400 700 800

Alanine Aminotransferase
Liquid Reagent

Order information COBAS INTEGRA Alanine Aminotransferase Calibrator f.a.s. Precinorm U Precipath U Precinorm U plus Precipath U plus 500 Tests 10 3 mL 20 5 mL 20 5 mL 10 3 mL 10 3 mL Cat. No. 20764957 System-ID 07 6495 7 Cat. No. 10759350 System-ID 07 3718 6 Cat. No. 10171743 System-ID 07 7997 0 Cat. No. 10171778 System-ID 07 7998 7 Cat. No. 12149435 System-ID 07 7999 7 Cat. No. 12149443 System-ID 07 8000 6 Indicates analyzer(s) on which cassette can be used

INTEGRA 400/ 400 plus

INTEGRA/ INTEGRA 700

INTEGRA 800

Intended use The cassette COBAS INTEGRA Alanine Aminotransferase (ALTL) contains an in vitro diagnostic reagent system intended for use on COBAS INTEGRA systems for the quantitative determination of the catalytic activity of ALT (EC 2.6.1.2; L-alanine: 2-oxoglutarate aminotransferase) in serum and plasma. This method sheet describes the application for ALT without pyridoxal phosphate activation (test ALTL, 0-495). The application for ALTL activated with pyridoxal phosphate is described in the method sheet Alanine Aminotransferase Pyridoxal Phosphate Activated (Liquid Reagent). Summary1,2 The enzyme alanine aminotransferase (ALT) has been widely reported as present in a variety of tissues. The major source of ALT is the liver, which has led to the measurement of ALT activity for the diagnosis of hepatic diseases. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase (AST) and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver-specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity. In patients with vitamin B6 deficiency, serum aminotransferase activity may be decreased. The apparent reduction in aminotransferase activity may be related to decreased pyridoxal phosphate, the prosthetic group for aminotransferases, resulting in an increase in the ratio of apoenzyme to holoenzyme. Test principle Method according to the International Federation of Clinical Chemistry (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 catalyzed by lactate dehydrogenase (LDH) to form L-lactate and NAD+.
ALT

The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by measuring the decrease in absorbance at 340 nm. Reagents - working solutions R1 Enzyme in vial A and B (liquid). R2 = SR NADH in vial C (liquid). Active ingredients Components TRIS L-Alanine LDH (microbial) Albumin (bovine) NADH 2-Oxoglutarate Sodium azide pH (37C) Concentrations R1 SR Test 224 100 1120 500 45 20 0.25 0.11 1.7 0.2 94 12 0.09 0.09 0.05 7.3 7.3

mmol/L mmol/L kat/L (1.2 kU/L) % mmol/L mmol/L %

Reagent R1 contains nonreactive stabilizers, reagent SR a nonreactive buffer. Please see cassette label for reagent filling volumes. Precautions and warnings Pay attention to all precautions and warnings listed in Chapter 1, Introduction, particularly point 6 (sodium azide). Reagent handling Ready for use. Storage and stability Shelf life at 2 to 8C INTEGRA 400 On-board in use at 10 to 15C INTEGRA 700/800 On-board in use at 8C See expiration date on cassette 12 weeks 12 weeks

L-Alanine + 2-oxoglutarate Pyruvate + NADH + H + 2003-11, V 2 EN

LDH

pyruvate + L-glutamate L-lactate + NAD+ 1/3

ALTL

Enzymes

400 700 800

Specimen collection and preparation Only the specimens listed below were tested and found acceptable. Serum (free from hemolysis): Collect serum using standard sampling tubes. Plasma (free from hemolysis): Li-heparin or EDTA plasma. Do not use other anticoagulants. When processing samples in primary tubes, follow the instructions of the tube manufacturer. Nonhemolyzed serum is the specimen of choice. Stability:5 1 day at 20-25C 1 day at 4-8C Calibration Calibrator Calibrator f.a.s. Use deionized water as zero calibrator. Linear regression Duplicate recommended Each lot

Calibration mode Calibration replicate Calibration interval

Traceability: This method has been standardized manually against the original IFCC formulation.6 Quality control Reference range Pathological range Control interval Control sequence Control after calibration Precinorm U or Precinorm U plus Precipath U or Precipath U plus 24 hours recommended User defined Recommended

Centrifuge samples containing precipitates before performing the assay. Materials provided See Reagents - working solutions section for reagents. Assay For optimal performance of the assay follow the directions given in this document for the analyzer concerned. Refer to the appropriate operators manual for analyzer specific assay instructions. Application for serum and plasma INTEGRA 400 test definition Measuring mode Abs. calculation mode Reaction mode Reaction direction Wavelength A/B Calc. first/last Test range with postdilution Postdilution factor Unit Pipetting parameters Diluent (H2O) 10 L 26 L 9 L Absorbance Kinsearch R1-S-SR Decrease 340/378 nm 39/64 0-700 U/L (0-11.7 kat/L) 0-7 000 U/L (0-117 kat/L) 10 recommended U/L

Calculation COBAS INTEGRA analyzers automatically calculate the analyte activity of each sample. For more details please refer to Chapter 7, Data Analysis, User Manual (COBAS INTEGRA 700), or to Data analysis in the online Help (COBAS INTEGRA 400/800). Conversion factor: U/L 0.0167 = kat/L

Limitations - interference Criterion: Recovery within 10% of initial value. Serum, plasma Hemolysis Erythrocyte contamination may elevate results, since ALT activities in erythrocytes are three to five times higher than those in normal sera. No significant interference. Lipemic specimens may cause high absorbance flagging. Choose diluted sample treatment for automatic rerun. Citrate and fluoride inhibit the enzyme activity. In vitro therapeutic drug interference on the assay was tested according to the recommendations of the Symposium of Drug Effects in Clinical Chemistry Methods (1996).7 Calcium dobesilate and doxycycline HCl cause artificially low ALT values at the tested drug level. Refer to Chapter 1, Introduction for a list of tested drugs and their concentration.

Icterus Lipemia

Anticoagulants 59 11 17 132 L L L L Drugs

R1 Sample SR Total volume

INTEGRA 700/800 test definition Measuring mode Abs. calculation mode Reaction mode Reaction direction Wavelength A/B Calc. first/last Test range with postdilution Postdilution factor Unit Pipetting parameters Diluent (H2O) 10 L 26 L 9 L Absorbance Kinsearch R1-S-SR Decrease 340/378 nm 54/97 0-700 U/L (0-11.7 kat/L) 0-7 000 U/L (0-117 kat/L) 10 recommended U/L

For diagnostic purposes, the results should always be assessed in conjunction with the patients medical history, clinical examination and other findings. Expected values8 37C Females Males up to 31 U/L up to 41 U/L (up to 0.52 kat/L) (up to 0.68 kat/L)

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges. Specific performance data6 Representative performance data on the COBAS INTEGRA analyzers are given below. Results obtained in individual laboratories may differ. 2/3 2003-11, V 2 EN

R1 Sample SR Total volume

59 11 17 132

L L L L

ALTL

Enzymes

400 700 800

Precision Reproducibility was determined using human samples and controls in an internal protocol (within run n = 20, between run n = 20). The following results were obtained. Mean CV within run CV between run Level 1 35 U/L (0.58 kat/L) 1.0% 1.5% Level 2 132 U/L (2.20 kat/L) 0.47% 1.9%

Analytical sensitivity (lower detection limit) 1 U/L (0.02 kat/L) The detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying three standard deviations above that of a zero sample (zero sample + 3 SD, within run precision, n = 30). Method comparison ALT values for human serum and plasma samples obtained on COBAS INTEGRA 700 with the cassette COBAS INTEGRA Alanine Aminotransferase (ALTL) were compared to those determined with commercially available reagents for ALT on COBAS INTEGRA and on an alternative clinical chemistry 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 kat/L). COBAS INTEGRA 236 Sample size (n) 1.000 Corr. coefficient (r) 0.998 (rs) Lin. regression y = 1.07x - 2.3 U/L y = 1.06x - 1.8 U/L Passing Bablok Alternative system 224 1.000 0.995 y = 0.98x + 0.8 U/L y = 0.98x + 0.9 U/L

References 1. 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: Tietz NW, ed. Fundamentals of Clinical Chemistry. 3rd ed. Philadelphia: WB Saunders 1987:346-421. 3. Bergmeyer HU, Hrder M, Rej R. Approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase. J Clin Chem Clin Biochem 1986;24:481-495. 4. ECCLS. Determination of the catalytic activity concentration in 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 Chem Clin Biochem 1996;34:385-386. 8. Fischbach F, Zawta B. Age-dependent reference limits of several enzymes in plasma at different measuring temperatures. Klin Lab 1992;38:555-561.
COBAS INTEGRA, Precinorm and Precipath are trademarks of a member 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 Mannheim for USA: US Distributor: Roche Diagnostics Corporation, Indianapolis, IN US Customer Technical Support 1-800-428-2336

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ALTL