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CHAPTER

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MOLECULAR TESTINGFOR BLOOD GROUPS INTRANSFUSION MEDICINEM. 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 molecu-

lar 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 PCR-

based assays for antigen prediction.

6. Summarize relevant regulatory issues.

KEY WORDS

Alleles

Blood group antigens

DNA to protein

Molecular testing

Prediction of blood groups

and in the understanding of the molecular bases asso-ciated with most blood group antigens and pheno-types enables us to consider the prediction of bloodgroup antigens using molecular approaches. Indeed,this knowledge is currently being applied to help re-solve some long-standing clinical problems that can-not be resolved by classical hemagglutination.

Blood group antigens are inherited, polymorphic,structural characteristics located on proteins, glyco-proteins, or glycolipids on the outer surface of theRBC membrane. The classical method of testing forblood group antigens and antibodies is hemaggluti-nation. This technique is simple and when done correctly, has a specificity and sensitivity that is appro-priate for the clinical care of the vast majority of patients. Indeed, direct and indirect hemagglutination testshave served the transfusion community well for, respectively, over 100 and over 50 years. However, insome aspects, hemagglutination has limitations. Forexample, it gives only an indirect measure of the po-tential complications in an at-risk pregnancy, it cannotprecisely indicate RHD zygosity in D-positive people,it cannot be relied upon to type some recently trans-fused patients, and it requires the availability of spe-cific reliable antisera. The characterization of genesand determination of the molecular bases of antigensand phenotypes has made it possible to use the poly-merase chain reaction (PCR)1 to amplify the preciseareas of deoxyribose nucleic acid (DNA) of interest todetect alleles encoding blood groups and thereby predict the antigen type of a person.

This chapter first provides an overview of the pro-cessing of DNA to a blood group antigen and then

A 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 byhemagglutination in the serum of a transfused pa-tient or pregnant woman. The astounding pace ofgrowth in the field of molecular biology techniques

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96 UNIT 3 Principles of Testing

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.

summarizes current applications of molecular ap-proaches for predicting blood group antigens in transfu-sion medicine practice for patients and donors, especiallyin those areas where hemagglutination has limitations.

FROM DNA TO BLOOD GROUPS

The Language of GenesDNA is a nucleic acid composed of nucleotide bases, asugar (deoxyribose), and phosphate groups. The nu-cleotide bases are purines (adenine [A] and guanine [G])and pyrimidines (thymine [T] and cytosine [C]). Thelanguage of genes is far simpler than the English lan-guage. Compare four letters in DNA or RNA (C, G, A,and T [T in DNA is replaced by U in RNA]) with 26 let-ters of the English alphabet. These four letters are callednucleotides (nts) and they form “words,” called codons,each with three nucleotides in different combinations.There are only 64 (4 � 4 � 4 � 64) possible codons ofwhich 61 encode the 20 amino acids and 3 are stopcodons. There are more codons (n � 61) than there areamino acids (n � 20) because some amino acids are en-coded by more than one codon (e.g., UCU, UCC, UCA,UCG, AGU, and AGC, all encode the amino acid calledserine). This is termed redundancy in the genetic code.

Essentials of a GeneFigure 7-1 shows the key elements of a gene. Exonsare numbered from the left (5�, upstream) to right (3�, downstream) and are separated by introns.

Nucleotides in exons encode amino acids or a “stop”instruction, while nucleotides in introns are not en-coded. Nucleotides in an exon are written in uppercase letters and those in introns and intervening se-quences are written in lower case letters. At the junc-tion of an exon to an intron, there is an invariantsequence of four nucleotides (AGgt) called the donorsplice site, and at the junction of an intron to an exonis another invariant sequence of four nucleotides(agGT) called the acceptor splice site. The splice sitesinteract to excise (or outsplice) the introns, therebyconverting genomic DNA to mRNA. A single strandof DNA (5� to 3�) acts as a template and is duplicatedexactly to form mRNA. Nucleotide C invariablypairs with G, and A with T. Upstream from the firstexon of a gene, there are binding sites (promoter re-gions) for factors that are required for transcription(from DNA to mRNA) of the gene. Transcription ofDNA always begins at the ATG, or “start,” transcrip-tion codon. The promoter region can be ubiquitous,tissue specific, or switched on under certain circum-stances. At the 3� end of a gene there is a “stop” tran-scription codon (TAA, TAG, or TGA) and beyondthat there is often an untranslated region. Betweenadjacent genes on a chromosome, there is an “inter-vening” sequence of nucleotides, which are not tran-scribed.

After the introns are excised, the resultantmRNA contains nucleotides from the exons of thegene. Nucleotides in mRNA are translated (frommRNA to protein) in sets of three (a codon) to producea sequence of amino acids, which form a protein. Liketranscription of DNA, translation of mRNA always

Protein COOH

Exon 2DNA Exon 1Exon 1 Exon 3 3

Transcriptionbinding site

5

Interveningsequence

Intron 1 Intron 2

Gene A Gene B

Upstream A DD A DUTR

Exon 2mRNA Exon 1

Met

3Gene A

5

NH2

Exon 3

Starttranslation

ATGStart

transcription

Stoptranscription

D = donor splice site (AGgt)

A = acceptor splice site (agGT)

UTR = untranslated region

NH = amino terminus2

COOH = carboxy terminus

Stoptranslation

AUG

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CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine 97

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 Knull, Gy(a�)

aNot uncommon in African Americans and Japanese.2

begins at the “start” codon (AUG) and terminates at a“stop” codon (UAA, UAG, or UGA). The resultantprotein consists of amino acids starting with methion-ine (whose codon is AUG) at the amino (NH2) termi-nus. Methionine, or a “leader” sequence of aminoacids, is sometimes cleaved from the functional pro-tein and thus, a written sequence of amino acids (ormature protein) does not necessarily begin with me-thionine.

DNA is present in all nucleated cells. For the pre-diction of a blood group, DNA is usually obtainedfrom peripheral white blood cells (WBCs), but alsocan be extracted from epithelial cells, cells in urinesediment, and amniocytes.

Molecular Bases of Blood GroupsAlthough many mechanisms give rise to a bloodgroup antigen or phenotype (Table 7-1), the major-ity of blood group antigens are a consequence of asingle nucleotide change. The other mechanismslisted give rise to a small number of antigens andvarious phenotypes. Figure 7-2 shows a short hy-pothetical sequence of double-stranded DNA to-gether with transcription (mRNA) and translation

(protein) products. The effect of a silent, missense,or nonsense single nucleotide change togetherwith examples involving blood group antigens areillustrated.

Effect of a Single Nucleotide Change on a Blood Group

Due to redundancy in the genetic code, a silent (synonymous) nucleotide change does not changethe amino acid and, thus, does not affect the antigenexpression. Nevertheless, because it is possible thatsuch a change could alter a restriction enzymerecognition site or a primer binding site, it is impor-tant to be aware of silent nucleotide changes whendesigning a PCR-based assay. In contrast, a missense(nonsynonymous) nucleotide change results in a different amino acid, and these alternative forms ofan allele encode antithetical antigens. Figure 7-2 illustrates this where “G” in a lysine codon (AAG) isreplaced by “C,” which gives rise to the codon forasparagine (AAC). The example of a missense nucleotide change shows that a “C” to “T” changeis the only difference between the clinically impor-tant blood group antigens k and K. A nonsense

TABLE 7-1 Molecular Events That Give Rise to Blood Group Antigens and Phenotypes

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nucleotide change transforms a codon for an aminoacid to a stop codon. Figure 7-2 and Table 7-2 giveexamples relative to blood groups.

Effect of Deletion or Insertion of Nucleotide(s)

A deletion of one nucleotide results in a �1 frameshiftand an eventual stop codon (see Fig. 7-2 and Table 7-2).Typically, this leads to the encoding of a truncatedprotein, but it can cause elongation. For example, a dele-tion of “C” close to the stop codon in the A2 allele results in a transferase with 21 amino acids more than inthe A1 transferase.2 Similarly, deletion of two nucleotidescauses a �2 frameshift and a premature stop codon.Deletion of a nucleotide also can cause a stop codon, butthere is no known example for a blood group.

An insertion of one nucleotide results in a �1frameshift and a premature stop codon (see Fig. 7-2and 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 noknown example for a blood group.

APPLICATIONS OF MOLECULARANALYSIS

The genes encoding 29 of the 30 blood group sys-tems (only P1 remains to be resolved) have beencloned and sequenced.3,4 Focused sequencing ofDNA from patients or donors with serologically defined antigen profiles has been used to determinethe molecular bases of variant forms of the gene.This approach has been extremely powerful becauseantibody-based definitions of blood groups readilydistinguish variants within each blood groupsystem. Details of these analyses are beyond thescope of this chapter but up-to-date details aboutalleles encoding blood groups can be found on theBlood Group Antigen Gene Mutation database at:

ADNAT

TA

GC

TA

CG

GC

AT

AT

GC

GC

AT

AT

GC

CG

AT

–3–5

mRNA Transcription product

Single nucleotide deletion

Missense 698 T>C in KEL exon 6Met193Thr = K/k

Nonsense 287G>A in FY exon 2Trp96Stop = Fy (a–b–)

Protein Translation productA

A

A

A

U

U

U

U

Met

Met

Met

Met

G

G

G

G

|

|

|

|

|

|

|

|

|

|

|

|

|

|

||

|

U

U

U

U

C

C

C

C

Ser

Ser

Ser

Ser

G

G

G

G

C/

A

A

A

A

A

A

A

A

Lys

Lys

Lys

Asn

G

G

G

G

G

G

G

G

A

A

A

A

Glu

Glu

Glu

A

A

A

A

G

G

G

G

C

C

C

C

Ala

Ala

Ala

A

A

A

A/C

/U

Stop

New Sequence261del G in O exon 6Frameshift → 116Stop = 0

Stop Codon

A

A

U

U

Met

Met

G

G

|

|

|

|

| |

U

C

C

G

G

A

A

A

A

G

G

G

G

A

A

A

A

G

G

C

C

A

A

UX

X

LysArg

Stop

Arg

New Sequence 307-308 ins T in CO exon 2Frameshift → Stop = Co(a–b–)

Stop Codon No example known

A

A

U

U

Met

Met

G

G

|

|

|

|

|

|

| |

|

U

U

C

C

Ser

Ser

G

G

GA

A

A

A

Arg

Lys

G

G

G

G

A

AStop

A

A

G

G

C

C

A

AUGly Ser

Silent 378 T>C in DO exon 2Tyr126Tyr = no change

Single nucleotide substitution

Single nucleotide insertion

Examples:

No example known

FIGURE 7-2 A hypothetical piece of DNA and the effect of single nucleotide changes. A short hypothetical sequenceof double-stranded DNA and the resultant transcription (mRNA) and translation (protein) products are shown. The figurealso shows the five amino acids that are determined by the codons in the DNA (PPaanneell AA). PPaanneellss BB through DD demonstratethe effect of three different types of single nucleotide changes, substitution (PPaanneell BB), deletion (PPaanneell CC), and insertion(PPaanneell DD), and the effects on the amino acids. Where available, examples of these various types of changes in bloodgroups are given.

A

B

C

D

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http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi. cgi?cmd=bgmut/systems, or by entering “dbRBC” ina search engine. While there are 30 blood group sys-tems, 34 associated gene loci, and 270 antigens, thereare close to 1,000 alleles that encode the blood groupantigens and phenotypes.

Techniques Used to Predict a Blood Group AntigenOnce the molecular basis of a blood group antigen hasbeen determined, the precise area of DNA can be ana-lyzed to predict the presence or absence of a bloodgroup antigen on the surface of an RBC. Fortunately, asthe majority of genetically defined blood group anti-gens are the consequence of a single nucleotidechange, simple PCR-based assays can be used to detecta change in a gene encoding a blood group antigen. In-numerable DNA-based assays have been described forthis purpose. They include PCR-restriction fragmentlength polymorphism (RFLP), allele-specific (AS)-PCRas single or multiplex assay, real-time quantitative

PCR (Q-PCR; RQ-PCR), sequencing, and microarraytechnology. Figure 7-3 illustrates readout formats forthese assays. Microarrays use a gene “chip,” which iscomposed of spots of DNA from many genes attachedto a solid surface in a grid-like array.5,6 Microarraysallow for multiple single nucleotide changes to be ana-lyzed simultaneously and overcome not only thelabor-intensive nature of hemagglutination but alsodata entry. This technology has great potential in trans-fusion medicine for the prediction of blood groups andphenotypes.

There are clinical circumstances where hemagglu-tination testing does not yield reliable results and yetthe knowledge of antigen typing is valuable to obtain.Molecular approaches are being employed to predictthe antigen type of a patient to overcome some of thelimitations of hemagglutination. Determination of a patient’s antigen profile by DNAanalysis is particularlyuseful when a patient, who is transfusion dependent, hasproduced alloantibodies. Knowledge of the patient’sprobable phenotype allows the laboratory to determine to which antigens the patient can and

Antigen/Phenotype Gene Nucleotide Change Amino Acid

Missense nucleotide change

S/s GYPB 143T > C Met29Thr

E/e RHCE 676C > G Pro226Ala

K/k KEL 698T > C Met193Thr

Fya/Fyb FY 125G > A Gly42Asp

Jka/Jkb JK 838G > A Asp280Asn

Doa/Dob DO 793A > G Asn265Asp

Nonsense nucleotide change

Fy(a–b–) FY 407G > A Try136Stop

D– RHD 48G > A Trp16Stop

Gy(a–) DO 442C > T Gln148Stop

Nucleotide deletion

D– RHD 711Cdel Frameshift → 245Stop

D– RHD AGAG Frameshift → 167Stop

Nucleotide insertion

Ael ABO 798-804Gins Frameshift → Stop

D– RH 906GGCTins Frameshift → donor splice site

change (I6 + 2t > a)

TABLE 7-2 Molecular Bases Associated with a Few Blood Group Antigens

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FIGURE 7-3 From DNA to PCR-based assay readouts. Schematic representation of DNA isolated from a nucleatedcell, 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 ex-tremely important to obtain an accurate medical his-tory for the patient because with certain medicaltreatments, such as stem cell transplantation andkidney transplants, typing results in tests usingDNA from different sources (such as WBCs, buccalsmears, or urine sediment) may differ. DNA analysisis a valuable tool and a powerful adjunct to hemag-glutination testing. Some of the more common clini-cal applications of DNA analyses for blood groupsare listed in Box 7-1.

Applications in the Prenatal SettingThe first application of molecular methods for the pre-diction of a blood group antigen was in the prenatalsetting, where fetal DNA was tested for RHD.7

Hemagglutination, including titers, gives only an in-direct indication of the risk and severity in hemolyticdisease of the fetus and newborn (HDFN). Thus, anti-gen prediction by DNA-based assays has particularvalue in this setting to identify a fetus who is not atrisk for HDFN, that is, antigen negative, so that ag-gressive monitoring of the mother can be avoided.

PCR reactionexpotential amplification

Real-Time PCR

Allele 21&2

Restrictionendonuclease

Target sequencewith primers

PCR-RFLP

Strong

SS-PCR

Analysis

150 bp

100 bp 50 bp

ChromosomeSupercoiledDNA strand

Double helixDNA strand

Cellnucleus

Targetsequence

SensePrimer

TaqPolymerase

AntisensePrimer

Allele 1Allele: 21

Sequencing Microarray

Negative

Predictionconfirmed by

hemagglutination

Certain criteria should be met before obtainingfetal DNA for analyses: the mother’s serum containsan IgG antibody of potential clinical significanceand the father is heterozygous for the gene encodingthe antigen of interest or when paternity is in doubt.It is helpful to know the ethnic origin and to concur-rently test both mother and father, in order to restrict the genes involved and to identify potentialvariants that could influence interpretation of thetest results. DNA analysis can be performed for anyblood group incompatibility where the molecularbasis is known.

Fetal DNA can be isolated from cells obtained bya variety of invasive procedures; however, the use ofamniocytes obtained by amniocentesis is the mostcommon source. Remarkably, free fetally derivedDNA can be extracted from maternal serum orplasma8,9 and RHD typing is possible after 5 weeksof 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, therebypermitting detection of the fetal RHD DNA. Further-more, anti-D is still notoriously clinically significantin terms of HDFN (reviewed in Avent and Reid14).

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For analysis of single nucleotide changes (e.g., K/k),a source of DNA consisting of mostly fetal DNA, forexample, amniocytes, is preferred.

Before interpreting the results of DNA analyses, itis important to obtain an accurate medical history andto 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 prena-tal diagnosis of a fetus not at risk of HDFN, the approach to molecular genotyping should err on theside of caution. Thus, the strategy for fetal DNAtyping should detect a gene (or part of a gene) whoseproduct is not expressed (when the mother will bemonitored throughout pregnancy), rather than fail todetect a gene whose product is expressed on the RBCmembrane (e.g., a hybrid gene).

When performing DNA analysis in the prenatalsetting, it is also important to always determine theRHD status of the fetus, in addition to the test beingordered. In doing so, if the fetus has a normal RHDthere is no need to provide Rh-negative blood for intrauterine transfusions.

Applications in the Transfusion SettingFor Transfusion-dependent Patients

Certain medical conditions, such as sickle cell dis-ease, thalassemia, autoimmune hemolytic anemia,and aplastic anemia, often require chronic bloodtransfusion. When a patient receives transfusions, thepresence of donor RBCs in the patient’s peripheralblood makes RBC phenotyping by hemagglutinationcomplex, time-consuming, and often inaccurate. Theinterpretation of RBC typing results of multitrans-fused patients, based on such things as number ofunits transfused, length of time between transfusionand sample collection, and size of patient (the “bestguess”), is often incorrect.15 Because it is desirable todetermine the blood type of a patient as part of theantibody identification process, molecular ap-proaches can be employed to predict the blood typeof patients, thereby overcoming this limitation ofhemagglutination.

For Patients Whose RBCs Have a Positive DAT

DNA-based antigen prediction of patients with au-toimmune hemolytic anemia, whose RBCs are coatedwith immunoglobulin, is valuable when available an-tibodies require the indirect antiglobulin test. Al-though useful for the dissociation of bound globulins,IgG removal techniques (e.g., EDTA-acid-glycine,chloroquine diphosphate) are not always effective atremoving bound immunoglobulin or may destroy theantigen of interest.2 The management of patients withwarm autoantibodies who require transfusion sup-port is particularly challenging, as free autoantibodypresent in the serum/plasma may mask the forma-tion of an underlying alloantibody. Knowledge of thepatient’s predicted phenotype is useful not only fordetermining which alloantibodies he or she is capableof producing, but also as an aid in the selection ofRBCs for heterologous adsorption of the autoanti-body. This phenotype prediction is extremely valu-able for the ongoing management of patients withstrong warm autoantibodies. Potentially, the pre-dicted phenotype could be used to precisely matchblood types, thereby reducing the need to performextensive serologic testing.

For Blood Donors

DNA-based assays can be used to predict the anti-gen type of donor blood both for transfusion and forantibody identification reagent panels. This is par-ticularly useful when antibodies are not available orare weakly reactive. An example is the Dombrock

Clinical Applications of DNA Analyses forBlood Group Antigens

• To type patients who have been recently transfused

• To type patients whose RBCs are coated with im-

munoglobulin (�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 ex-

pressed 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

BOX 7-1

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blood group polymorphism, where DNA-based as-says16–18 are used to type donors as well as patients forDoa and Dob in order to overcome the dearth of reliabletyping reagents. This was the first example where aDNA-based method surpassed hemagglutination.Although some antibodies are not known to causeRBC destruction, such as antibodies to antigens ofthe Knops blood group system, they are often foundin the serum/plasma of patients and attain signifi-cance by virtue of the fact that a lack of phenotypeddonors makes their identification difficult and time-consuming.19 DNA-based assays can be useful topredict the Knops phenotype of donors whose RBCsare used on antibody identification panels andthereby aid in their identification.

PCR-based assays are valuable to test donors toincrease the inventory of antigen-negative donors. Asautomated procedures attain fast throughput at lowercost, typing of blood donors by PCR-based assays israpidly becoming more widespread.20 With donortyping, the presence of a grossly normal gene whoseproduct is not expressed on the RBC surface wouldlead to the donor being falsely typed as antigen-positive, and although this would mean the potentialloss of a donor with a null phenotype, it would notjeopardize the safety of blood transfusion.

DNA analysis is useful for the resolution of appar-ent discrepancies, for example, the resolution of ABOtyping discrepancies due to ABO subgroups, and forreagent discrepancies that would otherwise poten-tially be reportable to the FDA. Another example is toclassify variants of RHD and RHCE.21

For Patients and Donors

Detecting Weakly Expressed AntigensDNA analysis can be useful to detect weakly ex-pressed antigens. For example, a patient with a weak-ened expression of the Fyb antigen due to the Fyx

phenotype (FY nt 265) is unlikely to make antibodiesto transfused Fy(b�) RBCs. In this situation, PCR-based assays can help determine which phenotypi-cally antigen-negative patients can be safelytransfused with antigen-positive RBCs. It has beensuggested that DNA assays can be used to detectweak D antigens in apparent D-negative donors toprevent possible alloimmunization and delayed trans-fusion reactions22 or to save true D-negative RBCproducts for true D-negative patients.

Limitations of DNA AnalysisWhen recommendations for clinical practice are basedon molecular analyses, it is important to rememberthat, in rare situations, a genotype determination will

not correlate with antigen expression on the RBC (seeTable 7-3).23–25 If a patient has a grossly normal genethat is not expressed on his or her RBCs, he or she could produce an antibody if transfused with antigen-positive blood. When feasible, the appropriateassay to detect a change that silences a gene should bepart of the DNA-based testing. Examples of such test-ing include analyses for the GATA box with FYtyping,26 presence of RHD pseudogene with RHD typ-ing,27 and exon 5 and intron 5 changes in GYPB withS typing.28

In addition to silencing changes that can impactantigen expression, there are other circumstances,both iatrogenic and genetic, that may impact the results of DNA analysis (see Table 7-4). With certainmedical treatments such as stem cell transplantationand kidney transplants, typing results may differ de-pending on the source of the DNA; therefore, it is ex-tremely important to obtain an accurate medicalhistory for the patient. These medical procedures, aswell as natural chimerism, can lead to mixed DNApopulations; therefore, the genotyping results will beimpacted by the source of the DNA used for testing.

Another limitation of DNA analysis is that not allblood group antigens are the consequence of a singlenucleotide change. Furthermore, there may be manyalleles per phenotype, which could require multipleassays to predict the phenotype. There are also someblood group antigens for which the molecular basis isnot yet known.

OTHER APPLICATIONS FORMOLECULAR ANALYSES

Molecular biology techniques can be used to transfectcells with DNA of interest and then grow the trans-fected cells in tissue culture. These cells, which express a single protein, and thus the antigens fromonly one blood group system, can be used to aid in theidentification of antibodies. Indeed, single-pass (Kell)and multi-pass (Duffy) proteins have been expressedin high levels in mouse erythroleukemic (MEL) cellsor 293T cells and detected by human polyclonal anti-bodies.29 Similar experiments have been performedwith antibodies to Lutheran antigens.30 Thus, it is the-oretically possible to produce a panel of cell lines ex-pressing individual proteins for development of anautomated, objective, single-step antibody detectionand identification procedure. Such an approachwould eliminate the need for antigen-matched, short-dated, potentially biohazardous RBC screening andpanel products derived from humans. As promisingas this approach is, some major hurdles are yet to be overcome; for example, antigens from all blood group

82043_ch07.qxd 11/13/09 4:40 PM Page 102


gens are proving difficult to express in adequate levels.

Transfected cells expressing blood group antigensalso can be used for adsorption of specific antibodiesas part of antibody detection and identification, orprior to crossmatching if the antibody is clinically insignificant. In addition, genes can be engineered toexpress soluble forms of proteins expressing antigensfor antibody inhibition, again as part of antibody detection and identification procedures, or prior tocrossmatching.31–33 For example, concentrated formsof recombinant CR1 (CD35) would be valuable to inhibit clinically insignificant antibodies in the Knopssystem, thereby eliminating its interference in cross-matching.

Recombinant proteins and transfected cells expressing blood group antigens have been used asimmunogens for the production of monoclonal anti-bodies. This approach has led to the successful production of murine monoclonal antibodies withspecificities to blood group antigens not previouslymade34,35 (see http://www.nybloodcenter.org). Suchantibodies are useful because the supplies of human

Event Mechanism Blood Group Phenotype

Transcription Nucleotide change in GATA box Fy(b�)

Alternative splicing Nucleotide change in splice site: S� s�; Gy(a�)

partial/complete skipping of exon

Deletion of nucleotides Dr(a�)

Premature stop codon Deletion of nucleotide(s) → frameshift Fy(a�b�); D�; Rhnull; Ge: �2,

�3, �4; Gy(a�); K0; McLeod

Insertion of nucleotide(s) → frameshift D�; Co(a�b�)

Nucleotide change 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 GP.Vw; GP.Hil; GP.TSEN

Gene conversion GP.Mur; GP.Hop; D- -; R0Har

Interacting protein Absence of RhAG Rhnull

Absence of Kx Weak expression of Kell antigen

Absence of amino acids 59–76 of GPA Wr(b�)

Absence of protein 4.1 Weak expression of Ge antigens

Modifying gene In(LU) Lu(a�b�)

In(Jk) Jk(a�b�)

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

systems must be expressed at levels that are at leastequivalent to those on RBCs and the detection systemshould have low background levels of reactivity. Importantly, the highly clinically significant Rh anti-

TABLE 7-3 Examples of Molecular Events Where Analyses of Gene and Phenotype Will Not Agree

82043_ch07.qxd 11/13/09 4:40 PM Page 103

research purpose, or is to be published, even as an ab-stract, then IRB approval is required. The type of re-search dictates whether the review is expedited orrequires full board approval. Influencing factors in-clude whether the sample is linked or unlinked,whether it exists or is collected specifically for the test-ing, and whether or not the human subject is at riskfrom the procedure.

SUMMARY

Numerous studies have analyzed blood samples frompeople with known antigen profiles and identified themolecular bases associated with many antigens.2 Theavailable wealth of serologically defined variants hascontributed to the rapid rate with which the geneticdiversity of blood group genes has been revealed. Ini-tially, molecular information associated with eachvariant was obtained from only a small number ofsamples and applied to DNA analyses with the as-sumption that the molecular analyses would correlatewith RBC antigen typing. While this is true in the ma-jority of cases, like hemagglutination, PCR-based as-says have limitations. Many molecular events result inthe DNA-predicted type and RBC type being appar-ently discrepant (some are listed in Table 7-3). Fur-thermore, analyses of the null phenotypes havedemonstrated that multiple, diverse genetic eventscan give rise to the same phenotype. Nonetheless,molecular analyses have the advantage that genomicDNA is readily available from peripheral blood leuko-cytes, buccal epithelial cells, and even cells in urine,


polyclonal antibodies are diminishing. Molecular ma-nipulations have been used to convert murine IgGanti-Jsb and anti-Fya to IgM direct agglutinins, whichare more practical in the clinical laboratory.36,37

REGULATORY COMPLIANCE

In addition to a knowledge of blood groups, theirmolecular bases, technical aspects of PCR-based as-says, and causes of possible discrepancies (be theytechnical, iatrogenic, or genetic), it is important to becognizant of issues of regulatory compliance. The lab-oratory director is responsible for ensuring accuracy ofresults regardless of whether the test is a laboratory-developed test (LDT; previously known as “home-brew”) or a commercial microarray for research useonly (RUO). Each facility should have a quality planthat includes test procedures, processes, validation,etc. According to the FDA, DNA testing cannot be usedas the sole means of determining the antigen statusand a disclaimer statement must accompany reportsgiving the prediction of blood types. As DNA testingto predict a blood group for the purpose of patient careis not used to identify or diagnose a genetic disease,but is doing a test in a different way (hemagglutinationvs. DNA assays) to achieve a similar result, informedconsent may not be required. Whether or not informedconsent should be obtained from the patient or donorto be tested depends on local laws.

If DNA-based testing is done strictly for patientcare, it is exempt from Institutional Review Board(IRB) approval. However, if testing is performed for a

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 test-

ing 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 applica-

ble 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)

Review Questions

82043_ch07.qxd 11/13/09 4:40 PM Page 104

and it is remarkably stable. The primary disadvantagesare that the type determined on DNA may not reflectthe RBC phenotype and certain assays can give falseresults. The prediction of blood group antigens fromtesting DNA has tremendous potential in transfusionmedicine and has already taken a firm foothold. DNA-based assays provide a valuable adjunct to the classichemagglutination assays. The high-throughput natureof microarrays provides a vehicle by which to increaseinventories of antigen-negative donor RBC productsand, in this aspect, change the way we practice transfu-sion medicine.

ACKNOWLEDGMENT

We thank Robert Ratner for help in preparing themanuscript and figures.

REFERENCES

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3. Lögdberg L, Reid ME, Lamont RE, et al. Human bloodgroup genes 2004: chromosomal locations and cloningstrategies. Transfus Med Rev. 2005; 19: 45–57.

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REVIEW QUESTIONS (continued)

8. In the transfusion setting, DNA analysis is a valuable ad-

junct 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 alloanti-

bodies a patient can produce

d. for patients with a positive DAT and a warm auto-

antibody

9. Which of the following statements is true about antigen

testing in a recently multiply-transfused patient?

a. Antigen typing by routine hemagglutination meth-

ods gives accurate results.

b. The transfused donor RBCs can be easily distin-

guished from the patient’s own RBCs.

c. DNA analysis is an effective tool for antigen prediction.

d. Antigen typing is not required to manage these

patients.

82043_ch07.qxd 11/13/09 4:40 PM Page 105

30. Ridgwell K, Dixey J, Parsons SF, et al. Screening humansera for anti-Lu antibodies using soluble recombinant Luantigens [abstract]. Transfus Med. 2001; 11(suppl 1): P25.

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24. Cartron JP, Bailly P, Le Van Kim C, et al. Insights into thestructure and function of membrane polypeptides car-rying blood group antigens. Vox Sang. 1998; 74(suppl 2):29–64.

25. Reid ME. Molecular basis for blood groups and func-tion of carrier proteins. In: Silberstein LE, ed. Molecularand Functional Aspects of Blood Group Antigens. Arling-ton, VA: American Association of Blood Banks; 1995:75–125.

26. Tournamille C, Colin Y, Cartron JP, et al. Disruption of aGATA motif in the Duffy gene promoter abolishes ery-throid gene expression in Duffy-negative individuals.Nat Genet. 1995; 10: 224–228.

27. Singleton BK, Green CA, Avent ND, et al. The presenceof an RHD pseudogene containing a 37 base pair dupli-cation and a nonsense mutation in Africans with the RhD-negative blood group phenotype. Blood. 2000; 95:12–18.

28. Storry JR, Reid ME, Fetics S, et al. Mutations in GYPBexon 5 drive the S�s�U�var phenotype in persons ofAfrican descent: implications for transfusion. Transfusion.2003; 43: 1738–1747.

29. Yazdanbakhsh K, Øyen R, Yu Q, et al. High level, stableexpression of blood group antigens in a heterologoussystem. Am J Hematol. 2000; 63: 114–124.

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