Titrations

Antibody Titration is a technique that might seem a bit old-school compared to some molecular methods, but it remains incredibly valuable, especially in monitoring pregnant patients. It’s less about identifying an antibody (we usually know what it is already) and more about measuring how much of it is present, or how strongly it reacts

Think of it as trying to figure out the “concentration” or “strength” of an antibody in the patient’s serum

The Core Purpose: Quantifying Antibody Strength

The primary goal of titration is to provide a semi-quantitative measure of the concentration of a specific antibody in a patient’s serum or plasma. We do this by determining the highest dilution of the serum that still produces a detectable reaction with red blood cells possessing the target antigen

Key applications include:

• Monitoring Pregnant Patients with Clinically Significant Antibodies: This is the MOST IMPORTANT use! Tracking the titer of IgG antibodies (like anti-D, anti-K, anti-c, etc.) known to cause Hemolytic Disease of the Fetus and Newborn (HDFN) helps assess the potential risk to the fetus. A significant rise in titer during pregnancy suggests increased antibody production and may indicate worsening fetal jeopardy
• Characterizing Antibodies: Helps differentiate certain types of antibodies, like High Titer, Low Avidity (HTLA) antibodies. These often show reactivity at very high dilutions but the reactions are consistently weak, and they are usually clinically insignificant
• Specific Serologic Investigations: Occasionally used to compare the reactivity of an antibody against different red cell samples (e.g., comparing reactivity against cells homozygous vs. heterozygous for the antigen to demonstrate dosage)

Important Note Titration results are generally NOT used to decide whether to transfuse antigen-negative blood. If a patient has a clinically significant antibody, they should receive antigen-negative units regardless of the titer

The Basic Procedure: Serial Dilution

1. Sample Patient serum or plasma known to contain the antibody of interest
2. Target Cells Washed red blood cells known to be positive for the antigen corresponding to the antibody being titrated (e.g., use D-positive cells for titrating anti-D). Ideally, use cells with a consistent antigen expression (e.g., R1r or RzR1 for anti-D, often homozygous cells like Jk(a+b-) for anti-Jka to maximize reactivity)
3. Serial Dilutions Prepare a series of doubling dilutions of the patient’s serum in a suitable diluent (usually saline). This typically starts with undiluted (neat) serum, then 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and so on, often up to 1:1024 or higher if needed
   • Consistency is key: Use precise pipetting techniques to ensure accurate dilutions!
4. Incubation Add an equal, consistent volume of the prepared target red cell suspension to each tube containing a serum dilution
5. Test Method Incubate all tubes using a standard Indirect Antiglobulin Test (IAT) procedure (often using LISS or saline as the medium, sometimes albumin, rarely PEG due to reading difficulties). The method chosen should be consistent for subsequent titrations on the same patient
6. Washing & AHG After incubation (usually 37°C), wash all tubes thoroughly and add Anti-Human Globulin (AHG)
7. Reading & Grading Centrifuge gently, resuspend the cell buttons, and examine macroscopically for agglutination. Crucially, grade the reaction strength in each tube (e.g., 0, w+, 1+, 2+, 3+, 4+)

Determining the Titer and Score

• Titer Endpoint: The titer is reported as the reciprocal of the highest dilution of serum that shows a 1+ macroscopic agglutination. For example, if the 1:64 dilution shows 1+ agglutination, but the 1:128 dilution is weaker (w+) or negative (0), the titer is 64
• Scoring (Semi-Quantitative): Because the endpoint can be subjective and small changes might be missed, a score is often calculated to provide a more sensitive measure, especially for monitoring pregnant patients. Each grade of agglutination is assigned a numerical value (scores vary slightly by institution, but a common system is):
  • 4+ = 12
  • 3+ = 10
  • 2+ = 8
  • 1+ = 5
  • w+ = 2 (or 3)
  • 0 = 0
  • (Hemolysis, if present and antibody-mediated, might be scored as 10 or 12)
  • The scores for all reactive tubes (including the w+ reactions below the 1+ endpoint) are summed to get the total score

Interpretation, Especially in HDFN Monitoring

• Baseline: An initial titer is established early in pregnancy if a significant antibody is found
• Monitoring: Titrations are repeated at intervals (e.g., monthly, bi-weekly). Crucially, for valid comparison, the current sample MUST be tested in PARALLEL with the previously frozen sample from the same patient, using the same target cells and the exact same technique and reader.
• Significant Change: A clinically significant rise in antibody level is generally considered to be:
  • A two-dilution (four-fold) increase in titer (e.g., from 16 to 64)
  • OR: an increase in score of 10 or more (this is often more sensitive than the titer endpoint alone)
• Critical Titer: This is a predetermined titer level for specific antibodies (e.g., often 16 or 32 for anti-D, but varies for other antibodies like anti-K where any level can be concerning) above which the risk of severe HDFN is high enough to warrant more intensive fetal monitoring (like Middle Cerebral Artery Doppler scans to assess for fetal anemia)

High Titer, Low Avidity (HTLA) Antibodies

• Titration helps identify these. They typically show:
  • Very high titers (e.g., ≥ 64, often much higher)
  • Weak reaction strength (often ≤ 1+) that persists even at high dilutions
  • Non-reactive or weakly reactive in PEG or enzyme methods
  • Usually clinically insignificant
  • Examples: Anti-Ch, Anti-Rg, Anti-JMH, Anti-Sda

Factors Affecting Reproducibility (CRITICAL!)

Titration results can be highly variable if not performed meticulously! Consistency is everything

• Pipetting Accuracy: Essential for accurate serial dilutions
• Red Cell Sample: Use cells from the same donor or with the same phenotype for parallel testing. Antigen strength can vary between donors
• Cell Suspension Concentration: Must be consistent
• Reagents: Use same method (LISS, saline), same AHG lot# for parallel tests
• Incubation: Same time and temperature
• Reading/Grading: VERY subjective! Ideally, the same technologist should read parallel titrations. Consistent technique for resuspension and reading is vital

Titrations are labor-intensive and require skill and consistency, but they provide invaluable information for managing pregnancies complicated by red cell antibodies. Remember that parallel testing is the gold standard for monitoring changes over time!

Key Terms

• Titration: A semi-quantitative method to determine the concentration or strength of an antibody by testing serial dilutions of serum/plasma against antigen-positive red cells
• Serial Dilution: A stepwise dilution of a substance (e.g., serum) by the same quantity each time (usually doubling dilutions like 1:2, 1:4, 1:8)
• Titer: The reciprocal of the highest dilution of serum/plasma showing a defined positive reaction (typically 1+ macroscopic agglutination)
• Score: A numerical value obtained by summing scores assigned to the agglutination grades of all reactive tubes in a titration. Provides a more sensitive measure of antibody strength than the titer endpoint alone
• Endpoint: The highest dilution producing the minimal defined positive reaction (1+)
• Parallel Testing: Testing a current patient sample simultaneously with a previously frozen sample from the same patient, using identical reagents and methods, to accurately assess changes in antibody level over time. Essential for HDFN monitoring
• Critical Titer: A predefined antibody titer level that, if reached or exceeded during pregnancy, indicates a high risk for severe HDFN and triggers closer fetal surveillance
• High Titer, Low Avidity (HTLA): A category of antibodies characterized by reactivity at very high dilutions but with weak reaction strength, typically clinically insignificant