The H & E Stain

The “Routine” Stain

Outline of Topics in this Presentation: Purpose/need to stain tissue under the microscope Nature of Hematoxylin dye/oxidation Mordant-dye-lake concept Different Hematoxylin formulas/mordant Summary of Hematoxylin staining theory Progressive v. Regressive staining Differentiation Bluing Counterstaining- the nature of Eosin dye Overview of H&E procedure Each step in more detail Automatic v. Hand-staining Quality control

The Need to Stain: Visualizing Tissue Elements The constituents of tissue are mostly transparent

and are visible under the microscope without staining only when extreme differences in refractive index exist (RI of unstained tissue = 1.53-1.54)

“Biological dyes” - highlight and differentiate tissue components and allow them to be seen under the microscope

H & E staining is the general tissue stain most commonly used in all histology laboratories

Hematein and Hematoxylin Hematoxylin is a natural dye extracted from the

wood of the haematoxylin campechianum, a type of logwood tree.

It is sold commercially as a mixture of Hematoxylin with other added chemicals

The active dye is not Hematoxylin itself, but rather Hematein

Hematoxylin can be considered as the dye precursor, and Hematein the oxidation product which acts as the active dye source

Hematoxylin can be considered a basic dye that will stain acidic tissue components such as nucleic acids (Basophilic)

Continuous Oxidation In Progress Hematoxylin solutions have a definite “shelf-life” and inferior

staining will result from overusing the solution. Hematein, which further oxidizes to a dark precipitate in this continuous process eventually exhausts the stain

Air exposure will cause oxidation, chemical oxidizers may also be used to speed oxidation to Hematein; such as sodium iodate, or potassium permanganate

You may wonder why a solution of Hematoxylin is used instead of the active Hematein; this is so that the supply of active dye may be replenished by the Hematoxylin and the “shelf-life” of the solution is extended

However despite this, there will always be some of the final oxidation product of Hematein, which forms a dark precipitate

This must be removed by filtering before use or it will deposit on the final slides.

Mordant Required Hematein is a weak basic dye that requires a

mordant to help link it to the desired tissue elements

A mordant is a metal with a valence of at least two. (2+ charge)

A mordant links the dye to the tissue by means of a covalent and coordinate bond (chelation)

The chelate is the complex formed from a mordant and a dye and is referred to in histology as a lake

Metal salts often used as mordants in histology include; iron and aluminum

Chemical Structures: Hematoxylin & Hematein

Hematoxylin Hematein

DNA and RNA stain by forming salt unions with the basic dye molecule; This attachment is dependant on the chelate formed by dye-mordant complex.

Note: Oxidation (loss of electron) is demonstrated by the loss of hydrogen and its electron from the Hematoxylin structure

A diagram showing how a mordant can be used to link the dye molecule to selected tissue elements

A Mordant-Dye “lake” using aluminum The mordant allows attachment where otherwise

there would only be a weak affinity The colored property of the dye (chromophore)

allows visualization of the site under the microscope.

Some Frequently Encountered Hematoxylin

Formulas and their most common uses: Aluminum mordant: includes Harris, Mayer’s, Erhlich’s, &

Delafield’s Iron mordant: Weigert’s, Lillie’s, & Heidenhain’s Iron Hematoxylins such as Weigert’s using a mordant such

as ferric chloride are stable only a short time due to the oxidation strength of this mordant

Iron Hematoxylins are used in techniques where aluminum Hematoxylin would be decolorized by subsequent steps in the procedure

Aluminum Hematoxylins are most often used for routine staining because decolorization in succeeding steps is not an issue, and the stability is greater due to less propensity for rapid oxidation making them more practical for extended use

To Summarize: Hematein alone has a weak affinity for tissue elements

For effective staining of nuclear material with Hematein you must have:

A mixture of Hematoxylin/Hematein-the dye precursor and active dye to replenish the active dye as oxidation occurs

A mordant, such as an aluminum or iron salt to link the dye to the nuclear material

A solvent, such as water in which to dissolve the dry powder dye which acts as a carrier of the dye

Additional Ingredients Added to Many Hematoxylin Solutions and their Functions

Oxidizing agents to convert the precursor hematoxylin to hematein

Acids to adjust pH, to extend shelf life Stabilizers to control the rate of oxidation Additions to the solvent to retard

evaporation and precipitation Additions such as glycerin, which retard

over-oxidation and discourage fungal growth

Progressive vs. Regressive Hematoxylin Methods: Nuclear staining using Hematoxylin may be accomplished

using progressive or regressive staining techniques

Some commonly used Hematoxylin formulas are most often used in either a progressive or regressive fashion

However, most Hematoxylins may be used progressively or regressively in various techniques

Exception: Iron mordant Hematoxylins are often preferred for special

staining techniques; being less resistant to decolorization

Progressive Staining

Progressive staining just means that the tissue is left in the staining solution just long enough to reach the desired endpoint

Frequent monitoring of stain quality may be needed to determine when staining is complete

The staining intensity is controlled by the time it is immersed in the solution

An example of progressive staining is H& E staining of frozen sections using Gill’s Hematoxylin

Regressive Staining Regressive staining involves deliberate

over-staining where the dye completely saturates all tissue elements

The tissue is then selectively de-stained using a process called differentiation

Harris Hematoxylin is popularly used regressively in many histology labs for routine H & E staining

Regressive staining is often preferred when very clear differentiation of tissue elements is desired

Differentiation is Selective De-staining:

Differentiation is achieved by using a dilute acid, most typically acid alcohol

In a well-stained slide, only the nuclei, calcium deposits and some mucins will be stained by the hematoxylin

Differentiation is affected because the ions in the differentiating solutions diffuse more rapidly than the dye molecule releasing loosely attached dye

The solution will also remove the dye more easily from some tissue elements than others (specificity)

Differentiation is halted by washing in water when the desired endpoint is reached

Note: with all Hematoxylin staining methods, progressive or regressive, the endpoint is the same

Bluing The nuclei will be stained the purplish

color of the acid dye Shifting the color range to blue provides a

much better contrast to the usual pinkish-red counter-stains

Bluing is the process of shifting the color from reddish to purple blue by the application of a weak alkaline solution

Bluing is utilized in both progressive and regressive techniques

Two Bluing Methods 1. The slides may be dipped into a weakly alkaline

solution such as ammonia water

2. The slides may also be washed in tap water, which may be slightly acid (pH 6.0-6.8) but this is still more alkaline than the Hematoxylin (pH 2.6-2.9) so bluing still occurs

Note: differentiation of the nuclei was accomplished by the acid alcohol and halted by previous water rising, so bluing is not needed for differentiation of the nuclei, only color shift

Next we will look at the Eosin Counter-Stain Eosin counter-staining is used to

demonstrate the general architecture of the tissue and to provide contrast to the now stained nuclei

Eosin is an acid dye that binds to the basic parts of the cell, i.e. the cytoplasm (Acidophilic)

An optimal Eosin stain will stain the cytoplasm and other tissue elements in three shades of pink

Considerations in the Application of the Eosin Counter-stain:

A mordant is not required The endpoint is not as sharp The shade of the color is important to provide

optimal contrast, and it can be adjusted by adding Acetic Acid which will make it redder

A too highly concentrated Eosin solution (dye density); will cause a blurring of the distinction between nuclear and non-nuclear elements

The solvent most commonly used solvent is 95% alcohol, so Eosin can be decolorized slowly by leaving the slides too long in alcohol solutions, weakening the color (undesirable)

Eosin: The Chemical Structure

The carboxylic salt present lends Eosin it’s acid character

Let’s review: We have been focusing on the dyes used

in H&E staining We have looked at some of the different

steps in the H&E procedure and their functions

Now we will review the whole staining procedure as a whole

We will see how all of the steps work together to create a well-stained slide

Major Steps in the Routine H & E

1. De-paraffinization-(removal of paraffin wax using xylene)

2. Hydration-(graded alcohols to water)3. Nuclear staining-using Hematoxylin4. Differentiation-Acid alcohol 5. Bluing-Ammonia Water6. Counterstaining-using Eosin7. Dehydration-(application of graded alcohol to

100% alcohol) 8. Clearing-Xylene (transition from alcohol to non-

aqueous reagents)9. Note: Water-rising steps are not shown

Step 1: De-Paraffinizing The slides have usually been heated before

staining to help bind the tissue to the glass slide and melt away excess paraffin

However, some paraffin still remains and must be removed so that the aqueous solution may reach and penetrate tissue components

This is accomplished by submersion in an organic solvent such as xylene or a xylene substitute, which dissolves and removes the remaining wax

The xylene must then be removed by concentrated alcohol since it is also not miscible with aqueous solutions

Step 2:Hydration The slides are then immersed in

graduating solutions of alcohol and water (from more concentrated to more dilute), until they reach a point where they can enter purely aqueous solutions

Most staining techniques move from 100% “absolute” alcohol, to 95%, and then can enter water since alcohol and water are miscible

Step 3:The Nuclear Staining

The slides are able to be stained with the water-based Hematoxylin solutions, as we have removed both the paraffin and the xylene with the alcohol treatment

The slides are immersed in the Hematoxylin solution

The staining time is established at each laboratory

Step 4: Differentiation Following a water rinse to remove excess

dye, the slides are immersed in the acidic differentiating solution

The timing of immersion must be established for each laboratory and procedure so that optimal differentiation is achieved

The differentiation process is halted by a water rinse

Step 5: Bluing

Bluing is the process of shifting the color from reddish to purple-blue by the application of a weak alkaline solution

Ammonia water (ammonium hydroxide + water) is often used; alternatively tap water may also be used if pH is suitable

Step 6 : Counter-stain

Eosin is used to contrast the cytoplasm with the nuclei

Eosin will stain in three shades of pink, provide contrast to the nuclear stain, and show many cytoplasmic and tissue elements

Dehydration: Step 7

After the eosin step, an alcohol rinse follows. This must be brief, as the Eosin stain can be diluted by the alcohol

95% alcohol is used first, followed by 100% alcohol

Complete dehydration is necessary, to remove any remaining water

Again timing is important here, so you don’t want to leave the slides in the alcohol

Step 8: Clearing When all the water is removed, the slide can be

immersed into xylene Incomplete dehydration will result in a cloudy

appearance to the final slide Just as in processing, xylene is used as a

transition between aqueous and non-aqueous Xylene is miscible with non-aqueous mounting

media, but will cause a “cloud” to form on the final if any water is present

Xylene is also ideal as a transition due to it’s refractive index, which produces a clear slide that allows light to pass well under the microscope

Xylene is also serves to activate the adhesive on cover-slippers using cover-slip tape.

Application of the Coverslip:Final step When satisfied with the final result, the histologist finishes

the slide by applying either a glass or tape cover-slip Mounting media is applied over to tissue and which serves

to “glue” the cover-slip to the slide, protecting the tissue from damage and exposure to air

Most routine staining uses xylene as a clearant Alternatively there are aqueous mounting media which

allow cover-slipping directly from water. Aqueous mounting/cover-slipping is often used when

exposure to alcohols or xylene will remove or dilute the stain applied to the tissue

In all cases the cover-slip must be applied to completely cover the tissue, removing all air bubbles and debris which may interfere with viewing under the microscope

The final result: a permanent microscopic “record” of the tissue sample

Automatic H & E or “Hand-staining” ?

Routine H & E staining can be accomplished by using either an automated H & E stainer or by manually executing the staining sequence

Both methods can produce high-quality slides The method chosen by each laboratory is often

determined by laboratory staffing, time constraints, specimen volumes and other factors

The stain procedure and optimal timing for each step must be established at each laboratory and will be outlined the lab’s routine H&E staining procedure

Quality Control The histologist is charged with monitoring the stain quality,

troubleshooting staining problems, and making adjustments when necessary

Many labs run an H&E control slide after daily changing of the staining set-up to avert any problems before a large number of slides are stained

An understanding of the H&E staining procedure is essential to help the histologist detect and correct problems

With experience, the histologist will learn to judge the desirable endpoints of both Hematoxylin and Eosin staining under the microscope

Use of a control slide and a quality control procedure will assist the histologist in producing high-quality and consistent H&E staining

The “Routine” Stain If everything has gone well, what we

should now have is a stained slide that clearly differentiates the nucleus and cytoplasm.

The “routine” H &E stain can provide very useful diagnostic information, and is sometimes the only slide needed for a diagnosis to be made.

H & E : liver tissue

Resources:

Bettelheim & March, Introduction to General, Organic & Biochemistry. 2nd Edition, Saunders Publishing. 1988

Sheehan & Hrapchak, Theory and Practice of Histotechnology. The C.V. Mosby Co., 1973

Milikin, Paul M.D., http://stainsfile.info/Stains/StainsFile/hematoxylin/hxintro.htm 2008

Information Prepared/compiled 3/12/2008 Joelle Weaver B.A., HTL(ASCP