Diagnostic Approach for Autoimmune Hemolytic Anemia
The diagnosis of autoimmune hemolytic anemia (AIHA) requires a systematic approach that includes laboratory evidence of hemolysis, confirmation of immune-mediated destruction with direct antiglobulin testing, and exclusion of alternative causes. 1
Initial Diagnostic Workup
Clinical Assessment
- Evaluate for symptoms including weakness, paleness, jaundice, dark-colored urine, fever, inability to perform physical activity, and heart murmur 1
- Obtain detailed history with special consideration of new medications and insect, spider, or snake bites 1
- Assess for underlying conditions that may cause secondary AIHA 2
Laboratory Evaluation for Hemolysis
- Complete blood count (CBC) with evidence of anemia and macrocytosis 1
- Peripheral blood smear to identify evidence of hemolysis 1
- Reticulocyte count (typically elevated) 1
- Lactate dehydrogenase (LDH) (elevated) 1
- Haptoglobin (decreased or absent) 1
- Bilirubin (direct and indirect) - elevated indirect (unconjugated) bilirubin 1
- Free hemoglobin assessment 1
Confirming Immune-Mediated Hemolysis
Direct Antiglobulin Test (DAT)
- The monospecific direct antiglobulin test is mandatory in the diagnostic workup of AIHA 3
- Test should be performed before initiating any treatment 1
- Determines presence of antibodies or complement on RBC surface 2
- Helps classify AIHA subtypes:
Additional Immunological Testing
- Indirect antiglobulin test to detect free autoantibodies in serum 1
- Autoimmune serology to identify underlying autoimmune disorders 1
Excluding Alternative Causes
Infectious Causes
- Evaluate for viral and bacterial (especially mycoplasma) causes of hemolysis 1
- Consider HIV, hepatitis testing 1
Hematological Disorders
- Paroxysmal nocturnal hemoglobinuria screening 1
- If no obvious cause is found, consider bone marrow analysis and cytogenetic analysis to evaluate for myelodysplastic syndromes 1
Metabolic/Enzymatic Disorders
Other Testing
- Protein electrophoresis and cryoglobulin analysis 1
- Workup for bone marrow failure syndrome if refractory, including B12, folate, copper, parvovirus, iron studies, and thyroid function 1
Classification of AIHA
Based on Thermal Reactivity and Antibody Type
- Warm AIHA (wAIHA): Antibodies most active at 37°C, typically IgG 2
- Cold agglutinin disease (CAD): Antibodies most active at 4°C, typically IgM 2
- Mixed AIHA: Features of both warm and cold AIHA 2
- Paroxysmal cold hemoglobinuria (PCH): Biphasic cold-reactive IgG antibody (Donath-Landsteiner test positive) 2
Based on Etiology
- Primary (idiopathic): No identifiable underlying cause 2
- Secondary: Associated with underlying conditions such as:
- Lymphoproliferative disorders
- Autoimmune diseases
- Infections
- Medications 2
Special Considerations
Drug-Induced Immune Hemolytic Anemia
- Evaluate common drug causes including ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin, lorazepam, diclofenac, etc. 1
- Appears as warm AIHA with DAT positive for IgG and/or C3d 2
DAT-Negative AIHA
- May occur due to IgG antibody below detection thresholds or non-detected IgM or IgA antibodies 2
- Consider more sensitive techniques when clinical suspicion is high despite negative standard DAT 4
Pitfalls and Caveats
- False-normal PK enzyme activity levels may occur due to:
- Markedly increased reticulocyte count
- Recent blood transfusions
- Incomplete removal of platelets and leukocytes 1
- Autoantibody titers and specificity may vary during the course of disease; seronegative individuals at diagnosis may express conventional autoantibodies later 1
- Repeated testing may be necessary if initial results are negative but clinical suspicion remains high 1
- Comprehensive autoimmune serology workup may not be available in all laboratories; consider sending samples to reference laboratories in cases of diagnostic uncertainty 1