2019 Histo 3 Special Stains to Pathology PDF

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© Built for Learning © Built for Learning Perform Histological Tests Histological Stain ‐ Quality Control Tissue © Leah Simmons - Built For Learning 1 2 © Built for Learning © Built for Learning Common Histochemical Stains Introduction to Histochemical Staining. Periodic Acid Shiff Ziehl Neelsen Oil Red O Gomoris reticulin Toludine Blue Grocott Methenanine Silver PAS w/Diastase Digestion Verhoeff’s Van Gieson PAS/Alcian Blue Schmorls Histochemistry: refers to the science of using chemical reactions between laboratory chemicals, often dyes and various components within tissue. Colloidal iron Masson Fontana It is primarily used to aid in the diagnosis of disease through the demonstration Perls Prussian Blue Congo Red of specific morphological features within tissue. Cell structures will stain based on the presence of specific chemical substances such as carbohydrates. Gram Twort Orcein 3 4 © Built for Learning © Built for Learning Quality Control in Staining 1. Cross check the test request sheets. Make sure the correct stain is performed by cross checking the test sheet. Like in any Laboratory Quality Control plays a major role in ensuring that the patient gets It is common to have multiple special stain requested on the one sample. the correct result and is treated accordingly. For histology laboratories to maintain their Some pathologists will even request different counter stains or accreditation with the The National Association of Testing Authorities (NATA) they must background stains to enhance what they are looking for. guarantee the accuracy of their results. http://www.nata.asn.au/ When it comes to staining there are several ways that we can be sure that this happens. 1. Cross check the test request sheet. 2. Label the slide before dewaxing. AB/H AB/PAS AB/AY 3. Always run control slides with the patients slides. International standards for histology 4. Participate in internal proficiency testing. laboratories include: 2. Label Slides before dewaxing 14332/13A PITCHFORK 5. Participate in external proficiency. ISO 15189 ‐ Medical Testing Label the slide correctly before dewaxing. AB/PAS ISO/IEC 17025 ‐ Laboratory Testing 12/7/13 It is common to dewax all of the slides together. ISO 9001 – Research They could by for up slides for up to 15 different stains in the one rack. GLP – Good Laboratory Practice 5 6 © Built for Learning © Built for Learning 3. Use control slides 3. Use control slides Always run a control slide with the slides you are testing, so you know that 2. Both positive and negative controls the stain has worked correctly. ‒ You will stain two control slides, and two patients slides There are two main types of control slides used. ‒ The controls are identical and have previously tested positive. 1. Known positive control slide ‒ One control and sample slide is put through the complete procedure, some steps prevent the stain from binding to the tissue and will be negative. ‒ You will stain two slides ‒ The other control and sample slides skip theses steps and will be positive. ‒ One has usually previously tested positive using more than one method of diagnosis or has normal pathophysiology. Eg. When staining for glycogen using PAS you need to use both positive and negative controls. The negative slide is treated with amylase to break down the glycogen, the Eg. Colloidal iron stain is used to demonstrate acid positive control skips this step. Stain name: PAS with Diastase digestion mucins often found in connective tissue as well as goblet cells throughout the gastrointestinal tract (GIT), Patients sample: GIT can be used as a positive control for this stain. If the controls work but the colour is not removed from the patients negative slide it is something other ‒ The other is the patients sample. than glycogen staining. ‒ If the control does not stain positive then the results are not valid and the test needs to be repeated regardless of whether the ‒ If the positive control is negative or the negative control is positive patients sample was positive or negative. then the results are not valid and the test needs to be repeated regardless of whether the patients samples were positive or negative. 7 8 Bacteria are The importance of controls clearly visible False Results The same bacteria, if and stain as if stained correctly, they are Gram should be negative. blue/black, i.e. However, this Gram positive is wrong! Gram’s technique for bacteria © Built for Learning © Built for Learning 4. Internal Proficiency Testing 4. External Proficiency Testing Like in all NATA accredited laboratories it is a requirement that all staff All NATA accredited laboratories are also required to participate in external undergo continuous competency assessment to ensure that they are proficiency testing programs to ensure that they are performing the test performing the test methods according to the procedure and achieving methods to industry standards. accurate and consistent results. 1. Slides are sent to the laboratory from an external proficiency testing program 1. A different stain is selected each time. provider ‐ The Royal College of Pathologists of Australasia http://www.rcpaqap.com.au/anatomical/. 2. One control slide is stain by each technician. 3. A pathologist reviews the slides and compares the staining results. 2. The requested stain is performed and the slides sent back for assessment and comparison with up to 800 laboratories. 4. If the stain does not work, that technician is not able to perform the stain on 3. The stains are graded with a mark from 1 to 5. patient samples until they have be retrained and assessed as competent. 5. If one stain is better than the others the technician is questioned about any 4. If the stain is assessed as unsatisfactory the test must be repeated, if it continues to fail NATA may remove accreditation from that test until the laboratory is able modifications they have made and the procedure may be reviewed and updated. to resolve the problems. Technician 1 Technician 2 Laboratory 1 Laboratory 2 Excellent stain Retraining 5 out of 5 Unsatisfactory 11 12 © Built for Learning © Built for Learning Carbohydrates (Sugars) Substance Identification Mucopolysaccharides (mucins): – Complex carbohydrates, consist of a long chain of polysaccharides (>10 sugars connected) and an amino group. – Produce mucus e.g Saliva – Slippery to touch Types of Carbohydrates Neutral Mucins: Fructose rich mucins Acid Mucins: A complex group that have a strong acid attached. Saccharides: Sucrose and glucose © Leah Simmons - Built For Learning Polysaccharides: Glycogen, starch, cellulose (chitin in insects) 13 14 © Built for Learning © Built for Learning Staining Mucopolysaccharides Periodic Acid Schiff (PAS) Structures and cell products stained magenta in colour Neutral Mucins – Periodic Acid Schiff (PAS) Neutral Mucins Basement membranes Acid Mucin stains – Colloidal Iron  Kidney – glomerula capsule  Inside glomerulus – Alcian Dyes  Outside of the tubules  Base of epithelial cells Glomerulus Glycogen  Skin ‐ hair follicles, epithelium, sweat glands  Liver‐ all columns of cells Fungi and parasite cell walls Amyloid Reticulin fibres PAS Colloidal Iron Alcian Blue Trichinella spiralis Cyst Fungi 15 16 © Built for Learning © Built for Learning Basic Mechanism of staining Colloidal Iron 1. Formation of Schiff Reagent Structures and cell products stained Coloured Clear Acid Mucins ‐ Blue in colour SO2 Parallel double bond was broken Goblet cells in the mucosa of GIT  Acid mucopolysaccharides are a good lubricant such as SO3‐ in saliva it also aids in the movement of waste in the bowel. Pararosaniline Leucosulphonic acid of Pararosaniline PAS w/ Enzyme digestion 2. Oxidation of the tissue by Periodic acid Acid Structures and cell products stained ‐ ve control = digested Tissue Aldehyde groups Glycogen – Magenta  Glycogen can be removed from 3. Schiff reaction with Aldehydes Schiff reagent + Aldehyde = Magenta tissue by treating it with an enzyme such as amylase found in saliva. Parallel double bond reformed  Glycogen should not stain in the enzyme treated sections + ve control = untreated 17 18 © Built for Learning © Built for Learning Alcian Blue(AB) / Periodic Acid Schiff (PAS) Alcian Blue(AB) / Periodic Acid Schiff (PAS) Structures and cell products stained Acid Mucins ‐ Blue in colour Mucosa neck cells secreting acid mucus  Acid mucopolysaccharides aid in digestion and can be found in the stomach, they also helps protect the body from pathogenic microorganisms that may have been consumed. Orcein Neutral Mucins ‐ Magenta in colour Basement membranes Goblet cells secreting neutral mucus  Neutral mucopolysaccharides line the mucosal surface of the stomach – protection from acid. Mixture of Acid and Neutral Mucins– Purple in colour 19 20 © Built for Learning © Built for Learning LIVER ‐ve control Colloidal Iron Glycogen stains Magenta in the +ve control. +ve control PAS w/ Enzyme Digestion Glycogen has been removed from the –ve control, so it is no longer staining magenta. Periodic Acid Schiff 21 22 Artefacts ‐ fixation derived pigment Pigment and Deposit Identification Formalin pigment from using unbuffered formalin Brownish pigment, often appears over RBC’s. Birefringent under polarized light. Confirmed by bleaching the pigment using saturated alcoholic picric acid. Mercury pigment from using mercury based fixatives Black pigment, often appears over the entire tissue. Confirmed by bleaching the pigment using an iodine/thiosulphate sequence. © Leah Simmons ‐ Built For Learning Formalin pigment – H&E Formalin pigment – Polarized Mercury artefact – H&E 23 24  Endogenous ‐ produced within tissue (INTERNAL/NATURAL)  Melanin: pigment found in skin Stain: Masson‐Fontana and Schmorls stains  Lipofuscin: hepatocytes, cardiac muscle, neurons and testis Stain: PAS, Schmorl’s or Masson‐Fontana  Hemosiderin (iron): liver deposits Lipofuscin in Muscle – H&E Bile pigment (Bilirubin – H&E) Copper ‐ Rhodanine stain Stain: Perl’s Prussian Blue stain  Copper: liver deposits Stain: Rhodanine stain Calcium – H&E  Bile: gall bladder pigment Stain: Hall’s technique  Calcium: Deposit in various locations Stain: Von Kossa Calcium – Von Kossa Melanin – Schmorl’s Melanin ‐ Masson‐Fontana Hemosiderin ‐ Perl’s Prussian Blue 25 © Built for Learning Endogenous Amyloid Bleaching technique to confirm Melanin Both positive and negative controls Amyloid is an extracellular deposit that usually develops in ‒ Both control slides are identical and have previously tested positive for Melanin. people suffering from chronic diseases such as Tuberculosis or ‒ The negative control slide and one of two patient slides are treated with Arthritis. The deposits are characterised by a lack of nuclei. Potassium permanganate and Oxalic acid (“Mallory’s Bleach”) to break down Congo Red stains amyloid brick red. the melanin. Melanin will not stain, giving a –ve result. ‒ The positive control slide and the patient’s second slide skip these steps. The Under polarized light amyloid has apple green birefringence melanin will stain, giving a positive result. (normal collagen is white) Polarizer Rotate left Specimen Patients sample: to alter light If the controls work Polarizer but the colour is not removed from the patients negative slide it is something other than Melanin staining. Untreated +ve Masson’s Fontana Bleach treated –ve Masson’s Fontana ‒ If the positive control is negative or the negative control is positive then Amyloid – Congo Red the results are not valid and the test needs to be repeated regardless of Amyloid – Congo Red under polarized light 27 whether the patients samples were positive or negative. 28 Exogenous – from external source (ENVIRONMENT)  Carbon: black deposits in lungs  Tattoo ink: Coloured pigment in skin  Asbestos: Brown elongated pigment in Lungs  Silica: Implants (e.g. breast) and lungs of miners Tattoo ink in skin – H&E Formalin artefact – H&E Formalin artefact – Polarized  Formalin: brown pigment sits over red blood cells, birefringent  Mercury: Black pigment over the entire tissue. Looking at lung?? Keep an eye out for carbon 29 Mercury artefact – H&E Asbestos in lung – H&E Asbestos in lung – Perl’s  © Built for Learning Endogenous and Exogenous Urate Crystals Gout is caused by excessive amounts of uric acid within tissues and body fluids. It results in the deposition of urate crystals in cells and tissues. CNS Tissue Recognition Tophi are large aggregates of urate crystals surrounded by large numbers of white blood cells (inflammation). Tophi are common in the cartilage of joints as well as in tendons, ligaments and soft tissue. White blood cells Urate Crystals © Leah Simmons - Built For Learning Gouty Tophus (10x Objective) – H&E Gouty Tophus (100x Objective) – H&E 31 32 © Built for Learning © Built for Learning The Nervous System Nervous System Cells The nervous system is the bodies cellular communication network. Neurons Nervous System Functions: Nucleus An electrically excitable cell that processes – Receives stimuli from sense organs, processes then sends a response and transmits information through electrical – Controls voluntary and involuntary actions of muscles. and chemical signals. – Transfers messages between different parts of the body. – Higher mental functions such as memory intelligence and movement Two major divisions 1. The Central Nervous System Brain ‐ control centre of the body Spinal Cord – connects the brain to the rest of the body 2. The Peripheral Nervous System Cranial and spinal nerves ‐ connect the brain and spinal cord to the rest of the body 34 Click link for image credit. 33 © Built for Learning © Built for Learning The Central Nervous System ‐ Brain The Central Nervous System ‐ Cerebellum Cerebellum: Brain: – The brain is the control centre of the body it is provided with nutrients by Primary role in coordination of voluntary movement, posture and balance. Cerebrial Spinal Fluid (CSF) and protected by the skull. Grey matter (Cortex) Molecular layer Unmyelinated axons Granular layer White matter myelinated axons Grey matter 36 Click link for image credit. (Cortex = molecular + granular) 35 © Built for Learning © Built for Learning Cerebellum Cells Cerebellum Cellular Layers Purkinje cells Pia Matter Molecular Unmyelinated axons Purkinje Granular White matter myelinated axons Granular cells 37 Grey matter 38 (Cortex = molecular + granular) © Built for Learning © Built for Learning The Central Nervous System ‐ Spinal Cord Spinal Cord Cells Motor Neurons Spinal Cord: The cylindrical bundle of nerve fibres and associated tissue which is enclosed in the spine and connects nearly all parts of the body to the brain. Central Canal White matter Grey matter Grey matter Spinal Nerves 39 Nissl Substance 40 Click link for image credit. Stain: Cresyl Fast Violet © Built for Learning © Built for Learning Staining Myelin and Nissl Substances CNS Techniques Myelin is a type of insulation that is formed by support cells wrapping around neural axons CNS myelinated by – Oligodendrocytes PNS myelinated by – Schwann cells in the PNS). Nissl Substance is the rough endoplasmic reticulin located in © Leah Simmons - Built For Learning the cell body of neurons. 42 41 Diagram from Kelley, Kaye and Pawlina, "Histology, a Text and Atlas," 4th ed., page 284. Neuron‐Ross4‐284.tif. © Built for Learning © Built for Learning Cresyl Fast Violet – Spinal Cord Cresyl Fast Violet for Nissl Substance Motor Neurons Nissl Substance (Nissl bodies/granuales) Cell body Dendrites Staining Outcomes Nuclei Nissl Substance = Violet “Owl eye” or “Fried egg” Nuclei = Violet 43 44 Nissl Substance Background = Colourless © Built for Learning © Built for Learning Weil’s Haematoxylin ‐ Cerebellum Weil’s Haematoxylin for Myelin Capillary with RBC Pia Matter Molecular Purkinje Unmyelinated Molecular layer Unmyelinated axons Granular White matter myelinated axons Nuclei in Granular layer Staining Outcomes Myelinated white matter = Black/Blue Nuclei = Black/blue Red Blood cells = Black/Blue Grey matter 45 Unmyelinated molecular layer = Yellow/brown 46 (Cortex = molecular + granular) Myelinated White matter © Built for Learning © Built for Learning Luxol Fast Blue for Myelin Luxol Fast Blue/Cresyl Fast Violet – Spinal Cord Nissl Substance White matter (Myelinated) Unmyelinated Molecular layer Nuclei in Granular layer Staining Outcomes Myelinated white matter = Blue Red Blood Cells = Colourless Unmyelinated molecular layer = Colourless Myelinated White matter Grey matter Nuclei = Pale Violet (w/Cresyl violet counterstain) 47 (Unmyelinated) Motor Neurons Myelin 48 Nissl Substance = Violet (w/Cresyl violet counterstain)  © Built for Learning Spiruiod in a Finch Microorganism stains Cystacanth Haematoxylin © Leah Simmons ‐ Built For Learning and 49 Heart Parasite Eosin 50 © Built for Learning © Built for Learning Giemsa Stain Periodic Acid Schiff (PAS) ‐ Parasites Magenta Parasite walls Blue micro‐organisms Pale pink background Pink nuclei and pale pink background Blue nuclei if a Haematoxylin counterstain was used. Shizont – H&E Coccidia Whip worm Gamonts – H&E Oocyst – H&E 51 52 © Built for Learning © Built for Learning Fungal Hyphae Gram stain Gram +ve bacteria stain blue/purple Brown – Hopps Gram +ve Gram ‐ve bacteria stain pink/red Background varies Periodic Acid Schiff Grocott Silver Technique Magenta fungi Pale pink background Fungi black Pale green background Gram +ve and Gram ‐ve Bacteria 53 54 © Built for Learning © Built for Learning Gram Stain Ziehl Neelsen Gram +ve bacteria stain blue/purple Gram ‐ve bacteria stain pink/red Background varies High power – JD H&E Gram Twort – Gram +ve Microbiology Gram Stain – Gram +ve Anthrax High power – JD ZN Acid Fast Bacteria AFB – Magenta Background ‐ Blue 55 56 © Built for Learning © Built for Learning Mycobacterium Helicobacter pylori Warthin‐Starry Spirochetes – Black Background ‐ yellow/brown Alcian Yellow / Toludine Blue Spirochetes – Blue Auramine/Rhodamine Ziehl Neelsen (ZN) Fluorescent Technique Acid fast technique Background – Blue Mucus – Yellow 57 58 © Built for Learning © Built for Learning Helicobacter pylori Toluidine blue All bacteria stain blue Immunohistochemistry Background ‐ blue Helicobacter Antigen – Brown Nuclei ‐ Blue Immunofluorescence Helicobacter Ag – fluoresce Orange Nuclei – yellow green 59 Leptospira 60 © Built for Learning © Built for Learning Orcein Stain Silver Impregnation Techniques Hepatitis B Hep B – IHC Hep C – IHC surface antigen Hep B Antigen – Red/brown/marron © Leah Simmons - Built For Learning Background – light version of the above 61 62 © Built for Learning © Built for Learning Silver Impregnation Method 1 Silver salt may react with tissue components to produce an insoluble silver salt. This can then be reduced to metallic silver by exposing it to U.V. light. Silver carbonate and silver phosphate are Grocott ‐ Fungi Masson‐Fontana ‐ Melanin Gomoris ‐ Reticulin formed. Silver Techniques Silver impregnation techniques depend on the clinical reduction of a silver salt so that a Von Kossa ‐ Calcium deposit of metallic silver is formed. An example of this type of reaction is the von The silver deposit is recognisable in the section Kossa method to demonstrate calcium. as black areas. Von Kossa ‐ Calcium 64 63 © Built for Learning © Built for Learning Silver Impregnation Silver Impregnation Method 2 – Argyrophil Reaction Method 3 – Argentaffin Reaction A deposit of metallic silver forms with the help of an added reducing agent. The There is no external reducer required, the reducing agent in most methods of this type tissue contains strong reducing substances is formalin, occasionally it is hydroquinone or that reduce the silver salt to metallic silver. pyrogallic acid. (without the reducer silver Silver  Reduced by the tissue  Metallic Silver binds with no colour). (black deposit) Masson Fontana Gomoris Reticulin Melanin and Examples of this method include reticulin fibres, neurofibrils and axons, glial cells argentaffin cells and fibres and spirochaetes. Examples of this type include melanin and the granules in the Kultschitzky cells of the intestinal mucosa and carcinoid tumours. 65 66 © Built for Learning © Built for Learning Cellular responses/adaptions to stress Histopathology Adaptive responses Hypertrophy – Increase in cell size Atrophy – Decrease in cell size Hyperplasia – Increase in cell number © Leah Simmons - Built For Learning Metaplasia – Change in cell type 67 © Built for Learning © Built for Learning Hypertrophy Atrophy Increase in cell size → increase in organ size Decrease in cell size → Decrease in organ size Physiological: Lack of hormone s mula on → Decrease in size of Physiological: Enlargement of uterus after giving birth, the uterine cells are broken down and muscle cells in response to exercise reabsorbed. Pathological: Enlargement of Pathological: Shrinkage of muscle cells in response to lack of use. cardiac muscle cells in response to increased demand (hypertension) Normal Atrophic Skeletal muscle Skeletal muscle Normal Hypertrophic cardiac muscle cardiac muscle © Built for Learning © Built for Learning Hyperplasia Metaplasia Increase in cell number – from cellular proliferation (replication) Change in cell type – Reversible change in which one adult cell type is replaced by another. Physiological: Increase in the number of glandular epithelial cells in breasts during pregnancy in preparation for milk productions. Pathological: Epithelial metaplasia – Columnar epithelium changes to squamous epithelium to protect itself from damage. Nonpregnant Pregnant Lactating Pathological: Proliferation of cells enlarging organs and/or forming tumours. These changes increases the risk of cancer development. © Built for Learning © Built for Learning Irreversible Cell Injury Causes of cell injury Apoptosis Necrosis Oxygen deprivation Programmed cell death Pathologic/cumulative cell damage Cells shrink Cells swell  Hypoxia – oxygen deficiency Cellular fragmentation Contents may leak out No Inflammation (mostly) Inflammation present (mostly)  Ischemia – loss of blood supply causing hypoxia Chemical exposure – poisons/toxins (consider formalin) Infectious agents – Bacteria, fungi, virus, parasite (What stains?) Immunological reactions – allergy or autoimmune Genetic factors – Sickle cell anaemia Nutritional factors ‐ risk factors in obesity, vitamin deficiency Aging – Decreased replication and repair Physical agents – trauma/burns, radiation, pressure change © Built for Learning © Built for Learning Inflammation Inflammation A protective response to: Chronic inflammation: Eliminate the initial cause of injury (prevent further damage) Prolonged response to Eliminate necrotic cells and tissue (clean up) persistent stimuli. Clear infection and initiate repair (Repair) New blood vessels form. Acute inflammation: Active inflammation and repair simultaneously. Rapid response to injury, Primarily mononuclear microbes or other foreign leucocytes (Lymphocytes substances designed to and macrophages). deliver leucocytes and plasma proteins to sites of injury. Fibrosis – Connective tissue may replace glands and Presence of PMNs other structures. Lots of RBC (congestion) e.g. TB Granuloma e.g. Pancreatitis © Built for Learning © Built for Learning Definitions Metastasis Oncology: The study of tumours and their treatment. Cancer: Uncontrolled, abnormal cell growth with the potential to invade or spread to other parts of the body. (Genetic & Epigenetic causes) Neoplasm: The presence of new abnormal growth of tissue. Tumour: New growth of cells originating from normal cells that have mutated (can be benign or malignant). Metastasis: The spread of cancer or other disease from one part of the body to another. Invasive: Spreading into surrounding tissues. Benign: Non‐cancerous, non‐invasive, non‐metastatic, slow growing Malignant: Cancerous, invasive, metastatic, rapid growth © Built for Learning © Built for Learning Invasive Melanoma Tumour Classification and Nomenclature Melanin in the dermis Proliferation down hair folicle Melanocytes Classification is base on: Tissue of origin e.g. “Renal” = Kidney , “Hepato” = Liver Microscopic (cellular origin)/clinical presentation Benign: “‐oma” on site of origin e.g. Fibroma = fibrous tissue, lipoma = fatty tissue e.g. Adenoma = glandular origin, Papilloma = wart like growth Exception: Polyp = projection from mucosal surface Malignant : “sarcoma” = mesenchymal origin (connective tissue) e.g. fibrosarcoma, liposarcoma “Carcinoma” = epithelial origin e.g. adenocarcinoma, squamous cell carcinoma © Built for Learning © Built for Learning Benign Prostatic Hyperplasia V’s Adenocarcinoma Normal V’s Abnormal Skin © Built for Learning Hallmarks of cancer 1. Self sufficient in growth signal ‐ growth is unregulated by body 2. Do not respond to growth inhibitory signals ‐ unregulated growth 3. Evasion of cell death ‐ survive conditions that trigger apoptosis 4. Limitless replication potential – cancer cells are immortal 5. Development of angiogenesis – new blood vessels form 6. Ability to invade local tissue and spread – can metastasise 7. Reprogramming of metabolic pathways – can switch from aerobic to anaerobic respiration for energy production. 8. Ability to evade the immune system © Built for Learning © Built for Learning Immunohistochemistry Immunohistochemistry Using antibodies to stain tissue Histochemistry relies on the interaction of dyes and reactive chemical groups present in cells and tissue. Immunohistochemistry uses antibodies to identify antigens which are present in cells, followed by a © Leah Simmons - Built For Learning colour reaction which reveals the location of those antigens. 85 © Built for Learning © Built for Learning What are Antibodies? Antibodies are Y‐shaped glycoproteins Also called immunoglobulins (Igs) or gamma globulins Produced by the immune system in response to Antigen (acquired immunity). Produced by plasma cells (which differentiate from B cells) Polyclonal antibodies: – Are Heterogeneous mixtures of Antibodies. – Are purified from serum – Recognise several epitopes (sites) on the antigen. – Can have cross reactive problems – Polyclonal antibodies are especially useful as the second labelled antibodies in the complex Ag‐Ab‐Ab‐*** Monoclonal antibodies: – Monospecific antibody to a single epitope on the antigen. – Are produced from a single clone. – Are very specific do not have cross reactive problems – Are especially useful as the first capture antibody Ag‐Ab © Built for Learning © Built for Learning Antigens of interest Structural proteins: cells are held together by filaments which are IHC V’s Immunofluorescence cell‐type specific and located in the cytoplasm – Epithelial cells: keratin – Muscle cells: actin, desmin – Connective tissue: vimentin Cell surface antigens are specific for different subclasses of lymphocytes: – CD45: Specific for lymphocytes – CD3: T cells CD = Cluster of Differentiation – CD20: B cells – CD68: Macrophages Other Intracellular antigens, cytoplasmic or nuclear – Proliferation markers – Hormone receptors, growth factor receptors, cytokines – Micro organisms‐ bacteria, virus © Built for Learning © Built for Learning IHC General Procedure Pre‐treatment – Unmasking the antigenic sites Fixation or processing may obscure antigens, which can be recovered Use FFPE sections (Formalin fixed, paraffin embedded) under certain conditions. Dewax sections and rehydrate Enzyme digestion Protein antigens can be digested by enzymes which expose the antibody Place sections in buffer binding site. Apply pre‐treatment if required Heat induced epitope retrieval (HIER) Blocking reagents Heating sections in certain buffers can restore many antigens which have been modified by fixation. Primary antibody ‐ Waterbath Detection system ‐ Pressure cooker ‐ Microwave Develop colour at antigen sites Counterstain and coverslip Visualization with microscope © Built for Learning © Built for Learning Blocking reagents – avoiding false positives Primary antibody Binding non‐specific proteins Identify the location of the antigen of interest in the section Antibodies may accidentally bind with other proteins resulting in false positives. – Concentrated antibodies need to be diluted before use. It is possible to apply a protein solution before applying the antibody to saturate – Antibody data sheets describe the antibody’s reactivity etc. these sites. – Use a section known to include the antigen as a control. Neutralise endogenous enzymes Detection system Enzymes used in the detection system may also be present in Consists of something to attach to the primary antibody and something cells, so apply the enzyme’s to make the antigen‐antibody complex visible. substrate to neutralise that – A second antibody is used to activity and prevent/reduce combine with the primary background staining. antibody. – An enzyme (e.g. horseradish peroxidase), attached to the second antibody, reacts with its substrate (H2O2,) which causes a visible precipitate to deposit at the reaction site. © Built for Learning © Built for Learning Counterstain Antibody to smooth muscle actin Haematoxylin is usually applied lightly to contrast with the cytoplasmic pattern (structural protein) brown precipitate from the enzyme reaction. If the counterstain is too intense positive staining may be obscured, so only nuclei are stained. Incubation times Primary antibodies usually bind to antigens in about 30 min. Secondary antibodies – about 20 min. Colour development takes 5‐10 min. Prepare substrate immediately before use. All washing steps use buffer. © Built for Learning © Built for Learning Smooth muscle cells Antibody to oestrogen hormonal receptor positive for Actin (structural protein) nuclear staining of breast cancer cells © Built for Learning © Built for Learning Membrane staining Cytoplasmic staining glandular epithelium glandular epithelium © Built for Learning © Built for Learning Lymphocytes around glandular epithelium, Cytoplasmic staining of duct epithelium, antibody to CD45, membrane staining antibody to keratin, enzyme treated (Cell surface Ag) (structural protein) © Built for Learning © Built for Learning Liver epithelium Antibody to cytokeratin antibody to keratin (structural protein) cytoplasmic pattern (structural protein) © Built for Learning © Built for Learning All lymphocytes are positive for CD45 B‐cells in lymph node (Cell surface Ag) antibody to CD79 (Cell surface Ag) © Built for Learning © Built for Learning Antibody to insulin (hormone) Antibody to Cryptococcus neoformans cytoplasmic pattern in beta cells of pancreas (Microorganisms) © Built for Learning © Built for Learning Antibody to HBsAg CMV infected cells cytoplasmic staining of infected cells nuclear and cytoplasmic staining © Built for Learning © Built for Learning Antibody to CD3 A solitary positive lymphocyte membrane staining of T‐cells (Cell surface Ag) © Built for Learning © Built for Learning Microscopic Differentiation - Haematoxylin H&E Quality Control 1. Do not let your slide dry out – The slide is ruined if it dries at this point. (black nuclei) 1. Good Balance the haematoxylin and the Eosin – not too pink, not too blue 2. Blue Nuclei – Crisp detailed nuclei in the smooth muscle cells 2. Detailed Nuclei ‐ Blue/purple nuclei with sharp edges and patterns (chromatin) inside 3. Almost clear background – No blue in the cytoplasm of the smooth muscle cells. 3. Good Eosin Differentiation ‐ 3 shades of pink (RBC, smooth muscle, connective tissue) (Cytoplasm in the smooth muscle cells pink, not purple/pink or blue) Smooth Muscle Nuclei 4. Good Haematoxylin differentiation Nuclei Smooth Muscle (Points 1, 2 and 3 combined) 5. Section Quality (Correct staining, no bubbles, not too much glue, coverslip covers the tissue) Find an Artery Find an Artery You must find a blood vessel You must find a blood vessel (artery or vein) to assess (artery or vein) to assess the haematoxylin differentiation. quality of your stain. Connective Red Blood Cells (RBC) Connective Tissue Red Blood Cells (RBC) Tissue