bilirubin and hemoglobin

Source of Bilirubin

About 80-85% derived from the heme moiety of hemoglobin released from aging erythrocytes processed in reticuloendothelial cells.

Bilirubin Transport Process

Bilirubin binds to albumin, is transported to the liver, conjugated with glucuronide, excreted into biliary canaliculi, then hydrolyzed in the gastrointestinal tract.

Conditions Causing Unconjugated Bilirubin Increase

Overproduction, impaired hepatic uptake, defective conjugation.

Conditions Leading to Elevated Conjugated Bilirubin Levels

Hepatocellular damage, bile flow obstruction.

Bilirubin Determination Method

Malloy Evelyn End Point Method (ELITECH system).

Bilirubin Measurement Principle

Sulfanilic acid and sodium nitrite form diazotized sulfanilic acid, which reacts with bilirubin to form reddish-purple azobilirubin; absorbance at 546 nm is proportional to bilirubin concentration.

Result of Absence of Accelerator in Bilirubin Testing

Only conjugated bilirubin reacts with diazotized sulfanilic acid.

Main Reagents for Bilirubin Determination

R1: Sulfanilic Acid, Hydrochloric Acid, Cetrimide; R2: Sodium Nitrite.

Precautions for Handling Bilirubin Reagents

R1 is an irritant; R2 contains sodium azide which can form explosive compounds.

Stability of Bilirubin Reagents and Samples

Reagents stable at 2–8°C; samples stable for 2 days room temp, 4 days at 4°C, longer storage requires freezing at -20°C.

Incubation Times for Total vs Direct Bilirubin Tests

Total Bilirubin: 5 minutes at 37°C, Direct Bilirubin: 50 seconds at 37°C.

Bilirubin Concentration Calculation Formula

Bilirubin = (A2−A1)Sample / (A2−A1)Standard × n.

Normal Reference Values for Bilirubin

Total Bilirubin: 0.3–1.2 mg/dL; Direct Bilirubin: <0.2 mg/dL.

Cyanmethemoglobin Method Principle

Potassium ferricyanide oxidizes hemoglobin to methemoglobin, which reacts with potassium cyanide to form cyanmethemoglobin measured at 540 nm.

Specimen for Hemoglobin Determination

3–5 mL whole blood in a purple-top (EDTA) tube.

Key Steps in Hemoglobin Determination

Mix 5 mL Drabkin’s reagent + 20 μL blood, incubate for 10 min, measure absorbance at 540 nm, calculate Hemoglobin = Absorbance × 36.8.

Reference Range for Normal Hemoglobin Levels

Male: 13.8–18 g/dL; Female: 11–16 g/dL.

Total vs Direct Bilirubin Incubation Timing

Total bilirubin is incubated for 5 minutes at 37°C; direct bilirubin for 50 seconds at 37°C.

Role of Potassium Cyanide in Cyanmethemoglobin Method

Converts methemoglobin to cyanmethemoglobin.

Bilirubin Detection Requirements

Detection requires an accelerator; samples are stable for 4 days at 4°C.