TITLE IN ENGLISH
ASSOCIATION OF DEL AND RhCcEe PHENOTYPE DISTRIBUTION AMONG 112
THAI Rh NEGATIVE BLOOD DONORS
Jairak Thongbut1,*, Pornlada Nuchnoi 2, Apapan Srisarin 3, Aungkura Supokawej3, Tasanee
Sakuldumrongpanich 4#
1
Master of science programme in Medical Technology (International programme)
, Faculty of Medical Technology, Mahidol University, Thailand
2
Center for Innovation Development and Technology Transfer, Faculty of Medical
technology, Mahidol University, Thailand
3
Department of Clinical Microscopy, Faculty of Medical technology, Mahidol University,
Thailand
4
National blood center, Thai Red Cross Society
*e-mail: mura_murah@yahoo.com, #e-mail: pornlada.nuc@mahidol.ac.th,
apapan.sri@mahidol.ac.th, aungura.jer@mahidol.ac.th, tasaneesakul@yahoo.com
Abstract
RhD is the most important and highly immunogenic antigen. An anti-D is the major cause of
hemolytic disease of the fetus and newborn (HDFN) and hemolytic transfusion reaction
(HTR). Many RhD variants influencing on D antigen expression have been documented such
as partial D, weak D, and DEL phenotype. DEL is the very weakly expressed D antigen
which is undetected by routine serologically test. The adsorption-elution technique need to be
used in order to detect DEL. Approximately 20% of Thai Rh negative donors were identified
as DEL. In this study we investigated the association of DEL and RhCcEe phenotype
distribution among Thai Rh negative blood donors. To understand the genetic background of
Thai Rh negative blood donors and apply for policy of blood transfusion safety program in
Thai. Samples were test with anti-D in three steps to exclude weak D and partial D.
RhD negative sample were then perform RhCcEe phenotyping. Only Rh negative with
RhC (+) sample were perform anti-D adsorption test and SSP-PCR for RHD1227A analysis.
Total 112 serologically RhD negative donors included of 49 Rh negative and 63 DEL. The
distributions of RhCcEe phenotype were demonstrated as follow. The Rh negative was
frequently found with ccee (65.31%) follow by Ccee (30.61%) and ccEe (4.08%) phenotype
respectively. DEL mostly found with Ccee (79.36%) follow by CCee (19.05%) and
CCEe phenotype (1.59%) respectively. The DEL is highly associated to RhC (+) phenotype
(80.77%). Among 78 Rh negative with RhC (+), 63 were identified as DEL using adsorptionelution test and RHD1227A analysis. Sixty-two samples were positive for adsorption-elution
test and one DEL sample which show negative for adsorption-elution test carried
RHD1227A allele. The SSP-PCR analysis demonstrated that 61 of 63 DEL samples carried
RHD1227A allele (96.83%) and 2 DEL samples carried non-RHD1227A allele. This data
provide the information that the identification of molecular marker associated DEL
phenotype is clinically important for donor screening in blood transfusion service.
Keywords: Rh DEL, DEL
Introduction
Rh (Rhesus) is one of the most clinically important blood group systems consisting of
two highly homologous genes, RHD and RHCE. In the Rh blood group system, RhD is the
most important and highly immunogenic antigen. The anti-D alloantibody produced from
RhD sensitization in Rh negative individual is the major cause of hemolytic disease of the
fetus and newborn (HDFN) and hemolytic transfusion reaction (HTR) (1). RhD is the highly
polymorphic blood group among different ethnic populations. The frequencies of Rh negative
are 15%-17% in Caucasian, 3%-5% in Black Africans and 0.1%-0.5% in Asian population (2,
3). The molecular mechanism mediated Rh negative phenotype mostly described by
RHD gene deletion resulting in no D antigen expression. However, the genetic heterogeneity
of RHD gene causes the variation of molecular mechanism underlying Rh negative among
racial populations. For Rh negative individuals, the frequency of RHD gene carriers were
approximately 0.6% in Caucasians (4), 10% in Africans (5), and 30% in Asians (6). These
RhD phenotype negative RHD genotype positive could be explained by multiple genetic
mechanisms; RHD pseudogene (RHDΨ), hybrid gene (Ccdes) in African (5) and DEL
phenotype, RHD(K409K) or 1227G>A in Asians (7). The Rh negative with RHD gene
detectable causes the Rh discrepancy between serology typing and molecular typing.
Nowadays, RhD variants mediated variation of D antigen expressions have been categorized
for practical purposes into 3 groups: partial D, weak D, and DEL. Basically, weak D and
patial D phenotype can be serologically detected using indirect antiglobulin test (IAT). While
DEL one type of weak D with the weakest D antigen expression show no reactivity using
IAT. The DEL blood group could only be detected using adsorption-elution test (8).
Furthermore, the DEL blood group is considerably common in Asian comparing to European
(1). Interestingly, approximately 30% of Asian with Rh negative were identified as DEL (7).
There are many allele associated DEL phenotype such as RHD(M295I) allele in European
population (1:6493) (4) and RHD(K409K) allele in Asian population (1:110) (9). The
RHD(K409K) called Asia type is a marker of DEL blood group in Asian (7). However, not
all DEL person in Asian carries RHD(K409K) allele. Moreover, in Caucasian difference DEL
allele were observed for example weak D type 4.3 is the most frequence DEL allele in Upper
Austria (72.09% in DEL individuals) (10) and RHD(IVS3+1G>A) is the most frequency
DEL allele in South-western Germany (34.04% in DEL individuals) (11). Thus the policies
for DEL detection should be adjusted depend on genetic background of certain countries.
Additionally, the RHD(K409K) allele was also associated with the Rh (C+) especially in
Asian. The incidence of DEL in Rh (C+) individuals determined by RHD(K409K) allele was
74.3% and adsorption elution test 76% in Taiwan (6). Thereby the combination of
RhC phenotype and RHD1227A analysis is now recognized as an essential tool for detection
of Asian DEL in transfusion practice (6, 12). Up to date, there are the large numbers of case
reports of DEL mediated induction of primary and secondary anti-D alloimmunization in
Rh negative recipients (13-15). The incidences of DEL case reports could be explained by the
mistyping of DEL as Rh negative. This leads to the immunization of anti-D in Rh negative
when received DEL blood. Prior to genomic-era, DEL detection was limited by conventional
serological test. In order to prevent anti-D alloimmunization, the discrimination between
DEL and true Rh negative is significantly important in blood transfusion practice (15).
Recently, the frequency of DEL in Thai population have been reported as 19.69% in
Rh negative group (12). In this study we investigated the association of DEL and
RhCcEe phenotype distribution among Thai Rh negative blood donors. To understand the
genetic background of Thai Rh negative blood donors and apply for policy of blood
transfusion safety program in Thai.
Methodology
Serologic Rh Phenotyping
The blood samples were tested for RhD phenotype using anti-D IgM (National Blood
Centre, Thai Red Cross Society) and analyzed by automate analyzer (PK 7200 or PK 7300®
Automated Microplate System, Beckman Coulter Inc., CA, USA). The result with flag “*”
were retested for conventional tube test with anti-D IgM/IgG (National blood centre,
Thai Red Cross society) in three steps; room temperature (RT), 37°C, and indirect
antiglobulin test (IAT) to exclude weak D or partial D phenotype. The serologically
Rh negative samples were sequentially serotyped for RhC, c, E, e antigen by monoclonal
IgM antibodies anti-C and anti-e (DiaClon, DiaMed GmbH) and monoclonal IgM antibodies
anti-c and anti-E (National Blood Centre, Thai Red Cross Society) according to the
manufacturer’s instruction.
Adsorption-elution test for DEL detection
The combination of adsorption-elution technique, RhC (+) phenotype and RHD1227A
analysis is an effective test for detection of DEL which can distinguish true Rh negative from
DEL (16). The Rh negative samples with RhC (+) phenotype were performed adsorptionelution test with anti-D polyclonal antibodies from human serum (DiaMed GmbH, Bio-Rad
Laboratories). 200 µl of RBCs were washed with normal saline solution (NSS) and added
equal volume of anti-D. The mixture were incubated at 37 0C for 1 hour and then washed the
RBCs with normal saline solution (NSS) thoroughly, keep the last washed supernatant for
testing. The eluate was prepared by In House Acid-glycine/EDTA technique (17). First,
prepared elution reagent by mix 1 ml of 0.1 M glycine-HCl buffer (pH 1.5) together with
250 µl of 10% EDTA into test tube (ratio of 0.1 M glycine-HCl buffer: 10% EDTA is 4:1).
Placed 200 µl of pack washed sensitized RBCs into test tube and add equal volume of elution
reagent to the sensitized RBCs. Mixed well and incubated at room temperature (22-24 0C) for
1 minute (caution: over incubation will cause damage to the RBCs). 1 M TRIS-NaCl 28 µL
were added before mixing and immediately centrifuged at 1,000 g for 1 minute to collect the
supernatant (eluate). Adjusted the eluate pH by 1 M TRIS-NaCl, the optimal pH should be
between 7.0-7.4 using pH paper. The eluated and last washed supernatants were used for
indirect anti-globulin test against Rh positive and Rh negative control cells (National Blood
Centre, Thai Red Cross Society). The reactivity was graded according to the criteria of
AABB (0, 1+, 2+, 3+ and 4+).
DNA extraction
Citrate phosphate dextrose adenine (CPDA-1) blood was used for DNA extraction.
The genomic DNA of DEL samples were extracted by QIAamp® DNA Blood Mini Kit
(QIAGEN GmbH, Hilden, Germany). 20 µl of QIAGEN Proteinase K was used for lysis
200 µl of whold blood together with 200 µl of Buffer AL. Mixed thoroughly by pulsevortexing for 15 seconds and incubated at 56 0C for 10 minutes. After that, briefly
centrifuged before add 200 µl of absolute ethanol. Mixed by pulse-vortexing for 15 seconds
and briefly centrifuged again. The mixture was carefully transferred into QIAamp Mini spin
column in collection tube before centrifuge at 8000 rpm for 1 minute. Placed the QIAamp
Mini spin column into clean microcentrifuge tube following by carefully added 500 µl of
Buffer AW1 and centrifuge at 8000 rpm for 1 minute. QIAamp Mini spin column was placed
into another microcentrifuge tube, and then carefully added 500 µl of Buffer AW2 before
centrifuge at 14000 rpm for 3 minutes. After placed the QIAamp Mini spin column into
another microcentrifuge tube, centrifuged at 14000 rpm for 1 minute to eliminate the
carryover of Buffer AW2. Placed the QIAamp Mini spin column into clean
1.5 microcentrifuge tube, then carefully added 200 µl of Buffer AE and incubated at room
temperature (15-25 0C) for 1 minutes. Finally, centrifuged at 8000 rpm for 1 minute, then
keep the eluting DNA at -20 0C.
RHD1227A analysis by Sequence specific primer-Polymerase Chain Reaction (SSP-PCR)
SSP-PCR technique was used for detecting RHD1227A allele in DEL samples using
previously published primer set (6). The nucleotides sequences of forward primers for
RHD1227A allele is 5’-GATGACCAAGTTTTCTGGAAA-3’ and for RHD1227G allele is
5’-GATGACCAAGTTTTCTGGAAG-3’. The reverse primer for both RHD1227A allele and
RHD1227G allele is 5’-GTTCTGTCACCCGCATGTCAG-3’. PCR reactions were
performed as previously described (7). A total volume of 10 µ L, each reaction containing
1 µ L of genomic DNA, 0.5 U of Taq DNA polymerase (Genet Bio, Korea), 200 µM of
dNTPs, primers, 2.5 mM MgCl2 and 10X Reaction buffer provided by the manufacturer.
Forty cycles were programmed on thermal cyclers (GeneAmp® PCR System 9700, Applied
Biosystems, Singapore) as follows: denaturation at 94 oC for 5 minutes, then 35 cycles of
30 seconds at 94 oC and 40 seconds at 68 oC and 30 seconds at 72°C. PCR products were
visualized on a 2.5% agarose gel with ethidium bromide staining
Results
Serologically Rh phenotype distribution among sample groups
A total 112 serologically Rh negative Thai blood donors divided into two categories.
Forty-nine Rh negative donors with fifteen RhC (+) donor (30.61%) and thirty-four were
RhC (-) phenotype (65.31%) and sixty-three DEL (100%). The Rh phenotype distributions of
112 serologically Rh negative are shown in Table 1. The Rh negative was frequently found
with ccee phenotype (65.31%) follow by Ccee phenotype (30.61%) and ccEe (4.08%)
respectively. DEL mostly found with Ccee phenotype (79.36%) follow by CCee phenotype
(19.05%) and CCEe phenotype (1.59%) respectively. The frequency of DEL identified in
RhC (+) is particularly high (80.77%).
Table 1. Rh phenotype distributions among 112 Rh negative donors used in this study.
Rh Phenotype
Rh negative (%)
DEL (%)
CCEe
0
1 (1.59)
CCee
0
12 (19.05)
CcEE
0
0
CcEe
0
0
Ccee
15 (30.61)
50 (79.36)
ccEE
0
N/D
ccEe
2 (4.08)
N/D
ccee
32 (65.31)
N/D
Total
49 (100%)
63 (100%)
C (+)
C (-)
*N/D = Not done in RhC (-) phenotype because of low prevalence of DEL.
** This result demonstrated the distribution of Rh phenotype among sample used in this study.
RHD1227A analysis in Rh negative with RhC (+) phenotype
All of 78 serologically Rh negative with RhC (+) phenotype were tested for
distinguish DEL from Rh negative using anti-D adsorption-elution test and SSP-PCR for
RHD1227A analysis. Among 78 Rh negative with RhC (+), sixty-three were identified as
DEL using adsorption-elution test and RHD1227A analysis. Among 63 DEL, sixty-two
samples were positive for adsorption-elution test. Interestingly, one DEL sample which show
negative for adsorption-elution test carried RHD1227A allele and CCee phenotype. The
SSP-PCR result was shown in figure 1. The SSP-PCR analysis demonstrated that 61 of 63
DEL samples carried RHD1227A allele (96.83%) and 2 DEL samples carried
non-RHD1227A allele (Ccee phenotype). Data are shown in Table 2.
Table 2. Results
of SSP-PCR for RHD1227A analysis and adsorption-elution of 78
RhD negative donors with RhC (+) phenotype.
Adsorption-elution (+)
Adsorption-elution (-)
n=62
n=16
Rh
Phenotype
RHD1227A
(+)
Total
RHD1227A (-)
RHD1227A (+)
RHD1227A (-)
CCEe
1 (1.28%)
0
0
0
1 (1.28%)
CCee
12 (15.38%)
0
1 (1.29%)
0
13 (16.67%)
Ccee
47 (60.26%)
2 (2.56%)
0
15 (19.23%)
64 (82.05%)
Total
60 (76.92%)
2 (2.56%)
1 (1.29%)
15 (19.23%)
78 (100%)
Figure 1 Results of sequence specific primer – polymerase chain reaction (SSP-PCR) determining
RHD1227A polymorphism. Lane1-4: The PCR products with 348 base pair (bp) separated on
2.5% agarose gel are shown. M: DNA ladder
Discussion and Conclusion
DEL is characterized by showing negative reaction at indirect anti-globulin test (IAT)
but positive using adsorption-elution test. However, the variation in techniques and reagents
or even the limitation of serology testing lead to low sensitivity for detection of DEL only by
serological testing. In current study, one sample missing by serologically anti-D adsorptionelution test but was detected at molecular level. Thus a combination of serological and
molecular testing could be a powerful tool for detection of DEL (6, 12).
The incidence of RhC (+) phenotype in Thai Rh negative donors were found 46.4%
(12). In this study, DEL is highly associated with RhC (+) phenotype (either CCee or Ccee
phenotype) (7). From 78 apparent Rh negative donors with RhC (+) phenotype, 63 donors
with DEL were detected (80.77%). This data impled that the detection of DEL in
RhC (+) phenotype is more appropriate and cost-effective than detection in all Rh negative
samples. In contrast to Caucasian population especially in upper Austria, DEL is mostly
identified in RhC (-) phenotype with ccee phenotype (72.09%) (10). In addition, DEL is
mostly associated with RhC/E (+) phenotype in South-Western Germany (11). This
underlined the heterogeneity of DEL among different ethnic groups. Therefore, the policy for
DEL detection should be adjusted depending on the genetic background of certain
populations.
Based on previous studies, RHD1227A allele (K409K) was commonly found in Asia
including China, Korea, Japan and Thai (7, 12, 18, 19). Our study using SSP-PCR for DEL
conformation, RHD1227A allele was found 96.83% in Thai blood donor with DEL.
However, two samples of DEL (positive for adsorption-elution test) were found negative with
RHD1227A analysis. This indicated that the other RHD genetic polymorphisms should be
investigated further.
The identification of molecular marker associated DEL phenotype is clinically
important for donor screening in blood transfusion service. The molecular testing for DEL
detection has been applied in routine laboratory in Europe using pool samples strategy (20).
In Asian, the DEL screening still not apply for donor screening program because short supply
of Rh negative pool in Asian. But the case reports of DEL red blood cell induced anti-D
production in Rh negative recipients still observed. The topic of anti-D production by DEL
red blood cell in Thai population is very interesting and important for study further.
Moreover, when consider the potential of DEL red blood cells that can induce anti-D
alloimmunization in Rh negative recipient. Thus, DEL unit should be transfer into
Rh positive pool to improve blood safety transfusion.
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Acknowledgements:
We thank National Blood Centre, The Thai Red Cross Society, Thailand for
supporting the blood samples and provided the instrument for this thesis.