Histopathology Tissue Processing

• These notes cover the introduction to tissue processing in histopathology and cytology, including surgical procedures for obtaining tissue, methods of fresh tissue examination, conventional tissue processing steps, factors affecting tissue processing, and methods of freezing.

Introduction to Tissue Processing

• Examination of tissue sections and smears is a better and more effective method of studying tissues.
• Tissues must be permanently preserved, stained, and mounted on glass slides with cover slips.
• The entire process is referred to as tissue processing.

Surgical Procedures to Obtain Specific Types of Tissue

• Various surgical procedures are used to obtain tissue specimens for examination.
• These procedures each have specific applications and methods.

Core Needle Biopsy

• Removes cells and a small amount of surrounding tissue.
• Offers additional information to assist in lesion examination.

Curetting

• Tissue scooped or spooned to remove tissue or growths from a body cavity.
• Common tissues are the endometrium or cervical canal.

Excision Biopsy

• Removes the entire area in question (entire lesion).

Fine Needle Biopsy

• Uses the smallest needle to remove cells from the area of abnormality.
• Sometimes not adequate to obtain a diagnosis, depending on the biopsied area.

Incisional Biopsy

• Only a portion of tissue is removed from the lesion.
• Further surgery is needed to remove the remaining cancerous tissue.

Shave Biopsy

• Small tissue fragments are "shaved" from the surface.
• This is usually done in the skin with either a small scalpel blade or a curved razor blade

Punch Biopsy

• The primary technique for obtaining a full-skin specimen requires surgical and suture-tying skills.
• A Yield size of an estimated 3-4 mm thanks to the circular blade that perforates the epidermis-dermis-subcutaneuous fat

Specimen Received for Processing

• Specimens can be preserved or fresh.
• Formalin: fixative used for preserving
  • Advantage - permanent

Fresh Specimens

• Advantage: Examination in living state
• Disadvantage: Not permanent, develop changes
• Enables observation of protoplasmic activities like motion, mitosis, and phagocytosis.
• Used for frozen sections for immediate microscopic evaluations.

Methods of Fresh Tissue Examination

Teasing and Dissociation

• Selected tissue specimen immersed in isotonic solution (NSS or Ringer's).
• Needle used to dissect tissue, and applicator stick used for tissue separation.
• The selected pieces of the tissue carefully transferred to a microscope slide and mounted as a wet preparation.
• Visualization achieved through wet preparation with supravital stain or unstained using phase contrast or brightfield microscopy.
• Advantage: Cells can be examined in the living state
• Disadvantage: Not permanent

Squash Preparation

• Small pieces of tissue are compressed between a slide and cover glass, by crushing, and then observed.
• Visualization: supravital stain is placed where a slide and cover glass meet, and then absorbed through capillary attraction

Smear Preparation

• Cellular materials spread lightly over the slide for examination.
• Platinum loop or appositional second slide
• Examination either fresh or by supravital stain.
• Useful for cytological examinations and cancer diagnosis.

Streak Method

• Applicator stick or platinum loop to spread material rapidly and gently in a direct or zigzag line on the slide; creating uniform sample distribution.

Spreading Method

• Selected material transferred to a clean slide and spread into a moderately thick film.
• Tease the mucous strands apart with an applicator stick.
• Advantage: Maintains cellular interrelationships
• Disadvantage: More tedious than streaking
• Recommended for: fresh sputum, bronchial aspirates, and thick mucoid secretions

Pull-Apart Method

• Involves placing a drop of secretion or sediment on one slide and facing it to another clean slide.
• The material is evenly dispersed over the surface of two slides.
• Slides slide in opposite directions until materials are separate in a single uninterrupted motion
• Used for immediate examination with or without vital stain

Touch Method

• Impression Smear
• Surface of freshly cut tissue brought into contact and pressed on clean slide.
• Visualization:
  • Unstained for phase contrast microscope
  • Stained for brightfield microscope
• Advantage: Maintains the intercellular relationship of cells

Conventional Tissue Processing

• Tissue processed for section cutting on a microtome.
• Steps:

Numbering

• Each specimen given an accession number
• Request form should be complete and have
  • Patient Information
  • History
  • Diagnosis
  • Site Of Origin For The Tissue Submitted
• Reject specimen if unlabeled, or if incomplete information on the requisition form

Fixation

• Immerse in a liquid fixing agent (Formalin/ formaldehyde)
• Tissue must be preserved and maintain its life-like state
• Fixative volume should be 10-20x more than the specimen

Decalcification (Optional)

• Process to remove calcium salts from the specimen, depending on the specimen.
• Agents: acids

Dehydration

• Reduce water content in the tissue to allow infiltration with wax
• Pass the tissue through increasing concentrations of dehydrating agents
• Ethyl alcohol
  • Removes water soluble proteins at low concentrations
  • DISSOLVES CERTAIN LIPIDS when Ethanol (100%) is increased

Clearing

• Dehydrated tissue transferred to a solvent miscible with ethanol and paraffin wax.
• Clearing agents impart transparency due to high refractive index
• Removes fat from tissue
• Xylene: Common because miscible with ethanol and paraffin wax

Impregnation/Infiltration

• Tissue infiltrated with paraffin wax + additives
• Keeps tissue in wax bath with molten paraffin
• Allows thin sectioning

Embedding

• Transfer the tissue to a mold filled with molten wax, cool, and solidify.
• Orientation of the block in mold is most important
• Place oriented tissues to the half-filled paraffin, before covering it with molten wax

Section Cutting

• Blocks are cut or sectioned and thin strips of varying thickness are prepared
• Use of microtome
• Specimens:
  • H&E: cut 3-5 µm thick
  • Amyloid deposits: cut 8-12 µm thick
  • Kidney biopsies: cut 2 µm thick

Floating Bath

• Place sections "floated out" on water in floatation bath to flatten them
• Use slides to pick it out, before thorough drying for staining

Staining

• Bring out the particular details of the tissue under study
• Haematoxylin and eosin stain: most common

Mounting

• Use adhesives to fix sections to slides
• Apply a mounting medium (syrupy fluid, viscous) between section and coverslip after staining , setting the section firmly, preventing movement of the coverslip.
• The mounting medium helps maintain easy storage, preserving of slides and image against distortion

Factors Affecting Processing Duration

• Tissue density and thickness: thicker tissues take longer
• Agitation: Increases fluid flow through tissue/cassettes
• Temperature:
  • 37-45°C for limited time can speed up fluid penetration and tissue processing protocols
  • High temperatures: will SHRINK and make materials hard and brittle
  • Low temperature: ↓ viscosity = ↓ rate of diffusion = ↑ processing time
• Pressure lowers the time for processing; improves with fatty and dense tissue

Frozen Section

• Rapid diagnosis of a pathologic process*
• Use microtome with CO2/cold chamber (cryostat) at -10 to -20°C.
• For lipids and nervous/muscles tissue
• Advantages:
  • less ventilation
  • rapid time