Introduction to Histology_e9ef0e34fe657b431db81d6354e8924a.pptx

INTRODUCTION TO HISTOLOGY DULCE ANN ROSS B. DALANGIN, MD, DPSP OBJECTIVES 1. Describe the importance of histology in medicine and biological sciences. 2. Explain the relationship between tissue structure and function. 3. Explain the various techniques used to prepare and examine tissue samples. 4...

INTRODUCTION TO HISTOLOGY DULCE ANN ROSS B. DALANGIN, MD, DPSP OBJECTIVES 1. Describe the importance of histology in medicine and biological sciences. 2. Explain the relationship between tissue structure and function. 3. Explain the various techniques used to prepare and examine tissue samples. 4. Discuss the principles behind staining methods and their applications. 5. Familiarize in the use of microscopy in histological studies. HISTOLOGY  The study of the tissues of the body and how these tissues are arranged to constitute organs  Involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. FIXATION  To preserve tissues permanently in as life-like a state as possible.  Should be carried out as soon as possible after removal of the tissues or soon after death (with autopsy) to prevent autolysis.  No perfect fixative, though formaldehyde comes the closest TYPES OF FIXATIVES: ALDEHYDES  Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues.  Cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost.  Formaldehyde is good for immunohistochemical techniques.  Formalin penetrates tissue well, but is relatively slow.  Standard solution: 10% neutral buffered formalin  A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues. TYPES OF FIXATIVES: ALDEHYDES - GLUTARALDEHYDE  Causes deformation of alpha-helix structure in proteins so is not good for immunohistochemical staining.  Fixes very quickly so is good for electron microscopy.  Penetrates very poorly, but gives best overall cytoplasmic and nuclear detail.  Standard solution: 2% buffered glutaraldehyde TYPES OF FIXATIVES: MERCURIALS  Unknown mechanism  Contain mercuric chloride  B-5 and Zenker’s  Penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail.  Best application: fixation of hematopoietic and reticuloendothelial tissues TYPES OF FIXATIVES: ALCOHOLS  Methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness.  Very good for cytologic smears because they act quickly and give good nuclear detail.  Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well. TYPES OF FIXATIVES: OXIDIZING AGENTS  Permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide  Cross-link proteins, but cause extensive denaturation.  Used very infrequently TYPES OF FIXATIVES: PICRATES  Fixatives with picric acid  Bouin's solution - unknown mechanism of action; does almost as well as mercurials with nuclear detail but does not cause as much hardness.  Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin. FACTORS AFFECTING FIXATION Buffering Penetration Volume Temperature Concentration Time interval FIXATION: BUFFERING  Best carried out close to neutral pH, in the range of 6-8.  Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue.  Buffers: phosphate, bicarbonate, cacodylate, and veronal.  Commercial formalin is buffered with phosphate at a pH of 7. FIXATION: PENETRATION  Depends upon the diffusability of each individual fixative, which is a constant.  Formalin and alcohol penetrate the best, and glutaraldehyde the worst.  Mercurials are somewhere in between.  Penetration into a thin section will occur more rapidly than for a thick section - Section the tissues thinly (2 to 3 mm) FIXATION: VOLUME  10:1 ratio of fixative to tissue  Change the fixative at intervals to avoid exhaustion of the fixative.  Agitation of the specimen in the fixative will also enhance fixation. FIXATION: TEMPERATURE  Increasing the temperature, as with all chemical reactions, will increase the speed of fixation  Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor. FIXATION: CONCENTRATION  Adjusted down to the lowest level possible  Formalin is best at 10%  Glutaraldehyde is generally made up at 0.25% to 4%. FIXATION: TIME INTERVAL  Time interval from removal of tissues to fixation  The faster you can get the tissue and fix it, the better.  Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline.  The longer waiting time, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.  TISSUE PROCESSING Fixation: Small pieces of tissue are placed in Dehydration: The tissue is transferred through solutions of chemicals that cross-link proteins a series of increasingly concentrated alcohol and inactivate degradative enzymes, which solutions, ending in 100%, which removes all preserve cell and tissue structure. water.  TISSUE PROCESSING Clearing: Alcohol is removed in organic Infiltration: The tissue is then placed in melted solvents in which both alcohol and paraffin paraffin until it becomes completely infiltrated are miscible. with this substance.  TISSUE PROCESSING Embedding: The paraffin-infiltrated tissue is Trimming: The resulting paraffin block is placed in a small mold with melted paraffin trimmed and allowed to harden. MICROTOME Used for sectioning paraffin-embedded tissues for light microscopy. The trimmed tissue specimen is mounted in the paraffin block holder, and each turn of the drive wheel advances the holder a controlled distance, generally from 1 to 10 μm. After each forward move, the tissue block passes over the steel knife edge and a section is cut at a thickness equal to the distance the block advanced. The paraffin sections are placed on glass slides and allowed to adhere, deparaffinized, and stained for light microscope study. TISSUE STAINING  The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections.  Before any staining can be done, the slides are "deparaffinized" by running them through xylenes to alcohols to water.  There are no stains that can be done on tissues containing paraffin.  Routine stain: hematoxylin and eosin (H and E) TISSUE STAINING: H&E STAINING  Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten.  Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus.  Eosin is an acidic dye with an affinity for cytoplasmic components of the cell.  Problem: overstaining, especially with decalcified tissues COVERSLIPPING  The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come.  The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over the section. DECALCIFICATION  Some tissues contain calcium deposits which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and paraffin.  Bone specimens  Mineral acids, organic acids, EDTA, and electrolysis  Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. Strong acids damage cellular morphology, so are not recommended for delicate tissues such as bone marrow.  Organic acids such as acetic and formic acid are better suited to bone marrow. They act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier.  EDTA can remove calcium and is not harsh but it penetrates tissue poorly and works slowly and is expensive in large amounts.  Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily use. ARTEFACTS IN HISTOLOGIC SECTIONS: FORMALIN-HEME PIGMENT  Fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices or vessels)  Confirmed by polarized light microscopy and the slide will look like many stars in the sky.  Most often seen in very cellular or bloody tissues, or in autopsy tissues, because this pigment forms when the formalin buffer is exhausted and the tissue becomes acidic, promoting the formation of a complex of heme (from red blood cells) and formalin.  Spleen and lymph node are prone to this artefact.  Making thin sections and using enough neutral-buffered formalin (10 to 1 ratio of fixative to tissue) will help.  If the fixative solution in which the tissues are sitting is grossly murky brown to red, then place the tissues in new fixative. ARTEFACTS IN HISTOLOGIC SECTIONS: DEZENKERIZED  The presence of large irregular clumps of black precipitate on slides of tissues fixed in a mercurial fixative such as B-5 suggests that the tissues were not "dezenkerized" prior to staining.  These black precipitates will also appear white with polarized light microscopy. ARTEFACTS IN HISTOLOGIC SECTIONS: POOR DEHYDRATION AND PARAFFIN INFILTRATION  Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hard to section on the microtome, with tearing artefacts and holes in the sections.  Final ethanol dehydrant solution should be at 100% concentration.  Toluene as a clearing agent is more forgiving of poorly dehydrated tissues, but it is more expensive and presents more of a health hazard than other non-xylene clearing agents. ARTEFACTS IN HISTOLOGIC SECTIONS: VENETIAN BLIND  Though alcohols such as ethanol make excellent fixatives for cytologic smears, they tend to make tissue sections brittle, resulting in microtome sectioning artefacts with chattering and a "venetian blind" appearance. ARTEFACTS IN HISTOLOGIC SECTIONS: BUBBLES  Bubbles under the coverslip may form when the mounting media is too thin, and as it dries air is sucked in under the coverslip.  Contamination of clearing agents or coverslipping media may also produce a bubbled appearance under the microscope. PROBLEMS IN TISSUE PROCESSING  "Floaters" are small pieces of tissue that appear on a slide that do not belong there  They have floated in during processing.  Floaters may arise from sloppy procedure on the cutting bench-- dirty towels, instruments, or gloves can have tissue that is carried over to the next case. SAFETY IN THE LABORATORY  The lab should be well-ventilated.  Every chemical compound used in the laboratory should have a materials safety data sheet on file that specifies the nature, toxicity, and safety precautions to be taken when handling the compound.  Contract with waste management company.  Tissues that are collected should be stored in formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to a large sink.  Every instrument used in the laboratory should meet electrical safety specifications and have written instructions regarding its use. SAFETY IN THE LABORATORY  Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose.  Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, and fire alarms should be within easy access.  A shower and eyewash should be readily available.  Laboratory accidents must be documented and investigated with incident reports and industrial accident reports. SPECIFIC HAZARDS  Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive.  Many reagent kits have sodium azide as a preservative. Flush solutions containing sodium azide down the drain with lots of water, or there is a tendency for the azide to form metal azides in the plumbing. These are also explosive.  Benzidine, benzene, anthracene, and naphthol containing compounds are carcinogens and should not be used.  Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers.  Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed. SPECIAL STAINS: MUCIN STAINS  Colloidal iron ("AMP") - Iron particles are stabilized in ammonia and glycerin and are attracted to acid mucopolysaccharides. It requires formalin fixation. Phospholipids and free nucleic acids may also stain. The actual blue color comes from a Prussian blue reaction. Tissue can be pre-digested with hyaluronidase to provide more specificity.  Alcian blue - The pH of this stain can be adjusted to give more specificity; a lot of background staining SPECIAL STAINS: MUCIN STAINS  PAS (periodic acid-Schiff) – most sensitive; Stains glycogen as well as mucins, but tissue can be pre-digested with diastase to remove glycogen.  Mucicarmine - Very specific for epithelial mucins; very insensitive SPECIAL STAINS: STAINS FOR BIOGENIC AMINES "Chromaffin" cells have cytoplasmic "Argentaffin" cells reduce a silver granules that appear brown when solution to metallic silver after fixed with a dichromate solution. formalin fixation. argentaffin reaction Chromaffin reaction is associated with is associated with carcinoid tumors of adrenal medulla or extra-adrenal the gut. Diazo (diazonium salts) paraganglion tissues 1. Diazo (diazonium salts) (pheochromocytomas) 2. Fontana-Masson 1. Modified Giemsa 3. Schmorl's 2. Schmorl's 4. Autofluorescence 3. Wiesel's  SPECIAL STAINS: MELANIN STAINS  Fontana-Masson ("melanin stain") method relies upon the melanin granules to reduce ammoniacal silver nitrate.  Schmorl's method uses the reducing properties of melanin to stain granules blue-green. SPECIAL STAINS: MELANIN STAINS DOPA-oxidase  The most specific method  an enzyme histochemical method  Requires frozen sections for best results  Paraffin sections of well-fixed tissues may be used.  The DOPA substrate is acted upon by DOPA-oxidase in the melanin-producing cells to produce a brownish black deposit. SPECIAL STAINS: FAT STAINS The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues. Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing, will remove lipids. It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood. SPECIAL STAINS: CONNECTIVE TISSUE STAINS  The trichrome stain helps to highlight the supporting collagenous stroma in sections from a variety of organs.  This helps to determine the pattern of tissue injury.  Trichrome will also aid in identifying normal structures, such as connective tissue capsules of organs, the lamina propria of gastrointestinal tract, and the bronchovascular structures in lung. SPECIAL STAINS: CONNECTIVE TISSUE STAINS  The reticulin stain is useful in parenchymal organs such as liver and spleen to outline the architecture.  Delicate reticular fibers, which are argyrophilic, can be seen.  A reticulin stain occasionally helps to highlight the growth pattern of neoplasms. SPECIAL STAINS: CONNECTIVE TISSUE STAINS  An elastic tissue stain helps to outline arteries, because the elastic lamina of muscular arteries, and the media of the aorta, contain elastic fibers.  The van Gieson method for elastic fibers provides good contrast. SPECIAL STAINS: HEMATOLOGIC STAINS  There are a variety of "Romanowsky-type" stains with mixtures of methylene blue, azure, and eosin compounds.  Giemsa and Wright stains  One property of methylene blue and toluidine blue dyes is metachromasia. This means that a tissue component stains a different color than the dye itself. For example, mast cell granules, cartilage, mucin, and amyloid will stain purple and not blue, which is helpful in identifying these components. SPECIAL STAINS: HEMATOLOGIC STAINS  The leukocyte alkaline phosphatase (LAP) stain is helpful in determining whether a high peripheral blood leukocytosis is a reactive process or a leukemia (chronic myelogenous leukemia, or CML).  The more differentiated cells in the reactive process will stain more readily with LAP, while leukemic cells will not.  The cells on a smear can be assessed and an "LAP score" can be generated. A high score generally indicates a "leukemoid reaction" or reactive condition (with an infection or other inflammatory process) while a low score suggests CML. SPECIAL STAINS: HEMATOLOGIC STAINS  The tartrate-resistant acid phosphatase (TRAP) stain has one major usefulness--to help diagnose a rare leukemia known as hairy cell leukemia.  Hairy cell leukemia - neoplastic B lymphocyte proliferation affecting mainly bone marrow and spleen.  There is pancytopenia, so the peripheral WBC count is not high.  The circulating hairy cells get their name from the cytoplasmic projections  Positive staining with TRAP helps make the diagnosis. SPECIAL STAINS: HEMATOLOGIC STAINS  The myeloperoxidase (MPO) stain is helpful to identify cytoplasmic granules characteristic of myeloid cells.  This is useful when there are large, immature white blood cells in the peripheral blood, and it is not clear whether they are of myeloid or of lymphoid origin.  Staining with MPO in this setting suggests a myeloid leukemia. SPECIAL STAINS: MICROORGANISMS  Bacteria appear on H and E as blue rods or cocci regardless of gram reaction. Colonies appear as fuzzy blue clusters.  Tissue gram stains are all basically the same as that used in the microbiology lab except that neutral red is used instead of safranin.  Gram positive organisms usually stain well, but gram negatives do not (because the lipid of the bacterial walls is removed in tissue processing).  Brown and Brenn (or the Brown and Hopps modification) is the method most commonly used. SPECIAL STAINS: MICROORGANISMS  Fungi stain blue with H and E and red with PAS. The most sensitive method for demonstrating them is Methenamine silver.  A Giemsa stain may help demonstrate donovan bodies and leishmania organisms in tissue sections. SPECIAL STAINS: MICROORGANISMS  For tertiary stage syphilis the best method is the Warthin-Starry.  The small, thin, spiral organisms are difficult to see and a careful search must be made.  In primary syphilis, a lesion called a "chancre" may be present on genitalia, and a scraping made from this lesion can be placed on a slide, and the organisms observed under darkfield microscopy (but you need a darkfield condenser on a microscope). SPECIAL STAINS: MICROORGANISMS  AFB stain uses carbol-fuchsin to stain the lipid walls of acid fast organisms such as M. tuberculosis.  The most commonly used method is the Ziehl-Neelsen method, though there is also a Kinyoun's method.  A modification of this stain is known as the Fite stain and has a weaker acid for supposedly more delicate M. leprae bacilli. SPECIAL STAINS: MICROORGANISMS  The most sensitive stain for mycobacteria is the auramine stain which requires a fluorescence microscope for viewing.  Acid fast: mycobacteria, cryptosporidium, isospora, and the hooklets of cysticerci SPECIAL STAINS: MICROORGANISMS  Gomori methenamine silver stain is used to stain for fungi and for Pneumocystis jiroveci (carinii).  The cell walls of these organisms are stained, so the organisms are outlined by the brown to black stain. SPECIAL STAINS: MICROORGANISMS  PAS (periodic acid-Schiff) is an all-around useful stain for many things.  It stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi.  A predigestion step with amylase will remove staining for glycogen.  PAS is useful for outlining tissue structures--basement membranes, capsules, and blood vessels.  It is very sensitive, but specificity depends upon interpretation. SUMMARY  Histology and methods in histology  Fixatives  Tissue processing  Artefacts  Safety in the laboratory  Special stains REFERENCES  Junqueira’s Basic Histology Text & Atlas Chapter 1  https://webpath.med.utah.edu/HISTHTML/HISTOTCH/HISTOTCH.html  https://webpath.med.utah.edu/HISTHTML/STAINS/STAINS.html