Intended use . Colorimetric system for creatinine determination in
blood and urine by end point reaction.
Test principle . Creatinine and other components of serum react to
alkaline picrate in alkaline solution yielding a red complex what is
photometrically measured.
The addition of an acid decrease the pH to 5.0 yielding the decomposition
of the creatinine picrate, the chromogen derived color remained unaltered
what is also a photometric measurement. The difference of the two
measurements yields the true value of creatinine.
Summary . Creatinine applies an end point procedure in order to
improve the method specificity and minimize the susceptibility to
1-3, 5
interferences .
The measurement procedure is calibrated with the NIST SRM 914 and
renders the results traceable to the IDMS (isotropic dilution, mass
spectrometry) definitive method, which complies the National Kidney
Disease Education Program (NKDEP) recommendations for
4
standardization of serum creatinine measurement .
All the direct methods that apply the Jaffe reaction are susceptible to a
constant systematic error, due to the plasmatic proteins ant other
chromogens interference. In order to minimize this error and increase the
Creatinine results accuracy, Labtest recommends the use of the
8,11
correction index that must be applied whatever are the found results .
Several studies have demonstrated that is possible minimize significantly
the interferences caused by icteric and lipemic samples in creatinine
3
measurement . The procedure with acidification step minimizes the
bilirubin negative interference, while the desproteinization procedure
removes interference of lipemic samples equivalent to a triglycerides
11
value of 250 up to 1800 mg/dL .
The measurement procedure is applied in automated and semi-
automated systems able to perform accurate measure of absorbance at
510 nm.
Methodology . Labtest.
Reagents
1. - Picric acid - Store at 15 - 25 ºC.
Reagent label bears expiration date. Picric acid 44.4 mmol/L.
2. - Buffer - Store at 15 - 25 ºC.
Reagent label bears expiration date. Sodium hydroxide (208 mmol/L),
sodium tetraborate (12.7 mmol/L) and surfactant. The reagent may
precipitate in low temperatures at 15 ºC. In this case, warm up at 37 ºC
and mix until it dissolves completely.
01 English - Ref.: 35
3. - Standard 4.0 mg/dL - Store at 15 - 25 ºC.
Reagent label bears expiration date. In order to avoid evaporation of the
Standard, keep the bottle tightly closed.
4. - Acid solution - Store at 15 - 25 ºC.
Acetic acid (11.4 mol/L).
Precautions and warnings
For in vitro diagnostic use.
Disposal of all waste material should be in accordance with local
guidelines.
The usual security cares should be applied on the reagent handling.
The reagents Buffer and Acid solution are corrosive. Avoid ingestion. In
case of eyes, skin and mucosa contact, immediately flush then with plenty
of water and get medical assistance.
The Buffer may result in ulcerations when ingested. In case of ingestion,
immediately offer a lot of water with lemon juice or vinegar. Do not induce
vomiting and get medical assistance.
In case of ingestion of Picric Acid, offer 4 glasses of water and if the
individual is conscious, induce vomiting and get medical assistance.
Storage and stability . Unopened reagents, when stored at
indicated temperature, are stable up to expiration date shown on the label.
Deterioration . Microbial or chemical contamination may decrease
reagents stability.
Specimen collection and preparation
Use serum or plasma (heparin, EDTA, fluoret, oxalate and citrate). The
analyte is reportedly stable for about 7 days at 2 - 8 ºC.
Glistab anticoagulant (Labtest Ref.: 29) allows the collection of only one
blood sample for the measurements of creatinine, glucose, and urea.
Urine of 24 hours and amniotic fluid must be stored at 2 - 8 ºC during the
period of collection until it is measured.
No known test method can offer complete assurance that human blood
samples will not transmit infectious diseases. Therefore, all blood
derivatives should be considered potentially infectious.
Interference
Proteins present in the samples yield a positive interference introducing a
constant systematic error. This error may be minimized applying a
correction index.
Since the urine has no proteins that may interfere, the correction index is Calculations
not applied to the calculation of concentration in urine samples. See
application of correction index on Calculations. A1 - A2
Creatinine (not corrected) = x 4 mg/dL
Creatinine determination in urine samples may be affected by the action of Astandard
high amount of reducers substances present in cases of ketoacidosis.
Boiling the urine sample for one minute eliminates partially these 4
According with NKDEP recommendations, the results should be reported
substances interference. The remaining interference is excluded in the with two places of decimals in order to avoid systematic errors due to
procedure with acidification step. making the results round, which may reach ± 6%.
For therapeutic control it is recommended to collect the sample at the Due the great reproductive results of the assays system, it is possible to
same time due circadian variations. use the factor method:
The aspirin in anti-inflammatory doses increases the creatinine value in
blood sample. Calibration factor = 4 /Astandard
Physical exercises increase the creatinine values.
Creatinine values are lower in individuals who have vegetarian diet. Creatinine (not corrected) = (A1 - A2) x Factor (mg/dL)

Bilirubin up to 5 mg/dL, hemoglobin up to 180 mg/dL and triglycerides up

Urinary creatinine
to 250 mg/dL do not interfere significantly.

Bilirubin values over 5 mg/dL interfere negatively in the reaction. Urinary Creatinine
Urinary
(mg/24 hours) = x UrineVolume (mL/24h)
Triglycerides values over 250 mg/dL provide false increased results. Creatinine
100
Materials required not provided mg/kg weight = mg/24 hours divided by body weight

1. A constant temperature water bath (37 ºC).
2. Photometer capable of measuring absorbance at 500 - 540 nm.
3. Pipettes to measure reagents and samples.
4. Timer.
Manual and direct assay procedure
Applying the correction index . Plasmatic proteins interference
5
that occurs in Jaffe reaction, introduce a constant error in the
measurement which is minimized by the correction index (0.25 mg/dL).
The obtained results with the calibration and the correction are
4
traceable to IDMS method and comply with the NKDEP
recommendations.

See notes 1, 2 and 3. Creatinine = Creatinine - Correction index

(corrected) (not corrected) (0.25 mg/dL)
Urine . Dilute the urine 1:25 with distilled water (0.2 mL of urine +
4.8 mL of distilled water) and multiply the result by the dilution factor (25).
Procedure with deproteinization
The water must have resistivity ≥1 megaohm, or conductivity
≤1 microsiems and silicates concentration must be <0.1mg/L. This must be applied to icteric and turbid samples.

Set up three tubes and proceed as follows: Mix 0.5 mL of serum or plasma to 1.0 mL of Picric Acid (n° 1),
homogenize and centrifuge 10 minutes.
Blank Unknown Standard
Set up three tubes and proceed as follow
Buffer (Nº. 2) 2.0 mL 2.0 mL 2.0 mL
Sample 0.25 mL
Distilled or deionized water 0.25 mL Blank Unknown Standard
Standard (Nº. 3) 0.25 mL Buffer (Nº. 2) 2.0 mL 2.0 mL 2.0 mL
Picric acid (Nº. 1) 0.5 mL 0.5 mL 0.5 mL Supernatant 0.75 mL
Distilled or deionized water 0.25 mL
Standard (Nº. 3) 0.25 mL
Mix and incubate in a water bath at 37 ºC during 10 minutes. Measure the
Picric acid (Nº. 1) 0.5 mL 0.5 mL
absorbance of the Unknown and Standard against Blank at 510 nm or
green filter (500 - 540 nm).
Absorbance will be A1. Mix and incubate in a water bath at 37 ºC during 10 minutes. Measure the
absorbance of the Unknown and Standard against Blank at 510 nm or
green filter (500 - 540 nm).
Acid solution (Nº. 4) 0.1 mL 0.1 mL
Absorbance will be A1.
Mix and let at room temperature for 5 minutes. Measure the absorbance of
the Unknown against Blank at 510 nm or green filter (500 - 540 nm). Acid solution (Nº. 4) 0.1 mL 0.1 mL
Absorbance will be A2.

02 English - Ref.: 35
Mix and let at room temperature for 5 minutes. Measure the absorbance of
the Unknown against Blank at 510 nm or green filter (500 - 540 nm).
Absorbance will be A2.
Use the same calculation proposed for the direct assay. Do not apply the
correction index.
In turbid samples in excess, it is not possible to get a clear supernatant in
the deproteinization procedure. In this case, it is not possible to measure
the creatinine.
Manual calibrations
Perform a new calibration after reagent lot change or when the internal
quality control indicates.
Quality control . For quality control use Qualitrol H Level 1 and
Qualitrol H Level 2 or other suitable control material. The limits and control
interval must be adapted to the laboratory requirements.
Each laboratory should establish corrective actions to be taken if values
fall outside the control limits.

Endogenous creatinine depuration. The laboratory must

Measurement/reportable range
inform the patient how to collect the correct urine within 24 hours.
The reaction is linear between 0.2 mg/dL and 12 mg/dL.
Use the proposed methodologies to measure creatinine in serum and
urine. If creatinine concentration exceeds 12 mg/dL, the sample must be diluted
with 0.85% NaCl. Multiply the result by the appropriate dilution factor.
Apply the obtained results in the following equation:
Expected values . Each laboratory should evaluate the
U
transferability of the expected range to its own patient population and, if
Depuration (mL/minute) = x VM 8,10
S necessary, estimate its own reference range .

U: Creatinine in urine (mg/dL) Serum/Plasma (mg/dL)*

S: Creatinine in serum (mg/dL) newborn 0.31 - 0.92
VM: Volume per minute (urinary volume of 24 hours, in mL, divided by
2 weeks - 1 year 0.16 - 0.39
1440).
1 - <3 years 0.17 - 0.35
PS: Depuration must be corrected to the patient's body surface that is
3 - <5 years 0.26 - 0.42
obtained by a nomogram correlating weight and height, or using the
following equation: 5 - < 7 years 0.29 - 0.48
0.425 0.725
A = W x H x 0.007184 7 - <9 years 0.34 - 0.55
9- <11 years 0.32 - 0.64
A = body surface (m )
2 11 - <13 years 0.42 - 0.71
W = weight (kg) 13 - <15 years 0.46 - 0.81
H = height (cm) Adults (women) 18 - 74 0.53 - 1.00
Adults (men) 18 - 74 0.70 - 1.20
Multiply the depuration value by 1.73 and divide by the patient body
surface.
* Corrected values with the correction index and IDMS traceable.
Glomerular de filtration rate . The NKDEP4 recommends that the
laboratories report the estimated glomerular filtration rate (eGFR) in all the There isn't established range for the age 15 - <18 years. It's suggested to
reports containing creatinine results. use the women and men adult range.
When the plasmatic creatinine results are corrected and traceable
to the IDMS, the following equations that apply creatinine (CREA), age Conversion mg/dL to IS Units: µmol/L = mg/dL x 88.4.
(18 - 70 years) and sex are used.
Urine (mg/kg/24 hours)
Women 2 - 3 years 6 - 22
-0.203
eGFR (mL/min/1.73m2) = 175*(CREA)-1.154*(Age) *0.742 >3 years 12 - 30
Adults (women) 16 - 22
Men Adults (men) 21 - 26
eGFR (mL/min/1.73m2) = 175*(CREA)-1.154*(Age)-0.203

4
According NKDEP recommendations, the eGFR must be reported as the 2
2
Creatinine Depuration (mL/minute/1,73 m )**
calculated value when the result is ≤60 mL/min/1.73m . When the Children 70 - 140
calculated value is higher than 60, must be reported as: higher than Adults (women) 88 - 128
2 2
60 mL/min/1.73m or > 60 mL/min/1.73m . Adults (men) 97 - 137

Calibration . Standard is traceable to the Standard Reference Material

(SRM) 914 of the National Institute of Standards and Technology (NIST). ** Established values for not corrected and not IDMS traceable results

03 English - Ref.: 35
 4
The NKDEP recommends calculate the glomerular filtration rate (eGFR) 3. It is suggested to consult “www.fxol.org/” in order to review
in substitution of Creatinine Depuration, using the creatinine IDMS physiopathological source and drugs interference in results and
traceable result after application of the correction index. methodology.

Performance characteristics References

Recovery studies . In two samples with creatinine concentrations of 1. Cook JGH. Clin Chim Acta 1971;32:485-6.
1.2 and 3.2 mg/dL were added different quantities of the analyte.
Subsequent analyses provided recoveries ranging from 93 to 98%. The 2. Yatzidis H. Clin Chem 1974;20:1131-34.
mean proportional systematic error at 3.0 mg/dL decision level was
3. Spencer K. Ann Clin Biochem 1986;23:1-25.
0.1 mg/dL or 3.7 %.
4. Meyers GL, Miller WG, Coresh J et al. Clin Chem 2006;52:5-18.
Method comparison . A group of 20 sera were assayed by the
Creatinine method and the Creatinine K method (traceable to IDMS 5. Heinegard D, Tiderström G. Clin Chim Acta 1973;43:305-10.
method). Serum creatinine values ranged from 0.59 - 4.40 mg/dL. The
comparisons yielded a correlation coefficient of 0.995 and regression 6. Sociedad Española de Bioquímica Clínica y Patologia Molecular,
equation was 1.003x + 0.00. The mean total systematic error Base de Datos de Variación Biológica. Avaiable in:
(proportional and constant) at 1.00 mg/dL, 1.20 mg/dL and 2.00 mg/dL <htttp://www.seqc.es/ar ticle/ar ticleview/330/1/170> (access
decision levels were 0.003 (0.30 %), 0.004 (0.30 %) and 0.006 (0.30 %), 2006/04).
respectively.
7. Basques JC. Especificações da Qualidade Analítica. Labtest
Diagnóstica 2005.
Imprecision - Within Run
8. Junge W, Wilke B, Halabi A, Klein G. Clin Chim Acta 2004;344:137-48.
Mean SD
N (%) CV
(mg/dL) (mg/dL) 9. Martensson A, Rustad P, Lund H, Ossowicki H. Scand J Clin Lab Invest.
Sample 1 20 2.0 0.04 2.0 2004;64:439-42.
Sample 2 20 2.8 0.04 1.3
10. Ceriotti F, Boyd JC, Klein G et al. Clin Chem 2008; 54:559-66.

11. Labtest: Data on file.

Imprecision - Run-to-Run

N
Mean SD
(%) CV
Presentation
(mg/dL) (mg/dL)
Product Reference Contents
Sample 1 20 2.0 0.05 2.5
Sample 2 20 2.8 0.06 2.3 1 X 50 mL
1 X 200 mL
35-100
1 X 10 mL
Analytical sensitivity . Detection limit: 0.3 mg/dL. The detection 1 X 10 mL
Creatinine
limit represents the lowest measurable creatinine concentration that can 1 X 250 mL
be distinguished from zero. It is calculated as two standard deviations of 1 X 1000 mL
35E-500
20 replicates of one sample without creatinine. 1 X 30 mL
1 X 50 mL
Matrix dilution effects . Two samples with values equal of 16.5
and 18.6 mg/dL were used to evaluate the system response on the matrix
dilutions with 150 mmol/L NaCl (0.85%). Recoveries were found a range Consumer information
of 103 and 104 %, using dilution factors that vary from 2 to 4.
[Warranty conditions]
Notes
Labtest Diagnóstica warrants the performance of this product under the
1. The material cleaning and drying are fundamental factors to the specifications until the expiration date shown in the label since the
reagent stability and to obtain correct results. application procedures and storage conditions, indicated on the label and
in this insert, have been followed correctly.
2. The deionized or distilled water in the laboratory to prepare reagents,
use in the measurements and for final glass washing must have resistivity
≥1 megaohm.cm, or conductivity ≤1 microsiems/cm and silicates
concentration must be <0.1mg/L.

04 English - Ref.: 35
 Labtest Diagnóstica S.A.
CNPJ: 16.516.296 / 0001 - 38
Av. Paulo Ferreira da Costa, 600 - Vista Alegre - CEP 33400-000
Lagoa Santa . Minas Gerais Brasil - www.labtest.com.br
Consumer Service e-mail: sac@labtest.com.br

Revision: May, 2009 Copyright by Labtest Diagnóstica S.A.

Ref.: 160311 Reproduction under previous autorization

05 English - Ref.: 35
 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

Ref.: 170309

06 English - Ref.: 35