Download Chemical Tests- Fecal Occult Blood

Prepared By:
Mr. Raed Z. Ahmed
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Fecal Occult Blood Test “ FOBT”
• Hematemesis: bleeding into the gastrointestinal tract may
be rapid with the vomiting of blood.
• Melaena: the passage of blood through the rectum.
• When the bleeding is chronic with only small amounts of
blood being passed in the feces.
• If the blood or it’s products is not recognized in the feces,
it is referred to Occult blood (hidden Blood).
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Principle of the Test
• Chemical tests to detect occult blood are based on the
principle that hemoglobin and it’s derivatives react in a
similar way to peroxidase enzymes (pseudo-peroxidase
activity).
• Chromogen such as guaiac, O- toluidine, 4-aminophenazone
or benzidine will be used as indicator for oxidation reaction.
• Hemoglobin and its derivatives catalyze the transfer of
oxygen from hydrogen peroxide to Guaiac, oxidation of the
colorless chromogen produces a blue color.
Hemoglobin + H2O2 + Guaiac Pseudo-peroxidase Oxidized guaiac+ H2O
O2
Blue color
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Reagents and method
• Reagents:
– Acetic acid 10% v/v
– Alcohol 95% v/v
– 4-aminophenazone (4-aminoantipyrine)
– Hydrogen Peroxide (H2O2) 10 vol. solution*
• Working 4-aminophenazone reagent:
– The amounts given are sufficient for 1 test with positive and negative
controls.
– Prepare fresh as follows:
• Alcohol 95% v/v -------------------------------------------------- 15 ml
• Acetic acid 10% v/v ------------------------------------------------ 1ml
• 4-aminophenazone -------------------------------------------------- 0.4g
• Dissolve the 4-aminophenazone in alcohol solution and immediately
before use add the acetic acid. Mix well
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Procedures
1.
2.
Dispense about 7ml of distilled water into a test tube
Add a sample of feces about 1gm., use a glass or plastic rod to
emulsify the feces.
3. Allow the fecal particles to settle or centrifuge the emulsified
specimen.
4. Take 3 completely clean tubes and label them as :
– T: Patient's test.
– Neg.: Negative control.
– Pos.: Positive control.
5. Add into each tube as follow:
– T ------ 5ml supernatant fluid from emulsified feces.
– Neg. ------ 5ml distilled water.
– Pos.: ------5ml distilled water in which 5ul of whole blood has
been mixed. Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue …………
6.
Add 5ml of working 4-aminophenazone reagent on top of the fluid in
each tube .Do Not Mix
7. Add 10 drops of the 10 vols. Hydrogen peroxide solution. Do Not
Mix, allow to stand for 1 minute.
8. Look for the appearance of a blue color where 4-aminophenazone
reagents meets the sample or control solutions.
– Neg. control: this should show no color change.
– Pos. control: this should show a positive reaction.
Color
Result
No color change
Negative
Pale blue
Positive +
Dark blue
Positive + +
Blue – black
Positive + + +
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
False Reactions
 False positive: 1
1. Aspirin and anti-inflammatory medications.
2. Red meat (contain Myoglobin)and fish.
3. Green vegetables (Melons), and Horseradish.
4. Menstrual and hemorrhoid contamination.
5. Some intestinal bacteria that produce peroxidase enzymes.
 False negative: 2
1. Vitamin C greater than 250 mg/dl
2. Iron supplements containing vitamin C
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Interpretation
• The commonest cause of positive occult blood tests in
tropical and other developing countries are Hookworm
infections, peptic ulcer, and bleeding from esophagus or
liver cirrhosis.
• Other causes include carcinoma in gastrointestinal tract,
erosive gastritis duo to alcohol or drugs, or swallowed
blood from nosebleeds.
• If the test is negative but there is high clinical suspicion,
a further two specimen should be tested to detect
bleeding which be intermittent.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Commercial Methods
• Therefore, to prevent false-positive reactions, the sensitivity
of the test must be increased.
• Many commercial testing kits are available for occult blood
testing with guaiac reagent.
• The kits contain guaiac impregnated filter paper, to which the
fecal specimen and hydrogen peroxide are added.
• Two or three filter paper areas are provided for application of
material taken from different areas of the stool, and positive
and negative controls are also included.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue …...
• Additional, more sensitive and specific methods, for the
detection of occult blood have been developed.
• Hemoquant, provides a fluorometric test for hemoglobin
and porphyrin.
• As hemoglobin progresses through the intestinal tract,
bacterial actions degrade it to porphyrin that the guaiac test
cannot detect, this can result in some false-negative results
from upper gastrointestinal bleeding when using the guaiac
test.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue …...
• iFOBT: The immunochemical fecal occult blood test,
Hemoccult ICT, is specific for the globin portion of human
hemoglobin and uses anti-human hemoglobin antibodies.
• Because Hemoccult ICT is specific for human blood in
feces, it does not require dietary or drug restrictions.
• It is more sensitive to lower GI bleeding that could be an
indicator of colon cancer or other gastrointestinal disease.
• Can be used for patients who are taking aspirin and other
anti-inflammatory medications.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013