by heating and urea testing. Placental alkaline phosphatase

is still more stable to heat than urea.

The test is conducted (when the total alkaline

phosphatase is raised) to distinguish between bone and

liver origin of alkaline phosphatase.

Clinical Relevance

1. Osteoblastic bone tumors, increase the bone alkaline

phosphatase in the blood serum; less than 25% is

thermostable in bone disease.

2. Liver diseases such as cancer and biliary obstruction

increase the liver isoenzyme, more than 25% in

thermostable in hepatic disease.

3. The intestinal isoenzyme may be increased in patients

with cirrhosis.

4. The placental isoenzyme is increased in some patients

with cancer (Carcino placental antigen) and normally

in pregnancy.

Enzymology 527

Acid Phosphatase

Clinical Significance

Request for the analysis of serum acid phosphatase is often

done in male patients with suspected prostatic cancer.

The increase of serum acid phosphatase activity in such

patients is found to be inhibited by tartrate. Acid phosphatase is also present in very high concentrations in semen,

a fact utilized in forensic medicine in the investigations of

rape offences.

Normal Values

Methods SI units

Bodansky 0.5–2 U/L 2.7–10.7 IU/L

King-Armstrong 0.1–5 U/L 0.2–8.8 IU/L

Bessey-Lowery-Brock 0.1–0.8 U/L 1.7–13.4 IU/L

Gutman 0.1–2 U/L 1.7–13.4 IU/L

Specimen

Serum is the most commonly used specimen; hemolyzed

serum specimens are contaminated with red cell acid

phosphatase and should be rejected.

Mod. King’s Method

(Courtesy: Tulip Group of Companies)

For the determination of Acid Phosphatase activity in

serum (For in vitro diagnostic use only).

Summary

Acid phosphatase (ACP) is an enzyme of the hydrolase

class of enzymes and acts in an acidic medium. It is

widely distributed and found in high concentrations

in the liver, RBCs and the prostate. Increased levels

of the prostatic fraction are associated with prostatic

carcinomas. Increased levels of the non-prostatic fraction

are associated with liver diseases, hyperparathyroidism,

and Paget’s disease.

Principle

ACP at an acidic pH hydrolyzes di-sodium 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 ACP present in the sample. Tartrate inhibits prostatic

ACP and the testing in its presence is done to find the non

prostatic ACP. The difference between the activities of the

total and non-prostatic ACP gives the activity of the prostatic

ACP.

Disodium phenyl phosphate ACP Phenol + + H2O pH 5.0 Disodium Hydrogen

 Phosphate

Phenol Alkaline Medium Red + Colored

4-Aminoantipyrine K3Fe(CN)6 Complex

Normal Reference Values

Total ACP activity : 1.0–4.0 KA units

Prostatic ACP activity : 0.0–0.8 KA units

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 Tests 25 Tests

L1: Buffer reagent 50 mL 125 mL

L2: Substrate reagent 5 mL 12.5 mL

L3: Color reagent 50 mL 125 mL

L4: Tartrate reagent 2 mL 2 mL

S : Phenol Standard (10 mg/dL) 5 mL 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

All reagents are ready to use.

Sample Material

Serum. Free from hemolysis.

The ACP, especially the prostatic fraction, is unstable in

a collected sample hence, the serum should be separated

from the clot, as soon as possible, and assayed. In case of a

delay in testing the serum should be acidified to a pH of 5.0

with 0.02 mL Acetate Buffer (5M) for each mL of serum.

Procedure

Wavelength/filter : 510 nm (Hg 546 nm)/green

Temperature : 37°C

Light path : 1 cm

Assay

Pipette into 5 clean dry test tubes labeled as blank (B),

standard (S), control (C), Test (T), and tartrate stable (TS).

Addition

Sequence

B

(mL)

S

(mL)

C

(mL)

T

(mL)

TS

(mL)

Distilled water 1.1 1.05 1.0 1.0 1.0

Buffer reagent

(L1)

1.0 1.0 1.0 1.0 1.0

Substrate

reagent (L2)

0.10 0.10 0.10 0.10 0.10

Mix well and allow to stand at 37°C for 3 minutes and add.

Contd...

528 Concise Book of Medical Laboratory Technology: Methods and Interpretations Tartrate reagent

(L4)

- - - - 0.02

Sample - - - 0.1 0.1

Phenol standard

(S)

- 0.05

Mix well and allow to stand at 37°C for 60 minutes and add.

Color 1.0 1.0 1.0 1.0 1.0

reagent (L3)

Sample - - 0.1 -

Mix well after each addition. Measure the absorbances

of the Blank (Abs.B), Standard (Abs.S), Control (Abs.C), Test

(Abs.T), and Tartrate Stable (Abs.TS) against Distilled water.

Calculations

 Abs. T-Abs. C

Total ACP activity in KA Units = _______________× 5.0

 Abs. S-Abs. B

 Abs. T - Abs. TS

Prostatic ACP activity = _______________ × 5.0

in KA units Abs. S - Abs. B

Linearity

If enzyme activity exceeds 40 KA.Units dilute the sample

with distilled water and repeat the assay. Multiply the

value obtained with an appropriate dilution factor.

Notes

In case of multiple samples to be assayed simultaneously,

only one Blank and Standard can be run for the entire

series, however for each sample, a Control, Test and

Tartrate Stable assay has to be run additionally. It has been

seen that in a collected sample ACP, especially the prostatic

form, may loose around 50% of its activity in an hour at RT.

System Parameters

Reaction : End point Abs Interval :

Wavelength : 510 nm Sample volume : 0.10 mL

Zero setting : Distilled water Reagent volume : 3.10 mL

Incubation

temperature

: 37°C Standard : Calculate

Incubation

time.

: 60 min Factor :

Delay time : — Factor : Increasing

Read time : — Linearity : 40 KA Units

No. of read : — Units : KA Units

Acid Phosphatase

(α Naphthyl Phosphate Kinetic Method)

(Courtesy: Tulip Group of Companies)

For the determination of acid phosphatase activity in

serum (For in vitro diagnostic use only).