HISTOPATHOLOGY TECHNIQUES
CLASSIFICATION OF
FIXATIVES
Based on the parts of tissue or
cells to be demonstrated
NATURE
OF FIXATIVES |
NAME
OF FIXATIVES |
Microanatomical
fixatives |
• 10%
formalin • Neutral
buffered formalin • Formal
saline • Buffered
Gluteraldehyde • Zenker
Fluid, Zenkers formol • Formal
Calcium |
Cytological
Fixatives |
|
Cytoplasmic
Fixative (pH ≥ 4.6) |
• Zenkers
fluid • Flemming
Fluid without acetic acid • Schaudinn
fluid • Regaud's
fluid • Formal
saline and formal calcium |
Nuclear
Fixative (pH ≤
4.6) |
• Carnoy's
fluid • Clarke
fluid • Flemming
Fluid • Newcomer's
fluid |
Histochemical
Fixatives |
• Formal
saline • Cold
acetone • Absolute
alcohol |
Based on fixative's ability to
fix by coagulating protein |
|
NATURE
OF FIXATIVES |
NAME
OF FIXATIVES |
Coagulant
Fixatives |
Mercuric
chloride, Chromium, Picric Acid Ethanol |
Non
Coagulant fixatives |
Formaldehyde,
osmium tetroxide, Pot. Dichromate, Acetic acid |
Based on number of compounds
mixed in a fixative |
|
NATURE
OF FIXATIVES |
NAME
OF FIXATIVES |
Simple
Fixatives |
|
Aldehydes |
• Formaldehyde • Gluteral
dehyde |
Oxidizing
Agents |
• Osmium
tetroxide • Potassium
dichromate • Potassium
permanganate |
Protein
Denaturing Agents |
• Acetic
acid • Ethyl
alcohol • Methyl
alcohol |
Unknown
Mechanism |
• Picric
acid • Mercuric
chloride |
Compound Fixatives - Neutral
buffered formalin - 10%
Formol saline - Zenker's
fluid, Zenker's formol |
|
Metallic Fixative |
|
NATURE
OF FIXATIVES |
NAME
OF FIXATIVES |
Mercuric
fixatives |
• Zenker's • Helly's • Heidenhain's
Susa • Schaudinn
fluid • B5
fixative |
Chromate
fixatives |
• Zenker's fluid • Orth's
fluid • Regaud's |
Lead
fixatives |
• Lillies
fluid • Lead
sub acetate |
OTHER FIXATIVES |
|
NATURE
OF FIXATIVES |
NAME
OF FIXATIVES |
Picric
acid containing fixatives |
• Bouin's
fixative • Gendre's
fixative • Brasil's
alcoholic picroformol |
Alcoholic
Fixatives |
Methyl
alcohol Ethyl
alcohol Carnoy's
fluid Clarke's
fluid |
Fixatives for
different target substances
S.N. |
Target
Substance |
Fixative |
|
Proteins |
Neutral
Buffered formalin |
|
Lipids |
Osmium
tetroxide Gluteraldehyde |
|
Glycogen |
Carnoy's fluid Bouin's fluid |
|
Carbohydrates |
Heidenhain's
Susa Buffered
Gluteraldehyde Formal calcium |
|
Biogenic
amines |
Bouin's fluid Neutral
buffered formalin |
|
Myelin
sheath |
Flemming's
fluid |
TIME OF FIXATION REQUIRED FOR
VARIOUS TISSUES
Tissue |
Fixative of
choice |
Time for
fixative |
Routine. |
Formalin |
10-12 hours |
GIT biopsies |
buffered
formaldehyde |
4-6 hours |
Testicular
biopsy and embryos |
Bouin's
fixative |
4-6 Hours. |
Liver Biopsy |
|
4-12 hours |
Bone marrow and
pituitary tissue |
Helly's fluid
(Zenker's formol) |
|
Spleen and
liver |
Buffered
formaldehyde, Zenker's fluid |
1-6 hours |
Lymph node |
B5 |
12-18 hours |
Mictocondria,
phosphatides and Nissil substance |
Carnoy's fluid |
1-2 hours |
Chromosome /
cell culture |
Clarke's fluid |
1-2 hours |
Smears for
H&E Staining |
Schaudinn's
fluid |
2-5 min |
Fibrous tissues |
Heidenhain's
Susa |
12-24 hr |
Whole Organs |
Neutral
Buffered Formalin |
|
Vapour
fixatives
1. Formaldehyde-
Vapour is obtained by heating paraformaldehyde at temperature between
50° and 80°C. Blocks of tissue require 3-5 hours whereas section
require ½- 1 hours.
2. Acetaldehyde-
Vapour at 80°C for 1-4 hours.
3. Glutaraldehyde-
50% aqueous solution at 80°C for 2 min to 4 hours. 4. Acrolein /chromyl
chloride- used at 37°C for 1-2 hours
Secondary
fixation:- Mercuric chloride for 4 hours
Post
chromation:- 3% potassium dichromate following normal fixation for
myelin and phospholipids demonstration ( for pre and post mordanting).
REMOVAL OF PIGMENT:
A. Formalin
pigment (Three methods)
Method1-Saturated
alcoholic picric acid followed by 30 minutes running tap water (15 minutes).
Method2-One
per cent sodium hydroxide in 70% ethanol followed by five minutes running tap
water (10 minutes).
Method3-Ten
volumes hydrogen peroxidase followed by five minutes running tap water (five
hours)
B. Mercuric
chloride pigment
Gram’s or
lugol’s iodine for 5 minute followed by treatment
with 5 % sodium thiosulphate for 5 min
C. Osmium
tetroxide deposit
Hydrogen
peroxide in 70% alcohol treatment followed by sunlight
bleaching followed by 0.5% potassium permanganate followed
by sulfurous acid treatment
DECALCIFICATION
1. Removal
of calcium by acids:- (a) Strong acid, (b) weak acid.
a. Strong
acid
Nitric acid-
5-10% aqueous solution used(Most commonly used)- 1-3 days
Formalin Nitric
acid- 1-3 days
Hydrochloric
acid (Von Ebner's Fluid): 5-10% aqueous solution : 3-5 Days
b. Weak
acid:- formic, acetic and picric acid (5-10% aqueous solution or with
additives like formalin)
2. Gooding
and Stelwart's fluid:- formic acid,Formalin and D/W- 2-7 Days
3. Evans
and Krajian fluid:- trisodium citrate,formic acid 2-7 Days
4. Ion
exchange resins
5. Chelating
agents- EDTA- 3 weeks
6. Electrolytic
method:- The electrolyte is HCL and Formic acid in D/W
Determination
of end point of decalcification
- X-ray method
- Chemical Method
:- uses 5% ammonium oxalate and 5% ammonium
hydroxide solution
Softening
of Hard tissues:-
- Perenyi's
fluid (10% HNO3+Absolute alcohol+0.5% Chromic acid)
- 4%
aqueous solution of phenol for 1-3 days (lendrum’s technique)
TISSUE PROCESSING
Steps:- Fixation,
Dehydration, Clearing , Embedding and mounting
1. Fixation
2. Dehydrating
agents:
- Ethanol
- Methanol
- Acetone-
missible with epoxy
- Isopropyl
alcohol
- Dioxane- Both
dehydrating and clearing agent
3. Clearing
agents
- Xylene:- Causes
brittleness to tissue on prolonged use
- Toluene
or Benzene : Does not make the tissue brittle
- Chloroform:
- Carbon
tetrachloride
- Cedar
wood oil (Histological): best clearing agent, does not harden the
tissue but expensive
4. Impregnation
a) Waxes
(Temperature 56-600c)
- Paraffin
wax
- Bee
wax
- Paraplast
- Bioloid
- Ester
wax
- Water
soluble waxes
b) Celloidin -
for hard and dense tissues such as bones and teeth and for large tissue
sections of the whole embryo
c) Nitrocellulose
Method:-
d) Dry
celloidin method is preferred for processing of whole eye sections
e) Gelatin
Impregnation - when dehydration is to be avoided and when tissues are
to be subjected to histochemical and enzyme studies
5. Embedding
- Leuckhart’s
Embedding Mold
- Compound
Embedding Unit
- Plastic
Embedding Rings and Base mold
Double
Embedding Method:- tissues are first infiltrated with
celloidin and subsequently embedded in paraffin wax. Eg cutting of large blocks
of dense firm tissues like the brain.
SECTION CUTTING
Types of
knieves
Plane wedge
: for paraffin and frozen sections.
Planoconcave :
celloidin section
Biconcave
: paraffin section cutting on rocking and sledge type of microtome.
Tool edge
: used with a heavy microtome for cutting very hard tissues like
undecalcified bone
Honing
Grinding of knife
on a hone to restore straight cutting edge and correct bevel.
Hones
- Belgian
black vein or Belgian yellow
- Arkansas
– Not very fast
- Aloxide
- Carborundum
- Plate
glass
Stroping
It is the process
of polishing an already fairly sharp edge. It removes burrs formed during
honing.
- Horse
neck leather
- Heart
woods
TYPES OF MICROTOME
1. Cambridge
Rocking microtome:- knife is fixed, block moves
2. Sliding
microtome:- Moving knife and fixed block
3. Rotary microtome:-
- Knife
is fixed and block moves.
- Most
commonly used
- Best
for serial sections
4. Freezing microtome(cryo
microtome):-
- For
frozen sections
- Useful
when lipids, enzymes, and neurological structures are to be demonstrated.
5. Base
sledge microtome
- Used
for cutting whole organ or large tissue blocks
TISSUE THICKNESS REQUIRED FOR
VARIOUS SAMPLES
Tissue Types |
Micron Requirement |
Brain and central nervous system
(CNS) tissue |
|
Tissue for amyloid
diagnosis employing Congo red stain |
|
Renal (kidney) biopsies |
|
Lymph nodes |
|
Faults in
section cutting, causes and their remedy
|
Fault |
Cause |
Remedy |
1 |
Tear or scratch
across the section or splitting of ribbon Sharpen the knife |
- Jagged
knife edge - Dirt or
hair on knife edge |
Clean the knife |
2 |
Tear or scratch
across part of section, |
- Calcium,
Carbon, or Suture etc., in the tissue or wax |
Examine block
under magnifying glass. If calcium is present, decalcify block. Remove suture
from the tissue with scalpel
point. If dust is in wax - Re-embed |
3 |
Holes in the
section. |
- Air
bubbles in the tissue or wax - A piece
of hard material in tissue - A soft
piece of tissue in block |
Re-embedd Reprocess
specimen Remove hard
material if possible |
4 |
Cracks across
the section parallel to knife |
- A blunt knife - Knife
tilt too small. - Block
too hard for thickness of specimen |
Sharpen knife Adjust tilt Warm block
slightly or re-embed in soft wax. |
5 |
Section shows
thin and thick horizontal lines (chatters) |
- A loose knife - A loose
block A blunt knife - Extremely
hard tissue |
Tighten knife
and/or block Sharpen the
knife Soften the
tissue if possible or embed in harden wax |
6 |
section cut
thick and thin alternative |
- Knife
tilt is too great and is compressing the block. |
Adjust tilt |
7 |
Section
compress at one end |
- Blunt
spot on the knife - A soft
spot in the wax, due to presence of clearing agent |
Move block
along the knife or sharp knife. Re infiltrate
tissue and re-embed |
8 |
Section curves
to one end. |
- Edge of
block is not parallel to knife. - A dull
spot on knife. |
Trim edges Move block
along knife or sharpen knife. |
9 |
Section curl as
the they are cut |
- Blunt knife - Sections
too thick - Too much
tilt to knife |
Sharpen knife Adjust
microtome Correct the tilt |
10 |
The tissue is
shrunken away from wax |
- Insufficient
dehydration |
Reprocess |
11 |
The tissue is
too soft when block is trimmed |
- Insufficient fixation |
Reprocess |
12 |
Specimen
crumbles and drops out of the wax leaving a rim of wax as a section |
- Insufficient infiltration - Overheated
paraffin bath causing tissue to become hard and brittle |
Re infiltrate
and re-embed Service the
paraffin bath |
13 |
Tissue is dried
out or mummified |
- Mechanical
failure of tissue processing machine or a
basket was out of balance and hung up. |
Place the
specimen in the following rehydration solution for 18-24 hrs. Sodium
Carbonate - 1.0 gm Dist. Water -
70.0 ml Absolute ethyl alcohol - 30.0 ml Re hydrate the reprocess |
14 |
Sections
crumble on cutting |
- Knife is blunt - Wax is
too soft; has crystallized due to slow cooling or contamination with water or
clearing agent. - Defective
processing e.g. incomplete fixation, dehydration, clearing or embedding. |
Sharpen knife. Re-embed and
block with fresh wax Reprocess |
15 |
Failure of
block to ribbon |
- Block
not parallel to ribbon - Paraffin
too hard. Knife tilted too much Sections too thick |
Correct the alignment Re-embed
Correct the tilt Adjust the
section thickness |
Staining
Some common terms used in
Histopathology staining
S.N. |
Term |
Meaning |
Example |
1. |
Premordanting |
The mordant is
applied first, followed by the dye. |
Heidenhain’s
iron hematoxylin |
3. |
Metachrome |
Mordant and dye
are mixed together then applied. |
Alum
hematoxylin solutions |
4. |
Progressive
staining |
Decolorization
is not done. |
Mayer’s,
Delafield’s, Gill’s, Harris’, Carrazzis' |
5. |
Regressive
staining |
The slide is
over stained and then decolourised. |
Delafield’s,
Harris’,Ehrlich’s |
6. |
Birefringence |
The colour
(different from original stain) shown by slides when viewed under polarized
light |
Amloyds: Pink
to red colour under plain light and Apple-Green colour under polarized light |
7. |
Argentaffin
cells |
the cells
having strong reducing groups reduce the ionic silver to metallic
silver by itself |
E.g. Melanin:
Massons Fontana stain |
8. |
Argyrophilic
cells |
Cells absorb
the silver ions but require the addition of a separate reducing agent in
order to converted silver into elemental silver |
Warthin-Starry
stain for spirochetes |
Hematoxylin
Extraction: extracted
by boiling the heart wood of Hematoxylon Campechianum in hot water.
Classification
of Hematoxylin
A. Based
on type of oxidation
1. Natural
oxidation:
§ Ehrlich's
Hematoxylin
§ Delafield's
Hematoxylin
2. Chemical
oxidation:
§ Sodium
or Potassium Iodate
§ Mercuric
chloride
B. Based
on the mordant used
1. Alum
haematoxylins: Mordant used are aluminum salts, either aluminum
potassium
sulphate (potashalum) or aluminum ammonium...
TYPES OF ALUM HEMATOXYLIN
a) Ehrilch’s
haematoxylin.
b) Mayer’s
haematoxylin.
c) Harris’s
haematoxylin.
d) Gill’s
haematoxylin.
e) Cole’s
f) Delafield's
g) Carazzi's
2. Iron
haematoxylins
3. Tungsten
haematoxylins
4. Molybdenum
haematoxylins
5. Lead
hematoxylins
6. Hematoxylin
without mordants
Progressive
Hematoxylins: Mayer’s, Delafield’s, Gill’s, Harris’, Carrazzis'
Regressive
Hematoxylins: Delafield’s, Harris’,Ehrlich’s
Type of
Hematoxylin |
Mordant Used |
Oxidant |
Uses |
Staining
time |
Remarks |
A. Alum
Hematoxylin |
|||||
Ehrilch’s |
Potassium alum |
Natural |
Nuclear stain
used with eosin. Fading of colour is much slower |
20-45 min |
|
Mayer’s |
Potassium or
ammonium alum |
Sodium iodate |
Celestine Blue
Hemalum method of Nuclear Staining |
Progressive:
5-10 min Regressive:10-20
min |
|
Harris’s |
Potassium alum |
Sodium iodate |
Routine
histopathology; Exfoliative Cytology and for staining of sex chromosome |
4-6 minutes |
|
Gill’s |
Aluminum
sulfate |
Sodium iodate |
Nuclear stain
used with eosin |
Progressive:5-15
min |
|
Cole’s |
Potassium or
ammonium alum |
Alcoholic
iodine |
Nuclear stain
used with eosin. used in sequence with Celestine Blue |
20-45 min Used with
Celestin Blue stain |
|
Delafield's |
Ammonium alum |
Natural |
Blood films for
spirochaetes, Protozoa |
|
|
Carazzi's |
Potassium alum |
Sodium iodate |
For Frozen
section |
Progressive: |
|
B. Iron
haematoxylins :- The iron acts both as mordant and oxidizer. (Resist
decolourization , used especially for acid stains e.g. Trichrome stains,
PTAH, Picrosirius Red Stain) |
|||||
Weigert’s |
Ferric chloride |
Natural |
Masson’s
Trichrome Stain, van Geison's stain |
Regressive:
20-30 min |
Differentiator: |
Heidenhain’s |
Ferric ammonium
sulphate |
Natural |
For Intestinal
parasites, for fungal mycelia in cytology; for mitochondria, muscle
striations, chromatin, myelin |
Regressive: 1
hour |
Differentiator:
5% iron alum |
Verhoeff’s |
|
|
Elastic fibres |
|
2% aquous
ferric chloride |
Loyez |
|
|
myelin |
|
|
C. TUNGSTUN
HEMATOXYLIN |
|||||
Mallory's
Phosphotungstic acid hematoxylin (PTAH) |
Phosphotungstic
acid |
Potassium
permanganate or Natural |
Nervous
tissues: |
Regressive:
Over night |
Chemically
ripened hematoxylin can be used only for 24 hr |
D. Molybdenum
Hematoxylin |
|||||
Thomas Phosphomolybdic acid
hematoxylin |
Phosphomolybdic acid |
|
Used for
argentafin cells |
|
Not Widely used |
E. Lead
Hematoxylin |
|||||
Solcia
Hematoxylin |
Lead salts |
No oxidation |
For secretory
granules in endocrine cells. |
|
|
F. Hematoxylin
without mordant |
|||||
Unripened
Hematoxylin |
….. |
…….. |
For
demonstration of minerals: Lead, Iron and Copper |
|
|
S.N. |
STAIN Method |
Used For |
Result |
1. |
Hematoxylin
& Eosin Stain |
Most routine
stain |
Nuclei: blue
(hematoxylin) |
2. |
Cresyl Fast
Violet |
NISSL |
|
3 |
Congo Red |
Amyloid (Mostly used in
patients with Alzheimer’s disease) |
pale orange-red
(apple green birefringence under polarized light) |
4 |
Luxol Fast
Blue |
Nerve fibers |
Myelin fibres:
blue to blue/green |
5 |
Masson's
Trichrome |
|
|
6 |
Mallory’s
Muscle Fiber |
|
|
7 |
Fungal elements Including Pneumo |
black (with
sharp margins and cleared center) |
|
8 |
Elastic fibre |
Elastic fibers
and cell nuclei: black (Verhoeff component: Iron Hematoxylin) |
|
9 |
Warthin-Starry
Silver impregnation |
Spirochaetes
and small bacilli |
Microorganism:
dark brown to black |
10 |
PTAH |
Intracellular
filaments |
|
11 |
Nissl
Staining |
Brain &
Spinal Cord |
|
12 |
Thionin |
Nissl |
|
13 |
Toluidine
Blue |
Mast
Cells |
|
14 |
4′,
6-diamidino-2-phenylindole (DAPI) |
stain DNA |
Nuclei: blue |
15 |
Pluripotent
cells: |
stem cell
membrane: red/purple |
|
16 |
visualize nerve
fibers. |
Axons, plaque
neurites, and tangles: black |
|
17 |
Lipids stain |
Neutral
triglycerides and lipids (frozen sections), lipoproteins (paraffin sections),
and lipofuscin: Bright red |
|
18 |
Sudan Black
B |
Lipids stain |
Neutral
triglycerides and lipids (frozen sections); some lipoproteins
(paraffin-embedded sections): blue-black stains
myeloblasts, but not lymphoblasts. |
19 |
Glycogen |
Glycogen and
other carbohydrates: magenta |
|
20 |
Periodic-acid
Schiff with Diastage |
To
differentiate mucin from glycogen |
Glycogen is
digested but mucin is not and stains Magenta |
21 |
Alcian Blue - pH-2.5 - pH
5.8 |
- Acid
Mucin - Neutral
Mucin |
- Blue |
22 |
Perl’s
Prussian Blue |
hemosiderin
(type of iron-storage complex found inside cells) deposits, for instance in
some liver diseases or hemolytic anemia. |
Iron deposits:
Blue or purple |
23 |
Mast cells |
Mast cell
granules and polysaccharides: violet |
STAINING METHODS
BY NATURE OF SUBSTANCES
Stain |
Substance |
Interpretation |
Amyloid |
||
Congo red under polarizing microscope |
Amyloid |
Green-birefringence |
Carbohydrates |
||
Periodic acid-Schiff (PAS) |
Glycogen,
mucin, mucoprotein, glycoprotein, fungi, basement membranes of glomeruli and tubules |
Magenta color |
Mucicarmine/Best’s carmine |
Epithelial mucin |
Red color |
Alcian blue |
Acid mucin |
Blue |
Lipids |
||
Sudan III |
Lipid |
Orange |
Oil Red O |
Red |
|
Osmium tetroxide |
Brown black |
|
Connective
tissue |
||
Van Gieson |
Extracellular collagen |
Red |
Masson’s trichrome |
Collagen, smooth muscle |
Collagen-blue, smooth muscle-red |
Phosphotungstic acid hematoxylin (PTAH) |
Cross striation
of skeletal muscles, glial filaments, fibrin |
Dark blue |
Verhoeff’s elastic |
Elastic fibers |
Black |
Microorganisms |
||
Gram’s stain |
Bacteria |
Gram+ve = blue Gram-ve = red |
Ziehl-Neelsen’s (Acid-fast) stain |
Tubercle bacilli and other acid-fast organisms |
Red |
Fite-Faraco |
Lepra bacilli |
Red |
Silver methanamine |
Fungi |
Black |
Pigments and
minerals |
||
Prussian blue stain (Perl’s stain) |
Hemosiderin |
Blue |
Masson Fontana |
Melanin |
Black |
Von Kossa |
Calcium |
Orange red |
Alzarine Red
S |
Black |
|
Rubeanic acid |
Copper |
Greenish-black |