H & e staining part 2

Hematoxylin & Eosin Staining

Presented by

Dr. Shrikant Sonune

Guided by

Dr Ashok Patil,

Dr Shilpa Kandalgaonkar,

Dr Mayur Chaudhary,

Dr Suyog Tupsakhare,

Dr Mahesh Gabhane.

Hematoxylin and eosin technique

Principle

H and E are principle stain for demonstration of nucleus and cytoplasm.

Alum acts as a mordant and the hematoxylin containing alum stains the

nucleus light blue which turns red in the presence of acid.

The cell differentiation is achieved by treating the tissue with acid

solution. The counterstaining is performed using eosin which imparts

pink color to cytoplasm


Removal of paraffin wax (dewaxing)

Removed with xylene (impermeable to stains)

2-3min of xylene immersion sufficient for sections of 10 µ thickness

First facilitated by warming the slides at 60 degrees oven to melt

the wax

Removal of xylene

Xylene is not miscible with water or low grade alcohols, hence dipped in

two changes of absolute alcohol

Hematoxylin and eosin technique…..

Hydration – after removal from xylene sections are

transferred to absolute alcohol for 1-2min until it becomes

opaque

Sections rinsed in second bath of alcohol, drained and taken

to water

Any pigments or deposits should be removed at this stage


Staining Slides immersed in hematoxylin (Mayer s, Harris, Gills) If regressive stain is used longer time is used to overstained

the structures

Differentiation Sections are dipped in acid alcohol, agitated and washed in

tap water

Observed under microscope If underdifferentiated- returned to acid alcohol If overdifferentiaited – retured to hematoxylin and

differentiated again


Blueing

Slides after draining off hematoxylin is transferred to water

for 10min. Sections when removed from hematoxylin or

acid alcohol are pink in color

Washing turns them blue

Counterstain

Transfer the slides to 1% aqueous eosin for 2min. Wash in

running water

Dehydration

Slides are taken through 3 stages of acid alcohol


11. Clearing

Sections transferred to xylene and left until clear

Tested for clarity by being held against a dark background

12. Mounting

Surplous xylene wiped off from slide surface

This step completed quickly to avoid section drying

Whole operation takes 5-10 seconds


Results

Cell nuclei – blue

Muscle fibres – red

Collagen fibres – pink

RBC – bright red

Theory of H & E staining

Hematoxylin:

Most commonly used stain.

It is derived from core of the logwood tree, Haematoxylon

compechianum,

It is not a true dye until partially oxidized (ripened)

----by exposure to air

----by chemical means with an oxidizing agent such as sodium

iodate or mercuric oxide.

HAEMATOXYLIN AND EOSIN

The hematoxylin and eosin stain is the most widely used histological stain because……

Its comparative simplicity

Ability to demonstrate clearly an enormous number of different tissue structures.

The hematoxylin --cell nuclei blue / black,

Eosin stains cell cytoplasm and most connective tissue fibres

The hematoxylin may be used in the demonstration of:

Intracellular substances

Chromosomes

Keratohyaline granules

Extracellular substances – Elastin

Minerals – calcium, copper etc.

Central nervous system – myelin, neuroglia

fibers.

Hematoxylin

Dark red color

Extracted from the heartwood of the tree Hematoxylin camechianum

The hematoxylin is extracted from log wood with hot water and then precipitated out from the aqueous solution using urea.

It is sold commercially as a crude mixture of hematoxylin and other, unidentified substance.

It comes as a brownish tan powder which is poorly soluble in water and somewhat more soluble in ethyl alcohol.

Hematoxylin itself is not a stain.

On oxidation it produces haematin - a poor dye but

Metallic mordant, forms the most powerful stain.

When aluminum salts– will stain blue

When ferric salt– will stain blue-black.

Ripening

This process of oxidation is often referred to as

ripening or maturing.

This can be carried out in two ways

1. Natural oxidation

2. Chemical oxidation

Natural oxidation:

Carried out by exposure to light and air.

Slow process

Resultant solutions seem to retain its staining ability for a long time.

Advantage

Ones oxidation has reached an acceptable level, the staining solution may be used, and it last for longer,

Disadvantage

In the planning and organization required ensuring that usable solution is always available. For example: Ehrlich’s and Delafield’s hematoxylin.

Chemical oxidation:

It is achieved by the addition of the oxidizing agents

such as mercuric oxide, sodium iodate and

potassium permanganate.

The use of chemical oxidizing agents converts the

hematoxylin to haematin almost instantaneously, so

these hematoxylin solutions are ready for use after

preparation.

Properties of chemically oxidized

hematoxylin Have a shorter useful life than the naturally oxidized

haematoxylins .

However, the possibility of over – oxidation has been clearly established that the production of oxyhaematein inhibits successful staining.

To prevent these, glycerol has been incorporated in many formulas. Glycerol acts as stabilizer by preventing over oxidation and reducing evaporation.

Properties

Haematin is anionic, having poor affinity for tissue.

It is an inadequate stain without the presence of

mordant. (most useful mordant are salts of

aluminium,)

Hematoxylin solution using Lead as a mordant are

occasionally used for demonstration of argyrophil

cells.

Classification according to which mordant is

used.

Alum haematoxylins Ehrlich’s

Mayer’s

Harris

Cole’s

Delafield

Carazzi’s

Classification according to which mordant is

used. Iron haematoxylins

Weigert

Heidenhain’s

Loyez

Verhoeff

Tungesten

Molybdenum

Lead

Hematoxylin without mordant

Alum hematoxylin:

Routinely used in the hematoxylin and eosin stain,

and produce good nuclear staining.

The mordant is aluminium, in the form of ‘potash

alum’- aluminium ammonium sulphate.

The nuclei 1st become a red colored,

Differentiation is carried out

Then, Converted to familiar blue-black when the

section is washed in weak alkali.

The alum hematoxylin can be used regressively or

progressively.

The tomes for hematoxylin staining and for satisfactory

differentiation will vary according to:

The type and age of alum hematoxylin used.

The type of tissue.

The personal preference of the pathologist.

The most commonly used hematoxylin are

Ehrlich’s, Mayer’s, Harris’s, Cole’s and Delafield’s

haematoxylins.

Carazzi’s hematoxylin is occasionally used,

particularly for urgent frozen sections.

Harris’s hematoxylin: (1900)

This is an alum hematoxylin which is traditionally chemically ripened with mercuric oxide (sodium or potassium iodatemay be used as substitutes for oxidation.).

It is a powerful and selective nuclear stain giving clear nuclear staining.

It is used widely as a nuclear stain in exfoliative cytology.

In routine histology practice it is used regressively, but in exfoliative cytology it may be used as a progressive stain.


Preparation of solution:

Hematoxylin - 1 g

Absolute alcohol - 10 ml

Ammonium or potassium alum - 20 g

Distilled water - 200 ml

Mercuric oxide - 0.5 g


Dissolve hematoxylin in alcohol

Add to it alum, previously dissolved in hot water.

The mixture is rapidly brought to boil

Mercuric oxide is then slowly and carefully added, when the solution turns dark purple.

The stain is rapidly cooled under tap water.

Filter before use.

Mayer’s hematoxylin: (1903)

A next widely used hematoxylin

Chemically ripened with sodium iodide.

It is more vigorous in action than Ehrlich’s

hematoxylin and gives little or no staining of muco-

polysaccharide material.

It is used as a nuclear counter stain in the

demonstration of glycogen (PAS, mucicarmine) in

various enzyme histological techniques.

The stain is applied for short period (progressive

stain usually 5-10 min.) until nuclei are stained, and

is then blued without any differentiation.

Differentiation might destroy or decolour the stained

cytoplasmic components. It can be used as a

regressive stain like any alum hematoxylin.

Mayer’s hematoxylin: (1903)


Hematoxylin - 1 g


Potassium or aluminium alum - 50 g

Sodium iodate - 0.2 g

Citric acid - 1 g

Chloral hydrate - 50 g

The hematoxylin, potassium alum, sodium iodate is

dissolved in distilled water by warming and stirring, or

by allowing to stand at room temperature overnight.

The chloral hydrate and citric acid are added, and the

mixture is boiled for 5 minutes, then cooled and

filtered.

Chloral hydrate acts as a preservative and citric acid

sharpens nuclear staining.

Ehrlich’s hematoxylin: (1886)

This is a naturally ripened alum hematoxylin, most commonly used in both normal and morbid histology.


Hematoxylin - 2 g


Glycerol - 100 ml


Glacial acetic acid - 10 ml

Potassium alum - 10 – 14 g

( in excess )

Dissolve the hematoxylin in the alcohol.

The incorporation of glycerol.

Finally, add potassium alum (till saturation).

The stain may be ripened naturally by allowing to stand in large flask, loosely stoppered with cotton wool.

Filter before use.

It may be partially oxidized

Stain used immediately by the addition of 0.3 g

sodium iodate to the above.

It also stains mucin in salivary glands, some muco-

polysaccharide substances such as cartilage, and

‘cement lines’ of bone etc. Ehrlich’s hematoxylin is

not ideal for frozen sections.

Delafield’s hematoxylin: (1885)

This is a naturally ripened alum hematoxylin, which has similar longevity to Ehrlich’s hematoxylin.


Solution A:

Hematoxylin - 4 g


Solution B:

Alumunium alum - 60 g


Solution C:

Glycerol - 100 ml


The hematoxylin is dissolved in 25 ml of alcohol

added to solution ‘B’ (alum solution).

Mixture is allowed to stand in light and air for 5 days and is then filtered.

Added to solution ‘C’.

Allowed to stand exposed to light and air for about 3-4 months or until the stain is sufficiently dark in color,

Then filtered and stained.

Cole’s hematoxylin: (1943)

This is an alum hematoxylin, artificially ripened with an alcoholic iodine solution. It has good keeping qualities and is suitable for use in sequence with celestine blue, unlike Ehrlich’s hematoxylin.


Hematoxylin - 1.5 g

1%iodine in absolute ethanol - 50 ml

Saturated aqueous potassium alum - 700 ml


The hematoxylin is dissolved in warm distilled water

and mixed with iodine solution.

The alum solution is added, and the mixture

brought to boil, then cooled quickly and filtered.

The solution is ready for immediate use,but may

need on occasion filtering after storage,

Carazzi’s hematoxylin: (1911)

This is an alum hematoxylin which is chemically

ripened using potassium iodate.


Hematoxylin -5 gm

Glycerol -100 ml

Potassium alum - 25 gm


Potassium iodate - 0.1 g

Hematoxylin is dissolved in the glycerol

and the alum is dissolved in most of the water overnight.

The alum solution is added slowly to solutition. Potassium iodate is dissolved in the rest of the water with gentle warming and is then added to the haematoxylin-alum-glycerol mixture.

The final staining solution is mixed well and is then ready for immediate use, it remains usable for about six months.

Carazzi’s hematoxylin may be used as a

progressive nuclear counter stain using a short

staining time followed by blueing in tap water.

It is particularly suitable since its pale and precise

nuclear stain, does not stain any of the cytoplasmic

components.

It is particularly used for the frozen section (due to

rapid staining)

Gill’s hematoxylin: (1974)

Preparation of solution:-


Ethylene glycol - 250 ml

Hematoxylin - 2 g

Sodium iodate - 0.2 g

Aluminium sulphate - 17.6 g

Glacial acetic acid - 20 ml

The reagents are added in the order given and the

mixture stirred for 1 hour at room temperature.

The solution is ready for immediate use.

Double or triple concentrations of ethylene glycol

may be used as preferred

Gill’s 1 (normal),

Gill’s 2 (double conc of ethylene glycol),

Gill’s 3 (triple conc of ethylene glycol).

Advantages

They are fast in action,

Stable for at least 12 months,

Produce little or no surface precipitate,

Their preparation does not involve boiling the

solution.

Staining times with alum haematoxylins:

It will vary according to following factors:

Type of hematoxylin used:- e.g. Ehrlich’s hematoxylin 20-45 min, Mayer’s hematoxylin 10-20 min.

Age of stain: as the stain ages the staining time will need to be increased.

Intensity of use of stain: A heavily used hematoxylin will loose its staining power rapidly and longer staining times will be necessary.

Progressively or regressively method.

e.g. Mayer’s hematoxylin used progressively 5-10 min, used regressively 10-20 min.

Pretreatment of tissues or sections

e.g. length of time in fixative or acid decalcifying solution or whether paraffin or frozen section.

Post treatment of sections

e.g. subsequent acid stains such as Van Gieson.

Personal preference.

Disadvantages of alum hematoxylin

Sensitivity to any subsequently applied acidic

staining solutions

examples are in the Van Gieson and other trichrome

stains.

The application of the picric acid – acid fuchsin

mixture in Van Gieson’s stain removes most of the

hematoxylin so that the nuclei are barely

discernible.

Nuclear staining can be achieved by using an iron –

mordanted hematoxylin e.g.Weigert’s hematoxylin,

Alternative is the combination of celestine blue

staining solution with an alum hematoxylin.

Celestine blue is resistant to the effects of acid and

ferric salt.

Strengthens the bond between the nucleus and alum

hematoxylin.

Celestine blue- alum hematoxylin:


Celestine blue solution

Celestine blue B -2.5 g

Ferric ammonium sulphate -25 g

Glycerol -70 ml


Ferric ammonium sulphate is dissolved in cold

distilled water with stirring.

The celestine blue B is added to this solution and

the mixture is boiled for few minutes.

Filtered

glycerin is added.

Filter before use.

Celestine blue B,

an oxazine dye,

Has little useful colouring property of its own.

It forms an additional strong mordant with certain

haematoxylins .

Celestine blue B is used as a preliminary to alum

hematoxylin staining.

Iron haematoxylin

Iron salts such as ferric chloride or ferric ammonium sulphate are used both as oxidizing agent and as mordant.

Demonstrating a much wider range of tissue structures than the alum haematoxylins,

The most common iron haematoxylins are

Heidenhain’s hematoxylin

Weigert’s hematoxylin

Verhoeff’s hematoxylin

Loyez hematoxylin

Over oxidation of the hematoxylin is a problem with these stains,

So it is usual to prepare separate mordant/ oxidant and hematoxylin solutions and mix them immediately before use

e.g. Weigert’s hematoxylin

To use them consecutively (e.g. Heidenhain’s and Loyez haematoxylins).

It may be used as

Differentiating fluid after hematoxylin staining,

Mordanting fluid before it.

Weigert’s hematoxylin (1904):

An iron hematoxylin used as a nuclear stain in techniques where acidic staining solutions are applied to the sections subsequently (e.g. Van Gieson stain).

In Van Gieson stain, picric acid is one of the constituents which have marked decolorizing action on nuclei stained with alum hematoxylin.

Weigert hematoxylin which is mordanted to iron salt (ferric chloride) has sufficient avidity to withstand this treatment.

Preparation:

The iron and hematoxylin solutions are prepared separately and are mixed immediately before use.

Solution A (stain) Solution B (mordant)

Hematoxylin – 1 g 30% aqueous ferric chloride

Absolute alcohol – 100 ml ( anhyd. ) -4 ml

Conc. HCl - 1 ml

Dist. Water - 95ml

The color of the mixture should be a violet black.

If muddy – brown, it must be discarded.

Heidenhain’s hematoxylin (1896):

Ferric ammonium sulphate as oxidant/ mordant.

The same solution is also used as a differentiating fluid.

The iron solution is used first

The section is treated with hematoxylin solution until it is over stained,

Then it is then differentiated with iron solution under microscopic control.

Heidenhain’s hematoxylin is a cytological stain.

It is used regressively

Requires careful differentiation, for which reason it is

only completely successful on thin sections.

It may be used to demonstrate

chromatin,

chromosomes,

nuclei,

centerosomes,

mitochondria,

muscle striations

myelin.

Heidenhain’s hematoxylin (1896):

Preparation:

Hematoxylin solution (stain)

Hematoxylin - 0.5g

Absolute alcohol- 10ml

Distilled water- 90ml

Iron solution (mordent and differentiator)

Ferric ammonium sulphate- 5g

Distilled water - 100ml

Dissolved hematoxylin in alcohol

add water.

Allow to ripen for a few weeks and store in a tightly

stoppered bottle.

Loyez hematoxylin (1910):

This is an iron hematoxylin in which ferric ammonium sulphate is used as the mordant.

The mordant and hematoxylin solution are used consecutively,

Differentiation is by Weigert’s differentiator (borax and potassium ferricyanide).

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

Verhoeff’s hematoxylin (1908):

Verhoeff’s hematoxylin is used to demonstrate

elastic fibers after all routine fixative.

Coarse fibres are intensely stained, but the staining

of fine fibers may be less than satisfactory.

The differentiation step is critical to the success of

this method.

Introduced by Mallory in 1897 for demonstration of neuroglia.

It is now widely used for other purposes (fibrin, muscle striation).

It is unique among hematoxylin stains in the number of structures that may be demonstrated, together with the two color staining (shades of blue and red) from the single solution.

Tungsten hematoxylin

Mallory first advocated a 1: 10 ratio of hematoxylin

to mordant, later a 1: 20 ratio should be used with

pure mordant due to earlier mordant was impure.

Later Turner et al proposed that this proportion is

too high and does not give the most satisfactory

staining.

Natural ripening of phosphotungstic acid

hematoxylin is slow and several chemical oxidizing

agents have been used to hasten the conversion of

hematoxylin to haematein. As an alternative to

chemical oxidation,

Haematein may be used initially, instead of

hematoxylin and provides a stain that is usable 24

hours after preparation.

Molybdenum hematoxylin:

Molybdenumic acid as the mordant are rare,

Technique which gained any acceptance was the

Thomas (1941) technique which was mentioned by

MacManus and Mowry (1964).

Demonstration of collagen and coarse reticulin,

More valuable and accepted for these connective

tissue fibre exist

Lead hematoxylin:

Hematoxylin solutions which incorporate Lead

salts have recently been used in the demonstration

of the granules in the endocrine cells of the

alimentary tract and other regions

Hematoxylin without a mordant

Freshly prepared hematoxylin solutions, used without a mordant,

Used to demonstrate various minerals in tissue sections – Mallory described a method for lead, iron and copper.

The basis of the Mallory methods is the ability of unripened hematoxylin to form blue black lakes with these metals.

Test for staining power of hematoxylin…

Adding few drops of hematoxylin to 50ml of tap

water will turn a bright, clear purple or blue violet

color.

Exhausted solutions will not be clear & bright & the

color will be rusty or green.

Hematoxylin Specific use

Harris;’s hematoxylin Exfoliative cytology

Mayers hematoxylin Nuclear counter stain in PAS

Ehrlichs hematoxylin Mucin & other mucopolysaccharide

Carazzis hematoxylin Progressive nuclear stain & frozen section

Celestine blue- alum hematoxylin Where subseqent acidic stains are to be used e.g. van Gieson stain

Weigerts hematoxylin Where subseqent acidic stains are to be used e.g. van Gieson stain

Heidenhains hematoxylin Differentiating cytological fluid stain

Loyes hematoxylin To demontrate myelin, frozen section

Tungsten hematoxylin Fibrin , & muscle straitions

Molybdenum hematoxylin Demonstration of collegen fibre

Lead hematoxylin Granules of endocrine cell

Hematoxylin without mordant Pigment lead , iron

Eosin

Eosin, a red dye,

Stains connective tissue and cytoplasm in varying intensity and shades (red to pink)

Eosin is derived from fluorescein and is available in following types:

Eosin Y (eosin yellowish, eosin water soluble)

Ethyl eosin (eosin S, eosin alcohol soluble)

Eosin B (eosin bluish, erythrosine B)

Eosin Y is most commonly used and is readily

soluble in water, less so in a alcohol thus it is

sometimes sold as ‘water and alcohol soluble’

Preparation

Eosin Y, water soluble 5gm.

Distilled water 1000ml.

Crystals of thymol added to inhibit the growth of fungi.

Alcohol soluble eosin is employed as a 0.5 % solution

in alcohol.

Eosin Y water &

Alcohol soluble 10gm

Distilled water 50ml

95%ethyl alcohol 940ml

In use, sections should be treated with 95% alcohol

before staining with alcoholic eosin, and the excess

stain washed out in the same solvent.

The addition of little acetic acid (0.5 ml to 1000 ml

stain) is said to sharpen the staining.

Ethyl eosin and eosin B are now rarely used,

although occasional old methods specify their use,

e.g. the Harris stain for Negri bodies.

Thank you……

Health & Medicine

H & e staining part 2