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Histotechniques
Histotechniques
Histotechniques
Histotechniques
Histotechniques
Histotechniques
Histotechniques
Histotechniques
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Histotechniques
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Histotechniques

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this is a ppt on histotechniques,, all techniques from receiving samples to block making to sectioning to staining are discusses in detail..useful for postgraduate pathology students and lab …

this is a ppt on histotechniques,, all techniques from receiving samples to block making to sectioning to staining are discusses in detail..useful for postgraduate pathology students and lab technicians

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  • 1. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniqueshttps://www.scribd.com/doc/200310129/Histotechniques HISTO -TECHNIQUES
  • 2. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques OVERVIEW • Definition • Specimen Receiving • Fixation • Grossing • Tissue processing • Embedding • Microtomy
  • 3. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Definitions • Histology – greek – histos + logia (A.F.J.K. MAYER 1819) – Study of microscopic anatomy of cells and tissues • Histopathology – Microscopic study of diseased tissue • Histotechnology – processing of tissues in such a manner as to enable microscopy / study of the tissue
  • 4. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques AIM • Obtaining thin enough sections of the tissue so as to enable light to pass through it
  • 5. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques SPECIMEN RECEIVING • Documented policy defining the minimum demographic details of the patient • Spill kit – 5% hypochrorite, gauze piece, yellow bag, gloves • Sample transportation – covered containers, in 10% buffered formalin, chutes • Unique identification number • Strict Sample rejection criteria
  • 6. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 7. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques
  • 8. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques
  • 9. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques “FIX AT A POINT IN TIME” This slide is not available in this preview
  • 10. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques TYPES OF FIXATION • Immersion • Perfusion – brain, spinal cord, lungs, embalming • Vapour • Spray • Microwave fixation/Stabilization • Freeze drying
  • 11. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques MICROWAVE PRINCIPLE This slide is not available in this preview
  • 12. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 13. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques FREEZE DRYING • Fresh tissue immersed in isopentane cooled by liquid nitrogen at -160° to -180° - Quenching • Tissue water removed under vacuum, absorbed by phosphurous pentoxide – sublimation • Put in embedding medium • Little chemical alteration, no loss of glycogen • Modification of this technique useful in enzyme histochemistry
  • 14. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques • Standard histological processing abolishes the activity of most enzymes • Freeze drying is the optimal method of tissue preservation maintaining tissue components in their native state. Conventionally, however, the freeze dried tissue specimens are fixed and embedded in wax after freeze drying. • Glycol methacrylate resin has been widely used as an alternative embedding medium to wax and the activities of a limited number of enzymes have been shown in tissue fixed in aldehyde and embedded in resin. Enzyme histochemistry performed on these resin sections gives good results.
  • 15. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques PHYSICAL METHODS CHEMICAL METHODS Heating Microwaving Freeze - drying Aldehydes – Formal dehyde, Glutaraldehyde Alcohol – Methyl alcohol, ethyl alcohol Methacarn Glacial acetic acid Bouin’s fluid Osmium tetroxide SIMPLE FIXATIVE COMPOUND FIXATIVE Formal dehyde Glutaryl dehyde Ethyl alcohol Zenker’s fluid Bouin’s fluid
  • 16. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 17. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Formulae  Formal Saline  40% Formaldehyde 100 ml  NACL 9 gm  Tap Water 900 ml  10% Buffered Formalin  40% Formaldehyde 10 ml  Sodium Hydrogen phosphate 0.4gm  Disodium Hydrogen phosphate 0.65gm  Tap Water 90 ml Buffered formalin prevents formation of pigment acid formaldehyde hematin formed from hemoglobin at acidic pH.
  • 18. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Formulae Zenker’s fluid  Distilled Water 1000 ml  Hg Cl2 50 gms  K-Dichromate 25 gms  NaSO4 10 gms • It provides excellent fixation of nuclear chromatin, connective tissue fibres and some cytoplasmic features but does not preserve delicate cytoplasmic organelles such as mitochondria. • Currently out of fashion because of the toxicity.
  • 19. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 20. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Glutaraldehyde • Used for electron microscopy with osmium tetroxide • Glutaraldehyde produces nuclear and cytoplasmic shrinkage while osmium produces swelling and balances it • Formalin is not useful for EM because methanol which is added to commercial preparations has denaturing action on tissue components.
  • 21. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 22. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Factors affecting fixation 1. Hydrogen ion Concentration & Buffers 1. Satisfactory fixation occurs at pH between 6 and 8. 2. Buffers like phosphate, bicarbonate, s-collidine and cacodylate are chosen. 2. Temperature 1. Higher temp causes faster fixation 2. Lower temperature preserves tissue better 3. A tissue of 4mm thickness will be adequately fixed in NBF 10 to 20 times the volume in about 8 hours at room temperature. Fixation time is shortened by 25-40% if temperature is raised to 40°c. 4. Faster fixation is obtained through agitation.
  • 23. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 24. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques 6. Duration of Fixation ideal is 8 hrs • over fixation causes hardening of tissue and loss of antigens 7. Volume change – Tissue fixed in formaldehyde and embedded in paraffin wax shrinks by 33%
  • 25. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Fixation Artefacts This slide is not available in this preview
  • 26. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Decalcification This slide is not available in this preview
  • 27. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques 4. Ion exchange resins – polystyrene 5. Electrolytic method – Electrolytic solution of HCl and formic acid used 6. Ultrasonic method 7. Surface decalcification – Achieved by inverting paraffin block in 5% HCl for one hour, top 30 micron is decalcified.
  • 28. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Factors affecting rate of decalcification This slide is not available in this preview
  • 29. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Assesment of decalcification • Needling, knifing, finger nailing • X ray examination is the best • Chemical method using ammonia
  • 30. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Trimming This slide is not available in this preview
  • 31. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Trimming
  • 32. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 33. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Tissue Processing This slide is not available in this preview
  • 34. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Tissue Processing  Principle : The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut and yet soft enough not to damage the knife or tissue.  Stages  Dehydration – to remove fixatives and water from tissue.  Clearing – replacing dehydrating fluid with a fluid that is totally miscible with dehydrating fluid and embedding medium  Impregnation – replacing clearing agent with the embedding medium  Embedding
  • 35. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Dehydration This slide is not available in this preview
  • 36. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Clearing  Hydrocarbons which have refractive indices similar to tissue proteins, end point noted by transparent appearance of tissues.  Criteria  Miscible with both dehydrating and embeding agent  Speedy removal of dehydrating agents  Easy removal by paraffin  Examples  Xylene  Toluene  Chloroform  Citrus fruit oils  Cedar wood oil
  • 37. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Impregnation • This slide is not available in this preview
  • 38. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Automated tissue processing Processing schedule This slide is not available in this preview
  • 39. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Important points Bone, skin and CNS tissues require three changes of wax Two changes of wax suffice for other organs
  • 40. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Embedding  Requirements  Embedding media  Cold plate / ice plate  Wax Dispenser  L-molds / ice trays / paper blocks / tissue tek system  Wooden blocks  Forceps  Spatula  Typed labels
  • 41. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Embedding
  • 42. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Embedding
  • 43. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 44. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Embedding media  Water insoluble media – Paraffin wax – Carbowax – Plastic embedding medium – Butyl methacrylate – Acrylic – Epoxy resin – Epon – Maraglas 655 – Polyester resin Water soluble media – Durcupan – Aquon – Glycol methacrylate
  • 45. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Important points • Size of the mould should be such that there is a margin of 1-2 mm around the tissue • Embed all bits of skin / cervical cone only few mm apart in the same block with parallel epithelial edges • Endometrial samples and cell blocks are best kept at centre of block and tamped to the bottom • Stand mount – cyst walls, gall bladder walls, ovarian wedges and skin sections – required edge kept at the bottom
  • 46. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 47. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Paraffin wax additives This slide is not available in this preview
  • 48. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Special embedding techniques • Double embedding – Impregnated in celloidin, blocked in paraffin wax – Used in making blocks of tissues with varying consistency such as eyes where retina is easily detached • Tissue mat and Bioloid – Thin walled and circular sections maintain their original shape due to elasticity of the media
  • 49. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques
  • 50. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques This slide is not available in this preview
  • 51. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Paraffin section cutting  Requirements:  Microtomes  Disposable blades  Water bath  Fine forceps  Small squirrel hair brush  Slide rack  Clean slides  Ice tray  Diamond pencil
  • 52. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Microtomes  Precision instruments that cut sections from the paraffin blocks, thin enough for examination under microscope.
  • 53. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Sliding microtome • Block is stationary, knife moves horizontally against it This slide is not available in this preview
  • 54. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Rocking microtome • The knife is fixed, blok of tissue moves through an arc and strikes against the knife
  • 55. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Sledge Microtome • Similar in function to sliding microtome
  • 56. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Rotatoty Microtome This slide is not available in this preview
  • 57. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Auto cut microtome
  • 58. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Vibrating knife microtome • Sections obtained without fixation /impregnation /freezing, useful for histochemistry • Sections are thick and not suitable for routine staining This slide is not available in this preview
  • 59. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Ultracut microtome • Used for cutting 500 to 600 A° thin sections for EM
  • 60. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques MICROTOME KNIFE • Vicker’s Hardness number – Important to use knives of gauranteed hardness – VHN = 1.72p the desirable hardness is 400-900 VHS D² Where D is the diagonal between corners of rectangular indentation, P is standard load
  • 61. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques one side is flat, other is concave used for cutting nitrocellulose blocks originally designed for frozen sections, now used for all microtomes Used to section hard tissues such as undecalcified bone
  • 62. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Knife materials This slide is not available in this preview
  • 63. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Sharpening of knives • Manual – Honing • Coarse • Fine – Stropping • Automatic
  • 64. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Honing • Honing is removing nicks and irregularities from the knife edge • Hones are sharpening stones – Examples – Belgian Vein Black and Arkansas • Lubricants used during the process to act as coolants and flow away fine particles
  • 65. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Honing • Abrasive powders – Diamond – Carbodundum (silicon carbide) – Aluminium oxide – Iron oxide – Ceric oxide – Chromium oxide
  • 66. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques To watch stropping and honing, search for the same on you tube… many videos are available • This slide is not available in this preview
  • 67. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Stropping • Polishing a fairly sharp knife • Strops are made of hide from the rump of horse
  • 68. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Knife Sharpening machine
  • 69. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Fine Cutting This slide is not available in this preview
  • 70. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Sections
  • 71. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Section adhesion This slide is not available in this preview
  • 72. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Difficulties commonly encountered in sectioning  Failure of block to ribbon  Too hard paraffin  Knife  Uneven Ribbons  Irregular knife edge  Knife not parallel to the block  Impure paraffin  Alternate thick and thin Ribbons  Too soft wax  Loose block or blade  Faulty microtome
  • 73. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Difficulties commonly encountered in sectioning (Contd.)  Tight coil of sections  Blunt blade  Too thick sections  Adherence of sections to knife  Dirty knife edge  Dull knife
  • 74. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Frozen sections & Cryostat This slide is not available in this preview
  • 75. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Cryostat
  • 76. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Cryostat This slide is not available in this preview
  • 77. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques Cryostat
  • 78. PREVIEW ONLY To download full presentation follow this link https://www.scribd.com/doc/200310129/Histotechniques

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