Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Creatinine – Jaffe’s method

     Introduction

Creatinine is a waste product formed in muscle from a high-energy storage compound, creatine phosphate. Creatine phosphate can be stored in muscle at approximately 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.

     Principle of the method

Creatinine present in serum or plasma directly reacts 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 using haemolysed or lipaemic samples. Stable for 12 hours at room temperature (25-35⁰C), one week at 2-8⁰C and for 3 months at –20⁰C.

     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 sodium tetraborate [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 C

Picric 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 water taken 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 and make 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).

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP.

     Procedure

The protocol of the procedure is described below.

Pipette the following into appropriately labelled 18 x 150 mm tubes

(Standards:S2 =2mg/dl, S4=4mg/dl, S6=6mg/dl & S8=8mg/dl)

Leave at room temperature (25-35⁰C) for 30 minutes. Set the spectrophotometer/ filter photometer to zero with blank at 505 nm/green filter and measure the absorbance of the other tubes. After measuring the absorbance pour the solutions back into the respective tubes. Then add 0.2 ml of 30% acetic acid to the test and QC tubes, mix well and leave at room temperature (25-35⁰C) for 5 minutes. Again set the spectrophotometer/filter photometer to zero with blank at 505nm/green filter and measure the absorbance of test and QC.

      Calculation and calibration graph

Subtract the second absorbance values of test and QC from the first set of values. Draw a calibration graph by plotting the absorbance values of standards against their respective concentrations. The measurable range with this graph is from 0.2 to 8.0 mg/dl. Plot the corrected absorbance of test and QC and read off the values of creatinine.

Once linearity is proved, it is enough if a single standard such as S6 is taken each time when patients’ samples are analysed and the results are calculated using the following formula

Serum Creatinine =   Test absorbance
                                -------------------------- x 6 mg/dl
                                 Standard absorbance     

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients’ results).

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 of the 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 day can be pooled with the preceding 10 or 20 mean values obtained in the previous days and between–day precision can be calculated and expressed as % CV. Ensure that this is well within the acceptable limit, i.e, 8%.

At least once a day analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials.

     Hazardous materials

Picric acid is poisonous and sodium hydroxide is caustic – avoid contact with skin and mucous membranes.

     Reference range and clinical interpretation

Serum/Plasma Creatinine: Male 0.7 – 1.4 mg%
Female 0.4 – 1.2 mg%

Serum creatinine concentration is related to muscle mass and the values are lower in children. Increased serum creatinine is associated with decrease in glomerular filtration rate (GFR), whether the cause is pre-renal, renal or post- renal. Pre-renal factors include conditions such as congestive heart failure, shock, diarrhoea, uncontrolled diabetes mellitus, use of diuretics, etc. Renal factors involve mainly damage to the glomeruli. Post-renal factors may be prostatic hypertrophy, calculi blocking the ureters or neoplasms compressing the ureters. The serum creatinine concentration is monitored closely after a renal transplantation because a rising concentration, even though small, may be an indication of graft rejection.

     Limitations

Ascorbic acid, uric acid, glucose, ketones and cephalosporin antibiotics, if present at high concentrations, may interfere in the assay causing falsely high values. Do not report the result from specimens with suspected interference. Inform the requesting physician of the problem.

     References

1.      Slot C. (1965) Scand J. Clin. Lab Invest. 17, 381 – 387.

2.      Seation B. & Ali A (1984) Med. Lab Sci., 41, 327- 336.