The purpose of the title is to accentuate the importance of the method of measurement in evaluating the condition of the liver and in Diagnostic Laboratory, as well as the procedures connected with the determination of serum ALT/SGPT level.
Introduction
Hepatic ALT and ASTAST are critical enzymes in the Diagnostic Laboratory and vital for the metabolism of amino acids. Another important parameter for evaluation of the stage of liver injury as well as the function of this organ is an increase of serum ALT/SGPT concentration. As you continue to read within this guide, we will illustrate a practical way of predicting the serum ALT/SGPT level.
Methods
Here are different researcher techniques utilized for ALT (Alanine Transaminase) estimation:
1. IFCC (International Federation of Clinical Chemistry) method
It is recommended as the first method of choice for the assessment of ALT activities and is founded on the enzymatic assay.2. UV-Vis spectrophotometry
Determines the amount of NADH reduced which is directly related to the activity of ALT enzyme and is evident from the OD at 340 nm.
3. Kinetic method
Measures the rate of ALT-catalyzed reaction, calculating enzyme activity from the slope of the absorbance vs. time curve.
4. Colorimetric method
Uses a chromogenic substrate, such as alanine, to measure ALT activity.
5. Fluorimetric method
Measures the fluorescence of a probe, such as NADH, to detect ALT activity.
6. Electrochemical method
Uses an electrode to detect the enzymatic reaction, measuring ALT activity.
7. Chromatographic method
Separates and detects ALT using techniques like HPLC or GC.
8. Immunoassay method
Estimates plasma ALT by using reagents that selectively react with the enzyme.
9. Point of care testing (POCT)
Non-serum assays for a rapid determination of ALT at the clinical site or the battle front.
10. Automated analyzer method
Uses automated instruments to measure ALT, often used in clinical laboratories.
Materials Needed
- Blood Specimen
- Clotting Tubes
- Microcentrifuge tubes
- Centrifuge
- ALT/SGPT assay kit
- Pipettes
- Spectrophotometer
- Calculator
Tips and Considerations
- Handle samples and reagents carefully to avoid contamination.
- Follow the manufacturer's instructions for assay kits and equipment.
- Use appropriate controls and calibrators.
- Consider interfering substances and hemolysis.
Step-by-Step Procedure
1. Sample Preparation
- Collect specimen in appropriate tubes
- Centrifuge the specimen for 5-10 mnts at low speed.
- Carefully pour the serum into microcentrifuge tubes.
2. Reagent Preparation
- Prepare working reagent for ALT/SGPT assay reagents as per the instructions provided by the manufacturer.
3. Assay Procedure
- Add a specified volume of serum to the assay mixture.
- Incubate the mixture at a specified temperature for a recommended time.
- Read the absorbance of assay at 340 nm or as per manufacturer guidelines.
4. Calculation
- To Calculate the levels of ALT/SGPT, follow the manufacturer's instructions.
The calculations for estimating serum ALT/SGPT levels requires the absorbance values measured from the assay procedure. Here's a step-by-step explanation for calculation:
4.1. Absorbance Measurement
Measure the absorbance of the sample and blank (control) using a spectrophotometer.
4.2. Calculate Delta Absorbance
Subtract the blank absorbance from the sample absorbance to get the delta absorbance (ΔA).
4.3. Calculate Enzyme Activity
Use the manufacturer's formula to calculate the enzyme activity (EA) from the delta absorbance. The formula typically includes a factor for the molar absorptivity (ε) of the enzyme:
EA (U/L) = (ΔA x ε x TV) / (t x V)
Where:
- EA = Enzyme activity (U/L)
- ΔA = Delta absorbance
- ε = Molar absorptivity (L/mol/cm)
- TV = Total volume (L)
- t = Incubation time (min)
- V = Sample volume (L)
4. Calculate ALT/SGPT Levels
- Convert the enzyme activity to ALT/SGPT levels (U/L) using a predetermined conversion factor or calibration curve.
Example Calculation
- Absorbance (Sample): 0.5
- Absorbance (Blank): 0.1
- Delta Absorbance (ΔA): 0.4 (0.5 - 0.1)
- Enzyme Activity (EA): 100 U/L (using manufacturer's formula)
- ALT/SGPT Level: 50 IU/L (using conversion factor or calibration curve)
Note: The exact calculations and formulas may vary depending on the specific assay kit and manufacturer's instructions.
Reference Range
The Diagnostic Laboratory as well as the individual's age and gender determine the ALT (Alanine Transaminase) reference range. Here are some general reference ranges for ALT:
- Newborns (0-1 month): 10-40 U/L
- Infants (1-12 months): 10-50 U/L
- Children (1-18 years): 10-45 U/L
- Adult males: 10-40 U/L
- Adult females: 7-35 U/L
- Pregnant females: 6-32 U/L
Troubleshooting
- High levels: Consider liver damage or disease.
- Low levels: Consider instrument error or sample hemolysis.
- Inconsistent results: Check sample handling and assay protocol.
Conclusion
In Clinical Laboratories, the IFCC method is widely used to estimate serum ALT/SGPT levels, which are crucial for determining liver function and damage. The change factor, communicated in units of U/L, addresses how much protein movement relating to a particular ALT/SGPT level. However, methods vary in principles, advantages, and limitations.
The IFCC method is considered the gold standard for ALT measurement due to its:
- High sensitivity and specificity
- Good precision and accuracy
- Wide linear range
- Minimal interference from other enzymes or substances
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