CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D.

BALCE, RMT

A. Introduction to Urinalysis
1. Composition of Urine: 95% water, 5% solutes
a. Organic components – ________, _____________, uric acid, ammonia, undetermined nitrogen, others
+ + 2+
b. Inorganic components – ___________, Na , K , P, Ca , phosphates, sulfates
2. Urine Volume
a. Daily output: Random, __________________ Average, __________________ D/N ratio, ________
b. Polyuria – abnormal increase in urine output; seen in ____________________________________
c. Oliguria – abnormal decrease in urine output; associated with dehydration, renal insufficiency, heart
disease, calculi, kidney tumors
d. Anuria/Anuresis – total suppression of urine production associated with severe acute nephritis, Hg
poisoning, obstructive uropathy, kidney failure
e. Nocturia – excretion of more than ______ mL urine at night with a specific gravity of ________
f. Diuresis – transitory increase in urine volume
3. Types of Urine Specimen/ Collection Techniques
a. First morning – routine screening, pregnancy test, detection of orthostatic proteinuria
b. Random – routine screening
c. 24-hour – quantitative chemical tests, hormone studies
d. 12-hour – _________________
e. Afternoon specimen (_____ pm) – ________________________________
f. Fasting/Second morning – diabetic screening/monitoring
g. 2-h Postprandial – diabetic monitoring
h. Glucose Tolerance – accompaniment to blood samples in GTT
i. Drug testing specimen – COC ___________________________; volume __________; acceptable
temperature _________ºC; addition of blueing agent to the ______________________
j. Midstream clean-catch – routine screening, bacterial culture
k. Catheterization – bacterial culture
l. Suprapubic aspiration – bacterial culture, cytology
m. Three-glass collection – diagnosis of _______________________
4. Specimen Collection and Handling
a. Specimen container for routine urinalysis – capacity of ___________
b. Minimum labeling requirements ________________________________________
c. All specimens must be analyzed within ________________________________
5. Methods of Specimen Preservation
Preservatives Comments
Refrigeration Bacteriostatic for 24 h; maintains acid pH up to 8; precipitates ____________
Phenol Does not interfere with routine tests; causes odor change
Toluene Does not interfere with routine tests; floats on surface and clings to pipettes
Thymol Preserves glucose and sediments well; interferes with acid precipitation tests
Formalin Excellent sediment preservative; interferes with reagent strip test for _______
Sodium fluoride Good preservative for drug analysis; inhibits reagent strip test for __________
Boric acid Preserves protein and formed elements well; interferes with ______________
Saccomanno fixative Preserves cellular elements; for cytology studies
6. Changes in Unpreserved Urine (>2 h)  Clarity
 Glucose
 Color
 Ketones
 Odor
 Bilirubin
 pH
 Urobilinogen
 Nitrite
 Cells
 Bacteria
 Casts
 Most crystals
 T. vaginalis

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

B. Laboratory Safety
1. Hand washing – best way to break the links in the chain of infection: ________________________
2. Decontamination of Body Fluid Spills
 Use 0.5% sodium hypochlorite or 10% bleach ( ___ part household bleach + ____ parts water)
3. Chemical Hazard
 MSDS _________________________________________
 Standard hazards identification system by the NFPA
RED = ____________ DEGREES OF HAZARD
0 = ________________
BLUE=____________ YELLOW=___________ 1 = ________________
2 = ________________
WHITE=____________ 3 = ________________
4 = ________________
4. Fire Hazard
 Classes of fire and extinguishing materials
Class Combustible materials Extinguishers
A Ordinary combustible materials
B Flammable liquids or gases
C Electrical equipment
D Combustible metals
 Sequence of actions in case of fire (RACE): ____________________________________________
 Correct use of fire extinguishers (PASS): ______________________________________________

C. Physical Examination of Urine

Color
1. Normal – varies from almost colorless, straw or light yellow to dark yellow, yellow-orange, or amber
2. Variations
Pathologic causes Nonpathologic causes
a. Colorless/ ____________________ Recent fluid consumption, diuresis
pale yellow ____________________
b. Amber/ orange ____________________ Acriflavine, phenazopyridine, nitrofurantoin, phenindione
c. Blue/green ____________________ Amitriptyline, methocarbamol, clorets, methylene blue
____________________
d. Pink/red ____________________ Beets, rifampin, menstrual contamination
____________________
____________________
e. Brown/black ____________________ Argyrols, methyldopa, levodopa, metronidazole
____________________

Transparency
1. Normal: Clear – no visible particulates, transparent
2. Variations
a. Hazy - few particulates, print easily seen through urine c. Turbid - print cannot be seen through urine
b. Cloudy – many particulates, print blurred through urine d. Milky – may precipitate or be clotted
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Specific Gravity
1. Normal Values: Random ___________________ 24-h ____________________
2. Methods
a. Urinometry – density measurement using a weighted float that is designed to sink to a level of 1.000
in distilled water; calibrated at ____°C; less accurate than other methods; requires ___ mL of urine
Corrections
 Temperature – adjust by ______ for every 3°C difference from calibration temperature
 Protein – subtract ______ for every g/dL; Glucose – subtract ______ for every g/dL
b. Refractometry – refractive index measurement; instrument is compensated between _______°C;
subject to interference by ______________
Corrections: protein and glucose only; temperature correction not done
Calibrating media: distilled water, SG ________; 5% NaCl, SG ________; 9% sucrose, SG _______
c. Harmonic Oscillation Densitometry – density measurement based on the change in the frequency of
sound waves oscillating through urine; previously used by Yellow IRIS

Odor
1. Normal – faint aromatic due to volatile acids; becomes ammoniacal as the specimen stands
2. Variations
a. Odorless – __________________ g. Sulfur odor – ____________________
b. Mousy – _________________ h. Fruity/ sweet – __________________
c. Rancid – ________________ i. Cabbage/ hops – ____________________
d. Sweaty feet – ______________________ j. Fecaloid – recto-vesicular fistula
e. Maple syrup/ caramel-like – __________ k. Bleach – contamination
f. Rotting fish – ___________________ l. Mercaptan – asparagus, garlic, and eggs

D. Chemical Examination of Urine

Parameter and Clinical significance Additional comments
Normal values
1. Specific Hypersthenuria: ______________________
Gravity Hyposthenuria: _______________________ Reagent strip detects ionized solutes only.
Random,
1.003-1.035 Isosthenuria: _________________________
2. pH Acid urine: Alkaline urine:
Random ____________________________________ ____________________________________
___________ ____________________________________ ____________________________________
First AM ____________________________________ ____________________________________
________ ____________________________________ ____________________________________

3. Protein Degrees of proteinuria: SSA Test/ Cold Protein Precipitation

a. Mild – ______ g/day
`

Degree of Turbidity Grade mg/dL

(<30 mg/dL or b. Moderate – ______ g/day
<150 mg/day) c. Heavy – _______ g/day No increase in turbidity
Noticeable turbidity
Types of proteinuria:
Distinct turbidity, no granulation
a. Pre-renal – IV hemolysis; muscle injury;
Turbidity with granulation
infection/inflammation; multiple myeloma
b. Renal (glomerular) – diabetic nephropathy, Turbidity with granulation
amyloidosis, glomerulonephritis, immune and flocculation
disorders, toxic agents, hypertension, Clumps of protein
strenuous exercise, pre-eclampsia, Detection of microalbuminuria
dehydration, orthostatic proteinuria a. Immunochemical tests _________________
c. Renal (tubular) – Fanconi syndrome, toxic b. Albumin excretion rate _________________
agents, severe viral infections c. Albumin/creatinine ratio ________________
d. Post-renal – lower UTI; injury or trauma

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

4. Glucose Types of Glucosuria: Clinitest

a. Hyperglycemia-associated – diabetes  Rgts: _______________________________
(<15 mg/dL) mellitus, endocrine disorders, pancreatic  False(+): ____________________________
disorders, CNS disorders, drugs, GDM ____________________________________
b. Renal-associated – Fanconi syndrome,  False (-): ____________________________
renal tubular dysfunction, tubular necrosis,
5. Ketones Ketone bodies: Acetone ____%, acetoacetic Acetest ________________________________
acid ____%, β-hydroxybutyric acid ____% _________________________________
Causes of Ketonuria: diabetes mellitus, Other Tests for Ketones:
starvation, fasting, weight reduction, strenuous a. Gerhardt’s, Lindeman’s – diacetic acid
exercise, malabsorption, pancreatic disorders, b. Legal’s, Rothera’s – diacetic acid and acetone
inborn errors of amino acid metabolism c. Osterberg, Hart’s test – β-hydroxybutyric acid
6. Blood a. Hematuria – renal calculi, tumors, trauma, Ammonium sulfate precipitation
glomerular disorders, anticoagulant therapy _____________________
b. Hemoglobinuria– hemolytic anemias,  Reagent: 2.8 g (NH4)2SO4
transfusion reactions, severe burns, malaria  Hemoglobin is precipitated by (NH4)2SO4 and
c. Myoglobinuria – rhabdomyolysis is not detected in the supernatant.
7. Bilirubin Bilirubinuria – hepatitis, cirrhosis, other liver Tests for Bilirubin:
disorders, and biliary obstruction a. Foam Shake Test
b. Oxidation tests _________________________
Differential diagnosis of jaundice
 (+) Result: rainbow array of colors: green
Urine Urine (biliverdin), blue (bilicyanin), yellow (choletelin)
Bilirubin UBG c. Ictotest
Hemolytic  Reagents: p-nitrobenzene-diazonium-p-
Hepatic toluene sulfonate, Na2CO3, boric acid, SSA
Obstructive  (+) Result: ___________________

8.Urobilinogen ↑ Urobilinogen – hemolytic disorders, Tests for Urobilinogen:

hepatitis, cirrhosis, and carcinoma a. Ehrlich’s Tube Test
(< 1.0 mg/dL  Never reported as negative b. Scwartz-Watson Differentiation Test
or <1.0 Ehrlich  Reagent strip lacks sensitivity
unit)

9. Nitrite UTI caused by nitrate-reducing bacteria e.g. Non-nitrate reducing bacteria:________________

___________________________________ No. of bacteria detected (sensitivity): __________
10. LE Advantages over nitrite: WBC types detected:_______________________
1. Detection of bacterial and nonbacterial UTI
Nonbacterial UTIs: _________________________
2. Inflammation of the urinary tract

Summary of Reagent Strip Testing

Test Principle Reagents Color reaction Sources of interference
1. Specific Polyelectrolytes, F↑: High concentration of protein
gravity bromthymol blue F↓: Highly alkaline urine
2. pH Methyl red; F↑: Old specimens
bromthymol blue F↓: Runover from adjacent pads
3. Protein Tetrabromphenol blue F(+):Highly alkaline urine,
or tetrachlorophenol phenazopyridine, QACs, antiseptics,
tetrabromosulfonphthalein chlorhexidine, loss of buffer, high SG
Buffer (pH 3.0) F(–):_____________________________
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

4. Glucose Glucose oxidase, F(+): ____________________________

peroxidase, F(–): ____________________________
KI or tetramethylbenzidine
5. Ketones Sodium nitroprusside F(+): Phthalein dyes, red urine, levodopa,
± glycine SH-containing medications
F(–): _____________________________
6. Blood Lysing agent F(+): _____________________________
Peroxide (OA) _____________________________
Tetramethylbenzidine F(–):  SG, crenated cells, formalin, nitrite,
captopril, ascorbic acid, unmixed spx
7. Bilirubin Dichloroaniline F(+): Highly pigmented urine, indican,
diazonium salt or phenazopyridine, metabolites of Lodine
dichlorobenzene F(–): _____________________________
diazoniumtetrafluoroborate _____________________________
_____________________________
8.Urobilinogen p-dimethylamino- F(+): _____________________________
benzaldehyde _____________________________
or 4-methoxybenzene F(–): Old specimens, formalin, nitrite
diazoniumtetrafluoroborate
9. Nitrite p-arsanilic acid F(+): _____________________________
tetrahydrobenzoquinolinol F(–): _____________________________
or sulfanilamide-hydroxy- _____________________________
tetrahydrobenzoquinoline _____________________________
10. LE Amino acid ester or F(+): Strong oxidizing agents, formalin,
indoxylcarbonic acid highly pigmented urine
ester and diazonium salt F(–):  protein, glucose, oxalic acid,
ascorbic acid, antibiotics
th
Detection of ascorbic acid – 11 pad in some reagent strip brands using ___________________________
Reagent strip readers – use the principle of __________________________ (Light reflection from the test
pads decreases in proportion to the intensity of color produced by the test substance)

E. Microscopic Examination of Urine

Sediment Examination Techniques
1. Standardizing Manual Microscopic Examination of Urine Sediment
a. Volume of urine examined ____________ d. Sediment preparation _________________
b. Speed of centrifugation ____________ e. Volume of sediment examined _____________
c. Length of centrifugation _____________ f. Sediment examination ________________________
2. Recommended Reporting Format
Parameter Manner of reporting Reference values
RBCs _____/HPF
WBCs ______________________________ _____/HPF
RTE/Oval fat bodies _____/HPF
Casts ______________________________ ___ hyaline or ___ granular/LPF
Squamous ECs None, rare, few, moderate, or many per LPF Few/LPF (higher in females)
____ ____ ____ _______ _______
Transitional ECs None, rare, few, moderate, or many per HPF
Crystals None, rare, few, moderate, or many per HPF
____ ____ ____ _______ _______ None/HPF
Bacteria None, rare, few, moderate, or many per HPF
____ ____ ____ _______ _______

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

3. Sediment Stains
Component/s Elements/structures stained
Sternheimer-Malbin ________________________ WBCs, epithelial cells, and casts
Metachromatic stain ________________________ WBC nuclei
Lipid stains ________________________ Triglycerides and neutral fats in cells and casts
Hansel stain ________________________ Eosinophilic granules
Prussian blue stain ________________________ Hemosiderin granules in cells and casts
Papanicolaou stain ________________________ Cellular elements for cytologic studies

4. Types of Microscope
Bright-field Routine urinalysis
Phase-contrast Elements with low RI _____________________________________
Polarizing Cholesterol-containing cells and casts; birefringent crystals
Dark-field Unstained specimens (e.g. T. pallidum)
Fluorescence Fluorescent microorganisms
Interference contrast 3-D image and layer-by-layer imaging; 2 types___________________________

Sediment Constituents
1. Cells
Red blood cells White blood cells Renal tubular ECs Transitional ECs

Normal: _____________ Normal: ___________ Singly or in syncytia after

Most significant EC type
Hematuria: glomerulonephritis, Pyuria: urinary tract urologic procedures e.g.
________ = tubular injury
renal calculi, malignancy infection or inflammation ______________________
Neutrophils: most common Oval fat bodies:__________
Ghost cells _____________
Glitter cells: sparkling; Bubble cells: _________ Abnormal morphology
Crenated cells ___________
Brownian movement Bilirubin-laden: __________ indicates _______________
Dysmorphic RBCs
Eosinophils: ________ Hemosiderin-laden: ______________________
_______________________
Mononuclears:___________ ___________________

Squamous ECs Bacteria Yeast cells Parasites

T. vaginalis –most common;
Abundant, irregular Gram-neg rods of family Small, refractile oval
may resemble a WBC or EC;
cytoplasm and a prominent Enterobacteriaceae, and structures
causes urethritis
central nucleus about the cocci esp. Staphylococcus Most common agent:
S. haematobium – ova have
size of an RBC and Enterococcus spp. _____________________
a terminal spine
Bacteriuria – indicates UTI Associated with diabetes,
E. vermicularis – common
Clue cells = ____________ when seen in conjunction immunocompromised state
contaminant from the
______________________ with pyuria and a positive and vaginal moniliasis
perianal area
urine culture (accompanied by WBCs)

2. Casts
a. Sites of formation: _______________________________________________
b. Factors that encourage formation: C_______________ A__________ S___________ T_______________
c. Sequence of formation and degeneration:

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

HYALINE CELLULAR GRANULAR WAXY

 Major component:  Types and significance  Normal no.: ___________  Appearance:
___________________ ____________________
 Significance of ↑ no.:
RBC: _______________
 RI _________________
_______________ ____________________
_____________________
 Normal no.: _________
 RI: _________________
WBC: ______________
 Significance of ↑ no.: _____________________
_______________  Significance:
___________________
____________________
___________________ _____________________
RTE: _______________
 Cylindroid – ________________________________________________________________________
 Fatty cast – _________________________________________________________________________
 Broad cast – ________________________________________________________________________
3. Crystals
a. Factors that encourage formation: ____________________________________________________
b. Identification techniques: ____________________________________________________________
c. Crystals in Acidic Urine
Crystal Appearance Solubility Significance
1) Uric acid Yellow-brown; pleomorphic; __________________ __________________________
birefringent __________________________
2) Amorphous “Brick dust” macroscopically; Commonly seen ff. refrigeration
urates granular microscopically
3) Acid urates Small brown spheres __________________ Rarely encountered and have
4) Sodium urates Colorless birefringent needles little clinical significance
5) Calcium oxalate Dihydrate – colorless envelope __________________ __________________________
Monohydrate – oval or dumbbell __________________________
6) Calcium sulfate Colorless needles or prisms __________________ No clinical significance
7) Hippuric acid Yellow-brown or colorless needles __________________ Ingestion of benzoic acid
8) Cystine Colorless, hexagonal plates; may __________________ __________________________
be confused with hexagonal UA
9) Cholesterol Notched rectangular plates; highly __________________ __________________________
birefringent
10) Leucine Yellow-brown oily-looking spheres __________________ Severe liver disease or _______
with concentric circles and radial
striations
11) Tyrosine Fine refractile needles that form __________________ Severe liver disease or _______
clumps or rosettes
12) Bilirubin Clumped yellow needles or granules __________________ Severe liver disease or _______

13) Radiographic Colorless, notched rhombic plates; __________________ Associated with SG > ________
Dye highly birefringent
14) Sulfonamide Needles, sheaves, or rosettes __________________ May cause tubular damage if
15) Ampicillin Colorless needles Refrigeration forms bundles crystals form in the nephron
d. Crystals in Alkaline Urine
Crystal Appearance Solubility Significance
1) Am. phosphates Milky white macroscopically Commonly seen ff. refrigeration
2) Ca phosphate Flat plates, needles or prisms __________________ __________________________
3) Triple phosphate Prism resembling _____________
UTI caused by urease-producing
4) Ammonium Yellow-brown spicule-covered __________________ bacteria; renal calculi
biurate spheres or _________________
5) Calcium Small, colorless, dumbbell or __________________ No clinical significance
carbonate spherical; may occur in clumps
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

F. Renal Physiology and Diseases

Renal Structure and Functions
1. Nephrons – basic functional units of the urinary system; 1-1.5 million per kidney
a. Cortical
b. Juxtamedullary
2. Renal Blood Flow
 Renal artery  afferent arteriole  glomerulus  efferent arteriole  peritubular capillaries & vasa recta
 Normal RBF: ______________ Normal RPF: _______________; evaluated using ___________
3. Glomerular Filtration
 Glomerulus: ___________________________________________________
 Characteristics of the glomerular filtrate: __________________________________________________
 Normal GFR: ____________; evaluated using ___________________
 Glomerular filtration barrier: ____________________________________
____________________________________
____________________________________
 Shield of negativity: _______________________________________
 Hydrostatic pressure differential: ________________
 RAAS

4. Tubular Reabsorption
 Substances that are actively transported:  Substances that are passively transported:
Glucose, amino acids, salts ________ Water _________________________
Chloride _________ Urea _____________
Sodium _________ Sodium _________
 Countercurrent mechanism – through selective reabsorption in the loop of Henle
DLH ______________________________________ ALH _________________________________
5. Tubular Secretion
 Elimination of unfiltered wastes _____________________
 Regulation of acid-base balance _____________________________

Renal Disorders
1. Glomerular Disorders
Disorder Etiology and Lab Findings
a. Acute Post-streptococcal GN Immune complex deposition 2° to streptococcal infection; ↑ ___________
b. Rapidly progressive GN Immune complex deposition 2° to immune disorders; ↑ _____________; ↓ ____
c. Goodpasture’s syndrome _______________________________________
d. Wegener’s Granulomatosis _______________________________________
e. Henoch-Schonlein Purpura Disruption of vascular integrity following viral respiratory infections; ↓__________
f. IgA Nephropathy Deposition of IgA on the glomerular membrane; ↑ _________
g. Membranous GN Immune complex deposition 2° to systemic disorders; +___________________
h. Membranoproliferative GN Cellular proliferation affecting the capillary walls or the glomerular basement
membrane; ↓ _________________________
i. Chronic Glomerulonephritis Marked decrease in renal function; ↑ __________________; ↓ ________
j. Nephrotic Syndrome Massive loss of protein and lipids due to disruption in electrical charges (shield
of negativity); ↑ ________________________
k. Minimal Change Disease Disruption of the podocytes following allergic reaction and immunization
l. Focal Segmental GS Disruption of podocytes associated with heroin/analgesic abuse and AIDS
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

2. Vesicotubulointerstitial Disorders
Disorder Etiology Lab findings
a. Acute Tubular Necrosis Damage to RTE by ___________________ ______________________________
b. Fanconi’s Syndrome Failure of PCT reabsorption ______________________________
c. Cystitis Ascending bacterial infection of the bladder ______________________________
d. Acute Pyelonephritis Reflux of urine untreated cystitis ______________________________
e. Chronic Pyelonephritis Structural abnormalities affecting urine flow ______________________________
f. Acute Interstitial Nephritis Drug-induced or allergic renal inflammation ______________________________

3. Renal Lithiasis
 Sites of calculi formation: _____________________________________________________________
Calculi type Incidence Description
a. Calcium oxalate ________ Very hard with dark color and coarse rough surface
b. Calcium phosphate Pale, light, and friable
c. Triple phosphate ________ Seen in x-ray film as ____________________________
d. Uric acid ________ Yellow to brownish red, moderately hard and translucent
e. Cystine ________ Yellow-brown and greasy resembling ___________________

4. Renal failure – end-stage renal disease due to pre-renal, renal, or post-renal causes; characterized by
____________________________________________________________________

G. Metabolic Disorders and Screening Tests

Major Disorders of Metabolism
Etiology Clinical manifestations Tests
a. Phenylketonuria ______________________ _______________________ FeCl3
_______________________ 2,4-DNPH
Guthrie test
b. Tyrosinuria Enzyme deficiency or liver Tyrosine and leucine crystals Nitrosonaphthol
disease in urine Microscopy
c. Alkaptonuria ______________________ Blackening of urine on Alkali test
standing; tissue pigmentation Silver nitroprusside
d. Melanuria Overproliferation of melanin- Blackening of urine upon Sodium nitroprusside
producing cells exposure to air (Acetest/ Thormahlen)
e. MSUD Deficiency in decarboxylases Maple syrup urine odor (↑ 2,4-DNPH
ketoacids), mental retardation Acetest
f. Isovaleric acidemia Isovaleryl CoA deficiency Sweaty feet odor of urine 2,4-DNPH
g. Propionic acidemia Error in converting valine, Vomiting, metabolic acidosis, Acetest
h. Methylmalonic isoleucine, threonine, and hypoglycemia, hyper-
p-nitroaniline
acidemia methionine to succinyl CoA ammonemia, ketonuria
i. Indicanuria ______________________ ______________ syndrome Obermayer’s test
______________________ Generalized aminoaciduria
j. Abnormal urinary Carcinoid tumors involving Vascular & bronchospastic Nitrosonaphthol
excretion of 5-HIAA the argentaffin cells symptoms (↑ serotonin)
k. Cystinosis ______________________ Deposition of cystine in CN nitroprusside
many areas of the body
l. Cystinuria ______________________ Sulfur odor of urine CN nitroprusside
Cystine crystals in urine
m. Homocystinuria ______________________ Failure to thrive, cataracts, CN nitroprusside
mental retardation Silver nitroprussde

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

n. Porphyrin disorders Inborn error of metabolism or Port wine color of urine, Hoesch test
acquired through erythrocytic psychiatric symptoms, Fluorescence test
and hepatic malfunctions cutaneous photosensitivity FEP
o. Mucopolysaccharide Inborn error of metabolism Mental retardation, skeletal Metachromatic staining
disorders abnormality, corneal deposits Acid albumin
of mucopolysaccharides CTAB test
p. Purine Disorder _______________________ Severe motor defects, Microscopy
(Lesch-Nyhan disease) _______________________ mental retardation, self-
mutilation, gout, calculi,
“orange sand in diapers”
q. Galactosemia/ Inborn error of metabolism Failure to thrive, liver Clinitest
Galactosuria disorders, mental retardation

H. Cerebrospinal Fluid
Specimen Considerations
1. Specimen collection
a. Lumbar puncture
b. Other methods: cisternal puncture, lateral cervical puncture or through ventricular cannulas
2. Volume collected: up to 20 mL distributed into 3-4 tubes
Tube Tests Storage
1 ______________________ __________________________
2 ______________________ __________________________
3 ______________________ __________________________
4 Additional tests Depends on test to be performed

Gross Examination
1. Viscosity
a. Normal: similar to that of water
b. Viscous CSF: metastatic mucin-producing adenocarcinoma, cryptococcal meningitis
2. Appearance
a. Normal: colorless and crystal clear
b. Variations:
1) Hazy, cloudy, turbid, milky – WBCs and microorganisms, damage to blood-brain barrier, MS
2) Oily – radiographic contrast media
3) Clotted – proteins in disorders affecting blood-brain barrier; traumatic tap
4) Pellicle formation – _________________________________
5) Bloody – traumatic tap or subarachnoid hemorrhage
Traumatic tap Hemorrhage
Distribution of blood ________________ _________________
Supernatant ________________ _________________
Clot formation ________________ _________________
D-dimer ________________ _________________
Erythrophagocytosis ________________ _________________

6) Xanthochromic – indicates presence of RBC degradation products or other pigments

Disorder Supernatant color
RBC lysis/Hb breakdown products Orange, pink, or yellow
Hyperbilirubinemia Yellow or yellow-green
CSF protein >150 mg/dL Yellow
Hypervitaminosis A (carotenoids) Orange
Meningeal metastatic melanoma Brown

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Chemical Examination
1. Total protein
a. Normal values: __________________________________________
b. Protein fractions
 Prealbumin, albumin, transferrin, α-globulins (haptoglobin, ceruloplasmin, α2-macroglobulin),
gamma globulins (IgG, IgA), tau protein (carbohydrate-deficient transferrin fraction)
 Not present in significant amounts: ___________________________________________
 Oligoclonal bands in electrophoresis: _____________________________________________
2. Albumin and IgG Measurements
a. values < 9 = intact BBB

b. values > 0.70 = IgG production within the CNS

3. Glucose
a. Normal values: _____________________
b. Important consideration: Blood should be drawn _____ prior to spinal tap to allow time for equilibration
4. Lactate
a. Normal values: _____________
b. Clinical significance: ___________________________________________________________
5. Glutamine
a. Normal values: ____________
b. Clinical significance: liver disorders Reye’s syndrome, disturbance of consciousness

Microscopic Examination
1. Total cell count – dilution, counting, and calculation are done using the same procedure as WBC count
2. WBC count
a. Normal values: <5/µL (adult), <30/µL (neonates)
b. Clarity and recommended dilutions: Slightly hazy ______ Hazy ______ Slightly cloudy ______
Slightly bloody ______ Cloudy, bloody, turbid ______

c. Calculation:

3. Differential Cell Count

Predominant cells seen in CSF
Type of Cell Clinical Significance
Lymphocytes and Monocytes Normal; ratio in adults _____________
Neutrophils Bacterial meningitis, cerebral hemorrhage
Erythrophages Intracranial hemorrhage
Blast forms Leukemias and lymphomas
Plasma cells Multiple sclerosis, lymphocyte reactions
Ependymal and choroidal cells Diagnostic procedures
Malignant cells Metastatic carcinomas, primary CNS carcinoma
Eosinophils Intracranial shunt malfunctions
nRBCs Bone marrow contamination during spinal tap

Differential diagnosis of meningitis

Bacterial Tubercular Fungal Viral
Chemical

Microscopic
Other lab findings

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

I. Synovial Fluid
Specimen Considerations
Specimen collection
a. Arthrocentesis – volume usually collected is about 25 mL
b. Tubes/Additives
1) Gram stain and culture __________________ 3) Chemical and serologic tests _____________
2) Cell counts ____________________________ 4) Glucose analysis ______________________

Gross Examination
Parameter Normal findings Abnormal findings Clinical significance of abnormal findings
1. Color Colorless to pale Green ____________________________________
yellow Deep yellow ____________________________________
Red-brown ____________________________________
2. Clarity Clear Turbid ____________________________________
Milky and opalescent ____________________________________
Ground pepper ____________________________________
3. Viscosity
a. Falling drop 4-6 cm string No string ____________________________________
b. Ropes test Clot formation No clot formation

Chemical Examination
Test Normal values Clinical significance of abnormal values
Glucose _____________ ____ Inflammatory or septic arthritis
Lactate _____________ ____ Septic arthritis
Total protein _____________ ____ Inflammatory and hemorrhagic arthritis
Uric acid _____________ ____ Gouty arthritis

Microscopic Examination
1. WBC count
a. Normal values: < 200 cells/µL
b. Dilutions: done only on turbid or bloody fluids using _____________________________________
2. Differential count – requires incubation of the fluid with _____________________ and cytocentrifugation
Cell/Inclusion Comments Clinical significance
Neutrophil Normally < 25% of the differential ________________________
Lymphocyte Normally 15% of the differential ________________________
Macrophage Normally seen ________________________
Synoviocyte May be multinucleated resembling a mesothelial cell None
LE cell Neutrophil containing characteristic ingested round body ________________________
Reiter cell Vacuolated macrophage with ingested neutrophils ________________________
Ragocyte Neutrophil with dark cytoplasmic granules ________________________
Cartilage cells Large, multinucleated cells ________________________
Rice bodies Microscopically show collagen and fibrin ________________________
Fat droplets Refractile globules stained with Sudan ________________________
Hemosiderin Inclusion within clusters of synovial cells ________________________

3. Crystal examination
a. Compensated polarizing microscope – used to demonstrate crystal polarization by placing a red
compensator between the crystal and the analyzer
b. Control for the polarization properties of MSU – _________________________________________
c. Negative birefringence – ___________________________________________________________
d. Positive birefringence – ____________________________________________________________
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Characteristics of synovial fluid crystals

Crystal Shape Birefringence Significance
Monosodium urate Needles _________________ ___________________
Calcium pyrophosphate Rhombic square, rods _________________ ___________________
Cholesterol Notched, rhombic plates Negative ___________________
Corticosteroid Flat, variable-shaped plates Positive and negative ___________________
Calcium oxalate Envelopes Negative ___________________
Calcium phosphate (apatite) Small particles None ___________________

Differential diagnosis of joint disorders

Group and nature Laboratory findings
Examples
of disorder Viscosity Glucose WBCs/µL PMNs (%)
I – Non-inflammatory ________________________ __________ ________ _____________ ________
II – Inflammatory
Immunologic ________________________ __________ ________ _____________ ________
Crystal-induced ________________________ __________ ________ _____________ ________
III – Septic ________________________ __________ ________ _____________ ________
IV – Hemorrhagic ________________________ __________ ________ _____________ ________

J. Serous Fluids
Specimen Considerations
1. Collection techniques: thoracentesis, pericardiocentesis, paracentesis
2. Collection tubes: a. Cell counts and differential __________
b. Microbiology and cytology procedures __________________
c. Chemistry tests _________________________
d. pH ____________________________________

Types of Effusions
Effusion Pathologic mechanisms Examples of disorders
Transudative ↓ Osmotic pressure __________________________________________________
↑ Hydrostatic pressure __________________________________________________
Exudative ↑ Capillary permeability __________________________________________________
↓ Lymphatic resorption __________________________________________________

Gross Examination
1. Appearance
a. Normal: clear and colorless to pale yellow
b. Turbid, white – microbial infection
c. Bloody
Parameters Hemothorax Hemorrhagic exudate
Distribution of blood ________________________ _________________________
Hematocrit ________________________ _________________________

d. Milky
Parameters Chylous Pseudochylous
Cholesterol crystals ________________________ _________________________
Triglycerides ________________________ _________________________
Sudan III staining ________________________ _________________________

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Chemical Examination
1. Light’s criteria – F:S protein ratio
LDH
F:S LDH ratio
2. Cholesterol
3. F:S cholesterol ratio
4. F:S bilirubin ratio
5. Serum-ascites albumin gradient

Test Significance Test Significance

Glucose ___ Rheumatoid inflammation and Amylase ___ Pancreatitis, esophageal
purulent infection rupture, GI perforation
Lactate ___ Bacterial infection ALP ___ GI perforation
Triglyceride ___ Chylous effusions ADA ___ Tubercular effusions
pH ___ Pneumonia; esophageal rupture BUN and creatinine ___ Punctured bladder

Microscopic Examination
Differential count: routinely performed to examine WBCs and demonstrate malignant cells
Serous fluid Cell types Significance
Pleural Mesothelial cells _____________ in tuberculosis
Plasma cells _____________ in tuberculosis
Eosinophils Hemothorax, pneumothorax, allergic reaction, parasitic infection
Peritoneal WBC >500 cells/µL Bacterial peritonitis, cirrhosis
Absolute granulocyte count >250 cells/µL – Bacterial peritonitis
RBC >100,000/µL Malignancy, intraabdominal bleeding (blunt trauma injury)
Psammoma bodies Benign tumors, ovarian and thyroid CA

K. Semen
Formation and Physiology
1. Spermatogenesis – promoted by _____; 64-day cycle; maturation series ______________________
2. Semen production and composition
Structure Function Contribution to semen volume
Testes __________________________
5% ____________________
Epididymis __________________________
Seminal vesicles Provide alkaline fluid and nutrients for sperm 60-70% ___________________
Prostate gland Secrete enzymes for coagulation and liquefaction 20-30% ___________________
Bulbourethral glands Neutralize prostatic fluid and vaginal acidity 5% ______________________

Specimen Considerations
1. Specimen collection
a. Masturbation – recommended method
b. Condom method – Silastic or nonlubricant-containing rubber or polyurethane condoms
c. Vaginal aspiration – for _________________________________
d. Coitus interruptus
2. Important considerations
a. Patient preparation – sexual abstinence of ____ days; must empty bladder before collection
b. Specimen container – prewarmed sterile glass or plastic containers
c. Transport – kept at 37 °C, delivered to the laboratory within __________ of collection
d. Fertility testing – ____ samples tested at 2-week intervals; 2 abnormal samples considered significant
e. Fructose test – __________________________________________________________________

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Gross Examination
Parameter Normal Abnormal Clinical significance of abnormal findings
1. Appearance _____________ Yellow Pyospermia, urine contamination, prolonged abstinence
Red/ rust color Bleeding
Turbid Infection
2. Volume _____________ <2 mL Improper functioning of one of the semen-producing organs
>5 mL Prolonged abstinence
3. Liquefaction _____________ >2 hours Deficiency in prostatic enzymes
4. Viscosity _____________ Clumped, stringy Deficiency in prostatic enzymes
5. pH _____________ >8.0 Infection within the reproductive tract
<7.2 Increased prostatic fluid
Microscopic Examination
1. Sperm concentration and count
 Dilution: 1:20 using sodium bicarbonate in formalin, saline, or distilled water
 Calculation: Sperm/mL = sperm counted (average of 2 sides) x 100,000
Sperm/mL = sperm counted (average of 2 sides) x 1,000,000
Sperm/ejaculate = sperm/mL x specimen volume
 Normal values: _________________________________________________
 Clinical significance of abnormal counts: azoospermia, oligospermia
2. Sperm motility
 Performed on well mixed, undiluted, liquefied semen ______________________
Grade Interpretation
 Normal motility:
4 Rapid, straight line motility
3 Slower speed, some lateral movement
______________________
2 Slow forward progression, noticeable lateral movement  Clinical significance of
1 No forward progression abnormal motility: midpiece
0 No movement and tail abnormalities
3. Sperm morphology
 Evaluation is done on a thin smear stained using Wright’s, Giemsa, or Papanicolaou (best stain)
 ______ sperm are evaluated under OIO for abnormalities in the head, midpiece, and tail
 Strict criteria: oval-shaped head (approx. ______ µm) tail approx. _____ µm long
acrosomal cap normal in size no big cytoplasmic droplet
 % Normal forms: _______________________________________

Additional Testing for Abnormal Semen Analysis

Routine semenalysis findings Additional test Clinical significance
Decreased motility with normal count _________________________ ________________________
Decreased count _________________________ ________________________
Decreased motility with clumping _________________________ ________________________
Normal analysis with continued infertility _________________________ ________________________

Function evaluated Sperm function test

Ovum penetration ___________________________________
Ability to penetrate partner’s midcycle cervical mucus ___________________________________
Membrane integrity and sperm viability ___________________________________
Ability to produce enzymes essential for ovum penetration ___________________________________

Chemical tests Normal values Significance of decreased values

Neutral α-glucosidase ≥20 mU/ejaculate ___________________________________
Zinc ≥2.4 µmol/ejaculate
Citric acid ≥52 µmol/ejaculate ___________________________________
Acid phosphatase ≥200 Units/ejaculate

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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

L. Pregnancy Testing
Types of Pregnancy Test
1. Human chorionic gonadotrophin hormone
 synthesized and secreted by the trophoblast cells of the developing placenta
 dimeric molecule
 serum concentration increases from <5 mIU/mL to >100,000 mIU/mL during the first trimester
2. Bioassays – introduction of hCG to a test animal produces changes in their reproductive organs (ovarian
hyperemia, corpora hemorrhagica, corpora lutea, spermatogenesis, oogenesis)
3. Immunoassays – detection of hCG using monoclonal antibodies
a. Agglutination Immunoassays – direct or agglutination-inhibition
b. Radioimmunoassay – most sensitive technique (5 mIU/mL); uses radiolabeled hCG
c. EIA – microtiter plate or ICT (commonly used in home-based pregnancy test; sensitivity _________)

Sources of Errors
1. False-negative: low titer or concentration of hCG; low sensitivity of test animal or assay method
2. False-positive: chlorpromazine, phenothiazine, heterophilic Ig, hCG-secreting tumors ______________

M. Amniotic Fluid
st rd
 Volume: approximately 35 mL during the 1 trimester, peaks during the 3 trimester (approx. 1 L)
↑ ______________ – results from failure of the fetus to begin swallowing; indicates fetal distress
↓ ______________ – due to increased fetal swallowing, urinary tract deformities, and membrane leakage

Specimen Considerations
1. Specimen collection
a. Amniocentesis– transabdominal or transvaginal; performed after the ______ week of gestation
b. Volume collected: 30 mL
2. Specimen handling
a. FLM tests – transported in ice and refrigerated up to 72 hours; filtration or low-speed centrifugation
b. Cytogenetic test – incubated at _____°C prior to analysis
c. Chemical testing – separated from cellular elements and debris ASAP
d. Bilirubin analysis – placed in amber bottles

Gross Examination
Appearance Significance
Colorless Normal (may show slight to moderate turbidity)
Blood-streaked Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Yellow __________________
Dark green __________________
Dark red-brown ___________________

Tests
1. Tests for Fetal Distress
Test Method Comments Normal values
a. Bilirubin Spectrophotometry ΔA450 plotted on a Liley graph to determine severity of HDN; ____________
Hgb and meconium interfere
b. AFP Immunoassay Screening test for NTDs ____________
c. AChE Spectrophotometry Confirmatory test for NTDs; affected by blood contamination ____________
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

2. Tests for Fetal Lung Maturity

Test Method Comments Normal values
a. L/S ratio Thin-layer _____________ used as internal standard; _________
chromatography affected by blood and meconium contamination
b. Amniostat-FLM Agglutination Uses antisera specific to ________________; _________
immunoassay not affected by blood and meconium
c. Foam stability index Modified foam-shake ________________ used as anti-foaming agent _________
d. Microviscosity Fluorescence- polarization _____________ used as internal standard _________
e. Lamellar body ct Resistance pulse counting Uses the ______ channel of hematology analyzers _________
f. OD at 650 nm Spectrophotometry Requires centrifugation at ______ g for ____ min _________

N. Feces
Specimen Considerations
1. Routine fecalysis – __________________________
2. Quantitative fecal fat analysis – _______________________________
Gross Examination
1. Color 2. Appearance/Consistency
a. Brown – normal (due to stercobilin/urobilin) a. Bulky, frothy – ________________________
b. Black – _______________________ b. Ribbon-like, slender – intestinal constriction
c. Red – ________________________ c. Mucoid, blood-streaked – colitis, dysentery
d. Pale yellow, white, or gray – ____________________ d. Small, hard – constipation
e. Green – biliverdin, oral antibiotics, green vegetables e. Watery – diarrhea

Chemical Examination
Test Method/principle Reagent Result Significance
FOBT Pseudoperoxidase activity Guaiac ______________ _____________________
Van de Kamer Titration of fatty acids NaOH ______________ _____________________
Apt test Alkali denaturation (fetal NaOH ______________ _____________________
vs maternal blood) ______________ _____________________
Trypsin Gelatin hydrolysis Gelatin on x-ray ______________ _____________________
Carbohydrates Copper reduction Clinitest ______________ _____________________

Microscopic Examination
Test Method/Principle Significance
Muscle fibers Examination of eosin-stained smear to visualize >10 undigested muscle fibers
muscle fiber striations indicate pancreatic insufficiency
Qualitative fecal Direct smear stained with Sudan III 60 large orange-red droplets
fats Smear heated with acetic acid and Sudan III 100 orange-red droplets (6–75 µm)
indicate malabsorption
Fecal neutrophils Examination of stained wet mount or dried smear 3/hpf indicates invasive condition

O. Sputum
Specimen Collection
1. Expectoration – first morning; may require induction using ________________________________
2. Bronchoalveolar lavage – infusion of saline followed by aspiration using a ______________________
3. Throat swab
4. Endotracheal aspiration
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 CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS RODERICK D. BALCE, RMT

Examination of Sputum
1. Color
a. Bright green – jaundice, caseous pneumonia, Pseudomonas infection, rupture of liver abscess
b. Red/bright red – recent hemorrhage (acute cardiac or pulmonary infarction, neoplasm invasion)
c. Rust-colored – decomposed hemoglobin ________________________________
d. Brown – congestive heart failure
e. Olive green/grass green – ______________________
f. Black – dust particles, carbon or charcoal, heavy smokers, anthracosis
2. Macroscopic Structures
a. ________________ – fragments of necrotic tissue seen in pulmonary gangrene, PTB, lung abscess
b. ________________ – yellowish or gray caseous materials that emit a foul odor when crushed
c. Pneumoliths/Broncholiths/Lung stones – calcificified materials seen in chronic PTB and histoplasmosis
d. Bronchial casts – branching tree-like casts seen in lobar pneumonia and fibrinous bronchitis
e. Mycetomas – rounded masses of fungal elements seen in Aspergillus infection
.

3. Microscopic Structures
a. _________________ – spirally twisted mucoid strands frequently coiled into little balls
b. Myelin globules – colorless, oval or pea-shaped; no significance but may be mistaken for __________
c. Elastic fibers – refractile fibers shed off during the cougning out process; indicates destructive disease
d. Charcot-Leyden crystals – hexagonal, needle-like or bipyramidal crystals; seen in ________________
e. _________________ – bronchial epithelial cells with vacuolated cytoplasm and ciliated borders
f. Heart failure cells/siderophages – hemosiderin-laden cells seen in CHF and alveolar hemorrhage
g. Microorganisms

P. Gastric Fluid
Specimen Considerations
1. Stimulants
a. Test meals – poor gastric stimulants (e.g. Ewald’s, Boa’s, Reigel’s, or Alcohol test meal)
b. Histamine – exerts unpleasant systemic effects on blood vessels and smooth muscles
c. Histalog/Betazole – histamine isomer with preferential effect on gastrin secretion
d. Pentagastrin – stimulant of choice resembling gastrin; more rapid response than Histalog
e. Insulin (hypoglycemia test) – used to determine completeness of vagotomy
f. Sham feeding
2. Evacuation tubes
a. ___________ – has a metal tip; swallowed by gravity; for both gastric and duodenal fluid collection
b. ___________ – has the smallest diameter; inserted through the nose

Gross Examination
1. Appearance a. Normal: colorless or pale gray and transluscent
b. Variations: green (old bile), yellow (fresh bile), red (blood), coffee brown (old blood)
2. Volume a. Normal: 20-80 mL after a test meal; 45-150 mL after chemical stimulation
b. Increased volume: hypomotility, pyloric obstruction, __________________ syndrome
c. Decreased volume: gastric hypermotility
Chemical Examination
1. pH Normal: 1.6-1.9
2. Gastric Acidity
a. Total Acidity: 40-70 mEq/L c. Basal acid output (BAO): _____________________________
b. Free HCl: 20-40 mEq/L d. Maximal acid output (MAO): ___________________________
3. Clinical Significance
a. Hyperchlorhydria – increased free HCl seen in ___________________
b. Hypochlorhydria – decreased free HCl seen in chronic gastritis, gastric ulcer, and stomach CA
c. Achlorhydria – absence of free HCl seen in _________________________
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