CM Handouts Clinical Micros

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CM-handouts - Clinical Microscopy
Medical Technology (Centro Escolar University)
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CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS Roderick D. Balce, RMT
CLINICAL
MICROSCOPY
ANALYSIS OF URINE,
OTHER BODY FLUIDS,
AND MISCELLANEOUS
SPECIMENS
Roderick D. Balce
Centro Escolar University

MEDICAL TECHNOLOGY
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CLINICAL MICROSCOPY
CONTENT ANALYSIS OF URINE AND OTHER BODY FLUIDS Roderick D. Balce, RMT
1. Introduction to Urinalysis 2 10. Pregnancy Tests 19

2. Quality Assurance 4 11. Cerebrospinal Fluid 20
3. Physical Examination of Urine 6 12. Semen 22
4. Chemical Examination of Urine 7 13. Synovial Fluid 24
5. Microscopic Examination of Urine 10 14. Serous Fluids 26
6. Urinalysis Automation 12 15. Amniotic Fluid 28
7. Renal Structure, Functions, and Diseases 13 16. Feces 29
8. Renal Calculi 16 17. Sputum and BAL 30
9. Urine Screening Tests 17 18. Gastric Fluid 31
1. Introduction to Urinalysis
A. History
 Urinalysis – marked the beginning of laboratory medicine; included observations of color, turbidity, odor,
volume, viscosity, and even sweetness
 5th century BC – Hippocrates wrote a book on uroscopy
 1140 AD – color charts were developed that described the significance of 20 different colors
 1627 – Thomas Bryant wrote a book about charlatans (pisse prophets) which inspired the passing of the
first medical licensure law in England
 1694 – Frederik Dekkers’ discovered albuminuria by boiling urine
 17th century – microscope was invented which led to the examination of urinary sediment and to the
development by Thomas Addis of methods for quantitating the microscopic sediment
 1827 – Richard Bright introduced the concept of urinalysis as part of routine patient examination
B. Utilities of Analysis
1. To aid in the diagnosis of diseases
2. To screen asymptomatic populations for undetected disorders
3. To monitor the progress of disease and the effectiveness of therapy
C. Specimen Considerations
1. Composition of Urine: 95% water, 5% analytes
a. Organic components – urea, creatinine, uric acid, ammonia, undetermined nitrogen, others
b. Inorganic components – Cl , Na , K , P, Ca , phosphates, sulfates
- + + 2+
2. 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 – Addis count
e. Afternoon specimen (2-4 pm) – urobilinogen determination
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 – collection requires stringent protocols (COC); temperature should be within
32.5- 37.7ºC; blueing agent added to the toilet water reservoir in unwitnessed collection
j. Midstream clean-catch – routine screening, bacterial culture
k. Catheterization – bacterial culture
l. Suprapubic aspiration – bacterial culture, cytology
m. Three-glass collection – diagnosis of prostatic infection
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3. Specimen Preservation
a. Methods of Preservation
Preservatives Comments
Prevents bacterial growth for at least 24 hours; preserves organized
sediments; maintains an acid pH up to about 8 hours; and does not interfere
Refrigeration
with chemical tests
Precipitates amorphous materials increasing the specific gravity
Phenol Does not interfere with routine tests; causes an odor change
Does not interfere with routine tests; floats on surface of specimens and
Toluene
clings to pipettes and testing materials
Preserves glucose and sediments well; interferes with acid precipitation tests
Thymol
for protein
Excellent sediment preservative; acts as a reducing agent, interfering with
Formalin
chemical tests for glucose, blood, LE, and copper reduction
Prevents glycolysis; is a good preservative for drug analyses; inhibits reagent
Sodium fluoride
strip tests for glucose, blood, and leukocytes
Preserves protein and formed elements well; does not interfere with routine
Boric acid
analyses other than pH; interferes with drug and hormone analyses
Saccomanno
Preserves cellular elements; for cytology studies
Fixative
b. Changes in Unpreserved Urine

Parameter Change Cause
Color Modified/ darkened Oxidation or reduction of metabolites
Odor Bacterial multiplication or breakdown of urea to ammonia
pH Bacterial breakdown of urea to ammonia/ loss of CO2
Increased
Nitrite Multiplication of nitrate-reducing bacteria
Bacteria Multiplication
Clarity Bacterial growth and precipitation of amorphous material
Glucose Glycolysis and bacterial use
Ketones Volatilization and bacterial metabolism
Bilirubin Decreased Photooxidation to biliverdin
Urobilinogen Oxidation to urobilin
Cells and
Disintegration in dilute alkaline urine
casts
4. Urine Volume (24-hour)

a. Average daily output: 1,200-1,500 mL
b. Variations
1) Polyuria – abnormal increase in urine output
 Clinical significance: diabetes mellitus, diabetes insipidus
2) Oliguria – abnormal decrease in urine output
 Clinical significance: dehydration, renal insufficiency, poorly compensated heart disease, calculi
formation, kidney tumors
3) Anuria/Anuresis – total suppression of urine production
 Clinical significance: severe acute nephritis, Hg poisoning, obstructive uropathy, kidney failure
4) Nocturia – excretion of more than 500 mL urine at night
5) Diuresis – transitory increase in urine volume

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2. Quality Assurance
A. Pre-analytical Aspects of Quality Assurance
1. Specimen Collection
a. Appropriate specimen container (capacity, need for a sterile or opaque container)
b. Minimum labeling requirements (patient’s name, date and time of collection)
c. Specimen type and volume required for each test
2. Specimen Receipt or Rejection
Criteria for Specimen Rejection
 Unlabeled or mislabeled containers  Containers with contaminated exteriors
 Request form incomplete or lacking  Inappropriate specimen type
 Nonmatching labels and requisition forms  Specimens of insufficient quantity
 Visibly contaminated specimens (feces or  Improperly transported specimens
toilet paper)  Incorrect urine preservative
3. Specimen Processing
 Immediate processing (within 2 hours for routine UA) to improve specimen TAT and prevent changes in
specimen integrity
 For timed specimens: adequate mixing, volume measurement, aliquoting
B. Analytical Aspects of Quality Assurance

1. Reagents
 Deionized water for reagent preparation: check pH and purity every week and bacterial count every
month
 Reagent strips: store in opaque container with dessicant at room temperature; check with positive and
negative controls every 24 hours (+ control: ± 1 color block, - control: water not acceptable)
2. Equipment
Routine performance checks, calibration, and preventive maintenance schedules
Equipment Frequency Checks performed
Centrifuges Daily or weekly Clean rotor, trunnions, and interior with suitable disinfectant.
Every 3 months Check speed and timer.
Microscopes Daily Clean and adjust if necessary (e.g. Kohler illumination, phase ring)
Annually Preventive maintenance and cleaning.
Reagent strip Daily Calibrate reflectance meter with standard reagent strip.
readers Daily or periodically Clean mechanical parts and optics.
Refractometers Every shift or daily Calibrate with distilled water (1.000) and at least one standard of
known SG e.g. 3% NaCl (1.015), 5% NaCl (1.022), 7% NaCl (1.035),
or 9% sucrose (1.034). Acceptable tolerance: target ± 0.001
3. Microscopy
Guidelines for Standardizing Microscopic Examination of Urine Sediment
a. Volume of urine examined 10, 12, or 15 mL
b. Speed of centrifugation 400 g
c. Length of centrifugation 5 minutes
d. Sediment preparation 0.5 or 1 mL left after decantation
e. Volume of sediment examined 20 µL or 0.02 mL
f. Sediment examination At least 10 LPFs and 10 HPFs
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4. Quality Control
a. Internal QC
 Two levels of commercial controls must be run and recorded at the beginning of each shift and if
reagents are changed, an instrument malfunction has occurred, or if test results are questionable
 QC data must be retained for 2 years and should be reviewed daily and monthly to detect errors.
b. External QC/Proficiency testing
 Analysis of lyophilized or ready-to-use specimens sent by a regulatory agency
C. Post-analytical Aspects of Quality Assurance

1. Critical or Panic Values
 Pathologic urine crystals, strongly positive test for glucose and ketones, presence of a reducing
substance other than glucose and ascorbic acid in an infant’s urine
2. Reporting and Interpretation of Results
 Reporting format must be standardized and reference values must be included in the results form
Sample standardized urine microscopic reporting format
Quantitate an average of 10 fields both under low and high power. Do not quantitate
budding yeast, mycelial elements, trichomonas, mucus threads, or sperm, but do note
their presence.
Epithelial cells: Rare (0–5), Few (5–20), Moderate (20–100), Many (>100) per LPF
Casts: 0–2, 2–5, 5–10, >10 per LPF
RBCs: 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 per HPF
WBCs:0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 per HPF
Crystals: Rare (0–2), Few (2–5), Moderate (5–20), Many (>20) per LPF or HPF
Bacteria: Rare (0–10), Few (10–50), Moderate (50–200), Many (>200) per HPF
D. Approaches to Quality Management

1. Total Quality Management
 focuses on teams, processes, statistics, and services that meet or exceed customer expectations
 strives to continually look for ways to reduce errors (defect prevention) by empowering employees to
assist in solving problems and understand their integral role within the system (universal responsibility)
2. Continuous Quality Improvement
 strives to continually improve practices and not just meet established quality standards
 patients are the ultimate customers of CQI but also include health-care providers, personnel in other
departments, and the patient’s family and friends
3. Six Sigma
 hands-on process with the single mantra of improvement (improved performance, improved quality,
improved customer and employee satisfaction)
 based on statistics and quantitative measurements which involve the determination of the number of
defects per million opportunities (DPMO) and the reduction of the same to near zero

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3. Physical Examination of Urine

A. Color
1. Normal – varies from almost colorless, straw or light yellow to dark yellow, yellow-orange, or amber
2. Variations
a. Colorless/pale yellow – recent fluid consumption, polyuria, DM, DI
b. Amber/ orange – bilirubin, acriflavine, phenazopyridine, nitrofurantoin, phenindione
c. Yellow-green – oxidation of bilirubin to biliverdin
d. Blue/green – Pseudomonas infection, amitriptyline, methocarbamol, clorets, indican, methylene blue
e. Pink/red – intact RBCs, hemoglobin, myoglobin, porphyrins, beets, menstrual contamination
f. Brown/black – methemoglobin, homogentisic acid, melanin, argyrol, methyldopa, levodopa,
metronidazole
B. Odor
1. Normal – faint aromatic due to volatile acids; becomes ammoniacal as the specimen stands
2. Variations
a. Ammoniacal (freshly voided) – UTI g. Mousy – PKU
b. Rancid – tyrosinuria h. Sweaty feet – isovaleric acidemia
c. Maple syrup/ caramel-like – MSUD i. Rotting fish – trimethyl aminuria
d. Sulfur odor – cystine disorders j. Fecaloid – recto-vesicular fistula
e. Fruity/ sweet – diabetes ketoacidosis k. Cabbage/ hops – methionine malabsorption
f. Mercaptan – asparagus, garlic, and eggs l. Bleach – contamination
C. Transparency
1. Normal: Clear – no visible particulates, transparent
2. Variations: 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
D. Specific Gravity
1. Normal Values: depend on the patient’s degree of hydration
2. Methods
a. Urinometry
1) Urinometer/Hydrometer - weighted float that is designed to sink to a level of 1.000 in distilled
water; calibrated at 20°C; less accurate than other methods; requires large volume of urine
2) Corrections
 Temperature – for every 3°C that the urine temperature is above or below the calibration
temperature, 0.001 is respectively added to or subtracted from the reading
 Protein – subtract 0.003 for every g/dL; Glucose – subtract 0.004 for every g/dL
b. Refractometry
1) Refractometer/ TS meter - measures refractive index; compensated between 15°C and 38°C
2) Corrections: protein and glucose only; temperature correction not done
c. Harmonic Oscillation Densitometry
1) Mass gravity meter – used by Yellow IRIS automated workstations to measure specific gravity
2) Principle: 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.

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4. Chemical Examination of Urine

A. Summary of Chemical Tests
Parameter
(Normal Clinical significance Additional comments
values)
1. Specific 1. Monitoring patient’s hydration.
Gravity 2. Detection of loss of renal concentrating
ability
3. Diagnosis of diabetes insipidus
4. Determination of unsatisfactory specimens
due to low concentration
2. pH 1. Respiratory or metabolic acidosis/ alkalosis Causes of acid urine: emphysema, diabetes
2. Renal tubular acidosis mellitus, starvation, diarrhea, dehydration, acid-
(Average, 6.0;
3. Renal calculi formation producing bacteria, high protein diet, medications
random, 4.5-
4. Treatment of UTI Causes of alkaline urine: hyperventilation,
8.0; fasting,
5. Precipitation/ identification of crystals vomiting, renal tubular acidosis, urease-producing
5.5-6.5)
6. Determination of unsatisfactory specimen bacteria, vegetarian diet, old specimens
3. Protein Degrees of proteinuria: Tests for protein:
a. Mild – < 1.0 g/day a. Heat and Acetic Acid Test
(<30 mg/dL or b. Moderate – 1.0-4.0 g/day  Grading: diffused cloud (+1); granular cloud
<150 mg/day; c. Heavy – > 4.0 g/day (+2); distinct flocculi (+3); large flocculi (+4)
Negative rgt b. SSA Test/ Cold Protein Precipitation
Types of proteinuria: `
strip test)
a. Pre-renal – intravascular hemolysis; muscle Grade Degree of Turbidity and Conc. in mg/dL
injury; severe infection and inflammation; Neg No increase in turbidity (<6)
multiple myeloma Trace Noticeable turbidity (6-30)
b. Renal (glomerular) – diabetic nephropathy, +1 Distinct turbidity, no granulation (30-100)
amyloidosis, glomerulonephritis, Turbidity with granulation, no flocculation
autoimmune disorders, toxic agents, +2
(100-200)
hypertension, strenuous exercise, pre-
Turbidity with granulation and flocculation
eclampsia, dehydration, orthostatic +3
(200-400)
proteinuria
+4 Clumps of protein (>400)
c. Renal (tubular) – Fanconi syndrome, toxic
agents, severe viral infections  False (+): mucin, uric acid, penicillin,
d. Post-renal – lower UTI; injury or trauma; tolbutamides, radiocontrast media,
menstrual contamination; prostatic fluid; sulfonamides, cephalosporins
spermatozoa; vaginal secretions  False (-): highly buffered alkaline urine
 Correlate with reagent strip results
c. Tests for microalbuminuria
 Micral test and Immunodip
 Significant values reported as AER
4. Glucose Types of Glucosuria: Tests for Glucose (Copper Reduction):
a. Hyperglycemia-associated – diabetes a. Benedict’s test
(<15 mg/dL; mellitus, endocrine disorders, pancreatic  Positive result: brick red precipitate
Negative rgt disorders, CNS disorders, disturbance in b. Clinitest
strip test) metabolism, liver disease, drugs, gestational  Rgts: CuSO4, NaOH, sodium citrate, Na2CO3
diabetes mellitus  False(+): other reducing sugars, ascorbic
b. Renal-associated – renal tubular acid, drug metabolites, cephalosporins
dysfunction, tubular necrosis, Fanconi  False (-): pass-through phenomenon
syndrome, osteomalacia, pregnancy  Correlate with reagent strip results
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5. Ketones Ketone bodies: Tests for Ketones:

Acetone – 2% a. Gerhart’s, Lindeman’s – diacetic acid
(Negative rgt Acetoacetic acid – 20% b. Frommer’s, Walhauster’s, Lange’s, Jackson-
strip test) β-hydroxybutyric acid – 78% Taylor’s, Rantzmann’s, Lieben’s – acetone
Causes of Ketonuria: diabetes mellitus, c. Legal’s, Rothera’s, Acetest, Ketostix – diacetic
starvation, fasting, weight reduction, strenuous acid and acetone
exercise, malabsorption, pancreatic disorders, d. Osterberg, Hart’s test – β-hydroxybutyric acid
inborn errors of amino acid metabolism
6. Blood a. Hematuria – renal calculi, glomerular Tests to differentiate hemoglobin and
disorders, pyelonephritis, tumors, trauma, myoglobin:
(Negative rgt toxic chemicals, anticoagulant therapy, a. Ammonium Sulfate Method
strip test) strenuous exercise, and menstruation  Reagent: 2.8 g (NH4)2SO4
b. Hemoglobinuria– hemolytic anemias,  Hemoglobin is precipitated by (NH4)2SO4
transfusion reactions, severe burns, and is not detected in the supernatant.
malaria,strenuous exercise b. Enzyme assays
c. Myoglobinuria – muscular trauma, crush c. Absorption Spectrophotometry
syndrome, prolonged coma, convulsions, d. Immunodiffusion Technique
muscle-wasting diseases, alcoholism, drug e. Electrophoresis
abuse, extensive exertion
7. Bilirubin 1. Diagnosis of hepatitis, cirrhosis, other liver Tests for Bilirubin:
disorders, and biliary obstruction a. Foam Shake Test
(Negative rgt 2. Determination is more significant when b. Oxidation Test (Gmelin or Fouchet’s method)
strip test) combined with serum bilirubin and urine  Acidic oxidation of bilirubin into a rainbow
urobilinogen array of colors: green (biliverdin), blue
Blood Urine Urine (bilicyanin), and yellow (choletelin)
Bilirubin Bilirubin UBG c. Diazotization Test (Ictotest)
Hemolytic Inc Neg +++  Reagents: p-nitrobenzene-diazonium-p-
Hepatic Inc +/- ++ toluene sulfonate, Na2CO3, boric acid, SSA
Obstructive Inc/N +++ Normal  (+) Result: blue to purple color
8.Urobilinogen 1. Only a small amount is normally found in Tests for Urobilinogen:
urine. a. Ehrlich’s Tube Test
(< 1.0 mg/dL 2. Useful for the early detection of liver  Reagent: p-dimethylaminobenzaldehyde
or <1.0 Ehrlich disease and for the diagnosis of hemolytic  (+) Result: cherry red color
unit) disorders, hepatitis, cirrhosis, and carcinoma b. Scwartz-Watson Differentiation Test
3. Absence in urine may indicate biliary  UBG - soluble in both chloroform and butanol
obstruction  PBG - insoluble in both
 ERCs- soluble only in butanol
9. Nitrite 1. Diagnosis of cystitis and pyelonephritis
2. Evaluation of antibiotic therapy
(Negative rgt
3. Monitoring of patients at high risk for UTI
strip test)
4. Screening of urine culture specimens
10. LE 1. Detection of bacterial and nonbacterial UTI
(Negative rgt 2. Inflammation of the urinary tract
strip test) 3. Screening of urine culture specimens
NOTES:
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B. Summary of Reagent Strip Testing

Regent strip Color
Principle Reagents Sources of interference
test reaction
M: Poly(methylvinyl ether)
Blue-green-
1. Specific pKa change of maleic anhydride, BTB F(+): High concentration of protein
yellow
gravity polyelectrolytes C: Ethyleneglycol-bis F(–): Highly alkaline urine
(45 s)
(aminoethylether), BTB
Double indicator Methyl red; Red-yellow- Runover from adjacent pads
2. pH
system bromthymol blue blue (60 s) Old specimens
F(+): Highly buffered alkaline urine,
M: Tetrabromphenol blue pigmented specimens, chlorhexidine,
Protein error of C: Tetrachlorophenol Blue-green phenazopyridine, QACs (detergents),
3. Protein
indicator tetrabromosulfon- (60 s) antiseptics, loss of buffer, high SG
phthalein F(–):Proteins other than albumin, high salt
concentration, microalbuminuria
M: Glucose oxidase, M: green- F(+): Oxidizing agents, detergents
Glucose peroxidase, KI brown F(–): Ascorbic acid, ketones, high SG, low
4. Glucose
oxidase reaction C: Glucose oxidase, C: yellow- temperatures, improperly preserved
peroxidase, TMB green (30 s) specimens
F(+): Phthalein dyes, highly pigmented
Sodium M: Sodium nitroprusside
Purple red urine, levodopa, medications
5. Ketones nitroprusside C: Sodium nitroprusside
(40 s) containing SH group
reaction and glycine
F(–): Improperly preserved specimens
M:Diisopropylbenzene F(+): Strong oxidizing agents, bacterial
Pseudo- dihydroperoxide TMB peroxidases, menstrual contamination
Blue-green
6. Blood peroxidase C: 2,5-dimethyl 2,5- F(–): High SG, crenated cells, formalin,
(60 s)
activity of heme dihydroperoxyhexane captopril, nitrite, ascorbic acid, unmixed
TMB specimen
F(+): Highly pigmented urine, indican,
M: 2,4-dichloroaniline
Tan, pink, phenazopyridine, metabolites of Lodine
diazonium salt
7. Bilirubin Diazo reaction or violet F(–): Specimen exposure to light,
C:2,6-dichlorobenzene
(30 s) ascorbic acid >25 mg/dL, high
diazoniumtetrafluoroborate
concentration of nitrite
F(+): PBG, indican, procaine,
M: paradiethylamino-
p-aminosalicylic acid, sulfonamides,
benzaldehyde
Red methyldopa, chlorpromazine, pigmented
8.Urobilinogen Ehrlich reaction C: 4-methoxybenzene
(60 s) urine
diazonium tetrafluoro-
F(–): Old specimens, formalin, high
borate
concentration of nitrite
F(+): Old specimen, highly pigmented
M: p-arsanilic acid,
urine
tetrahydrobenzoquinolinol
F(–):Nonreductase-containing bacteria,
C: sulfanilamide, 3- Pink
9. Nitrite Greiss reaction lack of urinary nitrate, insufficient contact
hydroxy- 1,2,3,4- (60 s)
time between bacteria and nitrate,
tetrahydro-
bacteria converting nitrite to nitrogen,
7,8-benzoquinoline
antibiotics, ascorbic acid, high SG
M:Derivatized pyerole F(+): Strong oxidizing agents, formalin,
10. Granulocytic amino acid ester, highly pigmented urine, nitrofurantoin
Purple
Leukocyte esterase diazonium salt F(–):  protein, glucose, oxalic acid,
(120 s)
esterase reaction C: Indoxylcarbonic acid ascorbic acid, gentamicin,
ester, diazonium salt cephalosporins, tetracyclines

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5. Microscopic Examination of Urine

A. Sediment Examination Techniques
1. Standardization of Manual Microscopic Technique (refer to QA notes)
2. Sediment Stains
Stain Action/Function
Delineates structure and contrasting colors of the nucleus and cytoplasm; identifies
Sternheimer-Malbin
WBCs, epithelial cells, and casts
Toluidine blue Enhances nuclear detail; differentiates WBCs and RTE cells
Lipid Stains: Oil Red Stains triglycerides and neutral fats orange red; identifies free fat droplets and lipid-
O and Sudan III containing cells and casts
Hansel stain Stains granules of urinary eosinophils
Prussian blue stain Stains structures containing iron; identifies hemosiderin granules in cells and casts
3. Microscopy
Type Function
Bright-field Used for routine urinalysis
Phase-contrast Enhances visualization of elements with low refractive indices e.g. hyaline casts
Polarizing 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 T. pallidum
Fluorescence Used to visualize naturally fluorescent microorganisms or those stained by a fluorescent dye
DIC Produces a three-dimensional microscopy-image and layer-by-layer imaging of a specimen
B. Sediment Constituents
1. Cells
Red blood cells White blood cells Renal tubular ECs Transitional ECs
Biconcave, anucleate discs, Granular, larger than RBCs, Vary in size and shape; Spherical, polyhedral, and
7 µm in diameter ~12 µm in diameter with eccentric nucleus caudate; with central nucleus
Normal: 0-2/hpf Normal: <5/hpf Abnormal morphology
Most clinically significant
Hematuria: glomerulonephritis, Pyuria: urinary tract indicates malignancy or
>2/hpf = tubular injury
renal calculi, malignancy infection or inflammation viral infection
Neutrophils: most common Bilirubin-laden: hepatitis Singly, in pairs, or in
Ghost cells in dilute urine Glitter cells:sparkling Hemosiderin-laden: clumps (syncytia) following
Crenated in hypertonic urine appearance and Brownian hemolytic conditions invasive urologic
Dysmorphic in glomerular movement Oval fat bodies: lipiduria, procedures e.g.
bleeding Eosinophils: AIN nephrotic syndrome catheterization
Mononuclears: graft rejection Bubble cells: ATN
Squamous ECs Bacteria Yeast cells Parasites

Abundant, irregular Gram – rods of family Small, refractile oval T. vaginalis –most common;
cytoplasm and a prominent Enterobacteriaceae, and structures; may resemble a WBC,
central nucleus about the cocci esp. Staphylococcus Candida albicans most transitional, or RTE cell;
size of an RBC and Enterococcus species common causes nonspecific urethritis
True yeast infection
No clinical significance Bacteriuria – indicates UTI S. haematobium – inhabits
accompanied by WBCs;
(except clue cells = covered when seen in conjunction the urinary bladder; ova
seen in diabetes,
with Gardnerella vaginalis with pyuria and a positive have a characteristic
immunocompromised state
coccobacilli) urine culture terminal spine
and vaginal moniliasis
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2. Casts (See Formation and degeneration of casts on page 32)

Hyaline cast Red blood cell cast White blood cell cast RTE cell cast
Orange-red under LPO; Granular appearance, Consists of RTE cells
Colorless with low refractive
more fragile and have a irregular borders, incorporated into the cast
index; prototype of all casts
more irregular shape multilobed nuclei matrix
Normal: 0-2/lpf Indicates Seen in pyelonephritis Advanced tubular
Seen with other pathological glomerulonephritis and (+WBCs and bacteria) and destruction, associated
casts in cases of AGN, damage to the capillary interstitial nephritis with exposure to toxic
CGN, APN, and CHF structure of the nephrons (+eosinophils) agents
Granular cast Waxy cast Broad cast Fatty cast

Matrix contains coarse or High RI; brittle consistency Highly refractile; matrix may
fine granules from the (appear fragmented with Renal failure cast; mostly contain few or many fat
disintegration of cellular jagged ends and granular and waxy formed droplets, and intact oval fat
casts, filtered proteins, or notches);homogenous, dark in the collecting ducts bodies may be attached to
lysosomes secreted by RTE pink with supravital stains the matrix
Extreme urine stasis, Extreme urine stasis and Seen in lipiduria in
Indicates stasis of urine flow indicating CRF; final phase destruction of the tubular conjunction with oval fat
of cast degeneration walls bodies and free fat droplets
3. Crystals
a. Normal and Iatrogenic Crystals in Acidic Urine
Uric acid Amorphous urates Acid urates Sodium urates Calcium oxalate
Macroscopically
Dihydrate – colorless,
Yellow-brown; resemble brick dust Small brown spheres;
Colorless birefringent octahedral envelope
pleomorphic; Microscopically appear may cluster in pairs
needles Monohydrate – oval or
birefringent as yellow-brown and triplets
dumbbell-shaped
granules
Soluble in alkali Soluble in alkali and heat; convert to uric acid crystals when acidified Soluble in dilute HCl
Increased in gout, Commonly seen in Ethylene glycol
Rarely encountered and have little clinical
leukemia, and Lesch- refrigerated poisoning, renal
significance
Nyhan syndrome specimens Calculi
Calcium sulfate Hippuric acid Radiographic dye Sulfonamide crystals Ampicillin crystals
Long, thin colorless Yellow-brown or Flat, colorless, Colorless to yellow-
Colorless needles that
needles or prisms colorless, needles, notched rhombic brown needles,
tend to form bundles
identical to calcium rhombic plates and plates; highly sheaves of wheat,and
following refrigeration
phosphate four-sided prisms birefringent rosettes
Soluble in hot water
Soluble in acetic acid Soluble in 10% NaOH Soluble in acetone ---
and alkali
Rarely seen; Rare; associated with Associated with a Indicates inadequate hydration among
no clinical foods containing markedly elevated SG patients being treated for UTI; may cause
significance benzoic acid (>1.040) tubular damage if crystals form in the nephron
b. Normal Crystals in Alkaline Urine

Amorph. phosphates Calcium phosphate Triple phosphate Ammonium biurate Calcium carbonate
Macroscopically Colorless, flat Prism shape Yellow-brown spicule-
Small, colorless,
appear milky white; rectangular plates or resembling “coffin covered spheres
dumbbell or spherical;
Microscopically thin prisms often in lids”; birefringent described as “thorny
may occur in clumps
granular in appearance rosette formations under polarized light apples”
Soluble in acetic acid Soluble in acetic acid
Soluble in dilute acetic acid
and heat with evolution of gas
Seen in refrigerated Common constituent Presence of urea-splitting bacteria (e.g.
No clinical significance
specimens of renal calculi Proteus, Pseudomonas)
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c. Abnormal Crystals
Cystine Cholesterol Leucine Tyrosine Bilirubin
Colorless,
Rectangular plates Yellow-brown spheres Fine colorless to Clumped needles or
hexagonal plates;
with a notch in one or that demonstrate yellow needles that granules with the
may be confused
more corners; highly concentric circles and frequently form characteristic yellow
with colorless uric
birefringent radial striations clumps or rosettes color
acid crystals
Soluble in acetic acid,
Soluble in ammonia, Soluble in hot alkali or Soluble in alkali or
Soluble in chloroform HCl, NaOH, ether,
dilute HCl alcohol heat
chloroform
Cystinuria; renal Lipiduria (nephrotic
Severe liver disease
calculi formation syndrome)
4. Urinary Sediment Artifacts

a. Starch granules – highly refractile spheres with a dimpled center; resemble fat droplets when polarized,
producing a Maltese cross formation
b. Oil droplets and air bubbles – highly refractile and may resemble RBCs
c. Pollen grains – spheres with a cell wall and occasional concentric circles; large size may cause the
microscopist to be out of focus
d. Hair and fibers – may be mistaken for casts but are longer, more refractile, and are able to polarize light
e. 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
6. Urinalysis Automation
Instrument Tests Principle
1. Semiautomated chemistry Urine chemistries Reflectance photometry
analyzers Specific gravity Reflectance photometry
Reflectance photometry or
Color
spectrophotometry
2. Fully automated chemistry
Clarity Light transmittance or light scatter
analyzers
Urine chemistries Reflectance photometry
Specific gravity Refractive index measurement
3. Automated Microscopy
Urine sediment analysis Flow cytometry
analyzers
Color, clarity, urine
Same as no. 2
chemistries
Refractive index measurement (old
4. Fully automated urinalysis
Specific gravity Yellow IRIS models used harmonic
systems or workstations
oscillation densitometry)
Flow cytometry or intelligent
Urine sediment analysis
microscopy system
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7. Renal Structure, Function, and

Diseases
A. Anatomy of the Kidney
1. Basic Structure (Refer to figures 1 and 2 on page 32)
2. Nephron – basic functional unit of the urinary system, 1-1.5 million per kidney
a. Types
1) Cortical – constitute approximately 85% of the nephrons; located in the cortex of the kidney
2) Juxtamedullary – have longer Henle’s loops that extend deep into the medulla of the kidney
b. Major Parts
1) Glomerulus (Renal Corpuscle) – highly specialized tuft of capillaries
2) Renal tubules
a) Bowman's capsule – forms the beginning of the renal tubules
b) Proximal convoluted tubule – reabsorption of water and essential substances; major site for
removal of nonfiltered substances
c) Loop of Henle – selective reabsorption
i. Descending limb – freely permeable to water but not to solutes
ii. Ascending limb – impermeable to water but permeable to solutes
d) Distal convoluted tubule – reabsorption of Na and H2O continues
e) Collecting duct – further concentration of the filtrate
B. Physiology of Urine Formation

1. Renal Functions
a. Renal Blood Flow
b. Glomerular Filtration
1) Glomerular Filtration Rate (GFR): 120 mL/min
2) Characteristics of the Glomerular Filtrate: SG of 1.010; protein- and cell-free
3) Factors Affecting Glomerular Filtration
a) Cellular Structure
i. Capillary Wall Membrane – made up of endothelial cells that are fenestrated
ii. Basement Membrane/ Basal lamina – further restriction of large molecules and blood cells
iii. Visceral Epithelium of Bowman’s capsule – podocytes with intertwining foot processes
b) Glomerular pressure – 75 mmHg; results from the smaller sizes of the efferent arteriole and
glomerular capillaries
c) Renin-Angiotensin-Aldosterone Mechanism – responds to changes in blood pressure and
plasma sodium content monitored by the juxtaglomerular apparatus
c. Tubular Reabsorption
1) Active Transport – glucose, amino acids, salts, chloride, sodium
2) Passive Transport – water, urea, sodium
3) Counter Current Mechanism – maintains the osmotic gradient of the medulla
d. Tubular Secretion
Major Functions:
1) Elimination of waste products not filtered by the glomerulus
+ – –
2) Regulation of acid-base balance through secretion of H that react with HCO3 , PO4 , and NH3
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C. Evaluation of Renal Functions

1. Glomerular Filtration Tests
a. Clearance Test – standard test to measure the filtering capacity of the glomeruli
b. Methods
1) Urea Clearance – earliest method because of presence in all urine specimens and the existence of
chemical methods of analysis
2) Radioisotope Clearance – involves injection of radionucleotides; not only determines GFR but also
enables visualization of filtration in one or both kidneys
3) β-2 microglobulin Clearance – dissociates from HLA at a constant rate and is rapidly removed
from the plasma; not reliable in patients with history of immunologic disorders or malignancy
4) Cystatin C Clearance – small protein (MW 13,359) produced at a constant rate by all nucleated
cells, readily filtered by the glomerulus, and reabsorbed and broken down by the renal tubular
cells; serum marker recommended for pediatric patients, diabetics, elderly, and critically ill patients
5) Inulin Clearance – reference method; extremely stable substance that is neither reabsorbed nor
secreted by the tubules; exogenous procedure
6) Creatinine Clearance – recommended method; endogenous procedure
a) Calculation
𝐔𝐕
Formula: 𝐂 = where, C=clearance in mL/min; V=urine volume in mL/min;
𝐏
U=urine creatinine in mg/dL; and P=plasma creatinine in mg/dL
𝐔𝐕 𝟏.𝟕𝟑
To adjust for body size: 𝐂 = 𝐱 where, A=body surface area
𝐏 𝐀
b) Normal Values: 75 – 112 mL/min (F); 85 – 125 mL/min (M)

2. Tubular Reabsorption Tests
a. Concentration Tests
1) Fishberg Test – patients are deprived of fluids for 24 hours prior to measuring the specific gravity
2) Mosenthal Test – compares the volume and SG of day and night samples
3) Osmometry – used to measure the osmolarity of serum and urine by comparing a colligative
property value of the sample with that of pure water
a) Normal Values: 275 – 300 mOsm (serum); 50 – 1, 400 mOsm (urine)
b) Clinical Osmometers – FP and VP osmometers
4) U:S Osmolarity Ratio – important in determining whether DI is caused by decreased ADH
production or lack of renal response to ADH
a) should be at least 1:1 under normal random conditions
b) should reach 3:1 after controlled fluid intake or ADH injection
5) Free Water Clearance – an expansion of the U:S Osmolarity Ratio; used to determine the ability of
the kidneys to respond to the state of body hydration
a) Calculation: CH2O = V − Cosm where, CH2O=free water clearance; V=urine volume in mL/min
b) Interpretation of Results
+ value: excess water is excreted
0 value: no renal concentration or dilution would be taking place
– value: less than the necessary amount of water is being excreted
3. Tubular Secretion and Renal Blood Flow Tests
a. Methods
1) Phenolsulfonphthalein (PSP) – not currently performed due to difficulty in standardization and
interpretation of results
2) Indigo Carmine Test – used by urologists as confirmatory test for unilateral kidney disease
3) Para-aminohippuric Acid Test (PAH) – used to measure effective renal plasma flow
4) Titratable Acidity and Urinary Ammonia – used to determine the ability of the kidneys to produce
+
an acid urine through the tubular secretion of H ions and the production and secretion of NH3 by
the cells of the DCT
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D. Renal Disorders
1. Glomerular Disorders
Disorder Etiology Urinalysis results Other tests
1. Glomerulonephritis
a. Acute or Post- Deposition of immune complexes, in Macroscopic hematuria, ASO titer, anti-
streptococcal conjunction with streptococcal proteinuria, RBC casts, group A strepto-
Glomerulonephritis infection, on the glomerular membrane granular casts coccal enzymes
b. Rapidly Progressive Deposition of immune complexes from Macroscopic hematuria, BUN, creatinin,
Glomerulonephritis systemic immune disorders on the proteinuria, RBC casts creatinine
glomerular membrane clearance
c. Goodpasture’s Attachment of a cytotoxic antibody Macroscopic hematuria, Anti-glomerular
Syndrome formed during viral respiratory proteinuria, RBC casts basement
infections to glomerular and alveolar membrane
basement membranes antibody
2. Vasculitis
a. Wegener’s Binding of antineutrophilic cytoplasmic Macroscopic hematuria, ANCA
Granulomatosis antibody to neutrophils in vessel walls proteinuria, RBC casts
b. Henoch-Schonlein Disruption of vascular integrity Macroscopic hematuria, FOBT
Purpura following viral respiratory infections proteinuria, RBC casts
3. IgA Nephropathy Deposition of IgA on the glomerular Early: Macroscopic or Serum IgA
membrane resulting from increased microscopic hematuria
levels of serum IgA Late: hematuria, casts,
proteinuria, glucosuria
4. Membranous Thickening of the glomerular Microscopic hematuria, ANA, HBsAg,
Glomerulonephritis membrane following IgG immune proteinuria FTA-ABS
complex deposition associated with
systemic disorders
5. Membranoproliferative Cellular proliferation affecting the Hematuria, proteinuria Serum
Glomerulonephritis capillary walls or the glomerular complement levels
basement membrane
6. Chronic Marked decrease in renal function Hematuria, proteinuria, BUN, creatinine,
Glomerulonephritis resulting from glomerular damage glucosuria, cellular, creatinine
precipitated by other renal disorders granular casts, waxy, clearance
and broad casts
7. Nephrotic Syndrome Disruption of the electrical charges that Microscopic hematuria, Serum albumin,
produce the tightly fitting podocyte heavy proteinuria, RTE cholesterol, TAG
barrier resulting in massive loss of cells, oval fat bodies, fat
protein and lipids droplets, fatty casts
8. Minimal Change Disruption of the podocytes following Heavy proteinuria, Serum albumin,
Disease allergic reactions and immunizations transient hematuria, fat cholesterol, TAG
droplets
9. Focal Segmental Disruption of podocytes associated Proteinuria, microscopic Drug test, HIV test
Glomerulosclerosis with heroin/analgesic abuse and AIDS hematuria
2. Vesicotubulointerstitial Disorders
10. Acute Tubular Damage to RTE cells caused by Microscopic hematuria, Hemoglobin,
Necrosis ischemia or toxic agents proteinuria, RTE cells, RTE hematocrit,
cell casts cardiac enzymes
11. Fanconi’s Syndrome Inherited in association with cystinosis Glucosuria, possible cystine Serum and urine
and Hartnup disease or acquired crystals electrolytes,
(exposure to toxic agents) chromatography
12. Cystitis Ascending bacterial infection of the Leukocyturia, bacteriuria, Urine culture
bladder microscopic hematuria, mild
proteinuria, increased pH
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13. Acute Pyelonephritis Infection of the renal tubules and Leukocyturia, bacteriuria, Urine and blood
interstitium related to interference of WBC casts, bacterial casts, cultures
urine flow or reflux of urine from the microscopic hematuria,
bladder, and untreated cystitis proteinuria
14.Chronic Pyelonephritis Recurrent infection of the renal Leukocyturia, bacteriuria, BUN, creatinine,
tubules and interstitium caused by WBC, bacterial, granular, creatinine
structural abnormalities affecting the waxy, and broad casts, clearance
flow of urine hematuria, proteinuria
15. Acute Interstitial Allergic inflammation of the renal Hematuria, proteinuria, Urine eosinophils,
Nephritis interstitium in response to certain leukocyturia, WBC casts BUN, creatinine,
medications creatinine
clearance
16. Renal Failure May be gradual progression from the Proteinuria, renal Creatinine
original disorder to chronic renal glycosuria, abundance of clearance, BUN,
failure or end-stage renal disease granular, waxy, and broad creatinine,
casts (telescoped sediment) electrolytes
8. Renal Calculi
A. Renal Lithiasis
17. Renal Deposition of renal calculi or kidney Microscopic hematuria Chemical
Lithiasis stones in the calyces and pelvis of the analysis of
kidney, ureters and urinary bladder kidney stones
B. Examination of Renal Calculi

1. Chemical Composition of Renal Calculi
a. Calcium oxalate or phosphate
b. Magnesium ammonium phosphate
c. Uric acid
d. Cystine
2. Qualitative Examination of Renal Calculi
a. Physical Examination (appearance, size/weight)
b. Chemical Examination
1) Pulverize the stone.
2) Ash a small portion over a hot burner.
3) Distribute the remaining pulverized stone into 7 tubes as follows:
Tube Reagents Positive Result
1 Uric Acid 20% sodium carbonate, Folin reagent Deep blue
2 Carbonate and Oxalate 10% HCl, MnO2 Bubble formation
3 Phosphate ammonium molybdate, 25% HNO3 Yellow
4 Calcium 10% HCl, 20% NaOH White cloud
5 Magnesium 10% HCl, 20% NaOH, p-nitrobenzene Blue
azoresorcinol
6 Ammonium 10% HCl, 20% NaOH, Nessler’s solution Orange-brown
7 Cystine 28% NH4OH, 5% NaCN, 5% sodium nitroprusside Red-purple
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9. Urine Screening Tests

A. Major Disorders of Metabolism
Abnormal urinary
Disorder Etiology Clinical manifestations
constituent
I. Amino Acid Disorders
A. Phenylalanine – Tyrosine Disorders
1. Phenylketonuria Phenylalanine hydroxylase Phenylalanine, Mousy odor of urine, mental
deficiency phenyllactic acid, retardation
phenylpyruvic acid
2. Tyrosinuria Tyrosine transaminase or p-hydroxyphenylpyruvic Tyrosine and leucine
p-hydroxy phenylpyruvic acid acid and p-hydroxy- crystals in urine; liver and
oxidase deficiency; liver phenyllactic acid renal disease (temporary or
disease or under- permanent)
development of the liver
3. Alkaptonuria Homogentisic acid oxidase Homogentisic acid Dark blue to black
deficiency pigmentation of cartilage
and connective tissues, liver
and cardiac disorders
4. Melanuria Overproliferation of melanin- Melanin Malignant melanoma
producing cells
B. Branched Chain Amino Acid Disorders
1. Maple Syrup Deficiency in decarboxylases Ketoacids Maple syrup odor of urine,
Urine Disease and other enzymes needed (α-ketoisovaleric; failure to thrive, mental
(MSUD) for the conversion of keto- α-ketoisocaproic; retardation
amino acids to fatty acids α-keto,β-methylvaleric)
2. Organic Acidemias
a. Isovaleric Isovaleryl CoA in the leucine Isovalerylglycine Sweaty feet odor of urine
Acidemia pathway
b. Propionic and Error in the metabolic Propionic and Early severe illness,
Methylmalonic pathway converting valine, methylmalonic acid vomiting, metabolic acidosis,
acidemias isoleucine, threonine, and hypoglycemia, increased
methionine to succinyl CoA serum ammonia, ketonuria
C. Tryptophan Disorders
1. Indicanuria Intestinal disorders Indican “Blue diaper syndrome”
Hartnup disease Generalized aminoaciduria
(Fanconi’s syndrome)
2. Abnormal Urinary Carcinoid tumors involving 5-HIAA Vascular and
Excretion of 5-HIAA the argentaffin cells of the (>25 mg/24 hours) bronchospastic symptoms
intestine (carcinoid syndrome)
D. Cystine Disorders
1. Cystinuria Defect in renal tubular Cystine, lysine, Sulfur odor of urine
reabsorption arginine, ornithine or Cystine crystals in urine
Cystine and lysine only Formation of urinary calculi
Fanconi’s syndrome
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2. Cystinosis Inborn error of metabolism Cystine Deposition of cystine

crystals in many areas of the
body
3. Homocystinuria Cystathionine β-synthase Homocystine and Failure to thrive, cataracts,
deficiency methionine mental retardation,
thromboembolic problems
II. Porphyrin Disorders Inborn error of metabolism or ALA, porphobilinogen, Port wine color of urine
acquired through erythrocytic uroporphyrin, Neurologic/psychiatric
and hepatic malfunctions or coproporphyrin symptoms
exposure to toxic agents Cutaneous photosensitivity
III. Mucopolysaccharide Inborn error of metabolism Dermatan sulfate, Mental retardation
Disorders keratan sulfate, Accumulation of
heparan sulfate mucopolysaccharides in the
cornea of the eyes
Abnormal skeletal structure
IV. Purine Disorders
* Lesch-Nyhan Disease Hypoxanthineguanine Uric acid Severe motor defects,
phosphoribosyl transferase mental retardation, tendency
deficiency toward self destruction,
gout, renal calculi, “orange
sand in diapers”
V. Carbohydrate Disorders/Meliturias
* Galactosuria Inborn error of metabolism Galactose Failure to thrive, liver
(GALT, galactokinase, or disorders, cataracts, severe
UDP-galactose-4-epimerase mental retardation
deficiency)
C. Summary of Screening Tests for Metabolic Disorders

Test Disorders Observation
Phenylketonuria and 5-HIAA Blue-green
Tyrosinuria Transient green
Alkaptonuria Transient blue
Ferric Chloride Tube Test
Melanuria Gray-black
MSUD Green-gray
Indicanuria Violet-blue with CHCl3
2,4-Dinitrophenylhydrazine Phenylketonuria, MSUD, Tyrosinuria, Acidemias Yellow
Phenylketonuria Green-gray
Phenistix
Tyrosinuria Transient green
Melanuria Red
Acetest
MSUD, Acidemias Purple
Tyrosinuria, MSUD Red
Nitrosonaphthol
5-HIAA Violet with HNO3
Cyanide nitroprusside Cystine disorders Red-purple
Alkaptonuria Black
Silver nitroprusside
Homocystinuria Red-purple
Clinitest Cystinosis, Melituria Orange-red
Sodium nitroprusside Melanuria Red
p-nitroaniline Methylmalonic academia Emerald green
CTAB Test Mucopolysaccharidoses White turbidity
Metachromatic staining Mucopolysaccharidoses Blue spot
Guthrie Test Phenylketonuria Growth of B. subtilis
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10. Pregnancy Testing

A. Types of Pregnancy Test
1. Immunoassays
a. Principle: Detection of hCG using monoclonal antibodies.
b. Methods
1) Agglutination Immunoassays – direct or agglutination-inhibition
2) Competitive Radioimmunoassay
a) Principle: serum hCG and the radiolabeled hCG compete for the binding of anti-hCG
b) Sensitivity – 5 mIU/mL
3) EIA (Sandwich ELISA)
a) Principle: Detection of hCG based on color indicator reaction mediated by an enzyme (e.g.
ALP); commonly used in home-based pregnancy tests
b) Sensitivity – 2-5 mIU/mL
4) Immunochromatography (lateral flow tests)
a) Principle: The labeled antibody-dye conjugate in the reaction zone binds to the hCG in the
specimen forming an antibody-antigen complex. This complex binds to the anti-hCG antibody
in the test zone and produces a colored band when the hCG concentration is equal to or
greater than 20 mIU/ml. In the absence of hCG, the reaction mixture continues flowing
through the absorbent device past the test zone allowing the binding of unbound conjugates
to the reagent in the control zone.
b) Interpretation of Results:
2. Bioassays
a. Principle: Introduction of hCG to a test animal produces characteristic changes in reproductive organs.
b. Methods
Test Animal Route of Injection Positive Result
1) Aschheim- Immature female Enlargement of the ovary; corpora
Subcutaneous
Zondek mouse hemorrhagica and corpora lutea
2) Friedmann/ Hyperemia of the uterus and
Female virgin rabbit Free marginal ear vein
Hoffman corpora hemorrhagica
Subcutaneous (into the Oogenesis (Extrusion of eggs 8-12
3) Hogben Female frog
dorsal lymph sac) hours after injection)
Intramuscular Spermatogenesis (Release of
4) Galli-Mainini Male frog
(gastrocnemius) sperm cells into the cloaca)
5) Frank-Berman Female virgin rat Subcutaneous Ovarian hyperemia
6) Kupperman Female virgin rat Intraperitoneal Ovarian hyperemia
B. Sources of Errors
1. False-positive: production of hCG in the pituitary; tumors characterized by significant amounts of hCG;
ectopic pregnancy and incomplete abortion; intake of chlorpromazine, phenothiazine, and aspirin
2. False-negative: low titer or concentration of hCG; low sensitivity of test animal or assay method; use of
toxic urine (bacterial contamination, increased electrolyte levels, salicylates, and barbiturates)
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11. Cerebrospinal Fluid

 Imperfect ultrafiltrate of plasma produced in the choroid plexuses within the ventricles of the brain at a
rate of approx. 500 mL/day maintaining a total volume of 90-150 mL in adults or 10-60 mL in neonates.
A. 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
Order of draw, tests, and manner of storage
Tube Tests Storage
1 Chemistry and immunology Frozen
2 Microbiology Room temperature
3 Cell count and differential Refrigerated
4 Additional tests Depends on test to be performed
B. Gross Examination
1. Appearance
a. Normal: colorless and crystal clear
b. Variations:
1) Hazy, cloudy, turbid, milky or purulent – WBCs and microorganisms, proteins in disorders that
affect blood-brain barrier, and production of IgG within the CNS
2) Oily – radiographic contrast media
3) Clotted – proteins in disorders affecting blood-brain barrier; traumatic tap
4) Bloody – traumatic tap or subarachnoid hemorrhage
Traumatic tap Hemorrhage
Distribution of blood Uneven Even
Supernatant Clear and colorless Xanthochromic
Clot formation Present Absent
Erythrophagocytosis Absent Present
D dimer test Negative Positive
5) Xanthochromic – indicates presence of RBC degradation products or other pigments
Supernatant color Associated diseases/disorders
Pink RBC lysis/hemoglobin breakdown products
Yellow RBC lysis/hemoglobin breakdown products
Hyperbilirubinemia
CSF protein > 150 mg/dL (1.5 g/L)
Orange RBC lysis/hemoglobin breakdown products
Hypervitaminosis A (carotenoids)
Yellow-green Hyperbilirubinemia (biliverdin)
Brown Meningeal metastatic melanoma
2. Viscosity
a. Normal: similar to that of water
b. Clinical significance of viscous CSF: metastatic mucin-producing adenocarcinomas, cryptococcal
meningitis, liquid nucleus pulposus
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C. Chemical Examination
1. Total protein
a. Normal values: 15 – 45 mg/dL or less than 1% of the plasma level
b. Methods:
1) Turbidimetry – based on TCA or SSA and sodium sulfate protein precipitation; uses benzethonium
or benzalkonium chloride as precipitating agents in automated methods and micromethods
2) Dye-Binding techniques – uses Coomassie brilliant blue or Ponceau S
3) Colorimetric-spectrophotometric methods – based on Lowry or Biuret method
4) Immunoassays – used to measure the amount of specific proteins e.g. MBP
5) Electrophoresis – detection of oligoclonal bands in the gamma region
c. Protein fractions
 Prealbumin, albumin, transferring, α-globulins (haptoglobin, ceruloplasmin, α2-macroglobulin),
gamma globulins (IgG, IgA), tau protein (carbohydrate-deficient transferrin fraction)
 Not present in significant amounts: fibrinogen, IgM, β-lipoprotein
2. Albumin and IgG Measurements
a. CSF/Serum albumin index– reflects the integrity or permeability of the blood-brain barrier;
values < 9 = intact BBB
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑛𝑖𝑛 (𝑚𝑔/𝑑𝐿)
𝐶𝑆𝐹/𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 𝑖𝑛𝑑𝑒𝑥 =
𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿)
b. CSF IgG index – calculated to measure IgG synthesis within the CNS
values > 0.70 = IgG production within the CNS
𝐶𝑆𝐹 𝐼𝑔𝐺 (𝑚𝑔/𝑑𝐿)/𝑠𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 (𝑔/𝑑𝐿)
𝐼𝑔𝐺 𝑖𝑛𝑑𝑒𝑥 =
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑚𝑔/𝑑𝐿)/𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿)
3. Glucose
a. Normal values: 50–80 mg/dL
b. Important consideration: Blood sample should be drawn about 2 hours prior to spinal tap to allow time
for equilibration between the blood and fluid glucose.
4. Lactate
a. Normal values: <25 mg/dL
b. Important consideration: Xanthochromic or hemolyzed fluid will produce falsely elevated results
Chemical results for the differential diagnosis of meningitis
Bacterial Tubercular Fungal Viral
Protein Markedly elevated Moderate to Moderate to Moderately
markedly elevated markedly elevated elevated
Glucose Markedly decreased Decreased Normal to Normal
(usually ≤40 mg/dL) (may be <45 mg/dL) decreased
Lactate > 35 mg/dL > 25 mg/dL > 25 mg/dL Normal
5. Glutamine
a. Normal values: 8 to 18 mg/dL
b. Clinical significance of elevated levels: liver disorders that result in increased blood and CSF
ammonia, Reye’s Syndrome, coma of unknown origin, and disturbance of consciousness
6. Enzymes
a. CK-BB isoenzyme – increases about 6 hours following ischemic or anoxic insult
b. LD – increased in patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis,
and subarachnoid hemorrhage
c. ADA – increased in tubercular meningitis (abundant in T lymphocytes)
d. Lysozyme – significantly increased in patients with both bacterial and tubercular meningitis
D. Microscopic Examination
1. Total cell count
 Dilution (using normal saline), counting, and calculation of the number per µL are done using the same
procedure as WBC count; Fuchs-Rosenthal–type chamber may be used
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2. WBC count
a. Normal values: <5/µL (adult), <30/µL (neonates)
b. Recommended Dilutions:
Amount of Diluent
Clarity Dilution Amount of sample
(3% acetic acid)
Slightly hazy 1:10 270 µL
Hazy 1:20 570 µL
30 µL
Slightly cloudy 1:100 2, 970 µL
Slightly bloody 1:200 5, 970 µL
Cloudy, bloody, turbid 1:10,000 1 mL of 1:100 dilution 9 mL
c. Calculation:
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑊𝐵𝐶/µ𝐿 =
𝑁𝑜. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑎𝑐𝑕 𝑠𝑞𝑢𝑎𝑟𝑒
3. Differential Cell Count
Predominant cells seen in CSF
Type of Cell Clinical Significance
Lymphocytes and Monocytes Normal (70:30 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
E. Microbiologic and Serologic Examination

1. Gram stain and culture
2. Limulus lysate test
3. Bacterial agglutination tests (immunochromatographic assay, latex agglutination)
4. Nucleic acid amplification tests (PCR and 16S rRNA sequencing)
5. Acid-fast stain or fluorescent antibody stains and culture
6. DOT ELISA
7. India ink or nigrosin stain
8. Latex agglutination test (detection of cryptococcal antigen)
12. Semen
 Composition: spermatozoa 5%, seminal fluid 60%–70%, prostatic fluid 20%–30%, and bulbourethral
secretions 5%
a. Masturbation – recommended method
b. Condom method – Silastic or nonlubricant-containing rubber or polyurethane condoms
c. Vaginal vault aspiration
d. Coitus interruptus
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2. Important considerations
a. Patient preparation – sexual abstinence of 2 to 5 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 1 hour of collection
d. Fertility testing – 2-3 samples tested at 2-week intervals; 2 abnormal samples considered significant
Parameter Findings Clinical significance
1. Appearance Gray-white, translucent Normal
Yellow Pyospermia, contamination, prolonged abstinence, medications
Red/ rust color Bleeding
Turbid Infection
2. Volume 2–5 mL Normal
<2 mL Improper functioning of one of the semen-producing organs
>5 mL Prolonged abstinence
3. Liquefaction 30 to 60 min Normal
>2 hours Deficiency in prostatic enzymes
4. Viscosity Pours in droplets Normal
Clumped, stringy, or gel-like Deficiency in prostatic enzymes
5. pH 7.2 to 8.0 Normal
>8.0 Infection within the reproductive tract
<7.2 Increased prostatic fluid
C. Microscopic Examination
1. Sperm concentration and count
a. Recommended procedure:
 Dilution: 1:20 using sodium bicarbonate in formalin, saline, or distilled water
 Both sides of a Neubauer counting chamber are loaded and counted.
 Calculation: When using the above procedure and counting in the typical WBC squares,
Sperm/mL = sperm counted (average of 2 sides) x 100,000
When using the above procedure and counting in the typical RBC squares,
Sperm/mL = sperm counted (average of 2 sides) x 1,000,000
To solve for total sperm count,
Sperm/ejaculate = sperm/mL x specimen volume
b. Normal values: >20 million/mL (concentration); >40 million/ejaculate (count)
c. Clinical significance of abnormal counts: azoospermia, necrospermia, oligospermia
2. Sperm motility
 Performed on well mixed, undiluted, liquefied semen within 1 hour of collection
 Speed and direction are both evaluated using approximately 20 hpfs
Grade Interpretation
4 Rapid, straight line motility
3 Slower speed, some lateral movement
2 Slow forward progression, noticeable lateral movement
1 No forward progression
0 No movement
b. Normal % motility: >50% within 1 hr, quality >2.0
c. Clinical significance of abnormal motility: midpiece and tail abnormalities
3. Sperm morphology
 Evaluation is done on a thin smear stained using Wright’s, Giemsa, or Papanicolaou (best stain)
 200 sperm are evaluated under OIO for abnormalities in the head, midpiece, and tail
 Strict criteria: oval-shaped head (approx. 5 x 3 µm) tail approx. 45 µm long
acrosomal cap normal in size no big cytoplasmic droplet
 % Normal forms: >14% (strict criteria), >30% (routine criteria)
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D. Additional Testing
Additional tests for abnormal semen analysis
Clinical significance of
Routine semenalysis findings Additional test
abnormal result
Decreased motility with normal count Eosin-nigrosin stain Necrospermia
Decreased count Fructose level determination Lack of support medium
MAR, Immunobead test,
Decreased motility with clumping Male antisperm antibodies
Agglutination with male serum
Normal analysis with continued infertility Agglutination with female serum Female antisperm antibodies
Sperm function tests
Test Function evaluated
Hamster egg penetration Ovum penetration
Cervical mucus penetration Ability to penetrate partner’s midcycle cervical mucus
Hypo-osmotic swelling test Membrane integrity and sperm viability
In vitro acrosome reaction Ability to produce enzymes essential for ovum
penetration
Chemical tests
Test Normal values Significance of decreased values
Neutral α-glucosidase ≥20 mU/ejaculate Disorder of the epididymis
Zinc ≥2.4 µmol/ejaculate
Citric acid ≥52 µmol/ejaculate Lack of prostatic fluid
Acid phosphatase ≥200 Units/ejaculate
13. Synovial Fluid

 Imperfect ultrafiltrate of plasma less than 3.5 mL in volume containing hyaluronic acid and a small
amount of protein produced by the synoviocytes
Specimen collection
a. Arthrocentesis – volume usually collected is about 25 mL
b. Tubes and tests performed
1) sterile heparinized tube – Gram stain and culture
2) sodium heparin or liquid EDTA tube – cell counts
3) nonanticoagulated tube – chemical and serologic tests
4) sodium fluoride tube – glucose analysis
1. Color
a. Normal: colorless to pale yellow
b. Variations: green tinge (septic arthritis), deep yellow (inflammatory and noninflammatory disorders),
red-brown (pathologic hemarthrosis)
2. Clarity
a. Normal: clear
b. Variations: turbid (leukocytes, fibrin, rice bodies, metal and plastic particles from patients with joint
prostheses, or cartilage fragments in osteoarthritis), milky and opalescent (crystals), ground pepper
appearance (pigmented cartilage fragments resulting from a metabolic disorder)
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3. Viscosity
a. Falling drop – ability to form a string 4–6 cm long
b. Ropes/Mucin clot test – 2% to 5% acetic acid is added to the fluid and the formation of clot is
observed; results are reported as good (solid clot), fair (soft clot), low (friable clot), and poor (no clot).
Clinical significance of synovial fluid chemistry results
Test Normal values Clinical significance
Glucose 70-110 mg/dL Decreased in inflammatory or septic arthritis
Total protein < 3 g/dL Increased in inflammatory and hemorrhagic arthritis
Lactate < 30 mg/dL Increased in septic arthritis
Uric acid 2–8 mg/dL Increased in gouty arthritis
Differential diagnosis of joint disorders
Group I Group II Inflammatory Group III Group IV
Finding
Noninflammatory Immunologic Crystal-induced Septic Hemorrhagic
Viscosity Good Poor Low Variable Low
Glucose Normal Decreased Decreased Decreased Normal
WBCs/µL <1000 2000–75000 Up to 100000 50000–100000 Equal to blood
PMNs (%) <30 >50 <70 >75 <50
1. WBC count
a. Normal values: < 200 cells/µL
b. Dilutions: done only on turbid or bloody fluids using 0.3% saline or 1% saponin in saline
c. Clinical significance of elevated counts: crystal-induced arthritis, chronic inflammatory arthritis, and
septic arthritis
2. Differential count
* Incubation of the fluid with hyaluronidase and cytocentrifugation are recommended.
Cells and inclusions seen in synovial fluid
Cell/Inclusion Comments/Clinical significance
Neutrophil Normally < 25% of the differential; increased in septic and crystal-induced arthritis
Lymphocyte Normally 15% of the differential; increased in nonseptic inflammation
Macrophage Normall seen; increased in viral infections
Synovial lining cell Normally seen; similar to macrophage but may be multinucleated resembling a
mesothelial cell
LE cell Neutrophil containing characteristic ingested round body; seen in lupus erythematosus
Reiter cell Vacuolated macrophage with ingested neutrophils; seen in Reiter syndrome
RA cell (ragocyte) Neutrophil with dark cytoplasmic granules containing immune complexes; seen in RA
Cartilage cells Large, multinucleated cells; seen in osteoarthritis
Rice bodies Macroscopically resemble polished rice; microscopically show collagen and fibrin; seen
in tuberculosis, septic and rheumatoid arthritis
Fat droplets Refractile globules stained with Sudan dyes; seen in traumatic injury and chronic
inflammation
Hemosiderin Inclusion within clusters of synovial cells; seen in pigmented villonodular synovitis
3. Crystal examination
a. Compensated polarized light – used to demonstrate crystal polarization by placing a red
compensator between the crystal and the analyzer
b. Betamethasone acetate corticosteroid – control slide for the polarization properties of MSU
c. Negative birefringence – yellow color under a compensated polarized light produced by a crystal
when it is aligned to the slow vibration of the compensated polarized light
d. Positive birefringence – blue color produced by a crystal when it is aligned to the slow vibration of
the compensated polarized light
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Characteristics of synovial fluid crystals

Crystal Shape Birefringence Significance
Monosodium urate Needles Negative Gout
Calcium pyrophosphate Rhombic square, rods Positive Pseudogout
Cholesterol Notched, rhombic plates Negative Chronic inflammation
Corticosteroid Flat, variable-shaped plates Positive and negative Injections
Calcium oxalate Envelopes Negative Renal dialysis
Calcium phosphate (apatite) Small particles None Osteoarthritis
D. Microbiologic and Serologic Examination

1. Septic arthritis: Gram stain and culture, PCR
2. Immunologic arthritis: Test for RF, ANA, and antibodies to B. burgdorferi; CRP and complement levels
14. Serous Fluids

 Ultrafiltrates of plasma contained within the pleural, pericardial, and peritoneal cavities with no
additional material from membrane cells.
1. Collection techniques: thoracentesis, pericardiocentesis, paracentesis
2. Volume collected: > 100 mL
3. Collection tubes: a. EDTA – cell counts and differential
b. sterile heparinized – microbiology and cytology procedures
c. nonanticoagulated or heparinized – chemistry tests
B. Differentiation of Transudates and Exudates

Transudate Exudate
Clarity Clear Depends on condition
Color Colorless to pale yellow Depends on condition
Spontaneous clotting No Possible
pH Alkaline Acidic
Specific gravity < 1.015 > 1.015
Glucose As in plasma level Lower than plasma level
Total protein < 3 g/dL > 3 g/dL
LDH < 200 IU/L > 200 IU/L
Pleural fluid cholesterol < 45 mg/dL > 45 mg/dL
PF:serum cholesterol ratio < 0.3 > 0.3
PF:serum bilirubin ratio < 0.6 > 0.6
Fluid:serum protein ratio < 0.5 > 0.5
Fluid:serum LD ratio < 0.6 > 0.6
Serum-ascites albumin gradient > 1.1 < 1.1
WBC count < 1000/µL > 1000/µL
C. Gross Examination
1. Appearance
a. Normal: clear and colorless to pale yellow
26

lOMoARcPSD|4642032
b. Variations:
1) Turbid, white – microbial infection
2) Bloody or milky
Clinical significance of bloody and milky effusions
Bloody Hemothorax Hemorrhagic exudates
Distribution of blood Uneven/streaked Even
Hematocrit >50% of blood hematocrit <50% of blood hematocrit
Milky Chylous Pseudochylous
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
Clinical significance of other abnormal gross presentations
Serous fluid Appearance Disease
Pleural Brown Rupture of amoebic liver abscess
Black Aspergillus infection
Viscous Malignant mesothelioma
Pericardial Grossly bloody Cardiac puncture, anticoagulant medications
Peritoneal Green Gallbladder, pancreatic disorders
D. Chemical Examination
Clinical significance of other chemistry tests
Serous fluid Test Significance
Pleural Glucose Rheumatoid inflammation and purulent infection
Lactate Bacterial infection
Triglyceride Chylous effusions
pH Pneumonia; esophageal rupture
Adenosine deaminase Tuberculosis and malignancy
Amylase Pancreatitis, esophageal rupture, and malignancy
Pericardial Adenosine deaminase Tubercular pericarditis
Peritoneal Glucose Tubercular peritonitis and malignancy
Amylase Pancreatitis and gastrointestinal perforation
Alkaline phosphatase Gastrointestinal perforation
BUN and creatinine Ruptured or punctured bladder
Adenosine deaminase Tubercular peritonitis
Differential count: routinely performed to examine WBCs and demonstrate malignant cells
Clinical significance of cells seen in serous fluids
Serous fluid Test Significance
Pleural Mesothelial cells Decreased in tuberculosis
Plasma cells Increased in tuberculosis
Peritoneal WBC >500 cells/µL Bacterial peritonitis, cirrhosis
RBC >100,000/µL Intraabdominal bleeding (blunt trauma injury)
Absolute granulocyte count >250 cells/µL Bacterial peritonitis
E. Microbiologic and Serologic Examination

1. Gram staining and culture
2. PCR
3. Acid-fast staining
4. Tumor markers and cytologic examination
5. Measurement of RF/SLE titer and complement levels
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15. Amniotic Fluid

st rd
 Volume: approximately 35 mL during the 1 trimester, peaks during the 3 trimester (approx. 1 L)
Polyhydramnios – results from failure of the fetus to begin swallowing; indicates fetal distress
Oligohydramnios – due to increased fetal swallowing, urinary tract deformities, and membrane leakage
th
a. Amniocentesis– transabdominal or transvaginal; performed after the 14 week of gestation
b. Volume collected: 30 mL
2. Specimen handling
a. Fluid for FLM tests – transported in ice and refrigerated up to 72 hours prior to testing; filtration or low-
speed centrifugation is recommended to prevent loss of phospholipids
b. Fluid for cytogenetic test – incubated at 37°C prior to analysis to prolong the life of the cells
c. Fluid for chemical testing – separated from cellular elements and debris ASAP
d. Fluid for bilirubin analysis – placed in amber bottles
Clinical significance of amniotic fluid appearance
Appearance Significance
Colorless Normal (may show slight to moderate turbidity)
Blood-streaked Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Yellow Hemolytic disease of the newborn
Dark green Meconium
Dark red-brown Fetal death
C. Tests
1. Tests for Fetal Lung Maturity
Normal
Test Method/Principle Comments
values
Thin-layer Sphingomyelin used as internal standard; greatly
1. L/S ratio ≥2.0
chromatography affected by blood and meconium contamination
Agglutination Uses antisera specific to phosphatidylglycerol; not
2. Amniostat-FLM Positive
immunoassay affected by blood and meconium contamination
3. Foam Stability Index Modified foam-shake 95% ethanol used as anti-foaming agent ≥47
4. Microviscosity Fluorescence- polarization Albumin used as internal standard ≥55 mg/g
5. Lamellar body count Resistance pulse counting Uses the platelet channel of hematology analyzers ≥32,000/mL
6. OD at 650 nm Spectrophotometry Requires centrifugation at 2000 g for 10 min ≥0.150
2. Tests for Fetal Distress
Test Method/Principle Comments Normal values
1. Bilirubin ΔA450 plotted on a Liley graph to determine seveity of
Spectrophotometry <0.025
ΔA450 HDN; Hgb and meconium interfere
2. AFP Immunoassay Screening test for NTDs <2.0 MoM
Confirmatory test for NTDs; greatly affected by blood
3. AChE Spectrophotometry Undetectable
contamination
28

lOMoARcPSD|4642032
16. Feces
A. Gross Examination
Significance of stool macroscopic characteristics
Color Clinical significance
Brown Normal (stercobilin, urobilin)
Black Upper GI bleeding, iron therapy, charcoal, bismuth
Red Lower GI bleeding, beets and food coloring, rifampin
Pale yellow, white, or gray Bile-duct obstruction, barium sulfate
Green Biliverdin, oral antibiotics, green vegetables
Appearance Clinical significance
Soft to well-formed Normal
Watery Diarrhea
Small, hard Constipation
Bulky, frothy Bile-duct obstruction, pancreatic disorders
Ribbon-like, slender Intestinal constriction
Mucoid, blood-streaked Colitis, dysentery, malignancy, constipation
B. Chemical Examination
Fecal chemistry tests
Test Method/principle Reagent Result Significance
Pseudoperoxidase
FOBT Guaiac Blue color GI bleeding
activity of hemoglobin
Fecal fat extraction and NaOH 1-6 g/day Normal
Van de Kamer
titration of fatty acids (titrant) >6 g/day Steatorrhea
Differentiation between Pink Presence of HbF
Apt test NaOH
fetal and maternal blood Yellow-brown Presence of HbA
Gelatin on an Clearing Normal
Trypsin Gelatin hydrolysis
x-ray film Absence of clearing Pancreatic insufficiency
Carbohydrates Copper reduction Clinitest Orange-red (0.5 g/dL) Carbohydrate intolerance
Significance of stool microscopic findings
Test Method/Principle Significance Other comments
Examination of eosin-stained >10 undigested muscle fibers Only fibers with visible
Muscle fibers smear to visualize muscle indicate pancreatic vertical and horizontal
fiber striations insufficiency striations are counted
Examination of direct smear 60 large orange-red droplets
Detects neutral fats only
Qualitative stained with Sudan III indicates malabsorption
fecal fats Examination of smear heated 100 orange-red droplets (6–75 Represents total fat
with acetic acid and Sudan III µm) indicates malabsorption content
Fecal Microscopic count of 3/hpf indicates invasive
70% sensitive
neutrophils neutrophils in stained smear condition
*Lactoferrin latex immunoassay – detects secondary granule component of neutrophils; remains sensitive in
refrigerated and frozen specimens
29

lOMoARcPSD|4642032
17. Sputum and BAL

Specimen collection
a. Expectoration – first morning; may require induction (10% NaCl, acetylcysteine, sterile distilled water)
b. Bronchoalveolar lavage – infusion of saline through a bronchoscope followed by aspiration
c. Throat swab/ Cough plate or cough swab method
d. Endotracheal aspiration
B. Gross Examination of Sputum

1. Color
a. Yellow/yellow-green – presence of pus (pulmonary TB, chronic bronchitis)
b. Bright green – jaundice, caseous pneumonia, Pseudomonas infection, rupture of liver abscess
c. Red/bright red – recent hemorrhage (acute cardiac or pulmonary infarction, neoplasm invasion)
d. Rust-colored – decomposed hemoglobin (lobar or pneumococcal pneumonia, pulmonary gangrene)
e. Brown – congestive heart failure
f. Olive green/grass green – chronic cancer
g. Black – dust particles, carbon or charcoal, heavy smokers, anthracosis
2. Macroscopic Structures
a. Cheesy masses – fragments of necrotic pulmonary tissue that range in size from pinpoint to pea-size;
seen in pulmonary gangrene, pulmonary TB, and lung abscess
b. Dittrich’s plugs – yellowish or gray caseous materials about the size of a pinhead that give a foul odor
when crushed; seen in bronchiectasis, putrid bronchitis, and bronchial asthma
c. Pneumoliths/Broncholiths/Lung stones – small white or gray fragments from the calcification of
infected and necrotic tissue within the bronchial cavity; seen in chronic PTB and histoplasmosis
d. Bronchial casts – white or gray branching tree-like casts from the bronchioles; seen in lobar
pneumonia and fibrinous bronchitis
e. Mycetomas – rounded masses of fungal elements seen in Aspergillus infection
C. Microscopic Examination of Sputum

Microscopic Structures
a. Curschmann’s spirals – spirally twisted mucoid strands frequently coiled into little balls
b. Elastic fibers – refractile fibers shed off during the cougning out process; indicates destructive disease
c. Crystals
1) Charcot-Leyden – hexagonal, needle-like or bipyramidal crystals; seen in bronchial asthma
2) Hematoidin – rhombic-shaped crystals; seen in pulmonary infarction and lung abscess
3) Cholesterol – notched plates; seen in lung abscess
d. Cells and Inclusions
1) Creola bodies – bronchial epithelial cells with vacuolated cytoplasm and ciliated borders
2) Carbon-laden or dust cells – contain black granules; seen in pneumoconioses
3) Heart failure cells/siderophages – hemosiderin-laden cells seen in CHF and alveolar hemorrhage
D. Microscopic Examination of Bronchoalveolar Lavage

1. Cell count
 Appropriate dilution for WBC and RBC counts using the Unopette system
 Both sides of the hemocytometer are counted and the average of the two sides is calculated using the
standard Neubauer formula
2. Differential count
 slides are prepared by cytocentrifugation; 500 to 1000 cells (or at least 300) are counted and classified
30

lOMoARcPSD|4642032
Cells and inclusions seen in BAL specimens

Cells/inclusions Clinical significance
Interstitial lung disease, drug reactions, pulmonary
Lymphocytes
lymphoma, and nonbacterial infections
Cigarette smokers, bronchopneumonia, toxin exposure,
Neutrophils
and diffuse alveolar damage
Asthma, drug-induced lung disease, parasitic infections,
Eosinophils
hypersensitivity, pneumonitis, and eosinophilic pneumonia
Erythrocytes, erythrophages, siderophages Alveolar hemorrhage
Bronchial epithelial cells Normal; more numerous in bronchial wash specimens
Sulfur granules Actinomyces infection
Langerhans cells Cigarette smokers, Langerhans cell histiocytosis
Fat droplets/ lipid-laden macrophages Fat embolism
Dust particle inclusions Pneumoconioses or asbestos exposure
E. Microbiologic Examination
1. Bacterial pathogens: M. tuberculosis, L. pneumophila, M. pneumoniae, Actinomyces spp.
2. Fungal pathogens: Pneumocystis jiroveci, Cryptococcus neoformans, Histoplasma capsulatum
3. Parasites: P. westermani, S. stercoralis, E. histolytica, E. granulosus
4. Viruses: Influenza A and B, respiratory syncytial virus
18. Gastric Fluid

Specimen collection
a. Evacuation/gastric tubes:
1) Rehfuss’ tube – has a metal tip; swallowed by gravity; for both gastric and duodenal fluid collection
2) Levine’s tube – has the smallest diameter; inserted through the nose
b. Stimulants:
1) Test meals – poor gastric stimulants (e.g. Ewald’s, Boa’s, Reigel’s, or Alcohol test meal)
2) Histamine – exerts unpleasant systemic effects on blood vessels and smooth muscles
3) Histalog/Betazole – histamine isomer with preferential effect on gastrin secretion
4) Insulin (hypoglycemia test) – used to determine completeness of vagotomy
5) Pentagastrin – stimulant of choice resembling gastrin; more rapid response than Histalog
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: 20-50 mL after a test meal; 45-150 mL after chemical stimulation
b. Increased volume: hypomotility, pyloric obstruction, Zollinger-Ellison syndrome
c. Decreased volume: gastric hypermotility
1. Gastric Acidity a. Total Acidity: 40-70 mEq/L b. Free HCl: 20-40 mEq/L
2. Clinical Significance
a. Hyperchlorhydria – increased free HCl seen in peptic ulcer
b. Hypochlorhydria – decreased free HCl seen in chronic gastritis, gastric ulcer, and stomach CA
c. Achlorhydria – absence of free HCl seen in pernicious anemia, advanced gastric ulcer, and pellagra
31

lOMoARcPSD|4642032
FIGURES
Fig 1. Sagittal section of the right kidney Fig 2. Parts of a nephron
Fig 3. Formation and degeneration of casts
32

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CM-handouts - Clinical Microscopy

Medical Technology (Centro Escolar University)

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Centro Escolar University

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1. Introduction to Urinalysis 2 10. Pregnancy Tests 19

2. Types of Urine Specimen/ Collection Techniques

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b. Changes in Unpreserved Urine

4. Urine Volume (24-hour)

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B. Analytical Aspects of Quality Assurance

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C. Post-analytical Aspects of Quality Assurance

D. Approaches to Quality Management

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3. Physical Examination of Urine

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4. Chemical Examination of Urine

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5. Ketones Ketone bodies: Tests for Ketones:

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B. Summary of Reagent Strip Testing

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5. Microscopic Examination of Urine

Squamous ECs Bacteria Yeast cells Parasites

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2. Casts (See Formation and degeneration of casts on page 32)

Granular cast Waxy cast Broad cast Fatty cast

b. Normal Crystals in Alkaline Urine

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4. Urinary Sediment Artifacts

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7. Renal Structure, Function, and

B. Physiology of Urine Formation

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C. Evaluation of Renal Functions

b) Normal Values: 75 – 112 mL/min (F); 85 – 125 mL/min (M)

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B. Examination of Renal Calculi

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9. Urine Screening Tests

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2. Cystinosis Inborn error of metabolism Cystine Deposition of cystine

C. Summary of Screening Tests for Metabolic Disorders

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10. Pregnancy Testing

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11. Cerebrospinal Fluid

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E. Microbiologic and Serologic Examination

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13. Synovial Fluid

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Characteristics of synovial fluid crystals

D. Microbiologic and Serologic Examination

14. Serous Fluids