AutoPure AST (GOT)

IFCC without pyridoxal phosphate activation

Introduction
1. AutoPure AST (GOT) is a reagent kit for direct quantitative determination
of aspartate transaminase (AST) in human serum and plasma on automated
clinical chemistry analyzers.
2. AutoPure AST (GOT) is a ready-to-use, two liquid reagent system.
Specimen Collection & Preservation
Collect sample using standard sampling tube. Although serum is preferred,
heparinized plasma or EDTA - plasma can also be used. Specimen with any
visible haemolysis is not acceptable, since erythrocytes contain
approximately ten times the normal concentration of AST (GOT) found in
serum. AST (GOT) in serum / plasma is stable for 7 days at 20 - 80C and for
30 days at -10 0 C. Centrifuge samples containing precipitate before
performing assay.

3. With AutoPure AST (GOT), the assay is linear upto 800 IU/I (13.36 μkat/I). Procedure
AutoPure AST (GOT) can be used on various automated analyzers. The
Principle procedure described below is for a Hitachi 902 auto-analyzer.
0 5 9 17 Assay point
α-ketoglutarate reacts with L-aspartate in the presence of AST (GOT) to
form oxaloacetate and L-glutamate.
0
37 C Measurement
A1 A2 at 700/340 nm.
AST (GOT)
Sample : 10 μl R2 : 50 μl
α-ketoglutarate + L-aspartate oxaloacetate + L-glutamate R1 : 250 μl


The increase in oxaloacetate is determined in an indicator reaction catalyzed Hitachi is a registered trademark of Roche Diagnostic, Indianapolis, IN

by malate dehydrogenase.
malate dehydrogenase
Calculations
+ +
oxaloacetate + NADH + H L-malate + NAD Fully automated systems automatically calculate the AST (GOT) activity of
each sample.
The conversion of NADH to NAD+ is proportional to the AST (GOT) activity
Results in μkat/l = Results in IU/I x 0.0167
in the specimen and is determined kinetically.

Abbreviations : AST = Aspartate transaminase Application Sheets

GOT = Glutamate oxaloacetate transaminase Application sheets for the popular fully automated analyzers are provided
along with the kit. For additional system applications, contact our local
Accurex representative.
Reagent Storage, Stability & Handling
AutoPure AST (GOT) is a ready-to-use, two liquid reagent system. Calibration
Shelf - Life Determine the factor using a calibrator. For calibration, it is recommended to
0 0
use C.f.a.s.* or any other suitable calibrator material.
Stable till the expiry date indicated on the label, when stored at 2 - 8 C.

On - Board Reagent Stability Calibration frequency :

R1 : 30 days at 20 - 80C after opening. Protect the reagent from light and
Re-calibration is recommended
contamination.
R2 : 30 days at 20 - 80C after opening. Protect the reagent from light and • Whenever the reagent lot is changed
contamination.
• As per the requirements of quality control procedures
Do not freeze the reagent.
*C.f.a.s. is the brand name of Roche Diagnostics, Germany

Components & Concentration of Working Solution Quality Control

Each batch of AutoPure AST (GOT) is assayed with at least six quality
Component Concentration
control sera — Precinorm** , Precipath** , Biorad l*** , Biorad II*** , Sero Low****
R1 and Sero High****prior to release.
• Tris buffer; pH 7.8 100 mmol/l To ensure adequate quality control, it is recommended that the laboratory
• L-aspartate ≥ 200 mmol/l should use a normal and an abnormal commercial reference control serum.
It should be realized that the use of quality control material checks both
• Malate dehydrogenase ≥ 400 IU/I reagent and instrument functions together. The value of these controls should
• Lactate dehydrogenase ≥ 1500 IU/I fall within the specified limits. If control values fall outside specified limits,
each of the below criteria should be cross-checked and corrected :
• Stabilizers, excipients & surface active agents.
• Proper instrument function - wavelength setting, light source and
R2 temperature control

• Tris buffer; pH 9.5 20 mmol/l • Cleanliness of probes & cuvettes

• α-ketoglutarate ≥ 10 mmol/l • Bacterial contamination of wash water used by the instrument

• NADH ≥ 0.18 mmol/l • Expiry date of the reagent kit

• Stabilizers, excipients & surface active agents ** brand name of Roche Diagnostics, Germany; ***brand name of Biorad, USA
****brand name of Sero, Norway; subject to availability of control serum

AOT-2009-05-00I
Expected Values 4. Penttila, I.M. et al, Scand, J. Clin. Lab. Invest 35, 275 / (1975).
5. Hafkenshelid, J.C.M. et al. J. Clin. Chem. Clin. Biochem. 17, 219
Serum/Plasma
(1979).

250C 300C 370C 6. In-house test data. Accurex Biomedical Pvt. Ltd., 2002.

IU/I Men Upto 18 Upto 25 Upto 37

Women Upto 15 Upto 21 Upto 31
μkat/l Men Upto 0.30 Upto 0.42 Upto 0.62
Women Upto 0.25 Upto 0.35 Upto 0.52

The following factors are used for conversion :

From 370C to 300C : 0.67
From 370C to 250C : 0.49

Note :
Expected range varies from population to population. It is therefore
recommended that each laboratory should establish its own normal range.
For diagnostic purposes, the AST (GOT) results should always be assessed
in conjunction with the patient’s medical history, clinical examinations and
other findings.

Performance Characteristics
Linearity
With AutoPure AST (GOT), the assay is linear upto 800 IU/I (13.36 μkat/l). AR. No.: I 41 AOT-2009-05-00I
Determine samples with higher concentrations via the rerun function.
On instruments without rerun function, manually dilute samples with higher
concentrations using 0.9% NaCl or distilled / deionized water (e.g. 1 + 2).
Multiply the result by the appropriate dilution factor (e.g. 3).

Interference
There is no significant interference in samples containing upto 60 mg/dl of
bilirubin. Haemolysis interferes due to AST activity from erythrocytes.

Precision
Reproducibility was determined using quality control sera as shown below:
n = 15
AST (GOT)
Within run Between run
Quality Control Material Mean SD %CV Mean SD %CV
IU/I IU/I IU/I IU/I IFCC without
Low Control Serum 37 1.29 3.5 37 1.27 3.5 pyridoxal phosphate activation
High Control Serum 162 2.21 1.4 158 2.83 1.8

Co-Relation Studies
A comparison of AST (GOT) determination using AutoPure AST (GOT) and
AutoZyme SGOT reagent gave the following co-relation (IU/I):
Linear Regression
y = -0.114 + 1.011x
r = 0.986
Sy.x = 1.88
No. of samples measured : 58
The sample concentrations measured were between 15 and 65 IU/I.

References
1. Tietz, N.W. ed. Clinical Guide to Laboratory Tests, 3 rd ed.
Philadelphia, Pa : W.B. Saunders, 1995 : 76 - 77.
2. Bergmeyer, H.U., Horder, M., Rej, R. Approved recommendation. (1985)
on IFCC methods for the measurement of catalytical concentration of
enzymes, Part 3. IFCC method for L - aspartate aminotransferase.
J. Clin. Chem. Clin. Biochem. 1986; 24 : 497 - 510.
3. Fischbach, F., Zawta, B. Age - depending Reference Limits of Several
Enzymes in plasma at Different Measuring Temperatures. Klin Lab.
1992; 38:555 - 561.