Initial Workup for Hemolytic Anemia
Begin with a complete blood count, peripheral blood smear, reticulocyte count, lactate dehydrogenase, haptoglobin, indirect bilirubin, and direct antiglobulin test (Coombs test) to confirm hemolysis and differentiate immune from non-immune causes. 1
Essential First-Line Laboratory Tests
The minimum diagnostic workup must include:
- Complete blood count (CBC) with red blood cell indices (MCV, RDW) to assess anemia severity and red cell characteristics 1
- Peripheral blood smear to identify schistocytes (suggesting microangiopathic hemolysis), spherocytes (autoimmune or hereditary spherocytosis), or other morphologic abnormalities 1, 2
- Reticulocyte count to assess bone marrow compensatory response—elevated in hemolysis unless marrow involvement, nutritional deficiency, or autoimmune suppression is present 1, 2
- Lactate dehydrogenase (LDH) as a marker of intravascular hemolysis, though note that 25% of autoimmune hemolytic anemia cases may have normal LDH 3, 2
- Haptoglobin (decreased in hemolysis due to binding free hemoglobin) 1, 2
- Indirect (unconjugated) bilirubin (elevated from red cell breakdown) 1, 2
- Direct antiglobulin test (DAT/Coombs test) with monospecific antisera—this is the cornerstone test to differentiate autoimmune hemolytic anemia from other causes 1, 4, 5
Critical History and Physical Examination Elements
Obtain specific information about:
- Drug exposure history: ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin 1
- Recent infections: mycoplasma, viral pathogens that can trigger hemolysis 1
- Insect, spider, or snake bites 1
- Family history of anemia, jaundice, splenomegaly, or early cholecystectomy (suggests hereditary hemolytic conditions) 1
- Neonatal jaundice history (suggests congenital hemolytic disorders) 1
- Physical findings: jaundice, splenomegaly, pallor, dark urine 1
Second-Tier Diagnostic Tests Based on Initial Results
If DAT is Positive (Autoimmune Hemolytic Anemia)
- Autoimmune serology to evaluate for underlying systemic autoimmune disease 1
- Protein electrophoresis and cryoglobulin analysis to exclude lymphoproliferative disorders 1
- Immunoglobulin levels if frequent infections or suspected immunodeficiency 4
- Imaging (ultrasound/CT) and bone marrow evaluation if lymphoproliferative disorder suspected 4
If DAT is Negative
Pursue non-immune causes:
- Glucose-6-phosphate dehydrogenase (G6PD) assay for enzyme deficiency 1
- Paroxysmal nocturnal hemoglobinuria (PNH) screening by flow cytometry 1
- Hemoglobin electrophoresis for hemoglobinopathies 1
- Osmotic fragility or eosin-5-maleimide binding test for hereditary spherocytosis 2
- DIC panel (PT/INR, fibrinogen, D-dimer) if microangiopathic features present 1
- ADAMTS13 activity if thrombotic thrombocytopenic purpura suspected (should be available urgently) 1
If Refractory or Unclear Etiology
- Bone marrow analysis with cytogenetic studies to evaluate for myelodysplastic syndromes or marrow failure 1
- Vitamin B12, folate, copper levels to exclude nutritional causes of inadequate reticulocyte response 1
- Parvovirus B19 testing if reticulocytopenia present 1
- Iron studies, thyroid function as part of comprehensive evaluation 1
- Methemoglobin level if cyanosis or oxidative drug exposure 1
Common Pitfalls to Avoid
Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and represents a poor prognostic factor—do not exclude hemolysis based on low reticulocyte count alone 2. Consider marrow involvement, nutritional deficiencies, infections, or autoimmune suppression of erythroid precursors 1, 2.
Normal LDH does not exclude hemolytic anemia—approximately 25% of autoimmune hemolytic anemia cases present with normal LDH levels 3. Rely on the complete constellation of findings including haptoglobin, bilirubin, and peripheral smear 3, 2.
DAT-negative autoimmune hemolytic anemia exists in approximately 5-10% of cases 4. Diagnosis requires exclusion of other hemolytic causes and may be supported by response to corticosteroids 4.
Thrombocytopenia accompanying hemolytic anemia suggests thrombotic microangiopathy (TTP, HUS, aHUS) or Evans syndrome—requires urgent evaluation with ADAMTS13 and consideration of complement-mediated TMA 1, 2.