Introduction to Histology and Histotechniques

# Welcome to MMS2200: Introduction to Histology and Histotechniques - Unit Coordinator: Emily Wong - Tutor: Sanjeev Adhakari # Learning Objectives - Develop a thorough understanding of safety protocols and potential hazards in histopathology laboratories. - Explore the specialised techniques of histotechnology and histology, and their role in histopathological analysis. - Gain knowledge of the complete histopathology workflow, from sample receival to fixation, grossing, processing, embedding, sectioning, and staining. # History of Histology and Histopathology - **17th Century:** - Robert Hooke (~1665): Advent of microscopy - Antonie van Leeuwenhoek (~1673): Staining of cells, plant-based dyes - **19th Century:** - 1867: Fixation (Formaldehyde is introduced for embalming) - Mid 1800s: Toluidine Blue is developed as one of the first synthetic dyes used for histology. - **Early 20th Century:** - 1922-1924: The Biological Stains Commission (BSC) is established to standardise dye nomenclature, ensuring consistency in histological staining methods. - Early 1900s: Several histological stains and techniques are named after pioneering researchers. - Golgi – Silver staining for nervous tissue. - Weigert – Elastic tissue and myelin stains. - Grocott – Methenamine silver stain for fungi. - Masson – Trichrome staining for connective tissue differentiation. # Historical Stains - Grocott Methenamine Silver (GMS): Stains a variety of fungal organisms - Golgi: Stain neurons - Masson’s trichrome: stain connective tissue - Weigert’s: stain elastic fibers # Histopathology Laboratory Workflow - Biopsy/Autopsy - Specimen receival and registration - Fixation or Freezing (frozen remains frozen) - Dissection & selection of blocks for examination - Processing to paraffin wax - Embedding in paraffin wax (blocking out) - Cutting sections - Staining – routine, specials, immunohistochemistry - Microscopy - Reporting - Turnaround time: Most routine samples take 24 hours; results expected within one day. - Technical staff primarily carries out processes. # Types of Samples/Specimens - **Histopathology – Routine Diagnosis:** - Tissue in 10% Neutral Buffered Formalin, at least 10x the volume of the specimens. - **Histopathology – Frozen sections (on request):** - Fresh tissue (i.e. no fixative) – delivered immediately to the laboratory. # Sample Preservation Before Fixation - Surgical Removal - Biopsies/Excision - Autopsy investigation # First Step: Fixation - Surgical/ Tissue specimens require immediate processing to maintain integrity. - Fixation is the first and most critical step in histopathology. - Specimen Collection from Surgery/Clinics - Proper handling: Place specimens immediately in a clean, labeled container. - Documentation: Ensure correct patient information, clinical history, and anatomical site are recorded. - Transportation: Minimise time between collection and fixation to prevent degradation. # Fixation/Preservation Objectives - Maintain clear and consistent morphological features - Retain tissue and cellular integrity - To analyse and examine the tissues/organs under the microscope for diagnosis with minimal loss/destruction of the structure. - Reduce/prevent further breakdown, autolysis of cells or enzymatic destruction of cellular and extracellular molecules - Protect tissue from micro-organisms invasion # Fixation Details - Chemicals used are called “fixatives” - Fixation was introduced over 150 years ago; many principles still apply today - Physical and Chemical Methods of Fixation - **Physical Methods:** - Heating - Freeze-drying - Microwave technology - **Chemical Methods:** - Coagulant: Alcohol (ethanol, methanol) and acetone - Cross-linking: Formaldehyde, glutaraldehyde, other aldehydes groups - Compound: Alcoholic formalin - The most common fixative used in diagnostic pathology is Formaldehyde in its 10% neutral buffered form # Fixation of Antigens - **No Fixation** – Some antigens require no fixation to preserve their structure. - **Formaldehyde** – The most commonly used fixative. It cross-links proteins, which can mask antibody binding sites, often requiring antigen retrieval to restore binding. - **Effect on Protein Structure** – Formaldehyde changes the 3D shape of proteins (tertiary & quaternary structure) but keeps the primary sequence (amino acids) and secondary structures (helices & sheets) intact. - **Other Fixatives** – Acetone, methanol, and ethanol may be recommended by the antibody manufacturer. - **Impact** – Any type of fixation alters antigen structure and may affect antibody binding. - **Protein Structures** 1. Primary Structure – The exact sequence of amino acids in a protein. 2. Secondary Structure – Local folding into helices or sheets, stabilized by hydrogen bonds. 3. Tertiary Structure – The overall 3D shape, formed by folding and coiling of the polypeptide chain. 4. Quaternary Structure – When multiple protein units (subunits) come together, like in dimers or larger complexes. # Fixation Specifics - 10% Neutral Buffered Formalin (NBF) is most commonly used: - penetrates the tissue rapidly - causes less shrinking - permits most special stains - Fixation is generally complete in about 1 hour per mm of tissue. - Small biopsies will thus take 1-2 hours to fix. - Larger tissue may take 5-10 hours or more # Factors Affecting Fixation - Buffering - Penetration - Volume - Temperature - Concentration - Time ## A. Buffering - Fixation is best carried out close to neutral pH, in the range of 6-8. - Common buffers include phosphate, bicarbonate, cacodylate and veronal. ## B. Penetration - Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. - Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurial's and others are somewhere in between. - Penetration into a thin section will occur more rapidly than for a thick section. ## C. Volume - The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. - It is better to change the fixative at intervals to avoid exhaustion of the fixative. - Agitation of the specimen in the fixative will also enhance fixation. ## D. Temperature - Increasing the temperature, as with all chemical reactions, will increase the speed of fixation. - Hot formalin will fix tissues faster. This is often the first step on an automated tissue processor. ## E. Concentration - Concentration of fixative should be adjusted down to the lowest level possible. - Formalin is best at 10%; glutaraldehyde is generally made up at 0.25 to 4% - Too high concentration may adversely affect the tissues and produce artefact similar to excessive heat. ## F. Time Interval - The time interval from removal of the tissue to the fixation is very important. The faster you can get the tissue and fix it, the better. - Artefact will be introduced by drying (use of saline solution to moist avoid this issue) - The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage will occur. # Key Notes on Fixation - **Fixation before arriving in the lab - Pre-laboratory** - Specimens should be transferred to a fixative within 1 hour of surgical excision. If this cannot be achieved, keep the specimen at 4°C including during transfer to the laboratory. - Proper fixation is essential for accurate histopathological diagnosis. - Delays or errors in fixation compromise diagnostic reliability. - Adhering to best practices ensures optimal tissue preservation for analysis. - The success of histopathology begins with correct specimen handling and fixation. # Safety in the Histology Lab - Working in a Histology Lab requires awareness of hazards - Chemical hazards - Physical hazards - Biological hazards # Occupational Health and Safety - Many fixatives, particularly formalin (formaldehyde-based solutions), glutaraldehyde, and other aldehydes, pose health and environmental hazards. - Procedures for appropriate handling, storage and disposal must be considered in the use of fixatives in the laboratory. - Use of personal protective equipment (PPE) (gloves, goggles, lab coats) - Proper ventilation and fume extraction ## Chemical Hazards - GHS (Global Harmonisation System) Pictograms - Documents of chemical hazards - Material Safety Data Sheets (MSDS) ## Chemical Incompatibility - Segregate appropriately, accreditation requirement - Storing in alphabetical order is not acceptable # Safety Issues Regarding Histology - Formaldehyde is an irritant, possible carcinogen - Acetone, xylene and other volatile solvents - Commonly used acids – HCl, H2SO4, acetic acid and others - Mercury is a toxic heavy metal. Fixatives and stains containing heavy metals must be contained and disposed of by registered contractors - Picric acid is explosive when dry - OsO_4 is highly toxic and corrosive. Use in a well-ventilated area (fume hood) - Diaminobenzidine (DAB) – carcinogenic - Fume hoods or exhaust ducting should be readily available # Classification of Formalin - 10% formalin: Not classed as a dangerous good but considered a hazardous substance - Avoid the use of concentrated formalin (37% formaldehyde) in the lab: - Category 2 carcinogen - Higher risk, requiring stricter handling procedures - Use commercially prepared 10% formalin instead of concentrated solutions # Safe Handling of Formalin - Avoid contact to skin and eyes. - Use personal protective equipment. - Gloves. - Safety glasses. - Clean up spillages. - Avoid inhalation of vapour. - Keep containers closed. - Ensure well-ventilated work spaces. - Store in well-ventilated areas, out of direct sunlight below 30°C. - Use of respirator masks may be considered if required. # Accidental Release Measures - Spills – Contain, Inform, Remove, Evacuate, call Emergency - Ensure spill kits are available (waste disposal bag, PPE wear, absorbent for soaking up the spills, formalin neutralising agent if it is a formalin spill) - Formaldehyde – large volumes - Acetonitrile – health consequences - Fire- fighting - First aid - Safety showers - Eye washes - Inhalation - Ingestion - Eye/Skin contact - Disposal - Use fume cupboards where applicable # Physical Hazards - Sharp blades are a major hazard - Glass - Electrical - Hot wax - Falls - Freezer burns - Latex sensitivity- contact dermatitis - Design of workplace - Ergonomic work areas prevent carpal tunnel syndrome, wrist issues from microtomes, back issues from sitting, tendonitis, trigger finger # Biological Hazards - Patient samples may be contaminated or infectious (e.g. Hep B/C, HIV, Tuberculosis) - Fresh samples, fresh frozen human samples, cryostat, an ideal source of infection - Needle-stick injuries - Bodily fluids - Cut from handling sharps - **Prevention:** - Vaccinations (Hep B) - Wear gloves, lab coats, and safety glasses # Prion Diseases - Caused by misfolded protein molecules forming beta-pleated sheets - **Transmission:** Ingestion of CNS tissue, organ transplantation, or inherited (familial). - Prion Protein (PrP): Normal form (PrP^c), disease-associated form (PrP^{Sc}). - **Examples:** Creutzfeldt-Jakob Disease (CJD), Bovine Spongiform Encephalopathy (BSE), Gerstmann-Sträussler-Scheinker syndrome (GSS), Scrapie, Fatal Familial Insomnia (FFI), Kuru. - **Diagnosis:** Immunohistochemical detection of PrP^{Sc} - **Decontamination:** Resistant to standard disinfection; requires 1–2N NaOH, strong sodium hypochlorite (1M or 2% available chlorine), or autoclaving at 134°C. - **Kuru Prevention:** Achieved through education and ending cannibalistic practices. - **Control Measures:** Isolation, containment, and incineration # Precaution/ Infection Risk - Universal precautions: - Formaldehyde fixation (2-8%) is a bactericide, tuberculocide, fungicide, virucide and sporicide - 70% ethanol inactivates many organisms – work benches and instruments - Hypochlorite (5-15%) inactivates many infectious agents very effectively - Steam autoclaving (121°C) is routinely used for instruments, glassware, clothing if necessary - Disposal of biological hazards in biohazard bags. # GHS Hazard Classifications - Health hazards, Physical hazards, Environmental hazards. - Acute toxicity, Skin corrosion/irritation, Serious eye damage/eye irritation, Sensitization, Germ cell mutagenicity, Carcinogenicity, Reproductive toxicity, Specific target organ system toxicity-single exposure, Specific target organ system toxicity-repeated exposure, Aspiration toxicity. - Explosives, Flammable gases, Flammable aerosols, Gases under pressure, Flammable liquids, Flammable solids, Self-reactive substances, Pyrophoric liquids, Pyrophoric solids, Self-heating substances, Substances which in contact with water emit flammable gases, Oxidizing liquids, Oxidizing solids, Organic peroxides, Substances corrosive to metal. - Acute aquatic toxicity, Chronic aquatic toxicity. # GHS Hazard Pictograms - Flame over circle: Oxidizers - Flame: Flammables, Self reactives, Pyrophorics, Self-heating, Emits flammable gas, Organic peroxides - Corrosion: Corrosives - Skull and crossbones: Acute toxicity (severe) - Health hazard: Carcinogen, Respiratory sensitizer, Reproductive toxicity, Specific target organ toxicity, Mutagenicity, Aspiration toxicity - Exclamation mark: Irritant, Dermal sensitizer, Acute toxicity (harmful), Narcotic effects, Respiratory tract irritation - Gas cylinder: Gases under pressure - Environment: Acute aquatic toxicity, Chronic aquatic toxicity # The Future - Many manufacturers looking for alternatives to using toxic chemicals - Better safety education - Duty of care to others # Unit Discussion - CANVAS - WORKSHOPS - ASSESSMENTS - CANVAS: check announcement - Assessment outline - Semester schedule - Work through exercises in the lab manual # Schedule | Week | Date commencing Monday | Lectures | Learning activities readings, class preparation, online tasks during laboratory sessions | Assessment | |------|-------------------------|----------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------| | 1 | 24 Feb | Introduction to the unit outline. Safety/Tissue fixation in the histology lab | Lab safety induction/ Lab Discussion- topic selection | | | 2 | 3 Mar | Primary Tissue Types Microscopy | A2.2: Weekly Discussion conducted in lab time | | | 3 | 10 Mar | Fixation, Processing and Embedding Microscopy & Introduction to the Microtome | A2.2: Weekly Discussion conducted in lab time | | | 4 | 17 Mar | Features of healthy human cell structures and cell types - H&E stain & Special Stain Introduction to Lab Report – Work instruction Write-up | Microscopy & Microtomy/ Lab Report – histology machine selection | A2.2: Weekly Discussion conducted in lab time | | 5 | 24 Mar | Quality Control and Artefacts Microtomy & H&E Staining | A2.2: Weekly Discussion conducted in lab time | | | 6 | 31 Mar | A1.1 Safety Theory Test | A1.1 Practical Assessment 1 | A1.1 Practical 1 conducted in designated lab time and ‘Safety Theory Test’ to be completed in lecture time. | | 7 | 7 Apr | Causes of injury to cells and tissues Feedback session/Wax embedding | A2.2: Weekly Discussion conducted in lab time | | | 8 | 14 Apr | Online lecture: Diseases and Neoplasms | No lab: prepare for writing report | | | | 21 Apr | Mid Semester Break – No classes | | | | 9 | 28 Apr | A2: Lab report – Work Instruction Write-up | Microtomy, Van Gieson Stain | A2.1: Lab Report to be completed in lecture time. | | 10 | 5 May | Changes in cell morphology caused through disease – Immunohistochemistry/frozen section | Microtomy, Staining and Embedding | A2.2: Weekly Discussion conducted in lab time | | 11 | 12 May | Changes in cell morphology caused through disease - Diagnostic Cytology | Microtomy and Staining | A2.2: Weekly Discussion conducted in lab time | | 12 | 19 May | Assessment discussion: feedback on assessments, skill test and final exam review. | Last practice session for final prac test | A2.2: Weekly Discussion conducted in lab time | | 13 | 26 May | A1.2 Practical Assessment 2 | Practical Assessment 2 | A1.2 Practical assessment to be conducted in the laboratory (Schedule to be announced) | | | Study Week Exams | | | A3: Final exam TBA Please check SIMO | # Assessment Outline | Time | Assessment | Description | Location | Weight (Total 100%) | |----------------------------|--------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------| | Weekly, from Week 2-12 | Lab discussion | Students will work in pairs to discuss selected histology topics and present their findings to the class. | In designated laboratory sessions | 10% | | Week 6 Wednesday 03/04/2025 | Practical/Theory Assessments (Safety) | Hands-on/ theory assessments focusing on safety procedures | In designated laboratory + lecture sessions
Schedule TBA on Canvas | 20%
10% practical
10% theory | | Week 9 Wednesday 30/04/2025 | Lab report- work instruction write-up | Write work instructions for histology machines to demonstrate understanding of equipment and processes. | In lecture session
60 minutes | 20% | | Week 13 Wednesday 28/05/2025| Practical Assessment (Microtomy and Staining Skills) | Hands-on practical assessment to evaluate microtomy and staining techniques. | In laboratory + lecture sessions
~Schedule TBA on Canvas | 20%
10% microtomy
10% staining | | Exam period 09/06/2025 – 20/06/2025 | End of Semester Examination | Written examination assessing overall knowledge. | Location and date TBA – run centrally | 30% | # Assessment 2.2 Laboratory Discussion - This assessment is designed to enhance students' understanding of key histology concepts while developing communication and critical thinking skills. - Each pair will select a topic in Week 1 and deliver a 5-minute presentation at the beginning of a lab session in their assigned week. - The talk should provide a clear, concise overview of the topic, its relevance to histology, and its real-world or clinical applications. - Students are encouraged to engage the class with a well-structured presentation, use visual aids where appropriate, and confidently respond to questions. - After the presentation, the presenters will answer at least one question from the instructor or peers to demonstrate their depth of understanding. - The assessment is graded out of 10 marks, with a focus on content accuracy, delivery, time management, and the ability to answer questions effectively. # Unit Discussion - CANVAS - PRACTICAL ASSESSMENTS # Histology Practical Assessments - Two Histology Practical Assessment – clear your diary!! - Linked to assessment. - Linked to your professional learning. - Linked to WIL – Work Integrated Learning. - Linked to your future.