What is the recommended initial workup for a patient with suspected hemolytic anemia?

Initial Workup for Suspected Hemolytic Anemia

Begin with a complete blood count (CBC) with differential, reticulocyte count, peripheral blood smear, lactate dehydrogenase (LDH), haptoglobin, indirect (unconjugated) bilirubin, and direct antiglobulin test (DAT/Coombs test) to confirm hemolysis and differentiate immune from non-immune causes. 1, 2, 3

Immediate Laboratory Tests (Minimum Workup)

The following tests should be ordered simultaneously when hemolytic anemia is suspected:

• CBC with differential and reticulocyte count: Assess degree of anemia, presence of thrombocytopenia (which suggests thrombotic microangiopathy), and reticulocytosis (confirms active hemolysis) 1, 3, 4

• Peripheral blood smear: Identify schistocytes (thrombotic microangiopathy), spherocytes (hereditary spherocytosis or autoimmune hemolytic anemia), sickle cells, or other morphologic abnormalities 1, 2, 3, 4

• LDH and haptoglobin: The combination of elevated LDH and decreased/absent haptoglobin is specific for hemolysis 2, 4, 5

• Indirect (unconjugated) bilirubin: Should be elevated in hemolysis 2, 4, 5

• Direct antiglobulin test (DAT/Coombs test): This is the single most critical test to distinguish immune from non-immune hemolysis and must be performed before initiating any treatment 3, 6, 4

Critical Additional Tests Based on Initial Findings

If Thrombocytopenia is Present (Anemia + Low Platelets)

This combination suggests life-threatening thrombotic microangiopathy and requires urgent additional testing:

• ADAMTS13 activity level: Must be obtained urgently; activity <10% indicates thrombotic thrombocytopenic purpura (TTP) requiring immediate plasma exchange 1, 3, 6

• Creatinine and BUN: Assess renal involvement to differentiate TTP from hemolytic uremic syndrome (HUS) 1, 3

• Stool culture for Shiga toxin-producing E. coli (STEC): Particularly in patients with diarrhea, as STEC-HUS typically appears 4-5 days after diarrhea onset 1

Critical pitfall: Schistocytes may be absent in early thrombotic microangiopathy, so their absence does not exclude the diagnosis 1, 3

If DAT is Positive (Immune Hemolysis)

• Indirect antiglobulin test: Detects free autoantibodies in serum 6

• Autoimmune workup: ANA, anti-dsDNA, ENA panel if systemic autoimmune disease is suspected 6

• Medication review: Evaluate for drug-induced hemolysis (cephalosporins, penicillins, NSAIDs, quinine/quinidine, rifampin, dapsone, fludarabine) 1, 2

• Infectious causes: Mycoplasma, viral serologies (cytomegalovirus, Epstein-Barr virus) 1

If DAT is Negative (Non-Immune Hemolysis)

Pursue hereditary causes and other non-immune etiologies:

• G6PD enzyme activity: Screen for glucose-6-phosphate dehydrogenase deficiency, especially in patients with oxidative stress triggers 1, 6

• Pyruvate kinase (PK) activity by spectrophotometry: Most common glycolytic enzyme defect; however, falsely normal levels occur with markedly elevated reticulocyte count, recent blood transfusions, or incomplete platelet/leukocyte removal 6

• Osmotic fragility testing or eosin-5-maleimide (EMA) binding test: For membranopathies such as hereditary spherocytosis 6

• Hemoglobin electrophoresis: Evaluate for hemoglobinopathies (sickle cell disease, thalassemia) 1, 4

• PNH flow cytometry screening: Consider in cases with clinical suspicion of paroxysmal nocturnal hemoglobinuria 1, 6

Extended Workup (When Cause Remains Unclear)

• Vitamin B12 and folate levels: Rule out megaloblastic causes of macrocytic anemia 1

• Iron studies (serum ferritin, transferrin saturation): Exclude iron deficiency as confounding factor 1

• Liver function tests: Assess hepatic contribution to hyperbilirubinemia 1

• Thyroid-stimulating hormone: Exclude thyroid dysfunction 1

• Protein electrophoresis and cryoglobulin analysis: Evaluate for paraprotein-related hemolysis 1

• Copper level: Part of bone marrow failure syndrome workup if refractory 1

• Next-generation sequencing (NGS) gene panel: Analyzing 68+ hemolytic anemia-related genes is the gold standard for hereditary hemolytic anemias when enzyme assays are equivocal 6

• Bone marrow biopsy with cytogenetic analysis: If no obvious cause identified and myelodysplastic syndrome is suspected 1

Key Diagnostic Pitfalls to Avoid

• LDH elevation alone is non-specific: Can occur in liver disease, myocardial infarction, kidney disease, and muscle damage; must be interpreted with haptoglobin and bilirubin 2

• Haptoglobin can be decreased in patients with mechanical heart valves without clinically relevant hemolysis 2

• Do not delay plasma exchange while waiting for ADAMTS13 results if TTP is strongly suspected clinically 3

• Wait 90-120 days after red blood cell transfusion before performing enzyme assays, as donor RBCs can cause false-negative results 6

• Reticulocyte count may be inappropriately low in early hemolysis, bone marrow suppression, or nutritional deficiencies (B12, folate, iron) 1