Serum is the preferred specimen but plasma (heparinized)
can also be used. Other anticoagulants inhibit the activity
also be used. Separate the serum promptly and store in a
refrigerator if immediate analysis is not possible. Red blood
cells are high in acid phosphatase concentration and hence,
a hemolyzed serum specimen is not acceptable for acid
phosphatase determination of serum. In case of increased
acid phosphatase activity due to hemolysis, check for
“tartrate-inhibition.” Acid phosphatase of red blood cells
is not “tartrate-labile” and hence is not inhibited. Alkaline
phosphatase (ALP) activity increases with storage, hence,
as a general rule, it is best to analyze ALP specimens the
same day they are drawn. Acid phosphatase (ACP) is best
stored in acid medium, hence, maintain an acid pH with
citric acid (“acid stabilizer”). Control sera must be treated
in the same way after reconstitution. ACP is measured in
vaginal washings in suspected rape cases and this must
Increased alkaline phosphatase activity may be related
to hepatobiliary and bone diseases. Very high alkaline
phosphatase activity in serum is seen in patients with bone
cancer, and marked increase also occur in obstructive
jaundice and biliary cirrhosis. Moderate elevations have
been noted in case of Hodgkin’s disease, congestive heart
failure, infective hepatitis and abdominal problems.
Total alkaline phosphatase SI Units
Adult age 20–60 4.5–13 U/dL 32–92 U/L
Children: Values remain high until epiphyses close
10 years 15–30 U/dL 107–213 U/L
Adults age 20–60 2–4 U/dL 10.7–21.5 U/L
Adults age 20–60 0.8–2.3 U/dL 13.3–38.3 U/L
Age 1–12 < 350 U/L < 5.95 µKat/L
Age >15 25–100 U/L 0.43–17.0 µKat/L
Age 1–12 < 350 U/L < 5.95 µKat/L
Age 12–14 < 500 U/L < 8.50 µKat/L
Heat inactivation After 16 minutes After 16 minutes
Liver isoenzyme 50–70 0.50–0.70
Bone isoenzyme 90–100 0.90–1.00
Intestinal isoenzyme 50–60 0.50–0.60
Trimester 1 to 1 month postpartum 50% of total
(Courtesy: Tulip Group of Companies)
For the determination of alkaline phosphatase activity in
Contd... serum (for in vitro diagnostic use only).
524 Concise Book of Medical Laboratory Technology: Methods and Interpretations Summary
Alkaline phosphatase (ALP) is an enzyme of the hydrolase
class of enzymes and acts in an alkaline medium. It is found
in high concentrations in the liver, biliary tract epithelium and
in the bones. Normal levels are age dependent and increase
during bone development. Increased levels are associated
mainly with liver and bone disease. Moderate increases are
seen in Hodgkin’s disease and congestive heart failure.
ALP at an alkaline pH hydrolyzes disodium phenyl
phosphate to form phenol. The phenol formed reacts with
4-aminoantipyrine in the presence of potassium ferricyanide,
as an oxidizing agent, to form a red colored complex. The
intensity of the color formed is directly proportional to the
activity of ALP present in the sample.
Disodium phenyl phosphate ALP Phenol
4-Aminoantipyrine K3Fe(CN)6 complex
Total ALP activity : 3.0–13.0 KA units
It is recommended that each laboratory establish its
own normal range representing its patient population.
L1: Buffer reagent 60 mL 120 mL
L2: Substrate reagent 6 mL 12 mL
L3: Color reagent 60 mL 120 mL
S: Phenol standard (10 mg/dL) 5 mL 5 mL
Contents are stable at 2–8°C till the expiry mentioned on
All reagents are ready to use.
Serum free from hemolysis. ALP is reported to be stable in
Wavelength/filter : 510 nm (Hg 546 nm)/green
Pipette into four clean dry test tubes labeled as Blank
(B), Standard (S), (C), Test (T).
Distilled water 1.05 1.00 1.0 1.0
Buffer reagent (L1) 1.0 1.0 1.0 1.0
Substrate reagent (L2) 0.10 0.10 0.10 0.10
Mix well and allow to stand at 37°C for 3 minutes and add.
Phenol standard (S) - 0.05 - -
Mix well and allow to stand at 37°C for 15 minutes and add.
Color reagent (L3) 1.0 1.0 1.0 1.0
Mix well after each addition. Measure the absorbances
of the blank (Abs. B), standard (Abs. S), control (Abs. C),
and test (Abs. T) against distilled water.
Total ALP activity in KA units = _______________ × 10
If enzyme activity 60 KA. Units dilute the sample with
distilled water and repeat the assay. Multiply the value with
In case or multiple samples to be assayed simultaneously,
only one blank and standard can be run for the entire
series, however for each sample a control and test assay
Reaction : End point abs Interval : —
Wavelength : 510 nm Sample volume : 0.05 mL
Zero setting : Distilled water Reagent volume : Calculate
Delay time : — Reaction slope Increasing
Read time : — Linearity : 60 KA
No. of read : — Units : KA units
Alkaline Phosphatase (DEA) (PNPP Kinetic Method)
(Courtesy: Tulip Group of Companies)
For the determination of alklaline phosphatase activity in
serum (For in vitro diagnostic use only).
Alkaline phosphatase (ALP) is an enzyme of the hydrolase
class of enzymes and acts in an alkaline medium. lt is found
in high concentrations in the liver, biliary tract epithelium and
in the bones. Normal levels are age dependent and increase
during bone development. Increased levels are associated
mainly with liver and bone disease. Moderate increases are
seen in Hodgkin’s disease and congestive heart failure.
ALP at an alkaline pH hydrolyzes p-Nitrophenylphosphate
to form p-Nitrophenol and Phosphate. The rate of ‘formation
of p-Nitrophenol is measured as an increase in absorbance
which is proportional to the ALP activity in the sample.
p-Nitrophenylphosphate p-Nitrophenol + Phosphate
Serum (Adults) : 80–290 U/L at 37°C.
(Children) : 245–770 U/L at 37°C.
It is recommended that each laboratory establish its
own normal range representing its patient population.
L1 : Buffer reagent 35 mL 80 mL
T1 : Substrate reagent 10 Nos 5 Nos
Contents are stable at 2–8°C till the expiry mentioned on
Working reagent : Dissolve 1 substrate tablet in 3.2 mL
(10 × 3 mL pack) or 15 mL (5 × 15 mL pack) of buffer reagent.
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