Laboratory Techniques Flashcards
Examples of techniques used in pathology are
• Light microscopy
• Histochemistry
• immunohistochemistry
• Electron microscopy
• Flow cytometry
• Tissue culture
• Molecular pathology techniques
LIGHT MICROSCOPY can be likened to what__________ is to the clinician
clinical examination
2 disadvantages of light microscopy
2 disadvantages:
• Resolution is limited to the wavelength of the light usually about 250nm
• Living tissue are transparent you therefore need to stain the tissues to see cellular details.This means that only dead tissues can be examined exception Janus stain a supravital stain for mitochondria in leucocytes. The are rapidly killed of by the stain.
Light microscopy disadvantages is overcome by
• Dark flied illumination
• Phase contrast microscopy
In histopathology for tissue to the examined they are first fixed and processed either by the___________ technique or by the___________ technique before being stained (Histochemistry) and examined under the light microscope
paraffin section
frozen section
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INTRODUCTION
TO PATHOLOGY. PROF.A.A.F.BANJO,FMCPATH
2
THE DEPARTMENT.
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• Names of Lecturers
• Prof.A.A.F. Banjo, FMCPath
• Prof. F.B.Abdulkareem, FMCPath • Prof.A.O. Daramola, FMCPath
• Prof C.C.Anunobi, FMCPath
• Dr. R. O.Akinde,FMCPath
• Dr. K. B. Badmus, FMCPath
• Dr N.A.Awolola, FMCPath
• Dr M.O. Odubanjo, FMCPath
• Dr O O Dawodu, FMCPath
3
THE DEPARTMENT(2).
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• Laboratory/technical Staff • Mr. Daniel Osuagwu
• Mrs.PhilomenaAwopetu • Mr. Mike Ojo
• Mr. Mike Nwakocha
• Administrative Staff • Mr.Okpara
• Mrs.Adeyemo • Mr. Odeyemi
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LOCATION.
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• White House CMUL • Seminar rooms
• Lecturers Offices
• Departmental Library
• Research and teaching laboratories
• Mortuary Building LUTH.
• Head of department’s office
• Museum
• Autopsy Rooms
• Demonstration Room
• Hospital Laboratories
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INTRODUCTION TO PATHOLOGY.
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• Pathology can be defined as the science that deals with the study of diseases and the body’s response to diseases
• Pathos(suffering),logos(study)
• It is a bridging discipline between basic medicine and clinical practice
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SCOPE OF PATHOLOGY.
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• Clinical pathology
• Experimental pathology
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SUBDIVISIONS OF CLINICAL PATHOLOGY.
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• Histopathology
• Autopsy pathology
• Forensic pathology
• Cytopathology
• Haematology and blood transfusion
• Microbiology
• Immunology
• Chemical pathology • Genetics
• Toxicology
• Molecular Pathology
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WHO IS A PATHOLOGIST?
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• Pathologists often help to determine which test is most effective for a complete diagnosis.
• A pathologist is first and foremost a doctor who undertakes an extra four to five years training in laboratory medicine and may sub specialize.
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ANATOMIC PATHOLOGY.
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• Cellular pathology/ morbid anatomy/ histopathology is devoted to the study of structural and functional changes that underlie diseases.
• It is divided into general pathology which deals with the basic reaction of the cells and tissues to abnormal stimuli that underlie disease and
• Systemic pathology which examines specific response of specialized organs and tissue to more or less well defined stimuli.
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PROCESSES IN PATHOLOGY.
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• There are four disease processes that form the core of pathology
1. Its cause or etiology
2. Pathogenesis or the mechanism of its
development.
3. Morphology or the structural alteration induced in the cells and organs of the body
4. Clinical syndromes or the functional consequences of the morphological changes.
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ETIOLOGY.
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• In ancient times and up till now in some parts of the world causes of disease or illnesses were attributed to the patient. He most have sinned or it was the effect of outside agents such as bad air, bad smells, colds, evil spirits or the Gods.
• Later the diseases where thought to arise from two major etiologic factors
• Intrinsic or genetic
• Acquired(physical,chemical;nutritional,infections)
• It is however now known that the theory of one disease one etiology is not valid
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PATHOGENESIS
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• This deals with the sequence of events in the response of the cell or tissues to the etiologic agent from initial stimulus to the ultimate expression of disease
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MORPHOLOGY.
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• This deals with the structural alterations in the cell or tissues that are either characteristic or diagnostic of etiologic process
• E.g.caseousnecrosis,gangrene
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CLINICAL SIGNIFICANCE
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• This examines how the nature of the morphologic changes and their distribution in different tissues or organs influence their normal function
• Symptoms,signsclinicalcourseandprognosis
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WHO IS A PATHOLOGIST?
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• A pathologist is a laboratory physician who examines tissues and is responsible for the accuracy of laboratory tests
• He interprets the results of these examinations and tests- information that is important for the patient’s diagnosis and recovery.
• Pathologists are problem-solvers.
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WHO IS A PATHOLOGIST?
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• Pathologists make it possible to apply scientific advances to improve the accuracy and efficiency of medical diagnosis and treatment. In general, the earlier a disease is detected and treated, the greater the chance of a cure and the more cost-effective the treatment.
• Pathology plays a particularly important role in preventive medicine by ruling out diseases or detecting them early.
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PATHOLOGIST’S ROLE IN THE
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DISEASE MANAGEMENT
ANALYSIS OF DISEASE
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• Chief complain
• History of present illness
• Past medical and dental history
• Patient examination
• Categorization of Disease(diagnosis) • Treatment plan
• Prognosis
• Treatment
ROLE IN THE DISEASE MANAGEMENT
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• The medical pathologist is involved with the categorization of disease
• He is a graduate of Medicine with specialization in pathology.
• Not involved in chief complaint history or treatment.
• Supervises the facility in which laboratory test are carried out.
PATHOLOGIST’S ROLE IN THE DISEASE
MANAGEMENT(2).
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• Spend time performing autopsies studying tissue removed for biopsy .
• Interpret results of analysis of body fluids, blood andstool forchemistry,hematologyand microbiology.
• Writes findings in reports that state the diagnosis of the disease in question and may include comments to help the referring physician
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ROLE IN THE DISEASE MANAGEMENT,(3)
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• Examines tissues and is responsible for the accuracy of laboratory tests
• Interprets the results of this examinations and test
• Together with the patient’s other doctors consult on which test to order, test results and appropriate treatments
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ROLE IN THE DISEASE MANAGEMENT,(4)
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• Problem solvers fascinated by the process of disease and eager to unlock medical mysteries e.g.. AIDS, diabetes etc.
• Involved in preventive medicine by ruling out disease or detecting them early e.g. cervical cancer, hyperlipidemia
• Interpret results of specialized tests.
RESPONSIBILITIES OF THE PATHOLOGIST
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• Bound by the code of conduct and ethics of the profession
• Has a duty to perform the test accurately, efficiently and to the highest profession standards.
• Are consultant to other doctors
• Advise on appropriate specimen collection bottle, best way to take a specimen
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TECHNIQUES IN PATHOLOGY
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• These are varied and rapidly increasing in number. Some of the commonly used techniques in the examination of tissues are:
• Light microscopy
• Histochemistry
• immunohistochemistry
• Electron microscopy
• Flow cytometry
• Tissue culture
• Molecular pathology techniques but to mention a
few
25
LIGHT MICROSCOPY.
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• This can be liken to what clinical examination is to the clinician. It however has 2 disadvantages
• Resolution is limited to the wavelength of the light usually about 250nm
• Living tissue are transparent you therefore need to stain the tissues to see cellular details.This means that only dead tissues can be examined exception Janus stain a supravital stain for mitochondria in leucocytes. The are rapidly killed of by the stain.
• This is over come by the use of • Dark flied illumination
• Phase contrast microscopy
26
LIGHT MICROSCOPY (2).
17/06/2019
• In histopathology for tissue to the examined they are first fixed and processed either by the paraffin section technique or by the frozen section technique before being stained (Histochemistry) and examined under the light microscope
• Fixation
27
FIXATION(1).
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• Properties.
• Preserves tissue by preventing autolysis by cellular enzymes and
decomposition by the actions of bacteria and molds
• Hardens tissue to allow thin sectioning
• Devitalize or inactivates infectious agents exception CJD infections on glass slides
• Stabilizes tissue components
• Enhances avidity for dyes
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FIXATION (2).
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• Undesirable effects
• Alteration of protein structure • Solubility of tissue components • Shrinkage of tissues
• DNA and RNA degradation
• Factors affecting fixation. • Volume
• Access of fixative to tissues • Time
• Temperature
• Buffer
• pH
29
FIXATION (3).
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• Types of fixatives.
• Aldehydes
• Alcohols
• Mercurial
• Oxidizing agents
• Picrate’s.
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FIXATION(4).
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• Most commonly used fixative is 10% buffered formal saline or Formalin
• Others
• Boiun’s solution for testis small biopsies will also decalcify
• Carnoy’s contains alcohol for rapid processing. Dissolves fat good for identifying lymph node
• B-5 for lymphoid tissues
31 GROSSING OF SAMPLES
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• This is key to getting a good and accurate result.
• Usually done by the pathologists or an Anatomic Pathology
assistant with the requisite training.
• Number of sections taken depends on the type of sample and the clinical indication.
32 TISSUE PROCESSING
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33 PROCESSING OF TISSUE
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• FormalinFixedParaffinEmbedded,(FFPE) • Frozen section
34
PROCESSING OF FFPE TISSUE.
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• Methods
• Can be manual or automated.
• This takes between 18-72 hours depending on method
• There are three basic steps in tissue processing. • Dehydration
• Clearing
• Infiltration
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AUTOMATIC TISSUE PROCESSOR
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TISSUE EMBEDDING
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TISSUE EMBEDDING
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38 MICROTOMY
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FLOATING OF TISSUE
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PARAFFIN SECTION ON GLASS SLIDE
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DRYING OF SLIDES IN THE OVEN
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42 H&E STAINING WORK STATION
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43 WHAT IS H&E?
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• The Haematoxylin and Eosin Stain
• Use
• In histology and histopathology
• Demonstrates a wide range of normal and abnormal tissue components
• Benefits
• Cheap, simple and easy to use
• First step in the diagnosis of disease particularly cancer
44
FROZEN SECTIONS
17/06/2019
• This is used to overcome some of the disadvantages of the paraffin wax technique such as removal of fat, and alteration of antigens .
• It is also used for quick diagnosis for the presence of diagnostic material in tissues and the examination of resection margins
• Sections are cut at –30oC
45
HISTOCHEMISTRY
17/06/2019
• Histochemistry is an important technique that is used for the visualization of biological structures.
• It is concerned with the identification and distribution of various chemical components of tissues through the use of stains, indicators as well as microscopy.
• The identification and distribution of chemical constituents of tissues is achieved through the exploitation of unique chemical environments in cells, heterologous expression techniques as well as enzymatic activities.
46 EXAMPLES OF HISTOCHEMICAL TECHNIQUES
17/06/2019
• Perls’s Reaction for detection of Ferric ions
• Von Kossa technique for calcium
• Sudan black technique for Lipids
• Congo Red for amyloid
• PAS/PASD reaction (Periodic Acid Schiff) for Carbohydrates •
47
IMMUNOHISTOCHEMISTRY.
17/06/2019
• Definition. It is a technique for the identification of cellular or tissue constituents(antigens) by means of antigen –antibody interactions, the site of the antibody binding being identified either by direct labeling of the antibody or by the use of a secondary labeling method
• There are two methods • Direct
• Indirect
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DIRECT METHOD
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• Simplest and shortest.
• The primary antiserum is conjugated directly with a tracer molecule such as the horseradish peroxidase
• Least sensitive method
• Antibody may need to be conjugated if not commercially available and conjugate may be low
• Large aggregates of antibody may cause background staining
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INDIRECT METHOD
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• The primary unconjugated antibody is allowed to bind to the antigen in tissue sections
• A second tracer conjugated antibody raised in another animal host and specific for the animal and immunoglobulin class of the primary antibody and allowed to bind to the primary antibody
• It is more sensitive than direct, rapid and more expensive
• A common conjugate second antibody can be used
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USE OF IMMUNOHISTOCHEMISTRY.
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• Immunohistochemistry is used to accumulate evidence for or against for or against specific diagnostic possibilities
• Most be used in panels, and interpreted based on the immunohistochemistry profile.
• Slides most be pretreated
• Factors affecting immunoreactivity.
• Type and duration of fixation
• Prior decalcification
• Temperature of baking the slide
• Length of time glass was made
• Antigen retrieval procedures
• Type of antibody
• Incubation time. Incubation period, dilution of antibody
• Methods of signal amplification
53
IMMUNOFLUORESCENCE
17/06/2019
• Detects antigens in tissue.
• Because signal is not amplified, it is better than IHC for the precise location of antigen antibody complexes and for determining deposition pattern of immune complexes
• Uses immune complex deposition in glomerular disease and bullous disease of the skin
54
IMMUNOFLUORESCENCE
17/06/2019
• Tissue may be snapped frozen or stored in fixative for IF
• There are 2 methods
• Direct immunofluorescence antibodies to
detect antigen in patients tissue
• Indirect uses control tissues to detect antibodies in patients serum
55
USES OF IMMUNOFLUORESCENCE
17/06/2019
• Skinbiopsiese.g..Lupus.Pemphigus,pemphigoid,dermatitis herpetiformis
• All diagnostic non transplant renal biopsies • Some transplant renal biopsies
• Evaluation of vasculitis in nerve biopsies
56 ELECTRON MICROSCOPY
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• Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological specimens using a beam of accelerated electrons.
• It is used in biomedical research to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes.
• Disadvantage:Electronmicroscopesareexpensivetobuild and maintain.
57
METHOD
17/06/2019
• Ultra structural details of tissues are lost rapidly so fresh tissue must be fixed quickly and well
• Fixative 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4
58
USES OF ELECTRON MICROSCOPY.
17/06/2019
• Diagnosis of child hood small round cells.
• Diagnosticrenalbiopsiesforglomerulardisease
• Difficulttoclassifytumors
• Nerveandmusclebiopsies
• Bullousskindiseases
• Ciliarydysmorphology
• Fineneedleaspirations
• Endomyocardialbiopsies(Adriamycintoxicity,amyloid)
• Liver biopsies for micro vesicular fat in acute fatty liver of pregnancy
• Small bowel biopsies
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MOLECULAR PATHOLOGY.
60 INTRODUCTION
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• Molecular pathology is an emerging discipline
within pathology which is focused in the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids.
61
TECHNIQUES.
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• Southern blotting
• Polymerase chain reaction
• Fluorescent in-situ hybridization
APPLICATIONS OF MOLECULAR PATHOLOGY.
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• Identification of Inherited diseases e.g. cystic fibrosis, hemochromatosis, Factor V leden, prothrombin 20210A, fragile X syndrome;
• Identification of genes conferring susceptibility to diseases e.g. BRCA1
• Identification of human tissues
63
APPLICATIONS OF MOLECULAR PATHOLOGY.
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• Detection of infectious diseases, identification of specific organisms, quantitation of viral infection
• Neoplasms.
• Identification of specific genetic aberrations associated with tumors, • Identification of clonality in haemato-lymphoid proliferations,
• Detection of minimal residual diseases after treatment.
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CYTOGENETICS.
65
METHOD OF SUBMITTING TISSUE
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• The tissue most be fresh, viable and relatively sterile
• If left overnight, mince and cover with culture medium
66
INDICATIONS.
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• Soft tissue tumors
• Mesotheliomas
• Unusual tumors
• Poorly differentiated tumors
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FLOW CYTOMETRY
68
INTRODUCTION.
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• Flow cytometry analysis disaggregated cells as the pass by stationary detectors
• It measures cell size, and cytoplasmic granularity and DNA content
• DNA content can be used to determine number of cells in S-phase
• Because cells are not visualized, it is important to only submit lesional tissues
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INDICATIONS FOR FLOW CYTOMETRY.
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• Indications for ploidy and S phase analysis
• Hydatidiform mole
• Carcinomas for prognosis
• Indications for cell surface markers analysis • Lymphomas
• Leukemias
70 BOOKS IN PATHOLOGY
17/06/2019
• Robins and Contran Pathologic basis of Disease 10th Edition • PathologyIllustratedbyFionaRoberts,ElaineMacDuff,
Robin Callander, and Ian Ramsden 8th edition,
• Underwood’s pathology: a clinician approach 7th Edition
• Rapid Review Pathology: by Edward F. Goljan MD 5th Edition
Properties of fixation
• Preserves tissue by preventing autolysis by cellular enzymes and
decomposition by the actions of bacteria and molds
• Hardens tissue to allow thin sectioning
• Devitalize or inactivates infectious agents exception CJD infections on glass slides
• Stabilizes tissue components
• Enhances avidity for dyes
Undesirable effects of fixation
• Alteration of protein structure
• Solubility of tissue components
• Shrinkage of tissues
• DNA and RNA degradation
Factors affecting fixation
• Volume
• Access of fixative to tissues
• Time
• Temperature
• Buffer
• pH
Types of fixatives
• Aldehydes
• Alcohols
• Mercurial
• Oxidizing agents
• Picrate’s.
• Most commonly used fixative is _______ or ________
• Others include
10% buffered formal saline or Formalin
• Boiun’s solution for testis small biopsies will also decalcify
• Carnoy’s contains alcohol for rapid processing. Dissolves fat good for identifying lymph node
• B-5 for lymphoid tissues
2 ways of processing tissue
Formalin Fixed Paraffin Embedded,(FFPE)
• Frozen section
In processing of FFPE Tissue
• Methods: Can be manual or automated.
• This takes between 18-72 hours depending on method
• There are three basic steps in tissue processing
. • Dehydration
• Clearing
• Infiltration
The basic stain are
Haematoxylin and Eosin Stain
