Alanine aminotransferase (ALT or sGPT) test|Kinetic (IFCC) method|Biosystems kit

Alanine aminotransferase (ALT or GPT) test

Liver Function Panel

Kinetic Technique (IFCC Method)

By BIOSYSTEMS kits

I) Introduction

1) Names:-

Formal name: Alanine Aminotransferase and abbreviated as (ALT)

Also known as: Serum Glutamic-Pyruvic Transaminase; SGPT; GPT; Alanine Transaminase.

2) What is the ALT?

Read about it in the introduction section of the ALT test|definition and patient education. CLICK HERE

3) Why it is required?

Read about it in the introduction section of the ALT test|definition and patient education. CLICK HERE

4) When should you get tested?

Read about it in the introduction section of the ALT test|definition and patient education. CLICK HERE

5) What are the needed preparations before the test?

Read about it in the introduction section of the ALT test|definition and patient education. CLICK HERE

II) Required sample:-

1) Sample type:-

Read about it in the required sample section of the ALT test general concept

2) Sample collection method:-

Read about it in the required sample section of the ALT test general concept

3) Criteria for sample rejection:-

Read about it in the required sample section of the ALT test general concept

III) The Test:-

Kinetic technique (IFCC METHOD)

By BIOSYSTEMS kits

1) Principle

Read it in the principle of the general concept of alt test.

2) Devices used:-

Read it in the general concept of alt test

3) Reagents:-

  • Composition:-
    • Reagent 1 (R1) (Substrate and BUFFER 7.3): Tris 150 mmol/L, L-alanine 750 mmol/L, lactate dehydrogenase > 1350 U/L, pH 7.3.
    • Reagent 2 (R2) (substrate): NADH 1.9 mmol/L, 2-oxoglutarate 75 mmol/L, Sodium hydroxide 148 mmol/L, sodium azide 9.5 g/L.
  • STORAGE
    • Store at 2-8ºC.
    • Reagents are stable until the expiry date shown on the label when stored tightly closed and if contaminations are prevented during their use.
  • Indications of deterioration:
    • Presence of particulate material, turbidity, absorbance of the blank lower than 1.400 at 340 nm (1 cm cuvette).
  • Reagent preparation
    • Working Reagent: Pour the contents of the R2 into R1 bottle. Mix gently.
      • Other volumes can be prepared in the proportion: Mix 4 vol or R1 + 1 Vol of R2. Mix gently. (because the cuvette is filled by 1 ml, you will mix 800µl R1 with 200µl R2 [4R1:1R2]).
      • Stable for 1 month at 2-8ºC.


4) Procedure:-



Note:- these procedures are for spectrophotometer users, not for semi-automated users

  1. Prepare the work solution as mentioned in the reagent in test tube.
  2. 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 suction system by keeping the tubing in a distilled water for 2 minutes.
  3. Zero spectrophotometer or photometer with distilled water or air at 340nm.
  4. Pipette 1.0ml of the prepared working reagent into appropriate tubes or cuvettes and incubate at 37oC or 30oC for five minutes in the water bath or in the spectrophotometer if it has built-in incubator (this incubation to bring the reagent to 37oC or 30oC).
  5. Transfer and mix well 0.10ml (100µl) of the sample if the incubation temperature of the reagent is 30oC and 0.05ml (50µl) of sample if it is 37oC to the incubated 1.0 ml working solution.
  6. Start the stopwatch immediately after sample addition and wait for one mintue delay. This delay to make the reaction happen and all the pyruvate is produced (NOTE:- the volume of the sample and the incubation time is recommended by the reagent supplier).
  7. After the one-minute delay, read and record absorbance after 1 minute at 340nm.
  8. Return tube to incubation.
  9. Repeat readings every minute for the next two minutes.
  10. Calculate mean absorbance difference/minute (ΔA/Min.).
  11. If the sample is hemolyzed, collect another sample and if it is icteric or lipemic, dilute the sample 1 in 10 with distilled water (read in how to make dilution and calculate the dilution factor. click here).

In the case, if you use a semi-automated analyzer, you will flush the suction system if it has a suction system or clean the cuvettes if it has not a suction system. Then choose the programmed procedure for ALT test (you program it according to the reagent supplier). In this case, you can prepare the working solution in a cuvette if it has not a suction system or tube if it has a suction system. Then place the cuvettes or tubes in the thermostable cell holder of the semi-automated analyzer or in the water bath, then read the blank and then add the sample, start reading immediately after adding the sample, where the analyzer starts its own timer and takes the 3 readings as scheduled then multiply the absorbance by the factor which we will calculate in the calculation section. (if you don’t make the analyzer start reading after adding the sample immediately, you will get false low result) DON’T forget to flush again at the end of the test if it has a suction system. IT IS EASIER THAN SPECTROPHOTOMETER

Question

In this procedure, we didn’t measure the standard although you tell us that there is a standard???

Answer:

Because in this procedure we didn’t measure the concentration of ALT but its activity where we measure the rate of increment of pyruvate but the standard in this case is used as control to detect the validity of the reagents.

5) Calculation:-

After you get the 3 absorbance values, you will take the average of the ΔA in the 3 readings:-

CMBX12

The ALT activity is calculated in the sample from the next equation:-

CMBX12

Read the calculation of ALT in the basics of Kinetic techniquesclick here to know how we get this equation and how to find out the measuring units.

(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 or 1.05ml (1.0 reagent + 0.05 sample) at 37oC

(Vs) the sample volume; 0.1 at 30oC or 0.5ml at 37oC

(106) 1000 to convert U/ml to U/L and 1000 for conversion from Millimolar absorptivity of NADH to molar absorptivity; the unit of the molar absorptivity.

Now, at 30oCalt biosystems

Now, at 37oC

alt biosystems 37

If the sample is lipemic and you dilute the sample 1 in 10, for example, at 30oC, you will add 0.9ml (900µl) distilled water to 0.1ml (100 µl) sample (1:10) then the sample volume after dilution will be 1ml and during the test you will take 0.1 ml of this diluted sample. The dilution is factor calculated form the following equation:-

dilution-factor

If you want to know how to calculate the dilution factor. Click here

You can multiply this dilution factor by that of the kits if you dilute the sample before the test (1746 x 10)
And at 37oC, you will dilute the serum as follow:- 450µl distilled water will be added to 50µl serum

6) Reference range:-

  • At 30oC :- Up to 29 U/L
  • At 37oC :- Up to 41 U/L

Concentrations in newborns and infant are slightly higher than in adults. Values in men are slightly higher than in women.

IV) Sources of errors:-

Read it in the sources of errors in the general concept of alt test.

V) Quality Control:-

Read it in the QC in the general concept of alt test.

VI) Interpretation of test results:-

Read about it in 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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