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, protecting against potentially disastrous immune reactions between your patient’s blood and donor red blood cells (RBCs). 

Key points of indirect antiglobulin test (IAT)

• The indirect antiglobulin (Coombs) test (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 (Coombs) test (DAT), including weak antibodies that may not cause visible agglutination.
• The indirect antiglobulin (Coombs) test (IAT) is essential for both pre-transfusion compatibility testing and prenatal antibody screening in pregnant women.
• By identifying incompatible antibodies, the indirect antiglobulin (Coombs) test (IAT) helps prevent potentially life-threatening hemolytic transfusion reactions.
• Different types of antihuman globulin (AHG) reagents can be used in the indirect antiglobulin (Coombs) test (IAT), offering varying degrees of sensitivity and specificity for different antibody types.
• Interpreting indirect antiglobulin (Coombs) test (IAT) results is crucial for making informed clinical decisions in blood transfusion and managing autoimmune hemolytic anemia.

Principle of indirect antiglobulin (Coombs) test (IAT)

The indirect antiglobulin test (IAT), or indirect 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 indirect antiglobulin (Coombs) test (IAT) investigates the serum surrounding them for the presence of free globulin like IgG, IgM or C3d. 

The indirect antiglobulin (Coombs) test (IAT) principle revolves around using RBCs, usually from a donor or a universal donor pool, and mix 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 indirect antiglobulin (Coombs) test (IAT) lies in its versatility. It detects a wider range of antibodies than the direct Coombs test (DAT), 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 indirect antiglobulin (Coombs) test (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

Result	Interpretation
Positive agglutination or hemolysis  in Test 1 only	Antibodies against the corresponding RBC type present
Positive agglutination or hemolysis in Test 1 and Test 2	Non-specific agglutination due to cold agglutinins or protein interactions
Negative agglutination in all tubes	No detectable antibodies against the tested RBCs
Negative agglutination after AHG phase and after addition of CCC	The 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 indirect antiglobulin (Coombs) test (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 (Coombs) 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 indirect antiglobulin (Coombs) test (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 indirect antiglobulin (Coombs) test (IAT) using gel cards in microplates, enabling high throughput and standardization.

Choosing the Right IAT Method

The choice of indirect antiglobulin (Coombs)test (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 Indirect Antiglobulin (Coombs) Test (IAT)

Despite the availability of alternative methods, the spin-tube indirect antiglobulin (Coombs) test (IAT) remains valuable due to:

• 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 indirect antiglobulin (Coombs) test (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.

Frequently Asked Questions (FAQs)

What is the difference between direct antiglobulin (Coombs) test (DAT) and indirect antiglobulin (Coombs) test (IAT)?

Both direct antiglobulin (Coombs) test (DAT) and indirect antiglobulin (Coombs) test (IAT) are blood tests used in immunohematology to detect antibodies related to red blood cells (RBCs). However, they differ in what they are looking for:

• DAT (Direct Antiglobulin (Coombs) Test): This test checks for antibodies that are already attached to the surface of RBCs. This can happen due to autoimmune diseases or past blood transfusions. A positive direct antiglobulin (Coombs) test (DAT) indicates that RBC destruction might be occurring.
• IAT (Indirect Antiglobulin (Coombs) Test): This test looks for free antibodies in the blood plasma that can potentially bind to RBCs. These antibodies haven’t attached yet but could cause problems during a blood transfusion or in a newborn with hemolytic disease.

Here’s a table summarizing the key differences:

Feature	Direct Antiglobulin (Coombs) Test (DAT)	Indirect Antiglobulin (Coombs) Test (IAT)
What it detects	Antibodies bound to RBCs	Free antibodies in blood plasma
Indicates	Current RBC destruction	Potential for future RBC destruction
Use cases	Autoimmune hemolytic anemia, transfusion reactions	Blood transfusion compatibility testing, hemolytic disease of the newborn

What is IAT used to detect?

IAT (Indirect Antiglobulin (Coombs) Test) is used to detect free antibodies in the blood plasma that can potentially bind to red blood cells (RBCs). These antibodies haven’t attached to RBCs yet, but their presence indicates a potential for future problems.

What is the purpose of indirect antiglobulin (Coombs) test?

The indirect antiglobulin test (IAT), also known as the indirect Coombs test, has two main purposes:

1. Blood Transfusion Compatibility Testing: Before receiving a blood transfusion, an indirect antiglobulin (Coombs) test (IAT) is performed to ensure compatibility between the recipient’s blood and the donor’s blood. The test detects pre-existing antibodies in the recipient’s plasma that could attack the donor’s red blood cells. A positive indirect antiglobulin (Coombs) test (IAT) indicates incompatibility, and the recipient would need blood from a different donor with compatible blood type. This helps prevent transfusion reactions that can be serious or even fatal.
2. Hemolytic Disease of the Fetus and Newborn (HDFN): In pregnant women, an indirect antiglobulin (Coombs) test (IAT) can be used to screen for HDFN. This condition occurs when a Rh-negative mother (lacks the Rh factor protein on red blood cells) carries a Rh-positive baby (inherits the Rh factor from the father). The mother’s immune system may develop antibodies against the baby’s Rh-positive red blood cells, potentially leading to anemia or other complications in the baby. A positive indirect antiglobulin (Coombs) test (IAT) in a pregnant woman might indicate a need for further monitoring and potential interventions to protect the baby.

Why is IAT used for antibody screening?

The indirect antiglobulin (Coombs) test (IAT) is a valuable tool for antibody screening because it offers several key advantages:

1. High Sensitivity: The indirect antiglobulin (Coombs) test (IAT) is very sensitive in detecting antibodies against red blood cells (RBCs). Even small amounts of antibodies can be picked up by the test, which is crucial for preventing potential transfusion reactions.
2. Antibody Specificity: While the initial indirect antiglobulin (Coombs) test (IAT) screening might not identify the specific antigen the antibody targets, further testing using the indirect antiglobulin (Coombs) test (IAT) with different types of RBCs (called an identification panel) can pinpoint the specific antigen-antibody interaction. This information is essential for selecting compatible blood for transfusion.
3. Non-Agglutination Detection: Some antibodies, particularly IgG, may not cause visible clumping (agglutination) of RBCs on their own. The indirect antiglobulin (Coombs) test (IAT) overcomes this limitation by employing an antiglobulin reagent (also called Coombs’ serum). This reagent acts as a bridge, binding to the patient’s antibody if it’s attached to an RBC, even if agglutination isn’t readily apparent. This ensures detection of a wider range of antibodies that could cause problems during a transfusion.
4. Safety and Efficiency: The indirect antiglobulin (Coombs) test (IAT) is a relatively simple and safe test to perform. It uses separated blood components (plasma or serum) instead of whole blood, allowing for easier manipulation and reducing the risk of errors. Furthermore, it’s an efficient way to screen for multiple potential transfusion reactions compared to testing compatibility with every possible blood type.

What causes false negative indirect antiglobulin (Coombs) test?

There are a few reasons why an indirect antiglobulin (Coombs) test (IAT) might produce a false negative result, meaning it misses antibodies that are actually present in the blood plasma. Here’s a breakdown of some potential causes:

• Antibody Type: Most commercially available indirect antiglobulin (Coombs) test (IAT) tests are designed to detect antibodies against IgG, complement C3, or both on red blood cells (RBCs). If the antibody in question is of a different type, like IgM or IgA, it might not be picked up by the test. In these uncommon situations, a specific test for the suspected antibody type or a quantitative DAT (direct antiglobulin test) might be necessary.
• Low Antibody Levels: In some rare cases, autoimmune hemolytic anemia can be caused by antibody levels that fall below the detection limit of the indirect antiglobulin (Coombs) test (IAT). This threshold is typically around 150 to 500 IgG molecules per RBC.
• Technical Issues: Although less common, technical errors during the test procedure can lead to false negatives. Factors like inadequate centrifugation, delays in adding reagents, or problems with the antiglobulin reagent itself could affect the test’s sensitivity.
• Interference from Rheumatoid Factor (RF): RF is an autoantibody (antibody against one’s own tissues) that can sometimes interfere with the indirect antiglobulin (Coombs) test (IAT). The presence of RF can alter the interaction between the patient’s antibody and the antiglobulin reagent, leading to a falsely negative result.

It’s important to note that interpreting indirect antiglobulin (Coombs) test (IAT) results should be done in conjunction with the patient’s clinical history and other relevant tests. If a high suspicion of antibody presence exists despite a negative indirect antiglobulin (Coombs) test (IAT), further investigation or a different type of antibody test might be warranted.

What does a positive test before a blood transfusion mean?

A positive indirect antiglobulin (Coombs) test (IAT) before a blood transfusion indicates that your blood plasma contains antibodies that have the potential to react with certain antigens on donor red blood cells (RBCs). This reaction could cause a transfusion reaction, which can be serious or even fatal.

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.