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Transcript of Creatinine estimation
Dr. Urender S. GhanghasMBBS, MD IIyr For BMLT, DMLTBIOCHEMISTRYSGT MEDICAL COLLEGEGURGOAN
1. Jaffe method(Manual method) Disadvantage: non creatinine chromagen interfere with the reaction e.g. protein , glucose, Ketones, Pyruvate, Bilirubin.2. Kinetic analyses modes(Automated method)
PFF + Saturated picrate acid
(alkaline media) NaOH
Creatinine picrate orange red complex (absorbed at 510 nm)
Serum or Plasma
Protein free filtrate: to avoid non creatinine chromagen interfering with the reaction
Saturated picrate acid Creatinine standard -1 mg%NaOH-5%Sodium tungstate solution 10%H2SO4
In a centrifuge tube take;
1. 1 ml of DW 2. 1 ml serum3. 1 ml of sodium tungstate4. 1 ml of H2SO4Mix and centrifuge for 5 min. to obtain clear supernatant PFF
BlankStand.TestSample :PFF------2 mlDW4 ml3 ml2 mlStandard (0.01mg/ml)--1 ml--NaOH1 ml1 ml1 mlSat. Picric acid1 ml1 ml1mlMixIncubate at Room Temp. for 10 min.Read the OD test & Stand. against the blank at 510 nm
Read the sample against its blank at 510 nmO.D. =..
According to Beer Lambert lawC T = ODT X CSTD
Serum Creatinine (mg%) = O.D Test X Amount of standard X 100 O.D Std. Volume of sample
Conc. = . mg/dl
KINECTIC METHOD :- they were developed in a quest both for specificity and for faster and automate analysis .Rate is measured:FasterMore specificMost widely used
Implemented on various automated instruments.JAFFES KINETIC ASSAYENZYMATIC KINETIC ASSAY
In this method also Jaffes reaction principle is used but defined experimental condition, by using special type of spectrophotometer or autoanalyser, equipped with a thermocuvette. Jaffes reaction is carried out at 30 degree centigrade as follows:
First reading is 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.
The result are calculated as follows-
O.D.T[80 sec]-O.D.T[20sec] X conc. of std. O.D std[80sec]-O.D std[20sec]
Dilute urine 1:100 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 Jaffes method.
BSTDilute Urine------3 mlDistilled water3 ml------Standard(0.01 mg/ml)---3 ml---NaOH 5%1 ml1 ml1 mlSat.Picric acid1 ml1 ml1 mlMix for 15 min. at room temp.Take OD at 520 nm. Calculate the result.
Increased values = (Azotemia)
Prerenal: systemic infection e.g. babesiosisRenal: e.g. Amyloidosis, Glomerulosclerosis and Polycystic disease Post renal: e.g. Ureteral obstruction
Decreased creatinine blood level
Muscle diseaseCachexiaPortosystemic shunts