TOPIC 2: FIXATION

.Fixation

Defined is the process or killing, penetration, and hardening of tissue

Fixation

most important a crucial step in tissue processing

Fixation

May also be defined as alteration of tissues by stabilizing protein so that tissues become resistant to further changes.

Fixation

First and most critical step in routine tissue processing.

Fixation

The process stopping all cellular activities so that cells can be viewed under the microscope.

Fixation

stabilization of proteins - Most important reaction in maintaining the morphology of tissue during fixation.

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

Primary aim of fixation

hardens and protects the tissue from trauma of further handling, so that it is easier to cut during gross examination.

Secondary aim of fixation

Fixation Method (Physical)

examples of this type of fixation is heating (-120C to -180C), microwave heating, cryopreservation.

Fixation Method (Chemical)

usually achieved by immersing specimen in fixative (immersion fixation) or, in the case of small animals or some organs such as a lung, perfusing or injecting the vascular system with fixative - perfusion fixation.

Heating Microwave heating Cryopreservation

What are examples of Physical methods of Fixation?

Immersion fixation Perfusion fixation

What are examples of Chemical fixation?

Heat fixation

not usually carried out in histopathology → done in microbiology.

Heat fixation

Each component is less soluble in water after heat fixation.

Heat fixation

Primarily used to accelerate other forms of fixation as well as the steps of tissue processing.

Microwave heating

speeds fixation and can reduce times for fixation of some gross specimens and histological sections from more than 12 hours to less than 20 minutes.

Microwave heating

Commercial glyoxal-based fixatives which do not form a vapor when heated at 55°C.

Microwave heating

introduced as an efficient method of microwave oxidation.

Cryopreservation

also known as freeze drying.

Freeze drying, quenching, 2mm, dessicatioin

A special way of preserving tissues by rapid freezing (__________) of fresh tissue (____ thick) and subsequently removing it from iced water (_____) by a physical process of transferring the still frozen tissue block in a vacuum at a higher temperature.

-20°C to -40°C.

Sublimation temperarure

Freeze substitution

Rossman's Formula/1% Acetone

Absolute Alcohol

Similar to freeze drying, the only variation is that the frozen tissue instead of being subjected to dehydration in an expensive vacuum drying apparatus, frozen tissue is fixed in_________________, and dehydrated in _________

Immediately fix tissue after removal.

Factor involved in fixation - Speed

1 mm/hr for formalin.

Factor involved in fixation - Rate of penetration

neutral, between pH 6 to 8.

Factor involved in fixation - pH

room temperature.

Factor involved in fixation - Temperature (routine tissue processing)

60C - use formalin.

Factor involved in fixation - Temperature (urgent biopsies/rapid fixation)

100C.

Factor involved in fixation - Temperature (Fixing tissues with Tuberculosis)

0-4C.

Factor involved in fixation - Temperature (Electron Microscope and Histochemistry)

40-42C.

Factor involved in fixation - Temperature (Automatic Tissue Processor)

The recommended size of the tissue is 2 cm² (except for lung specimen) and no more than 4 mm thick.

Factor involved in fixation - Thickness of section

Factor involved in fixation - Thickness of section (Electron Microscope)

1-2 mm thick.

4 mm thick.

Factor involved in fixation - Thickness of section (Light Microscopy)

slightly hypertonic but in reality, isotonic can be used usually at 400-450 mOsm.

Factor involved in fixation - Osmolality

10%.

Factor involved in fixation - Concentration (Formalin)

used at 3% in electron microscopy; 0.25% in immunochemistry.

Factor involved in fixation - Concentration (Glutaraldehyde)

20X the volume of tissue (20

Factor involved in fixation - Amount of fixative/volume (Routine tissue processing)

5-10X the volume of specimen.

Factor involved in fixation - Amount of fixative/volume (Osmium tetroxide)

20X the volume of specimen.

Factor involved in fixation - Amount of fixative/volume (Electron Microscope)

not less than 50X the volume of the specimen.

Factor involved in fixation - Amount of fixative/volume (Museum preparation)

ideal time to do

Factor involved in fixation - Duration of fixation

Adequate fixation

4-6 hours following removal from the body______ fixation

Prolonged fixation

can lead to tissue shrinkage, too much hardening, inhibit of chemical reaction.

mortuary ref (temp 4C)

Autopsy materials should be fixed as soon after death as possible. If not possible, body must be placed in a______________

cotton, soaked in fixative

Hollow organs (stomach, intestine) should be packed with _____________

gauze.

Air filled lungs may float in fixative, cover it with several layers of _____

intravascular perfusion, Ringer's lactate.

Human Brain must undergo _________using the reagent _________

dissected

Eyes should not be _______ before they are fixed.

Lendrum's method – immerse in 4% phenol, 1-3 days

Hard tissues (cervix, uterine, fibroid etc) must undergo _____________________________

Harden the tissues, makes cells resistant to damage

Increase optical differentiation of cells

acts as mordant/accentuator (facilitates easy staining)

Effects of fixatives in general

1 mm/hr.

Penetration rate

Fixation time

hours immersed in fixative.

Longer fixation time

tissue covered with mucus, blood, fats should have _____

Shorter fixation time

apply heat 56C to 37C to for a _____fixation time

Incomplete washing may lead to formation of Artifacts

Overfixation will render the tissue brittle, hard, cause shrinkage and swelling

Loss of substance soluble in fixing agent

Loss or inactivation of enzymes may result from wrong choice of fixative.

Problem in fixation

need for immediate examination (urgent biopsies)

type of specimen to be processed

tissue structure to be studied.staining technique

Factors to be considered when choosing the right fixative

It must be cheap, stable, and safe to handle

It must kill cells quickly and must inhibit bacterial decomposition and autolysis

It must make tissue resistant to damage and distortion, increasing optical differentiation of cells

It must produce minimum shrinkage of tissues. It must permit rapid and even penetration of tissues

It must harden the tissue

It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.

Characteristics of a good fixation

Additive fixation

Chemical constituent of the fixative is taken in and becomes a part of tissue.

Formaldehyde

Osmium tetroxide

Mercuric chloride

What are examples of Additive fixation?

Non-additive fixation

Fixing agent is not taken into the tissue but it alters the tissue composition thus, stabilizing the tissue which makes them unsuitable for bacteria decomposition.

acetone and alcohol.

All fixatives are additive fixation except for_____ and ______

Alcohol

Acetone Fixative

What are examples of Non-additive fixation?

Simple fixative

Made up of only one component of substance.

Compound fixative

Made up of 2 or more fixatives which have been added together.

10% Formol Saline

Bouine's Solution

What are examples of Compound fixatives?

Formaldehyde

NaCl

Distilled Water

10% Formol Saline contains what 3 components

formaldehyde

glacial acetic acid

picric acid

Bouine's Solution contains what 3 components

Formaldehyde

10% Formol Saline

10% Neutral Buffered Formalin (phosphate-buffered formalin)

Formol-corrosive (Formol Sublimate)

Alcoholic Formalin (Gendre's Fixative)

Glutaraldehyde

Glyoxal

What are examples of Aldehyde fixatives?

Mercuric Chloride

Chromate Fixative

Lead Fixative

What are examples of Metallic fixatives?

B-5 fixative

Heidenhain's Susa Solution

Zenker's Fluid

Zenker-Formol (Helly's Fluid)

What are examples of Mercuric Chloride fixatives?

Chromic acid

Regaud's Fluid

Orth's Fluid

Potassium Dichromate

What are examples of Chromate fixatives?

Bouin's Solution

Brasil's Alcoholic Picroformol

What are examples of Picric acid fixatives?

Isopropyl alcohol

Carnoy's Fluid

Ethyl alcohol

Methyl alcohol

Newcomer's Fluid

What are examples of Alcohol fixatives?

Fleming's Solutio

Fleming's Solution without Acetic Acid

What are examples of Osmium tetroxide fixatives?

Trichloroacetic Acid

not a routine fixative

need to be mixed with others to become a fixative

fixative and decalcifying agent.

Acetone

fix and dehydrate tissues

fixative and decalcifying agent

fixative for brain tissue specimen for rabies detection.

dissolve fats

evaporates

raw material used in making shabu

Disadvantages of Acetone

utilized in cold temperature

preservation of enzymes.

Uses of Acetone

Microanatomical fixatives

permit the general microscopic study of tissue structures.

Microanatomical fixatives

it should never contain osmium tetroxide because it inhibits hematoxylin.

10% Formol Saline

10% Neutral Buffered Formalin

Heidenhain's Susa

Formol Sublimate (Formol-corrosive)

Zenker's Solution

Zenker-Formol

Bouin's Solution

Brasil's Solution

What are examples of Microanatomical fixatives?

Histochemical fixatives

preserve the chemical constituents of cells and tissues.

10% Formol Saline

Absolute Ethyl Alcohol

Acetone

Newcomer's

Phosphatases

Lipases

What are examples of Histochemical fixatives?

Cytological fixatives

preserve nuclear/cytoplasmic parts and particular microscopic elements of the cell.

Nuclear fixatives

preserve the nuclear structures. It usually contains glacial acetic acid due to its affinity for nuclear chromatin.It should have a pH of equal or less than 4.6.

Bouin's

Flemming's with Glacial Acetic Acid

Carnoy's, Newcomer's

Heidenhain Susa

What are examples of Nuclear fixatives?

Cytoplasmic preservatives

Preserve the cytoplasmic structures. It should have no glacial acetic acid which destroys mitochondria and Golgi bodies. It should have a pH > 4.6.

Flemming's Fluid without Acetic Acid

Formalin with post chroming

Orth's Fluid Helly's

Flegaud's

What are examples of Cytoplasmic preservatives?

Formaldehyde

most widely used for all. 37%-40% solution - 100% formalin (concentrated form which is not used)

a gas produced by the oxidation of methyl alcohol and is soluble in water to the extent of 37%-40% weight in volume.

Formaldehyde

formalin is saturated solution of 37%-40% in water

the usual 10% formalin used in fixation of tissues is a 10% solution, containing about 40% weight of formaldehyde

formaldehyde fixation is performed at room temperature.

allergic rhinitis

sinusitis

excessive lacrimation.

formaldehyde fumes are irritating to the nose and eyes and may cause _________________________

violent explosion

concentrated solutions of formaldehyde must never be neutralized since this might precipitate _________

cheap

stable

readily available

tolerant fixative for colloid tissue photography

dermatitis

irritating fumes.

10% Formalin (Advantages)

prolonged storage

precipitation

turbidity (add 10% methanol).

10% Formalin (Disadvantages)

10% Formol Saline

recommended for fixation of general post-mortem and central nervous tissues for histochemical examination.

Formol Corrosive/Formol Sublimate

recommended for routine post mortem tissues.

Gendre's/Alcoholic Formalin

used to fix sputum, since it coagulates mucus.

10% Neutral Buffered Formalin

best general purpose tissue fixative.

7

formaldehyde is usually buffered at pH _ with the use of phosphate buffer.

37%-40%

In order to prepare a 10% solution of formalin, mix 1 part (100 mL) of concentrated solution (____) to 900 mL of tap water or a 1