What laboratory tests should be used to evaluate for hemolytic anemia?

Laboratory Evaluation for Hemolytic Anemia

The essential laboratory tests for evaluating hemolytic anemia include complete blood count (CBC) with peripheral blood smear, reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, bilirubin (total and indirect), and direct antiglobulin test (DAT/Coombs test). 1

Initial Laboratory Workup

First-line Tests

• Complete Blood Count (CBC) with differential and red cell indices

  • Provides hemoglobin level, mean corpuscular volume (MCV), and red cell morphology
  • Helps categorize anemia as microcytic, normocytic, or macrocytic 2

• Peripheral Blood Smear Examination

  • Essential for identifying morphological abnormalities
  • May show spherocytes, fragmented cells, or other abnormal RBC forms 3

• Reticulocyte Count

  • Elevated in hemolytic anemia (compensatory bone marrow response)
  • Reticulocytopenia in 20-40% of autoimmune hemolytic anemia cases indicates poor prognosis 4

Markers of Hemolysis

• Lactate Dehydrogenase (LDH)

  • Elevated in hemolysis, particularly marked increase in intravascular hemolysis 4

• Haptoglobin

  • Reduced or absent in hemolysis as it binds free hemoglobin 4

• Bilirubin

  • Elevated unconjugated (indirect) bilirubin 3

• Direct Antiglobulin Test (DAT/Coombs test)

  • Cornerstone for diagnosing autoimmune hemolytic anemia
  • Differentiates between warm (IgG) and cold (C3d) antibody types 5

Additional Specialized Tests

For Suspected Intravascular Hemolysis

• Hemosiderinuria
• Plasma free hemoglobin
• Serum ferritin (often elevated in chronic hemolysis) 4

For Suspected Enzyme Deficiencies

• Enzyme activity assays (e.g., PK enzyme activity for pyruvate kinase deficiency)
• Molecular testing (e.g., PKLR gene sequencing) 3

For Suspected Membrane Disorders

• Osmotic fragility test
• Ektacytometry 2

Diagnostic Algorithm

1. Confirm hemolysis with elevated reticulocytes, LDH, indirect bilirubin, and decreased haptoglobin 4

2. Determine if hemolysis is immune or non-immune:

   • Perform DAT (Coombs test)
   • If DAT positive → Autoimmune hemolytic anemia (AIHA)
   • If DAT negative → Consider non-immune causes 6

3. For DAT-positive cases, characterize the type:

   • IgG positive only → Warm AIHA
   • C3d positive only → Cold agglutinin disease or paroxysmal cold hemoglobinuria
   • Both IgG and C3d positive → Mixed AIHA 5

4. For DAT-negative cases, evaluate:

   • Blood smear for fragmented cells → Consider microangiopathic hemolytic anemia
   • Family history → Consider hereditary disorders
   • Medication history → Consider drug-induced hemolysis
   • Flow cytometry for CD55/CD59 → Consider paroxysmal nocturnal hemoglobinuria 3, 4

Important Considerations and Pitfalls

• Timing of sample collection is critical, especially for cold agglutinin disease (samples must be kept warm) 5

• Recent transfusions can interfere with test results, particularly enzyme assays and DAT 3

• Reticulocytosis may be inadequate/absent in cases of:

  • Bone marrow involvement
  • Iron/vitamin deficiency
  • Infections
  • Autoimmune reaction against bone marrow precursors 4

• False normal levels in enzyme assays can occur due to:

  • Markedly increased reticulocytes
  • Interference from normal donor red cells in recently transfused patients
  • Incomplete platelet and leukocyte removal 3

• Elevated markers like LDH, bilirubin, and reduced haptoglobin can be seen in conditions other than hemolysis, requiring careful interpretation 4

Follow-up Testing

• Repeat CBC in 2-4 weeks to assess response to interventions 1
• Monitor hemoglobin and iron studies monthly 1
• Consider hematology referral for persistent unexplained hemolysis 1

By systematically applying this laboratory approach, clinicians can effectively diagnose the specific cause of hemolytic anemia and guide appropriate treatment decisions to improve patient outcomes.