Stool Analysis

Bleeding into the GIT may be rapid with vomiting of blood (hematemesis) or passage of blood into rectum(melaena)

When bleeding is chronic with only small amount of blood being passed in feces, the blood or its breakdown product
is not recognized in feces & referred to as occult (hidden) blood.

Request for occult blood testing is usually made to investigate cause of iron deficiency anaemia.

Also used to assist in diagnosis of bleeding lesions of GIT such as;
(i) Peptic ulcer
(ii) Carcinoma of GIT
(iii) Diverticulosis

Occult blood can be detected in feces by two methods;
(i) Chemically using reagent preparation in the laboratory such as aminophenazone screen test or readymade
reagent in kit test such as Hema Screen
(ii) Immunological using ready reagent in kit test.

Stool is a waste product of digestion

Composition of Stool
(i) Indigestible food such as cellulose
(ii) Undigested food
(iii) Unabsorbed food
(iv) Water
(v) Mucus
(vi) Cells
(vii) Bacteria
(viii) Enzymes such as trypsin
(ix) Pigment such as stercobilinogen

Weight; – Wet stool is 60 – 250g/24hr

Odour;- Depends with bacteria action on protein to produce substances such as indole & Skatole which has foul
smell
Color;- Depends on diet diet, disease and drugs. E.g
 Meat produces hard dark brown stool
 Vegetables produces soft light colored stool
 Infant produces greenish soft stool due to biliverdin

Collection of feces
 Before collection of feces, the patient should be on diet free from meat and vegetable containing peroxidases for at
least 2 days before collecting specimen
 Three specimens are collected on different days
 Stool specimen should be collected in a clean, dry wide necked container
 The testing should be done as soon as possible after collection
 Refrigerate if delay is unavoidable at 2-8OC.
 No preservative should be added to feces.

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 NB:For correct test results, you must follow these instructions carefully.
Do not collect samples if you have bleeding hemorrhoids or blood in your urine. If you are having
your menstrual period, do not collect samples until after you stop bleeding.
Diet Guidelines
o For three days before and during stool collection period, avoid red meats (beef, lamb,and liver).
o Eat a well balanced diet including fiber such as bran cereals, fruits and vegetables.
Drug Guidelines
o For seven days before and during the stool collection period, avoid non-steroidal anti-inflammatory
drugs such as ibuprofen (Motrin, Advil), naproxen or aspirin (more than one adult aspirin a day).
o Acetaminophen (Tylenol) can be taken as needed.
o For three days before and during the stool collection period, avoid Vitamin C in excess of 250 mg
a day from supplements, citrus fruits and juices.

A request form
• Should be simple and clear
• Should provide essential information concerning the patient and clinical note regarding diagnosis & treatment
• Note details of drugs because some are known to interfere with chemical reactions of certain test.
• Form must specify actual test to avoid generalization
• Form must state date and time of specimen collection

Reasons for specimen rejection

 Unlabeled or identity of form does not match that of specimen
 Specimen not correct for test requested
 Collected in wrong container or incorrect anticoagulant
 Container leaking
 Evidence of contamination
 Blood samples appear hemolyzed or presence of clot
 Too long delay in specimen reaching laboratory or not transported correctly.

1) Bilirubin in stool
 Almost all bilirubin reaching intestines is converted to stercobilinogen by enteric bacteria
 Presence of bilirubin in stool indicate diarrhea
 Detected by fouchet’s test provided stool is emulsified with distilled water.
2) Stercobilin in stool
 Stercobilinogen is normally found in stool
 Stercobilinogen decreases in obstructive jaundice but increases in hemolytic jaundice
 Stercobilin can be detected in stool due to oxidation of stercobilinogen by atmospheric oxygen
 It is detected by schlesinger’s test provided stool is mixed /emulsified in 5% Hcl in ethernol & neutralized
with alkali (NaOH)
3) Occult blood in stool
 Term occult mean hidden blood
 Blood excreted in stool is not visible with naked eyes
 Occult blood is detected microscopically or chemically
 Causes of occult blood includes;
(i) Parasitic infection
(ii) Cancer of the stomach
(iii) Ulcers

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 Detection of Occult blood
 At least 2-3days before collecting feces, instruct the patient to obtain from;
• Red meat
• Fish
• Spinach
• Drugs
• Vitamins
• Fruit juices etc.
 Instruct the patient to eat plenty of vegetables, corn and non-citrus fruits.
 Presence of large amount of occult blood gives a stool a black tar-like appearance
 Fresh blood on the surface of the stool may be due to rectal or vaginal (menstrual) bleeding.
 This blood should not be regarded as positive occult blood as this will be misleading.

Principle of occult blood testing

The principle is based on the fact that hemoglobin and its derivatives react in a similar way to peroxidase
enzyme by catalyzing the transfer of an oxygen atom from a peroxide to a chromogen such as benzidine, O-
tolidine, guaiac or aminophenazone. Oxidation of the chromogen is indicated by production of a blue, blue-
green, pink or red color.
 Unfortunately, three chromogens, Benzidine, O-dianisidine and O-tolidine have been found to be
carcinogenic although they give satisfactory results

a) Okokit tablet test

A test kit supplied with; A diluents, Okokit tablet and test papers
Method
(i) Prepare a thin smear of the feces at the center of the test paper
(ii) Place one okokit tablet in the center of the smear
(iii) Two or three drops of diluents are supplied on the tablet
(iv) Add two more drops after 2 min
(v) Read the color change after 5 min
Results
(i) Negative - no color change around the tablet
(ii) Positive (+) - pale blue color around the tablet viewed from reverse side of test paper held
up to direct sunlight
(iii) Positive (++) – dark blue color around the tablet

b) Benzidine test
Benzidine is a chromogen which catalyzes the transfer of an oxygen atom from hydroperoxide producing
a distinct color
Method
(i) Add 0.5ml of glacier acetic acid to small amount of benzidine in the test tube
(ii) Add a few drops of hydrogen peroxide (10 vol)
(iii) Observe for a green or blue color
(iv) In the absence of green or blue color, add 3-5ml of a fecal suspension in saline

Results
Production of blue or green color is indicative of presence of blood in the feces.

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c) Haemoccult method (slide and tape test)
This method uses filter paper envelopes impregnated with guaiac resin
Method
(i) The filter paper is smeared thinly with feces and allowed to dry.
(ii) Two drops of hydrogen peroxide are added to the smear.
Results
Trace : Faint blue green in 1 min
+ : Light blue slowly
++ : Clear blue rapidly
+++ : Deep blue almost immediately

d) O-tolidine test
Hydrogen peroxide in presence of Hb yields oxygen which oxidizes O-tolidine (white) to blue green
compound.
Procedure
(i) Add a small piece of stool to 2mls of distilled water in a test tube
(ii) Emulsify the stool completely and inactivate it by boiling in a water bath for 20min.
(iii) Add 1 vol of cool inactivated stool to a mixture of 4 vol of O-tolidine reagent and 1 vol of 6 %
Hydrogen peroxide in a chemically clean test tube.
(iv) Mix and observe the color
Results
Blue/ green color indicates the presence of blood whose conc. is proportional to the intensity of the color

NB:- Do not inhale or handle O-tolidine carelessly because it is carcinogenic.

Also cool stool suspension before adding to other reagents to avoid getting false-positive results

e) Haematest
A tablet impregnated with O-tolidine reagent for detecting Hb & iron containing derivative in stool
f) Occultest
A reagent tablet with the same principle as O-tolidine but more sensitive than hematest

g) Guaiac test
Hydrogen peroxide in presence of Hb yields oxygen which oxidizes guaiac to blue compound.
Procedure
(i) Add a small piece of stool to 2mls of distilled water in a test tube
(ii) Emulsify the stool completely and inactivate it by boiling in a water bath for 20min.
(iii) Allow to cool, add 2mls of a freshly prepared mixture of equal volumes of 1% guaiacum solution
and 3% hydrogen peroxide
(iv) Mix and observe the color

Results
Immediate blue color indicates the presence of blood
NB: This method is free from carcinogenic hazards and all iron derivatives gives a positive reaction.
It is also less sensitive than O-tolidine.

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 h) Phenolpthlaein test
Hydrogen peroxide in presence of Hb yields oxygen which oxidizes Phenolpthalein to form a red color
Procedure
(i) Add a small piece of stool to 2mls of distilled water in a test tube
(v) Emulsify the stool completely and inactivate it by boiling in a water bath for 20min.
(ii) Allow to cool, add 5 drops of phenopthalein reagent and mix
(iii) Add 3 drops of 3% hydrogen peroxide and mix
(iv) Observe the color
Results
Immediate red color indicates the presence of blood
NB: This method is free from carcinogenic hazards and iron derivative gives a positive reaction

4) Trypsin in Stool
 Trypsin is an enzyme that digest protein in alkaline medium
 Its precursor trypsinogen is secreted by pancreatic juice is activated by enterokinase to trypsin
 Trypsin is normally found in stool in reasonably large amount
 Determination is based on its ability to digest gelatin (protein) in alkaline medium
 Decrease in trypsin is seen in;
• Pancreatic obstruction
• Fibrocystic disease (disease of young children characterized by fatty stool and repeated lung infection)

Determination of tryptic activity by X-ray film method

• Trypsin liquefies gelatin in alkaline media

Microscopic examination of feces

 Substances & organisms of no significance values includes;
 Starch granules
 Cellulose structures
 Crystals
 Fat
 Muscle fibres

5) Fat estimation in Stool

To estimate fat, look at the stool appearance and weight.

Steatorrhoea is impaired fat absorption

Steatorrhoea is commonly associated with;
 Post-infective malabsorption (tropical sprue)
 Giardiasis
 Strongyloidiasis
 Chronic pancreatitis

Stool contains 5g of fat /24hrs or 18Mmol of fat/24hrs

Stool containing lot of fat is ;
o Bulky
o Pale/Light colored
o Offensive odour

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 o Frothy in appearance
o Not easily flashed

Excess fat in feces may be inform of;
 Neutral fat globules (the unsplit fat in food before digestion)
 Fatty acid crystals (Slit fats not yet absorbed)
 Soapy flakes (Slit fats not yet absorbed)

Presence of neutral fat globules is indicative of lipase deficiency due to pancreatic disease

More than one specimen is collected for 3-5 days (about 72hrs)

Give the patient a carmine capsule which will impact some color to the stool

1st stool containing the marker is collected

On day 3 or 5, another marker of different color is administered

Continue collecting stool until the next marker is observed

1st stool containing the 2nd marker is not considered

Under microscope, fat stains red with Sudan III solution

Stool acidity test. For infants and children who can't undergo other tests, a stool acidity test may be used.
The fermenting of undigested lactose creates lactic acid and other acids that can be detected in a stool
sample in the diagnosis of Lactose intolerance.

Clinical significance

 Little firm, spherical masses of feces – dehydration, low fiber diet and constipation
 Clay colored stool – problem with biliary system, obstructive jaundice, cyst in bile duct, alcoholic hepatitis
 Narrow ribbon like stool – intestinal spasm, hemorrhoids, spasmic bowel, rectal narrowing, stricture
 Watery stool – diarrhea
 Large amount of foul smelly gray stool – steatorrhoea(fat malabsorption)
 Reddish stool – bleeding in lower GIT, beetroot consumption or Vaginal bleeding (Menstrual contamination)
 Black tarry stool – bleeding from upper GIT, bismuth and charcoal constipation
 Green stool – spinach ingestion, calomel, presence of biliverdin, also seen in patient taking oral antibiotics.

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