Instrumentation in Histopathology Flashcards
Based on PPT
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
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)
9-10 reagent positions and 2-3 wax positions
Capacity: 30-110 cassettes
Tissue-transfer processors (carousel-type)
Characterized by processing fluids pumped to and from a retort in which the tissues remain stationary
Fluid-transfer processors
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
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
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
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
Specimen holder
Specimen block
Knife
(Sometimes included : Copper mesh Grid)
Ratchet Feed wheel
Adjustment screws
Essential Parts of microtome
Cuts the block at a predetermined thickness.
It produces tissue ribbons that are connected to each other
Knife
Line up the tissue block in the proper position
Adjust the thickness for the sections
Adjustment screws
Rocking microtome
Rotary microtome
Sliding microtome
Freezing microtome
Ultrathin microtome
Kinds of Microtome
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)
Name derived from the rocking action of the crossarm
Rocking microtome
Can cut up to 10-12µm tissue sections
Rocking microtome
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
Disadvantage of this microtome:
Restrictions in size of tissue block that can be cut
Difficulty of reorienting the block
Rocking Microtome
Manually operated
Minot microtome
Invented by Minot in 1885-1886
Most common type used for both routine and research laboratories
Rotary Microtome
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
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
Manually operated
Discovered by Adams in 1789
Especially recommended for cutting extremely hard and rough tissue blocks
Most dangerous type of microtome
Sliding Microtome
Two kinds of sliding microtome
Base-sledge microtome
Standard sliding microtome
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
Favored in laboratories where very hard tissue or large blocks are usually sectioned
Base-sledge microtome
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
