optimizing hematoxylin-eosin, iron & reticulin - start · optimizing hematoxylin-eosin, iron...
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OptimizingHematoxylin-Eosin,Iron & Reticulin
Charlotte Bergström
Senior Application Specialist
Agilent Technologies
October 10, 2017
Confidentiality Label
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How Can We Help Our Users to Optimize?
▪ Hematoxylin-Eosin
▪ Iron
▪ Reticulin
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Dako CoverStainer solution
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Hematoxylin
Extracted from heartwood of the Haematoxylon
campechianum tree
H&E is the most widely used stain in Pathology
Forms strongly coloured complexes with certain metal ions
as Fe(III) and Al(III) salts, known as mordants
Combined with Eosin, Erytrosin, Phloxin, Saffron
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Hematoxylin
Connection, March 2010, Special Stains and H&E
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Eosin
Fluorescent red dye resulting from the reaction of bromine on
flourescein
Eosin comes in two different variations – Eosin Y (Eosin G)
and Eosin B
Counter stain to Hematoxylin
Can be used in combinations with other dyes
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Hematoxylin & Eosin Stain
Results:
Nuclear cellular elements - Blue
Cell cytoplasm, muscle fibers and
collagen should appear different shades
of pink
Red blood cells – Intense pink
Eosinophils granules – Intense Pink
Eosinophilic structures eg. Paneth cell
granules - Intense pink
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Crisp, blue, open Chromatin pattern
Dense Chromatin pattern
Well-defined Nuclear Membrane Acidophilic Nucleoli
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Red blood cells
Collagen
Smooth Muscle fibers
Elastic fibers
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Hematoxylin-Eosin
▪ Three different variations available
▪ Different types of Hematoxylin
▪ Different types of Eosin
▪ Progressive staining
▪ Regressive staining
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Optimizing H&E - Hematoxylin
Nuclei too dark
• Progressive staining – decrease the staining time
• Regressive staining – differentiate a bit longer
Nuclei too week
• Progressive staining – increase staining time
• Regressive staining – decrease time in differentiating step
Nuclei pink or purple
• Longer time in Bluing Step
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Optimizing H&E - Eosin
Eosin too week
• Increase staining time
• Use alcohol step before putting slides in Eosin
• Decrease time in alcohols
Eosin too strong
• Decrease staining time
• Increase time in alcohols during dehydration
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Artisan Link Pro: Dako’s staining solutionfor Special Stains
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31 stains avaliable
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Iron - Perls’ Technique
• Common method for demonstrating Iron
• Iron is stored in the body as hemosiderin in bone marrow,
spleen and liver
• Chemical reaction between potassium ferrocyanide and the
iron bound in the tissue
• A ferric ferrocyanide complex called Prussian Blue is formed
4FeCl3 + 3K4Fe(CN)6 => Fe4[Fe(CN)6]3 + 12KCl
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Perls’ Technique
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Optimization
Iron too week
• Increase time of the Potassium Cyanide solution
Iron too strong
• Decrease staining time in Potassium Cyanide solution
Counter stain too dark
• Decrease staining time
Counter stain too week
• Increase incubation time
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Silver Stains
• Some structures can bind silver ions
• Some can reduce the silver ions to metallic silver –
Argentaffin structures like melanin and enterochromaffin cells
• Some binds silver but can’t reduce silver ions to metallic
silver – Argyrophilic structures like Reticulin and nerve fibers
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Silver Stain for Reticulin
Common in Pathology labs
Used for liver, bone marrow,
lymph nodes and more
Reticulin fibers are Argyrophilic
Several different techniques
available; Gomori, Gordon and
Sweets’, Laidlaw’s
Reticular fibers are coated with
proteoglycans
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Silver Stain for Reticulin
• Potassium Permanganate oxidize the tissue to induce aldehyde groups in the carbohydrate matrix surrounding the reticular fibers
• Oxalic acid removes Permanganate deposits
• Iron Alun is used as an enhancer and makes the fibers more ”willing” to take up the silver salt
• Ammonium-silver-complex is reduced by the aldehyde groups, and submicroscopic amounts of metallic silver are deposited on the reticular fibers
• The silver salt is reduced by formalin to make the metallic deposits
• Gold chloride is toning the background deposits and enhances the black staining
• Unreduced silver is removed by Sodium thiosulphate
• Counterstain if wanted
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Reticulin Stain with counterstain NFR
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Retic Stain without counterstain
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Optimizing Retic Stain
Reticulin fibers too week
• Increase time in Iron Alun
• Short down wash step after Iron Alun
• Increase time in the Silver solution
• Increase time in Reduction step
• Increase time in Gold Chloride
Background too dark
• Increase time in Gold Chloride
Counter stain too week – increase staining time
Counter stain too strong – decrease staining time
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Questions?
October 10, 2017
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