FRESH TISSUE EXAMINATION

MARK LESTER B. CAUAN, RMT

FRESH TISSUE EXAMINATION

⚫May vary according to the structural and chemical

components of the cells to be studied, the nature and
amount of the tissue to be evaluated, and the need for
immediate examination of a tissue structure.
⚫Examination may be done on fresh or preserved
tissues, depending upon necessity.
FRESH TISSUE EXAMINATION

⚫Fresh tissues have the advantage of being examined in

the living state, thereby allowing protoplasmic
activities such as motion, mitosis, phagocytosis and
pinocytosis to be observed.
⚫Its use has been limited, however, because of the fact
that tissues examined in the fresh state are not
permanent and liable to develop changes that have
usually been observed after death.
METHODS OF FRESH TISSUE
EXAMINATION
Teasing or dissociation
⚫Selected tissue specimen is immersed in a watch glass
containing isotonic salt solution, carefully dissected or
separated, and examined under the microscope, either
unstained by phase contrast or bright field microscopy,
or stained with differential dyes
METHODS OF FRESH TISSUE
EXAMINATION
Squash preparation (Crushing)
⚫Small pieces of tissue not more than one mm in diameter
are placed in a microscopic slide and forcibly
compressed with another slide or with a cover glass.
⚫A vital stain may be placed at the junction of the slide
and the cover glass, and allowed to be absorbed by the
tissue through capillary attraction.
METHODS OF FRESH TISSUE
EXAMINATION
Smear preparation
⚫Examining sections or sediments whereby cellular
materials are spread lightly over a slide by means of a
wire loop or applicator, or by making an apposition
smear with another slide.
⚫Useful in cytological examinations, particularly for
cancer diagnosis.
Smear preparation

⚫Streaking
⚫ with an applicator stick or platinum loop,
the material is rapidly and gently applied in a
direct or zigzag line throughout the slide,
attempting to obtain a relatively uniform
distribution of secretion.
⚫Too thin or too thick smears have to be
avoided, since they make the tissues
unsuitable for examination.
 Smear preparation
Spreading
⚫A selected portion of the material is transferred to a
clean slide and gently spread into a moderately
thick fil by teasing the mucous strands apart with
an applicator stick.
⚫More tedious than streaking, but has the advantage
of maintaining cellular interrelationships of the
material to be examined.
⚫Recommended for smear preparations of fresh
sputum and bronchial aspirates, and also for thick
mucoid secretions
 Smear preparation
⚫Touch imprint (impression smear)
⚫The surface of a freshly cut piece if tissue is
brought into contact and pressed on to the surface
of a clean glass slide, allowing the cells to be
transferred directly to the slide for examination by
phase contrast microscopy or stained for light
microscopic study.
⚫It has the edge of added advantage in that the cells
may be examined without destroying their actual
intercellular relationship and without separating
them from their normal surrounding.
 Smear preparation
Pull-Apart
⚫Done by placing a drop of secretions or sediment
upon one slide and facing it to another clean slide.
The material disperses evenly over the surface of
the two slides
⚫Slight movement of the two slides are necessary to
initiate the flow of materials.
⚫The two slides are then pulled apart with a single
uninterrupted motion, and the specimen placed
under the microscope for immediate examination,
or applied with vital stain.
⚫Useful for thick secretions such as serous fluids,
concentrated sputum, enzymatic lavage samples
METHODS OF FRESH TISSUE
EXAMINATION
⚫Frozen section
⚫Utilized when rapid diagnosis of the tissue in question is
required, and is recommended when lipids and nervous
tissue elements are to be demonstrated.
⚫Very thin slices, around 10-15µ in thickness are cut from
a fresh tissue frozen on a microtome with CO2 or on
cryostat, a cold chamber kept at an atmospheric
temperature of -10 to -20 C .
⚫The frozen sections are then transferred to a slide, and
processed for light microscopic study.
Uses of frozen section

⚫Rapid diagnosis during surgery

⚫Diagnostic and research enzyme
histochemistry
⚫Diagnostic and research demonstration of
soluble substances such as lipids and
carbohydrates.
⚫Immunoflourescent and
immunohistochemical staining
⚫Some specialized silver stains, particularly in
neuropathy.
Frozen section
⚫The tissue for processing should be fresh,
and freezing should be done as quickly as
possible.
⚫Slow freezing can cause distortion of
tissue due to ice crystal artifacts.
The more commonly used method of
freezing include:
⚫Liquid nitrogen
⚫Isopentane cooled by liquid
nitrogen
⚫Carbon dioxide gas
⚫Aerosol sprays
Liquid nitrogen
Liquid nitrogen is generally used in histochemistry
and during operative procedures, and is the most
rapid of commonly available freezing agents.
⚫Its main disadvantage is that soft tissue is
liable to crack due to rapid expansion of the ice
within the tissue, producing ice crystals or
freeze artifacts.
⚫It also over cools urgent biopsy blocks,
causing damage to both block and blade if
sectioning is done at minus 70 c or below.
Liquid nitrogen
⚫Majority of non-fatty unfixed tissues are sectioned
well at temperatures between -10C and -25C.
⚫One problem with the use of liquid nitrogen is that
it causes a vapor phase to form around the tissue,
acting as an insulator that causes uneven cooling
of tissue, particularly of muscle biopsies, and
making diagnostic interpretation difficult. This
problem can be overcome by freezing the tissue in
Isopentane, OCT, Freon 2.2 that has a high
thermal conductivity.
Isopentane
⚫Isopentane is liquid at room temperature. A
pyrex glass beaker is usually suspended in a
flask of liquid nitrogen until half-liquid and
half solid stage is reached. The beaker is
removed from the liquid nitrogen when
crystals start forming on the side of the
beaker (approximately -170C), and the
tissue to be frozen is dropped into the
cooled liquid isopentane
Carbon dioxide gas

⚫Tissue blocks can also be frozen by

adapting a conventional freezing microtome
gas supply of carbon dioxide gas from a
CO2 cylinder, or by using a especially made
piece of equipment.
Aerosol sprays

⚫The use of aerosol sprays has

become increasingly popular in recent
years, and is adequate for freezing
small pieces of tissue except muscle.
PROCESSING OF TISSUES
⚫Fresh tissues are usually examined when there is
an immediate need for evaluation.
⚫A better and more effective means, however, of
studying tissues, whether normal or abnormal, is
by examination of their sections and smears which
have been permanently preserved, stained for
demonstration of specific structures, and mounted
on glass slides with coverslips for permanent
keeping.
Solid structures and tissues must be
preserved and carefully processed in the
following order:
⚫Fixation
⚫Dehydration
⚫Clearing
⚫Infiltration (impregnation)
⚫Embedding
⚫Trimming
⚫Section-cutting
⚫Staining
⚫Mounting
⚫Labeling
end