6 histotechniques
TRANSCRIPT
Histotechniques
PRINCIPLES AND PRACTICE
Capt Rishi PokhrelDr Swati PatilDr Kirti SolankeDr Anil Dwivedi
STAININGTISSUE PROCESSINGSECTION CUTTING
MICROSCOPY
INTRODUCTION TISSUE PROCUREMENT FIXATION
INTRODUCTION
Histo-techniques• Preparation of tissue for microscopic examination• Series of processes• Ultimate aim – to make tissue ‘visible’ as it is• Pathology Vs Anatomy• Steps vary
– types of tissue & microscopy– structure to be seen– stains to be used– time duration etc.
Steps Tissue procurement and preparation Fixation Dehydration Clearing Impregnation Embedding Section cutting Staining and mounting in slide
Source of tissues Post-mortem bodies Cadavers Tissue of patients from pathology lab Animal sacrifice Slaughter house/ butcher shop
Tissue Procurement
PM bodies : written consent form NOK Cadavers : no additional consent required Patient tissue : written consent From slaughterhouse / butcher : no ethical
issues, preserve bills
Legal & ethical issues
Using animals – Indian laws
CPCSEA has laid down specific rules regarding the use of animals
Anesthesia, trained vet. surgeon etc ? Dead or dying animals
Tissue preparation & precautions
Start fixation a.s.a.p. Prevent osmotic damage
do not dry wash with and immerse in NS
No unnecessary handling Remove excess blood, mucus etc. Cut with a sharp knife Marking of ‘cutting surface’ Labeling and putting in ‘cassette’ Instructions for mounting – wall, tube, stained surface etc.
FIXATION
Objectives of Fixation
Preserve tissue in ‘life like’ condition
Prevent autolysis, microbial decomposition and
petrification.
Coagulate tissue to prevent loss of easily diffusible
substances.
Render tissue unaffected to the harmful effects of
chemicals to be used in further processing.
Effects of fixation Tissue hardening – ease of cutting
Mordant action – Fleming's fluid for
safranine.
Optical differentiation – refractive index
Precipitation or coagulation of proteins
How do fixatives act?• Physical and chemical changes• Reactions with proteins– Aldehydes– Oxidizing agents, OsO4– HgCl2
• Reactions with nucleic acids– Carnoy’s fluid– Formaldehyde at raised temperature– Ethanol and methanol
• Reactions with lipids– Imidazoles– Cholesterol - digitonin
Methods of fixation
• Immersion – commonly used
• Perfusion – ideal method e.g. embalming
• Hyaluronidase pretreatment
FACTORS INVOLVED IN FIXATION
PHTEMPERATUREOSMOLALITY
PENETRATIONCONCENTRATIONDURATION
H+ concentration / PH
• Anoxia in tissue -> CO2 -> pH• General purpose : optimal pH 6-8• Exceptions– Gastric mucosa pH 5.5– Granules of adrenaline and noradrenaline pH 6.5
• Buffers maintain desired pH
Choosing buffers
• Buffer should not react chemically with fixative– Barbiturates with aldehyde
• Histochemistry : buffer should not react or inhibit incubation medium or enzymes– Phosphates react with G6PD– Phosphates combine with lead salts in metal precipitation methods
for phopotase.
• As close to tissue pH as possible – esp for Histochemistry
Temperature
• Routine : room temperature• Special– Electron microscope: 0-4 oC
• Low temperature – rate of decomposition• High temperature – better rate of fixation
Penetration of fixatives
• Rate of penetration varies with various factors.
– Glutaraldehyde > Formaldehyde
• Slow process; d = k √t
• Deeper tissue takes longer time, fixed tissue acts as
a barrier for diffusion of fixative
• Tissue slices should be thin 3-5 mm
Osmolality
• Close to tissue osmolality; higher side – (400 – 450
mOsm)
• Commonly used : NaCl
• Some exceptions e.g. pancreas ; cause shrinkage
Vehicles / additives
• (NH4)2S04 : stabilize proteins
• NaCl + (Na)2SO4 : binding of HgCl2 to proteins
• Alcian blue, ruthenium red, lanathum – preserve
ultrastructure of mucosubstances
• Tannic acid : enhanced microtubules and filaments
in EM
Detergents
• Used to dissolve lipids in cell membrane
• Immunoflorescent techniques – 150,000 Daltons
• Commonly used - saponin
Concentration of fixative
• Determined by– Cost
– Effectiveness
– Solubility
– Type of tissue and method of fixation
• Formalin – 5% for plant tissue
– 10% for animal tissue
– 50% for embalming
Time
• Formaldehyde – Too short – reversal of fixation– Too long
• shrinkage of tissue• Inhibit enzyme activity - Histochemistry
• Depends upon – Thickness of tissue– Temperature
• Optimal 24-48 hours
Fixation artifacts
• Primary or secondary
• Intrinsic or extrinsic
• Solidification of colloid material
• Volume change
• Pigments – formalin, HgCl2
• False localization – Histochemistry and
immunohistochemistry
Volume changes
• Size of cells and tissue in slide do not reflect their true size
• Routine preparation (formalin and paraffin wax) : tissue shrink by 33% (net)
• Not same for all components of tissue/cells– Collagen fibers swell
• Prolonged fixation - more shrinkage• Nuclei in frozen sections are bigger than routine
preparations.
• Size of tissue, 3-5 mm1
• Volume of fixative, 10X2
• Time – 24 hrs (formalin)3
Fixation : Golden rules
FIXATIVES & FIXING FLUIDSClassification
1. Coagulant and non coagulant
2. General and specific
3. Primary and secondary
4. Microanatomical and cytological
5. Simple and compound
6. Routine and special
7. Physical and chemical
8. Conventional and newer
COMMON FIXATIVES
Formaldehyde
• Non coagulant• Aldehyde - HCHO • Gas – soluble in water; max con. 40% by
weight• Sp. gravity : 1.124• On storage converted to formic acid &
paraformaldehyde.
Effects of formaldehyde
• Proteins• Carbohydrates• Lipids• Acidic and basic structures
40% formaldehyde gas in water
Formic acid, Paraformaldehyd
eAdd buffer
Formalin
100 % Formalin
Mix with distilled water
1:9 10% formalin
=
Hypotonic to tissue fluids
Add NaCl
Buffered, Normal
10% Formalin
Buffered normal formalin• 100 % formalin 100 ml
NaH2PO4 3.5 gms
• Buffer +
Na2HPO4 6.5 gms
• Salt NaCl - 8.5 gms
Mix in 900 ml of water
Disadvantages• Contact dermatitis
• Urea crystals – 5 gm• (NH4)2SO4 - 1gm • Water - 1l
• Irritant to eyes and resp. mucosa• Pigment formation in Hemoglobin• Pigments and turbidity• Not suitable for certain tissue – testis, bone
marrow, spleen.
Formalin pigments in slidesBefore staining :-
• Schridde’s method: leave slides for 30 mins– 200 ml 75 % alcohol + 1ml of 25% ammonia liquor
• Verocay’s method : leave sections for 10 mins– 100 ml of 80% ethanol + 1% KOH
Mercuric chloride
• White crystals• Saturated solution : 7% water, 33% alcohol• Extremely poisonous and corrosive to metals• Seldom used alone – compound fixative• Cytological fixative
Mercuric chloride
• Pros – cytological fixative– good fixative for proteins – shrinks but doesn‘t distort tissue– fixes both nucleus & cytoplasm
• Cons – mercury pigments– Corrosive and poisonous
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Potassium dichromate Used in compound fixatives or as mordant for lipids or lipoproteins
• Pros – excellent fixation of mitochondria, phospholipids & myelin– acts as a mordant for mitochondria – iron staining of mitochondria– iron-containing pigments better fixed at higher pH
• Cons – chromatin gets dissolved– mitochondria gets thickened– formation of insoluble precipitants– brittleness of tissues
Chromic acid• CrO3 mixed in water• Fixative for carbohydrate
Osmic acid• Used for cytoplasmic organelles• Demonstration of myelin• Expensive• Use in EM• Use and throw• Use goggles while handling – vapors irritant to eyes.
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Glacial acetic acid• Part of compound fixative• Good fixation for nuclei – precipitates nucleoproteins• Counteracts the shrinking effect of others.• pronounced swelling of collagen fibers• Distorts mitochondria & Golgi body
Ethyl alcohol :Histochemistry - alkaline phosphatase & lipasehardening & shrinkage Improper fixation of chromatin, mitochondria & Golgi
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Glutaraldehyde• Suitable for EM• Better than formaldehyde• cytological fixative• poor penetration – use small tissue • Expensive
Picric acid• good fixative for proteins, carbohydrates & glycogen• better staining• enhances results with cytoplasmic stains• much shrinkage & less hardening
Stock solutions
• Formalin 40%• Mercuric chloride 7%• Potassium dichromate 5%• Chromic acid 2%• Picric acid 1%• Osmium tetra oxide 2%• Acetic acid 100%• Ethanol 100%
Fixing fluids
Formalin mercuric chloride (formal-sublimate)
• Mercuric chloride 30 gms• Water 900 ml• 100% Formalin * 100 ml*Formalin added just before use as added solution is
unstable Uses• Secondary fixation following formalin• Excellent Microanatomical fixative• Cytoplasm preservation• Brilliant staining with acid dyes• Heidenhain’s susa - biopsy
Zenker’s fluidMercuric chloride 5gmPotassium dichromate 2.5gmSodium Sulphate 1gmWater 100mlAcetic acid* 5ml
Uses• Efficient micro anatomical fixative.• Better staining with cytoplasmic & fiber stains
Bouin’s fluid (formalin-picric-acetic)
• Picric acid 75ml• Formalin 25ml• Acetic acid 5ml
Uses• Micro anatomical as well as cytological fixative• Used for demonstration of chromosomes • Glycogen is well preserved.
Helly’s fluid (zenker-formal)
Acetic acid omitted & formalin 5 ml is added in Zenker’s fluid.
Uses• Testis, bone marrow, spleen, lymph node,
pituitary, pancreas
Carnoy’s fluid
• Absolute ethanol 60ml• Chloroform 30ml• Acetic acid 10ml
Uses• Rapid fixation• Not of much use for anatomy slides.
Sanfelice’s fluid
• Solution A Formalin 128ml Acetic acid 16ml• Solution B Chromic acid 100ml
• Mix 9ml A + 16ml B Uses• mitotic figures & chromosomes
• Flemming’s fluid : Cytological fixative, not used
• Lewitsky – Baker modification : tissue of
invertebrates
• Orth’s fluid : Mordant for mitochondria,
chromaffin reaction
Causes of poor fixation
• Less volume of fixation
• Less time
• Poorly penetrating fixative
• Specimen settling down on contained
• Wrong choice of fixative
Always necessary?
• Sclero proteins – nails
• Chitin, cellulose, inorganic salts,
• Frozen sections
Storage and preservation of tissue
• Formalin : store in fixative• 70% alcohol• Formal sublimate : 30% glycerin• 1% phenoxetol• Best : store a block rather than tissue
TAKE HOME MESSAGE
• Precautions on collecting and handling tissues• Legal implications - animals• Neutral normal 10% formalin is the best fixative in
most circumstances• Remember the exceptions
• Pancreas• Testis• Bone marrow and spleen
• Choose fixative as per requirement – tissue or cell, stains
• Storage of tissue
DISCUSSION
ONCE UPON A TIME IN
HAITI