FIXATION AND PRESERVATION

General Principles of fixation and preservation of Histological and cytological specimens

Definitions of Terms 

·         Histology is a study of structure, composition and function of tissue(s)

·         Histopathology is a study of abnormal or diseased tissue.

·         Cytology is a study of structure, composition and function of cells.

·         Cytopathology is a  study of abnormal or diseased cells

·         Biopsy is tissue specimen taken from a living body.

·         Autopsy/post mortem/necropsy is an examination of dead body to determine the cause of death.

·         Autopsy specimen is a tissue specimen taken from a dead body.

·         Autolysis is a cell death due self enzymes digestion

·         Putrefaction is a cell death due to bacterial action

·         Post mortem changes are the changes which occur after tissue death, can be due autolysis, putrefaction or both.

·         Fixation is a process of preserving tissue or cells using chemical agent(s) in as life-like manner as possible.

·          Fixative is a chemical agent that is used to preserve tissue or cells.

·         Decalcification is a process of removing calcium from bone and calcified tissue.

·         Tissue processing is a preparatory treatment of tissue before being sectioned, comprised of dehydration, clearing and infiltration.

·         Dehydration is a process of removing free water (not molecularly bound water) from the tissue.

·         Clearing is a process of replacing the dehydrating agent with a reagent that is miscible with paraffin wax.

·         Infiltration/impregnation is a processing of filling in tissue, intracellular and extracellular, spaces with a medium which supports it during sectioning.

·         Tissue blocking/ embedding/ casting is a process of enclosing the tissue in the infiltration medium used for processing and then allowing the medium to solidify.  

·         Microtomy/section cutting is a cutting of thin sections of tissue  for microscopic examination using a microtome.

·         Adhesive materials are materials which are thinly smeared on the microscope glass slided before mounting for the purpose of increasing adherence of tissue section to slide. E.g. Mayer’s egg-albumin.

·         Stain is a substance used to impart colour to tissue or cells, facilitate microscopic study and identification.

·         Staining is a process of imparting colour to tissue or cells so as to facilitate microscopic study and identification. 

·         Mounting/coverslipping is a process of covering the stained slide with coverslip using special media in between them.

·         Mountants are special media with gluing property used to facilitate adhering of coverslip to stained slide.

·         Slide smear a thin tissue or blood sample spread on a glass slide and stained for cytologic examination and diagnosis under a microscope

·         Cytological aspirates is a specimen obtained by sucking fluid from the body for the purpose of harvesting cells for investigation.

Classification of histological fixative

Histological fixatives are those fixatives which are used for the purpose of preserving general morphology and inclusion of the tissue.

Classification of can be based on either of the following:

1.      According to their constituents

2.      According to their mechanism of action or

3.      According to reaction with soluble proteins

1.      According to their constituent:

a.       Simple fixatives(individual)   

b.      Compound fixatives(two or more)

                                                              i.      Micro-anatomical fixatives

                                                            ii.      Cytological fixatives

2.      According to mechanism of action:

a.       Aldehydes

b.      Mercurials

c.       Alcohols         

d.      Oxidizing agents

e.       Picrates

3.      According to their reaction with soluble proteins.

a.       Coagulant

b.      Non-coagulant

1.  According their constituents, fixative are classified into:

A.    Simple fixatives(individual)

Is a fixative that contains individual fixing agent in it.

They include Formaldehyde,Glutaraldehyde, Picric acid, Acetic acid, Osmium tetroxide, Ethanol, Potassium dichromate, Chromic acid, Mercuric chloride,

Formaldehyde

Is gas with pungent odour, soluble in water to a maximum extent of 40% by weight.

Formaldehyde doesn’t precipitate proteins but slightly precipitates other components of cell. Does render albumin insoluble though cause slightly hardening and slightly shrinkage of tissue. It prevents subsequent hardening by alcohols.

Thought to act by forming cross-links between proteins.

Neither preserve nor destroy adipose tissue, good fixative of complex lipids but has no effect to neutral lipids.

           

Formalin(formaldehde 40%) it is nearly acid. Becomes acid on storage when oxidizes to formic acid. Acidic pH favour autolysis and cause precipitation of formol-heme pigment {formalin pigments} which can be seen in sites containing blood. A buffer prevents acidity.

           

Glutaraldehyde

Is a gas soluble in water. also used as vapour fixative. Other uses include chemical sterilization and disinfection

Act by forming cross-links between protein end groups.

Because of its low penetrations, only small blocks of tissue (1-2 mm) fix well at 1-4 ˚C. Fixed tissue specimen can be stored in buffer solution for many months.

 Picric acid,

Is bright yellow crystalline, explosive when dry or if heated. In solution colours everything yellow.

Picric acid coagulates all proteins including nucleoproteins and cause little hardening but much shrinkage of tissue.

Picric acid is stored under water since it is explosive when dry.

Acetic acid

Is a solution with pungent odour. It is used together with other fixatives; when used alone, picric acid cause tissue swelling.

Acetic does not fix nor destroy carbohydrates, and it does not fix lipids. It preserve well nucleoproteins.

Penetrates tissue thoroughly and rapidly but lyses red blood cells.

Osmium tetroxide

Osmium tetroxide is pale yellow crystalline powder. It react with proteins by forming cross-links, react with unsaturated lipids to form black compounds.

OsO₄  preserves lipid by stabilization. OsO₄ vapours preserve blood and tissue smears, fixed tissue specimen often crumble if embedded in paraffin wax.

It penetrates poorly into tissue, is effective for small (1-2mm) specimens.

It interferes with many staining procedures.

The hazards of Osmium tetroxide is that, if in contact with cornea of an eye can result into blindness.

Ethanol,

They cause shrinkage and hardening of tissues.

Coagulate protein but not nucleoprotein and dissolve many lipids.

Ethanol and methanol are good for cytological smears because they penetrate quickly and give good nuclear details.

Ethanol is preferentially used at 95% concentration as fixative.

It has reducing property  hence should not be mixed with strong oxidizing agents like chromic acid, potassium dichromate and osmium tetroxide.

Potassium dichromate

Potassium dichromate is an orange crystalline substance used at 2% solution with water, fixes tissue by oxidizing proteins.

If mixed with ethanol it forms insoluble lower oxide that can not be removed from issue.Tissue fixed in potassium dichromate must be washed thoroughly in water before commencing dehydration in alcohols.

Chromic acid

·         Chromic acid is a strong oxidizer hence used with other fixatives, but not alcohols and formalin.

·         Coagulate proteins and fixes carbohydrates.

·          If tissue is not washed well after fixation in chromic acid, an insoluble precipitates will be formed.

Mercuric chloride,

·         Mercuric chloride is a white crystalline substance, soluble in water and alcohol.

·         Coagulates protein and cause tissue shrinkage and hardening.

·          It is the best fixative for hematopoietic and reticuloendothelial tissues. Fixes both nucleus and cytoplasm.

·         Forms Mercuric pigments, which are radio-opaque, in tissues hence unsuitable for x-ray method as the way to determine the end point of decalcification

B.     Compound fixatives

These are fixative which contain more than one fixing agents in them. For example Bouin’s fluid consists of picric acid, glacial acetic acid and formaldehyde.

They have further been divided into:

                                                              i.      Micro-anatomical fixatives

Are used to preserve various layers of tissue and cells in relation to one another. They include 10% Formal-saline, 10% Neutral buffered Formalin, Haidenhan’s Susa, Zenker solution, Helly’s fluid, Bouin’s solution, and Gendre’s fluid.

                                                            ii.      Cytological fixatives

Are used for preservation of  the constituent elements of the cell.

Theses have further been divided into;

-Nuclear fixatives        - Carnoy’s fluid and Flemming’s fluid

-Cytoplasmic fixatives –Helly’s fluid, Flemming’s fluid without acetic     acid.

Classification of cytological fixative

Cytological  fixatives, in this context, are fixatives used for the purpose of preserving cytological materials. They can be classified into two based on its form;

1.      Liquid fixative;

a.       50:50 Ether-ethanol

Is prepared by mixing equal parts of ether and 95% ethanol.

b.      95% ethanol

Is prepared by adding 5 mls of ethanol to 95 mls of distilled water to every 100 mls.        

c.       Schaudinn’s fluid

Is  prepared by mixing  saturated mercuric chloride, absolute ethanol and glacial acetic acid at 66 mls, 33 mls and 1 ml respectively.

Normally cytological smears soon after being prepared are immersed in any of the fixatives above. Fixation is complete at about 5 minutes.

2.      Vapour/Spray fixatives;

a.       Formaldehyde

b.      Glutaraldehde

c.       Acetaldehyde

d.      Aerosol-spray fixatives

·         To fix cytological smears with above mentioned aldehydes, on has to keep them exposed to the fume/vapour of fixative for about 10 minutes with smear facing down but not in contact with solution.

·         For aerosol-spray (which are alcohol based) the cytological smear has to be sprayed with industrially made fixative and left to dry. Fixation is achieved after 3 minutes.

Key Points

In the histopathology and cytopathology various terms are used some of them include biopsy, autopsy, decalcifaication tissue processing, fixation and fixatives,

Histologicalm fixatives are classified into different groups:

1.      According to their constituent:

a.       Simple fixatives(individual)   

b.      Compound fixatives(two or more)

                                                              i.      Micro-anatomical fixatives

                                                            ii.      Cytological fixatives

2.      According to mechanism of action:

a.       Aldehydes

b.      Mercurials

c.       Alcohols         

d.      Oxidizing agents

e.       Picrates

3.      According to their reaction with soluble proteins.

a.       Coagulant

b.      Non-coagulant

Cytological fixatives are classified into two:

1.      Liquid fixative;

a.       50:50 Ether-ethanol

b.      95% ethanol   

c.       Schaudinn’s fluid

2.      Vapour/Spray fixatives;

a.       Formaldehyde

c.       Glutaraldehde

d.      Acetaldehyde

e.       Aerosol-spray fixatives

Evaluation

·         Differentiate biopsy from autopsy specimen

·         Distinguish histological fixatives from cytological fixatives

References:

·         F.J. Baker, R.E. Silverton, Introduction to Medical Laboratory Technology, 7th Edition, (2001) Oxford University Press;

·         F.I. Carson, Histotechnology, A Self Instructional Text, 3^rd edition (2009) ASCP Press

·         R.A.B Drurry, E.A. Wallington, Carleton’s Histological Technique, (1976) Oxford University Press

·         J.D Bancroft, M. Gamble, Theory and Practice of Histological Techniques,6^th edition,(2008) Churchill Livingstone Elsevier.