6 histotechniques


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  • In most text books available, Histotechniques are explained from the perspective of pathology. In anatomy, it the the same process but there is alteration in some steps. For example tissue procurement is seldom discussed because this step is crucial only to anatomists who is preparing a slide for teaching learning purpose unlike a pathologist who gets tissue from patient for tissue diagnosis. Similarly grossing is usually described in detail which is not very important to an anatomist.Decalcification for bone – addedFrozen section: fixation omitted
  • Though Histotechniques are done in various steps, they should not be viewed in isolation. Each step usually complement the other . For example fixation with osmium tetroxide will also stain the myelin. Fixation not only preserves the tissue but also fortifies it against harmful effects of chemicals used in further processing.Hardening of tissue by fixation aids in section cutting.
  • The first thing that is needed for Histotechniques is the tissue itself. In pathology tissues are available from patients which are to be processed and examined to provide tissue diagnosis. In anatomy tissue are processed at majority of times for preparation of slides for teaching learning process.Hence in anatomy we need source of ‘normal’ tissues. Easiest source of tissue is our set up is PM bodies. Certain organs need to be obtained from smaller animals because they are larger in human and sections of whole organs cannot be accommodated in glass slide. Commonly used animals are rabbit, dog, squirrel, gunniepigs etc. for luminated organs like large blood vessels, parts of GIT, brain and Genitourinary tract. For certain organs we need specific animals e.g. pig for liver. If few specific organs are needed tissue can be acquired from butchers instead of sacrificing the the whole animals.
  • The Committee for the Purpose of the Control & Supervision of Experimental Animals (CPCSEA). CPCSEA mentions that, animals should not be used for repetition of experiments or experiments whose results are already well established. Use of animals which are very old, diseased and dying , and ones dead due to some other experimentation is not clearly mentioned.
  • Unlike in pathology again processing of tissue is planned in Anatomy. If tissue needs to be transported, the best medium for transportation is fixative itself. Washing for removing excess blood, mucasetc should always be done with isotonic saline. These precautions are basically to prevent swelling or shrinkage of tissue and prevent crushing of tissue. Labeling and marking is necessary for correct orientation of tissue in block and subsequently in slide.Collapse of luminated structure e.g. can be prevented by stuffing them with paraffin wax, for its proper demonstration.
  • 1,3)Maintained in as lifelike as possible but certain changes are necessary. Coagulation of tissue is necessary but is a major artifact since living tissue are in fluid or semi-fluid state.Properties of a good fixative: cheap, easily available, non irritant, non- toxic, non-inflammable, easy to store, no change on storage etc.Ideally , fixed tissue should tell more about life rather than looking more ‘life like’
  • 5) Fixation aids in optical differentiation of cells and tissue constituents by altering their refractive indices, in various degrees. This is valuable since refractive index of some elements of cell is so close to that of surrounding structures making them invisible in living state when examined under ordinary microscope.5) Fixatives may have both facilitating and inhibiting action on dyes. Carmalum, a nuclear stain stains less strongly when fixed with formalin but stains strongly when mercuric chloride is used for fixation.5) Fixatives can act as mordant- direct link b/w tissue and stain, e.g. potassium dichromate if mixed with formalin aids in demonstration of myelin sheath with hematoxylin.
  • Aldehydes : react with basic amino acid residues of proteins, form cross links b/w protein molecules leading to polymerization and increased molecular weight. Max cross linking is formed by Glutaraldehyde followed by formaldehyde and hydroxyadipaldehydeOsO4; form cross links with proteins with rapid increse of viscosity followed by decrese in viscosity – secondary liquefactionHgCl2 ; commonly used as secondary fixatives, reacts with amino acid residues espthiol, imidazole, phospate and hydroxyl group. Special affinity for histidine. Ultrastrucural preservation poor.
  • What will the audience be able to do after this training is complete?Briefly describe each objective and how the audience will benefit from this presentation.
  • Apart from acidity of fixative, tissue is also acidic due to accumulation of CO2 following anoxia.
  • Chemical reaction will reduce effectiveness of both buffer and fixative.
  • Chemical reaction will reduce effectiveness of both buffer and fixative.
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  • For ideal penetration of tissue, coagulum formation makes tissue impermeable to fixatives.Time is inversely proportional to size of tissue.
  • Fixative is a pure chemical substance e.g. 100% formaldehyde or 40% formalin, fixing fluid is that is actually used for fixation , made by dilution of or mixing fixative with some other component. E.g. buffered normal 10% formalin.General ; suitable for most tissues, specific ; for a particular tissue or tissue component eg TestisCompound : When 2 different fixatives are used one after another e.g. Chromaffin reaction : aldehyde + potassium dichromate; potassium dichromate oxidese formaldehyde to formic acid ->Cr +++, enters into catacholamines. Mercuric chloride-formalin & Helly’s fluidPreserves orientation of tissue components Vs preservation of cell componentsSingle VS multiple componentsLight microscopy Vs Histochemistry, electron microscopy etc.Chemical : Aldehyde, oxidising agents, protein denaturating agents, other cross linking agents, Physical – heat, microwaveUnknown mechanism – HgCl2, picric acidNewer : Carbodiimides like demethylsuberimidate and p-benzoquinone etc.
  • Add slides to each topic section as necessary, including slides with tables, graphs, and images. See next section for sampletable, graph, image, and video layouts.
  • Add slides to each topic section as necessary, including slides with tables, graphs, and images. See next section for sampletable, graph, image, and video layouts.
  • Formic acid -> formalin pigmentsFormic acid : add borx to diluted formalin -> red color with phenolphthaline,Allow diluted formalin to stand on layer of calcium carbonate, addition of caco3 on conc formalin can cause explosion.Add 2% calcium acetate -> preservation of phospolipidsParaformaldehyde -> cause turbidity, filter with filter paper.
  • Good protein fixativeNo effect on carbs. – glycogen held by proteinsLipids preserved – not fixedFavors staining of acidic structures with basic dyes – nucleiDiminishes effects of acid dyes on basic structures
  • Turbidity can be removed by filtering with filter paper
  • Glacial -> solidifies at about 17 degrees.
  • Add a case study or class simulation to encourage discussion and apply lessons.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • 6 histotechniques

    1. 1. PRINCIPLES AND PRACTICE Capt Rishi Pokhrel Dr Swati Patil Dr Kirti Solanke Dr Anil Dwivedi
    2. 2. Histo-techniques• Preparation of tissue for microscopic examination• Series of processes• Ultimate aim – to make tissue ‘visible’ as it is• Pathology Vs Anatomy• Steps vary – types of tissue & microscopy – structure to be seen – stains to be used – time duration etc.
    3. 3. Steps Tissue procurement and preparation Fixation Dehydration Clearing Impregnation Embedding Section cutting Staining and mounting in slide
    4. 4. Tissue Procurement Source of tissues Post-mortem bodies Cadavers Tissue of patients from pathology lab Animal sacrifice Slaughter house/ butcher shop
    5. 5. Legal & ethical issues PM bodies : written consent form NOK Cadavers : no additional consent required Patient tissue : written consent From slaughterhouse / butcher : no ethical issues, preserve bills
    6. 6. Using animals – Indian laws CPCSEA has laid down specific rules regarding the use of animals Anesthesia, trained vet. surgeon etc ? Dead or dying animals
    7. 7. Tissue preparation & precautions Start fixation a.s.a.p. Prevent osmotic damage  do not dry  wash with and immerse in NS No unnecessary handling Remove excess blood, mucus etc. Cut with a sharp knife Marking of ‘cutting surface’ Labeling and putting in ‘cassette’ Instructions for mounting – wall, tube, stained surface etc.
    8. 8. Objectives of Fixation Preserve tissue in ‘life like’ condition Prevent autolysis, microbial decomposition and petrification. Coagulate tissue to prevent loss of easily diffusible substances. Render tissue unaffected to the harmful effects of chemicals to be used in further processing.
    9. 9. Effects of fixation Tissue hardening – ease of cutting Mordant action – Flemings fluid for safranine. Optical differentiation – refractive index Precipitation or coagulation of proteins
    10. 10. How do fixatives act?• Physical and chemical changes• Reactions with proteins – Aldehydes – Oxidizing agents, OsO4 – HgCl2• Reactions with nucleic acids – Carnoy’s fluid – Formaldehyde at raised temperature – Ethanol and methanol• Reactions with lipids – Imidazoles – Cholesterol - digitonin
    11. 11. Methods of fixation• Immersion – commonly used• Perfusion – ideal method e.g. embalming • Hyaluronidase pretreatment
    13. 13. H+ concentration / PH• Anoxia in tissue -> CO2 -> pH• General purpose : optimal pH 6-8• Exceptions – Gastric mucosa pH 5.5 – Granules of adrenaline and noradrenaline pH 6.5• Buffers maintain desired pH
    14. 14. Choosing buffers• Buffer should not react chemically with fixative – Barbiturates with aldehyde• Histochemistry : buffer should not react or inhibit incubation medium or enzymes – Phosphates react with G6PD – Phosphates combine with lead salts in metal precipitation methods for phopotase.• As close to tissue pH as possible – esp for Histochemistry
    15. 15. Temperature• Routine : room temperature• Special – Electron microscope: 0-4 oC• Low temperature –  rate of decomposition• High temperature – better rate of fixation
    16. 16. Penetration of fixatives• Rate of penetration varies with various factors. – Glutaraldehyde > Formaldehyde• Slow process; d = k √t• Deeper tissue takes longer time, fixed tissue acts as a barrier for diffusion of fixative• Tissue slices should be thin 3-5 mm
    17. 17. Osmolality• Close to tissue osmolality; higher side – (400 – 450 mOsm)• Commonly used : NaCl• Some exceptions e.g. pancreas ; cause shrinkage
    18. 18. Vehicles / additives• (NH4)2S04 : stabilize proteins• NaCl + (Na)2SO4 : binding of HgCl2 to proteins• Alcian blue, ruthenium red, lanathum – preserve ultrastructure of mucosubstances• Tannic acid : enhanced microtubules and filaments in EM
    19. 19. Detergents• Used to dissolve lipids in cell membrane• Immunoflorescent techniques – 150,000 Daltons• Commonly used - saponin
    20. 20. Concentration of fixative• Determined by – Cost – Effectiveness – Solubility – Type of tissue and method of fixation• Formalin – 5% for plant tissue – 10% for animal tissue – 50% for embalming
    21. 21. Time• Formaldehyde – Too short – reversal of fixation – Too long • shrinkage of tissue • Inhibit enzyme activity - Histochemistry• Depends upon – Thickness of tissue – Temperature• Optimal 24-48 hours
    22. 22. Fixation artifacts• Primary or secondary• Intrinsic or extrinsic• Solidification of colloid material• Volume change• Pigments – formalin, HgCl2• False localization – Histochemistry and immunohistochemistry
    23. 23. Volume changes• Size of cells and tissue in slide do not reflect their true size• Routine preparation (formalin and paraffin wax) : tissue shrink by 33% (net)• Not same for all components of tissue/cells – Collagen fibers swell• Prolonged fixation - more shrinkage• Nuclei in frozen sections are bigger than routine preparations.
    24. 24. Fixation : Golden rules1 • Size of tissue, 3-5 mm2 • Volume of fixative, 10X3 • Time – 24 hrs (formalin)
    25. 25. FIXATIVES & FIXING FLUIDS Classification1. Coagulant and non coagulant2. General and specific3. Primary and secondary4. Microanatomical and cytological5. Simple and compound6. Routine and special7. Physical and chemical8. Conventional and newer
    27. 27. Formaldehyde• Non coagulant• Aldehyde - HCHO• Gas – soluble in water; max con. 40% by weight• Sp. gravity : 1.124• On storage converted to formic acid & paraformaldehyde.
    28. 28. Effects of formaldehyde• Proteins• Carbohydrates• Lipids• Acidic and basic structures
    29. 29. Formalin40% formaldehyde gas in water = 100 % Formalin Hypotonic totissue fluids  Mix with distilled water 1:9 10% formalin  Formic acid, Paraformaldehyde Add buffer Add NaCl  Buffered,  Normal 10% Formalin
    30. 30. Buffered normal formalin• 100 % formalin 100 ml NaH2PO4 3.5 gms• Buffer + Mix in 900 ml of water Na2HPO4 6.5 gms• Salt NaCl - 8.5 gms
    31. 31. Disadvantages• Contact dermatitis • Urea crystals – 5 gm • (NH4)2SO4 - 1gm • Water - 1l• Irritant to eyes and resp. mucosa• Pigment formation in Hemoglobin• Pigments and turbidity• Not suitable for certain tissue – testis, bone marrow, spleen.
    32. 32. Formalin pigments in slidesBefore staining :-• Schridde’s method: leave slides for 30 mins – 200 ml 75 % alcohol + 1ml of 25% ammonia liquor• Verocay’s method : leave sections for 10 mins – 100 ml of 80% ethanol + 1% KOH
    33. 33. Mercuric chloride• White crystals• Saturated solution : 7% water, 33% alcohol• Extremely poisonous and corrosive to metals• Seldom used alone – compound fixative• Cytological fixative
    34. 34. Mercuric chloride• Pros – cytological fixative – good fixative for proteins – shrinks but doesn‘t distort tissue – fixes both nucleus & cytoplasm• Cons – mercury pigments – Corrosive and poisonous
    35. 35. Potassium dichromateUsed in compound fixatives or as mordant for lipids orlipoproteins• Pros – excellent fixation of mitochondria, phospholipids & myelin – acts as a mordant for mitochondria – iron staining of mitochondria – iron-containing pigments better fixed at higher pH• Cons – chromatin gets dissolved – mitochondria gets thickened – formation of insoluble precipitants – brittleness of tissues 40
    36. 36. Chromic acid • CrO3 mixed in water • Fixative for carbohydrate Osmic acid• Used for cytoplasmic organelles• Demonstration of myelin• Expensive• Use in EM• Use and throw• Use goggles while handling – vapors irritant to eyes.
    37. 37. Glacial acetic acid• Part of compound fixative• Good fixation for nuclei – precipitates nucleoproteins• Counteracts the shrinking effect of others.• pronounced swelling of collagen fibers• Distorts mitochondria & Golgi body Ethyl alcohol : Histochemistry - alkaline phosphatase & lipase hardening & shrinkage Improper fixation of chromatin, mitochondria & Golgi 42
    38. 38. Glutaraldehyde• Suitable for EM• Better than formaldehyde• cytological fixative• poor penetration – use small tissue• Expensive Picric acid• good fixative for proteins, carbohydrates & glycogen• better staining• enhances results with cytoplasmic stains• much shrinkage & less hardening 43
    39. 39. Stock solutions• Formalin 40%• Mercuric chloride 7%• Potassium dichromate 5%• Chromic acid 2%• Picric acid 1%• Osmium tetra oxide 2%• Acetic acid 100%• Ethanol 100%
    40. 40. Fixing fluids
    41. 41. Formalin mercuric chloride (formal-sublimate)• Mercuric chloride 30 gms• Water 900 ml• 100% Formalin * 100 ml*Formalin added just before use as added solution is unstableUses• Secondary fixation following formalin• Excellent Microanatomical fixative• Cytoplasm preservation• Brilliant staining with acid dyes• Heidenhain’s susa - biopsy
    42. 42. Zenker’s fluidMercuric chloride 5gmPotassium dichromate 2.5gmSodium Sulphate 1gmWater 100mlAcetic acid* 5mlUses• Efficient micro anatomical fixative.• Better staining with cytoplasmic & fiber stains
    43. 43. Bouin’s fluid (formalin-picric-acetic)• Picric acid 75ml• Formalin 25ml• Acetic acid 5mlUses• Micro anatomical as well as cytological fixative• Used for demonstration of chromosomes• Glycogen is well preserved.
    44. 44. Helly’s fluid (zenker-formal)Acetic acid omitted & formalin 5 ml is added in Zenker’s fluid.Uses• Testis, bone marrow, spleen, lymph node, pituitary, pancreas
    45. 45. Carnoy’s fluid• Absolute ethanol 60ml• Chloroform 30ml• Acetic acid 10mlUses• Rapid fixation• Not of much use for anatomy slides.
    46. 46. Sanfelice’s fluid• Solution A Formalin 128ml Acetic acid 16ml• Solution B Chromic acid 100ml• Mix 9ml A + 16ml B Uses• mitotic figures & chromosomes
    47. 47. • Flemming’s fluid : Cytological fixative, not used• Lewitsky – Baker modification : tissue of invertebrates• Orth’s fluid : Mordant for mitochondria, chromaffin reaction
    48. 48. Causes of poor fixation• Less volume of fixation• Less time• Poorly penetrating fixative• Specimen settling down on contained• Wrong choice of fixative
    49. 49. Always necessary?• Sclero proteins – nails• Chitin, cellulose, inorganic salts,• Frozen sections
    50. 50. Storage and preservation of tissue• Formalin : store in fixative• 70% alcohol• Formal sublimate : 30% glycerin• 1% phenoxetol• Best : store a block rather than tissue
    51. 51. TAKE HOME MESSAGE• Precautions on collecting and handling tissues• Legal implications - animals• Neutral normal 10% formalin is the best fixative in most circumstances• Remember the exceptions • Pancreas • Testis • Bone marrow and spleen• Choose fixative as per requirement – tissue or cell, stains• Storage of tissue
    52. 52. DISCUSSION