17 Identification of Alloantibodies toother than non-red-cell-stimulated anti-A or -B are called unexpected red cell alloantibodies and may be found in 0.3-38% of the population, depending

17 Identification ofAlloantibodies toRed Cell Antigens

17

Red cell alloantibodiesother than non-red-cell-stimulated anti-Aor -B are called unexpected red cellalloantibodies and may be found in 0.3-38% of the population, depending uponthe group of patients or donors studiedand the sensitivity of the test methodsused.1,2 In contrast to red cell autoanti-bodies, which may also react with redcells from other individuals (see Chapter18), alloantibodies react only with anti-gens present on red blood cells from per-sons other than the “self.” Non-self im-munization to red cell antigens mayresult from pregnancy or transfusion, orfrom injection with immunogenic mate-rial. In some instances no immunizingevent can be identified.

Significance ofAlloantibodiesAlloantibodies to red cell antigens maybe initially detected in any test that usesa serum [including ABO testing, the an-tibody detection test (antibody screen),

Copyright © 2002 by the A

or the crossmatch] or eluate. Ordinarily,once an antibody is detected, its specific-ity should be determined and its clinicalsignificance assessed.

A clinically significant red cell anti-body is one that shortens the survival oftransfused red cells or has been associ-ated with hemolytic disease of the new-born (HDN). The degree of clinical sig-ni f icance may vary, however, evenamong antibodies with the same speci-ficity. Some antibodies cause destruc-tion of incompatible red cells withinhours or even minutes, others decreasethe survival by only a few days, and somecause no discernible cell destruction.Antibodies of some specificities areknown to cause HDN; some may cause apositive direct antiglobulin test (DAT) inthe fetus without clinical evidence ofHDN; and others do not cause HDN.

Reported experience with other ex-amples of antibody with the same speci-ficity can be used in assessing clinicalsignificance. Table 14-2 summarizes theexpected reactivity and clinical signifi-cance of commonly encountered alloan-tibodies, and Marsh et al3 have published

349ABB. All rights reserved.

350 AABB Technical Manual

a review of these and other specificities.For some antibodies, few or no data ex-ist, and decisions must be based on thepremise that clinically significant anti-bodies are usually those active at 37 Cand/or by the indirect antiglobulin test(IAT). It is not necessarily true, however,that all antibodies active in vitro at 37 Cand/or by the IAT are clinically signifi-cant; not all antibodies serologically re-active only below 37 C are benign.

Antibodies encountered in pretransfu-sion testing should be identified, for as-sessment of the need to select antigen-negative blood for transfusion. Patientswith clinically significant antibodiesshould, whenever practical, receive redcells that have been tested and found tolack the corresponding antigen. In prena-tal testing, the specificity and immuno-globulin class of an antibody influence thelikelihood of HDN. While identification ofunexpected antibodies in donor blood isnot required, results of such testing areuseful for characterizing the units fortransfusion use and for procuring bloodgrouping reagents or teaching samples.

General ProceduresSpecimen RequirementsEither serum or plasma may be used forantibody identification; serum is usedmore widely, and that is the only termthat will be used in this chapter. Plasmais not suitable for detection of comple-ment activation. When autologous redcells are studied, the use of a sampleanticoagulated with EDTA avoids prob-lems associated with the in-vitro uptakeof complement components by red cellsthat may occur with a clotted sample. A10 mL aliquot of whole blood usuallycontains enough serum for identifyingsimple antibody specificities; more maybe required for more complex studies.


Medical HistoryIt is useful to know a patient’s clinicaldiagnosis, history of transfusions orpregnancies, and recent drug therapy. Inpatients who have had recent red celltransfusions, the circulating blood maybe so admixed with donor cells that spe-cial procedures are needed to separatethe autologous red cells for typing (seeMethod 2.15). Other procedures will benecessary for patients known to haveautoantibodies, which may be disease-associated or induced by drugs (seeChapter 18).

Reagents

Red Cell PanelsIdentification of an antibody to red cellantigens requires testing the serumagainst a panel of selected red cell speci-mens, usually eight or more. They areusually obtained from commercial sup-pliers, but institutions may assembletheir own by using red cells from localsources. Panel cells are (except in specialcircumstances) group O, allowing serumof any ABO group to be tested, and thephenotypes for antigens of the majorblood groups are recorded.

Each cell of the panel is from a differ-ent individual. The cells are selected sothat, taking all the cells into account, adistinctive pattern of positives and nega-tives exists for each of many antigens. Tobe functional, a reagent red cell panelmust make it possible to identify withconfidence those clinically significantalloantibodies that are most frequentlyencountered, such as anti-D, -E, -K, and-Fya. The phenotypes of the reagent redcells should be distributed such thateach of the common alloantibodies, if itis the only one in a serum, can be clearlyidentified and most others at least tenta-tively excluded. The pattern of reactivityfor most examples of single alloanti-bodies should not overlap with any

ABB. All rights reserved.

Chapter 17: Identification of Alloantibodies to Red Cell Antigens 351

other; eg, all of the K+ samples shouldnot be the only ones that are also E+. Tolessen the possibility that chance alonehas caused an apparently definitive pat-tern, there must be sufficient number ofred cell samples that lack, and sufficientred cell samples that carry, most of theantigens listed in Table 17-1.

Commercially prepared panels aregenerally issued every 2-4 weeks. Eachpanel contains different cells, with dif-ferent antigen patterns, so it is essentialto use the phenotype listing sheet thatcomes with the panel in use. Commer-cial cells usually come as a 2-5% suspen-sion in a preservative medium; they canbe used directly from the vial, althoughsome workers prefer to wash the cellswith saline and obtain a dry cell buttonto which the test serum is added. Forsolid phase methods, panels of dried redcell monolayers may be prepared andstored for long periods before use.4

Enhancement Media

Although the test system may consistsolely of serum and cells, most workers

Table 17-1. A Reagent Red Cell Pane

Sample#

RhPhenotype

Rhesus Kel

C Cw c D E e K

1 r′r + 0 + 0 0 + 02 R1

w + + 0 + 0 + +3 R1 + 0 0 + 0 + 04 R2 0 0 + + + 0 05 r″r 0 0 + 0 + + 06 r 0 0 + 0 0 + 07 r 0 0 + 0 0 + +8 r 0 0 + 0 0 + 09 r 0 0 + 0 0 + 0

10 R0 0 0 + + 0 + 0

+ Denotes presence of antigen; 0 denotes abse

Copyright © 2002 by the

use some kind of enhancement medium.Many different media are available, in-cluding low ionic strength saline (LISS),polyethylene glycol (PEG), and albumin.For the initial identification panels,most laboratories use the same enhance-ment method used in their routine anti-body detection and crossmatch tests. Ad-ditional enhancement techniques maybe employed for more complex identifi-cation. Enhancement techniques arediscussed later in this chapter.

Antiglobulin Reagents

Most antibody identification tests in-clude an antiglobulin phase. Either poly-specific or IgG-specific antiglobulin re-agents may be used . Po lyspeci f icreagents may detect, or detect morereadily, antibodies that bind comple-ment. While this may be advantageous insome instances, many workers prefer touse IgG-specific reagents to avoid un-wanted reactivity due to in-vitro comple-ment binding by cold-reactive antibod-ies.

l for Alloantibody Identification

l Duffy Kidd P Lewis MNS

Fya Fyb JkaJkb P1 Lea Leb M N S s

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

nce of antigen.

AABB. All rights reserved.


Autologous Control

It is important to know how a serumunder investigation reacts with autolo-gous red cells. This helps determinewhether alloantibody, autoantibody, orboth are present. Serum that reacts onlywith the reagent red cells usually con-tains only alloantibody, whereas reactiv-ity with both reagent and autologous redcells suggests the presence of autoanti-body, or autoantibody plus alloantibody.A patient with alloantibodies to antigenson recently transfused red cells mayhave circulating donor red cells coatedwith alloantibodies, that produce a posi-tive autocontrol, usually in a mixed-fieldpattern. This may be misinterpreted asbeing due to autoantibody. A detailedhistory of recent transfusions should beobtained for all patients with a positiveDAT.

An autocontrol provides useful infor-mation in antibody identification stud-ies, even if one has been previously per-formed, so that react ions withautologous and reagent red cells can becompared. When test methods used forantibody identification are differentfrom or additional to the initial autocon-trol method, an autologous control isessential. An autocontrol is not requiredin all circumstances, especially if the se-rum is not reactive by the method used.It may not be necessary to prepare en-zyme-treated autologous cells whentesting the serum against panel cellspretreated with enzyme.

The autologous control, in which se-rum and autologous cells undergo thesame manipulation as serum and re-agent cells, is not the same as a DAT. Ifthe autocontrol is positive in the an-tiglobulin phase, a DAT should be per-formed. If the DAT is positive, elutionstudies should be considered if the pa-tient has been recently transfused, ifthere is evidence of immune hemolysisand/or if the results of serum studies


prove inconclusive. A reactive DAT mayalso indicate the presence of autoanti-body. If autoantibody is detected in theserum, adsorption studies may be neces-sary to establish that it is not maskingcoexisting alloantibodies.

Basic AntibodyIdentification TechniquesTraditional serologic methods based onagglutination and performed in testtubes or microplates remain the mostcommonly used and are the primarytechniques discussed in this chapter.Other methods, which modify tradi-tional test endpoints or are not depend-ent on agglutination, are also available.These include solid phase, flow cy-tometry, and gel or column techniques,as well as some automated systems. Suchmethods may be used in addition to orinstead of the classical techniques dis-cussed below. Interpretation of reactionpatterns to identify antibody specificityare the same for all tests.

Initial ObservationsRed cell panels are usually employed toinvestigate a serum already known tocontain antibody, through reactivity de-tected in preliminary tests such as theantibody screening test or crossmatch.These initial tests provide important in-formation about the test phases at whichreactivity occurs, and the serum maysometimes be tested only by the tech-nique with which the antibody was origi-nally detected. For initial panels, how-ever, it is common to use the samemethods and range of phases used in theinitial procedures; typically these in-clude room temperature and 37 C test-ing, often with enhancement medium,and IAT. This ensures that the serum hasbeen tested against cells of various phe-



notypes and antigen strengths, andguards against missing weaker reactionsat some phases.

Knowing the phase of initial reactiv-ity may suggest possible specificities.Reactivity only at room temperature, forexample, suggests the possibility of anti-M, -P1, -I, -Lea, or -Leb. The phenotype ofthe originally-reactive antibody detec-tion cells may also provide clues as tospecificity, or help exclude specificities.If multiple panels are available, this in-formation can help in selecting the cellslikely to be informative.

If the patient has had antibodies pre-viously identified, this may affect panelselection. For example, if the patient isknown to have anti-e, it will not be help-ful to test the serum against a panel of10 cells, nine of which are e-positive.Testing a panel of selected e-negativecells will better reveal any newly formedantibodies.

Sometimes the patient’s phenotypeinfluences the selection of reagent cells.If the patient is D-negative and the se-rum is reactive with D-positive cells inthe screening test, an abbreviated panelof D-negative cells may be tested. Thiscan both confirm the presence of anti-Dand demonstrate the presence of addi-tional antibodies.5

Interpreting ResultsInterpretation of panel results can be acomplex process combining technicalknowledge and intuitive skills. Panel re-sults will include a range of positive andnegative results, each of which should beexplained by the final conclusion.

Positives and NegativesBoth positive and negative reactions areimportant in antibody identification.Positive reactions indicate the phase andstrength of reactivity, which can suggestspecificities expected to react in that


manner. Positive reactions also can becompared to the antigen patterns ex-pressed by the panel cells to help assignspecificity. Single alloantibodies usuallyyield definite positive and negative reac-tions that create a clear-cut antigen pat-tern with reagent red cell samples. Forexample, if a serum reacts only with cells4 and 5 of the reagent red cell panelshown in Table 17-1, anti-E is very likelypresent. Both reactive samples express Eand all nonreactive samples lack E.

Negative reactions are also importantin antibody identification, because theyallow at least tentative exclusion of anti-bodies to antigens expressed on the non-reactive cells. Exclusion of antibodies isan important step in the interpretationprocess and must be done to ensureproper identification of all antibodiespresent.

Exclusion or “Crossing Out”

A widely used first approach to the inter-pretation of panel results is to excludespecificities based on nonreactivity withthe serum tested. Such a system is some-times referred to as a “cross-out” or“rule-out” method. Once results havebeen recorded on the worksheet, the an-tigen profile of the first nonreactive cellis examined. If an antigen is present onthe cell and the serum did not react, thepresence of the corresponding antibodymay be at least tentatively excluded.Many workers will actually cross outthat antigen from the listing on thepanel sheet to facilitate the process. Af-ter all antigens present on that cell havebeen crossed off, interpretation pro-ceeds with the other nonreactive cellsand additional specificities are excluded.In most cases, this process will leave agroup of antigens that still have not beenexcluded.

Next, the cells reactive with the se-rum are evaluated. The pattern of reac-tivity for each nonexcluded specificity is



compared to the pattern of reactivity ob-tained with the test serum. If there is apattern that matches exactly, that ismost likely the specificity of the anti-body in the serum. However, if there areremaining specificities that have notbeen excluded, additional testing may beneeded to eliminate remaining possibili-ties and to confirm the specificity iden-tified. This requires testing the serumagainst additional cells.

Once tentative identification has beenestablished, testing the serum againstcells selected for specific antigenic char-acteristics gives more information thanusing an additional, unmodified panel.For example, if the pattern of positivereactions exactly fits anti-Jka, anti-K andanti-S may still not be excluded. Theserum should be tested against selectedcells, ideally with the three phenotypes:Jk(a–), K–, S+; Jk(a–), K+, S–; andJk(a+), K–, S–. The reaction pattern withthese cells should both confirm the pres-ence of anti-Jka and include or excludeanti-K and anti-S.

While the exclusion (cross-out) ap-proach often identifies simple antibodyspecificities, it should be consideredonly a provisional step. As discussed be-low, some antibodies may be mistakenlyexcluded if, for some reason, no reactiv-ity was obtained with a cell positive forthe antigen. On occasion, similar pat-terns may be obtained for different spe-cificities or when multiple antibodiesare present.

Computer programs are available thatcan interpret panel results with somesuccess. Such programs can utilize in-formation read by traditional manualmethods and entered by the user, or canbe coupled to automated test systemsthat directly transmit test results.

ProbabilityConclusive antibody identification re-quires serum to be tested against suffi-


cient reagent red cell samples that lack,and that carry, the antigen that appearsto correspond to the specificity of theantibody, to ensure that an observed pat-tern is not due to chance alone.

CalculationsThe traditional way to calculate theprobability that an antibody has beencorrectly identified is Fisher’s exactmethod, in which the numbers of posi-tive and negative results are comparedwith the numbers of cells that express orlack the corresponding antigen.6 [SeeTable 17-2 (A).] For this method, a prob-ability (p) value of 0.05 is generally ac-cepted as a minimum value for consider-ing an interpretation statistically valid.(See Table 17-3.) This means that chancealone would produce an identical set ofresults once in 20 similar studies. Mostbasic red cell panels have limited capac-ity for conclusive identification of somespecificities, especially when multipleantibodies are present. To meet thep≤0.05 statistical standard, tests withadditional cells are often necessary.

Fisher’s exact method has been chal-lenged as being too conservative and notaddressing population frequencies forantigens for which the cells have notbeen typed. Harris and Hochman7 havederived an alternative calculation to al-low for such antigens. [See Table 17-2(B).] Comparative p values by theirmethod are also given in Table 17-3. Thiscalculation allows more liberal interpre-tation of results, and may require lessactual testing to confirm specificity.

InterpretationMost workers rarely use either statisticalmethod in a conscious manner. A stand-ard approach (based on Fisher’s exactmethod) has been to require, for eachspecificity identified, three antigen-positive cells that do react and three an-


Table 17-2. Calculation of Probability

A. Fisher’s Exact Method6

The formula for calculating probability (p) is:(A+B)! × (C+D)! × (A+C)! × (B+D)!

N! × A! × B! × C! × D!

B. Modification of Harris and Hochman7

(A/N)A(B/N)B

A = number of positive reactions observed with antigen-positive red cell samplesB = number of positive reactions observed with antigen-negative red cell samplesC = number of negative reactions observed with antigen-positive red cell samplesD = number of negative reactions observed with antigen-negative red cell samplesN = number of cells tested! = factorial, the product of all the whole numbers from 1 to the number involved.

For example, 6! = 6 × 5 × 4 × 3 × 2 × 1 = 7201! = 10! = 1

Consult references and suggested readings for further details on calculations.

Table 17-3. Probability ValuesNo. Tested No. Positive No. Negative p (Fisher6) p (Harris and Hochman7)

5 3 2 0.100 0.0356 4 2 0.067 0.0226 3 3 0.050 0.0167 5 2 0.048 0.0157 4 3 0.029 0.0088 7 1 0.125 0.0498 6 2 0.036 0.0118 5 3 0.018 0.0058 4 4 0.014 0.0049 8 1 0.111 0.0439 7 2 0.028 0.0089 6 3 0.012 0.003

10 9 1 0.100 0.03910 8 2 0.022 0.00710 7 3 0.008 0.00210 6 4 0.005 0.00110 5 5 0.004 0.001


Copyright © 2002 by the AABB. All rights reserved.


tigen-negative cells that fail to react.This standard is not always possible, butworks well in practice, especially if cellswith strong antigen expression are avail-able. A somewhat more liberal approachis derived from calculations by Harrisand Hochman, whereby minimum re-quirements for a p value of 0.05 are metby having two positive and three nega-tive cells, or one positive and seven nega-tive cells (or the reciprocal of eithercombination). The possibility that theserum fails to react with antigen-posi-tive cells (or of some “false-positive” re-sults) must also be considered in deter-mining specificity.

Additional details on calculatingprobability may be found in the sug-gested readings by Race and Sanger andby Menitove.

Phenotype of Autologous Red CellsOnce an alloantibody has been identifiedin a serum, it is often helpful to demon-strate that the autologous red cells arenegative for the corresponding antigen.For example, if serum from an untrans-fused individual appears to contain anti-Fya but the autologous red cells have anegative DAT and type as Fy(a+), the dataare clearly in conflict and further testingis indicated.

Determination of the patient’s pheno-type can be difficult if the patient hasbeen transfused recently, generallywithin 3 months. If a pretransfusionspecimen is still available, these red cellsshould be used to determine the pheno-type. Alternatively, the patient’s own redcells can be separated from the trans-fused red cells and then typed. Proce-dures for this are given in Methods 2.15and 2.16. The use of potent blood group-ing reagents, appropriate controls, andobservation for mixed-field reactionsoften allows an unseparated specimen tobe phenotyped. If there is little uncer-tainty about antibody identification, ex-


tensive efforts to separate and type thepatient’s own red cells are not necessary.Antigen-negative blood can be selectedfor transfusion; for patients with clini-cally significant antibodies, an an-tiglobulin phase crossmatch is re-quired,8 and a compatible crossmatchwill provide additional confirmation ofantibody specificity. Definitive testingcan be done on the patient’s red cellsafter a period without transfusion or, ifthis is not possible, after an interval dur-ing which only antigen-negative bloodhas been given. If the chronically trans-fused patient is not aplastic, any anti-gen-positive red cells detected after pro-longed transfusion of antigen-negativeblood would presumably be the patient’sown.

Complex AntibodyProblemsNot all antibody identifications are sim-ple. The exclusion procedure does notalways lead directly to an answer andadditional approaches may be required.Figure 17-1 shows some approaches toidentifying antibodies in a variety ofsituations when the autocontrol is nega-tive. Additional approaches may beneeded if the autocontrol is positive;these are discussed later in this chapter.

Variations in Antigen ExpressionFor a variety of reasons, antibodies donot always react with all cells positivefor the corresponding antigen. Interpre-tation by exclusion may cause a givenspecificity to be crossed out if a cell isantigen-positive and the serum is nonre-active, despite the presence of the anti-body. Sometimes, this prevents discern-ment of any pattern, but in some cases itmay, by coincidence, yield a false patternof specificity. Technical error or weak


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Copyright © 2002 by the AABB. All rights reserved.


antibody reactivity are possible causes,and the strength of antigen expressionon tested red cells should be kept inmind. Antibody specificities should,when possible, be excluded only on thebasis of cells known to bear a strongexpression of the antigen.

ZygosityReaction strength of some antibodiesvaries from one red cell sample to an-other. This may be due to the phenome-non known as dosage, in which antibod-ies react preferentially with red cellsfrom persons homozygous for the genethat determines the antigen (ie, process-ing a “double dose” of the antigen). Redcells from individuals heterozygous forthe gene may express less antigen andmay react weakly or be nonreactive. Al-loantibodies vary in their tendency torecognize dosage. Many antibodies inthe Rh, Duffy, MN, and Kidd systemshave this trait.

Variation in Adults and InfantsSome antigens (eg, I, P1, Lea, and Sda) areexpressed to varying degrees on red cellsfrom different adult donors. This expres-sion is unrelated to zygosity; however,the antigenic differences can be demon-strated serologically. Certain antibodies,including those to I, Lea, Leb, Sda, Lua,Lub, Vel, Yta, Hy, McCa, Yka, Csa, Ch, andRg antigens, react more weakly withcord red cells than they do with red cellsfrom adults.

Changes with StorageBlood group antibodies may react lesswell with stored than with fresh redcells. The M and P1 antigens deteriorateduring storage more rapidly than mostothers; the rate varies among red cellsfrom different donors. Storage mediacan affect the rate of antigen deteriora-tion. Fya and Fyb antigens, for example,


may be weakened when the cells arestored in a suspending medium of lowpH and low ionic strength. The potentialloss of antigen reactivity must be consid-ered when using older cells for antibodyidentification.

Red cells from donor units are oftenfresher than commercial reagent cellsand have been stored in different me-dia. Some antibodies give stronger re-actions with suspensions of donor cellsthan with reagent cells; eg, antibodiesin the Knops system and antibodiesthat react with HLA antigens on redcells often react best with fresh donorcells and may not react at all withstored reagent cells. After frozen stor-age, reagent cells may give weaker re-actions with some antibodies. This cancause misleading patterns, especiallywhen an antibody is interpreted as rec-ognizing a high-incidence antigen, onthe basis of nonreactivity with one ortwo thawed specimens. Certain anti-bodies react more strongly or weaklywith cells from different commercialmanufacturers, whose suspending me-dia may differ in pH or other charac-teristics. Enhancement techniquesoften help resolve problems associatedwith variations in antigen expression(see Methods 3.2.3, 3.2.4, 3.2.5, and3.5.5).

The age and nature of the specimenmust also be considered when typingred cells. Antigens on cells from clot-ted samples tend to lose activity fasterthan cells collected in citrate antico-agulants such as ACD or CPD. Red cellsin donor units collected into these an-ticoagulants generally retain their an-tigens throughout the standard shelflife of the blood component. If red cellsare used that are collected into EDTAthey must be tested within 2 days ofcollection, although some studies havedemonstrated signi f icantly longerpreservat ion of most ant igens onEDTA-anticoagulated cells.10



No Discernible SpecificityFactors other than variation in antigenexpression may contribute to difficultyin interpreting results of antibody iden-tification tests. If the reactivity obtainedwith the serum is very weak and/or if thecross-out process has excluded all likelyspecificities, alternative approaches tointerpretation should be used.

Antigens Present in Common

Instead of excluding antigens on nonre-active cells, one can observe what anti-gens are common to the reactive cells.For example, if the cells reacting at roomtemperature are all P1-positive, yet notall the P1-positive cells react, the anti-body could be an anti-P1 that does notreact with cells having a weaker expres-sion of the antigen. (Such cells are some-times marked on the panel sheet as“+w”.) With this in mind, one could usea method to enhance anti-P1, such astesting at colder temperatures.

If all the reactive cells are Jk(b+), butnot all the Jk(b+) cells react, the reactiveones might all be Jk(a–b+), with a dou-ble-dose expression of the antigen. En-hancement techniques, such as en-zymes, LISS, or PEG, may then helpdemonstrate reactivity with all the re-maining Jk(b+) cells. Typing the pa-tient’s cells to confirm they lack the cor-responding antigen can also be veryhelpful.

Inherent Variability

Nebulous reaction patterns that do notappear to fit any particular specificity arecharacteristic of antibodies (such asanti-Bga) that react with HLA antigenson red cells. These antigens vary mark-edly in expression on red cells from dif-ferent individuals. Rarely, a pattern ofclear-cut reactive and nonreactive teststhat cannot be interpreted can result


from the incorrect typing of reagent redcells.

Unlisted Antigens

Sometimes a serum reacts with an anti-gen not routinely listed on the antigenprofile supplied by the reagent manufac-turer; Ytb is one example. Even thoughserum studies yield clear-cut reactiveand nonreactive tests, anti-Ytb may notbe suspected. In such circumstances it isuseful to ask the manufacturer for addi-tional phenotype information. If the ap-propriate blood grouping reagent isavailable, reactive and nonreactive redcell samples, as well as the autologousred cells, can be tested. However, theseproblems often have to be referred to animmunohematology reference labora-tory.

ABO Type of Red Cells Tested

A serum may react with many or all ofthe group O reagent red cell samples,but not with red cells of the same ABOphenotype as the autologous red cells.This occurs most frequently with anti-H,-IH, or -LebH. Group O and A2 red cellshave large amounts of H antigen; A1 andA1B red cells express very little H (seeChapter 12). Sera containing anti-H or-IH react strongly with group O reagentred cell samples, but autologous A1 orA1B red cells or donor cells used forcrossmatching may be weakly reactiveor nonreactive. Anti-LebH reacts stronglywith group O, Le(b+) red cells, but re-acts weakly or not at all with Le(b+) redcells from A1 or A1B individuals. Suchantibodies should be suspected when theantibody serum, which uses group O redcells, is strongly reactive, but serologi-cally compatible A1 or A1B donor samplescan be found without difficulty.



Multiple Antibodies

When a serum contains two or more al-loantibodies, it may be difficult to inter-pret the results of testing performed ona single panel of reagent red cells. Thepresence of multiple antibodies may besuggested by a variety of test results.

1. The observed pattern of reactive andnonreactive tests does not fit that ofa single antibody.When the exclusion approach fails toindicate a specific pattern, it is help-ful to see if the pattern matches anytwo combined specificities. For ex-ample, if the reactive cells (see Table17-1) are numbers 2, 4, 5, and 7,none of the specificities remainingafter crossing-out exactly fits thatpattern, but if both K and E are con-sidered together, a pattern is dis-cerned. Cells 2 and 7 react becauseof anti-K, cells 4 and 5, because ofanti-E. If the typing patterns for notwo specificities fit the reaction pat-tern, the possibility of more thantwo antibodies must be considered.The more antibodies a serum con-tains, the more complex the identi-fication and exclusion of specifici-ties will be, but the basic processremains the same.

2. Different red cell samples react atdifferent test phases.When reactivity occurs at severalphases, each phase should be evalu-ated separately. The pattern seen atroom temperature may indicate adifferent specificity from the patternof antiglobulin results. It is helpfulto know which specificities are mostlikely to be seen at which phase (seeTable 14-2).

3. Unexpected reactions are obtainedwhen attempts are made to confirmthe specificity of a suspected singleantibody.If a serum suspected of containinganti-e reacts with additional sam-


ples that are e-negative, another an-tibody may be present or the sus-pected antibody may not really beanti-e. Testing a panel of selectede-negative cells may help indicate anadditional specificity.

4. No discernable pattern emerges.Uni form or var iab le react ionstrengths may be observed, and dos-age or other variation in antigenstrength does not provide an expla-nation. Additional approaches andmethods of testing are usually indi-cated.Some helpful steps include:a. If strong positive results were ob-

tained, use the exclusion methodwith nonreactive cells to elimi-nate some specificities from in-itial consideration.

b. If weak or questionable positiveresults were obtained, test the se-rum against cells carrying astrong expression of antigenscorresponding to any suspectedspecificities, and combine thiswith methods to enhance reactiv-ity.

c. Type the patient’s red cells andeliminate from considerationspecificities corresponding to an-tigens present on the autologouscells.

d. Use methods to inactivate certainantigens on the red cells, eg, en-zyme treatment to render cellsnegative for Fya, Fyb, S.

e. Use adsorption/elution methodsto separate antibodies.

These and other methods that may behelpful are discussed below.

Antibodies to High-Incidence AntigensIf all reagent red cell samples are reac-tive, but the autocontrol is nonreactive,alloantibody to a high-incidence antigenshould be considered, especially if thestrength and test phase of reactions are


incidence antigen, such as anti-Wr . If


uniform for all cells tested. Antibodies tohigh-incidence antigens can be identi-fied by testing red cells of selected rarephenotypes, and by testing the patient’sautologous red cells with sera known tocontain antibodies to high-incidence an-tigens. Knowing the race or ethnic ori-gin of the antibody producer can help inselecting additional tests to be per-formed. Cells that are null for all anti-gens in a system (eg, Rhnull or Ko) ormodified red cells (eg, dithiothreitol-treated cells) can help limit possible spe-cificities to a particular blood group.

If cells negative for particular high-incidence antigens are not available,cells positive for lower-incidence allelescan sometimes be helpful. Weaker reac-tivity with Co(a+b+) cells than withcommon Co(a+b–) cells, for instance,might suggest anti-Coa. Antibodies tohigh-incidence antigens may be accom-panied by other antibodies to commonantigens, which can make identificationmuch more difficult.

Serologic Clues

Knowledge of the serologic charac-teristics of particular antibodies to high-incidence antigens can help in identifi-cation.

1. Reactivity in tests at room tempera-ture suggests anti-H, -I, -P1, -P, -Tja

(-PP1Pk), some-LW, -Ge, -Sda, or -Vel.2. Lysis of reagent red cells is charac-

teristic of anti-Vel, -P, -Tja, and -Jk3.It is also seen with some examples ofanti-H and -I.

3. Reduced or absent reactivity in en-zyme tests occurs with anti-Ch, -Rg,-Inb, or -JMH and is seen with someexamples of anti-Yta and -Ge2 or-Ge3.

4. Weak, nebulous reactions in the an-tiglobulin phase are often associatedwith anti-Kna, -McCa, -Yka, and -Csa.Complement-binding autoanti-bodies, such as anti-I or anti-IH,


give similar results when polyspeci-fic antiglobulin reagents are used.

5. Antibodies such as anti-U, -McCa,-Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and-Ata should be considered if the se-rum is from a Black individual be-cause the antigen-negative pheno-types occur almost exclusively inBlacks. Makers of anti-Kpb are al-most always White. Anti-Dib is usu-ally found among Asians, Hispanics,and Native Americans.

Interpreting a Positive DATWhen a patient produces antibody di-rected to a high-incidence antigen fol-lowing transfusion, the posttransfusionred cells may have a positive DAT, andboth serum and eluate may react with allcells tested. This pattern of reactivity isidentical to that produced by manywarm reactive autoantibodies, whichmay also appear after transfusion; thesetwo scenarios can be very difficult todifferentiate. A posttransfusion alloanti-body to a high-incidence antigen wouldbe expected to produce a DAT of mixed-field appearance, because only the trans-fused red cells would be coated with an-tibody. In practice, however, weaksensitization and mixed-field sensitiza-tion can be difficult to differentiate. Itmay be helpful to use cell separationprocedures to isolate autologous cellsfor testing.

Chapter 14 discusses additional sero-logic characteristics of antibodies react-ing with high-incidence red cell anti-gens. Problems with these antibodiesoften have to be referred to an immuno-hematology reference laboratory.

Antibodies to Low-Incidence AntigensReactions between a serum sample and asingle donor or reagent red cell samplemay be caused by an antibody to a low-

a



red cells known to carry low-incidenceantigens are available, the serum can betested against them, or the one reactivered cell sample can be tested with knownexamples of antibodies to low-incidenceantigens. A single serum often containsmultiple antibodies to low-incidence an-tigens, and the expertise and resourcesof an immunohematology referencelaboratory will be required to confirmthe suspected specificities.

Serologic StrategiesIf antibody to a low-incidence antigen issuspected, transfusion should not be de-layed while identification studies are un-dertaken. If antibody in the serum of apregnant woman is thought to be di-rected against low-incidence antigen,testing the father’s red cells can predictthe possibility of incompatibility withthe fetus, and identifying the antibody isunnecessary. If a newborn has a positiveDAT, testing of the mother’s serum or aneluate from the infant’s cells against thefather’s red cells (assuming they areABO-compatible) can implicate an anti-body to a low-incidence antigen as theprobable cause; identifying the antibodyis usually of little importance.

Some reference laboratories do notattempt to identify antibodies to low-in-cidence antigens, since they are oftenonly of academic interest and resourcescan better be devoted to problems ofgreater clinical importance. Identifica-tion may be made when time permits andsuitable reagents are available. It is oftenpractical to store individual specimensand then perform batch testing, to con-serve rare frozen samples of cells or an-tibodies.

Unexpected Positive ResultsWhen serum reacts with a panel cell des-ignated as positive for a low-incidenceantigen, further testing to exclude the


antibody is usually unnecessary. Forevery antigen of low incidence repre-sented on a panel there are many morethat are not represented and that are alsonot excluded by routine testing. Reactiv-ity against low-incidence antigens is notuncommon; although the antigens arerare, antibodies against some of the low-incidence antigens are much less rare.Presumably the testing is being per-formed because the serum containssome other antibody (-ies) and reactivitywith the cell expressing the low-inci-dence antigen is a coincidental finding.This may complicate interpretation ofthe panel results, but rarely requiresconfirmation of antibody specificity ortyping of donor blood to ensure the ab-sence of the antigen. If typing is desired,a negative crossmatch with the patient’sserum is sufficient demonstration thatthe antigen is absent. Many antibodies tolow-incidence antigens are reactive onlyat temperatures below 37 C and are ofdoubtful clinical significance.

When serum reacts only with red cellsfrom a single donor unit or reagent cell,the other possibilities to consider arethat the reactive donor red cells areABO-incompatible, have a positive DAT,or are polyagglutinable.

Antibodies to Reagent Componentsand Other Anomalous SerologicReactionsAntibodies to a variety of drugs and ad-ditives can cause positive results in anti-body detection and identification tests.The mechanisms are probably similar tothose discussed in Chapter 18.

Most of these anomalous reactionsare in-vitro phenomena and have noclinical significance in transfusion ther-apy other than causing laboratory prob-lems that delay provision of a neededtransfusion. Very rarely they may causeerroneous interpretations of ABO typingthat could endanger the patient.



Ingredients in the Preservative Solution

Antibodies that react with an ingredientin the solution used to preserve reagentred cells (eg, chloramphenicol, neomy-cin, tetracycline, hydrocortisone, EDTA,or various sugars) may agglutinate cellssuspended in that solution. Reactivitymay occur with cells from several com-mercial sources or may be limited tocells from a single manufacturer. Theautologous control is often nonreactive,unless the suspension of autologous redcells is prepared with the manufacturer’sred cell diluent or a similar preservative.Such reactions can often be circum-vented by washing the reagent cells withsaline before testing. The role of the pre-servative can often be confirmed by add-ing the medium to the autologous con-trol and converting a nonreactive test toa positive test. In some cases, however,washing the reagent cells does not cir-cumvent reactivity and the resolutionmay be more complex.

Ingredients in Enhancement Media

Antibodies reactive with ingredients inother reagents, such as commerciallyprepared LISS additives or albumin, cancause agglutination in tests using re-agent, donor, and/or autologous redcells. Ingredients that have been impli-cated include parabens (in some LISSadditives), sodium caprylate (in some al-bumins), and thimerosal (in some salinepreparations). LISS and other enhance-ment media may also dramatically in-crease the reactivity of some autoanti-bodies. Antibodies such as anti-I, -IH, or-Pr that ordinarily react only at coldertemperatures may even react in the an-tiglobulin phase when these enhance-ment media are used. Omitting the en-hancement medium, subst i tut ingplasma for serum, and/or using anti-IgGrather than polyspecific antiglobulin se-


rum will usually circumvent this reac-tivity.

In some cases antibodies dependentupon reagent ingredients will also showblood group specificity, eg, paraben-de-pendent anti-Jka, caprylate-dependentanti-c. The autocontrol may be reactiveif the patient’s own red cells carry theantigen, but the DAT should be negative.

Problems with Red CellsThe age of the red cells can cause anoma-lous serologic reactions. Antibodies ex-ist that react only with stored red cells,and these can cause agglutination of re-agent red cells by all techniques, andenhanced reactivity in tests with en-zyme-treated red cells. Such reactivity isnot affected by washing the red cells, andthe autocontrol is usually nonreactive.No reactivity will be seen in tests onfreshly collected red cells, ie, fromfreshly drawn donor or autologous bloodsamples.

Immunohematology ReferenceLaboratoriesWhen antibody problems cannot be re-solved (or when specially typed blood isneeded) immunohematology referencelaboratories can provide consultationand assistance, through their access torare donor files. (See Method 4.8.)

The Patient with a Positive Autocontrol

No Recent TransfusionsReactivity of serum with the patient’sown cells may indicate the presence ofautoantibody. (See Chapter 18.) If thisreactivity occurs at room temperature orbelow, the cause is often anti-I or othercold autoagglutinin. Reactivity of theautocontrol in the antiglobulin phaseusually signifies a reactive DAT and thepossibility of autoantibody. If, in addi-tion, the serum reacts with all cells



tested, autoadsorption or other specialprocedures may be necessary to deter-mine whether autoantibody in the se-rum is masking any significant alloanti-bodies. If the serum is not reactive orshows only weak reactivity, an eluatemay demonstrate more potent autoanti-body.

If the DAT is negative, but the auto-control is positive by IAT, an alternativeexplanation is required. Such results areunusual and may indicate antibody to areagent constituent causing in-vitro re-activity with all cells, including the pa-tient’s own. It may also indicate that thewrong cells were added to the test!

Cold Autoantibodies. Potent coldautoagglutinins that react with all cells,including the patient’s own, can createspecial problems, especially when reactiv-ity persists at temperatures above roomtemperature. Cold autoagglutinins may bebenign or pathologic. (See Chapter 18 fora more detailed discussion.)

There are two divergent objectives intesting a serum with a potent cold agglu-tinin. One is to determine if the thermalamplitude is high enough (usually 30 Cor above) that the antibody has clinicalsignificance; to do this, in-vitro autoad-sorption of the serum must be avoided.Keeping the freshly collected bloodwarm (37 C) until the serum is separatedusually provides an appropriately infor-mative specimen. The second objective,the common one outside of cold aggluti-nin syndrome, is to circumvent the cold-reactive antibody and allow detection ofmore important antibodies.

Procedures for the detection of al-loantibodies in the presence of cold-re-active autoantibodies are discussed inChapter 18 and include:

1. Prewarmed techniques, in which redcells and the serum to be tested areincubated at 30-37 C before they aremixed (see Method 3.3).

2. The use of anti-IgG rather than poly-specific antiglobulin serum.


3. Cold autoadsorption, to removeautoantibodies but not alloantibodies.

4. Heterologous adsorption with rab-bit red cells.

Dealing with Warm Autoantibodies.Patients with warm-reactive autoanti-body present in their serum create a spe-cial problem, because the antibody re-acts with virtually all cells tested. If suchpatients are to be transfused, it is impor-tant to detect any clinically significantalloantibodies that the autoantibodymay mask. Techniques are discussed inChapter 18 and Methods 6.4, 6.5 and 6.6.

Reactivity of most warm reactiveautoantibodies is greatly enhanced bysuch methods as PEG and enzymes, andto lesser extent by LISS and albumin. Itmay be advantageous to perform anti-body detection tests without the en-hancement media usually employed. Iftests are nonreactive, the same proce-dure can be used for crossmatching,without the need for adsorptions.

Recent Transfusions

If the autocontrol is positive in the an-tiglobulin phase, there may be antibody-coated cells in the patient’s circulation,causing a positive DAT, often of mixed-field reactivity. Elution may be helpful,especially when tests on serum are in-conclusive. For example, a recentlytransfused patient may have a positiveautocontrol and serum that reactsweakly with most but not all Fy(a+) redcells. It may be possible to confirm anti-Fya specificity by elution, which concen-trates into a small fluid, volume the im-munoglobulin molecules present insmall numbers on each of the red cellsin the starting preparation. It is rare fortransfused cells to make the autocontrolpositive at other test phases, but it canoccur, especially with a newly developingor cold-reactive alloantibody.

If the positive DAT does not have amixed-field appearance and, especially, if



the serum is reactive with all cells tested,the possibility of autoantibody shouldagain be considered. Detect ion ofmasked alloantibodies may require allo-geneic adsorptions.

Accurate phenotyping of red cells maybe difficult if the DAT is reactive in anypatient, whether or not there has beenrecent transfusion. A positive DAT willcause the cells to be reactive in any testrequiring addition of antiglobulin serumand with some reagent antibodies (nota-bly those in the Rh system) that includean enhancement medium. Many mono-clonal reagents can give valid phenotyp-ing results despite a positive DAT.

Selecting Blood forTransfusionOnce an antibody has been identified, itis also important to decide its clinicalsignificance. Antibodies reactive at 37 Cand/or by IAT are generally consideredclinically significant and those reactiveat room temperature and below are not;however, there are many exceptions. Forexample, anti-Ch, anti-Rg, and many ofthe Knops and Cost antibodies have littleor no clinical effect, despite reactivity byIAT. Anti-Vel, -P, and -Tja (-P+P1+Pk) mayreact only at cold temperatures yet maycause significant cell destruction in vivo.Comparison with documented cases inthe literature and consultation with im-munohematology reference laboratoriesshould provide guidance about previousexamples of similar specificities.

Phenotyping Donor UnitsRed-cell-containing components se-lected for transfusion to a patient withan antibody should, whenever possible,be tested and found to be negative forthe appropriate antigen, unless the an-tibody is known to lack clinical signifi-


cance. Even if the antibody should be nolonger detectable, the red cells of allsubsequent transfusions to that patientshould lack the antigen, to prevent asecondary immune response. The trans-fusion service must maintain records ofall patients in whom significant antibod-ies have been previously identified.8 Anantiglobulin crossmatch procedure isrequired if the serum contains, or haspreviously contained, a significant anti-body.

A potent example of the antibodyshould be used to identify antigen-nega-tive blood. Often, this is a commercialantiserum, but to save expensive or rarereagents, units can first be tested withthe patient’s serum. The absence of anti-gen, in nonreactive units, can then beconfirmed with the commercial reagent.Sufficiently potent antibodies in pa-tients’ specimens, that have reactivity asgood as, or better than, commercial an-tibodies, can be stored frozen for futureuse. If the antibody is of unusual speci-ficity or one for which commercial re-agents are not available, a stored samplecan be used to select units for transfu-sion at a later time, especially if the pa-tient’s later specimens lose reactivity. Ifa patient’s serum is to serve as a typingreagent, it should be well characterizedand retain its reactivity after storage,and appropriate negative and weaklypositive controls should be used at thetime of testing. The FDA has establishedthe following criteria for licensing somereagents,11 and these criteria may beuseful guidelines for evaluating in-house antisera.

1. Anti-K, anti-k, anti-Jka, anti-Fya,and anti-Cw: dilution of 1:8 to give atleast 1+ reaction.

2. Anti-S, anti-s, anti-P1, anti-M, anti-I, anti-c (saline), anti-e (saline), andanti-A1: dilution of 1:4 to give atleast 1+ reaction.

3. Most other specificities: undiluted,must give at least a 2+ reaction.



Reagents prepared from sera that meetthese dilution criteria will ordinarily beused undiluted.

Source of AntibodiesWhen selecting units for patients withclinically significant antibodies, someworkers recommend typing the chosenunits with antibodies from two differentsources, but others consider it unneces-sary, especially when potent reagents areavailable. Different lots of antibody fromthe same manufacturer and even reagentsfrom different manufacturers may nothave been prepared from different sourcematerial. For some specificities, donors ofsource plasma or the range of availablemonoclonal antibodies are limited; if sup-plies are scarce, manufacturers oftenshare the same resources.

Labeling UnitsIf a donor unit from a licensed bloodestablishment is to be labeled with theresults of special antigen typing and thatunit is shipped from the labeling facility,the FDA requires use of licensed (com-mercial) reagents.11 If no licensed re-agent is available, the unit may be la-beled with appropriate wording (eg,“Screened for Jsb; Js(b–) status not con-firmed with licensed reagent”). Exceptfor results of ABO and D typing, there isno requirement that results of antigentyping be on the label of donor units.

When to TestFor certain antibody specificities, spe-cial typing of donor units may not benecessary and the patient’s serum can beused to select serologically compatiblered cells. This is especially true for anti-bodies that characteristically react be-low 37 C (eg, anti-M, -P1, -A1) and do notordinarily exhibit an anamnestic re-sponse to the transfusion of antigen-positive red cells. Blood can be selected


without antigen typing for transfusionto patients with anti-Lea or -Leb, becausethese antigens are not intrinsic to thered cell and delayed transfusion reac-tions are not a concern (see Chapter 12).

It is rarely necessary to provide anti-gen-negative donor units as a prophylac-tic measure for patients whose cells lackan antigen but who do not have demon-strated antibody. However, special con-sideration is sometimes given to certainRh antibodies. When a patient of theR1R1 phenotype has anti-E detected inthe serum, some workers suggest thatdonor blood be negative for both the Eand c antigens,12 based on the assump-tion that the stimulus to produce theanti-E may also have stimulated an anti-c or anti-cE that remains undetected byroutine tests. For an R2R2 patient withdemonstrated anti-C, the use of C–e–donor blood must be considered. Whenan antibody has not been specificallydemonstrated, but cannot conclusivelybe excluded, it may be appropriate totransfuse blood that lacks the antigen.

Tests to Predict Clinical SignificanceCertain laboratory procedures have beenused to predict the significance of par-ticular antibodies. The mononuclearphagocyte assay, which quantifies roset-ting or phagocytosis of antibody-sensi-tized red cells, can be used to predict thein-vivo clinical significance of some an-tibodies. Determination of the IgG sub-class of an antibody may help predict thedestructive potential of either allo- orautoantibodies. The test for antibody-de-pendent cellular cytotoxicity (ADCC),which measures lysis of antibody-coatedcells, and the chemiluminescence assay,which measures the respiratory releaseof oxygen radicals after phagocytosis ofantibody-coated cells, have been helpfulin predicting in-vivo antibody reactivity,especially for predicting severity ofHDN. For cold-reactive antibodies, de-



termination of in-vitro thermal ampli-tude suggests the likelihood of causingin-vivo problems.

In-vivo tests may also be used toevaluate significance of a given antibody.The most common technique is infusionof radiolabeled, antigen-positive redcells, usually tagged with 51Cr. It is pos-sible to measure survival of 1 mL or lessof infused cells. Flow cytometry can alsobe used to measure the survival of in-fused cells, but a larger aliquot of redcells (about 10 mL) is generally re-quired. Small aliquots of incompatiblecells may have a faster rate of destruc-tion than an entire unit of red cells.

When Blood of Rare Type Is NeededBlood of rare type includes not only

units negative for high-incidence anti-gens but, often, blood negative for acombination of less exotic charac-teristics. When a patient has multipleantibodies, it can be helpful to deter-mine how rare compatible blood mightbe, especially when multiple units maybe required. To calculate this, the fre-quency of random donors negative forone antigen must be multiplied by thefrequency of donors negative for each ofthe other antigens. For example, if a se-rum contains anti-c, -Fya, and -S, andamong random donors 18% are c-nega-tive, 34% are Fy(a–), and 45% are S-negative, the frequency of compatibleunits would be: 0.18 × 0.34 × 0.45 =0.028. If the patient is group O, then,because 45% of random donors aregroup O, 1.3% (0.028 × 0.45) of randomdonors would be compatible with thepatient’s serum. If any of these threeantibodies occurred singly, finding com-patible blood would not be too difficult.Clearly, when all three are present, alarge number of random donors wouldbe necessary to provide even one unit.The preceding calculation uses frequen-cies in the Caucasian population. If the


donor population is predominantly of adifferent origin, frequencies for thatgroup would be appropriate. For mostblood suppliers the problem might beeased by previous records of donor phe-notypes or adequate numbers of unitsthat could be typed.

When units of rare (<1 in 5000) oruncommon (<1 in 1000) type areneeded, a Rare Donor File can be veryhelpful. These files, which can be ac-cessed only by personnel of an accreditedimmunohematology reference labora-tory, can identify blood suppliers knowneither to have units available (usuallyfrozen red cells) or to have the names ofsuitable donors who may be asked todonate (see Method 4.8).

Family members offer another poten-tial source of rare blood donors. Siblingsare often the best source of serologicallycompatible blood for patients with mul-tiple antibodies or antibodies to high-in-cidence antigens. The absence of high-incidence antigens usually reflectsinheritance, from each parent, of thesame rare blood group gene, and off-spring of the same parents are far morelikely to have the same two rare genesthan someone in the random donorpopulation. In most cases, blood fromthe patient’s parents or children (andsome siblings) will carry only a singledose of the relevant antigen; if transfu-sion is essential, and there is no alterna-tive to giving incompatible blood, theseheterozygous donors would be consid-ered preferable to random donors. Occa-sionally, blood from one parent or childalso lacks the high-incidence antigen.

In HDN or other alloantibody-associ-ated problems in infants, the mother isoften the logical donor. If the mother’s redcells are transfused, it is useful to retainthe plasma for use as a rare reagent.

If the clinical situation allows, autolo-gous transfusion should be considered forpatients for whom compatible blood is dif-ficult to find. For some patients with mul-



tiple antibodies for whom autologoustransfusion is not an option, it may benecessary to determine whether any ofthe antibodies is likely to be significantlyless destructive than the others and, in acritical situation, give blood incompat-ible for that antigen alone.

Frequency of Antibody TestingOnce an antibody has been identified ina patient’s serum, how frequently shouldantibody detection and identificationtests be performed? A primary antibodyresponse typically will produce detect-able antibody over a period of 2 weeks toseveral months, while a secondary im-mune response produces detectable an-tibody in a shorter time, usually within2-20 days. Shulman13 found that, in asmall number of patients, “new” anti-bodies could be detected within 1-2 daysafter transfusion. AABB Standards8 re-quires that, for a patient who has beenpregnant or received red cells within thepreceding 3 months, antibody detectionand compatibility tests be done on aspecimen obtained within 3 days of thenext scheduled transfusion. The transfu-sion service may consider testing afresher specimen when there have beenrepeated transfusions within the 3-dayperiod, especially if clinical evidencesuggests failure of transfused red cells tosurvive as expected.

It is rarely necessary to repeat identi-fication of known antibodies. The pri-mary concern of subsequent tests is thedetection of other antibodies. If theknown alloantibody is clinically signifi-cant, antigen-negative red cells must beselected for all future transfusions, evenif the antibody is no longer detectable,and there must be an antiglobulin cross-match, which serves as one means ofdetecting additional antibodies. It maybe useful, at intervals, to test one or twoantigen-positive red cells to confirmwhether the known antibody is still pre-


sent, so as to adopt the most informativeprocedure for further antibody detectionor identification tests.

Depending on the specificity of theknown antibody, repeated testing of thepatient’s serum against routine antibodydetection cells is often not informative.It is more useful to test against cellsnegative for the antigen(s) to which thepatient has antibody and positive forother major antigens. This allows detec-tion of most additional antibodies thatmight develop. Appropriate cells canusually be selected from available redcell panels. If the patient’s phenotype isknown, selection of appropriate testcells may be simplified. If the patient’scells are known to express a given anti-gen, the selected cells need not be posi-tive for that antigen since the corre-sponding ant ibody would not beanticipated. If appropriate cells are notavailable to detect additional antibodies,later serum specimens can be testedagainst cells retained from recentlytransfused units; these cells presumablycarry the antigens that would elicit mostnewly developing immune response.

Selected SerologicProceduresMany techniques and methods may beuseful in antibody identification. Someof the methods given here are used rou-tinely by many laboratories; others arealternatives that may apply only in spe-cial circumstances. It is important toremember that no single method is opti-mal for detecting all antibodies in allsamples. Any laboratory doing antibodydetection or identification should havestandard procedures for routine testing,and have access to at least some alterna-tive approaches. Additional proceduresare available in a variety of references.See Suggested Reading.



Enhancement Techniques

When a pattern of weak reactions fails toindicate specificity, or when the pres-ence of an antibody is suspected but can-not be demonstrated, use of the follow-ing procedures may be helpful. Anautologous control should be includedwith each test performed.

LISS, PEG, Polybrene®

The rationale for these procedures andsome technical details are discussed inChapter 11. Each may be used to en-hance reactivity and reduce incubationtime. Some antibodies react preferen-tially by one of these techniques. LISSmethods include the use of low ionicstrength saline for resuspension of testcells and also the more common use ofcommercial ly ava i lab le low ionicstrength additive media. The use of aLISS additive requires no preparatorystages. Commercially prepared LISS ad-ditives may include other enhancementcomponents besides low ionic strengthsaline. Commercially prepared PEG ad-ditives are also available and may con-tain additional enhancing agents. Be-cause LISS , PEG, and Polybrene®

enhance autoantibody activity, they maycreate problems with certain samples.

Table 17-4. Alteration of Antigens by V

Agent Antigens Us

Proteolytic Enzymes‡ M, N, S, Fya,Jea, Nya, JMH

DTT Yta, JMH, KnLutheran, Do

ZZAP (a combination of DTTand proteolytic enzymes)

Alteration of

*Modified from Wilkinson.14

†Some antigens listed may be weakened rather thshould be used with modified cells.‡Different proteolytic enzymes may have differen


Enzyme TechniquesTreatment of red cells with proteolyticenzymes enhances their reactivity withantibodies in the Rh, P, I, Kidd, Lewis,and some other blood group systems,and simultaneously destroys or weakensother antigens. See Table 17-4. Proce-dures for the preparation and use of pro-teolytic enzyme solutions are given inMethods 3.5 through 3.5.5.

Temperature ReductionSome alloantibodies (eg, anti-M, -P1)that react at room temperature reactbetter at lower temperatures; specificitymay only be apparent below 22 C. Anautocontrol is especially important fortests at cold temperatures, becausemany sera also contain anti-I or othercold-reactive autoantibodies.

Increased Serum-to-Cell RatioIncreasing the volume of serum incu-bated with a standard volume of red cellsmay enhance the reactivity of antibodiespresent in low concentration. One ac-ceptable procedure is to mix 5-10 vol-umes of serum with one volume of a2-5% saline suspension of red cells andincubate for 60 minutes at 37 C; periodicmixing during incubation promotes

arious Agents*

ually Denatured or Altered†

Fyb, Yta, Ch, Rg, Pr, Tn, Mg, Mia/Vw, Cla,, some Ge, Inb

a, McCa, Yka, LWa, LWb, Ge, all Kell,mbrock, and Cromer blood group antigensall the antigens listed above

an completely denatured. Appropriate controls

t effects on certain antigens.



contact between red cells and antibodymolecules. It is helpful to remove theserum before washing the red cells forthe antiglobulin test, because the stand-ard three or four wash phases may beinsufficient to remove all the unboundimmunoglobulin present in the addi-tional volume. Additional wash phasesare not recommended because boundantibody molecules may dissociate. In-creasing the serum-to-red cell ratio isnot appropriate in LISS, PEG, or othertests that require specific proportions ofserum and additive.

Increased Incubation Time

For some antibodies, a 15-minute incu-bation period is insufficient to achieveequilibrium and the observed reactionsmay be weak, particularly in saline oralbumin media. Extending incubation to60 minutes may improve reactivity andhelp clarify the observed pattern of reac-tions.

Extended incubation may have anegative effect when LISS or PEG areused. If incubation exceeds the recom-mended times for these methods, anti-body reactivity may be lost.

Alteration of pH

Decreasing the pH of the reaction sys-tem to 6.5 enhances the reactivity ofcertain antibodies, notably some exam-ples of anti-M.15 If anti-M specificity issuspected because the only cells aggluti-nated are M+N–, modifying the serum toa pH of 6.5 may reveal a definitive pat-tern of anti-M reactivity. The addition ofone volume of 0.1 N HCl to nine volumesof serum brings the pH to approximately6.5. The acidified serum should be addedto known M-negative cells as a controlfor nonspecific agglutination. Some ex-amples of anti-P may similarly benefitfrom a lower pH.16


Low pH, however, significantly de-creases reactivity of some antibodies.17 Ifunbuffered saline used for cell suspen-sions and for washing has a pH muchbelow 6.0, antibodies in the Rh, Duffy,Kidd, and MNS systems may lose reactiv-ity. Use of phosphate-buffered saline (seeMethod 1.6) can control pH and enhancedetection of antibodies poorly reactive atlower pH.18

Techniques to Isolate, Remove, orDepress Antibody ReactivityIt is sometimes useful to decrease oreliminate the reactivity of an antibody.This can be done by inhibiting the anti-body with specific substances, by physi-cally removing immunoglobulin mole-cules or by removing (or weakening)corresponding antigens from the redcells. Such methods can help confirmsuspected specificities and promoteidentification of additional antibodies.

Inhibition TestsSoluble forms of some blood group anti-gens exist in such body fluids as saliva,urine, or plasma, or can be preparedfrom other sources. These substancescan be used to inhibit reactivity of thecorresponding antibody. If, for example,a suspected anti-P1 does not give a de-finitive agglutination pattern, loss of re-activity after addition of soluble P1 sub-stance strongly suggests that this is thespecificity. A parallel control with salineis essential.

Inhibition can also be used to neutral-ize antibodies that mask the concomi-tant presence of nonneutralizable anti-bodies. The following soluble bloodgroup substances can be used in anti-body identification tests:

1. Lewis substances. Lea and/or Leb sub-stances are present in the saliva ofpersons who possess the Le gene. Lea

substance is present in the saliva of



Le(a+b–) individuals, and Le(a–b+)persons have both Lea and Leb sub-stances in their saliva. (See Method2.5.) Commercially prepared Lewissubstance is also available.

2. P1 substance. Soluble P1 substanceis present in hydatid cyst fluid andcan also be prepared from pigeonegg whites. Pigeon-derived P1 sub-stance is available commercially.

3. Sda substance. Soluble Sda bloodgroup substance is present in vari-ous body fluids; the most abundantsource is urine.19 To confirm anti-Sda specificity in a serum, urinefrom a known Sd(a+) individual (ora pool of urine specimens) can beused to inhibit reactivity. Urineknown to lack Sda substance, or sa-line, should be used as a negativecontrol. Urine should be dialyzedagainst phosphate-buffered saline tocorrect pH and salt concentrationsbefore use. Once dialyzed, Sd(a+)and Sd(a–) urine specimens can befrozen in small aliquots for futureuse. (See Method 4.7.)

4. Chido and Rodgers substances. Chand Rg antigens are epitopes of thefourth component of human com-plement (C4).20,21 Anti-Ch and -Rgreact by the IAT with the traceamounts of C4 present on normalred cells; if red cells are coated invitro with excess C422 (see Method4.4), these antibodies may cause di-rect agglutination. Anti-Ch and -Rgcan be inhibited by plasma from Ch-positive, Rg-positive individuals.(See Method 4.5.) This is a usefultest for rapid identification of anti-Ch and -Rg.

5. Blood Group Sugars. Sugars thatcorrespond to the immunodominantconfigurations of A, B, H, and someother red cell structures can be usedto inhibit antibodies. Inhibiting anti-A or -B may allow a serum to be testedagainst non-group O cells.


Inactivation of Blood Group Antigens

Certain blood group antigens can be de-stroyed or weakened by suitable treat-ment of the cells. (See Table 17-4.) Modi-f i ed ce l l s can be use ful both inconfirming the presence of suspectedantibodies and in detecting additionalantibodies. This can be especially helpfulif the antigen is one of high incidenceand antigen-negative cells are rare.

Proteolytic enzymes are commonlyused to alter red cell antigens. Ficin,papain, trypsin, and bromelin, the en-zymes most frequently used, remove M,N, S, Fya, Fyb, Xga, JMH, and some Geantigens. Depending on the specific en-zyme and method used, other antigensmay also be altered or destroyed. Anti-gens inactivated by one proteolytic en-zyme will not necessarily be inactivatedby other enzymes.

Sulfhydryl reagents such as 2-ami-noethylisothiouronium bromide (AET)or dithiothreitol (DTT) can be used toweaken or destroy antigens in the Kellsystem and some other antigens.23-25 Ananti-IgG reagent should be used in testswith DTT- or AET-treated red cells. ZZAPreagent, a mixture of proteolytic enzymeand DTT,26 denatures antigens in theKell system, as does treatment with gly-cine-HCl/EDTA. (See Methods 6.5 and5.4.) Sulfhydryl treatment alters redcells to induce a state of heightened sen-sitivity to the actions of complement.With glycine-HCl/EDTA treatment, anti-gens outside the Kell system that areoften destroyed by the sulfhydryl re-agents are left intact, although Era anti-gens may also be denatured.27

Chloroquine diphosphate can be usedto weaken the expression of Class I HLAantigens (Bg antigens) on red cells.28

Chloroquine removes the β2-micro-globulin light chain covalently associ-ated with the heavy chain of Class I HLAantigens. This deters reactivity withmost HLA-reactive antibodies, although



some may still react.29 Chloroquinetreatment also weakens some other anti-gens, including Rh antigens.

Adsorption

Antibody can be removed from a serumby adsorption to red cells carrying thecorresponding antigen. After the anti-body attaches to the membrane-boundantigens and the serum and cells areseparated, the specific antibody remainsattached to the red cells. It may be pos-sible to harvest the bound antibody byelution.

Adsorption techniques are useful insuch situations as:

1. Separating multiple antibodies pre-sent in a single serum.

2. Removing autoantibody activity topermit detection of coexisting al-loantibodies.

3. Removing unwanted antibody (oftenanti-A and/or anti-B) from a serumthat contains an antibody suitablefor reagent use.

4. Confirming the presence of specificantigens on red cells through theirability to remove antibody of corre-sponding specificity from previouslycharacterized serum.

5. Confirming the specificity of an an-tibody by showing that it can be ad-sorbed only to red cells of a particu-lar blood group phenotype (see page374).

Technical Considerations. Adsorp-tion serves different purposes in differ-ent situations; there is no single proce-dure that is satisfactory for all purposes.The usual serum-to-cell ratio used is onevolume of serum to an equal volume ofwashed, packed red cells. To enhance an-tibody uptake, the proportion of antigencan be increased by use of a larger vol-ume of cells. The incubation tempera-ture should be that at which the anti-body is optimally reactive. Pretreatingred cells with a proteolytic enzyme may


enhance antibody uptake and reduce thenumber of adsorptions required for com-plete removal of antibody. Since someantigens are destroyed by proteases, an-tibodies directed against these antigenswill not be removed by enzyme-treatedred cells.

In separating mixtures of antibodies,the selection of red cells of the appropri-ate phenotype is extremely important,and depends on the object of the separa-tion. If none of the antibodies in theserum have been identified, weakly reac-tive cells may be used, on the assump-tion they are reactive with only a singleantibody. The phenotype of the personproducing the antibody gives a clue as towhat specificities might be present, andcells intended to separate those particu-lar antibodies can be chosen. If one ormore antibodies have been identified,cells lacking those antigens are usuallychosen so that only one antibody is re-moved. Adsorption requires a substan-tial volume of red cells. Vials of reagentred cells usually will not suffice, andblood samples from staff members ordonor units are the most convenientsources.

Adsorption Procedure. Attention todetail is important for obtaining satis-factory test results.

1. Wash the selected red cells at leastthree times with saline. After thelast wash, centrifuge the red cells forat least 5 minutes, and remove asmuch of the supernatant saline aspossible. Additional saline may beremoved by touching the red cellmass with a narrow piece of filterpaper.

2. Mix appropriate volumes of thepacked red cells and serum, and in-cubate at the desired temperaturefor 30-60 minutes. Adsorption ismore effective if the area of contactbetween the red cells and serum islarge; use of a large-bore test tube(13 mm or larger) is recommended.



The serum/cell mixture should bemixed periodically throughout theincubation phase.

3. Centrifuge to pack the red cellstightly. Centrifuge at the incubationtemperature, if possible, to avoiddissociation of antibody from thered cell membranes.

4. Transfer the supernatant fluid,which is the adsorbed serum, to aclean test tube. If an eluate is to beprepared, save the red cells.

5. Test an aliquot of the adsorbed se-rum, preferably against an addi-tional aliquot of the cells used foradsorption, to see if all antibody hasbeen removed. If reactivity remains,repeat the adsorption process with afresh aliquot of washed, packed redcells. Multiple adsorptions may benecessary, but each successive ad-sorption increases the likelihoodthat the serum will be diluted andunadsorbed antibodies be weakened.

Elution

Elution frees antibody molecules fromsensitized red cells. Bound antibody maybe released by changing the thermody-namics of antigen-antibody reactions, byneutralizing or reversing forces of at-traction that hold antigen-antibodycomplexes together, or by disturbing thestructural complementarity between anantigen and its corresponding bindingsite on an antibody molecule. The usualobjective is to recover bound antibody ina usable form.

A variety of elution methods havebeen described. Selected procedures aregiven in Methods 5.1 through 5.6. Nosingle method is best in all situations.Use of heat or freeze-thaw elution is usu-ally restricted to the investigation ofHDN due to ABO incompatibility, be-cause these manipulations rarely workwell for antibodies outside the ABO sys-tem. Acid or organic solvent methods are


used for elution of warm-reactive auto-and alloantibodies.

Technical Considerations. Technicalfactors that influence the success of elu-tion procedures include:

1. Incorrect technique. Such factors asincomplete removal of organic sol-vents or failure to correct the tonic-ity or pH of an eluate may cause thered cells used in testing the eluate tohemolyze or to appear “sticky.” Thepresence of stromal debris may in-terfere with the reading of tests.Careful technique and strict adher-ence to protocols should eliminatesuch problems.

2. Incomplete washing. The sensitizedred cells must be thoroughly washedprior to elution, to prevent contami-nation of the eluate with residualserum antibody. If it is known thatthe serum does not contain anti-body, saline washing may not benecessary. Six washes with saline areusually adequate, but more may beneeded if the serum contains a high-titer antibody. To determine the effi-cacy of the washing process, super-natant fluid from the final washphase should be tested for antibodyactivity and should be inert.

3. Binding of proteins to glass sur-faces. If the eluate is prepared in thesame test tube that was used duringthe sensitization phase, antibodynonspecifically bound to the testtube surface may dissociate duringthe elution. Similar binding can alsooccur from a whole blood sample ifthe patient has a positive DAT andfree antibody in the serum. To avoidsuch contamination, the washed redcells should be transferred into aclean test tube before the elutionprocedure is begun.

4. Dissociation of antibody before elu-tion. IgM antibodies, such as anti-Aor -M, may spontaneously dissociatefrom the cells during the wash



phase. To minimize this loss ofbound antibody, cold (4 C) saline canbe used for washing. Although thisis not a concern with most IgG anti-bodies, some low-affinity antibodiescan also be lost during the washphase. If such antibodies are sus-pected, washing with cold LISS in-stead of normal saline may helpmaintain antibody association.

5. Instability of eluates. Dilute proteinsolutions, such as those obtained byelution into saline, are unstable.Eluates should be tested as soon af-ter preparation as possible. Alterna-tively, bovine albumin may be addedto a final concentration of 6% wt/voland the preparation stored frozen.Eluates can also be prepared directlyinto antibody-free plasma, 6% albu-min, or similar protein medium in-stead of into saline.

Applications. Elution techniques areuseful for:

1. Investigation of a positive DAT (seeChapter 18).

2. Concentration and purification ofantibodies, the detection of weaklyexpressed antigens, and the identifi-cation of multiple antibody specifi-cities. Such studies are used in con-junct ion wi th an appropriateadsorption technique, as describedabove and in Method 2.4.

3. Preparation of antibody-free redcells for use in phenotyping orautologous adsorption studies. Proce-dures used to remove cold- and warm-reactive autoantibodies from red cellsare discussed in Methods 6.1 and 6.4,and methods for autologous adsorp-tion of warm-reactive autoantibodiesare given in Chapter 18.

Combined Adsorption-Elution

Combined adsorption-elution tests canbe used to help identify weakly reactiveantibodies, to separate mixed antibodies


from a single serum, or to detect weaklyexpressed antigens on red cells. Theprocess consists of first incubating se-rum with selected cells, and then elutingantibody from the adsorbing red cells.Both the eluate and treated serum can beused for further testing. Unmodified redcells are generally used for adsorptionand subsequent elution. Elution fromenzyme- or ZZAP-treated cells may cre-ate technical problems.

Antibody Separation. Adsorption andelution may be used to separate a mix-ture of antibodies. This is useful in sev-eral situations:

1. If the serum contains one or moreunidentified antibodies, adsorption-elution can be used to isolate indi-vidual antibodies, which are theneasier to identify.

The serum under investigationshould be adsorbed until it nolonger reacts with the adsorbing redcells. The adsorbed serum is savedand an eluate prepared from the redcells used from the first adsorption.Cells from subsequent adsorptionscan also be used for elution, but thequantity of antibody may be signifi-cantly less. Both the adsorbed serumand eluate should be examined forantibody specificity. The goal is thatspecific antibodies will be apparentin either or both preparations, butthis does not always happen. Someantibodies (eg, anti-Kna and -McCa)are difficult to adsorb and elute. Theserum may lose all reactivity afteradsorption, yet the eluate reactswith all reagent red cell samples;this may indicate the presence of anautoantibody or an antibody to ahigh-incidence antigen that is vari-ably expressed on different red cells.

2. If the antibodies in a serum havealready been presumptively identi-fied, they may be separated by ad-sorption-elution, either to confirmthe specificities or to use one (or



more) antibody separately as a re-agent. This is especially useful whenrare antibodies are detected, sincethey often occur in serum that con-tains other antibodies as well.

Adsorption-elution is an effective wayto prepare antibodies, especially those tohigh-incidence antigens, for use withred cells of any ABO type. An eluate pre-pared from group O red cells known toreact with the antibody in questionshould be devoid of anti-A and -B andcontain only the desired specific anti-body. Antibodies purified in this mannercan be preserved frozen, providing theprotein concentration is adjusted, andused as a typing reagent.

This method is also useful with seracontaining antibodies to low-incidenceantigens. Such sera often contain anti-bodies to several different low-incidenceantigens. Since a cell positive for onelow-incidence antigen is usually nega-tive for others, eluates can be obtainedthat contain only a single specificity.

Detection of Weak Antigens. An anti-body that does not cause direct aggluti-nation may be adsorbed to antigen-posi-t i ve red cel l s . Occurrence of theantibody-antigen interaction can bedemonstrated by loss of antibody reactiv-ity in the adsorbed serum and sub-sequent recovery of the antibody fromadsorbing red cells.

Antigen detection by adsorption-elu-tion is usually best accomplished usinga low serum-to-cell ratio (eg, 2:1 or less).Care should be taken not to dilute theserum, and hence the antibody, with re-sidual saline from inadequately packedred cells. Determining the protein con-centrations of the serum by refrac-tometry before or after the adsorptionstep is an appropriate check for suchdilution. A reduction in titration score of10 or more, using the system describedin Method 4.2, is considered evidence forthe presence of the relevant antigen onthe adsorbing red cells. In critical stud-


ies, control adsorptions with red cellsknown to lack the antigen in questionshould be undertaken.

Detection of Weak Antibodies. Aweakly reactive antibody can be demon-strated by showing that it can be ad-sorbed and eluted by cells known to ex-press a part icular ant igen . Theadsorption-elution process can also beused to concentrate the antibody, mak-ing the eluate more reactive than theoriginal serum. A high serum-to-cell ra-tio (eg, 5:1 or more) should be used tosensitize the red cells. If the volume ofeluate is less than the volume of originalserum used to coat the red cells, activityshould be stronger in the eluate than inthe serum. For most methods, the vol-ume of eluate obtained can be controlledby varying the volume of saline or otherdiluent with which the red cells are in-cubated. Once prepared, the eluateshould be tested against uncoated redcells from the same sample used for ad-sorption, to establish whether elutionoccurred.

Use of Sulfhydryl Reagents

Sulfhydryl reagents, such as DTT and2-mercaptoethanol (2-ME), cleave thedisulfide bonds that join the monomericsubunits of the IgM pentamer. Intact 19SIgM molecules are cleaved into 7Ssubunits, which have altered serologicreactivity.30 The interchain bonds of 7SIg monomers are relatively resistant tosuch cleavage. (See Chapter 10 for thestructure of immunoglobulin mole-cules.) Sulfhydryl reagents are used todiminish or destroy IgM antibody reac-tivity. DTT also destroys certain red cellantigens. The applications of DTT and2-ME in immunohematology include:

1. Determining the immunoglobulinclass of an antibody. See Method 4.3.

2. Identifying specificities in a mixtureof IgM and IgG antibodies, particu-larly when an agglutinating IgM an-



tibody masks the presence of IgGantibodies.

3. Determining the relative amounts ofIgG and IgM components of a givenspecificity (eg, anti-A or -B).

4. Dissociating red cell agglutinatescaused by IgM antibodies (eg, thespontaneous agglutination of redcells caused by potent cold-reactiveautoantibodies). See Method 2.11.

5. Dissociating IgG antibodies fromred cells using a mixture of DTT anda proteolytic enzyme (ZZAP re-agent). See Method 6.5.

6. Converting nonagglutinating IgGantibodies into direct agglutinins.31

Commercially prepared chemicallymodified blood grouping reagentsfor use in rapid saline tube, slide, ormicroplate tests have been manufac-tured in this manner. See Chapter11.

7. Destroying selected red cell anti-gens (eg, those of the Kell, Dom-brock, Cartwright, Gerbich, and LWsystems) for use in antibody investi-gations. See Method 6.4.

Titration

The titer of an antibody is usually deter-mined by testing serial two fold dilutionsof the serum against selected red cellsamples. Results are expressed as the re-ciprocal of the highest serum dilutionthat causes macroscopic agglutination.Titration values can provide informationabout the relative amount of antibodypresent in a serum, or the relativestrength of antigen expression on redcells. Titration studies are usually per-formed in several situations.

Prenatal Studies. When the antibodyis of a specificity known to cause HDN orits clinical significance is unknown, theresults of titration studies may contrib-ute to the decision about performingamniocentesis. (See Chapter 21.)


Antibody Identification. Some anti-bodies that agglutinate virtually all re-agent red cell samples may produce anindication of specificity by demonstrat-ing reactivity of different strength withdifferent samples in titration studies.For example, potent autoanti-I may re-act in the undiluted state with both adultand cord red cells, but titration may re-veal reactivity at a higher dilution withadult I+ red cells than with cord redcells.

Most weakly reactive antibodies losereactivity when diluted even modestly,but some antibodies that give weak reac-tions when undiluted continue to reactat dilutions as high as 1 in 2048. Suchantibodies include anti-Ch, -Rg, -Csa,-Yka, -Kna, -McCa, -JMH, and other spe-cificities. When weak reactions are ob-served in indirect antiglobulin tests, ti-trat ion may be used to ind icatespecificity within this group. Not all an-tibodies of the specificities mentioneddemonstrate such “high titer, low avid-ity” characteristics. Thus, while demon-stration of these serologic charac-teristics may help point to certainspecificities, failure to do so does noteliminate those possibilities. Antibodiesof other specificities may also sometimesreact at high titers. Details of titrationare given in Method 4.2 and 4.5.

Separating Multiple Antibodies. Inserum samples containing more thanone specificity, titration results may sug-gest that one antibody reacts at higherdilutions than another. This informationcan allow the serum to be diluted beforetesting against a cell panel, effectivelyremoving one antibody and allowingidentification of the other.

Other Methods

Methods other than traditional tubetechniques may be used for antibodyidentification. Some are especially use-ful for identifying individual antibody



specificities, for dealing with small vol-umes of test reagents, for batch testingor for use with automated systems. Suchmethods include testing in capillarytubes, microplates, or by solid phase; en-zyme-linked immunosorbent assays;column agglutination; or gel tech-niques. Other methods useful in labora-tor ies with special ized equipmentinclude radioimmunoassay, immuno-fluorescence (including flow cytometricprocedures), immunoblotting, and im-munoelectrode biosensoring. Some ofthese methods are discussed in Chapter11.

References1. Giblett ER. Blood group alloantibodies: An as-

sessment of some laboratory practices. Trans-fusion 1977;17:299-308.

2. Walker RH, Lin DT, Hatrick MB. Alloimmuni-zation following blood transfusion. ArchPathol Lab Med 1989;113:254-61.

3. Marsh WL, Reid ME, Kuriyan M, Marsh NJ. Ahandbook of clinical and laboratory practicesin the transfusion of red blood cells. Moneta,VA: Moneta Medical Press, 1993.

4. Stone DL, Eatz RA, Rolih SD, et al. Red cellantibody identification by solid phase red celladherence utilizing dried RBC monolayers.Immunohematology 1990;6:12-17.

5. Shulman IA , Ca lderon C, Nelson JM,Nakayama R. The routine use of Rh-negativereagent red cells for the identification of anti-D and the detection of non-D red cell antibod-ies. Transfusion 1994;34:666-70.

6. Fisher RA. Statistical methods and scientificinference. 2nd ed. Edinburgh: Oliver andBoyd, 1959.

7. Harris RE, Hochman HG. Revised p values intesting blood group antibodies. Transfusion1986;26:494-9.

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

9. Brendel WL. Resolving antibody problems. In:Pierce SR, Wilson JK, eds. Approaches to sero-logical problems in the hospital transfusionservice. Arlington, VA: American Associationof Blood Banks, 1985:51-72.

10. Westhoff CM, Sipherd BD, Toalson LD. Red cellantigen stability in K3EDTA. Immunohema-tology 1993;9:109-11.


11. Code of federal regulations. Title 21 CFR660.25. Washington, DC: US GovernmentPrinting Office, 1996 (revised annually).

12. Shirey RS, Edwards RE, Ness PM. The risk ofalloimmunization to c (Rh4) in R1R1 patientswho present wi th ant i -E . Trans fus ion1994;34:756-8.

13. Shulman IA. Controversies in red blood cellcompatibility testing. In: Nance SJ, ed. Im-mune destruction of red blood cells. Ar-lington, VA: American Association of BloodBanks, 1989:171-99.

14. Wilkinson SL. Serological approaches totransfusion of patients with allo- or autoanti-bodies. In: Nance SJ, ed. Immune destructionof red blood cells. Arlington, VA: AmericanAssociation of Blood Banks, 1989:227-61.

15. Beattie KM, Zuelzer WW. The frequency andproperties of pH-dependent anti-M. Transfu-sion 1965;5:322-6.

16. Judd WJ. A pH dependent autoagglutinin withanti-P specificity. Transfusion 1975;15:373-6.

17. Bruce C, Watt AH, Hare V, et al. A serioussource of error in antiglobulin testing. Trans-fusion 1986;26:177-81.

18. Rolih S, Thomas R, Fisher F, Talbot J. Antibodydetection errors due to acidic or unbufferedsaline. Immunohematology 1993;9:15-18.

19. Morton J, Pickles MM, Terry AM. The Sda bloodgroup antigen in tissues and body fluids. VoxSang 1970;19:472-82.

20. O’Neil GJ, Yang SY, Tegoli J, et al. Chido andRodgers blood groups are distinct antigeniccomponents of human complement, C4. Na-ture 1978;273:668-70.

21. Tilley CA, Romans DG, Crookston MC. Local-ization of Chido and Rodgers to the C4d frag-ment of human C4 (abstract). Transfusion1978;18:622.

22. Judd WJ, Kreamer K, Moulds JJ. The rapididentification of Chido and Rodgers antibodiesusing C4d-coated red blood cells. Transfusion1981;21:189-92.

23. Advani H, Zamor J, Judd WJ, et al. Inactivationof Kell blood group antigens by 2-ami-noethylisothiouronium bromide. Br J Haema-tol 1982;51:107-15.

24. Branch DR, Muensch HA, Sy Siok Hian AL,Petz LD. Disulfide bonds are a requirement forKell and Cartwright (Yta) blood group antigenintegrity. Br J Haematol 1983;54:573-8.

25. Moulds J, Moulds MM. Inactivation of Kellb lood group ant igens by 2-amino-ethylisothiouronium bromide. Transfusion1983;23:274-5.

26. Branch DR, Petz LD. A new reagent (ZZAP)having multiple applications in immunohe-matology. Am J Clin Pathol 1982;78:161-7.

27. Liew YW, Uchikawa M. Loss of Era antigen invery low pH buffers. Transfusion 1987;27:442-3.



28. Swanson JL, Sastamoinen R. Chloroquinestripping of HLA A,B antigens from red cells(letter). Transfusion 1985;25:439-40.

29. Giles CM. Human leukocyte antigen (HLA)class I (Bg) on red cells studied with mono-clona l ant ibodies . Immunohematology1990;6:53-8.

30. Freedman J, Masters CA, Newlands M, et al.Optimal conditions for use of sulphydryl com-pounds in dissociating rbc antibodies. VoxSang 1976;30:231-9.

31. Romans DG, Tilley CA, Crookston MC, et al.Conversion of incomplete antibodies to directagglutinins by mild reduction. Evidence forsegmental flexibility within the Fc fragment ofimmunoglobulin G. Proc Natl Acad Sci USA1977;74:2531-5.

Suggested ReadingBoorman KE, Dodd BE, Lincoln PJ. Blood groupserology. 6th ed. Edinburgh: Churchill Living-stone, 1988.

Crookston MC. Soluble antigens and leukocyte re-lated antibodies. Part A. Blood group antigens inplasma: An aid in the identification of antibodies.In: Dawson RD, ed. Transfusion with “crossmatchincompatible” blood. Washington, DC: AmericanAssociation of Blood Banks, 1975:20-5.

Engelfriet CP, Overbeeke MAM, Dooren MC, et al.Bioassays to determine the clinical significance ofred cell antibodies based on Fc receptor-induceddestruction of red cells sensitized by IgG. Transfu-sion 1994;14:617-26.


Giles CM. An update on Rodgers and Chido, theantigenic determinants of C4. Immunohematol-ogy 1989;5:1-6.

Issitt PD. Applied blood group serology. 3rd ed.Miami: Montgomery Scientific Publications, 1985.

Judd WJ. Elution of antibody from red cells. In:Bell CA, ed. A seminar on antigen-antibody reac-tions revisited. Washington, DC: American Asso-ciation of Blood Banks, 1982:175-221.

Judd WJ. Methods in immunohematology. 2nd ed.Durham, NC: Montgomery Scientific Publications,1994.

Mallory D, ed. Immunohematology methods andprocedures. Rockville, MD: American Red Cross,1993.

Menitove JE. The Hardy-Weinberg principle: Se-lection of compatible blood based on mathematicprinciples. In: Fridey JL, Kasprisin CA, ChambersLA, Rudmann SV, eds. Numbers for blood bankers.Bethesda, MD: American Association of BloodBanks, 1995:1-11.

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

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

Rolih S. A review: Antibodies with high-titer, low-avidity characteristics. Immunohematology1990;6:59-67.

Telen MJ. New and evolving techniques for anti-body and antigen identification. In: Nance ST, ed.Alloimmunity: 1993 and beyond. Bethesda, MD:American Association of Blood Banks, 1993:117-39.


Documents

17 Identification of Alloantibodies toother than non-red-cell-stimulated anti-A or -B are called unexpected red cell alloantibodies and may be found in 0.3-38% of the population, depending