Fecal occult blood test: Principle, Procedure and Results

Bleeding into the gastrointestinal tract may be profuse with the vomiting of blood (haematemesis) or the passage of blood through the rectum (melaena). When the bleeding is chronic with only small amounts of blood being passed in the faeces, the blood (or its breakdown products) is not recognized in the faeces.

The fecal occult (hidden) blood test (FOBT) is a lab test used to check stool (feces) samples for hidden  blood or blood products that may arise due to small amount of bleeding in the gastrointestinal tract . Fecal occult blood is blood in feces that cannot be seen in stool or on toilet paper by yourself.  Usually we require approximately 200 cc of blood to be able to visually recognize bleeding. Using FOBT we can detect bleeding that is as little as 10 cc of blood in the stool.

Occult blood in the stool indicates gastro intestinal bleeding that may be because of helminth infestation (hookworm infection). Other conditions associated with internal gastro-intestinal bleeding are;

Uses of Occult Blood Testing 

Requests for occult blood testing are usually made to investigate the cause of iron deficiency anemia, or to assist in the diagnosis of bleeding lesions of the gastrointestinal tract, e.g. peptic ulcer, carcinoma, or diverticulosis.

Test Methods:

  1. Chemically using laboratory reagents:  e.g. Aminophenazone Test.
  2. Immunologically using ready-made reagents in kit tests (More expensive and usually less stable than aminophenazone test)

Aminophenazone Test: 

This test detects the presence of hemoglobin in test sample. Hemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to aminophenazone. Oxidation of the aminophenazone produces a blue color (Positive Occult Blood Test).

Reagents required: 

Preparation of working Aminophenazone reagent 

The amounts given are sufficient for 1 test with positive and negative controls. Prepare fresh as follows:

Dissolve the aminophenazone in the alcohol solution and immediately before use add the acetic acid. Mix well.

Points to remember: Preparing for the test

Procedure of Occult Blood Test (Chemical Method):

  1. Dispense about 7 ml of distilled water into a wide bore test tube.
  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 fecal particle to settle or centrifuge the emulsified specimen.
  4. Take three completely clean tubes and label them as;
    1. T: Patient’s Test
    2. Neg: Negative Control
    3. Pos: Positive Control
  5. Add to each tube as follows:
    1. T: 5ml supernatant fluid from emulsified faeces.
    2. Neg: 5 ml distilled water
    3. Pos: 5 ml distilled water in which about 50 microliter of whole blood has been mixed
  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 color where the aminophenazone reagent meets the sample or control solutions.

Report the test results

  1. No color Change: Negative Test
  2. Pale Blue: Positive +
  3. Dark blue: Positive + +
  4. Blue-black: Positive + + +


Interpretation of Results and Sources of Errors: 

A positive result means that the test has detected blood. This does not mean you have tested positive for cancer or any other illness. Its just an indication of internal bleeding which will require a formal evaluation of the patient’s gastrointestinal tract. Physician may request colonoscopy or endoscopy to examine to examine the entire lining of the gastro-intestinal tract. The commonest causes of positive occult blood tests in tropical and other developing countries are hookworm infection, peptic ulcer any other causes mentioned above.

False Positive result: A false positive reaction may occur if the faeces contains peroxidase-like substances. Such reactions may be avoided by dietary restrictions (read above-preparing for the test). Further testing and examinations should be performed by the physician to determine the exact cause and source of the occult blood in the stool.

A false negative reaction may be obtained if the faeces contains  a high concentration of ascorbic acid.

Note: If the test is negative but there is high clinical suspicion, two further specimens should be tested to detect bleeding which may be intermittent.