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Introduction. Basic histological techniques.
Histological staining (acid, basic, histochemistry).
Všeobecné lekárstvo
MUDr. Pavol Janega
MUDr. Svetoslav Štvrtina
MUDr. Peter Michalka Lucia Donárová
Ústav patologickej anatómie LFUK a UN pracovisko Staré mesto
Sasinkova 4, Bratislava
Prof. MUDr. Ľudovít Danihel, CSc.
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What is pathology?
• Processing biological material
tissue samples from living or dead organism
biopsy necropsy
• Determination of diagnosis or pathological process / cause of death
• Therapeutic management
GOAL
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SAMPLING BIOLOGICAL MATERIAL
BIOPSY
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CYTOLOGY
• excisional (dg, th) • incisional • core-cut (core needle)
Exfoliative: • spontaneous (body fluids) • mechanical (brushing...) Interventional: • needle aspiration
CYTOLOGY
• Advantages:
• fast
• cheap
• sampling of hardly accessible tissue
• Disadvantage:
• Not able to precisly classify and determine tumor type typify
• Not able to determine invasion
• False positivity and false negativity
• SCREENING METHOD!!!
• in a certain population to identify the possible presence of an as-yet-undiagnosed disease in individuals without signs or symptoms
• Sampling – representative
• sample marking
• sample labeling
• sampling artefacts
• Fixation – immediatly aftert sampling
• prevent autolysis and microbiological decomposition
• Adequate transport medium
• Send to path-lab
Sample labeling:
- age - insurance information - clinical diagnosis - anamnesis - symptoms of disease - sample desciption (what,
from where, side...)
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FIXATION
1. Fixation with loss of biological function - chemical, physical methods
- cross-linkages formed in the proteins
- denaturation of proteins - terciary structure change
- without any antigenicity change
2. Fixation preserving biological function - frozen sections
- biological and enzymatic activities of proteins will not change during this process
- intraoperative diagnostic procedures to guide the surgeon
- in techiques for recovery of DNA, mRNA, and proteins
- enyzmatic histochemistry
formalin 10%
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GROSS EXAMINATION
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TISSUE PROCESSING
Goal: processing into a form from which the thin microscopic sections can be prepared
Tissue in fixative solution (formalin)
Dehydration – removing the water and fixative solution (ethanol, methanol,...) - In series of concentrations 70%..90%..100%
Clearing – removing of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). (xylen, toluen, chloroform,...)
Impregnation – embedding medium
Tissue impregned in embedding medium (paraffin)
SECTIONING
- after the embedding the samples must be cut into sections that can be placed on a slide - microtome and ultramicrotomes
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SPECIMEN STAINING
ZÁKLADNÉ PRINCÍPY FARBENIA
• Acidofile stains:
• - Eosine, Azokarmine, Anilin blue
• Substrates: Cytoplasma, intercellular substances,...
• Bazofile stains:
• - Metyl blue, Toluidine, Hematoxyline
• Substrates: Chromatin, Ribozomes,...
• Impregantions
• - Salt of silver or gold
• - neuronal and glial cells
• BASIC STAINS:
HEMATOXYLIN AND EOSIN
OIL RED
PERIODIC ACID SCHIFF
ALCIAN BLUE
RETICULIN
GRAM STAIN
ACID FAST STAIN...
ENYZME HISTOCHEMISTRY
ENZYME HISTOCHEMISTRY
• perserve E activity during fixation and processing
• frozen sections
Tissue with active enyzme substrate
Intermediate product
percipitate
reactant
IMMUNOHISTOCHEMISTRY
IMMUNOHISTOCHEMISTRY
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• The using of immunological methods in addition to morphological evaluation in the diagnosis of diseases allows the demonstration of various proteins production in tissues
• Evaluation of the presence, localization and quantification of specific proteins (Ag) by a specific antibody (Ab)
• Significance
• The diagnosis (presence or absence of specific markers)
• The prognosis (prognostic markers)
• The therapy (proof of markers having a relationship to the effectiveness of treatment, affecting resistance, adequacy of the used treatment)
Method
• Reliable and reproducible
• Low costs
Currently, the gold standard of diagnosis
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* *
* *
primary antibody
secondary antibody (anti-rabbit, mouse, goat,...)
antigen
Fluorescent stain
fluorescence method
positive = flourescens in fluorescent microscope
primary antibody (rabbit, mouse, goat,...)
secondary antibody (anti-rabbit, mouse, goat,...)
antigen
Enzyme (peroxidase)
Dye (DAB) precipitate (brown color) in light microscope
enzymatic method
IMMUNOHISTOCHEMISTRY
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• Diagnostic markers (cytokeratins, vimentin...)
• Prognostic markers (Ki67...)
• Therapeutic markers (Her2...)
CYTOKERATINS
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• Epithelial cells
VIMENTIN
• Mezenchymal cells
AE1/3 cytokeratíny
KI67
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• Positive in proliferating cells in late G1,S,G2 and M phase, but not in G0 and early G1
phase
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ELECTRON MICROSCOPY
MOLECULAR PATHOLOGY