Faecal clinical chemistry tests

1. FAECAL CLINICAL CHEMISTRY TESTS
GATLUAK JAMES KEDOK JIEK JANY (BBLT, MUK)

2. Clinical chemistry faecal tests that may be requested include:
1. Testing faeces for occult blood.
2. Examining faeces for excess fat.

3. 1. Faecal Occult Blood Test (FOBT)
Specimen: Faeces.
Reference Value: Negative.
Method:
A) Chemical test (Guaiac based reagent test).
B) Immunological test.
FOBT is the qualitative detection of blood haemoglobin (Hb) in faecal specimens.
FOB refers to the small amount of blood in the stool which is not visually
detectable i.e. hidden (occult).

4. Why Measure FOBT?
1. This test is mainly performed for colorectal cancer screening (especially for
patients over 50 years).
2. It may also be performed in the evaluation of anaemia (iron deficiency).

5. Interpretation
Positive in:
1. Colorectal cancers: The earlier the bowel cancers are diagnosed, the better the
chance of cure.
2. GI ulcers:
3. Polyps: Small growths on the inner lining of the large intestine (colon) or
rectum.
4. Hemorrhoids: Swollen veins in small rectums (internal and external piles).
5. Inflammatory bowel diseases, Colitis.
6. Diverticulosis: Small pouches or sacs, push outwards through weak spots in the
wall of colon.

6. A) Chemical test for FOB (guaiac based reagents)
Chemical tests are not specific to haemoglobin.
These are the most used tests. They detect the peroxidase activity of haem, whether of human or
animal origin.
Guaiac based reagents are prepared in the laboratory, e.g. aminophenazone test, or ready-made
reagent in kit tests can be used e.g. Hema-Screen.
Principle of guaiac based reagent test
The tests are based on the principle that haemoglobin and its derivatives react in a similar way to
peroxidase enzymes i.e. they catalyze the transfer of an oxygen atom from a peroxide such as
hydrogen peroxide to a chromogen such as guaiacum, 2,6-dichlorophenolindophenol or
aminophenazone. Oxidation of the chromogen is shown by the production of a blue, blue-green, or
pink colour.
HB(peroxidase activity) + H₂O₂  O₂
Guaiac reagent(colourless) + O₂ (Oxidation) Oxidized guaiac(Blue colour) + 2H₂O

7. The test is performed on a paper slide that is coated with guaiac.
A small portion of stool (faecal) is applied to the paper using applicator stick.
A developer solution containing hydrogen peroxide (H₂O₂) is added to the paper.
One side of the card is marked for the application of the stool and the other is for the developer fluid.
If the blood is present in the specimen, the iron (Fe) in the Haemoglobin catalyzes the reaction between
quaiac in the paper and the hydrogen peroxide.
The completed reaction forms a blue colour.

8. Aminophenazone test
Haemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide
to aminophenazone. Oxidation of the aminophenazone produces a purple red colour.
Reagents
Acetic acid, 10% v/v
Alcohol, 95% v/v
Hydrogen peroxide (H₂O₂)
10 vols solution
A 10 vols (volume) hydrogen peroxide solution means that 1 volume will give 10 volumes
of oxygen at NTP on complete degradation. If the solution available is 100 vols, dilute 1 in
10 with distilled water to obtain a 10 vols solution. Store in a tightly stoppered dark
bottle away from light and heat.

9. Working 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 ................................................. 1 ml
4-Aminophenazone (4-aminoantipyrine) ...... 0.4 g
Accurate weighing is not necessary. For easy preparation, mark a small tube to hold
0.4 g of the chemical. Between use keep the marked tube attached to the bottle of
chemical with an elastic band.
Dissolve the aminophenazone in the alcohol solution and immediately before use
add the acetic acid. Mix well.

10. Method
1. Dispense about 7 ml of distilled water into a wide bore test tube.
A 10 ml syringe may be used to dispense the water.
2. Add a sample of faeces about 10–15 mm in diameter (taken from various parts of the
specimen). Using a glass or plastic rod, emulsify the faeces in the water.
3. Allow the faecal particles to settle or centrifuge the emulsified specimen.
4. Take three completely clean tubes and label them:
T – Patient’s test
Neg – Negative control
Pos – Positive control
5. Add to each tube as follows:
T ........ 5 ml supernatant fluid from emulsified faeces
Neg .. 5 ml distilled water
Pos .... 5 ml distilled water in which about 50 µl of whole blood has been mixed
A 5 ml syringe may be used to dispense the faecal supernatant fluid and distilled water. It is not
necessary to use a calibrated pipette.

11. 6. Layer 5 ml of working aminophenazone reagent on top of the fluid in each tube
(i.e. pipette down the side of 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 colour where the aminophenazone reagent
meets the sample or control solutions.
Report the results as follows:
No colour change Negative test
Pale red Positive +
Dark red Positive ++
Dark red purple Positive +++

12. Negative control:
This should show no colour change.
Positive control:
This should show a positive reaction.

13. Chemical tests for FOB can give false positives with:
High meat diets, peroxidase containing foods such as turnip, certain vegetables
(which contain a chemical with peroxidase properties).
Gastric irritants such as NSAIDS which can give positives because of minor
bleeding they induce.
High dose vitamin C or E supplements or citrus fruits can give false negatives due
to its anti-oxidant properties inhibiting the colour reaction.
In chemical tests, non-haemoglobin substances (as mentioned above) with
peroxidase activity can therefore cause false positive reactions. Other substances
can interfere with peroxidase activity resulting in false negative results. The
specificity of chemical tests can be improved by dietary restrictions.
Therefore, to improve the test specificity, supplements and foods that give false
positive or false negative results should be avoided for three days before the test.

14. B) Immunochemical test for FOB
This uses specific antibodies to detect human globin in FOB.
These tests detect the globin in the faeces rather than haem. By detecting globin,
the tests are both more sensitive and specific for lower gastrointestinal bleeding.
Procedure
The sample is mixed with buffer.
The buffer solution is then introduced into a test device.
The buffer solution will migrate through the test device.
Results are read after 5 minutes.

15. Chemical test Immunochemical test
Detection Haem Globin
Interference Meat, certain plant foods, Vitamin
C, NSAIDS
None
Origin of detected occult bleeding Entire GI tract Colorectal

16. FOBT Sensitivity
Considerable variation of sensitivity is shown by both chemical and immunological
occult blood tests.
Highly sensitive tests can be misleading because they detect trace amounts of blood
which can be found in normal faeces. Highly sensitive chemical tests can give false
positive reactions when faeces contain dietary substances which have peroxidase-
like activity.
Tests of low sensitivity can also be misleading because they may fail to detect small
amounts of blood which are pathological.
Using three cards, each on different days, is recommended to improve sensitivity.
Note:
This test is not enough to diagnose bowel cancer.
Further evaluation (e.g. colonoscopy) is required.

17. 2. Microscopical examination of faeces for fat
Fatty stools are pale in colour, bulky, float on water, and are often frothy with an
offensive odour.
Excess fat in faeces may occur as:
Neutral fat globules
Fatty acid crystals
Soapy flakes

18. Method
1. Make a thin preparation of the faeces in physiological saline on a slide. Cover with
a cover glass (avoid trapping air bubbles).
2. Examine the preparation for excess neutral fat globules, fatty acid crystals, and
soapy fats using the 10 and 40 objectives with the condenser iris closed sufficiently
to give good contrast.
Note: Constant focusing is necessary because fat does not sediment but tends to float
in the preparation.
Neutral fat globules
These are easily recognized because they are highly refractile, colourless, and variable
in size and shape with an oily look. They can be stained orange-red with a
concentrated alcoholic solution of Sudan III or a saturated solution of oil red 0 in
isopropanol (the stain can be run under one end of the cover glass). If a drop of
ethanol or diethyl ether is added, the fat globules will dissolve. Patients taking liquid
paraffin excrete droplets of oil which are identical in appearance to the fat globules
present in steatorrhoea.

19. Fatty acids
Fatty acids usually appear as groups of needle-like colourless crystals. They do not stain with Sudan III or oil
red 0, but melt easily if gentle heat is applied to the preparation. Fatty acid crystals dissolve in ethanol and
diethyl ether.
Soapy fats
Soaps also form masses of needle-like colourless crystals. They can be distinguished from fatty acid crystals
because they do not melt with heat and they do not dissolve in ethanol or diethyl ether unless first treated
with acetic acid. Soapy fats occur as flakes (soapy plaques).
Interpretation of microscopical findings
The presence of excess neutral fat globules in the faeces of a person taking a normal diet suggests lipase
deficiency due to pancreatic disease. Neutral fat is the unsplit form of fat which is found in food before it
is digested.
The presence of excess fatty acids and soapy fats (split fats that have not been absorbed) in faeces
suggests malabsorption.