ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

4. Sediment Examination (microscopic; serve as

- Urinalysis is the testing of urine with procedures the gold standard)
commonly performed in an expeditious, reliable,
accurate, safe, and cost-effective manner (CLSI).
- Manual procedure: 30 minutes
- Automated procedure: 6 minutes

HISTORY OF URINALYSIS
- Analyzing urine was actually the beginning of
laboratory medicine.
- References to the study of urine can be found in
the drawings of cavemen and in Egyptian
hieroglyphics, such as the Edwin Smith Surgical
Papyrus.
- They were able to obtain diagnostic information
from such basic observations as color, turbidity,
odor, volume, viscosity, and even sweetness - Converts approximately 170,000 mL of filtered
plasma to 1,200 mL to 1,500 mL of average daily
History urine output
• 5th BC: Hippocrates wrote a book on “Uroscopy”
• Middle Ages: Physicians were trained on the
“Art of Uroscopy”
• 1140 AD: Color charts were developed to
describe significance of 20 different colors
• 1627: Thomas Bryant published a book on
charlatans called “pisse prophets”
o Pisse prophets – prediction about health
without proper medical educational
background (quack doctors)
- 95% water + 5% solutes
o 1st medical licensure laws in England
- Variation in solute concentration may be due to:
• 1694: Frederik Dekker discovered albuminuria
o Dietary intake (ex. high sodium and
(white precipitates) by boiling urine.
glucose level)
• 17th century: invention of the microscope by o Physical activity (ex. high physical
Anton Van Leeuwenhoek; Thomas Addis activity → high metabolic processes)
developed quantifying the microscopic sediment o Body’s metabolism (results to high waste
(Addis count – quantitation of formed elements products)
in the urine) o Endocrine functions (ex. problem in
• 1827: Richard Bright introduced urinalysis as antidiuretic hormone)
part of a doctor’s routine patient examination o Body’s position (prolonged standing can
cause increase protein, orthostatic or
IMPORTANCE OF URINALYSIS postural proteinuria)
- Unique Characteristics of Urine:
o Readily available & easily collected SOLUTES
o Contains information which can be ➢ Organic Solutes
obtained by inexpensive lab tests about o Urea – major metabolic waste product
the body’s major metabolic functions ▪ Produced by the liver due to
breakdown of protein and
REASONS FOR PERFORMING URINALYSIS amino acids
- Aiding in diagnosis of disease ▪ The amount of urea accounts
- Screening asymptomatic population for nearly half of the total dissolved
undetected disorders (especially for metabolic solids in urine
diseases) ▪ Highest
- Monitoring the progress of disease & o Creatinine: product of muscular
effectiveness of therapy metabolism
o Uric acid: product of purine metabolism
4 PARTS OF ROUTINE URINALYSIS ➢ Inorganic Solutes: chloride (major inorganic solid
dissolved in urine), sodium, and potassium
1. Specimen Evaluation (accept or reject the
➢ Other substances present: hormones, vitamins,
specimen)
and medications
2. Physical Examination (check for the physical
➢ Formed elements present: Increased amounts
characteristic of the urine)
of formed elements, such as cells, casts, crystals,
3. Chemical Examination (detect the presence of
mucus, and bacteria, are often indicative of
different analytes and serves as a confirmatory
disease
for the physical examination)
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

COMPOSITION OF URINE SPECIMEN PRESERVATION

COLLECTED FOR 24 HOURS - Most routinely used method of preservation:
Organic
Amount Remark
o Refrigeration (2⁰C to 8⁰C)
Component o Decreases bacterial growth and
60%–90% of nitrogenous material;
25.0 to metabolism
Urea derived from metabolism of amino
35.0 g
acids into ammonia
Derived from creatine, nitrogenous
Creatinine 1.5 g
substance in muscle tissue
Common component of kidney stones;
0.4 to
Uric acid derived from catabolism of nucleic
1.0 g
acid in food and cell destruction
Benzoic acid is eliminated from the
Hippuric
0.7 g body in this form; increases with
acid
highvegetable diets
Carbohydrates, pigments, fatty acids,
Other
2.9 g mucin, enzymes, hormones;
substances
depending on diet and health

Inorganic
Amount Remark
Component
Prevents enzyme enolase
Sodium
15.0 g Principal salt; varies with intake
chloride (NaCl)
Occurs as chloride, sulfate, and
Potassium (K) 3.3 g
phosphate salts
Sulfate (SO4 2) 2.5 g Derived from amino acids
Occurs primarily as sodium
Phosphate
2.5 g compounds that serve as buffers
(PO4 3)
in the blood
Derived from protein metabolism
Ammonium and glutamine in kidneys; amount
0.7 g
(NH4) varies depending on blood and
tissue fluid acidity
Magnesium Occurs as chloride, sulfate,
0.1 g
(Mg2) phosphate salts
Composed of 50% ethanol
Occurs as chloride, sulfate, and 2% carbowax
Calcium (Ca2) 0.3 g
phosphate salts
Commercial Urine Transport Tubes
with Preservatives
- urine specimen is so readily available and easily
collected often leads to laxity in the treatment of
the specimen after its collection.
- Changes in urine composition take place not only
in vivo but also in vitro
o requiring correct handling procedures

SPECIMEN INTEGRITY
- specimens should tested within 2 hours
- A specimen that cannot be delivered and tested
within 2 hours should be refrigerated or have an
appropriate chemical preservative added.
- Notice that most of the changes are related to
the presence and growth of bacteria
SPECIMEN INTEGRITY
• Urine (biohazardous substance) Standard
Precautions
• Clean, dry, leak-proof, disposable containers
• Sterile containers for culture and sensitivity
• Properly applied screw-top lids
• Wide mouth (4 to 5 cm), flat bottom
• Clear plastic (50 mL to 100 mL capacity)
o 12 mL microscopic
o repeat analysis
o there should be room for swirling

SPECIMEN LABEL
• Patient’s name
• Identification number
• Date and time of collection
• Patient’s age and location
• Physician’s name
o Others (as required by institutional
protocol)
o Attached to the container, not to the lid
o Should not become detached if the
container is refrigerated or frozen
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
REQUISITION FORM
• Requisition form (manual or computerized)
• Match the information on the specimen label
• Additional information on the form:
o Method of collection
o Type of specimen
o Interfering medications
o Patient’s clinical information
o Time specimen is received in the
laboratory
SPECIMEN REJECTION
• Unlabeled containers
• Nonmatching labels and requisition forms
• Contaminated with feces or toilet paper
• Containers with contaminated exteriors
• Specimens of insufficient quantity (12 or 10-15
mL is enough to do the testing)
URINE CONFIRMATION
➢ Specific Gravity: 1.003-1.035
o Increased if the patient undergoes MRI
o Decreased in cases of adulteration
▪ 1.002 is still accepted if the
patient has diabetes insipidus
➢ pH: 4.0-8.0
➢ Temperature: 32.5°C to 37.5°C
➢ Creatinine: Approximately 50x that of plasma
RANDOM URINE
- most commonly received specimen because of
its ease of collection and convenience.
- may be collected at any time
- useful for routine screening tests to detect
obvious abnormalities
- may also show erroneous results resulting from
dietary intake or physical activity
Time Urine Collection Protocol
FIRST MORNING URINE
- ideal screening specimen
- preventing false-negative pregnancy tests and
for evaluating orthostatic proteinuria
- concentrated specimen (detection of chemicals
and formed elements that may not be present in
a dilute random specimen)
FASTING SPECIMEN (SECOND MORNING)
- A fasting specimen differs from a first morning
specimen by being the second voided specimen
after a period of fasting.
- This specimen will not contain any metabolites
from food ingested before the beginning of the
fasting period.
- It is recommended for glucose monitoring
TWO-HOUR POSTPRANDIAL SPECIMEN
- specimen is tested for glucose, and the results
are used primarily for monitoring insulin therapy
in persons with diabetes mellitus.
- A more comprehensive evaluation of the
patient’s status can be obtained if the results of
the 2-hour postprandial specimen are compared
with those of a fasting specimen and
corresponding blood glucose tests.
GLUCOSE TOLERANCE SPECIMENS
- The urine is tested for glucose and ketones, and
the results are reported along with the blood test
results as an aid to interpreting the patient’s
ability to metabolize a measured amount of
glucose and are correlated with the renal
threshold for glucose.
- Renal threshold for glucose: 160-180 mg/dL
- Collection of these specimens is an institutional
option.
- Usually performed for pregnant women
- The number of specimen varies with the length
of test for GTT includes:
o 1-hour postprandial
o 2-hour postprandial
o 3-hour postprandial
TIMED SPECIMEN
- Example of timed specimen includes:
o 24-hour: to quantitate amount of solute
in the urine (creatinine, urea, uric acid)
o 12-hour: usually used for addis count
(quantitation of formed elements in the
urine)
o 4-hour: for nitrite determination to
detect whether the bacteria found in the
urine can convert nitrate to nitrite
▪ Usually used for patients with
cystitis or infection (UTI)
o afternoon urine: used for urobilinogen
(2pm to 4pm)
- Measuring the exact amount of a urine. A
carefully timed specimen must be used to
produce accurate quantitative results
- Required when the concentration of the
substance to be measured changes with diurnal
variations and with daily activities such as
exercise, meals, and body metabolism.
• Provide patient with written instructions and
collection container (with preservative, if
required)
• At start time (e.g., 7 am on day 1), patient
empties bladder into toilet; afterward, all
subsequent urine throughout timed interval is
collected in the container provided
• At end time (e.g., 7 am on day 2), patient empties
bladder into collection container.
• Specimen is transported to laboratory, where
urine is mixed well and the volume is measured
and recorded.
• A sufficient volume (approximately 50 mL) is
removed for routine testing and possible repeat
or additional testing; the remainder is discarded.
CATHETERIZED SPECIMEN
- The most commonly requested test on a
catheterized specimen is a bacterial culture.
- Collected under sterile conditions by passing a
hollow tube (catheter) through the urethra into
the bladder.
- Invasive procedure because the urine will flow
directly from the bladder through the use of
catheter
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
- If a routine urinalysis is also requested, the
culture should be performed first to prevent
contamination of the specimen.
- A less frequently encountered type of
catheterized specimen measures functions in the
individual kidneys. Specimens from the right and
left kidneys are collected separately by passing
catheters through the ureters of the respective
kidneys.
2 Types of Catheterized Specimen
➢ Urethral: collect urine specimen up to the
urinary bladder
o Used to check if the patient has cystitis,
UTI, or urethral infection
o Kidney infection is uncertain
➢ Ureteral: collect urine specimen up to the ureter
o Used to check if the patient has
pyelonephritis (kidney infection)
o To locate whether the infection is on the
left, right, or both kidneys
MIDSTREAM CLEAN-CATCH SPECIMEN
- safer, less traumatic method for obtaining urine
for bacterial culture and routine urinalysis.
- is less contaminated by epithelial cells and
bacteria and, therefore, is more representative
of the actual urine than the routinely voided
specimen. Patients must be provided with
appropriate cleansing materials, a sterile
container
- Strong bacterial agents, such as
hexachlorophene or povidone-iodine, should not
be used as cleansing agents.
- Mild antiseptic towelettes are recommended
(Castile Soap Towelettes).
SUPRAPUBIC ASPIRATION
- Most invasive among the collection
- Introduction of a needle through the abdomen
into the bladder.
- Provides a sample for bacterial culture that is
completely free of extraneous contamination.
- Can also be used for cytologic examination
PROSTATITIS SPECIMEN
Three-Glass Collection
- Quantitative cultures are performed on all
specimens, and the first and third specimens are
examined microscopically.
- In prostatic infection, the third specimen will
have a white blood cell/high-power field count
and a bacterial count 10 times that of the first
specimen. Macrophages containing lipids may
also be present.
- It uses three glasses: (three urine sample is used)
o 1st glass: pre-massage
▪ Ex. first morning urine or
random urine
o 2nd glass: midstream clean-catch
▪ Serve as control
▪ Increased bacteria or WBC
indicates UTI
▪ Cannot rule out prostatitis
infection if positive (do not
proceed to 3rd specimen)
o 3 glass: post-massage
rd
▪ There should be presence of
bacteria and WBC (10x higher
compared to the first specimen)
Stamey-Mears four-glass localization method
- To locate the infection
- Prostatitis: highest in EPS → VB3 → VB1 or VB2
- UTI: highest in VB2 → VB1 → VB3 → EPS
- It uses four glasses:
o Initial voided urine (VB1): for urethral
infection
o midstream urine (VB2): to detect
whether the patient has urinary bladder
infection (cystitis)
o expressed prostatic secretions (EPS):
can be cultured and examine for white
blood cells
o Post-prostatic massage urine specimen
(VB3): pure prostatic secretion (urine
sample has prostatic secretion)
Pre- and post-massage test (PPMT)
- A positive result is significant bacteriuria in the
post-massage specimen of greater than 10 times
the pre-massage count
- No need to do the midstream clean-catch
DRUG SPECIMEN COLLECTION
- most vulnerable part of a drug-testing program.
- The chain of custody (COC) is the process that
provides this documentation of proper sample
identification from the time of collection to the
receipt of laboratory results.
- Individuals who undergo drug testing are called
clients
- Should be collected within the facility
o Waterless system collection room
o Has bluing agent to toilet water
reservoir to avoid tampering of sample
- that no tampering of the specimen occurred,
such as substitution, adulteration, or dilution
- collection may be witnessed or unwitnessed
- Volume: 30-45 mL
- Temperature: 32.5 to 37.5°C (near body
temperature)
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- 2 types of nephron:
o Cortical Nephrons: 85%, found on the
cortex of the kidneys
▪ function: removal of waste
products and reabsorption of
nutrients
o Juxtamedullary nephrons: have longer
Loop of Henle that extend deep into the
medulla of the kidney.
▪ function: concentration of the
urine or for the maintenance of
osmotic gradient

- Major organ of the excretory system

- Bean-shaped organ
- Order of urine formation:
o Glomerulus (filters blood)
o Renal tubules
▪ Proximal Convoluted Tubule
▪ Loop of Henle
▪ Distal Convoluted Tubule
o Collecting Duct (collects urine from the
nephrons)
o Calyx (has 12 minor calyces and 2-3
major calyces)
o Renal Pelvis (collects all the urine
formed during the filtration process)
o Ureter (approx. 25cm long that connects
kidney and urinary bladder)
o Urinary bladder (urine is being stored)
o Urethra (empties urine from the RENAL BLOOD FLOW
bladder)
- the one that receives blood from the heart is the
renal artery
- the kidney approximately receives 25% of blood
from the heart
- Blood enters the capillaries of the nephron
through the afferent arteriole. It then flows
through the glomerulus and into the efferent
arteriole.
o Varying sizes of arterioles help to create
hydrostatic pressure differential
important for glomerular filtration and
to maintain consistency and of
Nephrons glomerular capillary pressure and renal
- Functional unit of the kidney blood flow within the glomerulus
- It is responsible for the urine formation o Smaller size of efferent arteriole
- Each kidney contains approximately 1 to 1.5 increases the glomerular capillary
million pressure.
- Kidney has 2 distinct areas: - Before returning to the renal vein, blood from
o Renal cortex: lighter and outer portion the efferent arteriole enters the peritubular
o Renal medulla: darker and inner portion capillaries (where the reabsorption of important
- 5 distinct areas of nephron: substances that was filtered by the glomerulus)
o Blood vessels found on the Bowman’s and the vasa recta (salt and water reabsorption)
capsule that surrounds the nephron and flows slowly through the cortex and medulla
▪ Not part of the glomerulus, but of the kidney close to the tubules.
it is part of the renal tubule - The blood, together with the important
o Glomerulus is blood vessels or substances, will now go back to the circulation
capillaries that filters blood coming out from the kidney through the renal
o Peritubular capillaries are blood vessels vein
that surrounds PCT and DCT
o Blood vessel that located adjacently to
the Loop of Henle is called vasa recta
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- total renal blood flow: approx. 1,200 mL/min

- total renal plasma flow: 600 to 700 mL/min
- normal values of renal blood and plasma flow
depends on the body size (1.73m2)

GLOMERULAR FILTRATION
B. Hydrostatic and Oncotic pressure
- The glomerulus consists of a coil of
- Hydrostatic pressure is a force that pushes the
approximately eight capillary lobes, the walls of
fluid out of the blood capillaries
which are referred to as the glomerular filtration
- Oncotic pressure resist the hydrostatic pressure
barrier. It is located within Bowman’s capsule,
o Force that pushes the fluid into the
which forms the beginning of the renal tubule.
blood capillaries
o The purpose of bowman’s capsule is to
- An autoregulatory mechanism within the
catch the filtrate filtered by the
juxtaglomerular apparatus
glomerulus
o Juxtaglomerular cells (JG cells) – found
o The glomerulus acts as a sieve while the
on the afferent arteriole
bowman’s capsule acts as a basin
▪ Produces hormone renin when
- Substances that are non-filterable will go back to
blood pressure is low
the circulation by coming out from the efferent
▪ Constrict (↑BP) or dilate (↓BP)
arteriole
the afferent arteriole
- Non-selective filter of plasma substances with
o Macula Densa – found on the distal
MW of less than 70,000 Daltons
convoluted tubule
- fluid as it leaves the glomerulus shows the
▪ Sense the changes in the blood
filtrate to have a specific gravity of 1.010 and
pressure, especially when
confirms that it is chemically an ultrafiltrate of
decreased
plasma.
▪ It will send signal to JG cells to
produce hormone
Factors that influence the actual filtration process
A. Cellular structure of the capillary walls and
Bowman’s capsule
- Plasma filtrate must pass through 3 cellular
layers
1. Capillary wall membrane (endothelial
cells)
2. Basement membrane (basal lamina)
3. Visceral epithelium of the Bowman’s
capsule
- Endothelial cells of the capillary wall differ from
the others because they have pores
(fenestrated)
- Pores increases the cellular permeability but do
not allow large substances and blood cells C. Renin-Angiotensin-Aldosterone System (RAAS)
- Further restriction of large molecules occurs as - It is a hormone system within the body that is
the filtrate passes through the basement essential for the regulation of blood pressure
membrane and the thin membranes covering and fluid balance
the filtration slits formed by the intertwining foot - Mainly comprised of three hormones: Renin,
processes of the podocytes of the inner layer of Angiotensin, and Aldosterone
Bowman’s capsule.
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
- RAAS will be activated if the body has decreased
blood pressure, decreased sodium
concentration, or decreased water inside the
body
o The activation of RAAS should be
inhibited when blood pressure is high by
taking Losartan (ACE inhibitor)
- controls the regulation of the flow of blood to
and within the glomerulus.
- responds to changes in blood pressure and
plasma sodium content that are monitored by
the juxtaglomerular apparatus (juxtaglomerular
cells in the afferent arteriole and the macula
densa of the distal convoluted tubule)
- Low plasma sodium content decreases water
retention within the circulatory system
RAAS Cascade of Reaction
1. Renin produces the inert hormone angiotensin I
2. Angiotensin I passes through the lungs,
Angiotensin-converting enzyme (ACE) changes
it to the active form angiotensin II.
3. Angiotensin II corrects renal blood flow in the
following ways:
• Dilates the afferent arteriole and
constricts the efferent arteriole
• Stimulates sodium reabsorption in the
proximal and distal convoluted tubule
• Triggers the adrenal cortex to release
aldosterone (help in the final
reabsorption of sodium in the DCT)
• Triggers the hypothalamus to produce
antidiuretic hormone (Water will be
reabsorbed in the collecting ducts)
TUBULAR REABSORPTION
- The molecules that were filtered by the
glomerulus will come back to the circulation and
will be reabsorbed in the proximal convoluted
tubule.
Active transport
- It requires energy to transport important
substances from the renal tubule going to the
peritubular capillaries
o Vasa recta also has the capability to
reabsorb substances
- The substance to be reabsorbed must combine
with a carrier protein contained in the
membranes of the renal tubular epithelial cells.
- Active transport is responsible for the
reabsorption of glucose, amino acids, and salts in
the proximal convoluted tubule, chloride in the
ascending loop of Henle, and sodium in the distal
convoluted tubule.
Tubular Reabsorption
Substance Location
Active Glucose, amino Proximal convoluted tubule
transport acids, salts
Chloride Ascending loop of Henle
Sodium Proximal and distal convoluted
tubules
Passive Water Proximal convoluted tubule
transport Descending loop of Henle
Collecting duct
Urea Proximal convoluted tubule
Ascending loop of Henle
Sodium Ascending loop of Henle
Passive Transport
- Higher concentration → lower concentration
- The movement of molecules across a membrane
as a result of differences in their concentration
or electrical potential on opposite sides of the
membrane physical differences are called
gradients.
- The plasma concentration at which active
transport stops is termed the renal threshold
(maximal reabsorptive capacity)
o For glucose, the plasma renal threshold
is 160-180 mg/dL, and glucose appears
in the urine when the plasma
concentration reaches this level.
o Excess are secreted in the urine
- Knowledge of the renal threshold and the plasma
concentration can be used to distinguish
between excess solute filtration and renal
tubular damage.
- Renal concentration begins in the descending
and ascending loops of Henle, where the filtrate
is exposed to the high osmotic gradient of the
renal medulla
o Loop of Henle is important for sodium
and water reabsorption
o Descending loop of Henle: where the
water is reabsorbed so the osmotic
gradient in the renal medulla will be
maintained
o Ascending loop of Henle: where the salt
is reabsorbed
- Passive reabsorption of water takes place in all
parts of the nephron except the ascending loop
of Henle, the walls of which are impermeable to
water.
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
- Countercurrent mechanism – selective
reabsorption process to maintain the osmotic
gradient of the medulla
Tubular Concentration
- Renal concentration begins in the descending
and ascending loops of Henle, where the filtrate
is exposed to the high osmotic gradient of the
renal medulla.
- Water is removed by osmosis in the descending
loop of Henle, and sodium and chloride are
reabsorbed in the ascending loop.The sodium
and chloride leaving the filtrate in the ascending
loop prevent dilution of the medullary
interstitium by the water reabsorbed from the
descending loop.
- the actual concentration of the filtrate leaving
the ascending loop is quite low owing to the
reabsorption of salt and not water in that part of
the tubule.
- Reabsorption of sodium continues in the distal
convoluted tubule, but it is now under the
control of the hormone aldosterone, which
regulates reabsorption in response to the body’s
need for sodium
Collecting Duct Concentration
- The final concentration of the filtrate through
the reabsorption of water begins in the late
distal convoluted tubule and continues in the
collecting duct
- Reabsorption depends on the osmotic gradient
in the medulla and the hormone vasopressin
(antidiuretic hormone [ADH]), which will be
stored in the posterior pituitary gland.
- production of ADH is determined by the state of
body hydration
- chemical balance in the body is actually the final
determinant of urine volume and concentration.
- The concept of ADH control can be summarized
in the following manner:
↑Body Hydration ↓ADH ↑Urine Volume
↓Body Hydration ↑ADH ↓Urine Volume
TUBULAR SECRETION
- involves the passage of substances from the
blood in the peritubular capillaries to the tubular
filtrate
- two major functions:
o elimination of waste products not
filtered by the glomerulus
o regulation of the acid-base balance in
the body through the secretion of
hydrogen ions.
- Many foreign substances, such as medications,
cannot be filtered by the glomerulus because
they are bound to plasma proteins
- When these protein-bound substances enter the
peritubular capillaries, they develop a stronger
affinity for the tubular cells and dissociate from
their carrier proteins, which results in their
transport into the filtrate by the tubular cells
- The major site for removal of these nonfiltered
substances is the proximal convoluted tubule
GLOMERULAR FILTRATION TESTS
- The standard test used to measure the filtering
capacity of the glomeruli is the clearance test.
- a clearance test measures the rate at which the
kidneys are able to remove (to clear) a filterable
substance from the blood.
Clearance Test
➢ Urea – earliest glomerular filtration tests
because of its presence in all urine specimens
and the existence of routinely used methods of
chemical analysis
o 40% of urea is reabsorbed while 60% is
excreted
o Hard to assess renal function
o Depends on the diet of the patient
➢ Inulin Clearance – it is a polymer of fructose and
is not a normal body constituent because it is
exogenous. However, it must be intravenously
infused at a constant rate throughout the testing
period.
o Gold standard
o Therefore, although inulin was the
original reference method for clearance
tests, it is currently not used for
glomerular filtration testing
➢ Creatinine Clearance – Some creatinine is
secreted by the tubules, and secretion increases
as blood levels rise.
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
o Normally produced by the muscle and it
is not absorbed back to the circulation
o It requires 24-hr urine specimen
o Patient should have no heavy meat diet,
o Chromogens present in human plasma
react in the chemical analysis. Their
presence, however, may help counteract
the falsely elevated rates caused by
tubular secretion.
o Medications, including gentamicin,
cephalosporins, and cimetidine
(Tagamet), inhibit tubular secretion of
creatinine, thus causing falsely low
serum levels
Computations:
𝑪𝒓𝒆𝒂𝒕𝒊𝒏𝒊𝒏𝒆
=
𝑈𝑟𝑖𝑛𝑒 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 (𝑚𝑔/𝑑𝐿) 𝑥 𝑈𝑟𝑖𝑛𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿/𝑚𝑖𝑛)
𝑪𝒍𝒆𝒂𝒓𝒂𝒏𝒄𝒆 𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑒 (𝑚𝑔/𝑑𝐿)
Example given:
• 24-hr urine specimen: 1,440mL
• Urine creatinine: 120mg/dL
• Plasma creatinine: 1mg/dL
Solution:
120𝑚𝑔/𝑑𝐿 𝑥 1𝑚𝐿/𝑚𝑖𝑛
𝑪𝒓𝒆𝒂𝒕𝒊𝒏𝒊𝒏𝒆
= = 𝟏𝟐𝟎𝒎𝑳/𝒎𝒊𝒏
𝑪𝒍𝒆𝒂𝒓𝒂𝒏𝒄𝒆 1𝑚𝑔/𝑑𝐿
Normal Values:
• Male: 107-139 mL/min
• Female: 87-107 mL/min
Calculated Glomerular Filtration Estimates
Cockcroft and Gault
Variables: age, weight, and gender (if female, multiply
the computed value by x 0.85)
Modification of diet in renal disease
Note: no need to consider body weight
Variables: age, gender, ethnicity (if black, multiply the
computed value by x 1.1880), BUN, and serum albumin
Renal Function Test
- The development of simplified procedures
measuring the plasma disappearance of infused
substances, thereby eliminating the need for
urine collection, has enhanced interest in
exogenous procedures.
o Injection of radionucleotides such as
125I-iothalamate
o correlation between the GFR and plasma
levels of beta 2 microglobulin
o Beta 2 microglobulin
o Cystatin C
TUBULAR REABSORPTION TESTS
➢ Fishberg test (water deprivation test)
o Specific gravity: ≥1.026
➢ Mosenthal test – Throughout the years, various
methods have been used to produce water
deprivation, including the Fishberg and
Mosenthal concentration tests, which measured
specific gravity.
o Compare the day and night urine
specific gravity and volume
Renal Concentration Ability
➢ Osmolarity – affected only by the number of
particles present
➢ Specific gravity – depends on the number of
particles present in a solution and the density of
these particles
o Freezing Point Osmometers –
determine the freezing point of any
solution by supercooling and measuring
the amount of sample to approx. 27°C
o Vapor Pressure Osmometers – due
point (temperature at which water vapor
will be condensed to a liquid) should be
performed first.
▪ Low due point is parallel to the
decrease vapor pressure
Factors to consider because of their influence on
true osmolarity readings
➢ lipemic serum – displacement of serum water by
insoluble lipids produces erroneous results with
both vapor pressure and freezing point
osmometers.
➢ lactic acid – Falsely elevated if serum samples
are not separated or refrigerated within 20
minutes
➢ volatile substances – Vapor pressure
osmometers do not detect the presence of
volatile substances
TUBULAR SECRETION AND
RENAL BLOOD FLOW TESTS
- Total renal blood flow through the nephron must
be measured by a substance that is secreted
rather than filtered through the glomerulus
o p-aminohippuric acid (PAH) test
o phenolsulfonphthalein (PSP) dye
PAH Test
- Exogenous
- is loosely bound to plasma proteins, which
permits its complete removal as the blood
passes through the peritubular capillaries.
Titratable Acidity and Urinary Ammonia
- The ability of the kidney to produce an acid urine
depends on the tubular secretion of hydrogen
ions and production and secretion of ammonia
by the cells of the distal convoluted tubule
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

FACTORS AFFECTING URINE COLOR

1. Specimen Evaluation • the amount of the substance present
2. Physical Examination • the urine pH
3. Chemical Examination o some pigments when exposed to
4. Sediment Examination alkaline environment results darker
color
o Ex. alkaptonuria (melanin pigment gives
- Rough indicator of the degree of hydration dark brown color to the urine)
- Depends primarily on the body’s homeostasis • the structural form of the substance
and the degree of hydration
- Should correlate with the urine Specific Gravity LABORATORY CORRELATIONS OF URINE COLOR
- Low fluid intake = darker urine color = higher Normal Urine Color
amount of solute = increased specific gravity ➢ Colorless/Straw – recent fluid consumption
- High fluid intake = lighter urine color = lower ➢ Pale yellow
amount of solute = decreased specific gravity o Dilute random specimen
- Normal: Colorless (straw) to Deep Yellow o Polyuria
- Abnormal: Red/Red-Brown (Most Common) ▪ Diabetes mellitus: increased
glucose and water excretion
Recommendations for the Evaluation of Urine ▪ Diabetes insipidus: deficiency in
Physical Characteristics antidiuretic hormone
• Use a well-mixed specimen • Water will be excreted
• View through a clear container – plastic or glass. even the patient is
• View against a white background. already dehydrated
• Evaluate a consistent depth or volume of the ▪ Both has Increased water
specimen. excretions
• View using room lighting that is adequate and ▪ More than 2 liters per day
consistent. ➢ Dark Yellow
o Concentrated specimen
URINE PIGMENTS o Depends on the increased urochrome
o Dehydration
Urochrome o Strenuous exercise
- product of endogenous metabolism o First morning urine specimen
- Major pigment ▪ Pale yellow first morning urine if
- Yellow pigment the patient has diabetes
- It is a lipid-soluble pigment insipidus
- Increased metabolism = increased urochrome
o ex. hyperthyroidism, starvation, and Urine Color Chart
fever Urine Color Possible Meaning
- Constantly produced by the body Good hydration, overhydration, or mild
Clear
- Present normally in the body and is excreted in dehydration
the urine Pale Yellow Good hydration or mild dehydration
Mild or moderate dehydration or taking
- Patient with renal disease, failure, or CKD may Bright Yellow
vitamin supplements
not excrete urochrome Orange, Amber Moderate or severe dehydration
o The filtering capacity of kidney is Tea-Colored Severe dehydration or burned patients
damaged
o The urochrome will be deposited under Abnormal Urine Color
the skin (subcutaneous fats), causing ➢ Dark Yellow/Amber/Orange
yellowish pigmentation of skin o Presence of the abnormal pigment
bilirubin
Uroerythrin ▪ a yellow foam appears when the
- May deposit in amorphous urates and uric acid specimen is shaken, may contain
crystals hepatitis virus
- Pink pigment ▪ yellow foam sometimes due to
- Smaller in quantity compared to urochrome medications (Phenazopyridine)
- Does not always evident ▪ Bilirubin increases when there is
- Seen when sample is centrifuged a liver problem
- Has reddish or brick-red crystals ▪ It should be converted to
urobilinogen
Urobilin ▪ Bilirubin is sensitive to light
- Imparts an orange-brown color of urine that is o Photo-oxidation of large amounts of
not fresh excreted urobilinogen to urobilin (no
- Oxidized form of urobilinogen yellow foam when shaken)
- Old urine specimen
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

➢ Yellow-Green ➢ Purple
o Biliverdin (photo-oxidation of bilirubin) o Found in catheter bags
o Upon standing or improper storage, o Associated with purple bag syndrome
bilirubin may be oxidized to biliverdin and blue diaper syndrome (in infant
➢ Yellow-orange patients with indicanuria)
o Phenazopyridine (Pyridium) or o Presence of indican (water soluble)
azogantrisin (produce a yellow foam o Presence of indole gives purple color
when shaken) ▪ Tryptophan converts into
▪ Treatment for UTI indole, which further converts
o also interferes with chemical tests that into indican, causing indicanuria
are based on color reactions o Bacterial infection
o high consumption of food such as ▪ Klebsiella or Providencia species
vegetables rich in beta carotene ➢ Brown/Black
➢ Red/Pink/Brown o Methemoglobin: oxidized form of
o One of the most common causes of hemoglobin in acidic urine
abnormal urine color o Melanin
o How to differentiate: ▪ oxidation product of melanogen
▪ Intact RBC: cloudy red or pink (colorless)
▪ Hemolyzed RBCs (hemoglobin ▪ produced in excess when a
or myoglobin): clear red malignant melanoma is present
Plasma Examination Test o Homogentisic acid: metabolite of
Hemoglobin Red or pink phenylalanine (alkaptonuria)
Myoglobin Clear yellow
▪ The sample become alkaline
o Blood may range from pink to brown, upon standing
depending on o Medications: include levodopa,
▪ the amount of blood methyldopa, phenol derivatives, and
▪ the pH of the urine metronidazole (Flagyl).
▪ the length of contact
o oxidation of hemoglobin to
Urine color changes with commonly used drugs
methemoglobin (brown) Drug Effect
o Fresh brown (glomerular bleeding) Levodopa Cola-colored, due to myoglobin
o Alkaline urine with red blood cell gives Mepacrine (Atabrine) Yellow
reddish brown in color Methyldopa (Aldomet) Green-brown
➢ Porphyrin: Oxidation of porphobilinogen (port Metronidazole (Flagyl) Darkening, reddish brown
Phenazopyridine (Pyridium) Orange-red, acidic pH
wine, Burgundy red, Purplish Red)
Rifampin Bright orange-red
o Disorder in the porphyrin metabolism Riboflavin Bright yellow
o The pigment also deposited in teeth
o Patients before are thought to be
vampires (pale skin and bleeding mouth)
o Non-pathogenic causes
▪ Menstrual contamination
▪ Ingestion of highly pigmented
foods
▪ Medications (rifampin,
phenolphthalein, phenindione,
and phenothiazines)
▪ In genetically susceptible
persons, eating fresh beets
causes a red color in alkaline
urine
▪ Ingestion of blackberries can
produce a red color in acidic
urine
➢ Blue/Green
o Bacterial infections
▪ urinary tract infection by
Pseudomonas species, which
gives pyoveridin pigment
o Intestinal tract infections
o Ingestion of breath deodorizers (clorets)
o Excessive use of mouthwash
o Phenol derivatives produce green urine
on oxidation
o Medication (blue)
▪ Methocarbamol (Robaxin)
▪ Methylene blue
▪ Amitriptyline (Elavil)
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Other causes:
- Normal Range (24 hours): 600 mL to 2000 mL o Diabetes mellitus (glucose)
- Average (24 hours): 1200 to 1500 mL o Drugs (diuretic therapy, caffeine,
- Night:Day Ratio: 1:2 or 1:3 alcohol)
- Depends on: o Excessive fluid intake (IV administration,
o Amount of water the kidneys excrete compulsive water intake)
o Body’s state of hydration o Diabetes insipidus (ability to retain
- Factors that influence urine volume: water is lost)
o Fluid intake o Renal disease
o Fluid loss from non-renal sources o Drugs (lithium)
(diarrhea, 3rd degree burn, excessive
vomiting, and excessive sweating)
o Variations in the excretion of
Antidiuretic hormone (diabetic
insipidus)
o Need to excrete increased amount of
dissolved solids

OLIGURIA
- decrease in urine output
- Body enters a state of body dehydration Renal threshold: 160 to
180 mg/dL
- Ranges:
Hypothalamic Nephrogenic
o Infants: < 1mL/kg/hr complete deficiency ADH is present, but the cells in
o Children: <0.5mL/kg/hr of ADH the collecting duct are not
responsive to ADH stimulation
o Adults: <400mL/day
- Other causes: NOCTURIA
o Decreased renal blood flow - increase in the nocturnal excretion of urine
o Dehydration/water deprivation - range: >500mL/night
o Shock - causes:
o Decreased cardiac output (hypotension) o Pregnancy
o Renal disease o Chronic progressive renal failure
o Urinary tract obstruction
o Renal tubular dysfunction
o End-stage renal disease
o Nephrotic syndrome - Describes the overall visual appearance of a
o Edema urine specimen.
- Should be assessed at the same time with color
- Cloudiness of the urine caused by suspended
ANURIA particulate matter that scatters light
- Cessation of urine flow - Refers to the transparency of the specimen.
- May result from any serious damage to the
kidneys or from a decrease in the flow of blood
COLOR AND CLARITY PROCEDURE
to the kidneys
- According to Graff’s textbook, <100 mL per day • Use a well-mixed specimen
is also considered anuria • View through a clear container
- Causes: • View against a white background
o Acute renal failure • Maintain adequate room lighting
o Ischemic causes (shock, heart failure) • Evaluate a consistent volume of specimen
o Nephrotoxic causes (drugs, toxic agents) • Determine color and clarity
o Urinary tract obstruction (tumor,
blockages due to kidney stones, or URINE CLARITY
nephrolithiasis) Clarity Term
o Hemolytic transfusion Clear No visible particulates, transparent
Hazy Dew particulates, print easily seen through urine
Cloudy Many particulates, print blurred through urine
POLYURIA Turbid Print cannot be seen through urine
- an increase in daily urine volume Milky May precipitate or be clotted
- Ranges:
o Adults:
▪ > 2.5 L/day (Henry’s)
▪ > 2000 mL/24 hours (Strasinger)
o Children: 2.5–3 mL/kg/day
- often associated with diabetes mellitus and
diabetes insipidus
- artificially induced by diuretics, caffeine, or
alcohol
o all suppress the secretion of antidiuretic
hormone
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

CAUSES OF URINE TURBIDITY - Obsolete method because it requires large

Nonpathologic Pathologic volume of sample (10-15mL)
Squamous epithelial cells RBCs - There should be a correction to be done because
Mucus WBCs of the presence of certain solutes
Amorphous phosphates, - Calibration Temperature: 20°C
Bacteria
carbonates, urates - Requires Temperature correction:
Semen, spermatozoa Yeast
Fecal contamination Nonsquamous epithelial cells
o – 0.001 for every 3°C that the specimen
Radiographic contrast media Abnormal crystals temp is below the calibrating temp
Talcum powder Lymph fluid o + 0.001 for every 3°C that the specimen
Vaginal creams Lipids temp is above the calibrating temp
- Requires solute correction:
LABORATORY CORRELATIONS IN URINE TURBIDITY o 1 g/dL Glucose = – 0.004
➢ Acidic Urine o 1 g/dL Protein = – 0.003
o Amorphous urates
o Radiographic contrast media Example
➢ Alkaline Urine A specimen containing 1 g/dL protein and 1 g/dL glucose
o Amorphous phosphates, carbonates has a specific gravity reading of 1.030.
➢ Soluble with Heat Specimen Temp Reading: 26°C
o Amorphous urates, uric acid crystals Calculate the corrected reading:
➢ Soluble in Dilute Acetic Acid 1.030 – 0.003 (protein) = 1.027
o RBCs 1.027 – 0.004 (glucose) = 1.023
o Amorphous phosphates, carbonates 1.023 + 0.002 (temp) = 1.025
➢ Insoluble in Dilute Acetic Acid 1.025 = corrected specific gravity
o WBCs (add 2 drops of acetic acid)
o Bacteria, yeast REFRACTOMETER
o Spermatozoa - Determines the concentration of dissolved
➢ Soluble in Ether particles in a specimen
o Lipids - Measures refractive index (a comparison of the
o Lymphatic fluid, chyle velocity of light in air with the velocity of light in
a solution)
- Use only small volume of specimen (one or two
- Specific gravity (SG) is an expression of urine drops).
concentration in terms of density - Temperature corrections are not necessary
- Detects possible dehydration or abnormalities in (15°C and 38°C)
antidiuretic hormone. - solute correction is needed
- Measure of the density of the dissolved
chemicals in the specimen
- Direct methods using a urinometer (hydrometer)
or harmonic oscillation densitometry (HOD)
o To check the total solid present directly
from the urine sample
- Indirect methods using a refractometer or the
chemical reagent strip.
o To check the refractive index
(refractometer) and amount of ions
(chemical reagent strip) instead of
solutes
- Specific Gravity of Urine: 1.003 to 1.035
- Average Specific Gravity of Urine: 1.015 to 1.030

𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑈𝑟𝑖𝑛𝑒
SG =
𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑒𝑞𝑢𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑝𝑢𝑟𝑒 𝑤𝑎𝑡𝑒𝑟

Particle Changes to Colligative Properties

Normal Pure Effect of 1 Mole
Property
Water Point of Solute
Freezing Point 0°C Lowered 1.86°C
Boiling Point 100°C Raised 0.52°C
2.38 mm/Hg at Lowered 0.3 mm/Hg
Vapor Pressure
25°C at 25°C
Increased 1.7x109
Osmotic Pressure 0 mm/Hg
mm/Hg

URINOMETER
- Urinometry is less accurate than the other
methods currently available and is not
recommended by the CLSI
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Due to organic and inorganic substances

(byproducts of metabolism)
- Normal urine has a characteristic aromatic odor
that is typically faint and unremarkable
- Urine odor led to the discovery of
Phenylketonuria

Common Causes of Urine Odor

Odor Cause
Normal
Aromatic
Caused by urea
Bacterial decomposition, urinary tract
infection
Foul, ammonia-like
Normal when the urine sample standing
for a long time
Ketones (product of lipid metabolism)
Associated with diabetes mellitus,
Fruity, sweet starvation, and vomiting
Grapelike-odor, caramelized sugar, and
curry odor
Calibration Maple syrup Maple syrup urine disease
Mousy Phenylketonuria
SG Solution
Rancid Tyrosinemia
1.000 Water, distilled
Sweaty feet Isovaleric acidemia
1.015 NaCl, 0.513 mol/L (3% w/v)
Cabbage Methionine malabsorption
1.022 NaCl, 0.856 mol/L (5% w/v)
Bleach Contamination
1.034 Sucrose, 0.263 mol/L (9% w/v)
Rotting Fish Trimethylaminuria
Swimming pool Hawkinsinuria
HARMONIC OSCILLATION DENSITOMETRY

- accurate and precise in determining the urine

specific
- checks specific gravity up to 1.080
- one of the method used in semi-automated
station
- Based on the principle that the frequency of a
sound wave entering a solution changes in
proportion to the density of the solution
o High sound frequency = high solute
present
- Shifts in harmonic oscillation are measured, and
relative density is calculated
- Yellow iris system: an automated urinalysis work
station wherein it is used from chemical up to
microscopic (slideless microscope)
o Uses 6mL of sample: 4mL for slideless
microscope and 2mL for mass gravity
meter

Urine Specific Gravity Measurements

Method Principle
Urinometry Density
Refractometry Refractive Index
Harmonic Oscillation
Density
Densitometry
pKa changes of a
Reagent Strip
polyelectrolye
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

CARE OF THE REAGENT STRIP

- examine different substances or analytes that
are present in the urine sample
- Biochemical testing: Old method or traditional
method that requires larger volume of samples
and very tedious
- Advanced technology: reagent strip pads which
are impregnated with chemicals that will react
with the urine specimen
o Easier and faster to use
• Store with desiccant in an opaque, tightly closed
container.
• Store below 30°C/ Room temperature; do not
freeze.
• Do not expose to volatile fumes.
• Do not use past the expiration date.
• Do not use if chemical pads become discolored.
• Remove strip immediately prior to use.
• Do not touch the chemical pads.

QUALITY CONTROL OF REAGENT STRIPS

1. Specimen Evaluation - Strips must be checked with both positive and
2. Physical Examination negative controls a minimum of once every 24
3. Chemical Examination hours.
4. Sediment Examination - Testing is also performed when
o a New Bottle of reagent strips is opened
o questionable results are obtained
- Provide a simple, rapid means for performing - Distilled water is not recommended as a negative
medically significant chemical analysis of urine control
- Consist of chemical-impregnated absorbent pads o Negative control is supplied by the
attached to a plastic strip manufacturer
- A color-producing chemical reaction takes place o water is used as a blank
when the absorbent pad comes in contact with
urine.
- The reactions are interpreted by comparing the ➢ 4-parameter: glucose, protein, specific gravity,
color produced on the pad within the required and pH
time frame with a chart supplied by the ➢ 10-parameter: 4-parameter plus bilirubin,
manufacturer. ketones, blood, urobilinogen, nitrite, and
- Several colors or intensities of a color for each leukocyte esterase
substance being tested appear on the chart. By ➢ 11-parameter: 10-parameter plus ascorbic acid
careful comparison of the colors on the chart and (one of the most interfering substances present
the strip, a semiquantitative value of trace, 1+, in the urine)
2+, 3+, or 4+ can be reported.
- An estimate of the milligrams per deciliter Parameter Time Principle
present is available for appropriate testing areas. Double Sequential Enzyme
Glucose 30 secs
- Automated reagent strip readers also provide Reaction
Système International units Bilirubin 30 secs Diazo Reaction
Ketone 40 secs Sodium Nitroprusside
- 2 types of reagent strips: multistix and
Specific gravity 45 secs pka change of polyelectrolyte
chemstrips pH 60 secs Double indicator system
Protein (Sorensen’s) error of
Protein 60 secs
REAGENT STRIP TECHNIQUE indicators
Pseudoperoxidase activity of
Blood 60 secs
hemoglobin
Urobilinogen 60 secs Ehrlich reaction
Nitrite 60 secs Greiss’ reaction
Leukocyte 120 secs Leukocyte esterase

pH
- Important in the identification of crystals and
determination of unsatisfactory specimen
o Crystals:
1. Dip the reagent strip briefly into a well mixed
▪ Acidic pH: A. urate
uncentrifuged urine specimen at room
▪ Alkaline pH: A. phosphate
temperature
o Unsatisfactory:
2. Remove the excess urine by touching the edge of
▪ Too alkaline: urea is already
the strip to the container as the strip is
converted to ammonia
withdrawn
- Normal pH:
3. Blot the edge of the strip on a disposable reagent
o Random Specimen: pH 4.5 to 8.0
pad.
o First Morning: pH 5.0 to 6.0
4. Wait the specified length of time for reactions to
o After a meal: alkaline due to alkaline tide
take place.
- Unpreserved urine: pH 9.0 due to ammonia
5. Compare the colored reactions against the
- Use pH to check the acid-base content of the
manufacturer’s chart using a good light source
blood, patient’s renal function, presence of UTI,
patient’s dietary intake, and age of the specimen
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
Acidic Urine
• Diabetes Mellitus and Starvation
o Caused by increased ketones
o primary constituents of ketone are
acetoacetic acid and beta hydroxybutyric
acid
o there is an increased lipolysis
• High Protein Diet: increased amino acids
• Cranberry juice: contains ascorbic acid
• Emphysema
o Associated with difficulty in breathing
and hypoventilation
o Lungs cannot function normally
o Increased carbon dioxide resulting to
increased carbonic acid
• Dehydration: increased ketones
• Diarrhea: increased ketones
• Acid-producing bacteria: urinary tract infections
caused by E. coli
• Medication
Alkaline Urine
• Renal Tubular Acidosis
o refers to the blood pH
o renal tubules cannot excrete hydrogen
ions which accumulate in the blood
o insufficient hydrogen ion in the urine
results to alkaline urine
• Vegetarian Diet
• After meal
o Alkaline tide: most hydrogen ions will go
to the stomach to increase the acidity of
the hydrochloric acid to digest the food
o There will be a decreased amount of
hydrogen ion in the blood, resulting to
decreased amount of hydrogen ion
excreted in the urine
• Old specimen: associated with increased
ammonia
• Hyperventilation: there is an increased oxygen
and decreased carbon dioxide, which results to
decreased carbonic acid
• Urease-producing bacteria: splits the urea into
ammonia
Reagent Strip for pH
Principle: Double Indicator System
- It is double for wider range of spectrum
- It uses 2 indicators: methyl red and bromthymol
blue
- There are no interfering substances for pH
- Old specimen: common interference that results
to alkaline urine
Summary of Clinical Significance of Urine pH
• Respiratory (increased carbonic acid) or
metabolic (increased hydrogen ions) acidosis or
ketosis
• Respiratory (increased oxygen than carbon
dioxide, produces carbonic acid) or metabolic
(increased bicarbonate) alkalosis
• Defects in renal tubular secretion and
reabsorption of acids and bases (renal tubular
acidosis)
• Renal calculi formation (to know if a certain
substance can produce a renal stone)
• Treatment of urinary tract infections (to monitor
if a patient is having acidic or alkaline urine)
• Precipitation/identification of crystals
• Determination of unsatisfactory specimens
(associated with old specimen)
PROTEIN
- Most indicative of Renal Disease
- It is the least affected to changes due to bacterial
multiplication
- Produces white foam in the urine after shaking
- Albumin: Major serum protein found in the urine
o Normal values:
▪ <10 mg/dL or 100 mg/24 hours
(Strasinger)
▪ <150 mg/dL/24 hours (Henry)
- Other proteins: Serum and tubular
microglobulins (immunoglobulin), Tamm-
Horsfall protein produced by the tubules (mucus
threads), and proteins from prostatic, seminal,
and vaginal secretions.
Pre-Renal Proteinuria
- Also known as before or overflow proteinuria
- Caused by the conditions that affect the plasma
prior its reaching the kidney.
o Intravascular hemolysis (increased
hemoglobin)
o Muscular injury (increased myoglobin)
o Severe infection and inflammation
(increased acute phase reactants such as
C-reactive protein)
o Multiple Myeloma: proliferation of
immunoglobulin-producing plasma cells
(Bence Jones Proteins)
▪ Immunoglobulin light chains
(Kappa and lambda proliferates
in the urine)
▪ Can be identified using Serum
Immunoelectrophoresis or
precipitation
▪ precipitates at 40-60°C (cloudy)
and dissolves at 100°C (clear)
Renal Proteinuria (True Renal Disease)
A. Glomerular Proteinuria
➢ Diabetic Nephropathy
o Decrease glomerular filtration
▪ May lead to renal failure
o Indicator: Microalbuminuria
(proteinuria that is undetectable by
using the routine reagent strip)
o Albumin Excretion Rate (AER) in ug/min
or in mg/24hrs
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

▪ Normal: 0-20 ug/min Reagent Strip for Protein

▪ Microalbuminuria: 20 to 200 Principle: Double Indicator System
ug/min or 30 to 300 mg/24hrs - Protein or Sorensen’s error of indicators
▪ Clinical Albuminuria: >200 - Indicators produce specific color in response to
ug/min (detectable) particular pH level
➢ Orthostatic / Cadet / Postural Proteinuria – - If the indicator remains acid due to the presence
Proteinuria when standing due to increased of hydrogen ions, it is yellow or negative for
pressure to renal veins protein
o There is no problem in the kidney - If there is a presence of protein, the protein will
o Due to prolonged standing bind with hydrogen ions, removing it from the
o Differentiate orthostatic proteinuria indicator, which becomes alkaline and will have
from clinical proteinuria: blue green color
OP CP
FMS (–) (+)
2hrs after standing (+) (+)
o MICRAL test: rapid test used to detect
presence of microalbuminuria
- Indicators:
▪ Strip is employed using an
o Multistix: Tetrabromphenol blue
antibody-enzyme conjugate that
o Chemstrips: Tetrachlorophenol
binds albumin
tetrabromosulfophthalein
▪ Enzyme immunoassay
- Note: Indicator is sensitive to albumin
▪ Sensitivity: 0 to 10 mg/dL of
- Sensitivity of the reagent strip: >200 ug/min or
albumin
>200 mg/24hrs
▪ Interference: diluted urine
- Readings are reported corresponding to each
sample causes false negative
color change:
reaction
o Negative, Trace, 1+, 2+, 3+, and 4+
o Semiquantitative values of 30, 100, 300,
B. Tubular Proteinuria
or 2000 mg/dL
- Increased albumin is also present in disorders
affecting tubular reabsorption because the
normally filtered albumin can no longer be
reabsorbed.
- Causes of tubular dysfunction include exposure
to toxic substances and heavy metals, severe
viral infections, and Fanconi syndrome.
o problem with the reabsorptive capacity
of renal tubules
- The amount of protein that appears in the urine
following glomerular damage ranges from
slightly above normal to 4 g/day, whereas
markedly elevated protein levels are seldom
seen in tubular disorders.

Post Renal Proteinuria

- Protein can be added to a urine specimen as it
passes through the structures of the lower
urinary tract (ureters, bladder, urethra, prostate,
and vagina).
- Causes: Lower UTI/ inflammation, Injury or
trauma, Menstrual contamination, Prostatic
fluid/ spermatozoa, and Vaginal secretions
Sulfosalicylic Acid Precipitation Test (SSA)
- It is a biochemical reaction
- cold precipitation test that reacts equally with all
forms of protein
- 3 ml of 3% SSA + 3ml centrifuged urine = (+)
turbidity or cloudiness
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

GLUCOSE (DEXTROSE) Clinitest/ Benedict’s Test

- Most frequently tested in urine - Nonspecific test for reducing sugars: Galactose,
- Renal threshold – plasma concentration of a Lactose, Fructose, Maltose, Pentoses, Ascorbic
substance at which tubular reabsorption stops Acid, Certain Drug Metabolites, and Antibiotics
- If a patient has increased glucose in the urine, such as the Cephalosporin.
glucose will be reabsorbed by the urinal tubules. - Tests for reducing sugars aside from glucose
But after reaching the maximum reabsorptive except for sucrose
capacity of glucose, it will stop the reabsorption - Principle: Copper Reduction Test
- Renal threshold for glucose: 160-180 mg/dL - (+) result = brick red precipitates
- It is being detected in the urine of the patient has - Clinitest/Benedict’s Test:
certain metabolic problem o False positive: presence of ascorbic acid
- Other sugars in the urine can also be detected (copper sulfate will be reduced to
such as galactose, sucrose, and fructose. These copper oxide), uric acid
are being identified by thin-layer o False negative: oxidizer such as
chromatography detergent (prevents the reduction of
copper sulfate to become copper oxide)
CLINICAL SIGNIFICANCE OF URINE GLUCOSE
Hyperglycemia-associated Renal-associated
Increased Blood glucose Normal Blood glucose
Increased Urine glucose Increased Urine glucose
Causes: Impaired tubular reabsorption
• DM (insulin deficiency) of glucose
• Cushing’s Syndrome Causes: Fanconi syndrome – Clinitest Tablet Procedure (Henry’s)
(increased cortisol) defective tubular reabsorption - 5 drops of urine + 10 drops of H2O + Clinitest
• Pheochromocytoma of glucose and amino acids
tablet
(increased
catecholamines)
- The tablet contains:
• Acromegaly (increased o CuSO4 (main reacting agent)
growth hormone) o NaCO3 (remove the interfering oxygen)
• Hyperthyroidism o Sodium Citrate (prevents caramelization
(increased T3 or T4) of sugar)
o NaOH (for heat production)
Reagent Strip for Glucose - Pass-through Phenomenon occurs when >2 g/dL
Principle: Double Sequential Enzyme Reaction sugar is present
o Blue > Green > Yellow > Brick red > Blue
o To prevent pass through, use 2 drops of
urine

- Two enzymatic process

- Chromogens:
o O-toluidine (pink to purple)
o Multistix: Potassium iodide (blue to
green to brown)
o Aminopropylcarbazole (yellow to
orange-brown)
o Chemstrip: Tetramethylbenzidine
(yellow to green)
- Reported in terms of negative, trace, 1, 2, 3, and
4; however, the color charts also provide
quantitative measurements ranging from 100
mg/dL to 2 g/dL, or 0.1% to 2%. KETONES
- Reagent strips: - Result from an increased fat metabolism due to
o False positive: oxidizer such as detergent inability to metabolize carbohydrates.
o False negative: presence of ascorbic acid - In cases of diabetes mellitus, the glucose in the
(reducer) plasma is not utilized because of the deficiency
with insulin
- The body will get energy from the fats (lipolysis)
- There will be beta oxidation in order for the cells
to have energy
o Only 2 ATP is generated in beta oxidation
o Ketones are one of the metabolic
products of fat metabolism
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

KETONE BODIES o Myoglobinuria: the heme portion of

Beta-hydroxybutyric acid (major ketone but not myoglobin is toxic to renal tubules
78%
detected in reagent strip)
▪ Clear and red, but sometimes
Acetoacetic acid/ Diacetic acid (parent ketone and can
20% cola-colored (reddish brown)
be detected in reagent strip)
Acetone (can be detected in specific type of reagent
2%
strip)

Reagent Strip for Ketones

Principle: Sodium Nitroprusside / Legal’s Test

- Note: Acetone is detected only when glycine is

present
- Results are reported:
o Qualitatively: negative, trace, small (1),
moderate (2), or large (3)
o Semi-quantitatively: negative, trace (5
mg/dL), small (15 mg/dL), moderate (40
mg/dL), or large (80 to 160 mg/dL)
- Glycine is only present in chemstrip HEMOGLOBIN VS MYOGLOBIN
o Chemstrip can both detect acetoacetic Test Hemoglobin Myoglobin
acid and acetone Plasma Red of Pink Plasma Pale yellow plasma
o More preferred reagent strip for ketone Examination Test because because myoglobin
hemoglobin stays is excreted in the
bodies longer in plasma urine
- In multistix, it only detects acetoacetic acid
decreased Increased creatinine
haptoglobin kinase or aldolase
Blondheim Test Presence of There is no presence
(ammonium precipitated of precipitated
sulfate test) substance at the substance at the
bottom part of the bottom part of the
Procedure:
tube (hemoglobin) tube
Urine + 2.8g NH4
sulfate Check for the Check for the
(filter/centrifuge) presence of blood presence of blood
Test supernatant using a reagent using a reagent strip
for blood reagent strip (negative) (positive)
strip

Reagent Strip for Blood

Principle: Pseudoperoxidase activity of Hemoglobin

- Chromogen: Tetramethylbenzidine
o (–) yellow
o (+) Green (↑) to Blue (↑↑↑)
- Notes:
o Hemoglobin or Myoglobin: Uniform
green/blue
o Hematuria: Speckled or Spotted

- Tablet contains: Sodium nitroprusside, Glycine,

Disodium phosphate, and Lactose
- Acetest procedure detects the presence of
acetone or acetoacetic acid in the urine sample

BLOOD
- Present in the urine either in the form of:
o Hematuria: Intact red blood cells
▪ Cloudy and red urine
o Hemoglobinuria: Product of red blood
cell destruction, hemoglobin
▪ Clear and red urine
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- There are two things that can happen to the remaining 20% of
BILIRUBIN
urobilinogen formed.
- Early indication of liver disease o 99%: The majority will be absorbed by extrahepatic
- Amber urine with yellow foam circulation to be recycled through the liver and reexcreted.
- The only type of bilirubin that appears in the o 1%: The other very small quantity left will enter systemic
circulation and will subsequently be filtered by the kidney
urine is the water-soluble bilirubin (conjugated and excreted in the urine
bilirubin) - Approximately 200 to 300 mg of bilirubin is produced per day, and
- Clinical Significance: it takes a normally functioning liver to process the bilirubin and
o Hepatitis eliminate it from the body.
o Cirrhosis
o Biliary Obstruction (gallstone, Reagent Strip for Bilirubin
carcinoma) Principle: Diazo Reaction
o Other liver disorders

- (+) result = Tan or Pink to Violet

- Multistix: dichloroaniline diazonium salt
- Chemstrip: dichlorobenzene diazonium salt
- Other Test: Ictotest (tablet)
o More sensitive than reagent strips
o Less interference
o Contains p-nitrobenzene-diazonium p-
toluene sulfonate, SSA, sodium
carbonate, boric acid
o (+) blue to purple

UROBILINOGEN
- Bile pigment that result from Hemoglobin
degradation
- 99% found in the feces and 1% found in the urine
- Normal Value: <1 mg/dL or Ehrlich units
- Specimen: Afternoon urine (2 to 4pm)

- Approximately 126 days after the emergence from the

reticuloendothelial tissue, red blood cells are phagocytized and
hemoglobin is released.
- Hemoglobin is degraded to heme, globin, and iron. The iron is
bound by transferrin and is returned to iron stores in the liver or
bone marrow for reuse.
- The globin is degraded to its constituent amino acids, which are
reused by the body.
Clinical Significance
- The heme portion of hemoglobin is converted to bilirubin in 2 to • Early Detection of Liver Disease
3 hours. • Liver Disorders, hepatitis, cirrhosis, carcinoma
- Bilirubin is bound by albumin and transported to the liver.
- This form of bilirubin is referred to as unconjugated or indirect
• Hemolytic disorders
bilirubin.
o This form of bilirubin is insoluble in water and cannot be Reagent Strip for Urobilinogen
removed from the body until it has been conjugated by the
Principle: Ehrlich Reaction
liver.
- Once at the liver cell, unconjugated bilirubin flows into the - Reagent: p-dimethylaminobenzaldehyde
sinusoidal spaces and is released from albumin so it can be picked (PDAB) or Ehrlich’s reagent
up by a carrier protein called ligandin. Ligandin, which is located o It is not specific for urobilinogen
in the hepatocyte, is responsible for transporting unconjugated o It can also react with other Ehrlich’s
bilirubin to the endoplasmic reticulum, where it may be rapidly
conjugated.
reactive compound
- The conjugation (esterification) of bilirubin occurs in the presence o Used for multistix
of the enzyme uridyldiphosphate glucuronyl transferase - Chemstrip: 4-methoxybenzene-diazonium-
(UDPGT), which transfers a glucuronic acid molecule to each of tetrafluoroborate
the two propionic acid side chains of bilirubin to form bilirubin
o More specific for urobilinogen
diglucuronide, also known as conjugated bilirubin.
- This conjugated bilirubin is water soluble and is able to be - Note: Also (+) Ehrlich reactive compounds
secreted from the hepatocyte into the bile canaliculi. o Porphobilinogen, indican, p-
- Once in the hepatic duct, it combines with secretions from the aminosalicylic acid, sulfonamides,
gallbladder through the cystic duct and is expelled through the methyldopa, procaine, chlorpromazine
common bile duct to the intestines.
- Intestinal bacteria (especially the bacteria in the lower portion of
the intestinal tract) work on conjugated bilirubin to produce
mesobilirubin, which is reduced to form mesobilirubinogen and
then urobilinogen (a colorless product).
- Most of the urobilinogen formed (roughly 80%) is oxidized to an
orange-colored product called urobilin (stercobilin) and is
excreted in the feces.
o The urobilin or stercobilin is what gives stool its brown
color.
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Multistix results are reported as Ehrlich units Hoesch Test

(EU), which are equal to mg/dL, ranging from
normal readings of 0.2 and 1 through abnormal
readings of 2, 4, and 8.
- Chemstrip results are reported in mg/dL
- Note: Reagent strip tests cannot determine the
absence of urobilinogen, which is significant in
biliary obstruction.
Principle: Inverse Ehrlich Reaction
- Rapid screening test for Porphobilinogen (≥2
mg/dL)
- Procedure:
o 2 drops of urine + 2 ml Hoesch reagent
o Hoesch reagent: Ehrlich’s reagent in 6M
or 6N HCl
- (+) result is red

NITRITE
- Rapid Screening test for UTI
- Specimen: First Morning urine or 4-hour urine

Watson-Schwartz Test
- Differentiates urobilinogen, porphobilinogen,
and Ehrlich-reactive compounds
- Uses two extraction with organic solvents
o Chloroform Reagent Strip for Nitrite
o Butanol Principle: Greiss Reaction

- (+) result = Uniform pink

- Note:
o Pink spot/ edges are considered
negative
o (+) Nitrite corresponds to 100,000
organisms/ml
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

LEUKOCYTE - Reagent is sensitive to number of ions in the

urine specimen, indicator changes in relation to
ionic concentration
- Indicator: Bromthymol Blue
- Note:
o Add 0.005 to SG reading when pH is ≥ 6.5
due to interference with Bromthymol
blue indicator
o Not affected by Glucose, Protein and
Reagent Strip for Leukocyte Radiographic dye
Principle: Leukocyte Esterase

- (+) result = Purple

- Note:
o With esterase (neutrophils, eosinophils,
basophils, monocytes, histiocytes and
Trichomonas)
o Without esterase (Lymphocytes)
o Strip can even detect lysed WBC
o False positive: parasites such as
trichomonas (contains esterase)

SPECIFIC GRAVITY
- Density of the solution compared with the
density of similar volume of distilled water at a
similar temperature
- Normal SG: 1.003-1.035 (Random)
- SG < 1.003: Not urine
- SG > 1.040: Radiographic Contrast Media
ASCORBIC ACID
Ascorbic acid (≥5 mg/dl) + Phosphomolybdate
→ (+) Molybdenum blue
- A reducing agent that causes
o false-negative reactions on: Blood,
Bilirubin, Leukocyte, Nitrite and Glucose
o false-positive reactions on: copper
reduction test or benedict’s test
th
- 11 Reagent pad
- Gas Chromatography Mass Spectroscopy (GC-
MS) – More accurate quantitative method for
ascorbic acid
- Two type of chemical strips:
o C-stix: 10 seconds
o Stix: 60 seconds

• Isosthenuria: 1.010
• Hyposthenuria: <1.010
• Hypersthenuria: >1.010

Reagent Strip for Specific Gravity

Principle: pKa Change (dissociation constant) in
Polyelectrolytes
Blue → Green → Yellow
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Specimen: 12-hour urine

- Also known as sediment examination - Preservatives: Formalin
- Gold standard in routine urinalysis - Normal Count:
- To detect, identify, and quantitate insoluble o RBC: 0 to 500,000/12-hr urine
materials present in the urine o WBC: 0 to 1,200,000/12-hr urine
- Examine the urinary sediments and correlate the o Hyaline Cast: 0 to 5,000/12-hr urine
result of microscopic examination to physical
and chemical examination
- Disadvantage: least standardized and most time Technique Function
consuming Bright Field Microscopy For Routine urinalysis
Phase-contrast Enhances visualization of elements
Microscopy with low refractive indices (hyaline
cast)
• Determine the correctness of diagnosis of a renal Polarizing Microscopy Identification of cholesterol in oval
fat bodies, fatty casts, and crystals
system infection and disease (maltese cross appearance)
• Refrigerated urine preserve almost all cellular Dark-Field Microscopy Identification of T. Pallidum
elements, crystals, and cast, very well Fluorescence Microscopy Visualization of fluorescent
o Preheat the sample at 37°C to dissolve microorganisms or those stained by
fluorescent dyes
amorphous materials
Interference Microscopy 3-D microscopy image and layer by
• Fresh and concentrated urine: more accurate a. Nomarski (Differential) layer imaging system
results b. Hoffman (Modulation) Bright field microscopy can be
• Hypotonic urine causes lysis of cells and casts adapted
• Proper collection avoid extra debris of the - Abnormalities in the physical and chemical
urethral meatus, vaginal secretions portions of the urinalysis play a primary role in
• Proper use of subdued light in LPO before HPO: the decision to perform a microscopic analysis,
for accurate results thus the use of the term macroscopic screening,
• Examine the urine specimen within 1 to 2 hours also referred to as Chemical Sieving

• Average number: 10 representative fields (10

LPO and 10 HPO)
• Estimate (semiquantitative reporting): rare, few,
moderate, many
• RBC and WBC: Average/HPF
• Casts: Average/LPF
• Epithelial cells and crystals: occasional, rare or
few, moderate and abundant, too many to count
or too numerous to count, and plenty

CELLS
• Careless transfer of sediment (contamination)
1. RBCs (Hematuria)
• Too much light (retractile bodies cannot be seen
- Most commonly encountered cells in the urine
• Using the high power only
- Normal Value: 0-2 or 0-3/HPF
• Specimen dries up only on long standing (false
- Smooth, non-nucleated, biconcave disks
elements are seen)
- Hypertonic solution: Crenate / shrink
• Dirty equipment
- Hypotonic Solution: Swell (hemolyze) or ghost
• Scratches on slides
- Glomerular membrane damage: Dysmorphic
with projections
- Sources of error:
o Yeasts
(1500 rpm) o Oil droplets
o Air bubbles
o Calcium oxalate crystals
(1 drop) - Remedy: add 2% acetic acid to lyse the RBC

- Quantitative measurement of formed elements

of urine using hemacytometer
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

3. Epithelial Cells
➢ Squamous epithelial cells – Largest cell with
abundant, irregular cytoplasm and abundant
nucleus
o From the linings of vagina, female
urethra and lower portion of male
urethra
o Reporting: Estimate/LPF
o Variations: clue cells (largest)
▪ Squamous epithelial cell
covered with Gardnerella
vaginalis
▪ Associated with bacterial
vaginosis
➢ Transitional epithelial cells
o Also known as Urothelial cells
o It is also clinically significant
o Spherical, polyhedral, or caudate
centrally located nucleus
o Smaller than squamous epithelial cells
o Derived from the renal pelvis, ureter,
2. WBCs (Pyuria) urinary bladder and upper portion of the
- Normal values: 0-5 or 0-8 / HPF male urethra
- Larger than RBC o Reporting: Estimate/HPF
- Types of WBCs: ➢ Renal Tubular Epithelial Cells (RTE)
o Neutrophils (most predominant) o Most clinically significant epithelial cell
▪ Granulated and multilobulated o Origin: Nephron
▪ Hypotonic urine: swells (Glitter o Rectangular, polyhedral (DCT), cuboidal
cells) and undergo Brownian (CD), or columnar (PCT)
movement o Eccentric nucleus
▪ UTI: (+) WBC and (+) bacteria o Reporting: Average/HPF
o Eosinophils o >2 RTE/HPF indicates tubular injury
▪ Normal values: <1% o RTE cell variation:
▪ Significant: >1% Oval Fat Body Bubble Cells
▪ Associated with drug-induced • Lipid containing RTE cells • RTE cell with
interstitial nephritis (allergy • Seen in lipiduria nonlipid-
(nephrotic syndrome) filled
because of some antibiotics) • Identified by lipid stains vacuoles
▪ (+) WBC and (–) bacteria (Oil Red O, Sudan III or • Seen in
o Mononuclear cells: Lymphocytes, IV) for triglyceride and Tubular
Monocytes, Macrophages, Histiocytes polarizing microscope for Necrosis
cholesterol
ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

4. Bacteria
- UTI: Bacteria and WBC
- Enterobacteriaceae: most common cause of UTI
o E. coli (normal flora)
o Pseudomonas (nosocomial infection)
- Staphylococcus saprophyticus (2nd)
- Enterococcus (3rd)
- Reporting: Estimate/HPF
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

5. Yeasts
- True yeast infection: Yeast + WBC
- Small refractile oval structures that may or may
not bud
- Candida albicans: seen in DM and vaginal
moniliasis
- Reporting: Estimate/HPF

8. Mucus Thread
- Major constituent: Tamm Horsfall Protein
o Also known as Uromodulin
o comes from renal tubular epithelial cells
- Reporting: Estimate/LPF
- (+) protein
- Has low refractive index unlike with cotton fiber

6. Parasites
➢ Trichomonas vaginalis: Pear-shaped flagellate
with jerky motility
o agent of Ping pong Disease
o females are symptomatic
o sometimes mistaken as WBC when dead
o has rapid jerky tumbling motility
o (+) leukocyte esterase
➢ Schistosoma haematobium
o With terminal spine
o Hematuria
o Associated with bladder cancer
➢ Enterobius vermicularis ova: most common CASTS (CYLINDURIA)
fecal contaminant
- Unique to the kidney
o Lay eggs early in the morning
- Formed in the DCT and CD
- Major constituent: Tamm Horsfall Protein –
Produced by RTE CELLS
o Low urine flow (urinary stasis)
o Low pH
- Performed along the edges of the coverslip with
subdued light
- mimicking the shape of the renal tubules

Step-by-step analysis of the formation of the Tamm-

Horsfall protein matrix
1. Aggregation of Tamm-Horsfall protein into
7. Spermatozoa individual protein fibrils attached to the RTE cells
- After sexual intercourse 2. Interweaving of protein fibrils to form a loose
- (+) protein and increased specific gravity fibrillar network (urinary constituents may
become enmeshed in the network at this time)
3. Further protein fibril interweaving to form a
solid structure
4. Possible attachment of urinary constituents to
the solid matrix
5. Detachment of protein fibrils from the epithelial
cells
6. Excretion of the cast
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

1. Hyaline Cast (Prototype Cast)

- Beginning of all type of casts
- Normal Value: 0-2/LPF ➢ Bacterial Cell Cast
- Clinical Significance o Pyelonephritis: an upper urinary tract
o Physiologic infection (kidney)
▪ Stress
▪ Strenuous exercise
o Pathologic
▪ Glomerulonephritis
▪ Pyelonephritis
▪ Congestive Heart Failure

SUMMARY OF URINE CAST

➢ WBC Cast
o Inflammation within the nephron
o May be confused with Epithelial cells
o To differentiate:
▪ Phase contrast microscopy
▪ Supravital stain

2. Cellular Cast
➢ RBC Cast
o Bleeding within the nephron
o Clinical Significance ➢ Epithelial (RTE) Cell Cast
▪ Glomerulonephritis o Advanced Tubular Destruction
▪ Strenuous exercise o Renal Tubular Damage
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

4. Granular Cast
- From the disintegration of cellular casts
- Granules are derived from the lysosomes of RTE
cells during normal metabolism (nonpathologic)
- Clinical Significance
o Glomerulonephritis
o Pyelonephritis
o Stress
o Strenuous Exercise

3. Fatty Cast
- Not stained with Sternheimer-Malbin (pink)
- Identification:
o Triglycerides and Neutral Fats: Oil red O
and Sudan 3
o Cholesterol: polarizing microscope
- Clinical significance
o Nephrotic syndrome 5. Waxy Cast
- Longer than oval fat body - Final degenerative form of all types of cast
- Brittle, highly refractile, with jagged ends
- Clinical significance
o Stasis of urine flow
o Chronic renal failure
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

1. Normal Findings in Acid Urine

➢ Amorphous urates
o between pH 5.7 and 7.0
o small, yellow-brown granules, much like
sand
o occur in clumps resembling granular
casts
o frequently encountered refrigerated
specimen with a pink characteristic
(pink-orange)
▪ uroerythrin: cause of the pink
color.
o cloud-like appearance at the bottom of
the container
o Often referred to as “brick dust” –
6. Broad Cast
identified by their solubility in alkali or
- Also known as Renal failure cast their dissolution when heated to
- Indicates destruction widening of the tubular approximately 60°C
walls o Converted to uric acid upon the addition
- Any type of cast can be broad of concentrated acetic acid
- Clinical Significance o Indistinguishable with amorphous
o Extreme urine stasis phosphate when colorless
o Renal failure

➢ Acid Urates
o Sodium, potassium, and ammonium
salts of uric acid (Reported as “urate
crystals”)
o small, yellow-brown balls or spheres
o present when the urine pH is neutral to
slightly acidic
o misidentified as leucine crystals
▪ it has eccentric striations
▪ looks like scallop or pillowcase
o Acid urates appear as larger granules
and may have spicules similar to the
ammonium biurate crystals without
thorns
o acid urates dissolve at 60°C
o converted to uric acid crystals by the
CRYSTALS addition of glacial acetic acid.
- Crystals are identified on the basis of their
microscopic appearance and the pH at which
they are present.
- Factors that influence crystal formation:
o concentration of the solute in the urine
o the urine pH
o the flow of urine through the tubules.

➢ Sodium urates
o Colorless or yellowish slender prisms,
arranged in fan or sheaf-like or leaf – like
structures
o Peacock tail-like looking crystals
o needle-shaped and are seen in synovial
fluid during episodes of gout
o caused by dried urine sample
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

2. Abnormal metabolism of Acid Urine

(Crystals of metabolic origin)
➢ Cystine
o Colorless, highly refractive, rather thick,
hexagonal plates or quadrilateral
prisms
o Overflowing: High level of cystine
because the body lacks enzymes for
cystine metabolism
➢ Monosodium urates o Soluble in hydrochloric acid but insoluble
o appear as colorless to light-yellow in acetic acid
slender, pencil-like prisms o Associated with cystine calculi (kidney
o may be present singly or in small stones)
clusters, and their ends are not pointed o clinically significant and indicate disease,
o dissolve at 60°C that is, congenital cystinosis (overflow)
o reported as “urate crystals” or cystinuria (renal)
o very rare to be seen in urine ▪ overflow: increased in the blood
o can be observe in synovial fluid and increased in the urine
▪ renal: normal in the blood,
increased in the urine; problem
in the reabsorption of acid
o Disintegrating forms may be seen in the
presence of ammonia.
o The chemical confirmatory test for
cystine is based on the cyanide-
nitroprusside reaction.
▪ Sodium cyanide reduces cystine
to cysteine,
➢ Uric Acid Crystals ▪ the free sulfhydryl groups
o product of purine metabolism subsequently react with
o high purine-rich diet nitroprusside to form a
o occur in many forms; the most common characteristic purple color
form is the rhombic or diamond shape o They dissolve in alkali and hydrochloric
o Yellowish brown, Whet-stone-shaped or acid (pH <2)
rhombic plates or rosettes or quadrates, o Sullivan test: red color
sometimes colorless ▪ Folin’s reagent + sodium
o they occasionally have six sides and may dithionite under alkaline
require differentiation from colorless conditions
cystine crystals.
Characteristics Uric Acid Cystine
o may appear as cubes, barrels or bands
Color Yellow-brown colorless
and may cluster together to form Solubility in ammonia Soluble Soluble
rosettes Solubility in Dilute HCl Insoluble Soluble
o Does not dissolve by heat, HAC and HCI, Birefringence + –
but soluble if heated with sodium Cyanide Nitroprusside – +
hydroxide
o Warning of a possible danger of calculus
formation (kidney stones)
o present following the administration of
cytotoxic drugs (e.g., chemotherapeutic
agents) and with gout, patients with
Lesch-Nyhan syndrome (absence of
HGPRT enzyme necessary for the
metabolism of uric acid, which causes
orange sand diaper syndrome)

➢ Cholesterol crystals
o rarely seen unless specimens have been
refrigerated
o Large flat plates with one or more
corners cut off
o Contains fat globules
o Notched plates (Staircase pattern)
o Resembles crystals of radiographic dye
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
o They are associated with disorders
producing lipiduria, such as the
nephrotic syndrome
o seen in conjunction with fatty casts and
oval fat bodies
o soluble in chloroform and ether
3. Crystals of Iatrogenic Origin
➢ Leucine crystals
o Highly refractive, yellow, or brown
spheres with delicate radiating and
concentric striations
o Scallop-like crystals
o Evident in patients with liver disease
o can resemble fat globules
o when present, should be accompanied
by tyrosine crystals
o Insoluble in ether or HCI
o Salkowski test: blue color
➢ Tyrosine crystals
o Colorless, fine needles, grouped in
clusters or sheaves, crossing at various
angles
o Exist together with leucine cystals
o Clusters may appear black in the center
o Needles in clusters
o Soluble in ammonium hydroxide and
HCI, but not in acetic acid
o Leucine and tyrosine occur in acute
yellow atrophy and in other destruction
disease of the liver
o Morner reagent: green color
➢ Bilirubin crystals
o A present in hepatic disorders producing
large amounts of bilirubin in the urine
o appear as clumped needles or granules
with the characteristic yellow color of
bilirubin
o dissolve when alkali or strong acids are
added
➢ Sulfa Crystals:
o Sheaves of wheat
Sulfanilamide crystals – Large colorless,
needles, frequently gathered together in
sheaves or rosettes. Rarely appear in urine.
Sulfapyridine crystals – Brownish needles in
large conglomerated masses or rosettes.
Shaped like colorless arrowheads or
whetstones
Sulfathiazole crystals – Appear as brownish
sticks of wheat with central binding or rosettes
with radial striations or as colorless diamond
and hexagonal shaped plates. Sometimes
grouped in rosettes resembling uric acid
crystals
o Maybe mistaken with calcium
phosphate (crystal found in alkaline
urine)
o To differentiate:
▪ calcium phosphate: soluble in
acetic acid
▪ Sulfonmide: (+) lignin test
➢ Ampicillin crystals
o long, colorless, thin prisms or needles
o indicate large doses of ampicillin and are
rarely observed with adequate
hydration
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

➢ indinavir sulfate crystals investigation is required to determine

o slender feather-like crystals that whether:
aggregate into wing-like bundles, which ▪ the integrity of the urine
can also associate into a rosette-like or specimen has been
cross form compromised (improper
o only present in acid urine, indinavir and storage)
other antiviral drugs (e.g., acyclovir) ▪ in vivo formation is taking place
o dissolve in acetic acid or on heating to
approximately 60°C.
o always encountered in old specimens
o ammonium biurate crystals can be
converted to uric acid crystals with the
addition of concentrated hydrochloric
or acetic acid
o can resemble some forms of
4. Normal Metabolism of Alkaline Urine sulfonamide crystals
➢ Amorphous phosphates o One differentiates between them on the
o found in alkaline and neutral urine basis of:
o microscopically indistinguishable from ▪ urine pH
amorphous urates ▪ a sulfonamide confirmatory
o Resemble amorphous urates but are test
colorless ▪ the solubility characteristics of
o Dissolved in acetic acid, but not by heat the crystals
o resembles fine, colorless grains of sand
in the sediment
o the precipitate is white or gray

➢ Calcium Carbonate
o Colorless granules slightly larger than
amorphous phosphate appearing singly
or masses and often appearing in dumb-
bell forms
o resemble amorphous material
o exists in clusters or strand form
o misidentified as bacteria because of
their size and occasional rod shape.
o Dissolve in acetic acid with the evolution
of gas.
➢ Ammonium biurates
o Yellow to golden spherical body usually
with radial and concentric striations and
bearing long prismatic spicules
o Occur during ammoniacal fermentation
o Abnormal in freshly passed urine (has
renal tubular damage)
o Thorny-apple looking elements
▪ Can also exist in spikeless
o Inadequate hydration of the patient
o when ammonium biurate crystals are
encountered in a urine specimen,
➢ Calcium phosphates (Apatite)
o Large forms, thin, irregular, usually
granular, colorless plates
o Mistaken for squamous epithelial cells
o Sometimes called magnesium
phosphates
o The rosette forms may be confused with
sulfonamide crystals when the urine pH
is in the neutral range.
o Calcium phosphate crystals dissolve in
dilute acetic acid and sulfonamides do
not
o present in urine as dibasic calcium
phosphate (i.e., CaHPO4, calcium
monohydrogen phosphate) sometimes
called stellar phosphates
(hydroxyapatite)
o monobasic calcium phosphate (i.e.,
Ca[H2PO4]2, calcium biphosphate)
(Brushite)
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

➢ Calcium oxalate
o Colorless, mostly envelope (dihydrate),
or dumbbell-shape (monohydrate)
o Soluble in HCI but insoluble in HAC.
o derived from ascorbic acid (vitamin C),
an oxalate precursor or from oxalic acid.
o Foods notably the spinach, rhubarb,
berries and tomatoes
o Possibility of calculus
o Increased numbers of calcium oxalate
crystals are often observed following
ingestion of the oxalate precursor
ethylene glycol (antifreeze) and during
severe chronic renal disease.
o can resemble RBCs and may require
differentiation by polarizing microscopy

➢ Triple phosphates crystals or

Ammonium-Magnesium phosphates crystals
o NH4MgPO4
o A.k.a. Struvite
o Colorless modified prisms with oblique
ends
o Feathery or leaf-like forms
o Coffin-lid form
o Suggest stasis of urine in the kidney or
bladder or form calculi

URINARY SEDIMENT ARTIFACTS

➢ Starch granules
o Spheres with dimpled center
o “Maltese cross” formation on polarizing
microscope
➢ Others: Oil droplets, Air bubbles, Pollen grains,
Hair and fibers, Fecal contamination
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

PHENYLKETONURIA
➢ Overflow type: Result from the disruption of a - Presence of abnormal phenylalanine
normal metabolic pathway that causes increased metabolites in the urine
plasma concentrations of the non-metabolized - most well-known of the aminoacidurias
substances. - if undetected, results in Phenylpyruvic
➢ Renal type: caused by malfunctions in the oligophrenia
tubular reabsorption mechanism - Peculiar mousy odor of urine
- Ivan Følling in 1934 (Norway)
- Enzyme deficient: Phenylalanine hydroxylase
- Disruption of enzyme function - Ferric chloride tube test is commonly used as a
- failure to inherit the gene to produce an enzyme screening test for most amino aciduria
- lifetime medication (supportive treatment)
Tests for Phenylketonuria
Abnormal Metabolic Constituents or Conditions Test Comment Result
Detected in the Routine Urinalysis Phenylalanine • Increased 2-6 weeks normal results is
Color Odor Crystal Blood Level prior to the urinary lowered from 4
• Homogentesic • Phenylketonuria • Cystine excretion of mg/dL to 2 mg/dL
acid • Maple syrup • Leucine phenylpyruvic acid
• Melanin urine disease • Tyrosine • Detected as early as 4
• Indican • Isovaleric • Lesch-Nyhan hours after birth
• Porphyrins acidemia disease Phenylpyruvic • based upon the ferric permanent
• Cystinuria acid Urine Test chloride reaction bluegreen color
• Cystinosis performed by tube
• Homocystinuria test. (non-specific test)

- Ferric Chloride Tube Test:

Major Disorders of Protein and Carbohydrate
o Commonly used as a screening test for
Metabolism Associated with Abnormal Urinary
most aminoaciduria
Constituents Classified as to Functional Defect
o (+) result: stable green/blue green
Overflow Inherited Metabolic Renal
• Phenylketonuria • Infantile • Hartnup
- Reagent strip: phenistix strip contains ferric
• Tyrosinemia tyrosinemia disease ammonium sulfate, magnesium sulfate, and
• Alkaptonuria • Melanuria • Cystinuria cyclohexylsulfamic acid
• Maple syrup urine • Indicanuria - Guthrie (Bacterial Inhibition) Test: beta-2-
disease • 5- thienylalanine (inhibits the growth of the
• Organic acidemias Hydroxyindoleaceti
bacteria)
• Cystinosis c acid
• Porphyria • Porphyria o Media: Demain’s medium
• Mucopolysaccharid o Bacteria: Bacillus subtilis
oses o (+) result: growth of Bacillus subtilis
• Galactosemia
• Lesch-Nyhan
disease TYROSYLURIA/TYROSINEMIA
- Accumulation of excess tyrosine in the plasma
(tyrosinemia) producing urinary overflow
Amino Acid Disorders with Urinary Screening Tests - Inherited defect (absence of gene for the coding
• Phenylketonuria (PKU) of enzyme) or metabolic defect (accumulation
• Tyrosinemia because of liver disease)
• Alkaptonuria - Urine metabolites:
• Melanuria
o p-hydroxyphenylpyruvic acid
• Maple syrup urine disease
• Organic acidemias o p-hydroxyphenyllactic acid
• Indicanuria - urine may contain excess tyrosine
• Cystinuria - transitory tyrosinemia
• Cystinosis o most common
o seen in premature infants
Phenylalanine-Tyrosine Pathway o underdevelopment of the liver function
to produce enzyme for tyrosine
metabolism

Types of Tyrosyluria
Type Enzyme Deficiency Comment
Type 1 Fumarylacetoacetat Produces a generalized renal
e hydrolase (FAH) tubular disorder and
progressive liver failure in
infants soon after birth
Type 2 tyrosine Persons develop corneal
(most aminotransferase erosion and lesions on the
common) palms, fingers, and soles of
the feet believed to be
caused by crystallization of
tyrosine in the cells
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Type 3 p- result in mental retardation if MAPLE SYRUP URINE DISEASE

hydroxyphenylpyru dietary restrictions of
vic acid oxidase phenylalanine and tyrosine - amino acids involved are leucine, isoleucine, and
are not implemented valine
- Failure to inherit the gene for the enzyme
Tests for Tyrosyluria necessary to produce oxidative decarboxylation
Test Comment Result of keto acids results in their accumulation in the
nitrosonaphthol • Nonspecific, reacts with Orange- blood and urine
test (urinary compounds other than red color o α-ketoisovaleric
screening test) tyrosine and its metabolites.
o α-ketoisocaproic
Reagent: o α-keto-β-methylvaleric
• 2.63N nitric acid - strong odor of urine resembling maple syrup,
• 21.5% sodium nitrite
caramelized sugar, and curry odor
• 1-nitroso-2-napthol
Tandem mass • Used to confirm
spectrometry nitrosonaphthol test Tests for MSUD
(MS/MS) • Can confirm most of the Test Comment Result
inborn-error metabolism 2,4- • urine screening test most Yellow
dinitrophenylhy frequently performed for turbidity or
drazine (DNPH) keto acids ppt
MELANURIA reaction
- Increased urinary melanin → darkening of urine Interference:
• Large doses of ampicillin
after exposure to air
• a positive reagent strip test
- Malignant melanoma: over proliferation of result for ketones produce a
melanocytes positive DNPH result
- Albinism: absence of melanin
- 2 Metabolic pathways of converting tyrosine into Organic Acidemias
melanin: Point of Isovaleric Propionic Methylmalo
o Eumelanin Pathway: from brown to Reference Acidemia Acidemia nic Acidemia
black Characterist Sweaty feet No changes in No changes in
o Pheomelanin: from yellow to red ic odor odor of urine urine odor urine odor
Special Accumulation of immediate
characterist isovalerylglycine precursor to
Tests for Melanuria ic methylmaloni
Test Comment Result c acid
Ferric Chloride • Oxidation of chromogen Gray or Cause isovaleryl errors in the metabolic
Tube Test • Screening test black ppt coenzyme A pathway converting isoleucine,
sodium • Screening test Red color def. in the valine, threonine, and
nitroprusside leucine methionine to succinyl
Interference: add glacial Hac Purple pathway. coenzyme A
test
(reverts color greenish-black) Amber Test MS/MS p-nitroaniline
• Acetone method urine Test
• Creatinine (emerald
Tandem mass • Used to confirm the green)
spectrometry presence of melanin
(MS/MS)
TRYPTOPHAN DISORDERS
ALKAPTONURIA (ALKALI LOVER)
- One of the six original inborn errors of
metabolism described by Garrod in 1902
- Darkened urine after becoming alkaline from
standing at room temperature.
- occurs from failure to inherit the gene to
produce the enzyme homogentisic acid oxidase
- brown-stained or black-stained cloth diapers and
reddish-stained disposable diapers
- cola-colored sample

Tests for Alkaptonuria

Test Comment Result
FeCl3 Test • Screening test for transient deep
metabolic disorder blue color in test
tube - tryptophan is metabolized in the gut or intestine
Clinitest • Test for reducing Yellow ppt - it will be converted to indole by the normal flora
substance present, which will then be excreted in the feces
urinary • Screening test observe for in the form of skatole
homogentisic • add alkali to freshly darkening of the
acid Test color
- Serotonin is water insoluble, so it will not be
voided urine
(Ascorbic Acid evident in the urine.
interferes the o Serotonin is present in the urine in the
test) form of its metabolite, 5-
Ammoniacal • Addition of silver Black Urine
hydroxyindoleacetic acid (5-HIAA)
Silver Nitrate nitrate and ammonium
Test hydroxide to urine
• Screening test
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Indicanuria Mucopolysaccharide (glycosaminoglycans)

- increased amounts of tryptophan are converted Disorders
to indole (Hartnup disease) Point of Hurler Hunter Sanfilippo
- Excess indole is reabsorbed in the intestine → Reference syndrome syndrome syndrome
Characteristic skeletal structure is Mental
circulated to liver → converted to indican →
Abnormality abnormal and there is retardation
excreted in urine severe mental retardation
- Colorless when oxidized turns indigo blue
mucopolysaccharides
- Blue diaper syndrome accumulate in the cornea of
- FeCl3 Test → deep blue/violet color → the eye.
chloroform extraction Treatment Bone marrow transplants and gene replacement
therapy
5-Hydroxyindoleacetic Acid Test • acid-albumin (turbidity after 30 mins)
o (+) white ppt
- Serotonin: stimulation of smooth muscle • cetyltrimethylammonium bromide (CTAB)
o produced from tryptophan by the turbidity tests (turbidity after 5 mins)
argentaffin cells in the intestine o (+) white ppt
o argentaffinoma: over proliferation of • Metachromatic staining (blue spot)
argentaffin cells
- 5-HIAA: degradation product (urine metabolite) Purine Disorder (Lesch-Nyhan Disease)
- Foods rich in serotonin: bananas, pineapples, and Cause Characteristics Test
tomatoes Increased level of • severe motor Microscopic →
uric acid in the defects presence of
body • mental retardation increased uric
Tests for 5-Hydroxyindoleacetic Acid • tendency toward acid crystals in
Failure to inherit
Test Comment Result self-destruction pediatric urine
the gene to
Nitroso- • Normal daily excretion: 2- Purple to • Gout specimens
produce the
naphthol Test 8mg black color
enzyme • renal calculi
• argentaffin cell tumors: (depending
hypoxanthine • Presence of uric
Reagent:
>25mg/24h on 5-HIAA acid crystals
nitrous acid 1- guanine
level) resembling orange
nitroso-2- Requirement: phosphoribosyl-
transferase (HGPRT sand in diapers
naphthol • 24h sample
• Preservative: HCl or Boric enzyme)
Acid
• Strict diet Carbohydrate Disorder
• Hold medication
Melituria Comment Test
(phenothiazines and
pentosuria one of Garrod’s original six Test for
acetanilids) for 72h prior to
IEMs reducing
sample collection
galactosuria deficiency in any of three substance
(inability to enzymes, galactose-1- (Clinitest),
Cystine Disorders properly phosphate uridyl transferase except for
Point of Cystinuria Cystinosis metabolize (GALT), galactokinase and UDP- sucrose
Homocystinuria galactose to galactose-4-epimerase
Reference (Renal) (Overlow)
Characterist elevated a.a. crystalline increase in glucose)
ic cystine in deposits of homocystine Lactosuria seen during pregnancy and
urine cystine in throughout the lactation
(+) cystine many areas body. Fructosuria parenteral feeding and
crystals in of the body ingestion of large amounts of
urine fruit
Cause inability of the defect in Defect in
renal tubules the methionine
to reabsorb lysosomal metabolism
cystine membranes FOUR TYPES RENAL DISEASE
filtered by the
glomerulus. • Glomerular (immune mediated)
Test & Cyanide- Positive test (+) cyanide- • Tubular (infection)
Result nitroprusside results for nitroprusside test • Interstitial (infection)
Test (red- reducing plus • Vascular (reduction in the renal perfusion )
purple color) substances (+)
silvernitroprussid
e test CLASSIFICATION OF RENAL DISEASE
➢ Glomerular Disorders – disorders associated
Porphyrin disorder with the glomerulus
Disorder Comment Test o immunologic disorders
Porphyria Collective term for o sometimes, metabolic and hereditary
disorders of porphyrin
metabolism
disorders could also induce damage to
the glomerulus
Problem with the heme
o glomerular disorders are collectively
synthesis
Porphyrinuria observation of a red or Ehrlich reaction called as glomerulonephritides
port wine color to the (Watson-Schwartz) ➢ Tubular Disorders – affecting the renal tubules
urine after exposure to and fluorescence include those in which tubular function is
air Test disrupted as a result of actual damage to the
tubules and those in which a metabolic or
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
hereditary disorder affects the intricate
functions of the tubules.
o It may result from primary renal disease
or may induce secondarily
o Functions affected: Reabsorption and
secretion
➢ Hereditary and Metabolic Tubular Disorders –
Disorders affecting tubular function may be
caused by systemic conditions that affect or
override the tubular reabsorptive maximum
(Tm) for particular substances normally
reabsorbed by the tubules or by failure to inherit
a gene or genes required for tubular
reabsorption.
➢ Interstitial Disorders – disorders affecting the
interstitium also affect the tubules
(tubulointerstitial disease) due to close
proximity.
o Cause: pathogenic mechanisms such as
infection and allergic action
➢ Renal Failure – exists in both acute and chronic
forms
o Acute renal failure: sudden decrease in
the glomerular filtration rate and is
associated with azotemia (increased
BUN and creatinine level in the blood)
and oliguria. Nephron is morphologically
normal, but its function is abnormal.
o Chronic renal failure: slow and silent
decrease in the glomerular filtration rate
(<25mL/min); destroyed nephrons
under the microscope. It is associated
with progressive loss of renal function.
Decreased GFR is clinically recognizable
only after 80-85% of the renal function
has been loss.
▪ Electrolyte imbalance
▪ Decreased renal concentrating
ability (isosthenuria)
▪ Multiple formed elements are
seen
▪ Increased cells and casts
▪ Telescope specimen
➢ Renal Lithiasis – Renal calculi (kidney stones)
o Lithotripsy: non-surgical procedure
using high-energy shock waves, used to
break stones located in the upper
urinary tract into pieces that can then be
passed in the urine
▪ Disadvantage: not all kidney
stones are excreted
o Conditions in favor for the formation of
renal calculi:
▪ pH
▪ chemical concentrations (70%
contains calcium)
▪ urinary stasis
o examples of renal calculi:
▪ calcium oxalate
▪ triple phosphate (ammonium-
magnesium phosphate)
▪ uric acid
▪ phosphate crystals
SUMMARY OF GLOMERULAR DISORDERS
Acute glomerulonephritis
✓ Primary Urinalysis Result:
o Macroscopic hematuria
o Proteinuria
o RBC casts
o Granular casts
✓ Other significant tests:
o Antistreptolysin O titer
o Anti–group A streptococcal enzymes
✓ Etiology: Deposition of immune complexes,
formed in conjunction with group A
Streptococcus infection, on the glomerular
membranes
✓ Clinical Course:
o Rapid onset of hematuria and edema
o Permanent renal damage seldom occurs
o Occurs 1-2 weeks after the infection
Rapidly progressive glomerulonephritis
✓ Primary Urinalysis Result:
o Macroscopic hematuria
o Proteinuria
o RBC casts
✓ Other significant tests:
o BUN Creatinine
o Creatinine clearance
✓ Etiology: Deposition of immune complexes from
systemic immune disorders on the glomerular
membrane
✓ Clinical Course:
o Rapid onset with glomerular damage
and possible progression to endstage
renal failure
o Presence of antinuclear antibody (ANA)
in patients with systemic lupus
erythematosus (SLE)
o There is leukocytic infiltration because
there is a presence of antinuclear
antibody
Goodpasture syndrome
✓ Primary Urinalysis Result:
o Macroscopic hematuria
o Proteinuria
o RBC casts
✓ Other significant tests: Antiglomerular
basement membrane antibody
✓ Etiology: Attachment of a cytotoxic antibody
formed during viral respiratory infections to
glomerular and alveolar basement membranes
✓ Clinical Course: Hemoptysis and dyspnea
followed by hematuria Possible progression to
end-stage renal failure
Wegener’s granulomatosis
✓ Primary Urinalysis Result:
o Macroscopic hematuria
o Proteinuria
o RBC casts
✓ Other significant tests: Antineutrophilic
cytoplasmic antibody
✓ Etiology: Antineutrophilic cytoplasmic auto-
antibody (ANCA) binds to neutrophils in vascular
walls producing damage to small vessels in the
lungs and glomerulus
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
✓ Clinical Course: Pulmonary symptoms including
hemoptysis develop first followed by renal
involvement and possible progression to end-
stage renal failure
o FANA testing is used to differentiate
Wegener’s granulomatosis from
Goodpasture syndrome
OTHER GLOMERULAR DISORDERS
➢ Henoch-Schönlein purpura – occurs primarily in
children after upper viral respiratory infections.
o Disrupts vascular integrity because of
the decreased platelet count
o initial symptoms include the appearance
of raised, red patches on the skin
➢ Membranous glomerulonephritis – thickening
of the glomerular basement membrane resulting
from the deposition of immunoglobulin G
immune complexes.
➢ Membranoproliferative glomerulonephritis –
marked by two different alterations in the
cellularity of the glomerulus and peripheral
capillaries.
o Type 1 displays increased cellularity in
the subendothelial cells of the
mesangium, causing thickening of the
capillary walls
o Type 2 displays extremely dense
deposits in the glomerular basement
membrane.
➢ Chronic glomerulonephritis – an irreversible
condition because there is a marked decreased
in the renal function
o May be associated with renal failure
➢ IgA nephropathy – there is a deposition of IgA on
the glomerulus
o it is the most common cause of
glomerulonephritis.
o There are IgA complexes or aggregates
that is being trapped and engulfed by
the mesangial cells
➢ Nephrotic syndrome – Increased permeability of
the glomerular membrane is attributed to
damage to the shield of negativity and the
podocytes that produces a less tightly connected
barrier.
o The patient has albuminuria, lipiduria,
and edema
o Has lower oncotic pressure in the
capillaries resulting from the depletion
of plasma albumin increases fluid loss
into the interstitial spaces, which,
accompanied by sodium retention,
produces the edema.
➢ Minimal change disease (lipid nephrosis) –
produces little cellular change in the glomerulus
because of the disruption of podocyte
o Patients are usually children after an
allergic reaction or immunization
➢ Focal segmental glomerulosclerosis – there is a
disruption of podocytes.
o The affected area is localized only to a
particular area in the glomerulus
➢ Alport Syndrome – a genetic disorder of collagen
production affecting the glomerular basement
membrane.
o
o The glomerular basement membrane
has a lamellated appearance with areas
of thinning.
o Can cause presence of RBCs and albumin
SUMMARY OF METABOLIC AND
TUBULAR DISORDERS
Acute Tubular Necrosis
✓ Primary Urinalysis Result:
o Microscopic hematuria
o Proteinuria
o Renal tubular epithelial cells
o RTE cell casts
o Hyaline, granular, waxy, broad casts
✓ Other significant tests:
o Hemoglobin
o Hematocrit
o Cardiac enzymes
✓ Etiology: Damage to the renal tubular cells
caused by ischemia or toxic agents
o Ischemia: there are changes in the renal
tubule because of low oxygen level that
may cause shock
o Toxic agents: exposed to nephrotoxins
(antibiotics or certain chemicals can also
be toxic such as vancomycin)
✓ Clinical Course: Acute onset of renal dysfunction
usually resolved when the underlying cause is
corrected
o The patient has odorless urine
Fanconi Syndrome
✓ Primary Urinalysis Result:
o Glucosuria
o Possible cystine crystals
✓ Other significant tests:
o Serum and urine electrolytes
o Amino acid chromatography
✓ Etiology: Inherited in association with cystinosis
and Hartnup disease or acquired through
exposure to toxic agents
✓ Clinical Course: Generalized defect in renal
tubular reabsorption requiring supportive
therapy
o Characterize any conditions that
presents with generalized loss of
proximal tubular function (absorption
and secretion)
Nephrogenic diabetes insipidus
✓ Primary Urinalysis Result:
o Low specific gravity
o Polyuria
✓ Other significant tests: ADH testing
✓ Etiology: Inherited defect of tubular response to
ADH or acquired from medications
o Hypothalamic: complete deficiency of
ADH
o Nephrogenic: ADH is present, but the
aquaporin 2 in the collecting duct are not
responsive to ADH stimulation
✓ Clinical Course: Requires supportive therapy to
prevent dehydration
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Renal glucosuria Chronic Pyelonephritis

✓ Primary Urinalysis Result: Glucosuria ✓ Primary Urinalysis Result:
✓ Other significant tests: Blood glucose o Leukocyturia
✓ Etiology: Inherited autosomal recessive trait o Bacteriuria
o Failure to produce enzyme necessary for o WBC casts
the reabsorption of glucose in the o Bacterial casts
proximal convoluted tubule o Granular, waxy, broad casts
✓ Clinical Course: Benign disorder o Hematuria
o Proteinuria
SUMMARY OF INTERSTITIAL DISORDERS ✓ Other significant tests:
- Interstitial disorders are usually caused by o Urine culture
urinary tract infections (can involve upper and o Blood cultures
lower urinary tract) o BUN
o Lower UTI: involves the urethra o Creatinine
(urethritis) and urinary bladder (cystitis) o Creatinine clearance
o Upper UTI: ✓ Etiology: Recurrent infection of the renal tubules
▪ Acute pyelonephritis: Left and interstitium caused by structural
untreated cystitis abnormalities affecting the flow of urine
▪ Pyelitis: renal pelvis infection o Associated with continuous scarring or
▪ Pyelonephritis: infection in the fibrosis
kidneys o Renal calyces are dilate and deformed
✓ Clinical Course: Frequently diagnosed in
Cystitis
children; requires correction of the underlying
✓ Primary Urinalysis Result: structural defect Possible progression to renal
o Leukocyturia failure
o Bacteriuria
o Microscopic hematuria
Acute Interstitial Nephritis
o Mild proteinuria
o Increased pH ✓ Primary Urinalysis Result:
✓ Other significant tests: Urine culture o Hematuria
✓ Etiology: Ascending bacterial infection of the o Proteinuria
bladder o Leukocyturia
o Common in females (10x more often o WBC casts
than males) because they have shorter ✓ Other significant tests:
urethra and has close proximity of o Urine eosinophils
vagina to rectum o BUN
o Can also be caused by high hormones, o Creatinine
which can enhance the bacterial o Creatinine clearance
adherence to mucosa ✓ Etiology: Allergic inflammation of the renal
o 2 mechanisms of kidney infection: interstitium in response to certain medications
▪ movement of bacteria from the o acute allograph rejection
lower urinary tract to kidneys o Medications: penicillin, methicillin,
▪ hematogenous infection ampicillin, cephalosporins, NSAIDs,
(bacteria from the blood) sulfonamides, thiazide diuretics, and
o transitional epithelial cells are present non-steroidal drugs such as ibuprofen
✓ Clinical Course: Acute onset of urinary frequency ✓ Clinical Course: Acute onset of renal dysfunction
and burning resolved with antibiotics often accompanied by a skin rash Resolves
following discontinuation of medication and
treatment with corticosteroids
Acute Pyelonephritis
✓ Primary Urinalysis Result:
o Leukocyturia
o Bacteriuria
o WBC casts
o Bacterial casts
o Microscopic hematuria
o Proteinuria
✓ Other significant tests:
o Urine culture
o Blood cultures
✓ Etiology: Infection of the renal tubules and
interstitium related to interference of urine flow
to the bladder, reflux of urine from the bladder,
and untreated cystitis
o Increased renal tubular cells and casts
✓ Clinical Course: Acute onset of urinary
frequency, burning, and lower back pain
resolved with antibiotics
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Clear, colorless body fluid found within the tissue

that surrounds the brain and spinal cord;
Replaces the body fluid found outside the cells of
bilateral animals
- 3rd major body fluid

• Supply nutrients to the nervous system

• Remove metabolic wastes
• Produce a mechanical barrier to cushion the
brain and spinal cord against trauma

- CSF provides a physiologic system to supply

nutrients to the nervous tissue, remove
metabolic wastes, and produce a mechanical BLOOD BRAIN BARRIER
barrier to cushion the brain and spinal cord - Tight-fitting structure of the endothelial cells in
against trauma the choroid plexuses that prevents molecule
- It occupies the subarachnoid space between the passage
arachnoid and pia mater - Protect the brain from chemicals and other
substances circulating in the blood that could
MENINGES harm the brain tissue.
- Lines the brain and spinal cord o Also prevent the passage of helpful
- Provides supportive framework for the cerebral substances including Ig and medications
cranial vasculature - Disruption of the blood-brain barrier by diseases
such as meningitis and multiple sclerosis allows
Three Layers WBC, Proteins, and additional chemicals to enter
➢ Dura Mater – outer layer that lines the skull and the CSF.
vertebral canal - CSF is considered as ultrafiltrate of plasma
o Latin word: hard mother
➢ Arachnoid Mater – spider web-like which lines
the inner membrane - Lumbar puncture or spinal tap between the
➢ Pia Mater – thin membrane lining the surfaces of third, fourth, or fifth lumbar vertebra
the brain and spinal cord - Precautions:
o Latin word: gentle mother o measurement of intracranial pressure
▪ normal: 5 to 15 mmHg
▪ mild intracranial hypertension:
20 to 30 mmHg
o careful technique to prevent infection or
Neural Tissue damage
o Fluid collected: CSF (ultra filtrate)

CSF TUBES
• Tube 1: Chemistry/Serology (freezer temp.)
• Tube 2: Microbiology (room temp.)
• Tube 3: Hematology (refrigerated)
• Tube 4: Microbiology/Serology
CHOROID PLEXUSES
- Specialized ependymal cells in the area produces Note:
CSF If single tube is collected, the specimen should be send
- rate of CSF production: 20mL per hour first at the microbiology section, followed by the
- CSF total Volume hematology section, and last for chemistry and serology
o Adults: 90-150 mL section
o Neonates: 10-60 mL
- To maintain CSF normal value, the fluid is
reabsorbed back into the capillaries in the
Arachnoid granulations/villae
- Production of CSF via selective filtration under
hydrostatic pressure and active transport
secretion
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

CSF APPEARANCE
APPEARANCE CLINICAL SIGNIFICANCE
Crystal Clear Normal
↑ WBC (>200 /uL)
Hazy/ Turbid/ ↑ RBC (>400 / uL)
Cloudy/ Milky ↑ Lipids and Proteins
(+) Microorganisms
Due to hemoglobin degradation product
Xanthochromic
• Pink: Slight amount of oxyhemoglobin
(Pink/Yellow/
• Yellow: Oxyhemoglobin → Bilirubin
Orange)
• Orange: Heavy Hemolysis
Oily Radiographic Contrast Media
Protein and clotting factors
Clotted
Disrupted blood brain barrier
Pellicle Tubercular meningitis
↑ RBC (>6,000/uL)
Traumatic Tap (puncture of blood vessel)
Bloody
Intracranial Hemorrhage (bleeding within the
braincase)

TRAUMATIC TAP VS. INTRACRANIAL HEMORRHAGE

Traumatic Tap Intracranial Hemorrhage
Distribution of
Uneven Even
blood in 3 tubes
Clot Formation + (fibrinogen) –
Supernatant Clear Xanthochromic
Erythrophages – + PREDOMINANT CELLS IN THE CSF
- Predominant: Lymphocytes and Monocytes
- Occasional: Neutrophils
- Any cell count should performed Immediately o Lymphocytes-monocytes ratio in adults
- WBC and RBC begin to lysed within 1 hour (70:30)
- 40% of WBC begin to lysed within 2 hours o Neonates (inversed ratio)
- Formula: ▪ Up to 80% monocytes is
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 considered normal
𝑾𝑩𝑪 𝒄𝒐𝒖𝒏𝒕/𝒖𝑳 =
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑒𝑝𝑡ℎ - Pleocytosis: Increased number of normal cells in
the CSF
WBC COUNT o Increased white blood cell in the CSF
- Routinely performed
- Normal Values: OTHER CELLS
o Adults: 0-5 WBC/uL ➢ Eosinophil – parasitic and fungal (Coccidioides
o Neonates: 0-30 WBC/uL immitis)
- Diluting fluid: 3% acetic acid with methylene ➢ Macrophages
blue o remove cellular debris and foreign
CSF DILUTION objects such as RBC
Appearance Dilution o Appear within 2-4 hours after RBC enters
Appearance Dilution the CSF
Clear Undiluted o Indicates previous hemorrhage
Slightly Hazy 1:10
o Degradation of RBC- appearance of dark
Hazy 1:20
Slightly Cloudy 1:100 blue or black iron hemosiderin granules
Cloudy/ Slightly bloody 1:200 o Further degradation= Yellow
Bloody or Turbid 1:10,000 hematoidin- iron free, with hemoglobin
and unconjugated bilirubin
RBC COUNT
- Done only in cases of traumatic tap NON-PATHOLOGICALLY SIGNIFICANT CELLS
- To correct for WBC Count and total protein - Seen after pneumoencephalography and
concentration neurosurgery
o -1 WBC for every 700 RBC seen - Uniform in appearance
o -8 mg/dL Total protein for every 10,000 - Choroidal cells: linings of choroid plexus
RBCs/uL - Ependymal cells: linings of ventricles and neural
o -1 mg/dL Total protein for every 1,200 canal
RBCs/uL o It produces CSF
- Spindle-shaped cells: linings of arachnoid

- Performed on stained smear Malignant Cells of Hematologic Origin

- Specimen should be concentrated before - Lymphoblast, myeloblasts, and monoblasts
smearing by using the following methods: - Found in acute leukemias
o Cytocentrifugation (most common) - Lymphoma cells: indicates dissemination of
o Centrifugation lymphoid tissues
o Sedimentation
o Filtration
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Malignant cells of Nonhematologic Origin o Normal Value: <9

- Metastatic Carcinoma Cells: primarily from o Abnormal: >9
lungs, breast, renal, and gastrointestinal ▪ Correlates with the degree of
malignancies the damage
- Usually seen in clusters and have tendency to ▪ Index of 100 = complete damage
fuse the cell to BBB
- Cells from CNS tumors: ➢ IgG Index
o Astrocytomas o Assess the condition of IgG production
o Retinoblastomas within the CNS (e.g. Multiple Sclerosis)
o Medulloblastomas
𝐶𝑆𝐹 𝐼𝑔𝐺 (𝑚𝑔/𝑑𝐿) ÷ 𝑠𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 (𝑔/𝑑𝐿)
𝑰𝒈𝑮 𝒊𝒏𝒅𝒆𝒙 =
𝐶𝑆𝐹/𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝐼𝑛𝑑𝑒𝑥

o Normal Value: <0.77

o Abnormal: >0.77
▪ Indicative of IgG production
within the CNS
▪ Possible that the patient has
multiple sclerosis

CSF ELECTROPHORESIS
- Done in conjunction with serum electrophoresis
- For the detection of the oligoclonal bands
(Gamma Region)
- The presence of two or more oligoclonal bands
in the CSF but not in serum is valuable for the
diagnosis of Multiple Sclerosis
CSF PROTEIN - Other conditions:
CSF PROTEIN o Encephalitis
• Adult: 15-45 mg/dl
o Neurosyphilis
Normal Values • Infants: 150 mg/dl
• Immature: 500 mg/dl
o Guillain-Barre Syndrome
Damage to BBB (most common) o neoplastic syndrome
• Meningitis
Increased in • Hemorrhage Detection of TAU Protein
Production in immunoglobulins within the
CNS → Multiple Sclerosis - Isoelectric Focusing and Immunofixation test are
Decreased in CSF Leakage used to check the presence of TAU protein
Major CSF Protein Albumin - Normal: Low protein levels in the CSF
2nd Most Prevalent Prealbumin - Abnormal: High protein levels in the CSF
Protein - Method of choice when determining whether a
Alpha globulins Haptoglobin and ceruloplasmin
fluid is actually CSF
Beta transferrin (TAU Protein)
Beta-globulins • Carbohydrate-deficient transferrin
• Found in CSF but not in Serum
Gamma globulins IgG and some IgA (monomer)
• IgM
Not found in
• Fibrinogen
normal CSF
• B-Lipoprotein

Total Protein
Multiple Sclerosis
➢ Turbidimetric
Trichloroacetic acid Sulfosalicylic Acid Method - Autoimmune disorder
- Preferred method - Precipitates albumin - Most common demyelinating disease of the CNS
- Precipitates both - To precipitate globulin, add - The body produces immunoglobulin G to attack
albumin and globulins sodium sulfate the cells in the myelin sheath
➢ Dye Binding using Coomasie Brilliant Blue - Findings:
o Protein binds to dye → dye turns from o (+) Anti-myelin sheath autoantibody
red to blue o (+) Oligoclonal band in the CSF but not in
o Increased protein = increased blue color serum
▪ Short and less intense bands
Protein Fractions o (+) Myelin Basic Protein CSF
➢ CSF/Serum Albumin Index o Increased IgG
o Assess the integrity of the blood brain - Myelin Basic Protein
barrier o Protein Component of the lipid-protein
complex that insulate the nerve fibers
𝐶𝑆𝐹 𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 (𝑚𝑔/𝑑𝐿) o Presence of MBP in the CSF indicates
𝑪𝑺𝑭/𝑺𝒆𝒓𝒖𝒎
= destruction of Myelin Sheath
𝑨𝒍𝒃𝒖𝒎𝒊𝒏 𝑰𝒏𝒅𝒆𝒙 𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿)
o Used to monitor the course of Multiple
Sclerosis
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

▪
Neurological abnormalities: LD 2
>1
▪ Bacterial Meningitis: LD 5 > 4 > 3
>2>1
• LD 4 and 5 are seen in
neutrophils
➢ Creatine Kinase (CK) – Increase in stroke, MS,
Degenerative disorders, Brain tumors, Viral and
Bacterial meningitis, and seizures
➢ Aspartate Amonitransferase (AST) – Increase in
intracerebral and subarachnoid hemorrhage and
CSF GLUCOSE
bacterial meningitis
- Glucose is normally seen in the CSF because it o Not common in the CSF
serves as a fuel or the energy source of the brain
- Done in the conjunction with Blood Glucose
- Specimen for blood glucose should be drawn 2
hours prior to spinal tap - Identify the causative agent in Meningitis
- Normal values: - Confirmatory: 24 hours to 6 weeks
o 60-70% of blood glucose - Preliminary Diagnosis: Gram staining –
o 50-80 mg/dL CSF glucose organisms most frequently encountered include
- Increased: due to increased plasma glucose o Streptococcus pneumoniae (gram-
- Decreased: positive cocci)
o Bacterial meningitis o Haemophilus influenzae (pleomorphic
o Tubercular meningitis gram-negative rods)
o Fungal meningitis o Escherichia coli (gram-negative rods)
- Normal: Viral Meningitis o Neisseria meningitidis (gram-negative
cocci)
o Streptococcus agalactiae
CSF LACTATE
o Listeria monocytogenes may be
- There will be an increased lactic acid due to the encountered in newborns
tissue destruction within the CNS, causing tissue
hypoxia, which results to oxygen deprivation. Major Laboratory Results for the
- Lactic acid (lactate): end product of glycolysis Differential Diagnosis of Meningitis
- Normal values: 10-22 mg/dl Bacterial Viral Tubercular Fungal
Predominant Lymphocytes Lymphocytes
- Increased: WBC
Neutrophil Lymphocytes
Monocytes Monocytes
o Bacterial meningitis Protein
Glucose
↑
↓
↑
N
↑
↓
↑
↓
o Tubercular meningitis Lactate ↑ N ↑ ↑
(+) Gram stain Agents: Agent: MTB Agent: C.
o Fungal meningitis (+) Culture Enteroviruses (+) AFB neoformans
(+) Limulus • Poliovirus (+) Pellicle or (+) Gram stain
- Normal: Other Info Lysate test • Echovirus web-like Starburst pattern
o Viral meningitis • Coxsackievirus (+) India ink
(+) Immunologic
o offering a sensitive method for test

evaluating the effectiveness of antibiotic

therapy Limulus Lysate Test
- Detects Gram negative bacterial endotoxin in the
CSF GLUTAMINE body fluids and surgical equipment
- Product of Ammonia and Alpha-Ketoglutarate - Reagent: Blood horse-shoe crab (hemolyn)
- It is an indirect test for the presence of excess - Principle:
ammonia in the CSF o In the presence of endotoxin, the
Amoebocytes (WBC) will release lysate
- Normal value: 8-18 mg/dL
- Increased in: (protein)
o Liver disorder that causes blood and CSF o Blue color
ammonia o (+) presence of clot/clumping and
o Disturbance of consciousness (comatose erythrophages
patient)
o Reye’s syndrome AGENTS OF BACTERIAL MENINGITIS
Age Group Causative Agent
Birth to 1 month old Streptococcus agalactiae
CSF ENZYMES 1 month to 5 yrs old Haemophilus influenzae
➢ Lactate Dehydrogenase (LDH): 5 – 29 years old,
Neisseria meningitis
immunocompromised
Isoenzymes: LD1, 2, 3, 4, 5 >29 years old Streptococcus pneumoniae
o Serum LDH Infants, elderly,
Listeria monocytogenes
▪ Normal: LD 2 > 1 > 3 > 4 > 5 immunocompromised
▪ Flipped Pattern: LD 1 > 2
• Associated with Acute
Myocardial Infarction • Latex agglutination test and ELISA: detection of
o CSF LDH bacterial antigens
▪ Normal: LD 1 > 2 > 3 > 4 > 5
• VDRL: recommended by CDC for the detection of
neurosyphilis
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

COMPOSITION CONTAINER
- Contains bacteria, cellulose and other - clean, dry, non-breakable container, leakproof,
undigested foodstuff, gastrointestinal and screw-capped
secretions, bile pigments, cells from intestinal - multiple-day collection: large containers
walls, electrolytes and water
- Escherichia coli: major normal flora that can be TYPE AND AMOUNT COLLECTED
found in the intestine - pea-sized: FOBT, WBCs, qualitative fecal fat
- Bacteroides fragilis: major anaerobic bacteria o at least 3 grams
present in the gastrointestinal tract - 2- to 3-day fecal collection: quantitative tests
o Helps in the metabolism of certain o it is used to know the exact amount of
substances, which can also be found in fat in the stool
feces
- Around 100-200 g of stool is passed per day
o >200g per day and more than 3 times of SPECIMEN PRESERVATION
defecation per day indicates diarrhea • Refrigeration
- Normal conditions: 500-1500mL reaches the • Freezing in dry ice
large intestine and 150mL is excreted in the feces • 10% Formalin (2% and 5% can also be used)
- Frequency of defecation: below 3 times a day • Alcohol
- Composition: ¾ water and ¼ solid • 20% glycerin in saline (Cumming Method)
o Water: about 60-80% of fecal volume • Methiolate-Iodine Formaldehyde (MIF) Solution
o Solid: o Can’t be used for trophozoites because
▪ 30%, bacteria mostly non- iodine is toxic for these parasites
pathogenic • Polyvinyl alcohol (PVA) Fixative
▪ 50-60%, remains on the
intestinal secretions (food
residues such as seeds, fruits
and vegetable skins, hairs,
fibers, vegetable cells and
muscle fibers
▪ 10-20% fat droplets and other
soluble substances
• Bacteria • Electrolytes
• Cellulose • Water
• undigested foodstuffs • Trypsin
• GI secretions • Chymotrypsin
• bile pigments • Amino peptidase
• Cells from the intestinal • Lipase
walls • Bile salts

- Odor foul to offensive due to:

o Skatole
o Indole
o Butyric acid
- pH: neutral / slightly alkaline / slightly acidic
o 7.0 to 8.0 pH level
o Acidic feces: carbohydrate fermentation
o Alkaline feces: protein fermentation
▪ Occurs in the distal colon
▪ It can also produce potentially
toxic products such as ammonia,
amines, phenols, or sulfides.
- Normal Consistency: Soft to well-formed,
depending on the dehydration of the patient
- Presence of Mucus: large amount of mucus
indicates irritable bowel disease BRISTOL STOOL CHART
o Trace amount in the large intestine to
absorb more water
o Abundant in the small intestine to
prevent infiltration of microorganisms
Reasons for Fecalysis
• Early detection of GI bleeding • Inflammation
(colorectal cancer) • Diarrhea
• Liver and biliary duct disorders • Steatorrhea
• Maldigestion and • Detection of pathogenic
malabsorption bacteria and parasites
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

MUSCLE FIBERS
- Creatorrhea: abnormal excretion of undigested
FATS muscle fibers in the feces
- Normal: - Determination:
o 5 grams/day (fatty diet) o The patient should include meat in the
o 1-4 grams/day (free fat diet) diet for at least 3 days
- Steatorrhea: >6 g/day o Emulsified stool + 10% eosin → coverslip
o increased fats in the stool and stand for 3 minutes → count the
o Deficiency in lipase in Fibrocystic Disease number of undigested fibers per HPF
of the Pancreas - Abnormal: 10 undigested muscle fiber
o Deficiency of bile salts in obstructive o Parameter:
jaundice ▪ Undigested: striations in two
o Lymphatic Obstruction (in abdominal different directions
TB) ▪ Digested: no striation present
- Test: ▪ Partially digested: striations in
o Screening Test: microscopic examination one directions
of free fat globules o Causes:
o Definitive: fecal fat determination ▪ biliary obstruction
▪ gastrocolic fistulas
Qualitative Test ▪ pancreatic insufficiency (cystic
➢ Neutral Fat Stain (Triglycerides) fibrosis)
o Caused by maldigestion
o Suspension + 95% ETOH + Sudan III
o Orange Droplets (Neutral Fats or
Triglycerides)
o Steatorrhea: ≥ 60 droplets/hpf
➢ Split Fat Stain
o Caused by malabsorption
o Emulsified stool + 36% Acetic acid +
Sudan III
o Orange Droplets (Fatty Acids)
FECAL LEUKOCYTES
o Normal: 100 droplets (< 4 um)
o Slightly increased: 100 droplets (1-8 um) - invasive condition: > 3 neutrophils/ HPF
o Increased: 100 droplets (6-75 um) - has increased daily stool rate
- frequent defecation
Quantitative Test
➢ Van de Kamer Titration
o Gold standard for fecal fat
o For definitive diagnosis of steatorrhea
o Sample: 3-day stool
o Titration with NaOH
o Normal value: 1-6 g fats/day
o Steatorrhea: > 6 g fats/day Determination
o Acid steatocrit 1. Wet Preparation: Stool + Loeffler’s Methylene
▪ Feces and HCl are used Blue
▪ rapid test to estimate the 2. Dried Preparation: Stool + Wright’s/ Gram stain
amount of fat excretion 3. Lactoferrin latex agglutination test:
▪ screen for steatorrhea in • Lactoferrin: secondary granules of
pediatric populations neutrophils
▪ capillary tube: • (+) invasive bacterial pathogen
• 1st layer: fecal solids
• 2nd layer: fecal fats
➢ Other Quantitative test - Stool weight of >200g/day with increased
o Gravimetric liquidity and frequency of more than 3x a day
o Near-infrared reflectance spectroscopy - Duration of Illness:
o Nuclear magnetic resonance o Acute: <4weeks
spectroscopy o Chronic: >4weeks
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Laboratory Tests: o time required for gastric emptying

• Fecal Electrolytes ▪ Halftime: 35 to 100 minutes
o Fecal Osmotic Gap o RGE can be caused by disturbances in
290 - [2 (fecal sodium + fecal potassium)] gastric reservoir or transporting function
• Fecal Osmolality (near 290mOsm/serum Osm) o It is the hallmark of early dumping
• Stool pH (acidic stool when caused by bacteria) syndrome (EDS)

Two Types of Dumping Syndrome

SECRETORY DIARRHEA
➢ Early Dumping Syndrome (EDS)
- increased secretion of water and electrolytes
o 10-30mins. after meal
which override reabsorptive capacity of large
o nausea, vomiting, bloating, cramping,
intestine
diarrhea, dizziness, fatigue
- Enterotoxin-producing organisms:
➢ Late Dumping Syndrome (LDS)
• Escherichia coli • Staphylococcus
• Clostridium • Campylobacter o 2-3hrs. after meal
• Vibrio cholera • Protozoa o weakness, sweating, dizziness,
• Salmonella • Cryptosporidium hypoglycemia
• Shigella

- Stimulant laxatives such as tea Causes of Dumping Syndrome

- Hormones • Gastrectomy
- Inflammatory bowel disease: • Gastric bypass surgery
o Crohn’s disease • Post-vagotomy status
o Ulcerative colitis
o Lymphocytic colitis
• Zollinger-Ellison syndrome
o Diverticulitis • Duodenal ulcer disease
- Endocrine disorders • Diabetes mellitus
o Hyperthyroidism
o Zollinger-Ellison syndrome (problem in producing SUMMARY OF THE CLASSIFICTION OF DIARRHEA
acidity of the stomach)
o Vipoma (pancreatic tumor)
- Neoplasm
- Collagen-vascular disorders

OSMOTIC DIARRHEA
- Incomplete breakdown/reabsorption of food
- Retention of water and electrolytes in large
intestine
- Excessively watery stool
- Only monosaccharides are reabsorbed in the
intestine
- Maldigestion: impaired digestion FECAL OCCULT BLOOD TEST (FOBT)
- Malabsorption: impaired absorption
- Occult (hidden)
- Presence of unabsorbable solute
- Screening test for GI bleeding and possible
- ↑ stool osmolality
colorectal cancer
- ↓ fecal electrolytes
- Significant: >2.5 mL blood/150g of stool
- Sample: center portion of the stool
Conditions Associated
- Principle: Pseudoperoxidase activity of
• Disaccharide deficiency (Lactose intolerance)
Hemoglobin
• Malabsorption (Celiac sprue)
• Poorly absorbed sugars (lactose, sorbitol, mannitol)
• Laxatives
• Magnesium-containing antacids
- Chromogens:
• Amoebiasis
• Antibiotic administration o Benzidine (most sensitive)
▪ contains benzene (carcinogenic)
o Guiac (preferred)
ALTERED MOTILITY
o O-toluidine
- Hypermotility or slow motility - Interference of FOBT:
- It depends on the peristalsis movement of o False (+) FOBT
intestine ▪ Dietary Pseudoperoxidases
- Most common cause: Irritable Bowel Syndrome ▪ Red Meat
– nerves and muscle of bowel are extra sensitive ▪ Melon, broccoli, cauliflower,
- Other symptoms: cramping, bloating, flatus, horseradish
diarrhea, and Constipation (triggered by food, ▪ Aspirin and other anti-
chemicals, emotional stress, and exercise) inflammatory drugs (avoid for 7
- Rapid Gastric emptying (RGE) Dumping days)
Syndrome – hypermotility of the stomach and o False (-) FOBT
the shortened gastric emptying half-time, which ▪ Reducing agents: Ascorbic acid
causes the small intestine to fill too quickly with and iron therapy (>250mg/day)
undigested food from the stomach
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

APT TEST (FETAL HEMOGLOBIN) - Check the amount of D-xylose in the blood and
- Also known as “APT Downey Test” urine
- Differentiates fetal blood and maternal blood o Low D-xylose level in the blood and urine
- Specimen: infant stool or vomitus indicates malabsorption
- Hgb F is alkali-resistant - sugar is not digested but has to be absorbed to
- Hgb A is denatured by alcohol be present in urine
- Procedure: - Determination:
o Emulsified stool (centrifuged) → Add 1% o there should be no food intake for 8 to
NaOH to supernatant 12 hours
o Pink solution: (+) fetal blood o after 8 to 12 hours, drink the D-xylose
o Yellow brown supernatant: (+) maternal solution
blood o measure the D-xylose
- Normal D-xylose: pancreatitis
- Low urine D-xylose: malabsorption
Procedures o Bacterial overgrowth
1. Emulsify specimen in water o Intestinal resection
2. Centrifuge o Celiac disease
3. Divide pink supernatant into two tubes o Tropical sprue
o Lymphoma
4. Add 1% sodium hydroxide to one tube o Whipple disease
5. Wait 2 mins o G. lamblia infestation
6. Compare color with that in the control tube o Crohn’s disease
7. Prepare controls using cord blood and adult o Intestinal ischemia
blood
TESTS, MATERIALS, AND INSTRUMENTATION
FOR FECAL FAT ANALYSIS
PROCEDURE MATERIALS, INSTRUMENTATION
Proteolytic enzymes Sudan III Sudan stain, microscopy
Enzymes used for protein breakdown Steatocrit and Acid Hct centrifuge, gravimetric assay
Steatocrit
➢ Trypsin – Protein digesting enzyme Fecal Elastase I Immunoassay ELISA technique
o X-ray film test Near-Infrared NIRS spectrophotometer with
➢ Chymotrypsin – More sensitive indicator of less Reflectance specialized computer software
severe cases of pancreatic insufficiency Spectroscopy (NIRS)
o Spectrophotometric method Fecal fat extraction and titration of
Van De Kamer Titration
long chain fatty acid by NaOH
➢ Elastase I – Specific in pancreas (for severe cases Nuclear Magnetic Microwaved-dried specimen;
of exocrine pancreatic insufficiency) Resonance hydrogen nuclear magnetic
o ELISA testing Spectroscopy spectrophotometer

X-RAY FILM SUMMARY OF FECAL SCREENING TESTS

TEST METHODOLOGY/PRINCIPLE INTERPRETATION
- Detects trypsin enzyme, Microscopic count of Three per HPF
- Trypsin has the ability to digest gelatin Examination neutrophils in smear indicates
- Trypsin is negative in cystic fibrosis for stained with methylene condition
- Procedure: Neutrophils blue, Gram stain, or affecting
o Emulsified Stool + X-ray Film (with Wright’s stain intestinal wall
Microscopic examination of 60 large red-
gelatin) direct smear stained with orange droplets
o Clearing of the film: (+) Trypsin Sudan III. indicates
o No clearing of the film: (-) for trypsin malabsorption
Qualitative Microscopic examination of
- False Negative: intestinal degradation of trypsin, smear heated with acetic 100 orange-red
Fecal Fats
possible presence of trypsin inhibitors acid and Sudan III droplets
- False positive: proteolytic activity of bacterial measuring 6-
enzyme 75um indicates
malabsorption
Pseudoperoxidase activity Blue color
of hemoglobin liberates indicates
Occult Blood oxygen from hydrogen gastrointestinal
- Most valuable in assessing cases of infant
peroxide to oxidize guiac bleeding
diarrhea (ex. Lactose Intolerance) reagent
- Determination: Addition of sodium Pink color
o Clinitest hydroxide to hemoglobin indicates
▪ Test for reducing sugars APT Test containing emulsion presence of fetal
determines presence of blood
▪ carbohydrate intolerance: > 0.5 maternal or fetal blood
g/dL Emulsified specimen placed Inability to digest
o pH Trypsin on xray paper determines gelatin indicates
▪ Normal stool pH: 7.0 to 8.0 ability to digest gelatin lack of trypsin
▪ CHO disorders: <5.5 stool pH Addition of Clinitest tablet Reaction of
to emulsified stool detects 0.5g/dL reducing
presence of reducing substances
Clinitest
D-XYLOSE TEST substances suggests
carbohydrate
- D-xylose is an exogenous substance that is not
intolerance
digested, but it should be reabsorbed by the
intestine
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Method of Collection:
o Masturbation (most preferred)
Reasons for Fluid Analysis
o Coitus interruptus (withdrawal method)
• Fertility Testing o Condom Method
• Post vasectomy semen analysis ▪ use non-lubricant containing
• Forensic Analysis (Alleged Rape) rubber or polyurethane condom
o Vaginal vault aspiration
- Time of collection: preferably in the morning,
COMPOSITION brought to the lab within 30 minutes and
examined within 1 hour
Composition of Semen
Seminiferous tubules (Testes) - During transport: maintained by body
• Spermatogenesis temperature
• Sertoli cells: serve as nurse cells for - Preservative: Dulbecco's phosphate buffered
developing sperm cells. It secretes saline
5% Spermatozoa enzyme inhibin which inhibits FSH
- Take note of the time of specimen collection,
synthesis
Epididymis specimen receipt and liquefaction
• Sperm maturation (Sperm become o proteolytic enzymes, such as bromelain
motile) and alpha chymotrypsin, can be used to
Seminal vesicles liquify the specimen
60-70% Seminal • Provide nutrients for sperm and slightly
- Analysis should be done after liquefaction
Fluid alkaline fluid
• Rich in fructose for sperm motility (usually 30-60 minutes)
Acidic Fluid - Specimen awaiting analysis should be kept at
20-30% Prostatic Contains ACP, Zinc, Citric acid, and other 37°C
Fluid enzymes (proteolytic enzyme essential for
coagulation and liquefaction)
5% Bulbourethral Thick alkaline mucus (pre-cum)
gland Neutralize acidity from prostatic secretions Appearance
(Cowper’s gland ) and vagina Gray-white, translucent
Normal color Pearly white, colorless to creamy
white
STRUCTURE Odor Musty or bleach/ chlorox odor
➢ Seminiferous Tubules – spermatogenesis Increased white infection/ increased WBC
➢ Epididymis – Sperm maturation turbidity
Red Coloration Increased RBC
➢ Vas (Ductus) Deferens – Propel sperm to
Increased contamination, urine
ejaculatory ducts contamination or medication
➢ Seminal Vesicles – Provide nutrients for sperm Yellow Coloration
Caused by flavin due to increased
and fluid abstinence
➢ Prostate Gland – Provide enzymes and proteins
for coagulation and liquefaction Macroscopic Examination
➢ Bulbourethral Glands – Acid alkaline mucus to Normal 2 to 5 mL
Volume Increased in Increased abstinence
neutralize prostatic acid and vaginal acidity
Decreased in Infertility, incomplete collection
Normal Pour in droplets (highly viscous)
Increased Decreased sperm motility
Viscosity viscosity
Reporting 0 – watery
4 – gel-like
Normal 7.2 to 8.0 or 7.3 to 8.3 (slightly
alkaline)
pH
Increased pH Infection
Decreased pH Increased prostatic fluid
Specific Normal 1.033
Gravity

SPERM CONCENTRATION
- Abstinence of 2-3 days for fertility testing, 2-3
- Number of sperm per mL
samples must be examined at 2 weeks interval - Normal Value: 20-160 million/mL
with an abnormal samples considered significant
o More than 5 days of abstinence will
Methods
result to increased volume but
decreased motility ➢ Improved Neubauer Counting Chamber
- The patient must collect with an empty bladder o Dilution: 1:20
- Collect the entire ejaculate o Diluents: to immobilize the sperm
o unable to collect the first part will result ▪ Cold water (commonly used)
to decreased sperm count, increased pH, ▪ 5% NaHCO3 in 1% phenol
and the specimen will not liquefy ▪ 1% Formalin
o unable to collect the last part will result ▪ 1% formalin in trisodium citrate
to decreased specimen volume and ▪ 5% Sodium bicarbonate
increased sperm count, and the ▪ 0.5% chlorozene
specimen will not clot.
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

➢ Makler Counting Chamber

o For undiluted sample
o Uses heat to immobilize the sperm cells

Long Method for Sperm Count Concentration

Computation
(Standard Neubauer Calculation Formula)

# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 SPERM MORPHOLOGY

𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐. = Normal Values
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 0.1
- Examine at least 200 spermatozoa under OIF
Examples: - Routine criteria: > 30% normal forms or <50%
• 1 sperm counted, 2 WBC squares abnormal forms
- Kruger’s strict criteria: >14% normal forms or <
1 𝑥 20
= 100 𝑠/𝑢𝐿 𝑥 1,000 = 𝟏𝟎𝟎, 𝟎𝟎𝟎 𝒔/𝒎𝑳 70% abnormal
2 𝑥 0.1 o Measure the head, neck, and tail using a
• 1 sperm counted, 5 RBC squares micrometer
1 𝑥 20
= 1,000 𝑠/𝑢𝐿 𝑥 1,000 = 𝟏, 𝟎𝟎𝟎, 𝟎𝟎𝟎 𝒔/𝒎𝑳 Stains
0.2 𝑥 0.1
• Wright’s stain
Shortcut Method for Sperm Count Concentration • Giemsa stain
Computation • Hematoxylin
• Crystal violet
• 2 WBC squares
# of sperm counted x 100,000 = sperm in M/mL • Papanicolau’s stain
• 5 RBC squares
# of cells counted x 1,000,000 = sperm in M/mL Morphology
➢ Head
o Normal: oval shaped
SPERM COUNT
o Abnormal: poor ovum penetration
- Normal Value: ≥ 40 million/ejaculate
o 5um length and 3um width
- Formula:
o The size of the acrosomal cap should be
𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡 = 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐. 𝑥 𝑠𝑝𝑒𝑐𝑖𝑚𝑒𝑛 𝑣𝑜𝑙𝑢𝑚𝑒 1/2 of the sperm head
➢ Midpiece and Tail
SPERM MOTILITY o The midpiece contains mitochondria
- Performed undiluted, examine microscopically o abnormal: poor motility
(20 HPF) o 45um long (neck to tail)
- Depends on the type of sperm ➢ Varicocele
o Androsperm: produces male child o Hardening of the veins that drain the
▪ Highly motile but dies easily testes
o Gymnosperm: produces female child o Most common cause of male infertility
▪ Very slow but do not die easily o Sperm head: tapered
- Normal Values: > 50 % motile (within 1 hour)
- fresh specimen: 80% motile and 20% non-motile
- Quality: ≥2.0

Computer-Assisted Semen Analysis (CASA)

- Provides objective determination of both sperm
velocity and trajectory (direction of motion)
- Sperm concentration and morphology can also
be analyzed

Sperm Motility Grading

Grade WHO Criteria Methods of Examining Sperm Morphology
4.0 a Rapid, straight-line motility ➢ William de Gugan-Carpenters – seminal fluid is
3.0 b Slower speed, some lateral movement
smeared on a slide, air-dried and fixed with
Slow forward progression, noticeable lateral
2.0 b moderate heat, 0.5% chlorazene is applied for 2-
movement
1.0 c No forward progression 4mins, washed with distilled water, then with
0 d No movement 95% ethanol
o Stain used: Crystal Violet and Rose
Bengal
➢ Meaker’s Method
o stain used: Carbol Fuchsin and Bluish
Eosin
➢ Holbert’s Method
o stain used: Gentian Violet and Rose
Bengal
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Abnormal Sperm Head SPERM VIABILITY

➢ Modified Bloom’s Test
o Reagent: Eosin and Nigrosin
o Count 100 sperms
▪ Living sperm: unstained, bluish
white (75% or 50%)
▪ Dead sperm: red
➢ Seminal Fluid Fructose
o Normal Value: ≥13 umol/ejaculate
o Test within 2 hours
o Screening test
▪ Resorcinol test: (+) orange-red
color
▪ seliwanoff's test: qualitative

Antisperm Antibodies (Serological test)

Used if the patient and his wife doesn’t have ability to
conceive a baby (specimen: semen, cervical mucosa,
serum)
➢ Mixed Agglutination Reaction
o Detects the presence of IgG antibodies
o Semen sample + AHG + latex particles or
treated RBC’s coated with IgG
o Normal: <10% motile sperm attached to
the particles
➢ Immunobead test
o Detects the presence of IgG, IgM and IgA
o Demonstrates what area of the sperm
the autoantibodies are affecting
o Reporting: IgG tail antibodies, IgM head
antibodies
➢ Gelatin Agglutination Test
o Sperm + 10% gelatin + patient’s serum
o White particles in clear surroundings

Chemical Testing
Decreased Value
Analyte Normal Value
Indicates
Decrease
Fructose ≥ 13 umol/ejaculate
seminal fluid
Neutral α- Epididymis
≥ 20 mU/ejaculate
glucosidase disorder
Zinc ≥ 2.4 umol/ejaculate
Citric Acid ≥52 umol/ejaculate Decrease
Acid prostatic fluid
≥ 200 Units/ejaculate
phosphatase

Terminology Definition
Aspermia No ejaculate
Absence of sperm cells
• Underdeveloped sperm cells
Azoospermia • Obstruction from previous operation or
traumatic procedures
• Infection with gonorrhea
Necrospermia Immotile/ dead sperm cells (viability: <50%)
Decrease sperm concentration, decrease
number of sperm cells or presence of few
Oligospermia motile sperms seen in
• Hypotropic lesions
• hypothyroidism
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Microbial Testing Additional Testing for Abnormal Semen Analysis

Abnormal Possible
➢ Round Cells Test
Result Abnormality
o WBC and spermatids (immature sperm Decreased
cells) motility with Viability Eosin-nigrosin stain
o Normal value: < 1 million/mL normal count
▪ Infection: > 1 million WBC/ mL Decreased
Lack of seminal
vesicle support Fructose level
▪ disruption of spermatogenesis: count
medium
>1 million spermatids / mL Mixed agglutination
• problem in epididymis Decreased reaction and
Male antisperm
o Test for: motility with
antibodies
immunobead tests
▪ Chlamydia trachomatis clumping Sperm agglutination
with male serum
▪ Mycoplasma hominis
Normal analysis
▪ Ureaplasma urealyticum with continued
Female antisperm Sperm agglutination
antibodies with female serum
infertility

Sperm Function Test

➢ Hamster egg penetration – Sperms are
incubated with species non-specific hamster
eggs and penetration is observed microscopically
➢ Cervical mucus penetration – Observation of
sperm penetration ability of partner’s midcycle
cervical mucus
o in cases of acrosomal cap problems
➢ Hypo-osmotic swelling – Sperms exposed to
low-sodium concentration are evaluated for
membrane integrity and sperm viability
➢ In vitro acrosome reaction – Evaluation of the
acrosome to produce enzymes essential for
ovum penetration

Medico-Legal Tests
➢ Microscopic Examination
➢ Fluorescence under UV light
➢ Acid phosphatase determination
➢ Glycoprotein p30 (more specific)
➢ Florence Test
o Test for choline
o Reagents: iodine crystal + Potassium
Iodide
o (+) Dark browm rhombic crystals
➢ Barbiero’s test
o Test for spermin
o TCA + Picric Acid
o (+) yellow leaf-like crystals

Post-vasectomy Semen Analysis

➢ Vasectomy
o Cutting of vas deference so that the
ejaculate will not contain any sperm cell
o The only concern is the presence or
absence of sperm
o Done two months after vasectomy and
continued until 2 consecutive monthly
specimens show no sperm
o Centrifuge first to double check

Normal Values for Semen Analysis

Volume 2-5 mL
Viscosity Pour in droplets
pH 7.2- 8.0
Sperm concentration ≥ 20 million/ mL
Sperm count ≥ 40 million/ mL
Motility ≥ 50 within 1 hour
Quality ≥ 2.0 or a, b, c
>30% normal forms (routine criteria)
Morphology
> 14% normal forms (strict criteria)
Round cells < 1 million / mL
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
- It is a clear, colorless, and sometimes yellowish
fluid which is found in pregnant women
- It is usually produced during the first 12 days
following the conception
- Purpose: surround the growing fetus in the
uterus
- Problems in the amount of amniotic fluid inside
the uterus will lead to complication
- It is made up of fetal urine and fetal cells
- During the first trimester of pregnancy, the
amniotic fluid is mainly made up of maternal
plasma
- It is an ultrafiltrate of plasma
STRUCTURE OF THE PLACENTA
- 2 extraembryonic membrane:
o Amnion: inner membrane (thin sac)
▪ Covers directly the embryo
▪ When amnion ruptures, it will
release the amniotic fluid, which
signals the start of the
pregnancy’s delivery stage
o Chorion: outer membrane
▪ It is a support platform of the
fetus and the amnion
- Their function is for embryo’s overall
development
- They also play an important role in the
nourishment and breathing of the fetus
PRIMARY FUNCTIONS OF AMNIOTIC FLUID
• Provide cushion for the fetus
• Allow fetal movement
• Stabilizes fetal temperature exposure
• Infection control (IgG could pass through the
placenta coming from the mother)
• Proper lung development
• Exchanges water and chemicals among the fluid,
fetus, and maternal circulation
• Muscle and bone development
• Lubrication (to prevent webbing)
AMNIOTIC FLUID VOLUME
- During the first trimester, approximately 35 mL
of amniotic fluid is derived primarily from the
maternal circulation or maternal plasma
- As the fetus grows after the first trimester of
pregnancy, the major contributor of the amniotic
fluid will be the fetal urine and lung fluid
o Volume: 200 to 500mL per day
- Urine regulation:
o High urine = high fetal swallowing
o Lung fluid adds lung surfactant to the
amniotic fluid
o Phospholipids: one of the compositions
of amniotic fluid that could assess the
lung maturity
- Increased amniotic fluid peak at 800 to 1200 mL
in the third trimester is the result of fetal urine
Abnormal Amniotic Fluid Volume
➢ Polyhydramnios (>1200 mL)
o Increased amniotic fluid volume
o Causes: Decreased fetal swallowing of
urine and neural tube defects
o Acute Polyhydramnios – associated with
fetal edema, hydroxy fetalis, or fetal
heart failure
o Chronic Polyhydramnios – associated
with fetal disorders (ex. poor fetal
swallowing, toxemia during pregnancy,
and mother has diabetes)
➢ Oligohydramnios (<300 mL)
o Decreased amniotic fluid volume
o Could cause fecal distress syndrome
o Causes: Increased fetal swallowing of
urine, membrane leakage, and urinary
tract deformities
CHEMICAL COMPOSITION
- 98 to 99% water with some electrolytes during
the first trimester
- During the 12th to 14th week, the fluid may now
have the presence of metabolic products
- Composition similar to maternal plasma with
sloughed fetal cells
o Cells are used for cytogenetic analysis
- The fluid also contains biochemical substances
that are produced by the fetus, such as bilirubin,
lipids, enzymes, electrolytes, urea, creatinine,
uric acid, proteins, and hormones
- Fetal urine increases creatinine, urea, & uric acid
- Fetal age can be estimated by creatinine:
o <36 weeks = 1.5 to 2.0 mg/dL
o >36 weeks = >2.0 mg/dL
QUADRUPLE SCREENING TEST
- Done prior to performing Amniocentesis
- Check the hormones in the blood of the mother
- Performed during the second trimester (14th up
to 22nd week of gestation)
- It is used to evaluate whether the pregnancy has
increased chance of certain conditions such as
down syndrome or neural tube defects
Four Pregnancy Hormones
➢ Alpha-fetoprotein (AFP) – protein made by the
developing baby
o Check for neural tube defects (NTD) or
trisomy 21 (down syndrome)
➢ Human Chorionic Gonadotropin (hCG) –
hormone made by the placenta
➢ Unconjugated estriol (UE3) – hormone made by
the placenta and the liver of the baby
➢ Inhibin A – hormone made by the placenta
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

AMNIOCENTESIS ➢ Test for Hemolytic Disease of the Newborn:

- It is an invasive procedure because the amniotic o Detect the presence of bilirubin
fluid is obtained by needle aspiration into the o Protect from light
amniotic sac
- Two procedures: AMNIOTIC FLUID vs. MATERNAL URINE
ANALYTE AMNIOTIC FLUID MATERNAL URINE
o Transabdominal amniocentesis Less Reliable
(preferred) Protein + –
o Vaginal amniocentesis (may cause Glucose + –
leakage of amniotic fluid and More Reliable
contamination of bacteria) Urea <30 mg/dL > 300 mg/dL
Creatinine < 3.5 mg/dL > 10 mg/dL
- It is guided by an ultrasound
- It is done by OB-GYN and trained doctors
AMNIOTIC FLUID MATERNAL URINE
- Maximum of 30 mL collected in sterile syringes The watery and typically
- Discard first 2 to 3 mL for contamination The fluid, which surrounds the yellowish fluid, one of the chief
- Protect specimens from light for bilirubin fetus within the amnion means of eliminating excess
analysis for HDN at all times: amber tubes or water and salt
black plastic tube covers Occurs inside the amniotic
Produced by kidney
fluid
Watery hopefully, clear but
Normal urine is either colorless
sometimes yellow, green or
or pale yellow in color
with white specks
Has a strong "fish-like" odor
Odorless due to bacterial growth after
urination
Contains nutrients including
Contains mainly water,
proteins, carbohydrates, and
inorganic salts, proteins,
lipids, hormones, immune
hormones, and a wide range of
system cells, and the urine of
metabolites
baby
Normal level is about one
Normal range of urine is 0.6 to
quart by 36 weeks after
2.6 L per person per day
pregnancy
Serves as a cushioning liquid to
Responsible for excreting
the fetus and facilitates the
nitrogenous wastes, excess
exchange of nutrients and
salts, and other water-soluble
water between mother and
chemicals out of the body
baby
A true amniotic fluid leak The flow can be controlled by
cannot be controlled squeezing muscles

FERN TEST
- Detects ruptured amniotic membranes
- It is also used to diagnose early pregnancy
- Procedure:
o Vaginal Fluid → Slide (Air Dry for at least
5 to 7 minutes) → visualize under LPF
o (+) Fern-like Crystal (Amniotic Fluid)

SPECIMEN COLLECTION
➢ Method of Collection: Amniocentesis with
ultrasound Up to 30 mL is collected in sterile AMNIOTIC FLUID COLOR
Color Clinical Significance
syringe
Colorless Normal
➢ 2nd Trimester Amniocentesis: Assess genetic Traumatic Tap, trauma,
defects or chromosomal abnormalities (Ex. Blood-Streaked
intraamniotic hemorrhage
Trisomy 21 and Down’s Syndrome) Yellow HDNF (Bilirubin)
➢ 3rd Trimester Amniocentesis: Fetal Lung Dark-Green
Meconium
Maturity and Fetal Hemolytic Disease (first fetal bowel movement)
Dark Red Brown Fetal Death

SPECIMEN HANDLING
➢ Test for Fetal Lung Maturity
o Place on ice (deliver)
o Refrigerated or frozen
o Filtration: prevents loss of phospholipids
➢ Test for Cytogenetic Studies
o Room temperature or body temperature
DIFFERENT TESTS FOR DISORDERS
Test for HDNF
- Hemolytic Disease of the Fetus & Newborn
- Also known as Optical Density or OD 450
- To detect whether there is any hemolytic disease
- Absorbance of Amniotic Fluid
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
o Normal: Increased at 365nm and
decreased at 550 nm
o HDN: increased at 450 nm (bilirubin)
- Common cause: Rh incompatibility
- Results are plotted on a Liley Graph
o Zone I: nonaffected/ mildly affected
fetus
o Zone 2: Moderately affected fetus
(requires close monitoring)
o Zone 3: Severely affected fetus (requires
intervention)
- Usually checked during the 3rd trimester
- Interferences include: cells, mecomium, debris,
and hemoglobin
Test for Neural Tube Defects
- The neural tube forms the early brain and spine
- Alpha-fetoprotein (AFP) produced by the fetal
liver prior to 18 weeks’ gestation
- Increased levels in maternal blood or amniotic
fluid indicate possible anencephaly or spinal
bifida
- Increased levels are found when skin fails to
close over neural tissue
- Measure maternal blood first, then amniotic
fluid
- Alpha-fetoprotein could also reach the maternal
circulation
- Diseases:
o Spina bifida – the spine and the spinal
cord do not form properly
o Anencephaly – serious birth defect in
which the baby is born without the parts
of the brain and skull
- Screening Test: AFP
o Increased in neural tube defects
o Decreased in Down Syndrome
- Confirmatory Test: Acetylcholinesterase
(enzyme that hydrolyzes a neurotransmitter in
the body, acetylcholine)
Test for Fetal Lung Maturity
- The production of lung fluid containing
phospholipids can be used to assess or evaluate
whether the fetal lung is matured and ready for
delivery
- Most common complication of early delivery is
respiratory distress syndrome (RDS)
- Lack of lung surfactant, which keeps the alveoli
open during inhaling and exhaling
- Surfactant decreases the surface tension on the
alveoli so they can inflate more easily
- Many laboratory tests are available for FLM
Lecithin/Sphingomyelin (L/S Ratio)
- Reference Method
- Lecithin: for alveolar stability
o primary component of lung surfactant
o high during 3rd trimester (35th week)
- Sphingomyelin: serves as a control for the rise of
lecithin
o Produced at a constant rate
o Start to increase during the 26th week
- Mature fetal lungs: Ratio of > 2.0
- Cannot be done on a specimen contaminated by
blood or meconium
- It requires thin-layer chromatography
Amniostat-FLM
- Immunologic test for Phosphatidyl Glycerol
- Uses antisera against Phosphatidyl Glycerol (not
a major component of lung surfactant)
o The production of phosphatidyl glycerol
is parallel to lecithin
- Alternative for L/S ratio
- Not affected by blood or meconium
- Production of PG is delayed among diabetic
mother
Foam Stability Test (Foam or Shake Test)
- Bedside testing
- Amniotic Fluid + 95% Ethanol → Shake 15 secs →
Stand 15 mins
- Mature fetal lungs: (+) Foam/Bubbles
o Ethanol is an anti-foaming agent
o Continuous formation of bubbles in the
solution indicates sufficient amount of
lung surfactant
- Cannot be used with contaminated amniotic
fluid
Microviscosity
- The presence of phospholipids decreases
microviscosity
- Measured by Fluorescence Polarization
Lamellar Body Count
- Lamellar Bodies (a.k.a. Type II pneumocytes)
o Found in the lungs as dust cells
o Responsible for production of alveolar
surfactants
o Spherical in shape and contains granules
inside
o Storage form of lung surfactant
- Alternative method to detect the presence of
phospholipids
- adequate FLM: >32,000/uL lamellar body count
- hematology analyzer is used for counting
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

OD 650 nm - Specimen: 1st Morning Urine, random urine,

- Increased Lamellar = Increased OD (Absorbance) serum, or plasma
- An OD of ≥ 0.150 is equivalent to: - Responsible for rescuing and maintaining corpus
o L/S ratio of ≥ 2.0 luteum, which produces the hormone
o (+) Phosphatidylglycerol progesterone.
- Lamellar bodies absorbance: 650nm o It also produces estradiol and inhibit-A
hormones
Lamellar Bodies - Secreted immediately on implantation (6-8 days
- Approximately 90% phospholipid and 10% after conception)
protein - Doubling does not occur in ectopic pregnancy
- Secreted by the type II pneumocytes of the fetal - HCG decreases to nondetectable 2 weeks after
lung to the alveolar space at about 24 weeks of delivery
gestation
- Increase in amniotic concentration from 50,000 METHODS IN MEASURING HCG ACTIVITY
to 200,000/mL by the end of the third trimester ➢ Immunologic test
- OD of 150 at 650 nm correlates with L/S ratio of o Test kits are used
2.0 and the presence of PG o Direct or Agglutination Test (common)
- Lamellar bodies are red in platelet channel when o Indirect or Agglutination Inhibition/
using hematology analyzer Hemagglutination Inhibition
➢ Radioimmunoassay: competitive binding assay
➢ Radio Receptor Assay
➢ Immunometric Assay
➢ Bioassays: uses test animal and done in vivo

HCG Bioassays
Animal Mode of
Test Positive Result
Test For Fetal Age Used Injection
Formation of
≥ 2.0 mg/dL creatinine = 36 weeks Immature hemorrhagic
Ascheim-
female Subcutaneous follicles and
Zondek
Test for Fetal Well-Being and Maturity mice Corpus Lutea
Test Normal Values Significance (enlarged ovary)
Change of OD at Hyperemic Uterus
Bilirubin scan HDN Mature
450 nm (>0.25) and Corpora
virgin Marginal ear
Neural Tube Friedman hemorrhagia
Alpha-Fetoprotein < 2.0 female vein
Defects (small spots in
rabbit
Fetal Lung ovary)
L/S Ratio ≥ 2.0 Female
Maturity
Fetal Lung toad South Oogenesis
Amniostat-fetal Maturity/ Hogben African Lymph sac (Extrusion of
Positive Clawed eggs)
Lung Maturity Phosphatidyl
Glycerol 7 Frog
Foam Stability Fetal Lung Spermatogenesis
≥ 47 Galli- Male Frog
Index Maturity Subcutaneous (presence of
Mainini Male Toad
Microviscosity Fetal Lung sperm in urine)
≥55 mg Ovarian
(FLM-TDx) Maturity Immature
Optical Density Fetal Lung Frank- hyperemia
≥0.150 Female Subcutaneous
650nm Maturity Berman (enlarged red
Rats
Lamellar Body Fetal Lung ovary)
≥ 32,000/mL Ovarian
Count Maturity
Female Hyperemia
Kupperman Intraperitoneal
Rats (enlarged red
ovary)
- It is a hormone that is being produced by the
cytotrophoblast cells in the placenta of pregnant
women as a recognition of pregnancy
- It is detected using pregnancy test
- Male who are positive with pregnancy test have
a possibility of having seminoma or testicular
cancer
- HCG is important to thicken the uterine lining to
support the growing embryo and to tell the body
to stop menstruation
- There is a high HCG level after conception and it
will continue to rise about 10 weeks of
pregnancy
- Peaks during 1st trimester of pregnancy
(Increased blood, urine, amniotic fluid)
- Composed of 2 subunits (dimer):
o Alpha: HCG, LH, FSH, TSH
o Beta: unique for HCG
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

SPECIMEN COLLECTION
- It is also known as joint fluid analysis - Method of collection: arthrocentesis or joint
- Purpose: to diagnose the cause of joint aspiration
inflammation - Normal synovial fluid does not clot
- Synovial fluid is a thick liquid that is used to - Diseased joints may clot
lubricate joints and allow for ease of movement - 1mL is enough for laboratory analysis
- In joint diseases like arthritis, the joint produces - Volume:
a lot of synovial fluid, which causing o Normal: <3.5 mL
inflammation o Inflammation: >25 mL

SYNOVIAL FLUID Distribution

- Also known as joint fluid 1. Microbiology: Sodium heparin or SPS (gram-
- “Synovial,” Latin word for egg staining and culture)
- Viscous fluid circulating in the diarthroses 2. Hematology: Sodium heparin / liquid EDTA (cell
(movable joints) count)
o The viscosity resembles like egg white 3. Chemistry and other tests: non-anticoagulated
- Viscosity is due to hyaluronic acid produced by 4. Glucose analysis: sodium fluoride
synoviocytes
Note: Never use powdered anticoagulants because it
- An ultrafiltrate of plasma across the synovial
may interfere with crystal identification
membrane. The filtration is nonselective except
for the exclusion of high-molecular weight
proteins PHYSICAL EXAMINATION
- Synovium – tissue lining in the synovial tendon Color
which is usually seen in the movable joints Appearance Significance
Colorless to pale yellow Normal
Deeper Yellow Inflammation
Greenish tinge Bacterial Infection (septic arthitis)
Traumatic Tap or
Red
hemorrhagic arthritis
Milky (+) crystals

Clarity
Appearance Significance
Transparent or Normal
Clear
due to WBC / crystals, fibrin, free-floating
rice bodies (made up of collagen, looks like
Turbid shiny grain of rice, and is commonly
encountered in patients with rheumatoid
arthritis)
crystals (most common: monosodium
urates that causes gout and calcium
Opaque, milky
pyrophosphate dihydrate seen with
pseudogout)
Associated with ochronosis or ochronotic
Ground pepper
shards (degeneration of cartilage)
Oily, shimmering Radiographic Contrast Media (RCM)

Viscosity
- Normal: able to form a string (4-6 cm long)
- Ropes/Mucin Clot Test (Hyaluronate
Polymerization Test)
o Reagent: 2-5% acetic acid
Grading Appearance
Good Solid Clot
Fair Soft Clot
- Cartilages in the joints are avascular (they do not Low Friable Clot
have any blood vessels) Poor No clot
- Any damage on this membrane will produce pain
and stiffness on the joints, collectively called as - Some bacteria produces enzyme called
arthritis hyaluronidase, which degrades hyaluronic acid
o If the sample contains bacteria, it will not
FUNCTIONS OF SYNOVIAL FLUID produce clot and string
o False negative test
• Keeps the bones slightly apart
• Lubricates the joints
• Reduce friction between joints CELL COUNT
• Provides nutrients to the articular cartilage WBC Count
• Lessen the shock of joint compression occurring - Most frequently performed count
during activities such as walking and jogging - To check for septic arthritis
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

- Diluting Fluids: Causes of Crystal Information

o NSS with methylene blue 1. Metabolic Disorders
o Hypotonic solution (if RBC is present) 2. Decreased renal excretion that produce
o Saline with saponin (if RBC is present) increased blood levels of crystallizing chemicals
- Never use acetic acid for joint analysis because 3. Degeneration of cartilage and bones
it induces clot, which may cause falsely increase 4. Injection of medication (corticosteroid)
fluid viscosity
- For very viscous fluid Crystals
o Add a pinch of hyaluronidase to 0.5 mL
➢ Monosodium urate: needle-shaped rods
of fluid
o Most common cause of gout
o Add 1 drop of 0.05% hyaluronidase in
o 90% of acute urate gout
phosphate buffer per mL of fluid
➢ Calcium pyrophosphate dihydrate (CPPD):
(incubate at 37°C for 5 minutes)
rhomboids, rod, or rectangular
o Associated with degenerative arthritis
Differential Count ➢ Hydroxyapatite & basic calcium phosphate
Perform cytocentrifugation or thinly smeared slide and (BCP): small needles (apatite)
incubate it with hyaluronidase prior to preparation o Seen in apatite gout and osteoarthritis
➢ Neutrophils: 20% ➢ Calcium oxalate: bipyramidal envelopes
o Elevated: urate gout, rheumatoid ➢ Cholesterol crystals: notched rhombic plates
arthritis (septic arthritis and ➢ Corticosteroid: flat, variable-shaped plates
inflammatory disorders) o Accumulation of drug in the synovial
➢ Lymphocytes: 15% fluid
o Elevated: early rheumatoid arthritis, ➢ Lipid crystals: maltese-cross
viral arthritis, chronic infection, collagen
disorders (non-septic arthritis ) Special Types of Microscope
➢ Monocytes: 65%
➢ Polarizing Microscope – detects for the presence
o Elevated: viral arthritis, serum sickness,
or absence of birefringence
SLE, autoimmune disease
➢ Compensated Polarizing Microscope – confirms
➢ Eosinophils: 2%
the type of birefringence (positive or negative
o Elevated: rheumatic fever, metastatic
birefringence)
carcinoma, Lyme disease, parasitic
o Red compensator is placed between
infection, chronic urticaria, allergy
crystal and analyzer
o MSU: parallel = yellow = negative
Differential Count
birefringence
Cell Normal Value
RBC < 2,000 / uL o CPPD: opposite or perpendicular = blue
WBC < 150 to 200 / uL = positive birefringence
• 65% = Monocytes and Macrophages
WBC
• < 25% = Neutrophil
Differential
• < 15% = Lymphocytes

Positive Negative
No Birefringence
Birefringence Birefringence
• CPPD • Monosodium urates • Apatite
• Corticosteroids • Cholesterol crystals
• Corticosteroids
• Calcium oxalate

LE cell Reiter cell

CRYSTAL IDENTIFICATION
- Inflamed joints may be due to crystals or bacteria
- Accumulation of the crystals will lead to acute
inflammation or gout
- WBCs increases
- Crystal induced arthritis will result to increased
neutrophils and macrophages
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

CHEMICAL EXAMINATION IIb III

GROUP
(Crystal-induced) (Septic)
➢ Glucose Gout
Significance Microbial Infection
o Normal: < 10mg/dL Pseudogout
o ↑ in infection, inflammation Color and
Cloudy or milky fluid
Cloudy, yellow-green
o Most frequently tested in chemistry Clarity fluid
Viscosity low variable
section WBC Count Up to 100,000 / uL 50,000 – 100, 000/uL
o Done in conjuction with blood glucose (8 Neutrophils <70% >75%
hours fasting) Glucose Decreased Decreased
o Blood glucose – SF glucose = < 10 mg/dL others (+) Crystals
(+) Culture and Gram
(normal) stain
➢ Protein
IV
o Normal: < 3 g/dL GROUP
(Hemorrhagic)
o ↑ in inflammatory and hemorrhagic Traumatic injuries, coagulation
disorders Significance
deficiencies
➢ Lactate Color and Clarity Cloudy, red fluid
o Normal: same as plasma Viscosity Low
o ↑ in bacterial infection WBC Count Equal to blood
Neutrophils Equal to blood
➢ Uric Acid
Glucose normal
o Normal: same as plasma others (+) RBCs
o ↑ in gout
o Normal value is the same as blood uric
acid
➢ Lipid Droplet EXAMINATION OF SWEAT
o Trauma - Measurement of sweat electrolytes (sodium and
➢ Enzymes chloride) is performed to confirm Cystic Fibrosis
o LDH: ↑ in gout, RA - There is elevated sodium and chloride because
o ALP the sweat glands cannot to reabsorb them
➢ Mucin Clot Test - Cystic Fibrosis – Metabolic disease that affects
o inflammatory arthritis the mucus secreting glands of the body.
- It is a special test usually done in reference
MICROBIOLOGY laboratories
- Common organisms that infect the synovial fluid:
o S. aureus CYSTIC FIBROSIS
o Streptococcus - Common indicators
o Haemophilus o Family history
o Neisseria gonorrheae o Newborns with intestinal obstruction
o Appearance of pancreatic insufficiency
IMMUNOLOGIC/SEROLOGIC EXAMINATION in infants
- Autoantibody Detection (SLE, RA) o Respiratory distress in infants
o ANA: SLE o Confirmed by elevated sweat Na and Cl
o Rheumatoid Factor: RA - Signs and Symptoms
- Detection of autoantibodies to Borrelia o Noticeable salty sweat
burgdorferi (Lyme Disease) o Frequent respiratory infections and
o Also causes fluid accumulation chronic cough
- Complement levels: ↑inflammation, ↓bacterial o Bulky offensive greasy stool
and crystal-induced arthritis o Malnutrition
o There is an accumulation of antigen- o Male infertility (obstructive
antibody complex, complement system azoospermia)
will be activated, which will destroy the - Na and Cl over 70 mEq/L
cells o Consistent in 98% of patient with cystic
fibrosis
Laboratory Findings in Joint Disorders - Borderline: Na and Cl of 40 mEq/L
I IIa o Flame photometry: sodium is yellow
GROUP
(Non-inflammatory) (Immunologic) o Ion exchange chromatography:
Significance
Degenerative joint Immunologic separates Na and Cl
disorders (osteoarthritis) disorders (RA, SLE) o Manual or automated titration: for
Color and
Clarity
Clear, yellow fluid Cloudy, yellow fluid chloride
Viscosity Good Poor - Gibson and Cooke Pilocarpine (+ mild current)
WBC Count < 1,000 / uL 2,000-75,000 / uL Iontophoresis Technique
Neutrophils < 30% > 50%
Normal (similar to blood
Glucose
glucose)
Decreased TEST FOR CHLORIDE IN SWEAT
others (+) autoantibodies • Cotlove Chloridometer
• Microtitration with Mercuric Nitrate
• Cystic Fibrosis Analyzer
• Chloride Electrode Method
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

SEROUS FLUID - Occurs due to increased hydrostatic pressure or

- Also known as the serosal fluid low plasma oncotic pressure
- Originated from the Latin word “serosus” - Low in protein and LDH
meaning serum - Examples: Congestive Heart Failure, Nephrotic
- It is a body fluid that resembles serum Syndrome, Liver Cirrhosis, Malnutrition,
- Clear to pale yellow fluid seen in spaces Hypoalbuminemia
between organs and membranes which lines
them Exudates
- Increased quantity of serous fluid is indicative of - Produced by conditions that directly involve the
pathological conditions membrane of the particular cavity
o Liver cirrhosis: increased production of - Occurs due to inflammation and increased
serous fluid in the peritoneum capillary permeability
o Heart failure: increased fluid in the - High in protein and LDH
pericardium - Examples: Infection, malignancy, SLE,
- Some are not associated with pathological pneumonia, cancer, TB, autoimmune
conditions. Example is surgical complications.
- Purpose: reduce the friction from muscle
movement, removal of wastes, and exchange of
nutrients
- Formed as an ultrafiltrate of plasma
- Production & reabsorption is due to hydrostatic
& colloidal (oncotic) pressure
- Collected by needle aspiration procedure
- Accumulation of fluid: Effusion (Caused by
imbalance of fluid production and reabsorption)
- Ultrafiltration is controlled by the presence of
the membrane
o Parietal membrane: outer layer
o Visceral membrane: inner layer
- Serous fluid is located between the parietal and
visceral membrane

THREE TYPES OF SEROUS FLUIDS

SEROUS FLUIDS
➢ Pleural Fluid:
o Thoracentesis Pleural Fluid
o Seen in lungs - Abnormal accumulation occurs due to
o Normal volume: <30mL conditions that affect:
➢ Pericardial Fluid: o Transudative effusion
o Pericardiocentesis ▪ Capillary Hydrostatic Pressure
o Seen in heart ▪ Colloidal Pressure
o Normal volume: <50mL o Exudative effusion
➢ Peritoneal or Ascitic Fluid: ▪ Permeability
o Paracentesis or Peritoneocentesis ▪ Lymphatic Drainage
o Seen in abdominal cavity - Conditions caused by the accumulation of
o Normal volume: <100mL pleural fluid
o Congestive Heart Failure
o Hypoalbuminemia
SPECIMEN COLLECTION
o Pneumonia
• Anticoagulated tube (EDTA): cell count and o Carcinoma
differential count
• sterile tube (SPS): culture Appearance Significance
• heparinized tube: chemistry Normal clear and pale yellow
• non-anticoagulated: clotting Cloudy, Turbid, WBC And Bacterial infection, Immunologic
and White Disorder, Tuberculosis, and SLE
Note: Brown Ruptured amoebic liver abscess
• fluid should be retained for 7-10 days in case of Black Fungal infection, aspergillosis
further testing Malignancy, Traumatic Aspiration,
Hemothorax (Traumatic Injury),
• if the pH of the specimen needs to be checked, - uneven distribution of blood
it should be maintained anaerobically in ice - pleural fluid hematocrit is ≥ 1/2 of the
o anaerobe heparinized syringe is used whole blood hematocrit
- the effusion occurs from the inpouring
Blood
of blood from the injury
CLASSIFICATION OF EFFUSION Hemorrhagic effusion (bleeding caused by
Transudates other causes)
- even distribution of blood
- Effusion that forms due to systemic disorder - pleural fluid hematocrit is < 1/2 of the
disrupting the balance in the regulation of fluid whole blood hematocrit
filtration and reabsorption Chylous (Thoracic Duct Leakage)
Milky - increased lipid content (chylomicrons
and triglycerides)
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
- Lymph duct has leakage Appearance Significance
- Increased lymph fluid Pale Yellow and Clear transudates and normal
Pseudochylous material (Chronic Turbid infection and malignancy
Inflammatory) Milky chylous and pseudochylous effusion
Presence of hyaluronic acid due to Blood-streaked Frequently In Malignant Effusions
Viscous
malignant mesothelioma Accidental Cardiac Puncture, Misuse
Grossly Bloody
of Anticoagulant

Cell Count
- ↑ neutrophils: bacterial endocarditis
- malignant cells: Metastatic carcinoma

Chemistry Test
- ↓ glucose level: bacterial infection,
malignancies
o Increased lactate due to glucose
Cell Count metabolism
- Diagnosis of tuberculosis and bacterial - Perform gram stain and culture to confirm
infections bacterial infection
- Increased cells: - Increased adenosine deaminase: tuberculosis
lymphocytes and eosinophils - Endocarditis due to previous respiratory
neutrophils
plasma cells (<10%)
infection caused by haemophilus, streptococcus,
• Tubercular • Bacterial • Associated
effusion infection with allergic and staphylococcus
• Viral effusion • Pneumonia and parasitic
• Autoimmune • Pulmonary infection Peritoneal Fluid (Ascitic Fluid)
such as RA and infarction • Trauma
- Peritoneal lavage – lavage for detection of
SLE • Pancreatitis
abdominal injuries:
- Present of mesothelial cells
o Irrigate the peritoneum with normal
o Pleiomorphic shape
saline, then aspirate
o Normally seen in the lines of the serous
o Sensitive to intraabdominal bleeding
membrane
(> 100,000 RBC/uL)
o low mesothelial cells indicate that the
- Psammoma bodies
patient has tuberculosis
o Seen in peritoneal exudates
o Containing concentric striations of
Chemistry Test
collagen-like material
Glucose Decrease - Most common
- Tubercular & Rheumatoid o Seen in benign conditions, ovarian, and
inflammation thyroid malignancies
- Increased lactate due to glucose
metabolism
Triglyceride Increase Chylous effusion
pH ↓pH 7.2 - Need for chest tube drainage
- Empyema (puss filled pleural cavity)
- Unresponsive antibiotic treatment
to pneumonia
↑pH 7.4 - Malignancies
As low - Esophageal rupture
as pH
6.0
Adenosine > 40 u/L - Tuberculosis
deaminase
Amylase Increase - Pancreatitis

Serology Test
- tumor markers are checked
- most frequently performed in autoimmune
disorders
- ↑ immunoglobulin or ↓ complement
o Autoimmune disorder and
inflammatory reaction
- ↑ Carcinoembryonic antigen (CEA)
o Tumor marker associated with
malignancy
- CYFRA 21-1 (cytokeratin fragment)
o More specific
o Used for lung cancer, breast cancer,
urinary bladder cancer

Pericardial Fluid
- Abnormal effusion due to infections,
malignancy, or metabolic change
- Volume: 10-50mL
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
(needed for Vitamin B12 absorption, which is
essential for nucleic acid synthesis)
- An autoimmune disorder that has the presence
of anti-parietal cell or anti-intrinsic factor is
associated with pernicious anemia
o No HCl is produced
- Hydrochloric acid will activate pepsinogen
(produced by the chief cells) to produce pepsin
- Zollinger Ellison Syndrome – it is a gland-like
tumor caused by an adenoma on the islets of
Langerhans of the pancreas
o It also produces gastrin
DUODENAL FLUID o Increased hydrochloric acid
- Very clear fluid and is reach in enzymes - Pepsin will digest proteins (digestion of protein
- Fluid on the first part of the intestine starts at the stomach)
- Aspirating fluid from the duodenum to check
for signs of infection
SPECIMEN COLLECTION
- Biliary atresia: problem in the bile duct, usually
associated with pediatric patients - Method of Collection: gastric aspiration
- 1,200 to 1,500 mL/day - Gastric Tubes:
- pH level: 8.0-8.5 o Ventrol Levin tube: inserted through
o 145 mEq/L of bicarbonate ion – gives the nose
the alkalinity of the duodenal fluid o Rehfuss: inserted through the mouth
o Acidic gastric contents enter the
duodenum Macroscopic Examination
Color Significance
o Acidic pH stimulates the mucosal cells Pale gray with mucus Normal
to produce secretin Yellow-green Large amount of bile
o Secretin will then provoke the pancreas
to secrete bicarbonates Volume Significance
Few mL to 50 mL Normal (fasting specimen)
Secretin and Pancreozymin ≥ 50 mL Abnormal Fasting Specimen
20 to 16 mL up to 120 mL After Ewald’s test meat
➢ Secretin After alcohol test meal or after
o provokes the pancreas to secrete 45-150 mL
histamine accumulation
bicarbonate
o Stimulates watery pancreatic secretion Two type of specimen
with high bicarbonate content ➢ Basal Acid Output (BAO) – Total gastric
o Most sensitive test for impaired secretion during unstimulated fasting state
pancreatic function o 1hr collection: consist of four 15 min.
o Administered intravenously, then DF specimen (common)
bicarbonate is tested o 2hr collection: used for insulin
➢ Pancreozymin hypoglycemia test (uncommon)
o Provokes enzyme production by the ➢ Maximal Acid Output (MAO) – Total gastric
pancreas secretion after gastric stimulation
o 1hr collection: consist of four 15 min.
Pancreatic Cancer vs Chronic Pancreatitis specimen (common)
Pancreatic Cancer Chronic Pancreatitis
Decrease volume Decrease volume
▪ Stimulant: pentagastrin and
Normal bicarbonate Decrease bicarbonate histamine
Normal amylase Decrease amylase o 2hr collection: used for insulin
hypoglycemia test (uncommon)
GASTRIC FLUID ▪ Stimulant: histalog
- Fluid inside the stomach
GASTRIC STIMULANTS
- Highly acidic because of the HCl content Test Meals 1. Ewald’s – bread, tea, or water
- Rich in enzymes 2. Boa’s – oatmeal
3. Riegel’s – beef steak and mashed potato
Gastric Acid Secretion Chemical 1. Pentagastrin: most preferred (1hr)
Stimulants 2. Histamine (1hr)
3. Histalog (Betazole) (2hr)
4. Insulin: assess vagotomy procedure
Sham Feeding Fictitious Feeding (Sandwich)

BAO MAO
BAO/MAO
(mEq/hr) (mEq/hr)
Normal 2.5 25 10%
Pernicious
0 0 0
Anemia
Gastric
- G Cells of the stomach will produce gastrin once 1.0 4.0 25%
Carcinoma
a person eats food Duodenal Cancer 5 30 17%
- Gastrin substance will stimulate Parietal Cells to Zollinger-Ellison
18 25 72%
produce hydrochloric acid and intrinsic factor Syndrome
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)
Terminologies
Term Definition Significance Gastric juice
Test
Euchlorhydria Normal free HCl component
Hyperchlorhydria Increased free HCl Peptic Ulcer Free HCl: 20-80 1.Boa’s test: (+) result-red
Carcinoma of the meq/L 2.Gunzenberg’s test: reagents- vanillin, 95%
Hypochlorhydria Decreased free HCl ethyl alcohol; (+) result-purple red
Stomach
Achlorhydria No free HCl Pernicious Anemia Free Acidity + Na alizanine test: (+) result- violet color with
Achlorhydria: gastric fluid pH is > 3.5 (doesn’t fall even in gastric stimulation) Free HCl = bluish tinge
Anacidity: failure to produce pH < 6.0 following gastric stimulation Total Acidity
Lactic Acid 1.Uffelman’s test: (+) canary yellow
QUALITATIVE TEST FOR FREE HCl 2.Kelling’s test: (+) deep yellow
Dimethylaminoazobenzol (+) Cherry Red 3.Strauss test: 0.05% (+) light green; 0.01% (+)
Gunzberg (+) Purplish Red intense yellow green
Boas (+) Rose Red 4.Reitman (Gradwohl) test: (+) canary yellow
Bile Gmelin’s test: conc. HNO3; (+) band of colors
Renin 1.Reitman(Gradwohl): hydrogen peroxide +
Quantitative Test for Gastric Acidity milk
COMBINED HCL 2.Reigel’s test: hydrogen peroxide + milk +
FREE HCl TOTAL ACIDITY (BOUND TO phenolphthalein; (+) coagulation of milk
PROTEINS) Pepsin 1.Bray’s / Bauer’s test
TITRANT NaOH NaOH NaOH 2.Hammerschlag test
pH DAAB Blood Guiac/ Benzidine test (+) green to blue
Phenolphthalein Na Alizarin
indicator (Topfer’s rgt)
End Point Canary Yellow Faint Pink Violet
Normal DIAGNEX BLUE TEST
25-50° 50-75° 10-15°
Value
Diagnex Tubeless Test
- Uses azure dye combined with HCl
Tests for Lactic Acid
TEST REAGENTS ENDPOINT
- Stimulant: caffeine
Kelling’s FECl3 Yellow - HCl is measured in the intensity of the color of
Modified urine
FECl3 + phenol Yellow
Uffelmann’s - (+) intense blue = increased amount of HCl
Strauss FECl3 + ether Yellow - Test for gastric intubation
Lactic Acid: Indicative of advanced gastric cancer
- Without evacuation tube (alternative method)
- Specimen: Urine
Collection Method
Levin (Ventrol 1. smallest evacuation tube
- Principle:
Levin) 2. most commonly used o Azure Blue is given by mouth
3. propelled through mouth or nostrils o The presence of azure blue in urine
Boa’s / 1. Ideal for emptying or washing the stomach indicates the presence of Free HCl in
Ewald’s 2. For cases of poisoning the stomach
3. Flask is present at the tip
Rehfuss 1. Metallic tip
2. Propelled in mouth MICROSCOPIC ANALYSIS
Miller-Abbott 1. Mercurial tip is chilled
• Pus cells/WBC: Stomach abscess, chronic
2. People who are easy to vomit
Sawyer 1. Longest evacuation tube gastritis, gastric cancer
Kaslow 1. Softest evacuation tube • RBC: Ulcer or trauma
Jutte 1. Stylet tip • Yeast Cells: Fermentation in the stomach
because large amount of food have been
Stimulants (Test Meal) retained
Ewald’s 1. Routine test for gastric juice exam • Bacteria
2. “breakfast-test meal”
3. Content: 40 g of bread + 400 ml of water or tea
• Food residues
Dock’s 1. Modification of Ewald’s • Parasites
2. Content:40 g of shredded whole wheat biscuit +
400 ml of water or tea
Riegel’s a. Ideal for determining hypoacidity and achylia
b. Content: 100-150 g broiled beef steak + 150-
200g mashed potatoes + 400 ml bouillion soup
Fischer’s 1. Same value as Riegel’s but gives increased
acidity values
Heckman’s 1. 2% methylene blue, albumin, distilled water
Boa’s 1. Ideal for lactic acid determination
2. 1 tbsp of oatmeal in 1 drop of water + pinch of
salt
Stasis 1. Supplemented by barium meals
2. Undergoes fluoroscopic determinations
3. 2 ounces of half-cooked rice + 12 raisins
Lavine/ 1. Utilize 70% alcohol
Alcohol
Motor 1. Spinach or raisins + 400 ml water

Chemical Stimulants
• Histamine-Phosphatase
• Insulin
• Caffeine
• Pilocarpine & Acetylcholine
• Pentagastrin
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

PHYSICAL EXAMINATION
SPUTUM Volume
- Derived from alveoli, trachea, bronchi of the No specific volume
pulmonary tract. ➢ Small amount – not always normal; the sputum
- Normally produced in the upper and lower is still adhering on the lungs
respiratory tract o early PTB
- It is secreted by goblet cells found in the o acute bronchitis
bronchial lining o pneumonia
o Responsible for secretion of mucus from ➢ Over 100cc/24hrs
the bronchial tree o pulmonary edema: increased amount of
- Normal Condition: a mucus secretion of goblet water in the lungs
cells and other organs associated with o Broncheictasis: characterized by
respiratory epithelium bronchial dilatations of bronchi
- It should be differentiated from saliva based on ▪ widened, thickened, and
the presence of macrophage (dust cells or permanently damaged
carbon laden macrophage) o Lung abscess: lesion on the lungs
- Acceptable if: because of infection
o < 10 squamous epithelial cells /LPF ▪ Necrosis of the pulmonary tissue
o > 25 WBC /LPF ▪ Necrotizing pneumonia or lung
- Mixture of plasma, electrolytes, mucin and water gangrene – multiple small
abscess is formed
➢ Over 500cc/ 24hrs
o Amoebic abscess: infection caused by
parasite
➢ Over 1,000cc/ 24hrs
o severe bronchiectasis
o cavity TB: hole on lungs/ white spots on
the lungs
o chronic bronchitis
o acute edema of the lungs

Odor
Normal: Odorless (>24hours standing: offensive)
➢ Sweetish odor – candy smell
SPECIMEN COLLECTION o cavity TB
- Time Collection: Early Morning o Bronchiectasis
o highest concentration of bacteria (small o Pseudomonas infection
amount may be used for examination) ➢ Putrid Odor – presence of bacteria
- Collection: 3 consecutive days o Gangrene
- Other collection: o Necrotizing tumor
o 24-hour o Bacterial infection
o Throat swab ➢ Cheesy Odor
o Sputum induction (sodium chloride o Emphysemas: accumulation of pus cells
solution or NSS) in the cavity of the lung
o Tracheal aspiration ▪ Enlargement of alveoli
- Precautions: ▪ Doesn’t shrink and expand
o Deep cough o Carcinoma of the lungs
o Rinse the mouth properly ➢ Fecal odor
o Wide mouth bottle/container/sterile o Liver abscess

Color
Normal: Colorless; Contents: Mucus
➢ White/Yellow – increase in pus cell
➢ Gray color – influence of pus and epithelial cells
➢ Green color – presence of Pseudomonas
bacteria or Bile pigment (pyoverdine and
pyocyanin)
➢ Red color – presence of blood
o Hemoptysis: coughing out blood
▪ Bright red: alkaline reaction
▪ Dark brown or Dark red: acid
reaction
o Hematemesis: vomiting blood from
stomach
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

Causes of Blood Stained Sputum ➢ Lung stone (Pneumoliths or Bronchioliths) –

Appearance
Uniform rusty color, pus is
present
Uniform rusty color with no pus
Bright streaks in viscid sputum
Currant jelly appearance
Produces mucoid texture
Scant but persistent streaks in
mucoid sputum
Episodic occurrence of small
hemorrhages
Episodes of large hemorrhages
Spurious hemoptysis
Usual Causes fragment of calcified Lung tissue
Pneumococcal pneumonia o Calcified TB
Lobar pneumonia
o Histoplasmosis
Congestive Heart Failure
Klebsiella pneumoniae ➢ Bronchial Cast – whitish branching tree
o Diphtheria
➢ Cheesy Masses – fragment of Necrotic tissue/
Bronchogenic carcinoma Pulmonary tissue
o Cavity TB
Tuberculosis
o Lung gangrene
Cavitary TB, pulmonary
infarction, fungal pneumonia MICROSCOPIC EXAMINATION
Bleeding in nose/ nasopharynx

➢ Rusty / Anchovy sauce appearance – due to old

blood
o amoebic abscess
o cardiac pigments
o pneumonia
o gangrene
➢ Black color
o inhalation of dust/ dirt
o inhalation of charcoal/ carbon
o heavy smoker
➢ Olive green or grass green
o Cancer

pH
- Normal: 6.5-7.0 (almost neutral)
- Saliva’s pH: 6.2-7.2

Consistency OTHER CELLS/CRYSTAL

Normal: Viscous ➢ Elastic fibers: PTB, protein fiber
➢ Mucoid ➢ Hematoidin crystals: Bleeding (also present in
o Bronchial asthma stool sample)
o Acute bronchitis (SG 1.004-1.008) ➢ Heart failure cells: made up of hemosiderin
➢ Serous – combination of mucoid and phlegm (round, colorless structure) found in congestive
o Has greenish tinge heart failure
o Lung edema (SG Above 1.037) ➢ Myelin globules: spherical/pear shape (no
➢ Purulent clinical significance)
o Bronchiectasis o Blastomyces: has clinical significance
o cavity TB (SG 1.015-1.060) ➢ Yeast

Specific Gravity Microscopic Formed Elements in Sputum

➢ Mucoid: 1.004 to 1.008 Observations
➢ Purulent: 1.015 to 1.060 Numerous squamous epithelial
cells
➢ Serous: 1.037 or higher Numerous neutrophils (often
with intracellular or
MACROSCOPIC EXAMINATION extracellular organisms)
Structures Numerous eosinophils
➢ Curschmann’s spiral – yellowish/whitish spiral Numerous neutrophils,
mucoid threads abundant mucus, with few or
o Coiled mucus strand more organisms
PAS positive macrophage
o Bronchial asthma (CREOLA and charcot
PAS positive rounded bodies
leyden crystals are also seen) that take silver stain
Seen in immunocompromised
Lipid droplets in macrophages
Curschmann spiral (coiled
mucus filaments)
Charcot-Leyden Crystals
Dense, crystalline concretions
Usual Causes
Specimen is mostly saliva
Bacterial or Fungal Pneumonia
Asthma or pulmonary
hypersensitivity reaction
Chronic bronchitis or
bronchiectasis
Alveolar proteinosis
Pneumocystis carinii
Lipid or aspiration pneumonia
Asthma or obstruction of small
bronchi
Asthma
Broncholithiasis

➢ Dittrich’s plugs – yellowish/ grayish caseous 3 LAYERS OF SPUTUM

matter in a size of a Bean/Pin After 24 hours of standing
o Very small spherical-shaped structure ➢ Carbohydrate mucus layer: upper layer
usually seen in Broncheiciasis ➢ Opaque watery material: middle
o Head that produces a foul odor when ➢ Sediments: 3rd layer or bottom
crushed
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

RBC COUNT
- Diluted with isotonic saline
- Allow to settle at 5 minutes

- Alveolar Hemorrhage
o Phagocytosed RBC: alveolar hemorrhage
within 48 hours
o Hemosiderin-laden Macrophage: alveolar
hemorrhage >48 hours
- A method for obtaining cellular and ▪ orange-red sample
microbiological information from the lower
respiratory tract.
DIFFERENTIAL COUNT
- Collection: bronchoscopy or bronchoalveolar
washing - Prepared by cytocentrifugation
- Used to collect samples from the deepest part of - At least 300 cells, often 500 to 1000 cells are
the lungs counted and classified
- Saline infused by bronchoscope mixes with the - Cells seen:
bronchial contents o Macrophages
- High-Resolution Computerized Tomography o Lymphocytes
- Instillation Volume: 100 and 300 mL of Sterile o Neutrophils
saline in 20 to 50-mL aliquots o Eosinophils
- It should be processed within 30 minutes (STAT) o Ciliated columnar bronchial epithelial
- Should be placed in an ice box when transported cells, and squamous epithelial cells
- Use nutrient broth if ice box is not available (can Bronchioalveolar Lavage Cells
be viewed within 24 hours) Cells Normal Values
- Container: siliconized glass or non-cell adherent Macrophage
56-80%
(dust cells)
plastic
1-15%
- Uses: ↑ Interstitial Lung Disease, Drug Reaction,
o Evaluating immunocompromised Lymphocytes
Pulmonary Lymphoma, Non-Bacterial
patients (check for the presence of ) Infections
o Interstitial lung disease <3%
Neutrophils ↑ Cigarette Smokers, Bronchopneumonia,
o Airway diseases
Toxin Exposure, Diffused Alveolar Damage
o Suspected alveolar hemorrhage <1-2%
o 2 samples: The first aliquot is discarded Eosinophils ↑ Asthma, Hypersensitivity, Pneumonitis,
(sample consists of the subsequent 3 to Eosinophilic Pneumonia
5 aliquots) Ciliated Columnar 4-17%
Bronchial Epithelial ↑ More Numerous in Washing than in
▪ Bronchial sample
Cells Lavage
▪ Alveolar sample

HEMATOLOGY
➢ Color: colorless (normal)
o milky white or light brown-beige:
accumulation of phospholipid protein
complex seen in patients with
pulmonary alveolar proteinosis)
o red: glossy bloody; alveolar hemorrhage
o orange-red: older hemorrhagic
syndrome
➢ Clarity: clear, hazy, cloudy, turbid
➢ Presence of clots: presence of protein or there is
an alveolar leakage
o Check for fibrinogen (+ blood)
➢ Volume
➢ Cell counts and differential counts

WBC COUNTS AUTOMATED REAGENT STRIP READERS

- Diluted using a Unopette diluting system
- Lysis of RBC: 1/100 ammonium oxalate or the
1/20 glacial acetic acid
- Count all the cells in the 18 squares
- Spectrophotometric measurement of light
- Reflectance refractometry
o The intensity and concentration is
inversely proportional to the reflected
light
- Light-Emitted Diode (LED)
o provide the specific wavelength needed
for each test pad color reaction
o Example: Clinitek 50 or Clinitek Status
 ANALYSIS OF URINE AND BODY FLUIDS (LECTURE)

AUTOMATED URINALYSIS ANALYZERS

- Today, urinary screening is one of the most
frequently performed analyses in laboratory and
requires:
o Accurately detect and enumerate the
bacteria and particles present in urine
specimens
o Rapidly confirm whether a patient could
have a urinary tract infection (UTI), or
orientate towards renal pathologies.

SMART TECHNOLOGY
- SYSMEX – First corporation to develop machines
- The UF range of instruments is a fully automated,
urinary screening system which bases its
objective analysis on both physical and chemical
particle properties.
- It uses:
o Advanced flow cytometry technology
with hydrodynamic focusing
o Specific fluorescent dyes for bacteria
and sediment
o Three high-definition, reproducible
measurement signals: size, structure,
and fluorescence

TIME-SAVING
- Simply Load and Go
- Minimum hands-on time, maximum workflow
efficiency
- Results in approximately 1 minute
- Continuous loading function for immediate
processing of samples or series testing with up to
50 positions
- Bi-directional connection to LIS

Optical diode laser reading system

or UF-1000i/UF-500i

HIGH-QUALITY, STANDARDIZED RESULTS

- Accurate detection and enumeration of urine
particles (2 separate analysis channels for
bacteria and sediment)
- Reduced cross-contamination with an anti-
carryover function and sequential analysis mode
- Detection of low bacteria concentrations as of
10³ CFU/mL
- Barcode reader identification of samples and
reagents
- Daily Quality Control performed for all the main
parameters (high and low positive controls)
- Negative Predictive Value: 98.5%¹ for a cut-off of
100 bacterial particles/μl