Antibody TitrationPrincipleTitration is a semiquantitative method usedto determine the concentration of antibodyin a serum sample or to compare thestrength of antigen expression on differentred cell samples. The usual applicationsof titration studies are: 1) estimatingantibody activity in alloimmunized pregnantwomen to determine whether andwhen to perform more complex invasiveinvestigation of the fetal condition 2) elucidating autoantibodyspecificity 3) characterizingantibodies as high-titer, low-avidity,traits common in antibodies to antigensof the Knops and Chido/ Rodgers systems,Csa, and JMH and4) observing the effect of sulfhydryl reagentson antibody behavior, to determineimmunoglobulin class (IgG or IgM).See Method (below) for titration studies specificallyto assist in monitoring clinicallysignificant antibodies in the pregnant

woman.SpecimenSerum or plasma antibody to be titrated.Reagents1. Red cells that express the antigen(s)corresponding to the antibody specificity(ies), in a 2% to 5% saline suspension.Uniformity of cell suspensionsis very important to ensurecomparability of results.2. Saline. (Note: Dilutionsmay be madewith albumin if desired.)ProcedureThe master dilution technique for titrationstudies is as follows:1. Label 10 test tubes according to theserum dilution (eg, 1 in 1, 1 in 2, etc).A 1 in 1 dilution means one volumeof serum undiluted; a 1 in 2 dilutionmeans one volume of serum in a fi-nal volume of two, or a 50% solutionof serum in the diluent. 2. Deliver one volume of saline to all testtubes except the first (undiluted 1 in1) tube.3. Add an equal volume of serum to

each of the first two tubes (undilutedand 1 in 2).4. Using a clean pipette, mix the contentsof the 1 in 2 dilution severaltimes and transfer one volume intothe next tube (the 1 in 4 dilution).5. Continue the same process for all dilutions,using a clean pipette to mixand transfer each dilution. Removeone volume of diluted serum fromthe final tube and save it for use iffurther dilutions are required.6. Label 10 tubes for the appropriatedilutions.7. Using separate pipettes for each dilution,transfer 2 drops of each dilutedserum into the appropriatelylabeled tubes and add 2 drops of a2% red cell suspension. Alternatively,for convenience, add 1 drop ofa 3%-4% suspension of red cells assupplied by the reagent manufacturer,although this method is lessprecise.

8. Mix well and test by a serologic techniqueappropriate to the antibody9. Examine test results macroscopically;grade and record the reactions. Theprozone phenomenon may cause reactions to be weakerin the more concentrated serum preparationsthan in higher dilutions.To avoid misinterpretation of results,itmay be preferable to examine firstthe tube containing the most diluteserumand proceed through the moreconcentrated samples to the undilutedspecimen.Interpretation1. Observe the highest dilution thatproduces 1+ macroscopic agglutination.The titer is reported as the reciprocalof the dilution level, eg,32—not 1 in 32 or 1:32 (see Table3.7-1). If there is agglutination in thetube containing the most dilute serum,the endpoint has not beenreached, and additional dilutions

should be prepared and tested.2. In comparative studies, a significantdifference in titer is three or more dilutions.Variations in technique andinherent biologic variability can causeduplicate tests to give results thatdiffer by one dilution in either direction.Serum containing antibody at atrue titer of 32 may show, on replicatetests, the endpoint in the 1:32tube, the 1:64 tube, or the 1:16 tube.3. Titer values alone can be misleadingwithout also evaluating the strengthof agglutination. The observed strengthof agglutination can be assigned anumber and the sum of these numbersfor all tubes in a titration studyrepresents the score, another semiquantitativemeasurement of antibodyreactivity. The arbitrarily assignedthreshold for significance in comparingscores is a difference of 10 ormore between different test samples.See Table 3.7-1.4. Antibodies with high-titer, low-avidity

characteristics generally have a titergreater than 64, with most tubesshowing consistently weak reactivity.Table 3.7-1 shows the results obtainedwith three sera, each of which shows nomore agglutination after 1:256. The differencesin score, however, indicate considerablevariation in strength of reactivity.

NotesTitration is a semiquantitative technique.Technical variables greatly affect the resultsand care should be taken to achievethe most uniformpossible practices.1. Careful pipetting is essential. Pipetteswith disposable tips that canbe changed after each dilution arerecommended.2. Optimal time and temperature of incubationand time and force of centrifugationmust be used consistently.3. The age, phenotype, and concentrationof the test red cells will influence

the results. When the titers of severalantibody-containing sera are to becompared, all of them should betested against red cells (preferablyfreshly collected) from the same donor.If this is not possible, the testsshould use a pool of reagent red cellsfrom donors of the same phenotype.When a single serum is to be testedagainst different red cell samples, allsamples should be collected and preservedin the same manner and dilutedto the same concentration beforeuse.4. Completely reproducible results arevirtually impossible to achieve. Comparisonsare valid only when specimensare tested concurrently. In testswith a single serum against differentred cell samples, material from themaster dilution must be used for allthe tests.5. Measurements are more accurate withlarge volumes than with small volumes;a master dilution technique

(see above) gives more reliable resultsthan individual dilutions for asingle set of tests. The volume neededfor all planned tests should be calcu-lated and an adequate quantity ofeach dilution prepared.6. When performing a titration foranti-D for HDFN,

Titration Studies to Assist inEarly Detection of HemolyticDisease of the Fetus andNewbornPrincipleAntibody titration is a semiquantitativemethod of determining antibody concentration.Serial twofold dilutions of serumare prepared and tested for antibody activity.The reciprocal of the highest dilutionof plasma or serum that gives a 1+ reactionis referred to as the titer (ie, 1 in128 dilution; titer = 128).In pregnancy, antibody titration is performedto identify women with significantlevels of antibodies that may lead tohemolytic disease of the fetus and newborn(HDFN) and, for low-titer antibodies, to establisha baseline for comparison with

titers found later in pregnancy. Titration ofnon-Rh antibodies should be undertakenonly after discussion with the obstetricianabout how the data will be used in the clinicalmanagement of the pregnancy. The significanceof titers has been sufficiently establishedonly for anti-D (using a salinetechnique).SpecimenSerum for titration (containing potentiallysignificant unexpected antibodies to redcell antigens, 1 mL). If possible, test thecurrent sample in parallel with the mostrecent previously submitted (preceding)sample from the current pregnancy.Materials1. Antihuman IgG: need not be heavychain-specific.2. Isotonic saline.3. Volumetric pipettes, or equivalent:0.1- to 0.5-mL delivery, with disposabletips.4. Red cells: group O reagent red cells,2% suspension. (See note 1 regardingthe selection of red cells for testing.)Avoid using Bg+ red cells becausethey may result in falsely highvalues, especially with sera frommultiparous women.5. IgG-coated red cells.Quality Control1. Test the preceding sample in parallel

with the most recent sample.2. Prepare the dilutions using a separatepipette for each tube. Failure todo so will result in falsely high titersbecause of carryover.3. Confirm all negative reactions withIgG-coated red cells (see step 9 below).Procedure1. Using 0.5-mL volumes, prepare serialtwofold dilutions of serum in saline.The initial tube should contain undilutedserum and the doubling dilutionrange should be from 1 in 2 to1 in 2048 (total of 12 tubes). 2. Place 0.1 mL of each dilution intoappropriately labeled test tubes.3. Add 0.1 mL of the 2% suspension ofred cells to each dilution. Alternatively,for convenience, add 1 drop ofa solution of a 3% to 4% suspensionof red cells as supplied by the reagentmanufacturer, although thismethod is less precise.

4. Gently agitate the contents of eachtube; incubate at 37 C for 1 hour.5. Wash the red cells four times withsaline; completely decant the finalwash supernatant.6. To the dry red cell buttons thus obtained,add anti-IgG according to themanufacturer’s directions.

7. Centrifuge as for hemagglutinationtests.8. Examine the red cells macroscopically;grade and record the reactions.9. Add IgG-coated red cells to all negativetests; recentrifuge and examinethe tests for macroscopic agglutination;repeat the testing if the tests withIgG-coated red cells are nonreactive.InterpretationThe titer is reported as the reciprocal ofthe highest dilution of serum at which 1+agglutination is observed. A titer ≥16 (thisvalue may vary according to the laboratory)is considered significant and maywarrant further monitoring for HDFN.Notes1. The selection of the most suitablephenotype of red cells to use whenperforming titration studies for HDFNis controversial. Some workers selectred cells that have the strongest expressionof antigen, such as R2R2 foranti-D. Others select red cells with thephenotype that would be expectedin fetal circulation—ie, red cells thatexpress a single dose of the antigen,such as R1r for testing for anti-D.Whichever viewpoint is followed, itis important that the laboratory beconsistent and use red cells of thesame phenotype for future titrations

to test the same patient’s serum.2. Titration studies should be performedupon initial detection of the antibody;save an appropriately labeledaliquot of the serum (frozen at –20 Cor colder) for comparative studieswith the next submitted sample.3. When the titer (eg, >16) and the antibodyspecificity have been associatedwith HDFN, it is recommendedthat repeat titration studies be performedevery 2 to 4 weeks, beginningat 18 weeks’ gestation; save an aliquotof the serum (frozen at –20 C orcolder) for comparative studies withthe next submitted sample.4. When invasive procedures (eg, amniocentesis)have demonstrated fetalcompromise and are being used tomonitor the pregnancy, use the optimalmethod for follow-up of fetalwell-being. However, if initial studiesdo not show fetal compromise or theLiley curve result is borderline, additionaltitrations may be helpful as ameans of following the pregnancy ina less invasive manner.5. Each institution should develop apolicy to ensure a degree of uniformityin reporting and interpretingantibody titers.6. For antibodies to low-incidence antigens,

consider using putative paternalred cells, having establishedthat they express the antigen in question.7. Do not use enhancement techniques[albumin, polyethylene glycol, lowionic-strength saline (LISS)] or enzyme-treated red cells because falselyelevated titers may be obtained. Geltesting is not recommended.8. LISS should not be used as a diluentin titration studies; nonspecific uptakeof globulins may occur in serum-LISS dilutions.9. Failure to obtain the correct resultsmay be caused by 1) incorrect technique,notably, failure to use sepa-rate pipette tips for each dilution or2) failure to adequately mix thawedfrozen serum.References1. Issitt PD, Anstee DJ. Applied blood group serology.4th ed. Durham, NC: MontgomeryScientific Publications, 1998:1067-9.2. Judd WJ, Luban NLC, Ness PM, et al. Prenataland perinatal immunohematology: Recommendationsfor serologic management of thefetus, newborn infant, and obstetric patient.Transfusion 1990;30:175-83.3. Judd WJ. Methods in immunohematology.2nd ed. Durham, NC: Montgomery ScientificPublications, 1994.4. Judd WJ. Practice guidelines for prenatal andperinatal immunhematology, revisited. Transfusion2001;41:1445-52.