INTRO & FIXATION

Numbering, Enter details in logbook, Give spx to pathologist, gross examination

What are the steps before Tx processing?

Formalin

What is the commonly used fixation?

Nitric Acid

What is the commonly used Decalcification?

Ethanol

What is the commonly used Dehydration?

Xylene/Xylol

What is the commonly used Clearing?

Paraffin Wax

What is the commonly used Infiltration?

What is the size of the spx to be processed?

3x2 cm

Thickness of specimen

3-5 mm

Autotechnicon

Fixation - Dehydration - Clearing - Infiltration

Constant Tx Agitation

can help reagent penetration

Fixation

Most crucial step in tissue processing

To preserve Tx as close to the original state

Primary goal in Fixation

To harden and protect Tx from further trauma

Secondary goal in Fixation

Physical Methods of Fixation

Less preferred, Heat Fixation, Microwave technique, Cryopreservation/Freeze Dry

Heat Fixation

For bacteriological smears

Microwave technique

For neurochemical substances (i.e., acetylcholine)

Chemical Methods of Fixation

More preferred, Uses fixing agents or fixatives

Physical and Chemical Method

2 Methods of Fixation

Standard Volume

10-20x

Volume for Osmium Tetroxide

5-10x

Volume for Museum Preparation

not less than 50x

pH concentration

6.0-8.0

Temperature for Manual

RT

Temperature in Autotechnicon

40 degC

Temperature for EM & Histochem

0-4 degC

Thickness for Lung spx

1-2cm

Osmolality

slightly hypertonic

Duration of NBF

2-6 hours to 1 week

Duration for EM

3 hours

Penetration

1mm/hr

Formalin and Alcohol

Best Penetrating Fixative

Glutaraldehyde

Worst Penetrating Fixative

Urgency of the case, type of Tx to be processed, Tissue structure to be studied, Staining technique to be applied, Type of section to be made

Factors in choosing the right fixative

Failure to fix immediately/Insufficient fixation

Failure to arrest early cell autolysis

Wrong choice of fixative

Removal of substances soluble in fixing agents

Incomplete washing

Presence of artifact pigments on tissue sections

Incomplete Fixation

Tx are soft and feather-like in consistency

wrong choice of fixative

Loss/inactivation of enzymes needed for study

Overfixation

Shrinkage and swelling of cells & tissue structure

Prolonged fixation

Tx blocks are brittle and hard

Simple Fixative

made up of 1 component substance (acetone, glacial acetic acid, TCA, aldehydes, metallic, picric, osmium tetroxide, alcohol)

Compound

Combination of simple fixatives; most common added are glacial acetic acid and TCA

Additive

Binds with tissues through cross-link formation or molecular complexes

Formaldehyde, Osmium tetroxide, Mercuric chloride

Examples of Additive

Non-additive

Do not bind with tissues but involves tissue alteration. Stabilizes through water removal.

Acetone, Alcohol (Carnoy’s, Newcomer’s)

Examples of non-additive

Microanatomical

General microscopic study of tissue structures w/o altering structural pattern

10% Formol Saline, 10% NBF, Heidenhain’s Susa, Formol sublimate, Zenker’s solution, Zenker-formol, Bouin’s, Brasil’s

Fixatives under Microanatomical

Nuclear Fixatives

w/ glacial acetic acid

<_ 4.6

pH of nuclear fixatives

Heidenhain’s susa, Flemming’s, Necomer’s, Carnoy’s, Bouin’s

Example of Nuclear Foxatives

Cytoplasmic Fixatives

w/o glacial acetic acid

>4.6

pH of Cytoplasmic Fixative

Helly’s, Orth’s, Regaud’s, Flemming’s w/o acetic acid, Formalin w/ postchroming

Examples of Cytoplasmic Fixative

Histochemical

Preserve chemical components of tissues

10% Formol saline, absolute alcohol, newcomer’s, acetone, lead

Examples of Histochemical

Newcomer’s

for mucopolysaccharide

Acetone

for enzymes

Lead

for acid mucopolysaccharide

Commercial formaldehyde (formalin)

approx. 35%-40% solution

causes dermatitis, irritating fumes to eyes/nose, and asthma

Reagent-grade formaldehyde

contains 10% methanol

Used as preservative to retard decomposition to formic acid and prevent precipitation to paraformaldehyde

Has denaturing effect on proteins

37-40% Formaldehyde

Undiluted form and is also known as 100% formalin

10% Formalin

Precipitation of white paraformaldehyde formation is possible.

10% NBF/Phosphate-buffered Formalin

Best general tissue fixative

preparation is time-consuming

Sodium Phosphate

Buffer of 10%NBF

Calcium acetate formalin (Lillie’s)

Preserve phospholipids

10% Formol-saline

Diluted w/ NaCl or NSS

For post-mortem or autopsy tissues, CNS tissues, fats, and enzymes

Formol-corrosive

100% formalin and Mercuric chloride

Excellent for silver reticulum methods

For lipids, neutral fats, and phospholipids

Alcoholic formalin (Gendre’s)

Diluted w/ ethanol

For sputum preservation & microincineration techniques

Glutaraldehyde

Most effective aldehyde for protein cross-linking

Made up of 2 formaldehyde residues, linked by 3 carbon chains

Recommended for EM & Enzyme Histochem

expensive and reduces PAS [+] of reactive mucin

Immersion glutaraldehyde w/ glacial HAC and aniline oil

Remedy in glutaraldehyde

2.5% glutaraldehyde

Solution recommended for small tissue fragments

4% glutaraldehyde

Solution recommended for larger tissues with thickness less than 4mm

Karnovsky’s paraformaldehyde-glutaraldehyde and Aclrolein

used for EM and immunohistochem (IHC)

Mercuric Chloride

Most common metallic fixative

Excellent for Trichrome staining & Tx photography

Not used due to toxicity

Produces black granular deposits except for Heidenhain’s Susa

Wash w/ alcoholic iodine

Remedy for black granular deposits

Zenker’s Fluid

Mercuric chloride + glacial acetic acid

For fixing small pieces of liver, spleen, connective tissue fibers & nuclei

Zenker’s formol/Helly’s

For pituitary gland, bone marrow, and blood-containing organs (i.e, liver and spleen)

Better nuclear fixation and staining than Zenker’s

Preserves cytoplasmic granules better than Zenker’s

Brown pigments are produced

Immerse Tx in alcoholic picric acid

Remedy for brown pigments in Helly’s

Heidenhain’s SuSa

Recommended for tumor biopsies (especially of the skin)

Schaudinn’s/Sublimated Alcohol

Used on wet smears for cytologic examinations

Lillie’s B5

Contains anhydrous sodium acetate

For BM spx

Chromate

Highly corrosive to skin and mucus membrane

Regaud’s/Moller’s

Recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi-bodies, RBCs, and colloid-containing tissues

Hardens the tissues better and more rapidly than Orth’s

Prolonged fixation blackens the tissue pigments

Wash w/ Tap water

Remedy for blacken Tx in Regaud’s

Orth’s

Recommended for study of early degenerative processes and Tx necrosis

Demonstrates Rickettsia and other bacteria

1-2% Chromic Acid

It precipitates all proteins and adequately preserves carbohydrates

3% Potassium Dichromate

Preserves mitochondria and lipids (pH 4.5-5.2)

Lead

Recommended for acid mucopolysaccharides and connective tissue mucins

Bouin’s

Fixation of embryos, pituitary biopsies, and endometrial cutterings

Not suitable for fixing kidney structures, lipid, and mucus

Reduces or abolishes the Feulgen reaction

Brasil’s

Best routine fixative for glycogen

Hollande’s

For GIT biopsies and endocrine tissues

Methanol

100% - wet & dry smears, blood smears, BM tissues

10% - added to prevent precipitation of formaldehyde to white paraformaldehyde

Ethanol

70-100% - blood tissue films and smears

Isopropanol

95% - touch preparations

Carnoy’s

Most rapid fixative

Recommended for fixing chromosomes and lymph glands

Used w/ acetone in brain Tx for rabies diagnosis

Rossmann’s

Recommended for CT mucins & umbilical cord

Newcomer’s

Nuclear and Histochemical fixative

For Mucopolysaccharides and nuclear protein

Compatible w/ feulgen reaction

Osmium Tetroxide

It is a pale-yellow powder which dissolves in water to form strong oxidizing solution

Adequately fixes materials for ultra thin sectioning in EMZ

For myelin and peripheral nerves specimen

Inhibits Hematoxylin & makes counterstaining difficult

TCA

It precipitates proteins and nucleic acids

Softens dense fibrous tissues

Poor penetrating fluid, often incorporated in compound fixative