Calculate the change in absorbance ∆A for both the

standard and test.

For standard ∆AS = A2S – A1S

For test ∆AT = A2T – A1T

Calculations

 ∆AT

Creatinine in mg/dL = ________ × 2.0 ∆AS

 ∆AT

Urine creatinine in g/L = _______ × 1.0

 ∆AS

Urine creatinine =

 Urine creatinine in g/L × _______________________________

g/24 hours Volume of urine in liters 24 hours

Linearity

The procedure is linear upto 20 mg/dL of Creatinine. If

values exceed this limit, dilute the sample with distilled

water and repeat the assay. Calculate the value using the

proper dilution factor.

Note

The buffer reagent may turn milky or show white

precipitates at cold temperatures. This is not a deterioration of the reagent. Dissolve/clear the same by warming

the reagent to 37°C with gentle swirling before use. The

determination is not specific and may be affected by the

presence of large quantities of reducing substances.

Clinical Chemistry 475

As the test is temperature sensitive, it is essential to

maintain the indicated reaction timings and temperatures

meticulously during the test procedure.

System Parameters

Reaction : Fixed time kin Interval : 60 seconds

Wavelength : 520 nm Sample

volume

: 0.10 mL

Zero setting :  Distilled water Reagent

volume

: 1.00 mL

Incubation

temperature

: 30°C/37°C Standard : 2 mg/dL

Incubated

time

: - Factor : -

Delay time : 30 seconds React slope : Increasing

Read time : 60 seconds Linearity : 20 mg/dL

No. of read : 2 Units : mg/dL

Clinical Relevance

Causes of Raised Serum Creatinine Levels

All renal causes of uremia are usually associated with raised

serum creatinine values. Elevated BUN levels in a patient

with normal creatinine usually signal a nonrenal cause

for the uremia. With severe, permanent renal damage,

urea levels continue to climb, but creatinine values tend

to plateau. At very high creatinine levels, some is excreted

across the alimentary tract.

Decreased Creatinine Levels Occur in

Muscular dystrophy.

Interfering Factors

1. High levels of ascorbic acid can give a falsely increased

level.

2. Drugs influencing kidney function (diuretics and

dextran), chloral hydrate, marijuana, acetohexamide,

guanethidine, furosemide, chloramphenicol,

and sulfonamides can cause a change in blood

creatinine.

3. A diet high is roast meat will cause increased levels.

4. Many drugs may cause a change in the blood creatinine.

 A normal blood serum creatinine does not always

indicate unimpaired renal function. A normal value

cannot be used as standard for a patient who is known

to have existing renal disease.

Serum Bilirubin

Normal Values

SI units

Total bilurubin

1 month – adult < 1.5 mg/dL 1.7–20.5 µmol/L

Premature infant

Cord < 2.8 mg/dL < 48 µmol/L

24 hours 1–6 mg/dL 17–103 µmol/L

48 hours 6–8 mg/dL 103–137 µmol/L

3–5 days 10–12 mg/dL 171–205 µmol/L

Full-term infant

Cord < 2.8 mg/dL < 48 µmol/L

24 hours 2–6 mg/dL 34–103 µmol/L

48 hours 6–7 mg/dL 103–120 µmol/L

3–5 days 4–6 mg/dL 68–103 µmol/L

Direct bilirubin 0.0–0.3 mg/dL 1.7–5.1 µmol/L

Indirect bilirubin 0.1–1.0 mg/dL 1.7–17.1 µmol/L

Bilirubin (Mod Jendrassik and Grof’s Method)

(Courtesy: Tulip Group of Companies)

For the determination of direct and total bilirubin in serum

(for in vitro diagnostic use only).

Summary

Bilirubin is mainly formed from the heme portion of aged

or damaged RBCs. It then combines with albumin to form

a complex which is not water soluble. This is referred to

as indirect or unconjugated bilirubin. In the liver, this

bilirubin complex is combined with glucuronic acid into a

water soluble conjugate. This is referred to as conjugated

or direct bilirubin. Elevated levels of bilirubin are found in

liver diseases (hepatitis, cirrhosis), excessive hemolysis/

destruction of RBC (hemolytic jaundice) obstruction

of the biliary tract (obstructive jaundice) and in drug

induced reactions. The differentiation between the direct

and indirect bilirubin is important in diagnosing the cause

of hyperbilirubinemia.

Principle

Bilirubin reacts with diazotized sulfanilic acid to form

a colored azobilirubin compound. The unconjugated

bilirubin couples with the sulfanilic acid in the presence of

a caffein-benzoate accelerator. The intensity of the color

476 Concise Book of Medical Laboratory Technology: Methods and Interpretations formed is directly proportional to the amount of bilirubin

present in the sample.

Bilirubin + Diazotized Sulfanilic acid→ Azobilirubin

Compound

Normal Reference Values

Serum (Direct) : upto 0.2 mg/dL

(Total) : upto 1.0 mg/dL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 30 tests 75 tests

L1: Direct bilirubin reagent 75 mL 150 mL

L2: Direct nitrite reagent 4 mL 4 mL

L1: Total bilirubin reagent 75 mL 150 mL

L2: Total nitrite reagent 4 mL 4 mL

S : Artificial standard (10 mg/dL) 10 mL 10 mL

Storage/Stability

All reagents are stable at RT till the expiry mentioned on

the label.

Reagent Preparation

Reagents are ready to use. Do not pipette with mouth.

Sample Material

Serum. Bilirubin is reported to be stable in the sample for

4 days at 2–8°C protected from light as it is photosensitive.

Procedure

Wavelength/filter : 546 nm/yellow-green

Temperature : RT

Light path : 1 cm

Direct Bilirubin Assay

Pipette into clean dry test tubes labeled as Blank (B), and

Test (T):

Addition

Sequence

B

(mL)

T

(mL)

Direct bilirubin reagent (L1) 1.0 1.0

Direct nitrite reagent (L2) - 0.05

Sample 0.1 0.1

Mix well and incubate at RT for exactly 5 minutes.

Measure the absorbance of the test samples (Abs T)

immediately against their respective blanks.

Total Bilirubin Assay

Pipette into clean dry test tubes labeled as blank (B), and

test (T):

Addition

Sequence

B

(mL)

T

(mL)

Total bilirubin reagent (L1) 1.0 1.0

Total nitrite reagent (L2) - 0.05

Sample 0.1 0.1

Mix well and incubate at RT for 10 min. Measure the

absorbance of the test samples (Abs T) immediately

against their respective blanks.

Calculations

Total or direct bilirubin in mg/dL = Abs T × 13 (13 being

the factor).

Linearity

This procedure is linear upto 20 mg/dL. If values exceed

this limit, dilute the sample with distilled water and repeat

the assay. Calculate the value using the proper dilution

factor.

Note

In case, the exact wavelength is not available the artificial

standard (S) may be used. Measure the absorbance of

the artificial standard against distilled water with the

appropriate filter and keep the same for future calculations

by dividing the Abs T with the Abs. of the Std. × 10. Discard

the artificial standard after use.

In case of neonates where the sample quantity is a

limitation, and the samples have high bilirubin (above

3 mg/dL), only 0.05 mL/0.02 mL of the sample may be

used for bilirubin estimation. The calculation factor in

this case would be 24.9/60.5 respectively instead of 13. In

case of using the standard the value of the same would be

19.1/46.5 mg/dL respectively instead of 10 mg/dL.

System Parameters

Reaction : End point Interval :

Wavelength : 546 nm Sample

volume

: 0.10 mL

Zero setting : Sample blank Reagent

volume

: 1.05 mL

Incubation

temperature

: RT Standard :

Incubated time : 5 min/10 min Factor : 13

Delay time : — React slope : Increasing

Read time : — Linearity : 20 mg/dL

No. of read : — Units : mg/dL

Clinical Chemistry 477

Causes of Hyperbilirubinemia

Unconjugated (Indirect) Hyperbilirubinemia

I. Overproduction of bilirubin

 A. Hemolytic disorders.

 1. Congenital (e.g. hemoglobinopathies)

 2. Acquired (e.g. Coombs’ positive anemia)

 3. Liver disease (e.g. hepatitis and cirrhosis).

 B. Shunt hyperbilirubinemia

II. Defective uptake and storage of bilirubin

 A. Idiopathic unconjugated hyperbilirubinaemia.

 1. Hereditary-Gilbert’s syndrome.

 2. Acquired

 – Post-viral hepatitis.

 – Post-portacaval shunt.

 B. Decreased availability of cytoplasmic binding

proteins (Y and Z) in newborn and premature infants.

 C. Drugs (e.g. flavispidic acid).

III. Defective glucuronyl transferase activity.

 A. Deficiency.

 1. In newborn and premature infants

 2. Crigler-Najjar syndrome.

 B. Inhibition

 1. Abnormal steroids in breast milk or maternal

plasma (Lucey-Driscoll type).

 2. Drugs (e.g. novobiocin).

Conjugated (Direct) Hyperbilirubinemia

Defective excretion of conjugated bilirubin

A. Hereditary

 1. Dubin-Johnson syndrome

 2. Rotor syndrome.

B. Obstructive

 1. Intrahepatic cholestasis

 a. Cirrhosis (occasionally)

 b. Hepatitis (often)

 c. Alcoholic liver disease (occasionally)

 d. Drugs (e.g. chlorpromazine and methyltestosterone).

 e. Primary biliary cirrhosis.

 2. Extrahepatic obstruction.

 a. Gallstones

 b. Carcinoma of the bile duct, pancreas, ampulla

of Vater

 c. Bile duct stricture

 d. Biliary atresia.

Interfering Factors

1. A 1 hour exposure of the specimen to sunlight or

high intensity artificial light at room temperature will

reduce the bilirubin content.