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Andrew Lisowski, M.S., HTL (A.S.C.P.)

Science of H & E

“The desired end result of a tissue stained with hematoxylin and eosin is based upon what seems to be almost infinite factors. Pathologists have individual preferences for section thickness, intensities, and shades. The choice of which reagents to use must take into consideration: cost, method of staining, option of purchasing commercially-prepared or technician-prepared reagents, safety, administration policies, convenience, availability, quality, technical limitations, as well as personal preference.”

Hematoxylin and Eosin Staining

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Guidelines for Hematoxylin and Eosin StainingNational Society for Histotechnology

Why Do We Stain?

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In order to deliver a medical diagnosis, tissues must be examined under a microscope.Once a tissue specimen has been processed by a histology lab and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: when viewed under the microscope, everything appears in uniform dull grey color.

Unstained tissue H&E stained tissue

What Does "Staining" Do?

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Contrasts different cells

Highlights particular features of interest

Illustrates different cell structures

Detects infiltrations or deposits in the tissue

Detect pathogens

There are different staining techniques to reveal different structures of the cell

H&E stain showing extensive iron deposits

Placenta’s large blood vessels

Superbly contrasted GI cells

As its name suggests, H&E stain makes use of a combination of two dyes – hematoxylin and eosin.

It is often termed as “routine staining” as it is the most common way of coloring otherwise transparent tissue specimen. H&E is fast and relatively inexpensive method of assessing tissue morphology. First used almost 150 years ago, it is still used today with little changes.

What is H&E Staining?

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Staining does not produce color randomly; instead, the dyes exploit differences in the chemistry of the tissue to differentially dye various components.Ionic bonding is the most important type of bonding that occurs in histologic staining techniques. It involves electrostatic attraction between ions of opposite charge, one of which is in the tissue, and the second of which is in the dye. Hematoxylin is positively charged and can react

with negatively charged cell components, such as nucleic acids in the nucleus. These stain blue as a result.

Eosin is negatively charged and can react with positively charged components in the tissue, such as amino groups in proteins in the cytoplasm. These stain shades of red to pink as a result.

How is it Done?

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Example of H&E stain

Long history of use, staining method published by Böhmer & Fischer in 1875 H&E is the primary diagnostic technique for the evaluation of morphology and the changes

associated with the disease process H&E remains the most frequently used tissue stain worldwide with an estimated 2.5 to 3

million slides stained per day It is a useful all-purpose stain that is quick and easy to use, which may explain why it has

stood the test of time Customer expectations or preferences are extremely subjective

H&E Stain

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H&E Staining Procedure Step-by-Step

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Procedure involves several steps and regents divided into groups based on their function.

Deparaffinization or Dewaxing steps:

Typically done by solvents like xylene or xylene substitutes to remove paraffin from sections adhered to the glass slide.

Staining pathway

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H&E Staining Procedure: Hydration

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Hydration:

It is an introduction of water into the tissue section. This is done by passing the slides slowly through a series of decreasing concentrations of alcohols.

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H&E Staining Procedure: Staining

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Primary Staining:

Hematoxylin is used after deparaffinization and hydration. It stains the nucleus of the cell, specifically, the chromatin within the nucleus and the nuclear membrane. The nucleoplasm of the nucleus should remain unstained; this allows the staining pattern of the chromatin to be seen easily.

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H&E Staining Procedure: Differentiating

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Differentiating:

Achieved by strong or weak acid solutions depending on a staining method. In the regressive method, strong acid rinse removes the excess stain and all non-specific background. In a progressive method, weak acid solution removes.

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H&E Staining Procedure: Bluing

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Bluing Step:

Bluing reagents vary from ammonia solutions, tap water, Scott’s solution, Blue Buffer 8, lithium and magnesium carbonate solutions. Bluing changes the reddish-purple hematoxylin to a blue or purple-blue color. It is a pH dependent reaction and occurs in an alkaline solution.

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H&E Staining Procedure: Neutralizing

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Neutralizing Step:

When using an alcoholic eosin this step must contain 95% (or similar %) alcohol; when choosing an aqueous eosin, water is used. This is to saturate sections with the same diluent that makes up an eosin used.

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H&E Staining Procedure: Counterstaining

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Secondary Staining or Counterstaining:

Eosin stains nearly everything that hematoxylin will not stain. When applied correctly, eosin produces three different hues which can be used to differentiate various tissue elements; red blood cells stain dark reddish orange, collagen stains a lighter pastel pink, and smooth muscle stains bright pink.

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H&E Staining Procedure: Dehydration

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Dehydration:

Removal of water from the tissue section. Increasing concentrations of alcohol after eosin staining are to remove water from the tissue section. Concentration of alcohols following the stain is important. Since eosin is very soluble in water, it is easily removed from already stained section by alcohol that is less than 100%.

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H&E Staining Procedure: Clearing

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Clearing:

Displacement of alcohol from the tissue sections with the clearant (usually xylene or xylene substitutes) to assure miscibility when coverslipping with xylene, toluene or other petroleum-based mounting media.

Staining pathway

H&E Staining Methods: Differences

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Stains until the desired intensity is reached No differentiation Water wash followed by

bluing

Stains until the desired intensity is reached Differentiation in a weak acid Water wash followed by

bluing

1. Regressive Method

2. Progressive Method

3. Progressive Method with Regressive Element

Staining method is dictated by hematoxylin type used, personal preference or just convenience.

Hematoxylin purposely overstains the tissue sections

Differentiation in a strong acid, removing excessive stain Water wash followed by bluing

Staining Overview: Hematoxylin

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Hematoxylin comes from a logwood tree that grows in Central and South America. Hematoxylin alone is not technically a dye, and will not directly stain tissues. First, it needs to be oxidized to hematein. It is done by adding chemical oxidizing agents and also

done naturally in a process called ripening when hematoxylin is exposed to air. Hematoxylin ripens throughout its life.

Secondly, it needs to be complexed with a “mordant” (from French “mordre” – to bite, grip), typically aluminum ion, that helps it link to the tissue.

Hematoxylin in complex with aluminum is positively charged and can react with negatively charged cell components, such as nucleic acids in the nucleus. These stain blue as a result.

MordantOxidation

Hematoxylin binds to its target, i.e. nucleic acid, but also binds to large proteins creating unwanted background. While to some pathologists this is negligible, others prefer no background caused by hematoxylin.

Hematoxylin

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Target: DNA Background: mucins, large proteins

Tissue section

Nucleic acid (DNA)

Glass slide

Unwanted hematoxylin background or non-specific staining can be removed in a differentiating step by acidic solution.

Differentiation by Acid Solutions

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Differentiation breaks the bond between mordant and the tissue

Tissue sectionNucleic acid (DNA)

Glass slide

Over-differentiation may cause tissue loss or tissue lifting. Choice of differentiator (strong, medium or weak acid) and immersion time are

crucial for keeping tissue sample on the surface of the glass safely.

Differentiation by Acid Solutions

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Glass slide

Degree of tissue loss or tissue lifting might depend on slide type used (coated vs. non-coated)

Broken bonds

Properly done differentiation removes non-specific hematoxylin staining.

Removing the Background

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Background staining: goblet cells Background-free goblet cells

Example: Goblet cells in GI track contain mucins that are non-specifically stained with hematoxylin causing the background that can be removed with differentiator

Acid is the main and key component of a differentiator Acidity of acids is measured by “pK” value The lower pK value, the stronger is the acid By choosing the right differentiator for any staining method we can optimize the results

Choosing Optimal Differentiator

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Hydrochloric acid (Acid alcohol)

Maleic acid

Tartaric Acid

Citric acid

Acetic Acid

-8

-6

-4

-2

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6pK scale

Acid Strength (negative log)

Different acids with a wide pK range

Hematoxylin Bluing

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o Tap water

o Ammonia water

o Scott’s tap water

o Buffered solutions

pH may be unpredictable

pH may be too high (section loss may occur)

more effective at maintaining the optimal pH (less risk of tissue loss)

Bluing is necessary to convert hematoxylin reddish purple nuclear coloration to a crisp blue/purple. Changing the color to blue gives a much better contrast with the red (eosin) counterstain.

There are numerous formulations of bluing reagents available. Choose the right product with stable pH and gentle mode of action.

Eosin Y is a negatively charged synthetic dye that binds to positively charged molecules in tissue sample.

It is soluble in either water or alcohol so aqueous or alcoholic eosin version are common as counterstains.

Good quality eosin generates three shades of red but there are also variants that produce fourth color.

Eosin Staining

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Eosin moleculePowdered Eosin Y

H&E stained kidney

Good counterstain will not only contrast sharply with the blue nuclei, but it will allow the non-nuclear tissue components to be clearly differentiated from each other by three different shades of eosin.

Eosin Staining: Result

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2. Connective tissue paler shade of pink

3. Red blood cells intensly red

1. Cytoplasm/muscle pink-orange

Nuclei blue

Hematoxylin Staining:

o Hematein/aluminum binds to negatively charged molecules (nucleic acids) in cell and tissues

Differentiation Step:

o Uses acidic solution to remove excess of hematoxylin or/and background staining

Bluing Step:

o Necessary to convert nuclear coloration from reddish purple to a crisp blue/purple

Eosin Staining:

o Eosin binds to positively charged proteins in the cytoplasm, connective tissue, muscle and red blood cells staining them different shades of red

H&E Staining Summary

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190137 Rev A ∙ 02/2019

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