Antibody Identification Techniques Study Notes

Antibody Identification Techniques pt 2

Overview

  • Focus on complex antibody problems encountered during antibody identification (ID) in immunohematology.

  • The shift in focus is to the clinical lab and reference lab methods for identifying multiple antibodies.

  • Emphasis on the detective aspect of identifying antibodies using various techniques.

Techniques for Antibody Identification

Enzymes in Antibody Identification

  • Enzymes can enhance or eliminate specific antibody activities to aid identification.

  • Main Enzymes Used:

    • Ficin (from figs)

    • Papain (from papaya)

    • Bromelain (from pineapple)

    • Trypsin (from hog stomach)

  • Mechanism of Action:

    • Enzymes modify the surface of red blood cells by removing sialic acid residues from the red cell membrane.

    • They can denature or remove glycoproteins.

  • Important Considerations:

    • Perform enzyme treatments after the initial antibody panel to avoid eliminating clinically significant antibodies.

    • Panel Cells: Specific panel or screening cells treated with ficin or papain.

  • Antibody Response Table:

    • Enhanced Antibodies: R.H.I.P, Lewis, Kitt, and ABO.

    • Inactivated Antibodies: Duffy, M, N, S, XG.

    • Noteworthy: Clinically significant antibodies like Duffy or big S can be inactivated, posing a risk if unrecognized.

Pre-Warm Technique

  • Aimed at eliminating cold antibodies, particularly IgM, to prevent interference during testing.

  • Procedure:

    1. Warm patient's serum/plasma in a tube.

    2. Warm screening or antibody ID cells in a separate tube.

    3. Incubate both at 37°C for 10-15 minutes.

    4. Combine serum and red cell reagents, maintaining 37°C during testing.

  • Important Notes:

    • Ensure all clinically significant antibodies are ruled out before conducting this technique.

    • Previous incidents of inadvertently exhausting clinically significant antibodies have occurred.

Neutralization Techniques

  • Generally not performed in clinical blood banks; used to neutralize antibodies by combining them with corresponding soluble antigens.

  • Facilitates separation and identification of antibodies in the sample.

  • Example: Lewis antibodies are neutralized in saliva, indicating their less clinical significance.

Absorption Techniques

  • Absorption: Removal of antibodies from a sample by mixing with target antigen to allow binding.

  • Auto-Absorption: Uses the patient’s own red blood cells to absorb out antibodies.

  • Procedure for auto-absorption:

    1. Treat patient cells with a solution (often warm) to eliminate autoantibodies.

    2. Incubate treated red cells with the patient’s serum/plasma to remove related antibodies.

  • This assists in identifying clinically significant alloantibodies that may be obscured.

Elutions

  • Performed on DAT (Direct Antiglobulin Test) positive samples to clarify which antibodies are coating the red cells.

  • Purpose:

    • Concentrate and purify antibodies from the red cells by breaking bonds through specific methods.

  • Applications:

    • Useful in investigating hemolytic disease of the fetus and newborn, or during hemolytic transfusion reactions.

  • Methods Include:

    • Temperature changes

    • pH alterations

    • Using organic solvents (not common due to disposal concerns)

    • Extended incubation times if necessary.

Antigen Typing

  • Conducted for verifying the presence of antigens corresponding to identified antibodies, ensuring safe transfusions.

  • Procedure:

    1. If an antibody is identified, antigen typing must confirm it’s absent on the patient’s red cells.

    2. The same applies to blood donor units for transfusions to avoid reactions due to antigen-antibody interactions.

  • Quality Control:

    • Utilization of antigram panels and ensuring reagents' homozygosity to assure accurate results in typing.

  • Antigen typing is not routine; can go months without requiring this step in the blood bank.

Direct Antiglobulin Test (DAT) / Coombs Test

  • Identifies antibodies that are coating the red cells in vivo (s sensitization).

  • Uses polyspecific antihuman globulin reagent that encompasses anti-IgG and complement to assess what's on the patient’s red cells.

  • Interpretation:

    • If polyspecific is negative: no antibodies are coating the red cells.

    • If positive: investigate further into IgG or complement presence, as this would indicate potential causes of hemolytic reactions or pregnancy-related issues.

Antibody Titration

  • Measures strength/concentration of an antibody, particularly significant in pregnant women regarding potential hemolytic disease of the fetus and newborn.

  • Conducted through serial dilutions, observing reactivity orders.

  • Documenting Titers: reported as the last positive dilution, not in the form of ratios (reported as whole numbers).

  • Example of Serial Dilutions:

    • 1:1 (not diluted), to 1:2, 1:4, 1:8, 1:16, 1:32.

    • Always read from positive titer down to avoid mismatch errors.

Conclusion

  • Antibody identification in blood banking involves complex techniques and careful methodologies to ensure safety and accuracy in transfusion.

  • The diagnostics can be intricate, requiring specific laboratory knowledge and principles for effective handling.