MUST TO KNOW IN CLINICAL MICROSCOPY

URINALYSIS
Nephron Basic structural unit of kidney
1M/kidney
Urethra F: 3-4 cm
M: 20 cm
Urine formation (order) Glomerulus  Bowman’s capsule  PCT  Loop of Henle 
DCT  CD
PCT 65% of reabsorption
ADH Regulate H2O reabsorption in DCT and CD
Urine composition 95-97% H2O
3-5% solids
60g TS in 24 hrs
35g: Organic = Urea (major)
25g: Inorganic = Cl (#1) > Na+ > K+
Glomerular Filtration
Clearance tests Evaluate glomerular filtration
1. Urea clearance
2. Creatinine clearance = most common
3. Inulin clearance = gold standard
4. Beta2-microglobulin
5. Radioisotopes
Creatinine clearance Formula:
Cc = U x V x 1.73
P A
Normal values:
M = 107-139 mL/min
F = 87-107 mL/min
Tubular Reabsorption
st
Tubular Reabsorption 1 function to be affected in renal disease
Concentration tests Evaluate tubular reabsorption
Fishberg test (Old) Patient is deprived of fluid for 24hrs then measure urine
SG
(SG ≥ 1.026)
Mosenthal test (Old) Compare day and night urine in terms of volume and SG
Specific Gravity (New) Influenced by # and density of particles in a solution
Osmolarity Influenced by # of particles in a solution
Principle: Freezing point depression
- 1 Osm or 1000 mOsm/kg of H2O will lower the FP of H2O
(0’C) by 1.86’C
- FP = Osm
Example:
Determine Osm in mOsm/kg
Temp. = -0.90’C
Solution:
1000 mOsm/kg = _ _x____
-1.86’C -0.90’C

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 x = 484 mOsm/kg
Tubular Secretion and Renal Blood Flow
PAH test p-aminohippuric acid
PSP test Phensulfonphthalein test
Obsolete, results are hard to interpret
Methods of Collection
Midstream/Catheterized Urine culture
Suprapubic aspiration Anaerobic urine culture
3 glass technique For detection of prostatic infection
1. 1st portion of voided urine
2. Middle portion of voided urine: Serves as control for
kidney and bladder infection
-If (+), result for #3 is considered invalid
3. Urine after prostatic massage
Compare WBC and Bacteria of specimen 1 and 3
Prostatic infection: 1 < 3 (10x)
Pediatric specimen Wee bag
Drug Specimen Collection Chain of custody: step by step documentation of handling
and testing of legal specimen
Required amount: 30-45 mL
Temperature (urine): 32.5-35.7’C (w/in 4 mins)
Blueing agent  Toilet bowl (to prevent adulteration)
Types of Urine Specimen
Occasional/Single/Random Routine
Qualitative UA
24 hr 1st voided urine  discarded
w/ preservative
Ex. 8AM  8AM
12 hr Ex. 8AM  8PM
Addis count: measure of formed elements in the urine
using hemacytometer
Afternoon (2PM-4PM) Urobilinogen (alkaline tide)
4 hr Nitrite determination (1st morning/4 hr)
NO3  NO2 = (+) UTI
1st morning Pregnancy test (hCG)
Ideal specimen for routine UA
Most concentrated and most acidic = preservation of cells
and casts
Fasting/2nd morning Glucose determination
2nd voided urine after a period of fasting
Changes in Unpreserved Urine
Decreased
Clarity Bacterial multiplication
Precipitation of AU/AP
Glucose Glycolysis
Ketones Volatilization
Bilirubin Photooxidation

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Urobilinogen Oxidized to urobilin
RBC/WBC Disintegrate in alkaline urine
Increased
pH Urea ---(Urease)---> NH3
Bacteria Multiplication
Odor Urea ---(Urease)---> NH3
Nitrite Bacterial multiplication
Differentiate Contamination:  Bacteria
contamination from true True infection:  Bacteria and WBCs
infection
Preservation
Refrigeration 2-8’C
 SG (hydrometer/urinometer)
Precipitate AU/AP
Formalin Addis count
Boric acid Urine culture
Bacteriostatic to contaminants
Sodium fluoride Glucose
Sodium benzoate/ Substitute for sodium fluoride
Benzoic acid
Saccomanno’s fixative 50% ethanol + carbowax
Cytology (50mL urine)

Physical Examination of Urine

Volume NV:
24 hr = 600-1200 mL
Ave (24 hr) = 1200-1500 mL
Night: Day ratio = 1:2 to 1:3
Routine UA Vol = 10-15 mL (Ave: 12 mL)
-15 mL: for urinometry
-physical, chemical, microscopic exam
Polyuria  Urine volume
-Diabetes Mellitus:  vol,  SG
-Diabetes Insipidus:  vol,  SG
Oliguria Calculus/kidney tumors
Dehydration
Anuria Complete cessation of urine flow
Nocturia >500mL w/ SG <1.018
Pregnancy
Urine color Roughly indicates the degree of hydration
Should correlate w/ urine SG
 fluid intake: Dark urine,  SG
Urine pigments 1. Urochrome
-Major pigment (yellow)
-Production is directly proportional to metabolic
rate
- in thyrotoxicosis, fever, starvation

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 2. Uroerythrin
-Pink pigment
-May deposit in amorphous urates and uric acid
crystals
3. Urobilin
-Dark yellow/orange
-Imparts an orange-brown color to a urine w/c is not
fresh
Urine Color
Normal Colorless to deep yellow
Abnormal Red/red brown (most common)
Colorless/Pale Polyuria: DM/DI
yellow
Amber Bilirubin (yellow foam)
[Protein: white foam, concentrated urine]
Orange Pyridium (Tx: UTI)
-Yellow/orange foam
-Orange and viscous
Yellow-green Bilirubin ---(oxidized)---> Biliverdin
Yellow-brown
Green Pseudomonas aeruginosa
Blue-green Clorets, methylene blue, phenol
Indican (blue): Hartnup disease or Blue diaper
syndrome
Cloudy/Smoky red Hematuria (intact RBCs)
Clear red Hemoglobin, Myoglobin
Red/Purple/Burgu Porphyria
ndy red/ purplish (Lead poisoning: normal color)
red/Portwine
Brown/black Methemoglobin (acid urine)
Homogentisic acid: Alkaptonuria
-Urine darkens after a period of standing
-(-) Homogentisic acid oxidase
Urine Color Changes w/ Commonly Used Drugs
Cola-colored Levodopa (Tx: Parkinsonism)
Red  Brown (alkaline)
Yellow Mepacrine/Atabrine (Tx: Malaria, Giardiasis)
Red to brown Metronidazole/Flagyl (Tx: Trichomoniasis,
Amoebiasis, Giardiasis)
Methyldopa/Aldomet (Antihypertensive)
Orange-red (acid) Phenazopyridine/pyridium (Tx: UTI)
Bright orange-red Rifampin (Tx: TB) = all body fluids are red
(acid)
Bright yellow Riboflavin (Multivitamins)
Nubecula Faint cloud in urine after a period of standing
WBCs, epithelial cells and mucus
Bilifuscin Hemoglobin Köln = unstable

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(Dipyrrole) Red-brown urine
Clarity/Transparency/Turbidity
Clear Transparent, no visible particulates
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 clot
Bacteria Uniform turbidity NOT cleared by acidification or
filtration
Chyluria Lymph fluid in urine
Filariasis
Squamous  females
epithelial cells
Radiographic  SG by refractometer (>1.040)
contrast media Rgt strip: not affected by RCM
Vaginal cream Tx: Candida
Pseudochyluria
Laboratory Correlations in Urine Turbidity
Acidic urine AU
RCM
Alkaline urine AP
Carbonates
Soluble w/ heat AU
Uric acid
Soluble w/ dilute RBCs
acetic acid AP
Carbonates
Insoluble in dilute WBCs
acetic acid Yeasts
Spermatozoa
Bacteria
Soluble in ether Lipids
Lymph fluid
Chyle

Specific Gravity
SG Density of solution compared w/ density of similar
volume of distilled H2O at a similar temperature
NV = 1.003-1.035 (random)
SG <1.003 = not a urine except DI
Refractometer Based on refractive index:
(TS meter) RI = _light velocity in air_
light velocity in soln
Compensated to temperature (15-38’C)
Corrections:
a. 1g/dL glucose: (-0.004)
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 b. 1g/dL protein: (-0.003)
Calibrations:
a. Distilled H2O = 1.000
b. 5% NaCl = 1.022 ± 0.001
c. 9% Sucrose = 1.034 ± 0.001
Urinometer Requires temperature correction
a.  3’C calibration temperature (20’C) = (+0.001)
b.  3’C calibration temperature (20’C) = (-0.001)
Requires correction for glucose and protein (Rf/U)
Rf < U by 0.002 Refractometer reading is lower than the urinometer
reading by 0.002
Urinometer K2SO4 solution: 1L H2O + 20.29g K2SO4
calibration SG = 1.015
Isosthenuria SG = 1.010 (Glomerular filtrate)
Hyposthenuria SG < 1.010
Hypersthenuria SG > 1.010
Urine Odor
Aromatic/Odorles Normal
s
Ammoniacal Urea ---(Urease)---> NH3
Ex. UTI (Proteus:  urease)
Fruity, sweet DM (Ketones)
Rotten Trimethylaminuria
fish/Galunggong
Sweaty feet Isovaleric acidemia
Mousy Phenylketonuria
Cabbage Methionine malabsorption
Caramelized MSUD
sugar, curry
Bleach Contamination
Sulfur Cystine disorder

Chemical Examination of Urine

Specific Gravity
Principle (Rgt pKa dissociation constant
Strip)  concentration =  H+
Indicator: Bromthymol blue = () Blue  Green 
Yellow ()
Other info. Not affected by glucose, protein and RCM
Harmonic Frequency of soundwave entering a solution will
Oscillation change in proportion to the density (SG) of the
Densitometry solution
-Yellow IRIS (Automated): International Remote
Imaging System
pH
Normal Random = 4.5-8.0
1st morning = 5.0-6.0

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 pH 9.0 = Unpreserved urine
Acid urine DM (Ketone bodies)
Starvation (Ketone bodies)
High protein diet
Cranberry juice (Tx: UTI)
Alkaline urine After meal
Vomiting
Renal tubular acidosis: inability to produce an acid
urine
Vegetarian diet
Old specimen: Urea ---(Urease)---> NH3
Principle (Rgt Double indicator system (Methyl red & Bromthymol
Strip) blue)
MR + H+ ------------> BTB – H+
pH 4.0-6.0 pH 6.0-9.0
(Red-Yellow) (Yellow-Blue)
Protein
Protein White foam
Analyte indicative of renal disease
1. Albumin: major serum protein found in urine
2. Serum and tubular microglobulins
3. Tamm-Horsfall protein (Uromodulin)
4. Proteins derived from prostatic and vaginal
secretions
Normal value < 10 mg/dL or < 100 mg/24 hrs
< 150 mg/24 hrs (Henry)
Pre-renal Prior to reaching the kidney
proteinuria 1. Intravascular hemolysis (Hgb)
2. Muscle injury (Mgb)
3. Severe inflammation (APRs)
4. Multiple myeloma: proliferation of Ig-producing
plasma cells (BJP)
Bence-Jones protein: Ig light chains (Identical: κ-κ,
λ-λ)
-ID: Serum IEP
-Urine: precipitates at 40-60’C and dissolves at
100’C
-Seen in MM, macroglobulinemia, malignant
lymphoma
Renal proteinuria Glomerular and tubular disorders
1. Diabetic nephropathy
- Glomerular filtration  Renal failure
-Microalbuminuria: proteinuria not detected by
routine rgt strip
-Micral test: a strip employing Ab enzyme conjugate
that binds albumin (EIA)
2. Orthostatic/Postural/Cadet: proteinuria when
standing due to pressure to renal veins
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 -1st morning urine: (-) protein
-2 hrs after standing: (+) protein
3. Fanconi’s syndrome
Post-renal Lower UTI/inflammation
proteinuria
Principle (Rgt Protein (Sorensen’s) error of indicators
Strip) Indicator + Protein ---(buffered at pH 3.0)---> (-)
Yellow
(+)[] Green  Blue
[]
Sensitive to albumin
Indicators:
a. Tetrabromphenol blue
b. Tetrachlorophenol tetrabromosulfonphthalein
Sulfosalicylic acid Cold precipitation test that reacts equally to all
precipitation test types of protein
If (-) rgt strip, (+) SSA = presence of other
proteins

CSF protein = frequently tested

-Det: TCA (preferred) and SSA
SSA Reactions (Protein)
Negative No increase in turbidity < 6 mg/dL
Trace Distinct turbidity 6-30 mg/dL
1+ Noticeable turbidity w/ no 30-100 mg/dL
granulation
2+ Turbidity w/ granulation but 100-200 mg/dL
no flocculation
3+ Turbidity w/ granulation and 200-400 mg/dL
flocculation
4+ Clumps of protein > 400 mg/dL
Glucose
Glucose Most frequently tested in urine
Threshold substance
Renal threshold = 160-180 mg/dL
-Plasma concentration of a substance at w/c tubular
reabsorption stops and  amount of substance in
the urine
Other substances ID: TLC
in urine 1. Fructose (Levulose):  fruits, honey syrup
2. Galactose: infants (Galactosemia: enzyme
deficiencies)
-Galactose-1-uridyltransferase deficiency
-Galactokinase deficiency
3. Lactose
-During lactation
-Towards the end of pregnancy
-Patient on strict milk diet
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 -(+) Rubner’s test (Lead acetate)
4. Pentose
-Xylose, arabinose
-Xylulose:Benign pentosuria
5. Sucrose
- Intestinal disorders
-Nonreducing sugar
-(-) Copper reduction test
Hyperglycemia  Blood glucose,  Urine glucose
associated 1. DM
Glycosuria 2. Cushing’s syndrome/disease =  cortisol
3. Pheochromocytoma =  catecholamines
4. Acromegaly =  GH
5. Hyperthyroidism = T3/T4
Renal associated N-Blood glucose, impaired tubular reabsorption of
Glycosuria glucose
1. Fanconi’s syndrome: defective tubular
reabsorption of glucose and amino acids
Principle (Rgt Double sequential enzyme reaction:
Strip) -Glucose oxidase
-Peroxidase
Chromogen:
-KI (Brown)
-Tetramethylbenzidine (Blue)
Copper Reduction Blue tablet
test (Clinitest) Relies on the ability of glucose and other
substances to reduce CuSO4 to Cu2O in the
presence of alkali and heat
CuSO4 (Blue) -------------> Cu2O (Brick red)

Pass through Occurs if >2 g/dL sugar is present in urine

phenomenon Blue  Green  Yellow  Brick red  Blue (Pass
through)
To prevent, use 2 gtts urine (instead of 5 gtts) + 10
gtts H2O + Clinitest
(-) Glucose (+) Nonglucose reducing substance
oxidase
(+) Clinitest
1+ Glucose oxidase True glucosuria
(-) Clinitest Small amount of glucose present
4+ Glucose False (+)
oxidase Possible oxidizing agent interference on reagent
(-) Clinitest strip
Ketones
Ketones Result from increased fat metabolism due to
inability to metabolize CHO
78% BHA = major ketone but not detected
20% AA/Diacetic acid = parent ketone
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 2% Acetone
Significance Diabetic acidosis
Insulin dosage monitoring
Starvation
Vomiting
Principle (Rgt Legal’s test (Sodium nitroprusside reaction)
Strip) AAA + Sodium nitroprusside ---------
-----> (+) Purple
(Acetone) (Glycine)
Acetest Sodium nitroprusside
Glycine
Disodium phosphate
Lactose
Blood
Hematuria Cloudy red urine (Intact RBCs)
Renal calculi
GN
Strenuous exercise
Anticoagulants
Hemoglobinuria Clear red urine
Intravascular hemolysis
Myoglobinuria Clear red urine
Rhabdomyolysis
Hgb vs. Mgb 1. Plasma examination
-Hgb: Red/pink plasma, haptoglobin
-Mgb: Pale yellow,  CK,  Aldolase
2. Blondheim’s test (Ammonium SO4): Precipitates
Hgb
Urine + 2.8g NH4SO4 (80% Satd.) ---
(Filter/Centrifuge)---> Supernatant
Supernatant:
Red = Myoglobin = (+) Rgt strip
Clear w/ red ppt. = Hemoglobin = (-) Rgt strip
Hemolytic anemia 1 hr post transfusion urine = Hgb
Week after = Hemosiderin
Principle (Rgt Pseudoperoxidase activity of hemoglobin
Strip) Chromogen: TMB [(-) Yellow/(+){}Green  Blue
{}]
H2O2 + Chromogen ---(Heme)---> Oxidized
chromogen + H2O
Hgb/Mgb Uniform green/blue
Hematuria Speckled/spotted
Extravascular  Unconjugated bilirubin
lysis  Urine and fecal urobilinogen
Intravascular (+) Hemoglobinuria
lysis  Haptoglobin and Hemopexin
Bilirubin
Bilirubin Amber urine w/ yellow foam
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 Early indication of liver disease
Significance Hepatitis
Cirrhosis
Bile duct obstruction
Principle (Rgt Diazo Reaction:
Strip) Bilirubin diglucuronide + Diazo salt ---------> (+) Tan
or pink purple
Reagents:
2,4-Dichloroaniline diazonium salt
2,6-Dichlorobenzene diazonium salt
Ictotest (+) Blue to purple
Reagents:
p-nitrobenzene-diazonium-p-toluenesulfonate
SSA
Na2HCO3
Urobilinogen
Urobilinogen Afternoon specimen: 2PM-4PM (alkaline tide)
Small amount in normal urine (<1 mg/dL or EU)
Principle (Rgt Ehrlich’s reaction (PDAB):
Strip) Urobilinogen + PDAB ----------> (+) Cherry red
Watson-Schwartz For differentiating urobilinogen and porphobilinogen
test from other Ehrlich’s reactive compounds
1. Urobilinogen: soluble to both chloroform and
butanol
2. Porphobilinogen: insoluble to both chloroform and
butanol
3. Other Ehrlich’s reactive compounds: soluble to
butanol only
Hoesch test Rapid screening test for urine porphobilinogen
(Inverse Ehrlich (≥2mg/dL)
reaction) Hoesch reagent: Ehrlich reagent in 6M (6N) HCl
2 gtts urine + Hoesch rgt -------------> (+) Red
Urine Bilirubin and Urobilinogen in Jaundice
Urine Bilirubin Urobilinogen
Hemolytic (ex.
- 
SCD)
Hepatic /- N/
Obstructive /N (never report as

negative)
Nitrite
Nitrite 1st morning/4 hr urine
Detection of bacteriuria
(+) NO3: 100,000 orgs./mL
UTI: ≥100,000 or ≥1 x 105 CFU/mL
Principle (Rgt Greiss reaction:
strip) NO3 + Sulfanilamide + p-arsanilic acid +
tetrahydrobenzoquinoline
------------> (+)
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 Uniform pink
Pink spots/edges = (-)
Leukocytes
Significance UTI/inflammation
Screening of urine culture specimen
Leukocyte (+) Neutro/Eo/Baso/Mono
esterase (-) Lympho
Maybe (+): Histiocytes, Trichomonas

Principle (Rgt Indoxyl carbonic acid ester + Diazonium salt ---

strip) (LE)---> Indoxyl + Acid indoxyl
----------> (+) Purple
Strip can detect even lysed WBCs
Reading Time (Reagent Strips)
30 seconds Glucose
Bilirubin
40 seconds Ketones
45 seconds SG
60 seconds “PPBUN”
pH
Protein
Blood
Urobilinogen
Nitrite
120 seconds Leukocytes
Vitamin C 11th reagent pad
(Ascorbic acid) Reducing property
False (-) rgt strip: “BB LNG”
-Blood
-Bilirubin
-Leukocytes
-Nitrite
-Glucose
Rgt: Phosphomolybdate
Phosphomolybdate + Vitamin C (≥5 mg/dL) -------->
(+) Molybdenum blue

Sources of Error/Interference (Reagent Strips)

False-positive False-negative
SG High concentrations of Highly alkaline urines (>6.5)
protein
pH No known interfering substance
Runover from adjacent pads
Old specimens
Protein Highly buffered alkaline Proteins other than albumin
urine
Pigmented specimens,

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 phenazopyridine
Quarternary ammonium
compounds (detergents)
Antiseptics, chlorhexidine
Loss of buffer from
prolonged exposure of the
reagent strip to the
specimen
High specific gravity
Glucose Contamination by oxidizing High levels of ascorbic acid
agents and detergents High levels of ketones
High specific gravity
Low temperatures
Improperly preserved
specimens
Ketones Phthalein dyes Improperly preserved
Highly pigmented red urine specimens
Levodopa
Medications containing free
sulfhydryl grps

False-positive False-negative
Blood Strong oxidizing agents High specific gravity/crenated
Bacterial peroxidases cells
Menstrual contamination Formalin
Captopril
High concentration of nitrite
Ascorbic acid >25 mg/dL
Unmixed specimens
Bilirubin Highly pigmented urines, Specimen exposure to light
phenazopyridine Ascorbic acid >25 mg/dL
Indican (intestinal disorders) High concentrations of nitrite
Metabolites of Lodine
Urobilin Porphobilinogen Old specimens
ogen Indican Preservation in formalin
p-aminosalicylic acid
Sulfonamides
Methyldopa
Procaine
Chlorpromazine
Highly pigmented urine
Nitrite Improperly preserved Nonreductase-containing
specimens bacteria
Highly pigmented urine Insufficient contact time
between bacteria and nitrate
Lack of urinary nitrate
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 Large quantities of bacteria
converting nitrite to nitrogen
High concentrations of
ascorbic acid
High specific gravity
Leukocy Strong oxidizing agents High concentrations of
tes Highly pigmented urine, protein, glucose, oxalic acid,
nitrofurantoin ascorbic acid, gentamicin,
cephalosporins, tetracyclines

Microscopic Exam of Urine

Phase-contrast Visualization of elements w/ low refractive indices:
microscopy -Hyaline casts
-Mixed cellular casts
-Mucous threads
-Trichomonas
Polarizing ID of cholesterol in OFB, FC and crystals
microscopy
Interference Produces 3D microscopy-image and layer-by-layer
contrast imaging of a specimen
microscopy 1. Hoffman microscope: modulation contrast
microscope
2. Nomarski microscope: differential interference
contrast microscope
Sternheimer- Crystal violet and safranin
Malbin Nucleus and cytoplasm
ID: WBCs, ECs, casts
Toluidine blue Enhances nuclear detail
(Supravital) Differentiates WBCs and RTE
Lipid stains: Stain TG and neutral fats orange red
ORO and Sudan ID: free fat droplets and lipid-containing cells and
III casts
Gram stain Differentiates Gram (+) and Gram (-) bacteria
ID: bacterial casts
Hansel stain Eosin Y and Methylene blue
ID: Eosinophils
Prussian blue stain Stains structures containing iron
ID: yellow-brown granules of hemosiderin in cells
and casts
Sediment Constituents
RBCs NV = 0-2 or 0-3/hpf
Hypertonic: crenated, shrink
Hypotonic: Ghost cells, swell, hemolyzed
Dysmorphic: glomerular membrane damage, w/
projections, fragmented

Sources of error:
-Yeasts
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 -Oil droplets
-Air bubbles
-CaOx crystals
♫ Remedy: add 2% acetic acid
-RBCs: lysed
-Other cells: intact
WBCs NV = 0-5 or 0-8/hpf
Glitter cells (Hypotonic urine)
-Granules swell
-Brownian movement
>1% eosinophils: significant
- Drug-induced allergic reaction
- Inflammation of renal interstitium
Addis count Quantitative measure of formed elements of urine
using hemacytometer
Specimen: 12 hr urine
Preservative: Formalin
NV:
a. RBCs: 0-500,000/12 hr urine
b. WBCs: 0-1,800,000/12 hr urine
c. Hyaline Casts: 0-5000/ hr urine
Squamous Largest cell in the urine sediment
epithelial cells From linings of vagina, female urethra and lower
male urethra
♫ Variation: Clue cells:
-EC w/c are studded w/ bacteria (bacterial
vaginosis)
-Whiff/Sniff test: vaginal discharge + 10% KOH 
Fishy amine-like odor
-Culture: G. vaginalis = HBT medium
Transitional Spherical, polyhedral, or caudate w/ centrally
epithelial cells located nucleus
(Urothelial cells) Derived from the linings of the renal pelvis, ureter,
urinary bladder, male urethra (upper portion)
Not clinically significant in small numbers
Renal tubular Rectangular, polyhedral, cuboidal or columnar w/ an
epithelial cells eccentriac nucleus, possibly bilirubin stained or
hemosiderin laden
From nephron:
-PCT: rectangular, columnar/convoluted
-DCT: round/oval
>2 RTE/hpf: tubular injury
Oval fat body Lipid containing RTE cells
Lipiduria (Ex. nephrotic syndrome)
Cholesterol: Maltese cross
Bubble cells RTE cells w/ nonlipid containing vacuoles
Acute tubular necrosis
Yeast C. albicans (DM, vaginal moniliasis)
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T. vaginalis Flagellate w/ jerky motility
Pingpong disease
S. haematobium “Hematuria”
Specimen: 24 hr unpreserved urine
E. vermicularis Most common fecal contaminant
Casts Formed in the DCT and CD
(Cylindruria) ♫ Tamm-Horsfall protein (Uromodulin)
-Major constituent
-Glycoprotein secreted by RTE cells of DCT and CD
Hyaline casts NV = 0-2/lpf
Beginning of all types of casts (prototype cast)
a. Physiologic:
- Strenuous exercise (HC, GC, RC)
- Heat
b. Pathologic:
- GN
- PN
- CHF
RBC casts Bleeding w/in the nephron
a. GN
b. Strenuous exercise (HC, GC, RC)
WBC casts Inflammation w/in the nephron
Differentiates upper UTI (pyelonephritis, w/ cast)
from lower UTI (cystitis, no cast)
To differentiate from EC cast:
1. Phase contrast microscopy
2. Supravital stain
Seen in:
-PN
-AIN
Bacterial casts Pyelonephritis
Epithelial cell Renal tubular damage
casts Advanced tubular destruction
Coarse/Fine Formed from the disintegration of cellular cast
granular casts GN
PN
Strenuous exercise (HC, GC, RC)
Fatty casts Nephrotic syndrome: lipiduria
Not stained by Sternheimer-Malbin
Waxy casts Final degenerative form of all types of casts
Stasis of renal flow
Chronic renal failure
Brittle, highly refractile, w/ jagged ends
Broad casts “Renal failure casts”
Extreme urine stasis
Widening and destruction of tubular walls
Any type of cast can be broad
Sediment Urine  Centrifuge: 400 RCF for 5 mins  Decant
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preparation  Remaining: 0.5mL/1.0mL
Urine sediment: 20μL (0.02 mL)
-10 lpf
-10 hpf
-Reduced light
RCF 1.118 x 10-5 x radius (cm) x (rpm)2
Urine Crystals
Amorphous Yellow-brown granules
Urates Pink sediment (Uroerythrin)
(Normal) Mistaken as cystine crystals
(pH: acid) Rhombic, wedge, rosette, hexagonal, four-sided
plate (whetstone)
Lemon-shaped (Henry)
 Lesch-Nyhan syndrome: orange sands in diaper
 Gout
 Chemotherapy
Calcium Oxalate 1. Weddelite = dihydrate
(Normal) -Envelope/pyramidal
(pH: 2. Whewellite = monohydrate
acid/alkaline/neut -Oval, dumbbell
ral) -Ethylene glycol poisoning (antifreeze agent)
Most renal stones consist of CaOx
Amorphous White precipitate
Phosphates Granular appearance
(Normal)  After meal (alkaline tide)
(pH:
alkaline/neutral)
Ammonium Yellow-brown
Biurate Thorny apples
(Normal) Old specimen: due to the presence of urea-splitting
(pH: alkaline) bacteria
Triple Phosphate A.k.a. Magnesium ammonium phosphate
(Normal) Coffin lid, “Struvite”, staghorn appearance
(pH: alkaline) Presence of urea-splitting bacteria
Calcium Phosphate Colorless, flat rectangular plates or thin prisms
(Normal) often in rosette formation
(pH: Rosettes may resemble sulfonamides
alkaline/neutral) -To differentiate: CaPO4 dissolves in acetic acid
1. Calcium Phosphate = Apatite
2. Basic Calcium Phosphate = Hydroxyapatite
3. Calcium Hydrogen Phosphate = Brushite
Calcium Carbonate Small and colorless
(Normal) Dumbbell or spherical shapes
(pH: alkaline) Acetic acid: (+) Effervescence
Cystine Colorless hexagonal plates
(Abnormal) Cystinuria
(pH: acid)
Cholesterol Rectangular plate w/ notch in one or more corners
lec.mt 04 |Page | 150
(Abnormal) Staircase pattern
(pH: acid) Lipiduria (Nephrotic syndrome)
Resemble crystals of RCM, to differentiate
a. Patient history
b. Correlate w/ other UA results
c. RCM: SG by refractometer ≥1.040
Tyrosine Colorless to yellow needles
(Abnormal) Liver disease (more common)
(pH: acid/neutral) (+) Nitroso-naphthol
Leucine Yellow-brown spheres w/ concentric circles and
(Abnormal) radial striations
(pH: acid/neutral) Liver disease
Bilirubin Clumped needles or granules w/ yellow color
(Abnormal) (+) Diazo reaction
(pH: acid) Liver disease
Sulfonamide Colorless to yellow brown
(Abnormal) Deposits in nephrons
(pH: acid/neutral) Tubular damage
Needles, sheaves of wheat, rosette (res. CaPO4
rosette)
♫ Lignin test:
Newspaper = urine + 25% HCl  (+) Yellow orange
color
Ampicillin Massive doses
(Abnormal) Colorless needles
(pH: acid/neutral)
Uric Acid Cystine
Color Yellow brown Colorless
Solubility in NH3 Soluble Soluble
Solubility in dilute HCl Insoluble Soluble
Birefringence (Polarizing
+ -
microscope)
Cyanide-Nitroprusside
- +
test
Urinary Sediment 1. Starch granules (gloves):
Artifacts -Most common
-Maltese cross (O, FC, S)
-Dimpled center
2. Oil droplets
3. Air bubbles
4. Pollen grains = spheres w/ concentric circles
5. Hair and fibers = (+) Birefringence (polarizing
microscope)
6. Fecal contaminants

Renal Diseases
Cystitis Inflammation of urinary bladder
Infection
lec.mt 04 |Page | 151
 WBCs, RBCs, bacteria
NO CAST
Urethritis Inflammation of urethra
WBCs, RBCs
Usually NO BACTERIA on routine UA
a. Male: GS of urethral exudates [Gram (-)
diplococcic]
b. Female: pelvic exam for vaginitis and cervicitis
Glomerulonephriti Inflammation of the glomerulus
s Immune-mediated
RBCs, WBCs, RBC CASTS, WBC casts, hyaline and
granular casts
Pyelonephritis Infection of renal tubules
WBCs, RBCs, bacteria, RBC casts, WBC CASTS,
hyaline and granular casts
Acute Interstitial Infection of the renal interstitium
Nephritis RBCs, WBCs, WBC casts, NO BACTERIA
Renal carcinoma 1’ = RCC
2’ = Transitional CC
RBCs and WBCs
Nephrotic Massive proteinuria and lipiduria
syndrome a. Serum (Chemistry)
- Albumin, alpha1, beta and gamma globulins
- alpha2 (AMG)
b. Urine (CM)
- Albumin, alpha1, beta and gamma globulins
-(-) alpha2 (AMG)
-Oval fat bodies, fatty and waxy casts
Telescoped Simultaneous appearance of the elements of
sediments acute/chronic GN and nephrotic syndrome
 Cells and Casts
a. Lupus nephritis
b. SBE
UTI E. coli = 90% cases of UTI
S. saprophyticus = UTI among sexually active young
females
G. vaginalis = bacterial vaginosis
S. pyogenes = AGN and ARF
Viridans Streptococci = SBE
Rapidly Deposition of immune complex from systemic
progressive immune disorders on the glomerular membrane
(Crescentic) GN
Goodpasture Attachment of cytotoxic antibody to glomerular and
syndrome alveolar basement membrane
Wegener’s Antineutrophilic cytoplasmic autoantibody
granulomatosis
Henoch-Schönlein Occurse in children following viral respiratory
purpura infection
lec.mt 04 |Page | 152
 Decrease in platelets disrupts vascular integrity
Membranous GN Thickening of the glomerular membrane following
IgG immune complex deposition
Membranoprolifer Cellular proliferation affecting the capillary walls or
ative GN the glomerular basement membrane
Chronic GN Marked decrease in renal function resulting from
glomerular damage precipitated by other renal
disorders
IgA nephropathy Deposition of IgA on the glomerular membrane
(Berger’s disease)
Nephrotic Disruption of the electrical charges that produce
syndrome tightly fitting podocyte barrier
Minimal change Disruption of the podocytes occurring primarily in
disease (Lipoid children following allergic reaction and immunization
nephrosis)
FSGS Disruption of podocytes in certain areas of
glomeruli associated w/ heroin and analgesic abuse
and AIDS
Alport syndrome Lamellated and thinning of glomerular basement
membrane
Diabetic Most common cause of ESRD
Nephropathy Microalbuminuria
(Kimmelstiel-
Wilson disease)
Acute tubular Damage to the renal tubules caused by ischemia or
necrosis toxic agents
Fanconi syndrome Generalized defect in renal tubular reabsorption in
the PCT
Nephrogenic DI Inability of the renal tubules to respond to ADH
Neurogenic DI Inability of the hypothalamus to produce ADH
Renal glucosuria Inability of the renal tubules to reabsorb glucose
Cystitis Ascending bacterial infection of the bladder
Acute PN Infection of the renal tubules and interstitium
Chronic PN Recurrent infection of the renal tubules and
interstitium
Visicoureteral reflux: most common cause
-Reflux of urine from the bladder back into the
ureters

Screening for Metabolic Disorders

Aminoaciduria 1. Overflow type
 AA in blood
 AA in urine
Ex. PKU, alkaptonuria, MSUD

2. Renal type
N-AA in blood

lec.mt 04 |Page | 153

 Impaired tubular reabsorption of AA
Ex. Cystinuria (COLA), Fanconi’s syndrome
Phenylalanine-Tyrosine Disorders

Phenylalanine (-)

PAH PKU Phenylpyruvic acid

Tyrosine

Tyrosine transaminase (-)

p-Hydroxyphenylpyruvic acid Tyrosinemia

Tyrosyluria:

p-Hydroxyphenylpyruvic acid oxidase p-OHPPA

Homogentisic acid (-) p-OHPLA

Homogentisic acid oxidase Alkaptonuria

Maleylacetoacetic acid Homogentisic acid

Fumarylacetoacetic acid

Fumaric acid and Acetoacetic acid

Phenylketonuria Severe mental retardation

Mousy odor
(-) PAH
Screen: FeCl3  (+) Blue-green
Confirm: Guthrie test (Bacterial inhibition)
-B. subtilis
-Inhibitor: Beta2-thienylalanine (neutralized by
phenylalanine)
-Growth = (+) PKU
-No growth = (-) PKU
Tyrosyluria Rancid butter odor
(-) Tyrosine transaminase and p-OHPPA oxidase
Screen: FeCl3  (+) Transient green
Confirm: Nitroso-naphthol  (+) Orange-red
Alkaptonuria Urine darkens after a period of standing
(-) Homogentisic acid oxidase
 Homogentisic acid in blood and urine
FeCl3  (+) Transient blue
Clinitest/Benedict’s  (+) Yellow ppt.
Melanuria Overproliferation of melanocytes
FeCl3  Gray or black ppt.
Ehrlich’s  Red
Branched-Chain Amino Acid Disorders
lec.mt 04 |Page | 154
MSUD Accumulation of leucine, isoleucine and valine in
blood and urine
2,4-DNPH  (+) Yellow turbidity/ppt.
Organic acidemias 1. Isovaleric acidemia = sweaty feet
2. Propionic acidemia
3. Methylmalonic acidemia
Tryptophan Disorders
Indicanuria Intestinal disorder
Blue color
Hartnup disease: “Blue diaper syndrome”
Obermayer’s test: FeCl3  (+) Violet w/ chloroform
Argentaffinoma Carcinoid tumor involving argentaffin cells
 5-HIAA: metabolite of serotonin
FeCl3  (+) Blue-green (PKU)
Nitrosonaphthol  (+) Violet w/ HNO3
Be sure patient should avoid banana, pineapple,
tomatoes (serotonin-rich)
Cystine Disorders
Cystinuria (Renal Defect in renal tubular transport of:
type) -Cystine (least soluble  urine)
-Ornithine
-Lysine
-Arginine
Cystinosis Inborn error of metabolism
Cystine deposits in many areas of the body
Cyanide-nitroprusside  (+) Red-purple
Homocystinuria Defect in the metabolism of homocystine
Silver nitroprusside  (+) Red-purple
Brand’s Rxn: Cyanide-nitroprusside  (+) Red-purple
modification of
Legal’s
nitroprusside
Mucopolysaccharide Disorders
MPS Dermatan SO4
Keratan SO4
Heparan SO4
Clinical Alder-Reilly syndrome
significance Hurler syndrome = MPS  cornea of the eye
Hunter syndrome = Sex-linked recessive
Sanfilippo syndrome = Mental retardation only
CTAB (+) White turbidity
Purine Disorders
Lesch-Nyhan  Urinary uric acid crystals
disease
Porphyrias
D-ALA Glycine + Succinyl CoA ----(ALA synthetase)----> D-
ALA

lec.mt 04 |Page | 155

Porphobilinogen D-ALA ----(ALA synthetase)----> Porphobilinogen
Lead poisoning: inhibits ALA synthase
Uroporphyrinogen Porphobilinogen -----(Uroporphyrinogen synthase/
Uroporphyrinogen
cosynthase)----> Uroporphyrinogen
Acute intermittent porphyria: (-) Uroporphyrinogen
synthase
Congenital erythropoietic porphyria: (-)
Uroporphyrinogen cosynthase
Coproporphyrinog Uroporphyrinogen ---------(Uroporphyrinogen
en decarboxylase)-
--------------> Coproporphyrinogen
Porphyria cutanea tarda: (-) Uroporphyrinogen
decarboxylase
Protoporphyrinoge Coproporphyrinogen ---------(Coproporphyrinogen
n oxidase)---
----------------> Protoporphyrinogen
Hereditary coproporphyria: (-) Coproporphyrinogen
oxidase
Protoporphyrin IX Protoporphyrinogen ---(Protoporphyrinogen
oxidase)---> Protoporphyrin IX
Variegate porphyria: (-) Protoporphyrinogen oxidase
Heme Protoporphyrin IX + Fe2+ ----(Ferrocheletase)-------
-------> Heme
Lead poisoning: inhibits Ferrocheletase
Porphyrias Vampire disease
Disorders of porphyrin metabolism

Screening tests 1. Ehrlich reaction

(porphyria) = (+) D-ALA and porphobilinogen
2. Fluorescence at 550-600nm
= Uro/Copro/Protoporphyrin
= (+) Red/pink/violet
= (-) Blue
3. Free Erythrocyte Protoporphyrin (FEP)
= CDC recommended test for Lead poisoning
Specimens Urine: red/purple/portwine (normal: Lead poisoning)
Stool
Blood
Bile
Lead poisoning RBC inclusion coarse basophilic stippling
Qualitative Tests for Protein
(+) White Ring Heller’s
Robert’s
Spiegler’s
(+) Violet Biuret (Albumin)
(+) White Heat and acetic acid
turbidity/ SSA
lec.mt 04 |Page | 156
cloudiness Purdy’s
Potassium ferrocyanide
Picric acid
Kingsbury-Clark (Rgt: SSA)
(+) coagulum (24 Esbach’s
hrs) -Rgt: Picric acid + Citric acid
Tsuchiya’s
(+) coagulum Kwilecki’s
(72’C for 5mins) -Rgt: Esbach’s + 10% FeCl3
Qualitative Tests for Sugars
Benedict’s Reducing substances
Seliwanoff’s Rgt: Resorcinol
Fructose  (+) Red
Rubner’s Rgt: Lead acetate, NH3 H2O
Lactose  (+) Bright red w/ red ppt.
Glucose  (+) Red color w/ yellow ppt.
Bial Orcinol Pentose  (+) Green
Tauber’s Pentose  (+) Green
Others Osazone or phenylhydrazine (Kowarsky)
Nylander’s
Moore Heller
Borchardt’s
Qualitative Tests for Ketones
Frommer’s Acetone  (+) Purplish red ring
Rothera’s Acetone & AAA  (+) Purple ring
Lange Acetone & AAA  (+) Purple ring
Acetest/Ketostix Acetone  (+) Purple
Gerhardt’s AAA  Bordeaux red
Qualitative Tests for Bile Pigments
Gmelin Bile  (+) Play of colors
Smith Bile  (+) Emerald green
Harrison’s spot Bile  (+) Blue to green
Ictotest Bile  (+) Blue to purple mat
Wallace and Rgt: PDAB
Diamond Urobilinogen  (+) Cherry red
Schlesinger Rgt: Lugol’s iodine, Alc. Zinc acetate
Urobilin  (+) Greenish fluorescence
Qualitative Tests for Hemoglobin
Benzidine (+) Green-blue
Guiac (+) Blue
Ortho-toluidine (+) Blue
Qualitative Tests for Melanin
FeCl3 (Screening) (+) Black (after 24 hrs)
Thomahlen (+) Dark green or blue color (fresh urine)
Blackberg & (+) Brown to black ppt. (24 hr urine)
Wanger
Qualitative Tests for Chloride
lec.mt 04 |Page | 157
Fantus (+) Reddish ppt
Mercurimetric (+) Blue-violet colored complex
titration (Schales
& Schales)
Qualitative Test for Calcium
Sulkowitch (+) Precipitation

Renal Function Tests

Test for Clearance
Glomerular
filtration
Test for Tubular Concentration tests
reabsorption -Fishberg (old)
-Mosenthal (old)
-SG (new)
-Osmolality (new)
Fishberg test Patient deprived of fluid for 24 hrs = SG ≥1.026
Patient deprived of fluid for 12 hrs = SG ≥1.022
Test for Tubular PAH
Secretion and PSP
Renal Blood flow
Tests for NPN 1. Urea: Urease, DAM (NV = 6-17 g/24 hr urine)
2. Creatinine: Jaffe (NV = 1-2 g/24 hr urine)
3. Uric acid: Uricase, PTA (NV = 0.25-0.75 g/24 hr
urine)
BCR (BUN: Crea a. NV = 10:1
Ratio) -BUN: 90% excreted, 10% reabsorbed
-Crea: 99% excreted, 1% reabsorbed
b. Renal disease: Normal ratio
 BUN,  Crea
c. Pre- and Post-renal disease:  Ratio
 BUN, N-crea
Other Topics
Biohazard Symbol 4 circles
Top = Source
Left = Host
Right = Transmission
PPE Gloves
Fluid-resistant gowns
Eye and face shields
Plexiglas countertop shields
Disinfection of 1:5 or 1:10 dilution of sodium hypochlorite (daily)
sink
Sodium Effective for 1 month
hypochlorite soln.
Chemical spills on Flush the area w/ water for at least 15 mins  seek
skin medical attention

lec.mt 04 |Page | 158

 Do not neutralize chemicals
Always add acid To avoid the possibility of sudden splashing caused
 water by the rapid generation of heat in some chemical
reactions
Handwashing Best way to break the chain of infection
Clean between finger for at least 15 seconds
Downward
Sing “Happy Birthday”
Hazard 0 = Stable
Classification 1 = Unstable if heated
(Yellow = 2 = Violent chemical change
Reactivity) 3 = Shock and heat may deteriorate
4 = May deteriorate
Hazard OXY = Oxidizer
Classification ACID = Acid
(White = Specific ALK = Alkali
Hazard) COR = Corrosive
W = Use no water
 = Radiation
Hazard 0 = Normal material
Classification 1 = Slightly hazardous
(Blue = Health) 2 = Hazardous
3 = Extreme danger
4 = Deadly
Hazard 0 = Will not burn
Classification 1 = Above 200’F
(Red = 2 = Below 200’F
Flammability) 3 = Below 100’F
4 = Below 73’C
Types of Fire A = Wood, paper, clothing (ordinary combustibles)
B = Flammable liquids
C = Electrical equipment
D = Flammable metals
E = Detonation (Arsenal)
In case of Fire R = Rescue
A = Activate the alarm
C = Contain the fire
E = Extinguish
Fire extinguisher P = Pull the pin
A = Aim at the base of the fire
S = Squeeze handles
S = Sweep nozzle side to side
Urinalysis Actually the beginning of laboratory medicine
Hippocrates Uroscopy
Frederik Dekkers Discovered albuminuria by boiling urine
Thomas Bryant Pisse prophets (charlatans)
Thomas Addis Examination of urinary sediment
Richard Bright Introduced the concept of UA as part of doctor’s
lec.mt 04 |Page | 159
 routine patient examination
UA Defined as the testing of urine with procedures
(CLSI/NCCLS) commonly performed in an expeditious, reliable,
accurate, safe and cost-effective manner

Care of Reagent 1. Store w/ dessicant in an opaque, tightly closed

Strips container
2. Store below 30’C. Do not freeze
3. Do not expose to volatile fumes
4. Do not use past the expiration date
5. Do not use if chemical pads become discolored
6. Removed strips immediately prior to use
QC (Reagent 1. Test open bottles of reagent strips w/ known
Strips) positive and negative controls every 24 hr (some: at
the beginning of each shift)
2. Resolve control results that are out of range by
further testing
3. Test reagents used in backup tests w/ positive
and negative controls
4. Perform positive and negative controls on new
reagents and newly opened bottles of reagent strips
5. Record all control results and reagent lot
numbers
Resolution Ability to visualize fine details
(Microscope) Ability of the lens to distinguish two small objects
that are a specific distance apart
Parfocal Require only minimum adjustment when switching
(Microscope) among objectives
Centering and Provide optimal viewing of the illuminated field
Köhler illumination
(condenser)
Camel-hair brush Removes dust coating the optical surface of the
microscope
Quality Overall process of guaranteeing quality patient care
assessment and is regulated throughout the total testing
system
Quality system Refers to all of the laboratory’s policies, processes,
procedures, and resources needed to achieve quality
testing
Accreditation JCAHO (Joint Commission on the Accreditation of
agencies Healthcare Organizations)
CAP (College of American Pathologists)
AABB (American Association of Blood Banks)
AOA (American Osteopathic Association)
ASHI (American Association of Histocompatibility
and Immunogenetics)
COLA (Commission on Laboratory Assessment)
NCCLS (National New: CLSI (Clinical and Laboratory Standards
lec.mt 04 |Page | 160
Committee for Institute)
Clinical
Laboratory
Standards)
Policy for 1. Do NOT assume any information about the
Handling specimen or patient
Mislabeled 2. Do NOT relabel an incorrectly labeled specimen
Specimens 3. Do NOT discard the specimen until investigation
is complete
4. Leave specimen EXACTLY as you receive it; put in
the refrigeration for preservation until errors can
be resolved
5. Notify floor, nursing station, doctor’s office, etc.
of problem and why it must be corrected for
analysis to continue
6. Identify problem on specimen requisition with
date, time and your initials
7. Make person responsible for specimen collection
participate in solution of problem(s). Any action
taken should be documented on the requisition slip
8. Report all mislabeled specimens to the quality
assurance board
Preanalytical Test requests
Factors Patient preparation
Specimen collection, handling and storage
Analytical Factors Reagents
Instrumentation and equipment
Testing procedure
QC
Preventive maintentance
Access to procedure manuals
Competency of personnel performing the tests
Microscopic Quantitations
Crystals Bacteria Mucous
EC (lpf)
(hpf) (hpf) threads
None 0 0 0 -
Rare 0-5 0-2 0-10 0-1
Few 5-20 2-5 10-50 1-3
Moderate 20-100 5-20 50-200 3-10
Many >100 >20 >200 >10
Casts (lpf) None = 0
Numerical ranges = 0-2/2-5/5-10/>10
RBCs (hpf) None = 0
Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-
100/>100
WBCs (hpf) None = 0
Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-
100/>100
lec.mt 04 |Page | 161
 Quality Assurance Errors
Preanalytical Patient misidentification
Wrong test ordered
Incorrect urine specimen type collected
Insufficient urine volume
Delayed transport of urine to the laboratory
Incorrect storage or preservation of urine
Analytical Sample misidentification
Erroneous instrument calibration
Reagent deterioration
Poor testing technique
Instrument malfunction
Interfering substances present
Misinterpretation of quality control data
Postanalytical Patient misidentification
Poor handwriting
Transcription error
Poor quality of instrument printer
Failure to send report
Failure to call critical values
Inability to identify interfering substances
TQM Based on a team concept involving personnel at all
levels working together to achieve a final outcome
of customer satisfaction through implementation
CQI Improving patient outcomes by providing continual
quality care in a constantly changing health-care
environment
PDCA Plan-Do-Check-Act
PDSA Plan-Do-Study-Act

lec.mt 04 |Page | 162

 OTHER BODY FLUIDS
Cerebrospinal Fluid
CSF 1st noted by Cotugno
Not an ultrafiltrate of plasma
Na+, Cl-, Mg2+: CSF than in plasma
K+, Total Ca2+: CSF than in plasma
3rd major body fluid
Production Filtration
Active transport secretion
Functions Supply nutrients  nervous tissue
Remove metabolic waste
Provide mechanical barrier
CSF glucose 60-70% of blood glucose
Brain 1,500g (Henry)
Meninges (Sing. Three layers:
Meninx) 1. Dura Mater = outermost
- Meningeal layer next to the bone
2. Arachnoid mater (Arachnoidea) = spider web
♫ Subarachnoid space: where the CSF is flowing
3. Pia Mater = innermost
Layers Skin  Skull  Dura Mater  Arachnoid mater 
Subarachnoid space  Pia mater  Brain
Arachnoid villi/ Reabsorbs CSF
granulations If it can’t absorb CSF, CSF accumulates 
Hydrocephalus
Choroid plexuses Produce CSF at approximately 20 mL/hr
CSF Total Volume Adults:
5th ed. = 90-150 mL
4th ed = 140-170 mL
Neonates = 10-60 mL
Blood brain Between brain and blood
barrier Functions:
1. Protects brain from organisms
2. Shields brain from hormones and
neurotransmitters
3. Maintains homeostasis for brain
Circumventricular Regions of the brain where BBB is weak
organs 1. Pineal gland: melatonin, associated w/ circadian
rhythms
2. Neurohypophysis (posterior pituitary): ADH,
oxytocin
3. Area postrema: vomiting center of the brain
4. Subfornical organ
5. Vascular organ of the lamina terminalis
6. Median eminence
Specimen Lumbar tap: routine (collected by physician)
collection -Bet. 3rd, 4th and 5th lumbar vertebrae
-Fetal position
lec.mt 04 |Page | 163
 Cisternal puncture: suboccipital region
Ventricular puncture: infants w/ open fontanels
Precautions Measurement of intracranial pressure
Prevent infection (povidone iodine)
Prevent damage  neural tissue

Collection 3 Sterile Tubes

1. Chemistry/Serology (Frozen)
2. Microbiology (Room temp)
3. Hematology (Refrigerated)
[4. Microbiology]
Do not use glass tubes (Henry): Cells will adhere to
glass surface producing erroneous low counts on
Tube 3
Note Excess CSF ---(DO NOT)--> Discard
Left-over supernatant  Chemistry/Serology
CSF specimen  STAT
If STAT not possible, specimens are stored
Low volume Collected on 1 tube
specimen Microbiology  Hematology  Chemistry/Serology
CSF Appearance
Crystal clear Normal
Turbidity/cloudin WBC > 200μL
ess RBC > 400μL
(+) Microorganisms
RCM
Aspirated fat
 Protein (>45 but <150mg/dL)
Oily RCM
Hemolyzed/Blood RBCs
y
Clotted, pellicle  Protein
 Clotting factors
Xanthochromia Pink: very slight amount of HbO2
Orange: heavy hemolysis
Yellow: HbO2  Unconjugated bilirubin
RBC degradation products: most common cause
Causes of Visible HbO2 from artifactual RBC lysis
CSF Bilirubin in jaundiced patients
Xanthochromia CSF protein >150mg/dL
Merthiolate contamination
Carotenoids (orange)
Melanin (brownish): meningeal metabolic melanoma
Collection of CSF 2-5 days after traumatic tap
In normal neonates: because of immature BBB
CSF Protein NV = 15-45mg/dL
lec.mt 04 |Page | 164
 >45 but <150mg/dL (cloudy/turbid)
>150mg/dL (xanthochromic)
Intracranial Hemorrhage vs. Traumatic Tap
Intracranial/Cerebral
Traumatic Tap
Hemorrhage
Distribution of
Uneven (Tube 1 > 2 > 3) Even (Tube 1 = 2 = 3)
Blood
Clot + (Plasma Fibrinogen) -
Xanthochromia - +
(+) Clot Meningitis
(-) Blood Froin syndrome
Blockage of CSF circulation
(+) Weblike After overnight refrigeration (12-24 hrs)
pellicle TB meningitis
(+) D-dimer Indicates formation of fibrin at a hemorrhage site
Recent Clear supernatant
hemorrhage To examine a bloody fluid for xanthochromia:
Microhematocrit tube ---(Centrifuge)---> Examine
supernatant against white BG
Erythrophagocyto Indicates intracranial hemorrhage
sis/ Hemosiderin
granules
CSF Cell count Done immediately
WBCs and RBCs lyse w/in 1 hr
Refrigerate if cannot be processed immediately
WBC count Routinely performed on CSF
Diluting fluid: 3% Acetic acid
NV (adults) = 0-5 WBC/μL
NV (neonate) = 30 mononuclear WBCs/μL
Vol. of 1 square = 0.1 μL
Formula (Improved Neubauer counting chamber):
WBC count = No. of cells x Dilution factor
No. of sq. ctd x vol. of 1 sq.
-For diluted and undiluted specimen
-Neubauer counting chamber = No small RBC square
(Improved NCC: w/ small RBC squares)
-Four large squares (corner) and central large
square on both sides of the hemocytometer
Methylene blue = stains WBCs for better
differentiation
Total CSF cell WBC ct + RBC ct
count Diluting fluid: 0.85% (0.9%) NSS
RBC count = Not counted
RBC count Used for the correction of CSF WBC count and CSF
protein count when a traumatic tap has occurred
Clarity/Appearan Amount of Amount of
Dilution
ce Sample Diluent
Slightly Hazy 1:10 30 μL 270 μL
lec.mt 04 |Page | 165
Hazy 1:20 30 μL 570 μL
Slightly Cloudy 1:100 30 μL 2970 μL
Cloudy/Slightly
1:200 30 μL 5970 μL
Bloody
Bloody/Turbid 0.1 mL of 1:100
1:10,000 9.9 mL
dilution
CSF WBC Count WBC (added) = WBCBlood x RBCCSF or
Correction RBCBlood
PBS (normal) = -1 WBC/700 RBCs (CSF)
Differential Count Performed on stained smears
Specimen should be concentrated
Methods for Sedimentation
specimen Filtration
concentration Centrifugation
Cytocentrifugation
Cytocentrifugatio Fluid  Conical chamber
n Cells are forced into a monolayer w/in a 6mm
diameter circle on the slide
Addition of albumin:
 cell yield/recovery
 cellular distortion
CSF Differential Neonates (0-2 mos.)
count 50-90% monocytes
5-35% lymphocytes
0-8% neutrophils
Children (2 mos.-18 y.o.)
-Not yet established
Adults (>18 y.o.)
40-80% lymphocytes
15-45% monocytes
0-6% neutrophils
Cells in the CSF
Normal Lymphocytes
Monocytes
Neutrophils (occasional)
Adult Lymphocytes > Monocytes (70:30)
Neonates Monocytes > Lymphocytes
Pleocytosis  no. of normal cells
 amount of WBCs in any body fluid
Lymphocytes  viral, TB, fungal meningitis
Monocytes  multiple sclerosis
Neutrophils  bacterial meningitis, cerebral hemorrhage
 early cases of viral, TB, fungal meningitis
nRBCs BM contamination
(metarubricytes)
Eosinophils  helminthic parasitic infection
 fungal infection (C. immitis)

lec.mt 04 |Page | 166

  medications and shunts
Protozoans do not induce eosinophilia
Charcot-Leyden crystals (E. histolytica) 
eosinophils concentrate at the intestines and are
degraded
Plasma cells  multiple sclerosis
 lymphocytic reactions
Macrophages (+) RBCs
(+) RCM
Nonpathologically Choroidal cells: EC (choroid plexus)
significant cells Ependymal cells
Spindle-shaped cells: arachnoid
Malignant cells Hematologic:
-lymphoblasts
-myeloblasts
-monoblasts
Nonhematologic
-Astrocytomas
-Retinoblastomas
-Medulloblastomas
QC of CSF and Other Body Fluid Cell Count
Biweekly basis All diluents ---(check for)---> Contamination
Monthly basis Speed of cytocentrifuge should be checked w/ a
tachometer
CSF Protein
CSF protein Most frequently tested chemical test
NV:
Adults = 15-45 mg/dL
Infants = 150 mg/dL
Premature = 500 mg/dL
Albumin = majority
α-globulins = Haptoglobin
β-globulins = Tau transferrin (major)
γ-globulins = IgG (major), IgA (small amount)
IgM, fibrinogen, β-LPP = not found in normal CSF
Transthyretin TRANSports THYroxine and RETINol
(prealbumin) 2nd most prevalent
Tau Transferrin CHO-deficient β2-transferrin
Seen in CSF and not in serum
Electrophoresis: method of choice when
determining if a fluid is actually CSF
 CSF protein Damage: BBB (most common)
Production of Ig in CNS (multiple sclerosis)
 CSF protein CSF leakage
Recent puncture
Rapid CSF production
Water intoxication
Correction for If blood Hct and serum protein are normal:
lec.mt 04 |Page | 167
traumatic tap = -1 mg/dL protein/1,200 RBCs
Measurement of 1. Turbidimetric
Total CSF protein -Principle: precipitation of protein
a. TCA = precipitates albumin and globulins
(preferred)
b. SSA = precipitates albumin, add Na2SO4 to
precipitate globulins
2. Dye-binding technique
-Principle: protein error of indicators
-Coomassie Brilliant blue G250 (red)
-Beer’s law
-Protein binds to dye = red to blue
CSF IgG index In MS:  CNS IgG
CNS IgG must be differentiated from serum IgG
(damage to BBB)
 CSF IgG Damage to BBB
Active production w/in CNS (MS)
CSF/Serum _CSF Albumin (mg/dL)_
Albumin index Serum Albumin (g/dL)
a. Index <9 = Intact BBB
b. Index ≥9 = BBB is damaged
c. Index 100 = Complete damage to BBB
IgG index _CSF IgG (mg/dL)_
Serum IgG (g/dL)_____
CSF/Serum Albumin index
a. Index >0.70 = indicative of IgG production w/in
CNS (MS)
b. Index <0.70 = no active production w/in CNS
Oligoclonal bands ♫ Elevated IgG indices and presence of oligoclonal
bands – complementary findings useful in the
diagnosis of MS
Not normally present in CSF
Represents inflammation w/in the CNS
Not pathognomonic for multiple sclerosis
Oligoclonal bands Encephalitis
(not seen in Guillain-Barré syndrome
serum) Neurosyphilis
Neoplastic disorder
Multiple sclerosis
♫ Single band should not be interpreted as positive
Oligoclonal bands Leukemia
in serum but not Lymphoma
in CSF Viral infections
♫ Produces serum banding w/c can appear in the
CSF because of BBB leakage or traumatic tap
HIV Produces serum and CSF banding
Multiple sclerosis Symptoms:
-Abnormal sensations: numbness, tingling, pain,
lec.mt 04 |Page | 168
 burning, itching
-Mood swings
Myelin basic Protein component of the lipid-protein complex that
protein insulate the nerve fibers
Monitor the progress of MS
Identify individuals w/ MS who do not show
oligoclonal bands (approx. 10%)
Protein 14-3-3 In patients w/ dementia
Suggests CJD – caused by prions
“Mad cow disease” in cattle
Prions Live proteins
Infectious protein
Spongiform encephalopathy (CJD)
β-amyloid protein  CSF levels of microtubule associated Tau protein
42 and decreased levels of β-AP42 have been shown to
significantly increase the accuracy of Alzheimer’s
disease
Meningitis
S. agalactiae Neonates – 1 month
H. influenzae 1 month – 5 years old
N. gonorrhoeae 5 – 29 years old
S. pneumoniae >29 years old
L. monocytogenes Infants
Elderly
Immunocompromised patients

CSF Glucose
CSF Glucose NV = 60-70% of the plasma glucose concentration
A plasma glucose must also be run for comparison
Diagnostic significance:
- values
- CSF glucose values = result of  plasma glucose
 CSF glucose Bacterial, TB, fungal meningitis
N-CSF glucose Viral meningitis
CSF Lactate
CSF Lactate Frequently used to monitor severe head injuries
False elevations: Xanthochromia/hemolysis (RBC
contains lactate)
10-22 mg/dL Normal lactate
>35 mg/dL Bacterial meningitis
>25 Fungal and TB meningitis
<25 mg/dL Viral meningitis
CSF Enzymes
LD Serum: LD 2 > 1 > 3 > 4 > 5 [MI: LD 1 > 2 > 3 > 4 > 5]
CSF (normal): LD 1 > 2 > 3 > 4 > 5
Neurologic abnormalities: LD 2 > 1
Bacterial meningitis: LD 5 > 4 > 3 > 2 > 1

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CK-BB  Postcardiac arrest (poor prognosis)
<17 mg/mL = recovery
CSF Glutamine
CSF Glutamine Product of ammonia and α-ketoglutarate
NV = 8-18 mg/dL
Requested for patients w/ coma of unknown origin
>35 mg/dL Associated w/ some disturbances of consciousness
Reye’s syndrome Life threatening
 Glutamine in children
Degeneration of the liver  blood clotting problems
and bleeding
Gram Stain Concentrated specimen = often very few organisms
are present at the onset of disease
Organisms:
-S. pneumoniae
-H. influenzae
-E. coli
-N. meningitidis
-S. agalactiae (NB)
-L. monocytogenes (NB)
-C. neoformans
C. neoformans India ink
GS: Starburst pattern
-seen more often than a (+) India ink
Latex agglutination test
Limulus lysate Diagnosis of Gram (-) bacteria
test Reagent:
-Blood cells (Amoebocytes) of horse-shoe crab
(Limulus polyphemus)
-Amoebocytes: contain copper complex that gives
them blue color
Principle: In the presence of endotoxin,
amoebocytes (WBCs) will release lysate (protein) 
(+) Clumping/Clot formation
All materials must be sterile (Tap water:
endotoxin)
CSF serology Det. neurosyphilis
VDRL: recommended by Centers for Disease Control
and Prevention (CDC)
FTA-ABS
PAM (Henry) Naegleria fowleri
Acanthamoeba spp.
Acridine orange stain: useful to differentiate
amoeba (brick-red) from leukocytes (bright green)
L. monocytogenes The only Gram (+) that produces endotoxin
(-) Limulus lysate test
Seminal Fluid
Interstitial cells Secrete testosterone
lec.mt 04 |Page | 170
of Leydig Outside the seminiferous tubules
Seminiferous Site of spermatogenesis
tubule
Epididymis Stores and concentrates sperm
Sperm maturation
Prostate gland Zn3+, enzymes and proteins (coagulation and
liquefaction)
Seminal vesicles Fluid:  fructose
Vas deferens Transports sperm  ejaculatory duct
Bulbourethral Secretes alkaline mucus  neutralize prostatic and
(Cowper’s) gland vaginal acidity
Sertoli cells Serve as nurse cells for developing sperm cells
Inside the seminiferous tubules
Spermatogenesis Spermatogonia  1’ Spermatocytes  2’
Spermatocytes  Spermatids  Sperm
Round cells Either WBCs or spermatids
Seminal Fluid Composition
Seminal fluid (SV) 60-70%
Prostatic fluid 20-30%
Spermatozoa 5%
Bulbourethral 5%
gland
Chemical Composition of Seminal Fluid
ACP For liquefaction
Zn3+  in prostatic disease
Fructose Major nutrient of spermatozoa
+
K , citric acid, --
ascorbic acid
Proteolytic Liquefaction and coagulation
enzymes
Spermine and Inhibit growth of bacteria
Choline
Importance of To investigate the causes of infertility in marriages
Seminalysis To check the effectiveness of previous vasectomy
In medico-legal cases, where paternity is being
disclamed on the basis of male sterility
Sexual abstinence 2-3 days and not >5 days
 abstinence =  volume,  motility
Methods of Important: 1st portion of ejaculate
collection 1. Masturbation: best
2. Coitus interruptus (withdrawal method)
3. Common condom collection
-Condoms for sperm collection:
a. Silastic (Silicone rubber)
b. Polyurethane condoms: called the Male Factor Pak
4. Aspiration of semen from the vaginal vault after
coitus

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 5. Specimen should be delivered in the lab w/in 1 hr
(RT’)
Methods of Specimen kept at 37’C awaiting analysis
preservation For artificial insemination, it can be preserved in
frozen state and stored at
-85’C (seminal banks)
Fresh specimen is clotted
Semen Analysis
Liquefaction time 30-60 mins
If not yet liquefied after 2 hrs, use α-chymotrypsin
Normal values Color = grayish white
Volume = 2-5 mL
Viscosity = pour in droplets
pH = 7.2-8.0
Sperm concentration = >20,000,000/mL
Sperm count = >40,000,000/ejaculate
Motility = >50% w/in 1 hr
Motility quality = >2.0 or a, b, c after 1 hr
WBCs = <1,000,000/mL
♫ >1,000,000/mL = inflammation
Volume : incomplete collection/infertility
: prolonged abstinence
Yellowish semen Prolonged abstinence
Medication
Urine contaminationi
 White turbidity Infection ( WBCs)
Red coloration (+) RBCs
Viscosity 0 (watery)
4 (gel-like)
pH Too basic = infection
Too acidic =  prostatic fluid
Sperm Diluting fluid:
concentration 1. Cold H2O
2. Formalin
3. NaHCO3
4. 0.5% in chlorazene
5. 1% formalin in 3% trisodium citrate
1:20 = mechanical positive displacement pipette
Counting chamber 1. Neubauer counting chamber = diluted specimen
(WHO recommended)
2. Makler chamber = undiluted w/ heating processes
Purpose of To immobilize the sperm
Dilution
Det. sperm conc. 1. 5 RBC squares
(Short-cut) # sperms counted x 1,000,000 = sperms in
million/mL
2. 2 WBC squares
# sperms counted x 100,000 = sperms in million/mL
lec.mt 04 |Page | 172
Sperm count Sperm concentration x volume of specimen
Motility quality 4.0 (a) = Rapid motility
(20/hpf) 3.0 (b) = Slower speed, some lateral movement
2.0 (b) = Slow forward progression + lateral
movement
1.0 (c) = No forward progression
0 (d) = No movement at all
CASA Computer-Associated Semen Analysis
-Sperm concentration
-Sperm velocity and trajectory
Sperm morphology At least 200 sperms evaluated
1. Routine criteria = >30% normal morphology
2. Kruger’s strict criteria = >14% normal morphology
-measure head, neck, tail using micrometer
Head morphology Poor ovum penetration
abnormalities
Tail abnormality Poor motility
Sperm head Oval, approximately 5 μm x 3 μm wide
Tail 45 μm long
Midpiece Contains mitochondria
Connects head and tail
Acrosomal cap Ovum penetration
Covers approximately 2/3 of sperm nucleus and ½ of
the head
Tapered head Varicocele
-Common cause of male sterility
-Hardening of veins that drains the testes
Stains Giemsa
Papanicolau = method of choice
Wright’s
Fructose test If sperm count is low
Rgt: Resorcinol
End-color: Orange-red
Specimens should be tested in 2 hrs or frozen
 neutral-α- Epididymis disorder
glucosidase
Florence test Choline
(+) Dark brown rhombic crystals
Barbiero’s test Spermine
(+) Yellow leaf-like crystals
Spinbarkeit test Tenacity of mucus
Sim Huhner test Post-coital test
Test for the ability of sperm cells to penetrate the
cervical mucosa
Bloom’s/Eosin- If N-sperm count but  motility
Nigrosin/ Sperm Living sperm cells = bluish white
viability test Dead = red

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 NV = 75% living sperms
(25% dead)
Decreased (+) Male antisperm antibodies
motility w/ Blood testis barrier  disrupted
clumping Clumps of sperm
Normal (+) Female antisperm antibodies
seminalysis w/
continued
infertility
MAR Mixed agglutination reaction
Detect IgG antibodies
Immunobead test Detect IgG, IgA, IgM
Demonstrate area of the sperm the autoantibodies
are affecting
Hamster egg Sperm incubated w/ species non-specific hamster
penetration egg
Cervical mucus Observed sperm penetration ability
penetration
Hypo-osmotic Test for membrane integrity and viability of sperm
swelling
To determine Microscopic exam for sperm cells
whether semen is Enhance w/ xylene
present Examine  Phase microscope
Seminal glycoprotein p30: specific method
Aspermia No ejaculate
Azospermia Absence of sperm cells
Necrospermia Immotile/dead sperm cells
Oligospermia  sperm cells

Synovial Fluid
Synovial Latin: “Egg”
Fluid Diarthroses/joints
Arthrocentesis Method of collections
Synoviocytes Phagocytic cells
Secrete hyaluronic acid
Specimen Fluid  Syringe (heparin)
collection Micro: 3-5 mL in sterile tube, add 25 U heparin/mL
fluid
Hema: 3-5 mL, add 25 U heparin/mL fluid
-Do not use crystalline EDTA but liquid EDTA may
be accepted
Chem: 3-5 mL in red top and observe for clotting
NaF: glucose analysis
Normal Values Volume = <3.5 mL
Color = pale yellow
Clarity = clear
lec.mt 04 |Page | 174
 Viscosity = 4-6 cm long
Crystals = none present
Glucose = <10 mg/dL lower than the blood glucose
Lactate = <250 mg/dL
Total protein = <3 mg/dL
Uric acid = equal to blood value
Turbidity  WBCs
Milky (+) Crystals
Rope’s/Mucin clot Hyaluronate polymerization test
test 2-5% acetic acid
Grading:
-Good = solid clot
-Fair = soft clot
-Low = friable clot
-Poor = no clot at all
Cells in Synovial Fluid
WBC Total WBC count: most frequently tested
STAT, otherwise, refrigerate
Neubauer counting chamber
Clear fluids = counted undiluted
Diluting fluid = NSS
If necessary to lyse RBCs:
-Hypotonic saline (0.3%)
-Saline w/ saponin
Do not use WBC diluting fluid
Differential count 65% = Monocytes & Macrophages
<25% = Neutrophils
<15% = Lymphocytes
Very viscous Add hyaluronidase  0.5 mL of fluid or
specimen 1 drop of 0.05% hyaluronidase in PO4 buffer/mL of
fluid
37’C for 5 mins
LE cell Neutrophil
Reiter cells Vacuolated macrophage w/ ingested neutrophils
Ragocyte Neutrophil w/ dark cytoplasmic granules
Cartilage cells Large multinucleated cells
Rice bodies Macroscopically resembles polished rice
Ground pepper Ochronotic shards = debris
appearance of Metal and plastic joint prosthetic
synovial fluid
Hemosiderin Pigmented villonodular synovitis
Cartilage cells Osteoarthritis
Crystals
Normal No crystals
Abnormal Hydroxyapatite crystals
Cholesterol
Corticosteroid

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 MSU = needle-like (gout)
CPP = needle-like/rods (pseudogout)
MSU (-) Birefringence (yellow) on compensated (red)
polarizing microscopy
CPP (+) Birefringence (blue) on compensated (red)
polarizing microscopy
Joint Disorders
Group I: Non- OA
inflammatory Traumatic arthritis
Neuroarthropathy
Group II: Immunologic: RA, SLE
Inflammatory Crystal-induced: milky synovial fluid
Group III: Septic Yellow green
Low viscosity
>50,000 WBC/μL (highest)
(+) Culture: CAP = Haemophilus, N. gonorrhoeae
(gonococcal arthritis)
S. aureus: predominant pathogen in adult joint
infection
B. burgdorferi: Lyme disease (Stage 3: Lyme
arthritis)
Group IV: Sickle cell disease
Hemorrhagic Glucose = Blood
Serous Fluids
Serous fluids Fluids that are formed between the parietal and
visceral membrane
Ultrafiltrate of plasma
Provides lubrication
Effusion Increase in serous fluid amount between the
membranes
Either transudate or exudates
Transudate From disturbances of fluid production and
regulation between serous membranes
-Hypoproteinemia
-Nephrotic syndrome
-CHF
Testing of transudates is not necessary
Exudate Purulent fluid that forms in any body cavity as a
result of inflammatory processes
-Infection
-Malignancies
Specimen >100 mL is usually collected
collection EDTA = cell counts and differential
Heparin = chemistry, serology, microbiology,
cytology
Rivalta’s Differentiates transudates from exudates
test/Serosa Acetic acid + H2O + unknown fluid  heavy ppt. = (+)
mucin clot test exudate
lec.mt 04 |Page | 176
Fluid: Serum LD Most reliable differentiation between transudates
ratio and exudates (except peritoneal fluid: SAAG)
Fluid: Serum
protein ratio
Pleural Fluid
Thoracentesis Method of collection
Normal Clear/pale yellow
appearance
Turbid  WBCs (infection)
Bloody Hemothorax (traumatic injury):
= Pleural fluid Hct is >50% of WB Hct
Chronic membrane disease/Hemorrhagic effusion:
= Pleural fluid Hct < WB Hct
Milky Chylous (thoracic duct leakage) =  TG, (+) Sudan
III
Pseudochylous (chronic inflammation) = 
cholesterol, (-) Sudan III
CA 15-3 and CA Breast cancer
549
CYFRA 21-1 Lung cancer
(Cytokeratin
Fragment)
AMS Pancreatitis
Esophageal rupture
ADA TB
Malignancy
Mesothelial cells  TB
Glucose  infection
Lactate  bacterial infection
pH  in pneumonia
 in esophageal rupture
Pericardial Fluid
Pericardiocentesis Method of collection
Normal volume 10-50 mL
Mesothelial cells Produces pericardial fluid
Function Reduces friction
Normal Clear/pale yellow
appearance
Grossly bloody Accidental cardiac puncture
Misuse of anticoagulant medicatios
Milky Chylous/pseudochylous effusions
Bacterial >1000 WBCs/μL (% neutrophils)
endocarditis
Peritoneal Fluid (Ascites)
Paracentesis Method of collection
Peritoneal lavage Sensitive test for the detection of intra-abdominal
bleeding

lec.mt 04 |Page | 177

WBC <500/μL Normal
RBC >100,000/μL Blunt trauma cases
Serum Ascites Recommended to differentiate transudates from
Albumin Gradient exudates
(SAAG) a. >1.1 = Transudate
b. <1.1 = Exudate
Psammoma bodies Contains concentric striations of collagen materials
Ovarian and thyroid malignancies
(+) CA 125 Tumor of ovaries, fallopian tubes or endometrium
(-) CEA
Glucose  TB peritonitis
ALP Intestinal perforation
AMS Pancreatitis
GI perforation
BUN Ruptured bladder or accidental puncture of the
Creatinine bladder
Sweat Test
Cystic fibrosis Autosomal recessive
(mucoviscidosis) Pancreatic insufficiency
Respiratory distress
Intestinal obstruction
-Bulky offensive greasy stools (butter-like)
Gibson and Cooke  Sweat Cl- and Na+ = >70 mEq/L
Pilocarpine Borderline = 40 mEq/L
Iontophoresis Pilocarpine w/ mild current = stimulates sweat
glands
Sodium FEP, IEE
Chloride Manual or automated titration
Amniotic Fluid
During 1st 35 mL: derived from maternal circulation/plasma
trimester
After 1st Fetal urine
trimester
3rd trimester Peak: 1L amniotic fluid
Oligohydramnios  amniotic fluid
Urinary tract deformities
Membrane leakage
 fetal swallow of urine
Hydramnios/  amniotic fluid
Polyhydramnios  fetal swallow of urine
Neural tube defects
Amniocentesis Method of collection
2nd trimester: assess genetic defects
3rd trimester: FLM or HDN
Fern test Specimen: Vaginal fluid
Glass slide  air dry
(+) Fern-like crystals

lec.mt 04 |Page | 178

 (+) Amniotic fluid
Normal Clear
appearance
Green Meconium
Yellow Bilirubin (HDN)
Bloody/red Trauma, abdominal trauma, intra-amniotic
hemorrhage
Dark-brown/ Fetal death
Reddish-brown
Assessment of  OD at 450 nm
HDN OD reading at a Liley graph
1. Zone 1: observe fetus for stress
2. Zone 2: moderate disease
3. Zone 3: severe disease
Neural tube Ex. Spina bifida and Anencephaly
defects Screening: AFP
Confirmatory: Acetylcholinesterase
Fetal Lung Maturity
L/S ratio Reference method
>2.0 = mature fetal lungs
Disadvantage: can’t be done on specimen
contaminated w/ meconium
Lecithin Surfactant for alveolar stability
Surfactants Produced by type II alveolar pneumocytes (stored
as lamellar bodies = about the size of platelets)
Amniostat FLM Detect the presence of phosphatidyl glycerol
Advantage: not affected by blood or meconium
Foam stability Amniotic fluid + 95% ethanol  shake for 15 secs 
test stand (15 mins)
(Foam/Shake (+) Continuous line of bubbles
test) Can be done bedside
Fetal age Creatinine ≥2.0 mg/dL (36 weeks/9 months)
Gastric Fluid Analysis
Clinical Pernicious anemia
significance Peptic ulcer
Zollinger-Ellison disease
Parietal cells Produces HCl and intrinsic factor
Pepsinogen Produced by chief cells
Pepsinogen ---(HCl)---> Pepsin
Gastrin Produced by G-cells
Stimulates parietal cells to produce HCl
Zollinger-Ellison  gastrin
disease Gastric acid hypersecretion
Adenoma of the islets of Langerhans (pancreas) =
produce gastrin
Pernicious anemia “Dangerous” anemia
BAO/MAO = 0

lec.mt 04 |Page | 179

Specimen Collect gastric juice for 1 hr
collection 1. Levine tube = nose
2. Rehfuss tube = mouth
Basal Acid Output Total gastric secretion during unstimulated fasting
(BAO) state
Maximal Acid Total acid secreted in the hour after stimulation
Output (MAO)
Gastric stimulants 1. Pentagastrin = most preferred
2. Histamine
3. Histalog (Betazole)
4. Alcohols
5. Insulin = assess vagotomy procedure
-SHAM feeding = sandwich
Test Meals 1. Ewald’s meal = bread and tea/H2O
2. Boa’s = oatmeal
3. Riegel’s = mashed potato and beef steak
Yeast cells in Fermentation in stomach because large amounts of
Gastric fluid food have been retained
Quantitative Tests for Gastric Acidity (Topfer’s)
Free HCl Titration: NaOH
pH indicator: Dimethylaminoazobenzol
(+) Canary yellow
NV = 25-50O
Total Acidity Titration: NaOH
pH indicator: phenolphthalein
(+) Faint pink
NV = 50-75O
Combined HCl Titration: NaOH
(bound to pH indicator: sodium alizarin
proteins) (+) Violet
NV = 10-15O
Euchlorhydria Normal free HCl
Hyperchlorhydria  free HCl
Ex. peptic ulcer
Hypochlorhydria  free HCl
Ex. carcinoma of the stomach
Achlorhydria (-) free HCl
Ex. pernicious anemia [BAO/MAO = 0]
Diagnex tubeless Specimen: Urine
test Principle:
-Azure blue is given by mouth
-Presence of azure blue in urine indicates presence
of free HCl in stomach
Lactic acid Indicative of advanced gastric cancer
1. Modified Uffelmann’s
-Rgt: FeCl3 and phenol
-(+) Yellow
2. Strauss
lec.mt 04 |Page | 180
 -Rgt: FeCl3 and ether
-(+) Yellow
3. Kelling’s
-Rgt: FeCl3
-(+) Yellow
Fecalysis
Creatorrhea Undigested muscle fibers in feces
Stain: Eosin
a. Completely digested: no striations
b. Partially digested: striation in one direction
c. Undigested: striations in both directions
>10 undigested muscle fibers = bile duct
obstruction, cystic fibrosis
Celiac disease Most common cause of malabsorption in developed
countries
Steatorrhea  fat in stool
G. lamblia
Rotten egg odor
Melena Black tarry feces
Upper GI bleeding
Stool Color
Red Lower GIT bleeding
Tx: Rifampin
Black Upper GIT bleeding
Iron ingestion
Bismuth (antacids)
Charcoal
Green Biliverdin
Vegetables
Gray Bile duct obstruction
Mucus/RBCs Dysentery
Colitis
Malignancies
Consistency Variations
Rice watery Cholera
Pea soup Typhoid fever
Flattened/ribbon- Spastic colitis
like
Bulky/frothy Bile duct obstruction
Pancreatic disorders
Abundant fats
Butter-like Cystic fibrosis
Scybalous/Goat Constipation
droppings
Bristol Stool Chort
Type 1 Separate hard lumps, like nuts (hard to pass)
Type 2 Sausage-shaped but lumpy

lec.mt 04 |Page | 181

Type 3 Sausage, w/ cracks on surface
Type 4 Sausage, snake, smooth surface
Type 5 Soft blobs w/ clear cut edge
Type 6 Fluffy w/ ragged ends (mushy)
Type 7 Watery
Occult Blood
Occult blood “Hidden”
Screening for colorectal cancer
Significant: >2.5 mL blood/150g stool
Guaiac Least sensitive but the most preferred
Benzidine Most sensitive
O-toluidine
Principle Pseudoperoxidase activity of Hgb
(+) Blue
False (-) Vitamin C
False (+) Turnips, broccoli, cauliflower, banana, apple, melon,
horseradish (to avoid, 3 days free)
Aspirin, aspilet (promote GIT bleeding; to avoid, 7
days free)
Red meat (to avoid, should be 3 days free)
APT test Distinguish fetal blood and maternal blood in an
infant’s stool
Specimen: infant stool/vomitus
Rgt: 1% NaOH
HbF: alkali-resistant
Maternal Hgb: not alkali-resistant
(+) Pink = HbF
(-) Yellow brown = Maternal Hgb
Diarrhea Acute: <4 weeks
Chronic: >4 weeks
Secretory  solute secreted by the intestine
diarrhea Endotoxins
Osmotic diarrhea  amounts of osmotically active solutes in the lumen
(maldigestion)
 CHO in stool
Intestinal Secretory and osmotic diarrhea
hypermotility Laxatives
Emotions/stress
Cardiovascular drugs
Fecal enzymes 1. Trypsin
= X-ray paper
= Trypsin deficiency (CF): inability to digest gelatin
on the X-ray paper
2. Chymotrypsin
3. Elastase I = pancreas specificity
Fecal CHO Stool pH = 7.0-8.0
pH 5.5 = CHO disorders (lactose intolerance)

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 Clinitest: >0.5 g/dL = CHO intolerance
Follow up tests:
a. D-xylose: malabsorption
b. Lactose intolerance test: maldigestion
Fecal Leukocytes Primarily neutrophils
 Neutrophils:
-Salmonella
-Shigella
-Campylobacter
-Yersinia
-EIEC
(-) Fecal leukocytes:
-Parasites
-Viruses
-S. aureus
-Vibrio spp
Methylene blue For wet preparation
Faster procedure than Wright’s and GS (for dry
smears) but may be more difficult to interpret
Lactoferrin Latex Sensitive in refrigerated and frozen specimens
agglutination test
 amounts of Biliary obstruction
striated fibers Gastrocolic fistulas
Fecal fats NV = 1-6 g/day
Qualitative Fecal Sudan III = most routinely used
fat test Sudan IV
Oil red O
Split fat stain Free fatty acids and fatty acids from hydrolysis of
soaps and neutral fats
NV = 100 droplets (<4 μm)
Slightly increased = 100 droplets (1-8 μm)
Increased = 100 droplets (6-75 μm)
Quantitative Confirmative test for steatorrhea
Fecal fat test 3 day specimen
-intake of fat = 100g/day
Prior to and during collection  paint cans
Van de Kamer titration = NaOH
Duodenal Fluid
Duodenal fluid Physiologically acidic pH stimulates mucosal cells to
produce secretin
Secretin Stimulates watery pancreatic secretions w/
bicarbonate
Pancreozymin Provokes enzyme production of pancreas (AMS,
LPS)
Secreted by mucosal cells
(-) Pancreozymin = (-) AMS & LPS
Secretin test Most sensitive test for impaired pancreatic
function
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 Secretin is administered IV, then duodenal fluid
HCO3- is tested
Pancreatic cancer  volume
N-HCO3-
N-AMS
Chronic  volume
pancreatitis  HCO3-
 AMS
Sputum
1st morning Most ideal
24 hr Volume measurement:
 vol = bronchiectasis, lung abscess, edema,
gangrene, TB
 vol = bronchial asthma, acute bronchitis, early
pneumonia
Throat swab Pediatric patients
Tracheal aspirate Debilitated patients
Sputum Color
Transparent Normal
Mucus only
White/yellow (+) Pus
Gray (+) Pus, EC
Yellow-green TB, bronchiectasis
Green P. aeruginosa
(+) Bile
Red or Bright red Fresh blood (hemorrhage)
TB, bronchiectasis
Anchovy Old blood
sauce/rusty Pneumonia
brown
Rusty red Lobar pneumonia
Brown CHF
Black Heavy smokers
Anthracosis
Inhalation of dust, dirt, carbon, charcoal
Prune juice Pneumonia
Chronic lung cancer
Olive green/grass Chronic lung cancer
green
Causes of blood- a. Rusty, (+) pus = pneumococcal pneumonia
stained sputum b. Rusty, (-) pus = CHF
c. Bright streaks in viscid sputum = K. pneumoniae
d. Spurious hemoptysis = nosebleed
Heart failure cells Hemosiderin laden macrophages in the alveolar
spaces
Sputum Odor
Odorless Normal

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Foul/putrid Cavitary TB
Lung abscess
Gangrene
Advanced necrotizing tumors
Fruity P. aeruginosa
Sweetish TB, Bronchiectasis
Consistency
Mucoid Asthma and bronchitis
Serous/frothy Lung edema
Mucopurulent TB, bronchiectasis
Macroscopic Structures in Sputum
Bronchial casts Made of fibrin
Branching tree-like casts
Lobar pneumonia
Cheesy masses Fragments of necrotic pulmonary tissue
Pulmonary TB
Pulmonary gangrene
Dittrich’s plugs Grayish to yellowish material
Size of pinhead
Foul odor when crushed
Cellular, fatty acids, fat globles, some bacteria
Bronchiectasis
Chronic bronchitis
Bronchial asthma (3C’s)
Lung From calcified pulmonary tissue
stones/Pneumolith Histoplasmosis
s/ Broncholiths
Curschmann’s Mucoid threads that are twisted/coiled
spirals Bronchial asthma (3C’s)
Bronchitis
Layer formation Bronchiectasis
(3) Lung abscess
Gangrene
Top = Frothy mucus
Middle = opaque H2O material
Bottom = pus, bacteria, tissues
Microscopic Elements
PAS (+) Alveolar proteinosis
Macrophages
PAS (+) rounded P. carinii
bodies that take
silver stain
Elastic fibers TB
Charcot-Leyden From degeneration of eosinophils
crystals Bronchial asthma (3C’s)
Creola bodies Clusters of columnar cells
Bronchial asthma (3C’s)

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Fungi C. albicans
C. neoformans (Torulosis)
C. immitis
Parasites 1. Migrating larva (Heart  Lung) = “ASH”
-Ascaris
-Strongyloides
-Hookworm
2. P. westermani
3. E. granulosus (Pulmonary hydatid disease)
4. E. gingivalis
5. T. tenax
Bronchoalveolar Lavage
Clinical significane P. jiroveci
Macrophages (56- Most predominant
80%)
Lymphocytes (1-  interstitial lung disease, pulmonary lymphoma,
15%) nonbacterial infection
Neutrophils (<3%)  cigarette smokers, bronchopneumonia, toxin
exposure
Eosinophils (<1 to  hypersensitivity reaction (asthma)
2%)
Human Chorionic Gonadotropin
hCG Produced by cytotrophoblast cells of the placenta
 1st trimester of pregnancy
α-subunit = FSH, LH, TSH, hCG
β-subunit = unique to hCG
Specimen 1st morning urine
Bioassays
Ascheim-Zondek Female mice
Subcutaneous injection
(+) Corpora lutea, hemorrhagic follicles
Hogben Female toad
Lymph sac injection
(+) Oogenesis
Galli-Mainini Male frogs, male toads
Subcutaneous injection
(+) Spermatogenesis
Friedmann’s Mature virgin female rabbit
Marginal ear vein injection
Frank-Bermann Female rats
Intraperitoneal injection
(+) Ovarian hyperemia

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