Clinical Chemistry: Bilirubin and Hemoglobin Analysis

Bilirubin concentration

The increase in absorbance at 546 nm is proportional to bilirubin concentration.

Total Bilirubin

Reagent composition includes Sulfanilic Acid at 29 mmol/L.

Direct Bilirubin

Reagent composition includes Sodium Nitrite at 5.8 mmol/L.

Hydrochloric Acid

Reagent composition includes Hydrochloric Acid at 67 mmol/L.

Cyanmethemoglobin Method

A method used for hemoglobin determination.

Reagent R1

An irritant (Xi) that is irritating to eyes, respiratory system, and skin.

Reagent R2

Contains less than 0.1% sodium azide, which can react with copper and lead plumbing.

Warnings and Precautions

Includes instructions to avoid breathing vapor and to wear suitable gloves and eye/face protection.

Clinical Significance

Total and Direct Bilirubin 4+1 is significant for clinical testing.

Reference Values

Values that are used as a standard for comparison in clinical tests.

Interferences with protein

Can be avoided by rinsing with 0.1 N NaOH.

Detergent in Bilirubin Total 4+1 R1

Can lead to the formation of foam in the washing unit of some equipment.

Regulations for dangerous waste

Must be respected regarding elimination of hazardous materials.

S 23

Do not breathe vapor.

S 26

In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.

S 37.39

Wear suitable gloves and eye/face protection.

Sodium azide

Can form explosive metal azides when reacting with copper and lead plumbing.

Cleaning laboratory equipment

Use clean or single-use laboratory equipment only to avoid contamination.

Total and Direct Bilirubin Test

A test that measures the levels of bilirubin in the blood.

Sulfanilic Acid

A reagent used in the Total Bilirubin test.

Cetrimide

A reagent included in the Direct Bilirubin test.

Stability of reagents

When stored at 2-8°C and protected from light, the reagents are stable until the expiry date stated on the label.

Bilirubin production

Approximately 80-85% of the bilirubin produced is derived from the heme moiety of the hemoglobin released from aging erythrocytes in the reticuloendothelial cells.

Bilirubin transport

Bilirubin, bound to albumin, is transported into the liver where it is rapidly conjugated with glucuronide to increase its solubility.

Bilirubin excretion

Then it is excreted into biliary canaliculi and hydrolyzed in the gastrointestinal tract.

Unconjugated bilirubin increase causes

Unconjugated bilirubin increases in case of overproduction of bilirubin or disorders of bilirubin metabolism.

Acute/Chronic hemolytic anemia

A condition that can cause overproduction of bilirubin.

Gilbert's Syndrome

A disorder that causes impaired uptake of bilirubin by liver cells.

Crigler-Najjar Syndrome

A disorder that causes defects in the conjugation reaction of bilirubin.

Physiological jaundice of newborns

A condition that can lead to an increase in unconjugated bilirubin.

Conjugated bilirubin elevation causes

Conjugated bilirubin elevation occurs due to reduced excretion of bile or obstruction of the flow of bile.

Hepatocellular damage

A condition such as hepatitis or cirrhosis that can lead to conjugated bilirubin elevation.

Dubin-Johnson syndrome

A genetic disorder that can cause conjugated bilirubin elevation.

Rotor syndrome

A rare genetic disorder that can lead to conjugated bilirubin elevation.

Gallstones

Obstruction of the flow of bile most often produced by gallstones can lead to conjugated bilirubin elevation.

Sample protection

Protect samples from light before and after the analysis.

Sulfanilic acid reaction

Sulfanilic acid reacts with sodium nitrate (NaNO2) to form diazotized sulfanilic acid.

Bilirubin reaction

In the presence of accelerator (cetrimide), conjugated and unconjugated bilirubin react with diazotized sulfanilic acid to form azobilirubin (reddish-purple).

Sample stability at Room Temperature

If plasma and serum are separated and protected from light, samples are stable for 2 days at Room Temperature.

Sample stability at 4°C

If plasma and serum are separated and protected from light, samples are stable for 4 days at 4°C.

Sample freezing

For longer storage, freeze samples at -20°C.

Wavelength for analysis

Wavelength: 550 nm.

Temperature for analysis

Temperature: 37°C.

Reference values for total bilirubin

Standard Test: 0.3 - 1.2 mg/dL (5 - 21 μmol) for adults and children over 10 days.

Direct bilirubin reference value

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

Cyanmethemoglobin Method

A method where whole blood is diluted with a potassium ferricyanide-cyanide solution (Drabkin's solution) which oxidizes hemoglobin to methemoglobin, which is converted to cyanmethemoglobin.

Absorbance (A1)

The measurement taken after mixing the sample with Reagent 1 and incubating for 5 minutes.

Absorbance (A2)

The measurement taken after mixing the sample with Reagent 2 and incubating for 50 seconds.

Total Bilirubin Procedure

800 μL of Reagent 1 + 50 μL of Serum, incubated at 37°C for 5 minutes, followed by adding 200 μL of Reagent 2 and incubating again at 37°C for 5 minutes.

Direct Bilirubin Procedure

800 μL of Reagent 1 + 100 μL of Serum, incubated at 37°C for 5 minutes, followed by adding 200 μL of Reagent 2 and incubating at room temperature for 10 minutes.

Hemoglobin Determination

5 mL of Hemoglobin Reagent + 20 μL of whole blood, incubated at 37°C for 5 minutes, followed by adding 200 μL of Reagent 2 and incubating at room temperature for 10 minutes.

Spectrophotometer Reading

The absorbance is read at 540 nm after the final incubation.

Hgb Calculation

Hgb g/dL = Abs x 36.8.

Reference Values for Hemoglobin (Male)

Normal values range from 13.8-18 g/dL.

Reference Values for Hemoglobin (Female)

Normal values range from 11-16 g/dL.

Potassium Cyanide Role

Converts hemoglobin to methemoglobin.

Total Bilirubin Incubation Time and Temperature

Incubated for 5 minutes at 37°C after adding Reagent 2.

Direct Bilirubin Incubation Time and Temperature

Incubated for 50 seconds at 37°C after adding Reagent 2.

Calculation of Total and Direct Bilirubin

𝐴2−𝐴1 𝑆𝑎𝑚𝑝𝑙𝑒 / 𝐴2−𝐴1 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 × 𝑛.

Accelerator in Total Bilirubin Detection

Detection requires the presence of an accelerator (cetrimide).

Sample Stability for Bilirubin Detection

Samples for detection of total and direct bilirubin can be stable for up to 4 days at 4°C.

Incubation Color Change

Observe slight color change to peach when incubated.

Specimen for Total Bilirubin

3 mL Serum (Red Top Tube) protected from light.

Specimen for Direct Bilirubin

3 mL or 5 mL whole blood (Purple Top Tube).

Reverse Pipetting

For obtaining whole blood samples, use reverse pipetting since blood is viscous.

Normal Values Conversion Factor

𝑚𝑔/𝑑𝐿 × 17.10 = μ𝑚𝑜𝑙.

Total Bilirubin Detection Method

Malloy Evelyn, End Point.

Direct Bilirubin Detection Method

Colorimetric, End Point.