Comprehensive guide to Stool specimen Analysis

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stool specimen analysis

In the minds of the majority of laboratory personnel, stool specimens analysis is a “necessary evil.” Feces, on the other hand, as a byproduct of body metabolism, can provide useful diagnostic information. Routine stool examination includes macroscopic, microscopic, and chemical analyses to detect GI bleeding, liver and biliary duct disorders, maldigestion/malabsorption syndromes, pancreatic diseases, inflammation, and causes of diarrhea and steatorrhea early. The detection and identification of pathogenic bacteria, viruses, and parasites is also of diagnostic value; however, we will discuss these procedures later.

Physiology of Stool specimen

Bacteria, cellulose, undigested foodstuffs, GI secretions, bile pigments, intestinal wall cells, electrolytes, and water are all present in a normal stool specimen. In a 24-hour period, approximately 100 to 200 g of feces is excreted. The normal flora of the intestines is made up of many different types of bacteria. The strong odor associated with feces and intestinal gas is produced by bacterial metabolism (flatus). Carbohydrates that are resistant to digestion, particularly oligosaccharides, pass through the upper intestine unchanged but are metabolized by bacteria in the lower intestine, resulting in large amounts of flatus. When lactose from milk or lactose-containing substances is metabolized by intestinal bacteria in lactose intolerant people, excessive gas production occurs.

Although the digestive tract digests ingested proteins, carbohydrates, and fats, the small intestine is the primary site for the final breakdown and reabsorption of these compounds. Trypsin, chymotrypsin, amino peptidase, and lipase are digestive enzymes secreted into the small intestine by the pancreas. Bile salts produced by the liver aid in fat digestion. A lack of any of these substances results in an inability to digest and, as a result, reabsorb certain foods. Excess undigested or unreabsorbed materials then appear in the feces, and the patient exhibits maldigestion and malabsorption symptoms. Each day, approximately 9000 mL of ingested fluid, saliva, gastric, liver, pancreatic, and intestinal secretions enter the digestive tract, as shown in the image below. Only 500 to 1500 mL of this fluid reaches the large intestine under normal conditions, and only about 150 mL is excreted in the feces. Water and electrolytes are easily absorbed in both the small and large intestines, resulting in fecal electrolyte content comparable to plasma.

Fluid regulation in the gastrointestinal tract.
Fluid regulation in the gastrointestinal tract.

The large intestine can absorb approximately 3000 mL of water per day. When the amount of water that reaches the large intestine exceeds this limit, it is excreted along with the solid fecal material, resulting in diarrhea. Constipation, on the other hand, allows for more water to be reabsorbed from the fecal material, resulting in small, hard stools.

Diarrhea and Steatorrhea


Diarrhea is defined as a daily stool weight increase of more than 200 g, increased stool liquidity, and frequency of more than three times per day. Diarrhea can be classified using four criteria: illness duration, mechanism, severity, and stool characteristics. Acute diarrhea is defined as diarrhea that lasts less than four weeks, and chronic diarrhea is defined as diarrhea that lasts more than four weeks.

Secretory, osmotic, and intestinal hypermotility are the three main mechanisms of diarrhea. Fecal electrolytes (fecal sodium, fecal potassium), fecal osmolality, and stool pH are laboratory tests used to differentiate these mechanisms. Normal fecal sodium is 30 mmol/L and normal fecal potassium is 75 mmol/L, which is close to serum osmolality (290 mOsm/kg). The fecal osmotic gap is calculated using the fecal sodium and potassium results. The following formula is used to calculate the fecal osmotic gap:

Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)]

The osmotic gap is greater than 50 mOsm/kg in all forms of osmotic diarrhea and less than 50 mOsm/kg in secretory diarrhea. Electrolytes are higher in secretory diarrhea and lower in osmotic diarrhea. A fecal fluid pH of less than 5.6 indicates sugar malabsorption, resulting in osmotic diarrhea.

Secretory Diarrhea

Secretory diarrhea is caused by an increase in water secretion. Bacterial, viral, and protozoan infections cause increased water and electrolyte secretion, which overrides the large intestine’s reabsorptive ability, resulting in secretory diarrhea. These water and electrolyte secretions can be stimulated by enterotoxin-producing organisms such as E. coli, Clostridium, Vibrio cholerae, Salmonella, Shigella, Staphylococcus, Campylobacter, protozoa, and parasites such as Cryptosporidium. Drugs, stimulant laxatives, hormones, inflammatory bowel disease (Crohn disease, ulcerative colitis, lymphocytic colitis, diverticulitis), endocrine disorders (hyperthyroidism, Zollinger-Ellison syndrome, VIPoma), neoplasms, and collagen vascular disease are other causes of secretory diarrhea.

Osmotic Diarrhea

Osmotic diarrhea is caused by inadequate absorption, which causes osmotic pressure to build up across the intestinal mucosa. Incomplete breakdown or reabsorption of food causes increased fecal material to reach the large intestine, resulting in water and electrolyte retention (osmotic diarrhea), which causes excessive watery stool. Osmotic diarrhea is caused by maldigestion (impaired food digestion) and malabsorption (impaired nutrient absorption by the intestine). The presence of an unabsorbable solute raises the osmolality of the stool while decreasing the concentration of electrolytes, resulting in an increased osmotic gap. Disaccharidase deficiency (lactose intolerance), malabsorption (celiac sprue), poorly absorbed sugars (lactose, sorbitol, mannitol), laxatives, magnesium-containing antacids, amebiasis, and antibiotic administration are all causes of osmotic diarrhea. To assist in determining the cause of diarrhea, laboratory testing of feces is frequently performed.

Altered Motility

Altered motility refers to conditions characterized by either increased motility (hypermotility) or decreased motility (slow motility) (constipation). Both can be seen in irritable bowel syndrome (IBS), a functional disorder in which the bowel nerves and muscles are overly sensitive, resulting in cramping, bloating, flatus, diarrhea, and constipation. Food, chemicals, emotional stress, and exercise can all cause IBS.

Intestinal hypermotility is the abnormal movement of intestinal contents through the GI tract, which can result in diarrhea because normal absorption of intestinal contents and nutrients is not possible. It can be caused by enteritis, the use of parasympathetic drugs, or malabsorption complications. Rapid gastric emptying (RGE) dumping syndrome is characterized by hypermotility of the stomach and a shortened gastric emptying half-time, causing the small intestine to fill with undigested food from the stomach too quickly. It is a defining feature of early dumping syndrome (EDS). Gastric emptying half-time in healthy people ranges from 35 to 100 minutes, depending on age and gender. RGE is defined as a gastric emptying time of less than 35 minutes.  RGE can be caused by problems with the gastric reservoir or the transporting function. Changes in the motor functions of the stomach cause a large accumulation of osmotically active solids and liquids to be transported into the small intestine. Fundic tone, duodenal feedback, and GI hormones regulate normal gastric emptying. These are altered following gastric surgery, resulting in clinically significant dumping syndrome in about 10% of patients.

RGE is classified as early or late dumping based on how soon after a meal the symptoms appear. EDS symptoms appear 10 to 30 minutes after consuming a meal. Nausea, vomiting, bloating, cramping, diarrhea, dizziness, and fatigue are some of the symptoms. Late dumping is characterized by weakness, sweating, and dizziness and occurs 2 to 3 hours after a meal.  Hypoglycemia is a common side effect of dumping syndrome. Gastrectomy, gastric bypass surgery, post vagotomy status, Zollinger-Ellison syndrome, duodenal ulcer disease, and diabetes mellitus are the most common causes of dumping syndrome.


Steatorrhea (fecal or stool fat) detection aids in the diagnosis of pancreatic insufficiency and small-bowel disorders that cause malabsorption. The absence of bile salts, which aid pancreatic lipase in the breakdown and subsequent reabsorption of dietary fat (primarily triglycerides), results in an increase in stool fat (steatorrhea) of more than 6 g per day. Pancreatic disorders that reduce pancreatic enzyme production, such as cystic fibrosis, chronic pancreatitis, and carcinoma, are also linked to steatorrhea. Steatorrhea can occur in both maldigestion and malabsorption conditions and can be detected using the D-xylose test. D-Xylose is a sugar that does not need to be digested but must be absorbed in order to be present in urine. Steatorrhea indicates a malabsorption condition if urine D-xylose is low. Bacterial overgrowth, intestinal resection, celiac disease, tropical sprue, lymphoma, Whipple disease, Giardia lamblia infestation, Crohn disease, and intestinal ischemia are all causes of malabsorption. A normal D-xylose test results in pancreatitis.

Stool Specimen Collection

Collecting a fecal specimen, also known as a stool specimen, is a difficult task for patients. Detailed instructions and appropriate containers should be provided depending on the type of test and amount of feces required. Certain tests necessitate dietary restrictions prior to stool specimen collection.

Patients should be told to collect the specimen in a clean container, such as a bedpan or disposable container, and then transfer it to the laboratory container. Patients should be aware that the specimen must not be contaminated with urine or toilet water, as these may contain chemical disinfectants or deodorizers that interfere with chemical testing. Containers containing ova and parasite preservatives must not be used to collect specimens for other tests.

Random blood specimens suitable for qualitative testing and microscopic examination of leukocytes, muscle fibers, and fecal fats are typically collected in plastic or glass containers with screw-top lids similar to those used for urine specimens. In addition, material collected on a physician’s glove and samples applied to filter paper in occult blood testing kits are received.

Timed specimens are required for quantitative testing, such as fecal fats. A 3-day collection is the most representative sample due to the variability of bowel habits and the transit time required for food to pass through the digestive tract. These specimens are frequently collected in large containers to accommodate the specimen quantity and allow for emulsification prior to testing. When opening any stool specimen, take care to slowly release any gas that has accumulated within the container. Patients must be warned not to contaminate the container’s exterior.

Macroscopic Screening of Stool specimen

Changes in the brown color and formed consistency of normal stool are frequently the first indication of GI disturbances. Of course, the appearance of abnormal fecal color can also be caused by the consumption of highly pigmented foods and medications, so this must be distinguished from a possible pathologic cause.


The brown color of the feces is caused by the oxidation of stercobilinogen to urobilin in the intestine. Conjugated bilirubin formed during hemoglobin degradation travels through the bile duct to the small intestine, where intestinal bacteria convert it to urobilinogen and stercobilinogen. As a result, pale stools (acholic stools) may indicate a bile duct blockage. Pale stools are also linked to diagnostic procedures involving barium sulfate.

The presence of blood in a stool specimen is a major source of concern. The color can range from bright red to dark red to black depending on the area of the intestinal tract where the bleeding occurs. Blood from the esophagus, stomach, or duodenum takes about 3 days to appear in the stool; during this time, hemoglobin degradation produces the characteristic black, tarry stool. Similarly, blood from the lower GI tract appears faster and retains its original red color. Because ingestion of iron, charcoal, or bismuth often results in a black stool, and some medications and foods, including beets, result in a red stool, both should be chemically tested for the presence of blood.

Because of the oxidation of fecal bilirubin to biliverdin, patients taking oral antibiotics may have green stools. Increased consumption of green vegetables or food coloring results in green stools.

Appearance of stool

During a macroscopic examination, abnormalities other than color variations may be observed. The table below lists the most common abnormalities seen during a macroscopic examination. Examples include diarrhea’s watery consistency, constipation’s small, hard stools, and slender, ribbon-like stools, which indicate an obstruction in the normal passage of material through the intestine.

Pale stools associated with biliary obstruction and steatorrhea are bulky and frothy, with a foul odor. Stools may appear greasy and float.

The presence of mucus-coated stools indicates that the intestine is inflamed or irritated. Pathologic colitis, Crohn’s disease, colon tumors, or excessive straining during elimination can all result in mucus-coated stools. Blood-streaked mucus indicates damage to the intestinal walls, which could be caused by bacterial or amebic dysentery or cancer. Mucus should be reported if it is present.

Color/ AppearancePossible Cause
BlackUpper GI bleeding, Iron therapy, Charcoal Bismuth (antacids)
RedLower GI bleeding, Beets and food coloring, Rifampin
Pale yellow, white, grayBile-duct obstruction Barium sulfate
GreenBiliverdin/oral antibiotics Green vegetables
Bulky/frothyBile-duct obstruction Pancreatic disorders
Ribbon-likeIntestinal constriction
Mucus- or blood-streaked mucusColitis Dysentery Malignancy Constipation
Macroscopic Stool Characteristics

Microscopic Examination of stool specimen

Microscopic examination of fecal smears is carried out to detect the presence of leukocytes in the presence of microbial diarrhea as well as undigested muscle fibers and fats linked to steatorrhea.

Fecal Leukocytes

In conditions that affect the intestinal mucosa, such as ulcerative colitis and bacterial dysentery, leukocytes, primarily neutrophils, are found in the feces. As a preliminary test, microscopic screening is used to determine whether diarrhea is caused by invasive bacterial pathogens such as Salmonella, Shigella, Campylobacter, Yersinia, and enteroinvasive E. coli. Bacteria that cause diarrhea by producing toxins, such as Staphylococcus aureus and Vibrio spp., viruses, and parasites, do not usually cause the appearance of fecal leukocytes. As a result, the presence or absence of fecal neutrophils can provide diagnostic information to the physician prior to receiving the culture report.

Specimens can be examined as wet methylene blue preparations or as dried smears stained with Wright’s or Gram stain. Methylene blue staining is the quickest method, but it is more difficult to interpret. Dried preparations stained with Wright’s or Gram stains provide permanent slides for analysis. Another advantage of the Gram stain is the ability to distinguish between gram-positive and gram-negative bacteria, which could aid in the initial treatment. Fresh specimens must be used for all slide preparations. In a high-powered examination of preparations, as few as three neutrophils per high-power field can be indicative of an invasive condition. The detection of any neutrophils using oil immersion has approximately 70% sensitivity for the presence of invasive bacteria.

For detecting fecal leukocytes, a lactoferrin latex agglutination test is available that is sensitive in refrigerated and frozen specimens. Lactoferrin, a component of granulocyte secondary granules, indicates the presence of an invasive bacterial pathogen.

Muscle Fibers in stool specimen

Microscopic examination of the stool for undigested striated muscle fibers can aid in the diagnosis and monitoring of patients suffering from pancreatic insufficiency, such as cystic fibrosis. It is frequently ordered in conjunction with fecal fat microscopic examinations. In biliary obstruction and gastrocolic fistulas, the amount of striated fibers may be increased.

Slides for muscle fiber detection are made by emulsifying a small amount of stool in 10% alcoholic eosin, which enhances the striations of the muscle fibers. For exactly 5 minutes, the entire slide is examined, and the number of red-stained fibers with well-preserved striations is counted.

To correctly classify the fibers observed, care must be taken.

Undigested fibers show visible striations that run vertically and horizontally. Striations are only visible in one direction in partially digested fibers, and there are no visible striations in digested fibers. Only undigested fibers are counted, and the presence of more than ten is considered elevated.

Meat fibers present in fecal emulsion specimen using brightfield microscopy examination (×400).

To ensure a representative sample, patients should be instructed to consume red meat before the specimen is collected. Specimens must be examined within 24 hours of being collected.

Qualitative Fecal Fats

Microscopically, specimens from suspected cases of steatorrhea can be screened for the presence of excess fecal fat (steatorrhea). The procedure can also be used to track the progress of patients undergoing treatment for malabsorption disorders. In general, the correlation between the qualitative and quantitative fecal fat procedures is good; however, the quantitative procedure measures additional unstained phospholipids and cholesterol esters. Neutral fats (triglycerides), fatty acid salts (soaps), fatty acids, and cholesterol are all lipids found in feces under the microscope.

Sudan III readily stains neutral fats, which appear as large orange-red droplets near the edge of the cover slip. The presence of more than 60 droplets per high-power field can indicate steatorrhea; however, the split fat stain, which represents total fat content, can provide a better indication. The breakdown of neutral fats by bacterial lipase and spontaneous hydrolysis of neutral fats may reduce the neutral fat count, making comparison of the two slide tests to determine whether maldigestion or malabsorption is causing steatorrhea impossible.

Because soaps and fatty acids do not stain directly with Sudan III, a second slide must be examined after the specimen has been heated and mixed with acetic acid. Examining this slide reveals stained droplets that represent not only free fatty acids, but also fatty acids produced by the hydrolysis of soaps and neutral fats. The number and size of the fat droplets must be considered when examining this split fat slide.

In a high-power field, a normal specimen may contain up to 100 small droplets less than 4 m in diameter. The same number of droplets measuring 1 to 8 m is considered slightly increased, and 100 droplets measuring 6 to 75 m is considered significantly increased and is commonly seen in steatorrhea. Malabsorption is indicated by an increase in total fat on the second slide with normal fat content on the first slide, whereas maldigestion is indicated by an increase in neutral fat on the first slide.

Sudan III stains cholesterol after heating, and as the specimen cools, crystals form that can be identified microscopically.

Chemical Testing of Feces

Occult Blood

The detection of occult blood is by far the most commonly performed fecal analysis (hidden blood). As previously discussed, bleeding in the upper GI tract can result in a black, tarry stool, whereas bleeding in the lower GI tract can result in an overtly bloody stool. However, fecal occult blood testing (FOBT) is required because any bleeding in excess of 2.5 mL/150 g of stool is considered pathologically significant, and no visible signs of bleeding may be present with this amount of blood. The American Cancer Society recommends annual testing for occult blood because it has a high positive predictive value for detecting colorectal cancer in its early stages, especially for people over the age of 50. The guaiac, immunochemical, and fluorometric porphyrin quantification tests are all used to detect fecal occult blood. Immunochemical testing and fecal porphyrin quantification are more sensitive and specific than guaiac-based fecal occult blood tests.

Guaiac-Based Fecal Occult Blood Tests

The guaiac-based test for occult blood (gFOBT), which detects hemoglobin pseudoperoxidase activity, is the most commonly used screening test for fecal blood. The principle is the same as the reagent strip test for urinary blood, but a different indicator chromogen is used. The reaction uses hemoglobin’s pseudoperoxidase activity to oxidize a colorless compound to a colored compound by reacting with hydrogen peroxide.

Meat fibers present in fecal emulsion specimen using brightfield microscopy examination (×400).
Meat fibers present in fecal emulsion specimen using brightfield microscopy examination (×400).

To detect occult blood, several different indicator chromogens have been used. All react chemically in the same way, but their sensitivity varies. Unlike most chemical tests, guaiac, the least sensitive reagent, is preferred for routine testing. Given that a normal stool can contain up to 2.5 mL of blood, a chemical reactant that is less sensitive is understandably preferable. Furthermore, hemoglobin and myoglobin, as well as certain vegetables and fruits and intestinal bacteria, contain pseudoperoxidase activity. To avoid false-positive reactions, test sensitivity must be reduced, which can be done by varying the amount and purity of the guaiac reagent used in the test.

There are numerous commercial testing kits available for occult blood testing with guaiac reagent. The kits include guaiac-impregnated filter paper enclosed in a cardboard slide to which a fecal specimen and hydrogen peroxide are added. Two or three filter paper areas are provided for the application of material taken from various areas of the stool, as well as positive and negative controls. Taking samples from the center of the stool reduces the possibility of false-positive reactions due to external contamination. The patient is asked to collect samples from stool specimens collected three days in a row. With an applicator stick, the sample is placed on the front side of the slide, and the slide is closed. When pseudoperoxidase activity is present, adding hydrogen peroxide to the back of the filter paper slide containing stool results in a blue color with guaiac reagent.

The packaging of guaiac-impregnated filter paper in individually sealed containers has aided colorectal cancer screening by allowing patients to place a specimen on a filter paper slide at home and bring or mail it to the laboratory for testing. Unless specifically instructed by the kit manufacturer, specimens mailed to the laboratory should not be rehydrated before adding the hydrogen peroxide to avoid false-positive reactions. Before testing, specimens applied to paper in the laboratory should be allowed to dry. The specimens must be tested within 6 days of being collected. Before confirming a negative result, two samples from three different stools should be tested. To avoid the presence of dietary pseudoperoxidases in the stool, patients should be instructed to refrain from eating red meat, horseradish, melons, raw broccoli, cauliflower, radishes, and turnips for three days prior to specimen collection. To avoid GI irritation, aspirin and NSAIDs other than acetaminophen should be avoided for 7 days prior to specimen collection. Because ascorbic acid is a strong reducing agent that interferes with the peroxidase reaction, causing a false-negative result, vitamin C and iron supplements containing vitamin C should be avoided for 3 days before collections.

Immunochemical Fecal Occult Blood Test

The immunochemical fecal occult blood test (iFOBT) uses polyclonal anti-human hemoglobin antibodies to detect the globin portion of human hemoglobin. Because this method is specific for human blood in feces, no dietary or drug restrictions are required. It is more sensitive to lower GI bleeding, which could be an indication of colon cancer or another GI disease, and it can be used in patients who are taking aspirin or other anti-inflammatory medications. The iFOBT tests do not detect bleeding from other sources, such as a bleeding ulcer, which reduces the possibility of false-positive results. Hemoglobin from upper GI bleeding is immunochemically nonreactive because it is degraded by bacterial and digestive enzymes before reaching the large intestine. Lower GI bleeding, on the other hand, has little hemoglobin degradation, so the blood is immunochemically active. Collection kits, which are similar to those used for guaiac testing, such as the Hemoccult ICT test (Beckman Coulter Inc., Fullerton, CA), can be given to patients for home collection. The results may be read visually or by an automated photometric instrument, depending on the method used.

Porphyrin-Based Fecal Occult Blood Test

HemoQuant (SmithKline Diagnostics, Sunnyvale, CA) provides a fluorometric hemoglobin test based on the conversion of heme to fluorescent porphyrins. The test detects both intact hemoglobin and hemoglobin converted to porphyrins. Bacterial actions degrade hemoglobin to porphyrin, which the gFOBT cannot detect, making the HemoQuant test more sensitive to upper GI bleeding. The gFOBT can produce false-negative results due to upper GI bleeding. Furthermore, the presence of reducing or oxidizing substances, as well as the water content of the fecal specimen, have no effect on the porphyrin-based test. When non-human sources of blood (red meat) are present, false-positive results can occur with the porphyrin-based test; therefore, patients should be instructed to avoid red meat for 3 days before the test.

Quantitative Fecal Fat Testing

As a confirmatory test for steatorrhea, quantitative fecal fat analysis is used. As previously stated, quantitative fecal analysis necessitates the collection of at least a three-day specimen. Before and during the collection period, the patient must consume a controlled amount of fat (100 g/d). The sample is placed in a large, pre-weighed container. The specimen is weighed and homogenized prior to analysis. The specimen is kept cold to prevent bacterial degradation. The Van de Kamer titration is the most commonly used method for measuring fecal fat, though gravimetric, near-infrared reflectance spectroscopy, and nuclear magnetic resonance spectroscopy methods are also available. Fecal lipids are converted to fatty acids and titrated to a neutral endpoint with sodium hydroxide in the titration method. Titration measures approximately 80% of total fat content, whereas the gravimetric method measures all fecal fat. The titration/gravimetric method is time consuming and employs corrosive and flammable solvents. The hydrogen nuclear magnetic resonance spectroscopy (H NMR) method is a quick (5-minute) and safe method for analyzing quantitative fecal fat. The homogenized specimen is microwaved-dried and analyzed in this method. The results are consistent with the gravimetric method.

The fat content is expressed in terms of grams of fat or the coefficient of fat retention per 24 hours. Reference values based on a 100 g/d intake are 1 to 6 g/d or a fat retention coefficient of at least 95%. The fat retention coefficient is calculated as follows:

 Stool fat retention coefficient
Stool fat retention coefficient

Although the Van de Kamer titration is the gold standard for fecal fat, the acid steatocrit is a quick test for estimating fat excretion. It is similar to the microhematocrit test but less time-consuming than a 72-hour stool collection. In pediatric populations, the acid steatocrit is a reliable tool for monitoring a patient’s response to therapy and screening for steatorrhea.

Near-infrared reflectance spectroscopy (NIRS) is a quick method for detecting fecal fat that requires less stool handling by laboratory personnel. To eliminate day-to-day variability, the test requires a 48- to 72-hour stool collection, but it does not require reagents after homogenization of the sample. The outcome is based on the measurement and computation of signal data from fecal surface reflectance, which is scanned with infrared light between 1400 nM and 2600 nM wavelength. The results are computed using calibration obtained from known samples. The method measures water, fat, and nitrogen in grams per 24 hours.

APT Test (Fetal Hemoglobin)

As a result of swallowing maternal blood during delivery, neonates may have bloody stools and vomitus. The APT test may be requested if it is necessary to distinguish between the presence of fetal blood and maternal blood in an infant’s stool or vomitus.

The material to be tested is emulsified in water to release hemoglobin (Hb), and then 1 percent sodium hydroxide is added to the pink hemoglobin-containing supernatant after centrifugation. When alkali-resistant fetal hemoglobin (HbF) is present, the solution remains pink (HbF), whereas denaturation of maternal hemoglobin (HbA) results in a yellow-brown supernatant after 2 minutes. The APT test distinguishes not only between HbA and HbF, but also between AS, CS, and SS maternal hemoglobins and HbF. Because of the high concentration of HbF, the presence of maternal thalassemia major would result in incorrect results. Stool specimens should be tested as soon as possible. They may appear bloody, but they should not be black and tarry, as this would indicate that the hemoglobin has already denatured.

Fecal Enzymes

The pancreas supplies enzymes to the gastrointestinal tract, which are required for the digestion of dietary proteins, carbohydrates, and fats. Reduced production of these enzymes (pancreatic insufficiency) is linked to diseases like chronic pancreatitis and cystic fibrosis. Steatorrhea develops, and undigested food is found in the feces.

The proteolytic enzymes trypsin, chymotrypsin, and elastase I are the primary targets of feces analysis.

Fecal chymotrypsin is less susceptible to intestinal degradation and is a more sensitive indicator of less severe cases of pancreatic insufficiency. It is also stable in fecal specimens at room temperature for up to 10 days. Chymotrypsin can hydrolyze gelatin, but it is most commonly measured using spectrophotometric methods.

Elastase I is the enzyme form produced by the pancreas and is an isoenzyme of the enzyme elastase. It can be found in high concentrations in pancreatic secretions and is highly resistant to degradation. It accounts for approximately 6% of all pancreatic enzymes secreted. Fecal elastase I is pancreas-specific, with a concentration approximately five times that of pancreatic juice. It is not affected by motility or mucosal defects. Elastase I can be measured using an immunoassay. Elastase I is the enzyme form produced by the pancreas and is an isoenzyme of the enzyme elastase. It can be found in high concentrations in pancreatic secretions and is highly resistant to degradation. It accounts for approximately 6% of all pancreatic enzymes secreted. Fecal elastase I is pancreas-specific, with a concentration approximately five times that of pancreatic juice. It is not affected by motility or mucosal defects. Elastase I can be measured using an immunoassay kit and is a very sensitive indicator of exocrine pancreatic insufficiency. It is simple to carry out and only requires a single stool sample. Because the ELISA test employs monoclonal antibodies against human pancreatic elastase-1, the result is specific for human enzyme and is unaffected by pancreatic enzyme replacement therapy. In patients with steatorrhea, the test is sensitive in distinguishing between pancreatic and nonpancreatic causes.


Idiopathic lactase deficiency is common, with African, Asian, and southern European Greek populations being the most affected. Carbohydrate malabsorption or intolerance (maldigestion) is primarily assessed using serum and urine tests; however, an increased carbohydrate concentration can be detected using a copper reduction test on a fecal specimen.

Testing for fecal reducing substances can detect both congenital disaccharidase deficiencies and enzyme deficiencies caused by nonspecific mucosal injury. Fecal carbohydrate testing, which may be accompanied by a pH determination, is most useful in assessing cases of infant diarrhea. Normal stool pH ranges between 7 and 8; however, increased carbohydrate use by intestinal bacterial fermentation raises the lactic acid level and lowers the pH to less than 5.5 in carbohydrate disorders.

A Clinitest tablet (Siemens Healthcare Diagnostics, Inc., Deerfield, IL) and one part stool emulsified in two parts water are used for the copper reduction test. Carbohydrate intolerance is indicated by a result of 0.5 g/dL. The Clinitest on stools can tell the difference between diarrhea caused by abnormal excretion of reducing sugars and diarrhea caused by viruses and parasites. Because sucrose is not a reducing sugar, it cannot be detected using the Clinitest method. There is a link between a positive Clinitest and inflammatory necrotizing enterocolitis in premature infants. A positive result would be followed by more specific serum carbohydrate tolerance tests, the most common being the D-xylose test for malabsorption and the lactose tolerance test for maldigestion. Stool chromatography is available to identify the malabsorbed carbohydrate, but it is rarely required to diagnose sugar intolerance. Small-bowel biopsy specimens for histologic examination and disaccharidase enzyme activity testing distinguish primary from secondary disaccharidase intolerance.

The table below provides a summary of fecal screening tests.

Examination for neutrophilsMicroscopic count of neutrophils in smear stained with methylene blue, Gram stain, or Wright’s stainThree per high-power field indicates condition affecting intestinal wall
Qualitative fecal fatsMicroscopic examination of direct smear stained with Sudan III.Microscopic examination of smear heated with acetic acid and Sudan III60 large orange-red droplets indicates malabsorption.100 orange-red droplets measuring 6 to 75 µm indicates malabsorption
gFOBTPseudoperoxidase activity of hemoglobin liberates oxygen from hydrogen peroxide to oxidize guaiac reagentBlue color indicates gastrointestinal bleeding
iFOBTUses polyclonal anti-human antibodies specific for the globin portion of human hemoglobinPositive test and control lines indicate GI bleeding
APT testAddition of sodium hydroxide to hemoglobin-containing emulsion determines presence of maternal or fetal bloodPink color indicates presence of fetal blood
TrypsinEmulsified specimen placed on x-ray paper determines ability to digest gelatinInability to digest gelatin indicates lack of trypsin
Elastase 1Immunoassay using an ELISA testSensitive indicator of exocrine pancreatic insufficiency
ClinitestAddition of Clinitest tablet to emulsified stool detects presence of reducing substancesReaction of 0.5 g/dL reducing substances suggests carbohydrate intolerance
summary of stool screening tests.


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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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