AUBF Reviewer (HY)

MTLE Clinical Microscopy — High-Yield Reviewer

SAFETY

Chain of Infection: Infectious agent → Reservoir → Portal of Exit → Mode of Transmission → Portal of Entry → Susceptible Host

Handwashing: Best way to break chain of infection; scrub at least 15-20 seconds; visibly soiled = soap & water; not visibly soiled = alcohol-based sanitizer

Biohazard label color: Fluorescent orange; urine is the exception — pour down sink, flush, disinfect with 1:10 sodium hypochlorite daily

Disinfection: Eliminates pathogens except bacterial spores

NFPA Diamond: Red=Fire, Blue=Health, Yellow=Reactivity, White=Specific hazard; Scale 0–4 (4 = extreme)

Fire types: A=ordinary combustibles, B=flammable liquids, C=electrical, D=flammable metals, K=cooking oils

• ABC dry chemical = most common all-purpose extinguisher

• Water = Class A only

Chemical spills: Flush with water 15 minutes; NEVER neutralize on skin; ALWAYS add acid to water

RACE: Rescue → Alarm → Contain → Extinguish/Evacuate
PASS: Pull → Aim → Squeeze → Sweep

RENAL FUNCTION

Kidney weight: ~150g; measures 12.5 × 6 × 2.5 cm

Urine formation order: Glomerulus → PCT → Loop of Henle → DCT → Collecting Duct → Calyx → Renal Pelvis

Renal blood flow: 1,200 mL/min; plasma flow: 600–700 mL/min; kidneys receive 25% of cardiac output

Glomerulus: Non-selective filter; MW cutoff <70,000 daltons; SG of filtrate = 1.010; albumin blocked by negative charge at physiologic pH

Glomerular filtration barrier: Capillary endothelium → Trilayer basement membrane → Filtration diaphragm (podocytes)

PCT reabsorbs: 65% of all substances — salts, water, amino acids, glucose, urea

Renal threshold for glucose: 160–180 mg/dL

ADH (Vasopressin): Regulates water reabsorption in DCT & CD; deficiency = Diabetes Insipidus; excess = SIADH

Aldosterone: Regulates sodium reabsorption; sodium in → potassium out (DCT & CD)

RAAS: Low BP → Renin → Angiotensinogen → Angiotensin I → (ACE in lungs) → Angiotensin II → vasoconstriction + aldosterone + ADH release

Tubular secretion functions: Acid-base balance (secretes H⁺ as NH₄ and H₂PO₄) + eliminates non-filtered waste

RTA (Renal Tubular Acidosis): Failure to secrete H⁺ → alkaline urine despite acidic blood

RENAL FUNCTION TESTS

GFR tests (clearance):

• Urea = obsolete

• Creatinine = most common

• Inulin (MW 5,200 Da) = gold standard (reference method)

• Cystatin C (MW 13,000 Da)

• Beta₂-microglobulin = better marker of tubular function than GFR

Creatinine clearance formula: Ccr = (U × V) / P × (1.73/A)

• Normal: Male 107–139 mL/min; Female 87–107 mL/min

• ~7–10% of creatinine is tubularly secreted

Cockroft-Gault eGFR variables: Age, Sex, Body weight (×0.85 if female)

Tubular reabsorption tests (concentration tests):

• Fishberg (obsolete): fluid restriction → SG ≥1.022 (12 hr) or ≥1.026 (24 hr)

• Osmolality preferred over SG; normal urine = 1–3× serum osmolality (275–900 mOsm/kg)

Tubular secretion test: PAH test (reference method); PSP test = obsolete

URINALYSIS INTRODUCTION

Urine composition: 95–97% water; major organic solid = urea; major inorganic solid = chloride > sodium > potassium; principal salt = NaCl

Specimen types:

Drug specimen collection: Volume = 30–45 mL; container = 60 mL; temperature within 4 min = 32.5–37.7°C; blueing agent added to toilet

Specimen integrity: Test within 2 hours (Strasinger/Harr); ideally 30 min (Turgeon)

Changes in unpreserved urine:

• Increased: pH, bacteria, odor, nitrite

• Decreased: glucose, ketones, bilirubin, urobilinogen, RBCs/WBCs/casts

• Darkened: color

• Least affected: protein

Key preservatives:

PHYSICAL EXAMINATION

Normal volume: 600–2,000 mL/day; average 1,200–1,500 mL/day

Normal urine pigments:

• Urochrome = major yellow pigment (endogenous metabolism)

• Uroerythrin = pink; deposits on amorphous urates

• Urobilin = dark yellow/orange-brown; oxidized urobilinogen; present in old specimens

Key urine colors:

Urine clarity:

• View through newspaper against light source

• Clear → Hazy → Cloudy → Turbid → Milky

• Amorphous urates = pink sediment (acid urine); soluble in heat & alkali

• Amorphous phosphates = white (alkaline urine); soluble in dilute acetic acid

Urine odor:

CHEMICAL EXAMINATION — REAGENT STRIP

Ascorbic acid (11th pad): Causes false negatives in Blood, Bilirubin, Leukocytes, Nitrite, Glucose ("BB LNG"); + phosphomolybdate → molybdenum blue

SPECIFIC GRAVITY

Urinometer (Hydrometer):

• Calibration temp: 20°C

• Correction: ±0.001 per 3°C above/below calibration

• Glucose correction: −0.004 per 1 g/dL; Protein: −0.003 per 1 g/dL

• Read at bottom of meniscus; calibrate with K₂SO₄ → SG 1.015

Refractometer:

• Based on refractive index; compensated 15–38°C (no temp correction needed)

• Same glucose/protein corrections as urinometer

• Reads 0.002 lower than urinometer

Reagent strip SG:

• False (+) = high protein; False (−) = highly alkaline urine (pH >6.5)

• Add 0.005 to reading if pH ≥6.5

SG reference ranges:

• Random: 1.003–1.035

• First morning: >1.020

• Isosthenuria = 1.010

• SG >1.040 = radiographic dye

pH

Normal: Random = 4.5–8.0; First morning = 5.0–6.0; pH >9.0 = unpreserved urine

Acidic urine causes: DM, starvation, high protein diet, cranberry juice, emphysema, diarrhea

Alkaline urine causes: RTA, vegetarian diet, after meals (alkaline tide), vomiting, UTI with urease-producing bacteria, old specimens

PROTEIN

Normal: <10 mg/dL or <100 mg/day (Strasinger); <150 mg/day (Henry)

Proteins in normal urine: 1/3 albumin, 2/3 globulins

Categories of proteinuria:

• Pre-renal (overflow): hemoglobinuria, myoglobinuria, Bence-Jones protein (multiple myeloma — precipitates at 40–60°C, dissolves at 100°C)

• Renal: glomerular (microalbuminuria in diabetic nephropathy; orthostatic proteinuria) or tubular (Fanconi, heavy metals)

• Post-renal: lower UTI, trauma, vaginal contamination

Microalbuminuria (AER):

• Normal: 0–20 μg/min

• Microalbuminuria: 20–200 μg/min (30–300 mg/24hr)

• Clinical albuminuria: >200 μg/min

SSA (Sulfosalicylic Acid) test:

• Cold precipitation; reacts with ALL proteins equally

• 3% SSA + equal volume urine → 10 min incubation → cloudiness = positive

• Grades: Neg → Trace → 1+ (30–100) → 2+ (100–200) → 3+ (200–400) → 4+ (>400 mg/dL)

Strip (+) / SSA (−): Highly buffered alkaline urine (false+ strip) or non-albumin proteins absent
Strip (−) / SSA (+): Non-albumin proteins present (Bence-Jones, radiographic dye, drugs)

GLUCOSE

Renal threshold: 160–180 mg/dL

Reagent strip: Glucose oxidase + Peroxidase + chromogen; specific for glucose only; sensitivity = 100 mg/dL

• False (+) = oxidizing agents, detergents

• False (−) = ascorbic acid, ketones, high SG, low temp

Clinitest (Copper reduction): Nonspecific; detects all reducing sugars (glucose, galactose, lactose, fructose) — NOT sucrose

• Pass-through phenomenon: occurs at >2 g/dL sugar; Blue → brick-red → back to blue/green

Other sugars:

• Galactose = galactosemia (infants)

• Lactose = pregnancy/lactation

• Fructose = fructose intolerance, honey ingestion

• Pentose = benign pentosuria

• Sucrose = non-reducing (Clinitest negative)

KETONES

Renal threshold: 70 mg/dL

Ketone bodies: Beta-hydroxybutyric acid (78%, not detected by strip) > Acetoacetic acid (20%, detected by strip) > Acetone (2%)

Principle: Sodium nitroprusside (Legal's test); positive = purple

False (+): Phthalein dyes, levodopa, drugs with sulfhydryl groups
False (−): Improperly preserved specimens

Acetest tablet: Sodium nitroprusside + disodium phosphate + glycine + lactose → purple (30 sec)

BLOOD

Strip: Uniform green/blue = Hgb or Myoglobin; Speckled = intact RBCs (hematuria)

Blondheim's test (ammonium sulfate): Hgb precipitates (strip negative); Myoglobin stays in solution (strip positive)

False (+): Strong oxidizing agents, bacterial peroxidases, menstrual contamination
False (−): High SG, formalin, captopril, ascorbic acid >25 mg/dL, crenated cells

BILIRUBIN & UROBILINOGEN

Only conjugated bilirubin appears in urine (water-soluble)

Urobilinogen normal: <1 mg/dL; specimen = afternoon urine (2–4 PM)

Watson-Schwartz test: Differentiates UBG, porphobilinogen (PBG), other Ehrlich-reactive compounds using chloroform & butanol

Hoesch test: Inverse Ehrlich; rapid screening for PBG (>2 mg/dL)

Ictotest (confirmatory for bilirubin): More sensitive than strip; 10 drops urine + tablet + 2 drops H₂O → blue-purple (60 sec)

NITRITE & LEUKOCYTES

Nitrite: Greiss reaction; gram-negative bacteria convert nitrate → nitrite; (+) = 100,000 organisms/mL; collect first morning or 4-hour specimen; pink spots = NEGATIVE

Leukocytes: Leukocyte esterase; detects neutrophils, eosinophils, monocytes — NOT lymphocytes; strip can detect lysed WBCs; Trichomonas, Chlamydia, yeast, interstitial nephritis → pyuria without bacteriuria

MICROSCOPIC EXAMINATION

Sediment preparation:

1. 10–15 mL urine (recommended 12 mL)

2. Centrifuge at 400 RCF for 5 minutes

3. Decant; leave 0.5–1.0 mL

4. Transfer 20 μL to slide with 22×22 mm coverslip

5. Examine under reduced light; LPO first (detect casts); HPO for identification

Stains:

Cells — normal values:

• RBCs: 0–2 or 0–3/HPF; dysmorphic = glomerular damage

• WBCs: 0–5 or 0–8/HPF; glitter cells (hypotonic urine, pale blue on SM stain)

Epithelial cells (largest to smallest):

1. Squamous EC (30–50 μm) — vagina/lower urethra; clue cells = Gardnerella vaginalis

2. Transitional/Urothelial EC (20–30 μm) — renal pelvis to upper urethra

3. RTE cell (3–5× RBC) — most clinically significant; >2/HPF = tubular injury

   • Oval fat body = lipid-containing RTE; Maltese cross (cholesterol) on polarizing microscope; seen in nephrotic syndrome

   • Bubble cell = non-lipid vacuoles; acute tubular necrosis

Parasites:

• Trichomonas vaginalis = pear-shaped, jerky motility; most common parasite in urine; "ping-pong disease"

• Schistosoma haematobium = blood fluke with terminal spine; associated with bladder cancer

• Enterobius vermicularis = most common fecal contaminant

CASTS

Formed primarily in DCT and collecting duct; major constituent = uromodulin (Tamm-Horsfall protein); uniform diameter ~7–8× diameter of RBCs

Degeneration sequence (worst to best): RBC → WBC → RTE → Granular (coarse → fine) → Waxy → Hyaline

CRYSTALS

Normal acid (acidic urine):

• Amorphous urates = pink/brick dust; soluble in heat & alkali

• Uric acid = most pleomorphic (rhombic, whetstone, rosettes); ↑ gout, Lesch-Nyhan; soluble in alkali

• Calcium oxalate = most frequently observed; dihydrate (envelope) more common; monohydrate (dumbbell); ↑ ethylene glycol poisoning

Normal alkaline (alkaline urine):

• Triple phosphate (struvite) = colorless coffin-lid; urease-splitting bacteria; branching staghorn calculi

• Amorphous phosphates = white precipitate; most common cause of turbidity in alkaline urine

• Ammonium biurate = thorny apples; old specimens

Abnormal (always significant):

• Cystine = colorless hexagonal plates; cystinuria/cystinosis; (+) cyanide-nitroprusside test

• Cholesterol = rectangular plate with notched corners; nephrotic syndrome

• Leucine = yellow-brown oily spheres; liver disease

• Tyrosine = fine needles in clumps; liver disease (more common than leucine)

• Bilirubin = yellow needles/granules; liver disease

• Sulfonamide = sheaves of wheat; may cause tubular damage; (+) lignin test

• Ampicillin = colorless needles in bundles after refrigeration

Uric acid vs. Cystine differentiation:

• Uric acid = birefringent, insoluble in HCl, cyanide-nitroprusside negative

• Cystine = not birefringent, soluble in HCl, cyanide-nitroprusside positive

URINE SCREENING FOR METABOLIC DISORDERS

FeCl₃ reactions summary:

• Blue-green = PKU (and argentaffinoma)

• Transient green = tyrosinemia

• Transient blue = alkaptonuria

• Gray-black = melanuria

Cystinuria: Renal type; defective reabsorption of Cystine, Ornithine, Lysine, Arginine (COLA); (+) cyanide-nitroprusside (red-purple)

Porphyrias: Red/purple/port-wine urine (colorless in lead poisoning); if red urine + negative blood strip + diet/meds ruled out = consider porphyria

MPS screening: CTAB test and Acid albumin test → white turbidity; MPS paper test → blue color

RENAL DISEASES

Renal calculi — major constituent: Calcium oxalate (75%); triple phosphate = staghorn calculi; cystine = least common, resembles old soap

AMNIOTIC FLUID & hCG

hCG: Produced by syncytiotrophoblast; peaks 1st trimester; beta subunit is unique; home test cutoff = 25 mIU/mL

Amniotic fluid volume (3rd trimester): 800–1,200 mL; fetal urine = major contributor after 1st trimester

Fetal Lung Maturity (FLM) tests:

HDN test (ΔA450): Plotted on Liley graph; Zone I = mild; Zone II = moderate; Zone III = severe; normal AF = ↑ at 365 nm, ↓ at 550 nm; HDN = ↑ at 450 nm

Neural tube defects: ↑ AFP; confirmatory = acetylcholinesterase; AFP ↓ in Down syndrome

Fern test: Detects ruptured amniotic membranes; vaginal fluid air-dried → fern-like NaCl crystals = amniotic fluid

CSF

Normal volume: Adults = 90–150 mL; produced by choroid plexus at 20 mL/hour; reabsorbed by arachnoid villi; collected by lumbar puncture (L3–L4 adults; L4–L5 infants)

Normal pressure: 50–180 mmHg

Tube distribution: Tube 1 = Chemistry/Serology; Tube 2 = Microbiology; Tube 3 = Hematology

CSF appearance:

• Xanthochromia = oxyhemoglobin → bilirubin; Pink = slight, Yellow = moderate, Orange = heavy

• Traumatic tap: uneven blood (1>2>3), clot forms, clear supernatant

• Intracranial hemorrhage: even blood, no clot, xanthochromic supernatant, (+) erythrophages

Normal cell count: Adults = 0–5 WBCs/μL; Neonates = 0–30 WBCs/μL; predominant cells = 70% lymphocytes, 30% monocytes

CSF protein: Normal = 15–45 mg/dL; Albumin = major protein; Prealbumin = 2nd most prevalent; IgM, fibrinogen, lipids NOT found normally

CSF glucose: 60–70% of blood glucose (50–80 mg/dL); drawn 2 hours before LP

Differential diagnosis of meningitis:

Multiple sclerosis: (+) oligoclonal bands in CSF but NOT serum; ↑ IgG index (>0.77); (+) myelin basic protein

Limulus Amoebocyte Lysate (LAL) test: Detects gram-negative endotoxin; uses horseshoe crab blood (Limulus polyphemus)

SEMEN

Composition: 5% spermatozoa (testes) + 60–70% seminal fluid (seminal vesicles; fructose source) + 20–30% prostatic fluid (ACP, zinc; coagulation/liquefaction) + 5% bulbourethral glands

Abstinence: 2–3 days (not >7 days); deliver within 1 hour at room temp; analyze after liquefaction (30–60 min)

Normal values:

Motility grading: 4.0a = rapid straight-line; 3.0b = slower with lateral; 2.0b = slow forward; 1.0c = no forward; 0d = immotile

Stains: Morphology = Papanicolaou (choice); Viability = Eosin-Nigrosin (dead = red, live = unstained bluish-white)

Varicocele: Hardening of testicular veins; most common cause of male infertility; tapered sperm head

Fructose test (Resorcinol/Seliwanoff's): (+) orange-red; decreased = lack of seminal vesicle

Post-vasectomy: Done 2 months after; even ONE motile sperm = unsuccessful vasectomy

Medico-legal tests:

• Florence test (choline) → dark brown rhombic crystals (not specific)

• Barbiero's test (spermine) → yellow leaf-like crystals (very specific)

• Glycoprotein p30 (PSA) = most specific for semen detection

SYNOVIAL FLUID

Normal volume: <3.5 mL; viscosity forms string 4–6 cm long

Collection: Arthrocentesis; do NOT refrigerate (causes crystals); do NOT use powdered anticoagulants or lithium heparin (interfere with crystal ID)

Mucin (Ropes) clot test: 2–5% acetic acid; Good = solid clot → Poor/Very poor = no clot; identifies fluid as synovial

Do NOT use acetic acid as WBC diluent — causes mucin clot formation; use saline with methylene blue or saline with saponin

Crystal identification:

Lab findings by group:

SEROUS FLUIDS

Transudate vs. Exudate (most reliable criteria):

• Fluid:serum protein ratio <0.5 = transudate; >0.5 = exudate

• Fluid:serum LD ratio <0.6 = transudate; >0.6 = exudate

• Protein <3 g/dL = transudate; >3 g/dL = exudate

Collection methods: Pleural = thoracentesis; Pericardial = pericardiocentesis; Peritoneal = paracentesis

Normal volumes: Pleural <30 mL; Pericardial <50 mL; Peritoneal <100 mL

Chylous vs. Pseudochylous pleural fluid:

• Chylous: thoracic duct leakage; triglycerides >110 mg/dL; Sudan III (+++); ↑ lymphocytes

• Pseudochylous: chronic inflammation; cholesterol crystals present; triglycerides <50 mg/dL

SAAG: Serum albumin − Peritoneal albumin; >1.1 = transudate of hepatic origin

Rivalta's test: Acetic acid + water + fluid; heavy precipitation = exudate

Tumor markers:

• CEA = colon cancer

• CA 125 = ovarian cancer

• CA 15-3 = breast cancer

• CYFRA 21-1 = lung cancer

SPUTUM

Acceptable specimen: <10 squamous EC/LPF and >25 WBC/LPF

Preferred specimen: First morning (most concentrated)

Key microscopic findings:

BAL: Important for Pneumocystis jirovecii (immunocompromised); Grocott's methenamine silver stain = best for P. jirovecii cysts; predominant cell = alveolar macrophages (56–80%)

FECALYSIS

Normal stool: 75% water, 25% solids; 100–200 g/day; odor from indole and skatole

Key stool colors:

• Black = upper GI bleeding (melena), bismuth, iron

• Red = lower GI bleeding, beets

• Pale/white/gray = bile duct obstruction, barium sulfate

• Green = biliverdin, antibiotics

• Rice watery = cholera

Fecal fat (steatorrhea = >6 g/day):

• Screening = Sudan III microscopy; >60 droplets/HPF (neutral fat)

• Gold standard = Van de Kamer titration (72-hr stool, titration with NaOH; normal 1–6 g/day)

Fecal occult blood (gFOBT): Guaiac (preferred chromogen); pseudoperoxidase principle; significant = >2.5 mL blood/150g stool; screen for colorectal cancer

• False (+): red meat, melon, broccoli, NSAIDs

• False (−): Vitamin C >250 mg/dL

Apt-Downey test: Differentiates fetal (HbF) vs maternal (HbA) blood in neonatal stool; add 1% NaOH to supernatant; Pink = fetal; yellow-brown = maternal (HbA denatured by NaOH)

D-xylose test: Low urine D-xylose = malabsorption; normal = maldigestion

SWEAT TEST (Cystic Fibrosis)

Gibson & Cooke pilocarpine iontophoresis: Current = 0.16 mA for 5 minutes

Results:

• 40 mEq/L = borderline (repeat)

QUICK HIGH-YIELD MNEMONICS

• Chain of infection: Infectious agent, Reservoir, Exit, Mode, Entry, Susceptible host

• RACE/PASS: Fire emergency/extinguisher

• NFPA: "You Were Born Right" = Yellow, White, Blue, Red

• No SMS Ex's = NFPA hazard index 0–4

• BB LNG = false negatives from ascorbic acid (Blood, Bilirubin, Leukocytes, Nitrite, Glucose)

• COLA = cystinuria reabsorption defects (Cystine, Ornithine, Lysine, Arginine)

• 3 C's of asthma = Charcot-Leyden, Curschmann's spirals, Creola bodies

• Maltese cross = cholesterol (oval fat bodies, fatty casts, starch granules) on polarized microscopy