What is the Direct Antiglobulin Test (DAT)?
The Direct Antiglobulin Test (DAT), also known as the direct Coombs test, is a laboratory test that detects immunoglobulin (IgG) and/or complement (C3) bound to the surface of red blood cells in vivo, serving as the critical step to distinguish immune-mediated hemolysis from non-immune causes. 1
Primary Clinical Purpose
The DAT is used to classify hemolysis into immune versus non-immune etiologies by detecting antibodies or complement already attached to circulating red blood cells. 1 This test is essential when evaluating patients with:
- Suspected autoimmune hemolytic anemia (AIHA) 2
- Hemolytic disease of the fetus/newborn 1
- Drug-induced hemolytic anemia 1
- Transfusion-related hemolysis 1
When to Order DAT
DAT should be ordered as part of the baseline evaluation in any patient with suspected hemolysis, particularly when reticulocyte count is elevated and haptoglobin is decreased. 2 Specific clinical scenarios include:
- Chronic lymphocytic leukemia patients prior to treatment initiation, as part of serum chemistry evaluation 2
- Immune thrombocytopenic purpura (ITP) workup to exclude concurrent Evans syndrome (combined ITP and autoimmune hemolytic anemia) 2
- Evaluation of unexplained anemia with laboratory evidence of hemolysis (elevated LDH, indirect bilirubin, low haptoglobin, elevated reticulocytes) 2
- Before initiating immunosuppressive therapy in hematologic malignancies 2
Interpretation of Results
Positive DAT Results
A positive DAT indicates antibodies and/or complement are coating red blood cells, which can occur with:
- IgG positive alone: Most commonly seen in warm autoimmune hemolytic anemia 2, 3
- C3 (complement) positive alone: Rare finding that may indicate severe, refractory AIHA or cold agglutinin disease 3
- IgG + C3 positive: Suggests active immune-mediated hemolysis 1
Negative DAT with Hemolysis
A negative DAT does not exclude autoimmune hemolytic anemia. 4, 1 DAT-negative AIHA can occur due to:
- Low-affinity IgG autoantibodies that dissociate during room temperature washing 4
- IgA or IgM antibodies not detected by standard reagents 1
- Very low levels of red cell-bound IgG below detection threshold 1
When AIHA is strongly suspected clinically but DAT is negative, request cold-washed (4°C) red blood cells for repeat testing or use the DiaMed system with unwashed cells. 4
Critical Pitfalls to Avoid
False Positive Results
- Elevated serum IgG levels (hypergammaglobulinemia) can cause false positive DAT without true hemolysis 5
- Improper washing technique, centrifugation errors, or specimen agitation can produce false reactions 1
- Spontaneous red blood cell agglutination may mimic positive results 1
Always correlate DAT results with clinical findings and other hemolysis markers (reticulocyte count, haptoglobin, LDH, indirect bilirubin) before diagnosing immune hemolysis. 1
False Negative Results
- Standard room temperature washing removes low-affinity antibodies 4
- Delay in testing may allow antibody dissociation 1
- Inadequate sensitivity of reagents for detecting low levels of bound immunoglobulin 4
Integration with Blood Film and Reticulocyte Count
When evaluating hemolysis, the DAT should always be interpreted alongside:
- Blood film: Identifies spherocytes, schistocytes, or other morphologic abnormalities suggesting hemolysis mechanism 2
- Reticulocyte count: Elevated reticulocytes confirm bone marrow response to hemolysis and exclude production defects 2, 6
- Haptoglobin: Decreased haptoglobin with elevated reticulocytes is pathognomonic for hemolysis 7
The combination of positive DAT, elevated reticulocytes, decreased haptoglobin, and spherocytes on blood film confirms immune-mediated hemolytic anemia. 2, 7