Integrating Molecular Technologies for Red Blood Cell Typing andCompatibility Testing Into Blood Centers and Transfusion Services

Christopher D. Hillyer, Beth H. Shaz, Anne M. Winkler, and Marion Reid

Nucleic acid–based technology is now at a point wherethe field of transfusion medicine is ready for itswidespread application. In the donor center, genotypingof red blood cell (RBC) products provides phenotype-matched products for special patient populations orantigen-negative products for patients with alloantibo-dies. In the immunohematology reference laboratory,molecular technologies aid in discerning blood types inthe situation of a typing discrepancy and improvepretransfusion RBC testing reagents. In the hospitaltransfusion service, genotyping patients aids in provid-ing phenotype-matched RBC products. In prenataltesting, genotyping for RHD aids in the decision forRh immune globulin prophylaxis and predicting risk of

Transfusion Medicine Reviews, Vol 22, No 2 (April), 2008: pp 117-13

hemolytic disease of the fetus and newborn. Beforegenotyping is accepted as the universal standard forpretransfusion and donor testing, important limitationsof this technology must be addressed, including thefact that the genotype does not always predict thephenotype and the need for creating the ideal high-throughput platform. Clinical trials are needed toanswer important questions, and a donor and patientdatabase is needed. A stepwise plan for progressiveintroduction into the donor centers and transfusionservices must be established. In conclusion, the field oftransfusion medicine is ready to expand the use ofmolecular diagnostics.C 2008 Elsevier Inc. All rights reserved.

From the Department of Pathology and Laboratory Medicine,Center for Transfusion and Cellular Therapies, EmoryUniversity, Atlanta, GA; and Laboratory of Immunohematology,New York Blood Center, New York, NY.

Address reprint requests to Christopher D. Hillyer, MD,Department of Pathology and Laboratory Medicine, EmoryUniversity, 1364 Clifton Rd, Atlanta, GA 30321.

E-mail: chillye@emory.edu0887-7963/08/$ - see front mattern 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.tmrv.2007.12.002

FOR OVER A century, hemagglutination hasbeen the gold standard for red blood cell

(RBC) antigen detection in the determination of apatient's RBC phenotype. Hemagglutinationimplies that RBC antigen and antibody reagentsinteract, and this interaction has allowed the terms“serology” and “immunohematology” to becomecommon parlance. However, despite its relativelylow cost, ease of performance, and sensitivity andspecificity suitable for what we now consider to beoptimal patient care, hemagglutination-based deter-mination of RBC phenotype has its limitations,which are described below. In part, to mitigate theselimitations, nucleic acid–based technologies havebeen added to the armamentarium of methodsavailable in advanced immunohematology refer-ence laboratories, thus allowing the term “mole-cular immunohematology” to enter the bloodbanking lexicon. Molecular immunohematologyrefers to the use of genotyping applied to thegenes encoding RBC antigens. Thus, this techno-logy represents an indirect method for predictingan individual's RBC blood group phenotype.Initially assumed to be complex, costly, anddifficult to automate, it appeared that RBC bloodgroup antigen genotyping would not be ready, inthe reasonably foreseeable future, for routineapplication. However, advances in our under-standing of the relationship of RBC genotype tophenotype, detailed description of the sequenceand function of blood group genes and relatedsilencing elements, concepts of nano and chiptechnology applied to RBC nucleotide polymorph-

ism detection, and the concept that genotypingmight be a valuable complement to hemagglutina-tion, it is likely that genotyping technologies willsee widespread adoption in both the blood centerand the hospital transfusion service. This approachwill primarily be used initially in specializedclinical situations and in immunohematologyreference laboratories servicing more complexpatients. Thus, transfusion authorities are nowstarting to consider the use of molecular techni-ques in transfusion medicine as critical to advan-cing the understanding of blood group antigenpolymorphisms and to increasing blood safety byproviding better-matched, compatible homologousblood products for transfusion. The purpose of thisreview is to present the current status of molecularimmunohematology as applied to donor centers,reference laboratories, and transfusion services.We also consider what clinical trials and regulatoryaspects might be needed and the apparent path to astepwise implementation and adoption plan for thisimportant and emerging technology.

2 117

Table 2. Molecular Events That Give Rise to Blood GroupAntigens and Phenotypes3

Single-nucleotide substitution in coding sequences and intronicsequences involved in splicing (most blood group systems)Deletion of a gene, exon, or nucleotide(s) (ABO, MNS, Rh, Kell,Duffy, Dombrock, Gerbich, etc)Gene conversion or recombination events, which have beenrecognized for several blood groups, especially those encodedby clustered gene loci (namely, MNS, Rh and Ch/Rg)Insertion of a nucleotide(s) (Rh, Colton)Duplication of an exon (Gerbich)

118 HILLYER ET AL

RED BLOOD CELL ANTIGENS AND THEIR GENESAND GENOTYPING

There are approximately 270 serologically deter-mined RBC antigens. Initial recognition of theallelic nature of serologically defined antigendifferences led to the concept that single nucleotidepolymorphisms (SNPs), which lead to single aminoacid differences in RBC antigens, could be used toprecisely determine an individual's phenotype viaclear relationships between RBC antigen genotypeand phenotype. Well-known examples of thisconcept are Gly42Asp being the amino acid changeassociated with Fya/Fyb determinants, Thr193Metfor K/k, and Met29Thr for S/s. However, detailedserologic and molecular/genetic investigations overmany years also confirmed that some RBC antigengenes and their corresponding protein or carbohy-drate moiety antigens are not as simple as formerlythought. There can be multiple genetic variations toaccount for the same blood group phenotype. Forexample, there are over a 100 alleles for the 4 ABOphenotypes.1 This tremendous complexity ofalleles encoding RBC antigens, now confirmedwith genotyping, and the corresponding antibodyformation, is increasingly compounded by globali-zation of populations and heterogeneous RBCexposure through transfusion. Another example ofthis complexity is demonstrated by the currentunderstanding of the MNS blood group system,which is highly polymorphic and includes at least43 antigens. Many of the antigens are uncommon,resulting from an amino acid substitution or a

Table 1. Limitations of Serologic Immunohematology

Technical limitationsSubjective interpretationLabor intensive procedure requiring manual data entryRequires use of reliable antiseraCost of FDA-approved reagents is escalatingMany antisera used are not FDA approvedAntisera are often limited in volume, weakly reactive, orunavailable

Source material is a biohazard serving as a potential reservoirof infectious disease

Clinical limitationsTyping of RBCs from patients who have been recentlytransfused

Typing of RBCs from patients with autoantibodiesDoes not precisely determine RHD zygosity in D-positiveindividuals

Small number of donors are typed for a small number ofantigens, limiting antigen-negative or rare donor registries

rearrangement between GYPA and GYPB. Low-prevalence antigens in the MNS blood groupsystem include Cla, DANE, Dantu, ERIK, Far,HAG, He, Hil, Hop, Hut, MARS, Me, Mg, Mia,MINY, Mit, Mta, Mur, MUT, Mv, Nob, Nya, Or,Osa, Ria, sD, SAT, Sta, TSEN, Vr, and Vw. Thereare rare null phenotypes in this system that resultfrom gene deletions, namely, En(a-), which lacksMN antigens; U-, which lacks Ss antigens; andMkMk, which lacks both MN and Ss antigens.Finally, some antigens that are associated with theMNS system but are not numbered by theInternational Society of Blood Transfusion (ISBT)Working Party on Terminology for Red CellSurface Antigens are a consequence of alteredglycosylation at residues 2, 3, and 4 of glycophorinA (GPA). These include Tm, Sj, M1, Can, Sext, andHu. Thus, the genetic, protein and carbohydratecomplexity of antigenic differences has madeserologic determination of the wide range of RBCantigens quite challenging. These challenges can bedifferentiated into Technical Limitations and Clin-ical Limitations as summarized in Table 1.

To date, genes encoding 28 of the 29 establishedblood group systems have been cloned andsequenced; only the encoding of the P systemremains to be resolved.2 The variety of differentmolecular events that can occur in the generation ofblood group antigens is summarized in Table 2.3 Asabove, most of these occur because of a SNP, whichcan be exploited in the use of genotyping.

During the past 10 years, experimental immu-nohematology laboratories have implementedmolecular methods to identify specific SNPs inthe many genes that encode blood group antigens.In addition, there has been the development ofseveral different mass-scale genotyping technolo-gies to perform high-throughput blood groupprediction with the goal of mainstream application.

Table 3. Application and Implementation of MolecularTechnologies

Donor centerGenotype RBC productsProduct for special patient populations, such as sickle celldisease patients

Products for patients with multiple alloantibodiesRHD genotyping donors who are D-negative

Reference laboratoryReagent RBCs for antibody detectionGenotype to determine dosage of RBC antigensResolution of typing discrepanciesGenotype to predict presence or absence of an antigenwhen no antisera exists

Determination if new antibody is an autoantibody oralloantibody

Resolution of unusual serological findingsTransfusion serviceGenotype patientsRecently transfused patientsPatients with autoantibodiesD type of the patient to predict need for RhIg or D-negativeproducts

Providing genotyped matched productsPatients with SCDPatients with thalassemiaPatients with AIHAChronically transfused patients

Prenatal testingRHD type to predict need for RhIgGenotype fetal DNA to predict risk for HDFN

Abbreviations: SCD, sickle cell disease; AIHA, autoimmune

hemolytic anemia; HDFN, hemolytic disease of the fetus and

newborn.

119MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

The methods use amplification of the target genesequence by polymerase chain reaction (PCR)followed by analysis using restriction fragmentlength polymorphisms, real-time PCR, sequence-specific primer PCR either single or multiples,single base extension, high-throughput beadtechnology, or microarrays. These technologiesare recently reviewed in an article by Avent.4 Theapplication and implementation of moleculartechnologies in the blood center, referencelaboratory, and transfusion service is presentedin Table 3.

DONOR CENTER

Phenotyping RBC Products

In the United States, the current practice is to onlymatch for the ABO and D antigens, but there are 2situations when multiple antigen-negative RBCproducts are requested: (a) when recipients havecorresponding alloantibodies and (b) for special

chronically transfused patient populations, such aspatients with sickle cell disease (SCD).

Alloimmunization occurs in approximately 2% to6% of patients who receive RBC transfusions, butthe rate of alloimmunization may be as high as 36%in patients with SCD.2 Recipients with multiplealloantibodies impair the ability to provide antigen-negative, compatible RBCs for transfusion becauseblood banks must phenotype many times thenumber necessary to find an appropriate product.The numbers of products screened multipliesdepending on the RBC antigen prevalence and thenumber of negative antigens necessary, thus placinga burden on the donor center needing to identifysuch products. The burdens include laboratorytechnologist time, adequate RBC inventory, andappropriate reagents. Amajor goal of the mass-scalegenotyping process is to allow for the expansion ofphenotype/genotype matching for a greater numberof patients, thereby improving patient care.

There are patient populations that benefit fromreceiving phenotype-matched products, especiallythose who are chronically transfused or at increasedrisk for alloantibody formation. Phenotype-matched products can be limited to the C, E, andK antigens or extended to include Fya, Jka, Jkb, S,and other antigens. The fundamental reason tophenotype-match products is to prevent alloanti-body formation and the subsequent negativeconsequences of hemolytic transfusion reactions.The downside of this precise matching practice isthat it makes routine transfusion more difficult forboth the donor center and transfusion service.Therefore, currently phenotype-matching is onlyapplied to specific patient populations.

Donor centers currently screen and stock RBCproducts to keep a pool of frequently neededantigen-negative products. Usually, donor centersscreen products from repeat group O donors andfamily members of patients who have formedalloantibodies to high-prevalence antigens for rareblood types. Batch serologic screening is technol-ogist time-intensive to perform; typing reagents areexpensive; and appropriate controls are required.As a result, donor centers have algorithms to helpdetermine the likelihood of an antigen-negativeproduct based on limited phenotyping and donorrace and ethnicity.

The American Rare Donor Program (ARDP) is alist of over 30000 individuals compiled from AABBand the American Red Cross who are active blood

120 HILLYER ET AL

donors with a blood type that occurs in less that 1 in10000 people.5 This program supplies these rareRBCs all over the world, but mostly within theUnited States. It relies on the above methods to findthese products and the continued good will of thesedonors to maintain an adequate supply. In addition,the ISBT maintains a rare blood donor program,which is compiled and maintained by the Interna-tional Blood Group Reference Laboratory.6 The useof large-scale genotyping methods would enableincrease identification of these RBC products andcorresponding donors. Hashmi et al7 demonstratedthe above concept when they genotyped 2355 donorsusing the BeadChip to predict for the presence of K,k, Jka, Jkb, Fya, Fyb, M, N, S, s, Lua, Lub, Dia, Dib,Coa, Cob, Doa, Dob, Joa, Hy, LWa, LWb, Sc1, Sc2,and HgbS. They were able to identify 21 rare donors(Co(a−b+), Jo(a−), S−s−, and K+k−).

Red Blood Cell Product Genotyping

High-throughput molecular technologies in thefield of transfusion medicine make it possible todetermine multiple RBC antigen SNPs simulta-neously. There are multiple mass-scale genotypingplatforms in use and in development in NorthAmerica and Europe.Since 2000, the BloodGen project has aimed to

develop and standardize mass-scale moleculargenotyping using a microarray platform knownas the Bloodchip manufactured by ProgenikaBiopharma (Vizaya, Spain).4 This platformincludes genotypes for ABO, RHD, RHCE, KEL,FY, JK, DI, CO, MNS, and DO blood groups andselected human platelet antigen alleles. TheBloodGen project has performed multiple small-scale clinical trials, initially using samples fromindividuals with rare blood types. This exercisehas been repeated to optimize the design of theBloodchip, which is a tactic used by other high-throughput platforms as well. Currently, there is alarge clinical trial with the goal of bringing thisproduct to market.GenomeLab SNPstream by Beckmann Coulter

(Fullerton, CA) and BeadChip developed byBioArray Solutions (Warren, NJ) are also beingdeveloped for mass-scale genotyping of RBCdonors.4 Recently, the human erythrocyte antigenBeadChip array was used to determine 24 antigenswithin 10 blood group systems for 2355 Ameri-can RBC donors.7 The donors represented adiverse population of whites, African Americans,

Hispanics, and Asians. There was nearly completeconcordance of genotypes and serologic pheno-types; however, of the discordant results, con-firmatory testing including gene sequencing andrestriction fragment length polymorphism analysisfavored the molecular method (n = 16) or wasresolved by manual DNA analysis of GYPB exon5 mutations (n = 8). These clinical trialsconfirmed the importance of including relevantsilencing SNPs.

Mass-scale genotyping of RBC donors wouldenable increased provision of RBC antigen-matched products to recipients, especially thosespecial populations who are at an increased risk foralloimmunization. In addition, mass-scale genotyp-ing would expand the genotyped/phenotyped donordatabase and increase the number of rare donorproducts, which would improve patient care.

D Typing

The donor center must ensure that D-negativeproducts are appropriately labeled, such that arecipient of a D-negative product does not formanti-D in response to transfused RBCs. Currently,donor centers use typing reagents to detect thepresence of the D antigen, and to increase thesensitivity of these reagents, the test is brought tothe antiglobulin phase (weak D testing), whichtakes additional time, reagents, and controls.8 Ifthe donor's RBCs have a positive direct anti-globulin test, the antiglobulin phase will befalsely positive, and additional work must to bedone to determine the D type. Wagner et al9

screened 8442 D-negative blood donations byRHD sequence-specific primer PCR and detected5 D-positive donors. One donor was a D-positive,D-negative chimera with 94% D-negative RBCs.They traced 13 previously donated products to 2D-negative recipients who had formed an anti-Dalloantibody after transfusion. In addition, theyfound 45 D-negative yet RHD gene–positive(albeit silenced) samples, which mostly representednovel RHD genes.9 Approximately, 30 000D-negative donors in Germany have been RHDtested, and about 1 in 1000 express the D antigenand are subsequently removed from the D-negativedonor pool.10 In a recent study from Austria, 3 of2427 D-negative donors carried RHD (1 weak Dand 2 Del), which is an estimated incidence of0.12%.11 In conclusion, the use of moleculartyping will prevent mislabeling of 0.1% of donors

121MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

as D-negative and avoid potential alloimmuniza-tion in the recipient.

REFERENCE LABORATORY TESTING

Reagent RBCs in Antibody Detection

The detection and identification of RBC alloanti-bodies rely on the use of well-characterized reagentRBCs. Lack of hemagglutination between thepatient's plasma and RBCs ideally from a personhomozygous for the genes expressing the presenceof the antigen is used to exclude the correspondingalloantibody. Currently, serologic methods are usedfor phenotyping RBC reagents used for antibodyscreening and the identification of panel RBCs.There are blood groups were the determination ofhomozygosity is notoriously difficult, especiallyFya/Fyb and D. In addition, there are blood groupantigens were serologic testing cannot be per-formed; this is particularly true for low-prevalenceantigens. When low-prevalence RBC antigens arepresent on screening cells but are not identified, theymay lead to a positive antibody screen, and furtherworkup reveals no identifiable alloantibody. Thus,the patient's plasma must be crossmatched to RBCproducts often without the knowledge of thealloantibody's identity. From a logistic and patientcare perspective, the appropriate antigen-negativeproduct cannot be preselected in the future,especially when the reactivity disappears from thepatient's plasma or the screening RBCs lack theantigen. In addition, additional time and resourcesare often used without benefiting and, in fact,delaying patient care. At the Blood Center inSweden, 3 of 52 reagent RBCs, which had beenpredicted by phenotype to be from an RHDhomozygote, were in fact from a hemizygote bygenotype and thus expressed a single dose of D.12

Of the 74 Fya or Fyb RBC samples predicted byphenotype to be from a homozygote person, 7 werefound to be hemizygous by genotype due to thepresence of either the FY*X or the FY*O genes.13 Inaddition, reagent RBCs were genotyped for DO.Antibodies to Doa and Dob have resulted inhemolytic transfusion reactions, but the antibodiesare difficult to identify due to lack of adequatetyping reagents and, therefore, identification of theantigen on various RBC reagents used. GenotypingDNA from donors of reagent RBCs allows forimproved prediction over serology that the RBCscarry a double-dose expression of the antigen for use

to exclude the presence of RBC alloantibodies,possibly reducing the in finding of “nonspecific”reactivity, and for predicting reagent RBC antigenphenotype when no typing reagents are available.

Resolution of Typing Discrepancies

Red blood cell genotyping can be used to resolveweak hemagglutination reactions and typing dis-crepancies, most often encountered as a result ofvariation in the ABO and D phenotypes. The abilityto accurately determine an individual's antigenstatus would eliminate the use of antigen-negativeblood where the patient would be unlikely tobecome alloimmunized, the use of group O RBCsand AB plasma for transfusion in the situation ofABO typing discrepancy, or the loss of a product inthe donor setting because of the inability toappropriately label it. Olsson et al14 investigated324 RBC samples with ABO typing discrepancies.They investigated samples with acquired variantABO phenotypes from pregnancy, hematologicdisorders, and other medical conditions, sampleswith inherited ABO phenotypes due to known andunknown subgroup alleles, samples of relatives ofthese subgroup individuals, samples with unex-pected absence of or weakly reactive anti-A or anti-B, and samples of suspected chimerism. Thus,genotyping can aid in the differentiation betweensubgroup alleles and acquired weakened agglutina-tion and allows proper ABO identification of bothdonors and patients.

Providing Antigen-Negative Products When NoTyping Reagents Exists

Monoclonal antibodies prepared from human/mouse heterohybridoma cell lines or polyclonalantibodies from human plasma are used as typingreagents. These reagents usually meet Food andDrug Administration (FDA) potency requirements,but some RBC typing reagents are either notavailable or made from saved patient's plasmawith the desired antibody specificity. These human-derived antibodies may be only weakly reactive,formed in conjunction with other antibodies or inindividuals with anti-A and/or anti-B present,making their use difficult for some RBC samples.Antibodies, including anti-A and/or anti-B can beremoved through absorption, but this is a time-consuming process and may dilute the wantedantibody's strength. These typing reagents can bedifficult to obtain. If antisera are not available, then

122 HILLYER ET AL

a crossmatch with the patient's plasma to detect thepresence of the RBC antigen must be relied on. Ifthe patient's antibody titer is too low to detect, thenthere is no method to ensure that the products areantigen-negative and the patient may receiveantigen-positive products. Reid15 described theuse of genotyping to find needed RBC productswhen antiserum was not available for the Dom-brock antigens. DNA analysis allowed for thesuccessful transfusion of Doa and Dob antigen–negative products. The concept of genotypingrather than relying on rare antisera or patientcrossmatch results, if the antibody still persists, isvital to the prevention of possible hemolytictransfusion reactions in such patients.

Alloantibody Versus Autoantibody

When patients have been transfused and nopretransfusion specimen is available, then thedetermination of a newly identified antibody isagainst foreign cells (allo) or self cells (auto) can bedifficult. An example is a patient whose RBCs typeas D-positive and forms an anti-D after transfusionof D-positive products. The only way to determineif the antibody is to the transfused RBCs is toseparate the transfused from the patient's RBCs anddetermine to which the antibody binds. Usingmolecular methods, the patient's RHD genotypewould predict if their phenotype was one at risk atmaking anti-D. Such phenotypes are known aspartial D. In the case of an autoantibody, transfusionof antigen-positive products is possible, but in thecase of an alloantibody, it could lead to a hemolytictransfusion reaction and should be discouraged.

TRANSFUSION SERVICE

Genotyping the Patient

Recently transfused patient. Patients who aretransfusion dependent, such as those with SCD,thalassemia, and aplastic anemia, are difficult tophenotype when no pretransfusion sample isavailable. Because of the presence of circulatingdonor RBCs, which may persist for weeks,determination of an individual's phenotype bytraditional hemagglutination methods is complexdue to the presence of a mixed field population.With the use of time-consuming and labor-intensivetechniques, such as isolating reticulocytes or sicklecells (in individuals with SCD), the RBC phenotypemay be determined, but it may be inaccurate.

Molecular methods can overcome this limitation ofhemagglutination. Because transfusion has noeffect on somatic cells, the use of buccal epithelialcells and urine sediment as a source of DNA canprovide accurate results. It is also possible to obtainDNA from white blood cells present in theperipheral blood; however, some have criticizedthat interference from circulating donor white bloodcells may have an effect on the result. However,most PCR assays used in transfusion medicine donot detect posttransfusion DNA chimerism.16-18

Rozman et al16 demonstrated identical RBC typingon pretransfusion, posttransfusion, and buccalsamples in 8 patients after 26 multiple-transfusionevents. In addition, no differences were identifiedbetween the numbers of RBC products transfusedincluding those that were not leuko-reduced or thetime elapsed between transfusions and testing. Insummary, the determination of a recently transfusedpatient's RBC genotype is superior to usingtraditional hemagglutination techniques.

The patient with a positive direct antiglobulintest. Patients with circulating autoantibodies, withor without autoimmune hemolytic anemia (AIHA),can complicate RBC phenotyping. In patients withwarm autoantibodies, the use of indirect antiglobu-lin reactive reagents may result in false-positiveantigen typings unless the IgG can be removedfrom the patient's RBCs before testing. Fortunately,many RBC typing reagents now consist of mono-clonal antibodies allowing for antigen detection inthe direct agglutination phase.19 There are situa-tions when no direct agglutinating reagents areavailable, when the antigen is sensitive to the IgGremoval treatment, or when the IgG removal is noteffective.1 In such cases, genotyping provides anacceptable alternative by allowing the prediction ofa patient's RBC antigen status without the inter-ference of the autoantibody.

D status of the patient. Current D-typingreagents in use in both donor centers and transfu-sion services may have difficulty in determining thestatus of weak D or partial D individuals.Individuals with partial D, whether strong, variable,or weak expression of D, are at risk for forming ananti-D. These patients would benefit from receivingD-negative RBCs for transfusion and potentiallybenefit from Rh immune globulin (RhIg) prophy-laxis. Patient typing is usually performed with anIgM monoclonal anti-D reagent that does notdetect, in the direct phase, DVI, which is the most

Table 4. Weak D Types That Are Prevalent or Clinically Relevant10

Weak Dphenotype

Prevalencein

Germany(%)

Haplotypeassociation

Recommended management

RBC product transfusion RhIg in pregnancy

Type 1 0.2964 CDe D-positive NoType 2 0.0759 cDE D-positive NoType 3 0.0219 CDe D-positive NoType 4.0 0.0140 cDe D-positive NoType 4.1 0.0230 cDe D-positive NoType 4.2 Rare cDe D-negative YesType 5 0.0035 cDE D-negative YesType 11 N0.0009 CDe D-negative Yes

0.0009 cDeType 15 Rare cDE D-negative YesType 19 0.0670 CDe D-negative YesType 20 0.0240 cDE D-negative YesOther types Rare Variable D-negative Yes

123MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

common form of partial D in whites.20 The practiceof Flegel10 is to genotype all patients with serologicreactivity of 2+ or less in gel (about 1% of patients)to resolve their weak D type at the molecular level.Based on their molecular type, the patient istransfused with D-positive or D-negative RBCs asappropriate and is evaluated for the need for RhIgduring pregnancy (Table 4).10 This logical combi-nation of the use of phenotyping followed bygenotyping leads to improved patient care andpresumed less D alloimmunization. Unfortunately,at this time, there are no guidelines about the needto determine the weak D type or the transfusionmanagement of these patients. The increased useand data collection of molecular weak D typeswould create a better understanding of how to bestmanage these patients.

Providing Phenotyped-Matched RBCs

Sickle cell disease. SCD is the most prevalentgenetic disorder in the African American popula-tion.21 These individuals are usually homozygousfor the hemoglobin S gene, but such patients canalso carry HgbS along with β thal0, β thal+, andHgbC. SCD affects approximately 80000 peoplein the United States with a prevalence of 1 in 400in African Americans and a carrier rate of 1 in12.22 Patients with SCD live on average 45 yearssecondary to improved management, whichincludes the use of RBC transfusion.23 In a studylooking at adult and pediatric patients with SCD,46% of the children were transfused a mean of 24products, whereas 87% of the adults weretransfused a mean of 24 products over a 10-year

?

period.24 High alloimmunization rates are postu-lated to result from the antigenic disparity betweenAfrican Americans and whites, which is moststrikingly seen in transfused patients with SCD.Alloimmunization often makes finding compatibleRBC products difficult and increases the risk ofdelayed hemolytic transfusion reactions (DHTRs).Without the implementation of extended pheno-type matching in patients with SCD, studiesreported an alloimmunization rate in the range of19% to 43% in transfused patients with SCD.25

One study reported an alloimmunization rate of29% in pediatric and 47% in adult patients withSCD, with more females than males beingimmunized. The number of delayed hemolyticand/or serologic transfusion reactions was 8%(adult) and 9% (pediatric), and the incidence ofhyperhemolysis was 1.6% (adult) and 5.1%(pediatric).24 In contrast, multiple-transfused non-SCD, nonblack, chronic anemia patients witheither thalassemia major or pure red cell aplasiahad an alloimmunization rate of approximately5%.25 In a comprehensive study of the incidenceof, and risk factors associated with, alloimmuniza-tion in patients with SCD, Vichinsky et al25

suggested that the increased alloimmunizationrate in patients with SCD was likely due toantigenic differences between patients with SCD(most of whom were of African descent) and mostblood donors (most of whom were white).26,27 Themost common alloantibodies found are against theK, E, C, and Jkb antigens, which are related to theantigenic prevalence in donors versus patients withSCD (respectively, K 9% vs 2%, E 35% vs 24%, C

124 HILLYER ET AL

68% vs 28%, and Jkb 72% vs 39% are positive).25

Therefore, phenotype-matched RBCs for thehigh likelihood antigens (Rh and K) result indecreased alloimmunization.In addition to RBC antigen mismatch, a

recipient's inflammatory status may play a role inthe risk of alloimmunization.28 This concept hasbeen hypothesized for humans and demonstrated inmice by two separate studies.29,30 In patients withSCD, absolute steady-state neutrophil count hasbeen correlated with clinical severity.31 As acorrelate to this, patients with SCD who respondwell to hydroxyurea have a decrease in theirneutrophil count. Thus, as inflammation appearsto be associated with sickle exacerbation, which istreated with transfusion, patients with SCD may beselectively exposed to RBC alloantigens when in aninflamed state. However, it is currently unknown ifpatients with SCD with higher neutrophil counts areat increased risk of alloimmunization.Providing partially phenotype-matched RBC

products for the treatment of patients with SCDhas been advocated to minimize the likelihood ofRBC alloimmunization.32 One such study showeda decrease in the allomunization rate from 35% to0%, with the exclusive use of phenotype-matchedRBCs (C, c, E, e, K, S, Fya, and Fyb) in a 12.5-year period.33 Afenyi-Annan and Bandarenko32

surveyed hospitals in North Carolina about SCDtransfusion practices and demonstrated that only17% of prophylactically provided antigen-matchedproducts, which is in contrast to most majoracademic medical centers. Among the academiccenters, the protocol for antigen-matching wasinconsistent as to which antigens were matched;73% matched for E, 70% for K, 68% for C, 41%for c, and 41% for e. Therefore, providingphenotype-matched RBC products for SCD isinconstantly practiced yet important to the care ofthese patients.One study has applied genotyping to chronically

transfused patients with SCD. Castilho et al34 founddiscrepancies in 6 of 40 patients between theserologic RBC phenotype and phenotype predictedby the genotype in the patients with SCD but not thecontrol group. The serologic phenotype mistypeswere secondary to recent transfusion. The 6 patientswho then were switched to the correct antigen-matched RBCs had improved RBC survival withdiminished frequency of transfusions. This studyhighlights the use of genotyping patients with SCD

for antigen-matched RBC transfusions and theresulting improved patient care.

β-Thalassemia. Thalassemia is a hereditaryanemia resulting from defects in the β-globinchain, which can lead to a chronic severe anemia.35

The disease is clinically heterogeneous due togenotypically different mutations or compoundheterozygozity with other hemoglobinopathiesand to unknown individual patient factors. Patientswith thalassemia may require life-long RBCtransfusions to ameliorate the chronic anemia andto suppress the extramedullary hematopoiesis,which would otherwise lead to severe bonedeformities. RBC alloimmunization occurs at arate of 5% to 33% depending on the homogeneityof the population.36,37 Alloimmunization rateswere lowered by phenotype-matching for Rh andKell from 33% to 2.8% at Children's HospitalOakland and 23.5% to 3.7% at Aghia SophiaChildren's Hospital.36 In addition, the treatment ofthese patients can be complicated by the presenceof RBC autoantibodies. Because these patients aretransfused from birth, RBC phenotyping should beperformed on the initial pretransfusion sample orgenotyping must be used to adequately predict thepatient's RBC antigen type. Castilho et al38

genotyped 10 alloimmunized β-thalassemiapatients for E, e, K, k, Fya, Fyb, Jka, and Jkb

who had been receiving antigen-matched RBCtransfusions based on phenotype. In 9 of 10patients, there was a discrepancy between thehistorical phenotype and the phenotype predictedby the genotype, 5 in the Rh system, 3 in the Kiddsystem, and 1 in the Duffy system. The discoveryof these discrepancies aided in the identification ofalloantibodies and the selection of the correctantigen-matched products.38 Genotyping of RBCtransfusion-dependant patients is vital to prevent-ing and identifying alloimmunization and provid-ing appropriate antigen-matched products.

Autoimmune hemolytic anemia. During anacute presentation in a patient with newly diag-nosed AIHA, finding the appropriate RBC productfor transfusion can be a challenge, and closecommunication between the transfusion serviceand the treating physician is necessary. Because ofthe presence of a strong autoantibody, the antibodyscreen and identification panels will show panag-glutination, making it difficult to detect or excludeunderlying RBC alloantibodies. Absorption tech-niques using either donor or patient RBCs are

125MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

available at some hospital laboratories, but manymust send samples to reference laboratories forthese specialized tests, which are time consuming.Of patients with warm autoantibodies, 20% to 40%have clinically significant alloantibodies.39 Per-forming the RBC phenotype in these patients ishelpful because it focuses the antibody workup onthe possible alloantibodies the patient is capable offorming. In addition, if a complete phenotype canbe determined, then the transfusion service canprovide antigen-matched RBCs, which may pre-vent future alloimmunization and DHTRs as well ascircumvent the absorption studies. The JohnHopkins Hospital published their approach topatients with warm autoantibodies, which includeda phenotype for C, E, c, e, K, Jka, Jkb, Fya, Fyb, S,and s, if they were able to perform the phenotypeand providing antigen-matched as well as antigen-negative for any identified alloantibodies.39 Pheno-typing may not be possible if the IgG dissociationmethod is unsuccessful, when only antiglobulin-reactive antisera is available, if the antigen isdenatured by the dissociation method, or if thepatient has been recently transfused. During analy-sis of subsequent samples, if the serologic findingswere consistent with previous findings, thenphenotype-matched products were provided. Ofthe 20 patients, 12 could be fully phenotyped and 8patients could be partially phenotyped or phenotyp-ing was indeterminate. The patients receivedbetween 2 and 39 products, and none developednew alloantibodies during the study period of 13months. Garratty and Petz,40 in an accompanyingeditorial, reported 202 alloantibodies to 37 differentantigens detected in the sera of 418 patients withAIHA; 15% of these alloantibodies would not becovered in the Hopkins' protocol, but most of theseuncovered antibodies were to low-prevalence anti-gens and unlikely to cause hemolysis. A secondpotential criticism of this protocol is that theprevalence of phenotypes in these patients rangedfrom 0.0002 to 0.09, which may make it highlyunlikely to find a matched RBC product. Ifgenotyping where available, then prophylacticantigen-matching without future absorption studiescould have been performed on all the patients,simplifying future laboratory pretransfusion testingand improving delivery of appropriate RBCs fortransfusion in a more timely manner.

All patients. A pilot study from the Hong KongRed Cross demonstrated the feasibility of pheno-

typing all citizens, placing the results on a smartcard and providing phenotyped-matched products(for ABO, C, c, E, e, D, K, k, M, N, Fya, Fyb, Jka,and Jkb) without the need for pretransfusiontesting.41 These investigators were able to provide395 RBC products for 92 patients almost entirelyfrom the hospital, which stocks 300 products, andrarely needed products supplied from the HongKong Red Cross donor center, which stocks 4000products. For recipients with uncommon pheno-types, only 0.2%, the probability of finding aphenotype-matched product was 0.451 in thehospital inventory and 0.999 in the Red Crossinventory. The cost of phenotyping was offset byeliminating pretransfusion testing. The benefits ofa phenotype-matched system over the currentantibody screening system are the time saving ofnot having to collect blood samples or test themand the decrease of mismatched transfusionsecondary to the ability to electronically matchthe patient data and the RBC product data. Inconclusion, this practice is feasible and cost-effective if an adequate inventory is maintained inthe hospital and donor center, which is adequatelyrepresentative of the patient population, as well asreliable communication between the donor centerand transfusion service.

Prenatal Testing

RhIg use. Current D typing reagents havedifficulty in determining weak D or partial Dindividuals. Individuals with partial D, and somewith weak D, are at risk for forming anti-D. Thesepatients would benefit from receiving RhIg pro-phylaxis. Molecular testing of RHD, especially inpatients with weak expression of D, would aid inthe determination if RhIg is necessary (Table 3).10

Domen42 surveyed more than 3000 hospitals in theUnited States about their practices to test for weakD and administration of RhIg; 58% perform weak Dtesting and 31.8% had at least 1 patient with theweak D phenotype who formed an anti-D alloanti-body. Testing for weak D in pregnant women isoptional; the usual practice in the United States is toperform weak D test and not give RhIg to patientswho are clearly D-positive or weak D-positive.43

This prevents women with weak D antigens who donot need RhIg from receiving it but may lead towomen with partial D antigens, such as those withDVI, who theoretically need RhIg from notreceiving it. Another argument used for not treating

126 HILLYER ET AL

weak D women as RhIg candidates is that weak Dexpression will cause a false-positive rosette test forfetal maternal hemorrhage, but sending the samplefor Kleihauer-Betke acid elution method cancircumvent this. The addition of molecular testingfor weak D patients would aid in the decision of theuse of RhIg prophylaxis.Hemolytic disease of the fetus and newborn.

The management of a newly identified clinicallysignificant alloantibody in a pregnant womanrelies on serologic testing as well as phenotypingand rarely genotyping. If paternity is assured, thefather is phenotyped to assess for risk ofhemolytic disease of the fetus and newborn(HDFN). If the father's RBCs do not carry theantigen, then no further workup needs to beperformed. If the father is homozygous for thegene expressing the antigen, the fetus is at risk. Ifthe father is heterozygous for the gene expressingthe antigen, then the fetus has a 50% chance ofbeing at risk. Amniocentesis provides samples forfetal genotype (if needed), amniotic fluid spectralanalysis, and fetal lung maturity. Alternatively,fetal DNA can be extracted from the maternalplasma to help determine antigen status of thefetus, but currently, this is not routinely availablein the United States.44

PRACTICAL CONSIDERATIONS

Limitations

Most of the criticisms surrounding RBCantigen prediction by molecular methods havebeen the cost and the uncertainty of regulationsneeded to implement this technology. However,one of the biggest limitations to consider is thatgenotyping may not accurately predict theantigen type. In the donor setting, false-positiveresults for the prediction of the presence of aRBC antigen would eliminate the donor as beingsuitable for a patient requiring an antigen-negative product, but it would not jeopardizethe potential transfusion recipient. For example, aproduct falsely labeled Fy(b+) would have nonegative consequences for the recipient. How-ever, false-negative results could potentially leadto a hemolytic transfusion reaction. Therefore, asystem to confirm the absence of antigens isnecessary. Confirmation by hemagglutinationmethods, when suitable reagents are available,and/or a full crossmath is recommended.

In addition, medical procedures such as stem cellor bone marrow transplantation, certain solid organtransplants, and natural chimerism may lead tomixed DNA populations. In patients with a historyof transplantation, their genotyping results maychange over time in parallel with their chimerismstatus; this would be influenced by which cells wereused as the source of DNA. Therefore, an accuratepatient history should be obtained, and in transplan-tation patients, repeat samples may be necessary.

Another limitation is that not all blood groupantigens are a consequence of SNPs, which resultsin the need for more sophisticated testing metho-dologies or testing algorithms to use the genotypeto predict the phenotype. For example, silencingmutations can be detected in a gene, but the genemay not be expressed, and therefore, the individualwill phenotype as being negative for the antigen.

Cost

With the development of high-throughput sys-tems, the cost of genotyping has dramaticallydecreased. Costs surrounding the BloodGen projecthave been estimated to be one Euro per SNP with atleast 116 SNPs being analyzed. With mass-scaleapplication, the cost may decrease further.4

Direct cost comparisons between genotypingand phenotyping are difficult and will varydepending on the number of antigens and whichantigens to genotype versus phenotype. If noantisera are available for the corresponding anti-gen, then genotyping should be performed. ABO isstraightforward and inexpensive to phenotype yetrelatively complicated to genotype. In contrast, theD antigen is complex and may require an algorithmsuch as phenotype first then genotype only if weakD is identified in the transfusion service orgenotype all D-negative individuals at a donorcenter. For the S antigen, the appropriate antiseramay at times not be available; thus, genotypingcould be used.

A second aspect of cost is determined by theneed for repeat testing. Ideally, genotyping onlyneeds to be performed once. Reasons to repeatgenotyping are to ensure the results are correct andwere entered correctly, but other quality assurancesystems should be in place to prevent these errors.Another important consideration is that thegenotyping information be transferable frominstitution to institution nationally and internation-ally, which would eliminate the need for repeat

127MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

testing of patients and blood donors. Genotypinglikely could be provided in a cost-effective mannerif appropriate algorithms are followed and repeattesting is minimized.

Gold Standard

The blood bank community relies on FDAapproval for RBC typing reagents, which arecurrently used as the gold standard to identify thepresence of an antigen. As polymorphisms arediscovered through molecular technologies, theremust be a continual connection back to the presenceof an immunologic antigen; the RBC genotype mustbe connected to a RBC phenotype.

The gold standard for antibody screening is lessclear, although all methods are based on hemagglu-tination. Throughout the years, multiple platformshave been developed to enhance and ease antibodydetection, yet minimize false-positive reactions,defined as reactions seen but no alloantibodyidentified. The use of genotyped RBC reagentswill aid in choosing RBCs from people who arehomozygous for the gene encoding the requiredantigen and also to an extended phenotype tominimize false-positive reactions, which could besecondary to the presence of low-prevalence anti-gens. It has been proposed to use recombinant celllines expressing a single protein to enhance andsimplify antibody detection and identification.3 Theuse of improved reagents in conjunction withimproved antibody screening platforms couldimprove the sensitivity and specificity of theantibody identification testing.

Who to Genotype

Secondary to cost and resource limitations,genotyping of donors and patients could be rolledout to a selected group first. For example, onlydonors who have demonstrated a commitment tobecome a long-term blood donor are genotyped socost is not wasted on those who do not return andpatients who demonstrate repeated need fortransfusion or form multiple alloantibodies. How-ever, the goal should be once genotyping isestablished in the reference laboratories of donorand transfusion centers, widespread genotypingshould be conducted and applied universally.

Genotype for All Blood Groups

The cost of genotyping increases as the numberof SNPs needed to be tested for increases, but the

incremental cost is low. Of the genotypingplatforms available, they range in the number ofSNPs and blood group antigens tested, but all testfor the clinically significant RBC antigens. Out-side the major clinically significant RBC anti-gens, recipients who lack a high-prevalenceantigen are at high risk of antibody formation,whereas donors who have the presence of low-prevalence antigens may induce an immuneresponse in the recipient. Therefore, genotypingpanels should predict the presence of a low-prevalence antigen or the absence of a high-prevalence antigen, which can be performed bytesting for a single SNP.

Food and Drug Administration Approval

In the United States, the FDA regulates medicaldevices and biologics under the Federal Food,Drug, and Cosmetic Act and Public Health ServiceAct, respectively. Currently, reagents and medicaldevices used in the immunohematology laboratoryare regulated by the Federal Food, Drug, andCosmetic Act and are considered class I and IIdevices depending on control requirements, perfor-mance standards, and additional patient trackingand postmarket information. Additional regulationswill likely be imposed on new genotyping technol-ogies, and the application process is extensive andincludes clinical trials.

On September 25-26, 2006, in Bethesda, MD,a workshop cosponsored by the FDA/Center forBiologics Evaluation and Research, the Depart-ment of Health and Human Services/Office ofPublic Health and Science, and the NationalInstitutes of Health/National Heart, Lung andBlood Institute discussed the current status ofmolecular methods in the field of immunohema-tology and addressed potential questions andconcerns held by the FDA, which is highlightedin the July 2007 supplement of Transfusion.Specific questions included the amount of pre-market clinical trials to evaluate these moleculartechnologies, voluntary or mandatory usage ofgenotyping in only reference laboratories, transfu-sion services or both, and if this technology willbecome the universal standard. The workshopconcluded that genotyping presents a promisingfuture for the transfusion community; however,many questions remain unanswered and thedevelopment of standardization, clinical guide-lines, controls, and proficiency testing needs to be

128 HILLYER ET AL

established before widespread use can beadvocated.45

Quality Assurance

For the purposes of quality assurance, profi-ciency testing should be performed biannually asmandated by the Clinical Laboratory ImprovementAct. Multiple external proficiency testing pro-grams are available for RBC antigen genotypingincluding RHD genotyping from the College ofAmerican Pathologists Molecular GeneticsLaboratory survey, annual ISBT workshopsincluding various patient situations, and anexchange program provided by the Consortiumfor Blood Group Genes. In addition, in-housecomparison of serology to the molecular result canbe performed. Recently, the results from the 2006ISBT workshop were published, in which 6samples that included transfusion-dependentpatients; D, c, and K testing of fetal DNA fromamniotic fluid; and prenatal RHD testing frompregnant D-negative woman plasma for fetal DNAwere distributed to 41 laboratories throughout theworld.46 A high level of accuracy was obtained;however, suggestions for improvement includedformation of defined terminology for reportingblood group alleles and genotypes and a need formore standards and standardization includingcontrols, which are currently unavailable. Thenext ISBT workshop will be held in 2008 with theintent of distributing both relatively simple andmore complex genotypes.

Personal Identification Issues

With the introduction of the Health InsurancePortability and Accountability Act Privacy Rule tothe medical community in the United States in2003, regulations regarding Protected Health Infor-mation have been implemented with the intent ofprotecting an individual's personal and medicalinformation. The use of unique identifiers notlinked to a particular individual is mandatory. Thecreation of a donor or patient database, which canbe accessed by multiple institutions and indivi-duals, leads to questions of patient confidentialityand identification. However, with the use of uniqueidentifiers similar to those in use by the ARDP,anonymity could be maintained. In addition, thereare also security issues and informed consent issuesfor the storage of DNA, especially if it remainslinked to an individual.

Repeat Testing, NewMutations, and RareGenotypes

With the introduction of molecular methods tothe immunohematology laboratory, much hasbeen discovered regarding RBC antigens espe-cially of the identification of additional RBCantigen polymorphisms. As more individuals aregenotyped, new mutations and rare genotypeswill be uncovered. New alleles identified shouldbe added to the blood group antigen genemutation database maintained by the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects/mhc/xslcgi.fcgi?cmd=bgmut/home).3 The ISBT Working Party onTerminology for Red Cell Surface Antigens isaddressing the need for a system for namingblood group alleles.

Need for Irradiation

Currently, phenotype-matched RBC products areused in the treatment of a selected group of patients,such as patients with SCD, without knownoccurrence of transfusion associated-graft versushost disease (TA-GVHD). Because there is a moveto genotype-match more antigens between thedonor and recipient, it is possible that HLAmatching may parallel RBC matching, and thegenetic differences between the donor and recipientwill become less. This would theoretically lead toan increase risk of TA-GVHD, and irradiation maybe necessary. However, there have been nopublished studies investigating the probability ofHLA matching in parallel to RBC matching.

PROPOSED CLINICAL TRIALS

Phenotype-Matched RBCs VersusGenotype-Matched RBCs

A randomized control trial (RCT) should be donein patients with SCD to receive either phenotype-matched or genotype-matched RBCs. Such an RCTwould demonstrate the feasibility of performingthis testing and finding these products as well asdemonstrate the clinical benefit (frequency oftransfusion, alloimmunization rate, transfusionreaction rate, and product delay time).

Donor Genotyping Versus Phenotyping

A clinical trial should be done to investigate theconcordance between genotyping and phenotypingto determine the future need of phenotyping. Thegoal of this trial would be to create the stepwise

Fig 1. Stepwise process for the implementation of molecular testing in the donor center.

129MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

algorithm to ensure that antigen-negative productsare identified correctly. A second goal would be todetermine the need for the repeat genotyping ofdonors. To minimize costs, one time genotypingwould be preferred if methods were in place toensure the donor's identity.

Correlation Between HLA and RBC Antigens

To ensure that there is no increase risk of TA-GVHD, a clinical trial should be done to investigatepairs of unrelated RBC antigen-matched indivi-duals to see if there is a HLA similarity andtherefore a risk of TA-GVHD. This is important to

Fig 2. Stepwise process for the implementation o

perform before implementing genotype-matchingwithout the need for irradiated RBCs.

Genotyping to Eliminate the Need ForPretransfusion Testing

Small clinical trials have demonstrated thatphenotype matching can prevent alloimmunizationand therefore the need for antibody screening. Alarger-scale trial is needed to evaluate the risksand benefits of eliminating or minimizing pre-transfusion testing. The trial should followalloimmunization rates after genotype-matchedtransfusions, product delays for locating matched

f molecular testing in the transfusion service.

Fig 3. Donor and patient database development.

130 HILLYER ET AL

RBCs, transfusion reaction rates in comparison todecrease patient blood draws, product delays fortesting, and cost.

RhIg and D-negative RBCs Administration

A clinical trial should be done with one group ofpatients following an algorithm for molecular RHDtesting and appropriate transfusion practices basedupon such results versus current standard of care.The outcome would be to look at anti-D formationrates in both groups.

IMPLEMENTATION OF MOLECULAR TESTING

In the Donor Center

Mass-scale genotyped blood donors could beentered in a shared database, a larger- and wider-scale version of the database currently in use bythe ARDP. This would help the donor center finda particular product to distribute. (I) Repeat groupO donors would be genotyped, (II) D-negativedonors to ensure they are indeed D-negative, (III)all repeat donors, and (IV) eventually all donors(Fig 1). The database would allow recruitment ofindividuals whose blood type is needed ascompared with general recruitment methodsmostly used currently.

In the Transfusion Service

Molecular technologies have already been imple-mented in many hospital laboratories, for example,for the identification of human immunodeficiencyvirus, cytomegalovirus, hepatitis C, and chlamydia,and for HLA testing, and now should be introducedin a stepwise fashion in the transfusion service(Fig 2). The first phase of molecular testing in atransfusion service would be to create a detailedpatient database with antibody and molecularantigen typing information. The initial populationto test is patients with SCD who are alreadyphenotyped and are receiving phenotype-matchedRBCs. This would bring this process to a molecularlevel to produce improved RBC matching. Thesecond population for whom to add moleculartyping is other chronically transfused individualsand patients with warm autoantibodies. The thirdphase would be genotyping of RHD in patients withweak D phenotyping to aid in the determination ofRhIg use and D-negative RBCs for transfusion. Thefourth phase would be the genotyping of all patients.Ideally, these databases could be shared betweeninstitutions, or patients would receive this informa-

tion as a part of their Health Information System,which could be easily accessed when visitinganother institution.

Database With Patient and Donor Information

Patients or donors move from one hospital orcenter to the next; this occurs particularly in manyurban areas in the United States. This leads to repeattesting, and patient care can be delayed, or worse,incorrect products issued. An ideal database wouldhave individual data present such that it couldbe accessed by both donor centers and hospitals(Fig 3). There would be a continuous cycle betweendonors and patients as donors become patients andpatients become donors. On the transfusion serviceside, knowing the patient's blood type and pre-viously identified antibodies streamlines antibodyidentification workup. The combined databasewould aid in matching/pairing donors to patientsand limit donor exposure. It may also increase donorrecruitment because one reason that potentialdonors do not donate is that the process is not seenas volunteering and is too impersonal.47 Thisprocess of pairing a donor with a patient allows

131MOLECULAR TECHNIQUES IN TRANSFUSION MEDICINE

for personalization of the donation process. Themerging of donor and patient databases wouldimprove the care of patients plus give an additionalinspiration for individuals to donate.

If extended genotype-matched products can beensured for every transfusion, then the use ofantibody screening would be minimal. It isunlikely that all antigens can be matched, but itis possible that the common clinically significantantigens (D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, S,and s) could be matched for patients needing RBCtransfusions, leaving only the low-prevalenceantigens at risk to cause alloimmunization. Inaddition, patients should be identified for the lackof a high-prevalence antigen and, therefore, athigh risk of forming the corresponding alloanti-body; the logistics and likelihood of finding anappropriate antigen-matched product may makethis approach difficult.

CONCLUSIONS

The transfusion community has moved to anexciting time where new molecular technologies

are emerging and being implemented. With thesetools, patients can receive RBC antigen-matchedproducts, which will decrease the number ofDHTRs and decrease the time needed for antibodyidentification and finding the appropriate product.The application of molecular technologies willimpact all aspects of transfusion medicine. Donorcenters daily face problems due to the inability tofind the proper antigen-negative products or toappropriately label a D-negative product, and thetransfusion services struggle to identify alloantibo-dies, interpret ABO types in the face of an ABOtyping discrepancy, correctly identify patients atrisk for anti-D formation, and provide antigen-matched products to chronically transfusedpatients. There is a step-by-step process thatdonor centers and transfusion services must taketo reach the goal of providing genotype-matchedproducts to all and minimizing pretransfusiontesting. Molecular testing has entered this field,and the transfusion medicine specialist must findthe ideal and cost-effective way to use thispowerful tool.

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