WELCOME

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.

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.

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.

This afternoon we tell something about

(Basic principle) immunology

Fixation

. Antibodies

Immunology protocol

Immunoresponse

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

Immuun response

Types immuun response

Biologica immuun response

Experimentele immuun response Production of monoclonal, polyclonal antibodies

Respons on infectious diseaes Vaccination

Research at function/ action of immune system

Immunology

Basic principal

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.

Interaction between antigen and the fitting antibody

Immunology

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)

Fixation

After fixation you still need: materials with contrast

Conformation of antigens

Genetical information

Residual enzyme activity

Fixation

Fixation, what is important;

Duration of fixation

Kind of fixative

Temperature

Fixation methods

Physical Freeze

Chemical Coagulation (methanol, aceton)

Not coagulation, crosslinking (formalin)

Fixation

Chemical: Coagulation

(Ethanol, aceton)

Proteins, colloïdal solvability disappears, and condensate

Carbohydrates condensate Nucleic acids condensate Fat dissolves

FixationChemical: 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

FixationFormaldehyde vs Formaline

Formaldehyde Formaline

H OH

C

H OH

Fixation

Diffuse ability fixative

D = k • V t

D = diameter, half (radius) in mm

K = diffusioncoëfficiënt

T = time in houres

K for Formaline 0,78

Fixation

Example: tissue of 4x4x3 mm

Howlong takes it before the tissue is fixed by formalin?

AntibodiesProduction of:

Polyclonale antibodies

Monoclonal antibodies

Antibodies, derived from different cells or diversity of cells. Therefore they are a mixture of many different specifications.

Antibodies, derived of only one single B-lymphocyte (plasmacel).

Antibodies

AntibodiesAdvantages and disadvantages of polyclonal antibodies Advantages:

High harvest

Several clones plasmacells conformation change of antigens Production procedure more simple than monoclonale antibodies

Relative low costs

Multivalent interactions between antigens and polyclonal antibodies

AntibodiesAdvantages 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

AntibodiesAdvantages and disadvantages of monoclonal antibodies Advantages:

High, exclusife specificity, monospecific

No / few background

Immunisation with weak immunogens

Constant quality en quantity

AntibodiesAdvantages 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

AntibodiesHAT selectie

AntibodiesStructure antibody

Immunoglobulin monomer

AntibodiesDifferent Isotypes

The H- chains determine the antibody class (the Isotype)

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)

AntibodiesAffinity 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

AntibodiesCrossreactivity

Antibody reacts with other unrelated antigen:

The affinity of the determinant with which the crossreaction

happens is often lower

2 antigens with the same identical Ag determinant

2 antigens with the same structural similar Ag determinant

Immuno protocolRecommended 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 IgG and 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.

Immuno protocol

Immuno protocol

Immuno protocol

CD 30

Mib-1

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)

• 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

• 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•

• 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• Synaptophysin Terminal Deoxynucleotidyl Transferase (TdT) Thyroid-Stimulating Hormone (TSH) Thyroid Transcription Factor (TTF-1)• Vimentin• •

So that’s wants to tell

Thank you for the attention

Alie Wouda from the Netherlands