CREATININE REAGENT SET
Kinetic and endpoint
methods for the quantitative determination
of creatinine in serum.
INTRODUCTION
Creatinine, an anhydride of
creatine, is a waste product formed by
the spontaneous dehydration of kidneys.1 Most of the creatinine is
found in muscle tissue where it is present as creatine phosphate and serves as
a high energy storage reservoir for conversion to ATP. In- dependent of diet
serum creatinine concentrations depends almost entirely upon its excretion rate
by the kidneys. For this reason, its
elevation is highly specific for kidney diseases.2 The assay of creatinine has been based on the
reaction of creatinine with alkaline picrate as described by Jaffe. Further
modifications have developed the Jaffe reaction into a kinetic assay that is
fast, simple, and avoids interferences. In the endpoint method, acetic acid is
used to destroy the creatinine picrate complex, resulting in a loss of color,
the non creatinine serum constituents retain their picrate derived colors and
thus, the differences in absorbances gives the
creatinine concentration.3,4 This procedure is further
modified by Heinegard and Tiderstrom3 to eliminate the interference without acid
treatment.
PRINCIPLE
Alkali
Creatinine + Sodium Picrate
------> Creatinine - Picrate complex (yellow-orange)
Creatinine reacts with picric
acid in alkaline conditions to form a color complex which absorbs at 510
nm. The rate of formation of color is
proportional to the creatinine concentration in the sample. In the endpoint
method the difference in absorbance measurements after color formation yields a
creatinine value corrected for interfering substances.
REAGENTS
1. Creatinine
Picric Acid Reagent: a solution
containing 10mM
picric acid.
2. Creatinine
Buffer Reagent: a solution containing 10
mM.
sodium
borate, 240 mM, sodium hydroxide and surfactant.
*Important
Note: If the Creatinine Buffer Reagent
has been
subjected to cold temperatures white
precipitate may form.
Warm reagent to 37°C with
agitation to dissolve all the
precipitate before use.
3. Creatinine standard (5.0 mg/dl):
A solution containing creatinine in hydrochloric acid
with preservative.
PRECAUTIONS
1. This reagent is for "in vitro" diagnostic use only.
2. Creatinine Picric Acid Reagent is a strong oxidizing agent.
Avoid contact with skin. WIPE ANY
SPILLAGE SINCE PICRIC ACID IS EXPLOSIVE.
3. Creatinine buffer reagent is an alkali. Avoid ingestion and contact.
REAGENT PREPARATION
Combine equal volumes of
Creatinine Picric Acid Reagent and
Creatinine Buffer Reagent,
mix well.
REAGENT STORAGE
1. Both reagents are stored at room temperature
(18 - 25°C).
2. Combined (working) reagent is stable for up
to one (1) week.
REAGENT DETERIORATION
The reagent should be
discarded if:
1. Turbidity has
occurred; turbidity may be a sign of contamination.
2. The reagent fails to meet linearity claims or fails to recover
control values in the stated range.
SPECIMEN COLLECTION AND STORAGE
1. Serum is recommended.
2. Creatinine in serum is stable for
twenty-four (24) hours at refrigerated temperatures (2 - 8°C)
and several months when fro- zen (-20° C) and protected from evaporation and
contamination.
3. 24 hour urine specimens must be preserved with 15 grams of boric
acid.
INTERFERENCES
A number of substances affect
the accuracy of creatinine
determination. See Young et al5 for a comprehensive list.
MATERIALS PROVIDED
1. Creatinine Picric Acid Reagent.
2. Creatinine Buffer Reagent.
3. Creatinine Standard.
MATERIALS REQUIRED BUT NOT
INCLUDED
1. Pipetting
devices.
2. Timer.
3. Heating bath/rack.
4. Test tube/rack.
5. Vessel for combining reagents (glass or plastic).
6. Spectrophotometer with a
temperature controlled cuvette.
PROCEDURE (AUTOMATED)
Not available.
ASSAY PROCEDURE
1. Combine equal volumes of Creatinine Picric Acid Reagent and
Creatinine Buffer Reagent, mix well.
2. Label test vial, reagent blank, standard, control, and unknown
test tubes.
3. Pipette 3. 0 ml of working reagent into test tubes.
4. Transfer 0.1 ml (100 µl) of sample to its respective tube,
distilled water to reagent blank and mix.
5. Place all tubes in 37°C healing bath for fifteen (15) minutes.
6. Set wavelength of the spectrophotometer at 510 nm and zero the
instrument with the reagent blank. Read and record the absorbance of all tubes.
(Wavelength range:500-520).
7. Calculate creatinine value. See "calculations".
* USE TC - MUTI PURPOSE CALIBRATOR TO
REPLACE STANDARD.
ALTERNATE VOLUMES
If the spectrophotometer in
use requires a volume less than 3.0 ml for accurate reading, use 0.05 ml (50
µl) sample to 1.0 ml reagent. Perform as above.
CALCULATIONS
The creatinine value of the
unknown is determined by comparing its
absorbance change with that
of a known standard.
Abs. (Unknown)
mg/dl
= -------------------------- ´ Concentration of Standard
Abs. (Standard)
Where:
Abs.
= Absorbance
SAMPLE CALCULATION
If:
Abs./Unknown = 0.040
Abs./Standard = 0.150
Conc. of Standard =
5.0 mg/dl
Then
0.040
-------- ´ 5.0
= 1.3 mg/dl
creatinine
0.150
PROCEDURE LIMITATIONS
Albumin at a concentration of
10.0 gm/dl contributes 0.2 mg/dl
to the creatinine value, moderate hemolysis (0.2 gm/dl
Hgb), grossly icteric and lipemic samples will give elevated results. Acetoacetate above 10 mg/dl
will interfere with the results.
CALIBRATION
Use the aqueous standard
provided.(Also can use TC - muli purpose
calibrator to replace
standard)
QUALITY CONTROL
The integrity of the reaction
should be monitored by use of normal
and abnormal control sera
with known creatinine values.
EXPECTED VALUES6
Serum: Male 0.9 - 1.5 mg/dl
Female 0.7 - 1.37 mg/dl
PERFORMANCE
CHARACTERISTICS
Linearity: 25 mg/dl
Comparison: A
study performed between this procedure and a similar kinetic procedure yielded
a correlation coefficient of 0.99 with a regression equation of y = 0.96x +
0.06. Serum and control samples used in the study had creatinine values ranging
from 0.9 to 8.3 mg/dl.
Precision:
|
|
Within Run |
|
|
Mean |
S.D. |
C.V.% |
|
1.9 |
0.05 |
2.6 |
|
8.2 |
0.60 |
7.3 |
|
|
|
|
|
|
Run to Run |
|
|
Mean |
S.D. |
C.V.% |
|
2.0 |
0.20 |
10.6 |
|
8.0 |
0.40 |
4.6 |
REFERENCES
1. Henry.
J.B., Clinical Diagnosis and
Management by
Laboratory Method. 16th
ed. Saunders. Philadelphia. PA.
p.
263. (1974).
2. Vasilades. J. Clin. Chem. 22:1664 (1976).
3. Heinegard. D. and Tiderstrom. G., Clin. Chem. Acta 43:305
(1973).
4. Buffer.
A.R.. Clin. Chem. Acta 59:227 (1975).
5. Young. D.S.. et al., Clin. Chem. 21(1975).
6. Tietz. N.W.. Fundamentals of Clinical Chemistry. W.B.
Saunders. R.S., Phila. p.
1211(1976).
Date revised: 6/95