Introduction to Histology and Cytology - Flashcards (60)
Anatomic Pathology, Cytology, and Histology Foundations
Anatomic pathology encompasses the study of disease through tissue and cell analysis. It includes two closely related disciplines: cytopathology (cytology) and histopathology (histology). Cytology diagnoses are based on cellular characteristics observed in prepared slides, while histology examines tissue architecture. In many labs, cytology and histology are distinct units, yet they share foundational principles: assessment of morphologic features of cells or tissues and the preparation and examination of stained samples on glass slides by pathologists. Medical laboratory technicians contribute significantly by preparing specimens: accessioning, staining, cover slipping, and filing. This body of work underpins the diagnosis of diseases, including cancer, and forms the basis for further clinical decision-making.
Lab Roles and Workflow
Histology and cytology labs, though specialized, rely on similar skill sets in tissue handling and microscopic assessment. The histotechnologist (histotechnician) prepares samples for pathologist review, often via a sequence of steps from specimen receipt to slide preparation. The workflow typically includes accessioning, fixation, processing, embedding, sectioning, mounting, and staining. Safety, documentation, and quality control are integral, with attention to WHMIS guidelines for labeling, storage, and disposal of hazardous solutions. Proper handling of sharps, spill response, and ergonomic practices are essential to minimize occupational hazards. Understanding personal protective equipment (PPE) and the correct use of safety devices in histology labs is part of the foundational training.
Histology and Histopathology: Definitions and Scope
Histology: a branch of anatomy dealing with the minute structure and composition of tissues. It forms a core part of anatomical pathology by enabling microscopic examination to detect abnormal cellular changes, including malignancy.
Histopathology: diagnosis and study of diseases of tissues through microscopic examination.
Histology specimens are prepared from autopsy, surgical, or biopsy materials. Special stains highlight specific cell morphologies, and histotechnologists ensure samples are prepared for pathologist review.
Specimens include surgical resections, autopsies, biopsies, amputations, and large organ tissue fragments.
Histology Specimens and Processing Overview
Histology specimens come from surgical procedures, autopsies, and biopsies. They vary in size from small tissue biopsies to large organs or amputations. The standard processing chain aims to produce stained tissue slides suitable for microscopic evaluation by the pathologist. The typical end product is a stained tissue section of thickness 3-5 \,\mu\text{m} mounted on a glass slide and stained with hematoxylin and eosin (H&E) to reveal tissue architecture and cellular details. Tissue is fixed, dehydrated, cleared, infiltrated with paraffin, embedded into FFPE (formalin-fixed, paraffin-embedded) blocks, sectioned to thin ribbons, mounted on slides, and stained.
Specimen Receiving and Accessioning
Specimens arrive with a requisition and must be labelled according to standard operating procedures (SOPs). A surgical number is assigned by sequential numbering and recorded in a daily accession log. The surgical number is carried through all steps: it appears on the requisition, the specimen container, tissue cassettes, slides, and the pathologist’s report.
Requisition information includes: patient name, date, tissue type, site/body source, physician and patient location (in-patient or out-patient).
Specimen numbering example: S-14-4567-1A breakdown: S = surgical, 14 = year, 4567 = specimen number, 1A = unique identifier for the specimen. This format helps traceability from collection to diagnosis.
Specimen Rejection and Priority
Specimen rejection is governed by a formal policy and can result from labeling errors, missing or incorrect requisitions, incorrect fixative, insufficient fixative, specimen without fixative, leaking containers, or container size issues.
Priorities determine turnaround times (TAT):
Emergency/ED: immediate (now) with results delivered rapidly despite missing patient information.
STAT: within 1 \,\text{hour}.
ASAP: typically within 4 \,\text{hours}.
Routine: typically within 24 \,\text{hours}.
Transport, Packaging, and Handling of Specimens
Fresh specimens deteriorate quickly; rapid transport to the lab is essential. Temperature control is crucial; preferably refrigeration, but freezing must be avoided. When fixed specimens are involved, handling is somewhat less critical but still requires care.
Packaging guidance for transport and handling of biological materials includes four components (TDG framework):
1) A rigid, leak-proof specimen container; 2) Absorbent material; 3) A leak-proof secondary container with a seal; 4) An outer container.
If dry ice is used, do not seal the dry ice within the sealed secondary container; instead, place it between the primary container and the outer bag.
Specimens may be sent fixed or unfixed. UNFIXED specimens require rapid STAT processing or refrigeration with appropriate handling to preserve tissue integrity. UNFIXED specimens should be wrapped in towels soaked in physiological saline (0.9% NaCl).
Histology Specimen Processing: Fixation and Preparation
The primary goal of histology sample processing is to provide the pathologist with stained tissue slides suitable for microscopic evaluation. The core steps are:
Fixation: preserves tissue structure and stops autolysis and putrefaction; initiates stabilization of proteins;
Dehydration: removes water from tissue;
Clearing: replaces alcohol with a substance miscible with paraffin (e.g., xylene);
Infiltration: tissue is penetrated by paraffin wax;
Embedding: tissue is embedded in paraffin blocks (FFPE);
Trimming: blocks prepared for sectioning;
Sectioning: microtome cuts thin sections, typically 58-60^\circ\text{C} paraffin sections;
Mounting and staining: sections mounted on slides and stained (H&E is standard).
Most tissue is fixed using formalin-containing fixatives, with fixation lasting from hours to days depending on tissue type and size. A common target is approximately 1 \,\text{mm/hr} fixation penetration for diffusion-based fixation.
Fixation: Definition, Purpose, and Key Parameters
What fixation does
Fixation preserves cellular structures by stabilizing proteins and other tissue components, preventing autolysis and putrefaction, and hardening tissue for subsequent processing.
Chemical fixation typically stabilizes proteins by denaturing them, converting a gel-like colloidal tissue into a semi-solid gel that is easier to handle during processing.
Why fixation matters
Proper fixation preserves morphology and antigenicity for accurate histological interpretation. Poor fixation leads to artifacts and unreliable results.
Ischemic time
Ischemic time is the interval from tissue removal from the body to placement into fixative; minimizing this time improves fixation quality and downstream morphology.
Fixation time and tissue factors
Tissue size, density (dense or fatty), and the presence of a capsule affect penetration and fixation time. A general guideline is 1 \,\text{mm/hr} penetration, with larger specimens commonly fixed for 18-24 \text{ hours}.
Fixation methods
Chemical fixation: immersion in fixative solution.
Physical fixation: heating, microwave fixation, or cryo-preservation (freeze-drying). Microwave fixation is common in routine labs.
Some tissues require special approaches or longer fixation times due to structural complexity.
Fixative concentration and volume
The fixative volume should be at least 15-20\times the tissue volume to ensure proper penetration and fixation.
pH considerations
For electron microscopy and ultra-structural preservation, buffering to pH = 7.2-7.4 is often required.
Size and diffusion considerations
Large specimens or those with capsules require more time or pre-sectioning to allow fixative to penetrate all regions. Fragmentation or slicing aids penetration.
Choice of fixative: factors to consider
The structures to be demonstrated, long-term vs short-term stability, and the effect on tissue over time.
Common fixatives include: Formaldehyde, potassium dichromate, mercuric chloride, ethanol, picric acid, acetic acid, osmium tetroxide, and glutaraldehyde.
Fixation primarily targets proteins and aims to stabilize them for subsequent processing.
Formalin: The Universal Fixative (10% NBF)
What it is
10% neutral buffered formalin (NBF) is the most widely used fixative in histopathology. Formalin itself is a solution of formaldehyde in water; formaldehyde gas dissolves in water to form aqueous formalin with roughly 37-40\% by weight, which is used as a fixative solution.
The 10% NBF solution is typically about 3.7-4.0\% formaldehyde in buffered water.
Preparation
The standard recipe for 10% NBF involves buffering 40% formaldehyde to neutral pH with phosphate buffers to achieve a stable pH around neutral.
If 10% formalin becomes cloudy due to paraformaldehyde formation, it can be filtered or a small amount of methanol can be added.
Fixation time and tissue considerations
The fixation time depends on tissue size; a typical minimum is 6-8\text{ hours}, with longer times for larger or more dense tissues and for nervous system tissues where it is particularly effective.
Advantages
Tolerant fixative: specimens can remain in fixative for extended periods without damage; inexpensive with good penetration; well-suited for nervous system tissues; blocks do not require washing prior to processing; widely validated for H&E staining.
Disadvantages and safety
Safety concerns: formaldehyde vapors are irritants and carcinogenic; exposure can irritate eyes and mucous membranes. Ventilation and fume hoods are recommended, and PPE should be used.
Formalin pigment formation can occur if the solution becomes too acidic; remove pigment with alcohold picric acid or alkaline alcohol. Paraformaldehyde polymerization can occur; filtration removes it. Formalin is unsuitable for some electron microscopy preparations.
Formalin reactions with tissue components
Formalin acts as a non-coagulant, additive-tolerant fixative; it cross-links amino groups on proteins, stabilizing tissue while preserving overall morphology with minimal shrinkage compared to other fixatives.
Lipids are preserved but not fully insoluble; carbohydrates are not directly fixed but glycogen can be trapped within protein matrices (indirect fixation).
Formalin pigments and management
Acid hematin formalin pigment can form when formalin becomes acidic; remove with alcoholic picric acid or alkaline alcohol.
Safety and handling
Formaldehyde exposure requires PPE and fume hood use; avoid skin contact and inhalation; ensure adequate ventilation.
Other Fixatives and Alternatives
Zenker’s fluid
A fixative containing mercuric chloride; provides excellent nuclear and connective tissue staining, but corrodes metals (except nickel alloys) and can cause tissue shrinkage and pigment formation. It is toxic and requires thorough washing and post-processing steps (alcoholic iodine, sodium thiosulfate) to remove precipitates.
Bouin’s fluid
Contains picric acid; penetrates rapidly with slight tissue shrinkage and stable fixation, but picric acid is explosive when dry, and tissues can become hard and brittle after prolonged exposure. Lipids can be altered or reduced.
Brasil’s alcoholic picro-formal fixative
A routine fixative for glycogen demonstration; shares disadvantages with Bouin’s fixatives.
Common Reagents in Histology
Histology relies on a variety of reagents for fixation, dehydration, clearing, embedding, staining, and mounting. The Medical Laboratory Assistant should understand the use and safe handling of reagents, not just memorize recipes. Many reagents are purchased in solid form and stored in airtight, glass containers away from heat. Water used as a solvent is typically distilled, unless otherwise instructed. Labeling, filtration, and SDS awareness are essential.
Reagents must be stored in a cool, dark area, and proper quality control and SOPs must be followed. Toxic components require heightened safety measures and documentation.
Practical and Safety Considerations
Ergonomics: apply proper ergonomic principles to reduce strain during repetitive tasks such as microtomy and specimen handling.
Spill containment: have established spill clean-up procedures for biological and hazardous materials, with appropriate PPE and containment equipment ready.
Lab incidents: incidents must be documented and reviewed to prevent recurrence; emergency protocols should be understood and rehearsed.
Whmis compliance: labels, dates, storage, and disposal of hazardous chemicals must comply with WHMIS regulations.
Personal protective equipment: PPE selection depends on the hazard; gloves, lab coats, eye protection, and respiratory protection as needed.
Waste disposal: hazardous waste disposal procedures must be followed, including sharps disposal and chemical waste handling.
Specimen Logistics and the Pathology Workflow
Tissue processing aims to create FFPE blocks suitable for sectioning. The process often includes formalin fixation, dehydration through graded ethanol, clearing with xylene or substitutes, infiltration with paraffin, embedding to create solid blocks, trimming, sectioning (with microtome at proper temperatures, typically around 58-60^\circ\text{C} for paraffin), mounting sections on slides, and performing H&E staining. The end product is slides ready for pathologist review.
Fixed specimens (in fixative) require clear labeling and proper separation from unfixed specimens. In addition to the requisition, the tissue’s journey from collection to slide involves key decisions about fixation and processing to preserve morphology.
Practical Considerations in Fixation and Processing
Ischemic time minimization is critical to preserve the tissue’s architecture and molecular content.
The volume ratio for fixatives (≥ 15-20\times\text{ tissue volume}) affects penetration and fixation quality.
The pH of fixatives affects preservation and downstream imaging, with buffering important for electron microscopy:
For ultra-structural preservation, maintain fixative pH in the range 7.2-7.4\,.
Fixatives can be classified by reaction with proteins (additive vs non-additive), by whether they cause coagulation (coagulant vs non-coagulant), and by toleration time (tolerant vs intolerant):
Examples include Formaldehyde (additive, coagulant or non-coagulant depending on context) and Glutaraldehyde (strong cross-linking fixative).
Special Topics: Formalin Pigment and Polymerization
Formalin can form fixative pigments such as acid hematin when pH is too low. Neutralization and proper buffering prevent pigment formation. Paraformaldehyde, a polymerization product of formaldehyde, can occur in concentrated formalin and is typically removed by filtration. Methanol is sometimes added to prevent or slow polymerization in commercial preparations.
Safety: Formaldehyde Hazards and Controls
Formaldehyde exposure presents irritant and carcinogenic risks. Adequate ventilation, fume hood or cabinet use, and PPE are essential. Avoid skin contact and inhalation; work in a well-ventilated area and in a fume hood when possible.
The Pathology Context and Real-World Relevance
Histology enables pathologists to diagnose diseases by revealing tissue architecture and cellular details. The sample processing chain—from accessioning to FFPE block to H&E slide—must preserve morphology with minimal artifacts. Fixation quality is central to valid interpretation; poor fixation can obscure or mimic pathology, leading to diagnostic errors.
The interplay between pre-analytic variables (specimen handling, fixation, processing), analytic steps (sectioning, staining), and post-analytic interpretation (diagnosis) underpins clinical decision-making. Maintaining robust SOPs, safety practices, and accurate documentation ensures reliability, reproducibility, and ethical patient care.
Quick Reference: Key Numerical and Formula-like Details
Tissue section thickness: 3-5\;\mu\text{m}
Fixative-to-tissue volume: at least 15-20\times the tissue volume
Fixation penetration rate: 1\;\text{mm/hr}
Common fixative: 10\% \,NBF (which corresponds to roughly 3.7-4.0\% formaldehyde in buffered solution)
Embedding temperature for paraffin: 58-60^\circ\text{C}
Typical fixative pH targets: pH\approx 7.2-7.4 for electron microscopy requirements; general buffers aim for neutral pH
TATs: STAT ≈ 1\ hour; ASAP ≈ 4\ hours; Routine ≈ 24\ hours
Storage temperature for unfixed samples prior to fixation: commonly 4\,^{\circ}\text{C}; unfixed tissue is sometimes kept on ice or cold in certain collection settings
Common saline used for unfixed specimens: 0.9\% \text{NaCl}
Appendix: Quick Reference Lab Policies and Sources
Requisition and accessioning policies ensure consistent labeling and tracking across the workflow. A single surgical number ties requisition, container labels, tissue cassettes, slides, and the final report.
Specimen rejection policies protect test integrity and patient safety by ensuring proper labeling, fixative use, and handling. These policies should be documented in the SOPs.
Safety policies include WHMIS requirements for labels, dates, storage, and disposal; spill containment procedures; and documentation of safety incidents.
Video resources (for further context) include: Histopathology lab tour and practical demonstrations such as YouTube videos titled “Histopathology Laboratory Tour” and “In the Lab - Histology,” which illustrate real-world lab workflows.
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