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Coagulation and Transfusion Medicine / Weak D Types in Egyptians
Weak D Types in the Egyptian Population
Eiman Hussein, MD,1 and Jun Teruya, MD, DSc2
Key Words: Weak D alleles; Egyptian population; Anti-D alloimmunization
DOI: 10.1309/AJCP1T9FGZBHIQET
Abstract
Patients with the most common weak D types
1, 2, and 3 can be safely considered D positive. We
evaluated 1,113 Rh-negative Egyptian samples for
weak D expression to propose a cost-effective strategy
related to D variant testing. D variants were tested
using polymerase chain reaction with sequence-specific
priming. Fifty samples were D variants (4.5%): weak
D type 4.2 (32%), weak D type 4.0/4.1 (16%), and
weak D type 15 (2%). Fifteen (62.5%) of 24 samples
were identified serologically as partial D. We also
studied the probability of the development of anti-D
in 52 Rh-negative children with thalassemia who
were receiving units for which weak D was not tested.
Anti-D alloimmunization was observed in 63.5% of
patients with thalassemia. It is prudent to implement
weak D typing in Egyptian donors. Weak D variants
of Egyptians are significantly different compared with
Caucasians. Ethnicity must be taken into consideration
when developing clinical and prenatal strategies
related to D variants.
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The molecular basis of D antigen varies substantially in
different ethnic populations. Egypt has always been a country
characterized by a mix of races. Egyptian ethnicity comprises
an admixture of the indigenous African population, ancient
Egyptians, Jews, Greeks, Romans, Persians, those of Arab
ancestry, foreign invaders, immigrants, Turks, Armenians,
and other Mediterranean populations. Evidence suggests
that these different ethnic influences are distributed homogeneously in the delta and Nile valley, where the overwhelming
majority of Egyptians live (99.6%). Ethnic minorities include
Bedouins in the Eastern and Western desert and the Sinai
peninsula, as well as some Nubians clustered along the Nile
in upper (Southern) Egypt.
Numerous studies have characterized the different RHD
alleles in whites, Africans, and Asians,1-6 but none have been
conducted among the Middle Eastern population. Approximately 7% to 8% of Egyptians are serologically D negative.
D negative phenotype is prevalent in whites (15%-17%) and
less common in black Africans (5%) and Asians (0.5%). In
whites, the D-negative phenotype arises from deletion of the
entire RHD gene,7 whereas Africans and Asians often have
an inactive or silent RHD.8 Major advances in cloning of
the Rh system gene have challenged the way that D status is
assigned. RHD variants are classified into 3 groups: weak D,
DEL, and partial D alleles.9 They occur in an estimated 0.2%
to 1% of whites.10 In black Africans, the frequency of the D
variant is much greater, with the most prevalent being partial
D.11 In Asians, 10% to 30% of D-negative donors are of the
DEL phenotype as shown by studies conducted in China,
Japan, and Taiwan.5,12-15 Weak D primarily results from
single point mutations in the transmembranous or intracellular
regions of RHD, reflected in reduced quantities of the normal
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D antigen.16 More than 50 different molecular weak D types
have been described.17 Weak D types 1, 2, and 3 represent
more than 90% of all weak D in whites.
Patients who carry weak D types 1, 2, 3, and 4.1 are not
sensitized when exposed to the D antigen and can be safely
considered D positive, thus saving the use of some D-negative
units and Rh immune globulin prophylaxis during pregnancy.11 Anti-D alloimmunization has only been documented for
weak D types 4.0, 4.2 (DAR), 11, and 15.18
DEL RBCs express very low quantities of D antigen that
cannot be detected on routine serologic testing and can only
be detected with adsorption elution techniques.19 They are
less frequent in whites (0.27%) and are often found in Asian
ethnic populations.20 It is well established that RBC units with
DEL phenotype can cause alloimmunization in D-negative
recipients.20,21 Many blood centers in Europe have applied
programs to implement RHD molecular screening for DEL
donors.22,23 In contrast with weak D, partial D variants are
caused by mutations in the extracellular regions and replacement of RHD axons by their RHCE counterparts, leading to
altered or new epitopes.9 Patients with partial D may develop
anti-D when exposed to D antigen.
Usually transfusion of 200 mL or more of D-positive
RBCs causes allo–anti-D production in 75% to 80% of Rhnegative recipients within 2 to 5 months.24 More recent data
demonstrated an inverse correlation between the number of
transfused units and the probability of antibody formation,
underscoring the importance of transfusion of weak D. This
issue is controversial because not enough reports have been
published in the literature to give conclusive evidence.22 The
aim of this study was to determine the frequency of various
weak D types among Egyptians and evaluate the serologic
testing practice. We sought to propose a cost-effective, standard transfusion policy related to D variant testing and
administration of Rh immune globulin. This study was also
conducted to help assess the risk of anti-D development in
Rh-negative patients receiving RBC units for which weak D
was not tested.
Materials and Methods
Blood Samples
A total of 1,113 Rh-negative blood samples from voluntary donors, family replacement donors, and patients were
collected from Cairo University Blood Center and studied for
weak D expression. Cairo University Hospital is a large tertiary teaching hospital that serves patients from various parts
of the country.
Samples that did not react in the immediate spin (IS)
tube method were identified as Rh-negative. D variants were
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further tested using polymerase chain reaction (PCR) with
sequence-specific priming (SSP) (BAGene PCR-SSP, BAG
Health Care, Lich, Germany), designed to detect the more
common weak D types (1, 2, 3, 4.0/4.1, 4.2, 5, 11, 15, and 17).
An additional group of 24 samples with D variants was
also studied to estimate the incidence of partial D in the Egyptian population based on serologic techniques. We also studied the probability of anti-D development in 52 Rh-negative
children with β-thalassemia who received Rh-negative RBC
units for which weak D was not tested.
Serologic D Typing
Routine D typing was performed using monoclonal
blended DiaClone anti-D reagents (Diamed, Cressier sur
Morat, Switzerland) containing IgG and IgM antibodies (cell
lines TH-28 and MS-26), which react in the indirect antiglobulin test (IAT) with most weak D and partial D RBCs,
including DVI. It was performed using the IS tube method
according to the process described in the American Association of Blood Banks (AABB) Technical Manual following the
manufacturer’s recommendations.25
Samples that were not agglutinated using the tube method
were further tested with the IAT using the manufacturer’s recommendations (anti-IgG/C3d, polyspecific antihuman globulin, Millipore, Billerica, MA), and results were interpreted
microscopically. Appropriate controls were used. Samples
that were weakly agglutinated with the tube method or that
reacted only on the IAT were classified as D variants.
Molecular Study
DNA was isolated from peripheral blood with the PCR
purification kit following the manufacture’s instruction (QIAquick, Qiagen, Hilden, Germany).
Kit Design
The basic material for typing with the BAGene DNASSP kits is purified leukocytic DNA. The test uses the PCRSSP procedure.26,27 This method is based on the fact that
primer extension and hence successful PCR relies on an exact
match at the 3ʹ end of both primers. If the primers entirely
match the target sequence, amplification is produced, which is
subsequently visualized on agarose gel electrophoresis.
Partial D Identification
Another group of 24 samples with D variants were
studied serologically in gel cards with the advanced partial
D typing kit (ID-partial RHD-typing kit, Bio-Rad, Hercules,
CA), following the manufacturer’s recommendations. The
kit included commercially available panels of monoclonal
anti D (cell lines: LHM76/55 [IgG], LHM77/64 [IgG],
LHM70/45 [IgG], LHM59/19 [IgG], LHM169/80 [IgG], and
LDM1 [IgM]).
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Analysis of Patients With Thalassemia
Screening for anti-D alloantibody was performed on
patients with thalassemia using a gel method (Diamed-ID
microtyping system).
Results
Molecular Identification of Weak D Phenotypes
Using PCR-SSP
Fifty (4.5%) of 1,113 Rh-negative samples were classified as D variants. Molecular typing of the 50 D variants
revealed that 16 (32%) were weak D type 4.2, 8 (16%) were
weak D type 4.0/4.1, and 1 (2%) sample was weak D type 15
❚Table 1❚. The remaining 25 (50%) samples probably constituted partial D or other rare weak D types.
Serologic Identification of Partial D
Using a panel of 6 monoclonal antibodies, all samples
with partial D could be characterized according to the reactivity chart ❚Table 2❚. A total of 15 samples (62.5%) were
classified as partial D when 24 samples with D variants were
tested serologically with the ID-partial D typing kit. Nine
samples (60%) were partial DIII and 6 samples (40%) were
category DV. The remaining 9 (37.5%) of 24 D variants
were weak D.
Analysis of Patients With Thalassemia
A total of 52 Rh-negative children with β-thalassemia
were included in this study, of whom 29 (55.8%) were boys.
❚Table 1❚
Different Weak D Types Identified by SSP-PCR in 50 Samples
With D Variants
RHD Variant
No. (%)
Weak D type 4.2
Weak D type 4.0/4.1
Weak D type 15
Partial or other rare weak D
16 (32)
8 (16)
1 (2)
25 (50)
PCR, polymerase chain reaction; SSP, sequence-specific primary.
Patients ranged in age from 3 to17 years with a mean of 12.3 ±
5.0 years. All patients received regular ABO- and D-matched
transfusions for a mean of 6.6 ± 2.7 years (range, 2-13 years).
Patients exclusively received prestorage leukofiltered blood
transfusions for 4 years before the analysis. The vast majority of patients had long-term exposure to nonleukofiltered or
bedside leukofiltered blood (1-9 years). Transfusion of Rhnegative RBC units that were not tested for weak D caused
alloimmunization in 33 (63.5%) of 52 patients. Patients were
a mean of 15 ± 3.5 years old and received a mean of 247.7
± 44.8 RBC units (range, 212-298 units) for 10.2 ± 2.2 years
(range, 7-13 years). Patients with no anti-D received significantly fewer transfusions (P < .05) at a mean of 30 ± 71
transfusions (range, 10-144 units) for 7 ± 3 years (range, 2-8
years). A total of 18 (54.5%) of 33 alloimmunized patients
were girls, including 2 siblings.
Discussion
One of the major challenges in clinical transfusion practice is to avoid anti-D alloimmunization with the least possible costs while avoiding wastage of D-negative units and
Rh immune globulin. It is well documented that patients with
partial D phenotypes are at risk for production of anti-D. In
contrast, patients with the most common weak D types—1,
2, and 3—are not at any risk for sensitization when exposed
to D-positive RBCs and could safely receive transfusions of
D-positive blood without the need for Rh immune globulin prophylaxis.10 Anti-D alloimmunization has only been
documented for weak D types 4.0, 4.2 (DAR), 11, and 15.18
However, distinction among these phenotypes is impossible
serologically. Only molecular analysis will identify patients
with D variants who are at risk for anti-D production.
Serologic phenotyping is the standard test to assign
transfusion strategies. RBCs with D variants may react
differently depending on the typing method, the affinity of
anti-D, and the serologic cutoff.28 The monoclonal anti-D
has been in routine use at Cairo University Blood Center
since 2000. Repeated testing of all Rh-negative units by IS
tube test is typically carried out both before screening as
❚Table 2❚
Partial D Kit Reactivity Chart
Anti-D
Cell Line
1
2
3
4
5
6
LHM76/55
++– – ++
LHM77/64
–+–– ++
LHM70/45
++–– ––
LHM59/19
++++ +–
LHM169/80
++++ +–
LDM-1 ++++ +/–a–
a
DII
DIII
DIVa
DIVb
DV
DVI
DVII
DFR
DBT
DHAR
+ +––
+ +
––
+ –––
a
+/– – + –
+ +––
+
– + +/–a
A weaker reaction can be observed with this antibody in comparison with the other 5 serum samples.
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well as before the release of blood units. It has been reported
that high-potency monoclonal anti-D reagents can detect D
variants that are difficult to detect with less sensitive techniques.28 However, 4.5% of Egyptian samples were missed
on routine serotyping and were only detected with the IAT.
The monoclonal IgM and IgG blend clones are routinely
used in the United States, whereas most European centers do
not use monoclonal anti-D and do not perform IAT for weak
D.29 It has been noted that the current monoclonal Food and
Drug Administration–licensed anti-D serologic reagents are
designed not to react in direct testing with partial DVI RBCs,
which is the most common partial D in whites.3
According to AABB, weak D testing is not required for
patients or pregnant women but is mandatory in blood donor
and cord blood testing.31
Molecular analysis for blood groups was introduced more
than 10 years ago as an important aspect of immunogenetics.
Their clinical application since then has been evolving.32,33
RHD molecular strategy varies considerably between the
United States and Europe. In the United States, it is performed
to resolve discrepant serologic results, to aid complex antibody identification, and to differentiate allo- from autoantibodies. RHD genotyping in many European centers, predominantly in Germany, is used for D variant testing in patients and
pregnant women, in patients who have had a transfusion, and
in patients with D typing discrepancies; in donors for DEL
and D+/D– chimera; and to determine RHD zygosity.21
Because in most white patients RHD alleles are caused
by a few weak D–variant types, molecular assays of these
phenotypes would characterize most samples.3,9 Flegel21 recommended that patients carrying the prevalent weak D types
1, 2, and 3 can be classified as D positive, saving 3% to 5%
of all D-negative units. Likewise, when pregnant women are
genotyped to identify D variants, 3% to 5% of all anti-D injections can be spared. This service has been applied routinely in
Germany since 2001. Pham and colleagues34 recommended
that this strategy be applied in other countries to avoid wasting D-negative units. In our study, D variant prevalence was
4.5% of all D-negative samples (50/1,113). Molecular assay
of the 50 D variants revealed that weak D type 4.2 was the
most frequent (32%), followed by weak D type 4.0/4.1 (16%),
and weak D type 15 (2%). Only 8 (0.7%) of 1,113 cases were
weak D type 4.0/4.1, and the PCR-SSP platform was unable
to distinguish between weak D type 4.0 and 4.1. The remaining 25 samples were not identified and probably constituted
partial D or other rare weak D types.
To estimate the incidence of partial D in Egyptians,
another group of 24 samples with D variants were screened
serologically for detection of partial D with the advanced
partial D kits, which enable the characterization of many
known partial D phenotypes and facilitate differentiation of
weak D from partial D. In our study, 62.5% of D variants were
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characterized as partial D; the DIII category was the most
frequent, followed by DV. The most common partial D in
whites are DVI and DVII, and the most common in people of
African descent is probably DIII.35 The DV has been reported
in white, black, and Japanese people.6,36
Our data show that in the Egyptian population most D
variants with weak expression were partial D, resembling
black individuals.36 Wagner and colleagues10 proposed that
frequency data of D variants in specific populations, based on
DNA analysis, can be used as a basis to implement optimal
transfusion and obstetric programs.
Based on our data, with the routine use of the advanced
ID-partial D typing kit, 50% to 62.5% of patients with D
variant can be characterized serologically. Further implementation of weak D genotyping is necessary to appropriately
characterize the remaining RHD variants. This approach can
ensure blood transfusion safety and would allow a better use
of D-negative units and Rh immune globulin without adding
much to the costs.
Whether RBCs with D variants are capable of stimulating
antibody production when transfused to D-negative recipients
has been debated.22,37-40 For more than 60 years, it has been
known that transfusion of RBC units with D variants could
cause sensitization in D-negative recipients, but the first
molecular workup was described in 2000.41 Sensitization with
minor amounts of D antigen was reported previously.21,42,43
The only clinical trial to assess the risk of RBCs with
weak D to stimulate anti-D in D-negative recipients reported
no antibody production in 49 patients who received transfusions of 68 units that harbored D variants.44 In our group of
patients, transfusion of Rh-negative RBC units that were not
tested for weak D caused alloimmunization in 33 (63.5%) of
the 52 patients with thalassemia who received multiple transfusions. The rate of alloimmunization in our group of patients
was considerably high. Not all D-negative patients can make
anti-D when receiving D-positive RBC transfusions. Whether
an allo–anti-D is induced depends on the immunologic condition of the transfusion recipient.28 The immunomodulation
role of WBCs might have contributed to the high incidence
of anti-D observed in our patients. The vast majority of those
patients were exposed to nonleukofiltered blood or bedside
filtered blood, which is a suboptimal leukoreduction method.
D alloimmunization incidence also increased with the number of units transfused. A high incidence of anti-D has been
reported in African Americans and in individuals of mixed
ethnic backgrounds because of the common occurrence of
D variants. In a previous study conducted to determine the
prevalence of RBC alloimmunization in 272 Egyptian patients
with β-thalassemia, D alloimmunization was observed in
34.5% of all Rh-negative patients. In this study, 80% of
all anti-D developed in patients over 18 years.45 In a study
conducted at another university hospital in Cairo, 19.6% of
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patients with thalassemia were alloimmunized, of whom 13%
developed anti-D.46 A high incidence of anti-D in thalassemia
patients was also noted in another Iranian study.47 The high
incidence of anti-D in our group of transfusion-dependent
patients with thalassemia underscores the importance of
IAT in the D typing of Egyptian donors. Detection of all
D-negative donors who harbor clinically relevant D variants,
however, is impossible serologically.22 Reported prevalence
of D variants among apparently negative samples ranged
from 0.2% to 5.23%.4,23,41 Polin and colleagues48 proposed
that that all apparently D-negative donors should be screened
with genotyping methods to avoid missing potentially immunogenic D variants. Routine screening of first-time donors for
RHD has been implemented in some European centers.4,22,23
The rationale for this strategy is to eliminate the risk of D
sensitization and hence improve the safety of blood transfusions. The cost benefit of this policy has been debated for
years. The application of an affordable, automated, higher,
and faster throughput genotyping technology to transfusion
practice might help to resolve this contention. It could also
improve patient care, especially for those receiving long-term
transfusion therapy.
In summary, weak D variants among Egyptians are significantly different compared with whites, but were found to
be similar to those seen in black Africans. Population ethnicity
must be taken into consideration when developing transfusion
and prenatal strategies related to D variants. It is prudent to
systematically implement weak D typing in Egyptian donors.
From the 1Cairo University Blood Bank, Clinical Pathology
Department, Cairo University, Cairo, Egypt, and 2Departments
of Pathology and Immunology, Pediatrics, and Medicine, Baylor
College of Medicine and Texas Children’s Hospital, Houston, TX.
Address reprint requests to Dr Hussein: 6 Hussein Elezaby
St, Higazy, Cairo Alexandria Desert Road, Cairo, Egypt;
[email protected].
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Am J Clin Pathol 2013;139:806-811
DOI: 10.1309/AJCP1T9FGZBHIQET
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