TECO DIAGNOSTICS ALT (SGPT) LIQUID REAGENT

1268 N. Lakeview Ave.

Anaheim, CA 92807 (KINETIC METHOD)
1-800-222-9880

INTENDED USE REAGENT STORAGE

For the quantitative determination of alanine aminotransferase in serum Reagents are stable until the expiration date on their respective labels,
used in routine examination and monitoring of therapy and relapses. when properly stored at 2 - 8°C and protected from light. Reagents
should appear clear and colorless.
INTRODUCTION
The enzyme alanine aminotransferase is widely reported in a variety of REAGENT DETERIORATION
tissue sources. The major source of ALT is of hepatic origin and has 1. Discard if either appears cloudy or contains particulate matter.
led to the application of ALT determinations to the study of hepatic 2. The working reagent is stable for 2 weeks at 2-8°C. The working
diseases. Elevated serum levels are found in hepatitis, cirrhosis, and reagent should be discarded if the initial absorbance, read against
obstructive jaundice. Levels of ALT are only slightly elevated in distilled water at 340 nm, is below 1.000.
patients following a myocardial infarction.1
MATERIALS REQUIRED BUT NOT PROVIDED
UV methods for ALT determination were first developed by 1. Spectrophotometer capable of absorbance reading at 340 nm and 1
Wroblewski and LaDue in 1956.2 The method was based on the cm light path
oxidation of NADH by lactate dehydrogenase (LDH). In 1980, the 2. Constant temperature block or bath, 37°C, or temperature
International Federation of Clinical Chemistry recommended a controlled cuvette well
reference procedure for the measurements of ALT based on the 3. Accurate pipetting devices
Wroblewski and LaDue procedure.3 The ALT reagent conforms to the 4. Test tubes
formulation recommended by the IFCC. 5. Interval timer

PRINCIPLE SPECIMEN COLLECTION AND STORAGE

The enzymatic reaction sequence employed in the assay of ALT is as Non-hemolyzed serum is the specimen of choice. Whenever possible
follows: specimens should be separated and analyzed on the day of collection.
Store serum in stoppered tubes. About 10% ALT is lost 3 days at 4°C
ALT and in 1 day at 25°C.5
L-Alanine + 2-oxoglutarate Pyruvate + L-Glutamate
INTERFERING SUBSTANCES
LDH Hemolysis must be avoided as the concentration of ALT in red cells is
Pyruvate + NADH + H+ Lactate + NAD+ + H2O roughly 5 times that of serum.2 Bilirubin levels up to 40 mg/dL and
triglyceride levels up to 2000 mg/dL show no interference in this test.
The pyruvate formed in the first reaction is reduced to lactate in the Certain drugs and other substances are also known to affect ALT
presence of lactate dehydrogenase and NADH. The activity of ALT is values.5
determined by measuring the rate of oxidation of NADH at 340 nm.
Endogenous sample pyruvate is converted to lactate by LDH during the MANUAL PROCEDURE
lag phase prior to measurement. 1. Prepare ALT working reagent according to instructions.
2. Pipette 1.0 mL of working reagent into tubes labeled “controls”,
REAGENTS patient(s)”, etc.
ALT Liquid Reagents 1 and 2 come in separate containers, and both 3. Pre-incubate all tubes at 37°C for at least five minutes.
reagents are clear, colorless liquid in ready to use format. After 4. Zero spectrophotometer at 340 nm with distilled water.
combining ALT Liquid R1 (Buffer Reagent) and ALT Liquid R2 (Co- 5. Add 100 µL (0.10 mL) serum to its respective tube, mix gently
Enzyme) the working reagent contains: and turn to a thermo cuvette.
6. Read and record absorbance at 1 minute. Continue incubating at
L-Alanine 500 mmol/L 37°C and record absorbance again at 2 and 3 minutes. Rate should
LDH 1200 U/L be constant.
Tris Buffer, pH 7.5 100 mmol/L 7. Determine the average absorbance per minute (A/min), multiply
2 – Oxoglutarate 15 mmol/L by factor 1768 for results in U/L.
NADH (Disodium salt) 0.18 mmol/L 8. Repeat the procedure for each sample.
Stabilizers and Preservatives
NOTE: If cuvette is not temperature controlled, incubate samples at
WARNINGS AND PRECAUTIONS 37°C between readings.
Normal precautions exercised in handling laboratory reagents should be
followed. The reagents contain sodium azide, which may be toxic if AUTOMATED PROCEDURE
ingested. Sodium azide may also react with lead and copper plumbing Refer to appropriate application manual available.
to form highly explosive metal azides. Refer to Material Safety Data
Sheet for any updated risk, hazard, or safety information. QUALITY CONTROL
It is recommended that controls be included in each set of assays.
REAGENT PREPARATION Commercially available control material with established ALT values
The working reagent is prepared by mixing five (5) volumes of R1 with may be routinely used for quality control. The assigned value of the
one (1) volume of R2 in a disposable container. control material must be confirmed by the chosen application. Failure
Example: 25 ml R1 + 5 ml R2 to obtain the proper range of values in the assay of control material may
indicate either reagent deterioration, instrument malfunction, or REFERENCES
procedure errors. 1. Henry, J.B.: Clinical Diagnosis and Management by Laboratory
Methods, W.B. Saunders and Co., Philadelphia, PA, p-332-335
CALIBRATION (1974).
ALT activity is based on the "micromolar extinction coefficient" of 2. Wroblewski, F. and LaDue, J.S. Proc. Soc. Exper. Biol. and Med.
NADH at 340 nm (see "Results" section). The instrument 91:569 (1956).
manufacturer's calibration guidelines should be followed to calibrate 3. International Federation of Clinical Chemistry, J. Clin. Chem.
your analyzer. Assaying the ALT contents of a control serum with Clin. Bio. 18: 5231(1980).
known ALT values can be used to assure instrument calibration has 4. Bergmeyer, H.U. Principles of Enzymatic Analysis. Verlag
been performed correctly. Chemic, 1978.
5. Young D.S. Effects of drugs on clinical laboratory tests. AACC
RESULTS Press, Washington D.C., 1990.
Values are derived based on the "absorptivity micromolar extinction 6. Henry JB. Clinical Diagnosis and Management by Laboratory
coefficient" of NADH at 340 nm (0.00622). Units per liter (U/L) of Methods, 17th ed. WB Saunders Co., 1984, p 1437.
ALT/GPT activity is that amount of enzyme, which oxidizes one
µmol/L of NADH per minute. A524: 02/2018

A/Min Total Volume Manufactured by:

U/L = Absorptivity  Sample Volume TECO DIAGNOSTICS
1268 N. LAKEVIEW AVE.
A/Min 1.100 ANAHEIM, CA 92807
U.S.A.
U/L = 0.00622  0.100

U/L = A/Min  1768

LIMITATIONS
If the A/min. is greater than 0.342, dilute 1 part sample with 9 parts
isotonic saline and re-assay. Multiply the result by 10. ALT values for
neonatal patients have not been established with this procedure.
Grossly icteric or turbid specimen may require the use of a sample
blank.

EXPECTED VALUES
Normal Range: 3 - 35 U/L (37°C)
It is recommended that each laboratory establish its own range of
expected values, since differences exist between instruments,
laboratories, and local populations.

PERFORMANCE CHARACTERISTICS
1. Comparison: A group of 128 sera ranging in ALT activity from 7 -
625 U/L was assayed by the described ALT method and by a
similar commercially available ALT reagent. Comparison of the
results yielded a correlation coefficient of 0.999 and the regression
equation was y = 0.960 x + 3.2. (Comparison studies were
performed according to NCCLS Tentative Guideline, EP9-T.)
2. Precision:

Within-Run
Serum 1 Serum 2
Mean ALT (U/L) 25.1 116.0
Std. Deviation (U/L) 0.82 0.90
C.V. (%) 3.25 0.77

Total Precision
Serum 1 Serum 2
Mean ALT (U/L) 25.8 114.8
Std. Deviation (U/L) 1.13 0.8
C.V. (%) 4.40 0.69

Precision studies were performed according to NCCLS Tentative

Guideline, EP5-T.

3. Linearity: Linear to 500 U/L at 37°C. Performed according to

NCCLS Guideline EP6-P.
4. Sensitivity: Based on an instrument resolution of A = 0.001, the
method presented shows a sensitivity of 1.8 U/L.