R1 Diethanolamine (DEA) pH 10.

4 1 mmol/L Sample (µL) 20

Buffer
Magnesium chloride 0.5mmol/L 4. Mix, incubate for 1 minute.
Alkaline phosphatase 5. Read initial absorbance (A) of the sample, start
R2 p-Nitrophenylphosphate (pNPP) the stopwatch and read absorbances at 1 min
p-Nitrophenylphosphate. 10 mmol/L
Substrate intervals thereafter for 3 min.
kinetic. Liquid. DGKC 6. Calculate the difference between absorbances and
For In-Vitro diagnostic and professional use only
the average absorbance differences per minute
PREPARATION
(ΔA/min).
Store at 2-8C Working reagent (WR)
Mix: 4 vol. (R1) Buffer + 1 vol. (R2) Substrate
CALCULATIONS
INTENDED USE Stability:
ΔA/min x 3300 = U/L de ALP
For the quantitative determination of alkaline 1 month at 2-8°C or 10 days at room temperature.
Units: One international unit (IU) is the amount of
phosphatase in human serum heparinzed plasma.
enzyme that transforms 1µmol of substrate per
STORAGE AND STABILITY
minute, in standard conditions. The concentration is
PRINCIPLE OF THE METHOD  All the components of the kit are stable
expressed in units per litre of sample (U/L).
Alkaline phosphatase (ALP) catalyses the hydrolysis until the expiration date on the label when
stored tightly closed at 2-8°C, protected from Assay Conversion factor to
of p-nitrophenyl phosphate at pH 10.4, liberating
p-nitrophenol and phosphate, according to the light and contaminations prevented during temperature 25°C 30°C 37°C
following reaction: their use. 25°C 1.00 1.22 1.64
 Do not use reagents over the expiration
30°C 0.82 1.00 1.33
p-Nitrophenylphosphate + H2O ALP p-Nitrophenol date.
+ Phosphate  Signs of reagent deterioration 37°C 0.61 0.75 1.00
o Presence of particles and
The rate of p-nitrophenol formation, measured turbidity. Temperature conversion factors
photometrically, is proportional to the catalytic o Blank absorbance (A) at 405 nm > 1.30. To correct results to other temperatures multiply by
concentration of alkaline phosphatase present in the ADDITIONAL EQUIPMENT
sample  Spectrophotometer or colorimeter QUALITY CONTROL
measuring at 405 nm. Control sera are recommended to monitor the
CLINICAL SIGNIFICANCE  Thermostatic bath at 25°C, 30°C ,37°C (± 0.1°C) performance of assay procedures:
Alkaline phosphatase is an enzyme present in almost  Matched cuvettes 1.0 cm light path. If control values are found outside the defined range,
all weaves of the organism, being particularly high in  General laboratory equipment. check the instrument, reagents and technique for
bone, liver, placenta, intestine and kidney. Both SAMPLES problems.
increases and decreases of plasma ALP are of Serum or heparinzed plasma. Use unhemolyzed serum, Each laboratory should establish its own Quality
importance clinically. separated from the clot as soon as possible. Stability : 3 Control scheme and corrective actions if controls do
Causes of increased plasma ALP: Paget’s disease of bone, days at 2-8°C. not meet the acceptable tolerances.
obstructive liver disease, hepatitis, hepatotoxicity caused
by drugs or osteomalacia. PROCEDURE REFERENCE 25°C 30°C 37°C
1. Assay conditions: VALUES
Causes of decreased plasma ALP: Cretinism and vitamin C
deficiency. Wavelength 405nm
Children < 400 < 480 < 645 U/L
Clinical diagnosis should not be made on a single test Cuvette 1 cm light path
(1-14 years) U/L U/L
result; it should integrate clinical and other laboratory Constant temperature 25 ,30 ,37°C
2. Adjust the instrument to zero with distilled 60 - 170 73 - 98 - 279
data.
water or air. Adults U/L 207 U/L U/L
REAGENTS 3. Pipette into a cuvette:
WR (ml) 1.2 Factors affecting ALP activities in a normal
population include exercise, periods of repaid
growth in children and pregnancy. Mosby Co. St Louis. Toronto. Princeton
These values are for orientation purpose; each 1984; 1094-1098.
laboratory should establish its own reference 2. Rosalki S et al. Clin Chem 1993; 39/4:
range. 648-652.
3. Young DS. Effects of drugs on Clinical
PERFORMANCE CHARACTERISTICS Lab. Tests, 4th ed AACC Press, 1995.
Measuring range: 4. Young DS. Effects of disease on Clinical
From detection limit of 4 U/L to linearity limit of Lab. Tests, 4th ed AACC 2001.
825 U/L. 5. Burtis A et al. Tietz Textbook of Clinical
If the results obtained were greater than Chemistry, 3rd ed AACC 1999.
linearity limit, dilute the sample 1/10 with 6. Tietz N W et al. Clinical Guide to Laboratory
NaCl 9 g/L and multiply the result by 10. Tests, 3rd ed AACC 1995.

Precision:
Intra-assay Inter-assay Atlas Medical
(n=20) (n=20) William James House,
Mean 170 408 165 446 Cowley Road, Cambridge, CB4 4WX, UK
(U/L)
SD 3.9 7.4 2.26 5.17 Tel: +44 (0) 1223 858 910
CV (%) 4
2.3 5
1.8 1.37 1.16 Fax: +44 (0) 1223 858 524
0 2
Sensitivity: 1 U/L = 0, 0003 ΔA/min. PPI397A01
Accuracy: Rev B (12.10.2015)
Results obtained using Atlas reagents (y) did not Catalogue Number Store at
show systematic differences when compared with For In-Vitro Diagnostic
Caution
other commercial reagents (x). use
The results obtained using 50 samples were the Number of tests in the Read product insert
following: pack before use
Correlation coefficient (r):0.99. Lot (batch) number Manufacturer
Regression equation: y= 0.9995x - 1.1116
Fragile, handle with
The results of the performance characteristics Expiry date
care
depend on the analyzer used.
Manufacturer fax Do not use if
number package is damaged
INTERFERENCES
Manufacturer
Fluoride, oxalate, citrate and EDTA inhibit
telephone number
alkaline phosphate activity and should therefore
not be used as anticoagulants. Haemolyses
interferes due to the high concentration of
alkaline Phosphatase in red cells.
A list of drugs and other interfering substances with
acid Phosphatase determination has been reported
by Young et.

REFERENCES
1. 1.Wenger C. et al. Alkaline phosphatase.
Kaplan A et al. Clin Chem The C.V.