AUBF Reviewer

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CLINICAL MICROSCOPY — COMPREHENSIVE EXAM REVIEWER

PART 1: SAFETY IN THE CLINICAL LABORATORY

Chain of Infection

6 components: Infectious agent → Reservoir → Portal of Exit → Mode of Transmission → Portal of Entry → Susceptible Host

Routes of infection: inhalation, ingestion, direct inoculation/skin contact

Prevention

Handwashing = best way to break the chain of infection

• Wash with soap and water when hands are visibly soiled

• Use alcohol-based hand rub when hands are not visibly soiled

• Clean fingers, thumbs, under fingernails, wrists for at least 15–20 seconds

• Rinse hands in a downward position

Biological Waste Disposal

• All biological waste except urine → biohazard-labeled containers (fluorescent orange)

• Urine → pour into lab sink, avoid splashing, flush with water

• Empty urine containers → non-biohazardous waste

• Disinfect sink daily with 1:10 dilution of sodium hypochlorite (1 part bleach + 9 parts water; effective for 1 month)

• Disinfection eliminates pathogens except bacterial spores

Sharp Hazards → puncture-resistant containers; needle-stick must be reported to supervisor

Radioactive Waste → store in locked room until background count is down to 10 half-lives (radioiodine)

Chemical Spills

• Best first aid: flush with water for at least 15 minutes

• DO NOT neutralize chemicals on skin

• Always ADD ACID TO WATER (never water to acid — explosion risk)

NFPA Hazard Classification

Diamond: Red (Fire), Blue (Health), Yellow (Reactivity/Instability), White (Specific Hazard)

• Mnemonic: "You Were Born Right" (Yellow, White, Blue, Red — starting rightmost)

• Scale: 0 = No hazard → 4 = Extreme/Severe hazard

• Mnemonic for scale: "No SMS Ex's"

Fire

RACE: Rescue → Alarm → Contain → Extinguish/Evacuate PASS: Pull pin → Aim at base → Squeeze → Sweep side to side

• Dry chemical (ABC) = most common all-purpose

• Class D and E = trained personnel only

• Water (A) | Dry chemical (ABC) | CO₂ (BC) | Halon (BC)

Miscellaneous Hazards

• Ergonomic = work-related strain from repeated positions

• Cryogenic = extremely low temperatures

• Mechanical = centrifuges, autoclaves, refrigerators, glasswares

• Centrifuge accidents or improper stopper removal → aerosols

PART 2: RENAL FUNCTION

Urinary System Dimensions

• Kidney: ~150g; 12.5 × 6 × 2.5 cm

• Ureter: 25 cm

• Bladder: nerve reflex at ~150 mL

• Urethra: 4 cm (female), 24 cm (male)

• Urine enters bladder every 10–15 seconds

Nephron

• Basic structural and functional unit of the kidney

• 1–1.5 million nephrons per kidney

• Composed of glomerulus + renal tubules

Order of Urine Formation

Glomerulus → PCT → Loop of Henle → DCT → Collecting Duct → Renal Calyx → Renal Pelvis

Renal Blood Flow

• Kidneys receive 25% of total cardiac output

• Total Renal Blood Flow: 1,200 mL/min

• Total Renal Plasma Flow: 600–700 mL/min

Order: Renal artery → Afferent arteriole → Glomerulus → Efferent arteriole → Peritubular capillaries → Vasa recta → Renal vein

Glomerular Filtration

• Glomerulus = "working portion" of the kidney; resembles a sieve

• Non-selective filter of plasma substances with MW <70,000 daltons

• Glomerular filtrate SG = 1.010

• Albumin cannot pass — "shield of negativity" (negatively charged at physiologic pH)

• ~1% of filtered plasma is excreted as urine

Glomerular Filtration Barrier (3 layers):

1. Capillary endothelium (large open pores)

2. Trilayer basement membrane (lamina rara interna, lamina densa, lamina rara externa)

3. Filtration diaphragm (between podocytes)

Tubular Reabsorption

• 1st function affected in renal disease

• Renal threshold for glucose = 160–180 mg/dL

• PCT reabsorbs 65% of plasma substances

Key hormones:

• ADH (Vasopressin) = regulates water reabsorption in DCT and CD

  • ↑ Hydration → ↓ ADH → ↑ Urine volume

  • ↓ Hydration → ↑ ADH → ↓ Urine volume

  • Diabetes Insipidus = ADH deficiency; SIADH = ADH excess

• Aldosterone = regulates sodium reabsorption

Loop of Henle Memory Tips:

• "DAM" — Descending LH collects water

• "ASIN"-ding — Ascending LH reabsorbs ASIN (salt), NOT water

• Ascending LH is highly impermeable to water

RAAS (Renin-Angiotensin-Aldosterone System)

Angiotensinogen →(Renin)→ Angiotensin I →(ACE in lungs)→ Angiotensin II → Effects

Effects of Angiotensin II:

• Releases Aldosterone & ADH (↑ Na⁺ and water reabsorption)

• Vasoconstriction (↑ BP)

• Dilates afferent; constricts efferent arteriole

• Renin produced by Juxtaglomerular (JG) cells

Tubular Secretion

2 major functions:

1. Regulate acid-base balance (secretion of H⁺ as NH₄⁺ and H₂PO₄⁻)

2. Eliminate waste not filtered by glomerulus

Renal Tubular Acidosis (RTA) = failure to secrete H⁺ → alkaline urine

PART 3: RENAL FUNCTION TESTS

Tests for Glomerular Filtration (Clearance Tests)

• Urea = obsolete

• Creatinine = most common

• Inulin (MW 5,200 Da) = gold standard/reference method (not routine; injected)

• Beta₂-microglobulin (MW 11,800 Da) = better marker of tubular function

• Cystatin C (MW 13,000 Da)

Creatinine Clearance Formula: Ccr = (U × V) / P × (1.73 m² / A)

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

• 7–10% of creatinine is secreted by renal tubules

Cockroft-Gault eGFR: Ccr = [(140 - age)(body weight in kg)] / [72 × serum creatinine (mg/dL)] × 0.85 (if female) Variables: Age, Sex, Body weight

MDRD variables: Ethnicity, BUN, Serum albumin

Tests for Tubular Reabsorption (Concentration Tests)

• Fishberg test (obsolete) — fluid restriction; SG ≥1.022 after 12 hrs

• Mosenthal test (obsolete) — compare day/night urine

• Specific gravity (currently used)

• Osmolality (preferred; more precise than osmolarity)

  • NV = 1–3× serum = 275–900 mOsm/kg (serum = 275–300 mOsm/kg)

Tests for Tubular Secretion & Renal Blood Flow

• PAH (p-aminohippuric acid) = most commonly used reference method

• PSP test = obsolete

PART 4: INTRODUCTION TO URINALYSIS

Historical Notes

• Hippocrates = uroscopy; first documented importance of sputum

• Frederik Dekkers = albuminuria by boiling urine

• Thomas Addis = urine sediment examination

• Richard Bright = urinalysis as routine patient exam

• Ludwig Thudichum = urochrome

• Stanley Benedict = Benedict's reagent

• Archibald Garrod = alkaptonuria

• Ivan Følling = phenylketonuria

Urine Composition

• 95–97% water; 3–5% solids (60 g total solids/24 hrs)

• Organic solids (35 g): Urea (major) > Creatinine > hippurate, uric acid

• Inorganic solids (25 g): Chloride (major) > Sodium > Potassium; principal salt = NaCl

Types of Urine Specimen

Drug Specimen Collection:

• Required volume: 30–45 mL; Container capacity: 60 mL

• Temperature within 4 minutes: 32.5–37.7°C

• Blueing agent added to toilet to prevent adulteration

Containers: Wide base; opening ≥4 cm; 24-hr container holds up to 3 liters

Specimen Integrity

• Test within 2 hours (Strasinger/Harr); ideally within 30 minutes (Turgeon)

Changes in Unpreserved Urine:

Least affected after standing = protein

Urine Preservatives (Key Points)

• No ideal urine preservative exists

• Preservatives for 5-HIAA: Boric acid, HCl

PART 5: PHYSICAL EXAMINATION

Urine Volume

• Normal 24-hr: 600–2,000 mL; Average: 1,200–1,500 mL

• Night urine: <400 mL; Day:Night ratio = 2–3:1

Urine Color

• Normal: colorless to deep yellow

• Most common abnormal: Red/Red-brown

• Examine under good light source; look down through container against white background

Normal Pigments:

• Urochrome = major pigment (yellow); product of endogenous metabolism; ↑ in thyrotoxicosis, fever, starvation

• Uroerythrin = pink/red; derived from melanin; deposits in amorphous urates

• Urobilin = dark yellow/orange-brown; from oxidation of urobilinogen; present in old specimens

Key Color Associations:

Urine Clarity

• Method: Mix specimen → hold in front of light source → view through newspaper print

Causes of Turbidity:

• Nonpathologic: Squamous epithelial cells, amorphous urates (pink), amorphous phosphates (white/beige), vaginal cream, semen

• Pathologic: RBCs, WBCs, bacteria, yeast, abnormal crystals, lipids, chyluria

Solubility key:

• Soluble in heat → amorphous urates, uric acid

• Soluble in dilute acetic acid → RBCs, amorphous phosphates

• Insoluble in dilute acetic acid → WBCs, bacteria, yeast, sperm

• Soluble in ether → lipids, chyle

Urine Odor

PART 6: CHEMICAL EXAMINATION (REAGENT STRIP)

Quick Reference: Reading Times, Principle, Positive Color

Reagent Strip Technique:

• Dip briefly (<1 second) into well-mixed, uncentrifuged urine at room temperature

• Remove excess urine by touching edge to container

• Blot edge on absorbent pad

• Compare to manufacturer's color chart under good lighting

Storage: Opaque, tightly closed container with desiccant; store below 30°C; use within 6 months of opening; never freeze

1. Specific Gravity (SG)

• Influenced by number AND SIZE of particles

• Random urine: 1.003–1.035; 1st morning: >1.020; 24-hr: 1.016–1.022

• SG <1.003 = not a urine (except DI); SG >1.040 = radiographic dye

• Isosthenuria = 1.010; Hyposthenuria <1.010; Hypersthenuria >1.010

Methods:

Urinometry:

• Calibration temp: 20°C

• Temp correction: ±0.001 per 3°C

• Glucose correction: −0.004 per 1 g/dL

• Protein correction: −0.003 per 1 g/dL

• Volume required: 10–15 mL

• Calibration: K₂SO₄ solution → reads 1.015

• Read at bottom of meniscus; add with spinning motion

Refractometry:

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

• Still requires glucose/protein correction

• Reads 0.002 lower than urinometer

• Calibration: distilled water = 1.000; 3% NaCl = 1.015; 5% NaCl = 1.022; 9% sucrose = 1.034

Reagent Strip (pKa method):

• Polyelectrolyte releases H⁺ proportional to ionic concentration

• Reagents: bromthymol blue

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

• Add 0.005 when pH ≥6.5

• NOT affected by glucose, protein, or radiographic dye (Henry)

Harmonic Oscillation Densitometry (obsolete):

• Yellow IRIS; requires 6 mL (4 mL for microscope, 2 mL for SG)

2. pH

• Normal random: 4.5–8.0; first morning: 5.0–6.0

• pH >9.0 = unpreserved urine

• Blood pH <6.8 or >7.8 = death

• Alkaline tide = pH rises after meals (H⁺ withdrawn for HCl secretion)

• Cranberry juice = contains quinic acid → excreted as hippuric acid (treats UTI)

Strip: Double indicator — methyl red (pH 4–6) + bromthymol blue (pH 6–9)

3. Protein

• Most indicative of renal disease

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

• Normal urine protein = 1/3 albumin + 2/3 globulins

• Produces white foam when shaken

• Strip sensitive to albumin only

Categories:

• Pre-renal (overflow): hemoglobin (hemolysis), myoglobin (muscle injury), Bence-Jones protein (multiple myeloma — BJP precipitates at 40–60°C, dissolves at 100°C)

• Renal/glomerular: diabetic nephropathy (indicator = microalbuminuria), orthostatic proteinuria (only when standing), nephrotic syndrome

• Renal/tubular: Fanconi syndrome, heavy metals, viral infections (originally found in cadmium-exposed workers)

• Post-renal: lower UTI, menstrual contamination, prostatic fluid

Microalbuminuria: 20–200 μg/min (or 30–300 mg/24 hrs); undetectable by routine reagent strip Micral Test: Enzyme immunoassay; (−) white, (+) red at 60 seconds

SSA (Exton's Test): Cold precipitation test; reacts equally with all proteins; reagent = 3% SSA + sodium sulfate

Strip (+), SSA (−): Highly buffered alkaline urine → acidify to pH 5.0 and retest Strip (−), SSA (+): Proteins other than albumin (e.g., Bence-Jones); radiographic dye; drug metabolites (penicillins, cephalosporins)

Strip interferences:

• False (+): alkaline urine, Phenazopyridine, quaternary ammonium compounds, chlorhexidine

• False (−): proteins other than albumin, microalbuminuria

4. Glucose

• Renal threshold: 160–180 mg/dL

• Strip sensitivity: 100 mg/dL; detects glucose only

Strip (double sequential enzyme reaction): Glucose + O₂ →(glucose oxidase)→ Gluconic acid + H₂O₂ H₂O₂ + Chromogen →(peroxidase)→ Oxidized chromogen + H₂O

• Multistix chromogen = potassium iodide (blue → green → brown)

• False (+): oxidizing agents, detergents

• False (−): ascorbic acid, ketones, high SG, low temp, improperly preserved specimen

• First "dip and read" strip developed by Miles, Inc. in 1950

Copper Reduction Test (Clinitest/Benedict's):

• Nonspecific; detects ALL reducing sugars (glucose, galactose, lactose, fructose — NOT sucrose)

• CuSO₄ (blue) → Cu₂O (brick-red) in presence of reducing sugars

• Pass-through phenomenon: occurs when >2 g/dL sugar; Blue → Brick-red →→ Blue/green-brown (re-oxidation); prevent with 2 drops urine

• Tablet contains: CuSO₄, Na citrate, Na₂CO₃, NaOH

Interpretation:

• Glucose oxidase (+), Clinitest (−) = small amount of glucose or oxidizing agent interference

• Glucose oxidase (4+), Clinitest (−) = oxidizing agent interference on strip

• Glucose oxidase (−), Clinitest (+) = non-glucose reducing substance (e.g., ascorbic acid, galactose, lactose)

5. Ketones

• Result from increased fat metabolism

• Renal threshold: 70 mg/dL

• Seen in: Type I DM, vomiting, starvation, malabsorption

Ketone bodies:

• 78% = Beta-hydroxybutyric acid (major; NOT detected by strip)

• 20% = Acetoacetic acid (parent ketone; detected by strip)

• 2% = Acetone

Strip (sodium nitroprusside / Legal's test): Acetoacetic acid + acetone + Na nitroprusside + glycine → purple

• Acetone only detected if glycine present (Chemstrip)

• False (+): phthalein dyes, pigmented red urine, levodopa, drugs with sulfhydryl groups

• False (−): improperly preserved specimens

6. Blood

Blondheim's (Ammonium Sulfate) Test:

• Hemoglobin: precipitates → negative for blood on strip

• Myoglobin: does NOT precipitate → positive for blood on strip

Strip (pseudoperoxidase activity):

• Uniform green/blue = Hgb or Mb; Speckled = intact RBCs (hematuria)

• False (+): strong oxidizing agents, bacterial peroxidases, menstrual contamination

• False (−): high SG, crenated cells, formalin, captopril, nitrite, ascorbic acid >25 mg/dL, unmixed specimen

• Hgb >10 mg/dL → positive protein strip result

• Chemstrip contains iodate overlay that eliminates ascorbic acid interference

7. Bilirubin

• Only conjugated bilirubin (CB) appears in urine (water-soluble)

• Tea-colored/amber/beer-brown urine with yellow foam

• Early indication of liver disease (hepatitis, cirrhosis, biliary obstruction)

Strip (diazo reaction): CB + diazonium salt → azodye; (+) tan or pink to violet

• False (+): highly pigmented urine, phenazopyridine, indican

• False (−): light exposure, high nitrite, ascorbic acid >25 mg/dL

Ictotest: Confirmatory; more sensitive; (+) blue to purple at 60 seconds

8. Urobilinogen (UBG)

• Normal: <1 mg/dL or Ehrlich unit

• Specimen: Afternoon urine (2–4 PM)

• Bile pigment from hemoglobin degradation

Strip (Ehrlich reaction): UBG + PDAB → red

• Multistix = PDAB; Chemstrip = 4-methoxybenzene-diazonium-tetrafluoroborate (specific for UBG)

• False (+): Ehrlich-reactive compounds (porphobilinogen, indican, methyldopa), pigmented urine

• False (−): old specimens, formalin, high nitrite

Watson-Schwartz Test: Differentiates UBG, porphobilinogen (PBG), and other Ehrlich-reactive compounds using chloroform and butanol extraction

Hoesch Test: Inverse Ehrlich reaction; rapid screen for PBG (>2 mg/dL); 2 drops urine + 2 mL Hoesch reagent → (+) red

9. Nitrite

• Rapid screening test for UTI/bacteriuria

• Nitrate converters = generally Gram-negative bacilli (Enterobacteriaceae)

• Preferred specimen: 4-hour collection or first morning urine

• (+) Nitrite = ~100,000 organisms/mL

Strip (Greiss reaction):

• p-arsanilic acid + nitrite → diazonium salt → + tetrahydrobenzoquinolin → uniform pink

• Pink spots/edges = considered NEGATIVE

• False (+): improperly preserved specimens, highly pigmented urine

• False (−): non-reductase bacteria, insufficient contact time, no dietary nitrate, large bacterial quantities converting nitrite to nitrogen, antibiotics, high ascorbic acid, high SG

• Negative nitrite does NOT rule out UTI (Gram-positive cocci and yeasts lack nitrate reductase)

10. Leukocytes

• Detects WBC esterase; also detects lysed WBCs

• Cells with esterase: Neutrophil, Eosinophil, Basophil, Monocyte, Histiocyte, Trichomonas

• No esterase: Lymphocyte

• Trichomonas, Chlamydia, yeast, interstitial nephritis → pyuria without bacteriuria

• Strip takes 120 seconds (longest reading time)

Strip (leukocyte esterase → purple):

• False (+): strong oxidizing agents, formalin, highly pigmented urine, nitrofurantoin

• False (−): high protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines, inaccurate timing

11. Ascorbic Acid (Vitamin C)

• Strong reducing substance; 11th reagent pad

• Causes false-negative on: Blood, Bilirubin, Leukocytes, Nitrite, Glucose (mnemonic: "BB LNG")

• Strip: ascorbic acid (>5 mg/dL) + phosphomolybdate → molybdenum blue

• GC-MS = more accurate quantitative method

PART 7: MICROSCOPIC EXAMINATION

Urine Sediment Preparation

1. Transfer 10–15 mL (recommended 12 mL) to test tube

2. Centrifuge at 400 RCF for 5 minutes

3. Decant; leave 0.5–1.0 mL

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

5. Examine: 10 LPF and 10 HPF under reduced light

• RCF = 10⁵ × radius (cm) × RPM²

• If <12 mL available: centrifuge 3 mL

• If <3 mL: examine without centrifugation

• First examine under LPO for casts; use HPO for identification

Microscopic Techniques

Sediment Stains

Cells in Urine Sediment

RBCs:

• NV: 0–2 or 0–3/HPF; smooth, non-nucleated, biconcave discs

• Hypertonic urine → crenated; Hypotonic urine → ghost cells

• Glomerular damage → dysmorphic RBCs

• Errors: yeasts, oil droplets, air bubbles, monohydrate calcium oxalate

• Remedy: 2% acetic acid (lyses RBCs, not others)

WBCs:

• NV: 0–5 or 0–8/HPF; larger than RBCs

• Neutrophils: most predominant; multilobed; in hypotonic urine → glitter cells (Brownian movement of granules); dying cells form blebs (myelin forms)

• Eosinophils: NV 1%; significant >1% (drug-induced interstitial nephritis)

• ↑ Lymphocytes = renal transplant rejection

• ↑ Monocytes/histiocytes = chronic inflammation, radiation therapy

Epithelial Cells:

RTE cell variations:

• Oval fat body: lipid-containing RTE cell; seen in lipiduria (nephrotic syndrome); "Maltese cross" under polarizing scope (cholesterol); lipid stains for TAG and neutral fats

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

Bacteria:

• True UTI = bacteria + WBCs (bacteria alone = contamination or old specimen)

• Most common UTI cause: Enterobacteriaceae (E. coli)

• Differentiate from amorphous urates by motility

Yeasts:

• True infection = yeast + WBCs

• Candida albicans: seen in DM and vaginal moniliasis

• Small, refractile oval structures that may bud; branching forms in severe infection

Parasites:

• Trichomonas vaginalis: most frequent; pear-shaped flagellate; jerky motility; agent of Ping-Pong disease

• Enterobius vermicularis egg: most common fecal contaminant

• Schistosoma haematobium: terminal spine; causes hematuria; associated with bladder cancer

Bladder Cancer Markers: NMP (Nuclear Matrix Protein), BTA (Bladder Tumor Antigen)

Microscopic Quantitation (Strasinger)

• RBCs, WBCs, Casts: numerical ranges (0–2, 2–5, 5–10, etc.) per HPF/LPF

• Do NOT quantitate: budding yeast, mycelia, Trichomonas, sperm (just note presence)

Casts

• Unique to the kidney; represent a biopsy of an individual tubule

• Most difficult and most important urinary sediment constituent

• Primarily formed in DCT and collecting duct

• Major constituent: Uromodulin/Tamm-Horsfall protein (THP) produced by RTE cells

• Protein gels under: urine-flow stasis, acidity, presence of Na⁺ and Ca²⁺

• Cylindroids = same significance as casts (cast with a tail)

• Examine along coverslip edges with subdued light

Types of Casts:

Degeneration sequence (worst to best): Waxy → Broad → Granular (coarse → fine) → Cellular → Hyaline

Pseudoleukocyte cast = NOT a true cast (clump of leukocytes); seen in lower UTI — do NOT report as cast

PART 8: CRYSTALS

Normal Acid Crystals

Normal Alkaline Crystals

Abnormal Crystals

Uric Acid vs. Cystine:

• Cystine = colorless, soluble in dilute HCl, NOT birefringent, (+) cyanide-nitroprusside

• Uric acid = yellow-brown, insoluble in HCl, birefringent, (−) cyanide-nitroprusside

Maltese cross formation under polarizing microscope: Oval fat bodies, fatty casts, fat droplets, starch granules

PART 9: URINE SCREENING FOR METABOLIC DISORDERS

FeCl₃ (Ferric Chloride) Tube Test Results

Key Disorders

PKU (Phenylketonuria):

• Missing: phenylalanine hydroxylase

• ↑ Phenylpyruvic acid in urine; mousy odor (phenylacetic acid)

• Screening: FeCl₃ (blue-green), Phenistix (gray to gray-green), Guthrie test

• Confirmatory: Ion exchange HPLC

• Guthrie test: B. subtilis + B₂-thienylalanine; phenylalanine counteracts inhibition → growth = positive

Alkaptonuria:

• Missing: homogentisic acid oxidase

• Urine darkens upon becoming alkaline (standing at room temperature)

• Brown/black-stained cloth diapers; ochronosis

• Screening: FeCl₃ (transient blue), Clinitest (+), alkalinization of fresh urine

MSUD (Maple Syrup Urine Disease):

• Most common IEM in the Philippines

• Missing: branched-chain α-keto acid dehydrogenase (BCKD)

• ↑ Ketoacids of Leucine, Isoleucine, Valine

• Caramelized sugar/maple syrup/curry odor

• Screening: DNPH = (+) yellow turbidity/precipitate

PKU → Tyrosyluria → Alkaptonuria (progressive pathway blocks)

Cystinuria vs. Cystinosis:

• Cystinuria = renal type (defective tubular reabsorption of COLA: Cystine, Ornithine, Lysine, Arginine)

• Cystinosis = overflow type (inherited enzyme deficiency; cystine deposits throughout body)

• Both: (+) cyanide-nitroprusside (red-purple)

• Homocystinuria: (+) Silver-nitroprusside (red-purple)

Porphyrias:

• Urine = red, purple, port wine (colorless in lead poisoning)

• Lead poisoning inhibits ALA synthetase and ferrocheletase

MPS Disorders:

• Screening tests: Acid albumin test, CTAB test = (+) white turbidity; MPS Paper Test = (+) blue

• Hurler (Type I / Gargoylism): corneal deposits

• Hunter: sex-linked recessive

• Sanfilippo: mental retardation only

Lesch-Nyhan:

• Missing: HGPRT (hypoxanthine guanine phosphoribosyltransferase)

• ↑ Uric acid in blood and urine; "orange sand" in diapers

PART 10: RENAL DISEASES

Glomerular Disorders

Tubular Disorders

Interstitial Disorders

Renal Failure

• GFR <25 mL/min; azotemia (↑ BUN and creatinine)

• Isosthenuria; telescoped sediment (variety of cast types in same specimen)

Renal Calculi

PART 11: OTHER BODY FLUIDS

Amniotic Fluid & hCG

hCG:

• Produced by syncytiotrophoblast cells; peaks during 1st trimester

• α-subunit = shared with LH, FSH, TSH; β-subunit = specific for hCG

• Home test: enzyme immunoassay; cut-off: 25 mIU/mL; first morning urine; anti-hCG source: rabbit

Amniotic Fluid Volume:

• Normal (3rd trimester): 800–1,200 mL

• Polyhydramnios (>1,200 mL): decreased fetal swallowing, neural tube defects

• Oligohydramnios (<800 mL): increased fetal swallowing, membrane leakage, urinary tract deformities

Tests for Fetal Lung Maturity:

Test for HDN: OD 450 nm; plot on Liley graph (Zone I = mild, Zone II = moderate, Zone III = severe)

Neural Tube Defects:

• Screening: AFP (↑ in NTD; ↓ in Down syndrome)

• Confirmatory: Acetylcholinesterase

Fetal Age: Amniotic fluid creatinine >2.0 mg/dL = 36 weeks (9 months)

Sputum

• Acceptable specimen: <10 SEC/LPF and >25 WBC/LPF

• Sialic acid = most important single component of viscosity

• Preferred specimen: first morning (most concentrated)

Key Microscopic Findings:

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

Sweat (Cystic Fibrosis)

• Gibson and Cooke pilocarpine iontophoresis (0.16 mA for 5 minutes)

• Na⁺ and Cl⁻ >70 mEq/L = diagnostic for CF; 40 mEq/L = borderline

Cerebrospinal Fluid (CSF)

• 3rd major body fluid; produced by choroid plexus (20 mL/hour); reabsorbed by arachnoid villi

• Total volume: Adults 90–150 mL; Neonates 10–60 mL

• Lumbar puncture: L3–L4 (adults), L4–L5 (infants); normal pressure 50–180 mmHg

• Tube 1 = Chemistry/Serology (least affected by blood or bacteria); Tube 2 = Microbiology; Tube 3 = Hematology (least likely to contain cells from puncture)

CSF Appearance:

• Xanthochromia: pink (oxyhemoglobin), yellow (oxyhemoglobin → bilirubin), orange (heavy hemolysis)

• Bloody: RBCs >6,000/μL; differentiate traumatic tap vs. intracranial hemorrhage

CSF Cell Count:

• WBC NV: Adults 0–5/μL; Neonates 0–30/μL

• Normal differential: 70% lymphocytes, 30% monocytes (inverted in neonates)

• Pleocytosis = abnormal ↑ in CSF cells

• Diluting fluid for WBC count: acetic acid with methylene blue

CSF Protein:

• NV adults: 15–45 mg/dL; Infants: 150 mg/dL

• Major protein: Albumin; 2nd most prevalent: Prealbumin

• Beta₂-transferrin (tau) = found in CSF but NOT in serum

• NOT found in normal CSF: IgM, fibrinogen, lipids

• CSF/Serum Albumin Index >9 = abnormal BBB; >100 = complete BBB damage

• IgG Index >0.77 = IgG production within CNS (Multiple sclerosis)

Oligoclonal bands in CSF but NOT serum: Multiple sclerosis, neurosyphilis, encephalitis

Multiple Sclerosis findings: (+) anti-myelin sheath antibody, (+) oligoclonal bands in CSF not serum, (+) myelin basic protein (MBP), ↑ IgG index

CSF Glucose: 60–70% of blood glucose (50–80 mg/dL); draw blood 2 hours prior to spinal tap

CSF Lactate: NV 10–22 mg/dL; inversely proportional to glucose

Meningitis Differential:

Limulus Amoebocyte Lysate (LAL) Test: Detects Gram-negative endotoxin; reagent = horseshoe crab blood

Semen Analysis

Composition:

• 5% spermatozoa (seminiferous tubules → epididymis for maturation)

• 60–70% seminal fluid (seminal vesicles; rich in fructose for motility)

• 20–30% prostate fluid (ACP, zinc, citric acid; for coagulation/liquefaction)

• 5% bulbourethral glands (alkaline mucus; neutralizes acidity)

• Spermatogenesis + maturation = 90 days (Graff: 74 days)

Normal Values:

• Volume: 2–5 mL; Viscosity: pour in droplets; pH: 7.2–8.0

• Concentration: >20 million/mL; Count: >40 million/ejaculate

• Motility: >50% within 1 hour; Quality: >2.0; Morphology: >30% (routine) or >14% (Kruger's strict criteria)

Motility Grading (WHO):

• 4.0 = rapid straight-line; 3.0 = slower, some lateral; 2.0 = slow forward, noticeable lateral; 1.0 = no forward; 0 = no movement

Key Points:

• Abstinence: 2–3 days (not >7 days); prolonged = ↑ volume, ↓ motility

• Deliver within 1 hour; liquefy within 30–60 minutes at 37°C

• Failure to liquefy = prostatic enzyme deficiency

• Best collection method: masturbation

Sperm Morphology Stain of Choice: Papanicolaou's; use 45° angle for smears

• Head: 5 μm length × 3 μm width; acrosomal cap = ½ head = 2/3 nucleus

• Neck: 7 μm; Tail: 45 μm (includes neck)

• Varicocele = most common cause of male infertility; tapered head

Sperm Viability (Modified Blom's): Living = unstained/bluish white; Dead = red (eosin-nigrosin); NV: ≥50% living

Seminal Fructose: Seliwanoff's (Resorcinol) test = (+) orange-red; test within 2 hours or freeze

Medico-Legal Tests:

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

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

• Glycoprotein p30 (PSA) = most specific method to detect semen

Semen Terminology:

• Aspermia = no semen; Azoospermia = no spermatozoa; Oligozoospermia = decreased concentration

• Necrozoospermia = ↑ dead sperm; Leukospermia = ↑ leukocytes; Hematospermia = blood in semen

Leydig cells = testosterone; Sertoli cells = inhibin

Synovial Fluid

• Viscous due to hyaluronic acid polymerization (by synoviocytes)

• Normal: <3.5 mL in adult knee cavity; >25 mL = inflammation

• Normal: does NOT clot

• Viscosity: normal = 4–6 cm string; hyaluronic acid 0.3–0.4 g/dL

Mucin Clot Test: 2–5% acetic acid; Good (solid clot) → Fair → Low/Poor → No clot (less viscosity = more inflammation)

DO NOT use acetic acid as WBC diluting fluid (causes mucin clot formation)

Crystal Identification (Compensated Polarizing Microscope):

Laboratory Findings in Joint Disorders:

Serous Fluid

Transudate vs. Exudate:

Collection: 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; ↑ lymphocytes; TG >110 mg/dL; Sudan III +++

• Pseudochylous: chronic inflammation; mixed cells; cholesterol crystals present; TG <50 mg/dL

Tumor Markers:

• CEA = colon cancer; CA 125 = ovarian/uterine; CA 15-3 = breast; CYFRA 21-1 = lung cancer

Psammoma bodies: Concentric striations; associated with ovarian and thyroid carcinomas

Gastric Fluid

• Parietal cells = HCl + intrinsic factor

• Chief cells = pepsinogen

• G cells = gastrin

• Foveolar cells = mucus

Reference Values:

• Preferred stimulant: Pentagastrin

• Levin tube = inserted through nose

• Diagnex tubeless test = uses azure blue dye; specimen = urine

Fecalysis

• Normal stool: 100–200 g/day; 75% water, 25% solids

• Odor due to indole and skatole

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

• Red stool = hematochezia (lower GI bleeding)

• Pale/white/gray = bile duct obstruction

Fecal Fat:

• Steatorrhea = >6 g fat/day

• Qualitative: Sudan III with ethanol (neutral fats) or acetic acid (fatty acids); >60 droplets/HPF = steatorrhea

• Quantitative gold standard: Van de Kamer titration (3-day stool, NaOH titration)

Fecal Occult Blood (Guaiac):

• Principle: pseudoperoxidase activity of hemoglobin

• Chromogens (most to least sensitive): benzidine > guaiac (preferred) > o-toluidine

• False (+): red meat, melon, broccoli, cauliflower, horseradish (avoid 3 days); aspirin/NSAIDs (avoid 7 days)

• False (−): Vitamin C >250 mg/dL, iron supplements with Vitamin C

Apt-Downey Test: Differentiates fetal (HbF) vs. maternal (HbA) blood; 1% NaOH added to supernatant; Pink = HbF (alkali-resistant); Yellow-brown = HbA (denatured by NaOH)

X-ray Film (Gelatin) Test: Detects trypsin; clearing = (+); absence = cystic fibrosis

D-Xylose Test: Low urine D-xylose = malabsorption; Normal urine D-xylose = maldigestion

PART 12: QUALITY ASSURANCE

QC of Laboratory Equipment

Centrifuge calibration devices: Tachometer (speed), Stopwatch (timing), Strobe light

Addis Count

• Quantitative measure using hemacytometer; specimen = 12-hour urine; preservative = formalin

• NV: RBCs 0–500,000; WBCs + ECs 0–1,800,000; Hyaline casts 0–5,000 per 12-hr urine

QUICK-REFERENCE MNEMONICS

This reviewer covers all major topics in the uploaded Clinical Microscopy lecture notes by Sir Errol Coderes, RMT. Good luck on your MTLE!