1 / 39

WELCOME

WELCOME. MY INTRODUCTION.

parry
Download Presentation

WELCOME

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. WELCOME

  2. MY INTRODUCTION I am Alie Wouda and I am living in Holland. During 15 years I worked on the department of pathology. That is now 17 years ago, meanwhile I’m studying p e herbs, homeopathy and so on. Because my husband finished with his study medicine and became a Pathologist we moved from the north of the Netherlands to the south. In 1995 I started a practice of my own, because I was finished also.

  3. We were asked to make a start with the implementation of immuno-histochemistry in the department of Pathology of the KCKM in Moshi. We started two weeks ago and the results are promissing.

  4. Something about tumor cells First I tell you something about the tumor cells. When the pathologist doesn’t know which cell is seen microscopically, the cells might probably be of a primary mammacarcinoma or a metastasis of an other tumor he can perform an immuno-histochemical investigation. The difference is important because the treatment can be very different of the tumors.

  5. This afternoon we tell something about • Immunoresponse • (Basic principle) immunology • Fixation . Antibodies • Immunology protocol

  6. Immunoresponse WikipediaThe immune system is a defense system, the purpose is to destroy bacteria or changed cells of the own body. Defense against pathogens/ infectious bacteria: • Virusses • Bacteria • Fungi • Protozoa • parasites

  7. Immuun response Types immuun response • Biologica immuun response • Respons on infectious diseaes • Vaccination • Experimentele immuun response • Production of monoclonal, polyclonal antibodies • Research at function/ action of immune system

  8. Immunology Basic principal Interaction between antigen and the fitting antibody Wikipedia:Immunology is the biological science researching the immunonological system: the defencemechanisms which in organisms prevents the entering of organisms (bacteria) and cells of outside the body.

  9. Immunology

  10. Fixation Purpose fixation: • Make cells/tissue resistant for histochemical proceedings • Decreasing catabolic processes • autolyse • heterolyse (demolition by microorganisms) • Prevent extraction • Fixate 3D structure (of proteins)

  11. Fixation After fixation you still need: • materials with contrast • Conformation of antigens • Genetical information • Residual enzyme activity

  12. Fixation Fixation, what is important; • Kind of fixative • Duration of fixation • Temperature

  13. Fixation methods • Physical • Freeze • Chemical • Coagulation (methanol, aceton) • Not coagulation, crosslinking (formalin)

  14. Fixation Chemical: Coagulation (Ethanol, aceton) • Proteins, colloïdal solvability disappears, and condensate • Carbohydrates condensate • Nucleic acids condensate • Fat dissolves

  15. Fixation Chemical: Not coagulate, crosslinking(Formaline 3,7%) • Resistance against - extraction in organic solvents - conformation of changes, possibly made during tissue processing • Protection of antigenic determinants • Crosslinks incraeses the molecular density

  16. Fixation Formaldehyde vs Formaline Formaldehyde Formaline H OH C H OH

  17. Fixation Diffuse ability fixative D = k • Vt D = diameter, half (radius) in mm K = diffusioncoëfficiënt T = time in houres K for Formaline 0,78

  18. Fixation Example: tissue of 4x4x3 mm Howlong takes it before the tissue is fixed by formalin?

  19. Antibodies Production of: • Polyclonale antibodies Antibodies, derived from different cells or diversity of cells. Therefore they are a mixture of many different specifications. • Monoclonal antibodies Antibodies, derived of only one single B-lymphocyte (plasmacel).

  20. Antibodies

  21. Antibodies Advantages and disadvantages of polyclonal antibodies • Advantages: • High harvest • Several clones plasmacells • conformation change of antigens • Production procedure more simple than monoclonale antibodies • Multivalent interactions between antigens and polyclonal antibodies • Relative low costs

  22. Antibodies Advantages and disadvatages of polyclonal antibodies • Disadvantages: • Large laboratory animals ( goat, sheep, pig) • Stock antiserum is finite • There are also undesired AB present, so a greater chance for cross reactions • Respons of the laboratory animals on the immunisations are not consistant during a period of time

  23. Antibodies Advantages and disadvantages of monoclonal antibodies • Advantages: • High, exclusife specificity, monospecific • No / few background • Immunisation with weak immunogens • Constant quality en quantity

  24. Antibodies Advantages and disadvantages of monoclonal antibodies • Disadvantages: • Procedure is sensentive for loss of antigenicity • Low sensitivity at low concentration of antigen • (Too) high specificity • Loss of quality of hydromacells

  25. Antibodies HAT selectie

  26. Antibodies Structure antibody Immunoglobulin monomer

  27. Antibodies Different Isotypes The H- chains determine the antibody class (the Isotype)

  28. Antibodies Affinity = binding strength between 1 antibody en 1 epitope Avidity = total binding strength of a complex consists of antigens, different epitopes and their counterparts (antibodies)

  29. Antibodies Affinity is determined by: • Fitting • Size of the complementary area • Amount of interactions • Quality of the interactions Interactions: H-bridges, ion binding, hydrofobic interactions, induced dipoles, and van der Waals forces

  30. Antibodies Crossreactivity • Antibody reacts with other unrelated antigen: • 2 antigens with the same identical Ag determinant • 2 antigens with the same structural similar Ag determinant • The affinity of the determinant with which the crossreaction happens is often lower

  31. Immuno protocol Recommended Staining Protocol 1. Deparaffinize and rehydrate tissue section. 2. To reduce non-specific background staining due to endogenous peroxidase, incubate slide in hydrogen peroxide for 10-15 minutes. 3. Wash 2 times in PBS or TBS wash buffer. 4. If required, incubate tissue in digestive enzyme or perform appropriate HIER pre-treatment. 5. Wash 2 times in PBS or TBS wash buffer. 6. (Optional) Apply Pre-antibody Blocking Solution (NGS)and incubate for 5 minutes at room temperature to block non-specific background staining. 7. Wash 2 times in PBS or TBS wash buffer). 8. Apply primary mouse or rabbit or rat antibody and incubate according to manufacturer's protocol. 9. Wash 2 times in PBS or TBS wash buffer. 10. Apply Post-antibody Blocking and incubate for 15 minutes at room temperature. 11. Wash 2 times in PBS or TBS wash buffer. 12. Apply Poly-HRP-Goat anti Mouse/Rabbit IgGand incubate for 30 minutes at room temperature. 13. Wash 2 times in PBS or TBS wash buffer. 14. Incubate with peroxidase-compatible chromogen 15. Counterstain and coverslip.

  32. Immuno protocol

  33. Immuno protocol

  34. Immuno protocol CD 30 Mib-1

  35. Different types • Actin (Smooth Muscle) • Adrenocorticotropin (ACTH) • Albumin • Alpha-1-Antitrypsin • Alpha-1-Fetoprotein • AMACRprstate • Amyloid A • Androgen Receptor • Bax • BCL2 Oncoprotein • BCL6 Protein • Beta-Catenin • BRCA1 • CA 19-9 • CA 125 • Calcitonin • Caldesmon • Calponin • Calretinin • Carcinoembryonic • Antigen (CEA)

  36. CD1a • CD2 • CD3 • CD5 • CD5 • CD7 • CD8 • CD10 • CD14 • CD15 • CD19 • CD20cy • CD21 • CD23 • CD23 • CD30 • CD31, Endothelial Cell • CD34 Class II • CD35 • CD43 • CD3 • CD4 • CD44, Phagocytic Glycoprotein-1 • CD45, Leucocyte Common Antigen • CD45R0 • CD45RA • CD56 • CD57 • CD61, Platelet Glycoprotein IIIa

  37. CD68 • CD68 • CD68 • CD79α • CD79αcy • CD99, MIC2 Gene Products, Ewing's Sarcoma Marker • CD117, c-kit • CD138 • CD246, ALK Protein • CDX2 • Chorionic Gonadotropin (hCG) • Chromogranin A • Collagen IV • COX-2 • Cyclin D1 • Cytokeratin • Cytokeratin • Cytokeratin 5/6 • Cytokeratin 7 • Cytokeratin 10 • Cytokeratin 10/13 • Cytokeratin 17 • Cytokeratin 18 • Cytokeratin 19 • Cytokeratin 20 • Cytokeratin, High Molecular Weight • Cytokeratin, Wide Spectrum Screening • Cytomegalovirus • D2-40 • Desmin • E-Cadherin • EGFR epidermal growth factor receptor • Epithelial Membrane Antigen (EMA) • Epstein-Barr Virus, LMP • Estrogen Receptor α

  38. Glial Fibrillary Acidic Protein (GFAP) • Helicobacter Pylori • Hepatitis B Virus Core Antigen (HBcAg) • Hepatocyte • Herpes Simplex Virus Type 1 • Herpes Simplex Virus Type 2 • Human Immunodeficiency Virus (HIV), p24 • IgA, IgG, IgM, Kappa, Lambda • Inhibin α • Kappa Light Chains • Ki-67 Antigen proliferatie marker • Lambda Light Chains • Leukaemia, Hairy Cell • Melan-A • Mesothelial Cell • Neuron-Specific Enolase (NSE) • p53 Protein • Papillomavirus (HPV) • Parvovirus B19 • Placental Alkaline Phosphatase • Plasma Cell • Progesterone Receptor • Prostate-Specific Antigen (PSA) • Renal Cell Carcinoma Marker • S100 • Serotonin • Somatostatin • SynaptophysinTerminal Deoxynucleotidyl Transferase (TdT)Thyroid-Stimulating Hormone (TSH)Thyroid Transcription Factor (TTF-1) • Vimentin

  39. So that’s wants to tell Thank you for the attention Alie Wouda from the Netherlands

More Related