Alanine aminotransferase (ALT or sGPT) test|Colorimetric method|Spinreact kit


4) Procedure:-

  1. Calibration curve preparation:-

When you buy a reagent kit you should do a calibration curve if the kit recommends this. From this curve, you will get the concentration of the analyte we measure in the sample … Let’s see how you prepare a calibration curve and how you perform the test to know why you need to prepare a calibration curve.

Method:-

  1. In 5 clean tubes, pipettes (in ml) the following:
Tube12345
Water0.20.20.20.20.2
R1 (Substrate)1.00.90.80.70.6
Calibrator0.00.10.20.30.4
R2 (DNPH)1.01.01.01.01.0

As we see, we prepare different concentrations from the pyruvate and hence we will get different absorbance values for known concentrations. Therefore, if you measure the absorbance of your sample, you can get the corresponding concentration from the curve. Let’s continue to understand more.

  1. Start the timer immediately, Mix well, and allow standing for 20 min at room temperature.
  2. Add 10 ml of NaOH in each tube.
  3. Mix NaOH well, and start the timer and leave it for 5 minutes.
  4. Adjust the spectrophotometer at 505 nm then zero it using distilled water or deionized water.
  5. Then read the absorbance for each tube.
  6. Using the Microsoft Excel Sheets, plot a curve of the absorbance on the Y-axis and the concentration on the X-axis.
Tube12345
Alt activity in (WU/ml)0255083126
Alt activity in (U/l)012244062
Suppose that the abs value00.200.320.560.80

Units

– One Wróblewski unit (WU) of GPT is defined as the amount of enzyme that will form 4.82 x 10-4 µmole of Glutamate/min (25ºC).

– To convert WU into international units (U/L), multiply results by 0.482.

If you plot these values on a graph you will not get a straight line >>> so, you will need to make a trend line. It is recommended to use Microsoft Excel sheets. If you type the data of the table on Excel sheet and insert a chart using the (x,y) scattering chart type you will get the following image:-

alt colorimetric chart 1

But, you need to make the trend line, this is more accurate in excel. If you make the trend line, your chart will be as follow:-

alt colorimetric chart 2

Make sure that all the guidelines are found to be able to determine the activity of the enzyme accurately.

NOTE THAT:-

  • You should prepare a calibration curve when you use a new reagent.
  • You need a control serum (serum whose ALT activity is known) to check calibration graph

NOW, We are ready to measure the sample.

To use this method, you need to measure absorbance so you can use spectrophotometer or semi-automated analyzer that can store a calibration curve.

  1. Measuring the absorbance of the sample:-


  1. Clean the cuvettes if you use a spectrophotometer or semi-automated analyzer without suction system or flush the aspiration system of your semi-automated analyzer if it is with the suction system by keeping the tubing in a distilled water for 2 minutes.
  2. Zero the instrument at 505nm with distilled or deionized water.
  3. ­Pipette 0.5ml (500 µl) from R1 into a tube.
  4. Incubate in the water bath or at the incubation room of the spectrophotometer or at 37oC, for 5 min.
  5. Add 100µl of the patient’s serum or plasma.
  6. Start timing after adding serum or plasma, immediately.
  7. Mix well, continue incubation for 30 min.
  8. Add 0.5ml of R2.
  9. Mix well, then allow standing for 20 min at room temperature.
  10. Add 5ml of the NaOH.
  11. Mix well, let stand for 5 min at room temperature.
  12. Read the initial absorbance. (The color is stable at least one hour)
  13. Then use the calibration curve to get the activity of ALT.

The measuring limit: –

The measuring limit of ALT colorimetric assay from spinreact company is 126 U/L. In other words, if the ALT activity is more than 126 then, the kit can’t measure any higher activity of ALT which means you should dilute the sample 1/10 with NaCl (9 gm/l) and multiply the result by 10. To know how to prepare dilutions and how to calculate the dilution factor click here.

5) Calculation:-

Here, no calculations are required because we found the activity from the calibration curve.

6) Reference range:-

5-30 UW/ml (2-16 U/L)

 These values are for orientation purpose; each laboratory should establish its own reference range.

IV) Sources of errors:-

Read about it in topic of ALT test by colorimetric technique – the general conceptCLICK HERE

V) Quality Control:-

Read about it in topic of ALT test by colorimetric technique – the general conceptCLICK HERE

VI) Interpretation of test results:-

Read about it in topic of ALT test definition and Patient education.CLICK HERE

References:-

  • clevelandcliniclabs.com
  • labtestonline.com
  • spinreact.com
  • boisystems.es
  • Sankara Nethralaya’s Manual of Medical Laboratory Techniques
  • Wallach’s Interpretation of Diagnostic Tests Pathways to Arriving at a Clinical Diagnosis


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