Indirect Antiglobulin Test INTRODUCTION

The Indirect Antiglobulin Test (IAT), also known as the Indirect Coombs Test, is a laboratory test that detects antibodies in a patient’s blood serum that might trigger adverse events such hemolytic transfusion reactions or HDN. The test works by clumping red blood cells (RBCs) together when antibodies attach to RBC antigens.

The patient’s serum is mixed with a panel of RBCs with particular antigens for the IAT. Agglutination indicates a positive outcome if serum antibodies react with RBC antigens. IgG antibodies may pass the placenta and induce HDN in neonates.

The IAT indirectly tests RBCs by testing serum. In blood banking and transfusion medicine, it detects RBC antigen-antibody mismatches. This information ensures compatibility and reduces adverse responses during blood transfusions or in pregnant HDN-risk women.

HDN testing and pre-transfusion IATs are common. Immunohematology relies on it, usually done in a lab with specialised reagents. The IAT can assist choose suitable blood products for transfusion or manage pregnant HDN-risk patients.

This is simply an introduction to the Indirect Antiglobulin Test, which requires immunohematology and transfusion medicine skills to fully comprehend and interpret.

Purpose of Indirect Antiglobulin Test (IAT)

Indirect Antiglobulin Tests (IATs) detect antibodies in blood serum that may induce unpleasant effects. In blood banking and transfusion medicine, the test checks donor-recipient antibody compatibility and assesses pregnant women’s risk of hemolytic disease of the newborn (HDN).

IAT uses:

Blood compatibility testing: Before a blood transfusion, the IAT checks the recipient’s serum for antibodies that potentially react with the donor’s RBCs. Antibodies indicate donor-recipient incompatibility, which might cause transfusion reactions. Results assist choose suitable blood products to reduce adverse outcomes.

Crossmatching: The IAT mixes the patient’s serum with the donor’s RBCs to test compatibility. The IAT detects patient serum antibodies that may react with donor RBCs, suggesting an incompatible crossmatch.

HDN risk assessment in pregnant women: IAT. These antibodies may cross the placenta and target the baby’s RBCs, producing HDN. The IAT detects maternal antibodies and advises neonatal risk management.

The IAT detects serum antibodies that may cause transfusion reactions or HDN. The test findings guide clinical choices on blood product selection, transfusion compatibility, and patient and recipient safety.

Procedure of Indirect Antiglobulin Test (IAT)

The IAT requires multiple phases. General procedure:

Sample collection: Centrifuge a patient blood sample to extract serum from red blood cells (RBCs). The IAT will test serum antibodies.

Select a commercial RBC panel with known antigen profiles. These RBCs reflect population antigens. The panel should include RBCs with patient-specific antigens.

Incubation: Mix small aliquots of patient serum with panel RBCs in test tubes or microplate wells. Incubate tubes or microplates at 37°C (body temperature) for antibody-antigen responses.

Washing: Remove serum components from RBCs after incubation. Eliminating non-specific reactions and ensuring reliable findings requires this step.

Add Coombs (antiglobulin) reagent to washed RBCs. Human IgG and complement antibodies are in the Coombs reagent. It binds patient RBC IgG antibodies.

Incubation and washing: Incubate the tubes or microplates again to enable the Coombs reagent to react with RBC-bound IgG antibodies. Wash unbound Coombs reagent from RBCs.

Observe RBC agglutination in tubes or microplates. Agglutination implies the patient’s serum antibodies have bonded to RBCs. Negative reactions lack agglutination.

Agglutination affects findings interpretation. Negative results indicate no antibodies in the patient’s serum, whereas positive reactions show antibodies.

The IAT technique depends on the lab’s procedures and reagents. The IAT stages are described above.

Indication of Indirect Antiglobulin Test (IAT)

The Indirect Antiglobulin Test (IAT) has several clinical uses. Common IAT indicators include:

Pre-transfusion testing requires the IAT. It detects recipient serum antibodies that may react with donor RBCs. The IAT reduces transfusion reactions by recognising antibody-antigen incompatibility.

Fever, hemolysis, and other transfusion responses are investigated using the IAT. Testing the patient’s serum against the donor’s RBCs might reveal unexpected antibodies, revealing the transfusion reaction’s source.

HDN risk assessment in pregnant women relies on the IAT. It detects maternal blood antibodies that may penetrate the placenta and cause foetal hemolysis. The IAT detects antibodies against foetal RBC antigens, enabling pregnancy management and monitoring.

The IAT can distinguish AIHA subtypes. It determines whether the patient’s RBCs are coated with IgG antibodies, indicating warm AIHA, or if their blood includes antibodies that may bind to RBCs in the presence of complement, indicating cold AIHA.

Unexpected antibody identification: The IAT characterises unexpected antibodies found during standard blood typing or screening assays. The IAT helps identify antibody specificity and choose blood products for patients with complicated antibody profiles.

The IAT can evaluate autoimmune disorders such autoimmune hemolytic anaemia, immune thrombocytopenia, and autoimmune neutropenia. It helps diagnose and treat RBC, platelet, and neutrophil autoantibodies.

These are common IAT indicators. In immunohematology and transfusion medicine, the test helps identify maternal-fetal incompatibility, manage blood transfusions, and investigate antibody-mediated diseases.

Types of Indirect Antiglobulin Test (IAT)

Based on reagents and purpose, there are two kinds of indirect antiglobulin tests (IAT):

Routine IAT: Pre-transfusion testing detects unusual antibodies in a patient’s serum that may trigger transfusion reactions. The patient’s serum is tested against a panel of commercial RBCs with established antigen profiles. It detects antibodies in the patient’s serum that may react with the donor’s RBCs, suggesting incompatibility. IATs reduce transfusion responses by ensuring donor blood and recipient antibody compatibility.

The antibody identification panel is a specialised IAT designed to detect and characterise particular antibodies in a patient’s blood. It’s done when blood typing or screening tests reveal unexpected antibodies. The antibody identification panel uses a wider variety of RBCs with established antigen patterns to determine the patient’s blood antibodies’ specificity. By analysing the responses between the patient’s serum and the panel’s RBCs, the antibody or antibodies producing the reaction may be detected or narrowed down, helping choose compatible blood products and manage patients with complicated antibody profiles.

Immunohematology and transfusion medicine use these IAT types differently. The antibody identification panel characterises serum antibodies, whereas the regular IAT tests for transfusion compatibility. Both kinds are essential for safe blood transfusions and sophisticated antibody management.

Risk of Indirect Antiglobulin Test (IAT)

IATs are typically safe. Like every lab test, there are dangers and considerations:

Blood sample required: The IAT is invasive. Blood collection is usually painless, however the needle site may bruise. Sterilisation reduces infection risk.

False-positive or false-negative results: IAT interpretation takes skill and practise. Technical flaws or testing variances might cause false-positive or false-negative responses. Avoiding misunderstanding requires careful attention to detail and standardised practises.

Sensitization to RBC antigens or adverse responses during the IAT process are infrequent. Commercial reagents reduce the chance of adverse outcomes. However, the reagent manufacturer’s safety precautions must be followed.

Blood and possibly infectious materials: Blood samples and laboratory reagents pose little danger of infection. Wear PPE, implement infection control policies, and properly dispose of biohazardous objects.

It’s crucial to highlight that these hazards are usually modest and may be avoided by following standard laboratory practises, utilising quality-controlled reagents, and training and competence of the laboratory employees doing the IAT.

If you have concerns about the hazards of the IAT or any other medical treatment, see a healthcare practitioner or skilled laboratory expert who can provide you personalised advice.

Result of Indirect Antiglobulin Test (IAT)

Red blood cell agglutination (clumping) determines the Indirect Antiglobulin Test (IAT) findings. IAT outcomes interpretations:

Positive result: RBCs agglutinate when antibodies in the patient’s serum bind to them. Positive findings may indicate alloantibodies or autoantibodies. Agglutination pattern and RBCs may provide antibody specificity and clinical relevance.

Negative result: No agglutination means the patient’s serum lacks antibodies that react with the RBC antigens examined. A negative result does not rule out antibodies, since some may be present at low levels or not detectable by the test’s RBC antigen panel.

Positive and negative controls verify the IAT. Agglutination in the positive control indicates adequate test reagents and procedure. Non-specific responses should not agglutinate the negative control.

The patient’s clinical history, including transfusion, pregnancy, and alloantibody history, should be considered when interpreting IAT findings. The findings inform transfusion choices, compatibility testing, and HDN/transfusion reaction management.

IAT interpretation needs immunohematology and transfusion medicine competence. Laboratory specialists should interpret the findings.


In conclusion, immunohematology and transfusion medicine benefit from the IAT. It aids clinical decision-making. IAT findings:

Transfusion medicine requires the IAT to determine donor blood-recipient antibody compatibility. It detects antibody-antigen incompatibility that potentially cause transfusion reactions.

Antibody identification: An antibody identification panel helps the IAT identify and characterise individual antibodies in a patient’s serum. Selecting suitable blood supplies and treating complicated antibody profiles need this knowledge.

HDN risk assessment: The IAT is important in measuring pregnant women’s HDN risk. It identifies maternal antibodies that may induce foetal hemolysis and directs pregnancy surveillance.

Transfusion reaction investigation: The IAT finds unusual antibodies that may have caused a transfusion response. Preventing transfusion-related adverse effects requires this knowledge.

Autoimmune illness evaluation: The IAT may help diagnose RBC, platelet, or neutrophil-directed autoimmune disorders. It detects and characterises autoantibodies, aiding diagnosis and treatment.

IAT interpretation needs immunohematology and transfusion medicine competence. Accurate test interpretation and clinical choices depend on qualified laboratory workers.

The IAT helps determine blood compatibility, antibody detection, HDN risk assessment, and transfusion responses. It improves transfusion safety, high-risk pregnancy management, and antibody-mediated disease identification and treatment.


Q: How do DAT and IAT differ?
A: Immunohematology lab assays DAT and IAT. The DAT directly detects antibodies or complement components on the patient’s red blood cells (RBCs). The IAT detects non-RBC-attached antibodies in the patient’s serum. The IAT is mostly used for antibody identification, compatibility testing, and HDN risk assessment.

Q: How long do IAT results take?
A: The laboratory’s workload and testing methods determine the IAT’s turnaround time. The test and findings may take a few hours to two days. Emergency transfusions and important HDN evaluations may get accelerated processing and reporting.

Can the IAT detect all antibodies?
A: The IAT detects most clinically important serum antibodies. The IAT only employs RBCs with established antigen profiles. The panel may not identify antibodies to rare RBC antigens. Specialised testing or reference laboratory consulting may be needed.

Can the IAT type ABO blood?
A: The IAT does not routinely type ABO blood. ABO blood type is usually done via direct agglutination or forward and reverse typing. ABO typing tests RBCs for ABO antigens, whereas the IAT detects antibodies.

Can the IAT test organ transplant compatibility?
A: Organ transplantation compatibility testing does not employ the IAT. Blood banking and transfusion medicine use the IAT to determine donor-recipient antibody compatibility. HLA typing and crossmatching assess donor-recipient compatibility in organ donation.

Q: Are there any pre-IAT preparations?
A: Patients do not need to prepare for the IAT. It’s best to follow the doctor’s or lab’s directions. Inform the lab of any drugs or medical conditions that may affect test findings.

These responses are generic and may not cover all variations or conditions. Healthcare professionals or laboratory specialists can provide personalised and accurate Indirect Antiglobulin Test (IAT) information.

Myth vs FACT

Myth: The Indirect Antiglobulin Test (IAT) detects all serum antibodies.
Fact: The IAT detects most clinically important antibodies in serum. It employs a small panel of RBCs with known antigen signatures. The panel may not identify antibodies to rare RBC antigens. Specialised testing or reference laboratory consulting may be needed.

Myth: IATs are crossmatch tests.
Fact: IAT and crossmatch tests serve various objectives. Unlike the crossmatch test, the IAT detects antibodies in a patient’s serum that may react with RBCs. The crossmatch test checks for agglutination or other responses by mixing the donor’s RBCs with the recipient’s serum or plasma. The IAT is part of the crossmatch method but not the test.

Myth: A positive IAT prevents blood transfusions.
Fact: A positive IAT does not exclude blood transfusions. The IAT identifies antibodies in a patient’s serum, which may signal transfusion responses or compatibility concerns. However, antibody specificity, titer, patient clinical state, and compatible blood products determine the clinical importance and effect of identified antibodies. Healthcare professionals should examine all aspects while determining transfusion compatibility.

Myth: The IAT uses any blood sample.
The IAT needs serum or plasma, not whole blood. The patient’s blood sample must be treated to separate serum and plasma for the IAT. Centrifugation separates serum or plasma from RBCs. IAT testing uses serum or plasma.

Myth: The IAT diagnoses autoimmune disorders.
The IAT can identify RBC-directed antibodies, but it cannot diagnose autoimmune disorders. The IAT may identify antibodies affecting RBCs, however it does not diagnose autoimmune disorders. Clinical examination, laboratory testing, and other specific criteria are needed to diagnose autoimmune disorders.

These remarks are generic and may not apply to all situations. Healthcare professionals or laboratory specialists can provide personalised and accurate Indirect Antiglobulin Test (IAT) information.


Indirect Antiglobulin Test (IAT): A lab test that identifies antibodies in patient serum by mixing it with RBCs with known antigens.

Antibody: An immune system protein generated in response to antigens. Antibodies attach to antigens and activate immunological responses.

Antigen: A material that induces antibody formation. Antigens are red blood cell molecules in the IAT.

Alloantibody: An antibody generated in reaction to non-self antigens, usually through transfusions or pregnancy.

Autoantibody: An antibody that attacks one’s own cells or tissues, causing immunological responses.

Serum: The liquid part of blood after it has clotted and settled, containing red blood cells.

Plasma: Blood’s liquid part after an anticoagulant prevents clot formation.

Agglutination: Red blood cells clump together when antibodies attach to cell surface antigens.

Sensitization: When a person develops antibodies against certain antigens after being exposed to them.

Hemolytic Disease of the infant (HDN): Maternal antibodies penetrate the placenta and kill foetal red blood cells, causing infant anaemia and other problems.

Antibody Identification: Testing a patient’s serum against a panel of red blood cell antigens to identify particular antibodies.

Routine IAT: The pre-transfusion IAT detects unusual antibodies in a patient’s serum that may trigger transfusion reactions.

Before antibody identification or compatibility testing, a patient’s blood is screened for unexpected antibodies.

Positive Control: A sample or reagent anticipated to give a positive result in a laboratory test to validate the test technique.

Negative Control: A known sample or reagent that should not respond in a lab test to rule out non-specific responses.

Donor Units: Donated blood or blood products for transfusion.

Crossmatch: A test that mixes recipient serum or plasma with donor RBCs to detect compatibility and transfusion risk.

Compatibility Testing: Assessing the donor’s blood and the recipient’s serum or plasma for safe and effective blood transfusions.

Direct Antiglobulin Test (DAT): A laboratory test that detects antibodies or complement components on the patient’s red blood cells.

Transfusion Reaction: A moderate to severe reaction after a blood transfusion, involving fever, chills, shortness of breath, and hemolysis.

Panel Cells: Standardised red blood cells used in the IAT to represent several antigens for antibody identification and compatibility testing.

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