Sarah Hughes - Fresh Tissue and Additional Testing

Fresh Tissue and Additional Testing

Learning Objectives

  • Fresh Tissue Handling: Understanding how to deal with fresh tissue samples.
  • Cryotomy Principles: Knowing the principles behind cryotomy.
  • Reasons for Receiving Fresh Tissue: Understanding why fresh tissue samples are received in the lab.
  • Techniques for Fresh Tissue: Identifying techniques that can only be performed on fresh tissue.
  • Intra-operative Frozen Sections: Understanding the purpose of intra-operative frozen sections.
  • Tissue Freezing: Knowing how tissues are frozen and the effects of freezing on the tissue.
  • Frozen Section Production: Explaining the steps involved in producing a frozen section.
  • Frozen Section Advantages/Disadvantages: Listing the advantages, disadvantages, and risks associated with frozen sections.

Fresh Tissue Overview

  • Previous Lecture Reference: Previous lecture covered FFPE block production.
  • Focus: Current lecture focuses on fresh tissue and its handling.
  • Key Question: Primary difference between fresh and FFPE tissue processing?
  • Specimen Types:
    • Intra-operative frozen sections.
    • Breast resections.
    • Paediatric tumors.
    • Muscle biopsies.
    • Rectal biopsies.
    • Mohs surgery specimens.

Why Fresh Tissue?

  • Procedure-Driven: Fresh tissue handling is often dictated by specific procedures.
  • Lab-Controlled Fixation: Allows the lab to control the fixation process.
  • Additional Pre-treatment: Enables additional pre-treatment steps (e.g., for breast tissue).
  • Correct Fixation: Ensures correct fixation for specific analyses.
  • Additional Tests: Facilitates additional tests that require fresh tissue.

Intra-operative Frozen Section

  • Context: Patient is under anesthesia in the operating theater.
  • Purpose: Assessment of resection margins or unexpected nodules.
  • Turnaround Time (TAT): Critical TAT of 20 minutes from receipt in the laboratory.
  • Priority: Considered the most urgent specimen in the department.

Frozen Section Procedure

  1. Specimen Reception: Specimen is received in the lab and booked in.
  2. Pathologist Examination: Pathologist examines the specimen.
  3. Sampling: Area of concern is sampled.
  4. Rapid Freezing: Rapidly frozen onto a freezing medium.
  5. Sectioning: Sections are cut using a cryostat.
  6. Staining: Sections are stained.
  7. Reporting: Pathologist reports the findings.
  8. Fixation and Processing: Specimen is then fixed and processed to paraffin for permanent storage.

Specimen Arrival

  • Specimen arrives in the lab fresh and unfixed.

Freezing Methods

  • Orientation: Specimen may need orientation prior to freezing.
  • Embedding: Embedded on a freezing media.
  • Freezing: Freeze, the method depends on the specimen type and desired freezing rate.

Standard Freezing - Cryobar

  • Common Method: Most common freezing method.
  • Speed: Quick freezing method.
  • Temperature: Usually -20°C to -40°C. Cryobar in cryostat often set to -50°C.
  • Cost: Cheap and easy to use

Liquid Nitrogen (N2) Freezing

  • Temperature: -196°C rapid freezing on contact.
  • Storage: Used to store some fresh specimens.
  • Boiling: Boils on contact with the specimen.
  • Risks: Asphyxiation and freeze burns are potential risks.

Cryospray - Mohs Technique

  • Application: Specifically used for Mohs surgery specimens.
  • Process: Tissue is frozen onto a glass slide.
  • Orientation: Allows for orientation purposes.
  • Embedding: Inverted onto a chuck to embed onto freezing medium

Freezing Effects

  • Slow Freezing:
    • Results in large hexagonal ice crystals.
    • Intracellular ice crystals damage cell membranes.
  • Rapid Freezing:
    • Results in small cubic ice crystals.
    • Causes less distortion of the cell membrane.
  • Ice Crystal Artifact: Ice crystal formation can lead to artifacts in the tissue.
  • Cooling Rate: Rate of cooling depends on the rate of conduction.
  • Conduction Rate: Rate of conduction depends on the temperature difference between the tissue and the coolant.

Ice Crystal Artifact

  • Image provided showing ice crystal artifacts at 50 μm scale.

Cryotomy

  • Cryotomy Sectioning diagram with labels:
    • UV-C
    • Freeze shelf
    • Anti-roll
    • Cryobar/Peltier element
    • Chuck Holder
    • Knife guard and knife holder
    • Knife release

Staining

  • H&E Staining:
    • Hand stained.
    • Rapid technique.
    • Regressive staining is employed.
    • Uses Harris’ Hematoxylin.
    • Aims to be as close to paraffin staining as possible.

Mohs Surgery

  • Gold Standard: Gold standard for Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC).
  • Process:
    • Patient arrives in clinic.
    • Surgeon takes a small amount of cancerous tissue.
    • Sections are cut and examined.
    • If margins are positive, further layers are taken.
    • When patient is clear, reconstruction is performed on the same day.

Paediatric Tumors

  • Rarity: Relatively rare.
  • Wilms Tumor:
    • An aggressive renal tumor.
    • Occurs in childhood (1-4 years).
  • Mirror Sections:
    • Frozen sections and duplicate H&Es sent to specialist centers for diagnosis/confirmation.

Paediatric Tumours - Neuroblastoma

  • Urgency: Very urgent.
  • Description: Aggressive tumor arising from autonomic ganglia in the abdomen.

Rectal Biopsies

  • Application: Particularly in paediatric biopsies.
  • Diagnosis: Diagnosis of Hirschsprung’s disease.
  • Hirschsprung’s Disease:
    • Absence of ganglion cells in the anorectal region.
    • Diagnosis from rectal biopsy via Calretinin staining.
  • Pull-Through Procedure:
    • Aganglionic (diseased) section is removed.
    • Ganglionic (healthy) section is pulled through to the anus allowing normal bowel movements

Muscle Biopsies

  • Freezing: Frozen in isopentane.
  • Techniques: Used for EM (electron microscopy), EHC (enzyme histochemistry), and IHC (immunohistochemistry).
  • Necessity: Only demonstration method for certain conditions.
  • Complexity: Very complex disorders are investigated.

Advantages of Fresh Tissue Analysis

  • Quick turnaround time.
  • Cost-effective.
  • Compatible with other techniques.
  • Only method for demonstration of some methods (e.g., oil red o for fat).
  • Diagnostic sections can be rapidly produced.

Disadvantages of Fresh Tissue Analysis

  • Time pressure due to the urgency of diagnosis.
  • Morphology is often not as good as FFPE.
  • Storage can be difficult.
  • Difficult to cut due to the nature of frozen tissue.
  • Artifacts are common.

Risks and Hazards

  • Burns (cryogenic).
  • Sharps (knives and blades).
  • Biological hazards.
  • Chemical hazards.

Summary

  • Many specimens are received fresh, with intraoperative frozen sections being the most common.
  • Freezing is a delicate process to prevent damage to the tissue.
  • Cutting is based on microtomy, but at cold temperatures.
  • Many complex conditions are diagnosed using fresh tissue techniques.

Case Study 1 - Mohs Surgery

  • Presentation: Patient with nodule on temple.
  • Diagnosis: Punch biopsy confirmed Basal Cell Carcinoma (BCC).
  • Procedure: Mohs surgery performed.
  • First Layer: First layer taken with debulking.
  • Second Layer: Second layer taken with significantly more tissue to ensure clear margins.
  • Outcome: Specimen was not clear, requiring a slow Mohs procedure the following day.

Case Study 2 - Renal Biopsy

  • Patient: 18-year-old female with reduced renal function.
  • Symptoms: Elevated creatinine and C-reactive protein.
  • Procedure: Renal biopsy taken, with a portion used for immunofluorescence.
  • Immunofluorescence: Detects presence of antigens or immunoglobulins in the kidney.
  • Panel: IgG, IgA, IgM, IgG, C3, C1q, fibrinogen, λ light chains, and κ light chains.
Immunofluorescence Results
  • IgG: Shows buildup of plasma cells common in acute and chronic renal dysfunction.
  • IgA: Positivity indicates IgA nephropathy, potentially leading to blood in urine, swelling, and chronic kidney disease symptoms.

Case Study 3 - Pull-Through Procedure

  • Urgency: Specimen was clinically urgent.
  • Patient: 7-week-old boy with chronic constipation.
  • Diagnosis: Rectal biopsy showed no ganglionic cells.
  • Procedure:
    • Baby given general anesthesia (GA).
    • Specimens taken along the colon.
    • Serial sections cut for examination.
Surgical Procedure for Pull-Through
  • Diseased section (no nerve cells) of the large intestine and rectum is surgically removed.
  • Healthy section is connected to the anus to allow the child to have normal bowel movements.

NEQAS (National External Quality Assessment Scheme)

  • Submission: Submission of the first frozen section of the month every 2 months.
  • Schemes: Different schemes exist.
  • Mohs: Mohs scheme asks for different requirements to be demonstrated.
  • Focus: Frozen sections in general.

NEQAS standards

  • NEQAS Scoring:
    • 0 - Non submission
    • 1 - Fail: no staining demonstrated based of the method employed
    • 2 - Borderline fail: unsatisfactory demonstration based on the method employed, with expected staining results being inappropriate
    • 3 - Pass: appropriate demonstration based on the method employed and the expected staining results, although improvements need to be made in the staining
    • 4 - Good: good appropriate demonstration based on the method employed and the expected staining results
    • 5 - Excellent: excellent demonstration based on the method employed and the expected staining results

Quality Difference

  • Images showing good vs bad quality frozen sections at 50 μm scale.