pathology : uses , methods and techniques

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Importance of the study of pathology :

Importance of the study of pathology

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The medical model of human pathology is the study of diseases or abnormalities of the body. It is also the study of the change in tissues and organs over time which results in disease. “Pathos “ means suffering and “logos” means study

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Pathophysiology is the other word in consideration which is compromised of “pathos” meaning suffering and “physiology” meaning study of normal function and includes the study of disordered function or breakdown of homeostasis in diseases.

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Pathology is the precise study and diagnosis of disease. Pathology addresses 4 components of disease: cause/etiology, mechanisms of development (pathogenesis), structural alterations of cells (morphologic changes), and the consequences of changes (clinical manifestations)

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Pathology is a clinical knowledge service that is fundamental to modern medical practice and health care. Pathology is used in the diagnosis, treatment and management of an increasing range of clinical conditions. Pathology investigations are an integral part of the clinical consultation and procedural process with all health care decisions affecting diagnosis or treatment involve a pathology investigation 1 . Pathology is essential to the prevention, early detection, diagnosis and treatment of many of the leading causes of disease burden in– e.g. cancer, cardiovascular disease and diabetes.

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Helps detect diseases based on gross, microscopic, chemical, immunologic and molecular examination of organs, tissues, and whole bodies (autopsy) or based on laboratory analysis of bodily fluids such as blood and urine, and tissues using the tools of chemistry, microbiology, hematology and molecular pathology.

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Pathologists may also conduct autopsies to investigate causes of death. Pathologists examine tissue biopsies to determine if they are benign or cancerous. Some pathologists specialize in genetic testing that can, for example, determine the most appropriate treatment for particular types of cancer. In addition, a pathologist analyzes blood samples from a patient's annual physical and alerts their primary care physician to any changes in their health early, when successful treatment is most likely. Pathologists also review results of tests ordered or performed by specialists, such as blood tests ordered by a cardiologist, a biopsy of a skin lesion removed by a dermatologist, or a Pap test performed by a gynecologist, to detect abnormalities. Pathologists play a critical role on the patient care team, working with other doctors to treat patients and guide care.

Terms in Pathology :

Terms in Pathology Hematology : Deals on with diseases of Blood Chemical Pathology : Deals with analysis of Biochemical Constituents of Blood, urine, semen, CSF and other body fluids. Immunology: Detection of abnormalities of immune system. Clinical pathology is a medical specialty that is concerned with the diagnosis of disease based on the laboratory analysis of bodily fluids such as blood and urine, and tissues using the tools of chemistry, microbiology, hematology and molecular pathology.

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Dermatopathology is a subspecialty of anatomic pathology that focuses on the skin as an organ. Anatomical pathology or anatomic pathology is a medical specialty that is concerned with the diagnosis of disease based on the gross, microscopic, chemical, immunologic and molecular examination of organs, tissues, and whole bodies (autopsy). Same as histopatholo gy

Methods:

Methods 1 . Molecular Examination of Tissues : This is called histopathology and the study can be done in various ways as by studying the tissues removed from living body during surgery (surgical pathology ) , removed after death (forensic or autopsy pathology) done from extraction or in situ. It can also involves the study of cells either of the patients in the tissue or by the ones shed off from lesions ( cytopathology ). Cancers, causes of death , Ulcerations etc can be detected.

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2. Molecular Examination of Body fluids: The blood ,Urine, semen , vaginal fluid , serum all can provide data upon examination ( clinical pathology). Anemias , Thalasemias , Veneral Diseases can be found out. 3. Examination of Enzymes : This can yield the information about diseases and requires fresh tissues an is useful in diagnosing diseases e.g. ATPase in Myopathies , Cardiac Enzymes in Cardiac Arrest etc. 4 . Examination of Genetic Material like DNA and RNA is helpful in the detect of Down Syndrome , Turners Syndrome and other Genetic Diseases. (molecular pathology) 5. Examination of the Immune system (immunology) to detect the antigen – antitbody reaction and hence find out the related diseases.

Techniques:

Techniques The various techniques in the pathology are: Cellular storage and Histopathological Studies Autopsy Staining, Reagents and Solutions ( Histochemistry / hostological biochemistry) Blood Banking and testing Culture Media Microscopy Cryogenetics Immunofluorescence Flow Cytometry Morphometry Lasers DNA Microarrays Molecular Pathology Other new techniques

Cellular storage and Histopathological Studies :

Cellular storage and Histopathological Studies The tissues are taken out from living body. The tissues usually are removed by means of surgery – either major or minor . Histopathological studies involves techniques of Biopsy ,Paraffin Embedding and frozen section for rapid diagnosis. Biopsy of the tissue specimen so obtained is done .The process involves tissue cutting, gross description and selection of tissue for pathologic examination. Nowadays , the gross description is written with the help of dictaphone without aid of assistant to write it.

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Gross specimen photography/radiography may be used. For bones and calcified tissues, the decalcification is done to soften the tissue and observe it. The tissue goes onto automated tissue processors with fixation and paraffin impregnation or into plastic, metallic containers .Cryostat is used instead. All this makes the tissue ready for staining with Haematoxylin and Eosin to give the report and macroscopic and microscopic findings.

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Autopsy: The autopsy enlightens about the pathogenesis of diseases , reveals hazardous effects of therapy administered and settles the differences between antemortem and postmortem diagnosis. Block extraction of the abdominal and thoracic organs or in situ organ – by-organ dissection . This confirms the disease, cause of death , establish the final diagnosis, diagnosis of missed diseases and discovery of new ones.

Staining, Reagents and Solutions :

Staining, Reagents and Solutions The stains / dyes are used when there is need to demonstrate the specific substances or constituents of cells to confirm etiologic, histogenic , or pathogenetic substances. The staining depends on the solubility of stains with the tissues. It help in detection of the certain abnormalities in the tissue sample shown by discoloration, excess coloration and viewed under microscopes . Reagents are also of importance when it comes to microscopic examination of tissues.

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Some reagents are designed to preserve tissue by a process called fixation.Some others are use for toning and others for clearing the tissue or to take traces of mercury from already fixed tissue. Fixatives are histology reagents use to preserve tissue from degradation and to maintain the cell structure. One of the most popular fixative in histology is 10% neutral formalin for light microscopes. There are many others that have increased in popularity like: Davidson's for the fixation of the eyes. Bouin's Fluid is another well known fixative use for histology. Carnoy's fluids now can be used without chloroform, avoiding the health hazards of chloroform. Acetic Acid, 0.5% up to 10% is used for clearing or differentiation of the tissue after staining; also, Acid Alcohol is used for Clearing. Gold Chloride, 0.1% up to 5% is use for toning. Other reagents can be use to promote a reaction like: an oxidation reaction in those categories which have Potassium Permanganate 0.25% up to 3%. Periodic Acid 0.5% up to 5% reacts with Schiff's reagent to oxidize formaldehyde producing a magenta color for the demonstration of glycogen.

Blood Banking and testing :

Blood Banking and testing Blood banking, the process of collecting (donation), testing, processing, and storing blood for later use (transfusion), is a cornerstone of emergency and surgical medicine and is dependent on the clinical laboratory for ensuring the safe use of blood and its components. Involves Donating Blood, Protecting Supply, ensuring its proper use, and the Risks involved for donors as well as recipients.

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A number of laboratory tests must be completed before blood or blood products can be transfused: Determination of the blood type with a crossmatch . Screening for antibodies that may produce adverse effects if transfused. Screening for possible infectious agents that could be transmitted with transfusion. The following tests are manadatory on all units of blood collected for transfusion: ABO group and Rh type Screening for blood-group antibodies Serologic test for syphilis Serologic tests for human retroviruses /HIV Test for Sickle cell trait (for children) Serologic tests for hepatitis antigen , antibody and virus Test for antibody to cytomegalovirus (BT to Cancer patients) Testing

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Short-term storage of Blood Routine blood storage is 42 days or 6 weeks for stored packed red blood cells or pRBC (the most common blood product), and involves refrigeration but usually not freezing Long-term storage Long-term storage is relatively uncommon, compared to short-term storage. Cryopreservation of red blood cells is done to store rare units for up to 10 years. The cells are incubated in a glycerol solution which acts as a cryoprotectant ("antifreeze") within the cells. The units are then placed in special sterile containers in a freezer at very cold temperatures. The exact temperature depends on the glycerol concentration. Storage

Culture Media :

Culture Media A substance, either solid or liquid, used for the cultivation, isolation, identification, or storage of microorganisms. There are three main forms of media : liquid, semisolid, and solid. Liquids are usually some kind of broth contained in tubes, and the bacteria will grow suspended in this liquid. Media that is of semisolid or solid consistency usually contains a thickening agent called agar. Semisolid media is of a soft jelly-like consistency, is also usually in tubes, and the bacteria will also grow within it. Solid media is the consistency of a very firm gelatin type desert, but the bacteria will tend to grow only where it is inoculated on the media surface. Solid media that is provided in tubes often has a slanted surface and are referred to as “slants”. Solid media that is provided in Petri-style dishes are referred to as “plates”.

Microscopy :

Microscopy Microscope is the basic tool of the pathologist. It is an instrument which produces greatly enlarged images of minute objects. The usual type of microscope used is the light microscope which is further of simple and compound types. The simple microscope has simple hand magnifying lens of 2x and 200x . The compound microscope had battery of lenses which is fitted in a complex instrument . They can have simple eye piece (monocular) or two (binocular ) which has two eye pieces. Multi- headed microscopes are used as aid to teaching and for demo.

Variants of Light Microscope:

Variants of Light Microscope Dark Ground Illumination (DGI): Involves examination of unstained living organisms e.g. Treponema Pallidum . The Microorganisms are illuminated by an oblique ray of light which doesn't pass through the microbes. The condenser is blackened in the centre and light passes through its periphery illuminating the living microbes on a glass slide. Polarizing Microscope : This method is used for demonstration of Birefrigence ( ) . The light is made plane polarized. After passing through a disc , the rays of light vibrate in a single plane at right angle to each other. Two discs made up of prism are placed in the path of light , one below the object known as polarizer and another placed in the body tube is known as analyzer . The lower disc is rotated to make the light plane polarized.

Cryogenetics :

Cryogenetics Cryo -diagnostic techniques can be employed for chromosomal studies including leucocytes and tissue cultures for demonstration of sex- chormatin . Karyotyping is a procedure which is associated with the sequence of chromosomal alignment on the basis of size, centrometric location and banding pattern. The determination of karyotype of an individual is important tool and it involves:

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Cell Selection : Cells capable of growth and division are selected for cytogenetic analysis . E.g . From amniotic fluid, bone marrow. Cell culture is done in mitogen media e.g. PPD, pokeweed mitogen (PWM) – a substance which induces the mitosis process and dividing cells are then arrested in metaphase by the addition of colchicine , or colcemid which inhibit microtubule formation . This is then lysed by adding hypotonic solution. The metaphase cells are then fixed in methanol glacial acetic acid mixture. Staining is done to give bands various colors like Giemsa Banding , Quinacrine Banding for bands along chromosomes, Consecutive banding for consecutive heterochromatin, and reverse staining gives pattern opposite to those obtained by G- Banding. Microscopic Analysis : Chromosomes are then photographed by examining the preparations under microscope.

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Karyotyping By Giemsa Stain Spectral karyogram of a human female

Uses:

Uses Diagnosing the Chromosomal Numerical Abnormalities. E.g. Downs Syndrome, Klinefelters Syndrome. Chromosomal Structural Abnormalities. e.g. Philadelphia Chromosome, Deletion , Insertion. Cancers are characterized by multiple and complex chromosomal abnormalities including deletions, amplifications, inversions, translocations, especially in Leukemias , Lymphomas and Germ Cell tumors.

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Translocation Duplication Deletion

Immunofluorescence :

Immunofluorescence It is done to localize the antigenic molecules on the cells by microscopic examination. This is done by using specific antibody against antigenic molecule forming antigen- anitbody complex at the specific antigenic site which is made visible by employing a fluorochrome which has the property to absorb the radiation in the form of Ultraviolet light so s to be within the visible spectrum of light when examining.

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Fluorescence microscope is used based on the principle that the existing radiation from Ultraviolet light of shorter wavelength (360 nm) or blue light (400 nm) causes fluorescence of certain substances and thereafter reemits light of shorter wavelength. The substances that fluorescence naturally is primary fluorescence (auto-fluorescence) and the ones that have added dyes or chemicals called fluorochromes lead to secondary fluorescence. Mercury vapors and xenon gas lamps are used as source of light for fluorescence microscopy. A number of filters exist like heat absorbing, red light stopper, another to allow light of desired wavelength for excitation and collection of fluorescence wavelength with barrier filter to remove excess exciter light to protect the eyes. A dark-ground condenser is used so that no direct light falls into the object and also gives the dark contrast to the fluorescence.

Uses:

Uses For detection of auto-antibodies in serum e.g. smooth muscle antibodies( SMA), antinuclear antibodies (ANA) In renal diseases , to detect deposits of immunoglobulin, complement and fibrin in various types of glomerular diseases by frozen section. In skin diseases , the detection of deposits of Immunoglobulins by frozen section , esp. in dermato -epidermal junction and in upper dermis . E.g. in various bullous dermatosis . To study mononuclear cell surface using monoclonal antibodies and infective diseases . E.g. Viral Hepatitis.

Flow Cytometry :

Flow Cytometry Flow cytometry uses the principles of light scattering , light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5um to 40um diameter. Lasers are most often used as a light source. As your cells or particles of interest intercept the  light source they scatter light and fluorochromes are excited to a higher energy state. This energy is released as a photon of light with specific spectral properties unique to different fluorochromes. One unique feature of flow cytometry is that it measures fluorescence per cell or particle.

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Used for : Immuophenotyping : by detailed antigenic analysis of various hematopoietic neoplasm. Measurement of Proliferation – associated antigens e.g. Ki67 associated with ribosomal RNA transcription and hence, it inactivation can arrest cell proliferation. Measuring the nucleic acid content :measuring RNA content of reticulocytes. Diagnosis of Immunodeficiency e.g. CD4 count In case of AIDS. Diagnosis of auto-antibodies in ITP, autoimmune neutropenia .

Morphometry :

Morphometry The image analyzer is used to perform the measurement of architectural , cellular and nuclear features of the cells. The standard light microscope, computer system and image capture- analyser are the important part to take a digital image and do calculations based on the software installed. It does qualitative and quantitative analysis

Lasers :

Lasers New technique for carrying out the diagnostic in surgical pathology at molecular level involving dissection of single cell or part ( chromosomes) by laser and software usage. The isolated material can then be used for performing various tests.

DNA Microarrays :

DNA Microarrays New application of silicon chip technology to analyze large data involving human genes and hence, detection mutations, variations in size and shape of cells ( pleomorophism ) Fluorescent labeling is done of DNA segment and is hybridized with target sample. The scans for fluorescent signals emmitted and using bioinformatics for to see level of gene expression and genotyping (finding differences in genes)

Molecular Pathology :

Molecular Pathology Molecular techniques employ hybridization to see for the detection of abnormalities , diseases and with the use of probe to view it. E.g Genomic probes for region of DNA cells, Riboprobe for In vitro transcription. Insitu hybridization is done for localizing nucleic acid sequence in the intact cell. The Nucleic acid is may or may not be radioisotope marked and can be hence , viewed with probes : Radioactive (I125) or Non radioactive (Biotin) . Finds infections, tumors , chromosomal disorders (FISH – Fluorescent Insitu Hy .)

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Filter hYb . method involves extracting the target DNA / RNA onto immobilized on nylon or nitrocellulose filter. Involves various methods of analysis to generate data which is highly specific and sensitive for diagnosis of cancers, infections , genetic diseases and identity determination for transplantation , forensics. Polymerase chain reaction involves generating DNA STRANDS FROM ANOTHER. Involves heat denaturation of DNA at 94 degree for 60-90 seconds, annealing (altering) of primers to complementary sequences (55 degree for 30-120 sec) , extension (72 degree for 60-180 sec) . This involves repeated cycling and hence, large accumulation of target DNA for Next cycle. For diagnosis and research.

New methods:

New methods Mitotic Count Radioautography : Labelling and processing with thymidine in vitro for S-phase Microspectrometric : Feulgen staining and DNA content is seen. Immunohistochemistry Nucleolus Organizer region (NOR) seen in the chromosomal regions containing DNA and have affinity for silver , appearing black dots in stained yellow – brown background.

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