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82043_ch07.qxd 11/13/09 4:40 PM Page 95 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 95 82043_ch07.qxd 96 11/13/09 4:40 PM Page 96 UNIT 3 Principles of Testing 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. 82043_ch07.qxd 11/13/09 4:40 PM Page 97 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. 97 82043_ch07.qxd 98 11/13/09 4:40 PM Page 98 UNIT 3 Principles of Testing 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: 82043_ch07.qxd 11/13/09 4:40 PM Page 99 CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine 99 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 82043_ch07.qxd 11/13/09 100 4:40 PM Page 100 UNIT 3 Principles of Testing 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). 82043_ch07.qxd 11/13/09 4:40 PM Page 101 CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine 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 82043_ch07.qxd 102 11/13/09 4:40 PM Page 102 UNIT 3 Principles of Testing 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 82043_ch07.qxd 11/13/09 4:40 PM Page 103 CHAPTER 7 Molecular Testing for Blood Groups in Transfusion Medicine 103 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 82043_ch07.qxd 104 11/13/09 4:40 PM Page 104 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) 82043_ch07.qxd 11/13/09 4:40 PM Page 105 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. 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