1. Antibody Titration Mr . Mohammed A. Jaber

2. Principle • Titration is a semiquantitative 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 alloimmunized pregnant women to determine whether and when to perform more complex invasive investigation of the fetal condition.

3. Principle • Elucidating autoantibody specificity. • characterizing antibodies as high-titer, low-avidity • observing the effect of sulfhydryl reagents on antibody behavior, to determine immunoglobulin class (IgG or IgM). Titration studies specifically to assist in monitoring clinically significant antibodies in the pregnant woman.

4. 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 tile 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.

7. 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.

8. 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 multiparous women. • IgG-coated red cells.

9. 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.

10. Procedure • 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.

11. Procedure • 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.

12. 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.

13. 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. Others select red cells with the phenotype that would be expected in fetal circulation—ie, red cells that express a single dose of the antigen, such as R1r for testing for anti-D. Whichever viewpoint is followed, 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.

14. Notes • 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. • 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.

15. 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.

16. Notes • 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.

17. GRADING OF AGGLUTINATION REACTION +4 Single clump of agglutination with no free cells +3 Three or four individual clumps with few free cells +2 Many fairly large clumps with many free cells +1 Fine granular appearance visually, but definite small clumps (10-15 cells) per low power field +W 2 to 3 cells sticking together per low power field, uneven distribution Visually no agglutination - All cells are free H Hemolysis (partial or total) must be interpreted as positive