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MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – ANALYSIS OF URINE AND OTHER BODY FLUIDS
CLINICAL MICROSCOPY
Lecturer: Sir Errol Coderes, RMT
Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

SAFETY IN THE CLINICAL LABORATORY B. SHARP HAZARDS

- Sharp objects (needles, lancets, broken glassware)
A. BIOLOGICAL HAZARDS - Disposed of in puncture-resistant containers
- Potentially harmful organisms - Phlebotomists should carry these red, puncture-resistant
- Chain of infection containers in their collection trays
o Process of how microorganisms are transmitted from one person to another - An accidental needle-stick must be reported to the
o Essential in preventing the spread of infection supervisor
o Requires a continuous link between:
 Source C. RADIOACTIVE HAZARDS
 Mode of transmission - When procedures using radioisotopes are performed
 Susceptible host - Exposure to radiation during pregnancy presents a danger to the
o Routes of infection fetus
 Inhalation - The best method of radioactive waste disposal is to store the used
 Ingestion radioactive material in a locked, marked room until the background
 Direct inoculation or skin contact count is down to 10 half-lives for radioiodine

6 COMPONENTS OF THE CHAIN OF INFECTION D. CHEMICAL HAZARDS

1. Infectious agent
2. Reservoir
Chemical Spills
3. Exit (Portal of Exit)
4. Mode of transmission - BEST FIRST AID: Flush the area with amounts of water for at least
5. Entry (Portal of Entry) 15 minutes then seek medical attention.
6. Susceptible host - For alkali or acid burns in the eye, wash out eye thoroughly with
running water for 15 minutes (Turgeon)
- DO NOT NEUTRALIZE CHEMICALS that come in contact with the skin.
PREVENTION
- Acid spills on floors can be neutralized and then soaked up with wet rags or spill
1. PERSONAL PROTECTIVE EQUIPMENT pillows
- Gloves Chemical Handling
o For every patient, use new set of gloves (not applicable in the
Philippines) - ALWAYS ADD ACID TO WATER!!
- Fluid-resistant laboratory gowns o To avoid sudden splashing
o An EXPLOSION can occur if water is added to acid
- Eye and face shields
- Countertop shields
NATIONAL FIRE PROTECTION ASSOCIATION (NFPA)
2. PROPER HANDWASHING
HAZARDOUS MATERIALS CLASSIFICATIONN
- Hand contact is the primary method of infection transmission
- Handwashing = best way to break the chain of infection - Uses numbers from 0 to 4 to classify hazard severity, with 4 representing
- Handwashing procedure extremely hazardous
o Wet hands with warm water - Mnemonic: You Were Born Right: Yellow, White, Blue Red (starting rightmost)
o Apply antimicrobial soap
o Rub to form lather, create friction, and loosen debris
o Thoroughly clean between fingers, including thumbs, under fingernails
and rings, and up to the wrist for at least 15 (or 20) seconds
o Rinse hands in a downward position
o Dry with a paper towel
o Turn off faucets with a clean paper towel to prevent contaminations
o And don’t forget the handwashing song: Happy Birthday (sung twice)

KEEP IN MIND!
Hand Hygiene
- When the hands are visibly soiled: wash hands with soap and water
- When the hands are not visibly soiled: apply alcohol-based hand-rub (ex.
sanitizer)

3. DISPOSAL OF BIOLOGICAL WASTES

- All biological waste, except urine, must be placed in
appropriate containers labeled with the biohazard symbol.
- The accepted "BIOHAZARD" label is fluorescent orange.
(Henry)
- Discard urine by pouring it into a laboratory sink, avoid splashing, and then
flush with water.
KEEP IN MIND!
- Empty urine containers can be discarded as non-biologically hazardous
Degree of Hazards (Hazards Index)
waste
- 0 = No/Minimal Hazard
- Disinfection of the sink using a 1:5 or 1:10 dilution of sodium hypochlorite
- 1 = Slight Hazard
should be performed daily.
- 2 = Moderate Hazard
- Disinfection eliminates many or all pathogenic microorganisms, except
- 3 = Serious hazard
bacterial spores
- 4 = Extreme/Severe Hazard
- 1:10 dilution of sodium hypochlorite is prepared by adding 1part of sodium
hypochlorite to 9 parts of water (effective for 1 month; used for disinfecting
Mnemonic: “No SMS Ex’s”
countertops & spills.)
E. ELECTRICAL HAZARDS
- DO NOT OPERATE equipment with wet hands.
KEEP IN MIND! - All electrical equipment is grounded in a 3-pronged plug to avoid
- The basic outline of the biohazard symbol is a plain trefoil, which is three circles electric shock
overlapping each other equally like in a triple Venn diagram with the - If electrical shock occurs, never touch the person or the equipment
overlapping parts erased. The diameter of the overlapping part is equal to half involved.
the radius of the three circles. o Turn off the circuit breaker
o Unplug the equipment
o Move the equipment using a nonconductive glass or wood object

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F. FIRE /EXPLOSIVE HAZARDS RENAL FUNCTION

- Flammable chemicals should be stored in safety cabinets and
explosion-proof refrigerators in a remote area. URINARY SYSTEM
- Persons with burning clothes should be wrapped in the blanket to - Kidney: weighs approximately
smother the flames. 150g & measures 12.5 cm x 6 cm
- All laboratory personnel must be involved in laboratory fire drills at x 2.5 cm (length, width, depth)
least annually - Ureter: 25 cm long
- Bladder: when approximately 150
WHEN A FIRE IS DISCOVERED… mL urine accumulates, a nerve
- R (Rescue) = Rescue anyone in immediate danger reflex is initiated
- A (Alarm) = Activate the institutional fire alarm system - Urethra: 4 cm long in women and
- C (Contain) = Close all doors to potentially affected areas 24 cm long in men
- E (Extinguish/Evacuate) = Attempt to extinguish the fire, if possible; exit the - About every 10-15 seconds,
area small amounts of urine are
emptied into the bladder from the
TO OPERATE A FIRE EXTINGUISHER… ureters
- P = Pull the pin - Urine is actually a fluid biopsy of
- A = Aim at the base of the fire the kidney
- S = Squeeze handles
- S = Sweep nozzle side to side

TYPES OF FIRE AND FIRE EXTINGUISHER

Fire Type of Hazard Type of Extinguisher

type NEPHRON
A Ordinary combustibles: Water, dry chemical, loaded steam - Basic structural & functional unit of the kidney
paper, cloth, rubbish, plastic, - 1 to 1.5 million nephrons per kidney
wood - Consists of glomerulus and renal tubules
B Flammable liquids: grease, Dry chemical, carbon dioxide, halon foam
gasoline, paints, oil ORDER OF URINE FORMATION
C Electrical equipment and Dry chemical, carbon dioxide, halon 1. Glomerulus
motor switches 2. Proximal convoluted tubule (PCT
D Flammable metals: mercury, Metal X, sand; dry powder; fought by fire 3. Loop of Henle (LH)
magnesium, sodium, lithium fighters only 4. Distal convoluted tubule (DCT)
E Detonation (Arsenal fire) Allowed to burn out and nearby materials 5. Collecting duct (CD)
K Cooking media: grease, oils, Liquid designed to prevent splashing and ---------------------------------------------
fats cool the fire 6. Renal Calyx
- Water (A) | Dry chemicals (ABC) | Carbon dioxide (BC) | Halon (BC) 7. Renal Pelvis
- Class D and E fires should be handled only by trained personnel
- Dry chemical extinguishers (ABC) are the most common all-purpose
extinguishers

G. PHYSICAL HAZARDS

GENERAL PRECAUTIONS:
- Avoid running in rooms and hallways
- Watch for wet floors
- Bend knees when lifting heavy objects
- Keep long hair pulled back
- Avoid dangling jewelry
- Maintain clean, organized work area
- Wear closed-toe shoes

MISCELLANEOUS HAZARD INFORMATION

- Ergonomic hazards are work-related and include strain due to repeated positions
- Cryogenic hazards are hazards due to extremely low temperatures
- Mechanical hazards include centrifuges, refrigerators, autoclaves, homogenizers
and glasswares
- Centrifuge accidents or improper removal of rubber stopper from test tubes may
produce aerosols 1. RENAL BLOOD FLOW
- The kidneys receive 25% of the total cardiac output
REVIEW!!!
ORDER:
1. Clean fingers and wrist for at least: 4. Degree of hazard number 3: - Renal artery (blood in)
a. 15 seconds a. Slight hazard - Afferent arteriole (“Approaching”)
b. 30 seconds b. Serious hazard - Glomerulus
c. 45 seconds c. Moderate hazard - Efferent arteriole (“Exiting)
d. 60 seconds d. Extreme hazard - Peritubular capillaries (“Surrounding the renal tubules”)
- Vasa recta
2. When hands are not visibly soiled: 5. Type of fire for flammable - Renal vein (blood out)
a. Wash hands with soap and liquids
water a. Type A - Total Renal Blood Flow: 1200 mL/min
b. Apply sanitizer b. Type B - Total Renal Plasma Flow: 600 to 700 mL/min
c. Apply Benzalkonium chloride c. Type C
d. Wipe hands with paper towel d. Type D
2. GLOMERULAR FILTRATION
e. Type E
3. Yellow quadrant in the NFPA f. Type K
GLOMERULUS
hazard classification:
a. Health hazard - The "working portion" of the kidney
b. Fire hazard - Coil of approx. 8 capillary lobes (capillary tuft) located within the Bowman's
c. Specific hazard capsule
d. Reactivity hazard - Attached to the glomerular basement membrane are the podocytes (epithelial
cells)
- Resembles a sieve
- Non-selective filter of plasma substances with MW of <70,000 daltons
Answer key: A, B, D, B, B o <70kDa: Salts, water, amino acids, glucose, urea
- Glomerular filtrate (S.G. 1.010)
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o Albumin can pass through the glomerulus based on size but still cannot pass
through the Bowman’s capsule because of the shield of negativity; albumin at
physiologic pH is negatively charged (similar charges repel)
o At pH 4.9 the albumin becomes positively charged but this means the patient
would have to be at a very acidotic state (impossible/deadly)
- Approximately 1% of the filtered plasma volume is actually excreted as urine

GLOMERULAR FILTRATION BARRIER

- Capillary endothelium with its large open pores
- Trilayer basement membrane (lamina rara interna, lamina densa, lamina rara
externa)
- Filtration diaphragm found between the podocytes of Bowman's space

3. TUBULAR REABSORPTION
- 1st function to be affected in renal disease. 4. TUBULAR SECRETION
- When the plasma concentration of a substance that is normally completely
2 MAJOR FUNCTIONS:
reabsorbed reaches an abnormally high level, the filtrate concentration exceeds
the maximal reabsorptive capacity (Tm) of the tubules, and the substance begins - Regulation of the acid-base balance in the body through secretion of hydrogen
appearing in the urine ions (in the form of NH4 and H2PO4).
- Renal threshold is the plasma concentration at which active transport stops - Elimination of waste products not filtered by the glomerulus
o RENAL THRESHOLD FOR GLUCOSE = 160-180 mg/dL
PROXIMAL CONVOLUTED TUBULE
PROXIMAL CONVOLUTED TUBULE - Major site for removal of nonfiltered substances
- 65% of reabsorption of substances - H+ ions are secreted in exchange for Na+ ions, which are reabsorbed with HCO3-
- Reabsorbs salts, water, amino acids, glucose, and urea into the plasma

RENAL TUBULAR ACIDOSIS (RTA)

KEEP IN MIND!
- Failure to produce an acid urine due to inability to secrete hydrogen ions
- PCT, LH, DCT & CD alter urine concentration
- PCT is the major site (65%) of reabsorption of plasma substances BLOOD TUBULE URINE
- Renal concentration begins in the descending and ascending LH Waste W ↑ Waste
- The ascending LH is highly impermeable to water
pH: 7.40 H+ H+ ↑ H+ = ↓ pH
(acidic)
- "DAM" Collects water, so does the Descending LH (Sir James M.)
RTA ↑ H+ ↓ H+ ↓ H+
- ASIN"-ding loop = reabsorbs ASIN (salt), but NOT water (Doc Krizza A) (Renal Tubular ↓ pH (acidic) ↑ pH (alkaline)
Acidosis)
BLOOD TUBULE URINE
↑𝐴𝑙𝑑𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒 ↓ 𝐴𝑙𝑑𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒
S ← Salts ← S RENAL FUNCTION TESTS
↑𝐴𝐷𝐻 ↓ 𝐴𝐷𝐻
W ← Water ← W
100% 0%
A ← Amino acids ← -- I. TESTS FOR GLOMERULAR FILTRATION
<160 >160
G ← Glucose ← G - Clearance tests = used to evaluate glomerular filtration
40% 60%
U ← Urea ← U - Measure the rate at which the kidneys are able to remove a filterable substance
from the blood
TUBULAR REABSORPTION o Urea = obsolete
ACTIVE TRANSPORT PASSIVE TRANSPORT o Creatinine = MOST COMMON
Substance Location Substance Location o Inulin (MW: 5,200 Da) = Gold Standard; Reference method (Not routinely
performed because inulin needs to be injected into the body)
Glucose, amino PCT Water PCT, descending
o Beta2-microglobulin (MW: 11,800 Da) = better marker of renal tubular function
acids, salts LH, CD
than of GFR
Chloride Ascending LH Urea PCT, ascending
o Radioisotopes
LH
o Cystatin C (MW: 13,000 Da)
Sodium PCT and DCT Sodium Ascending LH
- Active transport is the movement of a substance across cell membranes into the CREATININE CLEARANCE FORMULA:
bloodstream by electrochemical energy 𝑈𝑉 1.73𝑚2
- Passive transport is the movement of molecules across a membrane by diffusion Ccr (ml/min) = 𝑥
𝑃 𝐴
because of a physical gradient
Where:
ANTI-DIURETIC HORMONE (ADH/VASOPRESSIN)
- Regulates water reabsorption in the DCT and CD - Ccr: Creatinine clearance
o ↑ Body Hydration = ↓ ADH = ↑ Urine volume - U: Urine creatinine (mg/dL)
o ↓ Body Hydration = ↑ ADH = ↓ Urine volume - P: Plasma creatinine
- Diabetes Insipidus (DI) = ADH deficiency - V: Urine volume (mL/min)
- Syndrome of inappropriate ADH secretion (SlADH) =ADH excess - A: Body surface area

ALDOSTERONE
SAMPLE PROBLEM
- Regulates sodium reabsorption
- Given the following, compute for creatinine clearance:
o Urine creatinine = 120 mg/dL
RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) o Plasma creatinine =1 mg/dL
𝑅𝑒𝑛𝑖𝑛 𝐴𝐶𝐸 o Urine volume in 24 hrs. = 1440 Ml
𝐴𝑛𝑔𝑖𝑜𝑡𝑒𝑛𝑠𝑖𝑛 → 𝐴𝑛𝑔𝑖𝑜𝑡𝑒𝑛𝑠𝑖𝑛 𝐼 → 𝐴𝑛𝑔𝑖𝑜𝑡𝑒𝑛𝑠𝑖𝑛 𝐼𝐼 → 𝐸𝑓𝑓𝑒𝑐𝑡𝑠 o Patient of average body surface area
↓ Na+, ↓BP lungs
𝑚𝑔 1440 𝑚𝐿
(120 )[ ] 2
- Effects of Angiotensin II (active form of Angiotensin) 𝐶𝑐𝑟 = 𝑑𝐿 1440 𝑚𝑖𝑛 𝑥 1.73𝑚 = 120 𝑚𝐿/𝑚𝑖𝑛
o Release of Aldosterone & ADH (↑ Sodium & water reabsorption) 1 𝑚𝑔/𝑑𝐿 1.73𝑚2
o Vasoconstriction (↑ blood pressure)
- NORMAL VALUE
o Corrects renal blood flow
o Male: 107-139 mL/min
o Female: 87-107 mL/min
- NOTE: Renin is produced by the Juxtaglomerular (JG) cells
- ACE: Angiotensin converting enzyme KEEP IN MIND!
- Creatinine clearance is a measure of the completeness of a 24-hour urine
- ACTIONS OF RAAS: collection
o Dilates the afferent arteriole & constricts the efferent arteriole - By far the greatest source of error in any clearance procedure utilizing urine is
o Stimulates sodium reabsorption in the PCT the use of improperly timed urine specimens
o Triggers the adrenal cortex to release aldosterone to cause sodium - Around 7-10% of creatinine is secreted by the renal tubules
reabsorption & potassium excretion in the DCT and CD - Recently, studies have shown the value and clinical usefulness of calculating
o Triggers release of anti-diuretic hormone by the hypothalamus to stimulate an "estimated" GFR (eGFR) to detect and monitor kidney disease.
water reabsorption in the CD
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ESTIMATED GFR FORMULA DEVELOPED BY COCKGROFT &GAULT: INTRODUCTION TO URINALYSIS

𝐶 (140−𝑎𝑔𝑒)(𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚𝑠) HISTORY OF URINALYSIS

𝑐𝑟= 𝑥 0.85 (𝑖𝑓 𝑓𝑒𝑚𝑎𝑙𝑒)
72 𝑥 𝑠𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 𝑖𝑛 𝑚𝑔/𝑑𝐿 - Hippocrates, Aristotle and the ancient Egyptians inferred diagnoses from urine
Variables: evaluation, but it was not until the Middle Ages that uroscopy reached diagnostic
- Age, Sex, and Body weight in kilograms dominance.
- A major reason for its rise to prominence was the publication of Johannes de
Ketham's Fasciculus Medicinae in 1491. This was the first illustrated medical book
MODIFICATION OF DIET IN RENAL DISEASE (MDRD) SYSTEM FORMULA printed. It depicts a urine wheel: a large circle surrounded by thin-necked, urine
(Strasinger, 5th Ed): flasks. This wheel shows how the color and consistency of urine could be matched
𝐺𝐹𝑅 = 170 𝑥 𝑠𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 −0.999 𝑥 𝑎𝑔𝑒 −0.176 𝑥 0.822 (𝑖𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑖𝑠 𝑓𝑒𝑚𝑎𝑙𝑒) to a diagnosis.
+0.318 - Disease was thought to result from the imbalance of humours, reflected by one of
𝑥 1.1880 (𝑖𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑖𝑠 𝑏𝑙𝑎𝑐𝑘) 𝑥 𝐵𝑈𝑁 −0.170 𝑥 𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛
the urine colors. In the corners of the urine wheel, four small circles contain
Variables: descriptions of the 4 temperaments/humors:
- Ethnicity 1. Sanguineous (blood)
- BUN 2. Choleric (yellow bile)
- Serum albumin 3. Phlegmatic (phlegm)
4. Melancholic (black bile)
MDRD-IDMS (ISOTOPE DILUTION MASS SPECTROPHOTOMETRY) HISTORICAL NOTE
TRACEABLE FORMULA: - The "taste test" of urine was used by the Babylonians and Egyptians to detect
diabetes
- Recommended by the National Kidney Disease Education Program (NKDEP) - Hindu physicians noticed that "honey urine" attracted ants
𝐺𝐹𝑅 = 175 𝑥 𝑠𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 −0.1154 𝑥 𝑎𝑔𝑒 −0.203 𝑥 0.742 (𝑖𝑓 𝑓𝑒𝑚𝑎𝑙𝑒) Scientist Discovery/Contribution
𝑥 1.202 (𝑖𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑖𝑠 𝑏𝑙𝑎𝑐𝑘) Hippocrates Uroscopy; first documented the importance of sputum
examination
Frederik Dekkers Albuminuria by boiling urine
II. TESTS FOR TUBULAR REABSORPTION Thomas Bryant Wrote a book about "pisse prophets" (charlatans)
- Concentration tests = used to evaluate tubular reabsorption (assess the ability of Thomas Addis Examination of urine sediment
the kidney to concentrate or dilute urine) Richard Bright Introduced urinalysis as part of doctor's routine patient
examination
Obsolete 1. Fishberg test Ludwig Thudichum Urochrome
tests - Patient is deprived of fluid for up to 24 hours Domenico Cotugno Cerebrospinal fluid
- Urine S.G. after 12-hour restricted fluid diet is about 1.022 or Ivan Følling Phenylketonuria
more Archibald Garrod Alkaptonuria
- Urine S.G. after 24-hour restricted fluid diet is about 1.026 or William Wollaston Cystine calculi
more
Stanley Benedict Benedicts reagent
2. Mosenthal test
- Patient maintains normal diet and fluid intake URINE COMPOSITION
- Compare day & night urine in terms of volume & S.G. - 95-97% water
- 3-5% solids (60 grams= Total Solids in 24 hours)
Recently 3. Specific Gravity (S.G.) - Total Solids (TS)
used tests - Influenced by the number & density of particles in a solution o 35 grams’ organic
 Urea (major)
4. Osmolality  Creatinine (2nd), hippurate, uric acid, CHO, pigments, fatty acids, mucins,
- Influenced by the number of particles in a solution enzymes, hormones
- More preferred than S.G. determination o 25 grams’ inorganic
- More precise than osmolarity because it does not vary with  Chloride (major) > Sodium > Potassium
temperature  NaCl - Sodium chloride (principal salt)
- Methods include freezing point osmometry &vapor pressure  Sulfate, phosphate, ammonium, magnesium, calcium
osmometry
- NV = 1-3x (275 to 900 mOsm/kg) than of serum (275 to 300 TYPES OF URINE SPECIMEN
mOsm/kg)
Random/ - For routine and qualitative urinalysis
Occasional/ - Ideal for cytology studies (ONLY IF with prior hydration, &
Single exercise 5 mins. before collection!)
III. TESTS FOR TUBULAR SECRETION & RENAL BLOOD FLOW First morning - Ideal specimen for routine urinalysis and pregnancy
- p-aminohippuric acid (PAH) test = most commonly used reference method testing (hCG)
- Phenolsulfonphthalein (PSP) test = obsolete; results are hard to interpret - Often preferred for cytology studies/cytodiagnostic urine
testing
REVIEW!!! - Most concentrated and most acidic - allows well
preservation of cells and casts
1. Part of the nephron that resembles 4. Variables in the creatinine - For evaluation of orthostatic proteinuria
a sieve clearance formula by - Patient voids before going to bed, and immediately on
a. PCT Cockgroft and Gault: rising from sleep collects urine specimen
b. Afferent arteriole I. Age 2nd morning/ - 2nd voided urine after a period of fasting
c. Glomerulus II. Sex Fasting - For glucose determination
d. Loop of Henle III. Urine creatinine 2-hour post- - For diabetic screening or monitoring
e. Collecting duct IV. Body weight prandial - Preferred for testing glucose
a. I, II, III Glucose - Optional with blood samples in glucose tolerance test
2. Substances reabsorbed by the b. I, III, IV tolerance test
PCT c. I, II, IV Fractional - At least 2 voided collection
a. Salts d. II, III, IV specimen - Series of blood and urine samples are collected at specific
b. Amino acids time intervals to compare concentration of a substance in
c. Glucose 5. Renal threshold for glucose urine with its concentration in the blood
d. All of the above a. 70-100 mg/dL - Used in the diagnosis of diabetes
b. >126 mg/dL Midstream - For routine screening and bacterial culture
3. Urine pH in renal tubular acidosis c. 160-180 g/dL clean-catch - Patient should thoroughly cleanse his glans penis or her
a. Acidic d. 160-180 mg/dL urethral meatus before collection
b. Alkaline e. 160-180 g/L Catheterized - May be urethral or ureteral
c. Neutral - For bacterial culture
d. Any of these Suprapubic - Abdominal wall is punctured, and urine is directly aspirated
aspiration from the bladder
- Bladder urine for anaerobic bacterial culture and urine
cytology
Answer key: C, D, B, C, D
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Pediatric - Use of soft, clear plastic bag with adhesive - Required urine volume: 30-45 mL
specimen - Sterile specimen obtained by catheterization or - Container capacity: 60 mL
suprapubic aspiration - Temperature (checked within 4 minutes): 32.5-37.7oC
- Urine collected from diapers is NOT recommended for - Added to the toilet water reservoir to prevent specimen
testing adulteration: Blueing agent (dye)
Three-glass - For prostatic infection
technique o First portion of voided urine KEEP IN MIND!
o Middle portion of voided urine - Urine containers should have a wide base, and has an opening of at least 4
o Urine after prostatic massage cm. The wide base prevents spillage, and a 4-cm opening is an adequate
- Examine the 1st and 3rd specimen microscopically, then target for urine collection.
compare the # of WBC and bacteria - 24-hr urine container should hold up to 3 liters and may be colored to protect
- Prostatitis = if the # of WBC and bacteria in the 3rd light sensitive analytes.
specimen is 10x GREATER than that of the 1st - Addition of urine before the start of 24-hour collection period causes false-
- 2nd specimen increased results
o CONTROL, for bladder & kidney infection - Failure to include urine at the end of 24-hour collection period causes false-
o If control is (+) for WBCs and bacteria, the results from decreased results
the 3rd specimen are considered invalid - When both routine UA and culture are requested, the culture should be
STAMEY-MEARS TEST FOR PROSTATITIS performed first.
- The four-glass method consists of bacterial cultures of the initial voided urine
(VB1), midstream urine (VB2), expressed prostatic secretions (EPS), and a SPECIMEN INTEGRITY
post- prostatic massage urine specimen (VB3). - Following collection, urine specimens should be delivered to the laboratory
- Urethral infection or inflammation is tested for by the VB1, and the VB2 tests promptly and tested within 2 hours (Strasinger, Harr); ideally within 30 minutes
for urinary bladder infection. The prostatic secretions are cultured and (Turgeon)
examined for white blood cells. - Physical, chemical and microscopic characteristics of a urine specimen begin to
- Having more than 10 to 20 white blood cells per high-powerfield is considered change AS SOON AS THE URINE IS VOIDED
abnormal.
Timed - For quantitative testing CHANGES IN UNPRESERVED URINE
specimen Increased Cause
24-hour (Ex: 8 AM → 8 AM) 1. pH Urea → (Urease) → Ammonia; loss of CO2
- At start time, patient empties bladder into toilet; then all 2. Bacteria Multiplication
subsequent urine is collected 3. Odor Urea → (Urease) → Ammonia
- At end time, patient empties bladder into collection 4. Nitrite Due to bacterial multiplication
container Darkened/Modified Cause
- Requires preservative - it depends on the test performed
5. Color Oxidation or reduction of metabolites (↑ Urobilin)
Decreased Cause
12-hour (Ex: 8 AM → 8 PM)
6. Clarity Bacterial multiplication, precipitation of amorphous
- For Addis count
material
4-hour 7. Glucose Glycolysis
- For nitrite determination 8. Ketones Volatilization and bacterial metabolism
- Urine remains in bladder for at least 4 hours before voiding 9. Bilirubin (CB) Light exposure/photo oxidation to biliverdin
10. Urobilinogen Oxidation to urobilin
Afternoon (2-4 PM) 11. RBCs/WBCs/Casts Disintegrate in dilute alkaline urine
- For urobilinogen determination 12. Trichomonas Become immobile or die, possible misidentification
DRUG - Chain of custody: process providing documentation of as WBCs
SPECIMEN proper sample ID from the time of collection to the receipt *Least affected urine parameter after standing = protein
COLLECTION of laboratory results

URINE PRESERVATIVES
- Ideal urine preservative does not exist (Strasinger)
- A preservative that best suits the needs of the required analysis should be chosen

Preservatives Advantages Disadvantages Additional information

Refrigeration  Does not interfere with chemical tests  Raises SG by hydrometer  Preservative of choice for routine
 Precipitates amorphous phosphates and urinalysis and urine culture (up to
urates 24hrs.)
 Prevents bacterial growth for 24 hrs.
Thymol  Preserves glucose and sediments well  Interferes with acid precipitation test for protein
Boric acid  Preserves protein & formed elements well  May precipitate crystals when used in large  Keeps pH about 6.00
 Does not interfere w routine analyses amounts  Bacteriostatic at 18 g/L;
other  For culture transport, C&S
 than pH  Interferes with drug& hormone analyses
Formalin  Excellent sediment preservative  Reducing agent, interferes with chemical tests  Rinse specimen container with formalin
(formaldehyde) for glucose, blood, to preserve cells and casts
 leukocytes & copper reduction  Preservative of choice for: Addis count
Toluene (Toluol)  Does not interfere with routine tests  Floats on urine surface; clings to pipettes &  Best all-around preservative (Turgeon)
testing materials
Sodium fluoride  Prevents glycolysis  Inhibits reagent strip tests for glucose, blood &  May use sodium benzoate instead of
 Good preservative for drug analysis leukocytes fluoride for reagent strip testing
 Specimens for Drug testing
Phenol  Does not interfere w/ routine tests  Causes an odor change  Use 1 drop/ounce of specimen
Commercial  Convenient when refrigeration is not  May contain one or more of the preservatives  Check tablet composition to determine
preservative tablets possible including sodium fluoride possible effects on desired tests
 Have controlled concentration to minimize
interference
Urine collection kits  Contains collection cup, C & S
preservative tube or UA tube
Gray C & S tube  Sample stable at room temperature for 48  Decreases pH; do not use if urine is below the  Preservative is boric acid and may not
hours minimum fill line be used for UA
 Preserves bacteria
Yellow plain UA tube  Use on automated instruments  Must refrigerate within 2hrs.  Round or conical bottom
Cherry red/ Yellow  Stable for 72 hours at room temperature;  Bilirubin & urobilinogen may be decreased if  Preservative is sodium propionate;
top tube instrument compatible specimen is exposed to light and left at room conical bottom
temperature
Saccomanno fixative  Preserves cellular elements  Used for cytology studies (50 mL urine)
(50% ethanol + 2%
carbowax)
*Preservatives for 5-HIAA (Serotonin) testing: Boric acid, HCl

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REVIEW!!! LABORATORY CORRELATIONS OF URINE COLOR

1. Major inorganic substance in urine 4. Disintegrate in alkaline
a. Sodium urine COLOR CAUSE
b. Potassium a. Amorphous Colorless to Recent fluid consumption, polyuria, dilute random specimen
c. Chloride phosphates pale yellow
d. Urea b. Bacteria Yellow Normal
e. Sodium chloride c. Casts Dark yellow to Concentrated specimen: strenuous exercise, dehydration, fever,
d. Struvite crystals amber burns
2. Required urine volume for drug testing: First morning specimen
a. 10-15 mL 5. Preservative for Addis Excessive urobilin, bilirubin, carotene
b. 60 mL count urine specimens Orange Bilirubin (yellow foam) = “Tea-colored urine”
c. 30-45 mL a. Boric acid Phenazopyridine (Pyridium): orange & viscous urine w/ orange
d. 50 mL b. Refrigeration foam
c. Saccomanno’s Acriflavin, Nitrofurantoin, Phenindione
3. Urine temperature for drug testing within fixative Yellow-green Bilirubin → (oxidized) → Biliverdin
4 minutes from the time of collection d. Formalin Yellow-brown
a. 35.5-37.7oC Green Pseudomonas infection
b. 32.5-35.7oC
Blue-Green Amitriptyline, Methocarbamol, Clorets, Methylene blue,
c. 32.5-37.5oC
Chlorophyll
d. 32.5-37.7oC
Phenol: initially brown, turns green when oxidized
e. 35.2-37.5oC
Indican
Pink/Red RBCs (Cloudy/ smoky red): Hematuria
Answer key: C, C, D, C, D Hemoglobin (Clear red)
Myoglobin (Clear red/reddish-brown/cola-colored/tea-colored)
Porphobilin (derived from porphobilinogen)
PHYSICAL EXAMINATION OF URINE Beets, menstrual contamination
Fuchsin (aniline dye from foods and candy)
URINE VOLUME Rifampin = all body fluids are red
- Normal range (24 hours) = 600 to 2,000 mL Burgundy/ Porphyrins
Purplish red,
- Average (24 hours) = 1,200 to 1,500 mL Portwine
- Night urine output = <400 mL Brown Methemoglobin (acidic urine)
- Day: Night ratio = 2-3:1 Black Homogentisic acid (alkaline urine): Alkaptonuria
- Container capacity (UA) = 50 mL Melanin (upon air exposure)
- Required for routine UA: 10 to 15 mL; average: 12 mL (for urinometry and reagent Phenol derivative, Argyrol, Methyldopa/Levodopa, Metronidazole
strip) (Flagyl)
Milky white Pyuria/leukocyturia (↑ WBCs)
URINE VOLUME TERMINOLOGIES
URINE COLOR CHANGES WITH COMMONLY USED DRUGS
Definition Causes Alcohol, ethyl Pale yellow or colorless (dieresis)
Polyuria Increased urine volume - Increased fluid intake Levodopa (Tx: Parkinsonism) Cola-colored (red then brown, alkaline)
- >2.000 mL/24 hrs. (in adults - - Diuretics, nervousness Nitrofurantoin (Tx: UTI) Yellow-brown
Henry) - Diabetes mellitus = ↑ SG Mepacrine [Atabrine] Yellow
- >2.5 L/day (in adults- - Diabetes insipidus = ↓ SG (antimalarial, Tx: Giardiasis)
Strasinger) Methyldopa [Aldomet] Green-brown
- 2.5-3.0 mL/kg/day (in (anti-hypertensive)
children) Metronidazole [Flagyl] Darkening, reddish brown
Oliguria Decreased urine volume - Dehydration (Tx: Trichomoniasis)
- <500 mL/24 hrs. (in adults - - Renal diseases Phenazopyridine [Pyridium] Orange-red, acid pH
Henry) - Renal calculi or tumor (Tx: UTI)
- <400 mL/day (in adults - Phenol poisoning Brown, oxidized to quinones (green)
Strasinger) Rifampin (Tx: TB) Bright orange-red
- <1 mL/kg/hr (in infants) Riboflavin [Multivitamins] Bright yellow
- <0.5 mL/kg/hr (in children)
Anuria Complete cessation of urine flow - Complete obstruction
- <100 mL/24 hrs (Graff) (stones, tumors) URINE CLARITY (TRANSPARENCY, TURBIDITY, CHARACTER)
- Toxic agents
- Decreased renal blood flow URINE CLARITY DETERMINATION
Nocturia Excretion of more than 500 mL of - Pregnancy - Thoroughly mix the specimen
urine at night - Renal diseases, bladder - Examine the specimen while holding in front of a light source
- S.G.<1.018 stones - View through a newspaper print
- Prostate enlargement
Diuresis Any increase in urine excretion - Excessive water intake Clarity Term
(polydipsia) Clear No visible particulates, transparent
- Diuretic therapy, hormonal Hazy Few particulates, print easily seen through urine
imbalance
Cloudy Many particulates, print blurred through urine
- Renal dysfunction, drug
ingestion Turbidity Print cannot be seen through urine
Milky May precipitate or be clotted
URINE COLOR
CAUSES OF URINE TURBIDITY
- Rough indicator of the degree of hydration
NONPATHOLOGIC PATHOLOGIC
- Should correlate with urine S.G.
- Normal: Colorless to deep yellow - Squamous epithelial cells - RBCs, WBCs
- Abnormal: Red / Red brown (most common) (females) - Bacteria (uniform turbidity not
- Amorphous urates (pink sediment) cleared by acidification or filtration)
URINE COLOR DETERMINATION - Amorphous phosphates & - Yeasts (↑ DM)
carbonates (white or beige - Non-squamous epithelial cells
- Examine the specimen under a good light source precipitate) - Abnormal crystals, lymph fluid
- Look down through the container against a white background (also works for - Vaginal cream, semen, fecal (chyluria), lipids
determining urine clarity but not the best way) contamination, radiographic
contrast media, talcum powder
NORMAL PIGMENTS IN URINE
Urochrome - Major pigment (yellow)
LABORATORY CORRELATIONS IN URINE TURBIDITY
- Lipid-soluble pigment that is a product of endogenous
Acidic Urine Amorphous urates, radiographic contrast media
metabolism
- Production is directly proportional to metabolic rate Alkaline Urine Amorphous phosphates, carbonates
- ↑ in thyrotoxicosis, fever, starvation Soluble with Heat Amorphous urates, uric acid crystals
Uroerythrin - Pink (or red) Soluble in Dilute Acetic Acid RBCS, amorphous phosphates, carbonates
- Derived from melanin metabolism Insoluble in Dilute Acetic WBCs, bacteria, yeast, spermatozoa
- May deposit in amorphous urates and uric acid crystals Acid
Urobilin - Dark yellow/orange-brown Soluble in Ether Lipids, Lymphatic fluid, chyle
- Derived from oxidation of colorless urobilinogen
- Present in old specimens

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URINE ODOR AUTOMATED REAGENT STRIP READERS

ODOR CAUSE PRINCIPLE = REFLECTANCE PHOTOMETRY

Aromatic, faintly Normal (due to presence of volatile acids from food) - Light reflection from the test pads decreases in proportion to the intensity of
Odorless Acute tubular necrosis (acute renal failure) color produced by the concentration of the test substance
Foul, ammoniacal UTI (ex. Proteus vulgaris), old urine - The darker the color of the reagent pad, the lesser the light reflection (and
Fruity, sweet Ketones (Diabetes Mellitus, starvation, vomiting) vice versa)
Caramelized sugar, Maple syrup urine disease (MSUD)
curry, maple syrup
Mousy, musty, barny Phenylketonuria (PKU) 1. SPECIFIC GRAVITY (S.G.)
Rancid butter Tyrosinemia - A measure of the amount of dissolved substances in a solution
Sweaty feet, acrid Isovaleric acidemia, glutaric acidemia - Density of solution compared with density of similar volume of distilled water at a
Menthol-like Phenol-containing medications similar temperature
Cabbage, hops Methionine malabsorption (Oasthouse syndrome) - Influenced by number and size of particles in a solution
Bleach Specimen adulteration or container contamination
Sulfur Cystine disorder REFERENCE RANGES
Rotting fish; galunggong Trimethylaminuria - Random urine: 1.003-1.035 (random)
Pungent, fetid Ingestion of onions, garlic, & asparagus - 1st morning urine: >1.020
(methylmercaptan), UTI (Brunzel)
- 24-hour urine: 1.016-1.022
Swimming pool Hawkinsinuria
Cat urine 3-hydroxy-3-methylglutaric aciduria
Tomcat urine Multiple carboxylase deficiency - If SG is <1.003 = Not a urine (except in D.I.)
o 1.001 = Water
- If SG is >1.040 = Radiographic dye present
REVIEW!!!
1. Urine volume produced in polyuria 4. Clear red urine - Isosthenuria = SG = 1.010
a. <500 mL a. Hemoglobin - Hyposthenuria = SG <1.010
b. 100 mL b. Myoglobin - Hypersthenuria = SG >1.010
c. 600-2000 mL c. RBCs
d. >2000 mL d. WBCs DETERMINATION
e. 1200-1500 mL e. A and B
f. A, B, C 1. URINOMETRY (URINOMETER /HYDROMETER)
2. Urine clarity determination
a. View through a newspaper print 5. Rotting fish urine odor - Calibration temperature: 20oC
b. Hold the specimen against a white a. Hawkinsinuria - Requires temperature correction:
background b. Trimethylaminuria o -0.001 for every 3oC that the specimen temp. is below the
c. Dim the room light c. Methionine calibration temp.
d. View through a dark container malabsorption o +0.001 for every 3oC that the specimen temp. is above the
d. Asparagus calibration temp.
3. Pink sediment in urine ingestion - Requires correction for glucose and protein:
a. Amorphous urates e. Rotting fish o 1g/dL Glucose = -0.004
b. Amorphous phosphates ingestion
o 1g/dL Protein = -0.003
c. Calcium oxalate crystals
d. Bilirubin crystals - Urine volume required: 10-15 mL
e. Squamous epithelial cells - CALIBRATION:
o Potassium sulfate (K2SO4) solution (20.29 g K2SO4 to 1L H2O)
Answer key: D, A, A, E, B o S.G. reading should be 1.015
- When using the urinometer, an adequate amount of urine is poured
into a proper-size container and the urinometer is added with a spinning motion.
CHEMICAL EXAMINATION The scale reading is then taken at the bottom of the urine meniscus.

READING URINE PRINCIPLE POSITIVE COLOR 2. REFRACTOMETRY (REFRACTOMETER, Rf/TS [Total Solids] METER)
TIME PARAMETER
30 seconds Glucose Double sequential Green to brown (KI STEPS IN USING THE REFRACTOMETER
enzyme reaction chromogen) 1. Put 1 or 2 drops of sample on the prism.
Bilirubin Diazo reaction Tan or pink to violet 2. Close the daylight plate gently
40 seconds Ketones Sodium Purple 3. Sample must spread all over the prism surface.
nitroprusside 4. Look at the scale through the eyepiece
reaction 5. Read scale where the boundary line intercepts it
45 seconds Specific gravity pKa change of Blue (1.000) to yellow
6. Wipe the sample from the prism clean w/ tissue &water
polyelectrolyte (1.030)
60 seconds Protein Protein error of Blue-green
indicators - Indirect method based on refractive index (RI)
pH Double indicator Orange (pH 5.0) to blue
𝑙𝑖𝑔ℎ𝑡 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑖𝑛 𝑎𝑖𝑟
system (pH 9.0) 𝑅𝐼 =
𝑙𝑖𝑔ℎ𝑡 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑖𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Blood Pseudoperoxidase Uniform green/blue
activity of (Hgb/Mb)
hemoglobin Speckled/spotted - Compensated to temperature (15-38oC)
(intact RBCs) - No need for temperature correction
Urobilinogen Ehrlich reaction Red - Requires correction for glucose and protein
Nitrite Greiss reaction Uniform pink
120 Leukocytes Leukocyte esterase Purple CALIBRATION:
seconds - Distilled/deionized H2O = 1.000 + 0.001
- 3% NaCl = 1.015 + 0.001
Reagent Strip Technique - 5% NaCl = 1.022 + 0.001
- Dip the reagent strip briefly (no longer than 1 second) into a well-mixed - 7% NaCl = 1.035 + 0.001
uncentrifuged urine specimen at RT. - 9% Sucrose = 1.034 + 0.001
- Remove excess urine by touching the edge of the strip to the container as the strip
is withdrawn, Sample Problem #1: (URINOMETRY)
- Blot the edge of the strip on a disposable absorbent pad. - Urine S.G. reading by urinometer is 1.025
- Wait the specified amount of time for the reaction to occur. o Urine temperature is 26oC
- Compare the color reaction of the strip pads to the manufacturer's color chart in o Urinometer calibration temp, is 20oC
good lighting. - What is the corrected SG?
26oC - 20oC = 6oC
Care of Reagent Strips 6oC / 3oC = 2
- Store with desiccant in an opaque, tightly closed container (in a cool dry area). 2 x 0.001 = 0.002
- Store below 300oC (room temperature); do not freeze. 1.025 + 0.002 = 1.027
- Once the container is opened, use strips within 6 months
- Do not expose to volatile fumes.
- Do not use past the expiration date.
- Do not use if chemical pads become discolored.
- Remove strips immediately prior to use.
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Sample Problem #2: (REFRACTOMETRY) - Alkaline tide occurs after meals due to withdrawal of H+ ions for the purpose
- Urine SG. reading by refractometer is 1.025 of secretion of HCI
- With 2g/dL glucose and 2g/dL protein - Cranberry Juice contains quinic acid that causes urinary excretion of hippuric
- Temperature: 37oC acid (antibacterial)
- What is the corrected SG?
2 g/dL glu x 0.004 = 0.008
2 g/dL pro x 0.003 = 0.006 REAGENT STRIP REACTION for pH (60 seconds)
0.008 + 0.006 = 0.014
1.025 – 0.014 = 1.011 Principle Double Indicator System

KEEP IN MIND! ↓ pH (↑H+) → ↑pH (↓H+)

- Both refractometer (Rf) & urinometer (U) require corrections for glucose & Methyl Red Bromthymol blue
protein pH 4.0-6.0 (Red to yellow) pH 6.0-9.0 (yellow to blue)
- Refractometer reading is lower than that of the urinometer by 0.002 (Rf <U by Reagents Methyl red, Bromthymol blue
0.002) Interferences - No known interfering substances
- Runover from adjacent pads, old specimens
SPECIFIC GRAVITY DILUTION - Correlations with other tests = Nitrite, Leukocytes,
- Specimens with very high S.G. readings can be diluted and retested Microscopic
- To obtain the actual S.G., multiply the decimal portion of S.G. by the dilution factor

Sample Problem:
- Urine specimen diluted 1:4 has a reading of 1.014. What is the actual S.G.
reading?
Actual SG = 0.014 x 4 = 0.056 = 1.056 3. PROTEIN
- Most indicative of renal disease
3. REAGENT STRIP REACTION for SPECIFIC GRAVITY (45 seconds) - Produces white foam in urine when shaken

REFERENCE RANGES:
Principle Blue (1.000) → Green → Yellow (1.030)
(↓ H+) (↑ H+) (↑↑↑ H+) - Normal urinary protein
o 10 mg/dL or <100 mg/day (Strasinger)
- The polyelectrolyte ionizes, releasing hydrogen ions in o <150 mg/day (Henry)
proportion to the number of ions in the solution - Mild/minimal proteinuria = <1 g/day
- The reagent is sensitive to the number of ions in urine - Moderate proteinuria = 1 to 3 or 4 g/day
- Indicator changes color in relation to ionic concentration - Large/heavy proteinuria = >3 or 4 g/day
Reagents - Multistix = Poly (methyl vinyl ether/maleic anhydride) "Proteins in normal urine consist of 1/3 albumin and 2/3 globulins."
bromthymol blue
- Chemstrip = Ethylene glycol diaminoethyl ether tetraacetic - Albumin
acid bromthymol blue o Major serum protein found in the urine
Interferences - False (+) = High concentration of protein (Strasinger) o <0.1% of plasma albumin enters the ultrafiltrate
- False (-) = Highly alkaline urine (>6.5) o 95-99% of all filtered protein is reabsorbed
Notes - Add 0.005 to reading when pH2 6.5 due to interference with - Other Proteins
bromthymol blue indicator o Serum and tubular microglobulins
- Not affected by glucose protein & radiographic dye (Henry) o Tamm-Horsfall protein (uromodulin)
o Proteins derived from prostatic and vaginal secretions

CATEGORIES OF PROTEINURIA

PRE-RENAL PROTEINURIA
4. HARMONIC OSCILLATION DENSITOMETRY (H.O.D.) ("BEFORE") OR OVERFLOW PROTEINURIA
- Obsolete method - Caused by conditions that affect the plasma prior to its reaching the kidney:
- Based on frequency of soundwave entering a solution changes in proportion to o Intravascular hemolysis = hemoglobin
the density of soln. o Muscle injury = myoglobin
- Ex: Yellow IRIS (International Remote Imaging System) o Severe infection & inflammation = ↑ APRs
- IRIS Diagnostics o Multiple myeloma
o Models 300 and 500 workstations  Proliferation of Ig-producing plasma cells (Bence-Jones protein)
o 6mL = required urine volume  BJP = Immunoglobulin light chains (identical: κ - κ, λ - λ)
 4 mL (of 6 mL) = for IRIS sIideless microscope  Tests = Serum electrophoresis, immunofixation electrophoresis
 2mL (of 6 mL) = for IRIS Mass Gravity Meter (for S.G. determination - by  Urine = precipitates at 40-60oC (cloudy) & dissolves at 100oC
using Harmonic oscillation densitometry) (clear)
 Interference due to other precipitated proteins can be re moved
2. pH by filtering the specimen at 100oC & observing the specimen for
- Acidity refers to the "sourness" of a solution, whereas alkalinity refers to its turbidity as it cools to between 40oC and 60oC.
"bitterness"
- Important in the identification of crystals and determination of unsatisfactory RENAL PROTEINURIA
specimens TRUE RENAL DISEASE
- A blood pH <6.8 or >7.8 will result in death A. GLOMERULAR PROTEINURIA
- Normal urine pH: - Diabetic nephropathy
o Random = 4.5-8.0 o Decreased glomerular filtration
o 1st morning = 5.0-6.0 o May lead to renal failure
- When pH is >9.0 = unpreserved urine o Indicator: Microalbuminuria = proteinuria undetectable by routine reagent
strip
Causes of - Diabetes Mellitus (↑ ketone bodies) o Albumin Excretion Rate (AER) = in ug/min or in mg/24 hours (Source: CC
Acidic - Starvation (↑ ketone bodies) by Marshal)
Urine - High protein diet  Normal AER = 0-20 ug/min
- Cranberry juice = a treatment for UTI  Microalbuminuria = 20 – 200 ug/min (or 30-300 mg/24hrs)
- Emphysema, dehydration, diarrhea, acid-producing bacteria  Clinical albuminuria = >200 ug/min
(E. coli), medications - Orthostatic/Cadet/Postural proteinuria
Causes of - Renal tubular acidosis o Proteinuria when standing due to increased pressure to renal veins
Alkaline - Vegetarian diet o Increased venous pressure causes renal congestion and glomerular
Urine - After meal = due to alkaline tide changes
- Vomiting o Monitored every 6 months and re-evaluated as necessary
- Old specimens, hyperventilation, presence of urease-
producing bacteria Orthostatic Proteinuria Clinical Proteinuria
First morning - +
2 hours after standing + +

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- Other Causes SULFOSALICYLIC ACID PRECIPITATION TEST (a.k.a. EXTON'S TEST)

o Nephrotic syndrome - A cold precipitation test that reacts equally with all forms of protein
o Toxic agents - Reagent = Exton's reagent (3% SSA + sodium sulfate)
o Dehydration - Procedure:
o Strenuous exercise o 3 mL of 3% SSA+ 3 mL centrifuged urine → 10 mins incubation → (+)
o Hypertension Cloudiness
o or 3 mL of 7% SSA + 1l mL centrifuged urine → 10 mins incubation → (+)
o Amyloidosis Cloudiness
o Pre-eclampsia
B. TUBULAR PROTEINURIA Grade Turbidity Range (mg/dL) Range
- Originally discovered in workers exposed to cadmium dust (a heavy metal) (Strasinger) (Henry)
- Normally filtered albumin can no longer be reabsorbed Neg No increase in turbidity <6 ≈ 5 mg/dL
o Fanconi's syndrome Trace Noticeable turbidity 6 - 30 ≈ 20 mg/dL
o Toxic agents / heavy metals If viewed from top, circle is visible in
o Viral infections test tube bottom
1+ Distinct turbidity with no granulation 30 – 100 ≈ 50 mg/dL
If viewed from top, circle not visible
MICRAL TEST in test tube bottom
- Test for microalbuminuria Can read newsprint through mixture
- A strip employing antibody-enzyme conjugate that binds albumin 2+ Turbidity with granulation but NO 100 – 200 ≈ 200
- Principle: Enzyme immunoassay flocculation mg/dL
- Reagents: Gold-labeled antibody, -galactosidase, Chlorophenol red Cannot read newsprint through
galactoside mixture
- Sensitivity: 0 -10 mg/Dl 3+ Turbidity with granulation AND 200 – 400 ≈ 500
- (-) = White; (+) = Red [60 seconds] flocculation mg/dL
4+ Clumps of protein > 400 ≈ 1.0 g/dL
- Interference: False (-) = Dilute urine

IMMUNODIP TEST FOR MICROALBUMINURIA

- Principle: Immunochromographics
- Sensitivity: 1.2 -8.0 mg/dL
- Reagents: Antibody coated blue latex particles
- Interference: False (-) = Dilute urine

ALBUMIN: CREATININE RATIO - CLINITEST MICROALBUMIN STRIPS/

MULTISTIX-PRO
- 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: COMPARISON OF REAGENT STRIP AND SSA PROTEIN TEST RESULTS
o Albumin = 10 - 150 mg/L (Brunzel, 4th Ed.)
o Creatinine = 10 - 300 mg/dL (0.9-26.5 mmol/L) Strip SSA Possible explanation
- Interferences Result result
o Visibly bloody/abnormally colored urine + - - Highly buffered alkaline with no albumin present - false-
o Creatinine = Cimetidine - False (+) positive reagent strip
- Highly buffered alkaline with albumin present false -
negative SSA test
- To differentiate, acidify urine to pH - 5.0 and retest
POST RENAL PROTEINURIA - + - Proteins other than albumin present
(“AFTER”) - False (+)
- Lower UTI / inflammation o Radiographic contrast media (delayed reaction),
- Menstrual contamination Drugs and/or drug metabolites (tolbutamide,
- Injury / trauma penicillins, cephalosporins, sulfonamídes)
- Vaginal secretions - Examine precipitate microscopically: drugs and
radiographic dye form crystalline precipitates; whereas
- Prostatic fluid / spermatozoa
protein precipitates are amorphous

REAGENT STRIP REACTION for PROTEIN (60 seconds) - Large volumes of urine can produce a negative protein reaction despite significant
proteinuria because the protein present is being excessively diluted...
Principle Protein (Sorensen’s) error of indicators - S.G. should be considered in evaluating urine protein because a trace protein in a
- Contrary to the general belief that indicators produce specific dilute specimen is more significant than in a concentrated specimen.
colors in response to particular pH levels, certain indicators
change color in the presence of protein even though the pH 4. GLUCOSE (DEXTROSE)
of the medium remains constant. - Most frequently tested in urine
- Renal threshold: plasma concentration of a substance at which tubular
Indicator + Protein → (+) Blue-green reabsorption stops
(Yellow) (-) Yellow - Other Sugars in Urine: (identified by TLC)
Reagents - Multistix = Tetrabromphenol blue, citrate buffer at pH 3.0 1. Fructose (Levulose) = ↑ fruits, honey, syrup, fructose intolerance
- Chemstrip = Tetrachlorophenol tetrabromosulfonphthalein, 2. Galactose = ↑ infants with galactosemia
3. Lactose (Glu + Gal) = ↑ during pregnancy, lactation, strict milk diet,
citrate buffer at pH 3.0 lactose intolerance
Interferences - False (+) 4. Pentose = ↑ fruits, benign essential pentosuria
o Highly buffered alkaline urine, pigmented specimen, (Xylulose, Arabinose)
Phenazopyridine, Quaternary ammonium compounds 5. Sucrose (Glu + Fru) = ↑ Intestinal disorders, sucrose intolerance; it is
(detergents), antiseptics, chlorhexidine, loss of buffer a non-reducing sugar
from prolonged exposure of the reagent strip to the
specimen, high S.G. CLINICAL SIGNIFICANCE OF URINE GLUCOSE
- False (-) HYPERGLYCEMIA-ASSOCIATED RENAL-ASSOCIATED
o Proteins other than albumin, microalbuminuria ↑ Blood glucose Normal Blood glucose
Notes - Indicator is SENSITIVE to Albumin ↑ Urine glucose ↑ Urine glucose
- Correlations with other tests = Blood, Nitrite, Leukocytes, Causes: Causes
Microscopic - Diabetes Mellitus - Impaired tubular reabsorption of
- Cushing's syndrome (↑ cortisol) glucose
- Phaeochromocytoma (↑ - Fanconi syndrome
catecholamines) o Defective tubular
- Acromegaly (↑ growth hormone) reabsorption of glucose and
- Hyperthyroidism (↑ T3, T4) amino acids

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- It is possible for an individual to have hyperglycemia without glucosuria when the 5. KETONES
glomerular filtration rate is decreased due to certain diseases. Only limited - Result from increased fat metabolism due to inability to metabolize carbohydrates
amounts of glucose are able to pass into the ultrafiltrate, and the tubules are able - Renal threshold = 70 mg/dL
to reabsorb all the glucose presented to them. - Seen in:
o Type I DM
REAGENT STRIP REACTION for GLUCOSE (30 seconds) o Vomiting
o Starvation
Principle Double Sequential Enzyme Reaction o Malabsorption
𝑖𝑛𝑠𝑢𝑙𝑖𝑛
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 →
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐻2 𝑂2 - Normal: Glucose → Cells → Energy
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒 𝑐𝑒𝑙𝑙𝑠
𝐻2𝑂2 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 + 𝐻2𝑂 - Type I DM: Fats → Ketones (β-oxidation) → Energy
Reagents Multistix = Glucose oxidase, Peroxidase, Potassium iodide (blue
to green to brown) KETONE BODIES
Chemstrip = Glucose oxidase, Peroxidase, Tetramethylbenzidine 78% Beta-hydroxybutyric acid
(yellow to green) - major ketone but not detected in reagent strip
Interferences - False (+) = Oxidizing agents, detergents 20% Acetoacetic acid (AAA)/ Diacetic acid
- False (-) = High levels of ascorbic acid, ketones, high S.G., - parent ketone (1st ketone body formed)
LOW TEMP, improperly preserved specimen - detected by the reagent strip
Notes - Glucose strip was the 1st "dip and read" reagent strip 2% Acetone
developed by Miles, Inc., in 1950
- Sensitivity = 100 mg/dL (detects glucose only) REAGENT STRIP REACTION for KETONES (40 seconds)
- Other chromogens:
o Aminopropylcarbazole (yellow to orange-brown) Principle Sodium nitroprusside reaction (Legal’s test)
o o-toluidine (pink to purple)
- Correlations with other tests = Ketones, Protein Acetoacetic acid (& Acetone) + Na nitroprusside (& Glycine) →
(+) Purple
Reagents Na nitroprusside/nitroferricyanide, Glycine (Chemstrip)
Interferences - False (+) = Pthalein dyes, pigmented red urine, levodopa,
drugs with sulfhydryl groups
- False (-) = Improperly preserved specimens
COPPER REDUCTION TEST (CLINITEST/ BENEDICT'S TEST) Notes - Acetone is detected only when glycine is present
Information - Nonspecific test for reducing sugars - Correlations with other tests = Glucose
(Glucose, Galactose, Lactose, Fructose but NOT Sucrose)
Principle Copper reduction

Reducing sugars
CuSO4 → (+) Cu2O ACETEST (Tablet)
(Copper sulfate) (Copper Oxide) 1 gtts urine + Acetest tablet → (+) Purple color after 30 seconds
[Blue] [Brick-red]
Reporting (-) = clear blue color, blue precipitate may form - Composition = Sodium nitroprusside, Disodium phosphate, Glycine and
(Benedict’s Tr = bluish-green color Lactose
test) 1+ = green color, green or yellow precipitate
2+ = yellow to green color, yellow precipitate 6. BLOOD
3+ = yellow-orange color, yellow-orange precipitate
4+ = reddish-yellow color, brick red or red precipitate HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA
False-positive Causes = Reducing agents (ascorbic acid, uric acid) Cloudy red urine Clear red urine Must be at least
CuSO4 → Cu2O 25 mg/dL to show clear red
False-negative Causes = Oxidizing agents (detergents) (red-brown) urine
CuSO4 → Cu2O - Sensitive early -
Seen in: - Seen in:
indicator of renal Intravascular Rhabdomyolysis
TIP!! disease hemolysis o Muscular trauma
- False-positive (same action as the test principle) o Transfusion o Crush syndromes
- False-negative (opposite of the test principle) Seen in: reactions o Extensive exertion
- Glomerulonephritis o Hemolytic o Cholesterol-
CLINITEST TABLET PROCEDURE - Renal calculi, anemia lowering statin
5 gtts urine + 10 gtts H2O + Clinitest tablet → Read reaction 15 secs after bubbling tumors o Severe burns medications
stops - Strenuous o Brown recluse - Heme portion of the
exercise, trauma spider bites myoglobin (more toxic)
Pass-through phenomenon: - Microscopic = No is toxic to the renal
- Occurs when> 28/dL sugar is present Microscopic: Intact RBCS seen tubules
- Blue → Green → Yellow → Brick-red →→→ Blue or Green-brown RBCs - Heme portion of the - >1.5 mg/dL = renal
- Due to re-oxidation of cuprous oxide to cupric oxide and other cupric hemoglobin is toxic failure risk
complexes (green) to the renal tubules
- To prevent pass through, use 2 gtts urine (use separate color chart to interpret *Lysis of RBCs in the urine usually shows a mixture of hemoglobinuria and hematuria.
the reaction)
The tablets contain: HEMOGLOBIN VS. MYOGLOBIN
TEST HEMOGLOBIN MYOGLOBIN
- CuSO4 = main reacting agent 1. Plasma examination Red/Pink plasma Pale yellow plasma
- Na citrate = for heat production (hemoglobin is not (myoglobin is rapidly
- NaCO3 = eliminates interfering O2 immediately excreted in urine)
- NaOH = for heat production excreted in urine)

SUMMARY OF GLUCOSE OXIDASE AND CLINITEST REACTIONS ↓ Haptoglobins ↑ CK and aldolase

GLUCOSE CLINITEST INTERPRETATION activity
OXIDASE 2. Blondheim’s test Precipitated Not precipitated
1+ positive Negative Small amount of glucose present (Ammonium sulfate test)
- No longer clinically useful Negative for blood Positive for blood in
4+ positive Negative Possible oxidizing agent interference on reagent
strip in the reagent strip the reagent strip
Procedure:
Negative Positive Non-glucose reducing substance present - Urine + 2.8g NH4 Sulfate
Possible interfering substance for reagent strip (ex: (80% satd.)
Ascorbic acid) - Allow the mixture to sit for 5
mins
- Filter/Centrifuge
- Test supernatant for blood
with a reagent strip

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REAGENT STRIP for BLOOD (60 seconds) WATSON-SCHWARTZ TEST

- Differentiate urobilinogen (UBG), porphobilinogen (PBG) and other Ehrlich-
Principle Pseudoperoxidase activity of hemoglobin reactive compounds (ERC)
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛
- Uses extraction with organic solvents: Chloroform & Butanol
𝐻2𝑂2 + 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 + 𝐻2𝑂 - “UCBU” = Urine – Chloroform; Butanol - Urine
(yellow) Pseudoperoxidase (↑ Green to Blue ↑↑)
Reagents - Multistix = Diisopropylbenzene dehydroperoxide
tetramethylbenzidine
- Chemstrip = Dimethyldihydroperoxyhexane
tetramethylbenzidine
Interferences - False (+) = Strong oxidizing agents, bacterial peroxidases,
menstrual contam.
- False (-) = High SG, crenated cells, formalin, captopril, high
concentrations of nitrite, ascorbic acid (>25 mg/a), unmixed
specimens
Notes - Uniform green /blue color = Hemoglobin/ Myoglobin
- Speckled/spotted = Hematuria (Intact RBCs)
- Chemstrip contains iodate overlay that eliminates ascorbic
acid interference
- Hemoglobin level of> 10 mg/dL produces a positive protein
reagent strip reaction
- Correlations with other tests = Protein, Microscopic

HOESCH TEST
- Inverse Ehrlich reaction: reagent volume is more abundant than urine volume
- Rapid screening test for porphobilinogen (> 2 mg/dL)
7. BILIRUBIN
Procedure:
- Conjugated bilirubin (CB) - water soluble - 2 gtts urine + 2 mL Hoesch reagent (Ehrlich's rgt in 6M or 6N HCl) → (+) Red
- 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)

REAGENT STRIP REACTION for BILIRUBIN (30 seconds)

Principle Diazo reaction

Bilirubin diglucoronide (CB) + Diazonium salt → Azodye

Reagents - Multistix = 2,4-dichloroaniline diazonium salt
- Chemstrip = 2,6-dichlorobenzene diazonium salt
Interferences - False (+) = Highly pigmented urines, phenazopyridine,
indican, metabolite of Lodine
- False (-) = Specimen exposure to light, high conc., of nitrite,
ascorbic acid (25 mg/dL)
Notes - (+) Tan or Pink to Violet
- Correlations with other tests = Urobilinogen

Condition Blood Urine Urine

Bilirubin Urobilinogen
ICTOTEST (Tablet) (CB)
10 gtts urine + Ictotest tablet + 2 gtts H2O → (+) Blue to purple color after 60 Extravascular hemolytic disease ↑ UB Negative +++
seconds (Pre-hepatic jaundice)
- Confirmatory test; more sensitive than strip with less interference Liver damage ↑ +/- ++
- Composition = p-nitrobenzene-diazonium p-toluenesulfonate, SSA, Na2CO3, (Hepatic jaundice) UB/CB
boric acid Bile duct obstruction ↑ CB +++ -/↓
(Post-hepatic or Obstructive jaundice) Strip = N
8. UROBILINOGEN (UBG)
Clinical Significance
- Bile pigment that resulted from hemoglobin degradation
- ... of Bilirubin = only CB can appear in the urine when the normal degradation cycle is
- Normal value = <1 mg/dL or Ehrlich Unit disrupted by bile duct obstruction (post-hepatic jaundice) (e.g., gallstones or cancer) or
- Specimen = Afternoon urine (2-4 PM) when the integrity or the liver is damaged (hepatic jaundice), allowing leakage of CB
into the circulation. Hepatitis and cirrhosis are common examples of conditions that
REAGENT STRIP for UROBILINOGEN (60 seconds) produce liver damage resulting in bilirubinuria. Not only does the detection of urinary
bilirubin provide an early indication of liver disease, but also its presence or absence
Principle Ehrlich reaction can be used in determining the cause of clinical jaundice. This determination can be
even more significant when bilirubin results are combined with urinary UBG. Jaundice
due to increased destruction of red blood cells does not produce bilirubinuria. This is
Urobilinogen (and Ehrlich-reactive compounds) + PDAB → (+) because the serum bilirubin is present in the unconjugated form and the kidneys cannot
Red excrete it.
Reagents - Multistix = p-dimethylaminobenzaldehyde (PDAB or Ehrlich - … of Urobilinogen (UBG) = Increased urine UBG (greater than 1 mg/dL) is seen in liver
reagent) disease and hemolytic disorders. Measurement of urine UBG can be valuable in the
- Chemstrip = 4-methoxybenzene-diazonium-tetrafuoroborate detection of early liver disease, however, studies have shown that when UBG tests are
routinely performed, 1% of the non-hospitalized population and 9% of a hospitalized
(specific for UBG) population exhibit elevated results. This is frequently caused by constipation.
Interferences - False (+) = Ehrlich-reactive comp, (porphobilinogen, indican, Impairment of liver function decreases the ability of the liver to process the UBG
methyldopa, procaine sulfonamides, p-aminosalicylic acid recirculated from the intestine The excess UBG remaining in the blood is filtered by the
chlorpromazine), pigmented urine kidneys and appears in the urine. The clinical jaundice associated with hemolytic
- False (-) = Old specimens, preservation in formalin, high disorders results from the increased amount of circulating UB. This UB is presented to
the liver for conjugation, resulting in a markedly increased amount of CB entering the
concentrations of nitrite intestines. As a result, increased UBG is produced, and increased amounts of UBG are
Notes - Correlations with other tests = Bilirubin reabsorbed into the blood and recirculated through the kidneys where filtration takes
place. In addition, the overworked liver does not process the reabsorbed UBG as
efficiently, and additional UBG is presented for urinary excretion. Although it cannot be
determined by reagent strip, the absence of UBG in the urine and feces is also
diagnostically significant and represents an obstruction of the bile duct that prevents
the normal passage of bilirubin into the intestine. An additional observation is the
production of pale (acholic) stools as the result of the lack of urobilin.
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9. NITRITE REVIEW!!!
- Significance: Rapid screening test of UTI or bacteriuria 1. Correct usage and storage of reagent 6. Reading time for blood
- Nitrate converters are generally Gram-negative bacilli, such as the strips, except: reagent pad
a. Closed tightly a. 30 seconds
Enterobacteriaceae b. Keep at room temperature b. 10 seconds
- Specimen: 4-hour collection or first morning urine (preferred) c. Blotted facing down the tissue c. 40 seconds
d. Stored in a dark container d. 60 seconds
REAGENT STRIP REACTION for NITRITE (60 seconds) e. 120 seconds
2. Principle of protein reagent strip f. 45 seconds
Principle Greiss reaction (Specific for nitrite) a. Greiss reaction
b. Diazo reaction 7. Positive color reaction
c. Legal’s test for bilirubin reagent pad:
p-arsanilic acid (or sulfanilamide) + Nitrite → Diazonium salt
d. Double indicator system a. Violet
Diazonium salt + Tetrahydrobenzoquinolin → (+) Uniform pink e. Ehrlich reaction b. Cherry red
Reagents - Multistix = p-arsanilic acid, tetrahydrobenzo(h)-quinolin-3-ol f. Error of indicators c. Uniform pink
- Chemstrip = Sulfanilamide, hydroxytetrahydro d. Blue
benzoquinoline 3. Normal value for albumin excretion rate
Interferences - False (+) = Improperly preserved specimens, highly (AER) 8. Specific gravity of 9%
pigmented urine a. 0-20 mg/min sucrose
- False (-) = Non-reductase-containing bacteria, insufficient b. 20-200 ug/min a. 1.010
contact time bet. bacteria & urinary nitrate, lack of urinary c. >200 mg/min b. 1.034
d. 0-20 ug/min c. 1.022
nitrate, large quantities of bacteria converting nitrite to e. 20-200 mg/min d. 1.000
nitrogen, antibiotics, high ascorbic acid, high SG e. 1.015
Notes - Pink spots/edges = considered NEGATIVE 4. SSA grading = 100-200 mg/dL
- (+) Nitrite corresponds to 100,000 organisms/mL a. 1+ 9. Urine volume required
- If the nitrite test area shows a negative reaction, UTI cannot b. 3+ by the Yellow IRIS:
be ruled out c. Trace a. 1 mL
- Some UTIs are caused by Gram (+) cocci & yeasts they lack d. 2+ b. 2 mL
nitrate reductase enzymes e. Negative c. 7 mL
f. 4+ d. 15 mL
- Dietary nitrate can be found in green vegetables
- Correlations with other tests = Protein, Leukocytes, 5. Reagents in the nitrite pad: 10. Percentage of diacetic
Microscopic a. p-dimethylaminobenzaldehyde acid
b. Sodium nitroferricyanide a. 20%
c. 4-methoxybenzene-diazonium- b. 78%
tetrafluoroborate c. 2%
d. Indoxylcarbonic acid ester d. 0%
e. Tetrahydrobenzoquinoline
10. LEUKOCYTES
- Significance:
o Urinary tract infection or inflammation Answer key: C, F, D, D, E, D, A, B, B, A
o Screening of urine culture specimens

REAGENT STRIP REACTION for LEUKOCYTES (120 seconds)

MICROSCOPIC EXAMINATION OF URINE
Principle Leukocyte esterase
𝑙𝑒𝑢𝑘𝑜𝑐𝑦𝑡𝑒 𝑒𝑠𝑡𝑒𝑟𝑎𝑠𝑒 - Abnormalities in the physical & chemical portions of urinalysis play a primary role
𝐼𝑛𝑑𝑜𝑥𝑦𝑙𝑐𝑎𝑟𝑏𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 𝑒𝑠𝑡𝑒𝑟 → 𝐼𝑛𝑑𝑜𝑥𝑦𝑙 + 𝐴𝑐𝑖𝑑 𝑖𝑛𝑑𝑜𝑥𝑦𝑙
+ 𝐷𝑖𝑎𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡 → (+) 𝑃𝑢𝑟𝑝𝑙𝑒 in the decision to perform a microscopic analysis, thus the use of the term
Reagents - Multistix = Derivatized pyrrole amino acid ester, Diazonium macroscopic screening (chemical sieving)
salt
- Chemstrip = Indoxylcarbonic acid ester, Diazonium salt URINE SEDIMENT PREPARATION
Interferences - False (+) = Strong oxidizing agents, formalin, highly 1. Transfer 10-15 mL of urine in a test tube (recommended volume = 12 mL)
pigmented urine, nitrofurantoin 2. Centrifuge tube at 400 RCF for 5 minutes
- False (-) = High concentrations of protein, glucose, oxalic 3. Decant urine (0.5 or 1.0 mL urine remains in the tube)
acid, ascorbic acid, gentamicin, cephalosporins, 4. Transfer 20 uL (0.02 ml) sediment to glass slide with 22 x 22 mm coverslip
tetracyclines, inaccurate timing 5. Examine microscopically (10 LPE 10 HPE, under reduced light)
Notes - With esterase: Neutrophil, Eosinophil, Basophil, Monocyte, KEEP IN MIND!
Histiocyte, Trichomonas - If <12 mL urine is available for microscopy, centrifuge 3 mL of it
- No esterase: Lymphocyte - If <3ml urine is available, examine sediment without centrifugation
- Strip can detect lysed WBCs - RCF = 105 x radius in centimeters x RPM2
- Trichomonas, Chlamydia, yeast, & interstitial nephritis - Slides are 1st examined under LPO to detect casts
produce pyuria w/o bacteriuria
- Use HPO for further identification of casts
- Correlations with other tests = Protein, Nitrite, Microscopic

ADDIS COUNT
- Quantitative measure of formed elements of urine using hemacytometer
- Specimen = 12-hour urine
11. ASCORBIC ACID (Vitamin C) - Preservative = Formalin
- Water-soluble vitamin
- Dietary sources include citrus fruits & vegetables (tomatoes, green peppers, Normal values:
cabbage, leafy greens) - RBCs = 0-500,000/12-hr urine
- Excreted as ascorbic acid or its principal metabolite, oxalate - WBCs & ECs = 0-1,800,000/12-hr urine
- Strong reducing substance - Hyaline casts = 0-5,000/12-hr urine
- Interferes with reagent strip that use hydrogen peroxide or diazonium salt
- Causes false-negative reactions on (“BB LNG”): QUICK FACTS ABOUT THE MICROSCOPE!
o Blood - First lens system = located in the objective & is adjusted to be near the specimen
o Bilirubin - Second lens system = located in the eyepiece (ocular lens)
o Leukocytes - Resolution = ability to distinguish 2 small objects that are a specific distance apart
o Nitrite - Parfocal = microscopes requiring minimum adjustment when switching objectives
o Glucose - Camel-hair brush = used to remove dust on the optical surface of the microscope
- 11th Reagent Pad: - Lens paper = Used to clean the optical surfaces of the microscope
o Ascorbic acid (>5 mg/dL) + Phosphomolybdate → (+) Molybdenum blue - Commercial lens cleaner = used to clean any contaminated lens
- Brands (Henry): - 10 remove oil on lens, use dry lens paper, then lens paper moistened w lens
o Cstix = 10 seconds reading time cleaner
o Stix = 60 seconds reading time - Using xylene to remove oil on lens is not recommended due to its toxic fumes
o Others = vChem, Urispec GP + A, and Merckoquant
- GC-MS = More accurate quantitative method (Henry)

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BASIC COMPONENTS OF THE STANDARD LIGHT MICROSCOPE SEDIMENT STAINS

(Steininger, 1992 p. 41)
Sternheimer - Malbin (SM) -
Delineates structure & contrasting colors of
(Crystal violet + Safranin O) the nucleus & cytoplasm
Available as KOVA & Sedi - Identifies WBCs, epithelial cells and casts
stain - Most commonly used stain
Toluidine blue - Enhances nuclear detail
(Supravital stain) - Differentiates WBCs from RTE cells
2% acetic acid - Lyses RBCs, enhances nuclei of WBCs
- Distinguishes RBCs from WBCs, yeast, oil
droplets & crystals
Lipid stains - Stains triglycerides and neutral fats orange-
(Oil Red O and Sudan III) red (cannot stain cholesterol)
- Identifies free fat droplets & lipid-containing
cells & casts
Gram stain - Differentiates Gram-positive & Gram negative
bacteria
- Identifies bacterial casts
Hansel stain - Stains eosinophilic granules
(Eosin Y + Methylene blue) - Identifies urinary eosinophils
Prussian blue (Rous test) - Stains structures containing iron
- Identifies hemosiderin granules
Phenathridine (orange) - Stains DNA
Carbocyanine (green) - Stains nuclear membranes. mitochondria &
cell membranes
*These stains are used by Sysmex UF-100 Urine Cell Analyzer (Strasinger, 5th Ed p.
262)

SEDIMENT CONSTITUENTS
Biological or Organized Sediment (Cells and Casts)

CELLS

1. RBCs (Hematuria)
o NV = 0-2 or 0-3/HPF
Diopter Rings Adjust for focusing difference between eyes o Smooth, non-nucleated, biconcave disks
Rubber Eyeguard Adjust for comfort o Hypertonic urine = Crenate/Shrink
Eyepiece Tube Clamp Screw Loosen to rotate head o Hypotonic urine = Swell/Hemolyze (Ghost cell)
Reverse Facing Nosepiece For ease in specimen manipulation o Glomerular membrane damage = Dysmorphic, with projections, fragmented
Revolving Nosepiece Use to rotate objectives o Sources of error = Yeasts, Oil droplets, Air bubbles, Monohydrate calcium
Objectives Lenses which form primary magnification (initial oxalate crystals
image of specimen) o Remedy = Add 2% acetic acid. It will lyse the RBCs but not the others
Field Diaphragm Aperture diaphragm which restricts area of
illumination
Field Diaphragm Control Adjusts size opening of field diaphragm 2. WBCs (Pyuria or Leukocyturia)
Ring o NV = 0-5 or 0-8/HPF
Coarse Focus Knob Brings slide into view o Larger than RBCs
Fine Focus Knob Sharpens image o Increased number indicates presence of infection or inflammation
Lamp Socket Holds light source o Neutrophils
Interpupillary Distance Scale Indicates distance between eyes  Most predominant
Eyepieces Rotate to adjust for interpupillary distance  Granulated and multilobed
Magnify image (x10) formed by objective lens  In hypotonic urine, they swell, and granules undergo Brownian
Slide Holder Holds slide in place movement, producing a sparkling appearance (Glitter cells)
X/Y Travel Knobs Moves slide on stage  When dying, form blebs & finger-like projections (myelin forms)
Condenser Focus Knob Focuses light onto slide o Eosinophils
Stage Holds specimen  Normal value = 1%
Stage Clamp Screw Loosen to remove stage  Significant = >1% (associated w/ drug-induced interstitial nephritis)
Condenser Control Ring Adjusts size opening of condenser o Mononuclear cells (Lymphocytes, monocytes, macrophages, histiocytes)
Condenser Aperture diaphragm that controls light
 Normally present in small numbers
Condenser Centering Centers the field of view
Screws  ↑ Lymphocytes = Renal transplant rejection
Brightness Control Dial Turns microscope on/off; adjusts light intensity  ↑ Monocytes, histiocytes = chronic inflammation & radiation therapy

KEEP IN MIND!
MICROSCOPIC TECHNIQUES Using Sternheimer-Malbin Stain:
Bright-field (BF) - For routine urinalysis - Glitter Cells (Pale Blue)
Microscopy - Leukocytes (Pale Pink)
Phase-contrast - Enhances visualization of translucent elements (1.e. with
(PC) Microscopy low refractive indices [e.g. casts]) 3. Epithelial Cells
- To convert BF into PC, replace objective lens & o Squamous epithelial cell (S.E.C.)
condenser with PC objective lens & PC condenser  Largest cell w/ abundant, irregular cytoplasm & prominent nucleus
Polarizing - Detects the presence or absence of birefringence  Cell size is about 30-50 um (5-7x the size of an RBC)
Microscopy - Birefringence is the ability of an element to refract light in
 The nucleus is about the size of an RBC
2 dimensions at 90o to each other
- For identification of cholesterol in oval fat bodies, fatty  From linings of vagina, female urethra & lower male urethra (more
casts and crystals commonly found in female patients)
- To convert BF into polarizing, add 2 filters (1 below the  Variation = Clue cells
condenser, 1 between objective & oculars)  S.E.C. covered with Gardnerella vaginalis
Dark-field (DF) - For identification of Treponema pallidum  Associated with bacterial vaginosis
Microscopy - To convert BF into DF, replace the condenser with a DF o Transitional epithelial (Urothelial/Bladder) cell (T.E.C.)
condenser that contains an opaque disk  Cell size is about 20-30 um (4-6x the size of an RBC)
Fluorescence - For visualization of fluorescent substances and  Spherical, polyhedral or caudate with centrally located nucleus
Microscopy microorganisms
 Derived from the renal pelvis, calyces, ureter, urinary bladder & upper
Interference- - 3-D microscopy-image & layer-by-layer imaging of a
contrast specimen male urethra
Microscopy - Bright-field microscopes can be adapted for  Increased following catheterization: may be seen singly, in pairs, or in
interference-contrast microscopy clumps (syncytia)
- Two types:  If exhibiting abnormal morphology: malignancy or viral infection
o Nomarski (Differential interference contrast)
o Hoffman (Modulation contrast)

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o Renal tubular epithelial (R.T.E.) cell REVIEW!!!

 Most clinically significant epithelial cell 1. Ten (10) calcium oxalate crystals were seen 3. Few bacteria
 Origin: Nephron per HPF. How do you report this finding? a. 0-10
 Size is 3-5x the size of RBCs a. Rare b. 10-50
 Rectangular, polyhedral, cuboidal or columnar w/ eccentric nucleus b. Few c. 50-200
 Sometimes bilirubin-stained or hemosiderin-laden c. Moderate d. >200
d. Many
 RTE cells from the DCT may be mistaken for WBCs
 >2 RTE/hpf indicates tubular injury 2. Nine (9) bacteria were seen per HPF. How do
 VARIATIONS you report this finding?
 Oval Fat Body (Renal tubular fat bodies) a. Rare
o Lipid containing RTE cell (may also be a b. Few
monocyte/macrophage) c. Moderate
o Seen in lipiduria (Ex. nephrotic syndrome) d. Many
o Identified by:
 Lipid stains (TAG and neutral fats) Answer key: C, B, B
 Polarizing microscope (Cholesterol - "MALTESE CROSS"
formation)
CASTS
 Bubble cell
o RTE cell with non-lipid vacuoles - Excretion is termed cylindruria
o Injured cells in which the endoplasmic reticulum has dilated - Unique to the kidney
prior to cell death - Represents a biopsy of an individual tubule
o Seen in acute tubular necrosis - The most difficult & the most important urinary sediment constituent
- Primarily formed in the = DCT and collecting duct
4. Bacteria - Major constituent = Uromodulin/THP (produced by RTE cells)
o True UTI = Bacteria + WBCs (If bacteria only = contamination or old - Other proteins such as albumin & immunoglobulins are also incorporated into the
specimen) cast matrix
o Enterobacteriaceae (Ex. E. coli) = most common cause of UTI - Cylindroids have the same significance as Casts (a cast with a tail)
o Staphylococcus, Enterococcus - Protein gels more readily under conditions of urine-flow stasis, acidity and
o Motility differentiates them from amorphous urates & phosphates presence of Na+ and Ca2+
- A cast structure should have an even & definite outline, parallel sides and two
5. Yeasts rounded ends
o True yeast infection = Yeast + WBCs (If yeast only = contamination) - Has uniform diameter (about 7-8x the diameter of RBCs)
o Small, refractile oval structure that may or may not bud - Examination is performed along the coverslip edges with subdued light
o Branched, mycelial forms are seen in severe infections
o Candida albicans = seen in DM and vaginal moniliasis
FORMATION OF CASTS
6. Parasites 1. Aggregation of uromodulin into individual protein fibrils attached to RTE cells
o Trichomonas vaginalis 2. Interweaving of protein fibrils to form a loose fibrillar network
 Most frequently encountered parasite in urine 3. Further protein fibril interweaving to form a solid structure
 Pear-shaped flagellate with jerky motility 4. Possible attachment of urinary constituents to the solid matrix
 Agent of Ping-Pong disease 5. Detachment of protein fibrils from the epithelial cells
 Reported as rare, few, moderate, or many per HPF 6. Excretion of the cast
 When not moving, may resemble WBC, T.E.C. or R.T.E. cell
o Enterobius vermicularis egg
 Most common fecal contaminant
o Schistosoma haematobium egg
 Blood fluke with terminal spine
 Causes hematuria
 Associated with bladder cancer
o Other parasites include: Trichuris, Strongyloides, Giardia, various amoebae
o Various insects or “bugs" (lice, fleas, bedbugs, mites, and ticks)

KEEP IN MIND!
- Urinary Bladder Cancer Markers (Specific)
o NMP = Nuclear Matrix Protein
o BTA = Bladder Tumor Antigen

7. Spermatozoa
o Oval, slightly tapered head 1. HYALINE CAST
o Long, flagella-like tail o Prototype cast (beginning of all types of cast)
o After sexual intercourse o Most frequently encountered & the most difficult cast to discover
o Colorless and translucent
8. Mucus Threads o Normal value = 0-2/LPF
o Has low refractive index o Physiologic Stress = strenuous exercise
o Major constituent: Tamm-Horsfall protein (Uromodulin) o Pathologic = Glomerulonephritis, pyelonephritis, CHF, CKD
MICROSCOPIC QUANTITATIONS (Strasinger)
2. RBC CAST
- Quantitate an average of 10 representative fields.
o Most fragile cast
- Do not quantitate budding yeast, mycelia elements, Trichomonas, or sperm,
o Indicates bleeding within the nephron
but do note their presence with the appropriate LIS code
o Easily identified by its orange-red color
Quantitated None Rare Few Moderate Many
o Significance = Glomerulonephritis, strenuous exercise
Epithelial cells per LPF 0 0-5 5-10 20-100 >100 o Blood Cast
Crystals per HPF 0 0-2 2-5 5-20 >20  Contains hemoglobin from lysed RBCs
(normal)  Homogeneous appearance with orange-red color
Bacteria per HPF 0 0-10 10-50 50-200 >200  Same significance as RBC cast
Mucus threads per LPF 0 0-1 1-3 3-10 >10
Casts per LPF 0 Numerical ranges: 0-2, 2-5, 5-10, >10 3. WBC/LEUKOCYTE/PUS CAST
RBCs per HPF 0 Numerical ranges: 0-2, 2-5, 5-10, 10- o Indicates inflammation or infection within the nephron
WBCs per HPF 0 25, 25-50, 50-100, >100 o Resembles RTE cast. To distinguish, use phase microscopy and supravital
Squamous epithelial cells Rare, few, moderate, or many per LPF stain.
Transitional epithelial cells, Rare, few, moderate, or many per HPF o Significance = Pyelonephritis, acute interstitial nephritis
yeasts o Pseudoleukocyte Cast
Renal tubular epithelial cells Average number per 10 HPFs  Not a true cast (DO NOT report as cast!)
Oval fat bodies Average number per HPF  Clump of leukocytes
Abnormal crystals, casts Average number per LPF  Seen in lower UTI

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4. EPITHELIAL (RTE) CAST Calcium Oxalate (Dihydrate and Monohydrate)

o Cells visible on the cast matrix are smaller, round and oval cells - The most frequently observed urinary crystal
o Significance = Advanced tubular destruction, tubular damage - Dihydrate (Weddellite) = more common; envelope, bipyramidal, octahedron
- Monohydrate (Whewellite) = oval/dumbbell
5. BACTERIAL CAST - ↑ Foods rich in oxalic acid (tomato, asparagus) and ascorbic acid
- Ascorbate when metabolized becomes oxalate and combines with calcium to
o Identified by performing Gram stain form calcium oxalate
o Significance = Pyelonephritis - ↑ Ethylene glycol (anti-freeze agent) or methoxyflurane poisoning (MH)
- Soluble in dilute HCl
6. GRANULAR CAST (COARSE AND FINE)
o Granules are derived from the lysosomes of RTE cells during normal
OTHER NORMAL ACID CRYSTALS
metabolism (nonpathologic)
o Cells disintegrate when the cast is retained in the tubule before being flushed Calcium Sulfate
out - “Cigarette-butt” appearance
o Finely granular cast has a sandpaper appearance - Soluble in acetic acid
o Significance = Glomerulonephritis, pyelonephritis, stress, strenuous exercise Hippuric Acid
- Yellow-brown/colorless elongated prism
7. FATTY CAST - Soluble in water and ether
o Fat globules not stained by Sternheimer-Malbin stain (only the cast matrix is Acid Urates (Na+, K+, NH4)
- Rare form of uric acid
stained)
- Brown spheres or clusters
o Identification: - Resembles ammonium biurate, leucine & sulfamethoxazole crystals
 TAG & neutral fats = Lipid stains - Turns into uric acid after adding acetic acid
 Cholesterol = Polarizing microscope (“Maltese cross”) - Soluble in heat and alkali
o Significance = Nephrotic syndrome, Toxic tubular necrosis, Diabetes mellitus, Monosodium or Sodium Urates
crush injuries - Rare form of uric acid
- Tiny, slender, colorless needles; spherulite or beachball (rare)
8. WAXY CAST - ↑ Acute gout (intracellular crystals) and chronic gout (extracellular crystals)
o Final degenerative form of all types of casts
o Brittle, highly refractile, with jagged ends NORMAL ALKALINE CRYSTALS
o Ground glass appearance Amorphous phosphates (Ca2+ & Mg2+)
o Significance = Stasis of urine flow, chronic renal failure - Most common cause of turbidity in alkaline urine
- Fine, or ‘lacy' white precipitate (macroscopic)
9. BROAD CAST - Granular in appearance (microscopic)
o Often referred to as renal failure cast (Strasinger) - Soluble in dilute acetic acid
o Indicates destruction (widening) of the tubular walls Ammonium biurate
o Any type of cast can be broad (most common are granular & waxy) - Alkaline counterpart of uric acid and amorphous urates
- Yellow-brown thorny apples
o 2-6x wider than ordinary cast - Seen in old specimens
o Significance = Extreme urine stasis, renal failure - ↑ Presence of urea-splitting bacteria (urea ammonia)
10. MISCELLANEOUS CASTS - Turns into uric acid after adding HCl or acetic acid
- Soluble in acetic acid with heat
o Pigmented Cast
Triple Phosphate (Magnesium Ammonium Phosphate, Struvite)
 Hyaline matrix with coloration due to pigment incorporation - Colorless, prism-shaped or coffin-lid; fern-leaf (Harr)
 Incorporated bilirubin (golden-brown) - Feathery appearance when they disintegrate
 Hemoglobin or myoglobin (yellow to red brown) - Presence of urea-splitting bacteria (urea ammonia)
o Mixed Cellular Cast - Soluble in dilute acetic acid
 Cast containing multiple cell types Magnesium Phosphate
 Glomerulonephritis (RBC& WBC casts) - Colorless, elongated rectangular or rhomboid plates
 Pyelonephritis (WBC & RTE casts or WBC and bacterial casts) - End or corner may be notched
o Crystal Cast - Edges can be irregular or eroded
 Casts containing urates, calcium oxalate and sulfonamides are - Soluble in acetic acid
occasionally seen Calcium Phosphate (Apatite)
- Dibasic calcium phosphate (stellar phosphate)
 Deposition of crystals in the tubule or collecting duct o Colorless, flat plates thin prisms in rosette form
SEDIMENT CONSTITUENTS – Chemical or Unorganized Sediment (Crystals) o Rosettes may resemble sulfonamide crystals
- Monobasic calcium phosphate
o Irregular, granular-appearing sheets or plates
CRYSTALS o Less common than dibasic form
- Other forms:
- Excretion is termed crystalluria o Hydroxyapatite (basic calcium phosphate)
- The most recognized but the most insignificant part of urine sediment (Turgeon) o Brushite (calcium hydrogen phosphate)
- Formed by precipitation of urine solutes (salts, organic compounds, medication) - Soluble in dilute acetic acid
- Presence of crystals in fresh urine is most frequently associated w/ concentrated Calcium Carbonate
specimen (↑ S.G.) - Small, colorless, dumbbell, tetrads or spherical-shaped
- Factors that contribute to crystal formation: - Forms gas (effervescence) after adding acetic acid
o pH - Misidentified as bacteria
o Solute concentration
o Temperature ABNORMAL ACID CRYSTALS
- Usually reported as rare, few, moderate or many per HPF Cystine
- Abnormal crystals may be averaged and reported per LPF - Colorless, refractile hexagonal plates, often laminated
- Mistaken as hexagonal uric acid crystals
- Abnormal crystals generally require confirmation before they are reported to the - ↑ in Cystinuria and Cystinosis
physician
Uric acid Cystine
NORMAL ACID CRYSTALS Color Yellow brown Colorless
Amorphous Urates (Ca2+, Mg2+, Na+, & K+ urates) Solubility in ammonia Soluble Soluble
- Fluffy orange or pink sediment (brick dust) due to uroerythrin Solubility in dilute HCl Insoluble Soluble
- Yellow brown granules (microscopic) Birefringence Birefringent Not birefringent
- Clumps resemble granular casts (termed "pseudocasts") Cyanide-nitroprusside reaction Negative Positive
- Turns into uric acid after adding acetic acid Cholesterol
- Turns into ammonium biurate after adding ammonium hydroxide - Rectangular plate with notch in one or more corners (staircase pattern)
- ↑ in Gout, Chemotherapy - Resembles crystals of radiographic dye
- Soluble in heat and alkali - in Nephrotic syndrome (lipiduria)
Uric Acid - Soluble in chloroform
- Product of purine metabolism
Radiographic dye (Meglumine diatrizoate, Renografin, Hypaque
- Most pleomorphic: Rhombic (diamond), 4-sided flat plate (whetstone), lemon-
- Flat, four-sided plates often with a notched corner
shaped - Other forms include-long8 thin prisms or rectangles
- Wedges, barrel, rosettes, irregular plates, laminated forms are also seen - Resembles cholesterol crystals. To differentiate:
- Hexagonal forms may be mistaken as cystine crystals o Check patient history
- Present at pH <5.7 if pH is >5.7, it is in its ionized form as urate o Correlate with other UA results (Ex: S.G. >1.040 using refractometer)
- ↑ in Lesch-Nyhan syndrome, Chemotherapy, Gout - Soluble in 10% NaOH
- Soluble in alkali
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Tyrosine URINE SCREENING FOR METABOLIC DISORDERS

- Fine colorless to yellow needles in clumps or rosettes
- ↑ in Liver disease (more commonly found than leucine) 2 CATEGORIES OF AMINOACIDURIA
- Soluble in alkali or heat OVERFLOW TYPE RENAL TYPE
Leucine - ↑ Amino acid in blood - Normal Amino acid in blood
- Yellow-brown oily-looking spheres w/concentric circles & radial striations - ↑ Amino acid in urine - ↑ Amino acid in urine
- Precipitated with tyrosine after adding alcohol - Examples: PKU, MSUD, cystinosis - Due to defective tubular
- May resemble fat globules reabsorption of amino acids
- ↑ in Liver disease - Examples: Cystinuria, Fanconi's
- Soluble in hot alkali or alcohol syndrome
Bilirubin
- Clumped granules or needles with bright yellow color (Strasinger) Inborn Error of Metabolism (EM)
- Reddish brown needles that cluster in clumps or spheres (Turgeon) - Failure to inherit a gene that codes for a particular enzyme.
- Granules and plates have been observed (Brunzel) - No gene = No enzyme
- ↑ in Liver disease
- Soluble in acetic acid, HCI, NaOH, acetone and chloroform I. PHENYLALANINE -TYROSINE DISORDERS

ABNORMAL ACID CRYSTALS 1. PHENYLKETONURIA

Sulfonamide
- Fan-shaped needles, sheaves of wheat. rosettes, arrowheads, petals, round- 𝑃ℎ𝑒𝑛𝑦𝑙𝑎𝑙𝑎𝑛𝑖𝑛𝑒 ℎ𝑦𝑑𝑟𝑜𝑥𝑦𝑙𝑎𝑠𝑒
𝑃ℎ𝑒𝑛𝑦𝑙𝑎𝑙𝑎𝑛𝑖𝑛𝑒 → 𝑇𝑦𝑟𝑜𝑠𝑖𝑛𝑒
shaped, whetstones
- Mistaken as calcium phosphate crystals. To differentiate: - The most well-known of the aminoacidurias
o Calcium phosphate: soluble in acetic acid - (-) gene that codes for phenylalanine hydroxylase
o Sulfonamide = (+) Lignin test & Diazo reaction - Other forms are due to lack of tetrahydrobiopterin
- Possible tubular damage (may deposit in nephrons - ↑ Phenylpyruvic acid (a ketone) in urine
- Currently, they are modified and their solubility is no longer a problem - “Mousy” odor of urine, sweat and breath odor (due to phenylacetic acid)
- Soluble in acetone - May lead to severe mental retardation
- Forms: - Screening tests:
o Sulfamethoxazole = brown rosettes or spheres w/ irregular radial o FeCl3 tube test = (+) blue-green color
striations o Phenistix strip = (+) gray to gray-green
o Acetylsulfadiazine = yellow-brown sheaves of wheat w/ eccentric binding o Guthrie bacterial inhibition test
o Sulfadiazine = yellow-brown sheaves of wheat w/ eccentric binding - Confirmatory test: Ion exchange HPLC
Ampicillin
- Colorless needles that tend to form bundles following refrigeration GUTHRIE BACTERIAL INHIBITION TEST:
- ↑ in massive doses of penicillin
- B. subtilis is cultured w/ B2-thienylalanine (TE)
- B2-TE Inhibits the growth of B. subtilis.
OTHER ABNORMAL CRYSTALS
Hemosiderin - Phenylalanine counteracts the action of B2-TE
- Coarse, yellow-brown granules - (+) result: growth, (-): no growth
- Resembles amorphous urates
- (+) Rous test (Prussian blue stain)
Acyclovir 2. TYROSYLURIA/TYROSINEMIA
- May be seen in alkaline urine - (-) gene that codes for:
- Colorless slender needles o Type 1: Fumarylacetoacetate hydrolase (FAH)
- Strongly birefringent with polarized light o Type 2: Tyrosine aminotransferase
Indinavir Sulfate o Type 3: p-hydroxyphenylpyruvic acid dioxygenase
- Slender colorless needles or slender rectangular plates - May also be seen in severe liver disease
- Feather-like crystals that aggregate into wing-like bundles
- Arranged in fan-shaped or starburst forms, bundles, or sheaves - “Rancid butter” urine odor
- Associated with renal blockage & stone formation in HIV-positive individuals - Screening Tests
o FeCl3 tube test = (+) transient green
o Nitroso-naphthol = (+) orange-red
URINARY SEDIMENT ARTIFACTS - Confirmatory Tests
o Chromatography
- Starch granules o Quantitative serum assay of tyrosine
o Spheres with dimpled center
o "Maltese cross" formation on polarizing microscope PHENYLALANINE AND TYROSINE METABOLISM
 Oval fat bodies
 Fatty casts
 Fat droplets
 Starch granules
- Oil droplets = mistaken for RBCs
- Air bubbles = mistaken for RBCs
- Pollen grains = spheres with cell wall & concentric circles
- Hair and fibers = mistaken for casts
- Fecal contamination

REVIEW!!!
1. Nomarski microscope is a type of what microscopy?
a. Phase contrast
b. Brightfield
c. Polarizing
d. Fluorescent
2. Oval fat bodies are seen in:
a. Acute glomerulonephritis
b. Interstitial nephritis
c. Nephritic syndrome
d. Nephrotic syndrome
3. Colorless needles that tend to form bundles following refrigeration:
a. Uric acid
b. Sulfonamide
c. Ampicillin
d. Leucine

4. What are the forms of triple phosphate crystal?

5. Sequence of cast degeneration starting from the worst

Answer key: A, D, C
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- State laws require that blood be collected 24 hours after birth and before the 2. ARGENTAFFINOMA
newborn leaves the hospital - Tumor of argentaffin or enterochromaffin cells - produce serotonin (carried by
- "Testing for may substances is now performed using tandem mass platelets) → metabolized into 5-HIAA
spectrophotometry (MS/MS). It is capable of screening the infant blood sample for - Screening tests
specific substances associated with particular inborn error of metabolism." o FeCl3 tube test = (+) Blue-green
o Nitrosonaphthol with nitrous acid = (+) Violet
3. ALKAPTONURIA - Patient must not eat bananas, pineapples, tomatoes, avocados, chocolates,
- (-) gene that codes for Homogentisic acid oxidase walnuts, & plums (they ↑ serotonin)
- ↑ Homogentisic acid in blood and urine
- Urine darkens after becoming alkaline from standing at room temperature IV. CYSTINE DISORDERS
- Brown - or black-stained cloth diapers - “Sulfur” urine odor
- Reddish-stained disposable (plastic) diapers - Treatment = D-Penicillamine
- Homogentisic acid causes black pigmentation in the connective tissues and ears
(ochronosis) 1. CYSTINURIA
- Screening Tests - Renal type of aminoaciduria
o FeCl3 tube test = (+) transient blue - Defective tubular reabsorption of:
o Clinitest = (+) yellow precipitate o Cystine (the only one which crystallizes; least soluble)
o Alkalinization of fresh urine o Ornithine
- Confirmatory Tests o Lysine
o Paper/thin-layer chromatography o Arginine
o Capillary electrophoresis - Tests for Cystinuria and Cystinosis
o Brand's modification of Legal's nitroprusside
4. MELANURIA  Reagent = Cyanide nitroprusside
- Caused by melanoma (tumor involving melanocytes)  (+) Red-purple color
- Tumors secrete 5,6-dihydroxyindole, which oxidizes to melanogen then to melanin o Thin layer or ion-exchange chromatography
- Urine darkens upon air exposure o High-voltage electrophoresis
- Deficient production of melanin results in albinism
- Screening Tests 2. CYSTINOSIS
o FeCl3 tube test = (+) Gray/black ppt - Inborn error of metabolism → Overflow type
o Sodium nitroprusside test = (+) Red - (-) gene that codes for an enzyme responsible for cystine metabolism
o Ehrlich test = (+) Red - Types = Nephropathic cystinosis, intermediate cystinosis, and ocular cystinosis
- Cystine deposits in many areas of the body (BM, cornea, lymph nodes & internal
II. BRANCHED-CHAIN AMINO ACID DISORDERS organs)

3. HOMOCYSTINURIA
- Defects in the metabolism of methionine (leads to ↑ homocystine)
- (-) gene that codes for the enzyme cystathione β-synthase
- Detected by the Silver-nitroprusside test = (+) Red-purple color
1. MAPLE SYRUP URINE DISEASE (MSUD)
- Most common IEM in the Philippines V. PORPHYRIN DISORDERS (PORPHYRIAS)
- (-) 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

2. ORGANIC ACIDEMIAS
- Isovaleric acidemia = "sweaty feet" urine odor due to isovalerylglycine
o Glutaric acidemia also presents with a sweaty feet urine
- Propionic acidemia
- Methylmalonic acidemia = detected using p-nitroaniline test = (+) Emerald green
color

III. TRYPTOPHAN DISORDERS

- Disorders of porphyrin metabolism

1. INDICANURIA - Urine color
- Indigo blue urine color (upon air exposure) o red, purple, burgundy-red, purplish red, “portwine”
- Seen in: o Colorless in = lead poisoning (Harr, Henry)
o Hartnup disease (“Blue diaper syndrome”)
o Intestinal disorders Consider it porphyria if:
- Screening test: Obermayer's test - Red-tinged urine
o FeCl3 + Urine + Chloroform = (+) Violet color - Negative for blood reagent strip
- Diet and medications ruled out

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Colorless Dark Red or Purple, REVIEW!!!

Nonfluorescent Intensely Fluorescent 1. Urine darkens after becoming alkaline, what is present?
- Porphobilinogen - Uroporphyrin a. Melanin
- D-aminolevulinic acid - Coproporphyrin b. Methemoglobin
- Uroporphyinogen - Protoporphyrin c. Homogentisic acid
d. Methyldopa
- Coproporphyrinogen 2. What is the positive result in the Guthrie bacterial inhibition test:
- Protoporphyrinogen a. Growth around the disk
b. No growth around the disk
SCREENING TESTS c. Lysis of colonies
Specimen = Urine, Stool, Blood, Bile d. Clearing of the medium
Ehrlich's reaction Detects D-ALA, porphobilinogen 3. Cetyltrimethylammonium bromide test is a screening test for:
a. Phenylalanine
Fluorescence at 550- Tests for uroporphyrin, coproporphyrin, & protoporphyrin b. Tryptophan
600 nm (+) Violet/Pink/Red fluorescence c. Mucopolysaccharides
Free erythrocyte CDC- CDC-recommended test for lead poisoning d. Porphyrins
recommended test for 4. Positive color in the MPS test?
lead protoporphyrin a. Red
(FEP) b. Blue
c. Yellow
Disorder Enzyme Deficient Elevated Clinical d. Green
5. Urine color in lead poisoning:
Compound/s Symptoms a. Portwine
Acute Uroporphyrinogen ALA Neurologic b. Blue
intermittent synthase Porphobilinogen Psychiatric c. Brown/black
porphyria d. Colorless
Porphyria Uroporphyrinogen Uroporphyrin Photosensitivity
cutanea tarda decarboxylase
Congenital Uroporphyrinogen Uroporphyrin Photosensitivity Answer key: C, A, C, B, D
erythropoietic cosynthase Coproporphyrin
porphyria RENAL DISEASE
Variegate Protoporphyrinogen Coproporphyrin Photosensitivity
porphyria oxidase Neurologic CLASSIFICATIONS OF RENAL DISEASE
Erythropoietic Ferrocheletase Protoporphyrin Photosensitivity - Glomerular disorders
protoporphyria o Majority are of immune origin (immune complexes, IgG, IgA)
Lead poisoning -- ALA Photosensitivity o Common: proteinuria, hematuria, casts
Protoporphyrin - Tubular disorders
- Port wine urine color is more prevalent in the erythropoietic porphyrias - Interstitial disorders
- Lead po1soning inhibits ALA Synthetase and ferrocheletase enzymes
GLOMERULAR DISORDERS
DISORDER AND ETIOLOGY FINDINGS
VI. MUCOPOLYSACCHARIDE [MPS] DISORDERS
1 Acute Post-Streptococcal - Macroscopic hematuria,
(MUCOPOLYSACCHARIDOSIS)
Glomerulonephritis proteinuria, dysmorphic
- Impaired metabolism of mucopolysaccharides or glycosaminoglycans (protein + - Deposition of immune complex, RBCs, RBC casts,
polysaccharides, located in the connective tissues) formed in conjunction of Group A granular casts,
- Frequently found in urine are dermatan sulfate, keratan sulfate and heparan Streptococcus (S. pyogenes) infection - (+) ASO titer
sulfate on the glomerular membranes
2 Rapidly Progressive (Crescentic)
1. HURLER SYNDROME Glomerulonephritis
o A.k.a. Gargoylism or MPS Type I - Deposition of immune complexes from - Macroscopic hematuria
o MPS accumulate in the cornea of the eye systemic immune disorders (ex: SLE) - Proteinuria
o (+) Skeletal abnormalities & mental retardation on the glomerular membrane - RBC casts
2. HUNTER SYNDROME - Cellular proliferation of epithelial cells
o Sex-linked recessive, rarely seen in females inside the Bowman's capsule form
o (+) Skeletal abnormalities & mental retardation "crescents”
3 Goodpasture Syndrome
3. SANFILIPPO SYNDROME - Deposition of antiglomerular basement - Macroscopic hematuria
o Mental retardation is the only abnormality membrane antibody to glomerular and - Proteinuria
alveolar basement - RBC casts
SCREENING TESTS 4 Wegener's Granulomatosis
- Acid albumin test = (+) White turbidity - Anti-neutrophilic cytoplasmic auto- - Macroscopic hematuria
- Cetyltrimethylammoniumbromide (CTAB) Test = (+) White turbidity antibody (ANCA) binds to neutrophils - Proteinuria
- Mucopolysaccharide (MPS) Paper Test = (+) Blue color in vascular walls producing damage to - RBC casts
small vessels in the lungs and
VII. PURINE DISORDER glomerulus
- Perinuclear ANCA (p-ANCA) forms
LESCH-NYHAN DISEASE when neutrophils are fixed in ethanol
- (-) gene that codes for the enzyme hypoxanthine guanine - Cytoplasmic ANCA (c-ANCA) forms
phosphoribosyltransferase when neutrophils are fixed with
- ↑ Uric acid in the blood and urine formalin
- “Orange sand” in diapers 5 Henoch Schönlein Purpura
- Occurs in children following viral - Macroscopic hematuria
VIII. CARBOHYDRATE DISORDERS respiratory infections - Proteinuria
- Glucose strip and (+) Copper reduction test - Decrease in platelets disrupts vascular - RBC casts
- Melituria = presence of any sugar in urine integrity
6 Membranous Glomerulonephritis (MGN)
1. Galactosemia/Galactosuria - Thickening of glomerular membrane - Microscopic hematuria
o Inability to metabolize galactose to glucose following IgG immune complex - Proteinuria
o Enzymes absent: deposition associated with systemic
 Galactose-1-phosphate uridyl transferase (GALT) disorders
 Galactokinase 7 Membranoproliferative
 UDP-galactose-4-epimerase Glomerulonephritis (MPGN)
o ↑ Galactitol, galactonate and galactose-1-phosphate - Cellular proliferation affecting the - Hematuria
o Associated with infant failure to thrive, liver disorders, cataracts and capillary walls or the glomerular - Proteinuria
severe mental retardation basement membrane, possibly
2. Glucosuria = Diabetes Mellitus immune-mediated
3. Lactosuria = seen during pregnancy and lactation - Glomeruli have visible lobular
4. Fructosuria = associated with parenteral feeding appearance
5. Pentosuria = associated with ingestion of large amounts of fruit - “Tram track”
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8 Chronic Glomerulonephritis INTERSTITIAL DISORDERS

- Marked decrease in renal function - Hematuria DISORDER AND ETIOLOGY FINDINGS
resulting from glomerular damage - Proteinuria, Glucosuria 1 Cystitis (Lower UTI)
precipitated by other renal disorders - Cellular & granular casts - Ascending bacterial infection of - WBCs, Bacteria, NO CAST
- Progression to renal failure - Waxy and broad casts the urinary bladder - Microscopic hematuria
9 IgA Nephropathy (Berger's Disease) - Acute onset of urinary - Mild proteinuria, increased pH
- Deposition of lgA on the glomerular - Early stages: Hematuria frequency and burning
membrane resulting from increased - Late stages: See chronic 2 Acute Pyelonephritis (Upper UTI)
levels of IgA GN - Infection of the renal tubules & - WBCs, Bacteria
10 Minimal Change Disease, MCD interstitium related to - WBC casts, bacterial casts,
(Nil Disease/Lipoid Nephrosis) interference of urine flow to the - Microscopic hematuria
- Little cellular changes in the - Heavy proteinuria bladder, reflux of urine from the
glomerulus - Transient hematuria bladder (vesicoureteral reflux)
- Glomeruli look normal by light - Fat droplets & untreated cystitis
microscopy 3 Chronic Pyelonephritis
- Electron microscopy reveals loss of - Recurrent infection of the renal - WBCs, Bacteria, WBC casts,
podocyte foot processes tubules & interstitium caused Bacterial casts, granular casts
- Disruption of podocytes primarily in by structural abnormalities - Waxy and broad casts
children following allergic reactions & affecting the flow of urine - Hematuria, proteinuria
immunizations 4 Acute Interstitial Nephritis
- Associated with HLA-B12 antigen - Allergic inflammation of the - Hematuria, proteinuria
11 Focal Segmental Glomerulosclerosis renal interstitium in response to - WBCs (↑ eosinophils, >1%)
(FSGS) certain medications - WBC casts, NO BACTERIA
- Disruption of podocytes in certain - Proteinuria
numbers and areas of glomeruli, - Hematuria RENAL FAILURE
others remain normal - ↓ Glomerular filtration rate (< 25 mL/min)
- IgM and C3 are evident on the sclerotic - Azotemia (↑ BUN & Creatinine)
areas (using IF) - Electrolyte imbalance
12 Diabetic Nephropathy (Kimmelstiel- - (-) renal concentrating ability → Isosthenuria
Wilson Disease) - Proteinuria & renal glycosuria
- Most common cause of ESRD - Microalbuminuria - Telescoped sediment
- Deposition of glycosylated proteins on - + Micral test o Variety of casts seen in the same specimen (cellular, coarsely granular, finely
the glomerular basement membranes granular, waxy)
caused by poorly controlled blood o Simultaneous appearance of the elements of acute, chronic GN, and
glucose levels nephrotic syndrome
13 Alport Syndrome o ↑ casts (granular, waxy, broad)
- Genetic disorder showing lamellated - See Nephrotic Syndrome
and thinning of glomerular basement RENAL CALCULI/RENAL LITHIASIS
membrane - May form in the calyces and pelvis of the kidney, ureters, and bladder
14 Nephrotic Syndrome - Lithotripsy uses high-energy shock waves to break kidney stones into pieces
- Disruption of the electrical charges that produce the tightly fitting - Conditions Favoring the Formation of Renal Calculi:
podocyte barrier resulting in massive loss of proteins & lipids o pH
- The rate of proteinuria in nephrotic syndrome is >3.5 g/day o Chemical concentration
- Occurs in patients with MCD (in children), MGN (in adults), FSGS and o Urinary stasis
MPGN - Primary UA Finding = Microscopic hematuria
BLOOD URINE Serum Findings:
- ↓ Albumin - ↑ Albumin - Albumin, α1, gamma - globulins Renal Calculi Information/Description
- ↓ Lipase - ↑ Lipase - α2-macroglobulin Calcium oxalate - Major constituent of renal calculi
- ↑ Lipids - ↑ Lipids - β-globulin (LDL) calculi - Very hard, dark in color with rough surface
- ↑ Apo B100 Uric acid & Urate - Associated w/ increased intake of foods w/ high purine
- ↑ LDL and Urinalysis Findings: calculi content, and w/ UKD
VLDL - Albumin, a1, B, gamma-globulins - Yellowish to brownish red & moderately hard
- ↑ Chole & - (-) a2-macroglobulin Cystine calculi - Seen in hereditary disorders of cystine metabolism
TAG - Oval fat bodies - Yellow-brown, greasy & resembles an old soap
- Fatty casts - Least common calculi
- Waxy casts Phosphate calculi - Pale & friable
Triple phosphate - Accompanied by urinary infections involving urea-
TUBULAR DISORDERS calculi splitting bacteria (Proteus vulgaris)
DISORDER AND ETIOLOGY FINDINGS - Branching/staghorn calculi resembling antlers of a
1 Acute Tubular Necrosis deer
- Damage to renal tubular cells caused by - Microscopic hematuria, Rare Calculi:
ischemia or toxic agents proteinuria - Sulfonamide calculi
- Urine odor = “odorless” - RTE cells, RTE casts - Silica calculi = ingestion of silica over a long period of time
- Hyaline, granular, waxy - Triamterene calculi = insoluble diuretic; mustard-colored stones
and broad casts - Adenine calculi = associated with inherited enzyme deficiency & hyperuricemia
2 Uromodulin-associated Kidney Disease - Xanthine calculi = associated with a genetic disorder w/ an absence of xanthine
(UKD) - RTE cells oxidase
- Inherited defect in the production of - Hyperuricemia
normal uromodulin by the renal tubules
and increased uric acid causing gout
- Normal uromodulin is replaced by
abnormal forms that destroy the RTE cells
3 Fanconi Syndrome
- Generalized failure of tubular reabsorption - Glucosuria
in the proximal convoluted tubule - Possible cystine
crystals (amino acid)
4 Diabetes Insipidus
- Neurogenic DI = hypothalamus fails to - Low specific gravity
produce ADH - Polyuria (>15 L/day) - Methods for Calculi Analysis
- Nephrogenic DI = renal tubules fail to o Optical crystallography
respond to ADH o Radiograph diffraction
5 Renal Glucosuria o Infrared spectroscopy
- (N) Blood glucose = ↑ Urine glucose - Glucosuria o Electron beam analysis
- Defective tubular reabsorption of glucose o Mass spectroscopy

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REVIEW!!! AMNIOTIC FLUID

1. Thickening of the glomerular membrane following IgG immune complex - Present in the amnion – a membranous sac that surrounds the fetus
deposition? - The placenta is the ultimate source of amniotic fluid water and solutes
a. Membranous GN
b. Berger’s disease PRIMARY FUNCTIONS OF AMNIOTIC FLUID
c. Chronic GN
d. Nephrotic syndrome 1. Cushion for the fetus
2. What is the indicator of diabetic nephropathy? 2. Stabilizes temperature
a. Lipiduria 3. Allows fetal movement
b. Hematuria 4. Proper lung development
c. Microalbuminuria
d. Glucosuria AMNIOTIC FLUID VOLUME
3. (+) WBC, bacteria, but no cast - From fetal urine and lung fluid
a. Cystitis - Normal = 800-1,200 mL (3rd trimester)
b. Pyelonephritis
- During 1st trimester, 35 mL of amniotic fluid is derived primarily from the maternal
c. Interstitial nephritis
d. Tubular necrosis circulation
4. Renal calculi: yellow-brown, greasy and resembles an old soap: - Fetal urine = major contributor to the AF volume after the 1st trimester of
a. Calcium oxalate pregnancy
b. Phosphate
c. Uric acid POLYHYDRAMNIOS OLIGOHYDRAMNIOS
d. Cystine ↑ amniotic fluid volume (<800 mL) ↓ amniotic fluid volume (>1,200 mL)
Causes: Causes:
Answer key: A, C, A, D - Decreased fetal swallowing of urine - Increased fetal swallowing of urine
- Neural tube defects (ex. Spina - Membrane leakage
AMNIOTIC FLUID & hCG bifida) - Urinary tract deformities
- Others: fetal structural anomalies, - Others: congenital malformations,
HUMAN CHORIONIC GONADOTROPIN (hCG) cardiac arrhythmias, congenital premature amniotic membrane
- Produced by the syncytiotrophoblast cells of the placenta infections, chromosomal rupture, umbilical cord
- Peaks during 1st trimester of pregnancy (↑ blood, urine, amniotic fluid) abnormalities compression
- Composed of 2 subunits:
o Alpha = hCG, LH, FSH, TSH (identical subunits) SPECIMEN COLLECTION
o Beta = Confers specificity for hCG - Method of collection = Amniocentesis
o Up to 30 mL collected in sterile syringe
HOME-BASED HCG PREGNANCY TEST KIT o 2nd trimester amniocentesis = Assess genetic defects (Ex: Trisomy 21/Down
- Principle: Enzyme-immunoassay syndrome)
- Specimen: 1st morning urine o 3rd trimester amniocentesis = Fetal lung maturity (FLM), Fetal hemolytic
- Cut-off point: 25 mIU/mL disease (HDN)
- Anti-hCG source: Rabbit - Quadruple screening tests prior to performing amniocentesis:
o Alpha-fetoprotein
CAUSES OF FALSE-POSITIVE AND FALSE-NEGATIVE PREGNANCY TESTS o Human chorionic gonadotropin (hCG)
FALSE-POSITIVE FALSE-NEGATIVE o Unconjugated estriol (UE3)
- Molar pregnancy, Midcycle LH - Too early (commonly) or too late o Inhibin A
surge (rarely) testing
- Hematuria or proteinuria - Dilute urine (low S.G) SPECIMEN HANDLING
- Malignancies (gynecologic & other) - Adulterated urine - Test for Fetal Lung Maturity = Placed on ice on delivery, kept refrigerated or frozen,
- Postpartum & post-abortion (up to - Ectopic pregnancy (rarely negative, Filtration prevents loss of phospholipids
4 weeks) almost always positive) - Test for Cytogenetic Studies = Kept at room temperature or at 37oC
- Chinese herbal medications - Impending or missed abortion - Test for HDN = Protected from light
- Perimenopausal (LH elevation) (rarely negative, almost always
- Premature ovarian failure (LH positive) AMNIOTIC FLUID VS. MATERNAL URINE
elevation) ANALYTE AMNIOTIC FLUID MATERNAL URINE
Less reliable Protein + -
HCG BIOASSAYS (BIOLOGICAL PREGNANCY TESTS) Glucose + -
Test Animal Used Mode of Injection Positive Result More reliable Urea (mg/dL) < 30 >300
Hogben Female frog Lymph sac (urine) Oogenesis Creatinine < 3.5 >10
Galli-Mainini Male frog Subcutaneous Spermatogenesis (mg/dL)
(urine/serum)
Friedmann/ Virgin female Marginal ear vein Corpora lutea & FERN TEST
Hoffman rabbit (urine) corpora - Detects ruptured amniotic membranes
hemorrhagica - Also used to diagnose early pregnancy (↑ estrogen)
Ascheim- Immature Subcutaneous Formation of - Procedure: Specimen (Vaginal Fluid) → Slide (Air Dry) → (+) Fern-like
Zondek female mice (urine) hemorrhagic follicles crystals = AMNIOTIC FLUID (Due to presence of sodium chloride and
& corpora lutea proteins)
Frank- Immature Subcutaneous Ovarian hyperemia
Berman female rats (urine)
Kupperman Female virgin Intraperitoneal Ovarian hyperemia AMNIOTIC FLUID COLOR
rat (urine) COLOR CLINICAL SIGNIFICANCE
Kelso Female virgin Subcutaneous Ovarian hyperemia Colorless Normal
rat (urine) Blood-streaked Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Yellow HDN (Bilirubin)
KEEP IN MIND! Dark-green Meconium = 1st fetal bowel movement (sign of distress)
- ELISA tests are very sensitive, giving positive reactions as early as 10 days Dark red-brown Fetal death
after conception
- Urine specimen for pregnancy testing should have a specific gravity of at least 1. TEST FOR FETAL LUNG MATURITY
1.015 - Respiratory distress syndrome
o Most frequent complication of early delivery
o 7th most common cause of morbidity and mortality in the premature infant
o Caused by insufficiency of lung surfactant (phospholipids) production & fetal
lung immaturity
- Surfactant normally appears in mature lungs and allows the alveoli to remain open
throughout the cycle of inhalation and exhalation
- Surfactant keeps the alveoli from collapsing by decreasing surface tension and
allows them to inflate with air more easily

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TEST INFORMATION REVIEW!!!

1. Lecithin/ - Reference method 1. Specimens for Fetal Lung Maturity (FLM) testing should be stored:
Sphingomyelin - Lecithin = for alveolar stability a. At room temperature or at 37oC
ratio - Sphingomyelin = serves as a control (due to constant b. Protected from light
production) c. Refrigerated or frozen
𝐿𝑒𝑐𝑖𝑡ℎ𝑖𝑛 o L/S ratio is measured using thin layer d. Any of these
𝑆𝑝ℎ𝑖𝑛𝑔𝑜𝑚𝑦𝑒𝑙𝑖𝑛 chromatography (TLC) 2. Cause of dark red-brown amniotic fluid?
↑ Numerator = ↑ Ratio
- Ratio of >2.0 = mature fetal lungs a. Meconium
o Cannot be done on a specimen contaminated by b. HDN
blood or meconium (falsely increases the ratio c. Intra-amniotic hemorrhage
[Strasinger]) d. Fetal death
2. Amniostat- - Immunologic test for phosphatidylglycerol (PG) 3. Alpha fetoprotein is decreased in
FLM o Not affected by blood or meconium a. Down syndrome
o Production of PG is delayed among diabetic b. Spina bifida
mothers c. Anencephaly
3. Foam stability - Amniotic fluid + 95% Ethanol → Shake for 15 secs → d. Hepatocellular carcinoma
(Foam/Shake Stand for 15 mins 4. Sub-unit unique for HCG?
test) - (+) Foam/Bubbles = MATURE FETAL LUNGS a. Alpha
(Presence of phospholipids) b. Beta
4. Microviscosity - The presence of phospholipids decreases c. Gamma
(Obsolete test) microviscosity d. Delta
o Microviscosity is the friction experienced by a 5. Hogben bioassay test for hCG uses what animal?
single particle undergoing diffusion because of its a. Male frog
interaction with its environment at the micrometer b. Female toad
length scale c. Female rabbit
- Measured by fluorescence polarization d. Female rat
o Surfactant to albumin (S/A) ratio is measured
o Dye bound to surfactant had longer fluorescence &
low polarization Answer key: C, D, A, B, B
o Dye bound to albumin had decreased fluorescence
& high polarization SPUTUM AND BRONCHOALVEOLAR LAVAGE
5. Lamellar body - Type ll pneumocytes produce alveolar surfactants
count (LBC) stored in the form of lamellar bodies
o Lamellar body diameter is similar to that of SPUTUM
platelets, therefore, LBC can be obtained with the - Not a sterile body fluid
use of platelet channel of hematology analyzers - From upper & lower (sterile) respiratory tract
o LBC can be done using impedance and/or optical - Tracheobronchial secretions (mixture of plasma, electrolytes, mucin & water)
scatter methods - Added with cellular exfoliations, nasal and salivary gland secretions and normal
o >32,000/uL lamellar body count = ADEQUATE oral flora
FLM - 95% water and 5% solids
6. OD 650 nm - ↑ Lamellar bodies = ↑ O.D. (Absorbance) - Secretions are viscoelastic - some of the properties of a liquid and some of a solid
- O.D. of >0.150 is equivalent to:
o L/S ratio of > 2.0 - Sialic acid - most important single component of sputum viscosity
o The presence of PG - Acceptable sputum specimen = <10 S.E.C./LPF and >25 WBC/LPF

SPECIMEN COLLECTION
2. TEST FOR FETAL AGE
- 1.5 to 2.0 mg/dL amniotic fluid creatinine = prior to 36 weeks' gestation - 1st morning = most preferred sample (most concentrated; routine)
- >2.0 mg/dL amniotic fluid creatinine = 36 weeks (9 months) - 24-hour sputum = for volume measurement
- Throat swab = for pediatric patients
3. TEST FOR HDN - Sputum induction = for non-cooperative patients
- Tracheal aspiration = for debilitated or unconscious patients
- A.k.a Optical Density (Absorbance) 450 - Specimen preservation methods = Refrigeration or 10% formalin
- Absorbance of amniotic fluid:
o Normal = ↑ at 365nm, ↓ at 550nm MACROSCOPIC EXAMINATION
o HDN = ↑ at 450 nm (bilirubin) Volume ↓ Bronchial asthma, acute bronchitis, early pneumonia, stage of
- Results are plotted on a Liley graph: healing
o Zone I = Non-affected or mildly affected fetus ↑ Bronchiectasis, lung abscess, edema, gangrene, tuberculosis,
o Zone II = Moderately affected fetus (requires close pulmonary hemorrhage
monitoring) Color Colorless or Made up of mucus only
o Zone III = Severely affected fetus (requires translucent
intervention)
White or yellow ↑ Pus (TB, bronchitis, jaundice,
- Interferences= cells, meconium, debris, and hemoglobin (peak absorbance at
pneumonia)
410nm)
Gray ↑ Pus & epithelial cells
- The oldest routinely performed lab test on AF evaluates the severity of fetal
Bright green or ↑ Bile; P. aeruginosa infection, lung
anemia due to HDN
greenish abscess
4. TEST FOR NEURAL TUBE DEFECTS (NTD) Red or bright red Fresh blood or hemorrhage, TB,
bronchiectasis
- Spina bifida ("'split spine”) is a birth defect where there is incomplete closing of
Anchovy sauce or Old blood, pneumonia, gangrene
the backbone & membranes around the spinal cord.
rusty brown
- Anencephaly is the absence of a major portion of the brain, skull, and scalp that
Prune juice Pneumonia, chronic lung cancer
occurs during embryonic development
- Screening test = Alpha-fetoprotein (AFP) Olive green or grass Cancer
o ↑ in Neural tube defects green
o ↓ in Down syndrome Black Dust or dirt, carbon, charcoal,
- Confirmatory test = Acetylcholinesterase anthracosis, smoking
- AFP is the major protein produced by the fetal liver during early gestation (prior to Rusty (with pus) Lobar pneumonia
18 weeks) (S. pneumoniae)
Rusty (without pus) Congestive heart failure
TESTS FOR FETAL WELL-BEING AND MATURITY Currant, jelly-like Klebsiella pneumoniae infection
Test Normal Values at Significance Odor Odorless Normal
Term Foul or putrid Lung gangrene, advanced necrotizing
Bilirubin scan ∆ A450 >.025 Hemolytic disease of the tumors
newborn Sweetish Bronchiectasis, tuberculosis
Alpha-fetoprotein <2.0 Multiples of Neural tube disorders Cheesy Necrosis, tumors, empyema
Median (MoM)
Fecal Liver abscess, enteric Gram-negative
L/S ratio >2.0 Fetal lung maturity
bacterial infection
Amniostat-FLM Positive Fetal lung maturity/
Phosphatidylglycerol Consistency Mucoid Asthma, bronchitis
Foam Stability Index >47 Fetal lung maturity Serous or frothy Lung edema
Microviscosity (FLM-TDx) >55 mg/g Fetal lung maturity Mucopurulent Bronchiectasis, tuberculosis with cavities
Optical Density 650 nm >0.150 Fetal lung maturity
Lamellar body count >32,000/uL Fetal lung maturity

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MACROSCOPIC STRUCTURES CLINICAL SIGNIFICANCE REVIEW!!!

Dittrich' s Yellow or gray material, size of a Bronchitis, bronchiectasis 1. What is the most preferred sample for 4. Majority of the
plugs pinhead Bronchial asthma routine sputum analysis? granulocyte seen in
Produces foul odor when crushed a. Tracheal aspiration bronchoalveolar
b. 1st morning sputum lavage is?
Pneumoliths Hard concretions in a bronchus Histoplasmosis (most c. 24-hour sputum a. Neutrophil
or (lung stones) common) d. Throat swab b. Macrophage
Broncholiths Yellow/white calcified TB Chronic tuberculosis c. Lymphocyte
structures/foreign material 2. Curschmann’s spirals, Creola bodies, d. Eosinophil
Bronchial Branching tree-like casts of the Lobar pneumonia, Charcot-Leyden crystals, and Dittrichs plugs
casts bronchi bronchitis, diphtheria are all associated with: 5. Sweat testing is
Layer 1st (top) = frothy mucus Bronchiectasis, lung a. Broncholithiasis used to diagnose?
formation 2nd (middle) = opaque, water abscess, gangrene b. Bronchiectasis a. Cystic fibrosis
c. Bronchitis b. A and D
(3 layers) material d. Bronchial asthma c. P. jirovecii
3rd (bottom) = pus, bacteria, pneumonia
tissues 3. In the sputum analysis, the only significance d. Mucoviscidosis
Foreign Bronchial calculi (calcium Pneumoconiosis of this finding is its resemblance to
bodies carbonate & phosphate) Blastomyces?
Asbestos bodies, silica particles a. Blue bodies
b. Curschmann’s spirals
MICROSCOPIC STRUCTURES CLINICAL c. Myelin globules
d. Alveolar macrophage
SIGNIFICANCE
Elastic fibers Slender fibrils w/ double contour & Tuberculosis Answer key: B, D, C, A, B
curled ends
Charcot- Colorless, hexagonal, double pyramid, Bronchial asthma (3
CEREBROSPINAL FLUID
Leyden often C’s)
crystals needle-like; arise from disintegration of - 3rd major body fluid
eosinophils - Functions:
o Supply nutrients to the nervous system
Pigmented Heart failure cells: hemosiderin-laden Congestive heart
o Remove metabolic waste
cells macrophage failure
o Produce a mechanical barrier to cushion the brain & Spinal cord against
Carbon-laden cells: angular black Heavy smokers
trauma
granules
Curschmann’s Coiled mucus strands Bronchial asthma (3
spirals Can also be observed macroscopically C’s)
Myelin Colorless globules occurring in a variety No significance
globules of sizes and bizarre forms Mistaken as
Blastomyces
Creola bodies Clusters of columnar epithelial cells Bronchial asthma (3
C’s)
Parasites Migrating larva: Ascaris, Strongyloides, Hookworm (heart-to-lung
migration)
E. histolytica, E. gingivalis, T. tenax, P. westermani, E.
granulosus, T. canis
Others Neoplastic cells, bacteria, fungi, leukocytes

BRONCHOALVEOLAR LAVAGE (BAL)

- 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
- Grocott's methenamine silver stain best delineates the cysts of Pneumocystis
jirovecii

CELLS SEEN IN BRONCHOALVEOLAR LAVAGE

56-80% Alveolar macrophages Most predominant
1-15% Lymphocytes Interstitial disease, pulmonary lymphoma,
nonbacterial infections
<3% Neutrophils Cigarette Smokers, bronchopneumonia,
toxin exposure
<1-2% Eosinophils Hypersensitivity reactions
4-17% Ciliated columnar bronchial epithelial cells

SWEAT MENINGES
- Line the brain and spinal cord
SWEAT TEST - 3 layers:
- Used to diagnose Cystic fibrosis (Mucoviscidosis) o Dura mater (Outer layer) = Lines the skull & vertebral canal
o Autosomal recessive metabolic disorder affecting the mucous secreting o Arachnoid mater (Spiderweb-like) = Filamentous inner membrane
glands of the body  Subarachnoid space (Below arachnoid) = Portion where CSF flows
o Associated with pancreatic insufficiency, respiratory distress & intestinal o Pia mater (Innermost layer) = Lines the surface of brain & spinal cord
obstruction
o ↑ Na+ & Cl- due to inability of the sweat glands to reabsorb them before the - CHOROID PLEXUS = produces CSF by selective filtration (at a rate of 20
sweat is secreted mL/hour)
- ARACHNOID VILLI/GRANULATIONS = reabsorbs CSF
GIBSON AND COOKE PILOCARPINE IONTOPHORESIS - BLOOD BRAIN BARRIER (BBB)
- Pilocarpine + mild current = induce sweat production o Protects brain from chemicals & other substances circulating in the blood that
- Application of 0.16 mA current for 5 minutes can harm the brain tissue
o Disruption of BBB allows WBCs, proteins & other chemicals to enter the CSF
Sweat Na+ and Cl- values: (Ex: Meningitis, Multiple sclerosis)
- >70 mEq/L= Diagnostic for CF
- 40 mEq/L= Borderline for CF (Repeat testing) CSF COLLECTION AND HANDLING
- Up to 20mL mL CSF can be collected using a manometer attached to a spinal
Sweat is tested for sodium and chloride needle
- Na+ = Flame photometry, Ion exchange electrode o Only if CSF pressure is normal (50-180 mmHg)
- Cl- = Manual or automated titration o If CSF pressure is high or low, only 1-2 mL can be removed
- Method of collection = Lumbar puncture (between L3-L4 [adults] or L4-L5 [infants])

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3 CSF TUBES RBC Count

- Done only in cases of traumatic tap
TEST STORAGE - To correct for WBC count & total protein concentration
Tube 1 Chemistry/Serology Freezing temperature o -1 WBC for every 700 RBCS seen
Tube 2 Microbiology Room temperature for 30 mins (CM o -8 mg/dL total protein concentration for every 10,000 RBCs/uL (Henry)
books) o -1 mg/dL total protein concentration for every 1,200 RBCs/uL (Strasinger)
35-37oC (Microbiology books)
Tube 3 Hematology Refrigeration temperature CSF DIFFERENTIAL COUNT
(Tube 4) Microbiology or Serology -- - Performed on stained smear
- Tube 1 = least affected by blood or bacteria introduced by spinal tap - Specimen should be concentrated before smearing by using the following
- Tube 2 = usually designated for microbiology methods:
- Tube 3= least likely contain cells introduced by the spinal tap o Cytocentrifugation
- Tube 4 = better exclusion of skin contamination or for additional tests o Centrifugation
- Cell counts may be performed on Tubes 1 & 4 to check for cellular contamination o Sedimentation
by puncture o Filtration
- Left-over supernatant fluid may also be used for additional chemical or serologic
tests CYTOCENTRIFUGE
- Excess fluid should not be discarded and should be frozen until there is no further - Fluid is added to conical chamber
use for it - Cells are forced into a monolayer within a 6mm diameter circle on the slide
- If 1 CSF tube only: Microbiology first, then Hema, then Chem/Serology - Addition of 30% albumin
o Increases cell yield or recovery
CSF TOTAL VOLUME o Decreases cellular distortion
- Adults = 90-150mL
- Neonates = 10-60 mL PREDOMINANT CELLS IN CSF
CSF APPEARANCE CLINICAL SIGNIFICANCE - Predominant = Lymphocytes and Monocytes
- Occasional = Neutrophils
Crystal clear Normal - Adults: (70:30 ratio)
Hazy/ Turbid/ Milky/ WBCs (>200/uL, RBCs (> 400/uL), lipids & protein, o 70% Lymphocytes
Cloudy microorganisms o 30% Monocytes
Xanthochromic Due to hemoglobin degradation products (most common) - Neonates (inversed ratio)
(Pink/Yellow/Orange) - Pink = Slight amount of oxyhemoglobin o Up to 80% (considered normal)
- Yellow = Oxyhemoglobin → Bilirubin - PLEOCYTOSIS
- Orange = Heavy hemolysis o Abnormal condition
Other causes: ↑ Carotene, ↑ Melanin, ↑ Protein (> 150 o Increased number of normal cells in the CSF
mg/dL), Rifampin
Bloody ↑ RBCs (>6,000/uL) PREDOMINANT CELLS SEEN IN CEREBROSPINAL FLUID
Traumatic tap (puncture of blood vessel) Type of cell Major Clinical Significance Microscopic Findings
Intracranial hemorrhage (bleeding within the braincase)
Lymphocytes Normal All stages of development
Monocytes Viral, tubercular & fungal may be found
TRAUMATIC TAP VS. INTRACRANIAL HEMORRHAGE meningitis Monocytes mixed with
Multiple sclerosis lymphocytes
Traumatic Tap Intracranial Hemorrhage Neutrophils Bacterial meningitis Granules may be less
Distribution of Uneven (1>2>3) Even (1=2=3) Early cases of viral, tubercular, prominent in blood
blood on 3 tubes & fungal meningitis Cells disintegrate rapidly
Clot formation (+) (-) Cerebral hemorrhage
Due to plasma CSF has no fibrinogen Macrophages RBCs in spinal fluid May contain phagocytized
fibrinogen Contrast media RBCs appearing as empty
Supernatant Clear Xanthochromic vacuoles or ghost cells,
(RBCs in CSF lyse after 2 hours) hemosiderin granules and
Erythrophages Absent Present hematoidin crystals
(Macrophages w/ (+) Hematoidin & Hemosiderin Blast forms Acute leukemia Lymphoblasts, myeloblasts,
ingested RBCs) or monoblasts
D-dimer Negative Positive Lymphoma Disseminated lymphoma Resemble lymphocytes with
cells cleft nuclei
CSF Appearance Clinical Significance Plasma cells Multiple sclerosis Traditional and classic forms
Oily Radiographic contrast media Lymphocyte reactions seen
Clotted Protein & clotting factors; meningitis, Froin syndrome, Ependymal, Diagnostic procedures Seen in clusters with distinct
blockage of CSF circulation choroidal, & nuclei and distinct cell walls
Pellicle Tubercular meningitis spindle-
shaped cells
Malignant Metastatic carcinomas Seen in clusters with fusing of
CSF CELL COUNT cells Primary CNS carcinoma cell borders & nuclei
- Any cell count should be performed IMMEDIATELY
o WBCs and RBCs begin to lyse within 1 hour CSF PROTEIN
o 40% WBCs disintegrate within 2 hours Normal values - Adults = 15-45 mg/dL (<1% or 1/200 that of serum
protein)
WBC Count - Infants = 150 mg/dL
- Routinely performed on CSF - Immature = 500 mg/dL
- Normal values: Increased in - Damage to the BBB (most common): meningitis,
o Adults = 0-5 WBCs/uL hemorrhage
o Neonates = 0-30 WBCs/uL - Production of immunoglobulins within the CNS (multiple
sclerosis)
KEEP IN MIND: CSF DILUTION! - Decreased normal protein clearance from the fluid
- Neural tissue degeneration
Decreased in - CSF leakage/trauma
Appearance Dilution - Recent puncture
Clear Undiluted - Rapid CSF production
Slightly Hazy 1:10 - Water intoxication
Hazy 1:20 Albumin - Major CSF protein
Slightly Cloudy 1:100 Prealbumin - 2nd Most Prevalent
Cloudy/Slightly Bloody 1:200 Alpha-globulins - Haptoglobin, ceruloplasmin
Bloody/Turbid 1:10,000 Beta-globulins - Beta2-transferrin (tau)
o Carbohydrate-deficient transferrin
o Found in CSF but not in serum
Gamma-globulins - IgG and some lgA
FORMULA FOR CSF WBC COUNT NOT found in - IgM, Fibrinogen, Lipids
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 normal CSF
𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑝𝑒𝑟 𝑢𝐿) =
𝑎𝑟𝑒𝑎 𝑥 𝑑𝑒𝑝𝑡ℎ 𝑓𝑎𝑐𝑡𝑜𝑟 (0.1)
WBC Diluting fluid
- Acetic acid with Methylene blue

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CSF PROTEIN DETERMINATION CSF GLUTAMINE

Turbidimetric 3% Trichloroacetic acid (TCA) Notes - Product of ammonia & alpha-ketoglutarate
- Preferred method - Indirect test for the presence of excess ammonia in the
- Precipitates BOTH albumin & globulins CSF
Total protein

Normal value - 8-18 mg/dL

3% Sulfosalicylic acid (SSA)
- Precipitates albumin only Increased - Disturbance of consciousness (coma)
- To precipitate globulins, add sodium sulfate (Na2SO4) - Reye's syndrome
Dye-binding Coomassie Brilliant Blue
- Protein binds to dye → Dye turns from red to blue CSF ENZYMES
- ↑ Protein = ↑ Blue color
CSF/Serum - Assess the integrity of the blood brain barrier 1. LACTATE DEHYDROGENASE (LDH)
𝑚𝑔
Albumin 𝐶𝑆𝐹 𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 ( ) - Serum LDH:
𝑖𝑛𝑑𝑒𝑥 = 𝑑𝐿
Index 𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 𝑔 o Normal = 2>1>3> 4>5 CSF LDH ISOENZYMES
𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )
𝑑𝐿 LD 1 & 2: Brain tissues
- Normal value = <9 o Flipped pattern (AMI) = 1>2
- Abnormal = >9 - CSF LDH: LD 2 & 3: Lymphocytes
- 9-14 = slight impairment o Normal pattern = 1>2>3>4>5 LD 4 & 5: Neutrophils
Protein fractions

- 15-30 = moderate impairment o Neurological abnormalities = 2>1

- >30 = severe impairment o Bacterial meningitis = 5>4>3>2>1 (↑ Neutrophils)
- 100 = complete damage to BBB
IgG index - Assess conditions with lgG production within the CNS 2. CK
(Ex: Multiple sclerosis)
𝑚𝑔 𝑔 - ↑ in stroke, MS, degenerative disorders, brain tumors, viral & bacterial meningitis,
[𝐶𝑆𝐹 𝐼𝑔𝐺 ( )] ÷ [𝑆𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 ( )]
𝐼𝑔𝐺 𝑖𝑛𝑑𝑒𝑥 = 𝑑𝐿 𝑑𝐿 seizures
𝑚𝑔 𝑔
[𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 ( )] ÷ [𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )]
𝑑𝐿 𝑑𝐿
3. AST
- Normal value = <0.77 (Brunzel: 0.30-0.70) - ↑ in intracerebral and subarachnoid hemorrhage, bacterial meningitis
- Abnormal = >0.77 (Old Strasinger: >0.77)
o Indicative of IgG production within the CNS (Ex: SEROLOGIC TESTING
Multiple sclerosis) - Latex agglutination test and ELISA= for detection of bacterial antigens
- VDRL = recommended by CDC for the detection of
CSF ELECTROPHORESIS
- For the detection of oligoclonal bands (in the y-region) DIFFERENTIAL DIAGNOSIS OF MENINGITIS
o These bands indicate immunoglobulin production Type ↑ WBC Type Other Info
- Done in conjunction with serum electrophoresis to ensure that banding is due to Bacterial Neutrophils Pro ↑ - (+) Gram stain
neurologic inflammation Glu ↓↓ - (+) Culture
- (+) 2 or more oligoclonal bands in CSF but NOT in serum is valuable for the
diagnosis of multiple sclerosis LAT ↑↑ - Limulus Lysate test (see details
below)
KEEP IN MIND! - Agents = S. agalactiae, S.
pneumoniae, N. meningitidis, L.
Oligoclonal Banding in Oligoclonal Banding in Oligoclonal monocytogenes, Gram-
Serum but NOT in CSF CSF but NOT in Serum Banding negative rods
(Ms. Neng) in Serum AND Viral Lymphocytes Pro ↑ - Agent = Enteroviruses
CSF Glu N - Poliovirus
- Multiple Sclerosis - Leukemia - HIV LAT N - Echovirus
(persistent) - Lymphoma - Coxsackievirus
- Neurosyphilis - Viral infections Tubercular Lymphocytes Pro ↑ - Agent = Mycobacterium
- Encephalitis - Bands may also
- Neoplastic disorders appear in GSF as a Monocytes Glu ↓ tuberculosis
- Guillain-Barré result of BBB leakage LAT ↑ - (+) AFB stain
syndrome or traumatic tap - (+) Pellicle or web-like clot
formation after 12-24 hour
refrigeration
MULTIPLE SCLEROSIS Fungal Lymphocytes Pro ↑ - Agent = Cryptococcus
- Demyelinating disorder Monocytes Glu ↓ neoformans
- Findings: LAT ↑ - (+) Gram stain = classic
o (+) Anti-myelin sheath autoantibody starburst pattern
o (+) Oligoclonal band in CSF but not in serum - (+) Latex agglutination test
o (+) Myelin basic protein (MBP) - (+) India ink stain = capsule
o ↑ IgG index
(unstained); background (black)
MYELIN BASIC PROTEIN (MBP) - Cryptococcal meningitis is now
- Protein component of the lipid-protein complex that insulate the nerve fibers commonly encountered in the
- Presence of MBP in CSF indicates destruction of myelin sheath clinical laboratory
- Used to monitor the course of multiple sclerosis
LIMULUS AMOEBOCYTE LYSATE (LAL) TEST
CSF GLUCOSE (GLU) - Detects Gram-negative endotoxin in body fluids & surgical instrument
Notes - Done in conjunction with blood glucose - Reagent: Blood of horseshoe crab (Limulus polyphemus); Hemocyanin = copper
- Specimen for blood glucose should be drawn 2 hours prior - Principle
to spinal tap (to allow time for equilibration between CSF o In the presence of endotoxin, the amoebocytes (WBCs) will release lysate
and plasma glucose) (protein)
Normal value - 60-70% of blood glucose (65% or 2/3) o (+) Clumping or clot formation
- (50-80 mg/dL)
Increased - Due to increased plasma glucose (not significant) REVIEW!!!
Decreased - Bacterial (markedly decreased), tubercular, and fungal 1. What are the three layers of the 6. Even distribution of blood in
meningitis meninges? CSF
- Due to alterations in the mechanisms of glucose transport - Dura mater a. Traumatic tap
- Arachnoid mater b. Intracranial
across the BBB
- Pia mater hemorrhage
- Due to increased use of glucose by the brain cells
Normal in - Viral meningitis 2. Portion of meninges where CSF 7. Normal CSF glucose and
flows: lactate?
CSF LACTATE (LAT) a. Dura mater a. Bacterial meningitis
Notes - Inversely proportional to glucose b. Choroid plexus b. Viral meningitis
- Sensitive method for evaluating the effectiveness of c. Arachnoid villi c. Fungal meningitis
d. Subarachnoid space d. Tubercular meningitis
antibiotic therapy
Normal value - 10-22 mg/dL 3. CSF is produced by: 8. Limulus lysate test detects:
Increased - Bacterial meningitis (>35 mg/dL) a. Dura mater a. Gram (+) exotoxin
- Tubercular and fungal meningitis (>25 mg/dL) b. Choroid plexus b. Gram (+) endotoxin
- Hypoxia c. Arachnoid villi c. Gram (-) exotoxin
Normal in - Viral meningitis d. Subarachnoid space d. Gram (-) endotoxin

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4. CSF is reabsorbed by: 9. Weblike clot or pellicle MACROSCOPIC EXAMINATION

a. Dura mater formation in the CSF is seen Appearance - Gray-white, translucent = Normal (with musty or bleach odor)
b. Choroid plexus in: - Increased white turbidity = Infection (↑ WBCs)
c. Arachnoid villi a. Bacterial meningitis - Red or brown coloration = ↑ RBCs, blood
d. Subarachnoid space b. Viral meningitis
c. Fungal meningitis - Yellow coloration = Urine contamination, medication, ↑
5. CSF tube number for microbiology: d. Tubercular meningitis abstinence (continence; ↑ flavin)
a. 1 Volume - Normal = 2-5 mL
b. 2 - Increased = ↑ abstinence
c. 3 - Decreased = Infertility, incomplete collection
d. Any of these Viscosity - Normal = Pour in droplets
- Abnormal = Threads >2 cm long
SEMEN - ↑ Viscosity = ↓ Sperm motility
- Reporting
STAGES OF SPERM REASONS FOR SEMINAL FLUID o 0 = Watery
MATURATION ANALYSIS o 4 = Gel-like
1.) Spermatogonium - Fertility testing o *May also be reported as low, normal or high
2.) Primary Spermatocyte - Postvasectomy semen analysis pH - Normal = 7.2 to 8.0
3.) Secondary Spermatocyte - Forensic analysis (alleged rape) - ↑ pH = Infection
4.) Spermatid - ↓ pH = ↑ Prostatic fluid
5.) Spermatozoon - pH should be measured within 1 hour of ejaculation

SPERM CONCENTRATION
- Normal value = > 20 (20-160) million sperms/mL
- Methods:
o Improved Neubauer Counting Chamber
 Dilution = 1:20
 Diluents: To immobilize sperm
 Formalin
Sodium bicarbonate (NaHCO3)
 Saline
 Distilled water
 Cold tap water (alternative)
o Makler Counting Chamber
 For undiluted specimen
 Uses heat to immobilize sperms
COMPOSITION OF SEMEN - Both sides of the hemocytometer are loaded and allowed to settle for 3 to 5
5% Spermatozoa - Seminiferous tubules (testes) minutes; then they are counted, and the counts should agree within 10%
o Site of – spermatogenesis
o Sertoli cells: nurse cells for developing sperms SHORTCUT METHOD LONG METHOD FOR SPERM
- Epididymis (Sperm Concentration CONCENTRATION COMPUTATION
o Site of sperm maturation (they becomes motile) Computation) (Standard Neubauer Formula)
- Spermatogenesis and sperm maturation take 90 2 WBC squares
days (Graff - 74 days) = # sperms counted x 100,000 𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝐿) =
60- Seminal fluid - Seminal vesicles
#𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
70% o Provide nutrients for sperm & fluid 5 RBC squares
= # sperms counted 𝑎𝑟𝑒𝑎 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)
o Secretions rich in fructose = for sperm motility
20- Prostate fluid - Acidic fluid that contains ACP, zinc, citric acid & other x 1, 000, 000
30% enzymes
- For coagulation and liquefaction
5% Bulbourethral - Secretes thick alkaline mucus
glands - Neutralizes acidity from the prostatic secretions &
vagina

SPECIMEN COLLECTION
- Abstinence of 2-3 days but not >7 days
o Prolonged abstinence = ↑ Volume, ↓ Motility
- Collect the entire ejaculate

If first portion is missing If last portion is missing

- ↓ Sperm count - ↑ Sperm count
- ↑ pH - ↓ pH
- Specimen will not liquefy - ↓ Volume
- Specimen will not clot

- Methods of collection:
o Masturbation = best (or self-production)
o Coitus interruptus = withdrawal method SAMPLE PROBLEMS FOR SPERM CONCENTRATION COMPUTATION
o Condom method = use non-lubricant-containing rubber or polyurethane (LONG METHOD)
condom Example 1: 𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝐿) =
- Specimen should be delivered to the lab within 1 hour of collection (at room - 1 sperm counted using 2 WBC
squares 1 𝑥 20 20 200
temperature) = = = 100/𝑢𝐿
- Take note of the time of specimen collection, specimen receipt, and liquefaction - Area of 1 WBC square = 1mm 1 (2) 𝑥 0.1 0.2 2
- Analysis should be done after liquefaction (usually 30-60 minutes) 100
o Failure to liquefy within 60 minutes may be caused a deficiency in prostatic = 𝑥 1000 = 100,000/𝑚𝐿
𝑢𝐿
enzymes Example 2: 𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝐿) =
o If sample fails to liquefy, treat w/ amylase/bromelain/α-chymotrypsin to break - 1 sperm counted using 5 RBC
up mucus squares 1 𝑥 20 20 2000
- Specimen awaiting analysis should be kept at 37oC = = = 1000/𝑢𝐿
- Area of 1 RBC square = 0.04 0.04 (5) 𝑥 1 0.02 2
mm 1000
= 𝑥 1000 = 1,000,000/𝑚𝐿
𝑢𝐿

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SPERM COUNT OTHER TESTS PERFORMED IN SEMEN

- Normal value = > 40 million sperms per ejaculate
Analyte Normal Value Decreased Values
𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡 = 𝑆𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛 𝑣𝑜𝑙𝑢𝑚𝑒
Indicate
- Sample Problem: Fructose > 13 umol/ejaculate Lack of seminal fluid
o Given the following, compute for the sperm count: Neutral α- > 20 mU/ejaculate Epididymis disorder

CHEMICAL
o Sperm concentration 20M sperms/mL glucosidase
o Sample volume 2 mL/ejaculate Zinc > 2.4 umol/ejaculate Lack of prostatic fluid
Citric acid > 52 umol/ejaculate
SPERM MOTILITY Acid phosphatase > 200 units/ejaculate
- Place a drop of semen in a slide & cover it w/ coverslip. Allow to settle for 1 min. - Decreased levels of glycerophosphocholine and L-carnitine also
Observe in 20 HPF. indicate an epididymis disorder
- Normal values - Decreased glutamyl transpeptidase level also indicates a lack of
o >50% motile (within 1hour) prostatic fluid
o Quality = >2.0 - Antisperm Antibodies = cause sperm agglutination; detected in semen,
cervical mucosa or serum
WHO CRITERIA 1. Mixed agglutination reaction (MAR)

IMMUNOLOGIC
Grading Sperm Motility Action o Detects the presence of lgG antibodies
4.0 a Rapid straight-line motility o Semen sample + AHG + latex particles or treated RBCs coated with
3.0 b Slower speed, some lateral movement IgG
2.0 b Slow forward progression, noticeable lateral movement o Normal = <10% motile sperm attached to the particles
1.0 c No forward progression 2. Immunobead test
0 d No movement o Detects the presence of IgG, IgM & lgA antibodies
o Demonstrates what area of sperm (head neck tail) the
autoantibodies are affecting
ALTERNATIVE SPERM MOTILITY GRADING CRITERIA o Normal = presence of beads on <50% of the sperm
Progressive Motility (PM) Sperm moving linearly or in a large circle - Routine aerobic and anaerobic cultures and tests for Chlamydia
Nonprogressive Motility (NP) Sperm moving with an absence of progression trachomatis, Mycoplasma hominis and Ureaplasma urealyticum
Immotility NO movement - Round cells = WBCs or spermatids (immature sperm cells) → use
peroxidase to differentiate them

MICROBIAL
Computer-Assisted Semen Analysis (CASA) o <1 million round cells/mL = Normal
- Determines sperm concentration, morphology, velocity & trajectory (direction of o >1 million WBCs/mL = infection
motion) o >1 million spermatids/mL = Disruption of spermatogenesis
𝑁𝑥𝑆
𝑅𝑜𝑢𝑛𝑑 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 =
100
SPERM MORPHOLOGY Where:
- Normal values: - N = # of spermatid or neutrophils
o Routine criteria = >30% normal forms - S = Sperm concentration (million/mL)
o Kruger's strict criteria = > 14% normal forms Tests for detection of semen:
 Measure the head, neck & tail using a micrometer - Microscopic exam
- Use 45o angle when preparing smears - Fluorescence under UV light
- Stains for Sperm Morphology: - Acid phosphatase (ACP) determination
o Papanicolaou's stain (stain of choice) - Glycoprotein p30 (a.ka. PSA) = more specific method to detect semen
MEDICO-LEGAL

o Wright's stain - Florence test (not specific)

o Giemsa stain o Test for choline (produced by the prostate gland; anti-bacterial)
- Head (length: 5 um, width = 3 um) o Reagents: Iodine crystals + Potassium iodide → (+) Dark brown
o Acrosomal cap rhombic crystals
 ½ of the head - Barbiero's test (very specific)
 2/3 of the nucleus o Test for spermine (produced by the prostate gland; anti-bacterial)
o Normal = oval-shape o Reagents: Saturated picric acid + Trichloroacetic acid → (+) Yellow
o Abnormal = poor ovum penetration leaf-like crystals
- Midpiece - ABO blood grouping
o Contains mitochondria - DNA analysis
- Neck (7 um) Vasectomy
- Tail (45 um) - Surgical cutting of vas deferens so that the ejaculate will not contain any
o Abnormal = poor motility sperm cell
o The sperm tail length already includes the neck length. The neck is the - Following a vasectomy, sperm count ideally should be zero within 12
POST-VASECTOMY

thickest part of the tail. weeks after the procedure

Post-vasectomy semen analysis

- The only concern is the presence or absence of sperm
- Done 2 months 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
Abnormal result Possible abnormality Test
Decreased motility Viability Eosin-Nigrosin
with normal count stain
ABNORMAL SEMENALYSIS

Decreased count Lack of seminal vesicle Fructose level

support medium
VARICOCELE Decreased motility Male antisperm MAR &
- Hardening of veins that drain the testes with clumping antibodies immunobead tests
- Most common cause of male infertility
- Sperm has a tapered head Sperm
agglutination with
male serum
SPERM VIABILITY (SPERM VITALITY) Normal analysis with Female antisperm Sperm
continued infertility antibodies agglutination with
Modified Blom's test female serum
- Tested within 1 hour of ejaculation
- Reagents = eosin and nigrosin
- Living sperms = unstained, bluish white Test Description
- Dead sperms = red Hamster egg Sperms are incubated with species-nonspecific
SPERM FUNCTION

- Normal value = 50% living sperms (Strasinger, 6th Ed, Brunzel) penetration hamster eggs & penetration is observed
microscopically
SEMINAL FLUID FRUCTOSE Cervical mucus Observing Sperm penetration ability of partner's
penetration midcycle cervical mucus
- Tested within 2 hours or frozen to prevent fructolysis
Hypo-osmotic Sperms exposed to low-sodium concentrations
- Screening test swelling are evaluated for membrane integrity & sperm
o Resorcinol test (a.k.a. Seliwanoff’s test) = (+) Orange-red color viability
In vitro acrosome Evaluation of the acrosome to produce enzymes
reaction essential for ovum penetration

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NORMAL VALUES FOR SEMEN ANALYSIS SPECIMEN COLLECTION

Volume 2-5 mL - Arthrocentesis = Method of collection
Viscosity Pour in droplets - Normal synovial fluid does not clot (diseased joints may clot)
pH 7.2-8.0 - Specimen Volume
Sperm concentration > 20 million/mL o <3.5 mL = Normal (adult knee cavity)
Sperm count > 40 million/ejaculate
o >25 mL = Inflammation
Motility > 50% within 1 hour
Quality > 2.0 or a, b, c - Distributed in the following tubes (CLSI):
Morphology > 30% normal forms (routine criteria) o Plain red top tube (no anticoagulant) = chemical & immunologic evaluation
> 14% normal forms (strict criteria)  Note: Sodium fluoride = glucose analysis
Round cells <1.0 million/mL o Microscopic examination
 Sodium heparin/Liquid EDTA for hematology or cell count
SEMEN TERMINOLOGIES  Do not use powdered anticoagulants & lithium heparin - interfere w/
Aspermia NO semen or ejaculate crystal identification
Azoospermia Absence of spermatozoa in the ejaculate  Do NOT refrigerate samples - this can produce additional crystals!
Hematospermia Presence of blood in the ejaculate o Sterile anticoagulant tube (heparin or SPS) = microbiological studies (GS and
Leukospermia Increased number of leukocytes in the ejaculate culture)
Necrozoospermia Increased number of immotile or dead spermatozoa in the
ejaculate COLOR AND CLARITY
Oligozoospermia Decreased sperm concentration Appearance Significance
Colorless to pale yellow Normal
KEEP IN MIND! Deeper yellow Inflammation
- Leydig cells secrete testosterone; Sertoli cells secrete inhibin Greenish tinge Bacterial infection
- Shortly after ejaculation, semen coagulates because of the action of a clotting Red Traumatic tap; hemorrhagic arthritis
enzyme, formed in the prostate, on a fibrinogen-like precursor substance that
is produced by the seminal vesicles. Turbid WBCS, synovial cell debris or fibrin
- When performing fertility testing, WHO recommends that 2 or 3 samples be Milky Presence of crystals
collected not <7 days or >3 weeks apart, with 2 abnormal samples considered
significant SYNOVIAL FLUID VISCOSITY
- Motile sperm can be detected for up to 24 hours after intercourse, whereas - Normal = forms a string that is 4-6 cm long
nonmotile sperm can persist for 3 days
- As the sperm die off, only the heads remain and may be present for 7 days - Normal hyaluronic acid level of 0.3 to 0.4 g/dL
after intercourse
Ropes or Mucin Clot Test (Hyaluronate Polymerization Test)
- Reagent = 2-5% acetic acid
REVIEW!!! - As the ability of the hyaluronate to polymerize decreases, the clot becomes
1. Cause of brown or red semen? 6. Sperm tail length less firm
a. Bile pigment - 45 um Grading (Strasinger, 6th edition) Grading (Strasinger, 3rd edition)
b. Continence Good = Solid clot Good = Solid clot
c. Blood 7. Sperm neck length Fair = Soft clot Fair = Soft clot
d. Urine contamination - 7 um Low = Friable clot Poor = Friable clot
Poor = No clot Very poor = No clot
2. Viscosity graded as 4 8. Stain of choice for sperm
a. Watery morphology:
b. Lumpy a. Eosin-Nigrosin - "Arfl Arf! Formation of a hecking mucin clot after adding acetic acid can be
c. Ropy b. Wright’s used to identify a questionable fluid as hecking synovial fiuid!"
d. Gel-like c. Giemsa
d. Papanicolau
3. Rapid, straight-line motility CELL COUNT
a. 4.0 9. Stain of choice for sperm
b. 3.0 viability
KEEP IN MIND!
c. 2.0 a. Eosin-Nigrosin
d. 1.0 b. Wright’s - DO NOT use acetic acid as WBC diluting fluid for synovial fluid cell count
e. 0 c. Giemsa - It can cause CLOT FORMATION
d. Papanicolau
4. Size of the acrosomal cap WBC COUNT
- 2/3 of the nucleus 10. Alternative diluent for semen - Most frequently performed count
- ½ of the head dilution - Diluting fluids:
a. Boiling water
o NSS with methylene blue
5. Size of the sperm head b. Cold tap water
- 3 um in width c. Saline with saponin o Hypotonic saline (0.3%) = lyses RBCs
- 5 um in length d. NSS with methylene blue o Saline with saponin = lyses RBCs
- For very viscous fluid: ↑ Hyaluronic acid
SYNOVIAL FLUID 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
- A.k.a. joint fluid
o Incubate at 37oC for 5 minutes
- "Synovial" = Latin word for “egg”; Resembles eggwhite
- Viscous fluid circulating in diarthroses (movable joints)
DIFFERENTIAL COUNT
- Viscosity is due to polymerization of hyaluronic acid produced by synoviocytes
Cell Normal value
- Arthritis affects production of hyaluronate and its ability to polymerize, thus
decreasing synovial fluid viscosity RBCs < 2,000/uL (Turgeon = ABSENT)
- Functions: WBCs < 200/uL
o Lubricates joints WBC Differential 65% = Monocytes & macrophages
o Reduce friction between bones <25% = Neutrophils
o Provides nutrients to the articular cartilage <15% = Lymphocytes
o Lessen shock of joint compression occurring during activities such as walking
and jogging CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID
Cell/Inclusion Description Significance
Neutrophil Polymorphonuclear leukocyte Bacterial sepsis
Crystal-induced
inflammation
Lymphocyte Mononuclear leukocyte Nonseptic inflammation
Macrophage Large mononuclear leukocyte, may Normal viral infections
(Monocyte) be vacuolated
LE cell Neutrophil containing ingested Lupus erythematosus
"round body”
Reiter cell Vacuolated macrophage with Reiter syndrome
ingested neutrophils Reactive arthritis
RA cell Neutrophil with dark cytoplasmic Rheumatoid arthritis
(Ragrocyte) granules containing immune Immunologic
complexes inflammation

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Rice bodies Macroscopically resemble polished Tuberculosis, septic and

rice rheumatoid arthritis
Microscopically show collagen &
fibrin
Ochronotic Debris from metal & plastic joint Ochronotic arthropathy
shards prosthesis Alkaptonuria, ochronosis
“Ground pepper” appearance
Cartilage cells Large, multinucleated cells Osteoarthritis
Synovial lining Similar to macrophage, but may be Normal
cell multinucleated, resembling a Disruption from
mesothelial cell arthrocentesis
Fat droplets Refractile intracellular & Traumatic injury
extracellular globules Chronic inflammation
Stain with Sudan dyes
Hemosiderin Inclusions within clusters of Pigmented villonodular
synovial cells synovitis

CRYSTAL IDENTIFICATION

CAUSES OF CRYSTAL FORMATION:

- Metabolic disorders
- Decreased renal excretion that produce increased blood levels of crystallizing
chemicals
- Degeneration of cartilage and bones
- Injection of medications (corticosteroid)

Crystal Shape Compensated Significance

Polarized Light CHEMISTRY TESTS
Monosodium Needles (-) birefringence Gout Glucose - Most frequently tested chemistry test
urate - Done in conjunction w/ blood glucose (8 hrs fasting)
Calcium Rhombic Square, (+) birefringence Pseudogout < 10 𝑚𝑔/𝑑𝐿
pyrophosphate rods 𝐵𝑙𝑜𝑜𝑑 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 − 𝑆𝐺 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 =
(𝑁𝑜𝑟𝑚𝑎𝑙)
Cholesterol Notched, rhombic (-) birefringence Extracellular - Example:
plates o 99 mg/dL - 90 mg/dL = 9 mg/dL (Normal)
Corticosteroid Flat, variable- (+) & (-) Injections o 99 mg/dL- 40 mg/dL = 59 mg/dL (Infection)
shaped plates birefringence
Lactate Normal value = < 250 mg/dL (increased in infection)
Calcium oxalate Envelopes (-) birefringence Renal dialysis
Apatite Small particles; No birefringence Osteoarthritis Protein Normal value = < 3 g/dL
(Calcium requires electron Increased in inflammatory & hemorrhagic disorders
phosphate) microscopy Uric acid Normal value = same as blood (increased in gout)

- POLARIZING MICROSCOPE MICROBIOLOGY TESTS

o Detects for the presence or absence of birefringence - Common organisms that infect the synovial fluid:
o Birefringence = ability to refract light in 2 directions o S. aureus = predominant
- COMPENSATED POLARIZING MICROSCOPE
o Streptococcus
o Confirms the type of birefringence (positive or negative BR)
o Red compensator is placed between crystal & analyzer o Haemophilus
- Owing to differences in the linear structure of the molecules in MSU & CPPD o N. gonorrhoeae = gonococcal arthritis
crystals, the color produced by each crystal when it is aligned w/ the slow vibration
can be used for identification SEROLOGIC TESTS
- A control slide for polarization of MSU can be prepared using betamethasone - Autoantibody detection (SLE, RA)
acetate corticosteroid - Detection of antibodies to Borrelia burgdorferi (Lyme disease)
MONOSODIUM URATE (MSU) CALCIUM PYROPHOSPHATE
DIHYDRATE (CPPD)
- The molecules in the MSSU - The molecules in the CPPD REVIEW!!!
crystals run parallel to the long axis crystals run perpendicular to the 1. Normal volume of synovial fluid in the knee 4. These are ground
of the crystal & when aligned w/ the long axis of the crystal, when cavity: pepper-like particles
slow vibration, the velocity of slow aligned w/ the slow axis of a. 3.5 mL seen in synovial fluid
light passing through the crystal is compensator, the velocity of fast b. 10 mL a. Rice bodies
not impeded as much as the fast light passing through the crystal is c. 20 mL b. Psammoma
light, which runs against the grain & much quicker, producing a blue d. 25 mL bodies
produces yellow color. This is a color & positive birefringence. c. Ochronotic
negative birefringence (subtraction 2. Why is acetic acid not used as WBC diluent shards
of velocity from the fast ray). on synovial fluid cell count? d. Pepper bodies
- It will form a mucin clot
KEEP IN MIND!
- When the crystals are aligned perpendicular to the slow vibration, the color is 3. Rice bodies are called so because they
reversed. resemble
a. Polished rice
b. Ground pepper
c. Cooked rice
d. Fried rice

LABORATORY FINDINGS IN JOINT DISORDERS

Group I IIa IIb III IV
Non-inflammatory Inflammatory Inflammatory Septic Hemorrhagic
(Immunologic) (Crystal-induced)
Significance Degenerative joint disorders Immunologic disorders Gout Microbial infection Traumatic injury,
(osteoarthritis) (RA, SLE, etc) Pseudogout Coagulation deficiencies
Color & Clarity Clear, yellow fluid Cloudy, yellow fluid Cloudy or milky fluid Cloudy, yellow-green fluid Cloudy, red fluid
Viscosity Good Poor Low Variable Low
WBC Count <1,000/uL 2,000-75,000/uL Up to 100,000/uL 50,000-100,000/uL Equal to blood
Neutrophils <30% >50% <70% > 75% Equal to blood
Glucose Similar to blood glucose Decreased Decreased Decreased Normal
Others -- (+) Autoantibodies (+) Crystals (+) Culture and gram stain (+) RBCs

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SEROUS FLUID METHOD OF COLLECTION

- Fluid between parietal & visceral membranes
- Function: To provide lubrication between 2 membranes as surfaces move against 3 P's (Pleural, Pericardial, Peritoneal fluid)
each other - NORMAL APPEARANCE = Clear, pale yellow
- Pleural fluid = Thoracentesis
- Pericardial fluid = Pericardiocentesis
- Peritoneal (ascitic) fluid = Paracentesis
NORMAL VOLUMES
- < 30 ml
- < 50 mL
- <100 mL
Specimen is distributed in the following tubes:
- EDTA = Cell counts and differential
- Sterile heparin or SPS = Microbiology and cytology
- Plain/heparin tubes = Chemistry (samples for pH must be maintained
anaerobically in ice)

PLEURAL FLUID

Appearance Significance
Clear, pale yellow Normal
Turbid, white Microbial infection (TB)
Brown Rupture of amoebic liver abscess
Black Aspergillosis
EFFUSION
Viscous Malignant mesothelioma (↑ hyaluronic acid)
- Accumulation of fluid between the membranes
Milky Chylous material, pseudochylous material
- Classified as exudate or transudate
Bloody Hemothorax, hemorrhagic effusion
TRANSUDATE
- Disruption of fluid production & regulation between membranes MILKY PLEURAL FLUID
- Changes in hydrostatic and oncotic pressure (HP, OP) Chylous effusion Pseudochylous effusion
- Examples: Hypoproteinemia (↓ Oncotic pressure), Congestive heart failure (↑ Cause Thoracic duct leakage Chronic inflammation
Hydrostatic pressure), Nephrotic syndrome (↓ Oncotic pressure) Appearance Milky/white Milky /green tinge /gold
paint
EXUDATE Leukocytes ↑ Lymphocytes Mixed cells
- Direct damage to the membrane of a particular cavity Cholesterol crystals Absent Present
- Examples: Infection, Inflammation, Malignancy Triglycerides >110 mg/dL <50 mg/dL
Sudan III staining (+++) (-)/weakly (+)

BLOODY PLEURAL FLUID

HEMOTHORAX HEMORRHAGIC EFFUSION
Distribution of blood Uneven Even
Hematocrit Pleural fluid Hct is >½ of Pleural fluid Hct is <½ of
whole blood Hct Whole blood Hct

In hemothorax, the A chronic membrane disease

effusion is actually effusion contains both blood
occurring from the and increased pleural fluid.
impouring of blood from
the injury.

SIGNIFICANCE OF CELLS SEEN IN PLEURAL FLUID

Cell Significance
Neutrophil Pneumonia, pancreatitis, pulmonary infarction
Eosinophil Pneumothorax, hemothorax, allergic reactions,
parasitic infections
Lymphocyte TB, viral infection, autoimmune disorders,
TRANSUDATES vs. EXUDATES malignancy
Transudate Exudate Mesothelial cell Normal and reactive forms have no significance
Appearance Clear Cloudy (Lines the serous membranes) Decreased in TB
Fluid:serum protein ratio (most reliable) <0.5 >0.5 Plasma cells TB
Fluid:serum LD ratio (most reliable) <0.6 >0.6 Malignant cells Primary adenocarcinoma, small cell carcinoma
WBC count <1,000/uL >1,000/uL
Spontaneous clotting No Possible SIGNIFICANCE OF TESTING OF PLEURAL FLUID
Pleural fluid cholesterol (mg/dL) <45-60 > 45-60 Test Significance
Pleural fluid:serum cholesterol ratio <0.3 >0.3 Glucose ↓ Rheumatoid inflammation, purulent infection
Pleural fluid:bilirubin ratio <0.6 > 0.6 Lactate ↑ Bacterial infection
Serum-ascites albumin gradient (SAAG) >1.1 <1.1 Triglyceride ↑ Chylous effusions
Glucose Equal to serum Possibly low pH ↓ Pneumonia not responding to antibiotics
↓↓↓ Esophageal rupture
Protein (g/dL) < 3.0 > 3.0
↓ Complicated parapneumonic effusion (loculated or
Rivalta’s test (-) (+)
associated with empyema)
Adenosine Tuberculosis, malignancy
RIVALTA'S TEST (SEROSAMUCIN CLOT TEST)
deaminase (ADA)
- Differentiates exudates from transudates Amylase ↑ Pancreatitis, esophageal rupture, malignancy
- Acetic acid + Water + Unknown fluid → (+) Heavy precipitation = EXUDATE
TUMOR MARKERS FOR EFFUSIONS OF MALIGNANT ORIGIN
SERUM-ASCITES ALBUMIN GRADIENT (SAAG)
Tumor marker Significance
- Recommended to detect transudates of hepatic origin
CEA (Carcinoembryonic antigen) Colon cancer
- SAAG Serum Albumin - Peritoneal Fluid Albumin
CA 125 Ovarian/ metastatic uterine cancer
- Transudate if SAAG is >1.1
CA 15-3, CA 549 Breast cancer
- Exudate if SAAG <1.1
CYFRA 21-1 Lung cancer, breast cancer (Henry)
(Cytokeratin Fragment) Urinary bladder cancer (Barh, Carpi, Berma)

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PERICARDIAL FLUID Basal Acid Output (BAO)

- Total gastric secretion during unstimulated, fasting state
SIGNIFICANCE OF PERICARDIAL FLUID TESTING - Duration of collection:
Appearance Significance o 1-hour collection (consists of four 15-minute specimens, but a single 1-hour
can be used): routinely performed
Clear, pale yellow Normal, transudate o 2-hour collection: for Insulin hypoglycemia test
Blood-streaked Infection, malignancy
Grossly bloody Cardiac puncture Maximum Acid Output (MAO)
Anticoagulant medications - Total gastric secretion after gastric stimulation
Milky Chylous and pseudochylous material - Duration of collection
Differential Significance o 1-hour collection (at 15-minute intervals) - when Pentagastrin and Histamine
are used
↑ Neutrophils Bacterial endocarditis o 2-hour collection-for Insulin hypoglycemia test and when Histalog is used
Malignant cells Metastatic carcinoma
Tests Significance GASTRIC STIMULANTS
Gram stain and culture Bacterial endocarditis - Test Meals
Acid-fast stain Tubercular effusion o Ewald's = bread, weak tea or water
Adenosine deaminase Tubercular effusion o Boa's = oatmeal
(ADA) o Riegel's = beef steak and mashed potato
- Chemical Stimulants
o Pentagastrin = most preferred
PERITONEAL (ASCITIC) FLUID o Insulin = to assess vagotomy procedure
o Histalog (Betazole)
SIGNIFICANCE OF PERITONEAL FLUID TESTING o Histamine
Appearance Significance - Sham Feeding (Fictitious feeding) = sandwich
Clear, pale yellow Normal
Turbid Microbial infection BAO (mEq/hr) MAO (mEq/hr) BAO/MAO Ratio
Green Gall bladder/pancreatic disorders Normal 2.5 25.0 10%
Blood-streaked Trauma, infection, malignancy Pernicious anemia 0 0 0
Mily Lymphatic trauma & blockage Duodenal ulcer 5.0 30.0 17%
WBC count Significance Zollinger-Ellison syndrome 18.0 25.0 72%
< 500 cells/uL Normal
> 500 cells/uL Bacterial peritonitis, cirrhosis MACROSCOPIC EXAMINATION
Differential Significance Color Significance
↑ Neutrophils Bacterial peritonitis, cirrhosis Pale gray (with mucus) Normal
Malignant cells Malignancy Yellow-green Large amounts of bile
Tests Significance Red Small amount of fresh blood
Peritoneal lavage > 100,000 RBCs /uL indicates blunt trauma injury (intra- Coffee ground Large amount of blood
abdominal bleeding) Volume Significance
CEA Malignancy of GI origin 20 – 50 mL Normal (fasting specimen)
CA 125 Malignancy of ovarian origin > 50 mL Abnormal (fasting specimen)
Glucose ↓ Tubercular peritonitis, malignancy 20-60 mL up to 120 mL After Ewald's test meal
Amylase ↑ Pancreatitis, Gl perforation 45-150 mL After alcohol test meal or histamine stimulation
BUN/Creatinine Ruptured/punctured bladder
Gram stain & Culture Bacterial peritonitis Term Definition Significance
Acid-fast stain Tubercular peritonitis Euchlorhydria Normal free HCI --
ADA Tubercular peritonitis Hyperchlorhydria Increased free HCl ZES, Peptic ulcer
Hypochlorhydria Gastric fluid pH >3.5 but falls after Carcinoma of the
PSAMMOMA BODIES gastric stimulation (Decreased free stomach
HCl)
- Contain concentric striations of collagen-like material Achlorhydria Gastric fluid pH >3.5 and does not fall Pernicious anemia
- Seen in benign conditions & associated with ovarian & thyroid carcinomas after gastric stimulation (Absence of
free HCl)
REVIEW!!!
Anacidity Failure to produce a pH <6.0 following Pernicious anemia
1. Effusion produced in congestive heart 4. Marker for lung cancer gastric stimulation
failure: a. CA 15-3
a. Transudate b. NMP
b. Exudate c. CA 549 DIAGNEX TUBELESS TEST (DIAGNEX BLUE TEST)
d. CYFRA 21-1 - Developed by Squibb
2. Normal appearance of serous fluids: - Specimen = Urine
a. Pale gray, slightly mucoid 5. Type of effusion in - Principle = uses Azure blue or Azure A
b. Red or pink empyema o A blue colored dye azure-A is complexed with an ion-exchange resin
c. Clear, pale yellow a. Pleural transudate o The granules are ingested by the patient in the morning
d. Deep yellow b. Pericardial exudate o The granules exchange the dye for H ions of the free HCl in the stomach
c. Peritoneal transudate o Azure-A is released in proportion to free HCl, w/cis absorbed in the blood &
3. Pleural fluid hematocrit = 26% d. Pleural exudate excreted in urine
Whole blood hematocrit = 50% e. Peritoneal exudate o The amount of dye excreted in urine is an indicator of gastric HCl secretory
a. Hemothorax f. Pericardial exudate activity
b. Hemorrhagic effusion
REVIEW!!!
1. Gastric tube inserted through the 4. BAO:MAO ratio in
GASTRIC FLUID nose: pernicious anemia
a. Levin tube a. 5.0
- Zollinger-Ellison Syndrome (ZES) b. Rehfuss tube b. 18.0
o Non-beta islet cell adenoma of the pancreas c. Edlich tube c. 0
o ↑↑ Gastrin = ↑↑ HCl = Hyperchlorhydria, hyperacidity d. Ewald’s tube d. 25.0
- Pernicious Anemia e. Lavacuator tube e. 10.0
o Anti-parietal cell Ab
o Anti-intrinsic factor Ab 2. Normal color of gastric fluid 5. Why does achlorhydria
o (-) HCl = Achlorhydria, Anacidity a. Clear, pale yellow occur in pernicious anemia
b. Yellow-green - Because parietal cells
CELLS IN THE STOMACH c. Orange which produce HCl
- Parietal cells = produce HCl and intrinsic factor d. Pale gray, mucoid are destroyed
- Chief cells = produce pepsinogen e. Coffee-ground
- Specialized G cells = produce gastrin 6. Specimen used in the
- Foveolar cells = produce mucus that protects the stomach wall from acid 3. Normal volume of gastric fluid from a Diagnex tubeless test
fasting patient: a. Urine
SPECIMEN COLLECTION a. 500 mL b. Blood
- Method of collection = Gastric aspiration b. 250 mL c. Stool
- Gastric tubes: (Henry, 17th Ed p.553) c. 0-20 mL d. Bile
o Levin tube = passed through the nose d. 20-50 mL
o Rehfuss = passed through the mouth e. 100 mL
o Others = Lavacuator tube, Ewald's tube, Edlich tube = passed through the
mouth
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FECALYSIS MUSCLE FIBERS

- Creatorrhea = Abnormal excretion of muscle fibers in feces
FECES - Determination:
- Normal feces contain bacteria, cellulose, undigested foodstuffs, Gastrointestinal o The patient should include meat in the diet
secretions, bile pigments, cells from the intestinal wall, electrolytes, water o Emulsified stool + 10% Eosin - Coverslip & stand for 3 minutes
- Around 100-200g of stool is passed per day o Count the number of undigested fibers (HPF)
- Human feces contains around 75% water & 25% solids - Completely digested: No striations
- The odor of feces is due to the presence of indole and skatole - Partially digested: Striation in one direction
- Undigested: Striation in both directions
MACROSCOPIC STOOL CHARACTERISTICS - Abnormal: >10 undigested muscle fibers (ex. Bile duct obstruction, Cystic fibrosis)
COLOR/APPEARANCE CLINICAL SIGNIFICANCE
Brown Normal (urobilin/stercobilin) FECAL LEUKOCYTES
Black Upper GI bleeding (melena), iron, charcoal, bismuth - >3 neutrophils/hpf = Invasive condition
Red Lower GI bleeding (hematochezia), beets, food o Diarrhea with WBCs = Salmonella, Shigella, Yersinia, Entero-invasive E. coli,
coloring, rifampin Campylobacter
Pale yellow, white, gray Bile duct obstruction (- Urobilin), barium sulfate o Diarrhea without WBCs = Toxin producing (S. aureus, V. cholerae), virus,
Green Biliverdin, oral antibiotics, green vegetables parasites
Blue Prussian blue, grape soda
Violet/purple Porphyria Determination:
Bulky/frothy Bile duct obstruction, pancreatic disorders, steatorrhea - Wet preparation = Stool + Loeffler's methylene blue
- Dried preparation = Stool +Wright's or Gram stain
Butter-like Cystic fibrosis (↑ mucus)
- Lactoferrin latex agglutination test
Mucus, blood-streaked Colitis, dysentery, malignancy, constipation o Lactoferrin = found in secondary granules of neutrophils = (+) Invasive
mucus bacterial pathogen
Ribbon-like Intestinal constriction
Rice watery Cholera GUAIAC FECAL OCCULT BLOOD TEST (gFOBT)
Pea-soup Typhoid Notes - Occult = “hidden”
Scybalous Constipation - Screen test for = colorectal cancer
("Goat droppings”) - Significant = >2.5 mL blood/150g stool
- Sample = center portion of the stool
𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛
Principle 𝐻2 𝑂2 + 𝐺𝑢𝑎𝑖𝑎𝑐 → 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝐺𝑢𝑎𝑖𝑎𝑐 (𝑏𝑙𝑢𝑒) + 𝐻2𝑂
(gFOBT) pseudoperoxidase

- Immunochemical FOBT (iFOBT) uses anti-human

hemoglobin antibodies
Chromogens - Considering that a normal stool can contain up to 2.5 mL of
blood, a less sensitive chemical reactant is understandably
more desirable (to avoid false positive reactions)
o Benzidine (most sensitive)
o Guaiac (preferred)
o O-toluidine
Interferences False (+) FOBT False (-) FOBT
Avoid for 3 days: Avoid for 7 days: Avoid for 3 days:

Red meat Aspirins *Vitamin C

Melon NSAIDs other (>250mg/dL)
Broccoli than paracetamol *Iron supplements
Cauliflower containing Vitamin C
Horseradish
Turnip Failure to wait
specified time after
sample is applied to
add the developer
reagent
- Type 1 = Separate hard lumps like nuts (hard to pass)
- Type 2 = Sausage-shaped but lumpy OTHER FECAL SCREENING TESTS
- Type 3 = Like a sausage but with cracks (Optimal)
- Type 4 = Like a sausage or snake, smooth & soft (Most optimal) Test Methodology/Principle Other Details
- Type 5 = Soft blobs with clear-cut edges (passed easily) - Differentiate fetal blood and Pink solution = (+) fetal
- Type 6 = Fluffy pieces with ragged edges, a mushy stool maternal blood blood (HbF)
(Apt-Downey Test)

- Type 7 = Watery, no solid pieces, entirely liquid - Specimen = infant stool/vomitus

- Bloody stools & vomitus are Yellow brown supernatant =
Apt Test

sometimes seen in neonates as a (+) maternal blood (HbA)

MICROSCOPIC EXAMINATION result of swallowing maternal blood
during delivery Remember:
FATS - HbF is alkali resistant
- Steatorrhea = Increased fats in stool (>6 g/day) Emulsified stool → Centrifuge → Add - HbA is denatured by
- Tests: 1% NaOH to supernatant NaOH
o Screening test = microscopic examination of feces for fat globules - Detects pancreatic enzyme called Clearing of film = (+) Trypsin
X-ray Film Test

o Definitive test = fecal fat determination trypsin

(Gelatin Test)

- When trypsin is present in the stool, No clearing of film = (-)

FECAL FAT DETERMINATION it digests the gelatin on the X-ray Trypsin
Qualitative Tests film leaving a clear area
1. Neutral Fat Stain 2. Split Fat Stain (Fatty acids) Absence of trypsin is seen
(Triglycerides) in: cystic fibrosis
Emulsified stool + 36% Acetic acid - Most valuable in assessing cases of - Clinitest of >0.5 g/dL =
+ Sudan III → Orange droplets
Carbohydrates

Stool suspension + 95% Ethanol infant diarrhea (Ex: Lactose Carbohydrate

+ Sudan III → Orange droplets (fatty acids) intolerance) intolerance
Fecal

(neutral fats/triglycerides) - Determination - 7.0 – 8.0 = Normal stool

Normal = 100 droplets (<4 um) o Clinitest: test for reducing pH
> 60 droplets/hpf = Steatorrhea Slightly increased = 100 droplets sugars - <5.5 = Stool pH in CHO
(1-8 um) o Fecal pH disorder
Increased = 100 droplets (6-75 um) - Immunoassay using an ELISA test - Sensitive indicator of
Elastase-1

Quantitative Tests exocrine pancreatic

Van de Kamer titration insufficiency
- Gold standard for fecal fat determination
- For definitive diagnosis of steatorrhea
- Titration with NaOH
- D-xylose is a pentose that is - Specimens = 2-hour
- Sample = 3-day stool (72 hours) absorbed without the help of blood & 5-hour urine
- Normal value = 1 – 6 grams of fats per day
D-xylose

pancreatic enzymes & is not - Low urine D-xylose =

test

- Steatorrhea = > 6 grams of fats per day

metabolized. This test differentiates malabsorption
- Acid Steatocrit= rapid test to estimate the amount or fat excretion (similar to
malabsorption & maldigestion - Normal urine D-xylose
microhematocrit test)
= maldigestion

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DIARRHEA - Bile
- Stool weight of >200 g/day with increased liquidity & frequency of >3x/day o Approximately 500-1,000 mL of bile enters the duodenum daily
- Acute diarrhea = <4 weeks o Yellow to brown or green & usually alkaline, with a pH of 7.0 to 8.5
- Chronic diarrhea = >4 weeks o Bile salts (sodium glycocholate & taurocholate), bilirubin, cholesterol,
- Major mechanisms are Secretory, Osmotic & Altered Motility. phospholipid & inorganic salts
- Laboratory tests used to differentiate these mechanisms are fecal electrolytes o ALP is the only enzyme present in significant amount in bile
(fecal Na+ and K+), fecal osmolarity and stool pH o Cholecystokinin -stimulates contraction of the gallbladder to increase bile flow
- Normal Fecal Osmolarity = 290 mOsm/kg - Intestinal secretion mixed with gastric secretion
- Normal Fecal Na+ level 30 mmol/L - Possibly, partially digested food
- Normal Fecal K+ level = 75 mmol/L
VAGINAL SECRETIONS (Strasinger, 6th Ed)
SECRETORY DIARRHEA
- Increased secretion of water and electrolytes, which override the reabsorptive CLINICAL FEATURES AND LABORATORY FINDINGS
ability of the large intestine Findings Normal Desquamative Atrophic Vaginitis
- Causes: Bacterial, viral and protozoan infections, drugs, laxatives, hormones, Inflammatory
inflammatory bowel disease, endocrine disorders, neoplasms, collagen vascular Vaginitis
disease Appearance White, flocculent Excessive Excessive
discharge purulent vaginal purulent vaginal
OSMOTIC DIARRHEA discharge, discharge,
- Retention of water and electrolytes in the large intestine due to incomplete vaginal erythema vaginal erythema
breakdown or reabsorption of food pH 3.8-4.2 >4.5 >4.5
- Causes: Maldigestion, malabsorption, disaccharidase deficiency (lactose WBCs 2+ 3+ to 4+ 3+ to 4+
intolerance), laxatives, antacids, amebiasis, antibiotics Lactobacilli Predominant Absent or Decreased
reduced
ALTERED MOTILITY Clue cells Absent
- Enhanced (hypermotility) or slow (constipation) motility Other cells Absent (except Occasional Occasional
- Causes: Irritable bowel syndrome (IBS), Rapid gastric emptying (RGE) dumping RBCs during parabasal or parabasal or
syndrome menstruation) basal cells; >1+ basal cells; >1+
RBCs RBCs
REVIEW!!! Other organisms Other lactobacilli 2+ gram-positive Increased gram
1. Bismuth causes which 4. Abnormal excretion of undigested
stool color muscle fibers in the feces: subgroups, cocci positive cocci and
a. Gray a. Creatorrhea occasional yeast gram negative
b. Black b. Steatorrhea rods; decreased
c. Green c. Diarrhea large rods
d. Brown d. Hematochezia Amine (Whiff test) Negative Negative Negative
e. Melena
2. Barium sulfate causes
which stool color? 5. Positive color in the Guaiac test: Findings Bacterial Candidiasis Trichomoniasis
a. Gray a. Blue vaginosis
b. Black b. Red
Appearance Thin, white to gray White, curd-like Yellow-green
c. Green c. Yellow
d. Brown d. Green vaginal discharge vaginal discharge frothy adherent
vaginal discharge
3. Gold standard for the 6. Pink solution in the Apt test indicates the increased in
definitive diagnosis of presence of: volume
steatorrhea: a. Maternal blood pH >4.5 3.8-4.5 >4.5
a. Van den Bergh b. Fetal blood WBCs Rare or absent 3+ to 4+
reaction c. Trypsin Lactobacilli Rare or absent Present
b. Van Handel- d. Fatty acids
Zilversmith Clue cells >20% Absent
method 7. What is the normal stool pH? Other cells -- Large clumps of
c. Van de Kamer a. 5.0 - 6.0 epithelial cells
titration b. 6.0 - 7.0 Other organisms Increase in small Budding yeast
d. Von Kossa Stain c. 7.0 - 8.0 curved bacilli, and
d. 8.0 - 9.0 coccobacilli & pseudohyphae
pleomorphic
bacilli
MISCELLANEOUS TOPICS Amine (Whiff) test Positive Negative
Confirmatory DNA probe DNA probe DNA probe or
APPEARANCE OF SEDIMENTs STAINED WITH tests Proline OSOM BVBLUE culture
STERNHEIMER-MALBIN STAIN (PER Handbook) aminopeptidase Rapid Test OSOM
Squamous Cells Pale purple with dark purple nuclei OSOM BVBLUE Trichomonas
Renal Cells Orange-purple cytoplasm and dark nuclei Rapid Test Rapid Test
Leukocytes Pale pink with purple nuclei
"Glitter" cells Pale blue
Erythrocytes May not stain at all or stain pale pink URINALYSIS AND BODY FLUIDS AUTOMATION
Yeast Cells Stain dark purple, or do not take the stain at all
Crystals Crystals do not stain
Hyaline/Waxy Casts May not stain at all or are pale pink
Granular Casts Have a pink matrix and purple granules
Red Blood Cell Casts Red-purple
Bacteria Bacteria vary in color
Spermatozoa Spermatozoa stain blue
Trichomonas Trichomonas stains pale blue the nucleus is purple
*Sternheimer-Malbin stain is available commercially under a variety of names,
including Sedi-Stain and KOVA stain

DUODENAL CONTENTS (Henry, 17h Ed p.558-560)

COMPOSITION
- Pancreatic exocrine secretion
o 1,500 mL/day, major contributor to duodenal content
o Colorless, clear, nonviscid alkaline solution with a pH approximately of 8.0
o 1-2% organic: enzymes & their precursors
o 1% inorganic: sodium (major cation), bicarbonate (major anion)
o Secretin & pancreozymin - hormones that stimulate pancreatic secretion

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REAGENT STRIP REACTION READINGS (Strasinger, Graff)

Parameter Reading Parameter Reading
Specific 1.000 Blood Negative
gravity 1.005 Non-hemolyzed trace
1.010 Non-hemolyzed moderate
1.015 Hemolyzed trace
1.020 1+ = Small
1.025 2+ = Moderate
1.030 3+ = Large
Protein Negative Bilirubin Negative
Tr = <30 mg/dL 1+ = Small
1+ = 30 mg/dL 2+ = Moderate
In UF-1000i Analyzer, particles in the urine are categorized on the basis of: 2+ = 100 mg/dL 3+ = Large
- Forward scatter 3+ = 300 mg/dL
- Impedance signals 4+ = >2000 mg/dL
- Fluorescence staining characteristics pH 5.0 Urobilinogen Normal
- Adaptive cluster analysis 6.0 - 0.2 mg/dL (E.U.)
- Side scatter = specific for detection of bacteria 6.5 - 1 mg/dL (E.U.)
7.0
- In UF-1000i automated urinalysis analyzer, bacteria can be quantitated with the 7.5 Abnormal
use of scattergram and histogram 8.0 - 2 mg/dL (E.U.)
8.5 - 4 mg/dL (E.U.)
- 8 mg/dL (E.U.)
Glucose Negative Leukocytes Negative
Tr = 0.1% (100 Trace
mg/dL) 1+ = Small
1+ = 0.25% (250 2+ = Moderate
mg/dL) 3+ = Large
2+ = 0.5% (500
mg/dL
3+ = 1.0% (1000
mg/dL)
4+ = 2.0% (>2000
mg/dL)
Ketones Negative Nitrite Negative
Tr = 5 mg/dL Positive
QUALITY ASSURANCE 1+ = Small (15
mg/dL)
EXAMPLES OF QUALITY ASSURANCE (Rodak, 4th Ed) 2+ = Moderate (40
mg/dL)
- Pre-analytical Variables
3+= Large (80 to 160
o Selection of assay relative to pattent need
mg/dL)
o Implementation of assay selection
o Patient identification and preparation
o Specimen collection technique SENSITIVITY OF URINE REAGENT STRIPS (Strasinger)
o Specimen transport, preparation and storage
Parameter Multistix Chemstrip
o Monitoring of specimen condition
pH 5.0 to 8.5 in 0.5 increments 5.0 to 9.0 in 1.0
- Analytical Variables
increments
o Assay validation and instrument selection
o Laboratory staff competence Protein 15 to 30 mg/dL albumin 6 mg/dL albumin
o External and internal quality control (DiaScreen 5 mg/dL)
- Post-analytical Variables Glucose 75 to 125 mg/dL (100 mg/dL) 40 mg/dL
o Accuracy in transcription and filing of results Ketones 5 to 10 mg/dL acetoacetic acid 9 mg/dL acetoacetic
o Content and format of laboratory report, narrative report, reference interval & acid; 70 mg/dL acetone
therapeutic range Blood 5 to 20 RBCs/mL, 0.015 to 0.062 5 RBCs/mL, hgb
o Timeliness in communicating critical values, patient and physician satisfaction mg/dL hgb corresponding to 10
o Turnaround time (Henry, Rodak, Turgeon), cost analysis RBCS/mL
- TQM (total Quality Management) = based on a team concept involving personnel Bilirubin 0.4 to 0.8 mg/dL bilirubin 0.5 mg/dL bilirubin
at all levels working together to achieve a final outcome of customer satisfaction Urobilinogen 0.2 mg/dL urobilinogen 0.4 mg/dL urobilinogen
through implementation Nitrite 0.05 mg/dL nitrite ion 0.05 mg/dL nitrite ion
- CQQ (Continuous Quality Improvement) = improving patient outcomes by Leukocytes 5 to 15 WBC/hpf 10 to 25 WBC/hpf
providing continual quality care in a constantly changing health-care environment Specific 1.000 to 1.030 1.000 to 1.030
- PDCA = Plan-Do-Check-Act gravity
- PDSA = Plan-Do-Study-Act
- PDMAI = Plan, Design, Measure, Assess, Improve

QUALITY CONTROL OF LABORATORY EQUIPMENT

Daily Basis Reagent strips; check the temperatures of refrigerators and
water baths
Weekly Basis Disinfection of centrifuges; check pH and purity meter
resistance of deionized water
Biweekly Diluents checked for contamination (examine in counting
Basis chamber under 4x magnification)
Monthly Basis Speed of cytocentrifuge checked with tachometer, & timing
checked with stopwatch
Every 3 Calibration of centrifuges (record appropriate centrifugal force)
Months
Annually Professional cleaning of the microscope
*Tachometer (Speed), Stopwatch (Timing), Strobe light = devices used to calibrate the
centrifuge

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