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Special techniques in histopathology Dr. A.A.F. Banjo, FMCPath Associate Professor / Consultant Department of Morbid Anatomy College of Medicine/Lagos University Teaching Hospital, Idi-araba, Lagos. Introduction. Special techniques are those that are used as an adjunct to routine histopathological techniques. There are a wide variety of special studies available to evaluate pathologic processes from simple histochemical stains to the investigation of gene expression It is important that the pathologist is familiar with the special studies available because the initial handling of the gross specimen can limit the types of studies that can be performed. Routine histopathological techniques A. B. C. D. E. F. G. H. Request for pathologic evaluation Specimen identification Specimen Accessioning Gross Examination Fixation - types of fixatives Tissue Processing Sectioning Staining A. Request for pathologic evaluation The pathologist plays an essential role in patient care as a diagnostician, patient advocate and the clinical teacher. He examines all tissues submitted for histopathological examination. These include All tissues. hair fingernails, toenails removed for cosmetic reasons are not included unless there are specific reasons All products of conception All medical devices implanted in the body and later removed(exclude iv catheters, endotracheal tubes) All foreign objects removed from the body including objects introduced by trauma, and bullets. Submitting specimen. The following information most be provided Identification of the patient Identification of the individual making the request Date of procedure and time if relevant Adequate clinical history Specimen identification,test required and any special handling required Timely and appropriate transport to the laboratory Instructions for disposal of the gross specimen. Adequate clinical history. Purpose of removal of the specimen and the type of specimen. Gross appearance and locations of any lesion present Prior diagnosis Prior or current treatment. Specific purpose of consultations Rush specimens Fixation(1). 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 Fixation (2). 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 Fixation (3). Types of fixatives. Aldehydes Alcohols Mercurials Oxidizing agents Picrates. Most commonly used fixative is 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 tisues Tissue processing. The tissue undergoes automatic processing through three basic steps. Dehydration Clearing Infiltration Problems with submitted tissue. Fatty tissues Tissue too thick Calcified tissues Hair Hard foreign material Multiple small tissue fragments Special techniques Histochemistry Enzyme histochemistry Immunohistochemistry Flow cytometry Cytogenetics Molecular biology Specimen radiography Histochemistry. Histochemistry. Almost all histochemical stains are suitable for formalin fixed tissues The commonly used ones are those for the detection of carbohydrates, mucin connective tissue, amyloid,lipids cells of the neuro-endocrine system, microorganisms, and neuro-pathological techniques Stains for connective tissue. This is the name applied to the tissues that connect and support the other tissues in the body. It consist of a cellular portion in an enveloping framework of non cellular substance The ratio of cells to intercellular substance varies from one type of connective tissue to other The nature of the intercellular substance varies according to function and it is classified into two main groups. Formed or fibrous type collagen, reticulin, elastic Amorphous or gel types glucosaminoglycans Structural glycoproteins Connective tissue Cells Fibroblast Fat cells Types of connective tissue Areolar Adipose Myxoid connective tissue Dense connective tissue Cartilage Bone Muscle Connective tissue stains Trichrome stain collective name for demonstration of muscle, collagen fibers, fibrin and erythrocytes. Three stains are employed one of which is a nuclear stain Factors affecting trichrome stain. Tissue permeability and dye molecular size Heat pH 1.5-3.0 Effects of fixation. Sub optimal results with prolonged fixation in formaldehyde. treat with picric acid or mercuric chloride to enhance staining Preferred fixative for trichrome Connective tissues stains Trichrome stain Van Gieson technique buffered formalin satisfactory Masson’s trichrome Demonstration of fibrin. Masson’s trichrome stain older deposits of fibrin like collagen. Martius Scarlet Blue technique differentiates between old and new fibrin. Early red,old yellow older blue Elastic tissue verhoeff’s method stains fine fibers less satisfactory Orcein simple Reticulin stain Reticulin two types Those using dyes Impregnation techniques Gordon and sweet, Gomori Hematologica;l stains Biologic Amines Cells that produce polypeptide hormones, active amines, or amine precursors (epinephrine, norepinephrine) can be found individually (Kulchitsky cell of GI tract) or as a group (adrenal medulla). Traditional classification of the staining patterns based upon the ability of the cells to reduce ammoniacal silver nitrate to metallic silver (black deposit in tissue section): Chromaffin Argentaffin Argyrophil (pre-reduction step necessary) The distinction bw chromaffin and argentaffin is artificial. Depends upon the fixative used. "Chromaffin" cells have cytoplasmic granules that appear brown when fixed with a dichromate solution. "Argentaffin" cells reduce a silver solution to metallic silver after formalin fixation. Either reaction can be produced depending upon which fixative was used. Chromaffin reaction - adrenal medulla or extraadrenal paraganglion tissues (pheochromocytomas) Argentaffin reaction-carcinoid tumors of the gut. pre-reduction step -called "argyrophil" Types of stains for argentaffin include: Types of stains for chromaffin include: Diazo (diazonium salts) Fontana-Masson Schmorl's Autofluorescence Modified Giemsa Schmorl's Wiesel's Types of stains for argyrophil include: Grimelius (Bouin's fixative preferred) Pascual's Amyloid. Congo red Polarising microscope Methyl voilet Immunofluorescence using thioflavine for cerebral plaques Amyloid Pretreatment with KMNO4 used to differentiate between AL and AA amyloid Carbohydrates(1) There are a variety of mucin stains trying to demonstrate one or more types of mucin Neutral mucins GI , prostate PAS, not with alcian blue collidal iron, mucicarmine or metachromatic dyes Acid simple or non sulphated mucin stain with PAS, alcian blue at pH 2.5. Colloidal iron and metachromatic dyes. Resist hyaluronidase digestion Acid simple mesenchymal mucin contain hyaluronic acid found in tissue stroma. Don't stain with PAS. Stains with alcian blue at pH 2.5,colloidal iron and metachromatic dyes digest with hyaluronidase carbohydrate Acid complex or sulpahated adenocarcinomas PAS + alcian blue at pH 1, colloidal iron positive, mucicarcimine, metachromatic stains resist digestion with hyaluronidase. Acid complex or connective tissue found in tissue stroma, cartilage, bone . Include chondroitin sulphate and keratin sulfate. PAS + alcian blue at pH 0.5 Mucin stains Most specific is murcicamine Most sensitive is PAS Alcian blue simple but has a lot of background staining . Colloidal iron unpredictable Pigments Artefact pigments Formalin Malarial Mercury dichromate Endogenous pigments Exogenous pigments Artefact pigments Formalin formed in formalin with acid pH. Occurs in blood rich tissues Microcrystalline structure birefregrent in polarized light Remove from unstained sections by treatment with picric acid Best to prevent by using buffered formalin fixative Malarial pigments. Similar to formalin Remove with treatment with saturated alcoholic picric acid for longer periods. Endogenous pigments Hemoglobin Bile pigments The lipofuscins Melanin and pseudo-melanin Chromaffin, argentaffin and argyrophyll Hemosiderin and iron Calcium Cupper Uric acid and urates. Hemoglobin. Appears as vivid orange- red in H&E Leuco-Patent blue V method for hemoglobulin Unreliable in tissues fixed for more than 36 hrs Hemoglobin dark blue Oxidase dark blue Nuclei acid red Use Based on the demonstration of peroxidase activity identification of cast in hemoglobinuria, GN Simple method Dunn-Thompson picric acid and acid fuschin technique neutral needs buffered Bilirubin,biliverdin and haemotoidin Fouchet –van Gieson technique Bile pigment blue green Muscle yellow Collagen red Use controls Treated with fouchet not counterstained with van gieson Gremelin is the only technique that will demonstrate both liver bile, gall bladder bile and hematoidin Lipofusin Early lipofusin Staining characteristics of lipids Sudan black lipofuschin black Ziehl –Neelson magenta Late lipofuschin Schmorl reaction melanin,liver bile, hematoidin Varying shades of blue melanin Masson’s fontana Confirm using Mallory bleach extraction method Iron Perls stain or prussian blue rection Always use a postive control Mercurial fixatives best for bone marrow Urates Present as sodium urates in tissues Tissue most be fixed iin 95% or absolute alcohol to prevent leaching of urates Methamine silver stains urates black Polarisable yellow with red plate, blue when axis is aligned at 90o to wave Cupper Stains: Rubeanic acid and Rhodamine Used to diagnose Wilson's disease which is a rare disease transmitted as an autosomal recessive disorder arising from def of caeruloplasmin calcium Only calcium that is bound to an anion can be measured. Alizarin red S method orange red at pH4.2 Useful to detect small amounts of calcium in Michaelis-Gutman bodies Can be used to measure serum calcium photomerically Von kossa demonstrates phosphates and carbonates present with calcium Exogenous pigments Asbestos ferruginous bodies Silica polarized light Carbon anthracotic pigments in lungs dd melanin Lead -Rhodizonate method lead salts red background green Beryllium solochrome auzine method 2 methods to differentiate between Al and Be Aluminuim and beryllium black Beryllium blue black Silver -Rhodamine method Fat stains Oil red O to identify neutral lipids and fatty acids Use fresh or formalin fixed sections cut on cryostat Micro-organism Bacteria H&E blue Fungi Gram negatives don't stain well with gram Brown and Brenn modification usually used Blue with H&E. red with PAS most sensitive is the Gomori methenamine silver stain or Gridley stain Spirochetes Warthin-starry silver stain AFB Zeihl Neelsen stain, Fite farraco for m.leprae most sensitive is auramine rhodamine stain AFB Fat removed by processing. Auramine rhodamine most sensitive GMS for pneumocystis carinii Lots of background artefacts. PAS for candida PAS around purpose stain Immunohistochemistry. Immunohistochemistry. 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 Direct method 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 Indirect method The primary uncongugated 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 expensive A common conjugate second antibody can be used Use of immunohistochemistry. 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 immunohistochemi9srty 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 retival procedures Type of antibody Controls. Positive and negative controls Negative controls Evaluation. Local of immunoreactivity Identification of immunoreactive cells Intensity of immunoreactivity Number of immunoreactive cells Common panels Toxoplasma The future-genomic immunohistochemistry To date, immunohistochemistry has largely focused on markers of cell type, as an aid to the diagnosis of specific tumours. More recently, it has been applied to a limited number of markers of cell proliferation, such as Ki-67 and PCNA, The future-genogenic immunohistochemistry To date, immunohistochemistry has largely focused on markers of cell type, as an aid to the diagnosis of specific tumours. More recently, it has been applied to a limited number of markers of cell proliferation, such as Ki-67 and PCNA, as prognostic factors in malignant tumours. Genomic immunohistochemistry Documentation of loss of expression of a protein due to specific mutations, usually truncation mutations. Loss of expression of E-cadherin in lobular breast cancer. Hereditary non-polyposis colon cancer arises because of mutations in genes for DNA repair, principally hMSH2 and hMLH1. Identification of proteins expressed as a consequence of specific translocations Expression of FLI-1 differentiates PNET/EWS from other small blue round cell tumours Genomic imunohistochmistry Identification of molecular targets for novel tumour therapies Expression of c-kit (CD117) is highly characteristic of gastrointestinal stromal tumours. The drug STI571 blocks the kinase pocket of c-kit and is highly effective in the treatment of GISTs. Identification of gene amplification and tumour therapy Identification of overexpression of the HER-2/neu protein to validate the use of trastuzumab (HerceptinTM) in the treatment of breast cancer patients. The HER-2/neu gene is overexpressed due to gene amplification Electron microscopy The only way to improve resolving power is to reduce substantially the wavelength of the light. This is achieved by the electromagnetic beam of the electron microscope. The beam is focused through the object suspended on its metal grid, and is magnified before striking a fluorescent screen to be transformed into a visible image The resolutions so far achieved in biology with transmission electron microscopy (TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. Electron microscopy. Uses of electron microscopy. Diagnosis of child hood small round cells. Diagnostic renal biopsies for glomerular disease Difficult to classify tumours Nerve and muscle biopsies Bullous skin diseases Ciliary dysmorphology Fine needle aspirations Endomyocardial biopsies(adriamycin toxicity, amyloid) Liver biopsies for microvesicular fat in acute fatty liver of pregnancy Method Ultra structural details of tissues are lost rapidly so fresh tissue must be fixed quickly and well Fixative 2% paraformaldehyde and 2.5% glutalraldehde in 0.1M cacodylate buffer at pH 7.4 Minimal change Disease Effacement of foot processes Basement membrane Red blood cells Immunofluorescence immunofluorescence Detects antigens in tissue. Because signal is not amplified, it is better than IM 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 Tissue may be snapped frozen or stored in fixative for IF Immunofluorescence. There are 2 methods Direct immunoflourescene antibodies to detect antigen in patients tissue Indirect uses control tissues to detect antibodies in patients serum Uses of immunoflorescence Skin biopsies e.g.. Lupus. Pemphigus, pemphigoid, dermatitis herpetiformis All diagnostic non transplant renal biopsies Some transplant renal biopsies Evaluation of vasculitis in nerve biopsies immunofluorescence The localization at the dermal-epidermal junction is typical, for immune complexes tend to be trapped along basement membranes. Diseases with this pattern may include systemic lupus erythematosus, discoid lupus erythematosus, and bullous pemphigoid. Tissue arrays Tissue microarrays are a method of re-locating tissue from conventional histologic paraffin blocks such that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic sections and placing the core into an array on a recipient paraffin block. This technology should not be confused with DNA microarrays where each tiny spot represents a unique cloned cDNA or oligonucleotide. In tissue microarrays, the spots are larger and contain small histologic sections from unique tissues or tumors. Advantages of tissue arrays amplification of a scarce resource, experimental uniformity decreased assay volume. does not destroy original block for diagnosis Technique Robotic technology is employed in the preparation of most arrays. The DNA sequences are bound to a surface such as a nylon membrane or glass slide at precisely defined locations on a grid. Using an alternate method, some arrays are produced using laser lithographic processes and are referred to as biochips or gene chips. Molecular genetic pathology. Applications of Molecular genetic pathology. 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 Detection of infectious diseases, identification of specific organisms, quantitation of viral infection Neoplasms. Identification of specific genetic aberrations associated with tumours, identification of clonality in hemato-lymphoid proliferations, detection of minimal residual diseases after treatment. Techniques. Southern blotting Polymerase chain reaction Fluorescent in-situ hybridization The FISH technique is shown with interphase nuclei in three panels. A specific DNA probe with a fluorescent tag identifies a specific region of a chromosome. Different colored fluorescent tags for probes allow identification of various abnormalities. DNA micro arrays-introduction Alterations in gene expression patterns or in a DNA sequence can have profound effects on biological functions. These variations in gene expression are at the core of altered physiologic and pathologic processes. DNA array technologies provide rapid and costeffective methods of identifying gene expression and genetic variations. DNA array-technique DNA microarrays typically consist of thousands of immobilized DNA sequences present on a miniaturized surface the size of a business card or less. Arrays are used to analyze a sample for the presence of gene variations or mutations (genotyping), or for patterns of gene expression, performing the equivalent of ca. 5,000 to 10,000 individual "test tube" experiments in approximately two days of time. Microarrays are distinguished from macroarrays in that the DNA spot size is smaller, allowing the presence of thousands of DNA sequences instead of the hundreds present on macroarrays. Types of DNA arrays The composition of DNA on the arrays is of two general types: Oligonucleotides or DNA fragments (approximately 2025 nucleotide bases). These arrays are frequently used in genotyping experiments. The sequences of alternate gene forms may be included for detection of mutations or normal variants (polymorphisms). Complete or partial cDNA (approximately 500-5,000 nucleotide bases). These arrays are generally used for relative gene expression analysis of two or more samples. Applications of DNA micro arrays. Diagnostics Custom Drug Selection Discovery of Therapeutic Targets Predict drug/toxin activity Accelerate FDA approval Determination of Pharmacologic Mechanism Future directions Custom designed DNA variation detection arrays will be used to scan the genome and detect single nucleotide polymorphisms (SNPs). The SNPs that are identified can be used in designing further genotyping chips for performing association and linkage analysis. DNA arrays will assist in the formation of information databases to assist in correlating genetic variation and gene expression with patient status, prognosis and responsiveness to treatment. DNA array analysis provides an excellent tool to enlarge our knowledge of genome function Cytogenetics. Indications. Soft tissue tumours Mesotheliomas Unusual tumours Poorly differentiated tumours Method of submitting tissue The tissue most be fresh, viable and relatively sterile If left overnight, mince and cover with culture medium Philadelphia chromosome "Philadelphia chromosome" of chronic myelogenous leukaemia, which is really a 9:22 translocation. Flow cytometry Introduction. Flow cytometry analysis disaggregated cells as they 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 Indications for flow cytometry. Indications for ploidy and S phase analysis Hydatidiform mole Carcinomas for prognosis Indications for cell surface markers analysis Lymphomas leukemias Tissue processing Submit single cell suspension Tissue most be viable Formalin fixed paraffin embedded tissues can also be used for DNA ploidy analysis but results are not satisfactory Cytologic preparations from surgical specimens. For intra operative diagnosis Infectious disease Neuropathology cases Tumours For special studies Advantage. Nuclear not cut therefore intact FISH and image analysis Specimen radiography. Preferable to patient radiograph A permanent record of the radiograph can be kept with the a case Reveals greater detail of the lesion There might be a significant time interval between removal of tissue and patient radiograph Indicates important sites to sample To confirm that clinical lesion was removed Indications for specimen radiography Tumors of bone and cartilage Tumours invading in to bone Avascular necrosis of bone All prosthetic heart valves to document the degree of calcification Breast biopsies or mastectomies performed for mammographic lesions that can not be located grossly Microbiologic cultures and smears Indications suspected infectious processes Suspected sarcoidosis as to exclude an infectious process Types of culture Routine Bacteria(aerobic), mycobacteria, fungi Special Anaerobic, salmonella,norcardia, N. gonorrhea.legionella, helicobacter Viral CMV,varicella-zooster,adenovirus herpes Influenza a B , RSV,para influenza require special techniques