FECALYSIS 9000 mL / day

Fluid regulation
ingested fluid, saliva, gastric, liver, pancreatic,
and intestinal secretions
Routine fecal examination 500 to 1500 mL reaches the large intestine
Large intestine = capable of absorbing
1. macroscopic approximately 3000 mL of water
2. microscopic DIARRHEA =When exceeded, its excreted as feces
3. chemical analyses CONSTIPATION = provides time for additional
• early detection of gastrointestinal (GI) water to be reabsorbed, result to small hard stool
bleeding 150 mL excreted in the feces
• liver and biliary duct disorders Water and readily absorbed in both the small and large
• maldigestion/malabsorption syndromes electrolytes intestines

• pancreatic diseases *fecal electrolyte content is similar to that of

plasma
• inflammation
• causes of diarrhea and steatorrhea

PHYSIOLOGY

➢ Normal fecal specimen contains:

• bacteria
• cellulose
• undigested foodstuffs
• GI secretions
• bile pigments
• cells from the intestinal walls
• electrolyte
• water
➢ 100 to 200 g = 24hrs DIARRHEA AND STEATORRHEA
➢ Strong odor = due to bacterial metabolism (associated
with feces and intestinal gas (flatus) Diarrhea
➢ Oligosaccharides (Carbohydrates) = resistant to digestion
→ increase in daily stool weight above 200 g, increased
in upper GI: metabolized by bacteria in the lower GI
liquidity of stools, and frequency of more than three
producing large amounts of flatus
times per day.
➢ alimentary tract = digestion of ingested proteins,
→ Diarrhea classification factors:
carbohydrates, and fats
1. Illness duration
➢ small intestine = primary site for the final breakdown and
Acute = <4 weeks
reabsorption
Chronic = >4 weeks
➢ Digestive enzymes (secreted by pancreas into small
2. Mechanism
intestine)
• trypsin, chymotrypsin, amino peptidase, and Secretory
lipase • increased secretion of water
➢ Bile salts = provided by the liver aid in the digestion of fats • Causes:
✓ Bacterial, viral, and protozoan infections
➢ Excess undigested or unabsorbed materials then appear in ✓ Enterotoxin-producing organisms stimulate secretion
the feces, and the patient exhibits symptoms of drugs, stimulant laxatives, hormones, inflammatory bowel disease
endocrine disorder, neoplasms, and collagen vascular disease
maldigestion and malabsorption
Osmotic
• poor absorption that exerts osmotic pressure across the intestinal
mucosa

• Mechanism:
1. Incomplete breakdown or reabsorption
2. increased fecal material to the large intestine
3. water and electrolyte retention
4. excessive watery stool
 Differential Features for Diarrhea
• Causes: Laboratory Test Osmotic Diarrhea Secretory Diarrhea
✓ disaccharidase deficiency Osmotic gap >50 Osm/kg <50 Osm/kg
✓ malabsorption Stool Na <60 mmol/L >90 mmol/L
✓ poorly absorbed sugars Stool output in 24 <200 g >200 g
✓ laxatives hours
✓ magnesium-containing antacids pH <5.6 >5.6
✓ amebiasis (malabsorption of sugar)
✓ antibiotic administration Reducing substances Positive Negative

Maldigestion = impaired food digestion

Malabsorption = impaired nutrient absorption by the intestine Laboratory test to differentiate:
✓ fecal electrolytes
Intestinal hypermotility Na = 30 mmol/L
• enhanced motility (hypermotility) or slow motility (constipation) K = 75 mmol/L
• excessive movement of intestinal contents through the GI tract Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)]
• normal absorption of intestinal contents and nutrients cannot occur
✓ fecal osmolality = 290 mOsm/kg
• seen in: irritable bowel syndrome (IBS) *Process specimens for
-extra sensitive nerves and muscles of bowel osmolality testing
-results to cramping, bloating, flatus, diarrhea, and constipation immediately*
• Cause: (Main)
✓ stool pH
✓ gastrectomy, gastric bypass surgery, post vagotomy status,
Zollinger-Ellison syndrome, duodenal ulcer disease, and diabetes 3. Severity
mellitus 4. Stool characteristics
• Cause: (other)
✓ Enteritis
✓ use of parasympathetic drugs
✓ complications of malabsorption
Steatorrhea (fecal fat)
Rapid gastric emptying (RGE) dumping syndrome
- shortened gastric emptying half-time (normal = 35 to 100 → useful in diagnosing pancreatic insufficiency and
minutes) (usually controlled by fundic tone, duodenal
feedback, and GI hormones) small-bowel disorders
- causes the small intestine to fill too quickly with undigested → Causes:
food from the stomach
✓ Absence of bile salts that assist pancreatic lipase ->
- hallmark of early dumping syndrome (EDS)
- Hypoglycemia is often a complication
increase in stool fat (>6 g per day)
1. early dumping (EDS) ✓ pancreatic disorders (cystic fibrosis, chronic
- symptoms begin 10 to 30 minutes following meal pancreatitis, and carcinoma) that decrease the
- nausea, vomiting, bloating, cramping, diarrhea, dizziness,
and fatigue production of pancreatic enzyme
2. late dumping → present in both maldigestion and malabsorption
- 2 to 3 hours after a meal
→ D-xylose test
- weakness, sweating, and dizziness
o Distinguish maldigestion and malabsorption
o sugar that does not need to be digested but
Common Fecal Tests for Diarrhea
Secretory Osmotic
does need to be absorbed to be present in the
• Stool cultures • Microscopic fecal fats urine
• Ova and parasite examinations • Muscle fiber detection o Low = malabsorption
• Rotavirus immunoassay • Qualitative fecal fats
• Fecal leukocytes • Trypsin screening o Normal = pancreatitis
• Microscopic fecal fats
• Muscle fiber detection SPECIMEN COLLECTION
• Quantitative fecal fats
• Clinitest
• D-xylose tolerance test → Random specimens suitable for qualitative testing
• Lactose tolerance test for blood and microscopic examination for
• Fecal electrolytes
leukocytes, muscle fibers, and fecal fats in plastic or
• Stool pH
• Fecal osmolality glass containers with screw-tops
→ quantitative testing, such as for fecal fats, timed
specimens are required.
→ most representative sample is a 3-day collection
MACROSCOPIC SCREENING MICROSCOPIC EXAMINATION OF FECE S

1. Fecal Leukocytes
2. Muscle fibers
3. Qualitative fecal fats

FECAL LEUKOCYTES

Neutrophils
→ Seen in n conditions that affect the intestinal
mucosa, such as ulcerative colitis and bacterial
dysentery
→ Microscopic screening “diarrhea is being caused
by invasive bacterial pathogens?”
→ invasive bacterial pathogens
✓ Salmonella, Shigella, Campylobacter,
Yersinia, and enteroinvasive E. coli
Color
→ toxin production of pathogen = do not
→ brown color = results from intestinal oxidation of cause the appearance of fecal leukocytes
stercobilinogen to urobilin ✓ Staphylococcus aureus and Vibrio
→ Pale (acholic stools) = blockage of the bile duct / spp., viruses, and parasites
barium sulfate
→ Blood = primary concern
✓ black, tarry stool = bleeding from upper GI
(degradation of hemoglobin)
✓ red stool = bleeding from lower GI
→ Green stools = oral antibiotics (oxidation of fecal
bilirubin to biliverdin)

Appearance

→ Pale stools = bulky and frothy, foul odor, and may Examination
appear greasy and may float
1. Wet preparations stained with methylene blue
→ mucus-coated stools = intestinal inflammation or
(fast but difficult to interpret)
irritation, may be caused by pathologic colitis, Crohn
disease, colon tumors, or excessive straining during
2. Dried smears stained with wright’s or gram stain
elimination
(provide permanent slides)
→ Blood-streaked mucus = damage to the intestinal
walls, possibly caused by bacterial or amebic Methylene Blue Stain for Fecal Leukocytes
dysentery or malignancy
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.

Result:
→ >3 neutrophils /HPF = invasive condition
→ Any neutrophil / Oil immersion = 70% sensitivity for
the presence of invasive bacteria
 3. lactoferrin latex agglutination test - detecting fecal
Falsely decrease = Breakdown of neutral fats by bacterial lipase and
leukocytes and remains sensitive in refrigerated spontaneous hydrolysis
and frozen specimens.
* comparison of the two slide tests to determine whether maldigestion or
malabsorption is causing steatorrhea*
Result (+) lactoferrin = invasive bacterial pathogen.

MUSCLE FIBERS Split Fat Stain - Soaps and fatty acids do not stain directly with Sudan III

Undigested striated muscle fiber 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.
→ Monitoring patients with pancreatic insufficiency,
4. Heat gently almost to boiling.
such as in cases of cystic fibrosis 5. Examine under high power.
→ Also seen in: biliary obstruction and gastrocolic 6. Count and measure the orange droplets per high power field.
Consider NUMBER and SIZE
fistulas. * Represent the free fatty acids, fatty acids produced by
hydrolysis of the soaps and the neutral fats*
Muscle Fibers
1. instructed to include red meat in their diet before collecting Result:
2. Emulsify a small amount of stool in two drops of 10% alcoholic Normal = < 4 µm in diameter 100 small droplets/HPF
eosin (enhances the muscle fiber striations) Slightly increase = 1 to 8 µm
3. Apply cover slip and let stand 3 minutes. Increased = 6 to 75 µm (common in STEATORRHEA)
4. Examine under high power for exactly 5 minutes.
5. Count the number of red-stained fibers with well-preserved * Cholesterol is stained by Sudan III after heating and as the specimen
striations UNDIGESTED FIBERS. cools forms crystals that can be identified microscopically
6. examined within 24 hours
Result: >10 = increased

Undigested = striations both vertically and horizontally

MALABSORPTION
Partially digested = striation in only one direction = Neutral fat stain (normal) & Increased Split fat stain
Digested = no visible striations

QUALITATIVE FECAL FATS MALDIGESTION

= Increased Neutral fat stain
Quantitative = additional unstained phospholipids and
cholesterol esters are measured

LIPIDS in feces

1. Neutral fats (triglycerides) - Neutral fat stain

2. Fatty acid salts (soaps) - Split fat stain
3. Fatty acids - Split fat stain
4. Cholesterol

Observed microscopically by:

→ staining with the dyes Sudan III, Sudan IV, or oil red O
→ Sudan III is the most routinely used
→ Two parts of staining procedure:
1. Neutral fat stain
2. Split fat stain - representing total fat content
(better indication)

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 often located near
the edge.

Result: >60 droplets/HPF = steatorrhea


CHEMICAL TESTING OF FECES
OCCULT BLOOD
→ > 2.5 mL/150 g of stool is considered pathologically
significant
→ no visible signs of bleeding may be present with this
amount of blood, fecal occult blood testing (FOBT) is
necessary
→ Annual testing for occult blood - high positive
predictive value for detecting colorectal cancer
→ Methods:
1. Guaiac-Based Fecal Occult Blood Tests (gFOBT)
2. Immunochemical Fecal Occult Blood Test (iFOBT)
3. Porphyrin-Based Fecal Occult Blood Test
GUAIAC-BASED FECAL OCCULT BLOOD TESTS
(GFOBT)
→ most frequently used screening test
→ Principle: detecting the pseudoperoxidase activity of
hemoglobin reacting with hydrogen peroxide to
oxidize a colorless compound to a colored compound
→ least sensitive reagent = guaiac
Why Guiac?
• normal stool can contain up to 2.5 mL of blood, a less
sensitive chemical reactant is understandably more
desirable
• to prevent false-positive reactions. Because
pseudoperoxidase activity is present from hemoglobin
and myoglobin in ingested meat and fish, certain
vegetables and fruits, and some intestinal bacteria.
→ commercial testing kits contains:
• 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, and positive and
negative controls
→ specimens collected on 3 consecutive days
gFOBT
1. sample is placed on the front side of the slide with an applicator
stick and the slide is closed
2. Adding hydrogen peroxide to the back of the filter paper
3. slide that contains stool produces a blue color with guaiac
reagent when pseudoperoxidase activity is present.
Prevent false positive:
→ obtain sample from the center of the stool (external
contamination)
→ specimens mailed to the laboratory should not be rehydrated
before adding the hydrogen peroxide
→ tested within 6 days of collection
→ avoid eating red meats, horseradish, melons, raw broccoli,
cauliflower, radishes, and turnips for 3 days before
→ Aspirin and NSAIDs other than acetaminophen should not be
taken for 7 days before
→ Menstrual and hemorrhoid contamination
Prevent false negative:
→ Vitamin C = >250mg/d (strong reducing agent)
→ iron supplements containing vitamin C 3 days before
→ Failure to wait specified time after sample is applied to add the
developer reagent
Notes:
→ Two samples from three different stools should be tested
before a negative result is confirmed.
IMMUNOCHEMICAL FECAL OCCULT BLOOD TEST
(IFOBT)
→ specific for the globin portion of human hemoglobin
and uses polyclonal anti-human hemoglobin
antibodies.
→ specific for human blood in feces
→ not require dietary or drug restrictions.
→ more sensitive to lower GI bleeding that could be an
indicator of colon cancer or other GI disease
PORPHYRIN-BASED FECAL OCCULT BLOOD TEST
→ porphyrin-based FOBT fluorometric test for
hemoglobin based on the conversion of heme to
fluorescent porphyrins.
→ measures both intact hemoglobin and the
hemoglobin that has been converted to porphyrins.
→ more sensitive to upper GI bleeding
→ not affected by the presence of reducing or oxidizing
substances or the water content
→ False positive - non-human sources of blood (red
meat) are present
* instructed to avoid red meat for 3 days before the
test*
QUANTITATIVE FECAL FAT TESTING
→ confirmatory test for steatorrhea
→ Specimen information:
• requires the collection of at least a 3-day
specimen
• regulated intake of fat (100 g/d) before and
during
• large, pre-weighed container
 • Before analysis, the specimen is weighed 4. Place the acid-homogenate mixture in a 75-µL plain hematocrit
capillary tube. Seal the end with wax.
and homogenized. 5. Centrifuge the capillary tube horizontally at 13,000 rpm for 15
• Refrigerating the specimen prevents any minutes in a microhematocrit centrifuge. This separates fat as
an upper layer overlying a solid fecal layer
bacterial degradation 6. Measure the length of the fat and solid layers using a
magnifying lens.
Methods: 7. Calculate the acid steatocrit in percent.
8. Calculate the fecal fat in grams per 24 hours
→ Van de Kamer titration
• gold standard The acid steatocrit in percent = (fatty layer length in cm)/[(fatty layer
length in cm) + (solid layer length)] × 100
• method routinely used for fecal fat
measurement Results:
• fecal lipids are converted to fatty acids and • <31% = considered normal
• >31% = indicates steatorrhea in adults.
titrated to a neutral endpoint with sodium • higher in infants and lowers with age
hydroxide • <10% indicates steatorrhea in children
• 80% of the total fat content is measured Adult
→ Gravimetric method Fecal fat in grams per 24 hours = [0.45 × (acid steatocrit in percent as a
whole number)] – 0.43
• measures all fecal fat
Children <15years
Fecal fat in grams per 24 hours = [0.1939 × (acid steatocrit in percent as a
whole number)] – 0.2174
→ hydrogen nuclear magnetic resonance spectroscopy
(1H NMR)
• A rapid (5 minutes) and safe procedure for
→ Near-infrared reflectance spectroscopy (NIRS)
analyzing quantitative fecal fat
• rapid procedure for fecal fat that requires
• homogenized specimen is microwaved-dried
less stool handling by laboratory personnel
and analyzed
• requires a 48- to 72-hour stool collection
• results correlate well with the gravimetric
• not require reagents after homogenization
method
• based on the measurement and computed
→ Reporting:
processing of signal data from reflectance
• fat content is reported as grams of fat or the
of fecal surface
coefficient of fat retention per 24 hours.
• infrared light between 1400 nM and 2600
• Reference value: (100 g/d intake)
nM
1 to 6 g/d or coefficient of fat retention of at
least 95% Tests, Materials, and Instrumentation for Fecal Fat Analysis
Procedure Materials, Instrumentation
Sudan III Sudan stain, microscopy
Steatocrit and acid Hematocrit centrifuge, gravimetric
steatocrit assay
Fecal elastase–I Immunoassay ELISA technique
Near-infrared reflectance NIRS spectrophotometer with
→ Acid steatocrit spectroscopy (NIRS) specialized computer software
• rapid test to estimate the amount of fat Van de Kamer titration Fecal fat extraction and titration of
long chain fatty acid by sodium
excretion
hydroxide
• a reliable tool to monitor a patient’s Nuclear magnetic Microwaved-dried specimen;
response to therapy and screen for resonance spectroscopy hydrogen nuclear magnetic
steatorrhea in pediatric populations spectrophotometer

Acid Steatocrit
1. Dilute 0.5 g of feces from a spot collection 1 to 4 with deionized
water.
2. Vortex for 2 minutes to homogenize the specimen.
3. Add a volume of 5 N perchloric acid equal to 20% of the
homogenate and then vortex the mixture for 30 seconds.
Confirm the pH to be <1.
APT TEST (FETAL HEMOGLOBIN) → strongly resistant to degradation
→ measured by immunoassay using the ELISA kit = very
→ Grossly bloody stools and vomitus are sometimes sensitive indicator of exocrine pancreatic
seen in neonates as the result of swallowing maternal insufficiency
blood during delivery → easy to perform and requires only a single stool
→ distinguish between the presence of fetal blood or sample
maternal blood in an infant’s stool or vomitus
→ Distinguish between maternal hemoglobins AS, CS, CARBOHYDRATES
and SS and HbF
→ increased carbohydrates = osmotic diarrhea
APT Test → carbs in feces is result of intestinal inability to
1. Emulsify specimen in water (release reabsorb carbohydrates, as is seen in celiac disease,
hemoglobin) or lack of digestive enzymes such as lactase resulting
2. Centrifuge.
in lactose intolerance
3. Divide pink supernatant into two tubes.
4. Add 1% sodium hydroxide to one tube. → Idiopathic lactase deficiency – common
5. Wait 2 minutes. → serum and urine tests
6. Compare color with that in the control tube. • Carbohydrate malabsorption or intolerance
7. Prepare controls using cord blood and adult (maldigestion) is primarily analyzed
blood.
→ copper reduction test
Result:
Pink = alkali-resistant fetal hemoglobin • increased concentration of carbohydrate can be
yellow-brown = denaturation of the maternal detected
hemoglobin • use clinitest tablet and one part stool emulsified
in two parts water
• 0.5 g/dL = carbohydrate intolerance
FECAL ENZYMES → Fecal carbohydrate testing
• most valuable in assessing cases of infant
→ Enzymes supplied to the gastrointestinal tract by the diarrhea and may be accompanied by a pH
pancreas are essential determination
→ Decrease production • Normal = 7-8
• Associated to chronic pancreatitis and cystic • Increase use of carbs -> increase lactic acid =
fibrosis below 5.5
• Steatorrhea occurs, and undigested food
appears in the feces
→ Primary enzymes:
• proteolytic enzymes trypsin, chymotrypsin,
and elastase I

Fecal chymotrypsin

→ resistant to intestinal degradation

→ sensitive indicator of less severe cases of pancreatic
insufficiency
→ stable in fecal specimens for up to 10 days at RT
→ capable of gelatin hydrolysis
→ measured by spectrophotometric methods

Elastase I

→ isoenzyme of the enzyme elastase

→ produced by the pancreas (pancreas specific enzyme)
(5x higher than in pancreatic juice)
→ high concentrations in pancreatic secretions (6%)