Fecal Occult Blood - Methodology

Methodology

There are four methods in clinical use for testing for occult blood in feces. These look at different properties, such as antibodies, heme, globin, or porphyrins in blood, or at DNA from cellular material such as from lesions of the intestinal mucosa.

• Fecal Immunochemical Testing (FIT), and immunochemical fecal occult blood test (iFOBT). FIT products utilize specific antibodies to detect globin. FIT screening is more effective in terms of health outcomes and cost compared with guaiac FOBT. The FIT tests are superior to low sensitivity gFOBT for colorectal cancer screening. Although FIT may be a consideration to replace gFOBT in colon cancer screening, high sensitivity gFOBT, such as Hemoccult SENSA, remains an accepted option alongside FIT in recent guidelines, being assessed as having similar overall performance characteristics to FIT. The number of fecal samples submitted for FIT may affect the clinical sensitivity and specificity of the methodology. This methodology can be adapted for automated test reading and to report quantitative results, which are potential factors in design of a widescale screening strategy. FOBT may have a role in monitoring gastrointestinal conditions such as ulcerative colitis.

• Stool guaiac test for fecal occult blood (gFOBT): – The stool guaiac test involves smearing some feces on to some absorbent paper that has been treated with a chemical. Hydrogen peroxide is then dropped on to the paper; if trace amounts of blood are present, the paper will change color in one or two seconds. This method works as the heme component in hemoglobin has a peroxidase-like effect, rapidly breaking down hydrogen peroxide. In some settings such as gastric or proximal upper intestinal bleeding the guaiac method may be more sensitive than tests detecting globin because globin is broken down in the upper intestine to a greater extent than is heme. There are various commercially available gFOBT tests which have been categorized as being of low or high sensitivity, and only high sensitivity tests are now recommended in colon cancer screening. Optimal clinical performance of the stool guaiac test depends on preparatory dietary adjustment.

• Fecal porphyrin quantification: – HemoQuant, unlike gFOBT and FIT, permits precise quantification of hemoglobin, and is analytically validated with gastric juice and urine, as well as stool samples. The heme moiety of intact hemoglobin is chemically converted by oxalic acid and ferrous oxalate or ferrous sulfate to protoporphyrin, and the porphyrin content of both the original sample and of the sample after hemoglobin conversion to porphyrin is quantified by comparative fluorescence against a reference standard; the specificity for hemoglobin is increased by subtracting the fluorescence of a sample blank prepared with citric acid to correct for the potential confounding effect of existing non-specific substances. Precise quantification measurement has been very useful in many clinical research applications.

• Fecal DNA test: – The PreGen-Plus test extracts human DNA from the stool sample and tests it for alterations that have been associated with cancer. The test looks at 23 individual DNA alterations, including 21 specific point alterations in the APC, KRAS and p53 genes, as well as testing BAT26, a gene involved in microsatellite instability (MSI). and a proprietary DNA Integrity Assay (DIA).

Additional methods of looking for occult blood are being explored, including transferrin dipstick and stool cytology.