What labs should be sent in cases of suspected hemolysis?

Laboratory Testing for Suspected Hemolysis

When hemolysis is suspected, immediately order a complete blood count with differential, reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, and fractionated bilirubin as your core diagnostic panel. 1, 2

Essential First-Line Laboratory Tests

The following tests confirm hemolysis and should be ordered together as an initial panel:

• Complete blood count (CBC) with differential and red cell indices to assess the degree of anemia, mean corpuscular volume, and identify any white blood cell abnormalities 1, 2
• Reticulocyte count to confirm increased bone marrow production in response to red cell destruction (typically >3-5% in hemolytic conditions) 1, 2
• Lactate dehydrogenase (LDH) as a sensitive marker released from lysed red blood cells (elevated in hemolysis) 1, 3
• Haptoglobin which binds free hemoglobin and becomes depleted during hemolysis (decreased levels confirm hemolysis) 1, 3
• Fractionated bilirubin to confirm predominantly unconjugated hyperbilirubinemia 1, 2
• Peripheral blood smear to identify abnormal red cell morphology such as schistocytes (microangiopathic hemolytic anemia), spherocytes (hereditary spherocytosis), or other diagnostic findings 2, 3

Critical Second Test: Direct Antiglobulin Test (DAT)

• Direct antiglobulin test (DAT/Coombs test) to differentiate immune-mediated from non-immune causes of hemolysis 2, 3

This single test determines your entire subsequent diagnostic pathway.

Additional Testing Based on DAT Results

If DAT is Positive (Immune-Mediated Hemolysis):

• Antibody identification panel to determine specific antibodies present 2
• Cold agglutinin testing if cold agglutinin disease is suspected 2
• Drug-induced antibody testing if medication exposure is relevant 2
• Autoimmune serology to evaluate for underlying autoimmune conditions 2
• PNH screening by flow cytometry (CD55/CD59 expression) for paroxysmal nocturnal hemoglobinuria 2

If DAT is Negative (Non-Immune Hemolysis):

• ADAMTS13 activity and inhibitor titer to evaluate for thrombotic thrombocytopenic purpura (TTP) 2
• G6PD enzyme assay for suspected glucose-6-phosphate dehydrogenase deficiency, especially in patients of African, Mediterranean, or Asian descent 1, 2
• Hemoglobin electrophoresis for suspected hemoglobinopathies or thalassemia 1
• Osmotic fragility test or eosin-5-maleimide (EMA) binding test for suspected hereditary spherocytosis or other membrane defects 1
• Pyruvate kinase enzyme activity if enzymopathy is suspected, followed by PKLR gene sequencing if decreased 4, 2

Critical Preanalytical Considerations to Avoid False Results

Proper specimen handling is crucial because in vitro hemolysis causes spurious results in over 60% of rejected samples. 1, 5

To prevent false hemolysis:

• Use a straight needle rather than butterfly needle during collection 1
• Avoid excessive tourniquet pressure during blood draw 1
• Process samples within 1 hour at room temperature 1
• Reject visibly hemolyzed samples and recollect to prevent spurious results 1

Special Circumstances Requiring Additional Testing

• If transfusion history exists: Wait minimum 50 days from last transfusion before performing enzyme assays, as donor RBC contamination affects results 4
• If severe intravascular hemolysis is suspected: Add free hemoglobin, hemopexin, hemoglobinuria, and hemosiderinuria to your panel 3, 6
• If neonatal hemolysis: Consider blood type incompatibility testing and expanded enzyme deficiency screening 4

Common Pitfalls to Avoid

• Do not assume low hemoglobin alone indicates hemolysis—you must demonstrate both red cell destruction (elevated LDH, low haptoglobin, elevated unconjugated bilirubin) AND increased production (reticulocytosis) 3, 6
• Do not interpret enzyme assays without knowing the reticulocyte count, as young red cells have higher enzyme activity and can mask deficiencies 4
• Do not order hemoglobin electrophoresis before checking ferritin—iron deficiency can mask thalassemia patterns 7