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.

 

Urea standard/serum/diluted urine 0.01 mL

Incubate at the assay temperature for 1 minute and add

Starter reagent (L2) 0.2 mL

Mix well and read the initial absorbance A for the

standard and test after exactly 30 seconds. Read another

absorbance A2 of the standard and test exactly 60 seconds

later. Calculate the change in absorbance ∆A for both the

standard and test.

Sample Start Assay

Pipette into a clean dry test tube labelled Standard (S) or

Test (T):

Addition (S)/(T)

Sequence 37°C / 30°C / 25°C

Working reagent 1.0 mL

Bring to assay temperature and add

Urea standard/serum/diluted urine 0.01 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. Calculate the change in absorbance ∆A for both the

standard and test.

For Standard ∆AS = A2S – A1S

For Test ∆AT = A2T – A1T

Calculations

 ∆A T

Urea in mg/dL = __________ × 40 ∆A S

Linearity

This procedure is linear upto 250 mg/dL. If values exceed

this limit, dilute the serum with normal saline (NaCL 0.9%)

and repeat the assay. Calculate the value using the proper

dilution factor.

Note

Plasma should not be collected with fluoride or heparin

salts as contamination by ammonia or ammonium salts

lead to erroneous results.

System Parameters

Reaction : Fixed time kin. Interval : 60 sec.

Wavelength : 340 nm Sample

volume

: 0.01 mL

Zero

setting

:  Distilled

Water

Reagent

volume

: 1.00 mL

Incubation

temperature

: 30°C/37°C Standard : 40 mg/dL

Incubated time — Factor :

Delay time : 30 seconds React slope : Decreasing

Read time : 60 seconds Linearity : 250 mg/dL

No. of read : 2 Units : mg/dL

Normal Values (general reference)

Adults: BUN is 8–18 mg%, Urea = 15–40 mg%

Adults over 60 years: May have a little higher values

normally. Low values may be found during pregnancy and

in full-term infants, whereas premature infants may have

slightly higher values than the adult range.

Clinical Relevance

Common Causes of Increased BUN or Uremia

Prerenal

¾ Reduced blood flow to kidney

¾ Shock, blood loss, dehydration

¾ Increased protein catabolism

¾ Crush injuries, burns, fever, hemorrhage into soft tissue

or body cavities, hemolysis.

Renal

¾ Acute renal failure

¾ Glomerulonephritis, malignant hypertension, nephrotoxic drugs or metals, renal cortical necrosis

¾ Chronic renal disease

¾ Glomerulonephritis, pyelonephritis, diabetes mellitus,

arteriosclerosis, renal tubular disease, collagen-vascular

diseases.

472 Concise Book of Medical Laboratory Technology: Methods and Interpretations Post-renal

¾ Ureteral destruction by stones, tumor, inflammation,

surgical trauma, obstruction of bladder neck or urethra

by prostate, stones, tumor, inflammation.

Decreased BUN is Associated with

a. Liver failure.

b. Negative nitrogen balance as may occur in malnutrition, excessive use of IV fluids and physiologic

hydremia of pregnancy.

c. Impaired absorption as in celiac disease.

d. Occasionally in nephrotic syndrome.

e. Overhydration.

Interfering Factors

1. A combination of a low protein and a high carbohydrate

diet cause a decreased BUN level.

2. The BUN is normally lower in children and women

because they have a smaller muscle mass than adult

men.

3. Increased BUN values occur in late pregnancy and

infancy because of increased use of protein.

4. Older people may have an increased BUN when their

kidneys are not able to concentrate urine adequately.

5. Decreased BUN values may normally occur earlier in

pregnancy because of physiologic hydremia.

6. Many drugs can cause increased BUN levels.

7. Drugs that may cause decreased BUN levels include

• Dextrose infusions

• Phenothiazines

• Thymol.

Comments

1. Ammonium oxalate should not be used as an

anticoagulant. Plasma can be used if it is obtained from

EDTA, citrate, potassium oxalate or heparin.

2. If the serum sample is very lipemic, prepare a special

blank tube by adding the phenol color reagent to

the urease before adding the serum. Set the zero

absorbance for the particular sample with this blank.

3. For urgent test__the incubation time can be reduced

to 5 minutes if the water bath temperature is raised to

55–56oC.

4. Plasma or serum preserved with fluoride cannot be

used as this inactivates the enzyme. Urea is stable in

frozen serum for months.

5. Make sure that there is no contamination by ammonia

or heavy metal ions.

6. For a small laboratory, commercially available multi/

monostep kits can be used.

Plasma or Serum Creatinine

Normal Values

Conventional units SI units

Jaffe, manual method 0.8–1.5 mg/dL 70–133 µmol/day

Jaffe, kinetic or enzymatic method

Adult

Female 0.5–1.1 mg/dL 44–97 µmol/L

Males 0.6–1.2 mg/dL 53–106 µmol/L

Eldery May be lower May be lower

Children

Cord blood 0.6–1.2 mg/dL 53–106 µmol/L

Newborn 0.8–1.4 mg/dL 71–124 µmol/L

Infant 0.7–1.7 mg/dL 62–150 µmol/L

Age 1 female ≤ 0.5 mg/dL ≤ 44 µmol/L

Age 1 male ≤ 0.6 mg/dL ≤ 53 µmol/L

Age 2–3 female ≤ 0.6 mg/dL ≤ 53 µmol/L

Age 2–3 male ≤ 0.7 mg/dL ≤ 62 µmol/L

Age 4–7 female ≤ 0.7 mg/dL ≤ 62 µmol/L

Age 4–7 male ≤ 0.8 mg/dL ≤ 71 µmol/L

Age 8–10 female ≤ 0.8 mg/dL ≤ 71 µmol/L

Age 8–10 male ≤ 0.9 mg/dL ≤ 80 µmol/L

Age 11–12 female ≤ 0.9 mg/dL ≤ 80 µmol/L

Age 11–12 male ≤ 1.0 mg/dL ≤ 88 µmol/L

Age 13–17 female ≤ 1.1 mg/dL ≤ 97 µmol/L

Age 13–17 male ≤ 1.2 mg/dL ≤ 106 µmol/L

Age 18–20 female ≤ 1.2 mg/dL ≤ 106 µmol/L

Age 18–20 male ≤ 1.3 mg/dL ≤ 115 µmol/L

Creatinine (Alkaline Picrate 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.

 

For the determination of urea in serum, plasma and urine

(for in vitro diagnostic use only)

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further reacts with a phenolic chromogen and

hypochlorite to form a green colored complex. Intensity of

the color formed is directly proportional to the amount of

urea present in the sample.

 Urease

Urea + H2O Ammonia + CO2

Ammonia + Phenolic chromogen + Hypochlorite

 Green colored complex

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 assays 3 × 75 assays

L1: Buffer reagent 75 mL 3 × 75 mL

L2: Enzyme reagent 7.5 mL 3 × 7.5 mL

L3: Chromogen reagent 15 mL 45 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/Stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use for the given procedure.

Working enzyme reagent: For the flexibility and

convenience in performing large assay series, a working

enzyme reagent may be made by pouring 1 bottle of L2

(Enzyme reagent) into 1 bottle of L1 (Buffer reagent). For

smaller series combine 10 parts of L1 (Buffer reagent) and

1 part of L2 (Enzyme reagent). Use 1 mL of the working

reagent per assay instead of 1 mL of L1 and 0.1 mL of L2

as given in the procedure. The working enzyme reagent is

stable for at least 4 weeks when stored at 2–8°C.

Working chromogen reagent: For larger volume cuvettes,

dilute 1 part of L3 (Chromogen reagent) with 4 parts of

fresh ammonia free distilled/deionised water. Use 1 ml

of working chromogen instead of 0.2 mL in the assay. The

working chromogen reagent is stable for atleast 8 weeks

when stored at 2–8°C in a tightly stoppered plastic bottle.

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50). Urea is reported to be

stable in the serum for 5 days when stored 2–8°C.

Procedure

Wavelength/filter : 570 nm (Hg 578 nm)/yellow

Temperature : 37°C/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)

Buffer reagent (L1 ) 1.0 1.0 1.0

Enzyme reagent (L2) 0.1 0.1 0.1

Distilled water 0.01 — —

Urea standard (S) 0.01 —

Sample — — 0.01

Mix well and incubate for 5 minutes at 37°C or 10 minutes at RT

(25°C)

Chromogen reagent (L3) 0.2 0.2 0.2

470 Concise Book of Medical Laboratory Technology: Methods and Interpretations Mix well and incubate for 5 minutes at 37°C or

10 minutes at RT (25°C). Measure the absorbance of the

Standard (Abs S), and Test sample (Abs T) against the

Blank, within 60 minutes.

Calculations

 Abs T

Urea in mg/dL = ________ × 40 Abs S

Urea nitrogen in mg/dL = Urea in mg/dL × 0.467

Linearity

This procedure is linear upto 250 mg/dL. Using the working

chromogen reagent (1 mL) the linearity is increased to

400 mg/dL. If values exceed this limit, dilute the serum

with normal saline (NaCL 0.9%) and repeat the assay.

Calculate the value using the proper dilution factor.

Note

Any contamination by ammonia or ammonium salts lead

to erroneous results, hence plasma should not be collected

with fluoride or heparin ammonium salts. The working

enzyme reagent is not stable at elevated temperatures and

should be stored back at 2–8°C immediately after use. The

chromogen reagent contains chlorine. The bottle should

be opened only when required and closed tightly after use

to prevent the loss of active chlorine.

System Parameters

Reaction : End point No. of read :

Wavelength : 570 nm Interval :

Zero setting :  Reagent blank Sample

volume

: 0.01 mL

Incubation

temperature

: 37°C/RT Reagent

volume

: 1.30 mL

Incubated

time

:  5 min + 5 min

or

10 min +10 min

Standard

factor

React slope

: 40 mg/dL

: Increasing

Delay time : Linearity : 250 mg/dL

Read time : .... Units : mg/dL

Urea (GLDH Kinetic Method)

(Courtesy: Tulip Group of Companies)

For the determination of urea in serum or plasma (for

in vitro diagnostic use only).

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further combines with a ketoglutarate and NADH

to form glutamate and NAD. The rate of oxidation of NADH

to NAD is measured as a decrease in absorbance in a fixed

time which is proportional to the urea concentration in the

sample.

 Urease

Urea + H2O + 2 H+ 2 NH4 + CO2

 GLDH

2 NH4

+ + 2 α Ketoglutarate + ↓

 2 NADH 2 L-glutamate + 2

 NAD+ + 2 H2O

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 mL 2 ×75 mL

L1: Enzyme reagent 60 mL 2 × 60 mL

L2: Starter reagent 15 mL 2 × 15 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme reagent).

This working reagent is stable for at least 10 days when

stored at 2–8°C. Alternatively for flexibility as much of

working reagent may be made as and when desired by

mixing together 4 parts of L1 (Enzyme reagent) and 1

part of L2 (Starter reagent). Alternatively 0.8 mL of L1 and

0.2 mL of L2 may also be used instead of 1 mL of the

working reagent directly during the assay.

Clinical Chemistry 471

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50 ). Urea is reported to be

stable in the serum for 5 days when stored at 2–8°C.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Substrate Start Assay

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

test (T):

Addition

Sequence

(S)/(T)

37°C/ 30°C/25°C

Enzyme reagent (L1) 0.8 mL