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.
The critical titer for anti-D (the level below which HDFN and hydrops fetalis are considered so unlikely that no further invasive procedures will be undertaken)

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.
antibody specificity
The property of antibodies which enables them to react with some antigenic determinants and not with others. Specificity is dependent on chemical composition, physical forces, and molecular structure at the binding site.
Specificity of Autoantibody
The specificity of autoantibodies associated with Warm Antibody Autoimmune Hemolytic Anemia (WAIHA) is complex. In routine tests, all cells tested are usually reactive. Some autoantibodies that have weaker or negative reactivity with cells of rare Rh phenotypes, such as D– – or Rhnull, appear to have broad specificity in the Rh system. Apparent specificity for simple Rh antigens (D, C, E, c, e) is occasionally seen, either as the sole autoantibody or as a predominant portion, based on stronger reactivity with cells of certain phenotypes. Such reactivity is often termed a “relative” specificity. Such relative specificity in a serum may be mistaken for alloantibody, but cells negative for the apparent target antigen can adsorb and remove the “mimicking” specificity.
Unusual Specificities. Apart from Rh specificity, warm autoantibodies with many other specificities have been reported, eg, specificities in the LW, Kell, Kidd, Duffy, and Diego systems.24 Dilution and selective adsorption of eluates may uncover specificity or relative specificity of autoantibodies. Patients with autoantibodies of Kell, Rh, LW, Ge, Sc, Lu, and Lan specificities may have depressed expression of the respective antigen and the DAT may be negative or very weakly positive.

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.
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. 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.

18. 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

20. 2+ W+ to 1+ Medium-sized Agglutinates-Clear Background
Medium-sized Agglutinates-Clear Background
Small agglutinates turbid background
Tiny agglutinates turbid background
No agglutination or Hemolysis

22. 4+ 3+ One Solid Agglutinate
Several large agglutinates-Clear Background