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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* *

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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