HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | LECTUREMODULE 1:

BSMLS 3A 1st SEMESTER | A.Y. 2022-2023

INSTRUCTOR: MR. RODS ANTHONY REYES

EXAMINATION OF FRESH TISSUE
Incisional Biopsy and Excisional Biopsy
 The tissues for histology/histopathology are
 An Incisional Biopsy takes out even more
usually obtained during surgery, biopsy, or
surrounding tissue. It takes out some of the
autopsy. They range from very large specimens or
abnormality, but not all. The doctor will slice into
whole organs to tiny fragments of tissue.
the lesion and remove only a portion of it. If the
 The following surgical procedures (Biopsy) are lesion is found to be cancerous, further surgery
usually performed to obtain the specific-types of may be needed to remove or excise the entire
tissue that are submitted to a lesion.
histology/histopathology laboratory for
 An Excisional Biopsy generally removes the entire
processing:
area in question.
o Fine needle aspiration
o Core needle biopsy
o Incisional biopsy
o Excisional biopsy
o Punch biopsy
o Shave biopsy
o Curettings
Fine Needle Aspiration
 The simplest, least invasive test and uses the
smallest needle to simply remove cells from the Punch Biopsy
area of abnormality. This is not always adequate to  Considered the primary technique for obtaining
obtain a diagnosis, depending on the area to be diagnostic full-thickness skin specimens. It requires
biopsied. basic general surgical and suture-tying skills and is
easy to learn. The technique involves the use of a
circular blade that is rotated down through the
epidermis and dermis, and into the subcutaneous
fat, yielding a 3- to 4- mm cylindrical core of tissue
sample.

Core Needle Biopsy

 Removes not only cells, but also a small amount of
the surrounding tissue. This provides additional
information to assist in the examination of the
lesion.
Shave Biopsy
 Where small fragments of tissue are “shaved”
from a surface (usually skin).

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | LECTUREMODULE 1:
BSMLS 3A 1st SEMESTER | A.Y. 2022-2023
INSTRUCTOR: MR. RODS ANTHONY REYES
Curettings
Where tissue is scooped or spooned to remove
tissue or growths from body cavity such as
endometrium or cervical canal.
Methods of Fresh Tissue Examination
 Fresh tissues are usually examined when there is
an immediate need for evaluation. 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. 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.
Teasing or Dissociation
 A process whereby a selected tissue specimen is
immersed in isotonic salt solution such as normal
saline or Ringer’s solution in a petri dish or watch
glass, carefully dissected with a needle and
separated by direct or zigzag spread using an
applicator stick.
 Selected pieces of the tissue are transferred
carefully to a microscope slide and mounted as a
wet preparation underneath a cover glass, care
being taken to avoid forming bubbles.
 It has the advantage of permitting the cells to be
examined in the living state.
Squash Preparation (Squashing)
 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. 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.
Smear Preparation
 The process of 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 a second slide to
obtain a relatively uniform distribution of
secretion.
o Too thin or too thick smears have to be
avoided, since they make the tissues less
suitable for examination.
o Smears may be examined either as fresh
preparations similar to that described for
teased preparations, or by using a
supravital staining technique.
GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
o Smear preparations can be made
permanent by fixing them while still wet,
staining them to demonstrate specific
structures and inclusions, and mounting
the cleared specimen beneath a cover
glass with a suitable mounting medium.
o The method of preparing the smear
differs depending on the nature of the
material to be examined.
i. Streaking
o 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.
ii. Spreading
o A selected portion of the material is
transferred to a clean slide and gently
spread into a moderately thick film
by teasing the mucous strands apart
with an applicator stick.
o It is a little more tedious than
streaking, but has the advantage of
maintaining cellular
interrelationships of the material to
be examined.
o It is especially recommended for
smear preparations of fresh sputum
and bronchial aspirates, and also for
thick mucoid secretions.
iii. Pull-Apart
o This is done by placing a drop of
secretion 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 in
opposite directions may be necessary
to initiate the 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.
iv. 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 to the
surface of a clean glass slide, allowing
 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | LECTUREMODULE 1:
BSMLS 3A 1st SEMESTER | A.Y. 2022-2023
INSTRUCTOR: MR. RODS ANTHONY
the cells to be transferred directly toREYES
the slide for examination by Phase
ice crystals artifacts. The more commonly used methods of
Contrast microscopy or staining for
freezing include:
light microscopic study.
o It has an added advantage in that the
cells may be examined without
destroying their intercellular
relationship.
Frozen Section
 This method is normally utilized when a rapid
diagnosis of the tissue in question is required,
and is especially recommended when lipids
and nervous tissue elements are to be
demonstrated.
 Frozen sections are usually done on muscle
and nerve biopsies as well as on surgically
removed tumors.
 Very thin slices, around 10-15 micrometer in
thickness are cut from a fresh tissue frozen on
a microtome with C02, or on a cryostat, a cold
chamber kept at an atmospheric temperature
of - 10° to -20° C. 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.
 For histochemistry, cryostat sections give
much faster results than paraffin sections.
However, the morphological detail and
resolution of frozen sections are usually
inferior compared to the quality of tissue that
has been embedded in paraffin.
 The advantage of the frozen section method is
rapid processing time with less equipment
requirement, and less need for ventilation in
the laboratory. The disadvantage is the
relatively poor quality of the final slide.
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 tissue for freezing should
GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
be fresh, and freezing should be done as
quickly as possible.
Slow freezing can cause distortion of tissue due to
1. Liquid nitrogen
o Liquid nitrogen is generally used in
histochemistry and during intraoperative
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 the rapid expansion
of the ice within the tissue, producing ice
crystals or freeze artifacts.
o It also overcools urgent biopsy blocks,
causing damage to both block and blade if
sectioning is done at -70°C or below.
o The tissue snap-frozen in liquid nitrogen
must therefore be allowed to equilibrate
to cryostat chamber temperature before
sectioning is attempted. The majority of
non-fatty unfixed tissues are sectioned
well at temperatures between -10oC and
-25°C.
o 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.
o This problem can be overcome by
freezing the tissue in Isopentane, OCT, or
Freon 2.2 that has a high thermal
conductivity.
2. Isopentane cooled by liquid nitrogen
o Isopentane 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. The beaker is
removed from the liquid nitrogen when
small crystals start forming on the side of
the beaker (approximately -170°C), and
the tissue to be frozen (affixed on a cork
disc, aluminum foil or cryostat chuck) is
dropped into the cooled liquid
isopentane.
o This is an excellent method for freezing
muscle tissue.
3. Carbon dioxide gas
 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | LECTUREMODULE 1:
BSMLS 3A 1st SEMESTER | A.Y. 2022-2023
o Tissue blocks can also be frozen by
adapting a
INSTRUCTOR:conventional freezingREYES
MR. RODS ANTHONY
microtome gas supply of carbon dioxide
gas from a C02 cylinder.
4. Aerosol sprays
o The use of aerosol sprays has become
increasingly popular in recent years, and
is adequate for freezing small pieces of
tissue except muscle. Quickfreezing spray
cans of fluorinated hydrocarbons (e.g.,
Cryokwik) have a distinct advantage of
rapidly freezing blocks of any type of
tissue.
Two Methods of Preparing frozen sections may be
resorted to:
Tissue blocks can be frozen by adapting a
conventional freezing microtome gas supply of carbon
dioxide gas from a C02 cylinder, or by using a specially
made piece of equipment known as cryostat.
1. Cold Knife procedure
o Almost any microtome can be utilized for
the purpose, provided means are made
available for freezing and maintaining the
specimen and the knife at low
temperatures, usually by utilizing the
carbon dioxide technique.
o A piece of filter paper soaked in gum
syrup is placed on the microtome stage,
and short bursts of C02 are applied,
freezing the filter paper to the stage. The
selected block of tissue, approximately 3-
5 mm. thick, is then oriented on the
stage, applied with a few drops of gum
syrup and frozen solid with several
intermittent bursts of CO2 , each for 1-2
seconds duration, at intervals of around 4
seconds. It should be frozen just to the
point where it will be firm enough to
section. The tissue is then lifted up to the
knife manually and trimmed until the
surface is flat. The surface is then warmed
with the finger until the hard frozen tissue
starts to thaw and becomes visible to the
naked eye. This is the DewLine, the point
at which sections may then be cut at 10
μm thickness.
o Sections do not form ribbons but rather
stick to the knife blade and should,
therefore, be removed with a camel hair
brush or finger moistened with water.
They are then transferred to a dish of
distilled water to separate, and picked up
individually for mounting and staining.
GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
The water dish is usually placed on a dark
or black background, in order to see the
sections which are usually colorless or
very light in color.
o Tissues that have been frozen too hard
will usually chip into fragments when cut.
The surface of the block may then be
softened by warming slightly with the ball
of the finger or thumb. Tissues that have
not been sufficiently frozen will cut thick
and crumble, and the block may come
away from the stage.
o Using a cold knife in a controlled cold
environment, optimum condition for
sectioning shall be provided for by the
following temperatures:
o Knife -40° to - 60°C
o Tissue -5° to - 10°C
o Environment 0° to - 10°C
2. Cryostat procedure (Cold Microtome)
o The cryostat consists of an insulated
microtome housed in an electrically
driven refrigerated chamber and
maintained at temperatures near -20°C,
where microtome, knife, specimen and
atmosphere are kept at the same
temperature.
o The optimum working temperature of
cryostat is -18 to -20°C.
o Preferably, the tissue block should be 2-4
mm. thick in order to minimize the risk of
the knife hitting the metal tissue block
holder.
o Majority of the sections can be cut in
isothermic conditions, where the
temperature for sectioning can be
accurately established and controlled.
o The tissue for freezing should be fresh,
and freezing should be done as quickly as
possible.
o The cryostat should be left on at all times
even when not in use, since it will require
several hours to reach operating
temperature from a room temperature
start. It takes at least one hour for a knife
to come down to operating temperature,
so that a spare knife should always be
kept inside the cryostat cabinet.
o The cryostat should be defrosted during
the weekend, including cleaning and
oiling of microtome with special low
temperature oil.
 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | LECTUREMODULE 1:
BSMLS 3A 1st SEMESTER | A.Y. 2022-2023

INSTRUCTOR: MR. RODS ANTHONY REYES

o Overall, cryostat sections provide the

simplest, quickest and least labor -
intensive method for producing frozen
sections, and are routinely used for
intraoperative and rapid diagnosis of
surgical specimen.
o It should be noted that cryostats cut only
individual sections, and do not form
ribbons, as in paraffin blocks.
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.
(Optimal Cutting Temperature) compound, Lab-
Tek Products, Division of Miles Laboratories is
especially recommended. It is marketed in
convenient 8 oz. plastic dispensers in three
temperature ranges, depending on the tissue
being cut:
o 5 to -15°C for brain, lymph nodes, liver,
spleen, uterine curetting, soft cellular
tumors;
o 15 to -25°C for non-fatty breast tissue,
ovary, prostate, tongue, and GI tract;
o 35°C for fatty breast and omental tissue.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA