Instrumentation in Histopathology Flashcards by MAREE NATHALEE DEUS

Order of tissue processing

(1) Numbering/Accessioning
(2) Fixation
(3) Decalcification
(4) Dehydration
(5) Clearing
(6) Impregnation
(7) Embedding
(8) Blocking
(9) Trimming
(10) Sectioning
(11) Staining
(12) Mounting
(13) Labelling

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Characterized by the transfer of tissues, contained within a basket, through a series of stationary reagents arranged in-line or in a circular carousel plan

Tissue-transfer processors (carousel-type)

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9-10 reagent positions and 2-3 wax positions
Capacity: 30-110 cassettes

Tissue-transfer processors (carousel-type)

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Characterized by processing fluids pumped to and from a retort in which the tissues remain stationary

Fluid-transfer processors

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10-12 reagent stations with adjustable between 30-45°C
3-4 paraffin wax stations with variable temperature settings between 48-68°C
Capacity: 100-300 cassettes

Fluid-transfer processors

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Basic instrument used that is capable of cutting section at a predetermined thickness by sliding the block into a cutting tool which is fixed and attached to the machine

Microtome

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A spring balance teeth or pawl is brought to contact with, and turns a ratchet feed wheel connected to a micrometer secret, which in turn rotated, moving the tissue block at a predetermined distance towards the knife for cutting sections at uniform thickness

Microtome Principle

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Holds the tissue
Moves while the knife is stationary
Movement is usually forward then downwards
Controlled by spring balance teeth/pawl and the ratchet feed wheel connected to the micrometer screw that is calibrated to move so that the section would be as thin as prescribed by the protocol of the microtome

Chunk/Block/Tissue block

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Specimen holder
Specimen block
Knife
(Sometimes included : Copper mesh Grid)
Ratchet Feed wheel
Adjustment screws

Essential Parts of microtome

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Cuts the block at a predetermined thickness.
It produces tissue ribbons that are connected to each other

Knife

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Line up the tissue block in the proper position
Adjust the thickness for the sections

Adjustment screws

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Rocking microtome
Rotary microtome
Sliding microtome
Freezing microtome
Ultrathin microtome

Kinds of Microtome

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Manually operated
Invented by Paldwell trefall in 1881
Simplest among the different types of microtome
Consists of a heavy base and two arms

Rocking Microtome

(Cambridge rocking microtome)

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Name derived from the rocking action of the crossarm

Rocking microtome

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Can cut up to 10-12µm tissue sections

Rocking microtome

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Used to cut small and large blocks of paraffin tissues
Not recommended for serial sections because tissues are cut in slightly curved planes

Rocking microtome

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Disadvantage of this microtome:
Restrictions in size of tissue block that can be cut
Difficulty of reorienting the block

Rocking Microtome

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Manually operated
Minot microtome
Invented by Minot in 1885-1886
Most common type used for both routine and research laboratories

Rotary Microtome

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Operated by the rotation of the flywheel
Causes reciprocal motion of the knife over the block
Thickness of the section being automatically regulated by the ratchet feed wheel

Rotary Microtome

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Difference of Rotary microtome from Rocking microtome

The knife and the block holder are brought together by upward and vertical motions (cuts sections in a perfectly flat plane)
Heavier and more stable
More complex in design and construction
More expensive
The blade is placed in a blade-up position which is relatively dangerous

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Manually operated
Discovered by Adams in 1789
Especially recommended for cutting extremely hard and rough tissue blocks
Most dangerous type of microtome

Sliding Microtome

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Two kinds of sliding microtome

Base-sledge microtome
Standard sliding microtome

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Consist of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections

Base-sledge microtome

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Favored in laboratories where very hard tissue or large blocks are usually sectioned

Base-sledge microtome

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Suited for sectioning specimens embedded in all forms of media

Base-sledge microtome

Comparatively more stable

Base-sledge microtome

The block remains stationary while the knife is moved backward and foward during the process of sectioning

Standard sliding microtome

Developed mainly for cutting celloidin-embedded tissue blocks

Standard sliding microtome

Inherently more dangerous

Standard sliding microtome

Base-sledge microtome Used in ALL FORMS of media Block is ___ Knife is ____

Moving Stationary

Common disadvantage of base-sledge microtome

slower than the rotary and rocking microtomes

Common tissues used in base-sledge microtome

Whole brain

Standard sliding microtome Block is ___ Knife is ___

Stationary Moving

Semi-automated Invented by Queckett in 1848

Freezing microtome

The stage for the block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe thru which carbon dioxide (freezing agent) passes from a cylinder

Freezing microtome

Designed for cutting frozen sections No fixative is used prior to using this microtome

Freezing microtome

A simple lever operated valve allows the release of rapid intermittent burst of carbon dioxide A second cooling device for lowering temperature of the knife to facilitate sectioning

Freezing microtome

Used to cut undehydrated tissues in a frozen state

Freezing microtome

When histological demonstration of fat is needed

Freezing microtome

When certain neurological structures are to be studied

Freezing microtome

When sensitive tissue constituents to be studied are damaged or destroyed by heat

Freezing microtome

Kind of freezing microtome

Cyrostat

Consists of microtome kept inside a cold chamber which has been maintained at a temperature between -5 to 30°C (-20°C) by an adjustable thermostat

Cyrostat

Capable of freezing fresh tissues within 2-3 minutes

Cyrostat

Can cut sections of 4µ with ease

Cyrostat

Advantages of Cyrostat

Provides a means of preparing thin sections of fresh frozen tissues for fluorescent antibody staining techniques (immunohistochemistry)a or histochemical enzyme studies (microstructure and enzyme/antigen integrity)

Most commonly used for rapid preparation of urgent tissue biopsises for intraoperative diagnosis

Cyrostat

Used to mount tissues in the Cyrotome

OCTs

Semi automated or automated Primarily used for cutting tissue sections at 0.5 micra for electron microscopy

Ultrathin microtome

The knife used for cutting the ultrathin sections consists mainly of selected fragments of broken plate glass

Ultrathin microtome

The specimen used is small, fixed in osmium tetroxide and embedded in plastic

Ultrathin microtome

Other kinds of microtome

Vibrating microtome Hand microtome Saw microtome Laser microtome Computerized microtome

Also called as "vibratome" Designed to cut sections from fresh and fixed materials from animal or botanical sources

Vibrating microtome

Uses high-speed vibrations produced by safety razor blades which provide the cutting power, allowing the resultant cut to be made with less pressure than would the stationary blade be

Vibrating microtome

Mostly used for rigid botanical materials For the hard stems of plants

Hand microtome

Designed to cut sections from very hard materials like undecalcified bone, teeth, glass and ceramics

Saw microtome

Samples are usually embedded in resin and are moved extremely slowly against a diamond coated saw (slices through the sample) rotating at 600 rpm

Saw microtome

Cannot produce very thin sections, the product is about 20 micra even using a diamond knife

Saw microtome

Used for tissue that are embedded in one of the tougher media

Saw microtome

Automatic microtome Designed for non-contact sectioning inside biological tissues without causing thermal damage

Laser microtome

Can produce tissue sections from 5-100µ (microns)

Laser microtome

Operates using a cutting action with the used of infrared laser

Laser microtome

The preparation of the sample through embedding, freezing, or chemical fixation is not required therefore minimizing the artifacts from the preparation methods in the tissue

Laser microtome

Automatic microtome Dual purpose microtome can produce: Rapid freezing sections for RFS, Routine paraffin sections for routine histopathology lab tests

Computerized microtome

Equipped with: Thermostatic switch semiconductor freezing cyroscalpel cyroplate

Computerized microtome

All of the accumulated paraffin and small pieces of tissues must be brushed away with soft brush and not allowed to stay in the microtome

Care of the microtome

After drying the machine and knife holder, the parts should be wiped with ___

xylol

Movable portions should be ___ thoroughly to prevent rusting

oiled

Microtome must always be ___ when not in use

covered

Trimming and section-cutting are done with a

microtome knife

Used for hard sharpening Done to remove gross nicks or irregularities heel to toe direction uses honing stones

Honing

Done to remove burns toe to heel direction uses horse leather

Stropping

Types of microtome knives

Plane-concave knife Biconcave knife Plane-wedge knife Disposable blades Glass knives Diamond knives

Usually 25mm in length one side of the knife is flat while the other is concave

Plane-concave knife

Flat side of the plane-concave knife is recommended for cutting _____ in a standard sliding microtome

Celloidin-embedded

Concave side of a plane-concave knife is used to cut ___ sections in a base-sledge, rotary, or rocking microtome

paraffin

Used cutting sections from a material that is too hard to cut with a biconcave knife (Could also be used for sapphire materials)

Plane-concave knife

Usually 120mm in length With both sides concave

Biconcave knife

Recommended for cutting paraffin embedded sections on a rotary microtome (also used on rocking microtomes and carbon dioxide-freezing microtomes)

Biconcave knife

Most recommended blade for routine cutting in microtomy

Biconcave knife

Not suitable for relatively hard materials which cause the edge to vibrate and produce the phenomenon known as chattering that destroys the microanatomy of the tissue

Biconcave knife

Usually 100mm in length have both sides straight

Plane-wedge knife

Recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin blocks using a base-sledge type or sliding microtome

Plane-wedge knife

Also used for paraffin blocks being cut in the rotary microtome

Plane-wedge knife

has more rigidity than biconcave knife and plane-concave knife

Plane-wedge knife

preferred or placed in a base ledge type or in a sliding microtome type when cutting paraffin

Plane-wedge knife

Knife angles

Wedge Angle Bevel Angle Clearance Angle Cutting Angle Rake angle

Refer to the position of the tissue block in reference to the knife

Angle

Flat side of the tissue block in reference to the bottom part of the knife would be known as the :

Clearange angle (0-15°)

Best angle to be used

5°

Angle of the edges in a wedge knife Body of the knife or blade Normally 15°

Wedge Angle

Angle of the very tip blade, between cutting facets Normally 27-32°

Bevel angle

Angle of the knife itself in reference to the tissue block (27-32°)

Bevel angle

Angle between the block face and the lower facet of the knife Dependednt on the tilt or knife holder Set between 3-8°

Clearance angle

When the clearance angle is too wide

The tip of the blade will scrape the block and chatter will result

When the clearance angle is too small

the body of the blade will scrape the block and skipped sections or poor ribboning will result

Angle between the block face and upper facet of the knife This is not an angle that can be adjusted on a microtome

Cutting angle

It is the result of the clearance angle and the upper bevel angle of the knife or blade

Cutting angle

90° minus the angle of the upper facet of the knife Subtract all the angles that have been set to 90°

Rake angle

Have sharp cutting edge that can cut 2-4µ thick sections with ease Cheaper to use

Disposable blades

Used for trimming and semi-thin sectioning of tissue blocks for electron microscopy Commercially prepared Should be prepared and stored in dust-free boxes with lids, just before use, to avoid contamination

Glass knives

Used to cut any type of resin block for electron microscopy Brittle and expensive but very durable They are already mounted in a metal bock designed to fit directly into the knife holder of the ultra-thin microtome when purchased

Diamond knives

Other equipment for tissue sectioning

Floatation water bath Drying oven or hot plate Microwave Forceps and squirrel/camel hairbrush Clean slides and coverslips

Floatation water bath Temperature : About __ below the melting point of the paraffin wax

10°

Floatation water bath A small amount of detergent may be added to water to ___ and allow ____

Reduce surface tension allow section to flatten out

Floatation water bath Capacity : ___ liters __ inches (diameter) x __ inches (height)

2 liters 11 inches (diameter) x 4 inches (height)

mostly used after microtomy to remove the wrinkles and crumpled areas of tissue ribbon

Floatation water bath

Temperature is set at the melting point of the wax to 2-5°C above* (*Gregorios)

Drying oven or hot plate

For rapid drying of tissue slides The unstained tissue slides should be put here For delicate tissues, a lower drying temperature must be used to avoid cracking and splitting of the sections

Drying oven or hot plate

To melt the paraffin Very seldom used Can be used to remove excess paraffin so that the only tissue is on the slide

Microwave

Both are needed for handling sections during cutting and removing folds and creases during "floating out" in water bath Both could be used for sectioning

Forceps and Squirrel/Camel Hairbrush

Mostly needed for embedding

Forceps

Used to fix sections in place

Forceps and Squirrel/Camel Hairbrush

Instrumentation in Histopathology Flashcards

Based on PPT (113 cards)