Rh Antibody Titration Test


 

1. Objective:

To determine the concentration (titer) of anti-D (Rh) antibodies in a patient's serum—especially in Rh-negative pregnant women—to assess risk of Hemolytic Disease of the Fetus and Newborn (HDFN).

2. Principle:
Serial dilutions of the patient’s serum are reacted with Rh-positive red cells. The highest dilution at which agglutination still occurs is the antibody titer. This provides a quantitative estimate of antibody strength.

3. Materials Required:

  • Patient's serum

  • Rh(D) positive red blood cells (usually R1R1)

  • Phosphate-buffered saline (PBS)

  • 2–5% red cell suspension

  • Incubator at 37°C

  • Centrifuge

  • Test tubes

  • Antihuman globulin (AHG or Coombs reagent)

4. Procedure:

  1. Prepare serial twofold dilutions of the patient’s serum (e.g., 1:1 to 1:1024).

  2. Add equal volume of 2–5% Rh(D)-positive RBC suspension.

  3. Incubate at 37°C for 30–60 minutes.

  4. Wash cells 3–4 times with PBS.

  5. Add AHG reagent, centrifuge, observe for agglutination.

  6. The highest dilution showing 1+ agglutination or stronger is recorded as the titer.

5. Result Interpretation:

  • Titer ≥16–32: Potential risk of HDFN, requires close monitoring.

  • Titer rising over time: Indicates active alloimmunization.

  • Stable or low titer: Lower risk, but monitor.

6. Clinical Relevance:

  • Helps guide obstetric management in Rh-incompatible pregnancies.

  • Determines need for intrauterine transfusion or early delivery.

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