INTERNAL POLICY AND PROCEDURE

IPP

DEPARTMENT
LABORATORY AND BLOOD BANK

TITLE/DESCRIPTION POLICY NUMBER

TEST PROCEDURE (ALTL) LAB-OPER-CHEM 6 Ver.1
)ALANINE AMINOTRANSFERASE(
EFFECTIVE DATE REVIEW DUE REPLACES NUMBER NO. OF PAGES
New 7
APPROVED BY APPLIES TO
HOSPITAL DIRECTOR ALL CHEMISTRY SECTION STAFF

:INTENDED USE .1
In vitro test for the quantitative determination of alanine aminotransferase (ALT) in human serum and plasma on
.COBAS INTEGRA 400 PLUS SYSTEMS
:CLINICAL SIGNIFICANCE .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

JCI CBAHI
Standards Page 1 of 7
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 .3
This assay follows the recommendations of the IFCC, but was optimized for performance and stability. 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
L-Alanine + 2-oxoglutarate( ALT) → pyruvate + L-glutamate
+Pyruvate + NADH + H+ (LDH) → L-lactate + NAD
The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by
.measuring the decrease in absorbance
:REAGENTS &WORKING SOLUTIONS .4
R1 TRIS buffer: 224 mmol/L, pH 7.6 (25°C); L-alanine: 1120 mmol/L; albumin (bovine): 0.25%; LDH
(microorganisms): ≥45 μkat/L; stabilizers; preservative
R2 2-Oxoglutarate: 94 mmol/L; NADH: ≥1.7 mmol/L; additives; preservative
:Storage and stability
Shelf life at 2-8°C: See expiration date. On-board in use and refrigerated on the analyzer: 12 weeks
:SPECIMEN COLLECTION AND PREPARATION .5
.Only the specimens listed below were tested and found acceptable
.Serum (free from hemolysis)
. .Plasma (free from hemolysis): Li-heparin and K2-EDTA plasma
Separate the serum or plasma from the clot or cells promptly. Centrifuge samples containing precipitates before
.performing the assay
:Stability
days at 15-25°C 7 days at 2-8°C >7 days at (-60)-(-80)°C7 3

JCI CBAHI
Standards Page 2 of 7
6. ASSAY:
For optimum performance of the assay, follow the directions given in this document for the analyzer concerned.
Refer to the appropriate operator manual for analyzer-specific assay instructions. The performance of applications
.not validated by Roche is not warranted and must be defined by the user
Application for serum and plasma
:Test definition
Assay type Rate A
Reaction time / Assay points 10 / 12-31
Wavelength (sub/main) 700/340 nm
Reaction direction Decrease
Units U/L
Reagent pipetting Diluent (H2O)
R1 59 μL 32 μL
R2 17 μL 20 μL
Sample volumes Sample Sample dilution
Sample Diluent (NaCl)
– – Normal 9 μL
Decreased 9 μL 15 μL 135 μL
– – Increased 18μL
:CALIBRATION .7
Calibrators S1: H2O
.S2: C.f.a.s
Calibration mode Linear
Calibration frequency 2-point calibration
after reagent lot change -
and as required following quality control procedures -

JCI CBAHI
Standards Page 3 of 7
Traceability: This method has been standardized against the original IFCC formulation, but without Pyridoxal,
using calibrated pipettes together with a manual photometer providing absolute values and the substrate-specific
.absorptivity
:QUALITY CONTROL .8
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained
should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values
fall outside the limits. Follow the applicable government regulations and local guidelines for quality control
:CALCULATION .9
.cobas systems automatically calculate the analyte concentration of each sample
Conversion factor: U/L x 0.0167 = μkat/L
:INTERFERENCES .10
.Criterion: Recovery within ±10% of initial value at an ALT activity of 30 U/L (0.5 μkat/L)
Icterus: No significant interference up to an I index of 60 (approximate conjugated and unconjugated bilirubin
.concentration: 1026 μmol/L (60 mg/dL))
Hemolysis: No significant interference up to an H index of 200 (approximate hemoglobin concentration: 124
μmol/L (200 mg/dL)). Contamination with erythrocytes will elevate results, because the analyte level in
erythrocytes is higher than in normal sera. The level of interference may be variable depending on the content of
.analyte in the lysed erythrocytes
Lipemia (Intralipid): No significant interference up to an L index of 150. There is poor correlation between the L
.index (corresponds to turbidity) and triglycerides concentration
.Lipemic samples may cause >Abs flagging. Choose diluted sample treatment for automatic rerun
.Drugs: No interference was found at therapeutic concentrations using common drug panels
Exception: Calcium dobesilate and Isoniazid cause artificially low ALT results. Cyanokit (Hydroxocobalamin)
may cause interference with results. In very rare cases gammopathy, in particular type IgM (Waldenström’s
.macroglobulinemia), may cause unreliable results
Special Wash Requirements: The use of special wash steps is necessary when certain test combinations are run

JCI CBAHI
Standards Page 4 of 7
.together
:EXPECTED VALUES .11
:Acc. to the optimized standard method (comparable to the IFCC method without pyridoxal phosphate activation)
.Males up to41 U/L
Females up to 33 U/L
.Calculated values: A factor of 1.85 is used for the conversion from 25°C to 37°C
Each laboratory should investigate the transferability of the expected values to its own patient population and if
.necessary determine its own reference ranges
:PRECISION .12
Reproducibility was determined using human samples and controls in an internal protocol (within-run n = 21,
:total n = 60). The following results were obtained
% Within-run Mean U/L SD U/L CV
Precinorm U 39.5 0.3 0.6
Precipath U 120 1.0 0.4
Human serum 1 113 0.5 0.4
Human serum 2 7.2 0.7 9.3
% Total Mean U/L SD U/L CV
Precinorm U 39.3 0.6 1.4
Precipath U 120 1.0 1.0
Human serum 3 24.0 0.6 2.6
Human serum 4 98.1 3.2 3.3
Measuring range: 5-700 U/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function
is a 1:10 dilution. Results from samples diluted by the rerun function are automatically multiplied by a factor of
.10
Lower detection limit: 5 U/L

JCI CBAHI
Standards Page 5 of 7
The lower 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 the lowest standard (standard 1 + 3 SD,
.within-run precision, n = 21)
13. REFERENCES :
Moss DW, Henderson AR, Kachmar JF. Enzymes. In: Tietz NW, ed. Fundamentals of Clinical Chemistry. 3rd .1
.ed. Philadelphia: WB Saunders 1987:346-421
Bergmeyer HU, Hørder M, Rej R. Approved recommendation (1985) on IFCC methods for the measurement of .2
catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase. J Clin Chem Clin Biochem
.1986;24:481-495
ECCLS. Determination of the catalytic activity concentration in serum of L-alanine aminotransferase (EC .3
.2.6.1.2, ALAT) Klin Chem Mitt 1989;20:204-211
Schumann G et al. IFCC Primary Reference Procedures for the Measurement of Catalytic Activity .4
Concentrations of Enzymes at 37°C – Part 4. Reference Procedure for the Measurement of Catalytic Activity
.Concentrations of Alanine Aminotransferase. Clin Chem Lab Med 2002;40(7):718-724
.Data on file at Roche Diagnostics .5
Report on the Symposium Drug effects in clinical chemistry methods, Breuer J, Eur J Clin Chem Clin Biochem .6
.1996;34:385-386
Sonntag O, Scholer A. Drug interferences in clinical chemistry: recommendation of drugs and their .7
.concentrations to be used in drug interference studies. Ann Clin Biochem 2001;38:376-385
Thefeld W, Hoffmeister H, Busch, E-W, Koller PU, Vollmar J. Referenzwerte für die Bestimmungen der .8
Transaminasen GOT und GPT sowie der alkalischen Phosphatase im Serum mit optimierten Standardmethoden.
.Dtsch Med Wschr 1974;99:343-351
Klein G, Lehmann P, Michel E, Regenauer H. Vergleich der IFCC-Methoden für ALAT, ASAT und GGT bei .9
.37°C mit den eingeführten Standardmethoden bei 25°C und 37°C. Lab Med 1994;18:403-404
Bablok W et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem .10
.1988;26:783-790

JCI CBAHI
Standards Page 6 of 7
14.APPROVAL PAGE:
NAME TITLE SIGNATURE DATE

PREPARED Dr/ Nagah Mohammed LAB. CONSULTANT

BY

/ .Mr LAB. DIRECTOR

REVIEWED
/Dr TQM DIRECTOR

BY

APPROVED /.Mr HOSPITAL DIRECTOR

BY

IPP HISTORY

Initial IPP: Version 1 Dated -----------------

Replaced by: Version --- Dated ----------------
Deleted: Date ----------------
Replaced by New IPP: IPP No. & Version ------------------------------ Dated --------

JCI CBAHI
Standards Page 7 of 7