ALKALINE PHOSPHATASE BIOCHEMISTRY REAGENT  

PRINCIPLE:

Alkaline phosphatase (ALP) catalyze the hydrolysis of 4- nitrophenyl phosphate (4-NPP) with the formation of free 4- nitrophenol and inorganic phosphate, acting the alkaline buffer as a phosphate-group acceptor.

The reaction is monitored kinetically at 405 nm by the rate of formation of 4-nitrophenol, proportional to the activity of ALP present in the sample.

REAGENT COMPOSITION:

R1 -ALP buffer. DEA buffer 1.25 mol/L pH 10.2, magnesium chloride 0.6 mmol/L. Biocides

R2 – ALP substrate. 4-NPP 50 mmol/L. Biocides

STORAGE AND STABILITY:

Store at 2-8ºC.

The Reagents are stable until the expiry date stated on the label.

REAGENT PREPARATION:

Working reagent.

Working reagent. Mix 4 mL of R1 + 1 mL of R2. Stable for 5 days at 20-25ºC or 30 days at 2-8ºC. Protect from light.

Discard the reagent if the blank presents an absorbance over 0.800 at 405 nm. against distilled water or if it fails to recover the declared values of control sera.

SAMPLES:

Serum or heparinized plasma, free of hemolysis. Other anticoagulants such as EDTA, oxalate and citrate inhibit the enzyme by complexing Mg⁺⁺ and should not be used.

Alkaline phosphatase in serum or plasma is stable for 7 days at 2- 8ºC.

INTERFERENCES:

• Bilirubin (>20 mg/dL) and triglycerides (>10 g/L) do not
• A list of drugs and substances winch cause changes in ALP levels or interfere with its measurement can be found .

MATERIALS REQUIRED:

• Photometer or spectrophotometer with a thermostatted cell compartment set at 25/30/37ºC, capable to read at 405
• Stopwatch, strip-chart recorder or
• Cuvettes with 1-cm
• Pipettes to measure reagent and

PROCEDURE:

1. Preincubate working reagent, samples and controls to reaction
2. Set the photometer to 0 absorbance with distilled
3. Pipette into a cuvette:
4. Working reagent: 1.0 ml
5. Sample or control: 20 mL
6. Mix gently by Insert cuvette into the cell holder and start stopwatch.
7. Incubate for 1 minute and record initial absorbance
8. Repeat the absorbance readings exactly after 1, 2 and 3
9. Calculate the difference between
10. Calculate the mean of the results to obtain the average change in absorbance per minute (DA/min
11. CALCULATIONS:U/L = DA/min x 2764 Samples with DA/min exceeding 0.250 at 405 nm should be diluted 1:2 with saline and assayed again. Multiply the results by 2.If results are to be expressed as SI units apply: U/L x 0.01667 = mkat/L

REFERENCE VALUES:

Serum, plasma:

It is recommended that each laboratory establishes its own reference range.

QUALITY CONTROL:

To ensure adequate quality control (QC), each run should include a set of controls (normal and abnormal) with assayed values handled as unknowns.

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CLINICAL SIGNIFICANCE:

Serum ALP measurements are of particular interest in the investigation of two groups of conditions: bone disease and hepatobiliary disease.

Among the bone diseases, the highest levels are found in Paget´s disease and in patients with osteogenic bone cancer, and moderate raises in osteomelacia and rickets, the latter falling to normal on treatment with vitamin D.

Physiological bone growth elevates ALP in serum of growing children and a transient elevation may be found during healing of bone fractures.

Causes of decreased plasma ALP level are: creatinine, vitamin D deficiency and hypophosphatasia, an hereditary bone disease.

The response to the liver to any form of biliary tree obstruction is to sinthesize more ALP. Intrahepatic obstruction of the bile flow by invading cancer or drugs raises serum ALP. Any drug that is hepatotoxic or induces cholestasis will greatly increase serum ALP. Well over 200 drugs have been shown to increase serum ALP in susceptible patients.⁴

ANALYTICAL PERFORMANCE:

• LInearity: Up to 800 U/L
• Precision
  

  U/L	Within-run
  Mean	49	186	301
  SD	0.7	2.8	5.2
  CV%	1.43	1.5	1.72
  N	21	21	21

• Replicates: 21 for each level. Instrument: PHILIPS
  • Sensitivity.Using this reagent and method an DA/min of 0.010 read at 405 nm is equivalent to 1,60 U/L of phosphatase activity.
  • Correlation.
    • This assay (y) was compared with a similar commercial method (x). The results were:

    N = 25      r = 0.999      y = 0.961x + 5.431

• REFERENCES:

• 1. German Society for Clinical Chemistry: Recommendations of the Enzyme Z. Klin. Chem. Klin. Biochem. 10 : 281 (1972).
  2. Young, D.S. Effects of Drugs on Clinical Laboratory Tests. 4^th AACC Press (1995).
  3. N.W. Clinical Guide to Laboratory Tests, 3^rd Edition.

  W.B. Saunders Co. Philadelphia, PA. (1995).

  4. Young, S., pestaner, L.G., and Gibberman, P. Clin. Chem. 21 : 246D-248D (1975).

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