Histopathology

LECTURE 1&2
INTRODUCTION TO TISSUE PROCESSING
aim- to embed tissue in a solid medium  Teasing or Dissociation: Tissue specimen is
 Histology is the microscopic study of the normal immersed in isotonic salt solution, carefully dissected,
tissues of the body. and spread using an applicator stick. Cells are
 histopathology is the microscopic study of tissues mounted as a wet preparation on a microscope slide,
affected by disease. either stained or unstained, for examination, allowing
 The procedures adopted for the preparation of observation of living cells. Preparations are not
material for such studies are known as histologic or permanent.
histopathologic techniques.  Squash Preparation (Crushing): Small tissue pieces
SURGICAL PROCEDURES TO OBTAIN THE are compressed between a microscope slide and
SPECIFIC-TYPES OF TISSUE: cover glass. Supravital stain may be used. This
 Fine needle aspiration method allows detailed examination but is also not
 is the simplest, permanent.
 least invasive test  Smear Preparation:
 uses the smallest needle to simply remove cells from o Streaking: Material is applied to a slide in a
the area of abnormality. zigzag line with an applicator stick or
 A core needle biopsy platinum loop for uniform distribution.
 removes not only cells, but also a small amount of the o Spreading: Material is transferred to a slide
surrounding tissue. and spread into a moderately thick film,
 An incisional biopsy maintaining cellular interrelationships,
 takes out even more surrounding tissue. It takes out  ideal for fresh sputum and thick
some of the abnormality, but not all. mucoid secretions.
 The doctor will slice into the lesion and remove only a o Pull-Apart: A drop of secretion is placed
portion of it. between two slides, then pulled apart,
 An excisional biopsy spreading the material evenly. The specimen
 generally removes the entire area in question. can be examined immediately or stained.
 Punch biopsy  Touch Preparation (Impression Smear): A freshly
 is considered the primary technique for obtaining cut piece of tissue is pressed onto a glass slide,
diagnostic full-thickness skin specimens transferring cells directly for examination.
 The technique involves the use of a circular blade that  This method preserves intercellular
is rotated down through the epidermis and dermis, relationships and is useful for phase
and into the subcutaneous fat, yielding a 3- to 4- mm contrast microscopy or staining.
cylindrical core of tissue sample. CONVENTIONAL TISSUE PROCESSING
 Shave biopsy This can be accomplished by preserving and carefully
 where small fragments of tissue are “shaved” from a processing solid structures and tissues in the following order:
surface (usually skin). 1. Numbering - complete request form
 Curettings lists the patient information, history and site of origin for the
 where tissue is scooped or spooned to remove tissue tissue submitted
or growths from body cavity such as reject if:
 endometrium or cervical canal. -unlabeled specimen
SPECIMEN RECEIVED PROCESSING -incomplete information in requisition form
-Incomplete specimen based on request form
 Specimens are usually received in fixative tissue accessioning
(preservative) but may also arrive fresh and require C: cytology specimen
immediate fixation. A: autopsy specimen
 Specimens are accessioned with a unique number to S: surgical specimen
ensure proper identification and minimize the risk of 2.Fixation - is crucial for preserving tissue specimens after
mislabeling. they arrive in the laboratory. Tissues are placed in a fixative,
 Autolysis occurs when tissues are removed from the commonly formalin, which hardens and preserves the tissue
body, breaking down proteins and cells through their by causing chemical and physical changes.
own enzymes, and is retarded by cold temperatures. 3. Decalcification (optional) - is the process of removing
 Fresh tissues allow for observation of living calcium salts from tissues, particularly bone, to soften them for
protoplasmic activities easier cutting and microscopic examination. This is typically
o not permanent and may undergo changes done using acidic solutions or chelating agents.
after death. 4. Dehydration - Water must be removed from tissue
o For frozen section specimens before paraffin wax infiltration, as wax is
 Preserved tissues, stained to demonstrate specific hydrophobic.
structures, are examined on glass slides mounted Ethanol Immersion: Specimens are immersed in ethanol
with coverslips for solutions of increasing concentration to gradually dehydrate
o permanent keeping. the tissue without causing excessive distortion.
o Volume – atleast 20 times Typical Dehydration Sequence for 4mm thick specimens:
o Formalin – common fixative  70% ethanol: 15 min
METHODS OF FRESH TISSUE EXAMINATION  90% ethanol: 15 min

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  100% ethanol: 15 min (repeated three times with 10. Staining - Tissue sections are nearly invisible under light
increasing duration) microscopy and require staining for contrast.
 To ensure complete dehydration, a superior fat Deparaffinization:
solvent such as acetone or isopropanol should be  Slides are dried at 60°C.
added before the final absolute ethanol, and  Xylene removes paraffin.
chloroform or trichloroethane used as the transition  Absolute alcohol removes xylene.
solvent. Rehydration: Prepares tissue for staining.
Staining Procedures:
5. Clearing - Dehydrated tissue is transferred to an  Use chemicals or dyes that bind to specific cell
intermediate solvent, fully miscible with both ethanol and components.
paraffin wax.  Different stains are used for different tissue
Clearing Agents: These agents provide optical clarity to the components.
tissue and remove fat, facilitating better wax infiltration. Contamination Risks:
Xylene Use: Tissue is typically immersed in xylene in a series  Deparaffinized tissue may fragment and contaminate
of steps to replace ethanol with xylene, which will later be staining solutions.
replaced by molten paraffin wax during embedding.  Changing staining fluids and baths frequently can
Typical Clearing Sequence for 4mm thick specimens: reduce contamination risk.
 Xylene: 20 min
 Xylene: 20 min 11. Mounting - The process of placing thin tissue sections
 Xylene: 45 min onto glass slides after they have been cut, typically from
paraffin blocks, to prepare them for microscopic examination.
6. Impregnation (Infiltration) - Cleared tissue is infiltrated Proper mounting ensures that the sections adhere to the
with histological wax, typically paraffin, which is liquid at 60°C slides, are free from wrinkles, and are ready for staining and
and solidifies at 20°C. analysis.
Wax Composition: Paraffin wax is mixed with additives like 12. Labeling
resins (e.g., styrene or polyethylene) to allow for thin AUTOMATIC TISSUE PROCESSING
sectioning on a microtome. This machine allows the specimens to be infiltrated with a
Microtome Sectioning: The wax allows sections to form sequence of different solvents finishing in molten paraffin wax.
ribbons that can flatten on a warm water bath. The specimens are in an aqueous environment to start with
Infiltration Sequence for 4mm thick specimens: (water-based) and must be passed through multiple changes
 Paraffin wax: 30 min of dehydrating and clearing solvents (typically ethanol and
 Paraffin wax: 30 min xylene) before they can be placed in molten wax.
 Paraffin wax: 45 min
Factors Impacting Tissue Processing and Infiltration:
7. Embedding - After wax infiltration, tissues are oriented in a 1. Tissue Density and Thickness: Denser and thicker
mold to determine the "plane of section" and ensure accurate tissues require longer processing times due to slower
sectioning. reagent diffusion, while spongy tissues process faster.
Critical Orientation: Proper orientation in the mold is crucial to 2. Agitation: Enhances fluid exchange around tissues
avoid missing or damaging diagnostically important tissue by keeping them in motion, preventing settling and
elements. ensuring maximum exposure to reagents.
Embedding Process: The mold is filled with molten wax, 3. Temperature: Higher temperatures speed up
solidified on a cold plate, and then removed as a solid tissue processing but must be controlled to prevent tissue
block ready for microtome sectioning. shrinkage or hardening, while lower temperatures
Mold Types: Stainless steel, ceramic, paper, plastic, and slow down diffusion.
aluminum foil molds can be used, depending on the 4. Vacuum and Pressure: Adjusting these settings can
microtome chuck. improve reagent infiltration and processing efficiency,
Double Embedding: Tissues are first embedded in a particularly in denser tissues
supporting medium (e.g., agar) and then in wax, useful for
handling small, delicate fragments like biopsies, ensuring
correct orientation, and preserving morphological detail for
histopathology.
8. Trimming - The process of cutting away excess tissue or
wax from an embedded tissue block to expose the area of
interest, making it ready for sectioning on a microtome. This
step ensures that only the relevant tissue is sectioned and
analyzed.
9. Section-Cutting (Microtomy) - Process of cutting
embedded tissues into thin sections using a microtome.
Section Thickness: Routine H&E sections are cut at 3-5 μm;
amyloid deposits at 8-12 μm; kidney biopsies at 2 μm.
Embedding Media: Paraffin for routine sections; epoxy or
acrylic resins for thinner sections and electron microscopy.
Microtome Knives: Standard metal or disposable blades for
paraffin; glass or diamond knives for plastic blocks and ultra-
thin sections.
Artifacts: Proper fixation and embedding minimize artifacts like
tearing, folding, or holes during sectioning.
Slide Preparation: Sections are floated on a warm water bath,
picked up on glass slides, and may be gently heated to
improve adhesion.

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