2Occult blood A fecal occult blood test (FOBT) is a non invasive test that detects the presence of hidden (occult) blood in the stool,not apparent to patient.Hidden blood in stool is often the first, and, warning sign that a person has colorectal disease, including colon cancer.
3Occult gastrointestinal bleeding: It is usually identified only by tests or, it may manifested as iron deficiency anemia.It is the bleeding that is clinically evident but from an obscure source.
4Causes of gastrointestinal bleeding: Mass lesion: Inflammation: carcinoma – adenoma>1cm at any site.Inflammation:erosive oesophagitis ,ulcer at any site, erosive gastritis, ulcerative colitis and crohn`s disease.Vascular disorder:vascular actasia at any site, portal hypertension gastropathy or colonpathy, varicoces ,haemangioma...
6Normally ml blood per day is lost in stools amounting to ~ 2 mg hemoglobin per gram of stool4-6 mg/gm, an amount that is typically not detected by occult blood tests.Hema- screen test will detect 10 mg of hemoglobin/gm of stool.Occult blood is commonly detected in the stool using a variety of FOBT.
7Anatomical level of lesion. Stool transit time. Stool mixing. Factors affecting the detection of faecal occult bloodAnatomical level of lesion.Stool transit time.Stool mixing.hemoglobin degradation in lumen.features of bleeding (intermittent or continuous bleeding).
8In screening programs an estimated 1-5% of the tested population have a positive fecal occult blood test. (1-2% false positive)Causes for a positive test are:2-10%: cancer (colorectal cancer, gastric cancer) .20-30% adenoma or polyps .Bleeding peptic ulcer .Angiodysplasia of the colon.
9The results of occult blood cannot be considered conclusive evidence of presence or absence of gastrointestinal bleeding or pathology.The test is preliminary screen not replace any other diagnostic methods (proctosigmoidoscopy, barium enema…)
11Intra-luminal haemoglobin degradation and faecal occult blood tests: In the upper gastro-intestinal tract, haemoglobin is cleaved to form heme and globin .Heme is converted to porphyrin and iron has been termed the "intestinal converted fraction" of haeme.
12intestinal converted fraction This fraction is not detected by guaiac-based tests but is detected by heme-porphyrin assay (HemoQuant test), which measures both heme and porphyrins and is therefore a highly accurate indicator of bleeding, regardless of the level.
13Guaiac based tests: Heme-porphyrin tests: Immunochemical tests: Three types of tests are used to detect faecal occult blood. these include:Guaiac based tests:HemeOccult (Hema-screen)Heme-porphyrin tests:HemoQuant testImmunochemical tests:HemeSelect testFlexSure OBT
14Occult blood test1. Guaiac based testHema-screen test
15PrincipleHema-screen is composed of guaiac impregnated paper (light tan in color) enclosed in a cardboard frame which permit stool sample application to one side ,and develoment and interpretation on reverse side.The hematin portion of hemoglobin can catalyzing oxidation of guaiac resulting in production of blue color.
16Guaiac-based tests detect the pseudo-peroxidase activity of heme, either as intact hemoglobin or as heme.Such activity converts colourless guaiac to a blue colour in the presence of an oxygen donor like hydrogen peroxide (H2O2) (developing reagent).It detect only hemoglobin released upon hemolysis of RBC by hydration
17specimens :Card with thin stool smear or5 g stool Container.Collection card or stool transport vial without preservative.
21Negative result positive result +ve &-ve monitor ( act as quality control) this provide assurance that guaiac paper and developer are reacting properly.
22Patient Preparation A high bulk, red meat free diet. Patient should not receive vitamin C (ascorbic acid), NSAIDs for 3 days prior to occult blood testing by guaiac.A high bulk, red meat free diet.restriction of peroxidase-rich vegetables (turnips, horseradish, artichokes, mushrooms, radishes, broccoli, bean sprouts, cauliflower, oranges, bananas, cantaloupes, grapes), has been recommended 3 days prior to guaiac testing, and during testing.Therapeutic iron causes false-positives with guaiac tests in more than half of healthy subjects.
23Special diet may be omitted initially, with dietary restriction imposed upon retesting of all positive results.Because gastrointestinal lesions may bleed intermittently and blood in feces is not distribute uniformly ,all positive tests regardless of diet , follow-up diagnostic procedures should be done.
24Smear two area from each stool sample. Recommendations for testing occult blood in stool using guaiac-based testsSmear two area from each stool sample.Test 3 successive evacuation, close to each other as possible.Test all samples within 4 days of collection.Do not rehydrate slide prior to development.Even one positive test should be considered a positive even without dietary restriction
25False-positive results: Intake of foods that contain peroxidase activity (uncooked fruits and vegetables).Drugs: Topical iodine, aspirin, NSAIDs.Non-human hemoglobins: red meat.RehydrationFalse-negative results:Vitamin C (Ascorbic acid) intake.Storage of slides.Improper sampling or developing.Lesion not bleeding at time of stool collection.Hemoglobin degradation by colonic bacteria.
26Guaiac-based test Advantage: Readily available.Convenient.Inexpensive.Can be performed in physician's office.DisadvantageDependant upon degree of hydration.Affected by storage.Affected by site of bleeding.Dietary restrictions required.
28Intestinal converted fraction is the fraction of heme converted to porphyrin during fecal transit, a phenomenon which leads to diminished guaiac sensitivity for a pathology more proximal to colon.
292. Heme-porphyrin testThe HemoQuant test includes the measurement of total fecal hemoglobin or porphyrin derived from heme, intestinal converted fraction (ICF) which is the performed porphyrin probably by bacteria during enterocolic transit.
30The heme-porphyrin test, HemoQuant measures hemoglobin derived porphyrin spectro-fluorometrically and allows exact measurement of total hemoglobin in stool.
31The amount of hemoglobin present in stools as determined by HemoQuant test can be graded as: Normal < 2 mg/gram of stool.Borderline 2- 4 mg/gram of stool.Increased > 4 mg/gram of stool.
32Heme-porphyrin test Advantage: No interference with dietary peroxidases or rehydration.Highly accurate indicator of bleeding, regardless of level.Disadvantage:Needs laboratory setup.Time consuming (~90 minutes required)False positive results with non-human haemoglobins.
33Since 2001, there are a new class of occult blood tests called Fecal Immunochemical Tests(FIT). These tests detect the globin in feces rather than heme. By detecting globin the tests are both more sensitive and specific but only for lower gastrointestinal bleeding.
34Globin in the upper gastro-intestinal tract is digested by pepsin ,pancreatic and intestinal proteases and is, thus, not detected by the immunochemical faecal occult blood tests.
353. Immunochemical testsuse antibodies directed against human globin epitopes and can detect colonic blood at a level as little as 0.3 ml of blood.not detect small quantities of blood from upper gastrointestinal tract.The tests are limited by loss of globin antigenicity at room temperature and require processing in laboratory.
36Tests usedHemeSelect test: is similar to enzyme linked immunosorbant assay (ELISA) and needs to be done in the laboratory,FlexSure OBT : is a simpler immunoassay which can be done in the physician's office.
37HemeSelect test (ELISA) A more complex reaction that uses monoclonal and/or polyclonal antibodies that detect the intact globin portion of human hemoglobin.If hemoglobin is present in the stool, the labeled antibody will attach to its antigens, creating a positive test result.For technical and commercial reasons, this test have not found wide usage among physicians.
38flexsure OBThandled brushtest pad cardtest strip+
39FlexSure OBTA long-handled brush is used to brush the surface of the stool, thus trapping dislodged occult blood and water on the bristles.The brush is then dabbed onto the test card where the water dries into a special pad. Once dry, the hemoglobin is considered stable and the card is sent to a laboratory for testing.
40Once the test card is received at the laboratory, an immunochromatographic test strip consisting of a test line and a control line is inserted into the test pad of the card. Liquid is added, causing any hemoglobin present to migrate up the test strip.If hemoglobin is present in the sample, it binds to a colloidal gold-conjugated polyclonal antibody against hemoglobin, and is labeled red.
42The control line has an antibody that reacts with the conjugate antibody, which also causes a line to form indicating the test has worked correctly.So positive result will produce two lines on the test strip, whereas a negative result produces only one line (the control line), The test is completed in approximately five minutes.
43Immunochemical test:Advantage: Highly specific, as it detects only human hemoglobinFlexSure OBT can be performed in physician's officeDietary restrictions are not requiredDetects as little as ~0.3 mg/gram of blood.Disadvantage:Needs laboratory setup (Hemoselect).Affected by storage (hemoglobin degradation)Affected by site of bleeding.
46Fecal DNA test (PreGen-Plus®): reported Four-Times More Sensitive than Fecal Occult Blood Test for Colorectal Cancer Screening
47PreGen-Plus works by isolating the human DNA from a single stool sample collected, and analyzing that DNA for alterations associated with, active colorectal cancer.
48colorectal cancers result from chromosomal instability, with acquired mutations progressively accumulating in the adenomatous polyposis coli (APC) and p53 tumor suppressor genes, and in the k-ras oncogene.
49PreGen-Plus consists of a panel of 23 individual tests each looking for the presence of DNA alterations in human DNA isolated from stool
50Research has shown that DNA is a good marker: It is stable in the stool, it is shed continuously.Through the use of amplification tests, such as polymerase chain reaction(PCR), it can be detected in minute amounts.
51PreGen-Plus does not require any special bowel preparation, stool handling or changes in diet prior to testing.It also does not carry risks associated with invasive tests, such as colon perforation, hemorrhage, or infection, or sedation.
52Ability to detect cancers proximal to the colon Ability to detect cancers proximal to the colon. aerodigestive cancers (e.g., lung, esophageal, stomach, pancreas, colorectal(The tests detect target mutations with a high degree of sensitivity and specificity.
55The other arises from malfunction to genes involved in DNA mismatch repair, manifested by microsatellite instability.Colorectal cancer may also be detectable by DNA markers associated with disordered apoptosis.
56CONCLUSIONSfecal blood appears to be a poor marker for colorectal neoplasia. Most cancers and the vast majority of polyps will be missed. Hemoccult and HemoQuant are similarly insensitive.