Books : ✓ strasinger ( 7th Ed -7

✓ Brunel

✓ Henrys
CLINICAL MICROSCOPY
SAFETY AND QUALITY ASSESSMENT

Terminologies:
JC Joint Commission
1. CDC- Centers for Disease Control and Prevention
-

2. OSHA- Occupational Safety and Health Administration CAP -

College of American Pathologists

3. CLSI-Clinical and Laboratory Standards Institute AABB -

American Association of Blood Banks

Association
4. PPE- Personal Protective Equipment AOA
-
American Osteopathic
5. UP- Universal Precautions ASHI -
American society of Histocompatibility t
Immunogenetics
6. BSI-Body Substance Isolation COLA -
Commission on
Laboratory Assessment
7. NFPA- National Fire Protection Association

TYPE OF SAFETY HAZARDS

A. SOURCE Infectious agents

B. Possible Injury bacterial, fungal, viral, prions, or parasitic infections
Biohazard Symbol ( Color : Fluorescent
Chain of Infection- A continuous link (6-link) on understanding on how microorganisms are Orange)
transmitted.
Component Example
Infectious agent Bacteria, fungi, parasites, viruses 4

(
Reservoir Animals, Humans, Fomites, Insects, Blood, Body fluids 6
IREMES
Portal of exit Nose, mouth, Mucous membranes 3
Mode of transmission Droplet, Airborne, Contact, Vector, Vehicle 5
Portal of entry Nose, mouth, mucous membranes, skin, Unsterile equipment 5
Susceptible Host Patients, Elderly, Newborns, Immuno-compromised, Healthcare workers 5

MODES OF TRANSMISSION

AIRBORNE /AEROSOL a. Centrifugation of unstoppered tubes

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. Pippeting with mouth
DIRECT INOCULATION a. Needlesticks
b. Broken glass
c. Animal bites
d. Small scratches
MUCOUS MEMBRANE infection may occur if the organism can directly enter through the mucous membranes such as
through the conjunctiva of the eye
ARHTROPODS / VECTOR Infectious source includes ticks, fleas, and mosquitos, which may harbor various microorganisms

Biologic Waste Disposal

urine
All biologic waste, EXCEPT _________, 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
_________dilution
1:10 t.la#ww of _______________________________
Sodium hypochlorite ( bleach/ 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

A. SOURCE- Needles/Syringe, lancet, broken glass wares

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 over-filled and must always be replaced when the
safe capacity mark is reached

Syringe facing northwest

I.K AYTONA 1
 /POISON
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.

15 minutes [
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 assoc with abnormal fetal development
.

Material Safety Data Sheets (MSDS)=Contains 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

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

A. SOURCE- Ungrounded or wet equipment; 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.
three
All electrical equipment must be grounded with _______________________.
pronged plug
When an accident involving electrical Shocks occur:
1. Turn off the circuit breaker
2 lightning bolt

2. Unplug the equipment

vertically positioned
3. Move the equipment using a nonconductive glass or wood object

/EXPLOSIVE
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 A Ordinary combustibles: Water, Dry chemicals, Steam
Ardinary Wood, paper, clothing/garments, plastic
Class B Flammable organic chemicals/liquids: Dry chemicals, carbon dioxide, foam, or halon
gasoline, paints, oil
Basa
Class C Electrical equipment: Dry chemicals, Carbon dioxide, or halon → preferred for
computer devices
Machines, motor switches, plugs
Euryente
Class D Combustible metals: Sand or dry powder, Metal X
Dick (Mati gas) (Hg, Mg, Na, and Li)
Dry chemicals for A, B, C
E- pal Class E Detonation or Arsenal fire Allowed to burn out and nearby materials are protected
Hawaii / Busing Class K Grease, oils, fats Liquid designed to prevent splashing and cool the fire.

I.K AYTONA 2
 Ergonomic Work ,

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

f) Best way to break Chain of Infection

HAND HYGIENE- includes both hand washing and using alcohol based antiseptic cleansers.
Hand contact is the primary method of infection transmission Hand Washing Songs:
A. Happy Birth day
Father of handwashing: Dr. Ignaz Semmelweis B. Twinkle-Twinkle little star
C. Alphabet song
Laboratory personnel must always sanitize hands
1. Before patient contact
2. After gloves are removed
3. Before leaving the work area
4. Anytime when hands have been knowingly contaminated
5. Before going to designated break areas
6. Before and after using bathroom facilities

not visibly soiled

ALCOHOL-BASED CLEANSERS- Used when hands are ______________________

HAND WASHING visibly soiled

Used when hands are _______________________________

CDC Hand Washing Procedure

1. Wet hands with warm water
2. Apply anti-microbial soap
3. Rub from a lather, 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

NSHED

stability

SUVSM

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

I.K AYTONA 3
 ADDENDUM
The last step in handwashing Turn off faucets with a clean paper towel to prevent recontamination
The most important step in handwashing Rubbing /Applying friction
Susceptible host Complete the chain of infection:
The source/causative agent transmission --_____________

RENAL FUNCTION

RENAL PHYSIOLOGY
A. The kidneys are bean shaped and are located on the posterior abdominal wall in the area known as the retroperitoneum. An
adult human kidney has a mass of approximately 150 g and measures roughly 12.5 cm in length, 6 cm in width, and 2.5 cm in
depth
B. Each kidney contains approximately _______________
I -1.5 millions functional units called nephrons
C. 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.
D. 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

1.
EXCRETORY FUNCTION
A. Glomerular filtration
B. Tubular reabsorption
C. Tubular secretion
2 Regulation of water balance in the body.
.

slow but complete

3. Regulation of acid-base balance → kidney compensation :
incomplete
4. Regulation of electrolytes lung compensation fast : but

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______
25% of the blood
pump.
Total renal blood flow: _____________________
1200mL/min
Renal plasma flow: ______________________
600 -700mL/min

FORCES INVOLVED IN GLOMERULAR FILTRATION

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. This hydrostatic blood pressure averages 55 mm Hg, approximately half of the mean arterial blood pressure,
c
pressure of 15 mm Hg that opposes filtration
An oncotic ((protein in blood and not in ultrafiltrate) pressure of 30 mm Hg caused by the higher protein concentration in
the plasma opposes glomerular filtration as well
The outcome of these three pressure differences is a net filtration pressure of 10 mm Hg, which favors the formation

An afferent arteriole at the vascular pole supplies blood individually to the glomerulus of each nephron

Order of Blood Flow In the Nephron

E
Renal Artery -7 Segmental Arteries → lobar Arteries → Inter lobar Arteries → Arcuate Arteries → Afferent Arterioles → Glomerulus → Efferent Arterioles → Peritubular capillaries
Order of Urine formation from the nephron Renal
Veins
←
Lobar
Veins
←
Inter 10bar
Veins
← Arcuate Veins (

GLOMERULAR FILTRATION
are referred
Walls

CHARACTERISTIC: f) as glomerular
barrier
filtration

The glomerulus consists of a coil of approximately eight capillary lobes referred to collectively as the capillary tuft. It
resembles as sieve
* doesn't pass through negatively
The glomerulus is located within the . charged particles
A non-selective filter for plasma substances with molecular weights of less than _____________
70,000 daltons
Normally, the fluid leaving the glomerulus has a specific gravity of 1.010
Analysis of the fluid as it leaves the glomerulus shows the filtrate to have a specific gravity of 1.010 and confirms that it is
chemically an ultrafiltrate of plasma.
Approximately 120 mL/min, or one fifth, of the renal plasma is filtered through the glomeruli forming what is
known as the ultrafiltrate, which is further processed as it travels through the nephron. The ultrafiltrate has the same
composition as blood plasma but it is normally free of protein except for about 10 mg/dL of low molecular-weight protein

I.K AYTONA 4
 secretes renin
Cellular Structure of Glomerulus: to

Plasma filtrate must pass through three cellular layers:

1. Capillary wall membrane (fenestrated )
2. Basement membrane
3.

The capillary wall of glomerulus is fenestrated

Intertwining foot processes ___________________
podocytes
____________
Shield of
Negativity - 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. juxta glomerular cells - found in the afferent arteriole, secretes the Renin enzyme
______________________
macula densa
b. ______________________ - found in the DCT, sensor of change in blood pressure

Decrease Blood Pressure = Dilation of afferent arteriole, Constriction of efferent arteriole

Increase Blood pressure = Constriction of afferent arteriole, Dilation of efferent arteriole
Triggered by
:

RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) → i. Low blood pressure

4 Low Nat in blood
System regulates the flow of blood to and within the glomerulus. The system responds to changes in blood pressure and
plasma sodium content that are monitored by the juxtaglomerular apparatus, which consists of the juxtaglomerular cells in
the afferent arteriole and the macula densa of the distal convoluted tubule
Controls the regulation of the flow of blood to and within the glomerulus.
Primary electrolyte affected when activated: Sodium

Functions:
1. Dilation of the afferent arteriole and constriction of the efferent arteriole
2. Stimulation of sodium reabsorption in the proximal convoluted 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. Trigger release of antidiuretic hormone by the hypothalamus to stimulate water reabsorption in the collecting duct

Stimulus: Decrease Blood Pressure/Low Plasma sodium

ANGIOGTENSINOGEN
cells
Renin → secreted by juxtaglomerular
ANGIOTENSIN I → inert
Angiotensin converting enzyme (ACE) = Lungs
ANGIOTENSIN II → active & potent increaser of BP

salt retention T Aldosterone water retention

y
hormone = A Retention
salt
of hormone

Sodium reabsorption at Aldosterone for Sodium

( Vasopressin)
ADH for water
DA/CE PCT
Dilate Afferent retention reabsorption
Constrict Efferent

TEST FOR GLOMERULAR FILTRATION

Clearance test =BEST INDICATOR OF OVERALL GLOMERULAR FUNCTION
1. Inulin clearance test
Gold Standard
_____________________
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
Creatinine is a waste product of muscle metabolism that is produced enzymatically by creatine phosphokinase
from creatine, which links with ATP to produce ADP and energy
3. Cystatin C small protein ( mol wt 13,359) produced at constant rate by all nucleated cells
- . .
a

4. Beta-2 Microglobulin dissociates from human leukocyte Ag at a constant rate

-

5. Radioisotopes I iothala mate 51 Cr EDTA 99 Tc DTPA

-
-

,
-

,
- -

6. Urea clearance test = earliest clearance test

Formula for the computation of GFR using the creatinine clearance test
¥×i¥o×i¥
T
C = Urine creatinine___ X volume of urine/24hours x 1.73 normal value
of urine w/in
Plasma creatinine A 24 hrs

1. By far the greatest source of error in any clearance procedure utilizing urine is the use of improperly timed urine
specimens
Brunel 2. Plasma/serum creatinine can be collected anytime within 24 hours of urine collection
Bishop 3. Specimen collection, therefore, must include both a 24-hour urine specimen and a serum creatinine value, ideally
collected at the midpoint of the 24-hour urine collection. The urine container must be kept refrigerated
throughout the duration of both the collection procedure and the subsequent storage period until laboratory analysis can
be performed.
4. A blood sample of 1 mL (minimum 0.5 mL) in a labeled tube, preferably stored in refrigerated or frozen temperature
5. The patient is required to drink at least 8 cups of liquid on the day of urine collection.

I.K AYTONA 5
 Disadvantage of using Creatinine
1. Some creatinine is secreted by the tubules, and secretion increases as blood levels rise
2. Medications, including gentamicin, cephalosporins, and cimetidine (Tagamet), inhibit tubular secretion of creatinine, thus
causing falsely low serum levels
3. Bacteria will break down urinary creatinine if specimens are kept at room temperature for extended periods, thus leads
to false low result
4. A diet heavy in meat consumed during collection of a 24-hour urine specimen will influence the results if the plasma
specimen is drawn before the collection period = false increase results
5. Not reliable indicator in athletes, persons involved in heavy exercise, and patients with muscle diseases
6. Drugs such as trimethoprim-sulfamethoxazole can increase serum creatinine level by approximately 0.4 to 0.5 mg/d
7. Creatinine clearance is affected by sex and race. Women have less muscle mass and a lower rate of creatinine
production in comparison to me n

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

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 female) × 1.212 (if patient is black)
Formula s A S E
MDRD-IDMS
Traceable formula
GFR = 175 × serum creatinine 1154
black/African-american)
× age 0.203 × 0.742 (if patient is female) × 1.202 (if patient is
4
S A
Other MDRD
formula s
E
6
/ B S
Parameters 4 PARAMETERS (SEAS) = serum creatinine, ethnicity, age, sex
included in MDRD 6 PARAMETERS (BASES) = BUN, Age, Serum creatinine, Ethnicity, Serum albumin ,Se×
A B S
Cockroft and gault formula
B-
A-
Body wt
Age
.

→ 5
s -
sex
s -
serum creatinine
CKD-EPI (Chronic Kidney eGFR (mL /min/1.73 m2) 141 x min(SCr/k,1)a x max(SCr/k,1) .209 x 0.993Age x (1.018 if
Disease Epidemiology 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 capability is often the first function affected in renal disease.
urine → UREA
URINE COMPOSITION Major organic substance in
Urganic
a. 95 % water Major inorganic substance in urine → a > Na > I Cha Nak
b. 5 % solutes -

TWO MECHANISMS 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 Reabsorb in PCT → U P sW¥G -

Salt, Water HA , Glucose, Urea

,

TYPE OF TRANSPORT Substance Location

-Glucose, Amino acid, Salts -Proximal convoluted tubule

Active Transport -Chloride -Ascending loop of Henle

-Sodium -PCT and DCT

-Water -PCT, DCT, DLH

Passive Transport -Urea (40% are reabsorbed) Proximal convoluted tubule and
ascending loop of Henle

-Sodium Ascending loop of Henle

I.K AYTONA 6
NOTE
ALH
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
Maximal Tubular reabsorptive capacity - Denoted Tm, the maximal rate of reabsorption of a solute by the tubular
epithelium per minute (milligrams per minute). Reabsorptive capacity varies with each solute and depends on the
glomerular filtration rate
The plasma concentration at which active transport stops is termed the renal threshold
Ex: Glucose renal threshold is _____________
160 180 -

mg/dl or equivalent to 350mg/min

Sodium renal threshold is 110 to 130 mmol/L

RENAL CONCENTRATION
Renal concentration begins in the descending and ascending loop of henle and the final concentration of urine
continues to the Collecting Duct.
Water is removed by osmosis in the descending loop of Henle, and sodium and chloride are reabsorbed in the ascending loop.
Osmolality - The movement of water across a semipermeable membrane in an attempt to achieve an osmotic equilibrium
between two compartments or solutions of differing osmolality (i.e., an osmotic gradient). This mechanism is passive, that is, it
requires no energy

Excessive reabsorption of water as the filtrate passes through the highly concentrated medulla is prevented by the water-
impermeable walls of the ascending loop. This selective reabsorption process is called the countercurrent mechanism and serves
to maintain the osmotic gradient of the medulla

Effect of Anti- Diuretic Hormone (Vasopressin) on Renal Concentration

ADH- hormone responsible for reabsorption of ___________
Huo in the distal convoluted tubules and collecting ducts of the
kidney.
→
hyposthenuria
→ hypersthene n'a

TEST FOR TUBULAR REABSORPTION

A. Osmolality test measures only the number of particles on solution.

- Major clinical uses of osmolarity include initially evaluating renal concentrating ability, monitoring the course of
renal disease, monitoring fluid and electrolyte therapy, establishing the differential diagnosis of hypernatremia
serum Osm :
and hyponatremia, and evaluating the secretion of and renal response to ADH. These evaluations may require
275 -300 MOSM
determination of serum in addition to urine osmolarity
Urine Osm : - The normal urine to serum ratio should be 1:1 to 3:1.
1400 MOSM
50
- Measurement of freezing point depression was the first principle incorporated into clinical osmometers, and many
-

instruments (supercooled then crystalline Crystallization raises temp Thermistor measures He corresponds to freezing paint )
.
. -

- The other instrument used in clinical osmometry is called the vapor pressure osmometer. The actual measurement
performed, however, is that of the dew point (temperature at which water vapor condenses to a liquid).
( sample is evaporated + them condensed Heat from condensation raises thermocouples to dew paint temp that is proportional to vapor pressure)
, . .

DIFFERNTIATING NEUROGENIC AND NEPHROGENIC DIABETES INSIPIDUS

controlled intake for 12 hours
The ratio of urine to serum osmolality in conjunction with procedures such →

Normal : 800 mosm

as controlled fluid intake and injection of ADH, is used to differentiate
whether it is neurogenic diabetes insipidus or nephrogenic diabetes insipidus 2 hrs
→ Normal 800
.
: mosm

3 :L ratio
ADH
Results makes
Urine osmolality >800mOm Neurogenic DI hypothalamus → injected
t
Urine: Serum ratio is 3:1 posterior → stores
Urine osmolality <400mOsm Nephrogenic DI pituitary secretes
gland
Urine: Serum ratio is 1:1

B. Specific gravity- measures the number and size of particles on solution.

polyuña polydipsia
, ,
polyphagia
C. Water deprivation test
Fish berg
________________ patients deprived of fluids for 24 hours before measuring Specific Gravity
________________
Mosenthal compared the volume and S.G of day and night urine samples

D. Free water clearance test

The free water clearance is determined by first calculating the osmolar clearance using the standard clearance
* uses formula
osmometer The calculation of the free water clearance is used to determine the ability of the kidney to respond to the state of
body hydration.
Calculating osmolar clearance indicates how much water must be cleared each minute to produce a urine with the
same osmolality as the plasma.

FORMULA
then subtracting the osmolar clearance value from the urine volume in mL/min.
YII.EE?-mxurineuoi
( mymint
.

2nd FORMULA =
Urine Volume -
Cos m

I.K AYTONA 7
 first morning urine
pH -151-0=6
RENAL SECRETION
Involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate
In contrast to tubular reabsorption, in which substances are removed from the glomerular filtrate and returned to the blood,
tubular secretion involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate
Two major functions of tubular secretion:
a. Elimination of waste products not filtered by the glomerulus
ions
Hydrogen
b. Regulation of acid-base balance in the body through the secretion of ____________________

Regulation of pH
Along with the lungs, the kidneys are the major regulators of the acid base content in the body. They do this through the secretion
of hydrogen in the form of ammonium ions, hydrogen phosphate, and weak organic acids, and by the reabsorption of bicarbonate
from the filtrate in the convoluted tubules.

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 and blood Ph is acidic)

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
It has the disadvantage of being exogenous, the chemical PAH meets the criteria needed to measure renal blood flow. This
nontoxic substance is loosely bound to plasma proteins, which permits its complete removal as the blood passes through
the peritubular capillaries.

C. Titratable acidity ( hydrogen ions 11-+1

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 information.

Hippocrates
Frederik Dekker Discovered albuminuria by boiling urine
Thomas Bryant
Thomas Addis Addis count
Richard Bright Introduced the concept of urinalysis as part
Thudicum Urochrome the pigment that causes yellow color of urine
Performed in
Urinalysis testing of urine with procedures commonly expeditious reliable
→ an

REASONS For Performing Urinalysis (CLSI)

, ,

accurate safe t cost effective

, ,
- manner .

1. Diagnosis of disease
2. Screening asymptomatic populations for undetected disorder
3. Monitoring the progress of disease organic
4. Monitoring the effectiveness of therapy solute g-
inorganic

URINE COMPOSITION
Urine consists of urea and other organic and inorganic chemicals dissolved in water.
Urine is normally 95% water and 5% solutes, although considerable variations in the concentrations of these solutes can
occur owing to the influence of factors such as dietary intake, physical activity, body metabolism, and endocrine
functions.
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
Nitrate A normal urine constituent. Nitrite
- abnormal
Others Carbohydrates, pigments, fatty acids, mucin, enzymes, hormones; may be present in small amounts
depending on diet and health

I.K AYTONA 8
 1- Urea
NOTE f) 2- Creatinine
Urea is the major organic component of urine ( urganic)
Chloride is the major inorganic component of urine followed by Sodium then Potassium →
i. chloride

A high urea and creatinine content can identify fluid as urine.

2. Na } chawak

KIT
3-

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 excretes two to three times more urine during the day than during the night

A. Oliguria decrease urine output Average

Less than 1mL/kg/hr in infants
Less than 0.5 mL/kg/hr in children
stras → Less than 400Mt 24 hrs / .

Less than _________________ Henry's → < 500Mt / 24 hrs .

Causes: Dehydration, vomiting, diarrhea, perspiration, severe burns 5

B. Polyuria- increase in daily urine output stras →

> 2.5424 hrs .

2.5 to 3ml/kg/day in children 's → > 2L / 24 hrs

Henry
.

_______________________
Causes: Diabetes mellitus, diabetes insipidus, diuretics, caffeine, alcohol 5

Analysis of urine in Differentiating between DM and DI

Due to defect in the pancreatic production of insulin

}÷÷÷:*
:

Diabetes Mellitus ___________Urine

Increase Specific gravity ✓ Polyana
Increase urine Glucose (glucosuria)
✓ Polydipsia
Due to decrease production or function of ADH
Diabetes Insipidus ___________Urine
Decrease Specific gravity

C. Nocturia- increase excretion of urine (>500ml) at night. Common among pregnant women, and urine has a specific gravity
of less than 1.018

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

Sometimes defined as being <100 mL/24 hour
Causes: damage to the kidneys, Renal stones, and renal tumors

SPECIMEN COLLECTION
2 hrs
Urine specimens should be delivered to the laboratory promptly and tested within _____________ .

Never discard a specimen before checking with a supervisor

I. CHARACTERISTICS OF CONTAINER
Clean, Dry, Leak-proof
With Screw top lids they are less likely to leak than snap-on lids
Wide mouth, and wide flat bottom
Made of sterile material

- the recommended container capacity is 50mL

___
- the required specimen volume for urine microscopic analysis is _____,
10 -15mL average of 12 ml

- Containers should stand upright, have an opening of at least 4 to 5 cm, and have a capacity of 50 to 100 ml

II. LABELS
Patient s name
Patient identification number
Date and time of collection
Additional information such as age, sex, etc.
*Labels must be attached to the BODY OF CONTAINER, not to the lid and should not become detached if the
container is refrigerated/frozen.

III. REQUISITION FORM

A requisition form must accompany specimens delivered to the laboratory

IV. POLICY FOR HANDLING MISLABELED SPECIMENS

Do NOT assume any information about the specimen or patient.
Do NOT relabel an incorrectly labeled specimen.
Do NOT discard the specimen until investigation is complete.
Leave specimen EXACTLY as you receive it; put in the refrigerator for preservation until errors can be resolved.
ted for analysis to continue.
Identify problem on specimen requisition with date, time, and your initials
Make person responsible for specimen collection participate in solution of problem(s). Any action taken should be
documented on the requisition slip.
Report all mislabeled specimens to the appropriate supervisor.

I.K AYTONA 9
 V. 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

Never discard a specimen before checking with a supervisor

VI. SPECIMEN PRESERVATION

*A specimen that cannot be delivered and tested within 2 hours should be refrigerated or have an appropriate
chemical preservative added

CHANGES IN UNPRESERVED URINE (Strasinger)

^PBaON -

C
Analyte Change Cause
Color Modified / Darkened Oxidation or reduction of 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 casts Decreased Disintegration in dilute alkaline urine
Trichomonads Decreased Loss of characteristic, motility and death
NOTE Protein/Albumin is least or not affected.

URINE PRESERVATIVES
Preservatives Advantages Disadvantages Additional Information
Refrigeration (2- Does not interfere with PRECIPITATES AMORPHOUS Prevents bacterial growth
*the easiest and most chemical tests CRYSTALS for 24 hours.
common → valid for Raises specific gravity by → can be used for culture t

24 hrs .

hydrometer sensitivity
Thymol Preserves glucose and Interfere with acid precipitation test
sediments well for protein
Boric acid *Preserves protein and May precipitate crystals when used Keeps pH at 6.0
formed elements well in large amounts -bacteriostatic at 18g/L
*Does not interfere with → can be used for culture t

routine analysis other than pH Interferes with drug and sensitivity

*Prevents bacterial growth and hormone analyses → used to
preserve protein &

metabolism 511-1 At

Formalin EXCELLENT SEDIMENT *Acts as reducing agent Can also be used for
PRESERVATIVE *Interfere with chemical tests for cytology (Brunzel)
glucose, blood, leukocyte esterase, → cellular analysis
and copper reduction blog
*False-negative reagent strip tests
for blood and urobilinogen
Toluene Does not interfere with Floats on surface of specimens and
To the routine test clings to pippete and testing
materials
Sodium Fluoride PREVENTS GLYCOLYSIS Inhibits reagent strip tests for May use sodium benzoate
GOOD FOR DRUG ANALYSIS glucose, blood, and leukocytes instead of fluoride for
big reagent strip testing
Phenol Does no interfere with Causes an odor change Use 1 drop per ounce of
routine test specimen
Gray C and S tube Preserves bacteria Decreases pH; do not use if urine is Preservative is boric acid
Sample stable at RT for 48hr below minimum fill line
Cherry red/ yellow top Stable for 72 hours Bilirubin and urobilinogen may be Preservative is sodium
tube principle based
: reflectance
on decreased if specimen is exposed propionate
photometry to light and left at RT
Yellow Plain UA FOR AUTOMATED Must refrigerate within Round or conical bottom
INSTRUMENTS 2 hours
Saccomanno fixative Preserves cellular elements Used for CYTOLOGICAL
(Ethanol + Carbowax) 5090 Ethanol 1- 2% Carbowax ( PEG ) EXAMINATION
Sodium carbonate Inexpensive Unacceptable for urinalysis testing For quantitative analysis of
Stabilizes porphyrins, porphyrins,
porphobilinogen, etc. porphobilinogen, etc.

Concentrated HCL can be used to preserve urine for catecholamines measurement

epinephrine
norepinephrine

I.K AYTONA 10
 TYPES OF URINE SPECIMEN
Random Specimen Most commonly received 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 specimen Ideal urine specimen for routine UA

Or 8-hour specimen The most concentrated specimen
Specimen that is ideal to test for substances that require concentration or incubation for
detection
These specimens are often preferred for cytology studies because the number of
epithelial cells present can be significant
Pregnancy Test
___________________
___________________
Glucose Monitoring
___________________
Orthostatic
proteinuria
Second morning/fasting spx For glucose or diabetic monitoring and screening
2- hour post prandial specimen Collect urine after 2 hours of meal
________________________
For Glucose Monitoring
Timed specimens (E.g 24 hours Quantitative Measurements of Analytes with Diurnal Variation ( Circadian Rhythm)
For ________________________
urine specimen) Urine specimen for clearance test
.

eg cortisol ( ACTA )

Analytes : Urine specimen for evaluation of fistulas

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✓ Electrolytes To obtain an accurate timed specimen, the patient must begin and end the
✓ catecholamines collection period with an empty bladder.
✓" ketosteroid All specimens should be refrigerated or kept on ice during the collection period and
-

Hydroxy
may also require addition of a chemical preservative.
On its arrival in the laboratory, a 24-hour specimen must be thoroughly mixed and the
p peak
level of urobiiinogen volume accurately measured and recorded 4 hrs urine → Nitrite testing
Afternoon urine (2pm to 4pm) Preferred for urobilinogen measurements
24 hrs unpreserved urine → schistosomiasis
12 hours urine specimen Ideal for screening microalbuminuria (Brunzel) ( Harr )
For determination of urine albumin, creatinine, and the albumin-to creatinine ratio
Catheterized specimen ______________
culture &
sensitivity
Note: If a routine urinalysis is also requested, the culture should be performed first to
prevent contamination of the specimen.
Midstream Clean catch Safer, less traumatic method for 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 aspiration For bacterial culture (especially for anaerobic microbes)
→ cellular
analysis /cytology cytology
_________________
Pediatric specimen We we bag → check bags every approx 15 mins until needed sample has been collected
.
.

Soft, clear plastic bags with hypoallergenic skin adhesive to attach to the genital area of
both boys and girls

SPECIMEN FOR PROSTATITIS

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)

Result Interpretation:
The first and third specimens are examined microscopically
If the third specimen will have a white cell / hpf count and bacterial count 10x 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.
The second specimen can be used for routine UA if additional testing is required
Quantitative cultures are performed on all specimens
Macrophages containing lipids may also be present

Pre and Post In the pre- and post-massage test (PPMT), a clean-catch midstream urine specimen is collected.
Massage Test A second urine sample is collected after the prostate is massaged positive result is significant
bacteriuria in the post-massage specimen of greater than 10 times the pre-massage count

Stamey-Mears Types of four Glass specimens

(Four glass) VB1 Initial voided urine, For bacterial cultures, Urethral infection or inflammation testing
VB2 Midstream urine and is used to tests for urinary bladder infection.
V13→ Voided
EPS Expressed prostatic secretion
Bladder
VB3 Post prostatic massage urine

The prostatic secretions are cultured and examined for white blood cells. More than 10 to 20 white
blood cells per high-power field is considered abnormal.

I.K AYTONA 11
 Drug Testing Specimen Collection
of
Chain custody or

___________________________-
chain of Evidence process that provides documentation of proper sample identification from the time of
collection to the receipt of laboratory results
The COC is a standardized form that must document and accompany every step of drug testing, from collector to courier to
laboratory to medical review officer to employer
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.
The collector adds bluing agent (dye) to the toilet water reservoir to prevent an adulterated specimen
Most common adulterant is water. prevent adulteration of sample

*Container capacity: ________

60mL
*Urine volume collected: _____________
30 -45mL
32.5 -37.7°C
*Urine Temperature: read within 4 minutes, range of ________________.
*The urine color is also inspected to identify any signs of contaminants.

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
urochrome
The yellow color of urine is caused by the presence of pigment, _____________.

Increased urochrome production:

a. Thyroid conditions
b. Fasting
c. Urine stands at room temperature
}OFT

Two additional pigments present in urine in much smaller quantities:

a. __________
Uroerythrin pink pigment, most evident in specimens that have been refrigerated, resulting in the precipitation of
amorphous urates.
Urobilin
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/Laboratory Correlation

Colorless -Recent fluid intake -Seen in random specimens
Pale yellow -Polyuria or Diabetes Insipidus -Increased 24 hours volume and low specific gravity
-Diabetes mellitus -Elevated specific gravity and positive glucose test result
-Dilute random specimen
Bright Yellow -RIBOFLAVIN (VITAMIN B2) Multivitamins
Dark Yellow -Concentrated specimen -After strenuous exercise or first morning specimen
-Dehydration -Fever or burns
-Acriflavine -for acriflavine, negative bile test results and possible green
fluorescence
-Carotene (may cause orange urine) -High consumption of vegetables and fruits that contain carotene
-Nitrofurantoin - Antibiotic administered for urinary tract infections
*Amber/Orange -Bilirubin -Yellow foam when shaken and positive chemical test for bilirubin
-Warfarin / Coumadin -Anticoagulant

Orange- yellow -Phenazopyridine (Pyridium) - drug commonly administered for urinary tract infection, produces
also a yellow foam when shaken

-Phenindione - anticoagulant, orange in alkaline, colorless in acid urine

Yellow-green -Bilirubin oxidized to biliverdin -colored foam in acidic urine and false negative test results for
bilirubin
Green -Pseudomonas infection - positive urine culture
Blue-green -Amitriptyline -antidepressant (blue urine color)
-Methocarbamol (Robaxin) -muscle relaxant (blue urine color)
AMMPCI -Methylene blue -fistulas
-Phenol -when oxidized (green urine color)
-Clorets -mouth deodorant (green urine color)
-Indican -bacterial infection, intestinal disorders

I.K AYTONA 12
 Yellow foam →
phenazopyridine ,
Bilirubin

white foam → Protein

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

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 refer
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 Possible Causes
Clear No visible particulates, transparent All solutes present are soluble (such as glucose and proteins)
Hazy Few particulates, print easily seen through urine RBC & WBC (varies with the substance and amount present)
Cloudy Many particulates, print blurred through urine Crystals, Microbes, Fat (lipids, chyle), epithelial cells
Turbid Print cannot be seen through urine Mucus, mucin, pus, radiographic dye, semen, contaminants
Milky May precipitate or be clotted Fats or lymph (lipiduria and chyluria)

Non-Pathologic Causes of Urine Turbidity Pathologic Causes of Urine Turbidity

-Squamous epithelial cells -RBCs - Nonsquamous epithelial cell
-Mucus -WBCs - Abnormality crystals
-Amorphous phosphates, carbonates, urates -Bacteria - Lymph fluid 8
-Semen, spermatozoa -Yeast - Lipids
-fecal contamination
-Radiographic contrast media
-Talcum powder
-Vaginal creams

LAB CORRELATION IN URINE TURBIDITY

Acidic urine Amorphous urates, radiographic contrast media
Alkaline urine SRAP Carbo Amorphous phosphates, carbonates
Soluble with heat t Amorphous urates, uric acid crystals
Soluble in dilute acetic acid RBCs, Amorphous phosphates, carbonates
Insoluble in dilute acetic acid WBCs, Bacteria, yeast, spermatozoa
Soluble in ether Lipids, lymphatic fluid c hyle

NOTE For checking of both clarity and color

Check urine with a white background with a good light source

I.K AYTONA 13
 URINE ODOR
Seldom of clinical significance and is not a part of the routine urinalysis

ODOR CAUSE
Aromatic Normal
Foul, ammonia-like, fetid Bacterial decomposition, urinary tract infection, old urine
Fruity, sweet Ketones, DM, Starvation, vomiting, strenuous exercise, diarrhea
Maple syrup Maple syrup urine disease, caramel sugar
Mousy odor, Barny or musty odor Phenylketonuria
Rancid Tyrosinemia
Sweaty feet Isovaleric academia
Cabbage, Hops Methionine malabsoprtion
* Bleach Contamination
* Odorless Acute tubular necrosis
Rotting fish Trimetylaminuria
* Pungent or distinctive odor Asparagus, Garlic, Onion ingestion, UTI(Brunzel), bacteruria, increase urinary amines
* Swimming pool Hawkinsinuria
Sulfur odor / rotten egg cystinuria
☒ Menthol-like Phenol-containing medications
* Mercaptan odor Asparagus, garlic, and egg

I.K AYTONA 14
URINE SPECIFIC GRAVITY
Specific gravity is defined 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
← average
-S.G of Normal random urine: _____________,
1. 002
-
1.035 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 pure water; 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 1.025 Indicates average solute and water intake and excretion
1.025 to 1.035 Concentrated urine; associated with dehydration, 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 methods Refractometer, reagent strip → most commonly used

Harmonic Oscillation Based on the principle that the frequency of a sound wave entering a solution changes in
Densitometry proportion to the density of the solution

It is rarely used today despite its ability to accurately and precisely determine urine specific gravity with
linearity up to 1.080 This method was initially used on a semiautomated urinalysis workstation known as
the Yellow IRIS

During testing, a portion of the urine sample is held in a U-shaped glass tube that has an
electromagnetic coil on one end and a motion detector on the other end. An electrical current applied to
the coil generates a sound wave of fixed frequency. This sonic oscillation is transmitted through the
specimen, and the frequency attenuation is measured. The frequency (the oscillating cycle period)
observed is directly proportionate to the sample density, and a microprocessor converts the
frequency to a corresponding specific gravity value.

Reagent strip The reagent strip reaction is based on 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 (Urinometer) The urinometer consists of a weighted 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 __________________
10 15 MI
-

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.
4G ← c. Subtract 0.004 for every 1 gram of glucose
Prothreein ← 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 3oC 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
3 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
KzS04 Potassium sulfate = S.G should be read at 1.015
Water = S.G should be read at 1.000

I.K AYTONA 15
 CM : •
CSF

sperm Analysis
•

Chem
Analysis
•

Microscopic Analysis
•

Refractometer (TS meter) It determines the concentration of dissolved particles in a specimen. It does this by measuring
Total Solid Meter
refractive index.
Refractive index is a comparison of the velocity of light in air with the
velocity of light in a solution(urine).
The refractometer provides the distinct advantage of determining specific gravity
using a small volume of specimen (one or two drops). I drop 50mL =

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- read_________________
I. 015 -1/-

0.001

1. 022 +/ 0.001
c. 5% NaCl read _________________
-

d. 9% Sucrose read _________________

1.034 -1/-0.001

Method Correction for temperature Correction for glucose Correction 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 → do not include whole number only decimal point, in multiplying : . 010
EXAMPLE: A specimen diluted 1:5 with a reading of 1.010 would have an actual S.G of ✗ 5
Toso
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
Vitamin C
11th parameter: __________________ → False Negative BBLNG ::

False Positive :(linitest

Reagent Strip Technique /Procedure
A
freaking
agent
Blood Bilirubin Leukocyte
, ,
esterase Nitrite , Glucose
,

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.
5. Com

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. The strip must be held close to the color chart without actually being placed on the chart.
e. Specimens that have been refrigerated must be allowed to return at room temperature prior to reagent strip testing, as the
enzymatic reactions on the strips are temperature dependent
f. Proper timing of reactions to take place.
anti moisture
-

Handling and Storing Reagent Strips f)

1. Reagent strips are packaged in opaque, tightly closed container with a desiccant (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
8. Specimens that have been refrigerated must be allowed to return to room temperature prior to reagent strip testing, as the
enzymatic reactions on the strips are temperature dependent.

I.K AYTONA 16
 Specimens must be returned to room temperature before chemical testing by reagent strips because the enzyme reactions on the
strips perform best at room temperature

water
QUALITY CONTROL OF REAGENT STRIPS f)
Reagent strips must be checked with both positive and negative controls a minimum of once every 24 hours. Many
laboratories perform this check at the beginning of each shift.

Distilled water is not recommended as a negative control because reagent strip chemical reactions are designed to perform
at ionic concentrations similar to urine. All readings of the negative control must be negative, and positive control readings should
agree with the published value

CONFIRMATORY TESTS
Confirmatory tests are defined as test using different reagents or methodologies to detect the same substances as detected by the
reagent strips with the same or greater sensitivity or specificity

Non-reagent strip testing procedures using tablets and liquid chemicals may be available when questionable results are obtained or
highly pigmented specimens are encountered.

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. Urinary pH is controlled primarily by dietary
regulation, although medications also may be used.
Urinary pH can be used for Determination of unsatisfactory specimens
Other clinical significance of measuring Urinary pH is for Treatment of urinary tract infections and Renal calculi formation and
prevention

Normal Urine pH 4. 5- 8.0

First morning urine pH 5.0 -
6.0
Improperly preserved specimen >9 or >8.5 (Strasinger, 6th edition)
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 (e. g. meat ) Presence of urease producing bacteria
Food rich in fats / lipids Alkaline tide (during and after following meals)
Presence of acid producing bacteria (E. coli) Presence of urease producing bacteria (Proteus spp. and Pseudomonas spp)
Medications such as Mandelamine and Citrus Food
Fosfomycintromethamine

NICE TO KNOW
Cranberry juice, which produces an acidic urine and has long been used as a home remedy for minor bladder infections because it
inhibits the colonization of certain urinary pathogens. People who are prone to frequent urinary tract infections are often advised to
drink cranberry juice or take over-the-counter cranberry pills MUST KNOW :
i.
principle
REAGENT STRIP REACTION (60 seconds) 2.
reagents
Principle ___________________________
Double indicator system 3.
positive rxn ( color)
4. reading time

Methyl red + H+ Bromthymol blue H+ 5. interferences

(Red to yellow) (green to blue)

pH 4.0 -6.0 pH 6.0-9.0 46 y O- .
MR. BB likes 69
Reagents Methyl Red and Bromthymol Blue
Source of Error / No known interfering substances, Run-over from adjacent pads, and Old specimens
interference

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 ___________
I. 002 1.035
-

Radiographic Contrast dye S.G = >1.040

Not A urine S.G = <1.002 exemption : 1.001 → associated with Diabetes Insipidus

REAGENT STRIP REACTION (45 seconds)

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

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

I.K AYTONA 17
 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 (100-500mg/dl), ketoacidosis
False negative: Highly alkaline urines (greater than pH 6.5 add 0.005 SG reading)

III.PROTEIN
Most Indicative of renal disease
Normal urine contains very little protein: usually, less than 10 mg/dL or 100 mg per 24 hours is excreted. (Strasinger)

Albumin is the major protein found in normal urine due to its low molecular weight
Other Proteins Normally found:
Serum and tubular microglobulins, Tamm-Horsfall protein (Uromodulin), protein derived from prostatic and vaginal
secretion
Produces a white foam in urine unique to kidney
mg/L

Pre Renal or Caused by conditions that affect the plasma prior to its reaching the kidney
Overflow
A. Hemoglobin =intravascular hemolysis
Proteinuria
B. Myoglobin = Muscle injury
C. Acute phase reactants = inflammation and infections
D. Bence Jones protein = multiple myeloma
BENCE JONES PROTEIN
An immunoglobulin light chains (kappa and lambda) found in cases of Multiple myeloma
Confirmatory Test: Serum electrophoresis
40°C -60°C and dissolves
Heat reactivity test in Urine: Coagulates/Precipitates at _____________
at____________
80 -

100 o C
Renal Proteinuria / I.Glomerular Proteinuria
True renal disease
-When the glomerular membrane is damaged, selective filtration is impaired, and increased amounts of
serum protein and eventually red and white blood cells pass through the membrane
and are excreted in the urine.
-The amount of protein that appears in the urine following glomerular damage ranges from slightly above
normal to 4 g/day

A. Diabetic Nephropathy / Kimmelstiel-

decreased GFR
associated with renal failure in persons with Type I and II Diabetes mellitu
Microalbuminuria
Indicator: __________________
Microalbuminuria is the presence of albumin in urine above the normal level but below the
detectable range of conventional urine dipstick methods.
The presence of microalbuminuria is also associated with an increased risk of cardiovascular
disease.

builds up in
B.Amyloidosis → abnormal protein called amyloid organs

C.Immune complexes found in SLE and Streptococcal glomerulonephritis

D.Toxic substances
E.Pre-eclampsia and Eclampsia → associated w/ pregnancy
F. Orthostatic / Cadet / Postural proteinuria
Urine Specimen Orthostatic Proteinuria Clinical Proteinuria
1st morning
negative positive
2hours after standing
positive positive
II.Tubular Proteinuria
Normally filtered albumin can no longer be reabsorbed
PCT
A. → impaired
B. Toxic agents/Heavy metals (such as cadmium dust)
C. Severe viral infections
Post- Renal Lower UTI/inflammations, Injury / Trauma, Menstrual Contamination, Prostatic fluid/ spermatozoa, and
Proteinuria Vaginal secretion

Benign Proteinuria
The discovery of protein, particularly in a random sample, is not always of pathologic significance, because several benign causes of
renal proteinuria exist. Benign proteinuria is usually transient and can be produced by conditions such as strenuous
exercise, high fever, dehydration, and exposure to cold.

shed
I.K AYTONA 18
 TESTS FOR MICROALBUMINURIA
Albumin Excretion Normal: 0-20 ug/min
Rate (AER) Microalbuminuria: 20 -200µg /min or 30-300 my / 24 hrs
24 hrs urine
. } .

Immunologic Micral-Test ImmunoDip

-
first morning spx . are Principle: Enzyme immunoassay Principle: Immunochromographics
recommended Sensitivity: 0 to 10 mg/dL Sensitivity: 1.2 to 8.0 mg/dL
Reagents: Gold-labeled antibody, Reagents: Antibody-coated blue latex particles
B-galactosidase, Chlorophenol red Interference: False-negative: Dilute urine
galactoside
NOTE NOTE
*Strips are dipped into the urine up to a Strips are individually packaged in specially designed
level marked on the strip and held for 5 containers. The container is placed in the urine
seconds specimen for 3 minutes
*reading time is 1minute Appearance Amount(mg/dl) Interpretation
*negative result is white color Darker bottom <1.2 Negative
*positive result is red band
Equal band colors 1.2 to 1.8 Borderline
Interferences: Darker top band 2 to 8 Positive
False-positive: Strong oxidizing agent(soap)
False-negative: Dilute urine
Albumin:Creatinine The Clinitek Microalbumin reagent strips and the Multistix Pro reagent strips (Siemens Healthcare
ratio Diagnostics, Deerfield, IN) provide simultaneous measurement of albumin/protein and creatinine that
permits an estimation of the 24-hour microalbumin excretion. The strips can be read manually or on
automated Clinitek instruments. Results from the Clinitek are automatically calculated. Results are
reported as normal or abnormal.

ABNORMAL A:C RATIO = 30 to 300 mg/g or 3.4 to 33.9 mg/mmol

ALBUMIN: CREATININE RATIO REAGENT STRIPS

Albumin strip Albumin reagent strips use the dye bis (3',3"-diiodo-4', 4"-dihydroxy-5',5"-
principle : dinitrophenyl)-3,4,5,6-tetrabromo sulphonphthalein (DIDNTB), which has a
Siemens Multistix Pro 10
Sorensen's error higher sensitivity and specificity for albumin.
→ no
urobilinogen of indicator DIDNTB strips can measure albumin between 8 and 15 mg/dL (80 to 150 mg/L)
protein high-protein error
.
→
without inclusion of other proteins.
-

Of indicator
protein DIDNTB
→ -
low =
dye binding-

( Albumin
binding of
Color range: Pale green to Aqua Blue
to su / phonphthalein
dye )
Interferences:
Highly buffered alkaline urine can be controlled by using paper treated with
bis- (heptapropylene glycol) carbonate
Falsely elevated results can be caused by visibly bloody urine, and
abnormally colored urines

Addition of polymethyl vinyl ether decreases the nonspecific binding of polyamino

acids to the albumin pad

Creatinine Principle: Pseudoperoxidase activity of copper-creatinine complexes

strip y
1. combines w/
creatinine y chromogen
Reagent: Copper sulfate (CuSO4), 3,3',5,5'-tetramethylbenzidine (TMB), and
diisopropyl benzene dihydroperoxide (DBDH)
TMB 2. peroxide that oxidizes chromogen

Color range: Orange (negative) to green to blue

DBDH
Results: Reported as 10, 50, 100, 200, 300 mg/dL, or 0.9, 4.4, 8.8, 17.7, or 26.5
mmol/L of creatinine

No creatinine readings are considered abnormal, as creatinine is normally present in

concentrations of 10 to 300 mg/dL. The purpose of the creatinine measurement is to
correlate the albumin concentration to the urine concentration, producing a
semiquantitative albumin:creatinine ratio (A:C) ratio

Interferences:
False increased: visibly bloody urine, presence of the gastric acid reducing medication
cimetidine (Tagamet), and abnormally colored urines

REAGENT STRIP REACTION FOR PROTEIN (60 seconds)

Principle
Tetra
¢
Indicator + protein ------------------------- protein + Hydrogen
(Yellow) (+) Blue-Green

I.K AYTONA 19
 NOTE f) Protons
a. Proteins mainly albumin accepts Hydrogen ions from the indicator
b. The test is more sensitive to albumin because albumin contains more amino groups to accept the
hydrogen ions than other proteins acid mixed w/ tetra
a. The pH of the medium remains constant (pH of 3 buffered with citrate) ←
b. Reagent strip is sensitive to Albumin only
c. The S.G of urine sample should be check because a trace protein in diluted sample is more
significant than in concentrated sample
d. Indicators appear yellow in the absence of protein; however, as the protein concentration
increases, the color progresses through various shades of green and finally to blue
Reagents Multistix = Tetrabromphenol blue
Chemstrip = Tetrachlorophenoltetrabromosulfonphthalein
Grading Grading Quantity of Albumin
Trace <30 mg/dl
1+ 30mg/dl
2+ 100mg/dl
3+ 300mg/dl
4+ 2000mg/dl
Interference False positive False negative
Highly buffered interference alkaline urine Proteins other than albumin
Pigmented specimens, phenazopyridine Microalbuminuria
Quaternary ammonium compounds (detergents)
Antiseptics, chlorhexidine
Loss of buffer from prolonged exposure of the
strip to the specimen reagent
High specific gravity

The specific gravity of the urine specimen should be considered in evaluating urine protein because a trace protein in a dilute
specimen is more significant than in a concentrated specimen.

SULFOSALICYLIC ACID (SSA) PRECIPITATION TEST Exton 's test

A cold precipitation test that reacts equally with all forms of protein.

PROCEDURE 3mL of 3% SSA (E 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 with no granulation T 30-100
2+ Turbidity with granulation, no flocculation TG 100-200
3+ Turbidity with granulation, and flocculation TGF 200-400
4+ Clumps of protein >400

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

MICROSCOPICALLY, WHAT IS THE SSA PATTERN IF PROTEINS CAUSE A POSITIVE REACTION? Amorphous
MICROSCOPICALLY, WHAT IS THE SSA PATTERN IF DRUGS AND RADIOGRAPHIC CONTRAST DYE
CAUSE A POSITIVE REACTION?
RC
Crystalline
PA
]
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
Specimens used: Fasting/8-Hours urine sample/ First morning urine / Second Morning urine/2hr post prandial
A first morning specimen does not always represent a fasting specimen because glucose from an evening meal may remain in
the bladder overnight, and patients should be advised to empty the bladder and collect the second specimen

CLINICAL SIGNIFICANCE OF URINE GLUCOSE

Hyperglycemia Associated Renal associated
Increase blood glucose, Increase Urine glucose Normal blood glucose, Increase Urine glucose
Causes: Causes: f) PCT : SWAG U -

Diabetes Mellitus and Gestational Diabetes Mellitus, , Advanced renal disease, Osteomalacia,
Pancreatitis and Pancreatic Cancer, Pheochromocytoma, Pregnancy, ESRD (End stage renal disease), Cystinosis
Acromegaly, Cushing syndrome, Hyperthyroidism, Liver
disease, Cerebrovascular accident / stroke catecholamine
GH cortisol

I.K AYTONA 20
 REAGENT STRIP REACTION FOR GLUCOSE (30 seconds)
Principle Double
Sequential Enigmatic Reaction

Glucose oxidase
Glucose + O2 -------------------------- gluconic acid + Hydrogen Peroxide
☒ Reference mtd for Glucose: specific for Glucose

Hexokinase Peroxidase
Hydrogen Peroxide + Chromogen----------------- Oxidized chromogen + Water

STEP: In the first step, glucose oxidase catalyzes a reaction between glucose and room air (oxygen) to produce
gluconic acid and peroxide. In the second step, peroxidase catalyzes the reaction between peroxide and
chromogen to form an oxidized colored compound that is directly proportional to the concentration of glucose.
Color charts provide quantitative measurements ranging from 100 mg/dL to 2 g/dL, or 0.1% to 2%.

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) and O-toluidine (pink to purple)
Grading
Grading Trace 1+ 2+ 3+ 4+
Amount of 1/10 g/dl (%) ¼ g/dl (%) 1/2 g/dl (%) 1 g/dl (%) 2g/dl (%)
glucose or or or or or
100 mg/dl 250 mg/dl 500 mg/dl 1000 mg/dl 2000 mg/dl
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
Iodate It is added by the manufacturers in the glucose reagent strip to minimize interference by ascorbic
acid. This chemical oxidizes ascorbic acid so that it cannot interfere with the oxidation of the
chromogen

Test Non-specific test for reducing sugars

a. Glucose, galactose, fructose, maltose, lactose, pentoses
Sucrose a non-reducing sugar and cannot be detected by this test
Clinical significance For the detection of inborn error of metabolism especially galactosuria in newborns in which there is a
lack of enzyme galactose -1- phosphate uridyltransferase
Component of the a. Copper sulfate- main reacting agent
Tablet b. Sodium carbonate eliminates interfering O2 (room air)
acts
[ c. Sodium citrate /citric acid for heat production
as
-

effervescent
d. Sodium hydroxide for heat production
-
alkaline medium

The tablet, when placed in a mixture of water and urine, the tablet is rapidly dissolved by the action
of sodium carbonate and citric acid which act as an effervescent. The sodium hydroxide provides the
alkaline medium necessary for the reaction, and the heat required is provided by the reaction of
sodium hydroxide with water and citric acid
drops
Procedure 5 drops
gtts urine + 10gtts distilled H2O + Clinitest tablet --- observe the reaction
Note: Wait 15 seconds after boiling (there is effervescent formation) has stopped and
gently shake the contents of the tube

milk * Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of sodium
lactose -
Glu 1- Gqlac hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium
sugar
malt
Maltose -
Glu 1- Glu carbonate to prevent room air from interfering with the reduction reaction.
sugar
table
Sucrose -
Fruc -1 Glu * Clinitest tablets are very hygroscopic and should be stored in their tightly closed packages. A
sugar strong blue color in the unused tablets suggests deterioration due to moisture accumulation,
as does vigorous tablet fizzing.

Pass through phenomenon:

-Occurs when greater than 2 g/dl sugar is present
-From blue > green > yellow > orange/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
* The sensitivity of Clinitest to glucose is reduced to a minimum of 200 mg/dL so the Clinitest
cannot be used as a confirmatory test for glucose.
Interference False Positive: Vufs
Reducing agents such as vitamin C, formalin, uric acid, and some drugs (cephalosporin)
False Negative:
Oxidizing agents such as detergent

Confirmatory
Inborn error of
for
metabolism
→ Tandem MS / MS I.K AYTONA 21
tests
Grading
Grading Color Sugar level Negative clear blue color, blue precipitate may form
negative Blue ¼% Trace-bluish-green color
GYOR 1+
2+
Green
Yellow
½%
¾%
1 + green color, green or yellow precipitate
2+yellow to green color, yellow precipitate
3+ Orange 1% 3+yellow-orange color, yellow-orange precipitate
4+ Brick red 2% 4+reddish-yellow color, brick red or red precipitate

SUMMARY OF GLUCOSE OXIDASE AND CLINITEST REACTIONS

Glucose Oxidase Clinitest Interpretation oxidizing agent → false positive reagent strips
4+ Negative -Oxidizing agent interference → false
negative Chini test
-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 sensitive
Negative Positive -Non glucose reducing substance
-Possible interfering substance such as reducing agent ( vitamin C)
-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. They are formed from beta oxidation of fats.
a. Inability to metabolize or utilize available carbohydrate ex. DM type1
b. Increased loss of carbohydrates ex. Vomiting
c. Inadequate intake of carbohydrate ex. Starvation and malabsorption/pancreatic disorder
d. Overuse of available carbohydrates frequent strenuous exercise

Ketone Bodies: p Butyrate

a. 78% Beta Hydroxybutyric acid major ketone but not detected in reagent strip
→ Acetate
b. 20% Acetoacetic acid (AAA) / Diacetic acid parent ketone
= c. 2 % Acetone detected only when glycine is present

When the blood ketone concentration exceeds 70 mg/dL (the renal threshold level), ketones are excreted in the urine

REAGENT STRIP REACTION FOR KETONES (40 seconds)

Principle Legal 's test ( sodium nitro prussiate rxn )
for
( acetone )
Acetoacetate and acetone + sodium nitroprusside + glycine-- (+) Purple

Reagents Sodium nitroprusside (nitroferricyanide), glycine (Chemstrip)

Reporting / Grading Grading Quantity
Negative --
Trace 5mg/dl
BKL -

purple 1+ (small) 15mg/dl

Bilirubin 2+ (moderate) 40mg/dl
ketone
leukocyte esterase 3+ (large) 80 to 160mg/dl
Interference False positive:
a. Phthalein dyes
b. Highly pigmented red urine
c. Levodopa
d. Medications containing free sulfhydryl groups including mercaptoethane sulfonate
sodium (MESNA) and captopril
False negative:
a. Improperly preserved specimens

ACETEST TABLET
Composition:
a. Sodium nitroprusside
b. Disodium phosphate
c. Lactose gives better color differentiation
The Acetest tablet test has been used as a confirmatory test for
questionable reagent strip results; however, it was primarily used for
testing serum and other bodily fluids and dilutions of these fluids for severe
ketosis
Read for 30 seconds
Report as negative, small (5 to10mg/dl), moderate (30 to
40mg/dl, or large (80 to 100mg/dl).
Acetest tablets are hygroscopic; if the specimen is not completely absorbed
within 30 seconds, a new tablet should be used.
Acetest can be used to test urine, serum, plasma, or whole blood
10 times more sensitive to diacetic acid than to acetone

I.K AYTONA 22
 g) rgt strip
reacts with heme component
VI.BLOOD
The finding of a positive reagent strip test result for blood indicates the presence of red blood cells, hemoglobin, or
myoglobin.
Any amount of blood greater than five cells per microliter of urine is considered clinically significant

HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA

Cloudy red urine Clear red urine Clear red urine
Presence of an intact RBC Uniform green / blue color in Heme portion of the myoglobin is
Produces a speckled/spotted reagent strip pad toxic to the renal tubules
pattern on reagent pad Hemoglobinuria may result from Uniform green / blue color in
the lysis of red blood cells reagent strip pad
Seen in cases of: produced in the urinary tract,
a. Glomerulonephritis particularly in dilute, alkaline Seen incases of:
b. Renal calculi urine a. Rhabdomyolysis
c. Pyelonephritis b. Prolonged coma
d. Tumors Seen in cases of: c. Convulsions
e. Trauma a. Transfusion reactions d. Extensive exertion
f. Anticoagulants b. Hemolytic anemias e. Muscle wasting diseases
g. Strenuous exercise c. Severe burns f. Cholesterol- lowering statin
h. Hypertension d. Infections: malaria, syphilis, medications
i. Cystitis mycoplasma, and C.perfringens g. Muscle ischemia: carbon
j. Exposure to toxic chemicals e. Strenuous exercise monoxide poisoning
☒ lysis of RBCs in f. Brown recluse spider bites h. Muscle infection(myositis)
urinary
tract shows i. Trauma
confirmatory
:
combination of hematuria t
Note j. Crush syndrome
✓ Microscopic hemoglobin uria while
intravascular hemolysis Hemoglobin must be present in the urine in an k. ALCOHOLISM
analysis shows no KBC
amount exceeding 10 mg/dL before it is l. Heroin abuse
detected by routine protein reagent strip tests

Reabsorption of filtered hemoglobin also results in the appearance of large yellow-brown granules of denatured ferritin called
hemosiderin in the renal tubular epithelial cells and in the urine sediment.

The heme portion of myoglobin is toxic to the renal tubules, and high concentrations can cause acute renal failure

Hemoglobin Versus Myoglobin

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

REAGENT STRIP REACTION FOR BLOOD (60 seconds)

Principle Pseudo of Heme ( Agb)
peroxidase activity
Oxidize
2.
Hemoglobin
Hydrogen peroxide + chromogen ----------------------------- oxidized chromogen + H20
1. reduce
Pseudoperoxidase

(-) yellow, (+) Green to Blue 5 tbcs 1mL

NOTE: Reagent strip tests can detect concentrations as low as five red blood cells per microliter

Through pseudoperoxidase activity of the heme moiety, peroxide is reduced and the chromogen becomes
oxidized, producing a color change on the reaction pad from yellow to green
Reagents Multistix = diisopropylbenzenedehydroperoxidetetramethylbenzidine
Chemstrip = dimethyldihydroperoxyhexanetetramethylbenzidine
Interference False positive:
a. Strong oxidizing agents
b. Vegetable and Bacterial peroxidases (e.g Escherichia coli)
c. Menstrual contamination
False negative:
a. High SG / Crenated cells

f
b. Formalin
c. Captopril
d. Ascorbic acid (>25mg/dl)
e. Unmixed specimen / failure to mix the specimen prior to testing
f. High concentration of nitrite (>10mg/dl)

I.K AYTONA 23
 difficult to interpret
✗
VII.BILIRUBIN
The appearance of bilirubin in the urine can provide an early indication of liver disease. It is associated with:
a. Hepatic jaundice = Hepatitis and Cirrhosis
b. Post hepatic jaundice = Biliary obstruction (gallstones, carcinoma)
Only the B2 or conjugated bilirubin is water soluble thus can be seen in urine and can be detected.
It produces an amber urine with yellow foam
Conjugated bilirubin is normally excreted in the bile into the duodenum, and normal adult urine contains only 0.02 mg of
bilirubin per deciliter. This small amount is not detected by the usual testing methods.
Excretion of bilirubin is enhanced by alkalosis
✓ polar
✓direct
Bilirubin, a highly pigmented yellow compound, is a
✓conjugated degradation product of hemoglobin. Under normal
conditions, the life span of red blood cells is approximately
✓ B2 120 days, at which time they are destroyed in the spleen and
liver by the phagocytic cells of the reticuloendothelial system.
The liberated hemoglobin is broken down into its component
parts: iron, protein, and protoporphyrin. The body reuses the
iron and protein, and the cells of the reticuloendothelial
system convert the remaining protoporphyrin to bilirubin.
The bilirubin is then released into the circulation, where it
binds with albumin and is transported to the liver. At this
point, the kidneys cannot excrete the circulating bilirubin
because not only is it bound to albumin, but it is also water
insoluble (unconjugated bilirubin).
/
In the liver, bilirubin is conjugated with glucuronic acid
by the action of glucuronyl transferase to form water-soluble
bilirubin diglucuronide (conjugated bilirubin). In the intestine,
intestinal bacteria reduce bilirubin to urobilinogen, which is
then oxidized and excreted in the feces in the form of
stercobilinogen and urobilin

REAGENT STRIP REACTION FOR BILIRUBIN (30 seconds)

Principle Diazo reaction
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

Reagent strip color reactions for bilirubin are more difficult to interpret than other reagent strip reactions and are
easily influenced by other pigments present in the urine

ICTOTEST (Tablet) FOR BILIRUBIN

A confirmatory test for bilirubin is the Ictotest
Ictotest is much more sensitive than the dipsticks, being able to detect as
little as 0.05 mg/dL
Components:
a. p-nitrobenzene-diazonium p-toluenesulfonate
b. SSA
c. Sodium carbonate
d. Boric acid
Positive reaction (+): Blue to purple color

VIII.UROBILINOGEN
A bile pigment that results 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.
mgldl : Ehrlich
1 : I
Clinical significance: urine urobilinogen greater than 1 mg/dl is seen in liver disease and hemolytic disorders.

CONSTIPATION CAN RAISE UROBILINOGEN LEVEL

1% of the non-hospitalized population and 9% of a hospitalized population exhibit elevated results. This is frequently
caused by constipation.

I.K AYTONA 24
 REAGENT STRIP REACTION FOR UROBILINOGEN (60 seconds)
Principle
indicator
Multistix: Uses Ehrlich reagent ✗
Urobilinogen + p-dimethlyaminobenzaldehyde ---------- red color
pdab ( Ehrlich ) (diazo)
faro coupling
- rxn

Chemstrip: uses 4-methyloxybenzene-diazonium- rxn)

Urobilinogen + diazonium salt ------- red azodye
Note Ehrlich-reactive compounds: porphobilinogen, indican, p-aminosalicylic acid, sulfonamides, methyldopa,
procaine, chlorpromazine ---
Interference False positive:
a.
b. Highly pigmented urine
False negative:
a. Old specimens
b. Preservation in formalin
c. Improperly preserved, allowing urobilinogen to be photo-oxidized to urobilin.
d. High concentration of nitrite

What would be the result of urobilinogen measurements if the reagent strip is performed at a higher temperature?
ANS: Falsely increased
RATIO: The sensitivity of the Ehrlich reaction increases with temperature, and testing should be performed at room temperature

What would be the result of urobilinogen measurements after following a meal?

ANS: normally highest after meal
RATIO: As a result of increased excretion of bile salts, urobilinogen results are normally highest following a meal

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
-URINE (TOP) Colorless Red Red
-CHLOROFORM (Bottom) Red Colorless Colorless
Butanol Extract
-BUTANOL (TOP) Red Colorless Red
-URINE (BOTTOM) Colorless Red Colorless

` C B C B C B

B
Red Colorless Red

u
Rea colorless colorless

soluble to both chloroform Insoluble to both chloroform soluble to Butanol

and Butanol and Butanol ONLY
HOESCH TEST (INVERSE EHRLICH REACTION) inhibit Urobilinogen
Rapid screening test for porphobilinogen (>2mg/dl) T
Procedure: dissolved in 6M or 6N HCL)- (+) Red on top of solution

Interpretation:
1. When the tube is shaken, the red color is seen throughout the solution. The test detects approximately 2 mg/dL of
porphobilinogen,
2. Urobilinogen is inhibited by the highly acidic pH.
3. High concentrations of methyldopa and indican, and highly pigmented urines, may produce false positive results.

correlate -10131
CORRELATION OF BILIRUBIN AND UROBILINOGEN p
Condition Blood Urine Bilirubin Urine Urobilinogen
Prehepatic jaundice Increase Unconjugated bilirubin negative +++
(Hemolytic disease)
Hepatic jaundice Increase both B1 and B2 +/- ++
(Liver damage)
Post hepatic jaundice Increase Conjugated Bilirubin +++ normal
(Bile duct obstruction)

I.K AYTONA 25
* Diazonium salt as reagent :
* Diazonium salt as
product
:
BUL → Bilirubin Urobilinogen , leukocyte esterase
Nitrite
,

IX.NITRITE
Provides a rapid screening test for the presence of UTI and bacteriuria.
The nitrite test also can be used to evaluate the success of antibiotic therapy and to periodically screen persons with recurrent
infections, patients with diabetes, and pregnant women, all of whom are considered to be at high risk for UTI
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 hours 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 Greiss Reaction
f) aromatic amine
p-arsanilic acid (or sulfanilamide) + Nitrite ----------- Diazonium salt

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

*Nitrite at an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a
diazonium compound that then reacts with tetrahydrobenzoquinolin compounds to produce a pink-
colored azodye
Reagents Multistix = p-arsanilic acid, tehtrahydrobenzoquinolin-3-ol
Chemstrip = sulfanilamide, hydroxytetrahydrobenzoquinoline indicator
-

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/Homogenous pink
Pink spots/edge is considered as NEGATIVE
Results are reported only as negative or positive.
Positive nitrite corresponds to ___________
100,000 organisms/ml

-It is for gram negative 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
NEGATIVE FOR LYMPHOCYTES → no esterase enzymes
Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain esterases
Infections involving trichomonads, mycoses (e.g., yeast), chlamydia, mycoplasmas, viruses, or tuberculosis cause leukocyturia
or pyuria without bacteriuria

REAGENT STRIP REACTION FOR LEUKOCYTES (120 seconds)

Principle Leukocyte Esterase

LE
Indoxylcarbonic acid ester--------- indoxyl + acid indoxyl + Diazonium salt - (+) Purple azodye

* The reagent strip reaction uses the action of LE to catalyze the hydrolysis of an acid ester embedded on
the reagent pad to produce an aromatic compound and acid. The aromatic compound then combines with
a diazonium salt present on the pad to produce a purple azodye.
Reagent Multistix = Diazonium salt, derivatized pyrrole amino acid ester
Chemstrip =Diazonium salt, Indoxylcarbonic acid ester
Sensitivity Multistix = 5 to 15 WBC/hpf indicator
Chemstrip = 10 to 25 WBC/hpf

I.K AYTONA 26
 Interference False positive:
a. Strong oxidizing agents
b. Formalin
c. Highly pigmented urine, nitrofurantoin, beets, phenazopyridine
d. False-positive results for leukocyte esterase are most often obtained on urine specimens
contaminated with vaginal secretions
False negative:
a. High concentration of protein (Greater than 500 mg/dl), high glucose , oxalic acid
(in acidified urine that has 4.4pH or below) and ascorbic acid
b. Antibiotics such as gentamicin, cephalosporins, tetracyclines,
c. Inaccurate timing
d.
High specific gravity
The LE reaction requires the longest time of all the reagent strip reactions (2 minutes). Trace readings may not be significant and
should be repeated on a fresh specimen.

ASCORBIC ACID
It is the 11th parameter
Causes False Negative result to BBLNG (Blood, Bilirubin, Leukocyte, Nitrite, Glucose)
Causes False Positive result to Clinitest

Ascorbic acid level that causes a negative reaction to Bilirubin and Nitrite
Ascorbic acid level that causes a negative reaction to glucose

Detergent/Soap / Quaternary Brand Reading time Positive result

Ammonium Compound : Stix 60 seconds Blue color
Cause False Positives in :
✓ Blood
C-stix 10 seconds Blue color
✓ Glucose
✓ Leukocyte
✓ Protein SUMMARY
Test Principle (+) result Reading time
rgt :

Bilirubin di chloro Diazonium

Diazo reaction Violet, tan, or pink 30 secs most sensitive

g-Reading
.
. . .

salt

\
Glucose Double sequential enzymatic reaction Potassium iodide 30secs regarding
yellow green Time
= blue-green to brown
-

Ketones
Sodium nitropmsside eaction) Purple 40secs
S.G brom pKa change of polyelectrolyte Diluted = blue 45secs
.

thymol blue
Concentrated =yellow
- . . .

pH MR BB -
. Double indicator system Acidic = red to yellow 60secs
Alkaline = green to blue
Protein Protein (Sorensen Blue-green 60secs
=
Tetra . ... .

Blood di tetramethyl benzidine

. . .. .

Pseudoperoxidase activity of hemoglobin Green to blue 60secs

urobilinogen Ehrli Red 60secs
Nitrite quinOlin
. . . . . ..
Greiss reaction Pink 60secs
Leukocyte Leukocyte esterase Purple 120secs -
Least sensitive
to Reading Time

MICROSCOPIC ANALYSIS OF URINE

ADDIS COUNT first procedure to standardize the quantitation of formed elements, used a hemocytometer
Specimen: _______________________
12 hrs urine
preserved w/ formalin .

NORMAL VALUE OF ADDIS COUNT

RBCs = 0 to 500,000 cells /ul
CREW
{ WBCs and Epithelial cells =0 to 1,800,000 cells /ul
Hyaline casts = 0 to 5000 cells/ul

Specimen Preparation

Urine 10 -15 ml Frequently used V01 . = 12mL

Centrifuge at 400 RCF for 5 minutes

Decant
24 or 12 - sediment cone .
factor
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)

To correct for differences in the diameter of centrifuge heads, RCF rather than revolutions per minute (RPM) is used.
Formula: RCF(g) = 1.118 x 10-5 x radius (cm) x RPM2

I.K AYTONA 27
 Normal value of Urinary eosinophil : < 1% # of Eosinophil counted hemolytic anemia =3 It
Formula =
✗ 10° ✓ hemoglobinemia
71%
Urinary eosinophil in Interstitial nephritis : Total WBC counted
✓ hemosiderinuria
allergic rxn / inflammation
of nephron ✓ hemoglobinuvia
SEDIMENT STAINS
STAIN ACTION FUNCTION
Sternheimer-Malbin(a supravital -Delineates structure and contrasting -Identifies WBCs, epithelial cells, and
Supravital stain consisting of Crystal violet and colors of the nucleus and cytoplasm casts CEW
safranino
stains
safranin) general stain for sediments
0.5%Toluidine blue -Enhances nuclear detail Differentiates WBCs and renal
(a metachromatic supravital stain) tubular epithelial (RTE) cells → eccentric
nucleus

2% acetic acid ( I -2 drops)

sediment because
Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs, yeast, oil
cannot be used for initial analysis
RBC are
lysed WBCs droplets, and crystals
Lipid Stains: Oil Red cannot stain Stain triglycerides and neutral fats Identifies free fat droplets and lipid
→ cholesterol
O and Sudan III orange-red containing cells and casts
dried heat fixed
Gram stain → use a

urine
-

sediment
Differentiates gram-positive and Identifies bacterial casts
of
preparation gram negative bacteria
Hansel stain → staining is
performed on a
Methylene blue + EosinY in methanol Identifies urinary eosinophils
dried of
smear of centrifuged cytocentrifuged prep 4 Wright Giemsa
Stains eosinophilic granules
Spx or .
can also use or
sediment stain

Prussian blue stain ( Rous test ) Stains structures containing iron Identifies yellow-brown granules of
confirm hemosiderinuvia hemosiderin in cells and casts
Sedi and KOVA stain Modified Sternheimer Malbin Hyaline cast appears as pink
contain
stabilizing chemicals that The dye is absorbed well by WBCs, Motile bacteria are unstained
prevent precipitation that occurred in epithelial cells, and casts, providing Non-motile bacteria stains purple
original stain clearer delineation of structure and T.vaginalis stains Light blue-green
contrasting colors of the nucleus and
cytoplasm

f) SternHeimer Maltin - ADDITIONAL INFORMATION

1. One disadvantage of its use is that in strongly alkaline urines, this stain can precipitate, which obstructs the visualization of
sediment components.
2. In Oil Red O and Sudan III, cholesterol and cholesterol esters do not stain and must be confirmed by polarizing
Microscopy
3. urinary eosinophils, but Hansel stain is preferred.

Commercial Systems for Urine Sediment Preparation

UriSystem The UriSystem tube is designed such that after centrifugation, it can be decanted with a quick smooth
motion and consistently retains 0.4 mL of urine for sediment resuspension.
KOVA System The KOVA System uses a specially designed pipette that snuggly fits the diameter and shape of the tube to
retain 1 mL of urine during decanting.
Count-10 System The Count-10 System offers several options to retain 0.8 mL for sediment resuspension

CYTODIAGNOSTIC URINALYSIS
Play an important role in the early detection of renal allograft rejection and in the differential diagnosis of renal disease.
Involves making a 10:1 concentration of a first morning urine specimen, followed by cytocentrifugation of the urine

MICROSCOPY

Technique Function and Description

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

Phase contrast Enhances visualization of elements with low refractive indices, such as hyaline casts,
diff from WBC thru motility
microscopy mixed cellular casts, mucous threads, and Trichomonas
.
-

for casts
undulating variations in light intensity or contrast
Trichomonas -
membrane
\
flagella Adaptation of a bright-field microscope with a phase-contrast/
objective lens and a/
matching

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 oval fat bodies, fatty casts, and crystals.
microscopy It uses halogen quartz lamp that produces light rays of many different waves
for high retractile A substance that rotates the plane of polarized light 90 degrees in a clockwise direction is said
used to diff .
cystine t to have positive birefringence.
uric acid
Substance that rotates the plane in a counterclockwise direction has negative
diff birefringence
'

frm
✓
Caox
-

monohydrate crystals Bright-field microscopes can be adapted for polarizing microscopy. Two polarizing filters
non
polarizing RBCs
-
Calcium
phosphate crystals from must be installed in a crossed configuration
non
polarizing bacteria

I.K AYTONA 28
Dark field Aids in identification of spirochetes such as Treponema pallidum
microscopy 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-image and layer-by-layer imaging of a specimen

contrast microscopy 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
what type of microscope : ← (Hoffman) and differential-interference contrast (Nomarski). Bright-field microscopes
l.
1cm
can be adapted for both methods.
2.
Bright field
-

Converting brightfield microscopy to differential interference contrast microscopy requires (1) a

y
polarizer placed between the light source and the condenser, (2)y a special condenser containing
modified Wollaston prisms for each objective, (3)-a Wollaston prism placed between the
objective and the eyepiece, and (4)/ an analyzer (polarizing filter) placed behind this Wollaston
prism and before the eyepiece

Fluorescence Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye
microscopy 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 system BODY
1. Oculars 1. Light source 1. Base
2. Objectives 2. Condenser 2. Body tube
3. Adjustment knobs 3. Stage field 3. Nose piece
4. Iris diaphragms

Initial/Primary magnification of sample Occurs in the OBJECTIVES

Final / second magnification of sample Occurs in the EYEPIECE

CARE OF MICROSCOPE
1. Carry microscope with two hands, supporting the base with one hand.
hair brush
f) camel
to dust
2. Always hold the microscope in a vertical position.
-
remove

3. Clean optical surfaces only with a good quality lens tissue and commercial lens cleaner.
4. Do not use the 10× and 40× objectives with oil.
Alternative -1 alcohol based cleanser
5. Clean the oil immersion lens after use.
-

cedar wood oil in 010

6. Always remove slides with the low-power objective raised. xylene → used to remove

not totally recommended

7. Store the microscope with the low-power objective in position and the stage centered.

TERMINOLOGIES
Aperture diaphragm Microscope component that regulates the angle of light presented to the specimen.
Birefringent/ doubly The ability of a substance to refract light in two directions.
refractile
Chromatic aberration Unequal refraction of light rays by a 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
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
Magnification Process of enlarging

I.K AYTONA 29
 LENS Individual magnification Total magnification (Eyepiece x Objective)
Eyepiece 10x --
Scanner 5x or 4x 50x or 40x
LPO 10x 100x
HPO 40x 400x
OIO 100x 1000x

controls amount of light

µ and angle of
light rays that pass
through spit lens

→ focuses the light on the

specimen & controls the light
for uniform illumination

→ controls
tight beam
reaching slide

Sediment Constituents Found in Urine

I. Red Blood Cells

Appear as smooth, non-nucleated, biconcave disk measuring approximately 7 um in diameter
Most difficult to recognize
The observation of microscopic hematuria can be critical to the- early diagnosis of glomerular disorders andr
malignancy
of the urinary tract andrto confirm the presence of renal calculi
The presence of not only RBCs but also hyaline, granular, and RBC casts may be seen following strenuous exercise
If the specimen is not fresh when it is examined, erythrocytes may appear as faint,
because the hemoglobin may dissolve out
They may become crenated in hypertonic urine and appear as small, rough cells with crinkled edges
t spikes
even

In Concentrated /Hypersthenuric urine: Crenated cells /ECHINOCYTES / Irregularly shaped > 1.010

In Diluted / Hyposthenuric urine: Ghost cells / Swollen RBC ( 1.010 , Alkaline urine

Dysmorphic or Distorted RBC vary in sizes, mainly they are acanthocytes, it is associated with
glomerular bleeding spikes uneven

Because their hemoglobin has been lost, ghost cells are difficult to see using brightfield microscopy; however,
they are readily visible with phase-contrast or interference contrast microscopy
When viewed from the side, RBCs have an hourglass shape; when viewed from above, they appear as disks
with a central pallor
Hypotonic and Alkaline urine promotes formation of ghost cells in urine
Normal RBC in normal urine is 0 2 cells/hpf ; more than 3 cells/hpf is considered abnormal
Source of identification error: Yeast cell, oil droplets, air bubbles
Look-alike crystal: Monohydrate calcium oxalate crystals
NOTE
and better
delineates the presence of cellular blebs and protrusions

I.K AYTONA 30
II.White Blood Cells
WBCs are larger than RBCs, measuring average of about 12-um in diameter
Pyuria or leukocytoruia- Term used to denote increase urinary WBCs and is associated with bacterial infection
(UTI), Interstitial nephritis, and SLE
Neutrophil is the predominant WBC found in urine
Neutrophils lyse rapidly in dilute alkaline urine and begin to lose nuclear detail.
In Hypotonic urine, white blood cell swells and become spherical balls that lyse as rapidly as 50% in 2 to 3 hours at
room temperature
Hypotonic Urine: Glitter Cells WBC with sparkling appearance due to Brownian movement of the
granules. When stained with Sternheimer-Malbin stain, these large cells stain light blue as opposed to the violet
color usually seen with neutrophils
In hypertonic urine, leukocytes become smaller as water is lost osmotically from the cells, but they do not crenate.
Another degenerative change in WBC is the development of numerous finger-like or wormlike projections protruding
from their surfaces. These long filaments, termed myelin forms, result from the breakdown of the cell membrane
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
Lymphocytes predominate in urine from patients experiencing renal transplant rejection.
Normal WBC in urine = 0-5 WBC/hpf for male, and 0-8 WBC/hpf for female

III.Epithelial Cells
non pathologic
-

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. They may appear as flagstone-shaped with distinct cell borders
The point of reference in microscopic analysis
They may occasionally appear folded, possibly resembling a cast, and will begin to disintegrate in urine that
is not fresh.
Increased amounts are more frequently seen in females.
Clue cells: pathologic squamous epithelial cell covered with the Gardnerella vaginalis coccobacillus
To be considered a clue cell, the bacteria should cover most of the cell surface and extend beyond the
edges of the cell. This gives the cell a granular, irregular appearance.

usually non pathologic

Transitional Epithelial (Urothelial) cells/ Bladder epithelial cells ✗
-

B.
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. The differences are caused by the ability to absorb large amounts of water.
They are two to four times as large as white cells. They may be round, pear-shaped, or may have taillike
projections. Occasionally, these cells may contain two nuclei.
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. Renal fragments are an indication of severe tubular injury with
basement membrane disruption.
RTE cells often resemble casts and they should be closely examined for the presence of a nucleus, as a
nucleus would not be present in a cast.

I.K AYTONA 31
 Tubular Injury: presence of more than 2 RTE/HPF
RTE cells are the most clinically significant of the epithelial cells and the presence of increased amounts
is indicative of necrosis of the renal tubules
They are the precursor of oval fat bodies ← retractile RTE highly
LOCATION OF RTE CELL APPEARANCE
PCT (Proximal convoluted tubules) Rectangular
DCT (Distal convoluted tubules) Round or Oval
Collecting duct Cuboidal and can be seen as large sheets of cells
I 2 3
Conditions producing tubular necrosis include exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin
toxicity,4viral infections (hepatitis B), pyelonephritis,
5 6
allergic reactions,7 malignant infiltrations,8salicylate poisoning, and9 acute
allogenic transplant rejection
1- normal RTE cells & oval fat bodies in Acute Tubular Necrosis

Bubble cells RTE cells containing large, nonlipid-filled vacuoles that is mainly associated with Acute tubular
necrosis. They appear to represent injured cells in which the endoplasmic reticulum has dilated prior to cell death

of oval fat bodies : i. RTE

IV. Oval Fat Bodies ←
precursor 2.
Monocytes / Macrophages
Lipiduria These are lipid-containing RTE cells
They are highly refractile RTE cells
They are usually seen in conjunction with free-floating fat droplets
When monocytes or macrophages have ingested lipoproteins and fat, these globular inclusions are distinctly
refractile. Called oval fat bodies, these cells are impossible to distinguish from renal tubular cells that can also
absorb fats
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, and in trauma cases that
cause release of bone marrow fat from the long bones
NOTE: In lipid-storage diseases, large fat-laden histiocytes may also be present in urine. They can be differentiated from
oval fat bodies by their large size. -Strasinger Macrophages w/ oval fat bodies are larger

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
The actual bacteria producing an UTI cannot be identified with the microscopic examination.

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

diff if motile :
VII.Parasites
.
non -

motility
look for : rapid
darting movement y
- i.
flagella
Trichomonas vaginalis most frequent parasite encountered in urine membrane
2. undulating

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

When not moving, Trichomonas is more difficult to identify and may resemble a WBC, transitional, or RTE cell. Use of phase
microscopy may enhance visualization of the flagella or undulating membrane.

I.K AYTONA 32
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.
Increase in numbers are found in cases of UTI.

X. Hemosiderin Granules
Hemosiderin granules are found in the urine sediment 2 to 3 days after a severe hemolytic episode (e.g.,
transfusion reaction, paroxysmal nocturnal hemoglobinuria).
Hemosiderin granules may be found free floating or within macrophages, casts, or tubular epithelial cells.
The Prussian blue reaction, also known as the Rous test, is used to identify hemosiderin in the urine sediment
and in tissues.
The urine sediment is suspended in a freshly prepared solution of potassium ferricyanide HCl and is allowed to
stand at room temperature for 10 minutes. After centrifugation and discarding of the supernatant, the
sediment is reexamined for the presence of coarse blue granules

URINARY CAST
CYLINDRURIA presence of urinary cast
Casts are the only elements found in the urinary sediment that are unique to the kidney.
The major constituent/mould /template/ matrix of cast is UROMODULIN which is secreted by the RTE Cells.
The protein (uromodulin) gels more readily under conditions of urine-flow stasis, acidity, and the presence of sodium and
calcium.
Uromodulin protein is found in both normal and abnormal urine
Casts are Formed in DCT, and Collecting duct
Examination of casts should be performed along the edges of the cover slip.
Cylindroids formed at the ALH and DCT with tapered end or have a tail at the other tail. They have the same
significance as casts (hyaline cast).
Cylindroids are product of incomplete cast formation, or cast disintegration.

CAST FORMATION
From least significant to the most significant

no
particular cell ✗
inside (
only solutes) in chronic kidney Disorders
found
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
Hypotonic and alkaline urine promotes the disintegration of casts in the urine sediment.
Acid pH, increased solute concentration, urine stasis, and increased plasma proteins (particularly albumin) enhance cast
formation
If the conventional glass-slide method is being used, casts have a tendency to locate near the edges of the cover slip;
therefore, low-power scanning of the cover-slip perimeter is recommended

I.K AYTONA 33
 Upper UTI / Pyelonephritis lower UTI / Cystitis / Urethritis
= affected is kidney =
urinary /
bladder urethra
WBC WBC
cast cast

HYALINE CAST
Most frequently seen cast which consists almost entirely of uromodulin
Pro cast
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
The morphology of hyaline casts is varied, consisting of normal parallel sides and rounded ends, cylindroid forms, and
wrinkled or convoluted shapes that indicate aging of the cast matrix
The accompanying dehydration of the protein fibrils and internal tension may account for the wrinkled and convoluted
appearance of older hyaline casts SHED
Physiologic increase: 1. Strenuous exercise 2. Dehydration 3. Heat exposure 4. Emotional stress
Pathologic increase: 1. Acute glomerulonephritis 2. Pyelonephritis 3. Chronic renal disease 4. Congestive
heart failure PACC

Sternheimer-Malbin stain and KOVA stain: Pink color

Normal value: 0-2 /low power field

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
They have also been observed in healthy individuals following participation in strenuous contact sports
As an RBC cast ages, cell lysis begins and the cast develops a more homogenous appearance, but retains the characteristic
orange-red color from the released hemoglobin. These casts may be distinguished as blood casts, indicating greater stasis
of urine flow

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)
They are also present in non-bacterial inflammations such as acute interstitial nephritis and may accompany RBC casts in
glomerulonephritis
WBC casts are visible under low-power magnification but must be positively identified using high power
Staining and the use of phase microscopy can be helpful to enhance the nuclear detail needed for differentiating WBC cast
from Epithelial cast

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 non-pathologic conditions appears to be from the lysosomes excreted by RTE cells during -
normal metabolism.
Granular casts are easily visualized under low-power microscopy. However, final identification should be performed using
high power to determine the presence of a cast matrix. in disease states
granules may represent
,

Pathologic increase: glomerulonephritis and pyelonephritis

disintegration of cellular casts t tubule cells or
Physiologic increase: strenuous exercise protein aggregates filtered by glomerulus

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
With supravital stains, waxy casts stain a homogenous, dark pink
Waxy casts are more easily visualized than hyaline casts because of their higher refractive index

I.K AYTONA 34
BROAD CAST
Often referred to as renal failure casts
Broad casts may result from tubular distension or, in the case of extreme urine stasis, from formation in the
collecting ducts
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
Bile-stained broad, waxy casts are seen as the result of the tubular necrosis caused by viral hepatitis
Two most commonly seen broad cast: granular and waxy

GRANULAR DIRTY BROWN CAST

Granular, dirty, brown casts representing hemoglobin degradation products such as methemoglobin may also be present
They are associated with the acute tubular necrosis often caused by the toxic effects of massive hemoglobinuria
that can lead to renal failure.
These dirty, brown casts must be present in conjunction with other pathologic findings such as RTE cells and a positive
reagent strip test for blood

OTHER CAST
Mixed cellular cast
-The presence of mixed elements in a cast may make identification more difficult. Staining or phase microscopy aids in the
identification. When mixed casts are present, there should also be homogenous casts of at least one of the cell types, and
they will be the primary diagnostic marker
Bilirubin cast → liver disease
Epithelial cast / RTE cast
tubular necrosis accompany
WBC casts in
pyelonephritis
represent presence of advance staining & use of
phase microscopy
tubular destruction help enhance nuclear detail
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

SOURCES OF ERROR
Cast Sources of error
Hyaline Cast Mucus, fibers, hair, increased lighting
RBC Cast RBC clumps
WBC Cast WBC clumps
Bacterial Cast Granular casts
Epithelial / RTE Cast WBC cast
Granular Cast Artifacts such as Clumps of small crystals, Fecal debris
Columnar RTE cells
Waxy Cast Fibers and fecal material
Fatty Cast Fecal debris
Broad Cast Fecal material, fibers

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
Solutes precipitate more readily at low temperatures. 1 Colder temp promotes .

Usually reported as rare, few, moderate, or many per hpf crystal formation
Abnormal crystals may be averaged and reported per lpf
→ All abnormal formed in acidic urine
crystals are

Crystals formed in acidic urine vowels Amorphous urates, Calcium oxalate, uric acid, and all abnormal crystals
Crystals formed in alkaline urine Amorphous phosphates, ammonium biurate, calcium carbonate, calcium phosphate, and
consonant
Triple phosphate

I.K AYTONA 35
 NORMAL URINARY CRYSTALS
Crystal Information Significance Solubility
Amorphous urates Brick dust / yellow brown granules None Alkali and heat
Pink sediment (uroerythrin)

Note! It will convert to uric acid crystals

with acidification with acetic acid
Uric acid Rhombic, wedge, hexagonal, four-sided flat Lesch Nyhan syndrome Alkali
plate(whetsone), lemon shaped, nipple Gout
shaped. SEEN IN VARIETY OF SHAPE Chemotherapy
Calcium oxalate Forms: and Dilute HCL
a.dihydrate (weddelite) common oxalic acid
envelope or pyramidal.

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

Rosette form may resemble sulfonamide Caphostite

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

ABNORMAL URINE CRYSTALS

Crystal Information Significance Solubility
Cysteine Colorless hexagonal plates Cystinuria Ammonia, dilute HCL
Mistaken as uric acid crystals cystinosis
Cholesterol Rectangular plates with a notch in one or Nephrotic syndrome Chloroform
more corners (staircase pattern)

Mistaken as Crystal of radiographic dye

Tyrosine Colorless to yellow needles in clumps or rosettes Liver disease Alkali or heat
Leucine Yellow brown spheres with concentric circles and Liver disease Hot alkali or alcohol
radial striations
Liver disease
Note! Leucine and tyrosine crystals may
crystals :
occur together; leucine may be
BLT
precipitated with tyrosine crystals if
alcohol is added to urine
Bilirubin Clumped needles or granules with bright yellow Liver disease Acetic acid, HCL, NaOH, ether,
color chloroform, and alkali 6
Sulfonamide Colorless to yellow brown needles, sheaves of Possible tubular Acetone
wheat, rosette, rhombics, whetstone damage

May be mistaken with calcium phosphate crystals

in rosette form.
Calcium phosphate: soluble to dilute acetic acid

Sulfonamide:
(+) lignin test (urine+25%HCL=Yellow)
Ampicillin Colorless needles that tend to form bundles Massive dose of Refrigeration forms bundles
following refrigeration penicillin

URIC ACID VERSUS CRSTALS

Uric acid Cystine
Color Yellow brown Colorless
Solubility in ammonia Soluble Soluble
Solubility in dilute HCL Insoluble Soluble

Birefringence Positive Negative

Cyanide nitroprusside reaction Negative positive

I.K AYTONA 36
 ADDITIONAL INFORMATION
The most common crystals seen in acidic urine are urates, consisting of amorphous urates, uric acid, acid
urates, and sodium urates. Microscopically, most urate crystals appear yellow to reddish brown and are the
only normal crystals found in acidic urine that appear colored.
Acid urates appear as larger granules and may have spicules similar to the ammonium biurate crystals seen in alkaline
urine
Sodium urate crystals are needle-shaped and are seen in synovial fluid during episodes of gout, but may also appear in the
urine.

RMT I.K AYTONA 37

 The most common form of calcium oxalate crystals is the dihydrate that is easily recognized as a colorless,
octahedral envelope or as two pyramids joined at their bases
Calcium oxalate crystals are sometimes seen in clumps attached to mucous strands and may resemble casts.
Calcium oxalate crystals are also associated with foods high in oxalic acid, such as tomatoes and asparagus, and
ascorbic acid, because oxalic acid is an end product of ascorbic acid metabolism
Phosphates represent the majority of the crystals seen in alkaline urine
Ammonium biurate crystals resemble other urates in that they dissolve at 60°C and convert to uric acid crystals when
glacial acetic acid is added
Cholesterol crystals are rarely seen unless specimens have been refrigerated, because the lipids remain in droplet form

urates @ 100°C
ppt
=

URINARY SEDIMENT ARTIFACTS

.

Oval fat fat droplets

1. Starch granules
OFFS C → starch
Spheres with dimpled center
-

← fatty cast
cholesterol
Resembles as fat droplet with se
2. Oil droplets → Casts Abnormal Crystals Mucus Threads Sq Epithelial
LPF → CAMS
.

, ,
,

3. Air bubbles
4. Pollen grains- spheres w/ a cell wall and concentric circles RFMOMA → TTBYN → Transitional Trichomonas Bacteria , , .

5. Hair and fibers mistaken as casts ms→LPF Yeast Normal crystals ,

6. Fecal contamination Ave # → RAWRCO → RTE cells,AbnÉm"ai crystals WBC

.
,TÉÉsc , ,

¥pf Casts Oval fat bodies

.

Epf PF

Manner of Reporting
RBC, WBC Average number per 10 HPF
Casts Average number per LPF
Squamous epithelial cells Rare, few, moderate, or many per LPF
Transitional epithelial cells Rare, few, moderate, or many per 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 crystals Rare, few, moderate or many per HPF
Abnormal crystals Average number per LPF

/ Squamous
Transitional 1

Reporting Mucus Normal Crystals Epithelial cells Bacteria

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 (2+) Easy to find; number present in FOV varies;
Many (3+) Prominent; large number present in all FOVs
Packed (4+) FOV is crowded by or overwhelmed with the elements

f) progressively irreversible
RENAL DISORDERS
Dysmorphic RBCs q pan characterized by hematuria
are &
proteinuria
GLOMERULAR DISORDERS t GFR =

DISORDER ETIOLOGY CLINICAL COURSE PRIMARY OTHER SIGNIFICANT

URINALYSIS TESTS
RESULT
Acute Deposition of immune Rapid onset of Macroscopic Antistreptolysin O titer
Anti DNase B
Glomerulonephritis complexes formed in hematuria and edema. hematuria, hyaline
-

casts
TBUN conjunction with group A Proteinuria, IWBCS ,
Anti- group A
Streptococcus infection, Permanent renal Hallmark -

Red Blood Cell Streptococcal enzymes

on the glomerular damage seldom occurs casts, oliguria
membranes Granular casts
Rapidly Deposition of immune Rapid onset with Macroscopic Blood Urea Nitrogen
progressive(crescentic) complexes from system glomerular damage and hematuria, Creatinine
glomerulonephritis immune disorders on the possible progression to Proteinuria, Creatinine clearance
I GFR fFDP , cnyoglobulins ,
glomerular membrane end-stage renal failure Red Blood Cell casts
deposition of IgA complexes SLE

Good pasteur Attachment of cytotoxic Hemoptysis and Macroscopic Anti- glomerular

syndrome antibody formed during dyspnea followed by hematuria, Basement membrane
viral respiratory infections hematuria. Proteinuria, Antibody
to glomerular and alveolar Red Blood Cell casts
basement membranes Possible progression to
end-stage renal failure

I.K AYTONA 38
 Antineutrophilic cytoplasmic Pulmonary symptoms Macroscopic Antineutrophilic
Granulomatosis autoantibody binds to including hemoptysis hematuria Cytoplasmic antibody
Granulomatosis with polyangiitis neutrophils in vascular develop first followed Proteinuria ( ANCA) I p
peñnnckar -

pattern
Granuloma -

producing inflammation walls producing damage to by renal involvement Red Blood Cell Casts directed to the cytoplasmic
primary
c-
of small vessels granular
small vessels in the lungs and possible 9 Serum creatinine
granules of neutrophil pattern
and glomerulus progression to end- f BUN ethanol treated Wen → ANCA
indirect
}
Px -

p
-

serum +
formalin/formaldehyde
stage renal failure
Immuno -

→ , Anca fixation
fixed
-

Nen

Henoch-Schonleinpurpura Occurs primarily in children Initial appearance of Macroscopic Stool occult blood
y -

following viral respiratory purpura followed by hematuria,

GI
bleeding infections; a decrease in blood in sputum and Proteinuria,(mild heavy) -

platelets disrupts vascular stools and eventual Red Blood Cell casts
joint bleeding integrity renal involvemen.

Complete recovery is
common, but many
progress to renal
failure.
IgA nephropathy Deposition of IgA on the Recurrent Early stages: Serum immunoglobulin A
glomerular membrane macroscopichematuria Macroscopic or
resulting from increased following exercise with microscopic
T IgA
levels of serum IgA slow progression to hematuria
serum
levels
chronic Late stages: See
glomerulonephritis chronic
glomerulonephritis
Membranous Thickening of the Slow progression to Microscopic Antinuclear Antibody
glomerulonephritis glomerular membrane Nephritic syndrome or hematuria : Hepatitis surface antigen
6 SLE Secondary syphilis following IgG immune possible remission Proteinuria 3 Flourescent treponemal
Sjogren syndrome Hep B complex deposition /Rare) antibody-absorption test
tendency towards thrombosis RBC casts
Gold t Mercury treatments malignancy associated with systemic (FTA-ABS)
disorders

Membranoproliferative Cellular proliferation Type

1
Noticeable progression Hematuria Serum complement levels
glomerulonephritis affecting the capillary walls to chronic 2
Proteinuria Type
l T
cellularity in the sub endothelial
-

Cells of
type mesangium causing thickening
or the glomerular type 2 glomerulonephritis to t scrum
complement levels
Of
capillary
the
walls
basement membrane, nephritis syndrome. Type 1 Type 2 -

extremely dense deposits in the glomerular

basement
PATTERN OF THE possibly immune mediated.
membrane tubules Baumann
,
&
capsule
type 3- Both sub epithelial tsubuudotuelial deposits

Chronic glomerulonephritis Marked decrease in renal Noticeable decrease in Hematuria Blood Urea Nitrogen
check t broadcast
function resulting from renal function Proteinuria Serum Creatinine
waxy glomerular damage progressing to renal Glucosuria Creatinine clearance
to differentiate from acute
precipitated by other renal failure. Cellular and Electrolytes
glomerulonephritis disorders IGER electrolyte imbalance granular casts
TBUN t creatinine levels Waxy & broad casts
non

immunologic
Nephrotic Syndrome Disruption of the Acute on set following Heavy proteinuria, Serum albumin
> 3.5g/
impaired shield of Negativity electrical charges that systemic shock 6 Microscopic day Cholesterol
podocytes
t

cannot produce the tightly fitting Gradual progression hematuria, Triglycerides

alpha 2- globulin
macro

pass podocyte barrier resulting from other glomerular Renal tubular cells,
in massive loss of protein disorders and then to Oval fat bodies
and lipids renal failure Fat droplets ( lipidic n' a)
① serum albumin
Fatty & waxy casts
T serum lipids
pronounced edema
Minimal change disease Disruption of podocytes shield t
of
Frequent complete Heavy proteinuria Serum albumin
(Lipid nephrosis) (nil disease)occurring primarily in negativity remission following Transient hematuria Cholesterol
children following allergic corticosteroid treatment Fat droplets Triglycerides
reactions and most common cause of
in children
nephrotic edema
BUN creatinine
syndrome normal t

immunizations HLA -1312 ,

Focal segmental Disruption of podocytes in May resemble nephrotic Proteinuria Drug of Abuse
glomerulosclerosis certain areas of glomeruli syndrome or minimal Microscopic HIV tests
immune deposits
IgM t , associated with heroin change disease. hematuria
and analgesic abuse and
acquired immunodeficiency
syndrome Hep viruses ,

Alport syndrome Genetic disorder showing Slow progression to to See Nephrotic

inherited disorder ← lamellated and thinning nephritic syndrome and syndrome
of
collagen production
tumigas
of glomerular basement end stage renal disease Micro albuminuria
sex-linked autosomal
membrane
or

genetic
Diabetic Nephropathy Most common cause of Vascular structure of (+)Microalbuminuria → early indicator of Diabetic

(Kimmelstiel- Wilson ESRD glomerulus develops (+)Micral test Nephropathy

disease) Deposition of glycosylated sclerosis

complication of DM proteins on the glomerular

basement membranes
caused by poorly controlled
blood glucose levels

I.K AYTONA 39
 NEPHROTIC SYNDROME LAB FINDINGS
URINE FINDINGS PLASMA OR BLOOD FINDINGS
1. Proteinuria 1. Increase A2-Macroglobulin in electrophoresis
III.
common
ngs
{2. Lipiduria 2. Hypoproteinemia and hypoalbuminemia
3. Hematuria 3. Hyperlipidemia
4. Cylindruria (fatty cast) 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 necrosis Damage to renal tubular cells caused by Microscopic hematuria, proteinuria
ischemic or toxic agents Hyaline, Granular, Waxy and Broad cast
RTE CELLS, RTE CASTS
-Conditions producing tubular necrosis include Odorless urine
exposure to heavy metals, drug-induced toxicity, Isosthenuria
hemoglobin and myoglobin toxicity, viral
infections (hepatitis B), pyelonephritis, allergic Bubble cells

reactions, malignant infiltrations, salicylate (t ) Granular Dirty Brown Cast

poisoning, and acute allogenic transplant
rejection
Fanconi syndrome Generalized failure of tubular reaction in Glucosuria, Cystinuria
the Proximal Convoluted tubule Proteinuria, phosphaturia
Urinary pH can be very Low due to the
failure to reabsorb bicarbonate
Diabetes Insipidus A.Neurogenic DI → more common Lows Specific gravity urine
-Failure of the hypothalamus to produce Polyuria
increased ADH) ADH
⑥ADH →
-Low ADH

b. Nephrogenic DI
-inability of the renal tubules to respond to
ADH
-Normal to increase ADH
-
Sex-linked recessive
of
Renal Glucosuria A- or
affinity Defective tubular reabsorption of glucose Normal Blood glucose
transporters are
Increase Urinary glucose
Interstitial
glucose
aureascd
-
autosomal recessive
Cystitis (Lower UTI) Ascending bacterial infection of the urinary WBCs, Bacteria
bladder Microscopic hematuria (no casts)
True UTI has WBC t Bacteria Mild proteinuria
Increased urine pH
Acute Pyelonephritis (Upper UTI) Infection of the renal tubules and WBCs, bacteria
interstitium related to interference of urine WBC cast, Bacterial Cast
flow to the bladder, reflux of urine from Microscopic hematuria, proteinuria
the bladder (Vesicoureteral reflux)
and untreated cystitis
Chronic Pyelonephritis Recurrent infection of the renal tubules WBC, Bacteria
has t broad casts and interstitium caused by structural WBC CAST, Bacterial cast
waxy
ccnginetal urinary structural defects abnormalities affecting the flow of urine Granular, Waxy, and Broad Cast
producing reflux nephropathy is the most
commonage Hematuria and Proteinuria
Acute Interstitial nephritis Allergic inflammation of the renal Hematuria, proteinuria
Drug-induced interstitium in the response to certain WBCs (Increase Eosinophil) 1%
medication WBC cast
medication binding to interstitial protein NO BACTERIA

PROXIMAL CONVOLUTED TUBULAR DYSFUNCTIONS

Impaired ability to reabsorb glucose Renal glucosuria
Impaired ability to reabsorb specific amino acids -Cystinuria (cystine and dibasic amino acids)
-Hartnup disease (mono amino-monocarboxylic amino acids or glycine)
Impaired ability to reabsorb sodium
Impaired ability to reabsorb bicarbonate Renal tubular acidosis type II
Impaired ability to reabsorb calcium Idiopathic hypercalciuria
Excessive reabsorption of calcium Hypocalciuric familial hypercalcemia
Excessive reabsorption of sodium
Excessive reabsorption of phosphate Pseudohypoparathyroidism

DISTAL CONVOLUTED TUBULAR DYSFUNCTIONS

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

I.K AYTONA 40
 ADDENDUM
Rapidly progressive Some forms may demonstrate increased fibrin degradation products, cryoglobulins, and the
(Crescentic) glomerulonephritis deposition of IgA immune complexes in the glomerulus
syndrome A cytotoxic autoantibody can appear against the glomerular and alveolar basement membranes
after viral respiratory infections Initial pulmonary complaints are hemoptysis and dyspnea,
followed by the development of hematuria
Henoch Schonlein purpura Disease occurs primarily in children after upper respiratory infections. As its name implies,
initial symptoms include the appearance of raised, red patches on the skin. Respiratory and
gastrointestinal symptoms, including blood in the sputum and stools, may be present
Membranous glomerulonephritis Disorders associated with membranous glomerulonephritis development include systemic lupus
erythematosus, Sjögren syndrome, secondary syphilis, hepatitis B, gold and mercury treatments,
and malignancy.
Membranoproliferative Marked by two different alterations in the cellularity of the glomerulus and peripheral capillaries.
Glomerulonephritis A. Type 1 displays increased cellularity in the subendothelial cells of the mesangium

B. Type 2 displays extremely dense deposits in the glomerular basement membrane. Many of
the patients are children, and the disease has a poor prognosis
Focal Affects only certain numbers and areas of glomeruli, and the others remain normal.
Symptoms may be similar to the nephrotic syndrome and minimal change disease owing to
damaged podocytes
IgA Nephropathy Patients usually present with an episode of macroscopic hematuria following an infection or
strenuous exercise.
Disorder may remain essentially asymptomatic for 20 years or more

ACUTE AND CHRONIC RENAL FAILURE

Acute renal Characterized clinically by a sudden decrease in the GFR, azotemia, and oliguria
failure (ARF) N
Usually reversible
progressive Primary causes of ARF include a sudden decrease in blood flow to the kidney (prerenal), acute
but glomerular and tubular disease (renal), and renal calculi or tumor obstructions (postrenal)
reversible Ischemic acute tubular necrosis is the most common cause of ARF

CAUSES OF ACUTE RENAL FAILURE

Pre renal Decreased blood pressure, decreased cardiac output, hemorrhage, burns, surgery,
septicemia
Renal Acute glomerulonephritis, acute tubular necrosis, acute pyelonephritis, acute interstitial
nephritis
Post Renal Renal calculi, Tumors
function Damage
I t
Chronic renal Progressive loss of renal function caused by an irreversible and intrinsic renal disease characterizes
failure (CRF). 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

RENAL STONES / RENAL LITHIASIS / RENAL CALCULI

Primary Microscopic Finding: Hematuria

Mostly formed during Summer season
Renal Calculi are found primarily in the renal calyces, pelvis, ureter, or bladder
Calculi may be of various sizes, commonly described as sand, gravel, or stone.

Conditions Favoring/Enhancing Formation of Renal calculi: → age does not affect formation
of kidney Stones
a. Urinary pH
b. Chemical/solute concentration
c. Urinary stasis
d. Metabolic disorders (ex. Gout, and inborn error of metabolism)
e. Endocrine disorders (ex. Hyperparathyroidism)
f. Infections (ex. UTI)
g. Nucleation (initial crystal deposition and formation)

I.K AYTONA 41
 in Alkaline urine :
Compound stone commonly found
→
Triple phosphate

RENAL CALCULI CHARACTERISTICS

calcium oxalate
Major constituent of renal calculi
Very hard, dark brown color with rough surface
Uric Acid Associated with increase intake of foods with high purine content, and uromodulin-associated kidney disease
Yellowish to brownish red and moderately hard → Lesch
Nyhan syndrome
Seen in hereditary disorders of cystine metabolism
cystine Yellow brown, greasy and resembles an old soap
Least common calculi (1-2%)
calcium phosphate 1Apatite ) Pale and friable

ADDITIONAL INFORMATION
1. Calcium oxalate or a mixture of oxalate and calcium phosphate is often found in stones ( 80%). Mixed calcium
phosphate, magnesium ammonium phosphate, and uric acid are the next most common constituents (3% to 10% each), and
these are followed by cystine stones (1% to 2%).
2. Males are more often affected with calcium stones than females, and children are not often affected with calcium stones.
3. Struvite (Triple phosphate) stones may become large, forming casts of the kidney pelvis and showing staghorns
4. staghorn calculi resembling the shape of the renal pelvis and smooth, round bladder stones with diameters of 2 or more inches

TELESCOPED SEDIMENTS → many sediments everytime you urinate

Simultaneous appearance of the elements of acute/chronic glomerulonephritis, ESRD and nephrotic syndrome
A telescoped sediment might therefore include red blood cells, red blood cell casts, cellular casts, broad waxy casts, lipid droplets,
oval fat bodies, and fatty casts
Such sediment may be found in collagen vascular disease (notably lupus nephritis) and subacute bacterial endocarditis.

ATHLETIC PSEUDONEPHRITIS → not all people

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

URINE SCREENING FOR METABOLIC DISORDERS

AMINOACIDURIA
Inborn
error of Metabolism
1. Over Flow Type →
INCREASE Amino acid in blood
INCREASE Amino acid in urine
Ex: PKU, MSUD, Cystinosis, Alkaptonuria
TANDEM MS/MS (Mass spectrometry)
-gold standard test for Newborn Screening
2. Renal Type -Specimen: Bloodspot
NORMAL amino acid in blood
INCREASE amino acid in urine
PCT)
Due to renal tubular defects ( impaired

PHENYLALANINE-TYROSINE DISORDERS
DISORDER Enzyme deficient Information Tests f) follow-up test
Phenylketonuria ____________________
Phenylalanine hydroxylase Mousy or musty odor FeCl3 tube test = (+) BLUE GREEN
- Inherited as an autosomal autosomal recessive trait Urine Phenistix strip = (+) gray to gray green
recessive disease, it is ( Barney odor) Guthrie bacterial inhibition test
characterized by increased urinary
excretion of phenylpyruvic acid (a
-associated with mental Bacillus subtilis is cultured with
ketone) and its metabolites retardation beta2 thienylalanine
inhibitor →
of Beta2-thienylalanine inhibits
blood or urine
.

- use B. subtilis
growth of B.subtilis
inhibitor → Phenylalanine counteracts the
of
Beta 2-
action of Beta2-thienylalanine
diagnosed by : thienylalanim (+) result: _____________
GROWTH
detection of
tyrosine & succinyl acetone genera hired renal tubular
Tyrosyluria/Tyrosinemia Type1: disorder liver failure in FeCl3 tube test = (+) TRANSIENT GREEN
-

T
progressive
-

Fumarylacetoacetate infants

diagnosed by : hydrolase Nitroso-naphtol = (+) Orange Red

testing for plasma tywsintr corneal erosions t lesions palms
as ,

Type 2: fingers soles of feet

- -

t
molecular diagnostic
+ ,

has f 4-hydroxy phenyl

also
Tyrosine aminotransferase
-
px -

pymvic acid
diagnosed by :
intellectual disability seiwires
Type 3: intermittent ataxia
-
, ,

of elevated levels =
t
presence
of plasma tyrosine presence
t

of 4-
hydroxy phenyl pymvicaad p-hydroxyphenylpyruvic
,

t
4- hydroxy phenyl lactic acid
4-
hydroxy phenyl acetic acid
acid dioxygenase
Alkaptonuria Homogentisic acid oxidase Urine darkens after FeCl3 tube test = (+) TRANSIENT BLUE
uit C is used for treatment
.
becoming alkaline from Clinitest = (+) Yellow ppt
standing at room
temperature

I.K AYTONA 42
 Melanuria -- melanoma secrete colorless Due to overproliferation FeCl3 tube test = (+) BLACK /GRAY PPT
Malignant
-

-Melanin is produced from precursor of melanin 5,6 dihydroxy indole

-
of melanocytes Sodium nitroprusside test = (+) Purple
tyrosine by melanocytes and is which oxidizes to milano gen & then to
Urine darkens upon air Ehrlich test (+) = Red
melanin
the pigment responsible for the exposure
color of hair, skin, and eyes.
Deficient production of melanin
results in albinism.
-
Melanoma → A production of melanin

5,6-dihydroxyindole A colorless precursor of melanin, which can be oxidized to melanogen and then to melanin, producing the
characteristic of dark urine.

Alkaptonuria An observation of brown-stained or black-stained cloth diapers and reddish-stained disposable

diapers indicate __________

FERRIC CHLORIDE TUBE TEST

TEST PROCEDURE POSITIVE RESULTS
1. Place 1 mL of urine in a tube. PKU Permanent Blue-Green
2. Slowly add five drops of 10% ferric chloride. Tyrosyluria Transient green
3. Observe color for a color appearance Alkaptonuria Transient blue
Melanuria Gray/Black precipitate

Nitroso-Naphthol Test for Tyrosine Homogentisic Acid Test (silver Nitrate

)
1. Place five drops of urine in a tube. 1. Place 4 mL of 3% silver nitrate in a tube.
2. Add 1 mL of 2.63N nitric acid. 2. Add 0.5 mL of urine.
3. Add one drop of 21.5% sodium nitrite. 3. Mix.
4. Add 0.1 mL 1-nitroso-2-napthol. 4. Add 10% NH4OH by drops.
5. Mix. 5. Observe for black color
6. Wait 5 minutes.
7. Observe for an orange-red color, indicating tyrosine
metabolites.

BRANCHED CHAIN AMINO ACID DISORDER → significant finding presence of ketonuria in :

newborns
Disorder Information Test
Maple syrup urine Disorder Amino acid that Increases in blood and urine 2,4 Dinitrophenylhydrazine (DNPH) in urine -110 gtts
Isoleucine )
]-0-2%2,4 DNPH -

autosomal recessive trait ________________________

LN ( leucine ,
Valine
(+) ____________________________
, Yellow precipitation wait to mins .

Plasma At
testingdetect
assess 9 of Butt

-Caramelized sugar / burnt sugar/ Curry odor / Maple confirmatory : a alto isoleucine

accumulation of isovaleryi glycine due to

deficiency syrup odor urine due to ketoacids in urine MASS
Spectrometry Plasma isoleucine 55mmol /( is most
of
isoualeryl coenzyme A specific t sensitive marker for
Organic acidemias T Msu ☐

1. Isovaleric acidemia → accumulation of

organic acids
2. Propionic acidemia Generalized symptoms of the organic acidemias include
+ 3. Methylmalonic early severe illness, often with vomiting accompanied p-nitroaniline test = (+) Emerald green
errors in the acidemia by metabolic acidosis; hypoglycemia; ketonuria; and
of isoleucine valine , threonine ,
metabolism
methionine to
succinyl
,

coenzyme A
increased serum ammonia

NOTE! In MSUD, the metabolic pathway begins normally, with the transamination of the three amino acids in the liver to the keto
acids (a -ketoisovaleric, a -ketoisocaproic, and a -keto-b -methylvaleric). Failure to inherit the gene for the enzyme necessary to
produce oxidative decarboxylation of these keto acids results in their accumulation in the blood and urine.

I.K AYTONA 43
Second metabolic pathway of tyrosine is responsible for the production of melanin, thyroxine, epinephrine, protein,
and tyrosine sulfate

TRYPTOPHAN DISORDERS
Disorder Information Test
Indicanuria Indigo blue color urine
(upon air exposure or oxidized) - FeCl3 purple

Seen in: Intestinal disorders, and

Hartnup disease → Blue Diaper Syndrome
Argentaffinoma Carcinoid tumor involving argentaffin FeCl3 tube test = (+) blue green
TRYPTOPHAN metaboliud
cells argentaffinoma 525mg / 24h Nitroso-naphthol w/ nitrous acid = (+) Violet
☒rgentaffin cells , serotonin 15 -
HIAA

Foods to avoid :
Produce 5-HIAA, a metabolite of to black 41mL urine
✓ Bananas ✓Chocolate serotonin normal
daily excretion :
24th come - NHYOH
✓ Plum 0.5mi of 590 silver nitrate
✓ Tomato
✓ walnuts Avoid ____________________
z
gang
-

wait to mins
✓ Avocado
✓ pineapples ✓ medications containing guaifenesin add 591--15 sodium nitvopwsside
NOTE! A second metabolic pathway of tryptophan is for the production of serotonin used in the stimulation of smooth muscles.
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 ✓cystinnña
's
legal nitwprusside -
positive for
✓
Cystinosis
CYSTINE DISORDERS ✓ Homocysteine
✓ Ketones
Disorder Information Test
Cystinuria Renal type aminoaciduria nitroprusside
Rgt: Cyanide nitroprusside = (+) Red-purple
Defective tubular reabsorption of:
amounts
1. 3 ml of urine in tube 4 Fgfts sodium .

normal COLA ( Cystine Ornithine Lysine, Arginine) 2.2mi sodium cyanide

→ -
in blood __________________________ , ,
3. wait 10min
hitwpmssidl ,

Cystinosis Inborn Error of metabolism s nitroprusside

nephwpathic
✗
nonnephwpathic(-) gene that codes for an enzyme responsible for
&
Rgt: Cyanide nitroprusside = (+) Red-purple
infantile late
onset cysteine metabolism → only homocysteine
is
positive
Homocystinuria Defects in the metabolism of methionine that leads to Silver-nitroprusside test = (+) Red-Purple
urine 41mL

increase homocysteine. Associated with methionine -Fresh urine should be used Tatts NHYOH
silver nitrate
cone

0.5Mt of 590
.

malabsorption ex
wait to mins
59TH sodium nitwpwsside
,
add

located
primarily ← MUCOPOLYSACCHARIDE DISRODERS → chondroitin sulfate Heparan sulfateKeradermatan
in connective tissue tan sulfate
sulfate
, ,
,

Disorder Information Test

Hurler syndrome Mucopolysaccharides accumulate in the Acid Albumin -

turbidity
scale
cornea of the eye A) white turbidity after zoning

)]
=

" "
skeletal CTA Bin citrate buffer
abnormality 590
bromide CCTAB
Cethyltvimethyl ammonium
[ Sex linked recessive, rarely seen in females
+ -

intellectual
disability -

= ( t ) white turbidity after 5- mins

Hunter syndrome
- meta chromatic staining spot test
4) Blue or Violet
Sanfilippo syndrome Mental retardation only =

T T
1st answer 2nd answer

NICE TO KNOW
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.
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 tumor
If a 24-hour sample is used for 5-HIAA measurement, it must be preserved with hydrochloric or boric acid.
The increased homocystine can result in failure to thrive, cataracts, mental retardation, thromboembolic problems, and death.
False positive results in cyanide nitroprusside test is associated with the presence of ketones and homocysteine
In mucopolysaccharidosis, the products most frequently found in the urine are dermatan sulfate, keratin sulfate, and heparan
sulfate.
up
of serum phenylalanine, the
In PKU, urinary excretion of phenylpyruvic acid may take 2 to 6 weeks before it will occur
The appearance of black urine from a patient of any age should be reported to a supervisor.
Generalized symptoms of the organic acidemias include early severe illness, often with vomiting accompanied by metabolic
acidosis; hypoglycemia; ketonuria; and increased serum ammonia
Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting isoleucine, valine, threonine,
and methionine to succinyl coenzyme
Under normal conditions, most of the tryptophan that enters the intestine is either reabsorbed for use by the body in
producing protein or is converted to indole by intestinal bacteria and excreted in the feces.

PURINE DISORDER sex-linked recessive

Lesch-Hyhan disease →
Lack Hypoxanthine-guanine phosphoribosyl transferase → needed for proper metabolism of urine
Increase uric acid in blood and urine
Patients suffer from severe motor defects, mental retardation, a tendency toward self-destruction, gout, and renal calculi
Development is usually normal for the first 6 to 8 months, and the first sign is often uric acid crystals resembling orange
sand in diapers ( sand diaper )
orange syndrome

I.K AYTONA 44
 → Problem in
production of heme
Disorder of porphyrin metabolism uro porphyrin
most
porphobitinogen
-

Urine color = Red/purple/Burgundy red/ Port wine/ Purplish red ALA , soluble
,

Normal or Colorless in = Lead poisoning Copw

less soluble
in urine
porphyrin
but found -

Screening test Specimen = urine, stool, blood, bile

protoporphyrin not seen in urine
-

a. detects D-ALA porphobilinogen

b. Fluorescence at 550-600nm test for uroporphyrin, coproporphyrin and protoporphyrin
(+) result: Violet/pink/red Fluoresence C-) faint blue fluorescence
-

c. Free Erythrocyte Protoporpyrin (FEP) CDC recommended test for lead poisoning
increased in porphyria
NOTE Lead poisoning inhibits ALA synthase and Ferrochelatase enzymes

Porphyria Elevated Compound(s) Clinical Symptoms Laboratory Testing

Acute intermittent porphyria ALA Porphobilinogen Neurologic/psychiatric Urine/Ehrlich reaction
Porphyria Cutanea Tarda Uroporphyrin Photosensitivity Urine fluorescence
Congenital Uroporphyrin and Coproporphyrin Photosensitivity Urine or feces fluorescence
erythropoietic porphyria
Variegate porphyria Coproporphyrin Photosensitivity/neurologic Bile or feces fluorescence
Erythropoietic Protoporphyrin Photosensitivity Blood FEP
protoporphyria Bile or feces fluorescence
Lead poisoning ALA and Protoporphyrin Neurologic Acetoacetic acid + urine/
Acquired porphyria Ehrlich reaction
Blood FEP

CARBOHYDRATES DISORDER lactation

Melituria = general term for the presence of urinary sugar
Lactosuria pregnancy t -

Fuuetosuvia parenteral feeding

Galactosuria, pentosuria, lactosuria, fructosuria, and glucosuria
-

Puntosuña ingestion of large amounts of fruit

-

Galactosuria = indicates inability to properly metabolize galactose to glucose such as in case of newborn errors
in !
deficiency transferase ( GALT)
i. Galactose -1 -

phosphate uvidyl
2. Ga lacto kinase (GALK)
3. UDP -

galactose 4- epimerase (GALE)

-

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
Latin word for synovia means 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 joint lubrication
Synoviocytes secrete a mucopolysaccharide containing ________________,
hyaluronic acid
and is responsible for viscosity ( approximately 44 of plasma concentration)
Beneficial tests :
FUNCTIONS ✓ WBC count ✓
Crystal examination
1. Lubricates joints ✓ Differential
2. Reduce friction between bones ✓ Gram stain
3. Provides nutrients to the articular cartilage ✓ culture
4. Lessen shock of joint compression occurring during activities such as walking and jogging

SPECIMEN COLLECTION
Arthrocentesis
Method: ___________________
If possible, patients should be fasting a minimum of 4 to 6 hours to allow for the equilibration of chemical constituents
(moistened
)
between plasma and synovial fluid (Brunzel, 3rd ed.) with
collect with :
syringe ,, heparin
Volume: Normal = 0.1 to 3.5 ml, Inflammation = >25 ml
container : Evacuated tube

REQUIRED TUBE TYPES FOR SYNOVIAL FLUID TESTS AND ORDER OF DISTRIBUTION
cellular components + supernatant is used for chemical or
immunologic
1. Chemistry and Serology = non-anticoagulated → observed for clotting centrifuged
analysis
to
,
remove

2. Hematology and cytology = sodium heparin or liquid EDTA → cell count differential count crystal identification
,
or

3. Microbiology= Sterilized heparin or SPS

CHEM
1 I 5

{ I.K AYTONA 45
 COLOR AND CLARITY
Colorless to pale yellow Normal
Turbid WBC, Synovial cell debris, and fibrin
Deeper yellow Inflammation
Greenish tinge Bacterial infection
Red Traumatic tap, hemorrhagic arthritis
Milky Crystals

VISCOSITY
4 to 6 cm long
Normal: able to form a string ___________ GRADING Interpretation
Test: Ropes/ Mucin clot test (Hyaluronate polymerization Test) Good Solid clot
not
→ Reagent: uses 2-5 % acetic acid → promotes polymerization of Hyaluronic
acid
Fair Soft clot
performed routinely
When added to a solution of 2% to 5% acetic acid, Low Friable clot
normal synovial fluid forms a solid clot surrounded by clear fluid Poor No clot

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
The total leukocyte count is the most frequently performed cell count on synovial fluid. Red blood cell (RBC) counts are
seldom requested. To prevent cellular disintegration, counts should be performed as soon as possible or the
specimen should be refrigerated. * Do not fluids that contain acetic acid
use

to dilute
synovial fluids because acetic acid
for lysis of RBCs can mucin clot
clumping
cause formation t cell
WBC COUNT
Most frequently performed count
Diluting fluid: NSS with methylene blue, Hypotonic saline 0.3%), Saline with saponin
Automated cell counters can be used for synovial fluid counts; however, highly viscous fluid may block the apertures, and the
presence of debris and tissue cells may falsely elevate counts. As described previously, incubating the fluid with hyaluronidase
decreases specimen viscosity. Analyzing scattergrams can aid in detecting tissue cells and debris
PMNS Mononuclear cells
f) differentiate
t

Methylene blue added to the normal saline stains the WBC nuclei, permitting separation of the RBCs and WBCs during
counts performed on mixed specimens

For very viscous fluid may need to be pretreated by adding a pinch of hyaluronidase to 0.5ml fluid or add 1 drop of 0.05%
hyaluronidase in phosphate buffer per ml of fluid and utes

CELL COUNT NORMAL VALUES

RBCs <2000 / ul line the
•

petri dish w/ moist paper t place the hemocytometer

ontwo small sticks to elevate it above
the moist
WBCs < 200 / ul Fill
paper
count both sides of the
hemocy meter for compatibility
•
&

WBC differential count Differential counts should be performed on cytocentrifuged preparations or on thinly smeared slides.
> i Neubauer Fluid should be incubated with hyaluronidase prior to slide preparation.
,

Monocyte and macrophages 65 %

Neutrophils <25 %
Lymphocytes <15 %

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 /Inclusion Description Significance
Neutrophil Polymorphonuclear leukocyte Bacterial sepsis
Crystal induced inflammation
Lymphocyte Mononuclear leukocyte Non-septic inflammation
Macrophage Large mononuclear leukocyte, may be vacuolated Normal
Viral infections
Synovial lining cell Similar to macrophage, but may be multinucleated resembling a Normal
mesothelial cell
LE cell Neutrophil containing characteristic ingested Lupus erythematosus
sides
nucleus pushed to
R
Reiter cell Vacuolated macrophage with ingested neutrophils Reiter syndrome
Non-specific inflammation
RA cell (Ragocyte) Neutrophil with dark cytoplasmic granules containing immune Rheumatoid arthritis
Rheumatoid arthritis cell
complexes (consists of precipitated rheumatoid factor ) Immunologic inflammation
Cartilage cells Large multinucleated cells Osteoarthritis

I.K AYTONA 46
 Rice bodies Macroscopically resembles polished rice Tuberculosis, septic and
Microscopically shows collagen and fibrin rheumatoid arthritis
They are fragments of degenerating proliferative synovial
cells or microinfarcted synovium
Fat droplets Refractile intracellular and extracellular globules stain with Sudan Traumatic injury
dyes Chronic inflammation
Hemosiderin Inclusions within clusters of synovial cells Pigmented villonodular synovitis
Ochronotic shards Ground pepper appearance Joint prosthesis, ochronosis

CRYSTAL IDENTIFICATION
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)

Ideally, crystal examination should be performed soon after fluid collection to ensure that crystals are not affected by
changes in temperature and pH
Both MSU and CPPD crystals are reported as being located extracellularly and intracellularly (within neutrophils); therefore,
fluid must be examined before WBC disintegration.
→ CPPD crystals are usually located within vacuoles of the neutrophils while MSU crystals lyse phagosome membranes and
classic rhomboid
shape should be therefore do not appear in vacuoles sticking through cytoplasm of cell
observed & confirmed
w/ red
compensated -stained smears
polarized microscopy 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 = (negative) Birefringence
- - parallel to slow vibration
-

Blue = perpendicular = (positive) Birefringence Bt perpendicular to slow vibration

-

Control slide for the polarization properties of MSU (monosodium urates) can be prepared using
betamethasone acetate corticosteroid.

NOTE!
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 Artifacts talcum powder
-
scratches on slides + cover
ships
starch from gloves
precipitated Anticoagulants
dust

Crystal Shape Compensated Polarized Light Significance

Monosodium urate Fine Needles Negative birefringence Gout
Calcium dihydrate Rhombic squares, rods Positive birefringence Pseudogout or chondrocalcinosis
Pyrophosphate
Cholesterol Notched, rhombic plates Negative birefringence Extracellular, chronic arthritis condition
corticosteroids after
such as Rheumatoid arthritis injections
i

Corticosteroid Flat, variables-shaped plates Positive & Negative birefringence Injections

Calcium oxalate Envelopes Negative birefringence Renal dialysis
Apatite Small particles; requires No birefringence Osteoarthritis
(Calcium Phosphate) electron microscopy
Basic Calcium Phosphate -
calcified cartilage degeneration
Pseudogout Most often associated with degenerative arthritis, producing cartilage calcification and endocrine
disorders that produce elevated serum calcium levels.

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

Normal Value = the difference between the Blood glucose and Synovial fluid glucose should be less than
10mg/dl
BLOOD GLUCOSE S.F GLUCOSE = <10mg/dl (normal)
Decreased in Type II and III arthritis or inflammatory disorder

To prevent falsely decreased values caused by glycolysis, specimens should be analyzed within
1 hour or preserved with sodium fluoride
Lactate Normal value = <250 mg/dl f in septic arthritis caused by gram-positive cocci &
gram-negative bacilli
for normal in N gonorrhea
Increase in infection
.

arthritis
T than 9mmol/L 181 my /44 indicate bacterial

I.K AYTONA 47
 GGT , Adenosine deaminase, muramidase , cytidine deaminase LD , AST
y ALP ,
,

Lactate or ACP Maybe requested to monitor the severity and prognosis of Rheumatoid arthritis
Protein Normal value = <3 g/dl ( approx Yz of serum value) .

Increased in inflammatory and hemorrhagic disorders

Uric acid Normal value = same as blood
Increase in gout

Joint Disorders
Group I. Non inflammatory IIa. Inflammatory IIb. Inflammatory- III. Septic IV. Hemorrhagic
Immunologic origin Crystal induced
Significance Degenerative joint Immunologic Gout Microbial infection Traumatic injury
disorder disorders Pseudogout Coagulation
deficiency
Color and Clear, yellow fluid Cloudy, yellow Cloudy or milky Cloudy, yellow- Cloudy, red fluid
Clarity fluid fluid green fluid
Viscosity Good Poor Low Variable Low
WBC count <1,000 / ul 2000-75000/ul Up to 100,000 /ul 50K-100K / ul Equal to blood
Neutrophils <30 % >50% <70% >75% Equal to blood
Glucose Similar to blood glucose Decreased Decreased Decreased Normal
Others +Auto antibodies + Crystals + Culture and GS + RBC
Associated Osteoarthritis Rheumatoid Crystal synovitis -Bacterial infection, -Trauma
diseases Osteochondritis arthritis, SLE, (gout, pseudogout) -Fungal infection, -Tumor
Osteochondromatosis ankylosing -Mycobacterial -Joint prosthesis
Traumatic arthritis Spondylitis, Lyme infection -Blood disease such
Neuroarthropathy arthritis as sickle cell disease
and hemophilia

MICROBIOLOGY → Gram stain & culture two most

important tests performed
are

Common organisms that infect the synovial fluid: → should include enrichment medium like chocolate Agar
1. Staphylococcus aureus → leading cause of ADULT joint infx
→ PCR -
beneficial for organisms that are hard to detect t culture

2. Streptococcus Borrelia burgdorferi -

Lyme disease arthritis

Mycobacterium tuberculosis osteo articular tuberculosis

3. Haemophilus
-

chlamydia trachomatis t N venereal arthritis

.
gonorrhea →
4. Neisseria gonorrheae

SEROLOGIC TEST
Auto antibody detection for RA, SLE
Detection of antibodies to Lyme disease
Borrelia burgdorferi

SEROUS FLUID
A Fluid between parietal membrane (lines the cavity wall) and
visceral membranes (covers the organ)
Serous fluids are formed from ultra-filtrate of plasma
Production and reabsorption are subject to hydrostatic
pressure and colloidal pressure (oncotic pressure) from the
t
capillaries that serve the cavities and the capillary permeability.

changes to Albumin -
major contributor

FUNCTION
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
Ex Maul
TRANSUDATE EXUDATE
Effusion caused by a systemic disorder that disrupts the fluid Effusion caused by direct damage to the membrane
production and regulation between membranes
Causes: Causes:
1. Hypoproteinemia = decrease oncotic pressure 1. Infection
}
+ increased capillary
salt + fluid
retention
←
2. Congestive heart failure- increase hydrostatic 2. Malignancy permeability
membrane
3. Nephrotic syndrome= decrease oncotic pressure 3. Inflammation
4. Malnutrition= decrease oncotic pressure 4. Lymphatic duct obstruction malignant tumors -
, lymphomas
- infx & inflammation
Protein losing ← 5. Hepatic cirrhosis = decrease oncotic pressure Thoracic duct
injury
-
-

enteropathy

I.K AYTONA 48
 TRANSUDATE VS EXUDATE
Transudate Exudate
most Appearance Clear Cloudy
reliable
Fluid: serum protein ratio <0.5 >0.5
-1£
test
diff in
Fluid: serum LD ratio <0.6 >0.6
.

pleural
fluid
WBC count <1,000 cell/ul >1,000 cell/ul
Spontaneous clotting No Possible
Pleural fluid cholesterol (mg/dl) <45-60 >45-60
Pleural fluid: serum cholesterol ratio <0.3 >0.3
Pleural fluid: bilirubin ratio <0.6 >0.6
F Serum ascites albumin gradient (SAAG) >1.1 <1.1 peritoneal fluid
-
serum Albumin
minus
ascite
Glucose Decrease Increase
Albumin
Negative Positive
Acetic acid + water + Unknown fluid
(+) heavy precipitation
RBC counts < 100,000µL 7100,0001µL
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

METHODS OF COLLECTION (3Ps (Pleural, Pericardial, Peritoneal fluid)

✗ RBC & WBC counts not routinely performed
are

Normal appearance should be clear, pale yellow ✓ Cell counts performed manually using a Neubauer counting chamber or by electronic cell counters

Fluid Aspiration technique (Collection) Organ located

Pleural fluid Thoracentesis Lungs
Pericardial fluid Pericardiocentesis Heart
Peritoneal or ascitic fluid Paracentesis Abdominal organs
✓Differential cell counts are performed routinely on serous fluids
Specimen Distribution
•
preferably Wright 's stained cyto centrifuged spx
, slides
prepared from
or sediment
1. EDTA = Cell count and differential count of centrifuged Spx .

Checked for WBCS normal t

malignant cells
2. Sterile heparin or SPS = Microbiology and cytology •
,

3. Plain tubes or heparin tubes= Chemistry

PLEURAL FLUID -

Appearance Significance
Clear, pale yellow Normal
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 leakage Chronic inflammation
Appearance Milky / white Milky / green tinge / gold paint
Leukocytes Increase lymphocyte Mixed cells
Cholesterol crystals → pseudo chylous Absent Present
Triglycerides → chylous >110 mg/dl <50 mg/dl
Sudan III staining +++ Negative or weakly +
Onset Sudden gradual
Chylomicrons present absent

HEMATOLOGY DIFFERENTIAL COUNT ON SEROUS FLUID → most

diagnostically significant hematology test

CELL REFERENCE VALUE

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

I.K AYTONA 49
 ☒ Malignant cells -
nuclear t cytoplasmic irregularities hyperchromic nucleoli
, ,

cellular
dumps w/ cytoplasmic molding & abnormal nucleus : cytoplasm ratios

* Malignant pleural effusions -

large ,
irregular adenocarcinoma cells small ,
or oat cell carcinoma cells

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 sarcoidosis ,

Mesothelial cell single small

large round cells
,
Normal and reactive forms have no significance
or c-
w/ cytoplasm round
abundant blue & ✗ cluster have
w/ uniform varying amounts of cytoplasm
(Characterized by fried egg appearance) purple cytoplasmDecrease in TB ← exudate
nuclei dark
covering the pleural
, ,

membranes
eccentric t
prominent nucleoli multinucleated nuclei
,

Plasma cells TB ( increased in plasma cells )

Malignant cells Primary adenocarcinoma, small cell carcinoma
Eosinophil Allergic reaction, parasitic infection, pneumothorax, and hemothorax
LE Cells
Systemic Lupus Erythematosus
parallel plasma
levels
c. 60
SIGNIFICANCE OF CHEMICAL TEST IN PLEURAL FLUID
mg loll C
ist
Glucose → most chemical test
common
Decrease in rheumatoid inflammation, purulent infection TB Malignant effusion esophageal rupture lupus plenritis , , , ,

Lactate Increase in bacterial infection

Triglyceride → confirm Increase in chylous effusion
of
presence
3
effusion
collected
anaerobically
chylous / 2
using hepariniudPh → most chemical test
common Decrease in pneumonia not responding to antibiotics, esophageal rupture, complicated
syringe &

pit should be compared parapneumonic effusion (empyema)

.

measured in
blood
in acidosis → pleural Microbiological
gas w/ blood pH ✓ 5 Enterobacteriaceae anaerobes
*Ph less than 7.2= need for test tube drainage
aureus ,
analyzer @
.
, ,

M tuberculosis
37°C → pleural fluid
pH at least 0.30 .

✓ PCR is sensitive test to detect

*Ph less than 6 = esophageal rupture allowing the influx of gastric fluid
a more
degrees lower significant
is
M tuberculosis .

Adenosine deaminase tuberculosis, malignancy

serological Test
→ most chemical test
common
-ADA levels higher than 40 U/L are highly indicative of tuberculosis. ✓ differentiate effusions from immunologic &

Amylase → most chemical test

common
increase pancreatitis, esophageal rupture, malignancy noniflammatay processes
✓ Antinuclear
antibody ( AWA ) t Rheumatoid factor ( RF)
elevated first in
pleural fluid performed frequently
are

✓tumor markers carci embryonic antigen (CEA)

PERICARDIAL FLUID
,
CAR 5C metastatic uterine
Milky -

chylous t
pseudochylous cancer )
CA 15.3,
,
CA 549 (breast cancer )
CYFRA 21-1 ( lung
APPEARANCE turbidity -
infxt malignancy SIGNIFICANCE ,
cancer )
Clear, pale yellow Normal, transudate
Blood-streaked , turbidity Infection, malignancy
Grossly bloody Cardiac puncture, anticoagulant medications
DIFFERENTIAL SIGNIFICANCE
Increase neutrophils (>1000 WBCs/ul) Bacterial endocarditis
Malignant cells Carinoembryonic Ag
,
Metastatic carcinoma
TEST SIGNIFICANCE
Gram stain and culture Bacterial endocarditis
Acid fast stain Tubercular effusion
Adenosine Deaminase Tubercular effusion

Normally, only a small amount (10 to 50 mL) of fluid is found between the pericardial serous membranes
Metabolic disorders such as uremia, hypothyroidism, and autoimmune disorders are the primary causes of transudates.
An effusion is suspected when cardiac compression (tamponade) is noted durin
peritonitis endocarditis pancreatitis, appendicitis fweutropwil , ,
-

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 ( lymphocytes predominant cell in TB t peritoneal carcinomatosis)
are

DIFFERENTIAL SIGNIFICANCE
Increase neutrophils Bacterial peritonitis
Malignant cells Malignancy
TEST SIGNIFICANCE
Peritoneal lavage >100, 000 RBCs/ ul indicates blunt trauma injury (intra-abdominal bleeding)
normal saline introduced
✗ te placed
focused assessment w/ sonography for trauma ( FAST) t
in
peritoneal lavage TEST computed by SIGNIFICANCE
tomography CCT)
sew CEA Malignancy of GI origin
CA 125 Malignancy of ovarian origin
Glucose Decrease in tubercular peritonitis, malignancy
Amylase Increase in pancreatitis, GI perforation Alkaline phosphatase G1 perforation -

BUN/ Creatinine Ruptured/ punctured bladder Bilirubin measured when leakage of bile into peritoneum is suspected after trauma
-

or

surgery
Gram stain and Culture Bacterial peritonitis
micro
Acid fast stain Tubercular peritonitis
ADA Tubercular peritonitis
intestinal perforation or -
most frequent causes

ruptured appendix + malignancy of exudative fluids

I.K AYTONA 50
 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. stabilize 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.
Third trimester
peak :

Normal volume range 800-1200 MI

Average normal volume 1000 Mt

POLYHYDRAMNIOS OLIGOHYDRAMNIOS
Increase amniotic fluid volume Decrease amniotic fluid volume
a. Decrease fetal swallowing a. Increase fetal swallowing
b. Neural tube defects (Ex. Spina bifida and anencephaly) b. Membrane leakage
CNS abnormal bite
c. Urinary tract deformities

SPECIMEN COLLECTION
AMNIOCENTESIS- term for the collection of amniotic fluid
A maximum of 30 mL of amniotic fluid is collected in sterile syringes. The first 2 or 3 mL collected can be contaminated
by maternal blood, tissue fluid, and cells and are discarded.
2ND trimester amniocentesis: Assess genetic defects (e.g., Down syndrome)
3rdtrimester amniocentesis: Assess Fetal lung maturity and Hemolytic disease of newborn
Nitramine

f
Normal vaginal pH Amniotic Fluid pH test
drop of
vaginal fluid + strip containing nitramine dye → blue if pH is > 6.0

4- 5 to 6.0 7. I -7.3 suggesting ruptured

membrane
Immunoassay insulin-like
Action prom - POCT that detects
growth placental -
✗ -

globulin
macro
ICPAMG -1 ) -
1000 -10,000 ✗ higher in amniotic fluid than in
factor binding protein l ( IGFBP 1) , also referred as
- - tmnisure Rom Test)
vaginal fluid
placental protein 12 ( PPR) normal 10,500 -350,000 ng 1mL
-

normal -
0.05 to 0.22
mg / me
Rom Plus detects both d- abnormal 3 my 1mL vaginitis
fetopwtcint / GFBP-1 → serum
- -
-

/MI
normal-2,800 -
U
normal : 55-242
V6 , 000 ng 1mL ( 33 -290
nglml)
2000 -

25000mg 1mL PROM

-

I.K AYTONA 51
SPECIMEN HANDLING

Test for fetal lung maturity Should be placed in ice for delivery to the laboratory and kept refrigerated.
Filtration prevents loss of lung surfactants such as phospholipids.
Test for cell culture and cytogenetic studies Maintained at room temp or body temp
Test for HDN OD 450 Specimen must be protected from light. This can be accomplished by placing the
specimens in amber-colored tubes, wrapping the collection tube in foil, or by use of a
black plastic cover for the specimen container
16th wk -
chromosome analysis
near end of 2nd trimester intrauterine
-
growth retardation
later in 3rd trimester - fetal distress
INDICATIONS OF PERFORMING AMNIOCENTESIS
14th In general, amniocentesis is a safe procedure, particularly when performed after the __ week of
gestation
15 to 18 week
th th -for fetal genetic assessment or genetic abnormality
amniocentesis -for women with three or more miscarriage
-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 weeks amniocentesis -for HDN, Fetal distress, fetal lung maturity, and infection

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
Used to evaluate premature rupture of the membranes

- fluid is amniotic fluid

due to &
protein NaCl

CREATININE LEVEL
Creatinine level
= provides a mean of determining fetal age
= creatinine level that is greater than 2mg/dl indicates fetal age greater than 36 weeks

AMNIOTIC FLUID COLOR

Colorless Normal
Blood streaked Traumatic tap, abdominal trauma, intra amniotic hemorrhage → use Neibauer betke
Yellow HDN Bilirubin
Dark green Meconium
Dark red brown 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 → measured by spectrophotometer → Spx must be covered at all times
Absorbance of amniotic fluid: → as little as 30 mins of exposure to
Normal = increase at 365nm, decrease at 550nm bilirubin
HDN = Increase at 450nm (maximum bilirubin absorption)
light , Marky decreases

Results are plotted on Liley graph:

Zone 1 Non affected or mildly affected fetus → ABO HDN
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 450

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

I.K AYTONA 52
 AFP major protein produced by fetal liver during early gestation ( beforeWks
-

TEST FOR NEURAL TUBE DEFECT 18 )

Neural tube defects, also known as spinal dysraphisms, are a category of neurological disorders related to malformations of
the spinal cord, such as spina bifida, anencephaly, meningocele, myelomeningocele and tethered spinal cord syndrome of
the developing fetus Fetal products maximal AFP between 12 & 15 wks gestation
'

AFP(alpha feto protein)

Screening test = ___________________ Down syndrome ↓ AFP =

Confirmatory = ___________________
Acetylcholinesterase Ache
↑ not perform
more
specific (do em
bloody Spx bee .
blood contains Ache)
FETAL LUNG MATURITY TEST
- Respiratory distress syndrome (RDS) is the most frequent complication of early delivery and is the seventh most
common cause of morbidity and mortality in the premature infant. It results from insufficient production of lung
surfactant
- Lung Surfactants act in two ways: They alter(decrease) the surface tension of the alveoli, preventing their collapse during
expiration, and they reduce the amount of pressure needed to reopen them during inspiration.

TEST INFORMATION
Lecithin / Sphingomyelin Reference method (gold standard )
ratio primary component of surfactants
Lecithin for alveolar stability
→ Produced @ constant rate after
about 26 weeks ' gestation
Sphingomyelin serves as a control
specimen preparation
:

Filtration
lecithin are
concentration increases while the sphingomyelin concentration remains constant
Measured using Thin Layer chromatography (TLC)
Ratio of > 2.0 = mature lungs
Cannot be done on specimen contaminated with blood (false increase) or meconium
(false increase value) -Strasinger 6th edition

Amniostat FLM Immunologic test for Phosphatidyl glycerol

etc )
Immunoassay ( ELISA ,
.

It based on agglutination of PG using polyclonal anti-PG antibodies; qualitative results

↑ PG mature lungs
Results are reported as negative (immature), or as low positive (mature) or high positive
-

✓ blood & meconium contamination (mature), based on the size of agglutinates and the degree of background clearance
is okay Not affected by blood or meconium
✗ not performed when Mother has Production of PG is delayed among diabetic patient
gestational diabetes
Considered as reference method (not primary , but alternative )
→ detected after 35 weeks
gestation
Foam stability / Shake Amniotic fluid + 95 % Ethanol --- shake for 15 seconds --- stand for 15 minutes
test → not used for spx w/ (+) Result = foam/bubbles/effervescence = mature lungs
0.5mi Amniotic fluid -1 gradient of Ethanol/fluid ratios
ranging
meconium & blood Mod
increasing amounts of 95% ETOH providing
: a

rangingfrom 0.42 -100.55mi in increments of 0.01mi * A Value Of 47 Indicates FLM

Microviscosity testing The presence of phospholipids decreases microviscosity
↓ polarization -
mature
lungs Measured by fluorescence polarization
↑ polarization / fluorescence immature
No longer used
-

lungs

Lamellar body count Lamellar bodies are densely packed layers of phospholipids that represent a storage form
1.7 -
7.3ft or I -

5µm of pulmonary surfactant.

blood false increase CBC They are secreted by the type II pneumocytes of the fetal lung @ about 24 wks'
gestation
due to platelets & false '

Specimens may be stored at 2°C to 8°C but never frozen enter amniotic fluid @ 26 wks
decrease due to entrapment of gestation
LBC in fibrin strand Uses hematology analyzer for counting f size of platelet to be counted in
analyzers :
They have similar size with platelets when counted
2- 20 Fem to liters

Mucus t meconium falsely

increase CBCS 32,000 / ml lamellar body count = adequate FLM
The advantages of lamellar body counting include: (1) rapid turnaround time, (2) low reagent
350,0001mL FLM
-

immature cost, (3) wide availability, (4) low degree of technical difficulty, (5) low volume of amniotic
< 15,0001mL
-

fluid required, and (6) excellent clinical performance.

OD 650nm Specimens are centrifuged at 2000 g for 10 minutes and examined using a
(Lamellar bodies) wavelength of 650 nm ☆ Rules out interference from Hgb
Specimen preparation :
Increase lamellar bodies = Increase O.D absorbance but hot meconium

centrifugation

I.K AYTONA 53
 SUMARRY OF TESTS FOR WELL BEING AND MATURITY
Test Reference values Significance
Bilirubin scan OD 450 >.025 Hemolytic disease of the newborn
Alpha feto protein <2.0 MoM (Multiples of median) Neural tube disorders
Lecithin sphingomyelin ratio Fetal lung maturity
Amniostat fetal lung maturity Positive Fetal lung maturity/phosphatidyl glycerol
Foam Stability Index Fetal lung maturity
Optical density 650 nm Fetal lung maturity
Lamellar body count Fetal lung maturity

ADDENDUM
Fetal epithelial cells A cell that indicate the genetic material and biochemical substances from the fetus that is
found in amniotic fluid. These cells can be separated, cultured and can be analyzed and
examined for chromosomal abnormalities thru karyotyping, FISH, fluorescent mapping
spectral karyotyping (SKY), and DNA testing.
Spectrophotometer Amniotic fluid bilirubin (O.D 450) is measured by ___using serial dilutions
Centrifugation at 2000rpm for 10minutes What is the Specimen preparation prior to measuring Lamellar bodies?

CEREBROSPINAL FLUID
The 3rd Major body fluid
1st - Blood
2nd Urine
FUNCTION
-

1. Supply nutrients to the CNS

2. Remove metabolic waste → where CSF flows
3. Produce a mechanical barrier to cushion the brain and
spinal cord against trauma

MENINGES
Lines the brain and spinal cord and composed of three 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
- The choroid plexuses are capillary networks that form the
CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion. Therefore,
the chemical composition of the CSF does not resemble an ultrafiltrate of plasma
- 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

Collection Lumbar puncture/tap (between the 3rd, 4th or 5th lumbar vertebrae) and cisternal puncture or
spinal tap
should be in
px
fetal / shrimp
C , S or
Before CSF is collected, the pressure should be between 90 and 180 mmHg; this is measured by allowing fluid
position
to rise in a sterile, graduated manometer
Volume collected Up to 20ml collected under normal pressure (approximately 15% of the estimated total CSF volume).
-When pressure is normal, 20 mL of specimen may be removed. On closing, the pressure should be between 10 and 30 mm
Hg. A marked decrease in pressure following this procedure suggests cerebellar herniation or spinal cord compression; thus,
no additional CSF should be collected

If the CSF pressure is less than or greater than normal, only 1 to 2 mL should be removed.

Not more than 2 ml can be removed when the pressure is greater than 200 mm Hg

I.K AYTONA 54
 Normal CSF Volume Adult: 90 to 150ml (adults), 10 to 60ml (neonates)
Distribution
TUBE # LAB SECTION STORAGE TEMPERATURE
1 Chemistry, Immunology, Serology Frozen
cells must be counted
* 2 Microbiology Room Temperature
w/in 1hr of collection

when is maintained 3 Hematology (Cell count), and Cytology studies Refrigerated Cup to 4 hrs)
spx .

@ room temperature 4 1st

→
Microbiology (Better exclude skin contamination)
answer
Depends ( RT if Micro ,
Frown if Seno)

or Serology (for additional serologic test)

1 tube only Micro Hema Chemistry/Serology

NOTE: Excess CSF should not be discarded and should be frozen until there is no further use of it.

APPEARANCE CLINICAL SIGNIFICANCE

CRYSTAL CLEAR Normal
Cloudy, Turbid, Milky Increase WBC that is greater than 200 /ul
Increase RBC that is greater than 400/ ul
Presence of microorganism
Increase Proteins and Lipid

Xanthochromic -Pink: Slight amount of oxyhemoglobin

> > colors -Yellow: Bilirubin FBI associated with Kern icterus
many
-Orange: In case of heavy hemolysis
associated w/ intracranial -Brownish: Methemoglobin formation
hemorrhage
OTHER CAUSES:
Increase dietary carotene
Increase rifampin intake → red-orange
Increase melanin
Normal neonate
Protein concentration exceeding 150mg/dl
Previous traumatic tap

Bloody / grossly bloody Greater than 6000 RBCs / ul due to:

no RBCs -
normal Traumatic tap
Intracranial hemorrhage / cerebral hemorrhage
Oily Radiographic contrast dye
Clotted Protein and clotting factors
Meningitis, Froin syndrome
Pellicle Tubercular meningitis
I
sample ref .
for 24
hrs of observe for
pellicles

progressively →

less bloody

Information Traumatic Tap ( improper collection) Intracranial Hemorrhage ( pathologic)

Distribution of blood in 3 tubes Uneven I> 253 Even 1--2=3

Clot formation Clot Negative

Supernatant Clear Xanthochromic → fluid
centrifuged in a

micro hematocrit tube t

Erythrophages ( macrophages w/ ingested RBCs) supernatant examined
against
D-dimer test through latex agglutinationimmunoassay a white background

I.K AYTONA 55
 CSF CELL COUNTING
CSF CELL COUNT corrects WBC counting t protein
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

CSF DILUTION Henry 's Brunel

Appearance Dilution
Clear Undiluted

Slightly hazy 1:10

Hazy 1:20

Slightly Cloudy 1:100

Cloudy/ Slightly bloody 1:200

Bloody/Turbid 1:10,000

WBC COUNT → most important cell count

Routinely performed on CSF
WBC diluting fluid
-3% glacial acetic acid (HAc) with Methylene Blue (Provides better differentiation between PMNs and Mononuclear cells)

Normal values: # cells counted dilution factor

✗
Adults = 0-5 WBCs/ ul
Neonates = 0-30 WBCs/ul total area counted ✗ 0 - I

To prepare a clear specimen that does not require dilution for counting, place four drops of mixed specimen in a clean tube.
Rinse a Pasteur pipette with 3% glacial acetic acid, draining thoroughly, and draw the four drops of CSF into the rinsed
pipette. Allow the pipette to sit for 1 minute, mix the solution in the pipette, discard the first drop, and load the
hemocytometer. -Strasinger
To enhance the visualization of white blood cell nuclei and to eliminate any RBCs present, the CSF is treated with glacial acetic
acid before the hemacytometer chambers are filled, and 3 to 5 minutes is allowed for RBC lysis. - Brunzel

WBC DIFFERENTIAL COUNT

Performed on a stained smear commonly used
Specimen must be concentrated prior to preparation of smear that can be achieved through cytocentrifugation (Most
preferred and widely used), centrifugation, filtration, or sedimentation
For centrifugation, the specimen is centrifuged for 5 to 10 minutes, supernatant fluid is removed and saved for

When the differential count is performed, 100 cells should be counted, classified, and reported in terms of percentage. If the
cell count is low and finding 100 cells is not possible, report only the numbers of the cell types seen.
When a differential cell count is performed using a cytospin slide or concentrated smear, the entire slide should be scanned
first using a low-power objective (10×). This scan will provide an overview of the cellularity of the specimen and will aid in
detecting abnormalities such as plasma cells, macrophages, hemosiderin-laden macrophages, malignant cells, or cell clumps
that can be few in number. Next, the differential cell count is performed using a 50× or a 100× oil objective

slowest
type ofcentrifuge
CSF CYTOCENTRIFUGATION
Procedure Spinal Fluid is added to the conical chamber, and as the specimen is centrifuged, cells present in the fluid are
forced into a monolayer within a 6-mm diameter circle on the slide. Fluid is absorbed by the filter paper blotter,
producing a more concentrated area of cells.
30% Albumin Addition of ___ to as little as 0.1mL of CSF can produce and adequate cell yield when processed with the
Cytocentrifuge. Adding albumin increases the cell yield and decreases the cellular distortion frequently seen
on cytocentrifuged specimens. PX CSF +
Control slide A daily control slide for bacteria should also be prepared using 0.2 mL saline and two drops of the
30% albumin
Recovery Chart NUMBER OF WBCs Counted in Number of Cells on Cytocentrifuge
Chamber Slide
0 0-40
1-5 20-100
6-10 60-150
11-20 150-250
20 250

I.K AYTONA 56
NORMAL WBC IN CSF
ADULT: 70% lymphocytes, 30% monocytes
Neonate: up to 80% monocytes

Currently, with the increased cell recovery obtained using cytospin preparations, neutrophil counts of less than 10% are
considered normal Brunzel

*Sundin parin po si stasinger na significant ang findings pag may neutrophil

RED BLOOD CELL COUNT

Not routinely done
Can be calculated by subtracting WBC count from Total Cell count
Done only in case of traumatic tap to correct Total Protein and WBC count

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

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

CELL TYPES AND CAUSES OF CSF PLEOCYTOSIS

Predominant Cell type Causes
Lymphocytes Normal, Viral meningitis, tubercular meningitis, fungal meningitis, Multiple sclerosis,
Guillain-Barré syndrome, Lymphoma HIV AIDS , ,

Monocytes Normal, Viral meningitis, tubercular meningitis, fungal meningitis, Multiple sclerosis, tumors
Neutrophils Bacterial meningitis, Cerebral hemorrhage, Amebic encephalomyelitis, Cerebral abscess, Repeated
lumbar puncture
Macrophages Tubercular meningitis, fungal meningitis, In response to RBCs and Lipids in spinal fluid, radiographic
Contrast media, Brain irradiation
Blasts Acute leukemia, lymphoma
Malignant cells (astrocytomas, retinoblastomas, and medulloblastomas) Metastatic carcinomas
Ependymal cells lining of ventricles - t neural
* Fusing of cell walls and nuclear irregularities and hyperchromatic nucleoli are seen in canal
-
less defined cell membranes t
clusters of malignant cells. Choroid -
from epithelial lining of choroid plexus in clusters
single in
Ependymal, choroidal, Diagnostic procedures clumps or nucleoli
present
- -
are
nucleoli absent t
-
are have uniform appearance
spindle shaped cells lining of
-

cells
and spindle shaped cells They are non- pathologically significant cells
-

arachnoid
in clusters w/ systemic
Plasma cells Multiple sclerosis, Lymphocyte reactions
-
t seen

malignancies
Eosinophils Parasitic infx , fungal infx (
primarily Coccidioidesimmitis) introduction of
,
foreign material including medications + shunts

CHEMICAL ANALYSIS OF SPINAL FLUID

CSF PROTEIN most
frequently test performed
→
on CSF

Normal values Adults = 15 45 mg 1dL

- < 190 of total protein in blood

Increased in Damage to the BBB

Meningitis
Hemorrhage

Increase Antibody production

Multiple sclerosis

OTHERS
Uremia, Diabetes, Cushing disease, CNS tumor, Myxedema, Polyneuritis, Connective tissue
disease, Neurosyphilis, Guillain Barre syndrome
Decreased in CSF leakage/trauma, water intoxication, Rapid CSF production, Recent puncture
Major CSF Protein HKA Albumin
2nd most prevalent Transthyretin → Pre albumin majority of Pre albumin goes to
-
CSF

Alpha globulin Haptoglobin, ceruloplasmin

Beta-globulins ______________________________
Tau Protein / Beta 2- Transferrin → Identies
-
CSF sample

Carbohydrate deficient transferrin Transferrin -

major B globulin present
Found on CSF not in serum
Gamma globulins IgG and some IgA
Not found in normal CSF IgM, Fibrinogen, Lipids

I.K AYTONA 57
 CSF PROTEIN DETERMINATION
Turbidimetric ______________________
Trichloroacetic acid
I Preferred method, it precipitates both albumin and globulin
in
nephelomety two most
automated instruments
commonly
used
______________________
SSA
manual It Precipitates albumin only
hntd of CSF
protein To precipitate globulins, add sodium sulfate
Determination
Dye binding Uses __________________________
Coo massie Brilliant Blue

CSF /Serum Albumin Assess the integrity of the blood brain barrier
Index

Normal value = <9

Abnormal value = >9
Correlates the degree of damage
Index of 100 = complete damage to BBB
IgG Index Assess condition with IgG production within the CNS (Multiple Sclerosis).
Indicative of IgG production within the CNS. autoimmune disorder of CNS
(f) Anti -

myelin sheath Ab

Normal value = <0.70

Abnormal value = >0.70

inflammation

regent
n
w/ in CNS

CSF ELECTROPHORESIS f) detect monoclonal gammopathy such as Multiple Myeloma

Done in conjunction with serum electrophoresis.
For the detection of oligoclonal bands. → OB MSNt located in gamma region of protein electrophoresis
The presence of 2 or more oligoclonal bands in CSF but not in serum is valuable for the diagnosis of:
Multiple sclerosis → T CSF protein T IgG index # oligoclonal band & Myelin basic protein
, ,

Neoplastic disorders
Encephalitis
Guillain-Barre syndrome
* Neuro syphilis

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 demyelination →

Used to monitor course of _____________________

multiple sclerosis
MBP may also provide a valuable measure of the effectiveness of current and future treatments
Immunoassay techniques are used for measurement

CSF GLUCOSE
Determination Done in conjunction with blood glucose.
Specimen for blood glucose should be drawn 2 hours prior to spinal tap
Normal values 60 to 7090 Of blood glucose
Increased Due to increase plasma glucose (not significant)
Decreased in Bacterial, Fungal, and Tubercular Meningitis
Normal in Viral Meningitis

CSF LACTATE
CSF lactate levels can be a valuable aid in diagnosing and managing meningitis cases
Normal value: 10-22 mg/dl (1.1 to 2.4 mmol/L)
Tissue destruction within the CNS owing to oxygen deprivation (hypoxia) increases CSF lactic acid levels. Therefore, elevated
CSF lactate is not limited to meningitis and can result from any condition that decreases oxygen flow to the tissues
Inversely proportional to glucose
CSF lactate levels remain elevated during initial treatment of meningitis but fall rapidly when treatment is successful, thus
offering a sensitive method for evaluating the effectiveness of antibiotic therapy.
Frequently CSF lactate levels are used to monitor patients with severe head injuries
RBCs contain high concentrations of lactate, and results that are falsely elevated may be obtained on xanthochromic or
hemolyzed fluid

Value
Bacterial meningitis Increased (>35mg/dl)
Fungal and tubercular meningitis Increased (>25mg/dl)
Viral meningitis Normal (remains lower than 25mg/dl)

I.K AYTONA 58
CSF GLUTAMINE
Product of ammonia and alpha ketoglutarate
Indirect test for the presence of excess ammonia in CSF
This is preferred over the direct measurement of CSF ammonia because the glutamine concentration remains more stable
than the volatile ammonia concentration in the collected specimen.
Normal value: 8 to 18 mg/dl
/coma (more than 35 mg/dl)
→ liver disorders

CSF ENZYMES

Lactate dehydrogenase CK BB -

LD1 and 2 = Brain tissue - elevate in

px w/ post cardiac arrest indicate
LD2 and 3 = Lymphocytes a
poor prognosis
LD4 and 5= Neutrophils levels less than
17mg 1dL predict recovery
-

Serum LDH
Normal = LD2>1>3>4>5
→
AMI Microbiology tests :

Flipped pattern = LD1>2>3>4>5 ☒ Renal Infarction

✓ take anywhere from 24 hrs in bacterial
meningitis
can
Hemolytic Anemia to
6 weeks in tubercular meningitis
CSF LDH
✓ Gram stain : concentrate
spx
-
centrifuge @ 1500 for 15 mins
Normal = LD 132737475
Neurological abnormalities= LD 2 > I
Bacterial meningitis= LD 5747372 > I

TYPES OF MENINGITIS
Bacterial Viral Tubercular Fungal
Predominant WBC Neutrophil Lymphocyte Lymphocyte and Monocyte Lymphocyte and Monocyte
Protein Increase Increase Increase Increase
Glucose decrease normal decrease decrease
Lactate Increase normal Increase Increase
Other information + gram stain Caused by Agent: mycobacterium Agent: Cryptococcus
+ culture smallest RNA virus tuberculosis neoformans
+limulus lysate test such as
Ex. Picornavirus +AFB stain + gram stain= classic
-Group B streptococcus , coxsackievirus, +pellicle/weblike clot starburst pattern
-H.Influenzae echovirus and formation after 12-24 hr + India ink
-N.Meningitidis poliovirus refrigeration +immunologic test for
-S.pneumoniae C.neoformans
-enterovirus

LIMULUS LYSATE TEST

Detects gram negative bacterial endotoxin carrying
p oxygen
Reagent: blood of horseshoe crab (blue blood due to
copper in hemocyanin)
Positive: clumping or clot formation

SEROLOGIC TESTING
Latex agglutination test and ELISA- for detection of bacterial antigens
CSF VDRL= recommended by CDC for detection of ______________________
Neurosyphilis

ADDENDUM
4 to 8 weeks Erythrophages can persist for ___ following a hemorrhage; they stain positive for hemosiderin and may
include hematoidin crystals.
2 hours RBCs must usually remain in the CSF for approximately______ before noticeable hemolysis begins
To differentiated PMNs What is the purpose of adding Methylene blue on the diluting fluid for CSF count?
and Mononuclear cell
Nucleated RBCs A Neutrophil found in CSF with PYKNOTIC nuclei may indicate a degenerating cell and can be mistaken as
Turbidimetry (SSA and TCA method) AND The two most routinely/commonly used manual
Dye Binding method (uses Coomassie brilliant blue) techniques for measuring total CSF protein
Gamma region Presence of oligoclonal band in CSF electrophoresis can be located at what region?
Silver staining In CSF electrophoresis by either IFE (Immunofixation electrophoresis) or IEF (Isoelectric focusing), what
staining technique is usually employed?

CSF Lactate It can be frequently tested on CSF sample to monitor severe head injuries.
Falsely elevated Xantochromic or Hemolyzed CSF can lead to ___________ CSF lactate values

I.K AYTONA 59
 SEMINAL FLUID

Reasons for Semen analysis

1. Fertility testing
2. Post vasectomy semen analysis
3. Forensic analysis

contains
seminiferous
tubules

Composition of semen
5% Spermatozoa Seminiferous tubules
meiosis
type of cellular division approx
-

spermatozoa }
pro tails

spermatogenesis ^°dM
'
" °

_____________________
site of spermatogonium →
lot 2°
2. spermatocytes → 3.
spermatids → 4.

_____________________
Sertoli cells known as nurse cells that supports the developing sperm
-

Epididymis → where spermatids migrate

_____________________
site of maturation

60-70 % Seminal fluid Produced by the Seminal vesicles

majority of semen Provides nutrients for sperm and fluid
Rich in fructose for sperm motility
20-30 % Prostate fluid Acidic fluid
Contain, ACP, Zinc, Citric acid, and other enzymes
For coagulation and liquefaction
5% Bulbourethral fluid Thick alkaline mucus
Neutralizes acidity from the prostatic secretion and vagina

SPECIMEN COLLECTION
1. Abstinence of 2- _________________________
3 days but not → prolonged abstinence : increase semen volume t decreased motility
longer than 7 days
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 ( WHOLE)
Methods: masturbation, coitus interruptus, condom method use a non-spermicidal, non-lubricant containing rubber or
least preferred
silastic condom Record :

✓ Px name t DOB
4. Specimen should be delivered to the laboratory within ____ 1 hour of collection at room temp ✓ Period of abstinence
✓
Completeness of Spx
5. Take note of the time of specimen collection, specimen receipt and liquefaction ✓ Difficulties w/ collection
6. Analysis should be done after liquefaction (usually _______ 30-60 minutes) ✓
Spx receipt .

7. If after 2 hours if the specimen has not liquefied, add -buffered saline, alpha chymotrypsin, or bromelain
to induce liquefaction
8. Specimen awaiting analysis should be kept at ____ 37°C

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 ejaculate missing Decrease sperm count, Increase PH, Specimen will not liquify
Last portion of ejaculate missing Increase sperm count, Decrease PH , Specimen will not clot, Decrease semen volume
Blue to yellow fluorescence
due to flavin MACROSCOPIC EXAMINATION
under uv light
( Wood 's lamp)
Appearance Gray- white, translucent, with musty or bleach odor =______________________
Normal
Increased white turbidity =______________________
Associated W/ Infection ( presence of WBC /pus)
Red or Brown coloration =______________________
presence of RBC
Yellow coloration =______________________
Prolonged abstinence urine contamination , medication or

Volume Normal = 2 to 5 ml
Increased = Prolonged Abstinence
Decreased = Infertility / Impotent
Improper or
Incomplete collection

I.K AYTONA 60
 f viscosity =L sperm motility
semen
Viscosity Normal = pour in droplets
Reporting: NOTE Droplets that form threads longer than 2 cm are
watery
0 = ____________ considered highly viscous and are recorded as abnormal
Get like / does not liquefy
4 = ____________
-

Ph Normal = __________
7- 2 to 8.0
measured w/in 1hr
w/in the reproductive tract
Increase = ___________
infx
.

of ejaculation
Decrease = increased prostatic fluid, ejaculatory duct obstruction, or poorly developed seminal vesicles

SPERM CONCENTRATION
Normal value = 20-250 million/ ml
Borderline = between 10-20 million/ml
Methods:
1. Improved Neubauer counting chamber common ✓

Dilution: 1:20 using a mechanical (positive displacement) pipette

Diluents: Formalin, Sodium bicarbonate, saline, distilled water
Purpose of diluents: To immobilize the sperm

2. Makler Counting chamber

For undiluted specimen
Uses heat to immobilize sperm cells

Formula for sperm concentration

2 WBC squares -_ 2

5 RBC 0.2
squares =
Always remember to convert your answer to sperm / ml (milliliters)
Both sides of the hemocytometer are loaded and allowed to settle for 3 to 5 minutes; then they are counted, and the counts
should agree within 10%

PRACTICE SOLVING
200 total sperm cells counted using a 1:20 dilution in the 5 RBC squares. What is the sperm concentration?
shortcut mtd
conc
: for 1 20 dilution only
sperm
.

200 ✗ 20 4000 for every 2 WBC cells ✗ 1001000

200,000µL 200,000,0001mL squares = sperm
i.
= = X 1000 =

0.2 ✗ 0 l -

0.02 z .for ever 5 RBC squares = sperm UHsilmiHion_

no thud to

multiply by low

to get me

SPERM COUNT
Normal value = > 40 million sperm / ejaculate
Formula: Sperm concentration x Specimen volume

SPERM MOTILITY
Specimen should be liquefied first
Normal value = >50% motile within 1 hour w/ rating 2.0

To provide continuity in reporting, laboratories should place a consistent amount of semen on a slide under the same size cover slip,
such as × 22 mm cover slip using a calibrated positive-displacement pipette, and allow it to settle for
1 minute. This procedure should be done in duplicate for accuracy. 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

GRADE WHO CRITERIA GRADE ALTERNATIVE GRADING

4.0 a Rapid, straight line motility Progressive motility (PM) Sperm moving linearly or in a large circle
3.0 b Slower speed, some lateral movement Non progressive motility (NP) Sperm moving with an absence of
progression
2.0 b Slow forward progression, noticeable Immotility (IM) No movement
lateral movement

FEI
1.0 c No forward progression
0 d No movement ②
I. 0
.
CASA (Computer Assisted Semen Analysis)
Provides objective determination of sperm velocity, trajectory, concentration and morphology

I.K AYTONA 61
:
 SPERM MORPHOLOGY 010
Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion. Smears are made by placing
n near the frosted end of a clean microscope slide
Air-dried slides are stable for 24 hours
At least 200 sperm should be evaluated and the percentage of abnormal sperm reported.

Acrosomal cap
Normal values:
-part of the sperm that contains enzyme
A. Routine criteria = 33090
for ovum penetration
B. > 14010
This criteria measures the head, neck and tail using a
1/2 Of head 2/3 of nucleus
-Size: _______________________
Micrometer or morphometry
,

Stains for sperm morphology:

a. Head = Length 5um, Width 3um
b. Giemsa stain Normal head appearance = oval
c. Shorr stain Tail = 45 um long
d. = best stain Midpiece: 7um
thickest part
of tail \,

An abnormally long neckpiece may cause the sperm head to bend backward and interfere with motility.
Abnormalities in head morphology are associated with poor ovum penetration, whereas neckpiece, midpiece, and tail
abnormalities affect motility

SPERM VIABILITY /Vitality

✓ large of vital but immobile cells
Modified B indicate
proportion
defective flagellum
a
may
Reagent = ___________________
Eosin Nig rossin stain
-

✓ high number of immotile t nonviable cells indicate epididymal

may
Count the number of dead cells in a 100 sperm using brightfield or phase contrast microscope pathology
Living sperm = unstained, bluish white (at least 50%)
tested whin 2 hrs Dead sperm = red with a purple background
or frown to prevent
f fmctohysis
SEMINAL FLUID FRUCTOSE → major source of energy
Tested within 2 hours or frozen to prevent fructolysis t seminal Fructose = Decrease
sperm concentration &

Screening test: Decrease Sperm Motility

Resorcinol test = (+) orange or orange-red color
Fructose normal
quantitative level is to
= > 13mmol
or
per ejaculate
measured-

using spectrophotometer mtds .

Low fructose levels are caused by abnormalities of the seminal vesicles, bilateral congenital absence of the vas deferens, obstruction of
the ejaculatory duct, partial retrograde ejaculation, and androgen deficiency

ANTISPERM ANTIBODIES
Detected in semen, cervical mucosa, or serum

1. Mixed agglutination reaction ( MAF)

Detects the presenceanti
of IgG antibodies
IgG human
globulin
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
Sperm are mixed with polyacrylamide beads known to be coated with either anti-IgG, anti-IgM, or anti- IgA.
Microscopic examination of the sperm shows the beads attached to sperm at particular areas.
Normal = beads on less than 50 % of sperm

Head-directed antibodies can interfere with penetration into the cervical mucosa or ovum, whereas tail-directed antibodies
affect movement through the cervical mucosa

I.K AYTONA 62
 CHEMICAL TESTING
Analyte Normal value Decreased Values Indicate
Fructose ejaculate Decrease seminal fluid
Neutral alpha glucosidase Epididymis problem
Zinc Decrease/lack prostatic fluid
Citric acid Decrease/lack prostatic fluid
Acid phosphatase ejaculate Decrease/lack prostatic fluid

MICROBIAL TESTING

ROUND CELLS ✓peroxidase positive granulocytes predominant

are the form of leukocyte in semen

t can be differentiated further from spermatogonia uns t

lymphocytes using a
peroxidase
These are WBCs and spermatids stain

Normal value = < 1,000,000 / ml

If greater than 1 million WBC/ ml = infection
If greater than 1 million spermatids/ml = disruption of spermatogenesis
This is a test for chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum

FORMULA FOR ROUND CELLS

C = NUMBER OF ROUND CELLS x SPERM CONCENTRATION

100 (mature sperm)

MEDICO LEGAL TESTING

Test for detection of semen:

1. Microscopic exam
2. Fluorescence under UV light
3. Acid phosphatase determination positively identify unknown fluid as semen
=

4. Glycoprotein p30 = more specific method

Prostatic specific Ag
5. ABO blood testing
6. DNA test
7. Florence test
choline
Test for _____
Reagent: Potassium iodide t Iodine -
Feeling Close si
-

(+) result: Presence of dark brown rhombic crystals

8. Barberi Bes
-

Test for spermine

_____
-

Reagent: TCA & Picric Acid

(+) result: Yellow Fern Leaf Shaped or Needles like crystals

Motile sperm can be detected for up to 24 hours after intercourse, whereas nonmotile sperm can persist for 3 days. As the sperm die
off, only the heads remain and may be present for 7 days after intercourse

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
Recommended testing includes examining a wet preparation using phase microscopy for the presence of motile and nonmotile
sperm. A negative wet preparation is followed by specimen centrifugation for 10 minutes and examination of
the sediment

VARICOCELE
Hardening of veins that drains the testes
Most common cause of male infertility
Sperm head = tapered head

ASSESSMENT OF SPERM CELLS

Sperm morphology make a smear in slide and stain it with P
Sperm Viability- Stain with Eosin Nigrosin
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

I.K AYTONA 63
 SPERM FUNCTION TEST
Test Description
Hamster egg penetration Sperms are incubated with species non-specific hamster eggs and penetration is observed
microscopically
Cervical mucus penetration Observation of sperm penetration ability of partners midcycle cervical mucus
Hypo-osmotic swelling Sperms exposed to low sodium concentrations are evaluated for membrane integrity and sperm
viability
In vitro acrosome reaction Evaluation of the acrosome to produce enzymes essential for ovum penetration

Abnormal result Possible abnormality Test

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

ADDENDUM
Ejaculatory duct Part of the male reproductive system that receive both the sperm from ductus deferens and fluid from
seminal vesicles
Flavin Responsible for the gray appearance of semen.
First Most of the sperm are contained in the ____ portion of the ejaculate, making complete collection
essential for accurate testing of both fertility and post-vasectomy specimens.
Sperm motility Presence of urine in semen sample may affect primarily sperm ____
Liquefying agent Purpose of -buffered Saline, and proteolytic enzymes such as alpha-chymotrypsin or
bromelain
Tests affected with an Increased semen viscosity Sperm motility, sperm concentration, anti-sperm antibody detection, and
and incomplete liquefaction measurement of biochemical markers
Midpiece It is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the
energy required by the tail for motility.
Oil immersion objective Sperm morphology is evaluated from a thinly smeared, stained slide under what objective
24 hours Slides for Seminal smear that are air-dried are stable for how many hours?
Retrograde ejaculation / Dry An uncommon but treatable condition in which semen is directed into the urinary bladder which
orgasm eventually can be found in urine instead of being ejaculated.
lack of prostatic fluid Decreased zinc, citric acid, glutamyl transpeptidase, and acid phosphatase indicates:
Disorder of the epididymis A decreased neutral a -glucosidase, glycerol-phosphocholine, and L-carnitine suggest
Spectrophotometry Methods that can be used to quantitate citric acid and zinc on seminal fluid?
Xylene A reagent that can be added to enhance the sperm under microscopic analysis using phase contrast
microscope
Motile sperm can be detected for up to 24 hours after intercourse, whereas nonmotile sperm can persist for 3 days. As the
sperm die off, only the heads remain and may be present for 7 days after intercourse
90 days The entire process of spermatogenesis takes approximately how many days?

FECALYSIS
NORMAL 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)
Digestive enzymes secreted into the small intestine by the pancreas include trypsin, chymotrypsin, amino peptidase, and
lipase
Water and electrolytes are readily absorbed in both the small and large intestines, resulting in a fecal electrolyte content that
is similar to that of plasma

Color/Appearance Clinical Significance

BROWN NORMAL DUE TO STERCOBILIN ( Urobilin )
→
BLACK UPPER GI BLEEDING (melena) black tarry stool
IRON THERAPY, CHARCOAL INGESTION
PALE YELLOW, WHITE GRAY, ACHOLIC STOOL, CHALKY, BILE DUCT OBSTRUCTION AND BARIUM SULFATE
CLAY-COLORED
GREEN BILIVERDIN, GREEN VEGETABLES, ORAL ANTIBIOTICS
RED LOWER GI BLEEDING, RIFAMPIN, FOOD COLORING
BULKY / FROTHY BILE DUCT OBSTRUCTION, STEATORRHEA, PANCREATIC
INSUFFICIENCY

I.K AYTONA 64
 RIBBON LIKE INTESTINAL CONSTRICTION
RICE WATERY CHOLERA
PEA SOUP TYPHOID
SYCBALOUS / GOAT DROPPING CONSTIPATION
BUTTER-LIKE CYSTIC FIBROSIS
MUCOID DYSENTERY, MALIGNANCY

BRISTOL STOOL CLASIFICATION

Type Characteristic
1 Separate hard lumps, like nuts
2 Sausage shaped but lumpy
3 Like a sausage but with cracks on its surface
4 Like a sausage or snake, smooth and soft
5 Soft blobs with clear cut edges
6 Fluffy pieces with ragged edges, a mushy stool
7 Watery, no solid pieces, entirely liquid

I.K AYTONA 65
 LABORATORY TESTS

I.TEST FOR FECAL FAT

STEATORRHEA= presence of increase fats in stool (>6g/day)

*Fecal characteristic: Greasy; foul odor; spongy consistency
*Fecal volume: Increased
*Causes:
1. Pancreatic insufficiency
2. Malabsorption 5.) Gardiasis
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

QUALITATIVE FECAL FAT STAIN

Neutral fat stain Split fat stain
Stain for Triglycerides Ethanol Stain for total fat content (including Fatty acids,
Procedure: emulsified stool + 95% ETOH + Sudan III 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

converted
lipids are
g. into
fatty
acids QUANTITATIVE FECAL FAT TEST
Van De Kamer Titration test reporting :
fat I -6g/d
grams of =

Gold standard test for fecal fat → confirmatory coefficient of 95% --

retention
Requires a 3-days stool sample (placed on a Paint Cans and must be homogenized prior to analysis) fat t
dietary fat fecal fat ✗ too
-

TitratedIwith NaOH 80% of total fat content

gravimetric mtd measures all fecal fat
dietary fat
-

to neutral
is measured
by titration hydrogen G- mins) & safe procedure
nuclear
rapid
a

magnetic resonance
spectroscopy
-

endpoint
D-XYLOSE TEST for
analyzing quantitative
fecal fat

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.
-hour blood
sample and a 5-hour urine specimen.
If D-xylose result is low/abnormal, the result indicates a malabsorption condition

I.K AYTONA 66
 II.FECAL LEUKOCYTES
Presence of neutrophils/hpf Indicates invasive condition (diarrhea)
Presence of at least 1 Neutrophil per OIF is significant

TEST
Wet preparation Stool + Methylene blue
Methylene blue staining is the faster procedure but may be more difficult to interpret
Methylene blue is used to differentiate Mononuclear cells and PMNs
Lactoferrin Latex A test for fecal WBC that gives a positive result in invasive bacterial pathogen
[ agglutination
s
component of
It remains sensitive in refrigerated and frozen specimens SSCYE
granulocyte 2° Positive in diarrhea with WBC: Salmonella, Shigella, Campylobacter, Yersinia, & enteroinvasive E. coli.
granules Negative in diarrhea without WBC: Staphylococcus aureus and Vibrio spp., viruses, and parasites
Dried preparation Stool stained with either Wright's or Gram stains provide permanent slides for evaluation.

III. FECAL OCCULT BLOOD TEST

Occult means ___
hidden

Screening test for __________

colorectal cancer

Significant value = >2.5ml blood / 150g stool

Sample must be obtained from the center portion of the stool to avoid false positive from external contamination
CHROMOGEN USED: Benzidine, Guaiac, O-toluidine

REACTION Based on pseudoperoxidase activity of Hemoglobin

Water + Guaiac ----------------------------------- oxidized guaiac(blue) + Water

Hgb 1- Hurt indicator
least sensitive
more preferred
INTERFERENCES OF GUAIC BASED FOBT
Result Interferences Avoided (days)
False (+) Aspirin and other NSAIDs 7 days (for aspirin & NSAIDs)
Red meats, horseradish, melons, raw broccoli, cauliflower, radishes, & turnips 3days
False (-) Reducing agent such as Vitamin C 3 days

IV. MUSCLE FIBERS

___________________
creatorrhea = Increase excretion of muscle fibers in feces
Presence of more than 10 undigested muscle fibers are associated with biliary obstruction, cystic fibrosis, and gastrocolic
fistulas BCG
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 visible striations. Fibers exhibit striations in only one direction Fibers have visible striations running
both vertically and horizontally
of maternal
>
[
presence
produce
w?¥
thalassemiaerroneous differentiate Hg Asics ,
ss & Hbf

V. APT TEST/ ALKALI DENATURATION TEST/ DOWNEY TEST

resist ✓Alkali denaturation Procedure
µ ✓ Acid
Elution 1. Emulsify specimen in water.
A test for Fetal hemoglobin
2. Centrifuge.
Used to Differentiate fetal blood from maternal blood
3. Divide pink supernatant into two tubes.
Discovered by Leonard Apt
4. Add 1% sodium hydroxide to one tube.
Specimen: infant stool, vomitus, emesis, or gastric aspirate
5. Wait 2 minutes.
Reagent:1% Sodium Hydroxide
6. Compare color with that in the control tube.
7. Prepare controls using cord blood and adult blood.
Pink supernatant Fetal blood with Hemoglobin F
Yellow brown supernatant Maternal blood with Hb A

VI.FECAL ENZYMES
Enzymes supplied to the gastrointestinal tract by the pancreas are essential for digesting dietary proteins, carbohydrates, and fats.
Decreased production of these enzymes (pancreatic insufficiency) is associated with disorders such as chronic pancreatitis and cystic
fibrosis. Steatorrhea occurs, and undigested food appears in the feces.

I.K AYTONA 67
 X-ray film test Detects trypsin enzyme (absent of trypsin is associated with cystic fibrosis)
Absence of trypsin has been screened for by exposing x-ray paper to stool emulsified in water. When
trypsin is present in the stool, it digests the gelatin on the paper, leaving a clear area.
Inability to digest the gelatin indicates a deficiency in trypsin production
Chymotrypsin more resistant to intestinal degradation and is a more sensitive indicator of less severe cases of
pancreatic insufficiency
Stable in fecal specimen up to 10 days at room temperature
Measured by spectrophotometry
Elastase 1 An enzyme form produced by the pancreas and accounts about 6% of all secreted pancreatic enzyme
Sensitive and specific test for exocrine pancreatic insufficiency
The test is specific in differentiating pancreatic from nonpancreatic causes inpatients with steatorrhea.
It is not affected by motility disorders or mucosal defects
It is measured by ELISA,
The ELISA test uses monoclonal antibodies against human pancreatic elastase-1; therefore, the result is specific for
human enzyme and not affected by pancreatic enzyme replacement therapy

VII.FECAL CARBOHYDRATES
Significant for assessing lactose intolerance
Normal stool pH: 7-8,
Stool pH with Carbohydrate Disorders = pH <5.5
Clinitest: a test for reducing sugar
0.5 g/dl indicates carbohydrate intolerance

fecal Formula : Osmotic [ 2(fecal sodium 1- fecal potassium)

gap
= 290 -

osmolality =close to serum 290 mosm/

leg
fecal Na 30 mmol
=
/L fecal K = 75mmol /L 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
Diarrhea lasting less than 4 weeks is defined as acute, and diarrhea persisting for more than 4 weeks is termed chronic
diarrhea
The major mechanisms of diarrhea are secretory, osmotic, and intestinal hypermotility

SECRETORY DIARRHEA Stool- with osmolality gap of <50mosm/kg

Caused by increased secretion of water and electrolytes, which override the reabsorptive ability of the
large intestine

CAUSES:
Viruses (e.g rotavirus), protozoa or bacteria, Colitis, Collagen vascular disease, hormones,
endocrine disorder (Hyperthyroidism, Zollinger Ellison Syndrome, VIPoma), inflammatory bowel
disease, and neoplasm
OSMOTIC DIARRHEA Stool- with osmol gap of >50mosm/kg. 775 mosmlkg Stress -

Caused by poor absorption that exerts osmotic pressure across the intestinal mucosa. Incomplete
breakdown or reabsorption of food presents increased fecal material to the large intestine, resulting in
water and electrolyte retention in the large intestine (osmotic diarrhea), which in turn results in excessive
watery stool

I.K AYTONA 68
 CAUSES:
Maldigestion, Malabsorption, Lactose intolerance, Ameobiasis , Antibiotics, Magnesium
containing antacids, Poorly absorbed sugars (lactose, sorbitol, mannitol), laxatives

ALTERED MOTILITY Stool with osmolal gap of >50mosm/kg.

Altered motility describes conditions of enhanced motility (hypermotility) or slow motility (constipation).
Both can be seen in irritable bowel syndrome (IBS), a functional disorder in which the nerves and
muscles of the bowel are extra sensitive, causing cramping, bloating, flatus, diarrhea, and constipation.
Normal Gastric emptying : IBS can be triggered by food, chemicals, emotional stress, and exercise.
controlled by :
fundic tone
duodenal feedback CAUSES:
GI hormones Gastric surgery (gastrectomy), Gastric bypass, Post vagotomy, Duodenal ulcer, DM Zollinger Ellison

Rapid gastric emptying (RGE) dumping syndrome describes hypermotility of the stomach and the shortened gastric emptying half-time,
which causes the small intestine to fill too quickly with undigested food from the stomach. It is the hallmark of earl dumping syndrome
(EDS). Healthy people have a gastric emptying half-time range of 35 to 100 minutes, which varies with age and gender.
A gastric emptying time of less than 35 minutes is considered RGE.

DIFFERENTIAL FEATURES FOR DIARRHEA

Laboratory test Osmotic Diarrhea Secretory Diarrhea
Osmotic gap >50 Osm/kg <50 Osm/kg
Stool Na <60 mmol/L >90 mmol/L
Stool output in 24 hours <200 g >200 g
pH <5.3 >5.6
Reducing substances Positive Negative

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.
It is produced from the tracheobronchial tree
DSSM
SPUTUM COLLECTION =
2 consecutive first morning sputum sample
First morning- Most preferred (routine) water
24 hour for volume measurement =
px should gargle w/
Throat swab for pediatric patients
Sputum induction- for non-cooperative patients
Tracheal aspiration- for debilitated patient

I.K AYTONA 69
 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 Putrid Lung gangrene, Advance necrotizing tumors
Sweetish Broncheictasis, tuberculosis
Cheesy Necrosis, tumors, emphysema
Fecal Liver abscess, Enteric gram-negative bacterial infection

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

Structures
*Yellowish or gray 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/ Pneumoliths Small, white or gray fragments of calcified TB tissue or calcified foreign
/ Broncholits matter
Bronchial cast Branching tree like casts of the bronchi
*Whitish or yellow wavy coiled threads
*Associated with bronchial asthma
Layer formation Top layer : 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 crystals - Colorless, hexagonal, double pyramid, pointed at both ends, and needle like
- Formed as a result of eosinophil degeneration
primary granule of
- Most significant
eosinophil
colorless
or
pink color - Associated with bronchial asthma
Pigmented cells - Heart failure cells: hemosiderin laden macrophages
- Carbon laden cells: angular black granules
- Coiled mucus strands
- 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
flung to heart
Parasites ASH migration - Ascaris, Hookwork, Threadworm, E.histolytica, Paragonimus westermani, Toxocara canis,
Entamoeba gingivalis, Trichomonas tenax, Echinococcus granulosus
Others that are stained - Neoplastic cells, Bacteria, Leukocyte
cells
pulmonary Langerhans
disease
related interstitial lung
or

Bronchial Asthma Macrophage -

smoking -

histioeytosis than bacterial infx

interstitial lung disease drug pulmonary lymphoma
.

=3 reactions
Lymphocytes -
, ,

lung disease
✓ Creola bodies -

lymphocyte = or > 2590 suggest granulomatous

nonspecific interstitial pneumonia
hypersensitivity pneumonitis
diff
- count ) 5090 or

✓ Curshmann
crystal Neutrophils -

cigarette smokers ,
bronchopneumonia
aspiration pneumonia suppurative
" in '

✓ Charcot Leyden suggest acute wing injury

or

neutrophil
-

= or > sogo ,

bacterial fungal ) hypersensitivity

disease , infx (parasitic
,

asthma drug induced

, ,

lung
,

Eosinophil
-
-
,

pneumonitis & eosinophilic pneumonia

eosinophil diff count > .
25% diagnoses eosinophilic lung disease
- or =

Mast cell diff count > 1% lymphocyte 350W neutrophil 3390 suggests
-

70
.
,
,
I.K AYTONA
hypersensitivity pneumonitis
 is discarded
first aliquot
installation volume too -300 mi of sterile saline f)
f) in 20 -
to 50 -
ml Aliquots f)
desired fluid volume for
analysis is lo w mL
-
( minimal v01 .
is 5m11

BRONCHOALVEOLAR LAVAGE
Provides a method of obtaining cellular and microbiological information from the lower respiratory tract transport
:

✓ RT during transport
Useful in evaluating immunocompromised patients, interstitial lung disease and airway diseases ✓ when delivery to laboratory
is
than 30 mins
Important diagnostic test for Pnuemocystis carinii in immunocompromised patients delayed larger
,

ice (404
AN transport an

A suitable respiratory specimen for culture and sensitivity ✓ centrifuge spx that will not be in a
t
analyzed immediately resin spend
nutrient rich ref
supplemented medium & -
-

hrs
CELLS SEEN IN BRONCHOALVEOLAR LAVAGE Elements and Viral inclusions seen in respiratory @ 4°C up to 24 .

Macrophage 56-80% specimens:

Lymphocyte 1-15% Toxoplasma gondii
Neutrophils <3% Legionella pneumophila
Eosinophil <1 2% Histoplasma capsulatum
Ciliated columnar bronchial epithelial 4-17% Mycoplasma pneumonia
cells Influenza A, B, and Respiratory syncytial virus

✓ cell viability - add Try pan blue ✓ Allow cells to settle for 5 mins
✓ ltemocytometny WBC - t.tw dilution of Ammonium oxalate
✓ RBC dilute w/ isotonic saline sol
-

to lyse RBCs .

using MLA pipette

SWEAT

Cystic Fibrosis / Mucoviscidosis

Autosomal recessive disorder
A metabolic disease that affects the mucous secreting glands of the body
Associated with pancreatic insufficiency, intestinal obstruction, and respiratory distress
There is chloride channel defect
chemical that
> sweat
f induces production
GIBSON AND COOKE PILOCARPINE IONTOPHORESIS
Sweat is tested for sodium and chloride
Sweat chloride and sodium values over 70 mEq/L are seen in 98% of patients with Cystic fibrosis
Borderline: 40 mEq/L

GASTRIC FLUID
G cells
SIGNIFICANCE
Determines whether or not a patient can secrete gastric fluid f makes
Measures amount of gastric acid that can be secreted by one with ulcer symptoms ( commands parietal
gastrin cells to secrete HCl )
Help determine the disturbed function of the GI system
t
CELLS OF THE STOMACH makes

parietal cells → HCl

to
1. Parietal cells- produces HCL and Intrinsic factor pepsinogen → pepsin
HCL- converts pepsinogen to pepsin that catalyzes the digestion of protein
Intrinsic factor responsible for Vitamin B12 absorption
Parietal cells will secrete HCL in response to Gastrin stimulation

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 _______ nose

b. Rehfuss = passed through the _______

month
c. Disposable plastic tubes are usually employed

I.K AYTONA 71
 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 (BAO) 1 hour collection (four 15minute specimens)
Requires 12 hour fasting
No gastric stimulant needed
Maximum acid output (MAO) 1 hour collection (four 15minute specimens)
With gastric stimulant

GASTRIC STIMULANTS
Test meals
Boas meal: oatmeal, meal for detection of lactic acid
oes, broiled beefsteak, bouillon
Chemicals Pentagastrin most preferred, it resembles true gastrin
Histamine → stimulate gastric acidity
Histalog
Insulin - assess successful vagotomy procedure
Sham Feeding Fictitious feeding
Sandwich is chew and then spit out

DIAGNEX 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.

1, 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 >3.5 but falls after gastric stimulation Carcinoma of stomach
Decrease free HCL
Achlorhydria Gastric fluid pH >3.5 and does not fall even after gastric stimulation Pernicious anemia
Absence of free HCL

QUALITATIVE TEST FOR FREE HCL

Dimethylaminoazobenzol Reagent: alcohol solution = (+) cherry red
Reagent: phloroglucin, Vanillin, Alcohol = (+) Purple red color
Boas Reagent: resorcinol, cane sugar, alcohol = (+) Purple red color

QUANTITATIVE TEST FOR GASTRIC ACIDITY

Free HCL
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

I.K AYTONA 72
 Combined HCL (Bound to Titrate with NaOH
proteins) pH indicator: sodium alizarin
Endpoint: Violet
Normal value: 10 15 degrees

LACTIC ACID TEST

Test Reagents Endpoint
FeCl3 + phenol Yellow
Strauss FeCl3 + ether Yellow
FeCl3 Yellow

QUALITY ASSESTMENT AND MANAGEMENT IN URINALYSIS LABORATORY

QC of laboratory equipment Daily

-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

* checking of speed of cyto centrifuge Monthly

-
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 (QA) Refers to the overall process of guaranteeing quality patient care and is regulated
throughout the total testing system
Quality system R
achieve quality testing.
Trend A gradual change in the mean
Shift An abrupt change in the mean
Total Quality Management (TQM) is a systematic problem-solving 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 control Refers to the materials, procedures, and techniques that monitor the accuracy,
precision, and reliability of a laboratory test.
External quality control External quality controls are used to verify the accuracy (ability to obtain the expected
result) and precision (ability to obtain the same result on the same specimen) of a test
and are exposed to the same conditions as the patient samples
Internal quality control Consists of internal monitoring systems built in to the test system and are called internal
or procedural controls. Internal controls monitor the sufficient addition of a patient
specimen or reagent, the instruments/reagents interaction, and, for lateral flow test
methods, whether the sample migrated through the test strip properly
Electronic Controls External quality control (EQC) uses a mechanical or electrical device in place of a liquid
QC specimen. This type of QC can be internal or an external component inserted into a
point of care (POC) instrument. EQC verifies the functional ability of a testing device,
but it does not verify the integrity of the testing supplies

I.K AYTONA 73
QUALITY ASSURANCE ERRORS IN CM (Strasinger, 5th edition)
PRE ANALYTICAL ERROR ANALYTICAL ERROR POST ANALYTICAL ERROR
Patient misidentification Sample misidentification Patient misidentification
Wrong test ordered Erroneous instrument calibration Poor handwriting
Incorrect urine specimen type Reagent deterioration Transcription error
collected Poor testing technique Poor quality of instrument printer
Insufficient urine volume Instrument malfunction Failure to send report
Delayed transport of urine to the Interfering substances present Failure to call critical values
laboratory Misinterpretation of quality control Inability to identify interfering
Incorrect storage or preservation data substances
of urine

In a clinical laboratory, a quality assessment program includes not only testing controls, referred to as quality control (QC), but also
encompasses preexamination variables (e.g., specimen collection, timing, handling, and storage), examination variables (e.g.,
reagent and test performance, instrument calibration and maintenance, personnel requirements, and technical competence),post-
examination variables (e.g., reporting of results and interpretation), and documentation that the program is being meticulously
followed

Electronic transmission is now the most common method for reporting results

The telephone is frequently used to transmit results of stat tests and critical values. Calls requesting additional results may be received
from personnel on hospital units and from healthcare providers. When telephoning results, confirm that the results are being reported
to the appropriate person. The time of the call and the name of the person receiving the results must be documented according to the

ADDENDUM
Home based 1. Principle of current PT test kit: IMMUNOLOGIC
pregnancy test (Enzyme immunoassay/immunochromatographic assay)
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
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 renal 1. Wipe off the stone(s) and describe in terms of size(mm), shape, color and hardness/texture
calculi 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 and bubbles and appear from the oxalate
bottom of the tube
Biologic test for
PT
th ed.)
TEST/METHOD ANIMAL USED POSITIVE RESULT
Ascheim-Zondek Immature female mice Formation of
hemorrhagic follicles
and corpora lutea
Friedman Mature virgin female rabbit Hyperemic uterus and
corpora hemorrhagica
Hogben Female toad (Xenopus laevis) south African clawed oogenesis
frog-carries eggs throughout the year

Galili- manini Male frog (Rana pipiens or Rana clamitans, leopard or spermatogenesis
grass frag) male toad (Bufo bufo or Bufo americanus)
Frank Berman Immature female rats Ovarian hyperemia
Kupperman Female rat Ovarian hyperemia

I.K AYTONA 74
Chemical test in Detected Name of test
urine
Calcium Sulkowitch
Chloride Fantus, Schales Schales
Bile pigments Smith, H Gmelin
Urobilin Schlesinger
urobilinogen Wallace and diamond
Ketones Lange, acetest, G
fructose Resorcinol, Seliwanoff, and
Qualitative test for Heat and acetic acid
protein
Quantitative test for Biuret, Kingsburry-clark, E K
protein
Sugars 1. Biacol orcinol
2. Benedicts
3. (glucose = red with red yellow, Lactose = red with red ppt)
4. Moore Heller
5.

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 chemistry These semi-automated instruments require the user to properly dip the reagent strip and place it onto a
Semiautomated platform.
analyzer
Dry Chemistry 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 productivity in the urinalysis laboratory
Microscopy Uncentrifuged urine is used, the time spent in handling and preparing concentrated urine sediment
analyzers for manual microscopy is eliminated
Increased standardization of the microscopic examination, which enhances the accuracy and
reproducibility (precision) of results

Iris iQ 200 It is an automated system that performs the microscopic examination of urine, as well
International as cell counts on body fluids
Remote
Uses patented technologies to capture and automatically classify digital images of urine
Imaging
System particles.

-SOFTWARE USED= AUTO PARTICLE RECOGNITION SOFTWARE OR Proprietary neural network

software

- APR Pre-classifies urine particles in the photographs based on size, shape, texture, and
contrast in to 1 to 12 categories. Using the computer monitor, the user can review results,
visually assess the particles present, and subclassify them into the 26 additional 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.
With each sample, the camera captures 500 frames, digitizes them, and sends them to a
computer for processing

I.K AYTONA 75
 Med tech

Note that the 12 auto-classified sediments are the primary parameters measured.

Sysmex -Principle: cell flow cytometry + impedance

UF- -Urine particles are identified and categorized by fluorescent staining characteristic, light
analyzers scatter, electrical impedance, and adaptive cluster analysis
/ Slideless
automated Fluorescent stain used
analyzers Carbocyanine Stain for RNA, cell membrane, cytoplasm
blue-green
Phenanthridine Stain for DNA, nucleic acid, nucleus, chromosomes
orange

Light source
Sysmex UF-100 model Argon laser
Sysmex UF-1000i model Red semi-conductor laser

For automated particle analysis, the UF-1000i analyzer requires a 4 mL sample volume;
however, if the instrument is used in the manual mode, only 1 mL of urine is required
Changes in the UF-1000i analyzer include a separate channel for bacterial analysis and
the monitoring of lateral or side scatter, which improves detection of bacteria

-bacteria is detected specifically by the side light scatter

-Results are displayed as SCATTERGRAM OR HISTOGRAM

UF 1000i Particle Detection Categories

Particles Enumerated RBCs, WBCs, Epithelial cells, Hyaline Cast, Bacteria
Flagged Particles Non hyaline(pathologic) cast, Crystals, small round cells
(transitional or renal cells), Yeast, Mucus, Sperm

microscopic review of the urine by the user.

Full automated Performing fully automated urinalyses requires combining a urine chemistry analyzer with a microscopy
Urinalysis analyzer.
system
iRICELL Urinalysis iChem Velocity urine chemistry + iQ200
Systems To perform a complete urinalysis, a minimum of 3 mL urine is poured into a barcode-
labeled tube. Specific tubes are not required; rather a variety of
tubes can be used, including commercial urinalysis tubes (e.g., KOVA, Vacuette, BD) or
disposable glass test tubes
CLINITEK AUWi ATLAS urine chemistry + Sysmex UF1000i
System For a complete urinalysis, 5 mL of uncentrifuged urine is poured into a barcode labeled
tube, which is placed into a 10-place sample rack. Tubes upto 16 mm wide can be used,
but they sample racks are placed
onto the system, and as each rack is moved to the sampling position of the ATLAS
analyzer, the barcoded sample tube is automatically identified
JUST A MIXTURE OF HARDWORK, DETERMINATION, FAITH,
AND A POSITIVE ATTITUDE
--END--

I.K AYTONA 76
Vaginal Secretions

of the conditions diagnosed by health-care

Vaginitis - one most common

for female px
provider .

characterized by abnormal vaginal discharge or odor

, pruritus vaginal
,

irritation , dysuria t
dyspareunia
-
most often ,
secondary to bacterial vaginosis ( BV) , trichomoniasis ,
or

vuluovaginal candidiasis

Spx Collection &

Handling
speculum moistened w/ warm water to visualize vaginal form.us
to collect epithelial
swabbing vaginal walls t vaginal pool cells
using
sterile , polyester tipped swabs
plastic shaft swabs
a
one or more - on or

Cotton swabs should not be used because it is toxic to W .

gonorrhoeae ,

wooden shaft be toxic to chlamydia trachomatis t calcium alginate

may
can inactivate herpes simplex virus ( HSV ) for viral cultures

place swab in tube w/ 0.5 to 1.0 ml of sterile physiological saline

 RT to
preserve motility
must be of
Spx kept @ T.

vaginalis +
recovery of
N .

gonorrhoeae
trachomatis t HSV be refrigerated to of normal flora
whereas
Spx .
for C- must
prevent overgrowth
Spx for T .
vaginalis examination should be examined w/ in 2 hrs of collection

Color &
Appearance
Normal white flocculent discharge
-

af a

of large rod-shaped gram-positive lactobacilli t SEQS

-

predominance ,
,

-
WBG & RBCs if menstruating
increased thin white BV
Abnormal -
,
homogenous ,
-
to -

gray discharge seen in

"
-
white cottage cheese like -
"

discharge for Candida infx .

discharge w/ T
- increased
yellow
-

green , frothy ,
adherent assoc . .

vaginalis
-

yellow opaque cervical discharge ,

C- trachomatis
,

Ph
should be performed before saline
placing swab into or KOH Sol .

interference : cervical mucus, Sernin t blood

,

Normal : 3.8 -4.2

Abnormal : about 4.5 -

Valvo
vaginal candidiasis
've
> 4.5 Bu , trichomoniasis
desquamate inflammatory vaginitis atrophic vaginitis
-

, ,

acidic
Lactic acid Hua ,
t
Estrogen production -

provides vaginal environment

Micwscopittoednres
Saline initial test Gram
wet mounts t Kott
screening stain confirmatory
-
-

cells + organisms are

quantified per HPF (40×1 three clean glass slides

cells t organisms are

reported per 010
(100-1)
Wet Mount

using low-power slide is scanned for an even distribution of cellular components ,

types t
numbers of epithelial cells ,
clumping of epithelial cells , the presence of
budding yeast or
pseudohyphae
counted
using high organisms reported pwhpf
& cells t
power are
-

Rare fewer than 10 cells /hpf

organisms
-
or

It -
fewer than 1 organism or cell / hpf
2-1 I -5
organism
or all
/ hpt
-

3-1 -
6-30
organism
or cell /npf
4-1
-
> 30
organism
or cell
/ hpf
 typical Constituents SEQS WBG RBCs due cells basal cells basal cells
para
-

, , ,
, , ,

bacteria motile
,
T .

vaginalis yeast , ,
t
hyphae / psendohypae
* SEQS
measure 25 to 70µm in diameter
prominent nucleus that is centrallylocated
" " t about the size of an KBC
exhibit a
polygonal flagstone appearance
large amount of irregular cytoplasm
lacking granularity w/
distinct
,
all
margins
Clue cells
*
distinguished by Cocco bacillus bacteria attached in clusters on the cell surface

Bacteria should cover at least 7590 of the epithelial all

"

granular irregular
"
sometimes
gives cell a ,
appearance described as
shaggy
* WBCS
measure 14 -16µm in diameter t exhibit a
granular cytoplasm
Normal - rare to 2-1

More than 3-1 suggests vaginal candidiasis , atrophic vaginitis ,

or infx
w/
Trichomonas chlamydia
, ,
N .

gonorrhoeae or HSV
* RBCs
smooth , hannu cheated , biconcave disks in
, measuring approx 7- 8mm diameter

present during menstruation or due to

desquamate've inflammatory process
a

distinguished from yeast cells by Kott ,

w/o lysis RBCs but not
yeasts
* Para basal cells

to t measure in diameter
round
oval-shaped 16 -40mm

ratio of is :L to marked
N :c I I :c
w/ basophilic granulation or a
morphia
basophils'c )
"

( blue
"
structures blobs

located in the luminal Squamous epithelium of vaginal mucosa

less cells be found if px

mature may is
menstruating or is
menopausal
increased ft w/ present large numbers of WBCS can indicate DN

* Basal cells
located deep in the basal
layer of vaginal stratified
epithelium
round ,
measure to -16mm in diameter N :c ratio is 1.2
,

distinguished from WBCS ,

by their round rather than honed nucleus

if present t
accompanied by WBCS faltered vaginal flora , suggests DN
* Bacteria

Lactobacillus spp the

largest portion of vaginal bacteria
.

normally comprise
appear as large gram-positive nonmotile rods t produce lactic acid
, ,

other bacteria
commonly present anaerobic streptococci diphtheroids coagulase negative staphylococci
: -

, ,

& a-
hemolytic streptococci
absent decreased numbers of lactobacilli relative to the number of SEQS
or
suggests
an alteration of normal flora

* Trichomonas vaginalis
atrial flagellated protozoan that can cause inflammation sink in wa r m

oval has 4 anterior

shaped ,
measures 15 -1018µm in diameter t
flagella &

an
undulating membrane that extends half the
length of the body
1-1×0 style bisects the trophozoite longitudinally J from w/ c
-

protrudes posterior end

enables the organism to attach to the vaginal mucosal t cause tissue damage

jerky motion of flagella t

undulating membrane is characteristic of T .

vaginalis
must be examined ASAP @ RT for maximum of
Spx .
or maintained 2 hrs .

* yeast cells
Candida albicans t Candida most fungal infix
non
spp cause
- .
.

in vaginal considered part of

occasional yeast secretions is normal flora

both (blastopore) uk long filaments that gun

appear
as
budding cells or as
hyphae are

& form a
mycelium
Pseudo hyphae -

multiple buds that do not detach t form chains also can be seen

yeast cells stain gram-positive

KOH Prep . t Amine test

performed by placing drop of the

saline
a
spx onto a clean slide t
adding a
drop of
for fishy
" "

1090 Kott sol . t checked immediately a amine odor &

reported as
positive or
negative
numbers of anaerobic bacteria in the that released into
increased vagina produce poly amines are

the vaginal fluid

results from trimethylamine volatilization KOH is added

odor
,
a
product of amines when

Positive result suggests BV caused by increased numbers of G. vaginalis in conjunction w/

spp t T vaginalis
examined for pseudohyphae
Mo billions
pg
.
.

+ Smaller blast oospores

After amine test , place cover

ship t
for 5 mins drop of / No glycerin my
)
a
rest may be t
an
heat
( applied he added to prevent deterioration
 Other diagnostic Tests

* Gram stain
standard in ID the
gold causative
organisms for Bu

scored Gram stain weighted combination of the ff

system is a .

morphotypes :
Lactobacillus acidophiles ( large gram-positive rods ) , G. vaginalis t Bacteroides
spp ( small
.

gram - variable or
gram-negative rods) & Mobiluncus
spp ( curved
.

gram variable
-
rods )
of BV
0 to 3 normal 4 -6 intermediate >7
diagnostic
-
-
,
,

* Culture

gold standard test for

detecting yeast t Trichomonas
up to 2 days for
more time consuming & requires a result

special Diamond medium is

req for
.

determining presence
of T .

vaginalis
* DNA Testing
DNA Probe testing system -

Affirm UP /I / → available for differential diagnosis of G. vaginalis ,

Candia
spp .
t T .

vaginalis
→ easy to perform t results are available in 1hr . w/ 9590 sensitivity
Trichomonas also detected by DNA
can be
probes amplified by PCR accurate
diagnostic mtd
detects nonviable
organisms
* POCT
Proline amino peptidase activity in vaginal secretions can be detected
by rapid antigen
tests to identify G. vaginalis
immuno test that
050M Trichomonas Rapid Test -

chromatographic strip detects T .

vaginalis

Ag from vaginal swabs in 10 mins

Trichomonas proteins are Solnbiliwd into the buffer

Visible blue line t red internal control line indicates
positive result
-

050M BVBLUE Test -

detects vaginal fluid sialidase , an
enzyme produced by
the bacterial pathogens BU such Gardnerellq Bacteroides
assoc .

w/ ,
as
,
,
Preto vella tmobiumars
takes the change
-
1 min to perform t is read
by examining in the color

of the solution : blue or

green is positive , yellow is
negative
Vaginal Disorders
Bacterial Vaginosis
-
most common cause of vaginitis
- occurs when there is imbalance in the ratio of normal vaginal
bacterial flora
-
as
vaginal pH becomes alkaline lactobacilli ,
an
replaced by an
overgrowth of
G. vaginalis ,
Mobil uncus
spp
.

,
Prevotella spp , Porphynmmas Pepto Streptococcus Mycoplasma
, ,

hominis Urea
t plasma spp .

malodor t increased vaginal discharge result from this mix of organisms t

after intercourse
is more apparent
-

According to Amsel's Diagnostic Criteria

,
3 of 4 features must be
present for diagnosis of Bu :

i. Thin white , homogenous discharge

,

fluid pH 54.5
2. vaginal
A positive amine ( whiff ) test
3.

4 .
Presence of due cells on
microscopic examination

Treatment : Metronidazole ( Flagyl ) ,

metronidazole get or
clindamycin cream

Trichomoniasis
transmitted by sexual intercourse

frequently occurs w/ ink

gonorrhea . of or
chlamydia
green
-
to yellow
-

frothy vaginal discharge ,

malodor
,
pruritus irritation dysuria
, , , dyspareuniat
vaginal mucosa
erythema
strawberry cervix

visualization of motile trichomonads , vaginal amine test firm KOH

pH > 4.5 &
positive prep
if wet Mount is negative for motile trichomonads, a
culture
using Diamond medium
for detection
or
commercially available
parch system
is recommended of T .

vaginalis
Treatment is metronidazole

Candidiasis
common cause of vaginitis
infx there is
occurs when a
change in the vaginal environment that permits overgrowth
of Candida + of
symptoms infx.
occur

broad-spectrum
conditions that in the
can cause
change vaginal environment include use of

estrogen replacement therapy

antibiotics
,
oral contraceptives ,
or
, hormonal changes
that occur
w/ pregnancy ,
ovulation t menopause
diabetic , iron deficient t.HN infx
increased iufx rates for those immunocompromised
.
-

. ,

typical clinical
symptoms : genital itching or
burning dyspareunia dysuria presence of an
, , ,
abnormal

thick white, curd like vaginal

,
-
discharge
Normal
pH negative
,
amine test ,
Gram stain will reveal budding yeast pseudohyphae
t forms ,

large # of WBCS ,
lactobacilli large ,
clumps of
epithelial cells

Treatment : over-the-counter or
prescription aww antifungal agents
 over -
the -
counter -
but caracole , clotrimawle.fi ocmaule micro narrow
,

recurrent infx -
oral fluconazole or intrauaginal bntoconauole,
nystatin ,
t

fercaracole be effective
may more

Desquamatiu Inflammatory Vaginitis

profuse purulent vaginal discharge vaginal erythema dyspareunia
, ,

be
heterogenous group of causes of DIV B hrmohytic gram-positive streptococci
,
can

cultured from most 20 to t estrogen

px , also occur
atrophic vaginitis as a result of

pH > 4.5 &

negative amine

large # of WBCS, RBCs, occasional parabasal t basal cells , SEQS t reduced

or absent lactobacilli that have been

replaced by gram-positive cocci
Treatment : 2% Clindamycin ,
hormone replacement therapy for px w/ DIV
20 to atrophic vaginitis
Atrophic Vaginitis
found in
postmenopausal women

thinning vaginal to reduced of both estrogen glycogen

of mucosa due
production t

vaginal dryness inflamed vaginal t

purulent
t soreness
, dyspareuuia ,
mucosa

discharge
pH 54.5 & negative amine test

large # of WBCS, RBCs, occasional parabasal t basal cells , SEQS t reduced

or absent lactobacilli that have been

replaced by gram-positive cocci +
gram-negative
rods
Treatment : estrogen replacement ; topical vaginal ointments are used ; recurrent
episodes
oral or transurtaneousc patch ) modes are more effective