What laboratory tests are used to diagnose and manage hemolysis (breakdown of red blood cells) in patients?

Laboratory Evaluation of Hemolysis

Core Diagnostic Tests

The diagnosis of hemolysis requires a combination of laboratory markers demonstrating red blood cell destruction and compensatory bone marrow response. 1, 2

Essential First-Line Tests

• Complete blood count (CBC) with differential and reticulocyte count to assess anemia severity and bone marrow response 3, 4, 5
• Peripheral blood smear to identify schistocytes (microangiopathic hemolysis), spherocytes (immune or membrane defects), or other diagnostic red cell morphologies 3, 4, 6, 1
• Lactate dehydrogenase (LDH) - elevated in hemolysis, particularly intravascular hemolysis; the combination of elevated LDH with decreased haptoglobin has 80% positive predictive value for hemolysis 4, 6, 1, 2
• Haptoglobin - decreased or undetectable in hemolysis as it binds free hemoglobin released from lysed red cells 3, 4, 6, 1
• Indirect (unconjugated) bilirubin - elevated due to breakdown of hemoglobin 3, 5, 1, 2
• Direct antiglobulin test (DAT/Coombs test) - differentiates immune-mediated from non-immune hemolysis with 90% sensitivity and 95% specificity 5, 6, 1, 2

Markers of Intravascular Hemolysis

When hemolysis occurs within blood vessels (rather than in the spleen/liver), additional markers become positive:

• Free hemoglobin in plasma (hemoglobinemia) - indicates severe intravascular hemolysis 1, 7
• Hemoglobinuria - dark-colored urine from filtered hemoglobin 3, 1, 7
• Hemosiderinuria - indicates chronic intravascular hemolysis 7, 2
• Decreased hemopexin - another hemoglobin-binding protein depleted in intravascular hemolysis 7

Algorithmic Approach to Laboratory Interpretation

Step 1: Confirm Hemolysis is Present

Look for the classic triad:

• Anemia with elevated reticulocyte count (typically >2.5%, or reticulocyte index >1.3) indicating bone marrow compensation 4, 1, 7
• Elevated LDH (often >500 U/L in significant hemolysis) 3, 4, 2
• Decreased haptoglobin (<25 mg/dL or undetectable) 3, 4, 6

Critical pitfall: Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases due to marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow precursors, and represents a poor prognostic factor. 2 Do not exclude hemolysis based solely on low reticulocyte count.

Step 2: Assess for Thrombotic Microangiopathy (TMA)

If thrombocytopenia is present alongside hemolysis, immediately evaluate for TMA as this is a medical emergency:

• Order ADAMTS13 activity level and inhibitor titer urgently - do not delay plasma exchange while awaiting results if TTP is strongly suspected clinically 3, 4, 6
• Review peripheral smear for schistocytes - presence of schistocytes >1% strongly supports TMA, though absence does not exclude early TMA due to low test sensitivity 4, 6
• Check platelet count, creatinine, and urinalysis for the TMA triad: microangiopathic hemolysis, thrombocytopenia, and organ (typically renal) involvement 4, 5
• Obtain prothrombin time, activated PTT, and fibrinogen to exclude disseminated intravascular coagulation (DIC) 3, 4

If ADAMTS13 activity <10%: Diagnosis is thrombotic thrombocytopenic purpura (TTP) - initiate therapeutic plasma exchange immediately and administer methylprednisolone 1g IV daily for 3 days. 3, 4 Mortality increases with delayed treatment.

If ADAMTS13 activity >10% with TMA: Consider atypical hemolytic uremic syndrome (aHUS) - begin eculizumab therapy urgently without waiting for genetic confirmation, and test complement levels (C3, C4, CH50). 4, 5, 6

Step 3: Differentiate Immune from Non-Immune Causes

Direct antiglobulin test (Coombs) is the cornerstone for this differentiation: 2

If DAT positive (immune-mediated):

• Autoimmune hemolytic anemia is confirmed 5, 1
• Consider drug-induced hemolytic anemia - evaluate common causative drugs (ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin) 3
• Assess for delayed hemolytic transfusion reaction if recent transfusion history 3

If DAT negative (non-immune):

• Examine peripheral smear morphology carefully - this is fundamental for diagnosis: 1, 2
  • Schistocytes → thrombotic microangiopathy 4, 1
  • Spherocytes → hereditary spherocytosis or membrane defects 1, 2
  • Bite cells/blister cells → G6PD deficiency 1
  • Sickle cells → sickle cell disease 1
• Screen for paroxysmal nocturnal hemoglobinuria (PNH) with flow cytometry 3
• Test glucose-6-phosphate dehydrogenase (G6PD) levels 3
• Consider red cell enzyme defects - measure pyruvate kinase activity if indicated 3

Step 4: Additional Context-Specific Testing

For suspected hereditary causes:

• Osmotic fragility testing for hereditary spherocytosis 1
• Hemoglobin electrophoresis for hemoglobinopathies 3
• Red cell enzyme assays (pyruvate kinase, G6PD) 3

For suspected infectious causes:

• Evaluate for viral/bacterial causes including mycoplasma 3
• Blood cultures if sepsis suspected 3

For chronic hemolysis:

• Ferritin levels - hyperferritinemia is associated with chronic hemolysis 2
• Protein electrophoresis and cryoglobulin analysis 3

For bone marrow evaluation (if refractory or unclear):

• B12, folate, copper levels 3
• Bone marrow biopsy with cytogenetic analysis to evaluate for myelodysplastic syndromes 3

Monitoring During Treatment

• Daily CBC with differential, LDH, haptoglobin, and creatinine during acute phase of TMA 4
• Weekly hemoglobin monitoring during steroid tapering for immune-mediated hemolysis 4, 6
• Serial monitoring of hemoglobin, hematocrit, LDH, and haptoglobin to assess disease progression 5, 6

Critical Pitfalls to Avoid

Do not dismiss TMA based on "rare" schistocytes alone - low schistocyte counts can occur in early or evolving TMA, and the test has low sensitivity. 4 Clinical context and other laboratory markers must guide decision-making.

Do not transfuse aggressively - RBC transfusion should only relieve symptoms or achieve hemoglobin 7-8 g/dL in stable, non-cardiac inpatients. 3, 4, 5 In TTP, platelet transfusion is generally contraindicated unless life-threatening bleeding. 4

Do not ignore confounding conditions - elevated LDH, bilirubin, and reticulocytes can occur in conditions other than hemolysis (liver disease, tissue injury, ineffective erythropoiesis), requiring comprehensive clinical correlation. 2

Recognize that haptoglobin phenotype (Hp 1-1) predicts higher risk of anemia in certain contexts like hepatitis C treatment, where hemolytic anemia with haptoglobin degradation is expected. 6