BLOOD GROUP GENOMICS
RHD and RHCE variant and zygosity genotyping via multiplex
ligation–dependent probe amplification
Lonneke Haer-Wigman, Barbera Veldhuisen, Remco Jonkers, Martin Lodén, Tracey E. Madgett,
Neil D. Avent, Masja de Haas, and C. Ellen van der Schoot
BACKGROUND: The presence of a D variant may
hamper correct serologic D typing, which may result in
D immunization. D variants can be determined via RHD
genotyping. However, a convenient single assay to
identify D variants is still lacking. We developed and
evaluated a multiplex ligation–dependent probe amplifi-
cation (MLPA) assay to determine clinically relevant
RHD and RHCE variant alleles and RHD zygosity.
STUDY DESIGN AND METHODS: We analyzed 236
cases (73 normal and 163 selected samples) with the
RH-MLPA assay, which is able to determine 79 RHD
and 17 RHCE variant alleles and RHD zygosity. To
confirm the results, mutations were verified by RHD
and/or RHCE exon–specific sequencing and RHD
zygosity was verified by quantitative real-time poly-
merase chain reaction (PCR) for 18 cases.
RESULTS: In 99% of the cases, the RH-MLPA assay
correctly determined whether a person carried only
wild-type RHD and RHCE alleles (n = 69) or (a) variant
RHD allele(s) and/or (a) variant RHCE allele(s)
(n = 164). In only three cases, including two new RHD
variant alleles, the variant allele was not identified, due
to lack of detecting probes. These were RHD*DCS2, a
new partial RHD allele, RHD*525T (Phe175Leu), and a
new D– null allele, RHD*443G (Thr148Arg). All RHD
(n = 175) and RHCE variant alleles (n = 79) indicated by
the RH-MLPA assay were confirmed by sequencing.
RHD zygosity was confirmed by quantitative PCR. Two
hematopoietic chimeras were recognized.
CONCLUSION: The RH-MLPA genotyping assay is a
fast, easy, and reliable method to determine almost all
clinically relevant RHD and RHCE variant alleles, RHD
zygosity, and RHD+/RHD– chimeras in blood donors,
blood recipients, and pregnant women.
T
he Rh blood group system is one of the most
complex and immunogenic blood group
systems in humans.1,2 Antibodies against the Rh
antigens can give severe hemolytic transfusion
reactions and/or hemolytic disease of the fetus and/or
newborn.3,4 Therefore, it is crucial to correctly determine
the Rh status in blood donors, blood recipients, and preg-
nant women to prevent immunization against the Rh
antigens.5-7
The antigens of the Rh blood group system are
encoded by two closely linked genes, RHD and RHCE, that
encode the homologous RhD and RhCE proteins, respec-
tively.8 The RhD protein carries the D antigens and the
RhCE protein carries the C, c, E, and e antigens.9-13
A wide genetic diversity exists in RHD and RHCE,
resulting in more than 250 RHD and RHCE variant alle-
les.14 These variant alleles have been classified into four
groups: the partial RHD alleles, alleles encoding the weak
D, and D-elution (Del) phenotypes, the D– null alleles and
the RHCE variant alleles, based on their phenotypic
expression (see Web resources). Individuals carrying a
partial RHD allele lack one or more D epitopes and are
prone to make alloantibodies against the epitopes they
miss.2,15 Individuals carrying a weak D or Del allele express
ABBREVIATIONS: MLPA = multiplex ligation–dependent probe
amplification; RHD-MPX = RHD multiplex; SNP = single-
nucleotide polymorphism.
From the Sanquin Research and Landsteiner Laboratory,
Academic Medical Centre, University of Amsterdam, and
MRC-Holland, Amsterdam, the Netherlands; and School of
Biomedical and Biological Sciences, Plymouth University,
Plymouth, United Kingdom.
Address reprint requests to: Lonneke Haer-Wigman,
Immunohematology Experimental, Sanquin Blood Supply,
Plesmanlaan 125, Amsterdam, 1066 CX, The Netherlands;
e-mail: L.Wigman@sanquin.nl
Received for publication March 16, 2012; revision received
July 17, 2012, and accepted August 20, 2012.
doi: 10.1111/j.1537-2995.2012.03919.x
TRANSFUSION 2013;53:1559-1574.
Volume 53, July 2013 TRANSFUSION 1559
HAER-WIGMAN ET AL.
the RhD protein, but in low amounts.2,15 In theory, indi-
viduals with a weak D or Del variant express all D epitopes;
hence they will not make anti-D.14,15 There have been,
however, reports describing alloanti-D formation in indi-
viduals with weak D variants.16-18 Both weak D and Del can
elicit an immune response in D– recipients.19,20 Individu-
als with a D– null allele lack the complete expression of the
RhD protein.14,15 D negativity is most often caused by the
entire deletion of the RHD gene (the RHD*01N.01 allele) in
the Caucasian population or the presence of the RHD*y
allele (RHD*Pseudogene) or the hybrid RHD*DIIIa-CE(3-
7)-D allele, formerly called (C)ceS Type 1 in the black
African population.21-23
Serology with monoclonal anti-D is currently the
standard method to determine the Rh status of blood
donors, recipients, and pregnant women.2,15,24 Serologic
typing will, in most cases, only reveal the presence of an
RhD variant, but cannot exactly determine which variant
is present.2,5 Since transfusion policy, as well as preventive
measurements during pregnancy, are based on the exact
nature of the RhD variant, it is important to correctly
determine which RhD variant is present.2,5,6,14 RHD and
RHCE variant alleles can be determined via genotyping of
the RHD and RHCE genes.5,25,26 Since molecular genotyp-
ing methods are increasingly applied for blood donor
typing and for noninvasive fetal RHD genotyping, the
number of cases with discrepant serologic and genotyping
results that require further molecular analysis is expand-
ing.5,25 A method to identify a broad range of clinically
relevant Rh variants (all frequently occurring variants and
all variants where the carrier is able to make alloanti-D) in
a convenient and cheap assay is still lacking.14,25
Only pregnant women who are D– or express an RhD
variant and who are carrying a D+ fetus are at risk for
anti-D formation and subsequently of anti-D–mediated
hemolytic disease of the fetus and/or newborn.3,6 Cur-
rently, the presence of a D+ fetus can be determined with
noninvasive fetal RHD typing.27,28 If the father is homozy-
gous for the RHD*01 allele (wild-type RhD) this test can be
omitted.6,14 Upon serologic typing of the C, c, E, and e
antigens of the father, a prediction of the paternal zygosity
can be made based on the most frequently occurring
RHD/RHCE haplotypes.6,29 Because the haplotype fre-
quencies differ between ethnic populations, these predic-
tions are not reliable without knowledge of the ethnic
background.6,29 The exact paternal zygosity can be det-
ermined by genotype-based methods; however, the
methods used at the moment are cumbersome and can be
confounded by genetic alterations, especially in the black
African population.30-33
We therefore developed a multiplex ligation–
dependent probe amplification (MLPA) genotyping assay.
This single assay detects mutations and copy number
variation of the RHD and RHCE genes and is easy to use, as
it only requires a thermocycler and capillary electrophore-
1560 TRANSFUSION Volume 53, July 2013
sis equipment. The aim of this study was to evaluate the
performance of the RH-MLPA genotyping assay in the
determination of clinically relevant RHD and RHCE
variant alleles and RHD zygosity.
MATERIALS AND METHODS
Materials
DNA samples from 73 random blood donors with normal
Rh serology were included after informed consent was
given (including 11 D– and 62 D+ samples; 44 C+, 60 c+, 27
E+, and 68 e+ samples).
Sanquin Diagnostics is the national reference labora-
tory for the analysis of the presence of Rh variants in blood
recipients, blood donors, and pregnant women. For this
study 97 cases were selected, which were analyzed
between 2003 and 2011. This selection included preferen-
tially a minimum of three samples for each known RHD
variant allele and all samples of which the specific RHD
variant was not yet determined. Twenty DNA samples
were provided by other blood group centers and were
selected for the presence of a specific RHD variant allele.
Forty-six cases positive for the RHD*y allele were obtained
from the screening program of D– pregnant women.
D serology
Serologic D typing was routinely preformed with two
anti-D reagents. A monoclonal anti-D reagent (immuno-
globulin [Ig]M clone MS201, Sanquin Reagents, Amster-
dam, the Netherlands) and a monoclonal blend reagent
(IgM clone TH28 and IgG clone MS26, Sanquin Reagents)
were used in a method with an immediate spin at room
temperature. All D– samples were also tested with the
monoclonal blend reagent in the indirect antiglobulin
test. In case of a discrepancy between the results of the
two reagents, the D-epitope pattern was determined with
an in-house panel of anti-D reagents and/or the extended
partial RhD typing set from Bio-Rad Laboratories B.V.
(Veenendaal, the Netherlands).
DNA isolation
Ethylenediaminetetraacetic acid–anticoagulated blood
was collected and genomic DNA was isolated from
white blood cells (WBCs) using a DNA extraction kit
(QIAamp DNA blood mini kit, Qiagen Benelux, Venlo, the
Netherlands).
MLPA
An MLPA assay specific for the analysis of RHD and RHCE,
their variants and zygosity was developed. This RH-MLPA
assay contains 17 RHD wild type, five RHCE wild type, 21

RHD mutation, and two RHCE mutation probe combina-
tions (Table 1; hybridizing sequences are shown in
Supplementary Table S1, available as supporting informa-
tion in the online version of this paper). So-called wild-
type probe combinations are used to determine the copy
number of wild-type RHD or RHCE sequence, while muta-
tion probe combinations detect the presence of a mutated
sequence. Twenty-five probe combinations were conven-
tionally designed and consisted of two probe pieces and
for the additional 20 probe combinations a three-piece
design was chosen (Table 1).
The RH-MLPA assay is able to distinguish between
51 RHD and 13 RHCE variant alleles. For another 28 RHD
and four RHCE variant alleles the assay can identify the
main type, but cannot discriminate between several sub-
types, for example, RHD*DOL1 or RHD*DOL3 (Table 2).
Since probes hybridizing to the same regions will
hamper each others’ binding, the probes of the RH-
MLPA had to be divided into three pools, p401, p402, and
p403 (Table 1).
The MLPA reaction was performed according to the
manufacturers’ protocol (MRC Holland, Amsterdam, the
Netherlands) on a thermocycler (Biometra T1, Wester-
burg BV, Leusden, the Netherlands; or Veriti, Applied Bio-
systems, Nieuwerkerk aan de IJssel, the Netherlands).
Supplementary Fig. S1 (available as supporting informa-
tion in the online version of this paper) shows a sche-
matic overview of the MLPA reaction. In short, 5 mL
containing 100 ng of DNA was denatured and 1.5 mL
probe mix and 1.5 mL SALSA MLPA dilution buffer were
added at 25°C. After hybridization at 60°C for between 16
and 20 hours, 1 mL of SALSA ligase-65, 1.5 mL of SALSA
ligase Buffer A, and 1.5 mL SALSA ligase Buffer B were
added and incubated for 15 minutes at 54°C. A poly-
merase chain reaction (PCR) was performed in a total
volume of 50 mL containing 4 mL of SALSA dilution buffer,
2 mL of SALSA enzyme dilution buffer, 2 mL of universal
primers, 0.5 mL of SALSA polymerase, and 10 mL of liga-
tion sample. PCR conditions were as follows: 5 minutes at
95°C, 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C
and 1 minute at 72°C, followed by 20 minutes at 72°C. A
mixture of 1.5 mL of MLPA sample, 8.5 mL of formamide
(Hi-Di, Applied Biosystems), and 0.5 mL of size standard
(GeneScan, 500-Liz, Applied Biosystems) was analyzed
on a genetic analyzer (Model 3130, Applied Biosystems).
Data analysis was performed using computer software
(Genemarker, Version 1.85, Softgenetics, State College,
PA). For each run two control samples hemizygously
positive for the RHD*01 allele, as determined via RHD
Exons 5- and 7-specific quantitative real-time PCR were
used as reference samples to determine zygosity. Supple-
mentary Fig. S2 (available as supporting information in
the online version of this paper) shows an example of the
results of an MLPA reaction. The total RH-MLPA assay
takes approximately 20 hours, including 16 hours of
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
hybridization and 2 hours of hands-on time. Between 1
and 16 samples can be analyzed simultaneously in a
single run consisting of all three RH-MLPA mixes.
RHD multiplex PCR
In our institution all samples that show unexpected weak
or negative reactions with D-typing reagents are cur-
rently analyzed using a multiplex PCR, simultaneously
detecting RHD Exons 3, 4, 5, 6, 7, and 9 and an internal
control.34 The RHD multiplex (RHD-MPX) PCR was per-
formed on a DNA engine thermocycler (Dyad, Bio-Rad
Laboratories B.V.) in a total volume of 50 mL, containing
10 to 1000 ng DNA, 5 mL of 10¥ FastStart Taq DNA poly-
merase buffer without MgCl2 (Roche, Woerden, the Neth-
erlands), 3 mL of MgCl2 25 mmol/L stock solution
(Roche), 1 mL of PCR-grade nucleotide mix (Roche), 15 mL
of primer mix (primer dilution between 0.1 and
1.2 mmol/L; Eurogentec, Maastricht, the Netherlands).
PCR conditions were as follows: 5 minutes at 95°C, 33
cycles of 1 minute at 95°C, 1 minute at 56°C, and 1
minute at 72°C, followed by 5 minutes at 72°C. The pres-
ence or absence of the RHD exons and internal control
was analyzed by gel electrophoresis on a 1.5% (wt/vol)
agarose gel (Invitrogen, Breda, the Netherlands).
PCR and sequence reaction
RHD and RHCE exon–specific sequencing was performed
using RHD- and RHCE-specific primers flanking each
exon. The primers were synthesized by Eurogentec and
sequences are shown in Supplementary Table S2 (avail-
able as supporting information in the online version of
this paper). The PCR was performed on a thermocycler
(Veriti, Applied Biosystems) in a total volume of 20 mL,
containing 50 to 150 ng DNA, 10 mL of 2¥ PCR master mix
(GeneAmp Fast, Applied Biosystems), 0.5 mmol/L forward
and reverse primer. PCR conditions were as follows: 10
seconds at 95°C, 35 cycles of 10 seconds at 95°C, and a
specific annealing-elongation temperature and time for
each primer set (listed in Supplementary Table S2), fol-
lowed by 1 minute at 72°C. PCR products were purified
using PCR product cleanup (ExoSAP-IT, GE Healthcare,
Eindhoven, the Netherlands), according to manufactur-
er’s protocol. The sequence reaction was performed on a
thermocycler (Veriti, Applied Biosystems) in a total
volume of 20 mL, containing 1 mL of purified PCR product,
1 mL of 2.5¥ polymerase mix (BigDye Terminator v1.1,
Applied Biosystems), 3.5 mL of 5¥ buffer (BigDye Termina-
tor, Applied Biosystems), and 0.25 mmol/L forward or
reverse primer. Sequence conditions were as follows:
25 cycles of 15 seconds at 95°C, 10 seconds at 50°C, and
4 minutes at 60°C. Sequence products were analyzed on
a genetic analyzer (3130, Applied Biosystems).
Volume 53, July 2013 TRANSFUSION 1561
1562
TABLE 1. Performance of RHD- and RHCE-specific probes from the RH-MLPA tested in 236 cases
Exon or Primary Secondary Probe Present Signal detected Mutation verfied
Probe name Gene intron ligation site* ligation site† size Use of probe/detecting in mix in tested cases by sequencing
D00_-132A RHD 5′ UTR –132A –173C 217 RHD*01 P401 222
MD01_008G RHD Exon 1 8G 149 RHD*weak D Type 3 P401 3 3
DCE01_048C RHD/RHCE Exon 1 48C 143 RHCE P402 191
MD01_048A RHD Exon 1 48A 144 RHD*48A P403 3 3
MCE01_122G RHCE Exon 1 122G 156 RHCE*CeCW P402 4 4
HAER-WIGMAN ET AL.

DCE02_IVS1-20G RHD/RHCE Intron 1 IVS1–20G 180 RHD*01 and RHCE*C P402 221
MD02_186T RHD Exon 2 186T 225C 241 RHD*DIIIa, RHD*DIII.4, RHD*DIVa.1, P401 14 5
RHD*DIVa.2, RHD*DIIIa-CE(3-7)-D
DCE02_203G RHD/RHCE Exon 2 201G/203G 224 RHD*01 and RHCE*C P403 220
MD02_270A RHD Exon 2 270A 214 RHD*270A P403 0 0
CE02_307C RHCE Exon 2 307C 269G 218 RHCE*c P402 208
MD02_329C RHD Exon 2 329C 158 RHD*DVII.1 P403 6 6
CE02_ins_C RHCE Intron 2 RHCE*C insertion 156 RHCE*C P401 123
D03_380T RHD Exon 3 380T 405C 167 RHD*01 P402 220

TRANSFUSION Volume 53, July 2013

MD03_410T RHD Exon 3 410T 383A 192 RHD*DIIIa, RHD*DIII.4, RHD*DIII.6, RHD*DIVa.2, P401 13 9
RHD*DOL3, RHD*DIIIa-CE(3-7)-D
MD03_446A RHD Exon 3 446A 419T 188 RHD*weak D Type 5 P403 4 3
D03_455A RHD Exon 3 455A IVS3+4T 175 RHD*01 P402 207
MD03_IVS3+1A RHD Intron 3 IVS3+1A 247 RHD*IVS3+1A P401 4 4
MD04_509C RHD Exon 4 509C 163 RHD*DOL1, RHD*DOL2, RHD*DOL3 P402 1 1
D04_514A RHD Exon 4 514A IVS3–5T 187 RHD*01 P401 168
D04_602C RHD Exon 4 602C 224 RHD*01 P402 196
MD04_609A RHD Exon 4 609A 130 RHD*y P401 46 5
D05_667T RHD Exon 5 667T 639C 211 RHD*01 P402 159
CE05_676G RHCE Exon 5 676G 712A 205 RHCE*e P401 230
CE05_676C RHCE Exon 5 676C 712A 211 RHCE*e P401 51
D05_697G RHD Exon 5 697G 192 RHD*01 P403 195
MCE05_733G RHCE Exon 5 733G 787A 204 RHCE*ceVS, RHD*DIIIa-CE(3-7)-D P402 19 10
D05_787G RHD Exon 5 787G IVS5+17C 181 RHD*01 P401 198
D05_800A RHD Exon 5 800A 148 RHD*01 P403 202
MD06_807G RHD Exon 6 807G 167 RHD*807G, RHD*y P403 46 4
MD06_809G RHD Exon 6 809G 192 RHD*weak D Type 1 P402 24 5
MD06_819A RHD Exon 6 819A IVS5-9T 169 RHD*DIIIa, RHD*DIII.6, RHD*weak D Type 4.0, P401 5 3
RHD*weak D Type 4.1, RHD*weak D Type 4.3
MD06_845A RHD Exon 6 845A 164 RHD*weak partial D Type 15 P403 5 4
MD06_872G RHD Exon 6 872G 132 RHD*weak D Type 4.3 P402 0 0
MD06_885T RHD Exon 6 885T 906G 174 RHD*weak partial D Type 11 P401 5 4
D06_932A RHD Exon 6 932A 237 RHD*01 P402 203
D07_941G RHD Exon 7 941G 174 RHD*01 P403 211
D07_989A RHD Exon 7 989A 143 RHD*01 P401 211
MD07_1025C RHD Exon 7 1025C 992A 151 RHD*DIV.3, RHD*DAR1, RHD*DAR2, RHD*weak P402 7 4
D Type 29
D07_1061T RHD Exon 7 1061T 181 RHD*01 P403 206
MD07_1063A RHD Exon 7 1063A IVS7+25T 222 RHD*DNB P401 3 3
MD08_1136T RHD Exon 8 1136T 211 RHD*DAU P403 5 5
MD09_1154C RHD Exon 9 1154C 1999A 236 RHD*weak D Type 2 P401 8 5
D09_1193A RHD Exon 9 1193A IVS9+2T 187 RHD*01 P402 219
MD09_1227A RHD Exon 9 1227A IVS9+46C 204 RHD*1227A P403 3 3
D10_1375C RHD Exon 10 1375C 162 RHD*01 P401 223
* Position as counted from ATG translation start site and nucleotide of the ligation site that is specific for the nucleotide detected by the probe.
† Position as counted from ATG translation start site and nucleotide of a second ligation site which is used to make the probe specific for RHD or RHCE.
 TABLE 2. RHD and RHCE variant alleles that can be determined using the RH-MLPA assay
Loss or gain of wild-type-probe combination(s) in specific variant allele

P402 D03_380T
P402 D05_667T

P402 D03_455A
P401 D04_514A
P403 D05_800A
P402 D06_932A
P401 D07_989A

P402 D04_602C
P403 D05_697G
P401 D05_787G
P403 D07_941G

P401 D00_-132A
P402 D09_1193A

P403 D07_1061C
P401 D10_1357C

P402 CE02_307C
P401 CE05_676C
P401 CE05_676G

P401 CE02_ins_C
P402 DCE02_-20C

P402 DCE01_048C
P403 DCE02_203G
Gain of mutation-probe combination(s)
Variant allele RHCE RHD in specific variant allele
RHD*DII/RHD*DIV.4 -
RHD*DIIIa - - - MD02_186T MD03_410T MD06_819A
RHD*DIIIb + - - - - - MD03_410T MD06_819A
RHD*DIII.3 - -
RHD*DIII.4/RHD*DIVa.2 - MD02_186T MD03_410T
RHD*DIII.6 - - - MD03_410T MD06_819A
RHD*DIVa.1 - MD02_186T
RHD*DIV.3 - - - - - MD07_1025C
RHD*DIV.5 - - - -
RHD*DIV.6 - -
RHD*DV.1 - -
RHD*DV.2 + - - - -
RHD*DV.3/RHD*DBS2 + - -
RHD*DV.4/RHD*DV.5/ -
RHD*DV.9
RHD*DV.6/RHD*DV.8 + - -
RHD*DV.7 + - - -
RHD*DVI.1 + - - - - - -
RHD*DVI.2 + - - - - - - -
RHD*DVI.3 + - - - - - - - - -
RHD*DVI.4 + - - - - - - - -
RHD*DVII.1 MD02_329C
RHD*DVII.2 + MD02_329C
RHD*DAR1 - - MD07_1025C
RHD*DAR2 - - - MD07_1025C
RHD*weak partial D type 4.0/ RHD*weak - - MD06_819A
partial D type 4.1
RHD*weak partial D type 4.3 - - MD06_819A MD06_872G
RHD*DAU0/ MD08_1136T
RHD*DAU1/
RHD*DAU2/
RHD*DAU3/
RHD*DAU6/
RHD*DAU7
RHD*DAU4 - MD08_1136T

Volume 53, July 2013

RHD*DAU5 - - MD08_1136T
RHD*DOL1/RHD*DOL2 - MD04_509C
RHD*DOL3 - MD03_410T MD04_509C
RHD*DBS1 + - - - -
RHD*DBT1 - - - - - - - -
RHD*DBT2 - - - - - - - - -
RHD*DCS1/RHD*DTO -
RHD*DFR1/RHD*DFR3 -

TRANSFUSION
RHD*DFR2 - -
RHD*DNB - MD07_1063A

1563
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
1564
TABLE 2. Continued
Loss or gain of wild-type-probe combination(s) in specific variant allele
HAER-WIGMAN ET AL.

P402 D03_380T
P402 D05_667T

P402 D03_455A
P401 D04_514A
P403 D05_800A
P402 D06_932A
P401 D07_989A

P402 D04_602C
P403 D05_697G
P401 D05_787G
P403 D07_941G

P401 D00_-132A
P402 D09_1193A

P403 D07_1061C
P401 D10_1357C

P402 CE02_307C
P401 CE05_676C
P401 CE05_676G

P401 CE02_ins_C
P402 DCE02_-20C

P402 DCE01_048C
P403 DCE02_203G
Gain of mutation-probe combination(s)
Variant allele RHCE RHD in specific variant allele
RHD*weak partial D type 11 MD06_885T
RHD*weak partial D type 15 MD06_845A
RHD*weak D type 1 MD06_809G

TRANSFUSION Volume 53, July 2013

RHD*weak D type 2 MD09_1154C
RHD*weak D type 3 MD01_008G
RHD*weak D type 5 MD03_446A
RHD*weak D type 14/RHD*weak D type -
40/RHD*weak D type 51
RHD*weak D type 29 - - MD07_1025C
RHD*weak D type 41 -
RHD*1227A MD09_1227A
RHD*IVS3+1A MD_IVS3+1A
RHD*01N.01 - - - - - - - - - - - - - - - - -
RHD*y - - MD04_609A MD06_807G
RHD*CE(1-9)-D + + - - - - - - - - - - - - - - - -
RHD*CE(2-7)-D + - - - - - - - - - - - - - -
RHD*CE(2-9)-D + + - - - - - - - - - - - - - - -
RHD*DIIIa-CE(3-7)-D - - - - - - - - - - - MD02_186T MD03_410T MCE05_733G
RHD*CE(3-7)-D + - - - - - - - - - - - -
RHD*CE(3-9)-D + - - - - - - - - - - - - -
RHD*CE(4-7)-D + - - - - - - - - - -
RHD*48A MD01_048A
RHD*270A MD02_270A
RHD*807G MD06_807G
RHD*941T -
RHCE*ceAR/RHCE*ceEK + - + +
RHCE*ceBI/RHCE*ceSM + -
RHCE*ceCF + + MCE05_733G
RHCE*ceHAR ⱖ1 - + + + +
RHCE*ceMO + +
RHCE*ceVS MCE05_733G
RHCE*ce-D(9)-ce +
RHCE*CeCW MCE01_122G
RHCE*CeFV - + +
RHCE*CeRN1 + +
RHCE*CeRN2 + + +
RHCE*CeVA ⱖ1 - + + + +
RHCE*Ce800A +
RHCE*cEFM + - +
RHCE*cE602C +

RHD Exons 5- and 7-specific quantitative
real-time PCR
Zygosity was determined with an RHD Exons 5- and
7-specific quantitative real-time PCR. Primers were devel-
oped to detect ALB35 (quantification of input DNA), SRY,36
and RHD Exons 527 and 7.37 The RHD Exons 5 and 7
primers and probes were synthesized by TIB MOLBIOL
(Berlin, Germany) and the primers for SRY and the ALB
were synthesized by Invitrogen. The real-time PCR was
performed on a sequence detection system (ABI PRISM
7000, Applied Biosystems) in a total volume of 25 mL, con-
taining 25 ng of DNA, 12.5 mL of universal primer PCR
master mix (Taqman, Applied Biosystems), 0.1 mmol/L of
the RHD Exon 5 and SRY probe or RHD Exon 7 and ALB
probe and 0.3 and 0.9 mmol/L of the RHD Exon 5 and SRY
forward and reverse primers, respectively, or 0.3 mmol/L of
the RHD Exon 7 and ALB forward and reverse primers.
Real-time PCR conditions were as follows: 2 minutes at
50°C, 10 minutes at 95°C, and 50 cycles of 15 seconds at
95°C and 1 minute at 60°C. Data analysis was performed
using computer software (Microsoft Office Excel 2003,
Microsoft, Amsterdam, the Netherlands).
Short-tandem-repeat multiplex PCR
The presence of chimerism was determined with genomic
DNA isolated from WBCs using short-tandem-repeat mul-
tiplex (Powerplex 16 HS system, Promega, Leiden, the
Netherlands). The reaction was performed according to
the manufacturer’s protocol.
RESULTS
Performance of RH-MLPA assay with plasmid DNA
First, all RH-MLPA wild-type (n = 22) and mutation probe
combinations (n = 23) were analyzed using a cloned DNA
plasmid sample, containing the genomic sequence of all
probes, as a template. For all probes correct signals were
detected (data not shown) and subsequently the perfor-
mance of the RH-MLPA was evaluated with a set of
genomic DNA samples.
Performance of RH-MLPA assay with
genomic DNA
Wild-type probe combinations
Next, the performance of the RH-MLPA assay was ana-
lyzed with DNA from 73 healthy donors with normal Rh
serology, representing all common Rh phenotypes. The
RHD wild-type probe combinations are able to detect the
copy number of all RHD exons, except that of Exon 8. No
probe combination for RHD Exon 8 was included, since
the sequence of RHD and RHCE Exon 8 is completely
identical. The probe combinations detecting wild-type
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
RHD Exon 2 also detect RHCE*C, because the RHD and
RHCE*C sequence of Exon 2 is completely identical.
RHCE*C genotyping is based on the detection of
the RHCE*C specific 109-nucleotide insertion in Intron
2 and 203G in Exon 2. RHCE*c genotyping is based on the
detection of 307C. RHCE*E and RHCE*e genotyping is
based on the detection of 676C and 676G, respectively. To
ensure RHCE specificity, the latter two probe combina-
tions consist of three probe pieces, creating a second
ligation site for the 712A RHCE-specific nucleotide. In all
D+ cases (n = 62) all RHD wild-type probe combination
signals were present, while in the D– cases (n = 11) all RHD
wild-type probe combination signals were absent. The
RH-MLPA RHCE*ce, RHCE*Ce, RHCE*cE, and RHCE*CE
genotyping results were completely concordant with
serology. All possible common RHD/RHCE haplotypes
were determined at least once (data not shown). In four
cases one or more mutation probe combination signals
were detected: two cases showed the presence of the
RHD*DVII.1 allele next to an RHD*01 allele (wild-type
RhD); similarly one case had an RHD*y allele next to an
RHD*01 allele and one case carried an RHCE*CeCW allele.
Sequencing of the involved exons in these cases con-
firmed the presence of the variant alleles detected by the
RH-MLPA.
Mutation probe combinations
The mutation probe combinations of the RH-MLPA were
selected to recognize, in combination with the wild-type
probe combinations, the majority of clinically relevant
RHD variant alleles and most frequently occurring RHCE
variant alleles in the Caucasian and black African popula-
tions. The performance of the mutation probe combina-
tions was evaluated with DNA from 97 cases, for whom
serology indicated the presence of an Rh variant and with
DNA from 66 cases, for whom the presence and/or
expressing of an Rh variant was already determined. With
this set of samples 19 RHD mutation probe combination
signals and two RHCE mutation probe combination
signals were obtained at least once. To confirm the pres-
ence of a mutation indicated by one of the mutation probe
combinations, the exons containing the respective muta-
tions were sequenced for at least three independent
samples (if available). All sequencing results were concor-
dant with the results of the RH-MLPA (Table 1). For two
RHD mutation probe combinations MD02_270A specific
for the RHD*270A allele and MD06_872G specific for the
RHD*weak partial D Type 4.3 allele no genomic DNA was
available.
The RHCE mutation probe combination MCE05_
733G was designed to determine whether an individual is
positive (733G) or negative (733C) for the VS antigen. Of
note, individuals carrying an RHCE*ceAR allele have the
733G mutation, but are negative for the VS antigen.38 We
designed the MCE05_733G with a second ligation site
Volume 53, July 2013 TRANSFUSION 1565
HAER-WIGMAN ET AL.
specific for the 787A RHCE wild-type single-nucleotide
polymorphism (SNP), which is present in all VS+ alleles
and absent in the RHCE*ceAR allele. Six of the nine cases
carrying a RHCE*ceAR allele were VS negative and indeed
these samples showed correctly the absence of signal for
MCE05_733G. In the three other cases the second RHCE
allele was a VS+ allele and as expected the MCE05_733G
signal was obtained.
Determination of RHD and RHCE variant alleles
using the RH-MLPA assay
In 160 of the above-described 163 individuals the
RH-MLPA indicated the presence of one or more variant
alleles. In these 163 cases a total of 172 RHD and 78 RHCE
variant alleles or variant allele groups were determined as
shown in Table 3.
The assigned RHD and RHCE variant allele(s) were in
all cases compatible with the initial serologic results
obtained for the cases. In addition, in 99 of the 163
selected samples, we performed an RHD-MPX PCR, in
which RHD Exons 3, 4, 5, 6, 7, and 9 are RHD specifically
amplified. The results of the RHD-MPX combined with the
results of the serology were concordant with the RHD vari-
ants concluded from the RH-MLPA, except for 11 cases.
In these cases, carrying the RHD*DV.5, RHD*DAU2,
RHD*DAU3, RHD*weak partial D Type 11, or RHD* weak
partial D Type 15 allele, the RHD-MPX results showed no
aberrant pattern, suggesting the presence of weak D vari-
ants, while the RH-MLPA indicated the presence of partial
RhD variants. Sequencing of the exons containing the
mutations showed that in these cases the RH-MLPA
assigned the correct RHD variant alleles (Table 3). In one
case the RH-MLPA indicated the presence of a new partial
RHD allele, containing the mutations of both RHD*DIIIb
and RHD*DNB on one allele (150T>C, 178A>C, 201G>A,
203G>A, 307T>C, and 1063G>A). Zygosity analysis showed
the presence of one RHD allele and sequencing confirmed
the presence of the mutations. Unfortunately, no red
blood cells (RBCs) were available for further serologic
typing. In 23 cases the RH-MLPA detected the presence of
a variant allele, but could not discriminate between differ-
ent subtypes (e.g., RHD*DAU alleles and RHD*weak
partial 4.0 or RHD*weak partial 4.1 alleles) and the
samples were sequenced to assign the specific RHD or
RHCE variant subtype (Table 3).
In three cases in which serology indicated the pres-
ence of an Rh variant, the RH-MLPA indicated the pres-
ence of a single normal RHD*01 allele. Sequencing of all
RHD exons revealed that one case concerned a hemizy-
gously present RHD*DCS2 allele (676G>C), for which no
RHD-specific probe combination is included. In the two
other cases two new RHD variant alleles were detected.
One new RHD variant allele has the 525C>T (Phe175Leu)
mutation and the other RHD variant allele has the 443C>G
1566 TRANSFUSION Volume 53, July 2013
(Thr148Arg) mutation. The latter was determined as D–
via the absorption-elution technique using a polyclonal
anti-D (anti-D bromelain, Sanquin Reagentia). This
patient was included in our series, because her current D
serology (D–) was discordant with a previously reported
D+ phenotype by the blood bank of Azerbaijan. Pro-
bably the donor was regarded as D+ because of the CE
phenotype (RhCcee). The new RHD*525T (Phe175Leu)
variant allele results in absence of expression of Epitope
1.2 (determined with monoclonal antibodies [MoAb]
LHM174/102 and LHM70/45 of the extended partial
RhD typing set of Bio-Rad Laboratories) and Epitope 2.2
(determined with MoAb 5C839). The LHM169/81 MoAb
(extended partial RhD typing set), which detects Epitope
1.1 showed the same strength of reactivity for both the
D+ control and the RBCs expressing the RHD*525T
(Phe175Leu) allele.40 Similarly, all other D epitopes tested
were normally present. The patient was typed positive for
the C, c, and e antigens. In two other cases no RHD variant
allele was detected, but an aberrant RHD copy number of
0.3 and 0.5, respectively, was detected. These samples are
further discussed below, see “RHD and RHCE zygosity”
results.
For three variant alleles a loss of signal of a wild-type
probe combination was obtained, whereas presence of
the RHD-specific nucleotide was shown by sequencing. In
three cases carrying the RHD*DNB or the new partial
RHD allele (150T>C, 178A>C, 201G>A, 203G>A, 307T>C,
1063G>A), the D07_1061T signal was absent. Apparently,
the 1063A mutation present in these alleles impaired the
complete hybridization and ligation of D07_1061T. In the
46 cases carrying the RHD*y allele, the D04_514A signal
was absent. The 37-bp duplication in intron 3 (IVS3-19)
present in the RHD*y allele prevents the hybridization of
the D04_514A probe combination, since the insertion
results in a shortening of the hybridization site to only 13
nucleotides instead of the normal 34 nucleotides. All 46
cases carrying the RHD*y allele were also associated with
an unexpected gain of the D09_1193A signal. The RHD
and RHCE Exon 9 were therefore PCR amplified in three
cases that were homozygous for the RHD*y allele. In these
cases RHCE Exon 9 could not be amplified, while RHCE
Exons 8 and 10 were normally amplified and sequencing
of these exons and surrounding introns showed RHCE
wild-type sequence. We therefore hypothesize that in this
RHCE variant allele, linked to the RHD*y allele, RHCE
Exon 9 is replaced by RHD Exon 9 resulting in a RHCE*ce-
D(9)-ce allele. This hybrid RHCE allele is normally
expressed since these three homozygous cases were all
serologically positive for the c and e antigens and also no
abnormal reaction patterns have been described for D–
black Africans. As no product was amplified in the RHCE
Exon 9 PCR, the break points of the mutated RHCE allele
are located outside the range of at least one of our RHCE
Exon 9 primers. To confirm the presence of this hybrid
 TABLE 3. RHD and RHCE variant alleles detected by the RH-MLPA assay in 163 DNA samples
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
Partial RHD alleles RHD*DIIIa RHD*DIIIa RHCE*ceVS 1
RHD*DIIIb New RHD variant 1
(150T>C, 178A>C,
201G>A, 203G>A,
307T>C, 1063G>A)
RHD*DIII.3 RHD*DIII.3 1
DIVa RHD*DIII.4 or RHD*DIVa.2 3
RHD*DIVa.2
RHD*DIV.4 RHD*DII or RHD*DIV.4 1
RHD*DIV.4
DVI t1 RHD*DVI.1 2
DVI t2 RHD*DVI.2 7
DVII RHD*DVII.1 4
DAR§ RHD*DAR1 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK
RHD*DAR1 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK and
RHCE*ceVS
RHD*DAR1 RHCE*ceAR or RHCE*ceAR 1
(homozygous) RHCE*ceEK (homozygous)
(homozygous)
RHD*DAR1 and RHCE*ceAR or RHCE*ceAR 1
RHD*DIIIa-CE(3-7)-D RHCE*ceEK
RHD*DAR2 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK
RHD*weak D Type 29 1
RHD*weak partial D RHD*weak partial D 3
Type 11 Type 11
RHD*DBT2 RHD*DBT2 1
RHD*DCS1 RHD*DCS1 1
DFR RHD*DFR1 1
RHD*DFR2 2
RHD*DOL1 RHD*DOL1 or RHCE*ceBI or RHD*DOL1 RHCE*ceBI 1
RHD*DOL2 RHCE*ceSM
RHD*DNB RHD*DNB 2
Partial D variant RHD*01 RHD*DCS1 1

Volume 53, July 2013

New variant 1
RHD*525T
RHD*DV.7 3
RHD*DAU5 1
RHD*weak partial D RHCE*ceVS RHD*weak partial D 1
Type 4.0 or Type 4.0
RHD*weak partial D

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Type 4.1

1567
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
1568
TABLE 3. Continued
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
RHD*weak partial D RHD*weak partial D 2
Type 4.0 or Type 4.0
HAER-WIGMAN ET AL.

RHD*weak partial D
Type 4.1 and
RHD*DIIIa-CE(3-7)-D
RHD*weak partial D RHCE*ceVS RHD*weak partial D 1
Type 4.0 or Type 4.0
RHD*weak partial D
Type 4.1 and
RHD*DIIIa-CE(3-7)-D
Weak D variant RHD*DV.4 or RHD*DV.5 1

TRANSFUSION Volume 53, July 2013

RHD*DV.5 or
RHD*DV.9
RHD*DAU0 or RHD*DAU2 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7
RHD*DAU3 1
RHD*DAU0 or RHCE*ce-D(9)-ce RHD*DAU3 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7 and
RHD*y
RHD*weak partial D 2
Type 11
RHD*weak partial D 5
Type15
Weak RHD and Del Weak D Type 2 RHD*weak D Type 2 7
alleles RHD*weak D Type2 1
and
RHD*DIIIa-CE(3-7)-D
RHD*weak D Type3 RHD*weak D Type3 1
RHD*weak D Type5 RHD*weak D Type5 1
Weak D variant RHD*weak D Type1 22
RHD*weak D Type1 1
and RHD*DVI.2
RHD*weak D Type1 1
and
RHD*DIIIa-CE(3-7)-D
RHD*weak D Type3 2
RHD*weak D Type5 3
 TABLE 3. Continued
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
RHD*1227A RHD*1227A 3
RHD*IVS3+1A RHD*IVS3+1A 2
Del variant RHD*IVS3+1A 2
D negative null alleles RHD*y RHD*y RHCE*ce-.D(9)-ce 30
RHD*y RHCE*ce-D(9)-ce and 4
RHCE*ceVS
RHD*y RHCE*ce-D(9)-ce and RHCE*ceAR 1
RHCE*ceAR or
RHCE*ceEK
RHD*y (homozygous) RHCE*ce-D(9)-ce 3
(homozygous)
RHD*y and RHCE*ce-D(9)-ce 5
RHD*DIIIa-CE(3-7)-D.
RHD*y and RHCE*ce-D(9)-ce and 1
RHD*DIIIa-CE(3-7)-D RHCE*ceCF
RHD*48A RHD*48A 3
D–¶ RHD*01 New variant 1
RHD*443G
RHD chimera Chimera Chimera 50% RHD 1
Weak D variant Chimera 30% RHD 1
RHCE variant alleles RHCE*CeCW RHCE*CeCW 3
ceHAR RHCE*ceHAR 1
RhCE variant RHD*DAU0 or RHCE*cE602C RHD*DAU0 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7
RHCE*CeRN1 1
* RHD and RHCE variant allele determination after serology and/or RHD-MPX PCR results.
† RHD and RHCE variant alleles that were chosen for their specific genotype.

Volume 53, July 2013

‡ Sequencing was performed when the RH-MLPA was not able to determine the specific subtype of the variant allele.
§ Serology and the RHD-MPX PCR are not able to distinguish between the DAR1, DAR2, and Weak D Type 29 variant alleles.
¶ This patient was included in our series, because her current D serology (D–) was discordant with a previously reported D+ phenotype by the blood bank of Azerbaijan.

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GENOTYPING OF RHD VARIANTS AND ZYGOSITY
HAER-WIGMAN ET AL.

Fig. 1. RH-MLPA results obtained with a mixture of hemizygous RHD*01 D+ DNA from a donor pheno- and genotyped as (CcDdEe)
mixed with homozygous D– RHD*01N.01 DNA typed as (ccddee). DNA was added in a dilution range from 64% to 0.25% RHD*01 in
RHD*01N. The ratio of the RHD wild-type probe combinations is calculated using the hemizygous RHD*01 (CcDdEe) sample as a
reference sample. For exons of which more than one set of RHD wild-type probe combinations is detected, the mean of the probes
is given. Error bars indicate the standard deviation. Two RHD wild-type probe combinations detected the presence of 0.25%
RHD*01. The presence of 2% RHD*01 is reliably detected by the RH-MLPA and the presence of 4% RHD*01 is detected by all RHD
wild-type probe combinations.

allele in cases which have only one RHD*y allele and
hence are heterozygous for the RHCE*ce-D(9)-ce allele, we
developed an MLPA probe combination for the RHCE-
specific 1193T SNP. Seven cases hemizygous for the
RHD*y allele and one case heterozygous for this allele
were tested with the CE09_1193T probe combination. In
all tested samples the CE09_1193T showed a copy number
of 1, indicating that these persons were indeed heterozy-
gous positive for the RHCE*ce-D(9)-ce allele (data not
shown).
RHD and RHCE zygosity
RHD and RHCE zygosity were determined for all 236 DNA
samples by RH-MLPA. Of these cases, 163 cases were
found to be hemizygous for the RHD gene (Dd), 58 cases
homozygous for the RHD gene (DD), and in 12 cases both
RHD genes were deleted (dd). To confirm the zygosity
determined by the RH-MLPA, 18 cases (seven DD and
eleven Dd) were compared with zygosity typing using
RHD Exons 5- and 7-specific quantitative real-time PCR.
The results were concordant between both assays.
For two cases the RH-MLPA detected an abnormal
RHD copy number of 0.5 and 0.3 for all RHD-specific
probe combinations, indicating the presence of, respec-
tively, 50 and 30% hemizygously RHD*01 (RhD+). D serol-
ogy showed a mixed field reaction in the case with an
RHD-copy number of 0.5 and the case with a copy number
of 0.3 was serologically determined as “weak D.” Further
genetic analysis using short-tandem-repeat multiplex
PCR showed that these samples were from individuals
with a hematopoietic chimerism. To evaluate the sensitiv-
ity of the RH-MLPA to detect hematopoietic chimerism,
DNA from a person hemizygous for RHD*01 (CcDdEe) was

1570 TRANSFUSION Volume 53, July 2013


mixed with DNA from a person homozygous for
RHD*01N.01 (ccddee). As shown in Fig. 1 the presence of
0.25% hemizygous RHD*01 DNA is detected by two RHD
wild-type probe combinations and the presence of 4%
hemizygous RHD*01 DNA is detected by all RHD wild-
type probe combinations. When 2% or more hemizygous
RHD*01 DNA is present the RH-MLPA can reliably detect a
hematopoietic chimerism and correct quantification of
the amount of the RhD chimerism is possible when 8% or
more hemizygous RHD*01 DNA is present.
In 207 of the 236 cases (88%) an RHCE copy number
of 2 was obtained for all RHCE-specific probe combina-
tions. In the other cases, a copy number of 2 was
obtained for all RHCE-specific probe combinations
except for the CE05_676C and CE05_676G probe combi-
nations that detect RHCE*E and RHCE*e, respectively.
The abnormal RHCE*E/RHCE*e Exon 5 copy number of 0,
1, or 3 could be explained in all cases. All cases with a
RHCE*ceAR, RHCE*ceBI, or RHCE*ceHAR allele have an
RHCE*E/RHCE*e Exon 5 copy number of 1 (heterozygous
variant allele) or 0 (homozygous variant allele), since
they all contain the 712G mutation, which impairs liga-
tion of the second ligation site specific for the 712A
RHCE wild-type SNP of CE05_676C or CE05_676G. In 20
cases an elevated RHCE*E/RHCE*e Exon 5 copy number
of 3 was obtained. All these cases carried next to their
RHCE alleles an RHD-CE-D hybrid allele that contained a
complete RHCE Exon 5. Therefore, the CE05_676G
(including the RHD*DV.7, RHD*DVI.2, and RHD*DIIIa-
CE(3-7)-D allele) or CE05_676C (RHD*DVI.1) was able to
bind to the hybrid allele, causing an elevated RHCE*E/
RHCE*e Exon 5 copy number.
DISCUSSION
In this study we showed that the RH-MLPA genotyping
assay is a reliable method to predict the Rh-phenotype
and to determine the majority of RHD and RHCE variant
alleles as well as RHD zygosity. The MLPA technique is
easy to use, as it only requires a thermocycler and capillary
electrophoresis equipment.
First, the specificity of the RHD and RHCE wild-
type and RHD and RHCE mutation probe combinations
was determined with plasmid DNA and subsequently
with genomic DNA. All RHD and RHCE wild-type and
mutation probe combinations detected the wild-type or
mutated sequence according to their design (Table 1). We
showed two exceptions of wild-type probe combinations
in which the loss of signal was not caused by mutation at
the ligation site, but due to a mutation close by the liga-
tion site: absence of D07_1061T in variant alleles with
1063G>A mutation and absence of D04_514A in RHD*y
alleles.
To evaluate the accuracy of the RH-MLPA assay its
performance was tested with a set of 163 cases that were
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
previously serologically typed for Rh and in some cases
already typed with an RHD-MPX PCR or by RHD exon–
specific sequencing. In 160 cases (98%) the RH-MLPA
assigned correctly if a wild-type and/or variant allele(s)
was (were) present. In one case sequencing revealed the
presence of a partial RHD allele (RHD*DCS2), for which no
probe was included in the RH-MLPA since it is extremely
rare.41 It is still possible to add an extra mutation probe for
detection of the RHD*DCS.2 allele. In the other two cases,
two new variant RHD alleles were found: a D– null allele
RHD*443G (Thr148Arg) and a partial RHD allele
RHD*525T (Phe175Leu). We therefore conclude that the
RH-MLPA can be used to determine an RHD and/or RHCE
variant in the vast majority of samples analyzed in a ref-
erence laboratory and only in few samples will follow-up
analysis be needed to show the presence of either a very
rare or a new variant allele. In all 254 variant alleles
detected by the RH-MLPA the correct variant or variant
group was assigned. In one case the RH-MLPA could even
determine a new partial RHD allele (150T>C, 178A>C,
201G>A, 203G>A, 307T>C, and 1063G>A). In 26 of the 254
variant alleles (10%) detected by the RH-MLPA, additional
sequencing was necessary to determine the specific sub-
group (Table 3). It is only in the RHD*DAU variant group
(n = 4) where determination of the subtype is clinically
relevant, because the RHD*DAU0 allele, with normal RhD
expression, should be discriminated from the other
RHD*DAU alleles.
A new RHD*443G (Thr148Arg) allele was detected in
this study. This seems to be a D– null allele, caused by a
mutation resulting in the substitution of a polar amino
acid (threonine) with a positively charged amino acid
(arginine) in the fifth transmembrane region of the RhD
protein. To confirm this unexpected profound effect an
RhD expression model should be performed.42
For the first time it was recognized that the RHD*y
allele is linked (in all 46 persons tested) to a hybrid
RHCE*ce variant allele containing RHD Exon 9 (RHCE*ce-
D(9)-ce). This could be determined with the RH-MLPA,
since it determines the RHD copy number of Exon 9. This
hybrid allele contains two mutations 1170C>T and
1193T>A, encoding for one amino acid change Glu398Val
at the intracellular C-terminal tail of the Rhce protein.
Serologic D and CE typing in persons homozygous posi-
tive for the RHD*y RHCE*ce-D(9)-ce haplotype showed
the normal presence of the c and e antigens and the
absence of all D epitopes. The amino acid change is
present in the intracellular tail of the protein and thus far
no effect on the expression of the RHCE epitopes has been
shown.21
The RH-MLPA assay is very accurate in determining
gene copy number variation. In the RH-MLPA the RHD
copy number is based on the signals derived from 17 RHD
wild-type probe combinations. This makes the RH-MLPA
more suitable for RHD and RHCE zygosity determination
Volume 53, July 2013 TRANSFUSION 1571
HAER-WIGMAN ET AL.

than real-time quantitative PCR30 or amplification of the
hybrid Rh box.31-33 Clinically, it is important to determine
RHD zygosity in fathers, to assess the need for fetal RHD
typing in RhD alloimmunized D– women. Furthermore,
since the RH-MLPA is highly accurate in determining exon
copy number, this is the first high-throughput assay that is
able to recognize hybrid RHD/RHCE alleles next to a
normal RHD gene.
Next to the “normal” zygosity scores of 0, 1, and 2, the
RH-MLPA is also able to reliably determine the presence of
hematopoietic chimerism of just 2% RHD*01 (RhD+) in an
RHD*01N.01 (RhD–) background. It is important to detect
RHD+/RHD– chimeras, because it has been shown that
transfusion of RBCs from a donor with a hematopoietic
chimerism of 6% RHD+ DNA was able to immunize two
D– recipients.43 The RH-MLPA can also reliably genotype
patients who received multiple nonleukoreduced RBC
units. After massive transfusions a (weak) signal derived
from donor WBCs might be detected with the RH-MLPA.44
Because the MLPA is a quantitative method, it will be
readily recognized if a signal is obtained from a minor
population of transfused WBCs.
Some variants were correlated with a loss or gain of
the RHCE*E/RHCE*e Exon 5 copy number. In the
RHCE*ceAR, RHCE*ceEK, RHCE*ceBI, and RHCE*ceHAR
alleles the correct binding of the CE05_676G and
CE05_676C probe combination is inhibited and there-
fore it is not possible to determine whether these
variant alleles contain the RHCE*E or RHCE*e variation.
Since the RHCE*ceAR, RHCE*ceEK, RHCE*ceBI, and
RHCE*ceHAR alleles result in partial e antigen expression,
an individual carrying a RHCE*ceAR, RHCE*ceEK,
RHCE*ceBI, or RHCE*ceHAR allele should always be deter-
mined as RHCE*e+, hence Rhe+.45-47 For the RHD*DVI
and RHD*DIIIa-CE(3-7)-D hybrid alleles in which the
RH-MLPA suggest an RHCE*E/RHCE*e Exon 5 copy
number of three it is known that the hybrid alleles result
in the expression of the e antigen (RHD*DVI.1 and
RHD*DIIIa-CE(3-7)-D allele) or the E antigen (RHD*DVI.1
allele).48 For these cases the presence of the RHCE*e
and/or RHCE*E SNP present in the hybrid allele should be
taken into account when determining the RhEe status. For
the precise determination of variant RHCE alleles and the
identification of rare RHCE variant alleles an additional
MLPA has to be developed, but as listed in Table 2 the
most frequently occurring RHCE variant alleles are recog-
nized with the current RH-MLPA.
In conclusion, the RH-MLPA genotyping assay cor-
rectly determines clinically relevant RHD and RHCE
variant alleles. Therefore, it can be used as a single and
rapid assay to facilitate comprehensive typing of RHD
and RHCE variants in blood recipients and blood donors,
thereby providing a safe basis for blood transfusion. The
need for extensive RHD genotyping, by for instance the
RH-MLPA assay, of blood recipients, blood donors, and
pregnant women is still growing, since molecular geno-
typing methods are increasingly applied and therefore
the number of cases with discrepant serologic and geno-
typing results that require further molecular analysis will
increase.
ACKNOWLEDGMENTS
We thank Peter Ligthart (Sanquin Diagnostic Services, Amster-
dam, the Netherlands) for his technical assistance, Karel de Groot
(MRC Holland, Amsterdam, the Netherlands) for assistance in
designing the MLPA probe mixes, Christof Weinstock (Institute
for Clinical Transfusion Medicine and Immunogenetics Ulm,
Ulm, Germany), Inge von Zabern (Institute for Clinical Transfu-
sion Medicine and Immunogenetics Ulm, Ulm, Germany), Geoff
Daniels (Bristol Institute for Transfusion Sciences, National
Health Service Blood and Transplant, Bristol, UK), Martin Pisacka
(Institute of Haematology and Blood Transfusion, Prague, Czech
Republic), and Yanli Ji (Guangzhou Blood Center, Guangzhou
Guangdong, China) for providing DNA samples with specific
RHD genotypes.
WEB RESOURCES
ISBT Working Party on Red Cell Immunogenetics and
Blood Group Terminology http://www.isbtweb.org/
working-parties/red-cell-immunogenetics-and-blood-
group-terminology/blood-group-terminology/blood-
group-allele-terminology/. Accessed 05/10/2012.
CONFLICT OF INTEREST
NDA is a member of the transfusion Medicine Advisory Board
for Grifols, SA. All other authors declare that they have no
conflicts of interest relevant to the manuscript submitted to
TRANSFUSION.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Schematic overview of the MLPA reaction.
Fig. S2. Example of analysis of mix P401.
Table S1. Hybridization sequences of the probes of the
RH-MLPA.
Table S2. Primer sequences for amplification and
sequencing of exons 1-10 of the RHD and RHCE gene.