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.