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An advanced diagnostic method in surgical pathology An advanced diagnostic method in surgical pathology

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Mexico Campeche Hemotoxylum campechianum Bloody red bark tree Hemotoxylum campechianum Bloody red bark tree Rosai : The H & E technique Old mistress apologue Mainstay of surgical pathology The H & E technique,1998 pathologica The H & E technique,1998 pathologica

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H&E Advantages Relatively quick Inexpensive Suitable for most situations Easy to master Allows accurate Dx of the large majority of specimens Nevertheless, Cannot answer all the questions Relatively quick Inexpensive Suitable for most situations Easy to master Allows accurate Dx of the large majority of specimens Nevertheless, Cannot answer all the questions

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SPECIAL TECHNIQUES Special stains Enzyme histochemistry Tissue culture Electron microscopy Immunohistochemistry vs Immunocytochemistry ! Flow cytometry Cytogenetics Molecular pathology Histometry Special stains Enzyme histochemistry Tissue culture Electron microscopy Immunohistochemistry vs Immunocytochemistry ! Flow cytometry Cytogenetics Molecular pathology Histometry

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Ag 1 st Ab 2 nd Ab Ag Tag Chromogene To detect target antigens cytoplasmic cytokeratin vimentin chromogranin A nuclear estrogen receptor progesterone receptor Ki-67 P-53 memebranous Her 2-neu E-cadherin EGFR Basic structure: Polypeptide Glycoprotein Lipoprotein

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Antigen The small site on an antigen to which a complementary antibody may specifically bind is called an epitope. This is usually one to six monosaccharides or 58 amino acid residues on the surface of the antigen.

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Antigen Because antigen molecules exist in space, specific three-dimensional antigenic conformation (e.g., a unique site formed by the interaction of two native protein loops or subunits), or the epitope may correspond to a simple primary sequence region. Such epitopes are described as conformational and linear.

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Antigen The range of possible binding sites is enormous, with each potential binding site having its own structural properties derived from: covalent bonds, ionic bonds and hydrophilic and hydrophobic interactions.

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Ag-Ab reaction For efficient interaction to occur between the antigen and the antibody: the epitope must be readily available for binding. If the target molecule is denatured, e.g. through fixation, reduction, osmolalaity changes, pH changes, temperature, chemical agents, the epitope may be altered and this may affect its ability to interact with an antibody.

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Characteristics of a Good Antigen Include: 1.Areas of structural stability and chemical complexity within the molecule. 2.Lacking extensive repeating units. 3.A minimal molecular weight of 8,00010,000 Daltons, although haptens with molecular weights as low as 200 Da have been used in the presence of a carrier protein. 4.The ability to be processed by the immune system.

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Characteristics of a Good Antigen Include: 5.Immunogenic regions which are accessible to the antibody- forming mechanism. 6.Structural elements that are sufficiently different from the host. 7.For peptide antigens, regions containing at least 30% of immunogenic amino acids: K, R, E, D, Q, N. 8.For peptide antigens, significant hydrophilic or charged residues.

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Class/ Subclass Heavy Chain Light ChainMolecular Weight (kDa) StructureFunction lgA 1 lgA 2 1212 or 150 to 600Monomer to tetramerMost produced lg; protects mucosal surfaces; resistant to digestion; secreted in milk. lgD or 150MonomerFunction unclear; Works with lgM in B-cell development. mostly B cell bound lgE or 190MonomerDefends against parasites; causes allergic reactions lgG lgG 2a lgG 2b lgG 3 lgG 4 1223412234 or 150MonomerMajor lg in serum; good opsonizer; moderate complement fixer (lgG 3 ); can cross placenta lgM or 900PentamerFirst response antibody; Strong complement fixer; Good opsonizer

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Monoclonal vs. Polyclonal 2nd Ab : Mouse / goat / rabbit anti- 1 st Ab 1st Ab : Mouse / goat / rabbit anti-human

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Antigen-Antibody Interaction The specific association of antigens and antibodies is dependent on hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. These are all bonds of a weak, non-covalent nature-Like antibodies.

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Antigen-Antibody Interaction Antigens can be multivalent, either through multiple copies of the same epitope, or through the presence of multiple epitopes that are recognized by multiple antibodies. Interactions involving multivalency can produce more stabilized complexes, however multivalency can also result in steric difficulties, thus reducing the possibility for binding.

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Antigen-Antibody Interaction All antigen-antibody binding is reversible, however, and follows the basic thermodynamic principles of any reversible bimolecular interaction: where KA is the affinity constant, Ab and Ag are the molar concentrations of unoccupied binding sites on the antibody or antigen respectively, and AbAg is the molar concentration of the antibody-antigen complex.

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Antigen-Antibody Interaction Affinity describes the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody arm interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

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Antigen-Antibody Interaction Avidity is perhaps a more informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: 1.antibody-epitope affinity; 2. the valence of both the antigen and antibody; 3.and the structural arrangement of the interacting parts.

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Polyclonal vs monoclonal 1.Polyclonal antibodies often recognize multiple epitopes, making them more tolerant of small changes in the nature of the antigen.

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Polyclonal vs monoclonal 2.Polyclonal antibodies are often the preferred choice for detection of denatured proteins

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Polyclonal vs monoclonal 3.Polyclonal antibodies may be generated in a variety of species, including rabbit, goat, sheep, donkey, chicken and others, giving the users many options in experimental design.

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Polyclonal vs monoclonal 4.Polyclonal antibodies are sometimes used when the nature of the antigen in an untested species is not known.

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Polyclonal vs monoclonal 5.Polyclonal antibodies target multiple epitopes and so they generally provide more robust detection.

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Monoclonal vs polyclonal 6.Because of their specificity, monoclonal antibodies are excellent as the primary antibody in an assay, or for detecting antigens in tissue, and will often give significantly less background staining than polyclonal antibodies.

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Monoclonal vs polyclonal 7.When compared to that of polyclonal antibodies, homogeneity of monoclonal antibodies is very high. If experimental conditions are kept constant, results from monoclonal antibodies will be highly reproducible between experiments.

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Monoclonal vs polyclonal 8.Specificity of monoclonal antibodies makes them extremely efficient for binding of antigen within a mixture of related molecules, such as in the case of affinity purification.

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 28 Basic concepts & Historical aspects

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 29 Basic Concept Immunohistochemistry is 1.The localization of antigens in tissue sections by the use of 2.labeled antibody as specific reagents through 3.antigen-antibody interactions 4.that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. colloidal gold.

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 30 There are numerous immunohistochemistry methods that may be used to localize antigens. The selection of a suitable method should be based on parameters such as : the type of specimen under investigation and the degree of sensitivity required.

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 31 1.Albert H. Coons 1944- 1955; fluorescent dye 2.Nakane and Pierce 1966 ; enzyme labels / peroxidase; Mason and Sammons 1978 alkaline phosphatase 3.Faulk and Taylor 1971; Colloidal gold by both light and electron microscopy level 4.Radioactive elements, and visualization by autoradiography. Immunohistochemistry Historical aspect

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 32 With the expansion and development of immunohistochemistry technique, enzyme labels have been introduced such as peroxidase (Nakane and Pierce 1966; Avrameas and Uriel 1966) and alkaline phosphatase (Mason and Sammons 1978).

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TO APPLY IHC OR ICC FOR ? 1.Diagnostic purpose. 1.Diagnostic purpose. 2.Prognostic purpose 2.Prognostic purpose 3.Therapeutic purpose 3.Therapeutic purpose 4.Preventive purpose? 4.Preventive purpose?

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Applications of IHC Diagnostic purpose:

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Applications of IHC Diagnostic purpose: IHC profile for metastatic carcinoma of unknown origin FemaleMale ER/PRPSA CK 7 CK 8 CK 20 CD X2 CEACEA TTF 1

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Diagnostic purpose of IHC CK 7 CK 8 CK 20 Dx ++- Follicular adenocarcinoma,Thyroid Adenocarcinoma,Pancreas -++ Hepatocellualr carcinoma -+- Squamous cell carcinoma CK 5/6 --- Thymoma CK 5/6 Thymoma CK 5/6

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Diagnostic purpose of IHC CK 8 CK 18 Dx ++ Follicular adenocarcinoma,Thyroid Papillary adenocarcinoma,Thyroid Adenocarcinoma,Pancreas Hepatocellualr carcinoma +-Trichoepithelioma -+Meningioma Collectind duct carcinoma --Thymoma Adenocarcinoma, Ampullary

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Applications of IHC Diagnostic purpose: an example IHC profile for prostatic carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 40 IHC in prostate Cancer Indications: Indications: 1- Distinction of Benign from Malignant High molecular weight cytokeratin (34 E12, CK5/6)High molecular weight cytokeratin (34 E12, CK5/6) Negative cytoplasmic marker (in basal cells) P63P63 Negative nuclear stain (in basal cells) AMACR (P504S)AMACR (P504S) Positive cytoplasmic marker (in tumor cells) Also positive in HGPIN, 31% of Bladder Ca. & 70% of Colorectal Ca.

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 41 HMW-CK (34 E12) Normal Glands Negative in Carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 42 AMACR (P504S) stain in Carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 43 P63 / AMACR cocktail Increasing IHC resolution:

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 44 34 E12 / P63 / AMACR 2-chromogen cocktail Increasing IHC resolution:

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 45 IHC in prostate Cancer (Cont.) Indications: Indications: 2- Differential Dx from urothelial carcinoma: PSA PSAP 34 E12 Leu7 CK5/6 PSA PSAP 34 E12 Leu7 CK5/6 Prostate Ca + + + - Urothelial Ca + +

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 46 IHC in prostate Cancer (Cont.) Indications: Indications: 3- Differential Dx in metastatic carcinoma: Bone Tumor: PSA stain

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Applications of IHC Prognostic purpose in breast carcinoma Poor prognostic markers Good prognostic markers ER(-)ER(+) PR(-)PR(+) pS2(-)pS2(+) Her-2 (3+) Her-2 (0/1+) Cathepsin D(+) Cathepsin D(-) P 53 >10% P 53 23%Ki-67

Potential for misdiagnosis (IHC) Antibodies (>28 commercially available) Technical performance Interpretation scoring artifacts

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Common problems in HER2 IHC Underestimation of expression: (over)fixation in NBF poor antigen retrieval (unmasking) choice of antibodies Overestimation alcoholic (post)fixation (check normal cells!) Cytoplasmic staining

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Who can interpret an IHC?

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Normal lobule 3+ breast carcinoma

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Lobular cancerization

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2+ breast carcinoma

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2+ breast carcinoma?

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Normal lobule

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Cytoplasmic staining

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What to do? Central Reference or large local laboratories high number of cases (>250 cases/year* or >100 cases/months [NSABP]) quality assurance controls (internal and external) automated IHC high level of training (technique/interpretation) Small laboratories (

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but There are labs capable of performing quality testing with lower volumes A high test volume does not ensure an accurate test result If an individual lab can properly validate an assay and perform acceptably in an external validation, then it should be permitted to offer the test Hsi ED & Tubbs RR: JCP 2004

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Applications of IHC therapeutic purpose Chemotherapy in breast carcinoma ER (+) PR (+) Tamoxifen / Femara ER (-) PR (+) Tamoxifen / Femara ER (+) PR (-) Tamoxifen / Femara Her-2 psoitive Trastazumab(Herceptin) Cost :300 000 000 Rials Her-2 negative --

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Applications Of IHC Therapeutic purpose: PharmaDx Abs Therapeutic purpose: PharmaDx Abs Chemotherapy in breast carcinoma Chemotherapy in breast carcinoma ER - / PR - / Her-2 - ER - / PR - / Her-2 - Triple negative breast carcinoma Treatment is completely different: Cisplatin Cisplatin

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Technical points

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Handling of Antibodies RTU Abs have a shorter shelf life RTU Abs have a shorter shelf life Upon receipt reagents should be stored promptly according to manufacturer s recommendation. Upon receipt reagents should be stored promptly according to manufacturer s recommendation. Record: lot No, expiration date, date of receipt, invoice number. Record: lot No, expiration date, date of receipt, invoice number.

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Storage Containers Containers: Containers: Negligible protein absorptivity Negligible protein absorptivity Polypropylene, polycarbonate, borosilicate glass Polypropylene, polycarbonate, borosilicate glass Clear & colorless containers Clear & colorless containers Labels should allow access for inspection Labels should allow access for inspection Solutions containing very low concentrations of protein (

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Storage Temperature Accurate and consistent temperature Accurate and consistent temperature Temperature alarm & emergency backup system Temperature alarm & emergency backup system Store RTU Abs & kits at 2-8 C Store RTU Abs & kits at 2-8 C Store concentrated Abs at -20 C in aliquots Store concentrated Abs at -20 C in aliquots Prevent from contamination, heat, excessive light exposure. Prevent from contamination, heat, excessive light exposure. Sterile, clean pipette tips Sterile, clean pipette tips Prompt return to storage temperature. Prompt return to storage temperature.

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Antibody Titer

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Antibody Dilution How to determine optimal dilution? Titration How to determine optimal dilution? Titration First select a fixed incubation time. First select a fixed incubation time. Make small volumes of experimental dilutions. Make small volumes of experimental dilutions. 100 to 400 microL per section 100 to 400 microL per section What is the optimal dilution? What is the optimal dilution? Peak in intensity Peak in intensity Minimal background Minimal background Maximal signal to noise ratio Maximal signal to noise ratio

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Effecte of pH & ion strength on Ab dilution All monoclonal Abs could be diluted higher & stained more intensly at pH 6.0. All monoclonal Abs could be diluted higher & stained more intensly at pH 6.0. IgG3 is an exception at pH 9.0 IgG3 is an exception at pH 9.0

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PBS suppress the reactivity of all monoclonal Abs Commercial Ab diluents are preferred. Commercial Ab diluents are preferred. PBS is usually used for dilution nevertheless it leads to less reaction and decrease in affinity, thus not recommended. PBS is usually used for dilution nevertheless it leads to less reaction and decrease in affinity, thus not recommended.

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Antibody Dilution Buffers Antibody dilution buffer is used for diluting primary and secondary antibodies as well as some detecting reagents. Primary Antibody Dilution Buffer 1% BSA (stabilizer and blocking) 0.1% cold fish skin gelatin (blocking) 0.05% sodium azide (preservative) 0.01M PBS, pH 7.2

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Antibody Dilution Buffers TBS as Ab diluent TBS as Ab diluent 1)TBS pH 7.6 used in primary antibody dilution buffer produces weaker staining; 2)Antibodies diluted using this buffer can be stored at 4 C for 6 months without reducing binding activity; 3) This buffer can not be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP.

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How to dilute? 1:10 dilution = one part of stock solution + nine parts of diluents. Then, two-fold serial dilutions

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How to dilute To prepare 1.0 mL of a 1:1000 dilution: To prepare 1.0 mL of a 1:1000 dilution: Step 1: 10 micro + 90 micro ------- > 1:10 Step 1: 10 micro + 90 micro ------- > 1:10 Step 2: 10 micro + 990 micro ----- > 1:100 Step 2: 10 micro + 990 micro ----- > 1:100 Final dilution is 1:1000 Final dilution is 1:1000

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Checkerboard titration Checkerboard titrations are used to determine the optimal dilution of more than one reagent simultaneously. 1.The optimal dilutions of the primary Ab & the streptavidin-HRP reagent are found. 2.While the dilution of the biotinylated link Ab is held constant. 3.Nine tissue sections are required for testing three dilutions. 4.If results achieved by use of several dilutions are identical or similar, reagent costs may become an additional factor in selecting optimal dilutions.

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Incubation Time Inverse relationship between incubation time & antibody titer. The higher the Ab titer, the shorter the incubation time required for optimal result. In practice: First set a suitable incubation time then determine the optimal dilution.

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Incubation Time The most widely used incubation time:10-30 min High concentration, high affinity, optimal pH, Optimal ion strength ------ > shorter incubation 24 hr incubation for economy purpose ----- > allow higher dilutions

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Incubation Time Low titer / low affinity Abs must be incubated for long periods in order to reach equilibrium. But nothing can be gained by prolonging primary Ab incubation beyond the time at which the tissue is saturated with antibody.

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Incubation Time Equilibrium is usually not reached before 20 min. Consistent timing is important. Inconsistent timing leads to variations in overall stain quality & intensity.

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Incubation temperature Equilibrium may achieved more quickly at 37C compare to RT. Incubation at 37C allows higher dilution + shorter incubation time ------ > consistency in incubation time becomes even becomes more crucial. Incubation at 37C ----- > increase background

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Incubation temperature 4C is used in combination with overnight or longer incubations. Slides incubated for extended periods, or at 37C should be placed in humidity chamber to prevent evaporation and drying of tissue sections. Tissue incubated at RT in a very dry or drafty environment will require the use of a humidity chamber.

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Specimen requirements Paraffin-embedded blocks vs. fresh frozen vs. cytospin prep. cytological slides. Paraffin-embedded blocks vs. fresh frozen vs. cytospin prep. cytological slides. Comprising the representative lesion. Not too small. Comprising the representative lesion. Not too small. Devoid of necrosis. Devoid of necrosis. Devoid of extensive hemorrhage. Devoid of extensive hemorrhage. Not too old Paraffin blocks < 3 years old. Not too old Paraffin blocks < 3 years old. Free from over fixation. Free from over fixation. Don t treat with overheated paraffin during embedding & Don t treat with overheated paraffin during embedding & processing. processing. Proper labeling. Proper labeling. Clear and precise IHC request. Clear and precise IHC request. Corresponding conventional pathology report. Corresponding conventional pathology report.

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Fixation

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Fixation Each tissue has finite amount of Ag Each tissue has finite amount of Ag Most steps in IHC process destroy some of the Ags Most steps in IHC process destroy some of the Ags particularly : fixation particularly : fixation

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Fixation The purpose of fixation: The purpose of fixation: 1. To change protein structure in order to preserve them from 1. To change protein structure in order to preserve them fromelution,degradation, and other modifications 2.To preserve the position of the Ag ; 2.To preserve the position of the Ag ; Nuclear Nuclear Cytoplasmic Cytoplasmic Membrane-bounded Membrane-bounded 3.Preserve secondary and tertiary structue 3.Preserve secondary and tertiary structue 4.To provide a target for Abs 4.To provide a target for Abs

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Fixation Elution of ER protein from nucleus to cytoplasm Elution of ER protein from nucleus to cytoplasm Elution of Cerb-b2 from membrane to cytoplasm Elution of Cerb-b2 from membrane to cytoplasm Therefore diagnostically the stain is useless. Therefore diagnostically the stain is useless. Poor or inadequate fixation Incorrect interpretation

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Fixation Tissue :A Neutral buffered formalin ER as target Ag Monoclonal Ab clone E15 Monoclonal Ab clone E45RT Result: negativeResult: positive Fixation may destroy specific epitopes thus may lead to improve two different reactions with two different monoclonal Abs

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What is the solution? Standardization of the fixative and fixation protocols would be an start. Standardization of the fixative and fixation protocols would be an start.

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Fixation & Ag retrieval Aldehyde cross link after formalin fixation AB Ag

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There is no one universal fixative that is ideal for the demonstration of all antigens. There is no one universal fixative that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories. The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.

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Some antigens will not survive even moderate Some antigens will not survive even moderate amounts of aldehyde fixation. amounts of aldehyde fixation. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocols After fixation, the sections can be processed using standard immunohistochemical staining protocols

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Ten percent neutral buffered formalin, pH 7 (10% NBF) Fresh&Buffered to pH of 7.0-7.6 Fresh&Buffered to pH of 7.0-7.6 Formalin 40% 100 mL Dibasic sodium phosphate, anhydrous, Na2HPo4 6.5 g Monobasic sodium phosphate, monohydrate, KH2Po4 4.0 g DW 900 mL

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Most common fixatives a) 4% paraformaldehyde in 0.1M phosphate buffer b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry)

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Other aldehyde-based fixative Glutaraldehyde 2% Glutaraldehyde 2% Act similarly to 10% NBF and are used much less frequently Act similarly to 10% NBF and are used much less frequently

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Mercuric chloride fixatives Used in the past Used in the past Mechanism: react with amino acid residues such as thiols, amino groups, imidazole, phosphate, and hydroxyl groups. Mechanism: react with amino acid residues such as thiols, amino groups, imidazole, phosphate, and hydroxyl groups. Fixation time : is short, as a positive point Fixation time : is short, as a positive point Highly toxic with special disposal procedures, as a negative point Highly toxic with special disposal procedures, as a negative point

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Mercuric chloride fixatives 1.B5 fixative 1.B5 fixative

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Mercuric chloride fixatives 2.Zenker s fixative 2.Zenker s fixative

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Alcoholic Fixatives Carnoy s Carnoy s In looking at lymphocytes using CD-specific markers In looking at lymphocytes using CD-specific markers In looking for immunoglubins such as IgG, IgA, IgM In looking for immunoglubins such as IgG, IgA, IgM

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1. Tissue not dry out : onto moist absorbent paper,in a covered container 2. Rapid delivery to the path. Lab. 3. Trimming and cut for fixation 4. Into blocks no more than 2 cm square by four mm thick Tissue pretreatment

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5. Thickness is important: 1. Penetration: ideally fast 2. Fixation : usually slow 6. Optimum fixation time: 6-12 hr 7. Over fixation can pose problems: increased cross linking 8. How to repair this damage? Heating the fixed tissue in boiling water to 95 degrees for 15-20 min Heating the fixed tissue in boiling water to 95 degrees for 15-20 min Tissue pretreatment

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Tissue & slide processing Once the tissue is well-fixed, subsequent steps seem to have little effect on antigen detection. Once the tissue is well-fixed, subsequent steps seem to have little effect on antigen detection. Variation in xylol processing, alcohol rehydration, wax temperature, time or formulation, instrumentation used etc, provide satisfactory results. Variation in xylol processing, alcohol rehydration, wax temperature, time or formulation, instrumentation used etc, provide satisfactory results.

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Tissue & slide processing No process should raise temperature to higher than 60C, No process should raise temperature to higher than 60C, as this will cause severe loss of antigenicity that may not be recoverable. as this will cause severe loss of antigenicity that may not be recoverable.

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Tissue & slide processing Tissue fixation medium must be replaced by wax, generally done through a series of incubations in increasing alcohol concentrations to 100 percent, followed by xylene and then hot wax. Tissue fixation medium must be replaced by wax, generally done through a series of incubations in increasing alcohol concentrations to 100 percent, followed by xylene and then hot wax. This is to provide stability of the tissue (wax) in order to make cutting the sections easier. This is to provide stability of the tissue (wax) in order to make cutting the sections easier.

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Tissue & slide processing Appropriate thickness: 3-4 microns Appropriate thickness: 3-4 microns No more than 5 microns No more than 5 microns

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Tissue & slide processing Commercially available slides with positive charge. Commercially available slides with positive charge. Albumin coated slides Albumin coated slides Silane coated slides Silane coated slides Poly-L-Lysine coated slides Poly-L-Lysine coated slides Sections that are not flat and that have non- adherent ridges likely will be digested or torn off the slide during immunostain. Sections that are not flat and that have non- adherent ridges likely will be digested or torn off the slide during immunostain.

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Tissue & slide processing : De-waxing protocol A.Circle & label the specimen with a diamond pencil B.Place in 60C oven for 30 minutes C.Transfer immediately to a fresh xylene bath for three minutes. D.Repeat step C above with a second xylene bath. E.Place in a fresh bath of absolute alcohol for three minutes. F.Repeat step E above with a second bath of absolute alcohol. G.Place in a bath with 95 percent ethanol for three minutes. H.Repeat step G with the second 95 percent ethanol bath. I.Rinse under gently running water. J.Do not let dry, store in buffer; begin required Ag treatment or immunostaining Note: 50 slides per 250 mL of xylene is the limit before the xylene Is no longer effective and residual wax begins causing artifacts in the final stained tissue.

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Procedure 1.3 micron slide sections37c48hr 2.XylolRT15min 3.XylolRT15min 4.Absolute alcoholRT15min 5.96 alcoholRT15min 6.70 alcoholRT15min 7.DWRT rinse/*2/5min 8.3%H2O2/methanol v/vRT30min 9.DWRT rinse/*2/5min 10.Citrate buffer pH=6microwave14min 11.CoolingRTgradually

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Procedure 12.PBSRTrinse/*2/5 min 13.Protein blockRT10min 14.1 st AbRT10-60min 15.PBSRTrinse/*2/5 min 16.2 nd AbRT30min 17.PBSRTrinse/*2/5 min 18.Detection systemRT30min 19.PBSRTrinse/*2/5 min 20.Chromogen DABRT10min 21.DWRTrinse 22.HematoxylineRT3 dips (10 sec)

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Citrate Buffer pH=6.0 2.1 gracid citric monohydarte 900mLDW 13mLNaOH 2 normal Up to 1000mL

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PBS 10x Na2HPo411.5 gr NaCl80 gr KCL2 gr KH2Po43.4 gr Up to 1000mL

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Antigen Retrieval Protocols Break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. Heat Induced Epitope Retrieval (HIER) Hydrochloric Acid Method (pH 1) Hydrochloric Acid Method (pH 1) Formic Acid Method (pH 2) Formic Acid Method (pH 2) Citrate Buffer Method (pH 6) Citrate Buffer Method (pH 6) Citrate-EDTA Buffer Method (pH 6.2) Citrate-EDTA Buffer Method (pH 6.2) EDTA Method (pH 8) EDTA Method (pH 8) Tris-EDTA Method (pH 9) Tris-EDTA Method (pH 9) TBS Method (pH 9) TBS Method (pH 9) Tris Buffer Method (pH 10) Tris Buffer Method (pH 10)

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Heat Induced Epitope Retrieval (HIER) Pressure cooker Pressure cooker Autoclave Autoclave Microwave Microwave Ag retrieval solutions: Ag retrieval solutions: 0.01 M citrate pH=610x 0.01 M citrate pH=610x 1 mM EDTA10x 1 mM EDTA10x 20 mM Tris/0.65 mM EDTA/0.0005% Tween 20 pH=910x 20 mM Tris/0.65 mM EDTA/0.0005% Tween 20 pH=910x

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Proteolytic Induced Epitope Retrieval (PIER) Proteinase K Method Proteinase K Method Trypsin Method Trypsin Method Pepsin Method Pepsin Method Pronase Method Pronase Method Protease Method Protease Method Frozen Section Epitope Retrieval SDS Method SDS Method Heating En Bloc Method Heating En Bloc Method

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Blocking: The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.

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Blocking: Background staining may be specific or non-specific. Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed.

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Non-immunological binding Non-immunological binding Inadequate or delayed fixation common in the center of large tissue blocks binding of the specific immune sera by hydrophobic and electrostatic non-specific background staining is non-immunological binding usually uniform and can be reduced by blocking those sites with normal serum. blocking those sites with normal serum.

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Blocking: Antibodies, specially polycolonal antibodies, are sometimes contaminated with other antibodies due to impure antigen used to immunize the host animal.

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Immunological binding impure antigen used to immunize the host animal contaminated polyclonal antibodieswith other antibodies contaminated polyclonal antibodies with other antibodies Non-specific Immunological binding

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Non-immunological background Peroxidase Block Endogenous peroxidase activity is found in many tissues and can be detected by reacting fixed tissue sections with DAB substrate. The solution for eliminating endogenous peroxidase activity is by the pretreatment of the tissue section with hydrogen peroxide prior to incubation of primary antibody.

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30% H2O2 ------------------------- 2 ml 30% H2O2 ------------------------- 2 ml Methanol --------------------------- 18 ml Methanol --------------------------- 18 ml Mix well and store at 4 C. Mix well and store at 4 C. Block sections for 20-30 minutes after primary antibody incubation. Block sections for 20-30 minutes after primary antibody incubation. Note: The solution must be fresh. Note: The solution must be fresh. Peroxidase Blocking Solution (3% H2O2 in Methanol)

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Endogenous alkaline phosphatase (AP) activity Many tissues also contain endogenous alkaline phosphatase (AP) activity and should be blocked by the pretreatment of the tissue section with levamisole if using AP as a label.

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Blocking: Some tissues such as liver and kidney have endogenous biotin. Some tissues such as liver and kidney have endogenous biotin. To avoid unwanted avidin binding to endogenous biotin if using biotin-avidin detection system, a step is necessary for these tissues by the pretreatment of unconjugated avidin which is then saturated with biotin. To avoid unwanted avidin binding to endogenous biotin if using biotin-avidin detection system, a step is necessary for these tissues by the pretreatment of unconjugated avidin which is then saturated with biotin.

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Avidin 0.001% in PBS Biotin 0.001% in PBS Store these blocking solution at 4 C. Incubate sections for 10-15 min each and rinse with PBS between steps. Recommended to block before primary antibody incubation. Avidin/Biotin Block

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Controls: Positive control Positive control To use the tissue of known positive as a control. If the positive control tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.

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Controls: Negative control is to test for the specificity of an antibody involved. First, no staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.

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Controls: Second, the staining must be inhibited by adsorption of a primary antibody with the purified antigen prior to its use, but not by adsorption with other related or unrelated antigens. Second, the staining must be inhibited by adsorption of a primary antibody with the purified antigen prior to its use, but not by adsorption with other related or unrelated antigens. This type of negative control is ideal and necessary in the characterization and evaluation of new antibodies but it is sometimes difficult to obtain the purified antigen, therefore it is rarely used routinely in immunohistochemical staining.

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Direct Method: Direct method is one step staining method, and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections:DFA This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.

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Indirect Method: Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised).

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Indirect Method: This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens.

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PAP Method (peroxidase anti-peroxidase method) Three layer method Rabbit antibody to peroxidase, coupled with peroxidase Unconjugated goat anti-rabbit gaba-globulin

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PAP Method (peroxidase anti-peroxidase method) The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.

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Avidin-Biotin Complex (ABC) Method Is standard IHC method. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies.

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Avidin-Biotin Complex (ABC) Method Three layers method. 1.The first layer is unlabeled primary antibody. 2.The second layer is biotinylated secondary antibody. 3.The third layer is a complex of avidin-biotin peroxidase. 4.The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.

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Labeled StreptAvidin Biotin (LSAB) Method Streptavidin, derived from streptococcus avidini, is a recent innovation for substitution of avidin. The streptavidin molecule is uncharged relative to animal tissue, unlike avidin which has an isoelectric point of 10, and therefore electrostatic binding to tissue is eliminated. In addition, streptavidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining. In addition, streptavidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining.

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Labeled StreptAvidin Biotin (LSAB) Method 1.The first layer is unlabeled primary antibody. 2.The second layer is biotinylated secondary antibody. 3.The third layer is Enzyme-Streptavidin conjugates (HRP- Streptavidin or AP-Streptavidin) A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method.

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Polymeric Methods: Polymeric Methods:

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Polymeric Methods: Dextran polymer technology. Binding of a large number of enzyme molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone. The benefits are many, including increased sensitivity, minimized non-specific background staining reduction in the total number of assay steps

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Polymeric Methods: Procedure: i) Application of primary antibody; i) Application of primary antibody; ii) Application of enzyme labeled polymer; iii) Application of the substrate chromogen. EnVision+ was developed after EnVision to provide increased sensitivity.

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Chromogen Substrate Solutions DAB-Peroxidase Substrate Solution (Brown) DAB-Peroxidase Substrate Solution (Brown) DAB-Peroxidase Substrate Soluiton (Gray) DAB-Peroxidase Substrate Soluiton (Gray) DAB-Peroxidase Substrate Solution (Black) DAB-Peroxidase Substrate Solution (Black) DAB-Peroxidase Substrate Solution (Blue) DAB-Peroxidase Substrate Solution (Blue) AEC-Peroxidase Substrate Solution (Red) AEC-Peroxidase Substrate Solution (Red) BDHC-Peroxidase Substrate Solution (Blue) BDHC-Peroxidase Substrate Solution (Blue) TMB-Peroxidase Substrate Solution (Blue) TMB-Peroxidase Substrate Solution (Blue) New Fuchsin Alkaline Phosphatase Substrate Sulution (Red) New Fuchsin Alkaline Phosphatase Substrate Sulution (Red) BCIP/NBT Alkaline Phosphatase Substrate Solution (Blue) BCIP/NBT Alkaline Phosphatase Substrate Solution (Blue)

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Protocol for DAB Peroxidase Substrate Solution DAB Peroxidase Substrate Solution Brown Final Dilution: 0.05% DAB - 0.015% H2O2 in 0.01M PBS, pH 7.2 0.05% DAB - 0.015% H2O2 in 0.01M PBS, pH 7.2 Stock Solutions: 1% DAB (20x) in Distilled Water: 1% DAB (20x) in Distilled Water: Add 0.1g of DAB (3,3 -diaminobenzidine tetrahydrochloride, Sigma) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and store at 20 C. Add 0.1g of DAB (3,3 -diaminobenzidine tetrahydrochloride, Sigma) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and store at 20 C. 0.3% H2O2 (20x) in distilled water: 0.3% H2O2 (20x) in distilled water: Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 C or aliquot and store at 20 C. Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 C or aliquot and store at 20 C.

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Counterstain Solutions Gill's Hematoxylin Solution (Blue) Gill's Hematoxylin Solution (Blue) Mayer's Hematoxylin Solution (Blue) Mayer's Hematoxylin Solution (Blue) Nuclear Fast Red Solution (Red) Nuclear Fast Red Solution (Red) Methyl Green Solution (Green) Methyl Green Solution (Green) PI Counterstain Solution (Fluorescent Red) PI Counterstain Solution (Fluorescent Red) DAPI Counterstain Solution (Fluorescent Blue) DAPI Counterstain Solution (Fluorescent Blue)

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 155

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TO APPLY IHC OR ICC FOR ? 1.Diagnostic purpose. 1.Diagnostic purpose. 2.Prognostic purpose 2.Prognostic purpose 3.Therapeutic purpose 3.Therapeutic purpose 4.Preventive purpose? 4.Preventive purpose?

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Applications of IHC Diagnostic purpose:

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Applications of IHC Diagnostic purpose: IHC profile for metastatic carcinoma of unknown origin FemaleMale ER/PRPSA CK 7 CK 8 CK 20 CD X2 CEACEA TTF 1

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Diagnostic purpose of IHC CK 7 CK 8 CK 20 Dx ++- Follicular adenocarcinoma,Thyroid Adenocarcinoma,Pancreas -++ Hepatocellualr carcinoma -+- Squamous cell carcinoma CK 5/6 --- Thymoma CK 5/6 Thymoma CK 5/6

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Diagnostic purpose of IHC CK 8 CK 18 Dx ++ Follicular adenocarcinoma,Thyroid Papillary adenocarcinoma,Thyroid Adenocarcinoma,Pancreas Hepatocellualr carcinoma +-Trichoepithelioma -+Meningioma Collectind duct carcinoma --Thymoma Adenocarcinoma, Ampullary

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Applications of IHC Diagnostic purpose: an example IHC profile for prostatic carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 162 IHC in prostate Cancer Indications: Indications: 1- Distinction of Benign from Malignant High molecular weight cytokeratin (34 E12, CK5/6)High molecular weight cytokeratin (34 E12, CK5/6) Negative cytoplasmic marker (in basal cells) P63P63 Negative nuclear stain (in basal cells) AMACR (P504S)AMACR (P504S) Positive cytoplasmic marker (in tumor cells) Also positive in HGPIN, 31% of Bladder Ca. & 70% of Colorectal Ca.

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 163 HMW-CK (34 E12) Normal Glands Negative in Carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 164 AMACR (P504S) stain in Carcinoma

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 165 P63 / AMACR cocktail Increasing IHC resolution:

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 166 34 E12 / P63 / AMACR 2-chromogen cocktail Increasing IHC resolution:

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 167 IHC in prostate Cancer (Cont.) Indications: Indications: 2- Differential Dx from urothelial carcinoma: PSA PSAP 34 E12 Leu7 CK5/6 PSA PSAP 34 E12 Leu7 CK5/6 Prostate Ca + + + - Urothelial Ca + +

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2003 By Default! A Free sample background from www.powerpointbackgrounds.com Slide 168 IHC in prostate Cancer (Cont.) Indications: Indications: 3- Differential Dx in metastatic carcinoma: Bone Tumor: PSA stain

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Applications of IHC Prognostic purpose in breast carcinoma Poor prognostic markers Good prognostic markers ER(-)ER(+) PR(-)PR(+) pS2(-)pS2(+) Her-2 (3+) Her-2 (0/1+) Cathepsin D(+) Cathepsin D(-) P 53 >10% P 53 23%Ki-67

Potential for misdiagnosis (IHC) Antibodies (>28 commercially available) Technical performance Interpretation scoring artifacts

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Common problems in HER2 IHC Underestimation of expression: (over)fixation in NBF poor antigen retrieval (unmasking) choice of antibodies Overestimation alcoholic (post)fixation (check normal cells!) Cytoplasmic staining

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Who can interpret an IHC?

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Normal lobule 3+ breast carcinoma

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Lobular cancerization

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2+ breast carcinoma

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2+ breast carcinoma?

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Normal lobule

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Cytoplasmic staining

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What to do? Central Reference or large local laboratories high number of cases (>250 cases/year* or >100 cases/months [NSABP]) quality assurance controls (internal and external) automated IHC high level of training (technique/interpretation) Small laboratories (

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but There are labs capable of performing quality testing with lower volumes A high test volume does not ensure an accurate test result If an individual lab can properly validate an assay and perform acceptably in an external validation, then it should be permitted to offer the test Hsi ED & Tubbs RR: JCP 2004

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Applications of IHC therapeutic purpose Chemotherapy in breast carcinoma ER (+) PR (+) Tamoxifen / Femara ER (-) PR (+) Tamoxifen / Femara ER (+) PR (-) Tamoxifen / Femara Her-2 psoitive Trastazumab(Herceptin) Cost :300 000 000 Rials Her-2 negative --

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Applications Of IHC Therapeutic purpose: PharmaDx Abs Therapeutic purpose: PharmaDx Abs Chemotherapy in breast carcinoma Chemotherapy in breast carcinoma ER - / PR - / Her-2 - ER - / PR - / Her-2 - Triple negative breast carcinoma Treatment is completely different: Cisplatin Cisplatin