A L A N I N E A M I N O T R A N S F E R A S E ( A LT / S G P T - I F C C U V K i n e t i c M e t h o d )

Intended Use The reagents are used for the quantitative determination of Alanine aminotransferase (ALT) in serum or plasma. For invitro diagnostic use only. Introduction Alanine aminotransferase(ALT) also referred to as serum glutamate pyruvate transferase (SGPT) is an enzyme involved in amino acid metabolism. ALT is found in liver, but a little amount also occurs in the kidneys, heart, muscles and pancreas. Low levels of ALT are normally found in the blood. However, higher concentration of ALT in blood is observed in liver disease, viral hepatitis, circulatory failure with shock, infectious mono-nucleosis, myopathy etc. ALT test is carried out along with the other tests including aspartate aminotransferase(AST), alkaline phosphatase (ALP), lactate dehydrogenase and bilirubin to assess various condition of body. Method IFCC - UV Kinetic Method. Principle alpha-Ketoglutarate + L-Alanine LDH Pyruvate +NADH +H
+

Avoid contact with skin and eyes. If spilled, thoroughly wash affected area with water. Do not swallow or inhale vapor, it may cause irritation to mouth, throat and stomach. Flush with plenty of water while disposing. Reagent Storage and Stability Unopened Reagents are stable till expiry mentioned on the O label when stored at 2-8 C. Reagent Preparation 1) Two Reagents Procedure: The reagents are ready for use. 2) One Reagent Procedure (Working solution) : Mix four volumes of Reagent R1 with one volume of Reagent R2.The working solution is stable for four weeks at 2 to 8C. Reagent Deterioration Reagents should be clear. Turbidity and/or precipitation may be because of reagent deterioration. Specimen Collection and Storage Serum or heparinized plasma can be used for the testing. Anticoagulants like EDTA, oxalates and citrates cannot be used, as they inhibit ALT activity.

PROCEDURE Two Reagent Procedure: Pipette into test tubes as per table given below:
Particulars Sample Volume Reagent R1 800 l Reagent R2 200 l Mix and incubate at 37C for 2 minutes then add Sample 100 I

One Reagent Procedure: Pre-warm working reagent at 37OC for two minutes prior to addition of sample.
Particulars Working Reagent Sample Sample Volume 1000 l 100 l

Mix and after incubation for 60 seconds at 37OC, measure the change of absorbance per minute (A/minute) during 180 seconds. Calculation ALT concentration in the sample can be calculated using the following formula: A = Rate of change of absorbance/minute ALT = Abs/min X Factor (1746)

SGPT

L-Glutamate + Pyruvate

L-Lactate + NAD+

Serum samples can be stored for 3 days at 2-8OC . Freezing of sample causes loss of enzyme activity, hence not recommended.

General Assay Parameters

Mode Wavelength 1 (nm) Wavelength 2 (nm) Blank with Sample Volume (l) Working Reagent (l) Delay Time (sec.) Read Time (sec.) No. of readings Incubation Temperature(C) Normal Level (IU/L) Linearity (IU/L) Factor Units Kinetic 340 Air or Water 50/100 500/1000 60 60 3 37 Upto 40 Upto 250 1746 IU/L

The rate of NADH consumption is measured photometrically and is directly proportional to the ALT concentration in the sample. Reagent Composition Reagent 1:
L-Alanine Lactate Dehydrogenase > 200 mmol/l >1500 IU/l

If the ALT concentration exceeds 250 IU/L, dilute the sample with normal saline and repeat the assay. The reportable results in this case shall be calculated by multiplying the results obtained with dilution factor. Reference value Serum Adult Male Adult Female : up to 40 IU/L : up to 31 IU/L

Reagent 2:
alpha-Ketoglutarate NADH >35 mmol/l 1.05 mmol/l

Precautions Following precautions should be taken: Avoid ingestion, do not pipette by mouth.

Reference range varies from population to population; therefore, each laboratory should establish its own normal range. 3

A L A N I N E A M I N O T R A N S F E R A S E ( A LT / S G P T - I F C C U V K i n e t i c M e t h o d )

Limitations 1.The reagent and sample volumes can be altered proportionately so that the sample:reagent ratio remains same. 2. Temperature has to be maintained constant throughout the reaction as the rate of color development is highly temperature sensitive. Quality Control The patient results obtained for each batch can be validated by using normal and abnormal control sera with assayed values for alanine amino transferase. Performance Linearity Limit: 250 IU/L Precision: Within run
Control No. of samples Mean (IU/L) S.D. C.V. % Control 1 20 20.60 0.91 4.43 Control 2 20 95.68 1.46 1.52

3. Chernecky CC, Berger BJ, eds. (2004). Laboratory Tests and Diagnostic Procedures, 4th ed. Philadelphia: Saunders http://health.med.umich.edu/healthcontent.cfm?xyzpdqabc =0&id=6&action=detail&AEProductID=HW%5FKnowledge base&AEArticleID=hw20645&AEArticleType=MedicalTest. Pack Presentation
Product Code/ Catalogue No. KGALT102.2.1 KGALT102.2.2 Pack Size* 5x20ml 5x50ml Reagent 1 4x20ml 4x50ml Reagent 2 1x20ml 1x50ml

*Pack size may vary on market demands. Revision No: (Ver: KGALT102.2/1) Date of Issue: 1st April 2010 Symbols Following symbols are used in the labeling of KEE GAD kits:

REF

Catalogue No. CE Mark

Batch No. Read instructions Storage temperature CONT Content Manufactured By

Between run
Control No. of samples Mean (IU/L) S.D. C.V. % Control 1 60 20.60 0.84 4.07 Control 2 60 95.74 1.73 1.81
ALT

In Vitro Diagnostics Expiry Date Product Name

References 1. National Committee for Clinical Laboratory Standards. User evaluation of Precision Performance of Clinical Chemistry Devices. NCCLS; 1984, NCCLS Publication. http://www. thermo.com/eThermo/CMA/PDFs/Product/productPDF_5 0918.pdf 2. Kennedy JW, Carey RN, Coolen RB, et al.Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline (EP5- A).Wayne, PA: The National Committee for Clinical Laboratory Standards, 1999.http:// www.qualtexlabs.org/pdf/ALT.pdf 4

Manufactured by: KEE GAD Biogen Pvt. Ltd. A-8, Third Floor, Naraina Industrial Area, Phase-II, New Delhi-110028 (India) 5