FRESH TISSUE EXAMINATION

Slide 2: You need to be aware

that not all specimen received
at the histopathology lab is
going to be processed fresh.
These are considerations in
deciding if a specimen is to be
processed fresh or preserved
Most commonly; specimen is
processed fresh because of 'stat'
requests. a result is needed urgently, just like in breast mass excisions where patient is allowed to wait in
the OR while a result is being made.
 As soon as the result is available (Benign or Malignant), the definitive surgical intervention is
done
 However fresh specimen because it is not initially fixed is not permanent and liable to change

METHODS OF FRESH TISSUE EXAMINATION

1. Teasing or Dissociation
2. Squash Preparation (Crushing)
3. Smear Preparation
4. Frozen Section
 You should also note that the process of teasing, squash, and smear are meant to distribute the cells
reasonably far from each other for better visualization and therefore better diagnosis

TEASING OR DISSOCIATION

 A process whereby a selected tissue specimen is immersed in a watch glass containing isotonic salt
solution, carefully dissected or selected and examined under the microscope
 Can be examined unstained via Phase Contrast Microscopy or stained via Bright Field Microscopy

SQUASH PREPARATION

 A process whereby small pieces (less than 1

mm) of tissue are placed on a glass slide and
forcibly compressed with another slide
 A vital stain may be placed at the junction of
the slide and the cover slip
SMEAR PREPARATION

 A process of examining sections or sediments, whereby cellular materials are spread lightly over a
slide
Methods of Smear Preparation
1. Streaking
2. Spreading
3. Pull Apart
4. Touch Preparation (touch preparation is the only one where intercellular relationship are
maintained)

STREAKING

 With an applicator stick or platinum loop the material is

rapidly spread and gently applied in a direct or zigzag
line throughout the slide

SPREADING

 A selected portion if the material is transferred to a clean slide and gently spread into a moderately
thick film by teasing the mucus strands with an applicator stick

PULL-APART

 Done by placing a drop of

secretion or sediment upon
one slide and facing it to
another clean slide
 Two slides are then pulled
apart with a single
uninterrupted motion

TOUCH PREPARATION

 Also called impression smear

 A special method of smear preparation whereby the surface of
freshly cut piece of tissue is brought into contact and pressed in
the surface of the slide
 May be stained for light microscopy or unstained for Phase Contrast Microscopy
 Cells are examined in their actual intercellular relationship

FROZEN SECTION
Purpose:
 For rapid diagnosis of tissue
 Recommended when lipids and nervous tissue elements are to be demonstrated
Method:
 Makes use of very thin slices 10-15u cut from fresh tissue frozen on a microtome with CO2 or on a
Cryostat
 Cryostat- cold chamber (-10 to -20 deg Celsius)
Slide 11 will show another method of fresh preparation- frozen section.
This involves the principle of rapid freeze. Note difference between
rapid freeze vs slow freeze.

For frozen section rapid freeze is attained most commonly by using

liquid nitrogen. the picture on the lower right is a freezing microtome, it
uses CO2. just like liquid nitrogen it causes the rapid freeze of a tissue
to harden it enough for sectioning. Rapid because it does not have to go
thru the process of fixation, dehydration, clearing, etc...

Applications
 Rapid pathologic diagnosis during surgery
 Diagnostic and research enzyme histochemistry
 Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates
 Immunofluorescent and immunohistochemical staining
 Some specialized silver stains in neuropathology
Rapid freeze: slow freezing causes distortion due to ice crystal artifacts
 Liquid nitrogen (most rapid and commonly available)
 Isopentane cooled by liquid nitrogen
 CO2 gas (freezing microtome)
 Aerosol sprays (not for muscle tissue)
Liquid nitrogen
 Overcools: cracks tissue, biopsy blocks (-70deg Celsiums)
 Sectioning requires equilibration at cryostat temp (-10 to 125 deg C)
 Vapor phase forms around the tissue causing uneven cooling making diagnostic interpretation difficult
Isopentane: liquid at room temperature
  Prevents vapor phase due to its high thermal conductivity
 equilibrated temperature should be -10 to -25 npt 125

PROCESSING OF TISSUES
Fresh vs Preserved tissue examination Fixation

Dehydration
 Most fresh tissue are very delicate
Clearing
o Easily distorted
Infiltration
(impregnation)
o Easily damaged
Embedding
 Preserved tissues provide the efficiency of:
Trimming
o Of staining for better demonstration Section Cutting

o Permanent keeping when mount on slides with coverslips Staining

Mounting
Slide 14: the challenge in fresh tissue examination is to create a tissue that is as life
like as possible because without initial fixation, they are easily distorted and damaged Labelling

FIXATION

PRIMARY AIM OF FIXATION

 To preserve the morphologic and chemical integrity of the cell in as life-like manner as possible.
 To prevent degeneration, decomposition, putrefaction, and distortion of tissues after removal
from the body.
SECONDARY AIM OF FIXATION
 To harden and protect the tissue from the trauma of further handling.
 Allows tissue to be more properly oriented in the cassette in preparation for paraffin embedding
and microtomy.

EFFECTS OF FIXATION

 They harden soft and friable tissues and make the handling and cutting of sections easier.
(accelerated by the action of alcohol)
 They make cells resistant to damage and distortion caused by hypotonic and hypertonic solutions.
 They inhibit bacterial decomposition.
 They increase optical differentiation of cells and tissue components.
 They act as mordants or accentuators to promote and hasten staining.
 They reduce the risk of infections.

FIXATION
FACTORS AFFECTING THE QUALITY OF FIXATION:
1. Buffers and pH (pH6-8)
2. Duration of fixation (2-6 hours, buffered formalin)
 Prolonged: shrinkage and hardening of tissue
3. Size of specimen
 1-2sqmm EM
 2sqcm Light Microscopy
 no more than 4mm thick
4. Temperature of Fixation
 0-4degC: EM, histochemistry
• Mast cells: room temp
 Chemical reactions are more rapid at higher temperatures
• Heat fixation in bacteriology
5. Concentration of fixative
 10% Formaldehyde
 3% Glutaraldehyde
6. Osmolality
 Hypertonic solutions: cell shrinkage
 Isotonic (340mOsm) and hypotonic fixatives: cell swelling and poor fixation
 Best: slightly hypertonic solutions (400-450mOsm)

PRACTICAL CONSIDERATIONS

1. SPEED: Tissue are placed in a fixative as soon as they are removed

 Prevent autolysis and putrefaction
2. Penetration:
 Formalin: 1 mm per hour, slows down as it goes deeper
3. Volume: 1 (VOlume should be 10-25X not 0-25X)
 0-25x the volume of the tissue
 Maximum effectiveness at 20x
4. Duration of fixation:
 Fibrous organs take longer
 Fixation time can be cut down by heat, vacuum, agitation or microwave
5. Refrigeration
 To slow down decomposition if the tissue cannot be fixed immediately
 Brain cells deteriorate very quickly, BM mitosis up 30 mins after death