What laboratory tests are indicated for a patient with suspected hemolytic anemia?

Laboratory Evaluation of Suspected Hemolytic Anemia

For any patient with suspected hemolytic anemia, immediately order: complete blood count with differential, reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, indirect bilirubin, peripheral blood smear, and direct antiglobulin test (DAT/Coombs). 1, 2

Initial Diagnostic Panel (First-Line Tests)

The following tests confirm hemolysis and guide further workup:

Core Hemolysis Markers

• Reticulocyte count: Elevated reticulocytes indicate bone marrow compensatory response to hemolysis 1, 2, 3
• LDH: Markedly elevated in hemolysis, particularly intravascular hemolysis 1, 2
• Haptoglobin: Decreased or absent; binds free hemoglobin released during hemolysis 1, 2
• Indirect (unconjugated) bilirubin: Elevated due to increased RBC breakdown 1, 2
• Peripheral blood smear: Essential to identify schistocytes, spherocytes, elliptocytes, and other RBC morphology abnormalities 1, 2

Critical Caveat on Schistocytes

Do not exclude hemolytic anemia based solely on absence of schistocytes, as this test has low sensitivity (particularly in early thrombotic microangiopathy) 4. The presence of anemia plus thrombocytopenia should always prompt immediate determination of haptoglobin, indirect bilirubin, and LDH levels 4.

Immune vs. Non-Immune Classification

Direct Antiglobulin Test (DAT/Coombs)

Perform DAT before initiating any treatment to differentiate immune from non-immune hemolysis 1, 5:

• DAT positive (IgG and/or C3d): Suggests autoimmune hemolytic anemia; proceed to evaluate for secondary causes including lymphoproliferative disorders, autoimmune diseases, infections, and drug-induced hemolysis 1, 5
• DAT negative: Consider hereditary causes (membranopathies, enzymopathies, hemoglobinopathies) or non-immune acquired causes 1, 5

Indirect Antiglobulin Test

• Detects free autoantibodies in serum 1

Additional First-Level Tests

When hemolysis is confirmed, obtain:

• Complete blood count with differential: Assess degree of anemia, thrombocytopenia (suggests thrombotic microangiopathy or Evans syndrome) 4, 2, 3
• Urinalysis: Detect hemoglobinuria/hemosiderinuria (indicates intravascular hemolysis) 2
• Serum electrolytes, BUN, creatinine: Assess renal involvement (critical for thrombotic microangiopathy diagnosis) 4

Second-Level Tests Based on DAT Results

If DAT Positive (Immune Hemolysis)

• Drug history: Comprehensive review of current and recent medications 5
• Autoimmune workup: ANA, anti-dsDNA, ENA panel if systemic autoimmune disease suspected 4
• Lymphoproliferative evaluation: Flow cytometry, lymph node imaging if indicated 5
• Infection screening: HIV, hepatitis panel, mycoplasma serology depending on clinical context 4, 5

If DAT Negative (Non-Immune Hemolysis)

For Hereditary Causes:

• Osmotic fragility test or EMA binding test: Diagnose hereditary spherocytosis and elliptocytosis 1
• Pyruvate kinase (PK) activity by spectrophotometry: Most common glycolytic enzymopathy; sensitivity 95%, specificity 98% 1, 3
  • Critical interpretation caveats: PK activity may be falsely normal with markedly elevated reticulocyte count, recent blood transfusions (wait minimum 90-120 days post-transfusion), incomplete platelet/leukocyte removal, or kinetically abnormal mutant enzymes 4, 1, 3
  • Reticulocytosis correction: Compare PK/hexokinase (HK) ratio to controls with similar reticulocyte counts 4
• G6PD enzyme activity: Diagnose glucose-6-phosphate dehydrogenase deficiency 1
• Hemoglobin electrophoresis: Exclude thalassemias and hemoglobinopathies 6

For Acquired Non-Immune Causes:

• ADAMTS13 activity: Must be obtained urgently if thrombotic microangiopathy suspected; severely deficient (<10 IU/dL) indicates thrombotic thrombocytopenic purpura (TTP) 4
• Stool culture for Shiga toxin-producing E. coli (STEC): Particularly in children with diarrhea; note that STEC-HUS typically appears 4-5 days after diarrhea onset, whereas concurrent diarrhea and HUS suggests atypical HUS 4
• PNH flow cytometry: Screen for paroxysmal nocturnal hemoglobinuria if clinical suspicion (sensitivity 90%, specificity 95%) 1

Advanced Molecular/Genetic Testing

Next-generation sequencing (NGS) gene panel analyzing 68+ hemolytic anemia-related genes is the gold standard for hereditary hemolytic anemias 1:

• PKLR gene sequencing: Confirms pyruvate kinase deficiency when enzyme assays are equivocal or confounded by reticulocytosis/transfusions 4, 1
• Comprehensive hemolytic anemia panel: Identifies membranopathies, enzymopathies, and rare hereditary causes 1
• DGKE, WT1, MMACHC genes: Consider in infants <1 year old with atypical HUS presentation 4

Essential Ancillary Studies

• Serum ferritin, transferrin saturation, iron studies: Exclude iron deficiency; assess for iron overload in chronic hemolysis 1, 3
• Vitamin B12 and folate levels: Rule out megaloblastic causes of macrocytic anemia 1
• Liver function tests: Assess hepatic contribution to hyperbilirubinemia 4
• Thyroid-stimulating hormone: Exclude thyroid dysfunction as contributing factor 4

Special Populations and Contexts

Pediatric Considerations

• Obtain detailed history of neonatal jaundice, transfusion history, and family history of hereditary conditions 1
• In first year of life, consider DGKE, WT1, and MMACHC gene mutations 4

Transfusion History

• Wait 90-120 days after last RBC transfusion before performing enzyme assays, as donor RBCs contribute normal enzyme activity and cause false-negative results 4, 1
• Mean erythrocyte loss is approximately 1%/day post-transfusion 4

Neurological Involvement

If neurological symptoms present (seizures, encephalopathy, motor symptoms, vision changes):

• EEG and brain MRI with FLAIR sequences: Bilateral symmetric hyperintensities of basal ganglia suggest thrombotic microangiopathy 4