1ST SEMESTER

ANALYSIS OF URINE AND BODY FLUIDS / laboratory SY 2023-2024 Aubf311

FECAL ANALYSIS
• part of the routine test in clinical microscopy • Split fat procedure
• main purpose: to asses if there is any gastrointestinal bleeding, o 36% acetic acid
liver disorders, malabsorption or maldigestion syndrome o Sudan III
• composition o Applicator sticks
o mainly made up of water – 60 to 80% o Slides and cover slips
o remaining ¼ comprises of solids (composed of remains of o Pea sized stool
food residues, intestinal secretions, bacteria, fat droplets and PRECAUTION BEFORE COLLECTION
other soluble substances)
• Ask the patient if he or she is taking any medication, food
PRE – ANALYTICAL supplements and vitamins as this will affect the result.
• Upon receiving the fecal specimen in the laboratory, the • Must refrain from eating red meat, food rich in pseudoperoxidase
receptionist checks if the container is properly labeled and as well as vitamin C.
records the information indicated. Patient should avoid the following things for at least 48 hours before
• The site of the experiment should be sterilized. The medical collection of stool:
technologist should wear proper personal protective equipment
before the experiment. • Mineral oils, bismuth, non absorbable anti diarrheal drugs,
antimalarial drugs, antibiotics, etc
MATERIALS • Patient should not have barium before stool examination
PHYSICAL EXAMINATION • Avoid iron containing drugs, meat, fish etc for at least 48 hours
before stool for occult blood
• only have to check the color and consistency of the stool sample
SPECIMEN COLLECTION
• Sterile container 1. CONTAINER – clean, dry, non-breakable container, leakproof,
• Applicator stick screw-capped
• Pea sized stool o multiple-day collection: large containers
CHEMICAL EXAMINATION 2. TYPE AND AMOUNT COLLECTED
o pea-sized: FOBT, WBCs, qualitative fecal fat
• Fecal Occult Blood Test ▪ or 3 grams of stool is enough
o Guaiac solution ▪ if watery – atleast 10mL should be examined
o Hydrogen peroxide o 2- to 3-day fecal collection: quantitative tests
o Glacial acetic acid o check if formed or watery
o Serologic pipettes ▪ formed – should be tested within 1 hour
o Filter paper ▪ watery – should be tested within 30 minutes
o Pasteur pipettes
o Applicator sticks
o Pea sized stool
• Acid Steatocrit Procedure
o screening test for fecal fat or if you want to estimate the
amount of fecal fat
o alternative to van de kamer titration SPECIMEN PRESERVATION
o if you only have very small amount of sample
• Refrigeration – most common
o 5N perchloric acid • Freezing in dry ice
o Deionized water • Formalin (2%, 5%, 10%)
o Capillary tubes o 10% formalin – all purpose fixative
o Pea sized stool • Alcohol
o Vortex mixer
• 20% glycerin in saline (Cumming Method)
MICROSCOPIC EXAMINATION • Methiolate – Iodine Formaldehyde (MIF) Solution
o Combination of fixative and stain
• Methylene blue stain procedure for fecal leukocytes
o Formaldehyde will fix the sample
o Loeffler methylene blue
o Merthiolate will stain the organism
o Applicator stick
o Disadvantage: cannot be used for trophozoites
o Slides and cover slips
o Can only be used for trophozoan cyst
o Pea sized stool
• Polyvinyl alcohol (PVA) Fixative
o used to stain fecal leukocytes or leukocytes during invasive o can preserve morphology both cyst and trophozoite
condition o toxic to helminths (eggs and larva)
• Muscle fiber procedure o has very low viscosity
o used to detect if able to digest muscle fibers
PHYSICAL EXAMINATION
o 10% eosin in alcohol
o Applicator sticks • observable characteristics
o Slides and cover slips • if the patient is unconscious, can collect from the bed pan or from
o Pea sized stool the diaper
• Neutral fat stain procedure o must collect from the mid portion of the stool, specially if the
o if able to digest fat patient is constipated
o Sudan III in 95% alcohol
o Applicator sticks
o Slides and cover slips
o Pea sized stool

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AUBF311 LAB 1st SEMESTER | FECAL analysis
1. Pass out stool in a bedpan lined with newspaper. Avoid admixture Score 2 – Firm, but not hard; should be
of urine, fibers, dirt, gauze threads and tissue papers. pliable; segmented appearance; little or
2. By means of an applicator stick, get a pea-size or marble-size of no residue left on ground when picked
stool from the mid-portion. If a portion is mucoid or bloody, collect up
from that portion.
3. In case of liquid stool, collect 10ml or 1/3 full of an eight ounce
container.
4. Examine the specimen immediately. Score 3 – Log-like; little or no
5. Note the color, odor and consistency. segmentation visible; moist surface;
MACROSCOPIC STOOL CHARACTERISTICS leaves residue, but holds form when
picked up.
Color / Appearance Clinical Significance
Light to dark Normal (Urobilin/ Stercobilin)
brown • urobilin or stercobilin is the oxidized
form of urobilinogen or stercobilinogen Score 4 – Very moist (soggy); distinct
• liver – gallbladder – bile duct – log shape visible; leaves residue and
intestines loses form when picked up.
• if there is blockage in the bile duct,
urobilinogen or bilirubin cannot pass - Most optimal
through the bile duct
o no color for the feces
o will become pale yellow or white Score 5 – Very moist but has distinct
gray shape; present in piles rather than as
Black Upper Gastrointestinal Bleeding distinct logs; leaves residue and loses
or Melena Iron therapy form when picked up.
Charcoal
Bismuth (antacids)
mostly caused by bleeding in the upper GIT Score 6 – Has texture, but no defi ned
(blood is already oxidized) shape; occurs as piles or as spots;
Red Lower Gastrointestinal Bleeding leaves residue when picked up
or hematochezia Beets and food coloring
Rifampin
mostly caused by bleeding in the lower GIT
Pale yellow, white Bile duct obstruction
gray Barium sulfate Score 7 – Watery, no texture, fl at;
occurs as puddles.
Green Biliverdin
Oral antibiotics
Green vegetables
Butter-like Cystic fibrosis
Bulky/ Frothy Bile Duct Obstruction
• bile duct is where bile pass through • Optimal – between 3 & 4
which is important for the breakdown CHEMICAL EXAMINATION
of fat
Pancreatic Disorders FECAL OCCULT BLOOD TEST (GUAIAC TEST)
• lipase (from the pancreas) – enzyme • most preferred as it is safe to use compared to benzidine
necessary for the breakdown of fats
• fats may accumulate 1. Mix 5 ml of hydrogen peroxide with 5 ml of guaiac solution.
Steatorrhea 2. Make the stool specimen acidic using glacial acetic acid (test
due to increase fat using litmus paper).
3. Apply a small portion of stool in the filter paper.
Mucus, Blood- Colitis
4. Add 2-3 drops of the prepared gum guaiac and hydrogen
streaked mucus Dysentery peroxide solution.
Malignancy 5. A blue color will appear in the filter paper in the presence of
Constipation blood.
Ribbon-like / Intestinal Constriction, spastic colitis, • In a test tube, prepare the color developer (combination of 5ml
flattened obstruction in lower colon, syphillis hydrogen peroxide, and 5ml of guaiac solution (combination
Rice watery Cholera (V. cholerae) 1g of guaiac, 5ml of ethanol – 95%))
Pea soup Typhoid (typhoid fever) • Hydrogen peroxide will serve as the substrate or the donor of
Scybalous (Goat Constipation, spastic colitis, decrease fluid oxygen
droppings) intake • Acidify first eh sample by adding glacial acetic acid
• Get a filter paper then smear the sample on a filter paper
• Add 2-3 drops of color developer and check for color reaction
Score 1 – Very hard and dry; requires and grade
much effort to expel from body; no o trace – faint blue color within 1 minute
residue left on ground when picked up. o 1+ - light blue green, gradually turns light blue
Often expelled as individual pellets. o 2+ - clear blue green, rapidly turns clear blue
o 3+ - deep blue, almost immediately forms
o 4+ - immediately naging deep blue after addition of the
color developer

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AUBF311 LAB 1st SEMESTER | FECAL analysis
FECAL OCCULT BLOOD TEST (BENZIDINE TEST) False (+) FOBT Dietary Pseudoperoxidases
• Carcinogenic • Red Meat
• Most sensitive • Melon, broccoli, cauliflower, horseradish
• Aspirin (gastric intestinal irritation) & other
1. 4 gm benzidine in 100 ml of glacial acetic acid anti inflammatory drugs= (avoid for 7 days)
2. Emulsify pea sized bit of feces in 5 ml of water. False (-) FOBT Reducing agents:
3. Mix 1 ml emulsion and 1 ml of reagent in test tube • Ascorbic acid (Vitamin C) and iron therapy
4. Add several drops of H2O2
o Ascorbic acid – reducing agent
5. Blue colour indicates positive reaction
• all test uses hydrogen peroxide and chromogen
• prepare 2 test tube (1st test tube for color developer) (2nd tube
for sample – emulsify first) (3rd tube, get 1ml from the tube one
and two -- which is the combination of color developer and
sample)
o composition of the color developer – 4 gm benzidine in
100 ml of glacial acetic acid or 1 gram in every 25mL of
glacial acetic acid

• for the rapid test card, we cannot grade on that – just qualitative
(positive or negative)

FECAL OCCULT BLOOD TEST (ORTHOTOLUIDINE TEST)

• Intermediate sensitive
• after benzidine, orthotoluidine is the next sensitive test
ACID STEATOCRIT PROCEDURE
1. Smear the stool on a filter paper with an applicator
2. Pipette a few drops of the reagent on to the filter paper • rapid test to estimate the amount of fat excretion
(orthotoluidine barium peroxide 200 mg+ glacial acetic acid 5 ml) • cannot quantify the amount, just estimate in percentage
3. After 30 sec examine for a blue colour • screening test for steatorrhea among pediatric patients
4. Blue green colour within 30 sec means positive test o we cannot ask them for 3days stool
o pea sized is enough for testing
• get a filter paper and directly smear the sample, without
diluting 1. 0.5g of feces from a spot collection is diluted 1 to 4 with deionized
• place a drop of orthotoluidine solution which is the combination water.
of orthotoluidine barium peroxide and glacial acetic acid 2. Vortex for 2 minutes to homogenize the specimen.
• after 30 seconds, examine for the blue color 3. A volume of 5 N perchloric acid equal to 20% of the homogenate
• no grading followed, but if i-grade, follow same as benzidine volume is added and the mixture is then vortexed for 30 seconds.
Confirm the pH to be <1.
4. Place the acid-homogenate mixture in 75 microliter plain
FECAL • Occult = hidden hematocrit capillary tube. Seal the end with wax.
OCCULT • Screening test for colorectal cancer 5. The capillary tube is centrifuged horizontally at 13000 rpm for 15
BLOOD TEST • Significant= >2.5 mL blood/ 150 g of stool minutes in a microhematocrit centrifuge. This separates fat as an
(FOBT) • Sample= center portion of the stool upper layer overlying a solid fecal fat.
Principle Pseudoperoxidase activity of Hemoglobin 6. The length of the fat and solid layers are measured using a
hemoglobin magnifying lens.
H2O2 + Guiac ------------> Oxidized Guiac (blue) 7. Calculate the acid steatocrit in percent.
+ H2O Pseudoperoxidase 8. Calculate the fecal fat in grams per 24 hours
• if there is blood in the sample, there is
hemoglobin • first thing to do is to emulsify the sample, using deionized
o hemoglobin has pseudoperoxidase water (1:4 – 1 part of stool, 4 parts of water)
activity • must ensure that it is homogenized since small amount
o pseudoperoxidase or peroxidase will o vortex mixer – used for very small amount of fluid
liberate the oxygen from the • after mag homogenized, add acid (5N perchloric acid – very
hydrogen peroxide and the liberated corrosive to use that is why sample must have a pH of <1)
oxygen will be donated to • mix, then place in a capillary tube (non heparinized tube –
chromogen, that is why guaiac is blue ring)
oxidized • fill the capillary tube with the mixture of the homogenate and
o the darker the color, the more acid up to ¾
pseudoperoxidase and oxygen is • put the clay then wax to seal the other end of capillary tube
donated by the guaiac or chromogen • centrifuge for 15 mins at 3,000 RPM using microhematocrit
o the darker the color the stronger the centrifuge
reaction • after 15 mins, there is separation of layers
Chromogens 1. Benzidine- most sensitive o pinaka baba – dark color layer which is the non fatty solid
2. Guaiac- preferred layer (SL)
3. O-toluidine o middle – intermediate liquid layer
o pinaka taas – Fatty layer
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AUBF311 LAB 1st SEMESTER | FECAL analysis
• to get the percentage, measure using microhematocrit reader
the fatty and solid layer FECAL LEUKOCYTES
= fatty layer_̲ x 100 =____% Determination
solid layer
• Normal: <10% fatty layer (no need to perform Van de Kamer) 1. Wet Preparation = Stool + Loeffler’s Methylene Blue
o >20% - steatorrhea 2. Dried Preparation= Stool + Wright’s/ Gram stain
▪ Van de Kamer titration must be used (amount of fat o pwede if formed stool
in g/24hrs) o still, Loeffler’s blue is better in staining leukocyte
3. Lactoferrin latex agglutination test
Lactoferrin = secondary granules of neutrophils
o (+) invasive bacterial pathogen
o Serologic test
o strong agglutination – more lactoferrin present
▪ more lactoferrin = more WBC = there is infection
MUSCLE FIBER PROCEDURE
• if the patient is suffering from maldigestion

1. Emulsify a small amount of stool in two drops of 10% eosin in

alcohol.
2. Coverslip and let stand for 3 minutes.
• SPECIMEN: Single stool
3. Examine under high power field for 5 minutes.
• It is preferable that patients have been on a diet containing 70- 4. Count the number of undigested fibers.
100g fat per day for at least 3 days prior to and during the
collection period.
• No laxatives should be given. (coffee, tea) • place a 2 drop of alcoholic eosin, in glass slide add very small
amount of stool and 2 drops of alcoholic eosin, place a cover
• Stool for Steatocrit testing is stable for seven days under
slip and stand for 3 mins
refrigeration. If longer term storage is necessary, it should be
frozen. • check for the presence of undigested muscle fibers in stool
(HPO)
• Specimen must not contain foreign material such as toilet paper.
o check for the striations
Reference Intervals o no striations – fully digested
• Units: expressed as percentages (%) o with striations (unidirectional) – partially digested
o with striations (both direction) – undigested
• Age >6 months:
o >10 undigested muscle fiber- maldigestion of muscle fiber
o <10% normal
/ creatorrhea
o 10-20% equivocal
o 20% abnormal (steatorrhoea)
• Abnormal
MICROSCOPIC EXAMINATION o 10 undigested muscle fiber
• UNDIGESTED STRIATED MUSCLE FIBER: biliary obstruction,
METHYLENE BLUE PROCEDURE FOR FECAL
gastrocolic fistulas, pancreatic insufficiency (cystic fibrosis)
LEUKOCYTES
• For fecal leukocytes we are looking for the presence of neutrophil
• It is normal to see rare amount of neutrophil but not in significant
number
o > 3/HPF indicates infection
1. Place mucus or a drop of liquid stool on a stool.
2. Add two drops Loffler methylene blue.
3. Mix with a wooden applicator stick.
4. Allow to stand 2-3 minutes.
5. Examine for neutrophils under high power. NEUTRAL FAT STAIN PROCEDURE
• most of the time performed if there is mucoidal or watery • looking for the presence of triglycerides
sample • normally, triglyceride is degraded or digested into glycerol or fatty
o if watery, place a drop of a liquified sample in the glass acid
slide and 2 drops of Loeffler’s methylene blue -- wet o if trig is still undigested – maldigestion
preparation
o mix then add cover slip, allow to stand for 2-3mins to 1. Homogenize one part stool with two parts water.
allow the cell to take up the stain 2. Mix emulsified stool with one drop 95% ethyl alcohol on slide.
o examine under HPO 3. Add two drops saturated Sudan III in 95% ethanol.
o blue cells that are multilobulated – neutrophils 4. Mix and coverslip.
o ≥ 3/HPF – invasive condition possibly infection 5. Examine under high power field.
6. Count orange droplets per high power field.

• homogenized the stool with water

• emulsify (1:2 water)
• add 1 drop of alcohol before maglagay ng stain (sudan III)
• mix then place a cover slip
• under the microscope you’ll see orange droplets (triglycerides)
• not after the size, but rather on the number of fat droplets
• ≥ 60 droplets/HPO – steatorrhea and maldigestion
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AUBF311 LAB 1st SEMESTER | FECAL analysis

SPLIT FAT STAIN PROCEDURE

• Check for fatty acid – product of digestion

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

2. Add two drops saturated Sudan III.
3. Mix and coverslip.
4. Heat gently almost to boiling.
5. Examine under high power.
6. Count and measure the orange droplets per high power field.

• emulsify the stool not with water but directly with the acetic
acid
• add 2 drops of sudan III
• another step:
o gently hit
o in the glass slide, papadaanin sa alcohol lamp until
magkaron ng konting usok
o then check under the microscope
• count 100 fatty acid
• not after the number but rather after with the size
o 6 to 75 um – steatorrhea, there is malabsorption POST – ANALYTICAL PHASE
Before leaving, the medical technologist must do all of the following:
• Returning of materials, slides and microscopes
• Disposal of wastes and disinfection with liquid Lysol or 10%
sodium hypochlorite of the area.

FECAL FAT DETERMINATION

Qualitative Test
1. Neutral Fat Stain 2. Split Fat Stain
(Triglycerides) Emulsified stool + 36% Acetic
Suspension + 95% ETOH + acid + Sudan III
Sudan III Orange Droplets (Fatty Acids)
Orange Droplets • Normal= 100 droplets (< 4
(Neutral Fats or Triglycerides) um)
≥ 60 droplets/hpf = Steatorrhea • Slightly increased= 100
droplets (1-8 um)
• Increased=100 droplets (6-
75 um)
Quantitative Test
Van de Kamer titration
• g/day - unit
• Gold Standard for fecal fats
• For definitive diagnosis of steatorrhea
• Sample= 3 day stool
• Titration with NaOH
• Normal value= 1-6 g fats/day
• Steatorrhea= > 6 g fats/day

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