CLINICAL SIGNIFICANCE PRINCIPLE

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).
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