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.

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(Manual method)
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 or Plasma
Protein free filtrate: to avoid non creatinine
chromagen interfering with the reaction

6.  Saturated picrate acid
 Creatinine standard -1 mg%
 NaOH-5%
 Sodium tungstate solution – 10%
 H2SO4

8.  In a centrifuge 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. 510 nm

11.  Read the sample against its blank at 510 nm
 O.D. =…………..

12.  According to Beer 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 this method 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 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.

17.  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]

18.  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 Jaffe’s method.

19. B S T
Dilute Urine --- --- 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 blood level
Muscle disease
Cachexia
Portosystemic shunts