When is a direct antiglobulin test (direct Coombs test) indicated, how is it performed, and what do positive or negative results imply for diagnosis and management?

Direct Antiglobulin Test (Direct Coombs Test): Indications, Performance, and Interpretation

Overview

The direct antiglobulin test (DAT) is indicated whenever immune-mediated hemolysis is suspected, and it functions as the critical branching point that separates immune from non-immune causes of red blood cell destruction. 1

Primary Clinical Indications

The DAT should be ordered in the following specific scenarios:

Hemolytic Anemia Evaluation

• Order DAT immediately when hemolysis is confirmed (elevated LDH, indirect bilirubin, reticulocytes, and decreased haptoglobin) to determine if the mechanism is immune-mediated 1
• The test detects immunoglobulin (IgG), complement (C3), or both bound to red blood cell membranes in vivo 2, 3

Neonatal Jaundice

• Strongly recommended in newborns with jaundice if the mother is Rh negative or has no prenatal typing to detect ABO or Rh incompatibility 4
• Essential for diagnosing hemolytic disease of the newborn 1, 2
• If mother is group O, Rh positive, DAT on cord blood is optional but close monitoring is recommended 4

Transfusion Reactions

• Indicated when transfusion-related hemolysis is suspected 3

Drug-Induced Hemolytic Anemia

• Should be performed when medications known to cause immune hemolysis are being used 3, 5

Thrombotic Microangiopathy Differentiation

• A negative DAT is diagnostically important in atypical hemolytic uremic syndrome (aHUS), where it helps differentiate non-immune microangiopathic hemolysis from immune-mediated destruction 6, 7

How the Test is Performed

The DAT involves the following technical steps:

• Patient red blood cells are washed with saline to remove unbound antibodies 8
• Anti-human globulin reagent (Coombs reagent) is added, which contains antibodies against human IgG and/or complement C3 2, 5
• If immunoglobulin or complement is coating the red cells, agglutination occurs, indicating a positive result 3, 5
• Modern variations include solid-phase methods (dot blot) and gel column technology (DiaMed system) that can detect low-affinity antibodies better than traditional tube methods 8, 9

Interpretation of Results

Positive DAT

A positive DAT indicates antibody-coated red blood cells and confirms immune-mediated hemolysis 4, 1

When DAT is positive with concurrent anemia and reticulocytosis:

• Confirms autoimmune hemolytic anemia (AIHA) 1
• Initiate prednisone 1-2 mg/kg/day immediately 1
• Consider transfusion only if hemoglobin <7-8 g/dL or patient is symptomatic 1
• Monitor weekly until stabilized 1
• If no response after 3 weeks, escalate to rituximab, IVIG, or splenectomy 1

In neonates, a positive DAT confirms:

• Hemolytic disease of the newborn from maternal-fetal blood group incompatibility 4, 2

Negative DAT

A negative DAT is normal in healthy individuals and excludes classic autoimmune hemolytic anemia 7, 1

When DAT is negative but hemolysis is present, redirect diagnosis toward:

Non-Immune Hemolysis

• Examine peripheral smear for schistocytes (suggests thrombotic microangiopathy) 7, 1
• Hereditary spherocytosis 1
• G6PD deficiency 1
• Mechanical hemolysis from prosthetic heart valves 1

Atypical Hemolytic Uremic Syndrome (aHUS)

• The combination of thrombocytopenia, hemolysis with schistocytes, renal dysfunction, and negative DAT strongly suggests aHUS 6, 7
• This is a diagnostic criterion for aHUS 7
• Requires urgent evaluation for complement-mediated thrombotic microangiopathy 6, 7

Critical Pitfalls and Technical Considerations

False-Negative Results

Low-affinity IgG antibodies can dissociate during routine room-temperature washing, causing false-negative DAT despite significant in vivo hemolysis 9

To avoid missing these cases:

• Use cold-washed (4°C) red blood cells when DAT is negative but clinical suspicion for AIHA remains high 9
• Consider gel column methods (DiaMed system) that use unwashed cells and are more sensitive for low-affinity antibodies 9
• An eluate prepared from cold-washed cells may reveal IgG autoantibodies even when standard DAT is negative 9

False-Positive Results

Technical errors causing false positivity include 3, 5:

• Improper washing technique
• Incorrect centrifugation
• Spontaneous red blood cell agglutination
• Over-agitation during interpretation

Context-Dependent Interpretation

The DAT result must always be interpreted alongside clinical findings and other laboratory data 3, 5:

• A positive DAT without hemolysis may represent clinically insignificant antibody binding
• A negative DAT does not exclude hemolysis—it redirects the differential diagnosis 1

Diagnostic Algorithm

1. Confirm hemolysis: Check LDH, indirect bilirubin, haptoglobin, reticulocyte count 7, 1
2. Order DAT to determine immune vs. non-immune etiology 1
3. If DAT positive: Diagnose immune-mediated hemolysis and initiate corticosteroids 1
4. If DAT negative with hemolysis:
   • Examine peripheral smear for schistocytes 7, 1
   • Evaluate platelets and renal function (thrombocytopenia + renal dysfunction + negative DAT = consider aHUS) 6, 7
   • Consider hereditary hemolytic anemias or mechanical causes 1
   • If high clinical suspicion for AIHA persists, repeat DAT with cold-washed cells 9