Haematoxylin & Eosin Stain

Prepared by: Ms. BR Tsauses

Anatomical Pathology 2A (ANP611S)

April 2020

Learning objective

• Demonstrate basic knowledge of the dyes and preparation techniques in order to troubleshoot and/ or modify procedures for specialized use.

Pre-learning Quiz

• Please take the pre-learning quiz before proceeding with the presentation.

Introduction

• H & E stain is the most widely used histological stain.

• Popular due to its comparative simplicity and ability to demonstrate clearly an enormous number of different tissue structures.

• Haematoxylin component stains cell nuclei blue-black and shows good intracellular detail.

• Eosin stains the cell cytoplasm and most connective tissue fibres in varying shades and intensities of pink, orange and red.

Eosin

• Eosin is the most suitable stain to combine with an alum haematoxylin to demonstrate the general histological architechture of a tissue.

• It has an ability to distinguish between cytoplasm of different types of cell and between the different types od connective tissue fibres and matrices, by staining them different shades of red and pink.

Eosin types

• Eosin Y )eosin yellowish, eosin water-soluble) – C.I. No. 45380 (C.I. Acid Red 87) – most widely used

• Ethyl Eosin (eosin S, eosin alcohol-soluble) C.I. No. 45386 (C.I. Solvent Red 45) – rarely used

• Eosin B (eosin bluish, erythrosine B) C.I. No. 45400 (C.I. Acid Red 91) –rarely used

Eosin Y

• Eosin Y is most widely used and is satisfactorily soluble in alcohol. It is sometimes sold as ‘water and alcohol soluble’.

• Usually used as a 0.5 or 1.0% solution in distilled water, with a crystal of thymol added to inhibit growth of fungi.

• Addition of little acetic acid is said to sharpen the staining.

Haematoxylin

• Extracted from heartwood (logwood) of the tree Hematoxyloncampechianum – originated in Mexican State of Campeche, but now mainly cultivated in West Indies.

• Hematoxylin is extracted from logwood with hot water, precipatedfrom aqueos solution using urea.

• Hematoxylin is not a stain by itself, major oxidization product is hematein (a natural dye responsible for color properties).

Production of Hematein from Hematoxylin• Production takes place in two ways:

• Natural oxidation (ripening) by exposure to light and air• Slow process, sometimes +/- 3-4 months• Resultatnt solution retain its staining ability for long time• E.g. Ehrlich’s and Delafield’s hematoxylin solutions

• Chemical oxidation• Chemical oxidizing agents converts hematoxylin to hematein almost instantaneously

– immediately ready for use after preparation• Demonstrate shorter useful life than naturally oxidized hematoxylins• E.g. Sodium iodide (Mayer’s hematoxylin), mercuric oxide (Harris’s hematoxylin)

• Hematein: Anionic, possess poor affinity for tissue, inadequate as nuclear stain without presence of a mordant.

Mordant

• Mordant/ metal cation confers a net positive charge to the dye-mordant complex and enables it to bind to anionic tissue sites, e.g. nuclear chromatin.

• Type of mordant used influences strongly the type of tissue components stained and their final colour.

• Most useful mordants for haematoxylin: salts of aluminium, iron and tungsten

• Haematoxylin solutions using lead as a mordant are occasionally used, e.g. in demonstration of argyrophil cells.

• Most mordants are incorporated into haematoxylin staining solutions.• Some haematoxylin stains require tissue section to be pre-treated with the

mordant before staining, e.g. Heidenhain’s iron haematoxylin.

Classification of hematoxylin solutions

Note: Classification based on type of mordant used.

• Alum hematoxylins

• Iron hematoxylins

• Tungsten hematoxylins

• Molybdenum hematoxylins

• Lead hematoxylins

• Hematoxylin without mordant

Alum Hematoxylins

• Comprises most of those used routinely in the H&E stain• Produces good nuclear staining• Mordant - aluminium in the form of:

• ‘potash alum’ also known a aluminium potassium sulphate• ‘ammonium alum’ also known as aluminium ammonium sulphate

• All stain the nuclei red, which is converted to blue-black when section is washed in a weak alkali solution.

• Tap water is usually alkaline enough to produce this colour change, however, following alkaline solutions are required:• saturated lithium carbonate, • 0.05% ammonia in distilled water• Scott’s tap water substitute

Alum Hematoxylins cont.

• May be used regressively, i.e.:• section is over-stained and then differentiated in acid alcohol, followed by

‘blueing’.

• Progressively, i.e.:• Stained for predetermined time to stain the nuclei adequately, but leave the

background tissue relatively unstained.

• Times for hematoxylin staining and for satisfactory differentiation will vary according to the type of tissue and personal preference of pathologist

Alum Hematoxylins cont.

• Most commonly used hematoxylins for routine H&E staining:• Ehrlich’s

• Mayer’s

• Harris’s

• Gill’s

• Cole’s

• Delafield’s

• Carazzi’s hematoxylin is occasionally used, especially for urgent frozen sectins.

Disadvantages of alum hematoxylins

• Their sensitivity to any subsequently applied acidic staining solutions• E.g. van Gieson and other trichrome stains

• Applocation of picric acid-acid fuchsin mixture in van Gieson’s stain removes most of the hematoxylin resulting in barely discernible nuclei.

• In this case, satisfactory nuclear staining can be achieved by using an iron-mordanted hematoxylin, e.g. Weigert’s hematoxylin – resistant to the effect of picric acid.

• Suitable and popular alternative: combination of a Celestine blue staining solution with alum hematoxylin.

• Celestine blue is resistant to effects of acid, and ferric salt in the prepared celestine blue strengthens bond between nucleus and alum hematoxylin to provide a strong nuclear stain that is reasonably resistant to acid.

Papanicolaou stain for cytological preparations

• Papanicolaou stain – universal stain for cytological preparations

• Harris’s hematoxylin is the optical nuclear stain and combination of OG 6 and EA 50 gives subtle range of green, blue and pink hues to the cell cytoplasm.

• It is vital that the transparent quality of the cytoplasmic stain, and the nuclear chromatin should be easily distinguished.

Iron hematoxylins

• Iron salts are used as oxidising agent and as mordant.

• Most commonly used iron salt’s:• Ferric chloride

• Feric ammonium sulphate

• Common iron hematoxylins:• Weigert’s hematoxylin

• Heidenhain’s hematoxylin

• Loyez hematoxylin for myelin

• Verhoeff’s hematoxylin for elastin fibres

Iron hematoxylins cont.

• Disadvantage:• Over-oxidation of hematoxylin• Techniques are more time-consuming and usually incorporates a differentiation stage

which needs microscopic control for accuracy.

• Remedy for over-oxidation:• Prepare separate mordant/ oxidant and hematoxylin solutions and mix them

immediately before use, e.g. Weigert’s hematoxylin or use them consecutively, e.g. Heidenhain’s and Loyez hematoxylins.

• Due to strong oxidizing ability of the solution containing iron salts, it is often used as a subsequent differentiating fluid after hematoxylin staining, as well as for mordanting fluid before it.

• Advantage:• Iron hematoxylins are capable of demonstrating a much wider range of tissue

structures than alum hematoxylins.

Weigert’s hematoxylin (Weigert 1904)

• Iron hematoxylin in which ferric chloride is used as the mordant/ oxidant.

• Iron and hematoxylin solutions are prepared separately and mixed immediately before use.

Heidenhain’s hematoxyin (Heidenhain 1896)

• This iron hematoxylin uses ferric ammonium sulphate as oxidant/mordant, and the same solution is used as differentiating fluid.

• Iron solution is used first, the section is then treated with the hematoxylinsolution until it is over-stained, and is then differentiated with iron solution under microscopic control.

• Heidenhain’s hematoxylin can be used to demonstrate many strictures according to the degree of differentiation.

• All components are black or dark gray-black after staining.

• Hematoxylin staining is removed progressively from different tissue structures at different rates using the iron alum solutions.

Heidenhain’s hematoxylin cont.

• Differentiation:• Mitochondria, muscle striations, nuclear chromatin and myelin can all be

demonstrated.

• Black colour disappears first from mitochondria, then from muscle striations, then from nuclear chromatin.

• Prolonged differentiation will remove stain from almost all structures, although red blood cells and keratin retain the stain the longest.

• To achieve more easily controllable differentiation: • Differentiating iron alum solution is diluted with an equal volume of distilled

water or an alcoholic picric acid solution.

Loyez hematoxylin (Loyez 1910)

• This iron hematoxylin uses ferric ammonium sulphate as mordant.

• Mordant and hematoxylin solutions are used consecutively, and differentiation is by Weigert’s differentiator, i.e. borax and potassium ferricyanide.

• It is used to demonstrate myelin and can be applied to paraffin, frozen, or nitrocellulose sections.

• Both methods are similar to that of Loyez.

• Heidenhain myelin stain (do not confuse with Heidenhain’s iron hematoxylin) is essentially the Loyez technique, but judicious selection of staining time removes the need for separate differentiation.

• Second variant is the short Weil technique in which the mordant and dye are mixed before use, rather than consecutively.

• Both these techniques are shorter than the Loyez.

Verhoeff’s hematoxylin (Verhoeff’s 1908)

• Used to demonstrate elastic fibres.

• Ferric chloride is included in the hematoxylin staining solution, together with Lugol’s iodine, and 2% aqueous ferric chloride is used as the differentiator.

• Coarse elastic fibres stain black.

• Other elastic methods may stain finer fibres, but for high contrast required for photomicrography the intense black staining produced by Verhoeff’s is ideal.

• Verhoeff’s is also used to stain elastin fibres as part of the Movat’spentachrome.

Verhoeff’s hematoxylin elastic stain

Tungsten hematoxylins

• Only one widely used tungsten hematoxylin, although many variants exist on the original Mallory phosphotungstic acid hematoxylin (PTAH) technique.

• Mallory (1897, 1900) combined hematoxylin with 1% aqueous phosphotungstic acid, the latter acting as the mordant,

• Hematoxylin can be oxidized chemically by using a potassium permanganate solution, again, the solution is usable within 24 hours.

• Most satisfactory method of preparation, albeit time-consuming, is to allow natural ripening of the tungsten hematoxylin solution in light and air.

• PTAH solution produced may take some months to ripen, but remain usable for years.

• Its use is applicable to both CNS material and general tissue structure, and to tissues fixed in any of standard fixatives.

• Staining times vary according to: • method of preparation, • fixative used and • tissue structure to be demonstrated

• Staining more accurate after section is treated with an acid dichromate solution, and after a Mallory bleach procedure (helps with differential staining).

Phosphotungstic Acid Hematoxylin (PTAH) Stain

Lead hematoxylins

• Hematoxylin solution incorporated lead salts – used in demonstration of granules in endocrine cells of the alimentary tract and other regions.

• Most practical diagnostic application – identification of endocrine cells in some tumors, research procedures such as in the localization of gastrin-secreting cells in stomach.

• It has largely been superseded by immunochemistry.

Hematoxylin without a mordant

• Freshly prepared hematoxylin solutions, used without a mordant, have been used to demonstrate various minerals in tissue sections.

• Mallory (1938) described a method capable of demonstrating iron and copper (Mallory & Parker 1939); these methods have now been superseded by techniques that are more specific.

• Basis of Mallory methods is the ability of unripened hematoxylin to form blue-black lakes with these metals.

Quality control in routine H&E staining

• Accurate diagnosis depends on the person examining stained microscope slides, usually H&E paraffin sections.

• Consistency of staining is vital to avoid difficult histological interpretation.

• Generally, automated staining machines allow accurate and consistent staining, differentiation and dehydration by adjusting times of each step.

• In contrast, variability in stains may require adjustment of staining times.

• In automatic staining, problems are usually associated with hematoxylinstaining as compared to eosin.

Quality control in routine H&E staining cont.

• Common variables:• Variation in batch number of hematoxylin• Change of supplier• pH differences• Different person following same preparation instructions may result in a

stain such as hematoxylin having slightly different staining properties each time it is made up

• Age of the stain• Degree of usage• Fixation, variations in processing schedules, section thickness and excessive

hot-plate temperatures may lead to variation in staining

Quality control in routine H&E staining cont.

• Quality control includes:

• Checking new stain batches for efficacy against current or earlier batches

• Adjusting staining times for uniformity

Difficult sections

• Problem of using hematoxylin as a nuclear counterstain when other acidic dyes are to be used, e.g. van Gieson.

• Similar problem experienced when trying to stain paraffin sections when tissue has been fixed for a long time in a formalin fixative that has gradually become more acidic.

• Tissues and/ or paraffin wax blocks sent from countries with hot climates compound the problem – tisssues may be fixed in poor quality unbuffered, non-neutral formalin fixative that deteriorates in the heat and progressively becomes more acidic.

• Major problem – getting adequate nuclear staining with hematoxylinwithout also staining the cytoplasm; thus giving a uniformly muddy purple to the finished section after applying eosin.

Difficult sections cont.

• Two ways in which diagnostically acceptable H&E can be obtained in this case:• Use of Celestine blue-alum hematoxylin sequence

• Use of an iron hematoxylin such as Weigert’s

Tips

• Reduced eosin staining intensity or decreased staining capacity is often the result of an increase in the pH of the eosin solution: • Carry over of alkaline tap water 48 • Inadequate rinsing to remove bluing agent • Contamination with bluing agent

• Addition of water to an alcoholic eosin can cause precipitation. When using an alcoholic eosin, an alcohol rinse should be placed prior to the eosin solution

• Depletion of hematoxylin dye content is a common staining issue

• Incomplete wax removal prior to staining will cause uneven staining

• Incubation time for sections: • 60°C for 30 minutes • 75°C for 18 minutes • 60°C for 10 minutes if the hotplate is used

Troubleshooting

• Progressive and Regressive staining should give the same results

• H&E stains must be filtered before use due to deposits that will appear on the tissue as crystals or debri

• Clean slides and coverslips must be used therefore don’t touch the flat surfaces with your fingers

• Tissue sections must be in the middle of the slide for easier microscopy

• Excess damaged sections must be removed before coverslipping

• Do smooth, gentle movements to prevent the tissue to be washed off

during the staining process

• Change solutions daily depending on the amount slides stained

• Recut section before staining if any problem is noticed e.g. folds, chatters, wrinkles

Self-activity

• Chapter 10, pages 175 – 183, 185 of your prescribed book:• Familiarize yourselves with the following:

• Different types of hematoxylins for routine H&E staining, including the preparation of the solution for each

• Staining times with alum hematoxylins

• Routine staining procedures using alum hematoxylins

• Papanicolaou formula & staining method

• Weigert’s iron hematoxylin (preparation of solutions)

• Heidenhain’s iron hematoxylin (preparation of solutions, method, results, notes)

• PTAH staining technique solution using hematein, potassium permanganate, naturally oxidized

• Table 10.2: Uses of hematoxylin stains

References

1. John D. Bancroft, Christopher Layton and S.Kim Suvarna, (2013), Bancroft’s Theory and Practice of Histological Techniques,7th Edition, Elsevier, China

2. J.A.Kiernan,(2015), Histological and Histochemical Methods, 5thEdition, Scion, UK