CLINICAL MICROSCOPY

GLANELA A. MANALOTO
SAFETY AND QUALITY ASSESSMENT
Terminologies:
1. CDC- Centers for Disease Control and Prevention
2. OSHA- Occupational Safety and Health Administration
3. CLSI-Clinical and Laboratory Standards Institute
4. PPE- Personal Protective Equipment
5. UP- Universal Precautions
6. BSI-Body Substance Isolation
7. NFPA- National Fire Protection Association

TYPE OF SAFETY HAZARDS

“BIOLOGIC HAZARD”
A. SOURCE – Infectious agents
B. Possible Injury – bacterial , fungal, viral or parasitic
infections

 Chain of Infection- A continuous link on understanding on

how microorganisms are transmitted.
Infectious agent- e.g Bacteria, fungi, parasites,
viruses
Reservoir- e.g Animals, Humans, Fomites, Insects, Blood
, Body fluids
Portal of exit –e.g Nose, mouth , Mucous membranes
Mode of transmission- e.g Droplet, Airborne, Contact,
Vector, Vehicle
Portal of entry-e.g Nose, mouth ,mucous membranes,
skin, Unsterile equipment
Susceptible Host- e.g Patients, Elderly, Newborns,
Immuno-compromised, Healthcare workers

MODE OF TRANSMISSION

AIRBORNE a. Centrifugation of unstoppered tubes

/AEROSOL b. Heating cultures of specimens too
rapidly
c. Sterilization of inoculating loops in
the bunsen burner flame
d. Leakage from a container that holds
contaminated specimens
e. Broken centrifuge and spills

INGESTION a. Failures to wash hand

b. Eating
c. Drinking
d. Smoking
e. Applying cosmetics
f. Pipeting with mouth
DIRECT a. Needlesticks
INOCULATION b. Broken glass
c. Animal bites
d. Small scratches
MUCOUS -infection may occur if the organism can
MEMBRANE directly enter through the mucous membranes
such as through the conjuctiva of the eye

ARHTROPODS / -infectious sources includes ticks, fleas,

VECTOR and mosquitos, which may harbor various
microorganisms

 Biologic Waste Disposal-

- All biologic waste, EXCEPT urine, must be placed In
appropriate containers labeled with biohazard symbol.
- All biological specimens, except urine, must be
sterilized or decontaminated before disposal
- Urine may be discarded by pouring it into a laboratory
sink under a Plexigas countertop shield. Care must be
taken to avoid splashing, and the sink should be
flushed with water after specimens are discarded.
Disinfection of the sink using a 1:5 or 1:10 dilution
of SODIUM HYPOCHLORITE should be performed daily.
- Empty urine containers can be discarded as non-
biologically hazardous waste.

A 0.5% bleach solution, prepared by adding 1 part household

bleach to 9 parts water (1/10 dilution), is stable for 1 week

“SHARP HAZARDS”
A. SOURCE- Needles/Syringe,lancet, broken glasswares
B. Possible Injury- cuts, punctures, or blood-borne pathogen
exposure

 All sharp objects must be disposed in puncture-resistant,

leak-proof container with the biohazard symbol.
 The biohazard sharp containers should not overfilled and
must always be replaced when the safe capacity mark is
reached
“CHEMICAL/POISON HAZARDS”
A. SOURCE- Preservatives and reagents
B. Possible Injury- Exposure to toxic, carcinogenic, or
caustic agents

 Hazardous chemicals should be labeled with a description

of their particular hazard, such as poisonous, corrosive,
flammable, explosive, teratogenic, or carcinogenic.
 In case of chemical spills, when skin contact occurs, the
best aid is to flush the area with large amount of water
for at least _________, then seek medical attention

 Material Safety Data Sheets (MSDS)- Contain the

information about the chemical hazards
1. Physical and chemical characteristics
2. Fire and explosion potential
3. Reactivity potential
4. Health hazards and emergency first aid procedures

5. Methods for safe handling and disposal

6. Primary route of entry

7. Exposure limits and carcinogenic potential

“RADIOACTIVE HAZARDS”
A. SOURCE- Equipment and radioisotopes
B. Possible Injury- Radiation exposure

 The amount of radiation exposure is related to a

combination of time, distance, and shielding.
Exposure to radiation during pregnancy presents a danger to
the fetus; personnel who are pregnant or think they may be
should avoid areas with this symbol

“ELECTRICAL HAZARDS”
A. SOURCE- Ungrounded or wet equipments; frayed cords
B. Possible Injury- Burns or shock

 Equipment should not be operated with wet hands

 Laboratory personnel should continually observe for any
dangerous conditions, such as frayed cords and overloaded
circuits, and report them to the supervisor.
 All electrical equipment must be grounded with
_______________________.

 When an accident involving electrical Shocks occur:

1. ______________________________________________
2. ______________________________________________
3. ______________________________________________

“FIRE/EXPLOSIVE HAZARDS”
A. Source : Open flames, organic chemicals
B. Possible Injury: Burns or dismemberment

 When a fire is discovered, people are expected to :

Rescue- anyone in immediate danger
Alarm- activate the institutional fire alarm system
Contain- close all doors to potentially affected areas
Extinguish/Evacuate- attempt to extinguished the fire, if
possible or evacuate, closing the door

 To operate fire Extinguisher:

P-ull the pin
A-im at the base of fire
S-queeze handles
S-weep nozzle side to side

Types Of Fire and Fire Extinguisher

Fire Extinguishing Material Extinguisher

Type
Class Ordinary combustibles: Water, Dry chemicals,
A Wood,paper,clothing/garments, Steam
plastic

Class Flammableorganicchemicals/liquids: Dry chemicals, carbon

B gasoline ,paints, oil dioxide, foam , or
halon
Class Electrical equipment: Dry chemicals, carbon
C Machines, motor switches, plugs dioxide, or halon

Class Combustible metals: Sand or dry powder,

D (Hg, Mg,Na , and Li) Metal X
 Dry chemicals
CLASS Detonation or Arsenal fire Allowed to burn out and
E nearby materials are
protected
Class Grease, oils, fats Liquid designed to
K prevent splashing and
cool the fire.

“PHYSICAL HAZARDS”
A. SOURCE- Wet floors, heavy boxes, patients
B. Possible Injury- Falls, sprains, or strains

 General Precautions
1. Avoid running in rooms and hallways
2. Watch for wet floors
3. Bend knees when lifting heavy objects
4. Keep long hair pulled back
5. Avoid dangling jewelry
6. Maintain a clean organized work area
7. Use a Closed-toed shoes

HAND HYGIENE- includes both hand washing and using alcohol

based antiseptic cleansers.

 Hand contact is the primary method of infection

transmission
 Father of handwashing: Dr. Ignaz Semmelweis
Hand Washing
 Laboratory personnel must always Songs:
sanitize hands a.Happy Birth
1. Before patient contact day
2. After gloves are removed b.Twinkle
3. Before leaving the work area Twinkle little
4. Anytime when hands have been star
knowingly contaminated c.Alphabet song
5. Before going to designated break
areas
6. Before and after using bathroom facilities

 ALCOHOL-BASED CLEANSERS- Used when hands are

______________________

 HAND WASHING – Used when hands are

_______________________________

CDC Hand Washing Procedure

1. Wet hands with warm water
2. Apply anti-microbial soap
3. Rub from alather, create friction, and loosen debris.
4. Thoroughly clean between fingers, including thumbs, under
fingernails and rings, and up to the wrist, for at least
15/20 seconds.
5. Rinse hands in a downward position
6. Dry with a paper towel
7. Turn off faucets with a clean paper towel to prevent
recontamination

Degree of Hazard
O No hazard
1+ Slight Hazard
2+ Moderate Hazard
3+ Serious Hazard
4+ Extreme Hazard

RENAL FUNCTION

Renal Physiology
a. Each kidney contains approximately _______________
functional units called nephrons
b. Cortical nephron- makes up approximately 85% of the total
nephron. Found mainly in the cortex of the kidney and are
responsible primarily for removal of waste products and
reabsorption of nutrients.
c. Juxtamedullary nephrons- have loops of Henle that extend
deep into the medulla of the kidney. Their primary
function is the concentration of urine

GENRAL FUNCTIONS OF THE KIDNEY

-EXCRETORY FUNCTION
a. Glomerular filtration
b. Tubular reabsorption
c. Tubular secretion
-Regulation of water balance in the body.
-Regulation of acid-base balance
-Regulation of electrolytes
-Regulation of Blood pressure through secretion of Renin
-Stimulates Erythropoiesis through secretion of EPO

Renal Blood flow

 The renal artery supplies blood to the kidney
 The human kidney receives approximately______ of the
blood pump.
 Total renal blood flow: _____________________
 Renal plasma flow : ______________________

Hydrostatic pressure – pressure that is created by the varying

sizes of the arterioles, which is important for glomerular
filtration and to maintain consistency of glomerular capillary
pressure and renal blood flow within the glomerulus.

Order of Blood flow In the Nephron

Renal artery→ Afferent arteriole→ glomerulus→efferent
arteriole→Peritubular capillaries→Vasa recta→ Renal vein

Order of Urine formation from the nephron

Glomerulus→ Bowman’s capsule → PCT → DLH →ALH→DCT→ Collecting
Ducts

GLOMERULAR FILTRATION

CHARACTERISTIC:
 The glomerulus consists of a coil of approximately eight
capillary lobes referred to collectively as the
capillary tuft
 The glomerulus is located within the Bowman’s capsule
  A non-selective filter for plasma substances with
Molecular weights of less than _____________
 Normally, the fluid leaving the glomerulus has a
specific gravity of 1.010

Cellular Structure of Glomerulus:

 Plasma filtrate must pass through three cellular layers:
1. Capillary wall membrane
2. Basement membrane
3. Visceral epithelium of Bowman’s capsule

Barrier’s that prohibits the filtration of Large molecules

 The capillary wall of glomerulus is fenestrated
 Intertwining foot processes –___________________-
 ________________ - repels molecules with a negative
charge even molecules are small enough to pass ( Example:
Albumin)

GLOMERULAR PRESSURE
 Juxtaglomerular apparatus- maintains the glomerular
blood pressure
a. ______________________ - found in the afferent
arteriole, secretes the Renin eznyme
b. __________________- found in the DCT, sensor of change
in blood pressure

↓ Blood Pressure = Dilation of afferent arteriole ,

Constriction of efferent arteriole
↑ Blood pressure = Constriction of afferent arteriole,
Dilation of efferent arteriole

Renin-Angiotensin-Aldosterone System
-Controls the regulation of the flow of blood to and within
the glomerulus.
-Functions:
1. Dilation of the afferent arteriole and constriction of
the efferent arteriole
2. Stimulation of sodium reabsorption in the proximal
convulated tubule
3. Triggers the adrenal cortex to release the sodium-
retaining hormone, aldosterone, to cause reabsorption of
sodium and excretion of potassium in the distal
convoluted tubule and collecting duct
4. Triggers release of antidiuretic hormone by the
hypothalamus to stimulate water reabsorption in the
collecting duct
Stimulus: Decrease BP/Low Plasma sodium

TEST FOR GLOMERULAR FILTRATION

Clearance test =BEST INDICATOR OF OVERALL GLOMERULAR FUNCTION

A.
1. Inulin clearance test
- _____________________
- Inulin is a polymer of fructose, is an extremely
stable substance that is not reabsorbed or secreted by
the tubules. It is not a normal body constituent,
 however, and must be infused by IV at a constant rate
throughout the testing period.
2. Creatinine clearance test = most commonly used
clearance test
3. Cystatin C
4. Beta 2 microglobulin
5. Radioisotopes
6. Urea clearance test = earliest clearance test

Formula for the computation of GFR using the creatinine

clearance test

C = Urine creatinine___ X volume of urine/24hours x

1.73
Plasma creatinine
A

NOTE  -By far the greatest source of error in any clearance

procedure utilizing urine is the use of improperly timed urine
specimens.

CYSTATIN C
A small protein (molecular weight 13,359) produced at a constant rate by all nucleated cells.
It is readily filtered by the glomerulus and reabsorbed and broken down by the renal tubular
cells. It has potential as a marker for long-term monitoring of renal function
 Its plasma concentration is inversely related to GFR. (Increase plasma cystatin C =
decrease GFR)
 The rate of production is not affected by muscle mass, sex, or race

Beta-2-microglobulin
It dissociates from human leukocyte antigens (MHC class I) at a constant rate and is rapidly
removed from the plasma by glomerular filtration. It is a better marker of reduced renal
tubular function than of glomerular function.

Estimated Glomerular Filtration Rate (eGFR)Computation

1. MDRD(Modification of Diet in Renal Disease) – most

frequently used formula
Original MDRD GFR = 173 × serum creatinine–1.154 × age–0.203 × 0.742 (if patient is
Formula female) × 1.212 (if patient is black)
MDRD-IDMS GFR = 175 × serum creatinine–1154 × age–0.203 × 0.742 (if patient is
Traceable female) × 1.202 (if patient is black/African-american)
formula

Other MDRD
formula

2.
Cockroft and
gault formula

3.
CKD-EPI (Chronic eGFR(mL /min/1.73 m2) 141 x
Kidney Disease min(SCr/k,1)a x max(SCr/k,1).209
Epidemiology x0.993Age x (1.018 if female) x (1.159
if Black)
Collaboration)
formula

TUBULAR REABSORPTION
 The body must not lose 120mL of water-containing
essential substances every minute.
 The loss of tubular function is capability is often the
first function affected in renal disease

Urine composition
a. 95 % water
b. 5 % solutes - Total solute in 24’hours = 60 grams
( 35 grams organic substances , 25 grams inorganic
substances )

TWO MECHANISM OF TUBULAR REABSORPTION:

Active transport – the substance to be reabsorbed must
combine with a carrier protein contained in the membranes
of the renal tubular cells. This transport requires
energy
Passive transport- the movement of molecules across a
membrane as a result of differences in their
concentration or electrical potential on opposite sides
of the membrane. It is Characterized by movement of a
substance from an area of higher concentration to one of
lower concentration.

Substance Location
-Glucose, Amino -Proximal
acid, Salts convoluted tubule
Active Transport
-Chloride -Ascending loop of
Henle
-Sodium
-PCT and DCT
-Water -PCT, DCT, DLH

Passive Transport -Urea(40%) Proximal convoluted

tubule and
ascending loop of
-Sodium Henle

Ascending loop of
Henle
Note 
- Passive reabsorption of water takes place in all parts of
the nephron except the ________.

- Sodium is actively transport in all part of the nephron

except in the Ascending loop of henle

-The plasma concentration at which active transport stops is

termed the renal threshold
Ex: Glucose renal threshold is _____________ mg/dl or
equivalent to 350mg/mi
RENAL CONCENTRATION
- Renal concentration begins in the descending and
ascending loop of henle and the final concentration of
urine continues to the Collecting Duct.

Effect of Anti- Diuretic Hormone (Vasopressin) on Renal

Concentration
 ADH- hormone responsible for reabsorption of ___________
in the distal convoluted tubules and collecting ducts of
the kidney.
↑ Body Hydration = ↓ADH = ↑ Urine Volume (Dilute)
↓ Body Hydration = ↑ADH = ↓Urine volume ( Concentrated)

TEST FOR TUBULAR REABSORPTION

A. Osmolality test – measures only the number of particles

on solution
The normal urine to serum ratio should be 1:1 tO 3:1.

B. Specific gravity- measures the number and size of

particles on solution

C. Water deprivation test

 ________________– patients deprived of fluids for 24

hours before measuring Specific Gravity
 ________________ – compared the volume and S.G of
day and night urine samples

RENAL SECRETION
 Involves the passage of substances from the blood in the
peritubular capillaries to the tubular filtrate
 Substances are removed from the glomerular
filtrate and returned to the blood
 Two major function of tubular secretion:
a. Elimination of waste products not filtered by the
glomerulus
b. Regulation of acid-base balance in the body through
the secretion of ____________________

Note  - a disruption of secretory function of the renal can

result in metabolic acidosis or renal tubular acidosis,
wherein the kidney is unable to produce an acid Urine ( In
short : urine is alkaline )

TEST FOR RENAL SECRETION AND BLOOD FLOW

 A. PSP(phenolsulfonphthalein) dye excretion test –
obsolete test
B. PAH(Para amino hippuric acid) test – most commonly used
C. Titrable acidity
D. Urinary ammonia

Measurement of urine pH, titratable acidity, and urinary

ammonia can be used to determine the defective function. The
tests can be run simultaneously on either fresh or toluene-
preserved urine specimens collected at 2-hour intervals from
patients who have been primed with an acid load consisting of
oral ammonium chloride. By titrating the amount of free H+
(titratable acidity) and then the total acidity of the
specimen, the ammonium concentration can be calculated as the
difference between the titratable acidity and the total
acidity

INTRODUCTION TO URINALYSIS

HISTORY AND IMPORTANCE

 References of the study of urine can be found in the
drawings of cavemen and in Egyptian hieroglyphics, such
as the Edwin smith surgical papyrus.
 Urine is a fluid biopsy of the kidney and provides a
“fountain” of information.

Hippocrates Wrote the book of “uroscopy”

Frederik Discovered albuminuria by boiling urine
Dekker
Thomas Bryant Published a book about “Pisse Prophets”
Thomas Addis Addis count
Richard Bright Introduced the concept of urinalysis as part
of a doctor’s routine patient examination
Thudicum Urochrome – the pigment that causes yellow
color of urine

REASONS For Performing Urinalysis ( CLSI)

1. Diagnosis of disease
2. Screening asymptomatic populations for undetected
disorder
3. Monitoring the progress of disease
4. Monitoring the effectiveness of therapy

URINE COMPOSITION
 Urine consists of urea and other organic and inorganic
chemicals dissolved in water.
 Urine is normally 95 % water and 5 % solutes
 The single most useful substance that identifies a fluid
as urine is its uniquely high creatinine concentration
(approximately 50 times that of plasma).
Urea Primary organic component.
Product of protein and amino
acid metabolism

Creatinine Product of creatine

metabolism by muscles

Uric acid Product of nucleic acid

breakdown in food and cells

Chloride Primary inorganic component.

Found in combination with
sodium and many other
inorganic substances

Sodium Primarily from salt, varies

by intake

Potassium Combined with chloride and

other salts

Phosphate Combines with sodium to

buffer the blood

Ammonium Regulates blood and tissue

acidity

Calcium Combines with chloride,

sulfate, and phosphate

NOTE 
- Urea is the major organic component of urine
- Chloride is the major inorganic component of urine
followed by Sodium then Potassium
- A high urea and creatinine content can identify fluid
as urine.

URINE VOLUME

 Urine volume depends on the amount of water that the

kidneys excrete.
 Factors that influence urine volume include –fluid
intake, fluid loss from non renal sources, variations in
the secretion of ADH, and need to excrete increased
amounts of dissolved solids, such as glucose or salts.
 Normal daily urine output is usually 1200 to 1500 mL, a
range of 600 to 2000 mL is considered normal

 The kidney excrete two to three times more urine during

the day than during the night
 A. __________ – decrease urine output
 Less than 1mL/kg/hr in infants
 Less than 0.5 mL/kg/hr in children
 Less than _________________
Causes : dehydration, vomiting, diarrhea, perspiration,
severe burns

B. ____________- increase in daily urine output

 2.5 to 3ml/kg/day in children
 _______________________
Causes: Diabetetes mellitus, diabetes insipidus,
diuretics, caffeine, alcohol

Analysis of urine in Differentiating between DM and DI

 Due to defect in the pancreatic
Diabetes production of insulin
Mellitus  ___________Urine Specific
gravity
 Increase urine Glucose
( glucosuria)
 Due to decrease production or
Diabetes function of ADH
Insipidus  ___________Urine Specific
gravity

C. ___________- increase excretion of urine at night. Common

among pregnant women , and urine has a specific gravity
of less than 1.018

D. ___________- cessation of urine flow, or no urine output.

Causes: damage to the kidneys, Renal stones, and renal

tumors

SPECIMEN COLLECTION
 Urine specimens should be delivered to the laboratory
promptly and tested within _____________
 Never discard a specimen before checking with a
supervisor

I. Characteristic of Containers Used for Urine collection

 clean
 Dry
 Leak proof
 With Screw top lids – they are less likely to leak
than snap-on lids
 Wide mouth
 Wide flat bottom
 Made of sterile material
 Sterile

- the recommended container capacity is ___

- the required specimen volume for urine microscopic
analysis is _____ , average of 12 ml
 II. Labels
 Patients name
 Patient identification number
 Date and time of collection
 Additional information such as age, sex, etc.
- Labels must be attached to the____________, not to the
lid and should not become detached if the container is
ref/frozen.

III. REQUISITION FORM

 A requisition form must accompany specimens
delivered to the laboratory

IV. WHEN TO REJECT SPECIMEN? 

1. Specimen in unlabeled containers
2. Non matching labels and requisition forms
3. Specimens contaminated with feces or toilet papers
4. Containers with contaminated exteriors
5. Specimens of insufficient quantity
6. Specimens that have improperly transported

V. SPECIMEN PRESERVATION
- A specimen that cannot be delivered and tested within 2
hours should be refrigerated or have an appropriate
chemical preservative added

URINE PRESERVATIVES
Preservative Advantages Disadvantage Additional
s s Information
Refrigeratio Does not Prevents
n interfere with PRECIPITATES bacterial growth
*the easiest chemical tests AMORPHOUS for 24 hours
and most CRYSTALS
common
Thymol Preserves Interfere
glucose and with acid
sediments well precipitatio
n test for
protein
Boric acid Preserves May Keeps pH at 6.0
protein and precipitate -bacteriostatic
formed elements crystals
well when used in
Does not large
interfere with amounts
routine
analyses other
than pH
Formalin EXCELLENT Acts as Can also be used
SEDIMENT reducing for
PRESERVATIVE agent, cytology(Brunzel
Interfere )
with
chemical
tests for
 glucose,
blood,
leukocyte
,esterase,
and copper
reduction
- False-
negative
reagent
strip tests
for blood
and
urobilinogen
Toluene Does not Floats on
interfere with surface of
routine test specimens
and clings
to pippete
and testing
materials
Sodium PREVENTS Inhibits May use sodium
Fluoride GLYCOLYSIS reagent benzoate instead
strip tests of fluoride for
ALSO GOOD FOR for glucose, reagent strip
DRUG ANALYSIS blood, and testing
leukocytes
Phenol Does no Causes an Use 1 drop per
interfere with odor change ounce of
routine test specimen
Gray C and Preserves Decreases Preservative is
S tube bacteria pH; do not boric acid
Sample stable use if urine
at RT for 48hr is below
minimum fill
line
Cherry red/ Stable for 72 Bilirubin Preservative is
yellow top hours and sodium
tube urobilinogen propionate
may be
decreased if
specimen is
exposed to
light and
left at RT
Yellow Plain FOR AUTOMATED Must Round or conical
UA INSTRUMENTS refrigerate bottom
within
2 hours
Saccomanno Preserves Used for
fixative cellular CYTOLOGICAL
elements EXAMINATION
Sodium Inexpensive Unacceptable For quantitative
carbonate • Stabilizes for analysis of
porphyrins, urinalysis porphyrins,
porphobilinogen testing porphobilinogen,
, etc. etc
CHANGES IN UNPRESERVED URINE
( Strasinger )
Analyte Change Cause
Color Modified / Oxidation or reduction of
Darkened metabolites
Ph Increased Breakdown of urea to ammonia
by urease-producing bacteria /
loss of CO2
Bacteria Increased Multiplication
Odor Increased Bacterial multiplication or
breakdown of urea to ammonia
Nitrite Increased Multiplication of nitrate
reducing bacteria
Clarity Decreased Bacterial growth, and
precipitation of amorphous
material
Glucose Decreased Glycolysis and bacterial use
Ketones Decreased Volatilization and bacterial
metabolism
Bilirubin Decreased Photo oxidation to
biliverdin / light exposure
Urobilinogen Decreased Oxidation to urobilin
RBC, WBC, and Decreased Disintegration in dilute
casts alkaline urine
Trichomonads Decreased Loss of characteristic
,motility and death
NOTE  Protein/Albumin is not affected.

Types of Urine Specimen

Random  ___________________________
Specimen  Easy to collect and convenient
 For Routine screening
 Can be collected at any time,
usually during daytime hours, and
without prior patient preparation
First Morning  Ideal urine specimen for routine UA
specimen  The most concentrated specimen
Or 8-hour  Specimen that is ideal to test
specimen for substances that require
concentration or incubation for
detection
 ___________________
 ___________________
 ____________________
Second morning  For glucose or diabetic monitoring and
/ fasting screening
specimen
2- hour post  Collect urine after 2 hours of meal
prandial  ________________________
specimen
Timed  For ________________________
specimens(E.g  Urine specimen for clearance test
24 hours urine  Urine specimen for evaluation of
specimen) fistulas
 To obtain an accurate timed specimen,
the patient must begin and end the
collection period with an empty bladder.
 All specimens should be refrigerated or
kept on ice during the collection period
and may also require addition of a
chemical preservative.
 On its arrival in the laboratory, a 24-
hour specimen must be thoroughly mixed
and the volume accurately measured and
recorded
Afternoon  Preferred for urobilinogen measurements
urine(2pm to
4pm)
12 hours urine  Ideal for screening microalbuminuria
specimen (Brunzel)
 For determination of urine albumin,
creatinine, and the albumin-to
creatinine ratio
Catheterized  ______________
specimen
Note: If a routine urinalysis is also
requested, the culture should be performed
first to prevent contamination of the
specimen.
Midstream  Safer , less traumatic method for
clean catch obtaining urine for bacterial culture
and routine urinalysis
 The specimen is less contaminated by
epithelial cells, and bacteria.
 Before collection of a midstream
clean catch specimen, the glans
penis of the male or the urethral
meatus of the female is thoroughly
cleansed and rinsed
Supra-pubic  For bacterial culture(especially for
aspiration anaerobic microbes)
 _________________
Three-glass  _________________
collection
3-SPECIMENS
1st sterile container- contain the first
urine passed
2nd sterile container- contain the midstream
portion of urine
3rd sterile container- contain a urine with
prostate fluid ( the prostate is massaged )

Results:
- The first and third specimens are
examined microscopically
- If the third specimen will have a
white cell / hpf count and bacterial
count 10 x that of the first specimen
 – positive for Prostatic infection
- The second specimen serves as control
for bladder and kidney infections and
should not be positive for bacteria.
Pediatric  We –we bag
specimen  Soft, clear plastic bags with
hypoallergenic skin adhesive to attach
to the genital area of both boys and
girls

Drug Testing Specimen Collection

 ___________________________- process that provides
documentation of proper sample identification from the
time of collection to the receipt of laboratory results
 For urine specimens to withstand legal scrutiny, it is
necessary to prove that no tampering of the specimen
occurred,
such as substitution, adulteration, or dilution.

Container capacity: ________

Urine volume collected: _____________
Urine Temperature : read within 4 minutes , range of
________________-

NOTE- If the specimen temperature is not within range, the

temperature should be recorded and the supervisor or employer
contacted immediately.

PHYSICAL EXAMINATION OF URINE

URINE COLOR
 The normal urine color includes pale yellow→ yellow→ dark
yellow
 The yellow color of urine is caused by the presence of
pigment, _____________.
 The actual amount of urochrome produced on the body is
dependent on the body’s metabolic state
 Increased urochrome production :
a. Thyroid conditions
b. Fasting
c. Urine stands at room temperature
 Two additional pigments present in urine in much smaller
quantities:
a. __________ – pink pigment, most evident in specimens
that have been refrigerated, resulting in the
precipitation of amorphous urates.
b. __________- orange brown color, an oxidation product of
the normal urinary constituent urobilinogen.
 The concentration of a normal urine specimen can be
estimated by urine Color

NOTE  - How to Check for Urine

Color
Care should be taken to examine the specimen under a good
light source, looking down through the container against a
white background
 Color Cause Clinical/Laborato
ry Correlation
Colorless -Recent fluid -Seen in random specimens
intake
Pale yellow -Polyuria or -Increased 24 hrs volume and
Diabetes low specific gravity
Insipidus -Elevated specific gravity
and positive glucose test
-Diabetes result
mellitus

-Dilute random
specimen
Bright -
Yellow RIBOFLAVIN(VITAM
IN B2)
Dark Yellow -Concentrated -After strenuous exercise or
specimen first morning specimen
-Fever or burns
-dehydration
-Negative bile test
-acriflavine results and possible
-CAROTENE
green fluorescence
*Amber/Orang -bilirubin -Yellow foam when shaken and
e positive chemical test for
bilirubin
Orange- -Phenazopyridine - drug commonly administered
yellow (Pyridium) for urinary tract infection,
produces also a yellow foam
when shaken
-Phenindione - anticoagulant, orange in
alkaline, colorless in acid
urine
Yellow-green -Bilirubin -colored foam in acidic urine
oxidized to and false negative test
biliverdin results for bilirubin
Green -Pseudomonas - positive urine culture
infection
Blue-green -amitriptyline -antidepressant
-methocarbamol -muscle relaxant
(robaxin) -fistulas
-methylene blue -when oxidized (green urine
-phenol color)
-clorets -mouth deodorant (green urine
-indican color)
-bacterial infection,
intestinal disorders

Red -RBCs -cloudy urine with positive

chemical results for blood
and visible RBCs when viewed
-Hemoglobin on the microscope
-clear urine with positive
-Myoglobin chemical test for blood; due
to intravascular hemolysis
-Beets -clear urine with positive
chemical test for blood;
-Rifampin muscle damage
-Menstrual -alkaline urine of
contamination genetically susceptible
 person
-medication for Tuberculosis
-cloudy specimen with RBCs,
mucus , and clots
Red- brown -myoglobin (25 -
mg/dl) -seen in acidic urine after
-RBCs oxidized standing
to methemoglobin -Foods, Candy
-Fuscin, aniline
dye
Port Wine -porphyrins/ -negative test for blood, may
Red-purple Porphyria require additional testing
-maybe colorless in Lead
poisoning
Brown Homogentisic -seen in alkaline urine after
acid standing
(Alkaptonuria)
Black -Melanin or -urine darkens on standing
Melanogen, and reacts with nitroprusside
Malignant and ferric chloride
melanoma - color disappears with
-Argyrol ( anti- ferric chloride
septic ) -antihypertensive drug
-Methyldopa or -darkens on standing, for
Levodopa parasitic infection
-Metronidazole -Interfere with copper
(Flagyl) reduction tests
-Phenol
derivates

Note!
- A purple staining may occur in catheter bags and is
caused by indicant in the urine or a bacterial
infection, frequently caused by Klebsiella or
Providencia species.

URINE CLARITY
 Clarity is a general term that refers to the
“Transparency or Turbidity “ of a urine specimen
 The specimen should be in a clear container
 The clarity of a urine specimen certainly provides a key
to the microscopic examination results, because the
amount of turbidity should correspond with the amount of
material observed under the microscope
 Clear urine is not always normal.
 Nubecula = Faint cloud in urine after standing due to
WBCs, epithelial cells and mucus
NOTE  How to Check for Urine Clarity
Visually examining the Mixed specimen while holding
it in front of a light source. View through a
newspaper print

Urine Clarity Reporting

Clarity Term
Clear No visible particulates, transparent
Hazy Few particulates, print easily seen through
urine
Cloudy Many particulates, print blurred through urine
Turbid Print cannot be seen through urine
Milky May precipitate or be clotted

Non Pathologic Causes of Pathologic Causes of Urine

Urine Turbidity Turbidity
-Squamous epithelial cells -RBCs -
-Mucus Nonsquamous epithelial cell
-Amorphous phopshates, -WBCs -
carbonates, urates Abnormality crystals
-Semen, spermatozoa -Bacteria - Lymph
-fecal contamination fluid
-Radiographic contrast media -Yeast - Lipids
-Talcum powder
-Vaginal creams

LAB CORRELATION IN
URINE TURBIDITY
Acidic urine Amorphous urates,
radiographic contrast media
Alkaline urine Amorphous phosphates,
carbonates
Soluble with heat Amorphous urates, uric acid
crystals
Soluble in dilute acetic acid RBCs, Amorphous phosphates,
carbonates
Insoluble in dilute acetic WBCs, Bacteria, yeast,
acid spermatozoa
Soluble in ether Lipids, lymphatic fluid
,chyle

NOTE  For checking of both clarity and color

Check urine with a white background with a good light
source

URINE ODOR
 Seldom of clinical significance and is not a part of the
routine urinalysis

ODOR CAUSE

Aromatic Normal

Foul, ammonia-like, Bacterial decomposition, urinary tract

fetid infection,old urine

Fruity, sweet Ketones, DM, Starvation, vomiting,

strenuous exercise, diarrhea

Maple syrup Maple syrup urine disease, caramel sugar

Mousy odor,Barny Phenylketonuria

or musty odor

Rancid Tyrosinemia

Sweaty feet Isovalericacademia

Cabbage, Hops Methionine malabsoprtion

Bleach Contamination

Odorless Acute tubular necrosis

Rotting fish Trimetylaminuria

Pungent or Asparagus, Garlic, Onion ingestion ,

distinctive odor UTI(Brunzel)

Swimming pool Hawkinsinuria

Sulfur odor cystinuria

Menthol-like Phenol-containing medications

Mercaptan odor Asparagus, garlic, and egg

URINE SPECIFIC GRAVITY

 Specific gravity is define as the density of a solution
compared with the density of a similar volume of
distilled water (S.G 1.000) at a similar temperature
 Specific gravity is influenced by the number of particles
present, and the size of the particles.
 The evaluation of urine concentration is included in the
routine urinalysis by measuring the specific gravity
 The specific gravity of the plasma filtrate entering the
glomerulus is 1.010
a. Isosthenuric- term to describe urine with S.G 1.010
b. Hyposthenuric/Diluted urine – term to describe urine
with S.G below 1.010
c. Hypersthenuric/Concentrated urine- term to describe
urine with S.G above 1.010

-S.G of Normal random urine: _____________, where most of the

random specimen falls between 1.015 -1.030.
-Abnormally high S.G results- above 1.040 – are seen in
patients who have recently undergone an intravenous pyelogram
(Ex. Radiographic contrast dye /X-ray film, Dextran, and other
Plasma expanders)

CLINICAL SIGNIFICANCE OF URINE SPECIFIC GRAVITY

RESULTS(Brunzel,3rd.)

S.G Indication / Cause

1.000 Physiologically impossible–same as purewater;
suspect adulteration of urine specimen
1.001-1.009 Dilute urine; associated with increased water
intake or water diuresis (e.g., diuretics,
Diabetes insipidus ,inadequate secretion/action
of ADH)
1.010 to Indicates average solute and water intake and
1.025 excretion
1.025 to Concentrated urine; associated with dehydration,
1.035 fluid restriction, profuse sweating, osmotic
diuresis
>1.040 Physiologically impossible; indicates presence
of iatrogenic substance (e.g., radiographic
contrast media, mannitol)
 METHODS FOR DETECTION URINE S.G

Direct methods Hydrometer,Harmonic oscillation

densitometry, falling drop
Indirect Refractometer, reagent strip
methods

Harmonic  Based on the principle that the

Oscillation frequency of a sound wave entering a
Densitometry solution changes in proportion to the
density of the solution

Example of instrument that uses HOD is

YELLOW IRIS(International remote imaging
system)
Reagent  The reagent strip reaction is based on
strip the change in the pKa(dissociation
constant) of a polyelectrolyte in an
alkaline medium
 S.G reading is not affected by
radiographic contrast dye, protein, and
glucose.

Hydrometer  The urinometer consists of a weighted

(Urinometer) float attach to a scale that has been
calibrated in terms of urine specific
gravity
 When using urinometer, an adequate
amount of urine is poured into a
proper-size container and the
urinometer is added with a spinning
motion. The scale reading is then taken
at the bottom of the urine meniscus.
 A major disadvantage of using a
urinometer to measure specific gravity
is that it requires a
__________________ of specimen
 It is less accurate that other methods
and is not recommended by the CLSI
 The urinometer reading needs to be
corrected for temperature, glucose and
protein.
 The calibrated temperature printed on
the instrument is usually about 20 oC.
 To Correct for the S.G :
a. Add 0.001 for every 30C above the
calibration temp.
b. Subtract 0.001 for every 3oC below
the calibration temp.
c. Subtract 0.004 for every 1 gram of
glucose
d. Subtract 0.003 for every 1 gram of
protein
Example:
A specimen that has been left at 29 oC has
been reported to contain 2g/dl of glucose
and 1g/dl of protein. The initial S.G was
1.035.Calculate the corrected S.G
 a. Temperature : add 0.001 for every 3 oC
so 0.001 x 3 = +0.003
b. Glucose : subtract 0.004 for every 1
g/dl so 0.004 x 2 = -0.008
c. Protein: subtract 0.001 for every 1g/dl
so 0.003 x 1 = -0.003

Formula 1.035 + 0.003 – 0.008 – 0.003 =

1.027 corrected SG

CALIBRATION
Potassium sulfate = S.G should be read at
1.015
Water = S.G should be read at 1.000

Refractometer  It determines the concentration of

( TS meter) dissolved particles in a specimen. It
does this by measuring refractive
index.
 Refractive index is a comparison of the
velocity of light in air with the
velocity of light in a solution.
 The refractometer provides the distinct
advantage of determining specific
gravity using a small volume of
specimen (one or two drops).
 Temperature corrections are not
necessary.
 Temperature is compensated between 15 oC
and 38 oC.
 Corrections for glucose and protein are
calculated.
Glucose = subtract 0.004 for each gram
Protein = subtract 0.003 for each gram

Example:
A specimen containing 1 g/dL protein and 1
g/dL glucose has a S.G reading of 1.030.
calculate the corrected reading

1.030 – [ 1(0.004)glucose +
1(0.003)protein ] = 1.023 corrected SG

 Calibration of the refractometer is

performed using a calibration screw.
a. Water – S.G should be read 1.000
b. 3% NaCl- readt_________________
c. 5% NaCl – read _________________
d. 9% Sucrose – read _________________

Method Correction for Correction Correction

temperature for glucose for protein
Urinometer yes yes yes
Refractometer No yes yes
Reagent strip No No No

S.G DILUTION

FORMULA : S.G x DILUTION = ACTUAL S.G

EX: A specimen diluted 1:5 with a reading of 1.010 would
have an actual S.G of
A. 1.050 B. 5.050 C.1.015 D.Prayers

CHEMICAL ANALYSIS OF URINE

 Reagent strips – provide, simple, rapid means for
performing medically significant analysis of urine
 Reagent strips consist of chemical-impregnated absorbent
pads attached to a plastic strip. A color producing
chemical reaction takes place when the absorbent pad
comes in contact with urine.
 A fresh, well-mixed, uncentrifuged specimen is
used for testing
 10 parameters: pH, protein, glucose, ketones, blood,
bilirubin, urobilinogen, nitrite, leukocytes, and S.G
 11th parameter : __________________

Reagent Strip Technique

Procedure
1. Dip the reagent strip briefly (no longer than 1 second)
into a well-mixed uncentrifuged urine specimen at RT.
2. Remove excess urine by touching the edge of the strip to
the container as the strip is withdrawn.
3. Blot the edge of the strip on a disposable absorbent pad.
4. Wait the specified amount of time for the reaction to
occur.
Compare the color reaction of the strip to the manufacturer’s
color chart in good lightning.

Errors from Improper Technique

a. Formed elements such as WBC and RBC sinks to the bottom
of the specimen and will be undetected in an unmixed
specimen
b. Allowing the strip to remain in the urine for an extended
period may cause leaching of reagents from the pads.
c. Run-over between chemicals on adjacent pads, producing
distortion of the colors. To ensure against run –over,
blot the edge of the strip with adsorbent paper and
holding the strip horizontally while comparing it with
color chart.
d. Specimens that have been refrigerated must be allowed to
return at room temp. prior to reagent strip testing, as
the enzymatic reactions on the strips are temperature
dependent
e. Proper timing of reactions to take place.
Handling and Storing Reagent Strips
1. Reagent strips are packaged in opaque, tightly closed
container with a dessicant(drying agent) to protect from
light and moisture.
2. Store below 30oC (room temp) ; do not freeze
3. Strips are removed just prior to testing, and the bottle
is tightly resealed immediately.
4. Do not expose to volatile fumes
5. Do not use past the expiration date
6. Do not use if chemical pads become discolored.
 7. Any strips showing evidence of deterioration,
contamination,or improper storage should be discarded

I.Ph
 Important in the identification of urinary crystals and
determination of unsatisfactory specimens.
 Important in aid of existence of systemic acid-base
balance disorders.
Normal Urine pH

First morning urine

pH

Improperly preserved >9 or >8.5 (6th edition)

specimen Note! Presence of detergent in the
urine container can cause alkalization
of urine

Causes of Acid Urine Causes of Alkaline Urine

Emphysema Renal tubular acidosis
Diabetes mellitus Hyperventilation
Starvation Vomiting
Dehydration Vegetarian diet
Cranberry juice Old specimens
High protein diet Presence of urease producing
Presence of acid producing bacteria
bacteria (E.coli) Alkaline tide (during and
Medications such as after following meals)
Mandelamine and Fosfomycintro
methamine

REAGENT STRIP REACTION (60 seconds)

Principle ___________________________

Methyl red + H+ → Bromthymol blue – H+

(Red to yellow) → (green to blue)
pH 4.0 -6.0 pH 6.0-8.0

Reagents Methyl red and bromthymol lue

II.SPECIFIC GRAVITY
 Density of a solution compared with density of similar
volume of distilled water at a similar temperature
 Influenced by number and size of particles on a solution.
 The reagent strip specific gravity test does not measure
the total solute content but only those solutes that are
ionic.

Normal random SG ___________

Radiographic S.G = >1.040

Contrast dye

Not A urine S.G = <1.002

REAGENT STRIP REACTION (45 seconds)

Principle Change in the pKa(dissociation constant) of a
 polyelectrolyte

Blue ---------- Green ---------- Yellow

↓H ↑H ↑↑↑ H

Reagents Multistix= poly (methyl vinyl ether/ maleic

anhydride) bromthymol blue
Chemstrip= Ethylene glycol diaminoethyl ether
tetra acetic acid, bromthymol blue
Sensitivity 1.000(Blue) to 1.030(Yellow)
Interference False positive: High concentration of protein
False negative: Highly alkaline urines (greater
than pH 6.5 –add 0.005 SG reading)

III.PROTEIN

 Most Indicative of renal disease

 Albumin is the major protein found in normal urine due to
its low molecular weight
Normal Value: ______________________ ,
(Henry’s________________)
 Other Proteins:
Serum and tubular microglobulins, Tamm-Horsfall protein
(Uromodulin), protein derived from prostatic and vaginal
secretion
 Produces a white foam in urine
 Clinical proteinuria = ≥30mg/dl or ≥300g/L
Pre –Renal or Renal Proteinuria / True Post- Renal
Overflow renal disease Proteinuria
Proteinuria
 Caused by I.Glomerular Proteinuria A. Lower
conditions A. Diabetic Nephropathy / UTI/inflammat
that Kimmelstiel-Wilson’s disease ions
affect the -decreased GFR B. Injury /
plasma -associated with renal Trauma
prior to failure in persons with Type C. Menstrual
its I and II Diabetes mellitus Contamination
reaching -Indicator: D. Prostatic
the kidney ___________________ fluid/
-Albumin Excretion Rate (AER) spermatozoa
a. Hemoglobin  Normal: 0-20 ug/min E. Vaginal
=intravasc  Microalbuminuria: secretion
ular ________________________
hemolysis _______
b. Myoglobin B.Amyloidosis
= Muscle C.Immune complexes
injury D.Pre-eclampsia
c. Acute E. Orthostatic / Cadet /
phase Postural proteinuria
reactants Orthostat Clinical
= ic Proteinur
inflammati Proteinur ia
on and ia
st
infections 1
d. Bence mornin
Jones g
protein = 2hours
multiple after
myeloma standi
ng
BJP –
immunoglobulin II.Tubular Proteinuria
light chains  Normally filtered
Test: serum albumin can no longer be
electrophoresis reabsorbed
A.Fanconi’s syndrome
Urine: B.Toxic agents/Heavy
Precipitates at metals(such as cadmium dust)
_______ and C.Severe viral infections
dissolves
at______oC

IMMUNOLOGIC TEST FOR MICROALBUMINURIA

Micral-Test ImmunoDip
 Principle: Enzyme  Principle:
immunoassay Immunochromographics
 Sensitivity: 0 to  Sensitivity: 1.2 to
10 mg/dL 8.0 mg/dL
 Reagents: Gold-  Reagents: Antibody-
labeled antibody,B- coated blue latex
galactosidase, particles
Chlorophenol red Interference: False-
galactoside negative: Dilute urine
Interferences:
False-positive:strong
oxidizing agent(soap)
False-negative: Dilute
urine
REAGENT STRIP REACTION FOR PROTEIN (60 seconds)

Principle ______________________________

Indicator + protein
------------------------- (+) Blue-green
(Yellow)
(-) Yellow

Note:
a. Proteins mainly albumin accepts Hydrogen
ions from the indicator
b. The ph of the medium remains constant (pH
of 3 buffered with citrate)
c. Reagent strip is sensitive to Albumin
only
d. The S.G of urine sample should be check
because a trace protein in diluted sample
is more significant than in concentrated
sample
Reagents Multistix = Tetrabromphenol blue
Chemstrip =
 Tetrachlorophenoltetrabromosulfonphthalein
Intereference
False positive False negative
 Highly buffered  Proteins other
interference than albumin
alkaline urine  Microalbuminuria
 Pigmented
specimens,
phenazopyridine
 Quaternary
ammonium
compounds
(detergents)
 Antiseptics,
chlorhexidine
 Loss of buffer
from prolonged
exposure of the
strip to the
specimen reagent
 High specific
gravity

SULFOSALICYLIC ACID (SSA) PRECIPITATION TEST

A cold precipitation test that reacts equally with all forms

of protein.

PROCEDURE – 3mL of 3% SSA(exton’s reagent) or 7%SSA + 3ml

centrifuged urine = (+) cloudiness

SSA GRADING
GRADE TURBIDITY PROTEIN RANGE (mg/dl)
Negative No increase turbidity <6
Trace Noticeable turbidity 6-30
1+ Distinct turbidity 30-100
with no granulation
2+ Turbidity with 100-200
granulation, no
flocculation
3+ Turbidity with 200-400
granulation, and
flocculation
4+ Clumps of protein >400

SSA INTERFERENCES
False  Radiographic contrast dye/x-ray film
increase/positive  Drugs (Tulbotamide,Penicillin,
Sulfonamide, cepalosphorin
 para-amino-salicylic acid/Salicylates
False  Highly alkaline urine
decrease/negative  Quaternary ammonium compounds (e.g
Detergents, and soap)

MICROSCOPICALLY, WHAT IS THE SSA PATTERN IF Amorphous

PROTEINS CAUSE A POSITIVE REACTION?
MICROSCOPICALLY, WHAT IS THE SSA PATTERN IF Crystalline
DRUGS AND RADIOGRAPHIC CONTRAST DYE CAUSE A
POSITIVE REACTION?
IV.GLUCOSE
 Most frequently performed chemical analysis on urine.
( due to monitoring of DM)
 Renal threshold- plasma concentration of a substance at
which tubular reabsorption stops
 Renal threshold for glucose = 160-180 mg/dl
CLINICAL SIGNIFICANCE OF URINE GLUCOSE

Hyperglycemia – Associated Renal – associated

Increase blood glucose ,
Increase Urine glucose
Causes :
a. DM
b. Pancreatitis
c. Pheochromocytoma
d. Acromegaly
e. Cushing syndrome
f. Hyperthyroidism
Normal blood glucose ,
Increase Urine glucose
Causes:
a. Fanconi’s syndrome
b. Advanced renal disease
c. Osteomalacia
d. Pregnancy

REAGENT STRIP REACTION FOR GLUCOSE (30 seconds)

Principle ___________________________-

Glucose + O2
--------------------------gluconic acid +
Hydrogen Peroxide

Hydrogen Peroxide + Chromoge-----------------

 Oxidized chromogen + water

Reagents Multistix = glucose oxidase, peroxidase,

Potassium iodide ( blue to green to brown)
Chemstrip = glucose oxidase, peroxidase,
tetramethylbenzidine (yellow to green)

Other chromogens: aminopropylcarbazole (yellow

to orange brown)
o-toluidine
( pink to purple )
Interference False positive :
a. Contamination of oxidizing agents and
detergents
False negative:
a. High levels of ascorbic acid
b. High levels of ketones
c. High SG
d. Low temp
e. Improperly preserved specimens

COPPER REDUCTION TEST ( CLINITEST / BENEDICT’S TEST)

Test Non-specific test for reducing sugars

a. Glucose,galactose, fructose, maltose etc.
Sucrose – a non-reducing sugar and cannot be
detected by this test
Clinical For the detection of inborn error of
significance metabolism especially galactosuria in newborns
in which there is a lack of enzyme galactose
-1- phosphate uridyltransferase
Component of a. _______________- main reacting agent
the Tablet b. _______________ – eliminates interfering
 O2
c. _______________ – for heat production
d. _______________ – for heat production
Procedure 5 gtts urine + 10gtts H2O + clinitest tablet

Pass through phenomenon:

-occurs when >2 g/dl sugar is present

-blue > green > yellow > brick red > green
brown
- to prevent pass through , use 2 gtts urine
Principle Copper Reduction

CuSO4(cupric sulfide) + reducing substance

-------- > Cu2O (cuprous oxide) + oxidized
substance -- color(+) BRICK RED
Interference False Positive:
a. Reducing agents such as vitamin C and
uric acid

False Negative:
a. Oxidizing agents such as detergent

SUMMARY OF GLUCOSE OXIDASE AND CLINITEST REACTIONS

Glucose Clinitest Interpretation

Oxidase
4+ Negative -Oxidizing agent interference
-False-positive reagent strip because of
contaminants (e.g., oxidizing agents,
peroxidases)
-False negative Clinitest due to
presence of radiographic contrast media
-Defective Clinitest tablets (e.g.,
outdated)
1+ Negative Small amount of glucose present since
reagent strip is more senstive
Negative Positive -Non glucose reducing substance
-Possible interfering substance such as
reducing agent
-Reagent strip interference (e.g., high
specific gravity, low urine temperature)
-Reagent strips defective (e.g.,
outdated, improperly stored)

V.KETONES
 Result from increased fat metabolism
a. Inability to metabolize or utilize available
carbohydrate – ex. DM
b. Increased loss of carbohydrates – ex. Vomiting
c. Inadequate intake of carbohydrate – ex. Starvation and
malabsoprtion
 Ketone Bodies:
a. 78% Beta Hydroxybutyric acid – major ketone but not
detected in reagent strip
b. 20% Acetoacetic acid (AAA) / Diacetic acid – parent
ketone
c. 2 % Acetone – detected only when glycine is present
REAGENT STRIP REACTION FOR KETONES (40 seconds)
Principle
 _________________________________

Acetoacetate and acetone + sodium

nitroprusside + glycine-- (+)Purple
Reagents sodium nitroprusside
glycine (Chemstrip)
Interference False positive :
a. Phalein dyes
b. Highly pigmented red urine
c. Levodopa
d. Medications containing free sulfhydryl
groups
False negative:
a. Improperly preserved specimens

ACETEST TABLET
Composition :
a. Sodium nitroprusside
b. Disodium phosphate
c. _________________ –gives better color differentiation

-read for 30 seconds

-report as negative, small, moderate, or large.
- Acetest tablets are hygroscopic; if the specimen is not
completely absorbed within 30 seconds, a new tablet should be
used.

VI.BLOOD
 The finding of a positive reagent strip test result for
blood indicates the presence of red blood cells,
hemoglobin, or myoglobin.

HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA

 Cloudy red  Clear red  Clear red
urine urine urine
 Presence of a  Uniform  Heme portion
intact RBC green / blue of the
 Produces a color in myoglobin is
speckled/spot reagent strip toxic to the
ted pattern pad renal tubules
on reagent  Uniform
pad green / blue
Seen in: color in
Seen in : a. Transfusion reagent strip
a. Glomeruloneph reactions pad
ritis b. Hemolytic
b. Renal calculi anemias
c. Pyelonephriti c. Severe burns Seen in:
s d. Infections:mal a. Rhabdomyolysis
d. Tumors aria, b. Prolonged coma
e. Trauma syphilis, c. Convulsions
f. Anticoagulant mycoplasma, d. Extensive
s and exertion
g. Strenuous C.perfringens e. Muscle wasting
exercise e. Strenuous diseases
h. Hypertension exercise f. Cholesterol-
i. cystitis f. Brown recluse lowering
spider bites statin
medications
g. Muscle
ischemia:
 carbon
monoxide
poisoning
h. Muscle
infection(myos
itis)

Hemoglobin Versus Myoglobin

Test Hemoglobin Myoglobin

1. Plasma Examination Red/ pink Pale yellow plasma
plasma due to
hemolysis
2. Blondheim’s test Precipitated by Not precipitated
(ammonium sulfate) ammonium by ammonium
Procedure: sulfate sulfate
a. 5 ml centrifuged
Urine + 2.8g Produce a clear Produce a red
Ammonium sulfate supernatant supernatant that
b. Mix and allow the that is is positive for
specimen to sit negative for blood reagent
for 5 minutes blood reagent strip
c. Filter or strip
centrifuged
d. Test the
supernatant with
blood reagent
strip

REAGENT STRIP REACTION FOR BLOOD (60 seconds)

Principle ___________________________
Hemoglobin
Hydrogen peroxide + chromogen
-------------------- oxidized chromogen + H20

Psuedoperoxidase

(-) yellow, (+) Green to Blue

Reagents Multistix =
diisopropylbenzenedehydroperoxidetetramethylbenz
idine
Chemstrip =
dimethyldihydroperoxyhexanetetramethylbenzidine

Interferen False positive:

ce a. Strong oxidizing agents
b. Bacterial peroxidases
c. Menstrual contamination
False negative:
a. High SG / Crenated cells
b. Formalin
c. Captopril
d. Ascorbic acid (>25mg/dl)
e. Unmixed specimen
f. High concentration of nitrite
( >10mg/dl)

VII.BILIRUBIN
 The appearance of bilirubin in the urine can provide an
early indication of liver disease.
 a. Hepatitis
b. Cirrhosis
c. Biliary obstruction (gallstones, carcinoma)
 Only the B2 or conjugated bilirubin is water soluble thus
can be seen in urine
 It produces an amber urine with yellow foam

REAGENT STRIP REACTION FOR BILIRUBIN (30 seconds)

Principle ________________
acid
Bilirubin glucuronide + diazonium salt
------------azodye (+) Tan or Pink to Violet
Reagents Multistix = 2,4-dichloroaniline diazonium salt
Chemstrip = 2,6-dichlorobenze diazonium
tetrafluoroborate
Interference False positive:
a. Highly pigmented urines such as
phenazopyridine
b. Indican
c. Metabolites of Lodine
False negative:
a. Specimen exposure to light
b. Ascorbic acid
c. High concentration of nitrite

ICTOTEST (Tablet) for `BILIRUBIN

Components:
a. p-nitrobenzene-diazonium p-toluenesulfonate
b. SSA
c. Sodium carbonate
d. Boric acid
(+) Blue to purple color

VIII.UROBILINOGEN
 A bile pigment that result from hemoglobin degradation
 Conjugated bilirubin is reduced by intestinal bacteria
into urobilinogen
 A small amount of urobilinogen – less than 1mg/dl or
Ehrlich unit – is normally found in the urine.
 Clinical significance: urine urobilinogen greater than 1
mg/dl is seen in liver disease and hemolytic disorders.

REAGENT STRIP REACTION FOR UROBILINOGEN (60 seconds)

Principle Ehrlich ‘s reaction

Multistix: Uses Ehrlich reagent

Urobilinogen + p-dimethlyaminobenzaldehyde
---------- red color

Chemstrip: uses 4-methyloxybenzene-diazonium-

tetrafluoroborate (more specific than
ehrlich’srxn
Urobilinogen + diazonium salt ------- red
azodye
Note Ehrlich-reactive compounds: porphobilinogen,
indicant, p-aminosalicylic acid, sulfonamides,
methyldopa, procaine, chlorpromazine --- also
gives positive reaction for Ehrlich’s reaction
Interference False positive :
a. Other Ehrlich’s compound
 b. Highly pigmented urine
False negative:
a. Old specimens
b. Preservation in formalin

WATSON-SCHWARTZ TEST
 Used to differentiate urobilinogen, porphobilinogen,
and other Ehrlich reactive compounds
 Uses extraction with organic solvents chloroform and
Butanol

Urobilinogen Porphobilinogen Other Ehrlich

Reactive
Compound
Chloroform
extract Colorless Red Red
-URINE (TOP) Red Colorless Colorless
-CHLOROFORM
(Bottom)
Butanol
Extract Red Colorless Red
-BUTANOL Colorless Red Colorless
(TOP)
-URINE
(BOTTOM)

HOESCH TEST (INVERSE EHRLICH REACTION)

 Rapid screening test for porphobilinogen ( >2mg/dl )
 Procedure:2 gtts urine + 2mL Hoesch reagent (Ehrlich’s
reagent in 6M or 6N HCL)---(+) Red

CORRELATION OF BILIRUBIN AND UROBILINOGEN

Condition Blood Urine Urine
Bilirubin Urobilinogen
Prehapatic Increase negative +++
jaundice Unconjugated
(Hemolytic bilirubin
diease)
Hepatic Increase both B1 +/- ++
jaundice and B2
(Liver damage)
Post hepatic Increase +++ normal
jaundice Conjugated
(Bile duct Bilirubin
obstruction)

IX.NITRITE
  Provides a rapid screening test for the presence of UTI
and bacteruria.
 It is not intended to replace the urine culture as the
primary test for diagnosing and monitoring bacterial
infection.
 Specimen used: 1st morning or 4 hour urine
 The chemical basis of the nitrite test is the ability of
certain bacteria to reduce nitrate, a normal constituents
of urine, to nitrite, which does not normally appear in
the urine.
REAGENT STRIP REACTION FOR NITRITE (60 seconds)
Principle _____________________

p-arsanilic acid (or sulfanilamide) + Nitrite

-----------Diazonium salt

Diazonium salt +
Tetrahydrobenzoquinolin------------ (+)
Uniform pink color

Reagents Multistix = p-arsanilic acid ,

tehtrahydrobenzoquinolin-3-ol
Chemstrip = sulfanilamide,
hydroxytetrahydrobenzoquinoline
Interference False positive:
a. Improperly preserved specimens
b. Highly pigmented urine
False Negative
a. Non reductase- containing bacteria
b. Insufficient contact time between
bacteria and urinary nitrate
c. Large quantities of bacteria
converting nitrite to nitrogen
d. Presence of antibiotics
e. Ascorbic acid
f. High specific gravity
Note Positive result should uniform pink
Pink spots / edges is considered as NEGATIVE
(+) nitrite corresponds to ___________
organisms/ml

-It is for gram (-) bacteria/bacilli which

are mostly nitrite positive
-enterobacteriaceae/coliform gives nitrite
positive result

X.LEUKOCYTES
 Significance: UTI/inflammation , Screening of urine
culture specimen, bacterial and non-bacterial infection
 It detects the presence of leukocyte that have been
lysed, particularly in dilute alkaline urine
 It offers a more standardized means for detection of
leukocytes
 The test is not designed to measure the concentration of
leukocytes, and it is recommended that quantitation
should be done by microscopic examination.
 LE test detects esterase found in
a. Neutrophil
b. Basophil
c. Eosinophil
d. Monocytes
 e. Trichomonas
f. Chlamydia
g. Yeast
h. Histiocytes
 Screening urine specimens using LE test should be
correlated with nitrite chemical reactions

REAGENT STRIP REACTION FOR LEUKOCYTES (120 seconds)

Principle Leukocyte Esterase

Leukocyte
esterase
Indoxylcarbonic acid ester
------------------------indoxyl + acid
indoxyl + Diazonium salt ------ (+)Purple
azodye

Reagent Multistix = Diazonium salt,

derivatizedpyrrole amino acid ester
Chemstrip =Diazonium salt, Indoxylcarbonic
acid ester
Sensitivity Multistix = 5 to 15 WBC/hpf
Chemstrip = 10 to 25 WBC/hpf
Interference False positive:
a. Strong oxidizing agents
b. Formalin
c. Highly pigmented urine, nitrofurantion
False negative:
a. High concentration of protein ( Greater
than 500 mg/dl), glucose, oxalic acid
and ascorbic acid
b. Antibiotics such as gentamicin,
cephalosphorins, tetracyclines,
c. Inaccurate timing

MICROSCOPIC ANALYSIS OF URINE

ADDIS COUNT – first procedure to standardize the quantitation
of formed elements, used a hemocytometer
 Specimen :
 RBCs = 0 to 500,000 cells /ul
 WBCs and Epithelial cells =0 to 1,800,000 cells /ul
 Hyaline casts = 0 to 5000 cells/ul

SEDIMENT STAINS

STAIN ACTION FUNCTION

Sternheimer- -Delineates -Identifies WBCs,
Malbin(a structure and epithelial cells, and
supravital stain contrasting casts
consisting of colors of the
Crystal violet nucleus and
and safranin) cytoplasm

0.5%Toluidine -Enhances Differentiates WBCs and

blue(a nuclear detail renal tubular epithelial
metachromatic (RTE) cells
supravital
stain)

2% acetic acid Lyses RBCs and Distinguishes RBCs from

enhances nuclei WBCs, yeast, oil
of WBCs droplets, and crystals
Lipid Stains: Stains Identifies free fat
Oil Red triglycerides droplets and lipid
O and Sudan III and neutral fats containing cells and
orange-red casts

Gram stain Differentiates Identifies bacterial

gram-positive casts
and gram
negative
bacteria

Hansel stain Methylene blue + Identifies urinary

EosinY eosinophils
Stains
eosinophilic
granules
Prussian blue Stains Identifies yellow-brown
stain structures granules of hemosiderin
containing iron in cells and casts

Specimen Preparation
Urine 10 -15 ml
↓
Centrifuge at 400 RCF for 5 minutes
↓
Decant
↓
Get the sediment (0.5-1.0mL)
↓
Place the sediment on the microscopic slide (20 ul or 0.02ml)
↓
Covered by glass cover slip (22x22mm)
↓
Observe under the microscope (Bright field –reduced
lightning )

MICROSCOPY

Technique Function and Description

Bright - Used for routine urinalysis
field - objects appear dark against a light
microscopy background
- most frequently used in the clinical
laboratory
- the oldest and most common type of
illumination system used on
microscopes
- all other types of microscope are
 adapted to bright-field

Phase - Enhances visualization of elements with

contrast low refractive indices, such as hyaline
microscopy casts, mixed cellular casts, mucous
threads, and Trichomonas
- Type of microscopy in which variations in
the specimen’s refractive index are
converted into variations in light
intensity or contrast
- Adaptation of a bright-field
microscope with a phase-contrast
objective lens and a matching
condenser. Two phase rings that
appear as “targets” are placed in
the condenser and the objective.
- Light passes to the specimen through
the clear circle in the phase ring
in the condenser, forming a halo of
light around the specimen
-
Polarizing - Aids in identification of cholesterol in
microscopy oval fat bodies, fatty casts, and
crystals.
- It uses halogen quartz lamp that
produces light rays of many
different waves
- A substance that rotates the plane
of polarized light 90 degrees in a
clockwise direction is said to have
positive birefringence.
- substance that rotates the plane in
a counterclockwise direction has
negative birefringence
- Bright-field microscopes can be
adapted for polarizing microscopy.
Two polarizing filters must be
installed in a crossed configuration

Dark field - Aids in identification of spirochetes such

microscopy as Treponema pallidum
- bright-field microscope is easily
adapted for dark-field microscopy by
replacing the condenser with a dark-
field condenser that contains an
opaque disk
- The specimen appears light against
the black background or dark-field

Interference - Produces a three-dimensional microscopy-

contrast image and layer-by-layer imaging of a
microscopy specimen
- Type of microscopy in which the difference
in optical light paths through the
 specimen is converted into intensity
differences in the specimen image.
- Two types of interference-contrast
microscopy are available: modulation
contrast (Hoffman) and differential-
interference contrast (Nomarski).
Bright-field microscopes can be
adapted for both methods.
Fluorescence - Allows visualization of naturally
microscopy fluorescent microorganisms or those
stained by a fluorescent dye
- Fluorescence microscopy uses two filters:
one to select a specific wavelength of
illumination light (excitation filter)
that is absorbed by the specimen, and
another (barrier filter) to transmit the
different, longer-wavelength light emitted
from the specimen to the eyepiece for
viewing

PARTS OF MICROSCOPE

Lens system Illumination BODY

system
1.Occulars 1.Light source 1.Base
2.Objectives 2.Condenser 2.Body tube
3.Adjustment 3.Stage field 3.Nose piece
knobs 4.Iris
diaphragms

Terminologies

Aperture diaphragm Microscopecomponent that regulates

theangle of light presented to
thespecimen.
Birefringent/ The ability of asubstance to refract
doubly refractile light in twodirections.
Chromatic Unequal refraction of light rays by a
aberration lens that occurs because the different
wavelengths of light refract or bend at
different angles
Condenser Microscope component that gathers and
focuses the illumination light onto the
specimen for viewing.
Eyepiece The microscope lens or system of lenses
located closest to the viewer’s eye. It
produces the secondary image
magnification of the specimen
Field diaphragm Microscope component that
controls/regulates the diameter of light
beams that strike the specimen and hence
reduces stray light.
Field of view The circular field observed through a
microscope
Köhler illumination Type of microscopic illumination in
which a lamp condenser (located above
the light source) focuses the image of
the light source (lamp filament) onto
 the front focal plane of the substage
condenser (where the aperture diaphragm
is located)
Mechanical stage Microscope component that holds the
microscope slide with the specimen for
viewing.
Objectives The lens or system of lenses located
closest to the specimen. The objective
produces the primary image magnification
of the specimen.
Parcenter Term describing objective lenses that
retain the same field of view when the
user switches from one objective to
another of a differing magnification
Parfocal Term describing objective lenses that
remain in focus when the user switches
from one objective to another of a
differing magnification.
Resolution Ability of a lens to distinguish two
points or objects as separate.
Cytocentrifugation A technique used to produce
permanent microscope slides of
urine sediment and body fluids.
The end result is a monolayer of
the urine sediment components with
their structural details greatly
enhanced by staining

Sediment Constituents Found On

Urine
I.Red Blood Cells
 Appear as smooth, non-nucleated, biconcave disk
measuring approximately 7 mm in diameter
 Most difficult to recognize
 Concentrated /Hypersthenuric urine : Crenated cells
/ECHINOCYTES
 Dilute / Hyposthenuric urine : ghost cells
 Dysmorphic or Distorted RBC – vary in sizes,mainly they
are acanthocytes, it is associated with glomerular
bleeding
 Source of identification error: Yeast cell, oil
droplets, air bubbles
II.White Blood Cells
 WBCs are larger than RBCs, measuring average of
about 12 mm in diameter
 Neutrophil is the predominant WBC found in urine
 Hypotonic Urine : Glitter Cells – WBC with
sparkling appearance due to Brownian movement
of the granules
 In hypertonic urine, leukocytes become
smaller as water is lost osmotically from
the cells, but they do not crenate.
 Eosinophil - The presence of urinary eosinophils
is primarily associated with drug-induced
interstitial nephritis; however, small numbers
of eosinophils may be seen with urinary tract
infection (UTI) and renal transplant rejection.
Eosinophils are not normally seen in the urine;
 therefore, the finding of more than 1%
eosinophils is considered significant
 Pyuria or leukocytoruia- Term used to denote
increase urinary WBCs and is associated with
bacterial infection, Interstitial nephritis ,
SLE

III.Epithelial Cells
A. Squamous Epithelial cell
 Originates from the linings of the vagina and
female urethra and the lower portion of the
male urethra.
 Squamous cells are the largest cells found in
the urine sediment. They contain abundant,
irregular cytoplasm and a prominent nucleus
about the size of an RBC
 The point of reference in microscopic
analysis
 Increased amounts are more frequently seen in
females.
 Clue cells : pathologic squamous epithelial
cell covered with the Gardnerella vaginalis
coccobacillus

B. Transitional Epithelial (Urothelial) cells/

Bladder epithelial cells
 Transitional epithelial cells originate from
the lining of the renal pelvis, calyces,
ureters, and bladder, and from the upper
portion of the male urethra.
 Transitional epithelial cells are smaller
than squamous cells and appear in several
forms, including spherical, polyhedral, and
caudate
 Spherical forms of transitional epithelial
cells are sometimes difficult to distinguish
from RTE cells. The presence of a centrally
located rather than eccentrically placed
nucleus, and supravital staining, can aid in
the differentiation.
 Increased numbers of transitional cells seen
singly, in pairs, or in clumps (syncytia)are
present following invasive urologic
procedures such as catheterization and are of
no clinical significance. An increase in
transitional cells exhibiting abnormal
morphology such as vacuoles and irregular
nuclei may be indicative of malignancy or
viral infection.

C. Renal Tubular Epithelial Cells (RTE cells)

 Renal tubular epithelial (RTE) cells vary in
size and shape depending on the area of the
renal tubules from which they originate.
 The cells from the proximal convoluted tubule
(PCT) are larger than other RTE cells. They
tend to have a rectangular shape and are
referred to as columnar or convoluted cells.
 Cells from the distal convoluted tubule (DCT)
are smaller than those from the PCT and are
round or oval.
 Collecting duct RTE cells are cuboidal and
 are never round. Along with the eccentrically
placed nucleus, the presence of at least one
straight edge differentiates them from
spherical and polyhedral transitional cells.
Cells from the collecting duct that appear in
groups of three or more are called renal
fragments. They are frequently seen as large
sheets of cells.
 Tubular Injury : presence of more than 2
RTE/HPF
 RTE cells are the most clinically significant
of the epithelial cells.
 They are the precursor of oval fat bodies

IV. Oval Fat Bodies

 These are lipid-containing RTE cells
 Identification of oval fat bodies is
confirmed by staining the sediment with Sudan
III or Oil Red O fat stains and examining the
sediment using polarized microscopy.
 Examination of the sediment using polarized
light results in the appearance of
characteristic Maltese cross formations
 They are present in disorders such as :
Nephrotic syndrome , DM, Severe tubular
necrosis
 Bubble cells – RTE cells containing,
nonlipid-filled vacuoles mainly associated
with Acute tubular necrosis

V.Bacteria
 They appear as small spherical and rod-shaped
structures
 Bacteria are not normally present in urine
 To be considered significant for UTI,
bacteria should be accompanied by WBCs.
 They are motile and is useful to
differentiate from similar appearance,
amorphous urates and phosphates.
VI.Yeast
 Yeast cells appear in the urine as small,
refractile oval structures that may or may
not contain a bud.
 In severe infections, they may appear as
branched, mycelial forms
 Yeast cells, primarily Candida albicans, are
seen in the urine of diabetic,
immunocompromised patients and women with
vaginal moniliasis.
 A true yeast infection should be accompanied
by the presence of WBCs.
 FAVORABLE URINE CONDITION : ACIDIC urine and
with glucose

VII.Parasites
 Trichomonas vaginalis – most frequent
parasite encountered in urine
 Schistosoma haematobium – bladder parasite,
associated with bladder tumors
 Enterobius vermicularis- most common
 contaminant ova
 Cyst of Giardia lamblia- observed in urine
sediment as the result of fecal
contamination of infected individuals
VIII. Spermatozoa
 Spermatozoa are easily identified in the
urine sediment by their oval, slightly
tapered heads and long, flagellalike tails
 Urine is toxic to spermatozoa; therefore they
rarely exhibit the motility observed when
examining a semen specimen.
 They are rarely of clinical significance
except in cases of male infertility or
retrograde ejaculation in
which sperm is expelled into the bladder
instead of the urethra.
 Laboratory protocols vary with regard to
reporting or not reporting the presence of
spermatozoa in a urine specimen

IX.Mucus
 Mucus is a protein material produced by the
glands and epithelial cells of the lower
genitourinary tract and the RTE cells.
 Mucus appears microscopically as thread-like
structures with a low refractive index
 Uromodulin / Tamm-Horsfall protein is the major
constituent or matrix of the mucus
 Mucus is more frequently present in female urine
specimens. It has no clinical significance when
present in either female or male urine.

URINARY CAST
 ______________– presence of urinary cast
 Casts are the only elements found in the urinary
sediment that are unique to the kidney.
 Formed in DCT, and Collecting duct
 Examination of casts should be performed along the
edges of the cover slip.
 ________________________ – major constituent of cast
matrix which is secreted by the RTE Cells.
 Cylindroids – formed at the ALH and DCT with tapered
end or have a tail at the other tail. It has the same
significance as casts.
 Cylindroids are product of incomplete cast formation,
or cast disintegration.

CAST FORMATION
- From least significant to the most significant.

Hyaline Cast→ Cellular cast→ Coarse granular cast → Fine

granular cast → Waxy cast → Broad Cast

Step by Step Analysis of the Formation of Tamm-

 Horsfall protein matrix
1. Aggregation of Tamm-Horsfall protein into individual
protein fibrils attached to the RTE cells
2. Interweaving of protein fibrils to form a loose
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

NOTE <3
1. Hypotonic and alkaline urine promotes the disintegration
of casts in the urine sediment.
2. Acid pH, increased solute concentration, urine stasis,
and increased plasma proteins (particularly albumin)
enhance cast formation
HYALINE CAST
 Most frequently seen cast
 Pro – cast
 The presence of zero to two hyaline casts per lpf is
considered normal
 Physiologic increase: 1. Strenuous exercise 2.
Dehydration 3. Heat exposure 4. Emotional stress
 Pathologic increase: 1.Acuteglomerulonephritis 2.
Pyelonephritis 3. Chronic renal disease 4. Congestive
heart failure
 Hyaline casts appear colorless in unstained sediments
and have a low refractive index similar to that of
urine; thus, they can easily be overlooked if
specimens are not examined under subdued light
 Steinheimer-Malbin stain and KOVA stain : Pink color
 Normal value : _______

RBC CAST
 Seen during bleeding in the nephron , especially
associated with glomerulonephritis
 RBC casts are easily detected under low power by their
orange-red color.
 They are more fragile than other casts and may exist
as fragments or have a more irregular shape as the
result of tightly packed cells adhering to the protein
matrix

WBC CAST
 The appearance of WBC casts in the urine signifies
infection or inflammation within the nephron.
 They are most frequently associated with
pyelonephritis and are a primary marker for
distinguishing pyelonephritis (upper UTI) from
Cystitis (lower UTI)
 Source of error: WBC clumps

BACTERIAL CAST
 Bacterial casts containing bacilli both within and
bound to the protein matrix are seen in
pyelonephritis.
 Their presence should be considered when WBC casts and
 many free WBCs and bacteria are seen in the sediment
 Confirmation of bacterial casts is best made by
performing a Gram stain on the dried or
cytocentrifuged sediment.

FATTY CAST
 Fatty casts are seen in conjunction with oval fat
bodies and free fat droplets in disorders causing
lipiduria.
 They are most frequently associated with the
nephrotic syndrome, but are also seen in toxic
tubular necrosis, diabetes mellitus, and crush
injuries
 Confirmation of fatty casts is performed using
polarized microscopy and Sudan III or Oil Red O fat
stains.

GRANULAR CAST
 Coarsely and finely granular casts are frequently
seen in the urinary sediment and may be of pathologic
or nonpathologic significance.
 The origin of the granules in nonpathologic
conditions appears to be from the lysosomes excreted
by RTE cells during normal metabolism.
 Pathologic increase : glomerulonephritis and
pyelonephtritis
 Physiologic increase: strenuous exercise

WAXY CAST
 Waxy casts are representative of extreme urine
stasis, indicating chronic renal failure.
 They are brittle, and highly refractive compared to
hyaline cast
 They often appear fragmented with jagged ends and
have notches in their sides
 Ground glass appearance, homogenous matrix, with
cracks or fissures from margins or along the length
of the cast

BROAD CAST
 Often referred to as renal failure casts, broad casts
like waxy casts represent extreme urine stasis.
 The presence of broad casts indicates destruction
(widening) of the tubular walls. Also, when the flow
of urine to the larger collecting ducts becomes
severely compromised, casts form in this area and
appear broad.
 All types of casts may occur in the broad form.
 Two most commonly seen broad cast: granular and waxy

OTHER CAST
 Mixed cellular cast
 Bilirubin cast
 Epithelial cast
LOOK-A-LIKES
1. Mucus threads = can be misidentified as hyaline cast
2. Cotton threads or diaper fibers = resembles waxy cast
3. Folded squamous epithelial cells
4. Aggregates of amorphous crystals
 CLASSIFICATION OF URINARY CASTS
(Brunzel, 3rd ed.)
Homogenous Hyaline and waxy cast
Pigmented Bilirubin, Myoglobin, and hemoglobin
Size Broad cast
inclusions Cellular inclusions: Red blood
cells,Leukocytes,Renal tubular epithelial
cells,Mixed cells ,Bacteria

Others: Granular,Fat globules—cholesterol,

triglycerides,Hemosiderin granules,
Crystals

URINARY CYRSTALS
 Crystals are formed by the precipitation of urine
solutes, including inorganic salts, organic
compounds, and medications (iatrogenic).
 Crystal formation is affected by changes in : pH,
Temperature, solute concentration
 Usually reported as rare, few, moderate, or many per
hpf
 Abnormal crystals may be averaged and reported per
lpf

NORMAL URINARY CRYSTALS

Amorphous Brick dust / yellow None Alkali

urates brown granules and
Pink sediment heat
(uroerythrin)

Note! It will convert

to uric acid crystals
with acidification with
acetic acid
Uric acid Rhombic, wedge, Lesch nyhan Alkali
hexagonal, four sided syndrome
flat plate(whetsone), Gout
lemon shaped, nipple chemotherapy
shaped
Calcium Forms: ↑food high Dilute
oxalate a.dihydrate(wheddelite) in ascorbic HCL
– envelope or pyramidal acid

b. monohydrate
(whewellite) – oval Ethylene
/dumbbell glycol
poisoning
Amorphous Granular in appearance None Dilute
phosphates White precipitate acetic
acid
Ammonium Thorny apples Presence of Acetic
biurate Seen in old specimens urea- acid
splitting with
bacteria heat
Triple Colorless, prism-shape Presence of Dilute
phosphate or “coffin lid” urea- acetic
/ Feathery appearance splitting acid
magnesium when they bacteria
ammonium disintegrate,Fern-leaf
phosphate/ shape, sheets or flakes Associated
struvite with Proteus
vulgaris
Calcium Colorless, flat plates, None Dilute
phosphate/ thin prisms often in acetic
apatite rosette form acid

Rosettes form may

resemble sulfonamide
crystals
Calcium Small, colorless, Gas
carbonate dumbbell, or spherical from
shaped acetic
acid

ABNORMAL URINE CRYSTALS

Crystal Information Significance Solubilit

y
Cysteine Colorless Cystinuria Ammonia,
hexagonal plates cystinosis dilute
Mistaken as uric HCL
acid crystals
Cholester Rectangular Nephrotic syndrome Chlorofor
ol plates with a m
notch in one or
more corners
(staircase
pattern)

Crystal of
radiographic dye
Tyrosine Colorless to Liver disease Alkali or
yellow needles heat
in clumps or
rosettes
Leucine Yellow brown Liver disease Hot
spheres with alkali or
concentric alcohol
circles and
radial
striations

Note! Leucine
and tyrosine
crystals may
occur together;
leucine may be
precipitated
with tyrosine
crystals if
alcohol is added
to urine

bilirubin Clumped needles Liver disease Acetic

or granules with acid,
bright yellow HCL,
color NaOH,
ether,
 chlorofor
m, and
alkali
Sulfonami Colorless to Possible tubular Acetone
de yellow brown damage
needles, sheaves
of wheat,
rosette

May be mistaken
with calcium
phosphate
crystals.
 Calcium
phosphate :
soluble to
acetic acid

 sulfonamide
:
(+) lignin
test (urine
+25%HCL=Yel
low )

ampicilli Colorless Massive Refrigerati

n needles that dose of on forms
tend to form penicill bundles
bundles in
following
refrigeration

Crystals formed in Amorphous urates, Calcium oxalate, uric

acidic urine acid, and all abnormal crystals
Crystals formed in Amorphous phosphates, amorphous
alkaline urine biurates, calcium carbonate, calcium
phosphate, and Triple phosphate

URIC ACID
VERSUS
CRSTALS
Uric acid Cystine
Color
Solubility in
ammonia
Solubility in dilute
HCL
Birefringence
Cyanide
nitroprusside
reaction

URINARY SEDIMENT ARTIFACTS

1. Starch granules
 spheres with dimpled center
 “maltese cross” formation on polarizing microscope

2. Oil droplets
3. Air bubbles
 4. Pollen grains- spheres w/ a cell wall and concentric
circles
5. Hair and fibers – mistaken as casts
6. Fecal contamination

Manner of Reporting
RBC, WBC Average number per 10 HPF
Casts Average number per LPF
Squamous epithelial Rare, few ,moderate, or many per
cells LPF
Transitional epithelial Rare, few, moderate, or many per
cells HPF
RTE cells Average number per 10 HPF
Oval fat bodies Average number per HPF
Bacteria, yeast Rare, few ,moderate, or many per
HPF
Trichomonas Rare, few ,moderate, or many per
HPF
Spermatozoa Present, based on laboratory
protocol
Mucus Rare, few ,moderate, or many per
LPF
Normal cyrstals Rare, few, moderate or many per
HPF
Abnormal crystals Average number per LPF

Reporting Mucus Crystals Epithelial Bacteria

cells
Rare 0-1 0-2 0-5 0-10
Few 1-3 2-5 5-20 10-50
Moderate 3-10 5-20 20-100 50-200
Many >10 >20 >100 >200

Qualitative terms and Descriptions

for field of views (FOVs)
Term Description
Rare (1+) Present, but hard to find
Few (1+) One (or more) present in almost every field of
view (FOV)
Moderate Easy to find; number present in FOV varies;
(2+) “more than few, less than many”
Many (3+) Prominent; large number present in all FOVs
Packed (4+) FOV is crowded by or overwhelmed with the
elements

RENAL DISEASES
GLOMERULAR DISORDERS
DISORD ETIOLOG CLINIC PRIMARY OTHER
ER Y AL URINALYS SIGNIFICANT
COURSE IS TESTS
RESULT
Acute Deposition Rapid Macroscopic Antistreptolysin O
Glomerulon of immune onset of hematuria, titer
ephritis complexes hematuria Proteinuria,
formed in and edema. Red Blood Anti- group A
conjunction Cell casts, Streptococcal
 with group Permanent Granular enzymes
A renal casts
Streptococc damage
us seldom
infection, occurs
on the
glomerular
membranes
Rapidly Deposition Rapid Macroscopic Blood Urea
progressiv of immune onset with hematuria, Nitrogen
e complexes glomerular Proteinuria, Creatinine
glomerulon from system damage and Red Blood Creatinine
ephritis immune possible Cell casts clearance
disorders progressio
on the n to end-
glomerular stage
membrane renal
failure
Good Attachment Hemoptysis Macroscopic Anti- glomerular
pasteur’s of and hematuria, Basement membrane
syndrome cytotoxic dyspnea Proteinuria, Antibody
antibody followed Red Blood
formed by Cell casts
during hematuria.
viral
respiratory Possible
infections progressio
to n to end-
glomerular stage
and renal
alveolar failure
basement
membranes
Wegener’s Antineutrop Pulmonary Macroscopic Antineutrophilic
Granulomat hilic symptoms hematuria Cytoplasmic
osis cytoplasmic including Proteinuria antibody
autoantibod hemoptysis Red Blood
y binds to develop Cell Casts
neutrophils first
in vascular followed
walls by renal
producing involvemen
damage to t and
small possible
vessels in progressio
the lungs n to end-
and stage
glomerulus renal
failure
Henoch- Occurs Initial Macroscopic Stool occult blood
Schonleinp primarily appearance hematuria,
urpura in children of purpura Proteinuria,
following followed Red Blood
viral by blood Cell casts
respiratory in sputum
infections; and stools
a decrease and
in eventual
platelets renal
disrupts involvemen
vascular .
integrity
Complete
 recovery
is common,
but many
progress
to renal
failure.
IgA Deposition Recurrent Early stages: Serum
nephropath of IgA on macroscopi Macroscopic immunoglobulin A
y the chematuria or
(Berger’s glomerular following microscopic
disease) membrane exercise hematuria
resulting with slow Late stages:
from progressio See chronic
increased n to glomeruloneph
levels of chronic ritis
serum IgA glomerulon
ephritis
Membranous Thickening Slow Microscopic Antinuclear
glomerulon of the progressio hematuria Antibody
ephritis glomerular n to Proteinuria Hepatitis surface
membrane ephritic antigen
following syndrome Flourescenttrepone
IgG immune or mal antibody-
complex possible absorption test
deposition remission (FTA-ABS)
associated
with
systemic
disorders

Membranopr Cellular Noticeable Hematuria Serum complement

oliferativ proliferati progressio Proteinuria levels
e on n to
glomerulon affecting chronic
ephritis the glomerulon
capillary ephritis
“TRAM walls or to
TRACKING the nephritis
PATTERN OF glomerular syndrome.
THE basement
GLOMERULUS membrane,
” possibly
immune
mediated.

Chronic Marked Noticeable Hematuria Blood Urea

glomerulon decrease in decrease Proteinuria Nitrogen
ephritis renal in renal Glucosuria Serum Creatinine
function function Cellular and Creatinine
resulting progressin granular clearance
from g to renal casts Electrolytes
glomerular failure. Waxy and
damage broad casts
precipitate
d by other
renal
disorders
Nephrotic Disruption Acute on Heavy Serum albumin
Syndrome of the set proteinuria, Cholesterol
 electrical following Microscopic Triglycerides
charges systemic hematuria,
that shock Renal tubular
produce the Gradual cells,
tightly progressio Oval fat
fitting n from bodies
podocyte other Fat droplets
barrier glomerular Fatty and
resulting disorders waxy casts
in massive and then
loss of to renal
protein and failure
lipids
Minimal Disruption Frequent Heavy Serum albumin
change of complete proteinuria Cholesterol
disease podocytes remission Transient Triglycerides
occurring following hematuria
primarily corticoste Fat droplets
in children roid
following treatment
allergic
reactions
and
immunizatio
ns
Focal Disruption May Proteinuria Drug of Abuse
segmental of resemble Microscopic HIV tests
glomerulos podocytes nephrotic hematuria
clerosis in certain syndrome
areas of or minimal
glomeruli change
associated disease.
with heroin
and
analgesic
abuse and
acquired
immunodefic
iency
syndrome
Alport Genetic Slow See Nephrotic
syndrome disorder progressio syndrome
showing n to to
lamellated nephritic
and syndrome
thinning of and end
glomerular stage
basement renal
membrane disease

Diabetic Most common (+)

Nephropath cause of Microalbuminu
y ESRD ria
(Kimmelsti Deposition (+) Micral
el- Wilson of test
disease) glycosylate
d proteins
on the
glomerular
basement
membranes
caused by
poorly
 controlled
blood
glucose
levels

NEPHROTIC SYNDROME LAB FINDINGS

URINE FINDINGS PLASMA OR BLOOD

FINDINGS
1. Proteinuri 1. Increase
a A2-
2. Lipiduria Macroglobulin
3. Hematuria in
4. Cylindruri electrophoresis
a(fatty cast ) 2. Hypoprot
einemia and
hypoalbuminemia
3. Hyperlip
idemia
4. INCREASE
PLASMA SODIUM
AND WATER LEVEL
DUE TO INCREASE
SODIUM AND
WATER
REABSORPTION IN
THE KIDNEY that
will eventually
lead to EDEMA

TUBULAR AND INTERSTITIAL DISORDERS

DISORDER ETIOLOGY FINDINGS

Acute tubular Damage to renal Microscopic
necrosis tubular cells hematuria,
caused by ischemic protienuria
or toxic agents Hyaline, Granular,
Waxy and Broad
cast
RTE CELLS, RTE
CASTS
Odorless urine
Isosthenuria
Fanconi syndrome Generalized failure Glucosuria
of tubular reaction Cystinuria
in the Proximal
 Convoluted tubule

Diabetis Insipidus a.Neurogenic DI Lows Specific

-failure of the gravity urine
hypothalamus to Polyuria
produce ADH
-Low ADH

b. Nephrogenic DI
-inability of the
renal tubules
to respond to ADH
-Normal to increase
ADH
Renal Glucosuria Defective tubular Normal Blood
reabsorption of glucose
glucose Increase Urinary
glucose
Cystitis (Lower Ascending bacterial WBCs, Bacteria
UTI) infection of the Microscopic
urinary bladder hematuria
Mild proteinuria
Increased urine pH
Acute Infection of the WBCs, bacteria
Pyelonephritis renal tubules and WBC cast,
(Upper UTI) interstitium Bacterial Cast
related to Microscopic
interference of hematuria,
urine flow to the proteinuria
bladder, reflux of
urine from the
bladder
(Visicoureteral
reflux) and
untreated cystitis
Chronic Recurrent infection WBC, Bacteria
Pyelonephritis of the renal WBC CAST,
tubules and Bacterial cast
interstitium caused Granular, Waxy,
by structural and Broad Cast
abnormalities Hematuria and
affecting the flow Proteinuria
of urine
Acute Interstitial Allergic Hematuria,
nephritis inflammation of the proteinuria
renal interstitium WBCs (Increase
in the response to Eosinophil)
certain medication WBC cast
NO BACTERIA

PROXIMAL CONVOLUTED TUBULAR

DYSFUNCTIONS
Impaired ability to Renal glucosuria
reabsorb glucose
Impaired ability to -Cystinuria (cystine and
reabsorb specific dibasicamino acids)
aminoacids -Hartnup disease (monoamino-
monocarboxylic amino acids or
 glycine)
Impaired ability to Bartter’s syndrome
reabsorb sodium
Impaired ability to Renal tubular acidosis type II
reabsorb bicarbonate
Impaired ability to Idiopathic hypercalciuria
reabsorb calcium
Excessive reabsorption Hypocalciuric familial hypercalcemia
of calcium
Excessive reabsorption Gordon’s syndrome
of sodium
Excessive reabsorption Pseudohypoparathyroidism
of phosphate

DISTAL CONVOLUTED TUBULAR

DYSFUNCTIONS
Impaired ability to Familial hypophosphatemia(vitamin D–
reabsorb phosphate resistant rickets)
Impaired ability to Idiopathic hypercalciuria
reabsorb calcium
Impaired ability to Renal tubular acidosis types, urine
acidify I and IV
Impaired ability to Renal salt-losing disorders, sodium
retain
Impaired ability to Nephrogenic diabetes
concentrate urine
Excessive reabsorption Liddle’s syndrome
of sodium

ACUTE AND CHRONIC RENAL FAILURE

Acute renal -characterized clinically by a sudden
failure (ARF) decrease in the GFR, azotemia, and
oliguria
-nephrons are “functionally” abnormal, no
histologic abnormality is usually present
- usually reversible
- Primary causes of ARF include a sudden
decrease in blood flow to the kidney
(prerenal), acute glomerular and tubular
disease (renal), and renal calculi or
tumor obstructions (postrenal)
- ischemic acute tubular necrosis is the
most common cause of ARF
Chronic renal -Progressive loss of renal function caused
failure (CRF). by an irreversible and intrinsic renal
disease characterizes chronic renal
failure (CRF).
-associated with azotemia, acid-base
imbalance, water and electrolyte
imbalance, and abnormal calcium and
phosphorus metabolism
-CRF progresses to an advanced renal
disease often termed end stage renal
disease or end-stage kidneys
- Urinalysis findings associated with end-
stage renal disease include a fixed
specific gravity (isosthenuria, at 1.010),
significant proteinuria, minimal to
 moderate hematuria, and the presence of
all types of casts, particularly waxy and
broad casts.
-The progression to end-stage renal
disease is characterized by a marked
decrease in the glomerular filtration rate
(less than 25 mL/min) steadily rising
serum BUN and creatinine values
(azotemia); electrolyte imbalance; lack of
renal concentrating ability producing an
isothenuric urine; proteinuria; renal
glycosuria; and an abundance of granular,
waxy, and broad casts, often referred to
as a telescoped urine sediment

TELESCOPED SEDIMENTS
 Simultaneous appearance of the elements of acute/chronic
glomerulonephritis and nephrotic syndrome
 Increase cells, casts, lipid droplets, and oval fat
bodies
Such sediment may be found in collagen vascular disease
(notably lupus nephritis) and subacute bacterial endocarditis.

ATHLETIC PSEUDONEPHRITIS
 Associated with strenuous exercise
such as marathon running
 Normal /physiologic condition characterized by
appearance of CELLS AND CASTS in urine
Positive in RBC, WBC, Granular cast , Hyaline cast.

URINE SCREENING FOR METABOLIC

DISORDERS
AMINOACIDURIA

1. Over Flow Type

 ___Amino acid in blood
 ___Amino acid in urine TANDEM MS/MS(MASS
Ex: PKU, MSUD, spectrometry)
Cystinosis,Alkaptonuria -gold standard test
for NBS
2. Renal Type
 ___Amino acid in blood
-Specimen: Bloodspot
 ___Amino acid in urine
 Due to defective tubular function
Ex: Cystinuria, Fanconi’s syndrome

PHENYLALANIN-TYROSINE DISORDERS
DISORDER Enzyme Information Tests
deficient
Phenylketonuria ______________ “Mousy odor FeCl3 tube
- Inherited as ______ urine” test = (+)
an autosomal _____________
-associated ___
recessive
with mental Phenistix
disease, it is retardation strip = (+)
characterized gray to gray
by increased green
urinary Guthrie
bacterial
excretion of
inhibition
phenylpyruvic test
acid (a  Bacillus
ketone) and subtilis
its is
metabolites cultured
with
beta2 –
thienyla
lanine
 Beta2-
thienyla
lanine
inhibits
growth
of
B.subtil
is
 Phenylal
anine
countera
cts the
action
of
Beta2-
thienyla
lanine
 (+)
result :
________
_____

Tyrosyluria/Tyro Type1: “Rancid FeCl3 tube

sinemia Fumarylacetoac Butter odor test = (+)
etate urine” ____________
hydrolase
Type 2: Nitroso-
Tyrosine naphtol = (+)
aminotransfera Orange Red
se
Type 3:
p-
hydroxyphenylp
yruvic
acid
dioxygenase
Alkaptonuria Homogentisic Urine FeCl3 tube
acid oxidase darkens test = (+)
after _____________
becoming ____
alkaline Clinitest =
from (+) Yellow
standing at ppt
room
temperature
Melanuria -- Due to FeCl3 tube
-Melanin is overprolifer test =
produced from ation of (+)
tyrosine by melanocytes _____________
melanocytes Urine ___
and is the darkens upon Sodium
pigment air exposure nitroprusside
responsible for test = (+)
the color of
hair, skin, and
Purple
eyes Ehrlich test
(+) = Red

Second metabolic pathway of tyrosine is responsible for the

production of melanin, thyroxine, epinephrine, protein, and
tyrosine sulfate.

BRANCHED CHAIN AMINO ACID DISORDER

Disorder Information Test
Maple syrup urine Increase in blood 2,4
Disorder and urine Dinitrophenylhydraz
 _______________ ine (DNPH)
_________ (+)
___________________
-Caramelized sugar / _________
burnt sugar/ Curry
odor / Maple syrup
odor urine due to
ketoacids in urine
Organic acidemias “Sweaty feet odor
1. Isovaleric urine”
academia
2. Propionic p-nitroaniline test
academia = (+) Emerald green
3. Methylmalonic
academia

TRYPTOPHAN DISORDERS
Disorder Information Test
Indicanuria Indigo blue color Obermayer’s test
urine (upon air - FeCl3 + Urine +
Tryptophan→ indole exposure) Chloroform → (+)
→ indican purple
Seen in : Intestinal
disorders, Hartnup
disease
Argentaffinoma Carcinoid tumor FeCl3 tube test =
involving (+) blue – green
argentaffin cells Nitrosonaphtol w/
 Produce 5- nitrous acid = (+)
HIAA , a Violet to black
metabolite of
serotonin
 Avoid
______________

CYSTINE DISORDERS
Disorder Information Test
Cystinuria Renal type aminoaciduria Brand’s
modification of
Defective tubular Legal’snitropruss
reabsorption of: ide
- _______________________ Rgt: Cyanide
___ nitroprusside =
(+)Red-purple
Cystinosis Inborn Error of metabolism Brand’s
(-) gene that codes for an modification of
enzyme responsible for Legal’snitropruss
cysteine metabolism ide
Rgt: Cyanide
nitroprusside =
(+)Red-purple
Homocystinur Defects in the metabolism of Silver cyanide
ia methionine that lead to nitroprusside=
increase homocysteine. (+)Red-purple
Associated with methionine
malabsor
MUCOPOLYSACCHARIDE DISRODERS
Disorder Information Test
Hurler Mucopolysaccharides
syndrome accumulate in the cornea
of the eye

Hunter Sex linked recessive,

syndrome rarely seen in females

Sanfilippo Mental retardation only

syndrome

NICE TO KNOW!

1. Serotonin is produced from tryptophan by the

argentaffin cells in the intestine and is carried
through the body primarily by the platelets. Normally,
the body uses most of the serotonin, and only small
amounts of its degradation product, 5-HIAA, are
available for excretion in the urine.
2. The normal daily excretion of 5-HIAA is 2 to 8 mg, and
excretion of greater than 25 mg/24 h can be an
indication of argentaffin cell tumors
3. The increased homocystine can result in failure to
thrive, cataracts, mental retardation, thromboembolic
problems, and death.
4. False positive results in cyanide nitroprusside test is
associated with the presence of ketones and homocystine
5. In mucopolysaccharidosis, the products most frequently
found in the urine are dermatan sulfate, keratin
sulfate, and heparan sulfate.

Purine Disorder
 Lesch-Hyhan disease
 Lack Hypoxanthine-guanine phosphoribosyl transferase
 Increase uric acid in blood and urine

Porphyrin Disorders (Porphyrias)

 Disorder of porphyrin metabolism
 Urine color = Red/purple/Burgundy red/ Port wine/
Purplish red
 Colorless in = Lead poisoning
 Screening test Specimen = urine, stool, blood, bile
a. Ehrlich’s reaction – detects D-ALA porphobilinogen
b. Fluorescence at 550-600nm – test for uroporphyrin,
coproporphyrin and protoporphyrin
(+)result: Violet/pink/red Fluoresence
 c. Free Erythrocyte Protoporpyrin (FEP) – CDC recommended
test for lead poisoning
Note: Lead poisoning inhibits ALA synthase and Ferrochelatase
enzymes

Porphyria Elevated Clinical Symptoms Laboratory

Compound(s) Testing
Acute ALA Neurologic/psychiat Urine/Ehrli
intermittent Porphobilinog ric ch reaction
porphyria en
Porphyria Uroporphyrin Photosensitivity Urine
cutanea fluorescenc
tarda e
Congenital Uroporphyrin Photosensitivity Urine or
erythropoiet and feces
ic porphyria Coproporphyri fluorescenc
n e
Variegate Coproporphyri Photosensitivity/ Bile or
porphyria n neurologic feces
fluorescenc
e
Erythropoiet Protoporphyri Photosensitivity Blood FEP
ic n Bile or
protoporphyr feces
ia fluorescenc
e
Lead ALA and Neurologic Acetoacetic
poisoning Protoporphyri acid +
n urine/
Ehrlich
reaction
Blood FEP

CARBOHYDRATES DISORDER
 Melituria = general term for the presence of urinary
sugar
 Galactosuria, pentosuria, lactosuria, fructosuria, and
glucosuria
 Galactosuria = indicates inability to properly
metabolize galactose to glucose such as in
case of newborn errors

SYNOVIAL FLUID
 Formed as a non-selective ultrafiltrate of plasma across
synovial membrane except for the exclusion of high
molecular weight protein.
 A.K.A= joint fluid
 “Synovial” = latin word for synovia means “egg or ovum”
 Viscous fluid circulating in diarthroses (movable joints)
 Synovial fluid viscosity comes from polymerization of the
hyaluronic acid and is essential for the proper joints
lubrication
 Synoviocytes secrete a mucopolysaccharide containing
________________, and is responsible for viscosity

FUNCTION
1. Lubricates joints
2. Reduce friction between bones
3. Provides nutrients to the articular cartilage
 4. Lessen shock of joint compression occurring during
activities such as walking and jogging

SPECIMEN COLLECTION
 Method : ___________________
 If possible, patients should be fasting a minimum
of 4 to 6 hours to allow for the equilibration of
chemical constituents between plasma and synovial
fluid (Brunzel, 3rd ed.)
 Volume : Normal = 0.1 to 3.5 ml , Inflammation - >25 ml
 REQUIRED TUBE TYPES FOR SYNOVIAL FLUID TESTS AND ORDER OF
DISTRIBUTION
1. Chemistry and Serology = non-anticoagulated
2. Hematology and cytology = sodium heparin or liquid EDTA
3. Microbiology= Sterilized heparin or SPS

GRADING Interpretation
COLOR AND CLARITY
 Colorless to pale yellow = Normal Good Solid clot
 Deeper yellow = inflammation
 Greenish tinge = bacterial infection Fair Soft clot
 Red = traumatic tap, hemorrhagic Low Friable clot
arthritis
Poor No clot
 Milky = presence of __________

VISCOSITY
 Normal : able to form a string ___________ long

Test: Ropes/ Mucin clot test (Hyaluronate polymerization
Test)
Reagent: uses 2-5 % acetic acid

TEST TO POSITIVELY IDENTIFY A SYNOVIAL FLUID

1. Ropes test = 1part of fluid + 4parts of
2%acetic acid  check for clot
2. Toluidine blue test = a few drops of the suspect
fluid are placed onto filter paper followed by
0.2% toluidine blue stain. If synovial fluid is
present, the drops of fluid will stain blue.
CELL COUNT

1. WBC count
 Most frequently performed count
 Diluting fluid : NSS with methylene blue, Hypotonic
saline , Saline with saponin
 For very viscous fluid add pinch of hyaluronidase to
0.5ml fluid or add 1 drop of 0.05% hyaluronidase in
phosphate buffer per ml of fluid ( incubate at 37’C
for 5 mins.)
 DIFFERENTIAL COUNT NORMAL VALUES
RBCs <2000 / ul
WBCs < 200 / ul
WBC differential count 65 % Monocyte and macrophages
<25 % Neutrophils
<15 % Lymphocytes
Note : An eosinophil of greater
than 2 %is associated with
allergic disease with arthritis,
hemorrhagic joint effusion, lyme
disease, parasitic arthritis, RA,
and tubercular arthritis

CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID

Cell Description Significance
/Inclusion
Neutrophil Polymorphonuclear Bacterial sepsis
leukocyte Crystal induced
inflammation
Lymphocyte Mononuclear Nonseptic inflammation
leukocyte
Macrophage Large mononuclear Normal
leukocyte, may be Viral infections
vacuolated
Synovial Similar to Normal
lining cell macrophage, but may
be multinucleated
resembling a
mesothelial cell
LE cell Neutrophil Lupus erythematosus
containing
characteristic
ingested “ round
homogenous body”
Reiter cell Vacuolated Reiter syndrome
macrophage with Non specific inflammation
ingested neutrophils
RA cell Neutrophil with dark Rheumatoid arthritis
(Ragocyte) cytoplasmic granules Immunologic inflammation
containing immune
complexes
Cartilage Large multinucleated Osteoarthritis
cells cells
Rice bodies Macroscopically Tuberculusosis, septic
resembles polished and rheumatoid arthritis
rice
Microscopically show
collagen and fibirn
Fat droplets Refractile Traumatic injury
intracellular and Chronic inflammation
extracellular
globules stain with
sudan dyes
Hemosiderin Inclusions within Pigmented
clusters of synovial villonodularsynovitis
cells
Ochronotic Ground pepper Joint prosthesis,
shards appearance ochronosis

2. CRYSTAL INDENTIFICATION
 Causes of crystal formation:
 a. Metabolic disorders
b. Decreased renal excretion that produce increased
blood levels of crystallizing chemicals
c. Degeneration of cartilage and bones
d. Injection of medications (corticosteroid)
 Polarizing microscope = detects for the presence or
absence of birefringence
 Compensated polarizing microscope = confirms the
type of birefringence (positive or negative)
 A red compensator is placed between
crystal and analyzer
 Yellow= parallel = (-) Birefringence
 Blue = perpendicular = (+) Birefringence

Specimens for crystal analysis should not be refrigerated

because they can produce additional crystals that can
interfere with the identification of significant
crystals. Avoid using powderized anticoagulant because it
can cause artifacts and may interfere in crystal
identification

Crystal Shape Compensated Significance

Polarized
Light
Monosodium Fine Needles Negative Gout
urate birefringenc
e
Calcium Rhombic Positive Pseudogout or
Pyrophosphate squares, rods birefringenc chondrocalcinosi
e s

Cholesterol Notched, Negative Extracellular,

rhombic plates birefringenc chronic
e arthritis
condition such
as Rheumatoid
arthritis
Corticosteroi Flat, Positive & Injections
d variables- Negative
shaped plates birefringenc
e
Calcium Envelopes Negative Renal dialysis
oxalate birefringenc
e
Apatite (Ca Smallparticles No Osteoarthritis
Phosphate) ; require birefringenc
electron e
microscopy

CHEMISTRY TEST
Glucose Most frequently test in chemistry.Done in
conjunction with blood glucose. Simultaneous blood
and synovial fluid samples should be obtained,
preferably after the patient has fasted for 8 hours to
allow equilibration between the two fluids

BLOOD GLUCOSE – S.F GLUCOSE = <10mg/dl

*Decreased in Type II and III arthritis or
inflammatory disorder
*Normal Value = the difference between the Blood
glucose and Synovial fluid glucose should be less
than 10mg/dl
Lactate Normal value = <250 mg/dl
Increase in infection
Protein Normal value = <3 g/dl
Increased in inflammatory and hemorrhagic disorders
Uric Normal value = same as blood
acid Increase in gout

GROUP I IIa IIb III IV

Non Inflammat Inflamma Septic Hemorrh
infammatory ory - tory- agic
Immunolog Crystal
ic induced
Signifi Degenerative Immunolog Gout Microbia Traumat
cance joint ic Pseudogo l ic
disorder disorders ut infectio injury
n Coagula
tion
deficie
ncy
Color Clear, Cloudy, Cloudy Cloudy, Cloudy,
and yellow fluid yellow or milky yellow- red
clarity fluid fluid green fluid
fluid
Viscosi Good Poor Low Variable Low
ty
WBC <1,000 / ul 2000- Up to 50K-100K Equal
count 75000/ul 100,000 / ul to
/ul blood
Neutrop <30 % >50% <70% >75% Equal
hils to
blood
Glucose Normal Decreased Decrease Decrease Normal
d d
Others +Auto + + + RBC
antibodie Crystals Culture
s and GS
Associa Osteoarthrit Rheumatoi Crystal - -Trauma
ted is d synoviti Bacteria -Tumor
disease Osteochondri arthritis s l -Joint
s tis , SLE, (gout, infectio prosthe
Osteochondro ankylos pseudogo n, sis
matosis ing ut) -Fungal -Blood
Traumatic infectio disease
Spondyl
arthritis n, such as
Neuroarthrop itis, - sickle
athy Lyme Mycobact cell
arthrit erial disease
is infectio and
n hemophi
lia

MICROBIOLOGY
 Common organisms that infect the synovial fluid:
1. Staphylococcus aureus
2. Streptococcus
3. Haemophilus
4. Neisseria gonorrheae

SEROLOGIC TEST
 Auto antibody detection for RA, SLE
 Detection of antibodies to Lyme disease
 SEROUS FLUID
 A Fluid between parietal membrane(lines the cavity wall)
and visceral membranes(covers the organ)
 Serous fluid are formed from ultra-filtrate of plasma
 Production and reabsorption are subject to hydrostatic
pressure and colloidal pressure (oncotic pressure) from
the capillaries that serve the cavities and the capillary
permeability.

FUNCTION:
1. To provide lubrication between the 2 membranes as the
surfaces move against each other

EFFUSION
 Accumulation of fluid between the membranes
 Classified as exudate or transudate
TRANSUDATE EXUDATE
Effusion caused by a systemic Effusion caused by direct
disorder that disrupts the damage to the membrane
fluid production and
regulation between membranes
Causes: Causes:
1. Hypoproteinemia 1. Infection
2. Congestive heart failure 2. Malignancy
3. Nephrotic syndrome 3. Inflammation
4. Malnutrition 4. Lymphatic duct
5. Hepatic cirrhosis obstruction

TRANSUDATE VS EXUDATE
Transudate Exudate
Appearance Clear Cloudy
Fluid: serum protein <0.5 >0.5
ratio
Fluid: serum LD <0.6 >0.6
ratio
WBC count <1,000 cell/ul >1,000 cell/ul
Spontaneous clotting No Possible
Pleural fluid <45-60 >45-60
cholesterol (mg/dl)
Pleural fluid: serum <0.3 >0.3
cholesterol ratio
Pleural fluid: <0.6 >0.6
bilirubin ratio
Serum ascites >1.1 <1.1
albumin gradient
(SAAG)
Glucose Decrease Increase
Rivalta’s test Negative Positive
Acetic acid + water
+ Unknown fluid ---
>(+)heavy
precipitation

Differentiation between ascitic fluid transudates and exudates is

more difficult than for pleural and pericardial effusions. The
serum-ascites albumin gradient (SAAG) is recommended over the
fluid:serum total protein and LD ratios for the detection of
transudates of hepatic origin.
METHOD OF COLLECTION
3Ps (Pleural, Pericardial, Peritoneal fluid)
- Normal appearance should be clear, pale yellow
1. Pleural fluid = _________________
2. Pericardial fluid = _________________
3. Peritoneal/Ascitic fluid = _________________

Specimen Distribution
1. EDTA = Cell count
2. Sterile heparin = Microbiology and cytology
3. Plain/heparin = Chemistry

PLEURAL FLUID
Appearance Significance
Clear, pale Normal
yellow
Turbid, white Microbial infection (TB)
Brown Rupture of amoebic liver abscess
Viscous Malignant mesothelioma (increase hyaluronic acid/)
Black Aspergillous
Milky Chylous material
Pseudochylous material
Bloody Hemothorax= due to traumatic aspiration
 Uneven distribution of blood, pleural fluid Hct
is>50% WB blood Hct

Hemorrhagic
 Pleural fluid Hct is <50% Whole blood Hct

CHYLOUS EFFUSION PSEUDOCHYLOUS

EFFUSION
Cause Thoracic duct Chronic
leakage inflammation
Appearance Milky / white Milky / green tinge
/ gold paint
Leukocytes Increase Mixed cells
lymphocyte
Cholesterol Absent Present
crystals
Triglycerides >110 mg/dl <50 mg/dl
Sudan III staining +++ Negative or weakly
+
Onset Sudden gradual
chylomicrons present absent

HEMATOLOGY DIFFERENTIAL COUNT ON SEROUS FLUID

CELL REFERENCE VALUE
Macrophages 64-80%
Neutrophil 1-2%
Lymphocyte 18-30%
Eosinophil <10%

SIGNIFICANCE OF CELLS SEEN IN PLEURAL FLUID

Cell Significance
Neutrophil ( normal is 1-2%) Pneumonia, pancreatitis,
pulmonary infarction
Lymphocyte (Normal is 18-30%) TB, viral infection,
autoimmune disorders,
malignancy
Mesothelial cell Normal and reactive forms
(Characterized by fried egg have no significance
appearance) Decrease in TB
Plasma cells TB
Malignant cells Primary adenocarcinoma, small
cell carcinoma
Eosinophil Allergic reaction, parasitic
infection, pneumothorax, and
hemothorax

SIGNIFICANCE OF CHEMICAL TEST IN PLEURAL FLUID

Glucose decrease in rheumatoid inflammation,
purulent infection
Lactate increase in bacterial infection
Triglyceride increase in chylous effusion
Ph decrease in pneumonia not responding
to antibiotics, esophageal rupture,
complicated parapneumonic effusion
(empyema)

*Ph less than 7.2= need for test tube

drainage
*Ph less than 6 = esophageal rupture
allowing the influx of gastric fluid
Adenosine deaminase tuberculosis, malignancy
Amylase increase pancreatitis, esophageal
rupture, malignancy

PERICARDIAL FLUID
APPEARANCE SIGNIFICANCE
Clear, pale yellow Normal, transudate
Blood-streaked Infection, malignancy
Grossly bloody Cardiac puncture,
anticoagulant medications
DIFFERENTIAL SIGNIFICANCE
Increase neutrophils Bacterial endocarditis
Malignant cells
TEST SIGNIFICANCE
Gram stain and culture Bacterial endocarditis
Acid fast stain Tubercular effusion
Adenosine Deaminase Tubercular effusion

PERITONEAL /ASCITIC FLUID

APPEARANCE SIGNIFICANCE
Clear, pale yellow Normal
Turbid Microbial infection
Green Gall bladder, pancreatic disorders
Blood streaked Trauma, infection, malignancy
Milky Lymphatic trauma and blockage
WBC COUNT SIGNIFICANCE
<500 cells/ ul Normal
>500 cells/ ul Bacterial peritonitis, cirrhosis
Note* bacterial peritonitis has an
absolute neutrophil count of
>250cells/ul or >50% of total WBC
Count
DIFFERENTIAL SIGNIFICANCE
Increase neutrophils Bacterial peritonitis
Malignant cells Malignancy
 TEST SIGNIFICANCE
Peritoneal lavage >100, 000 RBCs/ ul indicates blunt
trauma injury (intra-abdominal
bleeding)
CEA Malignancy of GI origin
CA 125 Malignancy of ovarian origin
Glucose Decrease in tubercular
peritonitis, malignancy
Amylase Increase in pancreatitis, GI
perforation
BUN/ Creatinine Ruptured/ punctured bladder
Gram stain and Culture Bacterial peritonitis
Acid fast stain Tubercular peritonitis
ADA Tubercular peritonitis

PSAMMOMA BODIES
 Contain concentric striations of collagen-like material.
 Seen in benign conditions and associated with ovarian and
thyroid carcinomas.

LIPOPHAGES
 Macrophage containing fat droplets.

AMNIOTIC FLUID
 Amniotic fluid is present in the amnion, a membranous sac
that surrounds the fetus

AMNIOTIC FLUID COMPOSITION

In the early stages of gestation, the water in amniotic
fluid is derived mostly from maternal serum; however, at 10
weeks, the fetus begins to produce urine which gets secreted
into the amniotic sac. During late gestation (the second and
third trimesters), as the amniotic fluid expands, fetal
urine becomes the largest source to the amniotic fluid. Lung
secretions, gastrointestinal secretions, and excretions from
the umbilical cord and placental surface contribute to the
composition of amniotic fluid as well; however, lung
secretions alone make up as much as one-third amniotic fluid

FUNCTIONS
The primary functions of the fluid are:
1. to provide a protective cushion for the fetus,
2. allow fetal movement,
3. stablize the temperature
4. to protect the fetus from extreme temperature changes,
and to permit proper lung development.

VOLUME
 Amniotic fluid volume is regulated by a balance between
the production of fetal urine and lung fluid and the
absorption from fetal swallowing and intramembranous
flow.
 The amount of amniotic fluid increases throughout
pregnancy, reaching a peak of approximately 1 L during
the third trimester, and then gradually decreases prior
to delivery.
 During the first trimester, the approximately 35 mL of
amniotic fluid is derived primarily from the maternal
circulation.
  After the first trimester, fetal urine is the major
contributor to the amniotic fluid volume.
POLYHYDRAMNIOS OLIGOHYDRAMNIOS
Increase amniotic fluid Decrease amniotic fluid
volume volume
a. Decrease fetal swallowing a. Increase fetal swallowing
b. Neural tube defects (Ex. b. Membrane leakage
Spina bifida and c. Urinary tract deformities
anecephaly

Test for fetal lung Should be placed in ice for

maturity delivery to the laboratory and
kept refrigerated.
Filtration prevents loss of lung
surfactants such as phospholipids.

Test for cell Maintained at room temp or body temp

culture and
cytogenetic studies
Test for HDN Specimen must be protected from light
INDICATIONS OF PERFORMING AMNIOCENTESIS
14th In general, amniocentesis is a safe procedure, particularl
when
performed after the __ week of gestation
th th
15 to 18 -for fetal genetic assesstment or genetic abnormality
week -for women with three or more miscarriage
amniocentesis -for neural tube defect
-for women who is carrier of metabolic disorder
-for earlier pregnancy or child with birth defect
-for abnormal triple marker screening test
20 to 42 -for HDN, Fetal distress, fetal lung maturity, and
weeks infection
amniocentesis

AMNIOTIC FLUID VS MATERNAL URINE

ANALYTE AMNIOTIC FLUID MATERNAL

URINE
Less Reliable
Protein + -
Glucose + -
More Reliable
Urea <30 mg/dl >300 mg/dl
Creatinine <3.5 mg/dl >10 mg/dl

FERN TEST
 A vaginal fluid is spread on glass and allowed to
completely air dry at RT
 positive result “ fern-like crystals” indicates the fluid
is amniotic fluid

CREATININE LEVEL
 Creatinine level >2 mg/dl = provides a mean of
determining fetal ag.

AMNIOTIC FLUID COLOR

Colorless Normal
Blood Traumatic tap, abdominal trauma, intra –
streaked amniotic hemorrhage
Yellow HDN Bilirubin
Meconium
Fetal death

Meconium = formed in the intestine from fetal

intestinal secretions and swallowed amniotic fluid.
Biliverdin is responsible for its dark green color.
It may be present in the amniotic fluid as a result
of fetal distress.

TEST FOR HDN

 A.K.A = O.D 450
 Absorbance of amniotic fluid:
Normal = increase at 365nm, decrease at 550nm
HDN = Increase at 450nm
 Results are plotted on Liley graph:
Zone 1 = non affected or mildly affected fetus
Zone 2 = moderately affected fetus (requires close
monitoring)
 Zone 3 = severely affected fetus (requires intervention
such as intrauterine or exchange transfusion)
INTERFERENCES IN O.D 45
Specimens contaminated with meconium will cause falsely low O.D450
values and are not acceptable for spectrophotometric analysis.
Specimens that are contaminated with blood are generally
unacceptable because maximum absorbance of oxyhemoglobin occurs at
410 nm and can interfere with the bilirubin absorption peak. This
interference can be removed by extraction with chloroform if
necessary
TEST Information
Lecithin /  Reference method
Sphingomyelin  Lecithin for alveolar stability
ratio  Sphingomyelin serves as a control
 Performed using Thin Layer
chromatography (TLC)
 Ratio of > 2.0 = mature lungs
 Cannot be done on specimen contaminated
with blood(False decrease value) or
meconium(false increase value)
Amniostat –FLM  Immunologic test for Phosphatidyl
glycerol
 It based on agglutination of PG using
polyclonal anti-PG antibodies;
qualitative results
 Results are reported as negative
(immature), or as low positive
(mature) or high positive
(mature), based on the size of
agglutinates and the degree of
background clearance
 Not affected by blood or meconium
 Production of PG is delayed among
diabetic patient
 Considered as reference method
Foam stability  Amniotic fluid + Ethanol --- shake for
/ Shake test 15 seconds --- stand for 15 minutes
 (+) = foam/bubbles = mature lungs
Microviscosity  The presence of phospholipids decrease
testing microviscosity
 Measured by fluorescence polarization
 No longer used
Lamellar body  Lamellar bodies also known as type II
count pneumocytes
 Responsible for production of alveolar
surfactants
 >32,000 / ml lamellar body count =
adequate FLM
 Uses hematology analyzer for counting
 Same size with platelets when counted
OD 650nm  Increase lamellar bodies = Increase O.D
absorbance
An O.D of ≥0.150 is equivalent to L/S ratio
of ≥2.0
Test Reference Significance
values
Bilirubin scan OD 450 Hemolyticdisease
>.025 of thenewborn
Alphafetoprotein <2.0 Neural tube
MoM(Multipl disorders
 es of
median)
Lecithinsphingomye ≥2.0 Fetal lung
lin ratio maturity
Amniostatfetal Positive Fetal lung
lung maturity maturity/phosphati
dyl glycerol
Foam Stability ≥47 Fetal lung
Index maturity
Optical density ≥0.150 Fetal lung
650 nm maturity
Lamellar body ≥32,000/mL Fetal lung
count maturity

CEREBROSPINAL FLUID
 3 RD
Major body fluid

Function:
1. Supply nutrients to the CNS
2. Remove metabolic waste
3. Produce a mechanical barrier to cushion the brain and
spinal cord against trauma

Meninges
 Lines the brain and spinal cord
 3 layers:
1. Dura mater = outer layer , lines the skull and
vertebral canal
2. Arachnoid mater = spider like , filamentous inner
membrane
3. Pia mater = innermost layer, lines the surface of the
brain and spinal cord
Note: Subarachnoid space = where CSF flows

CHOROID PLEXUS
 Specific part of the brain that produces CSF (Selective
filtration )
 20 ml/hr = rate of CSF production

ARACHNOID VILLI /GRANULATIONS

 Reabsorbs CSF

BLOOD BRAIN BARRIER

 A tightly fitting junctures structure of endothelial
cells in choroid plexus
 Protects the brain from chemicals and other substances
circulating in the blood that can harm the brain tissue
 Disruption of BBB allows WBC , protein and other
chemicals to enter CSF

CSF SPECIMEN COLLECTION AND HANDLING

 Up to 20 ml of CSF can be collected
 Method of collection : lumbar puncture - between the
3rd , 4th , or 5th lumbar vertebrae
 CSF total volume
 Adult : 90-150 ml
  Neonates : 10-60ml

CSF TUBES DISTRIBUTION

TUBE # LAB SECTION STORAGE TEMPERATURE

In case of 1 CSF Tube only = Micro – Hema – Chem/Sero

APPEARANCE CLINICAL SIGNIFICANCE

CRYSTAL CLEAR Normal

Cloudy, Turbid, Milky - Increase WBC >200 /ul

- Increase RBC >400/ ul
- Presence of microorganism
- Increase Proteins and Lipid

Xanthochromic -Pink : Slight amount of

oxyhemoglobin
-Yellow: Bilirubin
-Orange : In case of heavy hemolysis

OTHER causes:
- Increase dietary carotene
- Increase rifampin intake
- Increase melanin

Bloody / grossly >6000 RBCs / ul due to:

bloody Traumatic tap
Intracranial hemorrhage / cerebral
hemorrhage

Oily - Radiographic contrast dye

Clotted - Protein and clotting factors
- Meningitis, Froin syndrome
Pellicle - Tubercular meningitis

Traumatic Tap Intracranial

Hemorrhage
Distribution of
blood in 3 tubes
Clot formation
Supernatant
Erythrophages
D-dimer test
CSF CELL COUNT
 Performed immediately because WBCS and RBCs will begin
to lyse within 1 hour.
 40% of WBC disintegrates within 2 hours. If the
specimen is refrigerated, WBC lysis can be reduced
significantly to approximately 15%, but not completely
prevented. Similarly, RBCs do not demonstrate
significant lysis at 4° C; therefore the CSF collection
tube for cell counts should be refrigerated if the
count must be delayed for any reason

A. WBC count
 Routinely performed on CSF
 WBC diluting fluid – 3% acetic acid with methylene
blue
 Normal values:
Adults = 0-5 WBC/ ul
Neonates = 0-30 WBC/ul
CSF DILUTION
Appearance Dilution
Clear undiluted
Slightly hazy 1:10
Hazy 1:20
Slightly Cloudy 1:100
Cloudy/ Slightly 1:200
bloody
Bloody/Turbid 1:10,000

B. RED BLOOD CELL COUNT

-not routinely done
-done only in case of traumatic tap to correct Total
Protein and WBC count
 Subtract 1 WBC for every 700 RBC’s seen
 Subtract 8 mg/dl Total protein for every 10,000
RBCs/ul seen
 Subtract 1 mg /dl of TP for every 1,200 RBCs/ul seen

C. WBC DIFFERENTIAL COUNT

-performed on a stained smear
-specimen must be concentrated prior to preparation of
smear
-can be achieved through cytocentrifugation,
centrifugation, filtration, or sedimentation

NORMAL CELLS IN CSF

a. Lymphocyte
 b. Monocyte
-ADULT: 70% lymphocytes, 30% monocytes
-Neonate: up to 80% monocytes

PLEOCYTOSIS
 Abnormal condition
 Term for the increase in number of normal cells in CSF

CELL TYPES AND CAUSES OF CSF PLEOCYTOSIS

Predominant Cell Causes
type
lymphocytes Normal, Viral meningitis, tubercular
meningitis, fungal meningitis, Multiple
sclerosis, Guillain-Barré syndrome,
Lymphoma
Monocytes Normal, Viral meningitis , tubercular
meningitis, fungal meningitis, Multiple
sclerosis, tumors
Neutrophils Bacterial meningitis,Cerebral hemorrhage,
Amebic encephalomyelitis,Cerebral abscess,
Repeated lumbar puncture
Macrophages Tuburcular meningitis, fungal meningitis,
In response to RBCs and Lipids in spinal
fluid, radiographic Contrast media, Brain
irradiation
Blasts Acute leukemia, lymphoma
Malignant cells Metastatic carcinomas
Ependymal, Diagnostic procedures
choroidal, and
spindle shaped
cells
Plasma cells Multiple sclerosis, Lymphocyte reactions

CHEMICAL ANALYSIS OF CSF

CSF PROTEIN
Normal Adults =
values Infants =
Increased in Damage to the BBB


Production of Antibodies within the CSF

Decreased in CSF leakage
Major CSF
Protein
2nd most Pre albumin
prevalent
Alpha Haptoglobin, ceruloplasmin
globulin
Beta- ______________________________
globulins  Carbohydrate deficient transferrin
 Found on CSF not in serum
Gamma IgG and some IgA
globulins
Not found in IgM, Fibrinogen, Lipids
normal CSF

CSF PROTEIN DETERMINATION

Turbidemetric 1. ______________________
 Preferred method , precipitates
both albumin and globulin
2. ______________________

 Precipitates albumin only, to

precipitate globulins, add sodium
sulfate
Dye binding
CSF /Serum Assess the integrity of the blood brain
Albumin Index barrier

Normal value = <9

Abnormal value = >9
 Correlates the degree of damage
 Index 100 = complete damage to BBB
IgG Index Assess condition with IgG production within
the CNS (Multiple Sclerosis)

Normal value = <0.70

Abormal value = >0.70
 Indicative of IgG production within the
CNS

CSF ELECTROPHORESIS
 Done in conjuction with serum electrophoresis
 For the detection of oligoclonal bands
 The presence of 2 or more oligoclonal bands in CSF but
not in serum is valuable for the diagnosis of:
1.

2.

3.

4.

MYELIN BASIC PROTEIN

 Protein component of the lipid protein complex that
insulate the nerve fibers
 Presence of MBP in CSF indicates destruction of myelin
sheath
 Used to monitor course of ________

CSF GLUCOSE
Determination Done in conjunction with blood glucose
Specimen for blood glucose should be
drawn 2 hours prior to spinal tap
Normal values
Increased Due to increase plasma glucose (not
significant)
Decreased in
Normal in

CSF LACTATE
 Normal value : 10-22 mg/dl (1.1 to 2.4 mmol/L)
 Increase in :
  Normal in:

GLUTAMINE
 Product of ammonia and alpha ketoglutarate
 Indirect test for the presence of excess ammonia in CSF
 Normal value :
 Increase in: Reye’s syndrome , Disturbance of
consciousness(coma)

ENYZMES
A. Lactate dehydrogenase
LD1 and 2 = Brain trissue
LD2 and 3 = Lymphocytes
LD4 and 5= Neutrophils
Serum LDH
 Normal = LD2>1>3>4>5
 Flipped pattern = LD1>2

CSF LDH
 Normal
 Neurological abnormalities
 Bacterial meningitis

Bacterial Viral Tubercular Fungal

Predomin Neutrophil Lymphocyte Lymphocyte Lymphocyt
ant WBC and Monocyte e and
Monocyte
Protein Increase Increase Increase Increase
Glucose decrease normal decrease decrease
Lactate Increase normal Increase Increase
Other + gram Caused by Agent: Agent:
informat stain smallest RNA mycobacterium Cryptococ
ion + culture virus such tuberculosis cus
+limulus as Picorna neoforman
lysate virus +AFB stain s
test , +pellicle/web
Ex. coxsackievir like clot + gram
-Group B us, formation stain=
streptococ echovirus after 12-24 classic
cus and poli hr starburst
- voirus refrigeration pattern
H.Influenz + India
ae -enterovirus ink
- +immunolo
N.Meningit gic test
idis for
- C.neoform
S.pneumoni ans
ae

LIMULUS LYSATE TEST

 Detects gram negative bacterial endotoxin
 Reagent: blood of horseshoe crab
 Positive : clumping or clot formation
SEROLOGIC TESTING
 Latex agglutination test and ELISA- for detection of
bacterial antigens
 VDRL= recommended by CDC for detection of
______________________
 SEMINAL FLUID
Reasons for Semen analysis
1. Fertility testing
2. Post vasectomy semen analysis
3. Forensic analysis

Composition of semen
5% Spermatozoa 1. Seminiferous tubules
 _____________________
 _____________________
2. Epididymis
 _____________________
60-70 % Produced by the Seminal vesicles
seminal fluid  Provides nutrients for sperm and fluid
 Rich in fructose for sperm motility
20-30 % Acidic fluid
prostate fluid Contain, ACP, Zinc, Citric acid, and other
enzymes
For coagulation and liquefaction
5% Thick alkaline mucus
Bulbourethral Neutralizes acidity from the prostatic
fluid secretion and vagina

SPECIMEN COLLECTION
1. Abstinence of _________________________
2. In fertility testing WHO recommends two or three samples
be collected not less than 7 days or more than 3 weeks
apart, with two abnormal samples considered significant.
3. Collect the entire ejaculate
Methods: masturbation, coitus interruptus, condom method
– use a non-spermicidal, non-lubricant containing rubber
or silastic condom
4. Specimen should be delivered to the laboratory within
____ of collection at room temp
5. Take note of the time of specimen collection, specimen
receipt and liquefaction
6. Analysis should be done after liquefaction ( usually
_______ minutes )
7. If after 2 hours if the specimen has not liquefied, add
Dulbecco’s phosphate buffere saline, alpha chymotrypsin,
or bromelain to induce liquefaction
8. Specimen awaiting analysis should be kept at ____
9. Semen specimen are potential reservoir of HIV and
Hepatitis
10. Jelly-like granules (gelatinous bodies) may be
present in liquefied semen specimens and have no clinical
significance.

First portion of Decrease sperm count , Increase PH,

ejaculate missing Specimen will not liquify
Last portion of Increase sperm count, Decrease PH ,
ejaculate missing Specimen will not clot, Decrease semen
volume

MACROSCOPIC EXAMINATION
Appearance Gray- white , translucent , with musty or
bleach odor
=______________________
Increased white turbidity
=______________________
Red or Brown coloration
=______________________
Yellow coloration
=______________________

Volume Normal =
Increased =
Decreased =

Viscosity Normal = pour in droplets

Reporting :
0 = ____________
4 = ____________

Droplets that form threads longer than 2 cm

are considered highly viscous and are
recorded as abnormal
Ph Normal =
Increase =
Decrease =

SPERM CONCENTRATION
 Normal value = 20-160 million/ ml
 Methods :
1. Improved neubauer counting chamber
Dilution: 1:20 using a mechanical (positive
displacement) pipette
Diluents: Formalin, Sodium bicarbonate, saline,
distilled water

2. Makler Counting chamber

For undiluted specimen
Uses heat to immobilize sperm cells

Formula for sperm concentration

SPERM COUNT
 Normal value = > 40 million sperm / ejaculate
 Formula : Sperm count x Specimen volume
SPERM MOTILITY
 Specimen should be liquefied first
 Normal value = >50% motile within 1 hour
 Quality = ≥2.0
GRADE WHO CRITERIA GRADE ALTERNATIVE GRADING
4.0 Rapid, straight Progressive Sperm moving
a line motility motility(PM) linearly or in a
large circle
3.0 Slower speed, Non Sperm moving with
b some lateral progressive an absence of
movement motility(NP)
progression
2.0 Slow forward Immotility No movement
b progression, (IM)
 noticeable
lateral movement
1.0 No forward
c progression
0 No movement
d

 The percentage of sperm showing actual forward

movement can then be estimated after evaluating
approximately 20 high-power fields. An alternate
procedure is to examine 200 sperm per slide

CASA (Computer Assisted Semen Analysis)

 Provides objective determination of sperm velocity,
trajectory, concentration and morphology

SPERM MORPHOLOGY
 At least 200 sperm should be evaluated and the
percentage of abnormal sperm reported.
 Normal values:
 Routine criteria =
 Kruger’s strict criteria =
-measures the head, neck and tail using a micrometer

 Stains for sperm Acrosomal cap morphology:

 Wright’s stain -part of the
 Giemsa stain sperm that
 Papanicolau’s stain contains
 Head = Length 5um , Width enzyme for 3um
 Tail = 45 um long ovum
 Midpiece: 7um penetration
-Size:

SPERM VIABILITY
 Modified bloom’s test
 Reagent = ___________________
 Count the number of dead cell in a 100 sperm using
brightfield or phase contrast microscope
 Living sperm = unstained, bluish white (at least
50%)
 Dead sperm = red with a purple background

SEMINAL FLUID FRUCTOSE

 Tested within 2 hours or frozen to prevent fructolysis
 Screening test:
 Resorcinol test = (+) orange red color

ANTISPERM ANTIBODIES
 Detected in semen, cervical mucosa, or serum
1. Mixed agglutination reaction
 Detects the presence of IgG antibodies
 Semen sample + AHG reagent + latex particle or
treated RBCs coated with IgG
 Normal = <10% motile sperm attached to the particles

2. Immunobead test
 Detects the presence of IgG, IgM, IgA
  Demonstrate what area of the sperm the
autoantibodies are affecting
 Normal = beads on less than 50 % of sperm

CHEMICAL TESTING
Analyte Normal value Decreased Values
Indicate
Fructose ≥13 umol/ ejaculate Decrease seminal
fluid
Neutral alpha ≥20 mU/ ejaculate Epididymis problem
glucosidase
Zinc ≥2.4 umol / ejaculate Decrease/lack
prostatic fluid
Citric acid ≥52 umol/ ejaculate Decrease/lack
prostatic fluid
Acid ≥200 units/ ejaculate Decrease/lack
phosphatase prostatic fluid

MICROBIAL TESTING
 Round cells
 WBCs and spermatids
 Normal value =
 > 1 million WBC/ ml =
 >1 million spermatids/ml =
 Test for chlamydia trachomatis, Mycoplasma hominis and
Ureaplasma urealyticum
FORMULA FOR ROUND CELLS

C = N x S
100
N= Number of spermatids or WBC
S= Sperm concentration

MEDICO LEGAL TESTING

 Test for detection of semen:
1. Microscopic exam
2. Fluorescence under UV light
3. Acid phosphatase determination
4. Glycoprotein p30 = more specific method
5. Florence test
 Test for _____
 Reagent:
 (+)
6. Barbiero’s test (very specific)
 Test for _____
 Reagent:
 (+)
7. ABO blood grouping
8. DNA analysis

POST VASECTOMY SEMEN ANALYSIS

 Vasectomy – cutting of vas deferens so that the ejaculate
will not contain any sperm cell
 The only concern is the presence or absence of sperm
 Done 2 months after vasectomy and continued until 2
consecutive monthly specimen show no sperm

VARICOCELE
 Hardening of veins that drains the testes
 Most common cause of male infertility
  Sperm head = tapered head
ASSESTMENT OF SPERM CELLS
Sperm morphology – make a smear in slide and stain it with
papanicolau’s, Wright’s or Giemsa
Sperm Viability- stain with eosin nigrossin
Sperm Count – dilute with chilled tap water or formalin
bicarbonate solution; charge in a neubauer counting chamber
Sperm motility – place a drop of semen in a slide and cover
it with cover slip

SPERM FUNCTION TEST

Test Description
Hamster egg Sperms are incubated with species- non
penetration specific hamster eggs and penetration is
observed microscopically
Cervical mucus Observation of sperm penetration ability
penetration of partners midcycle cervical mucus
Hypo-osmotic Sperms exposed to lowsodium concentrations
swelling are evaluated for membrane integrity and
sperm viability
In vitro acrosome Evaluation of the acrosome to produce
reaction enzymes essential for ovum penetration

Abnormal result Possible Test

abnormality
Decreased motility Vitality Eosin-nigrosin
with normal count stain
Decreased count Lack of seminal Fructose level
vesicle support
medium
Decreased motility Male antisperm -MAR and Immunobead
with clumping antibodies test
-Sperm
agglutination with
male serum
Normal analysis Female antisperm Sperm agglutination
with continued antibodies with female serum
infertility

FECALYSIS

STOOL
 Contains bacteria, cellulose, and other undigested
foodstuffs, gastrointestinal secretions, bile pigments,
cells from intestinal walls, electrolytes and water.
 No BLOOD!
 Human passed stool around 100-200g per day
 Intestinal gas (flatus) and Odor = due to metabolism of
bacterial GI normal flora
 Small intestine: primary site for final breakdown and
reabsorption of fats, protein and carbohydrates
 Large intestine: absorbs water (maximum of 3L of Water)

Color/Appearance Clinical Significance

BROWN NORMAL DUE TO STERCOBILIN
BLACK UPPER GI BLEEDING
IRON THERAPY , CHARCOAL
INGESTION
PALE YELLOW, WHITE GRAY, BILE DUCT OBSTRUCTION
ALCHOLIC STOOL, chalky BARIUM SULFATE
GREEN BILIVERDIN, GREEN VEGETABLES,
ORAL ANTIBIOTICS
RED LOWER GI BLEEDING, RIFAMPIN,
FOOD COLORING
BULKY / FROTHY BILE DUCT OBSTRUCTION,
STEATORRHEA, PANCREATIC
INSUFFICIENCY
RIBBON LIKE INTESTINAL CONSTRICTION
RICE WATERY CHOLERA
PEA SOUP TYPHOID
SYCBALOUS / GOAT DROPPING CONSTIPATION
BUTTER-LIKE CYSTIC FIBROSIS
MUCOID DYSENTERY, MALIGNANCY

BRISTOL STOOL CLASIFICATION

1. Type 1 = separate hard lumps, like nuts
2. Type 2 = sausage shaped but lumpy
3. Type 3= like a sausage but with cracks on its surface
4. Type 4 = like a sausage or snake, smooth and soft
5. Type 5 = soft blobs with clear cut edges
6. Type 6 = fluffy pieces with ragged edges, a mushy stool
7. Type 7 = watery, no solid pieces, entirely liquid

LABORATORY TEST
I.FATS
________________ presence of increase fats in stool ( >6g/day)
*Fecal characteristic: Greasy; foul odor; spongy consistency
*Fecal volume: Increased
*Causes:
1. Pancreatic insufficiency
2. Malabsorption
3. Maldigestion
4. Absence of bile

malabsorption Inadequate intestinal absorption of

processed foodstuffs despite normal
digestive ability
maldigestion An inability to convert foodstuffs in the
gastrointestinal tract into readily
absorbable substances

TEST FOR FECAL FAT

1. QUALITATIVE FECAL FAT TEST

a. Neutral fat stain
Stain for Triglycerides
Procedure: emulsified stool + 95% ETOH + Sudan III
Steatorrhea = ≥60ORANGE DROPLETS/HPF

b. Split fat stain

Stain for total fat content (including Fatty acids,
soaps/fatty acid salts,and cholesterol)
Procedure : stool + 36% acetic acid + Sudan III + Heat
Steatorrhea = 100 droplets that are 6-75 um in size

Neutral Fat stain Split Fat stain Interpretation

Normal increased malabsorption
 Increased normal maldigestion

2. QUANTITATIVE FECAL FAT TEST

Van De Kamer Titration test
- Gold standard test for fecal fat
- Requires a 3 day stool sample ( placed on a Paint Cans
and must be homogenized prior to analysis)
- Titrated with NaOH
3. D-XYLOSE TEST
- A test that is useful to differentiate malabsorption and
maldigestion
- D-Xylose is a pentose sugar that does not need to be
digested but does need to be absorbed to be present in
the urine.
- The xylose absorption test involves the patient’s
ingestion of a dose of xylose, followed by the
collection of a 2-hour blood sample and a 5-hour
urine specimen.
- If D-xylose result is low/abnormal, the result indicates
a malabsorption condition

II.FECAL LEUKOCYTES
 Presence of ____ neutrophils/hpf = Invasive condition

TEST
1. Wet preparation = stool + Methylene blue
2. Lactoferrin latex agglutination test = (+) in invasive
bacterial pathogen
3. Dried preparation = stool + Wright’s/Gram stain

III.FECAL OCCULT BLOOD TEST

Occult means ___
Screening test for __________
Significant value = >2.5ml blood / 150g stool
Sample must be obtained from the center portion of the
stool to avoide false positive from external
contamination
 CHROMOGEN USED: Benzidine, Guaiac , O-toluidine

REACTION Based on pseudoperoxidase activity of Hemoglobin

Water + Guaiac -----------------------------------

oxidized guaiac(blue) + Water

FALSE POSITIVE FALSE NEGATIVE

 Red or rare cooked meats  Vitamin C
and fish
 Vegetables, such as
turnips, broccoli,
cauliflower, horseradish,
 Fruits, such as
cantaloupe, bananas,
pears, plums, melon
 Drugs such as aspirin and
NSAIDs

III. MUSCLE FIBERS

 ___________________ = Increase excretion of muscle fibers
in feces
  Digested fiber:
 Presence of more than 10 undigested muscle fibers are
associated with biliary obstruction, cystic fibrosis, and
gastrocolic fistulas
TEST
 Patient will undergo in a meat diet
 Procedure: Emulsified stool + 10% eosin in alcohol --
coverslip and stand for 3 mins then observed under HPF
for 5 minutes
 Count the number of undigested fibers
Digested fibers Partially digested Undigested fibers
fibers have no fibers exhibit fibers have
visible striations in visible
striations. only one striations
direction running both
vertically and
horizontally

IV. APT TEST/ ALKALI DENATURATION TEST/ DOWNEY TEST

 Differentiate fetal blood from maternal blood
 Discovered by Leonard Apt
 Specimen: infant stool, vomitus, emesis , or gastric
aspirate
 Reagent :1% Sodium Hydroxide
 Pink supernatant:
 Yellow brown supernatant:

V.FECAL ENZYMES

1. X-ray film test

- Detects trypsin enzyme (absent of trypsin is associated
with cystic fibrosis)

2. Chymotrypsin
- Stable up to 10 days at room temp
- Measured by spectrophotometry

3. Elastase -1
- Sensitive and specific test for exocrine pancreatic
insufficiency
- Test: ELISA

VI.FECAL CARBOHYDRATES
 Significant for assessing lactose intolerance
 Normal stool Ph: 7-8 , CHO Disorders = ph <5.5
 Clinitest : a test for reducing sugar , >0.5 g/dl
indicates carbohydrate intolerance

DIARRHEA
 Stool that weight more than 200g/day with increase liquid
and frequency of more than 3x a day
 Classified according to severity, mechanism, duration,
and stool characteristic

SECRETORY DIARRHEA OSMOTIC DIARRHEA ALTERED MOTILITY

Stool- with Stool- with osmol May be due to
osmolality gap of gap of hypermotility or
<50mosm/kg >50mosm/kg(High) slow motility

1. Due to 1. Maldigestion 1. Gastric

viruses, 2. Malabsorption surgery
protozoa or 3. Lactose 2. Gastric
bacteria intolerance bypass
2. Colitis 4. Ameobiasis 3. Post
3. Collagen 5. Antibiotics vagotomoy
vascular 4. Duodenal
disease ulcer
4. Zollinger 5. DM
Ellison 6. Zollinger
Ellison

SPUTUM AND BRONCHOALVEOLAR LAVAGE

SPUTUM
 From upper and lower respiratory tract
 Tracheobronchial secretions ( mixture of plasma,
electrolytes, mucin and water) added with cellular
exfoliations, nasal and salivary gland secretions and
normal oral flora.
SPUTUM COLLECTION
 First morning- Most preferred (routine)
 24 hour – for volume measurement
 Throat swab – for pediatric patients
  Sputum induction- for non-cooperative patients
 Tracheal aspiration- for debilitated patient

MACROSCOPIC EXAMINATION
Volume
Increase in : Broncheictasis, lung abscess,
edema, gangrene, tuberculosis, pulmonary
hemorrhage

Decrease in: Bronchial asthma, acute

bronchitis, early pneumonia

Odor
Odorless Normal
Foul or Lung gangrene, Advance
Putrid necrotizing tumors
Sweetish Broncheictasis, tuberculosis
Cheesy Necrosis, tumors, emphysema
Fecal Liver abscess, Enteric gram
negative bacterial infection

Color
Colorless or Made up of mucus only
translucent
White or yellow Pus is present
Gray Pus and epithelial cells
are present
Bright green and Presence of bile,
greenish Pseudomonas aeruginosa
infection
Red or bright Fresh blood, hemorrhage,
red TB , bronchiectasis
Anchovy sauce or Old blood, pneumonia,
rusty brown gangrene
Prune juice Pneumonia, Chronic lung
cancer
Olive Cancer
green/grass
green
Black Inhalation of dust or
dirt, carbon, charcoal,
anthracosis, heavy
smokers
Consistency A. Mucoid = asthma, and
bronchitis
B. Serours or frothy = lung edema
C. Mucopurulent = broncheictasis,
TB with cavities

Structures
Dittrich’ s  Yellowish or gray
plugs caseous matter, the size
of the pinhead or navy
bean
 Foul odor when crushed
 Occur in bronchial
asthma, chronic
bronchitis, healthy
persons and in TB
Lung stones/  Small,white or gray
Pneumoliths fragments of calcified
 / TB tissue or calcified
Broncholits foreign matter
Bronchial  Branching tree like
cast casts of the bronchi
Curschmann’s  Whitish or yellow wavy
spirals coiled threads
 Associated with
bronchial asthma
Layer Top layer
formation : frothy mucus
Second
layer :
opaque, water material
Bottom
layer : pus,
bacteria,and tissues

MICROSCOPIC EXAMINATION
Elastic fibers  Slender fibrils with double contour and
curled ends
 Found in abscess , gangrene of the
lung, and TB
Charcot-Leyden  Colorless, hexagonal, double pyramid,
crystals pointed at both ends, and needle like
 Formed as a result of eosinophil
degeneration
 Most significant
 Associated with bronchial asthma
Pigmented cells  Heart failure cells: hemosiderin laden
macrophages
 Carbon laden cells: angular black
granules
Curshmann’s  Coiled mucus strands
spirals  Can be found microscopically and
macroscopically.
 Associated with bronchial asthma
Creola bodies  Cluster of columnar cells that is
associated with Bronchial asthma
Myelin globules  Colorless, round, oval or pea shaped of
various sizes
 Little or no significance and mistaken
for Blastomyces
Yeast  During antibiotic treatment, they
maybe-seen in large numbers
 Examples are Candida albicans,
Cryptococcus neoformans, and Systemic
fungi
Parasites  Ascaris, Hookwork, Threadworm,
E.histolytica, Paragonimus westermani,
Toxocara canis, Entamoeba gingivalis,
Trichomonas tenax, Echinococcus
granulosus
Others that are  Neoplastic cells, Bacteria, Leukocyte
stained

BRONCHOALVEOLAR LAVAGE
 Provides a method of obtaining cellular and
microbiological information from the lower respiratory
tract
  Useful in evaluating immunocompromised patients,
interstitial lung disease and airway diseases
 Important diagnostic test for Pnuemocystis carinii in
immunocompromised patients.

CELLS SEEN IN BRONCHOALVEOLAR Elements and Viral

LAVAGE inclusions seen in
Macrophage 56-80% respiratory specimens:
Lymphocyte 1-15%  Toxoplasma gondii
Neutrophils <3%  Legionella pneumophila
Eosinophil <1 – 2%  Histoplasma capsulatum
Ciliated columnar 4-17%  Mycoplasma pneumonia
bronchial
epithelial cells  Influenza A, B, and
Respiratory syncytial
virus

SWEAT
GASTRIC FLUID
 2. Chief cells- produces pepsinogen that will be converted
to pepsin whenever HCL is present

3. Specialized G cells – produces Gastrin that stimulates

parietal cell to produce HCL.

SPECIMEN COLLECTION

 Gastric juice is obtained by insertion of a gastric tube

into the stomach
 Gastric tubes:
a. Levin tube = passed through the _______
b. Rehfuss = passed through the _______
c. Disposable plastic tubes are usually employed
 Specimen: Fasting specimen, few ml to 50ml average of 30
ml

 Normal appearance of gastric specimen: Pale gray with

mucus and no food particles

 Types of specimen:
Basal acid output  1 hour collection (four 15 minute
(BAO) specimens)
 Requires 12 hour fasting
 No gastric stimulant needed
Maximum acid  1 hour collection ( four 15 minute
output (MAO) specimens)
 With gastric stimulant

GASTRIC STIMULANTS

Test meals Ewald’s meal: bread and water or weak tea

Boas meal: oatmeal, meal for detection of
lactic acid
Riegel’s meal: mashed potates, broiled
beefsteak, bouillon
Chemicals  Pentagastrin – most preferred , it
resembles true gastrin
 Histamine
 Histalog
 Insulin - assess successful vagotomy
procedure
Sham Feeding  Fictitious feeding
 Sandwich is chew and then spit out

DIAGNEXT BLUE TEST/ TUBELESS TEST

 Specimen: Urine

 Principle: an ion exchange resin (Amberlite cation)

resin, coupled with a dye, azure blue, is given by mouth
after caffeine stimulation. In the presence of free HCL,
the azure blue is released from combination with the
resin in exchange for hydrogen ions. The azure blue is
rapidly adsorbed from the intestines and travels in the
blood to the kidneys and is excreted in urine. The
appearance of azure blue is then an indication that free
HCL is present in the stomach.
  Stimulant used: Test meals (Henry’s) , Histamine (other
books)
NOTE!
A. Zollinger Ellison syndrome
- Elevated gastrin levels
- Elevated BAO/MAO results (highest elevation)
B. Pernicious anemia
- Shows a zero BAO/MAO results
- Achlorydia ( absence of free HCL)

Euchlorhydria Normal free HCL ----

Hyperchlorhydria Increased free HCL Peptic ulcer

Hypochlorhydria Gastric fluid pH Carcinoma of

>3.5 but falls stomach
after gastric
stimulation
Decrease free HCL

Achlorhydria Gastric fluid pH Pernicious anemia

>3.5 and does not
fall even after
gastric
stimulation
Absence of free
HCL

Dimethylaminoazobenzol Reagent: alcohol solution = (+)

cherry red
Gunzberg’s Reagent: phloroglucin, Vanillin,
Alcohol = (+) Purple red color
Boas Reagent: resorcinol, cane sugar,
alchol = (+) Purple red color
Free HCL Topfer’s method
 Titrate with NaOH
 pH indicator: dimethyl
aminoazobenzol
 Endpoint : Canary yellow
 Normal value : 25-50 degrees or 0.1
or 0.2 HCL
Total acidity  Titrate with NaOH
 Ph indicator: phenolphthalein
 Endpoint: Faint pink
 Normal value: 50 -75 degrees
Combined HCL  Titrate with NaOH
(Bound to  pH indicator: sodium alizarin
proteins)  Endpoint: Violet
 Normal value: 10 – 15 degrees
LACTIC ACID TEST

Test Reagents Endpoint

Modified Uffelman’s FeCl3 + phenol Yellow
Strauss FeCl3 + ether Yellow
Kelling’s FeCl3 Yellow

QUALITY ASSESTMENT AND MANAGEMENT IN

URINALYSIS LABORATORY
QC of laboratory Daily
equipments  Check temperatures of
refrigerators and water baths

Weekly
 Disinfection of centrifuges
 Check pH and purity meter
resistance of deionized water used
for reagent preparation

Biweekly
 All diluents should be checked for
contamination

Monthly
 Speed of centrifuge should be
checked with a tachometer, and
timing should be checked with a
stop watch
 Check the bacterial count of
deionized water used for reagent
preparation

Every 3 months/ Quarterly

 Calibration of centrifuges
PDCA Plan – Do- Check- Act
PDSA Plan- Do- Study - Act
Quality assessment refers to the overall process of
(QA) guaranteeing quality patient care and
is regulated throughout the total
testing system
Quality system Refers to all of the laboratory’s
policies, processes, procedures, and
resources needed to achieve quality
testing.
Trend A gradual change in the mean
Shift An abrupt change in the mean
Total Quality is a systematic problem-solving
Management (TQM) approach using visual tools to identify
the steps in the process for meeting
customer satisfaction of quality care
in a timely manner at
 reduced costs
Turnaround time the time from receipt of the specimen
in the laboratory to reporting of
results to a patient care area or into
a data information system

QUALITY ASSURANCE ERRORS IN CM (Strasinger, 5th edition)

PRE ANALYTICAL ANALYTICAL ERROR POST ANALYTICAL

ERROR ERROR
 Patient  Sample  Patient
misidentificat misidentificat misidentificat
ion ion ion
 Wrong test  Erroneous  Poor
ordered instrument handwriting
 Incorrect calibration  Transcription
urine specimen  Reagent error
type collected deterioration  Poor quality
 Insufficient  Poor testing of instrument
urine volume technique printer
 Delayed  Instrument  Failure to
transport of malfunction send report
urine to the  Interfering  Failure to
laboratory substances call critical
 Incorrect present values
storage or  Misinterpretat  Inability to
preservation ion of quality identify
of urine control data interfering
substances

ADDENDUM
Home 1. Principle of current PT test kit :
based IMMUNOLOGIC (Enzyme
pregnancy immunoassay/immunochromatographic assay)
test kit 2. Detects the Beta hCG subunit of
glycoprotein/amino acid
3. It is an indirect test for the detection of
fetus
4. Sensitivity : a positive result if a minimum
of approximately 25mIU/ml hCG is present
(Stevens)
5. Presence of two color bands suggest a
positive result
6. Presence of one color band (control region)
suggest a negative result
7. Absence of two color bands suggest an
invalid result
8. A VERY FAINT LINE IN THE TEST AREA SUGGEST
TO REPEAT TEST AFTER 48hours
Test for 1. Wipe off the stone(s) and describe in terms
renal of size(mm), shape, color and
calculi hardness/texture
2. Powderized stone and dissolve in a small
amount of concentrated HCL
Foaming upon contact with HCL Carbonate
Magenta color Cysteine
Blue color Phosphate
Blue precipitate Magnesium
 Pale yellow color Calcium
Orange brown color Ammonium
Yellow orange color Uric acid
Black sediment which settles oxalate
and bubbles and appear from
the bottom of the tube
Biologic
test for TEST/METHOD ANIMAL USED POSITIVE RESULT
PT Ascheim- Immature female Formation of
(Henry’s Zondek mice hemorrhagic
19th ed.) follicles and
corpora lutea
Friedman Mature virgin Hyperemic uterus
female rabbit and corpora
hemorrhagica
Hogben Female toad oogenesis
(Xenopus laevis)
south African
clawed frog-
carries eggs
throughout the
year

Galili- Male frog(Rana spermatogenesis

manini pipiens or Rana
clamitans,
leopard or grass
frag) male
toad(Bufo bufo or
Bufo americanus)
Frank Immature female Ovarian
Berman rats hyperemia
Kupperman Female rat Ovarian
hyperemia
Chemical
test in Detected Name of test
urine Calcium Sulkowitch
Chloride Fantus, Schales schales
Bile pigments Smith, harrison’s spot,
ictotest, gmelin
Urobilin schlesinger
urobilinogen Wallace and diamond
Ketones Rothera’s, lange, acetest,
gerhardt’s
fructose Resorcinol, Seliwanoff, and
Borchardt’s
sugars 1. Biacol orcinol
2. Benedicts
3. Rubner’s (glucose = red
with red yellow, Lactose
= red with red ppt)
4. Moore Heller
5. Nylander’s
Qualitative test Heat and acetic acid, SSA,
for protein Purdy’s, picric acid,
Potassium ferricyanide,
Biuret, Heller’s, Spiegler’s
test
Quantitative Biuret, kingsburry-clark ,
test for protein esbach’s , kwilecki’s
 AUTOMATION AND INSTRUMENTATION IN CM
 A goal of the urinalysis laboratory is to maximize
productivity and testing quality, while keeping costs and
turnaround time at a minimum.

Urine These semi-automated instruments require the user

chemistry to properly dip the reagent strip and place it
Semi- onto a platform.
automated
analyzer REFLECTANCE PHOTOMETRY
1. When light strikes a matte or unpolished
surface (e.g., a reagent strip), some light
is absorbed, and the remaining light is
scattered or reflected in all directions.
The scattered light is known as diffuse
reflectance.
2. The relationship between reflectance and
concentration is not linear

Automated  Decrease labor costs and increasing

Microscop productivity in the urinalysis laboratory
y  uncentrifuged urine is used, the time spent
analyzers in handling and preparing concentrated urine
sediment for manual microscopy is eliminated
 increased standardization of the microscopic
examination, which enhances the accuracy and
reproducibility (precision) of results

Iris iQ - uses patented technologies to

200 capture and automatically classify
digital images of urine particles.
-SOFTWARE USED= AUTO PARTICLE
RECOGNITION SOFTWARE OR Proprietary
neural network software

- APR Preclassifies urine particles in

the photographs based on size,
shape, texture , and contrast in to
1 to 12 categories

-The field of view of the microscope

is coupled to a digital video camera,
and stroboscopic illumination freezes
the particles in motion as they stream
past, which ensures blur-free imaging
the field of view of the microscope is
coupled to a digital video camera, and
stroboscopic illumination freezes the
particles in motion as they stream
past, which ensures blur-free imaging
Sysmex -Principle : cell flow cytometry +
UF- impedance
analyzer -Urine particles are identified and
s / categorized by fluorescent staining
Slideles characteristic, light scatter,
s electrical impedance, and adaptive
automate cluster analysis
d
analyzer -fluorescent stain used
s Carbocyanin Stain for RNA , cell
membrane, cytoplasm
Phenanthridine Stain for DNA, nucleus,
 chromosomes

-bacteria is detected specifically

by the side light scatter
-Results are displayed as SCATTERGRAM
OR HISTOGRAM