Fresh Tissue Examination

Histology is the microscopic study of

the normal tissues of the body
Histopathology is the microscopic
study of tissues affected by disease.
The procedures adopted for the material
for such studies are known as
histologic or histopathologic
techniques.The tissues are usually
obtained during surgery, biopsy and
autopsy.

The following surgical procedures are ● An incisional biopsy takes out

usually performed to obtain the specific
types of tissue that are submitted to a
histology laboratory for processing:
● Fine needle aspiration is the
simplest, least invasive test and
uses the smallest needle to
simply remove cells from the area
of abnormality. This is not always
adequate to obtain a diagnosis,
depending on the area being
biopsied.
even more surrounding tissues. It
takes some out of the
abnormality, but not all. The
doctor will slice into the lesion
and remove only a portion of it. If
the lesion is found to be
cancerous, further surgery may
be needed to remove the lesion.
An excisional biopsy generally
removes the entire area in question.

● A core needle biopsy removes

not only cells, but also a small
portion of the surrounding tissue.
This provides additional
information to assist in the
examination of the lesion.
Punch biopsy is considered the
primary technique for obtaining
diagnostic full-thickness skin specimens.
It requires 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 3-4
mm cylindrical core of the tissue
sample.
Shave biopsy is where small fragments
of tissue are shaved from a surface
(usually a skin)
Curettings – where tissue is scooped or
spooned to remove tissue or growths
from the body cavity such as
endometrial or cervical canal.
Specimens are usually received in
fixative but sometimes they arrive fresh
and must be immediately fixed.
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.
METHODS OF FRESH TISSUE
EXAMINATION
Teasing or dissociation – is a process
whereby a selected tissue specimen is
immersed in isotonic salt solution such
as Normal saline solution 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 is
either stained with a supravital dye or
examined unstained by phase-contrast
microscopy.

Smear preparation – the method of

preparing the smear differs depending
Squash preparation (crushing) is a
on the nature of the material to be
process whereby small pieces of tissue
examined. Smears may be examined
(not more than one mm in diameter) are
either as fresh prep similar to that
placed in a microscopic slide and
described for teased preparations, or by
forcibly compressed with another slide
using a supravital staining technique.
or with a cover glass. If necessary, a
This is useful for preparing smears of
supravital stain may be placed at the
thick secretions like mucous fluids,
junction of the slide and the cover glass,
serous fluids, sputum, enzymatic lavage
and allowed to be absorbed by the
samples from GI tract, and blood
tissue through capillary attraction.
smears.

● 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.
● 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 an
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
especially recommended for
smear preparation of fresh
sputum and bronchial aspirates,
and also for thick mucoid
secretions.
● Pull-apart – this is done by
placing a drop of secretion or
sediment upon one slide and
facing it onto 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.
Touch preparation (Impression smear)
– 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.
FROZEN SECTIONS
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
sections. It is especially recommended
when lipids and nervous tissues are to
be demonstrated.
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.
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 is generally used in
histochemistry and during
intra-operative procedures, and is the
most rapid of the commonly available
freezing agents. 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 is liquid at room
temperature. 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
- is a refrigerated apparatus used
for fresh tissue microtomy.
- 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.
- Majority of the sections can be
cut in isothermic situations,
where the temperature for
sectioning can be accurately
established and controlled.
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 sections are
deemed the 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:
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
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.