What tests are used to diagnose hemolysis?

Diagnostic Tests for Hemolysis

The diagnosis of hemolysis requires a combination of laboratory tests including complete blood count with reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, bilirubin, and peripheral blood smear examination. 1

Core Diagnostic Tests for Hemolysis

First-Line Tests

• Complete blood count (CBC) - To assess anemia and red cell indices
• Reticulocyte count - Typically elevated in hemolysis as bone marrow compensates
• Lactate dehydrogenase (LDH) - Elevated in hemolysis, particularly intravascular hemolysis
• Haptoglobin - Decreased or absent in hemolysis as it binds free hemoglobin
• Bilirubin (direct and indirect) - Unconjugated (indirect) bilirubin is elevated
• Peripheral blood smear - To identify abnormal red cell morphology

Second-Line Tests

• Direct antiglobulin test (DAT/Coombs test) - To differentiate immune from non-immune causes
• Hemoglobinuria - Present in severe intravascular hemolysis
• Hemosiderinuria - Indicates chronic intravascular hemolysis

Diagnostic Algorithm for Hemolysis

1. Confirm hemolysis with core laboratory markers:

   • Decreased hemoglobin
   • Elevated reticulocyte count
   • Elevated LDH
   • Decreased haptoglobin
   • Elevated unconjugated bilirubin

2. Determine if hemolysis is intravascular or extravascular:

   • Intravascular: Marked elevation of LDH, hemoglobinemia, hemoglobinuria, hemosiderinuria
   • Extravascular: More modest LDH elevation, no hemoglobinuria

3. Determine if hemolysis is immune or non-immune:

   • Perform direct antiglobulin test (DAT)
   • Positive DAT suggests immune-mediated hemolysis
   • Negative DAT suggests non-immune causes

4. Examine peripheral blood smear for specific morphological abnormalities:

   • Spherocytes: Hereditary spherocytosis or immune hemolysis
   • Schistocytes: Microangiopathic hemolytic anemia
   • Sickle cells: Sickle cell disease
   • Bite cells: G6PD deficiency

Additional Tests Based on Clinical Context

For Suspected Immune Hemolysis

• Cold agglutinin titer
• Thermal amplitude testing
• Drug-dependent antibody testing

For Suspected Hereditary Hemolytic Anemia

• Hemoglobin electrophoresis (hemoglobinopathies)
• G6PD enzyme activity (G6PD deficiency)
• Osmotic fragility test (hereditary spherocytosis)
• Eosin-5-maleimide binding test (hereditary spherocytosis)

For Suspected Microangiopathic Hemolytic Anemia

• ADAMTS13 activity (TTP)
• Coagulation studies
• Complement testing (atypical HUS)

For Suspected Paroxysmal Nocturnal Hemoglobinuria

• Flow cytometry for GPI-anchored proteins

Clinical Pitfalls and Caveats

1. Reticulocyte count may be falsely low despite hemolysis in cases of:

   • Bone marrow suppression
   • Nutritional deficiencies (iron, B12, folate)
   • Infection
   • Autoimmune reaction against bone marrow precursors 2

2. Haptoglobin may be low in conditions other than hemolysis:

   • Liver disease
   • Congenital haptoglobin deficiency

3. LDH elevation is nonspecific and can occur in:

   • Liver disease
   • Myocardial infarction
   • Malignancy
   • Muscle trauma

4. Recent transfusion may mask laboratory evidence of hemolysis

5. Timing matters for hemodialysis patients - hemoglobin should be measured pre-dialysis, preferably before mid-week session 1

By systematically applying these diagnostic tests and considering the clinical context, the cause of hemolysis can be accurately identified, allowing for appropriate management to reduce morbidity and mortality associated with hemolytic conditions.