Tissue processing

Speaker : Rajat Mondal

Associate Professor (PATHOLOGY)
Tissue processing
Submitting the total or selected part of the
tissue to a series of processes for
preparation of slides for microscopic
examination for histopathological
diagnosis.
Manual/Automated
Aim: prevent decomposition/autolysis &
preserve tissue in a manner resembling its
original character as close as possible.
Steps of histopathology:

Labelling
Fixation
Dehydration
Clearing Tissue processing
Infiltration
Embedding
Factors affecting tissue procesing

Heat-45°C ideal
Agitation
Vacuum
viscosity
Concentration &
time interval of
specific steps
Nature of tissue
Nature of
examinations
needed
Labelling

Unique identification number/code

Must accompany the tissue
throughout the process
Manual/automated
Check requisition form for name,age,
short history,site of origin.
Fixation
Process by which tissue is fixed in a
physical/chemical state so that they
will withstand subsequent treatment
with reagents with minimum loss,
distortion or decomposition.
Physical- heat, microwave, freeze-
drying.
Chemical-coagulant, dehydrant,
cross-linking.
Ideal Fixative
Prevent autolysis & bacterial decomposition.
Preserve tissues in natural state & fix all
components.
Preserve tissue volume.
Avoid excessive hardness/brittleness.
Allow enhanced staining (mordant).
Nontoxic, non-allergic, environment-friendly, non-
flammable.
Recyclable, cost-effective.
Reversible.
Rapid onset of action & long shelf-life.
Factors affecting fixation

Buffer & pH
Temperature
Concentration of fixative & ionic
composition
Duration of fixation & volume of
specimen
Additives
Formalin/formaldehyde (H-CHO)

Most popular fixative

Saturated solution of formaldehyde
gas in water(40% by weight)-made
into 10% formol-saline.
Formation of additive compounds→
develop methylene bridges among
protein molecules→cross-linking.
10 times of specimen volume for 8
hours in room temperature.
Advantages: cheap,easy to
prepare,stable,good
penetration,reversible,good storage
property,
Disadvantages: allergic to mucosa,
dermatitis, asthma, formalin pigment.
 Some other Fixatives
Fixative Advantage Disadvantage
gluteraldehyde E-microscopy Background stain in
IHC, irreversible.
Osmium tetra-oxide E-microscopy, lipid in Black pigment
frozen.
Mercuric chloride/ Trichrome, Black pigment
zenker’s metachromatic stain.
Dichromate/ moller’s Chromafin reaction, PAS↓
phospholipids

Picrate/ bouin’s Glycogen, iron↓, brittle

conn.tissue stain.
Alcoholic/ carnoy’s Glycogen, Shrink, polarisation
nuclear stain
Fixatives in special circumstances

Electron microscopy- 2% gluteraldehyde

Immunohistochemistry- alcoholic formalin
Enzymatic study- carnoy’s fluid
Molecular biology-
10%NBF/4%formaldehyde in cryostat
Carbohydrate- carnoy’s/alcoholic formalin
lipid- 10%NBF
Iron-carnoy’s fluid
Post-fixation treatment

Picric acid
Carnoy’s fluid
Post-mordanting
Dehydration
Removal of intra- & extra-cellular water so
that it may be replaced by embedding
media.
Hydrophilic polar groups bind water
Graded ethanol-70%,95%,absolute
alcohol(start with 50% for delicate tisue)
isopropyl alcohol, methanol, acetone,
butanol,ethylene glycol.
Universal solvents-dioxane.
Clearing

Alcohol is removed from the tissue to

be replaced by a fluid which will
dissolve in the embedding media.It
also makes tissue transparent(clears).
Xylene(<4 hour)
Toluene
Chloroform( for more delicate tissues)
Limolene,clearite.
Ideal clearing agent
Miscible with both dehydrant and
infiltrating media
Low boiling point
Rapid in action
Minimal tissue damage
Recyclability
Flammability,toxicity
Cost.
Impregnation

Impregnation of tissues with a

medium to provide sufficient support
during cutting without distortion or
altering spatial orientation.
Paraffin wax
Paraplast
Carbowax
Resin
Embedding

Enclosing the processed sample in

correct orientation in a support
medium for thin sectioning.
Paraffin dispenser,Moulds
(Leuckhart’s L blocks/casettes),cold
plate,tissue holder.
Alternative-resin,agar,gelatin,methyl
methacrylate.
Molds

Tissue-tek molds

casettes Leuckhart’s L-blocks

Automated tissue processors
Tissue-transfer type &
enclosed vacuum
type.
Time for each
step,vacuum & heat is
programmed.
Rapid & overnight
schedules
Eliminate fluid &
fumes
Cost-effective
Rapid schedule
reagent time
10% formalin(2) 10 min each
70% alcohol 10 min
95% alcohol(2) 10 min each
100% alcohol(2) 10 min each
Xylene(2) 10 min each
Paraffin(2) 10 min each
Total 1hr 50 min
Overnight schedule
reagent time
10%formalin 1 hour each
(2)

50%alcohol/ 1hr
formalin

70%ethanol 30min
95%ethanol 30min each
(2)

100%ethanol 40min each

(2)

Xylene(2) 40min each

Paraffin(3/4) 30min each
Decalcification
Special treatment to remove calcium &
phosphate from hard tissues to make it
suitable for sectioning.
Inorganic acids-
HCl,HNO3(strong,rapid,more damaging)
Organic acids-formic acid, formalin-formic,
formic-sodium citrate (gentler, slower,
suitable for IHC).
Chelating agent-EDTA (very slow)
Decalcification
Factors affecting decalcification
Concentration
Temperature
Agitation
suspension
 Procedure
Place well-fixed 3mm thick bone tissue
suspended from a glass rod in 10%
formic acid-formalin solution
↓
Change fluid daily & check for
decalcification completion
(probing/chemical testing/radiologic)
↓
Average 5-7 days
Decalcification end-point test

Calcium oxalate test:5ml of

decalcifying
fluid+NH4OH→neutralise→add 5ml
ammonium oxalate → stand for 30
min.
White precipitate of calcium oxalate
indicates incomplete decalcification.
Processing after decalcification

Rinse in tap water/neutralise in 10%

aqueous sodium bicarbonate.
Dehydration & clearing same (time
may differ according to bony
proportion)
Embed in hard paraffin/plastic
polymer.
Surface decalcification
Incidental finding of
calcium in soft tissue
section→exposed
surface is kept face
down in 10% formic
acid for 15-30
minutes→rinse to
remove acid→reorient
& resection in
decalcified layer.