BIOLABO REAGENTS ALKALINE PHOSPHATASE
www.biolabo.fr
Colorimetric method
MANUFACTURER:
BIOLABO SA, Reagent for quantitative determination of alkaline phosphatase activity
02160, Maizy, France
[EC 3.1.3.1] in human serum and plasma
REF 80014 R1 2 x 56 mL R2 1 x 2,5 mL
R3 1 x 27 mL R4 1 x 27 mL
TECHNICAL SUPPORT AND ORDERS
Tel : (33) 03 23 25 15 50
IVD IN VITRO DIAGNOSTIC USE
Fax : (33) 03 23 256 256
CLINICAL SIGNIFICANCE (1) SAFETY CAUTIONS
Alkaline phosphatase (ALP) is found in many tissues, including bone,
liver, intestine, kidney, and placenta. Serum ALP measurements are of BIOLABO reagents are designated for professional, in vitro diagnostic
particular interest in the investigation of two groups of conditions : use.
hepatobiliary diseases (hepatitis, cirrhosis or malignacy) and bone • Use adequate protections (overall, gloves, glasses).
diseases associated with increased osteoblastic activity (child’s rickets • Do not pipette by mouth.
with D vitamin deficiency, Paget’s disease, hyperparathyroidism in the • In case of contact with skin or eyes, thoroughly wash affected areas
skeleton, metastasic carcinoma). with plenty of water and seek medical advice.
ALP determined by usual biochemical methods reflects total serum • Reagents contain sodium azide (concentration < 0.1%) which may
levels and does not distinguish the source of the isoenzyme. Clinicians react with copper and lead plumbing. Flush with plenty water when
must thererefore rely on other parameters of liver or other organ disposing.
function or a more specific determination of ALP to assess its source. • Material Safety Data Sheet is available upon request.
• Waste disposal : Respect legislation in force in the country.
All specimens should be handled as potentially infectious, in
PRINCIPLE (4) (5)
accordance with good laboratory practices using appropriate
Colorimetric determination of the ALP activity which reaction scheme is precautions. Respect legislation in force in the country.
as follows :
Alkaline phosphatase REAGENTS PREPARATION
Phenylphosphate Phenol + Phosphate Reagents are ready to use.
Free phenol liberated by hydrolysis of the substrate reacts then with
4-amino-antipyrine in the presence of alkaline potassium ferricyanide STABILITY AND STORAGE
to form a red-coloured complexe wich absorbance measured at 510 Store at 2-8°C and away from light.
nm is directly proportional to the ALP activity in the specimen. • Unopened :
Sodium arsenate incorporated in the reagent abolishes further enzyme Reagents are stable upon expiry date stated on the label.
activity and prevents the dilution of the colour inherent in earlier • Once reconstituted :
methods. Reagents are stable at least for 3 months when free from
contamination.
REAGENTS
SPECIMEN COLLECTION AND HANDLING (2)
Vial R1 SUBSTRATE-BUFFER Unhemolysed serum or heparinised plasma, immediately refrigerated.
ALP activity is stable in the specimen for :
Disodium Phenylphosphate 5 mmol/L
• 2-3 days at 2-8°C.
Carbonate-bicarbonate buffer pH 10 50 mmol/L • 1 month at –25°C.
Stabilizer
INTERFERENCES (3)
Vial R2 STANDARD
Avoid hemolysed serum.
Phenol corresponding to 20 U King and Kind For a more comprehensive review of factors affecting this assay refer to
the publication of Young D.S.
Vial R3 BLOCKING REAGENT (Toxic)
MATERIALS REQUIRED BUT NOT PROVIDED
4-Amino-antipyrine 60 mmol/L
1. Basic medical analysis laboratory equipment.
Sodium arsenate 240 mmol/L
2. Normal and pathological control sera.
R 45 : May cause cancer.
R 23/25 : Toxic by inhalation and ingestion.
S 28 : In case of contact with skin, wash promptly with copious
amount of water.
S 45 : If you feel unwell, seek medical advice (eventually, show him the
label)
Vial R4 DYE REAGENT
Potassium ferricyanide 150 mmol/L
Made in France Dernière version : www.biolabo.fr Version : 02/12/2011
MANUAL PROCEDURE
CALIBRATION
Let stand reagents and specimens at room temperature.
• Kit Standard (vial R2)
Prepare tubes as follows : Reagent Specimen Standard Assay
QUALITY CONTROL blank blank
• Assayed control sera referring to the same method. Reagent R1 2 mL 2 mL 2 mL 2 mL
• External quality control program.
It is recommended to control in the following cases : Incubate 5 minutes at 37°C.
• At least once a run.
• At least once within 24 hours. Specimen 50 JL
• When changing vial of reagent.
Reagent R2 (Standard) 50 JL
• After maintenance operations on the instrument.
If control is out of range, apply following actions : Let stand exactly 15 minutes at 37°C.
1. Check the temperature and repeat the test with the same control.
2. If control is still out of range, prepare a fresh control serum and Reagent R3 0,5 mL 0,5 mL 0,5 mL 0,5 mL
repeat the test.
3. If control is still out of range, use a new vial of Standard and repeat Mix well.
the test.
4. If control is still out of range, calibrate with a new vial of reagent. Reagent R4 0,5 mL 0,5 mL 0,5 mL 0,5 mL
5. If control is still out of range, please contact BIOLABO technical
support or your local Agent. Specimen 50 JL
Demineralised water 50 JL
EXPECTED VALUES (2)
Mix. Incubate 10 minutes at room temperature and away from light. Read
absorbances of the blank specimen, standard and assay at 510 nm against
Kind and King Units /dL IU/L (37°C) reagent blank.
Coloration is stable for 45 minutes away from light.
Birth 5-15 [36-107]
Premature (1.5 to 2 x adult value)
1 month 10-30 [71-213]
3 years 10-20 [71-142] CALCULATION
Calculate the result as follows :
10 years 15-30 [107-213]
Adults 4.5-13 [32-92]
Children : Values may be increased (up to threefold during puberty) ALP activity
Abs Assay - Abs Specimen blank
Each laboratory should establish its own normal ranges for the (Kind and King units/ 100 mL) = x 20
population that it serves. Abs Standard
LINEARITY
The assay is linear up to 40 Kind and King U (285 IU/L). Notes :
Above, reduce the volume of assay to : 1) One Kind and King unit is the quantity of
20 JL and multiply the result (§ CALCUL) by 2.5. enzyme which, on reaction’s conditions, liberates 1 mg of phenol in
10 JL and multiply the result (§ CALCUL) by 5. 15 minutes at 37°C.
Linearity limit depends on specimen/reagent ratio. 2) Result (IU/L) = 7,09 x Result (Kind and King
Unit/100 mL)
REFERENCES
(1) TIETZ N.W. Text book of clinical chemistry, 3rd Ed. C.A. Burtis, E.R.
Ashwood, W.B. Saunders (1999) p. 676-684 et p.1429-1431.
th
(2) Clinical Guide to Laboratory Test, 4 Ed., N.W. TIETZ (2006) p. 78-83
(3) YOUNG D.S., Effect of Drugs on Clinical laboratory Tests, 4th Ed. (1995)
P.3-26 à 3-35
(4) Kind P. R. N., King E. J, Estimation of plasma Phosphatase by
determination of hydrolysed phenol with amino-anti-antipyrine, J. Clin.
Path. (1954), 7, p.322-326
(5) Belfield A, Goldberg D. M., Revised assay for serum phenylphosphatase
activity using 4-amino-antipyrine, Enzyme (1971), 12, p.561-573
IVD REF LOT
Manufacturer Use by In vitro diagnostic Temperature limitation Catalogue number See insert Batch number Store away from light sufficient for dilute with
Made in France Dernière version : www.biolabo.fr Version : 02/12/2011
