| Topic # 2 | [MLS 419-LAB] Analysis of Urine and Other Body Fluids

P2: TYPES OF SPECIMENS AND PHYSICAL EXAMINATION OF

URINE
Professor: Mr. Christian Villahermosa, RMT, MSMT
Date:Jan. 29, 2024

TYPES OF SPECIMEN ○ If the patient voids at 7:30 am → not

included since the urine is formed before
RANDOM SPECIMEN
7am
● Easiest to collect ○ If the patient voids at 8 am → should be
● Most common specimen included (first sample)
● ideally collected midstream ○ In day 2 7am → the sample should not yet
○ Midstream be submitted, the patient should urinate for
■ Manner of collection of urine the final time
starting from the middle part of
the urine volume Summary
■ The first volume of urine contains ● First urine at day 1 → DISCARD
cells coming from the urethra, ● Urine after the time has ended at day 2 →
and tend to be large during a INCLUDED
microscopic exam MIDSTREAM CLEAN-CATCH SPECIMEN
● Can Cause Obsruction/ ● Safer, less traumatic method for obtaining urine for
hides important bacterial culture and routine urinalysis
sediments in the urine ● Less contamination
during examination ● Patients are provided with appropriate cleansing
● Actual time of voiding should be recorded materials (mild antiseptic towelettes or swabs), a
○ For one to be reminded that urine is only sterile container, and instructions
kept for 2 hours unpreserved ○ Cleaning the head of the penis or the folds
FIRST MORNING SPECIMEN of the labia
● Ideal screening specimen CATHETERIZED SPECIMEN
○ Concentrated specimen; assures detection ● Collection under sterile conditions by passing a
of chemicals and formed elements catheter through the urethra into the bladder
● Must be delivered to the lab within 2 hours or keep ● Commonly requested for bacterial culture
refrigerated ○ Not usually done for urine culture but a
● Essential to prevent false-negative urine pregnancy sample for patients who are in a coma
tests and to evaluate orthostatic proteinuria
○ Ideal for pregnancy test → the beta hCG,
which is the hormone tested in pregnancy
tests, has more chance to be detected in a
conc. urine
24-HOUR OR TIMED SPECIMEN
● To measure the exact amount of a urine
chemical, especially if a substance exhibits diurnal
variation SUPRAPUBIC ASPIRATION
○ To measure the exact amount of a urine ● External introduction of a needle through the
chemical → by the collection of all urine abdomen into the bladder
samples of a patient during the day ● Ideal for culture and cytology tests (urethra
■ since there are chemicals that bypass)
exhibit diurinal variation
● Used in clearance tests such as creatinine and urea
and hormones such as cortisol (dirunal variation)
● All specimens must be refrigerated or kept on ice
during collection
● In the lab, sample must be thoroughly mixed before
aliquoting
PROCESS OF COLLECTION OF A 24-HOUR URINE
● Example:
● Day 1 7 AM: patient voids and discards specimen;
collects urine for 24 hours 2-HOUR POST-PRANDIAL URINE
● Day 2 7 AM: patient voids and adds this urine to ● For monitoring diabetes mellitus
previously collected urine ● After meals → increase glucose, since insulin is just
starting to act

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● It takes insulin 2 hours to do its job PEDIATRIC SPECIMENS
○ 2 hours after meal, if one has a normally ● Neonates
functioning insulin and pancreas → blood ● Soft, clear plastic bags with hypoallergenic skin
glucose is normal adhesive (check every 15 minutes)
GLUCOSE TOLERANCE SPECIMEN ● Routine: ensure area is free from contamination,
● To correspond with the blood collection during GTT attach bag firmly over the genital area avoiding the
● Tests the patient’s ability to metabolize a measured anus
amount of glucose ● Microbiology: clean the area with soap and water,
● Compare plasma glucose and urine glucose dry the area, and firmly apply the sterile bag
(directly proportional) ● Samples must be transferred to a sterile urine
1. Extraction for fasting blood sugar and container upon submission to the laboratory
collect urine before drinking
2. After drinking → wait for an hour
(depending on the req form) → extraction
and collect urine
PROSTATITIS SPECIMENS
● To determine if there is infection in the prostate
glands
● Types:
○ Three-Glass Collection (3 specimens)
○ Pre- and Post-Massage Test (2 DRUG SPECIMEN COLLECTION
specimens) ● Urine collection is the most vulnerable part of drug
○ Meares-Stamey Test (4 specimens) testing
THREE-GLASS COLLECTION ● Chain of custody (COC): the process that
● Specimen 1: first urine voided documents sample identification from time of
● Specimen 2: midstream portion (Prostate collection to the receipt of results
massage) ● All personnel handling the specimen must be noted
● Specimen 3: remaining urine (w/ prostate fluid) ● May be ‘witnessed’ or ‘unwitnessed’
● Prostate infection: if the third specimen has a ADULTERATION
WBC/hpf and bacterial count 10 times that of the ● with a substance not normally present in the test
first specimen specimen
● NOTE: if the 2nd specimen is positive, the results ● e.g. bleach (slows Ab-Ag reaction), ammonia/liquid
are invalid (infected urine contamination) ● soap/table salt (increase pH), hydrogen peroxide
○ the 2nd specimen represents urine coming (destroys 50% of THC), vinegar (decreases pH)
from the bladder, if contaminated, one is DILUTION
not sure if the contamination is from the ● less than normal physiological constituents
bladder (2nd specimen) or the prostate (diuretics, water)
itself (3rd specimen) SUBSTITUTION
PRE- AND POST-MASSAGE TEST
● Commonly used PARAMETERS FOR VALIDITY TEST
● Specimen 1: midstream clean-catch prior to ● Color
massage (Prostate massage) ● Appearance: clear
● Specimen 2: urine sample after massage ● Odor: aromatic
● Positive result: bacteriuria in the post-massage ● Volume: 30-60 mL
(2nd specimen) specimen of greater than 10 times ● Temperature: 32-38 degrees C
the pre-massage count (specimen 1) ● pH: 4.0-9.0 (adulterated if <3 or >11)
MEARES-STAMEY TEST ● Specific Gravity: 1.003-1.030 (diluted: <1.003)
● Specimen 1/VB1: initial voided urine, 10 mL (test ● Creatinine: if < 20 mg/dL, sample is diluted
for urethral infection or inflammation) PHYSICAL EXAMINATION OF URINE
● Specimen 2/VB2: midstream urine (test for bladder STEPS IN URINALYSIS
infection) (Prostate massage) ● Specimen Collection
● Specimen 3/EPS: expressed prostatic secretions ● Sample Verification
○ Prostate fluid should be collected ● Physical Examination of Urine
separately ● Chemical Examination of Urine
● Specimen 4/VB3: post-prostatic massage urine ● Microscopic Examination of Urine
specimen, 10 mL ● Release of Results
● Target of a positive result: Specimen 3 and 4
(produced after massage)
● Abnormal result: more than 10-20 WBCs per hpf WHAT HAPPENS IN PHYSICAL EXAMINATION?
on EPS and/or VB3 ● Determination of urine: color, clarity (and specific
gravity)

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 ○ S.G. is a physical property of urine. SPECIFIC GRAVITY
However, it is included in the chemical ● Density of a solution compared with the density of a
examination of urine similar volume of distilled water (SG 1.000) at a
○ Odor: not part of routine physical similar temperature
examination but is a noticeable property ● Measures the density of the dissolved chemicals
■ Only if there is strange odor in urine
(maple syrup urine disease) → ● The higher the S.G. = More solutes present
report ● Evaluation of urine concentration
● Physical examination provides preliminary ○ Measures kidney’s ability to concentrate
information concerning disorders glomerular filtrate
● Can be used to confirm or to explain findings in ■ If a patient is dehydrated →
chemical and microscopic examination kidney reabsorbs water →
URINE COLOR excrete more solutes →
● Varies from almost colorless to black increased S.G.
● A noticeable change in urine color is often the ○ Determines adequacy of specimen
reason that a patient seeks medical advice concentration to ensure accuracy of
● Common urine colors: pale yellow, yellow, dark chemical tests
yellow ● Normal random specimens: 1.002-1.035
● Urochrome ○ Most random specimens: 1.015-1.030
○ the pigment that causes the yellow color ● SG of plasma filtrate leaving the glomerulus: 1.010
○ Normal waste product of the body ○ Isosthenuric: 1.010
● Color assessment should be done only once urine ○ Hyposthenuric: below 1.010
is transferred from container to test tube ○ Hypersthenuric: above 1.010
URINE CLARITY ● Influenced not only by the number of particles
● General term that refers to the transparency or present but also by their size
turbidity of a urine specimen ○ Presence of large molecules requires
● Visual examination of a mixed specimen while correction (in some methods of SG
holding it in front of a light source determination)
○ Paper with text should be at the back of ○ Even if proteins are only few in urine, but
the specimen since they are large → increased specific
● May be reported as: clear, hazy/slightly cloudy, gravity
cloudy, turbid, and milky MEASUREMENT OF SPECIFIC GRAVITY
● Urinometry
CLARITY DEFINITION
● Harmonic oscillation densitometry
Clear No visible particulates, transparent ● Osmolality
● Refractometry
Hazy Few particulates, print easily seen through ● Reagent strip
urine URINOMETRY
● Uses a urinometer
Cloudy Many particulates, print blurred through ● Principle: buoyancy
urine ● A weighted float attached to a calibrated scale
● Displaces a volume of liquid equal to its weight
Turbid Print cannot be seen through urine
● Designed to sink to a level of 1.000 in distilled water
Milky May precipitate or be clotted ● Additional mass causes the float to displace a
volume of urine smaller than that of distilled water
(will float higher in urine)
● The level to which the urinometer sinks represents
the specimen’s SG
● The more solutes present in the urine sample →
higher buoyancy → higher S.G.

URINE COLOR AND CLARITY PROCEDURE

● Evaluate an adequate volume of a specimen
● Use a well-mixed specimen
● View the urine through a clear container
● View the urine against a white background using
adequate room lighting
● Maintain adequate room lighting
● Evaluate a consistent volume of urine
● Determine color and clarity

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 STEPS IN URINOMETRY
● Allow urine to reach room temperature OSMOLALITY
● Fill the glass cylinder with urine up to 2/3 (at least
● The number of particles of solute per kilogram of
15 ml of urine)
solvent
● Make sure that there are no bubbles/foam in the
● Measurement of the colligative properties of a
glass cylinder.
solution and comparing their value with the value of
● Gently drop the urinometer into the container. Make
a pure solvent (osmometer)
sure the urinometer does not touch the bottom or
the sides of the container and allow it to float freely
● Impart a slight spin to the float as it is released
● Read the calibrated graduation at the lower
meniscus
DISADVANTAGE OF USING URINOMETER
● Not recommended by the CLSI because of the
following:
REFRACTOMETRY
○ Large volume of urine is needed
● Uses a refractometer
○ Turbid specimens are difficult to read
● Measures the refractive index (comparison of the
TEMPERATURE CORRECTION
velocity of light in air with the velocity of light in a
● Add 0.001 for every 3°C above the calibration
solution)
temperature
● Concentration of dissolved particles present in the
● Subtract 0.001 for every 3°C below the calibration
solution determines the velocity and angle at which
temperature
light passes through a solution
SOLVING
● The more conc. urine = more changes in the
refractive index
STEPS IN USING A REFRACTOMETER

LARGE SUBSTANCE CORRECTION

● Subtract 0.003 for each gram of protein present
● Subtract 0.004 for each gram of glucose present
SOLVING

HARMONIC OSCILLATION DENSITOMETRY

● Frequency of a sound wave entering a solution U.G. = S.G.// nD = refractive index
changes in proportion to the density of the solution
ADVANTAGE DISADVANTAGE
● Uses a U-shaped instrument with electromagnetic
coil in one end and motion detector in the other end ● Small volume of Correction for protein and
● Electric current is applied and allows sound waves specimen glucose
to pass through the solution ● No temperature
● Amount of sound frequency reflected = density corrections
● More solutes = more changes in the sound wave

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 CALIBRATION
● Use distilled water that should read 1.000
● Use 5% NaCl that should read 1.022 ± 0.001
● Use 9% sucrose that should read 1.034 ± 0.001
● Urine control samples with low, medium, and high
concentrations
REAGENT STRIP
● More convenient way
● Based on the change in pKa of a polyelectrolyte in
alkaline medium
● Polyelectrolyte ionizes, releasing hydrogen ions in
proportion to the number of ions in the solution
● More concentrated urine, more hydrogen ions
released, pH is decreased
● Indicator: bromthymol blue (measures the change in
pH)
○ Blue (1.000/alkaline) to green to yellow
(1.030/acid)
○ 0.005 intervals

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 | Topic #2 | [MLS 419-LEC] Analysis of Urine and Other Body Fluids

P2: Tubular Reabsorption and Secretion Part 1

Professor: Mr. Christian Villahermosa, RMT, MSMT
Date: January 22, 2024

INTRODUCTION ○ Against its gradient → it will not diffuse, it

● Once the blood enters the glomerulus from the is forcibly transported
afferent arteriole, it undergoes filtration. ○ A substance will move from an area of low
● The filtrate goes into the bowman’s space and will concentration to an area that is already
proceed to the proximal convoluted tubule. present in high concentrations
● The filtrate will undergo reabsorption and secretion ● Needs a carrier protein to transport substance
until it will form urine. and requires energy (e.g., hydrolysis of ATP)
○ The filtrate will be called urine if it no PASSIVE TRANSPORT
longer undergoes reabsorption and ● Movement is due to differences in the concentration
secretion. or electrical potentials (may be TRANSCELLULAR
TUBULAR REABSORPTION or PARACELLULAR)
● The body cannot lose 120-125 mL of water every ● Diffusion → a substance will naturally move from
minute an area of higher concentration to an area of lower
● Therefore, some substances (e.g., water, concentration to achieve equilibrium
electrolytes, glucose) in the urine need to be ● Does not need carrier protein and does not
REABSORBED require energy
● Tubular reabsorption refers to the return of most 2 PATHS THAT INVOLVE THE MOVEMENT OF
of the filtered water and many solutes from the SUBSTANCES INTO THE TUBULAR CELL
tubules to the bloodstream TRANSCELLULAR
○ About 99% of filtered water reabsorbed ● The substance will enter the cell and leave via that
■ If the kidney will not reabsorb → same cell until it reaches the peritubular capillaries
urination every minute PARACELLULAR
○ Proximal convoluted tubule cells make ● The substance will move from the lumen of the
the largest contribution tubule into the blood vessels via the tight junctions
● Reabsorption is highly selective (space between the cells)
○ Only essential substances are reabsorbed
FILTRATION, REABSORPTION, AND EXCRETION RATES
OF DIFFERENT SUBSTANCES BY THE KIDNEYS

Substances Amount Amount Amount % of filtered

filtered reabsorbed excreted load
reabsorbed

Glucose 180 180 0 100

(g/day)

Bicarbonate 4320 4318 2 >99.9 BULK FLOW/ULTRAFILTRATION

(mEq/day) ● Movement of substances from the tubular cell into
the peritubular capillaries
Sodium 25,560 25,410 150 99.4 ○ The substance crosses the interstitial fluid
(mEq/day) BRUSH BORDERS
● Fingerlike projections which contain microvilli
○ The presence of brush borders increases
Chloride 19,440 19,260 180 99.1
(mEq/day) surface area = increases reabsorption of
substances present in the lumen
Potassium 756 664 92 87.8 REABSORPTION OF SODIUM
(mEq/day) ● Sodium diffuses across the luminal membrane
(also called the apical membrane) into the cell down
Urea 46.8 23.4 23.4 50 an electrochemical gradient (passive transport)
(g/day)
○ Luminal membrane → membrane of the
tubular cells facing the lumen
Creatinine 1.8 0 1.8 0
(g/day) ● Sodium is transported across the basolateral
membrane against an electrochemical gradient by
ACTIVE TRANSPORT the Na+-K+ ATPase pump (Sodium-Potassium
● Movement of substance across a cell membrane ATPase pump)
and against an osmotic gradient (always ○ Basolateral membrane → membrane
TRANSCELLULAR) facing the interstitial fluid

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 SODIUM-POTASSIUM ATPASE PUMP ● Smaller proteins that goes in the proximal
● Uses ATP convoluted tubule → the kidney will reabsorb them,
● Its action is to release sodium since they are essential
○ Since sodium is extracellular, it should not PROTEINS
be present ● PCT reabsorbs proteins via pinocytosis
○ In return for releasing sodium → ○ Digestion into amino acids in vesicles
potassium enters the cell MEGALIN CUBILIN COMPLEX
● In the presence of the pump in the tubular cells → ● Found on the surface of the tubular cell (labelled as
expels sodium from the tubular cells into the M and C at the diagram below)
peritubular capillaries → back to the circulation ● Binds to any protein present in the filtrate
(reabsorbed) ○ When the protein binds to the complex →
undergoes pinocytosis → leads to the
entry of megalin and cubilin + protein
inside a vesicle
○ The vesicle will merge with a lysosome →
the proteins will be degraded into a simpler
amino acid
○ The amino acid will be sent back to the
REABSORPTION OF GLUCOSE circulation for recycling
● An example of secondary active transport
● Glucose is increased inside the cell
● Naturally, glucose would not enter the cell →
tubules will use transporters in order for it to enter
the cell
SGLT (SODIUM-GLUCOSE LINKED TRANSPORTER)
● “Entry point” WATER
● Carries glucose into the tubular cell cytoplasm
● Water reabsorption:
● Since glucose is elevated intracellularly, glucose
○ Due to aquaporins (found on the surface of
would not be attracted to the cell hence SGLT
the tubular cells) and tight junctions
should attract glucose.
■ water can either pass through
○ In order for sodium to diffuse into the cell,
intracellularly via aquaporin or
it has to pass through SGLT
tight junctions
○ When sodium enters the SGLT → It will go
○ Dependent on membrane permeability
out directly through the Na+-K+ ATPase
■ the different parts of our tubule of
pump
our nephron have their different
○ Since the pump would utilize ATP→ the
affinity to water
ATP released is enough to attract glucose
■ PCT/DLOH: always high due to
into the cell
abundant aquaporins
● Entry of glucose into the cell → active transport
● always reabsorb water
GLUT (GLUCOSE TRANSPORTER)
■ ALOH: always low (low surface
● Helps glucose diffuse out of the cell (facilitated area and less permeable tight
diffusion) junctions
● Glucose is in the cell, however, it must leave the cell ● impermeable to water
to go to the circulation (reabsorption) → it will not reabsorb
○ Glucose is increased intracellularly and water
decreased extracellularly → diffusion ● the tight junctions are
occurs tighter compare to PCT
○ Glucose will leave the cell via GLUT ● Has less surface area
● Exit of glucose from the cell → diffusion because there are less
brush borders

REABSORPTION OF PROTEINS AND WATER

● Albumin is the protein prioritised in the glomerulus
● Other smaller protein such as vitamins, hormones,
immune system products → they are still filtered by
the glomerulus

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 REABSORPTION OF UREA AND CHLORIDE LATE PCT
UREA ● Pars recta
● Half of the urea is reabsorbed ● sodium and chloride reabsorption
● Passive reabsorption via urea transporters LOOP OF HENLE
CHLORIDE THIN DESCENDING LOOP OF HENLE
● Transcellular passive diffusion ● Thin epithelial membranes with no brush
● Transported along with sodium due to lumen borders and few mitochondria
negative potential ○ Reabsorptive capacity is low
● Chloride will be reabsorbed only if there is a ● Simple diffusion of water and some solutes
negative potential in the lumen THICK ASCENDING LOOP OF HENLE
○ Negative potential: if sodium (cation) will ● Thick epithelial cells with high metabolic activity
easily diffuse into the cell → the lumen will ● Active reabsorption of sodium, potassium, and
become negative chloride via NKCC2 cotransporter
○ negatively charged lumen is not normal → ○ 1 sodium, 1 potassium, and 2 chloride at a
chloride will be reabsorbed time
■ since chloride is anion, the ● Note: Loop diuretics (furosemide, ethacrynic acid,
negative potential will decrease bumetanide) are inhibitors of NKCC2
ACTIVE TRANSPORT ○ diuretics can prevent sodium reabsorption
MAXIMAL REABSORPTIVE CAPACITY (Tm) ■ because they want sodium to
● Highest rate of reabsorption of a substance remain in the lumen and water
before it appears in urine; amount of solute would also remain → both of
reabsorbed per minute them will be urinated
● Unit: time/rate of reabsorption of all the nephrons in ● ALOH is impermeable to water
the kidney ○ As filtrate moves through the ascending
○ “How fast can the nephrons process a limb, NaCl moves out making the filtrate
solute?” more and more dilute
● Example: Glucose is at 350 mg/min. (once this is ● Significance: Keeping the renal medulla region of
reached, all nephrons have reached maximal the kidney at high osmolarity so water can move
capacity to reabsorb glucose) passively out of the filtrate in the CT
○ if beyond 350 mg/min → it will not be DISTAL CONVOLUTED TUBULE
processed → seen in the urine EARLY DISTAL TUBULE
RENAL THRESHOLD FIRST PORTION
● the plasma concentration of substances at which ● forms the macula densa
active transport of reabsorption stops SECOND PORTION
○ “What is the plasma concentration?” ● Reabsorber part of the DCT because of the
● Refers to the blood level of the substance presence of the Sodium-chloride co-transporter
○ Example: Glucose is at 160-180 mg/dL ● same reabsorptive characteristics of the thick ALOH
○ Note: When the plasma glucose ○ impermeable to water except for
concentration exceeds the transport electrolytes
maximum, reabsorption stops, and the ● Avid reabsorption of most ions
substance is excreted in urine. ○ Sodium-chloride co-transporter
● It is important because you have to correlate the ○ Note: Thiazide diuretics inhibit
positive urine glucose to the blood glucose of the sodium-chloride co-transporter
patient ● Virtually impermeable to water (requires ADH for
PARTS OF THE NEPHRON water reabsorption) and urea
PROXIMAL CONVOLUTED TUBULE ○ In special conditions, it can be permeable
● Major contributor of tubular reabsorption because it to water if there is ADH (vasopressin)
is highly metabolic (high number of mitochondria)
and contains many brush borders
● Substances reabsorbed (U.S.W.A.G):
○ Urea
○ Salt (Sodium and Chloride)
○ Water
○ Amino acid
○ Glucose
EARLY PCT
● Closest to the bowman’s capsule
● sodium reabsorption and co-transport with
glucose, amino acids, other solutes

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 | Topic # | [MLS 419-LEC] Analysis of Urine and Other Body Fluids
P1: DISTAL CONVOLUTED TUBULE, COLLECTING DUCT, AND CLEARANCE
TESTS
Professor: Mr. Christian Villahermosa, RMT, MSMT
Date: Jan 28, 2024

DISTAL CONVOLUTED TUBULE AND THE COLLECTING

DUCT
● The collecting duct is divided into two:
○ Cortical collecting duct → a part of the
collecting duct that is located in cortex and
functions similarly to the late distal tubule
○ Medullary collecting duct → a part of
the collecting duct that is located in the
renal medulla
LATE DISTAL TUBULE AND CORTICAL COLLECTING
TUBULES
● Both parts of the nephron contain Principal cells
MEDULLARY COLLECTING DUCT
PRINCIPAL CELLS
● Final site for urine processing
● Reabsorb sodium (controlled by aldosterone) via
● Permeability to water and urea is controlled by
Epithelial Sodium Channels (ENaC) and NKA
ADH
pump
○ Presence of aquaporins and urea
○ In normal cases, sodium reabsorption is
transporters (urea goes back to the DLOH)
minimal, however, if there is the presence
○ Urea is reabsorbed because the kidney’s
of aldosterone → reabsorption of sodium
renal medulla has to be preserved in a
○ Sodium to the blood vessels →
hypertonic environment → water can be
Sodium-Potassium ATPase pump
attracted and leave the collecting duct
● Secrete potassium via the Renal Outer Medullary
Potassium channel (ROMK)
○ When sodium is reabsorbed → negative
potential in the lumen → secretion of
potassium from the principal cells into the
lumen
● Note: Target of Potassium-Sparing Diuretics →
inhibits the activity of the principal cells
○ Needed by patients who need diuretic and
also experiencing hypokalemia
○ Two types: COUNTERCURRENT MECHANISM
■ Mineralocorticoid receptor ● What happens to the osmolarity of the filtrate as it
antagonists(e.g. Spironolactone) travels along the different parts of the nephron
● The drug will bind to the ● The renal medulla is hypertonic → water can easily
receptor of aldosterone be reabsorbed
→ Inhibits binding to the
principal cells
■ Sodium channel blockers (e. g.
Amiloride)
● Directly block epithelial
sodium channel
● Permeability to water depends on ADH (binds only
to aquaporin-2)
○ The late distal tubule and cortical collecting
tubules do not normally reabsorb water
○ If ADH is present → the tubular cells will
manifest aquaporin-2, which is the only
aquaporin that will respond to ADH → ● The filtrate that leaves the bowman’s space is 300
Water reabsorption mosm/L and enters the proximal convoluted tubule
● As it travels down the descending loop of Henle,
osmolarity increases up to 900 mosm/L
○ Since the DLOH can reabsorb water →
water is removed from the filtrate → what

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 remains inside is a very concentrated TUBULAR REABSORPTION
filtrate → increased osmolarity CONTROL OF WATER LOSS
● The highest increased level of osmolarity is found at ● How concentrated or diluted your urine is, depends
the “hairpin turn” since the movement/absorption is upon the body’s state of hydration:
slowed down ○ Dehydration = hypertonic urine (more
● The osmolarity is decreased at the ascending loop concentrated)
of henle since sodium/chloride is reabsorbed ■ High blood osmolarity in a
○ The NaCl removed from the ALOH dehydrated person → stim.
contributes to the osmolarity of the medulla Pituitary gland → release ADH →
○ When NaCl is removed → the osmolality of Increased aquaporin channels in
the medulla is increased → reabsorbs cell membrane → increased CD’s
water water permeability → less urine is
● The osmolality is also increased at the collecting formed
ducts since water is reabsorbed due to the ○ Drinking lots of water = hypotonic urine
presence of ADH (called water diuresis)
Summary of the concurrent mechanism ■ More water excretion
● Renal concentration begins in the loop of Henle, ■ Continuous urination → water
where filtrate is exposed to high gradient of the diuresis
renal medulla. TUBULAR SECRETION
● Water is reabsorbed through osmosis in the ● Passage of substances from the blood in the
descending loop of Henle peritubular capillaries into the tubules.
● Na and Cl are reabsorbed in the ascending loop
● Secretion happens due to:
of Henle
● Dilution of the highly concentrated medulla is ○ Elimination of waste products not filtered
prevented by the impermeable walls of the by the glomerulus (size and charge)
ascending loop to water. ○ Regulation of acid-base balance in the
● Therefore, most of the water delivered to this body through the secretion of hydrogen
segment remains in the tubule, despite ● Many foreign substances, such as medications, are
reabsorption of large amounts of solute.
not filtered by the glomerulus because they are
● Thus, the tubular fluid becomes very dilute and
results into diluted urine. bound to proteins, but when they travel to the
peritubular capillaries → loses their affinity to their
protein carriers→ they develop affinity to the
tubules, which causes them to dissociate from their
protein carriers = SECRETED
ACID-BASE BALANCE
● Regulates acid-base balance by eliminating either
hydrogen ions or bicarbonate ions
● Normal blood pH: 7.4 (excess acids must be
eliminated)
BICARBONATE IONS
● If alkalosis → the body will eliminate bicarbonate
ions, in exchange for the reabsorption of hydrogen
● Responsible for buffering capacity
○ Readily filtered and reabsorbed
HYDROGEN IONS
● If acidosis → the body will eliminate hydrogen ions,
in exchange for the reabsorption of bicarbonate
● Secreted by RTE cells into the filtrate
(sodium-hydrogen exchanger)
○ Prevents the filtered bicarbonate from
being excreted
● Hydrogen cannot be eliminated by itself, it has to
bind to a larger substance for it to be eliminated
● Excreted via:
○ Filtered phosphate ion
■ Hydrogen goes out the cell →
binds with hydrogen phosphate
→ to form dihydrogen phosphate
→ urine
○ Ammonia
■ Deamination of glutamine →
forms ammonia → hydrogen will

@mlstranses | 2
 bind to ammonia → to form
● Neither reabsorbed or ○ requires infusion at
ammonium ion secreted a constant rate (3-4
hours) because it is
not a normal body
constituent
● Impractical
RADIONUCLIDES
● Safe radioisotopes that is infused to the patient
● Sample: Plasma
● tested by their disappearance from the plasma,
thereby eliminating the need for urine collection.
CELLS THAT PERFORM ACID-BASE BALANCE ○ If 100mg of 99mTc-DTPA is infused to the
INTERCALATED CELLS patient, the same value should be seen
● Located in the Late distal tubules and cortical after collection
collecting tubules along with the principal cells ● Examples:
● Late distal tubules and cortical collecting tubules ○ 125I-iothalamate and 99mTc-DTPA
ALPHA-INTERCALATED CELLS (diethylene-triamine –pentaacetate)
● secrete hydrogen ions and reabsorb bicarbonate ○ 51Cr-EDTA
BETA-INTERCALATED CELLS
ADVANTAGES DISADVANTAGE
● secrete bicarbonate and reabsorb hydrogen ions
RENAL FUNCTION TESTS - GLOMERULAR FILTRATION Enables visualization of the More labor intensive and
RATE filtration in the kidneys costly
● About 1200 ml of blood (650 ml plasma) passes BETA-2-MICROGLOBULIN
through the kidneys every minute.
● forms part of the class I MHC present in leukocytes
● About 120-125 ml is filtered per minute by the
(11,800 kda)
kidneys & this is referred to as glomerular
○ REVIEW: MHC class I will connect
filtration rate (GFR).
antigen-presenting cells with cytotoxic t
GLOMERULAR FILTRATION TESTS
cell
CLEARANCE TESTS ● Dissociates at a constant rate from WBCs and is
● Gold standard rapidly removed from the plasma by the kidneys
● measures the rate by which the kidneys remove a
filterable substance from the blood.
● Sample used is 24-hour urine
CHARACTERISTICS OF AN IDEAL CLEARANCE
TEST SUBSTANCE
● The substance must be neither reabsorbed nor
secreted by the tubules
● The substance must be stable during the 24-hour
collection
● Substance’s availability in the body DISADVANTAGE
● Consistency of plasma level
● Availability of the test in the lab ● However, the test is not reliable in patients with
UREA immunologic disease or malignancy
● earliest glomerular filtration test ○ Patients with immunologic diseases are
prone to antigen presentation → more
ADVANTAGES DISADVANTAGE
beta-2-microglobulin production
● Also degrades in an acidic environment
● Present in all urine ● 50% of the filtered
specimens urea is reabsorbed by BETA TRACE PROTEIN
● Available lab methods the tubules
● Normally produced (endogenous)
● Endogenous ○ hydration needs to
○ From the normal be done ● A low-molecular weight protein isolated in the CSF
waste product of DISADVANTAGE
protein
metabolism
● Freely filtered and reabsorbed by the PCT
INULIN
TRYPTOPHAN GLYCOCONJUGATE
● polymer of fructose that is a prebiotic fiber
● Filtered freely and not reabsorbed
● considered as the gold standard in measuring
● Strong linear correlation with inulin clearance
GFR
DISADVANTAGE
ADVANTAGES DISADVANTAGE

● Highly stable ● Exogenous

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 ● Measured ONLY using HPLC (expensive and
time-consuming)

CYSTATIN C
● A small protein produced by all nucleated cells
● Sample: Serum (blood)
● Endogenous
● Readily filtered, reabsorbed, and broken down by
renal tubules
○ Normal levels in plasma: Low
■ Since the renal tubules can break
it down → content in blood is low/
zero → good GFR
○ Plasma level is inversely proportional to
GFR
○ Changes in serum concentration are used
as indirect estimate of GFR
● Potential marker for long-term monitoring of
renal function
ADVANTAGES DISADVANTAGE

● Constant in serum Higher analysis cost

levels
● Independent of age,
gender, and muscle
mass
● More sensitive to GFR
changes than serum
creatinine
CREATININE
● most widely used endogenous procedure
● Used in the philippines
● Not reabsorbed/secreted
● If the serum creatinine is increased → Kidney
failure (indirect testing)
DISADVANTAGE

● Secreted by tubules and secretion increases as

blood level increases
● Chromogens in the plasma can react in the
chemical analysis for creatinine
○ The machine might detect chromogens
as creatinine (false increase)
● Bacteria will break down urinary creatinine if
specimen is kept at room temp for extended
period
● A diet heavy in meat consumed during collection
of a 24hr urine will influence the creatinine level
● Not reliable in patients with muscle wasting
diseases
○ Increases creatinine
● Interference by medication (salicylate,
trimethoprim, cimetidine)

@mlstranses | 4
 | Topic # | [MLS 419-LAB] Analysis of Urine and Other Body Fluids

P3: Chemical Examination of Urine

Professor: Mr. Christian Villahermosa, RMT, MSMT
Date: February 6, 2024

CHEMICAL EXAMINATION OF URINE 3. Wait for the specified length of time (2 mins) for the
● Performed after the physical examination of urine reactions to take place and compare colored results
REAGENT STRIPS to the manufacturer’s chart under a good source of
● Multistix light
● Chemstrip
● Used in chemical examination of urine
● Chemical-impregnated absorbent pads attached
to a plastic strip
○ Has 10 parameters/absorbent pads
○ Each pad contains a specific chemical
targeting a specific analyte/solute present
in the urine
● A color-producing chemical reaction takes place ERRORS CAUSES BY IMPROPER TECHNIQUE
when the strip comes in contact with urine
● UNMIXED SPECIMEN
● Reactions are interpreted by comparing the color
○ Formed elements such as red and white
produced on the pad with the chart provided by
cells sink to the bottom of the specimen
the manufacturer (Semiquantitative)
and will be undetected in an unmixed
● Specific gravity and pH → reported by number
specimen → false negative results
OVERVIEW: PARAMETERS
● LEACHING
● Before: 4 parameters ○ Allowing the strip to remain in the urine for
● Current: 10 parameters an extended period may cause leaching of
○ pH reagents from the pad
○ Proteins ■ Each pad has chemicals attached
○ Glucose to it → urine is left to stand for a
○ Ketones long time → chemicals are
○ Blood released into the urine → no color
○ Bilirubin change
○ Urobilinogen ● RUN-OVER
○ Nitrite ○ If remained vertical → Excess urine
○ Leukocytes remaining on the strip after its removal
○ Specific gravity from the specimen can produce a run-over
between chemicals on adjacent pads,
producing distortion of color.
○ SOLUTION: To ensure against run-over,
blotting the edge of the strip on absorbent
paper and holding the strip horizontally
while comparing it with the chart
● TIMEFRAME
○ The timing for the reactions to take place
varies among manufacturers and ranges
from immediate reaction for pH to 120
seconds for the leukocyte esterase.
(60-120seconds)
■ Enough time for LE reaction to
occur
● LIGHT SOURCE
○ A good light source is essential for
accurate interpretation of color reactions
● READING
PROCESS ○ The strip must be held close to the color
1. Dip the reagent strip COMPLETELY but BRIEFLY chart without actually placing on the chart
into a well-mixed specimen ● DIFFERENT MANUFACTURERS
2. Remove the excess urine by running the edge of ○ Reagent strips and color charts from
the strip on the container/test tube when different manufacturers are not
withdrawing it from the specimen & blotting it interchangeable
horizontally on the absorbent medium.

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 ● REFRIGERATION ○ pH of 9.5-10 → contamination by
○ Specimens that have been refrigerated detergent
must be allowed to return to room
temperature prior to reagent strip testing
as the enzymatic reactions are REAGENT STRIP REACTION
temperature-dependent ● Change in color → Double Indicator System
HANDLING AND STORING OF REAGENT STRIPS ○ Methyl red
● Reagent strips must be protected from moisture, ■ Will react to pH of the urine that
volatile chemicals, heat and light ranges from 4 to 6 (acidic)
○ Close the bottle after taking → protected ○ Bromthymol blue
from heat and light ■ Will react to pH of the urine that
● Packaged into opaque containers with desiccant ranges from 6 to 9 (slighly acidic
● Strips are removed just prior to testing and bottle is to basic)
resealed immediately
● Stored at room temperature below 30 degree
Celsius (but never refrigerated)
● Must not be used beyond expiration date
● Care must be taken not to touch the chemical pads
when removing the strips from the container
● Visual inspection should be done every use to HANDLING/STORAGE
detect deterioration. Do not use if pads are ● Measurement of urine pH and acidity must always
discolored be made on freshly voided specimens.
QUALITY CONTROL OF REAGENT STRIPS ● The container should be filled to minimize the
● Performed during the analytical phase amount of dead space, and the urine covered
● Reagent strips must be tested with positive and tightly.
negative controls ● The container should be kept cold, preferably on
○ Minimum of once every 24 hours ice, but not frozen.
○ Every after new bottle is opened PROTEIN
○ Questionable results are obtained ● The presence of increased amounts of protein in
○ When there is concern about the integrity the urine can be an important indicator of renal
of the strip. disease.
● Record all control results ○ Albumin should not be present in the
● Distilled water should not be used as negative urine
control. The manufacturers will provide the controls.
ERROR SOURCES IN READING THE REAGENT STRIP
● Interfering substances in the urine REAGENT STRIP REACTION
● Technical carelessness ● Protein is tested under protein error of indicator
● Colorblindness ○ The protein pad contains an indicator and
VALUE AND APPLICATION it contains many hydrogen ions
● “Results should coincide with the physical ■ The protein, from the urine, reacts
examination results and must be suggestive of the with the indicator → The indicator
probable sediments that can be seen during will give up the hydrogen ions, so
microscopic exam.” that it could bind to the protein
REPORTING OF RESULTS ■ More protein → more hydrogen
● Depending on the test performed, the results are ions that is removed from the
reported in the following manner: indicator → color change
○ in concentration (mg/dL) ● ADDITIONAL INFO:
○ as small, moderate or large ○ Certain indicators can change color in the
○ using the plus system (1+,2+,3+,4+) presence of proteins even though the pH
○ as positive, negative or normal of the medium remains constant
○ specific gravity and pH ○ Proteins (amino group) accepts hydrogen
■ results are estimated in their ions from the indicator. Albumin has a lot
respective units of amino groups in its structure so it
THE 10 PARAMETERS accepts more hydrogen ions.
○ The protein pad is very sensitive to
pH
albumin
● The average adult on a normal diet excretes about
○ Other urine proteins such as gamma
50–100 mEq of hydrogen ions in 24 hours to
globulin, glycoprotein, ribonuclease,
produce urine of about pH 6.
lysozyme, hemoglobin, Tamm–Horsfall
○ pH 6 (acidic) → In normal conditions,
mucoprotein, and Bence-Jones protein are
tubules would secrete hydrogen ions
much less readily detected than albumin.
● In healthy individuals, urine pH may vary from 4.5–8
○ Below 4.5 → adulteration (e.g. vinegar)

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 ○ Therefore, a negative urinary dipstick KETONES
result does not necessarily rule out the ● Ketones, or ketone bodies are formed during the
presence of these proteins. catabolism of fatty acids
INDICATORS ○ The body will form ketones if fats are
○ Multistix indicator = Tetrabromophenol blue metabolized → since there are no more
○ Chemstrip indicator = carbohydrates
3’,3”,5’,5”-tetrachlorophenol-3,4,5,6- ● Ketones that are produced once fats are
tetrabromosulfonphthalein metabolised:
○ PLUS BUFFER ○ Acetoacetic acid (diacetic acid)
GLUCOSE ■ Parent ketone → can produce
● When the blood glucose exceeds the renal acetone and B-hydroxybutyric
threshold, the tubules cannot reabsorb all of the acid
filtered glucose, and so glycosuria occurs ○ B-hydroxybutyric acid
○ Acetone

REAGENT STRIP REACTION

● Only sensitive and specific to glucose REAGENT STRIP REACTION
○ To test maltose → benedict’s test ● Sodium nitroprusside reaction
● Follows the Double Sequential Enzymatic ● Measures acetoacetic acid
Reaction ○ If acetoacetic is the only one being
○ The pad for glucose contains chromogen, measured → the other two are also
glucose oxidase, and peroxidase present
● Acetoacetic acid in alkaline medium reacts with
sodium nitroprusside to yield a purple color.
○ The pad for ketone contains sodium
nitroprusside
● If glucose is present in the urine of the patient →
○ If acetoacetic acid is present in the urine
glucose oxidase will act on glucose → forms
→ reacts with sodium nitroprusside →
Gluconic acid and Hydrogen peroxide
color change

● H2O2 will now become the substrate

● H2O2 will react with the chromogen in the pad →
in the presence of peroxidase → forms an
oxidized chromogen (colored)
● Therefore, glucose = color change
○ The more glucose = more intense is the
color change OTHER REAGENT STROP BRANDS WITH THEIR
OTHER REAGENT STRIP BRANDS WITH THEIR SENSITIVITIES
CHROMOGENS

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 BLOOD HANDLING/STORAGE
● Blood in the urine sample is an indication of a ● Fresh urine
possible bleeding episode in the renal tract ● Avoid exposure to sunlight
● Blood has two rows FOAM TEST
○ Two rows for positive ● Physical test for bilirubin
○ One row for negative ● To correlate with a positive strip test for bilirubin
● Two reasons why there is blood in the urine: ● Shake vigorously the urine of the patient in a test
Hematuria (not lysed → intact RBC) versus tube → yellow foam → presence of bilirubin
Hemoglobinuria (lysed RBC) ○ yellow-brown to greenish brown urine that
may have a yellow foam

● Upper row → Hematuria

● Lower row → Hemoglobinuria
● Myoglobinuria
○ Comes from muscle destruction
REAGENT STRIP REACTION
● Pseudoperoxidase activity of Hemoglobin
UROBILINOGEN
● Urobilinogen represents a group of closely related
tetrapyrrole compounds formed from reduction of
bilirubin

● The pad for blood has hydrogen peroxide and

chromogen
● Urobilinogen is normally present in the urine but in
○ Pseudoperoxidase
concentrations of 1 Ehrlich unit or less per 100 mL
● Hemoglobin will act like peroxidase
of urine
○ If hemoglobin is present → will oxidize
○ Strip can still detect this level, lower than
chromogen → change in color (dark
this, not anymore
green)
○ Since normally found in the urine
OTHER REAGENT STRIP BRANDS WITH THEIR
■ Reporting (if color is same with
CHROMOGENS
the left) should be reported as
normal
● Urobilinogen instability is a problem
○ Fresh specimen
● When is the best time to collect urine for
urobilinogen testing?
○ Urobilinogen is best tested,two hours after
mid-day meal (2-4 pm)
○ Due to alkaline tide
■ The bicarbonate in the parietal
cells will be sent in the circulation
■ In exchange → chloride in the
circulation will go in the parietal
BILIRUBIN cells
● Bilirubin is a breakdown product of hemoglobin that ● Chloride is reabsorbed,
is formed in the reticuloendothelial cells of the in order for parietal cells
spleen, liver, and bone marrow to create hydrochloric
● B1 versus B2 acid → digestion
REAGENT STRIP REACTION
● Ehrlich Aldehyde Reaction
● Diazo Reaction/Azo-coupling
REAGENT STRIP REACTION ● Both produce pink to red colors if positive
● Diazo Reaction/Azo-coupling Reaction ○ Urobilinogen + diazonium salt = color
● Bilirubin reacts with change
2,4-dichloroaniline-diazonium salt (Multistix) or NITRITE
2,6-dichlorobenzene-diazonium-tetrafluoroborat ● The nitrite test is a rapid, indirect method for the
e in an acidic medium to produce an azo dye early detection of significant and asymptomatic
(tan/pink-violet) bacteriuria
○ Bilirubin + diazonium salt = color change

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 ● If nitrite positive and LE positive
○ Urinalysis (screeing) could not diagnose
UTI → culture
SPECIFIC GRAVITY
REAGENT STRIP REACTION
● The addition of a specific gravity testing area to
● Greiss reaction
reagent strips has eliminated a time-consuming
● Nitrite at an acidic pH reacts with an aromatic amine
step in routine urinalysis and has provided a
(para-arsanilic acid or sulfanilamide) to form a
convenient method for routine screening.
diazonium compound that then reacts with
tetrahydrobenzoquinolin compounds to produce a
pink colored azodye.
○ The pad of nitrite contains aromatic
amine and tetrahydrobenzoquinolin → REAGENT STRIP REACTION
waiting for nitrite in the urine ● Change in pKa (dissociation constant) of a
○ Nitrite in the urine → reacts with aromatic polyelectrolyte in an alkaline medium
amine → produces a diazonium ○ The pad of SG contains polyelectrolyte
compound ○ Polyelectrolyte → releases hydrogen,
○ Diazonium compound → reacts with equal to the number of ions in a solution
tetrahydrobenzoquinolin → color change ■ If there are 100 ions in the urine =
● Any degree of uniform pink color should be polyelectrolyte will give away 100
interpreted as a positive nitrite test hydrogen ions
○ suggesting the presence of 105 or more ○ More concentrated urine = More ions
organisms per milliliter. present = More hydrogen ions are
● First morning urine is the specimen of choice released
○ Since urine is concentrated ■ Since polyelectrolyte is pH
LEUKOCYTE ESTERASE dependent → the more hydrogen
● Detects the presence of esterase in the is released = the more it becomes
granulocytic white blood cells (Basophil, basic → change in color
Eosinophil, Neutrophil) ADDITIONAL REAGENT STRIP PARAMETER
○ Also present in monocyte and macrophage ASCORBIC ACID
● Esterases also are present in Trichomonas and
● Ascorbic acid may inhibit several reagent strip
histiocytes
reactions
● LE is not present in lymphocyte
○ Blood
REAGENT STRIP REACTION
○ Bilirubin
● Action of LE to catalyze the hydrolysis of an acid ○ Leukocyte esterase
ester ○ Nitrite
○ Glucose
REAGENT STRIP REACTION

● 10 seconds reading time

● Other reagent strip brands include:
○ Calcium, creatinine, and microalbumin
CONFIRMATORY TESTING
● Using different reagents or methodologies to detect
the same substance as detected by the reagent
● The pad of LE has ester strip with some or greater sensitivity and specificity
● In the presence of LE → acid ester will be ● Tablets and liquid chemicals
broken down to produce indoxyl or pyrole ● But not often used today due to increased
● Indoxyl or pyrole → reacts with diazonium sensitivity and specificity of reagent test strips
salt → purple color SULFOSALICYLIC ACID TEST FOR PROTEIN
● A positive LE test result is most frequently ● Sulfosalicylic acid testing
accompanied by: ● Advantage: can test for all protein
○ the presence of bacteria PROCESS
■ which may or may not produce a ● Add 3mL of 3% SSA to 3mL of centrifuged urine
positive nitrite reaction. ● Mix by inversion and observe for cloudiness
● if nitrite negative and LE positive ● Grade the turbidity
○ The bacteria that can reduce nitrate is
limited
○ There are bacteria that are not entero that
can cause UTI
○ It is possible nitrite negative → UTI could
be present

@mlstranses | 5
 OTHER CONVENTIONAL CHEMICAL TESTS
● Heller’s Ring Test for Protein
● Benedict’s Test for Glucose
● Rothera’s Test for Ketone bodies
● Gerhardt’s Test for Ketone Bodies
● Gmelin’s Test for Bile Pigments
● Smith Iodine Test for Bile Pigments
TABLET-BASED TESTS
● Clinitest
● Acetest
● Ictotest

@mlstranses | 6
 | Topic # 3 | [MLS 419-LEC] Analysis of Urine and Other Body Fluids
P3: Physical Examination of Urine,GFR, Tubular secretion/absorption, Introduction to
Urinalysis
Professor: Mr. Christian Villahermosa, RMT, MSMT
Date: February 5, 2024

PHYSICAL EXAMINATION OF URINE ● If pale yellow → diluted

● Includes the determination of urine color, clarity, → hydrated patient
and specific gravity ● If dark yellow →
○ Odor → not part of routine urinalysis but is concentrated →
a noticeable property dehydrated
■ Aromatic/Odorless ○ Dependent on body’s metabolic state
■ If there is a deviation on the ○ Gives a rough estimate of urine
normal smell of urine → Should concentration
be reported ● Additional pigments
○ Taste → not part of routine urinalysis and ○ Exist in smaller concentrations →
should not be done Overwhelmed by the presence of the
● Already performed by early physicians urochrome
● Provides preliminary information on disorders ● Uroerythrin: pink color in refrigerated specimens
● Can be used to confirm or to explain findings in the ○ Present in samples that have been
chemical and microscopic areas of urinalysis standing for quite some time
○ should not conclude based on physical ○ Specimens are not fresh/underwent
examination of urine alone preservation
■ perform chemical and ● Urobilin: orange-brown color to urine that is not
microscopic examination fresh
(confirmation) ○ If the urine sample is left to stand at room
PARAMETERS USED IN THE ANALYSIS OF URINE temperature for quite some time, the
yellow color changes to orange-brown in
URINE COLOR
color due to urobilin
● Varies from almost colorless to black
○ May be due to normal metabolic functions,
physical activity ingestion of food or drugs,
or pathologic conditions
● Not necessary to directly assume a pathologic
condition if the patient presents a urine sample
that is not common → may be due to underlying
factors stated above (dehydrated, medications) COLORS OF A URINE SAMPLE
HISTORY
● Early physicians used a ‘wheel of urine’ COLORLESS ● Common in random specimens
● Different urine colors correspond to a certain TO PALE ● Dilute random specimen (well
YELLOW hydrated)
description and a certain disease correlation
● Used glass flasks called ‘matula’ to view urine ○ Due to recent fluid
samples consumption
● Terms used before to remove subjectivity in the ● POLYURIA IN DIABETES
analysis of urine: INSIPIDUS
○ ‘white as well water’ ○ Observed if there is an
○ ‘ruddy as pure intense gold’ increased 24-hour volume
○ ‘black as dark horn’ of urine and low SG,
NORMAL URINE COLOR negative glucose test result
● Common descriptions: pale yellow, yellow, dark ○ In taste test → bland
yellow ● POLYURIA IN DIABETES
● Examine sample under a good light source MELLITUS
○ To properly differentiate the color ○ Observed if there is an
○ Examined on a test tube increased 24-hour volume,
● Urochrome: pigment responsible for the yellow high SG (presence of
color of urine glucose), and positive
○ Product of endogenous metabolism; glucose test result
produced at a constant rate ○ In taste test → urine tastes
■ Not varied → used to correlate like honey or sweet
different conditions based on the
intensity of the yellow color since
it is produced at a constant rate

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 DARK ● CONCENTRATED SPECIMEN ○ false-negative chemical
YELLOW TO ○ Observed in first morning test for bilirubin
AMBER urine ■ Since bilirubin is
● INTAKE OF B COMPLEX already oxidized
VITAMINS
○ Vitamin B2 (Riboflavin) → ● Indicate an active Pseudomonas
imparts a dark yellow GREEN infection
coloration to urine once it is ○ Confirmation through
metabolized positive urine culture
● DEHYDRATION
● ACRIFLAVINE: URINARY
ANTISEPTIC
○ Introduction of acriflavine in
the urinary tract
○ Urine tends to be very dark
yellow, which is commonly
mistaken with positive
bilirubin because they tend
to have the same color BLUE-GREEN ● AMITRIPTYLINE
○ Perform bile test ○ antidepressant
■ Acriflavin test ● METHOCARBAMOL (ROBAXIN)
results: Negative bile ○ Muscle relaxant (may be
test results and green-brown)
possible green ● CLORETS
fluorescence ○ Contain chlorophyll →
● NITROFURANTOIN excess amount →
○ Antibiotic prescribed in UTIs blue-green discoloration to
urine
● METHYLENE BLUE
DARK ● BILIRUBIN ○ used in fistulas
YELLOW ○ Yellow foam when shaken ○ Fistulas → abnormal
and positive chemical test pathways between the blood
for bilirubin vessels or organs
○ White foam and the color of ■ doctors inject
the urine is other than dark methylene blue dye
yellow → Protein is present to color the areas
where fistulas are
present
■ Elimination of
methylene blue dye
is through urine →
blue green urine
● PHENOL
○ When oxidized (people
exposed to benzene)
○ Phenol → end product of
benzene
ORANGE ● PHENAZOPYRIDINE
YELLOW ● INDICAN
(PYRIDIUM)
○ Bacterial infections,
● PHENINDIONE
intestinal disorders
○ Anticoagulant (rarely used)
○ Blue diaper syndrome
■ orange yellow in
alkaline urine BLUE DIAPER SYNDROME
■ colorless in acid ● A rare, genetic metabolic disorder characterized by
urine the incomplete intestinal breakdown of
tryptophan
DARK ● BILIRUBIN OXIDIZED TO ● If tryptophan is not broken down by the intestine →
YELLOW BILIVERDIN Intestinal bacteria will break down tryptophan,
GREEN ○ Not fresh → Oxidation upon resulting to increased amounts of indican (blue
prolonged standing and color)
improper storage ● Commonly observed in infants
○ Colored foam in acidic urine

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 black after standing
○ Wait for 2 hours to make
the urine entirely black
● MALIGNANT MELANOMA
○ Presence of melanin
oxidized from melanogen
○ Urine darkens on standing
and reacts with
nitroprusside and ferric
chloride
PINK ● HEMOGLOBIN
● MEDICATIONS
● RED BLOOD CELLS
○ Phenol derivatives
○ Blood in urine from urinary
○ Argyrol (antiseptic)
tract or from mestrual
○ Methyldopa or levodopa
contamination
○ Metronidazole (Flagyl)
RED ● Most common abnormal urine
color
● RED BLOOD CELL
○ Intact RBC → Cloudy urine,
positive chemical test for
blood, seen under the
microscope (intact RBC)
● HEMOGLOBIN
○ Clear urine, positive
chemical test for blood; TO CONSIDER: MUNCHAUSEN SYNDROME
intravascular hemolysis
● MYOGLOBIN ● A form of factitious disorder where a person
○ Found in conditions such as fabricates or induces an illness to assume the
myoglobinuria patient role
○ Clear urine, positive ● One method
chemical test for blood; due ○ Tampering with laboratory specimens
to muscle damage ● Variation
● BEETS
○ Munchausen syndrome by proxy
○ Plant product
○ alkaline urine of genetically ○ a person will induce or fabricate an illness
susceptible persons to a person that he/she is taking care of
● RIFAMPIN ● Example
○ TB drug ○ Artifactual / Factitious hematuria
● MESTRUAL CONTAMINATION (common)
○ Not advisable to submit
■ presence of red blood cells on a
urine sample if one is
having menstruation (2-3 patient's urine samples
days after menstruation) ■ People injure themselves and
allow drops of blood into their
PORT WINE ● Presence of Porphyrins in the urine
urine sample ○ Factitious protenuria
● Negative test for blood, may ■ Addition of egg white to the urine
require additional testing → elevated protein
● Be aware of highly variable results and/or
incompatible biochemical profiles
○ In the absence of lab workflow errors,
consider sample manipulation
URINE CLARITY
● Refers to the transparency or turbidity of a
specimen
● May be reported as clear, hazy/slightly cloudy,
RED ● RBCs oxidized to
BROWN methemoglobin cloudy, turbid, or milky
○ Seen in acidic urine after ● Freshly voided urine is usually clear
prolonged standing ○ Clear urine is not always normal
○ Positive chemical test for ● Precipitation of phosphates and carbonates may
blood cause white cloudiness
● Myoglobin ○ a cloudy or slightly cloudy urine sample is
● Rifampin
not always abnormal
● Clarity should correspond with the amount of
BROWN ● HOMOGENTISIC ACID
/BLACK (ALKAPTONURIA) material observed under the microscope
○ Excessive homogentisic ○ If sample is clear → few materials are
acid → Alkaline urine turns seen

@mlstranses | 3
 ○ If sample is cloudy → many materials ■ Calculated as number of mL of
(change in clarity) urine divided by the minutes used
NON-PATHOLOGIC TURBIDITY to collect the specimen
● Normal causes of a cloudy urine FORMULAS
● Presence of squamous epithelial cells and mucus 𝑈𝑉
● Specimens allowed to stand or that are refrigerated
○ White precipitate in alkaline pH:
𝐺𝐹𝑅 = 𝑃
amorphous phosphates and carbonates Whereas:U = Urine concentration of creatinine
○ Pink precipitate in acidic pH: amorphous V = Urine volume (24-hour urine)
urate P = Creatinine concentration in the blood
● Semen, spermatozoa, prostatic fluid
● Fecal contamination
● Radiographic contrast media: contrast materials
used in x-rays and CT scans; high SG (>1.040) SOLVING
● Talcum powder
Calculate the urine volume for a 24-hour specimen
● Vaginal creams
measuring 1440ml. Urine creatinine is 120mg/dL while
PATHOLOGIC TURBIDITY plasma creatinine is 1.0mg/dL
● Red blood cells
● White blood cells Step 1. Compute first for urine volume
● Bacteria
● Abnormal amounts of non-squamous epithelial cells Solution:
Urine volume = volume of urine in mL/minutes of
● Yeast
collection
● Trichomonads
● Abnormal crystals 1440 𝑚𝐿 1 ℎ𝑟
● Lymph fluid: Chyluria in filariasis (lymph fluid 𝑣= 24ℎ𝑟𝑠
𝑥 60 𝑚𝑖𝑛𝑠
= 1𝑚𝐿/𝑀𝑖𝑛
leaking into kidneys)
● Lipids
Step 2. Apply the formula
● Calculi
SPECIFIC GRAVITY 𝑈𝑉
● Measures the kidney’s ability to concentrate 𝐺𝐹𝑅 = 𝑃
glomerular filtrate by tubular reabsorption (120 𝑚𝑔/𝑑𝐿) (1𝑚𝐿/𝑚𝑖𝑛)
● Methods: 1 𝑚𝑔/𝑑𝐿
= 120 𝑚𝐿/𝑚𝑖𝑛
○ Urinometry: buoyancy
○ Refractometry: refractive index DISADVANTAGE OF THE FORMULA
○ Harmonic oscillation densitometry: ● Unfortunately, the formula is only applicable if the
sound waves patient has a normal body surface area of 1.73m^2
○ Osmolality: colligative properties ● Adjust by multiplying GFR by 1.73/A
○ Reagent strip: change in pH ○ A stands for the body surface of the patient
ODOR ○ Formula:
● Freshly voided urine: odorless to aromatic ■ log A = (0.007184) x (height in
● Not fresh urine: ammoniacal cm)0.725 x (weight in kg)0.425
● Bacterial infections: strong, unpleasant ○ Or use nomogram
● Ketones: sweet or fruity ● Requires urine collection for 24 hours
● Maple Syrup Urine Disease (MSUD): maple syrup NOMOGRAM FOR BODY SURFACE AREA
● Phenylketonuria: mousy
● Tyrosinemia: rancid butter
● Isovaleric acidemia: sweaty feet
● Methionine malabsorption: cabbage, hops
● Trimethylaminuria: rotting fish
● Bleach: contamination
COMPUTATION FOR GLOMERULAR FILTRATION RATE
● Most commonly used: creatinine
● Reported in ml/min
● Normal is 120ml/min → allows
allowances/compromise for the different body
surfaces of males and females
○ Males 107-139ml/min
○ Females 87-107ml/min
ESTIMATED GLOMERULAR FILTRATION RATE
● One must first know the following
● Does not measure the exact GFR but an estimate
○ Urine creatinine in mg/dL
of it
○ Plasma creatinine in mg/dL
○ Urine volume

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 COCKCROFT-GAULT FORMULA expected that the final urine is more concentrated or
● Can be used to estimate kidney function for CKD diluted depending on hydration
staging or whether to adjust or discontinue ○ If the patient is dehydrated → S.G. is
medications based on kidney function higher than 1.010
● Different formula for males and females since the ● Fluid deprivation tests evaluate the ability of renal
creatinine levels are different from one another tubular cells to selectively absorb and secrete
FORMULA FOR MALES solutes.
○ Measures the renal concentrating ability of
the kidneys
FLUID DEPRIVATION TEST
FORMULA FOR FEMALES ● Major test for tubular reabsorption
● Overnight water/fluid deprivation test for 12 hours (8
pm to 8 am)
● Urine is collected after 12 hours (8 am) →
measured using osmolarity
○ IF the urine osmolarity is above 800mOsm
or higher – NORMAL
■ Since the urine is concentrated
MDRD (MODIFICATION OF DIET IN RENAL ○ IF the urine osmolarity is below 800mOsm,
DISEASE)-IDMS FORMULA fluid restriction is continued for two more
● Measure serum creatinine via isotope dilution mass hours
spectrometry (reference method) ● After two hours (10 AM), urine and plasma are
● eGFR = 175 x serum creatinine -1154 x age -0.203 x collected and osmolarity is tested.
0.742 (if female) x 1.202 (if black) ○ Normal: if urine osmolarity is or above
WHEN INTERPRETING GFR, ONE MUST CONSIDER… 800mOsm or if the urine to serum
● It is determined not only by the number of nephrons osmolarity ratio is or greater than 3:1
but also by their functional capacity ○ (Note: normal evening meal but no water
○ GFR measures more, the ability of the or any fluids after)
nephrons to filter/form urine as opposed to ● If the test continues to be abnormal, additional
their number testing should be done to diagnose diabetes
○ Measures the capacity not the number insipidus
of nephrons ○ Patient is injected with ADH
○ Example: If one half of the nephrons are ○ Serum and urine are collected after 2 and
nonfunctional, GFR still remains normal if 4 hours
the remaining nephrons double their ● Results interpretation:
capacity. ○ If test becomes normal (> 800 mOsm)=
● Conclusion: Neurogenic/Cranial diabetes insipidus
○ It cannot detect early renal disease. It ■ The problem is at the posterior
can only evaluate the extent of nephron pituitary gland (no ADH)
damage. ○ If test result is below 400 mOsm or ratio is
■ If the glomerulus is damaged → 1:1 = Nephrogenic diabetes insipidus
the disease has already started ■ The kidney is unresponsive to
for a long time ADH
● the first indicator of a OTHER RENAL CONCENTRATION TESTS (USES SG)
kidney problem is the FISHBERG TEST
tubules ● Measure urine SG after fluid deprivation: greater
■ It is possible that the glomerulus than or equal to 1.025 is normal
is slowly damaged, but the GFR MOSENTHAL TEST
is normal ● Normal diet and fluid intake
■ If the GFR is abnormal → ● 24-hour urine: 12 hours (day), 12 hours (night)
glomerulus is damaged for a long ● Measure SG:
period ○ Urine volume of day urine must be greater
■ GFR is tested in patients that than night urine
have the disease ○ Night urine SG must be greater than or
○ It can also be used to determine if a equal to 1.020
person can be started on a medication.
TUBULAR REABSORPTION TEST (CONCENTRATION TUBULAR SECRETION AND RENAL BLOOD FLOW TEST
TEST) ● Test to measure tubular secretion of non-filtered
● Loss of tubular reabsorption capacity is the first sign substances and renal blood flow
of renal disease ● Traveling of substances from tubular capillaries to
● As previously mentioned, the ultrafiltrate that enters tubules and acid base balance
the tubules has a specific gravity of 1.010 and it is

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 PHENOLSULFONPHTHALEIN TEST ○ Thomas Addis: quantification of urine
● dye excretion sediments
○ If 100mg of phenolsulfonpthalein is infused ● Richard Bright (1827) introduced the concept of
in the patient → in the urine 100mg of urine examination as part of a doctor’s routine
phenolsulfonpthalein should be seen patient examination
● Can cause anaphylactic shock to patients WHY IS URINE TESTED
Ρ-AMINO HIPPURIC ACID TEST (PAH) ● Readily available
● Loosely bound to plasma proteins ● Easily collected
● Infused intravenously ● Contains information about the body’s major
● Performed in renal labs metabolic functions
ACID-BASE URINE COMPOSITION
TITRATABLE ACIDITY (DEPENDS ON PHOSPHATE IN ● 95% water, 5% solutes
THE FILTRATE) AND URINARY AMMONIA ● Urea: primary organic component
● Measures the ability of kidney to produce acid urine ● Other solutes
depends on tubular secretion/excretion of ammonia ○ Creatinine
by the cells of the DCT ○ Uric acid
AMMONIUM CHLORIDE TEST ○ Chloride: primary inorganic component
● measures the inability to produce acid urine (renal ○ Sodium
tubular acidosis) ○ Potassium
● Measurement of total hydrogen ion excretion in ○ Phosphate, ammonium, and calcium
urine after intake of oral ammonium chloride (urine URINE VOLUME
collection every 2 hours for 8-10 hours) ● Depends on the amount of water excreted by the
○ In the body, ammonium chloride will be kidney and the body’s state of hydration
broken down into urea and HCl ● Normal daily output: 600 mL-2000 mL/day (usually
○ HCl will acidify the blood 1200-1500 mL/day)
○ The urine pH of the urine should be acidic ● Oliguria: decrease in urine volume (<400 mL/day)
■ When HCl arrives in the tubules, ○ Anuria: cessation of urine flow due to
Hydrogen is secreted kidney damage, decrease in blood flow, or
○ If after intake, the urine pH is alkaline → obstruction
renal tubular acidosis ● Nocturia: increase in nocturnal excretion of urine
INTRODUCTION TO URINALYSIS (should precede and followed by sleep periods)
○ Nocturnal enuresis: bedwetting
HISTORY ● Polyuria: increase in urine volume (> 2500
● Urinalysis was the beginning of laboratory medicine mL/day)
● Early physicians ○ Diabetes mellitus and diabetes insipidus
○ Physical examination of urine ■ DM → polyuria (concentrated)
● Hippocrates wrote a book on ‘uroscopy’ in the 5th ● Due to polydipsia →
century BCE too much drinking of
○ Uroscopy → study of urine using physical water
examination ● Present in patients with
○ Included as part of medical training during DM because the plasma
the Middle Ages is very concentrated
● 1140 CE: urine color charts with 20 different colors (increased glucose) →
the body’s normal
mechanism is to activate
thirst receptor to dilute
plasma → urination
● In polyphagia → eating
too much → since
● Frederik Dekkers discovered albuminuria by glucose is not inside the
boiling urine in 1694 cell → body will intake
food
■ DI → Polyuria (diluted)
○ Diuretics, caffeine, or alcohol (suppress
ADH)
■ Diuretics → sodium and water is
released
■ Caffeine and alcohol suppresses
● ‘pisse prophets’ made predictions based on urine
ADH
samples
○ Their existence initiated the passage of the
first medical licensure laws in England
● 17th century: invention of the microscope

@mlstranses | 6
 | Topic # 1 | [MLS 419-LAB] Analysis of Urine and Other Body Fluids

M1: CHEMICAL EXAMINATION OF URINE PART 1

Professor: Mr. Christian Villahermosa, RMT, MSMT
Date: February 19, 2024

REAGENT STRIPS ● The normal range for pH in random urine is wide

● Provide a simple, rapid means for performing because the patient could have eaten before the
medically significant chemical analysis of urine test
○ pH ○ The diet could have changed the pH of the
○ Protein urine
○ Glucose ● Acidic pH for first-morning urine because the
○ Ketones tubules have time during sleep to eliminate
○ Blood hydrogen ions
○ Bilirubin ● pH will just measure the acidity or alkalinity of the
○ Urobilinogen urine → NO DISEASE CORRELATION
○ Nitrite ● NO NORMAL VALUES
○ Leukocyte esterase ○ Must be considered in conjunction with
○ Specific gravity other patient information
● If true positive → color should not fade CLINICAL SIGNIFICANCE
● Vitamin C interference (BBLNG) can be eliminated ● AIDS in determining the existence of systemic
by the addition of iodate on the strips. acid-base disorders of metabolic or respiratory
● Note: PPBUN (60 seconds) origin
○ In respiratory or metabolic acidosis
TEST TIME
■ not related to renal disorders
Glucose 30 seconds ■ the urine is acidic
● Hydrogen ions are
Bilirubin 30 seconds increased in the system
→ tubules would
Ketones 40 seconds eliminate more hydrogen
ions → seen in the urine
Specific gravity 45 seconds ○ In respiratory or metabolic alkalosis
■ not related to renal disorders
pH 60 seconds
■ the urine is alkaline
● Bicarbonate ions are
Protein 60 seconds
increased in the system
Blood 60 seconds → tubules would
eliminate more
Urobilinogen 60 seconds bicarbonate ions → seen
in the urine
Nitrite 60 seconds ○ Acid-base disorders are not related to
renal disorders
Leukocyte 120 seconds ■ There are certain conditions that
esterase
normal functions would not
pH happen
● The tubules are involved in the regulation of ■ Ex: If one has acidosis and have
acid-base balance in the body by: renal problems, the urine might
○ Secretion of hydrogen in the form of not be acidic
■ ammonium ions ■ In renal tubular acidosis, the
■ hydrogen phosphate kidney cannot form acidic urine
■ weak organic acids ● Therefore, in a patient
○ Reabsorption of bicarbonate with acidosis and renal
● May be controlled by diet and medications tubular acidosis, the
○ Thus, No normal value of pH, only a range urine could be alkaline
○ High-protein diet → acidic urine ● MANAGEMENT of urinary conditions that require a
○ vegetarian diet → alkaline urine specific pH to be maintained
■ (except cranberry juice) ○ In patients who ‘are prone to kidney stones
■ Their urine should not be acidic
FIRST MORNING URINE PH OF 5.0 to 6.0
→ must be maintained to an
A HEALTHY INDIVIDUAL
alkaline level
RANDOM URINE PH 4.5 to 8.0

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 ● Many kidney stone ● Historical screening test for BJP → not performed
crystals/ components anymore
precipitate in acidic urine ○ If protein coagulates (becomes turbid) at
○ Calcium oxalate 40℃ and 60℃
■ common cause of kidney stones ○ dissolves (clears) at 100℃
■ precipitates primarily in acidic ■ other proteins remain coagulated
urine ● Now: cases of multiple myeloma are easily detected
● Precipitation → start of by chemical methods and diagnosed by serum
stone formation, since electrophoresis (classic ‘M spike’ in the gamma
stones are an aggregate globulin region) and immunoelectrophoresis
of different precipitates RENAL PROTEINURIA
○ In patients with UTI ● The problem is the kidney itself
■ Urease-positive organisms prefer ● May be the result of either glomerular or tubular
alkaline urine damage
■ Urea-splitting organisms do not ○ Glomerular proteinuria
readily multiply in acidic urine ○ Microalbuminuria
PROTEINS ○ Orthostatic (postural) proteinuria
● MOST INDICATIVE OF RENAL DISEASE ○ Tubular proteinuria
○ Proteins, most especially albumin, are not GLOMERULAR PROTEINURIA
expected to be identified/tested in the ● presence of abnormal substances in the glomerular
reagent strip membrane may damage the glomerular
● Normal urine contains very little protein membrane
○ Low molecular weight serum proteins and ○ Since the glomerulus is damaged, it is now
proteins produced in the genitourinary tract able to facilitate the entry of proteins
■ Could be present in the urine of including albumin
the patient but not test positive for ● Damage may be due to
the reagent strip ○ Amyloid material in amyloidosis
○ The strip is sensitive to albumin (buildup can cause organ failure)
■ If other proteins are present in ■ A genetic condition wherein there
high amounts → positive is an increased deposition of
● Major serum protein found in urine: albumin amyloid material in the
○ Others: microglobulins; uromodulin glomerulus
(Tamm-Horsfall protein); proteins from ○ Toxic substances
prostatic, seminal, and vaginal secretions ■ Heavy metals (mercury) and viral
CLINICAL SIGNIFICANCE infections
● Note: proteinuria does not always signify renal ○ Immune complexes (main cause)
disease (more tests required to determine if the ■ If immune complexes are
protein present is pathologic or physiologic) increased due to the hyperactivity
● Causes of proteinuria can be grouped into three of the immune system → they will
major categories: be deposited into the glomerulus
○ Pre-renal ● increased pressure from the blood entering the
○ Renal glomerulus
○ Post-renal ○ The glomerulus will be overwhelmed
PRE-RENAL PROTEINURIA ○ May be reversible or transient
● Caused by conditions affecting the plasma prior (strenuous exercise and dehydration,
to reaching the kidney hypertension, pregnancy)
○ Proteins are increased in the circulation → MICROALBUMINURIA
entered the glomerulus because they are ● Mircal test
not albumin ○ Test to determine if there are small
● NOT INDICATIVE OF ACTUAL RENAL DISEASE amounts of albumin present in the urine
○ The kidney could be normal, but there is ○ The amount of urinary albumin is above
an increased level of protein in the plasma normal but lower than the reagent strip
● Caused by increased levels of LMW plasma specificity
proteins that exceed the normal reabsorptive ○ Why is this performed?
capacity of the tubules ■ The reagent strip may be
○ Hemoglobin sensitive to albumin, but it has a
○ Myoglobin → released during muscle minimum concentration before it
destruction becomes positive
○ Acute phase reactants → Inflammation ○ When is this performed?
BENCE-JONES PROTEIN ■ If the patient is exhibiting signs of
● Monoclonal immunoglobulin light chains excreted kidney damage/diagnosed with
by patients with multiple myeloma diabetes mellitus

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 ● Urinary albumin excretion of 30-300 mg/day, or ○ Orthostatic proteinuria:
20-200 μg/min ■ if specimen 1 is negative for
○ amount of urinary albumin greater than the protein
normal value, but also lower than what is ■ specimen 2 is positive for protein
detected by a conventional dipstick TUBULAR PROTEINURIA
● Common in patients with early-stage diabetic ● Failure of the tubules to reabsorb filtered
nephropathy leading to reduced glomerular albumin
filtration ● Causes: exposure to toxic substances and heavy
○ Poor glucose control inhibits metals, severe viral infections, Fanconi syndrome
N-deacetylase needed to form the ● Fanconi syndrome
heparan sulfate ○ a defect of proximal tubule leading to
■ Heparan sulfate → part of the malabsorption of various electrolytes and
basement membrane of the substances that are usually absorbed by
glomerulus the proximal tubule
○ Decreased proximal tubular albumin POST-RENAL
reabsorption ● The circulation and the kidneys are normal
MICROALBUMINURIA/ MICRAL TESTING ● PROBLEM: ureter to urethra
(MICRAL TEST REAGENT STRIPS) ● Bacterial and fungal infections and inflammations
● Contain gold-labeled antihuman albumin produce exudates containing protein
antibody-enzyme conjugate ○ If there is infection, there are
Process microorganism have been subsequent
1. Strips are dipped in urine for 5 seconds and immune response (?) which can produce
compared with the color chart after 1 minute proteins → when the urine goes through
2. Albumin in the urine binds to the antibody. The the ureter/urethra, proteins produced by
bound and unbound conjugates move up the strip the microorganisms will be added to the
by wicking action. urine → positive
● It binds because it is an antibody ● Presence of blood from injury or menstrual
against/specific to albumin contamination
● Albumin + antibody = bound conjugate ● Prostatic fluid and large amounts of spermatozoa
● Antibody alone = unbound conjugate SULFOSALICYLIC ACID TEST
3. Unbound conjugates are removed in a captive zone ● A cold precipitation test that reacts equally with all
by combining with albumin embedded in the strip. forms of protein
4. The urine albumin-bound conjugates continue up ● Add 3 ml of 3% SSA reagent to 3 ml centrifuged
the strip and reach an area containing enzyme urine
substrate. GLUCOSE
5. The conjugated enzyme reacts with the substrate, ● Most frequently performed chemical analysis on
producing colors ranging from white to red. urine
6. The amount of color produced represents the ● Early diagnosis of diabetes mellitus through blood
amount of albumin present in the urine. and urine glucose tests provides a greatly
improved prognosis
● Blood glucose levels fluctuate, and a non-fasting
normal person may have glycosuria following a
meal containing a high glucose content
● Ideal specimen:
○ fasting specimen
● For diabetes monitoring:
○ 2-hour post-prandial specimen
CLINICANCE SIGNIFICANCE
● Hyperglycemia-associated (High blood and
urine glucose)
○ Diabetes mellitus
○ Nondiabetic origin
■ Cushing syndrome
■ Pancreatitis
ORTHOSTATIC PROTEINURIA ■ Acromegaly
● Occurs following long periods spent in a vertical ■ Hyperthyroidism
position and disappears when a horizontal position ■ Pheochromocytoma
is assumed ■ Thyrotoxicosis
● Due to increased pressure on the renal vein ■ Severe stress
● Collect urine after waking up (specimen 1) and ● Renal-associated (Normal blood glucose and
another specimen after remaining in a vertical high urine glucose)
position for several hours (specimen 2) ○ End-stage renal disease

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 ○ Fanconi syndrome CLINICAL SIGNIFICANCE
■ May be caused by cystinosis ● Inability to metabolize carbohydrate
CLINITEST ○ Diabetes mellitus, mostly type 1 (diabetic
● Measurement of glucose by copper reduction ketoacidosis)
method ■ Since there is no insulin →
● A color change progressing from a negative blue unable to metabolize
(CuSO4) through green, yellow, and orange/red ● Increased loss of carbohydrate
(Cu2O) ○ Vomiting
● Inadequate intake of carbohydrate
○ Prolonged starvation
○ Malabsorption/pancreatic disorders
○ Severe alcoholism (alcoholic ketoacidosis)
DIABETIC KETOACIDOSIS
● Once carbohydrate stores become depleted and
gluconeogenesis cannot occur anymore,
ketogenesis is substantially increased, leading to
metabolic acidosis.
● Patients are usually very dehydrated from being
hyperglycemic
○ High glucose → osmotic diuresis
KETONES
■ Glucose is increased in the body
● Intermediate products of fat metabolism
→ The body will eliminate the
○ Acetone (2%)
glucose via urination → Polyuria
○ Acetoacetic acid (20%)
■ If excessive urination is not
○ β-hydroxybutyrate (78%)
corrected → dehydration
● Present only when the use of available
○ High glucose levels lead to osmotic
carbohydrates becomes compromised
diuresis, involving greater osmole
○ Fats are metabolized to supply energy in
concentrations that cause an increased
lieu of carbohydrates
osmotic pressure, which leads to reduced
○ Normally no measurable ketones are
water reabsorption in the kidneys.
produced
● Because of the acidosis, patients often breathe very
■ If there are measurable ketones
deeply and rapidly to eliminate carbon dioxide and
→ there is absorption of ketones
cause respiratory alkalosis (Kussmaul breathing)
■ If excessive → eliminated in the
and can experience respiratory distress due to the
urine
prolonged exertion of respiratory muscles.
■ Renal threshold: 70 mg/dL
● Patients can have breath that smells fruity or like
■ Also eliminated in the lungs
nail polish remover (acetone breath)
● Hyperventilation to
TREATMENT
eliminate carbon dioxide
● Inject insulin → correct hyperglycemia
FORMATION
● Bicarbonate → to correct acidosis
● Normal end products of fatty acid metabolism are
ALCOHOLIC KETOACIDOSIS
carbon dioxide and water
● Due to severe alcoholism
● Limited availability of carbohydrates forces the liver
● The pathophysiology of AKA starts with low
to oxidize fatty acids
glycogen stores and a lack of oral food intake
○ shifts the metabolism from carbohydrates
to fats and lipids
● Decreased oral intake causes decreased insulin
levels and increased counterregulatory
hormones such as cortisol, glucagon, and
epinephrine (stress hormones)
● Ethanol is metabolized to acetaldehyde and
acetyl-CoA, leading to an increased NADH/NAD+
ratio
● End result:
○ increased NADH/NAD+ ratio increases
lipid metabolism and increased breakdown
of lipids to ketoacids
CLASSIC TESTS FOR KETONE
● Ferric chloride test (Gerhardt’s test, 1865)
○ Discontinued due to many false-positive
reactions (salicylates)
● Normal fatty acid metabolism → CO2 and water
● Ketone in urine → acidosis

@mlstranses. | 4
● Nitroprusside test (Legal’s test, 1883; modified by
Rothera in 1908)
○ More sensitive to acetone and
acetoacetate than reagent strips
○ Acetoacetate: 1-5 mg/dL
○ Acetone: 10-25 mg/dL

REAGENT STRIP REACTIONS

● Uses sodium nitroprusside (nitroferricyanide) to
measure ketones
● Acetoacetate reacts with sodium nitroprusside to
produce a change in color from beige to purple
● The addition of glycine enables detection of
acetone (Chemstrip)
○ Acetoacetate: 5-10 mg/dL
○ Acetone: 50-70 mg/dL

REACTION INTERFERENCE
● False positive (difference: color fades rapidly on
standing)
○ Phthalein dyes (phenolsulfonphthalein)
○ Highly-pigmented red urine
○ Levodopa (medication for Parkinson’s
disease)
○ Medications containing free sulfhydryl
groups (MESNA, captopril)
● False negative
○ Improperly preserved specimens due to
rapid volatilization of ketones (remedy: test
immediately or refrigerate!)
ACETEST TABLETS
● Confirmatory test for questionable ketone strip
results
● May be used on other specimens
● Contains nitroprusside, glycine, and lactose
● 1 drop of specimen into the tablet and wait for 30
seconds
○ Acetoacetate: 5-10 mg/dL
○ Acetone: 20 mg/dL

@mlstranses. | 5
 | Topic #1.2 | [MLS 419-LEC] Analysis of Urine and Other Body Fluids

M1: CHEMICAL EXAMINATION OF URINE PART 2

Professor: Prof. Christian Villahermosa, RMT, MSMT
Date: March 4, 2024

BLOOD ● May also be due to post-strenuous exercise and

● Positive chemical test for blood is nonspecific: menstrual contamination
○ red blood cells (hematuria) ○ Strenuous exercise
■ Hematuria → intact RBCs in the ■ straining → blood vessels will
urine produce more
○ hemoglobin (hemoglobinuria) ■ After a period of rest → return to
■ Hemoglobinuria → Two reasons: normal
● Lysed RBCs in the urine ○ Menstrual contamination
(hematuria and ■ If one has menstruation → not
hemoglobinuria recommended for urine testing
● Hemoglobin goes inside HEMOGLOBINURIA
the glomerulus ● Hemoglobin in urine
(intravascular hemolysis) ● May due to the ff.
○ myoglobin (myoglobinuria) LYSIS OF RBC
■ Due to muscle injury ● The first reason why hemoglobinuria occurs is due
■ No connection with RBC/bleeding to the presence of RBCs which contains
but positive in the reagent strip hemoglobin
test ● The Lysis of RBC produced in the urinary tract
● Most accurate method in determining the particularly in dilute, alkaline urine
presence of RBCs in urine/more sensitive than ○ The lysis of RBC is due to the dilute urine
microscopic examination ○ The lysis occurred after reaching the
○ 5 RBCs/μL of urine → clinically significant kidney
(bleeding, injury) → detected by the ● RBCs in microscopic exam may be seen (hematuria
chemical test and hemoglobinuria)
■ but may not be detected ○ There is RBC in the urine (hematuria)
microscopically (too small) ○ However, there is Lysis of RBCs → release
CLINICAL SIGNIFICANCE of hemoglobin (hemoglobinuria)
● Hematuria INTRAVASCULAR HEMOLYSIS
○ cloudy or smoky red urine ● No RBC in microscopic exam (hemoglobinuria only)
■ Cloudy → presence of blood cells ● Hemoglobin goes into the glomerulus
(heavy solute) ○ The lysis of RBC happened before
○ In the reagent strip: speckled pattern reaching the kidney (no hematuria)
(yellow background, speckled green color) ● Normally: free hemoglobin forms a complex with
● Hemoglobinuria (clear red urine) haptoglobin → no Hgb in urine
○ Clear because these are only small ○ If all available haptoglobin is bound
proteins (increased hemoglobin) → free Hgb is
○ In the reagent strip: green filtered by glomerulus and could either be:
● Myoglobinuria (clear red-brown urine) ■ Excreted
○ Clear because these are only small ■ Processed by renal tubular cells
proteins into hemosiderin
HEMATURIA ● Hemosiderin
● Intact RBCs in the urine confirmation: prussian
● From trauma or damage of renal or blue
genitourinary origin
○ Renal calculi (kidney stones)
○ Glomerulonephritis
■ Inflammation → influx of blood
cells
○ Pyelonephritis
○ Tumors
○ Exposure to chemicals and anticoagulant
therapy Hemosiderin in concentrated urinary sediment after staining
● Why is RBC seen in the urine? with Prussian blue (Rous test)
○ Tissue damage → the blood vessel is ● Causes: hemolytic anemias, transfusion reactions,
exposed and damaged → RBC leaks into severe burns, strenuous exercise, brown recluse
the urine spider bites, infections (e. g. malaria)

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 ○ The change in the color strip is due to
myoglobin (supernatant), since
hemoglobin settles at the bottom
● Myoglobin present:
○ supernatant remains red
○ positive for blood
● Hemoglobin present:
○ red precipitate
○ negative for blood
REACTION INTERFERENCE

FALSE POSITIVE FALSE NEGATIVE

MYOGLOBINURIA
● Menstrual contamination ● High SG/crenated
● Myoglobin
● Strong oxidizing reagents RBCs
○ heme-containing protein found in muscle ● Vegetable peroxidase and ● Unmixed specimens
tissue bacterial enzymes (e.g., E. ● Formalin as
● Muscle destruction/rhabdomyolysis coli peroxidase) preservative
○ Trauma ○ In cases of oxidizing ● High concentrations of
○ Crush injuries and contact sports (common reagents and ascorbic acid > 25
cause) peroxidases → the mg/dL
enzymes would react to
○ Muscle ischemia (prolonged coma,
the pad → change in
alcoholism) color
■ Blood flow is not proper → death
of the tissue BILIRUBIN
○ Muscle infections ● Provides an early indication of liver disease
○ Myopathy from medications ● only conjugated bilirubin is detected in urine
○ Seizures/convulsions ○ Since conjugated bilirubin is water soluble
■ Due to increased muscle activity
○ Toxins from snake and spider bites
HEMOGLOBINURIA VS MYOGLOBINURIA

PARAMETER HEMOGLOBINURIA MYOGLOBINURIA

Urine color Pink, red, brown Pink, red, brown

Blood reagent Positive Positive

strip test
PREHEPATIC
Serum color Pink to red Pale yellow ● before reaching the liver (RBC)
(hemolysis) (normal) ● Thalassemia and pernicious anemia can cause
prehepatic jaundice
Serum chemistry tests ○ Since erythropoiesis is ineffective → they
are not proper RBCs → they must be
Haptoglobin Decreased to absent Normal destroyed
CLINICAL SIGNIFICANCE
Myoglobin Normal Increased

Free Increased Normal

hemoglobin

Creatine kinase Increased but <10 Increased but >10

times upper times upper
reference limit reference limit
HISTORICAL TEST: AMMONIUM SULFATE
PRECIPITATION
● 2.8 g of ammonium sulfate is added to 5 mL
centrifuged urine
● Mix and let the specimen sit for 5 minutes
● Filter/centrifuge urine and test supernatant with
reagent strip for blood
○ Dip the reagent strip to the supernatant
only
● Hemoglobin is larger and is precipitated

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 NORMAL BILIRUBIN PATHWAY POSTHEPATIC
1. RBC is degraded → unconjugated bilirubin will be
liberated → goes in the liver
2. In the liver, unconjugated → forms conjugated
bilirubin
3. Conjugated bilirubin leaves the liver via the bile
duct, reaches the small intestine → travels, and
reaches the distal ileum and the large intestine
4. The conjugated will be converted back to
unconjugated bilirubin
5. Metabolized by the intestinal bacteria →
urobilinogen
6. A portion of the Urobilinogen will be converted to
urobilin (stool color), and another portion will
recirculate (normal in urine)
a. Bilirubin is negative in urine
● Mechanical obstruction of the bile duct
i. since all bilirubin is converted into
● Urobilinogen is low because bilirubin was not
urobilinogen
converted to urobilinogen due to the obstruction
PREHEPATIC
● Bilirubin is increased → sent to the circulation → to
1. There is an increased BRC degradation
the kidney → increased in urine
2. Increased heme → increased unconjugated →
● Stool is pale because urobilinogen was formed →
converted into conjugated → urobilinogen
not converted to stercobilin
3. A portion is found in the stool, and a portion is
REACTION INTERFERENCE
reabsorbed
4. Increased urobilinogen output in urine FALSE POSITIVE FALSE NEGATIVE
a. The serum of the patient has increased
bilirubin ● Highly-pigmented urine ● Exposure to light
HEPATIC (e.g., phenazopyridine) ○ Photooxidation of
○ Urine color bilirubin
touches the pad → ● Ascorbic acid > 25
changes the color mg/dL
of the pad ● High nitrite
● Indican (intestinal concentrations
disorders) ○ It can prevent the
reaction of
bilirubin to the
diazonium salts of
the pad
ICTOTEST TABLETS
● Confirmatory test for bilirubin
○ Can detect 0.05 to 0.1 mg/dL of bilirubin
○ Reagent strip: 0.5 mg/dL lower limit of
detection
● The liver can neither conjugate, excrete properly the PROCEDURE
formed bilirubin and endorse to the intestine the 1. Add 10 drops of urine to an absorbent test mat
formed bilirubin 2. Place 1 Ictotest tablet to the moistened area
1. The liver conjugates bilirubin → sent to the 3. Add 2 drops of water to the tablet
circulation 4. After 30 seconds, the tablet is removed, and the
2. When the conjugated bilirubin reaches the blood → absorbent pad is observed for the development of
circulates and reaches the kidney →detected in the any purple or blue coloration, which indicates a
urine positive test

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 UROBILINOGEN ● Valuable in detecting initial bladder infection
● Normally present in urine in concentrations of 1 (cystitis)
mg/dL or less ○ Many UTIs start in the bladder and
● Best specimen for quantifying and monitoring: progress upward; may be asymptomatic
○ 2 hours after mid-day meal (2-4 PM) ○ Early detection of bacteriuria plus antibiotic
■ ‘Alkaline tide’; enhanced therapy can prevent pyelonephritis and
urobilinogen excretion in alkaline other complications
urine ● Can be used to evaluate antibiotic therapy
● Fresh urine is needed ● Can be used as periodical screen in persons at high
○ Labile in acid urine and easily risk for UTI
photo-oxidizes into urobilin ● Based on the ability of certain bacteria to reduce
CLINICAL SIGNIFICANCE nitrate (normal constituent) to nitrite
● Early detection of liver disease ○ Sensitivity: 100,000 organisms/mL
● Liver disorders, hepatitis, cirrhosis, carcinoma REACTION INTERFERENCE
(hepatic) ● Bacteria that lack nitrate reductase
● Hemolytic disorders (pre-hepatic) ○ Negative nitrite
CLASS EHRLICH’S REACTION ○ Nitrate reductase is found in many
● Old qualitative screening for urobilinogen Gram-negative bacteria that most
● Urobilinogen reacts with frequently cause UTI
pdimethylaminobenzaldehyde (Ehrlich’s reagent) in ○ Other organisms (non-nitrate-reducing
an acid medium to form a pink, magenta, or red bacteria, yeasts, T. vaginalis) that cause
color UTI are not detected
● Nonspecific test (many Ehrlich reactive substances) ● Insufficient contact time between bacteria and
PROCESS nitrate
● 1 part Ehrlich’s reagent + 10 parts urine in a tube ○ At least 4 hours for the bacteria to convert
● incubate for 5 minutes nitrate to nitrite
● Dark color → increased urobilinogen ○ First morning specimen is ideal
■ Enough time for the bacteria to
convert nitrate to nitrite
● Lack of urinary nitrate
○ Nitrate is commonly found in green
vegetables
○ Diet is not controlled pre-testing so
false-negative results may occur
● Large quantities of bacteria further reducing nitrite
to nitrogen
● Antibiotics which inhibit bacterial metabolism
LEUKOCYTE ESTERASE
● Normally, WBCs may be present in urine in small
numbers
REACTION INTERFERENCE ○ 20/HPF indicate a pathologic process
● A more standardized test than microscopic
FALSE POSITIVE FALSE NEGATIVE
examination of urine sediment
● Multistix ● Multistix ○ Can detect lysed WBCs which are not
○ Ehrlich reactive ○ old specimens, seen in microscopic examination
substances formalin ● Sensitivity: 10-25 WBCs/microliter
● Chemstrip preservation ○ Note: A negative result does not rule out
○ highly pigmented ● Chemstrip increased number of WBCs
urine ○ old specimens, CLINICAL SIGNIFICANCE
formalin
preservation, high ● Increased WBCs are indicators of UTI or
nitrite inflammation in the urinary tract
concentrations ○ Detects the presence of esterase in
granulocytes and monocytes (also present
NITRITE
in Trichomonas and histiocytes)
● Rapid screening test for the presence of UTI
● Infections caused by Trichomonas, Chlamydia,
○ REVIEW!
yeast, and inflammation of renal tissue produce
■ Some bacteria cannot convert
leukocyturia without bacteriuria
nitrate to nitrite
● Assessment of LE and nitrite tests can be
■ One can have UTI but the
cost-effective measures to determine the necessity
causative agent cannot convert
of performing urine culture
nitrate to nitrite; LE positive Nitrite
negative

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 REACTION INTERFERENCE

FALSE POSITIVE FALSE NEGATIVE

● Vaginal secretions ● High concentrations of

contamination protein, glucose,
● Strong oxidizing agents or oxalic acid, Vitamin C,
formalin in the container gentamicin,
● Highly –pigmented urine cephalosporins,
tetracyclines
● Inaccurate timing
(note: read LE after 2
minutes)

SPECIFIC GRAVITY
● Expression of solute concentration
● A fixed SG of 1.010 regardless of hydration implies
significant renal tubular dysfunction
● Reagent strip measures ionic or charged solutes
only

DIRECT AND INDIRECT SG MEASUREMENTS

DIRECT SPECIFIC GRAVITY METHODS
● Direct specific gravity methods determine the actual
or true density of urine, regardless of the solutes
present. All solutes are detected and measured
○ Examples: Urinometry, harmonic
oscillation densitometry
○ Other solutes are present because of other
abnormal processes unrelated to
concentrating ability
INDIRECT SPECIFIC GRAVITY METHODS
● Reagent strip and refractometry

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 | Topic # 1| [MLS 419-LAB] Analysis of Urine and Other Body Fluids

M1: MICROSCOPIC EXAMINATION OF URINE

Professor: Prof. Christian Villahermosa, RMT, MSMT
Date: February 20, 2024

MICROSCOPIC EXAMINATION OF URINE

STAIN DESCRIPTION
● Last step in urinalysis
● After the chemical test → centrifuge the urine Sternheimer-Malbin ● Consists of crystal violet
specimen Stain and Safranin O
SPECIMEN PREPARATION ● Absorbed well by WBCs,
● Specimens should be examined fresh or epithelial cells, and
adequately preserved casts
SPECIMEN VOLUME
● Standard amount collected: 10-15 mL, average 0.5% toluidine blue ● enhances nuclear detail
of 12 mL VOLUME OF SEDIMENT EXAMINED and differentiates WBCs
○ Note on the result form if the volume is ● Ideal: transfer the specimen using pipette and RTE
less than 10 mL ○ Other options: Pour the sample onto the
CENTRIFUGATION slide Oil Red O and ● Lipids (triglycerides,
● 20μL (0.02 mL) covered by a 22 x 22 mm glass Sudan III neutral fats, cholesterol)
● Speed of the centrifuge and the length of time
should be consistent at 5 min. cover slip
Gram Stain ● Used in Bacteriology for
○ RCF 400-450 g ○ If there is no cover slip → use the
differentiation of gram
SEDIMENT PREPARATION mouth of the tube to spread the urine negative and gram
● After centrifugation → Decant the sample (smear) → microscopy positive bacteria and
○ Uniform amounts should remain in the EXAMINATION OF THE SEDIMENT for casts
tube after decantation (0.5 and 1.0mL ● LPO (10 fields)
○ detects casts and ascertain general 2% acetic acid ● accentuates nuclei of
frequently used). WBCs and epithelial
composition of the sediment
● Resuspend the sediment with the liquid left in the ● HPO (10-20 fields) cells
tube (shake the tube) ○ confirms type of sediments found
○ Not done vigorously SEDIMENT STAINS Hansel Stain ● For the detection of
○ Resuspension is essential to provide ● Typically, the urine sample is examined eosinophils in
equal distribution of elements in the unstained (routine) drug-induced allergic
microscopic fields. reactions
● If needed to target specific sediment → stains ● Consists of methylene
● Increases overall visibility of sediment blue and eosin Y in
elements being examined methanol
● Imparts identifying characteristics to cellular ● Wright’s stain can be
structures used as an alternative

Prussian Blue Stain ● Confirms the presence

of hemosiderin
granules

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● NOTE:
○ WBCs in the urine is unstained → No
diff count
■ Reported as WBC, not
neutrophils, etc.
○ If eosinophil is present → they may
have a disease called “Acute interstitial
nephritis”
■ Inflammation of the tubulues of
the patient due to drug induced
allergic reactions
○ Hemosiderin → Destruction of RBC
■ Once hemoglobin will reach
the nephron → reabsorbed by
the tubules → becomes
hemosiderin
■ If hemosiderin is present →
Hemolytic anemic
● Due to malaria,
paroxysmal nocturnal
hemoglobinuria
CYTODIAGNOSTIC URINE TESTING
● play an important role in the early detection of
renal allograft rejection and in the differential
diagnosis of renal disease
● 10:1 concentration of first morning urine →
cytocentrifugation → Pap stain
● Provides additional method for detecting and
monitoring renal disease and malignancies of the
lower urinary tract

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SEDIMENT SEEN IMAGE DESCRIPTION
IN THE URINE

RED BLOOD NORMAL VALUES

CELLS ● 0-3/hpf
DESCRIPTION
● Typical form: smooth, non-nucleated biconcave disks; 7μm diameter
● Other forms:
○ Crenated cells in concentrated urine
■ Shrinked RBC due to the hypertonicity of the urine
○ Ghost cells in dilute urine
■ Seen in urine that is hypotonic
○ Dysmorphic in glomerular disorders
● Reporting depends on the lab protocol whether to report RBCs as one or differentiated with
one another
● Frequently confused with yeast cells, oil droplets, air bubbles, pus cells
● RBCs + RBC cast:
○ renal bleeding
● RBCs without cast or proteinuria:
○ contamination or bleeding below the kidney
RBC CORRELATION WITH OTHER TESTS
● Urine color
○ note that a normal-appearing urine can still have increased RBCs present
● Blood reaction
○ can be negative owing to ascorbic acid interference; degree of interference varies
with reagent strip brand
● Protein reaction
○ if the hemoglobin concentration exceeds 10 mg/dl, it will test positive
CLINICAL SIGNIFICANCE
● any condition that results in inflammation or that compromises the integrity of the vascular
system throughout the urinary tract can result in hematuria

WHITE BLOOD NORMAL VALUES

CELLS/ PUS ● 0-8 WBCs/hpf (Report any clumping)
CELLS DESCRIPTION
● Predominantly neutrophils
○ Eosinophils in acute interstitial nephritis due to drug hypersensitivity
○ Lymphocytes in renal allograft rejection
● ‘glitter cells’ in hypotonic urine
● May look like dead Trichomonads, crenated RBCs, and RTE
WBC CORRELATION WITH OTHER TESTS
● LE reaction
○ can be negative despite increased WBCs owing to excess hydration or when the
WBCs are lymphocytes
○ LE screening: 10-25 WBCs per microliter; amount of LE may be insufficient to
produce positive response

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 ● Nitrite reaction
○ Not all bacteria can convert nitrate to nitrite
○ Most UTI agents can convert nitrate to nitrite → positive nitrite and LE
● Physical examination: gray-white sediment, cloudy urine, foul odor
CLINICAL SIGNIFICANCE
● Inflammatory conditions (bacterial and non-bacterial)
● Renal diseases

EOSINOPHILS ● Primarily associated with drug induced interstitial nephritis, small numbers seen in UTI
and renal transplant rejection
● Not normally seen in the urine so presence is pathologically significant
● May be specifically requested
○ Cytocentrifuged and stained with Hansel’s stain or Wright’s stain

MONONUCLEAR ● Rare in urine

CELLS ● If present → endorse to the histopath section
● Lymphocytes, monocytes, macrophages, and histiocytes
● Their presence should be reported for cytodiagnostic urine test

SQUAMOUS ● Largest and most common cell found in the urine sediment
EPITHELIAL ● Not clinically significant
CELLS ● Centrally located nucleus
● Good reference for focusing the microscope
● Present in large amounts when collected inappropriately
○ Midstream urine → to avoid SEC
● Clue Cells
○ borders obscured with Gardnerella vaginalis

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TRANSITIONAL ● Located in the pelvis, ureter, and bladder
EPITHELIAL ○ If seen in the urine, something happened to these areas, since they are not usually
CELLS seen
● Also called urothelial cells
● Smaller than SEC with centrally located nuclei
● Syncytia
○ transitional cells in clumps following catheterization
● Caudate transitional cells
○ Tadpole-shaped cells that indicate disease in the renal pelvis
● If it exhibits abnormal morphology, this may indicate malignancy or viral infection and will thus
be referred for cytologic examination.

RENAL TUBULAR ● Tissue seen in the nephron

EPITHELIAL ○ PCT to DCT
CELLS ● Clinically significant
● Eccentrically located nucleus
● Smaller than TEC and SEC
● Commonly mistaken as pus cell
● Seen in:
○ Exposure to heavy metals
○ Drug-induced toxicity
○ Hemoglobin and myoglobin toxicity, viral infections
○ Pyelonephritis
○ Allergic reactions
○ Malignant infiltrations
○ Acute allogenic transplant rejection

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OVAL FAT BODY ● RTE cells or macrophages with absorbed lipids
● Damage to the glomerulus
● Highly refractile
● Identified through staining with Sudan III and Oil Red O (triglycerides only) using polarizing
microscopy
● Droplets composing of triglycerides, neutral fats and cholesterol appearing as Maltese cross
under polarizing microscopy
● Associated with lipiduria and nephrotic syndrome
● Referred to as “bubble cells”

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BACTERIA ● Not normally present in the urine
● UTI/contamination
● Common causative agent of UTI: Enterobacteriaceae (bacilli)
● May be present as cocci or bacilli
● Motility will differentiate the sediment from amorphous crystals
● May indicate UTI or urine contamination

YEAST ● Small, refractile oval structure that may or may not bud
● Mostly Candida albicans (budding and pseudohyphae) or Candida glabrata (no
pseudohyphae or may be phagocytized within WBCs)
● Clinical Significance:
○ contamination with vaginal secretions
○ diabetes mellitus
○ Pregnancy
○ women taking oral contraceptives
○ immunocompromised patients

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 PARASITES ● Trichomonas vaginalis frequently encountered
● Also: Schistosoma haematobium, Giardia lamblia, and Enterobius vermicularis (fecal
contamination)

SPERMATOZOA ● Easily identified in the urine by their oval, slightly tapered heads and long-flagella-like
tails
● May come from vaginal contamination or ejaculation
● Associated with positive protein reagent strip
● Laboratories not reporting its presence:
○ Cite lack of clinical significance
○ Possible legal consequences
● Only reported if:
○ Patients underwent vasectomy
■ No sperm → normal
○ Rape

MUCUS ● Protein material produced by the glands and epithelial cells of the lower genitourinary
tract and the RTE cells.
● Tamm Horsfall protein or Uromodulin is its major constituent
● Thread-like structures with low refractive index
● No clinical significance

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HEMOSIDERIN ● Present in urine 2-3 days after a severe hemolytic episode (transfusion reaction, paroxysmal
nocturnal hemoglobinuria

PRUSSIAN BLUE REACTION OR ROUS TEST

● The urine sediment is suspended in a freshly prepared solution of potassium ferricyanide HCl
● Hemosiderin iron causes the granules to stain Prussian blue

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ADDITIONAL PICTURES
RED BLOOD CELLS

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 WBC/PUS CELLS

SQUAMOUS EPITHELIAL CELLS

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TRANSITIONAL EPITHELIAL CELLS

BACTERIA

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YEAST

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 CASTS ■ Within the DCT, the movement 4. Once the casts have developed, they can
● Only sediments in the urine that are unique to is already slow (normal), if dislodge from the tubular lumen and travel
the kidney there are obstructions, the through the urinary tract, before being excreted
○ Casts are only seen in the urine movement will be slower → in the urine.
■ Since they are formed within filtrate will be steady at the
the lumens of the distal nephron → casts will form
convoluted tubule and ● Cylindroids
collecting ducts ○ casts that are well-formed at one end
● Shape is representative (assumes the shape) of but have a tail or tapered at the other
the tubular lumen end
○ parallel sides and rounded ends and ○ incomplete cast formation
may contain additional elements ○ formation of a cast in a tubule where the
present in the urine lumen width differs (naturally or from PROGRESSION OF THE CELLULAR CAST
● Low refractive index disease)
○ Each cast has a different R.I. ○ cast disintegration
● Dissolves quickly in dilute, alkaline urine CAST FORMATION
○ Samples must be processed 1. Precipitation of Tamm–Horsfall protein (also
immediately known as uromodulin) which is secreted by the
● Cylindruria epithelial tubule cells.
○ presence of urinary casts 2. Pre-existing condition (e.g., urine stasis) →
Why casts are formed? 3. Urine flow is slow → Aggregation of
● Major constituent: uromodulin Tamm–Horsfall protein into a protein matrix can
○ Uromodulin is a protein that is secreted then attract the adhesion of other tubular
constantly by the renal tubular particles (e.g., cells, bile, hemoglobin, albumin,
epithelium (RTE) → joins the urine immunoglobulins).
● If the patient is dehydrated and has normal urine ● If protein matrix is created and
flow → Mucous threads are seen in the urine eliminated in the urine → hyaline cast
○ However, if the flow of the urine is lower (basic; prototype cast)
than usual → the mucous ● However, if hyaline is formed and not
threads/uromodulin → stay longer in the excreted in the urine → the hyaline will
nephron → becomes a cast trap different sediments (e.g. cells) ● Starts as hyaline cast → Cellular cast
● Pre-existing conditions prior to cast formation: ○ Presence of RBCs due to
○ Increased in stress and exercise and glomerular damage → RBCs
under conditions of urine flow stasis, will be trapped in the hyaline
acidity, and the presence of sodium and cast → formation of RBC cast
calcium ● Thus, all cast start as hyaline cast
■ urine flow stasis (common) → ○ If hyaline cast is seen in the
slow movement of filtrate urine → no kidney problem
within the nephron due to since it was urinated
various causes such as ○ RBC cast, WBC cast →
dehydration, tumors, problem
obstruction, etc.

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 GRANULAR CAST
● Once the cellular cast is formed and it is not corrected (stays in the nephron) → the cells embedded within the cellular cast will degrade/ degenerate → the lysosome in the cell will
become granules → Formation of granular cast
WAXY CAST
● If the formation of the granular cast is still not corrected → granules will degrade → waxy cast
● Seen in end-stage renal disease/chronic kidney failure
○ The patient did not urinate regularly

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 CASTS

CAST SEEN IN IMAGE DESCRIPTION

THE URINE

HYALINE CAST NORMAL VALUE

● 0-2/LPF
DESCRIPTION
● Basic/ Prototype → first cast that is formed
● No inclusions in the matrix
● Consists almost entirely of uromodulin
● Most frequently seen cast
● Increased in strenuous exercise, dehydration, heat exposure, and emotional stress
● Refractive index similar to urine
○ Normal parallel sides and rounded ends
○ Cylindroid
○ Wrinkled or convoluted

RED BLOOD CELL DESCRIPTION

CASTS ● Not typically seen in the urine
● Hyaline cast + RBC
● Bleeding within the nephron
● RBCs are seen along with the cast
● Detected under LPO by their orange-red color

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 WHITE BLOOD DESCRIPTION
CELL CASTS ● Signifies infection or inflammation within the nephron
● Most frequently used to distinguish upper UTI (pyelonephritis) from lower UTI (cystitis)
○ Upper UTI → Kidney
○ Lower UTI → Bladder
■ Most UTI starts at the urethra, then it goes up
● The UTI should be treated will it is still at the bladder, so that it
does not reach the kidney
■ Presence of white blood cell casts → UTI is at the kidney already
● Observe for free WBCs

BACTERIAL DESCRIPTION
CASTS ● Difficult to identify because they resemble granular casts
○ Perform Gram stain on a dried or cytocentrifuged specimen

RENAL DESCRIPTION
EPITHELIAL CELL ● Denotes advanced tubular destruction producing urinary stasis with disruption of tubular
CASTS linings
● Accompanies WBC casts in cases of pyelonephritis

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 FATTY CASTS DESCRIPTION
● Seen in conjunction with oval fat bodies and free fat droplets in disorders causing
lipiduria and associated with nephrotic syndrome
○ Only in nephrotic syndrome → eliminate lipids in urine (lipiduria)
■ Nephritic syndrome → no lipiduria (Glomerulonephritis and pyelonephritis)
● Also seen in toxic tubular necrosis, diabetes mellitus, and crush injuries
● Highly refractile, confirmed through Sudan III and Oil Red O fat stains

MIXED CELLULAR DESCRIPTION

CASTS ● With multiple cell types
● Most frequently encountered:
○ RBC and WBC casts in glomerulonephritis
○ WBC and RTE cell casts and bacterial casts in pyelonephritis

GRANULAR DESCRIPTION
CASTS ● Occurs as Coarsely Granular Cast or Finely Granular Cast
● degeneration of cells in cellular casts
○ Granules are from lysosomes excreted by RTE cells
● When they remain in the tubules for extended periods, granules disintegrate, and the cast
develops a waxy appearance

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WAXY CASTS DESCRIPTION
● Representative of extreme urine stasis indicating chronic renal failure and tubular
destruction
● Seen in patients undergoing dialysis/waiting for transplant
● With higher refractive index
HYALINE CAST VS WAXY CAST

BROAD CASTS DESCRIPTION

● Not separate type of cast
● Type of granular and waxy casts
○ A variation of granular and waxy (e.g. Granular broad casts and Waxy broad casts)
● Often referred to as renal failure casts
● Represents urine stasis
● Indicates destruction (widening) of the tubule
● Most commonly seen broad casts: granular and waxy

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 URINARY CASTS
CRYSTAL FORMATION
● Formed by the precipitation of urinary solutes (inorganic salts, organic compounds, medications)
● Precipitation is subject to the following factors:
○ Low temperature (RT)
■ Some solutes precipitate at low temperatures
■ A urine left to stand for 2 hours would contain more crystals
○ Solute concentration
■ The more solutes = more precipitation
○ pH (affects solubility)
■ Precipitate at acidic or alkaline
ACIDIC CRYSTALS

CRYSTALS SEEN IMAGE DESCRIPTION

IN THE URINE

AMORPHOUS DESCRIPTION
URATES ● Yellow brown granules
○ If in acidic pH → reported as amorphous urates
○ If alkaline pH → reported as amorphous phosphates
● Occur in clumps resembling granular casts
● Appear after refrigeration producing characteristic pink sediment (uroerythrin)

URIC ACID DESCRIPTION

CRYSTALS ● Shapes vary: rhombic, four-sided flat plates (whetstones), wedges, and rosettes
● Yellow-brown in color
● May sometimes resemble cystine crystals
● Highly birefringent under polarized light
● Associated with high levels of purines and nucleic acids
○ Confirmation: Clin. chem results
● Increased uric acid in the blood → enters the glomerulus → urinated as crystals

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CALCIUM DESCRIPTION
OXALATE ● No specific pH
CRYSTALS ● But Frequently seen in acidic urine but can be found in neutral urine and rarely in alkaline
urine
● Most common form:
○ dihydrate
■ Commonly seen in people drinking milk and increase intake of vitamin C
■ colorless, octahedral envelope or as two pyramids joined together at their
bases
■ aka weddellite
● Less frequently encountered:
○ monohydrate
■ oval or dumbbell-shaped
Dihydrate
■ massive in ethylene glycol or antifreeze poisoning
■ aka whewellite
● May be related to renal calculi or increased vitamin C intake
○ Ascorbic acid, once metabolized → produce oxalic acid

Monohydrate

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 BASIC CRYSTALS

CRYSTALS SEEN IMAGE DESCRIPTION

IN THE URINE

AMORPHOUS DESCRIPTION
PHOSPHATES ● Granular in appearance and similar to amorphous urates and differentiated through pH

TRIPLE DESCRIPTION
PHOSPHATE ● Also called struvite or ammonium magnesium phosphate; “coffin lid” crystals
● Seen associated with the presence of urea-splitting bacteria
○ Urease positive bacteria → prefer alkaline pH

AMMONIUM DESCRIPTION
BIURATE ● Yellow-brown color; “thorny-apple” in shape (spicule-covered)
● Converts to uric acid crystals when added with acetic acid
● Frequently encountered in old specimens and associated with presence of ammonia
produced by urea-splitting bacteria
○ Recollect the sample

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 ABNORMAL URINE CRYSTALS
● Commonly found in acidic urine and rarely in neutral urine
● Identity should be confirmed through chemical tests and patient information (medications).
● Clinically significant when they precipitate in the renal tubules
● Present in:
○ Liver diseases
○ Inborn error of metabolism
○ Renal damage
CRYSTALS SEEN IMAGE DESCRIPTION
IN THE URINE

CYSTINE DESCRIPTION
CRYSTALS ● Found in patients with inborn error of metabolism (cystinuria) with a tendency to form
renal calculi at an early age
● Colorless, hexagonal plates
● Confirm with uric acid through cyanide nitroprusside test

CHOLESTEROL DESCRIPTION
CRYSTALS ● Present in patients with nephrotic syndrome
○ Rarely seen unless specimens have been refrigerated
● Resembles rectangular plates with a notch in one or more corners
● Associated with disorders producing lipiduria
● Highly birefringent

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RADIOGRAPHIC DESCRIPTION
DYE CRYSTALS ● Similar appearance with cholesterol crystals and highly birefringent
● To differentiate from cholesterol crystals:
○ Test specific gravity
● Urine specimen in this presence may have markedly elevated specific gravity when
measured with refractometer

CRYSTALS ASSOCIATED WITH LIVER DISORDERS

● Tyrosine, Leucine, and Bilirubin Crystals
CRYSTALS SEEN IMAGE DESCRIPTION
IN THE URINE

TYROSINE DESCRIPTION
CRYSTALS ● fine, colorless to yellow needles that frequently form clumps or rosettes
● Seen in conjunction with leucine crystals with positive chemical test for bilirubin
● Encountered in inherited disorders of amino acid metabolism

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 LEUCINE DESCRIPTION
CRYSTALS ● Yellow-brown spheres that demonstrates concentric circles and radial striations
● When seen is usually accompanied by tyrosine crystals

BILIRUBIN DESCRIPTION
CRYSTALS ● Present in hepatic disorders producing large amounts of bilirubin in the urine
● Appears as clumped needles or granules with the characteristic yellow color of bilirubin
● Positive chemical test for bilirubin
● In viral hepatitis that produces renal tubular damage, these crystals may be found
incorporated in the matrix of cast

SULFONAMIDE DESCRIPTION
CRYSTALS ● Commonly found in patients treated with UTI
● Also seen in patients with inadequate hydration
● Possibility of renal tubular damage in the nephron in fresh urine
● Shapes: needles, rhombics, whetstones, sheaves of wheat, and rosettes (colorless to yellow-
brown)
● Perform diazo reaction for confirmation

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AMPICILLIN DESCRIPTION
CRYSTALS ● Found following massive dosages of penicillin compounds without adequate hydration
● Colorless needles forming bundles after refrigeration
● Obtain patient’s history

URINARY SEDIMENT ARTEFACTS

● May be found in improperly collected urine or dirty containers
● Highly refractile
● STARCH GRANULES
○ Seen in powdered gloves with highly refractile spheres usually with dimpled center
○ Resemble fat droplets when polarized producing Maltese cross formation
○ Occasionally confused with RBCs
○ Consider reagent strip parameter results
ARTIFACTS

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ARTIFACTS SEEN IMAGE DESCRIPTION
IN THE URINE

OIL DROPLETS DESCRIPTION

AND AIR ● Highly refractile and resembles RBC
BUBBLES ● Oil droplets resulting from contamination by immersion oil or lotions and creams
● Air bubbles occur when using cover slip

POLLEN GRAINS DESCRIPTION

● Seasonal contaminants appearing as spheres with a cell wall and occasional concentric
circles

HAIR AND FIBERS DESCRIPTION

● From clothing and diapers may be occasionally mistaken for casts but larger and more
refractile

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 | Topic #2 | [MLS 419-LAB] Analysis of Urine and Other Body Fluids
M2: URINE SMEAR PREPARATION, READING, AND REPORTING OF URINE
Professor: Christian Villahermosa, RMT, MSMT
Date: March 12, 2024

STEPS IN URINALYSIS REPORTING THE MICROSCOPIC EXAMINATION

1. Specimen Collection ● Most sediments: assessed or enumerated using at
2. Sample Verification least 10 lpf or 10 hpf
3. Physical Examination of Urine ○ Some sediments are counted either only on
4. Chemical Examination of Urine lpf or hpf
5. Microscopic Examination of Urine ■ Cast → lpf
6. Release of Results ■ RBC → hpf
URINALYSIS RESULT ○ Mixed → Count using both lpf and hpf
MANNER OF REPORTING/COUNTING SEDIMENTS
RANGE
● Casts, RBCs, WBCs: counted as range (e.g. 0-2, 2-5,
5- 10)

SPECIMEN PREPARATION
● Specimens should be examined while fresh or
adequately preserved.
● The midstream clean catch specimen minimizes ● View 10 fields and write down the number of
external contamination of the sediment. sediment per field
SPECIMEN VOLUME ○ To create a range:
■ lowest number among the 10
● A standard amount of urine, usually between 10 and
fields + the highest number among
15 mL, is centrifuged in a conical tube.
the 10 fields
CENTRIFUGATION
● Example: Assessment of pus cells
● 5 minutes at a relative centrifugal force (RCF) of 400
○ Field 1: 2
SEDIMENT PREPARATION
○ Field 2: 7
● A uniform amount of urine and sediment should
○ Field 3: 5
remain in the tube after decantation.
○ Field 4: 6
● Volumes of 0.5 and 1.0 mL are frequently used.
○ Field 5: 6
COMMERCIAL URINE SEDIMENT PREPARATION
○ Field 6: 4
○ Field 7: 5
○ Field 8: 8
○ Field 9: 4
○ Field 10: 5
■ Result: 2-8/ hpf
● In practice, the lower and upper limit of the range must
not exceed 10
○ For example:
VOLUME OF SEDIMENT ■ 5-10/ hpf
● The recommended volume is 20 uL (0.02 mL) covered ■ 1-7/ hpf
by a 22 × 22 mm glass cover slip. ■ 1-10/ hpf
EXAMINING THE SEDIMENT ■ X 3 –24/ hpf
● Microscopic examination: observation of a minimum ■ X 1-15/ lpf
of 10 fields under both low (10 × ) and high (40 × ) ○ If more than 10 → not evenly distributed
● When the sediment is examined unstained, many sediments
sediment constituents have a refractive index similar DESCRIPTION
to urine: examined under reduced light when using ● Some sediments (mucus, crystals, bacteria):
bright field microscopy. ○ qualitative assessment in descriptive (rare,
few, moderate, many)

@mlstranses | 1
 ○ numeric terms (1+, 2+, 3+, 4+) per field of
view

● Rare →Ex: Only 2 sediments per fields or Field 1 (1),

Field 2 (1) , Other fields (0)
● Few →Ex: In 8 fields (present) but in the 2 remaining
fields (absent)
SEDIMENTS THAT VARY IN REPORTING
CASTS
● count in at least 10 lpf, get the average, and report
number of casts per lpf
○ Example: 3-5/ lpf, 6-10/ lpf, 0-2/ lpf
● Use hpf to identify casts by type
RBCS, WBCS, RTE, OVAL FAT BODIES
● count in at least 10 hpf, get the average, and report
number of cells per hpf
○ Example: 3-10/ hpf, 8-10/ hpf
SQUAMOUS CELLS, TRANSITIONAL CELLS, MUCUS
THREADS
● rare, few, moderate, or many per lpf
BACTERIA
● few, moderate, or many per hpf
● may be reported as ‘packed
YEAST, TRICHOMONAS
● rare, few, moderate, or many per hpf
● For trichomonas
○ If non-motile → Can assessed per field
○ If motile → Cannot be assessed per field
(reported as positive)
SPERM CELLS
● report only if present
CRYSTALS
● Reported as rare, few, moderate, or many per hpf
TOO NUMEROUS TO COUNT (TNTC)
● Only one field
● Also known as ‘packed field’
● No standard rule
● In practice, if more than 30/ hpf can be reported as
TNTC or if the field is packed with or overwhelmed
with the sediment
● Usually applied to RBCs, WBCs/pus cells
● In bacteria, report as ‘packed field’

@mlstranses | 2
 | Topic # 3 | [MLS 419-LEC] Analysis of Urine and Other Body Fluids

M3: RENAL DISORDERS

Professor: Christian Villahermosa, RMT, MSMT
Date: March 11, 2024

CLASSIFICATION DEPOSITION OF IMMUNE COMPLEXES/ ANTIBODIES

● Glomerular Disorders IN THE GLOMERULUS
● Tubular Disorders
● Interstitial Disorders
● Renal Failure
● Renal Lithiasis
GLOMERULAR DISORDERS
● May be damaged due to the following:
IMMUNOLOGIC
● Immune system components produce changes and
damage to the membranes
● can cause damage to the kidney due to:
○ Deposition of antibodies to the glomerulus
■ Autoimmune → The body
produces antibodies against its
glomerulus (deposited in the
glomerulus)
○ Trapped circulating immune complexes
(antigen + antibody)
■ Immune complexes are already
formed in the circulation (before ● The glomerular wall is compromised
the kidney) ○ Podocytes are not attached to the
■ Trapped immune complexes in the basement membrane due to the presence
of immune complexes → The filtration will
glomerulus → Antibodies will
stop → urine output is low (oliguria)
reach the glomerulus ● The integrity is also compromised
NON-IMMUNOLOGIC ○ Proteins and blood could enter
■ Proteinuria and Hematuria
PATHOGENESIS OF GLOMERULAR DISORDERS
ALGORITHM FOR THE DIAGNOSTIC CLASSIFICATION OF
● Effect Of Antibodies Inside The Glomerulus GLOMERULONEPHRITIS
IMMUNE-MEDIATED PROCESSES
● Circulating immune complexes trapped in the
glomerulus (main mode)
● Complement binding leads to injury
○ The presence of antibodies will trigger the
complement pathway → membrane attack
complex → lysis of the cell
ANTIBODIES AGAINST GLOMERULAR TISSUE
ANTIGENS OR NON-GLOMERULAR ANTIGENS IN THE
GLOMERULI
● Glomerular injury:
○ caused by chemical mediators and toxic
substances
● Immune reactions produce protease, free radicals,
and arachidonic acid metabolites, which induce local
inflammatory response and lead to damage
○ if antibodies are present, the entire immune
system will “visit” the glomerulus → induce SYNDROMES/MANIFESTATIONS THAT INDICATE
the release of proteinase, free radicals, etc. GLOMERULAR INJURY
● “What is going on with the patient”
→ damage the glomerulus
● Needs to be differentiated because medical
management differs
ASYMPTOMATIC HEMATURIA OR PROTEINURIA
● Has injury to the glomerulus but no symptoms
● Indication of kidney problem: Presence of hematuria
and proteinuria
● No heavy medical management

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 SYMPTOMATIC fluid part of the blood (plasma)
ACUTE NEPHRITIC SYNDROME will remain inside the blood vessel
● Hematuria (Present all the time) ■ Loss of proteins (urination) → no
● Proteinuria (Present but not heavy) force/pressure will hold the
● Oliguria plasma inside the blood vessels →
● Azotemia leave the blood vessels and stay in
● Edema the extravascular component
● Hypertension (tissues) → edema
● Rapidly Progressive/Crescentic Glomerulonephritis ○ Hypovolemia → worsens the swelling
○ Needs intense medical observation ■ Low pressure in the blood due to
○ Progressive loss of kidney function over a low blood volume
short period of time (crescents in most of ● The fluid is in the tissues
the glomeruli) ■ Activates the RAAS (water and
■ Crescent → in biopsy of salt retention) → water will retain
glomerulus there is crescent in the body → increase swelling
shape with light pink color ○ Treatment for edema: Diuretics
NEPHROTIC SYNDROME ○ decreased excretion of sodium and water,
● Increased permeability to plasma proteins (> 3.5 increased excretion of protein (note:
g/day) anasarca/generalized edema/massive
● Heavy proteinuria edema if whole body)
○ Intense loss of protein into the urine
○ Loss of albumin, immunoglobulin, LMW
proteins, complement
■ Prone to infection because there
is rapid loss of immunoglobulin
○ Liver can’t compensate protein loss
● Hyperlipidemia and lipiduria
○ Key difference between nephritic
syndrome
○ Lipiduria → urination of lipids
○ Increased liver synthesis of lipoproteins
and decreased catabolism (due to angptl-4)
■ angiopoietin like 4:
● Protein that prevents
lipase → no fat
breakdown → stay as
intact lipid in the blood
● Enhance the formation
TAG → inreased lipids in
the blood → increased
lipids in the urine
○ Physician needs to decrease the lipid profile
of the patient → Needs fat lowering drugs
● Edema
○ Accumulation of fluids in the tissues
■ Fluids in the plasma will leave the
blood vessels and go into the
neighbouring tissues
○ Two types
■ Pitting edema
● Applying pressure →
cause any lasting
indentation
■ Non-pitting edema
● Applying pressure →
doesn't cause any lasting
indentation
○ Main cause of edema: Hypoalbuminemia
■ Presence of hypoalbuminemia in
nephrotic syndrome due to
increase loss of proteins in the
urine
● → decrease oncotic
pressure (pressure
exerted by plasma
proteins) because they
attract water
■ If normal protein in the blood → the

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 GLOMERULAR DISORDERS MEMBRANOPROLIFERATIVE
ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS
GLOMERULONEPHRITIS ● Causes: immune complex formation in glomerulus or
● Most common deposition of complement; associated with
● The kidney was fine; however, there was an initial persistent hepatitis C, autoimmune diseases, and
event that had occurred that damaged the kidney cancer
● Symptoms appear 1-2 weeks following respiratory ● Increased cellularity in the subendothelial cells of
infections and 6 weeks after skin infection caused by the mesangium, causing thickening of the capillary
group A Streptococcus (S. pyogenes) that contain M walls and extremely dense deposits in the GBM
protein in the cell wall ○ The main difference with membranous
■ M protein (virulence factor) → glomerulonephritis, the mesangium also
resist phagocytosis thickens in membranoproliferative
○ There are different timeframes in glomerulonephritis
glomerulonephritis, depending on what ■ Mesangium
kind of strep infection occurred ● core of the glomerulus
■ If respiratory (strep throat) → It ● Holds the trunk of the
takes 1-2 weeks for glomerulus
glomerulonephritis to occur
■ If skin infection (impetigo) → it
takes 6 weeks for
glomerulonephritis to occur
● The immune system reacts against the strep →
Deposition of immune complexes on the glomerular
membranes → can cause glomerulonephritis,
specifically, antibodies against these two antigens
that is present in S. pyogenes:
○ Antigens:
■ nephritis-associated plasmin
receptor
■ streptococcal pyrogenic exotoxin
B
MEMBRANOUS GLOMERULONEPHRITIS
● Pronounced thickening of the GBM due to the
deposition of IgG immune complexes and
complement
○ Action of membrane attack complex is
responsible for the glomerular damage
● Associated with SLE, Sjogren syndrome, secondary
syphilis, hepatitis B, gold and mercury treatments,
and malignancy
● Major cause of nephrotic syndrome in adults
● Diagnosis: Biopsy

● Slow and progressive and may lead to chronic renal

failure
○ Progress to nephrotic syndrome and has
poor prognosis
MINIMAL CHANGE DISEASE
● Glomerulus looks normal in light microscopy but
there is loss of podocyte foot processes in electron
microscopy
● Onset following allergies, viral infection, or
immunization in genetically predisposed individuals
● Probably caused by a circulating cytokine associated
with T cell response that changed the charge and
podocyte integrity
○ Charge of the glomerulus → repels albumin
● Major cause of nephrotic syndrome in children

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 CHRONIC GLOMERULONEPHRITIS
● Progression of previously discussed glomerular
disorders
FOCAL SEGMENTAL GLOMERULOSCLEROSIS ● Depends on the amount and duration of the damage
● Affects only certain numbers and areas of glomeruli to the glomerulus
→ sclerosis/scar tissue
● Diffuse damage to podocytes (decrease in number)
● Often seen in heroin and analgesics abusers and
AIDS patients; also, in diabetic patients and reaction
to infection and toxicity
IMMUNOGLOBULIN A (IgA) NEPHROPATHY
● Due to the:
○ deposition of immune complexes containing
IgA in the glomerular membrane (initial
event)
○ in situ formation of immune complexes after
deposition of galactose-deficient IgA
■ The immune complex was formed
in the glomerulus
● Trapped and engulfed by mesangial cells leading to
activation of alternative complement pathway ANTIGLOMERULAR BASEMENT MEMBRANE
● May occur 1-2 days following mucosal infection DISEASE
○ Defect in immune regulation causes ● Autoimmune
excessive mucosal IgA synthesis in ● Cytotoxic autoantibody (antiglomerular basement
response to antigens membrane antibody) against the glomerular
HENOCH-SCHONLEIN PURPURA/IgA VASCULITIS basement membranes after viral respiratory
● Occurs primarily in children after respiratory infections
infections ● Attachment of autoantibody → complement
● Similar pathophysiology and renal presentation with activation → capillary destruction
IgA nephropathy BUT…HSP also shows signs of ● May progress to chronic glomerulonephritis, RPGN,
systemic vasculitis, younger patients, preceding and end-stage renal failure
infection, and abdominal complaints ● If with lung hemorrhage (Goodpasture’s syndrome)
○ Vasculitis → inflammation of blood vessels ○ Lung involvement → goodpasture’s
● Presence of raised, red patches on the skin
● Renal involvement is the most serious complication

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WEGENER GRANULOMATOSIS/GRANULOMATOSIS EOSINOPHILIC GRANULOMATOSIS WITH
WITH POLYANGIITIS POLYANGIITIS/CHURG-STRAUSS VASCULITIS
● Causes a granuloma-producing inflammation of the ● Like Wegener’s but with elevated eosinophils (Initial
small blood vessels of the kidney and respiratory event: severe asthma)
system ○ If one is asthmatic → formation of
● MICROSCOPIC POLYANGIITIS: with ANCA but w/o granuloma
granuloma formation ● Most accurate test is lung biopsy showing
● Due to the presence of antineutrophil cytoplasmic granulomas and eosinophils
antibody (ANCA) which binds to the neutrophils in ALPORT SYNDROME
the vascular walls → immune response → granuloma ● Most common hereditary nephritis
formation ● Inherited disorder of collagen production affecting
○ ANCA → antibodies against one’s the GBM
neutrophil ● The kidney is damaged due to the defect of Type 4
collagen which is a component of the basement
membranes of the kidney, eye, and cochlea
○ the structure of the basement membrane is
not complete
○ pxs may have eye problems or
sensorineural deafness
SYSTEMIC DISEASES AND GLOMERULAR DAMAGE
AMYLOIDOSIS
● Deposition of amyloid in the glomeruli
○ Amyloid → abnormal proteins that
accumulate in the glomeruli
● Primary amyloidosis (AL amyloidosis)
TESTING FOR ANCA
○ deposition of Ig light chains due to plasma
● Incubate patient’s serum with either ethanol or
cell dyscrasias
formalin-fixed neutrophils
■ The patient should have first a
○ ANCA will bind with the prepared
condition called plasma cell
neutrophil
dyscrasias (disorders involving
● Examine using indirect immunofixation
plasma cells)
○ p-ANCA:
● Most common plasma
■ antibodies form perinuclear
cell dyscrasia: multiple
pattern (directed against
myeloma → BM is
myeloperoxidase)
producing excessive
■ Common in Churg-Strauss and
abnormal plasma cells
microscopic polyangiitis
■ In multiple myeloma, the
■ Under the microscope: stains only
antibodies produced has abnormal
the nucleus
light chains (abnormal structure)
○ c-ANCA:
■ Since the structure of Ig light
■ granular pattern throughout the
chains are abnormal → they are
cytoplasm (directed against
not formed into actual antibodies
proteinase 3)
■ These light chains will be
■ Common in Wegener’s
deposited and accumulate and
■ Under the microscope: stains the
eventually form amyloid
entire neutrophil

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 SYSTEMIC LUPUS ERYTHEMATOSUS/LUPUS ACUTE TUBULAR NECROSIS
NEPHRITIS ● Tissue death → Tubules cannot reabsorb nor secrete
● SLE → affects many organs ○ Increased glucose/proteins in the urine
● Lupus nephritis → In the kidney ● May be due to decreased blood flow that causes lack
● An autoimmune disease in which organs and cells of oxygen presentation to the tubules (ischemic ATN)
undergo damage initially mediated by tissue-binding or the presence of nephrotoxic substances (toxic
autoantibodies and immune complexes followed by ATN) in the filtrate
destruction mediated by complement activation and TWO WAYS WHY ATN OCCURS:
release of cytokines/chemokines ● Ischemic ATN
● Common autoantibodies: ○ Loss of blood supply → decrease/absent
○ ANA (antinuclear antibody) and oxygen distribution in the renal tubular
anti-dsDNA epithelium → Necrosis
■ ANA ○ may be caused by shock, trauma, and
● best screening test surgical procedures
● 90% of SLE patients test ● Nephrotoxic substances
positive ○ More common
● It produces antibodies ○ include drugs (some antibiotics,
against your nucleus → cyclosporine), radiographic dye/contrast
cellular destruction agents, organic solvents, heavy metals,
○ Anti-double stranded DNA toxic mushrooms, and large amounts of
■ Antibodies created against the hemoglobin and myoglobin → necrosis
DNA FANCONI SYNDROME
● Kidney involvement is usually its most serious ● Generalized failure of tubular reabsorption in PCT
manifestation ○ Increase urine glucose and proteins
○ Microscopic hematuria and proteinuria ● May be inherited, acquired through exposure to toxic
● Common manifestation: Butterfly rash on the face agents, or as a complication of multiple myeloma and
● Causes of SLE: genetic conditions, environment, or renal transplant
epigenetics, which alter the immune response → RENAL TUBULAR ACIDOSIS
lead to the formation of immune complex → ● pH of the blood: acidic
deposited everywhere in the body ● Normally, if one has acidosis: urine pH is acidic →
secretion of hydrogens
● In RTA, Inability of tubules to secrete adequate
hydrogen ions despite normal GFR
○ Urine pH is alkaline
■ Worsens the acidosis
● Patients have acidosis but unable to produce an acid
urine
DEFECTS IN SODIUM REABSORPTION

Bartter’s syndrome inability of the PCT to reabsorb

sodium
● Blood pressure is low
DIABETIC NEPHROPATHY
Gordon’s syndrome excessive sodium reabsorption in the
● Thickening of GBM (due to the loss of heparan PCT
sulfate) and expansion of the mesangium due to the ● Blood pressure is high
accumulation of extracellular matrix
○ Heparan sulfate → preserve the negative Liddle’s syndrome excessive sodium reabsorption in the
charge of the filtration barrier DCT
● Blood pressure is high
■ If the negative charge is damaged
→ albumin can enter UROMODULIN KIDNEY DISEASE
● Presentation: nephrotic syndrome ● Uromodulin: only protein produced by the kidney
● Important to test for microalbuminuria 5-10 years ● Inherited disorder caused by an autosomal mutation
after the onset of diabetes in the gene that produces uromodulin
TUBULAR DISORDERS ○ Increased production of abnormal
● May be due to the ff: uromodulin
○ Direct damage/actual damage to tubules ○ Defective uromodulin cannot be exported,
(infection or toxic substances) and so accumulates inside renal tubule cells
○ if a metabolic or hereditary disorder affects and leads to cell death
the intricate functions of the tubules ○ The mutation also causes an increase in
serum uric acid

@mlstranses | 6
 URINARY TRACT INFECTION ● Urinalysis findings
● Lower UTI: ○ WBCs, bacteria, WBC casts
○ urethritis (urethra) and cystitis (bladder) (pathognomonic of upper UTI)
○ pain or burning sensation on urination, ■ WBC casts (upper UTI) → since
frequent urge to urinate the casts are formed in the
● Upper UTI: nephron (kidney)
○ pyelitis (renal pelvis) and pyelonephritis ● Bacteria multiply in the interstitium and cause acute
(nephron) inflammation
○ Started as lower UTI (not treated) ● Tubular necrosis
○ fever, back or flank pain ● Bacterial toxins and leukocyte enzymes cause the
● May also include prostatitis and asymptomatic formation of abscess
bacteriuria (no treatment needed) CHRONIC PYELONEPHRITIS
● Urinalysis findings: WBCs, bacteria, mild proteinuria ● Persistent inflammation of renal tissue and causes
and hematuria, increased pH permanent scarring
○ Mild proteinuria → post-renal → bacteria ● Mostly due to congenital urinary structural defects
releases enzymes producing reflux nephropathy (vesicoureteral reflux
○ Bacteria in the bladder/urethra → and intrarenal reflux) and chronic urinary tract
contribute to the release of proteins obstruction
● 10 times more common in females ○ intrarenal reflux → from pelvis to nephron
○ Short urethra with proximity to the vagina ACUTE INTERSTITIAL NEPHRITIS
and the rectum ● The body mounted an immune response against the
○ Hormones that enhance bacterial allergen
adherence to mucosa ● Cell-mediated immune response that causes
■ Hormones → ensure the vagina is damage to the interstitium and renal tubular
rich in bacterial normal flora epithelium (3-21 days after exposure to the
■ Hormones can also support the offending agent)
adherence pathogenic bacteria ● Major cause: allograft rejection of transplanted
○ Absence of prostatic fluid and its kidney
antibacterial action ● Other causes: antibiotics, NSAIDs (NO
○ ‘Milking’ of bacteria up the urethra during EOSINOPHILURIA), antiepileptic agents, diuretics,
sex and certain diseases
■ During sexual intercourse, the ● Note: INCREASED NUMBER OF EOSINOPHILS in
bacteria in the urethra can be differential analysis
pushed upwards ○ Presence of eosinophils because of the
● 85% of UTI is caused by Gram-negative rods (E. coli, allergic response
Proteus, Klebsiella, Enterobacter, Pseudomonas) ○ Confirmation: stain using hansel
○ Gram-positive agents include S. faecalis, S. stain/wright stain
saprophyticus and S. aureus ● Urinalysis findings
INTERSTITIAL DISORDERS ○ Hematuria, proteinuria, WBCs, WBC casts
ACUTE PYELONEPHRITIS without bacteria
● Most frequently occurs because of the ascending ■ WBC casts without bacteria →
movement of bacteria from a lower UTI into the tubules since it is due to allergy
and interstitium YEAST INFECTIONS
○ May also be due to hematogenous infection ● More common in women
■ The bacteria passes through the ● Yeasts like Candida albicans are normal flora in the
glomerulus (common in sepsis) GI tract and vagina and kept in check by the
● Conditions that interfere with the downward flow of bacterial flora
urine ○ Normal: more bacteria than yeast
○ The ascending of bacteria is prevented if ■ Yeast cannot act as a pathogen
there is constant flow of urine ○ Yeast is more than bacteria → yeast
○ Renal calculi, catheterization, sepsis, infection
pregnancy, DM, immunosuppressive ● Occurs when the bacterial flora is disrupted by
therapy antibiotics or pH changes
■ Low urine flow due to obstruction NEPHROLITHIASIS
→ bacteria is able to ascend ● Renal calculi or kidney stones
■ DM → glucose → food of bacteria ● Vary in size from barely visible to large, staghorn
○ Vesicoureteral reflux calculi
■ The urine flow is from bladder to ● When urine becomes supersaturated with insoluble
urether (reverse;ascending) materials because excretion rates are excessive
and/or because water conservation is extreme,

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 crystals form and may grow and aggregate to form a ■ less soluble in acidic urine
stone ■ Urine pH should be alkaline
○ Increased insoluble substances → one has ○ Urea-splitting organisms
to be hydrated → secreted in the urine ■ Prefer alkaline pH
● 75-85% of calculi are calcium oxalate or calcium ● Urinary stasis
phosphate ○ Increasing chances of supersaturation and
○ Magnesium ammonium precipitation
phosphate/struvite/staghorn (5%): ● Nucleation or initial crystal formation
■ accompanied by chronic urinary ○ Nucleation → substances that forms
infections involving urea-splitting crystals need something to attach to →
bacteria, usually Proteus species form crystals
■ Commonly, UTI is involved ○ occur on renal epithelium, other cell
■ Large by nature surfaces, cellular debris, bacteria, and
denatured or aggregated proteins
RENAL FAILURE
● May be a gradual progression from the original
disorder to chronic renal failure or end-stage renal
disease
ACUTE RENAL FAILURE
● Now known as acute kidney injury
● Sudden loss of renal function
○ Uric acid (5-10%):
● caused by sudden decrease in renal blood flow (25%),
■ food rich in purine and with
acute glomerular and tubular disease (65%, 99% of
uromodulin-associated kidney
cases is due to ATN), or renal calculi or obstructions
disease
(10%, high BCHP equates to low GFR)
○ Cystine (1%):
○ Common cause ATN → cell death
■ in conjunction with hereditary
HEMOLYTIC-UREMIC SYNDROME
disorders of cystine metabolism
○ Indinavir: ● One of the most common causes of acute renal
■ medication for HIV patients failure in children
■ poor solubility in physiologic pH ● Commonly occurs after ingestion of meat infected
● Urinalysis findings with verocytotoxin-producing E. coli, most often
○ Crystals in urine, microscopic hematuria serotype O157: H7
■ Hematuria → Trauma ○ Enterohemorrhagic
PATHOGENESIS OF STONES ● The toxin damages the endothelium, reducing nitric
oxide, promoting vasoconstriction and necrosis, and
● Conditions favoring the formation of renal calculi:
promoting thrombosis
○ Chemical concentration or supersaturation
CHRONIC RENAL FAILURE
of chemical salts in urine
○ Optimal pH ● Progressive loss of renal function caused by an
○ Urinary stasis irreversible and intrinsic renal disease and progresses to
○ Nucleation or initial crystal formation end-stage renal disease
● Chemical concentration/supersaturation ○ Marked decrease in GFR, slow but
○ Dehydration continuous (< 25 ml/min)
○ Dietary excess or increased intestinal ■ Oliguria
absorption ○ Steadily rising serum BUN and creatinine
■ Eating too much purine rich food values
→ prone to the formation of uric ■ Blood is not filtered → blood
acid crystals → stone formation recirculates
○ Medications ○ Electrolyte imbalance
○ Endocrine and metabolic disorders ■ Not reabsorbed
■ Increased PTH =more calcium in ○ Isosthenuric urine
the blood = more calcium urine ■ 1.010 SG regardless of hydration
● Optimum pH ■ Tubules are failing
○ Isohydruria: constant and unchanging urine ○ Proteinuria and glycosuria
pH ○ Abundance of granular, waxy, and broad casts
○ Inorganic salts: (telescoped urine sediment)
■ less soluble in neutral or alkaline
urine
■ Urine pH should be acidic
○ Organic salts:

@mlstranses | 8
 |Topic # 4| [MLS 419-LEC] Analysis of Urine and Other Body Fluids

M4: Urine Screening For Metabolic Disorders

Professor: Christian Villahermosa, RMT, MSMT
Date: March 30, 2021

METABOLIC DISORDERS
● Metabolism can be normal;
● Presence of abnormal metabolites in urine
however, tubular reabsorption is
○ Abnormal metabolites → substances which
abnormal → seen in the urine
are normally produced by the body but are
not supposed to be seen in a urine sample End result appearance of abnormal metabolic
In normal conditions: through a series of metabolic substances in the urine
reactions → exempts them from being present in the urine
OVERFLOW DISORDERS
sample
● Metabolic disturbances that produce substances
involved in protein, fat, and carbohydrate
In Abnormal conditions: such as genetic disorders, chemical
metabolism
exposures, and metabolic defects → metabolites are not
○ Vast number of involved enzymes
metabolised → seen in the urine
● Main cause: disruption of enzyme function
● Can be detected and monitored by additional ○ Failure to inherit the gene that produces
screening and confirmatory tests the enzyme
● May be detected by observations of alert lab ○ Organ malfunction from disease or toxic
personnel when performing urinalysis reactions
NEWBORN SCREENING TESTS
Produces color homogentisic acid, melanin, indican, ● Traditionally performed
change porphyrin to detect and monitor
● Colors change after a few newborns for any
hours → If tested inborn error of
immediately → color metabolism
change would not be seen ○ Uses blood
from heel prick
Produces unusual phenylketonuria, MSUD, isovaleric ○ The test is not
odor acidemia, cystinuria compulsory
● 29 tests performed in the expanded newborn
Produces crystals cystine, leucine, tyrosine, uric acid screening (before: 6)
● If tested “positive” during the screening:
● These disorders are characterized mostly by genetic
○ Recall for confirmatory testing
defects
○ Follow-up in continuity clinics (periodic
○ Early detection → early
check-up)
management/treatment
OVERFLOW VERSUS RENAL DISORDERS

Overflow result from disruption of a normal

disorders metabolic pathway
● Increased plasma concentrations
of the nonmetabolized substances
● Even if the tubule function is
normal → Since there is an
accumulation of substances in
excessive amounts → renal
thresholds are overwhelmed →
seen in the urine → detected by
the laboratory

Renal are caused by malfunctions in the tubular METABOLIC DISORDERS

disorders reabsorption mechanism ● Amino Acid Disorders
● In disorders of the tubules → ● Porphyrin Disorders
reabsorption would not occur → ● Mucopolysaccharide Disorders
excreted in the urine ● Purine Disorders
● Carbodhyrate Disorders

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 AMINO ACID DISORDERS ○ Accumulation of phenylalanine,
phenylpyruvic acid, and other acid
Phenylalanine-Tyrosine ● Phenylketonuria metabolites interfere with myelination and
disorders ● Tyrosyluria
other neurologic functions
● Melanuria
● Alkaptonuria ○ Mousy odor in urine may be detected
● Management: avoidance of food rich in
Branched-chain amino ● MSUD phenylalanine
acid disorders ● Organic acidemia URINE TEST FOR PHENYLKETONURIA: FERRIC
CHLORIDE TUBE TEST
Tryptophan disorders ● Indicanuria ● Detects phenylpyruvic acid
● 5-hydroxyindoleacetic acid ○ Indirect test for
phenylalanine
Cystine disorders ● Cystinuria
● Cystinosis ● 5 drops of 10% ferric
● Homocystinuria chloride to 1 mL of urine
● A permanent blue-green
PHENYLALANINE METABOLISM
color is positive

TYROSYLURIA
● Accumulation of excess tyrosine in the plasma
(tyrosinemia) produces urinary overflow
● Enzymes required in the metabolic pathway are not
produced

Tyrosinemia Type 1 Absence of fumarylacetate hydrolase

Tyrosinemia type 2 Absence of tyrosine aminotransferase

● If enzymes (e.g, Phenylalanine hydroxylase, Tyrosine
hydroxylase) are absent → accumulation of their Tyrosinemia type 3 Absence of p-hydroxyphenylpyruvic
substrate (e.g, Phenylalanine, tyrosine) acid dioxygenase
PHENYLKETONURIA
● Results into mental retardation if dietary restrictions
● Increased phenylalanine levels due to defective or of phenylalanine and tyrosine are not implemented
absent phenylalanine hydroxylase ○ Phenylalaline is included since, in the
○ Phenylalanine is converted instead into pathway, phenylalanine is converted into
phenylpyruvic acid (another pathway) tyrosine
which may also be detected URINE TEST FOR TYROSYLURIA:
ALTERNATIVE PHENYLALANINE PATHWAY NITROSO-NAPHTHOL TEST
● 5 drops of urine + 1 mL of 2.63 N nitric acid + 1 drop
of 21.5% sodium nitrite + 0.1
mLm1-nitroso-2-naphthol
● Mix and wait for 5 minutes
● Orange-red color indicates presence of tyrosine
metabolites
MELANURIA
● Melanin: product of a second metabolic pathway for
tyrosine
● Deficient production of melanin results in albinism
● Increased levels of melanin in the urine: melanuria
○ Proliferation of melanocytes which
produces melanoma
■ Cancer cells produce
5,6-dihydroxyindole which
oxidizes to melanogen and then to
melanin → Dark urine upon
standing
● The most important and sometimes the only ● Change in color takes a
manifestation of PKU is mental retardation few hours

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 BRANCHED CHAIN AMINO ACIDS METABOLISM

ALKAPTONURIA
● Failure to inherit the gene that codes for the enzyme
homogentisic acid oxidase
● Accumulation of :homogentisic acid in blood,
tissues, and urine
○ Brown-stained or black-stained or
red-stained diapers
○ Deposition of brown pigment in body
tissues like the ears (ochronosis)

MAPLE SYRUP URINE DISEASE (MSUD)

● a defect or deficiency of the branched chain
ketoacid dehydrogenase complex
● elevated quantities of leucine, isoleucine, valine, and
○ Deposits in the cartilage cause arthritis
their corresponding oxoacids accumulate in body
○ No developmental delays or cognitive
fluids
issues; only chronic pain and mobility
○ Leucine has a bigger chance to be damage
problems
● Urine darkens/turns to black upon standing
URINE TESTS FOR ALKAPTONURIA

Ferric chloride test ● deep blue color

Clinitest ● yellow precipitate

Homogentisic acid test ● mL 3% silver nitrate + 0.5 mL

urine
● Mix
● Add 10% NaOH by drops
● Observe for black color
● An increase in leucine may cause competitive
inhibition with other precursors of
neurotransmitters causing the neurologic
manifestations
CLASSICAL MSUD
● is the most severe and
common form with symptoms
of poor suck, lethargy, hypo and
hypertonia, opisthotonic
posturing, seizures, and coma
developing 4-7 days after birth
○ Odor of maple syrup
in urine may be
detected as soon as
neurological
symptoms appear

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 MSUD MANAGEMENT
2. Indole is converted to indican in the liver and
● dietary restriction of branched-chain amino acids (
excreted in urine
leucine, isoleucine, valine)
3. Urine is colorless then turns to indigo upon
SCREENING TEST: 2,4-DINITROPHENYLHYDRAZINE
exposure to air
TEST
● 1 mL of urine+ 10 drops of HARTNUP DISEASE
0.2% 2,4-DNPH in 2N HCl ● Caused by a mutation on the gene that
● Wait 10 minutes encodes for the B0AT1 protein
● Observe for yellow turbidity ○ The protein is a
or precipitate sodium-dependent
co-transport protein for the
absorption of neutral amino
acids (e.g., tryptophan)
ORGANIC ACIDEMIAS present at the apical surface of the small
● an important class of inherited metabolic disorders intestine and renal tubular cells
(IMD) arising due to a defect in intermediary metabolic ○ excessive excretion of neutral amino acids
pathways of carbohydrate, amino acids and fatty acid in urine and feces
oxidation ● Characterized by photosensitive skin rash and
● leads to accumulation of organic acids in tissues and neuropsychiatric symptoms
their subsequent excretion in urine ○ Niacin deficiency: pellagra-like skin
● Common organic acidemias eruptions (tryptophan is precursor for
○ Propionic acidemia niacin)
○ Methylmalonic acidemia 5-HIAA
○ Isovaleric acidemia ● Serotonin is produced from typtophan by argentaffin
■ Easy to detect in the lab since it is cells in the intestine
characterized by the odor of ● Only small amounts of its degradation product,
sweaty feet 5-hydroxyindoleacetic acid, are released in urine
TRYPTOPHAN METABOLISM ● Argentaffinoma
○ A carcinoid tumor that produces excess
serotonin and results into elevated urinary
levels of 5-HIAA
5-HIAA TESTS
● Addition of nitrous acid and 1-nitroso-2-naphthol
○ Change in urine color to purple to black
○ Dietary restrictions: no intake of food rich
in serotonin (pineapples, bananas,
tomatoes)
○ Drug restrictions: withhold drugs like
phenothiazines and acetanilids for 72 hours
prior to testing
CYSTINE DISORDERS
● Inherited disorders

3 TYPES

Cystinuria defect in the renal tubular transport

of amino acids
● No reabsorption of cystine

INDICANURIA Cystinosis inherited defect in the lysosomal

● Presence of intestinal disorders that prevent the membranes that results into cystine
metabolism of tryptophan accumulation in cell
○ Obstruction
Homocystinuria defect in the metabolism of
○ Presence of abnormal bacteria methionine
○ Hartnup disease
CYSTINURIA
PROCESS: ● inheritable, autosomal recessive genetic defect that
1. Excessive amounts of tryptophan is converted to affects the proximal renal tubular reabsorption of
indole and reabsorbed in the circulation cystine
○ Conversion is due to the bacteria
present in the intestine

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● also affects lysine, ornithine, and arginine, but only PORPHYRIN DISORDERS
cystine is clinically significant as it is the only amino ● Enzyme defect in the metabolism of heme
acid in this group that will form stones
○ Excreted in the urine
● Cystine crystals appear in urine
● Urine odor: sulfur/rotten egg
○ Early indicators of cystinuria
CYANIDE-NITROPRUSSIDE TEST FOR CYSTINE
● 3 mL of urine + 2 mL
sodium cyanide
● Wait 10 minutes
● Add 5 drops of 5%
sodium nitroprusside
● Observe for red-purple
color

CYSTINOSIS
● Originated prior to the kidney
● a rare autosomal recessive lysosomal storage ● Solubility of porphyrin compounds varies
disorder in which the amino acid cystine ALA, porphobilinogen, are the most soluble
accumulates in the lysosomes of cells and uroporphyrin
● Most common type: infantile nephropathic type →
leads to end-stage renal disease Coproporphyrin less soluble but still can be found in
○ Defective transport leads to accumulation urine (stool may be used)
and crystallization of cystine in the
lysosomes.
■ Cystine in the lysosome is not Protoporphyrin less soluble → not seen in urine
metabolized → accumulates in the (stool may be used)
cells
○ Affected cells suffer from mitochondrial
dysfunction, oxidative stress, and Bile more acceptable specimen to avoid
inflammation and, in the end, undergo false-positive results
apoptosis. ● Presence of urobilinogen in
○ Since lysosomes are part of every cell type urine and stool can cause
cystine accumulation occurs throughout false-positive results
the whole body, making cystinosis a ● Can be inherited or acquired
systemic disease. ● porphyrin precursors (porphobilinogen and
● Tx: aminothiol cysteamine δ-aminolevulinic acid) and the porphyrinogens
○ used for over 20 years now (uroporphyrinogen, coproporphyrinogen,
HOMOCYSTINURIA protoporphyrinogen) are colorless, nonfluorescent
● Deficiency of cystathionine synthase enzyme which compounds
is part of methionine metabolism ● their oxidative forms (uroporphyrin, coproporphyrin,
● Homocysteine accumulates and dimerizes to form protoporphyrin) are dark red or purple and intensely
the disulfide homocystine, which is excreted in the fluorescent
urine ○ Detected by the laboratory
○ prone to thrombosis and has adverse ● Red or port wine urine color is an indicator of possible
effects on connective tissue particularly the porphyria
eyes and skeleton; adverse neurologic ● Disorders that result in accumulation of the precursors
effects may be due to thrombosis or a direct (porphobilinogen and daminolevulinic acid) present
effect with primarily neurologic symptoms, because these
● Detected by cyanide-nitroprusside test followed by substances are neurotoxins
silver nitroprusside ● when porphyrins are the major accumulation products,
photosensitivity is the distinguishing clinical feature.
○ When porphyrins absorb light, they cause
toxic free radicals to form; this causes
cutaneous lesions (e.g., extensive blistering
or bullous lesions) or a burning sensation
with an inflammatory skin reaction.

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 VAMPIRES AND PORPHYRIA ● If the red color remains in the aqueous phase (now the
The myth of vampires started in Eastern Europe because bottom layer)
intermarriage is common → there is a higher chance of a ○ porphobilinogen is present.
genetic disorder to be manifested in the bloodline ● If the red color is in the butanol phase (the top layer),
(homogenous gene pool) ○ urobilinogen or other Ehrlich’s reactive
substances are present
● Photosensitivity
● Pale color SUMMARY
● Port wine urine/red-stained teeth
● Psychiatric symptoms Urobilinogen ● Soluble to chloroform and
● Inherited disorder butanol
● Red color in chloroform layer
PORPHYRIN DISORDER TESTS
and butanol layer
Hoesch Screening Test for Porphobilinogen
● 2 drops urine + 2 mL Hoesch reagent Porphobilinogen ● Insoluble to chloroform and
● Observe the top of the solution for the appearance of butanol
a red color ● Red color remains in the
● Shake the tube urine-acetate mixture
● Red color is seen throughout the solution, indicating
Other ● Insoluble to chloroform, soluble
the presence of porphobilinogen
Ehrlich-reactive to butanol
● Sensitive to porphobilinogen but subject to many substance Red color in urine-acetate layer
false-positive reactions in chloroform tube, butanol layer
○ Not specific → false-positive results in butanol tube
Watson-Schwartz Test
Can differentiate and isolate porphobilinogen, urobilinogen or
Other Ehrlich-reactive substance
PROCESS
● Equal parts (≈2 mL each) of urine and Ehrlich’s
reagent are mixed in a tube
● Add saturated sodium acetate (≈4 mL), and the
solution is mixed again
● A positive test results in the development of a
characteristic red or magenta color

Presence of red/magenta color → proceed to extraction

No red/magenta color → Stop the test (no presence of
porphobilinogen, urobilinogen, or Other Ehrlich-reactive
substance

● Extraction is performed by adding chloroform (≈2 to

5 mL) to the mixture, followed by vigorous shaking
○ After adding, two layers are formed:
■ Aqueous phase (top layer)
■ Chloroform phase (bottom layer)

Aqueous phase Chloroform phase MUCOPOLYSACCHARIDE DISORDERS

● caused by a deficiency of an enzyme responsible for
If the red color resides only in If the red color resides only in the degradation of a metabolic product, whose
the aqueous phase (the top the chloroform phase (the accumulation results in lysosomal malfunction and
layer), porphobilinogen or bottom layer), increased disease
another Ehrlich’s reactive amounts of urobilinogen are ● Accumulation of incompletely metabolized
substance is present, and a present. polysaccharide portions (acid mucopolysaccharides
butanol extraction must be or glycosaminoglycans) in the lysosomes of
performed connective tissues and excreted in urine
BUTANOL EXTRACTION ○ Dermatan sulfate
○ Keratan sulfate
To differentiate porphobilinogen, urobilinogen or Other
○ Heparan sulfate
Ehrlich-reactive substance
● Best known mucopolysaccharidoses: Hurler
● To perform the butanol extraction, the aqueous syndrome, Hunter syndrome, and Sanfilippo
phase (the top layer) is transferred to another tube syndrome
and an equal volume of butanol is added.
● The tube is shaken vigorously, and the phases are
allowed to separate.

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 HURLER SYNDROME
● molecular defect in Hurler disease is in the activity
of α-L-iduronidase
○ Abnormal skeletal structure
○ Severe mental retardation
○ ‘steamy’ cornea

● self-destructive biting of the lips and fingers

HUNTER SYNDROME ○ Patients bite with a ferocity that leads to
significant loss of tissue
● molecular defect is in the
○ Relative deficiency of GTP in HPRT
enzyme iduronate sulfatase
deficiency, resulting in decreased dopamine
● Patients with the severe form
receptor activation
may appear identical to those
● Excess uric acid is known to precipitate in kidneys
with Hurler disease except for
and joints, resulting in renal calculi and tophi
the absence of cloudy corneas
CARBOHYDRATE DISORDERS
● presence of nodular or pebbly
● Melituria: presence of sugar in urine
skin lesions, most
○ Primary concern: galactosuria
characteristically over the scapular area, the upper
○ Others: lactosuria (may be seen in
arms or the lateral aspects of the thighs
SANFILIPPO SYNDROME pregnancy and lactation), fructosuria
(parenteral feeding), pentosuria (ingestion
● Exclusive excretion of heparan sulfate
of large amounts of fruit)
● Has 4 types
GALACTOSEMIA
● degeneration of the central nervous system (CNS),
● an inborn error of carbohydrate metabolism
resulting in mental retardation and hyperactivity,
characterized by elevated levels of galactose and its
typically commencing during childhood
metabolites due to enzyme deficiencies
● associated with a progressive and severe loss of
● In classic galactosemia, the enzyme that is reduced
intellectual processes (such as speech) and motor
or missing is called galactose-1- phosphate uridyl
functions (including walking and swallowing) then
transferase (GALT) which enables the body to break
ultimately regress to a vegetative state until death
PURINE DISORDERS down galactose into glucose
Lesch-Nyhan disease
● deficiency of hypoxanthine-guanine
phosphoribosyltransferase, the enzyme is
responsible for recycling purines
● causes an increase in guanine and hypoxanthine,
which eventually gets converted into uric acid
● Characterized by hyperuricemia,
neurodevelopmental abnormalities with global
developmental delay, involuntary movements, and
self-injurious behavior ● Accumulation of galactose in the body is not good
● first sign is usually the appearance of orange crystals ● feeding problems, failure to thrive (most common
or orange sand in the diapers initial clinical symptom), hepatocellular damage,
● Massive excretion of urinary uric acid crystals bleeding, and sepsis in untreated infants which lead
○ alert: increased uric acid crystals in to mental retardation
pediatric urine specimens ● in approximately 10% of individuals, cataracts are
present
GALACTOSEMIA MANAGEMENT
● Dietary elimination of milk and milk products
containing lactose
○ Alternative: soy-based formula

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 | Topic #1 | [MLS 419-LAB] Analysis of Urine and Other Body Fluids

F1: Semen Analysis

Professor: Christian Villahermosa, RMT, MSMT
Date: April 22, 2024

REVIEW: SPERMATOGENESIS
Midpiece ● Where the mitochondria are found (energy)

Tail/flagellum ● Motility (travel from the cervix → fallopian tube)

○ Sperm cells move 1-4mm per minute

● The sperm cell would encounter “trials” while traveling to the egg cell
○ In the uterus, certain cells would try to phagocytose the sperm cell
○ In the fallopian tube, sperm cells would be repelled by the wave-like action
of the fimbriae
■ Only a few sperm cells would reach the egg cell and penetrate the
corona radiata

REVIEW!!
● Production of spermatogonium to spermatid → Testis
● Sperm cell → matures in the epididymis
○ The addition of the flagellum to the spermatid happens in the
epididymis

PHYSIOLOGY

Testes (seminiferous ● secretion of sperm

tubules)

Epididymis ● sperm maturation and development of flagella

● stores sperms
○ Sperm cells stay in the epididymis until there
is stimulus
THE SPERM CELL HAS THREE MAJOR PARTS:
Ductus deferens (vas ● transports sperm to the ejaculatory duct
Head ● The acrosome contains digestive enzymes which will help the deferens)
sperm to penetrate the corona radiata
● Where the nucleus is found → contains the 23 chromosome Seminal vesicle ● transport medium for the sperm

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 ● Sterile glass or plastic container
● Secretions in the seminal vesicle, which is rich in
fructose and flavin, will be added to the sperm ● Collected in a private room near the laboratory
○ Fructose (carbohydrate) and flavin → Energy ● Give a clear written and verbal instructions concerning the collection of semen
(needed by sperms to travel) ● Deliver to the laboratory within 1 hour after collection
● Record the patient’s name, the period of sexual abstinence, and the date and time of
Sperm + secretions of the seminal vesicle → will reach the ejaculatory duct → Prostate specimen collection
gland METHOD OF COLLECTION
1. Masturbation (highly recommended)
Prostate gland ● The secretions of the prostate gland are added to the
2. Coitus interruptus (only in exceptional cases)
mixture (sperm + secretions of the seminal vesicle)
3. Silastic seminal fluid collection device
● contains acid phosphatase, citric acid, zinc, and
proteolytic enzymes responsible for both the coagulation ● Silastic → condom but no spermicide
and liquefaction of the semen 4. Aspiration from the vaginal vault after coitus
○ Viscosity → protects the sperm cell from the
outside environment NOTE!!
● propels the sperm through the urethra ● Ordinary condoms are not recommended.
○ Contains spermicide
Bulbourethral glands ● secretes alkaline mucus (increases the pH) PRESERVATION
○ Neutralizes the acid pH of the vagina ● Awaiting analysis
● After the addition of the bulbourethral secretions →
○ Semen stored at body temperature
Formation of the semen
● For artificial insemination
○ frozen and stored for 1 year at -85 C at sperm bank
COMPOSITION OF SEMEN
MACROSCOPIC ANALYSIS
5% Spermatozoa

60-70% Seminal ● Fructose and Flavin

Fluid

20-30% Prostate ● Acid phosphatase, citric acid, zinc, and proteolytic

Fluid enzymes

5% Bulbourethral ● thick alkaline mucus

fluid
PURPOSE OF SEMENALYSIS
COLOR
● Evaluation of reproductive dysfunction in males— infertility testing
● Select donors for therapeutic insemination Gray-white, “Pearly-white”, Light NORMAL
● Monitor the success of surgical procedures: Yellow; Opaque
○ Varicocelectomy
○ Vasectomy Shades of yellow Correlate with flavin concentration
PREPARATION
Deep yellow Associated with certain drugs, over-abstinence, jaundice,
● Abstinence: 2-7 days (Note: No prolonged abstinence)
urine contamination
○ prolonged abstinence → more volume but affects the motility of the sperm

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 VOLUME
Brown or red Presence of red blood cells
● Normal volume: 2-5 mL
Increased white turbidity Presence of white blood cells and infection within the ● It can be measured by pouring the specimen into a clean graduated cylinder
reproductive tract calibrated in 0.1-mL increments
● Note:
LIQUEFACTION
○ Measuring volume by aspirating the sample from the specimen container
● After ejaculation, semen is typically a semi-solid coagulated mass as protection from
into a pipette or syringe is not recommended.
the acidic vaginal pH.
● Liquefies within 30 to 60 minutes after collection (Normal) Low volume High volume
● Failure of liquefaction within 60 minutes
○ deficiency in prostatic enzymes and should be reported. ● obstruction of the ejaculatory ● active exudation in cases of active
● If after 2 hours the specimen has not liquified → artificially induce liquefaction by duct, congenital bilateral absence inflammation of the accessory
of vas deferens, collection organs
adding the following:
problems (loss of fraction of the ● extended abstinence
○ Dulbecco’s phosphate-buffered saline ejaculate), partial retrograde
○ Proteolytic enzymes such as alpha-chymotrypsin or bromelain ejaculation (reverse), or androgen
■ Note that these treatments may affect biochemical tests, sperm deficiency
motility, and sperm morphology, so their use must be VISCOSITY
documented.)
● Aspirate into a wide-bore plastic disposable pipette
PROCEDURE
● Normal viscosity:
○ formation of small discrete droplets that do not appear like a clump or
stringy when falling by gravity
● Abnormal viscosity:
○ drop from a thread more than 2 cm long (viscous)
● Ratings:
○ 0 (watery)- 4 (gel-like)
● Note:
○ High viscosity can interfere with determination of sperm motility, sperm
concentration, detection of antibody-coated spermatozoa and
measurement of biochemical markers.
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 -(Strasinger)
● pH paper with a range of 4.0 to 10.0 should be used
○ mix the semen well
○ spread a drop onto pH paper
○ read within 30 seconds
○ compare the color with the calibration strip to read pH
● Normal pH:
○ 7.2 – 7.8

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● Increased pH:
SLIDE 2
○ infection ● Sperm: 200
● Decreased pH: ● PM:105 → 52.5%
○ increased prostatic fluid, ejaculatory duct obstruction, poorly developed ● NP: 65 → 32.5%
seminal vesicle ● IM: 30 → 15%
MICROSCOPIC ○ Get the average (PMxNP/2) = 42.5%
SPERM MOTILITY INTERPRETATION:
● total motility (PR+NP) (ref: 40%) → 42.4% (normal)
● assessed asap after liquefaction (preferably at 30 minutes, WHO) or within 1 hour
● progressive motility (PR) (32%) → 55% and 53% (normal)
following ejaculation
● assessed by checking sperm cells in 20 HPFs or 200 sperms in 2 replicates (2 slides) SPERM MORPHOLOGY
● Note: ● Head, midpiece, tail
○ Assess only intact sperm cells. ● Abnormalities:
● The lower reference limit for total motility (PR+NP) is 40%. ○ Head-poor ovum penetration
● The lower reference limit for progressive motility (PR) is 32%. ○ Midpiece and Tail - affects motility
● Head
○ oval-shaped; approx. 5 um long and
3 um wide
● Tail
○ approx. 45 um long (with axoneme)
● Acrosomal cap
○ encompasses half of the head;
cover 2/3 of sperm nucleus
● Midpiece
○ 7 um long; mitochondrial sheath
● Motility
○ 1-4 mm/min; Life span: 1-2 days at
body temp
● Normal values (count at least 200 sperm cells)

Routine criteria >30% normal

Kruger’s Strict Criteria >14% normal; measures

head, neck and tail using a
micrometer
CALCULATION

SLIDE 1
● Sperm: 200
● PM: 110 → 55%
● NP: 60 → 30%
● IM: 30 → 15%
○ Get the average (PMxNP/2) = 42.5%

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Semen smearing methods for sperm morphology STAINED

Common abnormalities of sperm heads and tails

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 SPERM CONCENTRATION/SPERM COUNT ● The addition of stain (crystal violet) to diluting fluid helps visualization using a
● “Total sperm number” vs. “sperm concentration” bright-field microscope.
● Normal values: ● Note:
○ 20-250 million sperm per milliliter ○ Only FULLY DEVELOPED sperm should be counted (Immature sperms and
● Note: WBCs must not be included)
○ allow the sperm cells to settle for 3-5 minutes before counting
>1 million WBCs/mL inflammation or infection
Sperm concentration Total sperm number/Sperm count
>1 million spermatids/mL disruption of spermatogenesis caused by
● number of spermatozoa per unit ● total number of spermatozoa in the viral infections, exposure to toxic chemicals,
volume of semen entire ejaculate and genetic disorders
● is a function of the number of ● is obtained by multiplying the Inclusion criteria of counting cells
spermatozoa emitted and the sperm concentration by the semen
volume of fluid diluting them. volume
● Normal values:
○ >40 million/ejaculate
Improved Neubauer Counting Chamber
● 1:20 dilution
○ 1mL sperm and 19mL Diluting fluid
● Diluting fluids:
○ cold water, formalin, Na bicarbonate, 0.5% chlorazene, 1% formalin in 3%
trisodium citrate
● 5 RBC squares: 4 corner and center squares of the large center square (same to
manual RBC count) CALCULATION
COMPUTATION:
● #sperm cells counted x 1,000,000 (mL)
● 2 WBC squares: only 2 corner large squares (same to manual WBC count)

COMPUTATION:
● #sperm cells counted x 100,000 (mL)

● To get the average (60) of sperms → count both sides of the

hemocytometer then divide it by two
○ EX. Side 1: 70 Side 2: 50 → average 60 sperms
Note: the counts on both sides of the hemocytometer should agree within 10%; if
not, repeat dilution and counting

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 Sperm Concentration-Related Terms and Sperm Count

Aspermia no ejaculate at all (anejaculation or retrograde ejaculation)

Oligospermia less than 20 million per milliliter

Necrospermia immotile/dead sperms

Azoospermia complete absence of sperm

OTHER TESTS AND TECHNIQUES
SPERM VITALITY
● Assessed within 1 hour of ejaculation. ANTISPERM ANTIBODIES
● Evaluated by eosin-nigrosin stain, preparing a ● Antibodies against the sperm
smear, and counting the number of dead cells ○ Can be produced by the male and the female
in 100 sperm ● Detected in semen, cervical mucosa, or serum.
○ Stained → determine which sperm ● Blood-testes barrier separates sperm from the male immune system.
cells are alive or dead ○ Males → protected from anti-sperm antibodies
● Living cells remain bluish white whereas dead ○ But when disrupted in surgery, vasectomy, trauma, or infection— antigens
cells stain red against a purple background on sperm produce an immune response →, clumps of sperm are observed in
● Normal vitality requires 50% or more living routine semenalysis.
cells ○ Sperm-agglutinating antibodies cause to stick in a head-to-head,
● Presence of a large proportion of vital but head-to-tail, or tail-to-tail pattern.
immobile cells may indicate a defective flagellum ● Damaged sperm may cause the production of antibodies in female.
● High number of immotile and nonviable cells may indicate epididymal pathology. ● In females, this is suspected if normal semen analysis is accompanied by continued
SEMINAL FLUID FRUCTOSE infertility.
● Low fructose levels are caused by abnormalities of the seminal vesicles, bilateral ● Mixing the semen with female cervical mucosa or serum and observing for
congenital absence of the vas deferens, obstruction of the ejaculatory duct, partial agglutination
retrograde ejaculation, and androgen deficiency . Agglutination of spermatozoa
● Screen for the presence of fructose using the resorcinol test that produces an orange ● Motile spermatozoa sticking to each other, head-to-head, tail-to-tail or in a mixed
color when fructose is present (qualitative) way.
● A normal quantitative level of fructose is equal to or greater than 13 μmol per ● Motility is vigorous with frantic shaking motion or with limited motion.
ejaculate. ● Any motile spermatozoa stuck to each other by their heads, tails or midpieces should
○ Need to use Spectrophotometric methods (quantitative) be noted.
● **Specimens for fructose levels should be tested within 2 hours of collection or ● Major type of agglutination (grades 1-4); site of attachment (grades A-E)
frozen to prevent fructolysis.

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 ● Normal:
○ < 10% of motile sperm attached to particles
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.
● Head- directed Ab:
○ interfere penetration into cervical mucosa or ovum
● 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 <50% of the sperm

MICROBIAL TESTING
● >1 million WBCs/ mL indicates infection within reproductive system, prostate.
(prostatitis)
● Aerobic/anaerobic cultures and test for Chlamydia trachomatis, Mycoplasma
hominis, Ureaplasma urealyticum
SCREENING FOR ANTISPERM ANTIBODIES CHEMICAL TESTING
Mixed Agglutination Reaction (MAR test) ● Decreased level of a substance → pinpoint where the damage is found
● screening procedure to detect IgG antibodies ● Spectrophotometric methods- quantitate cirtric acid and zinc
● Semen + IgG (AHG) + latex particles or treated RBCc coated with IgG
Decreased neutral alpha-glucosidase, suggest a disorder of epididymis.
● Bivalent AHG binds to both antibody on the sperm and antibody on latex particles or glycerophosphocholine and L-carnitine
RBCs, forming microscopically visible clumps of sperm and particles or cells.

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 ● Specimens tested at monthly intervals, beginning at 2 months postvasectomy and
Decreased zinc, citric acid, glutamyl indicate lack of prostatic fluid.
transpeptidase and acid phosphatase continues until two consecutive monthly 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
● Performed in specialized andrology laboratories
○ Hamster egg penetration assay
○ Cervical mucus penetration test
○ Hypo-osmotic swelling test
○ In-vitro acrosome reaction

SPECIAL CASE
● In cases of alleged rape
○ microscopic examination of the specimen for the presence of sperm with
xylene
● Motile sperm- detected for up to 24 hours after intercourse
● Nonmotile sperm- 3 days
● 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]) - more specific detection
○ Further information, perform ABO blood grouping and DNA analysis)
Medico-Legal Test
● Microscopic Exam in suspected material – enhanced with xylene
● Florence Test
○ Reagents: Iodine Crystal + Potassium Iodide
○ Detect the presence of Choline (part of the semen) = (+) brown rhombic
crystals
○ Not specific
● Barberio’s Test
○ Reagents: Saturated Picric Acid + TCA
○ Detects the presence of Spermine = (+) yellow leaf shaped crystals
○ Specific
● Acid Phosphatase
● Glycoprotein p30 = more specific method
● Hyperimmune sera Test or Precipitin Hektoen Test
POST VASECTOMY ANALYSIS
● concerned only in presence or absence of spermatozoa

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 | Topic #2| [MLS 419-LAB] Analysis of Urine and Other Body Fluids

F2: Fecal Analysis, Pregnancy Test, and Automation

Professor: Christian Villahermosa, RMT, MSMT
Date: April 30, 2024

ANATOMY OF THE DIGESTIVE SYSTEM

the small intestine → breaks down
carbohydrates into simpler sugars
● Pancreatic juice (trypsin, chymotrypsin, elastase, carboxypeptidase)
○ Contains proteases (trypsin, chymotrypsin, elastase,
carboxypeptidase) → breaks down proteins into amino
acids
● Pancreatic lipase and bile salts (gallbladder)
○ Lipase breaks down TAG into fatty acids
■ In order for lipase to break down TAG, it needs
Bile salts
● Bile salts → emulsify the fats (easy to
be broken down)
---------------------------------------------------------------------------------------------
In the small intestine → where digestion and absorption will occur
○ After being broken down, fatty acids, glucose, and amino
acids → absorbed by the small intestine into the
circulation
PHYSIOLOGY

Mouth ● Salivary amylase breaks down starch (polysaccharide) into glucose NOTE!!
● When it reaches the large intestine → No carbohydrates, lipids, and proteins
(monosaccharide)
○ Glucose (monosaccharide) only carbohydrate that is
Large ● Role: absorbs approx. 3 L of water
accepted by the cell to convert into ATP
intestine ● Prone to problems:
● Lingual lipase → produced by the tongue but not as strong as the
○ If water absorption is inhibited, or if inadequate time is
lipase produced by the pancreas
allowed for the absorption process, → water will be
excreted → diarrhea results
Stomach ● Protein digestion by pepsin and hydrochloric acid
○ Stationary bowel contents (or decreased intestinal
motility) permit increased water absorption, resulting in
Small ● Pancreatic amylase
constipation (small and hard stool)
intestine ○ Pancreas produces amylase and is present in the small
intestine due to:
NOTE!!
■ If there is a carbohydrate in the small intestine
● 100-200 g of feces excreted per 24 hours
→ the amylase, lipase, and proteases will leave
the pancreas via the pancreatic duct → enter

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 ○ Contains bacteria, cellulose, undigested foodstuffs, GI secretions, bile ○ Since lactose is a carbohydrate → bacteria from the large intestine will
pigments, cells from intestinal walls, electrolytes, and water ferment it → GAS
DIARRHEA VS. STEATORRHEA ● Accompanies conditions characterized by maldigestion or malabsorption
○ Maldigestion: inability to convert food into readily absorbable substances
DIARRHEA STEATORRHEA
○ Malabsorption: normal digestive ability but inadequate intestinal absorption
● An increase in daily stool weight ● Presence of fat in feces of the already processed food
above 200g ○ Due to pancreatic defect
● Increased liquidity of stools → cannot produce lipase
● Frequency of more than 3 times
per day

INTESTINAL HYPERMOTILITY
● Fast peristalsis of the intestine → the large intestine could not reabsorb the water →
diarrhea

CLASSIFICATION OF DIARRHEA
SECRETORY DIARRHEA
● Increased solute in the intestine (electrolytes were not absorbed) → many electrolytes
are left in the large intestine → attracts water (from BV to the large intestine)

STEATORRHEA
● Fecal fat excretion that exceeds 6-7 g per day
● Specimens are characteristically pale, greasy, bulky, spongy, or pasty, and are
extremely foul smelling
○ May be caused by pancreatic insufficiency or malabsorption
OSMOTIC DIARRHEA
■ pancreatic insufficiency → does not produce lipase → fats are not
● There is a presence of osmotically active substance in the large intestine
broken down
○ Came from the food that we eat/inherent disease
■ Malabsorption → TAG is digested but fatty acids are not absorbed
NOTE!! → diarrhea
● In lipase deficiency: TAG are not broken down → Intact TAG will travel the small ● Diagnosed by fecal fat determination
intestine and reach the large intestine → In the large intestine, TAG is an osmotically ○ Screening: qualitative fecal fat
active substance (large) → the lumen of the large intestine becomes concentrated ○ Confirmatory: quantitative fecal fat
compared to the BV → water will go to the large intestine SPECIMEN COLLECTION
● In lactase deficiency: cannot break down lactose to glucose and galactose → Intact ● Needs patient education regarding the importance of testing and proper collection
lactose reaches the large intestine → lumen becomes concentrated → attracts water → of fecal specimens
water will go to the large intestine ● Any clean, non-breakable container that is sealable and leakproof is acceptable
● Type and amount of specimen collected vary

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 ○ occult blood, white blood cells, or qualitative fecal fat: small amount
■ Barium sulfate is white → patient would defecate white stool
○ quantitative tests for the daily fecal excretion of any substance: 2-3 day
collection COLOR
● Contaminants to avoid ● Normal brown color (stercobilin) of feces results from bile pigments
○ urine, toilet tissue, or toilet water ● Intestinal anaerobic bacteria reduce it to the three colorless tetrapyrroles
● Closed containers of fecal specimens should be covered with a disposable tissue or collectively called the urobilinogens: stercobilinogen, mesobilinogen, and
toweling and slowly opened urobilinogen
○ Prevents spattering in case of sudden release of fecal contents in gas ○ Oxidized into urobilins—stercobilin, mesobilin, and urobilin— which are
formation orange-brown and impart color to the feces
MACROSCOPIC EXAMINATION ● Acholic/pale/clay-colored stool: post-hepatic obstruction/barium sulfate
● Primary concern: presence of blood in stool
○ Must be chemically tested

BRIGHT RED BLACK

may be caused by lower GI tract may be caused by upper GI tract

bleeding bleeding (esophagus to duodenum)
CONSISTENCY AND FORM
● Ranges from loose and watery stools (diarrhea) to small, hard masses (constipation)
● Normal: formed
● Soft: increased fecal water content
○ May be normal, related to laxatives, or can accompany gastrointestinal
disorders
● May be bulky and with undigested substances
MICROSCOPIC EXAMINATION

Fecal ● Aids in the differential diagnosis of diarrhea

leukocytes ○ Increased WBC → secretory (due to bacteria)
● Suggest that the intestinal wall is infected or inflamed
● Normally, leukocytes are not present in feces; the presence of
even a small number (1-3 per hpf) indicates an invasive and
inflammatory condition
● Can be stained using methylene blue (wet preparation) or
NOTE!! Wright’s or Gram stain (dry smears)
● Scybalum → Medical term for hardened constipation
● Grey colored stool Undigested ● Aids in the diagnosis and monitoring of pancreatic insufficiency
○ obstruction at the post hepatic → bilirubin did not reach the small intestine muscle ○ Normally, we can digest muscle fibers → broken down
→ no formation of urobilinogen fibers by pancreatic juices which contain proteases: Trypsin,
○ Barium enema → RADTECH procedure that involves injecting barium sulfate chymotrypsin, etc.
into the large intestine → to make anomalies clear under the x-ray ● Stools are emulsified in 10% alcoholic eosin (for enhancement)

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 ● Count the red-stained fibers ○ Heating → allow the soaps and fatty acids to absorb the
○ Undigested: visible striations both vertical and stain
horizontal ○ Count and measure orange droplets (100 or more
○ Partially digested: striations in one direction droplets measuring 6-75 micrometers is steatorrhea)
○ Digested: no striations

● increased amount of neutral fat (on the first slide)

○ Picture = Undigested ○ suggests maldigestion
● > 10 undigested fibers: reported as increased/creatorrhea ● increased amount of total fat (on the second slide)
○ indicates intestinal malabsorption
Qualitative ● Simple two-slide qualitative procedure can be used to detect
fecal fat increased fat in feces CHEMICAL EXAMINATION
○ Goal: determine if there is presence of neutral fats or Fecal Blood ● Bleeding anywhere in the gastrointestinal (GI) tract from the
total fat mouth (bleeding gums) to the anus (hemorrhoids) can result in
■ Neutral fats: TAG detectable blood in the feces
■ Total fat: TAG + others (fatty acid salts, fatty ● Melena: excretion of dark or black stools resulting from the
acids, and cholesterol) presence of large amounts of fecal blood (50 to 100 mL/day)
● Stains used: Sudan III (most common), Sudan IV, Oil Red O ● Hematochezia: Fresh blood on a stool
● Neutral fats are readily stained by Sudan III while soaps and fatty ● Hemoptysis: Coughing up blood (Hallmark of TB)
acids are not ● Hematemesis: vomiting blood (GIT problems)
○ Seen as orange-red droplets
PROCESS OCCULT BLOOD
● Slide 1: neutral fats are detected when several drops of ethanol ● To determine if a stool with a normal color contains blood
(95%) are added to a suspension of feces on a microscope slide, ● Small amount of blood in feces not visually apparent
stain is added, a coverslip is applied, and the wet preparation is ○ > 2.5 mL of blood/150 g of stool is considered pathologically significant
observed ( > 60/hpf is steatorrhea) ● Methods in testing fecal occult blood
● Slide 2: total fecal fats are detected when a drop of acetic acid is ○ Guaiac-based FOBT
added to a fecal suspension, stain is added, a coverslip is applied, ○ Immunochemical FOBT
heated gently almost to boiling, and examined ○ Porphyrin-based FOBT

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 GUAIAC-BASED FOBT FETAL HEMOGLOBIN IN FECES/APT TEST
● Based on the pseudoperoxidase activity of the heme moiety of hemoglobin ● presence of blood in the stool, emesis, or gastric aspirate from a newborn infant
● In the presence of an indicator and hydrogen peroxide, the heme moiety catalyzes ○ may have come from the GI tract of the neonate or could be maternal blood
oxidation of the indicator, which results in a color change (blue) that was ingested during delivery
● The collection of 3 fecal samples is the standard of practice to maximize test APT-DOWNEY TEST
sensitivity
○ instructed to sample several portions of a single stool specimen or, ideally,
to collect fecal material from stool samples on 3 different days
○ Used as a screening test for colon cancer

QUANTITATIVE FECAL FAT

● Definitive test for steatorrhea (confirmatory)
● 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
FALSE POSITIVE FOBT FECAL CARBOHYDRATES
● Diet is very important in compromising FOBT ● Unhydrolyzed disaccharides (pancreatic insufficiency) are osmotically active
● Food that contain peroxidase resulting in an osmotic diarrhea
○ Ingested meat and fish ● Lactose intolerance in adults is common
○ Horseradish, turnips, bananas, black grapes, pears, and plums ○ results from intestinal bacteria actively fermenting lactose in the intestinal
IMMUNOCHEMICAL FOBT lumen
● Use polyclonal antihuman antibodies directed against the globin portion of undegraded ○ large amounts of intestinal gas and diarrheal stools with low pH (5.0-6.0)
human hemoglobin ● Rapid qualitative fecal pH can be obtained by testing the supernatant of a diarrheal
● Highly specific for human blood in feces and do not have interference from dietary stool using pH paper
foodstuffs or medications ○ Normal stool pH: Alkaline (7-8)
● May be automated and photometric or manual and visual ○ Acidic pH → Unhydrolyzed disaccharides
PORPHYRIN-BASED FOBT ● Clinitest tablet test
● Heme present in the hemoglobin → converted into porphyrin → counted by the ○ Stool from lactose intolerance: Positive
machine ■ Clinitest detects reducing sugars
○ Advantage: Quantitation ● Most diagnostic test: Specific histochemical examination of the intestinal epithelium
● based on the chemical conversion of heme to intensely fluorescent porphyrins ● More convenient test: Oral tolerance test of specific disaccharides
● detection and quantitation of the total amount of hemoglobin in feces ○ Normal: Lactose converted by lactase to glucose and galactose
● hemoglobin from nonhuman sources such as red meats can cause a false-positive ■ Glucose level is measured
result ○ Increased glucose after 2 hours → lactose tolerance
● more expensive, time-consuming, and labor-intensive compared with other FOBTs

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 CARBOHYDRATES
can sometimes turn cancerous
Xylose absorption test
○ There is fertilization, but no fetus is formed
● Xylose is readily absorbed in the small intestine and freely filtered in the glomerulus ■ Egg cell is not viable
● Detects if there is malabsorption of carbohydrate
● Procedure Choriocarcinoma ● Malignant; usually grows from molar pregnancies that
○ Ingestion of xylose grow cancerous
○ Collect blood sample after 2 hours
○ Normally, the blood level of xylose is highest about 2 hours after ingestion Germ cell tumors: Males
■ Since it was absorbed
○ Positive: If xylose is detected in high levels in the blood Seminoma ● most common testicular germ cell tumor; painless
HUMAN CHORIONIC GONADOTROPIN enlargement of the testes; MALIGNANT (but often
treatable and curable)
● Pregnancy test measures HCG
● Very radiosensitive; peak incidence at 35 years of age
● Produced by the trophoblast cells of the newly developing embryonic tissues to prevent
the involution of the corpus luteum (continuously produce progesterone) Embryonal carcinoma ● second most common testicular germ cell tumor;
○ Progesterone → to prepare for pregnancy and to insert negative feedback younger age incidence than seminoma; MALIGNANT
against follicle stimulating hormone (often metastatic at presentation)
■ Prevents the formation of corpus luteum to corpus albicans →
initiates FSH → to start another cycle Testicular ● highly chemosensitive
● May be measured shortly after the blastocyst implants on the endometrium choriocarcinoma
○ Peaks at 10-12 weeks of pregnancy
○ Tested in: Blood, urine (FIRST MORNING), amniotic fluid Germ cell tumors: Females
● Composed of 2 subunits:
○ alpha Dysgerminoma ● MALIGNANT; analogous to seminoma
■ Can be seen in LH and FSH
Ovarian ● MALIGNANT
○ beta choriocarcinoma
■ BETA= unique to HCG (structural/functional properties of the
AUTOMATION
hormone)
■ Detected by pregnancy tests ● Goal: Improved reproducibility and color discrimination while increasing
● Quanti vs Quali HCG productivity and standardization for reporting urinalysis results.
○ Quali (normal pregnancy test) ● Standardize:
○ Quanti (machine can measure the amount of HCG present) ○ sample processing
○ analyze test strips
May also be used as a tumor marker: ○ perform urine sediment analysis
○ report results
Gestational ● Disorders of pregnancy characterized by abnormal
● CLASSIFICATIONS
trophoblastic diseases trophoblast cells growing inside the uterus after
conception ○ Semi-automated chemistry analyzers
● All GTDs produce HCG ○ Fully-automated chemistry analyzers
○ Automated urine cell analyzers
Hydatidiform ● there is an overproduction of trophoblast tissue that ○ Automated urine system
mole/molar pregnancy grows into abnormal masses that are usually benign but

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 SEMI-AUTOMATED URINE CHEMISTRY ANALYZERS AUTOMATED MICROSCOPY
● Test for chemical components of urine ● Provide standardized results in less than 1 minute
● Read and interpret the reagent strip results ● Cost-effective and improves turnaround time
● Eliminates bias in reagent pad color analysis and reading discrepancies ● Sysmex UF-1000i and iQ 200 (Iris Diagnostics
● leukocyte, nitrite, protein, blood, glucose, ketone, bilirubin, urobilinogen, pH, specific AUTOMATED URINALYSIS SYSTEMS
gravity, color, creatinine, and protein-to creatinine ratio ● Combination of urine chemistry analyzers and automated urine cell analyzers
● Patient identification, specimen color, clarity — entered manually or barcode reader ● Improved TAT and hands-on time will be reduced
● Positive results are flagged— requires confirmation testing or microscopic ● complete walkaway capability with minimal specimen handling from sampling
evaluation through results
● Minimal daily maintenance— cleaning reagent strip platform and emptying reagent ● bar-coded samples are automatically identified and processed
strip waste container.

FULLY AUTOMATED URINE CHEMISTRY ANALYZERS

● High-volume urinalysis laboratory
● User “walk-away” capability
● Ability to load many samples with the capability to insert a STAT specimen during the
run.
● Press the button to begin testing and samples move automatically through the
instruments

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 |Topic #1| [MLS 419-LEC] Analysis of Urine and Other Body Fluids

F1: Amniotic Fluid

Professor: Christian Villahermosa, RMT, MSMT
Date: April 1, 2024

AMNIOTIC FLUID COLLECTION: AMNIOCENTESIS

● Not commonly performed fluid in the laboratory ● Typically collected after 14 weeks of gestation
○ Can be done through ultrasound and other
means
● The fluid that bathes the fetus throughout its
gestation
● Protects the fetus while enabling fetal movement
● Fills the amnion
AMNION

Transabdominal amniocentesis

● It encloses the developing fetus and the amniotic

fluid
● A membrane composed of a single layer of cuboidal
epithelial cells
● Formation starts upon embryonation
● Enlarges along with the growth of the fetus
FORMATION OF THE AMNIOTIC FLUID
● Changes throughout fetal gestation
● Preferred
EARLY ● produced by the amnion and the ● With ultrasonic examination
PREGNANCY placenta from maternal plasma
○ To know where the fetus is located
○ continues until the end of
● Aspirate 10-20 ml in several sterile syringes
pregnancy
● Discard the first 2-3 ml (contains blood and tissues)
LATER STAGES ● Fetus contributes to the amniotic fluid Vaginal amniocentesis
OF PREGNANCY ○ Water-solute exchange between ● may be done but high risk of infection and possible
the fetus and the surrounding contamination of vaginal cells
medium Transport/storage
● Maternal plasma + Products of fetal
metabolism: For cell culture/chromosomal preserve in body or RT
○ Fetal swallowing and fetal studies
urination of the AF
■ Formation of the kidney →
For fetal lung maturity tests place in ice for delivery and
AF would contain urea and
creatinine refrigerated
○ Capillary exchange in the
pulmonary system
■ Inhalation and exhalation VOLUME
of AF 12 weeks gestation 25-50 ml
● Maternal plasma exchange
○ Plasma is from the blood of the
37 weeks gestation 800-1200 ml
mother
Increased volume in 37 weeks gestation due to → maternal
plasma + products of fetal metabolism

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Polyhydramnios ● > 1200 ml TRANSPARENCY
● Congenital fetal malformations
● Decreased fetal swallowing not very turbid Early pregnancy
○ Normally: Swallow AF → it
will be part of the structure turbid Later stages of pregnancy
→ urinate the excess (Some
AF is still in the body of the FERN TEST
fetus) ● Used to evaluate premature
rupture of membranes
Oligohydramnios ● < 800 ml (leakage)
● Premature rupture of amniotic ● Performed by collecting
membrane vaginal fluid → dry the
→ AF goes out the mother or
smear → microscopy
leak to nearby tissues
● Presence of ‘fern-like’
Anhydramnios ● no amniotic fluid crystals on a dried vaginal
○ As long the mother has fluid
blood → presence of ○ positive screen for AF
amniotic fluid CHEMICAL EXAMINATION
PHYSICAL EXAMINATION ● Fetal lung maturity tests (now rarely performed)
● Tests for fetal distress
COLOR
○ Hemolytic disease of the newborn/amniotic
fluid bilirubin
colorless or pale yellow Normal
○ Neural tube defects
yellow or amber Presence of bilirubin ○ Genetic testing (e.g., trisomy 21, 18, 13;
● Urinated by the fetus family history)
(HDN/HDFN) ○ Fetal infections (TORCH complex)
■ T → toxoplasmosis
green Presence of meconium ■ O → others (syphilis, hepatitis B)
● first stool of the infant
■ R → Rubella (german measles)
● gelatinous material that
forms in fetal intestine as ■ C → Cytomegalovirus
a result of swallowed AF ■ H → Herpes simplex
and fetal intestinal FETAL LUNG MATURITY TESTS
secretions ● To assess if the fetus can survive outside of the
● Medical emergency → uterus in cases of anticipated premature delivery
infant could swallow its
Respiratory distress syndrome
meconium (choke/sepsis)
● A common cause of death in newborns due to
pale pink to red Blood contamination insufficient surfactant production at the alveolar
surfaces
dark red-brown Fetal death

DIFFERENTIATION FROM MATERNAL URINE

The uterus is close to the urinary bladder, and AF has the same
color as urine → need to differentiate AF from maternal urine

To differentiate, two tests are involved

1. Reagent strip:
a. AF normally contains glucose (+) and
protein (+)
SURFACTANTS
If the patient has diabetes or nephrotic syndrome, perform the
second test ● Structure: 90% phospholipids, 10% protein
2. Measure Urea and Creatinine ○ Surfactants in the lungs are packaged into
a. Urine has higher urea and creatinine storage granules called lamellar bodies
levels (waste product → filtered) (secreted by type 2 pneumocytes)
● 50-100 times greater than plasma ● In reverse, the type 2 pneumocytes→ produce
b. Amniotic fluid has urea and creatinine
Lamellar bodies with surfactants
levels equal to plasma
NOTE!! ● Lamellar bodies release surfactants into the alveolar
● Creatinine comparison is challenging at ≥ 37 air space
weeks of gestation ○ Surfactants keep the alveoli from collapsing
○ The kidney of the fetus is already by reducing the surface tension (due to the
functional → releasing its own creatinine presence of water) during breathing

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 ● Use TLC or agglutination slide test
○ Amniostat-FLM: contains anti-PG
antibodies
○ Reporting: negative, low positive, and high
positive
■ High positive → mature lungs
● presence of
phosphatidylglycerol
● The reagent contains
antibodies against
phosphatidylglycerol =
agglutination

NOTE!!
● The alveolus contains alveolar fluid that facilitates gas
exchange
○ With fluid = faster FOAM STABILITY INDEX
● Without surfactants → there is increased surface ● Also known as the ‘shake test’
tension in the alveolus → when you inhale/exhale → it ● ONLY A SCREENING TEST!
will fully close → Opening will take a longer time for ● Amniotic fluid + 95% ethanol, then shake for 15
you to respire fully seconds = production of foam and bubbles that remain
● In the presence of surfactants → decrease surface stable for 15 minutes after shaking
tension → will not fully close ● Presence of foam:
● Surfactants are tested to determine if the infant’s lungs ○ sufficient amount of phospholipids
are compliant ● Falsely mature results:
FETAL LUNG MATURITY TESTS ○ if the AF is contaminated with blood or
● Lecithin/sphingomyelin ratio: reference method meconium
● Phosphatidylglycerol NOTE!!
● Foam stability index ● A ‘mature’ result on a contaminated specimen has no
● Lamellar body count value
● Fluorescence polarization assay: obsolete test ○ Phospholipid may be from the
LECITHIN/SPHINGOMYELIN RATIO blood/meconium
● Lecithin (major) and sphingomyelin: pulmonary ● An ‘immature’ result on a contaminated specimen is
surfactants clinically useful
● 34-36 weeks: ○ No phospholipid even if it is contaminated →
○ lecithin increases problem in the infant
○ sphingomyelin remains constant or
decreases (produced at about 26 weeks) LAMELLAR BODY COUNT
● Measured using TLC and there should be no blood or ● Storage of pulmonary surfactants
meconium contamination (false increase/positive) ● The number of lamellar bodies in AF correlates with
the number of phospholipids present in the lungs
< 2.0 immature lungs ● Counted via the platelet channels of automated
● Lecithin = sphingomyelin (1) hematology analyzers
● Advantages:
> or equal to 2.0 fetal lung maturity ○ low volume needed (0.5 ml), short TAT, low
● Lecithin > sphingomyelin cost
(normal) ● Fetal lung maturity:
FORMULA ○ > 50,000/microliter in an uncentrifuged
sample
𝐿𝐸𝐶𝐼𝑇𝐻𝐼𝑁
AMNIOTIC FLUID BILIRUBIN
𝑆𝑃𝐻𝐼𝑁𝐺𝑂𝑀𝑌𝐸𝐿𝐼𝑁
● Oldest routinely performed test on AF (for fetal
PHOSPHATIDYLGLYCEROL anemia due to HDFN)
● A phospholipid (a major component of surfactants) ● Measured using spectrophotometry
that enhances the spread of surfactants across the ● The optical density of the fluid is measured in
alveolar surface intervals between 350 nm and 580 nm
● Not detectable until 35 weeks gestation

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 ○ (Red line) Highest OD at 365 nm and
decreases linearly to 550 nm: normal
○ (blue line) If bilirubin is present, a rise in OD
is seen at 450 nm (wavelength of maximum
bilirubin absorption)

○ The difference between the OD of the

baseline and the OD at 450 nm is the AF
bilirubin concentration (also known as
△A450)
■ Normal: 0.2 absorbance
■ Peak: 0.5
● Difference = 0.3 → death
risk

NEURAL TUBE DEFECT

● Diseases due to the incomplete development of the
skin that covers the spine and brain
● Detected by measuring maternal serum
alpha-fetoprotein (AFP)
○ If increased in both maternal circulation
and amniotic fluid, it is indicative of NTDs
like anencephaly and spina bifida
(conditions when the skin fails to close over
the neural tissue)
● Reported in terms of multiples of the median (MoM)
○ Median is the laboratory’s reference level
for a given week of gestation
○ A fetus produces maximal AFP between
12-15 weeks and then its levels decline
○ A value greater than or equal to 2x the
median value is considered abnormal
■ Example: If the median is 40 and
the patient result is 120 →
abnormal
■ 80 below is normal
● Elevated results are followed by measurement of
amniotic acetylcholinesterase (neurotransmitter)
○ If the infant’s spine is exposed → release of
acetylcholinesterase

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 | Topic #2 | [MLS 419-LEC] Analysis of Urine and Other Body Fluids

F2: SEROUS FLUID

Professor: Christian Villahermosa, RMT, MSMT
Date: May 7, 2023

SEROUS FLUIDS 2. Parietal membrane

● Parietal pleura and parietal membrane of the pericardium →
PLEURAL FLUID From the peritoneal cavity that surrounds the lungs
closer to the chest wall compared to the heart and the lungs
PERICARDIAL FLUID From the pericardial cavity that surrounds the heart ● Parietal membrane of the peritoneum → closer to the abdominal
wall as opposed to its location to the abdominal organs
PERITONEAL FLUID From the peritoneal cavity that surrounds the abdominal ● Between the two membranes lies a cavity, wherein the serous fluids flow
organs FORMATION OF THE SEROUS FLUIDS
CAVITY LOCATIONS ● Nearby the cavities/spaces are capillaries which contain plasma and would leak out
from the blood vessel and into the space → forms a fluid
● 4 factors involved in formation
1. Permeability of capillaries in the parietal membrane (promotes formation of
serous fluids)
● Normally, leakage is in minute/small amounts
● In the event of pathologic conditions → increased permeability of
the capillaries (destroyed) → increased fluid in the cavity/space
2. Hydrostatic pressure in capillaries (promotes formation of serous fluids)
● Once water passes through the capillaries, some fluids leak out
into the cavity/space
3. Oncotic pressure in capillaries (control/maintain level of serous fluids)
● Pressure exerted by plasma proteins (attract water)
○ Sufficient number/conc. of plasma proteins → can take
away water from the space back into the capillaries
4. Absorption of fluid by the lymphatic system (control/maintain level of serous
fluids)
● Excess fluids are drained in the lymphatic system for excretion
● If the entry of fluid is uncontrolled → Increased fluid leakage and congest the vital
organs → Life threatening
● The organ surfaces have 2 types of membranes:
COLLECTION
1. Visceral membrane
● Paracentesis: percutaneous puncture of a body cavity for fluid aspiration
● directly adjacent to the vital organ
○ Thoracentesis (paracentesis of the pleural fluid)
○ lungs → Visceral pleura
○ Pericardiocentesis (paracentesis of the pericardial fluid)
○ heart → Visceral membrane of the pericardium
○ Peritoneocentesis (paracentesis of the peritoneal fluid)
○ Abdominal organs → Visceral membrane of the
peritoneum

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● For microscopic and microbiologic examination: use tubes with sodium heparin or
liquid EDTA
○ Some of the fluids have clots → The presence of clots → not examined
properly in the cell count
● For chemistry: red top
● For cytology: refrigerate
EFFUSION
● Accumulation of serous fluids, which suggests a pathologic process
○ Ascites: effusion in the peritoneal cavity (enlargement of the abdomen)
● Must be differentiated if exudate or transudate
● Common causes:
○ Increased capillary permeability and hydrostatic pressure
○ Decreased oncotic pressure and absorption in the lymphatics
TRANSUDATE VS. EXUDATE
● NOTE: Pericardial fluid effusion is not classified as transudate or exudate

TRANSUDATE EXUDATE

● Systemic diseases (close to the organ) ● Inflammatory conditions (e.g.

that increases hydrostatic pressure and pathogens, tumors, obstruction)
decreases oncotic pressure → that increase capillary permeability
accumulation of fluid and decrease absorption in the
● Cause: Non-inflammatory disease lymphatic system
processes
○ Congestive heart failure
■ Vasoconstriction →
narrow BV→ Increase
hydrostatic pressure →
accumulation of fluid
○ Liver cirrhosis
■ Liver does not produce
sufficient proteins →
low plasma proteins →
low oncotic pressure →
accumulation of fluid
○ Nephrotic syndrome
■ Too much excretion of PHYSICAL EXAMINATION
proteins → low protein
in circulation → low PALE YELLOW Normal (serous is an ultrafiltrate of the plasma)
oncotic pressure →
accumulation of fluid CLOUDY SEROUS FLUIDS Presence of WBCs, cells, lipids, or chyle

MILKY Differentiate chylous from pseudochylous effusions

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 BLOODY Differentiate traumatic tap and hemorrhagic effusions PERICARDIAL FLUID WBC count >1000 cells/ul → indicates pericarditis
(hemothorax)
PLEURAL FLUID RBC count > 10,000 cells/ul → indicates neoplasm or trauma
BROWN Rupture of amoebic liver abscess
PERITONEAL FLUID WBC count > 500 cells/ul with high neutrophils → indicates
BLACK Aspergillus infection (A. niger) bacterial peritonitis

VISCOUS Malignant mesothelioma (cancer of the mesothelial cells that ● Differential count: sedimentation via cytocentrifuged smear
cover most internal organs; may be pleural or peritoneal) ○ Count all nucleated cells both HPO and LPO
○ LE cells (neutrophils with phagocytized homogeneous nucleus) may be seen
CHYLOUS VS. PSEUDOCHYLOUS EFFUSIONS
○ Mesothelial cells
● Chylous
○ Malignant cells
○ Presence of chylomicrons → indicate chylous effusion
● Cytologic examination: 10-200mL of fluid when malignancy is suspected
■ Presence of chylomicrons in serous fluid is due to the obstruction
● Note: Differential count is not routinely performed in pericardial fluid as different
in the thoracic duct
conditions produce the same differential count
● When serous fluids are drained → they pass through the
DIAGNOSTIC PERITONEAL LAVAGE
thoracic duct → presence of obstruction → increased
● Introduction of normal saline into the peritoneal cavity to detect abdominal injuries
conc. of chylomicrons → Milky serous fluids
that have not yet resulted in fluid accumulation
● Pseudochylous
● Commonly used in patients with blunt abdominal trauma or any abdominal
○ Absence of chylomicrons but fats are present (cholesterol)
catastrophe
● RBC counts >100,000/ml are indicative of blunt trauma injuries

MICROSCOPIC EXAMINATION
● Total cell count
○ Use NSS as diluent

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 ● Lipids:
○ Aids in differentiating chylous and pseudochylous effusions
○ Exudate: > 0.3 serous fluid to serum cholesterol ratio
● pH
○ Applied determining in the resolution of Parapneumonic effusions (exudate
from pneumonia or lung abscess)
■ Collection: same with ABG (anaerobic in heparinized syringe,
transport with ice)
■ If >7.3 resolved
● Adenosine deaminase:
○ Increased in tuberculosis and malignancy
MICROBIOLOGIC EXAMINATION
● Gram stain
● Acid-fast stain
● Culture
○ Larger volume, greater chance of isolating an organism
○ Perform both aerobic and anaerobic culture

CHEMICAL EXAMINATION

TOTAL PROTEIN AND LDH RATIOS

TP RATIO: TP OF SEROUS FLUID/TP Transudate: < or equal to 0.5

OF SERUM
Exudate: >0.5

LDH RATIO: LDH OF SEROUS Transudate: < or equal to 0.6

FLUID/ LDH OF SERUM
Exudate: >0.6
● Glucose
○ Low glucose level is clinically significant
○ Exudate: <60 mg/dl or if the difference between serum and serous fluid
glucose is > 30 mg/dl
● Amylase:
○ abnormal if the value exceeds the ULN or if 1.5 or twice the serum value
● Carcinoembryonic antigen (CEA) and CA 125:
○ Malignant effusions of gastrointestinal (CEA) and ovarian (CA 125) origin

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