Fecal occult blood refers to a test that is done on feces to detect bleeding into the gastrointestinal tract. It is particularly used to detect upper gastrointestinal hemorrhage, i.e. melena (or digested blood), which can be more difficult to see grossly (i.e. occult) versus undigested frank blood from lower intestinal or colonic hemorrhage in feces, but the test does not discriminate between upper or lower gastrointestinal hemorrhage. It is more sensitive than visually examining feces for blood (frank or digested). Causes of gastrointestinal hemorrhage are many and varied, but include gastric and intestinal ulcers, bloodsucking parasites (e.g. Haemonchus contortus, which is a major problem in camelids), whipworm (Trichuris) infection in dogs and cats, bleeding tumors and intestinal varices (i.e. idiopathic dilation of vessels in the colonic mucosa). Fecal occult blood testing should be done in any animal with an iron deficiency anemia (i.e. microcytic hypochromic red blood cells) since chronic external hemorrhage from the gastrointestinal tract (which results in loss of iron and blood) is the most common cause of iron deficiency in adult animals. Studies in dogs have shown that positive fecal occult blood results can be obtained when dogs are orally given 20 mg/kg BW of hemoglobin, although positive results can be obtained with lesser amounts of fed hemoglobin (5 mg/kg BW) (Gilson et al 1990, Pierini et al 2020)
Most fecal occult blood tests are based on the detection of hemoglobin in feces, which can be done because the heme portion of hemoglobin has pseudo-peroxidase like activity, i.e. it is acts as an enzyme and can break down peroxidase-based substrates, yielding a distinctive color in a screen. The peroxidase-based fecal occult blood test, such as that used at Cornell University (which is the Coloscreen test® from Helena Laboratories) contains guaiac (guaiac denotes the name of the paper surface used in the test which has a phenolic compound, α-guaiaconic acid, that is extracted from the wood resin of Guaiacum trees) on which the feces is smeared. Then an ethanol-based hydrogen peroxide-containing developer is added, which oxidizes the α-guaiaconic acid to a blue quinone. Normally, when no blood is present, this oxidation occurs very slowly. Heme gives a positive blue result in about two seconds. Therefore, a positive test result is one where there is a quick and intense blue color change of the film. There are different fecal occult blood tests, which vary in their sensitivity and specificity to blood (Gilson et al 1990, Cook et al 1992).
False positive and negative results for fecal occult blood can occur.
- False positives: This can be seen if there are other substances in feces with peroxidase like activity, including enzymes in plants and meat-based diets (Cook et al 1992). Plants have catalases and the prosthetic group ferriprotoporphyrin IX (hemin) which has higher enzymatic activity than hemoglobin. False positives can be seen if the animal ingests or has a diet rich in plants with high endogenous peroxidase (beets, canteloupe, broccoli, cauliflower). We attempt to eliminate false positives due to catalase in plants by leaving the feces on the test developer for 48 hours. The plant peroxidases are neutralized by the ethanol developer, reducing false positives. Thus, we confirm positive reactions by a 48 hour testing period.
- False negatives: These can occur under the following conditions:
- Ingestion of citrus or vitamin C (due to anti-oxidant properties).
- Stored fecal samples due to degradation of hemoglobin (Young et al 1996).
- Colonic bacterial flora may remove iron, which is required for the pseudoperoxidase activity, particularly with delays in fecal transit (since removal of iron takes time) (Young et al 1990).
- Intermittent bleeding and.or testing single fecal sample is tested for occult blood in an animal with intermittent hemorrhage (the animal may have not bled at the time the feces were produced), so serial testing is recommended in animals suspected of suffering from gastrointestinal hemorrhage (although there is no standardized frequency or number of tests for serial analysis).
- Sampling of sections of feces that lack blood. This could potentially be offset by blending of fecal samples before analysis.
Other tests for fecal occult blood are available, mostly in humans. There are fluorescent-based tests in which ferric oxalate or oxalic acid converts the heme group to protophoryin, which is naturally fluorescent. There are also anti-globin antibody-based tests, which detect the globin chain in heme. However, species-specific antibodies must be used unless there is proven cross-reactivity with the animal hemoglobin in question. There is such a globin-based test available for horses, which also detects albumin in feces. The theory behind this combined equine anti-albumin and anti-globin test is that a positive reaction for albumin and globin would support protein and blood loss (e.g. ulcers) in the distal colon (albumin remains undigested) whereas a negative result for albumin and positive test for globin would indicate upper gastrointestinal hemorrhage, because albumin would be digested as it transits through the gastrointestinal tract. However, these globin- and albumin-based tests may be less sensitive than the highly sensitive guaiac tests since globins are broken down easier than heme.