Indirect Antiglobulin (Coombs) Test (IAT)

Introduction

The indirect antiglobulin test (IAT), also known as the indirect Coombs test, plays a crucial role in ensuring safe and compatible blood transfusions. This vital laboratory procedure delves into the world of antibodies, acting as a sentinel that guards against potentially disastrous immune reactions between your patient’s blood and donor red blood cells (RBCs)

Key points of IAT

  • The IAT detects antibodies against RBCs circulating in the serum, not directly bound to the RBCs themselves.
  • It identifies a wider range of antibodies than the direct antiglobulin test (DAT), including weak antibodies that may not cause visible agglutination.
  • The IAT is essential for both pre-transfusion compatibility testing and prenatal antibody screening in pregnant women.
  • By identifying incompatible antibodies, the IAT helps prevent potentially life-threatening hemolytic transfusion reactions.
  • Different types of AHG reagents can be used in the IAT, offering varying degrees of sensitivity and specificity for different antibody types.
  • Interpreting IAT results is crucial for making informed clinical decisions in blood transfusion and managing autoimmune hemolytic anemia.

Principle of IAT

Principle of IAT explained in a diagram
File:Coombs test schematic.png” by No machine-readable author provided. A. Rad~commonswiki assumed (based on copyright claims). is licensed under CC BY-SA 3.0.

The indirect antiglobulin test (IAT), or Coombs test, delves into the hidden world of potential blood incompatibilities. Unlike its direct counterpart – direct antiglobulin test (DAT), which scrutinizes red blood cells for attached antibodies, the IAT investigates the serum surrounding them for the presence of free globulin like IgG, IgM or C3d. 

The IAT’s principle revolves around a clever game of deception. We first coat RBCs, usually from a donor or a universal donor pool, with the patient’s serum. If any antibodies against RBCs are lurking within this serum, they latch onto the RBCs, forming invisible bonds. 

When antihuman globulin (AHG) is introduced, it binds to these antibody-coated RBCs, acting like a bridge that forms visible clumps, or agglutination. This agglutination reveals the presence of anti-RBC antibodies in the patient’s serum.

The power of the IAT lies in its versatility. It detects a wider range of antibodies than the direct Coombs test, including weak ones that might not cause visible clumping on the actual red blood cells. This makes it crucial for pre-transfusion compatibility testing, ensuring donated blood doesn’t trigger an immune reaction in the recipient. Additionally, the IAT plays a key role in diagnosing various immune-mediated hemolytic anemias, where antibodies attack the patient’s own RBCs, leading to their premature destruction.

Materials

  • Patient’s serum sample
  • Normal saline (0.9% NaCl) / Low ionic strength solution (LISS)
  • Bovine albumin solution (30%)
  • RBC suspension (3-5%):
    • For compatibility testing: Use reagent RBCs of various types (A, B, O, Rh)
    • For autoimmune hemolytic anemia (AIHA) investigation: Use patient’s own RBCs
  • Antihuman globulin (AHG) reagent: polyspecific or monospecific depending on test purpose
  • Test tubes
  • Centrifuge
  • Coombs control red cells – IgG coated red cell (CCC)

Protocol

  1. Centrifuge the patient’s serum sample and discard the clot.
  2. If using patient’s own RBCs for AIHA investigation, prepare a red cell suspension by washing 5 mL patient RBCs with normal saline 3 – 4 times, discard the supernatant and resuspend RBCs to a 3-5% suspension in saline.
  3. Label three test tubes for each antigen type being tested:
    1. Test tube 1: Serum + RBCs
    2. Test tube 2: Serum + RBCs + Bovine Albumin (30%)
    3. Test tube 3: Negative Control (RBCs + LISS or saline)
  4. Add two drops of patient’s serum and one drop of the respective RBC suspension to each tube.
  5. Add one drop of bovine albumin solution to the second tube only.
  6. Incubate all tubes at 37°C for 30 minutes or as per manufacturer’s instructions.
  7. Wash the red cells with normal saline 3 times, discard the supernatant completely after the final wash other than the red cell button.
  8. Add 2 drops of polyspecific AHG onto the red cells.
  9. Tilt the tube and gently swirl it to mix the AHG with the red cells, ensuring they are completely resuspended.
  10. Place the tube in the centrifuge and spin for 15-30 seconds at 900-1000 g or as per manufacturer’s instructions.
  11. Gently resuspend the cell buttons and observe for agglutination macroscopically..
  12. Grade agglutination strength (0 to 4+) if present.
  13. In the absence of agglutination after initial centrifugation, add Coombs control cells (CCC) and centrifuge as per manufacturer instructions. Observe and record any agglutination with CCC, confirming test validity.

Interpretation

ResultInterpretation
Positive agglutination or hemolysis  in Test 1 onlyAntibodies against the corresponding RBC type present
Positive agglutination or hemolysis in Test 1 and Test 2Non-specific agglutination due to cold agglutinins or protein interactions
Negative agglutination in all tubesNo detectable antibodies against the tested RBCs
Negative agglutination after AHG phase and after addition of CCCThe test is invalid and must be repeated

Additional Notes

  • Bovine albumin control: Helps differentiate specific antibody-mediated agglutination from non-specific agglutination due to cold agglutinins or protein interactions.
  • Positive agglutination: Requires further investigation to identify the specific antibody type and clinical significance.
  • Quality control: Perform Coombs control test to ensure AHG reagent reactivity and proper technique.

Function of Coombs Control Cells (CCC)

  • Quality control measure to ensure the validity of the IAT reagents and technique.
  • Consists of RBCs pre-coated with antibodies in the laboratory.
  • Should agglutinate when mixed with AHG reagent, demonstrating its ability to detect antibody-coated RBCs.
  • Negative CCC reaction with AHG suggests a technical error or reagent issue for example AHG was not added or inadequate, the reagent has expired or the AHG has been neutralized by free antibodies due to insufficient washing of the red cells.

Conclusion

Central Role of IAT in Immunohematology

The indirect antiglobulin test (IAT) is a cornerstone of blood bank practices, playing a crucial role in various immunohematological tests, including:

  • Antibody screening: Identifying the presence of any antibodies against red blood cells (RBCs) in a patient’s serum, potentially causing transfusion reactions.
  • Antibody identification: Characterizing the specific type of antibody present, guiding compatible blood selection for transfusions.
  • Antigen typing: Determining the blood group antigens present on RBCs, ensuring accurate crossmatching and transfusion compatibility.
  • Crossmatching: Checking for compatibility between a recipient’s serum and donor RBCs, minimizing the risk of hemolytic reactions.

Alternatives to the Spin-Tube Method

While the traditional spin-tube IAT remains the gold standard for its sensitivity and reliability, several alternative methods offer advantages in terms of efficiency and automation:

  • Solid-phase red cell adherence (SPRCA): RBCs are adhered to a solid surface, simplifying washing steps and enabling automated reading.
  • Column agglutination: RBCs and reagents flow through a column, offering rapid results and ease of use.
  • Gel card techniques: Miniaturized versions of the IAT using gel cards in microplates, enabling high throughput and standardization.

Choosing the Right IAT Method

The choice of IAT method depends on various factors, including:

  • Laboratory capabilities: Automation capabilities and resource availability.
  • Test volume and throughput: Need for high-volume testing efficiency.
  • Specific test requirements: Sensitivity needed for antibody detection or antigen typing.
  • Cost-effectiveness: Balancing test accuracy with resource limitations.

Ongoing Importance of IAT

Despite the availability of alternative methods, the spin-tube IAT remains valuable due to its:

  • High sensitivity: Ability to detect weak antibodies potentially missed by other methods.
  • Flexibility: Adaptability to various testing scenarios and antigen-antibody combinations.
  • Well-established protocols: Extensive experience and data accumulated over decades.

Therefore, the IAT remains a fundamental tool in blood banking, with both the traditional spin-tube method and newer automated techniques playing crucial roles in ensuring safe and effective blood transfusions.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.

References

  1. American Association of Blood Banks (AABB). Technical Manual, 21st Edition, 2023.
  2. Dean L. Blood Groups and Red Cell Antigens [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2005. 
  3. Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical Haematology: Expert Consult: Online and Print 12th Edition (Elsevier). 2016.

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