Antibody Titration Anti-A, Anti-B ,Anti-D

 

PRINCIPLE AND APPLICATIONS:   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.  If the concentration of the specific antibody is being determined, the cells must contain the known antigen and the procedure should be performed under the optimal conditions for that antibody.  The usual applications of titration studies are: estimating the 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

SAMPLE Either plasma and serum can be titrated.  In the case of a pregnant female, frozen serum or plasma from previous months should be run along with the fresh specimen to determine if there is a rise in titer.

REAGENTS, EQUIPMENT, AND SUPPLIES: Reagent cells known to have the antigen that will be reacting with the titered antibody.  If titering for anti-A or anti-B levels, A1 or B Cells would be used.  If titering for anti-D levels, O cells that are homozygous for D should be used. 12 x 75 mm tubes test tube rack marking pen dispo pipettes physiologic saline serofuge lighted agglutination viewer

PROCEDURE Label 10 tubes according to the serial dilution: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 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 0.3 ml of saline to tubes 2 through 512.  No saline in tube 1 Add 0.3 ml of serum to both tubes 1 and 2. Use a clean pipette to mix the 1/2 dilution several times and then transfer 0.3 ml to tube 4. Use a clean pipette to mix the 1/4 dilution several times and then transfer 0.3 ml to tube 8. Continue the process for all dilutions (512).  Remove 0.3 ml from tube 10 (512) and reserve in a clean tube if the titration needs to be continued. Label a new set of 10 tubes with the appropriate dilutions. Using a separate pipettes for each dilution, transfer 2 drops of each tube to the appropriate tubes. Add one drops 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

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. If testing a pregnant female, each month serum should be compared to the previous month. 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. Careful pipetting is essential. Cells with known antigens may have different reactivity and therefore the serum from each month must use the same cells Measurement is more accurate at larger dilution, therefore the larger dilution should be made before smaller volumes are used to test with the red cells antigen.

INTERPRETATION Observe the highest dilution that produces 1+ macroscopic agglutination The titer is reported as the reciprocal of the dilution level:  32 not 1/32 A rise in titer would need to be at least 2 dilution increase between the current specimen and the previous month. For identification of high-titer, low-avidity antibodies would generally have a titer of 64 or greater.