Histopathology I

Brief History of Microscopy

• 1590 — Zacharias Janssen (Netherlands) invented the first compound microscope
• 1665 — Robert Hooke coined the term "cell" after observing cork slices
• 1674 — Antonie van Leeuwenhoek observed living microorganisms (protozoa, bacteria) — first to see living cells
• 1830s — Joseph Jackson Lister improved lens design, reducing spherical aberration
• 1932 — Ernst Ruska developed the first Electron Microscope
• 1953 — George de Mestral inspired development of phase contrast microscopy

Basic Principle of Microscopy

• Microscopy uses lenses to magnify small objects that cannot be seen with the naked eye
• Two key properties: Magnification (making objects appear larger) and Resolution (ability to distinguish two points as separate)
• Resolution = 0.61λ / NA (where λ = wavelength of light, NA = numerical aperture)
• Light microscopes use visible light (400–700 nm) wavelength
• Electron microscopes use electron beams — much shorter wavelength = much higher resolution

Types & Classification of Microscopes

Parts of Compound Microscope

Care, Cleaning & Quality Control

• Clean lenses with lens tissue only — never use cloth or paper towels
• Use lens cleaning solution or xylene for oil immersion lens (remove immersion oil after use)
• Cover microscope when not in use to prevent dust accumulation
• Store in upright position, always carry with two hands (one on arm, one on base)
• QC: Check Köhler illumination regularly; calibrate with stage micrometer; check for lens scratches
• Never touch lens surfaces with fingers — fingerprint oils cause permanent damage

Reception of Histopathological Specimens

• All specimens must arrive with a properly filled request form: patient name, age, sex, clinical history, specimen site, surgeon's name
• Check: specimen is in correct fixative, container is properly labeled, quantity is adequate
• Assign a unique laboratory number — entered in the specimen register
• Reject specimens that are: unlabeled, inadequately fixed, too small, badly damaged
• Urgent biopsies (intraoperative) are processed as frozen sections for rapid diagnosis

Examination of Received Samples

• Record: size, shape, color, consistency, surface appearance of specimen
• Note any abnormal areas: nodules, ulcers, necrosis, hemorrhage
• Fresh specimens should be fixed immediately to prevent autolysis
• Ratio of fixative to tissue: minimum 10:1 (volume fixative to tissue)

Purpose of Fixation

• Prevents autolysis (self-digestion by own enzymes) and putrefaction (bacterial decomposition)
• Preserves tissue morphology and architecture as close to living state as possible
• Hardens tissue for sectioning (microtomy)
• Kills microorganisms — makes tissue safe to handle
• Allows staining — fixatives affect how dyes interact with tissue
• Preserves antigenicity for immunohistochemistry (IHC)

Methods of Fixation

• Physical Heat fixation — used for smears (blood films, sputum). Simple but can distort histology
• Physical Freeze drying — rapid, preserves enzymes and antigens well. Used for enzyme histochemistry
• Chemical Immersion fixation — tissue placed in fixative solution. Most common method in histopathology
• Chemical Perfusion fixation — fixative pumped through blood vessels. Used in animal studies for superior fixation
• Chemical Microwave fixation — rapid (minutes vs hours). Useful for urgent biopsies

Commonly Used Fixatives

Factors Affecting Quality of Fixation

• Penetration rate — formalin penetrates ~1 mm/hour. Large specimens must be sliced
• Volume ratio — minimum 10:1 fixative to tissue volume
• Temperature — higher temp speeds fixation but may cause artifacts. Standard: room temperature
• pH — neutral pH (7.0) prevents formalin pigment artifact (acid formalin = brown pigment in blood-rich tissues)
• Time — adequate fixation: 6–48 hours for most specimens. Over-fixation can harden tissue
• Concentration — 10% formalin = 4% formaldehyde (standard). Too concentrated causes surface hardening

Biopsy & Types of Biopsies

• Biopsy — removal of tissue from living patient for histopathological examination and diagnosis
• Core Core Biopsy — hollow needle removes a core of tissue. Used for breast, liver, kidney, prostate
• Skin Punch Biopsy — circular blade removes a plug of skin. 2–8 mm diameter. For skin lesions
• Skin Shave Biopsy — superficial skin lesions shaved with blade. Good for raised lesions
• Needle Fine Needle Aspiration (FNA) — thin needle aspirates cells. For cytology not histology. Rapid, minimal trauma
• Image Image-guided Biopsy — CT/ultrasound guided needle biopsy. For deep or small lesions
• Surgical Excisional Biopsy — entire lesion removed. Diagnostic + therapeutic. Gold standard
• Surgical Incisional Biopsy — part of large lesion removed for diagnosis only
• Endo Endoscopic Biopsy — forceps through endoscope. For GI tract, bronchial, bladder lesions
• Lap Laparoscopic Biopsy — minimally invasive. For intra-abdominal organs
• BM Bone Marrow Biopsy — trephine needle from posterior iliac crest. For hematological disorders
• Liquid Liquid Biopsy — detects circulating tumor DNA/cells in blood. Non-invasive. Emerging technology

Grossing Protocols

• Describe specimen: type, size (3 dimensions in cm), weight, color, consistency, surface, cut surface
• Ink surgical margins before cutting — to assess completeness of excision
• Sample representative sections: tumor, margins, adjacent normal tissue, lymph nodes
• Standard tissue cassette size accommodates sections of 2×1.5×0.3 cm maximum
• Label cassettes clearly with unique patient/specimen number

Decalcification of Bones/Hard Tissues

• Bone and calcified tissues must be decalcified before sectioning
• Acid decalcification (fast): 5–10% nitric acid or formic acid. 24–48 hours. Can damage tissue if over-decalcified
• Chelating agents (slow, gentle): EDTA (10–14 days). Best for IHC and molecular studies — preserves antigenicity
• Electrolytic method: Electrical current speeds up acid decalcification
• Endpoint test: X-ray or needle test — needle should pass through tissue without resistance

Purpose & Principle of Tissue Processing

• To replace water in tissue with a supportive medium (paraffin wax) that allows thin sectioning
• Tissue is infiltrated with paraffin so it can be cut at 3–5 µm thickness on a microtome
• Water and paraffin are immiscible — must remove water first using a series of reagents

Stages of Tissue Processing

• 1 Fixation — 10% NBF (already done before grossing)
• 2 Dehydration — ascending grades of alcohol (70% → 80% → 90% → 95% → 100% × 2). Removes water gradually to prevent tissue shrinkage
• 3 Clearing (Dealcoholization) — xylene (2 changes). Removes alcohol and makes tissue transparent and miscible with paraffin
• 4 Impregnation (Infiltration) — molten paraffin wax (60°C) × 2–3 changes. Paraffin replaces xylene in tissue spaces
• 5 Embedding — tissue oriented in mold with fresh paraffin, allowed to solidify. Creates paraffin block

Manual vs Automated Tissue Processing

Common Reagents Used

Principle of Embedding

• Tissue infiltrated with paraffin is placed in a mold with additional molten paraffin and allowed to solidify into a block
• The block supports the tissue during microtome sectioning
• Correct orientation is critical — determines the plane of section

Embedding Media & Their Properties

• Paraffin Wax — standard medium. MP 56–62°C. Good sections at 3–5 µm. Can be stored indefinitely
• Celloidin/Nitrocellulose — for large specimens (eye, brain). Very slow. Sections cut at 10–20 µm. Rarely used now
• Resin (Epoxy/Araldite) — for electron microscopy. Ultra-thin sections (50–100 nm). Very hard
• OCT (Optimal Cutting Temperature) compound — for frozen sections. Water-soluble gel. Rapid (minutes). Used intraoperatively
• Agar — to hold small or fragmented biopsies together during processing

Orientation of Tissues

• Flat orientation — skin biopsies, mucosal biopsies placed flat to show all layers
• On-edge orientation — tubular structures (intestine, fallopian tube) oriented to show lumen
• Endoscopic biopsies — small, fragile — wrap in lens tissue or agar-embed before processing
• Correct orientation ensures diagnostic sections show the relationship between epithelium and stroma

Properties of Paraffin Wax

• Melting point: 56–62°C (must be above room temp to remain solid during storage)
• Miscible with xylene (clearing agent) — allows replacement of xylene during infiltration
• Allows thin sections: 3–5 µm routine, 1–2 µm for special purposes
• Inert — does not react with tissue or stains
• Hard enough to support sectioning, yet soft enough to cut cleanly
• QC of paraffin blocks: check for air bubbles, incomplete infiltration, incorrect orientation, cassette label attached