Fixation Part 1

defined as the killing, penetration, and hardening of tissues.

FIXATION

First and most critical step in tissue processing

FIXATION

Preserve the morphologic and chemical integrity of the cell in a life-like manner as possible

FIXATION

EFFECTS OF FIXATIVES

● Hardens soft tissues in preparation for further tissue processing

● Render cells resistant to damage caused by chemicals used in further processing

● Inhibit decomposition caused by bacteria and fungi

● Minimize the risk of occupational infection

● Act as mordant for certain stains, thus promoting or hastening staining, or inhibit certain dyes

● Reduce the risk of infections during handling and actual processing of tissues

CHARACTERISTICS OF A GOOD FIXATIVE

● Cheap

● Stable

● Safe

● Quick

● Inhibits bacterial decomposition

● Produce minimum shrinkage

● Rapid and even penetration

● Hardens the tissue

● Makes cellular contents resistant to further processing

● Permit staining

True or False?
No single fixative has all the good characteristics. Each of them has its own advantages and disadvantages.

True.

FACTORS AFFECTING FIXATION

Fixative of Choice

Time

Tissue-to-Fixative Ratio

Penetration Rate

Thickness of Specimen

Tissue Components

pH

Temperature

Osmolality

Agitation, Vacuum

What is the Fixative of Choice?

10% Neutral Buffered Formalin

■ Morphologic criteria for dx have been established based on Formalin-Fixed Paraffin Embedded Specimen (FFPES)

This means that it must be performed as soon as possible; 20-30 mins after blood supply is cut off

Time

Ideal Tissue-to-Fixative Ratio

1:10 or 1:20 (tissue to fixative ratio)

Penetration Rate of Formalin?

Formalin: 1mm/hr (but slows down as it goes deeper into the tissue)

The Larger the specimen → ?

Longer fixation time, more fixative

Ideal thickness of spx for Light Microscopy

Microscopy: 2cm2 x 0.4cm

Ideal thickness of spx for Electron Microscopy

1-2mm2

Tissue Components that have longer fixation time

■ Fibrous Tissue

■ Mucus → wash with NSS

■ Fat → cut into thin slices → fixed longer

■ Blood → flushed out with saline

Tissue Components that have shorter Fixation time

■ Small of loosely textured tissues

Optimal pH for fixation?

6 to 8 pH (Use buffers)

True or False?

For Electron Microscopy: pH should match physiologic pH

True.

Higher temp in fixation → ?

faster fixation rate and autolysis

True or False?

○ Hypertonicity → cell swelling

○ Isotonicity and Hypotonicity → cell shrinkage

False.

○ Hypertonicity → cell shrinkage

○ Isotonicity and Hypotonicity → cell swelling

in Osmolality, one must maintain tissues at slightly hypertonic solution which is how many Milliosmoles?

400-450 mOsm

This factor Hastens fixation

Agitation, Vacuum

2 General Types of Fixatives

Based on Composition

Based on Action

2 Types of Fixatives based on Composition

Simple

Compound

3 Types of Fixatives based on Action

Microanatomical

Cytological

Histochemical

Types of Cytological Fixatives

Nuclear

Cytoplasmic

Fixative made of only one component

Simple Fixatives

Fixative consists of two or more components of fixatives

Compound Fixatives

General study of tissues w/o structure alteration

Microanatomical Fixatives

● pH ≤ 4-6

● Glacial acetic acid has affinity to nuclear chromatin

Nuclear Fixatives

● pH ＞ 4-6

● HAc destroys mitochondria and Golgi bodies

Cytoplasmic Fixatives

Preserves chemical constituents of cells and tissues

Histochemical Fixatives