CLINICAL MICROSCOPY

SAFETY AND QUALITY ASSESSTMENT

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

TYPE OF SAFETY HAZARDS

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

Chain of Infection- A continuous link (6-link) on understanding on how microorganisms are

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

MODE OF TRANSMISSION

AIRBORNE /AEROSOL a. Centrifugation of unstoppered tubes

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

Biologic Waste Disposal

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 of _______________________________ should be performed daily.
Empty urine containers can be discarded as non-biologically hazardous waste.

A 0.5% bleach solution, prepared by adding 1 part household bleach to 9 parts water (1/10 dilution), is stable for 1 week

“SHARP HAZARDS”
A. SOURCE- Needles/Syringe, lancet, broken 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

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“CHEMICAL/POISON HAZARDS”
A. SOURCE- Preservatives and reagents
B. Possible Injury- Exposure to toxic, carcinogenic, or caustic agents

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

❖ Material Safety Data Sheets (MSDS)=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

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

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

“ELECTRICAL HAZARDS”
A. SOURCE- Ungrounded or wet 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.
❖ All electrical equipment must be grounded with _______________________.

❖ When an accident involving electrical Shocks occur:

1. Turn off the circuit breaker
2. Unplug the equipment
3. Move the equipment using a nonconductive glass or wood object

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

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

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

❖ To operate fire Extinguisher:

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

Types of Fire and Fire Extinguisher

Fire Extinguishing Material Extinguisher
Type
Class A Ordinary combustibles: Water, Dry chemicals, Steam
Wood, paper, clothing/garments, plastic
Class B Flammable organic chemicals/liquids: Dry chemicals, carbon dioxide, foam, or halon
gasoline, paints, oil

Class C Electrical equipment: Dry chemicals, Carbon dioxide, or halon

Machines, motor switches, plugs

Class D Combustible metals: Sand or dry powder, Metal X

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

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

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

HAND HYGIENE- includes both hand washing and using alcohol based antiseptic cleansers.
❖ Hand contact is the primary method of infection transmission 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

❖ ALCOHOL-BASED CLEANSERS- Used when hands are ______________________

❖ HAND WASHING – Used when hands are _______________________________

CDC Hand Washing Procedure

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

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

I.K AYTONA 3

The last step in handwashing
The most important step in handwashing
Susceptible host
ADDENDUM
Turn off faucets with a clean paper towel to prevent recontamination
Rubbing /Applying friction
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 _______________ 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

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

Renal Blood flow

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

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,
and is the driving force behind glomerular filtration. The plasma ultrafiltrate already in Bowman’s space exerts a hydrostatic
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
of a plasma ultrafiltrate in Bowman’s space

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

Order of Blood Flow In the Nephron

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

Order of Urine formation from the nephron

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

GLOMERULAR FILTRATION

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

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

Barrier’s that prohibits the filtration of Large molecules

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

GLOMERULAR PRESSURE
➢ Juxtaglomerular apparatus- maintains the glomerular blood pressure
a. ______________________ - found in the afferent arteriole, secretes the Renin enzyme
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

RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS)

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
↓ Renin
ANGIOTENSIN I
↓ Angiotensin converting enzyme (ACE) = Lungs
ANGIOTENSIN II
↓

Sodium reabsorption at Aldosterone for Sodium

DA/CE ADH for water
PCT retention reabsorption

TEST FOR GLOMERULAR FILTRATION

Clearance test =BEST INDICATOR OF OVERALL GLOMERULAR FUNCTION
1. Inulin clearance test
_____________________
Inulin is a polymer of fructose, is an extremely stable substance that is not reabsorbed or secreted by the
tubules. It is not a normal body constituent, however, and must be infused by IV at a constant rate throughout
the testing period.
2. Creatinine clearance test
Most commonly used clearance test
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
4. Beta 2 microglobulin
5. Radioisotopes
6. Urea clearance test = earliest clearance test

Formula for the computation of GFR using the creatinine clearance test

C = Urine creatinine___ X volume of urine/24hours x 1.73

Plasma creatinine A

NOTE ☺
1. By far the greatest source of error in any clearance procedure utilizing urine is the use of improperly timed urine specimens
2. Plasma/serum creatinine can be collected anytime within 24 hours of urine collection
3. A blood sample of 1 mL (minimum 0.5 mL) in a labeled tube, preferably stored in refrigerated or frozen temperature
4. A 24-hour urine sample is collected from the patient to measure creatinine clearance. A plastic collection container is used to collect
urine. The collection starts with an empty bladder. At the start, the patient urinates into the toilet and flushes. The date and time get
recorded at the start of the collection. For the next 24 hours, the patient will collect urine and store into a container at room
temperature. Total urine collected for 24 hours gets sent to the laboratory for analysis. 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

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
MDRD-IDMS GFR = 175 × serum creatinine–1154 × age–0.203 × 0.742 (if patient is female) × 1.202 (if patient is
Traceable formula black/African-american)
Other MDRD
formula

Parameters 4 PARAMETERS (SEAS) = serum creatinine, ethnicity, age, sex

included in MDRD 6 PARAMETERS (BASES) = BUN, Age, Serum creatinine, Ethnicity, Serum albumin

Cockroft and gault formula

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 is capability is often the first function affected in renal disease.

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

TWO MECHANISM OF TUBULAR REABSORPTION:

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

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
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 _____________ 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 ___________ in the distal convoluted tubules and collecting ducts of the
kidney.
↑ Body Hydration = ↓ADH = ↑ Urine Volume (Dilute)
↓ Body Hydration = ↑ADH = ↓Urine volume (Concentrated)

TEST FOR TUBULAR REABSORPTION

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

- 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
and hyponatremia, and evaluating the secretion of and renal response to ADH. Thes evaluations may require
determination of serum in addition to urine osmolarity
- The normal urine to serum ratio should be 1:1 to 3:1.
- Measurement of freezing point depression was the first principle incorporated into clinical osmometers, and many
instruments
- 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).

DIFFERNTIATING NEUROGENIC AND NEPHROGENIC DIABETES INSIPIDUS

The ratio of urine to serum osmolality in conjunction with procedures such
as controlled fluid intake and injection of ADH, is used to differentiate
whether it is neurogenic diabetes insipidus or nephrogenic diabetes insipidus

Results
Urine osmolality >800mOm Neurogenic DI
Urine: Serum ratio is 3:1
Urine osmolality <400mOsm Nephrogenic DI
Urine: Serum ratio is 1:1

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

C. Water deprivation test

• ________________– patients deprived of fluids for 24 hours before measuring Specific Gravity
• ________________ – compared the volume and S.G of day and night urine samples

D. Free water clearance test

• The free water clearance is determined by first calculating the osmolar clearance using the standard clearance
formula
• 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.

𝑼𝒓𝒊𝒏𝒆 𝒐𝒔𝒎𝒐𝒍𝒂𝒍𝒊𝒕𝒚 𝒙 𝑼𝒓𝒊𝒏𝒆 𝒗𝒐𝒍𝒖𝒎𝒆

FORMULA =
𝑷𝒍𝒂𝒔𝒎𝒂 𝒐𝒔𝒎𝒐𝒍𝒂𝒍𝒊𝒕𝒚
then subtracting the osmolar clearance value from the urine volume in mL/min.

2nd FORMULA = 𝑂𝑠𝑚𝑜𝑙𝑎𝑟 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 − 𝑢𝑟𝑖𝑛𝑒 𝑣𝑜𝑙𝑢𝑚𝑒

I.K AYTONA 7
 RENAL SECRETION
➢ Involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate
➢ Substances are removed from the glomerular filtrate and returned to the blood
➢ Two major function of tubular secretion:
a. Elimination of waste products not filtered by the glomerulus
b. Regulation of acid-base balance in the body through the secretion of ____________________

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

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

Hippocrates Wrote the book of “uroscopy”

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

REASONS For Performing Urinalysis (CLSI)

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

URINE COMPOSITION
Urine consists of urea and other organic and inorganic chemicals dissolved in water.
Urine is normally 95% water and 5% solutes, 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.
Others Carbohydrates, pigments, fatty acids, mucin, enzymes, hormones; may be present in small amounts
depending on diet and health

I.K AYTONA 8
NOTE ☺
Urea is the major organic component of urine
Chloride is the major inorganic component of urine followed by Sodium then Potassium
A high urea and creatinine content can identify fluid as urine.

URINE VOLUME
Urine volume depends on the amount of water that the kidneys excrete.
Factors that influence urine volume include –fluid intake, fluid loss from non-renal sources, variations in the secretion of ADH,
and need to excrete increased amounts of dissolved solids, such as glucose or salts.
Normal daily urine output is usually 1200 to 1500 mL, a range of 600 to 2000 mL is considered normal
The kidney excretes two to three times more urine during the day than during the night

A. Oliguria – decrease urine output

• Less than 1mL/kg/hr in infants
• Less than 0.5 mL/kg/hr in children
• Less than _________________
Causes: Dehydration, vomiting, diarrhea, perspiration, severe burns

B. Polyuria- increase in daily urine output

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

Analysis of urine in Differentiating between DM and DI

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

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 ___

- the required specimen volume for urine microscopic analysis is _____, 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.
❖ Notify floor, nursing station, doctor’s office, etc. of problem and why it must be corrected 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)

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.

Preservatives
Refrigeration (2-8’C)
*the easiest and most
common
Thymol
Boric acid
Formalin
Toluene
Sodium Fluoride
Phenol
Gray C and S tube
Cherry red/ yellow top
tube
Yellow Plain UA
Saccomanno fixative
(Ethanol + Carbowax)
Sodium carbonate
URINE PRESERVATIVES
Advantages
Does not interfere with
chemical tests
Preserves glucose and
sediments well
*Preserves protein and
formed elements well
*Does not interfere with
routine analysis other than pH Interferes with drug and
*Prevents bacterial growth and hormone analyses
metabolism
EXCELLENT SEDIMENT
PRESERVATIVE
Does not interfere with
routine test
PREVENTS GLYCOLYSIS
GOOD FOR DRUG ANALYSIS
Does no interfere with
routine test
Preserves bacteria
Sample stable at RT for 48hr
Stable for 72 hours
FOR AUTOMATED
INSTRUMENTS
Preserves cellular elements
Inexpensive
Stabilizes porphyrins,
porphobilinogen, etc.
Disadvantages
PRECIPITATES AMORPHOUS
CRYSTALS
Raises specific gravity by
hydrometer
Interfere with acid precipitation test
for protein
May precipitate crystals when used
in large amounts
*Acts as reducing agent
*Interfere with chemical tests for
glucose, blood, leukocyte esterase,
and copper reduction
*False-negative reagent strip tests
for blood and urobilinogen
Floats on surface of specimens and
clings to pippete and testing
materials
Inhibits reagent strip tests for
glucose, blood, and leukocytes
Causes an odor change
Decreases pH; do not use if urine is
below minimum fill line
Bilirubin and urobilinogen may be
decreased if specimen is exposed
to light and left at RT
Must refrigerate within
2 hours
Unacceptable for urinalysis testing
Additional Information
Prevents bacterial growth
for 24 hours.
Keeps pH at 6.0
-bacteriostatic at 18g/L
Can also be used for
cytology (Brunzel)
May use sodium benzoate
instead of fluoride for
reagent strip testing
Use 1 drop per ounce of
specimen
Preservative is boric acid
Preservative is sodium
propionate
Round or conical bottom
Used for CYTOLOGICAL
EXAMINATION
For quantitative analysis of
porphyrins,
porphobilinogen, etc.

Concentrated HCL can be used to preserve urine for catecholamines measurement

I.K AYTONA 10

Random Specimen
First Morning specimen
Or 8-hour specimen
Second morning/fasting spx
2- hour post prandial specimen
Timed specimens (E.g 24 hours
urine specimen)
Afternoon urine (2pm to 4pm)
12 hours urine specimen
Catheterized specimen
TYPES OF URINE 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
Random urine “clean catch” with prior hydration = ideal for cytology
Ideal urine specimen for routine UA
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
___________________
___________________
___________________
For glucose or diabetic monitoring and screening
Collect urine after 2 hours of meal
________________________
For ________________________
Urine specimen for clearance test
Urine specimen for evaluation of fistulas
To obtain an accurate timed specimen, the patient must begin and end the
collection period with an empty bladder.
All specimens should be refrigerated or kept on ice during the collection period and
may also require addition of a chemical preservative.
On its arrival in the laboratory, a 24-hour specimen must be thoroughly mixed and the
volume accurately measured and recorded
Preferred for urobilinogen measurements
Ideal for screening microalbuminuria (Brunzel)
For determination of urine albumin, creatinine, and the albumin-to creatinine ratio
______________

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)
_________________
Pediatric specimen We –we bag
Soft, clear plastic bags with hypoallergenic skin adhesive to attach to the genital area of
both boys and girls

PROSTATITIS SPECIMENS
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.
EPS Expressed prostatic secretion
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

___________________________- 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.

*Container capacity: ________

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

NOTE ☺ - How to Check for Urine Color

Care should be taken to examine the specimen under a good light source, looking down through the container against a white
background

Color Cause Clinical/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)
-Methylene blue -fistulas
-Phenol -when oxidized (green urine color)
-Clorets -mouth deodorant (green urine color)
-Indican -bacterial infection, intestinal disorders
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

I.K AYTONA 12
 -Beets
-Rifampin
-Menstrual contamination
Red- brown -myoglobin (25 mg/dl)
-RBCs oxidized to methemoglobin
-Fuchsin, aniline dye
Port Wine -porphyrins/ Porphyria
/Burgundy red
Brown Homogentisic acid (Alkaptonuria)
Black Melanin or Melanogen, Malignant
melanoma
-Argyrol (anti-septic)
-Methyldopa or Levodopa
-Metronidazole (Flagyl)
-Phenol derivates
-alkaline urine of genetically susceptible person
-medication for Tuberculosis
-cloudy specimen with RBCs, mucus, and clots
-
-seen in acidic urine after standing
-Foods, Candy
-negative test for blood, may require additional testing
-maybe colorless in Lead poisoning
-seen in alkaline urine after standing
-urine darkens on standing and reacts with nitroprusside and ferric
chloride
- color disappears with ferric chloride
-antihypertensive drug
-darkens on standing, for parasitic infection
-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 refers to the “Transparency or Turbidity “ of a urine specimen
The specimen should be in a clear container
The clarity of a urine specimen certainly provides a key to the microscopic examination results, because the amount of
turbidity should correspond with the amount of material observed under the microscope
Clear urine is not always normal.
Nubecula = Faint cloud in urine after standing due to WBCs, epithelial cells and mucus

NOTE ☺ How to Check for Urine Clarity

Visually examining the Mixed specimen while holding it in front of a light source. View through a newspaper print

Urine Clarity Reporting

Clarity Term 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
-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 Amorphous phosphates, carbonates
Soluble with heat 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 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

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

CLINICAL SIGNIFICANCE OF URINE SPECIFIC GRAVITY RESULTS (Brunzel,3rd.)

S.G Indication / Cause
1.000 Physiologically impossible–same as 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

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 __________________ of specimen
• It is less accurate that other methods and is not recommended by the CLSI
• The urinometer reading needs to be corrected for temperature, glucose and protein.
• The calibrated temperature printed on the instrument is usually about 20 oC.
• To Correct for the S.G:
a. Add 0.001 for every 30C above the calibration temp.
b. Subtract 0.001 for every 3oC below the calibration temp.
c. Subtract 0.004 for every 1 gram of glucose
d. Subtract 0.003 for every 1 gram of protein

Example:
A specimen that has been left at 29 oC has been reported to contain 2g/dl of glucose and 1g/dl
of protein. The initial S.G was 1.035.Calculate the corrected S.G

a. Temperature: add 0.001 for every 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 for every 1g/dl so 0.003 x 1 = -0.003

Formula: 1.035 + 0.003 – 0.008 – 0.003 = 1.027 corrected SG

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

I.K AYTONA 15
 Refractometer (TS meter)
refractive index.
It determines the concentration of dissolved particles in a specimen. It does this by measuring
• 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).
• 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_________________
c. 5% NaCl – read _________________
d. 9% Sucrose – read _________________

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

CHEMICAL ANALYSIS OF URINE

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

Reagent Strip Technique /Procedure

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

Errors from Improper Technique

a. Formed elements such as WBC and RBC sinks to the bottom of the specimen and will be undetected in an unmixed
specimen
b. Allowing the strip to remain in the urine for an extended period may cause leaching of reagents from the
pads.
c. Run-over between chemicals on adjacent pads, producing distortion of the colors. To ensure against run –over, blot
the edge of the strip with adsorbent paper and holding the strip horizontally while comparing it with color
chart.
d. 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.

Handling and Storing Reagent Strips

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

QUALITY CONTROL OF REAGENT STRIPS

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
First morning urine pH
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 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

REAGENT STRIP REACTION (60 seconds)

Principle ___________________________

Methyl red + H+ → Bromthymol blue – H+

(Red to yellow) → (green to blue)
pH 4.0 -6.0 pH 6.0-9.0
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 ___________
Radiographic Contrast dye S.G = >1.040
Not A urine S.G = <1.002

REAGENT STRIP REACTION (45 seconds)

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

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

↓H ↑H ↑↑↑ H

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)
Normal urine contains <150mg/24hrs of protein (Henry’s)
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
Clinical proteinuria = ≥30mg/dl or ≥300mg/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
Heat reactivity test in Urine: Coagulates/Precipitates at _______ and dissolves
at______oC
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-Wilson’s disease

• decreased GFR
• associated with renal failure in persons with Type I and II Diabetes mellitu
• 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.

B.Amyloidosis
C.Immune complexes found in SLE and Streptococcal glomerulonephritis
D.Toxic substances
E.Pre-eclampsia and Eclampsia
F. Orthostatic / Cadet / Postural proteinuria
Urine Specimen Orthostatic Proteinuria Clinical Proteinuria
1st morning
2hours after standing

II.Tubular Proteinuria
• Normally filtered albumin can no longer be reabsorbed
A. Fanconi’s syndrome
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.

I.K AYTONA 18
 TEST FOR MICROALBUMINURIA
Albumin Excretion Normal: 0-20 ug/min
Rate (AER) Microalbuminuria:
Immunologic Micral-Test ImmunoDip
• Principle: Enzyme immunoassay • Principle: Immunochromographics
• 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.

NORMAL 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"-
dinitrophenyl)-3,4,5,6-tetrabromo sulphonphthalein (DIDNTB), which has a
higher sensitivity and specificity for albumin.
DIDNTB strips can measure albumin between 8 and 15 mg/dL (80 to 150 mg/L)
without inclusion of other proteins.

Color range: Pale green to Aqua Blue

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
Reagent: Copper sulfate (CuSO4), 3,3',5,5'-tetramethylbenzidine (TMB), and
diisopropyl benzene dihydroperoxide (DBDH)

Color range: Orange (negative) to green to blue

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 Protein (Sorensen’s) error of indicator

Indicator + protein -------------------------→ protein + Hydrogen

(Yellow) (+) Blue-Green

I.K AYTONA 19
 NOTE
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
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

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

PROCEDURE – 3mL of 3% SSA (Exton’s reagent) or 7%SSA + 3ml centrifuged urine = (+) cloudiness

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

SSA INTERFERENCES
False increase/positive
False decrease/negative
• Radiographic contrast dye/x-ray film
• Drugs (Tulbotamide, Penicillin, Sulfonamide, Cephalosporin)
• para-amino-salicylic acid/Salicylates
• 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 Crystalline
CAUSE A POSITIVE REACTION?

IV.GLUCOSE
Most frequently performed chemical analysis on urine (due to monitoring of DM).
Renal threshold- plasma concentration of a substance at which tubular reabsorption stops
Renal threshold for glucose = 160-180 mg/dl
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:
Diabetes Mellitus and Gestational Diabetes Mellitus, Fanconi’s syndrome, 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

I.K AYTONA 20
 REAGENT STRIP REACTION FOR GLUCOSE (30 seconds)
Principle
Glucose oxidase
Glucose + O2 --------------------------→gluconic acid + Hydrogen Peroxide

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

COPPER REDUCTION TEST (CLINITEST / BENEDICT’S TEST)

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)
c. Sodium citrate /citric acid – for heat production
d. Sodium hydroxide – for heat production

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
Procedure 5 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

* Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of sodium
hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium
carbonate to prevent room air from interfering with the reduction reaction.

* Clinitest tablets are very hygroscopic and should be stored in their tightly closed packages. A
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:
a. Reducing agents such as vitamin C, formalin, uric acid, and some drugs (cephalosporin)
False Negative:
a. Oxidizing agents such as detergent

I.K AYTONA 21
Grading
Grading Color Sugar level Negative clear blue color, blue precipitate may form
negative Blue ¼% Trace-bluish-green color
1+ Green ½% 1 + green color, green or yellow precipitate
2+ Yellow ¾% 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
4+ Negative -Oxidizing agent interference
-False-positive reagent strip because of contaminants (e.g., oxidizing agents, peroxidases)
-False negative Clinitest due to presence of radiographic contrast media
-Defective Clinitest tablets (e.g., outdated)
1+ Negative Small amount of glucose present since reagent strip is more sensitive
Negative Positive -Non glucose reducing substance
-Possible interfering substance such as reducing agent
-Reagent strip interference (e.g., high specific gravity, low urine temperature)
-Reagent strips defective (e.g., outdated, improperly stored)

V.KETONES
Result from increased fat metabolism. 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:
a. 78% Beta Hydroxybutyric acid – major ketone but not detected in reagent strip
b. 20% Acetoacetic acid (AAA) / Diacetic acid – parent ketone
c. 2 % Acetone – detected only when glycine is present

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

Acetoacetate and acetone + sodium nitroprusside + glycine--→ (+) Purple

Reagents Sodium nitroprusside (nitroferricyanide), glycine (Chemstrip)

Reporting / Grading Grading Quantity
Negative --
Trace 5mg/dl
1+ (small) 15mg/dl
2+ (moderate) 40mg/dl
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
 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
f. Brown recluse spider bites h. Muscle infection(myositis)
i. Trauma
Note j. Crush syndrome
Hemoglobin must be present in the urine in an k. ALCOHOLISM
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. Blondheim’s 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

Hemoglobin
Hydrogen peroxide + chromogen -----------------------------→ oxidized chromogen + H20
Psuedoperoxidase

(-) yellow, (+) Green to Blue

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
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
 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

Bilirubin, a highly pigmented yellow compound, is a

degradation product of hemoglobin. Under normal
conditions, the life span of red blood cells is approximately
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
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.
✓ 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 Ehrlich ‘s reaction

Multistix: Uses Ehrlich reagent

Urobilinogen + p-dimethlyaminobenzaldehyde ----------→ red color

Chemstrip: uses 4-methyloxybenzene-diazonium-tetrafluoroborate (more specific than ehrlich’s rxn)

Urobilinogen + diazonium salt -------→ red azodye
Note Ehrlich-reactive compounds: porphobilinogen, indican, p-aminosalicylic acid, sulfonamides, methyldopa,
procaine, chlorpromazine --- also gives positive reaction for Ehrlich’s reaction
Interference False positive:
a. Other Ehrlich’s compound
b. Highly pigmented urine
False negative:
a. Old specimens
b. Preservation in formalin
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 - Strasinger

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

HOESCH TEST (INVERSE EHRLICH REACTION)

➢ Rapid screening test for porphobilinogen (>2mg/dl)
➢ Procedure:2 gtts urine + 2mL Hoesch reagent (Ehrlich’s reagent 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.

CORRELATION OF BILIRUBIN AND UROBILINOGEN

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
 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

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
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 ___________ 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
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
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 (≥3g/dl), oxalic acid
(in acidified urine that has 4.4pH or below) and ascorbic acid
b. Antibiotics such as gentamicin, cephalosporins, tetracyclines,
c. Inaccurate timing

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 ≥25mg/dl
Ascorbic acid level that causes a negative reaction to glucose ≥50mg/dl

Brand Reading time Positive result

Stix 60 seconds Blue color
C-stix 10 seconds Blue color

SUMMARY
Test Principle (+) result Reading time
Bilirubin Diazo reaction Violet, tan, or pink 30 secs
Glucose Double sequential enzymatic reaction Potassium iodide 30secs
= blue-green to brown
Ketones Sod. Nitroprusside (Legal’s reaction) Purple 40secs
S.G pKa change of polyelectrolyte Diluted = blue 45secs
Concentrated =yellow
pH Double indicator system Acidic = red to yellow 60secs
Alkaline = green to blue
Protein Protein (Sorensen’s) error of indicator Blue-green 60secs
Blood Pseudoperoxidase activity of hemoglobin Green to blue 60secs
urobilinogen Ehrlich’s reaction Red 60secs
Nitrite Greiss reaction Pink 60secs
Leukocyte Leukocyte esterase Purple 120secs

MICROSCOPIC ANALYSIS OF URINE

ADDIS COUNT – first procedure to standardize the quantitation of formed elements, used a hemocytometer
• Specimen: _______________________

NORMAL VALUE OF ADDIS COUNT

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

Specimen Preparation

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

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
 SEDIMENT STAINS
STAIN ACTION FUNCTION
Sternheimer-Malbin(a supravital -Delineates structure and contrasting -Identifies WBCs, epithelial cells, and
stain consisting of Crystal violet and colors of the nucleus and cytoplasm casts
safranin)
0.5%Toluidine blue -Enhances nuclear detail Differentiates WBCs and renal
(a metachromatic supravital stain) tubular epithelial (RTE) cells
2% acetic acid Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs, yeast, oil
WBCs droplets, and crystals
Lipid Stains: Oil Red Stain triglycerides and neutral fats Identifies free fat droplets and lipid
O and Sudan III orange-red containing cells and casts
Gram stain Differentiates gram-positive and Identifies bacterial casts
gram negative bacteria
Hansel stain Methylene blue + EosinY Identifies urinary eosinophils
Stains eosinophilic granules
Prussian blue stain Stains structures containing iron Identifies yellow-brown granules of
hemosiderin in cells and casts
Sedi and KOVA stain Modified Sternheimer Malbin Hyaline cast appears as pink
The dye is absorbed well by WBCs, Motile bacteria are unstained
epithelial cells, and casts, providing Non-Motile bacteria stains purple
clearer delineation of structure and T.vaginalis stains Light blue-green
contrasting colors of the nucleus and
cytoplasm

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. Wright’s stain or Giemsa stain also distinguishes 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
sediment and Papanicolaou’s staining

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 microscope are adapted to bright-field

Phase contrast Enhances visualization of elements with low refractive indices, such as hyaline casts,
microscopy mixed cellular casts, mucous threads, and Trichomonas
Type of microscopy in which variations in the specimen’s refractive index are converted into
variations in light intensity or contrast
Adaptation of a bright-field microscope with a phase-contrast objective lens and a matching
condenser. Two phase rings that appear as “targets” are placed in the condenser and the
objective.
Light passes to the specimen through the clear circle in the phase ring in the condenser, forming
a halo of light around the specimen

Polarizing Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals.
microscopy It uses halogen quartz lamp that produces light rays of many different waves
A substance that rotates the plane of polarized light 90 degrees in a clockwise direction is said
to have positive birefringence.
Substance that rotates the plane in a counterclockwise direction has negative
birefringence
Bright-field microscopes can be adapted for polarizing microscopy. Two polarizing filters
must be installed in a crossed configuration

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
(Hoffman) and differential-interference contrast (Nomarski). Bright-field microscopes
can be adapted for both methods.
Converting brightfield microscopy to differential interference contrast microscopy requires (1) a
polarizer placed between the light source and the condenser, (2) 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.
2. Always hold the microscope in a vertical position.
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.
5. Clean the oil immersion lens after use.
6. Always remove slides with the low-power objective raised.
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 The microscope lens or system of lenses located closest to the viewer’s eye. It produces the
secondary image magnification of the specimen
Field diaphragm Microscope component that controls/regulates the diameter of light beams that strike the specimen
and hence reduces stray light.
Field of view The circular field observed through a microscope
Köhler illumination Type of microscopic illumination in which a lamp condenser (located above the light source) focuses
the image of the light source (lamp filament) onto the front focal plane of the substage condenser
(where the aperture diaphragm is located)
Mechanical stage Microscope component that holds the microscope slide with the specimen for viewing.
Objectives The lens or system of lenses located closest to the specimen. The objective produces the primary
image magnification of the specimen.
Parcenter Term describing objective lenses that retain the same field of view when the user switches from
one objective to another of a differing magnification
Parfocal Term describing objective lenses that remain in focus when the user switches from one objective to
another of a differing magnification.
Resolution Ability of a lens to distinguish two points or objects as separate.
Cytocentrifugation A technique used to produce permanent microscope slides of urine sediment and body fluids. The
end result is a monolayer of the urine sediment components with their structural details greatly
enhanced by staining
Magnification Process of enlarging or magnifying an object’s size without affecting its actual or physical size

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

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 and malignancy
of the urinary tract and to 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, colorless circles or “shadow cells,”
because the hemoglobin may dissolve out
They may become crenated in hypertonic urine and appear as small, rough cells with crinkled edges
In Concentrated /Hypersthenuric urine: Crenated cells /ECHINOCYTES / Irregularly shaped
In Diluted / Hyposthenuric urine: Ghost cells / Swollen RBC
Dysmorphic or Distorted RBC – vary in sizes, mainly they are acanthocytes, it is associated with
glomerular bleeding
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! Wright’s stain can be used to further analyze the dysmorphic RBCs. Analysis shows the cells to be
hypochromic 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

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.

B. Transitional Epithelial (Urothelial) cells/ Bladder epithelial cells

Transitional epithelial cells originate from the lining of the renal pelvis, calyces, ureters, and bladder,
and from the upper portion of the male urethra.
Transitional epithelial cells are smaller than squamous cells and appear in several forms, including spherical,
polyhedral, and caudate. 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.
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

I.K AYTONA 31
 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

Conditions producing tubular necrosis include exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin
toxicity, viral infections (hepatitis B), pyelonephritis, allergic reactions, malignant infiltrations, salicylate poisoning, and acute
allogenic transplant rejection

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

IV. Oval Fat Bodies

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

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

VII.Parasites
Trichomonas vaginalis – most frequent parasite encountered in urine
Schistosoma haematobium – bladder parasite, associated with bladder tumors
Enterobius vermicularis- most common contaminant ova
Cyst of Giardia lamblia- observed in urine sediment as the result of fecal contamination of infected individuals

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.
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

I.K AYTONA 32
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
Hyaline Cast→ Cellular cast→ Coarse granular cast → Fine granular cast → Waxy cast → Broad Cast

Step by Step Analysis of the Formation of Tamm-Horsfall protein matrix

1. Aggregation of Tamm-Horsfall protein into individual protein fibrils attached to the RTE cells
2. Interweaving of protein fibrils to form a loose fibrillar network (urinary constituents may become enmeshed in the
network at this time)
3. Further protein fibril interweaving to form a solid structure
4. Possible attachment of urinary constituents to the solid matrix
5. Detachment of protein fibrils from the epithelial cells
6. Excretion of the cast

NOTE
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

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
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

Sternheimer-Malbin stain and KOVA stain: Pink color

Normal value: 0-2 /low power field

I.K AYTONA 33
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.
Pathologic increase: glomerulonephritis and pyelonephritis
Physiologic increase: strenuous exercise

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

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
Epithelial cast / RTE cast

I.K AYTONA 34
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.
➢ Usually reported as rare, few, moderate, or many per hpf
➢ Abnormal crystals may be averaged and reported per lpf

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

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: ↑food high in ascorbic acid and Dilute HCL
a.dihydrate (weddelite) 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-shape or “coffin lid” Presence of urea-splitting Dilute acetic acid
magnesium Feathery appearance when they bacteria
ammonium phosphate/ disintegrate, Fern-leaf shape, sheets or
struvite flakes Associated with P. vulgaris
Calcium phosphate/ Colorless, flat plates, thin prisms often in None Dilute acetic acid
apatite rosette form

Rosettes form may resemble sulfonamide

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

I.K AYTONA 35
 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

Note! Leucine and tyrosine crystals may

occur together; leucine may be
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
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
Solubility in ammonia
Solubility in dilute HCL
Birefringence
Cyanide nitroprusside reaction

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.
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

URINARY SEDIMENT ARTIFACTS

1. Starch granules
➢ Spheres with dimpled center
➢ Resembles as fat droplet with “maltese cross” formation on polarizing microscope

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

Manner of Reporting
RBC, WBC Average number per 10 HPF
Casts Average number per LPF
Squamous epithelial 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

I.K AYTONA 37
 Reporting Mucus 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; “more than few, less than many”
Many (3+) Prominent; large number present in all FOVs
Packed (4+) FOV is crowded by or overwhelmed with the elements

RENAL DISORDERS

GLOMERULAR DISORDERS
DISORDER ETIOLOGY CLINICAL COURSE PRIMARY OTHER SIGNIFICANT
URINALYSIS TESTS
RESULT
Acute Deposition of immune Rapid onset of Macroscopic Antistreptolysin O titer
Glomerulonephritis complexes formed in hematuria and edema. hematuria,
conjunction with group A Proteinuria, Anti- group A
Streptococcus infection, Permanent renal 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
glomerular membrane end-stage renal failure Red Blood Cell casts

Good pasteur’s Attachment of cytotoxic Hemoptysis and Macroscopic Anti- glomerular

syndrome antibody formed during
viral respiratory infections
to glomerular and alveolar
basement membranes
Wegener’s Antineutrophilic cytoplasmic
Granulomatosis autoantibody binds to
neutrophils in vascular
walls producing damage to
small vessels in the lungs
and glomerulus
Henoch-Schonleinpurpura Occurs primarily in children
following viral respiratory
infections; a decrease in
platelets disrupts vascular
integrity
dyspnea followed by hematuria, Basement membrane
hematuria. Proteinuria, Antibody
Red Blood Cell casts
Possible progression to
end-stage renal failure
Pulmonary symptoms Macroscopic Antineutrophilic
including hemoptysis hematuria Cytoplasmic antibody
develop first followed Proteinuria
by renal involvement Red Blood Cell Casts
and possible
progression to end-
stage renal failure
Initial appearance of Macroscopic Stool occult blood
purpura followed by hematuria,
blood in sputum and Proteinuria,
stools and eventual Red Blood Cell casts
renal involvemen.

IgA nephropathy Deposition of IgA on the
(Berger’s disease) glomerular membrane
resulting from increased
levels of serum IgA
Membranous Thickening of the
glomerulonephritis glomerular membrane
following IgG immune
complex deposition
associated with systemic
disorders
Complete recovery is
common, but many
progress to renal
failure.
Recurrent Early stages: Serum immunoglobulin A
macroscopichematuria Macroscopic or
following exercise with microscopic
slow progression to hematuria
chronic Late stages: See
glomerulonephritis chronic
glomerulonephritis
Slow progression to Microscopic Antinuclear Antibody
Nephritic syndrome or hematuria Hepatitis surface antigen
possible remission Proteinuria Flourescent treponemal
antibody-absorption test
(FTA-ABS)

I.K AYTONA 38
Membranoproliferative Cellular proliferation Noticeable progression Hematuria Serum complement levels
glomerulonephritis affecting the capillary walls to chronic Proteinuria
or the glomerular glomerulonephritis to
“TRAM TRACKING basement membrane, nephritis syndrome.
PATTERN OF THE possibly immune mediated.
GLOMERULUS”
Chronic glomerulonephritis Marked decrease in renal Noticeable decrease in Hematuria Blood Urea Nitrogen
function resulting from renal function Proteinuria Serum Creatinine
glomerular damage progressing to renal Glucosuria Creatinine clearance
precipitated by other renal failure. Cellular and Electrolytes
disorders granular casts
Waxy & broad casts
Nephrotic Syndrome Disruption of the Acute on set following Heavy proteinuria, Serum albumin
electrical charges that systemic shock Microscopic Cholesterol
produce the tightly fitting Gradual progression hematuria, Triglycerides
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
Fatty & waxy casts

Minimal change disease Disruption of podocytes Frequent complete Heavy proteinuria Serum albumin
(Lipid nephrosis) occurring primarily in remission following Transient hematuria Cholesterol
children following allergic corticosteroid treatment Fat droplets Triglycerides
reactions and
immunizations
Focal segmental Disruption of podocytes in May resemble nephrotic Proteinuria Drug of Abuse
glomerulosclerosis certain areas of glomeruli syndrome or minimal Microscopic HIV tests
associated with heroin change disease. hematuria
and analgesic abuse and
acquired immunodeficiency
syndrome
Alport syndrome Genetic disorder showing Slow progression to to See Nephrotic
lamellated and thinning nephritic syndrome and syndrome
of glomerular basement end stage renal disease
membrane
Diabetic Nephropathy Most common cause of (+)Microalbuminuria
(Kimmelstiel- Wilson ESRD (+)Micral test
disease) Deposition of glycosylated
proteins on the glomerular
basement membranes
caused by poorly controlled
blood glucose levels

NEPHROTIC SYNDROME LAB FINDINGS

URINE FINDINGS PLASMA OR BLOOD FINDINGS
1. Proteinuria 1. Increase A2-Macroglobulin in electrophoresis
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
-Conditions producing tubular necrosis include RTE CELLS, RTE CASTS
exposure to heavy metals, drug-induced toxicity, Odorless urine
hemoglobin and myoglobin toxicity, viral Isosthenuria
infections (hepatitis B), pyelonephritis, allergic
reactions, malignant infiltrations, salicylate
poisoning, and acute allogenic transplant
rejection
Fanconi syndrome Generalized failure of tubular reaction Glucosuria, Cystinuria
in the Proximal Convoluted tubule Proteinuria, phosphaturia
Urinary pH can be very Low due to the
failure to reabsorb bicarbonate
Diabetes Insipidus A.Neurogenic DI Lows Specific gravity urine
-Failure of the hypothalamus to produce Polyuria
ADH
-Low ADH

b. Nephrogenic DI
-inability of the renal tubules to respond to
ADH
-Normal to increase ADH

I.K AYTONA 39
Renal Glucosuria Defective tubular reabsorption of glucose Normal Blood glucose
Increase Urinary glucose
Cystitis (Lower UTI) Ascending bacterial infection of the urinary WBCs, Bacteria
bladder Microscopic hematuria
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
and interstitium caused by structural WBC CAST, Bacterial cast
abnormalities affecting the flow of urine Granular, Waxy, and Broad Cast
Hematuria and Proteinuria
Acute Interstitial nephritis Allergic inflammation of the renal Hematuria, proteinuria
interstitium in the response to certain WBCs (Increase Eosinophil)
medication WBC cast
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 Bartter’s syndrome
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 Gordon’s syndrome
Excessive reabsorption of phosphate Pseudohypoparathyroidism

DISTAL CONVOLUTED TUBULAR DYSFUNCTIONS

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

Rapidly progressive
(Crescentic) glomerulonephritis
Good pasture’s syndrome
Henoch Schonlein purpura
Membranous glomerulonephritis
Membranoproliferative
Glomerulonephritis
Focal
IgA Nephropathy
ADDENDUM
Some forms may demonstrate increased fibrin degradation products, cryoglobulins, and the
deposition of IgA immune complexes in the glomerulus
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
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
Disorders associated with membranous glomerulonephritis development include systemic lupus
erythematosus, Sjögren syndrome, secondary syphilis, hepatitis B, gold and mercury treatments,
and malignancy.
Marked by two different alterations in the cellularity of the glomerulus and peripheral capillaries.
A. Type 1 displays increased cellularity in the subendothelial cells of the mesangium
(interstitial area of Bowman’s capsule), causing thickening of the capillary walls
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
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
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) Nephrons are “functionally” abnormal, no histologic abnormality is usually present
Usually reversible
Primary causes of ARF include a sudden decrease in blood flow to the kidney (prerenal), acute
glomerular and tubular disease (renal), and renal calculi or tumor obstructions (postrenal)
Ischemic acute tubular necrosis is the most common cause of ARF

I.K AYTONA 40
 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

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:

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)

RENAL CALCULI CHARACTERISTICS

Major constituent of renal calculi
Very hard, dark brown color with rough surface
Associated with increase intake of foods with high purine content, and uromodulin-associated kidney disease
Yellowish to brownish red and moderately hard
Seen in hereditary disorders of cystine metabolism
Yellow brown, greasy and resembles an old soap
Least common calculi (1-2%)
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
✓ 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
✓ 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

I.K AYTONA 41
 URINE SCREENING FOR METABOLIC DISORDERS

AMINOACIDURIA

1. Over Flow Type

✓ INCREASE Amino acid in blood
✓ NORMAL 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
✓ Due to renal tubular defects
Ex: Cystinuria, Fanconi’s syndrome

PHENYLALANINE-TYROSINE DISORDERS
DISORDER Enzyme deficient Information Tests
Phenylketonuria ____________________ Mousy or musty odor FeCl3 tube test = (+) BLUE GREEN
- Inherited as an autosomal Urine Phenistix strip = (+) gray to gray green
recessive disease, it is 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
▪ Beta2-thienylalanine inhibits
growth of B.subtilis
▪ Phenylalanine counteracts the
action of Beta2-thienylalanine
▪ (+) result: _____________

Tyrosyluria/Tyrosinemia
Alkaptonuria
Melanuria
-Melanin is produced from
tyrosine by melanocytes and is
the pigment responsible for the
color of hair, skin, and eyes.
Deficient production of melanin
results in albinism.
Type1: “Rancid Butter odor FeCl3 tube test = (+) TRANSIENT GREEN
Fumarylacetoacetate urine”
hydrolase Nitroso-naphtol = (+) Orange Red
Type 2:
Tyrosine aminotransferase
Type 3:
p-hydroxyphenylpyruvic
acid dioxygenase
Homogentisic acid oxidase Urine darkens after FeCl3 tube test = (+) TRANSIENT BLUE
becoming alkaline from Clinitest = (+) Yellow ppt
standing at room
temperature
-- Due to overproliferation FeCl3 tube test = (+) BLACK /GRAY PPT
of melanocytes Sodium nitroprusside test = (+) Purple
Urine darkens upon air Ehrlich test (+) = Red
exposure

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

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.

I.K AYTONA 42
 BRANCHED CHAIN AMINO ACID DISORDER
Disorder Information Test
Maple syrup urine Disorder Amino acid that Increases in blood and urine 2,4 Dinitrophenylhydrazine (DNPH)
________________________ (+) ____________________________

-Caramelized sugar / burnt sugar/ Curry odor / Maple

syrup odor urine due to ketoacids in urine
Organic acidemias “Sweaty feet odor urine”
1. Isovaleric acidemia
2. Propionic acidemia Generalized symptoms of the organic acidemias include
3. Methylmalonic early severe illness, often with vomiting accompanied p-nitroaniline test = (+) Emerald green
acidemia by metabolic acidosis; hypoglycemia; ketonuria; and
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.

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 Obermayer’s test
(upon air exposure or oxidized) - FeCl3 + Urine + Chloroform → (+) purple
Tryptophan→ indole → indican
Seen in: Intestinal disorders, and
Hartnup’s disease
Argentaffinoma Carcinoid tumor involving argentaffin FeCl3 tube test = (+) blue – green
cells Nitroso-naphthol w/ nitrous acid = (+) Violet
✓ Produce 5-HIAA , a metabolite to black
of serotonin
✓ Avoid ______________

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

I.K AYTONA 43
 CYSTINE DISORDERS
Disorder Information Test
Cystinuria Renal type aminoaciduria Brand’s modification of Legal’s nitroprusside
Rgt: Cyanide nitroprusside = (+) Red-purple
Defective tubular reabsorption of:
- __________________________
Cystinosis Inborn Error of metabolism Brand’s modification of Legal’s nitroprusside
(-) gene that codes for an enzyme responsible for Rgt: Cyanide nitroprusside = (+) Red-purple
cysteine metabolism
Homocystinuria Defects in the metabolism of methionine that leads to Silver-nitroprusside test = (+) Red-Purple
increase homocysteine. Associated with methionine -Fresh urine should be used
malabsorption
MUCOPOLYSACCHARIDE DISRODERS
Disorder Information Test
Hurler syndrome Mucopolysaccharides accumulate in the
cornea of the eye

Hunter syndrome Sex linked recessive, rarely seen in females

Sanfilippo syndrome Mental retardation only

NICE TO KNOW
1. Serotonin is produced from tryptophan by the argentaffin cells in the intestine and is carried through the body primarily by the platelets.
Normally, the body uses most of the serotonin, and only small amounts of its degradation product, 5-HIAA, are available for excretion in the urine.
2. The normal daily excretion of 5-HIAA is 2 to 8 mg, and excretion of greater than 25 mg/24 h can be an indication of argentaffin cell
tumors
3. If a 24-hour sample is used for 5-HIAA measurement, it must be preserved with hydrochloric or boric acid.
4. The increased homocystine can result in failure to thrive, cataracts, mental retardation, thromboembolic problems, and death.
5. False positive results in cyanide nitroprusside test is associated with the presence of ketones and homocystine
6. In mucopolysaccharidosis, the products most frequently found in the urine are dermatan sulfate, keratin sulfate, and heparan sulfate.
7. dietary changes that eliminate phenylalanine, a major constituent of milk, from the infant’s diet can prevent excessive buildup of serum
phenylalanine, thereby avoiding damage to the child’s mental capabilities
8. In PKU, urinary excretion of phenylpyruvic acid may take 2 to 6 weeks before it will occur
9. The appearance of black urine from a patient of any age should be reported to a supervisor.
10. Generalized symptoms of the organic acidemias include early severe illness, often with vomiting accompanied by metabolic acidosis; hypoglycemia;
ketonuria; and increased serum ammonia
11. Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting isoleucine, valine, threonine, and methionine to
succinyl coenzyme
12. 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
Lesch-Hyhan disease
Lack Hypoxanthine-guanine phosphoribosyl transferase
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

Porphyrin Disorders (Porphyrias)

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

Porphyria Elevated 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/
Ehrlich reaction
Blood FEP

I.K AYTONA 44
CARBOHYDRATES DISORDER
Melituria = general term for the presence of urinary sugar
Galactosuria, pentosuria, lactosuria, fructosuria, and glucosuria
Galactosuria = indicates inability to properly metabolize galactose to glucose such as in case of newborn errors

SYNOVIAL FLUID

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

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

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

REQUIRED TUBE TYPES FOR SYNOVIAL FLUID TESTS AND ORDER OF DISTRIBUTION
1. Chemistry and Serology = non-anticoagulated
2. Hematology and cytology = sodium heparin or liquid EDTA
3. Microbiology= Sterilized heparin or SPS

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

VISCOSITY
Normal: able to form a string ___________ long GRADING Interpretation
Test: Ropes/ Mucin clot test (Hyaluronate polymerization Test) Good Solid clot
▪ Reagent: uses 2-5 % acetic acid Fair Soft clot
▪ 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.

WBC count
Most frequently performed count
Diluting fluid: NSS with methylene blue, Hypotonic saline 0.3%), Saline with saponin

I.K AYTONA 45
 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

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 incubate at 37’C for 5 minutes

CELL COUNT NORMAL VALUES

RBCs <2000 / ul
WBCs < 200 / ul
WBC differential count Differential counts should be performed on cytocentrifuged preparations or on thinly
smeared slides. 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 Normal
vacuolated Viral infections
Synovial lining cell Similar to macrophage, but may be Normal
multinucleated resembling a mesothelial cell
LE cell Neutrophil containing characteristic ingested Lupus erythematosus
“round homogenous body”
Reiter cell Vacuolated macrophage with ingested Reiter syndrome
neutrophils Non-specific inflammation
RA cell (Ragocyte) Neutrophil with dark cytoplasmic granules Rheumatoid arthritis
containing immune complexes Immunologic inflammation
Cartilage cells Large multinucleated cells Osteoarthritis
Rice bodies Macroscopically resembles polished rice Tuberculosis, septic and rheumatoid
Microscopically shows collagen and fibirn arthritis
Fat droplets Refractile intracellular and extracellular Traumatic injury
globules stain with Sudan 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 medicat-ions (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
therefore do not appear in vacuoles
Crystals may be observed in Wright’s-stained smears
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
✓ Blue = perpendicular = (positive) Birefringence
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

I.K AYTONA 46
 Crystal Shape Compensated Polarized Light Significance
Monosodium urate Fine Needles Negative birefringence Gout
Calcium Rhombic squares, rods Positive birefringence Pseudogout or chondrocalcinosis
Pyrophosphate
Cholesterol Notched, rhombic plates Negative birefringence Extracellular, chronic arthritis condition
such as Rheumatoid arthritis
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

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
Increase in infection
Lactate or ACP Maybe requested to monitor the severity and prognosis of Rheumatoid arthritis
Protein Normal value = <3 g/dl
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
Common organisms that infect the synovial fluid:
1. Staphylococcus aureus
2. Streptococcus
3. Haemophilus
4. Neisseria gonorrheae

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

I.K AYTONA 47
 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
capillaries that serve the cavities and the capillary permeability.

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

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
2. Congestive heart failure- increase hydrostatic 2. Malignancy
3. Nephrotic syndrome= decrease oncotic pressure 3. Inflammation
4. Malnutrition= decrease oncotic pressure 4. Lymphatic duct obstruction
5. Hepatic cirrhosis = decrease oncotic pressure

TRANSUDATE VS EXUDATE
Transudate Exudate
Appearance Clear Cloudy
Fluid: serum protein ratio <0.5 >0.5
Fluid: serum LD ratio <0.6 >0.6
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
Serum ascites albumin gradient (SAAG) >1.1 <1.1
Glucose Decrease Increase
Rivalta’s test Negative Positive
Acetic acid + water + Unknown fluid
(+) heavy precipitation

Differentiation between ascitic fluid transudates and exudates is more difficult than for pleural and pericardial effusions. The serum-ascites albumin
gradient (SAAG) is recommended over the fluid:serum total protein and LD ratios for the detection of transudates of hepatic origin

METHOD OF COLLECTION

-3Ps (Pleural, Pericardial, Peritoneal fluid)

-Normal appearance should be clear, pale yellow

Fluid Aspiration technique (Collection) Organ located

Pleural fluid Thoracentesis Lungs
Pericardial fluid Pericardiocentesis Heart
Peritoneal or ascitic fluid Paracentesis Abdominal organs

Specimen Distribution
1. EDTA = Cell count and differential count
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

I.K AYTONA 48
 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 Absent Present
Triglycerides >110 mg/dl <50 mg/dl
Sudan III staining +++ Negative or weakly +
Onset Sudden gradual
Chylomicrons present absent

HEMATOLOGY DIFFERENTIAL COUNT ON SEROUS FLUID

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

SIGNIFICANCE OF CELLS SEEN IN PLEURAL FLUID

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

SIGNIFICANCE OF CHEMICAL TEST IN PLEURAL FLUID

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

*Ph less than 7.2= need for test tube drainage

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

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

PERITONEAL /ASCITIC FLUID

APPEARANCE SIGNIFICANCE
Clear, pale yellow Normal
Turbid Microbial infection
Green Gall bladder, pancreatic disorders
Blood streaked Trauma, infection, malignancy
Milky Lymphatic trauma and blockage
WBC COUNT SIGNIFICANCE
<500 cells/ ul Normal
>500 cells/ ul Bacterial peritonitis, cirrhosis
Note* bacterial peritonitis has an absolute neutrophil count of
>250cells/ul or >50% of total WBC Count
DIFFERENTIAL SIGNIFICANCE
Increase neutrophils Bacterial peritonitis
Malignant cells Malignancy
TEST SIGNIFICANCE
Peritoneal lavage >100, 000 RBCs/ ul indicates blunt trauma injury (intra-abdominal
bleeding)

I.K AYTONA 49
 TEST SIGNIFICANCE
CEA Malignancy of GI origin
CA 125 Malignancy of ovarian origin
Glucose Decrease in tubercular peritonitis, malignancy
Amylase Increase in pancreatitis, GI perforation
BUN/ Creatinine Ruptured/ punctured bladder
Gram stain and Culture Bacterial peritonitis
Acid fast stain Tubercular peritonitis
ADA Tubercular peritonitis

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

LIPOPHAGES
Macrophage containing fat droplets

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

AMNIOTIC FLUID COMPOSITION

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

FUNCTIONS
The primary functions of the fluid are:
1. to provide a protective cushion for the fetus,
2. allow fetal movement,
3. 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.

Normal volume range

Average normal volume

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
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

I.K AYTONA 50
SPECIMEN HANDLING

Test for fetal lung maturity
Test for cell culture and cytogenetic studies
Test for HDN
Should be placed in ice for delivery to the laboratory and kept refrigerated.
Filtration prevents loss of lung surfactants such as phospholipids.
Maintained at room temp or body temp
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

INDICATIONS OF PERFORMING AMNIOCENTESIS

14th In general, amniocentesis is a safe procedure, particularly when performed after the __ week of
gestation
15th to 18th week -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
➢ positive result “fern-like crystals” indicates the fluid is amniotic fluid

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
Yellow HDN Bilirubin
Meconium
Fetal death

Meconium = defined as a newborn’s first bowel. It is formed in the intestine from fetal intestinal secretions and swallowed amniotic
fluid. Biliverdin is responsible for its dark green color. It may be present in the amniotic fluid as a result of fetal distress.

TEST FOR HDN

A.K.A = O.D 450
Absorbance of amniotic fluid:
Normal = increase at 365nm, decrease at 550nm
HDN = Increase at 450nm (maximum bilirubin absorption)

Results are plotted on Liley graph:

Zone 1 Non affected or mildly affected fetus
Zone 2 Moderately affected fetus (requires close monitoring)
Zone 3 Severely affected fetus (requires intervention such as intrauterine or exchange transfusion)

INTERFERENCES IN O.D 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

TEST FOR NEURAL TUBE DEFECT

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
Screening test = ___________________
Confirmatory = ___________________

I.K AYTONA 51
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 at the alveolar surfaces in the newborn’s lungs.
- 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
ratio ▪ Lecithin for alveolar stability
▪ Sphingomyelin serves as a control
▪ Prior to 35 weeks’ gestation, the L/S ratio is usually less than 1.6 because large amounts of
lecithin are not being produced at this time. After 35 weeks’ gestation, the lecithin
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

▪ It based on agglutination of PG using polyclonal anti-PG antibodies; qualitative results
▪ Results are reported as negative (immature), or as low positive (mature) or high positive
(mature), based on the size of agglutinates and the degree of background clearance
▪ Not affected by blood or meconium
▪ Production of PG is delayed among diabetic patient
▪ Considered as reference method

Foam stability / Shake ▪ Amniotic fluid + 95 % Ethanol ---→ shake for 15 seconds ---→ stand for 15 minutes
test ▪ (+) Result = foam/bubbles/effervescence = mature lungs

Microviscosity testing ▪ The presence of phospholipids decreases microviscosity

▪ Measured by fluorescence polarization
▪ No longer used

Lamellar body count
▪ Lamellar bodies are densely packed layers of phospholipids that represent a storage form
of pulmonary surfactant.
▪ They are secreted by the type II pneumocytes of the fetal lung
▪ Specimens may be stored at 2°C to 8°C but never frozen
▪ Uses hematology analyzer for counting
▪ They have similar size with platelets when counted
▪ ≥32,000 / ml lamellar body count = adequate FLM
▪ The advantages of lamellar body counting include: (1) rapid turnaround time, (2) low reagent
cost, (3) wide availability, (4) low degree of technical difficulty, (5) low volume of amniotic
fluid required, and (6) excellent clinical performance.

OD 650nm ▪ Specimens are centrifuged at 2000 g for 10 minutes and examined using a wavelength
(Lamellar bodies) of 650 nm
▪ Increase lamellar bodies = Increase O.D absorbance
▪ An O.D of ≥0.150 is equivalent to L/S ratio of ≥2.0

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 ≥2.0 Fetal lung maturity
Amniostat fetal lung maturity Positive Fetal lung maturity/phosphatidyl glycerol
Foam Stability Index ≥47 Fetal lung maturity
Optical density 650 nm ≥0.150 Fetal lung maturity
Lamellar body count ≥32,000/mL Fetal lung maturity

Fetal epithelial cells
Spectrophotometer
Centrifugation at 2000rpm for 10minutes
ADDENDUM
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.
Amniotic fluid bilirubin (O.D 450) is measured by ___using serial dilutions
What is the Specimen preparation prior to measuring Lamellar bodies?

I.K AYTONA 52
 CEREBROSPINAL FLUID
The 3rd Major body fluid
FUNCTION
1. Supply nutrients to the CNS
2. Remove metabolic waste
3. Produce a mechanical barrier to cushion the brain and spinal
cord against trauma

MENINGES
Lines the brain and spinal cord 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
Volume collected Up to 20ml collected under normal pressure (approximately 15% of the estimated total CSF volume)
If the CSF pressure is less than or greater than normal, only 1 to 2 mL should be removed.
Normal CSF Volume Adult: 90 to 150ml (adults), 10 to 60ml (neonates)
Distribution
TUBE # LAB SECTION STORAGE TEMPERATURE
1 Chemistry, Immunology, Serology
2 Microbiology
3 Hematology (Cell count), and Cytology studies
4 Microbiology (Better exclude skin contamination)
or Serology (for additional serologic test)

1 tube only Micro → Hema → Chemistry/Seroloy

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

APPEARANCE CLINICAL SIGNIFICANCE

CRYSTAL CLEAR
Cloudy, Turbid, Milky
Normal
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

-Yellow: Bilirubin
-Orange: In case of heavy hemolysis
-Brownish: Methemoglobin formation

OTHER CAUSES:
Increase dietary carotene
Increase rifampin intake
Increase melanin
Normal neonate
Protein concentration exceeding 150mg/dl
Previous traumatic tap

I.K AYTONA 53
Bloody / grossly bloody Greater than 6000 RBCs / ul due to:
Traumatic tap
Intracranial hemorrhage / cerebral hemorrhage
Oily Radiographic contrast dye
Clotted Protein and clotting factors
Meningitis, Froin’s syndrome
Pellicle Tubercular meningitis

Traumatic Tap Intracranial Hemorrhage

Distribution of blood in 3 tubes
Clot formation
Supernatant
Erythrophages
D-dimer test

CSF CELL COUNTING

CSF CELL COUNT
✓ Performed immediately because WBCS and RBCs will begin to lyse within 1 hour.
✓ 40% of WBC disintegrates within 2 hours. If the specimen is refrigerated, WBC lysis can be reduced significantly to
approximately 15%, but not completely prevented. Similarly, RBCs do not demonstrate significant lysis at 4° C; therefore the
CSF collection tube for cell counts should be refrigerated if the count must be delayed for any reason

CSF DILUTION
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
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:
Adults = 0-5 WBCs/ ul
Neonates = 0-30 WBCs/ul

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 (using Wright’s stain)
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 additional
tests, and slides made from the suspended sediment are allowed to air dry and are stained with Wright’s stain. 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

I.K AYTONA 54
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.
Control slide A daily control slide for bacteria should also be prepared using 0.2 mL saline and two drops of the
30% albumin currently being used. The slide is stained and examined if bacteria are seen on a patient’s slide.
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

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

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 1 WBC for every 700 RBC’s seen
✓ Subtract 8 mg/dl Total protein for every 10,000 RBCs/ul seen
✓ Subtract 1 mg /dl of TP for every 1,200 RBCs/ul seen

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
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
* Fusing of cell walls and nuclear irregularities and hyperchromatic nucleoli are seen in
clusters of malignant cells.
Ependymal, choroidal, Diagnostic procedures
and spindle shaped cells They are non- pathologically significant cells
Plasma cells Multiple sclerosis, Lymphocyte reactions

CHEMICAL ANALYSIS OF SPINAL FLUID

CSF PROTEIN
Normal values Adults =
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

I.K AYTONA 55
Decreased in CSF leakage/trauma, water intoxication, Rapid CSF production, Recent puncture
Major CSF Protein
2nd most prevalent Pre albumin
Alpha globulin Haptoglobin, ceruloplasmin
Beta-globulins ______________________________
✓ Carbohydrate deficient transferrin
✓ Found on CSF not in serum
Gamma globulins IgG and some IgA
Not found in normal CSF IgM, Fibrinogen, Lipids

CSF PROTEIN DETERMINATION

Turbidimetric ______________________
Preferred method, it precipitates both albumin and globulin

______________________
It Precipitates albumin only
To precipitate globulins, add sodium sulfate
Dye binding Uses __________________________
CSF /Serum Albumin Assess the integrity of the blood brain barrier
Index
𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛
𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛
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

𝐶𝑆𝐹 𝐼𝑔𝐺⁄𝑆𝑒𝑟𝑢𝑚 𝐼𝑔𝐺

𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛⁄𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛

Normal value = <0.70

Abnormal value = >0.70

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

MYELIN BASIC PROTEIN

➢ Protein component of the lipid protein complex that insulate the nerve fibers
➢ Presence of MBP in CSF indicates destruction of myelin sheath
➢ Used to monitor course of _____________________
➢ 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
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
➢ 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
➢ Normal value: 10-22 mg/dl (1.1 to 2.4 mmol/L)
➢ 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.

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

I.K AYTONA 56
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
➢ Increase in: Reye’s syndrome, Disturbance of consciousness/coma (more than 35 mg/dl)

CSF ENZYMES
Lactate dehydrogenase
LD1 and 2 = Brain tissue
LD2 and 3 = Lymphocytes
LD4 and 5= Neutrophils

Serum LDH
Normal = LD2>1>3>4>5
Flipped pattern = LD1>2>3>4>5

CSF LDH
Normal =
Neurological abnormalities=
Bacterial meningitis=

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
➢ Reagent: blood of horseshoe crab
➢ Positive: clumping or clot formation

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

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 57
 SEMINAL FLUID

Reasons for Semen analysis

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

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

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

First portion of 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

MACROSCOPIC EXAMINATION
Appearance Gray- white, translucent, with musty or bleach odor =______________________
Increased white turbidity =______________________
Red or Brown coloration =______________________
Yellow coloration =______________________
Volume Normal =
Increased =
Decreased =
Viscosity Normal = pour in droplets
Reporting: NOTE Droplets that form threads longer than 2 cm are
0 = ____________ considered highly viscous and are recorded as abnormal
4 = ____________

I.K AYTONA 58
Ph Normal = __________
Increase = ___________
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
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

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?

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
➢ Quality = ≥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 10 μL
under a 22 × 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
1.0 c No forward progression
0 d No movement

CASA (Computer Assisted Semen Analysis)

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

SPERM MORPHOLOGY
➢ Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion. Smears are made by placing
approximately 10 μL of semen 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.

Normal values: Acrosomal cap

A. Routine criteria = -part of the sperm that contains enzyme
B. Kruger’s strict criteria = for ovum penetration
This criteria measures the head, neck and tail using a
Micrometer or morphometry -Size: _______________________

I.K AYTONA 59
Stains for sperm morphology:
a. Wright’s stain Head = Length 5um, Width 3um
b. Giemsa stain Normal head appearance = oval
c. Shorr stain Tail = 45 um long
d. Papanicolau’s stain = best stain Midpiece: 7um

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

SEMINAL FLUID FRUCTOSE

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

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

• Detects the presence of IgG antibodies
• Semen sample + AHG reagent + latex particle or treated RBCs coated with IgG
• Normal = <10% motile sperm attached to the particles

2. Immunobead test
• Detects the presence of IgG, IgM, IgA
• Demonstrate what area of the sperm the autoantibodies are affecting
• 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

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

I.K AYTONA 60
 MICROBIAL TESTING

ROUND CELLS
These are WBCs and spermatids
Normal value =
If greater than 1 million WBC/ ml =
If greater than 1 million spermatids/ml =
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

MEDICO LEGAL TESTING

Test for detection of semen:

1. Microscopic exam
2. Fluorescence under UV light
3. Acid phosphatase determination
4. Glycoprotein p30 = more specific method
5. ABO blood testing
6. DNA test
7. Florence test
✓ Test for _____
✓ Reagent:
✓ (+) result:
8. Barberio’s test (very specific)
✓ Test for _____
✓ Reagent:
✓ (+) result:

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

ASSESTMENT OF SPERM CELLS

Sperm morphology – make a smear in slide and stain it with Papanicolau’s, Wright’s or Giemsa
Sperm Viability- Stain with Eosin 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

SPERM FUNCTION TEST

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

Abnormal result Possible abnormality Test

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

I.K AYTONA 61
 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 Dulbecco’s phosphate-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
BLACK UPPER GI BLEEDING
IRON THERAPY, CHARCOAL INGESTION
PALE YELLOW, WHITE GRAY, ALCHOLIC STOOL, chalky BILE DUCT OBSTRUCTION AND BARIUM SULFATE
GREEN BILIVERDIN, GREEN VEGETABLES, ORAL ANTIBIOTICS
RED LOWER GI BLEEDING, RIFAMPIN, FOOD COLORING
BULKY / FROTHY BILE DUCT OBSTRUCTION, STEATORRHEA, PANCREATIC
INSUFFICIENCY
RIBBON LIKE INTESTINAL CONSTRICTION
RICE WATERY CHOLERA
PEA SOUP TYPHOID
SYCBALOUS / GOAT DROPPING CONSTIPATION
BUTTER-LIKE CYSTIC FIBROSIS
MUCOID DYSENTERY, MALIGNANCY

BRISTOL STOOL CLASIFICATION

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 62
 LABORATORY TEST

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
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
Stain for Triglycerides
Procedure: emulsified stool + 95% ETOH + Sudan III
Steatorrhea = ≥60ORANGE DROPLETS/HPF
Split fat stain
Stain for total fat content (including Fatty acids,
soaps/fatty acid salts, and cholesterol)
Procedure: stool + 36% acetic acid + Sudan III +
Heat
Steatorrhea = 100 droplets that are 6-75 um in size

Neutral Fat stain Split Fat stain Interpretation

Normal increased malabsorption
Increased normal maldigestion

QUANTITATIVE FECAL FAT TEST

Van De Kamer Titration test
Gold standard test for fecal fat
Requires a 3-days stool sample (placed on a Paint Cans and must be homogenized prior to analysis)
Titrated with NaOH

D-XYLOSE TEST

A test that is useful to differentiate malabsorption and maldigestion

D-Xylose is a pentose sugar that does not need to be digested but does need to be absorbed to be present in the urine.
The xylose absorption test involves the patient’s ingestion of a dose of xylose, followed by the collection of a 2-hour blood
sample and a 5-hour urine specimen.
If D-xylose result is low/abnormal, the result indicates a malabsorption condition

II.FECAL LEUKOCYTES
➢ Presence of ≥3 neutrophils/hpf Indicates invasive condition
➢ 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 ✓ It remains sensitive in refrigerated and frozen specimens
✓ Positive in diarrhea with WBC: Salmonella, Shigella, Campylobacter, Yersinia, & enteroinvasive E. coli.
✓ 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 ___
➢ Screening test for __________
➢ 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

I.K AYTONA 63
 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

➢ ___________________ = 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
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

V. APT TEST/ ALKALI DENATURATION TEST/ DOWNEY TEST Procedure

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

TEST
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
A result of ≥0.5 g/dl indicates carbohydrate intolerance

I.K AYTONA 64
 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

OSMOTIC DIARRHEA
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
Stool- with osmol gap of >50mosm/kg.
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

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.
IBS can be triggered by food, chemicals, emotional stress, and exercise.

CAUSES:
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

I.K AYTONA 65
 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

SPUTUM COLLECTION
First morning- Most preferred (routine)
24 hour – for volume measurement
Throat swab – for pediatric patients
Sputum induction- for non-cooperative patients
Tracheal aspiration- for debilitated patient

MACROSCOPIC EXAMINATION
Volume Increase in: Broncheictasis, lung abscess, edema, gangrene, tuberculosis, pulmonary hemorrhage
Decrease in: Bronchial asthma, acute bronchitis, early pneumonia

Odor
Odorless Normal
Foul or 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
Dittrich’ s plugs *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
Curschmann’s spirals *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

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 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
- Most significant
- Associated with bronchial asthma
Pigmented cells - Heart failure cells: hemosiderin laden macrophages
- Carbon laden cells: angular black granules
Curshmann’s spirals - 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
Parasites - Ascaris, Hookwork, Threadworm, E.histolytica, Paragonimus westermani, Toxocara canis,
Entamoeba gingivalis, Trichomonas tenax, Echinococcus granulosus
Others that are stained - Neoplastic cells, Bacteria, Leukocyte

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

CELLS SEEN IN BRONCHOALVEOLAR LAVAGE Elements and Viral inclusions seen in respiratory
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

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

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

SIGNIFICANCE
✓ Determines whether or not a patient can secrete gastric fluid
✓ Measures amount of gastric acid that can be secreted by one with ulcer symptoms
✓ Help determine the disturbed function of the GI system

CELLS OF THE STOMACH

1. Parietal cells- produces HCL and Intrinsic factor

❖ 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.

I.K AYTONA 67
 SPECIMEN COLLECTION

• Gastric juice is obtained by insertion of a gastric tube into the stomach

• Gastric tubes:
a. Levin tube = passed through the _______
b. Rehfuss = passed through the _______
c. Disposable plastic tubes are usually employed

• Specimen: Fasting specimen, few ml to 50ml average of 30 ml

• Normal appearance of gastric specimen: Pale gray with mucus and no food particles

Types of specimen
Basal acid output (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 Ewald’s meal: bread and water or weak tea
Boas meal: oatmeal, meal for detection of lactic acid
Riegel’s meal: mashed potatoes, broiled beefsteak, bouillon
Chemicals ✓ Pentagastrin – most preferred, it resembles true gastrin
✓ Histamine
✓ Histalog
✓ Insulin - assess successful vagotomy procedure
Sham Feeding ✓ Fictitious feeding
✓ Sandwich is chew and then spit out

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.

✓ Stimulant used: Test meals (Henry’s) 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
Gunzberg’s Reagent: phloroglucin, Vanillin, Alcohol = (+) Purple red color
Boas Reagent: resorcinol, cane sugar, alcohol = (+) Purple red color

I.K AYTONA 68
 QUANTITATIVE TEST FOR GASTRIC ACIDITY
Free HCL Topfer’s method
Titrate with NaOH
pH indicator: dimethyl aminoazobenzol
Endpoint: Canary yellow
Normal value: 25-50 degrees or 0.1 or 0.2 HCL

Total acidity Titrate with NaOH

Ph indicator: phenolphthalein
Endpoint: Faint pink
Normal value: 50 -75 degrees

Combined HCL (Bound to Titrate with NaOH

proteins) pH indicator: sodium alizarin
Endpoint: Violet
Normal value: 10 – 15 degrees

LACTIC ACID TEST

Test Reagents Endpoint
Modified Uffelman’s FeCl3 + phenol Yellow
Strauss FeCl3 + ether Yellow
Kelling’s FeCl3 Yellow

QUALITY ASSESTMENT AND MANAGEMENT IN URINALYSIS LABORATORY

QC of laboratory 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

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 Refers to all of the laboratory’s policies, processes, procedures, and resources needed to
achieve quality testing.
Trend A gradual change in the mean
Shift An abrupt change in the mean
Total Quality 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 69
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
facility’s policy.

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
TEST/METHOD ANIMAL USED POSITIVE RESULT
(Henry’s 19th ed.)
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 70
Chemical test in Detected Name of test
urine
Calcium Sulkowitch
Chloride Fantus, Schales Schales
Bile pigments Smith, Harrison’s spot, ictotest, Gmelin
Urobilin Schlesinger
urobilinogen Wallace and diamond
Ketones Rothera’s, Lange, acetest, Gerhardt’s
fructose Resorcinol, Seliwanoff, and Borchardt’s
Qualitative test for Heat and acetic acid, SSA, Purdy’s, picric acid, Potassium ferricyanide, Biuret,
protein Heller’s, Spiegler’s test
Quantitative test for Biuret, Kingsburry-clark, Esbach’s, Kwilecki’s
protein
Sugars 1. Biacol orcinol
2. Benedicts
3. Rubner’s (glucose = red with red yellow, Lactose = red with red ppt)
4. Moore Heller
5. Nylander’s

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
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 as cell counts on body fluids
Uses patented technologies to capture and automatically classify digital images of
urine 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

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

I.K AYTONA 71
 Sysmex UF- -Principle: cell flow cytometry + impedance
analyzers / -Urine particles are identified and categorized by fluorescent staining characteristic,
Slideless light scatter, electrical impedance, and adaptive cluster analysis
automated
analyzers Fluorescent stain used
Carbocyanin Stain for RNA, cell membrane, cytoplasm
Phenanthridine Stain for DNA, nucleus, chromosomes

Light source use

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
NOTE! Determining the specific identity of elements in “flagged” specimens requires a
manual 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 Systems iChem Velocity urine chemistry + iQ200
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 System ATLAS urine chemistry + Sysmex UF1000i
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 must be “lipless” for 10 samples to
fit in a rack. The 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

“THE SECRET RECIPE ON PASSING THE BOARD EXAM IS JUST A MIXTURE OF HARDWORK, DETERMINATION, FAITH,
AND A POSITIVE ATTITUDE”

R.M.T

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