Tissue Processing and Histology Techniques

Tissue Processing

• Haematoxylin and Eosin (H&E)-stained sections of formalin-fixed tissue are most commonly examined for histologic studies with a light microscope.
• There are 10 steps in tissue processing: receipt and identification, labeling, fixation, dehydration, clearing, infiltration, embedding, sectioning, staining, and mounting.

Automated Tissue Processor

• An automated tissue processor contains 12 stations.
• These stations include 1 jar of formalin, 7 jars with grades of alcohol, 2 jars of xylene, and 2 jars of molten paraffin wax.

### Fixation

• Fixation prevents tissue decomposition by terminating cell metabolism, preventing enzymatic degradation, killing microorganisms, and hardening the tissue.
• The most common fixative is formalin, a 37% aqueous solution of formaldehyde.

### Dehydration

• Involves removing water from tissue by passing it through increasing concentrations of alcohol.
• The process takes 7 hours, with 1 hour in each of the following solutions: 50% alcohol, 60% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, absolute alcohol, and absolute alcohol.
• Dehydration ensures the paraffin wax used for infiltration can easily mix with the tissue as it is not miscible with water.

Clearing

• This process removes the alcohol from the tissue and replaces it with a fluid that mixes with paraffin.
• Clearing agents penetrate the tissue, make it transparent, and harden it.
• Xylene is the most commonly used clearing agent.

Infiltration and Embedding

• Once clearing agents are removed, molten paraffin wax is used to fill the empty spaces in the tissue for 6-8 hours.
• The paraffin wax hardens the tissue and makes it easier to section.
• The tissue embedded in wax is transferred to a mould filled with molten wax, allowed to solidify, and then sectioned to obtain ribbons.

Sectioning

• Sectioning involves cutting the wax blocks into thin strips of varying thickness using a microtome.

Routine Staining (H&E)

• Staining highlights specific tissue details.
• The most commonly used stain is Haematoxylin and Eosin (H&E).
• Haematoxylin stains nuclei blue as it is a basic dye that stains acidic structures.
• Eosin stains cytoplasm pink as it is an acidic dye that stains basic structures.

Histology

• Histology is the study of tissues and organs using a microscope.
• It is often referred to as microscopic anatomy and involves the study of the detailed structure of tissues and organs using a light microscope or electron microscope (EM) (Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)).
• The word "histology" comes from the Greek words "histos" (tissue) and "logia" (science).

Cytology

• Cytology is the study of the structure and function of the cell.

Levels of Organization

• Cells are the smallest living units of the levels of organization.
• Tissues are comprised of many cells and an extracellular ground substance.
• The four basic types of tissues are: epithelial, connective, muscular, and nervous.
• Organs are made up of different tissues and have a specific shape, structure, and function.
• Systems are groups of organs that perform related functions.