Comprehensive Notes on Tissue Fixation 18

Tissue Fixation

This lecture discusses tissue fixation, its importance, and how it can lead to false results that impact clinical and research outcomes. It covers the steps from the operating table to microscope visualization, emphasizing the need to preserve tissues in a lifelike state.

Types of Specimens

Specimens include:

  • Cytological specimens: urine, cervical smears, fine needle aspirations

  • Tissue samples: punch needle biopsies, organs, amputation specimens

Tissue Fixation: The Cornerstone

Tissue fixation is critical. Without it, tissues degrade due to bacterial growth, putrefaction, and autolysis (self-destruction by enzymes).

  • Autolysis: Destruction of tissues by their own enzymes

Proper fixation is essential for accurate diagnosis, prognosis, and treatment because it allows for:

  • Hematoxylin and eosin (H&E) staining

  • Special stains

  • Immunostaining

  • Molecular analysis

Fixation prevents bacterial growth, shrinkage, expansion, and external artifacts. It hardens tissues, making them easier to handle, and must be cheap, available, and provide optical density for detailed visualization.

Examples of Well-Fixed vs. Poorly-Fixed Tissues

  • Lobular carcinoma of the breast: Shows nuclear detail and stromal tissue.

  • Squamous cervical cancer: Demonstrates differences between normal and cancerous cells.

  • Kidney: A well-fixed kidney shows clear glomeruli and convoluted tubules; a poorly fixed kidney lacks detail due to autolysis.

  • Brain tissue: Delayed fixation results in degraded, out-of-focus specimens.

  • GI tract and pancreas: Autolysis obscures cellular details like goblet cells and exocrine cells.

Autolysis vs. Necrosis

Distinguishing between autolysis and necrosis is crucial. Necrosis in tumors indicates poor prognosis due to nutrient supply and waste removal issues. Autolysis, resulting from poor fixation, can mimic necrosis, making differentiation difficult.

Impact on Biomarker Analysis

Proper fixation is essential for accurate biomarker analysis. Delayed fixation can lead to false negative results, affecting patient treatment decisions.

  • Estrogen receptor example: Delayed fixation in breast tissue samples reduces estrogen receptor staining, potentially denying life-saving treatment to patients. Tamoxifen is a treatment option that could be withheld due to improper specimen fixation leading to false negative results.

Achieving Optimal Fixation

Optimal fixation balances various needs. Over-fixation can impede staining and molecular biology techniques. The primary target is protein cross-linking, which stabilizes tissues.

  • Proteins are cross-linked to form a gel, maintaining tissue structure. Soluble proteins link to structural proteins, providing mechanical strength.

Historical Context

Tissue preservation dates back to the Egyptians, who used natural chemical salts for embalming. Persians and Syrians used honey and wax. The 19th century saw the development of synthetic dyes, enhancing understanding of tissue preservation. Virchow was a leading histologist.

Classification of Fixatives

Fixatives were initially classified as coagulant or non-coagulant. Later classifications include:

  • Aldehydes: Most common fixative (e.g., formaldehyde) all non-coagulant.

  • Oxidizing agents. eg osmium tetroxide. non-coagulant.

  • Protein denaturing agents: Primarily for cytological samples (e.g., acetone, alcohol). coagulant.

  • **Physical heats of microwaves **: Example of fixation method is cooking a steak.

  • Miscellaneous: Mercuric chloride, acids.

Aldehydes: Formaldehyde/Formalin

Formaldehyde is a gas diluted in water to create formalin (40%), then further diluted to 10% (4% formaldehyde) for lab use. It's buffered with salts to maintain osmotic balance.

NH2NH_2 groups on amino acids cross-link via a methylene bridge. Water precipitates out during this process. Acidic groups are unaffected, while basic groups (proteins) are blocked, reducing the affinity for acid dyes.

  • Lipids: Preserved but not fixed; they wash out during processing.

  • Carbohydrates: Glycogen can be visualised if closely associated with protein.

Glutaraldehyde

Useful for transmission electron microscopy (TEM), especially for nerve fibres. Lipids, such as myelin, can be preserved with osmium tetroxide.

Oxidizing Reagents: Osmium Tetroxide

Reacts with lipids, preserving the myelin sheath for detailed nerve examination via electron microscopy. Brain/Neuropathology centers often utilize electron microscopy.

Potassium Dichromate

Converts proteins to a gel. While DNA gets dissolved out, it can be used for pheochromocytoma tumours, where noradrenaline oxidised to a brown color, enabling visualization without staining.

Protein Denaturing Agents: Acetone and Methyl Alcohol, Ethyl Alcohol

Used mainly for cytological material. They precipitate proteins and dissolve lipids. They are quick and have relatively low toxicity but can cause tissue shrinkage. Alcohols in tissue processing act as secondary fixatives.

Temperature in Fixation

  • Heat: Speeds up chemical reactions. Heat fixation can be used for rapid specimens but can cause morphological changes. Microwave fixatives use controlled heating alongside chemical fixatives.

  • Cold: Useful for interpretive specimens Electron cryo-microscopy preserves tissue. Freezing is used for intraoperative specimens.

Mercury Chloride

A highly toxic secondary fixative that enhances morphology but prevents immunostaining and molecular biology. Mercury pigment may occur in tissue blocks when using mercury chloride.