Histotechniques

PRINCIPLES AND PRACTICE

Capt Rishi PokhrelDr Swati PatilDr Kirti SolankeDr Anil Dwivedi

STAINING

TISSUE PROCESSINGSECTION CUTTING

MICROSCOPY

INTRODUCTION TISSUE PROCUREMENT FIXATION

Introduction Dyes Types Equipments Steps H & E Special stain

Histotechnique-III

STAINING

STAINING Term 1st used by LEEUWENHOEK

in1719.used saffron for muscle fibre

GOPPERT& COHN in 1849 used carmine

GERLACH in1858 –selective nuclear staining for nerve cells,regressive stain-weak acetic acid

WALDEYER in 1863-used hematoxylin

Stains are colored substances which dye tissue

Dyes-staining agent whose chemical formula is known,mixture of very closely related compounds with alike properties

Stain-dyes which are metallic salts of animal and vegetable origin

DYESNATURAL

SYNTHETIC

CHEMICALLY

DYES

BASIC ACIDIC NEUTRAL

Acidic dyes-color acid is combined with non coloring metallic base(sodium & potassium). Eosin-Y,light green.Acid fuchsin is sodium salt of acidic sulphonated derivative of rosaniline

Basic dyes-color base is combined with non coloring metallic acid (acetate,chloride,sulphate radicle) Basic Fuchsin(colored rosaniline base and colorless acidic CI radicle &,haematoxylin

Neutral dyes- compounds of color base with color acid.neutral red.

SYNTHETIC DYES-organic compounds chromophore in its structure and has auxochrome radicle

Quinone AnilineBenzene

chromophore chromogen

auxochrome

Dye must ionize in solution to produce colored cations or anions –unite with proteins and other tissues to form colored compounds

haematoxyline

Haematoxylin oxidation Haematein

Stored for long time ;-ve charge possesed by haematein loses its affinity,mordanting is an essential

A mordant ammonium alum, potash alum ,iron alum(aluminium compound) forms lake with strong basic dye.

Water soluble lakes from aluminium compounds are blue.progressive staining

Lakes from ferric compounds-regressive staining

ALUM HAEMATOXYLINS

DELAFIELD’S

EHRLICH’S ACID

MAYER

HARRIS

Types of haematoxylins

Types of staining ROUTINE –with hematoxylin &eosin,

provides only little differentiation

SPECIAL-in selective instances,eg fuelgen.

VITAL-to stain living tissue eg tyrptan blue,lithium carmine for histiocytes.

Vital staining elective solubility Metallic impregnation- Staining with dyes

Types of staining processes

Ag(NH3)2OH

SUPRAVITAL-when stain is applied to a tissue which has already been removed before it is stained.(dissociation)e.g.-R.E. cells by trypan blue,lithium carmine stains histiocytes,alizarine stains bones red

INTRAVITAL-by injecting dye into the living org e.g. Janus Green stain mitochondria.

NON-VITAL-for fixed cells

VITAL STAIN

Substances that dissolve in tissue –lysochrome

Fat droplets electively stained in alcoholic solution if stain is more soluble in fat than in alcohol

ELECTIVE SOLUBILITY

Metallic compounds can be reduced by tissues to stable metallic state

Ammoniacal silver –deposited silver is stable after reduction.

Argentaffin cell-tyrosin derivative melanin,phenolic compound-

kultschitzky cells Argyrophil cells

METALLIC IMPREGNATION-

METACHROMASIA Certain basic dyes react with tissue

components such that their normal color changes from blue to red or purple

Reason-b’cos of presence of polyanions within the tissue

Metachromatic Dyes –thiazines-toludine blue

Eg metachr staining in cartilage,epithelial mucins,mast cell granules

Assist interaction of tissues & dyes Mercuric chloride,formaldehyde,ethyl

alcohol split chromatin-DNA & protein Trichloroacetic acid,picric acid,chromium

compounds facilitate-acidic dyes After fixation ethyle alcohol or acetic acid –

both acidic & basic dyes Blockage of carboxyl group with preserved

amino group-basic dyes

Effect of fixation upon staining

Progressive

staining

•Tissue sections placed in ascending soln of dye•Selective affinity of dye for different tissue•Less sharper

Regressive

staining

•Tissue over stained •Differentiated •Routinely used

Direct and indirect staining

Methyline blue, eosin Indirect-dye+mordant=colored

lake;combine with tissue-mordant-dye complex

Insoluble in ordinary acqeous or alcoholic solvent,allowing counterstaining & dehydration

De-staining basic dyes with- weakly acidic medium mordant

oxidizing agentdyes

Aqueous haematoxylin differentiated in acidified alcohol(1% HCl in 95% alcohol)

Eosin differentiated in alcohol 0.5%conc ammonium hydroxide

Differentiation/de-staining

Ripening of stain Well at room temperature some require refrigeration –schiff’s

reagent,aldehyde fuschin,methyl- green,azocarmine,silver nitrate.

Storage of stain

EQIPMENT AND MATERIALS

15cm deep sink,two taps,white background

A slide washing tray Staining rack-two stout glass

rods,4cm apart Bunsen burner

General staining procedures

Bright daylight Microscope Glass lidded jars-for stains,grooved to hold

6 slides-coplin jars Stainless steel racks-10-20 slides

COPLIN JAR

Slide Folder Rack

UniMailer

Slide Storage System

Removal of paraffin wax-two changes Removal of xylene with absolute alcohol Treatment with descending grades of

alcohol Water Staining Dehydration Clearing mounting

ACTUAL STAGES OF STAINING

Hydration

water

Differentiation+Blueing+ Dehydration

Mounting

eosin

H & E Staining procedure

• Xylene, decreasing concentration of alcohol-water I. Hydration

•over stained 2-20 minutesII. Staining with haematoxylin

•By acidified alcoholIII. Differentiation

•Water or lithium carbonate IV. Blueing•Increasing con. Of alcohol up to 95%V. Dehydration

• 0.5-1 % eosin in 90% alcohol 3 sec. to 1 min. VI. Staining with eosin

•Xylene VII. Clearing •With Canada balsamVIII. Mounting

MOUNTING-used between section and coverslip-1.resinous media(xylene preparation) 2.aqueous media(water preparations)-KAISER’S GLYSERINE-JELLYAPATHY’S MOUNTANT

1.RESINOUS MEDIA-xylene balsamcolophonium –terpentine

euparalxam

D.P.X. (distrene-polystyrene plasticizer-tricresyl phosphate,xylene

B.P.S.(butyl,phthalate,styrene

MOUNTING AND RINGING MEDIA

RINGING MEDIA-mount which fail to set completely hard sealed at margin

Solid media-paraffin wax,kronig’s cement Commercially available-cellulose adhesive

Durofix

Labelling of slides-

Fading of stained section-

carbohydrate

1- PERIODIC ACID SCHIFF'S (PAS )-

Principle: periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color.

(Periodic Acid cleaves sugars into aldehyde groups. Aldehydes react with Schiff Reagent- RED)

Amyloid ,BM,cartilage,cellulose,cerebrosides,epithelial mucins,fungi,glycogen,hyaline membrane fetal lung,lipochrome pigment,mucoid cells of ant lobe of pituitary,pancreatic zymogen granules,starch,thyroid colloid

Results:Glycogen: magenta (red)

H & E PAS

Feulgen Reaction: Active aldehyde group by breaking purine-

deoxyribose bond - DNA (not RNA) is cleaved by HCl, reacts

w/Schiff.

Acidic phosphate radicle is reason for basophilia-methyl green pyronin technique

DNA

Feulgen stain

Methyl Green Pyronin Stain

DNA: blue-green to green

RNA: pink to red

PURPOSE: Alcian blue stains acid mucus substances and acidic mucins.

PRINCIPLE: Alcian blue is a group of polyvalent basic dyes that are water

soluble. The blue color is due to the presence of copper in the molecule.

- Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides

and sulfated and carboxylated glycoproteins.

- It is believed to form salt linkages with the acid groups of acid

mucopolysaccharides.

RESULTS:

Acid mucins/mucus substances: blue cell nuclei:red

background:yellow

Alcian Blue pH 2.5 Acid Mucopolysaccharides

Purpose: To differentiate between neutral and acidic mucus

substances.

Routine stain for G.I. biopsies.

Results:

Acid mucus substances: blue

Neutral polysaccharides: magenta

Alcian Blue-PAS (PAb)

Acid mucus substances: blue

Neutral polysaccharides:

magenta

PURPOSE: acid mucopolysaccharides (mucin), which is a secretion

produced by a variety of epithelial cells and connective tissue cells.

The mucicarmine technique is also useful in determining the site of a primary

tumor in that finding mucin positive tumor cells.

Principle: aluminum is believed to form a chelation complex with the carmine,

changing the molecule to a positive charge allowing it to bind with the acid

substrates of low density such as mucins.

Results:Mucin: deep roseNuclei: blackOther tissue elements: yellow

Mucicarmine Stain - Mucin

Mucin: deep rose

Nuclei: black

Other tissue elements: yellow

SPECIFIC STAINS LIPIDS-SUDANlll,SUDAN4,SUDAN BLACK,OIL

RED O PROTEINS NUCLEOPROTEINS(DNA)-FUELGIN REACTION HEMOGLOBIN-BENZIDINE COPPER ASSOCIATED PROTEIN-ORCEIN FIBRIN-PTAH STAIN

OSMIUM TETRAOXIDE

VAN GIESON’S STAINING-1% acid fuchsin 10ml aqueous picric acid -100ml collagen fibre-red,deep red

nuclei-blue to blackother-bright lemon

yellow-epidermis olive –muscle & nerve

TAEZER-UNNA ORCEIN –for elastic fibreorcein-1g

alcohol-100mlHCl-1ml

elastic fibre-dark brown nuclei-blue

STAINING FOR CT

GOMORI’S STAINING-silver nitrate(10%sol) -20mlpotassium hydroxide(10%sol)-4ml

nuclei-greyreticulin fibre-blackcollagen fibre-greyish purple

Cont’ from CT………

Silver Stain

Stains Reticular Fibers and Basement Membrane Black.

MASSON’S TRICHROME-for collagen,hypophysis cerebri,thyroid glandmordant-phosphomolybdic acid 5g

phosphotungstic acid 5gdistilled water 200 ml

stain-weigert’s iron haematoxylinbiebrich scarlet in 1% acetic acid

2.5%fast green in 2.5% acetic acid result-nuclei –black

cytoplasm-pink to brownmuscle-redRBC-brilliant scarletmylinated nerve -red

Staining for muscle

Figure 2. Muscle and collagen demonstratedby Masson Trichrome in gastrointestinaltract. 20X

THIONIN STAINING-nissl substance,decalcified bone ,mucin ,mucopolysaccharide,mast cell,sex chromatin

staning sol A-lithium caronate 5.5g distilled water-1000ml sol B-thionine -0.25 g lithium carbonate-10ml nissle sub-bright blue

Staining for nerve cells

CAJAL’S GOLD SUBLIMATE METHOD-Astrocyte staining sol-distilled water 100ml1%aqueous gold chloride 20ml

mercuric chloride 1gresults-astrocytes-reddish purple to

black nerve cells-pale rose to violetnerve fibres-unstained or stained

pale DEL RIO HORTEGA ‘S METHOD-

Oligodendrrocyte,microgliafixation –FAB(formaline ammonium

bromide) preparation of ammoniacal siver carbonate- silver nitrate 5ml

sodium carbonate 15ml

Staining for neuroglia

cresyl violet Golgi's gold method.

HAPPY DASERA

The Azan-Mallory stain is one of several commonly used techniques in which three or more dyes are combined. These multiple-dye stains have the advantage of showing a large number of tissue structures. The Azan-Mallory's stain combines aniline blue, orange G (stains proteins) and acid fuchsin (stains DNA and RNA). Collagen-containing connective tissue is shown as blue, erythrocytes as orange, and chromatin, nucleoli, basophilic cytoplasm, and muscle cell cytoplasm as red. With azocarmine and aniline blue (Azan) stain, a combination of the basophilic dye (azocarmine) with aniline blue stains nuclei and basic structures are stained red and collagen, mucus, and cartilage matrix are stained blue

Figure 1. Weigert’s Iron Hematoxylindemonstrating nuclear detail prior tomuscle and collagen staining. 20X

Used to differentiate between collagen and smooth muscle in tumors, and the increase of collagen in diseases such as cirrhosis. Routine stain for liver and kidney biopsies. the name implies, three dyes are

employed selectively staining muscle, collagen fibers, fibrin, and erythrocytes. The general rule in trichrome staining is that the less porous tissues are colored by the smallest dye molecule; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule. Others suggest that the tissue is stained first with the acid dye, Biebrich Scarlet, which binds with the acidophilic tissue components. Then when treated with the phospho acids, the less permeable components retain the red, while the red is pulled out of the collagen. At the same time causing a link with the collagen to bind with the aniline blue.

COLLAGEN - MASSON'S TRICHROME STAIN(TRI)

The trichrome stain is utilized as the stain of choice of distinguishing

histologic changes in tumors, connective tissue diseases, muscle

and fibroblast tumors, renal diseases and dermatology cases. Even

the disciplines of forensics, archaeology and hematopathology

incorporate the trichrome stain for specific tissue entities and

structures. With the utilization of immunohistochemistry expressions,

the trichrome techniques still offer a great deal of diagnostic results

Cresyl Violet & Luxol Fast Blue

There are hundreds of other staining routines, most of which involve the use of gold or silver salts. Among the most elegant of these stains are the ones developed by Camillo Golgi (1843-1926) or Santiago Ramon y Cajal (1852-1934), who shared a Nobel prize for their work in 1906. These methods are especially useful for visualizing glial elements. Both these men are great figures of the history of the life sciences and the study of the nervous system in particular. Golgi developed several stains that are still used today, was the discoverer of several important nervous system structures, and won the Nobel Prize for his work. Golgi's stains comprise a set of methods for nerve cells and fibers; they're characterized by fixation in an aldehyde-osmium-dichromate solution, followed by impregnation with silver salts. As you can see here, the process renders the subject as several shades of golds, browns and blacks. Neuron somata are golden and their processes black. This stain permits the definition of much detailed information about the structure of the nervous system.

Nerve h & e

unmylinated

L S OF NERVE Mallory's connective tissue stain

PURKINJE CELLS H&E

Wilder’s retinaculin method

COLLAGEN FIBRE

Suprarenal capsule Masson’s fontana method for argentaffin

granules-direct reduction after non alcoholic fixation

Argyrophil cells-silver impregnation techniqes demonstrate argentaffin cells after alcoholic fixation

Diazo reaction for argentaffin granules-red Gibbs’ method

Adrenals

Romanowsky mixed methylene blue and eosin

Blood film

Heidenhain’s method and masson’s trichrome stain

embryo

Commence fixation with tissue intact ,bisect later on same day.

Acetic formaline-penetrate rapidly Acid fixation prevent “pink disease” Reticulin stains

Lymph nodes

Posterior pituitary-neurosecretory substance & hypothalamus is rich in cystine-acid alcian blue technique is better than gomori’s aldehyde fuchsin

pituitary

SPECIAL STAINS BASEMENT MEM- P.A.S CONNECTIVE TS FIBREa- COLLAGEN FIBRE- VAN GIESON’Sb- ELASTIC FIBRE- TAENZER UNNA ORCEIN

METHODc- RETICULIN FIBRE- GOMORI’S

TRICHROME,GOLGI SILVER/SILVER STAIN

Basic stain-base contains coloring substance combined with acidic radicle-Basic fuchsin

Acidic stain- Romanowsky-combination of polychrome

methylene blue and eosin Colorless leucobases-dyes can be reduced

easily

Masson's Trichrome Stain Muscles (red) Masson's Trichrome Stain Collagen (green or blue) Masson's Trichrome Stain Mucus (green or blue) Masson's Trichrome Stain Cytoplasm of most cells (pink) Masson's Trichrome Stain Glycogen (deep red or magenta) Periodic Acid Schiff (PAS) Reaction Contents of goblet cells (red or magenta) Periodic Acid Schiff (PAS) Reaction Basement membrane (positive or pink) Periodic Acid Schiff (PAS) Reaction Brush borders in kidney tubules (positive or pink) Periodic Acid Schiff (PAS) Reaction Elastic fibers (jet black) Verhoeff's Stain for Elastic Tissue Nuclei (gray) Verhoeff's Stain for Elastic Tissue Remaining structures (pink) Verhoeff's Stain for Elastic Tissue Fibrous c.t. (deep blue) Mallory-Azan Stain Mucus (deep blue) Mallory-Azan Stain Erythrocytes (red-orange) Mallory-Azan Stain Cytoplasm of liver (pink) Mallory-Azan Stain Cytoplasm of kidney (pink) Mallory-Azan Stain Nuclei (red) Mallory-Azan Stain Erythrocyte cytoplasm (pink) Mallory-Azan Stain Lymphocyte nuclei (dark purple-blue) Mallory-Azan Stain Lymphocyte cytoplasm (pale blue) Mallory-Azan Stain Monocyte nuclei (medium blue) Mallory-Azan Stain Monocyte cytoplasm (pale blue) Mallory-Azan Stain Neutrophil nuclei (dark blue) Mallory-Azan Stain Eosinophil nuclei (dark blue) Mallory-Azan Stain Eosinophil granules (bright pink) Mallory-Azan Stain Basophil granules (deep purple) Mallory-Azan Stain Platelets (light blue) Mallory-Azan Stain Myelinated fibers (blue-black) Cajal's and Del Rio Hortega's Methods (silver and gold) Unmyelinated fibers (blue-black) Cajal's and Del Rio Hortega's Methods (silver and gold) Neurofibrils (blue-black) Cajal's and Del Rio Hortega's Methods (silver and gold) General background (nearly colorless) Cajal's and Del Rio Hortega's Methods (silver and gold) Astrocytes (black) Cajal's and Del Rio Hortega's Methods (silver and gold) End product of stain (can be black, brown or gold) Cajal's and Del Rio Hortega's Methods (silver and gold) Lipids in general (black) Osmic Acid (Osmium Tetroxide) Stain Lipids in the myelin sheath of nerves (black) Osmic Acid (Osmium Tetroxide) Stain Elastic fibers (brown-reddish) Orcein Stain

Widely utilized techniques are the Masson, Gomori One Step, Martius Scarlet

Blue and Mallory. ionized acid dyes react with the ionized basic

tissues. fibrils of collagen stained blue, fibroglia, neuroglia and muscle fibers stained red and fibrils of elastin stained pink or yellow. The trichrome stain is also used to distinguish tumors that have arisen from muscle cells and

fibroblasts. Gomori’s trichrome is the trichrome stain of choice for

distinguishing histological changes that occur in neuromuscular diseases.