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Practical Blood Bank. Antibody Titration. Principle. Titration is a semi quantitative method used to determine the concentration of antibody in a serum sample or to compare the strength of antigen expression on different red cell samples. The usual applications of titration studies are:
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Practical Blood Bank Antibody Titration
Principle • Titration is a semi quantitative method used to determine the concentration of antibody in a serum sample or to compare the strength of antigen expression on different red cell samples. The usual applications of titration studies are: • Estimating antibody activity in alloimmunizedpregnant women to determine whether and when to perform more complex invasive investigation of the fetal condition. • characterizing antibodies as high-titer, low-avidity • Observing the effect of sulfhydryl reagents on antibody behavior, to determine immunoglobulin class (IgG or IgM).
Titration in pregnant women • Titration studies specifically to assist in monitoring clinically significant antibodies in the pregnant woman. • When the titer (eg, >16) and the antibody specificity have been associated with HDFN, it is recommended that repeat titration studies be performed every 2 to 4 weeks, beginning at 18 weeks’ gestation; save an aliquot of the serum (frozen at –20 C or colder) for comparative studies with the next submitted sample.
characterizing antibodies as high-titer, low-avidity • Speed and strength of agglutination is termed as avidity. • The test is done by mixing two drop of anti-serum with one drop of 40-50% cell suspension on a slide or cover and rocking gently at room temperature (RT). The time for a clearly visible reaction (+1) and then for strong (+4) reaction to occur are recorded with the help of stop watch.
Observing the effect of sulfhydryl reagents on antibody behavior, to determine immunoglobulin class (IgG or IgM).
Specimen • Serum for titration (containing potentially significant unexpected antibodies to red cell antigens, 1 mL). If possible, test the current sample in parallel with the most recent previously submitted (preceding) sample from the current pregnancy.
Materials • Antihuman IgG: need not be heavy chain- specific. • Isotonic saline. • Volumetric pipettes, or equivalent: 0.1- to 0.5-mL delivery, with disposable tips. • Red cells: group O reagent red cells, 2% suspension. (See note 1 regarding the selection of red cells for testing.) Avoid using Bg+ red cells because they may result in falsely high values, especially with sera from multi-parous women. • IgG-coated red cells.
Procedure • Using 0.5-mL volumes, prepare serial twofold dilutions of serum in saline. The initial tube should contain undiluted serum and the doubling dilution range should be from 1 in 2 to 1 in 2048 (total of 12 tubes). • Place 0.1 mL of each dilution into appropriately labeled test tubes. • Add 0.1 mL of the 2% suspension of red cells to each dilution. Alternatively, for convenience, add 1 drop of a solution of a 3% to 4% suspension of red cells as supplied by the reagent manufacturer, although this method is less precise.
Gently agitate the contents of each tube; incubate at 37 C for 1 hour. • Wash the red cells four times with saline; completely decant the final wash supernatant. • To the dry red cell buttons thus obtained, add anti-IgG according to the manufacturer’s directions. • Centrifuge as for hemagglutination tests. • Examine the red cells macroscopically; grade and record the reactions. • Add IgG-coated red cells to all negative tests; recentrifuge and examine the tests for macroscopic agglutination; repeat the testing if the tests with IgG-coated red cells are nonreactive.
Interpretation • The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed. • A titer ≥16 (this value may vary according to the laboratory) is considered significant and may warrant further monitoring for HDFN.
Notes • The selection of the most suitable phenotype of red cells to use when performing titration studies for HDFN is controversial. • Some workers select red cells that have the strongest expression of antigen, such as R2R2 for anti-D. • it is important that the laboratory be consistent and use red cells of the same phenotype for future titrations to test the same patient’s serum. • Titration studies should be performed upon initial detection of the antibody; save an appropriately labeled aliquot of the serum (frozen at –20 C or colder) for comparative studies with the next submitted sample.
Notes • Do not use enhancement techniques [albumin, polyethylene glycol, low ionic strength saline (LISS)] or enzyme-treated red cells because falsely elevated titers may be obtained. Gel testing is not recommended. • LISS should not be used as a diluents in titration studies; nonspecific uptake of globulins may occur in serum-LISS dilutions. • Failure to obtain the correct results may be caused by • incorrect technique, notably, failure to use separate pipette tips for each dilution or • failure to adequately mix thawed frozen serum.
NOTES AND PRECAUTIONS • If titrating anti-A, anti-B, or anti-A,B, the serologic technique is performed by the same method as ABO Typing • If titrating Rh, Kell, Duffy, or Kidd antibodies, the serologic technique includes a 37oC followed by antiglobulin testing. • Prozone phenomenon may occur so the first tubes may have a weaker reaction than the more diluted serum. AABB recommends reading the most dilute tubes first and then shake out the other tubes.
