HISTOPATHOLOGIC TECHNIQUES
Histological hazards and threats
Chemical hazard categories according to effects:
• 1. Human tissue destruction (visible):
•
2. Fire:
•
3. Causes cancer:
•
4. Interferes with metabolic processes
•
5. Embryo defects:
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6. Induces genetic mutations:
•
7. Causes explosion:
HAZMAT Diamond Symbol Position:
*Bottom:
*Top:
*Left:
*Right:
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CHEMICAL LABELING
Every chemical should be labeled with certain basic information, including:
• ________________ and, if a mixture, names of all ingredients;
• ________________ name and address if purchased commercially, or name of
person making the reagent;
• Date __________ or ______;
• ___________ date, if known;
• _____________________________________.
Other terms related to Chemical Hazards
• Are chemicals that cause reversible inflammatory effects at the site of contact
with living tissue, especially the skin, eyes and respiratory passages.
________________
•
Cause allergic reactions in some exposed workers, not just in hypersensitive
individuals. _________________
Notable Chemicals and associated hazards
• Explosive when dry:
• Explosive when old:
• May initiate or promote combustion and present a serious fire risk when in
contact with certain substances (MCS):
• Causes immediate death: COMU
• Classified as irritants and corrosives:
• Must not be poured onto the edge of a glass Dewar flask when filling because the
flask may break and implode:
• Causes aplastic anemia:
• Irritates the skin, eyes and nostrils:
• Deposits may precipitate in the cornea, causing blindness:
Examples of microscopy
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EXAMINATION OF FRESH TISSUES
***Histology versus Histopathology versus Histopathologic techniques
The following surgical procedures are usually performed to obtain the specific-types of
tissue that are submitted to a histology laboratory for processing:
*_____________: where tissue is scooped or spooned to remove tissue or growths from
body cavity such as endometrium or cervical canal.
*_____________: removes not only cells, but also a small amount of the surrounding
tissue.
*_____________: takes out even more surrounding tissue. It takes out some of the
abnormality, but not all.
*________________: the simplest, least invasive test and uses the smallest needle to
simply remove cells from the area of abnormality.
*________________: generally removes the entire area in question.
*________________: where small fragments of tissue are “shaved” from a surface
(usually skin).
*________________: is considered the primary technique for obtaining diagnostic fullthickness skin specimens. It requires basic general surgical and suture-tying skills and is
easy to learn.
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FRESH TISSUES
ADVANTAGE: examined in the living state, thereby allowing protoplasmic
activities such as:
a. M_________
b. M_________
c. P__________
d. P__________
- Its use has been limited, however, because of the fact that tissues examined in
the fresh state are ____ __________ and therefore, are liable to develop the
changes that have usually been observed after death.
POST-MORTEM changes:
1. d___________
2. p___________ / d____________
3. a________
___________ – a retrogressive pathologic process in cells in which the cytoplasm
undergoes ____________ while the nucleus is __________.
_____________ - the decomposition of organic matter under the influence of
microorganisms accompanied by the development of disagreeable odors.
_____________ - the destruction of the tissues (breaking down of the protein of
the cell) by enzymes which are produced by the tissues and eventually liquefy it.
- The first to occur among all post-mortem changes.
Methods of Fresh Tissue Examination:
1. ____________________- process whereby selected tissue specimen is immersed in a
watch glass containing ____________, carefully dissected or separated and examined
under the microscope, either unstained, by ______________________________, or
stained with ___________________.
2. ________________________-process where small pieces of tissues, not more than ___
are placed in a microscopic slide and forcibly compressed with another slide or with
coverglass.
- _______ are placed at the slide and coverslip junction and absorbed through capillary
action
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3. ______________- useful in cytology (Pap’s smear)
Technique
Material
Applicator stick or
platinum loop.
Process/ Important notes
*Rapid and gentle direct or
zigzag application to obtain
uniform distribution.
*______/________ are
unsuitable for exam.
Applicator stick is used to
Little more tedious than
tease the mucous strands to streaking, but has advantage
make a moderately thick
in maintaining
film.
_________________________.
Especially recommended for
_________, bronchial
aspirates and thick mucoid
secretions.
Slides are facing each other The material disperses
as a drop of secretion is
evenly over the surface of 2
sandwiched in-between.
slides. ________
______________ _______of
pulling apart is applied. It
is useful for serous fluids,
conc. sputum, enzymatic
GIT lavage and blood
smears
One slide. Cells may be
Special method where slide
examined w/o destroying
surface is in contact and
their actual intercellular
pressed on the site.
rel. w/o separating them
from their normal
environment.
4. ______________– normally utilized when a __________of the tissue in question is
required, and especially recommended when _____ and _______ tissue elements are to
be demonstrated.
FROZEN SECTIONS
• Immediate diagnosis is accomplished through the use of a frozen section,
especially in intra-operative pathology to help the surgeon in choosing his next
plan of action.
• Frozen sections are usually done on ______ and ______ biopsies as well as on
surgically removed tumors.
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A fresh tissue is frozen on:
*_______________
*_______________, a cold chamber kept at an atmospheric temperature of ___________.
Advantage?
Disadvantage?
Frozen sections, both fixed and unfixed, have many applications in histotechnology,
and are commonly used for:
1. Rapid pathologic diagnosis during surgery.
2. Diagnostic and research enzyme histochemistry.
3. Diagnostic and research demonstration of soluble substances such as lipids and
carbohydrates.
4. Immunofluorescent and immunohistochemical staining.
5. Some specialized silver stains, particularly in neuropathology.
The more commonly used methods of freezing include:
1. Liquid nitrogen
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
4. Aerosol sprays
•
_____________ is generally used in histochemistry and during intraoperative
procedures, and is the most rapid of the commonly available freezing agents.
•
Its main disadvantage is that soft tissue is liable to ______ due to the
_____________________________ within the tissue, producing ice crystals or
freeze artifacts.
•
It also overcools urgent biopsy blocks, causing damage to both block and blade
if sectioning is done at ______________.
•
The tissue snap-frozen in liquid nitrogen must therefore be allowed to equilibrate
to cryostat chamber temperature before sectioning is attempted.
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•
The majority of non-fatty unfixed tissues are sectioned well at temperatures
between ______________.
•
One problem with the use of liquid nitrogen is that it causes a vapor phase to
form around the tissue, acting as an _________ that causes
_______________________, particularly of muscle biopsies, and making
diagnostic interpretation difficult.
•
This problem can be overcome by freezing the tissue in ____________, ____, or
______that has a high thermal conductivity.
Two methods of preparing frozen sections may be resorted to:
1.
2.
BLANK SPACE FOR COLD KNIFE PROCEDURE BASICS
BLANK SPACE FOR CRYOSTAT BASICS
Mounting of Tissue Block
• Synthetic water-soluble glycols and resins are generally used as mounting media
for tissue blocks that need to be sectioned on a cryostat.
• The O.C.T. (________________) compound, L______________,
_______________________ is especially recommended.
• It is marketed in convenient 8 oz. plastic dispensers in three temperature ranges,
depending on the tissue being cut:
• __________ for brain, lymph nodes, liver, spleen, uterine curetting, soft cellular
tumors;
• __________for non-fatty breast tissue, ovary, prostate, tongue, and GI tract;
• ______for fatty breast and omental tissue.
• The cryostat is usually set at ______________.
• Preferably, the tissue block should be _____ mm. thick in order to minimize the
risk of the knife hitting the metal tissue block holder
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Special Processing Techniques
- methods that may be resorted to if chemical fixation of tissue blocks is to be
avoided:
1. Freeze-drying
2. Freeze-substitution
3. Fresh Frozen tissue sectioning
1. ______________
- by rapid ________/________and removing water/___________ by a
physical process from the still frozen tissue block without the use of any chemical
fixative.
- tissue size: 2mm thick
- complete processing time: 24-48 hours
- disadvantage:-time consuming
-_________
-more difficult to section
- advantage: -produces minimum shrinkage
-allows tissues to be processed in a fresh state
2. _______________
- similar to freeze-drying
- difference: tissue is fixed in __________or in __________
- advantage:
more ________
more suitable for routine purposes
•
•
•
•
•
Freeze-substitution is based on rapid freezing of tissues followed by solution
(“______________") of ice at temperatures well below 0°C.
A 1 mm to 3 mm specimen is thrown into ____ propane-isopentane that is super
cooled by liquid nitrogen to -175°C (with precautions).
Cryostat sections are cut 8-10 μm, and transferred to water-free acetone
(substituting fluid), and cooled to -__C for ___ hours to __ week in order to
dissolve ice slowly without distorting tissue structure.
The sections are floated onto coverslips or slides and allowed to dry for
subsequent histochemical staining.
This technique is relatively more economical and less time-consuming than
freeze-drying.
3. ___________________________
- requires that tissue be maintained in the _____ ______ ______ during cutting of
section
*All have the common principle of rapidly preserving the tissue block by freezing
(__________), to produce instant cessation of cellular activity thereby preventing
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chemical alteration of tissue constituents and displacement of cellular tissue
components.
*Freezing must be rapid, being accomplished within _______to prevent the
formation of ___ ______artifacts in tissue blocks, and produce optimum tissue
preservation.
*The freezing agent commonly employed is _______________.
*The use of i__________, p_______and p______and most recently of
________________________, which can be cooled to very low temperature(-1500C)
in order to retain the _______ of the freezing agents, have contributed much in
giving higher conductivity to this liquefied gas.
PRESERVED TISSUES
♦A better and more effective means of studying tissues whether normal or abnormal is
by examination of their sections and smears which have been ___________ preserved,
_____________________________and _____________________________for permanent
keeping.
***The aim of a good histopathological technique is to produce microscopic preparation
of tissue, usually stained, that __________________________________________________
OVERVIEW OF FIXATION
• It is important that tissues are handled carefully and appropriately fixed as soon
as possible after arriving in the laboratory.
• The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde
solution (formalin).
• __________, usually as a __________________ solution, is the most popular
fixative for preserving tissues that will be processed for paraffin embedding.
• If not carried out under optimal conditions or if fixation is delayed, a tissue
specimen can be irreversibly damaged.
FIXATION PROPER
• The first and most critical step in histotechnology is _________, a process that
preserves tissues from decay, thereby preventing ___________ or ____________.
•
Fixation should be carried out as soon as possible after removal of the tissues (in
the case of surgical pathology) or soon after death (in the case of autopsy) to
prevent autolysis.
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***When it comes to fixation, the following questions must be addressed:
1. Primary goal of fixation:
2. Secondary goal of fixation:
A fixative will initially produce a number of changes to the tissues in what is usually
an aqueous environment.
• S
• S
• H
• The tissues will undergo further changes during processing when they are
placed in a nonaqueous environment.
• For example, fixation in _____________________ will initially cause slight
swelling of tissue specimens.
• During processing, however, the specimen may shrink and lose ____________ of
its volume.
•
OBJECTIVES OF FIXATION
1.
2.
3.
Methods of Fixation
• Fixation of tissues can be achieved by physical or chemical means.
• Physical methods include _________, __________ and ______________________.
• Heat fixation is rarely used on tissue specimens, its application being confined to
_____________________.
• Chemical fixation is usually achieved by immersing the specimen in the fixative
solution (____________ fixation) or, in the case of small animals or some whole
organs such as a lung, by perfusing or injecting the vascular system with fixative
(___________ fixation).
There are two basic mechanisms involved in fixation:
1.
2.
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•
Fixative solutions may contain a single fixative agent dissolved in a solvent such
as water or alcohol or more commonly, a buffer solution to stabilize pH.
•
Some popular fixative solutions contain several different fixing agents in
combination, the rationale being that the defects in one agent can be
compensated for by the addition of another.
Benefits of Fixation
• Allows ____ sectioning of tissue by hardening tissue.
• Prevents autolysis and inactivates infectious agents (except _____ diseases)
• Improves cell _____ for special stains
Practical Considerations to Optimize Fixation of Tissue
• Usual time for fixation to be started:
•
Optimal fixative volume:
•
What to do with anatomical barriers?
•
Fixatives diluted or contaminated by body fluids:
•
Wick:
•
Prolonged fixation may result to:
•
Orientation:
Main Factors Involved in Fixation
1. Volume
2. Hydrogen Ion Concentration
3. Temperature:
4. Thickness of section
5. Osmolality
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6. Concentration
7. Duration of fixation
8. Time Interval
Effects of Fixatives in General
• They reduce the risk of infection during handling and actual processing of
tissues.
• They harden soft and friable tissues and make the handling and cutting of
sections easier. This is usually accelerated by the action of alcohol during the
dehydration process.
• They make the cells resistant to damage and distortion caused by the hypotonic
and hypertonic solutions used during tissue processing.
• They inhibit bacterial decomposition.
• They increase the optical differentiation of cells and tissue components thereby
rendering them more readily visible during examination.
• They may act as __________ or ____________ to promote and hasten staining, or
they may inhibit certain dyes in favor of another (e.g. formaldehyde intensifies
while osmium tetroxide inhibits _____________________).
Characteristics of a Good Fixative
• It must be cheap.
• It must be stable.
• It must be safe to handle.
• It must kill the cell quickly thereby producing minimum distortion of cell
constituents.
• It must inhibit bacterial decomposition and autolysis.
• It must produce minimum shrinkage of tissue.
• It must permit rapid and even penetration of tissues.
• It must harden tissues thereby making the cutting of sections easier.
• It must be isotonic, causing minimal physical and chemical alteration of the cells
and their constituents.
• It must make cellular components insoluble to hypotonic solutions and render
them insensitive to subsequent processing.
• It must permit the subsequent application of many staining procedures to
facilitate easier and more profitable examination.
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Types of Fixatives
4 Major Groups of Fixatives
1.
2.
3.
4.
According to COMPOSITION
• A. ___________________ -are made up of only one component substance.
•
B. ___________________ -are those that are made up of two or more fixatives
which have been added together to obtain the optimal combined effect of their
individual actions upon the cells and tissue constituents.
According to ACTION
• A. _____________________are those that permit the general microscopic study of
tissue structures without altering the structural pattern and normal intercellular
relationship of the tissues in question.
(10-10-HF-ZZ-BB)
•
B. _______________: are those that preserve specific parts and particular
microscopic elements of the cell itself.
*B.1. Nuclear (FBNCH)
*B.2. Cytoplasmic (HORFF)
Take Note:
• For RNA, the precipitant fixatives - _________________________- give the best
quantitative results using frozen tissues as the standard.
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•
C. _________________ - are those that preserve the chemical constituents of cells
and tissues. FANA
Secondary Fixation
***Is the process of placing an already fixed tissue in a second fixative in order:
• ____________________________________________________________________
• ____________________________________________________________________
• ____________________________________________________________________
• Secondary fixation may be done before dehydration and on deparaffinized
sections before staining, usually with 10% formalin or 10% formol saline as a
primary fixative.
• The tissue may have been placed in a primary fixative for storage, or may require
further fixation for special staining.
_______________
• Ia form of secondary fixation whereby a primarily fixed tissue is placed in
aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant
for better staining effects and to aid in cytologic preservation of tissues.
_____________________
• is the process of removing excess fixative from the tissue after fixation in order to
improve staining and remove artefacts from the tissues. Several solutions may be
used.
1. Tap water:
2. 50-70% alcohol:
3. Alcoholic iodine:
General Precautions in Handling and Fixation of Specimens
• 1. Proper label and identification.
• 2. Tissues fixed immediately after removal.
• 3. If not fixed immediately, ______________________________________________
• 4. Drying should be avoided to prevent _________ and __________ of tissue with
loss of cellular detail.
• 5. If placed in normal saline solution (NSS) during the operation, _________ may
occur before fixation is carried on.
• 6. Tissues should not be more than __ mm. thick except in lung edema (in which
case tissue slices may be ____ cm. thick), with minimum squeezing and handling.
• 7. Purulent material, exudates or transudates should be marked and kept for
possible ________, _______ and other ___________ examination
• 8. For prolonged fixation (e.g. museum preparation) volume of fixing fluid
should not be less than ___ times that of the tissue.
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9. Hollow organs (e.g. stomach, intestines) should be packed with
__________________________________________ before being immersed in
adequate fixing solution.
• 10. Air-filled lungs may float on fixative. To avoid this, the organ may be
______________________________ to maintain it under surface.
• 11. Human brains may be suspended by a cord tied under the
_________________ to prevent flattening.
• 12. Organs that are not supposed to be dissected before fixation:
_____________ must be injected before immersing the organ in the fixative.
• 13. Frozen sections may lead to formation of ice crystal artifacts.
• 14. Water should not be used for glycogen-containing tissues because glycogen is
_________ in water.
• 15. Purpose of mincing:
• 16. What to do with Hard tissues?
• 17. Tissues with large amount of blood:
• 18. What to do with hollow organs or specimens with natural cavities?
• 19. _______________ (________) of the specimen during its first few minutes in
fixative will facilitate penetration.
• 20. If a specimen is received in fixative of dubious quality, it must be
________________________.
• 21. Fixatives should be used only ______.
• 22. Avoid metal lids. Some fixatives are highly corrosive and will attack metals
(e.g., _____________________).
• 23. Some fixatives require that specimens be washed in water prior to processing
(e.g., ________________________).
•
Difficulties Caused by Improper Fixation:
1. Failure to arrest early autolysis of cells
• CAUSE: Failure to fix _____________ (the tissue was probably allowed to dry
before fixing); ____________ fixative
2. Removal of substances soluble in fixing agent
• CAUSE: _______________________________________
3. Presence of artefact pigments on tissue sections
• CAUSE: ________________________________________
4. Tissues are soft and feather-like in consistency
• CAUSE: _____________________
5. Loss or inactivation of enzymes needed for study
• CAUSE: _____________________
6. Shrinkage and swelling of cells and tissue structure
• CAUSE: _____________________
7. Tissue blocks are brittle and hard
• CAUSE: _____________________
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Other Fixative Details
• “_______________" may be found in surgical specimens particularly in liver
biopsies, associated with an intense eosinophilic staining at the center of the
tissue in H&E stained sections.
***Lipid fixation
***Carbohydrate Fixation
***Protein Fixation
***Fixation for Electron Microscopy
***Fixation for Enzyme Histochemistry
***Fixation for Immunofluorescence
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