MANUAL METHOD CLINICAL SIGNIFICANCE Increased levels are associated with substantially impaired renal function. PRINCIPLE In an alkaline medium, creatinine reacts with picrate to produce an ORANGE YELLOW colour. The coloration Is proportional to the creatinine concentration. REAGENTS SUPPLIED Cat. No. A 156 2x50 mL Composition 1. Picric Acid 50 mL Picric acid 30 mMol/L 2. Sodium Hydroxide 50 mL Sodium hydroxide 750 mMol/L 3. Standard 10 mL Creatinine 177 u.Mol/L (2 mGs/dL) Preparation of Working Reagent Reagent 1 ................................... 1 volume Reagent 2................................... 1 volume Mix well. Keep for 10 mine for maturation. Quality control of the reagent If the O.D. of reconstituted reagent at 510 nM is >0.300 against distilled water it must be discarded. STABILITY 18 months at 18-25°C away from light. The working reagent is stable for 7 days at 2-8°C and 24 hours at 28°C ± 5°C, away from light. SAMPLE Non-hemolysed serum or plasma (heparin or EDTA) No prior patient preparation is needed. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials). 1. Set the Colorimeter '0' Absorbance with dist. water at 510 nM (490-540 nM). 2. Pipette into optical cuvettes: I mL Sample or Standard ...................................................... 0.5 Working Reagent .......................................................... | 2.5 Perform the Test with Sample or Standard in the same way at R.T. On addition of working reagent in sample or standard start a stopwatch immediately and mix well. Place the tube in colorimeter or spectrometer. 3. After 20 seconds read the O.D. at 510 nM against Blank (i.e. OD^. 4. After exactly 80 seconds read again the O.D. (i.e. OD2) at the same wave length against Blank. NOTE: Reagent and Sample volume can be altered proportionately to accomodate different instruments cuvette size e.g. 4 mL reagent and 1 mL serum or diluted urine. Micro method for discrete analysers QUALITY CONTROL To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both Instrument and reagent function together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glasswares and accuracy of pipetting. It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly, laboratory findings should be established by clinical manifestations. URINE SAMPLE Fresh urine sample preferred Dilute urine 1 to 25 or 1 to 50 in distilled water. Perform test with this dilution as for the serum. Multiply result by dilution factor (25 or 50). SYSTEM PARAMETERS Reaction Fix-Time Temperature 28°C Wavelength 510 nM Standard Concentration Absorbance Range Kinetic ± 5°C (490-540 nM) 2 mGs/dL 0-1° A Cuvette Path Length _________________ 1 cM Reaction Time 60 seconds Delay Time 20 seconds Interval 60 seconds Number of readings 2 Linearity 9 mGs/dL Max. limit of blank rgt. 0.2°A WARNING This reagent system is for in vitro use only. This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incident wash off with plenty of water, or consult a physician. _______ Cuvette size.......................................... I 0.5 mL I 1.0 mL Working Reagent ................................... 500 ul 1000 \± Sample or Standard .............................. 50 u.L 100 u.L Zero set the instrument with Working Reagent Blank between each test. On addition of working reagent to sample mix well and take readings at 20 seconds (OD^ and 80 seconds (OD2) at 510 nM (490-540 nM). NOTE: Analysers equipped with flow-through cuvette must set "zero" with working reagent blank to obtain more reproducibility. Programme the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request. Bibliography 1.COOKJ.G.H. - Clin. Chem. Acta, 1971, 32, 485. 2. BARTELS H. BOHMER M., HEIERLI As with all diagnostic methods, the final diagnosis should not be C- Clin. Chem. Acta, 1972, 37,193. made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations. LIMITATIONS Alkaline picrate is sensitive to various metalic elements. In vitro addition of metalic compounds or contamination makes false results. This method highly depends on accuracy and precision of instrument. This assay Is linear to 9 mGs/dL. For levels higher than 9 mGs/dL (800 uMol/L), repeat the test diluting the sample with distilled water. Multiply the result by the dilution factor (i.e. 2 for a dilution of 1:1). BIOLAB DIAGNOSTICS (I) PVT. LTD. J-245, MIDC, Tarapur, BOISAR 401 501. Maharashtra, INDIA. E-mail : [email protected], [email protected] http://www.biolabdiagnostcs.com