San Pedro College
Department of Medical Laboratory Science
MANUAL OF HISTOPATHOLOGIC TECHNIQUES
ROUTINE PROCEDURES IN THE HISTOPATHOLOGY LABORATORY
RECEIVING
When receiving specimen, make sure to check the labels. It should include
patient’s name, age, sex, relevant clinical data, surgical findings, nature of
operation and name of tissue submitted. Serial numbers are assigned to each
sample. Each serial number is accompanied by a letter that signifies the type of
specimen submitted: A for autopsy specimen and S for surgical/biopsy specimen.
GROSS EXAMINATION
Under the pathologist’s trained eye, tissues are examined and each of their
physical macroscopic characteristics are noted. The specimen are cut into slices
that are about 2 cm2 and not more than 4 mm in thickness. Representative
sections are usually obtained from definite pathologic regions.
FIXATION
Fixation is a chemical process by which biological tissues are preserved in
a state (both chemically and structurally) as close to living tissue as possible. It
terminates any on-going biochemical reactions, and may also increase the
mechanical strength or stability of the treated tissues. The fixed tissue protected
from decay, thereby preventing autolysis or putrefaction. This requires a chemical
fixative that can stabilise proteins, nucleic acids and mucosubstances of the tissue
by making them insoluble.
DECALCIFICATION
The removal of calcium deposits is essential for good embedding
procedure. Decalcification is usually carried out between the fixation and
processing steps. Bone must obviously be processed in this way, but other tissues
may also contain calcified areas. A variety of agents or techniques have been
developed to decalcify tissues, each with advantages and disadvantages.
Immersion in solutions containing mineral acids, organic acids, or EDTA are the
predominant methods used. Electrolysis may also be used.
DEHYDRATION
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Specimens are transferred through increasing concentrations of hydrophilic
or water miscible fluids which dilute and eventually replace free water in the
tissues. Dehydration is necessary in all infiltration methods, except where tissues
are simply externally supported by an aqueous embedding medium. Choosing
the dehydrating agent to be used is determined by the nature of the task, the
embedding medium to be used, the processing method and even economic
factors.
CLEARING
Clearing is the transition step between dehydration and infiltration. A
chemical agent (solvent) that is miscible with the dehydrating and infiltrating
agents is used to facilitate the transition between dehydration and infiltration. The
term ‘clearing’ arises from the ability of some solvents with high refractive indices
to render anhydrous tissues transparent or clear.
INFILTRATION
This process involves the removal of the excess clearing agent from the
tissues and replaces it with a medium that will fill the natural cavities, spaces and
interstices of the tissues. This process provides the tissues with a firm consistency
resulting in better handling and cutting of the tissue sections. Usually, the medium
to be used for embedding will be the same medium used for infiltration.
EMBEDDING
Also known as casting or blocking, this process is done by placing the
infiltrated tissue, in a precisely arranged position, in a mold containing a medium
which is allowed to solidify. Embedded tissues are then prepared for sectioning or
cutting.
SECTIONING
This process refers to the cutting of the embedded tissues into uniformly thin
slices using the microtome.
STAINING
Staining is the process of adding colors or dyes to the thin tissue slices for
enhanced visualization and differentiation of cellular structures.
MOUNTING
It is the process of protecting tissue sections from physical damage by
coating it with a transparent medium then covering it with a glass slip.
San Pedro College
Department of Medical Laboratory Science
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MANUAL OF HISTOPATHOLOGIC TECHNIQUES
LEARNING ACTIVITY NO. 1
Instrumentation in Histotechnology
SPECIFIC LEARNING OBJECTIVES:
At the end of this activity, the Medical Laboratory Science student will be
able to:
1. Identify the different instruments used in the histopathology laboratory.
2. Explain the function of each instrument.
3. Appreciate the importance of proper handling and maintenance of each
histopathology laboratory instrument.
4. Demonstrate the use of each instrument.
5. Draw the identified laboratory equipments.
LEARNING CONTENT
In histopathology, diagnosis of diseases is done by examining the stained
morphologic cellular details of the specimen submitted and processed in the
laboratory. Needless to say, such study must involve the use of the microscope
which is the chief scientific tool for the procedure. However, the stained tissue
must pass through several processes before it is ready for microscopic evaluation.
These processes entail the use of a variety of instrumentations and appliances
present in a histopathology laboratory. Some of the common instruments include:
microtomes, cryostats, processors, stainers, water baths, microwave and dryers.
MICROTOMES
The earliest form of microtomy was the freehand sectioning of fresh or fixed
material using a sharp razor. Modern microtomes are precision instruments
designed to cut uniformly thin sections of a variety of material for detailed
microscopic examination.
flywheel
Block holder
Thickness scale
Knife stand clamp
Knife
Knife stand/holder
Figure 1. Parts of a microtome
Types of Microtomes
A. Rotary Microtome
• This type of microtome is generally used for cutting semi-thin to thin sections
of paraffin wax embedded material for light microscopy. Paraffin
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embedded tissues are normally cut between 3 to 5 µm. The operation of
the microtome is based upon the rotary action of a hand wheel activating
the advancement of a block towards a rigidly held knife. The block moves
up and down in a vertical plane in relation to the knife and therefore it cuts
flat sections. See figure 1.
B. Sliding Microtome
• These are designed for cutting large blocks of paraffin and resin embedded
material including whole organs, for light microscopy. The knife holding
clamps allow the knife to be offset to one direction, a major advantage
when sectioning large, hard blocks. They are not suitable for cutting very
hard resins such as araldite because of the risk of vibration.
A
Figure 2. (A) sliding microtome; (B) Rocking microtome
B
B
A. Rocking Microtome
• The razor of this microtome is fixed and the specimen to be sliced for
microscopic examination passes up and down in an arc of a circle across
the razor in a rocking motion. Fixed on to a table, the ribbons of specimen
fell to the desk top then were cut and mounted on to slides. The rocking
microtome was invented by Sir Horace Darwin (1851-1928), the son of
Charles Darwin.
D. Freezing Microtome
• This form of microtome is used for cutting thin to semi-thin (about 8-12µm)
sections of fresh, frozen tissue. The freezing microtome is equipped with a
stage upon which tissue can be quickly frozen using either liquid carbon
dioxide, from a cylinder, or a low temperature recirculating coolant. Some
cooling systems also allow the knife to be cooled at the same time. The
cutting action of the freezing microtome differs from those described
previously as in this case the knife is moved while the tissue block remains
static. Consistent, high quality, thin sections are very difficult to obtain with
this type of microtome.
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Figure 3. (A) Freezing microtome; (B) Cryostat
D. Cryostat
• A cryostat is primarily used for cutting sections of frozen tissue. It
commonly consists of a microtome contained within a refrigerated
chamber, the temperature of which can be maintained at a preset
level. The cryostat usually contains a rotary microtome although some
portable units utilize a rocking microtome. With the block, blockholder
and knife all at the same temperature and all other conditions for
cutting the material optimal, sections as thin as 1 micron are possible.
E. Ultramicrotome
• It is used to prepare ultrathin sections for light
and electron microscopy. Very small samples
of tissue are usually embedded in hard resin
before cutting. The cutting stroke is motor
driven to provide a regular, smooth motion
for sections of even thickness and constant
reproducibility. Knives are usually made from
glass, diamond or sapphire. The block is
brought to the knife edge under microscopic
control and as each section is cut it is floated
on to a water bath adjacent to the knife
edge.
Figure 4. Ultrathin microtome
FLOTATION BATH OR WATER BATH
• These are used for floating the paraffin ribbon.
Temperature depends on a degree of the personal
preference of the microtomist, but it is
recommended to maintain a temperature of 5 to
10 degrees below the melting point of the
paraffin used during embedding.
Figure 5. Floatation bath
SLIDE DRYERS
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Figure 6. Slide dryer
• These are used for drying the water that is
collected during the sectioning of tissue section.
The temperature used is between 5 to 10
degrees above the melting point of the paraffin.
Slides are left to dry for approximately 15-20
minutes, after they have been appropriately
drained. Not doing so can cause the bubbling
under the tissue sections. Overheating slides may
cause uneven staining as well as artifacts.
MICROWAVE OVEN
• The microwave oven is used to heat and speed
some
procedures.
Some
special
stains
techniques are performed in the microwave
oven. Heat induced epitope retrieval for
Immunohistochemistry is done in some occasions
in the microwave oven.
AUTOMATED STAINERS
• Linear types transfer slides from one container to
the next container with the same time allowed in
each container. Revolving types are similar to the
linear stainer and the time allowed in each can
be varied. Robotic ones are flexible with
computerized programming that allows the
continuous loading and use of the same solutions
at different timing.
Figure 7. Microwave oven
Figure 8. Automated stainer
EMBEDDING CENTERS
• It is a complete system designed for
embedding tissue in paraffin. It provides a
controlled heated environment (paraffin is
kept at 2 - 4ºC above its melting point) for
the processed cassettes and eliminates
xylene contamination.
Figure 9. Embedding center
PARAFFIN WAX DISPENSER
• Maintains the paraffin wax in liquid form and
aids in dispensing of wax into molds or casts.
TIME ALLOTMENT:
3 hours or 1 laboratory meeting
Figure 10. Paraffin wax dispenser
LEARNING RESOURCES:
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Microscope
Microtome
Paraffin dispenser
Tissue embedding molds
Tissue cassettes
Glossy magazine paper cut into 1/8 sheets
LEARNING STRATEGIES:
Lecture-Demonstration
PROCEDURE:
1. Review the parts of the microscope.
2. Listen to the instructor as he/she points out the parts of the microtome
and the paraffin dispenser. After the discussion, you will be given time
for a hands-on with the instruments. Make sure to review and identify the
parts of the microtome and dispenser on your own.
3. Once done with the machines, tissue molds and cassettes will be
introduced.
4. Practice making disposable molds using the sheets of glossy paper.
Follow the instructions below.Label the mold with your name, group
number and section then pass it to your instructor.
"To make paper trays proceed as follows. Take a
piece of stout paper or thin cardboard, of the shape
of the annexed figure; thin post-cards do very well
indeed. Fold it along the lines a a' and b b', then
along c c' and d d', taking care to fold always the
same way.
Then make the folds A A', B B' , C C', D D', still
folding the same way. To do this you apply A c
against A a, and pinch out the line A A', and so on
for the remaining angles. This done, you have an
imperfect tray with dogs' ears at the angles. To
finish it, turn the dogs' ears round against the ends
of the box, turn down outside the projecting flaps
that remain, and pinch them down."
LEARNING ACTIVITY NO. 1
STUDENT LEARNING EVALUATION
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Instrumentation in Histotechnology
Name : _______________________
Course/Yr./Sec.:_______________
Score: _____/30
Date: _________
Guide Questions:
Answer the following questions briefly. (30 points)
1. Name the optical components of the microscope. Give their functions. (10 pts)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
2. Enumerate the 5 types of microtomes. Identify the advantage(s) of each
microtome type. (10pts)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3. The cytospin is another instrument used in the histopathology laboratory. What
is it and what is its functions or uses?(10pts)
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
Rubrics for Scoring Guide Questions
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10 pts
8 pts
6pts
4 pts
Excellent
The answer
meets all of
the criteria:
1.Relevance
2. Clarity
3.Conciseness
4. Grammar
Good
The answer
meets most of
the criteria:
1. Relevance
2. Clarity
3.Conciseness
4. Grammar
Average
The answer
meets some of
the criteria:
1. Relevance
2. Clarity
3.Conciseness
4. Grammar
Fair
The answer
meets only a
few of the
criteria:
1. Relevance
2. Clarity
3.Conciseness
4. Grammar
1 pts
Needs
improvement
The answer
does not meet
any of the
following
criteria:
1. Relevance
2. Clarity
3. Conciseness
4. Grammar
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