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EDTA-Glycine Acid

EDTA-Glycine Acid, often shortened to EGA, is another reagent in our special toolkit, quite similar in purpose to Chloroquine Diphosphate (CDP), but using a different approach to achieve the same main goal: removing IgG antibodies stuck to red blood cells so we can phenotype them!

Think of EGA as a quick “acid wash” designed to pop off those bound IgG antibodies without destroying the underlying red cell antigens we need to identify

The Core Purpose: IgG Removal for Phenotyping (Primarily)

Like CDP, the main reason we use EGA is to dissociate bound IgG antibodies from red blood cells in vitro. This allows us to perform accurate red blood cell phenotyping on cells that originally had a positive Direct Antiglobulin Test (DAT) due to IgG coating

Why? Because that IgG coating interferes with phenotyping, potentially causing false positives or blocking antigen sites. EGA treatment aims to create DAT-negative (or at least weaker) cells suitable for testing

A secondary, less common use, is as an elution method itself. Since it works by lowering pH to dissociate antibody, the supernatant fluid after treatment contains the eluted antibody and can potentially be tested (after neutralization) – essentially performing an acid elution

Mechanism of Action: Low pH Dissociation + EDTA

EGA leverages two components:

  • Glycine-Acid: This provides a low pH environment (acidic). Similar to acid elution techniques, this low pH disrupts the electrostatic and hydrogen bonds holding the IgG antibody to the red cell antigen, causing the antibody to detach
  • EDTA (Ethylenediaminetetraacetic acid): This component is a chelating agent, meaning it binds calcium ions (Ca++). While the primary action is the low pH, EDTA’s presence helps prevent any in vitro complement activation during the procedure (though this is less critical here than in sample collection, as we’re usually starting with already-coated cells) and may contribute slightly to destabilizing certain interactions

Key Difference from CDP CDP works near neutral pH, while EGA relies on a temporary drop to acidic pH to achieve dissociation

The Procedure (Simplified)

  1. Wash Patient Cells As always with these techniques, start by thoroughly washing the patient’s EDTA red blood cells to remove all unbound plasma proteins
  2. Incubation with EGA Mix the washed packed cells with the EGA reagent solution
  3. Incubation Incubate the mixture, usually for a short period (often just 1-2 minutes, sometimes longer depending on protocol) at room temperature. The low pH works relatively quickly
  4. NEUTRALIZATION (CRITICAL STEP!): Immediately after the short incubation, the acidic reaction must be stopped and neutralized by adding a buffer solution (like Tris buffer or a specialized buffer). This brings the pH back up to physiological levels (~7.0) and is essential to prevent damage to the red cell antigens by prolonged acid exposure
  5. WASH Patient Cells (Extensively!): After neutralization, wash the EGA-treated cells thoroughly (e.g., 4-6 times) with saline to remove the EGA reagent, the buffer, and any dissociated antibody
  6. Quality Control (ESSENTIAL!): Before phenotyping:
    • Check the DAT: Perform a DAT (using Anti-IgG) on the treated cells. Aim for a negative result. If still positive, treatment may have been insufficient
    • Check Antigen Integrity: Test treated cells with known antisera to ensure common antigens weren’t damaged by the acid treatment or inadequate neutralization
  7. Phenotyping Use the washed, neutralized, DAT-negative (or weakly positive), QC-passed treated cells for antigen typing

When Might EGA Be Chosen?

  • As an alternative to CDP for removing IgG when phenotyping DAT-positive cells
  • Some labs find it faster than the typical 2-hour incubation for CDP
  • May sometimes be more effective than CDP at removing certain strongly bound IgG antibodies
  • When preservation of Kell, Duffy, MNS, Kidd, and Rh antigens is needed (similar to CDP, unlike ZZAP)

Advantages

  • Rapid: Often requires shorter incubation times than CDP
  • Effective IgG Dissociation: Generally good at removing bound IgG
  • Preserves Most Antigens: Like CDP, it avoids the enzyme/thiol destruction seen with ZZAP, preserving Kell, Duffy, MNS, etc
  • Can Double as Elution: The supernatant can potentially be used as an eluate

Limitations and Disadvantages

  • Requires Rapid & Effective Neutralization: This is the most critical step unique to EGA. Failure to neutralize properly after the acid exposure will damage red cell antigens, leading to false-negative typing results
  • Potential for Antigen Damage: Even with proper neutralization, the brief exposure to low pH might subtly affect some antigens more than CDP treatment does, although it’s generally considered safe for most common antigens
  • Incomplete Antibody Removal: Like CDP, may not remove all IgG if it’s very strongly bound
  • Extensive Washing Required: Both before and after treatment
  • QC is Paramount: Validating DAT negativity and antigen integrity post-treatment is non-negotiable
  • Less Effective for Complement: Primarily targets IgG dissociation

Comparison to CDP and ZZAP

  • EGA vs. CDP: Both gently remove IgG, preserving most antigens. EGA is faster but requires a critical neutralization step due to low pH. CDP is slower but avoids the acid step. Choice often depends on lab preference, protocols, and sometimes which one works better for a specific stubborn antibody
  • EGA vs. ZZAP: EGA preserves Kell, MNS, Duffy; ZZAP destroys them. ZZAP is better for complete antibody/complement removal regardless of antigen integrity; EGA is used when phenotyping for those specific antigen systems is required on DAT-positive cells

Key Terms

  • EDTA-Glycine Acid (EGA): A reagent solution used to dissociate IgG antibodies from red blood cells in vitro primarily through temporary exposure to low pH
  • Low pH Dissociation: Using an acidic environment to break the non-covalent bonds between antibodies and antigens
  • Neutralization: The critical step after EGA treatment where a buffer is added to restore the mixture to a physiological pH, preventing acid damage to antigens
  • Phenotyping DAT-positive Cells: The main application for EGA and CDP – determining the red cell antigen profile when cells are coated with IgG
  • Antigen Preservation: Maintaining the integrity of red cell antigens during treatment so they can be accurately typed afterwards