How do you diagnose hemolytic anemia?

Diagnosis of Hemolytic Anemia

Confirm hemolysis first with laboratory markers—elevated reticulocyte count, elevated LDH, decreased or absent haptoglobin, and elevated indirect bilirubin—then perform a direct antiglobulin test (DAT) to differentiate immune from non-immune causes before any treatment is initiated. 1

Step 1: Confirm Hemolysis with Laboratory Markers

The initial diagnostic workup requires specific laboratory evidence of red cell destruction:

• Elevated reticulocyte count indicates compensatory bone marrow response to accelerated red cell destruction 1, 2
• Elevated lactate dehydrogenase (LDH) reflects intracellular enzyme release from lysed red cells 1, 3
• Decreased or absent haptoglobin is the most sensitive early indicator of hemolysis, as it binds free hemoglobin and becomes rapidly consumed 1, 4
• Elevated indirect (unconjugated) bilirubin results from increased heme catabolism 1, 2
• Peripheral blood smear examination identifies red blood cell morphology abnormalities that guide further testing 1, 3

Critical caveat: Haptoglobin provides the most sensitive indicator of active hemolysis and should be obtained in all suspected cases, as it becomes reduced early in hemolytic processes 4. The combination of reduced haptoglobin with elevated LDH provides the most sensitive and specific evidence of active hemolysis 4.

Step 2: Perform Direct Antiglobulin Test (DAT) Before Treatment

The DAT must be performed before initiating any treatment to differentiate immune from non-immune hemolysis. 1

If DAT Positive (Immune-Mediated):

• Evaluate for secondary causes including lymphoproliferative disorders, autoimmune diseases, infections, and drug-induced hemolysis 1
• Perform autoimmune workup (ANA, anti-dsDNA, ENA panel) if systemic autoimmune disease is suspected 1
• Consider indirect antiglobulin test to detect free autoantibodies in serum 1

If DAT Negative (Non-Immune):

Proceed to evaluate hereditary and acquired non-immune causes through the following algorithm:

Step 3: Evaluate Red Cell Morphology on Peripheral Smear

The peripheral blood smear guides the differential diagnosis:

• Spherocytes suggest hereditary spherocytosis; confirm with osmotic fragility testing or eosin-5-maleimide (EMA) binding test 1, 4
• Schistocytes indicate microangiopathic hemolytic anemia; obtain ADAMTS13 activity urgently if thrombotic microangiopathy suspected 1
• Normal or near-normal morphology with mild anisocytosis/poikilocytosis suggests enzymopathy, particularly pyruvate kinase deficiency 4, 5
• Bite cells or blister cells suggest G6PD deficiency 1

Step 4: Targeted Testing Based on Clinical Context

For Suspected Enzymopathies:

Pyruvate kinase (PK) activity by spectrophotometry is the reference method for PK deficiency, the most common glycolytic enzyme defect 1, 4. However, multiple confounders cause falsely normal results:

• Markedly elevated reticulocyte count (young red cells have higher PK activity) 1
• Recent blood transfusions (donor RBCs mask deficiency for 90-120 days) 1, 5
• Incomplete platelet/leukocyte removal from the sample 1
• Kinetically abnormal mutant enzymes that appear normal on standard assays 1

Critical pitfall: Always obtain transfusion history before enzyme testing, as donor red cells can cause false-negative results for up to 120 days post-transfusion 5. Wait 90-120 days after transfusion before performing enzyme assays 1.

G6PD enzyme activity should be measured if oxidative stress triggers (drugs, infections) are present 1

For Suspected Membranopathies:

• Osmotic fragility testing or EMA binding test for hereditary spherocytosis and elliptocytosis 1
• Family history is typically positive, and splenomegaly is common 4

Step 5: Molecular/Genetic Testing When Indicated

Next-generation sequencing (NGS) gene panels analyzing 68+ hemolytic anemia-related genes represent the gold standard for hereditary hemolytic anemias. 1

Molecular testing is particularly valuable when:

• Enzyme assays are equivocal or confounded by reticulocytosis/transfusions 1
• PKLR gene sequencing confirms PK deficiency definitively 1
• Common causes have been excluded but hemolysis persists 6

Step 6: Additional Diagnostic Tests for Specific Scenarios

If Thrombotic Microangiopathy Suspected:

• ADAMTS13 activity must be obtained urgently; severely deficient activity (<10 IU/dL) indicates thrombotic thrombocytopenic purpura (TTP) 1
• Stool culture for Shiga toxin-producing E. coli in children with diarrhea 1
• Serum electrolytes, BUN, creatinine to evaluate renal involvement 1

If Paroxysmal Nocturnal Hemoglobinuria (PNH) Suspected:

• PNH flow cytometry screening has 90% sensitivity and 95% specificity 1

Exclude Confounding Factors:

• Serum ferritin, transferrin saturation, iron studies exclude iron deficiency 1
• Vitamin B12 and folate levels rule out megaloblastic causes 1
• Liver function tests assess hepatic contribution to hyperbilirubinemia 1
• Thyroid-stimulating hormone excludes thyroid dysfunction 1

Essential Clinical History Elements

Document the following specific details:

• Neonatal jaundice history suggests hereditary hemolytic disorders 1
• Transfusion history and timing affects enzyme assay interpretation 1, 5
• Drug exposures for drug-induced hemolysis 1
• Family history of anemia prompts investigation for hereditary disorders 4
• Presence of splenomegaly essentially rules out simple nutritional deficiency and indicates chronic hemolysis with splenic sequestration 4

Important consideration: Reticulocyte count may not be as elevated as expected in hereditary hemolytic anemias before splenectomy, because younger defective cells are preferentially sequestered in the spleen, creating a falsely reassuring reticulocyte response 4.