ISBT Science Series (2013) 8, 1–5

STATE OF THE ART 1A-H01-01 © 2013 The Author(s).

ISBT Science Series © 2013 International Society of Blood Transfusion

Molecular DNA-based testing for blood group antigens:

recipient–donor focus
C. M. Westhoff
Immunohematology and Genomics, New York Blood Center, New York, NY, USA

DNA-based testing for blood group antigens has become commonplace in a num-
ber of clinical situations to improve transfusion therapy for blood transfusion
recipients. These include typing for antigens in patients who are multiply trans-
fused to determine which blood group antibodies may be present, typing when
the red blood cells (RBCs) are coated with immunoglobulin, and typing for
uncommon antigens, especially when no serologic reagent is available. Other
clinical applications include resolving ABO typing discrepancies for patients
awaiting transplant, or determination of the original type of a bone marrow
transplant recipient, or confirmation of an ABO subgroup in a kidney donor.
Testing for the specific mutation associated with depressed Kell system antigens
in McLeod syndrome may also have value because different mutations appear to
be associated with prognosis.
Applications in prenatal medicine include discrimination between weak D and
partial D to determine if a pregnant woman is a candidate for Rh immune pro-
phylaxis, and assessing the risk for hemolytic disease of the fetus or newborn
and neonatal thrombocytopenia. Assessing risk includes testing the paternal sam-
ple for the target antigen, and if positive testing for gene copy number (zygosity)
and testing the fetus from amniocentesis or non-invasive maternal plasma
sample.
Donor centers are now able to screen large numbers of donors to label
antigen-negative RBC products. DNA typing will change transfusion practice by
allowing patients facing chronic transfusion therapy to have a more comprehen-
sive blood group antigen profile performed, and to receive donor units antigen-
matched for more than ABO and RhD to reduce alloimmunization. A patient who
makes an alloantibody to a RBC antigen is forever at risk for his or her lifetime;
for a serious transfusion reaction if ever requiring transfusion in an emergency,
and also for a high risk pregnancy if a woman of child bearing age. Prevention
of alloimmunization is an important advance in transfusion safety and is now
possible with electronic patient medical records and testing and labeling of blood
products for additional blood group antigens.
Key words: blood groups, blood group antigens, DNA based testing, molecular
matching, molecular testing

to direct testing by serological methods using a spe-

Introduction
Determination of blood group antigens with DNA
methods (genotyping) is an indirect method for predict-
ing an individual’s blood group phenotype, in contrast
cific antibody (phenotyping). The results of typing by
DNA are often reported as a ‘predicted’ type to distin-
guish the results from testing done by serological
methods.

Correspondence: Connie M. Westhoff, Director, Immunohematology and Genomics, New York Blood Center, 310 East 67th Street, New York, NY 10065,
USA E-mail: cwesthoff@nybloodcenter.org

1
2 C. M. Westhoff
Most blood group antigens result from single nucleo-
tide gene polymorphisms (SNPs) inherited in a straight-
forward Mendelian manner, making assay design and
interpretation fairly straightforward [1]. However, ABO
and Rh are more complex. There are over 100 different
alleles encoding the glycosyltransferases responsible for
ABO type, and a single point mutation in an A or B allele
can result in an inactive transferase, that is a group O
phenotype [2]. Next generation sequencing technology
would potentially allow routine ABO typing by DNA
methods. For the Rh system, testing for the common anti-
gens D, C/c and E/e is fairly straightforward in most indi-
viduals, but antigen expression is more complex in
diverse ethnic groups. There are over 200 RHD alleles
encoding weak D or partial D phenotypes (http://rhesus
base.atspace.com/), and more than 100 RHCE alleles
encoding weak, altered or novel hybrid Rh proteins [3,4].
RH genotyping, particularly in minorities, requires sam-
pling of multiple regions of the gene(s) and algorithms
for interpretation.
The most commonly used methods for determination of
red cell, human platelet antigen (HPA) and neutrophil
antigens use sequence-specific primer–polymerase chain
reaction (SSP-PCR) or allele-specific PCR. For manual
methods, gel electrophoresis is then used to separate the
PCR products for fragment size determination, or alterna-
tively, the assay may include digestion of the PCR prod-
ucts with a restriction enzyme, restriction fragment
length polymorphism, followed by electrophoresis and
visualization of the fragments. For resolution of discrep-
ancies and to identify new alleles, specialty referral labo-
ratories use methods similar to those used for high
resolution human leucocyte antigen typing, that is gene-
specific amplification of coding exons followed by
sequencing, or alternatively, gene-specific cDNA amplifi-
cation and sequencing. These methods are used to investi-
gate new alleles and resolve serological and molecular
discrepancies. Semi-automated approaches include real-
time PCR using florescent probes with quantitative and
qualitative automated read-out. Lastly, high-throughput
automated methods increase the number of target alleles
in the PCR reaction allowing determination of numerous
antigens in a single assay. Most platforms are based on
florescent bead technology.
Applications of DNA-based molecular testing
Clinical transfusion medicine
Type patients who have been recently transfused
In patients receiving chronic or massive transfusion, the
presence of donor red blood cells (RBCs) often makes RBC
typing by serological agglutination inaccurate. DNA typ-
ISBT Science Series © 2013 International Society of Blood Transfusion, ISBT Science Series (2013) 8, 1–5
ing overcomes this limitation and avoids time consuming
and cumbersome cell separation methods to isolate and
type the patient’s reticulocytes. DNA assays for blood
groups avoid interference from donor-derived DNA by
targeting and amplifying a region of the gene common to
all alleles. This allows reliable blood group determination
with DNA prepared from a blood sample collected after
transfusion. In transfusion-dependent patients who pro-
duce alloantibodies, an extended antigen profile is impor-
tant to determine additional blood group antigens to
which the patient can become sensitized.
Type RBCs coated with immunoglobulin [positive
direct antiglobulin test (DAT)]
In patients with RBCs coated with immunoglobulin, with
or without autoimmune haemolytic anaemia, the presence
of bound immunoglobulin often makes RBC typing by
serological methods invalid. Immunoglobulin G (IgG)
removal techniques are not always effective and can
destroy or weaken the antigen of interest. For patients
with serum autoantibody, DNA testing allows determina-
tion of an extended antigen profile to select antigen-neg-
ative RBCs for transfusion. This avoids the use of ‘least
incompatible’ blood for transfusion, and allows transfu-
sion of units ‘antigen-matched for clinically significant
blood group antigens’ to prevent delayed transfusion
reactions and circumvent additional alloimmunization.
Importantly, this approach can improve patient care and
testing turn-around time by eliminating the need for
repeat absorptions to remove the autoantibody to rule out
new underlying RBC alloantibodies.
Determination of D status
Altered expression of D antigen occurs in 2% of Cauca-
sians, <1% of Asians and approximately 4% of Black
and Hispanic groups. Routine serological D-typing
reagents cannot distinguish RBCs with weak D or partial
D, and distinction between these is of clinical impor-
tance because the latter are at risk for anti-D. RHD
genotyping strategies that sample multiple regions of
RHD can discriminate weak D and partial D phenotypes.
Females of child-bearing age with partial D would
potentially benefit from receiving D-negative RBCs for
transfusion to avoid future pregnancy complications and
be considered for Rh immune globulin (RhIG) prophy-
laxis. Many transfusion services error on the side of
caution and transfuse patients with D-negative RBCs if
the serological D-typing reaction strength is weaker than
expected (<3+ to 4+). This results in unnecessary use of
the limited D-negative blood supply. DNA testing to dis-
criminate weak D from partial D is important for patient
care and for responsible management of D-negative
blood resources.
© 2013 The Author(s).

Alloantibody versus autoantibody
For patients presenting with RBCs that type positive for
an antigen with an apparent antibody of the same speci-
ficity in the serum or plasma, a DNA-based investigation
is helpful for transfusion management. If the sample is
predicted to be antigen positive by SNP testing, it should
be further investigated by high resolution gene sequenc-
ing. The samples may have a novel amino acid change in
the protein carrying the blood group antigen. These result
in new epitopes and altered (or partial) expression of the
conventional antigen.
Sickle cell disease (SCD) and chronic transfusion
therapy
Alloimmunization is a serious complication of chronic
transfusion, particularly in patients with sickle cell dis-
ease and b-Thalassaemia requiring long-term transfusion
support. Antibody production can result in delayed hae-
molytic transfusion reactions (DHTR). Patients with SCD
undergoing a DHTR are at risk for life-threatening anae-
mia, pain crisis, acute chest syndrome and/or acute renal
failure, and patients may experience hyper-haemolysis, in
which haemoglobin levels drop below pre-transfusion
levels due to bystander haemolysis of the patients’ own
antigen-negative red cells. Diagnosis and management of
DHTR are difficult in this patient population as symptoms
mimic a painful episode and laboratory tests used to
diagnose DHTR, elevated lactate dehydrogenase and un-
conjugated bilirubin levels, are already abnormal. Alloim-
munization also results in significant delay in
transfusion, increases costs, is associated with risk for
production of additional antibodies and often results in a
chronic positive DAT with apparent warm autoantibody,
that is ‘panagglutinins’, which complicate further workups
and transfusion therapy.
Many programmes attempt to prevent or reduce the
risk and incidence of alloantibody production by trans-
fusing RBCs that are antigen matched for D, C, E and K
(and a few include Fya/b, Jka/b and S). The most fre-
quent antibodies encountered in patients with SCD
receiving units antigen matched for D, C, E and K, have
Rh specificities. These antibodies include anti-D, -C and
e in patients whose RBC type serologically as D+, C+
and e+. RH genotyping has revealed that these patients
have RHD and/or RHCE alleles with amino acid changes
that encode altered or partial antigens. For example,
approximately 23% of the SCD patients with C+ RBCs do
not have a conventional RhCe protein. In these patients,
the C antigen is expressed from a hybrid RHD gene that
has lost expression of D, but encodes a C epitope. More
than one-third of patients with this hybrid gene encoding
a C+ phenotype make anti-C or Ce. RH genotyping
can identify these patients who are better served on
© 2013 The Author(s).
ISBT Science Series © 2013 International Society of Blood Transfusion, ISBT Science Series (2013) 8, 1–5
Clinical application of blood group genomics 3
C rather than C+ transfusion protocols. Anti-D in D+
patients with SCD is associated with RHD alleles encod-
ing partial D antigens detected by RHD genotyping.
Anti-e in e+ patients with altered Rhce proteins are more
problematic to manage. Transfusion with e blood will
expose them to the E antigen, and most are E and at
risk for anti-E.
In summary, RH genotyping expands and extends
matching for Rh in this patient population and is impor-
tant for transfusion management.
Prenatal practice
Testing for Rh in pregnant women
Serological typing for Rh(D) cannot distinguish women
whose RBCs lack some epitopes of D (partial D) and who
are at risk for anti-D. RBCs with partial D type as D+,
some in direct tests and others by indirect tests, but these
women would potentially benefit from receiving RhIG
prophylaxis if they deliver a D+ fetus. Serological testing
cannot distinguish weak reactivity due to missing epi-
topes (partial D) from weak reactivity due to quantitative
reduced antigen (weak D). RHD genotyping can distin-
guish weak D from partial D to guide RhIG prophylaxis
and blood transfusion recommendations.
Haemolytic disease of the fetus and newborn
(HDFN)
The identification of a clinically significant alloantibody
in a pregnant woman relies on demonstration of an IgG
antibody by serological testing, but management of the
pregnancy is aided by testing of a paternal sample, and if
indicated, the fetus to assess risk for HDFN.
Paternal testing
Antigen Typing: The RBCs of father should be tested for
the corresponding antigen. If the RBCs are negative, the
fetus is not at risk. If the father is positive, zygosity test-
ing can determine if the father is homozygous or hetero-
zygous for the gene expressing the antigen, particularly
when there is no allelic antigen, or no antisera to detect
the allelic product.
Zygosity testing: DNA testing of paternal samples is
most often done when testing for possible HDFN due to
anti-D or anti-K. For maternal anti-K, testing the paternal
RBCs for expression of the allelic k antigen should be
performed, but many centres do not have a licenced
reagent available and genetic counsellors often request
DNA testing. For maternal anti-D, RHD zygosity testing
by DNA methods is the only method to determine the
paternal gene copy number. A number of different
genetic events cause a D-negative phenotype and multiple
4 C. M. Westhoff
assays must be done to accurately determine RHD zygos-
ity, especially in minority ethnic groups.
If the father is RHD homozygous, all children will be
D+ and monitoring of the pregnancy will be required. If
the father is heterozygous, the fetus has a 50% chance of
being at risk. The D type of the fetus should be deter-
mined to prevent invasive and unnecessary testing so the
mother need not be aggressively monitored or receive
immune modulating agents.
Fetal testing
Amniocentesis: To determine the fetal antigen status, fetal
DNA can be isolated from cells obtained by amniocentesis.
Non-invasive fetal testing from the maternal plasma:
The discovery that cell-free, fetal-derived DNA is present
in maternal plasma by approximately 5 weeks of gesta-
tion allows maternal plasma to be used as a source of
fetal DNA to determine the antigen status of the fetus.
This is particularly successful for D typing because the D-
negative phenotype in the majority of samples is due to
the absence of the RHD gene. Testing for the presence or
absence of a gene is less demanding than testing for a
single gene polymorphism or SNP. This approach is being
used in some European countries to test the maternal
plasma for the presence of a fetal RHD gene to eliminate
the unnecessary administration of antepartum Rh immune
globulin to the approximately 40% of D-negative women
who are carrying a D-negative fetus.
Neonatal alloimmune thrombocytopenia (NAIT)
A diagnosis of NAIT is based on demonstrating HPA-spe-
cific antibody in maternal serum and identifying an
incompatibility between the parents by HPA platelet
genotyping. Twenty-eight HPAs have been characterized,
but incompatibility in HPA-1 accounts for approximately
80% of all cases. Platelet genotyping is used to confirm
the HPA status of the mother and to type a paternal sam-
ple. If the father is homozygous for the target HPA, all
children will be positive and monitoring of the pregnancy
will be required. If the father is heterozygous, the fetus
has a 50% chance of being at risk. The HPA type of the
fetus should be determined to prevent invasive and
unnecessary testing. To determine the fetal HPA antigen
status, fetal DNA can be isolated from cells obtained by
amniocentesis. Non-invasive fetal testing from the mater-
nal plasma has been reported and may become more
readily available in the future.
Other clinical applications
McLeod syndrome
The McLeod phenotype, characterized by weak expression
of RBC Kell system antigens and absence of Kx antigen,
ISBT Science Series © 2013 International Society of Blood Transfusion, ISBT Science Series (2013) 8, 1–5
is encoded by the X-linked gene, XK. This X-linked syn-
drome is manifested only in males and is associated with
late onset of clinical or subclinical myopathy, neurode-
generation and central nervous system manifestations.
The syndrome may be under-diagnosed and the physical
characteristics, which often develop only after the fourth
decade of life, include muscular and neurological prob-
lems. Over 30 different XK gene mutations associated
with a McLeod phenotype have been found. Different XK
mutations appear to have different clinical effects and
may account for the variability in prognosis [5]. Sequenc-
ing of XK to determine the specific type of mutation in
individuals with McLeod phenotypes has clinical prognos-
tic value.
ABO typing discrepancies in patients
DNA testing is useful to resolve patient typing discrepan-
cies, confirm subgroup status and determine the original
blood type of patients massively transfused, or the origi-
nal blood type of transplant recipients by testing a buccal
sample. The ability to accurately determine an individ-
ual’s antigen status eliminates the use of group O RBCs
and AB plasma for transfusion in the situation of ABO
typing discrepancy. ABO genotyping can aid in the differ-
entiation of subgroup alleles, particularly to confirm A2
subgroup in kidney donors who may have been trans-
fused, or whose RBCs give discordant reactivity in sero-
logical testing with anti-A1 reagents. Accurate
determination of ABO often requires gene sequencing.
Donor centre applications
Typing for antigens for which there are no commer-
cial reagents
DNA-based typing has become the standard to identify
antigen-negative units for which there are no serological
reagents. One of the most often used is to type for Dom-
brock (Doa/b). Antibodies to these antigens are clinically
significant, but patient serum antibodies are often weak,
have poor avidity, disappear over time and are almost
always present with other blood group specificities that
interfere with screening donor units. As Dombrock anti-
bodies are difficult to detect, matching of patients with
donors for Dombrock antigens should be considered in
patients when survival of transfused red cells is compro-
mised and complex mixtures of antibodies are present,
even when no specific serum antibody to Dombrock anti-
gens is demonstrable by serological testing.
Confirming D type of donors
Donor centres must perform a test for weak D to attempt
to avoid labelling a product as D-negative that might
result in anti-D in response to transfused RBCs. It is well-
© 2013 The Author(s).

known that some donor RBCs with very weak D expres-
sion are not typed as D+ with current serological reagents
and are labelled as D for transfusion. The prevalence of
weak D RBCs not detected by serological reagents is
approximately 0
1% and although the clinical signifi-
cance has not been established, donor RBCs with weak D
expression have been associated with alloimmunization
[6]. RHD genotyping can improve donor testing by con-
firming D phenotypes [7].
High-throughput screening for donor inventory and
rare or uncommon antigens
The ability to screen for multiple minor antigens in a sin-
gle assay format has been a significant aid to donor cen-
tres to provide antigen-negative products and to provide
antigen-matched products for patients. Although DNA
methods are not yet Food & Drug Administration (USA)
licensed to label donor units, some are CE marked.
Resolving donor ABO discrepancies to retain donors
Donor samples with ABO typing results that are discor-
dant with results from a previous donation must be inves-
tigated. Donors with depressed antigen or antibody
expression and RBC reactions that do not match the
plasma reactions cannot be labelled for transfusion.
Determination of ABO by DNA-based methods can
resolve these, DNA methods offer the potential to be a
confirmatory test. Confirmatory testing by DNA would
avoid the loss of donor units and retain group O donors
with depressed antibody titres in the donor pool.
Discrepancies between serology (phenotype)
and DNA (genotype)
When differences between serological and DNA testing
occur, it is important to investigate. This can indicate the
presence of a novel allele or genetic variant, particularly
when testing individuals from diverse ethnic groups. The
primary cause of discrepancies between the serological
phenotype and DNA genotype when testing donors by
large-scale DNA typing has been traced to human record-
ing errors. Other common causes of discrepancies include
the presence in the sample of an FYX allele. This allele
encodes an amino acid change causing a Fy(b+w) weak
phenotype. The prevalence of FYX encoding Fy(b+w) in
Caucasians is as high as 2% [8].
DNA testing interrogates a single or few SNPs associ-
ated with antigen expression and cannot sample every
nucleotide in the gene. Consequently, discrepancies will
occur when typing patients who have rare or novel
© 2013 The Author(s).
ISBT Science Series © 2013 International Society of Blood Transfusion, ISBT Science Series (2013) 8, 1–5
Clinical application of blood group genomics 5
silencing mutations that cause loss of antigen expression.
These can be specific to a particular ethnic group. For
example, silencing mutations responsible for S-s-U- phe-
notypes are common in African ethnic groups, and mark-
ers for these should be included when typing these
populations. The Fy(a b ) phenotype found in African
Blacks is caused by a mutation in the promoter region of
FYB, which disrupts a binding site for the erythroid tran-
scription factor GATA-1 and results in the loss of Duffy
expression on RBCs [9]. For accuracy, the GATA mutation
must be included when typing for Duffy in African
groups. Importantly, expression of the protein on endo-
thelium is not altered and Fy(a b ) individuals with
GATA-1 mutations are not at risk for anti-Fyb. Silencing
mutations associated with loss of Kidd antigen expression
occur more often in Asians, whereas nucleotide changes
encoding amino acid changes that weaken Kidd expres-
sion are see in Blacks.
Disclosure
The author has no conflicts of interest to declare.
References
1 Westhoff C: Molecular testing for transfusion medicine. Curr
Opin Hematol 2006; 13:471–475
2 Storry JR, Olsson M: The ABO blood group system revisited: a
review and update. Immunohematology 2009; 25:48–59
3 Chou ST, Westhoff CM: Molecular biology of the Rh system:
clinical considerations for transfusion in sickle cell dis-
ease.Hematology Am Soc Hematol Educ Program 2009: 178–
184.
4 Chou ST, Westhoff CM: The role of molecular immunohematol-
ogy in sickle cell disease. Transfus Apheres Sci 2011; 44:73–79
5 Jung HH, Hergersberg M, Vogt M, et al. : McLeod phenotype
associated with a XK missense mutation without hematologic,
neuromuscular, or cerebral involvement. Transfusion 2003;
43:928–938
6 Wagner T, Kormoczi GF, Buchta C, et al. : Anti-D immuniza-
tion by DEL red blood cells. Transfusion 2005; 45:520–526
7 Flegel WA, von Zabern I, Wagner FF: Six years’ experience
performing RHD genotyping to confirm D- red blood cell units
in Germany for preventing anti-D immunizations. Transfusion
2009; 49:465–471
8 Reid ME, Lomas-Frances C, Olsson ML, editors: The Blood
Group Antigen FACTS book, 3rd edn. Elsevier, 2012
9 Tournamille C, Colin Y, Cartron JP, et al. : Disruption of a
GATA motif in the Duffy gene promoter abolishes erythroid
gene expression in Duffy-negative individuals. Nat Genet
1995; 10:224–228