National screening program
The Dutch screening program offers RBC antibody screen-
ing, free of charge, as part of the booking visit protocol
around the 12th week of pregnancy. This protocol includes
typing for ABO, and screening for human immunodefi-
ciency virus, hepatitis B, and syphilis. The obstetric care-
942 TRANSFUSION Volume 48, May 2008
giver (in the Netherlands, independent midwife [⫾80%],
general practitioner [⫾5%], or obstetrician [⫾15%]13) is
responsible for the application of the screening test. The
coverage of the RBC antibody screening is close to
100 percent.14 Certified Dutch laboratories (n = ⫾90)
process the screening test according to existing national
guidelines. Accepted screening tests are those with the
sensitivity of at least that of the bovine albumin indirect
antiglobulin test (IAT) to detect clinically relevant
antibodies.15
A positive screening result is checked by one of two
specialized national reference laboratories with our IAT in
the presence of polyethylene glycol or with the technique
used by the screening laboratory. After confirmation of the
positive screening result, the risk for HDFN is determined
by establishing the alloantibody specificity (occasionally
more than one) and by subsequent serologic typing of the
father for the antigen(s) against which the maternal
alloantibodies are directed. If the father has the corre-
sponding antigen, the pregnancy is considered “at risk for
HDFN” and monitored by repeated laboratory testing,
processed in reference laboratories (titer and antibody-
dependent cellular cytotoxicity test). The repeat frequency
depends on antibody specificity, test result, and duration
of pregnancy. If the father is heterozygous for an antigen
corresponding to a maternal alloantibody, a noninvasive
fetal typing with maternal plasma is offered (for D, c, E,
and K).
In pregnancies with M or N antibodies of the immu-
noglobulin M (IgM) class only, repeated laboratory testing
is performed in Weeks 24, 30, and 36, to detect a potential
switch to clinically relevant IgG-class antibodies (previous
guidelines).
If test results suggest a major risk (IAT titer in
phosphate-buffered saline ⱖ16 or antibody-dependent
cellular cytotoxicity test result ⱖ30%) for severe HDFN
(the need for IUT and/or neonatal blood transfusion),
women are referred to secondary care for clinical moni-
toring, by ultrasound and Doppler flow measurement.
Amniocentesis for fetal genotyping or Liley index is,
because of its risk of complications and antibody boost-
ing, only performed if the serologic analysis or the clinical
monitoring points to an increased risk.16 IUT treatment is
centralized at the Leiden University Medical Center in
Leiden.17
Definitions
The following definitions are used in this article:
1. Clinically relevant RBC alloantibodies: alloantibodies
that cross the placenta (IgG class) and that are
directed against an antigen expressed on fetal RBCs.
2. Index pregnancy (included in index-cohort study):
pregnancy with clinically relevant RBC alloantibodies
other than anti-D.
 HDFN CAUSED BY ANTIBODIES OTHER THAN ANTI-D

3. Pregnancy/fetus at risk of HDFN

associated with antibodies other Pregnancies with clinically Controls
relevant alloantibodies 481
than anti-D: pregnancy with clini- other than anti-D
cally relevant antibodies, other 1002
than anti-D, recognizing an antigen
expressed by fetal RBCs, the father
consent no consent
having the corresponding antigen,
900 102
or paternal antigen status
unknown. abortion abortion
< 16 < 16
4. Dominant alloantibody specificity weeks weeks
at screening (in patients with more 10 15
than one alloantibody specificity): 890 87
the alloantibody specificity associ-
fetal death fetal death
ated with a paternal antigen; if mul- >= 16 < >= 16 < 22
tiple alloantibodies are associated 22 weeks weeks
with paternal antigens, the highest 5 2
estimated risk for HDFN is anti-K, 885 85
-c, -E, -Rh antibodies other than
anti-c, -D, -E, -Duffy (Fy), -Kidd fetal death fetal death fetal death
>= 22 >= 22 >= 22 weeks
(Jk), -Ss, or other.6,7,18 weeks weeks 1
5. Controls (children): children, born 5 6
from pregnancies in which no 880 79 480
clinically relevant RBC alloanti- lost to no outcome
bodies were detected or children follow-up data:
born from pregnancies with clini- 1 12
†
(at risk)* (3 at risk)
cally relevant antibodies, who were
antigen negative. anti-D
twins twins
present
2 21 9

Study design live born pregnancies live born

Three related observational studies children with live born children
898 child(ren) 489
were conducted: 1) a procedural study 67
with laboratory registry data of positive
antibody screens; 2) a prospective neonatal
index-cohort study based on screen- death
1
detected index pregnancies (see Fig. 1);
and 3) a coverage validation study to
Fig. 1. Flow chart of pregnancies, included in index-cohort study (September 1,
retrospectively identify, at the national
2002-June 1, 2003, and October 1, 2003-July 1, 2004). *Anti-c, delivery abroad, no
level, cases of severe HDFN, undetec-
HDFN until Week 36. †Anti-c (delivery abroad), -Cw, -Jka.
ted by the screening program. The
entire study was discussed with and
approved by the relevant professional
organizations (obstetricians, midwives, general practitio- Inclusion of index pregnancies: selection of
ners, pediatricians, clinical laboratories). Representatives controls for the index-cohort study and data
from those organizations monitored the study process. collection methods
The study was approved by the ethical review board of
Included were two predefined cohorts of index pregnan-
the Academic Medical Center in Amsterdam. All women
cies (September 1, 2002, until June 1, 2003, and October
gave informed consent.
1, 2003, until July 1, 2004) with clinically relevant alloan-
tibodies, other than anti-D, recognized by first-trimester
screening and confirmed in either of the two reference
Laboratory registries laboratories (n = 1002). Consent for collection (by struc-
The two reference laboratories registered the numbers tured questionnaire) of detailed clinical data about the
and test results of all positive antibody screens. We used outcome of the pregnancy and after delivery (until
the results of 2003 and 2004. 6 weeks after birth) and collection of cord blood

Volume 48, May 2008 TRANSFUSION 943

KOELEWIJN ET AL.

was given by 900 women; an additional 90 women gave Data analysis

consent for collection of general HDFN-related informa-
tion via the obstetric caregiver (see Fig. 1). The women
without consent did not differ from those with consent,
regarding age (32.4 years vs. 32.3 years; p = 0.86), the
dominant RBC alloantibody specificity, and the prop-
ortion at risk for HDFN because the father had the
antigen(s) (56% vs. 59%; p = 0.60). If completion of the
questionnaire by the obstetric caregiver was incomplete,
data were collected by a telephone interview of the preg-
nant woman/mother by one of the investigators (JMK,
TGMV).
For comparative analysis of icterus, phototherapy,
and cord blood variables, we collected outcome data of
481 control pregnancies. The control group consisted of
412 pregnant women without antibodies, and of 69
women with M or N antibodies of the IgM class only,
without a switch to IgG during pregnancy. Controls were
randomly selected (1:1), only during the first 9-month
period, from the same obstetric practice as the pregnancy
under study, and matched for the duration of pregnancy
(⫾4 weeks; response 412/458, or 90%).
Serologic analysis of cord blood included: typing for
ABO and specific antigen testing (in index pregnancies)
and the direct antiglobulin test (DAT), according to the
existing guidelines.15 In the case of a positive DAT, an
eluate was made for identification of the alloantibody
specificity. Hemoglobin (Hb) and hematocrit (Hct) levels
were determined on a hematology analyzer (Cell-Dyn
4000, Abbott Diagnostics, Santa Clara, CA). Genotyping of
the child was performed, if no serologic typing result was
available, with DNA isolated from buccal swabs in those
index pregnancies where the father was heterozygous or
the paternal antigen was unknown.
Prevalence of clinically relevant RBC alloantibodies,
other than anti-D, at first-trimester screening
The denominator was based on vital statistics data (which
in the Netherlands include newborns from 24 weeks of
pregnancy onward), adjusted for the expected proportion
of abortions between screening and 24 weeks (32/1002,
3.19%) and for unregistered pregnancies (2%; G.J. Bonsel,
personal communication, 2005); calculations available
from the authors on request. The prevalence of additional
antibodies, detected later in pregnancy (after screening),
was estimated in all index pregnancies with consent,
based on laboratory monitoring (in index pregnancies at
risk of HDFN associated with antibodies other than
anti-D) or on cord blood analysis (all index pregnancies);
data were available in 794 of 900 pregnancies.
Outcome analysis
All outcomes were analyzed in pregnancies with a gesta-
tional age of at least 16 weeks.
Measures of diagnostic performance
Standard measures of performance (sensitivity, specificity,
etc.) were computed with their confidence intervals (CIs).
Statistical analysis
All data were analyzed with computer software (SPSS 11.0,
SPSS, Inc., Chicago, IL). Testing of differences between
categorical variables was performed by Pearson’s chi-
square or Fisher’s exact test. For testing of continuous
variables, the t test was used. All statistical tests were two-
tailed. Results were routinely checked for interaction with
ethnicity (based on racial classification as used in the
national obstetric registration).

RESULTS
Inclusion of patients for coverage validation study
Two independent epidemiologic case-finding strategies Prevalence of alloantibodies, other than anti-D, at
were applied, unrelated to the screening data. First, at the first-trimester screening
end of 2003 and the end of 2004, all Dutch obstetric part- The prevalence of a positive screen was 1,232 in 100,000
nerships (n = 116; response, 100%) and pediatric partner- (Table 1). The positive screen was not confirmed in the
ships (n = 112; response, 88% in 2003
and 98% in 2004) were contacted per-
sonally twice (by JMK or TGMV) to TABLE 1. First-trimester screening results, based on laboratory
registries in the two reference laboratories January 1, 2003, until
report any patient with severe HDFN, January 1, 2005
associated with antibodies other than Percentage of Prevalence
anti-D, in the past year (death, IUT, screen-positive number per
exchange transfusion, or blood transfu- Result Number samples 100,000*
Screen-positive pregnancies 4971 1232
sion in the first week of life). Second,
Positive screening, not confirmed 998 20 247
based on the records of Sanquin Blood Antibodies without a risk to mediate HDFN 2359 47 584
Supply (coverage of the Netherlands, HDFN-risk relevant antibodies 1614 32 400
Anti-D 335 7 83
100%), detailed hospital information on
Antibodies other than anti-D 1279 26 317
the transfusion indication for exchange
* Denominator of prevalence = the estimated number of pregnant women in week 12 in
transfusions was collected (response, 2003 and 2004 = 403,500.
543/759, 72% of units of blood).

944 TRANSFUSION Volume 48, May 2008


reference laboratories in 20 percent of pregnancies; in
47 percent the detected antibodies were without clinical
relevance and in 7 percent anti-D was found (Table 1).
The prevalence of pregnancies with clinically relevant
alloantibodies, other than anti-D, was 328 in 100,000
(Table 2). Anti-E was the most frequent alloantibody,
other than anti-D, followed by anti-K and anti-c. Alloanti-
bodies, other than anti-D, of more than one specificity
were found in 14 percent (140/1,002) of the index preg-
nancies, of which the combination of anti-c plus anti-E
(possible anti-cE) was the most frequent (47/192). The
estimated prevalence of pregnancies at risk of HDFN,
associated with antibodies other than anti-D, was 191 in
100,000, with anti-E as the most frequent alloantibody
specificity, followed by anti-c.
Additional clinically relevant antibodies detected
after the first screening, at one of the follow-up tests, were
present in 56 of 794 index pregnancies followed to term.
The mean gestational age at detection was 29 weeks
4 days. In four cases the additional antibodies were
detected in cord blood. In none of these 56 pregnancies
was a blood transfusion given between screening and
delivery. In 40 of these 56 pregnancies the child was
antigen positive for 43 additional maternal antibodies
detected after the first screen (anti-D, 2; anti-c, 16; anti-C,
3; anti-Cw, 1; anti-E, 4; anti-K, 1; anti-Fya, 5; anti-Fyb, 2;
anti-Jka, 4; anti-Jkb, 1; anti-S, 2; anti-s, 2) and in 16 preg-
nancies antigen negative for 17 additional antibodies
(anti-c, 1; anti-C, 1; anti-Cw, 3; anti-E, 1; anti-K, 2; anti-Kpa,
1; anti-Fya, 1; anti-S, 1; anti-Wra, 4; anti-Cob, 1; anti-Vw, 1).
Hence, in 40 women immunization occurred in the
current pregnancy or was boosted in the current preg-
nancy after immunization in the past.
Anti-c was the most frequent late-detected additional
antibody (17 of 56 pregnancies): in 13 of these 17 pregnan-
cies anti-c was found in addition to screen-detected
anti-E; 16 children were typed c+ and 1 c–; and 170 of 794
index pregnancies concerned c– women, without anti-c.
In 25 of these, the father was also c–, hence the prevalence
of “pregnancy-induced” anti-c was 14 percent in the 145
pregnancies that were at risk because the father was c+.
M or N antibodies of the IgM class showed a preva-
lence of 130 in 100,000 (95% CI, 112-148 in 100,000), with
no switch to IgG. In none of these pregnancies (including
negative controls) were other clinically relevant RBC anti-
bodies detected upon follow-up serologic monitoring, or
at birth in the cord blood.
HDFN in screen-detected index pregnancies
Severe HDFN, with a need for IUT or exchange transfusion
in the first week, occurred in 21 of 567 index pregnancies
at risk of HDFN associated with antibodies other than
anti-D; the HDFN was caused by anti-K, anti-c, or, less
frequently, by other Rh antibodies (Table 3). One severe
HDFN CAUSED BY ANTIBODIES OTHER THAN ANTI-D
case was caused by “late-detected” anti-c, detected in the
20th week of pregnancy during laboratory monitoring
because of screen-detected anti-E. IUTs were given to five
affected fetuses, 4 due to anti-K and 1 to anti-c (Table 3).
The first IUT was given in Weeks 21, 22, 25, 30 (all anti-K),
and 31 (anti-c). Complications of IUTs or exchange trans-
fusions occurred in 2 children: 1 child was born prema-
turely (Week 34) after 4 IUTs (anti-K); he was treated for
6 weeks at the neonatal intensive care and monitored at
home for irregular heart beating during the next 6
months. Another child suffered from necrotizing entero-
colitis after an exchange transfusion (anti-e). All children
with severe HDFN were confirmed to be in good health at
the age of 1 year. Assuming that no severe HDFN occurred
in pregnancies for which no outcome data were available,
the prevalence of severe HDFN associated with antibodies
other than anti-D, in all pregnancies, was 7 in 100,000, in
index pregnancies (2.1%) and in index pregnancies at risk
of HDFN, due to antibodies other than anti-D, 3.7 percent
(Table 4).
Icterus occurred in 25 percent and phototherapy was
given in 17 percent in case the child was born from an
index pregnancy at risk of HDFN due to antibodies other
than anti-D, compared to 10 percent icterus and 5 percent
treatment with phototherapy in children born from index
pregnancies not at risk for HDFN. Similarly, in controls,
icterus occurred in 10 percent and was treated with
phototherapy in 4 percent (data not shown). The mean
gestational age at delivery was 278.0 days (SD 10.7) in con-
trols, 275.3 days (SD 18.7) in cases not at risk (p = 0.008,
compared to controls); and 273.5 days (SD 19.4) in cases at
risk (p < 0.001, compared to controls; p = 0.165 compared
to cases not at risk).
Risk of occurrence of HDFN in index pregnancies,
related to antigen positivity of the child
In 85 percent of index pregnancies, the cord blood
samples (n = 716) were typed for the corresponding
antigen after birth or were genotyped with DNA from a
buccal swab (n = 47). In three children, the phenotype of
the child was not compatible with that of the purported
father: one child was “unexpectedly positive” (anti-E,
without clinical consequences) and two children “unex-
pectedly negative” (anti-S, anti-c plus -E).
Table 3 shows the outcome of severe HDFN, includ-
ing cord blood Hb and Hct levels and nontreated or
treated icterus. In K+ and c+ children, the risk for severe
HDFN was 26 and 10 percent, respectively. Children with
K-, c-, and Rh-antibodies other than anti-c, -D, or -E and
Duffy alloantibodies had icterus more often and photo-
therapy more often than did controls at birth. As expected,
antigen-negative children, born from index pregnancies
at risk for HDFN, showed a risk for occurrence of icterus
and need for phototherapy similar to that of children born
Volume 48, May 2008 TRANSFUSION 945
 TABLE 2. Prevalence of clinically relevant alloantibodies other than anti-D, identified upon first-trimester screening, related to the risk of occurrence of
HDFN because the father is positive for the corresponding antigen (September 1, 2002-June 1, 2003, and October 1, 2003-July 1, 2004)
Phenotype of the father At risk for HDFN
KOELEWIJN ET AL.

946 TRANSFUSION
Dominant alloantibody specificity Prevalence,* number Number Number Number Prevalence,* number
other than anti-D Number per 100,000 (95% CI) negative hetero† homo† Number?‡ Number per 100,000 (95% CI)
Kell antibodies 242 79 (69-89) 196 31 6 9 46 15 (11-19)
K 212
Kpa 4
K plus c 2
K plus other than anti-c or -D 24
Anti-c 152 50 (42-58) 6 63 74 9 146 48 (40-56)
c 93
c plus E 47

Volume 48, May 2008

c plus other than anti-D, -K, or -E 12
Anti-E 289 95 (84-105) 108 140 26 15 181 59 (51-68)
E 270
E plus other than anti-c, -D, or -K 19
Rh antibodies other than anti-c, -D, or -E 133 44 (36-51) 79 27 22 5 54 18 (13-22)
C 21
Cw 82
e 9
f 1
Combination of above 11
Combination of above plus other antibodies 9
Duffy antibodies 69 23 (17-28) 12 40 14 3 57 19 (14-23)
Fya 60
Fyb 1
Fya plus other 8
Kidd antibodies 43 14 (10-18) 1 16 25 1 42 14 (10-18)
Jka 39
Jkb 2
Jka plus other 2
Ss antibodies 53 17 (13-22) 10 26 10 7 43 14 (10-18)
S 43
s 5
S plus other 5
Antibodies other than Rh, Kell, Fy, 21 7 (4-10) 6 7 4 4 15 5 (2-7)
Jk, Ss antibodies
Other 20
Combination other 1
Total 1002 328 (307-348) 418 350 181 53 584 191 (176-207)
* Denominator of prevalence = the estimated number of pregnant women in Week 12 in study period, or 305,700.
† Hetero = heterozygously positive and homo = homozygously positive for at least one alloantibody specificity.
‡ ? = phenotype information unavailable.
 TABLE 3. Outcomes index pregnancies and controls, according to antigen status of the child (liveborn only, n = 1387)
Recognized
Dominant Severe HDFN§ Hb of cord blood Hct of cord blood Icterus|| Phototherapy
alloantibody (n = 1387) (n = 1020) (n = 913) (n = 1379) (n = 1384)
specificity Total, Number of Number of Number of Mean (SD), <Mean – <Mean – Days
other than anti-D* Number number (%) IUTs¶ ETs BTs g/dL** 2SD, % Mean (SD) 2SD, % Number % Number % mean
Antigen positive† 403
Anti-K 19 5 (26.3)a 4 1 0 14.9 (2.7) 8.3 0.47 (0.11)f 18.2g 7 37k 8 42a 3.4
Anti-c 118 12 (10.2)a 1 6 5 14.9 (1.9)c 11.4g 0.49 (0.08)c 8.5i 46 39a 39 33a 4.3m
Anti-E 95 2 (2.1)b 0 1 1 15.2 (1.7) 2.7 0.50 (0.06)g 1.5 26 27a 18 19a 2.4
Anti-Rh other than 40 2 (5.0)b 0 2 0 14.8 (1.5)d 2.9 0.49 (0.07)r 9.7 12 30a 8 20a 4.0
-c, -D, -E
Duffy antibodies 42 0 — — — 16.0 (2.0) 0.0 0.52 (0.08) 4.5 11 26b 6 14m 5.0n
Anti-A/-B‡ 30 0 — — — 14.7 (2.0)e 7.4 0.48 (0.11)h 12.0j 4 13 3 10 3.0
Other 59 0 — — — 15.2 (1.8) 4.2 0.51 (0.06) 0.0 11 19l 4 7 2.5
Controls and antigen 964 0 15.5 (1.7) 2.4 0.52 (0.07) 102 11 39 4 2.8
negative children†
Unknown antigen† 20†† 0 — — — No data No data 3 16 2 19
* Dominant alloantibody specificity: for ranking, see definitions.
† If the antigen typing result of the child was unknown, children of homozygously positive fathers were considered as antigen positive and children of antigen-negative fathers as antigen
negative.
‡ Detected in cord blood in controls and children antigen negative for all other maternal alloantibody specificities.
§ Severe HDFN: perinatal death, need for IUT, and/or exchange and/or top-up transfusion < 168 hours after birth because of RBC alloantibodies.
|| Recognized icterus: a score of “significant” on the icterus scoring list for at least 1 day on two or three variables (face, belly, sclerae). This scoring list was completed by the caregiver
during 6 days. If no scoring list was available, the retrospectively reported degree of icterus during the first week was used.
¶ IUT = one or more intrauterine transfusions, in some cases followed by ET and/or BT. ET = one or more exchange transfusions, no IUT, in some cases followed by BT. BT = blood trans-
fusion, 168 hours after birth, no IUT or ET.
** Measured in mmol/L; 1 mmol/L = 1.6 g/dL.
†† Maternal antibodies: anti-E (n = 7); Rh antibodies other than anti-c, -D, or -E (n = 2); or other (n = 11).
p values calculated for each alloantibody specificity compared to controls: a p < 0.001; b p = 0.002; c p = 0.001; d p = 0.014; e p = 0.019; f p = 0.028; g p = 0.035; h p = 0.004; i p = 0.011;
j
p = 0.032; k p = 0.003; l p = 0.05; m p = 0.009; n p = 0.016.

Volume 48, May 2008

TRANSFUSION 947
HDFN CAUSED BY ANTIBODIES OTHER THAN ANTI-D
KOELEWIJN ET AL.

TABLE 4. Prevalence of severe HDFN associated with antibodies other than anti-D, screen-detected (September
1, 2002-June 1, 2003, and October 1, 2003-July 1, 2004) and screen-undetected (January 1, 2003-January 1,
2005)
Severe HDFN*, caused by
alloantibodies other than anti-D
Antibodies responsible
Pregnancies ⱖ 16 weeks for HDFN anti-
Dominant alloantibody other Number at Number per 100,000
than anti-D, first-trimester screen All (number) risk for HDFN Number population† (95%-CI)
Anti-K
Screen detected 233 43 5 1.7 (0.2-3.1)
K 4
K plus c 1
Screen undetected None 0
Anti-c
Screen detected 147 142 12 4.0 (1.7-6.3)
c 10
E plus c 1
c plus A 1
Screen undetected 5 1.3 (0.2-2.4)
c 3
c plus E 2
Anti-E
Screen detected 281 174 2 0.7 (0-1.6)
c plus E‡ 1
E plus B 1
Screen undetected 2
E 2 0.5 (0-1.2)
Rh antibodies other than anti-c, -D, -E
Screen detected 132 53 2 0.7 (0-1.6)
Screen undetected e 1
C plus Jka 1 0
Duffy antibodies
Screen detected 69 57 None 0
Screen undetected None 0
Other
Screen detected 115 98 None 0
Screen undetected 1
Miltenberger 1 0.3 (0-0.8)
Total
Screen detected 977 567 21 7.0 (4.0-10.1)
Screen undetected 8 2.0 (0.6-3.4)
All 9.1 (6.1-12.1)
* Severe HDFN: for definition see Table 3.
† Prevalence in population: denominators: detected = 298,000, estimated number of pregnancies ⱖ 16 weeks in study period (1.5 years);
undetected = 393,500, estimated number of pregnancies ⱖ 16 weeks in 2003 and 2004 (2 years).
‡ Additional anti-c detected in Week 20.

from control pregnancies and from index pregnancies not
at risk. Hb and Hct in cord blood were significantly lower
for antigen-positive children with anti-c; Rh antibodies
other than anti-c, -D, or -E; and anti-A or anti-B. Cord
blood Hb results of children who were transfused antena-
tally were excluded; the Hb levels of these fetuses before
the first IUT varied from 5.1 to 8.0 g per dL.
no mortality in 13 pregnancies with unknown outcome)
and 0.20 percent (1/489), respectively. Causes of death in
19 index pregnancies (of which 14 at risk of HDFN) were
hypertensive disorders and/or abruptio placentae (n = 5),
preterm birth (n = 5), congenital malformations (n =
2), placental problems (n = 3), hydatidiform mole (n = 1),
twin-to-twin transfusion syndrome (n = 1), and unknown
(n = 2).

Perinatal mortality in index pregnancies and

controls
Although we did not observe perinatal mortality directly
caused by HDFN, perinatal mortality occurring at at least
22 weeks in children from index pregnancies was six times
higher than in controls: 1.21 percent (12/991, assuming
Severe HDFN in screen-undetected cases
The coverage validation study identified eight cases of
severe HDFN, all with a negative screen at the first
trimester; seven of eight infants needed an exchange
transfusion because of anti-c (n = 3), anti-c and anti-E

948 TRANSFUSION Volume 48, May 2008

 HDFN CAUSED BY ANTIBODIES OTHER THAN ANTI-D

TABLE 5. Diagnostic performance of first-trimester RBC alloantibody screen for detecting HDFN caused by
antibodies other than anti-D*
Positive predictive
value first-trimester
Dominant alloantibody specificity Severe HDFN† RBC alloantibody screen‡
first-trimester screen Sensitivity Specificity Severe HDFN Phototherapy§ Icterus§
Anti-K 100 11.6 (2.0-21.2) 21.7 (8.0-35.3) 18.0 (5.3-30.7)
Anti-c 76.2 (55.2-97.2) 8.5 (3.9-13.0) 26.0 (18.4-33.5) 24.8 (17.4-32.2)
Anti-E 57.1 (5.3-100) 1.1 (0-2.7) 10.5 (5.8-15.2) 13.7 (8.4-18.9)
Rh antibodies other than 100 3.8 (0-8.9) 6.4 (0-13.3) 10.2 (1.7-18.8)
anti-c, -D, or -E
Anti-Duffy No patients 0 9.7 (1.6-17.8) 12.9 (3.7-22.1)
Other 0 0 1.7 (0-4.4) 6.6 (1.4-11.9)
Total 77.8 (62.1-93.5) 99.82 (99.80-99.83) 3.7 (2.1-5.3) 13.2 (10.3-16.1) 15.2 (12.1-18.3)
* Data are reported as % (95% CI).
† Severe HDFN: for definition see Table 3.
‡ Positive test = clinically relevant antibodies other than anti-D with pregnancy at risk for HDFN (father antigen positive/antigen typing
unknown).
§ Adjusted for antibody-unrelated icterus and phototherapy.

(n = 2), or anti-E (n = 2), respectively (Table 4). One of the
children (anti-c plus anti-E) suffered from kernicterus
with a maximum bilirubin level of 1109 mmol per L and
showed permanent severe brain damage at 1 year.
Another child (anti-c) had an intracerebral bleeding,
caused by asphyxia, possibly related to the severe prenatal
anemia (Hb 2.4 mmol/L = 3.8 g/dL). At 1 year, the progno-
sis was still unclear. One child only needed a top-up trans-
fusion because of anti-Miltenberger, a low-incidence
antigen. In this child other unrelated morbidity contrib-
uted to the observed anemia. The prevalence of severe
HDFN with negative antibody screens was 2 in 100,000,
leading to a total prevalence of 9 in 100,000 (Table 4).
Diagnostic performance of the screening program
for detecting severe HDFN
The sensitivity of the current program for detecting severe
HDFN associated with antibodies other than anti-D was
77.8 percent (Table 5). The specificity was 99.8 percent.
The positive predictive value for detecting severe HDFN,
phototherapy, and icterus was 3.7, 13.2, and 15.2 percent,
respectively, depending on alloantibody specificity, the
highest positive predictive values being for anti-K. To
detect one fetus at risk for severe HDFN, the number of
pregnant women needed to be screened was approxi-
mately 15,000, the number of women needed to be
screened to detect an IUT-dependent case was 60,000, and
the number of women needed to be screened to detect
one case needing IUT and/or exchange transfusion was
20,000.
relevant antibodies other than anti-D (index pregnancies)
versus 1.7 percent (7/412) of the controls (p < 0.001). For 1
of the 55 mothers who received transfusions, the pretrans-
fusion serologic analysis showed additional antibodies,
not detected during laboratory monitoring at the refer-
ence laboratory (anti-Cw and anti-s). No antibodies, rel-
evant for transfusion, were detected in controls. The
sensitivity of the current program for detecting all
transfusion-relevant antibodies was 98 percent (65/66). In
the majority of women who received transfusions with
clinically relevant antibodies other than anti-D (36/55,
66%), the screening early in pregnancy was the first occa-
sion at which the RBC antibodies were detected in the
mother; in only 19 of 55 index pregnancies the presence of
the RBC antibodies was already known.
In 13 percent (7/55) of the women with clinically rel-
evant antibodies, other than anti-D, who received a blood
transfusion around delivery, the presence or specificity of
the antibodies was not known by the transfusion labora-
tory of the hospital where the transfusion was given. This
concerned anti-K (n = 2), anti-c, anti-c plus -E, anti-S,
anti-Cw, and anti-Vw, including one case in which the
specificity of the antibodies, detected by the antibody
screen, was known, but not that of additional antibodies
detected later in pregnancy.
Ethnicity
No role of ethnicity-related heterogeneity could be estab-
lished in any of the results.

Blood transfusion during pregnancy and delivery DISCUSSION

to the mother This is the first report of a large prospective cohort study
A blood transfusion during pregnancy or delivery was on the epidemiology of alloantibodies, other than anti-D,
given to 6.1 percent (55/897) of the mothers with clinically in pregnancy and on the effectiveness of a single general

Volume 48, May 2008 TRANSFUSION 949

KOELEWIJN ET AL.
first-trimester screening program for such antibodies. We
found a 1:80 prevalence of positive antibody screens and
a 1:300 prevalence of clinically relevant alloantibodies,
other than anti-D, primarily of the specificities anti-E, -K,
and -c; a 1:500 prevalence of pregnancies with alloanti-
bodies other than anti-D at risk of HDFN (father antigen
positive); a performance of moderate diagnostic value of
first-trimester screening to detect these at-risk pregnan-
cies (approx. 75% sensitivity, close to 100% specificity);
excellent outcome in detected cases; and, of eight cases of
HDFN due to antibodies other than anti-D, with negative
RBC antibody screens, at least one case with causally
related poor outcome. The sensitivity of detecting all anti-
bodies, relevant in case of blood transfusion to the mother
during delivery, was 98 percent.
Comparison of our results with other studies is ham-
pered by the fact that screening protocols are heteroge-
neous or nonexact or that the definitions of a clinically
relevant alloantibody is lacking and, most importantly, by
the use of preselected high risk study populations with an
unknown relation to the prevalence in the general popu-
lation level.19-24 Our study can best be compared with two
regional Swedish studies25,26 in unselected populations, in
which slightly different screening protocols were applied.
The prevalences of clinically relevant alloantibodies other
than anti-D in these studies were 0.24 percent (188/
778,300) and 0.15 percent (171/111,939), respectively; it is
not clear whether only pregnancies at risk were included,
but we assume that this is in fact the case, taking into
account the proportion of antigen-negative children and
the prevalence of alloantibodies of the different specifici-
ties. The prevalences of severe HDFN, due to antibodies
other than anti-D, were 0.03 and 0.02 percent, respec-
tively. These data fit well with our data.
The observed sensitivity of the Dutch screening
program for detecting severe HDFN, caused by antibodies
other than anti-D, with a single first-trimester screening
was approximately 75 percent, for which we could not
find comparable estimates. To increase sensitivity one
might consider a second screening in pregnancy. In the
literature, however, no cases of severe HDFN were ever
detected by a second screening of D+ women, most likely
due to the small numbers of women included.27-29 Simi-
larly, in one of our academic hospitals, we detected, by a
second screening of 5800 women before delivery, a
maximum of 10 cases of new alloantibodies, other than
anti-D, none of which caused HDFN (J.M. Koelewijn, per-
sonal communication, 2004).
In 7 percent of the index pregnancies, later in preg-
nancy “new” additional antibodies were detected, of
which 70 percent probably developed because of expo-
sure to antigen-positive fetal cells during the current
pregnancy. This confirms a high rate of further alloimmu-
nization in patients who are already alloimmunized, that
is, after blood transfusion30 and IUT.31
950 TRANSFUSION Volume 48, May 2008
The nationwide design with full collaboration of all
caregivers and laboratories allowed for an unbiased esti-
mate of all principal variables. The following caveats
can be recognized: 1) The retrospective identification of
screen-negative patients with severe HDFN may be
incomplete because of undiagnosed HDFN after death of
a fetus and unintentional incomplete responses. There-
fore, the calculated sensitivity of the screening may in fact
be slightly lower than reported. 2) The ultimate benefit of
RBC alloantibody screening also rests on the effectiveness
and side effects of treatment and the absence of overtreat-
ment. The observational data suggest sufficient effective-
ness and lack of serious side effects. IUT appeared safe (no
consequences observed at the age of 1 year) as was
already known from the literature,32 and overtreatment
with phototherapy is unlikely, given the same prevalence
of phototherapy in antigen-negative children of pregnan-
cies at risk of HDFN, caused by antibodies other than
anti-D, compared to children 1) from screen-positive
pregnancies not at risk or 2) from screen-negative
pregnancies.
In terms of the criteria of Wilson and Jüngner,12
HDFN, caused by antibodies other than anti-D, in our
study, concerns a nonnegligible perinatal entity with cur-
rently 7 to 8 severe cases per 100,000 pregnancies. Mortal-
ity and long-term morbidity due to severe HDFN are rare
in cases detected by screening. Without such a screening
program, fetal death due to severe intrauterine anemia is
likely to occur in approximately 2 in 100,000 pregnancies,
where now IUT is life-saving because the risk of occur-
rence of HDFN is detected in time. The effect on long-term
morbidity depends on adequate detection of icterus in the
newborn. The unfavorable outcome in at least 1 and prob-
ably 2 of 8 screen-undetected cases suggests that in the
absence of screening, 2 to 4 in 100,000 children are at risk
for a delayed diagnosis of hyperbilirubinemia and subse-
quent kernicterus. This is particularly true if postnatal
monitoring by a caregiver is limited, as is true in many
countries, the Netherlands included.
The fact that 1 case of severe HDFN, caused by anti-
bodies other than anti-D, is detected by screening 15,000
women is comparable to other pre- and postnatal screen-
ing programs, and justifies RBC antibody screening, if data
on remaining criteria (e.g., costs) essentially do not differ
from those of accepted programs. Furthermore, by screen-
ing in the first trimester anti-D will also be detected with a
similar benefit of early diagnosis of severe HDFN, caused
by antibodies other than anti-D. In particular, in our study,
anti-c was associated with the 25 percent “missed” cases
of severe HDFN.
At the size of a nationwide program, the sensitivity
would increase by a relevant degree, if a second screening
in screen-negative c– women only (approx. 18% of the
population33) would be introduced. A second screening
for all pregnant women is a common policy in many coun-

tries. Assuming that in that case all new c-related cases
will be identified, one patient with severe HDFN would be
detected by about 9000 second screens, a result that is
superior to the current first-round efficiency.
An additional benefit of the screening program is the
detection of RBC antibodies, relevant in case of transfu-
sion to the mother. Without screening, two-thirds of the
RBC antibodies will not be known at pretransfusion labo-
ratory testing. Screening can save time, needed for the
identification of antibody specificities. A procedure is
required guaranteeing transfer of information about
relevant antibodies to the transfusion laboratory.
We conclude that a single first-trimester RBC allo-
antibody screen detects the important health problem
of severe HDFN, associated with antibodies other than
anti-D (in particular by anti-K and anti-c), with a sensitiv-
ity of 75 percent. Assuming professional and organiza-
tional acceptability, this program seems justified as part
of routine prenatal care. Performance might even be
increased by introducing repeated testing of c– pregnant
women.
ACKNOWLEDGMENTS
We thank all pregnant women and caregivers who participated
in the study. The Royal Dutch Society of Midwives (KNOV), the
Dutch Society of Obstetrics and Gynecology (NVOG), the
National Society of General Practitioners (LHV), the Dutch
Society for Clinical Chemistry (NVKC), and the Dutch Society of
Paediatrics (NVK) provided generous and unconditional support
throughout this study. Index pregnancies were identified by
Sanquin Diagnostic Services (Amsterdam) and the Special Insti-
tute for Blood group Investigations (BIBO, Groningen; M.A.M.
Overbeeke and C.A.M. Hazenberg are acknowledged for making
data of their laboratory registries available for the study). Cord
blood testing was performed by the Clinical Chemistry Labora-
tory of the Academic Medical Center in Amsterdam (hematologic
tests) and by Sanquin Diagnostic Services (serologic tests). Dr
M.G.A.J. Wouters (obstetrician) and Ms N. Som (technician) of the
Free University Medical Center provided data on second screen-
ing results of a pregnancy cohort at the Free University Medical
Center. Ms H. Van der Kroon (medical student) piloted the blood
cord protocol. Ms M. Braamskamp (medical student) partici-
pated in data collection. Ms R. Smit and Ms S. Saraiva-
Duivenvoorden (medical students) participated in the coverage
validation study. The Leiden University Medical Center (Dr D.
Oepkes, Ms J. Verdoes) provided additional clinical data and
expert advice on the reported HDFN patients.
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