Section B Flashcards
Discuss five key factors that influence the effectiveness of tissue fixation
Fixative Volume: A 10:1 fixative-to-tissue ratio ensures complete penetration, too low may cause autolysis, too high isnt as bad but would be a waste of the fixative
* pH: Optimal at ~pH 6.8 to maintain protein integrity.
* Penetration Rate: Soft tissue (e.g., breast) fixes faster than dense tissue (e.g., colon). Carnoy’s
fixative is faster than formalin; glutaraldehyde is slower.
* Agitation: Gentle shaking enhances penetration and uniform fixation.
* Temperature: Elevated temperature accelerates fixation, Cold fixation slows enzymatic degradation but also slows down the penetration of the fixative.
- Time: Minimum 4 hours for small biopsies; up to 48 hours for larger tissues, Too short a time may lead to incomplete preservation, while too long can lead to tissue hardening or masking of antigens.
Tissue processing prepares fixed samples for embedding and sectioning by removing water and
replacing it with paraffin wax. Discuss the steps
Dehydration
Purpose: To remove water from tissues, since paraffin is hydrophobic and won’t mix with water.
Method: Tissues are passed through graded alcohols, typically in increasing concentrations:
70% → 80% → 95% → 100% ethanol (or isopropanol).
Duration: Several hours to allow gradual and complete water removal.
Note: Sudden dehydration (e.g., jumping straight to 100%) can cause tissue shrinkage or distortion.
Clearing
Purpose: To replace alcohol with a substance that is miscible with both alcohol and paraffin wax.
Common Agents: Xylene is standard; alternatives include toluene, limonene, or aliphatic hydrocarbons.
Process:
Usually 2–3 xylene changes, each lasting 20–30 minutes.
Clears the tissue and renders it transparent.
Note: Incomplete clearing can prevent proper paraffin infiltration.
Infiltration
Purpose: To impregnate tissues with molten paraffin wax.
Process:
Xylene is gradually replaced by paraffin wax at around 55–60°C.
Tissues are immersed in molten wax in multiple changes (usually 1–2 hours total).
Result: Tissue becomes fully saturated with wax, giving it support and rigidity.
Embedding: Tissues are placed in wax blocks for sectioning.
The Royal College of Pathologists outlines five specimen categories (A–E), each with a specific level of
sampling. Explain with examples
Category A: Small Biopsies
Definition: Very small tissue samples, usually ≤5 mm.
Example: Skin punch biopsy, prostate needle biopsy, endoscopic mucosal biopsy.
Entire tissue is embedded and examined.
Purpose: Ensure nothing is missed, as the whole specimen is available for histology.
Clinical Use: Often used in initial diagnosis
Category B: Visible Lesions
Definition: Small resected lesions or polyps with a clear gross abnormality.
Example: Colonic polyp, skin excision with a visible mole or wart.Screening colonoscopies, dermatological excisions.
Category C: Organs With No Obvious Lesion
Definition: Entire organ resections with no visible tumour or abnormality.
Example: Spleen removed for trauma or hematologic workup; non-neoplastic hysterectomy.
Category D: Tumour Resections
Definition: Large specimens with known or suspected tumours.
Example: Mastectomy, colectomy with carcinoma
Assess tumour type, grade, size, invasion, and margin status.
Provide staging information
E…
Haematoxylin & Eosin (H&E) Staining. Explain
Purpose:
To differentiate and visualize basic tissue structures by staining:
Nuclei: Blue to purple (via haematoxylin).
Cytoplasm, connective tissue, muscle fibers: Pink to red (via eosin).
Sections to Water
Process: Slides are transferred from xylene → 100% alcohol → 95% → 70% alcohol → water.
Purpose: This rehydrates the tissue, making it receptive to aqueous stains like haematoxylin.
Haematoxylin Staining
Stain: Basic dye that binds negatively charged (basophilic) structures, especially nucleic acids.
Result: Nuclei appear blue-purple.
Differentiation
Process: Brief immersion in acid alcohol (weak hydrochloric acid in ethanol).
Purpose: Removes excess haematoxylin to enhance contrast by clearing background staining.
Bluing
Reagent: Alkaline solution (e.g., tap water, ammonia water, or Scott’s tap water substitute).
Purpose: Converts haematoxylin from red-purple to a blue hue through alkalization.
Eosin Staining
Stain: An acidic dye that binds to positively charged (acidophilic) components like cytoplasmic proteins, collagen, and RBCs.
Result: Cytoplasm, muscle, and connective tissue appear pink to orange.
Dehydration
Process: Tissues are dehydrated again using increasing alcohol concentrations (70%, 95%, 100%).
Purpose: Prepares tissue for xylene clearing and permanent mounting.
Clearing
Reagent: Xylene or a substitute replaces alcohol.
Effect: Makes the tissue transparent and compatible with mounting media.
Safety measures in the lab
Personal Protective Equipment (PPE)
Essentials: Lab coat, gloves, and goggles (or face shield).
Purpose:
Protects skin and clothing from chemical splashes, biohazards, and glass breakage.
Gloves prevent direct contact with infectious material or chemical reagents.
Universal Precautions
Principle: Treat all biological specimens (e.g., blood, tissues) as if they are infectious.
Applies To: Handling of human samples regardless of clinical history.
Sample Labelling & Identification
Importance:
Accurate labelling ensures proper identification, traceability, and hazard awareness.
Specimens with known infectious agents (e.g., Mycobacterium tuberculosis) must be clearly marked.
Chemical Safety
Common Hazards:
Xylene: Flammable, toxic—requires fume hoods and proper storage.
alcohols
Handling of Hazardous Materials
SOP’s
Sharps, waste management
Spill management, reports prevent etc…
Mild dyskaryosis outcomes
Mild dyskaryosis refers to low-grade squamous intraepithelial changes (LSIL).
Microscopically:
Slight nuclear enlargement, irregular chromatin.
Nuclear changes occupy less than half the cell area.
f HPV Negative:
Indicates low risk of high-grade cervical disease.
Action: Return to routine 3-year recall for cervical screening (if age 25–49) or 5 years (if 50–64)
⚠️ If HPV Positive:
Indicates higher risk of persistent or progressive disease.
Action: Refer to colposcopy for further evaluation.
Colposcopic Assessment
Performed by a specialist to visually inspect the cervix under magnification after applying acetic acid and/or iodine.
Goals:
Assess extent and severity of changes.
Identify areas for targeted biopsy.
Biopsy & Histological Correlation
CIN1: Abnormal cells occupy the lower third of the epithelial thickness.
Associated with transient HPV infection.
If CIN1 Confirmed & Patient is Asymptomatic/Low Risk:
Conservative Management:
No immediate treatment.
Follow-up colposcopy or cytology + HPV test in 12 months…
Post-Treatment Follow-Up: Test of Cure
Conducted 6 months after LLETZ or other excisional treatment.
Includes:
HPV test
Cytology
Staining types
Basic Dyes
Charge: Positively charged (cationic).
Binding Target: Bind to acidic (basophilic) components of cells.
Tissue Targets:
Nucleic acids (DNA and RNA)
Ribosomes, nucleoli, rough ER
Common Dyes:
Haematoxylin: Stains nuclei blue or purple.
Acidic Dyes
Charge: Negatively charged (anionic).
Binding Target: Bind to basic (acidophilic) structures.
Tissue Targets:
Cytoplasmic proteins
Collagen, muscle fibers, red blood cells
Common Dyes:
Eosin: Stains cytoplasm pink.
Orange G: Used in PAP stain to highlight keratin.
Neutral Dyes
Composition: Contain both acidic and basic components.
Target: Stain both acidic and basic tissue elements simultaneously.
Common Dyes:
Romanowsky-type stains (e.g., Giemsa
used for cytology
Amphoteric Dyes
Definition: Can act as either acidic or basic, depending on pH of the staining solution.
Behaviour:
At low pH: Behave as basic dyes (bind acidic tissue).
At high pH: Behave as acidic dyes (bind basic tissue).
Examples:
Congo Red: Used to detect amyloid deposits in tissues.
Leuco Compounds
Definition: Dyes that are colorless
Often used in enzyme histochemistry and redox reactions
Leuco Crystal Violet: Used in Gram staining to identify bacterial morphology.
HPV description
Type: Small, non-enveloped DNA virus.
Size: ~55 nm in diameter.
Genome: Circular double-stranded DNA, ~8,000 base pairs.
Structure:
Early genes (E1–E7): Involved in viral replication and pathogenesis.
Late genes (L1, L2): Encode capsid proteins for viral assembly.
Risk Factor Effect
Early sexual activity Increased exposure to HPV
Multiple sexual partners Higher risk of HPV transmission
Unprotected sex Lack of barrier protection
HPV Infection:
Enters basal epithelial cells via microabrasions.
Infects transformation zone of cervix—where squamous and columnar epithelium meet.
Screening and Diagnosis
HPV DNA testing is now the primary screening tool in the UK and many countries.
More sensitive than cytology for detecting high-risk types.
Cervical smear (Pap test) identifies cytological abnormalities.
Colposcopy and biopsy are used to investigate abnormal screening results.
HPV Vaccination: Prevention of Cervical Cancer
Vaccines: Cervarix, Gardasil, and Gardasil 9.
Target high-risk HPV types
