Laboratory Tests for Diagnosing Hemolytic Anemia
Begin with a complete blood count, reticulocyte count, peripheral blood smear, and markers of hemolysis (LDH, haptoglobin, indirect bilirubin), followed immediately by a direct antiglobulin test (DAT) to differentiate immune from non-immune causes before initiating any treatment. 1, 2
Initial First-Line Laboratory Panel
The diagnostic workup requires a systematic approach starting with these mandatory tests:
Core Hemolysis Markers
- Complete blood count (CBC) to document anemia and assess severity 1, 2, 3
- Reticulocyte count (usually elevated, indicating bone marrow response to hemolysis) 1, 2, 4
- Peripheral blood smear to identify RBC morphology abnormalities, schistocytes, spherocytes, or other characteristic findings 1, 2, 4
- Lactate dehydrogenase (LDH) (elevated in hemolysis, particularly intravascular) 1, 2, 4
- Haptoglobin (decreased or absent in hemolysis) 1, 2, 4
- Indirect (unconjugated) bilirubin (elevated) 1, 2, 4
Critical Pitfall
Do not dismiss thrombotic microangiopathy based solely on absence of schistocytes, as this finding has low sensitivity. 1, 2
Second-Line Testing: Immune vs. Non-Immune Classification
Direct Antiglobulin Test (DAT/Coombs Test)
- Perform DAT before initiating any treatment to differentiate immune-mediated from non-immune hemolysis 1, 2, 5
- Indirect antiglobulin test detects free autoantibodies in serum 1
If DAT Positive (Immune-Mediated)
Evaluate for secondary causes:
- Autoimmune serology (ANA, etc.) for underlying autoimmune diseases 2
- Lymphoproliferative disorder workup (ultrasound/CT, bone marrow evaluation if indicated) 1, 5
- Drug history for drug-induced hemolysis 1, 2
- Infection screening 1, 5
If DAT Negative (Non-Immune)
Pursue hereditary and acquired non-immune causes:
Hereditary Causes
- RBC enzyme assays for enzymopathies:
- Osmotic fragility testing or eosin-5-maleimide (EMA) binding test for membranopathies (hereditary spherocytosis, elliptocytosis) 1
- Hemoglobin electrophoresis to exclude hemoglobinopathies 3
Important Caveats for Enzyme Testing
Do not interpret PK activity results without considering these confounders that cause falsely normal levels: 6, 1
- Markedly elevated reticulocyte count
- Recent blood transfusions (interference from donor RBCs)
- Incomplete platelet/leukocyte removal
- Kinetically abnormal mutant enzymes
Advanced/Confirmatory Testing
Molecular/Genetic Testing
- Next-generation sequencing (NGS) gene panels analyzing 68+ hemolytic anemia-related genes is the gold standard for hereditary hemolytic anemias 1, 7
- PKLR gene sequencing (Sanger or NGS) to confirm PK deficiency, particularly when enzyme assays are equivocal 6, 1
- Genetic testing enables definitive diagnosis and genetic counseling 6, 7
Additional Tests for Specific Conditions
- Free hemoglobin and hemosiderinuria for intravascular hemolysis (e.g., paroxysmal nocturnal hemoglobinuria) 4
- Flow cytometry for CD55/CD59 if PNH suspected 2
- Serum ferritin (hyperferritinemia associated with chronic hemolysis) 4
Exclude Common Confounders
Before finalizing hemolytic anemia diagnosis, exclude:
- Iron deficiency (serum iron, TIBC, ferritin) 3, 8
- Vitamin B12 and folate deficiency 3
- Chronic kidney or liver disease 5
- Thalassemia (hemoglobin electrophoresis, RBC distribution width) 3, 8
Clinical Context Requirements
Always obtain:
- Clinical history including neonatal jaundice, transfusion history, drug exposures 6
- Family history for hereditary conditions 6
- Timing of last blood transfusion (affects enzyme assay interpretation) 6
Special Consideration for Reticulocytopenia
If reticulocyte count is inappropriately low despite hemolysis (occurs in 20-40% of autoimmune hemolytic anemia cases), this indicates poor prognosis and suggests: 4
- Bone marrow involvement
- Concurrent iron/vitamin deficiency
- Infection
- Autoimmune reaction against bone marrow precursors