Creatinina Peo

University of Texas at El Paso
Clinical Laboratory Science
500 W University Ave
El Paso, TX 79902

CHEMISTRY LABORATORY


Copy _____of_____ Effective Date: _________

WHO Creatinine – Jaffe’s method


Summary and Principle
Creatinine is a waste product formed in muscle from a high-energy storage compound, creatine phosphate. Creatine phosphate can be stored in muscle atapproximately four times the concentration of adenosine triphosphate. In muscles it spontaneously undergoes degradation to form a cyclic anhyride-creatinine. The blood concentration of creatinine and its excretion in urine are remarkably constant in normal individuals. Therefore serum creatinine level is used as an indicator for assessing kidney function.
Creatinine present in serum or plasma directlyreacts with alkaline picrate resulting in the formation of a red colour, the intensity of which is measured at 505nm/green filter. Protein interference is eliminated using sodium lauryl sulphate. A second absorbance reading after acidifying with 30% acetic acid corrects for non-specific chromogens in the samples.
Specimen type, collection and storage
Serum or plasma can be used. Avoid usinghaemolysed or lipaemic samples. Stable for 12 hours at room temperature (25-350C), one week at 2-80C and for 3 months at –200C.
Materials and Reagents
All chemicals must be Analar grade

Reagent A
Into 400ml of distilled water taken in a 500 ml beaker add 4.4g of NaOH. Mix to dissolve, then add 9.5g trisodium phosphate [Na3PO412H2O], dissolve and then add 9.5g of sodiumtetraborate[Na2B4O710H2O]. After dissolving check that the pH is above 10, adjust if necessary by the dropwise addition of 1M NaOH. Transfer to a 500 ml volume flask and make up to 500ml with distilled water. Mix well. Stable for 3 months at 2-80C.



Reagent B
Dissolve 20g sodium lauryl sulfate in a final volume of 500ml distilled water. Stable for 6 months at room temperature (25-350C).
Reagent CPicric acid supplied commercially contains 50% by weight of water to ensure safety in transit. Therefore the amount of picric acid weighed out should be proportionally more than the amount of the required anhydrous picric acid.
For reagent C, 4.6g of anhydrous picric acid is required. Therefore weigh approximately 7.0g but not less than 6.0g moist picric acid and add to 500ml of distilled watertaken in a volumetric flask, mix and leave overnight at 370 C. Then filter and store in brown glass bottle at room temperature (25-350C). Stable for 1 year.
Working reagent
At the time of analysis freshly mix equal volumes of the above three reagents. After use discard any leftover working reagent.
Stock creatinine standard 100mg/dl
Dissolve 100 mg of pure creatinine in 0.1 M HCl andmake up to 100 ml with 0.1 M HCl in a volumetric flask. Stable for 6 months at 2-80 C.
Working creatinine standard
Dilute 2, 4, 6 and 8 ml of stock creatinine standard each to 100 ml with 0.1 M HCl to get creatinine concentrations of 2, 4, 6 and 8 mg/dl, respectively. Stable for 6 months at 2-80C.
30% (V/V) Acetic acid
Dilute 30ml of glacial acetic acid to 100ml with distilled water.Stable for 3 months at room temperature (25-350 C).
Precautions
Picric acid is poisonous and sodium hydroxide is caustic – avoid contact with skin and mucous membranes
Quality Control
Since creatinine is one of the most common analytes measured in a laboratory, inclusion of an internal QC (normal QC pool) with every batch of samples analysed in the day, is recommended, irrespective ofthe number of samples in a batch. Further, even when a single sample is analysed as an "emergency" sample at any time of the day or night, it is essential to include an internal QC. From the QC results obtained for the day, mean, standard deviation and %CV can be calculated to ensure that within-day precision is well within the acceptable limit, i.e, 5%.
The mean value of internal QC for the...