Stool examination:- Part 5 – Stool for Occult Blood, OB (Fecal occult blood)

Sample

1. This test is done on the stool.
2. The random sample can be taken, and a 3 mL quantity is enough.
   1. Avoid the outer portion and take a sample from the central area of the formed stool.
   2. Three consecutive stool samples are needed.
3. Collect the stool in a dry, sterilized wide-mouth container.
4. Instruct the patient to stop vitamin C, iron-containing drugs, meat, and vegetable for at least three days before the test.
5. Fresh stool testing is recommended.

Precautions

1. The special diet is recommended  48 to 72 hours before the test is performed.
2. Also, direct the patient to avoid the following foods:
   1. No, red meat. The chicken and fish were also stopped.
   2. Peroxidase-rich vegetables like turnip, spinach, horseradish, mushrooms, broccoli, beans, cauliflower, oranges, banana, cantaloupe, and grapes.
   3. No raw fruits.
   4. Avoid Vitamin C, which causes a false negative reaction by inhibiting the peroxidase reaction.
3. Avoid drugs like anticoagulants, aspirin, colchicine, nonsteroidal antiarthritics, iron preparation, and steroids for at least 7 days before the test.
4. Drugs that may cause false-positive results are:
   1. Colchicine, and iron.
   2. Oxidizing drugs like iodine, bromides,  boric acid, and rauwolfia derivatives.
5. Drugs that may cause false-negative results are vitamin C, etc.
6. Take H/O of bleeding gums.
7. Vigorous exercise.

Indications

1. This is a screening test for colorectal carcinoma.
2. This can be used for bleeding ulcers in the gastrointestinal tract.
3. This test can be included in the periodic medical checkup.
   1. This test is advised in older people after 40 asymptomatic to rule out GI malignancy.
4. This test is essential in hypochromic anemias because of ulcerative or neoplastic diseases.
5. Over the age of 50 years, this may be included in the annual check-ups.

Pathophysiology

1. Definition: There is a presence of hemoglobin in the stool, evidenced by the chemical test but not seen by the naked eye, hidden (occult).
   1. Occult blood is hidden, and it requires a chemical test for its detection.
2. Normally only minimal quantities of blood are passed into the gastrointestinal tract. This bleeding quantity is not significant enough to cause the occult blood test positive.
3. Normally 2 to 5 ml of blood passes in the stool per day, but this is not detectable.
   1. The healthy person passes roughly 2 ml of blood in 150 grams of stool.
   2. This amount will not show occult blood positive.
   3. This test will be positive when there is 5 mL of the blood passes in the stool per day.
      

      Criteria for the Occult blood positivity

   4.  Another reference says that the occult blood test can detect 2 ml of blood in the stool.
   5. Another source says >2 mg/g of the stool is positive, and <2 mg/g/day is seen in healthy people, and OB will be negative.
4. Occult blood will be positive when the tumors grow in the intestine’s lumen and if they ulcerate and give rise to bleeding.
5. Bleeding in the upper GI tract produces a black tarry stool.
6. While bleeding from the lower GI tract produces just blood in the stool.
7. Fecal occult blood should test for either heme or heme-derived porphyrins.
8. Heme has a peroxidase-like activity that is detected by the Guaiac dye test.
9. Stool with dark red to tarry black indicates a blood amount of 50 to 75 mL from the upper GI tract.
   

   Gastrointestinal bleeding and the role of occult blood test

Normal

• Occult blood normally is negative.

Procedure

Chemical method:

1. Advises patients to take plenty of vegetables, corn, and non-citrous fruits.
2. Modification of the benzidine and 0-tolidine test because these are carcinogenic.
   1. Take 95% alcohol 15 mL.
   2. Dissolve 4- aminophenazone 0.4 grams.
   3. Add Acetic acid 10% 1 mL.
   4.  H2O2 (Hydrogen peroxide)1 volume and 10 ml of water.
      1. Take 10 to 15 mL of D.water and emulsify the stool (10 mm in diameter).
      2. Now centrifuge and take the clear emulsified fluid.
      3. Take three tubes and label those as a patient, Negative control, and positive control.
      4. Add 5 mL of emulsified stool material to the patient tube.
      5. Add distle water to the negative control.
      6. Add one drop of blood to the positive control.
3. Now layer 5 mL of the mixed aminophenazone prepared reagent above the suspension and don’t mix. Just layer it over the suspension.
4. Now add 10 drops of Hydrogen peroxide 10 vols and don’t mix (dilute 1 mL of H₂O₂ with 10 mL of D.water.).
5. Check the result as follows.
   

   Occult blood test result interpretation

6. Every time you have to make a fresh sample to run this test.
   1. The false-positive test is seen if the patient’s stool has a peroxidase-like substance.
   2. The false-negative reaction is seen in the case of ascorbic acid excess in the stool.
      1. In the case of suspected cases, repeat the test two more times.

Guaiac test principle and interpretations:

1. The Guaiac test’s disadvantage is that it may react with non-Hb peroxidase present in the stool like vegetables and meat.
2. Vitamin C inhibits the Guaiac reaction.
3. Guaiac reaction test sensitivity is 40%, so many times; it can not detect the tumors in the early stages.
4. A more sensitive reaction developed, which has sensitivity and specificity of about 80% and 94%, respectively.
   1. A drop of water  (rehydration) is added to the slide before testing in the Guaiac test.
   2. This step increases the sensitivity but decreases the specificity.
   3. More cancers are detected after the rehydration, but a more false-positive result leading to further investigation like barium meal study or sigmoidoscopy.
5. The principle of the Guaiac test:
   1. The stool sample is applied to the guaiac impregnated paper.
   2. Then add the developer solution consists of H2O2 (hydrogen peroxide) and denatured alcohol.
      

      Occult blood guaiac reagent and the principle

      

      Principle of occult blood guaiac test

6. Interpretation of the Guaiac test:
   
   1. Any appearance of the blue color is a positive test.
   2. The intensity of color development does not matter, whether it is a week or strong.
   3. If the paper before the test is turned blue or blue-green should be discarded.
   4. American cancer society recommends three consecutive samples for colorectal cancer screening.
7. False-positive OB test is seen in:
   1. Ingested meat.
   2. Peroxidase-rich vegetables like turnip, horseradish, mushroom, broccoli, beans, sprouts, cauliflower, oranges, bananas, cantaloupe, and grapes.
   3. Drugs may lead to bleeding like anticoagulants, aspirin, iron preparation, nonsteroidal antiarthritic drugs, and steroids.
   4. White cells and bacteria also cause a false positive test.
   5. Other drugs causing false-positive tests are boric acid, colchicine, bromides, and iodine.
8. A false-negative OB test is seen in:
   1. Vit C may cause a false negative when taken more than 250 mg/day.
   2. Foods and juices are rich in vitamin C.
   3. Oxidants also cause a false-negative result.
   4. An iron supplement containing vitamin C >250 mg.

Causes of positive OB test seen in:

1. Gastrointestinal tumors.
   1. Rectal carcinoma.
   2. Gastric carcinoma.
2. Inflammatory bowel disease.
3. Diverticulosis.
4. Varices.
5. Ischemic bowel disease.
6. Arteriovenous malformations of the GI tract.
7. Hemorrhoids.
8. Blood is swallowed from the oral cavity or nasopharynx.
9. Adenoma.
10. Peptic ulcer.
11. Gastritis.
12. Amyloidosis.
13. Kaposi’s sarcoma.

Other tests for the detection of colorectal carcinomas:

The fluorometric method:

1. This method is specific for heme and porphyrins.
   1. Value < 2 mg/g of the stool is negative.
   2. A value> 2 mg/g of the stool is positive for colorectal carcinoma.

Immunological kits methods:

1. These are sensitive to detect the human hemoglobin component of globin.
2. A monoclonal antibody against the hemoglobin is used. These antibodies are specific for hemoglobin.
   1. Mostly agglutination is reported positive by the presence of coated antibodies or latex particles.
   2. These tests are called the fecal immunochemical tests for fecal occult blood.
   3. The disadvantage is that it may not detect bleeding from the upper GI tract because the globin is digested or degraded by the time it gets into feces.

DNA stool method:

1. This is a new technique to detect precancerous and malignant tumors.
2. The benign lesion does not bleed, and these may be missed by Guaiac or other tests in occult blood screening.
3. All precancerous lesions shed cells with abnormal DNA, so precancerous lesions can be removed before they transform into malignant conditions.

For the detection of colorectal cancer, can advise:

1. Occult blood in the stool.
2. Sigmoidoscopy.
3. Colonoscopy.

Advantage

• When screening started at the age of 50 years, it will reduce death by colorectal malignancies. In one reference, this may be a 60% reduction.
• NOTE. Please see more details in another stool part.

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