400 700 800

Alanine AminotransferaseLiquid Reagent

Order information

COBAS INTEGRA® 500 Tests Cat. No. 20764957 Indicates analyzer(s) on which cassette can be usedAlanine Aminotransferase System-ID 07 6495 7

Calibrator f.a.s. 10 × 3 mL Cat. No. 10759350System-ID 07 3718 6

Precinorm® U 20 × 5 mL Cat. No. 10171743System-ID 07 7997 0

Precipath® U 20 × 5 mL Cat. No. 10171778System-ID 07 7998 7

Precinorm® U plus 10 × 3 mL Cat. No. 12149435

ARGETNI/004

sulp004

/ARGETNIARGETNI

007

ARGETNI008

System-ID 07 7999 7Precipath® U plus 10 × 3 mL Cat. No. 12149443

System-ID 07 8000 6

Intended useThe cassette COBAS INTEGRA Alanine Aminotransferase (ALTL)contains an in vitro diagnostic reagent system intended for use onCOBAS INTEGRA systems for the quantitative determination ofthe catalytic activity of ALT (EC 2.6.1.2; L-alanine: 2-oxoglutarateaminotransferase) in serum and plasma. This method sheetdescribes the application for ALT without pyridoxal phosphateactivation (test ALTL, 0-495). The application for ALTL activatedwith pyridoxal phosphate is described in the method sheet AlanineAminotransferase Pyridoxal Phosphate Activated (Liquid Reagent).

Summary1,2

The enzyme alanine aminotransferase (ALT) has been widelyreported as present in a variety of tissues. The major source ofALT is the liver, which has led to the measurement of ALT activityfor the diagnosis of hepatic diseases. Elevated serum ALT is foundin hepatitis, cirrhosis, obstructive jaundice, carcinoma of theliver, and chronic alcohol abuse. ALT is only slightly elevated inpatients who have an uncomplicated myocardial infarction.Although both serum aspartate aminotransferase (AST)and ALT become elevated whenever disease processesaffect liver cell integrity, ALT is the more liver-specificenzyme. Moreover, elevations of ALT activity persist longerthan elevations of AST activity.In patients with vitamin B6 deficiency, serum aminotransferaseactivity may be decreased. The apparent reduction inaminotransferase activity may be related to decreased pyridoxalphosphate, the prosthetic group for aminotransferases, resultingin an increase in the ratio of apoenzyme to holoenzyme.

Test principleMethod according to the International Federation of ClinicalChemistry (IFCC), but without pyridoxal-5´-phosphate.3,4

ALT catalyzes the reaction between L-alanine and 2-oxoglutarate.The pyruvate formed is reduced by NADH in a reaction catalyzedby lactate dehydrogenase (LDH) to form L-lactate and NAD+.

ALTL-Alanine + 2-oxoglutarate pyruvate +

L-glutamateLDH

Pyruvate + NADH + H+ L-lactate + NAD+

The rate of the NADH oxidation is directly proportionalto the catalytic ALT activity. It is determined by measuringthe decrease in absorbance at 340 nm.

Reagents - working solutionsR1 Enzyme in vial A and B (liquid).R2 = SR NADH in vial C (liquid).

Active ingredients

Components ConcentrationsR1 SR Test

TRIS 224 100 mmol/LL-Alanine 1120 500 mmol/LLDH (microbial) ≥45 ≥20 µkat/L (≥1.2 kU/L)Albumin (bovine) 0.25 0.11 %NADH ≥1.7 ≥0.2 mmol/L2-Oxoglutarate 94 12 mmol/LSodium azide 0.09 0.09 0.05 %pH (37°C) 7.3 7.3

Reagent R1 contains nonreactive stabilizers, reagent SRa nonreactive buffer.Please see cassette label for reagent filling volumes.

Precautions and warningsPay attention to all precautions and warnings listed in Chapter 1,Introduction, particularly point 6 (sodium azide).

Reagent handlingReady for use.

Storage and stability

Shelf life at 2 to 8°C See expiration date on cassetteINTEGRA 400On-board in use at 10 to 15°C 12 weeks

INTEGRA 700/800On-board in use at 8°C 12 weeks

2003-11, V 2 EN 1 / 3 ALTL

Enzymes

400 700 800

Specimen collection and preparationOnly the specimens listed below were tested and found acceptable.Serum (free from hemolysis): Collect serum usingstandard sampling tubes.Plasma (free from hemolysis): Li-heparin or EDTA plasma.Do not use other anticoagulants.When processing samples in primary tubes, follow theinstructions of the tube manufacturer.Nonhemolyzed serum is the specimen of choice.

Stability:5 1 day at 20-25°C1 day at 4-8°C

Centrifuge samples containing precipitates beforeperforming the assay.

Materials providedSee “Reagents - working solutions” section for reagents.

AssayFor optimal performance of the assay follow the directions given inthis document for the analyzer concerned. Refer to the appropriateoperator’s manual for analyzer specific assay instructions.

Application for serum and plasma

INTEGRA 400 test definition

Measuring mode AbsorbanceAbs. calculation mode KinsearchReaction mode R1-S-SRReaction direction DecreaseWavelength A/B 340/378 nmCalc. first/last 39/64Test range 0-700 U/L (0-11.7 µkat/L)

with postdilution 0-7 000 U/L (0-117 µkat/L)Postdilution factor 10 recommendedUnit U/L

Pipetting parameters

Diluent (H2O)R1 59 µL 10 µLSample 11 µL 26 µLSR 17 µL 9 µLTotal volume 132 µL

INTEGRA 700/800 test definition

Measuring mode AbsorbanceAbs. calculation mode KinsearchReaction mode R1-S-SRReaction direction DecreaseWavelength A/B 340/378 nmCalc. first/last 54/97Test range 0-700 U/L (0-11.7 µkat/L)

with postdilution 0-7 000 U/L (0-117 µkat/L)Postdilution factor 10 recommendedUnit U/L

Pipetting parameters

Diluent (H2O)R1 59 µL 10 µLSample 11 µL 26 µLSR 17 µL 9 µLTotal volume 132 µL

Calibration

Calibrator Calibrator f.a.s.Use deionized water as zerocalibrator.

Calibration mode Linear regressionCalibration replicate Duplicate recommendedCalibration interval Each lot

Traceability: This method has been standardized manuallyagainst the original IFCC formulation.6

Quality control

Reference range Precinorm U or Precinorm U plusPathological range Precipath U or Precipath U plusControl interval 24 hours recommendedControl sequence User definedControl after calibration Recommended

CalculationCOBAS INTEGRA analyzers automatically calculate the analyteactivity of each sample. For more details please refer to Chapter 7,Data Analysis, User Manual (COBAS INTEGRA 700), or to Dataanalysis in the online Help (COBAS INTEGRA 400/800).

Conversion factor: U/L × 0.0167 = µkat/L

Limitations - interferenceCriterion: Recovery within ±10% of initial value.Serum, plasma

Hemolysis Erythrocyte contamination may elevateresults, since ALT activities in erythrocytesare three to five times higher than those innormal sera.

Icterus No significant interference.

Lipemia Lipemic specimens may cause highabsorbance flagging. Choose diluted sampletreatment for automatic rerun.

Anticoagulants Citrate and fluoride inhibit the enzymeactivity.

Drugs In vitro therapeutic drug interferenceon the assay was tested according to therecommendations of the Symposium ofDrug Effects in Clinical Chemistry Methods(1996).7 Calcium dobesilate and doxycyclineHCl cause artificially low ALT values atthe tested drug level. Refer to Chapter 1,Introduction for a list of tested drugs andtheir concentration.

For diagnostic purposes, the results should always beassessed in conjunction with the patient’s medical history,clinical examination and other findings.

Expected values8

37°C Females up to 31 U/L (up to 0.52 µkat/L)Males up to 41 U/L (up to 0.68 µkat/L)

Each laboratory should investigate the transferability ofthe expected values to its own patient population and ifnecessary determine its own reference ranges.

Specific performance data6

Representative performance data on the COBAS INTEGRAanalyzers are given below. Results obtained in individuallaboratories may differ.

ALTL 2 / 3 2003-11, V 2 EN

Enzymes

400 700 800

PrecisionReproducibility was determined using human samples andcontrols in an internal protocol (within run n = 20, betweenrun n = 20). The following results were obtained.

Level 1 Level 2Mean 35 U/L 132 U/L

(0.58 µkat/L) (2.20 µkat/L)CV within run 1.0% 0.47%CV between run 1.5% 1.9%

Analytical sensitivity (lower detection limit)1 U/L (0.02 µkat/L)The detection limit represents the lowest measurable analytelevel that can be distinguished from zero. It is calculated as thevalue lying three standard deviations above that of a zero sample(zero sample + 3 SD, within run precision, n = 30).

Method comparisonALT values for human serum and plasma samples obtained onCOBAS INTEGRA 700 with the cassette COBAS INTEGRAAlanine Aminotransferase (ALTL) were compared tothose determined with commercially available reagents forALT on COBAS INTEGRA and on an alternative clinicalchemistry system. Samples were measured in duplicate.Sample size (n) represents all replicates.Values ranged from 4 to 496 U/L (0.07 to 8.27 µkat/L).

COBAS INTEGRA Alternative systemSample size (n) 236 224Corr. coefficient (r) 1.000 1.000

(rs) 0.998 0.995Lin. regression y = 1.07x - 2.3 U/L y = 0.98x + 0.8 U/LPassing Bablok y = 1.06x - 1.8 U/L y = 0.98x + 0.9 U/L

References1. Sherwin JE. Liver function. In: Kaplan LA, Pesce AJ, eds.

Clinical Chemistry, theory, analysis, and correlation.St. Louis: Mosby 1984:420-438.

2. Moss DW, Henderson AR, Kachmar JF. Enzymes. In: TietzNW, ed. Fundamentals of Clinical Chemistry. 3rd ed.Philadelphia: WB Saunders 1987:346-421.

3. Bergmeyer HU, Hørder M, Rej R. Approved recommendation(1985) on IFCC methods for the measurement of catalyticconcentration of enzymes. Part 3. IFCC method for alanineaminotransferase. J Clin ChemClin Biochem 1986;24:481-495.

4. ECCLS. Determination of the catalytic activity concentrationin serum of L-alanine aminotransferase (EC 2.6.1.2,ALAT) Klin Chem Mitt 1989;20:204-211.

5. Tietz NW, ed. Clinical Guide to Laboratory Tests. 3rd ed.Philadelphia, PA: WB Saunders 1995:20-21.

6. Data on file at Roche Diagnostics.7. Breuer J, Report on the Symposium “Drug Effects

in Clinical Chemistry Methods”. Eur J Clin ChemClin Biochem 1996;34:385-386.

8. Fischbach F, Zawta B. Age-dependent reference limitsof several enzymes in plasma at different measuringtemperatures. Klin Lab 1992;38:555-561.

COBAS INTEGRA, Precinorm and Precipath are trademarks of amember of the Roche Group.Significant additions or changes are indicated by a change bar in the margin.©2003 Roche Diagnostics

Roche Diagnostics GmbH, D-68298 Mannheimfor USA: US Distributor:Roche Diagnostics Corporation, Indianapolis, INUS Customer Technical Support 1-800-428-2336

2003-11, V 2 EN 3 / 3 ALTL