Laboratory and Special Testing for Suspected Autoimmune Hemolytic Anemia
Order a complete blood count with peripheral smear, reticulocyte count, LDH, haptoglobin, direct and indirect bilirubin, and direct antiglobulin test (DAT) as your initial diagnostic panel, followed by autoimmune serology and evaluation for secondary causes. 1
Initial Laboratory Panel
Core hemolysis markers should be obtained immediately:
- Complete blood count (CBC) with evidence of anemia and macrocytosis 1
- Peripheral blood smear examination for evidence of hemolysis, spherocytes, and agglutination 1
- Reticulocyte count to assess bone marrow response 1
- Lactate dehydrogenase (LDH) - elevated in hemolysis 1
- Haptoglobin - decreased in intravascular hemolysis 1
- Direct and indirect bilirubin - indirect bilirubin elevated in hemolysis 1
- Free hemoglobin in plasma 1
Essential Immunohematologic Testing
The direct antiglobulin test (DAT) is the cornerstone diagnostic test:
- Direct antiglobulin test (DAT/Coombs test) - positive in most AIHA cases, identifies IgG and/or C3d coating on red blood cells 1, 2
- Direct agglutinin test to characterize antibody type 1
- Autoimmune serology to identify specific autoantibodies 1
The DAT pattern determines AIHA subtype: IgG-positive suggests warm AIHA, C3d-only suggests cold agglutinin disease or paroxysmal cold hemoglobinuria, and both IgG and C3d positive indicates mixed AIHA. 2
Evaluation for Secondary Causes
A thorough workup must exclude underlying conditions and alternative diagnoses:
- Detailed medication history evaluating for drug-induced hemolysis (ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin, lorazepam, diclofenac) 1
- Viral and bacterial infectious workup including mycoplasma and other causes of hemolysis 1
- Protein electrophoresis to evaluate for lymphoproliferative disorders 1
- Cryoglobulin analysis 1
- Paroxysmal nocturnal hemoglobinuria (PNH) screening by flow cytometry 1
Additional Specialized Testing
When initial workup is unrevealing or specific conditions are suspected:
- DIC panel including PT/INR and PTT if disseminated intravascular coagulation is suspected 1
- Glucose-6-phosphate dehydrogenase (G6PD) level to exclude enzymatic hemolysis 1
- Methemoglobinemia assessment 1
- Bone marrow aspiration and biopsy with cytogenetic analysis if no obvious cause identified or to evaluate for myelodysplastic syndromes 1
Workup for Refractory or Atypical Cases
If hemolysis persists despite treatment or presentation is atypical:
- Vitamin B12 and folate levels 1
- Copper level 1
- Parvovirus B19 testing 1
- Iron studies (ferritin, transferrin saturation) 1
- Thyroid function tests 1
- Infection screening 1
Critical Pitfalls to Avoid
DAT-negative AIHA occurs in approximately 5-10% of cases due to low antibody levels below detection threshold or presence of IgA/IgM antibodies not routinely tested. 2 If clinical suspicion remains high with negative DAT, consider specialized immunohematology consultation and more sensitive testing methods. 2, 3
Always obtain blood chemistry panel to assess renal function and electrolytes, as hemolysis can cause acute kidney injury from hemoglobinuria. 1
Coombs testing should be specifically considered in patients with chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or history of autoimmune disease, as these populations have higher AIHA risk. 1
Coordinate with blood bank before transfusions in suspected immune-mediated hemolysis to ensure appropriate crossmatching and minimize transfusion reactions. 1