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 Procedure: Hydration 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 Hydration: A-alcohol 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. 9 H&E Staining Procedure: Staining 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 Primary Staining: A-alcohol 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. 10 H&E Staining Procedure: Differentiating 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 Differentiating: A-alcohol 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. 11 H&E Staining Procedure: Bluing 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 Bluing Step: A-alcohol 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. 12 H&E Staining Procedure: Neutralizing 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 Neutralizing Step: A-alcohol 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. 13 H&E Staining Procedure: Counterstaining 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 Secondary Staining or Counterstaining: A-alcohol 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. 14 H&E Staining Procedure: Dehydration 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 Dehydration: A-alcohol 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%. 15 H&E Staining Procedure: Clearing 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 Clearing: A-alcohol 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. 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 .