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a.-ketoglutarate reacts with L-alanine in presence of GPT (ALT) to form pyruvate and L-glutamate. The increase in pyruvate is determined in an indicator reaction catalyzed by lactate dehydrogenase. The conversion of NADH to NAD at 340 nm. is proportional to the activity of GPT (ALT) in serum/plasma and is determined kinetically as rate of decrease in absorbance.
PREPARATION OF WORKING SOLUTION
Prepare working solution by mixing Reagent R, and Reagent R, in the ratio 4: 1 as per requirement.
REAGENT STORAGE STABILITY
COMPONENTS & CONCENTRATION OF WORKING SOLUTION
Tris buffer, pH 7.4
SPECIMEN COLLECTION & PRESERVATION
Blood should be collected in a clean dry container. Although serum is preferred, plasma with heparin or EDTA can be used. Samples with any visible haemolysis are not acceptable. GPT (ALT) activity in serum/plasma is stable for 1 week at 2 - 8 degree C and 1 month at -20 degree C. The samples should be brought to room temperature prior to use.
Prewarm at 37 degree C the required amount of working solution before use.
Perform the assay as given below :
Mix and aspirate. After the initial delay of 60 seconds, record the absorbance of the test at an interval of 30 seconds for the next 90 seconds at 340 nm. Determine the mean change if absorbance per minute and calculate test results.
Conversion factors :
Following factors can be used for conversion of IU/I from one
Temperature to another :
At 25 degree C
At 30 degree C
At 37 degree C
To ensure adequate quality control, it is recommended that each batch should include normal and abnormal commercial reference control serum. It should be realized that the use of quality control material checks both instrument and reagent functions together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glassware and accuracy of pipetting.