1.0     INTENDED USE

This reagent is intended for the quantitative determination of alanine aminotransferase (ALT) in serum.

 

2.0     BACKGROUND

2.1     METHOD AND HISTORY

Henley and Pollard (10.1) and Wroblewski and Ladue (10.2) developed kinetic procedures for assaying alanine aminotransferase, formerly called glutamate pyruvate transaminase (GPT).  The procedures were based on the oxidation of NADH by lactate dehydrogenase (LDH).  Henry et. al.(10.3) developed an optimized method and this procedure is a modification of the method of Henry.  The NADH concentration and LDH concentration are increased to improve linearity and completely remove interference from endogenous pyruvate in the sample.

2.2     TEST PRINCIPLE

The ALT catalyzes the conversion of L‑alanine and 2‑oxoglutarate to pyruvate and L‑glutamate. Then LDH catalyzes the oxidation of NADH to NAD.

                                                ALT

          2‑oxoglutarate + L‑alanine --------> L‑glutamate + Pyruvate

                                                LDH

          Pyruvate + NADH + H⁺ ---------> Lactate + NAD⁺

The rate of decrease in absorbance of the reaction mixture at 340 nm, due to the oxidation of NADH is directly proportional to the ALT activity.

2.3     CLINICAL SIGNIFICANCE

The activity of alanine aminotransferase (ALT, GPT) in serum is clinically useful in the differentiation of hepatic disorders, especially when its activity is compared to that of aspartate aminotransferase (AST, GOT.)  In cases of hepatic necrosis, the activity of serum ALT is typically greater than that of AST, whereas in cirrhosis and metastatic carcinoma, the reverse often occurs.  In cases of myocardia infarction, however, ALT levels may be within the reference interval while AST may frequently be 20-30 times greater.

 

3.0     SPECIMEN COLLECTION AND HANDLING

3.1     PATIENT PREPARATION

          No special patient preparation is required.

3.2     SPECIMEN COLLECTION

Fresh, clear, unhemolyzed serum is the preferred specimen.  No interference is experienced with plasma from commonly used anticoagulants.

          Use a standard venipuncture tube to draw patient sample.

The amount of sample required will depend on the analyzer used.  The amount of serum required is in the range of 5-200 µl.  Call Biotron's technical service department at 1-800-595 8766 for the recommended sample volume for your analyzer.

Record the patient's name, date and time of sample collection and preparation.

3.3     SPECIMEN STORAGE

Specimens for analysis should be stored at 2 to 8°C (refrigerated) and are stable for up to 7 days.  Specimens may be stored at -20 to 0°C (frozen) for longer periods.

It is recommended that testing be done as soon as possible after sample collection and preparation.  If testing cannot occur immediately, store the sample properly using the guidelines above.

 

4.0     MATERIALS                                                           (10 X 10 ml)

                                                                                              (6 X 50 ml)

                                                                                            (6 X 500 ml)

          Reagents necessary for the determination of ALT are included in the kit.

4.1     REAGENT

ALT reagent contains, after reconstitution with deionized water:

NADH                                                                            ³ 0.4 mM

lactate dehydrogenase (lactobacillus leichmannii)           ³ 800 U/L

2-oxoglutarate                                                                    13 mM

dL-alanine                                                                          13 mM

tris buffer                                                        100 mM (pH = 7.3)

preservative

4.2     WARNINGS AND PRECAUTIONS

For In Vitro Diagnostic Use.  Not for Internal use in Humans or Animals.  In Vitro Diagnostics reagents may be hazardous.  Avoid ingestion and skin or eye contact.

4.3     REAGENT PREPARATION

Add 10 ml of the deionized water to each of the required number of vials of ALT Reagent.  Replace the rubber stopper and allow 5 minutes for reconstitution.  Swirl gently until the contents of the vial are completely dissolved.  Record the date and time of reconstitution.

4.3.2

Add 50 ml of the deionized water to each of the required number of vials of ALT reagent.  Replace the rubber stopper and allow 5 minutes for reconstitution.  Swirl gently until the contents of the vial are completely dissolved.  Record the date and time of reconstitution.

.

4.3.3 

Add 500 ml of the deionized water to each of the required number of vials of ALT reagent.  Replace the rubber stopper and allow 5 minutes for reconstitution.  Swirl gently until the contents of the vial are completely dissolved.  Record the date and time of reconstitution.

 

4.4     REAGENT STORAGE AND STABILITY

Unopened reagents included in the kits are stable at 2-8° C (refrigerated) until the expiration date stated on the labels.  The reconstituted reagent is stable at 2-8° C (refrigerated) for 14 days or at 18-26° C (room temperature) for 8 hours.

The reagent solutions should be clear.  Cloudiness indicates contamination and the reagent should be discarded.  The initial absorbance of the reagent read against distilled water at 340 nm should be at least 1.100 to be considered suitable for use.

4.5     ADDITIONAL MATERIALS REQUIRED

4.5.1  Spectrophotometer capable of reading absorbance at 340 nm.

4.5.2  1 cm cuvettes or a flow cell capable of transmitting light at 340 nm.

4.5.3  Test tubes capable of holding 4 ml.

4.5.4  Pipettes capable of delivering 3.0 ml and 200 µl; cylinders capable of measuring 10 ml.

4.5.5  Timer with one minute increments.

4.5.6  Constant temperature source which can be adjusted to 30°C or 37°C.

4.5.7  Normal and abnormal control for quality control.

 

5.0     TEST PROCEDURE

          The following is a general procedure for use on a manual instrument.

5.1     PROCEDURE CONDITIONS

Wavelength                                                                       340 nm

Temperature                                                             30°C or 37°C

Pathlength                                                                          1.0 cm

Mode                                                                                  Kinetic

Reaction time                                                                  2 ‑ 4 min

Sample volume                                                                     200 µl

Reagent volume                                                                   3.0 ml

Total volume                                                                       3.2 ml

Sample to reagent ratio                                                           1/15

5.2     INSTRUMENT

Any instrument capable of reading absorbance accurately with a sensitivity of 0.001 absorbance at 340 nm may be used.  The band width should be 10 nm or less, stray light 0.5% or less, and the wavelength accuracy within 2 nm.

5.3     CALIBRATION

No reagent calibration is necessary as this procedure is standardized based on the millimolar absorptivity of NADH which is taken as 6.22 at 340 nm under the test conditions described.

5.4     PROCEDURE

5.4.1  Prepare the required volume of ALT working reagent.  (See 4.3 Reagent Preparation section.)

5.4.2  Into separate test tubes pipette 200 µl of serum to be assayed.

5.4.3  Add 3.0 ml of working reagent, mix, and incubate for one to two minutes at 30°C or 37°C.  The lag time will be decreased if the reagent is prewarmed to the incubation temperature.

5.4.4  Record the decrease in absorbance at 340 nm at one minute intervals until the absorbance change is constant.

5.5     CALCULATION AND RESULTS

          ALT (U/L) =

          DA/min X assay volume (ml) X 1000

          -------------------------------------------------------- = DA/min X 2572

          6.22 X light path (cm) X sample volume (ml)

          DA/min = change in absorbance per minute

          Assay volume = total reaction volume expressed in ml

          1000 = converts U/ml to U/L

          6.22 = absorbance coefficient of NADH at 340 nm

          Light path = length of the light path expressed in cm (usually 1)

          Sample volume = sample volume expressed in ml

          2572 = factor derived from constants in the equation

          Example:  ALT (U/L) =

          .017 X 3.2 X 1000

          --------------------------- = .017 X 2572 = 44 U/L

          6.22 X 1 X 0.2

          0.017 = change in absorbance per minute

          3.2 = total reaction volume expressed in ml

          1.0 = length of the light path expressed in cm

          0.2 = sample volume expressed in ml

 

6.0     INTERPRETATION OF RESULTS

6.1     EXPECTED VALUES (10.5)

          The range of expected values is:

8‑20 U/L  (30° C)

12‑31 U/L  (37° C)

These values are suggested guidelines.  It is recommended that each laboratory establish the normal range for the area in which it is located.

6.2     MEDICAL ALERT VALUES (10.7)

Each laboratory should establish low and high values beyond which the patient would require immediate attention by a physician.  If a "medical alert value" is reached, always repeat the test to confirm the result and notify a physician if the result is confirmed.

6.3     LIMITATIONS OF PROCEDURE

This procedure measures total ALT.  Red blood cells contain high concentrations of ALT, therefore, hemolysis can elevate results.  A summary of the influence of drugs on clinical laboratory test may be found by consulting Young, D.S., et. al. (4).

 

7.0     QUALITY CONTROL

Standard practice for quality control should be applied to this system.  Commercially available lyophilized controls can be used to monitor the daily acceptable variations.  Normal and abnormal controls should be assayed at the beginning of each run of patient samples, whenever a new reagent or a different lot number is being used, and following any system maintenance.

A satisfactory level of performance is achieved when the analyte values obtained are within the "acceptable range" established by the laboratory.

 

8.0     CALIBRATION PROCEDURES

No reagent calibration is necessary as this procedure is standardized based on the millimolar absorptivity of NADH which is taken as 6.22 at 340 nm under the test conditions described.

 

9.0     PERFORMANCE CHARACTERISTICS

9.1     PRECISION

The estimates of precision shown below were obtained from assays of human control serum.

Within-Run

          In this study, 30 replicates of 2 control sera were run.

          Mean (U/L)                    SD (U/L)  CV (%)

               25.0                          ±1.4                         5.6

               84.5                          ±4.0                         4.6

Between-Run

          In this study, two control sera were assayed twice a day for 10 days.

          Mean (U/L)                    SD (U/L)  CV (%)

               44.6                          ±2.7                         6.0

               77.8                          ±4.4                         5.6

9.2     CORRELATION

A correlation study was done comparing this method (y) a similar UV alanine aminotransferase procedure (x).  47 samples with a range from 6 to 109 U/L were assayed.  Linear regression analysis gave the following result.

          Number of  Regression Equation                     Correlation

          Samples            y=Biotron, x=Comparative                   Coefficient

               47         y = 1.008 x - 1.038                      .977

9.3     LINEARITY

This procedure is linear to 350 U/L.  Procedures on automated instruments which use greater than one to fifteen dilution factor will have an extended linearity.  A sample with an alanine aminotransferase activity exceeding the linearity limit should be diluted with 0.9% saline and reassayed incorporating the dilution factor in the calculation of the result.

9.4     SENSITIVITY

An absorbance change of 0.001 DA/min corresponds to approximately 2.6 U/L ALT activity.

 

10.0   REFERENCES

10.1   Henley, K.S., Pollard, H.M., J.Lab. and Clin.Med.,46,785(1955)

10.2   Wroblewski, F., Ladue, J.S., Proc. Soc. Exp. Biol. Med. 91,569(1956)

10.3   Henry, R.J., Chiamori, N., Gobub, O.J. and Berkman, S.,  Am. J. Clin. Pathology, 34,381(1960)

10.4   Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC Press (1990).

10.5   Tietz, N.W. (editor) Clinical Guide to Laboratory Tests (1983), W.B. Saunders Company, Toronto. p. 16.

10.6   Clinical Diagnosis, 15th ed., J. Davidson and J.B. Henry, Eds, W.B. Saunders Company, Philadelphia, PA 1974.

10.7   G.J. Kost, "Critical Limits for Urgent Clinician Notification at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704