MLS 419: AUBF LAB Composition of Semen

FINALS WEEK 1 – LESSON 1

Semen Analysis 5% Spermatozoa
INTRODUCTION 60-70% Seminal Fluid Fructose and Flavin
Spermatogenesis 20-30% Prostate Fluid Acid phosphatase, citric
- Formation of Spermatozoa (Sperm) in acid, zinc, and proteolytic
seminiferous tubules enzymes
- Spermatogenesis begins with spermatogonia →
undergo mitosis → some spermatogonia remain 5% Bulbourethral fluid Thick alkaline mucus
near the basement membrane of seminiferous Chemical Components
tubules in an undifferentiated stage to serve as a • Spermine and choline
reservoir of cells for future cell division and • Acid phosphatase
subsequent sperm production • Fructose – energy for the flagella to propel them
- The rest of spermatogonia lose contact with the thru the female reproductive tract
basement membrane and squeeze through the tight o w/o fructose the sperm cannot swim and
junction of the blood testis barrier → undergo will remain immotile
developmental changes and differentiate into • Flavin – responsible for the gray appearance of the
primary spermatocytes until they become sperm semen
cells. • Potassium, Citric Acid and Ascorbic acid
• Ergothionine
Physiology • Phosphorylcholine
• Proteolytic enzyme
o Coagulation and liquefaction of the semen
following the ejaculation

Purpose
- Evaluation of reproductive dysfunction in males –
INFERTILITY TESTING
- Select donors for therapeutic insemination
- Monitor success of surgical procedures:
o Varicocelectomy
o Vasectomy

Sample Collection
• PREPARATION
1. Abstinence: 2 – 7 days (Note: No prolonged
abstinence)
2. Sterile glass or plastic container (warm)
• Collected in a private room near the laboratory
- In order to limit the exposure of the semen to
fluctuation in temperature and the time
between the collection and analysis.
• Give a clear written and verbal instructions
concerning the collection of semen
• Deliver to the laboratory within 1 hour after
!
- Testes (seminiferous tubules) – secretion of collection
• Record the patient’s name, and birth date, the
sperm; Paired glands
- Epididymis – sperm maturation and development period of sexual abstinence, the completeness of
of flagella; store sperms the sample, difficulties with collection, and the time
- Ductus deferens (vas deferens) – transports specimen collection and specimen receipt
sperm to the ejaculatory duct
- Seminal vesicle – transport medium for the sperm;
• METHODS OF COLLECTION
rich in fructose and flavin. - Masturbation
o Produces the major fraction of semen - Coitus interrupts
- Prostate gland – propels the sperm through the - Common condom collection
urethra; contains acid phosphatase, citric acid, - Silastic condom collection
zinc, and proteolytic enzymes responsible for - Aspiration from the vaginal vault after coitus
both the coagulation and liquefaction of the semen; NOTE:
muscular gland. - If married, collection from sex is better than
- Bulbourethral glands – secretes alkaline mucus masturbation because of the degree of
o Located below the prostate gland arousal.
- Note: These glands contain the fraction of semen - The first part of the ejaculate is mostly
and mix together in ejaculation collected because it is more concentrated

• PRESERVATION
 ➢ Awaiting analysis LIQUEFACTION
▪ semen stored at body temperature • After ejaculation, semen is typically a semi-solid
(incubator) coagulated mass.
➢ For artificial insemination • Liquefy within 30 to 60 minutes (RT) after
▪ Frozen and stored for 1 year at -85degC at collection (Normal)
sperm bank o Rarely, it may take 60 minutes or more
• In liquefaction, semen becomes more homogenous
SEMENALYSIS PARAMETERS and quite watery; small areas of coagulation remain
• Liquefaction (final stage).
• Appearance • Failure of liquefaction within 60 minutes
• Volume o Deficiency in prostatic enzymes and
• Viscosity should be reported.
• pH o If complete liquefaction is not observed
• Sperm Concentration and Count after 60 minutes, this should be reported.
• Motility • Note:
• Morphology 1. Normal Liquefied semen samples may
contain jelly like granules (gelatinous
Parameters Reference Values
bodies) - no clinical significance
Volume 2 to 5 mL 2. Presence of mucus strands may interfere
with semen analysis
Viscosity Pours in droplets **Semen taken from home are normally liquefied by the
time they arrived in the lab**
pH 7.2 to 8.0

Sperm > 20 million/mL If after 2 hours the specimen has not liquefied:
Concentration o Dulbecco’s phosphate-buffered saline
o Proteolytic enzymes such as alpha-
Sperm Count > 40 million/ejaculate chymotrypsin or bromelain
Motility > 550% within 1 hour
o “these treatments may affect biochemical
t e s t s , s p e r m m o t i l i t y, a n d s p e r m
Quality > 2.0 or a, b, c morphology, so their use must be
documented.”
Morphology > 14% Normal forms (strict criteria)

> 30% normal forms (routine criteria)

Round Cells < 1.0 million/mL

APPEARANCE
COLOR
Gray-white, Pearly-
white, Light Yellow, NORMAL
Opaque

Shades of Yellow Correlate with flavin concentration

Associated with certain drugs, over

Deep Yellow abstinence, jaundice, urine
!
contamination

Brown or Red Presence of red blood cells

Increased white Presence of white blood cells and

turbidity infection within the reproductive tract
➢ Less opaque, if the sperm concentration is
very low
➢ If required, the specimen culturing is
performed prior to continuing with the semen
analysis
 pH
• Reflects the balance between the pH values of
different accessory gland secretions.
• Measured after liquefaction (preferably after 30
minutes) – (WHO) or within 1 hour of ejaculation
due to loss of CO2 occurs after the production –
(Strasinger)
• pH paper range 6.0 to 10.0 should be used
⎯ mix the semen well
⎯ spread a drop onto pH paper
⎯ read within 30 seconds
▪ wait for the color of the
impregnated zone to be uniform
⎯ compare the color with the calibration
!
strip to read pH
VOLUME
• Normal volume: 2-5 mL • Normal pH: 7.2- 8.0
o the volume of the ejaculate is contributed
• Increased pH: Infection
• Decreased pH: increased prostatic fluid,
mainly by the seminal vesicle and prostate
ejaculatory duct obstruction, poorly develop seminal
glands, and small amount from the
vesicle
bulbourethral gland and epididymis.
• It can be measured by pouring the specimen into a
INITIAL MICROSCOPIC EXAMINATION
clean graduated cylinder calibrated in 0.1 – mL • Phase-contrast microscope
increments • Scanning at a total magnification of 100x (10x
o Precise specimen volume is essential in
objective lens with 10x ocular lens)
any evaluation of semen because it allows • Provides overview
the total number of spermatozoa and non
o Mucus strand formation
sperm cells in the ejaculate to be
o Sperm aggregation or agglutination
calculated.
• Note: Measuring volume by aspirating the sample o Presence of cells other than spermatozoa
(Epithelial cells, round cells, and isolated
from the specimen container into a pipette or
sperm head or tails)
syringe is not recommended • Then be observed at 200x or 400x magnification
o Assessment of a sperm motility
Low Volume High Volume o Determination of the dilution required for
accurate assessment of sperm number
- Obstruction of the - active exudation in
ejaculatory duct cases of active Making a wet preparation
- congenital bilateral inflammation of the • Mix the semen sample well
absence of vas accessory organs • Remove an aliquot of semen immediately after
deferens - extended abstinence mixing, allowing no time for the spermatozoa to
- collection problems settle out of suspension.
(loss of fraction of the • Remix the semen sample before removing replicate
ejaculate) aliquots.
- partial retrograde
ejaculation or androgen
deficiency
VISCOSITY
• Consistency of the fluid
• Aspirating into a wide-bore plastic disposable pipette "
• Normal viscosity: form small discrete droplets that • Place a standard volume of semen, e.g. 10uL, onto
do not appear clump or stringy when falling by gravity a clean glass slide
by a pipette and observing the length of any thread. • Cover it with a coverslip, e.g. 22 mm × 22 mm for
• Abnormal viscosity: drop from a thread more than 10uL, to provide a chamber approximately 20um
2 cm long (viscous) deep. The weight of the coverslip spreads the
• Ratings: 0 (watery) – 4 (gel like) sample.
o Viscosity can also be reported as low, • Take care to avoid the formation and trapping of air
normal, or high. bubbles between the coverslip and the slide.
• Note: High viscosity can interfere with determination • Assess the freshly made wet preparation as soon
of sperm motility, sperm concentration, detection of as the contents are no longer drifting.
antibody-coated spermatozoa and measurement of
biochemical markers.
Aggregation of spermatozoa Cellular elements other than spermatozoa
• Adherence either of immotile spermatozoa to each • Epithelial cells in genitourinary tract
other or of motile spermatozoa to mucus strands, • Leukocyte and immature germ cells (collectively
non-sperm cells or debris is non-specific referred as round cells)
aggregation and should be recorded. • Examined the stained smear at 1000x magnification
• Can be more precisely identified and quantified by
detecting peroxidase activity or the antigen CD45

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• Motile spermatozoa sticking to each other, head-to-
head, tail-to-tail or in a mixed way.
• Motility is vigorous with frantic shaking motion but
sometimes are so agglutinated with limited motion
• Any motile spermatozoa stick to each other by their
heads, tails or midpieces should be noted.
• Major type of agglutination (grades 1-4); site of
attachment (grades A-E)

!
GRADE DEGREE DESCRIPTION
< 10 spermatozoa per agglutinate,
1 Isolated many free spermatozoa

10 – 50 spermatozoa per
2 Moderate agglutinate, free spermatozoa

Agglutinates of > 50 spermatozoa,

3 Large some spermatozoa still free

All spermatozoa agglutinated and

4 Gross agglutinates interconnected

Sperm Motility
- Assessed ASAP after liquefaction (preferably at 30
minutes, WHO) or within 1 hour following ejaculation
- Normal: 60% or higher progressively motile
sperm

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 Sperm Morphology
1. Mix the semen sample well
2. Remove an aliquot of semen immediately after
• Head. Neckpiece, midpiece,
tail
mixing, allowing no time for the spermatozoa to • Abnormalities:
settle out of suspension.
o Head – poor ovum
3. Remix the semen sample before removing a
penetration
replicate aliquot.
o N e c k p i e c e ,
4. For each replicate, prepare a wet preparation
Midpiece, and Tail –
approximately 20 um deep
5. Wait for the sample to stop drifting (within 60 affects motility
• Head – oval-shaped; approx.
seconds).
6. Examine the slide with phase-contrast optics at 5um long and 3 um wide
• Tail – approx. 45 um long
×200 or ×400 magnification.
7. Assess approximately 200 spermatozoa per
• Critical to ovum penetration:
Acrosomal cap – encompass
replicate for the percentage of different motile
half of the head; cover 2/3 of
categories.
8. Compare the replicate values to check if they are sperm nucleus
• Neckpiece – attaches the head to the tail and the
acceptably close. If so, proceed with calculations;
midpiece.
if not, prepare new samples. • Midpiece – 7 um long; thickest part because it is
Categories of Sperm Movement surrounded with mitochondrial sheath
o Examination of sperm morphology is also
Spermatozoa moving actively, essential, because sperm that are
Progressive motility (PR) either linearly or in a large circle, morphologically incapable for fertilization
regardless of speed
also results to infertility
All other patterns of motility with an • NORMAL VALUES:
Nonprogressive Motility (NP) absence of progression o Routine Criteria: > 30% normal
o Kruger’s Strict Criteria: > 14% normal;
Immotility (IM) No movement
measures head, neck and tail
Note: **When discussing sperm motility, it is important to ▪ Requires the use of stage
specify total motility (PR + NP) or progressive motility (PR). micrometer or morphometry
o Normally at least 70% of sperm
Must watch! 26:50 sperm motility under microscope demonstrate normal morphology

Semen smearing methods for sperm morphology

(a) “Feathering” method for undiluted semen. The semen
drop (S) spreads along the back edge of the angled slide
and is pulled forwards over the slide to form the smear. (b)
Pipette method for washed samples. A drop of the sperm
suspension (SS) is spread over the surface of the slide by
pushing the horizontal pipette (P).

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Common abnormalities of Sperm Heads and Tails

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Note: **Images are available in Stras page 211, chapter 10

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 Considerations:
• Additions of stain (crystal violet) to diluting fluid
helps visualization using bright-field microscope.
• Note: Only FULLY DEVELOPED sperm should be
counted
o Immature and WBCs – “round cells” must
not be included)
• > 1 million WBCs/mL – inflammation or infection
• > 1 million spermatids/mL – disruption of
spermatogenesis caused by viral infections,
exposure o toxic chemicals, and genetic disorders.
Inclusion Criteria:
• Count only whole spermatozoa (w/ the head and
tail)
• Whether or not sperm cell is counted is determined
by the location of its head (*the location of its tail is
unimportant)
o Sperm cell is counted if its head lies on the
boundary line
• To avoid counting the same sperm cells in adjacent
square
o A sperm cell with its head on the line
dividing two squares should be only, if that
line is one of the two perpendicular
boundary line
o for example: cells may be counted if most
of the sperm head lies on the lower or left
center boundaries = which form an 

“L-shape”
o DO NOT COUNT cells on the upper right
" center boundary line
Sperm Concentration
• “total sperm number” vs. “sperm concentration”
• Sperm concentration
⎯ Number of spermatozoa per unit volume of
semen and is a function of the number of
spermatozoa emitted and the volume of
fluid diluting them.
• Total sperm number
⎯ Total number of spermatozoa in the entire
ejaculate and is obtained by multiplying the
sperm concentration by the semen volume
⎯ NORMAL VALUES: > 40 million/ejaculate
• Normal values: 20-250 million sperm per milliliter
⎯ Concentration of 10-20 million/milliliter is
considered as borderline
• Makler counting chamber – undiluted semen, heat
Sample:
instead (inaccurate results)
• Improved Neubauer Counting Chamber
⎯ 1:20 dilution (common) 1. Using a 1:20 dilution, an average of 60 sperm are
⎯ Diluting fluids: cold water (most counted in the five RBC counting squares on the
common), formalin, Na bicarbonate, 0.5% both sides of the hemocytometer. Calculate the
chlorazene, 1% formalin in 3% trisodium sperm concentration per milliliter and the total
citrate sperm count in a specimen with a volume of 4mL.
⎯ Sperm are counted in the same manner of Ans:
counting in the CSF. 60 sperm counted x 1,000,000 = 60,000,000 sperm/mL
60,000,000 sperm/mL x 4 mL = 240,000,000 sperm/
✓ 5 RBC squares: 4 corner and ejaculate (NORMAL)
center squares of the large center
square (same to manual RBC count)
✓ Computation: # sperm cells Counting Sperm Cells in WBC Squares
counted x 1,000,000 (mL) • The middle of the three lines define the square’s
✓ 2 WBC squares: only 2 corner boundary (black line-left panel). All spermatozoa
large squares (same to manual within the central square are counted, as well as
WBC count) those with their heads between the twi inner lines
✓ Computation: #sperm cells (white circles). Do not count those whose heads lie
counted x 100,000 (mL) between the outer two lines (black circles)
 Staining Principles : Traditional Fixation and Sequential
• Sperm cells (spermatozoon) with most of its head Staining
lying on the central line is COUNTED ONLY IF that to fix the cells, it also hydrates them
Ethanol
line is the lower of or left-hand of the square (white
to rehydrate the fixed smears to permit
circles, middle panel) BUT NOT IF it is the upper or Graded ethanol water-soluble haematoxylin staining
right hand line of the square (black circles-right
to rehydrate dried smears to permit
panel) purified water water-soluble haematoxylin staining
• ❗ Always use the L-shaped pattern in counting
haematoxylin to stain the nucleus blue
your cells
to remove unbound nuclear haematoxylin
tap water
to remove non-specifically bound dye
acidic ethanol from the cytoplasm (destaining)
to reduce the acidity and return blue
tap water colour to the nucleus
to return blue color to the nucleus (if tap
Scott’s solution water is insufficient)
to dehydrate smears to permit ethanol-
Ethanol soluble Orange G/EA-50 staining
Sample:
to stain the cytoplasm pink
Orange G
1. Using a 1:20 dilution, an average of 600 sperm to stain the cytoplasm pink
EA-50
are counted in the 2 WBC counting. Calculate the
sperm concentration per milliliter and the total to dehydrate the stained smears
sperm count in a specimen with a volume of 2mL. Graded ethanol gradually to permit the use of ethanol-
soluble mountants
Solution: 
 to permit the use of ethanol-insoluble
Xylene

 mountants

 After staining the slides can be viewed MOUNTED or

 UNMOUNTED (with/without coverslip) 






 STEPS IN PAPANICOLAOU STAINING:
Answer: 120,000,000 sperm/ejaculate

Related Terms
• ASPERMIA = no ejaculate at all
• OLIGOSPERMIA = less than 20 million per mL
• NECROSPERMIA = immotile/dead sperms
• AZOSPERMIA = complete absence of sperm

Staining Methods
once the semen stains has been air-dried they must
be fixed and stained to highlight the details of the
spermatozoa

Different Stains:
• Papanicolaou Stain (recommended)j
• Shorr or Diff Quick Stain (recommended)
• Wright’s Stain
• Giemsa Stain 


for ALL THESE STAINS:

• the head is stained pale blue (acrosomal
region) and is stained dark blue (post
acrosomal region)
• Midpiece = red stain
• Tail = blue or reddish
the excess residual cytoplasm usually located
behind the head and around the midpiece is
stained pink or red in papanicolaou stain and
reddish-orange in Shorr or Diff Quick Stain
STEPS IN SHORR STAINING: SEMINAL FLUID FRUCTOSE—————————————
• Low fructose levels are caused by abnormalities of
the seminal vesicles, bilateral congenital absence
of the vas deferens, obstruction of the ejaculatory
duct, partial retrograde ejaculation, and androgen
deficiency .
• Screened for the presence of fructose using the
resorcinol test that produces an orange color
when fructose is present
• A normal quantitative level of fructose is equal to
or greater than 13 µmol per ejaculate.
• determined using Spectrophotometric methods.
Specimens for fructose levels should be tested
within 2 hours of collection or frozen to prevent
fructolysis.
• Procedure:
1. prepare reagent (50 mg resorcinol in 33 mL
concentrated HCl diluted to 100mL with
water)
2. mix 1mL of semen with 9 mL of reagent
3. boil
4. observe for orange red color


ANTISPERM ANTIBODIES——————————————-
• may be present in both men and women
• Detected in semen, cervical mucosa, 

or serum : possible cause of infertility
• Blood-testes barrier separates sperm from the male
immune system. (When disrupted in surgery,
PAS STAINED SEMEN vasectomy, trauma, infection— antigens on sperm
produce an immune response)
• Damage sperm may cause the production of
antibodies in female.
• In male, clumps of sperm are observed in routine
semenalysis.
o Sperm-agglutinating antibodies cause to
SHORR STAINED SEMEN stick in a head-to-head, head-to-tail, or tail-
to-tail pattern.
o Agglutination grading: “few”, “moderate”,
or “many”
• In female, normal semen analysis accompanied by
continued infertility.
o Mixing the semen with female cervical
H-E STAINED SEMEN mucosa or serum and observing for
agglutination

Tests for Antisperm Antibodies

MIXED AGGLUTINATION REACTION (MAR TEST)
• screening procedure to detect IgG antibodies
OTHER TESTS & TECHNIQUES • Semen + IgG (AHG) + latex particles or treated
RBCc coated with IgG
SPERM VITALITY——————————————————- • Bivalent AHG binds to both antibody on the sperm
• Assessed within 1 hour of ejaculation. and antibody on latex particles or RBCs, forming
• Evaluated by eosin-nigrosin stain, preparing a microscopically visible clumps of sperm and
smear, and counting the number of dead cells in particles or cells.
100 sperm using a brightfield or phase-contrast • Normal: < 10% of motile sperm attach to
microscope. particles
• Living cells remain bluish white whereas dead
cells stain red against the purple background
• Normal vitality requires 50% or more living cells
• Presence of a large proportion of vital but immobile
cells may indicate a defective flagellum
• High number of immotile and nonviable cells may
indicate epididymal pathology
IMMUNOBEAD TEST
• more specific to detect IgG, IgM, and IgA
antibodies and demonstrates what area of sperm
(head, neckpiece, midpiece, or tail) the
autoantibodies are affecting.
o Head- directed Ab: interfere penetration into
cervical mucosa or ovum
o Tail-directed Ab: affect movement through
the cervical mucosa

• Sperm + polyacrylamide beads (coated with anti-

IgG, anti-IgM, anti-IgA)
• Microscopic examination -beads attach to sperm at
particular areas
• Reported as “IgM tail antibodies, “IgG head
antibodies,” and so forth
• Normal: presence of beads on less than <50%
of the sperm

MICROBIAL TESTING————————————————- *TCA = Trichloroacetic acid

• >1 million WBCs/ mL indicates infection within
reproductive system, prostate. POST VASECTOMY ANALYSIS——————————/,.—-
• Routine Aerobic/anaerobic cultures and test for • much less involved procedure when compared with
Chlamydia trachomatis, Mycoplasma hominis, infertility analysis
Ureaplasma urealyticum = most frequently • concern only in presence or absence of
performed spermatozoa
• the length of time required for complete sterilization
CHEMICAL TESTING————————————,,,,,,,,——- can vary greatly among patients and depends on
• Decreased neutral alpha-glucosidase, both time and number of ejaculations
glycerophosphocholine and L-carnitine suggest a • finding viable sperm in a post-vasectomy patient is
disorder of epididymis. not uncommon and care should be taken not to
• Decreased zinc, citric acid, glutamyl overlook even a single sperm
transpeptidase and acid phosphatase indicate lack • Specimens are routinely tested at monthly
of prostatic fluid. intervals, beginning at 2 months postvasectomy
• Spectrophotometric methods- quantitate cirtric acid and continuing until two consecutive monthly
and zinc specimens show no spermatozoa
• Wet preparation using phase microscopy
• Negative wet preparation is followed by specimen
centrifugation for 10 minutes and examination of
the sediment.

SPERM FUNCTION TEST————…………………———,,,

advances in assisted reproduction and in vitro fertilization
have resulted in a need for more sophisticated semen
analyses to assess not only the characteristics of sperm but
also the functional ability
SPECIAL CASE
• In cases of alleged rape -microscopically examining
most commonly performed in specialized andrology
the specimen for the presence of sperm with xylene
laboratories:
and examine under phase microscopy.
• Hamster egg penetration assay
• Motile sperm- detected for up to 24 hours after
intercourse • Cervical mucus penetration test
• Hypo-osmotic swelling test
• Nonmotile sperm- 3 days
• In-vitro acrosome reaction
• As sperm die off, heads remain and present for 7
days after intercourse.
• Seminal fluid contains high concentration of
prostatic acid phosphatase.
• Seminal glycoprotein p30 (prostatic specific antigen
[PSA]) - present even in the absence of sperm;
more specific detection
• Further information, perform ABO blood grouping
and DNA analysis)
 MLS 419: AUBF LAB  Small intestine
FINALS WEEK 2 – LESSON 2 o Pancreatic amylase – break down
FECAL ANALYSIS starch that has been initially broken
down by salivary amylase
o Pancreatic juice (trypsin,
chymotrypsin, elastase,
Why should we analyze feces??? carboxypeptidase)
• Feces provides valuable diagnostic information o Pancreatic lipase and bile salts
• Excreted from the body  Large intestine
• Feces is formed in the digestive system o Absorbs approx. 3 L of water
→ provide clues or important info on  100-200 g of feces excreted per 24 hours
what is going on inside digestive system o Contains bacteria, cellulose, undigested
• Ex: Formed vs Watery Stool foodstuffs, GI secretions, bile pigments,
• Formed: normal conditions; no cells from intestinal walls, electrolytes,
pathologic conditions; water is and water
sufficiently reabsorbed by large ▪ Physical appearance of stool –
intestine indication of water content
• Watery: pathologic condition • Formed/ Hard – few
(salmonella, vibrio cholerae, water
shigella) → cause a significant • Soft – enough water
damage in the intestinal • Loose/ Watery – too
epithelium → release/ secretion
much water
of water than what the large
intestine can reabsorb Diarrhea and steatorrhea
• Macroscopic, microscopic, and chemical  Diarrhea
examination o An increase in daily stool weight above
• Macroscopic: color, consistency
200g
• Microscopic: possibility of abnormal cells o Increased liquidity of stools (loose/
being present (WBCs/ RBCs)
watery)
• Chemical: look into the presence of fat, o Defecation: frequency of more than 3
enzymes, carbohydrates → times per day
maldigestion or malabsorption  Steatorrhea
• Detection and identification of pathogenic o Presence of fat in feces
bacteria, viruses, and parasites
• Bacteria: E. coli – most common; highly Diarrhea
toxigenic • Major mechanisms: secretory, osmotic, and
• Vibrio cholerae – responsible for intestinal hypermotility
many epidemics in the past
• Tests performed:
• Fecal electrolytes (sodium, potassium)
Anatomy of the digestive system
• Fecal pH
• Fecal osmolality

Secretory diarrhea
• characteristic of infection with various
enterotoxin-producing organisms → cause
damage → derange normal absorptive
mechanisms (absorption of water) of small
intestine
 Vibrio cholerae, Salmonella, Shigella,
Escherichia coli, Clostridium,
Staphylococcus, protozoa, virus (most
common cause) release substances that
stimulate electrolyte-rich intestinal
secretions
• damage to intestinal mucosal due to drugs or
disease
Physiology • Secretion of water exceeds absorption
 Mouth  Too much water is released by small
o Salivary amylase breaks down starch intestine → overwhelm the absorptive
(polysaccharide) capacity of large intestine
 Stomach • Due to prolonged opening of Chloride channels
o Protein digestion by pepsin
  Small intestine has Chloride channels → Steatorrhea
in the presence of these organisms, • Fecal fat excretion that exceeds 6-7 g per day
chloride channels will remain open → • fecal specimens are characteristically pale,
water will be secreted in excessive greasy, bulky, spongy, or pasty and are
amounts extremely foul smelling
 Small intestine has villi where absorption • May be caused by:
happens → villi contains pores and • pancreatic insufficiency –
channels for absorption and secretion of problem in lipase secretion
electrolytes → in the presence of • malabsorption
enterotoxin-producing organisms, • Diagnosed by fecal fat determination
chloride channels are open for a long • May occur simultaneously with diarrhea
period → water is excreted • Patient history can provide information
• Widespread destruction of the absorptive that directly relates to the cause of the
epithelium of small intestine patient’s condition
 Result into inefficient water absorption
• Increased solute secretions by the intestine Specimen collection
cause increased fluid volume sent to the large • Needs patient education regarding the
intestine; the resultant fluid volume exceeds the importance of testing and proper collection of
absorptive capacity of the large intestine fecal specimens
• Digestive & absorptive capacity is quite normal • any clean, non-breakable container that is
but there is damage sealable and leakproof is acceptable
• type and amount of specimen collected vary
Osmotic diarrhea  occult blood, white blood cells, or
• accompanies conditions characterized by qualitative fecal fat: small amount
maldigestion or malabsorption  quantitative tests for the daily fecal
 Maldigestion: inability to convert excretion of any substance: 2-3 day
foodstuffs into readily absorbable collection (stool sample)
substances o rarely requested
 Malabsorption: normal digestive ability o larger container
but inadequate intestinal absorption of • Contaminants to avoid
the already processed foodstuffs  urine, toilet tissue, or toilet water
• Excessive solute present in the lumen of • closed containers of fecal specimens should be
intestine covered with a disposable tissue or toweling and
 Osmosis: water will go to an area where slowly opened
there is high solute concentration  Prevents spattering in case of sudden
 If lumen is filled with undigested & release of fecal contents in gas
unabsorbed food products, water from formation
the small intestine will be secreted →
exceed absorptive capacity of large Macroscopic examination
intestine
• abnormally increased quantity of foodstuffs to
the large intestine that cause the retention of
large quantities of water and electrolytes in the
intestinal lumen
• There is a problem in digestion and absorption
of processed food → results to excessive water
going out to the lumen of intestine

Intestinal hypermotility
• transit time for intestinal contents is too short to
allow normal intestinal absorption to occur
• intestinal contents (bolus) must be
adequately exposed to the mucosal
epithelium and must be retained long
enough to allow proper absorption to
occur
• increase in intestinal motility decreases the time
allowed for the intestinal absorptive processes
• Can be caused by laxatives, chemicals, nerves,
hormones, secretory/osmotic diarrhea*, and
emotions (nervousness)
*diarrhea: excrete substances present in the intestines
 Color  Lactoferrin – substance being tested;
• normal brown color of feces results from bile component secondary granules of
pigments granulocytes
• Intestinal anaerobic bacteria subsequently  Can perform on refrigerated and frozen
reduce it to the three colorless tetrapyrroles specimens
collectively called the urobilinogens: In the lab, report the presence of WBCs in an unstained
stercobilinogen, mesobilinogen, and manner.
urobilinogen - Turnaround time: 1 hr
 Oxidized into urobilins—stercobilin, - Staining takes time and there are many
mesobilin, and urobilin—which are specimens
orange-brown and impart color to the - Additional supplies → expensive
feces
• Acholic/pale/clay-colored stool: posthepatic Undigested muscle fibers
obstruction/ addition of barium sulfate (radiologic • Aids in the diagnosis and monitoring of
exam) pancreatic insufficiency
 Problem in transport of bile pigment o Meat is not digested properly
from the liver • Stools are emulsified in 10% alcoholic eosin (for
• Primary concern: presence of blood in stool enhancement)
 Bright red: may be caused by lower GI • Count the red-stained fibers
tract bleeding o Undigested: visible striations both
 Black: may be caused by upper GI tract vertical and horizontal
bleeding (esophagus to duodenum) o Partially digested: striations in one
 Must be chemically tested direction (vertical or horizontal)
 There are normal conditions that can o Digested: no striations
cause a bright red/ black color of stool o > 10 undigested fibers: reported as
sample increased/ creatorrhea
• Ex: if you ate Dinuguan • Creatorrhea: increased number of undigested
meat fibers
Consistency and Form
• ranges from loose and watery stools (diarrhea) Qualitative fecal fat
to small, hard masses (constipation)  simple two-slide qualitative procedure can
• Normal: formed be used to detect increased fat in feces
• Soft: increased fecal water content  Neutral fats (TAG), fatty acid salts, fatty
 May be normal, related to laxatives, or acids, and cholesterol
can accompany gastrointestinal  Stains used: Sudan III (most common),
disorders Sudan IV, Oil Red O
• May be bulky and with undigested substances  Neutral fats are readily stained by Sudan III
(leaf or seeds) while soaps and fatty acids are not
• Seen as orange-red droplets
Microscopic examination • Count number of fat globules
 Slide 1
Fecal leukocytes (WBCs) • neutral fats are detected when
• aids in the differential diagnosis of diarrhea several drops of ethanol (95%) are
 observed in secretory diarrhea added to a suspension of feces
• Suggest that the intestinal wall is infected or (emulsified stool – 1 part stool: 2
inflamed parts water) on a microscope slide,
• Normally, leukocytes are not present in feces; stain is added (Sudan III), a
hence the presence of even a small number (1-3 coverslip is applied, and the wet
per high-power field) indicates an invasive and preparation is observed (> 60/ hpf is
inflammatory condition steatorrhea)
• can be stained using Wright’s or Gram (stain  Slide 2
pathogens which caused the presence of • Total fecal fats are detected when
leukocytes) stain (dry smears) or methylene blue a drop of acetic acid is added to a
stain (wet preparation) fecal suspension, stain is added, a
 not normally done in routine exam coverslip is applied, heated (stain
 better mode of action → properly other fats) gently almost to boiling,
determine number and examined
 can be mistaken for cyst of E. histolytica  Count and measure orange droplets
• Lactoferrin latex agglutination test – indirect (100 or more droplets measuring 6-75
test for a leukocyte micrometers is steatorrhea)
• Increased numbers of globules, as well as *Filter paper – 2 or 3; smear on A and B
extremely large globules (i.e., 40 to 80 mm), are *on the other side, add hydrogen peroxide → elicit
common with steatorrhea change in color (positive)
• increased amount of neutral fat (on the first
slide) suggests maldigestion FOBT – not diagnostic of any disease; just indicates the
• increased amount of total fat (on the second presence of occult blood
slide) indicates intestinal malabsorption
Immunochemical FOBT
Chemical examination • use polyclonal antihuman antibodies directed
against the globin portion of undegraded human
Fecal Blood hemoglobin
• Bleeding anywhere in the gastrointestinal (GI) • highly specific for human blood in feces and do
tract from the mouth (bleeding gums) to the anus not have interference from dietary foodstuffs or
(hemorrhoids) can result in detectable blood in medications
the feces • may be automated and photometric or manual
• bleeding in the lower GI tract: bright red blood; and visual
brown with blood droplets/ streaks
• bleeding in the upper GI tract: black Porphyrin-based FOBT
• Melena: excretion of dark or black stools • based on the chemical conversion of heme to
resulting from the presence of large amounts of intensely fluorescent porphyrins
fecal blood (50 to 100 mL/day) • detection and quantitation of the total amount of
• Alarming - hemorrhoids, anal fissures, hemoglobin in feces
cancer • hemoglobin from nonhuman sources such as
red meats can cause a false-positive result
Occult blood • more expensive, time-consuming, and labor-
• small amount of blood in feces not visually intensive compared with other FOBTs
apparent
 > 2.5 mL of blood/ 150 g of stool is Fetal Hemoglobin in feces/APT Test
considered pathologically significant • if newborn infant has defecated or vomited blood
• Methods in testing fecal occult blood • differentiate the source of blood – infant
 Guaiac-based FOBT (most common) or mother
 Immunochemical FOBT • presence of blood in the stool, emesis, or gastric
 Porphyrin-based FOBT aspirate from a newborn infant
• may have come from the GI tract of the
Guaiac-based FOBT neonate or could be maternal blood that
• based on the pseudoperoxidase activity of the was ingested during delivery
heme moiety of hemoglobin
• In the presence of an indicator and hydrogen
peroxide, the heme moiety catalyzes oxidation of
the indicator, which results in a color change
(blue-green/ dark blue)
• the collection of 3 fecal samples is the standard
of practice to maximize test sensitivity
• instructed to sample several portions of
a single stool specimen (1 sample; 3
different parts – most common) or, ideally,
to collect fecal material from stool
samples on 3 different days

Hgb F – resistant to alkaline determination

Adult Hgb (Hgb A) – sensitive to alkaline determination
*Thalassemia major: high Hgb F in mother → erroneous
result
 Quantitative fecal fat “You have brains in your head…you know what you know.”
• Definitive test for steatorrhea
• patient collects all feces excreted for 2 to 3 days
• A portion of the homogenized fecal specimen is
removed for chemical analysis of the lipid
content by gravimetry, titrimetry (van de Kamer
titration), or nuclear magnetic resonance
spectroscopy
• Values less than 95% indicate steatorrhea in
people 3 years and older

Fecal Enzymes
Rarely performed
• Fecal trypsin
o Exposing x-ray paper stool emulsified in
water
o Inability to digest gelatin indicates
trypsin deficiency; detects only severe
cases
• Fecal chymotrypsin
o More resistant to intestinal degradation;
more sensitive indicator of less severe
cases of pancreatic insufficiency;
measured by spectrophotometry
• Elastase I - recommended
o Isoenzyme released by pancreas
o Measured using ELISA test; strongly
resistant to degradation; very sensitive
indicator of exocrine pancreatic
insufficiency

Fecal carbohydrates
• unhydrolyzed disaccharides are osmotically
active, they cause large amounts of water to be
retained in the intestinal lumen, resulting in an
osmotic diarrhea
• Lactose intolerance in adults is common
• result from intestinal bacteria actively
fermenting lactose in the intestinal
lumen
• lactase in brush border is
defective
• large amounts of intestinal gas and
diarrheal stools with low pH (5.0-6.0)
• rapid qualitative fecal pH can be obtained by
testing the supernatant of a diarrheal stool using
pH paper
• Clinitest tablet test
• Specific histochemical examination of the
intestinal epithelium (biopsy)
• oral tolerance test using specific sugars
• an increase less than 20 mg/dL above
the patient’s fasting glucose level
indicates deficiency of the enzyme
• xylose absorption test
• differentiate carbohydrate malabsorption
from carbohydrate maldigestion
• xylose: not digested but absorbed →
should be excreted in the urine
• malabsorption: urine xylose is
low
 4. Ascheim-Zondek
MLS 419: AUBF LAB • AU = Immature female MICE
MIDTERMS WEEK 3 – LESSON 3 • MOI = urine is injected subcutaneously
Miscellaneous Body Fluids • PR = Formation of hemorrhagic follicles and
corpora lutea
• Sen = 1-6 IU/mL
HUMAN CHORIONIC GONADOTROPIN
5. Frank-Berman
• hormone produced by the cytotrophoblast and • AU = Immature female RATS
syncytiotrophoblast
• MOI = urine is injected subcutaneously
• production of HCG peaks during 1st trimester of • PR = Ovarian hyperemia
pregnancy
• hyperemia: accumulation of blood; increased blood
• the hormone can be identified in the ff body fluids: flow in the ovaries (of female rats)
- Blood (plasma/serum)
- urine • Sen = 1 IU/mL
- amniotic fluid
Composed of 2 subunits: 6. Kupperman
• Alpha = similar in TSH, LH, FSH, HCG • AU = Female Virgin rat
- these 4 hormones, their alpha-subunits look alike • MOI = urine is injected Intraperitoneally (dorsal
- which is why we cannot use the alpha-subunit for portion of the rat)
identification of HCG • PR= Ovarian Hyperemia
• BETA = unique for HCG • Sen= 1 IU/mL
- beta-subunit is used for testing/test-kits of HCG
- that is why we call it the “beta-human chorionic 7. Kelso
gonadotropin hormone” • AU= Female virgin rat
First (1st) Morning Specimen = used as the best • MOI= urine injected Subcutaneously
specimen for Pregnancy Testing that aims to detect • PR= Ovarian hyperemia
HCG in urine • Sen= 1 IU/mL
- since it is a more concentrated sample = higher
chance of being detected/ identified SWEAT TEST
BIOASSAYS FOR THE DETECTION OF HCG IN
URINE • Primarily used to diagnose CYSTIC FIBROSIS
• Cystic Fibrosis : Autosomal recessive genetic
• bioassays = use of animals disease
Codes: - caused by mutations in one single gene located
• AU = Animal used in the long arm of chromosome 7
• MOI = Mode of Injection - Metabolic disease that affects the mucous
• PR = Positive result (presence of HCG) secreting glands of the body
• Sen = Sensitivity - the pathology lies in the mucous (exocrine)
1. Hogben glands of the body
• Animal used = Female frog - in cystic fibrosis the body produces thick and
• (Delivery) Mode of Injection = the urine of a sticky mucous that can clog the lungs, pancreas
suspected pregnant female is injected in the and other organs affected.
Lymph sac of the frog • Associated with:
• Positive result = Oogenesis - pancreatic insufficiency
• Sensitivity = 75-100 IU/mL - respiratory distress
2. Galii-Mainini - intestinal obstruction
• AU = Male frog Cystic fibrosis is a disease with multi-system
• MOI = Urine/Serum injected in the skin of the frog involvement (multiple organs are affected)
(subcutaneous)
• PR = Spermatogenesis • Diagnosis = accurate measurement of the sweat
• Sensitivity = Variable (will depend on the lab and chloride concentration
their reference values)
GIBSON AND COOKE PILOCARPINE
3. Friedman/Hoffman
IONTOPHORESIS = used for the detection of cystic
• AU = Virgin Female Rabbit fibrosis ; determines the sweat chloride concentration
• MOI = Urine is injected in the Marginal ear vein
(big vein on the ear) of rabbit • Pilocarpine + mild current = stimulates sweat
glands to induce sweating of the patient so you can
• PR = formation of the Corpora lutea and corpora collect sweat for testing
hemorrhagica Pilocarpine = medication that allows a person
• Sen = 10-15 IU/mL to sweat
Corpora lutea : dynamic endocrine glands mild current = kuryente ng konti
within the ovary that plays an integral role in
regulation of the menstrual cycle and early When you have collected your sweat sample you
pregnancy (Ncbi) have to check the chloride and sodium levels in the
corpora hemorrhagica = bleeding of the sweat
corpora lutea 

 sweat sodium is used as a quality control (QC) REFERENCE VALUES
since discordant values between both ions suggest
that there will be problems/ or there are problems
related to the collection or analysis BASAL ACID OUTPUT men: 0 - 5 mmol/hour
- in short SODIUM has NO DIAGNOSTIC VALUE (BAO) women: 0 - 4 mmol/hour
MAXIMAL ACID men: 5 to 26 mmol/hour
• Expected (normal) values: < 60mmol/L OUTPUT (MAO) women: 7 to 15 mmol/hour
• infants younger than 6 months: < 30 mmol/L
- Borderline for CF: 30-59 mmol/L 

GASTRIC STIMULANTS
(in infants less than 6 mo.)
Test Meals: food used to clinically stimulate gastric
GASTRIC FLUID secretion
• fluid found the stomach; important in digesstion • Ewald’s = bread and water
• best fluid to represent the status/condition of the • Boa’s = oatmeal
stomach
• as mechanical digestion begins (chewing) gastric fluid • Riegel’s = beef steak and mashed potato
is also being prepared
• Heckman’s = egg albumin, water, methylene blue
how do you digest food after chewing? • Stasis = rice, raisins
- food will travel through the esophagus and it will
go to the stomach • Fischer’s = Ewald’s meal + hamburg stock
- gastric fluid in the stomach contains: secretions, • Lavine’s = ethyl alcohol, methylene blue
enzymes, & acid that will help digest the food
• Motor = spinach or raisins + water
CELLS OF THE STOMACH • Salzer = beef, lambchop, milk, rice, egg
• Dock’s = biscuit
1. Specialized G cells = Produce gastrin
• gastrin: tells the parietal cells to do their job Chemical Stimulants: chemicals used to stimulate
gastric secretions
2. Parietal Cells= Produce HCL and Intrinsic Factor • Pentagastrin – most preferred
• HCl promotes acidity in the stomach and acidity for • Insulin – assess vagotomy procedure
stomach is needed to digest food
vagotomy procedure: procedure done in the
• Intrinsic Factor – needed for Vitamin B12 absorption
- pernicious anemia : problem with intrinsic cranial nerve
factor production; manifested by depleted • Histalog (Betazole)
stomach acidity • Histamine
3. Chief Cells = Produce pepsinogen Sham Feeding
• Pepsinogen: inactive enzyme (zymogen) • Fictitious Feeding: you give food and you
- Pepsinogen is converted to Pepsin (activated recover the food before it has been wholly altered
form) due to the acidity of the stomach-with by the digestive processes
the help of Hydrochloric acid • Food used: sandwich
• Pepsin: enzyme needed to digest proteins Usually done in experiments when you want to
study the gastric fluid status in the digestive
SPECIMEN COLLECTION (GASTRIC FLUID) process and psychological processes that affect
digestion
Method of collection: aspiration
1. Levin Tube = passed through the nose REPRESENTATIVE NORMAL AND ABNORMAL
2. Rehfuss = passed through the mouth GASTRIC ANALYSIS RESULTS

TYPE OF SPECIMEN – DURATION OF COLLECTION BAO mEq/hr MAO mEq/hr BAO/MAO

ratio
1 hour collection = Four
BASAL ACID OUTPUT NORMAL 2.5 25.0 10%
specimens collected evey 15
(BAO) - total gastric
minutes (but a single 1-hour
secretion during Pernicious
can be used) 0 0 0
unstimuted, fasting state Anemia
(walang laman ang tiyan)
2 hour collection = For Duodenal
insulin hypoglycemia test 5.0 30.0 17%
Ulcer
1 hour collection = Four Zollinger- 18.0 25.0 72%
specimens collected evey 15 Ellison
MAXIMAL ACID OUTPUT minutes; used when
(MAO) - total gastric Pentagastrin and Histamine Pernicious Anemia - can have little HCl or none at all
secretion after are used Duodenal Ulcer - ulcer is associated with hyperacidity,
gastric stimulation increased acid even without stimulation
2 hour collection = For
insulin hypoglycemia test Zollinger-Ellison - tumor in the pancreas that can
and when Histolog is used produce their own gastrin
MACROSCOPIC EXAMINATION (GASTRIC FLUID) SPUTUM
Color • comes from deep expectoration from the lungs
• Pale Grey/Mucus – normal • From both Upper Respiratory Tract and Lower RT
▪ Yellow-Green – large amount of bile • Mixture of plasma, electrolytes, mucin and water
mucin: make up the mucoid structure of the
▪ Red – small amounts of fresh blood sputum
▪ Coffee ground – Large amount of blood
COLLECTION
Volume
▪ Few mL – 50 mL = normal fasting specimen MANNER INDICATION
▪ >50 mL = Abnormal fasting specimen
1st morning Most preferred (routine)
▪ 20-60 mL up to 120 mL = after Ewald’s test meal
▪ 45-150 mL = after alcohol test meal or histamine 24-hour For volume measurement
stimulation For pediatric patients ; because it is hard
Throat swab
to tell kids to expectorate
TERMINOLOGIES (GASTRIC FLUID)
Sputum induction For non-cooperative patients
• Euchlorhydria = Normal free HCl
Tracheal aspiration Debilitated patient (comatose patient)
• Hyperchlorhydria = Increased free HCl = peptic
ulcer SPUTUM MACROSCOPIC EXAMINATION
• Hypochlorhydria = Gastric fluid pH > 3.5 but falls Volume:
after gastric stimulation (decreased free HCl) • decreased in: bronchial asthma, acute bronchitis,
early pneumonia, stage of healing
• Achlorhydria = Gastric fluid > 3.5 and does not fall
even after gastric stimulation (absence of free HCl) • increased in: bronchiectasis, lung abscess, edema,
gangrene, PTB, pulmonary hemorrhage
- Pernicious Anemia
• Anacidity = Failure to produce pH <6.0 following Odor: Normal if odorless
gastric stimulation = Pernicious Anemia
• foul or putrid = lung gangrene, advance
QUALITATIVE TESTS FOR FREE HCL necrotizing tumors
• sweetish = bronchiectasis, PTB
• Dimethylaminoazobenzol: cherry red (positive) • cheesy = necrosis, tumors, empyema
• Gunzberg • fecal = liver abscess, enteric Gram Negative
- Reagents: Phloroglucin, vanillin, alcohol bacterial infections
- purple red color (positive) Color:
• Boas • colorless or translucent = made up of mucus only
- Reagents: Resorcinol, Cane Sugar, Alcohol (normal)
- Rose red color (positive)
• White or yellow = purulent sample; ↑ pus
(pulmonary tuberculosis, bronchitis, jaundice,
QUALITATIVE TESTS FOR FREE HCL pneumonia)
• Gray = ↑ pus and epithelial cells
Combined HCl
FREE HCl Total Acidity (bound to • Bright green or greenish = ↑ bile; Pseudomonas
proteins) aeruginosa infection, lung abscess
Titrant NaOH NaOH NaOH
Pseudomonas aeruginosa: ca secrete
pH Dimethylaminoazobenzol green pigment
(Topfer’s reagent) Phenolphthalein Na alizarin
Indicator • Red or bright red = fresh blood or hemorrhage,
Endpoint Canary Yellow Faint Pink Violet pulmonary tuberculosis, bronchiectasis
• Anchovy sauce or rusty brown = old blood,
Normal 25-50 mEq/L 50-75 mEq/L 10-15 mEq/L pneumonia, gangrene
Value
• Prune juice = pneumonia, chronic lung cancer
• Olive green or grass green = lung cancer
Lactic Acid – may sometimes be tested in the gastric • Black = inhalation of dust or dirt, carbon/charcoal,
anthracosis, smoking
fluid; indicative of advanced gastric cancer/gastric
stagnation • Rusty with pus = lobar pneumonia; caused by
gastric stagnation: no emptying of the Streptococcus pneumoniae
stomach • Rusty without pus = congestive heart failure
Test Reagents Endpoint • Currant jelly-like = K. pneumoniae
Klebsiella pneumoniae: causes very
Modified Uffelmann’s FeCl3 + pheno Yellow hemorrhagic type and necrotizing
Strauss FeCl3 + ether YELLOW pneumonia; causing severe destruction of
cells & cell death = clot like/jelly sputum
Kelling’s FeCl3 YELLOW
Consistency: Pigmented cells
• Mucoid = asthma, bronchitis - Heart failure cells: hemosiderin-laden
• Serous or frothy = lung edema macrophages; found in Congestive heart failure
- Carbon-laden cells: angular black granules;
• Mucopurulent (mucoid + pus) = bronchiectasis, found in heavy smokers
pulmonary tuberculosis with cavities (butas sa
lungs) Curschmann spirals
bronchiectasis = enlargement of the airway - Coiled mucus strands
- Can also be observed
macroscopically and
SPUTUM MACROSCOPIC STRUCTURES microscopically
no need for microscope since they are large enough to - Bronchial asthma
be seen by the naked eye
Dittrich’s plug Myelin globules
- yellow or gray material, pinhead - Colorless globules occurring
size in a variety of sizes and
- Foul odor when crushed bizarre forms
- Bronchitis, bronchiectasis, - No clinical significance BUT
may be mistaken as the
bronchial asthma fungus Blastomyces
dermatitidis (since both are
Lung stones (“pneumoliths” or “bronchitis stones”) globular)
- Yellow or white calcified TB structures or foreign
material Epithelial cells (“Creola bodies”)
- Chronic bronchitis - cluster of ciliated columnar
cells
Bronchial casts ciliated columnar cells:
- Branching tree-like casts of the lining of the respiratory
tract
bronchi
- Lobar pneumonia - Bronchial asthma: increased
sloughing of the epithelial cells
(Streptococcus pneumoniae),
bronchitis, diphtheria
diphtheria: alot of discharge produced Fungi
by the neck that may drip down the - C. albicans, C. neoformans, C. immitis, H.
lungs capsulatum, B. dermatitidis, A. fumigatus
Layer formation – found during bronchiectasis, lung
abscess, gangrene Parasites
has 3 layers: - Migrating larva: A. lumbricoides, S. stercoralis,
- 1st or top layer – frothy mucus Hookworm (ASH)
- 2nd or middle layer- opaque, water material - E. histolytica, E. gingivalis, T. tenax, P.
westermani, E. granulosus, T. canis
- 3rd or bottom layer – pus, bacteria, tissues
- Others: neoplastic cells, bacteria, leukocytes
Foreign bodies if saliva no leukocytes
- bronchial calculi –made up of calcium carbonate
and phosphates
- Asbestos bodies, silica particles BRONCHOALVEOLAR LAVAGE (BAL)
- pneumoconiosis (i.e. Black lung disease) • A procedure in which a bronchoscope is passed
through the mouth or nose into the lungs and fluid
(saline) is flushed into a small part of the lung and
SPUTUM MICROSCOPIC STRUCTURES then recollected for examination
Lavage means washing
Elastic fibers
• Washings of the lungs will provide sampling of the
- Slender fibrils with double contour and curled ends components of the epithelial lining fluid of the lungs
- pulmonary tuberculosis (PTB)
in the BAL you can see:
- Body cells
Charcot-leyden crsytals (specifically lung
cells)
- Colorless, hexagonal, double
pyramid, often needle-like - Microorganisms
- Bronchial asthma: allergic - Pneumocystis
condition (primary pathogen)
- degratory products of eosinophils Important diagnostic test
for P. jirovecii (P. carinii)
in immunocompromised
patients
 P. jirovecii (P. carinii) = usually harmless
fungus but can cause pneumonia in
immunocompromised individuals like AIDS
patients

Macrophage
• 56-80% - most predominant
• specifically Alveolar macrophage (“Dust Cells”)

Lymphocyte
• 1-15% - interstitial diseases, pulmonary lymphoma,
nonbacterial infections

Neutrophil
• < 3% - cigarette smokers, bronchopneumonia, toxin
exposure

Eosinophil
• <1-2% - hypersensitivity reactions

Ciliated columnar bronchial epithelial cells

• 4-17%

Other microorganisms seen in BAL

• Pneumocystis jirovecii, Toxoplasma gondii,
Strongyloides stercoralis, Legionella pneumophila,
• Cryptococcus neoformans, Histoplasma
capsulatum, Mycobacterium tuberculosis,
Mycoplasma pneumoniae, influenza A and B
viruses, and respiratory syncytial virus.

Cytologic structures
• Sulfur granules (actinomycetes infection),
hemosiderin-laden macrophages, Langerhans cells,
cytomegalic cells, fat droplets seen in fat embolism,
and lipid-laden alveolar macrophages.

* Hi po! Sir Koudai says: Read more of BAL at the

appendix of Stransinger - DONT overthink this and take
them as they are - Study Well. Stay safe. God bless :> *

“Some people can’t believe in themselves until someone else

believes in them first.”
I believe in you 😊
🔅🔅🔅
Good luck and God Bless!
 MLS 419: AUBF LAB
FINALS WEEK 4 – LESSON 4
AUTOMATION IN URINALYSIS

URINALYSIS AUTOMATION
- Several instruments have been developed to partially or
completely automate routine urinalysis
- In addition to enhancing the workflow, automation can
standardize some aspects of manual urinalysis
- Most of these instruments can be interfaced with
Laboratory Information System (LIS), thereby
facilitating reports and results retrieval
- In general, equipment in the laboratory is necessary to
ensure an accurate, reliable, and timely testing. It
helps to maintain a high level of laboratory performance
- Goal: Improved reproducibility and color discrimination
while increasing productivity and standardization for
reporting urinalysis results.
- Standardize:
o Sample processing
o Analyze test strips
o Perform urine sediment analysis
o Report results
- Features:
1. Online computer capability
o With Laboratory Information System (LIS)
 Interface that manages inputting,
processing, and storing of data
operations
2. Bar coding
o For identification of samples
3. Manual entry of color
4. Clarity
5. Microscopic results
6. Flagging of abnormal results
7. Storing of patients and control results
8. Minimal calibration
9. Cleaning
10. Maintenance

CLASSIFICATIONS
1. Semi-automated analyzers
o The operation still depends on the medical
technologist – for specimen mixing, tests, strip
dipping, and microscopic results input SEMI-AUTOMATED URINE CHEMISTRY ANALYZERS
2. Fully-automated chemistry analyzers - Test for chemical
o The sample tubes of urine are placed on a rack components of urine
or a carousel and move automatically through - Read and interpret the
the instrument reagent strip results
o Still operated by the medical technologist - Eliminates bias in reagent
3. Automated urine cell analyzers pad color analysis and
o Mixing, aspiration, dilution, and staining of urine reading discrepancies
to classify the urine sediment particles - Leukocyte, nitrite, protein, blood, glucose, ketone,
4. Automated urine system bilirubin, urobilinogen, pH, specific gravity, color,
o Performs a complete urinalysis that includes the creatinine, and protein-to-creatinine ratio
physical, chemical, and microscopic parts of a - For small-medium volume laboratories and
routine urinalysis physician’s offices
o Also called as the “walk-away machines” - Self-calibrating and perform automatic checks (Auto-
because the medical technologist will just feed Checks)
the urine samples and let the machine perform o Identify strip type and humidity exposure
all the testing in the urine. The medical - Manually dipped  Placed in strip  Reader 
technologist will just come back once the Analyzing the strip  Dispose strip in waste container 
processing is done. Results are transmitted to the LIS
- Patient identification, specimen color, clarity – o Reflectance photometry – the diffused light
entered manually or barcode reader illuminates a reaction mixture in a carrier and the
- Positive results are flagged – requires confirmation reflected light is measured
testing or microscopic evaluation - Specific gravity – refractive index methodology
- Minimal daily maintenance – cleaning reagent strip o Measuring the concentration of a solute in an
platform and emptying reagent strip waste container. aqueous solution
- Clarity – transmitted or scattered light
- Integrated bar-coded sample identification
Dip the reagent strip into a well-mixed urine sample
- Abnormal ranges to be selected – microscopic
examination or confirmatory testing will be identified and
flagged.
Blot the strip to remove excess urine - Patient results, quality control results, calibrations –
stored for visual display, print-out, or transmission to a
laboratory computer system.

Place the strip onto the reagent strip platform

Press the analyzer

AUTOMATED MICROSCOPY
- Provide standardized results
in less than 1 minute
- Cost-effective and improve
turnaround time
- Sysmex UF-1000i and iQ
200 (Iris Diagnostics)
FULLY AUTOMATED URINE CHEMISTRY ANALYZERS Sysmex UF 1000i
- High-volume urinalysis
laboratory
- User “walk-away”
capability
- Ability to load many samples - Laser-based flow
with the capability to insert a cytometry
STAT specimen during the run. - Forward light
- Press the button to begin testing and samples move scatter, side scatter,
automatically through the instruments fluorescence
staining
Specimen Identification characteristics, and
adaptive cluster
analysis to identify
Aspirates urine sample and dispenses directly onto stained urine
reagent strip sediment particles
- 4 mL of uncentrifuged urine
- 2 channels:
Analyze by reflectance photometry 1. Urine particle analysis
2. Bacteria staining and detection
- You can easily detect the contaminators, inflammations,
and bacterial and mycotic infections
Dispose strip test automatically to waste box - Staining the internal components of the cells
- In the bacterial channel, the diluent stabilizes the pH
- Leukocytes, ketones, protein, glucose, nitrite, blood, and lyses the non-bacterial particles
urobilinogen, pH, bilirubin, color, clarity, creatinine, and - The stain is specific to the RNA in a bacterial cell,
protein. eliminating the non-specific staining of the debris
- Color – reflectance photometry or spectrophotometry - Passes through the flow cell, where it is
hydrodynamically focused and presented to a red semi-
conductor laser (635 nm)
- Identified by measuring the height and width of the iQ 200 Automated Urine Microscopy Analyzer
fluorescent and light scatter signals, which are presented - Produces a shorter turnaround time with standardized
in scattergrams and histograms. results
- Digital imaging and
Auto-Particle
Recognition
- Preclassify urine
particles in
uncentrifuged urine
based on size and shape
- By leveraging the digital
flow morphology technology using the auto-particle
recognition software, the urine particles are identified and
- The width of the fluorescent signal measures cellular characterized on the screen to virtually eliminate the
inclusions and the width of forward light scatter measures need for manual microscopy
the length of cells - Can be used also for body fluid cell counts – add body
- Resulting values are presented in quantitative cells per fluids software module
microliter and cells per high- or low-powered field - Mixes sample and aspirates approx. 1 mL of urine to a
planar flow cell
- 500 digital photomicroscopic images taken per sample

- Strobe light passes through the collimator in a single

beam
- Red blood cells - Specimen is surrounded by lamina – orient the particles
(RBCs), white blood for microscopic viewing
cells (WBCs), - Digital video camera takes 500 pictures as the specimen
epithelial cells, hyaline passes through the flow cell
casts, and bacteria. - Digital images are sent to the computer
- The signal intensity, - Auto-Particle Recognition (APR) software
signal width, signal - Preclassifies urine
waveform area, signal particles based on size,
waveform of forward shape, texture, and
scattered light, side contrast into 12
fluorescence light, categories: RBCs, WBCs,
side scattered light, WBC clumps, Hyaline
and polarized side casts, unclassified
scattered light of each particle obtained in the flow casts, SECs, non-SECs, bacteria, yeast, crystals,
cytometry are analyzed and classified by applying mucus, and sperm.
differentiation of algorithms that take this data into - Results reviewed easily and reclassified by the operator
account without the need for manual microscopy.
- Flagged particles include pathologic casts, crystals, - Subclassify 27 additional categories to indicate the
small round cells (renal tubular epithelial cells or specific types of crystals, casts, epithelial cells, yeast
transitional epithelial cells), sperm, with pseudohyphae, trichomonads, and oval fat bodies
mucus, and yeast-like cells, and must - You can add free text comments to the report as needed
be confirmed by manual microscopy.
sedMAX (77Elektronika)
- Performs automated microscopy
with digital imaging
- URISED 3 PRO
o Professional automated
urine sediment analyzer
with a revolutionary new
optical system combining the bright-field and
phase contrast microscopy
o Offers a uniquely advanced method for
visualization and recognition of formed elements
in urine samples
- By automating the traditional gold standard method - Bar-coded tubes are placed into the 10-position rack and
of sediment analysis, it increases reliability and improves moved to the iChem VELOCITY (mixed and the urine is
workflow aspirated)
- Decreases the turnaround time - Upon completion of the physical and chemical analysis,
- Requires 2 mL of urine centrifuged in a special cuvette the rack moves across the connecting bridge to the iQ
to produce a monolayer of urine sediment 200 for microscopy testing
- Sediment is analyzed by a bright-field microscope and - The sample can be reflected to urine microscopy based
digital camera upon the urine chemistry results
- Categorize 15 particle images based upon size and - The results are reported directly to the LIS, thereby
shape using image-processing software minimizing medical technologists’ follow-up
- Capability to zoom the viewed images
- Interpretation of the sediments is similar to manual
microscopic smears

AUTOMATED URINALYSIS SYSTEMS

- Combination of urine chemistry
analyzers and automated urine cell
analyzers
- Improved TAT and hands-on time will
be reduced
- Complete walk-away capability “Often when you think you’re at the end of something,
with minimal specimen handling from you’re at the beginning of something else.” – Fred Rogers
sampling through results
- Bar-coded samples are automatically identified and
processed
- Using the similar sample racks and moving on a
conveyor system, samples are easily transferred from
one instrument to the next
- Can independently perform both physical and chemical
testing, microscopy analysis, and a combination of both.
- A complete urinalysis report can be sent directly to the
LIS or printed out, thereby reducing clerical errors

CLINITEK AUWi System (Siemens)

- The Clinitek Atlas (automated urine chemistry analyzer)
+ Sysmex UF-1000i (automated urine cell analyzer) –
CLINITEK AUWi System (Siemens)
- You can walk away with confidence since they offer
complete integrated urinalysis for unattended operation
from start to finish
- Minimum of 5 mL of urine
- Rack advances to the ATLAS analyzer (identified,
mixed, aspirated, and
tested for physical and
chemical components)
- Sample travels across a
connecting bridge to the
UF-1000i for microscopic
analysis
- The instrument automatically reflects the samples
requiring the sediment analysis, thereby reducing the
time associated with manual microscopic analysis
- Automatic verified results are reported directly to the LIS,
which minimizes the medical technologists’ follow-up

iRICELL Automated Urinalysis Systems

- Combines urine chemistry and microscopy in a fully
automated walk-away cell that is easy to use and
maintain
- Optimizes and advances urine analysis and body fluid
testing in the laboratory with digital morphology
technology that uses an APR to deliver standardized
and accurate results
- Minimum of 4 mL of urine is required