2. Contrast media 24 hours before measurement may

cause an altered reaction.

3. A high fat meal may cause decreased bilirubin levels

by interfering with the clinical reactions.

4. Air bubbles and shaking of the specimen may cause

decreased levels.

5. Foods (carrots, etc.) and drugs increase the yellowish

hue in the serum.

6. Refer to the Many drugs can interfere with Bilirubin

tests for a listing of the many drugs that may interfere

with testing for bilirubin.

7. Hemolyzed blood will falsely elevate bilirubin level.

Comments

1. In severe obstructive jaundice with formation of

biliverdin, low results for the degree of jaundice will

be obtained since biliverdin does not react with the

diazo reagent and cannot be determined.

2. An unusual source of otherwise unexplained elevated

serum bilirubin has been described following 48 hours

fasting. A normal bilirubin value from 0.68 mg% may

rise to the abnormal range at 1.87 mg%.

Icterus Index

The icterus index is a measure of the degree of icterus

(yellowish-green color) in a plasma or serum specimen

in cases of jaundice. This is just a screening test for

hyperbilirubinemia. Substances other than bilirubin

in the serum (carotene, xanthophyll, hemoglobin, etc.)

may contribute to the icterus index, therefore, limiting its

clinical utility. The test is now considered to be obsolete.

Reagents

A. Potassium dichromate solution

 1. Stock solution (1%)

 Dissolve 1 g of potassium dichromate in 70 mL of

water placed in a 100 mL volumetric flask. Add 2

drops of sulfuric acid and dilute to 100 mL mark

with distilled water. Store in a glass-stoppered

brown/amber colored bottle.

 2. Working standard solution (0.1%)

 Pipette 10 mL of the stock solution into a 100 mL

volumetric flask and dilute to 100 mL mark with

distilled water.

B. Saline (0.9% NaCL) isotonic.

Method

A. Dilute the serum specimen ten times with saline (1 mL of

serum mixed with 9 mL of saline) in a test tube and mix.

B. Transfer the diluted serum into a cuvette and read

absorbance at 420 to 460 nm. If too dark, dilute further

478 Concise Book of Medical Laboratory Technology: Methods and Interpretations and multiply the final reading with the dilution factor

utilized here.

C. Determine the icterus index from the calibration curve.

Multiply the result by dilution factor. If the serum is

diluted ten times, the dilution factor is 10.

Calibration Curve

A. Prepare three concentrations of the standard by diluting appropriate quantities of the stock solution of

potassium dichromate in three 100 mL volumetric

flasks

 1. Five mL stock mixed with 95 mL of water (1:20).

This corresponds to 5 units.

 2. 25 mL of stock solution made to 100 mL with water

(1:4 dilution). This corresponds to 25 units.

 3. 50 mL of stock solution made to 100 mL with water

(1:2 dilution). This corresponds to 50 units.

B. Read the absorbance of each working standard

solution corresponding to 5, 25 and 50 units at 420 to

460 nm using water as blank.

C. Tabulate the results with the units of icterus index and

the corresponding absorbance values.

D. Plot a calibration curve and use this for the determination of icterus index.

TOTAL PROTEINS

Biuret Method

(Courtesy: Tulip Group of Companies)

For the determination of total proteins in serum and

plasma (for in vitro diagnostic use only).

Summary

Proteins are constituents of muscle, enzymes, hormones

and several other key functional and structural entities

in the body. They are involved in the maintenance of the

normal distribution of water between blood and the tissues.

Consisting mainly of albumin and globulin the fractions

vary independently and widely in diseases. Increased

levels are found mainly in dehydration. Decreased levels

are found mainly in malnutrition, impaired synthesis,

protein losses as in hemorrhage or excessive protein

catabolism.

Principle

Proteins, in an alkaline medium, bind with the cupric ions

present in the biuret reagent to form a blue-violet colored

complex. The intensity of the color formed is directly

proportional to the amount of proteins present in the

sample.

Proteins + Cu++→ Blue violet colored complex

Normal Reference Values

Serum and plasma : 6.0–8.0 g/dL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 150 mL 2 × 150 mL

Carton 1

L1: Biuret reagent 150 mL 2 × 150 mL

Carton 2

S: Protein standard ( 8 g/dL) 5 mL 5 mL

Storage/Stability

Carton 1 : Biuret reagent is stable at RT till the expiry

mentioned on the label.

Carton 2 : Protein standard is stable at 2–8°C till the

expiry mentioned on the label.

Reagent Preparation

Reagents are ready to use. Protect from bright light.

Sample Material

Serum or plasma. Proteins are reported to be stable in the

sample for 6 days at 2–8°C.

Procedure

Wavelength/filter : 550 nm (Hg 546 nm)/yellow-green

Temperature : RT/37°C

Light path : 1 cm

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

standard (S), and test (T)

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Biuret reagent (L1) 1.0 1.0 1.0

Distilled water 0.02

Protein standard (S) - 0.02

Sample - - 0.02

Mix well and incubate at 37°C for 10 minutes or at RT

for 30 minutes. Measure the absorbance of the standard

(Abs S), and test sample (Abs T) against the blank, within

60 minutes.

Calculations

 Abs T

Total proteins in g/dL = _________ = × 8 Abs S

Linearity

This procedure is linear upto 15 g/dL. If values exceed

this limit, dilute the sample with distilled water and

Clinical Chemistry 479

repeat the assay. Calculate the value using the proper

dilution factor.

Note

Do not use if the reagent shows turbidity or black

precipitates.

System Parameters

Reaction : End point Interval :

Wavelength : 550 nm Sample vol : 0.02 mL

Zero setting : Reagent blank Reagent vol : 1.00 mL

Incubation

temperature

: 37°C/RT Standard : 8 g/dL

Incubated

time

: 10 mm/30 min Factor :

Delay time : React slope : Increasing

Read time : Linearity : 15 g/dL

No. of read : Units : g/dL

SERUM ALBUMIN

Determination of Serum Albumin (BCG Method)

(Courtesy: Tulip Group of Companies)

For the determination of albumin in serum or plasma (for

in vitro diagnostic use only).

Summary

Albumin consists of approximately 60% of the total

proteins in the body, the other major part being globulin.

It is synthesized in the liver and maintains the osmotic

pressure in blood. Albumin also helps in the transportation

of drugs, hormones and enzymes. Elevated levels are

rarely seen and are usually associated with dehydration.

Decreased levels are seen in liver diseases (hepatitis,

cirrhosis). Malnutrition, kidney disorders, increased fluid

loss during extensive burns and decreased absorption in

gastrointestinal diseases.

Principle

Albumin binds with the dye bromocresol green in a

buffered medium to form a green colored complex. The

intensity of the color formed is directly proportional to the

amount of albumin present in the sample.

Albumin + Bromocresol green→ Green albumin BCG

complex.

Normal Reference Values (Albumin)

Serum, plasma (albumin) : 3.7–5.3 g/dL

Globulin : 2.3–3.6 g/dL

A/G Ratio : 1.0–2.3

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 150 mL 2 × 150 mL

Carton 1

L1: BCG reagent 150 mL 2 × 150 mL

Carton 2

S: Albumin standard (4 g/dL) 5 mL 5 mL

Storage/Stability

Carton 1 : BCG reagent is stable at RT till the expiry

mentioned on the label.

Carton 2 : Albumin Standard is stable at 2–8°C till the

expiry mentioned on the label.

Reagent Preparation

Reagents are ready to use. Protect from bright light.

Sample Material

Serum, EDTA plasma. Albumin is reported to be stable in

the sample for 6 days at 2–8°C.

Procedure

Wavelength/filter : 630 nm (Hg 623 nm)/Red

Temperature : RT

Light path : 1 cm

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

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

BCG reagent (L1) 1.0 1.0 1.0

Distilled water 0.01 - -

Albumin Standard (S) - 0.01 -

Sample - - 0.01

Mix well and incubate at RT for 5 minutes. Measure

absorbance of the standard (Abs S), and test sample (Abs T)

against the blank.

Calculations

 Abs T

Albumin in g/dL = ________ × 4 Abs S

Globulin in g/dL = (Total proteins) — (Albumin)

 (in g/dL) (in g/dL)

 Albumin in g/dL A/G Ratio = _________________

 Globulin in g/dL

Linearity

The procedure is linear upto 7 g/dL. If values exceed this

limit, dilute the sample with distilled water and repeat

480 Concise Book of Medical Laboratory Technology: Methods and Interpretations the assay. Calculate the value using the proper dilution

factor.

Note

Gross hemolysis, ampicillin and heparin interfere with

the results. Elevated bilirubin and lipemic samples may

have a slight effect on accuracy. For grossly lipemic

samples run a sample blank by adding 0.02 mL sample

in 2 mL distilled water. Read the absorbance against

DW and substract the blank absorbance from the test

absorbance.

System Parameters

Reaction : End point Interval :

Wavelength : 630 nm Sample

volume

: 0.01 mL

Zero setting :  Reagent

blank

Reagent

volume

: 1.00 mL

Incubation

temperature

: RT Standard : 4 g/dL

Incubated time : 5 minutes Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 7 g/dL

No. of read : —- Units : g/dL

Normal Values

Total Proteins

SI units

Adults 6.0–8.0 g/dL 60–80 g/L

Children

Premature 4.3–7.6 g/dL 43–76 g/L

Newborn 4.6–7.4 g/dL 46–74 g/L

Infant 6.0–6.7 g/dL 60–67 g/L

Child 6.2–8.0 g/dL 62–80 g/L

Specimen Collection and storage

1. Serum is the specimen of choice.

2. Avoid excessive hemolysis since every 100 mg/dL

of hemoglobin corresponds to about 100 mg/dL of

albumin.

3. Albumin in serum is reported stable for one week at

room temperature (18–30°C) and approximately one

month when stored in the refrigerator (2–8°C) and

protected against evaporation.

Clinical Relevance

Causes of Hypoalbuminemia

Reduced synthesis

¾ Malnutrition

¾ Malabsorption syndromes

¾ Chronic inflammatory diseases

¾ Acute hepatitis (lasting 14 days or more)

¾ Chronic liver disease

¾ Genetic abnormalities.

Increased Loss

¾ Nephrotic syndrome

¾ Massive burns

¾ Protein-losing enteropathy.

Increased catabolism

¾ Massive burns

¾ Widespread malignancy.

Multifactorial

¾ Cirrhosis

¾ Congestive heart failure

¾ Pregnancy.

Increased albumin levels are generally not observed

(When albumin concentration decreases there is a relative

increase in globulins. However, there is a definite rise in

globulins in mono/polyclonal gammopathies).

Disorders Associated with Polyclonal

Gammopathies

Chronic liver disease

¾ Nutritional cirrhosis

¾ Primary biliary cirrhosis

¾ Chronic active hepatitis

¾ Viral hepatitis.

Collagen diseases

¾ Rheumatoid arthritis

¾ Systemic lupus erythematosus

¾ Sjögren’s syndrome

¾ Felty’s syndrome

¾ Polymyositis

¾ Scleroderma.

Chronic Infections

¾ Tuberculosis

¾ Osteomyelitis

¾ Deep fungi

¾ Syphilis

¾ Bronchitis.

Miscellaneous

¾ Metastatic carcinoma

¾ Cystic fibrosis

¾ Recovery from trauma.

 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.

  Elevated levels

are found in renal dysfunction, reduced renal blood flow

(shock, dehydration, congestive heart failure) diabetes

acromegaly. Decreased levels are found in muscular

dystrophy.

Clinical Chemistry 473

Principle

Picric acid in an alkaline medium reacts with creatinine to

form a orange colored complex with the alkaline picrate.

Intensity of the color formed is directly proportional to the

amount of creatinine present in the sample.

Creatinine + Alkaline picrate→Orange colored complex

Reference Values

Serum Urine, 24 hours collection

Males : 0.6–1.2 mg% 1.1–3.0 g

Females : 0.5–1.1 mg% 1.0–1.8 g

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 15 Tests 35 Tests

L1: Picric acid reagent 60 mL 140 mL

L2: Buffer reagent 5 mL 12 mL

S: Creatinine standard (2 mg/dL) 5 mL 5 mL

Storage/Stability

All reagents are stable at RT till the expiry mentioned on

the labels.

Reagent Preparation

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

Sample Material

Serum or Urine

Creatinine is stable in serum for 1 day at 2–8°C

Urine of 24 hours collection is preferred. Dilute the

specimen 1:50 with distilled/deionised water before the

assay.

Procedure

Wavelength/filter 520 nm (Hg 546 nm)/green

Temperature RT

Light path 1 cm

Deproteinization of Specimen

Pipette into a clean dry test tube

Picric acid reagent (L1 ) 2.0 mL

Sample 0.2 mL

Mix well and centrifuge at 2500–3000 rpm for 10 minutes

to obtain a clear supernatant.

Color Development

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

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Supernatant - - 1.1

Picric acid reagent (L1) 1.0 1.0

Distilled water 0.1

Creatinine standard (S) - 0.1

Buffer reagent (L2 ) 0.1 0.1 0.1

Mix well and keep the test tubes at RT for exactly 20

minutes. Measure the absorbance of the standard (Abs S),

and test sample (Abs T) against the blank.

Calculations

 Abs T

Creatinine in mg% = _______ × 2.0

 Abs S

 Abs T

Urine creatinine in g/L = ______ × 1.0 Abs S

Urine creatinine g/24 h = Urine creatinine in g/L ×

Volume of urine in 24 hours in

liters.

Linearity

The procedure is linear upto 8 mg% 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

Maintain the reaction time of 20 minutes as closely as

possible since a longer incubation causes an increase in

the values due to the reaction of pseudochromogens. The

determination is not specific and may be affected by the

presence of large quantities of reducing substances in the

sample. The reaction is temperature sensitive and all the

tubes should be maintained at a uniform temperature.

System Parameters

Reaction : End point Interval :

Wavelength : 520 nm Sample

Volume

: 0.20 mL

Zero setting :  Reagent

blank

Reagent :  Volume

1.10 mL

Incubation

temprature

: RT Standard : 2 mg/dL

Incubated time : 20 minutes Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 8 mg/dL

No. of read : — Units : mg/dL

474 Concise Book of Medical Laboratory Technology: Methods and Interpretations Creatinine (Mod Jaffa’s Kinetic Method)

(Courtesy: Tulip Group of Companies)

For the determination of creatinine in serum and urine

(for in vitro diagnostic use only).

Summary

Creatinine is the catabolic product of creatinine phosphate

which is used by the skeletal muscle. The daily production

depends on muscular mass and it is excreted out of the body

entirely by the kidneys. Elevated levels are found in renal

dysfunction, reduced renal blood flow (shock, dehydration,

congestive heart failure) diabetes acromegaly. Decreased

levels are found in muscular dystrophy.

Principle

Picric acid in an alkaline medium reacts with creatinine to

form a orange colored complex with the alkaline picrate.

Intensity of the color formed during the fixed time is

directly proportional to the amount of creatinine present

in the sample.

Creatinine + Alkaline Picrate → Orange colored complex.

Reference Values

Serum Urine in 24 hours collection

Males : 0.6–1.2 mg% 1.1–3.0 g

Females : 0.5–1.1 mg% 1.0–1.8 g

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 2 × 35 mL 2 × 75 mL

L1: Picric acid reagent 35 mL 75 mL

L2: Buffer reagent 35 mL 75 mL

S: Creatinine standard (2 mg/dL) 5 mL 5 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.

Working reagent: For larger assay series a working reagent

may be prepared by mixing equal volumes of picric acid

reagent and buffer reagent. The Working reagent is stable

at RT (25–30oC) for at least one week.

Sample Material

Serum or Urine

Creatinine is stable in serum for 1 day at 2–8°C.

Urine of 24 hours collection is preferred. Dilute the

specimen 1:50 with distilled/deionised water before the

assay.

Procedure

Wavelength/filter : 520 nm (Hg 492 nm)/green

Temperature : 30°C/37°C

Light path : 1 cm

Pipette into a clean dry test tube labeled standard (S) or

test (T):

Addition

Sequence

(S)/(T)

30°C/37°C

Picric acid reagent (L1) 0.5 mL

Buffer reagent (L2) 0.5 mL

Bring reagents to the assay temperature and add

Creatinine standard (S)/sample/diluted urine 0.1 mL

Mix well and read the initial absorbance A1 for the

standard and test after exactly 30 seconds. Read another

absorbance A2 of the standard and test exactly 60 seconds

later.