lab report on alkalyne phosphatase test

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Prof NJAGI/Mr ELIAKIM
HANDING DATE: /3/2016
SUBJECT: PRACTICAL REPPORT ON ALKALYNE PHOSPHATASE TEST.
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2. TEST PRINCIPLE/CLINICALSIGNIFICANCE
Alkaline phosphatases are a group of enzymes that split off a terminal phosphate group from an organic
ester in alkaline solution. Their optimum pH is usually around pH 10, but this varies with the particular
substrate and isoenzyme. Alkaline phosphatase is widely distributed in almost every tissue in the body
and serum ALP levels are of interest in the diagnosis of hepatobiliary disorder and bone disease. Most of
the ALP in the adult serum is from the liver or bilary tract. Normal ALP levels are age-dependent and
levels are elevated during periods of active bone growth. Serum ALP activity is raised in all bone
disorders accompanied by increased osteoblastic activity. This includes Paget's disease (osteitis
deformans), osteoblastic tumors with metastases, hyperparathyroidism when there is mobilization of Ca
and P from the bone, rickets and osteomalacia. Moderate elevations of ALP (not involving the liver or
bone) may be attributed to Hodgkin's disease, congestive heart failure, abdominal bacterial infections, and
pregnancy. Low levels are found in hypophosphatasemia, dwarfism, hypothyroidism, and pernicious
anemia. Isoenzymes of ALP have been identified in the liver, bone, intestinal mucosa, placenta, and bile.
Isoenzymes can be separated by acrylamide electrophoresis or by their ability to withstand denaturation
when exposed to heat. The liver isoenzyme is heat stable and the bone isoenzyme is heat labile.
In this method, ALP catalyzes the hydrolysis of ρ-Nitrophenyl phosphate (pNPP) to ρ-Nitrophenol. pNPP
is colorless but ρ-Nitrophenol has a strong absorbance at 405 nm. The rate of increased absorbance at 405
nm is proportional to the enzyme activity. The procedure is standardized by means of the millimolar
absorptivity of ρ-Nitrophenol (18.75 at 405 nm) under the specified conditions. The results are based on
the change in absorbance per unit of time. One International Unit (IU/L) is defined as the amount of
enzyme that catalyzes the transformation of one micromole of substrate per minute under specified
conditions.
REQUIRED REAGENTS/EQUIPMENT
1. Spectrophotometer
2. Spectrophotometer cuvettes
3. Distilled deionized water
4. Physiological (0.9% NaCl) saline
5. Pipettes
6. Alkaline phosphatase reagent obtained from Pointe Scientific, Inc. Reconstitute reagent with 15
mL distilled water. Swirl gently to dissolve. When reconstituted as described, the reagent contains
p-Nitrophenylphosphate 10.0 mM, Magnesium ions 1.0 mM, Buffer (pH 10) , activator and
binder. The un reconstituted reagent is stored at 2-8°C. Once reconstituted, the reagent is stable
for 48 hours at 25°C and for 30 days at 2-8°C.
7. Paraffin squares
8. Heating block or water bath 37°C
9. Timer

3. PROCEDURE
1. Turn on the spectrophotometer and let warm up for at least 15 minutes.
2. Set the wavelength to 405 nm.
3. Label cuvettes.
4. Add 1.0 mL of distilled deionized water to cuvette 1.
5. Add 1.0 mL of Alkaline phosphatase reagent to each control and patient cuvettes.
6. Incubate all cuvettes at 37°C for 5 minutes.
7. Add 25 uL of each control and patient sample to their respective cuvettes.
8. Mix each by inversion using the paraffin squares to prevent spillage.
9. Incubate control and patient cuvettes at 37°C for 1 minute.
10. After one minute, place cuvette 1 in the spectrophotometer and set the absorbance to read 0.000.
11. Read and record the absorbance for each of the control and patient cuvettes after every 1minute
for 4 readings.
12. Return the control and patient cuvettes to 37°C and repeat readings for each cuvette every minute
for the next two minutes.
13. Calculate the average absorbance difference per minute (ΔAbs/min).
14. Calculate the amount of enzyme using the following calculation:
OUTCOMEAND CALCULATIONS
time Absorbance Change in absorbance
1minute
2nd
minute
3rd
minute
4th
minute
0.459
0.458
0.457
0.454
0.001
0.001
0.003
Total=0.005
ΔAbs/min = Average absorbance change per minute=0.005/3
=1.667 x 10-3
Hence Concentration of serum ALP=
(1.667 x 103
) x2754
=4.59U/L

4. REFERENCE INTERVALS
The reference range for alkaline phosphatase is as follows:
Serum or Plasma Age Group IU/L
0-5 Y
5-10 Y
10-12 Y
12-16 Y
>16 Y
Neonates
60-321
110-360
103-373
67-382
60-170
Upto 600
Therefore the alkaline phosphatase level is low in our sample.
DISCUSIONS AND CONCLUSION
High ALP usually means that either the liver has been damaged or a condition causing increased bone cell
activity is present.
If other liver tests such as bilirubin, aspartate aminotransferase (AST),or alanine aminotransferase
(ALT) are also high, usually the increased ALP is coming from the liver. If GGT or 5'-nucleotidase is also
increased, then the high ALP is likely due to liver disease. If either of these two tests is normal, then the
high ALP is likely due to a bone condition. Likewise, ifcalcium and/or phosphorus measurements are
abnormal, usually the ALP is coming from bone.
If it is not clear from signs and symptoms or from other routine tests whether the high ALP is from liver
or bone, then a test for ALP isoenzymes may be necessary to distinguish between bone and liver ALP.
ALP in liver disease
ALP results are usually evaluated along with other tests for liver disease. In some forms of liver disease,
such as hepatitis, ALP is usually much less elevated than AST and ALT. When the bile ducts are blocked
(usually by gallstones, scars from previous gallstones or surgery, or by cancers),ALP and bilirubin may
be increased much more than AST or ALT. ALP may also be increased in liver cancer.
ALP in bone disease
In some bone diseases,such as Paget's disease,where bones become enlarged and deformed, or in certain
cancers that spread to bone, ALP may be increased.
If a person is being successfully treated for Paget's disease,then ALP levels will decrease or return to
normal over time. If someone with bone or liver cancer responds to treatment, ALP levels should
decrease.
Moderately elevated ALP may result from other conditions, such as Hodgkin's lymphoma, congestive
heart failure,ulcerative colitis, and certain bacterial infections.

5. Low levels of ALP may be seen temporarily after blood transfusions or heart bypass surgery. A
deficiency in zinc may cause decreased levels. A rare genetic disorder of bone metabolism called
hypophosphatasia can cause severe,protracted low levels of ALP. Malnutrition or protein deficiency as
well as Wilson disease could also be possible causes for lowered ALP.
Pregnancy can increase ALP levels. Temporary elevations are also seen with healing fractures.
Children and adolescents normally have higher ALP levels than adults because their bones are growing,
and ALP is often very high during a growth spurt, which occurs at different ages in boys and girls.
Some drugs may affect ALP levels. For example, oral contraceptives may decrease levels while anti-
epileptics may increase levels.
LIMITATIONS
1. EDTA,oxalate, fluoride, and citrate are known inhibitors of ALP and specimens collected in
these anticoagulants should be avoided.
2. ALP levels in serum or plasma rise significantly when stored at 2-8ºC or frozen.
3. This methodology measures total alkaline phosphatase irrespective of tissue or organ of origin.
Further tests may be necessary to assist in differential diagnosis.

6. REFERENCES
1. Burtis, Carl A et al. Tietz Fundamentals of Clinical Chemistry, 6th ed. Saunders: St Louis,
Missouri, 2008.
2. Naser,Najih. Clinical Chemistry Laboratory Manual. Mosby: St Louis, Missouri, 1998.
3. Pointe Scientific, Inc package insert. February 2009.