This working reagent is stable for at least 15 days when

stored at 2–8°C.

The substrate is light and temperature sensitive. Take

adequate care, especially after reconstitution.

Sample Material

Serum. Free from hemolysis. ALP is reported to be stable

in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 405 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

(mL)

Working Reagent 1.0

Incubate at the assay temperature for 1 minute and add

Sample 0.02

Mix well and read the initial absorbance A0 after minute

and repeat the absorbance reading after every 1, 2, and

3 minutes. Calculate the mean absorbance change per

minute (∆A/min).

Calculations

ALP activity in U/L = ∆A/min. × 2754.

Temperature Conversion Factors

Assay

Temperature

Desired

25°C

Reporting

30°C

Temperature

37°C

25°C 1.00 1.22 1.64

30°C 0.82 1.00 1.33

37°C 0.61 0.75 1.00

Linearity

The procedure is linear up to 700 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.250, use only the

value of the first 2 minutes to calculate the result, or dilute

the sample 1 + 9 with normal saline (NaCI 0.9%) and repeat

the assay (Results × 10).

Note

Samples having a very high activity show a very high initial

absorbance. If this is suspected then dilute the sample and

repeat the assay.

System Parameters

Reaction : Kinetic Interval : 30

Wavelength : 405 nm Sample volume : 0.02 mL

Zero setting : Distilled

 water

Reagent volume : 1.00 mL

Incubation

temperature

: 37°C Standard : —

Incubation

time

: — Factor : 2754

Delay time : 30 sec Factor : Increasing

Read time : 120 sec Linearity : 700 U/L

No. of read : 4 Units : U/L

526 Concise Book of Medical Laboratory Technology: Methods and Interpretations Clinical Relevance

Elevated Levels

1. Liver disease (correlates with abnormal liver function

tests). An elevation of serum alkaline phosphatase is

often associated with elevated SGOT/SGPT and raised

bilirubin.

 a. Marked increases

 i. Obstructive jaundice (gallstones obstructing

major biliary ducts, accompanies elevated

bilirubin).

 ii. Space occupying lesions of the liver such as

cancer and abscesses.

 iii.Hepatocellular cirrhosis.

 iv. Biliary cirrhosis.

 b. Moderate increases

 1. Hepatitis

 2. Cirrhosis of liver.

2. Bone disease

 a. Marked increases

 i. Paget’s disease

 ii. Metastatic bone disease

 iii.Osteitis deformans.

 b. Moderate increases

 i. Osteomalacia (in osteoporosis, no increase in

ALP)

 ii. Rickets.

3. Other diseases

 a. Hyperparathyroidism (accompanied by

hypercalcemia)

 b. Infectious mononucleosis.

Reduced Levels

1. Hypophosphatasia (markedly reduced)

2. Malnutrition

3. Hypothyroidism

4. Pernicious anemia

5. Scurvy

6. Milk-alkali syndrome

7. Placental insufficiency.

Interfering Factors

1. Many drugs produce mild to moderate elevations of

ALP, e.g.

a. Phenothiazine tranquillizers

b. Methyltestosterone

c. Oral contraceptives

d. Allopurinol

e. Methyldopa

f. Procainamide

g. Tolbutamide

h. Isoniazid

i. PAS

j. Erythromycin

k. Oxacilin

l. Ergoesterol.

2. Young children, pregnant women in the third trimester,

and all women have physiologically high levels of

alkaline phosphatase.

3. The level is slightly increased in older people.

4. After IV administration of albumin, there is sometimes

a marked increase lasting for several days.

5. Drugs that may cause decreased levels

a. Fluoride

b. Oxalates

c. Phosphates

d. Propranolol

e. Vitamin D.

Alkaline Phosphatase Isoenzymes

Normal Values

AP-1, Alpha 2: Values are reported as weak, moderate,

or strong

AP-2, Beta 1: Values are reported as weak, moderate,

or strong

AP-3, Beta 2: Values are reported as weak, moderate,

or strong

The isoenzymes of alkaline phosphatase (ALP) are

produced by various tissues. AP-1, Alpha 2 is heat labile

and is produced in the liver and by proliferating blood

vessels. AP-2, Beta 1 is heat stable and is produced by bone

and placenta. The intestinal isoenzymes AP-3, Beta 2 is

present in small quantities in Group O and B individuals

AP-1 and 2 can be distinguished partially in the laboratory