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    (Modified Jaffe's Kinetic Method & End Point Method) For The Determination of Creatinine in Serum and Urine (For in Vitro Diagnostic Use Only)

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    Dinesh Sreedharan
    This document describes procedures for determining creatinine levels in serum and urine using a modified Jaffe's kinetic method. Creatinine forms a colored complex with picric acid in an alkaline solution. The intensity of the color is directly proportional to the creatinine concentration and can be measured spectrophotometrically. Normal creatinine levels in serum and 24-hour urine collections are provided along with details on sample collection, reagent preparation, assay parameters, and calculations.

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    (Modified Jaffe's Kinetic Method & End Point Method) For The Determination of Creatinine in Serum and Urine (For in Vitro Diagnostic Use Only)

    Uploaded by

    Dinesh Sreedharan
    58 views1 page
    This document describes procedures for determining creatinine levels in serum and urine using a modified Jaffe's kinetic method. Creatinine forms a colored complex with picric acid in an alkaline solution. The intensity of the color is directly proportional to the creatinine concentration and can be measured spectrophotometrically. Normal creatinine levels in serum and 24-hour urine collections are provided along with details on sample collection, reagent preparation, assay parameters, and calculations.

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    Crea

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    This document describes procedures for determining creatinine levels in serum and urine using a modified Jaffe's kinetic method. Creatinine forms a colored complex with picric acid in an alkaline solution. The intensity of the color is directly proportional to the creatinine concentration and can be measured spectrophotometrically. Normal creatinine levels in serum and 24-hour urine collections are provided along with details on sample collection, reagent preparation, assay parameters, and calculations.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    58 views1 page

    (Modified Jaffe's Kinetic Method & End Point Method) For The Determination of Creatinine in Serum and Urine (For in Vitro Diagnostic Use Only)

    Uploaded by

    Dinesh Sreedharan
    This document describes procedures for determining creatinine levels in serum and urine using a modified Jaffe's kinetic method. Creatinine forms a colored complex with picric acid in an alkaline solution. The intensity of the color is directly proportional to the creatinine concentration and can be measured spectrophotometrically. Normal creatinine levels in serum and 24-hour urine collections are provided along with details on sample collection, reagent preparation, assay parameters, and calculations.

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    CREATININE ASSAY PROCEDURE

    (Modified Jaffe's Kinetic Method & End point Method) FIXED - TIME KINETIC METHOD:
    For the determination of Creatinine in serum and urine Pipette into test tubes labeled Standard (S) And Test (T)
    (For In vitro Diagnostic Use Only) Addition Sequence Standard (µl) Test(µl)
    Working reagent 1000 1000
    Standard 50 -
    SUMMARY:
    Sample - 50
    Creatinine is the catabolic product of creatine 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 Mix well and read absorbance of S and T against distilled water at 520nm:
    kidneys. Initial absorbance A0 - Exactly after 30 sec
    Final absorbance A1 - Exactly after 90 sec. After Ao
    PRINCIPLE: Determine A for Standard (S) and Test (T)
    Alkali AS = AS1- AS0
    Creatinine + Sodium Picrate Creatinine Picrate-Complex AT = AT1- AT0
    (Yellow - Orange) END POINT METHOD:
    Intensity of the colour formed is directly proportional to the amount of creatinine present in the Pipette into test tubes labeled Standard (S) and Test (T) :
    sample. Addition Sequence S(µl) T(µl)
    Working reagent 1000 1000
    CONTENTS: Standard (s) 100 -
    PACK SIZE PICRIC ACID (A1) BUFFER REAGENT(A2) STOPPING SOLUTION(A3) STANDARD (S) Sample - 100
    Mix well and keep at RT for 5 min. Read absorbance A 0 for S and T against distilled water at 520
    2x50ml 50ml 50ml 6ml 5ml nm or with green filter (505-570 nm.)
    2x100ml 100ml 100ml 12ml 5ml Stopping Solution (A3) 50 50
    Mix well and keep at RT for 5 min.Read absorbance A1 for S and T against distilled water at 520
    STORAGE AND STABILITY: nm or with green filter (520-570 nm.). Determine A for S and T AS = AS0 -AS1
    All reagents are stable at RT (below 30°C) till the expiry mentioned on the labels. AT = AT0- AT1
    CALCULATIONS:
    REAGENT PREPARATION: a) Serum Creatinine in mg / dl = ( AT / AS) x 2
    Mix one Volume of Reagent (A1) with one volume of Reagent (A2) (According to the b) Urine Creatinine in gm / L = ( AT / AS) x 2
    requirement) the working reagent is stable for 1 week when stored in dark at R.T (below 30°C). c) Urine Creatinine in gm / 24 hrs. = b x24 hrs Urine volume collected in Litres.
    NORMAL REFERENCE VALUE:
    SAMPLE MATERIAL: Serum Urine, 24 hrs collection
    Serum or Urine Males 0.6-1.2mg/dl 1.1-3.0 gm
    Creatinine is stable in serum for 1 day at 2-8°C Females 0.5-1.1 mg/dl 1.0-1.8 gm
    It is recommended that each laboratory establish its own normal range representing its patient
    Urine of 24 hours collection is preferred. Dilute the specimen
    population.
    1:50 with distilled/deionised water before the assay.
    LINEARITY:
    GENERAL SYSTEM PARAMETER: The procedure is linear upto 20mg/dl of creatinine .If the values exceed this limit, dilute the
    sample with distilled water repeat the assay. Calculate the value using the proper dilution factor.
    Reaction Two Point or fixed time Sample Vol. 50 µl NOTES:
    Mode Kinetic/ End point Maintain the reaction time of 20 min, as closely as possible since a longer incubation causes an
    Wavelength 520 nm Reagent Vol. 500 µl(A1) + 500 µl(A2) increase in the values due to the reaction or pseudo chromogen the determination is not specific
    and may be affected by the presence of large quantities of reducing substance in the sample.
    Blank Distilled water Standard 2 mg / dl The reaction is temperature sensitive and all the tubes should be maintained at a uniform
    Incub. Temp 37°C React. Slope Increasing temperature

    Delay Time 30 Sec. Linearity 20 mg/dl REFERENCE :


    1. Bones, R.W. etal .(1945) J.Bio. Chem 158,581.
    Read Time 90 Sec Units mg/dl 2. Toro.G. etal. (1975) practical clinical chem.

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