What are the diagnostic criteria for autoimmune hemolytic anemia?

Diagnostic Criteria for Autoimmune Hemolytic Anemia

The diagnosis of autoimmune hemolytic anemia requires demonstrating both hemolysis and its immune-mediated nature through a positive Direct Antiglobulin Test (DAT), followed by classification of the specific AIHA subtype and exclusion of secondary causes. 1, 2

Essential Diagnostic Components

Step 1: Establish Evidence of Hemolysis

The initial workup must document anemia with laboratory markers of red blood cell destruction:

• Complete blood count (CBC) showing anemia with macrocytosis 1
• Reticulocyte count (typically elevated, reflecting bone marrow compensation) 1, 2
• Lactate dehydrogenase (LDH) (elevated due to intracellular enzyme release) 1
• Haptoglobin (decreased or absent, as it binds free hemoglobin) 1
• Bilirubin (elevated indirect/unconjugated bilirubin from heme catabolism) 1
• Peripheral blood smear to identify spherocytes, polychromasia, and other evidence of hemolysis 1
• Free hemoglobin assessment in plasma or urine 1

Step 2: Confirm Immune-Mediated Destruction

The Direct Antiglobulin Test (DAT) is mandatory and must be performed before initiating any treatment. 1, 2 This test detects antibodies and/or complement bound to red blood cells:

• Monospecific DAT is essential to identify whether IgG, C3d, or both are coating the RBCs 2
• Indirect antiglobulin test detects free autoantibodies circulating in serum 1

Important caveat: DAT-negative AIHA occurs in 3-10% of cases due to low antibody levels below detection thresholds, or presence of IgM or IgA antibodies not routinely tested. 3, 4 In these cases, diagnosis relies on excluding other causes of hemolysis and demonstrating response to corticosteroids. 3

Step 3: Classify the AIHA Subtype

Classification depends on DAT pattern and antibody characteristics:

• Warm AIHA (wAIHA): DAT positive for IgG with or without C3d; antibodies react optimally at 37°C 4, 2
• Cold Agglutinin Disease (CAD): DAT positive only for C3d; monoclonal IgM antibodies react at cold temperatures 4
• Cold Agglutinin Syndrome (CAS): DAT positive only for C3d; polyclonal IgM antibodies, typically secondary to infections 4
• Mixed AIHA: DAT positive for both IgG and C3d 4
• Paroxysmal Cold Hemoglobinuria (PCH): DAT positive only for C3d; caused by biphasic Donath-Landsteiner IgG antibody 4
• Drug-Induced Immune Hemolytic Anemia (DIIHA): DAT shows IgG and/or C3d; temporal relationship with drug exposure 4

Step 4: Exclude Secondary Causes

Secondary AIHA accounts for a significant proportion of cases and requires systematic investigation. 3, 5

Mandatory exclusion workup:

• Viral and bacterial infections: HIV, hepatitis, mycoplasma 1
• Drug exposure: Evaluate for ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin 1
• Lymphoproliferative disorders: Ultrasound/CT imaging and bone marrow evaluation if clinically indicated 1, 3
• Autoimmune diseases: Autoantibody screening (ANA, anti-dsDNA, ENA) in patients with systemic symptoms 1, 3
• Immunodeficiencies: Immunoglobulin levels if frequent infections present 3

Additional differential diagnoses to exclude:

• Paroxysmal nocturnal hemoglobinuria (PNH): Flow cytometry screening 1
• Glucose-6-phosphate dehydrogenase (G6PD) deficiency 1
• Methemoglobinemia 1
• Myelodysplastic syndromes: Bone marrow analysis and cytogenetic studies if no obvious cause found 1
• Nutritional deficiencies: B12, folate, copper, iron studies 1
• Chronic organ dysfunction: Liver and kidney disease evaluation 3

Clinical Presentation Considerations

Patients typically present with:

• Weakness, paleness, jaundice 1
• Dark-colored urine (hemoglobinuria) 1
• Fever, inability to perform physical activity 1
• Heart murmur (from high-output cardiac state) 1

Obtain detailed medication history and exposure to insect, spider, or snake bites. 1

Critical Diagnostic Pitfalls

• False-normal enzyme activity levels may occur with markedly elevated reticulocyte counts, recent blood transfusions, or incomplete removal of platelets and leukocytes 1
• Autoantibody titers vary during disease course: Seronegative patients at diagnosis may express autoantibodies later; repeated testing may be necessary if clinical suspicion remains high 1
• DAT-negative cases are more common in severe presentations including ICU admissions and may require multiple treatments rapidly 3
• Comprehensive autoimmune serology may not be available in all laboratories: Consider reference laboratory testing in diagnostically uncertain cases 1

When to Suspect Specific Subtypes

• CAD/CAS: Symptoms worsen with cold exposure; acrocyanosis present 4, 2
• PCH: Typically follows viral infections in children; self-limiting course 4
• DIIHA: Temporal relationship between drug initiation and hemolysis onset 4
• Secondary AIHA: Lymphadenopathy, splenomegaly, systemic symptoms, or known underlying conditions 3, 5