Pointe Alkaline Phosphatase

(Liquid) Reagent Set

Intended Use Specimen Collection and Storage

For the quantitative determination of alkaline phosphatase in human serum. 1. Use non-hemolyzed serum (plasma should not be used since anticoagulant
For in vitro diagnostic use only. Rx Only agents inhibit alkaline phosphatase activity).8,9
2. Serum samples should be stored at 2-8°C and run within two days.10
Clinical Significance 3. Specimen collection should be carried out in accordance with NCCLS M29-
Serum alkaline phosphatase estimations are of interest in the diagnosis of T2.11 No method can offer complete assurance that human blood samples
two groups of conditions; hepatobiliary disease and bone disease associated will not transmit infection. Therefore, all blood samples should be considered
with increased osteoblastic activity.1 potentially infectious.

Test Summary Interferences

1. Young, et al8 provide a list of drugs and other substances that interfere with
Alkaline phosphatase in serum is determined by measuring the rate of
the determination of ALP activity.
hydrolysis of various phosphate esters under specified conditions. ρ-
2. The method is not influenced (< 10%) by hemoglobin values up to 500mg/dl,
Nitrophenyl Phosphate is one such phosphate ester and was introduced as a
bilirubin levels up to 20mg/dl and lipemia / Triglycerides (Intralipid used to
substrate by Fujita in 1939.2
simulate) to 1000mg/dl. The studies were performed on the Hitachi 717™
Bessey, Lowry, and Brock published an endpoint procedure in 19463 while
analyzer following a modification of the guidelines contained in NCCLS
Bowers and McComb reported a kinetic procedure in 1966.4 The kinetic
document EP7-P.12
procedure has undergone several modifications and been recommended for
routine analysis.5,6 This liquid reagent is based on the recommended
method of the AACC.7 Materials Provided
Alkaline Phosphatase R1 Reagent.
Principle Alkaline Phosphatase R2 Reagent.
Alk. Phos.
ρ-NPP + H2O ----------------------- ρ -Nitrophenol + H3PO4 Materials Required but not Provided
1. Accurate pipetting devices (1.0 ml and 25 ul)
ρ-Nitrophenyl phosphate is hydrolyzed to ρ-nitrophenol and inorganic 2. Test tubes/rack
phosphate. The rate at which the p-NPP is hydrolyzed, measured at 405 3. Timer (To measure one minute intervals)
nm, is directly proportional to the alkaline phosphatase activity. 4. Spectrophotometer able to read at 405 nm
5. Heating bath/block (37°C)
Reagent Composition 6. Controls to monitor the validity of the reaction
After combining R1 and R2 as directed the reagent contains: AMP Buffer
(pH 10.45), ρ-NPP ≤16mM, Magnesium ions ≥1.0mM, activators and Procedure (Automated-General)
preservatives. Wavelength: 405 nm
Assay Type: Kinetic
Reagent Preparation Sample/Reagent Ratio: 1:41
Reaction Direction: Increasing
Mix 5 Parts of R1 Reagent with 1 part R2 Reagent.
Temperature: 37°C
Lag Time: 60 seconds
Reagent Storage and Stability Read Time: 60 seconds
Store reagent set at 2-8°C. The reagents are stable until the expiration date Low Normal: 35 IU/L
if stored as directed. Protect from direct light and avoid microbial High Normal: 123 IU/L
contamination. NOTE: The R2 reagent is temperature sensitive and can be For technical assistance concerning this product, contact the manufacturer’s
affected by prolonged exposure to room temperature. Return reagent to 2- Technical Service Department.
8°C as soon as possible after use.
Procedure (Manual)
Precautions 1. Prepare working reagent according to instructions.
1. This reagent set is for in vitro diagnostic use only. 2. Pipette 1.0 ml of reagent into appropriate tubes and pre-warm at 37°C for
2. Do not ingest any material, toxicity not determined. five minutes.
3. Do not use if the initial absorbance of the working reagent is greater 3. Zero spectrophotometer with water at 405 nm.
than 1.0 at 405 nm or if the reagent fails to meet the stated parameters 4. Transfer 0.025 ml (25 ul) of sample to reagent, mix and incubate at 37°C for
of performance. one minute.
4. Reagent should not be used if it fails to recover stated values in control 5. After one minute, read and record absorbance. Return tube to 37°C. Repeat
sera or shows evidence of microbial contamination. readings every minute for the next two minutes.*
5. All specimens and controls should be handled in accordance with good 6. Calculate the average absorbance difference per minute. (∆Abs/Min.)
laboratory practices using appropriate precautions as described in the
7. The ∆Abs/Min multiplied by the factor 2187 (See Calculation) will yield
CDC/NIH Manual, “Biosafety in Microbiological and Biomedical
results in IU/L.
Laboratories,” 2nd Ed., 1988, HHS Publication No. (CDC) 88-8395.
8. Samples with values above 1000 IU/L should be diluted with an equal
volume of saline, re-assayed and the results multiplied by two.

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 Pointe Alkaline Phosphatase
(Liquid) Reagent Set

*NOTE: If the spectrophotometer utilized is equipped with a temperature 4. Sensitivity: The sensitivity for this product was investigated by reading the
controlled cuvette, the reaction mixture may be left in the cuvette while the change in absorbance at 405nm for a saline sample and serums with known
absorbance readings are taken. concentrations. Ten replicates were performed. The results of this
investigation indicated that, on the analyzer used, the liquid alkaline
Limitations phosphatase reagent showed little or no reagent drift on a zero sample.
1. This methodology measures total alkaline phosphatase irrespective of Also, that an absorbance change of 0.0003 was approximately equivalent to
tissue or organ of origin. Further tests may be necessary to assist in 1 IU/L of alkaline phosphatase activity.
differential diagnosis.
2. Samples with values exceeding 1000 IU/L should be diluted with an
References
1. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders co., p 603
equal volume of saline and re-assayed multiplying the results by two.
(1982).
2. Fujita, H., J. Biochem, (Japan) 30:69 (1969).
Calibration 3. Bessey, O.A., Lowry, O.H., Brock, M.J., J. Biol. Chem. 164:321 (1964).
The procedure is standardized by means of the millimolar absorptivity of p- 4. Bowers, G.N., Jr., McComb, R.B., Clin. Chem. 12:70 (1966).
nitrophenol (18.75 at 405nm) under the specified conditions. Results are 5. The Committee on Enzymes of the Scandinavian Society for Clinical
based on the change in absorbance per unit of time; all parameters must be Chemistry and Clinical Physiology, Scand. J. Clin. Lab. Invest 32:291 (1974).
known and controlled. 6. Wilkinson, J.H., et al, Clin. Chem. 15:487 (1969).
7. Tietz, N.W., et al, Clin. Chem. 29:751 (1983).
Calculations 8. Young, D.S., et al, Clin. Chem. 21:1D (1975).
One international Unit (IU/L) is defined as the amount of enzyme that 9. Demetriou, J.A., Drewes, P.A., Gin, J.B., Clinical Chemistry: Principles and
catalyzes the transformation of one micromole of substrate per minute under Technics, 2nd Ed., Hagerstown (MD), Harper & Row, p. 927 (1974).
specified conditions. 10. Rej., R., Clin. Chem. 23:1903 (1977).
11. NCCLS document “Protection of Laboratory Workers from Infectious Disease
(IU/L) = ∆Abs./Min. x 1000 x 1.025 = ∆Abs./min. x 2187 Transmitted by Blood, Body Fluids, and Tissue”, 2nd Ed. (1991).
18.75 x 1 x .025 12. NCCLS document “Interference testing in Clinical Chemistry”, 2nd Ed. (1992).
Where ∆Abs./Min. = Average absorbance change per minute
1000 = Conversion of IU/ml to IU/L Symbol Key
1.025 = Total reaction volume (ml)
Use by (YYYY-MM-DD) Lot and batch code
18.75 = Millimolar absorptivity of ρ-nitrophenol
. 025 = Sample Volume (ml) Catalog number Manufacturer
1 = Light path in cm
In vitro diagnostic medical device Temperature limitation
Example: If your ∆Abs/min. = 0.06
Consult instructions for use Rx Only: Prescription Use Only
Then 0.06 x 2187 = 131 IU/L

NOTE: If test parameters are altered the factor has to be recalculated using CE mark Authorized representative in the European Community
the above formula.

SI Units: To convert to SI Units (nkat/L) multiply IU/L by 16.67. Manufactured by MedTest Dx: Pointe Brand
A7516 5449 Research Drive
Canton, MI 48188
Quality Control
The validity of the reaction should be monitored using control sera with
known normal and abnormal ALP activities and should be run with every
working shift in which ALP assays are performed. It is recommended that Manufactured by MedTest Dx: Pointe Brand
each laboratory establish their own frequency of control determination. 5449 Research Drive, Canton, MI 48188
Expected Values
Adults 35-123 IU/L at 37°C. This reference range is based on a study European Authorized Representative:
performed by the manufacturer using samples from 783 apparently healthy Obelis s.a.
adults. Children have a higher normal value. It is strongly suggested that Boulevard Général Wahis 53
each laboratory establish its own normal range. 1030 Brussels, BELGIUM
Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: [email protected]
Performance (Data generated using the Technicon RA500™analyzer)
1. Assay Range: 0-1000 IU/L
2. Correlation: A study performed between the present procedure and a Certified Performance Guarantee
similar methodology resulted in a correlation coefficient of 0.981 with MedTest Dx certifies that all of our products are manufactured according to
are a regression of y= 1.2x + 4.5 (n=95, range = 35-375, Sy.x = 9.86. specified parameters. Any product not meeting specifications through their listed
3. Precision: expiration date will be remedied immediately without charge.
Within Run Run to Run
Mean S.D. C.V.% Mean S.D. C.V.%
94 1.93 2.1 95 1.26 1.3 Rev. 08/19 P803-A7516-01
319 1.26 1.3 315 3.26 1.2