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CHAPTER
7
MOLECULAR TESTING
FOR BLOOD GROUPS IN
TRANSFUSION MEDICINE
M. E. REID AND H. DEPALMA
OBJECTIVES
After completion of this chapter, the reader will be able to:
1. Explain the basics of the structure and processing of a
gene.
2. Discuss mechanisms of genetic diversity and the molecular bases associated with blood group antigens.
3. Describe applications of PCR-based assays for antigen
prediction in transfusion and prenatal settings.
4. Describe some instances where RBC and DNA type may
not agree.
5. Delineate the limitations of hemagglutination and of PCRbased assays for antigen prediction.
6. Summarize relevant regulatory issues.
KEY WORDS
Alleles
Blood group antigens
DNA to protein
A
Molecular testing
Prediction of blood groups
blood group antigen is a variant form of a
protein or carbohydrate on the outer surface of
a red blood cell (RBC) that is identified when an
immune response (alloantibody) is detected by
hemagglutination in the serum of a transfused patient or pregnant woman. The astounding pace of
growth in the field of molecular biology techniques
and in the understanding of the molecular bases associated with most blood group antigens and phenotypes enables us to consider the prediction of blood
group antigens using molecular approaches. Indeed,
this knowledge is currently being applied to help resolve some long-standing clinical problems that cannot be resolved by classical hemagglutination.
Blood group antigens are inherited, polymorphic,
structural characteristics located on proteins, glycoproteins, or glycolipids on the outer surface of the
RBC membrane. The classical method of testing for
blood group antigens and antibodies is hemagglutination. This technique is simple and when done
correctly, has a specificity and sensitivity that is appropriate for the clinical care of the vast majority of patients.
Indeed, direct and indirect hemagglutination tests
have served the transfusion community well for,
respectively, over 100 and over 50 years. However, in
some aspects, hemagglutination has limitations. For
example, it gives only an indirect measure of the potential complications in an at-risk pregnancy, it cannot
precisely indicate RHD zygosity in D-positive people,
it cannot be relied upon to type some recently transfused patients, and it requires the availability of specific reliable antisera. The characterization of genes
and determination of the molecular bases of antigens
and phenotypes has made it possible to use the polymerase chain reaction (PCR)1 to amplify the precise
areas of deoxyribose nucleic acid (DNA) of interest to
detect alleles encoding blood groups and thereby
predict the antigen type of a person.
This chapter first provides an overview of the processing of DNA to a blood group antigen and then
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summarizes current applications of molecular approaches for predicting blood group antigens in transfusion medicine practice for patients and donors, especially
in those areas where hemagglutination has limitations.
FROM DNA TO BLOOD GROUPS
The Language of Genes
DNA is a nucleic acid composed of nucleotide bases, a
sugar (deoxyribose), and phosphate groups. The nucleotide bases are purines (adenine [A] and guanine [G])
and pyrimidines (thymine [T] and cytosine [C]). The
language of genes is far simpler than the English language. Compare four letters in DNA or RNA (C, G, A,
and T [T in DNA is replaced by U in RNA]) with 26 letters of the English alphabet. These four letters are called
nucleotides (nts) and they form “words,” called codons,
each with three nucleotides in different combinations.
There are only 64 (4 ⫻ 4 ⫻ 4 ⫽ 64) possible codons of
which 61 encode the 20 amino acids and 3 are stop
codons. There are more codons (n ⫽ 61) than there are
amino acids (n ⫽ 20) because some amino acids are encoded by more than one codon (e.g., UCU, UCC, UCA,
UCG, AGU, and AGC, all encode the amino acid called
serine). This is termed redundancy in the genetic code.
Essentials of a Gene
Figure 7-1 shows the key elements of a gene. Exons
are numbered from the left (5⬘, upstream) to right
(3⬘, downstream) and are separated by introns.
Transcription
binding site
DNA
5
Upstream
Nucleotides in exons encode amino acids or a “stop”
instruction, while nucleotides in introns are not encoded. Nucleotides in an exon are written in upper
case letters and those in introns and intervening sequences are written in lower case letters. At the junction of an exon to an intron, there is an invariant
sequence of four nucleotides (AGgt) called the donor
splice site, and at the junction of an intron to an exon
is another invariant sequence of four nucleotides
(agGT) called the acceptor splice site. The splice sites
interact to excise (or outsplice) the introns, thereby
converting genomic DNA to mRNA. A single strand
of DNA (5⬘ to 3⬘) acts as a template and is duplicated
exactly to form mRNA. Nucleotide C invariably
pairs with G, and A with T. Upstream from the first
exon of a gene, there are binding sites (promoter regions) for factors that are required for transcription
(from DNA to mRNA) of the gene. Transcription of
DNA always begins at the ATG, or “start,” transcription codon. The promoter region can be ubiquitous,
tissue specific, or switched on under certain circumstances. At the 3⬘ end of a gene there is a “stop” transcription codon (TAA, TAG, or TGA) and beyond
that there is often an untranslated region. Between
adjacent genes on a chromosome, there is an “intervening” sequence of nucleotides, which are not transcribed.
After the introns are excised, the resultant
mRNA contains nucleotides from the exons of the
gene. Nucleotides in mRNA are translated (from
mRNA to protein) in sets of three (a codon) to produce
a sequence of amino acids, which form a protein. Like
transcription of DNA, translation of mRNA always
Gene A
Exon 1
Intron 1
Intron 2
Exon 3
Exon 2
D
A
D
A
Intervening
sequence
Gene B
Exon 1
UTR
Stop
transcription
ATG
Start
transcription
3
D
Gene A
mRNA
5
Exon 1
AUG
Start
translation
3
Exon 2 Exon 3
Stop
translation
D = donor splice site (AGgt)
A = acceptor splice site (agGT)
UTR = untranslated region
NH2 = amino terminus
Protein NH2
COOH
COOH = carboxy terminus
Met
FIGURE 7-1 The anatomy of a gene. Schematic representation of a hypothetical gene, showing transcription of
DNA to mRNA and translation from mRNA to the corresponding protein.
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CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine
begins at the “start” codon (AUG) and terminates at a
“stop” codon (UAA, UAG, or UGA). The resultant
protein consists of amino acids starting with methionine (whose codon is AUG) at the amino (NH2) terminus. Methionine, or a “leader” sequence of amino
acids, is sometimes cleaved from the functional protein and thus, a written sequence of amino acids (or
mature protein) does not necessarily begin with methionine.
DNA is present in all nucleated cells. For the prediction of a blood group, DNA is usually obtained
from peripheral white blood cells (WBCs), but also
can be extracted from epithelial cells, cells in urine
sediment, and amniocytes.
Molecular Bases of Blood Groups
Although many mechanisms give rise to a blood
group antigen or phenotype (Table 7-1), the majority of blood group antigens are a consequence of a
single nucleotide change. The other mechanisms
listed give rise to a small number of antigens and
various phenotypes. Figure 7-2 shows a short hypothetical sequence of double-stranded DNA together with transcription (mRNA) and translation
(protein) products. The effect of a silent, missense,
or nonsense single nucleotide change together
with examples involving blood group antigens are
illustrated.
Effect of a Single Nucleotide Change
on a Blood Group
Due to redundancy in the genetic code, a silent
(synonymous) nucleotide change does not change
the amino acid and, thus, does not affect the antigen
expression. Nevertheless, because it is possible that
such a change could alter a restriction enzyme
recognition site or a primer binding site, it is important to be aware of silent nucleotide changes when
designing a PCR-based assay. In contrast, a missense
(nonsynonymous) nucleotide change results in a
different amino acid, and these alternative forms of
an allele encode antithetical antigens. Figure 7-2
illustrates this where “G” in a lysine codon (AAG) is
replaced by “C,” which gives rise to the codon for
asparagine (AAC). The example of a missense
nucleotide change shows that a “C” to “T” change
is the only difference between the clinically important blood group antigens k and K. A nonsense
TABLE 7-1 Molecular Events That Give Rise to Blood Group Antigens and Phenotypes
Molecular Mechanism
Example for Blood Group
Single nucleotide changes in mRNA
Multiple (see Fig. 7-2 and Table 7-2)
Single nucleotide change in a transcription site
T > C in GATA of FY
Single nucleotide change in a splice site
ag > aa in Jk(a⫺b⫺)
Deletion of a nucleotide(s)
Multiple (see Fig. 7-2 and Table 7-2)
Deletion of an exon(s)
Exon 2 of GYPC in Yus phenotype
Deletion of a gene(s)
RHD in some D-negative people
Insertion of a nucleotide(s)
37-bp insert in RHD⌿ in somea D-negative people
(see Fig. 7-2 and Table 7-2)
Insertion (duplication) of an exon(s)
Exon 3 of GYPC in Ls(a⫹)
Alternative exon
Exon 1 in I-negative people
Gene crossover, conversion, other recombination events
Many hybrid genes in MNS and Rh systems
Alternative initiation (leaky translation)
Glycophorin D
Absence/alteration of a required interacting protein
RhAG in regulator Rhnull, and Rhmod
Presence of a modifying gene
InLu in dominant Lu(a⫺b⫺)
Unknown
a
Knull, Gy(a⫺)
2
Not uncommon in African Americans and Japanese.
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DNA
A
A T G T C G A A G G A A G C A –3
T A C A G C T T C C T T C G T –5
mRNA
Protein
A U G |U C G | A A G |G A A |G C A
M e t Se r Lys Glu Ala
Transcription product
Translation product
Examples:
Single nucleotide substitution
Silent
B
Missense
Nonsense
C
A U G | U C G/ | A A G | G A A | G C A
M e t Se r Lys Glu Ala
C
/ |G A A |G C A
A U G |U C G | A A G
M e t Se r Asn Glu Ala
U
/ A A |G C A
A U G |U C G | A A G |G
M e t Se r Lys St op
378 T>C in DO exon 2
Tyr126Tyr = no change
698 T>C in KEL exon 6
Met193Thr = K/k
287G>A in FY exon 2
Trp96Stop = Fy (a–b–)
Single nucleotide deletion
New Sequence
U C G A |A G G | A A G | C A
A U G |X
Arg Lys
Met
Arg
261del G in O exon 6
Frameshift → 116Stop = 0
Stop Codon
C G A |A G G A A G C A
A U G |U X
M e t St op
No example known
C
Single nucleotide insertion
D
New Sequence
Stop Codon
G
A U G | U C G | A A |G G A | A G C | A
M e t Se r Arg Gly
Se r
U
A U G | U C G | A A G | G A |A G C A
M e t Se r Lys St op
307-308 ins T in CO exon 2
Frameshift → Stop = Co(a–b–)
No example known
FIGURE 7-2 A hypothetical piece of DNA and the effect of single nucleotide changes. A short hypothetical sequence
of double-stranded DNA and the resultant transcription (mRNA) and translation (protein) products are shown. The figure
Panel A). Panels B through D demonstrate
also shows the five amino acids that are determined by the codons in the DNA (P
Panel B), deletion (P
Panel C), and insertion
the effect of three different types of single nucleotide changes, substitution (P
Panel D), and the effects on the amino acids. Where available, examples of these various types of changes in blood
(P
groups are given.
nucleotide change transforms a codon for an amino
acid to a stop codon. Figure 7-2 and Table 7-2 give
examples relative to blood groups.
Effect of Deletion or Insertion of Nucleotide(s)
A deletion of one nucleotide results in a ⫺1 frameshift
and an eventual stop codon (see Fig. 7-2 and Table 7-2).
Typically, this leads to the encoding of a truncated
protein, but it can cause elongation. For example, a deletion of “C” close to the stop codon in the A2 allele
results in a transferase with 21 amino acids more than in
the A1 transferase.2 Similarly, deletion of two nucleotides
causes a ⫺2 frameshift and a premature stop codon.
Deletion of a nucleotide also can cause a stop codon, but
there is no known example for a blood group.
An insertion of one nucleotide results in a ⫹1
frameshift and a premature stop codon (see Fig. 7-2
and Table 7-2). Insertion of two nucleotides causes a
⫹2 frameshift and a premature stop codon. Insertion
of a nucleotide can cause a stop codon, but there is no
known example for a blood group.
APPLICATIONS OF MOLECULAR
ANALYSIS
The genes encoding 29 of the 30 blood group systems (only P1 remains to be resolved) have been
cloned and sequenced.3,4 Focused sequencing of
DNA from patients or donors with serologically
defined antigen profiles has been used to determine
the molecular bases of variant forms of the gene.
This approach has been extremely powerful because
antibody-based definitions of blood groups readily
distinguish variants within each blood group
system. Details of these analyses are beyond the
scope of this chapter but up-to-date details about
alleles encoding blood groups can be found on the
Blood Group Antigen Gene Mutation database at:
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TABLE 7-2 Molecular Bases Associated with a Few Blood Group Antigens
Antigen/Phenotype
Gene
Nucleotide Change
Amino Acid
S/s
GYPB
143T > C
Met29Thr
E/e
RHCE
676C > G
Pro226Ala
KEL
698T > C
Met193Thr
FY
125G > A
Gly42Asp
JK
838G > A
Asp280Asn
DO
793A > G
Asn265Asp
Fy(a–b–)
FY
407G > A
Try136Stop
D–
RHD
48G > A
Trp16Stop
Gy(a–)
DO
442C > T
Gln148Stop
D–
RHD
711Cdel
Frameshift → 245Stop
D–
RHD
AGAG
Frameshift → 167Stop
Ael
ABO
798-804Gins
Frameshift → Stop
D–
RH
906GGCTins
Frameshift → donor splice site
change (I6 + 2t > a)
Missense nucleotide change
K/k
a
Fy /Fy
b
Jka/Jkb
a
Do /Do
b
Nonsense nucleotide change
Nucleotide deletion
Nucleotide insertion
http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.
cgi?cmd=bgmut/systems, or by entering “dbRBC” in
a search engine. While there are 30 blood group systems, 34 associated gene loci, and 270 antigens, there
are close to 1,000 alleles that encode the blood group
antigens and phenotypes.
Techniques Used to Predict a Blood
Group Antigen
Once the molecular basis of a blood group antigen has
been determined, the precise area of DNA can be analyzed to predict the presence or absence of a blood
group antigen on the surface of an RBC. Fortunately, as
the majority of genetically defined blood group antigens are the consequence of a single nucleotide
change, simple PCR-based assays can be used to detect
a change in a gene encoding a blood group antigen. Innumerable DNA-based assays have been described for
this purpose. They include PCR-restriction fragment
length polymorphism (RFLP), allele-specific (AS)-PCR
as single or multiplex assay, real-time quantitative
PCR (Q-PCR; RQ-PCR), sequencing, and microarray
technology. Figure 7-3 illustrates readout formats for
these assays. Microarrays use a gene “chip,” which is
composed of spots of DNA from many genes attached
to a solid surface in a grid-like array.5,6 Microarrays
allow for multiple single nucleotide changes to be analyzed simultaneously and overcome not only the
labor-intensive nature of hemagglutination but also
data entry. This technology has great potential in transfusion medicine for the prediction of blood groups and
phenotypes.
There are clinical circumstances where hemagglutination testing does not yield reliable results and yet
the knowledge of antigen typing is valuable to obtain.
Molecular approaches are being employed to predict
the antigen type of a patient to overcome some of the
limitations of hemagglutination. Determination of a
patient’s antigen profile by DNA analysis is particularly
useful when a patient, who is transfusion dependent, has
produced alloantibodies. Knowledge of the patient’s
probable phenotype allows the laboratory to
determine to which antigens the patient can and
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Cell
nucleus
Target sequence
with primers
Double helix
DNA strand
Taq
Sense
Primer Polymerase
Antisense
Primer
Target
sequence
PCR reaction
expotential amplification
Chromosome
Supercoiled
DNA strand
Restriction
endonuclease
Analysis
150 bp
100 bp
50 bp
PCR-RFLP
Allele: 1 1&2 2
SS-PCR
Allele 1
Real-Time PCR Sequencing
Allele 2
Microarray
Prediction
confirmed by
hemagglutination
Strong
Negative
FIGURE 7-3 From DNA to PCR-based assay readouts. Schematic representation of DNA isolated from a nucleated
cell, with a particular sequence targeted and amplified in PCR amplification. The readout formats of some of the
various techniques available to analyze the results are shown.
cannot respond to make alloantibodies. It is extremely important to obtain an accurate medical history for the patient because with certain medical
treatments, such as stem cell transplantation and
kidney transplants, typing results in tests using
DNA from different sources (such as WBCs, buccal
smears, or urine sediment) may differ. DNA analysis
is a valuable tool and a powerful adjunct to hemagglutination testing. Some of the more common clinical applications of DNA analyses for blood groups
are listed in Box 7-1.
Applications in the Prenatal Setting
The first application of molecular methods for the prediction of a blood group antigen was in the prenatal
setting, where fetal DNA was tested for RHD.7
Hemagglutination, including titers, gives only an indirect indication of the risk and severity in hemolytic
disease of the fetus and newborn (HDFN). Thus, antigen prediction by DNA-based assays has particular
value in this setting to identify a fetus who is not at
risk for HDFN, that is, antigen negative, so that aggressive monitoring of the mother can be avoided.
Certain criteria should be met before obtaining
fetal DNA for analyses: the mother’s serum contains
an IgG antibody of potential clinical significance
and the father is heterozygous for the gene encoding
the antigen of interest or when paternity is in doubt.
It is helpful to know the ethnic origin and to concurrently test both mother and father, in order to
restrict the genes involved and to identify potential
variants that could influence interpretation of the
test results. DNA analysis can be performed for any
blood group incompatibility where the molecular
basis is known.
Fetal DNA can be isolated from cells obtained by
a variety of invasive procedures; however, the use of
amniocytes obtained by amniocentesis is the most
common source. Remarkably, free fetally derived
DNA can be extracted from maternal serum or
plasma8,9 and RHD typing is possible after 5 weeks
of gestation.8,10–13 The RHD type is the prime
target because, at least in the majority of Caucasians,
the Rh-negative mother has a deleted RHD, thereby
permitting detection of the fetal RHD DNA. Furthermore, anti-D is still notoriously clinically significant
in terms of HDFN (reviewed in Avent and Reid14).
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BOX 7-1
Clinical Applications of DNA Analyses for
Blood Group Antigens
• To type patients who have been recently transfused
• To type patients whose RBCs are coated with immunoglobulin (⫹DAT)
• To identify a fetus at risk for hemolytic disease of
the fetus and newborn (HDFN)
• To determine which phenotypically antigen-negative
patients can receive antigen-positive RBCs
• To type donors for antibody identification panels
• To type patients who have an antigen that is expressed weakly on RBCs
• To determine RHD zygosity
• To mass screen for antigen-negative donors
• To resolve blood group A, B, D, and e discrepancies
• To determine the origin of engrafted leukocytes in
a stem cell recipient
• To type patient and donor(s) to determine the
possible alloantibodies that a stem cell transplant
patient can make
• To determine the origin of lymphocytes in a patient
with graft-versus-host disease
• For tissue typing
• For paternity and immigration testing
• For forensic testing
• Prediction of antigen type when antisera is unavailable
• Identify molecular basis of a new antigen
For analysis of single nucleotide changes (e.g., K/k),
a source of DNA consisting of mostly fetal DNA, for
example, amniocytes, is preferred.
Before interpreting the results of DNA analyses, it
is important to obtain an accurate medical history and
to establish if the study subject is a surrogate mother,
if she has been impregnated with nonspousal sperm,
or if she has received a stem cell transplant. For prenatal diagnosis of a fetus not at risk of HDFN, the
approach to molecular genotyping should err on the
side of caution. Thus, the strategy for fetal DNA
typing should detect a gene (or part of a gene) whose
product is not expressed (when the mother will be
monitored throughout pregnancy), rather than fail to
detect a gene whose product is expressed on the RBC
membrane (e.g., a hybrid gene).
When performing DNA analysis in the prenatal
setting, it is also important to always determine the
RHD status of the fetus, in addition to the test being
ordered. In doing so, if the fetus has a normal RHD
there is no need to provide Rh-negative blood for
intrauterine transfusions.
101
Applications in the Transfusion Setting
For Transfusion-dependent Patients
Certain medical conditions, such as sickle cell disease, thalassemia, autoimmune hemolytic anemia,
and aplastic anemia, often require chronic blood
transfusion. When a patient receives transfusions, the
presence of donor RBCs in the patient’s peripheral
blood makes RBC phenotyping by hemagglutination
complex, time-consuming, and often inaccurate. The
interpretation of RBC typing results of multitransfused patients, based on such things as number of
units transfused, length of time between transfusion
and sample collection, and size of patient (the “best
guess”), is often incorrect.15 Because it is desirable to
determine the blood type of a patient as part of the
antibody identification process, molecular approaches can be employed to predict the blood type
of patients, thereby overcoming this limitation of
hemagglutination.
For Patients Whose RBCs Have a Positive DAT
DNA-based antigen prediction of patients with autoimmune hemolytic anemia, whose RBCs are coated
with immunoglobulin, is valuable when available antibodies require the indirect antiglobulin test. Although useful for the dissociation of bound globulins,
IgG removal techniques (e.g., EDTA-acid-glycine,
chloroquine diphosphate) are not always effective at
removing bound immunoglobulin or may destroy the
antigen of interest.2 The management of patients with
warm autoantibodies who require transfusion support is particularly challenging, as free autoantibody
present in the serum/plasma may mask the formation of an underlying alloantibody. Knowledge of the
patient’s predicted phenotype is useful not only for
determining which alloantibodies he or she is capable
of producing, but also as an aid in the selection of
RBCs for heterologous adsorption of the autoantibody. This phenotype prediction is extremely valuable for the ongoing management of patients with
strong warm autoantibodies. Potentially, the predicted phenotype could be used to precisely match
blood types, thereby reducing the need to perform
extensive serologic testing.
For Blood Donors
DNA-based assays can be used to predict the antigen type of donor blood both for transfusion and for
antibody identification reagent panels. This is particularly useful when antibodies are not available or
are weakly reactive. An example is the Dombrock
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blood group polymorphism, where DNA-based assays16–18 are used to type donors as well as patients for
Doa and Dob in order to overcome the dearth of reliable
typing reagents. This was the first example where a
DNA-based method surpassed hemagglutination.
Although some antibodies are not known to cause
RBC destruction, such as antibodies to antigens of
the Knops blood group system, they are often found
in the serum/plasma of patients and attain significance by virtue of the fact that a lack of phenotyped
donors makes their identification difficult and timeconsuming.19 DNA-based assays can be useful to
predict the Knops phenotype of donors whose RBCs
are used on antibody identification panels and
thereby aid in their identification.
PCR-based assays are valuable to test donors to
increase the inventory of antigen-negative donors. As
automated procedures attain fast throughput at lower
cost, typing of blood donors by PCR-based assays is
rapidly becoming more widespread.20 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 the potential
loss of a donor with a null phenotype, it would not
jeopardize the safety of blood transfusion.
DNA analysis is useful for the resolution of apparent discrepancies, for example, the resolution of ABO
typing discrepancies due to ABO subgroups, and for
reagent discrepancies that would otherwise potentially be reportable to the FDA. Another example is to
classify variants of RHD and RHCE.21
For Patients and Donors
Detecting Weakly Expressed Antigens
DNA analysis can be useful to detect weakly expressed antigens. For example, a patient with a weakened expression of the Fyb antigen due to the Fyx
phenotype (FY nt 265) is unlikely to make antibodies
to transfused Fy(b⫹) RBCs. In this situation, PCRbased assays can help determine which phenotypically antigen-negative patients can be safely
transfused with antigen-positive RBCs. It has been
suggested that DNA assays can be used to detect
weak D antigens in apparent D-negative donors to
prevent possible alloimmunization and delayed transfusion reactions22 or to save true D-negative RBC
products for true D-negative patients.
Limitations of DNA Analysis
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 (see
Table 7-3).23–25 If a patient has a grossly normal gene
that is not expressed on his or her RBCs, he or
she could produce an antibody if transfused with
antigen-positive blood. When feasible, the appropriate
assay to detect a change that silences a gene should be
part of the DNA-based testing. Examples of such testing include analyses for the GATA box with FY
typing,26 presence of RHD pseudogene with RHD typing,27 and exon 5 and intron 5 changes in GYPB with
S typing.28
In addition to silencing changes that can impact
antigen expression, there are other circumstances,
both iatrogenic and genetic, that may impact the
results of DNA analysis (see Table 7-4). With certain
medical treatments such as stem cell transplantation
and kidney transplants, typing results may differ depending on the source of the DNA; therefore, it is extremely important to obtain an accurate medical
history for the patient. These medical procedures, as
well as natural chimerism, can lead to mixed DNA
populations; therefore, the genotyping results will be
impacted by the source of the DNA used for testing.
Another limitation of DNA analysis is that not all
blood group antigens are the consequence of a single
nucleotide change. Furthermore, there may be many
alleles per phenotype, which could require multiple
assays to predict the phenotype. There are also some
blood group antigens for which the molecular basis is
not yet known.
OTHER APPLICATIONS FOR
MOLECULAR ANALYSES
Molecular biology techniques can be used to transfect
cells with DNA of interest and then grow the transfected cells in tissue culture. These cells, which
express a single protein, and thus the antigens from
only one blood group system, can be used to aid in the
identification of antibodies. Indeed, single-pass (Kell)
and multi-pass (Duffy) proteins have been expressed
in high levels in mouse erythroleukemic (MEL) cells
or 293T cells and detected by human polyclonal antibodies.29 Similar experiments have been performed
with antibodies to Lutheran antigens.30 Thus, it is theoretically possible to produce a panel of cell lines expressing individual proteins for development of an
automated, objective, single-step antibody detection
and identification procedure. Such an approach
would eliminate the need for antigen-matched, shortdated, potentially biohazardous RBC screening and
panel products derived from humans. As promising
as this approach is, some major hurdles are yet to be
overcome; for example, antigens from all blood group
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TABLE 7-3 Examples of Molecular Events Where Analyses of Gene and Phenotype Will Not Agree
Event
Mechanism
Blood Group Phenotype
Transcription
Nucleotide change in GATA box
Fy(b⫺)
Alternative splicing
Nucleotide change in splice site:
partial/complete skipping of exon
Deletion of nucleotides
S⫺ s⫺; Gy(a⫺)
Deletion of nucleotide(s) → frameshift
Insertion of nucleotide(s) → frameshift
Nucleotide change
Fy(a⫺b⫺); D⫺; Rhnull; Ge: ⫺2,
⫺3, ⫺4; Gy(a⫺); K0; McLeod
D⫺; Co(a⫺b⫺)
Fy(a⫺b⫺); r⬘; Gy(a⫺); K0; McLeod
Amino acid change
Missense nucleotide change
D⫺; Rhnull; K0; McLeod
Reduced amount of protein
Missense nucleotide change
Fyx; Co(a⫺b⫺)
Hybrid genes
Crossover
Gene conversion
GP.Vw; GP.Hil; GP.TSEN
GP.Mur; GP.Hop; D- -; R0Har
Interacting protein
Absence of RhAG
Absence of Kx
Absence of amino acids 59–76 of GPA
Absence of protein 4.1
Rhnull
Weak expression of Kell antigen
Wr(b⫺)
Weak expression of Ge antigens
Modifying gene
In(LU)
In(Jk)
Lu(a⫺b⫺)
Jk(a⫺b⫺)
Premature stop codon
systems must be expressed at levels that are at least
equivalent to those on RBCs and the detection system
should have low background levels of reactivity.
Importantly, the highly clinically significant Rh anti-
TABLE 7-4 Limitations of DNA Analysis
Iatrogenic
Stem cell transplantation
Natural chimera
Surrogate mother/sperm donor
Genetic
Not all polymorphisms can be
analyzed
Many alleles per phenotype
Molecular basis not yet known
Beware of possible silencing
changes that can affect antigen
expression (Rh and RhAG, Band 3,
and GPA dominant Lu(a⫺b⫺))
Not all alleles in ethnic populations
are known
Dr(a⫺)
gens are proving difficult to express in adequate
levels.
Transfected cells expressing blood group antigens
also can be used for adsorption of specific antibodies
as part of antibody detection and identification, or
prior to crossmatching if the antibody is clinically
insignificant. In addition, genes can be engineered to
express soluble forms of proteins expressing antigens
for antibody inhibition, again as part of antibody
detection and identification procedures, or prior to
crossmatching.31–33 For example, concentrated forms
of recombinant CR1 (CD35) would be valuable to
inhibit clinically insignificant antibodies in the Knops
system, thereby eliminating its interference in crossmatching.
Recombinant proteins and transfected cells
expressing blood group antigens have been used as
immunogens for the production of monoclonal antibodies. This approach has led to the successful
production of murine monoclonal antibodies with
specificities to blood group antigens not previously
made34,35 (see http://www.nybloodcenter.org). Such
antibodies are useful because the supplies of human
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UNIT 3 Principles of Testing
polyclonal antibodies are diminishing. Molecular manipulations have been used to convert murine IgG
anti-Jsb and anti-Fya to IgM direct agglutinins, which
are more practical in the clinical laboratory.36,37
REGULATORY COMPLIANCE
In addition to a knowledge of blood groups, their
molecular bases, technical aspects of PCR-based assays, and causes of possible discrepancies (be they
technical, iatrogenic, or genetic), it is important to be
cognizant of issues of regulatory compliance. The laboratory director is responsible for ensuring accuracy of
results regardless of whether the test is a laboratorydeveloped test (LDT; previously known as “homebrew”) or a commercial microarray for research use
only (RUO). Each facility should have a quality plan
that includes test procedures, processes, validation,
etc. According to the FDA, DNA testing cannot be used
as the sole means of determining the antigen status
and a disclaimer statement must accompany reports
giving the prediction of blood types. As DNA testing
to predict a blood group for the purpose of patient care
is not used to identify or diagnose a genetic disease,
but is doing a test in a different way (hemagglutination
vs. DNA assays) to achieve a similar result, informed
consent may not be required. Whether or not informed
consent should be obtained from the patient or donor
to be tested depends on local laws.
If DNA-based testing is done strictly for patient
care, it is exempt from Institutional Review Board
(IRB) approval. However, if testing is performed for a
research purpose, or is to be published, even as an abstract, then IRB approval is required. The type of research dictates whether the review is expedited or
requires full board approval. Influencing factors include whether the sample is linked or unlinked,
whether it exists or is collected specifically for the testing, and whether or not the human subject is at risk
from the procedure.
SUMMARY
Numerous studies have analyzed blood samples from
people with known antigen profiles and identified the
molecular bases associated with many antigens.2 The
available wealth of 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 and applied to DNA analyses with the assumption that the molecular analyses would correlate
with RBC antigen typing. While this is true in the majority of cases, like hemagglutination, PCR-based assays have limitations. Many molecular events result in
the DNA-predicted type and RBC type being apparently discrepant (some are listed in Table 7-3). Furthermore, analyses of the null phenotypes have
demonstrated that multiple, diverse genetic events
can give rise to the same phenotype. Nonetheless,
molecular analyses have the advantage that genomic
DNA is readily available from peripheral blood leukocytes, buccal epithelial cells, and even cells in urine,
Review Questions
1. True or false? The process of changing DNA to RNA is
called translation.
2. True or false? A single nucleotide change can give rise
to a null blood group phenotype.
3. True or false? A blood group can be predicted by testing DNA extracted from WBCs.
4. A single nucleotide change can cause which of the
following:
a. no change in the codon for an amino acid
b. a stop codon
c. a change from one amino acid to another
d. all of the above
5. A PCR-based assay:
a. has limitations
b. gives a prediction of a blood group
c. amplifies a specific sequence of DNA
d. all of the above
6. For antigen prediction in the neonatal setting, the most
common source of fetal DNA is:
a. amniocytes
b. fetal RBCs
c. cord blood
d. endothelial cells
7. Antigen prediction by DNA analysis is:
a. indicated only for patient testing and is not applicable for donor testing
b. used to determine weakly expressed antigens
c. used to predict antigens when licensed FDA antisera
are not available
d. b and c
(continued)
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CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine
105
REVIEW QUESTIONS (continued)
8. In the transfusion setting, DNA analysis is a valuable adjunct to hemagglutination testing for all of the following
circumstances except:
a. for patients with a negative DAT and no history of
transfusion
b. for patients who require chronic RBC transfusions
c. for predicting antigens to determine what alloantibodies a patient can produce
d. for patients with a positive DAT and a warm autoantibody
and it is remarkably stable. The primary disadvantages
are that the type determined on DNA may not reflect
the RBC phenotype and certain assays can give false
results. The prediction of blood group antigens from
testing DNA has tremendous potential in transfusion
medicine and has already taken a firm foothold. DNAbased assays provide a valuable adjunct to the classic
hemagglutination assays. The high-throughput nature
of microarrays provides a vehicle by which to increase
inventories of antigen-negative donor RBC products
and, in this aspect, change the way we practice transfusion medicine.
ACKNOWLEDGMENT
We thank Robert Ratner for help in preparing the
manuscript and figures.
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