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
Pseudo-peroxidase
O2
Oxidized guaiac+ H2O
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 ------------------------------------------------ 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.
5.
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.
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.
CONTINUE …………
6.
7.
8.
Add 5ml of working 4-aminophenazone reagent on top of
the fluid in each tube .Do Not Mix
Add 10 drops of the 10 vols. Hydrogen peroxide solution.
Do Not Mix, allow to stand for 1 minute.
Look for the appearance of a blue color where 4aminophenazone 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.
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.
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