Hemolysis

Overview of Hemolytic Indicators

Definition: Laboratory tests used to identify and quantify the presence of hemolysis (RBC destruction) in vivo (within the body)
Clinical Significance:
- Diagnosis of Hemolytic Anemia: Helps confirm the presence of hemolytic anemia and distinguish it from other causes of anemia
- Assessment of Hemolysis Severity: Helps evaluate the degree of RBC destruction
- Differentiation of Hemolytic Anemia Types: Helps differentiate between intravascular and extravascular hemolysis
- Monitoring Response to Therapy: Used to assess the effectiveness of treatment for hemolytic anemia
Key Indicators:
- Haptoglobin
- Lactate Dehydrogenase (LDH)
- Unconjugated (Indirect) Bilirubin
- Urine Hemoglobin and Hemosiderin
Methods: These tests are typically performed using spectrophotometric or immunochemical methods on serum, plasma, or urine samples

Haptoglobin

Definition: An alpha-2 globulin protein synthesized in the liver that binds free hemoglobin (Hb) released into the plasma
Function:
- Binding to Free Hemoglobin: Haptoglobin binds to free hemoglobin, forming a stable hemoglobin-haptoglobin complex
- Clearance of Hemoglobin: The hemoglobin-haptoglobin complex is rapidly cleared from the circulation by the liver via the CD163 receptor on macrophages
- Prevents Kidney Damage: Binding to haptoglobin prevents free hemoglobin from being filtered by the kidneys, which can cause kidney damage
- Antioxidant Activity: Haptoglobin has antioxidant properties, protecting tissues from oxidative damage caused by free hemoglobin
Measurement:
- Immunoturbidimetric Assay: The most common method
  - Antibodies specific to haptoglobin are added to the sample
  - The antibodies bind to haptoglobin, forming immune complexes
  - The turbidity (cloudiness) of the solution is measured, which is proportional to the haptoglobin concentration
- Nephelometry: Similar to immunoturbidimetry, but measures the light scattered by the immune complexes
Reference Range:
- Varies depending on the laboratory and the assay used
- Typical reference range: 30-200 mg/dL (adults)
Interpretation:
- Decreased Haptoglobin:
  - Increased hemolysis: Haptoglobin is consumed as it binds to free hemoglobin
  - Liver disease: Impaired haptoglobin synthesis
  - Congenital haptoglobin deficiency
- Elevated Haptoglobin:
  - Acute phase reaction: Haptoglobin is an acute phase reactant, so levels can increase during inflammation or infection
  - Corticosteroid use

Lactate Dehydrogenase (LDH)

Definition: An intracellular enzyme present in many tissues, including red blood cells, liver, muscle, and heart
Function: Catalyzes the interconversion of lactate and pyruvate
Isoenzymes: LDH exists as five isoenzymes (LDH1-LDH5), each with a different tissue distribution:
- LDH1: Heart, red blood cells
- LDH2: Heart, red blood cells
- LDH3: Lungs, lymphocytes
- LDH4: Liver, muscle
- LDH5: Liver, skeletal muscle
Measurement:
- Enzymatic Assay: Measures the rate of conversion of lactate to pyruvate or pyruvate to lactate
- Spectrophotometry: The change in absorbance of NADH (nicotinamide adenine dinucleotide) is measured at a specific wavelength
Reference Range:
- Varies depending on the laboratory and the assay used
- Typical reference range: 100-225 U/L (adults)
Interpretation:
- Elevated LDH:
  - Hemolysis: LDH is released from damaged red blood cells
  - Tissue Injury: LDH is released from damaged tissues (e.g., heart, liver, muscle)
  - Malignancy: Some cancers can cause elevated LDH levels
  - Pulmonary Embolism
- Isoenzyme Analysis (Less Common): May be performed to identify the source of LDH elevation
  - Elevated LDH1 and LDH2: Suggests cardiac or RBC damage
  - Elevated LDH5: Suggests liver or muscle damage

Unconjugated (Indirect) Bilirubin

Definition: A yellow pigment formed from the breakdown of heme (the iron-containing portion of hemoglobin)
Formation and Metabolism:
1. Heme is converted to biliverdin by heme oxygenase
2. Biliverdin is converted to unconjugated bilirubin by biliverdin reductase
3. Unconjugated bilirubin is transported in the blood bound to albumin
4. In the liver, unconjugated bilirubin is conjugated with glucuronic acid by UDP-glucuronosyltransferase (UGT)
5. Conjugated bilirubin is excreted into the bile and then into the intestines
6. In the intestines, bilirubin is converted to urobilinogen by bacteria
7. Urobilinogen is either:
  - Excreted in the feces (as stercobilin, which gives feces its brown color)
  - Reabsorbed into the bloodstream and excreted in the urine (as urobilin, which gives urine its yellow color)
Measurement:
- Diazo Reaction: Bilirubin reacts with a diazo reagent to form a colored product
- Spectrophotometry: The intensity of the color is measured at a specific wavelength
- Direct Bilirubin: Reacts directly with the diazo reagent
- Total Bilirubin: Requires the addition of an accelerator (e.g., methanol) to react with both conjugated and unconjugated bilirubin
- Indirect Bilirubin: Calculated by subtracting the direct bilirubin from the total bilirubin (Indirect = Total - Direct)
Reference Range:
- Varies depending on the laboratory and the assay used
- Typical reference range:
  - Total Bilirubin: 0.3-1.0 mg/dL
  - Direct Bilirubin: 0.0-0.3 mg/dL
  - Indirect Bilirubin: 0.2-0.8 mg/dL
Interpretation:
- Elevated Unconjugated (Indirect) Bilirubin:
  - Increased bilirubin production due to hemolysis
  - Impaired bilirubin uptake or conjugation by the liver
  - Examples: Hemolytic anemia, Gilbert syndrome, Crigler-Najjar syndrome
- Elevated Conjugated (Direct) Bilirubin:
  - Impaired excretion of conjugated bilirubin from the liver
  - Biliary obstruction (e.g., gallstones, tumor)
  - Liver disease (e.g., hepatitis, cirrhosis)

Urine Hemoglobin and Hemosiderin

Urine Hemoglobin:
- Normally, there is very little or no hemoglobin in the urine
- In intravascular hemolysis, free hemoglobin is released into the plasma
- Haptoglobin becomes saturated, free hemoglobin is filtered by the kidneys
- Some hemoglobin is reabsorbed by the renal tubules
- Excess hemoglobin is excreted in the urine (hemoglobinuria), causing the urine to appear red or brown
- Measured using a urine dipstick test
- False-positive results can occur with myoglobinuria or certain medications
Urine Hemosiderin:
- Hemosiderin is an iron-storage complex derived from hemoglobin
- In chronic intravascular hemolysis, the renal tubular cells reabsorb hemoglobin and store it as hemosiderin
- Hemosiderin is eventually sloughed off into the urine (hemosiderinuria)
- Urine hemosiderin can be detected using a Prussian blue stain on a urine sediment sample

Summary Table of Hemolytic Indicators

Test	Principle	Expected Result in Hemolysis	Notes
Haptoglobin	Binds free hemoglobin	Decreased	Decreased in both intravascular and extravascular hemolysis; also decreased in liver disease
LDH	Intracellular enzyme released with cell lysis	Elevated	Elevated in both intravascular and extravascular hemolysis; also elevated in tissue injury and malignancy
Unconjugated Bilirubin	Breakdown product of heme	Elevated	Elevated in both intravascular and extravascular hemolysis; may also be elevated in liver disorders
Urine Hemoglobin	Detects free hemoglobin in urine	Positive (in intravascular hemolysis)	Only positive in intravascular hemolysis; may have false positives due to myoglobin or other interfering substances
Urine Hemosiderin	Detects iron in urine	Positive (in chronic intravascular hemolysis)	Seen in chronic intravascular hemolysis; takes time to develop

Differential Diagnosis of Hemolytic Anemias

Hemolytic Anemia:
- Elevated Reticulocyte Count: Increased bone marrow response
- Elevated Indirect Bilirubin and LDH: Release of intracellular contents
- Decreased Haptoglobin: Consumption by free hemoglobin
Extravascular Hemolysis: (e.g., Hereditary Spherocytosis, Warm Autoimmune Hemolytic Anemia)
- Spherocytes on Peripheral Smear:
- Positive DAT (in AIHA):
- Splenomegaly:
Intravascular Hemolysis: (e.g., TTP, PNH)
- Schistocytes on Peripheral Smear:
- Hemoglobinuria:
- Hemosiderinuria:

Key Terms

Hemolysis: Destruction of red blood cells
Intravascular Hemolysis: RBC destruction within blood vessels
Extravascular Hemolysis: RBC destruction outside blood vessels (primarily in the spleen)
Haptoglobin: Protein that binds free hemoglobin
Lactate Dehydrogenase (LDH): Enzyme released from damaged cells
Bilirubin: Product of heme breakdown
Unconjugated (Indirect) Bilirubin: Bilirubin that has not been processed by the liver
Hemoglobinuria: Hemoglobin in the urine
Hemosiderinuria: Hemosiderin (iron) in the urine

Indices

Special Stains