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
PA
Normal values:
M = 107-139 mL/min
F = 87-107 mL/min

Tubular Reabsorption

Tubular Reabsorption 1st 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
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

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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

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

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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
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 yellow Polyuria: DM/DI

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

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Red/Purple/Burgundy Porphyria
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 (acid) Rifampin (Tx: TB) = all body fluids are red

Bright yellow Riboflavin (Multivitamins)

Nubecula Faint cloud in urine after a period of standing

WBCs, epithelial cells and mucus
Bilifuscin (Dipyrrole) Hemoglobin Köln = unstable
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 epithelial cells 🡩 females

Radiographic contrast 🡩 SG by refractometer (>1.040)

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

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 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 (TS meter) Based on refractive index:

RI = _light velocity in air_
light velocity in soln
Compensated to temperature (15-38’C)
Corrections:
a. 1g/dL glucose: (-0.004)
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 calibration K2SO4 solution: 1L H2O + 20.29g K2SO4

SG = 1.015

Isosthenuria SG = 1.010 (Glomerular filtrate)

Hyposthenuria SG < 1.010

Hypersthenuria SG > 1.010

Urine Odor

Aromatic/Odorless Normal

Ammoniacal Urea ---(Urease)---> NH3

Ex. UTI (Proteus: 🡩 urease)

Fruity, sweet DM (Ketones)

Rotten fish/Galunggong Trimethylaminuria

Sweaty feet Isovaleric acidemia

Mousy Phenylketonuria

Cabbage Methionine malabsorption

Caramelized sugar, curry MSUD

Bleach Contamination
Sulfur Cystine disorder

Chemical Examination of Urine

Specific Gravity

Principle (Rgt Strip) pKa dissociation constant

🡩 concentration = 🡩 H+
Indicator: Bromthymol blue = (🡫) Blue 🡪 Green 🡪 Yellow (🡩)

Other info. Not affected by glucose, protein and RCM

Harmonic Oscillation Frequency of soundwave entering a solution will change in proportion to

Densitometry the density (SG) of the solution
-Yellow IRIS (Automated): International Remote Imaging System

pH

Normal Random = 4.5-8.0

1st morning = 5.0-6.0
pH 9.0 = Unpreserved urine

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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 Strip) Double indicator system (Methyl red & Bromthymol 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 proteinuria Prior to reaching the kidney
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
-1st morning urine: (-) protein
-2 hrs after standing: (+) protein
3. Fanconi’s syndrome

Post-renal proteinuria Lower UTI/inflammation

Principle (Rgt Strip) Protein (Sorensen’s) error of indicators

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 types of
precipitation test protein If (-) rgt strip, (+) SSA = presence of other proteins

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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 granulation 30-100 mg/dL

2+ Turbidity w/ granulation but no flocculation 100-200 mg/dL

3+ Turbidity w/ granulation and flocculation 200-400 mg/dL

4+ Clumps of protein > 400 mg/dL


Glucose
Other substances in urine
Hyperglycemia
associated Glycosuria
Renal associated Glycosuria
Principle (Rgt Strip)
Copper Reduction test
(Clinitest)
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
ID: TLC
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
-(+) Rubner’s test (Lead acetate)
4. Pentose
-Xylose, arabinose
-Xylulose:Benign pentosuria
5. Sucrose
-🡩 Intestinal disorders
-Nonreducing sugar
-(-) Copper reduction test
🡩 Blood glucose, 🡩 Urine glucose
1. DM
2. Cushing’s syndrome/disease = 🡩 cortisol
3. Pheochromocytoma = 🡩 catecholamines
4. Acromegaly = 🡩 GH
5. Hyperthyroidism = 🡩T3/T4
N-Blood glucose, impaired tubular reabsorption of glucose
1. Fanconi’s syndrome: defective tubular reabsorption of glucose and
amino acids
Double sequential enzyme reaction:
-Glucose oxidase
-Peroxidase
Chromogen:
-KI (Brown)
-Tetramethylbenzidine (Blue)
Blue tablet
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)
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Pass through phenomenon Occurs if >2 g/dL sugar is present in urine
Blue 🡪 Green 🡪 Yellow 🡪 Brick red 🡪 Blue (Pass through)
To prevent, use 2 gtts urine (instead of 5 gtts) + 10 gtts H2O + Clinitest

(-) Glucose oxidase (+) Nonglucose reducing substance

(+) Clinitest

1+ Glucose oxidase True glucosuria

(-) Clinitest Small amount of glucose present

4+ Glucose oxidase False (+)

(-) Clinitest Possible oxidizing agent interference on reagent 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
2% Acetone

Significance Diabetic acidosis

Insulin dosage monitoring
Starvation
Vomiting

Principle (Rgt Strip) Legal’s test (Sodium nitroprusside reaction)

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 Strip) Pseudoperoxidase activity of hemoglobin

Chromogen: TMB [(-) Yellow/(+){🡫}Green 🡪 Blue {🡩}]
H2O2 + Chromogen ---(Heme)---> Oxidized chromogen + H2O

Hgb/Mgb Uniform green/blue

Hematuria Speckled/spotted

Extravascular lysis 🡩 Unconjugated bilirubin

🡩 Urine and fecal urobilinogen

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Intravascular lysis (+) Hemoglobinuria
🡫 Haptoglobin and Hemopexin

Bilirubin

Bilirubin Amber urine w/ yellow foam

Early indication of liver disease

Significance Hepatitis
Cirrhosis
Bile duct obstruction

Principle (Rgt Strip) Diazo Reaction:

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 Strip) Ehrlich’s reaction (PDAB):
Urobilinogen + PDAB ----------> (+) Cherry red

Watson-Schwartz test For differentiating urobilinogen and porphobilinogen 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 (Inverse Rapid screening test for urine porphobilinogen (≥2mg/dL)
Ehrlich 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 strip) Greiss reaction:

NO3 + Sulfanilamide + p-arsanilic acid + tetrahydrobenzoquinoline
------------> (+) Uniform
pink
Pink spots/edges = (-)

Leukocytes

Significance UTI/inflammation
Screening of urine culture specimen

Leukocyte esterase (+) Neutro/Eo/Baso/Mono

(-) Lympho
Maybe (+): Histiocytes, Trichomonas

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Principle (Rgt strip) Indoxyl carbonic acid ester + Diazonium salt ---(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 (Ascorbic acid) 11th reagent pad

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 protein Highly alkaline urines (>6.5)

pH No known interfering substance

Runover from adjacent pads
Old specimens

Protein Highly buffered alkaline urine Proteins other than albumin

Pigmented specimens,
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 agents High levels of ascorbic acid

and detergents High levels of ketones
High specific gravity
Low temperatures
Improperly preserved specimens
Ketones Phthalein dyes Improperly preserved specimens
Highly pigmented red urine
Levodopa
Medications containing free sulfhydryl grps

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False-positive False-negative

Blood Strong oxidizing agents High specific gravity/crenated cells

Bacterial peroxidases Formalin
Menstrual contamination Captopril
High concentration of nitrite
Ascorbic acid >25 mg/dL
Unmixed specimens

Bilirubin Highly pigmented urines, Specimen exposure to light

phenazopyridine Indican (intestinal Ascorbic acid >25 mg/dL
disorders) High concentrations of nitrite
Metabolites of Lodine

Urobilinogen Porphobilinogen Old specimens

Indican Preservation in formalin
p-aminosalicylic acid
Sulfonamides
Methyldopa
Procaine
Chlorpromazine
Highly pigmented urine

Nitrite Improperly preserved specimens Nonreductase-containing bacteria

Highly pigmented urine Insufficient contact time between bacteria
and nitrate
Lack of urinary nitrate
Large quantities of bacteria converting
nitrite to nitrogen
High concentrations of ascorbic acid
High specific gravity

Leukocytes Strong oxidizing agents High concentrations of protein, glucose,

Highly pigmented urine, nitrofurantoin oxalic acid, ascorbic acid, gentamicin,
cephalosporins, tetracyclines

Microscopic Exam of Urine

Phase-contrast microscopy Visualization of elements w/ low refractive indices:
-Hyaline casts
-Mixed cellular casts
-Mucous threads
-Trichomonas

Polarizing microscopy ID of cholesterol in OFB, FC and crystals

Interference contrast Produces 3D microscopy-image and layer-by-layer imaging of a

microscopy specimen 1. Hoffman microscope: modulation contrast microscope
2. Nomarski microscope: differential interference contrast microscope

Sternheimer-Malbin Crystal violet and safranin

Nucleus and cytoplasm
ID: WBCs, ECs, casts

Toluidine blue (Supravital) Enhances nuclear detail

Differentiates WBCs and RTE

Lipid stains: Stain TG and neutral fats orange red

ORO and Sudan III ID: free fat droplets and lipid-containing cells and 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

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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
-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 epithelial cells Largest cell in the urine sediment

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 epithelial Spherical, polyhedral, or caudate w/ centrally located nucleus

cells (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 eccentriac

epithelial cells 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)

T. vaginalis Flagellate w/ jerky motility

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Pingpong disease

S. haematobium “Hematuria”
Specimen: 24 hr unpreserved urine
E. vermicularis Most common fecal contaminant

Casts (Cylindruria) Formed in the DCT and CD

♫ 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 casts Renal tubular damage

Advanced tubular destruction

Coarse/Fine granular casts Formed from the disintegration of cellular cast

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 preparation Urine 🡪 Centrifuge: 400 RCF for 5 mins 🡪 Decant 🡪 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

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Urine Crystals

Amorphous Urates Yellow-brown granules

(Normal) Pink sediment (Uroerythrin)
(pH: acid) Mistaken as cystine crystals
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)
(pH: acid/alkaline/neutral)
-Envelope/pyramidal
2. Whewellite = monohydrate
-Oval, dumbbell
-Ethylene glycol poisoning (antifreeze agent)
Most renal stones consist of CaOx

Amorphous White precipitate

Phosphates (Normal) Granular appearance
(pH: alkaline/neutral) 🡩 After meal (alkaline tide)

Ammonium Biurate Yellow-brown

(Normal) Thorny apples
(pH: alkaline) Old specimen: due to the presence of urea-splitting bacteria

Triple Phosphate A.k.a. Magnesium ammonium phosphate

(Normal) Coffin lid, “Struvite”, staghorn appearance
(pH: alkaline) Presence of urea-splitting bacteria

Calcium Phosphate
(Normal)
(pH: alkaline/neutral)
Colorless, flat rectangular plates or thin prisms often in rosette
formation Rosettes may resemble sulfonamides
-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

(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 radial

(Abnormal) striations Liver disease
(pH: acid/neutral)

Bilirubin Clumped needles or granules w/ yellow color

(Abnormal) (+) Diazo reaction
(pH: acid) Liver disease

lec.mt 04 |Page | 147

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 Artifacts 1. Starch granules (gloves):
-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
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

Glomerulonephritis Inflammation of the glomerulus

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 Nephritis Infection of the renal interstitium

RBCs, WBCs, WBC casts, NO BACTERIA

Renal carcinoma 1’ = RCC

2’ = Transitional CC
RBCs and WBCs

Nephrotic syndrome Massive proteinuria and lipiduria

a. Serum (Chemistry)
-🡫 Albumin, alpha1, beta and gamma globulins
-🡩 alpha2 (AMG)
b. Urine (CM)
-🡩 Albumin, alpha1, beta and gamma globulins
-(-) alpha2 (AMG)

lec.mt 04 |Page | 148

-Oval fat bodies, fatty and waxy casts
Telescoped sediments Simultaneous appearance of the elements of 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 progressive Deposition of immune complex from systemic immune disorders on

(Crescentic) GN the glomerular membrane

Goodpasture syndrome Attachment of cytotoxic antibody to glomerular and alveolar

basement membrane

Wegener’s granulomatosis Antineutrophilic cytoplasmic autoantibody

Henoch-Schönlein purpura Occurse in children following viral respiratory infection

Decrease in platelets disrupts vascular integrity

Membranous GN Thickening of the glomerular membrane following IgG immune

complex deposition

Membranoproliferative GN Cellular proliferation affecting the capillary walls or 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 syndrome Disruption of the electrical charges that produce tightly fitting podocyte barrier

Minimal change Disruption of the podocytes occurring primarily in children following

disease (Lipoid 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 Nephropathy Most common cause of ESRD

(Kimmelstiel-Wilson Microalbuminuria
disease)

Acute tubular necrosis Damage to the renal tubules caused by ischemia or 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

lec.mt 04 |Page | 149

2. Renal type
N-AA in blood
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

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

lec.mt 04 |Page | 150

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 type) Defect in renal tubular transport of:
-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 modification Rxn: Cyanide-nitroprusside 🡪 (+) Red-purple

of Legal’s
nitroprusside

Mucopolysaccharide Disorders

MPS Dermatan SO4

Keratan SO4
Heparan SO4

Clinical significance Alder-Reilly syndrome

Hurler syndrome = MPS 🡪 cornea of the eye
Hunter syndrome = Sex-linked recessive
Sanfilippo syndrome = Mental retardation only

CTAB (+) White turbidity

Purine Disorders

Lesch-Nyhan disease 🡩 Urinary uric acid crystals

Porphyrias

D-ALA Glycine + Succinyl CoA ----(ALA synthetase)----> D-ALA

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

Coproporphyrinogen Uroporphyrinogen ---------(Uroporphyrinogen

decarboxylase)---------------> Coproporphyrinogen Porphyria cutanea tarda:
(-) Uroporphyrinogen decarboxylase

Protoporphyrinogen Coproporphyrinogen ---------(Coproporphyrinogen

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

lec.mt 04 |Page | 151

Screening tests (porphyria)
1. Ehrlich reaction
= (+) 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 turbidity/ Heat and acetic acid

cloudiness SSA
Purdy’s
Potassium ferrocyanide
Picric acid
Kingsbury-Clark (Rgt: SSA)

(+) coagulum (24 hrs) Esbach’s

-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

lec.mt 04 |Page | 152

Ictotest Bile 🡪 (+) Blue to purple mat

Wallace and Diamond Rgt: PDAB

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 & Wanger (+) Brown to black ppt. (24 hr urine)

Qualitative Tests for Chloride

Fantus (+) Reddish ppt

Mercurimetric (+) Blue-violet colored complex

titration (Schales &
Schales)

Qualitative Test for Calcium

Sulkowitch (+) Precipitation

Renal Function Tests

Test for Glomerular Clearance

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 Renal PSP
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 Ratio) a. NV = 10:1

-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 sink 1:5 or 1:10 dilution of sodium hypochlorite (daily)

lec.mt 04 |Page | 153

Sodium hypochlorite soln. Effective for 1 month

Chemical spills on skin Flush the area w/ water for at least 15 mins 🡪 seek medical
attention Do not neutralize chemicals

Always add acid 🡪 water To avoid the possibility of sudden splashing caused 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 Classification 0 = Stable

(Yellow = Reactivity) 1 = Unstable if heated
2 = Violent chemical change
3 = Shock and heat may deteriorate
4 = May deteriorate

Hazard Classification OXY = Oxidizer

(White = Specific Hazard) ACID = Acid
ALK = Alkali
COR = Corrosive
W = Use no water
✇ = Radiation

Hazard Classification 0 = Normal material

(Blue = Health) 1 = Slightly hazardous
2 = Hazardous
3 = Extreme danger
4 = Deadly

Hazard Classification 0 = Will not burn

(Red = Flammability) 1 = Above 200’F
2 = Below 200’F
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 routine patient examination

UA (CLSI/NCCLS) Defined as the testing of urine with procedures commonly performed in

an expeditious, reliable, accurate, safe and cost-effective manner

lec.mt 04 |Page | 154

Care of Reagent Strips 1. Store w/ dessicant in an opaque, tightly closed 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 Strips) 1. Test open bottles of reagent strips w/ known 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 (Microscope) Ability to visualize fine details

Ability of the lens to distinguish two small objects that are a specific
distance apart

Parfocal (Microscope) Require only minimum adjustment when switching among objectives

Centering and Köhler Provide optimal viewing of the illuminated field

illumination (condenser)
Camel-hair brush Removes dust coating the optical surface of the microscope

Quality assessment Overall process of guaranteeing quality patient care 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 agencies JCAHO (Joint Commission on the Accreditation of 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 Institute)

Committee for Clinical
Laboratory Standards)

Policy for Handling 1. Do NOT assume any information about the specimen or
Mislabeled Specimens patient 2. Do NOT relabel an incorrectly labeled specimen
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 Factors Test requests

Patient preparation
Specimen collection, handling and storage

Analytical Factors Reagents

Instrumentation and equipment
Testing procedure
QC
Preventive maintentance

lec.mt 04 |Page | 155

Access to procedure manuals
Competency of personnel performing the tests

Microscopic Quantitations

EC (lpf) Crystals (hpf) Bacteria (hpf) Mucous 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

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 | 156
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. Meninx) Three layers:

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 barrier Between brain and blood

Functions:
1. Protects brain from organisms
2. Shields brain from hormones and neurotransmitters
3. Maintains homeostasis for brain

Circumventricular organs Regions of the brain where BBB is weak

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 collection Lumbar tap: routine (collected by physician)

-Bet. 3rd, 4th and 5th lumbar vertebrae
-Fetal position
Cisternal puncture: suboccipital region
Ventricular puncture: infants w/ open fontanels

Precautions Measurement of intracranial pressure

Prevent infection (povidone iodine)
Prevent damage 🡪 neural tissue

lec.mt 04 |Page | 157

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 specimen Collected on 1 tube

Microbiology 🡪 Hematology 🡪 Chemistry/Serology

CSF Appearance

Crystal clear Normal

Turbidity/cloudiness WBC > 200μL

RBC > 400μL
(+) Microorganisms
RCM
Aspirated fat
🡩 Protein (>45 but <150mg/dL)

Oily RCM

Hemolyzed/Bloody RBCs

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 CSF HbO2 from artifactual RBC lysis

Xanthochromia Bilirubin in jaundiced patients
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

>45 but <150mg/dL (cloudy/turbid)
>150mg/dL (xanthochromic)

Intracranial Hemorrhage vs. Traumatic Tap

Traumatic Tap Intracranial/Cerebral Hemorrhage

Distribution of Blood Uneven (Tube 1 > 2 > 3) Even (Tube 1 = 2 = 3)

Clot + (Plasma Fibrinogen) -

Xanthochromia - +

(+) Clot Meningitis

(-) Blood Froin syndrome
Blockage of CSF circulation

(+) Weblike pellicle After overnight refrigeration (12-24 hrs)

TB meningitis

(+) D-dimer Indicates formation of fibrin at a hemorrhage site

Recent hemorrhage Clear supernatant

lec.mt 04 |Page | 158

To examine a bloody fluid for xanthochromia:
Microhematocrit tube ---(Centrifuge)---> Examine supernatant against white BG

Erythrophagocytosis/ Indicates intracranial hemorrhage

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 count WBC ct + RBC ct

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/Appearance Dilution Amount of Sample Amount of Diluent

Slightly Hazy 1:10 30 μL 270 μL

Hazy 1:20 30 μL 570 μL

Slightly Cloudy 1:100 30 μL 2970 μL

Cloudy/Slightly Bloody 1:200 30 μL 5970 μL

Bloody/Turbid 1:10,000 0.1 mL of 1:100 dilution 9.9 mL

CSF WBC Count Correction WBC (added) = WBCBlood x RBCCSF or

RBCBlood
PBS (normal) = -1 WBC/700 RBCs (CSF)

Differential Count Performed on stained smears

Specimen should be concentrated

Methods for specimen Sedimentation

concentration Filtration
Centrifugation
Cytocentrifugation

Cytocentrifugation Fluid 🡪 Conical chamber

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 count Neonates (0-2 mos.)
50-90% monocytes
5-35% lymphocytes
0-8% neutrophils
Children (2 mos.-18 y.o.)
-Not yet established

lec.mt 04 |Page | 159

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 (metarubricytes) BM contamination

Eosinophils 🡩 helminthic parasitic infection

🡩 fungal infection (C. immitis)
🡩 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

lec.mt 04 |Page | 160

Transthyretin (prealbumin) TRANSports THYroxine and RETINol
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:

traumatic tap = -1 mg/dL protein/1,200 RBCs
Measurement of Total 1. Turbidimetric
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 Albumin index _CSF Albumin (mg/dL)_

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 (not Encephalitis

seen in serum) Guillain-Barré syndrome
Neurosyphilis
Neoplastic disorder
Multiple sclerosis
♫ Single band should not be interpreted as positive

Oligoclonal bands in Leukemia

serum but not in CSF Lymphoma
Viral infections
♫ Produces serum banding w/c can appear in the CSF because of BBB
leakage or traumatic tap

lec.mt 04 |Page | 161

HIV Produces serum and CSF banding
Multiple sclerosis Symptoms:
-Abnormal sensations: numbness, tingling, pain, burning,
itching -Mood swings

Myelin basic protein Protein component of the lipid-protein complex that 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 42 🡩 CSF levels of microtubule associated Tau protein 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

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

lec.mt 04 |Page | 162

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 test Diagnosis of Gram (-) bacteria

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 of Leydig Secrete testosterone

Outside the seminiferous tubules

Seminiferous tubule Site of spermatogenesis

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 vaginal acidity

(Cowper’s) gland

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%

lec.mt 04 |Page | 163

Spermatozoa 5%

Bulbourethral gland 5%

Chemical Composition of Seminal Fluid

ACP For liquefaction

Zn3+ 🡫 in prostatic disease

Fructose Major nutrient of spermatozoa

K+, citric acid, ascorbic --

acid

Proteolytic enzymes Liquefaction and coagulation

Spermine and Choline Inhibit growth of bacteria

Importance of Seminalysis To investigate the causes of infertility in marriages

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 collection Important: 1st portion of ejaculate

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
5. Specimen should be delivered in the lab w/in 1 hr (RT’)

Methods of preservation Specimen kept at 37’C awaiting analysis

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
🡩 White turbidity
Red coloration
Viscosity
pH
Sperm concentration
Counting chamber
Purpose of Dilution
Det. sperm conc. (Short-cut)
Sperm count
Motility quality (20/hpf)
CASA
Sperm morphology
Head morphology
Prolonged abstinence
Medication
Urine contaminationi
Infection (🡩 WBCs)
(+) RBCs
0 (watery)
4 (gel-like)
Too basic = infection
Too acidic = 🡩 prostatic fluid
lec.mt 04 |Page | 164
Diluting fluid:
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
1. Neubauer counting chamber = diluted specimen
(WHO recommended)
2. Makler chamber = undiluted w/ heating processes
To immobilize the sperm
1. 5 RBC squares
# sperms counted x 1,000,000 = sperms in million/mL
2. 2 WBC squares
# sperms counted x 100,000 = sperms in million/mL
Sperm concentration x volume of specimen
4.0 (a) = Rapid motility
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
Computer-Associated Semen Analysis
-Sperm concentration
-Sperm velocity and trajectory
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
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-glucosidase Epididymis disorder

Florence test Choline

(+) Dark brown rhombic crystals

Barbiero’s test Spermine

(+) Yellow leaf-like crystals

lec.mt 04 |Page | 165

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-Nigrosi If N-sperm count but 🡫 motility

n/ Sperm viability test Living sperm cells = bluish white
Dead = red
NV = 75% living sperms
(25% dead)

Decreased motility w/ (+) Male antisperm antibodies

clumping Blood testis barrier 🡪 disrupted
Clumps of sperm

Normal seminalysis (+) Female antisperm antibodies

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 penetration Sperm incubated w/ species non-specific hamster egg

Cervical mucus penetration Observed sperm penetration ability

Hypo-osmotic swelling Test for membrane integrity and viability of sperm

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 collection Fluid 🡪 Syringe (heparin)

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
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

lec.mt 04 |Page | 166

Turbidity 🡩 WBCs

Milky (+) Crystals

Rope’s/Mucin clot test Hyaluronate polymerization 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 specimen Add hyaluronidase 🡪 0.5 mL of fluid or

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 synovial Metal and plastic joint prosthetic
fluid

Hemosiderin Pigmented villonodular synovitis

Cartilage cells Osteoarthritis

 Crystals

Normal No crystals

Abnormal Hydroxyapatite crystals

Cholesterol
Corticosteroid
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-inflammatory OA
Traumatic arthritis
Neuroarthropathy

Group II: Inflammatory Immunologic: RA, SLE

Crystal-induced: milky synovial fluid

Group III: Septic Yellow green

Low viscosity
>50,000 WBC/μL (highest)

lec.mt 04 |Page | 167

(+) 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: Hemorrhagic Sickle cell disease

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 collection >100 mL is usually collected

EDTA = cell counts and differential
Heparin = chemistry, serology, microbiology, cytology

Rivalta’s test/Serosa Differentiates transudates from exudates

mucin clot test Acetic acid + H2O + unknown fluid 🡪 heavy ppt. = (+) exudate

Fluid: Serum LD ratio Most reliable differentiation between transudates and exudates
Fluid: Serum protein ratio (except peritoneal fluid: SAAG)

Pleural Fluid

Thoracentesis Method of collection

Normal appearance Clear/pale yellow

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 549 Breast cancer

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

 lec.mt 04 |Page | 168
Normal volume 10-50 mL

Mesothelial cells Produces pericardial fluid

Function Reduces friction

Normal appearance Clear/pale yellow

Grossly bloody Accidental cardiac puncture

Misuse of anticoagulant medicatios

Milky Chylous/pseudochylous effusions

Bacterial endocarditis >1000 WBCs/μL (🡩% neutrophils)

Peritoneal Fluid (Ascites)

Paracentesis Method of collection

Peritoneal lavage Sensitive test for the detection of intra-abdominal bleeding

WBC <500/μL Normal

RBC >100,000/μL Blunt trauma cases

Serum Ascites Recommended to differentiate transudates from exudates

Albumin Gradient a. >1.1 = Transudate
(SAAG) 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 bladder

Creatinine

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 Iontophoresis Borderline = 40 mEq/L
Pilocarpine w/ mild current = stimulates sweat glands

Sodium FEP, IEE

Chloride Manual or automated titration

Amniotic Fluid

During 1st trimester 35 mL: derived from maternal circulation/plasma

After 1st trimester Fetal urine

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

lec.mt 04 |Page | 169

Glass slide 🡪 air dry
(+) Fern-like crystals
(+) Amniotic fluid

Normal appearance Clear

Green Meconium

Yellow Bilirubin (HDN)

Bloody/red Trauma, abdominal trauma, intra-amniotic hemorrhage

Dark-brown/ Fetal death

Reddish-brown

Assessment of HDN 🡩 OD at 450 nm

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 defects Ex. Spina bifida and Anencephaly
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 test Amniotic fluid + 95% ethanol 🡪 shake for 15 secs 🡪 stand (15
(Foam/Shake test) mins) (+) Continuous line of bubbles
Can be done bedside

Fetal age Creatinine ≥2.0 mg/dL (36 weeks/9 months)

Gastric Fluid Analysis

Clinical significance Pernicious anemia

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 disease 🡩 gastrin

Gastric acid hypersecretion
Adenoma of the islets of Langerhans (pancreas) = produce gastrin

Pernicious anemia “Dangerous” anemia

BAO/MAO = 0

Specimen collection Collect gastric juice for 1 hr

1. Levine tube = nose
2. Rehfuss tube = mouth

Basal Acid Output (BAO) Total gastric secretion during unstimulated fasting state

Maximal Acid Output Total acid secreted in the hour after stimulation
(MAO)

Gastric stimulants 1. Pentagastrin = most preferred

 2. Histamine

lec.mt 04 |Page | 170

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 Gastric fluid Fermentation in stomach because large amounts of 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 proteins) pH indicator: sodium alizarin
(+) 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 test Specimen: Urine

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
-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

lec.mt 04 |Page | 171

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-like Spastic colitis

Bulky/frothy Bile duct obstruction

Pancreatic disorders
Abundant fats

Butter-like Cystic fibrosis

Scybalous/Goat droppings Constipation

Bristol Stool Chort

Type 1 Separate hard lumps, like nuts (hard to pass)

Type 2 Sausage-shaped but lumpy

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
 lec.mt 04 |Page | 172
(+) Pink = HbF
(-) Yellow brown = Maternal Hgb

Diarrhea Acute: <4 weeks

Chronic: >4 weeks

Secretory diarrhea 🡩 solute secreted by the intestine

Endotoxins

Osmotic diarrhea 🡩 amounts of osmotically active solutes in the lumen

(maldigestion) 🡩 CHO in stool

Intestinal hypermotility Secretory and osmotic diarrhea

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)
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 striated Biliary obstruction
fibers Gastrocolic fistulas

Fecal fats NV = 1-6 g/day

Qualitative Fecal fat test Sudan III = most routinely used

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 Fecal fat test Confirmative test for steatorrhea

3 day specimen

lec.mt 04 |Page | 173

-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

Secretin is administered IV, then duodenal fluid HCO3-is tested

Pancreatic cancer 🡫 volume

N-HCO3-
N-AMS

Chronic pancreatitis 🡫 volume

🡫 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 sauce/rusty Old blood

brown Pneumonia

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 green Chronic lung cancer

Causes of a. Rusty, (+) pus = pneumococcal pneumonia

blood-stained b. Rusty, (-) pus = CHF
sputum 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

Foul/putrid Cavitary TB

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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/Pneumoliths/ Histoplasmosis
Broncholiths

Curschmann’s spirals Mucoid threads that are twisted/coiled

Bronchial asthma (3C’s)
Bronchitis

Layer formation (3) Bronchiectasis

Lung abscess
Gangrene
Top = Frothy mucus
Middle = opaque H2O material
Bottom = pus, bacteria, tissues

Microscopic Elements

PAS (+) Macrophages Alveolar proteinosis

PAS (+) rounded P. carinii

bodies that take
silver stain

Elastic fibers TB

Charcot-Leyden crystals From degeneration of eosinophils

Bronchial asthma (3C’s)
Creola bodies Clusters of columnar cells
Bronchial asthma (3C’s)

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)

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4. E. gingivalis
5. T. tenax

Bronchoalveolar Lavage

Clinical significane P. jiroveci

Macrophages (56-80%) Most predominant

Lymphocytes (1-15%) 🡩 interstitial lung disease, pulmonary lymphoma, nonbacterial infection

Neutrophils (<3%) 🡩 cigarette smokers, bronchopneumonia, toxin exposure

Eosinophils (<1 to 2%) 🡩 hypersensitivity reaction (asthma)

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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