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Intended use . System for determination of creatinine in serum, Methodology . Enzymatic Trinder.
plasma, and urine samples by end-point reaction.
Reagents
[For in vitro diagnostic use.]
1. 1 - Reagent 1- Store at 2 - 8 ºC.
Test principle . Creatinine present in sample is converted into Contains buffer pH 7.4, creatine amidinohydrolase £60 IU/mL, sarcosine
creatine by creatinine amidohydrolase. The creatine produced is oxidase £17 IU/mL, ascorbate oxidase <7 IU/mL, and N-ethyl-N-
hydrolyzed to sarcosine and urea by creatine amidinohydrolase. Next, the sulfopropryl-m-toluidine £0.21 mg/mL.
enzyme sarcosine oxidase causes the oxidative demethylation of
sarcosine, yielding glycine, formaldehyde and hydrogen peroxide. In
2. 2 - Reagent 2- Store at 2 - 8 ºC.
presence of peroxidase, hydrogen peroxide reacts with N-ethyl-N-
Contains buffer pH 7.3, creatinine amidohydrolase £670 IU/mL,
sulfopropryl-m-toluidine (ESPMT) and 4-aminoantipyrine, yielding a
peroxidase £91 IU/mL, 4-aminoantipirine £0.9 mg/mL, and sodium azide
quinoneimine with maximum absorbance at 546 nm. The color intensity
<0.1%
of the reaction product is directly proportional to the creatinine
concentration in sample. The reagents must be kept out of their storage temperature for only the
time necessary to obtain the volume to be used in tests. Avoid direct sun
light exposure.
Creatinine amidohydrolase
Creatinine + H2O Creatine The unopened reagents, when stored at indicated temperature, are stable
up to the expiration date shown on the label. Upon handling, reagents and
the calibrator may be submitted do microbial or chemical contamination,
Creatine amidinohydrolase
which may cause a reduction in reagent stability.
Creatine + H2O Sarcosine + Urea
A Standard Operating Procedure (SOP) must be created to establish Reaction Type End-point
adequate procedures for sample collection, preparation, and storage. The Reaction Direction Increasing
errors due to bad sampling can be more damaging than the ones which Primary Wavelength 546 nm
may occur during the analytical procedure. Secondary Wavelength 800 nm
Temperature 37 ºC
Use serum or plasma (heparin, EDTA, fluoride, oxalate, and citrate) 2 Points
samples. The analyte is stable for 7 days at 2 - 8 ºC. The Glistab Labtest Calibration Point 0: NaCl 0.85% or Deionized water
(Ref. 29) allows for collection of only one sample for determination of Point 1: Calibra H
creatinine, glucose and urea.
Calibration Model Linear
24-hour urine samples must be centrifuged. The urine sample must not Sample Volume* 6 mL
receive any preservative, and must be stored under refrigeration during R1 Volume* 270 mL
the collection period and after it is received by the laboratory. 300 seconds after incubating R1
Reading 1 (Abs 1)
at 37 ºC + sample
Since no known test method can offer complete assurance that human
R2 Volume* 90 mL
samples will not transmit infectious diseases, all samples should be
300 seconds after incubating R1
considered potentially infectious and handled accordingly. Reading 2 (Abs 2)
at 37 ºC + sample + R2
Disposal of all biological waste material should be in accordance with
local guidelines. * Sample and reagent volumes can be modified proportionally without
any loss in test performance, and the calculation procedure remains the
Interference same. In case of volume reduction it is crucial to observe the minimal
necessary volume for photometric reading.
Concentrations of triglycerides up to 1000 mg/dL, bilirubin (conjugated
and unconjugated) up to 16 mg/dL, hemoglobin up to 400 mg/dL, Calibration
ascorbic acid up to 40 mg/dL, creatine up to 20 mg/dL, cefpiramide up to
100 mg/dL, cefotaxime up to 100 mg/dL, and ceftraixone up to Automated systems
100 mg/dL do not interfere in sample testing. Dobutamine and 2-point Calibration
methyldopa interfere negatively in the reaction. Point 0: Reagent blank - deionized water or NaCl 150 mmol/L (0.85%).
Point 1: Calibrator - Calibra H Labtest series.
Samples with bilirubin, hemoglobin, and triglycerides levels higher than
the ones indicated above must be diluted with NaCl 150 mmol/L (0.85%) The creatinine concentration in the Calibra H material is traceable to the
prior to being tested. Standard Reference Material (SRM) 914 from the National Institute of
Standards and Technology (NIST).
Samples with azide may yield inaccurate results for creatinine caused by
insufficient creatine conversion. Calibration frequency
When the internal quality control indicates so;
To determine the approximate concentration of hemoglobin in a sample, When using a new reagent lot;
dilute 0.05 mL of sample in 2.0 mL of NaCl 150 mmol/L (0.85%) and When using new bottle of reagent from the same lot if a new calibration
measure the absorbance at either 405 or 415 nm, subtracting the zero has been performed for the prior reagent bottle.
absorbance value with deionized water.
Calculation . According to recommendations of NKDEP the results
must be reported with two decimal places to avoid systematic errors
Procedure
caused by rounding of results, which may reach ±6%.
To determine the creatinine concentration in urine, dilute the sample 1:5
(0.2 mL of urine + 0.8 mL of NaCl 150 mmol/L). Multiply the result DAbs of Test or Calibrator = Abs 2 - Abs 1
obtained by 5.
DAbs Test
Creatinine (mg/dL) = x Calibrator conc. mg/dL
DAbs Calibrator
Urine Creatinine
U
Clearance = x MV (mL/minute) Serum/Plasma (mg/dL)*
S Newborn 0.31 - 0.92
2 weeks - 1 year 0.16 - 0.39
U: urine creatinine (mg/dL)
1 - <3 years 0.17 - 0.35
S: serum creatinine (mg/dL)
MV: minute volume (24-hour urine volume in mL divided by 1440). 3 - <5 years 0.26 - 0.42
5 - <7 years 0.29 - 0.48
Note: The clearance results must be corrected according to the patient's 7 - <9 years 0.34 - 0.55
body surface area, which is obtained via a nomogram that correlates 9 - <11 years 0.32 - 0.64
weight and height, or using the equation below: 11 - <13 years 0.42 - 0.71
0.425 0.725
13 - <15 years 0.46 - 0.81
A=W xH x 0.007184 Adults (women) 18 - 74 years 0.53 - 1.00
2 Adults (men) 18 - 74 years 0.70 - 1.20
A = body surface area (m )
W = weight (kg)
H = height (cm) * Intervals established for results traceable to the IDMS method.
Multiply the clearance value by 1.73 and divide the result by the patient's There are no intervals established for patients between 15 and 18 years
body surface area. old. It is suggested to use the intervals established for adult men and
women.
Glomerular filtration rate . The NKDEP strongly recommends
Conversion of mg/dL to SI units: mmol/L = mg/dL x 88.4
that laboratories report the estimated glomerular filtration rate (eGFR) for
all creatinine results.
When the results for plasma creatinine are traceable to the IDMS method,
Urine (mg/Kg/24 hours)
the following equations are applied, which use creatinine (CREA), age
2 - 3 years 6 - 22
(18 to 70 years) and sex.
> 3 years 12 - 30
Women Adults (women) 16 - 22
eGFR (mL/min/1.73m2) = 175 * (CREA) -1.1154 * (Age) -0.203 * 0.742 Adults (men) 21 - 26
Men
eGFR (mL/min/1.73m2) = 175 * (CREA) -1.1154 * (Age) -0.203 2
Creatinine Clearance (mL/min/1.73m )**
According to recommendations from the NKDEP, eGFR must be Children 70 - 140
reported as calculated value when the result is equal o less than Adults (women) 88 - 128
2
60 mL/min/1.73m . When the calculated value is higher than 60, Adults (men) 97 - 137
2
it must be reported as either higher than 60 mL/min/1.73m or
2 **Intervals established for results traceable to the IDMS method.
>60 mL/min/1.73m .
Operating interval . The reaction is linear between 0.0 mg/dL and The NKDEP recommends the calculation of glomerular filtration rate
150 mg/dL. For higher concentrations, dilute the sample with NaCl (eGFR) instead of creatinine clearance, using the creatinine result
150 mmol/L (0.85%), perform a new test, and multiply the result traceable to the IDMS method.
obtained by the dilution factor used.
Using the regression equation, the following systematic errors (bias) were
found for the Enzymatic Creatinine method: Methodology sensitivity . A sample containing no creatinine was
used to evaluate the assay's detection limit. The value found was
0.19 mg/dL, which corresponds to the mean value of 9 assays plus two
Decision levels Creatinine estimated Systematic errors
for creatinine using the regression estimated based on standard deviations. Using the standard's absorbance as parameter, the
evaluation equation creatinine decision levels photometric detection limit was equal to 0.04 mg/dL, which corresponds
to a difference in absorbance equal to 0.001.
mg/dL mg/dL mg/dL %
1.00 1.00 0.0029 0.29
1.20 1.20 0.0024 0.20
Effect of matrix dilution . A sample with concentration of
101.60 mg/dL was used to evaluate the system response to matrix
2.00 2.00 0.0003 0.015
dilution using NaCl 150 mmol/L. Using dilution factors ranging from 1.25
to 5, the recovery values found were between 99.5% and 100.7%
Using the regression equation, the following systematic errors (bias) were
found for the Enzymatic Creatinine method:
2. MYERS. G. L. et al. Recommendations for Improving Serum Creatinine Labtest Diagnóstica warrants the performance of this product under the
Measurement: A Report from the Laboratory Working Group of the specifications until the expiration date shown in the label provided that the
National Kidney Disease Education Program. Clinical Chemistry, v. 52, procedures and storage conditions indicated on the label and in this insert
n. 1, p. 5-18, 2006. have been followed correctly.
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