IHC - IHC PDF

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Summary

Fresh Tissue in HistologyReasons why we receive fresh tissue1. To allow the lab control over how the tissue is treated Colon and breast tissue  Allows correct and best fixation  Ensures resection margins are preserved and clear. preservation allows identification of tumour.  Resection margins = m...

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Fresh Tissue in Histology Reasons why we receive fresh tissue 1. To allow the lab control over how the tissue is treated - Colon and breast tissue  Allows correct and best fixation  Ensures resection margins are preserved and clear. preservation allows identification of tumour.  Resection margins = margin of apparently non-tumorous tissue around tumour that has been surgically removed)  Colons are pinned to cork  Breasts are “toast racked” - Lymphomas  Lymph Node received whole  DAB specimen onto slide for FISH  4 pieces frozen in liquid nitrogen – thin slice rapidly processed  3.5 hours wait for results  Process remainder as normal for IHC 2. To enable specific techniques - Renal biopsies - Cytology  Any body fluid or cell aspirate

 Non-invasive, can be repeated, rapid  Specific clinics  Prepared using cytospins  Suitable for special stains and immuno - Paediatric tumours E.G.  Wilms tumour Aggressive renal tumour Childhood 1-4 years Photographed, mirror sections taken Frozen tissue and duplicate H&E’s sent to Cardif  Neuroblastoma Aggressive tumour arising from autonomic ganglia in abdomen Very urgent! 3 needle cores Frozen tissue and duplicated - H&E’s sent to Manchester  n-myc assessment – prognostic factor, over expression of n-myc due to lack of apoptosis = poor prognosis - Histochemistry specimens  Rectal biopsies - Paediatric biopsies e.g. diagnosis of Hirschsprung’s disease - absence of ganglion cells in anorectal region. Stained with acetyl cholinesterase which stain neurons and nerves. Results in pull through surgery – lining of diseased part of clon stripped away + normal section pulled through colon from inside and attached to the anus.  Muscle biopsies - Frozen in isopentane - only demonstrative method of muscle fibres. Very complex disorders diagnosed

3. -

Intraoperative Diagnosis Frozen sections used for intraoperative diagnostic sections and assessment of resection margins Patient is still in theatre Prepared using cryotomy - use of ice (instead of paraffin) as a supporting media to enable sections to be cut.  Mohs Micrographic Surgery - Gold standard for treating BCCs & SCCs  functionally important areas to preserve healthy tissue  Single outpatients visit  All margins examined so highest  survival rate

Know how tissues are frozen and the effect that this has on the tissue Tissues are frozen via: 1. 2. -

Deep Freeze Most common method Relatively quick Usually -20-40 degrees Used in cryostats Cheap, easy, does not require special reagents Liquid Nitrogen -196 O C rapid freezing on contact Use for all fresh tissue to be stored Can boil on contact Isopentane for muscle biopsies

Effect of Freezing -

Slow freezing = large hexagonal ice crystals. Intracellular ice crystals damage cell membrane Rapid freezing = small cubic ice crystals Less distortion of cell membrane Ice crystal artefacts looks like white spots Rate of cooling depends upon the rate of conduction. Rate of conduction depends upon the temperature diference between the tissue and the coolant

Explain the steps involved in producing a frozen section 1. 2. 3. -

Tissue frozen Section cut Using a cryostat- an enclosed refrigerated microtome with a temperature control (dif tissues cut at dif temps) Usually 20 degrees – adjustable Section cut at 5µm - adjustable Anti-roll plate Disposable blades Sections picked up onto normal slides – melts and sticks to slide Section stained H&E – rapid hand stained technique Alcohol fixed first then progressive staining with Harris Haematoxylin, water, Scott’s blue reagent, water, Eosin and finally DCM 4. Reported by pathologist 5. Specimen fixed and processed to paraffin wax

List the advantages, disadvantages and risks associated with frozen sections Advantages

Disadvantages

- Produces diagnostic sections within 40 mins - Quick and easy - Cost efficient

- Morphology is not as good as paraffin processed sections - More difficult to cut - Artefacts

Risks Burns - Liquid Nitrogen Blades - Cryostat blade - Cutting up specimen

Decontamination of cryostats Formaldehyde sterilization - Defrost then sterilize - Needs to cool

- Compatible with many subsequent techniques - Only available method for the demonstration of some substances (oil red o stain for lipids)

- Storage

down again - Overnight / immediate UV sterilization - UVC light source - No warming or defrosting - No chemicals involved - Only sterilizes exposed surfaces

Immunohistochemistry What controls are necessary and why they are used Positive -

Known to contain relevant tissue antigen Confirms the antibody is working Better to have low expression E.g. Liver from hep b positive liver for hep b control E.g. Colon for CEA, CK20 (tumour marker for lower half of body)

Internal - Structure within test tissue that will stain positively - E.g. Peripheral nerves and melanocytes s100 (polyclonal antibody) Negative - Test section that has had primary antibody omitted

- Detects any not specific interactions Or - Known not to contain the target antigen - E.g. Normal liver for hep b antibody - Not used for routine work

Effect of fixation on IHC and how antigen retrieval overcomes this How IHC works - Tissue Preparation Fixation - secondary investigation - fixation optimised for routine H&E stain - formalin fixation is followed by processing to paraffin wax - FFPE tissue prod. satisfactory results for most tissue Ag detection - processes of fixation in formalin changes protein structures by crosslinking amino acid side-chains – denatures enzymes - Excessive fixation of tissue samples can produce characteristic:  masking of antigenic binding sites due to excessive crosslinking amino acid side chains and formalin pigment

 edge efect ➝ result of inaccurate fixation ➝ alcohol = fixative instead of dehydrator - part paraffin and part alcohol fixation - frozen sections used when detection wanted

Antigen Retrieval 1. -

new method of preserving the immunoreactivity of the antigen under investigation/unmasking antigens split into two groups: enzyme digestion and HMAR Enzyme Digestion Performed in a water bath at 37 degrees Improves immunoreactivity of formalin-fixed material by selectively breaking protein links to reveal hidden epitopes without causing uncontrolled change in protein structure - Incubation of the tissue section with enzyme will need to vary depending on the antigen e.g. rate of hydrolysis slower in presence of an acid residue on either side of site of cleavage. Proline residue on carboxyl side lead to no cleavage. - Enzymes that are available to use include: Trypsin One of the most commonly used enzymes. Derived from porcine pancreas and achieves protein digestion by cleavage of peptide bonds on the C-terminal side of lysine and arginine amino acid residues.

Chymotrypsin It is produced in the pancreas and exerts its proteolytic efect by cleaving peptides at the carboxyl side of tyrosine, tryptophan and phenylalanine—three amino acids containing phenyl rings.

Used on its own it is a much slower proteolytic agent than Aids in conversion of antigenic precursors to the trypsin, but many commercial trypsin products actually immunoreactive form, and to digest protein aggregates, which may block access to the antigen = greatly increases Ab- contain a small amount of chymotrypsin to ofer a wider

Ag binding Under-digestion

spectrum of digestive activity.

- Failure to demonstrate any or all of the antigen Over-digestion -

Morphological damage (membrane disruption) Sections detach from slides Excessive background staining Cross-reactivity of antibody with inappropriate tissue components Digestion of antigen under investigation

Advantages - Only way of demonstrating some epitopes e.g. renal Disadvantages - must be expertly controlled - can destroy some epitopes completely - mainly replaced by HMAR

2. HMAR (heat-mediated antigen retrieval)

- Introduced by Shi et al. in 1991 using microwave heating heavy metal in solutions = produced greater sesitivty in subsequent staining - Cattoretti et al. Improved on this by using citrate bufer to demonstrate majority of antigens under investigation - many diferent bufers available (dependant on each lab) - citrate • Tris - EDTA • commercial solutions - Time required for efficacious antigen retrieval could be shortened as temp of solution increased. Most common HMARs below: Pressure Cooker Microwave Lead to antigen retrieval by one or more: allow the retrieval - Removal of methylol groups allowing solution to be heated to increased accessibility of antibody to 120ºC, enabling a shorter incubation epitope period (often just 2–4 - Loss of ‘blocking’ proteins minutes) - Renaturation of protein molecules previously denatured by formalin - Removal of residual embedding medium (particularly with resin-embedded tissue).

Advantages - reliable - cheap

Advantages - reliable - uses less bufer

New Automated Methods Fully automated strainers perform dewaxing, antigen retrieval, antibody incubation and detection using proprietary reagents. These machines use a combination of heat and chemical solutions to achieve antigen retrieval and constant replenishment of bufer, which removes the risk of dried tissue sections. E.G. Pascal PT Module OMNIS Advantages Disadavantages

disadvantages - quick cooling - requires longer cooling time time - hot and cold spots = lead to variation in the - can cope with retrieval efect large batches - done in smaller batches to prevent Disadvantages evaporation of bufer - health and safety, hazardous to user Blocking Biotin Block blocks endogenous biotin activity using sequential incubations with avidin and biotin endogenous biotin found in - Liver - Kidney - adipose tissue overcome by biotin free systems

Peroxidase Block hydrogen peroxide suppresses endogenous peroxidase activity endogenous peroxidase activity found in - erythrocytes - granulocytes - muscle - liver - kidney Horseradish peroxidase or alkaline phosphatase activity used to inactivate/inhibiting endogenous biotin activity - preventing false positives

Normal Serum Block Used to block nonspecific hydrophobic background blocks hydrophobic binding sites in the tissue Must have identical proteins to the secondary antibody to prevent nonspecific binding of the secondary antibody e.g. polyclonal 1o Ab / NSS / SR 2o Ab

Difference between monoclonal and polyclonal antibodies Primary Antibodies approx. 150 diferent antibodies in routine use lyophilised/liquid concentrates stirred at 4 degrees/frozen diluted as required into Polyclonal and Monoclonal Abs Polyclonal Antibodies Monoclonal Antibodies Reacts with various epitopes on the antigen against which they are Reacts with one specific epitope on the antigen against which raised they are raised - Initiates immune response - Produced from rabbits - B lymphocytes from spleen / lymph nodes harvested - Immunised with purified immunogen bearing the fuses lymphocytes with non-secreting mouse myeloma antigen of interest cells - Rabbit mounts an immune response = antibodies are - myeloma cells give the hybridoma cells longevity in produced and harvested culture - Produces numerous clones of plasma cells - Non-reactive B cells and myeloma cells are removed - Each clone has a slightly diferent specificity to the epitopes - antibody producing hybridoma cells are cultured on the immunogen - Can result in cross reaction with other molecules - Can be removed by purification -

Advantages : - Less batch to batch variation than with larger animals - Recognises more than one epitope on an antigen Disadvantages: - Recognises more than one epitope on an antigen - Cross reaction

Advantages - High homogenicity - No batch to batch or lot to lot variation Disadvantages - Targeted epitope must survive fixation - Target epitope must be unique to one antigen - Cross reactivity cannot be removed

Detection Methods Direct Relies on enzymelabelled primary antibody reacting with tissue antigen. One step procedure in which antibody binds to antigen and the final reaction is visualised by the appropriate

Indirect

Avidin Biotin Method

Dako Envision / FLEX Chromagens Polymer technique A two-step A more sophisticate Based on a unique, DAB procedure. First, and sensitive 3 step patented, labelled - Brown end an unconjugated procedure. polymer technology product primary antibody avidin-biotin methods which - insoluble in is applied and are used due to the enables a large number alcohol and binds to the fact that avidin or of enzyme molecules organic solvents antigen if streptavidin have a (HRP or AP) to be - can be used with present. strong affinity for biotin bound to a secondary copper / nickel The tissue section (have 4 binding sites antibody via a dextran sulphate/ osmium is then rinsed in a for biotin). backbone. tetroxide to bufer increase staining A second enzyme Sequence is: intensity labelled antibody 1. The primary -

Counterstain Stains the background tissue Allows localisation of the end product Any routine counterstain Mayer’s Haematoxylin

substrate and chromogen reaction

binds to the secondary antibody (directed against the primary primary antibody antibody. If has fluorescein polyclonal the label attached anti-rabbit; if - visualised monoclonal then using a fluorescence anti-mouse), a microscope two-layered complex is produced, which can be visualized using the appropriate substratechromogen final reaction product

Advantages - very quick, simple technique

Advantages disadvantages

antibody is applied 2. followed by a secondary biotinylated antibody raised against the primary 3. The tertiary layer consists of either an avidin-biotin complex (ABC) or a streptavidinbiotin complex (StABC) 4. The most commonly used enzyme labels are horseradish peroxidase or alkaline phosphatase Advantages Advantages - complete kit - ability to capture - ready-to-use numerous reagents enzyme

AEC - red end product - alcohol soluble - requires aqueous mount - non-permanent prep - susceptible to further oxidation in light therefore it will fade Fast red TR - alcohol soluble - requires aqueous mount - long technique - involves dangerous chemicals - e.g. malignant melanoma

disadvantages - requires special fluorescence microscopy - does not provide colour or contrast with surrounding cellular structures and morphology

molecules = - increased larger final sensitivity reaction product - faster than other - greater techniques sensitivity - no wastage - used in research - reduction in - good EQA results operator error Disadvantage - elimination of - endogenous endogenous biotin is present biotin in a wide variety - can be used with of tissue, and automation high levels are found in the Disadvantages - expensive (£800 liver, kidney and per kit) paracortical histiocytes in lymphoid tissue

Immunohistochemistry is the demonstration of antigens in tissue sections or smears by the use of specific antigen / antibody interactions which culminate in the attachment of a marker to the antigen – can demonstrate: -

Lymphocyte subtypes Cytokeratins of various types Glandular diferentiation markers Infectious organisms Proliferation markers and many more

What is positive IHC staining - When tissue has taken up colour of antibody as has antigen is present = Ab-Ag complex - Examples are:  BAPP stain  ER stain Diferent detection methods: - P63 nuclear staining

- S100 cytoplasmic staining

- PIN 4 double staining - two colours red and brown

- HER2 membrane staining

Examples of some antibodies, what they stain and the conditions that they can be used to diagnose Primary antibody Ae1/3 Βapp BerEp4 CA19.9 CA125 CALDESMON CALRETININ CAM CD45 CK5/6 CK7 CK20 CKIT (CD117) CHROMAGRANIN CMV DESMIN EMA ER HBME1 HMB45 HSV1

What it stains Pan cytokeratin, epithelium, adenocarcinoma, carcinoma Βeta amyloid precursor protein, senile plaques, Alzheimer’s disease Epithelial marker Epithelium, breast, kidney, colon, prostate. Gastrointestinal carcinomas Ovarian carcinoma Smooth muscle Mesothelioma Glandular epithelium, adenocarcinoma, carcinoma Leucocytes (T and B cells) Mesothelioma Breast, upper half of body Colon, lower part of the body CKit positive tumours and mast cells Neuroendocrine tumours, small cell carcinoma of the lung Cytomegalovirus Mesothelial cells, Rhabdomyosarcoma, Rhabdomyoma, Leiomyomas, leiomyosarcomas, Ewings sarcoma Epithelium, carcinoma, adenocarcinoma Oestrogen receptor Mesothelial cells, mesothelioma Malignant Melanoma Herpes Simplex Virus

MIC2 PR RCC S100 SV40 THROMBOMODULIN TTF

Ewings Sarcoma Progesterone receptor Renal Cell Carcinoma Malignant Mesothelioma SV40 antigen Normal mesothelial cells, mesothelioma Thyroid Transcription Factor, Type II clara cells in the lung

Explain the use of IHC in diagnosis -

Confirmation of tumour type suspected on H&E Identification of adjacent tumour spread Identification of distant metastases Confirmation of tumour subtype Identification of tumours of unknown aetiology Prognostic information about the likely response of the tumour to drug treatment Evaluation of inflammatory states and autoimmune diseases Identification of aetiological agents...