Science of H & E

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

1 Hematoxylin and Eosin Staining

“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.”

Guidelines for Hematoxylin and Eosin Staining National Society for Histotechnology

2 Why Do We Stain?

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

3 What Does "Staining" Do?

. Contrasts different cells . Highlights particular features of interest . Illustrates different cell structures . Detects infiltrations or deposits in the tissue . Detect pathogens

Superbly contrasted GI cells Placenta’s large blood H&E stain showing extensive vessels iron deposits

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

4 What is H&E Staining?

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.

5 How is it Done?

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.

Example of H&E stain

6 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

7 H&E Staining Procedure Step-by-Step

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

Staining pathway

X X A A A A A A X X Eosin Bluing Water rinse Water Water rinse Water Water rinse Water Water rinse Water Hematoxylin Differentiator

X-solvent Deparaffinization or Dewaxing steps: A-alcohol

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

8 H&E Staining X Hydration the slides the slides an is It X A P : introduction of water into the tissue section. This is done by passing passing by done is This section. tissue the into waterintroduction of rocedure: Hydration slowly throughconcentrations decreasingslowly of series a of A A

Water rinse

Hematoxylin

Water rinse Staining pathway

Differentiator

Water rinse

Bluing

Water rinse A

Eosin A alcohols. A A X - - alcohol solvent X X 9 H&E Staining X Primary Staining: this allows the staining pattern of the chromatin to be seen seen be to chromatin the of pattern staining the allows this the of unstained; nucleus should remain nucleoplasm The membrane.nuclear specifically, nucleus cell, the of the nucleus chromatinthe the and within hydration. and the stains It deparaffinization after used is Hematoxylin X A P rocedure: Staining A A

Water rinse

Hematoxylin

Water rinse Staining pathway

Differentiator

Water rinse

Bluing

Water rinse A

Eosin easily. A A A X - - alcohol solvent X X 10 H&E Staining X Differentiating: removes. -specific non background.In a progressive acidmethod, solution weak regressive the In method, strong acid rinse removes all excess the and stain Achievedstrong by solutions acid onor weak stainingadepending method. X A P rocedure: Differentiating A A

Water rinse

Hematoxylin

Water rinse Staining pathway

Differentiator

Water rinse

Bluing

Water rinse A

Eosin A A A X - - alcohol solvent X X 11 H&E Staining X Bluing Step: Bluing Bluing varyreagentsBluing from a reddish Buffer 8, lithium and magnesium carbonate solutions. dependent dependent X A P purple -purple rocedure: Bluing A reaction and occurs in an alkaline solution. reaction occurs and solution. in an alkaline A hematoxylin to a blue or or blue hematoxylin to a

Water rinse

Hematoxylin mmonia Water rinse Staining pathway

Differentiator solutions, tap water, Scott’s solution,

blue purple-blue Water rinse

Bluing

Water rinse color. A Bluing changes changes Bluing

is a pH is aIt Eosin A A A X the the - - alcohol solvent X Blue X 12 H&E Staining Procedure:Staining Neutralizing X Neutralizing Step: sections alcohol; when choosing This eosin, watersaturateused. is antoaqueousis When alcoholicusing an%) (or eosin this similar must containstep 95% X A with the same diluent that makes up an eosin used. eosin used. anmakes up that diluent the same with A A

Water rinse

Hematoxylin

Water rinse Staining pathway

Differentiator

Water rinse

Bluing

Water rinse A

Eosin A A A X - - alcohol solvent X X 13 H&E Staining Procedure:Staining Counterstaining X Secondary Staining or Counterstaining: Staining or Secondary Eosin pink orange, collagen lighterstainsa pastel pink,and smoothmuscle stainsbright differentiate correctly X . A stains , eosin, produces threedifferent whichused huescanto be A various tissue elements;red blood cells reddishstaindark nearly everything thathematoxylin will stain.not When applied A

Water rinse

Hematoxylin

Water rinse Staining pathway

Differentiator

Water rinse

Bluing

Water rinse A

Eosin A A A X - - alcohol solvent X X 14 H&E Staining X Dehydration: soluble soluble that is less that is less than 100%. Concentration alcohol Removal X A P in in after eosin eosin staining after rocedure: Dehydration of water from the tissue section. Increasingtissue concentrationsfromthewater of of water, it is easily removed from alreadystained section by alcohol A of alcohols following the of A

Water rinse

Hematoxylin are are Water rinse Staining pathway to removewaterfrom the tissue section.

Differentiator

stain Water rinse

is important. Since eosin important.is Bluing

Water rinse A

Eosin A A A X is veryis - - alcohol solvent X X 15 H&E Staining Procedure:Staining Clearing X Clearing: xylene, toluene toluene xylene, or xylene substitutes) xylene to Displacement X A A of alcohol from the tissue sections with the clearant the with sections fromtissue the alcohol of A based mounting or other petroleum mounting -based

Water rinse

Hematoxylin

Water rinse Staining pathway assure miscibility when coverslipping with with assurecoverslipping when miscibility Differentiator

Water rinse

Bluing

Water rinse media. A

Eosin A (usually (usually A A X - - alcohol solvent X X 16 H&E Staining Methods: Differences

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

1. Regressive Method

Hematoxylin purposely Differentiation in a strong Water wash followed by bluing overstains the tissue sections acid, removing excessive stain

2. Progressive Method

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

3. Progressive Method with Regressive Element

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

17 Staining Overview: Hematoxylin

. 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.

Oxidation Mordant 18 Hematoxylin

. 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.

Nucleic acid (DNA) Tissue section Glass slide

Target: DNA Background: mucins, large proteins

19 Differentiation by Acid Solutions

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

Nucleic acid (DNA) Tissue section Glass slide Differentiation breaks the bond between mordant and the tissue

20 Differentiation by Acid Solutions

. 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.

Broken bonds

Glass slide

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

21 Removing the Background

. Properly done differentiation removes non-specific hematoxylin staining.

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

22 Choosing Optimal 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

pK scale 6

. Hydrochloric acid (Acid alcohol) 4

. Maleic acid 2

. Tartaric Acid 0

. Citric acid -2

-4 . Acetic Acid -6 Acid Strength (negative log) -8

Different acids with a wide pK range

23 Hematoxylin Bluing 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.

pH may be unpredictable o Tap water

o Ammonia water pH may be too high (section loss may occur)

o Scott’s tap water more effective at maintaining the optimal pH (less risk of tissue loss) o Buffered solutions

24 Eosin Staining

. 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.

Powdered Eosin Y Eosin molecule

25 Eosin Staining: Result

. 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.

1. Cytoplasm/muscle  pink-orange

2. Connective tissue  paler shade of pink

3. Red blood cells  intensly red

. Nuclei  blue

H&E stained kidney

26 H&E Staining Summary

. 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

27 This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. Although every effort has been made to report faithfully the information, Leica Biosystems cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.

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190137 Rev A ∙ 02/2019