Massons Trichrome Stain

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Massons Trichrome Stain

  1. 2. MASSONS TRICHROME STAIN PRESENTED BY: MEHTAB SHAH B.S MT 3 Rd year (SDLS(LNH) HISTOPATHOLOGY
  2. 3. SPECIAL STAINING <ul><li>Special stains are used to identify certain normal and abnormal substance present in the cells an tissue, which cannot be differentiated by routine haemotoxylin and eosin staining. </li></ul>
  3. 4. INTRODUCTION <ul><li>Masson's trichrome stain is modified from Masson in (1929). </li></ul><ul><li>Masson's trichrome is a three-colour staining protocol used in histology . </li></ul><ul><li>This stainis used to distinguishes between collagen and smooth muscle in tumours. </li></ul>
  4. 5. Muscle <ul><li>Muscle cells are specialized for contractility, and they can be classified according to their microscopic appearance into 3 categories: </li></ul><ul><li>Smooth muscle which is found primarily in tubular organs. </li></ul><ul><li>Skeletal muscle which is generally attached to bones. </li></ul><ul><li>Cardiac muscle which is found in the wall of the heart. </li></ul>
  5. 6. SMOOTH MUSCLE (involuntary) <ul><li>Is developed from mesenchymal cells. </li></ul><ul><li>cell is spindle-shaped with an elongated centrally located nucleus. </li></ul><ul><li>Microscopically; cells appear eosinophillic the presence of glycogen. </li></ul><ul><li>The cytoplasm contains bundles of myofilaments called myofibrils. </li></ul><ul><li>Surrounded by the sarcoplasm. </li></ul><ul><li>The centrally situated nuclei with their fine chromatin pattern stain with hemotoxylin and have elongated appearance: they may contain one or two nucleoli. </li></ul>
  6. 7. COLLAGEN <ul><li>These connective tissue protein provide tensile strength. </li></ul><ul><li>There are at least 27 types of collagen. </li></ul><ul><li>Type 1,2,3.4 and 11 are the fibrillar collagens and most abundant. </li></ul><ul><li>The collagen of skin and bone is mostly type 1,while cartilage collagen is mostly type 2. </li></ul><ul><li>Type 4 forms sheets instead of fibrils and is major collagen found basement membrane. </li></ul>
  7. 8. Contd …………….. <ul><li>Other collagen may form extra cellular matrix meshwork, participate in cellular anchorage. </li></ul>
  8. 9. PRINCIPLE <ul><li>Trichrome procedure are three dyes </li></ul><ul><li>Sections are first stained with an acid dye such as Biebrich scarlet. </li></ul><ul><li>All acidophilic tissue elements such as cytoplasm. muscle and collagen will bind with the acid dyes. </li></ul><ul><li>The section are then treated with phosphotungstic and phosphomolydic acid. </li></ul>
  9. 10. Contd……………………. <ul><li>Because cytoplasm is much less permeable than collagen the phosphotigstic and phophomolydic acids cause the Biebrich scarlet to diffuse out of the collagen but not out of the cytoplasm. </li></ul><ul><li>Phosphotigstic and phosphomolydic have numerous acidic groups. </li></ul><ul><li>Finally a dye of large particle size such as light green is used that is able to enter collagen. </li></ul>
  10. 11. FIXATIVE Bouin’s solution is preferred 10% neutral buffered formalin may be used TECHNIQUE Cut paraffin section at 4 microns
  11. 12. REAGENT REQUIRMENT <ul><li>Weigerts iron haematoxylin </li></ul><ul><li>5% phosphomolydic acid </li></ul><ul><li>5% phosphotungstic acid </li></ul><ul><li>2% light green </li></ul><ul><li>Biebrich scarlet acid fushin solution </li></ul>
  12. 13. PROCEDURE <ul><li>Depparaffinize section in xylene </li></ul><ul><li>Clean xylene by alcohol </li></ul><ul><li>Rinse with distilled water </li></ul><ul><li>weigerts iron hemotoxylin </li></ul><ul><li>5-10 mint; </li></ul>
  13. 14. Contd……………………… <ul><li>Rinse well in d/w </li></ul><ul><li>Biebrich scarlet acid fuchin </li></ul><ul><li>5-10 mint; </li></ul><ul><li>Rinse in d/w </li></ul><ul><li>Phosphotungstic – phosphomolydic acid solution </li></ul><ul><li>10-15 mint; </li></ul>
  14. 15. Light green solution 2-5mint; Dehydrate in absolute alcohol Clear in xylene Mount in synthetic resin Microscopy
  15. 16. Quality control and safety <ul><li>Practically every tissue has an internal control so no other control are needed, if a control is desired ,uterus, small intestine, appendix or fallopian tube will provide good material. </li></ul><ul><li>Wear gloves, lab coat. avoid contact and inhalation </li></ul>
  16. 17. Results <ul><li>Nuclei…………………………….black, blue </li></ul><ul><li>Muscle.cytoplasm,fibrin………..red </li></ul><ul><li>Collagen………………………...green </li></ul>
  17. 19. Nuclei red, collagen green, cytoplasm red
  18. 20. Infracted rat heart showing damaged cardiac tissue in blue.
  19. 21. Terminal ileum with increase in subepithelial collagen table in a patient with collagenous colitis

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