COMPILED NOTES!!!

Urinalysis and Body Fluids

Sixth Edition
Summarized by: Nigelle Villasenor

Compiled by: MarL and Rohaynah

Oct, 2017
 Chapter 2 | Introduction to Urinalysis

HISTORY AND IMPORTANCE

 Analyzing Urine – beginning of laboratory medicine

o Edwin Smith Surgical Papyrus – historical study of urine
 Examining a bladder-shaped flask of urine
 Hippocrates (5th century BCE) – wrote Uroscopy
 1140 AD – color charts were developed that described the significance of 20 different colors
 Frederick Dekkers – discovered albuminuria by boiling urine (1694)
 Pisse Prophets (charlatans) – compromised the credibility of urinalysis
o Thomas Bryant (1627) – wrote a book about the pisse prophets
 Thomas Addis (17th century) – examination of urinary sediment with the invention of
microscope
 Richard Bright (1827) – introduced the concept of urinalysis as part of a doctor’s routine patient
examination
 2 Unique Characteristics of a Urine Specimen:
1. Urine is a readily available and easily collected specimen
2. Urine contains information
 Clinical and Laboratory Standards Institute (CLSI) – defines urinalysis as “the testing of urine with
procedures commonly performed in an expeditious, reliable, accurate, safe, and cost-effective
manner.”

URINE FORMATION

 Urine – ultrafiltrate of plasma formed by the kidneys

 Filters 170,000 mL of essential substances

URINE COMPOSITION

 Urine – 95% Water and 5% Solutes

Primary Composition in Normal Urine

Component Comment
Urea Primary organic component. Product of protein
and amino acid metabolism. (Liver)
Creatinine Product of creatinine metabolism by muscles
Uric Acid Product of nucleic acid breakdown in food and
cells
Chloride Primary inorganic component. Found in
combination with sodium (table salt) and many
other inorganic substances
Sodium Primarily from salt, varies by intake
Potassium Combined with chloride and other salts
Phosphate Combines with sodium to buffer the blood
Ammonium Regulates blood and tissue fluid acidity
Calcium Combines with chloride, sulfate, and phosphate
 Chapter 2 | Introduction to Urinalysis
Creatinine – also a product of purine metabolism

URINE VOLUME

 Urine excretion = Body’s hydration

 Factors that influence urine volume:
1. Fluid Intake
2. Fluid Loss from nonrenal sources
3. Variations in the secretion of antidiuretic hormone
4. Need to excrete increased amounts of dissolved clots
 Daily Urine Output – 1,200 to 1,500 mL; 600-2,000mL is considered normal
 Oliguria – a decrease in urine output
o May lead to Anuria (cessation of urine flow)
 Clinical Significance: Severe acute nephritis, Hg poisoning, obstructive uropathy,
kidney failure
o May be seen in Blood Transfusion Reaction; Renal Schemia, Stone, Disease
o Clinical Significance: Dehydration, Renal insufficiency, poorly compensated heart
disease, calculi formation, kidney tumors
 Nocturia – increase in the nocturnal excretion of urine (>500 mL at night)
 Polyuria – increase in daily urine volume (>2.5 L/day in adults; >2.5-3.0 mL/kg/day in children)
o Often associated with diabetes mellitus and diabetes insipidus
 Diabetes Mellitus - ↑specific gravity, ↓insulin = ↑glucose
 Diabetes Insipidus - ↓specific gravity, ↓ADH
o May be artificially induced by diuretics, caffeine, or alcohol (all suppresses ADH)
 Polydipsia – compensates fluid loss; increased ingestion of water (can be seen in DM)
 Diuresis – transitory increase in urine volume
 Dysuria – ex. UTI, cystitis

SPECIMEN COLLECTION

 Gloves – should be worn at all time when handling urine

 Containers – must be clean, dry, leak-proof and disposable
o Should have a wide mouth (for female)
o Should have a wide flat bottom to prevent overturning
o Should be made of a clear material
o Capacity – 50 mL (allows 12 mL)
o Sterile containers – if 2 hours have passed the specimen collection
 Labels
o Should be labeled with the patient’s name, identification number, date and time of
collection
o Should be attached to the container and not on the lid
 Chapter 2 | Introduction to Urinalysis
 Requisitions – this form can be manual or computerized)
o May include method of collection, type of specimen, possible interfering medications,
and the patient’s clinical information.
o The time the specimen is received in the laboratory should be found in this form

SPECIMEN REJECTION

 Unacceptable situations include:

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

SPECIMEN HANDLING

 Specimen Integrity
o Should be delivered to the laboratory within 2 hours
 Cannot be delivered within 2 hours = should be refrigerated/preserved

Changes in Unpreserved Urine

Analyte Change Cause
Color Modified/Darkened Oxidation/Reduction of
metabolites
Clarity Decreased Bacterial growth and
precipitation of amorphous
material
Odor Increased Bacterial multiplication causing
breakdown of urea to ammonia
pH Increased Breakdown of urea to ammonia
by urease-producing
bacteria/loss of CO₂
Glucose Decreased Glycolysis and bacterial use
Ketones Decreased Volatilization and bacterial
metabolism
Bilirubin Decreased Exposure to light/photo
oxidation to biliverdin
Urobilinogen Decreased Oxidation in urobilin
Nitrite Increased Multiplication of nitrate-
reducing bacteria
Red and white blood cells and Decreased Disintegration in dilute alkaline
casts urine
Bacteria Increased Multiplication
Trichomonas Decreased Loss of motility, death
 Chapter 2 | Introduction to Urinalysis

 Specimen Preservation
o Refrigeration (2:C to 8:C) – most routinely used method of preservation
o The specimen must return to room temperature before chemical testing by reagent
strips

Urine Preservatives
Preservatives Advantages Disadvantages Additional Information
Refrigeration Does not interfere with Precipitates amorphous Prevents bacterial
chemical tests phosphates and urates growth for 24 hours
Maintains an acid pH up
to 8 hours
Boric Acid Prevents bacterial Interferes with drug and Keeps pH at about 6.0
(cloudy specimen) growth and metabolism hormone analyses Can be used for urine
Preserves protein and culture transport
formed elements well Does not interfere with
routine analysis other
than pH
Phenol Does not interfere with Causes an Odor change
routine tests
Toluene Does not interfere with Floats on surface of
routine tests specimens and clings to
pipettes and testing
materials
Thymol Preserves glucose and Interferes with acid
sediments well precipitation tests for
protein
Formalin Excellent sediment Acts as a reducing Rinse specimen
(formaldehyde) preservative agent, interfering with container with formalin
chemical tests for to preserve cells and
glucose, blood, casts
leukocyte esterase, and
copper reduction
Sodium fluoride Good preservative for Inhibits reagent strip Prevents glycolysis
drug analyses tests for glucose, blood,
and leukocytes
Commercial Convenient when Check tablet
preservative tablets refrigeration not composition to
possible. determine possible
Have controlled effects on desired tests
concentration to
minimize interference
Urine Collection Kits Contains collection cup,
(Becton, Dickinson, transfer straw, culture
Rutherford, NJ) and sensitivity (C&S)
preservative tube, or
 Chapter 2 | Introduction to Urinalysis

UA tube
Light gray and gray C&S Sample stable at room Do not use if urine is Preservative is boric
tube temperature (RT) for 48 below minimum fill line acid, sodium borate and
hours; prevents sodium formate.
bacterial growth and Keeps pH at about 6.0
metabolism
Yellow UA Plus tube Use on automated Must refrigerate within Round or conical
instruments 2 hours bottom, no preservative
Cherry red/yellow Stable for 72 hours at Must be filled to Preservative is sodium
Preservative Plus tube RT; instrument- minimum fill line. propionate, ethyl
compatible Bilirubin and paraben, and
urobilinogen may be chlorhexidine; round or
decreased if specimen conical bottoms
is exposed to light and
left at RT
Saccomanno Fixative Preserve cellular For cytology studies
elements

TYPES OF SPECIMENS

Types of Urine Specimens

Type of Specimen Purpose Additional Information
Random Routine Screening Most commonly received specimen.
May be collected at any time
First Morning Routine Screening Ideal screening specimen.
Pregnancy Tests Concentrated specimen.
Orthostatic Protein Evaluates Orthostatic Proteinuria
24-hour (or timed) Quantitative Chemical Must begin and end with an empty bladder
Tests, Hormone Studies
Catheterized Bacterial Culture Passes hollow tube (catheter) through the
urethra
Midstream Clean-Catch Routine Screening Alternative to Catheterized Specimen
Bacterial Culture
Suprapubic Aspiration Bladder urine for bacterial
culture
Cytology

 Prostatitis Specimen
o Three-Glass Collection
 Chapter 2 | Introduction to Urinalysis

 Male mid-stream clean-catch -> first urine is not discarded, but collected in a
sterile container -> Midstream portion is collected in another sterile container ->
Prostate is then massaged so that prostate fluid will be passed with the
remaining urine into a third sterile container
 Prostatic Infection = third specimen will have WBC/high-power field count and
bacterial count 10 times that of the first specimen
 2nd specimen – used as a control for bladder and kidney infection
o Stamey-Mears Test – 4 glass method: Initial Voided Urine (VB1), Midstream Urine (VB2),
Expressed Prostatic Secretions (EPS), and a post-prostatic massage urine specimen (VB3)
 Midstream Clean-Catch – cleanse glans penis (for male) and urinary meatus (for female) with
antiseptic towelette (or with soap and water)
 Pediatric Specimens – a soft, clear plastic bag with hypoallergenic skin adhesive to attach to the
genital area is needed
 Drug Specimen Collection
o Chain of Custody (COC) – process that provides this documentation of proper sample
identification from the time of collection to the receipt of laboratory results
o May be “witnessed” or “unwitnessed”
 “Witnessed” – collection of 30 to 45 mL of urine
o Must be taken within 4 minutes to confirm the specimen hasn’t been adulterated
o Blueing agent added to the toilet water reservoir in unwitnessed collection
o Temperature – 32.5:C to 37.7:C
 2 hours post prandial & Fasting Specimen – to diagnose DM
 Glucose Tolerance Test (GTT) – may include fasting, half-hour, 1-hour, 2-hour, and 3-hour
specimens
 12-hour – for Addis count
 Afternoon specimen (2-4 pm) – urobilinogen determination
 Chapter 4 | Physical Examination of Urine

Physical Examination includes:

a.) Color
b.) Clarity
c.) Specific Gravity
d.) Odor

COLOR
Normal Urine Color

 It may vary from colorless to black

 Urochrome – pigment that causes the yellow color of urine
o Termed by Thudichum 1864
o Product of endogenous metabolism
o Increased amount in thyroid conditions and fasting states
o Increased if stands at room temperature
 Dilute urine = pale yellow
 Concentrated urine = dark yellow
 Uroerythrin – a pink pigment
o Evident in specimens that have been refrigerated, resulting in the precipitation of
amorphous urates (it attaches to urates to produce a pink color)
 Urobilin – an oxidation product of urobilinogen
o Imparts an orange-brown color to urine that is not fresh

Abnormal Urine Color

1.) Dark Yellow/Amber/Orange

 Can be caused by the presence of the bilirubin
 Bilirubin – detected during chemical examination
o Suspected if yellow foam appears when specimen is shaken (white foam = ↑protein)
o May also contain hepatitis virus
 Photo-oxidation of:
o Urobilinogen to Urobilin -> Yellow-Orange Urine (no yellow foam)
o Bilirubin -> Yellow-Green (caused by Biliverdin)
 Phenazopyridine (brand name = Pyridium) – causes Yellow-Orange specimens – interferes with
chemical tests
o Azo-gantrisin – to people who have urinary tract infection (UTI)
o Also contains yellow foam
2.) Red/Pink/Brown
 Red urine if blood is present, but may range from pink to brown depending on:
o Amount of blood, pH of urine, and length of contact
 Brown Urine – produced when RBCs remain in an acidic urine
 Chapter 4 | Physical Examination of Urine

o Due to the oxidation of hemoglobin to methemoglobin

o May indicate glomerular bleeding
 RBCs in urine = red and cloudy
 Hemoglobin & Myoglobin = red and clear
o Hemoglobinuria – results from in vivo breakdown of RBCs is accompanied by red plasma
o Myoglobin – breakdown of skeletal muscle; reddish-brown color
 Red = Urine with porphyrins – results from the oxidation of porphobilinogen to porphyrins
o Referred to as having the color of port wine
 Medications that may produce red urine: rifampin, phenolphthalein, phenindione, and
penothiazines
3.) Brown/Black
 Melanin – oxidation product of melanogen produced in excess when a malignant melanoma is
present
 Homogentisic Acid – metabolite of phenylalanine
o Imparts black color to alkaline urine
o Alkaptonuria – inborn-error of metabolism
 Medications that may produce brown/black urine: levodopa, methyldopa, phenol derivatives,
and metronidazole
 Cola-colored urine – may be seen with rhabdomyolysis
4.) Blue/Green
 Urinary tract infection by Pseduomonas species
 Ingestion of breath deodorizers (Clorets) = Green Urine
 Medications that may produce blue urine: methocarbamol (Robaxin), methylene blue, and
amitriptyline (Elavit)

Laboratory Correlation of Urine Color

Color Cause Clinical/Laboratory Correlation
Colorless Recent Fluid Consumption Observed in Random Specimens
Pale Yellow Polyuria/Diabetes insipidus ↑24-hr volume and ↓specific
gravity
Diabetes Mellitus ↑specific gravity and positive
glucose test
Dilute Random Specimen Recent fluid consumption
Dark Yellow Concentrated Specimen May be normal after exercise or
in first morning specimen
B Complex Vitamins
Dehydration Fever/Burns
Bilirubin Yellow foam when shaken
Acriflavine Negative bile test result and
possible green fluorescence
Nitrofurantoin Antibiotic administered for UTI
Orange-Yellow Phenazophyridine (Pyridium) Drug administered for UTI
Phenindione Anticoagulant, Orange in alkaline
 Chapter 4 | Physical Examination of Urine

urine, colorless in acid urine

Yellow-Green Bilirubin oxidized to biliverdin Colored foam in acidic urine and
false-negative test for bilirubin
Green Pseudomonas infection Positive Urine Culture
Blue-Green Amitriptyline Antidepressant
Methocarbamol (Robaxin) Muscle Relaxant, Green-brown
Clorets
Indican Bacterial infection, intestinal
disorders
Methylene Blue Fistulas
Phenol When oxidized
Pink RBCs Cloudy Urine
Red Hemoglobin Clear Urine; Intravascular
hemolysis
Myoglobin Clear Urine; Muscle damage
Beets Alkaline Urine
Rifampin Tuberculosis Medication
Menstrual Contamination Cloudy specimen
Fuscin, Aniline Dye Foods, candy
Port Wine Porphyrins Negative test for blood
Acquired (Normal)
Inherited (Port Wine)
Red-Brown RBCs oxidized to methemoglobin Acidic Urine after standing
Myoglobin
Bilifuscin (Dipyrrole) Unstable Hemoglobin
Brown Homogentisic Acid Alkaline Urine after standing
(alkaptonuria)
Black Malignant Melanoma Urine darkens on standing and
reacts with nitroprusside and
ferric chloride
Melanin/Melanogen
Phenol derivatives Interfere with copper reduction
tests
Argyrol (antiseptic) Color disappears with ferric
chloride
Methyldopa or levodopa Antihypertensive
Metronidazole (Flagyl) Darkens on standing, intestinal
and vaginal infections
Klebsiella providencia – urine purple color

CLARITY
 Clarity – refers to the transparency/turbidity of a urine specimen – examine in front of light
source

 Chapter 4 | Physical Examination of Urine

Normal Clarity

 Clear; midstream clean-catch specimen

 White cloudiness (alkaline pH) – precipitation of amorphous phosphates and carbonates

Nonpathologic Turbidity

 Hazy – presence of squamous epithelial cells and mucus

 Turbid - Stand or refrigerated (thick; precipitation of amorphous phophates, carbonates, urates)

Nonpathologic Causes of Urine Turbidity: Squamous epithelial cells; Mucus; Amorphous phosphates,
carbonates, and urates; semen, spermatozoa; Fecal contamination; Radiographic Contrast Media;
Talcum powder; Vaginal creams

Pathologic Causes of Urine Turbidity: RBCs, WBCs, Bacteria, Yeast, Nonsquamous epithelial cells,
Abnormal crystals (tyrosine and leucine), Lipids (Lipiduria), Lymph fluid (Chyluria; associated with
Wuchereria bancrofti[filariasis])

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

Appearance Cause Remarks

Cloudy Phosphates, Carbonates Soluble in dilute acetic acid
Urates, Uric Acid Dissolve at 60°C and in alkali
Leukocytes Insoluble in dilute acetic acid
Red cells (‘smoky’) Lyse in dilute acetic acid
Bacteria, Yeasts Insoluble in dilute acetic acid
Spermatozoa Insoluble in dilute acetic acid
Prostatic Fluid
Mucin, Mucous threads May be flocculent
Calculi, ‘gravel’ Phosphates, oxalates
Clumps, pus tissue
Fecal contamination Recto-vesical fistula
Radiographic Dye In acid urine
Milky Many neutrophils (pyuria) Insoluble in dilute acetic acid
Fat
Lipiduria, Opalescent Nephrosis, crush injury
Chyluria, Milky Lymphatic Obstruction
Emulsified Paraffin Vaginal Creams
 Chapter 4 | Physical Examination of Urine

SPECIFIC GRAVITY
 Urea (20%), Sodium Chloride (25%), Sulfate, and Phosphate – contribute most of the specific
gravity of normal urine
 1.010 – specific gravity of the plasma filtrate entering the glomerulus
o Isosthenuric – at 1.010
o Hyposthenuric – below 1.010
o Hypersthenuric – above 1.010
 1.002 to 1.035 – normal random specimen range
 <1.002 – not urine
 Specific Gravity – density of a solution compared with the density of a similar volume of distilled
water (sg 1.000) at a similar temperature
 Urine – water with dissolved chemicals

Urinometry

 Urinometer/Hydrometer – weighted float that is designed to sink to a level of 1.000 in distilled

water; calibrated at 20°C
o For every 3°C above/below the calibration temperature, add/subtract 0.001 respectively
o Protein – subtract 0.003 per g/dL Glucose – subtract 0.004 per g/dL

Refractometry

 Determines the concentration of dissolved particles in a specimen by measuring refractive index

 Temperature correction is not required
o Refractive Index – comparison of the velocity of light in air with the velocity of light in a
solution
o Temperature = 15:C to 38:C
o Subtract 0.003 for each gram of protein present
o Subtract 0.004 for each gram of glucose present
o Above 1.040 = abnormally high
o 5% NaCl – used to check further calibration
 >1.040 – seen in patients who have undergone intravenous pyelogram (caused by the excretion
of radiographic contrast media; dextran or other high mw intravenous fluids – tested by reagent
strip chemical test or osmometry

Harmonic Oscillation Densitometry

 Sound waves of specific frequency are generated at one end of the tube and as the sound waves
oscillate through urine, their frequency is altered by the density of the specimen
 Mass gravity meter – used by Yellow IRIS automated workstations to measure specific gravity
 Chapter 4 | Physical Examination of Urine

Osmolality

 Affected only by the number of particles present

 Osmole – 1g molecular weight of a substance divided by the number of particles into which it
dissociates
 mOsm (milliosmole) – unit of measure
 Colligative property – mathematically related to the number of particles in the solution
 A₂O Advanced Automated Osmometer – uses freezing point depression to measure osmolality

Reagent Strip Chemical Test

 Based on the change in pKa (dissociation constant of a polyelectrolyte in an alkaline medium)

 Release hydrogen equal to the ions in the solution
 Indicator – bromthymol blue
o Blue – alkaline/1.000 Green Yellow – acid/1.030

Particle Changes to Colligative Properties

Property Normal Pure Water Point Effect of 1 Mole of Solute
Freezing Point 0:C Lowered 1,86:C
Boiling Point 100:C Raised 0.52:C
Vapor Pressure 2.38 mm/Hg at 25:C Lowered 0.3 mm/Hg at 25:C
Osmotic Pressure 0 mm/Hg Increased 1.7 x 10⁹ mm/Hg
ODOR
 Not part of routine urinalysis
 Breakdown of urea = ammonia odor

Possible Causes of Urine Odor

Odor Cause
Aromatic (due to volatile acids) Normal
Foul, ammonia-like Bacterial decomposition, UTI
Fruity, sweet Ketones (diabetes mellitus, starvation, vomiting)
Maple syrup/Caramel-like Maple syrup urine disease
Mousy Phenylketonuria
Rancid Tyrosinemia
Sweaty feet Isovaleric acidemia (and glutaric acidemia)
Cabbage, hops Methionine malabsorption
Bleach Contamination
Rotting Fish Trimethylaminuria
Pungent Onion
Sulfur Odor Cystine disorders
Mercaptan Asparagus, Garlic, and Eggs
Fecaloid Recto-vesicular fistula
 Chapter 5 | Chemical Examination of Urine

REAGENT STRIPS

 Chemical analysis of urine including pH, protein, glucose, ketones, blood, bilirubin, urobilinogen,
nitrite, leukocytes, and specific gravity
 2 Major types (brands):
o Multistix and Chemstrip
 It consists of chemical-impregnated absorbent pads attached to a plastic strip (color-producing
chemical is seen when it comes in contact with urine)
 Reagent Strip Technique: Dip reagent strip in urine → Remove excess urine by running the edge
of the strip on the container → Blot horizontally → Wait for the reaction → Compare
 Errors caused by improper technique:
o Formed elements will sink to the bottom of the specimen and will be undetected in an
unmixed specimen
o Allowing the strip to remain in the urine for an extended period
o Excess urine remaining on the strip
o Timing for reactions (60 to 120 seconds; leukocyte esterase – 120 seconds)
o A good light source is needed
o The strip must be held close to the chart without actually being placed on the chart
o Reagent strips and color charts from different manufacturer
o Refrigerated specimens must be returned to room temperature first
 Packaged in opaque containers with a desiccant (to protect from light and moisture)
 Storing: stored at room temperature below 30:C (but never refrigerated)
 Distilled water ≠ Negative Control

pH

 Kidneys (and Lungs) – major regulators of the acid-base content in the body
o Secretes hydrogen in the form of ammonium ion, hydrogen phosphate, and weak
organic acids
o Reabsorbs bicarbonate from the filtrate in the convoluted tubules
 pH 5.0 to 6.0 (slightly acidic) – pH of first morning urine specimen
 Alkaline Tide – more alkaline pH after meals
 pH 4.5 to 8.0 – normal random samples
 Precipitation (dependent on pH) of Inorganic chemicals -> Urinary Crystals + Renal Calculi
o Calcium Oxalate – constituent of renal calculi, precipitates in acidic and not alkaline
urine
 Acidic urine – can be valuable in treating urinary tract infection
o Urea-splitting organisms do not multiply as readily in an acidic medium; these organisms
maintain the alkalinity of the specimen
 High-protein and High-meat diets = produces acidic urine
 Chapter 5 | Chemical Examination of Urine

 Fruits and Vegetables = produces alkaline urine (due to bicarbonate)

o *Exception: Cranberry Juice – acidic urine, home remedy for minor bladder infections
 Medications: methenamine mandelate (Mandelamine) and fosfomycin tromethamine (Monurol)
– metabolized to produce an acidic urine
 pH of freshly excreted urine does not reach above 8.5 in normal or abnormal conditions

Causes of Acid and Alkaline Urine

Acid Urine Alkaline Urine
Emphysema Hyperventilation
Diabetes Mellitus Vomiting
Starvation Renal Tubular Acidosis
Dehydration Presence of Urease-producing bacteria
Diarrhea Vegetarian diet
Presence of acid-producing bacteria (E. coli) Old specimens
High-protein diets
Cranberry Juice
Medications

Clinical Significance of Urine pH

Respiratory or Metabolic Acidosis/Ketosis
Respiratory or Metabolic Alkalosis
Defects in Renal Tubular Secretion and Reabsorption of Acids and Bases – Renal Tubular Acidosis
Renal Calculi formation and prevention
Treatment of Urinary Tract infections
Precipitation/Identification of Crystals
Determination of Unsatisfactory specimens

 Reagent Strip Reaction:

 Multistix – pH 5.0 to 8.5 in 0.5 increments
 Chemstrip – pH 5.0 to 9.0 in 1.0 increments
o Indicators: Methyl Red and Bromthymol Blue
 Methyl Red: Red to Yellow at pH 4-6
 Bromthymol Blue: Yellow to Blue at pH 6-9
o Interference: No known interfering substances, Run-over from adjacent pads, Old
specimens
o Correlation with other tests: Nitrite, Leukocyte, Microscopic
 Chapter 5 | Chemical Examination of Urine

PROTEIN

 Most indicative of renal disease

 Normal = <10 mg/dL or 100 mg per 24 hours is excreted
 Albumin – major serum protein found in normal urine
 Other proteins:
o Serum and tubular microglobulins
o Tamm-Horsfall protein (uromodulin) produced by the renal tubular epithelial cells
 Routinely produced in the distal convoluted tubule
o Prostatic Protein, Seminal Protein, and Vaginal Secretion
 Proteinuria – indicated at 30 mg/dL
o Prerenal Proteinuria
 Not indicative of actual renal disease
 Caused by ↑low-molecular-weight plasma (hemoglobin, myoglobin) and the
acute phase reactants
 Bence Jones Protein
 Monoclonal Immunoglobulin Light Chains
 Seen in persons with multiple myeloma (proliferative disorder of the
immunoglobulin-producing plasma cells)
 Coagulates at 40:C and 60:C, then dissolves at 100:C
 Diagnosed by serum electrophoresis and immunoelectrophoresis
o Renal Proteinuria
 Glomerular Proteinuria
 Causes of Proteinuria due to glomerular damage: Amyloid material,
toxic substances, and the Immune complexes found in lupus
erythematosus and streptococcal glomerulonephritis
 Microalbuminuria
 Its presence is associated with cardiovascular disease
 Can cause development of diabetic nephropathy leading to reduced
glomerular filtration
 24-hour urine specimen is needed
 Significant = 30-300mg albumin or 20-200ug/min AER (albumin
excretion)
 Orthostatic (Postural) Proteinuria
 Occurs following periods spent in a vertical posture
 Present = first specimen is (-), second specimen is (+)
 Tubular Proteinuria
 ↑Albumin; normally filtered Albumin cannot be reabsorbed
 A cause may be Fanconi Syndrome
 Slightly above normal to 4 g/day
o
 Chapter 5 | Chemical Examination of Urine

o Postrenal Proteinuria
 Protein can be added to a urine specimen as it passes through the structures of
the lower urinary tract (ureters, bladder, urethra, prostate, and vagina)

Clinical Significance of Urine Protein

Prerenal Renal Tubular Disorders Postrenal
Intravascular hemolysis Glomerular disorders Fanconi Syndrome Lower Urinary Tract
infections/inflammation
Muscle Injury Immune complex Toxic agents/Heavy Injury/Trauma
disorders Metals
Acute phase reactants Amyloidosis Severe Viral Infections Menstrual
contamination
Multiple Myeloma Toxic agents Prostatic
Fluid/Spermatozoa
Diabetic nephropathy Vaginal Secretions
Strenuous exercise
Dehydration
Hypertension
Pre-eclampsia
Orthostatic/Postural
proteinuria

 Reagent Strip Reaction

o Uses the principle “protein error of indicators” – produces a visible colorimetric reaction
o Indicator changes color in the presence of protein
o Protein area of the strip contains:
 Tetrabromophenol blue (Multistix)
 3’,3”,5’,5”-tetrachlorophenol
 3,4,5,6-tetrabromosulfonphthalein (Chemstrip)
 Acid Buffer (to maintain pH at constant level)
o At pH 3, Indicator (Yellow) + Protein → (Blue) → (Green)
 Reaction Interference:
o False Positive - Alkaline Urine, Phenazopyridine, Quaternary ammonium compounds,
Antiseptic, Chlorhexidine
o False-negative: Proteins beside albumin, microalbuminuria
 Correlations with other tests: Blood, Nitrite, Leukocyte, Microscopic

o Sulfosalicylic Acid Precipitation Test – cold precipitation test that reacts equally with all
forms of protein
 Must be performed on centrifuged specimens
 False-Positive: mucin, uric acid, penicillin, tolbutamides, radiocontrast media,
sulfonamides, cephalosporins
 False-Negative: Alkaline Urine
 Chapter 5 | Chemical Examination of Urine

SSA Turbidity
Grade Turbidity Protein Range (mg/dL)
Negative No increase in turbidity Less than 6
Trace Noticeable turbidity 6-30
1+ Distinct turbidity, no 30-100
granulation
2+ Turbidity, granulation, no 100-200
flocculation
3+ Turbidity, granulation, 200-400
flocculation
4+ Clumps of protein Greater than 400

o Testing for Microalbuminuria

 Immunochemical assays: Micral-Test and ImmunoDip
 First Morning Specimen is required
 Micral-Test – contain a gold-labeled antihuman albumin antibody-enzyme
conjugate
 Amount of color represents amount of albumin
o Bound conjugates – reacts with substrate (white to red)
 Reagents: gold-labeled antibody, B-galactosidase, Chlorophenol red
galactoside
 Interference: Dilute Urine (false negative)
 ImmunoDip – strips are individually packaged in specially designed containers
 Reagent: Antibody-coated blue latex particles
 Principle: Immunochromographics
 Interference: Dilute Urine (false negative)
o Albumin:Creatinine Ratio
 Sensitivity: 10 to 150 mg/L
o Albumin reagent strip – uses dye bis (3’,3”-diiodo-4’,4”-dihydroxy-5’,5”-dinitrophenyl)-
3,4,5,6-tetrabromosulphonphthalein (DIDNTB)
 Interference: Alkaline Urine
 Controlled by using paper treated with bis-(heptapropylene glycol)
carbonate
 Polymethyl vinyl ether – decreases the nonspecific binding of polyamino
acids to the albumin pad
 Color: Pale green to Aqua Blue
o Creatinine reagent strip
 Principle: prseduoperoxidase activity of copper-creatinine complex
 CuSo₄ + Creatinine → Cu(CRE) peroxidase
 DBDH + TMB –(Cu(CRE) peroxidase) oxidized TMB + H₂O (orange to
green to blue)
 Chapter 5 | Chemical Examination of Urine

 Reagents: Copper Sulfate (CuSO₄), 3,3’,5’,5’-tetramethylbenzidine (TMB), and

diisopropyl benzene dihydroperoxide (DBDH)
 Sensitivity: 10 to 300 mg/dL
 Interference: Gastric acid-reducing medication cimetidine (Tagamet)

GLUCOSE

 Hyperglycemia – elevated blood level of glucose

 Renal Threshold: 160-180 mg/dL
 2 hours after meal = diabetes monitoring; Fasting Specimen – for screening tests
 Glycosuria – having a meal with high glucose content
 Gestational diabetes – hyperglycemia during pregnancy and disappears after delivery
o Prone to developing type 2 diabetes
 Glycogenesis – converts glucose to glycogen for storage – function of insulin
 Glycogenolysis – breakdown of glycogen to glucose
o Hormones involved: glucagon, epinephrine, cortisol, thyroxine, GH
o Epinephrine – strong inhibitor of insulin
 Renal glycosuria – reabsorption of glucose is compromised
o May be seen in end-stage renal disease, cystinosis and Fanconi syndrome

Clinical Significance of Urine Glucose

Hyperglycemia-associated Renal-Associated
Diabetes Mellitus Fanconi Syndrome
Pancreatitis Advanced renal disease
Pancreatic Cancer Osteomalacia
Acromegaly Pregnancy
Cushing syndrome
Hyperthyroidism
Pheochromocytoma
Central nervous system damage
Stress
Gestational diabetes

 Reagent Strip (Glucose Oxidase) Reaction

o Glucose + O₂ --(Glucose Oxidase) Gluconic Acid + H₂O₂
o H₂O₂ + chromogen –(Peroxidase) Oxidized Colored Chromogen + H₂O
 Chromogen:
 Potassium Iodide (green to brown) (Multistix)
 Tetramethylbenzidine (yellow to green) (Chemstrip)
o Interference:
 False-Positive: Contamination by oxidizing agents and detergents
 False-Negative: ↑Ascorbic Acid, ↑Ketones, ↑Specific Gravity, ↓Temperature,
Improperly preserved specimens
 Chapter 5 | Chemical Examination of Urine

o Correlation with other tests: Ketones, Protein

 Copper Reduction Test (Clinitest)
o CuSO₄ (Cupric Sulfide) + Reducing Substance –(heat, alkali) Cu₂O (Cuprous Oxide) +
Oxidized substance → Color (blue/green, yellow into orange/red)
o Benedict’s Solution – contains copper sulfate, sodium carbonate, and sodium citrate
buffer (+ Sodium Hydroxide [Clinitest tablet]) – brick red precipitate
o Pass through – high glucose level – passes through the orange/red stage, and returns to
a green-brown color
 Use 2 drops instead of 5 drops of urine
o Does not provide a confirmatory test for glucose
o Interference: Reducing Sugars (Galactose, Lactose, Fructose, Maltose, Pentose),
Ascorbic Acid, Drug Metabolites, Antibiotics (ex. Cephalosporin)
 Galactose – most clinically significant – lack of galactose-1-phosphate uridyl
transferase
 “inborn error of metabolism” – galactose in urine

KETONES

 Represents 3 intermediate products of fat metabolism:

o Acetone (2%)
o Acetoacetic acid (20%)
o Β-hydroxybutyrate (78%)
 Ketonuria – shows a deficiency in insulin
o Early indicator of insufficient insulin dosage in type 1 diabetes

Clinical Significance of Urine Ketones

Diabetic Acidosis Strenous exercise
Insulin dosage monitoring Vomiting
Starvation Inborn errors of amino acid metabolism
Malabsorption/pancreatic disorders
 Sodium Nitroprusside (nitroferricyanide) reaction – used to measure ketones
o Acetoacetic acid (+acetone) + Na nitroprusside (+glycine) –(alkaline) purple color
 Acetest tablet test – used as a confirmatory test for questionable reagent strip results
o Provides Na nitroprusside, glycine, disodium phosphate, lactose
o Also tested for severe ketosis
o Hygroscopic – if the specimen is not completely absorbed within 30 seconds, a new
tablet should be used

Reaction Interference (False-positive)

Phthalein cyes
Highly pigmented red urine
Levodopa
Medications containing free sulfhydryl groups
 Chapter 5 | Chemical Examination of Urine

Tests for Ketones

Gerhart, Lindeman Diacetic acid
Frommer, Walhauster, Lange, Jackson-Taylor, Acetone
Rantzmann, Lieben
Legal, Rothera, Acetest, Ketostix Diacetic Acid and Acetone
Osterberg, Hart B-hydroxybutyric acid
Blood

 Hematuria – presence of intact RBCs in the urine

o Cloudy Red Urine
 Hemoglobinuria – presence of RBC destruction in the urine
o Clear Red Urine
o May result from the lysis of RBCs produced in the urinary tract, particularly in dilute,
alkaline urine
o May also result from intravascular hemolysis and the subsequent filtering of hemoglobin
o Formation of Hemoglobin-Haptoglobin = prevents glomerular filtration of hemoglobin =
Normal Condition
 Free hemoglobin exceeds haptoglobin, hemoglobin is available for filtration →
Reabsorption of filtered hemoglobin produces hemosiderin
 Hemosiderin
o Large yellow-brown granules of denatured ferritin
 When blood is >5 cells/mL ≠ Visual Examination
 Myoglobin – a heme-containing protein found in muscle tissue
o Produces a clear red-brown urine
o Heme portion – toxic to the renal tubules; ↑(Heme) = Acute Renal Failure
 Myoglobinuria – may be seen in rhabdomyolysis (muscle destruction)

Clinical Significance of a Positive Reaction for Blood

Hematuria Hemoglobinuria Myoglobinuria
Renal Calculi Transfusion Reactions Muscular trauma/Crush
Syndromes
Glomerulonephritis Hemolytic Anemias Prolonged Coma
Pyelonephritis Severe Burns Convulsions
Tumors Infections/Malaria Muscle-wasting diseases
Trauma Strenuous exercise/RBC trauma Alcoholism/overdose
Exposure to toxic chemicals Brown Recluse Spider Bites Drug abuse
Anticoagulants Extensive exertion
*Strenuous Exercise Cholesterol-lowering statin
medications
*Menstruation
*Non-pathologic

Reagent Strip Reaction

Hydrogen Peroxide + Chromogen Oxidized Chromogen (green-blue color) + H₂O

 Chapter 5 | Chemical Examination of Urine

Chromogen:

 Multistix: Diisopropylbenzene dihydroperoxidase + 3,3’,5,5’-tetramethylbenzidine

 Chemstrip: dimethyldihydroperoxyhexane + tetramethylbenzidine

Presence of free hemoglobin/myoglobin = negative yellow through green through positive blue-green

Reaction Interference: Menstrual Contamination, Strong Oxidizing Detergents, Vegetable Peroxidase,

Bacterial Enzymes (including E. coli peroxidase), >25 mg/dL Ascorbic Acid (vit. C), Urine with ↑sp gr,
Formalin is used as preservative, Captopril, Nitrite

*Bold = False-positive Underline = False-negative

 Precipitation test – used to differentiate hemoglobin and myoglobin in the urine

o 2.8g ammonium sulfate + 5 mL of urine
o Myoglobin is present = Red supernatant + Positive reagent strip for blood
o Hemoglobin is present = Red precipitate + Supernatant that is negative for blood

Bilirubin

 An early indicator for liver disease

 A highly pigmented yellow compound
 Degradable product of hemoglobin

Reticuloendothelial system

 Has phagocytic cells that destroys RBC after 120 days

 Happens in the spleen and liver

Clinical Significance of Urine Bilirubin

Hepatitis Liver Disorders
Cirrhosis Biliary obstruction
(gallstones, carcinoma)

Unconjugated Bilirubin Conjugated Bilirubin

Water Insoluble Water Soluble
Non-polar Polar
Indirect reacting Direct reacting

Urine Bilirubin and Urobilinogen in Jaundice

Urine Bilirubin Urine Urobilinogen
Bile duct obstruction +++ Normal
Liver damage + or - ++
Hemolytic disease Negative +++
 Chapter 5 | Chemical Examination of Urine

Reagent Strip Reaction

 Diazo Reaction

Bilirubin glucuronide + diazonium salt Azodye (tan or pink to violet)

o Diazonium Salt:
 Multistix: 2,4-dichloroaniline diazonium salt
 Chemstrip: 2,6-dichlorobenzene-diazonium salt

Reaction Interference
False-positive False-negative
Urine pigments (phenazopyridine) Specimen exposure to light (converts to biliverdin)
Indican (intestinal disorder) Ascorbic acid
Metabolites of Lodine Nitrite

 Ictotest Tablets (Diazotization test)

o Confirmatory test for Bilirubin
o Reagents: p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and
boric acid
o Presence of Bilirubin: Blue-to-purple color
 Oxidation Test (Gmelin or Fouchet’s method)
o Acidic oxidation of bilirubin into a rainbow array of colors: Green (Biliverdin), Blue
(Bilicyanin), and Yellow (Choletelin)
 Foam Shake test

Urobilinogen

 May be reabsorbed from the intestine into the blood or recirculates to the liver and is excreted
back into the intestine through the bile duct (oxidized to urobilin)
 Stercobilin and Urobilin – responsible for the brown color of feces
 May be found in the urine when it passes through the kidney and is filtered by the glomerulus.
(1 mg/dL or Ehrlich unit [EU] is found in the urine)

Clinical Significance of Urine Urobilinogen

Early detection of liver disease
Liver disorders, hepatitis, cirrhosis, carcinoma
Hemolytic disorders

Multistix (Ehrlich’s aldehyde reaction):

Urobilinogen + p-dimethylaminobenzaldehyde light to dark pink (red)

 Chapter 5 | Chemical Examination of Urine

Chemstrip (azo-coupling reaction):

Urobilinogen + diazonium salt red azodye

Reaction Interference
False-positive False-negative
Porphobilinogen, indican, p-aminosalicylic acid, Improper preservation
sulfonamides, methyldopa, procaine, and High concentration of nitrite
chlorpromazine compounds Formalin
 Tests for Urobilinogen:
o Ehrlich’s Tube test
 Reagents: p-dimethylaminobenzaldehyde
 Positive result: Cherry Red color
o Scwartz-Watson Differentiation test
 UBG – soluble in both chloroform and butanol
 PBG – insoluble in both
 ERCs – soluble only in butanol

Nitrite

 Screening test of Urinary Tract Infection (UTI)

 Detects bacteriuria
 Tests should be performed on first morning specimen
 Cystitis – initial bladder infection

Clinical Significance of Urine Nitrite

Cystitis Monitoring of patients at high risk for UTI
Pyelonephritis Screening of urine culture specimens
Evaluation of antibiotic therapy

Reagent Strip Reactions (Greiss reaction)

p-arsanilic acid (Multistix)/sulfanilamide (Chemstrip) + Nitrite (NO₂) Diazonium salt (nitrite)

Diazonium salt + tetrahydrobenzoquinolin Pink azodye

 Interference
o False-Negative: Nonreductase-containing bacteria, Insufficient contact time between
bacteria and urinary nitrate, Lack of urinary nitrate, Large quantities of bacteria
converting nitrite to nitrogen, Antibiotics, Ascorbic Acid, High Specific Gravity
o False-Positive: Improperly preserved specimens, Highly pigmented urine
 Correlation with other tests: Leukocyte Esterase, Protein, Microscopic
 Chapter 5 | Chemical Examination of Urine

LEUKOCYTE ESTERASE

 Requires microscopic examination of the urine sediments

o Detects presence of esterase in granulocytes and monocytes
o Neutrophil – most frequently associated with bacterial infections
 Normal values: 0-2 to 0-5 per high-power field
o Women > Men (due to vaginal contamination)
 Trichomonas, Chlamydia, yeast, inflammation of renal tissues – may produce leukocyturia

Clinical Significance of Urine Leukocytes

Bacterial and nonbacterial UTI
Inflammation of the Urinary Tract
Screening of urine culture specimens

Reagent Strip Reaction

 Requires the longest time (2 minutes) among all the reagent strip reaction

Derivatized pyrrole amino acid ester (Multistix)/ Indoxyl + Acid indoxyl

Indoxyl + Diazonium Salt Purple azodye

Reaction Interference
False-positive False-negative
Strong Oxidizing agents High concentration of protein, glucose, oxalic acid,
Formalin ascorbic acid, gentamicin, cephalosporins, and
Highly pigmented urine; nitrofurantoin tetra cyclines

SPECIFIC GRAVITY

 Based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium

 Polyelectrolyte ionizes → Release Hydrogen ions in proportion to the solution (↑conc =
↑release)

Clinical Significance of Urine Specific Gravity

Monitoring patient hydration and dehydration Diabetes insipidus
Loss of renal tubular concentrating ability Determination of unsatisfactory specimens due to
low concentration
 Chapter 5 | Chemical Examination of Urine

 Indicator – Bromthymol Blue

o Blue: 1.000 (Alkaline)
o Green
o Yellow: 1.030 (Acid)
 Reagents:
o Multistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue
o Chemstrip: Ethylene glycol diaminoethyl ether tetraacetic acid, bromthymol blue

Reaction Interference
False-positive False-negative
High concentration of protein Highly alkaline urine (greater than pH 6.5)
 Chapter 6 | Microscopic Examination of Urine

Macroscopic Screening

 Abnormalities in the physical and chemical portions of the urinalysis play a primary role in the
decision to perform a microscopic analysis

Macroscopic Screening and Microscopic Correlations

Screening Test Significance
Color Blood
Clarity Hematuria vs Hemoglobinuria/Myoglobinuria
Confirm pathologic or nonpathologic cause of
turbidity
Blood RBCs, RBC casts
Protein Cast, cells
Nitrite Bacteria, WBCs
Leukocyte Esterase WBCs, WBC casts, bacteria
Glucose Yeast

 Specimen Preparation
o Should be fresh or adequately preserved
o Formed elements (RBCs, WBCs, Hyaline Casts) disintegrate in Dilute Alkaline Urine
o Refrigeration = precipitation of amorphous urates and phosphates
o Warming to 37:C = may dissolve crystals
o Midstream Clean-Catch Specimen – minimizes external contamination
 Specimen Volume
o 10 and 15 mL (centrifuged in a conical tube)
o 12-mL – frequently used – multiparameter reagent strips are easily immersed in this
volume
 Centrifugation
o 5 minutes at 400 relative centrifugal force (RCF) = produces an optimum amount of
sediment
 RCF is used over revolutions per minute (RPM)
 To convert RPM to RCF:
o RCF = 1.118 x 10⁻⁵ x radius in centimeters x RPM²
 Sediment Preparation
o 0.5 and 1.0 mL – frequently used – amount after decantation
o = Concentration Factor
 Sediment Concentration Factor – relates to the probability of detecting
elements present in low quantities
 Urine should be aspirated off rather than poured off
 Volume of Sediment Examined
o Glass-slide method volume = 20 microliter (0.02 mL) covered by 22x22 cover slip
 Commercial Systems
 Chapter 6 | Microscopic Examination of Urine

o KOVA, Urisystem, Count-10, Quick-Prep Urinalysis System, CenSlide 2000 Urinalysis

System, R/S Workstations
o CenSlide and R/S Workstations (Closed Systems)
 CenSlide – permits direct reading of the urine sediment
 R/S Workstations – consist of a glass flow cell into which urine sediment is
pumped, microscopically examined, and then flushed from the system
 Examining the Sediment
o Observation of a minimum of 10 fields under both low (10x) and high (40x) power
o Sediments should be examined under reduced light when using bright-field microscopy
 Reporting the Microscopic Examination
o Cast – average number per low power field (lpf)
o RBCs and WBCs – average number per 10 high power field (hpf)
o Epithelial cells, crystals, and other elements – Rare, few, moderate, and many
 Correlating Results

Routine Urinalysis Correlations

Microscopic Elements Physical Chemical Exceptions
RBCs Turbidity +Blood Number
Red Color +Protein Hemolysis
WBCs Turbidity +Protein Number
+Nitrite Lysis
+LE
Epithelial Cells Turbidity Number
Casts +Protein Number
Bacteria Turbidity ↑pH Number and Type
+Nitrite
+Leukocytes
Crystals Turbidity pH Number and Type
Color +Bilirubin

Addis Count – developed by Addis (1926), it is the first procedure to standardize the
quantitation of formed elements in the urine microscopic analysis

Sediment Examination Techniques

 Sediment Stains
 Chapter 6 | Microscopic Examination of Urine

Urine Sediment Stain Characteristics

Stain Action Function
Sternheimer-Malbin Delineates structure and contrasting Identifies WBCs, epithelial cells,
(consists of Crystal colors of the nucleus and cytoplasm and casts
Violet and Safranin O)
0.5% Toluidine Blue Enhances nuclear detail Differentiates WBCs and Renal
Tubular Epithelial (RTE) cells
2% Acetic Acid Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs,
WBCs yeast, oil droplets, and crystals
Lipid Stains: Oil Red O Stain triglycerides and neutral fats Identify free fat droplets and lipid-
and Sudan III orange-red; containing cells and casts
Do not stain cholesterol
Gram Stain Differentiates gram-positive (blue) Identifies bacterial casts
and gram-negative (red) bacteria
Hansel Stain Methylene Blue and Eosin Y stains Identifies urinary eosinophils
eosinophilic granules
Prussian Blue Stain Stains structures containing iron Identifies yellow-brown granules
of hemosiderin in cells and casts

Expected Staining Reactions of Urine Sediment Constituents

Elements in Urinary Usual Distinguishing Comments
Sediment Color
RBCs Neutral – pink to
purple
Acid – pink (unstained)
Alkaline – purple
Nuclei Cytoplasm
WBCs (dark-staining Purple Purple Granules
cells)
Glitter Cells Colorless or light blue Pale blue or gray Some glitter cells
(Sternheimer-Malbin exhibit Brownian
positive cells) movement
Renal Tubular Dark shade of blue- Light shade of blue-
Epithelial Cells purple purple
Bladder Tubular Blue-purple Light purple
Epithelial Cells
Squamous Epithelial Dark shade of Orange- Light purple or blue
Cells purple
Inclusions and Matrix
Hyaline Casts Pale pink/pale purple Very uniform color;
slightly darker than
mucous threads
Coarse granular Dark purple granules in
inclusion casts purple matrix
Finely granular Fine dark purple
inclusion casts granules in pale pink or
 Chapter 6 | Microscopic Examination of Urine

pale purple matrix

Waxy Casts Pale pink/pale purple Darker than hyaline
casts, but of a pale
even color; distinct
broken ends
Fat Inclusion Casts Fat globules unstained Rare; present if
in a pink matrix polarized light
indicates double
refraction
Red cell inclusion casts Pink to orange-red Intact cells can be seen
in matrix
Blood (hemoglobin) Orange-red No intact cells
casts
Bacteria Motile: Do not stain Motile organisms are
Nonmotile: purple not impaired
Trichomonas vaginalis Light blue-green Motility in unimpaired
in fresh specimens
when recommended
volumes of stain are
used; immobile
organisms also
identifiable
Mucus Pale pink or pale blue
Background Pale pink or pale
purple

Cytodiagnostic Urine Testing – for the detection of malignancies of the lower urinary tract (Papanicolaou
stain)

Urinalysis Microscopic Techniques

Bright-field microscopy Used for routine urinalysis
Phase-contrast microscopy Enhances visualization of elements with low
refractive indices, such as hyaline casts, mixed
cellular casts, mucous threads, and Trichomonas
Polarizing Microscopy Aids in identification of cholesterol in oval fat
bodies, fatty casts, and crystals
Dark-field Often used for unstained specimens; and aids in
identification of Treponema pallidum
Fluorescence Microscopy Allows visualization of naturally fluorescent
microorganisms or those stained by a fluorescent
dye including labeled antigens and antibodies
Interference-contrast Produces a three-dimensional microscopy image
and layer-by-layer imaging of a specimen
 Chapter 6 | Microscopic Examination of Urine

Red Blood Cells

 Smooth, non-nucleated, biconcave disk. 7mm

 Crenated (irregular) – concentrated/hypersthenuric urine – cells shrink due to water loss
o May resemble the granules seen in WBCs
 Ghost cell – dilute/hyposthenuric urine – large empty cells
 Dysmorphic – associated with glomerular bleeding, seen after strenuous exercise
 Presence of RBC in urine = Macroscopic Hematuria – urine is cloudy with a red to brown color
 ↑RBC = Hematuria: glomerulonephritis, renal calculi, malignancy
 Normal = 0-2/hpf
 Close resemblance: Yeast Cells, Oil Droplets, Air Bubbles
 Reporting: Average number per 10 hpfs
 Complete Urinalysis Correlation: Color and Reagent Strip Blood Reaction

White Blood Cells

 Neutrophil – predominant WBC found in the urine sediment

o Contains granules and are multi-lobed
o Lyse rapidly in dilute alkaline urine
 Glitter cells – neutrophils exposed to hypotonic urine
o Sparkling appearance produced from the Brownian movement of the granules
o No pathologic significance
 Eosinophil – may be seen with UTI and Renal Transplant Rejection, Interstitial Nephritis
 Mononuclear cells – lymphocytes, monocytes, macrophages, and histiocytes
o Lymphocyte – smallest WBC; may resemble RBC
o Graft rejection
 ↑urinary WBC = pyuria (inflammation/infection in the genitourinary system)
o Pyelonephritis, cystitis, prostatitis, and urethritis – bacterial causes of pyuria
o Glomerulonephritis, interstitial nephritis, lupus erythematosus, tumor - nonbacterial
 Normal = <5/hpf, 12mm in diameter
 Close resemblance: Renal Tubular Epithelial (RTE) cells
 Reporting: Average number per 10 hpfs
 Complete Urinalysis Correlation: Leukocyte Esterase, Nitrite, Specific Gravity, pH

Epithelial Cells

 Squamous Cell
o Largest cell found in the urine sediment
o Contain abundant, irregular cytoplasm and a prominent central nucleus about the size
of an RBC
o Originate from the linings of the vagina, female urethra, and the lower portion of male
urethra
o No pathologic significance
 Chapter 6 | Microscopic Examination of Urine

o Midstream clean catch = less squamous cells

o Clue cells – vaginal infection by Gardnerella vaginalis
o Close resemblance: Casts (folded cells) (rarely encountered)
o Reporting: Rare, few, moderate, or many per lpf
o Complete Urinalysis Correlation: Clarity
 Transitional Epithelial (Urothelial) Cell
o Can be Spherical, Polyhedral, or Caudate
o Centrally located nucleus
o Originate from the lining of the renal pelvis, calyces, ureters, and bladders, and from the
upper portion of the male urethra
o ↑Transitional Cells = Syncytia – present following invasive urologic procedures (ex.
catheterization)
 Malignancy or viral infection
o Close resemblance: RTE cells (for spherical forms)
o Reporting: Rare, few, moderate, or many per hpf
o Complete Urinalysis Correlation: Clarity and Blood (if malignancy associated)
 Renal Tubular Epithelial (RTE) Cell
o Rectangular (columnar or convoluted) shaped from Proximal Convoluted Tubule
 Close resemblance: Cast (no nucleus)
o Oval or round shaped from Distal Convoluted Tubule
 Close resemblance: WBCs and Spherical Transitional Epithelial Cells
o Cuboidal shaped from Collecting Duct
o Eccentrically located nucleus
o Presence = Tissue destruction (necrosis), Tubular Injury (2 or more RTE)
o Hemoglobinuria -> RTE absorbs hemoglobin -> converted to yellow-brown Hemosiderin
o Reporting: Average number per 10 hpfs
o Complete Urinalysis Correlation: Leukocyte Esterase and Nitrite (pyelonephritis), Color,
Clarity, Protein, Bilirubin (hepatitis), Blood
 Oval Fat Bodies
o Lipid-containing, Highly refractile RTE cells
o Nephrotic syndrome -> Glomerular damage -> Lipiduria
o Seen in Severe tubular necrosis and diabetes mellitus
o Bubble Cells – RTE cells containing large, nonlipid-filled vacuoles
 In cases of Acute Tubular Necrosis
o Close resemblance: Starch and crystal particles
o Reporting: Average number per hpf
o Complete Urinalysis Correlation: Clarity, Blood, Protein, Free fat droplets/fatty casts

Bacteria

 Small spherical and rod-shaped structures

 Presence = UTI
 Chapter 6 | Microscopic Examination of Urine

 Enterobacteriaceae – frequently associated with UTI

o Staphylococcus and Enterococcus – may cause UTI
 Close resemblance: Amorphous phosphates and urates
 Reporting: Few, moderate, or many per hpf, the presence of WBCs may be required
 Complete Urinalysis Correlation: pH, nitrite, Leukocyte Esterase, WBCs

Yeast

 Small, oval, refractile structures with buds and/or mycelia

 Candida albicans – seen in the urine of diabetic patients, immunocompromised patients, and
women with vaginal moniliasis
 Close resemblance: RBCs
 Reporting: Rare, few, moderate, or many per hpf, the presence of WBCs may be required
 Complete Urinalysis Correlation: Glucose, Leukocyte Esterase, WBCs

Parasites

 Trichomonas vaginalis – most frequent parasite encountered in the urine

o Pear-shaped flagellate with an undulating membrane
o Sexually transmitted pathogen
 Schistosoma haematobium – bladder parasite – ova with a terminal spine
 May cause nonspecific urethritis
 Close resemblance: WBC, transitional, or RTE cells
 Reporting: Rare, few, moderate, or many per hpf
 Complete urinalysis Correlation: Leukocyte Esterase, WBCs

Spermatozoa

 Tapered oval head with long, thin tail

 Close resemblance: None
 Reporting: Present, based on laboratory protocol
 Complete Urinalysis Correlation: Protein

Mucus

 Single or clumped threads with a low refractive index

 No clinical significance
 Uromodulin – major constituent of mucus
o A glycoprotein excreted by the RTE cells of the DCT and upper collecting ducts
 Close resemblance: Hyaline casts
 Reporting: Rare, few, moderate, or many per lpf
 Chapter 6 | Microscopic Examination of Urine

Casts

 Formed within lumens of DCT and collecting ducts

 Only elements found in the urinary sediment that are unique to the kidney
 Dissolve quickly in dilute, alkaline urine
 Uromodulin – major cast constituent
 Cast Formation: Aggregation of Uromodulin protein into individual protein fibrils attached to the
RTE cells → Interweaving of protein fibrils to form a loose fibrillar network → Further protein
fibril interweaving to form a solid structure → Possible attachment of urinary constituents (ex.
albumin and Ig) to the solid matrix → Detachment of protein fibrils from the epithelial cells →
Excretion of the Cast
o Blocks Lumen = ↓Urinary Flow in the tubules
 Cylindruria – presence of urinary casts at the junction of the ascending loop of Henle and the
DCT
 Reporting: Average number per 10 lpf

Hyaline Cast

 Colorless with low refractive index, homogenous matrix, prototype of all casts
 May normal parallel sides and rounded cells, cylindroid forms, and wrinkled or convoluted
 Normal: 0-2/lpf
 Close resemblance: Mucus, fibers, hair, increased lighting
 Complete Urinalysis Correlation: Protein, Blood (Exercise), and Color (Exercise)
 Clinical Significance: Pathologic - Glomerulonephritis, Pyelonephritis, Chronic Renal Disease,
Congestive Heart Failure; Non-pathologic – strenuous exercise, dehydration, heat exposure,
emotional stress

RBC Cast

 Orange-red color, cast matrix containing RBCs

 May exist as fragments or have a more irregular shape
 Close resemblance: RBC clumps
 Complete Urinalysis Correlation: RBCs, Blood, Protein
 Clinical Significance: Glomerulonephritis, Strenuous exercise
o Glomerular damage - associated with proteinuria and dysmorphic erythrocytes
o Hemoglobinuria/Myoglobinuria – homogenous orange-red or red-brown casts
 Associated with Acute Tubular Necrosis

WBC Cast

 Cast matrix containing WBCs

 Close resemblance: WBC clumps
 Complete Urinalysis Correlation: WBCs, protein, Leukocyte Esterase
 Chapter 6 | Microscopic Examination of Urine

 Clinical Significance: Pyelonephritis, Acute interstitial nephritis (+eosinophils)

 Primary marker for distinguishing Pyelonephritis (upper UTI) from cystitis (lower UTI)

Bacterial Cast

 Bacilli bound to protein matrix

 Close resemblance: Granular Casts
 Complete Urinalysis Correlation: WBC cast (pyelonephritis), WBCs, Leukocyte Esterase, Nitrite,
Protein, Bacteria
 Clinical Significance: Pyelonephritis

Epithelial Cell Cast

 RTE cells attached to protein matrix

 Bilirubin-stained RTE – Hepatitis
 Close resemblance: WBC cast
 Complete Urinalysis Correlation: Protein, RTE cells
 Clinical Significance: Renal Tubular Damage; Advanced Tubular Destruction (exposure to toxic
agents); Viral Infections, Allograft Rejection

Fatty Cast

 Fat droplets and oval fat bodies attached to protein matrix

 Close resemblance: Fecal Debris
 Complete Urinalysis Correlation: Protein, Free fat droplets, Oval fat bodies
 Clinical Significance: Nephrotic Syndrome, Toxic Tubular Necrosis, Diabetes Mellitus, Crush
Injuries

Mixed Cellular Cast

 Glomerulonephritis – RBC + WBC casts

 Pyelonephritis – WBC + RTE cell/Bacterial casts

Granular Cast

 Coarse and fine granules in a cast matrix

 Lysosome (excreted by RTE cells) – where the granules in nonpathologic condition originates
 Becomes Waxy when stayed in the tubules in extended period
 Close resemblance: Clumps of small crystals and fecal debris, Columnar RTE cells
 Complete Urinalysis Correlation: Protein, Cellular Casts, RBCs, WBCs
 Clinical Significance: Glomerulonephritis, Pyelonephritis, Stress and Exercise, Stasis of Urine Flow

Waxy Cast

 Highly refractile cast with jagged ends and notches; brittle consistency
 Chapter 6 | Microscopic Examination of Urine
 Final phase of cast degeneration
 Close resemblance: Fibers and fecal material
 Complete Urinalysis Correlation: Protein, Cellular Casts, Granular Casts, WBCs, RBCs
 Clinical Significance: Stasis of Urine Flow, Chronic Renal Failure
 Supravital stain = homogenous dark pink

Broad Cast

 Renal Failure Cast

 Wider than normal cast matrix
 Close resemblance: Fecal material, fibers
 Complete Urinalysis Correlation: Protein, WBCs, RBCs, Granular Casts, Waxy Casts
 Clinical Significance: Extreme Urine Stasis, Renal Failure, Destruction of Tubular Walls

Urinary Crystals

 Rarely of clinical significance

 Reporting: Rare, few, moderate, or many per hpf (lpf for abnormal)
 Formed by the precipitation of urine solutes (precipitates more readily at room temperature)
 Organic and iatrogenic compounds crystalize more easily in an acidic pH
 Inorganic salts are less soluble in neutral and alkaline solutions

Major Characteristics of Normal Urinary Crystals

Crystal pH Color Appearance
Uric Acid Acid Yellow-Brown (rosettes,
(may be seen as wedges)
rhombic, whetstones)
(polarizes)
Amorphous Urates Acid Brick dust or yellow
(dissolved if warmed) brown

Calcium Oxalate Acid/neutral (alkaline) Colorless (dihydrate -

(reagent: HCl) envelopes,
(polarizes) monohydrate-
oval/dumbbell)
Amorphous Phosphates Alkaline/neutral White-colorless
(seen in refrigerated
specimens) (acetic acid)

Calcium Phosphate Alkaline/neutral Colorless

 Chapter 6 | Microscopic Examination of Urine

Triple Phosphate Alkaline Colorless (“coffin lids”)

(ammonium (disintegrate →
magnesium phosphate) feathery)
(polarizes)
Ammonium Biurate Alkaline Yellow-brown (“thorny
apples”)

Calcium Carbonate Alkaline Colorless (dumbbells)

(polarizes)

 Uric Acid – seen in ↑purine, leukemia, Lesch-Nyhan syndrome, Gout

 Sodium urates – needle-shaped – seen in Gout
 Calcium Oxalate (monohydrate) – has a close resemblance with nonpolarizing RBCs
o May resemble Casts
o Clinical Significance: Ethylene Glycol (antifreeze) poisoning, Renal Calculi (associated
with foods high in oxalic acid [tomatoes, asparagus, ascorbic acid])
 Alkaline Crystals – white precipitate – soluble in dilute acetic acid
 Calcium Oxalate – Whedellite (dihydrate); Whewellite (monohydrate)
 Calcium Phosphate (rosette form) – has a resemblance with sulfonamide crystals (in neutral)
 Calcium Carbonate – may resemble Amorphous material
 Hippuric Acid – Yellow-brown or colorless, needles, rhombic plates and four-sided prisms
o Rare; associated with foods containing benzoic acid

Abnormal Urine Crystals

Major Characteristics of Abnormal Urinary Crystals

Crystal (rgt) pH Color/Form Disorders Appearance
Cystine Acid Colorless Inherited
(Ammonia/dilute (Hexagonal Cystinuria
HCl) (polarizes) Plates)

Cholesterol Acid Colorless Nephrotic

(Chloroform) (notched Syndrome
(polarizes) Plates)

Leucine Acid/Neutral Yellow Liver

(rgt: (concentric Disease
Alkali/Alcohol) Circles)

Tyrosine Acid/Neutral Colorless-yellow Liver

(rgt: (needles) Disease
Alkali/Alcohol)
 Chapter 6 | Microscopic Examination of Urine

Bilirubin (acetic Acid Yellow Liver

acid, HCl, NaOH, Disease
ether,
chloroform)
Sulfonamides Acid/Neutral Varied Infection
(Acetone) (colorless- Treatment,
yellow, brown Inadequate
needles, sheaves Hydration
of wheat,
rosettes)
Radiographic Acid Colorless Radiographic
Dye (flat plates) Procedure,
(Cholesterol, ↑SG (1.040)
10% NaOH)
(polarizes)
Ampicillin Acid/Neutral Colorless Infection
(needles) Treatment,
Inadequate
Hydration

 Cholesterol – close resemblance: Radiographic Dye

 Cystine – close resemblance: Colorless Uric Acid Crystals
o Confirmation: Cyanide-Nitroprusside test

Urinary Sediment Artifacts

 Starch granules – highly refractile spheres with a dimpled center, resembles fat droplets when
polarized, producing a Maltese cross formation

 Oil droplets and air bubbles – highly refractile and may resemble RBCs

 Pollen grains – spheres with a cell wall and occasional concentric circles; large size may cause
the microscopist to be out of focus

 Hair and Fibers – May be mistaken for casts but are longer, more refractile, and are able to
polarize light

 Fecal artifacts – from improperly collected specimens or due to the presence of a recto-vesical
fistula; may appear as plant and meat fibers or as brown amorphous materials in a variety of
sizes and shapes
 Chapter 7 | Renal Disease

GLOMERULAR DISORDERS

 Formed mostly from immunologic disorders (Immune Complex) (ex. ↑ in immunoglobulin A)

o Components of the immune system (complement, neutrophils, lymphocytes,
monocytes, and cytokines) are then attracted to the area, producing changes and
damage to the membrane
 Non-immunologic disorders:
o Exposure to chemicals and toxins that affects the tubules
o Disruption of the electrical membrane charges (nephrotic syndrome)
o Deposition of amyloid material from systemic disorders that may involve chronic
inflammation and acute-phase reactants
o Basement membrane thickening associated with diabetic nephropathy

Glomerulonephritis

 Refers to a sterile, inflammatory process that affects the glomerulus

1. Acute Poststreptococcal Glomerulonephritis (AGN)

o A disease marked by the sudden onset of symptoms consistent with damage to the
glomerular membrane
i. Fever
ii. Edema (most noticeably around the eyes)
iii. Fatigue
iv. Hypertension
v. Oliguria
vi. Hematuria
o Symptoms usually occur in children following respiratory infections from group A
streptococcus that contain M protein in the cell wall
o Nephrogenic stains of streptococcus forms immune complexes → deposited on the
glomerular membranes
o Secondary complications (hypertension and electrolyte imbalance) are managed until
immune complexes are cleared from blood = no permanent kidney damage
o Blood Urea Nitrogen (BUN) may increase in acute stages but returns to normal
o Tests: ASO Titer, anti-group A streptococcal enzymes
2. Rapidly Progressive (Crescentic) Glomerulonephritis
o More serious form of acute glomerulonephritis
o Has much poorer prognosis, often terminating in renal failure
o Deposition of immune complex (IgA) in the glomerulus
i. Can lead to an immune systemic disorder - Systemic Lupus Erythematosus (SLE)
 Macrophages damages capillary walls → Releases cell and plasma into
Bowman’s capsule → Production of crescentic formations containing
macrophages, fibroblasts, polymerized fibrin → causes permanent
damage to the capillary tufts
 Chapter 7 | Renal Disease

o Some form may demonstrate ↑fibrin degradation products, cryoglobulins

o Tests: BUN, Creatinine, Creatinine clearance
3. Goodpasture Syndrome
o A cytotoxic antibody can appear against the glomerular and alveolar basement
membranes after viral respiratory infections → Complement activation → Capillary
destruction
o Antiglomerular basement membrane antibody – test to detect the autoantibody
o Initial pulmonary complaints: Hemoptysis and Dyspnea → then Hematuria
o Can progress to chronic glomerulonephritis and end-stage renal failure
4. Wegener Granulomatosis (Vasculitis)
o Causes a granuloma-producing inflammation of the small blood vessels primarily of the
kidney and respiratory system
o Antineutrophilic cytoplasmic antibody (ANCA) – key to diagnosis
i. Antibodies bind to neutrophil (located in vascular walls) → may initiate immune
response and the resulting granuloma formation
o ANCA Test – incubate serum with ethanol or formalin/formaldehyde-fixed neutrophils
→ examine using indirect immunofixation → detect antibodies attached to neutrophils
i. Fixed in ethanol = perinuclear pattern (p-ANCA)
ii. Fixed with formalin/formaldehyde = granular pattern (c-ANCA)
o Pulmonary symptoms – hemoptysis → renal involvement → end-stage renal failure
5. Henoch-Schonlein Purpura (Vasculitis)
o Disruption of vascular integrity following viral respiratory infections
o Occurs primarily in children after upper respiratory infections
o Initial symptom: Raised, red patches on the skin
o Respiratory and Gastrointestinal symptom: Blood in sputum and stool
o Renal involvement – most serious renal complication
o Test: FOBT (fecal occult blood testing)
6. Membranous Glomerulonephritis
o Deposition of IgG Immune complex → thickening of glomerular basement membrane
o Disorders: Lupus erythematosus, Sjogren syndrome, Secondary Syphilis, Hepatitis B,
Gold and Mercury Treatments, Malignancy
o Slow progression to: Nephrotic Syndrome or Possible remission, Thrombosis
o Tests: Antinuclear antibody, Hepatitis B surface antigen, Fluorescent treponemal
antibody-absorption test (FTA-ABS), Serum IgA
7. Membranoproliferative Glomerulonephritis
o Type 1 – increased cellularity in the subendothelial cells of the mesangium (interstitial
area of Bowman’s Capsule) → thickening of capillary walls
i. Progress to nephrotic syndrome
o Type 2 – Dense deposits in the glomerular basement membrane
i. Experience symptoms of Chronic Glomerulonephritis
o Test: Serum Complement levels
8. Chronic Glomerulonephritis
 Chapter 7 | Renal Disease

oMarked decrease in renal function resulting from glomerular damage precipitated by

other renal disorders
o Worsening symptoms: fatigue, anemia, hypertension, edema, oliguria
o ↓GFR = ↑BUN, ↑Creatinine, Electrolyte imbalance
9. Immunoglobulin A Nephropathy
o Also known as Berger disease
o Most common cause of glomerulonephritis
o IgA deposited into glomerular membrane
o Disorder: Mucosal Infection (most common in children and young adults)
o Test: Serum IgA

Nephrotic Syndrome

 Massive proteinuria (>3.5 g/day), ↓serum albumin, ↑serum lipid, and pronounced edema
 Disorder: Acute Onset → Systemic Shock (decrease pressure and flow of blood to the kidney)
 Damage to the shield of negativity and the podocytes → produces a less tightly connected
barrier → high mw proteins and lipids may pass into the urine
 Albumin – primary protein depleted from circulation; ↓Albumin = ↑Lipid (by the liver)
 Test: Serum Albumin, Cholesterol, TAG

1. Minimal Change Disease

o Also known as Lipid Nephrosis
o Damages podocytes and shield of negativity
o Allergic reactions, recent immunization, possession of human leukocyte antigen-B12
(HLA-B12) have been associated with this disease
o Responds to Corticosteriods → Frequent Complete Remission
2. Focal Segmental Glomerulosclerosis (FSGS)
o Disruption of Podocytes associated with heroin/analgesic abuse and AIDS
o Symptoms: Similar to nephrotic syndrome and minimal change disease
o Immune deposits: IgM and C3
o Test: Drug Test, HIV Test

Disorder Primary Urinalysis Results

Acute Glomerulonephritis Macroscopic hematuria, Proteinuria, Oliguria, RBC
Casts (dysmorphic), Hyaline and Granular Casts,
WBC
Rapidly Progressive Glomerulonephritis Macroscopic Hematuria, Proteinuria, RBC Casts
Goodpasture Syndrome Macroscopic Hematuria, Proteinuria, RBC Casts
Wegener Granulomatosis Macroscopic Hematuria, Proteinuria, RBC Casts,
Increased Serum Creatinine and BUN
Henoch-Schonlein Purpura Macroscopic Hematuria, Proteinuria, RBC Casts
Membranous Glomerulonephritis Microscopic Hematuria, Proteinuria
Membranoproliferative Glomerulonephritis Hematuria, Proteinuria, Decreased Serum
Complement Levels
 Chapter 7 | Renal Disease

Chronic Glomerulonephritis Hematuria, Proteinuria, Glucosuria, Cellular and

Granular Casts, Waxy and Broad Casts
IgA Nephropathy Early: Macroscopic/Microscopic Hematuria
Late: Hematuria, Casts, Proteinuria, Glucosuria
Nephrotic Syndrome Heavy Proteinuria, Microscopic Hematuria, Renal
Tubular Cells, Oval Fat Bodies, Fat Droplets, Fatty
and Waxy Casts
Minimal Change Disease Heavy Proteinuria, Transient Hematuria, Fat
droplets
Focal Segmental Glomerulosclerosis Proteinuria, Macroscopic/Microscopic Hematuria

TUBULAR DISORDERS

Acute Tubular Necrosis

 Decreased Blood flow → Lack of oxygen in tubules (Ischemia) or Presence of Toxic Substance →
Damage to RTE cells
 Disorders: Shock, Trauma, Surgical Procedures
 May cause Shock: Cardiac Failures, Sepsis involving toxigenic bacteria, anaphylaxis, massive
hemorrhage, contact with high voltage electricity
 Toxic substances: Aminoglycoside antibiotics, antifungal agent amphotericin B, cyclosporine,
radiographic dye, organic solvents (ethylene glycol, heavy metals, toxic mushrooms)
 Tests: Hemoglobin, Hematocrit, Cardiac Enzymes

Hereditary and Metabolic Tubular Disorders

 Affect or override the Tubular Reabsorptive Maximum (Tᵐ)

1. Fanconi Syndrome
o Failure of tubular reabsorption in the PCT
o Disorders: Cystinosis and Hartnup disease (acquired through exposure to toxic agents)
o Test: Amino Acid Chromatography, Serum and Urine Electrolytes
2. Alport Syndrome
o Inherited disorder of collagen production affecting the glomerular basement membrane
o Can be inherited as a sex-linked or autosomal genetic disorder
o Males are severely more affected than females
o Disorders: Macroscopic Hematuria → Microscopic Hematuria, Abnormalities in hearing
and vision
o Genetic Disorder: Lamellated appearance with areas of thinning
o Slow progression to Nephrotic Syndrome and End-stage Renal Disease
3. Uromodulin-Associated Kidney Disease
o Uromodulin – a glycoprotein and the only protein that is produced by the kidney
 Chapter 7 | Renal Disease

i. Produced by the PCT and DCT

o Autosomal mutation in the gene that produces uromodulin
o ↑Uric Acid = Gout
o Renal Failure → Kidney Transplantation
4. Diabetic Nephropathy
o Most common cause of End-stage renal disease
o Glomerular Membrane Thickening + ↑Proliferation of Mesangial cells + ↑deposition of
cellular and noncellular material within the glomerular matrix = Accumulation of solid
substances around the capillary tufts
o Associated with deposition of glycosylated proteins
o May develop sclerosis
o Microalbuminuria – important to detect the onset of diabetic nephropathy
5. Nephrogenic Diabetes Insipidus
o Can be inherited as a sex-linked recessive gene (defect of tubular response to ADH) or
acquired from medications (lithium and amphotericin B)
o Complication of Polycystic Kidney Disease and Sickle Cell Anemia
6. Renal Glycosuria
o Affects the reabsorption of glucose
o Inherited as an autosomal recessive trait
o Disorder: Benign Disorder; Test: Blood Glucose

Disorder Primary Urinalysis Results

Acute Tubular Necrosis Microscopic Hematuria, Proteinuria, RTE cells, RTE
cell casts, Hyaline/Granular/Waxy/Broad casts
Fanconi Syndrome Glucosuria, Possible Cystine Crystals
Uromodulin-associated Kidney Disease RTE cells
Nephrogenic Diabetes Insipidus ↓specific gravity, polyuria
Renal Glucosuria Glucosuria

INTERSTITIAL DISORDERS

 Tubulointerstitial disease – disorders affecting the renal interstitium and renal tubules
 Urinary Tract Infection (UT) – most common renal disease
o Lower – Urethra and Bladder
o Upper – Renal Pelvis, Tubules, Interstitium
 Cystitis – infection of the bladder
o Can progress to a more serious upper UTI if left untreated
o Common in women and children
o Symptoms: Urinary frequency and burning
 Treated with antibiotics
o Test: Urine Culture
 Chapter 7 | Renal Disease

1. Acute Pyelonephritis
o Ascending movement of bacteria from a lower UTI into the renal tubules and
interstitium = interfere with downward flow of urine (or reflux of urine)
o Symptoms: Urinary frequency, burning on urination, lower back pain
 Treated with antibiotics
o Vesicouretral reflux (reflux of urine from the bladder back into the ureters), Pregnancy,
Renal Calculi – enhances ascending movement of bacteria
o Test: Urine and Blood Cultures
2. Chronic Pyelonephritis
o Can result in permanent damage to the renal tubules and possible progression to
chronic renal failure
o Often diagnosed in children
o Recurrent infection of the renal tubules and interstitium
o Tests: BUN, Creatinine, Creatinine clearance
3. Acute Interstitial Nephritis
o Inflammation of the Renal Interstitium then Inflammation of the Renal Tubules
o Initial symptoms: Skin rash and fever
o Associated with allergic reactions (eosinophils may be present)
 Medications: Penicillin, Methicillin, Ampicillin, Cephalosporines, Sulfonamides,
NSAIDs, Thiazide Diuretics
 Treatment [and discontinuation of medications]: Corticosteroids
o Tests: Urine Eosinophils, BUN, Creatinine, Creatinine clearance

Disorder Primary Urinalysis Results

Cystitis Leukocyturia, Bacteriuria, Microscopic Hematuria,
Mild Proteinuria, ↑pH
Acute Pyelonephritis Leukocyturia, Bacteriuria, Microscopic Hematuria,
Proteinuria, WBC casts, Bacterial casts
Chronic Pyelonephritis Leukocyturia, Bacteriuria, Hematuria, Proteinuria,
WBC casts, Bacterial casts, Granular/Waxy/Broad
casts
Acute Interstitial Nephritis Hematuria, Proteinuria, Leukocyturia, WBC casts

RENAL FAILURE

 ↓Glomerular Filtration Rate (<25mL/min)

 ↑BUN and ↑Creatinine = Azotemia
 Other characteristics: Electrolyte Imbalance, Lack of Renal Concentrating Ability (isosthenuric
urine), Proteinuria, Renal Glycosuria, Abundance of Granular/Waxy/Broad casts (Telescoped
Urine Sediments)
 Acute Renal Failure (ARF) – exhibits a sudden loss of renal function; reversible

 Chapter 7 | Renal Disease

Causes of Acute Renal Failure

Prerenal Renal Postrenal
↓Blood Pressure/Cardiac Acute Glomerulonephritis Renal Calculi
Output Acute Tubular Necrosis Tumors
Hemorrhage Acute Pyelonephritis
Burns Acute Interstitial Nephritis
Surgery
Septicemia

RENAL LITHIASIS

 Renal calculi (kidney stones) may form in the calyces and pelvis of the kidney, ureters, bladder
 Renal Lithiasis – renal calculi vary in size
o Staghorn calculi – resembles shape of renal pelvis and smooth, round bladder stones
 Urinalysis Result: Microscopic Hematuria
 Test: Chemical Analysis of Kidney Stones
 Lithiotripsy – a procedure using high-energy shockwaves, can be used to break stones located in
the upper urinary tract; Surgery – can also be done to remove stones
 Analysis – X-ray crystallography
 75% of renal calculi are composed of Calcium Oxalate or Calcium Phosphate
o Other constituents: Magnesium Ammonium Phosphate (Struvite), Uric Acid, Cystine
 Calcium Calculi – Diet
 Struvite – urinary infections; urine pH is 7.0 or higher
 Uric Acid Calculi – Increased food intake with high purine content
 Cystine Calculi – Hereditary
 Qualitative Examination of Renal Calculi
o Physical Examination (appearance, size/weight)
o Chemical Examination
 Pulverize the stone → Ash a small portion over a hot burner → Distribute the
remaining pulverized stone into 7 tubes as follows:
Tube Reagents Positive Result
Uric Acid 20% Sodium Carbonate, Deep Blue
Folin reagent
Carbonate and Oxalate 10% HCl, MnO₂ Bubble formation
Phosphate Ammonium Molybdate, Yellow
25% HNO₃
Calcium 10% HCl, 20% NaOH White Cloud
Magnesium 10% HCl, 20% NaOH, p- Blue
nitrobenzene
azoresorcinol
Ammonium 10% HCl, 20% NaOH, Orange-Brown
Nessler’s solution
Cystine 28% NH₄OH, 5% NaCN, Red-Purple
5% Sodium Nitroprusside
 Chapter 8 | Metabolic Disease

OVERFLOW VERSUS RENAL DISORDERS

 Overflow disorders – disruption of normal metabolic pathway = ↑plasma concentration of the

nonmetabolized substances
o Nonmetabolized substances – override the reabsorption of the renal tubules or are not
normally reabsorbed from the filtrate
 Inborn error of metabolism (IEM) – failure to inherit the gene to produce a particular enzyme

Abnormal Metabolic Constituents or Conditions Detected in the Routine Urinalysis

Color Odor Crystals
Homogentisic Acid Phenylketonuria Cystine
Melanin Maple Syrup Urine Disease Leucine
Indican Isovaleric Acidemia Tyrosine
Porphyrins Cystinuria Lesch-Nyhan disease
Cystinosis
Homocystinuria

Major Disorders of Protein and Carbohydrate Metabolism Associated With Abnormal Urinary
Constituents, Classified by Functional Defect
Overflow Inherited Metabolic Renal
Phenylketonuria Infantile tyrosinemia Hartnup disease
Tyrosinemia Melanuria Cystinuria
Alkaptonuria Indicanuria
Maple Syrup Urine Disease 5-hydroxyindoleacetic acid
Organic acidemias Porphyria
Cystinosis
Porphyria
Mucopolysaccharidoses
Galactosemia
Lesch-Nyhan disease

NEWBORN SCREENING TESTS

 Performed primarily to detect IEMs

 Unmetabolized toxic food ingredients buildup: Blood > Urine
 Heel puncture – initially tested
 Tandem Mass Spectrophotometry (MS/MS) – capable of screening the infant blood sample for
specific substances associated with IEMs

AMINO ACID DISORDERS

Phenylalanine-Tyrosine Disorders

 Phenylketonuria
o Most well-known of the aminoacidurias
 Chapter 8 | Metabolic Disease

o Occur in 1 of every 10,000-20,000 births

o Urine = ↑keto acids (phenylpyruvate) = mousy odor
 Identified by Ivan Folling (1934) in Norway
 Conversion of phenylalanine to tyrosine is disrupted
o Caused by failure to inherit the gene to produce the enzyme phenylalanine hydroxylase
o Abnormal Urinary Constituent: Phenylalanine, Phenyllactic acid, Phenylpyruvic acid
o Can be detected early
 If detected, dietary changes eliminate phenylalanine (major constituent of milk)
to prevent buildup of serum phenylalanine
 Aspartame – contains large amount of phenylalanine
 Undetected → Mental Retardation
o Phenylketonuria → Urinary Excretion of Phenylpyruvic acid
o Blood should be collected 24 hours after birth
 Cut-off level for normal results became 4mg/dL to 2 mg/dL = presence of PKU
 May occur mostly on boys
o Urine testing with Ferric Chloride – follow-up test
 Also to detect Phenylpyruvic acid = Blue-green color
 Tyrosyluria
o Degradation products – p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid
o Transitory tyrosinemia (in premature infants) – underdevelopment of the liver function
required to produce the enzymes necessary to complete the tyrosine metabolism
 Tyrosine and Leucine crystals may be seen in the microscope
o Hereditary Disorders of Tyrosinemia
 Type 1 – deficiency of enzyme Fumarylacetoacetate hydrolase (FAH)
 Produces a generalized renal tubular disorder and progressive liver
failure in infants soon after birth
 Type 2 – deficiency of enzyme Tyrosine aminotransferase
 Crystallization of tyrosine in the cells = Corneal Erosion and Lesions
 Type 3 – deficiency of enzyme p-hydroxyphenylpyruvic acid dioxygenase
 Mental retardation
o Nitroso-Naphthol Test – test for tyrosine
 Reagents: Nitric Acid, Sodium Nitrite, 1-nitroso-2-napthol
 Positive result: Orange-red color
 Melanuria
o Second metabolic pathway of tyrosine
 Produces: Melanine, thyroxine, epinephrine, protein, and tyrosine sulfates
o Melanin – pigment responsible for the dark color of hair, skin, and eyes
 Albinism – absence of melanin
o ↑Urinary Melanin = Proliferation of melanocytes → Malignant Melanoma
 Malignant Melanoma – secretes 5,6-dihydroxyindole → Melanogen → Melanin
o Melanin + Sodium Nitroferricyanide (acetone reagent strip) → Red Color
 Chapter 8 | Metabolic Disease

 Alkaptonuria
o Described by Garrod (1902)
o Urine darkens standing at room temperature; alkaptonuria; “alkali lover”
o Deficiency of enzyme Homogentisic acid oxidase
 Homogentisic acid may accumulate in the absence of this enzyme
 Brown- or black-stained cloth diapers and reddish-stained disposable diapers
 In later life, brown pigment deposits in body tissues (particularly in ears)
 Deposits in cartilage → Arthritis
o May develop cardiac and liver disorders
o Tests for Homogentisic Acid:
 Ferric Chloride Test → Deep Blue
 Clinitest → Yellow precipitate
 Add alkali to urine → Darkening of the color
 Interference: Ascorbic Acid
 Paper and thin layer chromatography – to quantitate homogentisic acid
 Silver Nitrate Test
 Reagents: Silver Nitrate, NH₄OH
 Positive Result: Black Color

BRANCHED-CHAIN AMINO ACID DISORDERS

 Amino acid with methyl group (CH3)

 2 Major Group of Disorder:
o Accumulation of early amino acid degradation products (seen in Maple Syrup Urine
disease)
o Accumulation of organic acids (organic acidemias)
 Presence of Ketonuria in a newborn
1. Maple Syrup Urine Disease
o Tested by MS/MS; caused by IEM
o Amino acids involved: Leucine, Isoleucine, Valine
 Seen in liver
 Transaminated into keto acids (α-ketoisovaleric, α-ketoisocaproic, α-keto-β-
methylvaleric)
 Maple Syrup Urine Disease odor = accumulation of keto acids in the urine
 Other clinical manifestations: Failure to thrive, Mental Retardation
o Test: 2,4-dinitrophenylhydrazine (DNPH) test
 Positive result: Yellow turbidity or precipitate
2. Organic Acidemias
o Symptoms: Severe illness, Hypoglycemia, Ketonuria, ↑serum ammonia
o Disorders: Isovaleric, Propionic, and Methylmalonic acidemia
 Isovaleric acidemia – “sweaty feet” odor – accumulation of isovalerylglycine –
deficiency of isovalerylcoenzyme A in the Leucine pathway
 Chapter 8 | Metabolic Disease

 Propionic and Methylmalonic acidemia – errors in the metabolic pathway

converting isoleucine, valine, threonine, methionine to succinyl coenzyme A
 Propionic acid – immediate precursor to methylmalonic acid
 Clinical Manifestations: Early severe illness, vomiting, metabolic
acidosis, hypoglycemia, ↑serum ammonia, ketonuria

TRYPTOPHAN DISORDERS

 Indicanuria
o Normal condition: Tryptophan is either reabsorbed or converted to Indole for excretion
o Disorders: Obstruction, Abnormal Bacteria, Malabsorption syndrome, Hartnup disease
(↑amount of Tryptophan are converted to Indole) (inherited intestinal disorder)
 Indole (reabsorbed from Intestine into bloodstream) → Liver → Indican →
excreted in the urine (colorless) → exposure to air → Indigo blue
 Early diagnosis of Hartnup disease – “blue diaper syndrome”
 Hartnup disease → Fanconi Syndrome (generalized aminoaciduria)
 Good prognosis – Proper diet supplements (ex. Niacin)
 5-hydroxyindoleacetic Acid
o Metabolic Pathway – used in production of Serotonin – stimulation of smooth muscles
 Serotonin – produced from tryptophan by the argentaffin cells in the cells and is
carried through the body primarily by platelets
 Serotonin – major constituent of food (bananas, pineapples, tomatoes)
 Serotonin → 5-HIAA
 Carcinoid tumors involving argentaffin (enterochromaffin) cells → ↑Serotonin
→ ↑5-HIAA
 Clinical Manifestations: Vascular and Bronchospastic syndrome (carcinoid)
o Test: Silver Nitroprusside Test
 Specimen: Random or First Morning Specimen
 24-hour Urine – must be preserved with boric or hydrochloric acid
 Reagents: Nitrous acid, 1-nitroso-2-napthol
 Positive result: Purple-black
 Interference: Medications (including phenothiazines and acetanilids)
o Normal excretion: 2-8 mg
 >25mg/24 hour = Argentaffin cell tumor

CYSTINE DISORDERS

 Cystinuria
o Inherited; defect in the renal tubular transport of amino acids
o ↑amount of Cystine in the urine = Cystine is not reabsorbed
o 2 Modes of Inheritance:
 Cystine, Lysine, Arginine, Ornithine are not reabsorbed
 Cystine, Lysine are not reabsorbed
 Chapter 8 | Metabolic Disease

o Disorder: Renal Calculi

o Test: Cyanide-Nitroprusside Test
 Specimen: First Morning Specimen
 Reagent: NH₄OH, Sodium Cyanide, Sodium Nitroprusside
 Postivive Result: Red-purple
 False Positive: Presence of Ketones and Homocystine
 Cystinosis
o Genuine IEM
o 2 Categories:
 Nephropathic
 Infantile cystinosis – rapid progression to renal failure
o Findings: Polyuria, generalized aminoaciduria, positive Clinitest,
lack of urinary concentration
 Late-onset cystinosis – gradual progression to renal failure
 Nonnephropathic – may cause ocular disorders
o Defect in Lysosomal Membrane → Prevents release of cystine → Crystal deposits (in the
Cornea, Bone Marrow, Lymph nodes, and Internal Organs) → Affects PCT → Defect in
renal tubular absorption (Fanconi Syndrome)
 Homocystinuria
o Defect in the metabolism of Methionine = ↑Homocystine = Cataracts, Mental
Retardation, Thromboembolic problems, death
o Screening for homocystine is included in newborn screening programs
o Etiology: Cystathionine β-synthase deficiency
o Test: Silver Nitroprusside
 Reagents: NH₄OH , Silver Nitrate, Sodium Nitroprusside
 Positive Result: Red-purple

PORPHYRIN DISORDERS

 Porphyrin – intermediate compounds in the production of heme – may be IEM

 Primary porphyrins – Uroporphyrin, Coproporphyrin, Protoporphyrin
 Precursors – α-amminolevulinic acid (ALA) and Phorphobilinogen
 ALA, Phorphobilinogen, Uroporphyrin – appears in urine
 Coproporphyrin and Protophorphyrin – seen in feces
 Bile – specimen used to avoid false-positive interference
 Analysis of whole blood – to detect free erythrocytes protoporphyrin (FEP) – screening test for
lead poisoning
 Porphyrias – disorders of porphyrin metabolism
o Acquired: Lead poisoning, excessive alcohol intake, iron deficiency, chronic liver disease,
renal disease
o
 Chapter 8 | Metabolic Disease

Common Porphyrias
Porphyria Elevated Compounds Clinical Symptoms Laboratory Testing
Acute Intermittent ALA Porphobilinogen Neurologic/Psychiatric Urine/Ehrlich reaction
Porphyria
Porphyria cutanea Uroporphyrin Photosensitivity Urine Fluorescence
tarda
Congenital Uroporphyrin Photosensitivity Urine or Feces
Erythropoietic Coproporphyrin fluorescence
Porphyria
Varigata Porphyria Coproporphyrin Photosensitivity/Neurologic Bile or Feces
fluorescence
Erythropoietic Protoporphyrin Photosensitivity Blood FEP
Protoporphyria Bile or Feces
Fluorescence
Lead Poisoning ALA Neurologic Acetoacetic acid +
Protoporphyrin Urine/Ehrlich reaction
Blood FEP
 Porphyrinuria – red or port wine urine color after exposure to air
o Staining of the teeth
 Screening Test:
o Ehrlich Reaction – to detect ALA and phorpobilinogen
 Acetyl acetone – to convert ALA to phorpobilinogen
o Fluorescence under ultraviolet light in the 550- to 600-nm range
 Uses Glacial Acetic Acid and Ethyl Acetate
 Negative: Faint blue fluorescence
 Positive: Violet, pink, or red fluorescence
 Presence of interfering substance → remove organic layer → add 0.5 mL HCl
 Acid layer (Porphyrins) – produces bright orange-red fluorescence
 Watson-Schwartz test – differentiates the presence of urobilinogen and porphobilinogen
 Hoesch Screening Test – for porphobilinogen
o Positive: Top of the solution = Red color

MUCOPOLYSACCHARIDE DISORDERS

 Mucopolysaccharides/Glycosaminoglycans – located in the connective tissue - IEM

 Products found in the urine: Dermatan sulfate, Keratan sulfate, Heparan sulfate
 Mucopolysaccharidoses
o Hurler Syndrome – accumulates in the cornea of the eye
 Skeletal structure is abnormal and there is severe mental retardation
o Hunter Syndrome – sex-linked recessive
 Skeletal structure is abnormal and there is severe mental retardation
o Sanfilippo Syndrome – mental retardation
 Bone Marrow Transplants and Gene Therapy - treatment
 Chapter 8 | Metabolic Disease

 Screening test: Acid-Albumin and Cetyltrimethylammonium bromide (CTAB) turbidity test &
Metachromatic Staining Spot Test
o Positive: White Turbidity

PURINE DISORDERS

 Lesch-Nyhan disease – massive excretion of urinary uric acid crystals

o Patients suffer from severe motor defects, mental retardation, tendency toward self-
destruction, gout, and renal calculi
 Deficiency of enzyme Hypoxanthine Guanine Phosphoribosyltransferase
 Resembles orange sand in diapers

CARBOHYDRATE DISORDERS

 Melituria - ↑urinary sugar - IEM

o Pentosuria – one of the Garrod’s six original IEMs
o Galactosuria – deficiency of galactose-1-phosphate uridyl transferase (GALT),
Galactokinase, UDP-galactose-4-epimerase
 GALT deficiency – causes the severe symptoms associated with galactosemia
o Lactosuria – seen during pregnancy and lactation
o Fructosuria – associated with parenteral feeding

Test Disorder Observation

Ferric Chloride Tube Test Phenylketonuria and 5-HIAA Blue-green
Tyrosinuria Transient Green
Alkaptonuria Transient Blue
Melanuria Gray-Black
MSUD Green-Gray
Indicanuria Violet-Blue with CHCl₃
2,4-Dinitrophenylhydrazine Phenylketonuria, MSUD, Yellow
Tyrosinuria, Acidemias
Phenistix Phenylketonuria Green-Gray
Tyrosinuria Transient Green
Acetest Melanuria Red
MSUD, Acidemias Purple
Nitrosonaphthol Tyrosinuria, MSUD Red
5-HIAA Violet with HNO₃
Cyanide Nitroprusside Cystine disorders Red-Purple
Silver Nitroprusside Alkaptonuria Black
Homocystinuria Red-Purple
Clinitest Cystinosis, Melituria Orange-Red
Sodium Nitroprusside Melanuria Red
p-nitroaniline Methylmalonic acidemia Emerald Green
CTAB Test Mucopolysaccharidoses White Turbidity
Metachromatic Staining Mucopolysaccharidoses Blue Spot
Guthrie Test Phenylketonuria Growth of B. subtilis
 Chapter 8 | Metabolic Disease

Pathway of Heme Formation

Normal Pathway Enzyme affected Stage affected
1. Glycine and Succinyl-CoA Aminolevulinic acid synthetase
2. γ-Aminolevulinate (ALA) Aminolevulinic acid synthetase ALA Hydratase deificiency
(Lead exposure) porphyria
3. Porphobilinogen Uroporphyrinogen synthase Acute Intermittent porphyria
4. Hydroxymethylbilane Uroporphyrinogen cosynthase Congenital erythropoietic
porphyria
5. Uroporphyrinogen (UPG) Uroporphyrinogen Porphyria cutanea tarda
decarboxylase
6. Coproporphyrinogen Coproporphyrinogen oxidase Hereditary coproporphyria
(CPG)
7. Protoporphyrinogen Protoporphyrinogen oxidase Variate porphyria
8. Protoporphyrin IX Ferrochelatase (Lead exposure) Erythropoietic protoporphyria
9. Heme
 Chapter 9 | Cerebrospinal Fluid

FORMATION AND PHYSIOLOGY

 Meninges – lines the brain and the spinal cord

o 3 layers:
 Dura meter – outer layer; lines the skull and vertebral canal
 Arachnoid – filamentous (spider-like) inner membrane
 Subarachnoid (30%) – between the arachnoid and pia meter
 Pia meter – lines the surface of the brain and spinal cord; neural tissue
 CSF – produced in the choroid plexuses of the two lumbar ventricles and the third and fourth
ventricles; discovered by Cotugno; acts as cushion fluid and mechanical barrier
o Adult = 20 mL/hr; 500 mL/day; 0.2-0.7 mL/minute
o Reabsorbed in the arachnoid granulations/villae = to maintain 90-150 mL for adults and
10-60 mL for children
 Prevents reflux of the fluid; One-way valve
 Choroid Plexuses (70%) – capillary networks that forms the CSF through selective filtration
o Also forms the CSF through active transport secretion of choroidal epithelial cells
o Endothelial cells – have tight-fitting junctures; prevents passage of many molecules,
prevents entry of foreign bodies, maintains homeostasis
 “Blood-brain barrier” (BBB)
 Disorders: Meningitis and Multiple Sclerosis (Ig)
 2 Components:
o Choroid Plexus – lined by choroidal epithelial cells which
connect with each other; with fenestrated capillary
o Capillary endothelial cell – held together by Intercellular Tight
Junction

SPECIMEN COLLECTION AND HANDLING

 Collected by lumbar puncture, cisternal puncture (occipital bone), or ventricular puncture

 Collected in 3 sterile tubes (up to 20 mL)
o Tube 1 – for chemical and serologic tests
o Tube 2 – for microbiology laboratory
o Tube 3 – for cell count; hematology
o A 4th tube may be drawn for microbiology to exclude skin contamination or for
additional serologic tests
 Excess fluid shouldn’t be discarded; should be frozen
 STAT basis
o If not possible, Tube 3 are refrigerated; Tube 2 remains at room temp; Tube 1 are frozen
 Chapter 9 | Cerebrospinal Fluid

APPEARANCE

 Xanthochromia – used to describe CSF supernatant that is pink, orange, or yellow

o RBC degradation/lysis – most common factor
 Pink – small amount of oxyhemoglobin
 Orange – heavy hemolysis; Hypervitaminosis A (carotenoids)
 Yellow – ↑unconjugated bilirubin; CSF Protein >150 mg/dL
 Brown – Meningeal Metastatic Melanoma
 Red-Orange - Medications

Clinical Significance of CSF appearance

Appearance Cause Major Significance
Crystal Clear/Colorless Normal
Hazy, Turbid, Milky, Cloudy WBCs (cloudy) Meningitis
Microorganisms Meningitis
Protein (or Lipids) Disorders affecting BBB
Production of IgG within CNS
Oily Radiographic Contrast Media Non-pathologic
Bloody/Hemolyzed RBCs Hemorrhage
Traumatic Tap (non-pathologic)
Xantochromic Hemoglobin Old Hemmorhage
Lysed cells from traumatic tap
Bilirubin RBC degradation
Elevated Serum Bilirbuin level
Carotene ↑serum levels, non-pathologic
Protein Disorders affecting BBB
Melanin Meningeal melanosarcoma
Clotted Protein Disorders affecting BBB
Clotting Factors Introduced by traumatic tap
Pellicle Protein Disorders affecting BBB
Clotting Factors Tubercular meningitis

TRAUMATIC COLLECTION (Tap)

 3 Examinations to determine whether the blood resulted from hemorrhage or traumatic tap
o Uneven Blood Distribution (Traumatic tap)
 Traumatic = Heavy contamination on Tube 1; Streaks of blood may be seen
o Clot Formation
 Traumatic = Fibrinogen clot; Intracranial Hemorrhage = No fibrinogen clot
 Froin Syndrome – clot formation but no bloody fluid
o Xanthochromic Supernatant (Intracranial Hemorrhage)
 Intracranial Hemorrhage = macrophages containing ingested RBCs
(Erythrophages) or hemosiderin granules
 D-dimer by latex agglutination – fibrin formation = Traumatic
 Chapter 9 | Cerebrospinal Fluid
CELL COUNT

 Leukocyte (WBC) count – routinely performed – four corners

 Normal adult CSF = 0-5 WBCs/microliter; children = 30
o 200-400 WBCs/microliter = Clear (undiluted)
 CSF WBC count = cells/microliter =
 WBC Count
o Diluent - 3% Glacial Acetic Acid – lyses the RBC
o Methylene Blue – stains WBC
Clarity Dilution Amount of Sample Amount of Diluent
Slightly Hazy 1:10 270 microliter
Hazy 1:20 30 microliter 570 microliter
Slightly Cloudy 1:100 2,970 microliter
Slightly Bloody 1:200 5,970 microliter
Cloudy, Bloody, Turbid 1:10,000 1 mL of 1:100 dilution 9 mL

 RBC Count (if bloody specimen)

o RBC (CSF) = Total # of Cell – WBC (CSF)
o WBC added =
 Dilution =

 WBC (Blood) =
 RBC (Blood) = Number of RBCs x Dilution x Depth Correction Factor x Area Correction Factor
[ ]
 CSF Protein Added =
o Hct [SI unit] – multiply by .01

DIFFERENTIAL COUNT ON A CSF SPECIMEN

 Performed on a stain smear

 Count 100 cells
 Methods for specimen concentration
o Sedimentation and Filtration – not routinely performed
o Centrifugation – 5-10 minutes; Wright’s stain
o Cytocentrifugation
 Albumin – ↑cell yield and ↓cellular distortion
 Cellular distortion: Cytoplasmic vacuoles, nuclear clefting, prominent
nucleoli, indistinct nuclear and cytoplasmic borders, cellular clumping
that resembles malignancy

 Daily control slide for bacteria: 0.2 mL Saline + 30% Albumin
 Chapter 9 | Cerebrospinal Fluid

Cytocentrifuge Recovery Chart

Number of WBCs counted in chamber Number of Cells on Cytocentrifuge slide
0 0-40
1-5 20-100
6-10 60-150
11-20 150-250
20 250

 CSF Cellular Constituents

o Adult Lymphocyte-Monocyte Ratio = 70:30 (children = 30:70)
o Pleocytosis (↑leukocyte, monocyte, neutrophil) = abnormal
 Also abnormal = findings of immature leukocytes, eosinophils, plasma cells,
macrophages, ↑tissue cells, malignant cells
1. Neutrophils
o Contains Cytoplasmic Vacuoles after Cytocentrifugation; loses granules
o May increase following CNS hemorrhage, repeated lumbar punctures, injection of
medications or radiographic dye
o Reference Value
 <8 – neonates
 <6 – adults
o Pyknotic nuclei = degenerating cells
 Resembles Nucleated Red Blood Cells (nRBCs) (1%)
o Seen as a result of bone marrow contamination during spinal tap
2. Lymphocytes and Monocytes
o Reactive lymphocytes - ↑dark blue cytoplasm and clumped chromatin
3. Eosinophils
o ↑eosinophils = parasitic infections, fungal infections (primarily Coccidioides
immitis), and introduction of foreign material into CNS
o Intracranial Shunt malfunctions
4. Macrophages
o Purpose in CSF: Remove cellular debris and foreign objects
o Appear within 2-4 hours after RBC enters CSF and after repeated taps
o More cytoplasm than monocyte
5. Nonpathologically Significant Cells
 Appear in clusters
 Seen after diagnostic procedures such as pneumoencephalography and
neurosurgery
o Choroidal Cells – from the lining of epithelial lining of choroid plexus
 Seen singularly and in clumps
 No nucleoli, nuclei has uniform appearance
o Ependymal Cells – from the lining of the ventricles and neural canal
 Less defined cell membrane and in clusters; has nucleoli
 Chapter 9 | Cerebrospinal Fluid

o Spindle-shaped Cells – lining cells from the arachnoid

 In clusters; seen with systemic malignancies
6. Malignant Cells of Hematologic Origin
o Lymphoblast, Myeloblast, Monoblast
o Prominent nucleoli
7. Malignant Cells of Nonhematologic Origin
o Cells from primary CNS tumors: Astrocytomas, Retinoblastomas, Medulloblastomas

Predominant Cells seen in CSF

Type of Cell Major Clinical Significance Microscopic Findings
Lymphocytes & Monocytes Normal All stages of development may
Viral, tubercular, fungal be found (Lymphocytes)
meningitis Found mixed with lymphocytes
Multiple Sclerosis (Monocytes)
HIV and AIDS (Lymphocytes)
Neutrophils Bacterial Meningitis Granules may be less prominent
Early cases of viral, tubercular, than in blood
fungal meningitis Cells disintegrate rapidly
Cerebral Hemorrhage
Macrophages RBCs in spinal fluid May contain phagocytized RBCs
appearing as empty
vacuoles/ghost cells, dark blue
or black iron-containing
hemosiderin granules, and
yellow hematoidin crystals
Blast forms Acute Leukemia Lymphoblasts, Myeloblasts,
Monoblasts
Lymphoma Cells Disseminated Lymphomas Resemble lymphocytes with cleft
nuclei
Plasma Cells Multiple Sclerosis Traditional & Classic forms seen
Lymphocyte Reactions Reactive Lymphs
Ependymal, Choroidal, and Diagnostic Procedures Seen in clusters with distinct
Spindle-shaped cells nuclei and distinct cell walls
Malignant Cells Metastatic Carcinomas Seen in clusters with fusing of
Primary CNS carcinoma cell borders and nuclei

CHEMISTRY TESTS

 Filtration of Plasma → CSF

1. Cerebrospinal Protein
o Most frequently performed chemical test; Method dependent
o Reference Value: 15-45 mg/dL, method dependent
 Higher Values in infants and people over age 40
o Albumin – makes up most of the CSF protein
 Prealbumin – second most prevalent fraction in CSF
 Chapter 9 | Cerebrospinal Fluid

 α-globulins – includes haptoglobin, ceruloplasmin, and α2-macroglobulin

 Transferrin – major β constituent
1. “tau” – carbohydrate-deficient transferrin fraction – seen in CSF; not in
serum
 γ-globulins – IgG (+ small amount of IgA)
o Not present in significant amoutns: Fibrinogen, IgM, β-lipoprotein
o Cause of ↑CSF protein: Damaged BBB (↑Albumin), Ig production within CNS, ↓normal
protein clearance, neural tissue degeneration
o ↓CSF Protein = CSF Leakage
Clinical Causes of Abnormal CSF Protein Values
Elevated Results Decreased Results
Meningitis* Myxedema CSF leakage/trauma
Hemorrhage* Cushing Disease Recent puncture
Primary CNS tumors Connective tissue disease Rapid CSF production
Multiple Sclerosis* Polyneuritis Water intoxication
Guillain-Barre syndrome Diabetes
Neurosyphilis Uremia
Polyneuritis
*Most significant

o Methods:
 Turbidimetry (Nephelometry) – precipitating agents: Benzethonium or
Benzalkonium Chloride
 Dye-binding techniques – uses Coomassie brilliant blue or Ponceau S
 Colorimetric-Spectrophotometric methods – based on Lowry or Biuret method
 Immunoassays – used to measure the amount of specific proteins (ex. MBP)
o Calculations: CSF/serum albumin index = ; CSF (mg/dL) and Serum (g/dL)
 <9 = intact BBB

IgG Index = *>0.70 = IgG production in CNS

o Electrophoresis and Immunophoretic Techniques

 To detect oligoclonal bands – represents inflammation in the CNS
 Located in the gamma region, indicates Ig production
 Presence of 2 or more in the CSF = Multiple Sclerosis (↑IgG)
 For low protein levels – CSF immunofixation electrophoresis (IFE) and Isoelectric
Focusing (IEF) followed by Silver Staining
o Myelin Basic Protein (MBP) – indicates destruction of Myelin Sheath; demyelination
 Myelin Sheath – protects the axons of the neurons
 Immunoassay = Measurement of MBP = Monitors Multiple Sclerosis
2. CSF Glucose
o Reference Value: 60-70%
 If plasma glucose is 100 mg/dL, CSF glucose reference value is 65 mg/dL
 Chapter 9 | Cerebrospinal Fluid

o Blood Glucose should be drawn out 2 hours before spinal tap for equilibration
o ↓CSF Glucose+ ↑WBC count (neutrophils) = Bacterial Meningitis
o ↓CSF Glucose+ ↑WBC count (lymphocytes) = Tubercular Meningitis
o Normal CSF Glucose + ↑Lymphocytes = Viral Meningitis
3. CSF Lactate
o Reference Value: 10-24 mg/dL
 >35 mg/dL = Bacterial Meningitis
o Hypoxia (oxygen deprivation) = ↑CSF lactic acid
o False↑: Xanthochromic and Hemolyzed fluid
4. CSF Glutamine
o Reference Value: 8-18 mg/dL
 >35 mg/dL = Some disturbance of consciousness
o Glutamine – produced from ammonia and α-ketoglutarate by the brain cells
 To remove toxic waste ammonia from CNS
 ↑ammonia = ↓α-ketoglutarate = Coma
o ↑Glutamine = Liver disorders, Reye’s Syndrome
5. Enzymes
o CK-BB isoenzyme – increases in about 6 hours following ischemic or anoxic insult
o LD - ↑ in leukemia, lymphoma, metastatic carcinoma, bacterial meningitis,
subarachnoid hemorrhage
o ADA - ↑ in tubercular meningitis (abundant in T lymphocytes)
o Lysozyme - ↑ in bacterial and tubercular meningitis

MICROBIOLOGY TESTS

 To identify causative agent in meningitis

 CSF culture – confirmatory
 Gram Stain
o Should be performed on concentrated specimens
o Centrifuge for 15 minutes at 1,500g
o Blood cultures should be taken (because the causative agent is often present in both
CSF and Blood)
o False positive: Precipitated stain, Debris
o Bacteria detected: Streptococcus pneumonia, Haemophilus influenzae, Escherichia
coli, Neisseria meningitides
 Streptococcus agalactiae and Listeria monocytogenes – may be encountered
in newborns
 Acid-Fast or Fluorescent Antibody Stain
o Not routinely performed unless tubercular meningitis is suspected
 India Ink Preparation
 Chapter 9 | Cerebrospinal Fluid

o To detect Crytpococcus neoformans (starburst pattern) – occurring in AIDS

 Latex Agglutination – to detect C. neoformans antigen in serum and CSF
o False-positive: Rheumatoid Factor
o Pretreatment: Incubation with DTT or Pronase, Boiling with EDTA
o Enzyme Immunoassay – produces fewer false-positive results
 Lateral Flow Assay (LAF) – rapid method for detecting C. neoformans
o Uses a reagent strip coated with monoclonal antibodies that reacts with the
polysaccharide capsule of C. neoformans
 Enzyme-Linked Immunosorbent Assay (ELISA) – another rapid method of detection
 Bacterial Antigen Test (BAT) – should be used in combination with results from hematology
and clinical chemistry for diagnosing meningitis
 Naegleria fowleri – opportunistic parasite found in ponds, small lakes, and chlorinated
swimming pools
o Enters Nasal passages and migrates along the olfactory nerves to invade the brain
o Wet Preparation – Motile Trophozoites
o Cytocentrifuged specimen (+ ↑WBC and no bacteria) – Nonmotile Trophozoites

Major Laboratory Results for Differential Diagnosis of Meningitis

Bacterial Viral Tubercular Fungal
WBC count Elevated Elevated Elevated Elevated
WBC Neutrophil Lymphocytes Lymphocytes and Lymphocytes and
Monocytes Monocytes
Protein count Marked ↑ Moderate↑ Moderate-Marked Moderate-Marked
↑ ↑
Glucose level Marked ↓ Normal ↓ Normal-↓
Lactate level >35 mg/dL Normal >25 mg/dL >25 mg/dL
Positive Gram Pellicle Formation Positive India Ink
Stain and BAT with C.
neoformans
Positive
immunologic test
for C. neoformans

SEROLOGIC TESTING

 To detect presence of neurosyphilis

o Penicillin – to reduce neurosyphilis
 Recommended Diagnosis – Venereal Disease Reasearch Laboratories (VDRL)
o More sensitive – Fluorescent Treponemal Antibody-Absorption (FTA-ABS)
o Less sensitive – Rapid Plasma Regain (RPR)
 Chapter 10 | Synovial Fluid

PHYSIOLOGY

 Synovial Fluid – “Joint Fluid”

o Viscous liquid found in the cavities of the movable (diarthroses) or synovial joints
o Bones:
 Lined with smooth articular cartilage
 Separated by a cavity containing the synovial fluid
 Smooth articular cartilage + Synovial Fluid = Reduce friction between
the bones during joint movement
o The joint is enclosed in a fibrous joint capsule lined by the synovial membrane
 Synovial membrane – contains specialized cells – Synoviocytes
 Synoviocytes – secretes a mucopolysaccharide (containing Hyaluronic
Acid + small amount of proteins[1/4]) into the fluid
o Hyaluronic Acid – gives viscosity
o Provides lubrication in the joints
o Provides nutrient to the (vascular-deficient) articular cartilage and lessens the shock of
joint compression
o Ultrafiltrate of plasma across the synovial membrane
 Filtration = Nonselective (except for the exclusion of high-molecular-weight
proteins = Have chemical concentrations similar to plasma
o Arthritis – damage to the articular membranes → pain and stiffness to the joints
o Tests: WBC count, Differential count, Gram Stain, Culture, Crystal Examination

Normal Synovial Fluid Values

Volume <3.5 mL
Color Colorless to Pale Yellow
Clarity Clear
Viscosity Able to form a string 4-6 cm long
Leukocyte count <200 cells per microliter
Neutrophils <25% of the differential
Crystals None present
Glucose:Plasma difference <10 mg/dL lower than blood glucose level
Total Protein <3 g/dL
 Chapter 10 | Synovial Fluid

Classification of Joint Disorders

Group Classification Pathologic Significance Laboratory Findings
I. Non-inflammatory Degenerative Joint Disorders, Clear, Yellow Fluid
Osteoarthritis Good Viscosity
WBCs <1000 microliter
Neutrophils <30%
Similar to Blood Glucose
II. Inflammatory Immunologic Disorders, Cloudy, Yellow Fluid
(Immunologic origin) Rheumatoid arthritis, Systemic Poor Viscosity
Lupus Erythematosus, WBCs 2000-75000 microliter
Scleroderma, Polymyositis, Neutrophils >50%
Ankylosing Spondylitis, ↓ Glucose Level
Rheumatic Fever, Lyme arthritis Possible autoantibodies present
Inflammatory Gout, pseudogout Cloudy or Milky Fluid
(Crystal-induced origin) Low Viscosity
WBC up to 100,000 microliter
Neutrophils <70%
↓ Glucose Level
Crystals present
III. Septic Microbial infection Cloudy, Yellow-Green Fluid
Variable Viscosity
WBCs 50,000-100,000 microliter
Neutrophils >75%
↓ Glucose Level
Positive Culture and Gram Stain
IV. Hemorrhagic Traumatic injury, tumors, Cloudy, Red Fluid
hemophilia, Other coagulation Low Viscosity
disorders, anticoagulant WBCs equal to blood
overdose Neutrophils <50%
Normal Glucose Level

SPECIMEN COLLECTION AND HANDLING

 Collected by Needle Aspiration (Arthrocentesis)

o Collected in a syringe moistened with heparin
o Amount varies with size of joints and the extent of fluid buildup
 Normal Fluid = doesn’t clot; Diseased joint – may contain fibrinogen and will clot
 Powdered Anticoagulants – may produce artifacts; may interfere with crystal analysis
o Crystal analysis ≠ Refrigeration; (may produce additional crystals)

Required Tube for Synovial Fluid Tests

Synovial Fluid Test Required Tube Type
Gram Stain and Culture Sterile Heparinized/Sodium Polyanethol Sulfonate
Cell Counts Heparin/Liquid EDTA
Glucose Analysis Sodium Fluoride
All other tests Nonanticoagulated (must be centrifuged)
 Chapter 10 | Synovial Fluid
COLOR AND CLARITY

 “Synovial” – came from the Latin word for egg, ovum

o Resembles egg white
 Noninflammatory/Inflammatory effusions – Deep Yellow
 Septic Arthritis/Bacterial Infection – Green Tinge
 Pathologic Hemarthrosis (bleeding in the joints) – Red-brown
 Presence of blood from a hemorrhagic arthritis must be distinguished from blood from a
traumatic aspiration
o Observe uneven blood distribution or a single streak
 Tubidity = WBC, synovial cell debris, fibrin, rice bodies, metal and plastic particles from patients
with joint prostheses, cartilage fragments in osteoarthritis
 Milky [and opalescent] = Crystals present
 Ground Pepper appearance = Pigmented cartilage fragments resulting from a metabolic disorder

VISCOSITY

 From polymerization of the hyaluronic acid and is essential for the proper joints lubrication
 To measure – Falling Drop – ability to from a string 4-6 cm long
 Arthritis – affects hyaluronate production and its ability to polymerize = ↓fluid viscosity
 Test: Ropes/Mucin clot Test – not routinely performed
o Synovial Fluid + 2-5% Acetic Acid = Solid Clot surrounded by clear fluid
 ↓Polymerization = Clot becomes less firm + ↑Turbidity of surrounding fluid
o Manner of Reporting: Good (solid clot), Fair (soft clot), Low (friable clot), and Poor (no
clot)

CELL COUNT

 WBC count – most performed

 If fluid is very viscous, add 1 drop of 0.05% hyaluronidase in phosphate buffer per milliliter
 Clear fluid – can be counted undiluted; Turbid/Bloody Fluid – should be diluted first
 WBC diluting fluid – cannot be used – contains acetic acid – formation of mucin clots
 Diluent: Normal Saline, or 0.3% Hypotonic Saline with Saponin – to lyse RBC
o With Methylene Blue – stains the WBC nuclei
 Technique: Line the petri dish with moist paper and place the hemocytometer on 2 small sticks
 Counting procedure:
o <200 WBCs/microliter, count all 9 large squares
o >200 WBCs/microliter (above count), count the 4 corner squares
o >200 WBCs/microliter (above count), count the 5 small squares used for RBC count
 Interference: Highly viscous fluid, debris and tissue cells (false↑)
 <200 WBCs/microliter = Normal; <2,000 RBCs/microliter = Normal
 >100,000 = Severe infections (Crystal-induced/Chronic Inflammatory/Septic arthritis)
 Chapter 10 | Synovial Fluid

DIFFERENTIAL COUNT

 Performed on Thin Smear; incubated with hyaluronidase; cytocentrifugation

 Mononuclear Cells (Monocytes, Macrophages, Synovial Tissue Cells) – primary cells seen
 Normal Count: Neutrophil (<25%) and Lymphocyte (<15%), Macrophage (<65%)

Cells and Inclusions seen in Synovial Fluid

Cell/Inclusion Description Significance
Neutrophil Polymorphonuclear Leukocyte Bacterial Sepsis
Crystal-induced inflammation
Lymphocyte Mononuclear leukocyte Nonseptic inflammation
Macrophage (monocyte) Large Mononuclear leukocyte, Normal
may be vacuolated Viral infections
Synovial Lining Cell Similar to Macrophage, but may Normal
be multinucleated, resembling a Disruption from arthrocentesis
mesothelial cell
LE cell Neutrophil containing Lupus erythematosus
characteristic ingested “round
body”
Reiter cell (Neutrophage) Vacuolated Macrophage with Reactive Arthritis (infection in
ingested Neutrophil another part of the body)
RA cell (ragocyte) Neutrophil with dark cytoplasmic Rheumatoid Arthritis
granules containing immune Immunologic inflammation
complexes
Cartilage cells Large, multinucleated cells Osteoarthritis
Rice bodies Macroscopically resembles Tuberculosis
Polished Rice Septic and Rheumatoid Arthritis
Microscopically show Collagen
and Fibrin
Fat droplets Refractile intracellular and Traumatic Injury
extracellular globules Chronic inflammation
Stain with Sudan dyes
Hemosiderin Inclusions within clusters of Pigmented villonodular synovitis
synovial cells

CRYSTAL IDENTIFICATION

 Causes of Crystal Formation: Metabolic Disorders; ↓Renal Excretion → ↑blood levels of

crystallizing chemicals; Degeneration of cartilage and bone; Injection of Medications into a joint
 Causes of Gout:
o ↑Serum Uric Acid resulting from impaired metabolism of purines
o ↑consumption of high-purine-content foods, alcohol, and fructose
o Chemotherapy treatment of leukemias (destruction of cells → ↑Uric Acid)
o ↓Renal excretion of uric acid
 Chapter 10 | Synovial Fluid

 Pseudogout – most often associated with degenerative arthritis, producing cartilage calcification
and endocrine disorders that produce ↑serum calcium levels
 Interference: Artifacts – Talcum powder and starch from gloves, precipitated anticoagulants,
dust, scratches on slides and cover slip

Characteristics of Synovial Fluid Crystals

Crystal Shape Image Compensated Significance
Polarized Light
Monosodium Needles Negative Gout
Urate (MSU) birefringence

Calcium Rhomboid Positive Pseduogout

Pyrophosphate Square, [short] birefringence
dehydrate (CPPD) rods
Cholesterol Notched, Negative Extracellular,
Rhomboid plates birefringence Chronic
inflammation
Corticosteroid Flat, variable- Postive and Injections
shpaed plates Negative
birefringence
Calcium Oxalate Envelopes Negative Renal dialysis
birefringence

[Hydroxy] Apatite Small particles No Osteoarthritis;

(calcium Require electron birefringence Calcified Cartilage
phosphate) microscopy degeneration

 Slide Preparation
o Fluid is examined as an unstained wet preparation
o Under LPO and HPO; may be observed with Wright’s-stained smears
o MSU Crystals – may be extracellular or located within the cytoplasm of neutrophils
o CPPD Crystals – usually located within vacuoles of neutrophils
 Rhomboid shape – observed in Red Compensated Polarized Microscopy
 Crystal Polarization
o Direct Polarization → Red-Compensated Polarized Light (for confirmation) →
Prepare control slide with Betamethasone Acetate Corticosteroid (for MSU)
o MSU and CPPD – may polarize light
 MSU – more highly birefringent; appears brighter against dark background
o Red Compensator – placed in microscope between crystal and analyzer
 Separates light ray into slow-moving and fast-moving vibrations
 Produces red background
o Identification of Crystals = Colors produced by each crystals when it is aligned with
the slow vibration
 MSU color – yellow color; slow light; parallel to the long axis of crystals
 Chapter 10 | Synovial Fluid

 Negative birefringence – subtraction of velocity from the fast ray

 CPPD color – blue color; fast light; perpendicular to the long axis of crystals

CHEMISTRY TEST

 Glucose Determination - most frequently performed test

o Indicate inflammatory (group II) or septic (group III) disorders
o Sample: fasted for 8 hours
o Normal synovial fluid glucose should not be more than 10 mg/dL lower than the blood
value

Clinical Significance of Synovial Fluid Chemistry Results

Test Normal Values Clinical significance
Glucose 70-110 mg/dL ↓ in Inflammatory and Septic Arthritis
Total Protein <3 g/dL ↑ in Inflammatory and Hemorrhagic Arthritis
Lactate <30 mg/dL ↑ in Septic Arthritis
Uric Acid 2-8 mg/dL ↑ in Gouty Arthritis
Hyaluronidase – Normal Value: 0.3-0.4 g/dL

MICROBIOLOGIC TEST

 Gram Stain and Culture – most performed – to diagnose Septic Arthritis

o PCR can also be used
 Chocolate agar – enrichment medium
 Bacteria seen: Staphylococcus, Streptococcus, Haemophilus, Neisseria gonorrhoeae

SEROLOGIC TEST

 Synovial fluid analysis – confirmatory

 Rheumatoid Arthritis and Systemic Lupus Erythematosus – diagnosed in the serology laboratory
by detecting an autoantibody
 Arthritis – frequent complication of Lyme disease (through Tick Bites)
 Borrelia burgdorferi – causes arthritis
 Chapter 11 | Semen

 Semen
o 4 Fractions contributed by the testes
 Epididymis, Seminal Vesicles, Prostate Gland, Bulbourethral glands
 Testes (paired glands in the scrotum)
o Contain Seminiferous Tubules - for the secretion of sperm; production of spermatozoa
(in the epithelial cells of seminiferous tubules)
 Sertoli cells – provides support and nutrient for the germ cells as they undergo
mitosis and meiosis (spermatogenesis)
 Epididymis (where immature sperms [non-motile] enter)
o Where the sperm matures and develops flagella; 90 days; storage
o Stored until ejaculation to Ductus deferens (vas deferens) to the ejaculatory ducts
 Ejaculatory Ducts
o Receives both the sperm from the ductus deferens and fluid from the seminal vesicles
 Seminal Vesicles – transport medium for the sperm; 60-70%
 Fluid: fructose and flavin
o Fructose – metabolized for the motility of sperm as it propels
through the female reproductive tract
o Flavin – Gray appearance of semen
 Prostate Gland (below the bladder)
o Surrounds the upper urethra
o Aids in propelling the sperm through the urethra by contractions during ejaculation
o Produces acidic fluid (20-30%)
 Contains acid phosphatase, citric acid, zinc, proteolytic enzymes
 Responsible for both the coagulation and liquefaction of the semen following
ejaculation
 Bulbourethral gland (5%)
o Neutralization; alkaline mucus will neutralize acidity from the prostate secretions
 No neutralization = sperm motility would be diminished

SPECIMEN COLLECTION

 Most of the sperm are contained in the first portion of the ejaculate
o Without first part = false↑ pH, will not liquefy
o Without last part = ↓volume, false↑ sperm count, false↓ pH, no clot
 Complete collection – accurate testing for fertility and postvasectomy
 Sexual abstinence of at least 2 days and not more than 7 days
o Prolonged abstinence = ↑volume, ↓motility
o Fertility testing – two or three samples collected not less than 7 days or more than 3
weeks apart
 2 abnormal samples = significant
 Chapter 11 | Semen

 Warm sterile glass or plastic containers

 Should be kept at room temperature and delivered to the laboratory within 1 hour of collection
o Storage - 37°C
 Should be collected by masturbation; if not possible, Silastic or Nonlubricant-containing rubber
or polyurethane condoms
 Not reliable:
o Ordinary Condoms – has spermicides
o Coitus interruptus – first portion is ejaculated; ↓pH
 Handling
o All are potential reservoirs for HIV and hepatitis viruses
o Discarded as biohazard waste

SEMEN ANALYSIS

 Appearance
o Gray-white color, translucent, musty odor
o Low concentration = clear
o White turbidity = presence of WBCs; infection within the reproductive tract
 WBCs – must be differentiated with immature sperm (spermatids)
 Leukocyte Esterase reagent strip – to screen for the presence of WBCs
o Red color = presence of RBCs, bleeding
o Yellow color = contamination, prolonged abstinence, medications, pyospermia
o Urine is toxic to sperm
 Liquefaction
o Fresh specimen (clotted) should liquefy within 30-60 minutes after collection
 >60 minutes = prostatic enzyme deficiency
 >2 hours – add Dulbecco’s phosphate buffered saline or proteolytic enzymes
such as alpha-chymotrypsin or bromelain
 Contents: Potassium Chloride, Potassium dihydrogen phosphate,
Magnesium Chloride Hexahydrate, Sodium Chloride, Disodium
Hydrogen phosphate heptahydrate, D-glucose
 Calcium Chloride dehydrate – to prevent precipitation
 Bromelain – Jelly-like granules (gelatinous bodies) present; insignificant
o Interference: Mucus Strands
 Volume
o Normal range = 2-5 mL
o Measurement = graduated cylinder (0.1-mL increments)
o ↑volume = abstinence
o ↓volume = infertility; improper functioning of seminal vesicles
 Viscosity – consistency of the fluid
 Chapter 11 | Semen

o Incomplete liquefaction = clumped and highly viscous

o Normal = Small discrete droplets; do not appear clumped or stringy
 >2cm = highly viscous = abnormal
o ↑viscosity – affects sperm motility, concentration, antisperm antibody detection,
measurement of biochemical markers
o Ratings: 0 (watery) – 4 (gel-like); low, normal, high
 pH
o Normal = 7.2 to 8.0
o Should be measured within 1 hour; prolonged time = loss of CO₂
o ↑pH = infection within reproductive tract
o ↓pH = increased prostatic fluid, ejaculatory duct obstruction, poorly developed seminal
vesicles
 Sperm Concentration and Sperm Count
o Reference value: >20 to 250 million sperm per milliliter
 10-20 million – borderline
o Normal ejaculated sperm count: >40 million per ejaculate
 Total Ejaculated Sperm Count = (Sperm concentration) x (volume)
o Neubauer Counting Chamber – dilutes then counts the specimen
 Dilution – 1:20 prepared using a mechanical (positive-displacement) pipette
 Immobilizes the sperm before counting
 Contains Sodium Bicarbonate and Formalin (preserves the cell)
 Saline and distilled water can also be used
o Counted in four corners using Neubauer hemocytometer
 Both side of hemocytometer are loaded and allowed to settle for 3-5 minutes
 Charge: Both sides (up and down)
 Performed using Phase or bright-field microscopy (enhancement: Crystal Violet)
 Only count fully developed sperms; spermatids + WBC (round cells) are not
counted
 >1 million WBC = Inflammation/Infection of reproductive organs
 >1 million spermatids = Disruption of spermatogenesis
 9 Square with WBC at the 4 sides and RBC at the center
 RBC (middle) – has a 5x5 square (with each square containing 16 sq.)
 Sperm Motility
o Should be assessed using a liquefied specimen within 1 hour of specimen collection
o Evaluate 20 high-power fields OR 200 sperms per slide; evaluate speed and direction
 At room temperature or 37°C
o Normal = 50% with 2.0 rating after 1 hour
o Computer-assisted semen analysis (CASA) – determination of both sperm velocity and
trajectory (direction of motion)
 Also analyzes Sperm Concentration and Morphology
 Chapter 11 | Semen

Sperm Motility Grading Alternative Sperm Motility Grading Criteria

Grade WHO criteria Sperm Motility Action
4.0 A Rapid, straight-line Progressive Sperm moving linearly or
motility Motility (PM) in a large circle
3.0 B Slower speed, some Nonprogressive Sperm moving with an
lateral movement Motility (NP) absence of progression
2.0 B Slow forward Immotility (IM) No movement
progression, noticeable
lateral movement
1.0 C No forward progression
0 D No movement

 Sperm Morphology
o Normal = Oval-shaped (Head: 5 micrometer; Tail: 45 micrometers)
o Structures:
 Head – has acrosomal cap (for ovum penetration) at the tip that covers half of
the head and 2/3 of the nucleus
 Neckpiece – attaches head to the tail and the midpiece
 Midpiece (7 micrometers) – thickest part of the tail
 Surrounded by Mitochondrial sheath (produces energy for motility)
 Tail – for motility
o Evaluation: Thin smear (10 microliters) and oil immersion; 200 sperms
 Stains: Wright’s, Giemsa, Papanicolou, Shorr
 Air-dried slides – stable for 24 hours
 Abnormalities:
 Head – double heads, giant and amorphous heads, pinheads, tapered
heads, constricted heads
 Tail – doubled, coiled, bent
 Neckpiece – may cause the head to bend and interfere with motility
o Kruger’s Criteria: >30% normal forms (routine criteria); >14% normal (strict criteria)
 Use Morphometry or Stage Micrometer
o Calculating Round Cells (Spermatids and WBCs)
 Peroxidase-positive granulocytes – predominant form of leukocyte in semen
 Formula:

ADDITIONAL TESTING

Additional Testing for Abnormal Semen Analysis

Abnormal Result Possible abnormality Test
↓Motility with Normal Count Vitality, Necrospermia Eosin-nigrosin stain
↓Count Lack of seminal vesicle support Fructose level
medium
↓Motility with Clumping Male antisperm antibodies Mixed agglutination reaction and
 Chapter 11 | Semen

immunobead tests
Sperm agglutination with male
serum
Normal analysis with continued Female antisperm antibodies Sperm agglutination with female
infertility serum

 Sperm Vitality
o ↓Sperm Vitality = Normal Sperm Concentration + ↓Motility
o Eosin-negrosin stain → Smear →Count dead cells in 100 sperm
 Living cells – Bluish White
 Dead cells – Red against purple background
o Bright-field or Phase-contrast microscopy
o Normal: 50% or more living cells
o Immotile and nonviable = epididymal pathology
 Seminal Fluid Fructose
o ↓Sperm Concentration = ↓Fructose level
 Abnormalities of the seminal vesicles, bilateral congenital absence of the vas
deferens, obstruction of ejaculatory duct, partial retrograde ejaculation,
androgen deficiency
o Test: Resorcinol Test (Orange-Red Color)
 Should be tested within 2 hours
o Normal: 13 millimoles per ejaculate or greater
 Spectrophotometric Methods
 Antisperm Antibodies
o Male antisperm antibodies (clumps) – frequently encountered over women
o Blood-testes barrier – separates sperm from the male immune system
 Damaged barrier → from Surgery, Vasectomy Reversal (vasovasostomy),
trauma, infection → Damages the sperm → Produce antibodies in female
o Sperm-agglutinating antibodies (male) – cause sperm to stick to each other
 Reported as few, moderate, or many in microscopic examination
o Tests to detect:
 Mixed Agglutination Reaction (MAR) – detects IgG
 Incubated with IgG antihuman globulin (AHG) and a suspension of latex
particles or treated RBCs coated with IgG
 Normal: <10%
 Immunobead test – detects IgG, IgM, IgA and show structures being affected
o Head-directed antibodies – interfere with penetration
o Tail-directed antibodies – interfere with movement
 Sperm + Polyacrylamide beads coated with IgG/M/A
 Normal: <50%

 Chapter 11 | Semen

 Microbial and Chemical Testing

o To test for: To test for: Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma
urealyticum
o ↓ α-glucosidase, free L-carnithine, glycerophosphocholine – disorder in epididymis
o ↓zinc, citric acid, glutamyl transpeptidase, acid phosphatase – lack of prostatic fluid
o May be used to determine presence of semen in cases of alleged rape
 Motile Sperms – 24 hours
 Nonmotile sperms – 3 days
 Head – 7 days
 Seminal fluid = ↑Prostatic Acid Phosphatase = Detected
 Seminal glycoprotein p30 (Prostatic Specific Antigen [PSA]) – present even in the
absence of sperm

Reference Semen Chemical Values

Neutral α-glucosidase ˃20 mU/ejaculate
Zinc ˃2.4 mU/ejaculate
Citric Acid ˃52 micromol/ejaculate
Acid Phosphatase ˃200 units/ejaculate

 Postvasectomy Semen Analysis

o Only concern is the presence or absence of spermatozoa
o Routinely tested at monthly intervals, beginning at 2 months until 2 consecutive months
show no spermatozoa
o Wet preparation using Phase microscopy; cuts Vas Deferens
 Sperm Function Test

Sperm Function Tests

Test Description
Hamster Egg Penetration Sperm are incubated with species-nonspecific
hamster eggs and is observed microscopically;
Ovum Penetration
Cervical Mucus Penetration Observation of sperm’s ability to penetrate
partner’s midcycle cervical mucus
Hypo-osmotic Penetration Sperm exposed to low-sodium concentrations
are evaluated for membrane integrity and sperm
viability
In vitro acrosome reaction Evaluation of the acrosome to produce enzymes
essential for ovum penetration
 Chapter 11 | Semen

Calculation:

 WBC
o Long Method
 = (WBC/microliter) x 1,000 = WBC/mL

o Short Method
 (Cell Count) x (100,000) = Sperm Concentration (millions/mL)
o Total Ejaculated Sperm Count = (Sperm Concentration) x (Sperm Volume)

 RBC
o Long Method
 = (RBC/microliter) x 1,000 = RBC/mL

o Short Method
 (Cell count) x (1,000,000) = Sperm Concentration (millions/mL)
o Total Ejaculated Sperm Count = (Sperm Concentration) x (Sperm Volume)

 Dilution
o


 Chapter 12 | Serous Fluid

 Serous Fluid
o Fluid found between the parietal membrane (lines the cavity wall) and the visceral
membrane (covers the organ)
o Provides lubrication between the 2 membranes

FORMATION

 Ultrafiltrates of plasma
 Hydrostatic Pressure and Colloidal Pressure (Oncotic Pressure) – serve the cavities and capillary
permeability
o Hydrostatic Pressure – causes fluid to enter between membranes
o Filtration of plasma ultrafiltrate = ↑Oncotic Pressure (reabsorption) = Maintains the
normal volume of fluid between the membranes
 Effusion – disruption of fluid formation and reabsorption = ↑Fluid between membranes

Pathologic Causes of Effusions

↑Capillary Hydrostatic ↓Oncotic Pressure ↑Capillary Permeability Lymphatic
Pressure Obstruction
Congestive Heart Failure Nephrotic Syndrome Microbial Infections Malignant tumors,
Salt and Fluid retention Hepatic Cirrhosis Membrane inflammations lymphomas
Malnutrition Malignancy Infection and
Protein-losing Inflammation
enteropathy Thoracic Duct Injury

SPECIMEN COLLECTION AND HANDLING

 Collected by Needle Aspiration (>100mL)

o Thoracentesis (Pleural)
o Pericardiocentesis (Pericardial)
o Paracentesis (Peritoneal)
 EDTA tube – used for cell and differential count
 Sterile Heparinized or Sodium Polyanethol Sulfonate (SPS) – used for microbiology and cytology
 Centrifugation – recovery of microorganisms and abnormal cells
 Plain or Heparin tubes – Chemistry test – on clotted specimens
 Specimens for pH – must be maintained anaerobically on ice

TRANSUDATES AND EXUDATES

 Transudate – effusions that form because of a systemic disorder that disrupts the balance in the
regulation of fluid filtration and reabsorption (ex. Changes in Hydrostatic and Oncotic Pressure)
 Exudate – produced by conditions that directly involve the membranes of the particular cavity
(ex. Infection and Malignancies)
 Chapter 12 | Serous Fluid

Laboratory Differentiation of Transudates and Exudates

Transudate Exudate
Appearance Clear Cloudy
Fluid: Serum Protein ratio <0.5 >0.5
Fluid: Serum LD ratio <0.6 >0.6
WBC count <1000/uL >1000/uL
Spontaneous Clotting No Possible
Pleural Fluid Cholesterol <45 to 60 mg/dL >45 to 60 mg/dL
Pleural Fluid: Serum Cholesterol <0.3 >0.3
ratio
Pleural Fluid: Bilirubin ratio <0.6 >0.6
Serum-ascites albumin gradient >1.1 <1.1
pH; SG; LDH Alkaline; <1.015; <200 IU/L Acidic; >1.015; >200 UI/L
Glucose As in plasma level Lower than plasma level
Total Protein <3 g/dL >3 g/dL

GENERAL LABORATORY PROCEDURES

 Microbiology and Cytology – for examination of effusions from exudative origin

 RBC and WBC count – not routinely performed
o >1000 WBC/uL and >100,000 RBC/uL = exudate
 Neubauer Counting Chamber – counts serous fluid cells
 Manual Cell count – includes nucleated cells
 Differential Count – Wright’s stained, cytocentrifuged specimen

PLEURAL FLUID

 Located between the parietal pleural membrane lining the chest wall and the visceral pleural
membrane covering the lungs

Correlation of Pleural Fluid Appearance and Disease

Appearance Disorder
Clear, pale yellow Normal
Turbid, white Presence of WBC
Microbial Infection (tuberculosis)
Rheumatoid Arthritis
Bloody Hemothorax (traumatic injury) (fluid Hct >50%; uneven/streaked distribution)
Hemorrhagic effusion (<50%; even), pulmonary embolus, tuberculosis,
malignancy
Milky Chylous and Pseudochylous material
Brown Rupture of amoebic liver abscess
Black Aspergillus
Viscous Malignant Mesothelioma (↑Hyaluronic Acid)
 Chapter 12 | Serous Fluid

Chylous Effusion Pseudochylous Effusion

Cause Thoracic Duct Damage Chronic Inflammation
Lymphatic Obstruction
Appearance Milky/White Milky/Green Tinge
Leukocytes Predominantly Lymphocytes Mixed Cells
Cholesterol Crystals Absent Present
Triglycerides >110 mg/dL <50 mg/dL
Sudan III staining Strongly Positive Negative/Weakly Positive

Significance of Cells seen in Pleural Fluid

Cells Significance
Macrophages (64-80%)
Neutrophils (1-2%) Bacterial Infection (Pneumonia)
Pancreatitis
Pulmonary infarction
Lymphocytes (18-30%) Tuberculosis, Viral Infection, Malignancy,
Autoimmune Disorders (RA and SLE)
Eosinophils Trauma, Allergic reactions, Parasitic Infections
(>10%)
Mesothelial Cells No Clinical Significance (Normal &Reactive forms)
Tuberculosis (↓Mesothelial cells)
Pneumonia and Malignancy (↑Mesothelial cells)
Plasma Cells Tuberculosis
Malignant Cells Primary adenocarcinoma and Small-cell carcinoma
Metastatic Carcinoma
 Mesothelial Cells
o Pleiomorphic; resembles Lymphocytes, Plasma Cells, and Malignant Cells
o Single small or large round cells with abundant blue cytoplasm and round nuclei with
uniform dark purple cytoplasm – Normal Form
o Clusters; have varying amount of cytoplasm, eccentric nuclei, and prominent nucleoli;
multinucleated, resembling malignant cells – Reactive Form

Characteristics of Malignant Cells

Increased Nucleus:Cytoplasm (N:C) ratio. ↑Ratio = Poorly differentiated
Irregularly distributed nuclear chromatin
Variation is size and shape of nuclei
Increased number and size of nucleoli
Hyperchromatic nucleoli
Giant cells and multinucleation
Nuclear molding
Cytoplasmic molding (community borders)
Vacuolated Cytoplasm, Mucin production
Cellular crowding, phagocytosis
 Chapter 12 | Serous Fluid

Significance of Chemical Testing of Pleural Fluid

Test Significance
Glucose ↓ in Rheumatoid inflammation, Purulent
infection, Tuberculosis = <60 mg/dL
Lactate ↑ in Bacterial infection
Triglyceride ↑ in Chylous Effusions
pH ↓ in Pneumonia not responding to antibiotics =
<7.2; need for chest-tube drainage
Mark ↓ with Esophageal Rupture = 6.0
Adenosine Deaminase (ADA) ↑ in Tuberculosis and Malignancy = >40 U/L
Amylase ↑ in Pancreatitis, Esophageal Rupture, Malignancy
 Microorganisms associated with pleural effusions
o Staphylococcus aureus, Enterobacteriaceae, anaerobes, Mycobacterium tuberculosis
o Gram stain, Culture, Acid Fast stain – performed
 Serologic Testing – to differentiate effusions of immunologic and noninflammatory
o Test for Antinuclear antibody (ANA) and Rheumatoid Factor (RF) – performed
 Tumor markers – detected – for malignant origins
o Carcinoembryonic Antigen (CEA), CA 125 (Metastatic Uterine Cancer), CA 15.3 and CA
549 (Breast Cancer), CYFRA 21-1 (lung cancer)

PERICARDIAL FLUID

 Only small amount is found between the pericardial serous membranes; 10-50 mL
 Infection (Pericarditis), Malignancy, Trauma-producing exudates → Changes in Membrane
permeability → Pericardial Effusions
 Metabolic Disorders (Uremia, Hypothyroidism, Autoimmune disorders) → Transudates
 Cardiac Compression (Tamponade) – suspects for effusion

Significance of Pericardial Fluid Testing

Test Significance
Appearance
Clear, pale yellow Normal, transudate
Turbid Infection, Malignancy
Blood-Streaked Infection, Malignancy
Grossly Bloody Cardiac Puncture, Anticoagulant Medications
Milky Chylous and Pseudochylous Material
Additional Testing
Increased Neutrophils Bacterial Endocarditis
Malignant Cells Metastatic Carcinoma
Carcinoembryonic Antigen Metastatic Carcinoma
Gram Stain and Culture Bacterial Endocarditis (Streptococcus,
Staphylococcus, adenovirus, coxsackievirus)
Acid-Fast stain Tubercular Effusion (AIDS)
Adenosine Deaminase Tubercular Effusion (AIDS)
 Chapter 12 | Serous Fluid

PERITONEAL FLUID

 Ascites/Ascitic fluid – fluid between peritoneal membranes

 Hepatic Disorders (Cirrhosis) → Ascitic Transudates
 Serum-ascites Albumin Gradient (SAAG) – to detect transudates of hepatic origin
o Recommended over the Fluid: Serum total protein and LD ratio

Significance of Peritoneal Fluid Testing

Test Significance
Appearance
Clear, pale yellow Normal
Turbid Microbial Infection
Green/Dark Brown Bile, gallbladder, pancreatic disorders
Blood-Streaked Trauma, tuberculosis, infection, malignancy
Milky (Chylous and Pseudochylous present) Lymphatic trauma and blockage
Peritoneal Lavage >100,000 RBCs/uL = Blunt Trauma Injury
WBC count
<500/uL Normal
>500/uL Bacterial peritonitis, cirrhosis
Differential Bacterial peritonitis, malignancy
Carcinoembryonic Antigen Malignancy of Gastrointestinal origin
CA 125 Malignancy of Ovarian origin
Glucose Decreased in Tubercular peritonitis, malignancy
Amylase Increased in Pancreatitis, Gastrointestinal
perforation
Alkaline Phosphatase Increased in Gastrointestinal perforation
Blood Urea Nitrogen/Creatinine Ruptured or punctured bladder
Gram Stain and Culture Bacterial Peritonitis
Acid-fast Stain Tubercular Peritonitis
Adenosine Deaminase Tubercular Peritonitis

 Neutrophil count - >250 cells/uL or >50% of the total WBC count = Infection/Bacterial Peronitis
 Lymphocytes – predominant cell in tuberculosis
 Other cells present: Leukocytes, Abundant Mesothelial Cells, and Macrophages (including
Lipophages)
 Microorganisms present: Bacteria, Yeast, Toxoplasma gondii
 Psammoma bodies – contains concentric striations of collagen-like material
o Seen in benign conditions; associated with ovarian and thyroid malignancies
 Chapter 13 | Amniotic Fluid

 Amniotic Fluid
o Product of fetal metabolism

PHYSIOLOGY

 Present in the Amnion – a membranous sac that surrounds the fetus

o Involved in the exchanges of water and chemicals between the fluid, the fetus, and the
maternal circulation
o Produces peptides, growth factors, and cytokines
 Provides a protective cushion for the fetus, Allow fetal movement, Stabilize the temperature to
protect the fetus from extreme temperature changes, and Permit proper lung development

 Volume is regulated by a balance between production of fetal urine and lung fluid and the
absorption from fetal swallowing and intramembranous flow
o Intramembranous flow – absorption of amniotic fluid water and solutes into the fetal
vascular system
 Pregnancy (3rd trimester) = 800-1,200 mL (Peak) → decreases prior to delivery
o >1,200 mL = Polyhydramnios
 Indication of Fetal Distress, often associated with Neural Tube Disorder
 Secondarily associated with Fetal Structural Anomalies, Cardiac Arrhythmias,
Congenital infections, Chromosomal abnormalities
o <800 mL = Oligohydramnios
 Causes: ↑Fetal Swallowing, Urinary Tract deformities, Membrane leakages
 May be associated with Congenital Malformations, Premature rupture of
amniotic membranes, Umbilical Cord compression → decelerated heart rate →
fecal death
 st
1 trimester – 35 mL of amniotic fluid is derived primarily from the maternal circulation
o Latter third to half of pregnancy – fetus secretes lung liquid to expand the lungs with
growth
o Used to bath/wash the lungs, pulmonary and alveolar content (during each episode of
fetal respiratory movement): Lecithin, Sphingomyelin, Phosphatidyl glycerol = Index of
Fetal Lung Maturity
 st
After 1 trimester, Fetal Urine = major contributor to the amniotic fluid volume
o Fetal urine production → Fetal swallowing of amniotic fluid – regulates the increase in
fluid from fetal urine
o Fetal Swallowing = Fetus → Swallows Amniotic fluid → Absorbed through GIT →
Reexcreted by the Kidneys from the blood into fetal urine → Back to Amniotic fluid
 Chapter 13 | Amniotic Fluid

 Placenta – ultimate source of amniotic fluid water and solutes

 Contains small amount of sloughed fetal cells from skin, digestive system, urinary tract
o Provide basis for Cytogenetic analysis
 Fluid has biochemical substances (bilirubin, lipids, enzymes, electrolytes, urea, creatinine, uric
acid, proteins, hormones) – tested to determine the health and maturity of fetus
 Neural Tube Defect = allows fetal CSF to enter amniotic fluid
o Biochemical Markers: Alpha-fetoprotein and Acetylcholinesterase
 Fetal Urine Production = ↑Creatinine, Urea, Uric Acid; ↓Glucose and Protein
o Before 36 weeks’ gestation, Creatinine = 1.5-2.0 mg/dL
o After 36 weeks’ gestation, Creatinine = >2.0 mg/dL

 Needed to analyze for the differentiation of amniotic fluid from maternal urine (To determine
possible premature membrane rupture or accidental puncture of the maternal bladder)
o Creatinine – lower in amniotic fluid than in maternal urine
 In amniotic fluid, <3.5 mg/dL
 In urine, 10 mg/dL
o Urea – lower in amniotic fluid than in maternal urine
 In amniotic fluid, <30 mg/dL
 In urine, 300 mg/dL
o Glucose
o Protein
 Fern test – to differentiate amniotic fluid from other body fluids
o Used to evaluate premature rupture of the membranes
o Vaginal Fluid on Slide → Positive: “fern-like” crystals present due to protein and NaCl

Tests for Fetal Well-being and Maturity

Test Reference Values at term Significance
Bilirubin Scan ΔA450 >.025 Hemolytic disease of the Newborn
Alpha-fetoprotein <2.0 MoM Neural Tube Disorders
Lecithin-Sphingomyelin ratio >2.0 Fetal Lung Maturity
Amniostat-Fetal Lung Maturity Positive Fetal Lung Maturity/Phosphatidyl
Glycerol
Foam Stability Index >47 Fetal Lung Maturity
Optical Density 650nm >0.150 Fetal Lung Maturity
Lamellar Body Count >32,000/mL Fetal Lung Maturity

SPECIMEN COLLECTION

 Amniocentesis – recommended for neural tube defects

o Performed after the 14th week gestation
o Chromosome analysis – collection at 16 weeks’ gestation
 Chapter 13 | Amniotic Fluid

o Intrauterine Growth Retardation – near the end of 2nd trimester

o Fetal Distress and Maturity – performed in the 3rd trimester
 Ultrasonography – estimates the gestational age of the fetus
 Fetal Epithelial cells in amniotic fluid – indicate the genetic material of the fetus and the
biochemical substances the fetus has produced
o Examined for chromosome abnormalities by:
 Karyotyping
 Fluorescence in situ hybridization (FISH)
 Fluorescent mapping spectral karyotyping (SKY)
 DNA testing
o Biochemical substances – evaluated by thin-layer chromatography

INDICATIONS FOR PERFORMING AMNIOCENTESIS

Amniocentesis may be indicated at 15-18 weeks’ gestation for the following conditions:
 Mother’s age of 35 or older at delivery
 Family History of Chromosome abnormalities, such as Trisomy 21 (Down Syndrome)
 Parents carry an abnormal chromosome rearrangement
 Earlier pregnancy or child with birth defect
 Parent is a carrier of a metabolic disorder
 Family history of genetic diseases such as Sickle Cell disease, Tay-Sachs disease, Hemophilia,
Muscular dystrophy, Sickle Cell anemia, Huntington Chorea, Cystic Fibrosis
 Elevated maternal serum alpha-fetoprotein
 Abnormal triple marker screening test
 Previous child with a neural tube disorder such as Spina bifida, or Ventral wall defects
(Gastroschisis)
 Three or more miscarriages
Amniocentesis is indicated later in the pregnancy (20-42 weeks) to evaluate:
 Fetal Lung Maturity
 Fetal Distress
 HDN caused by Rh blood type incompatibility
 Infection

 Amniocentesis – collects amniotic fluid; Needle aspiration into amniotic sac

o Transabdominal amniocentesis – frequently performed
o Vaginal amniocentesis – may be performed but carries greater risk of infection
o 30 mL – collected in sterile syringes
 First 2 or 3 mL – may contaminated by Maternal Blood, Tissue Fluid, Cells;
discarded
 Transferred to a sterile plastic container
 Chapter 13 | Amniotic Fluid

SPECIMEN HANDLING AND PROCESSING

 Fluid for Fetal Lung Maturity (FLM) test – placed in ice for delivery and kept refrigerated
 Bilirubin – protected from light
o Placed in amber-colored tubes, wrapping the tube in foil, or by use of black plastic cover
 Specimen for Cytogenetic/Microbial studies – processed aseptically and maintained at room or
body temperature
 Centrifugation and Filtration – to separate fluid from cellular elements and debris

COLOR AND APPEARANCE

Amniotic Fluid Color

Color Significance
Colorless Normal (Turbid if cell debris present)
Blood-Streaked Traumatic Tap, Abdominal Trauma, Intra-amniotic hemorrhage
Yellow Hemolytic disease of the Newborn (bilirubin)
Dark Green Meconium; Fetal Distress
Dark Red-brown Fetal Death

 May exhibit slight to moderate turbidity (normal; from cellular debris)

 Meconium – newborn’s first bowel movement; formed in the intestine
 Kleihauer-Betke test – determines source of blood; test for fetal hemoglobin

TESTS FOR FETAL DISTRESS

 Hemolytic Disease of the Newborn

o Production of anti-Rh antibody in postpartum mothers
o Initial exposure to foreign red cell antigens occurs during:
 Gestation
 Delivery of the Placenta
 Previous pregnancy
o Antibodies enter fetal circulation through placenta → Bind to antigen on cells → Cells
are destroyed → Appearance of Unconjugated Bilirubin in amniotic fluid
 Amniotic fluid Bilirubin – measured by Spectrophotometric analysis using Serial
Dilutions
 Optical Density (OD) is measured in intervals (365-550 nm)
 Normal = OD is highest at 365 nm and decreases linearly at 550 nm
 Bilirubin present = a rise in OD at 450 nm
 ΔA450 (absorbance difference) - Plotted on a Liley graph
o Liley graph 3 zones:
 Zone I – mildly affected fetus
 Zone II – moderate hemolysis
 Zone III – severe hemolysis
 Chapter 13 | Amniotic Fluid

 Intervention through induction of labor or intrauterine exchange

transfusion must be considered
o Interference: Cells, hemoglobin, Meconium (false↓ΔA450), other debris, blood
 410 nm – maximum absorbance of oxyhemoglobin
 Chloroform – to remove interference
 Control - 1:10 saline
 Neural Tube Defects (NTD)
o To detect NTD:
 Maternal Serum Alpha-fetoprotein (MSAFP) blood test
 High-resolution ultrasound
 Amniocentesis
o ↑AFP = Neural Tube Defects (Anencephaly and Spina Bifida)
 AFP – major protein produced by fetal liver during early gestation (<18 weeks)
 Maximal at 12-15 weeks’ gestation
 Reported in terms of Multiples of the Median (MoM)
 Measurement of Amniotic Acetylcholinesterase (AChE) – confirmatory test
 Not performed on bloody specimen; blood contains AChE

TEST FOR FETAL MATURITY

 Fetal Lung Maturity (FLM)

o Respiratory Distress Syndrome (RDS) – most frequent complication of early delivery and
is the 7th most common cause of morbidity and mortality in the premature infant
 Caused by ↓Lung surfactant production and structural immaturity of the fetal
lungs
 Surfactant – allows alveoli (air sacs of lungs) to remain open; decreases
its surface tension
 Lecithin-Sphingomyelin (L/S) Ratio – reference method
o Lecithin – primary component of the surfactants (phospholipids, neutral lipids, proteins)
 Produced at a low constant rate until the 35th week of gestation
o Sphingomyelin – a lipid that serves as a control on which to base the rise in lecithin
 Produced at a constant rate after 26 weeks’ gestation
o After 35 weeks’ gestation, L/S ratio = <1.6 L=2.0 → Prevent alveolar collapse
o Interference (false↑): Fluid with blood, Meconium
o Thin-layer chromatography – quantitative measurement
 Phosphatidyl Glycerol (PG)
o Detected after 35 week’s gestation
o Production is delayed in maternal diabetes
o Amniostat-FLM test – uses antisera specific to phosphatidylglycerol
o Test: Immunologic Agglutination Test
 Reported as: Negative (Pulmonary Immaturity), Positive (Normal), Low/High
Positive (Pulmonary Maturity)
 Chapter 13 | Amniotic Fluid

 Foam Stability Index

o “Foam”/”Shake” test – mechanical screening test
 Amniotic Fluid + 95% Ethanol → Shake (15 minutes) → Stand (15 minutes)
 Result (normal): Continuous line of bubbles around the outside edge →
sufficient amount of Phospholipid (to reduce surface tension)
o Foam Stability Index
 Amniotic Fluid + increasing amounts of 95% Ethanol ranging from 0.42-0.55 mL
in 0.01-mL increments → Shake (15 minutes) → Stand (15 minutes)
 >47 = Fetal Lung Maturity
 Interference (false mature): Blood and Meconium
 Lamellar Bodies
o Surfactant (90% Phospholipid + 10% Protein) → Packaged into → Lamellar Bodies
(Layered Storage Granules)
o Secreted by type II pneumocytes of the fetal lung at about 24 weeks of gestation
o Enters amniotic fluid at 26 weeks of gestation; Concentration – from 50,000 to 200,000
per mL by the end of the 3rd trimester
o Lamellar Bodies amount = Fluid Phospholipid amount
o Presence = ↑OD (OD at 650nm)
 Specimens are centrifuged at 2,000g for 10 minutes, examined at 650 nm
 Principle: Spectrophotometry
o Diameter: 1.7-7.3 fL or 1-5 um; similar to small platelets
o Lamellar Body Counting (LBC)
 Can be obtained using the Platelet Channel of automated hematology analyzers
using either optical or impedance methods
o Interference: Amniotic fluid with whole blood, meconium, mucus
o Storage: 2-8°C; but not frozen
o Reporting: Lamellar bodies per microliter
 >50,000/uL LBCs = FLM
 <15,000/uL LBCs = Immature

 Microviscosity: Fluorescence Polarization Assay

o Measures the surfactant to albumin (S/A) ratio Albumin – internal standard
o Principle: Phospholipids decreases the microviscosity of the amniotic fluid
o Measure the polarization of a fluorescent dye that combined with both surfactants and
albumin in the amniotic fluid
 >55 mg/g = FLM
 40-54 mg/g = Indeterminate
 <39 mg/g = Immature lungs
o Interference: with blood, meconium, suspected maternal urine, icteric sample
 Chapter 14 | Fecal Analysis

PHYSIOLOGY

 Approximately 100 to 200 grams of feces is excreted in a 24-hour period

 Carbohydrates (Oligosaccharides) that are resistant to digestion pass through the upper
intestine unchanged but are metabolized by bacteria in the lower intestine → produce large
amount of flatus (strong odor)
o Excessive gas production = Lactose-intolerant people
 Alimentary Tract – where digestion of ingested protein, CHO, and fats takes place
 Small Intestine – primary site for the final breakdown and reabsorption of compounds
o Digestive Enzymes: From Pancreas – trypsin, chymotrypsin, amino peptidase, lipase
 Liver – digestion of fats
o Bile salts – aids in the digestion
 Ingestion:
o Food and Drink – 2000 mL
o Saliva – 1500 mL
o Gastric Secretions – 1500 mL
o Bile from liver – 1000 mL
o Pancreas – 1000 mL
o Intestinal Secretions (water and electrolytes) – 2000 mL
o Mucous Secretions – 200 mL
 Only 500-1500 mL of these ingestions reaches the large intestine
 150 mL is excreted in the feces
 Large Intestine – capable of absorbing approximately 3000 mL of water
 Constipation – provides time for additional water to be reabsorbed from the fecal material,
producing small, hard stools

DIARRHEA AND STEATORRHEA

Diarrhea

 Increased in daily stool weight above 200g, increased liquidity of stools, frequency of more than
3 three times a day
 4 Factors: Illness duration, Mechanism, Severity, Stool Characteristics
 <4 weeks = Acute Diarrhea; >4weeks = Chronic Diarrhea
 Major Mechanisms: To differentiate mechanisms:
o Secretory - fecal electrolytes (Na = 30; K = 75) (mmol/L)
o Osmotic - fecal osmolality (290 mOsm/kg)
o Intestinal Hypermotility - stool pH
 Osmotic Gap = 290 – [2 (fecal sodium + fecal potassium)]
 Chapter 14 | Fecal Analysis

1. Secretory Diarrhea
o Increased secretion of water
o Enterotoxin-producing organisms (E. coli, Clostridium, V. cholerae, Salmonella, Shigella,
Staphylococcus, Campylobacter, protozoa, Cryptosporidium) – can stimulate water and
electrolyte secretion
o Other causes: Drugs, Stimulant Laxatives, Hormones, Inflammatory Bowel Disease
(Crohn disease, Ulcerative colitis, Lymphocytic colitis, diverticulitis), Endocrine disorders
(hyperthyroidism, Zollinger-Ellison syndrome, VIPoma), neoplasms, and collagen
vascular disease
2. Osmotic Diarrhea
o Poor absorption that exerts osmotic pressure across the intestinal mucosa
o Water and electrolyte retention in the large intestine; excessive watery stool
o Maldigestion and Malabsorption contribute to osmotic diarrhea
o Causes: Disaccharide deficiency (lactose intolerance), Malabsorption (celiac sprue),
poorly absorbed sugars (lactose, sorbitol, mannitol), laxatives, magnesium-containing
antacids, amebiasis, antibiotic administration

Common Fecal Tests for Diarrhea

Secretory Osmotic
Stool Cultures Microscopic Fecal Fats
Ova and Parasite Examinations Muscle Fiber detection
Rotavirus immunoassay Qualitative/Qualitative Fecal Fats
Fecal Leukocytes Trypsin Screening
Clinitest
D-Xylose tolerance test
Lactose tolerance test
Fecal Electrolytes
Stool pH
Fecal Osmolality

Differential Features for Diarrhea

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

3. Altered Motility
o Enhanced (hypermotility) or slow (constipation) motility
i. Both are seen in Irritable Bowel Syndrome (IBS) – nerves and muscles of bowel
are sensitive
 Chapter 14 | Fecal Analysis

o Intestinal Hypermotility – excessive movement of intestinal contents through the GI

Tract – can be caused by enteritis, parasympathetic drugs, malabsorption
i. Rapid Gastric Emptying (RGE) dumping syndrome – causes the small intestine to
fill to quickly with undigested food from the stomach – hypermotility
 Hallmark of Early Dumping Syndrome (EDS)
 Healthy people – gastric emptying half-time of 35-100 minutes
a. <35 = RGE
b. Gastric Emptying – controlled by: Fundic Tone, Duodenal
feedback, GI hormones
 Early (10-30 minutes) and Late (2-3 hrs) dumping
 Hypoglycemia – often a complication of dumping
 Main cause of dumping syndrome: Gastrectomy, Gastric bypass surgery,
post vagotomy status, Zollinger-Ellison syndrome, duodenal ulcer
disease, and diabetes mellitus

Steatorrhea (fecal fat)

 Useful in detecting pancreatic insufficiency

 Absence of Bile Salts (assists pancreatic lipase) → ↑stool fat → Exceeds 6g per day
 Cystic Fibrosis, Chronic Pancreatitits, Carcinoma – decreases production of pancreatic enzymes
 D-Xylose test
o Low D-Xylose = Malabsorption
 Causes: Celiac disease, tropical sprue, lymphoma, Whipple disease, Giardia
lamblia infestation, Crohn disease, and intestinal ischemia

SPECIMEN COLLECTION

 Should not be contaminated with urine or toilet water

 Should not use containers with preservatives for ova and parasites
 Random specimen suitable for qualitative testing and microscopic examination
 Timed specimen suitable for quantitative testing
 3-day collection – most representative sample

MACROSCOPIC SCREENING

 Color – brown – oxidation of stercobilinogen to urobilin

Color/Appearance Possible Cause

Black Upper GI bleeding
Iron therapy
Charcoal
Bismuth (antacids)
Red Lower GI bleeding
 Chapter 14 | Fecal Analysis

Beets and Food coloring

Rifampin
Pale (acholic stools) yellow, white, gray Bile-duct obstruction
Barium Sulfate
Green Biliverdin/oral antibiotics
Green vegetables
Bulky/Frothy (foul odor) (may appear greasy and Bile-duct obstruction
may float) Pancreatic Disorders
Ribbon-like Intestinal constriction
Mucus- or blood-streaked mucus Colitis (mucus)
Crohn disease (mucus)
Colon tumors (mucus)
Dysentery (blood)
Malignancy (blood)
Constipation (blood)

MICROSCOPIC EXAMINATION OF FECES

1. Fecal Leukocytes
o Primarily Neutrophils
o Seen in the feces in conditions that affect the intestinal mucosa
o Examined as wet preparations stained with Methylene Blue
o Dry preparation with Wright’s or Gram stain
o 3 Neutrophils/HPF = Invasive condition
o Lactoferrin latex agglutination test – to detect fecal leukocytes
2. Muscle Fibers
o Can be useful in monitoring patients with pancreatic insufficiency
o ↑amount = Biliary obstruction and Gastrocolic fistulas
i. >10 (only undigested) = reported increased amount
o Emulsify with 10% alcoholic eosin (enhances muscle fiber striations)
i. Undigested fibers – visible striations
ii. Partially digested fibers – striations in one direction
iii. Digested fibers – no visible striations
o Red meat is needed to produce a sample
3. Qualitative Fecal Fats
o Can be seen in patients with steatorrhea
o Stained with Sudan III (routinely used), Sudan IV, or Oil Red O
i. Neutral Fats (triglycerides) – large orange-red droplets
 >60 droplets/hpf = steatorrhea
ii. Split Fat stain – total fat content
o Soap (fatty acid salts) and fatty acids – do not stain with Sudan III directly
i. Smear – heat and mix with acetic acid and Sudan III (stains cholesterol)
 100 orange-red droplets (6 to 75 mm) = malabsorption
 Chapter 14 | Fecal Analysis

CHEMICAL TESTING OF FECES

Occult Blood

 Detection of Occult Blood (Hidden Blood) – most frequently performed fecal analysis
 Bleeding in excess of 2.5 mL/150 g of stool = pathologically significant → Fecal Occult Blood
Tesing (FOBT) → Tested annually → high positive predictive value for detecting colorectal cancer

1. Guaiac-Based Fecal Occult Blood Tests (gFOBT)

o Most frequently used screening test for fecal blood
o Reaction: Hemoglobin + H₂O₂ + Guaiac (colorless) → (pseudoperoxidase) → Oxidized
Guaiac (Blue) + H₂O
2. Immunochemical Fecal Occult Blood Test (iFOBT)
o Specific for globin portion of hemoglobin
o Uses polyclonal anti-human hemoglobin antibodies
o More sensitive to lower GI bleeding
o Do not detect bleeding from other sources
3. Porphyrin-Based Fecal Occult Blood Test
o Measures both intact hemoglobin and the hemoglobin that has been converted to
porphyrins
o HemoQuant test – more sensitive to upper GI bleeding
i. Interference: Red Meat (false-positive)

gFOBT interference
False-Positive False-Negative
Aspirin and anti-inflammatory medications Vitamin C > 250 mg/d
Red Meat Iron supplements containing vitamin C
Horseradish Failure to wait specified time after sample is
Raw broccoli, cauliflower, radishes, turnips applied to add the developer agent
Melons
Menstrual and Hemorrhoid Contamination

Quantitative Fecal Fat Testing

 Confirmatory test for Steatorrhea

 3-day specimen → Must maintain regulated intake of fat (100 g/d)
 Van de Kamer titration – routinely used; gold standard
o Lipids → Fatty Acids → (titrated to neutral endpoint with NaOH)
 Hydrogen Nuclear Magnetic Resonance Spectroscopy method – more rapid (5 minutes);
microwave-dried specimen
 Coefficient of fat retention = (dietary fat – fecal fat)/(dietary fat) x 100
 Acid Steatocrit – rapid test to estimate the amount of fat excretion; hematocrit centrifuge,
gravimetric assay
 Chapter 14 | Fecal Analysis

 Near-infrared reflectance spectroscopy (NIRS) – rapid procedure for fecal fat that requires less
stool handling
o Scanned with infrared light between: 1400 nM and 2600 nM

APT Test (Fetal Hemoglobin)

 To distinguish between the presence of fetal blood or maternal blood in an infant’s stool
 Addition of 1% Sodium Hydroxide to Hemoglobin-containing emulsion determines presence of
fetal or maternal blood
o Pink Color – presence of Fetal Blood
o Yellow-brown supernatant – presence of Maternal Blood

Fecal Enzyme

 Chymotrypsin – capable of gelatin hydrolysis

 Trypsin Test – emulsified specimen placed on x-ray paper determines ability to digest gelatin
o Inability to digest = Lack of trypsin
 Elastase 1 – immunoassay using an ELISA test
o Sensitive indicator of exocrine pancreatic insufficiency

Carbohydrate

 ↑Carbohydrate = Osmotic Diarrhea

o Lowers stool pH into 5.5 (Normal stool pH = 7.0 to 8.0)
 Copper Reduction Test – can detect increased concentration of carbohydrates
o Addition of Clinitest Tablet to emulsified stool detects presence of reducing substance
 Reaction of 0.5 g/dL reducing substances suggests CHO intolerance
 Sucrose – not detected – not a reducing sugar
 Chapter 15 | Vaginal Secretions

 Vaginitis – characterized by abnormal vaginal discharge or odor, pruritus, vaginal irritation,

dysuria, dyspareunia
o Secondary to Bacterial Vaginosis (BV), vulvovaginal candidiasis, trichomoniasis
o Microscopic method
 Saline wet mount examination – initial screening test
 Potassium Hydroxide (KOH) examination – initial screening test
 Gram Stain – gold standard – confirmatory test
o Other tests: litmus pH levels, DNA probe testing, Culture, Point of Care test kits
 Tests are also performed to detect Placental α1-microglobulin (PAMG-1) protein – to diagnose
Ruptured fetal membranes, or fetal fibronectin enzyme to assess risk of preterm delivery

Clinical Features and Laboratory Findings in Vaginitis

Findings Bacterial Candidiasis Trichomoniasis Desquamative Atrophic
Vaginosis Inflammatory Vaginitis
Vaginitis
Appearance Thin, Homogenous, White, curd-like Yellow-Green Excessive purulent Excessive purulent
White-Gray Vaginal Vaginal discharge Frothy adherent vaginal discharge, vaginal discharge,
discharge vaginal discharge vaginal erythema vaginal erythema
increased in
volume
pH >4.5 3.8-4.5 >4.5 >4.5 >4.5
WBCs Rare/Absent 3+ to 4+ 2+ to 4+ 3+ to 4+ 3+ to 4+
Lactobacilli Rare/Absent Present Absent/Present Absent/Reduced Decreased
Clue cells >20% Absent Absent/Present
Other cells Large clumps Occasional Occasional
of epithelial parabasal/basal parabasal/basal
cells cells cells
>1+ RBCs >1+ RBCs
Other Increase in small Budding yeast Trichomonas 2+ gram-positive Increased gram-
organisms curved bacilli, cells and frequently cocci positive cocci and
coccobacilli, and pseudohyphae associated with gram-negative
pleiomorphic bacilli other organisms rods; decreased
large rods
Amine Positive Negative Positive Negative Negative
(Whiff)
Test
Other tests Confirmatory Tests: Confirmatory Confirmatory
DNA probe, Proline Tests: DNA probe, Tests: DNA Probe
amino peptidase, OSOM BVBLUE or Culture, OSOM
OSOM BVBLUE Rapid Rapid Test Trichomonas
Test, Affirm VPIII Rapid Test
 Chapter 15 | Vaginal Secretions

SPECIMEN COLLECTION AND HANDLING

 Speculum (moistened with warm water) – used to visualize the vaginal fornices
o Lubricants – shouldn’t be used
 Collected with one or more Sterile Polyester-tipped Swabs on the vaginal walls and vaginal pools
then place in a tube (with 0.5-1 mL of sterile physiologic saline)
o Cotton swabs shouldn’t be used
 Cotton – toxic to Neisseria gonorrhoeae
 Wood in shaft – toxic to Chlamydia trachomatis
 Calcium Alginate – can inactivate herpes simplex virus (HSV)
 Other preparation: Dilute with 1-2 drops of NSS, second sample is placed in 10% KOH solution
 Storage:
o Room Temperature - Trichomonas vaginalis and Neisseria gonorrhoeae
 T. vaginalis – should be examined within 2 hours
o Refrigerated – Chlamydia trachomatis and Herpes Simplex Virus

COLOR AND APPEARANCE

 Bacterial Vaginosis – white-gray discharge

 Candida infections – white “cottage cheese”-like discharge
 T. vaginalis infection – yellow-green, frothy, adherent discharge
 C. trachomatis infection – yellow, opaque cervical discharge

Normal Findings in Vaginal Secretions

Appearance White, flocculent discharge
pH 3.8 to 4.2
Amine (Whiff) test Negative
WBCs 2+
Lactobacilli Predominant
Clue cells Absent
Other cells Absent (except RBCs during menses)
Other organisms Other lactobacilli subgroups, occasional yeast

DIAGNOSTIC TESTS

 pH test – performed before placing the swab into saline or KOH solution
o Commercial pH paper with narrow pH range – recommended – to evaluate in 4.5 range
o Interference: Cervical mucus, semen, blood
o Lactobacilli → Lactic Acid (pH 3.8-4.5) → suppresses overgrowth of Mobiluncus,
Prevotella, Gardnerella vaginalis
o Hydrogen Peroxide and Estrogen Production – also helps to keep an acidic environment
 Microscopic Procedures
o Wet mount examination – quantified per high power field (hpf) (40x)
 Chapter 15 | Vaginal Secretions

 Examine slides at LPO (10x) and HPO (40x) with bright-field microscope
 Interference: Oil droplets from intravaginal medications
 Gram stain, cells, and organisms – oil immersion field (100x)
Quantitation Scheme for Microscopic Examinations
Rare <10 organisms or cells/slide
1+ <1 organism or cell/hpf
2+ 1-5 organisms or cells/hpf
3+ 6-30 organisms or cells/hpf
4+ >30 organisms or cells/hpf

Constituents found in Vaginal Fluid (Wet Mount)

 Squamous Epithelial Cell

o 25-70 um in diameter
o Polygonal “flagstone” appearance
o Centrally located nucleus
o Originate from linings of vagina and female urethra
o Clumps = ↑Yeast
 Clue Cell – abnormal variation of squamous epithelial cell
o “shaggy” appearance
o Caused by Gardnerella vaginalis
 WBC
o 14-16 um in diameter with granular cytoplasm, multi-lobed (PMN)
o >3+ = Candidiasis, Atrophic vaginitis, infections with Trichomonas,
Chlamydia, Neisseria gonorrhoeae, Herpes simplex
 RBC
o 7-8 um in diameter, smooth non-nucleated biconcave disks
o Present during menstruation or desquamative inflammatory process
o Resembles Yeast cell (use KOH to differentiate)
 Parabasal Cell
o 16-40 um in diameter, round-oval shaped
o Nucleus-Cytoplasm ratio = 1:1-1:2
o With marked basophilic granulation or marked basophilic structures (blue
blobs) in the surrounding cytoplasm
o Located in the luminal squamous epithelium of vaginal mucosa
o ↑parabasal cell (plus ↑WBC) = Desquamative Inflammatory vaginitis
 Basal Cell
o 10-16 um diameter, round, 1:2 N:C
o ↑basal cell (plus ↑WBC) = Desquamative Inflammatory vaginitis
 Bacteria
o Lactobacillus spp. – comprises large portion of the vaginal fluid
 Chapter 15 | Vaginal Secretions

o ↓Lactobacilli = ↑Mobiluncus, Prevotella, Porphyromonas, Bacteroides,

Gardnerella vaginalis, Peptostreptococcus, Enterococcus, Mycoplasma
hominis, Ureaplasma urealyticum
 Trichomonas vaginalis
o Atrial flagellated protozoan, 5-18 um in diameter
o Has 4 anterior flagella, undulating membrane, axostyle
o “jerky” motion
o Nonmotile resembles WBC
o Examined as soon as possible/Maintained at RT for 2 hours
 Yeast Cells
o Appears as Budding Yeast Cells (Blastophores) or as Hyphae (long filaments
that grow and form a mycelium)
o Pseudohyphae – multiple buds that do not detach and form chains – may
also be seen

o KOH Preparation and Amine (Whiff) Test

 1 drop of Saline Specimen + 1 drop 10% KOH → “fishy” odor
 Reported as Positive (presence) or Negative (absence)
 After amine test, place cover slip to exclude air bubbles → rest for 5 minutes →
(10% glycerin may be added to prevent deterioration) → examine slide
 LPO – Yeast pseudohyphae
 HPO – smaller blastophores
o Gram Stain
 0-3 = Normal
Nugent’s Gram Stain Criteria to Diagnose Bacterial Vaginosis
Lactobacillus Gardnerella, Bacteroides Curved Gram-variable rods Points
morphocytes
4+ 0 0 0
3+ 1+ 1+ or 2+ 1
2+ 2+ 3+ or 4+ 2
1+ 3+ 3
0 4+ 4
Note: Points are added according to the morphotypes seen. Add the points for all three columns
for a final sum. >7 = Bacterial Vaginosis

o Culture
 Gold standard for detecting Yeast and Trichomonas
 Time-consuming; requires 2 days for result
 Diamond’s medium – culture medium for T. vaginalis
o DNA Testing
 To diagnose G. vaginalis, Candida spp., and T. vaginalis
 Trichomonas – detected by DNA probe amplified by PCR
 1 hour for results; 95% sensitivity
 Chapter 15 | Vaginal Secretions

o Point of Care Tests

 Proline aminopeptidase activity – to identify G. vaginalis
 OSOM Trichomonas Rapid test – detects T. vaginalis from swabs in 10 minutes
 Positive: Visible Blue Line and Red Internal Control Line
 OSOM BVBLUE test – detects Sialidase
 Sialidase – enzyme produced by Gardnerella, Bacteroides, Prevotella,
and Mobiluncus
 Positive: Blue or Green
 Negative: Yellow

VAGINAL DISORDERS

 Bacterial Vaginosis (BV)

o Most common cause of vaginitis; imbalance in the ratio of normal vaginal bacterial flora
o Associated with new or multiple sex partners, frequent douching, use of intrauterine
devices, pregnancy, and a lack of the protective lactobacilli
o Risk factor for the premature rupture of membranes and preterm labor for pregnant
women
o Complications: Pelvic Inflammatory Disease, Endometritis, Sexually Transmitted
Infections (STIs)
o Gram stain – gold standard
o Affirm VPIII – detects G. vaginalis
o Treatment: Metronidazole (flagyl), Metronidazole gel, Clindamycin cream
 Trichomoniasis
o “Strawberry Cervix” because of punctuate hemorrhages
o 60-70% - sensitivity of wet mount examination
o Treatment: Metronidazole
 Candidiasis
o Vulvovaginal candidiasis – caused by the yeast Candida
 C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei
o Conditions that can change the vaginal environment: Broad-spectrum antibiotics, Oral
contraceptives, Estrogen Replacement Therapy
o Treatment: Over-the-counter or prescription azole antifungal agents
 Ex. Butoconazole, Clotrimazole, Tioconazole, Miconazole
o Not acquired through sexual intercourse
 Desquamative Inflammatory Vaginitis
o Β-hemolytic Gram-Positive Streptococci – can be cultured
o Treatment: 2% Clindamycin, Hormone Replacement Therapy
 Atrophic Vaginitis
o Found in postmenopausal women
o Caused by thinning of vaginal mucosa (↓Estrogen and Glycogen production)
o Treatment: Estrogen Replacement
 Chapter 15 | Vaginal Secretions

ADDITIONAL VAGINAL SECRETION PROCEDURES

Vaginal pH 7.0 + Positive Fern test = Amniotic Sac rupture

 Fetal Fibronectin (fFN) Test
o Preterm delivery (delivery before the 37 weeks’ gestation) – leading cause of neonatal
mortality and morbidity
 Presence of fFN during 24-34 weeks’ gestation
 Symptoms: Vaginal Secretions, Vaginal bleeding, Uterine contractions,
Abdominal or back discomfort, Pelvic pressure, Cramping
o Fetal Fibronectin – adhesive glycoprotein in the extracellular matrix at the maternal and
fetal interface within the uterus
 Elevated during the first 24 weeks of pregnancy then diminishes
o Interference: Creams, Soaps, Disinfectants
o Method: Solid-phase Enzyme Linked Immunosorbent Assay (ELISA) or Lateral flow, solid-
phase immunochromatographic assay (at 550 nm)
 AmniSure test
o PAMG-1 – present in high levels in amniotic fluid and low levels in blood
 Marker for fetal membrane rupture
 Normal: 0.05 to 0.22 mg/mL
 3 mg/mL = Vaginitis
 2,000-25,000 mg/mL = Fetal membrane rupture
o Uses immunochromatographic device
o Positive Result: Two lines
 Appendix B | Bronchoalveolar Lavage

 For obtaining cellular/immunologic/microbiologic information from the lower respiratory tract

 Useful in evaluating:
o Immunocompromised patients
o Interstitial lung disease (infectious/non-infectious/immunologic/malignant)
o Airway diseases
o Suspected alveolar hemorrhage
o Pulmonary alveolar proteinosis
o Langerhans cell histiocytosis
o Dust exposure
 Often used in conjunction with high-resolution computerized tomography (HRCT)
 Bronchoscopy
o Fiber-optic bronchoscope is guided into a bronchopulmonary segment (right middle or
lingular lobe)
o Optimal Targets:
 Areas of alveolar ground glass opacity
 More prominent nodular profusion
 Fine reticulation
o Aliquots of sterile normal saline are instilled into the alveolar spaces → To mix with
bronchial contents → Aspirated for cellular examination and culture
 Instillation volume: 100-300 mL (sterile saline) in 20- to 50-mL aliquots
 First aliquot is discarded
o Desired volume of analysis: 10-20 mL (min. 5 mL)
o Typical Recovery Range: 50-70% Optimal: >30%
 Low (<25%) – caused by fluid retention in the lung = Chronic Obstructive Lung
disease
 Recorded on requisition form
 Transport: Room Temperature, within 30 minutes
o >30 minutes – transport on ice (4°C)
o Specimen that will not be analyzed – centrifuge → resuspend in a nutrient-
supplemented medium → refrigerated at 4°C for up to 24 hours; ↑24 hours = invalid
 Cell count – performed within 1 hour (3 hours if in nutrient-supplemented medium)
 Diagnostic Tests: Differential Cell Count, Microbiologic Studies, Cytopathology

Appearance of BAL fluid

Clear (colorless) Normal
Milky White Pulmonary Alveolar Proteinosis (should be
centrifuged)
Light brown-beige Pulmonary Alveolar Proteinosis
Red/Bloody Acute Diffuse Alveolar Hemorrhage
Orange-Red Older Hemorrhagic Syndrome
Clotted Should be noted if present
 Appendix B | Bronchoalveolar Lavage

WHITE AND RED BLOOD CELL COUNTS

 May be diluted to facilitate counting using a hemocytometer

 Trypan Blue – to determine cell viability
 BMP LeukoChek system – for WBC count
o Diluent – Ammonium Oxalate (1:100) – to lyse RBCs
o Allow to settle for 5 minutes
o Count all cells in the 18 squares on both sides and calculate the average of the two sides
o Formula: WBC/cmm =
o Difference (between highest and lowest): not more than 15 cells
 MLA pipette – for RBC count
o Diluent – isotonic Saline
o Formula: RBC/cmm =
o Difference (between highest and lowest): not more than 30 cells
 Differential Count
o Slides are prepared by cytocentrifugation
o 500-1000 cells (min. 300 cells) – counted and classified

Cells Seen in BAL fluid

Macrophages Contains variety of phagocytized Smoking-related interstitial lung
(56-80%) materials (hemosiderin; disease
golden/brown/black pigments Pulmonary Langerhans cell
inclusions; foamy cells) histiocytosis
Lymphocytes Increased in Interstitial Lung >25% - Granulomatous Lung
(1-15%) disease, Drug Reactions, Disease
Pulmonary Lymphoma, >50% - Hypersensitivity
Nonbacterial infections Pneumonitis or Nonspecific
Interstitial Pneumonia
CD4/CD8 ratio Immunologic analysis is ↑ = Sarcoidosis or Connective
performed by flow cytometry Tissue Disorders
Normal = Tuberculosis or
Malignancies
↓ = Hypersensitivity
Pneumonitis, Silicosis, Drug-
induced disease, HIV infection
Neutrophils Primary granulocyte seen >50% = Acute Lung Injury,
(<3%) Elevated in cigarette smokers, Aspiration Pneumonia,
bronchopneumonia, toxin Suppurative Infection
exposure, diffuse alveolar
damage
Eosinophils Elevated in Asthma, Drug- >25% = Eosinophilic Lung
(<1%) induced Lung disease, Infections, Disease
Hypersensitivity, Pneumonitis,
Eosinophilic Pneumonia
 Appendix B | Bronchoalveolar Lavage

Mast Cells >1% + >50% Lymphocyte + >3%

Neutrophil = Hypersensitivity
Pneumonitis

ERYTHROCYTES

 Phagocytized Erythrocytes – Alveolar Hemorrhage within 48 hours

 Hemosiderin-Laden Macrophages – Alveolar Hemorrhage older than 48 hours

EPITHELIAL CELLS

 Ciliated Columnar Bronchial Epithelial Cells (4-17%) – more numerous in Bronchial wash
specimens

FUNGI, VIRUSES, AND BACTERIA

 BAL – diagnostic test for Pneumocystis carinii in immunocompromised patients

o Amorphous material (LPO) and Organisms (HPO) seen microscopically
o Performed on ventilator-assisted patients
 Cryptococcus neoformans – opportunistic pathogen in patients with AIDS
o Cup-shaped organism; Close resemblance: Yeast Cells
 Other organisms:
o Parasite – Toxoplasm gondii, Strongyloides stercoralis, P. westermani, E. histolytica, E.
granulosus,
o Bacteria – Legionella pneumophila, Mycobacterium tuberculosis, Mycoplasma
pneumoniae, Actinomyces spp.
o Virus – influenza A and B viruses, respiratory syncytial virus
o Fungi – Histoplasma capsulatum, C. neoformans, Pneumocystis jiroveci

CYTOLOGY

 Period Acid Schiff Stain/Oil Red O Stain – to diagnose pulmonary alveolar proteinosis or
aspiration

Cells observed
Sulfur Granules (Actinomycetes) Actinomyces infection
Hemosiderin-Laden Macrophages
Langerhans cell Cigarette smokers, Langerhans cell histiocytosis
Cytomegalic cell
Fat droplets (with Oil Red O stain) Fat embolism
Lipid-laden alveolar Macrophages (with Sudan III) Fat embolism
Dust particle inclusions Pneumoconises or Asbestos exposure