Lesson+2+Histology+and+Pathology+tools.pdf

Familiarization on the apparatuses and different steps in tissue processing used in histotechnique HISTOLOGICAL TOOLS INTRODUCTION  Histology is the study of microscopic anatomy of cells and tissues of plants and animals.  Performed by examining a thin slice of tissue under a lig...

Familiarization on the apparatuses and different steps in tissue processing used in histotechnique HISTOLOGICAL TOOLS INTRODUCTION  Histology is the study of microscopic anatomy of cells and tissues of plants and animals.  Performed by examining a thin slice of tissue under a light or electron microscope enhanced through the use of histological stains.  Histotechniques constitute the backbone of diagnostic pathology enabling the diagnosis of diseased tissue possible for proper management.  Histopathology, the microscopic study of diseased tissue, is an important tool in  microscopy Biopsy Tissue Processing It includes : Gaining Histo Chem S Immuno cyto lgtodnun MICROSCOPY BIOPSY TISSUE PROCESSING STAINING HISTOCHEMISTRY AND CYTOCHEMISTRY IMMUNOCYTOCHEMISTRY  MICROSCOPE ❑Microscope: It is the instrument that is used to see objects not visible to naked eye. ❑Microscopy: It is the study of a fine structure of an object under a microscope. ❑Microscope is the most commonly used piece of apparatus in the laboratory. It produces magnified images of minute objects. ❑ The microscope magnifies as well as resolves Magnification power is the degree of image enlargement. Magnification power of the microscope = Magnifying power of objective x Magnification power of eyepiece. Resolving power is the capacity of the optical system to produce separate image of objects very close to each other.  HISTORY In 1595, Zacharias Janssen built the first microscope. Magnified 3-9 times the original size. ❑The credit of invention of light microscope goes to Anton van Leeuwenhoek (1632-1723) who is now known as Father of microscopy. He was the first to observe and describe microbes which he referred to as ANIMALCULES under the microscope. ❑Robert Hooke made a copy of Leeuwenhoek's light microscope and then improved upon his design. Max Knoll Ernst Ruska ❑The electron microscope was introduced in the 1930's by Germans, Max Knoll and Ernst Ruska.  TYPES OF MICROSCOPE (A) LIGHT MICROSCOPE Microscopes utilizing visible light 1. Simple microscope 2. Compound microscope µ Dark Dark field microscope Phase contrast microscope Polarizing microscope Interference microscope Microscopes utilizing ultraviolet light 1. Fluorescence microscopy (B) ELECTRON MICROSCOPE Scanning electron microscope Transmission electron microscope Freeze fracture microscopy SIMPLE MICROSCOPE: A simple microscope is a microscope that uses only one lens for magnification. It is the original design of the light microscope. Even though it is now considered pre-historic, the use of a single, convex lens for viewing is still found in simple magnification devices, such as magnifying glass and the loupe. Magnification power of hand lens is from 20X to 200X.  COMPOUND MICROSCOPE The compound microscope consists essentially of two or more double convex lenses fixed in the two extremities of a hollow cylinder. The lower lens (nearest to the object) is called the objective; the upper lens (nearest to the eye of the observer), the eyepiece. The cylinder is mounted upright on a screw device, which permits it to be raised or lowered until the object is in focus, i.e., until a clear image is formed. PARTS: A) STAND B) BODY C) OPTICAL SYSTEM D) LIGHT/ILLUMINATION SYSTEM  STAND Base or foot: It is usually shaped like a horse shoe. It imparts stability to the microscope. 1. Body: It carries the body tube, stage, sub-stage and a mirror. A. Body tube: It is attached to the limb. It is of 2 TYPES. External tube which carries at its lower end a revolving nosepiece having objective lenses of different magnification. Internal tube carries at its upper end eyepieces. B Stage: It is the large plate below the tube on which you have to place the slide which you wish to see. C. Sub-stage: It is placed below the stage. It carries the condenser. MECHANICAL ADJUSTMENTS 1. Focusing mechanisms: Screws for coarse adjustment: Used to bring the section into focus with a 10X objective. It is controlled by a pair of large knobs. Screws for fine adjustment : Required to focus when using higher magnification objectives and oil immersion lenses. This is usually controlled by two smaller knobs on each side of the body.  Optical system CONDENSER It brings the parallel light rays into focus on the plane of the section or slide. It is mounted on the sub-stage. An iris diaphragm, situated below the condenser, can be used to restrict the glare produced by too much light. OBJECTIVE LENS They are fitted to the nose piece. It is near the object and produces an initial magnified image of the object. Their magnifying power is measured as the times of its diameter they magnify. The four objective lens commonly used have magnification of 4x, 10x ,40x, 100x with color coding as red, yellow, light blue and white respectively for rapid identification. Ocular or eye peice Mononuclear - 1 eyepiece. Binocular – 2 eyepiece Their magnification can be 5x, 10x,15x MIRROR It is located below the condenser. Its function is to direct the light rays from a light source to the condenser and optical field of the microscope. The mirror has two sides: one has a plane mirror (sunlight) and the other with a concave mirror (artificial light). The plane mirror is used to direct the light to the condenser to give a uniform illumination. In day light or when there is no condenser in the microscope a concave mirror is used to direct the light to the slide. LIGHT SOURCE Daylight: It is best to use reflected sunlight of a dull white background. It is not sufficient for oil immersion lens and it is not available during evening or night. Electric light: A built-in source of illumination of 60 watt electric lamp placed 18” away from the microscope is sufficient for most routine work. In the absence of electricity, a battery lamp or an oil lamp can be utilized. Best however are POLARISING MICROSCOPE This method is used for demonstration of birefringence eg amyloid, foreign body, hair etc. Birefringent objects rotate the light rays and therefore appear bright in a dark background. polariser analyser FLUORESCENT MICROSCOPE This method is used for demonstration of naturally occurring fluorescent material and other non fluorescent substances or micro- organisms after staining them with some fluorescent dyes Principle: Fluorescent substances are those substances which absorb light of a shorter wavelength and high energy and emit light of longer wavelength and less energy. Many substances have the property of emitting visible light when irradiated by invisible rays, like when ultraviolet light is focused upon them, such a specimen glows and can be observed by emitted fluorescence. The phenomenon is called fluorescence. Uses 1. For identifying mycobacterium tuberculosis, amyloid,lipids, elastic fibers. 2. It is used extensively in fluorescent antibody techniques used in parasitology and bacteriology. 3. It is also used widely in histopathology of kidney; skin etc. where immune/auto-immune basis of disease is expected. 4. Widely used in cytogenetics. Fluorescent antibody technique- USE IN PERIO Principle: Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. Method 1. Antibody to which a fluorescent dye is attached is referred to as labeled antibodies. 2. Labeled antibodies are mixed with suspension of bacteria and preparation is examined under microscope. 3. Bacterial cells that have combined with the labeled antibody will only be visible in the microscopic ELECTRON MICROSCOPE Most electron microscopes used to study biological material can magnify objects up to 1 million times. Although this does not make atoms visible, it does allow researchers to distinguish individual molecules of biological importance. Drawback Since no living specimen can survive under their high vacuum, they cannot show the ever-changing movements that characterize a living cell. PRINCIPLE By the substitution of an electron beam for light rays, a much greater degree of resolution can be obtained, since at an acceleration of 50,000 volts electrons have a wavelength of only 0.001nm; therefore a theoretical resolving power of 0.0005nm could be attained, which would enable molecules to be seen. ELECTRON MICROSCOPE It is used to study ultrastructural details of the tissues and cells. Tissue is fixed in 4% glutaraldehyde at 4 degree celsius for 4 hrs. Ultrathin microsections with thickness of 100 nm are cut with diamond knives. TYPES: 1) TRANSMISSION ELECTRON MICROSCOPY (TEM) : helps to visualize cell’s cytoplasm and organelles. 2) SCANNING ELECTRON MICROSCOPY (SEM) : helps in the study of cell surface.  TRANSMISSION ELECTRON MICROSCOPY (TEM) TEM gives two dimensional image of the tissue. TEM helps visualize cell’s cytoplasm and organelles. Unfortunately the degree of correction that is currently feasible with TEM lenses permit a resolution of 0.25nm, but this is still a thousand times greater than that possible with the light microscope. A further difficulty with TEM is that, since electrons have poor penetrating power, the sections to be examined must be very thin, less than 50nm thick. This necessitates the use of special hard embedding media (plastics) and special ultra microtomes to cut such thin sections. Steel knives cannot be used to cut these sections; either glass or diamond knives are used. Principle and Method In TEM, the electrons beam passes right through many parts of specimen. The filament of electron microscope emits beam of electrons. By means of magnetic coil, electrons are focused onto the object and electrons passing through object are then received by another magnetic coil acting as objective lens which gives a magnified image of the object. These electrons are then received by third magnetic coil acting as ocular or projection lens which further magnifies the primary image. This final image can be visualized on a fluorescent screen or recorded on a photographic plate. As the electron cannot be seen so this image is formed on the fluorescent screen that converts the energy of the electrons into light. SCANNING ELECTRON MICROSCOPY (SEM) In this 3-D image is produced. The image is produced on cathode ray oscillograph which can also be amplified. SEM helps in the study of cell surface. SEM can also be used for fluorescent antibody techniques. Resolution up to 50 nanometers can be achieved. APPLICATION: Secondary electrons are most valuable for showing morphology and topography of samples and imaging. Backscattered electrons are valuable for revealing chemical composition of samples. Diffracted –backscattered electrons determine crystalline structure and orientation of minerals in samples. Photons are used for elemental analysis. In SEM, accelerated electrons scans the surface of specimen instead of passing through it. These accelerated electrons carries significant amount of kinetic energy. As the electron beam hits the specimen energy is dissipated as variety of signals, these signals include: Secondary electrons Backscattered electrons Diffracted-backscattered electrons Photons Heat Detectors collect these signals and send to a screen and final image is produced. Electron micrographs are obtained by photographing the image.  BIOPSY It is the removal of tissue from living organism for the purposes of microscopic examination and diagnosis. PURPOSE: Diagnosis of pathologic lesions. Grading of tumors. Diagnosis of metastatic lesions. For evaluation of recurrence. It is useful for therapeutic assessment of lesion by differentiating between benign and malignant lesions. To ensure obtaining a proper specimen for the pathologist, following points must be considered: 1. Do not paint the surface of the area to be biopsied with iodine or a highly coloured antiseptic. 2. If using infiltration anesthesia, do not inject local anesthesia solution directly into the lesion. Instead, inject around the periphery of the lesion. 3. Use a sharp scalpel to avoid tearing of the tissues. 4. Remove a border of normal tissue with the specimen, if at all possible. 5. Use care not to mutilate the specimen when holding it with the forceps. 6. Fix the tissue immediately upon removal in 10% formalin or 70% alcohol. If the specimen is thin, place it upon a piece of glazed paper and drop into fixative to prevent curling of tissues. TYPES OF BIOPSY EXCISIONAL INCISIONAL PUNCH BIOPSY BIOPSY BIOPSY BRUSH CORE EXFOLIATIVE BIOPSY NEEDLE CYTOLOGY BIOPSY  EXCISIONAL BIOPSY It is the total excision of a small lesion for microscopic examination. INDICATIONS : a) Small lesion ( less than 1 cm). b) 2-3mm normal tissue surrounding the lesion is also excised. c) When the lesion is sessile or pedunculated. d)Tissues which are freely movable and located above the mucosa  INCISIONAL BIOPSY It is performed by removing a wedge shaped specimen of the pathological tissue along with surrounding normal zone. Biopsy - deep and narrow biopsy rather than a broad and shallow one, because superficial changes may be different from those deeper in the tissue. INDICATIONS: a) If the lesion is large (more than 1 cm) and diffuse and extend deeply into the surrounding tissue so that total removal cannot be obtained easily with LA.  PUNCH BIOPSY It is rarely used in the oral cavity as most of the oral lesions are easily accessible. In this technique, a sharpened hollow tube is rotated until underlying bone or muscle is reached. The tissue is then removed in the same manner as excisional and incisional biopsy.  BRUSH BIOPSY Biopsy obtained by abrading the surface of a lesion with a brush with stiff bristles to obtain cells and tissues for microscopic examination. Effective in obtaining tissue samples from inaccessible areas.  CORE NEEDLE BIOPSY Common needle biopsy procedures include fine-needle aspiration and core needle biopsy. It may be used to take tissue or fluid samples from muscles, bones, and other organs, such as the liver or lungs  EXFOLIATIVE CYTOLOGY It is the study of cells which exfoliate or abrade from the surface. It is not a substitute but an adjunct to surgical biopsy. The cytologic smear is reported in 5 classes: I : NORMAL II: ATYPICAL III: INTERMEDIATE IV: SUGGESTIVE OF CANCER V : POSITIVE FOR CANCER Oral exfoliative cytology is still controversial, particularly in premalignant conditions Normal oral squamous epithelium continuously sheds the most superficial cells If the area is affected by malignant or other disease, the deeper cells lose their cohesiveness and are exfoliated at the same time as the superficial cells. Exfoliative cytology has not been a very diagnostic or useful screening method for oral cancer because hyperkeratosis and keratin itself interfere with cell obtainment and a greater proportion of diagnostic cells are below the surface (most at the basement membrane level) Class 1 (normal) indicates that only normal cells were observed Class 2 (atypical) indicates presence of minor atypical but no evidence of malignant change Class 3 (intermediate) – this is an in between cytology that separates cancer from non cancer diagnosis biopsy is recommended. Class 4 (suggestive of cancer) a few cells with malignant characteristics or many cells with borderline characteristics biopsy is mandatory Class 5 (positive for cancer) cells that are obviously malignant  TISSUE PROCESSING STEPS INCLUDE: FIXATION DEHYDRATION CLEARING IMPREGNATION EMBEDDING AND BLOCKING SECTION CUTTING ROUTINE STAINING Tissue sample Sample of a tissue is called tissue block which is obtained by biopsy. If a sample is taken from cadaver it is essential to remove it promptly after death to avoid post mortem degeneration. Use Sharp instrument to avoid distortion of microscopic features. After obtaining sample it must be immersed  FIXATION Any tissue removed from the body starts decomposing immediately because of loss of blood supply and oxygen, accumulation of products of metabolism of action of autolytic enzymes and putrefaction by bacteria. This process of decomposition is prevented by fixation. EFFECTS: Prevention of putrefaction and autolysis. Hardens the tissue which helps in section cutting. Makes cell insensitive to hypertonic or hypotonic solution. COMMONLY USED FIXATIVES: 1. FORMALIN 2. GLUTRALDEHYDE 3. PICRIC ACID 4. ALCOHOL 5. OSMIUM TETRAOXIDE  DEHYDRATION This is a process in which water from the tissues and cells is removed so that this space so created is subsequently taken up by wax. Dehydration is carried out by passing the tissues through a series of ascending grades of alcohol (70%,80%,95% and absolute alcohol). If ethyl alcohol is not available isopropryl alcohol or acetone can be used. Purpose of dehydration is to remove excess fixative. Done in two stages: Stage 1 (Stage of gradual dehydration): Formalin fixed tissue block is passed through increasing strength of alcohol to absolute alcohol. Stage2 (clearing stage): Purpose is to remove dehydrating agent. This is the process in which alcohol from the tissues and cells is removed and is replaced by a fluid in which wax is soluble.It also makes the tissue transparent. Xylol is used for this purpose because alcohol does not act as paraffin solvent so it is necessary to replace alcohol with xylol which is miscible both with alcohol and paraffin. Alcohol dehydrated block is passed through xylol until all alcohol has been replaced by xylol. Other clearing agents are benzene and chloroform. IMPREGNATION This is the process in which empty spaces in the tissues and cells after removal of clearing agent are taken up molten by paraffin wax. This harden the tissue which helps in section cutting. Impregnation is done in molten paraffin wax which has the melting point ranging from 54-62 degree. EMBEDDING AND BLOCKING ❖Embedding is a process in which tissue sample are placed into mould along with liquid embedding material (agar / gelatin /wax) which is then hardened.. Purpose: to facilitate the sectioning of the tissue into slices sufficiently thin to allow the passage of light through them (approx 4 to 10 micrometre in routine practice). Most common medium used traditionally is paraffin. Xylol permeated tissue block is passed through warm paraffin which dissolve readily in xylol. Melted wax fills in tissue spaces which hardens on cooling and renders the block ready for sectioning. ▪ METALLIC MOULDS PLASTIC MOULDS TECHNIQUE Select a mould having sufficient room for tissue and at least a 2 mm surrounding margin of wax. Fill it with molten paraffin wax Lift tissue with warm forceps and place it at bottom of mould Gently press & orient it so that surface to be sectioned is kept flat. Insert a label. When thin layer forms over surface, cool and submerge it under water. After solidification, remove from mould. Trimmed with razor blade in such a way that edges are parallel. Ready to be sectioned on microtome.  SECTION CUTTING ❖ Technique of section cutting is MICROTOMY. ❖ Equipment used for cutting section is MICROTOME. ❖Microtome is used to cut extremely thin slices of material known as SECTIONS TYPES OF MICROTOMES: ❖ ROTARY : most commonly used ❖ SLIDING ❖ FREEZING ❖ ROCKING ❖ BASE-SLEDGE ROTARY MICROTOME STAINING ❖There are innumerable tissue stains but commonly used stain is HAEMATOXYLIN AND EOSIN. ❖HAEMATOXYLIN – natural dye obtained from log wood of tree. INACTIVE PRODUCT NATURAL SUNNLIGHT OXIDATION BY ADDING OXIDANTS KMnO4, SODIUM IODATE ACTIVE INGREDIENT (HAEMATEIN) TECHNIQUE Dried slides placed vertically. Sections are firstly deparaffinised ( removal of wax by placing slide in xylene for 10-15 min. Pass slide through descending grades of alcohol ( absolute alcohol , 95 % , 80 % , 60% ) & then to water (as haematoxylin is water based dye) Place slide in haematoxylin stain for 8-10 min. Rinse in water.. For differentiation (i.e. selective removal of excess dye from sect ion)put in SCOTT’S TAP WATER containing NaHCO3 and MgS O4. Counter stain with 1 % aqueous sol. Of Eosin for 30 sec – 1 min. Dip in tap water. Before mounting , dehydration of section through ascending grade of alcohol and finally cleared in xylene , 2-3 dips in each solution. Mount in DPX ( DEXTRENE POLYSTYRENE XYLENE ) / CANADA BALSAM.  Frozen sections When rapid confirmation of nature or spread of diseased tissue is required(during surgery) they can be prepared in great haste by another procedure. When a fresh tissue is rapidly frozen, the matter within the tissue turns into ice and in this state the tissue is firm, the ice acting as embedding medium. Therefore, sections are produced without the use of dehydrating solution, clearing agent or wax embedding. Everything present in the living tissue is still present in the frozen sections but it is necessary to examine these sections promptly to avoid fixation. Merits 1. This is a quick diagnostic procedure (10minutes). 2. Every type of staining can be done. 3. There is minimal shrinkage of tissues as compared to paraffin sections. 4. Lipids and enzymes which are lost in routine paraffin sections can be demonstrated. Demerits 1. It is difficult to cut serial sections. 2. It is not possible to maintain tissue blocks for future use. 3. Sections cut are thicker. 4. Structural details tend to be distorted due to lack of embedding medium.  Preparation of ground * sections of teeth or bone Decalcification of bone and teeth often obscures the structures. Teeth in particular are damaged because tooth enamel, being about 96% mineral substance, is usually completely destroyed by ordinary methods of decalcification. Undecalcified teeth and undecalcified bone may be studied by making thin ground sections of the specimen. The equipment used for making ground sections includes a laboratory lathe, a coarse and a fine abrasive lathe wheel, a stream of water, a wooden block, adhesive tape, and a camel hair brush. The finished ground section is then mounted on a microscope slide. Note: Teeth used for making ground sections should not be use  HISTOCHEMISTRY AND CYTOCHEMISTRY Cytochemistry is a science of localizing chemical components of cells and organelles on histological sections by using various techniques. Histochemistry has contributed greatly to the understanding of structure-function relationships. Histochemistry Histological technique Enzyme histochemistry used for studying Immunocytochemistry chemistry of tissues In situ hybridization and cells Application: Histochemical methods depend on a reasonably straightforward staining reaction of a dye for an organic molecule. For example: 1. Sudan black and oil red O stains are widely used for revealing the presence and location of lipids within tissues. 2. The nucleic acids DNA and RNA are demonstrable histochemically (with the Feulgen reaction and with toluidine blue and hematoxylin staining, respectively). ENZYME HISTOCHEMISTRY: It is used by both the histologist and the pathologist to localize enzymatic activity within tissue sections. E.g. acid and alkaline phosphatases, esterases, and the dehydrogenases, oxidases, and ATPases of various metabolic cycles. The general principle of enzyme tissue chemistry involves incubation of the tissue slice or section in a medium containing: A substrate specific to the enzyme system under study. A buffer chosen to maintain the ph optimum of the enzyme system. Any cofactors required by the enzyme.  IMMUNOCHEMISTRY It is the application of immunologic techniques to the cellular pathology. It is a powerful technique for the detection of tissue and cellular components, particularly protein molecules. It involves coupling, and subsequently visualizing, a labeled antibody to the component molecule of interest within a tissue section or cell population under study. PRINCIPLE To detect the status and localisation of particular antigen in the cells (membrane, cytoplasm or nucleus) by use of specific antibodies which are then visualised by chromogen. This helps in confirming a specific infection. TECHNIQUES 1. Peroxidase –antiperoxidase (PAP) in which PAP reagent is linked to primary antibody by a bridging antibody. 2. Avidin-biotin conjugate (ABC) immunoenzymatic technique in which biotinylated secondary antibody serves to link the primary antibody to large performed complex of avidin, biotin, peroxidase The coupling site within the tissue is revealed microscopically by tagging the antibody either: 1. With a colored molecule visible in the light microscope. 2. With a molecule of specific shape or electron opacity that can be recognized under the electron microscope. 3. GOLD/PROTEIN A labeling 4. FLUORESCENT labeling APPLICATION 1. Tumors of uncertain histogenesis 2. Prognostic marker in cancer. 3. Prediction of response to therapy. 4. Infections  HYBRIDIZATION TECHNIQUE Working of cell is imp to understand and this requires that technique that permit analysis of molecules involved in process of information flow from DNA to protein. Differrnt types of hybridization: Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid support, generally nylon or nitrocellulose membranes. There are different blotting procedures depending on the type of molecule being transferred. When DNA fragments are transferred the procedure is called a Southern blot. With Northern blotting, RNA molecules are transferred. With Western blotting, protein molecules are transferred. SOUTHERN ANALYSIS –characterizes and quantifies the presence of DNAof a specific gene in the presence of all other genes in a eukaryoticorganisms. NORTHERN ANALYSIS –Identifies and quantifies specific messenger RNA transcripts in the presence of all RNA transcripts expressed with in a single cell type WESTERN ANALYSIS- detects a single protein species from among all other proteins expressed in a single cell or tissue. It is based on high affinity and specificity between antibodies and antigens BASICS OF HYBRIDIZATION PROCEDURE All blotting procedures begin with a standard process called gel electrophoresis. During this step, DNA, RNA, or proteins are loaded on to an agarose or polyacylamide gel (that functions like a molecular sieve) and are then run through an electric field. wo types of gels are commonly used to separate molecules according to size and/or charge: agarose gels are used to separate DNA and RNA, and polyacrylamide gel electrophoresis (PAGE) is used to separate proteins. Shorter molecules move faster and migrate further than longer ones because the shorter molecules migrate more easily through the pores of the gel.  Transfer is initiated when the gel is retrieved from the electrophoresis apparatus and the nylon / nitrocellulose membrane is laid on top of the gel. The objective now is to transfer the bands of molecules found in the gel to the membrane. The molecules are immobilized (fixed) on the membrane. Short fragments of DNA that have a complementary nucleotide sequence to the molecule being analyzed are normally used as probes in Southern and Northern blots. Proteins/antigens that react with the proteins/antibodies being analyzed are used  Conclusion Microscope plays a very important role in viewing organisms and structures which are not visible to naked eye. A basic understanding of working and principles of various types of microscopes enable us to have a complete knowledge of the any subject at molecular level. Moreover other special techniques act as an adjunct. As the science is advancing, one has to keep himself updated with the latest techniques and armamentarium  REFERENCES HISTOLOGY by Ronald A. Bergman, Adel K. Afifi, Paul M. Heidger JR Medical laboratory technology methods and interpretations by Ramnik Sood Textbook of histology and A practical guide , 2nd edition, byJ.P. Gunasegaran Pathology practical book, 2nd edition, by Harsh Mohan. Basic Histology (9th Edition) Lange Shafer’s Oral Pathology (5th Edition) Immunihistochemistry (Eldem Sadikoglou) Molecular Hybridization Technique Of Nucleic Acid: ISSN 1843-6099 Oral Histology (Antonio Nancy 6th Edition) Thank you for listening! Any question? Stay healthy and safe ☺ ZFM