Human Hemoglobin Cat No. H-7309). This corresponds to a concentration of 100 to 200 μg/g hemoglobin/g of feces. No Prozone Effect up to a hemoglobin concentration of 1000 mg/L has been observed.
2. Retrieve the required number of test cards to perform
3. Label the cards with correct patient identity.
4. Open the sample application windows labeled A and
B respectively, to expose the reactive surfaces of the
5. By using the sample applicator provided in the kit,
spread a very thin layer of stool on the reactive surfaces
on the window A; similarly, on window B; from a
6. Wait until the smeared sample has dried completely.
8. Open the result window and add one drop of developer to fields RA and RB (the reverse side of the
sample smeared on the sample application windows)
9. Observe for color change exactly at 2 minutes.
10. Even if one of the field’s has a blue color, the test is
¾ No blue color indicates absence of occult blood in the
¾ Trace blue coloration indicates presence of
approximately 5 mg/dL of occult blood in the stool.
¾ Strong blue coloration indicates significantly more than
5 mg/dL of occult blood in the stool (Figs 7.1 to 7.4).
118 Concise Book of Medical Laboratory Technology: Methods and Interpretations Remarks
1. Stool samples collected during menstrual bleeding,
constipation induced bleeding, bleeding hemorrhoids or when rectal medication is used may cause
2. Hands, collection containers and test area should be
kept free of blood as they may cause false positive
3. Certain medications may induce gastrointestinal
bleeding and cause false positive reactions, hence
should be avoided during and prior to the testing
4. Diet containing exogenous peroxidases may induce
5. Dosages of vitamin C more than 250 mg per day will
cause a false negative result.
6. If the test is developed before the sample smear dries
completely on the test card, the results obtained may
7. Only use the sample applicators provided in the kit for
Rapid Immunochromatographic Device: Testing of
Cancheck-FOBT is a rapid, qualitative, two-site sandwich
immunoassay for the detection of fecal occult blood
Colorectal cancer (CRC) is a major cause of death from
cancer. The risk of CRC increases with age, with an
approximate doubling of the incidence in each decade
from 40 to 80 years of age. It has been estimated that the
lifetime risk of developing CRC is 1:50. Fecal Occult Blood
Test (FOBT) provides the most cost-effective way to screen
for CRC. It has been reported that screening for CRC by
FOBT decreases CRC mortality by 15–33%. FOBT is the test
to detect the presence of occult blood in the feces. Small
amounts of blood is present in the feces of normal healthy
individuals due to bleeding from the gastrointestinal tract
like bleeding gums and bleeding from minor abrasions. The
presence of small amounts of blood in feces may not alter the
color or appearance of the stool. The detection of fecal occult
blood can be useful in detecting bleeding resulting from
gastrointestinal disorders such as colitis, polyps, colorectal
carcinomas and diverticulitis. Benzidine and guaiac tests
for fecal occult blood detect the peroxidase activity of heme,
either as intact hemoglobin or as free heme. Hence, to avoid
false positives, for the week before the test, patients need to
follow a diet that excludes red meat, turnips, horseradish,
broccoli, radishes, cauliflower, cantaloupes and other melons
and supplemental vitamin C. Unlike Guaiac tests, Cancheck
- FOBT is a third generation immunochromatographic test
that is not affected by peroxidase activity.
Cancheck-FOBT utilizes the principle of immunochromatography, a unique two-site immunoassay on a
nitrocellulose membrane. The conjugate pad contains
two components—monoclonal anti-human hemoglobin
antibody conjugated to colloidal gold and rabbit IgG
conjugated to colloidal gold. As the test specimen flows
through the membrane assembly of the device, the
highly specific monoclonal anti-human hemoglobin
antibody-colloidal gold conjugate complexes with the
human hemoglobin in the specimen and travels on
the membrane due to capillary action along with the
rabbit IgG colloidal gold conjugate. This complex moves
further on the membrane to the test region (T) where it is
immobilized by another specific monoclonal anti-human
hemoglobin antibody coated on the membrane leading
FIG. 7.2: Cancheck FOBT negative result
FIG. 7.3: Cancheck FOBT positive result
FIG. 7.4: Canceck FOBT invalid results
Specimen Collection and Preparation
1. Cancheck-FOBT uses human feces as specimen.
2. Collect feces in a clean dry container.
3. Though fresh specimen is preferable, in case of delay
in testing, it may be stored at 2–8°C for maximum up
. 4. Refrigerated specimens must be brought to room
5. Label the specimen collection bottle with specimen
6. Unscrew and remove the cap (with attached sampling
stick) of the specimen collection bottle ensuring that
the extraction buffer is not spilt.
7. Take representative amounts of feces specimen from
different portions of the sample by introducing the
sampling stick at 3–4 different places in the feces
8. Wipe the sampling stick with an absorbent or tissue
paper. The sample taken up by the grooves is sufficient
9. Reinsert the sampling stick into the bottle and screw
10. Shake the specimen collection bottle so that there is
proper homogenization of feces in buffer solution.
Testing Procedure and Interpretation of Results
1. Bring the kit components of device to room temperature
2. Open a foil pouch by tearing along the “notch".
3. Remove the testing device. Once opened, the device
4. Label the device with specimen certification.
5. Place the testing device on a flat horizontal surface.
6. Hold the specimen collection bottle in an upward
position and break the tip off.
7. Invert the bottle and holding the dropper vertically,
carefully dispense exactly two drops of specimenbuffer mixture into the specimen port.
8. Observe the development of visible colored band at
9. Positive results may be observed within 5 minutes,
depending on the concentration of occult blood in the
10. Do not read and interpret after 5 minutes.
11. In negative specimens only the control band (C) would
Presence of one colored band at Control (C) region
indicates absence of Occult blood or the concentration of
Occult blood in the specimen is below the detection limit
of 200 μg/L of feces suspension.
to formation of a colored band. If occult blood level is
equal to or higher than the 200 µg/L of feces suspension,
the test is positive. The absence of this colored band in
the test region indicates a negative test result. The rabbit
IgG-colloidal gold conjugate and unbound complex, if
any, move further on the membrane and are subsequently
immobilized by the anti-rabbit antibodies coated on the
membrane at the control region (C), forming a colored
band. The control band formation is based on the ‘Rabbit/
anti-Rabbit globulin’ system. Since it is completely
independent of the analyte detection system, it facilitates
formation of consistent control band signal independent
of the analyte concentration. This control band acts as a
procedural control and serves to validate the test results.
Reagents and Materials Supplied
A. Individual pouches, each containing :
1. Device membrane assembly pre-dispensed with
monoclonal anti-human hemoglobin colloidal
gold conjugates, rabbit IgG colloidal gold conjugate, monoclonal anti-human hemoglobin
antibody and anti-rabbit antiserum coated at the
B. BUF: 0.1 M Tris, 1% sodium chloride, 0.5% Brij 35,
The sealed pouches in the test kit and the kit components
may be stored between 4–30°C for the duration of shelf life
as indicated on the pouch/carton. Do not freeze.
1. Read the instructions carefully before performing the
2. For in vitro diagnostic use only. Not for medicinal use.
3. Do not use the kit beyond expiry date and do not re-use
4. Do not intermix reagents from different lots.
5. Handle all specimens as if potentially infectious.
Follow standard biosafety guidelines for handling and
disposal of potentially infectious material.
6. If desiccant color at the point of opening the pouch
has turned from blue to pink or colorless, another test
7. Specimen extraction buffer contains sodium azide
(0.1%), avoid skin contact with this reagent. Azide may
react with lead and copper in the plumbing and form
highly explosive metal oxide. Flush with large volumes
of water to prevent azide build-up in the plumbing.
kept free of blood to avoid false positive results.
6. Since benzidine and guaiac-based tests suffer from
nonspecific interference of peroxidase activity, exact
one-to-one correlation of the results of such tests with
a 3rd generation immunochromatographic test like
Cancheck-FOBT may not be observed.
7. Cancheck-FOBT should only be used as a screening
test. As with all diagnostic tests, a definitive clinical
diagnosis should not be based on the result of a
single test, but should only be made by the physician
after all clinical and laboratory findings have been
In a normal diet, fat in the stool will be up to 20% of total
Lipids measured as fatty acids: 2–5 g/24 h
It is raised in malabsorption syndromes, the commonest
example being steatorrhea. Fecal fat quantitation can be
¾ Isotopic techniques (radioisotopes)
¾ Electrical capacitance method
¾ Titrimetric method of Van de Kamer.
Fats and fatty acids are converted to soap by boiling with
alcoholic potassium hydroxide. After cooling, excess
hydrochloric acid is added to convert soaps to fatty acids.
These are extracted with petroleum ether. An aliquot is
evaporated, taken up in neutral alcohol, and titrated with
sodium hydroxide. Fats are calculated as fatty acids.
This has replaced the titrimetric method now. An aliquot
of fecal suspension is extracted with solvent consisting
chiefly of chlorinated benzenes. The extract is filtered and
its electrical capacitance is measured and compared with
standards of triolein similarly treated.
To quantitate fat excretion, there must be known dietary
intake and timed stool collection. The usual technique
involves a diet containing 100 g of fat daily, with a 3-day
stool collection to measure total fat excretion. Excretion of
more than 6 g per day is abnormal and values may range
If concentration of Occult blood in specimen is above 200
μg/L of feces suspension, two colored bands appear at Test
(T) and Control (C) regions. The intensity of the test band
may be more or less than the Control band, depending
upon the concentration of Occult blood in specimen.
Invalid Result Cancheck-FOBT: The test is invalid if
no band is visible at 5 minutes. The test should also be
considered invalid if only the test band appears and no
control band appears. Verify the test procedure and repeat
The detection limit of Cancheck -FOBT is up to 200 μg/L
of feces suspension, i.e. equivalent to 100–200 μg/g of feces.
The detection limit of Cancheck-FOBT device is up to
200 μg/L feces suspension, (calibrated against Sigma
Human Hemoglobin Cat No. H-7309). This corresponds to
a concentration of 100 to 200 μg/g hemoglobin/g of feces.
No Prozone Effect up to a hemoglobin concentration of
Cancheck -FOBT is highly specific to human hemoglobin
and does not cross-react with the following:
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