Study Notes on Compound Fixatives and Transport Solutions

Fixation of Tissues Part 2

Compound Fixatives

Introduction

  • Compound fixatives: These are combinations of simple fixatives that work synergistically to mitigate negative effects from individual components.

  • Morphologic preservation: The combination of formalin and osmium tetroxide fixation is known to create a state resembling life, essential for accurate tissue evaluation.

  • Staining methods: Most are validated for formalin-fixed, paraffin embedded tissue (FFPE), necessitating considerations for evaluating slides fixed with compound fixatives.

  • Safety regulations: Adhere to safety protocols relevant to each individual component of compound fixatives.

Types of Compound Fixatives

Mercury-Based Fixatives

  1. B-5 Fixative

    • Components: Mercuric chloride, sodium acetate, distilled water.

    • Working solution: Combine stock with formaldehyde in a ratio of 10:1.

    • Applications: Suitable for hematopoietic or lymphoreticular tissues (e.g., bone marrow, lymph nodes).

    • Advantages: Provides superior nuclear detail compared to formalin; excellent for identification of many antigens (especially for immunohistochemistry).

    • Disposal: Must remove mercury pigment using iodine followed by sodium thiosulfate.

    • Hazards: Mercury is toxic; consider zinc as an alternative.

  2. Zenker and Helly Fixatives (Zenker-Formol)

    • Stock Solution: Contains mercuric chloride, potassium dichromate, sodium sulfate, and distilled water.

    • Working Solutions:

      • Zenker working: Combine stock with glacial acetic acid (to lyse red blood cells, enhances nuclear staining).

      • Helly working: Combine stock with formaldehyde (must be prepared fresh, unstable in storage).

    • Challenges: Care must be taken to process tissues properly to prevent the formation of chrome pigments from potassium dichromate; retain quality by washing tissues before dehydration.

    • Use Cases: Indicated in methodologies for staining muscle striations, such as Mallory PTAH staining.

    • Limitations: Heavy metals may interfere with silver staining; susceptible to overhardening if tissues are not handled properly.

Chrome Based Fixatives

  1. Orth Fixative

    • Components: Potassium dichromate, sodium sulfate, and water.

    • Working solution: Combine stock with formaldehyde right before application.

    • Indications: Preferred for the demonstration of chromaffin granules in adrenal medulla cells (useful for diagnosing pheochromocytoma).

    • Concerns: Potassium dichromate necessitates careful washing of tissues before dehydration to avoid chrome pigment formation; not suitable for long-term storage.

Aldehyde Based Fixatives

  1. 4CF-1G Fixative

    • Components: Formaldehyde, glutaraldehyde, sodium hydroxide, phosphate buffer.

    • Indications: Serves dual purposes - primary routine processing and stability for secondary electron microscopy (EM) processing lasting up to 3 months at 4°C.

    • Considerations: Lower glutaraldehyde concentration minimally interferes with periodic acid-Schiff (PAS) staining; note potential impacts on immunohistochemistry due to crosslinking effects.

  2. Davidson Fixative

    • Components: 95% ethanol, 10% neutral buffered formalin, glacial acetic acid, distilled water.

    • Applications: Used for eye and testes specimens.

    • Properties: Balances the shrinkage from alcohol with the swelling from acetic acid, facilitating microtomy.

    • Precautions: Prolonged fixation of eye tissue may lead to excessive swelling artifacts; following fixation, transfer tissues to 70% alcohol or 10% neutral buffered formalin (NBF).

Aqueous Compound Fixatives

  1. Picric Acid Based Fixatives

    • Bouin's Fixative:

      • Components: Picric acid, formaldehyde, glacial acetic acid.

      • Effects: Lyses red blood cells, dissolves iron and calcium deposits, and can produce formalin pigment.

      • Considerations: If not removed, picric acid deteriorates staining abilities over time. Yellow coloration removal requires 50%-70% alcohol or a blend with lithium carbonate.

      • Limitations: Unsuitable for ultrastructural studies or nucleic acid preservation; transfer to 70-80% alcohol for long-term storage.

      • Best Uses: Excellent for gastrointestinal (GI) tract and endocrine tissues.

    • Gendre Fixative:

      • Components: Saturated alcoholic picric acid, formaldehyde, glacial acetic acid.

      • Props: Excellent for glycaemic preservation due to the alcoholic solution.

      • Removal of yellow color: It is advised to wash the tissue with 80% alcohol.

    • Hollande Fixative:

      • Components: Picric acid, copper acetate, formaldehyde, acetic acid, and distilled water.

      • Advantages: Suitable for GI biopsies; better stabilizing effects for red blood cells as compared to Bouin's.

      • Cautions: Must wash out from tissues before processing to avoid precipitation of salts and insoluble precipitates.

    • Zamboni Fixative:

      • Components: Saturated aqueous picric acid, paraformaldehyde, phosphate buffer, and distilled water.

      • Identity: Also called buffered picric acid-formaldehyde (PAF).

      • pH Level: Target a final pH of 7.3.

      • Availability: While stable, not commonly used; effective general fixative for both standard and electron microscopy applications.

Non-Aqueous Compound Fixatives

  1. Carnoy's Fixative

    • Components: Absolute ethanol, chloroform, glacial acetic acid.

    • Effects: Rapid action; not recommended for fixation longer than 4 hours; excellent for cytological specimens due to RBC lysis.

    • Advantages: Preserves glycogen, promotes good nuclear preservation, and extracts lipids.

    • Drawbacks: Causes pronounced shrinkage and hardening of tissues; chloroform is a carcinogenic agent requiring fume hood usage.

  2. Clarke's Fixative

    • Components: Absolute alcohol, glacial acetic acid.

    • Benefits: Historically significant; excellent for microanatomical preservation for light microscopy applications.

Transport Solutions

Michel Transport Medium

  • Components: Citric acid, ammonium sulfate, N-ethylmaleimide, magnesium sulfate, distilled water.

  • Indications: Ideal for unfixed small tissues held for several days or transported over long distances; frequently used for kidney biopsies requiring fresh tissue (for immunofluorescence or subsequent fixation).

  • Storage: Maintain pH between 7.0-7.2 for physiological relevance to ensure specimen viability.

PBS – 10% Sucrose

  • Composition: Phosphate-buffered saline, sucrose, distilled water.

  • Applications: Suitable for holding small fresh tissue pieces intended for immunofluorescence studies.

References

  • Carson, F.L., Cappellano, C. (2020). Histotechnology, A Self-Instructional Text (5th ed.). ASCP Press.