HISTOPATH Lab Mod 2

• Histopathology involves examining tissues to understand diseases and their root causes
• Pathologists diagnose diseases by examining small tissue samples through a process called biopsy
• Histopathologists study tissues processed by histotechnicians to come up with a diagnosis based on the patient's history
• The end goal of histopathology is to produce a quality tissue section for diagnosis
• Tissue preparation involves placing samples in fixative reagent for preservation and hardening
• Surgical tools like knives and forceps are used in the macroscopic examination of tissues by pathologists
• Tissue sections are placed in cassettes for further processing after fixation
• Tissue processing involves embedding tissue in wax for sectioning
• Embedding rings are used to hold and identify tissue sample blocks during processing
• Tissue sections are mounted on slides before staining
• A floatation/water bath is used to float paraffin ribbons and prepare tissue sections for mounting on glass slides
• Microtomes are used to cut thin sections of tissue samples for slide preparation
• Excess paraffin on slides or tissues is removed using a stretching table
• Coplin jars and staining dishes are used for staining tissue slides
• Automated stainers can perform staining protocols without supervision and ensure reproducible results
• In single-celled organisms, substances can easily enter the cell due to a short distance
• In multicellular organisms, the distance substances need to cross is larger because of a higher surface area to volume ratio
• Multicellular organisms require specialised exchange surfaces for efficient gas exchange of carbon dioxide and oxygen
• The rotary microtome requires the rotation of a fine advance hand wheel by 360 degrees to move the specimen vertically past the cutting surface and return it to the starting position
• Steps to operate the microtome:

  1. Clamping the specimen:

  • Rotate the hand wheel until the specimen clamp is in the uppermost section
  • Activate the hand wheel lock and pull the specimen holder forward
  • Mount the tissue block horizontally or vertically as required

  2. Clamping the knife/disposable blade:

  • Lower the knife guard on the knife holder
  • Rotate the clamping lever downwards (unclamped)
  • Carefully insert the blade from the side
  • Rotate the clamping lever upwards once the blade is in position (clamped)
  • Raise the knife guard into position once the blade is securely clamped

  3. Adjusting the clearance angle: Secure the blade at the appropriate clearance angle (5O-10O)

  4. Orienting the specimen:

  • Run the specimen to the rear end position by turning the coarse hand wheel
  • Loosen the clamping lever and slide the knife holder base until it is almost in front of the specimen
• Steps to operate the microtome (continued):

  5. Move the specimen holder to the upper end position by turning the hand wheel and engaging the hand wheel lock

  6. Trimming the specimen:

  • Use the coarse feed wheel for horizontal movement towards and away from the knife
  • Set a large sectioning thickness (e.g., 50u)
  • Trim the specimen by rotating the coarse hand wheel and hand wheel simultaneously until the desired plane is reached

  7. Sectioning:

  • Set the desired thickness using the adjustment knob
  • Use a different area of the cutting edge for trimming and sectioning
  • Turn the hand wheel evenly in a clockwise direction for sectioning
• Microtomes have a feed mechanism to advance the specimen or knife to a predetermined thickness for sectioning and can produce serial sections
• Microtome knives:

  • Steel knives: common, heat-treated for hardening, fully hardened for durability
  • Glass knives: sharp but fragile, used in cryotomes
  • Diamond knives: expensive, precision ground for thin sections, long lifespan
  • Disposable knives/blade: special holders in modern microtomes, replaced standard steel knives
• Knife profiles:

  1. Biconcave knife: recommended for paraffin-embedded sections

  2. Planoconcave knife: extremely sharp, used for different types of tissue blocks

  3. Plane-Wedge knife: recommended for frozen sections or tough specimens

  4. Wedge-shaped knife: rigid, used for harder materials
• Angles of knives:

  • Bevel angle: maintained by a knife back, sharper with smaller angles (27O-32O recommended)
  • Cutting angle: optimum at 15O for maximum tissue penetration and minimum distortion
  • Rake angle: higher rake angles suitable for soft tissue
• Perfect and optimum cutting angle is 15°
• Rake angle:

  • The angle between the upper level of the knife and the perpendicular line from the block
  • High rake angles are suitable for soft tissues and need to be reduced for harder tissues
• Clearance angle:

  • The angle formed by a line drawn along the block surface and the lower level of the knife
  • Clearance angle of 2° to 4° is used for paraffin sections, and 5° to 7° for frozen sections
• A good cutting edge should be made of good quality steel

  • Too soft cutting edges are likely to become dull easily
  • Too hard edges are likely to produce nicks or jagged edges and irregularities on the knife edge, thereby producing tears or striation on the tissue sections during cutting
• A good cutting edge must be able to cut good sections from a paraffin wax block about 2-3μ thick without any serration noted on examination
• A good cutting edge must be sharp enough to cut good sections from a paraffin wax block at 4μ thick without causing serrations
• The sharpness of the knives must be maintained to produce quality tissue sections
• Honing:

  • Refers to the grinding of the cutting edge of the knife on a hard abrasive surface to sharpen it
  • Involves the removal of gross nicks on the knife edge (coarse honing) and grinding the cutting edge of the knife on a stone (honing proper) to acquire an even edge
• Types of hones:

  1. Belgium yellow: Used for manual sharpening when the cutting edge has been rendered blunt or nicked

  2. Arkansas: Gives a more polishing effect than the Belgium yellow

  3. Fine carborundum: Much coarser than the first two types and is used only for badly nicked knives, followed by either one of the first two stones
• Honing Procedure:

  • Secure the microtome knife with its back and holder
  • Wipe the surface of the hone clean with a smooth cloth moistened with xylene
  • Cover the surface of the hone with xylene or soapy water
  • Place the knife on one end of the hone
  • Draw the heel of the knife obliquely with the cutting knife edge facing away from the operator until the toe is reached
  • Turn the knife edge over on its back and draw the other surface forward, producing double strokes
  • Repeat 20-30 times in each direction or until all the teeth in the knife edge have been removed
• Cold Ischemia:

  • Lack of oxygen once tissue sample is removed from the patient's body before metabolic processes stop by fixation
  • Steps to preserve tissue integrity include immediate transportation to the laboratory for proper processing
  • Larger tissues can be sectioned serially; uteri and intestines can be opened to expose mucosal linings; small biopsies should be quickly immersed in fixative
• Fixation:

  • Tissue removed for histopathology examination must be fixed right away for better preservation
  • Properly preserved tissues are more resistant to processing artifacts
• Properly Filled-up Surgical Pathology Request:

  • Accompanied by clinician's request for diagnosis, patient's history, physical and laboratory findings, imaging findings, pre-operative and post-operative diagnosis
  • Clinical correlation is essential for accurate diagnosis
• Accessioning Procedure:

  • Specimens must be entered into surgical pathology database via accessioning process
  • Attention to accurate and unambiguous identification of samples is crucial
  • Unlabeled specimens are unacceptable for accessioning
• Gross Examination of Specimens:

  • Identification of specimen and components is crucial for diagnosis
  • Criteria for rejection include discrepancies between requisition and specimen labels, leaking containers, absent clinical data, and mislabeled specimens