MUST TO KNOW IN CLINICAL MICROSCOPY

URINALYSIS
Nephron Basic structural unit of kidney 1M/kidney
Urethra F: 3-4 cm M: 20 cm
Urine formation (order) Glomerulus → Bowman’s capsule → PCT → Loop of Henle → DCT → CD
PCT 65% of reabsorption
ADH Regulate H2O reabsorption in DCT and CD
Urine composition 95-97% H2O
3-5% solids
60g TS in 24 hrs
35g: Organic = Urea (major)
25g: Inorganic = Cl (#1) > Na+ > K+
Glomerular Filtration
Clearance tests Evaluate glomerular filtration
1. Urea clearance
2. Creatinine clearance = most common
3. Inulin clearance = gold standard
4. Beta2-microglobulin
5. Radioisotopes
Creatinine clearance Formula:
Cc = U x V x 1.73
P A
Normal values:
M = 107-139 mL/min
F = 87-107 mL/min
Tubular Reabsorption
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
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)
 Bacteriostatic to contaminants
Sodium fluoride Glucose
Sodium benzoate/ Benzoic acid Substitute for sodium fluoride
Saccomanno’s fixative 50% ethanol + carbowax
Cytology (50mL urine)
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 from Contamination:  Bacteria
true infection True infection:  Bacteria and WBCs
Preservation
Refrigeration 2-8’C
 SG (hydrometer/urinometer)
Precipitate AU/AP
Formalin Addis count
Boric acid Urine culture

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 Yellow-brown Bilirubin ---(oxidized)---> Biliverdin

Green Pseudomonas aeruginosa

Blue-green Clorets, methylene blue, phenol
Indican (blue): Hartnup disease or Blue diaper syndrome

Cloudy/Smoky red Hematuria (intact RBCs)

Clear red Hemoglobin, Myoglobin
Red/Purple/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 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 media  SG by refractometer (>1.040)
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 acid RBCs

AP
Carbonates

Insoluble in dilute acetic acid WBCs

Yeasts
Spermatozoa
Bacteria

Soluble in ether Lipids

Lymph fluid Chyle

Specific Gravity
SG Density of solution compared w/ density of similar volume of distilled H2O at a similar
temperature NV = 1.003-1.035 (random)
SG <1.003 = not a urine except DI

Refractometer (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 the density (SG) of the
Densitometry solution
-Yellow IRIS (Automated): International Remote Imaging System
pH
Normal Random = 4.5-8.0 1st morning = 5.0-6.0 pH 9.0 = Unpreserved urine
Acid urine DM (Ketone bodies)
Starvation (Ketone bodies)
High protein diet
Cranberry juice (Tx: UTI)
Alkaline urine After meal
Vomiting
Renal tubular acidosis: inability to produce an acid urine
Vegetarian diet
Old specimen: Urea ---(Urease)---> NH3
Principle (Rgt 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 precipitation Cold precipitation test that reacts equally to all types of protein If (-) rgt strip, (+)
test SSA = presence of other proteins

CSF protein = frequently tested -Det: TCA

(preferred) and SSA

SSA Reactions (Protein)

Negative No increase in turbidity < 6 mg/dL
Trace Distinct turbidity 6-30 mg/dL
1+ Noticeable turbidity w/ no 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
-(+) Rubner’s 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. Cushing’s 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. Fanconi’s 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 (Clinitest) 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)

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
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 Ehrlich Rapid screening test for urine porphobilinogen (≥2mg/dL)
reaction) Hoesch reagent: Ehrlich reagent in 6M (6N) HCl
2 gtts urine + Hoesch rgt -------------> (+) Red

Urine Bilirubin and Urobilinogen in Jaundice

Urine Bilirubin Urobilinogen
Hemolytic (ex. SCD) - 
Hepatic /- N/
Obstructive  /N (never report as negative)
Nitrite
Nitrite 1st morning/4 hr urine
Detection of bacteriuria
(+) NO3: 100,000 orgs./mL
UTI: ≥100,000 or ≥1 x 105 CFU/mL

Principle (Rgt 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

Principle (Rgt strip) Indoxyl carbonic acid ester + Diazonium salt ---Strip can detect even lysed WBCs
(LE)---> Indoxyl + Acid indoxyl ----------> (+) Purple

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 detergents High levels of ascorbic acid
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
 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) Metabolites of Lodine Ascorbic acid >25 mg/dL
High concentrations of nitrite
Urobilinogen Porphobilinogen Indican Old specimens
p-aminosalicylic acid Preservation in formalin
Sulfonamides
Methyldopa
Procaine
Chlorpromazine
Highly pigmented urine

Nitrite Improperly preserved specimens Highly Nonreductase-containing bacteria

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 acid,
Highly pigmented urine, nitrofurantoin 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 microscopy 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
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

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 cells Rectangular, polyhedral, cuboidal or columnar w/ an eccentriac nucleus, possibly bilirubin
stained or hemosiderin laden From nephron:
-PCT: rectangular, columnar/convoluted
-DCT: round/oval
>2 RTE/hpf: tubular injury
Oval fat body Lipid containing RTE cells
Lipiduria (Ex. nephrotic syndrome) Cholesterol: Maltese cross
Bubble cells RTE cells w/ nonlipid containing vacuoles Acute tubular necrosis
Yeast C. albicans (DM, vaginal moniliasis)
T. vaginalis Flagellate w/ jerky motility
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

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 Presence of urea-
(pH: alkaline) 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 Cystinuria

(Abnormal)
(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 Liver disease
(Abnormal)
(pH: acid/neutral)
Bilirubin Clumped needles or granules w/ yellow color (+) Diazo
(Abnormal) reaction

(pH: acid) Liver disease

Sulfonamide Colorless to yellow brown
(Abnormal) Deposits in nephrons
(pH: acid/neutral) Tubular damage
Needles, sheaves of wheat, rosette (res. CaPO4 rosette) ♫ Lignin test:
Newspaper = urine + 25% HCl → (+) Yellow orange color

Ampicillin Massive doses

(Abnormal) Colorless needles
(pH: acid/neutral)
 Uric Acid Cystine
Color Yellow brown Colorless
Solubility in NH3 Soluble Soluble
Solubility in dilute HCl Insoluble Soluble
Birefringence (Polarizing microscope) + -
Cyanide-Nitroprusside test - +
Urinary Sediment 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)

-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 (Crescentic) Deposition of immune complex from systemic immune disorders on the glomerular membrane
GN

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 (Berger’s Deposition of IgA on the glomerular membrane

disease)

Nephrotic syndrome Disruption of the electrical charges that produce tightly fitting podocyte barrier
Minimal change disease (Lipoid Disruption of the podocytes occurring primarily in children following allergic reaction and
nephrosis) 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 Microalbuminuria
(Kimmelstiel-Wilson 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

2. Renal type
N-AA in blood
Impaired tubular reabsorption of AA
Ex. Cystinuria (COLA), Fanconi’s syndrome

Phenylalanine-Tyrosine Disorders

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
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 of Legal’s Rxn: Cyanide-nitroprusside → (+) Red-purple

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

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/ cloudiness Heat and acetic acid
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
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 titration (Schales & (+) Blue-violet colored complex
Schales)

Qualitative Test for Calcium

Sulkowitch (+) Precipitation

Renal Function Tests

Test for Glomerular filtration Clearance
Test for Tubular reabsorption Concentration tests
-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 and PAH PSP

Renal Blood flow
Tests for NPN 1. Urea: Urease, DAM (NV = 6-17 g/24 hr urine)
2. Creatinine: Jaffe (NV = 1-2 g/24 hr urine)
3. Uric acid: Uricase, PTA (NV = 0.25-0.75 g/24 hr urine)
BCR (BUN: Crea 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)

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 (Yellow = 0 = Stable

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 (Blue = 0 = Normal material

Health)
1 = Slightly hazardous
2 = Hazardous
3 = Extreme danger
4 = Deadly
Hazard Classification (Red = 0 = Will not burn
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

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 illumination Provide optimal viewing of the illuminated field
(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 Committee for New: CLSI (Clinical and Laboratory Standards Institute)
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, 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
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

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/ granulations Reabsorbs CSF

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
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
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

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 Monocytes  viral, TB, fungal meningitis
 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 significant cells Choroidal cells: EC (choroid plexus)
Ependymal cells
Spindle-shaped cells: arachnoid

Malignant cells Hematologic:

-lymphoblasts
-myeloblasts
-monoblasts
Nonhematologic
-Astrocytomas
-Retinoblastomas
-Medulloblastomas
QC of CSF and Other Body Fluid Cell Count
Biweekly basis All diluents ---(check for)---> Contamination
Monthly basis Speed of cytocentrifuge should be checked w/ a tachometer
CSF Protein
CSF protein Most frequently tested chemical test NV:
Adults = 15-45 mg/dL
Infants = 150 mg/dL
Premature = 500 mg/dL Albumin =
majority α-globulins = Haptoglobin
β-globulins = Tau transferrin (major)
γ-globulins = IgG (major), IgA (small amount) IgM, fibrinogen,
β-LPP = not found in normal CSF

Transthyretin (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 traumatic tap If blood Hct and serum protein are normal: = -1 mg/dL
protein/1,200 RBCs

Measurement of Total CSF protein 1. Turbidimetric

-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

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 in Encephalitis

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

Oligoclonal bands in serum but not Leukemia

in CSF Lymphoma
Viral infections
♫ Produces serum banding w/c can appear in the CSF because of BBB leakage or traumatic tap

HIV Produces serum and CSF banding

Multiple sclerosis Symptoms:
-Abnormal sensations: numbness, tingling, pain, 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 βAP42 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

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 (Cowper’s) gland Secretes alkaline mucus → neutralize prostatic and vaginal acidity

Sertoli cells Serve as nurse cells for developing sperm cells Inside the
seminiferous tubules

Spermatogenesis Spermatogonia → 1’ Spermatocytes → 2’ Spermatocytes → Spermatids → Sperm

Round cells Either WBCs or spermatids

Seminal Fluid Composition
Seminal fluid (SV) 60-70%
Prostatic fluid 20-30%
Spermatozoa 5%
Bulbourethral 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 Prolonged abstinence

Medication
Urine contaminationi

 White turbidity Infection ( WBCs)

Red coloration (+) RBCs
Viscosity 0 (watery) 4
(gel-like)

pH Too basic = infection

Too acidic =  prostatic fluid

Sperm 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. Kruger’s strict criteria = >14% normal morphology
-measure head, neck, tail using micrometer

Head morphology abnormalities Poor ovum penetration

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

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-Nigrosin/ Sperm If N-sperm count but  motility
viability test Living sperm cells = bluish white
Dead = red
NV = 75% living sperms
(25% dead)

Decreased motility w/ clumping (+) Male antisperm antibodies

Blood testis barrier → disrupted Clumps of sperm

Normal seminalysis w/ continued (+) Female antisperm antibodies

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 semen is Microscopic exam for sperm cells
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
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 appearance of Ochronotic shards = debris
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/μL (highest)
 (+) Culture: CAP = Haemophilus, N. gonorrhoeae (gonococcal arthritis)
S. aureus: predominant pathogen in adult joint infection
B. burgdorferi: Lyme disease (Stage 3: Lyme arthritis)

Group IV: 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 mucin clot Differentiates transudates from exudates

test Acetic acid + H2O + unknown fluid → heavy ppt. = (+) exudate

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

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

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 Albumin Gradient Recommended to differentiate transudates from exudates a. >1.1 =
(SAAG) 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
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/ Reddish-brown Fetal death

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 (Foam/Shake Amniotic fluid + 95% ethanol → shake for 15 secs → stand (15 mins)
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 (MAO) Total acid secreted in the hour after stimulation

Gastric stimulants 1. Pentagastrin = most preferred

2. Histamine

3. Histalog (Betazole)
4. Alcohols
5. Insulin = assess vagotomy procedure
-SHAM feeding = sandwich

Test Meals 1. Ewald’s meal = bread and tea/H2O

2. Boa’s = oatmeal
3. Riegel’s = mashed potato and beef steak
Yeast cells in 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

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 O-toluidine Most sensitive
Principle Pseudoperoxidase activity of Hgb (+) Blue
False (-) Vitamin C
False (+) Turnips, broccoli, cauliflower, banana, apple, melon, horseradish (to avoid, 3 days free)
Aspirin, aspilet (promote GIT bleeding; to avoid, 7 days free)
Red meat (to avoid, should be 3 days free)
APT test Distinguish fetal blood and maternal blood in an infant’s stool
Specimen: infant stool/vomitus
Rgt: 1% NaOH
HbF: alkali-resistant
Maternal Hgb: not alkali-resistant
(+) Pink = HbF
(-) Yellow brown = Maternal Hgb
Diarrhea Acute: <4 weeks Chronic: >4
weeks

Secretory 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 agglutination Sensitive in refrigerated and frozen specimens

test

 amounts of striated fibers Biliary obstruction 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

-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 brown Old blood 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 sputum a. Rusty, (+) pus = pneumococcal pneumonia
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
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 stones/Pneumoliths/ From calcified pulmonary tissue 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 bodies that take P. carinii
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)
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