1. Antibody Titration

2. Definition
Antibody titration is a determination of the concentration of a specific antibody in the patient's serum or to determine the strength of antigen expression on different red cell samples. 

3. Applications of titration studies
antibody activity of an alloimmunized pregnant female.
attempting to determine if there is any specificity to an autoantibody
characterizing antibodies that may be high titer, low avidity antibodies

4. SAMPLE
Either plasma or serum can be titrated.

5. REAGENTS, EQUIPMENT, AND SUPPLIES
Reagent cells known to have the antigen that will be reacting with the titered antibody.
12 x 75 mm tubes
test tube rack
marking pen
disposable pipettes
physiologic saline
serofuge
lighted agglutination viewer

6. PROCEDURE
Label 5 tubes according to the serial dilution: 1, 2, 4, 8, 16 and the patient identification. The first tube will be undiluted serum. Tube 2 will be a 1/2 dilution, 4 will be a 1/4 dilution. 
Add 50 µl of saline to tubes 2 through 16.  No saline in tube 1
Add 50 µl of serum to both tubes 1 and 2.
Use a clean pipette to mix the 1/2 dilution several times and then transfer 50 µl to tube 4.
Use a clean pipette to mix the 1/4 dilution several times and then transfer 50 µl to tube 8.

7. PROCEDURE
Continue the process for all dilutions. Remove 50 µl from tube 5 (32) and reserve in a clean tube if the titration needs to be continued.
Add 25 µl of specific red blood cells.
Mix well and test by the appropriate technique for the specific antibody.
Examine test results macroscopically, grade and record the reactions

8. Interpretation of Antibody Titrations
Sample No.
Antibody Dilution
1/1
1/2
1/4
1/8
1/16
Titer 1 4+ 3+ 2+ 1+ 8 2
4+ 
3+  3
 2+
1+  w  4 

9. 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.