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13 The Rh System

13

The Rh system is com-plex, and certain aspects of its genetics,nomenclature, and antigenic interac-tions are not fully understood. Thischapter avoids exhaustive theoreticalconsiderations, and instead concen-trates on commonly encountered obser-vations, problems, and solutions.

The D Antigen and ItsHistorical ContextRh Positive and Rh NegativeThe unmodified descriptive terms Rhpositive and Rh negative refer to thepresence or absence of the red cell anti-gen D. An earlier name for D, Rho, is nolonger used and remains of only histori-cal interest. This chapter uses the CDEnomenclature originally proposed byFisher and Race,1 which has been able toaccommodate our present under-standing of the genetics and biochemis-try of this complex system. The Rh-Hrterminology of Wiener is presented only

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in its historical context, as moleculargenetic evidence to date does not sup-port Wiener’s one-locus theory.

Discovery of DThe first human example of the antibodyagainst the D antigen was reported in1939 by Levine and Stetson,2 who foundit in the serum of a woman whose fetushad hemolytic disease of the newborn(HDN) and who experienced a hemolyticreaction after transfusion of her hus-band’s blood. In 1940, Landsteiner andWiener3 described an antibody obtainedby immunizing guinea pigs and rabbitswith the red cells of Rhesus monkeys; itagglutinated the red cells of approxi-mately 85% of humans tested, and theycalled the corresponding determinantthe Rh factor. In the same year, Levineand Katzin4 found similar antibodies inthe serum of several recently deliveredwomen, and at least one of these seragave reactions that paralleled those ofthe animal anti-Rhesus sera. Also in1940, Wiener and Peters5 observed anti-bodies of the same specificity in the se-

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256 AABB Technical Manual

rum of persons whose red cells lackedthe determinant, who had received ABO-compatible transfusions in the past.Later evidence established that the anti-gen detected by animal anti-Rhesus andhuman anti-D were not identical, but bythat time the Rh blood group system hadalready received its name.

Clinical SignificanceOther than the A and B antigens, D is themost important red cell antigen in trans-fusion practice. In contrast to A and B,however, persons whose red cells lack theD antigen do not regularly have the corre-sponding antibody. Formation of anti-Dalmost always results from exposure,through transfusion or pregnancy, to redcells possessing the D antigen. A high pro-portion of D-negative persons who receiveD-positive blood do produce anti-D.

The D antigen has greater immuno-genicity than virtually all other red cellantigens; more than 80% of D-negativepersons who receive a D-positive trans-fusion are expected to develop anti-D. Toprevent this, the blood of all recipientsand all donors is routinely tested for Dto ensure that D-negative recipients areidentified and given D-negative blood.

Soon after anti-D was discovered,family studies showed that the D antigenis genetically determined; transmissionof the trait follows an autosomal domi-nant pattern. With only a few interestingexceptions, persons who have the genefor D will have D antigen detectable ontheir red cells.

Other Important AntigensBy the mid-1940s, four additional anti-gens, C, E, c, and e, had been recognizedas belonging to what is now called the Rhsystem. Subsequent discoveries havebrought the number of Rh-related anti-gens to over 50 (Table 13-1), many ofwhich exhibit both qualitative and quan-


titative variations. The reader should beaware that these exist, but in most trans-fusion medicine settings, the five princi-pal antigens (D, C, E, c, e) and theircorresponding antibodies account formore than 99% of clinical issues involv-ing the Rh system.

Genetic and BiochemicalConsiderationsAttempts to explain the genetic controlof Rh antigen expressions have beenfraught with controversy. Wiener7 pro-posed a single locus with multiple allelesdetermining surface molecules that em-body numerous antigens. Fisher andRace8 inferred from the existence of an-tithetical antigens the existence of recip-rocal alleles at three individual butclosely linked loci. Tippett’s prediction9

that two closely linked structural loci onchromosome 1 determine production ofRh antigens is presently considered to becorrect.

Rh GenesTwo highly homologous genes on theshort arm of chromosome 1 encode thenonglycosylated polypeptides that ex-press Rh antigenic activity.10,11 One, des-ignated RHD, determines the presenceof a membrane-spanning protein thatconfers D activity on the red cell. D-posi-tive individuals possess one or two exam-ples of this gene. D-negative personshave no genetic material at this site; theabsence of an antithetical allele explainswhy, after decades of searching, serolo-gists have never found a d antigen.

At the other, adjacent locus, the geneRHCE determines the C, c, E, and e an-tigens; its alleles are RHCe, RHCE,RHcE, and RHce.12 Much effort has goneinto determining if different proteins,resulting from alternative splicing of

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Table 13-1. Equivalent Notations in the Rh Blood Group SystemNumericalDesignation CDE Rh-Hr Other

NumericalDesignation CDE Rh-Hr Other

Rh1 D Rho Rh29 total RhRh2 C rh′ Rh30 DCor Goa

Rh3 E rh″ Rh31 hrB

Rh4 c hr′ Rh32* TrollRh5 e hr″ Rh33Rh6 ce,(f) hr Rh34 HrB BastiaanRh7 Ce rhi Rh35 1114Rh8 Cw rhw1 Rh36 Bea

Rh9 Cx rhx Rh37 Evans†

Rh10 ces hrv V Rh39 “C-like”Rh11 Ew rhw2 Rh40 Targett (Tar)Rh12 G rhG Rh41 “Ce-like”Rh17 Hro Rh42 Ces rhs ThorntonRh18 Hr Rh43 CrawfordRh19 hrs Rh44 NouRh20 es VS Rh45 RivRh21 CG Rh46 SecRh22 CE rhy Jarvis Rh47 DavRh23 Dw Wiel Rh48 JALRh26 “c-like” Deal Rh49 STEMRh27 cE rh Rh50 FPTTRh28 hrH Hernandez Rh51 MARThe table is compiled from the findings of the ISBT Working Party on Terminology for Red CellSurface Antigens6

*Rh32 is a low-incidence antigen that is a product of the predominately Black gene RN, theother products of which include a reduced expression of C and e.†Rh37 is the low-incidence antigen Evans, which occurs in association with the · D ·haplotype. This is similar to –D–, except for the presence of the Evans antigen and a lesserexaltation of D activity.

Chapter 13: The Rh System 257

mRNA, carry C/c and E/e or if a singlepolypeptide expresses both sites.12-14 Re-cent evidence derived from transfectionstudies15 suggest that both C/c and E/ereside on a single polypeptide product.

Biochemical and StructuralObservationsThe products of both RHD and RHCE areproteins of 416 amino acids that, model-


ing studies suggest, traverse the red cellmembrane 12 times and display onlyshort exofacial loops of amino acids onthe exterior. The polypeptides are fatty-acid acylated and, unlike most bloodgroup-associated proteins, carry no car-bohydrate residues. Within the red cellmembrane, the Rh polypeptides complexwith glycoproteins that have partial ho-mology with the Rh polypeptides but areencoded at a locus on chromosome 6.16



Considerable homology exists be-tween the products of RHD and RHCE;the products of the different alleles ofRHCE are even more similar.17 C and cdiffer from one another in only fouramino acids, at positions 16, 60, 68, and103, of which only the difference be-tween serine and proline at 103 appearsto be critical. The presence of proline oralanine at position 226 appears to be thesole characteristic that distinguishes Efrom e. The D polypeptide, by contrast,possesses 36 amino acids that will beperceived as foreign by D-negative indi-viduals.

The study of Rhnull red cells, whichentirely lack Rh antigens, reveals thatthe Rh proteins are part of a large mem-brane complex, in which the presence ofthe Rh proteins appears to be essentialfor correct expression or presentation ofother constituents. The glycoproteinsthat bear the LW, Duffy, and U antigensall seem to require the presence of Rhproteins for full expression. Rhnull cellslack all the LW antigens, are negative forFy5 of the Duffy system, and have weak-ened expression of the antigens carriedon glycophorin B (S, s, and U).18

Rh TerminologyThree systems of nomenclature were de-veloped before the recent advances inour understanding of the genetics of Rh.Each of these systems of nomenclaturehave been used to convey genetic andserologic information about the Rh sys-tem.

System NotationsThe Rh-Hr terminology derives from thework of Wiener,7 who believed the imme-diate gene product to be a single entityhe called an agglutinogen. Wiener’s con-cept was that each agglutinogen is char-acterized by numerous individual sero-


logic specificities, called factors, identi-fied by individual specific antibodies.Current biochemical and serologic datado not support this theory.

CDE terminology was introduced byBritish workers, Fisher and Race,1 whopostulated three sets of closely linkedgenes (C and c, D and d, and E and e).Both gene and gene product have thesame letter designation, with italics usedfor the name of the gene. Although thistheory does not fully explain some of theobserved Rh antigenic profiles, it does,however, provide the easiest way to com-municate research and serologic find-ings at present.

Rosenfield and coworkers19 proposed asystem of nomenclature based simply onserologic observations. Symbols were notintended to convey genetic information,merely to facilitate communication ofphenotypic data. Each antigen is given anumber, generally in order of its discoveryor its assignment to the Rh system. Thepresence of an antigen on a red cell speci-men is indicated by the appropriatenumber placed after the system designa-tion, Rh followed by a colon; a minus signbefore a number indicates that the antigenhas been tested for and found to be absent.This system is cumbersome for verbalcommunication, but well-suited for writ-ten communication without genetic infer-ence, and for computerized data entry. Ta-ble 13-1 lists the antigens currentlyincluded in the Rh system. Table 13-2shows the most common combinations ofantigens, expressed as haplotypes. Table13-3 shows reaction patterns achieved bytesting cells with antibodies to the fiveprincipal antigens, and the descriptiveterms used for phenotypes in three sys-tems of nomenclature.

Phenotypic and Genetic NotationsFor informal designation of phenotype,particularly in conversation, many work-ers use a shorthand system based on Wie-



ner’s Rh-Hr notation. This does not fit intoany system of nomenclature, but theseshorthand symbols convey information ina convenient and efficient fashion.

Table 13-2. The Principal RhGenes (or Gene Complexes)

Fisher-Race Terminology

HaplotypeGene

CombinationAntigenic

Specificities

R1 CDe C,D,er ce c,eR2 cDE c,D,ERo cDe c,D,er′ Ce C,er″ cE c,ERz CDE C,D,Ery CE C,E

Table 13-3. Determination of Some RhTests with the Five Principal Rh Blood

Reagent

Anti-D Anti-C Anti-E Anti-c Anti

+ + 0 + ++ + 0 0 ++ + + + ++ 0 0 + ++ 0 + + ++ 0 + + 0+ + + 0 ++ + + + 0+ + + 0 00 0 0 + +0 + 0 + +0 0 + + +0 + + + +

*Shorthand terminology


Haplotype NotationsThe phenotype notations convey haplo-types with the single letters R and r, inroman type, for the haplotypes that pro-duce or do not produce D, respectively.Subscripts or, occasionally, superscripts,indicate the combinations of other anti-gens present. For example, R1 indicates C,D, and e together; R2 indicates c, D, and E;r indicates c and e; Ro indicates c, D, ande; and so on. Phenotypes appearing to em-body homozygous expression of a singlehaplotype have only one letter; othershave two.

Determining PhenotypeIn clinical practice, five blood typing re-agents are readily available: anti-D, -C,-E, -c, and -e. Routine pretransfusionstudies include only tests for D. Otherreagents are used principally in the reso-

Phenotypes from the Results ofTyping Reagents

Phenotypes in Three Nomenclatures

-e Rh-Hr* CDE Numerical

R1r CcDe Rh:1,2,–3,4,5R1 CDe Rh:1,2,–3, –4,5

R1R2 CcDEe Rh:1,2,3,4,5R0 cDe Rh:1,–2,–3,4,5R2r cDEe Rh:1,–2,3,4,5R2 cDE Rh:1,–2,3,4,–5

RzR1 CDEe Rh:1,2,3,–4,5RzR2 CcDE Rh:1,2,3,4,–5Rz CDE Rh:1,2,3,–4,–5r ce Rh:–1,–2,–3,4,5

r′r Cce Rh:–1,2,–3,4,5r″r cEe Rh:–1,–2,3,4,5r′r″ CcEe Rh:–1,2,3,4,5



lution of antibody problems or in familystudies. The assortment of antigens de-tected on a person’s red cells constitutesthat person’s Rh phenotype.

Inferring GenotypeIdentifying antigens does not always al-low confident deduction of genotype.Presumptions regarding the most prob-able genotype rest on the frequencieswith which particular antigenic combi-nations derive from an individualgenome complex. For simplicity, the re-mainder of this chapter uses CDE termi-nology to express haplotypes, eg, CDErather than RHD, RHCe.

Inferences about genotype are usefulin population studies and in the investi-gation of disputed parentage. Suchanalyses are also used to predict whetherthe sexual partner of a woman with Rhantibodies is likely to transmit the genesthat will result in offspring negative orpositive for the particular antigen.

Serologic Testing for RhAntigen ExpressionTo determine whether a person has genesthat encode C, c, E, and e, the red cells aretested with antibody to each of these anti-gens. If the red cells express both C and cor both E and e, it can be assumed that thecorresponding genes are present in theindividual. If the red cells carry only C orc, or only E or e, the person is assumed tobe homozygous for the particular allele.Titration studies can sometimes docu-ment this assumption because the amountor dose of antigen on the red cells fromhomozygotes often is greater than whenthe genome includes only a single copy.Tests for the D antigen indicate only itspresence or absence; titration results todemonstrate dosage have not given reli-able information.

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Expression of D

D-negative persons either lack RHD,which encodes for the D antigen, or,much more rarely, have a nonfunctionalD gene. Most D-negative persons are ho-mozygous for RHce, the gene encodingc and e; less often they may have RHCeor RHcE, which encode C and e or c andE, respectively. The RHCE gene that pro-duces both C and E is quite rare.

The genotype of a D-positive personcannot be determined serologically; dos-age studies are not effective in showingwhether an individual is homozygous orheterozygous for RHD. An individual’s Dgenotype can be assigned only by infer-ence from the antigens associated withthe presence of D. Recent techniques forcloning the Rh genes may eventually al-low highly accurate determination of Dgenotype.

Interaction between genes results inso-called “position effect.” If the interac-tion is between genes, or the product ofgenes, on the same chromosome it iscalled a cis effect. If a gene or its productinteracts with one on the opposite chro-mosome, it is called a trans effect. Exam-ples of both effects were first reported in1950 by Lawler and Race,20 who noted asa cis effect that the E antigen producedby cDE is quantitatively weaker than Eproduced by cE. They noted as trans ef-fects that both C and E are weaker whenthey result from the genotype CDe/cDEthan when the genotypes are CDe/ce orcDE/ce, respectively.

Effect of Race

A person whose red cells are of the phe-notype CDe most likely has the genotypeCDe/CDe, and will transmit a gene en-coding D to all offspring. A less likelyalternative genotype would be CDe/Ce.Racial origin influences deductionsabout genotype because the frequenciesof Rh genes differ by race. For example,

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a White person with the phenotype cDewould probably be cDe/ce, but a Blackperson of the same phenotype, cDe/cDeand cDe/ce are almost equally likely.

Effect of Gene FrequencyThe phenotype CcDEe (line 3 of Table13-3) can arise from any of several geno-types. In any population, the most prob-able genotype is CDe/cDE. Both thesehaplotypes encode D; a person with thisphenotype will very likely be homozy-gous for the D gene, although heterozy-gous for the actual combination of genespresent on the two chromosomes. Someless likely alternative genotypes couldrender the person heterozygous at the D

Table 13-4. Frequencies of the More Comm

Phenotype GenotypeGenotype

Frequency (

CDE CDE Rh-hr Whites Bla

CDe/ce R1r 31.1 8CcDe CDe/cDe R1R0 3.4 15

Ce/cDe r′R0 0.2 1

Cde CDe/CDe R1R1 17.6 2CDe/Cde R1r′ 1.7 0

cDEe cDE/ce R2r 10.4 5cDE/cDe R2R0 1.1 9

cDE cDE/cDE R2R2 2.0 1cDE/ce R2r″ 0.3 <0

CDe/cDE R1R2 11.8 3CcDEe CDe/cE R1r″ 0.8 <0

Ce/cDE r′R2 0.6 0

cDe cDe/ce R0r 3.0 22cDe/cDe R0R0 0.2 19

For the rare phenotypes and genotypes not showlisted at the end of this chapter.

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locus, for example, CDe/cE, cDE/Ce, orCDE/ce, but these are uncommon in allpopulations. An even less likely possibil-ity is cDe/CE. Table 13-4 gives the fre-quencies of the more common geno-types in D-positive persons. The figuresgiven are for Whites and Blacks. In otherracial groups, the likelihood of beingheterozygous for D is reduced becauseabsence of RHD is so uncommon.

Weak Expression of DDifferent D-positive red cell specimensmay have differing reactivity with anti-Dreagents. Most D-positive red cells show

on Genotypes in D-Positive Individuals

%) Likelihood of Zygosity for D (%)

White Blacks

cks Homo- Hetero- Homo- Hetero-

.8

.0 10 90 59 41

.8

.9 91 9 81 19

.7

.7 10 90 63 37

.7

.2 87 13 99 1

.1

.7

.1 89 11 90 10

.4

.9 6 94 46 54

.4

n in this table consult the reference books

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clear-cut macroscopic agglutination af-ter centrifugation with reagent serumand can readily be classified as D-posi-tive. Red cells that are not immediatelyor directly agglutinated cannot as easilybe classified. For some D-positive redcells, demonstration of the D antigenrequires prolonged incubation with theanti-D reagent or addition of antiglobu-lin serum after incubation with anti-D.These cells are considered D-positive,even if there has been need for an addi-tional step in testing.

In the past, red cells that requiredadditional steps for demonstration of Dwere classified as Du. The term Du is nolonger used; red cells that carry weakforms of D are classified as D-positive,and may be described as “weak D.” Mono-clonal anti-D reagents may cause directagglutination of some D-positive cellsthat would have been considered weak Dafter use of polyclonal reagents. Addi-tionally, they may detect hitherto unrec-ognized epitopes of D or, occasionally,fail to react with variant configurationsof the D antigen.

Weak D Due to Transmissible Genes

Weak D phenotypes can result from sev-eral different genetic circumstances.Some examples of RHD appear to encodeweak expression of the D antigen. Thisquantitative characteristic follows aregular pattern of Mendelian dominantinheritance. Weak D expression of thistype is fairly common in Blacks, oftenoccurring as part of a cDe haplotype.Transmissible genes for weak D expres-sion are considerably less common inWhites, but may be seen as part of anunusual CDe or cDE haplotype.

On testing with most anti-D reagents,most red cell samples with weak D anti-gens due to a transmissible gene eitherfail to react or react very weakly in directagglutination tests, but react strongly


when antiglobulin serum is added to thesystem.

Weak D as Position EffectPerhaps the best-known example of po-sition effect is weakening of the D anti-gen by C gene trans to the D gene. Redcells from some persons of the genotypeCDe/Ce have weakened expression of D,a suppressive effect exerted by C in thetrans position to an unremarkable D onthe opposite chromosome. Similar de-pression of D can be seen with otherD-positive haplotypes accompanied byCe. For example, the CDe/ce and CDe/Cegenotypes encode for the same antigens,but D expression is often perceptiblyweaker on cells from the latter individ-ual. Many weak D phenotypes reportedin the early literature would, with cur-rently available reagents, appear as nor-mal D. Weak D antigens that have quali-tative differences will be discussed laterin this chapter.

Significance of Weak D in Blood DonorsTransfusion to D-negative recipients ofblood with weak expression of the D an-tigen has long been proscribed, based onthe possibility that such red cells couldelicit an immune response to D. Thispossibility may be more apparent thanreal, as weak forms of the D antigen seemto be substantially less immunogenicthan normal D-positive blood. Transfu-sion of a total of 68 units of blood withweak D to 45 D-negative recipients failedto stimulate production of a single ex-ample of anti-D.21 Although 15 of the 45recipients were receiving the level of im-munosuppression available in the late1950s, one person in the series madeanti-E and a second made anti-K. A re-port22 of anti-CD produced in responseto CDue red cells can be better explainedas anti-G than as anti-C and an anti-Delicited by the cells described as Du.



More important than the potentialimmunogenicity of red cells with weak-ened expression of D is the possibilityof such cells experiencing accelerateddestruction if transfused to a recipientwith circulating anti-D. Hemolytictransfusion reactions were reported inthe early literature, but it is probablethat the responsible cells would, withcurrently available reagents, have beenconsidered straightforwardly D-posi-tive. Hemolytic disease of the newbornhas also been reported, but not re-cently.23

Significance of Weak D in Recipients

The transfusion recipient with weak Dis sometimes a topic of debate. Mostsuch patients can receive D-positiveblood without risk of immunization,but if the weak D expression reflectslack of one or more D epitopes, thepossibility exists that transfusion of D-positive blood could elicit alloanti-D.The same possibility exists, however,for epitope-deficient persons whosered cells react strongly with anti-D re-agents . AABB Standards for BloodBanks and Transfusion Services24 re-quires donor blood specimens to betested for weak expression of D and belabeled as D-positive if the test is posi-tive, but recipients’ specimens neednot be tested with anti-D by any proce-dure other than direct agglutination.Currently available, licensed anti-D re-agents are sufficiently potent that mostpatients with weak D are found to beD-positive. The few patients classifiedas D-negative, whose D-positive statuswould have been detected by antiglobu-lin testing, can receive D-negativeblood without problems. Some workersconsider this practice wasteful of D-negative blood and prefer to test poten-tial recipients for weak D, and thenissue D-positive blood.


If D-positive blood is given to recipi-ents of the weak-D phenotype, it is im-portant to safeguard against careless orincorrect interpretation of tests. D-negative recipients erroneously classi-fied as D-positive, possibly because of apositive direct antiglobulin test (DAT),run the risk of immunization to D ifgiven D-positive blood. Individualswhose weakly expressed D antigen is de-tectable only in the antiglobulin test willordinarily be classified as D-negative re-cipients. If they give blood, however,they will be classified as D-positive at thetime of blood donation. Personnel inblood centers and transfusion servicesshould be prepared to answer questionsfrom puzzled donors or their physicians.This can present special problems inautologous donations, when the D-nega-tive patient’s own blood is labeled asD-positive. In this case, confirmation ofthe patient’s D status by antiglobulintesting resolves the apparent discrep-ancy between recipient and donor types.

Partial DThe concept that the D antigen consistsof multiple, individually determinedconstituents arose from observationsthat some people with D-positive redcells produced alloanti-D that was non-reactive with their own cells. MostD-positive persons who produce al-loanti-D have red cel ls that reactstrongly when tested with anti-D, al-though some have test results that sug-gest weak D. Red cells lacking parts ofthe D antigen complex have been re-ferred to in the past as “D mosaic” or “Dvariant.” Current terminology indicatessuch red cells, deficient in componentsof D, are more appropriately described asexpressing “partial D.”

Tests of various monoclonal anti-Dreagents with red cells of various D cate-gories confirm that the D antigen com-prises many epitopes. Numbered D cate-



gories and other partial-D phenotypescan now be defined in terms of their Depitopes. Lomas, McColl, and Tippett25

established nine D epitopes, expandingon the seven epitopes demonstrated in1989.26 Complete exposition of the D an-tigen, its sum and its parts, has not yetbeen achieved.

Quantitative vs Qualitative DifferencesRed cells lacking part of the D antigenmay appear phenotypically similar tothose that reflect inheritance of a genefor weak D expression unless tested withselected anti-D. The D antigen of theepitope-deficient types is qualitativelydifferent from normal; red cells of a per-son with a gene that encodes weak ex-pression of D simply have fewer exam-ples of the normal D polypeptide. Thedistinction can seldom be made se-rologically, unless the epitope-deficientperson makes alloanti-D. Red cells ofmany partial-D phenotypes, althoughepitope-deficient, react as strongly withmost anti-D reagents as do normal cells.In such cases, the qualitative abnormal-ity becomes apparent only if the personmakes alloanti-D. Such alloanti-D is in-distinguishable from the anti-D made byD-negative individuals except that it isnonreactive with D-positive red cellsthat lack the same epitope.

Spurious Alloanti-DNot all persons who are D-positive andproduce what appear to be anti-D shouldbe assumed to have epitope-deficient redcells. Weakly reactive anti-LWab or anti-LWa may react with D-positive cells butnot with D-negative cells. A D-positiveperson whose antibody is a weakly reac-tive anti-LWa may be indistinguishableon initial serologic testing from an indi-vidual with a partial-D antigen who hasmade anti-D to missing epitopes. (Seesection on LW in Chapter 14.) Anti-LW


can easily be differentiated from anti-Din tests with sulfhydryl-treated red cells;the LW antigen is destroyed by sulfhy-dryl reagents and D is not.

Other Rh AntigensNumbers up to 51 have been assigned tored cell antigens (see Table 13-1); someof the numbers have been rescinded orthe antigens reassigned, but of the cur-rently included 44, most beyond D, C, c,E, and e (Rh:1-5) are rarely encounteredin routine blood transfusion therapy.

Cis Product AntigensThe membrane components that displayRh activity have numerous possible an-tigenic subdivisions. Each gene or genecomplex determines a series of interre-lated surface structures, of which someportions are more likely than others toelicit an immune response. The polypep-tides determined by the genes in thehaplotype CDe express determinants ad-ditional to those defined as D, C, and e.These include Ce, a cis product that al-most always accompanies C and e whenthey are encoded by the same haplotype.The Ce antigen is absent from red cellson which the C and e were encoded bydifferent haplotypes, for example, in aperson of the genotype CDE/ce. Similarcis product antigens exist for c and edetermined by the same haplotype (theantigen called ce or f), for c and E (cE),and for C and E (CE).

Although antibodies directed at cisproduct antigens are encountered infre-quently, it would not be correct to con-sider them rare. Such antibodies may bepresent, unnoticed in serum containingantibodies of the more obvious Rh speci-ficities; only adsorption with red cells ofselected phenotypes would demonstratetheir presence. Anti-f (ce) may be pre-sent, for example, as a component of



some anti-c and anti-e sera, but its pres-ence would have little practical signifi-cance. The additional antibody shouldnot confuse the reaction patterns givenby anti-c and anti-e, since virtually anycell that reacts with anti-f would expressboth c and e.

Anti-Ce is frequently the true speci-ficity of the apparent anti-C that acDE/cDE person produces after receiv-ing D-positive blood. This knowledgecan be helpful in establishing an individ-ual’s Rh genotype. If anti-Ce is the pre-dominant specificity in a reagent anti-C,however, the individual whose C antigenresulted from a CDE haplotype may bemistyped unless test methods and con-trol red cells are chosen carefully.22

DeletionsRare genes exist that either fail to encodeRh material or encode Rh material lack-ing activity at the CcEe sites. Some por-tions of the surface configuration are notdetectable, leaving D (or C/c and D) asthe only remaining site(s). Red cells thatlack C/c and/or E/e antigens may showexceptionally strong D activity, an obser-vation by which such red cells havesometimes been recognized during rou-tine testing with anti-D. The –D– pheno-type may be identified in the course ofstudies to investigate an unexpected an-tibody. Such persons may have alloanti-body of complex specificity, because theperson’s red cells lack all the epitopesexpressed on the CcEe polypeptide. Asingle antibody with anti-Hro (anti-Rh17) specificity is often made by per-sons of this rare phenotype, althoughsome such sera have been reported tocontain apparently separable specifici-ties, such as anti-e.

The •D• phenotype is similar in mostrespects to –D–, except that the D anti-gen is not exalted to the same degree. •D•

red cells may be agglutinated weakly bysome examples of serum from immu-


nized Rh-deletion persons if the serumcontains anti-Rh47 in addition to anti-Rh17.23 The •D• gene encodes Rh47. Adistinguishing characteristic of •D• redcells is that they possess a low-incidenceantigen known as Rh37 (Evans).

The Antigen G and Cross-ReactionsThe G antigen is almost invariably pres-ent on red cells possessing either C or D.Antibodies against G appear superficiallyto be anti-C+D, but the anti-G activitycannot be separated into anti-C and anti-D. The fact that G appears to exist as anentity common to C and D explains thefact that D-negative persons immunizedby C–D+ red cells sometimes appear tohave made anti-C as well as anti-D. Itmay also explain why D-negative personswho are exposed to C+D– red cells maydevelop antibodies appearing to containan anti-D component. Rare red cellshave been described that possess G butlack D altogether and show greatly di-minished, altered, or absent expressionof the C antigen C(– –eG). The D–G+phenotype occurs in Blacks but evi-dently is not quite the same as when itoccurs in Whites. Red cells also existthat express at least a part of D but lackG entirely.23

Variant AntigensThere must be innumerable subtle dif-ferences in composition among variousRh gene products. Although red cellsfrom most people give straightforwardreactions with common antibodies,some cells give atypical reactions andothers stimulate the production of anti-bodies that do not react with red cells ofcommon Rh phenotypes. It has beenconvenient to consider C and c, and Eand e, as antithetical antigens at specificsurface sites. This scheme can be ex-panded to include variant antigens thatseem to occupy the same surface site but



have been determined by genes, possiblyconversion or misalignment products,that encode proteins that differ from thecommon Rh determinants. For example,several variant forms of the e antigenhave been identified, described as hrS orhrB (Rh19 and Rh31). Most people havered cells with all epitopes of the Rh anti-gens, but some lack some of the epitopesof certain Rh antigens, such as D, C, V,VS, Hr, and Hro.

Red cells lacking one or both of theepitopes of e are sometimes found inBlacks. Diminished C and markedly di-minished e activity characterize theproducts of the RN gene complex, whichalso encodes a low-incidence antigen,Rh32. Genes that encode weakly ex-pressed antigens may be designated byparentheses, ie, (C)D(e). Among Whites,a weakened e antigen is among the prod-ucts of genes that encode the low-inci-dence Rh33 and the Rh36 (Berrens, Bea)antigens. Antigens that behave as if theyhad an antithetical relationship to C/c orE/e have been found, mainly in Whites.The most common is Cw, found in 2% ormore of some White populations. Table13-1 lists the antigens that belong to theRh system and contribute to the advanc-ing complexity of the system and its no-menclature.

Rhnull Syndrome and Rhmod

Genetic DeterminationThe literature reports at least 43 personsin 14 families whose red cells appear tohave no Rh antigens; others are knownbut have not been reported. The pheno-type, described as Rhnull, may be pro-duced by at least two different geneticmechanisms. In the more common regu-lator type of Rhnull, the absence of a verycommon regulator gene , X1r, wasthought to prevent expression of the per-son’s perfectly normal genes at the Rh


locus on chromosome 1. Such personsappear to transmit normal Rh genes totheir offspring, in a manner roughlyanalogous to that in which the A or Btransferases are transmitted by people ofthe Bombay phenotype. These Rhnull per-sons were considered to be homozygousfor Xor, a rare allele of X1r, that segre-gates independently of genes of the Rhsystem. In some cases, parents or off-spring of people with the regulator typeof Rhnull show overall depression of theirRh antigens.

The other form of Rhnull was thoughtto arise through homozygosity for anamorphic gene at the Rh locus itself, r,which appears to have no products de-tectable with Rh testing reagents. Re-cent studies, however, suggest Rhnull

gene complexes could direct low levelsof Rh polypeptides to be produced.27 Thistype of Rhnull phenotype is considerablyrarer than the regulator type. Parentsand offspring of this type of Rhnull areobligate heterozygotes for the amorph.

Red Blood Cell AbnormalitiesWhatever the genetic origin, red cellslacking Rh antigens have membrane ab-normalities that shorten their survival.The severity of hemolysis and resultinganemia varies among affected persons,but stomatocytosis, shortened red cellsurvival, and variably altered activity ofother blood group antigens, especially S,s, and U, have been consistent features.

Serologic ObservationsA few Rhnull probands were recognizedbecause their serum contained Rh anti-bodies. Some came to light, however,when routine Rh phenotyping of theirred cells revealed the absence of any Rhantigens, and in three cases, the discov-ery resulted from deliberate testing forRh antigens in patients with morpho-log ica l ly abnormal red cel l s and



hemolytic anemia. Immunized Rhnull

people have produced antibodies varyingin specificity from apparently straight-forward anti-e or anti-C to several exam-ples that reacted with all red cells testedexcept those from other Rhnull people.This antibody, considered to be “anti-to-tal Rh,” has been given the numericaldesignation anti-Rh29.

Rhmod

The Rhmod phenotype represents lesscomplete suppression of Rh gene expres-sion. As for the regulator type of Rhnull,an unlinked recessive modifier gene isthought to be responsible; it has beennamed XQ. Unlike Rhnull red cells, thoseclassified as Rhmod do not completely lackRh and LW antigens. Rhmod red cells showmuch reduced and sometimes varied ac-tivity, depending on the Rh system genesthe proband possesses and on the po-tency and specificity of the antisera usedin testing. Sometimes the Rh antigenshave sufficiently weakened expressionthat only adsorption-elution techniqueswill demonstrate their presence. As inRhnull, hemolytic anemia is a feature ofthe Rhmod condition. It may be appropri-ate to think of the two abnormalities asbeing essentially similar, differing onlyin degree.

Rh Antibodies in aPatient’s SerumExcept for some non-red-cell-stimulatedexamples of anti-E, anti-Cw, and antibod-ies to rare low-incidence antigens, mostRh antibodies result from exposure tohuman red cells through pregnancy ortransfusion. D is the most potent immu-nogen, followed by c and E. Although afew examples of Rh antibodies behave assaline agglutinins, most react best inhigh-protein, antiglobulin, or enzyme


test systems. Even sera containing po-tent saline-reactive anti-D are usuallyreactive at higher dilutions in antiglobu-lin testing. Some workers find enzymetechniques especially useful for detect-ing weak or developing Rh antibodies.

Detectable antibody usually persistsfor many years. If serum antibody levelsfall below detectable thresholds, sub-sequent exposure to the antigen charac-teristically produces a rapid secondaryimmune response. With exceedinglyrare exceptions, Rh antibodies do notbind complement when they combinewith their antigens, at least to the extentrecognizable by techniques currentlyused.

Dosage EffectAnti-D seldom shows any difference inreactivity between red cells from indi-viduals homozygous or heterozygous forRHD, but D expression seems to varysomewhat with the accompanying al-leles of the genotype. For example, redcells from a cDE/cDE individual carrymore D antigen sites than red cells froma CDe/CDe person, and may show highertitration scores with anti-D. Dosage ef-fects can sometimes be demonstratedwith some antibodies directed at the E,c and e antigens and occasionally, at theC antigen. Quantifying the antigen sitesrequires specialized techniques such asradioisotope labeling, flow cytometry,immunoferritin localization, or auto-mated procedures.

Concomitant AntibodiesSome Rh antibodies tend to occur inconcert. For example, the CDe/CDe per-son manifesting immune anti-E has al-most certainly been exposed to c as wellas E. Anti-c may be present in additionto anti-E, although substantially weakerand possibly undetectable at the timethe anti-E is found, and transfusion of



seemingly compatible E–c+ blood mayelicit an immediate or somewhat delayedhemolytic reaction. Generally, it is not asound practice to select donor bloodnegative for all or most of the antigensabsent from the recipient’s cells, butsome workers feel that the CDe/CDe re-cipient with detectable anti-E is a casethat merits special consideration. Sinceanti-c occurs frequently with anti-E inimmunized people whose red cells areE-negative and c-negative,28 some selectblood of the patient’s own Rh phenotypefor transfusion even when the presenceof anti-c cannot be demonstrated by testprocedures routinely used. Anti-E lessconsistently accompanies anti-c, as thepatient can easily have been exposed to cwithout being exposed to E. There islittle clinical value in pursuing anti-E ina serum known to contain anti-c, as thevast majority of c-negative donor bloodwill be negative for the E antigen.

Rh Typing TestsRoutine Rh typing for donors and pa-tients involves only the D antigen, andtechniques to demonstrate weak D arerequired only for donor blood.24 Tests forthe other Rh antigens are performedonly for defined purposes, such as iden-tifying unexpected Rh antibodies, ob-taining compatible blood for a patientwith an Rh antibody, investigating dis-puted parentage or other family studies,selecting a panel of phenotyped cells forantibody identification, or evaluatingwhether a person is likely to be homozy-gous or heterozygous for RHD.

In finding compatible blood for a re-cipient with a comparatively weak Rhantibody, tests with potent blood group-ing reagents more reliably confirm theabsence of antigen than mere demon-stration of a compatible crossmatch.Testing the patient’s phenotype may pro-

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vide confirmation of the antibody speci-ficity and suggest which other Rh anti-bodies could also be present.

Prudent Use of ResourcesRoutine testing for Rh antigens otherthan D is not recommended. Besides theexpenditure of time and money involved,excessive testing wastes scarce re-sources. Reagent anti-e sera, in particu-lar, should be used sparingly as raw ma-terial suitable for manufacture is oftenscarce. In most cases, red cells negativefor the E antigen can be considered e-positive without actual testing. Thispractice preserves precious supplies ofanti-e for cases with specific indications,such as evaluating E-positive bloodspecimens or investigating a suspecteddeletion at the E/e sublocus.

Routine Testing for DUntil recently, high-protein anti-D re-agents of human, polyclonal origin andsuitable for slide, tube, or microplate testswere used for most routine testing. Morerecently, monoclonal anti-D reagents havebecome widely available. Tests may em-ploy red cells suspended in saline, in se-rum, or in plasma, but permissible testconditions should be confirmed by read-ing the manufacturer’s directions beforeuse. Recommended test procedures mayvary somewhat among manufacturers.

Slide tests produce optimal resultsonly when a high concentration of redcells and protein are combined at a tem-perature of 37 C. Because the slide testmust be read within 2 minutes, the redcells and serum on the glass slide mustreach 37 C quickly. The viewing surfaceused to perform slide tests should bekept lighted at all times to maintain atemperature of 45-50 C, permittingrapid warming of the test mixture. Theslide test has the serious disadvantagethat drying of the reaction mixture can

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cause the red cells to aggregate, whichmay be misinterpreted as agglutination.Another disadvantage is the risk thatspecimens will spill during manipula-tion. Procedures for microplate tests aresimilar to those for tube tests but verylight suspensions of red cells are used.Representative procedures for tube,slide, and microplate tests are given inMethods 2.6, 2.7, and 2.8. If there is anindication to test for weak D, an an-tiglobulin test should be performed.

A reliable test for weak D expressioncannot be performed on a slide. Red cellswith weak D, if they are agglutinated atall on a slide test, will invariably exhibitweaker agglutination than that seenwith normal D-positive cells. This dis-tinction may not be recognized, how-ever, especially if the reagent has highpotency and avidity, unless normalD-positive cells and weak D cells aretested in parallel and results compared.

High-Protein ReagentsSome anti-D reagents designated for use inslide, rapid tube, or microplate tests containhigh concentrations of protein (20-24%)and other macromolecular additives. Suchreagents are nearly always prepared frompools of human sera and give rapid, reliableresults when used in accordance withmanufacturers’ directions.

Because the macromolecular mediummay cause red cells coated with immu-noglobulin to aggregate spontaneously,antisera with these additives may pro-duce false-positive reactions. A false-positive result due to spontaneous ag-gregation could cause a D-negativepatient to receive D-positive blood andbecome immunized. To detect this kindof spurious results when the reagent hashigh-protein formulation, the red cellsmust be simultaneously tested with animmunologically inert reagent, identi-cal in formulation to the anti-D in usebut lacking the antibody component. If


red cells exhibit aggregation in the con-trol test, the results of the anti-D testcannot be considered valid. In mostcases the presence or absence of D canbe determined, with other reagents, asdetailed later in this chapter.

The Control for High-Protein Reagents

High-protein reagents may give false-positive results in several other circum-stances. Factors in the patient’s serummay affect the test if the test is per-formed on unwashed red cells suspendedin the patient’s own serum or plasma.Strong autoagglutinins, abnormal se-rum proteins that promote rouleaux for-mation, or antibodies directed at an ad-di t ive in the reagent may causeaggregation that can be mistaken for ag-glutination by anti-D. The best way todetect possibly invalid reactions is touse, as the immunologically inert con-trol, the diluent used to manufacture theparticular anti-D reagent. This materialwill contain all the additives present inthe reagent except for the active anti-body. It should, therefore, potentiatespontaneous aggregation to the same de-gree as the active anti-D.

Manufacturers offer their individualdiluent formulations for use as controlreagents; if patients’ red cells are typedwith high-protein reagents, the testsmust be controlled with this material.The nature and concentration of addi-tives differ significantly among reagentsfrom different manufacturers. The con-trol reagent from one manufacturer maynot produce the same pattern of false-positive reactions as that of another;tests on patients’ cells must use a con-trol suitable for the testing procedure.Using 22% or 30% bovine albumin de-tects even fewer false positives, becausereagent solutions of bovine albumin lackthe other high-molecular-weight poten-tiators that manufacturers use in high-protein reagents.



Misleading Results with High-ProteinReagents

False Positives. False-positive test re-sults with high-protein reagents in-clude:

1. Cellular aggregation resulting fromimmunoglobulin coating of the pa-tient’s red cells or serum factors thatinduce rouleaux will give positiveresults in both the active and thecontrol tubes. Serum factors can beeliminated by thoroughly washingthe red cells (with warm saline ifcold agglutinins are present or sus-pected) and retesting the washed redcells. The original high-protein re-agent may be used, concurrentlywith a new control tube, if themanufacturer’s directions state thatthe product is suitable for use withsaline-suspended cells. If the cells inthe control test remain unaggluti-nated and the anti-D test gives apositive result, the red cells areD-positive. If agglutination still oc-curs in the control tube, the mostlikely explanation is immunoglobu-lin coating of the red cells, whichmay then be tested with a saline-re-active reagent.

2. Rouleaux, simulating agglutination,may occur if red cells and anti-D areincubated together for too long be-fore the test is read. This may occurrapidly, particularly on a warm slideon which evaporation causes drying,which further increases the proteinconcentration of the reaction mix-ture. It is important to follow themanufacturer’s recommendation tointerpret the test within a limitedperiod, usually no more than 2 min-utes.

False Negatives. False-negative testresults with high-protein reagents in-clude:

1. Too heavy a red cell suspension inthe tube test or too weak a suspen-


sion in the slide test may weakenagglutination. To achieve the 40-50% cell suspension required forslide testing, it may be necessary tocentrifuge blood from a severelyanemic patient and remove some ofthe plasma before testing the redcells.

2. Saline-suspended red cells may re-act poorly with some Rh reagents,and must not be used for slide test-ing.

3. Red cells possessing weakly ex-pressed D antigen may not react wellwithin the 2-minute limit of theslide test or upon immediate cen-trifugation in the tube test.

Low-Protein ReagentsThe low-protein, saline-reactive Rh re-agents in current use are formulatedpredominantly with monoclonal anti-bodies. Immunoglobulin-coated redcells can usually be successfully typedwith low-protein Rh reagents that con-tain saline-agglutinating antibodies andno additives that promote spontaneouscellular aggregation. If such additivesare present, the fact should be stated inthe “Reagent Description” section of thepackage insert.

Monoclonal Source Anti-DMonoclonal anti-D reagents are madepredominantly from human IgM anti-bodies, which require no potentiatorsand agglutinate most D-positive redcells from adults and infants in a salinesystem. Monoclonal anti-D reagentsusually promote reactions stronger thanthose with polyclonal IgG reagents, butthey may fail to agglutinate red cells ofsome partial-D categories. Adding smallamounts of polyclonal anti-D to themonoclonal antibodies provides a re-agent that will react with partial-D redcells in antiglobulin tests. Just the right



amount of polyclonal material must beadded. If the reagent contains too muchpolyclonal material, IgG molecules mayattach to the D sites and block the reac-tivity of the monoclonal IgM compo-nent. If there is too little polyclonal anti-D, the reagent may fail to detect partialD.

Licensed monoclonal /polyclonalblends can be used in slide, tube, or mi-croplate tests, and are as satisfactory ashigh-protein or chemically modified re-agents in antiglobulin tests for weak D.False-negative findings can result, how-ever, if tests using monoclonal reagentsare incubated in excess of a manufac-turer’s product directions. These re-agents, prepared in a low-protein me-dium, can be used to test red cells witha positive DAT, provided those tests arenot subjected to antiglobulin testing.

Control for Low-Protein Reagents

Most monoclonal/polyclonal blended re-agents have a total protein concentra-tion approximating that of human se-rum. False-positive reactions due tospontaneous aggregation of immuno-globulin-coated red cells occur no moreoften with this kind of reagent than withother saline-reactive reagents. False-positive reactions may occur in any sa-line-reactive test system if the serumcontains cold autoagglutinins or a pro-tein imbalance causing rouleaux and thered cells are tested unwashed. It is sel-dom necessary to perform a separatecontrol test, however. Absence of sponta-neous aggregation can usually be dem-onstrated by observing absence of agglu-tination by anti-A and/or anti-B in thecell tests for ABO. For red cell specimensthat show agglutination in all tubes (ie,give the reactions of group AB, D-posi-tive), a concurrent control must be per-formed on patients’ cells; this is not re-quired when donors’ cells are tested. Asuitable control is to centrifuge a sus-


pension of the patient’s red cells withautologous serum or with 6-8% bovinealbumin at the same time the anti-D testis centrifuged. If the test is one of severalrequiring incubation before centrifuga-tion, any negative result on a test per-formed concurrently serves as an ade-quate control. A separate control tubewould be required only for a red cellspecimen that gives positive reactionswith all the Rh reagents, ie, is typed asD+C+E+c+e+.

Control for Medium-Protein Reagents

Some manufacturers offer reagents con-taining chemically modified IgG in a di-luent with a protein concentration be-tween that of human serum and that ofhigh-protein reagents. This type of re-agent should not be confused with thoseformulated in a low-protein diluent, be-cause at least some immunoglobulin-coated cells may experience spontane-ous aggregation in the higher proteinconcentration. The incidence of false-positive reactions is not as high as withhigh-protein reagents, but for patients’specimens, a suitable parallel control isneeded. The need for a control will bestated in the manufacturer’s instruc-tions.

Testing for D in Hemolytic Disease ofthe Newborn

Because red cells from an infant suffer-ing from HDN are coated with immuno-globulin, a saline-reactive reagent isusually necessary for Rh testing. Occa-sionally, the infant’s red cells may be soheavily coated with antibody that all an-tigen sites are occupied, leaving noneavailable to react with a saline-reactiveantibody of appropriate specificity. This“blocking” phenomenon should be sus-pected if the infant’s cells have a stronglypositive DAT, and are not agglutinated by



a saline-reactive reagent of the samespecificity as the maternal antibody.

Anti-D is the specificity responsiblefor nearly all cases of blocking by mater-nal antibody. It is usually possible toobtain correct typing results with a sa-line anti-D after 45 C elution of the ma-ternal antibody from the cord red cells.(See Method 2.14.) Elution liberatesenough antigen sites to permit red celltyping, but must be performed cau-tiously because overexposure to heatmay denature or destroy Rh receptors.

Tests for Antigens Other Than DReagents are readily available to test forthe other principal Rh antigens: C, E, c,and e. These are formulated as eitherlow-protein (chemically modified ormonoclonal) or high-protein reagents.High-protein reagents of any specificityhave the same problems with false-posi-tive results as high-protein anti-D andrequire a comparable control test. Ob-servation of a negative result in the con-trol test for anti-D does not determinethe tests for other Rh antigens, becauseresults with anti-D are usually obtainedafter immediate centrifugation and testsfor the other Rh antigens are generallyincubated at 37 C before centrifugation.A valid control procedure must be per-formed concurrently with the test, usingthe same duration and conditions of in-cubation, and be interpreted simultane-ously with the actual test.

Rh reagents may give weak or nega-tive reactions with red cells possessingvariant antigens. This is especially likelyto happen if anti-e is used to test the redcells from Blacks, among whom variantsof e are relatively common. It is impos-sible to obtain anti-e reagents that reactstrongly and consistently with the vari-ous qualitative and quantitative variantsof e. Variable reactivity with anti-C re-agents may occur if the CDE or CEhaplotypes are responsible for the ex-


pression of C on red cells. Variant E andc antigens have been reported but areconsiderably less common.

Whatever reagents are used, themanufacturer’s directions must be care-fully followed. The indirect antiglobulintechnique must not be used unless themanufacturer’s instructions state explic-itly that the reagent is suitable for thisuse. The pools of human sera used toprepare reagents for the other Rh anti-gens have a significant risk of containingantiglobulin-reactive, contaminatingspecificities. Positive and negative con-trols should be tested in parallel with thered cells under study. Red cells selectedfor the positive control should be knownto have a single dose of the antigen con-cerned or be known to show weak reac-tivity with the reagent. If these reagentsare used regularly, they should be in-cluded in the routine quality assuranceprogram.

Additional Considerations in RhTestingThe following limitations are commonto all Rh typing procedures, includingthose performed with high-protein re-agents.

False-Positive ReactionsThe following circumstances can pro-duce false-positive red cell typing re-sults.

1. The wrong reagent was inadver-tently used.

2. An unsuspected antibody of anotherspecificity was present in the re-agent. Antibodies for antigens hav-ing an incidence of less than 1% inthe population may occasionally bepresent and cause false-positive re-actions, even when the manufac-turer’s directions are followed.Though the antigens occur infre-quently, antibodies to them are com-



paratively common, even in personswith no history of pregnancy ortransfusions.

For crucial determinations, manyworkers routinely perform replicatetests, using reagents from differentsources. This reduces the likelihoodof a false classification, because dis-crepant reactions would alert the se-rologist to the need for further test-ing. Replicate testing is not anabsolute safeguard, however, be-cause reagents from different manu-facturers may not necessarily derivefrom different sources. The scarcityof donors or of clones producing ac-ceptable titers of such relatively un-common specificities as anti-C andanti-e, in particular, may cause thesame source of raw material to beused by several manufacturers. Dif-ferent manufacturers’ reagents pre-pared from the same polyclonalsource material may, in fact, containthe same contaminating antibody.

3. Polyagglutinable red cells may beagglutinated by any reagent con-taining human serum. Although an-tibodies that agglutinate these sur-face-altered red cells are present inmost adult human sera, polyagglu-tinins very rarely cause problemswith reagents. Aging, dilution, andvarious steps in the manufacturingprocess tend to eliminate these pre-dominantly IgM antibodies.

4. Autoagglutinins and abnormal pro-teins in the patient’s serum maycause false-positive reactions whenunwashed red cells are tested.

5. Reagent vials may become contami-nated with bacteria, with foreignsubstances, or with reagent from an-other vial. This can be prevented bythe use of careful technique and theperiodic inspection of the vials’ con-tents. Bacterial contamination maynot, however, cause recognizableturbidity because the refractive in-


dex of bacteria is similar to that ofhigh-protein reagents.

False-Negative ReactionsThe following circumstances can pro-duce false-negative red cell typing re-sults.

1. The wrong reagent was inadver-tently used.

2. The reagent was not added to thetube, either because of oversight orbecause the drops of fluid ran downthe outside of the tube. It is goodpractice to add serum to all thetubes before adding the red cells andany enhancement media.

3. A specific reagent failed to reactwith a variant form of the antigen.

4. A reagent that contains antibody di-rected predominantly at a cis-prod-uct Rh antigen failed to give a reli-ably detectable reaction with redcells carrying the individual anti-gens as separate gene products. Thisoccurs most often with anti-C sera.

5. The reagent was used incorrectly be-cause the manufacturer’s directionswere not followed.

6. The red cell button was shaken soroughly during resuspension thatsmall agglutinates were dispersed.

7. Contamination, improper storage,or outdating cause antibody activityto deteriorate. Chemically modifiedIgG antibody appears to be particu-larly susceptible to destruction byproteolytic enzymes produced bycertain bacteria.

References1. Race RR. The Rh genotypes and Fisher’s the-

ory. Blood 1948; special issue 2:27-42.2. Levine P, Stetson RE. An unusual case of in-

tragroup agglutination. JAMA 1939;113:126-7.

3. Landsteiner K, Wiener AS. An agglutinablefactor in human blood recognized by immune



sera for rhesus blood. Proc Soc Exp Biol NY1940;43:223.

4. Levine P, Katsin EM. Isoimmunization inpregnancy and the variety of isoagglutininsobserved. Proc Soc Exp Biol NY 1940;43:343-6.

5. Wiener AS, Peters HR. Hemolytic reactionsfollowing transfusions of blood of the homolo-gous group, with three cases in which thesame agglutinogen was responsible. Ann In-tern Prn Med 1940;13:2306-22.

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7. Wiener AS. Genetic theory of the Rh bloodtypes. Proc Soc Exp Biol Med 1943;54:316-9.

8. Fisher RA, Race RR. Rh gene frequencies inBritain. Nature 1946;157:48-9.

9. Tippett P. A speculative model for the Rh bloodgroups. Ann Hum Genet 1986;50:241-7.

10. Colin Y, Chérif-Zahar B, Le Van Kim C et al.Genetic basis of RhD-positive and RhD-nega-tive blood group polymorphisms as deter-mined by southern ana l ys i s . Blood1991;78:2747-52.

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22. Van Loghem JJ. Production of Rh agglutininsanti-C and anti-E by artificial immunizationof volunteer donors. Br Med J 1947;ii:958-9.

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24. Klein HG, ed. Standards for blood banks andtransfusion services, 17th ed. Bethesda, MD:American Association of Blood Banks, 1996.

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26. Lomas C, Tippett P, Thompson KM, et al. Dem-onstration of seven epitopes of the Rh antigenD using monoclonal anti-D antibodies and redce l l s f rom D categor ies . Vox Sang1989;57:261-4.

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Issitt PD. An invited review: The Rh antigen e, itsvariants, and some closely related serological ob-servations. Immunohematology 1991;7:29-36.

Issitt PD. The Rh blood group. In: Garratty G, ed.Immunology of transfusion medicine. New York:Marcel Dekker, 1994:111-47.

Issitt PD. The Rh blood group system 1988: Eightnew antigens in nine years and some observationson the biochemistry and genetics of the system.Transfus Med Rev 1989;3:1-12.

Moulds JJ. Rhnulls: Amorphs and regulators. In:Walker RH, ed. A seminar on recent advances in



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Reid ME, Ellisor SS, Frank BA. Another potentialsource of error in Rh-Hr typing. Transfusion1975;15:485-8.


Vengelen-Tyler V, Pierce S, eds. Blood group sys-tems: Rh. Arlington, VA: American Association ofBlood Banks, 1987.

White WD, Issitt CH, McGuire D. Evaluation of theuse of albumin controls in Rh typing. Transfusion1974;14:67-71.


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