1.0 INTENDED USE
This reagent is intended for
the quantitative determination of creatinine in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Jaffe (10.1) first described
the reaction of Creatinine with an alkaline picrate solution to form a red
color in 1886. Because of its
simplicity, precision, and low cost, the Jaffe reaction has been used
extensively for the colorimetric determination of Creatinine. The Biotron method is a modification of the
Jaffe method using a kinetic measurement of the color formation.
2.2 TEST PRINCIPLE
Creatinine reacts with picric
acid in an alkaline medium to produce a red colored complex.
Creatinine + Alkaline Picrate -----> Creatinine-picrate
complex
The rate of formation of this
complex, in a selected time interval, is directly proportional to the
concentration of creatinine in the sample.
2.3 CLINICAL SIGNIFICANCE
(10.4)
Creatinine is not actively
reabsorbed from the renal tubules and is excreted via the urine. In normal individuals, the level of
creatinine in serum remains low and constant.
When the glomerular filtration rate decreases, as in cases of renal
insufficiency, the level of serum creatinine will elevate. Since the serum creatinine is less affected
by diet and hydration than is the level of urea nitrogen, creatinine is
believed to be of greater clinical specificity.
At the onset of renal insufficiency, the serum creatinine increases more
slowly following recovery of renal function.
Since the creatinine in serum
is derived from the creatinine of muscles, increased serum creatinine levels
are seen in muscle-wasting disorders such as muscular dystrophy. Increased levels are also found after muscle
trauma such as can occur following strenuous exercise. In general, serum creatinine levels directly
correlate with muscle mass, i.e., a large muscle mass being reflected in higher
serum creatinine levels.
3.0 SPECIMEN COLLECTION AND
HANDLING
3.1 PATIENT PREPARATION
No special patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh, clear, unhemolyzed
serum is the preferred specimen. Plasma
prepared with lithium heparin may be used.
Use a standard venipuncture tube to draw patient sample.
The amount of sample required
will depend on the analyzer used. The
amount of serum required is in the range of 5-200 µl. Call Biotron'stechnical service department at
1-800-595 8766 for the recommended sample volume for your analyzer.
Record the patient's name,
date and time of sample collection and preparation.
3.3 SPECIMEN STORAGE
Serum or plasma samples may
be stored for 7 days at 2-8°C. Samples
may be stored indefinitely at -20°C. (10.2)
Frozen samples should be
thawed at room temperature, and mixed completely before analysis. Thawed samples should not be refrozen.
It is recommended that testing be done as soon as possible
after sample collection and preparation.
If testing cannot occur immediatedly, store the sample properly using
the guidelines above.
4.0 MATERIALS
(2 X 125 ml)
Reagents necessary for the
determination of creatinine are included in the kit.
4.1 REAGENT
Creatinine Reagent contains: picric acid 22 mmol/L
Creatinine Base Reagent contains: sodium hydroxide 450 mmol/L
4.1.1 Standard/Control/Calibrator
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic
Use. Not for Internal use in Humans or
Animals. In Vitro Diagnostics reagents
may be hazardous. Avoid ingestion and
skin or eye contact.
4.3 REAGENT PREPARATION
The working reagent is
prepared by mixing equal volumes of creatinine reagent and creatinine base
reagent. Mix well before using. Record the date and time of reconstitution.
4.4 REAGENT STORAGE AND
STABILITY
When stored at 18-26°C
unopened reagents are stable until the expiration date printed on the label. The working creatinine reagent is stable at
18-26°C for 10 days.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 Spectrophotometer capable of reading absorbance at 510 nm.
4.5.2 1 cm cuvettes or a flow cell capable of transmitting light at 510
nm.
4.5.3 Test tubes capable of holding 3 ml.
4.5.4 Pipettes capable of delivering 2 ml and 200 µl.
4.5.5 Deionized or distilled water for preparing the reagent blank.
4.5.6 Timer for a 1 minute intervals.
4.5.7 Calibrator .
4.5.8 Normal and abnormal controls for quality control.
5.0 TEST PROCEDURE
The following is a general procedure for use on a manual
instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 510
nm
Temperature 18-26°
C or 37° C
Pathlength 1.0
cm
Mode kinetic
Reaction time 1.3
- 2.3 minutes
Sample volume 200
µl
Reagent volume 2.0
ml
Total volume 2.2
ml
Sample to reagent ratio 1/10
5.2 INSTRUMENT
Any instrument capable of
reading absorbance accurately with a sensitivity of 0.001 absorbance at 510 nm
may be used. The band width should be 10
nm or less, stray light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
The creatinine assay is calibrated by
referencing the absorbance of the unknown sample to the absorbance of the
calibrator.
5.4 PROCEDURE
5.4.1 Prepare the required volume of Creatinine working reagent (see 4.3
Reagent Preparation Section)
5.4.2 Into separate test tubes pipette 200 µl of distilled water,
calibrator or serum to be assayed.
5.4.3 Add 2.0 ml of working reagent and incubate for 20 seconds.
5.4.4 Record the absorbance of the calibrator at 510 nm at 20 seconds
(As1) and at 80 or 140 seconds (As2). Also record the absorbance of each serum
at 510 nm at 20 seconds (A1) and at 80 or 140 seconds (A2).
5.5 CALCULATION AND RESULTS
A2 - A1
Creatinine (mg/dl) = --------------- X concentration of
calibrator
As2 - As1
A1 = initial absorbance of unknown
A2 = final absorbance of unknown
As1 = initial absorbance of calibrator
As2 = final absorbance of calibrator
Example:
0.192 - 0.172
Creatinine = -------------------- X 6.0 mg/dl = 0.98 mg/dl
0.180 - 0.057
with A1 = 0.172, A2 = 0.192
and As1 = 0.057, As2 = 0.180, concentration of calibrator = 6.0 mg/dl
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.3)
The range of expected values is: 0.7 - 1.4 mg/dl
These values are suggested
guidelines. It is recommended that each
laboratory establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES
(10.6)
Each laboratory should
establish low and high values beyond which the patient would require immediate
attention by a physician. If a
"medical alert value" is reached, always repeat the test to confirm
the result and notify a physician if the result is confirmed.
6.3 LIMITATIONS OF PROCEDURE
Icteric sera can give low
values for creatinine. Studies have
shown that creatinine values are depressed when bilirubin is 10 mg/dl or
higher.
For a list of drugs which may affect creatinine, see Young
(10.3.)
7.0 QUALITY CONTROL
Standard practice for quality
control should be applied to this system.
Commercially available lyophilized controls can be used to monitor the
daily acceptable variations. Normal and
abnormal controls should be assayed at the beginning of each run of patient
samples, whenever a new reagent or a different lot number is being used, and
following any system maintenance.
A satisfactory level of
performance is achieved when the analyte values obtained are within the
"acceptable range" established by the laboratory.
8.0 CALIBRATION PROCEDURES
The creatinine assay is
calibrated by referencing the absorbance of the unknown sample to the
absorbance of the calibrator. Refer to
your instrument manual for more details.
Calibration is required with
the use of a new lot of reagent, any system maintenance or whenever indicated
by quality control data.
9.0 PERFORMANCE
CHARACTERISTICS
9.1 PRECISION
The estimates of precision
shown below were obtained from assays of human control serum.
Within-Run
In this study, 15 replicates of 2 control sera were run.
Mean (mg/dl) SD
(mg/dl) CV (%)
1.88 ±
0.05 2.88
10.1 ±
0.08 0.76
Between-Run
In this study, 5 runs were
made, each run consisting of 5 replicates of 2 control sera.
Mean (mg/dl) SD
(mg/dl) CV (%)
1.91 ±
0.03 1.70
10.3 ±
0.07 0.70
9.2 CORRELATION
A correlation study was done
on the Technicon RA-500 system at 37° C comparing this method and a similar
creatinine method. The samples range
between 1.1 mg/dl and 16.6 mg/dl.
Number Regression
Equation Correlation
of Samples y=Biotron,
x=Comparative Coefficient
30 y
= 1.003 x - 0.0432 0.999
9.3 LINEARITY
This procedure is linear
through 12 mg/dl beyond which the specimen should be diluted 1 to 1 with 0.9%
saline. Reassay the specimen and
multiply the results by 2.
10.0 REFERENCES
10.1 Jaffe, M., Hoppe Selyer's Z. Physiol. Chem. 10,391-400 (1886)
10.2 Kaplan, L., Pesce, A., Clinical Chemistry Theory, C.V. Mosby Co.,
Princeton, NJ (1984)
10.3 Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 3rd
ed., Washington DC, AACC Press (1990).
10.4 Clinical Diagnosis, 15th ed., I. Davidsohn and J.B. Henry, Eds.,
W.B. Saunders, Philadelphia, PA, 1974, p.594.
10.5 Kaplan, A., Szabo, L.L., Clinical Chemistry: Interpretations and
Techniques, 2nd ed., Lea and Febiger, Philadelphia, PA 1983.
10.6 G.J. Kost, "Critical Limits for Urgent Clinician Notification
at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
Rev
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