Molecular Testing for Blood Groups in Transfusion Medicine

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CHAPTER 7

MOLECULAR TESTING FOR BLOOD GROUPS IN TRANSFUSION MEDICINE

M. E. REID AND H. DEPALMA

OBJECTIVES and in the understanding of the molecular bases associated with most blood group antigens and pheno- After completion of this chapter, the reader will be able to: types enables us to consider the prediction of blood group antigens using molecular approaches. Indeed, 1. Explain the basics of the structure and processing of a this knowledge is currently being applied to help re- gene. solve some long-standing clinical problems that can- 2. Discuss mechanisms of genetic diversity and the molecu- not be resolved by classical hemagglutination. lar bases associated with blood group antigens. Blood group antigens are inherited, polymorphic, 3. Describe applications of PCR-based assays for antigen structural characteristics located on proteins, glyco- prediction in transfusion and prenatal settings. proteins, or glycolipids on the outer surface of the 4. Describe some instances where RBC and DNA type may RBC membrane. The classical method of testing for not agree. blood group antigens and antibodies is hemagglutination. This technique is simple and when done 5. Delineate the limitations of hemagglutination and of PCR- correctly, has a specificity and sensitivity that is appro- based assays for antigen prediction. priate for the clinical care of the vast majority of patients. 6. Summarize relevant regulatory issues. Indeed, direct and indirect hemagglutination tests have served the transfusion community well for, respectively, over 100 and over 50 years. However, in KEY WORDS some aspects, hemagglutination has limitations. For example, it gives only an indirect measure of the po- Alleles Molecular testing tential complications in an at-risk pregnancy, it cannot Blood group antigens Prediction of blood groups precisely indicate RHD zygosity in D-positive people, DNA to protein 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 blood group antigen is a variant form of a and phenotypes has made it possible to use the poly- protein or carbohydrate on the outer surface of merase chain reaction (PCR)1 to amplify the precise aA red blood cell (RBC) that is identified when an areas of deoxyribose nucleic acid (DNA) of interest to immune response (alloantibody) is detected by detect alleles encoding blood groups and thereby hemagglutination in the serum of a transfused pa- predict the antigen type of a person. tient or pregnant woman. The astounding pace of This chapter first provides an overview of the pro- growth in the field of molecular biology techniques cessing of DNA to a blood group antigen and then

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summarizes current applications of molecular ap- Nucleotides in exons encode amino acids or a “stop” proaches for predicting blood group antigens in transfu- instruction, while nucleotides in introns are not en- sion medicine practice for patients and donors, especially coded. Nucleotides in an exon are written in upper in those areas where hemagglutination has limitations. 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 FROM DNA TO BLOOD GROUPS sequence of four nucleotides (AGgt) called the donor splice site, and at the junction of an intron to an exon The Language of Genes is another invariant sequence of four nucleotides (agGT) called the acceptor splice site. The splice sites DNA is a nucleic acid composed of nucleotide bases, a interact to excise (or outsplice) the introns, thereby sugar (deoxyribose), and phosphate groups. The nu- converting genomic DNA to mRNA. A single strand cleotide bases are purines (adenine [A] and guanine [G]) of DNA (5Ј to 3Ј) acts as a template and is duplicated and pyrimidines (thymine [T] and cytosine [C]). The exactly to form mRNA. Nucleotide C invariably language of genes is far simpler than the English lan- pairs with G, and A with T. Upstream from the ﬁrst guage. Compare four letters in DNA or RNA (C, G, A, exon of a gene, there are binding sites (promoter re- and T [T in DNA is replaced by U in RNA]) with 26 let- gions) for factors that are required for transcription ters of the English alphabet. These four letters are called (from DNA to mRNA) of the gene. Transcription of nucleotides (nts) and they form “words,” called codons, DNA always begins at the ATG, or “start,” transcrip- each with three nucleotides in different combinations. tion codon. The promoter region can be ubiquitous, ϫ ϫ ϭ There are only 64 (4 4 4 64) possible codons of tissue speciﬁc, or switched on under certain circum- which 61 encode the 20 amino acids and 3 are stop stances. At the 3Ј end of a gene there is a “stop” tran- ϭ codons. There are more codons (n 61) than there are scription codon (TAA, TAG, or TGA) and beyond ϭ amino acids (n 20) because some amino acids are en- that there is often an untranslated region. Between coded by more than one codon (e.g., UCU, UCC, UCA, adjacent genes on a chromosome, there is an “inter- UCG, AGU, and AGC, all encode the amino acid called vening” sequence of nucleotides, which are not tran- serine). This is termed redundancy in the genetic code. scribed. After the introns are excised, the resultant Essentials of a Gene mRNA contains nucleotides from the exons of the gene. Nucleotides in mRNA are translated (from Figure 7-1 shows the key elements of a gene. Exons mRNA to protein) in sets of three (a codon) to produce are numbered from the left (5Ј, upstream) to right a sequence of amino acids, which form a protein. Like (3Ј, downstream) and are separated by introns. transcription of DNA, translation of mRNA always

Transcription binding site Gene A Intervening Gene B sequence 5 Intron 1 Intron 2 3 DNA Exon 1 Exon 2 Exon 3 Exon 1 Upstream D A D A D UTR ATG Stop Start transcription transcription

Gene A 5 3 mRNA Exon 1 Exon 2 Exon 3

AUG Stop D = donor splice site (AGgt) Start translation translation A = acceptor splice site (agGT) UTR = untranslated region

NH2 = amino terminus COOH = carboxy terminus Protein NH2 COOH 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. 82043_ch07.qxd 11/13/09 4:40 PM Page 97

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begins at the “start” codon (AUG) and terminates at a (protein) products. The effect of a silent, missense, “stop” codon (UAA, UAG, or UGA). The resultant or nonsense single nucleotide change together protein consists of amino acids starting with methion- with examples involving blood group antigens are ine (whose codon is AUG) at the amino (NH2) termi- illustrated. nus. Methionine, or a “leader” sequence of amino acids, is sometimes cleaved from the functional pro- Effect of a Single Nucleotide Change tein and thus, a written sequence of amino acids (or on a Blood Group mature protein) does not necessarily begin with methionine. Due to redundancy in the genetic code, a silent DNA is present in all nucleated cells. For the pre- (synonymous) nucleotide change does not change diction of a blood group, DNA is usually obtained the amino acid and, thus, does not affect the antigen from peripheral white blood cells (WBCs), but also expression. Nevertheless, because it is possible that can be extracted from epithelial cells, cells in urine such a change could alter a restriction enzyme sediment, and amniocytes. recognition site or a primer binding site, it is important to be aware of silent nucleotide changes when Molecular Bases of Blood Groups designing a PCR-based assay. In contrast, a missense (nonsynonymous) nucleotide change results in a Although many mechanisms give rise to a blood different amino acid, and these alternative forms of group antigen or phenotype (Table 7-1), the major- an allele encode antithetical antigens. Figure 7-2 ity of blood group antigens are a consequence of a illustrates this where “G” in a lysine codon (AAG) is single nucleotide change. The other mechanisms replaced by “C,” which gives rise to the codon for listed give rise to a small number of antigens and asparagine (AAC). The example of a missense various phenotypes. Figure 7-2 shows a short hy- nucleotide change shows that a “C” to “T” change pothetical sequence of double-stranded DNA to- is the only difference between the clinically impor- gether with transcription (mRNA) and translation tant 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(aϪbϪ) 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(aϩ) 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(aϪbϪ)

Unknown Knull, Gy(aϪ) aNot uncommon in African Americans and Japanese.2 82043_ch07.qxd 11/13/09 4:40 PM Page 98

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A T G T C G A A G G A A G C A–3 DNA T A C A G C T T C C T T C G T –5 A mRNA A U G|U C G|A A G|G A A |G C A Transcription product Protein Met Ser Lys Glu Ala Translation product Examples: Single nucleotide substitution C Silent A U G|U C G/ |A A G|G A A |G C A 378 T>C in DO exon 2 Met Ser Lys Glu Ala Tyr126Tyr = no change C B Missense A U G|U C G|A A G/ |G A A |G C A 698 T>C in KEL exon 6 Met Ser Asn Glu Ala Met193Thr = K/k U Nonsense A U G|U C G|A A G|G/ A A |G C A 287G>A in FY exon 2 Met Ser Lys Stop Trp96Stop = Fy (a–b–)

Single nucleotide deletion A U G|XU C G A |A G G | A A G |C A 261del G in O exon 6 New Sequence Frameshift → 116Stop = 0 C Met Arg Arg Lys A U G U C G A A G G A A G C A Stop Codon | X | No example known Met Stop

Single nucleotide insertion G A U G|U C G|A A |G G A |A G C|A New Sequence 307-308 ins T in CO exon 2 D Met Ser Arg Gly Ser Frameshift → Stop = Co(a–b–) U A U G U C G A A G G A A G C A Stop Codon | | | | No example known Met Ser Lys Stop 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 also shows the ﬁve amino acids that are determined by the codons in the DNA (Panel A). Panels B through D demonstrate the effect of three different types of single nucleotide changes, substitution (Panel B), deletion (Panel C), and insertion (Panel D), and the effects on the amino acids. Where available, examples of these various types of changes in blood groups are given.

nucleotide change transforms a codon for an amino of a nucleotide can cause a stop codon, but there is no acid to a stop codon. Figure 7-2 and Table 7-2 give known example for a blood group. examples relative to blood groups. APPLICATIONS OF MOLECULAR Effect of Deletion or Insertion of Nucleotide(s) ANALYSIS A deletion of one nucleotide results in a Ϫ1 frameshift and an eventual stop codon (see Fig. 7-2 and Table 7-2). The genes encoding 29 of the 30 blood group sys- Typically, this leads to the encoding of a truncated tems (only P1 remains to be resolved) have been protein, but it can cause elongation. For example, a dele- cloned and sequenced.3,4 Focused sequencing of tion of “C” close to the stop codon in the A2 allele DNA from patients or donors with serologically results in a transferase with 21 amino acids more than in defined antigen profiles has been used to determine 2 the A1 transferase. Similarly, deletion of two nucleotides the molecular bases of variant forms of the gene. causes a Ϫ2 frameshift and a premature stop codon. This approach has been extremely powerful because Deletion of a nucleotide also can cause a stop codon, but antibody-based definitions of blood groups readily there is no known example for a blood group. distinguish variants within each blood group An insertion of one nucleotide results in a ϩ1 system. Details of these analyses are beyond the frameshift and a premature stop codon (see Fig. 7-2 scope of this chapter but up-to-date details about and Table 7-2). Insertion of two nucleotides causes a alleles encoding blood groups can be found on the ϩ2 frameshift and a premature stop codon. Insertion Blood Group Antigen Gene Mutation database at: 82043_ch07.qxd 11/13/09 4:40 PM Page 99

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

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)

http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi. PCR (Q-PCR; RQ-PCR), sequencing, and microarray cgi?cmd=bgmut/systems, or by entering “dbRBC” in technology. Figure 7-3 illustrates readout formats for a search engine. While there are 30 blood group sys- these assays. Microarrays use a gene “chip,” which is tems, 34 associated gene loci, and 270 antigens, there composed of spots of DNA from many genes attached are close to 1,000 alleles that encode the blood group to a solid surface in a grid-like array.5,6 Microarrays antigens and phenotypes. allow for multiple single nucleotide changes to be analyzed simultaneously and overcome not only the Techniques Used to Predict a Blood labor-intensive nature of hemagglutination but also Group Antigen data entry. This technology has great potential in transfusion medicine for the prediction of blood groups and Once the molecular basis of a blood group antigen has phenotypes. been determined, the precise area of DNA can be ana- There are clinical circumstances where hemagglu- lyzed to predict the presence or absence of a blood tination testing does not yield reliable results and yet group antigen on the surface of an RBC. Fortunately, as the knowledge of antigen typing is valuable to obtain. the majority of genetically defined blood group anti- Molecular approaches are being employed to predict gens are the consequence of a single nucleotide the antigen type of a patient to overcome some of the change, simple PCR-based assays can be used to detect limitations of hemagglutination. Determination of a a change in a gene encoding a blood group antigen. In- patient’s antigen profile by DNAanalysis is particularly numerable DNA-based assays have been described for useful when a patient, who is transfusion dependent, has this purpose. They include PCR-restriction fragment produced alloantibodies. Knowledge of the patient’s length polymorphism (RFLP), allele-specific (AS)-PCR probable phenotype allows the laboratory to as single or multiplex assay, real-time quantitative determine to which antigens the patient can and 82043_ch07.qxd 11/13/09 4:40 PM Page 100

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Cell nucleus Target sequence with primers Double helix DNA strand Taq Sense Antisense Polymerase Primer Primer Target sequence PCR reaction Chromosome expotential amplification Supercoiled DNA strand

Restriction endonuclease Analysis

150 bp

100 bp 50 bp Prediction confirmed by PCR-RFLP SS-PCR Real-Time PCR Sequencing Microarray hemagglutination

Allele:1 1&2 2 Allele 1 Allele 2 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 ex- Certain criteria should be met before obtaining tremely important to obtain an accurate medical his- fetal DNA for analyses: the mother’s serum contains tory for the patient because with certain medical an IgG antibody of potential clinical significance treatments, such as stem cell transplantation and and the father is heterozygous for the gene encoding kidney transplants, typing results in tests using the antigen of interest or when paternity is in doubt. DNA from different sources (such as WBCs, buccal It is helpful to know the ethnic origin and to concur- smears, or urine sediment) may differ. DNA analysis rently test both mother and father, in order to is a valuable tool and a powerful adjunct to hemag- restrict the genes involved and to identify potential glutination testing. Some of the more common clini- variants that could influence interpretation of the cal applications of DNA analyses for blood groups test results. DNA analysis can be performed for any are listed in Box 7-1. blood group incompatibility where the molecular basis is known. Applications in the Prenatal Setting Fetal DNA can be isolated from cells obtained by a variety of invasive procedures; however, the use of The ﬁrst application of molecular methods for the pre- amniocytes obtained by amniocentesis is the most diction of a blood group antigen was in the prenatal common source. Remarkably, free fetally derived setting, where fetal DNA was tested for RHD.7 DNA can be extracted from maternal serum or Hemagglutination, including titers, gives only an in- plasma8,9 and RHD typing is possible after 5 weeks direct indication of the risk and severity in hemolytic of gestation.8,10–13 The RHD type is the prime disease of the fetus and newborn (HDFN). Thus, anti- target because, at least in the majority of Caucasians, gen prediction by DNA-based assays has particular the Rh-negative mother has a deleted RHD, thereby value in this setting to identify a fetus who is not at permitting detection of the fetal RHD DNA. Further- risk for HDFN, that is, antigen negative, so that ag- more, anti-D is still notoriously clinically signiﬁcant gressive monitoring of the mother can be avoided. in terms of HDFN (reviewed in Avent and Reid14). 82043_ch07.qxd 11/13/09 4:40 PM Page 101

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Applications in the Transfusion Setting BOX 7-1 For Transfusion-dependent Patients Clinical Applications of DNA Analyses for Blood Group Antigens Certain medical conditions, such as sickle cell disease, thalassemia, autoimmune hemolytic anemia, • To type patients who have been recently transfused and aplastic anemia, often require chronic blood • To type patients whose RBCs are coated with im- transfusion. When a patient receives transfusions, the munoglobulin (ϩDAT) presence of donor RBCs in the patient’s peripheral • To identify a fetus at risk for hemolytic disease of blood makes RBC phenotyping by hemagglutination the fetus and newborn (HDFN) complex, time-consuming, and often inaccurate. The • To determine which phenotypically antigen-negative interpretation of RBC typing results of multitrans- patients can receive antigen-positive RBCs fused patients, based on such things as number of • To type donors for antibody identiﬁcation panels units transfused, length of time between transfusion • To type patients who have an antigen that is ex- and sample collection, and size of patient (the “best pressed weakly on RBCs guess”), is often incorrect.15 Because it is desirable to • To determine RHD zygosity determine the blood type of a patient as part of the • To mass screen for antigen-negative donors antibody identiﬁcation process, molecular ap- • To resolve blood group A, B, D, and e discrepancies proaches can be employed to predict the blood type • To determine the origin of engrafted leukocytes in of patients, thereby overcoming this limitation of a stem cell recipient hemagglutination. • To type patient and donor(s) to determine the possible alloantibodies that a stem cell transplant patient can make For Patients Whose RBCs Have a Positive DAT • To determine the origin of lymphocytes in a patient with graft-versus-host disease DNA-based antigen prediction of patients with au- • For tissue typing toimmune hemolytic anemia, whose RBCs are coated • For paternity and immigration testing with immunoglobulin, is valuable when available an- • For forensic testing tibodies require the indirect antiglobulin test. Al- • Prediction of antigen type when antisera is unavailable though useful for the dissociation of bound globulins, • Identify molecular basis of a new antigen 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 For analysis of single nucleotide changes (e.g., K/k), warm autoantibodies who require transfusion sup- a source of DNA consisting of mostly fetal DNA, for port is particularly challenging, as free autoantibody example, amniocytes, is preferred. present in the serum/plasma may mask the forma- Before interpreting the results of DNA analyses, it tion of an underlying alloantibody. Knowledge of the is important to obtain an accurate medical history and patient’s predicted phenotype is useful not only for to establish if the study subject is a surrogate mother, determining which alloantibodies he or she is capable if she has been impregnated with nonspousal sperm, of producing, but also as an aid in the selection of or if she has received a stem cell transplant. For prena- RBCs for heterologous adsorption of the autoanti- tal diagnosis of a fetus not at risk of HDFN, the body. This phenotype prediction is extremely valu- approach to molecular genotyping should err on the able for the ongoing management of patients with side of caution. Thus, the strategy for fetal DNA strong warm autoantibodies. Potentially, the pre- typing should detect a gene (or part of a gene) whose dicted phenotype could be used to precisely match product is not expressed (when the mother will be blood types, thereby reducing the need to perform monitored throughout pregnancy), rather than fail to extensive serologic testing. detect a gene whose product is expressed on the RBC membrane (e.g., a hybrid gene). When performing DNA analysis in the prenatal For Blood Donors setting, it is also important to always determine the DNA-based assays can be used to predict the anti- RHD status of the fetus, in addition to the test being gen type of donor blood both for transfusion and for ordered. In doing so, if the fetus has a normal RHD antibody identification reagent panels. This is par- there is no need to provide Rh-negative blood for ticularly useful when antibodies are not available or intrauterine transfusions. are weakly reactive. An example is the Dombrock 82043_ch07.qxd 11/13/09 4:40 PM Page 102

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blood group polymorphism, where DNA-based as- not correlate with antigen expression on the RBC (see says16–18 are used to type donors as well as patients for Table 7-3).23–25 If a patient has a grossly normal gene Doa and Dob in order to overcome the dearth of reliable that is not expressed on his or her RBCs, he or typing reagents. This was the first example where a she could produce an antibody if transfused with DNA-based method surpassed hemagglutination. antigen-positive blood. When feasible, the appropriate Although some antibodies are not known to cause assay to detect a change that silences a gene should be RBC destruction, such as antibodies to antigens of part of the DNA-based testing. Examples of such test- the Knops blood group system, they are often found ing include analyses for the GATA box with FY in the serum/plasma of patients and attain signifi- typing,26 presence of RHD pseudogene with RHD typ- cance by virtue of the fact that a lack of phenotyped ing,27 and exon 5 and intron 5 changes in GYPB with donors makes their identification difficult and time- S typing.28 consuming.19 DNA-based assays can be useful to In addition to silencing changes that can impact predict the Knops phenotype of donors whose RBCs antigen expression, there are other circumstances, are used on antibody identification panels and both iatrogenic and genetic, that may impact the thereby aid in their identification. results of DNA analysis (see Table 7-4). With certain PCR-based assays are valuable to test donors to medical treatments such as stem cell transplantation increase the inventory of antigen-negative donors. As and kidney transplants, typing results may differ de- automated procedures attain fast throughput at lower pending on the source of the DNA; therefore, it is ex- cost, typing of blood donors by PCR-based assays is tremely important to obtain an accurate medical rapidly becoming more widespread.20 With donor history for the patient. These medical procedures, as typing, the presence of a grossly normal gene whose well as natural chimerism, can lead to mixed DNA product is not expressed on the RBC surface would populations; therefore, the genotyping results will be lead to the donor being falsely typed as antigen- impacted by the source of the DNA used for testing. positive, and although this would mean the potential Another limitation of DNA analysis is that not all loss of a donor with a null phenotype, it would not blood group antigens are the consequence of a single jeopardize the safety of blood transfusion. nucleotide change. Furthermore, there may be many DNA analysis is useful for the resolution of appar- alleles per phenotype, which could require multiple ent discrepancies, for example, the resolution of ABO assays to predict the phenotype. There are also some typing discrepancies due to ABO subgroups, and for blood group antigens for which the molecular basis is reagent discrepancies that would otherwise poten- not yet known. tially be reportable to the FDA. Another example is to 21 classify variants of RHD and RHCE. OTHER APPLICATIONS FOR MOLECULAR ANALYSES For Patients and Donors Detecting Weakly Expressed Antigens Molecular biology techniques can be used to transfect DNA analysis can be useful to detect weakly ex- cells with DNA of interest and then grow the trans- pressed antigens. For example, a patient with a weak- fected cells in tissue culture. These cells, which ened expression of the Fyb antigen due to the Fyx express a single protein, and thus the antigens from phenotype (FY nt 265) is unlikely to make antibodies only one blood group system, can be used to aid in the to transfused Fy(bϩ) RBCs. In this situation, PCR- identiﬁcation of antibodies. Indeed, single-pass (Kell) based assays can help determine which phenotypi- and multi-pass (Duffy) proteins have been expressed cally antigen-negative patients can be safely in high levels in mouse erythroleukemic (MEL) cells transfused with antigen-positive RBCs. It has been or 293T cells and detected by human polyclonal anti- suggested that DNA assays can be used to detect bodies.29 Similar experiments have been performed weak D antigens in apparent D-negative donors to with antibodies to Lutheran antigens.30 Thus, it is the- prevent possible alloimmunization and delayed trans- oretically possible to produce a panel of cell lines ex- fusion reactions22 or to save true D-negative RBC pressing individual proteins for development of an products for true D-negative patients. automated, objective, single-step antibody detection and identiﬁcation procedure. Such an approach Limitations of DNA Analysis would eliminate the need for antigen-matched, short- dated, potentially biohazardous RBC screening and When recommendations for clinical practice are based panel products derived from humans. As promising on molecular analyses, it is important to remember as this approach is, some major hurdles are yet to be that, in rare situations, a genotype determination will overcome; for example, antigens from all blood group 82043_ch07.qxd 11/13/09 4:40 PM Page 103

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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(bϪ)

Alternative splicing Nucleotide change in splice site: SϪ sϪ; Gy(aϪ) partial/complete skipping of exon Deletion of nucleotides Dr(aϪ)

Premature stop codon Deletion of nucleotide(s) → frameshift Fy(aϪbϪ); DϪ; Rhnull; Ge: Ϫ2, Ϫ3, Ϫ4; Gy(aϪ); K0; McLeod Insertion of nucleotide(s) → frameshift DϪ; Co(aϪbϪ)

Nucleotide change Fy(aϪbϪ); rЈ; Gy(aϪ); K0; McLeod

Amino acid change Missense nucleotide change DϪ; Rhnull; K0; McLeod

Reduced amount of protein Missense nucleotide change Fyx; Co(aϪbϪ)

Hybrid genes Crossover GP.Vw; GP.Hil; GP.TSEN Har Gene conversion GP.Mur; GP.Hop; D- -; R0

Interacting protein Absence of RhAG Rhnull Absence of Kx Weak expression of Kell antigen Absence of amino acids 59–76 of GPA Wr(bϪ) Absence of protein 4.1 Weak expression of Ge antigens

Modifying gene In(LU) Lu(aϪbϪ) In(Jk) Jk(aϪbϪ)

systems must be expressed at levels that are at least gens are proving difficult to express in adequate equivalent to those on RBCs and the detection system levels. should have low background levels of reactivity. Transfected cells expressing blood group antigens Importantly, the highly clinically significant Rh anti- 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 TABLE 7-4 Limitations of DNA Analysis insignificant. In addition, genes can be engineered to express soluble forms of proteins expressing antigens for antibody inhibition, again as part of antibody Iatrogenic Stem cell transplantation detection and identification procedures, or prior to Natural chimera crossmatching.31–33 For example, concentrated forms Surrogate mother/sperm donor of recombinant CR1 (CD35) would be valuable to inhibit clinically insignificant antibodies in the Knops Genetic Not all polymorphisms can be system, thereby eliminating its interference in cross- analyzed matching. Many alleles per phenotype Recombinant proteins and transfected cells Molecular basis not yet known expressing blood group antigens have been used as Beware of possible silencing immunogens for the production of monoclonal anti- changes that can affect antigen bodies. This approach has led to the successful expression (Rh and RhAG, Band 3, production of murine monoclonal antibodies with and GPA dominant Lu(aϪbϪ)) specificities to blood group antigens not previously Not all alleles in ethnic populations made34,35 (see http://www.nybloodcenter.org). Such are known antibodies are useful because the supplies of human 82043_ch07.qxd 11/13/09 4:40 PM Page 104

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polyclonal antibodies are diminishing. Molecular ma- research purpose, or is to be published, even as an ab- nipulations have been used to convert murine IgG stract, then IRB approval is required. The type of re- anti-Jsb and anti-Fya to IgM direct agglutinins, which search dictates whether the review is expedited or are more practical in the clinical laboratory.36,37 requires full board approval. Influencing factors include whether the sample is linked or unlinked, whether it exists or is collected specifically for the test- REGULATORY COMPLIANCE ing, and whether or not the human subject is at risk from the procedure. In addition to a knowledge of blood groups, their molecular bases, technical aspects of PCR-based assays, and causes of possible discrepancies (be they SUMMARY technical, iatrogenic, or genetic), it is important to be cognizant of issues of regulatory compliance. The lab- Numerous studies have analyzed blood samples from oratory director is responsible for ensuring accuracy of people with known antigen profiles and identified the results regardless of whether the test is a laboratory- molecular bases associated with many antigens.2 The developed test (LDT; previously known as “home- available wealth of serologically defined variants has brew”) or a commercial microarray for research use contributed to the rapid rate with which the genetic only (RUO). Each facility should have a quality plan diversity of blood group genes has been revealed. Ini- that includes test procedures, processes, validation, tially, molecular information associated with each etc. According to the FDA, DNA testing cannot be used variant was obtained from only a small number of as the sole means of determining the antigen status samples and applied to DNA analyses with the as- and a disclaimer statement must accompany reports sumption that the molecular analyses would correlate giving the prediction of blood types. As DNA testing with RBC antigen typing. While this is true in the ma- to predict a blood group for the purpose of patient care jority of cases, like hemagglutination, PCR-based as- is not used to identify or diagnose a genetic disease, says have limitations. Many molecular events result in but is doing a test in a different way (hemagglutination the DNA-predicted type and RBC type being appar- vs. DNA assays) to achieve a similar result, informed ently discrepant (some are listed in Table 7-3). Fur- consent may not be required. Whether or not informed thermore, analyses of the null phenotypes have consent should be obtained from the patient or donor demonstrated that multiple, diverse genetic events to be tested depends on local laws. can give rise to the same phenotype. Nonetheless, If DNA-based testing is done strictly for patient molecular analyses have the advantage that genomic care, it is exempt from Institutional Review Board DNA is readily available from peripheral blood leuko- (IRB) approval. However, if testing is performed for a cytes, buccal epithelial cells, and even cells in urine,

Review Questions

1. True or false? The process of changing DNA to RNA is c. ampliﬁes a speciﬁc sequence of DNA called translation. d. all of the above 2. True or false? A single nucleotide change can give rise 6. For antigen prediction in the neonatal setting, the most to a null blood group phenotype. common source of fetal DNA is: 3. True or false? A blood group can be predicted by test- a. amniocytes ing DNA extracted from WBCs. b. fetal RBCs 4. A single nucleotide change can cause which of the c. cord blood following: d. endothelial cells a. no change in the codon for an amino acid 7. Antigen prediction by DNA analysis is: b. a stop codon a. indicated only for patient testing and is not applica- c. a change from one amino acid to another ble for donor testing d. all of the above b. used to determine weakly expressed antigens 5. A PCR-based assay: c. used to predict antigens when licensed FDA antisera a. has limitations are not available b. gives a prediction of a blood group d. b and c

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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 ad- 9. Which of the following statements is true about antigen junct to hemagglutination testing for all of the following testing in a recently multiply-transfused patient? circumstances except: a. Antigen typing by routine hemagglutination meth- a. for patients with a negative DAT and no history of ods gives accurate results. transfusion b. The transfused donor RBCs can be easily distin- b. for patients who require chronic RBC transfusions guished from the patient’s own RBCs. c. for predicting antigens to determine what alloanti- c. DNA analysis is an effective tool for antigen prediction. bodies a patient can produce d. Antigen typing is not required to manage these d. for patients with a positive DAT and a warm auto- patients. antibody

and it is remarkably stable. The primary disadvantages 8. Nelson M, Eagle C, Langshaw M, et al. Genotyping fetal are that the type determined on DNA may not reflect DNA by non-invasive means: extraction from maternal the RBC phenotype and certain assays can give false plasma. Vox Sang. 2001; 80: 112–116. results. The prediction of blood group antigens from 9. Lo YMD. Fetal DNA in maternal plasma: application to testing DNA has tremendous potential in transfusion non-invasive blood group genotyping of the fetus. Transfus Clin Biol. 2001; 8: 306–310. medicine and has already taken a firm foothold. DNA- 10. Avent ND, Finning KM, Martin PG, et al. Prenatal based assays provide a valuable adjunct to the classic determination of fetal blood group status. Vox Sang. hemagglutination assays. The high-throughput nature 2000; 78: 155–162. of microarrays provides a vehicle by which to increase 11. Lo YMD, Hjelm NM, Fidler C, et al. Prenatal diagnosis inventories of antigen-negative donor RBC products of fetal RhD status by molecular analysis of maternal and, in this aspect, change the way we practice transfu- plasma. N Engl J Med. 1998; 339: 1734–1738. sion medicine. 12. Faas BH, Beuling EA, Christiaens GC, et al. Detection of fetal RHD-specific sequences in maternal plasma. Lancet. 1998; 352: 1196. ACKNOWLEDGMENT 13. Bischoff FZ, Nguyen DD, Marquez-Do D, et al. Nonin- vasive determination of fetal RhD status using fetal We thank Robert Ratner for help in preparing the DNA in maternal serum and PCR. J Soc Gynecol Investig. 1999; 6: 64–69. manuscript and figures. 14. Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000; 95: 375–387. 15. Reid ME, Rios M, Powell VI, et al. DNA from blood sam- REFERENCES ples can be used to genotype patients who have recently received a transfusion. Transfusion. 2000; 40: 48–53. 1. Mullis KB, Faloona FA. Specific synthesis of DNA in 16. Rios M, Hue-Roye K, Lee AH, et al. DNA analysis for the vitro via a polymerase-catalyzed chain reaction. Methods Dombrock polymorphism. Transfusion. 2001; 41: 1143–1146. Enzymol. 1987; 155: 335–350. 17. Wu G-G, Jin Z-H, Deng Z-H, et al. Polymerase chain 2. Reid ME, Lomas-Francis C. Blood Group Antigen Facts- reaction with sequence-specific primers-based genotyping Book. 2nd ed. San Diego: Academic Press; 2004. of the human Dombrock blood group DO1 and DO2 3. Lögdberg L, Reid ME, Lamont RE, et al. Human blood alleles and the DO gene frequencies in Chinese blood group genes 2004: chromosomal locations and cloning donors. Vox Sang. 2001; 81: 49–51. strategies. Transfus Med Rev. 2005; 19: 45–57. 18. Reid ME. Complexities of the Dombrock blood group 4. Daniels G, Castilho L, Flegel WA, et al. International So- system revealed. Transfusion. 2005; 45(suppl): 92S–99S. ciety of Blood Transfusion Committee on Terminology 19. Moulds JM, Zimmerman PA, Doumbo OK, et al. Molec- for Red Cell Surface Antigens: Macao report. Vox Sang. ular identification of Knops blood group polymor- 2009; 96(2): 153–156. phisms found in long homologous region D of 5. Cuzin M. DNA chips: a new tool for genetic analysis. complement receptor 1. Blood. 2001; 97: 2879–2885. Transfus Clin Biol. 2001; 8: 291–296. 20. Hashmi G, Shariff T, Zhang Y, et al. Determination of 24 6. Petrik J. Microarray technology: the future of blood test- minor red blood cell antigens for more than 2000 blood ing? Vox Sang. 2001; 80: 1–11. donors by high-throughput DNA analysis. Transfusion. 7. Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal deter- 2007; 47: 736–747. mination of fetal RhD type by DNA amplification. 21. Westhoff CM. The structure and function of the Rh anti- N Engl J Med. 1993; 329: 607–610. gen complex. Semin Hematol. 2007; 44: 42–50. 82043_ch07.qxd 11/13/09 4:40 PM Page 106

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