Creatinine estimation

FOR BMLT AND DMLT

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%
H
2SO
4

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 H
2
SO
4
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 = OD
T X C
STD

OD
STD
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 water3 ml --- ---
Standard(0.01
mg/ml)
--- 3 ml ---
NaOH 5% 1 ml 1 ml 1 ml
Sat.Picric acid1 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