First, we allow a delay for 1 min and take the first absorbance reading. Then, we read the absorbance every 1 minutes for 3 minutes. Thus, we take 4 absorbance reading (one after the delay period [Adelay] and 3 for the next 3 minutes; A1, A2 and A3 respectively). Then, the ΔA/min is measured as follow: –
ΔA1/min (For the first minute) = A1 – Adelay
ΔA2/min (For the first minute) = A2 – A1
ΔA3/min (For the first minute) = A3 – A2
The result of these will be negative values. But, we neglect the negative sign because the activity in the final result will be a negative value, which doesn’t make sense.
Then take the average of the ΔA in the 3 readings: –
The AST activity is calculated in the sample from the next equation:-
- (ε) is the molar absorptivity of NADH at 340nm; equals 6300 (i.e. measuring the amount of light absorbed per mole when the light pass through a path length of 1 cm)
- (l) is the cuvette path length; 1 cm
- (Vt) the total volume of the reaction; 1.1ml (1.0 reagent +0.10 sample) at 30oC.
- (Vs) the sample volume; 0.1 at 30oC.
- (106) to convert mol to μmol. HOW?
You can then calculate the factor from the values we said here.
To know how 106 is the result of converting from mol to μmol, read how you can conclude the measuring unit which is U/L in the basics of the kinetic technique. We give and example of ALT test which is the same as AST.
6) Reference range:-
As specified by your reagent supplier.
- Hemolyzed serum due to some errors during phlebotomy process produces false high results because RBCs contain 15 times the AST activity in serum.
- The sample stands at room temperature for a long time. (The test should be performed within 3 hours).
- Incubation temperature exceeds 60oC leading to denaturation of enzyme and hence no activity.
- pH less than or more than the optimal pH can denature the enzyme.
- Usage of turbid expired reagent or when its blank absorbance exceeds the allowed value by the supplier of your reagent.
- Usage of a dirty or unclean cuvette or glassware or forgetting to flush your semi-automated analyzer before performing the test with distilled water.
- The sample Exposure to the direct light because the UV light inhibits the activity of the enzyme and the blue or red light can increase the activity of the enzyme.
- It is better to ask your patient if he take ascorbic acid or vitamins drugs containing vitamin C and write this in your report.
- Hemolyzed samples shouldn’t be accepted and a new one should be ordered.
- The test shouldn’t be performed within 3 hours if the serum or plasma left at room temperature because most enzymes don’t have good stability.
- Serum or plasma should be separated as soon as possible after blood clotting because the loss of CO2 increases the pH rapidly leading to enzyme inactivation.
- You should perform the test at the temperature recommended by your reagent supplier.
- You should perform the test at the pH recommended by your reagent supplier.
- You shouldn’t use expire or turbid reagent or when the absorbance of its blank exceeds the value recommended by your reagent supplier.
- You should clean your glassware and cuvettes well or flush your semi-automated analyzer with distilled water.
- Your should protect the serum or plasma from direct light.
- Sankara Nethralaya’s Manual of Medical Laboratory Techniques
- Wallach’s Interpretation of Diagnostic Tests Pathways to Arriving at a Clinical Diagnosis