Fecal Occult Blood Test (FOBT): Procedure, 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 a small amount of bleeding in the gastrointestinal tract. Fecal occult blood is blood in feces that cannot be seen in the 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;

  • Oesophagitis (inflammation of the esophagus; often associated with acid reflux)
  • Esophageal cancer
  • Gastratitis
  • Stomach ulcer or cancer
  • Arterie venous malformation
  • Colon polyp
  • Colon cancer
  • Diverticulosis
  • Ulcerative colitis and
  • Hemorrhoid

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 the test sample. Hemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to aminophenazone. Oxidation of aminophenazone produces a blue color (i.e. positive occult blood test).

Reagents Required

  • Acetic acid 10% v/v
  • Alcohol, 95% v/v
  • Hydrogen peroxide 10 vols solution

Preparation of working Aminophenazone reagent 

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

  • Acetic acid 10% v/v: 1 ml
  • Alcohol, 95% v/v: 15 ml
  • 4 Aminophenazone (4-Aminoantipyrine):0.4g

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

Preparing for the test

  • Any source of blood will give a positive test. Blood from another source, like bleeding hemorrhoids or menstrual period, will interfere with the test.
  • Do not eat red meat before testing for the occult blood test. Red meat in the diet can give false-positive test results.
  • Do not eat cantaloupe,  fish, uncooked broccoli, turnip, radish, horseradish for 3 days before the test. Patient should eat plenty of vegetables, corn, and non-citrus fruits.  Any fruit juices containing ascorbic acid, iron, barium sulphate, cimetidine also has to be avoided 2/3 days before testing.
  • Avoid drugs like aspirin, anti-inflammatory drugs, cough medicines for the 3 days before the test.
  • Taking vitamin C supplements may give false negative results. It is recommended not to take Vitamin C supplements for the 3 days before 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 feces about 10-15 mm in diameter (taken from various parts of the specimen). Using a glass or plastic rod, emulsify the feces 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 feces.
    2. Neg: 5 ml distilled water
    3. Pos: 5 ml distilled water in which about 50 microliters 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 + + +


  • Negative Control: This should show no color change.
  • Positive Control: This should show a positive reaction.

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. It is 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 the entire lining of the gastrointestinal 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.


  1. Kaur K, Zubair M, Adamski JJ. Fecal Occult Blood Test. [Updated 2023 Apr 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537138/ 
  2. Narula, N., Ulic, D., Al-Dabbagh, R., Ibrahim, A., Mansour, M., Balion, C., & Marshall, J. K. (2014). Fecal occult blood testing as a diagnostic test in symptomatic patients is not useful: a retrospective chart review. Canadian journal of gastroenterology & hepatology, 28(8), 421–426. https://doi.org/10.1155/2014/189652

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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