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Blood Bank

Neutralization

Neutralization and Inhibition techniques are clever little tricks we use in the blood bank, mostly during antibody identification, when we suspect certain antibodies are present and want to confirm their identity or get them “out of the way” so we can see other antibodies

Think of it like adding a specific “decoy” substance to the patient’s serum. If the antibody we suspect is present, it will bind to the decoy instead of the reagent red cells, effectively neutralizing or inhibiting its activity

The Core Purpose: Confirming Specificity or Removing Interference

The main goals of neutralization/inhibition are:

  • Confirming Antibody Specificity: If adding a substance known to contain a soluble form of a specific blood group antigen causes the antibody reactivity to disappear, it strongly confirms that the antibody is directed against that antigen
  • Removing Interfering Antibodies: Some antibodies (especially those against high-frequency antigens or substances present in plasma) can mask the presence of other, potentially more significant, alloantibodies. Neutralizing the interfering antibody can allow the underlying antibodies to be detected

The Basic Principle

  1. Suspicion You suspect a particular antibody specificity based on initial panel results (e.g., reactions consistent with anti-Lea, anti-Leb, anti-P1, anti-Ch/Rg, anti-Sda)
  2. Neutralizing Substance Obtain a substance known to contain the soluble form of the corresponding antigen (or a structurally similar substance)
  3. Incubation Mix the patient’s serum/plasma with the neutralizing substance and incubate (usually at room temperature or 37°C, depending on the antibody)
  4. Testing Test the treated (neutralized) serum/plasma against reagent red cells known to be positive for the suspected antigen
  5. Control Crucially, test a control sample of patient serum/plasma mixed with an inert substance (like saline) in parallel against the same reagent red cells
  6. Comparison Compare the reactivity of the neutralized serum to the control serum

Interpretation

  • Neutralization Confirmed: If the reactivity with the antigen-positive cells is significantly reduced or completely abolished in the tube containing the neutralized serum compared to the control tube (which should still show reactivity), then neutralization has occurred. This confirms the suspected antibody specificity
  • No Neutralization: If the reactivity remains the same in both the neutralized serum tube and the control tube, then the antibody present is likely not the one targeted by the neutralizing substance

Common Neutralization/Inhibition Scenarios

These techniques are most useful for antibodies directed against antigens that exist in soluble form in body fluids or can be obtained from other sources:

Lewis System Antibodies (Anti-Lea, Anti-Leb)

  • Neutralizing Substance: Commercial Lewis Substance (prepared from pooled plasma or saliva known to contain soluble Lea and Leb glycoproteins) or sometimes saliva from individuals secretor-tested for Lewis phenotype
  • Why it Works: Lewis antigens are primarily plasma antigens that adsorb onto red blood cells. Soluble forms are abundant in plasma and secretions (like saliva) of individuals with the corresponding Lewis genes
  • Use: Helps confirm anti-Lea or anti-Leb specificity, especially when they might be interfering with detecting other antibodies

P1 Antibody (Anti-P1)

  • Neutralizing Substance: Commercial P1 Substance (often derived from hydatid cyst fluid, which is rich in P1-like structures) or pigeon egg whites (contain P1-like substance)
  • Why it Works: P1 antigen is also present in soluble form
  • Use: Confirms anti-P1 specificity. Anti-P1 is a common, usually clinically insignificant cold antibody that can sometimes interfere with testing

Sda Antibody (Anti-Sda - Sid)

  • Neutralizing Substance: Urine from most individuals (especially Sda-positive individuals). Guinea pig urine is often used as a commercial source
  • Why it Works: The Sda antigen is present in high concentrations in urine
  • Use: Confirms anti-Sda specificity. Anti-Sda often causes characteristic small, refractile, mixed-field agglutinates that can be observed microscopically. Neutralization helps confirm this often tricky-to-identify antibody

Chido/Rodgers Antibodies (Anti-Ch, Anti-Rg)

  • Neutralizing Substance: Pooled normal human plasma/serum (plasma contains soluble C4 complement components, Ch and Rg antigens are located on C4d)
  • Why it Works: Ch/Rg antigens are determinants on the C4 component of complement, which circulates in plasma
  • Use: Confirms anti-Ch or anti-Rg specificity. These are common High Titer, Low Avidity (HTLA) antibodies, often weakly reactive with most panel cells. Neutralization helps distinguish them from other weakly reactive antibodies

Important Considerations & Controls

  • Specificity of Substance: Ensure the neutralizing substance is specific for the antigen in question
  • Dilution Control: The addition of the neutralizing substance dilutes the patient’s serum. The saline control (patient serum + saline) is essential to ensure that any reduction in reactivity isn’t simply due to this dilution effect
  • Potency of Substance: The neutralizing substance must be potent enough to effectively inhibit the antibody
  • Not Universally Applicable: Only works for antibodies whose corresponding antigens exist in a soluble form that can be readily obtained and safely used in the lab
  • Interpretation Caution: Complete neutralization is ideal, but significant inhibition (e.g., reduction in reaction strength by 2 grades or more) is often sufficient for confirmation

Key Terms

  • Neutralization/Inhibition: A technique used to confirm antibody specificity by adding a soluble form of the corresponding antigen (or a similar substance) to serum/plasma, which binds the antibody and prevents it from reacting with reagent red blood cells
  • Soluble Blood Group Substances: Blood group antigens that exist in a soluble form in body fluids like plasma, saliva, or urine (e.g., Lewis, P1, Sda, Ch/Rg)
  • Lewis Substance: Commercially prepared reagent containing soluble Lea and Leb antigens, used to neutralize anti-Lea and anti-Leb
  • P1 Substance: Commercially prepared reagent (often from hydatid cyst fluid) containing soluble P1 antigen, used to neutralize anti-P1
  • Urine (for Sda): Used as a source of soluble Sda antigen to neutralize anti-Sda
  • Pooled Plasma/Serum (for Ch/Rg): Used as a source of soluble C4 (carrying Ch/Rg determinants) to neutralize anti-Ch and anti-Rg
  • Dilution Control: A control tube prepared by mixing patient serum/plasma with an inert substance (like saline) in the same volume as the neutralizing substance, to account for any reduction in antibody reactivity due solely to dilution