(FINAL) CC1 LAB L5

Heme catabolism

Bilirubin

• Definition: End-product of __________ from senescent RBCs.

250-300

Bilirubin

• Normal adult production: ~__________ mg bilirubin/day.

Liver function and bile excretion

Bilirubin Clinical Significance

• Evaluates __________.

• Assesses hemolytic, hepatic, or obstructive causes of jaundice.

• Monitors neonatal hyperbilirubinemia.

Jaundice, hyperbilirubinemia

Bilirubin Clinical Significance

• Evaluates liver function and bile excretion.

• Assesses hemolytic, hepatic, or obstructive causes of __________.

• Monitors neonatal __________.

Biliverdin

Bilirubin Metabolism

1. Formation: Heme → __________ → Unconjugated bilirubin (UB).

Albumin

Bilirubin Metabolism

2. Transport: Unconjugated bilirubin (UB) binds to __________ in plasma.

UDP-glucuronyl transferase (UGT1A1)

Bilirubin Metabolism

3. Conjugation: In hepatocytes via __________ → bilirubin mono- & diglucuronides (water-soluble).

Conjugated bilirubin

Bilirubin Metabolism

4. Excretion: __________ secreted into bile → intestine → converted to urobilinogen/stercobilin (excreted in feces or urine).

Urobilinogen/stercobilin

Bilirubin Metabolism

4. Excretion: Conjugated bilirubin secreted into bile → intestine → converted to __________ (excreted in feces or urine).

Unconjugated (Indirect)

Types of Bilirubin: __________

• Solubility: Lipid-soluble

• Bound to: Albumin

• Measured as: Calculated: Total – Direct

• Clinical Example: Hemolysis, Gilbert’s

Conjugated (Direct)

Types of Bilirubin: __________

• Solubility: Water-soluble

• Bound to: Free

• Measured as: Directly measured

• Clinical Example: Hepatitis, Obstruction

Delta

Types of Bilirubin: __________

• Solubility: Covalently bound conjugated bilirubin

• Bound to: Albumin

• Measured as: Part of “direct” fraction

• Clinical Example: Persistent after obstruction

Total Bilirubin (TB)

CL Tests for Bilirubin: __________

• Measures both conjugated & unconjugated forms.

• Requires accelerator (e.g., caffeine or methanol).

Direct Bilirubin (DB)

CL Tests for Bilirubin: __________

• Measures conjugated fraction only.

• Reacts directly with diazo reagent.

Indirect Bilirubin

CL Tests for Bilirubin: __________

• Calculated: TB – DB.

Urine Bilirubin & Urobilinogen (qualitative)

CL Tests for Bilirubin: __________

• Screen for conjugated bilirubin (positive in hepatocellular or obstructive jaundice).

• Watson–Schwartz or Ehrlich’s reagent for urobilinogen

Jendrassik–Grof Reaction

Bilirubin LM: __________ (Reference Method)

• Principle: Bilirubin reacts with diazotized sulfanilic acid to form azobilirubin, a blue-green compound measured spectrophotometrically.

• Reaction: Bilirubin + Diazo reagent → Azobilirubin (blue-green)

• Measured at 600 nm (total) or 540 nm (direct)

600, 540

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

• Principle: Bilirubin reacts with diazotized sulfanilic acid to form azobilirubin, a blue-green compound measured spectrophotometrically.

• Reaction: Bilirubin + Diazo reagent → Azobilirubin (blue-green)

• Measured at ____ nm (total) or ___ nm (direct)

Blank and Test

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: __________.

2. Add serum (0.2 mL) and reagents as specified in protocol.

3. For Total Bilirubin, include accelerator; for Direct, omit accelerator.

4. Incubate for 10 min (room temperature, protected from light).

5. Add alkaline tartrate reagent to stop the reaction.

6. Measure absorbance spectrophotometrically at 600 nm.

0.2

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: Blank and Test.

2. Add serum (___ mL) and reagents as specified in protocol.

3. For Total Bilirubin, include accelerator; for Direct, omit accelerator.

4. Incubate for 10 min (room temperature, protected from light).

5. Add alkaline tartrate reagent to stop the reaction.

6. Measure absorbance spectrophotometrically at 600 nm.

Total Bilirubin, Direct

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: Blank and Test.

2. Add serum (0.2 mL) and reagents as specified in protocol.

3. For __________, include accelerator; for __________, omit accelerator.

4. Incubate for 10 min (room temperature, protected from light).

5. Add alkaline tartrate reagent to stop the reaction.

6. Measure absorbance spectrophotometrically at 600 nm.

10

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: Blank and Test.

2. Add serum (0.2 mL) and reagents as specified in protocol.

3. For Total Bilirubin, include accelerator; for Direct, omit accelerator.

4. Incubate for ___ min (room temperature, protected from light).

5. Add alkaline tartrate reagent to stop the reaction.

6. Measure absorbance spectrophotometrically at 600 nm.

Alkaline tartrate reagent

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: Blank and Test.

2. Add serum (0.2 mL) and reagents as specified in protocol.

3. For Total Bilirubin, include accelerator; for Direct, omit accelerator.

4. Incubate for 10 min (room temperature, protected from light).

5. Add __________ to stop the reaction.

6. Measure absorbance spectrophotometrically at 600 nm.

600

Bilirubin LM: Jendrassik–Grof Reaction (Reference Method)

1. Prepare two tubes: Blank and Test.

2. Add serum (0.2 mL) and reagents as specified in protocol.

3. For Total Bilirubin, include accelerator; for Direct, omit accelerator.

4. Incubate for 10 min (room temperature, protected from light).

5. Add alkaline tartrate reagent to stop the reaction.

6. Measure absorbance spectrophotometrically at ___ nm.

Heparinized plasma

Bilirubin Specimen Collection & Handling

• Specimen: Serum (preferred) or __________.

RBC lysis

Bilirubin Specimen Collection & Handling

• Collection: Avoid hemolysis; __________ increases bilirubin production.

50% in 1 hr

Bilirubin Specimen Collection & Handling

• Protection from light: Bilirubin degrades rapidly in sunlight or fluorescent light (up to __________)

24, −20

Bilirubin Specimen Collection & Handling

• Storage: Stable ___ h refrigerated; long-term—freeze at ___°C.

Icteric, hemolyzed, or lipemic samples

Bilirubin Specimen Collection & Handling

• Reject: __________ that interfere with optical reading.

Hemolysis

Sources of Error: Bilirubin

• Effect on Result: False ↑ or ↓

• Preventive Measure: Avoid rough handling

Light exposure

Sources of Error: Bilirubin

• Effect on Result: False ↓

• Preventive Measure: Protect from light

Lipemia

Sources of Error: Bilirubin

• Effect on Result: Turbid sample affects absorbance

• Preventive Measure: Ultracentrifuge or blank correction

Improper reagent prep

Sources of Error: Bilirubin

• Effect on Result: Erratic readings

• Preventive Measure: Follow SOP strictly

Old/unstable reagents

Sources of Error: Bilirubin

• Effect on Result: Low color intensity

• Preventive Measure: Use fresh reagents

Hemolysis, Gilbert’s, Crigler–Najjar

Interpretation of Results: Bilirubin

• Pattern: ↑ Indirect only

• Total (mg/dL): ↑

• Direct (mg/dL): N

• Indirect: ↑

• Probable Cause: __________

Obstructive/cholestatic jaundice

Interpretation of Results: Bilirubin

• Pattern: ↑ Direct only

• Total (mg/dL): ↑

• Direct (mg/dL): ↑

• Indirect: N

• Probable Cause: __________

Hepatocellular disease

Interpretation of Results: Bilirubin

• Pattern: Mixed (both ↑)

• Total (mg/dL): ↑↑

• Direct (mg/dL): ↑

• Indirect: ↑

• Probable Cause: __________

Physiologic jaundice, prematurity

Interpretation of Results: Bilirubin

• Pattern: Neonatal ↑ Indirect

• Total (mg/dL): ↑

• Direct (mg/dL): N

• Indirect: ↑↑

• Probable Cause: __________