ENZYMATIC CREATININE Ref.

: 127
Instruction for use

Intended use . System for determination of creatinine in serum, Methodology . Enzymatic Trinder.
plasma, and urine samples by end-point reaction.
Reagents
Professional use.

[Only for in vitro diagnostic use.]

1. 1 - Reagent 1- Store at 2 - 8 ºC.
Contains buffer pH 7.4; creatine amidinohydrolase £ IU/mL; sarcosine
oxidase £17 IU/mL; ascorbate oxidase <7 IU/mL; and N-ethyl-N-
Test principle . Creatinine present in sample is converted into
sulfopropryl-m-toluidine £0.21 mg/mL.
creatine by creatinine amidohydrolase. The creatine produced is
hydrolyzed to sarcosine and urea by creatine amidinohydrolase. Next, the
enzyme sarcosine oxidase causes the oxidative demethylation of 2. 2 - Reagent 2- Store at 2 - 8 ºC.
sarcosine, yielding glycine, formaldehyde and hydrogen peroxide. In Contains buffer pH 7.3; creatinine amidohydrolase £670 IU/mL;
presence of peroxidase, hydrogen peroxide reacts with N-ethyl-N- peroxidase £91 IU/mL; 4-aminoantipirine £0.9 mg/mL and sodium azide
sulfopropryl-m-toluidine (ESPMT) and 4-aminoantipyrine, yielding a <0.1%
quinoneimine with maximum absorbance at 546 nm. The color intensity
of the reaction product is directly proportional to the creatinine The reagents must be kept out of their storage temperature for only the
concentration in sample. time necessary to obtain the volume to be used in tests. Avoid direct sun
light exposure.

Creatinine amidohydrolase The unopened reagents, when stored at indicated temperature, are stable
Creatinine + H2O Creatine up to the expiration date shown on the label. Upon handling, reagents and
the calibrator may be submitted do microbial or chemical contamination,
which may cause a reduction in reagent stability.
Creatine amidinohydrolase
Creatine + H2O Sarcosine + Urea Precautions and warnings
The usual security cares should be applied to the reagent handling. They
Sarcosine oxidase must not be pipetted by mouth aspiration. Avoid ingestion and in case of
Sarcosine + H2O + O2 Glycine + Formaldehyde + H2O2 contact with eyes, wash them with plenty of water and seek medical help.

Reagent 2 contains sodium azide as preservative. Avoid ingestion. In case

Peroxidase of contact with eyes, immediately flush eyes with plenty of water and get
2H2O2 + ESPMT + 4-aminoantipyrine Quinoneimine + 4H2O medical assistance.
Sodium azide may react with lead and copper plumbing and yield highly
explosive metal azides. On disposal, flush with a large volume of water to
Summary . Enzymatic Creatinine Labtest uses the enzymes creatinine prevent azide accumulation.
aminohydrolase, creatine amidinohydrolase, and sarcosine oxidase
coupled with the Trinder reaction to determine the creatinine Materials required not provided
concentration in serum, plasma, and urine samples. The enzymatic
methodology makes the analyte determination more specific, eliminating 1. Analyzer capable of measuring absorbance accurately at 546 nm
interference from plasma proteins and other chromogens commonly (540 - 550 nm).
observed in direct method based on the Jaffé reaction. 2. Calibrator from the Calibra H Labtest series.
The calibration material indicated is calibrated with NIST SRM 914, which 3. Pipets to measure samples and reagents.
makes the method traceable to the IDMS (isotopic dilution mass
spectrometry) definitive method. Therefore, the method meets the
recommendations from the National Kidney Disease Education Program
(NKDEP) for standardization of creatinine determination in serum.

01 English - Ref.: 127

 Specimen collection and preparation Parameter Bi-Reagent Application
Reaction Type End-point
A Standard Operating Procedure (SOP) must be created to establish Reaction Direction Increasing
adequate procedures for sample collection, preparation, and storage. The Primary Wavelength 546 nm
errors due to bad sampling can be more damaging than the ones which Secondary Wavelength 800 nm
may occur during the analytical procedure. Temperature 37 ºC
2 Points
Use serum or plasma (heparin, EDTA, fluoride, oxalate, and citrate)
Calibration Point 0: NaCl 0.85% or Deionized water
samples. The analyte is stable for 7 days at 2 - 8 ºC. The Glistab Labtest
Point 1: Calibra H
(Ref. 29) allows for collection of only one sample for determination of
Calibration Model Linear
creatinine, glucose and urea.
Sample Volume* 6 mL
24-hour urine samples must be centrifuged. The urine sample must not R1 Volume* 270 mL
receive any preservative, and must be stored under refrigeration during 300 seconds after incubating R1
the collection period and after it is received by the laboratory. Reading 1 (Abs 1)
at 37 ºC + sample
R2 Volume* 90 mL
Since no known test method can offer complete assurance that human
samples will not transmit infectious diseases, all samples should be 300 seconds after incubating R1
Reading 2 (Abs 2)
considered potentially infectious and handled accordingly. at 37 ºC + sample + R2

Disposal of all biological waste material should be in accordance with * Sample and reagent volumes can be modified proportionally without
local guidelines. any loss in test performance, and the calculation procedure remains the
same. In case of volume reduction it is crucial to observe the minimal
necessary volume for photometric reading.
Interference
Concentrations of triglycerides up to 1000 mg/dL, bilirubin (conjugated Calibration
and unconjugated) up to 16 mg/dL, hemoglobin up to 400 mg/dL,
ascorbic acid up to 40 mg/dL, creatine up to 20 mg/dL, cefpiramide up to Automated systems
100 mg/dL, cefotaxime up to 100 mg/dL, and ceftraixone up to 2-point Calibration
Point 0: Reagent blank - deionized water or NaCl 150 mmol/L (0.85%).
100 mg/dL do not interfere in sample testing. Dobutamine and
Point 1: Calibrator - Calibra H Labtest series.
methyldopa interfere negatively in the reaction.

Samples with bilirubin, hemoglobin, and triglycerides levels higher than The creatinine concentration in the Calibra H material is traceable to the
the ones indicated above must be diluted with NaCl 150 mmol/L (0.85%) Standard Reference Material (SRM) 914 from the National Institute of
prior to being tested. Standards and Technology (NIST).

Samples with azide may yield inaccurate results for creatinine caused by Calibration frequency
insufficient creatine conversion. When the internal quality control indicates so;
When using a new reagent lot;
To determine the approximate concentration of hemoglobin in a sample, When using new bottle of reagent from the same lot if a new calibration
dilute 0.05 mL of sample in 2.0 mL of NaCl 150 mmol/L (0.85%) and has been performed for the prior reagent bottle.
measure the absorbance at either 405 or 415 nm, subtracting the zero
absorbance value with deionized water. Calculation . According to recommendations of NKDEP the results
must be reported with two decimal places to avoid systematic errors
caused by rounding of results, which may reach ±6%.
Procedure
DAbs of Test or Calibrator = Abs 2 - Abs 1
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 Test
obtained by 5. Creatinine (mg/dL) = x Calibrator conc. mg/dL
DAbs Calibrator

Urine Creatinine

Urine Creatinine (mg/dL)

Urine Creatinine = x Urine volume
(mg/24 h) 100 (mL/24 h)

mg/kg weight = mg/24 hours divided by body weight.

02 English - Ref.: 127

Endogenous creatinine clearance . Inform the patient so they It is recommended to use the products Qualitrol H - Labtest as internal
can collect a 24-hour urine sample. quality control. It is recommended to meet the specification proposed by
the NKDEP for coefficient of variation £4% and systematic error £5%.
Determine the creatinine concentration in serum and urine. The serum
sample can be obtained in any moment during the period of urine Expected values . These values should be used only for orientation
collection. purposes. Each laboratory should evaluate the transferability of the
expected values to its own patient population and, if necessary, estimate
Apply the results found to the equation below: its own reference interval.

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.

Internal quality control . The laboratory must keep an internal

quality control program with well-defined regulations, objectives,
procedures, criteria of quality specifications and tolerance limits,
corrective actions and registration of activities. Control materials should
be used for measurement imprecision monitoring and determination of
calibration deviation.

03 English - Ref.: 127

Performance characteristics Decision levels Creatinine estimated Systematic errors
for creatinine using the regression estimated based on
Recovery studies . Two samples with creatinine concentrations evaluation equation creatinine decision levels
equal to 20.32 mg/dL and 40.64 mg/dL received 20.32 mg/dL of mg/dL mg/dL mg/dL %
creatinine, yielding recovery rates between 99.5% and 100.3%. The 30 30 0.025 0.083
proportional systematic error, estimated based upon the decision level 100 100 0.333 0.333
equal to 1.0 mg/dL, is 0.001 mg/dL or 0.1%. 500 498 2.093 0.419

Method comparison . The Enzymatic Creatinine method was

compared against a similar method, and the following results were Imprecision
obtained:

For serum samples: Imprecision - Within Run

N Mean SD CV (%)
Comparative Enzymatic Sample 1 20 1.10 0.0144 1.31
Method Creatinine Sample 2 20 4.66 0.0397 0.85
Sample Nature Serum
Sample Number 50 50
Concentration interval Imprecision - Run-to-Run
0.47 - 4.4 0.46 - 4.4
(mg/dL)
Estimate mean (mg/dL) 1.02 1.02 N Mean SD CV (%)
Enzymatic Creatinine = 0.9974 x Sample 1 21 0.56 0.0053 0.96
Regression equation
Comparative + 0.0055 Sample 2 21 1.19 0.0146 1.23
Correlation coefficient 0.9996 Sample 3 21 5.66 0.0339 0.60

Using the regression equation, the following systematic errors (bias) were Methodology sensitivity . A sample containing no creatinine was
found for the Enzymatic Creatinine method: 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
standard deviations. Using the standard's absorbance as parameter, the
Decision levels Creatinine estimated Systematic errors
photometric detection limit was equal to 0.04 mg/dL, which corresponds
for creatinine using the regression estimated based on
to a difference in absorbance equal to 0.001.
evaluation equation creatinine decision levels
mg/dL mg/dL mg/dL %
Effect of matrix dilution . A sample with concentration of
1.00 1.00 0.0029 0.29
101.60 mg/dL was used to evaluate the system response to matrix
1.20 1.20 0.0024 0.20
dilution using NaCl 150 mmol/L. Using dilution factors ranging from 1.25
2.00 2.00 0.0003 0.015 to 5, the recovery values found were between 99.5% and 100.7%

Notes
For urine samples:
1. The material cleaning and drying are fundamental factors to the
reagent stability and to obtain correct results.
Comparative Enzymatic
Method Creatinine
2. The clinical laboratory is aimed at providing accurate and precise
Sample Nature Urine
results. Use of inappropriate quality water is a potential cause of analytical
Sample Number 50 50
bias. The water used in the laboratory should have the appropriate quality
Concentration interval for each application. Thus, to prepare reagents, use in the measures and
11.73 - 146.6 11.79 - 146.2
(mg/dL) for use in the final rinsing of the flasks, the water should have resistivity
Estimate mean (mg/dL) 134.87 134.41 ³1 megaohm.cm or conductivity £1 microsiemens/cm and silicate
Enzymatic Creatinine = 0.9956 x concentration <0,1 mg/L. When the deionizing column is with its
Regression equation
Comparative + 0.1070 capacity saturated, occurs release of several ions, silicates and
Correlation coefficient 0.9998 substances with large oxidation or reduction power that deteriorate the
reagents in a few days or even hours, thus changing the results
unpredictably. Consequently, it is essential to establish a quality control
Using the regression equation, the following systematic errors (bias) were program for the water.
found for the Enzymatic Creatinine method:

04 English - Ref.: 127

References Application procedures using Enzymatic Creatinine are available for
various automated instruments.
1. JUNGE, W. et. al. Determination of reference intervals for serum
creatinine, creatinine excretion and creatinine clearance with an Customer information
enzymatic and a modified Jaffé method. Clinica Chimica Acta, v. 344,
n. 1-2, p. 137-48, 2004. [Warranty conditions]

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.

3. MARTENSSON, A. et. al. Creatininium reference intervals for corrected

methods. Scandinavian Journal of Clinical and Laboratory
Labtest Diagnóstica S.A.
Investigation, v. 64, p. 439-442, 2004.
CNPJ: 16.516.296 / 0001 - 38
4. CERIOTTI, F. et. al. Reference Intervals for serum Creatinine Av. Paulo Ferreira da Costa, 600 - Vista Alegre - CEP 33240-152
Concentrations: Assessment of Available Data for Global Application. Lagoa Santa . Minas Gerais Brasil - www.labtest.com.br

Clinical Chemistry, v.54, n. 3, p. 559-566, 2008. Customer Service e-mail: [email protected]

5. Labtest: Data on file.

Edition: December, 2011 Copyright by Labtest Diagnóstica S.A.
Revision: March, 2019 Reproduction under previous autorization
Ref.: 280122(01)
Presentation
Product Reference Content

Enzymatic
Creatinine
127-1/216
()( 1 X 162 mL
1 X 54 mL
2 X 54 mL
127-2/72

Enzymatic Creatinine
Labmax 560/400
127-2/72
)() 2 X 18 mL
2 X 54 mL
2 X 18 mL

05 English - Ref.: 127

 Símbolos utilizados com produtos diagnósticos in vitro
Símbolos usados con productos diagnósticos in vitro
Symbols used with ivd devices

Conteúdo suficiente para < n > testes Risco biológico

Contenido suficiente para < n > tests Riesgo biológico
Contains sufficient for < n > tests Biological risk

Data limite de utilização (aaaa-mm-dd ou mm/aaaa) Marca CE

Estable hasta (aaaa-mm-dd o mm/aaaa) Marcado CE
Use by (yyyy-mm-dd or mm/yyyy) CE Mark

Material Calibrador Tóxico

Material Calibrador Tóxico
Calibrator Material Poison

Material Calibrador Reagente

Material Calibrador Reactivo
Calibrator Material Reagent

Limite de temperatura (conservar a) Fabricado por

Temperatura limite (conservar a) Elaborado por
Temperature limitation (store at) Manufactured by

Representante Autorizado na Comunidade Europeia Número do lote

Representante autorizado en la Comunidad Europea Denominación de lote
Authorized Representative in the European Community Batch code

Consultar instruções de uso Controle

Consultar instrucciones de uso Control
Consult instructions for use Control

Número do catálogo Controle negativo

Número de catálogo Control negativo
Catalog Number Negative control

Adições ou alterações significativas Controle positivo

Cambios o suplementos significativos Control positivo
Significant additions or changes Positive control

Produto diagnóstico in vitro Controle

Dispositivo de diagnóstico in vitro Control
In vitro diagnostic device Control

Liofilizado Corrosivo
Liofilizado Corrosivo
Lyophilized Corrosive

Período após abertura Uso veterinário

Período post-abertura Uso veterinario
Period after-opening Veterinary use

Instalar até
Instalar hasta
Install before Ref.: 140214

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