Unit 2 histology

Stages of Tissue Processing

• Includes key processes essential for histology:

  • Fixation

  • Dehydration

  • Clearing

  • Infiltration

  • Embedding

Gross Examination

• Performed by:

  • Pathologist

  • Pathologist assistant

  • Technologist

Process of Gross Examination

• Description of specimen and cutting into thin pieces about 3 to 5 mm thick.

• Specimens are placed into labeled cassettes.

• The gross room requires:

  • Sufficient size for safe work

  • Adequate illumination and ventilation

  • Exhaust fan to remove formalin vapors.

• Small specimens must be wrapped in lens paper or placed between sponges to prevent falling through cassettes.

• Cassettes enclose tissue and have holes for processing reagents to enter.

• Specimens suspected of malignancy are marked with colored ink for clarity during examination.

Decalcification

• Required for tissue containing calcium salts (e.g., bone, kidney).

• Essential because untreated tissues result in poor processing outcomes.

  • Torn sections and compromised microtome cutting edges.

• Tissue must be adequately fixed before decalcification to avoid cell morphology deterioration.

• Over-decalcification leads to tissue distortion and poor staining quality.

• Recommended practice involves checking tissue every 2-3 hours in decalcifying fluid with a ratio of 50 to 100:1 (fluid to tissue).

Decalcifying Agents

• Formic acid:

  • A weak organic acid (25% solution).

  • Gentle on tissues but slow.

• Gooding Stewart’s Fluid:

  • A combination of formic acid and formalin.

• Nitric acid or HCl:

  • Strong inorganic acids, can cause tissue damage, monitor carefully.

• EDTA:

  • A chelating agent, slow but gentle (may take several weeks).

End-Point of Decalcification

• Over-decalcification can irreversibly damage specimens.

• Detection methods include probing tissue pliability, chemical testing for calcium content in the decalcifying solution, and X-ray inspection.

Dehydration (Tissue Processor)

• Controlled removal of water to allow tissue infiltration with wax.

• Alcohol series progression:

  • 70% Ethanol

  • 85% Ethanol

  • 95% Ethanol

  • Absolute (100%) alcohol.

• Gradual water removal prevents shrinkage and distortion.

• Alternative dehydrators:

  • Acetone: Fast but poses fire hazards.

  • Dioxane: Toxic fumes; typically used without clearing.

Clearing (Tissue Processor)

• Removal of the dehydrating agent (ethanol) using a miscible agent for paraffin wax embedding.

• Common agents include:

  • Xylene

  • Toluene

• Any residual water in tissue leads to cloudiness post-clearing.

Wax Impregnation (Tissue Processor)

• Replacement of clearing agent with embedding medium (paraffin wax).

• Temperature: 56-60°C for melting wax.

• Duration: Tissues immersed for 3-4 hours.

• Paraplast as common reagent, a mixture of paraffin wax and plastic polymers.

Tissue Processor Steps

• Sequential steps include fixation, dehydration, clearing, and wax infiltration.

• Maintenance and reagent changes according to usage and guidelines are essential for accurate results and diagnosis. MLTs/MLATs are responsible for reagent changes.

Embedding

• The process surrounds tissues in a support medium for safe sectioning.

• Preceding steps ensure correct tissue orientation in solidification medium.

• Importance of proper alignment:

  • Misplacement can lead to loss of diagnostically relevant tissue areas during microtomy.

Embedding Agents/Media

• Paraplast: Common embedding medium, provides elasticity, reduces damage during cutting.

• Resins: For delicate tissues; enhance structural integrity when embedding.

• Cellulose: Used with electron microscopy.

Microtomy

• Cutting embedded tissues for slide preparation:

  • Utilizes microtome with sharp disposable blades.

  • Sections typically range from 3 to 5 microns.

• Tissue sections floated on water bath (40-45°C) before slide placement.

• Proper adherence techniques:

  • Charged slides enhance tissue adherence.

Sectioning and Staining

• Tissue deparaffinization or rehydration is necessary prior to staining.

• Common Stain: Hematoxylin and Eosin (H&E)

  • Other special stains used depending on diagnostic requirements.

• Steps for Hematoxylin staining include:

  1. Staining times based on the type of Hematoxylin.

  2. Over-staining followed by differentiation via acid-alcohol.

Final Staining Procedure

• Rehydration steps include:

  1. Changes of xylene to 100% and 95% ethanol, then to deionized water for staining.

• The staining can be regressively or progressively applied.

• Final results display:

  • Hematoxylin coloring nuclei blue/purple and Eosin stains cytoplasm red/pink.

Cover Slipping

• Critical for preserving tissue sections; protects and enhances viewing under the microscope.

• Steps include placing mounting media and ensuring no air bubbles under coverslip.

• Methods of cover slipping include manual and automated options.

  • Automated systems protect laboratory workers from fumes and enhance efficiency.

Frozen Sections – Cryostat Microtomy

• Used in urgent scenarios (e.g., surgical rooms).

• Tissue is frozen quickly for immediate analysis.

• Provides rapid results while preserving cellular structure during analysis.

Common Issues in Staining

• May relate to improper procedures—such as incomplete staining or excessive differentiation leading to inadequate cellular visibility.

• Quality checks essential to ensure proper outcomes for histopathological examinations.

Conclusion

• Tissue processing, including all outlined steps, is critical for accurate diagnostics in pathology. Consistent adherence to procedures and techniques is necessary to ensure proper outcomes in histological evaluations.