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

Alanine aminotransferase (ALT or GPT) test

Liver Function Panel

Kinetic Technique (IFCC Method)

By spinreact 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:-

Note that there are many types of the kits of ALT in spinreact company. We choose the one of the references 41280&4128.

  • Composition:-
    • Reagent 1 (R1) (Substrate and BUFFER 7.8): Tris 100 mmol/L, L-alanine 500 mmol/L, lactate dehydrogenase 1200 U/L, pH 7.8.
    • Reagent 2 (R2) (Substrate): NADH 0.18 mmol/L, 2-ketoglutarate 15 mmol/L.
  • STORAGE
    • Store at 2-8ºC.
    • Reagents are stable until the expiry date shown on the label when stored tightly closed, protected from light and contaminations prevented during their use.
  • Indications of deterioration:
    • Presence of particulate material, turbidity, absorbance of the blank lower than 1.0 at 340 nm (1 cm cuvette).
  • Reagent preparation
    • Working Reagent: 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 21 days at 2-8ºC or 72 hours at room temperature (15-20oC).


4) Procedure:-


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

  1. Prepare the work solution as mentioned in the reagent.
  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 250C or 37°C or 30oC  (fixed temperatures) for one minutes in the water bath or in the spectrophotometer if it has built-in incubator. (this incubation to bring the reagent to 37oC).
  5. Transfer 0.10ml (100µl) of the sample to the incubated 1.0 ml working solution.
  6. Start the stopwatch immediately after sample addition and wait for one-minute 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. Repeat readings every minute for the next two minutes.
  9. Calculate mean absorbance difference/minute (ΔA/Min.).
  10. If the sample is hemolyzed, collect another sample and of it is icteric or lipemic, dilute the sample 1 in 10 with distilled water (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.

Spinreact calculate the factor, at 30oC

alt biosystems

But in spinreact kit the factor is 1750 not 1746

Then it gives factors to convert from temp to another;

  • To correct results to other temperatures multiply by:
Assay

temperature

Conversion factor to
25ºC 30ºC 37ºC
25ºC

30ºC

37ºC

1,00

0,76

0,55

1,32

1,00

0,72

1,82

1,39

1,00

How to use this table? If you perform the test at 250C and want to convert the value of the result to the value at 30OC, then you should to multiply the result by 1.32

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 1 ml 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)

6) Reference range:-

  25oC 30oC 37oC
Men (up to) 22 U/L 29 U/L 40 U/L
Women (up to) 18 U/L 22 U/L 22 U/L

Normal newborns have been reported to show a reference range of up to double the adult, attributed to the neonate’s hepatocytes. These values decline to adult levels by approximately 3 months of age.

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

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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