CM Flashcards
Chain of infection requires continuous link bet?
Source
Mode of transmission
Susceptible host
what are the routes of infection
Inhalation
Ingestion
Direct inoculation or skin contact
6 COMPONENTS OF THE CHAIN OF INFECTION (IREMES)
- Infectious agent
- Reservoir
- Exit (Portal of Exit)
- Mode of transmission
- Entry (Portal of Entry)
- Susceptible host
_____ is the primary method of infection transmission
_____ best way to break the chain of infection
hand contact
handwashing
Handwashing procedure:
~ Rub to form lather, create ___, and loosen debris
~ Thoroughly clean between fingers, including thumbs, under fingernails and rings, and up to the wrist for at ________
~ Rinse hands in a ___ position
handwashing song:
friction
least 15 (or 20) seconds
downward
Happy Birthday (sung twice)
When hands are visibly soiled use:
when hands are not visibly soiled, use:
soap and water
alcohol based hand rub
The accepted “BIOHAZARD” label is
fluorescent orange
a _____ dilution of sodium hypochlorite
should be performed ___.
1:5 or 1:10; daily
1 part sodium hypochlorite, 9 parts water
The basic outline of the biohazard symbol is a ___, 3 circles interlapping with one another
plain trefoil
where are sharps disposed?
An accidental needle-stick must be reported to the
___
in red, puncture-resistant containers
supervisor
The best method of radioactive waste disposal is to
store the used radioactive material in a locked, marked room until 10 half-lives for radioiodine
BEST FIRST AID: Flush the area with amounts of water for at least
____ then seek medical attention.
it also applies for alkali or acid burns in the eye
DO NOT ___ CHEMICALS that come in contact with the skin.
15 minutes
NEUTRALIZE
NFPA mnemonic
Degree of Hazards (Hazards Index) mnemonic
You Were Born Right: Yellow, White, Blue Red (starting rightmost)
“No SMS Ex’s”
Flammable chemicals should be stored in ____ and ____ in a remote area.
safety cabinets; explosion-proof refrigerators
WHEN A FIRE IS DISCOVERED…(mnemonic)
TO OPERATE A FIRE EXTINGUISHER…
RACE (rescue, alarm, contain, extinguish/evacuate)
PASS (pull, aim, squeeze, sweep)
fire type and extinguisher type:
Ordinary combustibles: paper, cloth, rubbish, plastic, wood
A; Water, dry chemical, loaded steam
fire type and extinguisher type:
Flammable liquids: grease, gasoline, paints, oil
B; Dry chemical, carbon dioxide, halon foam
fire type and extinguisher type:
Electrical equipment and motor switches
C; Dry chemical, carbon dioxide, halon
fire type and extinguisher type:
Flammable metals: mercury, magnesium, sodium, lithium
D; Metal X, sand; dry powder; fought by fire fighters only
fire type and extinguisher type:
Cooking media: grease, oils, fats
K; Liquid designed to prevent splashing and
cool the fire
fire type:
Water | Dry chemicals | Carbon dioxide | Halon
Water (A)
Dry chemicals (ABC)
Carbon dioxide (BC)
Halon (BC)
Ureter: __ cm long
Bladder: when approximately ___ urine accumulates, a nerve
reflex is initiated (makes u have to pee)
Urethra: ___ long in women and __ long in men
About every __, small amounts of urine are
emptied into the bladder
25; 150 mL; 4 cm in female and 24 cm in male; 10-15 seconds
basic unit of kidney? how many per kidney?
The kidneys receive ___ of the total cardiac output
1 to 1.5 million nephrons
25%
order of urine formation
- Glomerulus
- Proximal convoluted tubule (PCT
- Loop of Henle (LH)
- Distal convoluted tubule (DCT)
- Collecting duct (CD)
~~~~ - Renal Calyx
- Renal Pelvis
The “working portion” of the kidney
- Coil of approx. ____ (capillary tuft) located within the Bowman’s capsule
- Attached to the glomerular basement membrane are the podocytes (epithelialcells)
- Resembles a ___
- Non-selective filter of plasma substances with MW of ___
Approximately ___ of the filtered plasma volume is actually excreted as urine
glomerulus; 8 capillary lobes; sieve; <70,000 daltons; 1%
- Capillary endothelium with its large open pores
- Trilayer basement membrane
- found between the podocytes of Bowman’s space
GLOMERULAR FILTRATION BARRIER;
lamina rara interna, lamina densa, lamina rara externa
Filtration diaphragm
65% of reabsorption of substances
it reabsorbs what? (mnemonic)
PCT
SWAGU (salts, water, amino acids, glucose, and urea)
1st function to be affected in renal disease.
tubular reabsorption
RENAL THRESHOLD FOR GLUCOSE
160-180 mg/dL
Renal concentration begins in the descending and ascending LH
- The ascending LH is highly impermeable to __
“DAM” Collects water, so does the Descending LH
ASIN”-ding loop = reabsorbs ASIN (salt), but NOT water
water
is the movement of a substance across cell membranes into the
bloodstream by electrochemical energy
Active transport
is the movement of molecules across a membrane by diffusion
because of a physical gradient
Passive transport
Regulates water reabsorption in the DCT and CD
ADH or vassopressin
what ADH does to the body
it increases water retention when dehydrated and inverse if there’s too much water intake
Diabetes Insipidus (DI) = ?
Syndrome of inappropriate ADH secretion (SlADH) = ?
ADH deficiency
ADH excess
Regulates sodium reabsorption
aldosterone
Effects of Angiotensin II (active form of Angiotensin) (3)
renin is produced by? how about ACE?
- release of Aldosterone & ADH (↑ Sodium & water reabsorption)
- vasoconstriction (↑ blood pressure)
- corrects renal blood flow
Juxtaglomerular (JG) cells; lungs
Major site for removal of nonfiltered substances
H+ ions are secreted in exchange for Na+ ions, which are reabsorbed with HCO3 into the plasma
PCT
2 major functions of tubular secretion
- Regulation of the acid-base balance in the body through secretion of hydrogen ions (in the form of NH4 and H2PO4).
- Elimination of waste products not filtered by the glomerulus
TESTS FOR GLOMERULAR FILTRATION
- Urea = obsolete
- Creatinine = MOST COMMON
- Inulin (MW: 5,200 Da) = Gold Standard; Reference method (Not routine bc inulin needs to be injected into the body)
- Beta2-microglobulin = better marker of renal tubular function
than of GFR - Radioisotopes
- Cystatin C (MW: 13,000 Da)
used to evaluate glomerular filtration
Measure the rate at which the kidneys are able to remove a filterable substance
from the blood
Clearance tests
CREATININE CLEARANCE FORMULA:
Ccr (ml/min) = (UV/P) x 1.73m2/A (body surf)
Urine vol = mL/min (1440)
is a measure of the completeness of a 24-hour urine collection
Greatest source of error in any clearance is the use of ___
Around ___ of creatinine is secreted by the renal tubules
Creatinine clearance
improperly timed urine specimens
7-10%
ESTIMATED GFR FORMULA DEVELOPED BY COCKGROFT & GAULT
Ccr=[[(140−age)(body weight in kilograms)]/
[(72 x serum creatinine in mg/dL)]] x 0.85 (if female)
MODIFICATION OF DIET IN RENAL DISEASE (MDRD) SYSTEM FORMULA
GFR = 170 x serum creatinine^(−0.999) x age^(−0.176) x 0.822 (if patient is female) x 1.1880 (if patient is black) x BUN^(−0.170) x serum albumin^(+0.318)
MDRD-IDMS (ISOTOPE DILUTION MASS SPECTROPHOTOMETRY TRACEABLE FORMULA:
Recommended by the National Kidney Disease Education Program (NKDEP)
GFR = 175 x serum creatinine^(−0.1154) x age^(−0.203) x 0.742 (if female) x 1.202 (if patient is black)
TESTS FOR TUBULAR REABSORPTION:
used to evaluate tubular reabsorption (assess the ability of the kidney to concentrate or dilute urine)
Concentration tests
TESTS FOR TUBULAR REABSORPTION:
- Patient is deprived of fluid for up to 24 hours
- Urine S.G. after 12-hour restricted fluid diet is about 1.022 or more
- Urine S.G. after 24-hour restricted fluid diet is about 1.026 or more
Fishberg test
TESTS FOR TUBULAR REABSORPTION:
- Patient maintains normal diet and fluid intake
- Compare day & night urine in terms of volume & S.G.
Mosenthal test
TESTS FOR TUBULAR REABSORPTION:
Influenced by the number & density of particles in a solution
Specific Gravity (S.G.)
TESTS FOR TUBULAR REABSORPTION:
Influenced by the number of particles in a solution
Osmolality
TESTS FOR TUBULAR REABSORPTION:
More preferred than S.G. determination
- More precise than osmolarity because it does not vary with temperature
- Methods include freezing point osmometry & vapor pressure osmometry
- NV = 1-3x (275 to 900 mOsm/kg) than of serum (275 to 300 mOsm/kg)
Osmolality
TESTS FOR TUBULAR SECRETION & RENAL BLOOD FLOW:
most commonly used reference method
p-aminohippuric acid (PAH) test
TESTS FOR TUBULAR SECRETION & RENAL BLOOD FLOW:
obsolete; results are hard to interpret
Phenolsulfonphthalein (PSP) test
A major reason for urinalysis’ rise to prominence was the publication of ____’s book ____ in 1491. This was the first illustrated medical book printed.
Johannes de Ketham; Fasciculus Medicinae
4 temperaments/humors:
- Sanguineous (blood)
- Choleric (yellow bile)
- Phlegmatic (phlegm)
- Melancholic (black bile)
The “taste test” of urine was used by the ____ to detect diabetes
___ physicians noticed that “honey urine” attracted ants
Babylonians and Egyptians
Hindu
SCIENTIST & DISCOVERY:
Uroscopy; first documented the importance of sputum examination
Hippocrates
SCIENTIST & DISCOVERY:
Albuminuria by boiling urine
Frederik Dekkers
SCIENTIST & DISCOVERY:
Wrote a book about “pisse prophets” (charlatans)
Thomas Bryant
SCIENTIST & DISCOVERY:
Examination of urine sediment
Thomas Addis
SCIENTIST & DISCOVERY:
Introduced urinalysis as part of doctor’s routine patient examination
Richard Bright
SCIENTIST & DISCOVERY:
Urochrome
Ludwig Thudichum
SCIENTIST & DISCOVERY:
Cerebrospinal fluid
Domenico Cotugno
SCIENTIST & DISCOVERY:
Phenylketonuria
Ivan Folling
SCIENTIST & DISCOVERY:
Alkaptonuria
Archibald Garrod
Cystine calculi
William Wollaston
SCIENTIST & DISCOVERY:
Benedicts reagent
Stanley Benedict
URINE COMPOSITION:
___ water
___ solids (60 grams= Total Solids in 24 hours)
Total Solids (TS)
95-97%
3-5%
URINE COMPOSITION:
35 grams’ organic:
25 grams’ inorganic:
ORG: Urea (major), Creatinine (2nd), hippurate, uric acid, CHO, pigments, fatty acids, mucins, enzymes, hormones
INORG: Chloride (major) > Sodium > Potassium, NaCl - Sodium chloride (principal salt) Sulfate, phosphate, ammonium, magnesium, calcium
For routine and qualitative urinalysis
Ideal for cytology studies (ONLY IF with prior hydration, &
exercise 5 mins. before collection!)
Random/Occasional/Single
Ideal specimen for routine urinalysis and
pregnancy testing (hCG)
- Often preferred for cytology studies/cytodiagnostic urine testing
- Most concentrated and most acidic (allows preservation of cells and casts)
- For evaluation of orthostatic proteinuria
First morning
2nd voided urine after a period of fasting
For glucose determination
2nd morning/Fasting
For diabetic screening or monitoring
- Preferred for testing glucose
2-hour post-prandial
Optional with blood samples in glucose tolerance test
Glucose tolerance test
-At least 2 voided collection
-Series of blood and urine samples are collected at specific time intervals to compare concentration of a substance in
urine with its concentration in the blood
-Used in the diagnosis of diabetes
Fractional specimen
- For routine screening and bacterial culture
- Patient should thoroughly cleanse his glans penis or her urethral meatus before collection
Midstream clean-catch
- May be urethral or ureteral
- For bacterial culture
Catheterized
- Abdominal wall is punctured, and urine is directly aspirated from the bladder
- Bladder urine for anaerobic bacterial culture and urine cytology
Suprapubic aspiration
- Use of soft, clear plastic bag with adhesive
- Sterile specimen obtained by catheterization or suprapubic aspiration
- Urine collected
Pediatric
specimen
For prostatic infection
o First portion of voided urine
o Middle portion of voided urine
o Urine after prostatic massage
- Examine the 1st and 3rd specimen microscopically, then
compare the # of WBC and bacteria
- Prostatitis = if the # of WBC and bacteria in the 3rd specimen is 10x GREATER than that of the 1st-
2nd specimen
o CONTROL, for bladder & kidney infection
o If control is (+) for WBCs and bacteria, the results from
the 3rd specimen are considered invalid
Three-glass technique
The four-glass method consists of bacterial cultures of the initial voided urine (VB1), midstream urine (VB2), expressed prostatic secretions (EPS), and a post- prostatic massage urine specimen (VB3).
what is the abnormal value?
STAMEY-MEARS TEST FOR PROSTATITIS
Having more than 10 to 20 white blood cells per high-powerfield
For quantitative testing.
24h, 12h, 4h or afternoon
Timed Specimen
24-hour: requires preservative (type depends on test)
12-hour: For Addis count
4-hour: For nitrite determination
afternoon: For urobilinogen determination
process providing documentation of
proper sample ID from the time of collection to the receipt of laboratory results
Chain of custody
- Required urine volume: ___
- Container capacity: ___
- Temperature (checked within 4 minutes): ___
- Added to the toilet water reservoir to prevent specimen
adulteration: __
Urine containers should have a wide base, and has an opening of at least ___
24-hr urine container should hold up to __ and may be __ to protect light sensitive analytes.
30-45 mL
60 mL
32.5-37.7degC
Blueing agent (dye)
4cm
3 liters; colored
SPECIMEN INTEGRITY:
Following collection, urine specimens should be delivered to the laboratory promptly and tested ___ (Strasinger, Harr); ideally within __ (Turgeon)
within 2 hours; 30 minutes
- Physical, chemical and microscopic characteristics of a urine specimen begin to change
AS SOON AS THE URINE IS VOIDED
CHANGES IN UNPRESERVED URINE:
Increased
- pH Urea → (Urease) → Ammonia; loss of CO2
- Bacteria Multiplication
- Odor Urea → (Urease) → Ammonia
- Nitrite → Due to bacterial multiplication
CHANGES IN UNPRESERVED URINE:
Darkened/Modified Cause
- Color - Oxidation or reduction of metabolites (↑ Urobilin)
CHANGES IN UNPRESERVED URINE:
Decreased
Clarity - Bacterial multiplication, precipitation of amorphous material
7. Glucose - Glycolysis
8. Ketones - Volatilization and bacterial metabolism
9. Bilirubin (CB) - Light exposure/photo oxidation to biliverdin
10. Urobilinogen - Oxidation to urobilin
11. RBCs/WBCs/Casts - Disintegrate in dilute alkaline urine
12. Trichomonas - Become immobile or die, possible misidentification as WBCs
Least affected urine parameter after standing
protein
URINE PRESERVATIVES:
Does not interfere with chemical tests
Raises SG by hydrometer
Precipitates amorphous phosphates and urates
Preservative of choice for routine urinalysis and urine culture (up to
24hrs.)
Prevents bacterial growth for 24 hrs.
Refrigeration
URINE PRESERVATIVES:
Preserves glucose and sediments well
Interferes with acid precipitation test for protein
Thymol
URINE PRESERVATIVES:
Preserves protein & formed elements well Does not interfere w routine analyses
other than pH
May precipitate crystals when used in large amounts
Keeps pH about 6.00
Bacteriostatic at 18 g/L;
For culture transport, C&S
Interferes with drug& hormone analyses
Boric acid
URINE PRESERVATIVES:
Excellent sediment preservative Reducing agent, interferes with chemical tests for glucose, blood, leukocytes & copper reduction
Rinse specimen container with formalin to preserve cells and casts
Preservative of choice for: Addis count
Formalin (formaldehyde)
URINE PRESERVATIVES:
Does not interfere with routine tests Floats on urine surface; clings to pipettes & testing materials
Best all-around preservative (Turgeon)
Toluene (Toluol)
URINE PRESERVATIVES:
Prevents glycolysis
Good preservative for drug analysis
Inhibits reagent strip tests for glucose, blood & leukocyte
May use sodium benzoate instead of fluoride for reagent strip testing
Specimens for Drug testing
Sodium fluoride
URINE PRESERVATIVES:
Does not interfere w/ routine tests
Causes an odor change
Use 1 drop/ounce of specimen
Phenol
URINE PRESERVATIVES:
Convenient when refrigeration is not possible
Have controlled concentration to minimize interference
May contain one or more of the preservatives including sodium fluoride
Check tablet composition to determine possible effects on desired tests
Commercial preservative tablets
URINE PRESERVATIVES:
Contains collection cup, C & S preservative tube or UA tube
Urine collection kits
URINE PRESERVATIVES:
Sample stable at room temperature for 48 hours
Preserves bacteria
Decreases pH; do not use if urine is below the minimum fill line
Preservative is boric acid and may not be used for UA
Gray C & S tube
URINE PRESERVATIVES:
Use on automated instruments
Must refrigerate within 2hrs.
Round or conical bottom
Yellow plain UA tube
URINE PRESERVATIVES:
Stable for 72 hours at room temperature; instrument compatible
Bilirubin & urobilinogen may be decreased if specimen is exposed to light and left at room temperature
Preservative is sodium propionate; conical bottom
Cherry red/ Yellow top tube
URINE PRESERVATIVES:
Preserves cellular elements
Used for cytology studies (50 mL urine)
Saccomanno fixative (50% ethanol + 2% carbowax)
URINE VOLUME:
Normal range (24 hours) = ____
- Average (24 hours) = ___
- Night urine output = ___
- Day: Night ratio = ___
- Container capacity (UA) = __
- Required for routine UA: ___ ; average: ___ (for urinometry and reagent strip)
600 to 2,000 mL.
1,200 to 1,500 mL
<400 mL
2-3:1
50 mL
10 to 15 mL; 12 mL
Increased urine volume
>2.000 mL/24 hrs. (in adults - Henry)
causes:
- Increased fluid intake
- Diuretics, nervousness
- Diabetes mellitus = ↑ SG
- Diabetes insipidus = ↓ SG
Polyuria
Decreased urine volume
- <500 mL/24 hrs. (in adults - Henry)
- <400 mL/day (in adults - Strasinger)
- <1 mL/kg/hr (in infants)
- <0.5 mL/kg/hr (in children)
causes:
- Dehydration
- Renal diseases
- Renal calculi or tumor
Oliguria
Complete cessation of urine flow
<100 mL/24 hrs (Graff)
causes:
- Complete obstruction
(stones, tumors)
- Toxic agents
- Decreased renal blood flow
Anuria
Excretion of more than 500 mL of urine at night
S.G.<1.018
causes:
- Pregnancy
- Renal diseases, bladder stones
- Prostate enlargement
Nocturia
Any increase in urine excretion
causes:
- Excessive water intake (polydipsia)
- Diuretic therapy, hormonal
imbalance
- Renal dysfunction, drug
ingestion
Diuresis
- Rough indicator of the degree of hydration
- Should correlate with urine S.G.
- Normal: Colorless to deep yellow
- Abnormal: Red / Red brown (most common)
urine color
NORMAL PIGMENTS IN URINE:
- Major pigment (yellow)
- Lipid-soluble pigment that is a product of endogenous
metabolism
- Production is directly proportional to metabolic rate
- ↑ in thyrotoxicosis, fever, starvation
Urochrome
NORMAL PIGMENTS IN URINE:
- Pink (or red)
- Derived from melanin metabolism
- May deposit in amorphous urates and uric acid crystals
Uroerythrin
NORMAL PIGMENTS IN URINE:
- Dark yellow/orange-brown
- Derived from oxidation of colorless urobilinogen
- Present in old specimens
Urobilin
LABORATORY CORRELATIONS OF URINE COLOR:
Recent fluid consumption, polyuria, dilute random specimen
Pyuria/leukocyturia (↑ WBCs)
Colorless to pale yellow
Milky white
LABORATORY CORRELATIONS OF URINE COLOR:
Concentrated specimen: strenuous exercise, dehydration, fever,
burns; First morning specimen; Excessive urobilin, bilirubin, carotene
Methemoglobin (acidic urine); Homogentisic acid (alkaline urine): Alkaptonuria; Melanin (upon air exposure); Phenol derivative
Dark yellow to amber
Brown Black
LABORATORY CORRELATIONS OF URINE COLOR:
Bilirubin → (oxidized) → Biliverdin
Porphyrins
Yellow-green Yellow-brown
Burgundy/Purplish red Portwine
LABORATORY CORRELATIONS OF URINE COLOR:
RBCs (Cloudy/ smoky red): Hematuria, Hemoglobin (Clear red)
Myoglobin (Clear red/reddish-brown/cola-colored/tea-colored)
Porphobilin (derived from porphobilinogen)
Beets, menstrual contamination
Fuchsin (aniline dye from foods and candy)
Rifampin = all body fluids are red
Pink/Red
URINE COLOR CHANGES WITH DRUGS:
(Tx: Parkinsonism) Cola-colored (red then brown, alkaline)
Brown, oxidized to quinones (green)
Levodopa (Tx: Parkinsonism)
Phenol poisoning
No visible particulates, transparent
Clear
Few particulates, print easily seen through urine
Hazy
Many particulates, print blurred through urine
Cloudy
Print cannot be seen through urine
Turbid
May precipitate or be clotted
Milky
Amorphous urates, radiographic contrast media
Amorphous phosphates, carbonates
Acidic Urine
Alkaline Urine
Soluble with Heat
Soluble in Dilute Acetic Acid
Insoluble in Dilute Acetic Acid
Soluble in Ether
Amorphous urates, uric acid crystals
RBCS, amorphous phosphates, carbonates
WBCs, bacteria, yeast, spermatozoa
Lipids, Lymphatic fluid, chyle
URINE ODOR:
Normal (due to presence of volatile acids from food)
Aromatic, faintly
URINE ODOR:
Acute tubular necrosis (acute renal failure)
Odorless
URINE ODOR:
UTI (ex. Proteus vulgaris), old urine
Foul, ammoniacal
URINE ODOR:
Ketones (Diabetes Mellitus, starvation, vomiting)
Fruity sweet
URINE ODOR:
Maple syrup urine disease (MSUD)
Caramelized sugar, curry, maple syrup
URINE ODOR:
Phenylketonuria (PKU)
Mousy, musty, barny
URINE ODOR:
Tyrosinemia
Rancid butter
URINE ODOR:
Isovaleric acidemia, glutaric acidemia
Sweaty feet, acrid
URINE ODOR:
Phenol-containing medications
Menthol-like
URINE ODOR:
Methionine malabsorption (Oasthouse syndrome)
Cabbage, hops
URINE ODOR:
Specimen adulteration or container contamination
Bleach
URINE ODOR:
Cystine disorder
Sulfur
URINE ODOR:
Trimethylaminuria
Rotting fish; galunggong
URINE ODOR:
Pungent, fetid Ingestion of onions, garlic, & asparagus, (methylmercaptan), UTI (Brunzel)
Pungent, fetid
URINE ODOR:
Hawkinsinuria
Swimming pool
URINE ODOR:
3-hydroxy-3-methylglutaric aciduria
Cat urine
URINE ODOR:
Multiple carboxylase deficiency
Tomcat urine
CHEMICAL EXAMINATION:
Double sequential enzyme reaction
Green to brown (KI chromogen)
30 seconds
Glucose
CHEMICAL EXAMINATION:
Diazo reaction
Tan or pink to violet
30 seconds
Bilirubin
CHEMICAL EXAMINATION:
Sodium nitroprusside reaction
Purple
40 seconds
Ketones
CHEMICAL EXAMINATION:
pKa change of polyelectrolyte
Blue to yellow
45 seconds
Specific gravity
CHEMICAL EXAMINATION:
Protein error of indicators
Blue-green
60 seconds
Protein
CHEMICAL EXAMINATION:
Double indicator system
Orange to blue
60 seconds
pH
CHEMICAL EXAMINATION:
Pseudoperoxidase activity of hemoglobin
Uniform green/blue - Speckled/spotted
60 seconds
Blood
CHEMICAL EXAMINATION:
Ehrlich reaction
Red
60 seconds
Urobilinogen
CHEMICAL EXAMINATION:
Greiss reaction
Uniform pink
60 seconds
Nitrite
CHEMICAL EXAMINATION:
Leukocyte esterase
Purple
120 seconds
Leukocytes
- Dip the reagent strip briefly (___) into a well-mixed uncentrifuged urine specimen at RT.
- Remove excess urine by touching the edge of the strip to the container as the strip is withdrawn,
- Blot ___ of the strip on a disposable absorbent pad.
- Wait the specified amount of time for the reaction to occur.
- Compare the color reaction
no longer than 1 second
the edge
AUTOMATED REAGENT STRIP READERS:
What is the principle?
REFLECTANCE PHOTOMETRY
- A measure of the amount of dissolved substances in a solution
- Density of solution compared with density of similar volume of distilled water at a
similar temperature - Influenced by number and size of particles in a solution
Specific gravity
- Isosthenuria = SG ___
- Hyposthenuria = SG ___
- Hypersthenuria = SG ___
- If SG is <1.003 = Not a urine (except in D.I.)
- 1.001 = __
- If SG is >1.040 = ___
=1.010
<1.010
>1.010
Water
Radiographic dye present
Calibration temperature: ___
- Requires temperature correction:
o ___for every 3oC that the specimen temp. is below the
calibration temp.
o ___ for every 3oC that the specimen temp. is above the
calibration temp.
- Requires correction for glucose and protein:
o 1g/dL Glucose = ___
o 1g/dL Protein = ___
- Urine volume required: ___
CALIBRATION:
o ___ solution (20.29 g K2SO4 to 1L H2O)
o S.G. reading should be __
URINOMETRY (URINOMETER /HYDROMETER)
20degC
-0.001
+0.001
correction:
-0.004 - glucose
-0.003 - protein
10-15 mL
Potassium sulfate (K2SO4); 1.015
REFRACTOMETER CALIBRATION:
- Distilled/deionized H2O =
- 3% NaCl =
- 5% NaCl =
- 7% NaCl =
- 9% Sucrose =
1.000 +/- 0.001
1.015 +/- 0.001
1.022 +/- 0.001
1.035 +/- 0.001
1.034 +/- 0.001
Blue (1.000) → Green → Yellow (1.030)
- The polyelectrolyte ionizes, releasing hydrogen ions in
proportion to the number of ions in the solution - The reagent is sensitive to the number of ions in urine
- Indicator changes color in relation to ionic concentration
REAGENT STRIP REACTION for SPECIFIC GRAVITY (45 seconds)
Reagents:
Multistix = Poly (methyl vinyl ether/maleic anhydride) “bromthymol blue”
Chemstrip = Ethylene glycol diaminoethyl ether tetraacetic
acid “bromthymol blue”
Interferences:
- False (+) = High concentration of protein (Strasinger)
- False (-) = Highly alkaline urine (>6.5)
it is not affected by?
SG
Not affected by glucose protein & radiographic dye (Henry)
Obsolete method
Based on frequency of soundwave entering a solution changes in proportion to the density of soln.
- Ex: Yellow IRIS (International Remote Imaging System)
- IRIS Diagnostics
6mL = required urine volume
- 4 mL (of 6 mL) = for IRIS sIideless microscope
- 2mL (of 6 mL) = for IRIS Mass Gravity Meter (for S.G. determination - by using Harmonic oscillation densitometry)
HARMONIC OSCILLATION DENSITOMETRY (H.O.D.)
Acidity refers to the “sourness” of a solution, whereas alkalinity refers to its “bitterness”
Important in the identification of crystals and determination of unsatisfactory specimens
- A blood pH <6.8 or >7.8 will result in death
- Normal urine pH:
o Random = 4.5-8.0
o 1st morning = 5.0-6.0
- When pH is >9.0 = unpreserved urine
pH
- Diabetes Mellitus (↑ ketone bodies)
- Starvation (↑ ketone bodies)
- High protein diet
- Cranberry juice = a treatment for UTI
- Emphysema, dehydration, diarrhea, acid-producing bacteria (E. coli), medications
Causes Acidic Urine
- Renal tubular acidosis
- Vegetarian diet
- After meal = due to alkaline tide
- Vomiting
- Old specimens, hyperventilation, presence of urease-
producing bacteria
Causes Alkaline Urine
Double Indicator System
Methyl Red ——————-> Bromthymol blue
pH 4.0-6.0 (Red to yellow) pH 6.0-9.0 (yellow to blue)
REAGENT STRIP REACTION for pH (60 seconds)
reagents of pH
Reagents Methyl red, Bromthymol blue
Most indicative of renal disease
Produces white foam in urine when shaken
PROTEIN
Normal urinary protein:
o ____ (Strasinger)
o <150 mg/day (Henry)
- Mild/minimal proteinuria = __
- Moderate proteinuria = __
- Large/heavy proteinuria = __
“Proteins in normal urine consist of __ albumin and ___ globulins.”
Major serum protein found in the urine?
10 mg/dL or <100 mg/day
<1 g/day
1 to 3 or 4 g/day
>3 or 4 g/day
1/3; 2/3
Albumin
Caused by conditions that affect the plasma prior to its reaching the kidney:
o Intravascular hemolysis = hemoglobin
o Muscle injury = myoglobin
o Severe infection & inflammation = ↑ APRs
o Multiple myeloma
PRE-RENAL PROTEINURIA (“BEFORE”) OR OVERFLOW PROTEINURIA
Decreased glomerular filtration
May lead to renal failure
Indicator: Microalbuminuria = proteinuria undetectable by routine reagent strip
Diabetic nephropathy
Albumin Excretion Rate (AER) in ug/min or in mg/24 hours
Normal = ____
Microalbuminuria = ___ (or 30-300 mg/24hrs)
Clinical albuminuria = __
0-20 ug/min
20 – 200 ug/min
>200 ug/min
Proteinuria when standing due to increased pressure to renal veins
Increased venous pressure causes renal congestion and glomerular changes
Monitored every 6 months and re-evaluated as necessary
Orthostatic/Cadet/Postural proteinuria
- Originally discovered in workers exposed to cadmium dust (a heavy metal)
- Normally filtered albumin can no longer be reabsorbed
o Fanconi’s syndrome
o Toxic agents / heavy metals
o Viral infections
TUBULAR PROTEINURIA
- Test for microalbuminuria
- A strip employing antibody-enzyme conjugate that binds albumin
- Principle: ___
- Reagents: Gold-labeled antibody, -galactosidase, Chlorophenol red
galactoside - Sensitivity: __
- (-) = White; (+) = Red [60 seconds]
- Interference: False (-) = Dilute urine
Micral test
Enzyme immunoassay
0 -10 mg/Dl
Principle: Immunochromographics
- Sensitivity: 1.2 -8.0 mg/dL
- Reagents: Antibody coated blue latex particles
- Interference: False (-) = Dilute urine
IMMUNODIP TEST FOR MICROALBUMINURIA
Principle: Sensitive albumin tests related to creatinine conc. to correct for patient hydration
- Reagents
o Albumin: diiodo-dihydroxydinitrophenyl tetrabromosulfonphthalein
o Creatinine: copper sulfate, tetramethylbenzidine,
diisopropylbenzenedihydroperoxide
Sensitivity:
o Albumin = ___
o Creatinine = ___ (0.9-26.5 mmol/L)
Interferences
o Visibly bloody/abnormally colored urine
o Creatinine = Cimetidine - False (+)
ALBUMIN: CREATININE RATIO - CLINITEST MICROALBUMIN STRIPS/
MULTISTIX-PRO
10 - 150 mg/L
10 - 300 mg/dL
- Lower UTI / inflammation
- Menstrual contamination
- Injury / trauma
- Vaginal secretions
- Prostatic fluid / spermatozoa
POST RENAL PROTEINURIA
(“AFTER”)
Principle: Protein (Sorensen’s) error of indicators
- Contrary to the general belief that indicators produce specific colors in response to particular pH levels, certain indicators change color in the presence of protein even though the pH of the medium remains constant.
Indicator + Protein → (+) Blue-green
REAGENT STRIP REACTION for PROTEIN (60 seconds)
PROTEIN REAGENT:
Indicator is SENSITIVE to ___
Multistix = “Tetrabrom”phenol blue, citrate buffer at pH 3.0
- Chemstrip = Tetrachlorophenol “tetrabrom”osulfonphthalein,
citrate buffer at pH 3.0
Albumin
- A cold precipitation test that reacts equally with all forms of protein
- Reagent = Exton’s reagent (3% SSA + sodium sulfate)
Procedure:
o 3 mL of 3% SSA+ 3 mL centrifuged urine → 10 mins incubation → (+) Cloudiness
o or 3 mL of 7% SSA + 1l mL centrifuged urine → 10 mins incubation → (+) Cloudiness
SULFOSALICYLIC ACID PRECIPITATION TEST (a.k.a. EXTON’S TEST)
EXTON GRADE:
No increase in turbidity
Neg
EXTON GRADE:
Noticeable turbidity
If viewed from top, circle is visible in
test tube bottom
Trace
EXTON GRADE:
Distinct turbidity with no granulation
If viewed from top, circle not visible
1+
EXTON GRADE:
Turbidity with granulation but NO
flocculation
Cannot read newsprint through
mixture
2+
EXTON GRADE:
Turbidity with granulation AND
flocculation
3+
EXTON GRADE:
Clumps of protein
4+
- Highly buffered alkaline with no albumin present
- false-positive reagent strip
- Highly buffered alkaline with albumin present false-negative SSA test
- To differentiate, acidify urine to pH - 5.0 and retest
Strip Result: +
SSA result: -
Proteins other than albumin present
- False (+)
~ Radiographic contrast media (delayed reaction), Drugs and/or drug metabolites (tolbutamide, penicillins, cephalosporins, sulfonamÌdes)
- Examine precipitate microscopically: drugs and radiographic dye form crystalline precipitates; whereas
protein precipitates are amorphous
Strip Result: -
SSA result: +
- Most frequently tested in urine
- Renal threshold: plasma concentration of a substance at which tubular reabsorption stops
GLUCOSE (DEXTROSE)
CLINICAL SIGNIFICANCE OF URINE GLUCOSE:
↑ Blood glucose
↑ Urine glucose
Causes:
- Diabetes Mellitus
- Cushing’s syndrome (↑ cortisol)
- Phaeochromocytoma (↑ catecholamines)
- Acromegaly (↑ growth hormone)
- Hyperthyroidism (↑ T3, T4)
HYPERGLYCEMIA-ASSOCIATED
CLINICAL SIGNIFICANCE OF URINE GLUCOSE:
Normal Blood glucose
↑ Urine glucose
Causes
- Impaired tubular reabsorption of
glucose
- Fanconi syndrome - Defective tubular
reabsorption of glucose and
amino acids
RENAL-ASSOCIATED
REAGENT for GLUCOSE (30 seconds)
Multistix = Glucose oxidase, Peroxidase, Potassium iodide (blue to green to brown)
Chemstrip = Glucose oxidase, Peroxidase, Tetramethylbenzidine (yellow to green)
Interferences:
- False (+) = Oxidizing agents, detergents
- False (-) = High levels of ascorbic acid, ketones, high S.G.,
LOW TEMP, improperly preserved specimen
GLUCOSE (rgt strp)
Glucose strip was the 1st “dip and read” reagent strip developed by Miles, Inc., in 1950
- Sensitivity = ____ (detects glucose only)
100 mg/dL
Nonspecific test for reducing sugars
(Glucose, Galactose, Lactose, Fructose but NOT Sucrose)
COPPER REDUCTION TEST (CLINITEST/ BENEDICT’S TEST)
Reporting (Benedict’s test):
clear blue color, blue precipitate may form
-/neg
Reporting (Benedict’s test):
bluish-green color
trace
Reporting (Benedict’s test):
green color, green or yellow precipitate
1+
Reporting (Benedict’s test):
yellow to green color, yellow precipitate
2+
Reporting (Benedict’s test):
yellow-orange color, yellow-orange precipitate
3+
Reporting (Benedict’s test):
reddish-yellow color, brick red or red precipitate
4+
GLUCOSE STRIP TEST
False-positive Causes = ___ (ascorbic acid, uric acid)
False-negative Causes = ___ (detergents)
Reducing agents;
Oxidizing agents
CLINITEST TABLET CONTENT:
- ___ = main reacting agent
- ___ = for heat production
- ____ = eliminates interfering O2
- ___ = for heat production
Pass-through phenomenon:
- Occurs when __ sugar is present
To prevent pass through, use ___ urine (use separate color chart to interpret
the reaction)
CuSO4
Na citrate
NaCO3
NaOH
> 28/dL
2 gtts
glucose oxidase: 1+ positive
clinitest: negative
Small amount of glucose present
glucose oxidase: 4+ positive
clinitest: negative
Possible oxidizing agent interference on reagent strip
glucose oxidase: negative
clinitest: positive
Non-glucose reducing substance present
Possible interfering substance for reagent strip (ex: Ascorbic acid)
Result from increased fat metabolism due to inability to metabolize carbohydrates
- Renal threshold = ___
Seen in:
o Type I DM
o Vomiting
o Starvation
o Malabsorption
KETONES; 70 mg/dL
KETONE BODIES:
___ - Beta-hydroxybutyric acid; major ketone but not detected in reagent strip
___ - Acetoacetic acid (AAA)/ Diacetic acid; parent ketone (1st ketone body formed); detected by the reagent strip
__ - Acetone
78%
20%
2%
REAGENT STRIP for KETONES (40 seconds)
Na nitroprusside/nitroferricyanide, Glycine (Chemstrip)
Interferences:
False (+) = Pthalein dyes, pigmented red urine, levodopa, drugs with sulfhydryl groups
- False (-) = Improperly preserved specimens
Notes
- Acetone is detected only when __ is present
- Correlations with other tests = Glucose
Ketone
glycine
test for ketone (tablet)
1 gtts urine + ___ → (+) Purple color after 30 seconds
- Composition: Sodium nitroprusside, Disodium phosphate, Glycine and
Lactose
ACETEST TABLET
BLOOD:
Cloudy red urine
- Sensitive early indicator of renal
disease
Seen in: - Glomerulonephritis
- Renal calculi, tumors
- Strenuous exercise, trauma
Microscopic: Intact RBCs
Hematuria
BLOOD:
Clear red urine
Seen in:
- Intravascular, hemolysis, transfusion
reactions, Hemolytic anemia, Severe burns, brown recluse spider bites
- Microscopic = No RBCS seen
- Heme portion of the hemoglobin is toxic to the renal tubules
Hemoglobinuria
BLOOD:
Must be at least 25 mg/dL to show clear red (red-brown) urine
Seen in:
Rhabdomyolysis, Muscular trauma, Crush syndromes, Extensive exertion, Cholesterol-lowering statin medications
- Heme portion of the myoglobin (more toxic) is toxic to the renal tubules
- ___ = renal failure risk
Myoglobinuria; >1.5 mg/dL
HEMOGLOBIN VS. MYOGLOBIN:
tests used to differentiate?
Plasma examination & Blondheim’s test
(Ammonium sulfate test)
Principle Pseudoperoxidase activity of hemoglobin
REAGENT STRIP for BLOOD (60 seconds)
Multistix = Diisopropylbenzene dehydroperoxide “tetramethylbenzidine”
- Chemstrip = Dimethyldihydroperoxyhexane “tetramethylbenzidine”
blood rgt
BLOOD RGT STRIP:
- Uniform green/blue color = __
- Speckled/spotted = ___
- Chemstrip contains __ overlay that eliminates ascorbic acid interference
- Hemoglobin level of ___ produces a positive protein reagent strip reaction
- Correlations with other tests = Protein, Microscopic
Hemoglobin/ Myoglobin
Hematuria (Intact RBCs)
iodate
>10 mg/dL
___ - water soluble type
- Early indication of liver disease
- Tea-colored/amber/beer brown urine with yellow foam
- Clinical significance:
o Hepatitis
o Cirrhosis
o Biliary obstruction (gallstones, carcinoma)
BILIRUBIN
Conjugated bilirubin (CB)
Principle: Diazo reaction
Bilirubin diglucoronide (CB) + Diazonium salt → Azodye
REAGENT STRIP REACTION for BILIRUBIN (30 seconds)
Multistix = 2,4-“dichloro”aniline diazonium salt
- Chemstrip = 2,6-“dichloro”benzene diazonium salt
Bilirubin strip reagents
- False (+) = Highly pigmented urines, phenazopyridine, indican, metabolite of Lodine
- False (-) = Specimen exposure to light, high conc., of nitrite, ascorbic acid (25 mg/dL)
Notes - (+) ___ (positive color)
BILIRUBIN:
Tan or Pink to Violet
test for bilirubin? (tablet)
ICTOTEST Tablet
10 gtts urine + __ tablet + 2 gtts H2O → (+) Blue to purple color after 60 seconds
- Confirmatory test for bilirubin; more sensitive than strip with less interference
- Composition = p-nitrobenzene-diazonium p-toluenesulfonate, SSA, Na2CO3,
boric acid
ICTOTEST
- Bile pigment that resulted from hemoglobin degradation
- Normal value = <1 mg/dL or Ehrlich Unit
- Specimen = Afternoon urine (2-4 PM)
UROBILINOGEN (UBG)
Principle: Ehrlich reaction
Urobilinogen (and Ehrlich-reactive compounds) + PDAB → (+)Red
REAGENT STRIP for UROBILINOGEN (60 seconds)
Multistix = p-dimethylaminobenzaldehyde (PDAB)
- Chemstrip = 4-methoxybenzene-diazonium-tetrafuoroborate
(specific)
urobilinogen reagents
Differentiate urobilinogen (UBG), porphobilinogen (PBG) and other Ehrlich-
reactive compounds (ERC)
- Uses extraction with organic solvents: Chloroform & Butanol
- “UCBU” = Urine – Chloroform; Butanol - Urine
WATSON-SCHWARTZ TEST
WATSON-SCHWARTZ TEST
__: red /
__: red \
__: red on top layers only
__: red all layers
chloroform always on: __; butanol on: __
urobilinogen
porphobilinogen
ehrlich reactive
urobilinogen/porphobilinogen
chlor: bottom; but: top
Inverse Ehrlich reaction: reagent volume is more abundant than urine volume
- Rapid screening test for porphobilinogen (> 2 mg/dL)
Procedure:
- 2 gtts urine + 2 mL reagent (Ehrlich’s rgt in 6M or 6N HCl) → (+) Red
HOESCH TEST
Significance: Rapid screening test of UTI or bacteriuria
- ___ converters are generally Gram-negative bacilli, such as the Enterobacteriaceae
- Specimen: ___ or ___ urine (preferred)
NITRITE; Nitrate
4-hour collection; first morning
p-arsanilic acid (or sulfanilamide) + Nitrite → Diazonium salt
Diazonium salt + Tetrahydrobenzoquinolin → (+) Uniform pink
REAGENT STRIP REACTION for NITRITE (60 seconds)
Reagents - Multistix = p-arsanilic acid, tetrahydrobenzo(h)-“quinolin”-3-ol
- Chemstrip = Sulfanilamide, hydroxytetrahydrobenzo”quinoline”
nitrite reagent
Pink spots/edges = considered ___
- (+) Nitrite corresponds to 100,000 organisms/mL
- If the nitrite test area shows a negative reaction, UTI cannot be ruled out
- Some UTIs are caused by Gram (+) cocci & yeasts they lack nitrate reductase enzymes
- Dietary nitrate can be found in __
- Correlations with other tests = Protein, Leukocytes, Microscopic
Nitrite
NEGATIVE
green vegetables
Significance:
o Urinary tract infection or inflammation
o Screening of urine culture specimens
LEUKOCYTES
Principle:
Leukocyte esterase
Indoxylcarbonic acid ester leukocyte esterase → Indoxyl + Acid indoxyl + Diazonium salt → (+) Purple
REAGENT STRIP REACTION for LEUKOCYTES (120 seconds)
Reagents
- Multistix = Derivatized pyrrole amino acid “ester”, Diazonium salt
- Chemstrip = Indoxylcarbonic acid “ester”, Diazonium salt
leukocyte rgts
With esterase: Neutrophil, Eosinophil, Basophil, Monocyte, Histiocyte, Trichomonas
- No esterase: ___
- Strip can detect lysed WBCs
- Trichomonas, Chlamydia, yeast, & interstitial nephritis produce ___
- Correlations with other tests = Protein, Nitrite, Microscopic
Lymphocyte
pyuria w/o bacteriuria
11 reagent pad on a strip
- Water-soluble vitamin
- Dietary sources include citrus fruits & vegetables (tomatoes, green peppers, cabbage, leafy greens)
- Excreted as ascorbic acid or its principal metabolite, oxalate
- Strong reducing substance
- Interferes with reagent strip that use hydrogen peroxide or diazonium salt
ASCORBIC ACID (Vitamin C)
ascorbic acid causes false-negative reactions on urine strip:
(“BB LNG”):
o Blood
o Bilirubin
o Leukocytes
o Nitrite
o Glucose
11th Reagent Pad:
o Ascorbic acid (>5 mg/dL) + Phosphomolybdate → (+) ____
Brands (Henry):
o Cstix = __ seconds reading time
o Stix = __ seconds reading time
o Others = vChem, Urispec GP + A, and Merckoquant
- GC-MS = More accurate quantitative method (Henry)
Molybdenum blue
10
60
URINE SEDIMENT PREPARATION:
1. Transfer ___ of urine in a test tube (recommended volume = ___)
2. Centrifuge tube at 400 RCF for ___
3. Decant urine (0.5 or 1.0 mL urine remains in the tube)
4. Transfer 20 uL (0.02 ml) sediment to glass slide with 22 x 22 mm coverslip
5. Examine microscopically (___, under reduced light)
10-15 mL; 12 mL
5 minutes
10 LPE 10 HPE
URINE SEDIMENT PREPARATION:
- If __ urine is available for microscopy, centrifuge ___ of it
- If __ urine is available, examine sediment without centrifugation
- RCF formula = ___
- Slides are 1st examined under __ to detect casts
- Use __ for further identification of casts
<12 mL; 3 mL
<3ml
10^5 x radius in centimeters x RPM^2
LPO
HPO
- Quantitative measure of formed elements of urine using hemacytometer
- Specimen = __
- Preservative = __
ADDIS COUNT
12-hour urine
Formalin
QUICK FACTS ABOUT THE MICROSCOPE!
First lens system = located in the __ & is adjusted to be near the specimen
- Second lens system = located in the __ (ocular lens)
- ___ = ability to distinguish 2 small objects that are a specific distance apart
- __ = microscopes requiring minimum adjustment when switching objectives
- Camel-hair brush = used to remove dust on the optical surface of the microscope
- __ = Used to clean the optical surfaces of the microscope
- __ = used to clean any contaminated lens
- 10 remove oil on lens, use dry lens paper, then lens paper moistened w lens cleaner
- Using ___ to remove oil on lens is not recommended due to its toxic fumes
objective
eyepiece
Resolution
Parfocal
Lens paper
Commercial lens cleaner
xylene
Adjust for focusing difference between eyes
Adjust for comfort
Loosen to rotate head
For ease in specimen manipulation
Use to rotate objectives
which form primary magnification (initial
image of specimen)
Aperture diaphragm which restricts area of illumination
Diopter Rings
Rubber Eyeguard
Eyepiece Tube Clamp Screw
Reverse Facing Nosepiece
Revolving Nosepiece
Objectives Lenses
Field Diaphragm
Adjusts size opening of field diaphragm
Brings slide into view
Sharpens image
Holds light source
Indicates distance between eyes
Rotate to adjust for interpupillary distance; Magnify image (x10) formed by objective lens
Holds slide in place
Moves slide on stage
Field Diaphragm Control Ring
Coarse Focus Knob
Fine Focus Knob
Lamp Socket
Interpupillary Distance Scale
Eyepieces
Slide Holder
X/Y Travel Knobs
Focuses light onto slide
Holds specimen
Loosen to remove stage
Adjusts size opening of condenser
Aperture diaphragm that controls light
Centers the field of view
Turns microscope on/off; adjusts light intensity
Condenser Focus Knob
Stage
Stage Clamp Screw
Condenser Control Ring
Condenser
Condenser Centering
Screws
Brightness Control Dial
MICROSCOPIC TECHNIQUES:
- For routine urinalysis
Bright-field (BF) Microscopy
MICROSCOPIC TECHNIQUES:
- Enhances visualization of translucent elements (1.e. with low refractive indices [e.g. casts])
- To convert BF into this, replace objective lens & condenser with objective lens & condenser intended for this microscope
Phase-contrast (PC) Microscopy
MICROSCOPIC TECHNIQUES:
- Detects the presence or absence of birefringence
- Birefringence is the ability of an element to refract light in 2 dimensions at 90° to each other
- For identification of cholesterol in oval fat bodies, fatty casts and crystals
- To convert BF into this, add 2 filters (1 below the condenser, 1 between objective & oculars)
Polarizing Microscopy
MICROSCOPIC TECHNIQUES:
- For identification of Treponema pallidum
- To convert BF into this, replace the condenser with a condenser that contains an opaque disk intended for this type of mc
Dark-field (DF) Microscopy
MICROSCOPIC TECHNIQUES:
- For visualization of fluorescent substances and microorganisms
Fluorescence Microscopy
MICROSCOPIC TECHNIQUES:
- 3-D microscopy-image & layer-by-layer imaging of a specimen
- Bright-field microscopes can be adapted for this type of microscopy
- what are the two types of this microscope?
Interference-contrast Microscopy
- Nomarski (Differential interference contrast)
- Hoffman (Modulation contrast)
SEDIMENT STAINS:
- Delineates structure & contrasting colors of the nucleus & cytoplasm
- Identifies WBCs, epithelial cells and casts
- Most commonly used stain
Sternheimer - Malbin (SM) (Crystal violet + Safranin O)
Available as KOVA & Sedi stain
SEDIMENT STAINS:
- Enhances nuclear detail
- Differentiates WBCs from RTE cells
Toluidine blue (Supravital stain)
SEDIMENT STAINS:
- Lyses RBCs, enhances nuclei of WBCs
- Distinguishes RBCs from WBCs, yeast, oil droplets & crystals
2% acetic acid
SEDIMENT STAINS:
- Stains triglycerides and neutral fats orange-red (cannot stain cholesterol)
- Identifies free fat droplets & lipid-containing cells & casts
Lipid stains (Oil Red O and Sudan III)
SEDIMENT STAINS:
- Differentiates Gram-positive & Gram negative bacteria
- Identifies bacterial casts
Gram stain
SEDIMENT STAINS:
- Stains eosinophilic granules
- Identifies urinary eosinophils
Hansel stain (Eosin Y + Methylene blue)
SEDIMENT STAINS:
- Stains structures containing iron
- Identifies hemosiderin granules
Prussian blue (Rous test)
SEDIMENT STAINS:
- Stains DNA
Phenathridine (orange)
SEDIMENT STAINS:
- Stains nuclear membranes. mitochondria & cell membranes
Carbocyanine (green)
SEDIMENT CONSTITUENTS:
o NV = 0-2 or 0-3/HPF
o Smooth, non-nucleated, biconcave disks
o Hypertonic urine = __
o Hypotonic urine = __
o Glomerular membrane damage = dysmorphic, with projections, fragmented
o Sources of error = Yeasts, Oil droplets, Air bubbles, Monohydrate calcium oxalate crystals
o Remedy = Add ___. It will lyse the RBCs but not the others
RBCs (Hematuria)
Crenate/Shrink
Swell/Hemolyze (Ghost cell)
2% acetic acid
SEDIMENT CONSTITUENTS:
o NV = 0-5 or 0-8/HPF
o Larger than RBCs
o Increased number indicates presence of infection or inflammation
o __
Most predominant
Granulated and multilobed
In hypotonic urine, they swell, and granules undergo Brownian movement, producing a sparkling appearance (Glitter cells)
When dying, form blebs & finger-like projections (myelin forms)
o __
Normal value = 1%
Significant = >1% (associated w/ drug-induced interstitial nephritis)
o __ (Lymphocytes, monocytes, macrophages, histiocytes)
Normally present in small numbers
↑ Lymphocytes = __
↑ Monocytes, histiocytes = __
WBCs (Pyuria or Leukocyturia)
Neutrophils
Eosinophils
Mononuclear cells
Renal transplant rejection
chronic inflammation & radiation therapy
SEDIMENT CONSTITUENTS:
what is the largest cell w/ abundant, irregular cytoplasm & prominent nucleus? __
Cell size is about 30-50 um (5-7x the size of an RBC)
The nucleus is about the size of an RBC
From linings of vagina, female urethra & lower male urethra (more commonly found in female patients)
Variation = __
–> S.E.C. covered with Gardnerella vaginalis
–> Associated with bacterial vaginosis
Epithelial Cells:
Squamous Epithelial Cells SEC
Clue cells
SEDIMENT CONSTITUENTS:
Cell size is about 20-30 um (4-6x the size of an RBC)
Spherical, polyhedral or caudate with centrally located nucleus
Derived from the renal pelvis, calyces, ureter, urinary bladder & upper male urethra
Increased following catheterization: may be seen singly, in pairs, or in clumps (syncytia)
If exhibiting abnormal morphology: malignancy or viral infection
Transitional epithelial (Urothelial/Bladder) cell (T.E.C.)
SEDIMENT CONSTITUENTS:
Most clinically significant epithelial cell
Origin: __
Size is 3-5x the size of RBCs
Rectangular, polyhedral, cuboidal or columnar w/ eccentric nucleus
Sometimes bilirubin-stained or hemosiderin-laden
RTE cells from the DCT may be mistaken for WBCs
>2 RTE/hpf indicates tubular injury
Renal tubular epithelial (R.T.E.) cell
Nephron
SEDIMENT CONSTITUENTS:
o True UTI = __
o __ = most common cause of UTI
o Staphylococcus, Enterococcus
o Motility differentiates them from
amorphous urates & phosphates
Bacteria
Bacteria + WBCs (If bacteria only = contamination or old specimen)
Enterobacteriaceae (Ex. E. coli)
SEDIMENT CONSTITUENTS:
o True infection = __
o Small, refractile oval structure that may or may not bud
o Branched, mycelial forms are seen in __
o __= seen in DM and vaginal moniliasis
Yeasts
Yeast + WBCs (If yeast only = contamination)
severe infections
Candida albicans
SEDIMENT CONSTITUENTS:
o __ - Most frequently encountered parasite in urine
Pear-shaped flagellate with jerky motility
Agent of ___
Reported as rare, few, moderate, or many per HPF
When not moving, may resemble WBC, T.E.C. or R.T.E. cell
o ___ - Most common fecal contaminant
o ___ - Blood fluke with terminal spine
Causes hematuria
Associated with ___
o Other: Trichuris, Strongyloides, Giardia, various amoebae
o Various insects or “bugs” (lice, fleas, bedbugs, mites, and ticks)
Parasites:
Trichomonas vaginalis; Ping-Pong disease
Enterobius vermicularis egg
Schistosoma haematobium egg; bladder cancer
SEDIMENT CONSTITUENTS:
o Oval, slightly tapered head
o Long, flagella-like tail
o After sexual intercourse
Spermatozoa
SEDIMENT CONSTITUENTS:
o Has low refractive index
o Major constituent: Tamm-Horsfall protein (other name:__)
Mucus Threads
Uromodulin
Using Sternheimer-Malbin Stain:
- (Pale Blue)
- (Pale Pink)
Glitter Cells
Leukocytes
VARIATION OF RTE CELLS:
o Lipid containing RTE cell (may also be a
monocyte/macrophage)
o Seen in lipiduria (ie.nephrotic syndrome)
o Identified by:
Lipid stains (TAG and neutral fats)
Polarizing microscope (Cholesterol - “MALTESE CROSS” formation)
Oval Fat Body (Renal tubular fat bodies)
VARIATION OF RTE CELLS:
o RTE cell with non-lipid vacuoles
o Injured cells in which the endoplasmic reticulum has dilated prior to cell death
o Seen in acute tubular necrosis
Bubble cell
Urinary Bladder Cancer Markers (Specific)
o NMP = Nuclear Matrix Protein
o BTA = Bladder Tumor Antigen
MICROSCOPIC QUANTITATIONS:
quantitated: per LPF
0; 0-5; 5-10; 20-100; >100
Epithelial cells
MICROSCOPIC QUANTITATIONS:
quantitated: per HPF
0; 0-2; 2-5; 5-20; >20
Crystals (normal)
MICROSCOPIC QUANTITATIONS:
quantitated: per HPF
0; 0-10; 10-50; 50-200; >200
Bacteria
MICROSCOPIC QUANTITATIONS:
quantitated: per LPF
0; 0-1; 1-3; 3-10; >10
Mucus threads
MICROSCOPIC QUANTITATIONS:
quantitated: per LPF
none: 0
numerical ranges: 0-2, 2-5, 5-10, >10
Casts
MICROSCOPIC QUANTITATIONS:
quantitated: per HPF
Numerical ranges:
0-2, 2-5, 5-10, 10-25, 25-50, 50-100, >100
RBCs & WBCs
MICROSCOPIC QUANTITATIONS:
Rare, few, moderate, or many per LPF
Squamous epithelial cells
MICROSCOPIC QUANTITATIONS:
Rare, few, moderate, or many per HPF
Transitional epithelial cells, yeasts
MICROSCOPIC QUANTITATIONS:
Average number per 10 HPFs
Renal tubular epithelial cells
MICROSCOPIC QUANTITATIONS:
Average number per HPF
Oval fat bodies
MICROSCOPIC QUANTITATIONS:
Average number per LPF
Abnormal crystals, casts
CASTS:
- Excretion is termed __
- Unique to which organ?
- Represents a biopsy of 1 tubule
- The most difficult & the most important urinary sediment constituent
- Primarily formed in the = ___
- Major constituent = __
- Other proteins such as albumin & immunoglobulins are also incorporated into the cast matrix
- __ have the same significance as Casts (a cast with a tail)
- A cast structure should have an even & definite outline, parallel sides and two
__
- Has uniform diameter (about 7-8x the diameter of RBCs)
- Examination is performed along the __ with subdued light
cylindruria
kidney
DCT and collecting duct
Uromodulin/THP (produced by RTE cells)
Cylindroids
rounded ends
coverslip edges
order of cast degeneration?
hyaline> cellular casts> degenerating cellular> coarsely granular> finely granular> waxy
o Prototype cast (beginning of all types of cast)
o Most frequently encountered & the most difficult cast to discover
o Colorless and translucent
o Normal value = 0-2/LPF
o Physiologic Stress = __
o Pathologic = Glomerulonephritis, pyelonephritis, CHF, CKD
HYALINE CAST
strenuous exercise
o Most fragile cast
o Indicates bleeding within the nephron
o Easily identified by its __ color
o Significance = Glomerulonephritis, strenuous exercise
___:
Contains hemoglobin from lysed RBCs
Homogeneous appearance with orange-red color
Same significance as RBC cast
RBC CAST
orange-red
blood cast
o Indicates inflammation or infection within the nephron
o Resembles ___. To distinguish, use phase microscopy and supravital stain.
o Significance = Pyelonephritis, acute interstitial nephritis
o __:
Not a true cast (DO NOT report as cast!)
Clump of leukocytes
Seen in lower UTI
WBC/LEUKOCYTE/PUS CAST
RTE cast
Pseudoleukocyte Cast
o Cells visible on the cast matrix are smaller, round and oval cells
o Significance = Advanced tubular destruction, tubular damage
EPITHELIAL (RTE) CAST
o Identified by performing Gram stain
o Significance = Pyelonephritis
BACTERIAL CAST
o Granules are derived from the lysosomes of RTE cells during normal metabolism (nonpathologic)
o Cells disintegrate when the cast is retained in the __ before being flushed
out
o Finely granular cast has a __ appearance
o Significance = Glomerulonephritis, pyelonephritis, stress, strenuous exercise
GRANULAR CAST (COARSE AND FINE)
tubule
sandpaper
o not stained by Sternheimer-Malbin stain (only the cast matrix is stained)
o Identification:
TAG & neutral fats = __
Cholesterol = __ (“Maltese cross”)
o Significance = Nephrotic syndrome, Toxic tubular necrosis, Diabetes mellitus, crush injuries
FATTY CAST
Lipid stains
Polarizing microscope
o Final degenerative form of all types of casts
o Brittle, highly refractile, with jagged ends
o __ appearance
o Significance = __ of urine flow, chronic renal failure
WAXY CAST
Ground glass
Stasis
o Often referred to as renal failure cast
o Indicates destruction (widening) of the tubular walls
o Any type of cast can be broad (most common are granular & waxy)
o 2-6x wider than ordinary cast
o Significance = Extreme urine stasis, renal failure
BROAD CAST
MISCELLANEOUS CASTS
o ___:
Hyaline matrix with coloration due to pigment incorporation
Incorporated bilirubin (golden-brown)
Hemoglobin or myoglobin (yellow to red brown)
o ___:
Cast containing multiple cell types
Glomerulonephritis (RBC& WBC casts)
Pyelonephritis (WBC & RTE casts or WBC and bacterial casts)
o ___:
Casts containing urates, calcium oxalate and sulfonamides are occasionally seen
Deposition of crystals in the tubule or collecting duct
Pigmented Cast
Mixed Cellular Cast
Crystal Cast
CRYSTALS:
- Excretion is termed __
- The most recognized but the most insignificant in urine sediment
- Formed by precipitation of urine solutes (salts, organic compounds, medication)
- Presence of crystals in fresh urine is most frequently associated w/ concentrated specimen (↑ S.G.)
- Factors that contribute to crystal formation: ____ (3)
- Usually reported as rare, few, moderate or many per HPF
- Abnormal crystals may be averaged and reported per LPF
- Abnormal crystals generally require confirmation before they are reported to the physician
crystalluria
pH, Solute concentration and Temperature
NORMAL ACID CRYSTALS:
Fluffy orange or pink sediment (brick dust) due to uroerythrin
- Yellow brown granules (microscopic)
- Clumps resemble granular casts (termed “__”)
- Turns into uric acid after adding __
- Turns to ammonium biurate after adding ammonium hydroxide
- ↑ in Gout, Chemotherapy
- Soluble in __
Amorphous Urates (Ca2+, Mg2+, Na+
, & K+ urates)
pseudocasts
acetic acid
heat and alkali
NORMAL ACID CRYSTALS:
- Product of purine metabolism
- Most pleomorphic: Rhombic (diamond), 4-sided flat plate (whetstone), lemon-shaped
- Wedges, barrel, rosettes, irregular plates, laminated forms are also seen
- Hexagonal forms may be mistaken as cystine crystals
- Present at pH <5.7 if pH is >5.7, it is in its ionized form as urate
- ↑ in __ syndrome, Chemotherapy Gout
- Soluble in __
Uric Acid
Lesch-Nyhan
alkali
NORMAL ACID CRYSTALS:
- The most frequently observed urinary crystal
- __ = more common; envelope, bipyramidal, octahedron
- __ = oval/dumbbell
- ↑ Foods rich in __ (tomato, asparagus) and ascorbic acid
- Ascorbate when metabolized becomes oxalate and combines with calcium to
form calcium oxalate
- ↑ Ethylene glycol (anti-freeze agent) or methoxyflurane poisoning (MH)
- Soluble in __
Calcium Oxalate (Dihydrate and Monohydrate)
Dihydrate (Weddellite)
Monohydrate (Whewellite)
oxalic acid
dilute HCl
OTHER NORMAL ACID CRYSTALS:
___:
- “Cigarette-butt” appearance
- Soluble in acetic acid
___:
- Yellow-brown/colorless elongated prism
- Soluble in water and ether
___:
- Rare form of uric acid
- Brown spheres or clusters
- Resembles ammonium biurate, leucine & sulfamethoxazole crystals
- Turns to uric acid after adding acetic acid
- Soluble in heat and alkali
___:
- Rare form of uric acid
- Tiny, slender, colorless needles; spherulite or beachball (rare)
- ↑ Acute gout (intracellular crystals) and chronic gout (extracellular crystals)
Calcium Sulfate
Hippuric Acid
Acid Urates (Na+, K+, NH4)
Monosodium or Sodium Urates
NORMAL ALKALINE CRYSTALS:
- Most common cause of turbidity in alkaline urine
- Fine, or ‘lacy’ white precipitate (macroscopic)
- Granular in appearance (microscopic)
- Soluble in ___
Amorphous phosphates (Ca2+ & Mg2+)
dilute acetic acid
NORMAL ALKALINE CRYSTALS:
- Alkaline counterpart of uric acid and amorphous urates
- Yellow-brown thorny apples
- Seen in __
- ↑ Presence of __ bacteria (urea ammonia)
- Turns into uric acid after __
- Soluble in __
Ammonium biurate
old specimens
urea-splitting
adding HCl or acetic acid
acetic acid with heat
NORMAL ALKALINE CRYSTALS:
- Colorless, prism-shaped or coffin-lid; fern-leaf
- Feathery appearance when they disintegrate
- Presence of __ bacteria (urea ammonia)
- Soluble in __
Triple Phosphate (Magnesium Ammonium Phosphate, Struvite)
urea-splitting
dilute acetic acid
NORMAL ALKALINE CRYSTALS:
- Colorless, elongated rectangular or rhomboid plates
- End or corner may be notched
- Edges can be irregular or eroded
- Soluble in __
Magnesium Phosphate
acetic acid
NORMAL ALKALINE CRYSTALS: ____
- __: (stellar phosphate)
o Colorless, flat plates thin prisms in rosette form
o Rosettes may resemble __ crystals
- ___:
o Irregular, granular-appearing sheets or plates
o Less common than dibasic form
- Other forms:
o Hydroxyapatite (basic calcium phosphate)
o Brushite (calcium hydrogen phosphate) - Soluble in ___
Calcium Phosphate (Apatite)
Dibasic calcium phosphate
sulfonamide
Monobasic calcium phosphate
dilute acetic acid
NORMAL ALKALINE CRYSTALS:
- Small, colorless, dumbbell, tetrads or spherical-shaped
- Forms gas (effervescence) after adding __
- Misidentified as __
Calcium Carbonate
acetic acid
bacteria
ABNORMAL ACID CRYSTALS:
- Colorless, refractile hexagonal plates, often laminated
- Mistaken as hexagonal uric acid crystals
- ↑ in __ and __
- soluble in ___ and ___
Cystine
Cystinuria and Cystinosis
ammonia and dilute HCl
ABNORMAL ACID CRYSTALS:
- Rectangular plate with notch in one or more corners (staircase pattern)
- Resembles crystals of radiographic dye
- in Nephrotic syndrome (lipiduria)
- Soluble in __
Cholesterol
chloroform
ABNORMAL ACID CRYSTALS:
- Flat, four-sided plates often with a notched corner
- Other forms include-long thin prisms or rectangles
- Resembles __ crystals. To differentiate:
o Check patient history
o Correlate with other UA results (Ex: S.G. ___ using refractometer)
- Soluble in ___
Radiographic dye (Meglumine diatrizoate, Renografin, Hypaque)
cholesterol
>1.040
10% NaOH
ABNORMAL ACID CRYSTALS:
- Fine colorless to yellow needles in clumps or rosettes
- ↑ in Liver disease (more commonly found than leucine)
- Soluble in __
Tyrosine
alkali or heat
ABNORMAL ACID CRYSTALS:
- Yellow-brown oily-looking spheres w/ concentric circles & radial striations
- Precipitated with tyrosine after adding __
- May resemble fat globules
- ↑ in Liver disease
- Soluble in ___
Leucine
hot alkali or alcohol
alcohol
ABNORMAL ACID CRYSTALS:
- Clumped granules or needles with bright yellow color (Strasinger)
- Reddish brown needles that cluster in clumps or spheres (Turgeon)
- Granules and plates have been observed (Brunzel)
- ↑ in Liver disease
- Soluble in ___ (5)
Bilirubin
acetic acid, HCI, NaOH, acetone and chloroform
ABNORMAL ACID CRYSTALS:
- Fan-shaped needles, sheaves of wheat. rosettes, arrowheads, petals, round-shaped, whetstones
- Mistaken as ___.
To differentiate:
o Calcium phosphate: soluble in acetic acid
o Sulfonamide = (+) __ (2)
- Possible tubular damage (may deposit in nephrons)
- Soluble in ___
Forms:
o __ = brown rosettes or spheres w/ irregular radial
striations
o __ & ___ = yellow-brown sheaves of wheat w/ eccentric binding
Sulfonamide
calcium phosphate crystals
Lignin test & Diazo reaction
acetone
FORMS:
Sulfamethoxazole
Acetylsulfadiazine & Sulfadiazine
ABNORMAL ACID CRYSTALS:
- Colorless needles that tend to form bundles following refrigeration
- ↑ in massive doses of __
Ampicillin
penicillin
OTHER ABNORMAL CRYSTALS:
- May be seen in alkaline urine
- Colorless slender needles
- Strongly birefringent with polarized light
Acyclovir
OTHER ABNORMAL CRYSTALS:
- Coarse, yellow-brown granules
- Resembles amorphous urates
- (+) Rous test (Prussian blue stain)
Hemosiderin
OTHER ABNORMAL CRYSTALS:
- Slender colorless needles or slender rectangular plates
- Feather-like crystals that aggregate into wing-like bundles
- Arranged in fan-shaped or starburst forms, bundles, or sheaves
- Associated with renal blockage & stone formation in HIV-positive individuals
Indinavir Sulfate
URINARY SEDIMENT ARTIFACTS:
___:
o Spheres with dimpled center
o “Maltese cross” formation on polarizing microscope (mnemonic; 4)
- __ (2) = mistaken for RBCs
- __ = spheres with cell wall & concentric circles
- __ = mistaken for casts
- Fecal contamination
Starch granules
maltese cross: OFFS
Oval fat bodies
Fatty casts
Fat droplets
Starch granules
Oil droplets & Air bubbles
Pollen grains
Hair and fibers
URINE SCREENING FOR METABOLIC DISORDERS: (aminoaciduria)
- ↑ Amino acid in blood
- ↑ Amino acid in urine
- Examples: PKU, MSUD, cystinosis
OVERFLOW TYPE
URINE SCREENING FOR METABOLIC DISORDERS: (aminoaciduria)
Normal Amino acid in blood
- ↑ Amino acid in urine
- Due to defective tubular
reabsorption of amino acids
- Examples: Cystinuria, Fanconi’s
syndrome
RENAL TYPE
- Failure to inherit a gene that codes for a particular enzyme.
- No gene = No enzyme
Inborn Error of Metabolism (EM)
PHENYLALANINE -TYROSINE DISORDERS:
- Most well-known of the aminoacidurias
- (-) gene that codes for phenylalanine hydroxylase
- Other forms are due to lack of tetrahydrobiopterin
- ↑ Phenylpyruvic acid (a ketone) in urine
- “Mousy” odor of urine, sweat and breath odor (due to phenylacetic acid)
- May lead to severe mental retardation
- Screening tests:
o FeCl3 tube test = (+) blue-green color
o Phenistix strip = (+) gray to gray-green
o Guthrie bacterial inhibition test
- Confirmatory test: __
PHENYLKETONURIA
Ion exchange HPLC
PHENYLALANINE -TYROSINE DISORDERS:
- (-) gene that codes for:
o Type 1: Fumarylacetoacetate hydrolase (FAH)
o Type 2: Tyrosine aminotransferase
o Type 3: p-hydroxyphenylpyruvic acid dioxygenase
- May also be seen in severe liver disease
- “Rancid butter” urine odor
- Screening Tests
o FeCl3 tube test = (+) transient green
o Nitroso-naphthol = (+) orange-red
- Confirmatory Tests
o Chromatography
o Quantitative serum assay of tyrosine
TYROSYLURIA/TYROSINEMIA
PHENYLALANINE -TYROSINE DISORDERS:
(-) gene that codes for Homogentisic acid oxidase
- ↑ Homogentisic acid in blood and urine
- Urine darkens after becoming alkaline from standing at room temperature
- Brown - or black-stained cloth diapers
- Reddish-stained disposable (plastic) diapers
- Homogentisic acid causes black pigmentation in the connective tissues and ears (ochronosis)
- Screening Tests
o FeCl3 tube test = (+) transient blue
o Clinitest = (+) yellow precipitate
o Alkalinization of fresh urine
- Confirmatory Tests
o Paper/thin-layer chromatography
o Capillary electrophoresis
ALKAPTONURIA
PHENYLALANINE -TYROSINE DISORDERS:
Caused by melanoma (tumor involving melanocytes)
- Tumors secrete 5,6-dihydroxyindole, which oxidizes to melanogen then to melanin
- Urine darkens upon air exposure
- Deficient production of melanin results in albinism
- Screening Tests
o FeCl3 tube test = (+) Gray/black ppt
o Sodium nitroprusside test = (+) Red
o Ehrlich test = (+) Red
MELANURIA
BRANCHED-CHAIN AMINO ACID DISORDERS:
- Most common IEM in the Philippines
- (-) Gene that codes for the enzyme complex known as branched-chain α-keto acid dehydrogenase (BCKD)
- ↑ Ketoacids of Leucine, Isoleucine and Valine
- “Caramelized sugar/Maple syrup/Curry” urine odor
- Presence of ketonuria in a newborn is significant
- Screening Test
o 2,4-dinitrophenylhydrazine (DNPH) = (+) Yellow turbidity/precipitate
- Confirmatory Test
o Gas or thin-layer chromatography
o Nuclear magnetic resonance spectro
MAPLE SYRUP URINE DISEASE (MSUD)
BRANCHED-CHAIN AMINO ACID DISORDERS:
- Isovaleric acidemia = “__” urine odor due to isovalerylglycine
o Glutaric acidemia also presents with this urine smell
- Propionic acidemia
- Methylmalonic acidemia = detected using p-nitroaniline test = (+) Emerald green color
ORGANIC ACIDEMIAS
sweaty feet
TRYPTOPHAN DISORDERS:
- Indigo blue urine color (upon air exposure)
- Seen in: ___ (“Blue diaper syndrome”) and Intestinal disorders
- Screening test: Obermayer’s test
o FeCl3 + Urine + Chloroform = (+) Violet color
INDICANURIA
Hartnup disease
TRYPTOPHAN DISORDERS:
- Tumor of argentaffin or enterochromaffin cells - produce serotonin (carried by platelets) → metabolized into 5-HIAA
- Screening tests
o FeCl3 tube test = (+) Blue-green
o Nitrosonaphthol with nitrous acid = (+) Violet
- Patient must not eat bananas, pineapples, tomatoes, avocados, chocolates, walnuts, & plums (they ↑ serotonin)
ARGENTAFFINOMA
- “Sulfur” urine odor
- Treatment = D-Penicillamine
CYSTINE DISORDERS:
CYSTINE DISORDERS:
- Renal type of aminoaciduria
- Defective tubular reabsorption of: (mnemonic)?
- Tests for Cystinuria and Cystinosis
o Brand’s modification of Legal’s nitroprusside
Reagent = Cyanide nitroprusside
(+) Red-purple color
o Thin layer or ion-exchange chromatography
o High-voltage electrophoresis
CYSTINURIA
COLA:
o Cystine (the only one which crystallizes; least soluble)
o Ornithine
o Lysine
o Arginine
CYSTINE DISORDERS:
- Inborn error of metabolism → Overflow type
- (-) gene that codes for an enzyme responsible for cystine metabolism
- Types = Nephropathic cystinosis, intermediate cystinosis, and ocular cystinosis
- Cystine deposits in many areas of the body (BM, cornea, lymph nodes & internal
organs)
CYSTINOSIS
CYSTINE DISORDERS:
- Defects in the metabolism of methionine (leads to ↑ __)
- (-) gene that codes for the enzyme cystathione β-synthase
- Detected by the Silver-nitroprusside test = (+) Red-purple color
HOMOCYSTINURIA
homocystine
Disorders of porphyrin metabolism
- Urine color: red, purple, burgundy-red, purplish red, “portwine”
o Colorless in = lead poisoning (Harr, Henry)
Consider it porphyria if:
- Red-tinged urine
- Negative for blood reagent strip
- Diet and medications ruled out
PORPHYRIN DISORDERS (PORPHYRIAS)
Dark Red or Purple,
Intensely Fluorescent
- Uroporphyrin
- Coproporphyrin
- Protoporphyrin
CDC-recommended test for lead poisoning
Free erythrocyte protoporphyrin
(FEP)
ENZYME DEF: Uroporphyrinogen synthase
ELEVATED COMP: ALA Porphobilinogen
SYMPTOMS: Neurologic Psychiatric
Acute intermittent porphyria
PORPHYRIN DISORDERS:
ENZYME DEF: Uroporphyrinogen decarboxylase
ELEVATED COMP: Uroporphyrin
SYMPTOMS: Photosensitivity
Porphyria cutanea tarda
PORPHYRIN DISORDERS:
ENZYME DEF: Uroporphyrinogen cosynthase
ELEVATED COMP: Uroporphyrin Coproporphyrin
SYMPTOMS: Photosensitivity
Congenital erythropoietic porphyria
PORPHYRIN DISORDERS:
ENZYME DEF: Protoporphyrinogen oxidase
ELEVATED COMP: Coproporphyrin
SYMPTOMS: Photosensitivity, Neurologic
Variegate porphyria
PORPHYRIN DISORDERS:
ENZYME DEF: Ferrocheletase
ELEVATED COMP: Protoporphyrin
SYMPTOMS: Photosensitivity
Erythropoietic protoporphyria
PORPHYRIN DISORDERS:
ENZYME DEF: –
ELEVATED COMP: Protoporphyrin
SYMPTOMS: Photosensitivity
Lead poisoning
Port wine urine color is more prevalent in the ___ porphyrias
- Lead poIsoning inhibits __ and __
erythropoietic
ALA Synthetase and ferrocheletase enzymes
MUCOPOLYSACCHARIDE [MPS] DISORDERS (MUCOPOLYSACCHARIDOSIS):
o A.k.a. Gargoylism or MPS Type I
o MPS accumulate in the cornea of the eye
o (+) Skeletal abnormalities & mental retardation
HURLER SYNDROME
MUCOPOLYSACCHARIDE [MPS] DISORDERS (MUCOPOLYSACCHARIDOSIS):
o Sex-linked recessive, rarely seen in females
o (+) Skeletal abnormalities & mental retardation
HUNTER SYNDROME
MUCOPOLYSACCHARIDE [MPS] DISORDERS (MUCOPOLYSACCHARIDOSIS):
o Mental retardation is the only abnormality
SANFILIPPO SYNDROME
Impaired metabolism of mucopolysaccharides or glycosaminoglycans (protein + polysaccharides, located in the connective tissues)
- Frequently found in urine are dermatan sulfate, keratan sulfate and heparan sulfate
mucopolysaccharide disorders
SCREENING TESTS
- Acid albumin test = (+) White turbidity
- Cetyltrimethylammoniumbromide (CTAB) Test = (+) White turbidity
- Mucopolysaccharide (MPS) Paper Test = (+) Blue color
for mucopolysaccharide disorders
PURINE DISORDER:
- (-) gene that codes for the enzyme hypoxanthine guanine
phosphoribosyltransferase
- ↑ Uric acid in the blood and urine
- “___” in diapers
LESCH-NYHAN DISEASE
Orange sand
detected by: Glucose strip and (+) Copper reduction test
___ = presence of any sugar in urine
CARBOHYDRATE DISORDERS
Melituria
CARBOHYDRATE DISORDER:
o Inability to metabolize galactose to glucose
o Enzymes absent:
Galactose-1-phosphate uridyl transferase (GALT)
Galactokinase
UDP-galactose-4-epimerase
o ↑ Galactitol, galactonate and galactose-1-phosphate
o Associated with infant failure to thrive, liver disorders, cataracts and severe mental retardation
Galactosemia/Galactosuria
CARBOHYDRATE DISORDER:
__ = Diabetes Mellitus
__= seen during pregnancy and lactation
Glucosuria
Lactosuria
CARBOHYDRATE DISORDER:
__ = associated with parenteral feeding
__ = associated with ingestion of large amounts of fruit
Fructosuria
Pentosuria
GLOMERULAR DISORDERS:
Deposition of immune complex,
formed in conjunction of Group A
Streptococcus (S. pyogenes) infection
on the glomerular membranes
Macroscopic hematuria,
proteinuria, dysmorphic
RBCs, RBC casts,
granular casts,
- (+) ASO titer
Acute Post-Streptococcal
Glomerulonephritis
GLOMERULAR DISORDERS:
- Deposition of immune complexes from
systemic immune disorders (ex: SLE)
on the glomerular membrane
- Cellular proliferation of epithelial cells
inside the Bowman’s capsule form
“crescents”
- Macroscopic hematuria
- Proteinuria
- RBC casts
Rapidly Progressive (Crescentic)
Glomerulonephritis
GLOMERULAR DISORDERS:
- Deposition of antiglomerular basement
membrane antibody to glomerular and
alveolar basement
- Macroscopic hematuria
- Proteinuria
- RBC casts
Goodpasture Syndrome
GLOMERULAR DISORDERS:
- Anti-neutrophilic cytoplasmic auto-
antibody (ANCA) binds to neutrophils in vascular walls producing damage to small vessels in the lungs and glomerulus
- Perinuclear ANCA (p-ANCA) forms
when neutrophils are fixed in ethanol
- Cytoplasmic ANCA (c-ANCA) forms
when neutrophils are fixed with
formalin
- Macroscopic hematuria
- Proteinuria
- RBC casts
Wegener’s Granulomatosis
GLOMERULAR DISORDERS:
- Occurs in children following viral
respiratory infections
- Decrease in platelets disrupts vascular
integrity
- Macroscopic hematuria
- Proteinuria
- RBC casts
Henoch Schonlein Purpura
GLOMERULAR DISORDERS:
- Thickening of glomerular membrane
following IgG immune complex
deposition associated with systemic
disorders
- Microscopic hematuria
- Proteinuria
Membranous Glomerulonephritis (MGN)
GLOMERULAR DISORDERS:
- Cellular proliferation affecting the
capillary walls or the glomerular
basement membrane, possibly
immune-mediated
- Glomeruli have visible lobular
appearance
- “Tram track”
- Hematuria
- Proteinuria
Membranoproliferative
Glomerulonephritis (MPGN)
GLOMERULAR DISORDERS:
- Marked decrease in renal function
resulting from glomerular damage
precipitated by other renal disorders
- Progression to renal failure
- Hematuria
- Proteinuria, Glucosuria
- Cellular & granular casts
- Waxy and broad casts
Chronic Glomerulonephritis
GLOMERULAR DISORDERS:
- Deposition of lgA on the glomerular
membrane resulting from increased
levels of IgA
- Early stages: Hematuria
- Late stages: See chronic
GN
IgA Nephropathy (Berger’s Disease)
GLOMERULAR DISORDERS:
- Little cellular changes in the
glomerulus
- Glomeruli look normal by light
microscopy
- Electron microscopy reveals loss of
podocyte foot processes
- Disruption of podocytes primarily in
children following allergic reactions &
immunizations
- Associated with HLA-B12 antigen
- Heavy proteinuria
- Transient hematuria
- Fat droplets
Minimal Change Disease, MCD
(Nil Disease/Lipoid Nephrosis)
GLOMERULAR DISORDERS:
- Disruption of podocytes in certain
numbers and areas of glomeruli,
others remain normal - IgM and C3 are evident on the sclerotic
areas (using IF) - Proteinuria
- Hematuria
Focal Segmental Glomerulosclerosis
(FSGS)
GLOMERULAR DISORDERS:
- Most common cause of ESRD
- Deposition of glycosylated proteins on
the glomerular basement membranes
caused by poorly controlled blood
glucose levels
- Microalbuminuria
- Micral test
Diabetic Nephropathy (Kimmelstiel-
Wilson Disease)
GLOMERULAR DISORDERS:
- Genetic disorder showing lamellated
and thinning of glomerular basement
membrane
- See Nephrotic Syndrome
Alport Syndrome
GLOMERULAR DISORDERS:
- Disruption of the electrical charges that produce the tightly fitting
podocyte barrier resulting in massive loss of proteins & lipids
- The rate of proteinuria in nephrotic syndrome is >3.5 g/day
- Occurs in patients with MCD (in children), MGN (in adults), FSGS and MPGN
Nephrotic Syndrome
TUBULAR DISORDERS:
- Damage to renal tubular cells caused by
ischemia or toxic agents
- Urine odor = “odorless”
- Microscopic hematuria, proteinuria
- RTE cells, RTE casts
- Hyaline, granular, waxy
and broad casts
Acute Tubular Necrosis
TUBULAR DISORDERS:
- Inherited defect in the production of
normal uromodulin by the renal tubules and increased uric acid causing gout
- Normal uromodulin is replaced by
abnormal forms that destroy the RTE cells
- RTE cells
- Hyperuricemia
Uromodulin-associated Kidney Disease
(UKD)
TUBULAR DISORDERS:
- Generalized failure of tubular reabsorption
in the proximal convoluted tubule
- Glucosuria
- Possible cystine
crystals (amino acid)
Fanconi Syndrome
TUBULAR DISORDERS:
- Neurogenic DI = hypothalamus fails to
produce ADH
- Nephrogenic DI = renal tubules fail to
respond to ADH
- Low specific gravity
- Polyuria (>15 L/day)
Diabetes Insipidus
TUBULAR DISORDERS:
- (N) Blood glucose = ↑ Urine glucose
- Defective tubular reabsorption of glucose
- Glucosuria
Renal Glucosuria
INTERSTITIAL DISORDERS:
- Ascending bacterial infection of
the urinary bladder
- Acute onset of urinary
frequency and burning
- WBCs, Bacteria, NO CAST
- Microscopic hematuria
- Mild proteinuria, increased pH
Cystitis (Lower UTI)
INTERSTITIAL DISORDERS:
- Infection of the renal tubules &
interstitium related to interference of urine flow to the bladder, reflux of urine from the bladder (vesicoureteral reflux)
& untreated cystitis
- WBCs, Bacteria
- WBC casts, bacterial casts,
- Microscopic hematuria
Acute Pyelonephritis (Upper UTI)
INTERSTITIAL DISORDERS:
- Recurrent infection of the renal tubules & interstitium caused by structural abnormalities affecting the flow of urine
- WBCs, Bacteria, WBC casts, Bacterial casts, granular casts
- Waxy and broad casts
- Hematuria, proteinuria
Chronic Pyelonephritis
INTERSTITIAL DISORDERS:
- Allergic inflammation of the
renal interstitium in response to
certain medications
- Hematuria, proteinuria
- WBCs (↑ eosinophils, >1%)
- WBC casts, NO BACTERIA
Acute Interstitial Nephritis
↓ Glomerular filtration rate (< 25 mL/min)
- Azotemia (↑ BUN & Creatinine)
- Electrolyte imbalance
- (-) renal concentrating ability → isosthenuria
- Proteinuria & renal glycosuria
- Telescoped sediment
o Variety of casts seen
o Simultaneous appearance of the elements of acute, chronic GN, and nephrotic syndrome
o ↑ casts (granular, waxy, broad)
RENAL FAILURE
- May form in the calyces and pelvis of the kidney, ureters, and bladder
- Lithotripsy uses high-energy shock waves to break kidney stones into pieces
- Conditions Favoring the Formation of Renal Calculi:
o pH
o Chemical concentration
o Urinary stasis - Primary UA Finding = __ hematuria
RENAL CALCULI/RENAL LITHIASIS
Microscopic
Renal Calculi:
- Major constituent of renal calculi
- Very hard, dark in color with rough surface
Calcium oxalate calculi
Renal Calculi:
- Associated w/ increased intake of foods w/ high purine content, and w/ UKD
- Yellowish to brownish red & moderately hard
Uric acid & Urate calculi
Renal Calculi:
- Seen in hereditary disorders of ___ metabolism
- Yellow-brown, greasy & resembles an old soap
- Least common calculi
Cystine calculi
Renal Calculi:
Pale & friable
Phosphate calculi
Renal Calculi:
- Accompanied by urinary infections involving urea-splitting bacteria (Proteus vulgaris)
- Branching/staghorn calculi resembling antlers of a deer
Triple phosphate
calculi
Rare Calculi:
- Sulfonamide calculi
- __ - ingestion of silica over a long period of time
- ___ = insoluble diuretic; mustard-colored stones
- __ = associated with inherited enzyme deficiency & hyperuricemia
- __ = associated with a genetic disorder w/ an absence of xanthine
oxidase
Silica calculi
Triamterene calculi
Adenine calculi
Xanthine calculi
Produced by the syncytiotrophoblast cells of the placenta
- Peaks during 1st trimester of pregnancy (↑ blood, urine, amniotic fluid)
- Composed of 2 subunits:
o Alpha = hCG, LH, FSH, TSH (identical subunits)
o Beta = Confers specificity for hCG
HUMAN CHORIONIC GONADOTROPIN (hCG)
Principle: Enzyme-immunoassay
- Specimen: __
- Cut-off point: 25 mIU/mL
- Anti-hCG source: __
HOME-BASED HCG PREGNANCY TEST KIT
1st morning urine
rabbit
RESULT IN PREGNANCY TEST:
- Molar pregnancy, Midcycle LH surge
- Hematuria or proteinuria
- Malignancies (gynecologic & other)
- Postpartum & post-abortion (up to
4 weeks)
- Chinese herbal medications
- Perimenopausal (LH elevation)
- Premature ovarian failure (LH elevation)
false positive
RESULT IN PREGNANCY TEST:
- Too early (commonly) or too late
(rarely) testing
- Dilute urine (low S.G)
- Adulterated urine
- Ectopic pregnancy (rarely negative,
almost always positive)
- Impending or missed abortion
(rarely negative, almost always
positive)
false negative
HCG BIOASSAYS:
Animal Used: Female frog
Mode of Injection: Lymph sac (urine)
Positive Result: Oogenesis
Hogben
HCG BIOASSAYS:
Animal Used: Male frog
Mode of Injection: Subcutaneous (urine/serum)
Positive Result: Spermatogenesis
Galli-Mainini
HCG BIOASSAYS:
Animal Used: Virgin female rabbit
Mode of Injection: Marginal ear vein
(urine)
Positive Result: Corpora lutea & corpora hemorrhagica
Friedmann/ Hoffman
HCG BIOASSAYS:
Animal Used: Immature female mice
Mode of Injection: Subcutaneous (urine)
Positive Result: Formation of hemorrhagic follicles & corpora lutea
Ascheim-Zondek
HCG BIOASSAYS:
Animal Used: Immature female rats
Mode of Injection: Subcutaneous (urine)
Positive Result: Ovarian hyperemia
Frank-Berman
HCG BIOASSAYS:
Animal Used: Female virgin rat
Mode of Injection: Intraperitoneal (urine)
Positive Result: Ovarian hyperemia
Kupperman
HCG BIOASSAYS:
Animal Used: Female virgin rat
Mode of Injection: Subcutaneous (urine)
Positive Result: Ovarian hyperemia
Kelso
HCG NOTES:
ELISA tests are very sensitive, giving positive reactions as early as __
after conception
- Urine specimen for pregnancy testing should have a specific gravity of at least
__
10 days
1.015
- Present in the amnion – a membranous sac that surrounds the fetus
- The __ is the ultimate source of amniotic fluid water and solutes
AMNIOTIC FLUID
placenta
PRIMARY FUNCTIONS OF AMNIOTIC FLUID (4)
- Cushion for the fetus
- Stabilizes temperature
- Allows fetal movement
- Proper lung development
AMNIOTIC FLUID VOLUME:
- From fetal urine and lung fluid
- Normal = ____ (3rd trimester)
- During __ trimester, __ of amniotic fluid is derived primarily from the maternal circulation
- __ = major contributor to the AF volume after the 1st trimester of pregnancy
800-1,200 mL
1st; 35 mL
Fetal urine
↑ amniotic fluid volume (>1200 mL)
Causes:
- Decreased fetal swallowing of urine
- Neural tube defects (ex. Spina bifida)
- Others: fetal structural anomalies,
cardiac arrhythmias, congenital infections, chromosomal abnormalities
POLYHYDRAMNIOS
↓ amniotic fluid volume (<800 mL)
Causes:
- Increased fetal swallowing of urine
- Membrane leakage
- Urinary tract deformities
- Others: congenital malformations, premature amniotic membrane rupture, umbilical cord compression
OLIGOHYDRAMNIOS
Method of collection for amniotic fluid = __
o Up to 30 mL collected in sterile syringe
o __ amniocentesis = Assess genetic defects (Ex: ___/Down syndrome)
o __ amniocentesis = Fetal lung maturity (FLM), Fetal hemolytic disease (HDN)
Amniocentesis
2nd trimester; Trisomy 21
3rd trimester
Quadruple screening tests prior to performing amniocentesis?
o Alpha-fetoprotein
o Human chorionic gonadotropin (hCG)
o Unconjugated estriol (UE3)
o Inhibin A
SPECIMEN HANDLING:
Test for __ = Placed on ice on delivery, kept refrigerated or frozen, filtration prevents loss of phospholipids
- Test for __ = Kept at room temperature or at 37oC
- Test for __ = Protected from light
Fetal Lung Maturity
Cytogenetic Studies
HDN
AMNIOTIC FLUID VS. MATERNAL URINE:
protein and glucose (less reliable): __
urea and creatinine in mg/dL (reliable): __
AMN: (+); URN: (-)
AMN: urea - <30mg/dL; crea - <3.5mg/dL
URN: urea - >300mg/dL; crea >10 mg/dL
- Detects ruptured amniotic membranes
- Also used to diagnose early pregnancy (↑ estrogen)
- Procedure: Specimen (Vaginal Fluid) → Slide (Air Dry) → (+) Fern-like crystals = AMNIOTIC FLUID (Due to presence of sodium chloride and proteins)
FERN TEST
AMNIOTIC FLUID COLOR:
Normal
Colorless
AMNIOTIC FLUID COLOR:
Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Blood-streaked
AMNIOTIC FLUID COLOR:
HDN (Bilirubin)
Yellow
AMNIOTIC FLUID COLOR:
Meconium = 1st fetal bowel movement (sign of distress)
Dark-green
AMNIOTIC FLUID COLOR:
Fetal death
Dark red-brown
o Most frequent complication of early delivery
o __ most common cause of morbidity and mortality in the premature infant
o Caused by insufficiency of ___ (phospholipids) production & fetal lung immaturity
- It normally appears in mature lungs and allows the alveoli to remain open throughout the cycle of inhalation and exhalation
- It keeps the __ from collapsing by decreasing surface tension and allows them to inflate with air more easily
Respiratory distress syndrome
7th
lung surfactant
alveoli
TESTS FOR FLM:
- Reference method for FLM
- __ = for alveolar stability
- __ = serves as a control (due to constant
production)
o ratio is measured using thin layer
chromatography (TLC)
- Ratio of __ = mature fetal lungs
o Cannot be done on a specimen contaminated by __ or __ (falsely increases the ratio
Lecithin/Sphingomyelin ratio
Lecithin
Sphingomyelin
>2.0
blood or meconium
TESTS FOR FLM:
Immunologic test for phosphatidylglycerol (PG)
o Not affected by blood or meconium
o Production of PG is delayed among diabetic mothers
Amniostat-FLM
TESTS FOR FLM:
- Amniotic fluid + 95% Ethanol → Shake for 15 secs → Stand for 15 mins
- (+) Foam/Bubbles = MATURE FETAL LUNGS (Presence of phospholipids)
Foam stability (Foam/Shake test)
TESTS FOR FLM:
- The presence of phospholipids decreases
__
o __ is the friction experienced by a single particle undergoing diffusion because of its interaction with its environment at the micrometer length scale
- Measured by fluorescence polarization
o Surfactant to albumin (S/A) ratio is measured
o Dye bound to surfactant had longer fluorescence & low polarization
o Dye bound to albumin had decreased fluorescence & high polarization
Microviscosity (Obsolete test)
TESTS FOR FLM:
- Type ll pneumocytes produce alveolar surfactants stored in the form of lamellar bodies
o Lamellar body diameter is similar to platelets, therefore, LBC can be obtained with the use of platelet channel of hematology analyzers
o LBC can be done using impedance and/or optical scatter methods
o >32,000/uL lamellar body count = ADEQUATE FLM
Lamellar body count (LBC)
TESTS FOR FLM:
- ↑ Lamellar bodies = ↑ O.D. (Absorbance)
- O.D. of >0.150 is equivalent to:
o L/S ratio of > 2.0
o The presence of PG
OD 650 nm
TEST FOR FETAL AGE:
___ amniotic fluid creatinine = prior to 36 weeks’ gestation
__ amniotic fluid creatinine = 36 weeks (9 months)
1.5 to 2.0 mg/dL
>2.0 mg/dL
TEST FOR HDN:
A.k.a __ (Absorbance) 450
- Absorbance of amniotic fluid:
o __ = ↑ at 365nm, ↓ at 550nm
o __ = ↑ at 450 nm (bilirubin)
- Results are plotted on a __ graph:
o __ = Non-affected or mildly affected fetus
o __ = Moderately affected fetus (requires close monitoring)
o __ = Severely affected fetus (requires
intervention)
- Interferences= cells, meconium, debris, and hemoglobin (peak absorbance at 410nm)
- The oldest routinely performed lab test on AF evaluates the severity of fetal anemia due to HDN
Optical Density
Normal
HDN
Liley:
Zone I
Zone II
Zone III
TEST FOR NEURAL TUBE DEFECTS (NTD):
__ (“split spine”) is a birth defect where there is incomplete closing of the backbone & membranes around the spinal cord.
- __ is the absence of a major portion of the brain, skull, and scalp that
occurs during embryonic development
- Screening test = __
o __ in Neural tube defects
o __ in Down syndrome
- Confirmatory test = __
- AFP is the major protein produced by the fetal liver during early gestation (prior to
18 weeks)
Spina bifida
Anencephaly
Alpha-fetoprotein (AFP)
increased
decreased
Acetylcholinesterase
TESTS FOR FETAL WELL-BEING AND MATURITY:
- Bilirubin scan ∆ A450 >.025 ____
- Alpha-fetoprotein <2.0 Multiples of
Median (MoM) ___
- L/S ratio >2.0 ____
Hemolytic disease of the newborn
Neural tube disorders
Fetal lung maturity
- Not a sterile body fluid
- From upper & lower (sterile) respiratory tract
- Tracheobronchial secretions (mixture of plasma, electrolytes, mucin & water)
- Added with cellular exfoliations, nasal and salivary gland secretions and normal
oral flora - __ water and 5% solids
- Secretions are viscoelastic - some of the properties of a liquid and some of a solid
- ___ - most important single component of sputum viscosity
- Acceptable sputum specimen = ___
SPUTUM
95%
Sialic acid
<10 S.E.C./LPF and >25 WBC/LPF
SPECIMEN COLLECTION:
__ = most preferred sample (routine; conc)
- __ sputum = for volume measurement
- ___ = for pediatric patients
- __ = for non-cooperative patients
- __ = for debilitated or unconscious patients
- Specimen preservation methods = __ or __
1st morning
24-hour
Throat swab
Sputum induction
Tracheal aspiration
Refrigeration or 10% formalin
MACROSCOPIC EXAMINATION: (VOL)
Bronchial asthma, acute bronchitis, early pneumonia, stage of healing
Bronchiectasis, lung abscess, edema, gangrene, tuberculosis, pulmonary hemorrhage
decreased
increased
MACROSCOPIC EXAMINATION: (COLOR)
Made up of mucus only
↑ Pus (TB, bronchitis, jaundice, pneumonia)
↑ Pus & epithelial cells
↑ Bile; P. aeruginosa infection, lung abscess
Fresh blood or hemorrhage, TB,
bronchiectasis
Old blood, pneumonia, gangrene
Pneumonia, chronic lung cancer
Cancer
Dust or dirt, carbon, charcoal,
anthracosis, smoking
Lobar pneumonia (S. pneumoniae)
Congestive heart failure
Klebsiella pneumoniae infection
Colorless or translucent
White or yellow
Gray
Bright green or greenish
Red or bright red
Anchovy sauce or rusty brown
Prune juice
Olive green or grass green
Black
Rusty (with pus)
Rusty (without pus)
Currant, jelly-like
MACROSCOPIC EXAMINATION: (ODOR)
- Normal
- Lung gangrene, advanced necrotizing
tumors
- Bronchiectasis, tuberculosis
- Necrosis, tumors, empyema
- Liver abscess, enteric Gram-negative, bacterial infection
Odorless
Foul or putrid
Sweetish
Cheesy
Fecal
MACROSCOPIC EXAMINATION: (CONSISTENCY)
- Asthma, bronchitis
- Lung edema
- Bronchiectasis, tuberculosis with cavities
Mucoid
Serous or frothy
Mucopurulent
MACROSCOPIC STRUCTURES:
Yellow or gray material, size of a pinhead
Produces foul odor when crushed
CS: Bronchitis, bronchiectasis
Bronchial asthma
Dittrich’s plugs
MACROSCOPIC STRUCTURES:
Hard concretions in a bronchus (lung stones)
Yellow/white calcified TB structures/foreign material
CS: Histoplasmosis (most common)
Chronic tuberculosis
Pneumoliths or Broncholiths
MACROSCOPIC STRUCTURES:
Branching tree-like casts of the bronchi
CS: Lobar pneumonia,
bronchitis, diphtheria
Bronchial casts
MACROSCOPIC STRUCTURES: (sputum)
1st (top) = frothy mucus
2nd (middle) = opaque, water
material
3rd (bottom) = pus, bacteria,
tissues
CS: Bronchiectasis, lung abscess, gangrene
Layer formation (3 layers)
MACROSCOPIC STRUCTURES: (sputum)
Bronchial calculi (calcium carbonate & phosphate)
Asbestos bodies, silica particles
CS: Pneumoconiosis
Foreign bodies
MICROSCOPIC STRUCTURES: (sputum)
Slender fibrils w/ double contour &
curled ends
SC: Tuberculosis
Elastic fibers
MICROSCOPIC STRUCTURES: (sputum)
Colorless, hexagonal, double pyramid,
often needle-like; arise from disintegration of eosinophils
SC: Bronchial asthma (3C’s)
Charcot-Leyden crystals
MICROSCOPIC STRUCTURES: (sputum)
Heart failure cells: hemosiderin-laden
macrophage
Carbon-laden cells: angular black
granules
CS: Congestive heart failure, heavy smokers
Pigmented
cells
MICROSCOPIC STRUCTURES: (sputum)
Coiled mucus strands
Can also be observed macroscopically
CS: Bronchial asthma (3
C’s)
Curschmann’s spirals
MICROSCOPIC STRUCTURES: (sputum)
Colorless globules occurring in a variety
of sizes and bizarre forms
SC: No significance; Mistaken as Blastomyces
Myelin globules
MICROSCOPIC STRUCTURES: (sputum)
Clusters of columnar epithelial cells
SC: Bronchial asthma (3 C’s)
Creola bodies
MICROSCOPIC STRUCTURES: (sputum)
Migrating larva: Ascaris, Strongyloides, Hookworm (heart-to-lung migration)
E. histolytica, E. gingivalis, T. tenax, P. westermani, E. granulosus, T. canis
Parasites
- A procedure for collecting the cellular milieu of the alveoli by use of a bronchoscope through which saline is instilled into distal bronchi and then withdrawn
- Important diagnostic test for Pneumocystis carinii (P. jirovecii) in
immunocompromised patients - ___ stain best delineates the cysts of Pneumocystis jirovecii
BRONCHOALVEOLAR LAVAGE (BAL)
Grocott’s methenamine silver
CELLS SEEN IN BRONCHOALVEOLAR LAVAGE:
__ Alveolar macrophages Most predominant
__ Lymphocytes Interstitial disease, pulmonary lymphoma,
nonbacterial infections
__ Neutrophils Cigarette Smokers, bronchopneumonia, toxin exposure
__ Eosinophils Hypersensitivity reactions
__ Ciliated columnar bronchial epithelial cells
56-80%
1-15%
<3%
<1-2%
4-17%
- Used to diagnose Cystic fibrosis (Mucoviscidosis)
o __ metabolic disorder affecting the mucous secreting glands of the body
o Associated with pancreatic insufficiency, respiratory distress & intestinal obstruction
o ↑ Na+ & Cl- due to inability of the sweat glands to __ them before the sweat is secreted
SWEAT TEST
Autosomal recessive
reabsorb
- Pilocarpine + mild current = induce sweat production
- Application of 0.16 mA current for 5 minutes
Sweat Na+ and Cl- values:
- __ mEq/L= Diagnostic for CF
- __ mEq/L= Borderline for CF (Repeat testing)
GIBSON AND COOKE PILOCARPINE IONTOPHORESIS
> 70
40
Sweat is tested for sodium and chloride
- __ = Flame photometry, Ion exchange electrode
- __ = Manual or automated titration
Na+
Cl-
3rd major body fluid
- Functions:
o Supply nutrients to the nervous system
o Remove metabolic waste
o Produce a mechanical barrier to cushion the brain & Spinal cord against
trauma
CEREBROSPINAL FLUID
MENINGES: Line the brain and spinal cord
- 3 layers:
o __ (Outer layer) = Lines the skull & vertebral canal
o __ (Spiderweb-like) = Filamentous inner membrane
__ (Below arachnoid) = Portion where CSF flows
o __ (Innermost layer) = Lines the surface of brain & spinal cord
Dura mater
Arachnoid mater
Subarachnoid space
Pia mater
__ = produces CSF by selective filtration (at a rate of 20 mL/hour)
- __ = reabsorbs CSF
- ___:
o Protects brain from chemicals & other substances circulating in the blood that
can harm the brain tissue
o Disruption of this allows WBCs, proteins & other chemicals to enter the CSF (Ex: Meningitis, Multiple sclerosis)
CHOROID PLEXUS
ARACHNOID VILLI/GRANULATIONS
BLOOD BRAIN BARRIER (BBB)
CSF COLLECTION AND HANDLING:
Up to __ CSF can be collected using a __ attached to a spinal needle
o Only if CSF pressure is __ (50-180 mmHg)
o If CSF pressure is high or low, only ___ can be removed
- Method of collection = __ (between L3-L4 [adults] or L4-L5 [infants])
20mL; manometer
normal
1-2 mL
Lumbar puncture
3 CSF TUBES:
Chemistry/Serology; Freezing temperature
tube 1
Microbiology; Room temperature for 30 mins (CM books)
35-37oC (Microbiology books)
tube 2
Hematology; Refrigeration temperature
Microbiology or Serology
Tube 3
(Tube 4)
__ = least affected by blood or bacteria introduced by spinal tap
- __ = usually designated for microbiology
- __ = least likely contain cells introduced by the spinal tap
- __ = better exclusion of skin contamination or for additional tests
- __ may be performed on Tubes 1 & 4 to check for cellular contamination by puncture
- Left-over supernatant fluid may also be used for additional chemical or serologic
tests
- Excess fluid should not be discarded and should be frozen until there is no further
use for it
- If 1 CSF tube only: Microbiology first, then Hema, then Chem/Serology
Tube 1
Tube 2
Tube 3
Tube 4
Cell counts
CSF TOTAL VOLUME:
Adults = __
Neonates = __
90-150mL
10-60 mL
CSF APPEARANCE:
Normal
Crystal clear
CSF APPEARANCE:
WBCs (>200/uL, RBCs (> 400/uL), lipids & protein,
microorganisms
Hazy/ Turbid/ Milky/Cloudy
CSF APPEARANCE:
Due to hemoglobin degradation products (most common)
__ = Slight amount of oxyhemoglobin
__ = Oxyhemoglobin → Bilirubin
__ = Heavy hemolysis
Other causes: ↑ Carotene, ↑ Melanin, ↑ Protein (>150mg/dL), Rifampin
Xanthochromic
Pink
Yellow
Orange
CSF APPEARANCE:
↑ RBCs (>6,000/uL)
Traumatic tap (puncture of blood vessel)
Intracranial hemorrhage (bleeding within the braincase)
Bloody
CSF Appearance:
__ - Radiographic contrast media
__ - Protein & clotting factors; meningitis, Froin syndrome, blockage of CSF circulation
__ - Tubercular meningitis
Oily
Clotted
Pellicle
Any cell count should be performed IMMEDIATELY with this specimen
o WBCs and RBCs begin to lyse within 1 hour
o 40% WBCs disintegrate within 2 hours
CSF CELL COUNT
- Routinely performed on CSF
- Normal values:
o Adults = 0-5 WBCs/uL
o Neonates = 0-30 WBCs/uL
WBC Count
CSF DILUTION:
Clear
Undiluted
CSF DILUTION:
Slightly Hazy
1:10
CSF DILUTION:
Hazy
1:20
CSF DILUTION:
Slightly Cloudy
1:100
CSF DILUTION:
Cloudy/Slightly Bloody
1:200
CSF DILUTION:
Bloody/Turbid
1:10,000
FORMULA FOR WBC COUNT
WBC Diluting fluid?
WBC count (per uL) = (# of cells counted x dilution) / (area x depth factor (0.1))
- Acetic acid with Methylene blue
Done only in cases of traumatic tap
- To correct for WBC count & total protein concentration
o -1 WBC for every ___ RBCS seen
o -_ mg/dL total protein concentration for every 10,000 RBCs/uL (Henry)
o -1 mg/dL total protein concentration for every __ RBCs/uL (Strasinger)
RBC Count for CSF
700
8
1,200
- Performed on stained smear
- Specimen should be concentrated before smearing by using the ff
methods:
o Cytocentrifugation
o Centrifugation
o Sedimentation
o Filtration
CSF DIFFERENTIAL COUNT
- Fluid is added to conical chamber
- Cells are forced into a monolayer within a 6mm diameter circle on the slide
- Addition of __ albumin
o Increases cell yield or recovery
o Decreases cellular distortion
CYTOCENTRIFUGE
30%
PREDOMINANT CELLS IN CSF
- Predominant = __ and __
- Occasional = Neutrophils
- Adults: (___ ratio)
o __ - Lymphocytes
o __ - Monocytes
- Neonates (inversed ratio)
o Up to 80% (considered normal)
___ : Abnormal condition and Increased number of normal cells in the CSF
Lymphocytes and Monocytes
70:30
70%
30%
PLEOCYTOSIS
CSF PROTEIN:
normal value:
Adults = __ (<1% or 1/200 that of serum protein)
__= 150 mg/dL
Immature = __
15-45 mg/dL
Infants
500 mg/dL
CSF PROTEIN:
Damage to the BBB (most common): meningitis, hemorrhage
- Production of immunoglobulins within the CNS (multiple sclerosis)
- Decreased normal protein clearance from the fluid
- Neural tissue degeneration
INCREASED
CSF PROTEIN:
- CSF leakage/trauma
- Recent puncture
- Rapid CSF production
- Water intoxication
DECREASED
CSF:
__ - Major CSF protein
__ - 2nd Most Prevalent
__-globulins - Haptoglobin, ceruloplasmin
Beta-globulins - ___
o Carbohydrate-deficient transferrin
o Found in CSF but not in serum
Gamma-globulins - __ and some __
NOT found in normal CSF: IgM, fibrinogen and lipids
Albumin
Prealbumin
Alpha
Beta2-transferrin (tau)
IgG; lgA
CSF PROTEIN DETERMINATION: TP
3% Trichloroacetic acid (TCA)
- Preferred method
- Precipitates BOTH albumin & globulins
3% Sulfosalicylic acid (SSA)
- Precipitates __
- To precipitate globulins, add sodium sulfate (Na2SO4)
Turbidimetric
albumin only
CSF PROTEIN DETERMINATION: TP
Coomassie Brilliant Blue
- Protein binds to dye → Dye turns from red to blue
- ↑ Protein = ↑ Blue color
Dye-binding
CSF PROTEIN DETERMINATION: protein fraactions
- Assess the integrity of the blood brain barrier
- Normal value = <9; Abnormal = >9
- 9-14 = slight impairment
- 15-30 = moderate impairment
- >30 = severe impairment
- 100 = complete damage to BBB
CSF/Serum Albumin Index
CSF PROTEIN DETERMINATION: protein fractions
Assess conditions with lgG production within the CNS
- Normal value = <0.77 (Brunzel: 0.30-0.70)
- Abnormal = >0.77 (Old Strasinger: >0.77)
o Indicative of IgG production
IgG index
- For the detection of oligoclonal bands (in the y-region)
o These bands indicate immunoglobulin production - Done in conjunction with serum electrophoresis to ensure that banding is due to neurologic inflammation
- (+) 2 or more oligoclonal bands in CSF but NOT in serum is valuable for the diagnosis of multiple sclerosis
CSF ELECTROPHORESIS
Oligoclonal Banding in Serum but NOT in CSF (mnemonic)
(Ms. Neng):
- Multiple Sclerosis (persistent)
- Neurosyphilis
- Encephalitis
- Neoplastic disorders
- Guillain-Barre syndrome
Oligoclonal Banding in CSF but NOT in Serum
OB in both serum and CSF?
- Leukemia
- Lymphoma
- Viral infections
- Bands may also appear in GSF as a result of BBB leakage or traumatic tap
-HIV
- Demyelinating disorder
- Findings:
o (+) Anti-myelin sheath autoantibody
o (+) Oligoclonal band in CSF but not in serum
o (+) Myelin basic protein (MBP)
o ↑ IgG index
MULTIPLE SCLEROSIS
- Protein component of the lipid-protein complex that insulate the nerve fibers
- Presence of MBP in CSF indicates destruction of myelin sheath
- Used to monitor the course of multiple sclerosis
MYELIN BASIC PROTEIN (MBP)
Done in conjunction with blood glucose
Specimen for blood glucose should be drawn ___ prior
to __ (for equilibration)
Normal value - ___ of blood glucose (65% or 2/3); (50-80 mg/dL)
CSF GLUCOSE (GLU)
2 hours; spinal tap
60-70%
Inversely proportional to glucose
Sensitive method for evaluating the effectiveness of antibiotic therapy
Normal value - 10-22 mg/dL
CSF LACTATE (LAT)
Product of ammonia & alpha-ketoglutarate
Indirect test for the presence of excess ammonia in the CSF
Normal value - 8-18 mg/dL
INC IN: Disturbance of consciousness (coma) & Reye’s syndrome
CSF GLUTAMINE
CSF ENZYMES:
- Serum: Normal = 2>1>3> 4>5; Flipped pattern (AMI) = 1>2
- CSF: Normal pattern = 1>2>3>4>5; Neurological abnormalities = 2>1; Bacterial meningitis = 5>4>3>2>1 (↑ Neutrophils)
__:
- ↑ in stroke, MS, degenerative disorders, brain tumors, viral & bacterial meningitis, seizures
__:
- ↑ in intracerebral and subarachnoid hemorrhage, bacterial meningitis
SEROLOGIC TESTING:
- Latex agglutination test and ELISA= for detection of bacterial ags
- VDRL = recommended by CDC for syphilis
LACTATE DEHYDROGENASE (LDH)
CK
AST
- Detects Gram-negative endotoxin in body fluids & surgical instrument
- Reagent: Blood of horseshoe crab (Limulus polyphemus); __ copper
- Principle: In the presence of endotoxin, the amoebocytes (WBCs) will release lysate (protein); (+) Clumping or clot formation
LIMULUS AMOEBOCYTE LYSATE (LAL) TEST
hemocyanin
REASONS FOR SEMINAL FLUID ANALYSIS
- Fertility testing
- Postvasectomy semen analysis
- Forensic analysis (alleged rape)
COMPOSITION OF SEMEN:
5% __ - Seminiferous tubules (testes)
o Site of spermatogenesis:
o __: nurse cells for developing sperms
- ___: Site of sperm maturation (they become motile)
- Spermatogenesis and sperm maturation take __ (Graff - 74 days)
Spermatozoa
Sertoli cells
Epididymis
90
days
COMPOSITION OF SEMEN:
__ - Seminal fluid
Seminal vesicles:
o Provide nutrients for sperm & fluid
o Secretions rich in __ = for sperm motility
60-70%
fructose
COMPOSITION OF SEMEN:
20-30% - __
Acidic fluid that contains ACP, zinc, citric acid & other enzymes
For coagulation and __
Prostate fluid
liquefaction
COMPOSITION OF SEMEN:
__ - Bulbourethral glands
- Secretes thick __ mucus
- Neutralizes acidity from the prostatic secretions & vagina
5%
alkaline
SPECIMEN COLLECTION:
- Abstinence of __ but not __
o Prolonged abstinence = ↑ Volume, ↓ Motility
- Collect the entire ejaculate
2-3 days; >7 days
Methods of collection for semen:
o __ = best (or self-production)
o __ = withdrawal method
o Condom method = use non-lubricant-containing rubber or polyurethane condom
Masturbation
Coitus interruptus
SEMEN COLLECTION:
Specimen should be delivered to the lab within __ (at room temp)
- Take note of the time of specimen collection, specimen receipt, and liquefaction
- Analysis should be done after liquefaction (usually 30-60 minutes)
o Failure to liquefy within __ may be caused a deficiency in prostatic
enzymes
o If sample fails to liquefy, treat w/ __ to break up mucus
- Specimen awaiting analysis should be kept at __
1 hour of collection
60 minutes
amylase/bromelain/α-chymotrypsin
37degC
MACROSCOPIC EXAMINATION SEMEN:
__ = Normal (with musty or bleach odor)
- Increased white turbidity = __ (↑ WBCs)
- Red or brown coloration = ↑ RBCs, blood
- __ = Urine contamination, medication, ↑ abstinence (continence; ↑ flavin)
Gray-white, translucent
Infection
Yellow coloration
MACROSCOPIC EXAMINATION SEMEN:
- Normal = __
- Increased = ↑ abstinence
- Decreased = Infertility, incomplete collection
Volume
2-5 mL
MACROSCOPIC EXAMINATION SEMEN:
- Normal = Pour in droplets
- Abnormal = Threads __ long
- ↑ Viscosity = ↓ ____
- Reporting
o 0 = __
o 4 = __
o *May also be reported as low, normal or high
Viscosity
>2 cm
Sperm motility
Watery
Gel-like
MACROSCOPIC EXAMINATION SEMEN:
pH - Normal = __
- ↑ pH = __
- ↓ pH = ↑ __
- pH should be measured within 1 hour of ejaculation
7.2 to 8.0
Infection
Prostatic fluid
SPERM CONCENTRATION
- Normal value = __ sperms/mL
- Methods:
o ___
Dilution = 1:20; Diluents: To immobilize sperm (formalin, sodium bicarbonate (NaHCO3), Saline, Distilled water, Cold tap water)
o ___
For undiluted specimen
Uses __ to immobilize sperms
- Both sides of the hemocytometer are loaded and allowed to settle for __; then they are counted, and the counts should agree within __
> 20 (20-160) million
Improved Neubauer Counting Chamber
Makler Counting Chamber
heat
3 to 5 minutes
10%
SPERM CONCENTRATION FORMULAS:
shortcut?
long method?
SHORTCUT:
2 WBC squares = # sperms counted x 100,000
5 RBC squares = # sperms counted x 1, 000, 000
LONG:
Sperm concentration (mL) = #sperms counted x dilution/
area x depth (0.1)
SPERM COUNT:
- Normal value = __ sperms per ejaculate
Sperm count = Sperm concentration x Specimen volume
> 40 million
SPERM MOTILITY:
- Place a drop of semen in a slide & cover it w/ coverslip. Allow to settle for 1 min. Observe in __.
- Normal values: __ motile (within 1hour), Quality =
20 HPF
>50%; >2.0
WHO CRITERIA FOR SPERM MOTILITY:
Rapid straight-line motility
Slower speed, some lateral movement
Slow forward progression, noticeable lateral movement
No forward progression
No movement
4.0 a
3.0 b
2.0 b
1.0 c
0 d
ALTERNATIVE SPERM MOTILITY GRADING CRITERIA:
Sperm moving linearly or in a large circle
Sperm moving with an absence of progression
NO movement
Progressive Motility (PM)
Nonprogressive Motility (NP)
Immotility
SPERM MORPHOLOGY
- Normal values:
o Routine criteria = __ normal forms
o Kruger’s strict criteria = __ normal forms
Measure the head, neck & tail using a micrometer
- Use __ angle when preparing smears
- Stains for Sperm Morphology:
o __ (stain of choice)
o Wright’s stain
o Giemsa stain
- Head (length: __, width = 3 um)
o Acrosomal cap:
__ of the head
__ of the nucleus
o Normal = oval-shape
o Abnormal = poor __
- Midpiece:
o Contains __
- Neck (__)
- Tail (__)
o Abnormal = poor motility
o The sperm tail length already includes the __ length. The neck is the thickest part of the tail.
> 30%
14%
45°
Papanicolaou’s stain
5 um
1⁄2
2/3
ovum penetration
mitochondria
7 um
45 um
neck
- Hardening of veins that drain the testes
- Most common cause of male infertility
- Sperm has a tapered head
VARICOCELE
SPERM VIABILITY (SPERM VITALITY):
- Tested within 1 hour of ejaculation
- Reagents = eosin and nigrosin
- Living sperms = unstained, __
- Dead sperms = __
- Normal value = __ living sperms (Strasinger, 6th Ed, Brunzel)
Modified Blom’s test
bluish white
red
50%
- Tested within 2 hours or frozen to prevent fructolysis
- Screening test
o Resorcinol test (a.k.a. Seliwanoff’s test) = (+) Orange-red color
SEMINAL FLUID FRUCTOSE
OTHER TESTS PERFORMED IN SEMEN:
Fructose: > 13 umol/ejaculate –> __
Neutral α-glucosidase: > 20 mU/ejaculate –>
Zinc: > 2.4 umol/ejaculate
Citric acid: > 52 umol/ejaculate –>
Acid phosphatase: > 200 units/ejaculate
Lack of seminal fluid
Epididymis disorder
Lack of prostatic fluid
Routine aerobic and anaerobic cultures and tests for Chlamydia
trachomatis, Mycoplasma hominis and Ureaplasma urealyticum
- Round cells = WBCs or spermatids (immature sperm cells) → use
peroxidase to differentiate them
o <1 million round cells/mL = Normal
o >1 million WBCs/mL = infection
o >1 million spermatids/mL = Disruption of spermatogenesis
Round cell count = N x S /100
MICROBIAL
SPERM TEST:
__ (not specific)
o Test for choline (produced by the prostate gland; anti-bacterial)
o Reagents: Iodine crystals + Potassium iodide → (+) Dark brown
rhombic crystals
__ (very specific)
o Test for spermine (produced by the prostate gland; anti-bacterial)
o Reagents: Saturated picric acid + Trichloroacetic acid → (+) Yellow
leaf-like crystals
Florence test
Barbiero’s test
- Surgical cutting of vas deferens so that the ejaculate will not contain any sperm cell
- Following this procedure, sperm count ideally should be zero within __ after the procedure
semen analysis
- The only concern is the presence or absence of sperm
- Done __ after vasectomy & continued until 2 consecutive monthly
specimen show no sperm
- If wet preparation is negative, centrifuge specimen for 10 minutes and examine the sediment
- The presence of even a single “motile” spermatozoon is evidence of an unsuccessful vasectomy
Vasectomy
12 weeks
Post-vasectomy
2 months
NORMAL VALUES FOR SEMEN ANALYSIS:
Volume:
Viscosity:
pH:
Sperm concentration:
Sperm count:
Motility:
Quality:
Morphology:
Round cells
2-5 mL
Pour in droplets
7.2-8.0
> 20 million/mL
> 40 million/ejaculate
> 50% within 1 hour
> 2.0 or a, b, c
> 30% normal forms (routine criteria)
> 14% normal forms (strict criteria)
<1.0 million/mL
SPERM NOTES:
- Leydig cells secrete __; Sertoli cells secrete __
- Shortly after ejaculation, semen coagulates because of the action of a clotting enzyme, formed in the prostate, on a fibrinogen-like precursor substance that is produced by the seminal vesicles.
- When performing fertility testing, WHO recommends that __ samples be collected not __, with __ samples considered
significant
- Motile sperm can be detected for up to __ after intercourse, whereas nonmotile sperm can persist for __
- As the sperm die off, only the heads remain and may be present for __ after intercourse
testosterone; inhibin
2 or 3
<7 days or >3 weeks apart; 2 abnormal
24 hours; 3 days
7 days
A.k.a. joint fluid
- “_” = Latin word for “egg”; Resembles eggwhite
- Viscous fluid circulating in diarthroses (movable joints)
- Viscosity is due to polymerization of _ produced by synoviocytes
- Arthritis affects production of hyaluronate and its ability to polymerize, thus decreasing synovial fluid viscosity
- Functions:
o Lubricates joints
o Reduce friction between bones
o Provides nutrients to the articular cartilage
o Lessen shock of joint compression occurring during activities such as walking and jogging
SYNOVIAL FLUID
Synovial
hyaluronic acid
SPECIMEN COLLECTION:
- __ = Method of collection
- Normal synovial fluid does not clot (diseased joints may clot)
- Specimen Volume
o __ = Normal (adult knee cavity)
o __ = Inflammation
- Distributed in the following tubes (CLSI):
o Plain red top tube (no anticoagulant) = chemical & immunologic evaluation
Note: Sodium fluoride = glucose analysis
o Microscopic examination
__ for hematology or cell count
Do not use powdered anticoagulants & lithium heparin - interfere w/ crystal identification
Do NOT __ samples - this can produce additional crystals!
o Sterile anticoagulant tube (__ or __) = microbiological studies (GS and culture)
Arthrocentesis
<3.5 mL
>25 mL
Sodium heparin/Liquid EDTA
refrigerate
heparin or SPS
COLOR AND CLARITY (SYNOVIAL FLUID)
Colorless to pale yellow:
Inflammation:
Bacterial infection:
Traumatic tap; hemorrhagic arthritis:
WBCS, synovial cell debris or fibrin:
Presence of crystals:
Normal
Deeper yellow
Greenish tinge
Red
Turbid
Milky
SYNOVIAL FLUID VISCOSITY
Normal = forms a string that is __ long
Normal hyaluronic acid level of __
4-6 cm
0.3 to 0.4 g/dL
- Reagent = 2-5% acetic acid
- As the ability of the hyaluronate to polymerize decreases, the clot becomes less firm
Grading (Strasinger, 6th edition)
Good = Solid clot
Fair = Soft clot
Low = Friable clot
Poor = No clot
Grading (Strasinger, 3rd edition)
Good = Solid clot
Fair = Soft clot
Poor = Friable clot
Very poor = No clot
- “Arfl Arf! Formation of a hecking mucin clot after adding acetic acid can be used to identify a questionable fluid as hecking synovial fiuid!”
Ropes or Mucin Clot Test (Hyaluronate Polymerization Test)
CELL COUNT for synovial fluid:
DO NOT use __ as WBC diluting fluid for synovial fluid cell count - It can cause CLOT FORMATION
acetic acid
Most frequently performed count for synovial fluid
- Diluting fluids:
o NSS with methylene blue
o Hypotonic saline (0.3%) = lyses RBCs
o Saline with saponin = lyses RBCs
- For very viscous fluid: ↑ Hyaluronic acid
o Add a pinch of hyaluronidase to 0.5 mL fluid; or
o Add 1 drop of 0.05% hyaluronidase in phosphate buffer per mL of fluid
o Incubate at __ for 5 minutes
WBC COUNT
37degC
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Polymorphonuclear leukocyte
Bacterial sepsis, Crystal-induced inflammation
Neutrophil
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Mononuclear leukocyte
Nonseptic inflammation
Lymphocyte
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Large mononuclear leukocyte, may be vacuolated
Normal viral infections
Macrophage
(Monocyte)
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Neutrophil containing ingested “round body”
Lupus erythematosus
Vacuolated macrophage with ingested neutrophils
Reiter syndrome, Reactive arthritis
LE cell
Reiter cell
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Neutrophil with dark cytoplasmic granules containing immune
complexes
Rheumatoid arthritis, Immunologic inflammation
Macroscopically resemble polished rice, Microscopically show collagen & fibrin
Tuberculosis, septic and rheumatoid arthritis
Debris from metal & plastic joint prosthesis, “Ground pepper” appearance
Ochronotic arthropathy, Alkaptonuria, ochronosis
RA cell (Ragrocyte)
Rice bodies
Ochronotic shards
CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID:
Large, multinucleated cells
Osteoarthritis
Similar to macrophage, but may be multinucleated, resembling a mesothelial cell
Normal disruption from arthrocentesis
Refractile intracellular & extracellular globules, Stain with Sudan dyes
Traumatic injury, Chronic inflammation
Inclusions within clusters of synovial cells
Pigmented villonodular synovitis
Cartilage cells
Synovial lining cell
Fat droplets
Hemosiderin
CRYSTAL IDENTIFICATION:
__:
o Detects for the presence or absence of birefringence
o Birefringence = ability to refract light in 2 directions
__:
o Confirms the type of birefringence (positive or negative BR)
o Red compensator is placed between crystal & analyzer
POLARIZING MICROSCOPE
COMPENSATED POLARIZING MICROSCOPE
SYNOVIAL CRYSTAL:
The molecules in the crystals run parallel to the long axis of the crystal & when aligned w/ the slow vibration, the velocity of slow light passing through the crystal is not impeded as much as the fast light, which runs against the grain & produces yellow color. This is a negative birefringence (subtraction of velocity from the fast ray).
MONOSODIUM URATE (MSU)
SYNOVIAL CRYSTAL:
The molecules in the crystals run perpendicular to the
long axis of the crystal, when aligned w/ the slow axis of
compensator, the velocity of fast light passing through the crystal is much quicker, producing a blue color & positive birefringence.
CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD)
SEROUS FLUID:
__:
- Accumulation of fluid between the membranes
- Classified as exudate or transudate
__:
- Disruption of fluid production & regulation between membranes
- Changes in hydrostatic and oncotic pressure (HP, OP)
- Examples: Hypoproteinemia (↓ Oncotic pressure), Congestive heart failure (↑ Hydrostatic pressure), Nephrotic syndrome (↓ Oncotic Pressure)
__:
- Direct damage to the membrane of a particular cavity
- Examples: Infection, Inflammation, Malignancy
EFFUSION
TRANSUDATE
EXUDATE
Fluid:serum protein ratio (most reliable)
Fluid:serum LD ratio (most reliable)
Serum-ascites albumin gradient (SAAG)
Rivalta’s test
TRANS:<0.5, <0.6, >1.1, (-)
EXU: >0.5, >0.6, <1.1, (+)
SEROUS FLUID: TRANSUDATES vs. EXUDATES
__:
- Differentiates exudates from transudates
- Acetic acid + Water + Unknown fluid → (+) Heavy precipitation = EXUDATE
__:
- Recommended to detect transudates of hepatic origin
- Serum Albumin - Peritoneal Fluid Albumin
- Transudate is >1.1
- Exudate <1.1
RIVALTA’S TEST (SEROSAMUCIN CLOT TEST)
SERUM-ASCITES ALBUMIN GRADIENT (SAAG)
FLUIDS:
3 P’s (Pleural, Pericardial, Peritoneal fluid)
- NORMAL APPEARANCE = Clear, pale yellow
- Pleural fluid = __
- Pericardial fluid = __
- Peritoneal (ascitic) fluid = __
NORMAL VOLUMES
- < 30 ml
- < 50 mL
- <100 mL
Specimen is distributed in the following tubes:
- __ = Cell counts and differential
- __ = Microbiology and cytology
- __ = Chemistry (samples for pH must be maintained anaerobically in ice)
Thoracentesis
Pericardiocentesis
Paracentesis
EDTA
Sterile heparin or SPS
Plain/heparin tubes
MILKY PLEURAL FLUID: Chylous effusion vs Pseudochylous effusion
CHYLE: Thoracic duct leakage, Milky/white, ↑ Lymphocytes, (+++) for sudan III, >110 Triglycerides
PSEUD: Chronic inflammation, Milky /green tinge /gold paint, Mixed cells
BLOODY PLEURAL FLUID: HEMOTHORAX VS HEMORRHAGIC EFFUSION
HEMOTHO: Uneven; Pleural fluid Hct is >1⁄2 of whole blood Hct
HE: Even, Pleural fluid Hct is <1⁄2 of Whole blood Hct
- Contain concentric striations of collagen-like material
- Seen in benign conditions & associated with ovarian & thyroid carcinomas
PSAMMOMA BODIES
GASTRIC FLUID:
__:
o Non-beta islet cell adenoma of the pancreas
o ↑↑ Gastrin = ↑↑ HCl = Hyperchlorhydria, hyperacidity
__:
o Anti-parietal cell Ab
o Anti-intrinsic factor Ab
o (-) HCl = Achlorhydria, Anacidity
Zollinger-Ellison Syndrome (ZES)
Pernicious Anemia
CELLS IN THE STOMACH
- __ = produce HCl and intrinsic factor
- __ = produce pepsinogen
- __ = produce gastrin
- __ = produce mucus that protects the stomach wall from acid
Parietal cells
Chief cells
Specialized G cells
Foveolar cells
SPECIMEN COLLECTION
- Method of collection = Gastric aspiration
- Gastric tubes: (Henry, 17th Ed p.553)
o __ = passed through the nose
o __ = passed through the mouth
o Others = Lavacuator tube, Ewald’s tube, Edlich tube = passed through the mouth
Levin tube
Rehfuss
__:
- Total gastric secretion during unstimulated, fasting state
- Duration of collection:
o 1-hour collection (consists of four 15-minute specimens, but a single 1-hour
can be used): routinely performed
o 2-hour collection: for Insulin hypoglycemia test
__:
- Total gastric secretion after gastric stimulation
- Duration of collection
o 1-hour collection (at 15-minute intervals) - when Pentagastrin and Histamine
are used
o 2-hour collection-for Insulin hypoglycemia test and when Histalog is used
Basal Acid Output (BAO)
Maximum Acid Output (MAO)
GASTRIC STIMULANTS:
__:
o Ewald’s = bread, weak tea or water
o Boa’s = oatmeal
o Riegel’s = beef steak and mashed potato
__:
o Pentagastrin = most preferred
o Insulin = to assess vagotomy procedure
o Histalog (Betazole)
o Histamine
__: (Fictitious feeding) = sandwich
- Test Meals
- Chemical Stimulants
- Sham Feeding
- Normal contains bacteria, cellulose, undigested foodstuffs, Gastrointestinal secretions, bile pigments, cells from the intestinal wall, electrolytes, water
- Around __ is passed per day
- Human’s contains around 75% water & 25% solids
- The odor is due to the presence of __ and __
FECES
100-200g
indole and skatole
MACROSCOPIC STOOL CHARACTERISTICS: COLOR
Bile duct obstruction (- Urobilin), barium sulfate
Bile duct obstruction, pancreatic disorders, steatorrhea
Cystic fibrosis (↑ mucus)
Cholera
Typhoid
Intestinal constriction
Constipation
Pale yellow, white, gray
Bulky/frothy
Butter-like
Rice watery
Pea-soup
Ribbon-like
Scybalous
(“Goat droppings”)
- Type 1 = Separate hard lumps like nuts (hard to pass)
- Type 2 = Sausage-shaped but lumpy
- Type 3 = Like a sausage but with cracks (Optimal)
- Type 4 = Like a sausage or snake, smooth & soft (Most optimal)
- Type 5 = Soft blobs with clear-cut edges (passed easily)
- Type 6 = Fluffy pieces with ragged edges, a mushy stool
- Type 7 = Watery, no solid pieces, entirely liquid
BRISTOL STOOL CHART
FATS
- ___ = Increased fats in stool (>6 g/day)
- Tests:
o Screening test = microscopic examination of feces for fat globules
o Definitive test = fecal fat determination
Steatorrhea
Quantitative Tests for fats in stool:
- Gold standard for fecal fat determination
- For definitive diagnosis of steatorrhea
- Titration with NaOH
- Sample = __ (72 hours)
- Normal value = 1 – 6 grams of fats per day
- Steatorrhea =__ of fats per day
- Acid Steatocrit= rapid test to estimate the amount or fat excretion (similar to microhematocrit test)
Van de Kamer titration
3-day stool
> 6 grams
MUSCLE FIBERS
- __ = Abnormal excretion of muscle fibers in feces
- Determination:
o The patient should include meat in the diet
o Emulsified stool + 10% Eosin - Coverslip & stand for 3 minutes
o Count the number of undigested fibers (HPF)
- Completely digested: No striations
- Partially digested: Striation in one direction
- Undigested: Striation in both directions
- Abnormal: __ (ex. Bile duct obstruction, Cystic fibrosis)
Creatorrhea
>10 undigested muscle fibers
___:
- >3 neutrophils/hpf = Invasive condition
o Diarrhea with WBCs = Salmonella, Shigella, Yersinia, Entero-invasive E. coli, Campylobacter
o Diarrhea without WBCs = Toxin producing (S. aureus, V. cholerae), virus, parasites
Determination:
- Wet preparation = Stool + Loeffler’s methylene blue
- Dried preparation = Stool +Wright’s or Gram stain
- Lactoferrin latex agglutination test
o __ = found in secondary granules of neutrophils = (+) Invasive bacterial pathogen
FECAL LEUKOCYTES
Lactoferrin
Notes - Occult = “hidden”
- Screen test for = colorectal cancer
- Significant = __ blood/150g stool
- Sample = __ portion of the stool
POSITIVE COLOR:
GUAIAC FECAL OCCULT BLOOD TEST (gFOBT)
>2.5 mL
center
blue
Avoid for 3 days:
Red meat
Melon
Broccoli
Cauliflower
Horseradish
Turnip
Avoid for 7 days:
Aspirins!!!
NSAIDs other than paracetamol
False (+) FOBT
Avoid for 3 days:
*Vitamin C (>250mg/dL)
*Iron supplements containing Vitamin C
Failure to wait specified time after sample is applied to
add the developer reagent
False (-) FOBT
OTHER FECAL SCREENING TESTS:
Differentiate fetal blood and maternal blood
- Specimen = infant stool/vomitus
- Bloody stools & vomitus are sometimes seen in neonates as a
result of swallowing maternal blood during delivery
Emulsified stool → Centrifuge → Add 1% NaOH to supernatant
Pink solution = (+) fetal blood (HbF)
Yellow brown supernatant = (+) maternal blood (HbA)
Remember:
- ___ is alkali resistant
- __ is denatured by NaOH
APT test (Apt-Downey Test)
HbF
HbA
- Stool weight of >200 g/day with increased liquidity & frequency of >3x/day
- Acute diarrhea =
- Chronic diarrhea =
- Major mechanisms are Secretory, Osmotic & Altered Motility.
- Laboratory tests used to differentiate these mechanisms are fecal electrolytes (fecal Na+ and K+), fecal osmolarity and stool pH
- Normal Fecal Osmolarity = 290 mOsm/kg
- Normal Fecal Na+ level 30 mmol/L
- Normal Fecal K+ level = 75 mmol/L
DIARRHEA
<4 weeks
>4 weeks
__:
- Increased secretion of water and electrolytes, which override the reabsorptive ability of the large intestine
- Causes: Bacterial, viral and protozoan infections, drugs, laxatives, hormones, inflammatory bowel disease, endocrine disorders, neoplasms, collagen vascular disease
__:
- Retention of water and electrolytes in the large intestine due to incomplete breakdown or reabsorption of food
- Causes: Maldigestion, malabsorption, disaccharidase deficiency (lactose intolerance), laxatives, antacids, amebiasis, antibiotics
__:
- Enhanced (hypermotility) or slow (constipation) motility
- Causes: Irritable bowel syndrome (IBS), Rapid gastric emptying (RGE) dumping syndrome
SECRETORY DIARRHEA
OSMOTIC DIARRHEA
ALTERED MOTILITY
QUALITY CONTROL OF LABORATORY EQUIPMENT:
__: Reagent strips; check the temperatures of refrigerators and water baths
__: Disinfection of centrifuges; check pH and purity meter resistance of deionized water
__: Diluents checked for contamination (examine in counting
chamber under 4x magnification)
__: Speed of cytocentrifuge checked with tachometer, & timing
checked with stopwatch
__: Calibration of centrifuges (record appropriate centrifugal force)
__: Professional cleaning of the microscope
Daily Basis
Weekly Basis
Biweekly Basis
Monthly Basis
Every 3 Months
Annually
devices used to calibrate the centrifuge
Tachometer (Speed), Stopwatch (Timing), Strobe light
