CLINICAL MICROSCOPY

ANALYSIS OF URINE AND BODY

FLUIDS

James Ryan A. Mendoza. RMT, MLS

(ASCPi)
 LEARNING
OBJECTIVES

• FULL COVERAGE OF CLINICAL MICROSCOPY FOR THE

MEDICAL TECHNOLOGISTS BOARD EXAM
• REVIEW ON URINALYSIS AND OTHER BODY FLUIDS
• THEORETICAL CONCEPTS, LABORATORY CORRELATIONS
TO VARIOUS PATHOLOGIC CONDITIONS
• RATIONALIZATION AND REINFORCEMENT
SAFETY AND QUALITY
ASSURANCE
HEALTH INSTITUTIONS
• CDC – Centers for Disease Control and Prevention
• HICPAC – Healthcare Infection Control and Practices
Advisory Committee
• CLSI – Clinical and Laboratory Standards Institute
• OSHA – Occupational Safety and Health Administration
THE ROLE OF HEALTH INSTITUTIONS

• CONSIDERS THE BIOSAFETY AND

BIOSECURITY OF THE SOCIETY, LABORATORY
PERSONNEL AND GENERAL POPULATION
 WHAT IS BIOSAFETY?
• The application of combinations of laboratory
practices and procedures, laboratory facilities,
and safety equipments to prevent the accidental
release of potentially infectious hazardous
microorganisms within the laboratory or the
community
 WHAT IS BIOSECURITY?

• The application of combinations of laboratory

practices and procedures, laboratory facilities, and
safety equipments to prevent the potential access of
people into these potentially infectious hazardous
microorganisms
BIOLOGICAL HAZARDS
• CHAIN OF INFECTION – the process of how microorganisms are transmitted.

• INFECTIOUS AGENT
• RESERVOIR
• EXIT (PORTAL OF)
• MODE OF TRANSMISSION
• ENTRY (PORTAL OF)
• SUSCEPTIBLE HOST
 IN 1987

• CDC instituted “UNIVERSAL PRECAUTIONS”

•“All Patients are considered to be possible
carriers of blood borne pathogens”
UNIVERSAL
PRECAUTIONS
• WEAR GLOVES – when collecting blood or handling blood
contaminated other bodily fluids

• WEAR FACE SHIELDS - When dangers of blood

splashing on mucous membranes are present or when
disposing needles and sharp objects in puncture resistant
containers
BODY SUBSTANCE ISOLATION

• Wear gloves at all times

• Disadvantage of BSI – don’t promote

handwashing after removing gloves unless
visual contamination is present
STANDARD PRECAUTIONS
• 1. Handwashing and use of Alcohol based
antiseptic forming cleansers
• 2. Gloves
• 3. Mouth, Nose and Eye Protection
• 4. Gown
• 5. Patient Care Equipment
• 6. Environmental Control
• 7. Linens
• 8. Occupational Health/ Blood
Borne Pathogens
• 9. Patient Placement
• 10. Respiratory Hygiene and
Cough Etiquette
SEQUENCE
OF
WEARING
AND
REMOVING
PPE’s
from CDC
DISPOSAL OF BIOLOGICAL
WASTES
GREEN BLACK YELLOW YELLOW

NON NON INFECTIOUS CHEMICAL

INFECTIOUS INFECTIOUS PATHOLOGICA WASTES
WET WASTES DRY WASTES L WASTES INCLUDING
HEAVY METALS
DISPOSAL OF BIOLOGICAL
WASTES
ORANGE RED WHITE LIGHT BLUE

RADIOACTIV SHARPS AND SOILED MATERIALS

E WASTES PRESSURIZED LINENS FOR
CONTAINERS AUTOCLAVING
NATIONAL FIRE
PROTECTION ASSOCIATION
(HAZARDOUS MATERIALS
CLASSIFICATION)
HAZARDOUS MATERIAL
CLASSIFICATION
YELLOW WHITE BLUE RED
REACTIVITY/ SPECIFIC HEALTH FLAMMABILIT
STABILITY HAZARD HAZARD Y
0 – Stable OXY – Oxidizer 0 - Normal Material 0 – will not burn
1 – Unstable when Heated ACID - Acid 1 - Slightly 1 – above 200 F
2 – Violent chemical change ALK – Alkali hazardous 2 - below 200 F
3 – Shock and Heat. COR – Corrosive 2 - Hazardous 3 – below 100 F
May deteriorate W – Use no water 3 - Extreme danger 4 - below 73 F
4 – May Deteriorate Radiation 4 - Deadly
DEGREES OF
HAZARD

0 1 2 3 4
NO/ SLIGHT MODERAT SERIOU EXTREME
MININAL HAZARD E S
HAZARD
DEALING WITH FIRE
HAZARDS
FIRE TYPE OF HAZARD EXTINGUISHER
TYPE
TYPE A Ordinary combustibles, paper, Water, Dry Chemical,
clothes, rubbish, plastics and wood Loaded Steam
Type B Flammable Liquids, Grease, Dry Chemical, Carbon
Gasoline, Paints Oil Dioxide, Halon Foam
Type C Electrical Equipment and motor Dry Chemical, Carbon
switches Dioxide, Halon
DEALING WITH FIRE
HAZARDS
Type D Flammable Metals, Mercury, Metal X, fought by fire fighters
Magnesium, Sodium, Lithium only

Type E Detonation (Arsenal Fire) Allowed to burn out and nearby

materials are protected

Type K Cooking material, grease, oil and Liquid designed to prevent

fat splashing and cool the fire
WHAT TO DO IN FIRE ACCIDENTS?
 DEALING WITH ELECTRICAL
HAZARDS

• Do not operate machinery with wet hands

• All electrical equipment must be
grounded with 3 pronged plugs
• Never touch an electrically shocked
person or the equipment
 DEALING WITH ELECTRICAL
HAZARDS

• Turn off the circuit breaker

• Unplug the equipment
• Move the equipment using a
nonconductive glass or wood
object
 DISPOSAL OF SHARPS

• NEEDLES ARE CAPPED

AND MUST BE
PLACED IN
PUNCTURE-
RESISTANT
CONTAINERS
 RADIOACTIVE HAZARDS

• When procedures using

radioisotopes are performed
• Exposure to radiation during
pregnancy presents a danger to a
fetus.
PHYSICAL HAZARDS

• 1. No running inside the

• 5. Don’t wear excessive jewelry
laboratory premises
• 6. Maintain a clean and well-
• 2. Watch for wet floors
organized area
• 3. Bend knees when lifting
• 7. Wear closed-toed shoes
objects
• 8. No eating and drinking inside
• 4. Keep long hair pulled
the laboratory
INTRODUCTION TO
URINALYSIS
URINALYSIS

• defined by Clinical and Laboratory Standards

Institute (CLSI) as “the testing of urine with
procedures commonly performed in an
expeditious, reliable, accurate, safe and cost
efficient manner.”
• Aims to assist in the diagnosis of diseases, serves
as a screening asymptomatic populations for
undetected disorders and serves to monitor the
progress of disease and effectiveness of therapy.
HISTORY OF CLINICAL MICROSCOPY
Egyptian Hieroglyphics
Hippocrates @ 5th Century BCE
Frederik Dekkers @ 1694
Thomas Addis
Thomas Bryant @ 1627
Richard Bright @ 1827
Edwin Smith Surgical Papyrus
Book on Uroscopy
Albuminuria – demonstrated by boiling urine
Examination and Methods of Quantification of
Urinary Sediments
Published a book about Charlatans or “Pisse
Prophets”
- publishing of his work led to the passing of the
first medical licensure laws in England
Concept of Urinalysis as a part of the physician’s
routine patient examination.
2 UNIQUE CHARACTERISTICS OF
URINE SPECIMEN FOR PATIENT
EVALUATION

• Urine is readily available and easily collected

• It contains information, which can be obtained by
inexpensive lab tests about many of the body’s major
metabolic functions.
CHANGES IN UNPRESERVED
URINE
INCREASED RATIONALE
pH Urea is split by bacteria, release of Ammonia
Bacteria Multiplication, metabolic activity
Odor Ammonia
Nitrite Nitrate converted by bacteria
DARKENED/MODIFI RATIONALE
ED
Color Oxidation/ Reduction of Metabolites
CHANGES IN UNPRESERVED
URINE
DECREASED RATIONALE
Clarity Bacteria multiplies, substances (like amorphous
crystals) precipitate
Glucose Glycolysis, Utilized by Bacteria
Ketones Volatile
Bilirubin Photo oxidation
Urobilinogen Oxidized into Urobilin
RBC, WBC and Disintegration in Alkaline Urine
their casts
TYPES OF URINE
SPECIMEN
Random Sample Routine Urinalysis
First Morning Ideal specimen for routine UA and pregnancy
Urine for well preservation of cells and casts
For evaluation for Orthostatic Proteinuria
Second Morning/ 2nd voided urine after a period of fasting; for
Fasting glucose determination
2 Hours Post For diabetic screening / monitoring
Prandial
Glucose Tolerance Optional with blood samples in glucose
tolerance test
Fractional Specimen At least 2 voided collection
Series of blood and urine samples are collected
at specific time intervals to compare the
concentrations of a substance in urine with its
concentration in the blood (used in the
diagnosis of diabetes)
Midstream Clean Routine UA and Bacteriologic sampling
Catch
Catheterized Bacterial Culture
Suprapubic Aspiration Anaerobic Bacterial culture, urine cytology
Pediatric Specimen Use of Soft, plastic bag with adhesive
Sterile specimen, obtained by catheterization or
suprapubic aspiration
 3 GLASS
TECHNIQUE
FOR DETECTION
OF PROSTATITIS

CONTROL
2ND 1ST TUBE
TUBEAND
TUBE (+) BACTERIA >>1ST
3RD TUBE
WBC
3RD TUBE
BACTERIA
RESULTS FROM BACTERIA
3RD AND
TUBE ISAND WBC
INVALID
WBC
(+) URINARY
CONTROL FOR KIDNEY TRACT
AND BLADDER
(+) PROSTATITIS
INFECTION INFECTION
PPMT – PRE AND
POST MASSAGE
TEST

POST MASSAGE
SAMPLE
10X BACTERIURIA
(+) PROSTATITIS
TIMED
SPECIMENS
24 Hours Specimen CLEARANCE TESTS
12 Hours Specimen ADDIS COUNT
4 Hours Specimen NITRITE
DETERMINATION
Afternoon – (2PM- UROBILINOGEN
4PM) DETERMINATION
 SAMPLING FOR DRUG
TESTING

• CHAIN OF CUSTODY – the process that

provides documentation of proper sample
identification from the time of collection to the
receipt of the laboratory
• Required Urine Volume
30ML - 60 ML (National Reference Lab)
30ML- 45ML (Strasinger)
• Temperature – within 4 minutes 32.5 C to 37.7 C
 SPLIT SAMPLING

BOTTLE A = SENT BY THE SDTL TO

THE NATIONAL REFERENCE
LABORATORY (NRL) FOR
BOTTLE BOTTLE CONFIRMATORY TEST
A B BOTTLE B = KEPT BY THE SDTL FOR
15 DAYS IN CASE OF RETEST OR
(+) CHALLENGE TEST

BOTH BOTTLES = 30 ML URINE

SINGLE SAMPLING

IF THE SINGLE SAMPLE IS TESTED

POSITIVE FOR DRUG METABOLITES.
THE ENTIRE BOTTLE IS SENT BY THE
60 ML URINE SDTL TO THE NATIONAL REFERENCE
LABORATORY FOR CONFIRMATORY
TESTING.
 URINE SAMPLING FOR DRUG
TESTING

• Container
60 ML Polyethylene Bottle (Single)
30 ML Polyethylene Bottle (Split)
• Blueing agent (Dye) is added to toilet water reservoir
to prevent specimen adulteration
RENAL ANATOMY
AND PHYSIOLOGY
 FUNCTIONS OF THE URINARY
SYSTEM

• Excretion – kidneys are the major excretory organs of the

body
• Blood volume control – regulating the volume of water
removed from the blood to produce urine
• Ion concentration regulation – the release of Hydrogen
Ions, reabsorption of Bicarbonate ions
• ph regulation – kidneys work with the lungs to
buffer the acidity or alkalinity of the blood
• Red blood cell concentration – kidneys are the
source of erythropoietin
• Vitamin D synthesis – kidneys, along with
skin and liver participates in Vitamin D
production.
RENAL
FUNCTIONS
• Single Kidney = 1 to 1.5 million
nephrons.
• NEPHRONS – are the basic structural
2 and functional unit of the kidneys
TYPES
Cortical Nephrons – comprises the 85% of the
total number of nephrons in the kidney.

Primary function is the removal of metabolites and

reabsorption of the nutrients
RENAL
FUNCTIONS
Juxtamedullary
Nephrons – they have
longer Loops of Henle that
extend deep into the
medulla
Primary of the iskidney
function for the concentration of the urine
 RENAL BLOOD
FLOW
A. RENAL BLOOD
FLOW TOTAL RENAL BLOOD
2. Renal Artery FLOW = 1200ml/min
3. Afferent Arterioles
4. Glomerulus
5. Efferent Arteriole TOTAL RENAL
6. Peritubular PLASMA FLOW =
Capillaries
600ml/min
 ORDER OF URINE
FORMATION
1. Glomerulus
2. Proximal Convoluted
Tubule
3. Descending Loop of Henle
& Ascending Loop of Henle
4. Distal Convoluted Tubule
5. Collecting Ducts
6. Calyx
7. Renal Pelvis

GLOMERULAR FILTRATION

influenced by the cellular structures of the

capillary walls and Bowman’s Capsule,
Hydrostatic and Oncotic Pressures and the Renin-
Angiotensin-Aldosterone System.
 GLOMERUL
US
the tuft of capillaries having approximately 8 coiled
lobes enclosed in Bowman’s Capsule.
• It resembles a sieve and is a non-selective filter of
plasma substances with molecular weight less than
70,000 Daltons
 SHIELD OF NEGATIVITY
- The glomerulus has a negative shield that
repels negatively charged substances
(particularly Albumin). Thereby preventing
Albuminuria and Protein Wastage
 CHON
- - -
-
CHON

-
CHON

- -
CHON

- -
CHON

- (+) -
- -
SHIELD OF
NEGATIVITY
- -
PROTEINURIA
 GLOMERULAR
PRESSURE
Hydrostatic Pressure resulting from
the smaller size of Efferent arterioles
and glomerular capillaries enhances
Filtration.
•Pressure is needed to overcome
opposition of fluids within the
Bowman’s Capsule and the Oncotic
pressure of unfiltered plasma proteins in
glomerular capillaries
 JUXTAGLOMERULAR
APPARATUS

• consists of Juxtaglomerular cells of the Afferent

Arterioles and Macula Densa of the Distal
Convoluted Tubule. Maintains the glomerular blood
pressure at a relatively constant rate regardless of
fluctuations in systemic blood pressure
•Has an autoregulatory
mechanism by either
increasing/decreasing the sizes of
the afferent and efferent
arterioles

AUTOREGULATORY
MECHANISM
 SYSTEMIC BLOOD PRESSURE -
HIGH

PREVENTS BLOOD OVERFLOW

GLOMERULAR BLOOD FLOW PREVENTS EXCESS
= INCREASED RATE FILTRATION
PREVENTS GLOMERULAR
 STRESS

SYSTEMIC BLOOD PRESSURE -

LOW

GLOMERULAR BLOOD FLOW GLOMERULAR BLOOD POOLING

= DECREASED RATE PREVENTS TOXIC WASTE BUILD
UP
RENIN-ANGIOTENSIN-ALDOSTERONE
SYSTEM (RAAS)

• responds to the changes in Blood Pressure,

Plasma Sodium concentration (that is also
monitored by the Juxtaglomerular Apparatus)
• RAAS will be triggered once the body
experiences the cascade of lows
 CASCADE OF LOWS

• Decrease in Plasma Sodium

• Decrease in H2O Retention
• Decrease in Blood Volume
• Decrease in Blood Pressure
RENIN-ANGIOTENSIN-ALDOSTERONE
SYSTEM (RAAS)

ACE – ANGIOTENSIN CONVERTING

ENZYME
 ANGIOTENSIN II

• Corrects Renal Blood Flow

• Aldosterone and ADH are released
• Increased Na+ & H2O Reabsorption
• Increase Blood Volume
• Increase Blood Pressure
TAKE NOTE

• Analysis of the fluid as it

• The only difference between the
leaves the glomerulus shows
compositions of the filtrate and
the filtrate to have a specific
the plasma is the absence of
gravity of 1.010 and confirms
plasma proteins or any protein
that it is chemically an
bound substances and the cells.
ultrafiltrate of plasma.
 TUBULAR
REABSORPTION
THE FIRST FUNCTION TO BE AFFECTED IN RENAL
DISEASES.
• ACTIVE TRANSPORT – o PASSIVE TRANSPORT –
requires the carrier proteins movement of molecules across a
membrane as a result of differences
contained in the membranes of
in their concentrations or electrical
the renal tubular epithelial cells. potential on opposite sides of the
An energy requiring process in membrane
the form of ATP
 TUBULAR REABSORPTION

ACTIVE TRANSPORT PASSIVE TRANSPORT

SUBSTANCE LOCATION SUBSTANCE LOCATION
Glucose, Amino PCT Water PCT, DLH, CD
Acid
Salts
Chloride ALH Urea PCT, ALH
Sodium PCT and DCT Sodium ALH
PROXIMAL CONVOLUTED
TUBULE

- 65% of reabsorption of substances

- Reabsorbs Salts, Water, Amino
Acids, Glucose and Urea
 COUNTERCURRENT
MECHANISM
• prevents the overabsorption of
water by maintaining the Osmotic
gradient of the medulla of the
kidneys. It is essential for the final
concentration of The filtrate when it
reaches the collecting duct
 COLLECTING DUCT
CONCENTRATION

• influenced by the action of the Vasopressin/ Anti-Diuretic Hormone

• HIGH LEVELS OF ADH increases the permeability of the capillaries
allowing an increase in water reabsorption
• LOW LEVELS OF ADH decreases/ or renders the collecting duct
impermeable to water.
• THEREFORE, the ADH value is determined by the body’s hydration
status.
 TUBULAR
SECRETION
secretion of waste products that aren’t filtered by the glomerulus
- Regulating Acid-Base balances by secreting Hydrogen Ions.
- Many foreign substances (ex. Medications) can’t be filtered by the
glomerulus because they are protein-bound.
- Major Site of removal of these non-filtered substances is the PCT.
 ACID-BASE BALANCE
- 100% Bicarbonate is reabsorbed and this occurs in the PCT.
- PCT is also the site of Ammonia production from the breakdown of
Glutamine.
- Ammonia reacts with the Hydrogen ions to form the Ammonium ion.
- Bicarbonate is prevented to be excreted by secretion of Hydrogen Ions
by the renal tubular cells into the filtrate
 METABOLIC ACIDOSIS/ RENAL
TUBULAR ACIDOSIS
- Inability of the kidneys to produce acidic
urine
•Excessive H+ in blood.
RENAL FUNCTION
TESTS
GLOMERULAR
FILTRATION
• normal functioning is determined through Clearance tests.
• It measures the ability of the kidneys to remove a filterable
substance in the blood.
• Substances tested must be neither reabsorbed nor secreted by
the tubules.
• The substance must be stable in urine during a 24 hours
collection.
 CLEARANCE
TESTS
•ENDOGENOUS CLEARANCE – the
substance is readily available in the body
•EXOGENOUS CLEARANCE – the
substance is infused into the bloodstream
 CREATININE
CLEARANCE
•Creatinine is a waste product of muscle
metabolism
•Produced enzymatically by Creatine
Phosphokinase which links with ATP to
produce ADP and Energy
 CREATININE CLEARANCE

• Crea. Clearance = Urine Creatinine (Volume in Ml/Minutes) X 1.73

Plasma Creatinine A

• MALES: 107 – 139 ml/min 1.73 = Constant Body

Surface Area
• FEMALES: 87 – 107 A = BSA of the patient
ml/min Minutes = 1440
COCKGROFT AND GAULT FORMULA
(Estimated Glomerular Filtration Rate)

(140 – Patient’s Age)

72 X Serum Creatinine (mg/dl)

Result is multiplied to 0.85 if the patient is Female

Variables to consider: Age, Gender, Serum Crea

CREATININE
CLEARANCE
INCREASED DECREASED
High Cardiac Input Impaired Kidney Function
Pregnancy Shock and Dehydration
Burns Hemorrhage
Carbon Monoxide Poisoning Congestive Heart Failure
INULIN
CLEARANCE GOLD STANDARD CLEARANCE
TEST

•Inulin – a polymer of •It must be

fructose. It is extremely administered
stable substance that is not intravenously at
reabsorbed or secreted by a constant rate
the tubules. throughout the
testing period
 CYSTATIN
C
• a small molecular weight protease inhibitor and is produced
constantly by all nucleated cells.
- Readily filtered by the glomerulus and reabsorbed and broken down
by renal tubular cells (PCT)
- Completely reabsorbed by the Proximal Convoluted Tubule, hence its
presence in the urine denotes damage of the tubule.
• The Indirect Estimate of GFR. (Because renal clearance of this
substance cannot be measured.)
 CYSTATIN C

- Specimen of choice is Serum or Plasma

(Fasting Requirements aren’t needed)
- Increased Serum Cystatin C = Acute and
Chronic Renal Failure and Diabetic Nephropathy
 CLINICAL SIGNIFICANCE OF CLEARANCE TESTS

GFR is determined not only by the number of functioning

nephrons but also knowing the functional capacity of these
nephrons.
GFR does not lie in detection of early renal diseases, instead,
it is used to know the extent of nephron damage in a known
renal disease.
 CLINICAL SIGNIFICANCE OF CLEARANCE TESTS

- To monitor the effectiveness of a treatment designed to

prevent further nephron damage
- And to determine the feasibility of administration of
medications (which can build up into toxic levels in the
blood if the GFR is markedly decreased)
TUBULAR REABSORPTION
TESTS

Concentration tests are used to evaluate reabsorption capacity

of the tubules

• SPECIFIC GRAVITY – Influenced by the number and density

of particles in a solution
• OSMOLARITY – influenced by the number of particles in the
solution
 TUBULAR REABSORPTION
TESTS

• Principle of Freezing Point Depression – 1

Osm or 1000 mOsm/ kg of water will lower
the freezing point of water by 1.86 Celcius.
• Used to determine the Osmolarity of urine
OBSOLETE REABSORPTION
TESTS

• FISHBERG TEST - patient is deprived of fluid

for 24 hrs then urine SG is measured. (SG should
be ≥1.026)
• MOSENTHAL TEST – compares the day and
night urine volume and SG.
TUBULAR SECRETION & RENAL
BLOOD FLOW
•PAH test – (P-aminohippuric acid)
•PSP test – (Phenolsulfonaphthalein)
•* All these substances aren’t normally
absorbed by the glomerulus, but it is secreted
entirely.
PHYSICAL ANALYSIS OF
URINE
 PHYSICAL
EXAMINATION

• URINE VOLUME
• NORMAL RANGE in 24 hours = 600 -
2000ml
• AVERAGE = 1200 – 1500ml
• Night to Day Ratio = 1:2 to 1:3
• Routine Urinalysis Required Volume = 10-15 ml
 VARIATIONS IN URINE
VOLUME

• POLYURIA – Increased in Urine Volume

• Production of >2000 ml/24 hrs (Henry) Or 2.5L/24hrs (Strasinger)
• Causes
- Increased Fluid Intake
- Diuretics (Caffeine, Alcohol, Thiazides)
- Diabetes Mellitus and Diabetes Insipidus
3 PATHOLOGIC STATES
RESULTING TO
POLYURIA
 DEFECTIVE HORMONAL
REGULATION
OF VOLUME HOMEOSTASIS

• Diabetes Insipidus – deficiency of Anti-Diuretic Hormone

(Arginine Vasopressin) or Unresponsiveness to the hormone
(Nephrogenic)
- Excessive thirst and water intake occur with marked polyuria
or nocturia. (Up to 15 Liters of urine may be produced in a
day)
 DEFECTIVE RENAL SALT AND WATER
ABSORPTION
- Due to Diuretic agents
- Abnormality of renal tubules resulting to Sodium wasting or impairment
of countercurrent mechanism
- Progressive Chronic Renal Failure – the functioning tissue is diminished.
The ability to concentrate urine is gradually lost.
- To excrete daily renal water and solute load, an increase in urine volume per
residual nephron results to urine eventually becomes isosmotic with plasma
ultrafiltrate.
 OSMOTIC DIURESIS
- Diabetes Mellitus with Hyperglycemia
•Excessive amounts of glucose is excreted,
causing a solute diuresis
DIABETES MELLITUS VS. DIABETES
INSIPIDUS

DIABETES MELLITUS DIABETES INSIPIDUS

- A defect of in either pancreatic production
of insulin or function of insulin
- Resulting to High Plasma Glucose and
kidneys don’t reabsorb excessive glucose
- RENAL THRESHOLD OF 160 – 180
mg/dl
- Necessitating excretion of increased
amounts of water to remove the dissolved
glucose
- Urine may appear diluted, but it has
- Decrease of ADH function
or production
- Water is not reabsorbed
from plasma filtrate
- Urine is truly diluted,
having LOW Specific Gravity.
 OLIGURI
• Decreased Urine volume
• Production of <500 ml/ 24hrs (Henry)
• <400 ml/24hrs (Strasinger)
• Causes - Dehydration, Renal Calculi or Tumor
DISEASE CORRELATIONS
WITH OLIGURIA
 PRE – RENAL
DISEASE
• Loss of intravascular volume attributed to bleeding, dehydration,
prolonged diarrhea, vomiting, excessive sweating and severe burns.
• Third Spacing – Shifting in intravascular fluids to extracellular
spaces
• Other conditions associated are Congestive Heart Failure, Sepsis,
Anaphylaxis, Renal Artery Embolic Occlusions
 POST RENAL
DISEASE
• BILATERAL HYDRONEPHROSIS – results from high
grade or long standing obstruction of the urinary tract
• Prostatic Hyperplasia or Carcinoma
• Bilateral Ureteral Obstruction - Stones, Clots, sloughed
tissues
• Urethral Obstruction – Stricture or valves
 RENAL PARENCHYMAL DISEASE
• Acute Glomerulonephritis
• Interstitial Nephritis
• Acute Tubular Necrosis (ATN)
• ATN – most common cause is Renal Ischemia (attributed to
Heart Failure or Hypotension)
• ATN – attributed to Hemoglobinuria/Myoglobinuria
• Nephrotoxic agents – some antibiotics, Mercury, Cadmium,
Carbon Tetrachloride and Glycerol.
CHRONIC RENAL
FAILURE
•The progressive and irreversible loss of renal function
attributed to several diseases
•Hypertensive & Diabetic Associated Nephrosclerosis,
Chronic Glomerulonephritis, Polycystic Kidney disease and
other urologic diseases)
•Urine SG is Low
•Proteinuria, Cast and renal cells may be evident
PYELONEPHRITIS/ INTERSTITIAL
NEPHRITIS

•Tubular dysfunction with polyuria in early

stage of the disease and later progresses into
oliguria of Chronic Renal Failure
URINE COLOR
• rough indicator of hydration status
• Should correlate with the urine Specific
Gravity
• Normal color – is colorless to deep yellow
• Abnormal – Red or Red brown (Most
URINARY
PIGMENTS
1. UROCHROME Yellow Pigment; the major pigment; Its production is
directly proportional to metabolic rate
Increased in Starvation, Thyrotoxicosis and Fever
2. Pink Pigment; may deposit in amorphous urates and
UROERYTHRIN uric acid crystals

3. UROBILIN Dark yellow/Orange pigment; Imparts that color to a

urine that’s not fresh. *Bacteria has converted
Urobilinogen into Urobilin
CLINICAL SIGNIFICANCE OF URINE
COLOR Recent Fluid intake
Colorless
Pale Yellow Polyuria, Diluted random Specimen
Dark Yellow Concentrated Urine, Strenous exercise, First morning Urine
Amber Dehydration, Fever, Burns
Orange Bilirubin *With yellow foam when shaken
Phenazopyridine (Pyridium);
Orange and viscous urine with orange foam
Acriflavin, Nitrofurantoin, Phenindione
Yellow green to Bilirubin oxidized into Biliverdin
Yellow Brown
Green Pseudomonas infection
 Indican, Amitriptyline, Methocarbanol, Clorets, Methylene
Blue-Green Blue, Phenol and Chlorophyll
Pink, Red RBCs (Cloudy / Smoky) Intact RBCs
Hemoglobin – Clear Red; Intravascular Hemolysis
Myoglobin - Clear Red or reddish brown – Muscle Damage
Beets, Rifampin, Menstrual Contamination
Burgundy/ Purplish/ Porphyrins
Portwine
Brown or Black Methemoglobin in acidic urine
Homogentisic Acid (Alkaline Urine)
Alkaptonuria (Upon Long Standing)
Melanin – (Upon Exposure to Air)
Phenol Derivatives, Argyrol, Methyldopa/Levodopa,
Metronidazole (Flagyl)
 HEMATURIA VS.
HEMOGLOBINURIA

HgB HgB

INTAC HgB LYZED

HgB
T RBCS HgB
RBCS
HgB
HgB
Hg
HgB B
CLINICAL SIGNIFICANCE OF URINE ODOR
Aromatic Normal (Volatile acids)
Foul, Ammoniacal UTI (ex. Proteus vulgaris)
Fruity or Sweet Ketones (Diabetes mellitus, Starvation or
vomiting)
Caramel, Curry or Maple Syrup Urine Disease (Leucine,
Maple Syrup Isoleucine and Valine)
Mousy, Musty Phenylketonuria (PKU)
Rancid Butter Tyrosinemia
Sweaty Feet, Acrid Isovaleric Acidemia, Glutaric Acidemia
URINE ODOR
Cabbage Methionine Malabsorption
Hops Methionine Malabsorption and Oasthouse
Urine Disease
Bleach Contamination
Sulfur Cystine Disorders
Rotting Fish Trimethylaminuria
Pungent Ingestion of Onions, garlic and asparagus
Swimming pool Hawkinsinuria
CLARI TERM
TY
Clear No visible particulates, transparent
Hazy Few Particulates, print easily seen through urine
Cloudy Many Particulates, print blurred through urine
Turbid Print cannot be seen through urine
Milky May precipitate or be clotted
CHEMICAL
EXAMINATION
GLUCOSE 30s
PRINCIPLE :
DOUBLE
SEQUENTIAL
ENZYME REACTION
REPORTING
• MULTISTIX AND
NEGATIVE
TRACE CHEMSTRIP REAGENT
1+ GLUCOSE OXIDASE AND
2+ PEROXIDASE
M = POTASSIUM IODIDE
3+
4+
C=
 PRINCIPLE: DIAZO
BILIRUB 30s REACTION

IN
REPORTING • M = 2,4 DICHLOROANILINE DIAZONIUM SALT
QUALITATI • C = 2,6 DICHLOROBENZENE DIAZONIUM
VE SALT
NEGATIVE REPORTING NEGATIVE, 1+, 2+, 3+
SMALL
MODERATE POSITIVE RESULT: TAN, PINK OR
LARGE VIOLET
 PRINCIPLE :
KETONE 40s
LEGAL’S TEST OR
SODIUM
BODIES NITROPRUSSIDE
• MULTISTIX & CHEMSTRIP = SODIUMTEST
NITROPRUSSIDE
• C = ADDITION OF GLYCINE TO DETECT
SEMIQUANTITATIVE QUALITATIVE
ACETONE
REPORTING REPORTING POSITIVE
NEGATIVE NEGATIVE
TRACE (5mg/dl) TRACE RESULT:
SMALL (15mg/dl) SMALL (1+)
MODERATE (40mg/dl)
LARGE (80-160mg/dl)
MODERATE (2+)
LARGE (3)
PURPLE
 PRINCIPLE :
SPECIFIC 45s pKa Change of a
GRAVITY Polyelectrolyte
• M = POLY METHYL VINYL ETHER MALEIC ANHYDRIDE
• C = ETHYLENEGLYCOLDIAMINOETHYLETHER TETRAACETIC
ACID
CHROMOGEN: BROMTHYMOL Dissociation Constant of a
Polyelectrolyte in an alkaline
BLUE medium
SPECIFIC GRAVITY READING
SHADES OF GREEN TO More Ions = More Hydrogen
YELLOW Ions are released
pH 60s PRINCIPLE: DOUBLE INDICATOR
SYSTEM

• M AND C = METHYL RED AND BROMTHYMOL

BLUE

pH READING
METHYL RED = RED TO YELLOW AT 4-6
BROMTHYMOL BLUE = YELLOW TO BLUE AT
6-9
PROTEI 60s
PRINCIPLE : SORENSEN’S
PROTEIN ERROR OF INDICATORS

N
• M = TETRABROMPHENOL BLUE
• C = TETRACHLOROPHENOL
QUALITATIVE REPORTING
TETRABROMOSULFONTHTHALEIN TRACE
NEGATIVE, TRACE, 1+, 2+, 3+, <30 mg/dl SEMIQUANTITATI
4+ VE REPORTING
30mg/dl, 100mg/dl,
CHROMOGEN: BROMPHENOL 300mg/dl, 2000mg/dl
BLUE
RESULTS: SHADES OF GREEN TO
BLUE
 BLOO 60s
PRINCIPLE:
PSEUDOPEROXIDASE ACTIVITY
OF HEMOGLOBIN
D
• M = DIISOPROPYLBENZENE REPORTIN
DEHYDROPEROXIDE G
• C= DIMETHYLDIHYDROPEROXYHEXANE NEGATIVE
TRACE
CHROMOGEN : SMALL
TETRAMETHYLBENZIDINE MODERATE
RESULTS:
LARGE
FREE HEMOGLOBIN / MYOGLOBIN : UNIFORM GREEN
OR BLUE-GREEN 1+
INTACT RBCS: SPECKLED/SPOTTED ABSORBENT PAD 2+
3+
 PRINCIPLE :
UROBILINOGE 60s EHRLICH’S
REACTION
N
• M = P-DIMETHYLAMINOBENZALDEHYDE
• C = 4 METHOXYBENZENE DIAZONIUM
TETRAFLUOROBORATE
RESULTS REPORTING BY UNIT
MULTISTIX: LIGHT TO DARK MULTISTIX: EHRLICH UNIT
PINK CHEMSTRIP: mg/dl (More Specific)
CHEMSTRIP: WHITE TO PINK
BEST ANSWER: RED
>1mg/dl Urine Urobilinogen – seen in hepatic disorders and hemolytic
episodes
 NITRI 60s PRINCIPLE REACTION
: GREISS

• MTE
= P- ARSANILIC ACID, TETRAHYDROBENZO(H)-QUINOLINE-3-
OL
• C = SULFANILAMIDE, HYDROXYTETRAHYDRO
BENZOQUINOLINE
RESULT – EQUIVALENT TO DETECTS THE
100,000 BACTERIA PER ML BACTERIA WITH
POSITIVE RESULT : UNIFORM REDUCTASE
PINK ENZYME
REPORTED AS POSITIVE OR
LEUKOCYT 120s PRINCIPLE : LEUKOCYTE
ESTERASE
ES
• M = DERIVATIZED PYRROLE AMINO ACID ESTER, DIAZONIUM
SALT
• C = INDOXYLCARBONIC ACID ESTER, DIAZONIUM SALT

REPORTING
TRACE, SMALL, MODERATE, LARGE OR 1+. 2+. 3+

POSITIVE RESULT :
 PURPLE
AUTOMATED REAGENT STRIP
READER

• PRINCIPLE: REFLECTANCE PHOTOMETRY

- Light reflection from the test pads decrease in proportion to the
intensity of color produced by the concentration of the test
substance
• Inversely proportional (Increase Light Reflection Decrease
Color Intensity) AND VICE VERSA
REFLECTANCE
PHOTOMETRY
SPECIFIC GRAVITY
• Density of solution compared with density of
similar volume of distilled water at a similar
temperature
• Influenced by number and size of particles in a
solution
SPECIFIC
GRAVITY
NORMAL SG 1.003 - 1.035 ISOSTHENURIA 1.010
(RANDOM)

When SG <1.003 Not a urine (Except in HYPOSTHENUR <1.010

Diabetes Insipidus) IA

When SG >1.040 Radiographic Contrast HYPERSTHENU >1.010

media RIA
Intravenous Pyelogram (Left Picture)
Retrograde Cystogram (Right Picture)
GRAVITY
• URINOMETRY/URINOMETER/HYDROMETE
R
CALIBRATION TEMPERATURE =
20°C
TEMP. CORRECTION (± 0.001 ↕ 3°C)
-0.004 FOR EVERY 1g/dl of Glucose
-0.003 FOR EVERY 1g/dl of Protein
Calibration Reagents = Potassium Sulfate solution (20.29 grams of K2SO4 to 1 liter
Water) (SG of 1.015)
DETERMINATION OF SPECIFIC
GRAVITY
REFRACTOMETER (TOTAL SOLIDS
METER)
• Indirect Method based on Refractive Index
• Compensated to temperature (15-38 Celsius)
• Requires correction for Glucose and Protein (same in
urinometry)
Calibration reagents include
Distilled Water (SG of 1.000)
5% Sodium Chloride (1.022 ± 0.001)
9% Sucrose (1.034 ± 0.001)
DETERMINATION OF SPECIFIC
GRAVITY

• HARMONIC OSCILLATION DENSITOMETRY

• - Based on the frequency of a soundwave entering a
solution changes in proportion to the density of the
solution.
• Ex. Yellow IRIS (International Remote Imaging System)
ACIDITY AND ALKALINITY OF
URINE

• pH - Important in the identification of crystals

and determination of unsatisfactory specimens
• Normal Random Ph = 4.5 - 8.0
• 1st Morning = 5.0 - 6.0
ACIDIC URINE ALKALINE URINE
Diabetes Mellitus Renal Tubular Acidosis
Starvation Vegetarian Diet
High Protein Diet After Meals
Cranberry Juice Old Specimen
Emphysema, Dehydration, Hyperventilation
Diarrhea, presence of Acid- Presence of urease
producing bacteria (E.coli) and producing bacteria
medications
URINE
PROTEIN

• PROTEIN - most OTHER PROTEINS

indicative of renal diseaseo Serum and Tubular Microglobulins
o Tamm-Horsfall Protein (Uromodulin)
• Albumin – Major Serum o Protein derived from prostatic and
Protein found in the urine vaginal secretions
(White foam)

Normal Value of
<10mg/dl or <100mg/day (Strasinger) or <150mg/24hrs (Henry)
PRE-RENAL PROTEINURIA
(OVERFLOW
PROTEINURIA)
• Conditions that affect the plasma prior to reaching the kidney
• A. Intravascular Hemolysis – Hemoglobin
• B. Muscle Injury – Myoglobin
• C. Severe infection and malignancy – increase of APRs
 PRE-RENAL
PROTEINURIA
• D. Multiple Myeloma – malignant plasma cells release defective
antibodies in form of free light chains called Bence-Jones Protein
(identical as Kappa-Kappa or Lambda-Lambda)
• Tests for BJP – Serum Electrophoresis, immunofixation
electrophoresis
• Urine= precipitates at 40-60 Celsius and dissolves at 100 Celsius
RENAL
PROTEINURIA A. GLOMERULAR
PROTEINURIA
• 1. Diabetic Nephropathy – decreased in glomerular filtration and
that may lead to renal failure.
• Indicator is = Microalbuminuria
• *Not detected by routine urinalysis reagent strip.

ALBUMIN EXCRETION RATE = ug/min or in mg/24 hours

Normal AER = 0-20 ug/min
Microalbuminuria = 20-200 ug/min or 30-300 mg/24 hrs
Clinical Albuminuria - >200 ug/min
RENAL
PROTEINURIA
• ORTHOSTATIC/ POSTURAL PROTEINURIA
• Proteinuria when standing due to increased pressure
to renal veins
• Compare protein from First morning sampling and 2
hours after standing.
• Clinical Proteinuria will show both positive reactions.
RENAL
PROTEINURIA
• B. TUBULAR PROTEINURIA - normally filtered
albumin can no longer be reabsorbed.
• 1. Fanconi’s Syndrome
• 2. Toxic agents and heavy metals
• 3. Severe viral infections
POST RENAL
PROTEINURIA

• Attributed to lower urinary tract

• 1. Lower UTI or Inflammations
• 2. Injury or Trauma
• 3. Menstrual contamination
• 4. Prostatic fluid or spermatozoa
• 5. Vaginal Secretions.
SORENSEN’S PROTEIN ERROR OF
INDICATORS

•Protein changes the color of the reagent

regardless of what pH the urine is.
•Reagent Strip is sensitive to Albumin only.
 SULFOSALICYLIC ACID
PRECIPITATION
GRADE TURBIDITY PROTEIN RANGE
(MG/DL)
NEGATIVE No increase in turbidity <6
TRACE Noticeable turbidity 6-30
1+ Distinct Turbidity with no granulation 30-100
2+ Turbidity with granulation but no 100-200
flocculation
3+ Turbidity with granulation and flocculation 200-400
4+ Clumps of protein >400
URINE GLUCOSE - MOST FREQUENTLY TESTED SUGAR IN
URINE.
CLINICAL SIGNIFICANCE OF URINE GLUCOSE
HYPERGLYCEMIA RENAL ASSOCIATED
ASSOCIATED
HIGH BLOOD GLUCOSE NORMAL BLOOD GLUCOSE
HIGH URINE GLUCOSE HIGH URINE GLUCOSE
Causes: Impaired Tubular Reabsorption of Glucose
Diabetes Mellitus Cause
Cushing’s Syndrome - Fanconi’s Syndrome – the inability of the renal
Acromegaly tubules to reabsorb glucose and amino acids.
Pheochromocytoma
Hyperthyroidism
• GLUCOSE- RENAL THRESHOLD OF 160 – 180 mg/dl
• The concentration of a substances at which the tubular reabsorption
stops.
 CLINITEST
PROCEDURE
5 DROPS OF URINE + 10 DROPS OF WATER + CLINITEST
TABLET
• PASS THROUGH PHENOMENON Occurs when >2 g/dl of sugar is
present
- Blue > Green > Yellow > Brick Red > Blue or Green – Brown
- To prevent this phenomenon, use 2 drops of urine
TABLET CONSTITUENTS
CuSO4 – Main Reacting Reagent
NaCO3 – Eliminates interfering
Oxygen
Na Citrate – for Heat production
NaOH – for heat production
KETONE
BODIES
• the byproduct of fatty acid metabolism due to the inability to metabolize
carbohydrates
• Seen in Diabetes Mellitus, Vomiting, Starvation, Malabsorption
TOT KETONE BODIES
AL
78% Beta- Hydroxybutyric Acid Major Ketone body but is not
detected in reagent strips
20% Acetoacetic Acid / Diacetic Parent Ketone
Acid
2% Acetone Detected in the presence of
Glycine
ACETEST TABLET
TEST

• CONTAINS
• SODIUM
NITROPRUSSIDE
• DISODIUM PHOSPHATE
• LACTOSE
 BLOOD
HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA
Cloudy Red Urine Clear Red Urine Clear Red Urine
Seen in Seen in HTR, Hemolytic Seen in Rhabdomyolysis
Glomerulonephritis and Anemia, Intravascular Heme portion of the
in Renal Calculi Hemolysis myoglobin protein that
is toxic to the tubules
Microscopic Analysis – *Pose more damage
may show intact RBCs risk than Hemoglobin

PLASMA
EXAMINATION
Blondheim’s Test
(Ammoniun Sulfate Test)
* Urine + 2.8 grams of
Ammonium Sulfate (80%
satd.)
Filter then centrifuge
Test the supernatant for
blood with the reagent strip
HEMOGLOBIN VERSUS MYOGLOBIN
HEMOGLOBIN MYOGLOBIN
Red/Pink Plasma – Pale Yellow
evidence of Hemolysis Increase in CK and
Marked Decrease of Aldolase
Haptoglobin
Precipitated Hemoglobin at No precipitation occurred
the bottom of the tube Urine is still Clear Red
NEGATIVE for Blood POSITIVE for Blood
Reagent Strip Reagent Strip
 BLOOD RGT STRIP

(NH4)2SO4

NEGATIVE FOR BLOOD REAGENT

STRIP
Hgb Hgb (+) HEMOGLOBINURIA
Hgb Hgb
Hgb
BLONDHEIM’S
 TEST

BLOOD RGT STRIP

Mb (NH4)2SO4

Mb
Mb
POSITIVE FOR BLOOD REAGENT
STRIP
Mb
Mb
(+) MYOGLOBINURIA

BLONDHEIM’S
 TEST

BILIRUBIN
• the conjugated type of Bilirubin is detected in this test.
• Conjugated Bilirubin, Water Soluble Bilirubin, Direct Bilirubin
• Early indication of liver disease
• Demonstrated by formation of yellow foam in an Amber-colored
Urine
• Seen in diseases like Hepatitis, Cirrhosis, and Biliary Obstructions
(Gallstones, Carcinoma)
 HEMOGLOBIN
120 HEME GLOBIN

DAYS IRON PORPHYRIN

RING

Heme Oxygenase
UNCONJUGATED
BILIRUBIN BILIVERDIN
ALBUMIN Biliverdin Reductase

UDPGT UROBILINOGEN
UROCHROME
CONJUGATED
BILIRUBIN

STERCOBILIN
HEMOLYTI
C DISEASE

UNCONJUGATED
BILIRUBIN

UROBILINOGEN

UDPGT
CONJUGATED
BILIRUBIN
UROCHROME

STERCOBILIN
COMPARISON BETWEEN B1
and B2
BILIRUBIN 1 BILIRUBIN 2
• Unconjugated Bilirubin • Conjugated Bilirubin
• Water Insoluble • Water Soluble
• Non- Polar Bilirubin • Polar Bilirubin
• Indirect Reacting • Direct Reacting
• Hemobilirubin • Cholebilirubin
• Slow reacting • One Minute/ Prompt Bilirubin
• Pre Hepatic Bilirubin • Post Hepatic/ Hepatic/ Obstructive/ Regurgitative
 UROBILINOGEN

• UROBILINOGEN – Bile pigment that results

from Hemoglobin degradation in the intestines
• Normal Value of <1 mg/dl or Ehrlich Unit
• Specimen of Choice is Afternoon Urine sample
(2pm – 4PM)
 U B U B U B
C B

C U C U C U
EHRLICH
UROBILINOGE PORPHOBILINOGE REACTIVE
N N COMPOUNDS
WATSON – SCHWARTZ TEST

•Urine Layer remains Red = the analyte

is insoluble to the extracting agents
•Extracting Layers turns Red= the
reagent has extracted the analyte from
Urine
 NITRITE
RAPID SCREENING TEST FOR URINARY TRACT
INFECTION
SPECIMEN OF CHOICE IS 4 HOURS URINE
• CLINICAL SIGNIFICANCE OF NITRITE
• Cystitis, Pyelonephritis
• Evaluation of antibiotic therapy
• Monitoring of patients at high risk for urinary tract infection
• Screening of urine culture specimens
NITRITE Reductase is found in the gram-negative bacteria
(Enterobacteriaceae) that most frequently cause UTIs.

• Bacteria that lack the enzyme reductase do not possess

the ability to reduce nitrate to nitrite.
• Non–nitrate-reducing gram positive bacteria and yeasts,
however, cause a significant number of infections, and
the nitrite test does not detect the presence of these
organisms.
 LEUKOCYTE
ESTERASE

• LEUKOCYTE ESTERASE – Significance includes detecting

Urinary Tract Infections and Screening of Urine Culture specimens
• Cells that possess the Esterase enzyme – Neutrophils, Eosinophils,
Basophils, Monocytes, Histiocytes and Trichomonas
• Cells that do not possess the Esterase enzyme - Lymphocytes,
erythrocytes, bacteria, and renal tissue cells
ASCORBIC ACID
• a reducing agent that causes false negative results to various
parameters
• 11th Reagent Pad – Ascorbic Acid (≥5 mg/dl) +
Phosphomolybdate = (POSITIVE) Molybdenum Blue
• Gas Chromatography – Mass Spectrometry = provides more
accurate and quantitative method
FALSE NEGATIVE RESULTS
(Blood, Bilirubin, Leukocyte Esterase, Nitrite and
 MICROSCOPIC
ANALYSIS OF URINE
 MICROSCOPIC TECHNIQUES
BRIGHTFIELD Routine Urinalysis
MICROSCOPY
PHASE CONTRAST Enhances the visual of elements with low refractive index
MICROSCOPY
POLARIZING To identify Cholesterol in oval fat bodies, fatty casts and
MICROSCOPY crystal
DARK FIELD To identify Treponema pallidum
MICROSCOPY
FLUORESCENCE To visualize fluorescent organisms or those stained with
MICROSCOPY fluorescent dyes.
INTERFERENCE- 3D microscopy image and layer-by-layer imaging of the
CONTRAST specimen
Bright Field Microscopy can be adapted
A. Nomarski (Differential)
SEDIMENT STAINS
STAIN ACTION FUNCTION
Sternheimer-Malbin Delineates structures and Identifies WBC, Epithelial
(Crystal Violet + Safranin 0) contrasting colors of nucleus cells and Casts
and cytoplasm
Toluidine Blue Enhances nuclear detail Differentiates WBC and
(Supravital) RTE cells
2% Acetic Acid Lyses RBC, enhances WBC’s Distinguishes RBC from
nuclei WBC, Yeast cells, oil
droplets and crystals
Lipid Stain (Oil Red O & Stains Triglycerides and Free Fat Droplets and lipid
Sudan III) Neutral Fats orange red containing cells and casts
*Cholesterol can’t be stained
SEDIMENT STAINS
STAIN ACTION FUNCTION
Gram Stain Gram Positive and Bacterial casts
Gram Negative
differential stain
Hansel Stain (Eosin Eosinophillic Urinary Eosinophils
Y + Methylene Blue) granules
Prussian Blue Stains Iron containing Yellow Brown
structures granules of
hemosiderin in cells
and casts
 URINARY
SEDIMENTS
 RED BLOOD
CELLS
SEEN IN HEMATURIA (NV OF 0-2 OR 0-
3/HPF)
- Smooth, non-nucleated, biconcave discs
- Hypertonic Urine – RBCs are crenated
- Hypotonic Urine – RBCs are hemolyzed, swollen (Ghost cell)
- Glomerular Membrane Damage – Dysmorphic RBCs
- Sources of Error – Yeast cells, Oil droplets, Air bubbles, Calcium
Oxalate Crystals
- Remedy – Add 2% acetic acid = RBCs will lyze.
RED BLOOD CELLS
 WHITE BLOOD
CELLS SEEN IN PYURIA (NV OF 0-5 OR 0-
8/HPF)
Increased WBCs indicate an
inflammatory or infectious process
Neutrophils – Most Predominant WBC –
granulated and multi-lobed, in hypotonic
urine, it tends to swell and undergo
Brownian Movement producing a sparkling
appearance (Glitter cells)
Mononuclear cells – (Lymphos,
Eosinophils – NV of <1% (Demonstrated by Hansel Monos, Macros, histiocytes) –
stain) present in small number
- Significant in Drug induced Interstitial Nephritis
WHITE BLOOD CELLS
 EPITHELIAL
CELLS
- SQUAMOUS EC = largest cell with
abundant irregular cytoplasm and
prominent nucleus
- From lining of the vagina, female urethra
and lower portion of the male urethra
• Variation= Clue Cells (are Squamous
ECs covered with Gardnerella vaginalis)
 TRANSITIONAL CELL/
UROTHELIAL CELL
- spherical, polyhedral or caudate
with centrally located nucleus
- Lining of renal pelvis, ureter,
urinary bladder and upper
portion of the male urethra
- Increased numbers are seen
following catheterization
 Most Clinically Significant
RENAL EC, originated from the
Nephron
TUBULAR
VARIATIONS EC > 2 RTE cells/hpf – suggests
tubular injury
- Oval Fat Body (Lipid Containing
RTE) seen in Lipiduria (Nephrotic
Syndrome)
- Identified through Lipid Staining
(TAG and Neutral fats) and
Polarizing Microscope (Cholesterol
showing the characteristic Maltese
Bubble Cell – RTE with non-lipid filled
Cross Appearance)
vacuoles, seen in acute tubular necrosis
 MICROORGANISMS
BACTERIA – Urinary Tract
Infection
Most Common UTI is caused by
Enterobacteriaceae (Escherichia
coli)
- Staphylococcus saphrophyticus
and Enterococcus
 YEAST – True Yeast Infection is

PARASITES
• Trichomonas vaginalis (most frequently seen in urine) –
Agent of Ping Pong Disease (Pear-shaped flagellate)
• Schistosoma haematobium ova – Blood fluke ova with terminal
spine, causes Hematuria, Associated with Bladder cancer
(Specimen is 24 hours unpreserved urine)
• Enterobius vermicularis ova – most common fecal contaminant
PARASITES IN URINE
•SPERMATOZOA – after sexual
intercourse or masturbation.
•MUCOUS THREADS – made up of
Tamm – Horsfall protein (the matrix for
casts)
SPERMATOZOA AND MUCUS
THREADS
CYLINDRURIA – CAST
FORMATION

- The structure that is unique to the kidney

- Forms in the Distal Convoluted Tubules and
the Collecting Ducts
- Major Constituent is Tamm – Horsfall Protein
 CASTS FORMATION
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
3. Further protein fibrils interweaving to form a solid structure
4. Possible attachment of urinary constituents to the solid matrix
5. Detachment of protein fibrils from the epithelial cell
6. Excretion of the cast.
SEQUENCE OF CAST
FORMATION

•HYALINE CAST
•CELLULAR CAST
•COARSE GRANULAR
CAST
•FINE GRANULAR CAST
TYPES OF CASTS

RBC CAST RBC CAST WBC CAST RTE CAST

FATTY CAST GRANULAR CAST WAXY CAST BROAD CAST

 URINARY CASTS
TYPE INFORMATION CLINICAL SIGNIFICANCE
HYALINE Prototype cast, Beginning of all casts Seen in strenuous exercise and
CAST stress
Seen in Glomerulonephritis,
Pyelonephritis and Congestive
Heart Failure
RBC CAST Bleeding within the nephron Glomerulonephritis
Strenuous exercise
WBC CAST Inflammation within the nephron Pyelonephritis
May be confused with EC cast Acute Interstitial Nephritis
Differentiate using Phase Contrast
Microscopy and Supravital stain
RTE CAST Tubular Injury Advanced Tubular Destruction
 Renal Tubular Damage

BACTERIAL Gram stain to differentiate Pyelonephritis

CAST
GRANULAR Derived from lysosomes of RTE cells Glomerulonephritis
CAST during normal metabolism Pyelonephritis
Stress and Strenuous Exercise
FATTY CAST Not stained by Sternheimer-Malbin stain Nephrotic Syndrome
Identified through Oil Red O and Sudan (Lipiduria)
III (Triglycerides and Neutral Fats)
And Maltese Cross appearance (for
Cholesterol)
WAXY CAST Final Degenerative form of all types of Stasis of Urine Flow
casts Chronic Renal Failure
BROAD CAST Renal Failure Cast Extreme Urine Stasis
Widening of the tubular walls, any type Renal Failure
of casts can be broad
 URINARY
CRYSTALS
• formed by the precipitation of urine solutes (salts, organic
compounds, medications)

• Factors that contribute to crystal formation (Temperature, Solute

Concentration, Ph of the urine)

• Usually reported in qualitative manner. Pathologic crystals may be

averaged and reported per lpf.
 CRYSTALS IN ACIDIC URINE
INFORMATION SIGNIFICANCE SOLUBILIT
Y

AMORPHOU Brick Dust, Yellow Brown Alkali and

Granules, Pink Sediment Heat
S URATES (Uroerythrin)
Product of Purine Metabolism Lesch-Nyhan Syndrome Alkali
URIC ACID
Rhombic, Wedge, Hexagonal, four
Chemotherapy (increased
sided plate (Whetstone), Lemon metabolism of cell nuclei)
Shaped
Can be mistaken as Cystine Gout
crystals
DiHydrate (Wheddelite) Increase in Food rich in Dilute
CALCIUM ascorbic acid (Tomato/
- Envelope shaped HCL
OXALATE MonoHydrate (Whewellite)
Asparagus)
Ethylene Glycol Poisoning
- Oval, Dumbell
 URIC ACID
CRYSTALS

AMORPHOUS URATES
CALCIUM OXALATE
DIHYDRATE
 CRYSTALS IN ALKALINE URINE
AMORPHOUS Granular in Appearance Dilute Acetic Acid
PHOSPHATES
AMMONIUM BIURATE Yellow Brown Presence of Acetic Acid with heat
Thorny Apples Urea-splitting
Seen in Old Specimens bacteria
TRIPLE PHOSPHATE/ Colorless, Prism shaped or Coffin Lid shaped Presence of Dilute Acetic acid
MAGNESIUM Feathery appearance when they disintegrate Urea-splitting
AMMONIUM bacteria
PHOSPHATE/
STRUVITE
CALCIUM PHOSPHATE/ Colorless, flat plates, thin prisms, often in rosette forms Dilute Acetic Acid
APATITE Rosettes may resemble Sulfonamide crystals
Other forms
- Hydroxyapatite – Basic Ca Phosphate
- Brushite – Calcium Hydrogen Phosphate
CALCIUM CARBONATE Small, colorless, dumbbell or spherical shaped Gas from Acetic Acid
Formation of Gas after adding Acetic acid
 TRIPLE
PHOSPHATE

CALCIUM CARBONATE AMMONIUM

BIURATE
 ABNORMAL CRYSTALS IN URINE
INFORMATION SIGNIFICANCE SOLUBILITY
CYSTINE Colorless, Hexagonal plates, Cystinosis, Cystinuria Ammonia, Dilute HCl
mistaken as Uric Acid crystal
CHOLESTER Rectangular plates with notched Nephrotic Syndrome Chloroform
OL edges, resembled crystals from (Lipiduria)
radiographic contrast dye
RADIOGRAPH How to differentiate with 10% NaOH
IC DYE Cholesterol
- Patient history
- Correlate other other
parameters
- SG of >1.040 in
refractometry
TYROSINE Colorless to Yellow Needles in Liver Disease Alkali or Heat
clumps or needles
CYSTINE TYROSINE CHOLESTEROL

LEUCINE BILIRUBIN AMPICILLIN

URIC ACID VS. CYSTINE
URIC ACID CYSTINE
COLOR YELLOW COLORLESS
BROWN
AMMONIA SOLUBLE INSOLUBLE
DILUTE. HCl INSOLUBLE SOLUBLE
BIREFRINGENCE BIREFRINGENT NOT
CYANIDE NITROPRUSSIDE NEGATIVE POSITIVE
REACTION
LEUCINE Yellow Brown Spheres with Liver Disease Hot Alkali, or
Concentric circles and radial striations Alcohol
BILIRUBIN Clumped needles or granules with Liver Disease Acetic Acid,
bright yellow color HCl, NaOH,
Ether,
Chloroform
SULFONAMI Colorless, to Yellow Brown needles, Possible tubular Acetone
DE sheaves of wheat, rosette damage
May be mistaken as calcium phosphate May deposit in LIGNIN TEST
crystal nephrons Newspaper:
To Differentiate Urine + 25%HCl
Ca Phosphate: Soluble in Acetic Acid = Yellow
Sulfonamide: LIGNIN TEST
(POSITIVE)
AMPICILLIN Colorless needles that tend to form Massive Refrigeration
 ARTEFACTS
- Starch Granules – Spheres with dimpled center; shows maltese
cross on polarizing microscope too.
- Oil Droplets, Air bubbles, Pollen grains (spheres with wall and
concentric circles
- Hair and fibers – mistaken as casts
- Fecal contamination
SCREENING FOR
ERRORS OF
METABOLISM
 AMINOACIDURI
A
• Presence of amino acids in the urine resulting
from excess amino acid in plasma brought by
Enzyme Deficiency or Over excretion of Amino
acids due to abnormal metabolism
 RENAL TYPE
AMINOACIDURIA

• NORMAL Amino Acid in Blood

• INCREASE Amino Acid in Urine
• Defective Tubular reabsorption of certain
amino acids
 RENAL TYPE AMINOACIDURIA
PLASMA KIDNEY URINE

AMINO ACID

AMINO ACID

AMINO ACID
OVERFLOW TYPE
AMINOACIDURIA
• INCREASE Amino Acid in Blood
• INCREASE Amino Acid in Urine
• excess amino acid in the plasma causing the hypersecretion of
the substance in the urine
• Enzymes are normally defective (Low or absent)
OVERFLOW TYPE
AMINOACIDURIA
PLASMA KIDNEY URINE
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
AMINO ACID
OVERVIEW OF PHENYLALANINE/TYROSINE
METABOLISM
PHENYLKETONURIA
Phenylalanine Hydroxylase deficiency

• Mental Retardation is the Major Clinical Finding

• Mousy/Musty odor of urine/sweat
• GUTHRIE BACTERIAL INHIBITION
• ION – EXCHANGE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY – CONFIRMATORY TEST
GUTHRIE BACTERIAL INHIBITION
TEST

NO
PKU
PKU

THIENYLALANINE BACTERIAL
INHIBITOR
TYROSLYLURIA
Rancid Butter odor of urine
Transitory Tyrosinemia
- Seen on premature/Low weight infant
- Excess Para hydroxy phenylpyruvic acid
- Excess Para Hydroxy phenyl lactic acid in urine

CONFIRMATORY – CHROMATOGRAPHY / QUANTITATIVE

SERUM ASSAY FOR TYROSINE
 TYROSLYLURIA
• Hereditary Tyrosinemia
- very rare genetic disorder, also marked with glucosuria,
proteinuria, ketonuria and absence of phosphate
- Also, known as Tyrosinosis
- Excess in Para Hydroxy Phenylpyruvic acid
- Loss in Para Hydroxy Phenyl Lactic Acid
ALKAPTONURIA
HOMOGENTISIC ACID OXIDASE
DEFICIENCY

• Urine Darkens after becoming alkaline from exposure to room temp.

• CONFIRMATORY – PAPER – THIN LAYER CHROMATOGRAPHY,
CAPILLARY ELECTROPHORESIS
• Increase in Homogentesic acid – due to deficiency of the enzyme
• Normally, Homogentesic Acid is converted to Maleyl acetic acid
ALKAPTONURIA

Patients suffer from Ochronosis – dark blue to brown

pigmentations deposition in Connective tissues and cartilages
which can’t be clinically diagnosed until arthritis should be
MELANURIA
• Due to overproliferation of Melanocytes
• Urine darkens upon exposure to air
 MAPLE SYRUP URINE
DISEASE
Carboxylase Enzyme Deficiency

• Increased amino acids particularly:

• LEUCINE, ISOLEUCINE, VALINE
• Characteristic Caramelized Sugar, Maple Syrup
Odor of Urine
• 2,4 Dinitrophenylhydrazine (DNPH) test
• (+) Yellow turbidity or precipitate
• *Confirmed through Amino Acid chromatography
 INDICANURIA
• Indican is derived from Tryptophan.
• Normally, Tryptophan is absorbed as is,
or metabolized as indole (bacterial
action). Indole is oxidized into Indican
(in the liver)
• Blue Diaper Syndrome – Hartnup
Disease
• Indican is found in urine
• Malabsorption of amino acid indican
OVERVIEW OF TRYPTOPHAN
METABOLISM
ARGENTAFFINOMA5-HIAA (5-HYDROXYINDOLEACETIC
ACID)

• Carcinoid tumor of argentaffin/ enterochromaffin cells

• FeCl3 Tube = (+) Blue-Green
• Nitrosonaphthol with Nitrous Acid = (+) Violet
• *Avoid eating bananas, pineapples and tomatoes
 CYSTINE DISORDERS
DISORDER INFORMATION TEST
Cystinuria Renal Type of Brand’s Modification of Legal’s
Aminoaciduria Nitroprusside
Defective tubular Rgt used is CYANIDE NITROPRUSSIDE
reabsorption of (+) Red-Purple Color
“COLA”
Cystinosis Inborn Error of Metabolism - Lack of Gene coding for enzymes
needed in Cystine metabolism
Cystine crystal deposition on Lyzosome of cells in kidneys, eyes, bone
marrow and spleen
Most affected organ is the kidney
Differentiating Cystinuria from Cystinosis - Cystinosis has Recurrent
stone formation
Homocystinuria Defective Metabolism of Homocystine;
 MUCOPOLYSACCHARIDE DISORDERS
Impaired metabolism of Mucopolysaccharides or glycosaminoglycans
(proteins + polysaccharides, located in Connective tissues)

DISORDERS INFORMATION TEST

Hurler Mucopolysaccharides accumulate in the Acid Albumin Test = (+)
Syndrome cornea of the eyes White Turbidity
Cetyltrimethylammonium
Hunter Sex-Linked Recessive Disorder, rarely Bromide
Syndrome seen in females (CTAB) Test = (+) White
Sanfilippo Mental Retardation is the only Turbidity
Syndrome abnormality
RENAL
DISORDERS
Acute Post Deposition of Immune Complexes, formed in Macroscopic Hematuria
Streptococcal conjunction of Group A Streptococcus Proteinuria, Dysmorphic RBCs and
Glomerulonephritis infection on glomerular membranes RBC cast
Granular Casts and (+) ASO titer

Rapidly Progressive Immune complexes deposited on glomerular Macroscopic Hematuria

(Crescentic) membranes Proteinuria
Glomerulonephritis Epithelial Cells proliferate inside Bowman’s RBC casts
Capsule to form crescents

Good Pasture Deposits of Antiglomerular Basement Macroscopic Hematuria

Syndrome Membrane Antibody to glomerular and alveolar Proteinuria
basement membrane RBC casts

Wegener’s Anti-neutrophillic Cytoplasmic Auto-Antibody Macroscopic Hematuria

Granulomatosis binds to Neutrophils in vascular walls producing Proteinuria
damage to small vessels in the lungs and RBC casts
glomerulus
Henoch Schonlein Purpura Occurs in children following Viral Macroscopic Hematuria
Respiratory infections Proteinuria
Decrease in Platelets disrupting RBC casts
vascular integrity
Membranous Thickening of Glomerular membrane Microscopic Hematuria
Glomerulonephritis following IgG deposition Proteinuria
Membranoproliferative Cellular Proliferation affecting Capillary Hematuria
Glomerulonephritis walls or the glomerular basement Proteinuria
membrane, possibly immune mediated
Chronic Glomerulonephritis Marked Decrease in renal function Hematuria; Proteinuria
resulting from glomerular damage Glucosuria; Cellular,
precipitated by other renal disorders. Granular,
Progression to Renal Failure Waxy and Broad Casts
 IgA Deposition on the Early Stages:
IgA Nephropathy glomerular membrane Macro/Microscopic
(Berger’s Disease) resulting from Hematuria
increased levels of IgA Late Stages:
Same as Chronic
Glomerulonephritis

Nephrotic Syndrome Disruption of the electrical charges that produce the

tight fitting podocyte barrier resulting in massive loss of
protein and lipids
CLINICAL FINDINGS
SERUM URINE

DECREASE INCREASE MICROSCOPIC

Albumin, Alpha, Albumin, Lipid
Gamma Globulins Alpha, Beta, FINDINGS
Gamma
OVAL FAT BODIES,
INCREASE DECREASE = WAXY CASTS
LIPID, APO B 100 Alpha 2 Proteins
(VLDL AND LDL)
Minimal Change Little Cellular Changes in the Heavy Proteinuria
Disease (Lipid glomerulus; Disruption of podocytes Transient Hematuria
Necrosis) primarily in children following Fat Droplets
allergic reactions & immunization
Focal Segmental Disruption of podocytes in certain Proteinuria and Hematuria
Glomerulosclerosis numbers and areas of the glomeruli,
others remain normal
Diabetic Most common cause of ESRD Microalbuminuria
Nephropathy Deposition of Glycosylated Proteins (+) Micral Test
(Kimmelstiel – on the glomerular basement
Wilson Disease) membrane caused by poorly
controlled blood glucose levels
Alport Syndrome Genetic Disorder showing lamellated See Nephrotic Sydrome
and thinning of glomerular
basement membrane
 TUBULAR DISORDERS
Damage to the renal tubular cells caused by Microscopic Hematuria,
Ischemia or toxic agents Proteinuria, RTE cells
Acute Tubular and RTE Cast
Necrosis Hyaline, Granular,
Waxy, Broad Cast
Fanconi Syndrome Generalized Failure of tubular reabsorption in the Glucosuria
proximal convoluted tubule Possible Cystine Crystal
Diabetes Insipidus Neurogenic DI Low Specific Gravity
- Hypothalamus fails to produce ADH Polyuria
Nephrogenic DI
- Inability of the renal tubules to respond to
ADH
Renal Glucosuria Normal Blood Glucose Glucosuria
Increase Urine Glucose
Due to defective tubular reabsorption of glucose
DISORDER ETIOLOGY FINDINGS
Cystitis Ascending bacterial infection of the WBCs, Bacteria
urinary bladder Microscopic Hematuria
Mild Proteinuria
Increased Ph
NO CAST
Acute Infection of Renal tubules and WBCs and WBC cast,
Pyelonephritis interstitium. Related to interference of Bacteria and Bacterial Cast
urine flow to the bladder, reflux of the Microscopic Hematuria and
urine from the bladder (vesicoureteral Proteinuria
reflux) and untreated cystitis
Chronic Pyelonephritis
Acute Interstitial Nephritis
Recurrent Infection of the
renal tubules and
interstitium caused by the
structural abnormalities
affecting the flow of urine
Allergic Inflammation of
the renal interstitium
response to certain
medications
WBCs and WBC cast,
Bacteria and Bacterial cast,
Granular, Waxy and Broad
cast, Hematuria and
Proteinuria
Hematuria, Proteinuria
WBCs (Increased
Eosinophils)
WBC cast
NO BACTERIA
RENAL
STONES
RENAL STONE

• Conditions that favor the formation of renal calculi are

• Ph, Chemical Concentration, Urinary Stasis
• Primary Microscopic Finding – Microscopic
Hematuria
CALCULI SIGNIFICANCE
Calcium Major constituent of Renal calculi. Very hard, dark brown in color
Oxalate with rough edges
Uric Acid Associated with increased intake of foods with high purine
content. Yellowish to brownish red and moderately hard
Cystine Seen in Hereditary disorders of cystine metabolism. Yellow
Brown, Greasy & resembles an old soap, least common calculi (1-
2%)
Phosphate Pale and Friable
Triple Accompanied by urinary infections involving urea-splitting bacteria
Phosphate
Calcium Oxalate Uric Acid Cystine

RENAL
 STONES
• Sulfonamide calculi
• Silica calculi – Ingestion of Silica over a long period of
time
• Triamterene calculi – Insoluble diuretic, mustard colored
stones
• Adenine Calculi – Associated with Inherited Enzyme
Deficiency and Hyperuricemia
• Xanthine Calculi – Associated with a genetic disorder with
an absence of xanthine oxidase
PREGNANCY TESTS
WHY IT IS
PERFORMED?
• Confirm Early Normal Pregnancy
• Completeness of Abortion (Miscarriages)
• Differs pregnancy from trophoblastic diseases
• Hydatidiform moles
• Choriocarcinoma
• Chorioepithelioma
• Teratoma (males <30 years old) leading to Testicular Cancer
POSITIVE – Beta Human Chorionic
Gonadotrophic Hormone
•Made by cytotrophoblastic cell of a developing placenta
•Secreted after 2-3 days (implantation of embryo)
and detectable in 8-10 days
•Increase in First Trimester and Decrease after 3 first months
BIOASSAYS FOR
PREGNANCY
 A. Gali Mainini (Male Frog)
• Positive result is production of very
motile banana shaped sperm cells
• 2-4 hours
• - inject urine in frog’s thigh
(subcutaneous, intramuscularly)
• Observe for Spermatogenesis for 30
minutes to 4 hours
• Species of Rana pipiens,
• Bufo americanus, Bufo bufo
 B. Hogben’s Test (Female
Frog)
• Positive result is exclusion of
ova for 8-12 hours.
• Species of Xeropus laevis
(South African Cloved
frog/toad)
• Disadvantage: Female Frogs are
sensitive and requires longer
 incubation

C. Hyperemia
• use of female rabbits, rats or mice. Observe for enlargement of
uterus or ovaries
• 1. Friedmann’s Test – Use of Virgin female rabbits, use 15 ml
urine, inject intravenously
• - incubate for 30-48 hours, sacrifice the rabbit and observe for
changes: full or enlarged ovaries
• - 1-6 Corpora hemorrhagica (red spots in ovaries) & Corpora
• 2. Hoffman’s Test – same with Friedmann’s, except
that serum sample is used.
• 3. Kelso, Frank Perman and Kupperman – uses
rats, incubation is 16-24 hours (Kelso – Frank) and 24
hours (Kupperman)
• - inject urine sample subcutaneously
• 4. Aschiem Zundek – uses Mice, incubation is 100-
120 hours
CHEMICAL PREGNANCY
TEST
Less accurate test, based on the presence of hormones (NON –HCG)

1. Pregnandiol test – Use to determine the presence of

progesterone
(+) deep orange or dark brown color, (-) amber color
- Get 5 ml of urine sample, add few drops of pregnandiol
reagent – observe for color change

2. Iodine Test/ Lugol’s reaction – (+) deep orange or

dark brown; - detecting Epinephrines
CLINICIAN’S PREGNANCY
TEST
A. Prostigmen – Withdrawal Bleeding Test – Physician
injects prostigmen; observe for bleeding
(-) bleeding within 2 weeks (+) No bleeding at all
B. Skin Colostrum Test – subcutaneous injection of
colostrum in the forearm; observe for redness
(-) Redness/Swelling (+) No Redness/Swelling
C. Premodas Oral – patient will take oral drug for 2 days,
observe for bleeding within 5 days (-) negative
FINISHED IN DAY 1
AFTERWORD
OTHER BODILY FLUIDS
By: James Ryan A. Mendoza, RMT, MLS (ASCPi)
BEFORE THE SESSIONS…
 OBJECTIVES FOR
TODAY

• Discuss the Importance of Laboratory Testing and its Relevance in Medicine.

• CEREBROSPINAL FLUID
• SEMINAL FLUID
• SYNOVIAL FLUID
• SEROUS FLUIDS (3P’s)
• AMNIOTIC FLUID
• GASTRIC FLUID
• Other Bodily Fluids (Sputum, Sweat, Stool)
CEREBROSPINAL FLUID
CEREBROSPINAL FLUID

• 3RD Major Body fluid

• Functions include:
• Supplies nutrients to the nervous system, removal of
metabolic wastes and produces a mechanical barrier to
cushion the brain and spinal cord against trauma.
 ANATOMY OF THE
BRAIN

• MENINGES – lining of the brain and the spinal cord consisted

of 3 layers
• Dura Mater (Latin for Hard mother) – the outer layer lining the
skull and the vertebral canal
• Arachnoid Mater – the “spider-like” filamentous inner
membrane
• - Subarachnoid Space – located between the Arachnoid Mater
and the Pia Mater, this is where the CSF flows
• Pia Mater (Latin for Gentle mother) – the innermost layer
lining the surface of the brain and the spinal cord.
CHOROID PLEXUS - the site for CSF production

• - Adults produce approximately 20 ml of CSF per hour

• 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
ARACHNOID GRANULATIONS/ VILLAE
CSF VOLUME
90-150 ml in adults and10-60 ml in infants.

• site of CSF reabsorption to maintain volume. Same rate of production is

as same as reabsorption.
• The cells of the arachnoid villae serve as a one-way valve that responds
to the pressure of CNS and prevents reflux of the fluid.
• Reabsorbed CSF returns to the blood circulation.
 BLOOD-BRAIN
BARRIER

• tight-fitting endothelial cells found on the choroid plexus.

• The barrier protects the brain from chemicals and other substances
circulating in the blood that can harm the brain tissue.
• Disruption of the BB barrier allows WBCs, protein and other
chemicals to enter the CSF (examples of conditions are Meningitis
and Multiple Sclerosis)
 SPECIMEN COLLECTION

• Up to 20 ml of fluid can be collected

• Lumbar tap between 3rd, 4th or 5th lumbar vertebrae.
• Intracranial pressure and opening pressure must be
considered before collecting the adequate volume of CSF.
STANDARD COLLECTION TUBES FOR
CSF

#1 – the least affected by RBC’s and bacteria

attributed to tap procedure
CHEM BACTE HEMA #2 – bacterial contamination is least
SERO
#3 – cytospin cellular differentials and
hemocytometer chamber counts. It should
1 2 3
contain no cells from surrounding tissues.

Physical examination of CSF may be done in this tube.

An optional #4 is provided as additional microbiology and serology sample.
TAKE
NOTE:

• CSF TESTINGS ARE PERFORMED ON A STAT! BASIS

(Rationale: Rapid Cellular degeneration)
• If delays are inevitable, CSF samples are stored in a specific manner
• #1 – Frozen Order of Usage
• #2 – stored in Room temperature 1st for Microbiology
• #3 – Refrigerated. 2nd for Hematology
• ***If ONE tube is only available 3rd for Chemistry and
Serology.
 TAKE NOTE:

• ALL SECTIONS OF THE LABORATORY SHOULD

NOT DISCARD THEIR SAMPLES EVEN AFTER THE
TESTS ARE DONE.
• All leftover supernatant fluids are saved for additional/follow
up chemistry and/or serology tests. It must be stored at
freezing temp until no further tests are required.
 CHARACTERISTICS OF CSF
APPEARANCE CAUSED BY CLINICAL SIGNIFICANCE
Crystal Clear Normal
Hazy, Turbid, Milky, WBCs, RBCs Meningitis (Increased WBC >200/ul)
Cloudy *Pleocytosis – causes (Increased RBC >400/ul)
Cellular Turbidity
Microorganisms Meningitis
Lipids and Protein BB barrier has been compromised
Protein IgG production within the CNS
Oily Radiographic contrast
media
Bloody RBCs Traumatic tap, hemorrhage (>6000/uL)
Pink to Red RBC Lysis 4–10 hours after subarachnoid hemorrhage.
Traumatic tap.
Xanthochromi Melanin Meningeal Melanosarcoma
a (Brown Methemoglobin, Subdural or Intracranial hematoma
color)
(Hubbarb)
Clotted Protein and Clotting factors BB barrier is compromised
Fibrinogen introduced to CSF from traumatic tap
Froin Syndrome - caused by meningeal irritation (e.g.
during spinal meningitis) and CSF flow blockage by
tumor mass or abscess. Blockage of CSF circulation
through subarachnoid hemorrhage
Pellicle Clotting Factors Tubercular Meningitis
DEALING WITH TRAUMATIC TAPSBB– barrier
Proteins A grossly bloody
has been CSF aspirate is an
compromised.
indication of an intracranial hemorrhage or a traumatic tap.

-3 visual inspections are observed to see if the cause of the bloody sample is indeed
from trauma or intracranial bleeding.
 DEALING WITH TRAUMATIC
TAPS

1. BLOOD DISTRIBUTION
• Intracranial Hemorrhage – evenly distributed
• Traumatic Tap – gradual decrease of blood, heaviest concentration at #1

2. CLOT FORMATION – Traumatic taps contaminate CSF by introducing plasma

coagulation factors (Fibrinogen)
• Visually inspected for clots
• Microscopic clumping of RBCs
DEALING WITH TRAUMATIC
TAPS

3. SUPERNATANT INSPECTION
• Clear Supernatant indicates traumatic tap
• Colored Supernatant/ Xanthochromic Supernatant – RBC lysis, Old
hemorrhage.
• Proper inspections include centrifugation in a microhematocrit tube and
reading against a white background
 DEALING WITH TRAUMATIC TAPS

• Erythrophages – are Macrophages with ingested RBCs. Indicative of

Intracranial Hemorrhage
• Hemosiderin granules seen in Intracranial hemorrhage
• D - Dimer Assay - Detects D-dimer by latex agglutination immunoassay
indicates fibrin formation at a hemorrhage site.
 CSF CELL
COUNTING

TOTAL CSF WBC NORMAL VALUE

ADULT: 0-5 WBCs/ul

NEONATE: 0-30 WBCs/ul
• Any cell count should be performed IMMEDIATELY.
• - WBCs and RBCs begin to disintegrate within 1 hour.
• - 40% WBCs disintegrate within 2 hours.
• - Routine Cell Counting is performed on WBCs
CSF CELL COUNTING

• WBC DILUTING FLUID – 3% Acetic Acid (RBC hemolyzing agent)

• METHYLENE BLUE – Stain for WBCs (Better differentiation bet.
PMN and MN)
• WRIGHT STAIN – WBC differential stain (Hubbarb)
CSF DIFFERENTIAL COUNT

• performed on a stained smear

• PREDOMINANT CELLS IN THE CSF:
• Predominantly: Lymphocytes and Monocytes.
• Occasionally: Neutrophils
• Adults: 70:30 ratio (Lympho to Mono)
• Neonates: Up to 80% Monocytes (considered as normal)
CSF DIFFERENTIAL COUNT

• -specimens must be concentrated before smearing by:

a. Cytocentrifugation
• Fluid is added to a conical chamber
• Cells are forced into a monolayer within a 6mm diameter circle on a slide
• Addition of Albumin to increase cell recovery and decreases cell distortion
b. Centrifugation
c. Sedimentation
d. Filtration
CSF CELL
COUNTING

• RBC COUNTING – done only in cases of a traumatic tap

• PLEOCYTOSIS – an increased number of normal cells in CSF
considered as abnormal.
• counting RBCs corrects for WBC counts and total protein concentration

• Subtract 1 WBC for every 700 RBCs seen

• Subtract 8 mg/dl Total protein concentration for every 10,000 RBCs/ul
(Henry)
• Subtract1 mg/dl Total protein concentration for every 1,200 RBCs/ul
 (Strasinger)
CELLS SEEN IN
CSF
TYPE OF CELL MAJOR CLINICAL SIGNIFANCE MICROSCOPIC FINDING
SEEN
Lymphocytes and Normal All stages of development may be found
Monocytes Viral, Tubercular and Fungal meningitis Monocytes mixed with Lymphocytes
Multiple Sclerosis
Neutrophils Bacterial Meningitis Granules may be less prominent in blood
Early cases of Viral, Tubercular and Cells disintegrate rapidly
Fungal Meningitis
Cerebral Hemorrhage
Eosinophils Parasitic/Fungal infections Coccidioides immitis
Macrophages RBCs in spinal fluid, contrast media May contain phagocytized RBCs appearing
as empty vacuoles or ghost cells,
hemosiderin granules, and hematoidin
crystals
TYPES OF CELL SEEN MAJOR CLINICAL MICROSCOPIC FINDING
SIGNIFICANCE
Blast forms Acute Leukemia Lymphoblasts, Myeloblasts or
monoblasts
Lymphoma cells Disseminated Lymphoma Resembles Lymphocytes with
cleft nuclei
Plasma cells Multiple Sclerosis, Lymphocytes Traditional and classic forms seen
reactions Reactive Lymphs
Ependymal, Choroidal and Diagnostic procedures Seen in clusters with distinct
Spindle-shaped cells nuclei and distinct cell walls
Malignant cells Metastatic carcinoma, primary CNS Seen in clusters with fusing of cell
Carcinoma borders and nuclei
CHEMICAL ANALYSIS
CSF
PROTEIN
• Most frequently tested in chemical testing in CSF.
• Normal CSF protein have very small amount.
• Adults have 15-45 mg/dl protein
• Infants have 150mg/dl protein.
• Immature infants have 500mg/dl protein.

INCREASED in Meningitis, Hemorrhage (BB barrier has been compromised)

Production of IgG within CNS (Multiple Sclerosis)
DECREASED in CSF Leakage
 CSF PROTEIN

• ALBUMIN is the Major CSF Protein

• PREALBUMIN is the 2nd Most Prevalent
• Alpha Globulins – Haptoglobulin and Ceruloplasmin
• Beta Globulins – B2 Transferrin or TAU Protein (Carbohydrate
Deficient Transferrin, found only in CSF)
• Gamma Globulins – primarily IgG and some IgA
• NOT FOUND IN NORMAL CSF - IgM, Fibrinogen and Lipids
CSF PROTEIN DETERMINATION

• TURBIDIMETRIC METHODS – adapted to automation instrumentation

in form of nephelometry
• TRICHLOROACETIC ACID (TCA) – preferred method for precipitation
of albumin and globulins (+) White Ptt.
• SULFOSALICYLIC ACID (SSA) – precipitates Albumin only. Add
Sodium Sulfate (Na2SO4) to precipitate Globulins (+) White Ptt.
 CSF PROTEIN DETERMINATION

• DYE BINDING TECHNIQUES

• COOMASSIE BRILLIANT BLUE – Protein binds to the dye, change of color
from Red to Blue
• Increased Protein = Increased Blue Color.
• GLOBULIN
• ROSE JONES’ and Nonne Apelts’ – Uses Ammonium sulfate;
• (+) result is Grayish-white (Rose Jones’)
• (+) result is cloudy white ptt. (Nonne Apelts’)
• PANDY’S – commonly used for globulin; (+) result is Bluish-white cloudiness.
 PROTEIN
FRACTIONS

• Diagnosis of Neurologic disorders cannot be easily be done through

sole reliance to total protein concentrations. Therefore, it is important
to establish individual protein fraction values correlated to certain
disorders.

• ELECTROPHORESIS – performed in conjunction with

Serum Protein Electrophoresis
• For diagnosis of Multiple Sclerosis - (+) 2 or more of IgG
bands in Gamma region
• “Oligoclonal Banding”
 (+) Anti-Myelin Sheath Autoantibody
MULTIPLE SCLEROSIS
(+) Myelin Basic Protein (MBP)

• a Demyelinating disorder, caused by an autoantibody

• (+) in Oligoclonal bands in CSF BUT not in serum
• Increased IgG index
• *MBP – Protein component of the lipid-protein complex
that insulate the nerve fibers, presence of MBP indicates
destruction of myelin sheathes and is used to monitor the
course of Multiple Sclerosis
CSF
ELECTROPHORESIS

*Multiple Sclerosis, Neurosyphilis, Encephalitis,

Neoplastic Disorders and Guillain – Barre Syndrome

• “MS. NENG” – Conditions that produce Oligoclonal Banding on Gamma regions for CSF
only.
• CSF/SERUM ALBUMIN INDEX – Assess the integrity of the Blood Brain Barrier
• NORMAL VALUE is <9 ABNORMAL is >9
*Correlates the degree of damage.
*An Index of 100 means Complete Damage of BB barrier
• IgG INDEX – assess the conditions with IgG production within
the CNS (ex. In Multiple Sclerosis)
• NORMAL VALUE is <0.77
• ABNORMAL IS >0.77
*Indicates the IgG production within the CNS
 CSF GLUCOSE (NV= 60-70% of Blood Glucose)
CSF GLUCOSE CLINICAL SIGNIFICANCE
INCREASE Hyperglycemia, Traumatic taps
MODERATE CNS Leukemia, Meningeal Carcinomatosis, Subarachnoid Hemorrhage, Partially
DECREASE treated Bacterial or Fungal Meningitis (while CSF Lactate will be High)
MARKED Bacterial, Tubercular and Fungal Meningitis
DECREASE
NORMAL Viral Meningitis, Neurosyphilis, Brain or cord tumor, cerebral thrombosis,
multiple sclerosis, polyneuritis
ARTIFACTUAL Delayed Fluid analysis that contains large numbers of metabolizing cells.
-Done in conjunction with Blood Glucose. Specimen for Blood Glucose must be
DECREASE
collected 2 Hours prior to CSF collection.
-No fasting. Normal value is 60-70% of the Blood Glucose
CSF GLUCOSE
• Glucose metabolism is an active process that continues after the
sample has been aspirated. Therefore, immediate testing must be
performed in samples suspected to contain microorganisms or
granulocytes.

• Dramatic drop in CSF Glucose occurs in purulent meningitis.

 CSF LACTATE (10-22mg/dl)
CSF LACTATE CLINICAL SIGNIFICANCE
INCREASE Bacterial Meningitis (>35mg/dl)
Tubercular Meningitis and Fungal Meningitis (>25mg/dl)
Tissue Destruction within CNS owing to Oxygen deprivation
(Hypoxia) Cerebral Edema, Hydrocephalus, Brain Abscess
NORMAL Viral Meningitis
INCREASED Lactate values elevation persists after antibiotic therapy.
VALUE Differential diagnosis on inadequately treated meningitis.
PERSISTS
-CSF Lactate is inversely
ARTIFACTUA proportional
Xanthochromic, to the
Hemolyzed CSF Glucose
fluid.
-Lactate reflects local glycolytic activity
L INCREASE
 CSF LACTATE
• Severe Systemic Lactic Acidosis increases CSF Levels in parallel, but an
isolated increase of CSF Lactate indicate an increased glucose metabolism of
microorganisms and leukocytes
• Lactate elevations are not limited to meningitis and can result from conditions
in which the brain tissue is deprived of oxygen
• it is used to monitor severe head trauma, RBCs contain high lactate
concentrations and falsely elevated results can be expected in xanthochromic
or hemolyzed samples
CSF GLUTAMINE (8-18mg/dl)

• Product of Ammonia and Alpha-Ketoglutarate.

• This process serves to remove the toxic metabolic waste product
ammonia from the CNS
• Indirect test for the presence of Excess Ammonia in CSF
• INCREASED GLUTAMINE in REYE’S SYNDROME and
Disturbance of Consciousness (Coma)
CSF GLUTAMINE
• Normally, Ammonia is converted back to Urea.
Ammonia is neurotoxic and is therefore eliminated in the
liver.
• Liver diseases hinder the normal detoxification process,
leading to increasing Ammonia concentrations in the
blood.
 CSF GLUTAMINE

• Ammonia is toxic in the body.

• In the Brain (Reye’s syndrome), Ammonia is converted to
Glutamine
• Glutamine in the brain compromises the Kreb’s Cycle
leading to coma due to lack of ATP in the brain. Ammonia
increases CNS pH.
CSF GLUTAMINE

• Elevated Ammonia in Plasma is Neurotoxic.

• It leads to Encephalopathy.
• Ammonia lowers Gamma Aminobutyric Acid (GABA), a
critical important neurotransmitter in CNS, by reacting
with glutamic acid to form glutamine via reversal of
glutaminase-catalyzed reactions
 CSF ENZYMES
• LACTATE DEHYDROGENASE – differentiates traumatic puncture from
intracranial hemorrhage

LDH PATTERNS (LDH CLINICAL SIGNIFICANCE

(SAMPLE) Isoenzymes)
SERUM LDH 2>1>3>4>5 NORMAL
1>2>3>4>5 (Flipped) Acute Myocardial Infarction

CSF LDH 1>2>3>4>5 NORMAL

2>1>3>4>5 Neurological Abnormalities

5>4>3>2>1 Bacterial Meningitis

CSF ENZYMES

• CREATINE KINASE – 3 isoforms, CK-MM for

muscles, CK-MB for the Cardiac tissues and CK-BB for
the Brain. (Brain damage)

• ADENOSINE DEAMINASE – for TB Meningitis

 CSF MICROBIOLOGY

• CSF culture sample stained with bacterial stains (Gram’s and

AFB) and Fungal stains (India Ink and Lactophenol Cotton Blue)
to demonstrate microorganism’s morphological characteristics

• A CSF giving Positive Culture tests is a confirmatory feature. But

other techniques like immunoassays provide a rapid screening
result.
 MENINGITIS-CAUSING MICROORGANISMS

• Cryptococcus neoformans
• Coccidiodes immitis
• Mycobacterium tuberculosis
• Haemophilus influenzae
• Neisseria meningitidis
• Streptococcus pneumoniae
• Staphylococcus aureus (Hubbarb)

Other microorganisms seen/isolated in CSF – Treponema pallidum, Naegleria fowleri,

Streptococcus agalactiae, Trypanosoma spp. Listeria monocytogenes, Eschericia coli,
Capnocytophaga
 AGENTS OF BACTERIAL MENINGITIS

AGENTS OF BACTERIAL AGE GROUP AFFECTED

MENINGITIS
Streptococcus agalactiae 0-1 month old
Haemophilus influenzae 1 month to 5 years old
Neisseria meningitidis 5-29 years old
Streptococcus pneumoniae >29 years old
Listeria monocytogenes Infants, elderly, immunocompromised
 OTHER ASSAYS

• LIMULUS LYSATE TEST – detects Gram Negative Bacterial Endotoxin

(Neisseria meningitidis)
• Reagent - Blood of Horseshoe crab or Limulus polyphemus
• (Blue blood caused by Hemocyanin, a copper -containing blood pigment)
• Principle: In the presence of endotoxin, the amoebocytes (WBC’s) will
release the lysate (+) clumping or clot formation
 TAKE
NOTE:

• Viral meningitis caused by Poliovirus, Echovirus, Coxsackievirus,

Enterovirus
• Mycobacterium tuberculosis in CSF show AFB and Pellicle/web-like clot
formation after 12-24 hours refrigeration
• Cryptococcus neoformans show Starburst pattern in Gram Stain, Halo in
India Ink preparation (Negative stain)
FINISHED IN CSF
SEMINAL FLUID ANALYSIS
SEMINAL FLUID ANALYSIS

The purpose of Semen analysis is for

• fertility testing
• post-vasectomy analysis
• qualifications for artificial insemination programs
• determining quality in semen/sperm banking
• alleged rape cases and paternity.
COMPOSITION OF SEMEN
SEMINAL FLUID
5% Spermatozoa Produced in the Seminiferous tubules of the testicles in a process
called Spermatogenesis
Sertoli cells serve as nurse cells for developing sperm cells
Sperm maturation occurs in the Epididymis (sperms become motile)
60-70% Seminal Produced in the Seminal vesicles
Fluid Provides nutrients for sperm and the fluid
Rich in fructose sugar – energy for motility
20-30% Prostate Acidic fluid that contains Acid Phosphatase, Zinc, citric acid and
Fluid other enzymes
For coagulation and liquefaction
5% Cowper’s gland Thick alkaline mucus that neutralizes acidity from the prostatic
 secretions and the vagina.

SPECIMEN
COLLECTION
• Important part of Analysis
• A. Sexual Abstinence – 2-3 days not more
than 5 days.
• B. Collection of the entire ejaculate

Methods of Collection include:

o Masturbation (most convenient)
o Coitus interruptus (withdrawal technique)
o Condom technique (use of non-lubricant containing rubber or silastic
condom)
SPECIMEN COLLECTION

• C. Specimen should be delivered to the laboratory within 1 hour of

collection at room temperature

• D. Take note of time of specimen collection, specimen receipt and

liquefaction

• E. Analysis should be done after liquefaction (usually 30-60 minutes)

• F. Specimen awaiting analysis should be kept at 37C.

 TAKE NOTE:

• Most of the sperm are contained in the first portion of the ejaculate,
making complete collection essential for accurate testing of both fertility
and post-vasectomy specimens.
• *MISSING FIRST PORTION OF THE EJACULATE – Decreased
Sperm Count, pH falsely increased, sample will not liquefy
• *MISSING LAST PORTION OF THE EJACULATE – Decreased
Semen volume, Sperm count falsely increased, pH falsely decreased,
sample will not coagulate.
LABORATORY EXAMINATION
APPEARANCE

• Pearly white, Grayish-White, Translucent (Normal)

• with musty or bleach odor
• Increased White Turbidity – Infection *Increase WBCs
• Red or Brown coloration – RBCs or Blood
• Yellow Coloration – Prolonged Abstinence, urine, drugs.
• *Flavin deposition
VOLUME
• NORMAL: 2-5ml
• 1ml (Abnormal *Harr)
• can reach to 15ml
• Increased in prolonged abstinence
• Decreased in Infertility, Incomplete collection
• Volume depends on various physiologic factors like hydration status,
frequency of ejaculation etc.
 VISCOSITY:

Viscosity Grading from 0 to 4 (0 – Watery) (4- Gel Like)

• VISCOSITY:
• NORMAL: Pours in droplets “creamy gelatinous”
• Increased Viscosity – decrease in sperm motility
Ph

• Normal pH is 7.2-8.0
• Increased pH indicates infection
• Decreased pH indicates more prostatic fluid
is present
SPERM REAGENTS: Papanicolau’s stain (the best
MORPHOLOGY stain), Wright’s or Giemsa

• ROUTINE CRITERIA of >30% Normal Forms

• KRUGER’S STRICT CRITERIA of >14% Normal forms
• -measure the head, neck and the tail using micrometer
• Head – normal shape is oval
• Midpiece – contains the mitochondria
• Tail – motility apparatus
SPERM MORPHOLOGY

• TAKE NOTE: Head of the Sperm cell measures

5um in length and 3um in width. The Tail
measures 45um. Acrosomal cap occupies 1/2 of
the sperm’s head and covers 2/3 of the sperm’s
nucleus
 SPERM MORPHOLOGY

Acrosomal cap occupies 1/2 of the sperm’s head and

covers 2/3 of the sperm’s nucleus

3µm

5µm 45µm
SPERM MOTILITY
• reading at 20 HPF; on 37C or room temperature, wet mount
prepared on pre-warmed slide and cover slip.

PERCENT MOTILITY
95% Immediately after ejaculation
50% Normal Within 1 hour
25-40% After 3-6 hours
0% After 12 hours
 Quality ≥2.0

SPERM MOTILITY
GRADING
MOTILITY GRADING (WHO CRITERIA)
GRADE DESCRIPTION
4.0 a Rapid- straight line motility
3.0 b Slower speed, some lateral movement
2.0 b Slow forward progression, noticeable lateral movement
1.0 c No Forward progression
0 d No movement
SPERM VIABILITY
• MODIFIED BLOOM’S TEST
• REAGENTS: Eosin and Nigrossin Stain
• *Living Sperm – Unstained, Bluish-white (75%)
• *Dead Sperm – Red Colored sperm

CONDITIONS OBSERVED IN VIABILITY TESTING

NECROSPERMIA – presence of dead spermatozoa
OLIGOSPERMIA – decreased number of spermatozoa
AZOOSPERMIA – absence of spermatozoa
CHEMICAL TESTS FROM
SEMEN
ANALYTE NORMAL VALUE DECREASED VALUES INDICATE
FRUCTOSE ≥13umol/ejaculate Lack of seminal fluid volume (Strasinger)
Ejaculatory Duct Obstruction or Pathology of
the Vas Deferens (Hubbarb)
NEUTRAL α- ≥20mU/ejaculate Disorders of the Epididymis
GLUCOSIDASE
ZINC ≥2.4umol/ejaculate Lack of Prostatic fluid / Prostatitis (Hubbard)
CITRIC ACID ≥52umol/ejaculate Lack of Prostatic fluid / Prostatitis (Hubbarb)
ACID ≥200 U/ ejaculate Lack of Prostatic fluid
PHOSPHATASE
SEMEN FRUCTOSE – tested within 2 hours for frozen to prevent fructolysis
SCREENING TEST- Resorcinol test (+) Orange-Red color
PROSTATE SPECIFIC ANTIGEN (Bishop)

• 28kD glycoprotein produced only in epithelial

cells of acini and ducts of prostatic ducts in the
prostate.
• A serine protease of Kallikrein gene family
 PROSTATE SPECIFIC ANTIGEN
(Bishop)

• Function is the regulation of Seminal Fluid viscosity. And

it is instrumental in dissolving cervical mucus cap
allowing sperm to enter. However, PSA as a means of
diagnosing Prostate cancer is still of a controversy.
• Why? PSA also increases in conditions like in Benign
Prostatic Hypertrophy and Prostatitis.
ACID PHOSPHATASE AND PSA FOR
DIAGNOSIS OF PROSTATE CANCER (Bishop)

• After surgical treatment of Prostate Cancer,

ACP levels fall faster than PSA (Prostate
Specific Antigen) and plasma levels are
expected to be undetectable following
complete removal of the tumor.
ACID PHOSPHATASE AND PSA FOR
DIAGNOSIS OF PROSTATE CANCER (Bishop)

• ACP is indeed an indicator of Cancer of the

Prostate, but it is replaced by PSA.

• Why? – Prostatic ACP is elevated but in majority

of cases wherein the cancer has metastasized.
 MICROBIAL ANALYSIS

• ROUND CELLS – Leukocytes and Spermatids

(Immature Sperm cells)

NORMAL VALUE: <1 Million/ml

>1 Million WBCs/ml – indicate an infection
>1 Million Spermatids/ml – Disrupted Spermatogenesis
MICROBIAL ANALYSIS

• *Peroxidase – positive granulocytes are the predominant

WBCs in semen, therefore it can also be further
differentiated from Spermatogenic cells and
Lymphocytes by Peroxidase stain.

TESTS FOR Chlamydia trachomatis, Mycoplasma

hominis and Ureaplasma urealyticum
 ANTISPERM
ANTIBODIES

• Detected in semen, cervical mucosa or serum.

• Presence of Male Anti-sperm antibodies can be observed
during a routine semen analysis.
• Sperm-agglutinating antibodies cause sperm to stick to
each other in a head-to-head, head-to-tail, or tail to-tail
pattern.
 ANTISPERM
ANTIBODIES

• The agglutination is graded as “few,” “moderate,” or

“many” on microscopic examination.
• Cause of male antisperm antibodies – compromised Blood
– Testes barrier.
• (Sperm cells exposed to male’s immune system)
ANTISPERM ANTIBODIES

• MIXED AGGLUTINATION REACTION – detects the

presence of IgG antibodies
• Semen Sample + AHG reagent + Latex particles or treated RBCs
coated with IgG
• Normal = <10% motile sperm cells attached to the particles
 ANTISPERM
ANTIBODIES

• IMMUNOBEAD TEST – Detects the presence of IgG, IgM and IgA

antibodies
• Demonstrates what area of the sperm (head, tail or neck) the autoantibodies
are affecting.
• Sperm are mixed with polyacrylamide beads known to be coated with
either anti-IgG, anti-IgM, or anti-IgA.
ANTISPERM ANTIBODIES

• Microscopic examination of the sperm shows the beads attached to sperm

at particular areas.

• Depending on the type of beads used, the test could be reported as “IgM
tail antibodies,” “IgG head antibodies,” and so forth. The presence of
beads on less than 50% of the sperm is considered normal as defined by
the WHO
ANTISPERM ANTIBODIES
POST – VASECTOMY SEMEN ANALYSIS

• VASECTOMY - the surgical procedure of cutting the Vas

Deferens so that the ejaculate will not contain any sperm cell
• The only concern is the presence or absence of sperm cells
• Done 2 months after vasectomy and continued until 2
consecutively monthly specimens show no sperm.
 MEDICO-LEGAL SEMEN TESTING

• Microscopic exam
• Fluorescence under UV Light (Green – Live sperm / Orange – Dead sperm)
• Acid Phosphatase
• Glycoprotein p30 – more specific method
• ABO Blood Grouping
• DNA Analysis
 MEDICO-LEGAL SEMEN TESTING

• FLORENCE TEST (not specific) – tests for Choline

• (Reagents: Iodine Crystals + KI)
• (+) Dark Brown Rhombic Crystals

• BARBIERO’S TEST (very specific) – tests for Spermine

• (Reagents: Saturated Picric Acid + TCA)
• (+) Yellow leaf-like crystals
SPERM
CONCENTRATION
NV: 20-160 Million/ml
Methods include
• IMPROVED NEUBAUER
COUNTING CHAMBER (Dilution of
1:20)
• Diluents include: Formalin, Sodium
Bicarbonate, Saline, Distilled Water or
Cold tap water.
SPERM COUNT
NV= ≥40 Million/ejaculate
*1:20 Dilution & 5 RBC Squares used ~ #Cells X 1,000,000
*1:20 Dilution & 2 WBC Squares used ~ #Cells X 100,000

• MAKLER COUNTING CHAMBER (no dilutions required)

• Uses heat to immobilize the sperm cells.
• *Calculations for Sperm Concentrations depend on the dilution and the
size and number of the squares used.
TESTING FOR ABNORMAL SEMEN ANALYSIS
ABNORMAL RESULT POSSIBLE CAUSE TEST
DECREASED Viability Eosin – Nigrosin Stain
MOTILITY W/
NORMAL COUNT
DECREASED COUNT Lack of seminal vesicle Fructose levels
support medium
DECREASED Male Antisperm Mixed Agglutination
MOTILITY W/ antibodies reactions
CLUMPING Immunobead test
Sperm agglutination with
male serum
NORMAL ANALYSIS Female antisperm Sperm Agglutination
SPERM FUNCTION TESTS
HAMSTER EGG Sperms incubated with species non-specific
PENETRATION hamster eggs and penetration is observed
microscopically
CERVICAL MUCUS Observe for sperm penetration ability of
PENETRATION partner’s midcycle cervical mucus
HYPO-OSMOTIC SWELLING Sperms are exposed to low sodium
concentrations are evaluated for membrane
integrity and sperm viability.
IN VITRO ACROSOME Evaluation of the acrosome to produce
REACTION enzymes essential for ovum penetration.
FINISHED IN
SEMEN ANALYSIS
SYNOVIAL FLUID
 SYNOVIAL
FLUID

• viscous fluid lubricating all moveable joints (diathroses)

• “Synovial” is latin for Eggwhite. Viscosity due to
Hyaluronic acid produced by the synoviocytes
• Functions: to lubricate joints, reduces friction between
bones, provides nutrients to the articular cartilages and
lessens the shock of joint compression during activities like
walking or jogging
SPECIMEN
COLLECTION ATHROCENTES
IS
• (Normal Synovial fluid does not clot)
• *Diseased joints may clot
• NORMAL VOLUME: <3.5 ml (> 25ml may indicate inflammation)
• Tubes are also provided for various laboratory tests
• #1 for Microbiology (Sodium Heparin)
• #2 for Hematology (Sodium Heparin/ Liquid EDTA)
 SPECIMEN COLLECTION

• *Do Not use POWDERED ANTICOAGULANTS and LITHIUM

HEPARIN
• for they may interfere with crystal identification.

• #3 for Chemistry and other tests –without anticoagulants

• #4 for Glucose Analysis – Sodium fluoride
 VISCOSITY

• SYNOVIAL FLUID VISCOSITY – formation of string

4 to 6 cm long from the tip of a syringe
• TEST: ROPE/MUCIN CLOT TEST
(HYALURONATE POLYMERIZATION)
• Reagent: 2-5 Acetic Acid (not used in WBC counting)
VISCOSITY

• Synovial fluid viscosity comes from polymerization of the

hyaluronic acid and is essential for the proper joints lubrication.
Arthritis affects both the production of hyaluronate and its
ability to polymerize, thus decreasing the fluid viscosity.

• Formation of a mucin clot after adding acetic acid can be used

to identify a questionable fluid as synovial fluid.
CELL COUNT NO ACETIC ACID FOR WBC
COUNTING

• Total WBC count is the most frequently performed

cell count on synovial fluid
• RBC count are seldom requested
• Diluting fluids used include:
• NSS with Methylene Blue
 CELL COUNT

• Hypotonic Saline (0.3%) and Saline with Saponin

• For very viscous fluid – Add a pinch of Hyaluronidase to
0.5 ml fluid or Add 1 drop of 0.05% Hyaluronidase in
Phosphate buffer per ml of fluid (incubate at 37 C for 5
mins)
NORMAL SYNOVIAL
FLUID

DIFFERENTIAL COUNTS VISCOSITY GRADING

RBCs - <2,000/uL Strasinger Strasinger Interpreted
WBCs- <200/uL 5th 3rd
WBC Diff.
Good Good Solid Clot
(65%) Monocytes and
Macrophages; Fair Fair Soft Clot
(<25%) Neutrophils; Low Poor Friable Clot
(<15%) Lymphocytes Poor Very Poor No Clot
CELLS IN SYNOVIAL
FLUID
CELL/INCLUSI DESCRIPTION SIGNIFICANCE
ON
Synovial lining Similar to macrophage, but may be Normal
cell multinucleated, resembling a mesothelial cell Disruption from
arthrocentesis
LE cell Neutrophil containing characteristic ingested Lupus Erythematosus
“round body”
Reiter cell Vacuolated macrophage with ingested Reactive arthritis
neutrophils (infection in
another part of the body)
RA cell (ragocyte) Neutrophil with dark cytoplasmic granules Rheumatoid arthritis
CELLS IN SYNOVIAL
FLUID
CELL/INCLUS DESCRIPTION SIGNIFICANCE
ION
Cartilage cells Large, multinucleated cells Osteoarthritis
Rice bodies Macroscopically resemble polished Tuberculosis
rice Septic and
Microscopically show collagen and rheumatoid arthritis
fibrin
Fat droplets Refractile intracellular and Traumatic injury
extracellular globules Chronic
Stain with Sudan dyes inflammation
Hemosiderin Inclusions within clusters of synovial Pigmented

SYNOVIAL FLUID
CRYSTALS

• Monosodium Urate (MSU)

• Calcium Pyrophosphate DiHydrate (CPPD)

• COMMON CAUSES OF GOUT include: Increased serum uric acid

resulting from impaired metabolism of purines; increased consumption of
high-purine-content foods, alcohol, and fructose; chemotherapy treatment of
leukemias; and decreased renal excretion of uric acid.
 SYNOVIAL FLUID
CRYSTALS

• CAUSES OF PSEUDOGOUT: most often associated with degenerative

arthritis, producing cartilage calcification and endocrine disorders that produce
elevated serum calcium levels
• Causes of crystal formation include: Metabolic disorders, Decreased renal
excretion of substances that increases blood levels of crystallizing chemicals,
Degeneration of bones and cartilages and injection of medications like
corticosteroids.
 SYNOVIAL FLUID
CRYSTALS

• BIREFRINGENCE is determined through the use of POLARIZING MICROSCOPY (to

see its absence or presence)

• COMPENSATED POLARIZING MICROSCOPY is used to determine if the

Birefringence is Positive or Negative

• *Red compensator is placed between crystal and analyzer.

SYNOVIAL FLUID
CHEMISTRY
CHEMICAL RATIONALE
TESTS
Glucose Most frequently tested chemistry test done in conjunction
with blood glucose (8 hours fasting)
*Blood Glucose – Synovial Fluid Glucose = <10mg/dl
(Normal)
Lactate Normal value of <250 mg/dl
Increased in infections
Protein Normal value of <3g/dl
Increased in Inflammatory and Hemorrhagic disorders
Uric Acid Normal Value is as same as Blood Uric Acid
 Increased in Gout
JOINT
DISORDERS
LABORATORY FINDINGS IN JOINT DISORDERS *Strasinger. 5th Edition.
GROUP I IIa IIb III IV
NON- INFLAMMATORY INFLAMMATOR SEPTIC HEMORRHAGI
INFLAMMATO (IMMUNOLOGIC) Y C
RY (CRYSTAL-
INDUCED)
SIGNIFICANCE Degenerative Joint Immunologic Gout and Microbial Traumatic injury,
Disorders diseases (RA, SLE, Pseudogout infection Coagulation
(Osteoarthritis) etc) deficiencies
COLOR/CLARI Clear, Yellow Cloudy, Yellow Cloudy or Milky Cloudy, Cloudy, Red
TY Yellow-green
VISCOSITY Good Poor Low Variable Low
WBC COUNT <1,000/ul 2,000-75,000/ul Up to 100,000/ul 50,000 to Equal to Blood
100,000/ul
NEUTROPHILS <30% >50% <70% >75% Equal to Blood
GLUCOSE Normal (Similar Decreased Decreased Decreased Normal

FINISHED IN SYNOVIAL FLUID

SEROUS FLUID
3P’s
(Peritoneal, Pericardial, Pleural)
 SEROUS FLUID

• SEROUS FLUID – the fluid found in between parietal and visceral

membranes
• Functions: to lubricate the spaces between 2 membranes as the surfaces move
against each other.
• EFFUSION – accumulation of fluid between the membranes
• (*Classified as either a Transudate or an Exudate)
 TRANSUDATE VS. EXUDATE

• TRANSUDATE – Effusion caused by a systemic disorder that disrupts the fluid production
and regulation between membranes. This results from excess filtration of blood serum across
a physically intact vascular wall due to disruption of reabsorption. This occurs in systemic
diseases that alter the hydrostatic pressure of the capillaries

• CAUSES: Hypoproteinemia, Congestive Heart Failure (Increased Hydrostatic pressure) and

Nephrotic syndrome, Hepatic Cirrhosis (Hubbarb)
 TRANSUDATE VS. EXUDATE

• EXUDATE – Effusion caused by direct damage to the membrane. Active

accumulation of fluid within body cavities associated with inflammation of the
membranes and vascular wall damage. Exudates are closer to serum in
chemical composition

• CAUSES: Infection and Malignancy

 SPECIMEN
COLLECTION

• SPECIMEN DISTRIBUTION
• EDTA for Cell counting and Differential counts
• STERILE HEPARIN TUBES for Microbiology and Cytology
• PLAIN HEPARIN TUBES for Chemistry (Specimens for pH
determination must be anaerobically in ice)
TRANSUDATE VS. EXUDATE *Strasinger 5th ed. Insert from Polansky, 2nd ed
TRANSUDATE EXUDATE
APPEARANCE CLEAR CLOUDY
FLUID:SERUM PROTEIN RATIO <0.5 >0.5
FLUID:SERUM LDH RATIO <0.6 >0.6
WBC COUNT <1,000/UL >1,000/UL
DIFFERENTIAL COUNT (Predominant cells) MN PMN
SPONTANEOUS CLOTTING NO POSSIBLE
PLEURAL FLUID CHOLESTEROL (mg/dl) <45-60 >45-60
PLEURAL FLUID:SERUM CHOLESTEROL RATIO <0.3 >0.3
<0.6 >0.6
PLEURAL FLUID: BILIRUBIN RATIO
SERUM – ASCITES ALB. GRADIENT >1.1 <1.1
GLUCOSE DECREASED INCREASED
TYPES OF SEROUS
FLUID
SEROUS SOURCE METHOD OF
FLUIDS COLLECTION
Peritoneal fluid The peritoneum enclosing Paracentesis/
abdominal organs. Peritoneocentesis
Pleural fluid The pleural cavity Thoracentesis
enclosing the lungs.
Pericardial The pericardium enclosing Pericardiocentesis
fluid the heart.
PLEURAL
FLUID
PLEURAL FLUID
APPEARANCE SIGNIFICANCE
Clear, pale yellow Normal
Turbid, white TB infection, microbial
Brown Rupture of amoebic liver abscess
Black Aspergillosis
Viscous Malignant mesothelioma
(Increased hyaluronic acid)
MILKY PLEURAL FLUID
CHYLOUS PSEUDOCHYLOUS

CAUSE Thoracic Duct Leakage Chronic Inflammation

APPEARANCE Milky/White Milky/ Green tinge/ Gold
paint
WBC Increase Lymphocytes Mixed cell
CHOLESTEROL Absent Present
CRYSTAL
TRIGLYCERIDE >110 mg/dl <50mg/dl
SUDAN III +++ -/weakly +
BLOODY PLEURAL
FLUID

•HEMOTHORAX ~ there is an uneven

distribution of blood
•HEMORRHAGIC EFFUSION ~ there
is even distribution of blood
BLOODY PLEURAL FLUID

• *To differentiate further, use the HEMATOCRIT as basis. (Get the 50% of Blood
Hematocrit and compare to the pleural fluid)

• *PLEURAL FLUID Hct is GREATER than 50% Whole Blood Hct = the bloody fluid
originates from HEMOTHORAX

• *PLEURAL FLUID Hct is LESSER than 50% Whole Blood Hct = the bloody fluid
originates from HEMORRHAGIC EFFUSION
 BLOODY PLEURAL
FLUID

PLEURAL WHOLE PLEURAL WHOLE

BLOOD BLOOD
22% Hct 50% Hct 40% Hct 50% Hct
HEMORRHAGIC HEMOTHORAX
EFFUSION
 PLEURAL FLUID
ANALYSIS
SIGNIFICANCE OF CELLS SEEN IN
CHEM. ANALYSIS PLEURAL FLUID
PLEURAL FLUID
CELL SIGNIFICANCE Glucose ↓Rheumatic inflammation, Purulent
infection
Neutrophil Pneumonia, Pancreatitis,
Pulmonary infarction Lactate ↑Bacterial Infection
Lymphocyte TB, Viral infection, Triglyceride ↑Chylous Effusion
autoimmune disorders,
pH ↓Pneumonia not responsive to
malignancy
antibiotics, ↓↓↓ Esophageal rupture
Mesothelial cell Normal and Reactive ↓ Empyema
(Lines the forms have no
serous significance. Decreased in Adenosine Deaminase Tuberculosis, Malignancy
membranes TB Amylase ↑Pancreatitis, Esophageal rupture
Plasma cells TB Malignancy
Malignant cells Primary Adenocarcinoma,
 Small Cell Carcinoma

PERICARDIAL FLUID
SIGNIFICANCE OF PERICARDIAL FLUID TESTING
APPEARANCE SIGNIFICANCE
Clear, Pale Yellow Normal, Transudate
Blood-Streaked Infection, Malignancy
Grossly-Bloody Cardiac puncture, Anticoagulant medication
DIFFERENTIAL COUNTS SIGNIFICANCE
Increased Neutrophils Bacterial Endocarditis
Malignant Cells Metastatic Carcinoma
TESTS SIGNIFICANCE
Gram stain and Culture Bacterial Endocarditis
Acid-Fast Stain Tubercular Effusion
PERITONEAL
FLUID
SIGNIFICANCE OF PERITONEAL FLUID TESTING
APPEARANCE SIGNIFICANCE
Clear, Pale Yellow Normal
Turbid Microbial infection
Green Gall bladder, Pancreatic disorder
Blood-streaked Trauma, Infection, Malignancy
Milky Lymphatic trauma, blockage
WBC COUNT SIGNIFICANCE
>500 cells/ul Bacterial Peritonitis, Cirrhosis
DIFFERENTIAL SIGNIFICANCE
↑Neutrophils Bacterial Peritonitis
PERITONEAL FLUID
TESTS
OTHER TESTS FOR PERITONEAL FLUID
Peritoneal lavage >100,000 RBCs/ul indicate blunt trauma injury
(Intra-abdominal bleeding)
CEA GI Carcinoma
CA 125 Ovarian cancer
GLUCOSE ↓Tubercular Peritonitis, Malignancy
AMYLASE ↑Pancreatitis., GI Perforations
BUN/CREATININE Ruptured/Punctured Bladder
GRAM STAIN/CULTURE Bacterial Peritonitis
ACID FAST STAIN Tubercular Peritonitis
INSERTS

• SUBACUTE BACTERIAL ENDOCARDITIS

• Causative agents for Subacute Endocarditis
(Haemophilus aphrophilus, Actinobacillus
(Aggregatibacter), Actinomycetes, Cardiobacterium
hominis, Eikenella corrodens, Kingella kingae)
FINISHED IN SEROUS FLUID
AMNIOTIC FLUID
 AMNIOTIC FLUID ANALYSIS

• functions include: Provides cushion to the fetus,

allows fetal movement, stabilizing the temperature
and permits proper lung development.
VARIATIONS IN VOLUME

• POLYHYDRAMNIOS – INCREASE IN VOLUME

• Caused by decreased fetal swallowing of urine and Neural tube defects
(spina bifida)

• OLIGOHYDRAMNIOS – DECREASE IN VOLUME

• Caused by increased fetal swallowing of urine, membrane leakage and
deformities
SPECIMEN COLLECTION AND
HANDLING
• AMNIOCENTESIS Up to 30 ml collected in sterile syringe

AMNIOTIC FLUID VS. MATERNAL URINE

ANALYTE AMNIOTIC MATERNAL
FLUID URINE
LESS Protein PRESENT ABSENT
RELIABLE Glucose PRESENT ABSENT
MORE UREA <30mg/dl >300mg/dl
RELIABLE CREATININE <3.5mg/dl >10mg/dl
AMNIOTIC FLUID
TESTS

• FETAL LUNG MATURITY – Place on ice, frozen

; cold (Filtration prevents loss of phospholipids)
• CYTOGENETIC STUDIES – place on body
temperature/ room temp.
• HDFN – protect from light
• FERN TEST: Detects ruptured amniotic membranes,
it is also used to diagnose early pregnancy due to
increased Estrogen
• *Specimen (Vaginal fluid) placed on Slide (Air Dry) =
Fern like crystals indicates Amniotic fluid
AMNIOTIC FLUID COLOR

COLOR SIGNIFICANCE
COLORLESS NORMAL
BLOOD-STREAKED TRAUMATIC TAP, ABDOMINAL TRAUMA
INTRA-AMNIOTIC HEMORRHAGE

YELLOW BILIRUBIN (HDFN)

DARK GREEN MECONIUM
DARK RED BROWN FETAL DEATH
NEURAL TUBE DEFECTS

• SPINA BIFIDA – “split spine” a birth

defect where there is an incomplete
closing of the backbone and membranes
around the spinal cord.
• Anencephaly – is the absence of a major
portion of the brain, skull and scalp that
occurs during embryonic development
 ALPHA FETO
PROTEIN

• Specimen of Choice is Amniotic Fluid.

• Decrease values indicate Down Syndrome,
• Increase values indicate Neural Tube Defects (the
incomplete development of the fetus’ spine)
 AFP AFP
AFP
AFP
AFP
AFP AFP

AFP

AFP AFP

SPINA AMNIOTIC
BIFIDA SAC
TESTS FOR AMNIOTIC
FLUID

• SCREENING TESTS
• ALPHA FETOPROTEIN
• ↑ in Neural Tube Defects
• ↓ in Down Syndrome

• CONFIRMATORY TESTS
• ACETYL-CHOLINESTERASE
FETAL LUNG MATURITY
TEST RATIONALE
LECITHIN/SPHINGOM Lecithin – Alveolar Stability
YELIN RATIO Sphingomyelin – serves as a control
Ratio of >2.0 indicates Fetal Lung Maturity
Cannot be done on a specimen contaminated
by blood or meconium
AMNIOSTAT-FLM Immunologic test for Phosphatidyl glycerol
Not affected by Meconium or blood
Production of PG is delayed among diabetic
mothers
FOAM Amniotic fluid + Ethanol ---Shake for 15 seconds---
STABILITY
Stand for 15 minutes
Mature Fetal Lungs = Indicated by Formation of Bubbles

MICROVISCOSIT The presence of phospholipids decrease microviscosity;

Y
measured by Fluorescence Polarization. Microviscosity is
the friction experienced by a single particle undergoing
diffusion because of its interaction with its environment
at the micrometer length scale
TEST RATIONALE

LAMELLAR BODY Centrifuge @ 45 g for 10 minutes @ 4C

COUNT
TYPE II Pneumocytes – Lost in strong centrifugation (results to
Falsely decreased L/S ratio)
Produce alveolar surfactants which are stored in the form of
lamellar bodies
The cells have similar diameter with platelets, therefore, LBC
can be obtained with the use of platelet channel of hematology
analyzers
>32,000/ml = indicate adequate FLM
Increased LBC = Increased OD
OD 650NM O.D of ≥0.150 is equivalent to L/S ratio of ≥2.0 and the presence of
Phosphatidyl glycerol
SUMMARY (AMNIOTIC
FLUID)
FETAL WELL-BEING AND MATURITY
BILIRUBIN Absorbance at 450 >0.025
ALPHA FETO PROTEIN <2.0 multiples of median (MoM)
L/S RATIO ≥2.0
AMNIOSTAT- FLM Positive
FOAM STABILITY ≥47
INDEX
MICROVISCOSITY ≥55mg/g
TESTING FOR FETAL AGE = >2.0 mg/dl amniotic fluid creatinine in
O.D 650NM ≥0.150
36 weeks or 9 months
LAMELLAR BODY ≥32,000ml
COUNT
 HUMAN CHORIONIC GONADOTROPHIN

• Produced by the cytotrophoblastic cells of a developing placenta

• Peaks during 1st Trimester of Pregnancy
• Composed of 2 subunits
• Alpha (along with HCG, LH, FSH, TSH)
• Beta (specific for HCG)
• Home Based Test kit : Principle of Enzyme Immunoassay
FINISHED IN AMNIOTIC FLUID
GASTRIC FLUID
GASTRIC FLUID
ANALYSIS

• testing of gastric fluid aims to detect the ff: Existence of diseases like Zollinger-Ellison
Disease and Pernicious anemia.

• CELLS OF THE STOMACH

• PARIETAL CELLS – produces HCl and intrinsic factors (IF)
• - (IF) needed for the Vitamin B12 absorption
• CHIEF CELLS – produces pepsinogen
• G CELLS – produces gastrin
 SPECIMEN COLLECTION
TYPE OF SPECIMEN DURATION OF COLLECTION
BASAL ACID OUTPUT 1 hour collection (Four 15-minute interval samples, but a
(BAO) – Fasting state single 1 hour can be used
2 hours collection (for insulin hypoglycemia test)
MAXIMUM ACID 1 hour collection (at 15 minute intervals) – when
OUTPUT (MAO) – Pentagastrin and Histamine are used
After Gastric stimulation 2 hour collection – for insulin hypoglycemia test and when
Histalog is used
BAO – Total Gastric Secretion during unstimulated fasting state
MAO – Total Gastric Secretion after gastric stimulation
• GASTRIC FLUID COLLECTION (Henry, 17th)
• LEVIN TUBE – tube passing through the nose
• REHFUSS TUBE – tube passing through the mouth
 GASTRIC STIMULANTS
GASTRIC STIMULANTS (From N.D. Moraleta, Clinical Microscopy)
TEST MEALS EWALD’S = Bread, Weak Tea, Water FISCHER’S – Ewald’s Meal + Hamburger
BOA’S = Oatmeal Stock
RIEGEL’S = Beef Steak, Mashed LAVINE’S – Ethyl Alcohol, Methylene
Potato Blue
HECKMANN’S = Egg Albumin, MOTOR – Spinach or Raisins and Water
Water, Methylene Blue SALZER – Beef, Lamb chops, Milk, rice,
DOCK’S – Biscuit egg
STASIS – Rice, Raisins
CHEMICAL PENTAGASTRIN – most preferred HISTALOG – Betazole
STIMULANT INSULIN – Vagotomy procedure HISTAMINE – gastric stimulant
SHAM FICTITIOUS FEEDING (Sandwich)
FEEDING
REPRESENTATIVE NORMAL AND ABNORMAL GASTRIC FLUID RESULTS (Strasinger, 3rd Ed)
BAO (mEq/hr) MAO (mEq/hr) BAO/MAO
Normal 2.5 25.0 10%
Pernicious Anemia 0 0 0
Duodenal Ulcer 5.0 30.0 17%
Zollinger - Ellison 18.0 25.0 72%
PERNICIOUS
Syndrome ANEMIA ~ an autoimmune disorder, characterized by auto-antibody
mediated cellular destruction of Parietal cells and Intrinsic factors. Destroyed Parietal cells
lead to a condition called ACHLORHYDRIA (No HCl)

ZOLLINGER-ELLISON DISEASE ~ an adenoma of islet of pancreas. The Adenoma

secretes excessive Gastrin, which later on leads to excessive HCl production. Therefore,
creating a Hyperacidity environment in the stomach

VAGOTOMY ~ surgical division of the Vagus nerve. The nerve that stimulates hunger
MACROSCOPIC EXAMINATION
COLOR SIGNIFICANCE
Pale Yellow with Mucus Normal
Yellow-Green Large amounts of bile
Red Small amount of fresh blood
Coffee ground Large amount of blood
VOLUME SIGNIFICANCE
20-50ML Normal in Fasting specimens
>50ML Abnormal in fasting specimens
20-60ML up to 120ML After Ewald’s test meal
45-150ML After alcohol test meal or histamine stimulation
GASTRIC FLUID CONDITIONS

TERM RATIONALE SIGNIFICANCE

EUCHLORHYDRIA Normal Free HCl
HYPERCHLORHYD Increased Free HCl Peptic Ulcers
RIA

HYPOCHLORHYDR pH >3.5 but falls after gastric stimulation Stomach Carcinoma

IA (Decreased Free HCl)

pH >3.5 and doesn’t fall even after Pernicious Anemia

ACHLORHYDRIA gastric stimulation (Absent Free HCl)

ANACIDITY Failure to produce a pH of <6.0 Pernicious anemia

following gastric stimulation
DIAGNEX TUBELESS TEST Urine Specimen

• Azure B orally taken, exchanged from an Amberlite cation

resin by H+ ions from HCl after Caffeine Stimulation.
• The Dye is rapidly absorbed in GIT and excreted in Urine.
• (+) Azure Blue in the Urine indicate presence of Free HCl
FINISHED IN GASTRIC FLUID
OTHER BODY FLUIDS
 SPUTUM

• SPUTUM – produced 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.
• BRONCHOALVEOLAR LAVAGE – a procedure for
collecting the cellular milieu of the alveoli by use of a
bronchoscope through which saline is instilled into the
distal bronchi and then withdrawn

Important diagnostic test for

Pneumocystis jiroveci in immunocompromised patients.
 SWEAT ANALYSIS
• for the diagnosis of Cystic Fibrosis. An autosomal recessive metabolic disorder
affecting the mucous secreting glands of the body leading to pancreatic insufficiency,
Respiratory distress and intestinal obstruction
• GIBSON AND COOKE PILOCARPINE IONTHOPHORESIS
• Pilocarpine + Mild Electric current = Induces Sweat Production
• Sweat is tested for Sodium and Chloride
• Both Na and Cl are elevated due to inability of sweat glands to reabsorb them before
secretion
 FECAL ANALYSIS

• FECES – contains bacteria, cellulose and other undigested

foodstuffs, gastrointestinal secretions, bile pigments, cells from
intestinal walls, electrolytes and water.
• Around 100-200 grams of stool are passed per day.
• Human Feces contain around 75% water and 25% solids
MACROSCOPIC STOOL CHARACTERISTICS
COLOR/APPEARAN CLINICAL SIGNIFICANCE
CE
Brown Normal
Black Upper GI Bleeding
Iron Therapy, Charcoal, Bismuth (antacids)
Red Low GI Bleeding
Beets and food coloring, Rifampin
Pale yellow, white, gray Bile Duct Obstruction
Barium Sulfate
Green Biliverdin, Oral Antibiotics, Green vegetables
Blue Prussian Blue, Grape Soda
APPEARANCE CLINICAL SIGNIFICANCE
Bulky/frothy Bile Duct obstruction
Pancreatic disorders
Steatorrhea
Butter-like Cystic Fibrosis
Mucus Colitis, Dysentery, Malignancy
Blood-streaked mucus Constipation
Ribbon like Intestinal constriction
Rice watery Cholera
Pea soup Typhoid
Scybalous Constipation
MICROSCOPIC ANALYSIS

• FATS – A condition called STEATORRHEA

(Increased Fat in stool >6 grams per day)
• TESTS:
• Screening test – Microscopic examination for fat
globules
• Definitive test – Fecal Fat determination
 QUALITATIVE FECAL FAT
NEUTRAL FAT STAIN SPLIT FAT STAIN (FATTY ACID)
(TRIGLYCERIDES) Emulsified Stool + 36% Acetic Acid + Sudan
Stool suspension + 95% Ethanol + Sudan III
III ↓
↓ Orange Droplets (Fatty Acid)
Orange droplets (Neutral
fats/Triglycerides) Normal = 100 droplets (<4um)
≥60 Droplets/hpf = Steatorrhea Slight increase = 100 droplets (1-8um)
Increased = 100 droplets (6-75um)
Van De Kamer Sample required is 3-day
Titration stool (72 hours)
-Gold Standard for Fecal Normal = 1-6 grams/day
Fat determination Steatorrhea = >6
-For Definitive diagnosis grams/day
of steatorrhea
-Titration with NaOH
 MICROSCOPIC ANALYSIS

• MUSCLE FIBERS – a condition called CREATORRHEA

• (abnormal excretion of muscle fibers in feces)

• COMPLETE DIGESTION – no striations

• PARTIAL DIGESTION – striation in one direction
• UNDIGESTED FIBER – striations both directions
• ABNORMAL = >10 undigested muscle fibers
MICROSCOPIC ANALYSIS

• FECAL LEUKOCYTES - ≥3 Neutrophils/hpf = invasive

condition
• Determined by:
• Wet preparation – Stool + Loeffler’s Methylene Blue
• Dried preparation – Stool +Wright’s/Gram’s stain
• Lactoferrin Latex Agglutination test (Lactoferrin = Secondary
Granules of Neutrophils = (+) Invasive Bacterial Pathogen
DIARRHEA correlated to WBC

• Diarrhea with WBC’s caused by Salmonella, Shigella,

Yersinia, Enteroinvasive E. coli and Campylobacter

• Diarrhea without WBC’s caused by Toxin producing

bacteria (S.aureus, V. cholerae) virus and parasites
 FECAL OCCULT BLOOD TEST
(FOBT)
• Screening test for Colorectal Carcinoma
• Significant = >2.5 ML blood per 150 grams of stool
• Center portion of stool

• Principle of Pseudoperoxidase activity of Hemoglobin

• Chromogens : Benzidine (Most Sensitive), Guaiac (Preferred) and O-Toluidine
• False (+) – Dietary Pseudoperoxidases like Red Meat (avoid for 3 days), Melon,
Broccoli, Cauliflower, Horseradish (avoid for 3 days), Aspirin and other anti-
inflammatory drugs (avoid for 7 days) (Oxidizing agents result to False (+))
• False (-) Ascorbic acid (Reducing Agents)
 CHEMICAL TESTS

• APT TEST
• Emulsified stool → centrifuge→ Add 1% NaOH to supernatant
• Pink Solution: HgB F Brown-Yellow: HgB A

• APT TEST (APT-DOWNEY TEST) Differentiates fetal blood and maternal blood
• Specimen used is infant’s stool or vomitus
• Hemoglobin F is alkali resistant (giving Pink solution)
• Hemoglobin A is denatured by NaOH (Brown solution)
CHEMICAL TESTS

• X-RAY FILM TEST

• Emulsified stool + X-Ray film
• Clearing of Film = (+) Trypsin
• No Clearing of Film = (-) Trypsin
CHEMICAL TESTS

• FECAL CARBOHYDRATES – Most valuable in assessing

cases of infant diarrhea

• Clinitest – test for reducing sugars.

>0.5 g/dl indicates Carbohydrate Intolerance

• pH – Normal stool has 7.0-8.0 pH , CHO disorders = <5.5

 DIARRHEA
• Stool weight of ≥200 g/day with increased liquidity and frequency of
more than 3x a day.

• Acute Diarrhea - < 4 weeks

• Chronic Diarrhea - > 4 weeks

• Major Mechanisms are Secretory, Osmotic and Altered Motility.

Laboratory tests used to differentiate these are fecal electrolytes (Fecal
Sodium and Fecal Potassium), Fecal Osmolarity and stool Ph
SECRETORY DIARRHEA

• increased secretion of water and electrolytes, which

override the reabsorption ability of the large intestine
caused by Bacterial, viral and protozoan infections,
drugs, laxatives, hormones, inflammatory bowel
diseases, endocrine disorders, neoplasms and collagen
vascular disease
OSMOTIC DIARRHEA

• retention of water and electrolytes in the large

intestine due to incomplete breakdown or
reabsorption of food caused by maldigestion,
malabsorption, disaccharidase disorders
(Lactose intolerance), laxatives, antacids,
amoebiasis and antibiotics
ALTERED MOTILIY

•enhanced (hypermotility) or slow

(constipated) motility caused by Irritable
Bowel syndrome (IBS), Rapid
(accelerating) gastric emptying (RGE)
dumping syndrome.
MISCELLANOUS
Finished In Clinical
Microscopy
Afterword
“Have I not commanded you? Be strong
and courageous. Do not be frightened, and
do not be dismayed, for the Lord your God
is with you wherever you go.”
~Joshua 1:9
He replied, “Because you have so little faith.
Truly I tell you, if you have faith as small as a
mustard seed, you can say to this mountain,
‘Move from here to there,’ and it will
move. Nothing will be impossible for you.”
~Matthew 17:20
Thank You
All For
Listening!
God Bless All Of You!
All For The Greater
Glory Of God