What is the typical hemolytic (breakdown of red blood cells) workup?

Hemolytic Workup

The hemolytic workup should include: CBC with reticulocyte count, lactate dehydrogenase (LDH), unconjugated bilirubin, haptoglobin, direct antiglobulin test (DAT/Coombs), and peripheral blood smear—these tests confirm hemolysis and differentiate immune from non-immune causes. 1

Initial Laboratory Tests to Confirm Hemolysis

The first step is establishing that hemolysis is actually occurring:

• Reticulocyte count is typically elevated (>2%), indicating the bone marrow is responding appropriately to red cell destruction 1, 2
• Lactate dehydrogenase (LDH) is elevated due to release from lysed red cells 1, 3
• Unconjugated (indirect) bilirubin is increased from breakdown of hemoglobin 1, 2
• Haptoglobin is decreased or absent because it binds free hemoglobin released during hemolysis 1, 3
• Complete blood count (CBC) confirms anemia and determines if it is normocytic or macrocytic (hemolysis typically presents as normocytic or macrocytic) 1, 2

Common pitfall: Reticulocytosis may be absent or inadequate in 20-40% of autoimmune hemolytic anemia cases, particularly when there is concurrent bone marrow involvement, nutritional deficiency, or autoimmune reaction against marrow precursors—this is a poor prognostic sign. 3

Distinguishing Immune from Non-Immune Causes

Once hemolysis is confirmed, the next critical step is determining the mechanism:

• Direct antiglobulin test (DAT/Coombs test) is essential to distinguish immune-mediated hemolysis from non-immune causes 1, 2
• A positive DAT indicates antibodies or complement on red cell surfaces, suggesting autoimmune hemolytic anemia, drug-induced hemolysis, or transfusion reaction 2, 4
• A negative DAT points toward hereditary causes (membranopathies, enzymopathies, hemoglobinopathies) or non-immune extrinsic causes 5, 2

Peripheral Blood Smear Evaluation

Peripheral blood smear examination is mandatory as red cell morphology provides crucial diagnostic clues:

• Spherocytes suggest hereditary spherocytosis or autoimmune hemolytic anemia 1, 4
• Schistocytes (fragmented cells) indicate microangiopathic hemolytic anemia or mechanical trauma 2, 4
• Sickle cells point to sickle cell disease 4
• Bite cells or blister cells suggest G6PD deficiency with oxidative stress 2
• Echinocytes may be seen in pyruvate kinase deficiency, especially post-splenectomy 5

Important caveat: In pyruvate kinase deficiency and other enzymopathies, red cell morphology is often unremarkable with only nonspecific anisocytosis and poikilocytosis, making these conditions easy to miss. 5

Additional Testing Based on Initial Results

For Suspected Immune Hemolysis (DAT Positive):

• Evaluate for underlying autoimmune disorders, malignancies, or drug exposures 2, 4
• Consider warm vs. cold antibody testing based on clinical presentation 2

For Suspected Hereditary Hemolysis (DAT Negative):

• Red cell enzyme assays for pyruvate kinase deficiency and G6PD deficiency are essential 1
• Molecular/genetic testing for specific mutations when hereditary hemolytic anemia is suspected 1
• Osmotic fragility testing may be helpful but can be normal or altered in pyruvate kinase deficiency, limiting its utility 5

For Suspected Intravascular Hemolysis:

• Hemoglobinuria and hemosiderinuria indicate severe intravascular hemolysis 3, 6
• Flow cytometry for paroxysmal nocturnal hemoglobinuria (PNH) screening when intravascular hemolysis is present 1
• Marked LDH elevation is typical of intravascular hemolysis 3

Supplementary Studies

• Iron studies (serum ferritin, transferrin saturation) may be disproportionately elevated in chronic hemolysis, even without transfusion history 5, 1
• Vitamin B12 and folate levels rule out other causes of macrocytic anemia 1
• Bone marrow examination is indicated when there is suspicion of infiltrative disease, ineffective erythropoiesis, or unexplained reticulocytopenia 1

Critical Timing Considerations

Recent blood transfusion significantly affects test results, particularly the DAT and enzyme assays, potentially obscuring the diagnosis. 1 Ideally, obtain pre-transfusion samples when hemolysis is suspected, though this should not delay life-saving transfusion in severe cases.

Family History and Special Populations

• Obtain detailed family history to identify potential hereditary causes, as many enzymopathies and membranopathies follow autosomal recessive or dominant patterns 1
• In transfusion-dependent patients with undiagnosed anemia, consider rare enzymopathies like pyruvate kinase deficiency, which may present with features mimicking congenital dyserythropoietic anemias 5, 1