AST GOT ok - Biotécnica

AST / GOT
11.007.00
INTENDED USE
Kit intended to determination of AST/GOT activity in serum.
PRINCIPLE OF THE METHOD
The Aspartate Transaminase (AST or GOT) catalyses the transference of amino
group from aspartate to alpha-ketoglutarate. This transference produces
oxaloacetate and glutamate. The activity is then determined by coupling this
reaction to Malate Dehydrogenase (MDH) enzyme that transforms the
oxaloacetate product in malate consuming NADH, which is measured at 340 nm.
AST
L - aspartate + alpha-ketoglutarate
oxaloacetate + NADH + H+
LDH
oxaloacetate + L-Glutamate
Malate + NAD+
REAGENT COMPOSITION
Tris buffer 100 mM pH 7.8; L-aspartate 240 mM, Sodium
azide 0.09% w/v; Malate Dehydrogenase > 600 U/L;
Lactate dehydrogenase > 1200 U/L.
NADH 0,18 mM; alpha–ketoglutarate 12 mM Sodium azide
0.09% w/v
STORAGE AND STABILITY
•
The reagents are stable up to the date stated on the label.
•
Do not use reagents over the expiration date.
•
Store at 2 to 8 ºC.
•
Do not freeze and protect from light.
ADDITIONAL EQUIPMENT
Spectrophotometer or photometer with a thermostatized cuvette able to
read at 340 nm.
Pipettes and micropipettes.
Clock or chronometer.
Assay tubes.
WARNINGS AND PRECAUTIONS
•
The kit is intended for in vitro diagnostic use only.
•
All specimens and controls should be handled as potentially infective.
•
Wear the individual protection equipment accordingly to Good Laboratory
Practice.
•
Discard the reactions surplus according to Good Laboratory Practice in a
proper place for potentially infective material.
•
The information for Disposing, Security and First Aid are described in the
Manual Safety Data Sheet (MSDS) of this product available at
www.biotecnica.ind.br or calling for 55-35-3214-4646.
•
Do not combine reagents from different batches.
•
Do not exchange the caps from different reagents in order to avoid cross
contamination.
•
Do not use the reagent if it displays any sign of contamination.
•
The reagents must be kept out of specified temperatures only the time
necessary for the realization of the tests.
•
Use glass pipettes and disposable tips for each sample, control,
standard/calibrator and reagent.
SAMPLE – PREPARATION AND STABILITY
Serum
The AST is stable for 3 days in serum if stored at 2 to 8 ºC.
TECHNICAL PROCEDURE
A) REAGENT PREPARATION
Work reagent
Mix in proportion: 4 parts of R 1 + 1 part of R 2.
Homogenize it gently. The Work Reagent is stable for two weeks at 2 to 8 ºC.
NOTE: The absorbance of the reagent must be higher than 0,800 at 340 nm, with
the instrument adjusted to zero with distilled or deionized water.
B) PROCEDURE
1. Heat the reagent for 3 minutes at 37 oC.
2. Pipette in the assay tube:
Work Reagent
Sample
Volume
1.0 mL
100 µL
3. Homogenize and insert it immediately in the thermostatized cuvette at 37 ºC.
4. After 1 min. note the initial absorbance (A0) and read again after exactly 1, 2
and 3 minutes (A1, A2 and A3, respectively).
CALIBRATION/CALCULATION PROCEDURES
Using the measured absorbances, calculate the mean variation of absorbance
per minute (∆A/min):
(∆A/min) = (A1 - A0) + (A2 - A1) + (A3 - A2)
3
The activity of the AST is then calculate by multiplying the ∆A/min by the factor
below:
AST (U/L) = ∆A/min x 1746
BIOTÉCNICA IND.COM. LTDA. Rua Ignácio Alvarenga nº 96, Vila Verônica,
Varginha MG BRASIL CEP: 37026-470 Tel/fax: +55 35 3214 4646
www.biotecnica.ind.br
Example:
A0 = 1.268; A1 = 1.228; A2 = 1.189; A3 = 1.152
(∆A/min.= (1.268 – 1.228) + (1.228 – 1.189) + (1.189 – 1.152)
3
AST (U/L) = 0.039 x 1746 = 68.1 U/L
Note: To calculate the Factor for automatic analyzers use the absorption
coefficient of NADH = 6.3
SENSITIVITY AND LINEARITY
Methodological Sensitivity:
Calculated method: 1.746 U/L
Linearity: 440 U/L.
For values higher than 440 U/L, dilute the sample with NaCl (0.9%) solution,
repeat the assay and multiply the obtained result by the dilution factor.
TECHNICAL LIMITATIONS
•
Anticoagulants
Fluoride and oxalate inhibits the enzyme and may interfere in the reaction.
•
Hemolyzed, Jaundice and Lipemic Sera
Hemoglobin > 180 mg/dL
Bilirubin > 19 mg/dL
Triglyceride > 350 mg/dL
•
Other interferences
False High results: Serum with high concentration of endogen keto acids.
False Low results: Salycilate (aspirin), patients in haemodialysis, with
hypovitaminosis and other pathologies related to the Pyridoxal phosphate
deficiency.
QUALITY CONTROL
Any clinical laboratory must keep an internal quality control program, which
defines the aids, procedures and criteria for the tolerance limits, corrective actions
and registration of the activities. Also, it must be kept a defined system for
verification of the analytical variability that occurs in any measuring system.
The use of controls to evaluate the imprecision of the analysis must be a routine
practice in the lab. It’s suggested to use a control within the reference range and
other control within the clinical significance range. The application of the Multiple
Rules of Westgard for evaluation of the control state is recommended.
The lab must participate of external quality control programs.
For the internal quality control of the lab it’s indicated the use of the calibrator
serum and control sera below:
Calibrator Serum - Autocal H
13.002.00
Normal Control Serum – Quantinorm
13.003.00
Pathological Control Serum – QuantiAlt
13.004.00
REFERENCE RANGES
MEN
WOMEN
ACTIVITY AT 37ºC
Up to 37 U/L
Up to 31 U/L
These values are intended for orientation only. It’s recommended that each lab
establishes its own reference range.
Conversion for International System of Units (SI):
AST (U/L) X 0.017 = AST (µKat)
PERFORMANCE CHARACTERISTICS
Intra-Assay
The realization of 20 determinations of the same sample at the same day showed
a Coefficient of Variation of 3.69%.
Inter-Assay
The realization of 10 determinations of the same sample at different days showed
a Coefficient of Variation of 2.39%.
Analytical Specificity
A comparison with a reference method showed a correlation coefficient (r) of
0.998 obtained from ambulatory samples. The result equation of the linear
regression is Y = 1.0216 X – 0.6448.
CLINICAL SIGNIFICANCE
The Glutamate Oxaloacetate Transaminase (GOT) or Aspartate Transaminase
(AST) is an intracellular enzyme present in high quantities in cardiac and skeletal
muscles, liver, kidney and brain.
Elevated levels of AST help the diagnostic of cardiac, hepatic and muscle
diseases. The activity of this enzyme in the myocardial infarction is elevated
within the first 12 hours, reaching a peak in 24 hours and come back to normal in
the fifth day. Discrete elevations are observed in pregnancy. Elevated levels are
also found in hepatic necrosis, hemolytic anemias, acute pancreatitis, cirrhosis,
hepatites, obstructive jaundice, mononucleosis, hipothyroidism, brain necrosis
and trauma, severe burns, muscle distrophies, skeletal muscle injuries, cardiac
cateterism and angioplasty.
Several common drugs may increase the level of AST (isoniazide, erithromycin,
progesterone, anabolic steroids, etc.), being then used in monitoring therapies
which uses hepatotoxical drugs.
Revision: 01 – JUN/2011
OBSERVATIONS
1. The cleaning and drying of the lab material are fundamental factors for the
stability of the reagents to reach correct results.
The water used in cleaning must be recent and free of contaminating agents. The
cleaning of the glassware must be done with neutral detergent. The rinsing must
be exhaustive being the last ones with distilled or deionised water.
2. The water used in the lab must have the specified quality for each application.
Thus, to prepare reagents the water must be of Type II, with resistivity > 1
megaohms/cm or conductivity < 1 microsiemens and silicate concentration < 0.1
mg/L. For the initial rinsing of the glassware, the water may be of Type III, with
resistivity > 0.1 megaohms/cm or conductivity < 10 microsiemens. In the final
rinse, the water must be of Type II. Saturated deionising columns releases
alkaline water, several ions and oxidizing or reducing agents that deteriorate the
reagents in a few days or even hours, changing the results in an unpredictable
way. Finally, it’s imperative to establish a program for the quality control of the
water.
PRESENTATIONS
Presentation 1
Presentation 2
R1
R2
R1
R2
1 x 40 m L
1 x 10 m L
1 x 200 mL
1 x 50 mL
50 tests of
1 mL
250 tests
of 1 mL
QUALITY ASSURANCE
Before being approved for use BioTécnica reagents are tested in the Quality
Control Department. The quality of the reagents is assured up to the expiring date
stated in the label of the external packaging, since it is stored and transported in
the specified conditions.
The quality control data concerning this product are available at
www.biotecnicaltda.ind.br
AUTOMATION
This product is compatible to the most types of biochemical automatic analysers.
The applications are available at www.biotecnicaltda.ind.br
CUSTOMER TECHNICAL SERVICE
Any technical doubt on handling this product or this procedure, contact us
calling +55 35 3214 4646, your local distributor or sending an e-mail:
[email protected]
REFERENCES
•
SCIENTIFIC DIVISION, WORKING GROUP ON ENZYMES. International
Federation of Clinical Chemistry IFCC methods for measurement of
catalytic concentration of enzymes Clin. Chim. Acta. v. 281, v.1-2, p.S5S39, 1999.
•
YOUNG, D.S. Effects of drugs on clinical laboratory tests - vol. 2, 5 ed.
Washington DC: AACC Press, 2000.
•
WESTGARD, J. O. et al. A multi-rule shewhart chart quality control in
clinical chemistry. Clin. Chem. v.27 p.493-501, 1981.
TECHNICAL RESPONSIBILITY
Dr. Gilson Sério Pizzo - CRF MG - 5310
ANVISA REGISTRATION NUMBER
80027310186
TABLE OF INTERNATIONAL SYMBOLS
SYMBOL
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EXPLANATION
Consultar Instruções de Uso
Consult instructions for use
Consultar la metódica
Código
Code
Código
Número de lote
Batch code
Denominación de lote
Para uso diagnóstico in vitro
For in vitro diagnostic medical device
Para uso em diagnostico in vitro
Conteúdo do kit
Contents of kit
Contenido del estuche
Conteúdo suficiente para <n> testes
Contains sufficient for <n> tests
Contenido suficiente para <n> ensayos
Calibrador
Calibrator
Calibrador
Controle
Control
Control
Padrão
Standard
Patrón
Reagente e seu número/abreviação
Reagent and its number/abbreviation
Reactivo e su número/abreviación
Limite de temperatura
Temperature limitation
Temperatura límite
Data limite de utilização (último dia do mês)
Use by (last day of the month)
Estable hasta (ultimo día del mes)
Fabricado por
Manufactured by
Elaborado por
Risco biológico
Biological risk
Riesgo biológico
Corrosivo
Corrosive
Corrosivo
Tóxico
Toxic
Tóxico
Inflamável
Flammable
Inflamable
Nocivo / Irritante
Harmful / Irritant
Nocivo / Irritante
Material reciclável
Recyclable material
Material reciclable
Não descartar diretamente no ambiente
Dispose properly
Desechar adecuadamente

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