bilirubin and hemoglobin

🔶 Total and Direct Bilirubin

Flashcard 1
Q: What is the main source of bilirubin in the body?
A: About 80–85% of bilirubin is derived from the heme moiety of hemoglobin released from aging erythrocytes processed in reticuloendothelial cells.

Flashcard 2
Q: How is bilirubin transported and processed in the body?
A: Bilirubin binds to albumin, transported to the liver, conjugated with glucuronide to increase solubility, excreted into biliary canaliculi, then hydrolyzed in the gastrointestinal tract.

Flashcard 3
Q: What conditions cause an increase in unconjugated bilirubin?
A:

• Overproduction (e.g., acute/chronic hemolytic anemia)

• Impaired hepatic uptake (e.g., Gilbert’s syndrome)

• Defective conjugation (e.g., Crigler-Najjar syndrome, physiological jaundice of newborns)

Flashcard 4
Q: What conditions lead to elevated conjugated bilirubin levels?
A:

• Hepatocellular damage (e.g., hepatitis, cirrhosis, Dubin-Johnson syndrome, Rotor syndrome)

• Bile flow obstruction (e.g., gallstones, tumors)

🔶 Bilirubin Determination — Method and Principle

Flashcard 5
Q: What method is used for bilirubin determination?
A: Malloy Evelyn End Point Method (ELITECH system).

Flashcard 6
Q: Describe the principle of bilirubin measurement.
A:

• Sulfanilic acid + NaNO₂ → Diazotized sulfanilic acid

• Diazotized sulfanilic acid reacts with bilirubin (with cetrimide accelerator) to form reddish-purple azobilirubin.

• Absorbance at 546 nm is proportional to bilirubin concentration.

Flashcard 7
Q: What happens in the absence of an accelerator (cetrimide)?
A: Only conjugated bilirubin reacts with diazotized sulfanilic acid.

🔶 Reagents, Precautions, and Storage

Flashcard 8
Q: What are the main reagents for bilirubin determination?
A:

• R1: Sulfanilic Acid, Hydrochloric Acid, Cetrimide

• R2: Sodium Nitrite

Flashcard 9
Q: What are some important precautions when handling reagents?
A:

• R1 is an irritant (Xi): Avoid inhalation and eye contact; wear protective equipment.

• R2 contains sodium azide: Can form explosive compounds with lead/copper plumbing.

Flashcard 10
Q: How stable are bilirubin reagents and samples?
A:

• Reagents: Stable at 2–8°C, protected from light, until expiry.

• Samples:

  • 2 days at room temp

  • 4 days at 4°C

  • Longer storage requires freezing at -20°C.

🔶 Bilirubin Testing Procedure

Flashcard 11
Q: What are the key incubation times for Total vs Direct Bilirubin tests after adding R2?
A:

• Total Bilirubin: 5 minutes at 37°C

• Direct Bilirubin: 50 seconds at 37°C

Flashcard 12
Q: What is the formula for calculating bilirubin concentration?
A:

Bilirubin=(A2−A1)Sample(A2−A1)Standard×n\text{Bilirubin} = \frac{(A2 - A1)_{\text{Sample}}}{(A2 - A1)_{\text{Standard}}} \times nBilirubin=(A2−A1)Standard​(A2−A1)Sample​​×n

Where n = standard concentration.

🔶 Bilirubin Reference Values

Flashcard 13
Q: What are the normal reference values for bilirubin?
A:

• Total Bilirubin: 0.3–1.2 mg/dL (5–21 μmol/L)

• Direct Bilirubin: <0.2 mg/dL (3.4 μmol/L)

🔷 Hemoglobin Determination — Cyanmethemoglobin Method

Flashcard 14
Q: What is the principle behind the cyanmethemoglobin method?
A:

• Potassium ferricyanide in Drabkin's reagent oxidizes hemoglobin to methemoglobin.

• Methemoglobin then reacts with potassium cyanide to form cyanmethemoglobin.

• The intensity of cyanmethemoglobin is measured photometrically at 540 nm.

Flashcard 15
Q: What specimen is needed for hemoglobin determination?
A: 3–5 mL whole blood in a purple-top (EDTA) tube.

Flashcard 16
Q: Outline the key steps in hemoglobin determination.
A:

• Mix 5 mL Drabkin’s reagent + 20 μL blood.

• Incubate for 10 min at room temperature (protected from light).

• Measure absorbance at 540 nm.

• Calculate Hemoglobin (g/dL) = Absorbance × 36.8.

Flashcard 17
Q: What is the reference range for normal hemoglobin levels?
A:

• Male: 13.8–18 g/dL

• Female: 11–16 g/dL

🔷 Review Questions (Based on Exam Type Questions)

Flashcard 18
Q: In bilirubin testing, total bilirubin is incubated for ___ at ___ after adding R2, while direct bilirubin is incubated for ___ at ___.
A: 5 minutes at 37°C (Total) and 50 seconds at 37°C (Direct).

Flashcard 19
Q: In the cyanmethemoglobin method, what is the role of potassium cyanide?
A: It converts methemoglobin to cyanmethemoglobin.

Flashcard 20
Q: Does bilirubin detection require an accelerator? Are samples stable for 4 days at 4°C?
A:

• Yes, detection needs cetrimide (accelerator).

• Yes, samples are stable for up to 4 days at 4°C.