AUBF: ANALYSIS OF URINE AND OTHER BODY FLUIDS

LECTURE 9: FECAL ANALYSIS

1st SEMESTER | S.Y. 2024-2025

▪ Early detection of GI bleeding

▪ Liver and biliary duct disorders
▪ Maldigestion/ malabsorption syndromes
▪ Inflammation
▪ Causes of diarrhea
▪ Steatorrhea
▪ Identification of pathogenic bacteria and parasites

NORMAL FECAL COMPOSITION

- ¾ water
- ¼ bacteria cellulose, and other undigested foodstuffs, GI secretions, bile pigments, cells from the
intestinal walls, electrolytes
▪ Final breakdown of ingested proteins, carbohydrates and fats takes place in the small intestine
where they are reabsorbed.
▪ Digestive enzymes (trypsin, chymotrypsin, amino peptidase and lipase) are secreted by the pancreas
into the small intestine.
▪ Bile salts that help in fat digestion are provided by the liver.
▪ ANY DEFICIENCY OF THESE SUBSTANCES will result to maldigestion or malabsorption and the
excess undigested or unabsorbed material will appear in feces.
▪ Carbohydrates especially oligosaccharides, that are resistant to digestion pass through the upper
intestine unchanged but are metabolized by bacteria in the lower intestine, producing large amounts
of flatus.
▪ Excessive gas production also occurs in Lactose Intolerant persons when the intestinal bacteria
metabolize the lactose from consumed milk or lactose containing substances.
▪ Large intestine - capable of absorbing 3,000mL of water
▪ When this is exceeded= DIARRHEA
▪ When fecal material stays longer in the LI, provides time for additional water absorption=
constipation

DIARRHEA
- Increased daily stool weight above 200g
- increased liquidity
- Frequency of more than 3x per day

CABAY, J.B. | BMLS3 | PLT COLLEGE, INC.

Classified based on 4 Factors
1. duration of illness
a) acute
b) chronic
2. mechanism
a) secretory
b) osmotic
c) altered motility
3. Severity
4. Stool characteristics
- Diarrhea -loose stools, occurs when the intestine does not complete absorption of electrolytes
and water from luminal contents.
- This happens when a nonabsorbable, osmotically active substance is ingested ("osmotic
diarrhea")
- when electrolyte absorption is impaired ("secretory diarrhea").

A. DURATION OF ILLNESS
a) acute = < 4 weeks
b) chronic = >4 weeks
B. MECHANISM
a) secretory- caused by enterotoxin-producing bacteria, virus & protozoa which induce increased
secretion of water and electrolytes into the LI overriding its resorptive ability
- Causes: E. coli, Clostridium, V. cholerae, Salmonella, Shigella, Staph., Campylobacter,
protozoa, parasites (cryptosporidium)
b) Osmotic- caused by incomplete breakdown or reabsorption of food which draws excessive water
in the LI
- Causes:
disaccharide deficiency (lactose intolerance) magnesium-containing antacids

malabsorption (celiac sprue) Amebiasis

poorly absorbed sugars (lactose, sorbitol, mannitol) antibiotic

Laxatives

c) Altered motility
- seen in irritable bowel syndrome (IBS)
- Rapid Gastric Emptying (RGE)/ dumping syndrome: hypermotility of stomach
TESTS TO DIFFERENTIATE THE MECHANISMS OF DIARRHEA:
- Fecal electrolytes (Na and K)- used to calculate fecal osmotic gap
- Osmotic gap= 290-12[2(Na + K)]
- Secretory= <5omosm/kg & increased electrolytes
- Osmotic= >50m0sm/kg & negligible electrolytes
- Osmolality = 290 mOsm/kg
- pH=<5.6 malabsorption of sugar=Osmotic

STEATORRHEA
✓ Increased in stool fat that exceeds 6g/day due to absence of bile salts or decreased pancreatic
enzyme (pancreatic DO)
✓ Feces appears pale, greasy, bulky or pasty.
✓ Has very strong odor.
✓ D-xylose test
- low - steatorrhea is due to malabsorption
- normal - indicates pancreatitis

CAUSES OF MALDIGESTION
- Decrease level of pancreatic enzymes in pancreatitis and pancreatic CA
- Decrease bile acid formation as in obstructive jaundice

Causes of malabsorption: Diseases that damage intestinal mucosa as in:

Tropical sprue Intestinal resection G. lamblia infestation

Celiac disease Lymphoma Crohn's disease

Bacterial overgrowth Whipple disease Intestinal ischemia

SPECIMEN COLLECTION AND HANDLING

Methods
- Routine- collected in screw-capped top clean container
- 3-day collection- quantitative test
- Cammidge - scraping from diaper
- Jallife - insertion of thick-walled glass in rectum
- Physician's gloves applied to filter paper (FOBT)
- Specimen must not contaminate with urine or toilet water, may contain chemical disinfectants
PRESERVATION
✓ Physical- refrigeration
✓ Chemical
- Formalin
- 95% ethanol
- Glycerol in NSS, MIF, PVA

PHYSICAL EXAMINATION
✓ Quantity - 100-250 g/day Variation
✓ Color - light to dark brown ▪ Quantity
✓ Consistency - soft to well- formed - Increase CHO - increase output
✓ Odor - foul to offensive - Increase meat - decrease output
✓ pH - 7.0 to 8.0 ▪ Color variation -

APPEARANCE/ CONSISTENCY VARIATION

✓ Watery:
- pea-soup - typhoid
- fever rice-water - cholera
✓ Small, hard/ goat droppings/ scybalous - constipation
✓ Slender/ flattened, ribbon-like= intestinal constriction
✓ Bulky, frothy, greasy and may float = biliary obstruction and steatorrhea
✓ Mucus- coated stools= intestinal inflammation or irritation, pathologic colitis, excessive straining
during defecation
✓ Blood- streaked mucus = damage to intestinal walls, bacterial or amebic dysentery or malignancy
✓ Small caliber - Hirschsprung's disease (massive enlargement of intestine)

ODOR
✓ Due to SKATOLE, INDOLE, BUTYRIC ACID
✓ PUTRID - ulcerated and malignant tumors of the lower bowel
✓ EXTREMELY FOUL- putrefaction due to undigested protein
✓ SOUR/RANCID- gas formation & fermentation of CHOs, due to unabsorbed fatty acids

pH
✓ Acidic feces -CHO fermentation
✓ Alkaline feces - CHON fermentation
MICROSCOPIC EXAMINATION
✓ Fecal leukocytes - primarily Neutrophils
✓ (+) Neutrophils = if diarrhea is caused by INVASIVE BACTERIA like Salmonella, Shigella,
Campylobacter, Yersinia, enter invasive E. coli
✓ (-) Neutrophils = if diarrhea is caused by TOXIN-PRODUCING BACTERIA such as S. aureus, Vibrio
spp, Viruses and Parasites

METHODS
▪ WET PREPARATION
- Stained with methylene blue
- Faster but more difficult to interpret
▪ DRIED PREPARATION
- Stained with Wright's or Gram's stain
- Provide permanent slides for evaluation
- Additional advantage of observation of Gram positive and Gram-negative bacteria which may aid
in the choice of initial treatment
- All slide preparations must be performed on fresh specimens.
- RESULT:
o As few as 3 neutrophils/PF can be indicative of an invasive condition
o Finding of any neutrophil in OIF has 70% sensitivity

LACTOFERRIN LATEX AGGLUTINATION TEST

- Detection of fecal leukocytes remains sensitive in refrigerated and frozen specimens
- Lactoferrin is a component of granulocyte secondary granules, its presences are indicative of
BACTERIAL PATHOGEN

MUSCLE FIBERS
- HELPFUL in the diagnosis and monitoring of patients with PANCREATIC INSUFICIENCY
- Increased amount is also seen in BILIARY OBSTRUCTION and GASTROCOLIC FISTULAS
- Ordered in conjunction with microscopic exam for fecal fats

METHODS
✓ Slides are prepared by emulsifying small amount of stool in 10% ALCOHOLIC EOSIN (enhances
muscle fiber striations)
✓ Entire slides must be examined within 5 MINUTES
✓ RED- STAINED FIBERS with well-preserved striations are counted
- UNDIGESTED FIBERS - Have visible striations running both vertically and horizontally.
- PARTIALLY DIGESTED - have striations in only one direction
- DIGESTED FIBERS- have no visible striations
✓ ONLY UNDIGESTED FIBERS are counted
✓ Presence of >10 is reported as increased
✓ Patient should include red meat in the diet prior to the specimen collection.
✓ Specimens should be examined within 24 hours of collection

QUALITATIVE FECAL FATS

✓ TEST for EXCESS FECAL FATS in cases of
✓ MONITOR PATIENTS UNDERGOING TREATMENT
✓ FOR MALABSORPTION DISORDER
✓ FAT STAINING with the following dyes:
✓ SUDAN III
✓ SUDAN IV
✓ OIL RED O

LIPIDS OBSERVED IN FECES

▪ NEUTRAL FATS - TRIGLYCERIDES
- stained by Sudan III - LARGE ORANGE RED DROPLETS seen at the edge of the cover slip
- >60 droplets/PF = STEATORRHEA
▪ FATTY ACID SALT (SOAPS) AND FATTY ACIDS
- Do not stain directly with Sudan IlI
- A second slide must be examined after the specimen has been mixed with acetic acid and
heated
- Stained droplets represent the free fatty acids and fatty acids liberated from the soaps and
neutral fats
- Both number and size of FAT DROPLETS are noted
- Normal - 100 small fat droplets, <4um in size/HPF
- Slightly increased- 100 droplets, 1-8um/HPF
- Increased - 100 droplets, 6-75um/HPF
▪ CHOLESTEROL- stained by Sudan III after heating and as specimen cools forms crystals that can be
identified microscopically

CHEMICAL EXAMINATION
- OCCULT (HIDDEN) BLOOD or FECAL OCCULT BLOOD TESTING (FOBT)
- MOST FREQUENTLY PERFORMED chemical screening test
- Necessary bec. any bleeding in excess of 2.5ml/150g of stool is PATHOLOGICALLY SIGNIFICANT
and no visible signs of bleeding may be present
- Melena: Large amount of blood (50-100 ml/day) turning stool black or tarry
- FOBT
o Used as mass screening procedure for the early detection of colorectal cancer
 o Recommended by American Cancer Society ages >50 y/o
o PRINCIPLE: PSEUDOPEROXIDASE ACTIVITY OF HEMOGLOBIN reacting with H202 to oxidize a
colorless compound to a colored compound
- INDICATOR CHROMOGENS
✓ BENZIDINE - most sensitive
✓ ORTHO-TOLUIDINE
✓ GUM GUAIAC - least sensitive

GUM GUAIAC - preferred for routine testing

✓ a less sensitive chemical reactant is desirable because a normal stool can contain upto 2.5ml of
blood
✓ False POSITIVE REACTION due to:
- Hemoglobin and myoglobin in ingested meat and fish
- Certain vegetables and fruits
- Some intestinal bacteria
Prevented by decreasing the sensitivity by varying the amount and purity of guaiac reagent used in the
test

PROCEDURES TO AVOID FALSE POSITIVE REACTION

✓ Obtain sample from the center of the stool
✓ Specimen applied to paper should be allowed to dry prior to testing
✓ Specimen should be tested within 6 days of collection
✓ Specimens mailed to the lab should not be rehydrated prior to adding H202.
✓ 2 samples from 3 different stools should be tested before a negative result is confirmed
✓ Foods and medications to be avoided
- Red meats, horseradish, melons, raw broccoli, cauliflower, radishes and turnips for 3 days prior
to specimen collection - this prevents the presence of dietary pseudo peroxidases in stool.
✓ ASPIRIN AND SAIDS other than acetaminophen should not be taken for 7 days prior to specimen
collection - to prevent possible Gl irritation
✓ VITAMIN C AND IRON SUPPLEMENTS CONTAINING
- VIT C should be avoided for 3 days prior to specimen collection - ascorbic acid is a strong
reducing agent that interferes with the peroxidase reaction.
 Summary of interferences in occult blood testing
FALSE POSITIVE FALSE NEGATIVE
✓ Aspirin and anti-inflammatory drugs ✓ Vitamin C- >250mg/dL
✓ Red meat ✓ Iron supplements containing vit. C
✓ Horseradish
✓ Raw broccoli, cauliflower, radishes, turnips
✓ Melons
✓ Menstrual and hemorrhoid contamination

HEMOQUANT
✓ More sensitive and specific
✓ Provides a fluorometric test for hemoglobin and porphyrin (cannot be detected by guaiac)
✓ As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that
the guaiac test cannot detect
✓ This can result in some false negative results from upper Gl bleeding when using the guaiac test

HEMOCCULT ICT
✓ IMMUNOCHEMICAL FECAL OCCULT BLOOD TEST (IFOBT)
✓ Specific for the globin portion of human hemoglobin and uses anti human hemoglobin antibodies.
✓ Does not require dietary or drug restrictions
✓ Can be used in patients taking in aspirin and NSAIDS
✓ More specific to lower Gl bleeding (indicator of colon CA or other GI diseases)
✓ In upper Gl bleeding, hemoglobin is immunochemically nonreactive due to bacterial and enzymatic
degradation.
✓ In lower GI bleeding, little Hgb degradation, therefore, blood is immunochemically active
✓ Does not detect bleeding from other sources such as bleeding ulcer, thus decreasing the chance for
false positives

QUANTITATIVE FECAL FAT TESTING

✓ Used as confirmatory test for steatorrhea
✓ Requirements:
- Collection of at least a 3-day specimen
- Patient must maintain a regulated intake of FAT (100g/dl) prior to and during the collection
period
- Care in opening container of specimen to slowly release gas that has accumulated within the
PRESERVATION: Refrigerate the specimen to prevent any bacterial degradation

METHODS
▪ VAN DE KAMER TITRATION
- GOLD STANDARD FOR FECAL FAT
- Routinely used for fecal fat measurement
- Fecal lipids are converted to fatty acids and content is reported as grams of fat or the coefficient
of fat retention per 24 hrs.
▪ GRAVIMETRIC METHOD: NORMAL VALUES (based on 1100g/dL intake) or at least 95% coefficient
of fat retention
▪ Other tests for Fecal Fat
o ACID STEATOCRIT
- Rapid test to estimate the amount of fat excretion
- Similar to the microhematocrit test
- More convenient than a 72- hour stool sample
- Reliable tool to monitor a patient's response to therapy and screen for steatorrhea in
pediatric
▪ NEAR-INFRARED REFLECTANCE SPECTROSCOPY
- Rapid procedure for fecal fat that requires less stool handling by lab personnel
- Quantitates water, fat and nitrogen in grams/24 hrs Requires a 48 to 72 hr stool collection to
exclude day to day variability
- Does not require reagents after homogenization of the sample.
- Result is CALCULATED from calibration derived from known samples and is based on the
measurement and computed processing of signal data from reflectance of fecal surface, which is
scanned with infrared light between 1400 nM and 2600 nM wavelength.

FECAL ENZYMES
- Focused on the proteolytic enzymes TRYPSIN, CHYMOTRYPSIN AND ELASTASE I
- Essential for digestion of dietary proteins, CHO and fats.
- Supplied to the GIT by the Pancreas

CLINICAL SIGNIFICANCE
- Pancreatic insufficiency seen in Chronic Pancreatitis and Cystic Fibrosis
- Steatorrhea occurs and presence of undigested food in the feces

TRYPSIN (GELATIN TEST)

- Absence is demonstrated by exposing ray paper to stool emulsified in water
- If trypsin is present - it will digest the gelatin on the ray paper= CLEAR AREA
- If trypsin is absent - inability to digest the gelatin = NO CHANGE
- INSENSITIVE PROCEDURE- detects only severe cases of pancreatic insufficiency

▪ FALSE POSITIVE - proteolytic activity of bacteria on enzymes (old specimen)

▪ FALSE NEGATIVE
- intestinal degradation of trypsin
- possible presence of trypsin inhibitors in the feces

CHYMOTRYPSIN
- MORE SENSITIVE indicator of less severe cases of pancreatic insufficiencies.
- MORE RESISTANT to intestinal degradation
- REMAINS STABLE in fecal specimens for up to 10 days at room temperature
- Capable of gelatin hydrolysis but is most frequently measured by SPECTROPHOTOMETRIC
METHODS

ELASTASE I
- Elastase I isoenzyme is present in high concentrations in pancreatic secretions. It is strongly
resistant to degradation and accounts for about 6% of all secreted pancreatic enzymes.
- Pancreas specific and its concentration is about 5 times higher than in pancreatic juice.
- It is not affected by motility DO or mucosal defects.
- VERY SENSITIVE INDICATOR of exocrine pancreatic insufficiency.
- Easy to perform and requires only a single stool sample.
- Measured by immunoassay using ELISA kit
o Uses monoclonal antibodies against human pancreatic elastase I, therefore, the result is specific
for human enzymes and not affected by pancreatic enzyme replacement therapy.
o The test is SPECIFIC IN DIFFERENTIATING PANCREATIC FROM NON-PANCREATIC CAUSES IN
PATIENTS WITH STEATORRHEA.

CARBOHYDRATES
- Present in feces as a result of intestinal inability to reabsorb CHO (as in CELIAC disease) or caused
by lack of digestive enzymes (such as lactase, which results in lactose intolerances).
- Increase amount in stool produces OSMOTIC DIARRHEA caused by the osmotic pressure of the
unabsorbed sugar in the intestine drawing in fluid

COPPER REDUCTION TEST (CLINITEST)

✓ Detects CONGENITAL DISACCHARIDASE DEFICIENCIES and ENZYME DEFICIENCIES due to
nonspecific mucosal injury.
✓ Most valuable in assessing cases of infant diarrhea and maybe accompanied by a pH determination.
- Normal stool pH: 7.0 to 8.0
 - (+) CHO disorders, pH: <5.5 due to increased use of CHO by intestinal bacterial fermentation
which increase lactic acid levels and lowers the pH.
✓ Performed using a CLINITEST tablet and one part stool emulsified in two parts water
✓ A result of 0.5g/dL is indicative of CHO: POSITIVE CLINITEST IN PREMATURE - has correlation with
INFLAMMATORY NECROTIZING ENTEROCOLITIS
- Can distinguish between diarrhea caused by abnormal excretion of reducing sugars and diarrhea
caused by various viruses and parasites.
- SUCROSE IS NOT DETECTED because it is not a reducing sugar

Serum CHO Tolerance Test STOOL CHROMATOGRAPHY SMALL BOWEL BIOPSY &
DISACCHARIDASE ENZYME
ASSAY
- Used following a positive To identify the malabsorbed Differentiate primary from
fecal clinitest CHO but rarely necessary for secondary disaccharidase
- D-xylose test for the diagnosis of sugar intolerance
malabsorption intolerance
- Lactose Tolerance Test for
maldigestion

APT TEST FOR FETAL HEMOGLOBIN

✓ Distinguishes between the presence of fetal blood or maternal blood in an infant's stool or vomitus
✓ Grossly bloody stools and vomitus are sometimes seen in neonates as a result of swallowing
maternal blood during delivery.
✓ APT test distinguishes not only between fetal hemoglobin and hemoglobin A but also between
maternal hemoglobins AS, CS and SS genotypes, and fetal hemoglobin.
✓ PRINCIPLE
- Material to be tested is emulsified in water (to release hemoglobin) and centrifuged
- 1% SODIUM HYDROXIDE is added to the pink hemoglobin- containing supernatant.
- FETAL HEMOGLOBIN: the solution remains pink because HbF is alkali resistant.
- MATERNAL Hgb: produces a yellow-brown supernatant after standing for 2 minutes
- CONTROLS: cord blood and adult blood