Tissue fixation, Processing

and H&E staining

Saikat Mitra
Moderator: Dr. Aravind Sekar
OUTLINE
 Tissue Fixation
 Tissue processing
 H&E staining
TISSUE FIXATION

A substance that fixes the tissue at a point in time by

minimizing the loss or enzymatic destruction of cellular and
extracellular molecules, maintaining macromolecular structures
and protecting tissues from destruction by microorganisms
AIMS OF FIXATION
 To preserve the tissue nearest to its living state
 To prevent any change in shape and size of the tissue at the
time of processing
 To prevent any autolysis
 To make the tissue firm to hard
 To prevent any bacterial growth in the tissue
 To make it possible to have clear stain
 To have better optical quality of the cells
IDEAL FIXATIVE
 Safe
 Rapid and effective fixation
 Prevent autolysis
 High quality and consistent staining
 Preserve cellular and subcellular components
 Devitalize infectious agents
 Applicability in a wide range of tissues
 Long shelf life
 Cost effective
METHODS OF FIXATION

CLASSIFICATION

PHYSICAL CHEMICAL

FREEZE CROSS-
HEAT MICROWAVE COAGULANT COMPOUND
DRYING LINKING
PHYSICAL FIXATION
 Freeze-drying:
 In this technique the tissue is cut into thin sections and then
rapidly frozen into a very low temperature [Liq nitrogen,
isopentane]. Subsequently the ice within the tissue is removed
with the help of vacuum chamber in higher temperature (−30 °C).
 Advantages:
 Excellent for enzyme study
 No change of proteins
 No shrinkage of tissue
 Preservation of glycogen
 PHYSICAL FIXATION
 Microwave Fixation: Electromagnetic wave with frequencies between 300 MHz
and 300 GHz creates electromagnetic field, and the dipolar molecules rapidly
oscillate generating heat by kinetic motion.

 Advantages:
 Uniform heat production
 No volume change of tissue
 Preservation of the tissue antigen

 Disadvantages:
 Tissue in the formalin for microwave fixation may produce toxic gas and
overhead hood is required.
 Heat injury may occur from microwave.

 Applications:
 In routine surgical pathology laboratory
 Urgent processing of special biopsies
CHEMICAL FIXATIVES
CROSS-LINKING FIXATIVES:
 Better called covalent additive fixatives
 Aldehydes, chloral hydrate, glyoxal, Hgcl2, Zncl2, OsO4.
 MOA: Cross-linking within/between proteins and nucleic
acids
FORMALDEHYDE
 Accidental discovery by Butlerov in 1859
 Practical aspects in 1868 by Van Hoffman
 Ferdinand Blum – use in histopathology
 Pure H-CHO: Vapor
 Pure H-CHO+H2O 37-40% H-CHO
(Formalin) : Commercially available
 For use: 10% solution of formalin which is neutral and
buffered ( 10%NBF)
 pH: 7.2-7.4
 Buffers: Sodium phosphate, monobasic monohydrate and
Sodium phosphate, dibasic anhydrous
 WHY “NBF”
 5-10% of commercially available formalin has formic acid
 Unbuffered aqueous solution: pH 5.0-5.5

Acid formalin

Amide groups
become
charged, Formalin
hence rate of Preservation of
pigment
cross-linking immune
recognition
slows
CONSTITUENTS OF NBF
 Tap water: 900mL
 Formalin (40% formaldehyde): 100mL
 Sodium phosphate, monobasic, monohydrate: 4g
 Sodium phosphate, dibasic, anhydrous: 6.5g
 pH: 7.2-7.4
MECHANISM OF ACTION

H-CHO+H20
HOCH2OH
Washing for
(Methylene hydrate)
24hrs
removes 50%
of reactive
Reactive groups
hydroxymethyl
groups
while washing
for 4 weeks
removes upto
90%
Primary and characteristic reaction
 Cross-linking: AT rich regions and increases with
temperature
 Reacts with C=C and –SH bonds in unsaturated lipids
 Does not interact with carbohydrates
FORMALIN
PREPARATIONS
 Carson’s modified Millonig’s phosphate buffered formalin –
Sodium hydroxide is present – better for EM
 Formal (10% formalin), calcium acetate - lipids
 Formal (10% formalin), saline
 Formal (10% formalin), zinc, unbuffered - IHC
 Formalin buffered saline
 Formalin buffered zinc
GLUTARALDEHYDE
 Buffer: Cacodylate
 EM: Excellent preservation of ultrastructural details
 Poor penetration
 Can result in false positive PAS staining (Free aldehyde
group)
OSMIUM TETROXIDE
 Osmium tetroxide (OsO4) is mainly used as a fixative in
electron microscopy.
 The compound causes oxidation of unsaturated bonds in the
biological tissue particularly lipid.
 It converts the unsaturated fatty acid into a stable product
known as glycol osmate.
 FACTORS AFFECTING
QUALITY OF FIXATION
Rate of formalin pH: 7.2-7.4
pH: PO CO Tris,
4
3-,
3
2-

penetration : acetate, cacodylate

1mm/hr

DURATION & SIZE

10 times TEMPERATURE
d = k√t
volume

0.5cm cuts for Temp: Fixation

fixation

CONCENTRATION ADDITIVES CaCl2, K-

thiocyanate, Ammo.
sulphate
400-450mOsm
COAGULANT FIXATIVES
DEHYDRANT ACIDIC
COAGULANT COAGULANT
FIXATIVE FIXATIVE

Glacial acetic
Ethyl alcohol
acid

Trichloroacetic
Acetone
acid

Picric acid
fixative
MECHANISM OF ACTION
DEHYDRANT ACIDIC COAGULANT
COAGULANT FIXATIVE FIXATIVE

CHANGE OF CHARGES
DEHYDRATION ON IONISABLE SIDE
CHAINS OF PROTEINS

DESTABILIZATION OF
DESTABILIZATION OF
ELECTROSTATIC AND
HYDROPHOBIC BONDS HYDROGEN BONDING

DISRUPTION OF
DISRUPTION OF TERTIARY
TERTIARY STRUCTURE
STRUCTURE OF PROTEIN OF PROTEIN
 NOTABLE FEATURES
DEHYDRANT COAGULANT ACIDIC COAGULANT
FIXATIVE FIXATIVE

 Ethanol: Precipitates, glycogen  Glacial acetic acid, picric acid

esp. glycogen storage disorders; and TCA: Components of
used in enzyme studies as it compound fixatives Eg; Carnoy
keep them in original state fixative (AA,ethanol and
chloroform)
 Acetone: Demonstration of
tissue enzymes esp.  Picric acid: Gives brilliant
phosphatase , lipase contrast to trichrome stain
BOUIN’S SOLUTION
 Saturated aqueous picric acid: 40% formaldehyde: Glacial
acetic acid = 3: 1: 0.2
 Small biopsies, connective tissue stains & glycogen
 Prolonged fixation : RBC lysis, dissolves calcium & iron,
brittleness
 Degradation of DNA & RNA
 Picric acid explosive if dry
SPECIAL FIXATIVES
MERCURIC FIXATIVES DICHROMATE FIXATIVES
 Act chiefly by combining with the  Oxidising agents
acidic group of proteins and
phosphoric acid group of nucleoprotein  Chromate ions forms complexes
 Zenker’s solution: Congested with water which interacts with
specimen, Trichrome stain hydroxyl and carboxyl groups
 B5 fixative of proteins

 Helly’s solution  Mitochondrial fixation:

preserves phosphatides
 Schaudinn’s solution
 Ohlmacher’s solution  Chromaffin reaction
 Carnoy – Lebrun solution
FIXATION ARTEFACTS
 Formalin pigment: Insoluble brownish-black granular refractile
birefringent pigment due to reaction of formalin with
haemoglobin derivatives.
 Removed by: 1.8% picric acid in absolute ethanol
 Mercury pigments: Dark-brown irregular deposit.
 Removed by: Lugol’s iodine followed by sod. thiosulphate
 .Dichromate deposit: This deposit may occur after dichromate
fixation if the tissue is not washed properly.
 Removed by: 1% acid alcohol
 Tissue shrinkage
 TISSUE PROCESSING
 Technique designed to remove all extractable water from the
tissue, replacing it with a support medium that provides
sufficient rigidity to enable sectioning of the tissue without
damage or distortion
STAGES OF PROCESSING
DEHYDRATION

CLEARNIG

IMPREGNATION

EMBEDDING &
BLOCKING

SECTION
CUTTING
FACTORS AFFECTING RATE
OF PROCESSING
AGITATION

HEAT VISCOSITY

VACUUM
 DEHYDRATION
 Removal of unbound water and aqueous fixatives from the
tissue components
 Always done in a graded series of reagents of increasing
concentration
 DEHYDRATION
• Ethyl alcohol: Expensive, not easily available
• Propanol
• Butanol: Plant and animal histology
• Glycol
• Acetone: Rapid onset, poor penetration; removes lipid
• Dioxane can be used without clearing, but has toxic fumes
 CLEARING
• Process of removing dehydrating solutions, making the tissue
components receptive to the infiltrating medium
• Xylene: The commonest clearing agent, causes marked
shrinkage of cells; ideal for blocks < 5mm thick
• Toluene: More tolerant of small amounts of water left in the
tissues, but is 3 times more expensive than xylene
• Chloroform: Slowest, health hazard;
• Cedar wood oil
• Benzene ; toxic
 IMPREGNATION
 Permeating the tissue in a support medium
 Holds the tissue in the final blocks
 Easy handling & storage of tissues
 Impregnation done 2-4 degrees above melting point of
wax(60 degree).
 EMBEDDING
 Enclosing of properly processed, correctly oriented specimen
in a support medium that provides external support during
microtomy

 Paraffin wax:
 Mixture of long chain hydrocarbons
 Melting point proportional to consistency
 May contain additives: Stearic acid, ceresin, DMSO.

Volume of embedding media should be atleast 25 times the volume of tissue

 EMBEDDING
 Paraffins can be purchased that differ in melting point, for
various hardnesses, depending upon the way the
histotechnologist likes them and upon the climate (warm vs.
cold)
 Embedding is done 5-7 degree above melting point of wax
 OTHER WAXES:
• Water soluble polyethylene glycol
• Ester wax
• Polyester wax
• Microcrystalline wax
 RESINS
• Acrylic (Glycol methacrylate)
• Epoxy (Araldite 502)
 Agar
 Gelatin
 Celloidin
TISSUE TRANSFER /
Dip and Dunk processor
FLUID TRANSFER/vacuum
infiltration processor
EMBEDDING
 Embedding, the final stage of the process, is carried out on
an embedding station
 The tissues, trimmed prior to processing to fit inside
cassettes (25 x 20 x 4 mm maximum), may have shrunk
considerably after the process and are orientated in the wax
mould to present required features/planes on the final cutting
surface
Embedding station
L-MOLD BASE MOLDS
MICROTOMY
 TYPES:
 Rotary microtome
 Rocking microtome
 Base sledge microtome
 Sliding microtome
 Cryomicrotome
 Ultramicrotome
 Laser microtome
 Knives are either of the standard thick metal variety or thin
disposable variety (like a disposable razor blade)
 The former type allows custom sharpening to one's own
satisfaction, but is expensive
 PLANE WEDGE KNIFE or HIGH PROFILE BLADES is
used in our lab for larger tissue
 Plastic blocks (methacrylate, araldite, or epon) are sectioned
with glass or diamond knives
 Cutting
Cutting thickness:
Liver : 2.5 microns
Kidney : 3 microns
Large tissues: 3- 4 microns
Thick sections (myelin, muscle) : 5-6 microns
Congo red : 8 micron
Semi thin: 1 micron
Ultra thin : 60 nanometer
 A glass knife can cut section down
to about 1 micron
 Ultrathin sections for electron
microscopy (1/4 micron) are best
done with a diamond knife which is
very expensive
 Cut sections are floated onto a warm
water bath to smooth out the
creases and then lifted out onto a
glass microscope slide and allowed
to dry on a hot plate set just below
the melting point of the wax
 Temp of water floatation bath is 6-8 degree below the
melting point of wax
 Fix the section at 56 - 60 degree to just melt the wax
 Cool at room temperature
 The glass slides are then placed in a warm oven for about 15
minutes to help the section adhere to the slide
 Once the sections are dried onto the slide, they can be
stained
 All tissues are routinely stained with the haematoxylin and
eosin method
 Stained slides are mounted under a glass coverslip using a
synthetic mounting medium such as DPX
 DECALCIFICATION
INORGANIC • Aqueous HNO3 5-10%
ACID • Perenyi’s fluid
DECALCIFIERS • Formalin - HNO3

• Aqueous HCOOH
ORGANIC ACID • HCOOH-Formalin
DECALCIFIERS • Buffered HCOOH

• Aqueous EDTA pH 7.0-7.4

CHELATING • Formalin EDTA
AGENTS
Perenyi’s fluid:
10% Nitric acid – 40 ml
Absolute ethanol – 30 ml
Chromic acid, 0.5% - 30 ml

5% Nitric acid – Routine Decal

Perenyi’s fluid -- Surface Decal
 ARTEFACTS
Causes: Improper fixation,
 type of fixative,
 poor dehydration
 paraffin infiltration,
 improper reagents, and
 poor microtome sectioning
 Staining- basics
 The embedding process must be reversed in order to get the paraffin
wax out of the tissue and allow water soluble dyes to penetrate the
sections

 what is bringing section to water (B.S.T.W)???

It is dewaxing the section and hydrating it

 Why is it done?
To allow water soluble dyes to penetrate the
sections
Bringing section to water
- wax removal
Most dyes are water or alcohol based ( not miscible
in wax)
Step 1: removal of wax.
- section is kept in hot air oven at 70 degrees for
two minutes.
This is done to melt the wax, so that it will take less
time to de-wax in Xylene bath.
Bringing section to water
- wax removal
 Now dip the section in three consecutive Xylene chambers, two
minutes in each chamber

 Xylene will dissolve the wax

 NOTE:
-The sections must be kept perfectly VERTICAL
for proper wax removal
Substitutes for Xylene: Toluene, Benzene, Chloroform,
Cedar wood oil, Methyl salicylate
Bringing section to water
- hydration
Ethanol is used to gradually hydrate the section and to
remove the excess of Xylene

Absolute Distilled
alcohol 90% alcohol 70% alcohol water
(20 sec) (20 sec)
(20 sec) (2 min)

Must always be gradual

Haematoxylin and Eosin staining
 Haematoxylin
 A basic dye and has affinity for the nucleic acids of
the cell nucleus.
Introduced by Waldeyer (1862), but not successful.
In 1865, Bhomer added “ mordant “ to haematoxylin.
In 1886, Ehrlich added 4% acetic acid .
Adding of acetic acid enhances the nuclear staining.
Source : Heartwood (log wood) tree
Haematoxylum campechianum from Mexican
state of Campeche
 Haematoxylin
Haematoxylin by itself does not have staining property
 Its oxidation product “ haematin” is the actual staining
compound
Haematoxylin -Ripening
The process of oxidizing haematoxylin to haematin is
called RIPENING.
Methods of ripening
Natural oxidation Chemical oxidation
By exposure to natural light and air By adding sodium iodide (Mayer’s
haematoxylin) or mercuric oxide
(Harris haematoxylin)
Slow process (6 to 8 weeks) Ready to use instantly
Retains stain for very long time Short lived staining since the
since it is completely oxidized. continuing oxidation destroys much
of haematin to a colourless
component.
Eg: Ehrlich’s haematoxylin
Delafield’s haematoxylin
Haematoxylin - mordant
 Haematin is anionic and has a poor affinity for nucleus
(anionic)
 So, nuclear staining needs a MORDANT (positive charge) to
be added – so net positive charge is obtained
 The cations used as mordants are:
Aluminium
Iron
Tungsten
Lead
Molybdenum
Haematoxylin -types

Haematoxylins are classified based on the mordant

used:
Alum haematoxylins (Aluminium
ammonium sulphate)
Iron haematoxylins
Tungsten haematoxylins
Molybdenum haematoxylins
Lead haematoxylins
Haematoxylins without mordants
Alum haematoxylins
 Good nuclear stain
 All stain the nuclei RED, which is then converted to blue
colour by washing the section in weak alkali . This process is
called blueing
 According to Mayer, the alkaline fluid precipitates the
mordant, which in turn carries haematin with it and makes the
nucleus blue.
Blueing
Bluing agents:
 Running tap water
 Hot water
 2% NaHCo3

 1% LiCo3

 Scott’s tap water (Mag sulphate, sod. Bicarbonate and tap water)
 Marble chips
 1% ammonia vapor
 Aluminium solutions
Progressive and regressive staining
 Regressive staining:
The slides are left in the dye for a set period of time and then
taken back through a solution such as acid-alcohol that removes
part of the stain.
Best for large batches of slides to be stained.
 Progressive staining:
The slide is dipped in the dye until the desired intensity of
staining is achieved, such as with a frozen section.
This is simple for a single slide, but difficult for batch
processing.
Alum haematoxylins
1. Ehrlich’s :
-also stains bone and mucopolysaccharides of cartilage.
-suitable for tissues exposed to acid

-not ideal for frozen sections usually

2. Carazzi’s haematoxylin (KI ripened)

Alum haematoxylins

3. Delafield’s haematoxylin : regressive stain – natural oxidised

4. Mayer’s haematoxylin :

- ripened by sodium iodide

-progressive stain

- used as counter stain for glycogen, IHC

Alum haematoxylins
5. Harris’s haematoxylin:
- ripened by mercuric oxide
-precise and selective nuclear staining
-progressive in exfoliative cytology
-Regressive in histopathology
6. Cole’s haematoxylin:
- better results when combined with Celestine blue -
colour fades
- cannot be used in combination with other acid
dyes
- not used in exfoliative cytology.
7. Gill’s Haematoxylin:
Harris’ hematoxylin

Hematoxylin powder 8gms

Mercuric oxide 4gm

Potassium alum 160gm

Distilled water 1600ml

Absolute alcohol 80 ml
Alum haematoxylins
Cole’s 20 to 45 min
Delafield’s 15 to 20 min
Ehrlich’s ( progressive) 20 to 45 min
Mayer’s Progressive: 10 to 20 min
Regressive : 5 to 10 min
Harris’s Progressive: 30 sec
Regressive : 5 to 15 min

Carazzi’s Progressive: 1 to 2 min

Regressive : 45 sec
Frozen: 1 min
Gill’s ( regressive) 5- 15 min
Iron haematoxylins
 Iron salts used as both oxidant and mordant

Disadvantages:

- Over oxidation due to strong oxidizing property

haematoxylin and the salt must be mixed just before using

- Time consuming
Iron Haematoxylins
1. Weigert’s Haematoxylin:
- FeCl3

- Used as a nuclear stain where acidic staining has to be a done

subsequently Eg: EVG, MT
Iron Haematoxylins
2. Heidenhain’s Haematoxylin:
- Ferric ammonium sulfate (used for differentiation
also)
- Used for: mitochondria, muscle striations, nucleus
and myelin
Iron Haematoxylins
3. Loyez haematoxylin
- for myelin

4. Verhoeff’s haematoxylin
- for elastic fibers
Tungsten Haematoxylins
 Phospho Tungstic Acid Haematoxylin (PTAH)

-Mordant : Phospho Tungstic acid

Muscle striations
Neuroglia Dark blue
Fibrin
amoeba
Nuclei
Cilia Blue
RBC
Myelin Light blue
Collagen
Osteoid Deep red- brown
Cartilage
cytoplasm Pale pink- brown
Molybdenum Haematoxylins
Rarely used
Only in Thomas technique

Collagen Violet to black

Coarse reticulin
Argentaffin cells Black
Nucleus Pale blue
Paneth cells orange
Lead Haematoxylins
To demonstrate granules in endocrine cells of GIT,
mainly for gastrin secreting cells
Haematoxylins without mordant
To demonstrate Lead, Iron and Copper in tissue
sections.

Concept: Unripened haematoxylin will form blue-

black lakes if these minerals are present in the tissue
Eosin
 Eosin
Xanthane dyes
Eosin is an acidic dye with an affinity for
cytoplasmic components of the cell.
Advantages:
- Best counter stain for alum haematoxylins
-Can distinguish between cytoplasm of different
cells and between different connective tissues and
matrices.
 Eosin
Commercially available types:
- Eosin Y ( yellow)
- Eosin B (Blue, used in haematology
staining)
- Ethyl Eosin ( not used)
 Eosin Y

Soluble in water and alcohol.

Usually used as 0.5% to 1% solution with thymol
(anti- fungal) and acetic acid ( for sharp staining)
Phloxin B is added to increase the range of colour
shades.
Over use of Phloxin will result in too bright staining.
 Eosin
 Eosin is attracted to tissue proteins by ionic forces, and then
held in place by van der Waals forces

 In sites where the bound eosin molecules are close together,

red blood cells,
granules of eosinophils
Paneth cells,
the colour is shifted from red towards orange

 Cytoplasm is red or dark pink

 Eosin
Collagen fibers, which contain relatively less protein
and more water than cytoplasm, are lighter pink

Eosin should impart at least three colours to a

correctly stained section
 Eosin
Counterstaining with eosin changes the colour of
haem alum-stained nuclei from blue to purplish

This additive colour change may be due to attraction

of eosin anions to positively charged amino acid side
chains of basic nucleoproteins.
 Eosin
Eosin differentiation:
1. Washing in tap water
2. During dehydration with alcohol
Staining process
AUTOMATED STAINER
PROTOCOL
STEP REAGENT DURATION
1 Drying station 4.00
2 Xylene 2.00
3 Xylene 2.00

4 Xylene 2.00

5 Ethanol 100% 00.30

6 Ethanol 90% 00.30

7 Ethanol 70% 00.30

8 Water station 00.10

9 haematoxylin 6.00
10 Water station 00.10
11 Acid alcohol 1% 00.05
12 water 00.10
13 Scott’s tap water 02.00
14 water 00.10
15 Eosin 1% 00.10
16 Water station 00.05
17 Ethanol 70% 00.10
18 Ethanol 90% 00.10

19 Ethanol 100% 00.10

20 Drying station 2.00
21 Xylene 10
22 Xylene 10
Acid Alcohol 1% (differentiation)

•70% Alcohol with 1% HCl

Differentiation is done in regressive staining
to remove extra stain from cytoplasm

Why ACID - ALCOHOL mixture???

- Alcohol by itself is a weak differentiating
agent
- Though acid is a strong differentiating
agent, it is corrosive
 Mounting
To make the stained slides permanent.

The refractive index of the mountant and cover slip

must be approximate to dried protein ( 1.53 to 1.54)
 Mountants
- types-
Aqueous mountants:
Refractive Index: 1.40 to 1.45
Examples:
1. Karo Corn Syrup
2. Laevulose
3. Farrant’s Medium
4. Glycerine Jelly
 Mountants
- types-
Synthetic resins:
1. DPX (Dibutylphthalate Polystyrene Xylene)
2. Neutral Balsam
3. Acidic Balsam
4. Xylene Damar
Thank You