COD 11592 COD 11593 COD 11598 ALKALINE PHOSPHATASE

50 mL 200 mL 500 mL (ALP) - AMP

STORE AT 2-8ºC
Reagents for measurement of ALP concentration ALKALINE PHOSPHATASE (ALP) - AMP
Only for in vitro use in the clinical laboratory 2-AMINO-2-METHYL-1-PROPANOL BUFFER (IFCC)

PRINCIPLE OF THE METHOD Values at 25ºC are obtained from those at 30ºC by using a conversion factor. Concentrations in
growing children are higher and highly variable.These ranges are given for orientation only; each
Alkaline phosphatase (ALP) catalyzes in alkaline medium the transfer of the phosphate group
laboratory should establish its own reference ranges.
from 4-nitrophenylphosphate to 2-amino-2-methyl-1-propanol (AMP), liberating 4-nitrophenol.
The catalytic concentration is determined from the rate of 4-nitrophenol formation, measured at QUALITY CONTROL
405 nm1.
It is recommended to use the Biochemistry Control Serum level I (cod. 18005, 18009 and
ALP
4 – Nitrophenylphosphate + AMP AMP – phosphate + 4 - Nitrophenol 18042) and II (cod. 18007, 18010 and 18043) to verify the performance of the measurement
procedure.
Each laboratory should establish its own internal Quality Control scheme and procedures for
CONTENTS
COD 11592 COD 11593 COD 11598
corrective action if controls do not recover within the acceptable tolerances.

A. Reagent 1 x 40 mL 1 x 160 mL 4 x 100 mL METROLOGICAL CHARACTERISTICS

B. Reagent 1 x 10 mL 1 x 40 mL 2 x 50 mL
− Detection limit: 1.0 U/L = 0.017 µkat/L
COMPOSITION − Linearity limit: 1200 U/L = 20 µkat/L. For higher values dilute sample 1/2 with distilled water
and repeat measurement.
A. Reagent: 2-Amino-2-methyl-1-propanol 0.4 mol/L, zinc sulfate 1.2 mmol/L,
N-hydroxyethylethylenediaminetriacetic acid 2.5 mmol/L, magnesium acetate 2.5 mmol/L, pH − Repeatibility (within run):
10.4. Mean Concentration CV n
B. Reagent: 4-Nitrophenylphosphate 60 mmol/L. 61 U/L = 1.02 µkat/L 1.0 % 20
244 U/L = 4.07 µkat/L 0.7 % 20
STORAGE
Store at 2-8ºC. − Reproducibility (run to run):
Reagents are stable until the expiry date shown on the label when stored tightly closed and if Mean Concentration CV n
contaminations are prevented during their use. 61 U/L = 1.02 µkat/L 3.4 % 25
Indications of deterioration: 244 U/L = 4.07 µkat/L 1.1 % 25
− Reagents: Presence of particulate material, turbidity, absorbance of the blank over 1.200 at
405 nm (1 cm cuvette). − Trueness: Results obtained with this reagent did not show systematic differences when
compared with reference reagents. Details of the comparison experiments are available on
REAGENT PREPARATION request.
Working Reagent: − Interferences: Lipemia (triglycerides < 10 g/L) and bilirubin (< 20 mg/dL) do not interfere.
Hemoglobin (> 2.5 g/L) interfere. Other drugs and substances may interfere3.
• Cod. 11592 and 11593: Transfer the contents of one Reagent B vial into a Reagent A bottle.
Mix gently. Other volumes can be prepared in the proportion: 4 mL Reagent A + 1 mL These metrological characteristics have been obtained using an analyzer. Results may vary if a
Reagent B. Stable for 2 months at 2-8ºC. different instrument or a manual procedure are used.
• Cod. 11598: Transfer 25 mL of one Reagent B vial into a Reagent A bottle. Mix gently. Other DIAGNOSTIC CHARACTERISTICS
volumes can be prepared in the proportion: 4 mL Reagent A + 1 mL Reagent B. Stable for 2
months at 2-8ºC. Alkaline phosphatase catalyzes the hydrolysis of organic phosphate monoesters at alkaline pH.
The enzyme is present in practically all tissues of the body, especially at or in the cell
ADDITIONAL EQUIPMENT membranes, and it occurs at particularly high concentrations in placenta, intestinal epithelium,
kidney tubules, osteoblasts and liver.
− Analyzer, spectrophotometer or photometer with cell holder thermostatable at 25, 30 or 37ºC
and able to read at 405 nm. The form present in the sera of normal adults originates mainly in the liver and bone.
− Cuvettes with 1 cm light path. Elevated serum ALP is found in patients with bone disease associated with increased
osteoblastic activity (Paget’s disease, primary and secondary hyperparathyroidism, bone
SAMPLES tumors, rickets, osteomalacia, bone fractures) and also in patients with hepatobiliary disease
(obstructive jaundice, hepatitis, hepatotoxicity caused by drugs, liver cancer). Physiological
Serum and plasma collected by standard procedures. changes, such as bone growth and pregnancy, may cause increases in ALP levels4,5.
Alkaline phosphatase in serum or plasma is stable for 7 days at 2-8ºC. Heparin may be used as
anticoagulant. Clinical diagnosis should not be made on the findings of a single test result, but should integrate
both clinical and laboratory data.
PROCEDURE
NOTES
1. Bring the Working Reagent and the instrument to reaction temperature.
1. These reagents may be used in several automatic analysers. Instructions for many of them
2. Pipette into a cuvette: (Note 1)
are available on request.
Working Reagent 1.0 mL
Sample 20 µL BIBLIOGRAPHY
3. Mix and insert the cuvette into the photometer. 1. IFCC primary reference procedures for the measurement of catalytic activity concentrations
of enzymes at 37 °C. Part 9. Reference procedure for the measurement of catalytic
4. Record initial absorbance and at 1 minute intervals thereafter for 3 minutes.
concentration of alkaline phosphatase. Clin Chem Lab Med 2011; 49:1439-1446.
5. Calculate the difference between consecutive absorbances, and the average absorbance
difference per minute (∆A/min). 2. Rosalki SB, Foo AY, Burlina A, at al. Multicenter evaluation of iso-ALP test kit for
measurement of bone alkaline phosphatase activity in serum and plasma. Clin Chem 1993;
CALCULATIONS 39:648-652.
The ALP catalytic concentration in the sample is calculated using the following general formula: 3. Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.

Vt x 106 4. Friedman and Young. Effects of disease on clinical laboratory tests, 4th ed. AACC Press,
∆A/min x = U/L 2001.
ε x I x Vs 5. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. Burtis CA, Ashwood
ER, Bruns DE. WB Saunders Co, 2005.
The molar absorbance (ε) of 4-nitrophenol at 405 nm is 18450, the lightpath (l) is 1 cm, the total
reaction volume (Vt) is 1.02, the sample volume (Vs) is 0.02, and 1 U/L are 0.0166 µkat/L. The
following formulas are deduced for the calculation of the catalytic concentration:

x 2764 = U/L
∆A/min
x 46.08 = µkat/L

REFERENCE VALUES
Reaction temperature men women

25ºC, up to 75 U/L = 1.25 µKat/L 68 U/L = 1.13 µKat/L

30ºC, up to2 87 U/L = 1.45 µKat/L 80 U/L = 1.33 µKat/L
37ºC, up to2 115 U/L = 1.92 µKat/L 105 U/L = 1.75 µKat/L

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Quality System certified according to
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