TM12 - ABO, H, and Lewis Blood Groups and Structurally ... · PDF fileT he ABO, H, P, I, and Lewis blood group antigens reside on structurally related carbohydrate mole-cules, and

12 ABO, H, and Lewis BloodGroups and StructurallyRelated Antigens

12

The ABO, H, P, I, andLewis blood group antigens reside onstructurally related carbohydrate mole-cules, and reflect the activity of geneti-cally determined glycosyltransferase en-zymes. Carbohydrate molecules carrysugars that may determine several anti-gens, providing an opportunity for inter-action between genetic products in sev-eral systems. The antigens appear whentransferases add individual sugars tosites on short chains of sugars (oligosac-charides) that often are part of other,much larger molecules. The added sug-ars are called immunodominant becausethey confer specific antigenic activity onthe oligosaccharide chains.

Oligosaccharides are chains of sugarsthat can be attached to either protein (gly-coprotein), sphingolipid (glycosphin-golipid), or lipid (glycolipid) carrier mole-cules. When attached to proteins,oligosaccharides are linked either to theamide nitrogen of asparagine via an N-ace-tylglucosamine (GlcNAc) or to the hy-droxyl oxygen of serine or threonine via anN-acetylgalactosamine (GalNAc). Glyco-proteins are associated with the mem-

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branes of red cells and other cells. Thebody’s serous and mucous secretionscontain soluble glycoproteins withblood group antigen activity. In glyco-sphingolipids, structurally similar oli-gosaccharides are attached via glucose(Glc) to ceramide residues. Glycosphin-golipids form part of the membranes ofred cells, of most endothelial cells, andsome epithelial cells. Soluable forms arepresent in plasma as glycolipids, but arenot secreted in body fluids.

The ABO SystemA series of tests reported by Karl Land-steiner in 1900 led to the discovery of theABO blood groups and to the develop-ment of routine blood grouping proce-dures.1 Landsteiner tested blood sam-ples from himself and several colleaguesby combining each serum specimen witha suspension of red cells from each per-son. Noting agglutination in some mix-tures but not in others, he was able toclassify the blood samples into one ofthree groups, now named A, B, and O.

229ABB. All rights reserved.

230 AABB Technical Manual

Landsteiner recognized that the pres-ence or absence of only two antigens, Aand B, was sufficient to explain the threeblood groups and he predicted that afourth group should exist. AB was dis-covered in 1902 by his pupils von Decas-tello and Sturli. He also demonstratedthat each person’s serum contained an-tibody against the antigen absent fromthat person’s red cells.

The first blood group system to bediscovered, ABO remains the most sig-nificant for transfusion practice. It is theonly system in which the reciprocal (orantithetical) antibodies (see Table 12-1)are consistently and predictably presentin the sera of normal people who havehad no exposure to human red cells. Be-cause of these antibodies, transfusion ofABO-incompatible blood may cause se-vere intravascular hemolysis as well asthe other manifestations of an acutehemolytic transfusion reaction (seeChapter 25). Testing to detect ABO in-compatibility between a recipient andthe donor is the foundation on which allpretransfusion testing is based.

Antigens of the ABO System

Biochemical and Genetic ConsiderationsGlycosphingolipids carrying A or B oli-gosaccharides are integral parts of themembranes of red cells, epithelial cells,and endothelial cells (Fig 12-1) and are

Table 12-1. Routine ABO GroupingReaction of Cells

Tested WithReaction of Se

Tested Again

Anti-A Anti-B A1 CellsB

Cells0 0 + ++ 0 0 +0 + + 0+ + 0 0

+ = agglutination; 0 = no agglutination


also present in soluble form in plasma.Secreted body fluids such as saliva con-tain glycoprotein molecules that may, ifthe person possesses an Se gene, carryidentical oligosaccharides. A and B oli-gosaccharides unattached to carrier pro-tein or lipid molecules are also found inmilk and urine.

Genes at three separate loci (ABO, Hh,and Sese) control the occurrence and thelocation of the A and B antigens. Threecommon alleles (A, B, and O) are locatedat the ABO locus on chromosome 9. The Aand B genes encode glycosyltransferasesthat produce the A and B antigens, respec-tively.2 The O gene is considered to benonfunctional because its protein productdetermines no detectable blood group an-tigen. The red cells of group O personslack A and B, but carry an abundantamount of H antigen, the precursor mate-rial on which A and B antigens are built.

Family studies have shown that thegenes at the remaining two loci, Hh andSese (secretor), are on chromosome 19and are closely linked.3 Each locus hastwo recognized alleles, of which one hasno demonstrable product and is consid-ered an amorph. The active allele at theH locus, H, produces a transferase en-zyme that acts at the cellular level toform the antigen on which A or B arebuilt. The amorph, h, is very rare.

The Se gene is directly responsible forthe expression of H (and indirectly re-sponsible for the expression of A and B)

rumst

Interpre-tation

Frequency (%) inUS Population

OCells

ABOGroup Whites Blacks

0 O 45 490 A 40 270 B 11 200 AB 4 4

ABB. All rights reserved.

Figure 12-1. Diagrammatic representation of some membrane glycoproteins and glycosphin-golipids that carry blood group antigens.

Chapter 12: ABO, H, and Lewis Blood Groups 231

on the glycoproteins in epithelial secre-tions such as saliva. Eighty percent ofthe population are described as secre-tors; they have inherited the Se gene andtheir secreted glycoproteins express H,which can be converted to A and/or B ifthey also have the A or B gene. Theamorph is called se.

The oligosaccharide chains to whichthe A or B immunodominant sugars areattached may exist as simple repeats of afew sugar molecules linked in linear fash-ion. They can also exist as more complexstructures, with many sugar residueslinked in branching chains. Differencesbetween infants and adults in cellular A, B,and H activity may be related to thenumber of branched structures present on


cellular membranes at different ages.The red cells of infants are thought tocarry predominantly linear oligosaccha-rides, which have only one terminus towhich the H (and subsequent A or B)sugars can be added. In contrast, the redcells of adults appear to carry a highproportion of branched oligosaccha-rides. Branching creates additional ex-pressions of oligosaccharide for conver-sion to H and then to A and B antigens.

A and B genes do not produce antigensdirectly; their products are enzymes,called glycosyltransferases, that add spe-cific sugars to oligosaccharide chains thathave been converted to H by the fucosyltransferase produced by the H gene. Hantigens are constructed on oligosaccha-



ride chains that end in Type 1 or Type 2linkages between the sugars β-D-galac-tose (Gal) and N-acetylglucosamine(GlcNAc) (see Fig 12-2). In Type 1 chains,the number 1 carbon of Gal is linked to thenumber 3 carbon of GlcNAc; in Type 2chains, the number 4 carbon of GlcNAc isthe acceptor for Gal. Oligosaccharides ofType 1, carrying A, B, and H activity, arewidely distributed in the body. Glycolipidswith Type 1 A, B, and H are present inplasma and on endodermally derived tis-sues such as the epithelial lining of thegut. Glycoproteins with Type 1 chains con-tribute A, B, and H activity to body fluidsand secretions. Unattached Type 1 oli-gosaccharides can be found in milk andurine. In saliva, glycoproteins of Type 1and Type 2 carry A, B, and H.

A, B, and H antigens on the red cellsurface are formed on Type 2 chains pre-sent in highly branched oligosaccha-rides attached to integral proteins of thered cell membrane, notably bands 3 and

Figure 12-2. Type 1 and 2 oligosaccharidechains differ only in the linkage between theGlcNAc and the terminal Gal.


4.5. The remaining Type 2 antigens arebound to glycolipids. Recent studies in-dicate that more complex core mole-cules, called Type 3 and Type 4 chains,may also be involved.4 The number ofpotential A, B, and H sites on red cells isthought to be in excess of two million.5

The H gene produces a transferasethat adds fucose (Fuc) to the number 2sugar of the galactose that constitutesthe terminal sugar of Type 1 and Type 2chains (see Fig 12-3). The A and B genetransferases can attach their immuno-dominant sugars to the number 3 sugarof the same galactose only if fucose is

Figure 12-3. Gal added to the subterminal Galconfers B activity; GalNAc added to the sub-terminal Gal confers A activity to the sugar.Unless the fucose moiety that determines Hactivity is attached to the number 2 carbon,galactose does not accept either sugar on the3 carbon.



already attached, ie, the core chain hasbeen converted to H. Attachment of theA- or B-defining sugar diminishes the Hconfiguration in a reciprocal manner;the expression of A or B and of H areinversely proportional.

Initially, it was believed that additionof A and B sugars to the terminal galac-tose halted chain growth, ie, that A or Bantigens could not serve as acceptor sub-strates. The discovery of glycolipids inwhich two A structures appeared linkedin tandem altered this view6 (see Fig 12-4).

ABO Genes at the Molecular Level

Yamamoto and Hakomori have shownthat A and B genes differ from one an-other by seven single-base substitu-tions.7 This results in four possibleamino acid substitutions (at positions176, 235, 266 and 268) in the proteinchains of the A and B transferases. Sub-stitutions at positions 266 and 268 aremost significant in determining sugar-nucleotide specificity, although substi-tution at position 235 also has some ef-fect. The alleles of A and B that result insubgroups (phenotypes of A and B thatexpress aberrant levels of antigen) mayhave additional mutations that result inenzymes with slightly different abilitiesto convert H antigen. A single nucleotidedeletion was found in A2 genes close tothe carboxyl terminus of the A trans-ferase coding sequence. As a result offrame-shifting, the glycosyltransferasespecified by A2 possesses an extra domain

Figure 12-4. Type 3 A antigen structure.


of 21 amino acids; this domain seemsresponsible for the low transferase activ-ity and restricted substrate recognitionof this enzyme.8

The base sequence of the O gene ap-pears to be similar to that of A except fora single base deletion at position 2589 inthe coding region close to the N-termi-nus of the resulting protein. This singledeletion shifts the reading frame to pro-duce a premature stop codon, resultingin a truncated protein with no trans-ferase activity.8 Thus, of the three pri-mary genes in the ABO system, two arefunctional and one determines a non-functional product.

Persons who lack the Se gene haveType l and Type 2 chains in their secre-tions that express no H, A, or B activity.It is thought that the H and Se geneseach encode a fucosyltransferase. Theenzyme produced by H acts primarily onType 2 chains, which predominate on thered cell membranes; the enzyme pro-duced by Se prefers (but does not limitits action to) Type 1 chains, and actsprimarily in the secretory glands. Stud-ies performed on the secretions of indi-viduals with the rare Oh phenotype sup-port the concept that two types of Hantigen exist.10 Individuals who lackboth H and Se genes (genotype hh andsese) have no H and, therefore, no A orB antigens on their red cells or in theirsecretions. However, H, A, and B anti-gens are found in the secretions of somehh individuals who appear, through fam-ily studies, to possess at least one Segene.



Direct agglutination tests are used todetect A and B antigens on red bloodcells. Reagent antibodies frequently pro-duce weaker reactions with red cellsfrom newborns than with red cells fromadults. Although they can be detected onthe red cells of 5-6 week-old embryos, Aand B antigens are not fully developed atbirth, presumably because the branch-ing oligosaccharide structures developgradually. By the time a person is 2-4years old, A and B antigen expression isfully developed, and remains fairly con-stant throughout life.

Subgroups

Subgroups are ABO phenotypes that dif-fer in the amount of antigen carried onred cells and, in secretors, present in thesaliva (see Table 12-2). Subgroups aremore commonly encountered and moresignificant for A than for B. The twoprincipal subgroups of A are A1 and A2.

Table 12-2. Serologic Reactions Observe

Red BloodCell

Phenotype

Reaction of Cells WithKnown Antiserum to

A B A,B H A1

A1 4+ 0 4+ 0 4+Aint 4+ 0 4+ 3+ 2+A2 4+ 0 4+ 2+ 0A3 2+mf 0 2+mf 3+ 0Am 0/± 0 0/± 4+ 0Ax 0/± 0 1+/2+ 4+ 0Ael 0 0 0 4+ 0B 0 4+ 4+ 0B3 0 1+mf 2+mf 4+Bm 0 0 0/± 4+Bx 0 0/± 0/2+ 4+

1+ to 4+, agglutination of increasing strength; ±of agglutination; 0, no agglutination.*Modified from Beattie11

†The occurrence of anti-A1 is variable in these panti-A1; A3 persons usually do not, but a few A3been found.


The A1 and A2 genes determine differenttransferases, which mediate both quan-titative and qualitative differences be-tween red cells of the A1 and A2 pheno-types. Red cells from A1 and A2 personsboth react strongly with reagent anti-Ain direct agglutination tests. The sero-logic distinction between Al and A2 cellsderives from tests with reagent anti-A1

prepared from group B human serum orwith the lectin of Dolichos biflorusseeds. Under prescribed testing condi-tions, anti-A1 reagents agglutinate A1 butnot A2 red cells. Approximately 80% ofgroup A or group AB individuals have redcells that are agglutinated by anti-A1 andthus are classified as A1 or A1B. The re-maining 20%, whose red cells are agglu-tinated by anti-A but not by anti-A1, arecalled A2 or A2B.

Anti-A1 occurs as an alloantibody inthe serum of 1-8% of A2 persons and22-35% of A2B persons.11 Anti-A1 cancause discrepancies in ABO testing and

d in Persons With A and B Phenotypes*

Reaction of Serum AgainstReagent Red Blood Cells

Saliva ofSecretorsContainsA1 A2 B O

0 0 4+ 0 A & H0 0 4+ 0 A & H† 0 4+ 0 A & H† 0 4+ 0 A & H0 0 4+ 0 A & H

2+/0 0/1+ 4+ 0 H2+/0 0 4+ 0 H4+ 4+ 0 0 B & H4+ 4+ 0 0 B & H4+ 4+ 0 0 B & H4+ 4+ 0 0 H

, weak agglutination; mf, mixed-field pattern

henotypes. A2 persons frequently haveindividuals with anti-A1 in the serum have



incompatibility in crossmatches with A1

or A1B red cells. Anti-A1 usually reactsbetter or only at temperatures well be-low 37 C, and is considered clinicallyinsignificant unless there is reactivity at37 C. Routine testing with anti-A1 is un-necessary for donors or recipients. Thisinformation is useful only when workingwith blood samples that contain anti-A1.

Subgroups weaker than A2 occur in-frequently and, in general, are charac-terized by decreasing numbers of A anti-gen sites on the red cells and a reciprocalincrease in H antigen activity. Classifica-tion of weak A subgroups is generallybased on the:

1. Degree of red cell agglutination byanti-A and anti-A1.

2. Degree of red cell agglutination byanti-A,B.

3. Degree of red cell agglutination byanti-H (Ulex europaeus).

4. Presence or absence of anti-A1 in theserum.

5. Presence of A and H substances inthe saliva of secretors.

See Table 12-2 for the serologic charac-teristics of A phenotypes.

Red cells of the Ax, Ael, Aint, or A3 sub-groups are seen only rarely in transfu-sion practice. Ax red cells are charac-teristically not agglutinated by humananti-A from group B persons, but areagglutinated by anti-A,B from group Opersons (see following section on anti-bodies). Ax red cells may react with somemurine monoclonal anti-A reagents, de-pending on which monoclonal antibodyis selected for the reagent. Ael red cellsare not agglutinated by anti-A or anti-A,B of any origin, and presence of A an-tigen is demonstrable only by adsorptionand elution studies. Aint red cells reactmore weakly than A1 red cells with anti-A1, yet more strongly with anti-H than A2

red cells; Aint phenotype is detected onlyif anti-A1 testing is performed and quan-tified. A3 red cells produce a charac-teristic mixed-field pattern of small ag-


glutinates among many free red cells intests with anti-A and anti-A,B. Weak sub-groups of A such as Ax, Am, and Ael cannotreliably be identified on the basis ofblood typing tests alone. Saliva studies,adsorption/elution studies, and familystudies provide confirmatory informa-tion.

Subgroups of B are even less commonthan subgroups of A. Criteria for theirdifferentiation resemble those for sub-groups of A (see Table 12-2). Amino acidsubstitutions have been reported insome of the glycosyltransferases codedby the weak subgroup genes.8 Wide-spread use of monoclonal anti-A andanti-B reagents will lessen the detectionof weak A or B subgroups because mono-clonal antibodies are selected for re-agent use based on their ability to agglu-tinate cells with weak or aberrantantigen expression.

Antibodies to A and BOrdinarily, individuals possess antibod-ies directed toward the A or B antigenabsent from their own red cells (see Ta-ble 12-1). This predictable complemen-tary relationship permits ABO testing ofserum as well as of red cells (see Methods2.2 and 2.3). One hypothesis for the de-velopment of these antibodies is basedon the fact that the configurations thatconfer A and B specificities on moleculesof the red cell membrane also exist inother biologic entities, notably bacterialcell walls. Bacteria are widespread in theenvironment, and their presence in in-testinal flora, dust, food, and otherwidely distributed agents ensures a con-stant exposure of all persons to A-likeand B-like antigens. Immunocompetentpersons react to the environmental anti-gens by producing antibodies to thosethat are absent from their own systems.Thus, anti-A is produced by group O andgroup B persons and anti-B is producedby group O and group A persons. Group



AB people, having both antigens, makeneither antibody. This “environmental”explanation for emergence of anti-A andanti-B remains a hypothesis that has notbeen proved.

Time of AppearanceAnti-A and anti-B can generally be de-tected in serum after the first fewmonths of life. Occasionally infants canbe found who already produce these an-tibodies at the time of birth, but mostantibodies present in cord blood are ofmaternal origin. Antibody productionincreases until reaching adult level at5-10 years of age and declines later inlife. Elderly people ordinarily have loweranti-A and anti-B levels than those seenin young adults. Anti-A and anti-B test-ing on serum from newborns or infantsyounger than 4-6 months cannot be con-sidered valid because some or all of theinfant’s antibodies are acquired by pla-cental transfer of maternal IgG anti-Aand anti-B.

Reactivity of Anti-A and Anti-BIgM is the predominant immunoglobu-lin class of anti-A produced by group Bindividuals and anti-B produced bygroup A individuals, although smallquantities of IgG molecules are also pre-sent. IgG is the dominant class of anti-Aand anti-B of group O serum. BecauseIgG readily crosses the placenta and IgMdoes not, the infants of group O mothersare at higher risk for ABO hemolytic dis-ease of the newborn (HDN) compared tothose of group A or B mothers.

The distinguishing features of IgMand IgG anti-A and anti-B are given inTable 12-3 . Both immunoglobul inclasses preferentially agglutinate redcells at room temperature (20-24 C) orbelow, and efficiently activate comple-ment at 37 C. The complement-mediatedlytic capability of these antibodies be-


comes apparent if serum testing in-cludes an incubation phase at 37 C.Some patients or donors are foundwhose serum causes hemolysis of ABO-incompatible red cells at temperaturesbelow 37 C. Hemolysis due to ABO anti-bodies should be suspected when the su-pernatant fluid of the reverse groupingtest is pink to red or when the cell buttonis absent or reduced in size. Hemolysismust be interpreted as a positive result.Because the hemolysis is complement-mediated, it will not occur if reagent redcells are suspended in solutions thatcontain EDTA or other agents that pre-vent complement activation, or if plasmais used for testing.

Anti-A,B (Group O Serum)

Serum from group O individuals con-tains an antibody designated as anti-A,Bbecause it reacts with both A and B redcells and the anti-A and anti-B specifici-ties cannot be separated by differentialadsorption. When group O serum is in-cubated with group A or group B cells,eluates prepared from each adsorbingcell exhibit reactivity against both A andB test cells. Saliva from secretors ofeither A or B substance inhibits the ac-

Table 12-3. DistinguishingCharacteristics of IgM and IgGAnti-A and Anti-B

IgM IgG

Reactions enhanced—with enzyme-treated rbcs—by lowering temperatures

yesyes

yesno

Readily inhibited bysoluble A or B antigens yes no

Inactivated by 2-ME or DTT yes noPredominant in non-

immunized group A andB donors yes no



tivity of this antibody against both A orB red cells.

Group O serum has been used to pre-pare potent grouping reagents capable ofagglutinating either A or B cells. Re-agent preparations containing blendedmonoclonal antibodies also agglutinateA or B cells, and either form of anti-A,Breadily distinguishes group O red cellsfrom the other three groups. Anti-A,Bcontaining blended monoclonal anti-bodies may react as well as, or betterthan, natural human anti-A,B with redcells of weak A phenotypes, dependingon the monoclonal preparations se-lected.

Anti-A1

The anti-A of group B serum appears,from simple studies, to contain separa-ble anti-A and anti-A1. Native group Bserum agglutinates A1 and A2 red cells;after adsorption with A2 red cells, groupB serum reacts only with A1 red cells. Iffurther tests are performed, however, thedifferences in A antigen expression be-tween A1 and A2 red cells appear to bequantitative rather than qualitative.Anti-A1 is sometimes found in the serumof persons with A2 or other subgroup Aphenotypes.

A reliable anti-A1 reagent can bemanufactured from the lect in ofDolichos biflorus (see Method 2.10). Theplant extract will react with both A1 andA2 red cells, but the appropriately dilutedreagent preparation will not agglutinateA2 cells and thus constitutes an anti-A1.

Routine Testing for ABOTests that use anti-A and anti-B to deter-mine the presence or absence of the an-tigens are often described as direct or redcell tests. The use of reagent A1 and B redcells to detect anti-A and anti-B in serumis called serum testing. Routine tests ondonors and patients must include both


red cell and serum tests, each serving asa check on the other.12,13 To confirm theABO type of donor units that have al-ready been labeled, or to test blood ofinfants less than 4 months of age, ABOtesting on red cells only is permissible.Procedures for ABO typing by slide,tube, and microplate tests are describedin Methods 2.1, 2.2, and 2.3.

Some ABO red cell typing reagentsare prepared from pools of sera fromindividuals who have been stimulatedwith A or B blood group substances toproduce antibodies of high titer. OtherABO grouping reagents are manufac-tured from monoclonal antibodies de-rived from cultured cell lines. Both typesof reagents agglutinate most antigen-positive red cells on direct contact, evenwithout centrifugation. Slide testingcannot, however, be recommended be-cause of the likelihood of spillage andcontact with blood. Anti-A and anti-B inthe serum of most patients and donorsare usually too weak to agglutinate redcells without centrifugation, so serumtests should be performed by tube ormicroplate techniques, not on slides.

B(A) Phenotype

In 1984, Yates et al14 reported that a10,000-fold concentrate of purified Bgene-specified galactosyltransferase,isolated from the sera of group B indi-viduals has the capacity, in vitro, to at-tach small amounts of A sugar to humanmilk fucosyllactose, a blood group H-like structure. High levels of galactosyl-transferase associated with the B genecan attach the A-determining sugar, Gal-NAc, to a fucosylated substrate mole-cule, both in vitro14 and in vivo.15 Redcells of some group B individuals wereagglutinated by an FDA-licensed anti-Areagent that contained a particular mur-ine monoclonal antibody MHO4. Thesegroup B individuals had excessively highlevels of B gene-specified galactosyl-



transferase, and the designation B(A)was given to this blood group pheno-type.15

Recognition of the B(A) phenotype isusually made with the discriminatingmonoclonal anti-A reagent, with whichthe B(A) red cells show varying reactiv-ity. The majority of examples reactweakly, and the agglutinates are fragileand easily dispersed, although some ex-amples have reacted as strongly as 2+.15

Serum from these individuals aggluti-nates both A1 and A2 cells, so except fornewborns and immunocompromised pa-tients, serum testing should distinguishthis phenomenon from the AB pheno-type in which a subgroup of A is accom-panied by anti-A1. Studies of the trans-ferase or the gene sequence shouldprovide conclusive proof. The trans-ferase for GalNAc should be present inthe serum from the AsubB individual andabsent in the B(A) sample. Comparedwith B transferase, the transferase deter-mining B(A) exhibits a single amino acidsubstitution at the second of the fouramino acid substitution sites (aa 235)that discriminate between A1 and Btransferases. The amino acid residue itpossesses is glycine, which characterizesthe A1 transferase.8

Nonroutine ABO TestingAdditional reagents may be used for ABOtesting, especially anti-A,B for red celltests and A2 and O red cells for serumtests. Some workers use anti-A,B in rou-tine tests in the belief that it is moreeffective than anti-A or anti-B in detect-ing weakly expressed antigens. This isnot true. Of the weak subgroups unde-tected by anti-A or anti-B, only Ax isdetected by human anti-A,B, and thenonly if the serum and cells are incubatedat room temperature for 10-60 minutes.Some monoclonal blends labeled anti-A,B react well with red cells of weaksubgroups, on immediate-spin tests. If


the manufacturer’s directions recom-mend using anti-A,B for the detection ofweak subgroups, it means that its reac-tivity against Ax red cells has been dem-onstrated.12 AABB Standards for BloodBanks and Transfusion Services13 doesnot require special techniques to detectweak subgroups because the absence ofexpected serum antibodies usually dis-tinguishes these specimens from groupO specimens (see Table 12-2).

Some commercially marketed sets ofserum testing cells contain A2 red cellsin addition to A1 and B red cells. The A2

red cells are intended to facilitate therecognition of anti-A1 in the serum ofspecimens exhibiting a subgroup of A.Because most A specimens do not con-tain anti-A1, routine use of this reagentis inappropriate unless discrepancies be-tween red cell and serum tests are en-countered. A2 cells for differential test-ing can be selected by testing donor orpatient samples with anti-A1, althoughhaving a readily available reagent may bemore convenient.

Manufacturers of ABO reagents pro-vide, with each reagent package, detailedinstructions for use of the reagent. Test-ing details may vary from one manufac-turer to another, and it is important tofollow the directions supplied with thespecific reagent in use.

Discrepancies Between Red BloodCell and Serum TestsTable 12-1 shows the results and inter-pretations of routine red cell and serumtests for ABO. A discrepancy exists whenthe results of red cell tests do not com-plement that of serum tests, and the ex-pected two positives and two negativesare not observed. When a discrepancy isencountered, the discrepant resultsshould be recorded, but interpretationmust be delayed until the discrepancy isresolved. If the specimen is from a donorunit, the unit may not be released for



transfusion until the discrepancy is re-solved. When the blood is from a poten-tial recipient, it may be necessary to ad-minister group O red cel ls o f theappropriate Rh type before the investiga-tion is completed. It is important to ob-tain a sufficient amount of the patient’spretransfusion blood to complete any ad-ditional studies that may be required.

Red cell and serum test results may bediscrepant because of intrinsic problemswith red cells or serum, because of test-related problems, or because of technicalerrors. Discrepancies may be signaledeither because negative results are ob-tained when positive results are ex-pected, or positive results are foundwhen tests should have been negative.

False NegativesFalse-negative test results may occur be-cause of failure to:

1. Add reagent or test serum to a tube.2. Identify hemolysis as a positive reac-

tion.3. Use the appropriate ratio of serum

(or reagent) to red cells.4. Centrifuge tests sufficiently.5. Incubate tests at temperatures of 20-

24 C or below.6. Interpret or record test results cor-

rectly.

False PositivesFalse-positive test results may occur be-cause of:

1. Overcentrifugation of tubes.2. Use of contaminated reagents, red

cells, or saline.3. Use of dirty glassware.4. Incorrect interpretation or record-

ing of test results.

Specimen-Related Problems in TestingRed CellsGrouping tests on red cells may give un-expected results for many reasons.

Copyright © 2002 by the AA

1. A patient who has received red celltransfusions or a bone marrowtransplant may have circulating redcells of more than one ABO group,and thus constitute a transfusion ortransplantation chimera.

2. Red cells from individuals with vari-ant A or B genes may carry poorlyexpressed antigens. Antigen expres-sion may be weakened on the redcells of some persons with leukemiaor other malignancies. Agglutina-tion tests with reagent anti-A andanti-B may fail to give expected re-actions.

3. Inherited or acquired abnormalitiesof the red cell membrane can lead towhat is called a polyagglutinablestate. The abnormal red cells can beunexpectedly agglutinated by re-agent anti-A, anti-B, or both.

4. Abnormal concentrations of serumproteins, the presence in serum ofmacromolecules or, in cord bloodsamples, the presence of Wharton’sjelly may cause nonspecific aggrega-tion of serum-suspended cells thatsimulates agglutination.

5. Exceptionally high concentrationsof A or B blood group substances inthe serum can combine with reagentantibodies and neutralize them toproduce an unexpected negative re-action against serum- or plasma-suspended red cells.

6. Serum may contain antibodies tothe dyes used to color anti-A andanti-B reagents. These antibodiescan cause false-positive agglutina-tion reactions if serum- or plasma-suspended red cells are used in test-ing.

7. A patient with potent cold-reactiveautoagglutinins may have red cellsso heavily coated with antibody thatthey agglutinate spontaneously inthe presence of d i luent , inde-pendent of the specificity of the re-agent antibody.

BB. All rights reserved.


Specimen-Related Problems in TestingSerum

Serum grouping tests are also subject tofalse results.

1. Small fibrin clots that may be mis-taken for agglutinates may be seenin ABO tests if plasma or incom-pletely clotted serum is used.

2. Abnormal concentrations of pro-teins, altered serum protein ratios,or the presence of intravenous con-trast materials of high molecularweight plasma expanders can causenonspecific red cell aggregation thatis difficult to distinguish from trueagglutination.

3. Antibodies other than anti-A andanti-B in a test sample can aggluti-nate reagent A1 or B red cells if theycarry the corresponding antigen.

4. Antibodies to constituents of the di-luents used to preserve reagent A1

and B red cells can agglutinate thecells independent of ABO antigensand antibodies.

5. Patients who are immunodeficientdue to disease or therapy may havesuch depressed immunoglobulinlevels that there is little or no ABOagglutinin activity. Samples fromelderly patients whose antibody lev-els have declined with age or frompatients whose antibodies have beengreatly diluted by plasma exchangeprocedures may also have unexpect-edly weak agglutinins.

6. Negative or weak results are seenin serum tests from infants under4-6 months of age. Serum fromnewborns is not usually tested,since antibodies present are gener-ally passively transferred from themother.

7. Very high-titer complement-bind-ing anti-A and anti-B may cause somany C1 molecules to associate withthe red cell surface that binding tothe membrane antigens is sterically


hindered and agglutination does notoccur. This phenomenon has beenreported in serum grouping tests us-ing red cells suspended in diluentsthat lack EDTA.16

8. If the patient has received a bonemarrow transplant of a compatiblebut dissimilar ABO group, serum an-tibodies will not agree with red cellantigens. For example, a group Aindividual transplanted with groupO marrow will have circulatinggroup O red cells, but produce onlyanti-B in the serum.

9. Recent transfusion with plasmacomponents containing ABO agglu-tinins may cause unexpected reac-tions.

Resolving ABO DiscrepanciesThe first step in resolving an apparentproblem should be to repeat the tests onthe same sample. If initial tests wereperformed on red cells suspended in se-rum or plasma, repeat testing should usea saline suspension of washed cells. If thediscrepancy persists, the following pro-cedures can be incorporated into the in-vestigation.

1. Obtain a new blood specimen fromthe donor unit or patient and testthe new sample. This should resolvediscrepancies due to mislabeled orcontaminated specimens.

2. Wash the test and reagent red cellsseveral times to remove any serumor chemical constituents that maybe causing unexpected positive reac-tions.

3. Test the red cells with anti-A,B, anti-A1, or anti-H as appropriate for theindividual problem.

4. If anti-A1 is suspected, test the se-rum against several examples ofgroup A2 red cells.

5. Review results of the antibodyscreening test against group O redcells to detect interfering effects



from nonspecific cold-reactive al-loantibodies.

6. Incubate tests for 30 minutes atroom temperature to facilitate thedetection of weak antigens or anti-bodies. Tests can be subjected toeven lower temperatures, but paral-lel tests with group O and autolo-gous cells should be observed to ruleout interference by broadly reactiveagglutinins, such as anti-I or anti-H,that react with the red cells of alladults.

Resolving Discrepancies Due toAbsence of Expected Antigens

Red cells of most A or B people arestrongly agglutinated (3–4+) by the cor-responding reagent antibody, and the se-rum of these samples usually stronglyagglutinates A1 or B reagent red cells(2–4+). The cause of a discrepancy cansometimes be inferred from the strengthof reactions obtained in red cell or serumgrouping tests. For example, serum thatstrongly agglutinates group B red cellsbut not A1 cells probably comes from agroup A person, even though the redcells are not agglutinated by anti-A oranti-B. A or B antigens may be weaklyexpressed on cells from individuals whohave inherited variant alleles, or withdisease-related antigen depression. Thefollowing procedures can be used to en-hance detection of weakly expressed an-tigens.

1. Incubate washed red cells with anti-A and anti-A,B for 30 minutes atroom temperature to increase asso-ciation of antibody with the scantamount of antigen. Incubating thetest system at 4 C may further en-hance antibody attachment, buttests performed at 4 C must be con-trolled with group O and autologousred cells to ensure that reactions ob-served are due to anti-A and anti-B


and not to other cold-reactive agglu-tinins.

2. Treat the patient’s red cells with aproteolytic enzyme such as ficin, pa-pain, or bromelain. Enzyme treat-ment increases the antigen-anti-body reaction with anti-A or anti-B.In some instances, reactions be-tween reagent antibody and red cellsexpressing antigens will become de-tectable at room temperature within30 minutes if enzyme-treated redcells are employed. Enzyme-treatedgroup O red cells must be tested inparallel as control for the specificityof the ABO reaction.

3. Incubate an aliquot of red cells atroom temperature or at 4 C withhuman anti-A or anti-B (as appro-priate) to adsorb antibody to thecorresponding red cell antigens.Anti-A1 lectin or monoclonal re-agents should not be used for ad-s o r p t i o n / e l u t i o n s t u d i e s , an dgroup O red cells should be sub-jected to parallel adsorption andelution to serve as a control. Fol-lowing incubation, wash the redcells thoroughly and prepare aneluate. (See Method 2.4.) Test theeluate against group A1, B, and Ocells. If the red cells carried the Aantigen, the eluate will agglutinateA1, but not B or O red cells. Eluatesprepared from group B red cellswill agglutinate only other B redcells. The eluate prepared fromgroup O cells should be nonreac-tive. Activity in the control groupO eluate invalidates results ob-tained with the patient’s red cells,and can mean either the anti-A oranti-B serum contained other anti-bodies, or that the adsorption/elu-tion procedure was not performedcorrectly.

4. Test the saliva for the presence of Hand A or B substances (see Method2.5 for hemagglutination-inhibition



tests for salivary antigens). Salivatests help resolve ABO discrepanciesonly if the person is a secretor, butthis may not be known until aftertesting is completed.

Resolving Discrepancies Due toUnexpected Reactions with Anti-A andAnti-BRed cell grouping tests sometimes giveunexpected positive reactions. For ex-ample, reagent anti-A may weakly ag-glutinate red cells from a sample inwhich the serum gives reactions ex-pected of a normal group B or O. Vari-ant alleles at the ABO locus or prob-lems unrelated to the actions of ABOgenes may be responsible. The follow-ing paragraphs describe some eventsthat can cause unexpected reactions inABO grouping tests and the steps thatcan be taken to identify them.

Acquired B Phenotype. A serum con-taining strong anti-B and red cells ag-glutinated strongly by anti-A and weaklyby anti-B suggests the acquired B state.The acquired B phenotype arises whenmicrobial deacetylating enzymes modifythe A antigen by altering the A-deter-mining sugar (N-acetylgalactosamine)so that it resembles the B-determininggalactose. Only A1 red cells exhibit ac-quired B activity in vivo. The acquired Bantigen develops at the expense of A, soa concomitant decrease in the strengthof A may be seen in the acquired B state.If sufficiently numerous, acquired B an-tigens cause the red cells to be aggluti-nated by human anti-B. Most red cellswith acquired B antigens react weaklywith anti-B, but occasional examples areagglutinated quite strongly. Behaviorwith monoclonal anti-B reagents varieswith the particular clonal products used.Acquired B antigens have been observedwith increased frequency in tests withcertain FDA-licensed monoclonal anti-Bblood grouping reagents.17 To confirm


that group A1 red cells carry the acquiredB structure:

1. Check the patient’s diagnosis. Ac-quired B antigens are usually associ-ated with tissue conditions that al-low colonic bacteria to enter thecirculation, but acquired B antigenshave been found on the red cells ofapparently normal blood donors.17

2. Test the patient’s serum againstautologous red cells. The individ-ual’s anti-B will not agglutinate hisor her own red cells that carry theacquired B.

3. Test the red cells with monoclonalanti-B reagents for which the manu-facturer’s instructions give a de-tailed description. Unlike human-source some monoclonal antibodiesdo not react with the acquired Bphenotype; this information may beincluded in the package insert.

4. Test the red cells with human anti-Bserum that has been acidified to pH6.0. Acidified human anti-B nolonger reacts with the acquired Bantigen.

5. If the patient is a secretor, test salivafor the presence of A and B. Secre-tors whose red cells carry acquiredB structures will have A, but not B,substance in their saliva.

6. Treat the red cells with acetic anhy-dride, which reacetylates the surfacemolecules and markedly diminishesthe reactivity of acquired B red cells.Reactivity of normal group B anti-gens is not affected by acetic anhy-dride.

Acquired A-Like Antigens. ABO dis-crepancies can be seen in the conditionknown as Tn polyagglutination, in whichdefective synthesis of oligosaccharidesnormally present on sialoglycoproteinmolecules leave abnormal antigenicstructures (cryptantigens) exposed onthe red cell surface. The residual sugaruncovered is N-acetylgalactosamine, thesugar that confers A specificity. A so-



matic mutation on hematopoietic stemcells results in a permanent populationof cells described as Tn-activated. GroupO or group B cells that have been Tn-ac-tivated behave as if they have acquired anA antigen, which reacts with human ormonoclonal anti-A reagents. The A-likeantigen of Tn-activated red cells can bedifferentiated from the A produced bythe A-gene transferase if red cells aretreated with proteolytic enzymes beforetesting. Proteolytic enzymes degrade themolecule that expresses the cryptan-tigen, abolishing the reactivity withanti-A.

Other forms of polyagglutination mayinterfere in ABO grouping tests. SeeMethod 2.10 to classify polyagglutinablered cells using lectins.

Mixed-Field Agglutination. Occa-sional samples are encountered thatcontain two distinct, separable popula-tions of red cells. Usually this reflectsrecent transfusion of group O red cellsto a non-O recipient or receipt of a bonemarrow transplant of an ABO group dif-ferent from the patient’s own. Red cellmixtures also occur in a condition calledblood group chimerism, resulting eitherfrom intrauterine exchange of erythro-poietic tissue by fraternal twins or frommosaicism arising through dispermy. Inall such circumstances, ABO red celltests may give a mixed-field pattern ofagglutination. Mixed-field reactions dueto transfusion last only for the life of thetransfused red cells. After bone marrowtransplantation, the mixed-field reac-t ion usual ly d isappears when thepatient’s own red cells are no longer pro-duced. Persistent mixed-cell popula-tions do occur in some bone marrowrecipients.18 Mixed-field reactions thatarise through blood group chimerismpersist throughout the life of the indi-vidual.

Mixed-field agglutination is charac-teristic of the reaction between A3 redcells and reagent anti-A. If the aggluti-


nated red cells are removed and the re-maining red cells again tested with anti-A, mixed-field agglutination occurs inthe residual, previously nonagglutinatedpopulation.

Antibody-Coated Red Blood Cells.Red cells from infants with HDN, orfrom adults suffering from autoimmuneor alloimmune conditions, may be soheavily coated with IgG antibody mole-cules that they agglutinate spontane-ously in the presence of reagent diluentscontaining high protein concentrations.Usually this is at the 18-22% rangefound in some anti-D reagents, butsometimes the sensitized red cells alsoagglutinate in ABO reagents with pro-tein concentrations of 6-12%. Gentleelution at 45 C can be used to removemuch of this antibody from the red cellsso they can be tested reliably with anti-Aand anti-B.

Red cells from a specimen containingcold-reactive IgM autoagglutinins mayagglutinate spontaneously in salinetests. The antibodies can usually be re-moved by incubating the cell suspensionbriefly at 37 C and then washing severaltimes with saline warmed to 37 C. If theIgM-related agglutination is not dis-persed by this technique, the red cellscan be treated with the sulfhydryl com-pound dithiothreitol (DTT) (see Method2.12).

Resolving Discrepancies Due toUnexpected Serum Reactions

The following paragraphs describe someevents that can cause unexpected or er-roneous serum test results, and the stepsthat can be taken to resolve them.

1. Immunodeficient patients may notproduce detectable levels of anti-Aand anti-B. These antibodies are ab-sent from the serum of newbornsand may be absent or very weak inserum from normal elderly persons.



2. Abnormally high concentrations ofanti-A and anti-B have caused pro-zone reactions that lead to false-negative results. In these instancesthe ABO group can be inferred by redcell tests, by dilution of the serum,or by the use of EDTA (2.5%)-treatedserum.16

3. Anti-A1 in the serum of individualsof A2, A2B, or other subgroups agglu-tinate A1 reagent red cells. To dem-onstrate this as the cause of the dis-crepancy:a. Test the serum against several ex-

amples of group A1, A2, and O redcells, preferably three of each.Only if the antibody agglutinatesall A1 red cells and none of the A2

or O red cell samples can it becalled A1.

b. Use anti-A1, either Dolichos bi-florus lectin or absorbed humangroup B serum, to demonstratethat the individual’s cells belongto a non-A1 subgroup.

Most examples of anti-A1 reactonly at temperatures below 30 Cand are considered clinically in-significant. Some examples havereactivity at 37 C and should beconsidered clinically significant;only A2 or O red cells should beused for transfusion.

4. Cold-reactive autoagglutinins, suchas anti-I and anti-IH can, if suffi-ciently strong, agglutinate red cellsof all adults, including autologouscells and reagent red cells. With fewexceptions, agglutination caused bythe cold-reactive autoagglutinin isweaker than that caused by anti-Aand anti-B. The following can bedone when such reactivity interferesto the point that interpretation ofserum tests is difficult.a. Warm serum and reagent red

cells to 37 C before mixing andtesting. Read serum tests at 37 Cand convert them to the an-


tiglobulin phase if necessary.Weakly reactive examples of anti-A or anti-B may not be detectedby this method, however, because37 C is above the reactive opti-mum for these antibodies. Ingroup A and B individuals, ABOantibodies are predominantly ofthe IgM class, which will not bedetected in routine antiglobulintests that employ anti-IgG re-agents. Examples of anti-A andanti-B with little or no IgG com-ponent can go undetected underthese conditions.

b. Remove the cold-reactive autoag-glutinin from the serum using acold autoadsorption method asdescribed in Method 6.1. The ad-sorbed serum can then be testedagainst A1 and B reagent red cells.

c. Treat the serum with DTT (seeMethod 4.3) and use the treatedserum for reverse-grouping tests.Because DTT destroys the agglu-tinating activity of IgM antibod-ies, tests that use DTT-treated se-rum must be converted to theantiglobulin phase, in which IgGforms of the antibodies will bedetected. Because many group Aor B persons do not producemore than minute quantities ofIgG anti-A or anti-B, negativetests should be interpreted withcaution.

5. Unexpected alloantibodies that reactat room temperature, such as anti-P1 or anti-M, may agglutinate thered cells used in serum tests if theycarry the corresponding antigen.Ordinarily, one or more of the re-agent cells used in the antibody de-tection test will also be agglutinatedat room temperature, but a rare se-rum may react with an antigen onthe serum testing cells that is notpresent on cells used for antibodydetection. Steps to determine the



correct ABO type of sera containingother cold-reactive alloantibodiesinclude:a. Raising the temperature to 30-37

C before mixing the serum andcells. If the thermal optimum ofthe alloantibody is below thetemperature at which anti-A andanti-B react, this may resolve thediscrepancy.

b. Identify the alloantibody, as de-scribed in Chapter 17, and testthe reagent A1 and B cells to de-termine which, if either, carriesthe corresponding antigen. Ob-tain A1 and B red cells that lackthe antigen and use these for se-rum testing.

c. If the antibody detection test isnegative, test the serum againstseveral examples of A1 and B redcells. The serum may contain anantibody directed against an anti-gen of low incidence, which willbe absent from most randomlyselected A1 and B red cells.

6. Serum from patients with abnor-mal concentrations of serum pro-teins, or altered serum protein ra-tios or who have received plasmae x p a n d e r s o f h i gh m o le cu l a rweight, can aggregate reagent redcells and mimic agglutination.Some of these samples cause ag-gregation of the type described asrouleaux. Rouleaux formation iseasily recognized on microscopicexamination if the red cells assumewhat has been described as a “stackof coins” formation. More often,the aggregates appear as irregu-larly shaped clumps that closely re-semble antibody-mediated aggluti-nates. The results of serum testscan often be corrected by dilutingthe serum 1:3 in saline, to abolishits aggregating properties, or byusing a saline replacement tech-nique (see Method 3.4).


The H SystemThe H system has two genes, H and h,and one antigen, H, which serves as theprecursor molecule on which A and Bantigens are built. On group O red cells,there is no A or B, and the membraneexpresses abundant H. The amount of Hantigen is, in order of diminishing quan-tity, O>>A2 >>B>>A2B>>A1>>A1B. Asfor A and B configurations, H-like anti-gens are found in nature. Individuals ofthe rare Oh phenotype, whose red cellslack H, have anti-A and anti-B, and apotent and clinically significant anti-Hin their serum. Occasionally, group A1,A1B, or (less commonly) B persons haveso little unconverted H antigen on theirred cells that they produce anti-H, butthis form of anti-H is relatively weak,virtually always reacts at room tempera-ture or below, and is not consideredclinically significant.

Oh PhenotypeThe term “Bombay” has been used forthe phenotype in which red cells lack H,A, and B, because examples of such redcells were first discovered in Bombay,India. The symbol Oh has been selectedto denote the phenotype because resultsof routine ABO typing tests mimic thoseof group O persons. Oh red cells are notagglutinated by anti-A, anti-B, or anti-A,B, and the serum contains strong anti-A and anti-B. The Oh phenotype becomesapparent when serum from the Oh indi-vidual is tested against group O red cells,and strong, immediate reactions occur.The anti-H of an Oh individual reactsover a thermal range of 4-37 C with allred cells except those of other Oh people.Oh patients must be transfused with onlyOh blood because their non-red-cell-stimulated antibodies rapidly destroycells with A, B, or H antigens. The Oh

phenotype is demonstrated by the ab-sence of reaction when cells are exposed



to the anti-H lectin of Ulex europeaus. Ifother examples of Oh red cells are avail-able, further confirmation can be ob-tained by demonstrating compatibilityof the serum with Oh red cells and noothers.

Para-Bombay Phenotypes, Ah, Bh, andABh

Ah, Bh, and ABh red cells lack serologi-cally detectable H antigen but carrysmall amounts of A and/or B antigen,depending on the individual’s genes atthe ABO locus. Tests with anti-A or anti-B give weak reactions, but the cells arenonreactive with anti-H lectin or withanti-H serum from Oh persons. The para-Bombay phenotype is thought to reflectthe presence of variant H genes that pro-duce only minute amounts of H antigen,all of which undergoes conversion to Aor B by the products of the A and B genes,respectively. The serum of Ah and Bh peo-ple contains anti-H in addition to theexpected anti-A or anti-B.

The Lewis SystemThe Lewis system has two genes, Le and le,and two main antigens, Lea and Leb. TheLewis antigens are not intrinsic to redcells, but are expressed on glycosphin-golipids that are adsorbed from plasma tothe red cell membranes. Their presence inplasma (and consequently on red cells)depends on the product of the Le gene, afucosyltransferase that adds Fuc inα(1→4) linkage to the subterminalGlcNAc of Type 1 oligosaccharides (see Fig12-2). In Type 2 chains, the number fourcarbon is not available for fucose attach-ment so Type 2 chains never express Lewissystem activity. The oligosaccharides pres-ent on molecules intrinsic to the red cellmembrane are all Type 2.


Genes and AntigensAntigens of the Lewis system are con-structed by addition of fucose moieties tothe Type 1 oligosaccharide. The product ofthe Le gene attaches fucose in α(1→4)linkage to the subterminal GlcNAc; thisconfiguration has Lea activity. The Se genedetermines a transferase that attaches afucose in α(1 → 2) linkage to the terminalGal, but only if the adjacent GlcNAc isalready fucosylated. The configurationwith two fucose moieties has Leb activity.Thus Leb reflects the presence of both theLe and Se genes; Le without Se results inLea activity; Se without Le has no conse-quences in terms of Lewis system activity.

Plasma glycosphingolipids are ad-sorbed to the red cell membrane; if Lewisactivity is present, adsorption will conferLewis reactivity on the circulating cells.Leb is adsorbed preferentially over Lea, sothe individual possessing both Le and Segenes will have red cells that express Leb

but not Lea. Red cells from persons withLe but not Se will express Lea. The individ-ual homozygous for the amorphic allele lewill have no Lewis reactivity on red cellsor on Type 1 oligosaccharides in otherbody sites. Table 12-4 shows phenotypes ofthe Lewis system and their frequencies inthe population. Red cells that type asLe(a+b+) are only rarely found when hu-man antisera are used in blood grouping.Such red cells are seen more frequentlywhen more potent monoclonal anti-Lea

and anti-Leb reagents are used.19

Table 12-4. Phenotypes andFrequencies in the Lewis System

Reactionswith Anti-

Adult PhenotypeFrequency %

Lea Leb Phenotype Whites Blacks+ 0 Le(a+b–) 22 230 + Le(a–b+) 72 550 0 Le(a–b–) 6 22+ + Le(a+b+) rare rare



Lewis AntibodiesLewis antibodies occur almost exclu-sively in the serum of Le(a–b–) individu-als, usually without known red cellstimulus. Those people whose red cellphenotype is Le(a–b+) do not make anti-Lea because small amounts of uncon-verted Lea are present in their saliva andplasma. It is unusual to find anti-Leb inthe serum of a Le(a+b–) individual, butanti-Leb may exist along with anti-Lea inthe serum of Le(a–b–) individuals. Lewisantibodies are predominantly IgM anddo not cross the placenta. Because ofthis, and because Lewis antigens arepoorly developed at birth, the antibodieshave not been implicated in HDN. Lewisantibodies may bind complement, andfresh serum that contains anti-Lea (orinfrequently anti-Leb) may hemolyze in-compatible red cells in vitro. Hemolysisis more often seen with enzyme-treatedred cells than with untreated red cells.

Serologic CharacteristicsMost Lewis antibodies agglutinate sa-line-suspended red cells of the appropri-ate phenotype. The resulting aggluti-nates are often fragile and are easilydispersed if red cell buttons are not re-suspended gently after centrifugation.Agglutination sometimes is seen afterincubation at 37 C, but rarely of the

Table 12-5. Serologic Behavior of theand P Blood Groups

In-VitroSaline

Antibody Hemolysis 4 C 22 Canti-Lea some most most santi-Leb rare most mostanti-P1 occ. most someanti-P some most some santi-P1+P+Pk some most some s


strength seen in tests incubated at roomtemperature. Some examples of anti-Lea,and less commonly anti-Leb, can be de-tected in the antiglobulin phase of test-ing. Sometimes this reflects comple-ment bound by the antibody; in othercases, antiglobulin reactivity resultsfrom an IgG component of the antibody.

Sera with anti-Leb activity can be di-vided into two categories. The morecommon type reacts best with Le(b+) redcells of group O and A2; these antibodieshave been called anti-LebH. Antibodiesthat react equally well with the Leb anti-gen on red cells of all ABO phenotypesare called anti-LebL. Anti-LebH, but notanti-LebL, can be neutralized by salivafrom group O persons who are secretorsof H substance, but are Le (a–b–). Table12-5 lists the serologic behavior of thecommon Lewis system antibodies.

Recognition of Other Antigens

Two other antibodies (anti-Lec and -Led)have been given Lewis designations butthe determinants with which they reactare glycosphingolipids with Type 1 oli-gosaccharides from Le (a–b–) persons.Anti-Lec reacts with unsubstituted Type1 chains, on which neither the Gal northe GlcNAc is linked to fucose. Anti-Led

reacts with Type 1 chains from lele per-

Principal Antibodies of the Lewis

Albumin Enzyme AssociatedWith

37 C AGT 37 C AGT HDN HTRome many most most no fewfew some some some no noocc. rare some few no rareome some some some no ?ome some some some rare ?



sons who possess Se; the configurationis H antigen expressed on Type 1 chains.

Transfusion Practice

Lewis antigens readily adsorb to andelute from red cell membranes. Trans-fused red cells shed their Lewis antigensand assume the Lewis phenotype of therecipient within a few days of enteringthe circulation. Lewis antibodies in arecipient’s serum are readily neutralizedby Lewis blood group substance in donorplasma. For these reasons, it is exceed-ingly rare for Lewis antibodies to causehemolysis of transfused Le(a+) or Le(b+)red cells. However, antibodies that causehemolysis in vitro or that give strongreactions in the antiglobulin phase ofthe crossmatch have been associatedwith posttransfusion hemolysis.

It is not considered necessary to typedonor blood for the presence of Lewisantigens before transfusion or whencrossmatching for recipients with Lewisantibodies. Reactions seen on cross-matches with anti-IgG reagents, with orwithout prewarming, provide a good in-dex of transfusion safety.20

Lewis Antigens in Children

Red cells from newborn infants usuallyreact with neither human anti-Lea noranti-Leb and are considered to beLe(a–b–). Some can be shown to carrysmall amounts of Lea when tested withpotent monoclonal or goat anti-Lea re-agents. Reliable Lewis grouping ofyoung children may not be possible, astest reactions may not reflect the correctphenotype until approximately 6 years ofage. Among children, the incidence ofLe(a+) red cells is high and that ofLe(b+) red cells low. The phenotypeLe(a+b+) may be transiently observed inchildren whose phenotypes as adults willbe Le(a–b+).


Cord red cells are agglutinated by anantibody called anti-Lex, which aggluti-nates adult red cells of Le(a+b–) andLe(a–b+), but not Le(a–b–) pheno-types.21 In serologic tests, this antibodybehaves as if it contains an inseparablecombination of anti-Lea and -Leb. Thedeterminant it defines has been calledLex, which is present on the majority ofcord red cells and on adult red cells thatexpress either Lea or Leb. Anti-Lex is nota more potent or more avid form of anti-Lea. The Lex determinant is the Type 2oligosaccharide to which Fuc is attachedin α(1→3) linkage to GlcNAc; it repre-sents a class of antigens that are devel-opmentally regulated embryonic oli-gosaccharides.22 The sialylated form ofLex appears to be a ligand for the adhe-sion receptor called endothelial leuko-cyte adhesion molecule 1 (ELAM-1) orE-selectin, and thus may play a role incertain metastatic cancers.23

The I/i Antigens andAntibodiesThe unexpected antibodies most fre-quently encountered in serologic testsperformed at room temperature are anti-H and anti-I. The antigens reside in thesubterminal portions of the oligosaccha-rides that eventually are converted to H,A, or B antigens and are found on mem-brane-associated glycoproteins and gly-cosphingolipids. Both Type 1 and Type 2oligosaccharide chains include multipleunits o f Galβ(1→4)GlcNAcβ(1→3).24

Two of these disaccharide units linked in astraight chain oligosaccharide constitutethe i antigen. On the red cells of adultsmany of these linear chains are modifiedby the addition of branched structuresconsisting of Galβ(1→4)GlcNAc linkedin β(1→6) to a galactose residue internalto the repeating sequence. The branch-ing configuration confers I specificity.



Different examples of anti-I appear torecognize different portions of thebranched oligosaccharide chain.

Fetal red cells carry few branched oli-gosaccharides and, therefore, are rich in iand poor in I. During the first 2 years oflife, I antigen gradually increases at theexpense of i. The red cells of most adultsare strongly reactive with anti-I and reactweakly or not at all with anti-i. Rare adultsexist whose red cells are I–negative. Theirsera usually contain anti-I, but activitymay be so weak as to require enzyme tech-niques for detection.

Antibodies to I/iAnti-I characteristically agglutinates, atroom temperature, red cells from nearlyall adults, but does not react with redcells from cord blood or from I-negativeadults. If tests are performed at 4 C,serum from many I-positive people canbe shown to have autoanti-I. Anti-I is acommon autoantibody, but it usually be-haves as a cold-reactive agglutinin, act-ing within a narrow thermal range and,even at 4 C, at a titer no higher than 64.Anti-I assumes pathologic significancein cold agglutinin disease or mixed-typeautoimmune hemolytic anemia, inwhich it behaves as a complement-bind-ing antibody with a high titer and widethermal range (see Chapter 16) .Autoanti-i is less often implicated insymptomatic disease than anti-I. On rareoccasions, anti-i may be seen as a rela-tively weak cold autoagglutinin reactingonly at 4-10 C. Patients with infectiousmononucleosis often have transient butpotent anti-i.

Table 12-6 illustrates the serologic be-havior of anti-I and anti-i at 4 C and 22 C.Reaction strengths should be consideredrelative; clear-cut differences in reactivitybetween the two are seen only with weakerexamples of the antibodies. Titration stud-ies may be needed to differentiate strongexamples of the antibodies.


Serum containing anti-I or anti-i issometimes reactive at the antiglobulinphase of testing. Such reactions rarelyindicate antibody activity at 37 C; rather,the reaction is between anticomplementin a polyspecific antiglobulin reagentand complement components boundwhen serum and cells interact at lowertemperatures. The complement remainsbound to cell surfaces after the antibodydissociates during 37 C incubation. Theproblem can usually be avoided either bywarming the serum and red cells to 37 Cbefore combining them and then keep-ing them strictly at 37 C for all phases oftesting, including centrifugation andwashing, or by omitting any potentiator(if used) and using anti-IgG instead ofpolyspecific antihuman globulin.

Complex ReactivitySome antibodies appear to recognizebranched oligosaccharides that havebeen further modified by, for example,transferases for H and P1. Anti-IH occursquite commonly in the serum of A1 indi-viduals; it reacts strongly with red cellsthat have high levels of H as well as I.Thus, it reacts very little with adultgroup A1 cells or cord cells of any group,and very strongly with adult group Ocells. Anti-IH should be suspected whenserum from a group A patient causesdirect agglutination of all cells used for

Table 12-6. Serologic Behavior ofthe I Blood Group Antibodies withSaline Red Cell Suspensions

Anti-I Anti-i4 C Iadult 4+ 0-1+

icord 0-2+ 3+iadult 0-1+ 4+

22 C Iadult 2+ 0icord 0 2-3+iadult 0 3+



antibody detection, but is compatiblewith all or most group A donor blood.

The P Blood Group andRelated AntigensThe first antigen of the P blood groupwas discovered by Landsteiner andLevine in 1927, in a series of animalexperiments that led also to the discov-ery of M and N. Originally called P, thename of the antigen was later changed toP1. The designation P has since been reas-signed to an antigen present on almostall human red cells. The InternationalSociety for Blood Transfusion has re-cently made an effort to classify P anti-gens in a logical manner (see Table 12-7). The definitive antigen in this systemis P1. The P, Pk, and Luke (LKE) antigens,formerly considered part of the P sys-tem, have been assigned to the globosidecollection of antigens. Another antigen,p, has not yet been assigned to a sys-tem.25 Red cells lacking P1, but shown topossess P, are of the P2 phenotype. P1 ispresent on the red cells of approximately80% of Whites and 94% of Blacks.

BiochemistryThe antigenic determinants of the bloodgroup P system are chemically based on

Table 12-7. Phenotypes and Frequenc

Reactions With Anti-

P1 P Pk PP1

+ + 0 +0 + 0 +0 *0* 0 0+ 0 + +0 0 + +

*Usually negative, occasionally weakly positive.


Galα(1→4)Gal. P1 antigen [Galα(1→4)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glcβ(1→1′)Cer] is present on red cells ofthe P1 and P1

k phenotype. P antigen[GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glcβ(1→1′)Cer] , a l so termed glo-botetrasylceramide or globoside, is pres-ent on P1 and P2 red cells. Pk antigen[Galα(1→4)Galβ(1→4)Glcβ(1→1′)Cer],also termed globotriaosylceramide orCTH, is present in low amounts on P1

and P2 and is enriched in cells of the rarephenotype P1

k and P2k. LKE, for Luke

antigen, has a structure the same as thestage-specific embryonic antigen 4[NeuAcα(2→3)Galβ(1→3)GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glcβ(1→1′)Cer]and is present in almost all red cells exceptthose of the rare phenotypes p or Pk andin about 2% of P-positive red cells. Thep red cells are devoid of P1, P, Pk and LKEantigens, but enriched in lactosyl-ceramide [Galβ(1→4)Glcβ(1→1 ′ )Cer].

All the antigens described above areexclusively expressed in glycolipids onhuman red cells, not on glycoproteins.26

The antigens are built through the addi-tion of sugars to precursor glycosphin-golipids, a process thought to be analo-gous to the development of the A, B, andH antigens. Pk determinants are formedwhen Gal is added in α(1→4) linkage tothe terminal Gal residues of lactosyl-ceramide.27 Subsequently, this structurecan be converted to P globoside by the

ies in the P Blood Group

Phenotype Frequency %

Pk Phenotype Whites Blacks

P1 79 94P2 21 6p

P1k All extremely rare

P2k



addition of GalNAc in β(1→3) linkage.Neither Pk nor P serve as substrate for P1.Instead, Gal is added in α(1→4) linkageto a different structure called paraglo-boside to form P1. Paragloboside is theprecursor on which cellular A, B, H, I,and i antigens are constructed. The pantigen results from addition of NeuNActo the terminal galactose in α(2→3). Thegene specifying the responsible trans-ferase is thought to be independent ofthe P system. The biochemical struc-tures of these antigens are shown in Fig12-5.

Figure 12-5. Biochemical structures of P antig


Rare PhenotypesSeveral rare phenotypes are associatedwith the P blood groups and these areshown in Table 12-7. The Pk phenotypeoccurs when the Pk antigen is not con-verted to P. Individuals whose red cellshave Pk instead of P consistently makea strong alloreactive anti-P that reactsin tests with P1+P+ (P1 phenotype) andP1–P+ (P2 phenotype) red cells. Thebiphasic hemolysin of paroxysmal coldhemoglobinuria (PCH) is often, butnot always, of this specificity. Unlikethe IgM anti-P in the serum Pk persons,

ens.28



the autoreactive anti-P found in peoplewith PCH is IgG.29

Very rare people lack P1, P, and Pk. Redcells from these individuals are said to beof the p phenotype. Characteristically, apotent hemolytic IgM antibody withanti-P1+P+Pk specificity is found in theirserum. The antibody, formerly calledanti-Tja, has caused hemolytic transfu-sion reactions and, occasionally, HDN.29

There is a curious association betweenanti-P1+P+Pk and abortions occurringearly in pregnancy in p women.30

Anti-P1

The serum of P2 persons commonly con-tains anti-P1. If sufficiently sensitivetechniques are applied, it is likely thatanti-P1 would be detected in the serumof virtually every P2 person. The anti-body reacts optimally at 4 C but mayoccasionally be detected at 37 C. As anti-P1 is nearly always IgM, it does not crossthe placenta and has not been reportedto cause HDN, but has, rarely, been re-ported to cause hemolysis in vivo.29,31

The strength of the P1 antigen variesamong different red cell samples, andantigen strength has been reported todiminish when red cells are stored.These characteristics sometimes createdifficulties in identifying antibody speci-ficity in serum with a positive antibodyscreen. An antibody that reacts weakly inroom temperature testing can often beshown to have anti-P1 specificity by in-cubation at lower temperatures or by theuse of enzyme-treated red cells. Hydatidcyst fluid or P1 substance derived frompigeon eggs inhibits the activity of anti-P1. Inhibition may be a useful aid toantibody identification, especially if it ispresent in a serum with antibodies ofother specificities.

It has recently been shown that redcells of P1

k and P2k phenotypes can be

agglutinated by the meningitis-causingbacterium Streptococcus suis32; the P


antigen has been shown to serve as areceptor for parvovirus B19, whichcauses erythema infectiosum.33 Indeed,persons of p phenotype who lack glo-boside are naturally resistant to infec-tion with this pathogen. These recentfindings implicate the P blood group sys-tem in the pathogenesis of these dis-eases.33

References1. Landsteiner K. Zur Kenntnis der antifermen-

tativen, lytischen und agglutinierendenWirkungen des Blutserums und der Lymph.Zbl Balk 1900;27:367.

2. Yamamoto F, Clausen H, White T, et al. Mo-lecular genetic basis of the histo-blood groupABO system. Nature 1990;345:229-32.

3. Larsen RD, Enst LK, Nair RP, Low JB. Molecu-lar cloning sequence, and expression of a hu-man GDP-L-fucose: β-D-galactose 2-α-L-fuco-syltransferase cDNA that can form the H bloodgroup antigen. Proc Natl Acad Sci USA1990;87:6674-8.

4. Anstee DJ. Blood-group active surface mole-cules of the human red blood cell. Vox Sang1990;58:1-20.

5. Bermeman Z, van Bockstaele DR, Uytten-broeck WM, et al. Flow cytometric analysis oferythrocyte blood group A antigen densityprofiles. Vox Sang 1991;61:265-74.

6. Clausen H, Levery SB, Nudelman E, et al.Repetitive A epitope (type 3 chain A) definedby group A1-specific monoclonal antibody TH-1: Chemical basis of qualitative A1 and A2 dis-tinction. Proc Natl Acad Sci USA 1985;82:1199-203.

7. Yamamoto F, Hakomori S. Sugar-nucleotidedonor specificity of histo-blood group A and Btransferases is based on amino acid substitu-tions. J Biol Chem 1990;265:19257-62.

8. Yamamoto FI. Review: Recent progress in themolecular genetic study of the histo-bloodgroup ABO system. Immunohematology1994;10:1-7.

9. Dzik W. DNA testing of blood group polymor-phisms. In: Silberstein LE, ed. Molecular andfunctional aspects of blood group antigens.Bethesda, MD: American Association of BloodBanks, 1995:1-40.

10. Mulet C, Cartron JP, Badet J, Salmon C. Activ-ity of α-2-L-fucosyltransferase in human seraand red cell membranes. A study of commonABH blood donors, rare “Bombay” and



“Parabombay” ind iv i dua l s . FEBS Lett1977;84:74.

11. Beattie KM. Discrepancies in ABO grouping.In: A seminar on problems encountered inpretransfusion tests. Washington, DC: Ameri-can Association of Blood Banks, 1972:129-65.

12. Code of federal regulations. Title 21 CFR, part660.26. Washington, DC: US GovernmentPrinting Office, 1992 (revised annually).

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

14. Yates AD, Feeney J, Donald ASR, Watkins WM.Characterisation of a blood group A-activetetrasaccharide synthesized by a blood-groupB-gene-specified glycosyltransferase. Car-borhydr Res 1984;130:251-60.

15. Beck ML, Yates AD, Hardman J, Kowalski MA.Identification of a subset of group B donorsreactive with monoclonal anti-A reagent. AmJ Clin Pathol 1989;92:625-9.

16. Judd WJ, Steiner EA, Oberman HJ. Reverseand typing errors due to prozone: How safe isthe immediate spin crossmatch? (abstract).Transfusion 1987;27:527.

17. Beck ML, Kowalski MA, Kirkegaard JR, KorthJL. Unexpected activity with monoclonal anti-B reagents. Immunohematology 1992;8:22-3.

18. Branch DR, Gallagher MT, Forman SJ, et al.Endogenous stem cell repopulation resultingin mixed hematopoietic chimerism followingtotal body irradiation and marrow transplan-tation for acute leukemia. Transplantation1982;34:226-81.

19. Longworth C, Rolih S, Moheng M, et al. Mousemonoclonal anti-Lea and anti-Leb as routinegrouping reagents (abstract). Transfusion1985;25:446.

20. Waheed A, Kennedy MS, Gerhan S. Transfu-sion significance of Lewis system antibodies:Report on a nationwide survey. Transfusion1981;21:542-5.

21. Arcilla MC, Sturgeon P. Lex, the spurned anti-gen of the Lewis blood group system. Vox Sang1974;26:425-38.

22. Stoolman, LM. Adhesion molecules control-ling lymphocyte migration. Cell 1989;56:907-10.

23. Polley MJ, Phillips ML, Wayner E, et al. CD62and endothelial cell-leukocyte adhesion mole-cule 1 (ELAM-1) recognize the same carbohy-drate ligand, sialyl-Lewis X. Proc Natl Acad SciUSA 1991;88:6224.

24. Feizi T. The blood group Ii system: A carbohy-drate antigen system defined by naturallymonoclonal or oligoclonal autoantibodies ofman. Immunol Commun 1981;10:127-56.

25. Daniels GL, Anstee DJ, Cartron JP, et al. Bloodgroup terminology 1995. From the ISBT work-ing party on terminology for red cell surfaceantigens. Vox Sang 1995;69:265-79.


26. Yang Z, Bergstrom J, Karlsson KA. Glycopro-teins with Galα4Gal are absent from humanerythrocyte membranes, indicating that gly-colipids are the sole carriers of blood group Pactivities. J Biol Chem 1994;269:14620-4.

27. Naiki M, Marcus DM. An immunochemicalstudy of human blood group P1, P and Pk

glycosphingolipid antigens. Biochemistry1975;14:4837-41.

28. Anstall HB, Blaycock RC. The P blood groupsystem: Biochemistry, genetics and clinical sig-nificance. In: Moulds JM, Woods LL, eds. Bloodgroups: P, I, Sda and Pr. Arlington, VA: AmericanAssociation of Blood Banks, 1991:1-19.

29. Mollison PL, Engelfriet CP, Contreras M. Bloodtransfusion in clinical medicine. 9th ed. Oxford:Blackwell Scientific Publications, 1993.

30. Cantin G, Lyonnais J. Anti-PP1Pk and earlyabortion. Transfusion 1983;33:350-1.

31. Chandeysson PL, Flyte MW, Simpkins SM,Holland PV. Delayed hemolytic transfusion re-action caused by anti-P1 antibody. Transfusion1981;21:77-82.

32. Haataja S, Tikkanen K, Liukkonen J, et al.Characterization of a novel bacterial adhesionspecificity of Streptococcus suis recognizingblood group P receptor oligosaccharides. JBiol Chem 1993;268:4311-7.

33. Brown KE, Hibbs JR, Gallinella G, et al. Resis-tance to parvovirus B19 infection due to lackof virus receptor (erythrocyte P antigen). NEngl J Med 1994;330:1192-6.

Suggested ReadingBeck ML. Blood group antigens acquired de novo.In: Garratty G, ed. Blood group antigens and dis-ease. Arlington, VA: American Association of BloodBanks, 1983:45-66.

Beck ML. The I blood group collection. In: MouldsJM, Woods LL, eds. Blood groups: P, I, Sda and Pr.Arlington, VA: American Association of BloodBanks, 1991:23-52.

Hakomori SI. Blood group ABH and Ii antigens ofhuman erythrocytes: Chemistry, polymorphismand their developmental change. Semin Hematol1981;18:39-47.

Hanfland P, Kordowicz M, Peter-Katalinic J, et al.Immunochemistry of the Lewis blood-group sys-tem: Isolation and structure of Lewis-c active andrelated glycosphingolipids from plasma of blood-group O Le(a–b–) nonsecretors. Arch BiochemBiophys 1986;246:655-72.

Judd WJ. Methods in immunohematology. Miami,FL: Montgomery Scientific Publications, 1988.



Lowe JB. Carbohydrate-associated blood group an-tigens: The ABO, H/Se, and Lewis loci. In: GarrattyG, ed. Immunobiology of transfusion medicine.New York: Marcel Dekker, Inc. 1994:3-36.

Race RR, Sanger R. Blood groups in man. 6th ed.Oxford: Blackwell Scientific Publications, 1975.

Salmon C, Cartron JP, Rouger P. The human bloodgroups. New York: Masson Publishing, 1984.


Watkins WM. The glycosyltransferase products ofthe A, B, H and Le genes and their relationship tothe structure of the blood group antigens. In:Mohn JF, Plunkett RW, Cunningham RK, LambertRM, eds. Human blood groups. Basel: Karger,1977:134-42.


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TM12 - ABO, H, and Lewis Blood Groups and Structurally ... · PDF fileT he ABO, H, P, I, and Lewis blood group antigens reside on structurally related carbohydrate mole-cules, and