Chemical fixatives

CHEMICAL FIXATIVES

Group B ⎸ Histopathology

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Chemical fixatives

• stabilize the proteins, nucleic acids and internal components of the tissue by making them insoluble
• neutral buffered formalin does not provide adequate fixation
• fixatives other than alcohol or formalin may be preferred

2 MAJOR GROUPS OF CHEMICAL FIXATIVES

Classified acc. to mechanism of action:

Crosslinking Fixatives (eg. Aldehydes) - that act by creating covalent chemical bonds between proteins in tissue Precipitating (or Denaturing) Fixatives (eg. Alcoholic fixatives) - that act by reducing the solubility of protein molecules

CROSSLINKING FIXATIVES – ALDEHYDES

• most commonly used fixative in histology is formaldehyde

• cross-linkage does not harm the structure of proteins, so that antigenicity is not lost

• the standard solution is 1O% neutral buffered formalin or approximately s.7%-4.O% formaldehyde in phosphate- buffered saline

• its effects are reversible with excess water and the presence of buffer

Other benefits:

• long term storage

• good tissue penetration

• immunohistochemistry techniques

• formaldehyde vapor can be used as a fixative for cell smears

BUFFERED FORMALIN

CHEMICALFIXATIVES

FORMALDEHYDE & FORMALIN

It is most widely used fixative for routine histology. This fixative can effectively prevent autolysis and provide excellent preservation of tissue and cellular morphology. It is also considered the fixative of choice for many other procedures that require paraffin embedding. Lastly, it usually contains about 10% methanol.

They are often used interchangeably. However, the chemical composition of each fixative is different

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FACTORS THAT INFLUENCE FORMALIN FIXATION

Post-Mortem/Post-Surgital Interval

Composition of Fixative

Fixation Time

Volume of Fixative

DELAY IN FIXATION

How to minimize it?

How to minimize it?

Inadequate fixation Too much fixation

Post-Fixation Storage

Tissue Thickness

Temperature

Mixture of Fixatives

Two aldehyde fixative mixtures:

Karnovsky's paraformaldehyde-glutaraldehyde solution: Best known mixture of fixative

Acrolein: a mixture with glutaraldehyde or formaldehyde

P R E C A U T I O N S

11

10

17

16

15

14

13

12

19

18

10% Formal-Saline

The solution should have a pH of 6.8

10% Neutral Buffered Formalin (NBF)

Advantages

Fixation time

Formula

Disadvantages

Formol – Corrosive (Formol – Sublimate)

Karnovsky’s Fixative

Paraformaldehyde

Zinc formalin (unbuffered)

Glutaraldehyde

• One of the popular aldehyde for fixation
• It is made up of two formaldehyde residues, linked by three carbon chains.
• Larger than formaldehyde
• Rate of diffusion across membranes is lower than formaldehyde
• Thicker tissue sample may be hampered (reduce the size of the tissue sample)

What is it?

• Causes rapid and irreversible changes
• Fixes quickly (fixes well at 4oC)
• Well suited for electron microscopy
• Gives best overall cytoplasmic and nuclear detail

Advantages

• causes deformation of alpha-helix structure in proteins
• not good for immunohistochemical staining
• It penetrates very poorly
• Must be cold and buffered and not more than 3 months old

Disadvantages

Precipitating (Alcohol) Fixatives

Alcohol are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness

• Good for cytologic smears
• Generally can act both as a fixative and as a dehydrating agent.
• Methanol, ethanol, acetone – used for studying nucleic acid

100% Methyl alcohol (Methanol)

DISADVANTAGES:

• Penetration is slow.

• If left in fixative for more than 48 hours, the tissues maybe over hardened and difficult to cut.

ADVANTAGES:

• It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues.

• It fixes and dehydrates at the same time.

90% Isopropyl alcohol

DISADVANTAGES

ADVANTAGES

• It dissolves fats and lipids.

-General rule: Alcohol-containing fixatives are contraindicated when lipids are to be studied.

• Glycogen granules move towards the poles or ends of the cell (polarization).

• Tissues left in alcohol for too long will shrink.

• It causes polarization of glycogen granules.

• It produces considerable hardening and shrinkage of tissue.

• Hemosiderin preserves less than buffered formaldehyde.
• It is a strong reducing agent.

-mixed chromic acid -potassium dichromate -osmium tetroxide

• Lower concentration (70%-80%) will cause RBC hemolysis and inadequately preserve leukocytes

• It preserves but does not fix glycogen.

• It fixes blood, tissue films and smears.

• It preserves nucleoproteins and nucleic acids.

• Fixes tissue pigments very well.

• Ideal for small tissue fragments.

• May be used as both as fixative and dehydrating agent.

Ethyl Alcohol

Carnoy's fixative

DISADVANTAGES

ADVANTAGES

• It produces RBC hemolysis, dissolves lipids and can produce excessive hardening and shrinkage.

• Causes considerable tissue shrinkage

• Suitable only for small pieces of tissue due to slow penetration.

• Dissolves acid-soluble cell granules and pigments

• Tends to harden tissue excessively and distorts tissue morphology

• Dissolves fat, tissue, and myelin

• Leads to polarization

• Most rapid fixative

-Paraffin processing within 5 hours

• Fixes and dehydrates at the same time.

• Permits good nuclear staining and differentiation.

• Preserve Nissl granules and cytoplasmic granules as well.

• Preserves nucleoproteins and nucleic acids.

• Excellent fixative for glycogen.

• Very suitable for small tissue fragments such as curettings and biopsy materials

• Following fixation for an hour, tissues may be transferred directly to absolute alcohol-chloroform mixture.

• Also used to fix brain tissue for diagnosis of rabies

• Has been used on frozen section and smears.

• Produce fair result after conventional processing If fixation time is kept very short

• It preserves nucleic acids but extracts lipids.

• Tissues can be transferred directly into 95% ethanol.

Clarke's solution

Alcoholic formalin

• Combines a denaturing fixative with the additive and cross-linking effects of formalin

• Sometimes used during processing to complete fixation following primary incomplete fixation

• Can be used for fixation or post fixation of large fatty specimens

• Can be placed directly to 95% ethanol for processing

Form0l-Acetic alcohol

• It is a faster acting agent than an alcoholic formalin due to the presence of acetic acid

• Sometimes used to fix diagnostic cryostat sections

• If used for primary fixation, the specimens can be placed directly into 95% ethanol for processing

Fixatives

Gendre's fixatives

Newcomer’s fluid

Applications: Enhance immunoperoxidase studies on the tissues and in electron microscopy.

Fixation time: 12 – 18 hours at 3 C

Metallic fixative

Mercuric Chloride

Zenker's solution

FORMULA:Mercuric chloride 5 gm Potassium dichromate 2.5 gm 1OO ml Distilled water 5 ml Acetic acid, glacial Fixation time: 12-24 hours

Zenker-Formol (Helly’s) Solution

Practical applications

Advantages

Disadvantages

Practical applications

1. This solution is popular for fixation of hematopoietic, bone marrow biopsies and lymphoid tissue. 2. It produces excellent nuclear detail, provides good results with many special stains 3. Sections will require the removal of mercury pigment prior to staining. 4. Over-fixation hardens the tissue

Lillie’s B-5 Fixative

most commonly used for bone marrow biopsies

Heidenhain’s Susa Solution

Precautions

Advantages

Disadvantages

CHEMICAL FIXATIVES

OXIDIZING AGENTS

Osmium Tetroxide (0smic Acid; OSO₄)

A pale-yellow powder which dissolves in water (up to 6% at 2O°C) to form a strong oxidizing solution. It is traditionally used in electron microscopy both as a fixative and a heavy metal stain. It is also a good fixative and excellent stain for lipids in membranous structures and vesicles. Lastly, it functions as a secondary fixative by reacting with lipids.

Precautions

Disadvantages

Procedure

Advantages

Fleming's solution

Advantages

Disadvantages

CHEMICAL FIXATIVES

CHROMATE FIXATIVES

CHROMIC FIXATIVES

POTASSIUM DICHROMATE

CHROMIC ACID

1. It fixes but does not precipitate cytoplasmic structures.2. It preserves lipids. 3. It preserves mitochondria

It is used in 1-2% aqueous solution, usually as a constituent of a compound fixative. Also, it precipitates all proteins and adequately preserves carbohydrates. Lastly, it is a strong oxidizing agent; hence, a strong reducing agent must be added to chrome-containing fixatives before use in order

Regauld's (Muller's) fluid

FORMULA: 8O ml Potassium dichromate (3%) & 2O ml Strong formaldehyde (4O%) (To be added just before use).

Advantages

Fixation time: 12-48 hours

Disadvantages

Orth's formula

FORMULA: 100ml Potassium dichromate (2.5%), Sodium sulfate (optional) 1gm, & 10ml Strong formaldehyde (40%)

Advantages

Fixation time: 36-72 hours

Disadvantages

CHROMIC FIXATIVES

CHEMICAL FIXATIVES

PICRIC ACID FIXATIVES

PICRIC ACID FIXATIVES

Picrates Includes fixatives with picric acid

How to remove excess picrate?

How to remove the yellow color?

What is Picric acid?

Removed from the sections when the paraffin was has been removed

What are immersed in picric acid solution/Bouin’s fluid before Trichrome stains?

What is Bouin's solution

Hollande's solution

Practical application: It is recommended for gastro-intestinal tract specimens and fixation of endocrine tissues. It also produces less lysis than Bouin’s Solution

Formula:

Copper acetate: 25 gmPicric acid: 40 gm 40% formaldehyde: 100 ml Acetic acid: 15 ml Distilled water: 1000 ml Fixation time: 4 – 18 hours

Gendre’s solution

FORMULA:95% Ethanol saturated with 800 ml picric acid 150 ml formaldehyde (40%) 50 ml Acetic acid glacial Fixation time: 4 - 18 hours

Brasil's Alcoholic Picroformol Fixative

FORMULA:2040 ml Formaldehyde (37%) Picric acid 80 gm 6000 ml Ethanol or isopropyl alcohol Trichloroacetic acid 65 gm

Glacial Acetic acid

• It is an undiluted acetic acid in which it is a water-free (anhydrous) acetic acid that freezes and solidifies at about 16°C.

• How it fixes: it does not have much effect on proteins other than to enable swelling by absorption of water. Its major effect is to precipitate DNA. With that, it is valuable for the preservation of nuclei and is often added to fixatives.

• It is incorporated into other fixatives to form a compound solution (approx. 5% concentration)

Advantages:

Disadvantages:

1. When combined with potassium dichromate, the lipid-fixing property of the latter is destroyed2. It is contraindicated for cytoplasmic fixation because it destroys the mitochondria and golgi elements of cells3. Concentrated acetic acid is corrosive cause skin to burn, permanent eye damage, and irritation of eye damage4. Latex gloves offer no protection thus those made of nitrile rubber are worn.

1. It fixes and precipitates nucleoproteins2. It precipitates chromosome and chromatin materials3. It causes the tissue to swell especially those containing collagen

LEAD FIXATIVES

• Used in 4% aqueous solution of basic lead acetate

• The primary reduction product precipitate for the visualization of the activity of glutamic oxaloacetic transaminase in tissue section is the lead acetate which is stable at slightly alkaline pH

• For the tissue fixation concentrations, acetone provides a slight inhibitory effect on glutamic oxaloacetic transaminase activity while for the glutaraldehyde-formaldehyde mixture results to marked reduction of activity.

TRICHLOROACETIC ACID

• This reagent is used for the precipitation of proteins and nucleic acids.
• It may also be used as decalcifier and fixative in microscopy
• Addition to a final concentration of 10% will precipitate most proteins from the solution
• 5% solution of ice cold TCA is used for the precipitation of nucleic acids

Advantages & Disadvantage

Acetone

What are its Advantages and Disadvantages?

What's Acetone?

Michel's solution

Comparison of different fixatives

SU M M A R Y

Fixative of choice for different substances

SU M M A R Y

Eureka!

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-Group B