Hemolytic Screen: Essential Laboratory Tests
A hemolytic screen is a panel of laboratory tests used to confirm the presence of hemolysis and includes reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, fractionated bilirubin, complete blood count (CBC) with differential, and peripheral blood smear as core components. 1, 2
Core Hemolysis Markers (First-Line Testing)
The American College of Pathologists identifies three essential tests to confirm hemolysis: 1
- Reticulocyte count – Elevated levels (typically >3-5%) indicate increased bone marrow production in response to red cell destruction 1
- Lactate dehydrogenase (LDH) – A sensitive marker released from lysed red blood cells 1, 2
- Haptoglobin – Binds free hemoglobin and becomes depleted during hemolysis; decreased or absent levels confirm hemolysis 1, 2
Complete Initial Workup
Beyond the core markers, the hemolytic screen includes: 1, 3
- Complete blood count (CBC) with differential – Assesses the degree of anemia and identifies white blood cell abnormalities 1, 2
- Fractionated bilirubin – Confirms predominantly unconjugated (indirect) hyperbilirubinemia characteristic of hemolysis 1, 2
- Peripheral blood smear – Identifies abnormal red cell morphology (schistocytes, spherocytes, etc.) that suggests specific causes 2, 3, 4
Critical Next Step: Immune vs. Non-Immune Differentiation
The direct antiglobulin test (DAT, also known as direct Coombs test) must be performed before initiating any treatment to differentiate immune from non-immune hemolysis. 2, 3, 4
- DAT-positive results suggest autoimmune hemolytic anemia and warrant antibody identification, cold agglutinin testing, or drug-induced antibody testing 3
- DAT-negative results prompt evaluation for hereditary causes (membranopathies, enzymopathies, hemoglobinopathies) or non-immune acquired causes (thrombotic microangiopathies, infections, oxidative stress) 2, 3
Critical Preanalytical Considerations
Proper specimen handling is crucial, as in vitro hemolysis causes spurious results in over 60% of cases. 1
- Use a straight needle rather than butterfly for collection 1
- Avoid tourniquet pressure during collection 1
- Process samples within 1 hour at room temperature 1
- Reject visibly hemolyzed samples to prevent false results 1
Second-Line Testing Based on Initial Results
If DAT is positive, evaluate for: 2, 3
- Secondary causes of autoimmune hemolytic anemia (lymphoproliferative disorders, autoimmune diseases, infections, drug-induced) 2
- Autoimmune serology (ANA, anti-dsDNA, ENA panel) if systemic disease suspected 2
- Paroxysmal nocturnal hemoglobinuria (PNH) flow cytometry screening 2, 3
If DAT is negative, consider: 1, 2
- G6PD enzyme assay for suspected enzyme deficiency, especially in patients of African, Mediterranean, or Asian descent 1
- Hemoglobin electrophoresis for suspected hemoglobinopathies or thalassemia 1
- Osmotic fragility or eosin-5-maleimide (EMA) binding test for suspected membrane defects (hereditary spherocytosis, elliptocytosis) 1, 2
- Pyruvate kinase (PK) enzyme activity for suspected enzymopathy 1, 2
- ADAMTS13 activity if thrombotic microangiopathy suspected 2
Important Interpretation Caveats
Enzyme assays should never be interpreted without knowing the reticulocyte count, as young red cells have higher enzyme activity and can mask deficiencies. 1, 2
- Wait a minimum of 50 days (preferably 90-120 days) from the last transfusion before performing enzyme assays, as donor RBC contamination affects results 1, 2
- Recent blood transfusions, incomplete platelet/leukocyte removal, and kinetically abnormal mutant enzymes can all cause falsely normal enzyme levels 2
Advanced Diagnostic Testing
When conventional testing is inconclusive: 2