Chapter 1: FRESH TISSUE EXAMINATION
Learning Objectives:
1.
2.
3.
4.
5.
To be able to differentiate histotechnology and
histotechnologist.
To be able to differentiate the advantages and
disadvantages of fresh tissue examination.
To be able to know the basis for method used in
fresh tissue examination.
To be able to name and differentiate the methods of
fresh tissue examination.
To be able to understand the fresh frozen method.
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Chapter 1:
FRESH TISSUE EXAMINATION
Histotechnology
- is the art and science
performed by the histotechnologist to produce a
tissue section of good quality that will enable the
pathologist to diagnose the presence of disease.
The tissue may be done fresh or preserved.
Advantage of fresh tissue examination: The
specimen may be in living state, therefore may
observe protoplasmic activities (motility, mitosis,
phagocytosis, pinocytosis).
Disadvantage: not permanent, and therefore
liable to changes
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Chapter 1:
FRESH TISSUE EXAMINATION
The choice of tissue examination method
would depend on the following conditions;
1. Cell Structure and chemical components
2. Amount and nature of tissue
3. Urgency of result
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Chapter 1:
FRESH TISSUE EXAMINATION
The methods of fresh tissue examination:
1. Teasing or dissociation – selected tissue is in isotonic solution,
dissected and examined under the microscope.
2. Squash Preparation (Crushing) – less than 1 mm tissue pieces are
compressed (stained when necessary), and examined under
the microscope.
3. Smear preparation – spread cellular materials (secretions,
sediments) are examined. useful for cytologic examinations
a)
b)
c)
d)
4.
Streaking – using an applicator stick by direct or zigzag spread
Spreading – teasing (sputum, bronchial aspirates, thick mucoid
secretions) by applicator stick and spread in circular spread.
Pull-Apart – thick secretions (gastric lavage, serous fluids, blood)
are dispersed evenly on two slide surfaces.
Touch Preparation (Impression) - freshly cut tissue surface is
brought into contact to slide. Cells are examined in their actual
intercellular relationship.
Frozen Section – for rapid diagnosis during surgery, and
demonstration of lipids and nervous tissue elements.
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Frozen tissue – 10 -15 u in thickness cut from frozen tissue on a microtome with CO2 or on cryostat.
A cryostat is cold chamber kept at an atmospheric temperature of -10 to -20 deg. C. The frozen
sections are then transferred to a slide, and examined under light microscopy.
Sequence of fresh frozen tissue examination by cryostat
a
b
c
d
e
The tissue should be fresh and processed quickly. Slow freezing can cause tissue distortion due to
ice artifacts. The more commonly used methods of freezing include;
1.
2.
3.
4.
Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays
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Chapter 2: Fixation and Fixatives
Learning objectives:
1. To understand the process of autolysis
2. To determine the contributing factors in autolysis.
3. To discuss why certain tissues are affected severely by
autolysis.
4. To know the objectives of fixation and qualities to
serve its objective.
5. To understand the factors involve in fixation.
6. To know the types of fixative.
7. To understand the properties of formaldehyde.
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Chapter 2:
Fixation and Fixatives
Once tissues are removed from the body, they undergo
a process of self-destruction or autolysis. Soon after tissue
death, the intracellular enzymes break down the protein and
then the cell eventually undergo liquefaction. – Autolysis.
1.
2.
3.
4.
Properties of Autolysis:
independent of a bacterial action
retarded by cold
accelerated at 30 degree C temperature, inhibited at 50
degrees Celsius
more severe in tissue which are rich in enzymes (e.g. liver,
brain, and kidney) and less rapid in elastic and collagen
tissues.
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Chapter 2:
Fixation and Fixatives
Autolysed tissue presentations:
1.
2.
3.
“washed-out” appearance with swelling of cytoplasm,
eventually to a granular, homogenous mass which fails to take
up stains.
The nuclei of autolytic cells may show some of the changes
including condensation (pyknosis), fragmentation (karyorrhexis)
and lysis (karyolysis).
There may be diffusion of intracellular importance like
glycogen. The epithelium may desquamate from its basement
membranes.
Dead tissue may allow bacteria to proliferate. When these bacteria
decompose, it can mimic those of autolyzed tissue.
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Chapter 2:
a)
b)
Fixation and Fixatives
The objective of fixation;
To preserve cells and tissue constituents in as close to life-like state.
To ready for further processing without or minimal tissue change.
Fixative should have the following qualifications to serve its
objective;
a) Arrest autolysis and bacterial decomposition
b) Stabilizes cellular and tissue constituents.
c) Preservation
of
tissue
substances
and
proteins
(for
Immunohistochemisatry techniques to augment diagnosis)
Fixation is therefore the first step and the foundation in a sequence
of events that culminate in the final examination of ta tissue section.
Each fixative has specific properties and disadvantages. Their varied
effects to tissues would require careful selection of fixative when specific
cellular substances is to be studied.
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Chapter 2:
Fixation and Fixatives
Factors involve in Fixation:
1. Temperature – standardized fixation is carried out at room temperature. However, for electron microscopy
and some histochemical procedure, fixation is usually at 0 – 4 degrees Celsius. Lower temperatures autolysis is
slowed down allowing a more life-like appearance of tissues.
2. Size of specimen – the penetration of fixative is inversely directly relative to size. In which, larger tissue size
will be fixed slowly. Thus, large specimens are opened and washed of contaminant or sliced thinly. . The
fixative should be at least 20 times the volume of the specimen.
3. Change in volume – the volume changes in tissue with fixation, as the tissue shrinks by 33%. This is evident
as the tissue show larger nuclei and cells in frozen sections (unfixed).
4. pH and Buffers – The hydrogen ion concentration varies between fixatives, but in general maintained
between 6 – 8. The buffer systems maintain this physiological range of pH level (e.g.; phosphate, acetate,
bicarbonate). The buffer should have the following properties;
a. do not interfere with fixative
b. do not inhibit enzymes
5. Osmolality – the fixative solutions; osmotic pressure may be affected by the addition of buffer. The best
result is obtained by using slightly hypertonic solutions (isotonic solutions adjusted by using sucrose).
6. Concentration of fixative
7. Duration of fixation
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Chapter 2:
Fixation and Fixatives
Types of fixative according to action:
A. Microanatomical fixatives – are those the permit the general microscopic study of tissues structures without altering
the structural pattern and normal intercellular relationship of the tissues in question.




10% formol saline
10% neutral buffered formalin
Zenkers solution
Bouin’s solution
B. Cytological fixatives – are those that preserve specific parts and particular microscopic elements of the cell itself.
1. Nuclear fixative – are those that reserve nuclear structure (e.g. chromosome) in particular. They
usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin. Usually have pH
of 4.6 or less




Flemmings
Carnoys
Newcomers
Heidenhain’s susan
2. Cytoplasmic – are those that preserve cytoplasmic structures in particular. They never contain glacial
acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm. Ph of more than 4.6.



Kelly flemmings
Regauds’s fluid
Orth’s fluid
C. Histochemical fixative - preserve chemical constituents of cells and tissues.




Formol saline 10%
Absolute ethyl alcohol
Acetone
Newcomer’s fluid
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Chapter 2:
Fixation and Fixatives
Formaldehyde (Formalin)
Properties:
1. The most widely used and routine fixative particularly for paraffin
embedded sections.
2. It is a gas with a very pungent odor.
3. The commercially available solution of formaldehyde (formalin) contains
35 – 40% gas by weight. However, pure stock solution of 40% formalin is
unsatisfactory.
4. Formaldehyde is commonly used as 4% solution, giving 10% formalin for
fixation. Therefore, it should be diluted 1:10.
5. Formaldehyde is usually buffered to pH 7 with phosphate buffer.
6. It is thought that formaldehydes form cross-links between proteins,
creating a gel, thus retaining cellular constituent.
7. It is a forgiving fixative – requires a relatively short fixation time (24 hours)
but can be used for long term usage with no deleterious effects on tissue.
8. Prepared by adding 100 ml of 40% formaldehyde to 900 ml distilled water
with 4 g sodium phosphatase (monobasic) and 6.5 g sodium phosphate
(dibasic anhydrous).
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