Handling fresh histological specimens

Handling fresh histological specimens

Handling fresh histological specimens is a complex and meticulous process requiring attention to detail at every step. Each phase, from collection to reporting, plays a vital role in ensuring the accuracy and reliability of the histopathological diagnosis. Adhering to best practices and protocols is essential for high-quality outcomes in histopathology.

1. Collection of Specimens

Preparation

• Personnel Training: Ensure the surgical team is trained in proper specimen handling techniques.
• Aseptic Technique: Utilize sterile gloves and instruments to minimize contamination. The surgical site should be properly disinfected before tissue collection.

Tissue Selection

• Representative Sampling: Select representative areas, especially if a suspected lesion exists. It may be helpful to obtain samples from multiple sites within a lesion.

Labelling

• Comprehensive Labels: Include patient ID, specimen type, date and time of collection, clinician’s name, and relevant clinical history. This ensures accurate tracking and minimizes errors.

2. Transporting Specimens

Transport Medium

• Saline or Specialized Media: Use sterile saline for fresh tissues. For specific analyses (e.g., microbiology), consider appropriate transport media.
• Avoiding Deterioration: For fragile tissues, like those intended for immunohistochemistry, use a medium that maintains antigenicity.

Temperature Control

• Room Temperature: Most tissues are best transported at room temperature. Refrigeration may be appropriate for certain specimens but should be done carefully to avoid freezing.
• Immediate Transport: Aim to deliver the specimen to the lab within 30 minutes to 1 hour post-collection.

3. Gross Examination

Initial Assessment

• Visual Inspection: Document size, shape, colour, and consistency. Note any visible lesions, necrosis, or abnormalities.

Photography

• Documentation: Take photos of the specimen and any notable features. This aids in both diagnosis and future reference.

4. Sectioning

Technique

• Sterile Instruments: Use sterile blades and instruments to avoid contamination.
• Handling Fragile Samples: Use forceps designed for minimal damage to delicate tissues.

Orientation

• Correct Orientation: Identify and mark the orientation (e.g., anterior/posterior) using orientation pins or ink. This ensures accurate sectioning and interpretation.

5. Fixation

Fixative Selection

• Formalin: The most commonly used fixative. Ensure it is buffered and fresh to maintain pH.
• Alternative Fixatives: Consider glutaraldehyde or other specialised fixatives for specific applications (e.g., electron microscopy).

Fixation Process

• Submersion: Ensure the specimen is fully submerged in fixative. Large specimens may require slicing to facilitate adequate penetration.
• Timing: Optimal fixation time varies; 6-24 hours is typically recommended. Consider the size and type of tissue when determining fixation duration.

6. Processing

Dehydration

• Ethanol Series: Gradually transition through increasing concentrations (e.g., 70%, 80%, 90%, 100%) to remove water. Each step lasts about 1-2 hours, depending on the tissue type.

Clearing

• Xylene or Alternatives: Replace alcohol with xylene (or a less toxic alternative like limonene) to make the tissue transparent. This step typically takes several hours.

Embedding

• Infiltration: Immerse the cleared tissue in molten paraffin (usually around 60°C). Allow it to infiltrate thoroughly, which may take several hours to overnight.
• Block Molding: Pour the paraffin into moulds and place the tissue in them, allowing it to solidify.

7. Sectioning for Microscopy

Microtome Operation

• Microtome Settings: Adjust settings for the thickness of sections (typically 3-5 µm). Use a disposable blade for clean cuts.
• Mounting: Place sections onto glass slides using an adhesive (e.g., albumin) or charged slides to improve tissue adhesion.

8. Staining

Routine Stains

• Hematoxylin and Eosin (H&E): This is the most common staining method. Hematoxylin stains nuclei blue, while eosin stains cytoplasm pink.
• Special Stains: Use additional stains (e.g., periodic acid-Schiff, Gomori’s trichrome) for specific tissue components or conditions.

9. Quality Control

Review Process

• Pathologist Examination: Pathologists should review all slides for clarity, quality, and diagnostic adequacy. This may include repeating staining or processing if necessary.

Documentation

• Record Keeping: Maintain thorough documentation of processing steps, times, and deviations from standard protocols.

10. Archiving and Storage

Storage Conditions

• Temperature and Humidity Control: Store paraffin blocks and slides in a cool, dry environment to prevent degradation.
• Access and Retrieval: Establish a systematic archiving system for easy retrieval, often utilizing databases or lab information systems.

11. Reporting

Diagnostic Process

• Microscopic Evaluation: Pathologists perform detailed analyses of stained sections, looking for cellular morphology, architectural patterns, and other diagnostic features.
• Differential Diagnosis: Consider various potential diagnoses based on findings, including histological criteria.

Communication

• Reporting Format: Provide a structured report that includes patient demographics, clinical history, findings, and a definitive diagnosis.
• Consultation with Clinicians: Discuss findings with the clinical team as necessary, particularly for complex cases or unexpected results.

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Cryo and frozen sections

Cryo and frozen sections are critical histopathological techniques, particularly for rapid diagnosis during surgical procedures and for preserving certain tissue characteristics. Here’s a detailed overview of these methods, including the processes for fresh and fixed tissues and the concept of freeze-drying.

1. Frozen Section Technique

Purpose

• Rapid Diagnosis: The frozen section technique is often used during surgeries (intraoperative consultations) to provide immediate pathological evaluation, helping guide surgical decisions.
• Preservation of Antigenicity: This method is crucial for preserving tissue antigens for immunohistochemical studies.

Process

1. Preparation of Fresh Tissue
2. Collection: Tissue should be collected as soon as possible after surgical removal.
3. Transport: To maintain tissue integrity, transport the specimen on ice or in a cold medium.
4. Freezing
5. Cryostat Use: A cryostat is a specialized microtome maintained at low temperatures, typically -20°C to -30°C.
6. Embedding: Fresh tissue may be embedded in an optimal cutting temperature (OCT) compound to stabilize the sample and facilitate sectioning. The tissue should be oriented correctly within the OCT.
7. Freezing Process: The specimen is rapidly frozen in the cryostat. This rapid freezing prevents the formation of ice crystals that can damage the tissue architecture.
8. Sectioning
9. Microtome Settings: Adjust the cryostat to cut sections of about 5-10 micrometers.
10. Slide Preparation: Sections are mounted on glass slides and typically stained immediately (often using H&E or other rapid staining techniques).
11. Diagnosis
12. Microscopic Evaluation: The pathologist examines the stained sections, providing a preliminary diagnosis that may guide surgical decisions.
13. Frozen Section of Fixed Tissue

Purpose

• Diagnostic Flexibility: Previously fixed tissues are sometimes sectioned in frozen form, particularly when additional analyses are needed.

Process

1. Preparation of Fixed Tissue
2. Fixation: Tissues are typically fixed in formalin or other fixatives. After fixation, tissues may be further processed for freezing.
3. Washing: Fixed tissues should be thoroughly washed to remove excess fixative, which can interfere with staining.
4. Freezing
5. Cryostat Use: As with fresh tissues, fixed tissues can also be sectioned using a cryostat.
6. Embedding: OCT embedding may help stabilize the tissue during sectioning.
7. Sectioning and Staining
8. Sectioning: Similar techniques apply, with sections cut and mounted on slides.
9. Staining: Immediate staining is crucial for diagnostic evaluation.
10. Freeze-Drying

Purpose

• Long-Term Preservation: Freeze-drying (lyophilization) is primarily used for preserving biological samples and maintaining structural integrity and biochemical properties for extended periods.
• Storage and Transport: It allows for easier storage and transport of sensitive biological materials.

Process

1. Preparation
2. Sample Selection: Choose tissues or biological samples suitable for freeze-drying.
3. Pre-Treatment: Samples may be pre-frozen in liquid nitrogen or at -80°C.
4. Freezing
5. Rapid Freezing: Freeze the samples quickly to prevent ice crystal formation, similar to the frozen section technique.
6. Sublimation
7. Primary Drying: In a vacuum chamber, the frozen samples undergo sublimation, where ice converts directly to vapour, bypassing the liquid phase. This is done at low temperatures and reduced pressure.
8. Secondary Drying: Further drying occurs to remove any residual moisture, ensuring the sample is completely dry.
9. Storage

• Vacuum-Sealed: Freeze-dried samples are often stored in vacuum-sealed containers or desiccators to prevent moisture reabsorption.

Applications

1. Frozen Sections: Used primarily in intraoperative settings, tumour margin assessments, and specific immunohistochemical analyses.
2. Freeze-Drying: Useful in research, vaccine development, and preservation of tissue samples for later use in various analyses.

Advantages and Disadvantages

Advantages

• Frozen Sections:
  • Rapid turnaround time for diagnoses.
  • Preservation of cellular morphology and antigenicity.
• Freeze-Drying:
  • Long-term preservation of biological specimens.
  • Reduced weight and volume for storage.

Disadvantages

• Frozen Sections:
  • Limited to small tissue samples and may have artifacts due to freezing.
  • Requires specialized equipment and trained personnel.
• Freeze-Drying:
  • Potential loss of some biochemical properties during the process.
  • More complex than simple freezing.