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