Rh Antibody Titration
Rh Antibody Titration

Table of Contents

Rh Antibody Titration


Specimen needs clotted (serum) or anti-coagulated (plasma) of the antibodies to be titer.


Rh Antibody Titration is a semi quantitative technique use to help to determine the concentration of antibodies in a serum specimen or to equate the strength of antigen expression on different red blood cell specimens.


  1. Label 10 test tubes according to the serum dilution (e.g. 1:1 and 1: 2). 1 in 1 means 1 volume of serum undiluted. 1 in 2 dilutions mean one volume (100 ul) of serum in a final volume of two, or a 50% solution of serum in the diluent.
  2. Deliver one volume of saline (100 ul) to all test tubes except the first (undiluted 1 in 1) tube.
  3. Add an equal volume (100 ul) of serum to each of the first two tubes. (Undiluted and 1 in 2).
  4. Using clean pipette, mix the contents of the 1 in 2 dilution several times and deliver one volume (100 ul) in to a next tube (1 in 4 dilutions).
  5. Continue the same process for all dilutions, using clean pipette to mix and transfer each dilution. Remove one volume (100 ul) of diluted solution from the final tube and save it for use if further dilution is required.
  6. Add 100 ul, of 5% cell suspension of “O” Positive cells to each tube and incubate at 37oC for 45 minute.
  7. Wash three times with normal saline.
  8.  Add two drops of anti-human globulin (AHG) reagent in each tube.
  9. Incubate for 5 minutes at 37oC.
  10. Centrifuge at 3500 rpm for 15-30 seconds.
  11. Examine the test result macroscopically grade and record the reactions in all tubes.


Observe the highest dilution that produces (1+) agglutination. The titer is reported as the reciprocal of the dilution level (e.g. 1 in 32 is 1:32). Presence of agglutination in the tube having the most dilution of serum, the end point has not been gotten, and further dilutions must be prepared/organized and tested.



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