Histopathology Lecture Notes - Introduction to Histochemistry Part 2 PDF

Allied Health Sciences Lect. 2 Introduction for Histopathological examination part 2 By / Dr. Doaa Elshaer  Histopathological Examination A. Routine Hematoxylin and Eosin stain B. Ancillary techniques 1. Special stains 2. Immunohistochemistr...

Allied Health Sciences Lect. 2 Introduction for Histopathological examination part 2 By / Dr. Doaa Elshaer  Histopathological Examination A. Routine Hematoxylin and Eosin stain B. Ancillary techniques 1. Special stains 2. Immunohistochemistry 3. Immunofluorescence 4. Electron microscopy 5. Molecular studies 6. Cytogenetic studies 7. Flow cytometry 8. Polymerase chain reaction  Fixation  Principle of Fixation: results in denaturation and coagulation of protein in the tissues. The fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other.  Aim of fixation: Prevent Autolysis and putrefaction of tissues  Media: 10% Neutral buffered formalin  However, mineralized samples such as bone or tooth may require decalcification before it can be processed.  The volume of the fixative should be in excess of 20 times the volume of the tissue.  In practice, the tissues are fixed for 6 to 24 hours.  MOST COMMON SIMPLE FIXATIVES 1. Formaldehyde: Formalin is most commonly used fixative. It is cheap, penetrates rapidly and does not over- harden the tissues.  If formalin is kept standing for a long time, a large amount of formic acid is formed due to oxidation of formaldehyde and this tends to form artefact which is seen as brown pigment in the tissues. To avoid this buffered formalin is used. 2. Absolute alcohol it may be used as a fixative as it coagulates protein. Due to its dehydrating property it removes water too fast from the tissues and produces shrinkage of cells and distortion of morphology. It penetrates slowly and over-hardens the tissues. Used for cytological specimin fixation 3. Osmium tetraoxide It gives excellent preservation of cellular details, hence used for electron- microscopy. 4. Picric acid  It precipitates proteins and combines with them to form picrates.  Owing to its explosive nature when dry; it must be kept under a layer of water. Tissue fixed in picric acid also require thorough washing with water to remove colour.  Tissue can not be kept in picric acid more than 24 hrs.  MOST COMMON COMPOUND FIXATIVES 1. Formal saline  It is most widely used fixative.  Tissue can be left in this for long period without excessive hardening or damage.  Tissues fixed for a long time occasionally contain a pigment (formalin pigment).  This may be removed in sections before staining by treatment with picric alcohol or 10% alcoholic solution of sodium hydroxide.  The formation of this pigment can be prevented by neutralizing or buffering the formal saline.  Fixation time – Optimum 24 hours at room temperature 2. Zenker’s fluid  It contains mercuric chloride, potassium-di-chromate, sodium sulphate and acetic acid.  Advantages – even penetration, rapid fixation  Disadvantages – After fixation the tissue must be washed in running water to remove excess dichromate. Mercury pigment must be removed with Lugol’s iodine. 3. Zenker’s formal (Helly’s fluid) In stock Zenker’s fluid, formalin is added instead of acetic acid. Advantages – excellent microanatomical fixative especially for bone marrow, spleen & kidney. 4. Bouins fluid It contains picric acid, acetic acid and 40% formaldehyde. Advantages – (a) Rapid and even penetration without any shrinkage. (b) Brilliant staining by trichrome method. It is routinely used for preservation of testicular biopsies.  Most Commonly used Fixatives in the Laboratory  10% Formalin Formaldehyde (40%) - 10 ml Distilled water - 90 ml  Formal Saline  Formaldehyde (40%)- 100 ml  Distilled Water- 900 ml  Sodium Chloride- 9 gm  10% Buffered Formalin  Formaldehyde (40%)- 10 ml  Sodium dihydrogen phosphate- 0.4 gm  Disodium hydrogen phosphate - 0.65 gm  Distilled water- 90 ml The advantage of this fixative is that it prevents the formation of formalin pigment  Bouin’s solution  Saturated picric acid (1.2 gm/ 100 ml)- 750 ml  Formaldehyde (40%)- 250 ml  Acetic acid- 50 ml  Points to Remember 1. 10% buffered formalin is the commonest fixative. 2. Tissues may be kept in 10% buffered formalin for long duration. 3. Volume of the fixative should be at least ten times of the volume of the specimen. The specimen should be completely submerged. 4. Special fixatives are used for preserving particular tissues. 5. Formalin vapors cause throat/ eye irritation hence mask/ eye glasses and gloves should be used. 6. Tissues should be well fixed before dehydration. 7. Penetration of fixatives takes some time. It is necessary that the bigger specimen should be given cuts so that the central part does not remain unfixed. 8. Mercury pigment must be removed with Lugol’s iodine. 9. Biopsies cannot be kept for more than 24 hours in bouin’s fluid without changing the alcohol. 10. Glutaraldehyde and osmion tetraoxide are used as fixatives for electron microscopy.  Tissue Processing Aim of tissue processing: › Sufficient rigidity › Cut into thin sections Dehydration Clearing Impregnation Water molecule Dehydrating agent is Tissue is infiltrated is removed from replaced by clearing agent with a supporting tissue. medium Dehydration:  Removes free or unbound water molecule.  It is mandatory because the supporting media (paraffin) not miscible with water.  Media: Methanol or ethyl alcohol.  Graded alcohol: 50, 70, 90 and 100% Clearing:  Removal of dehydrating agent (e.g: alcohol) to facilitate infiltration of paraffin wax.  Make tissue clear and improves microscopic examination. Media: Xylene. Impregnation/infiltration:  Clearing agent is removed by molten wax through process of diffusion.  Media: Paraffin wax  Embedding  Occur after processing.  Processed tissue is put into mould and paraffin wax is poured. Aim:  Give support to the tissue  Prevent distortion of tissue during cutting  Preserve tissue for archival uses   Microtome  Block is now prepared  Tissue embedded blocks are cut into thin sections (4-5 Micron).  Trouble shooting for poor sections Faults occurring during section cutting  Section shows thin and thick horizontal lines (chatters): Cause - A loose knife A loose block A blunt knife Remedy- Tighten knife and/or block Sharpen the knife  Holes in the section: Cause - Air bubbles in the tissue or wax , A piece of hard material in tissue, A soft piece of tissue in block Remedy- Re-embed Remove hard material if possible Reprocess specimen Faults due to poor processing  The tissue is shrunken away from wax: Cause - Insufficient dehydration Remedy- Reprocess  The tissue is too soft when block is trimmed: Cause - Insufficient fixation Remedy- Reprocess   Histochemical stain  Tissue section is colourless, thus stains are required.  Histochemical stain causes main chemical reaction that demonstrate the tissue elements of interest.  Stains have specific affinity with different tissue proteins and color them differently. Routine stain Special stains Hematoxylin and Eosin stain Carbohydrates Lipids  Commonly used. Nucleic acid and proteins  Hematoxylin: Nuclear stain (Bluish ) Pigments  Eosin: Cytoplasmic stain (Pink) Connective tissue stain Amyloid staining Microbial organisms Basic Steps of Hematoxylin and Eosin stain   Mounting and coverslip  Protective cover over the tissue  Make a permanent bond between the cover slip and slides.  Avoid Fading of staining or any further physical damage.   Special Stains Introduction:  Demonstration of various cellular products like carbohydrates, proteins, lipids and pigments for the diagnosis of certain diseases.  Demonstration of extracellular material for example amyloid for amyloidosis  Identification of microbial organisms  Estimation of DNA content of cell Classification: Carbohydrates Lipids Nucleic acid and proteins Pigments Connective tissue stain Amyloid staining Microbial organisms