Pointe Liquid ALT (SGPT)

Reagent Set

Intended Use Interferences

For the quantitative determination of Alanine Aminotransferase in serum. 1. A number of drugs and substances affect ALT activity. See Young, et al.8
Rx Only 2. Bilirubin to at least 30 mg/dl, and hemoglobin to at least 400 mg/dl, have
been found to have a negligible effect on this procedure.
Clinical Significance
ALT is widely distributed in tissues with the highest concentrations found in Materials Provided
the liver and kidneys. Even so, ALT is considered more liver-specific than
ALT (SGPT) Reagents R1 and R2.
AST. Elevated levels of ALT are often only observed in liver diseases such
as cirrhosis, hepatitis, or metastatic carcinoma. However, there can be
elevated levels of ALT with infectious mononucleosis, muscular dystrophy, Materials Required but not Provided
and dermatomyositis.1 1. Accurate pipetting devices.
2. Test tubes/rack.
Method History 3. Timer.
UV methods for ALT determination were described by Henley2 in 1955 and 4. Spectrophotometer able to read at 340 nm. (UV)
Wroblewski and La Due3 in 1956. The procedure was improved and 5. Heating bath/block (37°C).
optimized by Henry et al4 in 1960. In 1974, the Scandinavian Society for
Clinical Chemistry5 recommended optimized reaction conditions. The Test Procedure (Automated)
International Federation of Clinical Chemistry (IFCC)6 published a proposed Wavelength: 340 nm
recommended method in 1980 utilizing the LDH-NADH coupled assay. The Assay Type: Kinetic
procedure described herein is based on that method. Sample/Reagent Ratio: 1:10
Reaction Direction: Decreasing
Principle Temperature: 37°C
ALT Lag Time: 60 seconds
L-Alanine + α-Ketoglutarate ----------------------- Pyruvate + L-Glutamate Read Time: 60 seconds
LDH Low Normal: 5 U/L
Pyruvate + NADH + H+ ---------------------------- L-Lactate + NAD+ +H2O High Normal: 34 U/L
ALT catalyzes the transfer of the amino group from L-alanine to α- Application Parameters for various automated instruments are available. Please
ketoglutarate resulting in the formation of pyruvate and L-glutamate. Lactate contact the Technical Service Department for specific information.
dehydrogenase catalyzes the reduction of pyruvate and the simultaneous
oxidation of NADH to NAD. The resulting rate of decrease in absorbance is Procedure (Manual)
directly proportional to ALT activity. 1. Reconstitute reagent according to instructions.
2. Pipette 1.0ml of reagent into appropriate tubes and pre-warm at 37°C for five
Reagents minutes.
After combining R1 and R2 the reagent contains: L-alanine 500mM, α- 3. Zero spectrophotometer with water at 340nm.
ketoglutaric acid 15mM, LDH(microbial) >2000IU/L, NADH >0.18mM, Buffer 4. Transfer 0.10ml (100ul) of sample to reagent, mix and incubate at 37°C for
100mM, pH 7.5±0.1, Sodium azide 0.2%, Stabilizers. one minute.
5. After one minute, read and record absorbance. Return tube to 37°C.
Reagent Preparation Repeat readings every minute for the next two minutes.
Prepare working reagent by mixing 5 parts of R1 reagent with 1 part R2 6. Calculate mean absorbance difference/minute (∆Abs./Min.).
reagent. (e.g. 250 ul R1 with 50 ul R2 reagent.) 7. The ∆Abs./Min. multiplied by the factor 1768 (See Calculation) will yield
results in IU/L.
Reagent Storage
1. Store reagents at 2-8°C. Procedure Notes
2. Working reagent is stable for 48 hours at room temp. (15-30°C) and for 1. If the spectrophotometer being used is equipped with a temperature
14 days when refrigerated (2-8°C). controlled cuvette, the reaction mixture may be left in the cuvette while the
absorbance readings are taken.
Reagent Deterioration
2. A very low final reading, together with a small absorbance change between
Do not use reagent if:
readings could indicate a very high ALT level. Dilute and re-assay as
1. The initial absorbance at 340nm is below 0.800.
necessary.
2. The reagent fails to meet stated parameters of performance.

Precautions Limitations
1. Turbid or highly icteric samples may give readings whose initial absorbance
1. This reagent set is for in vitro diagnostic use only.
exceeds the capabilities of the spectrophotometer. More accurate results
2. The reagent contains sodium azide as a preservative. Do not ingest.
may be obtained by using 0.05ml (50ul) of sample and multiplying the final
May react with lead and copper plumbing to form highly explosive metal
answer by two.
azides. Upon disposal, flush with a large volume of water to prevent
2. Samples with values above 500 IU/L should be diluted 1:1 with saline, re-
azide build up.
assayed and the results multiplied by two.
Specimen Collection and Storage Calibration
1. Hemolyzed samples cannot be used as red cells contain ALT.7
The procedure is standardized by means of the millimolar absorptivity of NADH
2. ALT in serum is stable for three days at room temperature (15-30°C),
taken as 6.22 at 340nm under the test conditions described.
seven days refrigerated (2-8°C), and thirty days frozen (-20°C).7

5449 Research Drive ● Canton MI 48188 ● USA ● Phone: 734-487-8300 ● Toll Free: 800-445-9853 ● Fax: 734-483-1592 ● www.medtestdx.com
 Pointe Liquid ALT (SGPT)
Reagent Set

Calculation 5. The Committee on Enzymes of the Scandinavian Society for Clinical

One international Unit (IU/L) is defined as the amount of enzyme that Chemistry and Clinical Physiology, Scand. J. Clin. Lab. Invest 32:291 (1974).
catalyzes the transformation of one micromole of substrate per minute under 6. Clinica Chimica Acta 105:145F-172F (1980).
specified conditions. 7. Henry, R.J., Clinical Chemistry: Principles and Technics, Harper & Row, NY,
P522 (1968).
ALT (IU/L) = ∆Abs./Min. x 1.10 x 1000 = ∆Abs./min. x 1768 8. Young, D.S., et al, Clin. Chem. 21:1D (1975).
6.22 x 0.10 x 1.0 9. Henry, J.B., Clinical Diagnosis & Management by Laboratory Methods, W.B.
Where ∆Abs./Min. = Average absorbance change per minute Saunders Co., Philadelphia, P1437 (1984).
1000 = Conversion of IU/ml to IU/L
1.10 = Total reaction volume (ml) Symbol Key
6.22 = Millimolar absorptivity of NADH
0.10 = Sample Volume (ml)
1.0 = Light path in cm Use by (YYYY-MM-DD) Lot and batch code

Example: If the average absorbance change per minute = 0.12 then 0.12 x Catalog number Manufacturer
1768 = 212 IU/L
In vitro diagnostic medical device Temperature limitation
NOTE: If test parameters are altered the factor has to be recalculated using
the above formula. Consult instructions for use Rx Only: Prescription Use Only

SI Units: To convert to SI Units (nkat/L) multiply IU/L by 16.67. CE mark Authorized representative in the European Community

Quality Control
The validity of the reaction should be monitored using control sera with
known normal and abnormal ALT (SGPT) values. These controls should be Manufactured by MedTest Dx: Pointe Brand
run at least with every shift in which ALT (SGPT) assays are performed. It is A7526 5449 Research Drive
Canton, MI 48188
recommended that each laboratory establish their own frequency of control
determination.

Expected Values9 Manufactured by MedTest Dx: Pointe Brand

4 to 24 IU/L (30°C) 5449 Research Drive, Canton, MI 48188
4 to 36 IU/L (37°C)
Since the expected values are affected by age, sex, diet, and geographical European Authorized Representative:
location, each laboratory is strongly urged to establish its own reference Obelis s.a.
range for this procedure. Boulevard Général Wahis 53
1030 Brussels, BELGIUM
Performance Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: [email protected]
1. Linearity: 0-500 IU/L.
2. Comparison: Studies between the present method and a similar
method yielded a correlation coefficient of 0.999 and a regression Certified Performance Guarantee
equation of y=0.96x + 3.2. (n=128, range=7-625 IU/L) MedTest Dx certifies that all of our products are manufactured according to
3. Precision: specified parameters. Any product not meeting specifications through their listed
expiration date will be remedied immediately without charge.
Within Run (n=20) Run to Run (n=20)
Mean S.D. C.V.% Mean S.D. C.V.%
30 0.7 2.3 31 0.7 2.3
116 0.8 0.7 118 1.7 1.4
381 3.1 0.8 382 2.5 0.9
4. Sensitivity: The sensitivity for this reagent was investigated by reading
the change in absorbance at 340nm for a saline sample and serums
with known concentrations. Ten replicates were performed. The
results of this investigation indicated that, on the analyzer used, the
ALT (SGPT) reagent showed little or no reagent drift on a zero sample. Rev. 08/19 P803-A7526-01
Under the reaction conditions described, 1 U/L ALT activity gives a
∆Abs/Min. of 0.0004.

References
1. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders co., p
674 & 675 (1982).
2. Henley, K.S., Pollard, H.M., J. Lab. Clin. Med. 46:785 (1955).
3. Wroblewski, F., La Due, J.S., Proc. Soc. Exp. Biol. Med. 91:569 (1956).
4. Henry, R.J., et al, Am. J. Clin. Path. 34:381 (1960).