An alkaline phosphatase level test (ALP test) measures the amount of alkaline phosphatase enzyme in your bloodstream. The test requires a simple blood draw and is often a routine part of other blood tests.
(PNPP Kinetic Method
Kinetic reaction (Increasing)
Alkaline Phosphatase in serum catalyse the hydrolysis of p. nitrophenyl phosphate to p. nitrophenol and phosphate. p. nitrophenol is yellow coloured compound. As the reaction progresses the rate of absorbance increases which is proportional to the activity of Alkaline Phophatase in the sample. This reaction takes place in alkaline medium and in presence of magnesium ions. The change in absorbance is meusared at 405 nm.
- R 1 : PNPP Substrate
- 2-Amino-2-methylpropanol —– > 1.2 mol/L
- Magnesium ———— > 7.2 mmol/L
- Zinc Sulfate ————– > 3.6 mmol/L
- HEDTA —————— > 7.2 mmol/L
- R 2 : AMP buffer
- 4-Nitrophenyl Phosphate —– > 171.6 mmol/L
Test Requirements :
- Test Tubes
- Bio-Chemistry analyzer
- SGOT Test Kit
Reagent Preparation and Stability :
4 Part of R1 (0.8 ml)
1 Part of R2 (0.2 ml)
and Mix well
Avoid direct exposure to light. Stability of working reagent: 7 days at 2 – 8 °C.
Specimen collection and handling:
- Unhaemolysed Serum, heparinized plasma
- Stability: 10 days at 2 – 8° C.
Analyzer Parameter Required :
|Reaction Type||Kinetic reaction (Increasing)|
|Delay Time||30 sec|
|Interval Time||30 sec|
|No. Of reading||4|
|Zero Setting||Deionised Water|
|Light Path||1 cm|
Pipette into clean dry test tube labeled as (T) and Calibrator Label (c) and QC Label (QC) or as your required:
|Specimin||(T)||(C) / (QC)|
|Working Reagent||1.0 ml (1000 µl)||1.0 ml (1000 µl )|
|Pt. Sample||0.02 ml (20 µl )||–|
|Calibrator or QC||–||0.02 ml(20 µl )|
|Mix well and read the initial absorbance after 30 Seconds and repeat the absorbance reading after every 30 Seconds, upto 12 Seconds. Calculate the mean absorbance change per minute (Δ OD/min.).|
CALCULATION : ALP activity (IU/L) = D A/min x 2712
NOTE: Samples having a very high activity show a very low initial absorbance. If this is suspected then dilute the sample and repeat the assay
The procedure is linear upto 1500 IU/L.
if the activity exceeds this limit dilute the sample with normal saline (NaCl 0.9 %) and multiply result by dilution factor.
QUALITY CONTROL :
For accuracy it is necessary to run known controls with every assay.
Normal Value :
- Children (3-15 yrs) : 104 – 390 IU / Ltr
- Adults : 25 – 147 IU / Ltr
Possible References Used