What lab studies should be ordered when suspecting hemolytic (breakdown of red blood cells) anemia?

Laboratory Studies for Hemolytic Anemia Evaluation

When suspecting hemolytic anemia, a comprehensive panel of laboratory tests should be ordered including complete blood count with peripheral smear, reticulocyte count, lactate dehydrogenase, haptoglobin, bilirubin, and direct antiglobulin test as the core diagnostic workup. 1, 2

First-Line Laboratory Tests

Essential Markers of Hemolysis

• Complete blood count (CBC) - To assess hemoglobin, hematocrit, RBC indices, and RBC morphology
• Reticulocyte count - Typically elevated in hemolysis (compensatory response)
• Lactate dehydrogenase (LDH) - Usually elevated due to RBC destruction
• Haptoglobin - Typically decreased or absent (binds to free hemoglobin)
• Bilirubin (total and indirect) - Elevated indirect (unconjugated) fraction
• Peripheral blood smear - To identify abnormal RBC morphologies (spherocytes, schistocytes, etc.)
• Direct antiglobulin test (DAT/Coombs test) - To differentiate immune from non-immune causes 1, 2, 3

Second-Line Laboratory Tests

For Immune-Mediated Hemolysis (if DAT positive)

• Antibody specificity testing - To identify specific autoantibodies
• Cold agglutinin titer - If cold-reactive antibodies are suspected
• Thermal amplitude testing - To determine the thermal range of antibody activity

For Non-Immune Hemolysis

• Hemoglobin electrophoresis - To identify hemoglobinopathies
• RBC enzyme assays - For suspected enzymopathies (e.g., pyruvate kinase deficiency)
• Osmotic fragility test - For suspected membrane disorders
• Flow cytometry for CD55/CD59 - To rule out paroxysmal nocturnal hemoglobinuria (PNH)
• ADAMTS13 activity - If thrombotic thrombocytopenic purpura (TTP) is suspected 1

Additional Tests Based on Clinical Context

For Suspected Thrombotic Microangiopathies

• Platelet count - Often decreased in TTP and hemolytic uremic syndrome (HUS)
• Coagulation studies - PT, PTT, fibrinogen
• Renal function tests - BUN, creatinine
• Stool testing for verocytotoxin-producing E. coli - If STEC-HUS is suspected 1

For Suspected Metabolic Causes

• Vitamin B12 and folate levels - To rule out megaloblastic anemia with ineffective erythropoiesis
• Iron studies - Ferritin, transferrin saturation to assess for concurrent iron deficiency 2

Important Diagnostic Considerations

1. Timing of sample collection is critical:

   • Collect samples before blood transfusions when possible
   • For enzyme assays, samples should be processed within specific timeframes (e.g., PK enzyme assay may be stable at 4°C for up to 21 days) 1

2. Interpretation pitfalls:

   • Normal LDH does not exclude hemolysis (present in up to 25% of AIHA cases) 4
   • Recent transfusions can affect test results (especially enzyme assays)
   • High reticulocyte count can mask abnormal RBC indices 1, 3

3. Diagnostic algorithm:

   • Any case of anemia plus thrombocytopenia should prompt immediate testing for haptoglobin, indirect bilirubin, and LDH 1
   • If schistocytes are present with thrombocytopenia, urgent ADAMTS13 testing is required to rule out TTP 1
   • The absence of schistocytes does not exclude early TMA 1

Special Situations

Refractory Hemolytic Anemia

In cases of severe refractory hemolytic anemia not responding to first-line treatments, additional testing may be required to guide therapy options such as plasma exchange, rituximab, or splenectomy 5.

Pediatric Considerations

In children with hemolytic anemia and diarrhea, testing for Shiga toxin-producing E. coli is essential, particularly when diarrhea and HUS appear concurrently or with a short interval between them 1.

By systematically ordering and interpreting these laboratory studies, clinicians can efficiently diagnose hemolytic anemia, determine its etiology, and initiate appropriate treatment to reduce morbidity and mortality associated with this condition.