Graff's Textbook of Urinalysis and Body Fluids, Fecal Analysis, Atlas of Fecal Analysis
This chapter describes routine clinical testing of feces and the information this testing gives clinicians. While laboratory workers might prefer testing other specimens, sometimes testing feces is necessary for detection of a medical disorder. Testing feces gives important information related to gastrointestinal disorders and infections and several other medical conditions. Macroscopic, microscopic, and chemical testing of feces is routinely performed in the core laboratory for detection of colon cancer, steatorrhea and other malabsorptive digestive disorders, or maldigestion disorders. Tests are also performed in the microbiology department for various stool pathogens; bacterial, viral, and parasitic, as well as for their toxins.
GASTROINTESTINAL TRACT PHYSIOLOGY AND FECAL FORMATION
The digestive system is composed of the alimentary canal, a continuous tube from the mouth to the anus including the esophagus, the stomach, the intestines, the colon, and the accessory digestive organs, including the mouth, much of the pharynx, the teeth, tongue, salivary glands, liver, gall- bladder, and pancreas. The gastrointestinal (GI) tract contains and processes food from ingestion, through digestion, and elimination.
Food and water ingested becomes chyme in the stomach and small intestines. Enzymes, starting with salivary amylase and including stomach enzymes and many pancreatic enzymes, are added to the food along the pathway. Chyme is a mixture of digestive secretions and partially digested food. After chyme has remained in the large intestine for 3–10 hours, it normally becomes solid or semisolid and is then called feces. Along the way, much of the water, nutrients, vitamins, and electrolytes are adsorbed into the circulatory sys- tem. Despite about 9 L of water entering the small intestine from ingestion and digestive fluids, only about 0.1 L of water is excreted in feces daily, as most is absorbed via osmosis in the small intestines, with only about one tenth of the volume, about 0.9 L, also being reabsorbed in the large intestine.
Because of the limited ability of the colon to absorb water, if a large volume of water is presented to the colon from the intes- tines, a large volume of liquid diarrhea is the result.
DISORDERS OF THE GASTROINTESTINAL TRACT
Diarrhea is a common disorder of the GI tract. In diarrhea the frequency and volume of bowel movements are increased and the bowel movements are more liquid. Diarrhea is associated with infectious agents, toxins, malabsorption, and a variety of GI disturbances. Diarrhea is caused by a large volume of fluid being presented to the large intestine (a) due to increased secretions or (b) due to increased amounts of osmotically active substances remaining in the GI tract or (c) because of increased intestinal movement (hypermotility) resulting in decreased intestinal absorption. Diarrhea can result in dehydration and critical electrolyte imbalances.
Malabsorption is a state of abnormal digestion or absorption of a single nutrient or of multiple nutrients through the GI tract that may lead to malnutrition or anemia. If osmotically active substances are not absorbed it can result in diarrhea. Fats, meat fibers, and carbohydrates all may be improperly digested with malabsorption. Maldigestion is impaired digestion which is caused by lack of digestive enzymes.
Colorectal cancer is a relatively common cancer of the GI tract. In colon cancer, increased blood loss in the GI tract is detected by occult blood testing. The term “occult blood” refers to hidden blood or small amounts of fecal blood that are often not visible to the naked eye. These are some of the conditions that can be detected by fecal analysis. In addition, various diseases of the accessory organs can also be detected through fecal testing. An example of this is cystic fibrosis, a hereditary disease affecting mucous secretion in the pancreas and lungs. In cystic fibrosis or other pancreatic insufficiency, there are decreased pancreatic digestive enzymes such as trypsin, chymotrypsin, and elastase I, resulting in maldigestion. These enzymes can be tested in feces.
FECAL SPECIMEN COLLECTION
The patient needs proper directions on the collection of a stool sample and he or she should be provided with the proper sample collection container. For most fecal specimens, the container simply needs to be clean, dry, sealable, and leakproof. Patients must understand that the fecal sample should not be contaminated with urine or water. Toilet water often contains strong oxidizing cleaners that interfere with laboratory testing, protozoa can be destroyed by urine contamination, and even not adhering to recommended diet recommendations can adversely affect many fecal specimens. If the patient has had enemas or barium sulfate for radiological examinations, these can adversely affect stool samples for microscopy, making them difficult to read.
The type of collection container and the amount of specimen to be collected depend upon the tests to be performed. Special specimen containers are available for fecal occult blood, fecal parasite examinations, and some other fecal exam- inations. Timing of specimen collection is important in some tests, such as for parasite examinations or quantitative testing. If quantitative specimens are required, a 3-day fecal collection is recommended as 24 hours is often insufficient for foods to be processed through the alimentary canal. Use a secure lid for the fecal specimen as gas in the fecal specimen can build up, causing the lid to pop open, possibly with some force.
During many GI tract infections, diarrhea causes an increased number of bowel movements. Determining the time to symptom development and the type of symptoms, such as the frequency of eliminations can assist the physician in determining the causative agent or toxin.
FECAL ANALYSIS METHODS
The laboratory can screen for a variety of fecal characteristics that can aid healthcare providers in diagnosing and monitoring their patients. From simple gross observations to microscopic examinations and chemical determinations, laboratory testing gives clues to the functioning of the GI tract and diseases that may arise in the digestive system.
GROSS EXAMINATION
The gross appearance of the feces provides some clues to possible GI disorders. The consistency, whether formed, hard- ened, or liquid, and color change from the normal dark brown alert the healthcare provider to abnormalities. Black color may indicate older blood from the upper GI tract, whereas bright red blood is more likely to be from the lower GI tract. Bright red blood in feces is known as hematochezia. A very pale stool (called an acholic stool) often indicates a biliary obstruction. Another common reason for a pale stool is the presence of barium sulfate from a barium enema performed for radiological testing. This barium sulfate will interfere with some fecal examinations, especially microscopic examinations for fat, fibers, or parasites. A ribbonlike fecal specimen could be associated with GI tract obstruction. The normal fecal specimen is dark brown; due to the oxidation of urobilinogen in the intestines, the color changes to orange brown urobilin.
The presence of blood-streaked mucus or mucus with pus or eosinophils often accompanies bacterial or amebic dysentery. Dysentery is associated with damage to the intestinal wall due to invasion by these organisms.
MICROSCOPIC EXAMINATION
Fecal leukocytes, especially neutrophils, are commonly asso- ciated with dysentery or invasion of the intestinal wall. In amebic infections, eosinophils are also often present. Wet preparations made with methylene blue are used to detect fecal leukocytes or alternatively, dried smears of the stool sample can be stained with Gram stain or Wright stain to examine for leukocytes. The Wright stain improves differentiation of these cells. The presence of even a few leukocytes can be indicative of an invasive condition.
Another type of test that can be used to detect fecal leukocytes is a latex agglutination test for lactoferrin, an enzyme found in the granules of granulocytes, also indicative of fecal leukocytes (Fig. 13-1).
Figure 13-1. Fecal leukocytes. A. Gram stain, 1000x, fecal leukocytes.
B. Wright stain, 1000x, fecal leukocytes.
C. Wet preparation, Shigella dysentery or bacterial dysentery with fecal
white blood cells, red blood cells, and bacteria.
OCCULT BLOOD
Occult blood means “hidden blood” as it is often present but not visible in the dark stool sample, requiring laboratory test- ing for detection. Fecal blood is found in infection, in trauma, and in colorectal cancer. Because fecal blood is a frequent and an early symptom of colorectal cancer, the American Cancer Society recommends annual screening on all individuals older than 50 years of age. Early diagnosis is associated with a good prognosis and this testing is beneficial in this early diagnosis.
In addition to colorectal cancer, inflammatory conditions, infectious agents, ulcers, hemorrhoids, and even bleeding gums can cause a positive occult blood test. The excretion of large amounts of blood in the upper GI tract may cause the stool to be dark or black in color. A very large amount of fecal blood is called melena and is associated with a black, tarry stool. Lower GI tract bleeds tend to have a brighter red color blood, if visible.
The most common method for detection of fecal blood involves a guaiacimpregnated paper in a cardboard holder. Patients should be instructed to avoid red meat, fish, bananas, cantaloupe, pars, plums, turnips, horseradish,
Figure 13-2. Guiac fecal occult blood test.
broccoli, cauliflower, aspirin, and vitamin C for the days preceding testing as these substances interfere with guaiac testing. The patients must be instructed to collect several portions of the same stool sample to maximize blood detection. A thin layer of these stool portions is applied to the front of the guaiac card as directed. If hemoglobin or another peroxidase or pseudoperoxidase is present in the feces in sufficient amounts, the guaiac paper will develop (usually turns blue) when hydrogen peroxide developer is applied.
H2O2 colorless indicator—(pseudo) peroxidase or hemoglobin -> S colored H2O oxidized indicator
or hemoglobin
Methods other than guaiac are available for detecting fecal blood including benzidine, orthotolidine, and even immunological methods for hemoglobin. A problem with these tests is a higher degree of sensitivity that leads to many false positives. The less sensitive guaiac seems to give the best results under most circumstances (Fig. 13-2).
FETAL HEMOGLOBIN (APT TEST)
Newborns may excrete stools or vomitus containing blood. This blood can originate either from the maternal blood ingested at delivery or from the newborn’s own gas- trointestinal tract. Differentiating between these two sources of blood is important for the newborn’s survival. Black, tarry stool samples are not acceptable for this test as the hemoglobin degradation has taken place. This test determines whether the hemoglobin present is hemoglobin A (maternal) or hemoglobin F (fetal) in origin. The stool or vomitus is mixed with water to yield a pink supernatant. The supernatant is removed and then alkalinized with dilute sodium hydroxide. If the pink color remains after adding the alkali, the blood contains fetal hemoglobin. If the pink color changes to yellow or brown within 2 minutes, the hemoglobin in the sample is maternal hemoglobin.
Figure 13-3. Steatorrhea with fecal fat Sudan III stain for neutral fats.
FECAL FAT TESTING
Conditions that decrease the production of pancreatic enzymes such as cystic fibrosis, pancreatic insufficiency, pancreatitis, and pancreatic carcinoma, as well as the absence of the bile salts that assist lipases in fat breakdown, cause steatorrhea (an increase in fecal fat). Steatorrhea is also present in malabsorption. Malabsorption can be caused by bacterial overgrowth, intestinal resection, celiac disease, tropical sprue, lymphoma, Crohn disease, Whipple disease, and giardiasis. In steatorrhea, the stool sample has a pale and greasy appearance and a foul smell.
Before any fecal fat testing, it is important for the patient to have a diet with a normal amount of fat intake. Mineral oils and many creams can cause false-positive results in fecal fat testing and must be avoided (Figs. 13-3 and 13-4).
Both qualitative and quantitative fecal fat analyses are available.
Figure 13-4. Steatorrhea with fecal fat Sudan III stain for fatty acids.
Qualitative Fecal Fat Testing
Qualitative tests are performed microscopically for triglycerides (neutral fats), fatty acid salts (soaps), fatty acids, and cholesterol. These fats can be stained with Sudan III, Sudan IV, and oil red O. Two procedures are used to detect these different lipids.
Neutral fats are detected qualitatively by staining with Sudan III in 95% ethanol in a wet preparation and microscopic observation for the number and size of fat globules. A normal stool will have less than 60 medium or small-sized orange-red fat globules/high power field. Soaps and fatty acids do not stain directly with the Sudan III stain and the same wet preparation must also have acetic acid and be heated prior to staining and reading microscopically. Normal samples will have less than 100 orange-red fat globules/ high power field and they should not exceed 4 m. A normal amount of neutral fecal fat with an increased amount of soaps and fatty acids is associated with intestinal malabsorption, whereas an increased amount of neutral fats on the first slide is associated with maldigestion or impaired digestion leading to steatorrhea.
Quantitative Fecal Fat Testing
If qualitative fecal fat testing is positive, confirmatory quan- titative fecal fat analysis is performed. Generally, this is performed in the chemistry department and it is often a reference test. For accurate testing, the patient must maintain a
100 g/day fat intake both before and during specimen col- lection. Forty-eight- to seventy-two-hour fecal collections are necessary for these tests. Fecal fat analysis can be performed by the Van de Kramer titration, by the acid steat- ocrit, or by near-infrared spectroscopy.
MEAT FIBERS
Increased undigested food such as meat fibers and veg- etable fibers correlate to maldigestion especially in cystic
Figure 13-5. Creatorrhea. Undigested meat fiber. Note the clearly defined muscle striations.
Figure 13-6. Creatorrhea. Undigested meat fibers tangled.
The clear striations help identify these meat fibers
fibrosis patients and in hypermotility. Increased meat fibers can be recognized microscopically as they are rectangular with the cross striations that are characteristic of muscle fibers. If no striations are seen the fibers are not counted as they may be digested. More than 10 fibers/high power field is considered increased. Meat fiber examination may be done together with microscopic fecal fat analysis. Eosin in 10% ethanol can be used to assist in identifying these meat fibers with striations in a microscopic wet preparation. An increase in the number of meat fibers is creatorrhea (Figs. 13-5 and 13-6).
FECAL CARBOHYDRATES IN MALABSORPTION
Disaccharides are osmotically active and they trigger movement of a large amount of water to the intestines when they are located in the GI tract, resulting in osmotic diarrhea. The presence of carbohydrates in the feces produces increased osmosis with a great increase in f luids and electrolytes resulting in diarrhea. Fecal carbohydrates are present in celiac disease, due to inability to reabsorb carbohydrates, and in the lack of sugar digesting enzymes as in lactose intolerance and in congenital disaccharidase deficiencies. Fecal carbohydrate testing is most useful in infant diarrhea to assess fecal diarrhea and inf lammatory necrotizing enterocolitis. The copper reduction test (generally performed as the Clinitest) is used to detect the significant reducing sugars. If this test is positive in the Clinitest, the infant may be tested by other more specific serum tests for carbohydrate tolerance.
If carbohydrates are not reabsorbed, the pH of the feces decreases from a normal fairly neutral pH of between 7 and 8 to a pH below 5.5.6 Fecal pH is also usually tested using pH paper along with the copper reduction test.
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
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