Histology 4 - Introduction to Histological Methods PDF

Important relation between the morphology of a cell and its role —> different tissues = morphological different cells The information needed for a protein to function is contained in its tertiary structure (3d arrangement) Hierarchical structure —> DNA determines RNA —> RNA determines proteins’ st...

Important relation between the morphology of a cell and its role —> different tissues = morphological different cells The information needed for a protein to function is contained in its tertiary structure (3d arrangement) Hierarchical structure —> DNA determines RNA —> RNA determines proteins’ structures Changes in environmental conditions may lead to a change in the function of a cell Some cells have a xed shape while others can go through conformational changes dictated by the environment, the compression excreted by continuous cells or the relationships between the cells and the extra-cellular matrix (es blood cells change their structure and function according to the quantity of water they retain) Histology = study of cells structure and their changes All anatomical systems can be identi ed based on the appearance of their cells (es: islets of Langerhans are only found in the pancreas) A study brought on in 2015 tried to correlate the different morphologies of blood vessels to breast cancers types and stages State —> pattern con guration of a system at a particular instant liverstates ofthecell'smorphologydueto illness change Six steps of the histological image Sampling (taking a sample from the tissue) Fixation (block the degradation of the tissue avoiding necrosis) Processing (preparation of the tissue before embedding) Embedding Cutting (usually between 2-10 microns) Staining (making the targeted cells visible) SAMPLING Gathering of matter from the body to aid the process of medical diagnosis and evaluate the situation —> allows to state the best treatment, a suggestion for further tests or other procedures Sampling allows u to state the type of a tumour, its histological stage and the degree to which it extends Sampling techniques —> aspiration (es: amniocentesis), frozen section (used for an immediate diagnosis during surgery —> fresh tissue is frozen and cut in a cryostat) and scraping (es: Pap test —> scraped off of uterine cervix cells) To determine the stage of a cancer it is crucial to use a longitudinal section of the cell (no cross section) —> patched diseases develop in certain spots and not in others so having the sample from the right spot is crucial Biopsy —> preparation that maintains the natural tissue structure of the sample (individual cells and their spatial organisation) Samples for cytopathology —> only individual cells without maintaining their spatial conformation Fine needle aspiration —> different needles used to extract cells from different tissues. Sometimes this technique is guided by X-rays or ultrasounds FIXATION Blocking of the process of necrosis to analyse a sample which is as similar to as it was in the body. Fixation converts the semi- uid consistency of cells to a semi-solid one. Immersion —> The tissue is immersed in a xative (usually formalin) of a volume at a minimum of 10-15 times greater than the volume of the tissue to be xed. The xative diffuses through the tissue and hardens it. Perfusion —> The xative reagent is injected in the heart with its volume matching the cardiac output —> the xative spreads along the whole body and the tissues don’t die as long as they’re xed —> this is the method used when the structure needs to be maintained Delays in xation and mistakes in concentration lead to a poor xation with tissue loss —> a tissue can be over- xed or under- xed and this can lead to a fallout analysis Freezing —> used when an immediate analysis is needed —> the tissues are placed in a cryoprotective embedding medium and then snap frozen in isopentane cooled by liquid nitrogen. Advantages —> better preservation of antigenicity (maintains structure), minimal exposure to xatives, no exposition to organ solvents, much faster. Disadvantages —> lack of morphological detail (presence of water in the tissue which is otherwise removed with other methods), presence of potential biohazard (there may be alive pathogens in the tissue) Paraformaldehyde preserves the structure while methanol doesn’t PROCESSING Diffusion of various reagents into and out of the porous tissue (no ATP needed). The tissues are dehydrated, cleared (removal of the fat tissue) and in ltrated (adding of reagents instead of water, they preserve the structure) EMBEDDING Paraf n embedding —> standard method, it preserves the structure better than the frozen section —> the tissue is surrounded by paraf n wax which cools down and provides support for the section cutting CUTTING Video A microtome is used to cut the slices in thin sections. In 10 microns of tissue you might have two layers of cells and thus see an overlay —> the thinner is the section the easier it is to analyse it. STAINING Pre-staining —> wax is removed through the use of xylene (melts the wax) and then the tissue is rehydrated by immersing it in decreasing concentrations of alcohol Histochemistry —> general stains that bind to tissue structures without recognising any speci c structure Immunohistochemistry (IHC) —> conventional dye which can be seen with a normal microscope. Stain has antibodies that recognise speci c antigens and that bind to them (thus staining them) Immuno uorescence —> The stain allows the tissue to emit light HISTOCHEMISTRY (IHC) Principal staining technique used —> two reagents are used —> ematoxylin (acidophilus, binds to the nuclei and stains them with a blue color) and eosin (basophilic, stains the tissues in red) Trichrome stain —> three color components —> selective recognition of connective tissues Silver staining —> developed by Camillo Golgi —> useful to identify intracellular and extracellular components such as DNA and proteins through the deposition of silver on the targets. IMMUNOCHEMISTRY The antibodies used can be polyclonal (mix of clones of antibodies that can bind to different antigens of a speci c protein) or monoclonal (one clone that binds to a speci c antigen of a speci c protein). Polyclonal have a higher chance of interacting but it can result in a tricky staining (can bind to antigens similar to each other) POLYCLONAL Uman tibodiesableto recognizetheantigen injected MONOCLONAL Used continue toproliferate seeidentical thecellscanthen beputin culture The samples are incubated with the antibodies to which chromogen is bound (to stain the place where the antibodies have bound). A secondary antibody bound to chromogen can also be used —> it binds to the primary antibody  amplification ofthesignee Cstainitory better b ecause abinasontheupperpertogsenerant i tworeaconseitssaap otherwise DAB —> 3,3 Diaminobenzidine —> most used with IHC staining as a chromogen—> it gets oxidised by hydrogen peroxide thanks to the enzyme HRP (horseradish peroxidase) and forms a brown precipitate at the location of the HRP —> can be visualised through the use of a light microscopy Tissue microarrays (TMAs) consist of paraf n blocks in which up to 1000 separate tissue cores are assembled in array to allow multiplex histological analysis Human blood smear —> used to recognised the composition of blood monocytes O heritrocytes of precursors macrophages beanshaped nucleus Olalymphocytes bananabignocens msn.am gets