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![ The result are calculated as follows-
Mg/dl, Creatinine=
O.D.T[80 sec]-O.D.T[20sec] X conc. of std.
O.D std[80sec]-O.D std[20sec]](https://image.slidesharecdn.com/creatinine-estimation-151008095551-lva1-app6892/75/Creatinine-estimation-17-2048.jpg)
Creatinine estimation
This document describes the Jaffe method for measuring serum creatinine levels. It discusses how creatinine reacts with picric acid in an alkaline solution to form an orange-red complex, which is measured spectrophotometrically. It notes that the kinetic method is now more widely used as it is faster and more specific. The kinetic method measures the rate of change in absorbance over time to account for interfering substances. Normal creatinine levels and causes of increased or decreased levels are also mentioned.
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Creatinine estimation
- 1. Dr. Urender S.Ghanghas MBBS, MD IIyr For BMLT, DMLT BIOCHEMISTRY SGT MEDICAL COLLEGE GURGOAN
- 2. Principle Sample Reagents Procedures Wavelength Reading Calculation Normal values Interpretation
- 3. 1. Jaffe method(Manualmethod) Disadvantage: non creatinine chromagen interfere with the reaction e.g. protein , glucose, Ketones, Pyruvate, Bilirubin……. 2. Kinetic analyses modes(Automated method)
- 4. PFF +Saturated picrate acid (alkaline media) NaOH Creatinine picrate orange red complex (absorbed at 510 nm)
- 5. Serum orPlasma Protein free filtrate: to avoid non creatinine chromagen interfering with the reaction
- 6. Saturated picrateacid Creatinine standard -1 mg% NaOH-5% Sodium tungstate solution – 10% H2SO4
- 8. In acentrifuge tube take; 1. 1 ml of DW 2. 1 ml serum 3. 1 ml of sodium tungstate 4. 1 ml of H2SO4 Mix and centrifuge for 5 min. to obtain clear supernatant PFF
- 9. Blank Stand. Test Sample:PFF --- --- 2 ml DW 4 ml 3 ml 2 ml Standard (0.01mg/ml) -- 1 ml -- NaOH 1 ml 1 ml 1 ml Sat. Picric acid 1 ml 1 ml 1ml Mix Incubate at Room Temp. for 10 min. Read the OD test & Stand. against the blank at 510 nm
- 10.
- 11. Read thesample against its blank at 510 nm O.D. =…………..
- 12. According toBeer Lambert law C T = ODT X CSTD ODSTD Serum Creatinine (mg%) = O.D Test X Amount of standard X 100 O.D Std. Volume of sample Conc. = …………. mg/dl
- 14. KINECTIC METHOD:- they were developed in a quest both for specificity and for faster and automate analysis .Rate is measured: Faster More specific Most widely used Implemented on various automated instruments. JAFFE’S KINETIC ASSAY ENZYMATIC KINETIC ASSAY
- 15. In thismethod also Jaffe’s reaction principle is used but defined experimental condition, by using special type of spectrophotometer or autoanalyser, equipped with a thermocuvette. Jaffe’s reaction is carried out at 30 degree centigrade as follows:
- 16. First readingis recorded at 20 sec. Most of the interfering non-creatinine chromogens react with alkaline picrate reagent fast and are recorded after 20 seconds. Creatinine and alkaline picrate react relatively slowly after 20 seconds,upto 80 seconds.Hence second reading is noted after 80 seconds. Same procedure is used for a standard.
- 17. The resultare calculated as follows- Mg/dl, Creatinine= O.D.T[80 sec]-O.D.T[20sec] X conc. of std. O.D std[80sec]-O.D std[20sec]
- 18. Dilute urine1:100 with distilled water so that the creatinine values come within the range of Lambert-Beer’ s law and the interfering substances get diluted. Urine creatinine is then estimated by using Jaffe’s method.
- 19. B S T DiluteUrine --- --- 3 ml Distilled water 3 ml --- --- Standard(0.01 mg/ml) --- 3 ml --- NaOH 5% 1 ml 1 ml 1 ml Sat.Picric acid 1 ml 1 ml 1 ml Mix for 15 min. at room temp. Take OD at 520 nm. Calculate the result.
- 20. Increased values =(Azotemia) 1. Prerenal: systemic infection e.g. babesiosis 2. Renal: e.g. Amyloidosis, Glomerulosclerosis and Polycystic disease 3. Post renal: e.g. Ureteral obstruction
- 21. Decreased creatinine bloodlevel Muscle disease Cachexia Portosystemic shunts