Download Overview of molecular methods in immunohematology

Overview of molecular methods in immunohematology
Marion E. Reid
BACKGROUND
B
lood group antigens are inherited, polymorphic, structural characteristics located on proteins, glycoproteins, or glycolipids on the
outside surface of the red blood cell (RBC)
membrane. RBCs carrying a particular antigen can, if
introduced into the circulation of an individual who lacks
that antigen, elicit an immune response. It is the antibody
from such an immune response that causes problems in
transfusion incompatibility, maternal-fetal incompatibility, and autoimmune hemolytic anemia.
The classical method of testing for blood group antigens and antibodies is hemagglutination. This technique
is simple, inexpensive, and when carried out correctly,
has a specificity and sensitivity that are appropriate for
the clinical care of the vast majority of patients. However,
hemagglutination, which is a subjective test, has certain
limitations: 1) it does not reliably predict a fetus at risk of
hemolytic disease of the newborn (HDN); 2) it is difficult
to type RBCs from a patient who recently received a
transfusion or those that are coated with IgG; 3) it does
not precisely indicate RHD zygosity in D+ people; 4) a
relatively small number of donors can be typed for a relatively small number of antigens, thereby limiting
antigen-negative inventories; 5) it requires the availability of specific reliable antisera; 6) it is labor-intensive as
is the manual data entry; 7) the source material is expensive and diminishing; 8) the cost of commercial reagents
(Food and Drug Administration [FDA]-approved) is escalating; 9) many antibodies are not FDA-approved and are
characterized (often partially) by the user; and 10) some
antibodies are limited in volume, weakly reactive, or not
ABBREVIATION: IRB = Institutional Review Board; SCD = sickle
cell disease.
From the Laboratory of Immunohematology, New York Blood
Center, New York, New York.
Address reprint requests to: Marion E. Reid, PhD, Laboratory of Immunohematology, New York Blood Center, 310 East
67th Street, New York, NY 10021; e-mail: mreid@
nybloodcenter.org.
doi: 10.1111/j.1537-2995.2007.01304.x
TRANSFUSION 2007;47:10S-16S.
10S TRANSFUSION
Volume 47, July 2007 Supplement
available. The understanding of the molecular bases
associated with many blood group antigens and phenotypes enables us to consider the identification of blood
group antigens and antibodies using molecular
approaches. Screening donors by deoxyribonucleic acid
(DNA) testing would conserve antibodies for confirmation by hemagglutination of predicted antigen negativity.
The purpose of this overview was to discuss how molecular approaches can be used in transfusion medicine,
especially in those areas where hemagglutination is of
limited value.
BLOOD TRANSFUSION SUPPORT IN
PATIENTS WITH SICKLE CELL DISEASE
(SCD)
The Stroke Prevention Trial II corroborated the efficacy of
continuous transfusions for preventing strokes in patients
with SCD. So clear were the results that the National Heart,
Lung, and Blood Institute aborted the 6-year trial after
only 2 years. Of the proposed 100 patients, only 79 had
been enrolled, 41 of whom were selected to discontinue
transfusion. Of these, 14 reverted to high-risk transcranial
Doppler ultrasound profiles and resumed transfusion. Of
these 14 patients, 2 had suffered a stroke and received a
transfusion, and 6 others resumed transfusion for other
reasons.1 In contrast, none of the 38 patients who continued to receive transfusions had strokes or reverted to a
high-risk state. The National Heart, Lung, and Blood Institute accordingly issued an alert to inform and advise physicians who treat children with SCD that interruption of
transfusions for primary stroke prevention is not recommended. Unfortunately, one major risk of chronic transfusion therapy, whether given for stroke prevention or for
other indications, is blood group alloimmunization with
incidence rates of approximately 20 percent or higher,
compared with 5 percent in other transfusion-dependent
patient populations.2-6 Patients with SCD often produce
many alloantibodies to blood group antigens, which
makes the provision of appropriate antigen-negative
blood very problematic. Our ability to test a large number
of donors for minor antigens is restricted by laborintensive test procedures and data entry, and limited supplies or unavailability of typing grade antisera. Currently,
patients with alloantibodies to multiple blood group
MOLECULAR METHODS IN IMMUNOHEMATOLOGY
PCR-based assays can be of value in the
prenatal setting in identifying the fetus
who is not at risk of HDN (i.e., predicted
Antigen typing a patient
• To identify a fetus at risk for HDN
to be antigen-negative) so that the
• When antibody is weak or not available, e.g., anti-Doa, -Dob, -Jsa, -V/VS
mother need not be aggressively moni• Who has recently received a transfusion
tored. Sources of fetal DNA include
• Whose RBCs are coated with immunoglobulin (+DAT)
• To distinguish an alloantibody from an autoantibody
amniocytes and maternal plasma.
• To detect weakly expressed antigens (e.g., Fyb with the FyX phenotype); where the
DNA-based typing should be conpatient is unlikely to make antibodies to transfused antigen-positive RBCs
sidered when a mother’s serum con• Identify molecular basis of unusual serologic results, especially Rh variants
• To determine zygosity, particularly RHD
tains an immunoglobulin G (IgG)
Antigen typing for donors
alloantibody that has been associated
• To screen for antigen-negative donors
with HDN and the father’s antigen
• When antibody is weak or not available, e.g., anti-Doa, -Dob; -Jsa, -Jsb; -V/VS
• Mass screening to increase antigen-negative inventory and to find donors whose RBCs
status for the corresponding antigen is
lack a high-prevalence antigen
heterozygous, indeterminable, or he is
• To resolve blood group A, B, D, C, and e discrepancies
not available for testing. For prenatal
• To detect genes that encode weak antigens
• To type donors for reagent RBCs (antibody screening and identification panels)
diagnosis of a fetus at risk of HDN, the
Other applications
approach to DNA typing should err on
• One-step, automated, objective antibody detection and identification
the side of caution. Thus, the strategy
• Use of transfected cells as immunogens for production of monoclonal antibodies
• Conversion of IgG monoclonal antibodies to IgM direct agglutinins
should be to detect a gene even if the
product is not expressed on the RBC
membrane rather than fail to detect a
antigens, or to an antigen of high prevalence, may have to
gene whose product is expressed, because this could
wait for matched RBC components to be obtained
result in inadequate monitoring throughout pregnancy.
through a national or even international Rare Donor RegAnother potential pitfall is the possibility of contaminaistry, or to undergo surgery with fewer units than the
tion by maternal DNA.
optimal. The unavailability of compatible blood may
As the incidence of genes varies substantially in difextend hospital in-days (often in intensive care) and conferent populations, it can be helpful to know the ethnicity
tributes to morbidity and mortality. If we are to transfuse
of the parents. Also, to limit the gene pool, it is helpful to
these patients effectively, we must find better ways of
test DNA from the parents. An important practical considreducing the risk of transfusion reactions, hyperhemolysis
eration is to determine whether the mother has undersyndrome, and alloimmunization.
gone medical procedures such as artificial insemination,
The genes encoding 28 of the 29 blood group systems
in vitro fertilization, or whether she is a surrogate mother.
(only the P system remains to be resolved) have been
The RHD type is a prime target because anti-D is
cloned and sequenced,7 and the molecular bases of blood
notoriously clinically significant in terms of HDN
group antigens and phenotypes have been determined.8
(reviewed in Avent and Reid10). DNA analysis for the preAnalysis of DNA involves amplification of the target
diction of fetal D phenotype is based on detecting the
sequence of nucleotides, followed by analysis by such
presence or absence of portions of RHD. In Europeans, the
techniques as polymerase chain reaction-restriction fragmolecular basis of the D– phenotype is usually associated
ment length polymorphism (PCR-RFLP), allele-specific
with deletion of the entire RHD, but several other molecuPCR, real-time PCR, sequencing, and microchip. The
lar bases have been described.11 Approximately one-third
results of DNA testing can be used to predict the presence
of D– Japanese have an intact but inactive RHD,12 and as
of blood group antigens in various scenarios, and to
many as 10 percent of Japanese donors who had RBCs
express antigens in heterologous systems not only to
nonreactive with anti-D have the Del phenotype.13
detect and identify blood group antibodies in a single,
Approximately one-quarter of D– African Americans have
objective, automated assay but also as immunogens for
an RHD pseudogene (RHDY), which does not encode the
the production of monoclonal antibodies (Table 1).9 For
D antigen,14 and many others have a hybrid RHD-CE-D
the first time in the history of blood transfusion, microgene (e.g., the r’S phenotype). To predict the RhD antigen
chip technology makes it feasible to contemplate precisely
type by DNA analysis requires probing for multiple singlematching the antigen-negative status of a donor to that of
nucleotide polymorphisms.15 Establishing the fetal K
a patient destined to receive chronic transfusions.
genotype is also of great clinical value in determining
whether a fetus is at risk for severe anemia, because the
DNA ASSAYS TO IDENTIFY A FETUS AT
strength of the mother’s anti-K does not correlate with the
RISK FOR ANEMIA OF THE NEONATE
severity of the infant’s anemia.16
Hemagglutination, including titers, gives only an indirect
When performing DNA analysis in the prenatal
indication of HDN risk and severity. Antigen prediction by
setting, it is also important to always determine the RHD
TABLE 1. Clinical applications of molecular analyses
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REID
status of the fetus, in addition to the test being ordered. In
so doing, if the fetus has a normal RHD, there is no need to
provide Rh- blood for interuterine transfusions. This is
especially true if the mother has anti-c and fetal DNA is
being typed for RHCE*c.
DNA TYPING FOR BLOOD GROUP
ANTIGENS FOR PATIENTS
When a patient receives a transfusion, the presence of
donor RBCs in the patient’s peripheral blood makes RBC
phenotyping by conventional hemagglutination techniques complex, time-consuming, and possibly inaccurate. Indeed, the “best guess” of a patient’s antigen type
based on the strength of hemagglutination, the number of
RBC components transfused, the length of time since
transfusion, the estimated blood volume of the patient,
and the prevalence of the antigen in question is more
often inaccurate than accurate.17 To overcome this
problem, PCR-based assays using DNA isolated from
white blood cells (WBCs), buccal smear, or urine sediment
can be used to predict the antigen type of patients.18-21
DNA-based antigen typing of patients with autoimmune hemolytic anemia, whose RBCs are coated with
immunoglobulin (have a positive direct antiglobutin test
[DAT]), is valuable when 1) direct agglutinating antibodies, or murine monoclonal antibodies, are not available; 2)
antisera are weakly reactive; 3) antigen is sensitive
to the IgG removal treatment; and 4) diagnostic antibodies
require the indirect antiglobulin test and IgG removal
techniques are not effective at removing bound immunoglobulin. DNA-based assays are also useful as a tool to
distinguish alloantibodies from autoantibodies and to
identify the molecular basis of unusual serologic results.
When recommendations for clinical practice are
based on molecular analyses, it is important to remember
that, in rare situations, a genotype determination will not
correlate with antigen expression on the RBC.22 If a patient
has a grossly normal gene that is not expressed, he or she
could produce an antibody following transfusion of
antigen-positive blood. When feasible, the appropriate
assay to detect a mutation that silences a gene should be
part of the DNA-based testing, e.g., GATA box and FY-265
analyses with FY typing,23 presence of RHD pseudogene
with RHD typing,15 and exon 5 analysis with GYPBS typing.24 In addition, it is important to obtain an accurate
medical history of the patient because with certain
medical treatments, such as stem cell transplantation,
results of DNA typing may differ from results obtained by
hemagglutination.
DNA TESTING FOR SCREENING
BLOOD DONORS
PCR-based assays can be used to predict the antigen type
of the donor blood for transfusion and for antibody iden12S TRANSFUSION
Volume 47, July 2007 Supplement
tification reagent panels.25 This is particularly useful
when antibodies are not available or are weakly reactive.
Examples are Doa/Dob, Jsa, and V/VS where DNA-based
assays are being used to type patients and donors to
overcome the dearth of reliable typing reagents. PCRbased assays are also useful for confirming whether an
antigen is truly present in a double dose, especially S, D,
Fya, Fyb, Jka, and Jkb. In our laboratory, DNA-based assays
were found to be valuable for differentiating specific
Knops antigen negativity from a low copy number of CR1
(CD35).
With donor typing, the presence of a grossly normal
gene whose product is not expressed on the RBC surface
would lead to the donor being falsely typed as antigenpositive, and although this would mean loss of an antigennegative donor, it would not jeopardize the safety of blood
transfusion. As automated procedures attain higher and
faster throughput at lower cost, typing of blood donors by
DNA-based assays is likely to become more widespread.
Screening for rare donors by analysis of DNA is valuable
for typing for “minor” antigens and Rh variants. Those
antigens that are predicted to be negative should be confirmed by hemagglutination. In this manner, precious
antibodies are conserved for the confirmation of DNA
typing predictions.
Why not determine routine ABO and RHD by
testing DNA?
Like all donors, antigen-negative donors must have their
ABO and Rh type determined. However, for several
reasons DNA analysis is not the method of choice for
routine ABO and D determination of donor blood. They
include the following: the naturally occurring anti-A or
anti-B in the plasma of most people who lack the corresponding antigens provides a built-in check when performing ABO typing by hemagglutination; potent,
well-standardized monoclonal reagents are available for
ABO and D typing; hemagglutination is relatively simple
and rapid; and systems are in place to test and record,
relatively efficiently, the ABO and D type of a donor. In
both ABO and Rh systems, there are few antigens and
many alleles. In the ABO system, there are four primary
phenotypes (A, B, AB, O) but well over 100 alleles. In the Rh
system, D is one antigen but there are close to 200 alleles
known. In both scenarios, it is highly likely that more
alleles exist and await detection. Furthermore, RBCs with
a weak expression of the D antigen are almost always C+ or
E+. Thus, the fear of transfusing apparently D– RBCs that
actually express some serologically nondetectable D
antigen can be more easily overcome by transfusing D– C–
E– RBCs (which are usually truly D–) than by using DNA
assays to detect the multiple alleles involved in the weak D
phenotypes. For routine ABO and D determination, DNA
testing is more time-consuming, more expensive, prone
MOLECULAR METHODS IN IMMUNOHEMATOLOGY
to misinterpretation, and not an improvement over
hemagglutination.
DNA analysis for ABO and Rh types can be of value
in the resolution of ABO and D discrepancies, to show
that a discrepancy is due to a genetic variant and not to
technologist error or reagent failure and thus, not an
FDA reportable error. ABO genotyping can also be useful
for distinguishing an acquired phenotype from an inherited one without having to perform laborious family
studies. Many Rh phenotypes cannot easily be defined by
serologic methods, either because suitable panels of
monoclonal antibodies are not readily available or
because the antibodies are not available in the needed
strength or volume. DNA assays may be useful for defining some and precisely match the D and e antigen
status of a donor to a recipient, especially those with
SCD.
Testing for donors lacking Do antigens
RBC typing for Doa, Dob, Hy, and Joa antigens of the Dombrock blood group system is notoriously difficult because
the corresponding antibodies, although clinically significant, are often weakly reactive, available only in small
volume, and present in sera containing other alloantibodies.26 At the New York Blood Center, PCR-RFLP analysis is
used to type donors selected to lack certain combinations
of antigens [e.g., C–, E–, K–, Fy(a–), Jk(b–)] for DOA and
DOB for patients who have antibodies to such multiple
antigens, in addition to anti-Doa or anti-Dob. Donors
whose RBCs react weakly or do not react with human
polyclonal anti-Gya for DO-HY and DO-JO are also tested.
This has provided us with a larger inventory of valuable
Hy– and Jo(a–) donors. Because of the dearth of appropriate antisera, testing for polymorphisms in the Dombrock
system by DNA analysis surpasses hemagglutination for
antigen typing.
DNA typing for low-prevalence antigens
Patients in New York often need Js(a–), V–, VS–, Go(a–), or
DAK– RBC components. Providing RBC products for these
patients is difficult because patients make antibodies to
these antigens in addition to several others, e.g., anti-C,
-E, -K, -Fya, and -Jkb. While these immunogenic antigens
are of low prevalence in Caucasian donors, they are
present on up to 20 percent of RBCs from African American donors. As a natural consequence of transfusing Rhand K-matched RBC components to patients, as in the
Stroke Prevention Trials, the patients have made antibodies to these “low prevalence” antigens. These antigens are
not on antibody screening RBCs; the corresponding antibodies are not available to screen for donors; and the
crossmatch is not always reliable for their detection. PCRbased assays provide a tool to mass screen donors, thereby
increasing the antigen-negative inventory and improving
patient care.
As an illustration, we had a patient whose serum contained anti-U, -C, -E, -K, plus -VS, and -Jsa. Of 95 eligible U–
C– E– K– donors in the center’s inventory, four had been
typed as VS–, Js(a–), 27 were VS+ or Js(a+), and 64 had not
been typed for either antigen. Anti-VS and anti-Jsa were not
available in sufficient volume for typing. PCR-based
methods, which do not require special reagents, can be
used to screen for antigen-negative donors. Current
manual methods are time-consuming and labor intensive;
however, the prospect of using microchips to screen large
numbers of donors is appealing. This case is not unique,
and many such examples throughout the United States
exist.
DNA typing for high-prevalence antigens
As anti-Lub, -Yta, -Sc1, -LW, and -Coa are inconsistently
available, testing DNA is a desirable alternative. The ready
availability of anti-k, -Kpb, -Jsa, -Fy3, and -Jk3 often makes
hemagglutination the method of choice for these antigens. On the other hand, if the appropriate singlenucleotide polymorphisms can be added to a microchip at
little incremental cost, all of the above antigens could be
predicted. Detection of Vel–, Lan–, At(a–), or Jr(a–) donors
is restricted to hemagglutination because the molecular
bases of these antigens are not yet known. Detection of
null phenotypes such as Rhnull, K0, Gy(a–), Ge–, or McLeod
is complex because of multiple molecular bases associated with these phenotypes.8
LIMITATIONS OF DNA ANALYSIS
Testing by DNA analyses does have technical, medical,
and genetic pitfalls.27 Medical pitfalls include recent
transfusions, stem cell transplantation, and natural chimerism. In these scenarios, results of testing DNA may
not agree with hemagglutination results. In addition,
stem cell transplantation and natural chimerism may
cause the results of testing DNA from somatic cells to
differ from the results of testing DNA from WBCs. Thus,
when embracing DNA testing, it is important to obtain
an accurate medical history. There are many genetic
events that cause apparent discrepant results between
hemagglutination and DNA test results;28 the genotype is
not the phenotype.
The majority of DNA-based assays will detect a
grossly normal gene that is not expressed and this can
lead to a donor being falsely identified as antigenpositive. This would mean that a valuable antigennegative (e.g., null) donor would be lost to the inventory,
but would not jeopardize the safety of a patient receiving
blood transfusion. Confirmation by hemagglutination of
predicted antigen negativity is recommended using a
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TRANSFUSION 13S
REID
reagent antibody, if available, or by crossmatching using
a method optimal for the detection of the antibody or
antigen in question.
Not all blood group polymorphisms can be easily
analyzed, for example, if a large number of alleles encode
one phenotype (e.g., ABO, Rh, and null phenotypes in
many blood group systems); or alleles with a large deletion
(e.g., Ge–) or alleles encoded by a hybrid gene (e.g., in the
Rh and MNS systems); or when the molecular basis is not
yet known (e.g., Vel, Lan, Jra). Additionally, there is a high
probability that not all alleles in all ethnic populations are
known.
As discussed earlier, the molecular bases associated
with a large number of antigens have been reported.
However, in many cases, the analysis has been restricted
to a relatively small number of people with known
antigen profiles. This information is being applied to
DNA typing with the assumption that such analysis will
correlate with RBC antigen typing in all populations.
However, we are still in our infancy of understanding
gene polymorphisms in different ethnic groups and
their significance to the expression of blood group antigens. A much larger number of people from a variety of
ethnic backgrounds needs to be analyzed to establish
more firmly the correlation between genotype and the
blood group phenotype. Until such data are available,
caution should be exercised when recommending
clinical practice based on DNA typing for blood group
antigens.
MICROCHIP SCREENING FOR
ANTIGEN-NEGATIVE DONORS
Microchip technology simultaneously performs multiple
assays on one sample; thereby a large number of antigens
can be predicted on a large number of donor samples. The
results are analyzed and interpreted by computer, and can
be directly downloaded to a donor database, which will
reduce data entry errors inherent in manual systems.29,30
The cost of the approach will depend on how much the
manufacturers charge for kits they develop. An added cost
of doing DNA testing could be the expense of investigating
any discrepancies. High-throughput technologies have
the potential to dramatically increase inventories of
antigen-negative blood.
POSSIBLE USES OF PHENOTYPICALLY
MATCHED BLOOD
If antigen-negative inventories were sufficient, the following uses of antigen-matched blood could be
contemplated:
•
•
•
•
TYPING BY DNA ASSAYS: CONCERN
AND CONSENT
Whether a study is considered exempt, expedited, or
requires a full review by the Institutional Review Board
(IRB) depends on several factors, namely, whether the
samples are being tested for clinical purposes or research,
whether the samples already exist or are collected specifically for the study, whether they are unlinked or linked,
and whether there is a risk or not to the human subject. If
a sample is tested for patient care, no IRB approval is
needed.
Donor consent may or may not be needed. This will
depend on the wording of the local Donor Registration
Form with regard to how the testing for blood groups antigens will be performed.
There is much concern that testing DNA will reveal
unwanted information about a donor. However, the assays
are not considered “genotyping” but rather are a procedure to antigen type by DNA analysis. According to the
New York State, as this DNA testing is not used to identify
or diagnose a genetic disease, informed consent is not
required. The interpretations do not differ from what can
be presently accomplished by hemagglutination.
14S TRANSFUSION
Volume 47, July 2007 Supplement
•
•
•
to match antigen profiles in chronically transfused
patients who are immune responders, especially
those with SCD disease;
to match unusual Rh phenotypes, especially in
African Americans (hrB–, hrS–, etc.);
to predict the type of antigens for which there is no
antibody (e.g., V/VS, Goa, DAK, Jsa, Doa, Dob);
to match for Jka and Jkb if the patient has been
exposed, to prevent transfusion reactions and deaths
due to anti-Jka or anti-Jkb. Reports by the FDA in the
United States and the Serious Hazards of Transfusion
study in the UK have revealed that a handful of
patients die annually after receiving transfusion of
antigen-positive blood;
to transfuse RBC components with weak expression
of the RhD antigen to patients with similarly weak
RhD expression (e.g., match for Del and Dweak);
to transfuse patients with antibodies to high prevalence antigens; and
to transfuse patients with autoimmune hemolytic
anemia to eliminate labor-intensive procedures that
are required to ensure that there are no underlying
clinically significant antibodies.
OTHER CONSIDERATIONS
To apply molecular approaches to clinical situations,
several areas of knowledge are needed, for example, a
knowledge of molecular techniques, of gene structure and
processing, of the molecular bases of blood groups, of
hemagglutination techniques, of the expression of blood
group antigens, of factors that may affect the interpretation of genotype (e.g., chimeras), and of regulatory com-
MOLECULAR METHODS IN IMMUNOHEMATOLOGY
pliance (Good Laboratory Practices, IRB, FDA), as well as
an ability to correlate DNA and serologic results to the
clinical problems being addressed.
Numerous studies have analyzed blood samples from
people with known antigen profiles and identified the
molecular bases associated with many antigens. The available wealth of stockpiled serologically defined variants
has contributed to the rapid rate with which the genetic
diversity of blood group genes has been revealed. Initially,
molecular information associated with each variant was
obtained from only a small number of samples. This information was applied to DNA analyses with the hopeful
assumption that the molecular analysis would correlate
with RBC antigen typing. With the gathering of more information it became obvious that many molecular events
result in the genotype and phenotype being apparently
discrepant and that more than one genetic event can give
rise to the same phenotype. This is especially true for null
phenotypes, e.g., for Rh,8,31 Kell,26,32 and Kidd systems,32
and the p phenotype.33-34
Other considerations include establishing the extent
of testing alleles for each antigen, e.g., GATA and nt FY-265
with FY typing, and whether to use the results without
confirmation by hemagglutination if it is unlikely to harm
the patient. If we had a simple, inexpensive way of positively identifying a donor at subsequent donations, DNA
typing of a donor could be performed only once. Of value
would be a system of automated DNA preparation and
positive sample identification from the beginning of the
process to the end.
Hemagglutination is the gold standard technique to
type RBCs for the presence or absence of blood group
antigens. PCR-based assays, used as an adjunct to hemagglutination, will be a powerful tool that could radically
change approaches used to support patients in their
transfusion needs.
3. Heddle NM, Soutar RL, O’Hoski PL, et al. A prospective
study to determine the frequency and clinical significance
of alloimmunization post-transfusion. Br J Haematol 1995;
91:1000-5.
4. Redman M, Regan F, Contreras M. A prospective study of
the incidence of red cell allo-immunisation following
transfusion. Vox Sang 1996;71:216-20.
5. Aygun B, Padmanabhan S, Paley C, Chandrasekaran V.
Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions.
Transfusion 2002;42:37-43.
6. Rosse WF, Gallagher D, Kinney TR, et al. Cooperative study
of sickle cell disease. Transfusion and alloimmunization in
sickle cell disease. Blood 1990;76:1431-7.
7. Lögdberg L, Reid ME, Lamont RE, Zelinski T. Human blood
group genes: chromosomal locations and cloning strategies. Transfus Med Rev 2005;19:45-57.
8. Reid ME, Lomas-Francis C. Blood group antigen factsbook,
2nd edition. San Diego, CA: Academic Press, 2004.
9. Yazdanbakhsh K. Applications of blood group antigen
expression systems for antibody detection and identification. Transfusion 2007;47(Suppl):85S-8S.
10. Avent ND, Reid ME. The Rh blood group system: a review.
Blood 2000;95:375-87.
11. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet 2001;2:10.
Available from http://www.biomedcentral.com/1471-2156/
2/10.
12. Okuda H, Kawano M, Iwamoto S, et al. The RHD gene is
highly detectable in RhD-negative Japanese donors. J Clin
Invest 1997;100:373-9.
13. Chang JG, Wang JC, Yang TY, et al. Human RhDel is caused
by a deletion of 1,013 bp between introns 8 and 9 including exon 9 of RHD gene. Blood 1998;92:2602-4.
14. Singleton BK, Green CA, Avent ND, et al. The presence of
an RHD pseudogene containing a 37 base pair duplication
ACKNOWLEDGMENT
15.
Robert Ratner is acknowledged for his help in the preparation of
the manuscript.
16.
17.
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