What is the pathophysiologic basis of Warm Autoimmune Hemolytic Anemia (AIHA) versus Cold AIHA and what laboratory tests are used to differentiate between them?

Pathophysiology and Laboratory Differentiation of Warm vs Cold AIHA

Pathophysiologic Mechanisms

Warm AIHA is mediated by IgG antibodies that bind optimally at body temperature (37°C) and cause extravascular hemolysis through macrophage-mediated phagocytosis in the spleen and liver, while Cold AIHA is mediated by IgM antibodies that bind at lower temperatures (typically <30°C) and cause complement-mediated intravascular hemolysis. 1, 2

Warm AIHA Pathophysiology

• IgG autoantibodies coat red blood cells at body temperature and are recognized by Fc receptors on splenic and hepatic macrophages 1, 3
• Destruction occurs via antibody-dependent cellular cytotoxicity through macrophage-mediated phagocytosis, resulting in extravascular hemolysis 4, 2
• The spleen and lymphoid organs are the primary sites of red cell destruction 4
• Complement activation may occur but is typically less prominent than in cold AIHA 2

Cold AIHA (Cold Agglutinin Disease) Pathophysiology

• IgM autoantibodies bind to red blood cells at temperatures below 30°C, typically in peripheral circulation 1, 2
• These antibodies activate the complement cascade, leading to C3d deposition on red cell surfaces 1, 2
• Hemolysis occurs through two mechanisms: direct complement-mediated intravascular hemolysis and complement-coated red cell removal by hepatic macrophages 3, 2
• Cold agglutinins must have high thermal amplitude (active at >30°C) and high titer (≥1:64) to be clinically significant 2

Laboratory Tests to Differentiate Warm from Cold AIHA

Direct Antiglobulin Test (DAT/Coombs Test) - The Cornerstone

The DAT pattern is the single most important test to distinguish these entities: 1, 2

• Warm AIHA: DAT positive with anti-IgG antisera (typically strongly positive) 1, 2

  • May also show weak C3d positivity in some cases 2
  • The IgG pattern is the defining feature 1

• Cold AIHA: DAT positive with anti-C3d antisera (strongly positive) 1, 2

  • Anti-IgG is typically negative 2
  • May show weak IgM positivity, but IgM often elutes during washing steps 2

Cold Agglutinin Titer

• Essential for Cold AIHA diagnosis: Titer must be ≥1:64 at 4°C to be clinically significant 2
• Warm AIHA: Cold agglutinin titer is low or absent 5
• The titer should be performed at both 4°C and 37°C to assess thermal amplitude 2

Thermal Amplitude Testing

• Determines the highest temperature at which the antibody remains active 2, 5
• Cold AIHA: Antibodies active at ≥30°C are pathogenic 2
• Warm AIHA: Antibodies optimally reactive at 37°C 1, 5

Additional Distinguishing Laboratory Features

Immunoglobulin Pattern:

• Warm AIHA: Elevated polyclonal IgG (hypergammaglobulinemia may be present) 1
• Cold AIHA: May have monoclonal IgM spike on serum protein electrophoresis 2

Hemolysis Pattern:

• Warm AIHA: Predominantly extravascular hemolysis with elevated indirect bilirubin, low haptoglobin, elevated LDH, spherocytes on blood smear 3, 2
• Cold AIHA: May show intravascular hemolysis with hemoglobinuria, hemoglobinemia, and red cell agglutination on blood smear at room temperature 2

Critical Diagnostic Pitfalls

DAT-Negative AIHA

• Approximately 5% of AIHA cases are DAT-negative despite clinical hemolysis 1, 2
• Causes include: IgA autoantibodies, low-affinity IgG, warm IgM antibodies, or IgG below detection threshold 1, 2
• Requires specialized testing with anti-IgA antisera or more sensitive techniques 2

Atypical Presentations

• Warm IgM AIHA: IgM antibodies active at 37°C (rare variant) 2
• Mixed AIHA: Both warm IgG and cold IgM antibodies present simultaneously 2, 5
• IgA-mediated AIHA: Requires specific anti-IgA antisera for detection 1, 2

Technical Considerations

• Blood samples for cold agglutinin testing must be kept at 37°C until serum separation to prevent in vitro agglutination 2
• Room temperature handling can cause false-positive cold agglutinin results 2

Clinical Implications for Treatment Selection

The distinction between warm and cold AIHA is crucial because treatment strategies differ fundamentally: 5

• Warm AIHA: Corticosteroids are first-line therapy (effective in 70-85% of patients), followed by rituximab or splenectomy for refractory cases 1, 6, 5
• Cold AIHA: Corticosteroids have minimal efficacy; rituximab is the recommended first-line treatment 1, 6, 5
• Splenectomy is contraindicated in Cold AIHA but remains an option for warm AIHA 2, 5