Kinetic UV method
Kinetic UV method based on IFCC recommendations
Aspartate aminotransferase (AST) catalyzes the transfer of the amino group from L-aspartate to -ketoglutarate to yield oxaloacetate and L-glutamate. Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate with simultaneous oxidation of NADH+ to NAD. The resulting rate of decrease in absorbance at 340 nm is directly proportional to the AST activity. Lactate dehydrogenase (LDH) is added to prevent interference from endogenous pyruvate which is normally present in serum.
- R 1 : SGOT Enzyme Reagent
- Buffer Tris, pH = 7.5 —– 125 mmol/L
- L- aspartate ———— 600 mmol/L
- LDH ————– ≥ 0.9 U/L
- MDH —————— ≥ 0.6 U/L
- Detergent, preservative.
- R 2 : SGOT Substrate Reagent
- NADH —– 0.18 mmol/L
- α -ketoglutarate —- 15 mmol/L
- Detergent, preservative.
Test Requirements :
- Test Tubes
- Bio-Chemistry analyzer
- SGOT Test Kit
Reagent Preparation and Stability :
4 Part of R1 (0.8 ml)
1 Part of R2 (0.2 ml)
and Mix well
Avoid direct exposure to light. Stability of working reagent: 3 days at 2 – 8 °C.
Specimen collection and handling:
Analyzer Parameter Required :
|Reaction Type||Kinetic (decreasing)|
|Wavelength||340 nm (334nm – 365nm)|
|Delay Time||60 sec|
|Interval Time||60 sec|
|No. Of reading||2|
|Zero Setting||Against air or Deionised Water|
|Light Path||1 cm|
Pipette into clean dry test tube labeled as (T) and Calibrator Label (c) and QC Label (QC) or as your required:
|Specimin||(T)||(C) / (QC)|
|Working Reagent||1.0 ml||1.o ml|
|Pt. Sample||0.1 ml||–|
|Calibrator or QC||–||0.1 ml|
|Mix well and read the initial absorbance after 1 min and repeat the absorbance reading after every 1, & 2 mins. Calculate the mean absorbance change per minute (Δ OD/min.).|
CALCULATION : SGOT activity (U/L) = D A/min. x 1746
NOTE: Samples having a very high activity show a very low initial absorbance as most of the NADH is consumed prior to the start of the measurement. If this is suspected then dilute the sample and repeat the assay
The procedure is linear upto 300 U/L.
if the activity exceeds this limit dilute the sample with normal saline (NaCl 0.9 %) and multiply result by dilution factor.
QUALITY CONTROL :
For accuracy it is necessary to run known controls with every assay.
Normal Value :
Serum : < 40 U/L
Note: Each Laboratory or Each Company Kits should establish it’s own normal range.
- Gram-positive bacteria are the genus of bacteria family and a member of the phylum Firmicutes. […]
- One of the ways I protect paraffin slides is to dip in hot paraffin and […]
- Unstained paraffin sections offer very low contrast and therefore cannot be evaluated microscopically in routine […]
- The objective of this step is to cut 4–5 Mm-thick sections from paraffin blocks. This […]
- Once tissue samples are infiltrated by paraffin, they are removed from the cassettes and carefully […]
- After fixation, tissue samples need to be properly trimmed to reach the adequate size and […]
- Urine culture results should be interpreted in conjunction with clinical symptoms of urinary tract infection […]
- A urine culture is a test that can detect bacteria in your urine. This test […]