Download Testing For Fecal Occult blood

Faculty of health sciences
Medical Laboratory Sciences Department
Medical parasitology lab.
Testing For Fecal Occult blood
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).
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
Oxidized guaiac+ H2O
Pseudo-peroxidase
O2
Blue color
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 as PH for reaction
------------------------------------------------ 1ml
• 4-aminophenazone ------------------------------------------------- 0.4g
• Dissolve the 4-aminophenazone in alcohol solution and immediately before use add the
acetic acid. Mix well
Procedures
1.
2.
3.
4.
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.
Allow the fecal particles to settle or centrifuge the emulsified specimen.
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.
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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 + + +
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
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.
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.
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• 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.
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.
Strongyloides stercoralis
• Adults lives in the small intestine (duodenum and jejunum), fertilized females
are deeply embedded in the mucosa, where they also oviposit.
• Male have a curved
tail associated with two spicules.
• Female have straight tail without spicules.
• In contrast to the Anclystoma spp., both sexes have short buccal cavity.
• Infective stage: Filariform larvae.
• Diagnosis:
• Based on recovery of the rhabditiform larvae passed in stool.
• If diarrhea is present, eggs may also be recovered.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
S. Stercoralis Adult Male
spicules
spicules
S. Stercoralis Adult Female
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Ancylostoma duodenalae (Hookworm)
• Inhabit human intestine ( jejunum, ileum, and rarely duodenum ) and cause
Anclystomiasis.
• Male shorter than female and have copulatory bursa and two spicules.
• Female is long and has pointed end.
• Both sexes have long buccal cavity with two pairs of teeth.
• Infective stage: Filariform larvae.
• Diagnosis:
– Based on finding ova in fresh stool sample.
– In old sample, larvae present and must be differentiated from larvae of
Strongyloides stercoralis.
Hookworm Eggs
Hook worm eggs like insects or mites egg, therefore must be differentiated
Mites egg
Plant material
Hook worm egg
Hookworm Adult
Female
Male
Ancylostoma duodenalae copulatry burasa
Ancylostoma duodenalae buccal capsule
Comparison between S. stercoralis and
Hookworm
Aspect
Anclystoma spp.
(Hook worm)
Strongyloides stercoralis
Rhabditiform larvae
Buccal cavity
Esophagus
Genital permordium
Long
Short
One – third of the body
One – third of the body
Absence
Prominent
Filariform larvae
Esophagus
Tail
larvae
One – third of the body
Half of the body
Pointed
Notched
Striated sheath
Unsheathed
Strongyloides stercoralis Larvae
Rhabditiform
Filariform
Hookworm Larvae
Filariform
Rhabditiform