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 Bowmans 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
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 (0C) by 1.86C
- FP = Osm
Example:
Determine Osm in mOsm/kg
Temp. = -0.90C
Solution:
1000 mOsm/kg = _ _x____
-1.86C -0.90C
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): 32.5-35.7C (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 Contamination: Bacteria
from true infection True infection: Bacteria and WBCs
Preservation
Refrigeration 2-8C
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
Saccomannos 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 red/ Porphyria
purplish red/Portwine (Lead poisoning: normal color)
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 Kln = 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 acetic RBCs
acid AP
Carbonates
Insoluble in dilute acetic WBCs
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 (15-38C)
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. 3C calibration temperature (20C) = (+0.001)
b. 3C calibration temperature (20C) = (-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 the
Densitometry 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: -, -)
-ID: Serum IEP
-Urine: precipitates at 40-60C and dissolves at 100C
-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. Fanconis syndrome
Post-renal proteinuria Lower UTI/inflammation
Principle (Rgt Strip) Protein (Sorensens) 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 protein
precipitation test 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
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 in 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
-(+) Rubners test (Lead acetate)
4. Pentose
-Xylose, arabinose
-Xylulose:Benign pentosuria
5. Sucrose
- Intestinal disorders
-Nonreducing sugar
-(-) Copper reduction test
Hyperglycemia associated Blood glucose, Urine glucose
Glycosuria 1. DM
2. Cushings syndrome/disease = cortisol
3. Pheochromocytoma = catecholamines
4. Acromegaly = GH
5. Hyperthyroidism = T3/T4
Renal associated Glycosuria N-Blood glucose, impaired tubular reabsorption of glucose
1. Fanconis syndrome: defective tubular reabsorption of glucose and amino
acids
Principle (Rgt Strip) Double sequential enzyme reaction:
-Glucose oxidase
-Peroxidase
Chromogen:
-KI (Brown)
-Tetramethylbenzidine (Blue)
Copper Reduction test Blue tablet
(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)

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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) Legals 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. Blondheims 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) Ehrlichs reaction (PDAB):
Urobilinogen + PDAB ----------> (+) Cherry red
Watson-Schwartz test For differentiating urobilinogen and porphobilinogen from other Ehrlichs
reactive compounds
1. Urobilinogen: soluble to both chloroform and butanol
2. Porphobilinogen: insoluble to both chloroform and butanol
3. Other Ehrlichs 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 and High levels of ascorbic acid
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, phenazopyridine Specimen exposure to light
Indican (intestinal disorders) Ascorbic acid >25 mg/dL
Metabolites of Lodine High concentrations of nitrite
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, oxalic
Highly pigmented urine, nitrofurantoin acid, ascorbic acid, gentamicin, cephalosporins,
tetracyclines

Microscopic Exam of Urine

Phase-contrast microscopy
Polarizing microscopy
Interference contrast
microscopy
Sternheimer-Malbin
Toluidine blue (Supravital)
Lipid stains:
ORO and Sudan III
Gram stain
Hansel stain
Prussian blue stain
Visualization of elements w/ low refractive indices:
-Hyaline casts
-Mixed cellular casts
-Mucous threads
-Trichomonas
ID of cholesterol in OFB, FC and crystals
Produces 3D microscopy-image and layer-by-layer imaging of a specimen
1. Hoffman microscope: modulation contrast microscope
2. Nomarski microscope: differential interference contrast microscope
Crystal violet and safranin
Nucleus and cytoplasm
ID: WBCs, ECs, casts
Enhances nuclear detail
Differentiates WBCs and RTE
Stain TG and neutral fats orange red
ID: free fat droplets and lipid-containing cells and casts
Differentiates Gram (+) and Gram (-) bacteria
ID: bacterial casts
Eosin Y and Methylene blue
ID: Eosinophils
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 cells Spherical, polyhedral, or caudate w/ centrally 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 epithelial Rectangular, polyhedral, cuboidal or columnar w/ an eccentriac nucleus,
cells 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: 20L (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)
Uric 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) -Envelope/pyramidal
(pH: acid/alkaline/neutral) 2. Whewellite = monohydrate
-Oval, dumbbell
-Ethylene glycol poisoning (antifreeze agent)
Most renal stones consist of CaOx
Amorphous Phosphates White precipitate
(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 Colorless, flat rectangular plates or thin prisms often in rosette formation
(Normal) Rosettes may resemble sulfonamides
(pH: 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
(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 striations
(Abnormal) Liver disease
(pH: acid/neutral)
Bilirubin Clumped needles or granules w/ yellow color
(Abnormal) (+) Diazo reaction

lec.mt 04 |Page | 147

(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 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
lec.mt 04 |Page | 148
 -(-) alpha2 (AMG)
-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 the
(Crescentic) GN glomerular membrane
Goodpasture syndrome Attachment of cytotoxic antibody to glomerular and alveolar basement
membrane
Wegeners granulomatosis Antineutrophilic cytoplasmic autoantibody
Henoch-Schnlein 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
(Bergers disease)
Nephrotic syndrome Disruption of the electrical charges that produce tightly fitting podocyte barrier
Minimal change disease Disruption of the podocytes occurring primarily in children following allergic
(Lipoid nephrosis) reaction and immunization
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), Fanconis 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/Benedicts (+) Yellow ppt.
Melanuria Overproliferation of melanocytes
FeCl3 Gray or black ppt.
Ehrlichs 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
lec.mt 04 |Page | 150
 Blue color
Hartnup disease: Blue diaper syndrome
Obermayers 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
Brands modification of Rxn: Cyanide-nitroprusside (+) Red-purple
Legals 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 Hellers
Roberts
Spieglers
(+) Violet Biuret (Albumin)
(+) White turbidity/ Heat and acetic acid
cloudiness SSA
Purdys
Potassium ferrocyanide
Picric acid
Kingsbury-Clark (Rgt: SSA)
(+) coagulum (24 hrs) Esbachs
-Rgt: Picric acid + Citric acid
Tsuchiyas
(+) coagulum Kwileckis
(72C for 5mins) -Rgt: Esbachs + 10% FeCl3
Qualitative Tests for Sugars
Benedicts Reducing substances
Seliwanoffs Rgt: Resorcinol
Fructose (+) Red
Rubners Rgt: Lead acetate, NH3 H2O
Lactose (+) Bright red w/ red ppt.
Glucose (+) Red color w/ yellow ppt.
Bial Orcinol Pentose (+) Green
Taubers Pentose (+) Green
Others Osazone or phenylhydrazine (Kowarsky)
Nylanders
Moore Heller
Borchardts
Qualitative Tests for Ketones
Frommers Acetone (+) Purplish red ring
Rotheras Acetone & AAA (+) Purple ring
Lange Acetone & AAA (+) Purple ring
Acetest/Ketostix Acetone (+) Purple
Gerhardts AAA Bordeaux red
Qualitative Tests for Bile Pigments
Gmelin Bile (+) Play of colors
Smith Bile (+) Emerald green
lec.mt 04 |Page | 152
Harrisons spot Bile (+) Blue to green
Ictotest Bile (+) Blue to purple mat
Wallace and Diamond Rgt: PDAB
Urobilinogen (+) Cherry red
Schlesinger Rgt: Lugols 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 titration (+) Blue-violet colored complex
(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 Secretion PAH
and Renal Blood flow PSP
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
lec.mt 04 |Page | 153
Disinfection of sink 1:5 or 1:10 dilution of sodium hypochlorite (daily)
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 200F
2 = Below 200F
3 = Below 100F
4 = Below 73C
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 doctors 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 30C. 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 Khler 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 laboratorys 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 Committee New: CLSI (Clinical and Laboratory Standards Institute)
for Clinical Laboratory
Standards)
Policy for Handling 1. Do NOT assume any information about the specimen or patient
Mislabeled Specimens 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, doctors 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

lec.mt 04 |Page | 155

 Preventive maintentance
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 cant 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 > 200L
RBC > 400L
(+) 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/L
NV (neonate) = 30 mononuclear WBCs/L
Vol. of 1 square = 0.1 L
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 L 270 L
Hazy 1:20 30 L 570 L
Slightly Cloudy 1:100 30 L 2970 L
Cloudy/Slightly Bloody 1:200 30 L 5970 L
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
-globulins = Haptoglobin
-globulins = Tau transferrin (major)
-globulins = IgG (major), IgA (small amount)
IgM, fibrinogen, -LPP = not found in normal CSF
lec.mt 04 |Page | 160
Transthyretin (prealbumin) TRANSports THYroxine and RETINol
2nd most prevalent
Tau Transferrin CHO-deficient 2-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 traumatic If blood Hct and serum protein are normal:
tap = -1 mg/dL protein/1,200 RBCs
Measurement of Total CSF 1. Turbidimetric
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)
-Beers 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 seen Encephalitis
in serum) Guillain-Barr syndrome
Neurosyphilis
Neoplastic disorder
Multiple sclerosis
Single band should not be interpreted as positive
Oligoclonal bands in serum Leukemia
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)
-amyloid protein 42 CSF levels of microtubule associated Tau protein and decreased levels of -
AP42 have been shown to significantly increase the accuracy of Alzheimers
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 -ketoglutarate
NV = 8-18 mg/dL
Requested for patients w/ coma of unknown origin
>35 mg/dL Associated w/ some disturbances of consciousness
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Reyes 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 (Cowpers) Secretes alkaline mucus neutralize prostatic and vaginal acidity
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 37C awaiting analysis
For artificial insemination, it can be preserved in frozen state and stored at
-85C (seminal banks)
Fresh specimen is clotted
Semen Analysis
Liquefaction time 30-60 mins
If not yet liquefied after 2 hrs, use -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
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Sperm concentration 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
Counting chamber 1. Neubauer counting chamber = diluted specimen
(WHO recommended)
2. Makler chamber = undiluted w/ heating processes
Purpose of Dilution To immobilize the sperm
Det. sperm conc. (Short-cut) 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 count Sperm concentration x volume of specimen
Motility quality (20/hpf) 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
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. Krugers 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 m x 3 m wide
Tail 45 m long
Midpiece Contains mitochondria
Connects head and tail
Acrosomal cap Ovum penetration
Covers approximately 2/3 of sperm nucleus and of the head
Tapered head Varicocele
-Common cause of male sterility
-Hardening of veins that drains the testes
Stains Giemsa
Papanicolau = method of choice
Wrights
Fructose test If sperm count is low
Rgt: Resorcinol
End-color: Orange-red
Specimens should be tested in 2 hrs or frozen
neutral--glucosidase Epididymis disorder
Florence test Choline
(+) Dark brown rhombic crystals
Barbieros 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
Blooms/Eosin-Nigrosin/ If N-sperm count but motility
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 w/ (+) Female antisperm antibodies
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 whether Microscopic exam for sperm cells
semen is present Enhance w/ xylene
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
Ropes/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
37C 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 appearance Ochronotic shards = debris
of synovial fluid Metal and plastic joint prosthetic
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/L (highest)
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 (+) 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
Rivaltas test/Serosa mucin Differentiates transudates from exudates
clot test Acetic acid + H2O + unknown fluid heavy ppt. = (+) exudate
Fluid: Serum LD ratio Most reliable differentiation between transudates and exudates (except
Fluid: Serum protein ratio 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 (Cytokeratin Lung cancer
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
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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/L (% neutrophils)
Peritoneal Fluid (Ascites)
Paracentesis Method of collection
Peritoneal lavage Sensitive test for the detection of intra-abdominal bleeding
WBC <500/L Normal
RBC >100,000/L Blunt trauma cases
Serum Ascites Albumin Recommended to differentiate transudates from exudates
Gradient (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 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
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 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: cant 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 mins)
(Foam/Shake test) (+) 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
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 3. Histalog (Betazole)
4. Alcohols
5. Insulin = assess vagotomy procedure
-SHAM feeding = sandwich
Test Meals 1. Ewalds meal = bread and tea/H2O
2. Boas = oatmeal
3. Riegels = 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 (Topfers)
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 Uffelmanns
-Rgt: FeCl3 and phenol
-(+) Yellow
2. Strauss
-Rgt: FeCl3 and ether
-(+) Yellow
3. Kellings
-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

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 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 infants stool
Specimen: infant stool/vomitus
Rgt: 1% NaOH
HbF: alkali-resistant
Maternal Hgb: not alkali-resistant
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Diarrhea
Secretory diarrhea
Osmotic diarrhea
Intestinal hypermotility
Fecal enzymes
Fecal CHO
Fecal Leukocytes
Methylene blue
Lactoferrin Latex
agglutination test
amounts of striated Biliary obstruction
fibers
Fecal fats
Qualitative Fecal fat test
Split fat stain
Quantitative Fecal fat test
(+) Pink = HbF
(-) Yellow brown = Maternal Hgb
Acute: <4 weeks
Chronic: >4 weeks
solute secreted by the intestine
Endotoxins
amounts of osmotically active solutes in the lumen (maldigestion)
CHO in stool
Secretory and osmotic diarrhea
Laxatives
Emotions/stress
Cardiovascular drugs
1. Trypsin
= X-ray paper
= Trypsin deficiency (CF): inability to digest gelatin on the X-ray paper
2. Chymotrypsin
3. Elastase I = pancreas specificity
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
Primarily neutrophils
Neutrophils:
-Salmonella
-Shigella
-Campylobacter
-Yersinia
-EIEC
(-) Fecal leukocytes:
-Parasites
-Viruses
-S. aureus
-Vibrio spp
For wet preparation
Faster procedure than Wrights and GS (for dry smears) but may be more
difficult to interpret
Sensitive in refrigerated and frozen specimens
Gastrocolic fistulas
NV = 1-6 g/day
Sudan III = most routinely used
Sudan IV
Oil red O
Free fatty acids and fatty acids from hydrolysis of soaps and neutral fats
NV = 100 droplets (<4 m)
Slightly increased = 100 droplets (1-8 m)
Increased = 100 droplets (6-75 m)
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 blood-stained a. Rusty, (+) pus = pneumococcal pneumonia
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
Foul/putrid Cavitary TB
lec.mt 04 |Page | 174
 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
Dittrichs plugs Grayish to yellowish material
Size of pinhead
Foul odor when crushed
Cellular, fatty acids, fat globles, some bacteria
Bronchiectasis
Chronic bronchitis
Bronchial asthma (3Cs)
Lung stones/Pneumoliths/ From calcified pulmonary tissue
Broncholiths Histoplasmosis
Curschmanns spirals Mucoid threads that are twisted/coiled
Bronchial asthma (3Cs)
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 bodies P. carinii
that take silver stain
Elastic fibers TB
Charcot-Leyden crystals From degeneration of eosinophils
Bronchial asthma (3Cs)
Creola bodies Clusters of columnar cells
Bronchial asthma (3Cs)
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)
lec.mt 04 |Page | 175
 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
-subunit = FSH, LH, TSH, hCG
-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
Friedmanns Mature virgin female rabbit
Marginal ear vein injection
Frank-Bermann Female rats
Intraperitoneal injection
(+) Ovarian hyperemia

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