Histotechniq ues PRINCIPLES AND PRACTICE Capt Rishi Pokhrel Dr Swati Patil Dr Kirti Solanke Dr Anil Dwivedi
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.