PS1_Histo_Lect5_part 1_Power Point Presentation.pptx

PATHOLOGICAL SCIENCE 1 H&E Staining & Artefacts Lecture 5 Miss Yulia Humrye, BSc (Hon) [email protected] HAEMATOXYLIN AND EOSIN (H&E) STAINING … MECHANISM OF STAINING Direct method: Tissue and dyes contain charges Acidic elements in the tissue (which have negative charges) are attracted...

PATHOLOGICAL SCIENCE 1 H&E Staining & Artefacts Lecture 5 Miss Yulia Humrye, BSc (Hon) [email protected] HAEMATOXYLIN AND EOSIN (H&E) STAINING … MECHANISM OF STAINING Direct method: Tissue and dyes contain charges Acidic elements in the tissue (which have negative charges) are attracted to basic dyes (which have positive charges) = basophilic Basic elements in the tissue (which have positive charges) are attracted to acid dyes (which have negative charges) = acidophilic MECHANISM OF STAINING Indirect method: Uses intermediary to link the tissue and dye This link is called a mordant When mordant is combined with a dye = production of a dye lake The colour of the lake is dependent on the specific mordant used DIFFERENTIATION  Progressive staining = up to the point for the desired depth of colouration to be achieved  Regressive staining = overstaining of tissue with subsequent removal of the stain  Agents will remove or change dyes so that the individual tissue elements they stain look different CHARGES ON THE DYE AND TISSUE ARE OPPOSITE AND THEREFORE ATTRACT  Nuclei = acidic (-ve charges) = basophilic Reacts with haematoxylin = basic dye = blue/black  RBCs, cytoplasm, muscle, collagen = basic (+ve charges) = acidophilic Reacts with eosin = acid dye = red/shades of pink Haematoxylin – is not a dye, it requires oxidation to haematein (not to be confused with haematin – formalin pigment). Oxidising agents: Natural ripening (slow), mercuric oxide (low quality for partial effect) iron salts (immediate effect). e.g. Haematoxylin plus ammonium alum produces a blue lake (alum haematoxylin) Haematoxylin plus iron alum produces a black lake (iron haematoxylin) HAEMATOXYLIN & EOSIN STAIN = H&E  Routine histological stain = morphology stain Shape, size, space in-between arrangements (architecture), distribution of colours (basophilic = purple, eosinophilic = pink), pigments, other organisms, depositions CAN WE SEE ALL OF THE CELL ORGANELLES ON H&E? WHAT CELL ORGANELLES CAN WE SEE ON H&E? HISTOPATHOLOGY  Molecular Diagnostics in histopathology  In most cases diagnosis is made from examination of routine stains – e.g. H&E  Additional dye-based stains can be used – e.g. PAS  Where diagnosis is difficult immunohistochemistry is used to identify different cell types – e.g. CKs, CDX2 HISTOLOGICAL STAINS PAS H&E IHC (DAB) SUMMARY • The Hematoxylin and Eosin combination is the most common staining technique used in histology. • The diagnosis of most malignancies is based largely on this procedure. • Customer expectations or preferences are extremely subjective H&E STAINING  For routine diagnosis, the use of H&E is by far preferred for viewing cellular and tissue structure detail by pathologists.  This stain demonstrates such a broad range of cytoplasmic, nuclear, and extracellular matrix features, nearly all teaching texts use H&E images.  The format of staining procedures is easily reproduced and the reagents resilient enough to allow for large numbers of slides to be stained consistently before reagents need to be changed.  In the past, stains and their components were routinely made by the laboratory.  Many laboratories find ordering their stains to be the easiest way to ensure consistent and repeatable quality. HAEMATOXYLIN  Haematoxylin is used to illustrate nuclear detail in cells. Depth of coloration is not only related to the amount of DNA in the nuclei, but also to the length of time the sample spends in haematoxylin.  The dye itself is extracted from the tree Haematoxylon campechianum - "bloodwood" (haima being Greek for blood and xylon for wood). HAEMATOXYLIN  Oxidation of the haematoxylin produces hematein, which is the actual dye used in an H&E stain.  Addition of the mordant improves the ability of the hematein to attach to the anionic (negatively charged) components of the tissues.  Haematoxylins are typically classified by the mordant used before staining.  Mordant aids the bonding of the hematein to the anionic tissue component, which is most commonly chromatin.  The most common mordant used in routine histology is aluminum ammonium sulfate (ammonium alum or alum). Mayer’s, Gill’s, Harris’ haematoxylins.  Mordant causes the nuclei to be red in colour, which is then changed to the more familiar blue colour when the sample is later rinsed with a weakly basic solution. HARRIS HEMATOXYLIN • Harris hematoxylin: – Common hematoxylin used in histology – Strong, regressive stain • Harris hematoxylin produces well delineate crisp nuclear staining. • Harris may require filtering to remove precipitates. HARRIS HEMATOXYLIN Because of the strength of Harris hematoxylin, most labs use an agressive differentiating solution: – 1.0% or 0.5% HCl in 70% alcohol is commonly used – Sections typically are exposed to these strong differentiation solutions for 2-10 seconds EOSIN  Eosin is the most commonly used counterstain that distinguishes between the cytoplasm and nuclei of cells.  A red crystalline dye composed of the Potassium, Sodium, or Lead salt of tetrabromofluorescein.  Eosin is not permanent and fades rapidly in sunlight.  Eosin Y is the most commonly used form of eosin and may be used in both water and alcohol.  Other eosin mixtures are sometimes used, such as EA50 and EA65. These stains are primarily used for cytology, and in addition to eosin Y, include light green, yellowish, and Bismarck brown. • The overall coloration of the stained specimen is the result of the balance of the intensity of the alum-hematoxylin and eosin. • Remember – Alum-hematoxylin can stain cytoplasm – Eosin Y can stain nuclear basic protein THE STAINING PROCEDURE FOR H&E FOLLOWS A BASIC PROTOCOL: • Dewaxing • Rehydration • Haematoxylin • Differentiation • Bluing • Eosin • Dehydration • Clearing • Cover-slipping Colon H&E. Note the balanced coloration in this section of colon. The microvilli on the columnar epithelium are very crisp. Photo credit: Stanley Hansen EXAMPLE OF H&E STAINING SCHEDULE Reagents: Harris’ Haematoxylin 1% Eosin 1% Acid Alcohol (HCl in 70% alcohol) Water Alcohol Xylene DPX EXAMPLE OF H&E STAINING SCHEDULE 1. Immerse the slide in xylene to remove the wax – 5 minutes 2. Rinse in 100% alcohol – 1 minute 3. Rinse in 95% alcohol – 30 seconds 4. Rinse in 70% alcohol – 30 seconds 5. Rinse in 50% alcohol – 30 seconds 6. Wash well in water 7. Stain with Harris’ haematoxylin – variations 5 or 8 or 10 minutes 8. Wash sections in alkaline reagent (tap water or Scott’s water, or Saturated Lithium Carbonate) to “blue” the section – time will vary depend on the reagent used 30 sec to up to 5 min (tap water) H&E STAINING SCHEDULE 9. Differentiate in 1% acid alcohol for few seconds 10. Rinse in water and “blue” in alkaline reagent (see number 8) 11. Stain with Eosin – 5-10 minutes 12. Rinse in tap water – 1 minute If alcohol eosin use alcohols to dehydrate: 14. Rinse in 70% alcohol – 30 sec 15. Rinse in 95% alcohol – 30 sec 16. Rinse in 100% alcohol – 30 sec 18. Clear in xylene for 1 minute and mount in DPX using a coverslip glass. If aqueous eosin dry the slide (totally dry), then Clear in xylene for 1 minute and mount in DPX using a coverslip glass. **Alternatively dry mount can be performed – Air dried stained slides with an application of DPX or XPF on coverslip glass (no xylene) H&E RESULTS Results Nuclei deep blue Cytoplasm pink/purplish pink Connective tissue pale pink Muscle fibres, RBCs and eosinophilic granules deep pink/red Calcium dark blue Mucin grey/blue H&E IN PRACTICE Good nuclear detail. Clean pink/red background To achieve an optimum balance: staining time/concentration of the nuclear stain and the level of differentiation using acidalcohol H&E STAINING VARIABILITY DUE TO THICKNESS OF CUT SECTIONS At 1 micron At 2 micron At 3 micron At 4 micron H&E STAINING VARIABILITY DUE TO THICKNESS OF CUT SECTIONS At 3 micron At 4 micron At 5 micron At 6 micron PICTORIAL – H&E METHOD … H&E METHOD H&E METHOD CONTINUED H&E METHOD CONTINUED H&E METHOD CONTINUED