Lecture Notes in Fixation (Must Know in Tissue Fixation - Histopathology)

FIXATION (read the book)
Most important, initial and the most crucial step in tissue processing, and must be carried out adequately
because if not, the other steps that follow would be inadequate.
Defined as the killing, penetration and hardening of tissues
Alteration of tissues by stabilizing protein so that tissues become resistant to further changes
There are several reasons why fixation is done:
Primary goal of fixation:
a. Preserve cells & tissues as close to the original as possible (preserve the morphologic and chemical integrity
of the cell in as life-like a manner as possible)
Secondary goals:
a. Harden tissues to facilitate easy cutting (kase lahat ng tinatanggal sa katawan ay malambot so for easy
cutting, you harden it)
b. Protect tissue from trauma/damage of further handling because tissues are exposed to different reagents
Other objectives:
a. Prevent breakdown of cellular elements (autolysis, putrefaction, etc.)
b. To coagulate/precipitate protoplasmic substances
Fixatives – Reagents used to fix tissues
Primary goal and Secondary goals: Fixation methods

BENEFITS OF FIXATION/EFFECTS OF FIXATION (pg. 85)
1. Prevents autolysis, reduced risk of infection except prion diseases)
2. Allows thin sectioning
3. Acts as mordant thus facilitating staining
2 FIXATION METHODS (in general)
1. PHYSICAL METHOD
3 types of physical method fixation:
a. HEAT FIXATION – Heat is applied; Not usually carried out in histopathology because it is done in:
• Microbiology section = To fix bacterial smear
• Histopathology section = for frozen sections
b. MICROWAVE TECHNIQUE – acetylcholine
• Not a common method
• Recommended for fixing neurochemical substances in grain like acetylcholine
c. CRYO-PRESERVATION (freeze drying) – Sa libro to; has some applications in histochemistry but
is not usually applied to diagnostic tissue specimens.

2. CHEMICAL METHOD
Specimens are immersed/subject in chemical fixatives
Reagents are used
- immersion fixation
- perfusion fixation
Paraformaldehyde and osmium tetroxide can be used to vapor-fix freeze-dried tissues.

, CHEMICAL FIXATIVES
SIMPLE FIXATIVES COMPOUND FIXATIVES
1. Formalin
2. Mercuric chloride MICROANATOMICAL CYTOLOGICAL HISTOCHEMICAL
3. Osmic acid 1. Formal saline 1. Cold acetone
4. Picric acid 2. Neutral buffer formalin NUCLEAR CYTOPLASMIC 2. Ethanol
5. Acetone 3. Zenker’s fluid Carnoy’s Champy’s
6. Ethyl alcohol 4. Bouin’s fluid fluid fluid
7. Osmium tetroxide
8. Osmic acid



2 TYPES OF SPECIMEN / TISSUE PROCESSING
MANUAL - Kailangan maraming lalagyan
PROCESSING - Ikaw maglilipat ng specimen from 1 reagent to another then ikaw din magooras
- Not used anymore kase duh?




- So dito, kuha ka bote, formalin, babad mo tissue spx for 24hrs, then lipat mo sa
lalagyan with dehydrating agent (70%-80%-90% alcohol), then lipat sa Xylene or
Chloroform for clearing, then lipat sa paraffin wax for infiltration.
AUTOMATED – Use ➢ Used today kasi syempre matic
of autotechnicon ➢ Rapid penetration is ensured because of continuous mixing or agitation
➢ Autotechnicon:
o Has heat application; speeds up tissue processing
o Automatic tissue processor that can do the first four steps of tissue
processing (fixation, dehydration, clearing, & infiltration)

2 TYPES OF AUTOTECHNICON
➢ Specimen is transferred from 1 reagent to another
automatically
Tissue transfer ➢ Lalagay mo nalang yung spx sa 1st container, set the timer then
type/Dip & matic na
Dunk ➢ Consist of:
10 one-liter o Purpose: Where the tissue specimen
capacity reagents are placed
beakers
Cover plate o Purpose: Used to cover all beakers
o There is only 1 cover plate for all 10 beakers
o Di sya lapat na lapat sa bibig ng beaker kaya
maaamoy mo parin yung reagents.
Transfer arm o Purpose: Facilitates the transfer of specimen
from one reagent to another