HISTOPATHOLOGIC TECHNIQUES Sir Ian Kit E.

Bautista, RMT

FRESH TISSUE EXAMINATION

HISTOPATHOLOGIC TECHNIQUES • Curettings

o where tissue is scooped or spooned to remove
• Histology tissue or growths from body cavity such as
o is the microscopic study of the normal tissues of endometrium or cervical canal.
the body while Histopathology is the microscopic
study of tissues affected by disease.
o The procedures adopted for the material for such
studies are known as histologic or
histopathologic techniques.
o The tissues are usually obtained during surgery,
biopsy and autopsy.

The following surgical procedures are usually performed

to obtain the specific types of tissue that are submitted to
a histology laboratory for processing:

• Fine needle aspiration

o is the simplest, least invasive test and uses the
smallest needle to simply remove cells from the
area of abnormality.
o This is not always adequate to obtain a diagnosis,
depending on the area being biopsied.
• Core needle biopsy
o removes not only cells, but also a small portion of
the surrounding tissue.
o This provides additional information to assist in
the examination of the lesion.
• Incisional biopsy
o takes out even more surrounding tissues.
o It takes some out of the abnormality, but not all.
o The doctor will slice into the lesion and remove
only a portion of it.
o If the lesion is found to be cancerous, further
surgery may be needed to remove the lesion.
• Specimens are usually received in fixative but
sometimes they arrive fresh and must be immediately
fixed.
• Fresh tissues
o are usually examined when there is an immediate
need for evaluation.
o Fresh tissues have the advantage of being
examined in the living state, thereby allowing
protoplasmic activities such as motion, mitosis,
and phagocytosis to be observed.
o Its use is limited however, because of the fact that
tissues examined in the fresh state are not
permanent, and therefore, are liable to develop
the changes that have usually been observed
after death.

METHODS OF FRESH TISSUE EXAMINATION

• Teasing or dissociation
• Excisional biopsy
o is a process whereby a selected tissue specimen
o generally removes the entire area in question.
is immersed in isotonic salt solution such as
• Punch biopsy
Normal saline solution or Ringer’s solution in a
o is considered the primary technique for obtaining
petri dish or watch glass, carefully dissected with
diagnostic full-thickness skin specimens.
a needle and separated by direct or zigzag
o It requires basic general surgical and suture tying
spread using an applicator stick.
skills and is easy to learn.
o Selected pieces of the tissue are transferred
o The technique involves the use of a circular blade
carefully to a microscope slide and mounted as a
that is rotated down through the epidermis and
wet preparation underneath a cover glass, care
dermis, and into the subcutaneous fat, yielding a
being taken to avoid forming bubbles. It is either
3-4 mm cylindrical core of the tissue sample.
stained with a supravital dye or examined
• Shave biopsy
unstained by phase-contrast microscopy.
o is where small fragments of tissue are shaved
from a surface (usually a skin)

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• Squash preparation(crushing)
o is a process whereby 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.
o If necessary, a supravital 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.
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• Smear preparation
o the method of preparing the smear differs
depending on the nature of the material to be
examined.
o Smears may be examined as either as fresh
prep similar to that described for teased
preparations, or by using a supravital staining
technique. This is useful for preparing smears of
thick secretions like mucous fluids, serous fluids,
sputum, enzymatic lavage samples from GI tract,
and blood smears.
o Streaking
▪ with an applicator stick or a 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.
o Spreading
▪ A selected portion of the material is
transferred into a clean slide and gently
spread into moderately thick film by teasing
the mucous strands with applicator stick.
▪ It is a little more tedious than streaking, but
has the advantage of maintaining cellular
interrelationships of the material to be
examined.
▪ It is specially recommended for smear
preparation of fresh sputum and bronchial
aspirates, and also for thick mucoid
secretions.
o Pull-apart
▪ this is done by placing a drop of secretion or
sediment upon one slide and facing it unto
another clean slide.
▪ The material disperses evenly over the
surface of the two slides.
▪ Slight movement of the two slides in opposite
directions may be necessary to initiate flow of
materials.
▪ The two slides are then pulled apart with a
single uninterrupted motion, and the
specimen is placed under the microscope for
immediate examination, or applied with vital
stains.
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• FROZEN SECTIONS
o At times during the performance of surgical
procedures, it is necessary to get a rapid
diagnosis of a pathologic process. Immediate
diagnosis is accomplished through the use of
frozen section. It is especially recommended
when lipids and nervous tissues are to be
demonstrated.
o A fresh tissue is frozen on a microtome with CO2,
or on a cryostat, a cold chamber kept at an
atmospheric temperature of -10C to -20C. The
thin frozen sections are mounted on a glass slide,
fixed immediately and briefly in liquid fixative, and
stained using similar staining techniques as in
traditional wax embedded sections.

Frozen sections, both fixed and unfixed, have many

applications in histotechnology, and are commonly
used for:
a. undehydrated tissues for rapid diagnosis
b. histological demonstration of fats
c. for certain neurological structures
d. for sensitive tissue constituents damaged by
heat
e. Diagnostic and research enzyme
histochemistry
f. immunohistochemical and immunofluorescent
staining

• Touch preparation (Impression smear)

o this is a special method of smear preparation
whereby the surface of a freshly cut piece of
tissue is brought into contact and pressed on the
surface of a clean glass slide, allowing the cells
to be transferred directly to the slide for
examination phase-contrast microscopy or
staining for light microscopic study.

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• The tissue for freezing 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 methods of
freezing include:
• Liquid Nitrogen
• Isopentane cooled by liquid nitrogen
• Carbon dioxide gas
• Aerosol sprays
• Liquid nitrogen
o is generally used in histochemistry and during
intra-operative procedures, and is the most rapid
of the commonly available freezing agents.
o Its main disadvantage is that soft tissue is liable
to crack due to rapid expansion of ice within the
tissue, producing ice crystals or freeze artifacts.
• Isopentane
o is liquid at room temperature.
o A Pyrex glass beaker containing isopentane is
usually suspended in a flask of liquid nitrogen
until half-liquid and half-solid stage is reached.
This is an excellent method for freezing muscle
tissue.
• Tissue blocks can also be frozen by adapting a
conventional freezing microtome gas supply of
Carbon dioxide gas from a CO2 cylinder.
• The use of aerosol sprays has become increasingly
popular in recent years, and is adequate for freezing
small pieces of tissue except muscle.
• Quick-freezing spray cans of fluorinated
hydrocarbons (Cryokwik) have a distinct advantage
of rapidly freezing blocks of any type of tissue.
• Fresh, completely unfixed tissues, or tissues that
have been briefly treated with formalin may not
require embedding anymore; instead they may be
frozen and cut in a freezing microtome or cryostat.
• Cryostat or Cold Microtome
o is a refrigerated apparatus used for fresh tissue
microtomy.
o consist of microtome (rotary microtome) kept
inside a cold chamber w/c maintains the
temperature between -5 to -300C (average -
200C) by an adjustable thermostat.
o Majority of the sections can be cut in isothermic
situations, where the temperature for sectioning
can be accurately established and controlled.
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• Thermostat: capable of freezing fresh tissues w/in 2-
3 minutes
• Cutting sections of 4 Micra
• Provides sections for fresh tissue examination esp.
Fluorescent Antibody Staining techniques or
Histochemical enzymes studies
• Also used for intraoperative diagnosis
SPECIAL PROCESSING TECHNIQUES
• For histochemical evaluation involving enzyme
studies, the tissue needs to be chemically active,
and the important chemical constituents should not
have been removed, altered or displaced.
• In most instances, frozen section is deemed most
ideal and preferred means of preserving tissues in
order to avoid complete or partial loss of enzymes
consequent to chemical fixation.
• Difficulties, however, arise in obtaining thin and serial
sections of uniform thickness; since cut sections of
tissue tend to disintegrate and cannot be easily
handled without prior fixation. These disadvantages
will have to be considered in determining the
necessity and advisability of such sections.
• In addition to fresh frozen tissue sectioning, there are
methods that may be resorted to, if chemical fixation
of tissue blocks is to be avoided, namely:
o Freeze drying
▪ Special way of preserving tissues by rapid
quenching of fresh tissue at -160C and
subsequently removing ice molecules without
using any fixative
▪ Rapid freezing (quenching): -160deg.C
▪ Sublimation: vacuum at -40deg.C
▪ Technique is generally time-consuming and
expensive.
▪ More difficult to section than ordinary paraffin
blocks
o Freeze substitution
▪ It is a process of dehydration, performed at
temperatures low enough to avoid the
formation of ice crystals and to circumvent
the damaging effects observed after ambient
temperature dehydration.
▪ Use of chemical fixative
▪ Fixative for frozen tissue: ROSSMAN’S
FORMULA/ 1% ACETONE
▪ Dehydration of tissue: ABSOLUTE
ALCOHOL
▪ This technique is relatively more economical
and less time-consuming than freeze drying.
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