FIXATION - Lec & Lab

Numbering

• Includes entering the details of the specimen in a log book

• Classification as to whether it is a surgical, autopsy or cytologic specimen is likewise indicated

• Gross description is done by the pathologist

Fixation

• It is defined as the killing, penetration and hardening of tissues

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

• Used to preserve cells and tissues as close as possible to the original

Heat Fixation

This is not usually performed in histopathology, but used in the frozen section

Microwave Technique

• Increases movement of molecules thereby accelerating fixation, staining, decalcification, immunohistochemistry and EM

• May be used for neurochemical substances in brain like acetylcholine

Room temp

Routine Fixation temperature is carried out at?

0-4 deg C

Fixation temperature for EM & Histochemistry

40 deg C

Fixation temperature for Autotechnicon

Rapid fixation of urgent biopsies

Formalin @ 60 deg C is used for?

Fixing tissues with TB

Formalin @ 100 deg C is used for?

1-2 square mm

Thickness size in EM

2 square cm

Thickness size in LM

More than 4-5 mm

Tissues should not be ___________ thick except in processing lung specimens

Open or thinly sliced

Large solid tissues like uterus must be?

Slightly hypertonic

Osmolality of tissues must be?

Cell shrinkage

Effect of hypertonic osmolality causes?

Cell swelling

Effect of hypotonic osmolality causes?

Isotonic solution

It may be use as holding solutions for tissues to be transported to frozen sections or kidney biopsies for special processing

0.25%

The concentration of glutaraldehyde in immuno-EM

20-30 mins following interruption of blood supply

This is the ideal time to perform fixation

Fixation time

It refers to the period the tissue is exposed to formalin

• Large tissues

• Presence of mucus

• Presence of blood

• Presence of fat and cold temperature

Fixation is retarded by:

It can inactivate enzymes

Consequence of cold temperature in fixation?

• Use of smaller and thin tissues

• Agitation

• Heat

Factors that accelerate fixation time:

20x

For maximum effectiveness, the volume of the fixative must be ____ the volume of the tissue

20:1

Ratio of fixative to tissue

1mm/hr

Formalin diffuses into the tissue at a rate of approximately?

50-100x

Required volume of Osmium tetroxide is ____ the specimen volume

Fixative-soaked cotton

Hollow organs like the stomach and intestine should be packed in?

Lendrum’s Method

Hard tissues (cervix, uterine, fibroid etc) must undergo which method?

4% aqueous phenol

Give the solution and concentration used in Lendrum’s Method

Overfixation

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

Additive

• Type of fixative that is absorbed by the tissues

• It stabilizes tissue proteins

Non-additive

• Type of fixative that is not absorbed by the tissues

• Involves alteration of tissue components

• Formaldehyde

• Mercuric chloride

• Chromium trioxide

• Picric acid Glutaraldehyde

• Osmium tetroxide

• Zinc sulfate or chloride

Examples of additives:

• Acetone

• Alcohol

Examples of non-additives:

Cytological fixative

Type of fixative used to preserve parts of the cell

Nuclear fixative

Type of fixative that contains glacial acetic acid

• Flemming’s

• Carnoy’s

• Bouin’s

• Newcomer’s

• Heidenhain’s Susa

Examples of nuclear fixatives:

• Flemming’s without HAc

• Helly’s Formalin with post chroming

• Regaud’s / Moller’s Orth’s

Examples of cytoplasmic fixatives:

Microanatomical fixative

Type of fixative which allows the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues.

• 10% formol saline

• 10% neutral buffered formalin (NBF)

• Heidenhain’s Susa

• Formol sublimate/formol corrosive

• Zenker’s

• Bouin’s

• Brasil’s

Examples of microanatomical fixatives:

Histochemical fixative

Type of fixative used to preserve chemical components of tissues like enzymes

• 10% formol saline

• Absolute ethyl alcohol

• Acetone

• Newcomer’s

Examples of histochemical fixatives:

100% formalin

37-40% formalin is also known as?

10% formalin

It is a diluted form of the concentrated solution of formaldehyde

Formaldehyde / Formalin

• Cheap, easy to prepare and readily available

• Recommended for mailing specimens and for colored tissue photography

Brown

Formalin on prolonged skin contact can cause what pigment color?

Kardasewitsch method

MORSE TYPE:

• 70% ethanol

• 28% ammonia water

Lillie’s Method

MORSE TYPE:

• Hydrogen peroxide

• 28% ammonia water

Picric acid Method

MORSE TYPE:

Saturated alcoholic picric acid

10% methanol

This is added in case precipitation of white paraformaldehyde happens in prolonged storage

10% formol saline

It is diluted with distilled water with NaCl

Used for post-mortem tissues and for CNS tissues.

10% Neutral Buffered Formalin or Phosphate Buffered formalin

It is used for fixing tissues with iron pigments and for elastic fibers

Formol corrosive

• It is also known as formol sublimate

• It is recommended for lipids, neutral fats and phospholipids

Formaldehyde + Mercuric chloride

Give the composition of formol corrosive

Alcoholic Formalin

It is also known as Gendre’s solution

It is used in fixing sputum specimens and for micro-incineration techniques

• 95% ETOH

• Picric acid

• Glacial HAc

Give the composition of Gendre’s solution

Glutaraldehyde

It is recommended for enzyme histochemistry and EM

2.5%

Glutaraldehyde concentration for small tissue fragments

4%

Glutaraldehyde concentration for large tissues > than 4mm thick

Glyoxal

• Fast-acting fixative that is supplied as a 40% aqueous solution

• Surgical specimens are fixed within 4-6 hrs & small biopsy specimens within 45 min

Mercuric chloride

This is the most common metallic fixative and is excellent for trichrome staining

Zenker’s Fluid

• It contains Mercuric chloride and glacial acetic acid.

• It is used for fixing liver, spleen, CT fibers, and nuclei

Zenker’s Formol

• It is also known as Helly’s fluid

• It contains potassium dichromate and 40% formaldehyde

• It preserves the pituitary gland, BM, and other blood-containing organs

Heidenhain’s Susa

• It contains TCA, glacial HAc, and formalin

• It is used for preserving tumor skin biopsies

B5

• It has anhydrous sodium acetate

• It is used for preserving the bone marrow

• Schaudinn’s

• Ohlmacher’s

• Carnoy –Lebrun solution

Other metallic fixatives:

Chromic acid (1-2%)

It is used for preserving carbohydrates

Potassium dichromate (3%)

It is used for preserving lipids and mitochondria

Regaud’s

• It is also known as Muller’s Fluid

• It is used for chromatin, mitochondria, mitotic figures, golgi bodies and RBC containing colloid tissues

Orth’s fluid

• It is used for early degenerative processes and tissue necrosis

• Also used for Rickettsia and other bacteria

Lead fixative

Type of fixative used for acid mucopolysaccharide and tissue mucine

Picric acid

• It can act as fixative, as stain, and as decalcifying agent

• It is excellent for glycogen demonstration but can stain tissues yellow

Bouin’s solution

It is recommended for fixation of embryos, pituitary biopsies and endometrial curettings.

It abolishes Feulgen reaction

Hollande’s solution

It is used for GIT biopsies & endocrine tissues

Glacial Acetic Acid

• It is a compound fixative, and is recommended for nucleoproteins

• It solidifies at 17 degC and contraindicated for cytoplasmic fixation

Trichloroacetic acid (TCA)

It can be used as both fixative and decalcifying agent

Acetone

• It is used at ice-cold temperatures (-5 to 4 degC)

• It is recommended for preservation of enzymes and for fixing brain tissues to diagnose rabies

• It is also used as raw material for making SHABU

Alcohol fixative

• Type of fixative that rapidly denatures and precipitates proteins

• It can act both as fixative and dehydrating agent

• It causes glycogen polarization

Methyl alcohol

It is used for fixing wet and dry smears, blood smears and BM tissues

Isopropyl alcohol (95%)

It is used for touch preparations

Ethyl alcohol (70-100%)

It is used for blood, tissue films and smears

Carnoy’s fluid

This is the most rapid fixative and is recommended for fixing chromosomes.

Newcomer’s

• It is classified both as nuclear and histochemical fixative

• It can preserve mucopolysaccharides

Rossmann’s solution

It is used for fixing CT mucins & umbilical cord

Osmium tetroxide

• This fixative is used for electron microscopy and for fixing myelin and peripheral nerves

• It is also used for processing neurological tissues

Flemming’s

It is the most common chrome osmium acetic acid fixative, and is excellent for nuclear structures

• 4% formaldehyde

• Formol saline

Fixatives for Enzyme Histochemistry: