Faculty of health sciences

Medical Laboratory Sciences Department

Medical parasitology lab.

Testing For Fecal Occult blood

Fecal Occult Blood Test “ FOBTFecal Occult Blood Test “ FOBT””

• Hematemesis: bleeding into the gastrointestinal tract may be rapid with the vomiting of blood.

• Melaena: the passage of blood through the rectum.

• When the bleeding is chronic with only small amounts of blood being passed in the feces.

• If the blood or it’s products is not recognized in the feces, it is referred to Occult blood (hidden Blood).

Principle of the TestPrinciple of the Test

• Chemical tests to detect occult blood are based on the principle that hemoglobin and it’s derivatives react in a similar way to peroxidase enzymes (pseudo-peroxidase activity).

• Chromogen such as guaiac, O- toluidine, 4-aminophenazone or benzidine will be used as indicator for oxidation reaction.

• Hemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to Guaiac, oxidation of the colorless chromogen produces a blue color.

Hemoglobin + H2O2 + Guaiac Oxidized guaiac+ H2OO2

Pseudo-peroxidase

Blue color

Reagents and methodReagents and method• Reagents:

Acetic acid 10% v/v Alcohol 95% v/v 4-aminophenazone (4-aminoantipyrine) Hydrogen Peroxide (H2O2) 10 vol. solution*

• Working 4-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 as PH for reaction ------------------------------------------------

1ml • 4-aminophenazone ------------------------------------------------- 0.4g• Dissolve the 4-aminophenazone in alcohol solution and immediately before use add the

acetic acid. Mix well

ProceduresProcedures 1. Dispense about 7ml of distilled water into a test tube

2. Add a sample of feces about 1gm., use a glass or plastic rod to emulsify the feces.

3. Allow the fecal particles to settle or centrifuge the emulsified specimen.

4. Take 3 completely clean tubes and label them as :

– T: Patient's test.

– Neg.: Negative control.

– Pos.: Positive control.

5. Add into each tube as follow:

– T ------ 5ml supernatant fluid from emulsified feces.

– Neg. ------ 5ml distilled water.

– Pos.: ------5ml distilled water in which 5ul of whole blood has been mixed.

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6. Add 5ml of working 4-aminophenazone reagent on top of the fluid in 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 4-aminophenazone reagents meets the sample or control solutions.

– Neg. control: this should show no color change.

– Pos. control: this should show a positive reaction.

ColorResult

No color changeNegative

Pale bluePositive +

Dark bluePositive + +

Blue – blackPositive + + +

False ReactionsFalse Reactions

False positive: 1

1. Aspirin and anti-inflammatory medications.

2. Red meat (contain Myoglobin)and fish.

3. Green vegetables (Melons), and Horseradish.

4. Menstrual and hemorrhoid contamination.

5. Some intestinal bacteria that produce peroxidase enzymes. False negative: 2

1. Vitamin C greater than 250 mg/dl

2. Iron supplements containing vitamin C

InterpretationInterpretation

• The commonest cause of positive occult blood tests in tropical and other developing countries are Hookworm infections, peptic ulcer, and bleeding from esophagus or liver cirrhosis.

• Other causes include carcinoma in gastrointestinal tract, erosive gastritis duo to alcohol or drugs, or swallowed blood from nosebleeds.

• If the test is negative but there is high clinical suspicion, a further two specimen should be tested to detect bleeding which be intermittent.

Commercial MethodsCommercial Methods

• Therefore, to prevent false-positive reactions, the sensitivity of the test must be increased.

• Many commercial testing kits are available for occult blood testing with guaiac reagent.

• The kits contain guaiac impregnated filter paper, to which the fecal specimen and hydrogen peroxide are added.

• Two or three filter paper areas are provided for application of material taken from different areas of the stool, and positive and negative controls are also included.

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• Additional, more sensitive and specific methods, for the detection of occult blood have been developed.

• Hemoquant, provides a fluorometric test for hemoglobin and porphyrin.

• As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that the guaiac test cannot detect, this can result in some false-negative results from upper gastrointestinal bleeding when using the guaiac test.

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• iFOBT: The immunochemical fecal occult blood test, Hemoccult ICT, is specific for the globin portion of human hemoglobin and uses anti-human hemoglobin antibodies.

• Because Hemoccult ICT is specific for human blood in feces, it does not require dietary or drug restrictions.

• It is more sensitive to lower GI bleeding that could be an indicator of colon cancer or other gastrointestinal disease.

• Can be used for patients who are taking aspirin and other anti-inflammatory medications.

Strongyloides stercoralisStrongyloides stercoralis

• Adults lives in the small intestine (duodenum and jejunum), fertilized females are deeply embedded in the mucosa, where they also oviposit.

• Male have a curved tail associated with two spicules.• Female have straight tail without spicules.• In contrast to the Anclystoma spp., both sexes have short buccal cavity.• Infective stage: Filariform larvae.

• Diagnosis:

• Based on recovery of the rhabditiform larvae passed in stool.

• If diarrhea is present, eggs may also be recovered.

Raed Z. Ahmed, Medical Parasitology Lab.,2012

S. Stercoralis Adult MaleS. Stercoralis Adult Male

spicules

spicules

S. Stercoralis Adult FemaleS. Stercoralis Adult Female

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Ancylostoma duodenalae (Hookworm)Ancylostoma duodenalae (Hookworm)

• Inhabit human intestine ( jejunum, ileum, and rarely duodenum ) and cause Anclystomiasis.

• Male shorter than female and have copulatory bursa and two spicules.

• Female is long and has pointed end.

• Both sexes have long buccal cavity with two pairs of teeth.

• Infective stage: Filariform larvae.

• Diagnosis:– Based on finding ova in fresh stool sample.

– In old sample, larvae present and must be differentiated from larvae of Strongyloides stercoralis.

Hookworm EggsHookworm Eggs Hook worm eggs like insects or mites egg, therefore must be differentiated

Mites egg Hook worm eggPlant material

Hookworm AdultHookworm Adult

MaleFemale

Ancylostoma duodenalae copulatry burasaAncylostoma duodenalae copulatry burasa

Ancylostoma duodenalae buccal capsuleAncylostoma duodenalae buccal capsule

Comparison between S. stercoralis and Comparison between S. stercoralis and HookwormHookworm

Strongyloides stercoralis LarvaeStrongyloides stercoralis Larvae

Rhabditiform

Filariform

Hookworm LarvaeHookworm Larvae

Filariform Rhabditiform