Fecal

• Specimen Collection


  1. Stool specimen

  2. Clean container: bedpan or disposable container, and transfer the specimen to the laboratory container

  3. Must not be contaminated with urine or toilet water, which may contain chemical disinfectants or deodorizers that can interfere with chemical testing

  4. Random specimens suitable for qualitative testing for blood and microscopic examination for leukocytes, muscle fibers, and fecal fats are usually collected in plastic or glass containers with screw-tops similar to those used for urine specimens

  5. Material collected on a physician's glove and samples applied to filter paper in occult blood testing kits are also received

  6. For quantitative testing, such as for fecal fats, timed specimens are required

  7. Because of the variability of bowel habits and the transit time required for food to pass through the digestive tract, the most representative sample is a 3-day collection
• Microscopic Examination of Feces

  1. Fecal Leukocytes

  2. Neutrophils, are seen in the feces in conditions that affect the intestinal mucosa, such as ulcerative colitis and bacterial dysentery

  3. As a preliminary test to determine whether diarrhea is being caused by invasive bacterial pathogens including Salmonella, Shigella, Campylobacter, Yersinia, and enteroinvasive E. coli

  4. Bacteria that cause diarrhea by toxin production, such as Staphylococcus aureus and Vibrio spp., viruses, and parasites usually do not cause the appearance of fecal leukocytes

  5. Specimens can be examined as wet preparations stained with methylene blue or as dried smears stained with Wright's or Gram stain

  6. As few as three neutrophils per high-power field can be indicative of an invasive condition

  7. A lactoferrin latex agglutination test is available for detecting fecal leukocytes and remains sensitive in refrigerated and frozen specimens
• Methylene Blue Stain for Fecal Leukocytes

  1. Place mucus or a drop of liquid stool on a slide

  2. Add two drops of Löffler methylene blue

  3. Mix with a wooden applicator stick

  4. Allow to stand for 2 to 3 minutes

  5. Examine for neutrophils under high power

  
• Muscle Fibers


  • Patients should be instructed to include red meat in their diet before collecting the specimen
  • Undigested striated muscle fibers can be helpful in diagnosing and monitoring patients with pancreatic insufficiency, such as in cases of cystic fibrosis
  • May also be seen in biliary obstruction and gastrocolic fistulas
• Slides for muscle fiber detection


  1. Emulsify a small amount of stool in 10% alcoholic eosin, which enhances the muscle fiber striations

  2. Examine the entire slide for exactly 5 minutes

  3. Count the number of red-stained fibers with well-preserved striations

  4. Only undigested fibers are counted, and the presence of more than 10 is reported as increased
• Fecal Fats


  • Specimens from suspected cases of steatorrhea can be screened microscopically for the presence of excess fecal fat (steatorrhea)
  • Neutral fats (triglycerides), fatty acid salts (soaps), fatty acids, and cholesterol
  • Their presence can be observed microscopically by staining with the dyes Sudan III, Sudan IV, or oil red O; Sudan III is the most routinely used
  • Neutral fats are readily stained by Sudan III and appear as large orange-red droplets, often located near the edge of the cover slip
  • Observation of more than 60 droplets/high-power field can indicate steatorrhea
  • Soaps and fatty acids do not stain directly with Sudan III
  • Normal specimens may contain as many as 100 small droplets, less than 4 μm in diameter, per high-power field
  • 100 droplets measuring 6 to 75 μm is increased and commonly seen in steatorrhea
  • Cholesterol is stained by Sudan III after heating and as the specimen cools forms crystals that can be identified microscopically

  
• Neutral Fat Stain


  1. Homogenize one part stool with two parts water

  2. Mix emulsified stool with one drop of 95% ethyl alcohol on slide

  3. Add two drops of saturated Sudan III in 95% ethanol

  4. Mix and apply cover slip

  5. Examine under high power

  6. Count orange droplets per high-power field
• Split Fat Stain


  1. Mix emulsified stool with one drop of 36% acetic acid

  2. Add two drops of saturated Sudan III

  3. Mix and apply cover slip

  4. Heat gently almost to boiling

  5. Examine under high power

  6. Count and measure the orange droplets per high-power field
• Guaiac-Based Fecal Occult Blood Tests

  • The most frequently used screening test for fecal blood is the guaiac-based test for occult blood (gFOBT) based on detecting the pseudoperoxidase activity of hemoglobin
  • To prevent the presence of dietary pseudoperoxidases in the stool, patients should be instructed to avoid eating red meats, horseradish, melons, raw broccoli, cauliflower, radishes, and turnips for 3 days before specimen collection
  • Aspirin and NSAIDs other than acetaminophen should not be taken for 7 days before specimen collection to prevent possible GI irritation
  • Vitamin C and iron supplements containing vitamin C should be avoided for 3 days before collections, because ascorbic acid is a strong reducing agent that interferes with the peroxidase reaction, causing a false-negative result

  
• Commercial testing kits


  • The kits contain guaiac-impregnated filter paper enclosed in a cardboard slide, 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
  • Adding hydrogen peroxide to the back of the filter paper slide that contains stool produces a blue color with guaiac reagent when pseudoperoxidase activity is present
• False-Positive


  • Aspirin and anti-inflammatory medications
  • Red meat
  • Horseradish
  • Raw broccoli, cauliflower, radishes, turnips
  • Melons
  • Menstrual and hemorrhoid contamination
• False-Negative


  • Vitamin C >250 mg/d
  • Iron supplements containing vitamin C
  • Failure to wait specified time after sample is applied to add the developer reagent
• Quantitative Fecal Fat Testing

  • Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea
  • Requires the collection of at least a 3-day specimen
  • The patient must maintain a regulated intake of fat (100 g/d) before and during the collection period
  • The specimen is collected in a large, preweighed container
  • Before analysis, the specimen is weighed and homogenized
  • The method routinely used for fecal fat measurement is the Van de Kamer titration: fecal lipids are converted to fatty acids and titrated to a neutral endpoint with sodium hydroxide

  
• APT Test (Fetal Hemoglobin)

  1. Emulsify specimen in water

  2. Centrifuge

  3. Divide pink supernatant into two tubes

  4. Add 1% sodium hydroxide to one tube

  5. Wait 2 minutes

  6. Compare color with that in the control tube

  7. Prepare controls using cord blood and adult blood

  
• Fecal Enzymes


  • Decreased production of these enzymes (pancreatic insufficiency) is associated with disorders such as chronic pancreatitis and cystic fibrosis
  • Steatorrhea occurs, and undigested food appears in the feces
  • Enzymes trypsin, chymotrypsin, and elastase I
  • Chymotrypsin is most frequently measured by spectrophotometric methods
  • Elastase I can be measured by immunoassay using the ELISA kit
• Carbohydrates


  • The presence of increased carbohydrates in the stool produces osmotic diarrhea from the osmotic pressure of the unabsorbed sugar in the intestine drawing in fluid and electrolytes
  • Seen in celiac disease, or lack of digestive enzymes such as lactase resulting in lactose intolerance
  • Copper reduction test on the fecal specimen
  • Fecal carbohydrate testing is most valuable in assessing cases of infant diarrhea and may be accompanied by a pH determination
  • Normal stool pH is between 7 and 8; however, increased use of carbohydrates by intestinal bacterial fermentation increases the lactic acid level and lowers the pH to below 5.5 in cases of carbohydrate disorders
  • The copper reduction test is performed using a Clinitest tablet and one part stool emulsified in two parts water