Calculation

Alkaline PHOSPHATASE liquid To calculate the enzymatic activity in the sample, multiply ∆A/min by the proper
factor from the following table.
Kinetic method - S.C.E. Mono-reagent procedure Bi-reagent procedure
Liquid reagents - Ready to use 5430 6774

R1: 4 x 50 ml + R2: 1 x 50 ml REF 7012 Reference values

25°C 30°C 37°C
Intended use Adults 60 ÷ 180 U/L 80 ÷ 210 U/L 100 ÷ 275 U/L
Kit for quantitative determination of Alkaline Phosphatase ALP (EC 3.1.3.1.) in
serum and plasma according to S.C.E. method. Children 110 ÷ 720 U/L 145 ÷ 950 U/L 180 ÷ 1200 U/L
Each laboratory should define its own reference values for this method.
Principle
Alkaline phosphatase hydrolizes p-nitrophenylphosphate in phosphate and Quality control
p-nitrophenol. The absorbance variation in time unit measured at 405 nm is A quality control program is recommended for all clinical laboratories.
proportional to the enzymatic activity in the sample. Control sera in normal and high ranges for each assay are recommended.
The obtained values should be included within the manufacturer’s accepted
Sample ranges for the method being used.
Serum, heparinized plasma. Do not use anticoagulants like EDTA, citrate and
oxalate as they inhibit the enzyme. Avoid hemolyzed samples. Performance characteristics
Sensitivity: the sensitivity of the method is 5 U/L.
Pack Contents
Linearity: up to 2500 U/L (at 37°C).
Reagents REF 7012 Quantity Risk For higher values, dilute samples 1:10 with saline solution and multiply the result
by 10.
REAGENT 1 7012R1 4 x 50 ml * Within-run precision:
REAGENT 2 7012R2 1 x 50 ml Level 1 Level 2
Average [U/L] 182,6 510,9
Reagents DS 4,0 10,2
REAGENT 1 DEA/HCl buffer (pH 10,0)1 mol/L, magnesium chloride 0,5 mmol/L CV % 2,2 2,0
REAGENT 2 Glycine buffer (pH 9,5) 30 mmol/L, p-nitrophenylphosphate Between-run precision:
50 mmol/L, sodium azide 15 mmol/L
Stability: reagents are ready to use. Store at 2-8°C and protect from light to keep Level 1 Level 2
the reagents stable up to the expiration date on the label. Do not freeze. Once Average [U/L] 194,2 490,7
opened reagents are stable for 2 months at 2-8°C, if contamination is avoided. DS 9,9 28,5
Keep bottles tightly closed when not in use. Do not use turbid reagents. CV % 5,1 5,9

Preparation of working reagent Interferences: up to 30 mg/dl bilirubin does not interfere.

(only for mono-reagent procedure) Normal detergents inhibit the enzyme; it is recommended to use disposal
Mix 4 volumes of Reagent 1 with 1 volume of Reagent 2. products.
Stability: 5 days at 20-25°C or 4 weeks at 2-8°C, stored in a closed bottle Correlation against a reference method: the correlation of the method (Y)
protected from light.
against a reference method (X) gives this equation:
Manual Assay Procedure Y = 1,0583X + 2,8975 r = 0,9973

Analysis: increasing kinetic Warnings and precautions

Wavelength: 405 nm
Cuvette: 1 cm optical path - (*) dangerous reagents are marked by an asterisk. Refer to safety data
Temperature: 25, 30, 37°C sheet
Rate Time: 3 minutes - wear proper protective clothes - do not pipette by mouth
Measurement: against air or distilled water - disposal residual reagents and waste according to local laws
Sample/Reagents (bi-reagent): 1/100/25 - read the kit instructions before performing the test
Sample/Reagent (mono-reagent): 1/100 - use only the reagents contained in the kit and recommended reagents
- do not use reagents of different lots
- do not use reagents that have expired
Bi-reagent procedure - do not use reagents from other manufacturers
Let reagents necessary to perform the test reach the chosen temperature for the - chemistry analyzer parameters are available.
analysis.
Pipette in cuvette: References
1 Scandinavian Society for Clinical Chemistry, Committee on Enzymes.
Sample 10 µl Recommended methods for the determination of four enzymes in blood. Scan.
J. Clin. Lab. Invest. 33, 291, (1974).
Reagent 1 1,0 ml 2 German Society for Clinical Chemistry, Standard method for determination of
Alkaline Phpsphatase (AP) activity. J. Clin. Chem. Clin. Biochem. 10, 290
Mix and incubate at the chosen temperature for 1 minute at 37°C. Add:
(1972).
Reagent 2 250 µl 3 NCCLS Document, “Procedures for the collection of arterial blood specimens”,
Approved Standard, 3rd Ed. (1999).
Mix and pour in the test cuvette. Incubate at the test temperature for 1 minute. 4 EU-Dir 1999/11 Commission Directive of 8 March 1999 adapting to technical
Read initial absorbance and repeat reading at constant intervals of 1 minute, for progress the principles of good laboratory practice as specified in Council
3 minutes. Calculate the average value of the absorbance variations per minute Directive 87/18/EEC
(∆A/min).

Reaction volumes can be proportionally varied without any change in calculation.

Mono-reagent procedure
Let the working reagent necessary to perform the test reach the chosen
temperature for the analysis.
Pipette in cuvette:
Issue 01 - Sep 2006 RR
Sample 10 µl

Working reagent 1,0 ml

Mix and pour in the test cuvette. Incubate at the test temperature for 1 minute.
Read initial absorbance and repeat reading at constant intervals of 1 minute, for
3 minutes. Calculate the average value of the absorbance variations per minute Manufactured by: FAR srl
(∆A/min). Via Fermi, 12 - 37026 Pescantina - VERONA - ITALY
tel. +39 045 6700870/6700871 – fax +39 045 7157763
Reaction volumes can be proportionally varied without any change in calculation. website: http://www.fardiag.com e-mail: fardiag@fardiag.com