Aspartate Aminotransferase (AST or sGOT) test|Kinetic method|Spinreact kit

Aspartate Aminotransferase (AST or sGOT) test

Liver Function Panel

KineticTechnique

By spinreact kits

I) Introduction

1) Names:-

Formal name: Aspartate Aminotransferase (AST)

Also known as: Serum Glutamic-Oxaloacetic Transaminase; SGOT; GOT; Aspartate Transaminase.

2) What is the ALT?

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

3) Why it is required?

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

4) When should you get tested?

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

5) What are the needed preparations before the test?

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

II) Required sample:-

1) Sample type:-

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

2) Sample collection method:-

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

3) Criteria for sample rejection:-

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

III) The Test:-

Kinetic technique (IFCC METHOD)

By SPINREACT kits

1) Principle

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

2) Devices used:-

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

3) Reagents:-

Note that there are many types of the kits of AST in spinreact company. We choose the one of the references 41270&41272&41273.

  • Composition:-
    • Reagent 1 (R1) (Substrate and BUFFER 7.8): Tris 80 mmol/L, Lactate dehydrogenase (LDH) 800 mmol/L, Malate dehydrogenase (MDH) 600 U/L, L-Aspartate 200 mmol/l, pH 7.8.
    • Reagent 2 (R2) (Substrate): NADH 0.18 mmol/L, ɑ-ketoglutarate 12 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:
    • The presence of particulate material, turbidity and the absorbance of the blank lower than 1.0 at 340 nm (1 cm cuvette).
  • Reagent preparation
    • Working Reagent: Mix 4 vols 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. Bring the working solution and the temperature to the reaction temperature.
  3. 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 has a suction system, by keeping the tubing in a distilled water for 2 minutes.
  4. Zero the spectrophotometer or photometer with distilled water or air at 340nm.
  5. 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 minute in the water bath or in the spectrophotometer if it has a built-in incubator. (this incubation to bring the reagent to 37oC).
  6. Transfer 0.10ml (100µl) of the sample to the incubated 1.0 ml working solution.
  7. Start the stopwatch immediately after sample addition and wait for a one-minute delay. This delay is to get the first absorbance from which the first-minute reading will be subtracted (see the calculation section for more information) (NOTE: – the volume of the sample and the incubation time is recommended by the reagent supplier).
  8. Read the absorbance of the one-minute delay, then read the absorbance every one minute for 3 minutes intervals (3 readings total). And you should return the solution to the incubation after each reading to keep the temperature of the solution at 37oC.
  9. Calculate mean absorbance difference/minute (ΔA/Min.). Then multiply it by the factor that we will calculate in the next section.
  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 AST test or ALT test (you program it according to the reagent supplier) (either program are allowed because they have the same parameters) … In this case, you can prepare the working solution in a cuvette, if it has not a suction system, or a 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 to reach the reaction temperature, then read the blank and then add the sample, then the analyzer starts its own timer and takes the 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

5) Calculation:-



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 readings (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, however, we will neglect the negative sign because it is not important in clinical diagnosis and it doesn’t make sense to see activity with negative sign.

Then take the average of the 3 ΔA: –

average of the 3 delta a

The AST activity is calculated in the sample from the next equation: –

ast calculation

Read more about the calculation of AST in the basics of Kinetic techniques. click 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)

(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 calculates the factor:

ast spinreact factor

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 25ºC 25ºC
 25ºC
25ºC
25ºC
1.00
0.73
0.48 
1.37
1.00
0.65
2.08
1.54
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 multiply the result by 1.37

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 calculate the dilution factor from the following equation:-

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

You can multiply this dilution factor calculated by the factor of the kits if you dilute the sample before the test.

6) Reference range:-

25ºC 30ºC 37ºC
Men (Up to) 19 U/L 26 U/L 38 U/L
Women (Up to) 16 U/L 22 U/L 31 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 about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

V) Quality Control:-

Read about it in topic of AST test by Kinetic technique – the general conceptCLICK HERE

VI) Interpretation of test results:-

Read about it in the topic of AST 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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