TOP 1 HISTOLOPATHOLOGIC TECHNIQUES EXAM

TOP 1 EXAM

Fixation

• One of the most critical step in histotechnology

• Process that preserves tissues from decay, thereby preventing autolysis and putrefaction

• The process should be carried out as soon as possible after removal of tissue from the body

Autolysis and putrefaction

What does fixation prevent?

Goal of fixation

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

• Harden and protect the tissue from the trauma of further handling.

Objectives of fixation

• To preserve the tissue (stops all cellular activities)

• To prevent breakdown of cellular elements (prevent autolysis and putrefaction)

• To coagulate or precipitate protoplasmic substances (renders tissue components insoluble)

1. Hydrogen ion concentration

2. Temperature

3. thickness of the section (block)

4. Osmolality

5. Concentration of solutions

6. Duration of fixation

Main factors involved in fixation

1. Speed

2. Penetration

3. Volume

4. Duration of fixation

Practical considerations of fixation

Hydrogen ion Concentration

(pH 6 to 8)

for routine surgical specimens, room temperature.

Temperature

Thickness of the section (block):

Electron microscopy: 1 to 2 mm²

• Light microscopy: 2 mm² (recommended / 4-10mm)

• Measurements should not be compromised to obtain full penetration and satisfactory fixation.

• Large tissues like the uterus should be opened or sliced thinly.

Hypertonic

causes cell shrinkage.

Isotonic and Hypotonic

causes cell swelling and poor fixation.

For best results,

use slightly hypertonic solutions with an osmolality of 400 - 450 mOsm (isotonic solutions are 340 mOsm).

Concentration of solutions

Formaldehyde is most commonly used as 10% solution while glutaraldehyde is used as 3% solution. Low concentration (0.25%) of glutaraldehyde is ideal for immunoelectron microscopy.

Prolonged fixation:

may cause cell shrinkage and hardening of tissues.

Incomplete fixation:

softening of tissues, very hard to cut during section - cutting.

Speed

specimens are placed in fixative as soon as it is removed from the body. This is done to prevent autolysis and putrefaction.

Penetration

formalin diffuses into tissues at a rate of approximately 1 mm per hour and slows down as it goes deeper. However, this varies with different types of tissues.

Volume

the ideal fixative to tissue ratio is 20:1

Duration of fixation

the usual length of time for fixation is 24 hours but some tissues take longer times to fix than others.

Simple Fixatives:

made up of only one component substance

Compound Fixatives:

made up of two or more fixatives

Microanatomical Fixatives

Permit general microscopic study of tissue structures and normal intercellular relationship of tissues
- for general microscopic study of tissue structures.

Cytological Fixatives:

preserve specific cellular components

Nuclear fixatives

preserves nuclear structures of the cell. They usually contains glacial acetic acid (pH 4.6 or less) which fixes nucleoprotein.

Cytoplasmic fixatives

Useful for preservation of cytoplasmic details or structures (pH greater than 4.6). This must never contain glacial acetic acid because it destroys mitochondria and Golgi bodies.

Histochemical fixative

Preserve chemical constituents of the cell

Lipids:

mercuric chloride or potassium dichromate

Phospholipids:

Baker's formol-calcium

Cholesterol:

digitonin

Carbohydrates:

alcoholic fixatives

Glycogen:

Rossman's fluid or cold absolute alcohol

Proteins:

neutral buffered formol saline or formaldehyde vapor

Electron microscopy:

double fixation

Electron Cytochemistry:

Karnovsky's paraformaldehyde_glutaraldehyde solution

Acrolein:

mixture of glutaraldehyde or formaldehyde
rapid penetration, preserves morphology and enzyme activity at low concentrations v immersion fixation of surgical biopsies.

Aldehyde fixatives

for routine paraffin sections, electron microscopy, histochemistry and enzyme studies

Formaldehyde (Formalin)

• gas produced by the oxidation of methyl alcohol

• buffered atph 7 to 8

— hypoxia in tissues leads to acidity which favors the

formation of Formalin heme pigments (black, polarizable

deposits)

• Pure Stock: 40% formalin

• Dilution: 1:10 (10% solution); 1:20 (5% solution)

Paraformaldehyde

white precipitate due to prolonged storage
may be removed by filtration or addition of 10% methanol

10% Formol-Saline

• central nervous tissues and general post-mortem tissues for histochemical

examination

• ideal with most stains including silver impregnation

• duration of fixation: more than 24 hours (slow fixative)

10% Neutral Buffered Formalin

• MOST WIDELY USED FIXATIVE IN ROUTINE

HISTOLOGY

• preservation and storage of surgical, post-mortem and research specimens

• best fixative for frozen sections

• best fixative for iron pigments and elastic fibers

Formol-Corrosive

• routine post-mortem tissues

• for lipids, especially neutral fats and phospholipids

• no need for washing-out

Glutaraldehyde

* made up of two formaldehyde residues linked by three

carbon chains

• used in conjunction with osmium tetroxide

» Fixation time: ½ hour to 2 hours

ADVANTAGES
a. stable effect on tissues
b. preserves plasma
c. less tissue shrinkage
d. recommended for enzyme histochemistry & EM
e. it does not cause dermatitis and less irritating to the nose
DISADVANTAGE
| b. penetrates tissues more slowly
| c. tends to make tissue proteins better more brittle

Alcoholic Formalin

= used to fix sputum

« for the demonstration of immunoperoxidase activity

Mercuric Chloride

most common metallic fixative
V tissue photography
V tissues contain black precipitates of mercury (except Susa)

Zenker’s Fluid

fixing small pieces of liver, spleen, connective tissues fibers and nuclei

De-zenkerization:

removal of mercuric deposits in tissues

Zenker-formol (Hellys solution)

pituitary gland, bone marrow and blood containing organs (spleen and liver)

*Fixation time: 4-24 hours

Heidenhain's Susa Solution

v for tumor biopsies

v excellent cytologic fixative

*after using This, transferred the tissue to a high grade alcohol to avoid undue swelling.

Fixation Time: 3-12 hours

B-5 Fixative

for bone marrow biopsies

Osmium Tetroxide(Osmic acid; Os04)

v used in EM both as fixative and a heavy metal

stain.

v excellent stain for lipids in membranous structure

and vesicles.

Chromic acid

preserves carbohydrates

Potassium Dichromate

preserves lipids and mitochondria

Regauds Fluid (Mullers Fluid)

v demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissue.
*Fixation time: 12-48 hours

Orth's Fluid

v study of early degenerative processes and tissue

necrosis

v demonstrates Rickettsia and other bacteria

*Fixation Time: 36-72 hours

LEAD FIXATIVES

v demonstration of acid mucopolysaccharides

PICRIC ACID FIXATIVES

are soluble in water, therefore, tissues should not be washed in running tap water (use 70% alcohol instead)

Bouin’s Solution

v fixation of embryos and pituitary biopsies.

Brasil's Alcoholic Picroformol Fixative

v fixative for glycogen

GLACIAL ACETIC ACID

v fixation of nucleoproteins

Ethanol (95%)

- Preserves but does not “fix” glycogen granule

Methanol

- for dry and wet smears, blood and bone marrow samples

Isopropyl Alcohol (95%)

- for touch preparation

Carnoy’s fluid

- most rapid fixative
- for chromosomes, lymph glands, urgent biopsies and brain tissue for the diagnosis of rabies

.Newcomer’s

- demonstration of mucopolysaccharides and nucleoprotein

Gendre’s fixative

alcoholic form of bouins solution

Alcoholic Formalin

used for fixation or post-fixation of large fattyspecimens(particularly breast).

Flemming's (chrome-osmium HAc)

- Permanently fixes fats and recommended for fixing nuclear structures

. Flemming's w/o acetic acid

- For cytoplasmic structure

TRICHLOROACETIC ACID

v may also be used as a weak decalcifying agent

ACETONE

v use at cold temperature (-5 to 4 °C)
v fixation of brain tissues for rabies diagnosis

WASHING OUT

- The process of removing excess fixative from the

tissue after fixation in order to improve staining

and remove artefacts from the tissues.

- Several solutions may be used: tap water, 50 -

70% alcohol and alcoholic iodine.