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CODE REV ENREV PRODNAME LANG Replace Spaces with Underline Page 1 D01297_06_Insert_ALT_GPT (IFCC)_MU EN TEST PROCEDURE Konelab™ / T Series Refer to the Reference Manual and Application Notes for an automated procedure on your Konelab analyzer. Any application which has not been validated by Thermo Fisher Scientific ALT/GPT (IFCC) Oy cannot be performance guaranteed and therefore must be evaluated by the user. Materials provided 981361 7 x 50 ml Reagents as described above. 981769 8 x 20 ml Materials required but not provided Pyridoxal-5’-phosphate (code 981839) for 3-reagent method. THIS PACKAGE INSERT IS APPLICABLE FOR USE Controls as indicated below. OUTSIDE THE US. ANY REFERENCE TO THE KONELAB SYSTEMS ALSO REFERS TO THE T SERIES. Calibration The response dA/min is converted to result units by a calculation factor. INTENDED USE Or use eCal (981830) as a calibrator according to the instructions given in the package For the in vitro quantitative determination of alanine aminotransferase (L-Alanine: insert of eCal (981830). 2-Oxoglutarate Aminotransferase (ALT), EC 2.6.1.2) activity in human serum or plasma on Konelab analyzers. All test results must be interpreted with regard to the clinical context. Quality Control Use quality control samples at least once a day and every time a new bottle of reagent is SUMMARY(1) used. Alanine aminotransferase (ALAT/ALT), formerly called glutamic pyruvic transaminase Available controls: (GPT), and Aspartate aminotransferase (ASAT/AST), formerly called glutamic oxalacetic Nortrol, code 981043 transaminase (GOT), belong to a group of enzymes, the aminotransferases or Abtrol, code 981044 transaminases, which catalyze the reversible transformation of α-keto acids into amino The Control intervals and limits must be adapted to the individual laboratory requirements. acids by transfer of amino groups. The results of the quality control samples should fall within the limits pre-set by the Both AST and ALT are normally present in human plasma, bile, cerebrospinal fluid and laboratory. saliva, but they are not found in urine unless a kidney lesion is present. In viral hepatitis and other forms of liver disease associated with hepatic necrosis, serum AST and ALT CALCULATION OF RESULTS level are elevated even before the clinical signs and symptoms of disease appear. Levels The results are calculated automatically by the Konelab analyzer as follows: of both enzymes may reach values as high as 100 times the upper reference limit, although 20- to 50- fold elevations are most frequently encountered Activity in U/l = dA/min x Factor Although serum levels of both AST and ALT become elevated whenever processes affect liver cell integrity, ALT is the more liver-specific enzyme. Increased AST levels can occur TV x 1000 in connection with damages of heart or skeletal muscle as well as of liver parenchyma. Theoretical factor = - ⎯⎯⎯⎯⎯- Serum elevation of ALT activity is rarely observed in other conditions than parenchymal 6.3 x SV x P liver disease. where: TV= Total reaction volume in ml PRINCIPLE OF THE PROCEDURE SV= Sample volume in ml ALT catalyzes the transfer of the amino group from alanine to oxoglutarate with the 6.3= Extinction coefficient of NADH at 340 nm formation of glutamate and pyruvate. Pyruvate is reduced to lactate by lactate P= Cuvette pathlength = 1 dehydrogenase (LDH). Conversion factor μkat/l = (U/l)/60 ALT If eCal (981830) is used as a calibrator, the results are calculated automatically by the L-Alanine + 2-Oxoglutarate <⎯⎯> L-Glutamate + Pyruvate Konelab analyzer using a calibration curve. LDH LIMITATIONS OF THE PROCEDURE Pyruvate + NADH + H+ <⎯⎯> D-Lactate + NAD+ Interference In this same reaction an equivalent amount NADH is oxidized to NAD. The resulting Criterion: Recovery within ± 10% of initial values. decrease in absorbance at 340 nm is followed and is directly proportional to the activity of ALT in serum. 1-reagent method: The IFCC reference method includes pyridoxal –5’-phosphate (P-5´-P) at a level of Conjugated bilirubin: No interference found up to 420 μmol/l (24.6 mg/dl). 0.1 mmol/l, which has been shown to be sufficient to activate apo ALT in 5 minutes (2). Unconjugated bilirubin: No interference found up to 600 μmol/l (35.1 mg/dl). Hemolysate: No interference found up to 10 g/l of hemoglobin. REAGENT INFORMATION Lipemia: No interference found up to 2 g/l of Intralipid® (trademark of Fresenius Kabi AB). Reagent A: Enzyme reagent There is a poor correlation between turbidity and triglycerides concentration. Code: 981361 7 x 40 ml 3-reagent method: Code: 981769 8 x 16 ml Conjugated bilirubin: No interference found up to 420 μmol/l (24.6 mg/dl). Reagent B: Substrate Unconjugated bilirubin: No interference found up to 600 μmol/l (35.1 mg/dl). Code: 981361 7 x 10 ml Hemolysate: No interference found up to 8 g/l of hemoglobin. Code: 981769 8 x 4 ml Lipemia: No interference found up to 2 g/l of Intralipid®. For other interfering substances, please refer to the reference 4. Concentrations EXPECTED VALUES (2) Reagent A: Enzyme reagent Tris buffer, pH 7.15 137.5 mmol/l Male: < 45 U/l (< 0.74 μkat/l) at 37 °C L-Alanine 700 mmol/l Female: < 34 U/l (< 0.56 μkat/l) at 37 °C LDH > 1650 U/l The quoted values should serve as a guide only. It is recommended that each laboratory verify this range or derives a reference interval for the population that it serves. NaN3 < 0.1 % MEASURING RANGE Reagent B: Substrate 2-oxoglutarate 82.5 mmol/l 1-reagent method: NADH 1.0 mmol/l 4 - 300 U/l (0.07 – 5.00 μkat/l) NaN3 < 0.1 % Extended measuring range after secondary dilution: Note: Liquid pyridoxal-5’-phosphate is not included in the kit. It can be ordered separately 4 – 1800 U/l (0.07 – 30.00 μkat/l) by code number 981839. 3-reagent method: 5 - 250 U/l (0.08 – 4.17 μkat/l) Precautions Extended measuring range after secondary dilution: For in vitro diagnostic use only. Exercise the normal precautions required for handling all 5 – 1500 U/l (0.08 – 25.00 μkat/l) laboratory reagents. The reagents contain sodium azide as preservative. Do not swallow. Avoid contact with PERFORMANCE CHARACTERISTICS skin and mucous membranes. Detection limit Preparation 1-reagent method: Code: 981361, 981769 2 U/l (0.03 μkat/l) reserved. hts g 1-reagent method (without P-5´-P): 3-reagent method: Add the contents of Reagent B to the bottle of Reagent A. Cap vial and mix. The monoreagent must 2 U/l (0.03 μkat/l) be protected from light! The detection limit represents the lowest measurable concentration/activity that can be 3-reagent method (with P-5´-P): distinguished from zero. It is calculated as the concentration of zero sample + 3 SD (within Reagents are ready for use. run, n=24). Note: Check that there are no bubbles in the bottleneck or on the surface of the reagent when you insert the reagent vials or vessels in the Konelab analyzer. Imprecision (result unit U/l) Storage and Stability 1-reagent method: Mean Mean Mean Reagents in unopened vials are stable at 2…8 °C until the expiration date printed on the 33 U/l 90 U/l 108 U/l label when protected from light. SD CV% SD CV% SD CV% Refer to the Application Notes of your Konelab analyzer for the on board stability of reagents. Within run 0.6 1.8 0.6 0.6 0.9 0.8 Between run 0.5 1.5 0.9 1.0 1.0 0.9 SPECIMEN COLLECTION Total 1.0 3.0 1.8 2.0 2.7 2.4 Sample Type 3-reagent method: Clear serum or heparin plasma can be used. Mean Mean Mean Precautions 31 U/l 89 U/l 109 U/l SD CV% SD CV% SD CV% Human samples should be handled and disposed of as if they were potentially infectious. Within run 0.6 1.8 0.6 0.7 1.0 0.9 Between run 0.2 0.7 0.8 0.9 0.8 0.8 Storage (3) Total 0.8 2.6 1.5 1.7 2.2 2.0 The sample can be stored for 3 days at 20…25 °C, for 7 days at 4…8 °C or for 2 days at –20 °C. A precision study was performed according to guidelines in the NCCLS Document EP5-A using Konelab 60 during 20 days, with the number of measurements being n = 80. Template: D01162_4 © 2008 Thermo Fisher Scientific Inc. All ri All Inc. Scientific Fisher Thermo © 2008 D01162_4 Template: Page 2 D01297_06_Insert_ALT_GPT (IFCC)_MU Method comparison REAGENZDATEN REAGENZ A: Enzymreagenz 1-reagent method: Bestellnr.: 981361 7 x 40 ml A comparison study was performed using the NCCLS Document EP9-A as a guideline and Bestellnr.: 981769 8 x 16 ml Konelab two-reagent method as a reference. REAGENZ B: Substrat Linear regression (result unit U/l): Bestellnr.: 981361 7 x 10 ml y = 0.75 x + 2.8 Bestellnr.: 981769 8 x 4 ml r = 0.988 n = 60 The sample concentrations were between 7 and 190 U/l. Konzentrationen 3-reagent method: REAGENZ A: Enzymreagenz A comparison study was performed using the NCCLS Document EP9-A as a guideline and Tris-Puffer, pH 7.15 137.5 mmol/l Konelab two-reagent method as a reference. L-Alanin 700 mmol/l Linear regression (result unit U/l): LDH > 1650 U/l y = 1.00 x + 0.2 NaN3 < 0.1 % r = 1.00 REAGENZ B: Substrat n = 65 2-Oxoglutarat 82.5 mmol/l The sample concentrations were between 10 and 155 U/l.
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