OptimizingHematoxylin-Eosin,Iron & Reticulin

Charlotte Bergström

Senior Application Specialist

Agilent Technologies

October 10, 2017

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How Can We Help Our Users to Optimize?

▪ Hematoxylin-Eosin

▪ Iron

▪ Reticulin

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Dako CoverStainer solution

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

Hematoxylin

Connection, March 2010, Special Stains and H&E

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

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

Crisp, blue, open Chromatin pattern

Dense Chromatin pattern

Well-defined Nuclear Membrane Acidophilic Nucleoli

Red blood cells

Collagen

Smooth Muscle fibers

Elastic fibers

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

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

Artisan Link Pro: Dako’s staining solutionfor Special Stains

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31 stains avaliable

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

Perls’ Technique

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

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

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

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

Reticulin Stain with counterstain NFR

Retic Stain without counterstain

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

Questions?

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