Instrumentation and Automation in Histotechnology

Use of the Microscope in Histotechnology

• The microscope is a fundamental instrument in the histopathology laboratory, utilized for distinct purposes depending on the role of the professional.

• Pathologists are the primary figures authorized and qualified to interpret and read tissue slides to provide a medical diagnosis.

• Histotechnologists, medical technologists, and histotechnicians use the microscope primarily for quality control. This involves checking the quality of the slides for physical defects such as burs, cracks, or other technical flaws before they are submitted to the pathologist.

• Proper handling involves specific protocols for carrying and storing the device to ensure its longevity and accuracy.

The Microtome and Its Essential Components

• The microtome is critical for the sectioning of tissue. It consists of three essential parts:

  • Block Holder (Chuck): Holds the tissue block securely in position.

  • Knife and Knife Carrier: Responsible for the actual cutting of the tissue sections.

  • Adjustment Mechanisms: Includes the pawl, ratchet, feed wheel, and adjustment screws. These components line up the tissue block in the proper position relative to the knife and adjust the thickness of the tissue for successive cuts to ensure firm, uniform sections.

Historical and Modern Types of Microtomes

• Rocking Microtome (Cambridge Microtome):

  • Invented by Caldwell Trefall in 1881.

  • It is the simplest type of microtome.

  • Utilized for cutting serial sections of large blocks of paraffin-embedded tissues.

• Rotary Microtome (Minot Microtome):

  • Invented by Charles Sedgwick Minot in 1885 and 1886.

  • This is the most common type found in laboratories today.

  • It is used for sectioning small paraffin-embedded tissues.

• Sliding Microtome:

  • Developed by Adams in 1789.

  • It is identified as the most dangerous type of microtome because of its movable, exposed blade.

  • Specifically used for celloidin-embedded sections.

  • Subtypes include:

    • Base-Sledge Microtome: Used for all forms of media, particularly hard tissues or large blocks. It is ideal for resin-embedded decalcified bone. In this type, the block holder moves while the knife remains stationary or stagnant.

    • Standard Sliding Microtome: Developed for celloidin-embedded blocks. Unlike the base-sledge, the block holder is stationary while the knife moves forward and backward. This makes it more dangerous than the base-sledge design.

• Freezing Microtome:

  • Invented by Queckett in 1848.

  • It utilizes carbon dioxide ($CO_2$) for cooling.

  • It is used to cut undehydrated tissue in a frozen state (unembedded tissues).

  • This instrument is largely considered obsolete in modern practice.

• Cryostat (Cold Microtome):

  • Commonly used for rapid preparation and urgent tissue examination (e.g., during surgery in the operating room).

  • It consists of a microtome—usually a rotary type—housed within a cold chamber.

  • It is used for fresh tissue microtomy without the need for fixation, dehydration, clearing, or embedding.

  • The chamber temperature is maintained between $-5^{\circ}C$ and $-30^{\circ}C$.

  • The average or optimum temperature for cutting is $-20^{\circ}C$.

  • It is capable of freezing fresh tissues within 2 to 3 minutes for rapid diagnosis.

• Ultrathin Microtome:

  • Used to produce extremely thin sections for electron microscopy.

  • Utilizes diamond or glass knives.

  • Tissues must be embedded in plastic resins.

  • Section thickness ranges from $0.5$ to $1.0\,\mu m$. Ultrathin sections for electron microscopy are cut at $500$ to $1,200\,\text{Angstroms}$.

Microtome Knives and Characteristics

• Plain Concave Knife:

  • Length: $25\,mm$.

  • Used in base-sledge, rotary, and rocking microtomes.

  • The less concave side is used for celloidin-embedded blocks.

  • The more concave side is used for paraffin-embedded blocks.

• Biconcave Knife:

  • Length: $100\,mm$.

  • The knife is concave on both sides.

  • Used primarily in rotary microtomes for cutting paraffin-embedded tissues.

• Plain Wedge Knife:

  • Length: $100\,mm$.

  • Both sides of the knife are straight (wedged).

  • Used in base-sledge or sliding microtomes for frozen sections or very hard, tough paraffin-embedded tissues.

• Disposable Knives:

  • More common in modern laboratories because they eliminate the need for sharpening and are cheaper than conventional steel knives.

  • Magnetic Blades: Particularly suitable for cryostat microtomy.

  • Glass Knives: Used for trimming and semi-thin sectioning of tissue blocks for electron microscopy.

  • Diamond Knives: Extremely expensive but very durable; used for cutting ultrathin sections for electron microscopy and can be used on any type of resin block.

Maintenance of Knife Sharpness: Honing and Stropping

• Honing:

  • The process of removing gross nicks on the knife edge to sharpen it.

  • Direction: Edge first, from "heel to toe" (direction of a letter "H").

  • Standard procedure: 20 to 30 double strokes.

  • Types of hones:

    • Belgium Yellow: Yields the best results in honing.

    • Arkansas: Provides a polishing effect.

    • Fine Carborundum: Coarser than the other two; used for badly nicked knives.

• Stropping:

  • The process of polishing and removing the burr formed during honing.

  • Direction: "Toe to heel" (the counterpart to honing).

  • Standard procedure: 40 to 100 double strokes using leather.

Microtome Geometry and Cutting Angles

• Bevel Angle: The angle formed between the cutting edges of the knife. It is maintained at $27^{\circ}$ to $32^{\circ}$. This angle is held constant with the help of a knife back during sharpening.

• Cutting Angle: The perfect and optimum angle for tissue cutting is $15^{\circ}$. This provides maximum penetration while minimizing tissue distortion.

• Clearance Angle: The angle formed between the surface of the tissue block and the cutting edge of the knife.

  • General range: $0^{\circ}$ to $15^{\circ}$.

  • Specific range to prevent uneven sections (alternate thin and thick sections/rhythmic variations): The knife should be inclined at a $5^{\circ}$ to $10^{\circ}$ clearance angle.

• Rake Angle: The angle formed perpendicular to the sample ($90^{\circ}$ angle).

Automatic Tissue Processing

• Autotechnicon (Automatic Tissue Processor):

  • A common example is the Leica Autotechnicon.

  • It automates the stages of fixation, dehydration, clearing, and infiltration.

  • The primary advantage is constant agitation, which ensures even processing.

  • Processing typically lasts between 12 to 24 hours.

  • In manual processing, fixation is the most critical step; however, in automatic tissue processing, dehydration is considered the most critical stage.

• Station Cycles (Example 12-Jar Setup):

  • Stations 1 & 2: 10% Formalin (Fixation).

  • Stations 3 to 6: Ascending grades of ethyl alcohol ($70\%$ down to $95\%$) (Dehydration).

  • Stations 7 & 8: Acetone (two changes).

  • Stations 9 & 10: Chloroform, xylene, or xylol (Clearing).

  • Stations 11 & 12: Liquid (melted) paraffin wax (Infiltration).

• Fluid Transfer Processor (Vacuum Type):

  • A closed system where tissues remain in a single container and reagents are periodically pumped in and drained out.

  • Uses vacuum pressure to make the system faster and more efficient.

  • Prevents the tissue from drying out due to the closed environment.

Specialized Embedding and Preparation Apparatus

• Vacuum Embedding Apparatus: Used for tissue impregnation. It operates under negative atmospheric pressure. This is the fastest of the three impregnation methods (physical, chemical, radiologic).

• Flotation Bath (Water Bath): Used to float out and flatten paraffin ribbons after sectioning.

  • Temperature: Set between $45^{\circ}C$ and $50^{\circ}C$.

  • The temperature should be approximately $6^{\circ}C$ to $10^{\circ}C$ lower than the melting point of the specific wax used for embedding.

• Paraffin/Drying Oven: Used for drying out tissues and infiltration.

  • The temperature must be maintained at $2^{\circ}C$ to $5^{\circ}C$ above the melting point of the paraffin used for impregnation.

  • For infiltration, it is regulated at $55^{\circ}C$ to $60^{\circ}C$.

  • For drying, it is typically maintained at approximately $56^{\circ}C$ to $60^{\circ}C$ for two hours.

• Hot Plate: Not recommended for drying tissues as it causes a risk of overheating and dust contamination, which can obscure the tissue during microscopic viewing.

Slides and Staining Equipment

• Slide Specifications:

  • Routine size: $76 \times 15\,mm$.

  • Thickness: $1.0$ to $1.2\,mm$.

  • Frosted slides are preferred to allow for the write-in of accession numbers or ID numbers using a lead pencil (pencil is used because ink from pens may be removed by reagents).

  • Refractive Index: The ideal slide has a refractive index of $1.518$, which is optimized for light microscopy.

• Staining Apparatus:

  • Coplin Jars: Slotted staining dishes that hold 5 to 19 slides.

  • Metal or Glass Staining Racks: Used to transport groups of slides.

  • Advanced Automated Tissue Stainers: These are high-speed, automated systems that manage the entire staining sequence and drying process, reducing exposure to toxic chemicals.