PS1_Histo_Lect2_part 1_Power Point Presentation.ppt

Pathological Sciences 1 Surgical cut-up and Fixation Lecture 2 Miss Yulia Humrye, BSc (Hon) [email protected] SM 3.28b Contents           Cellular injury, necrosis, apoptosis Tissue and cells preservation and fixation Characteristics of fixatives Standard methods of fixation For...

Pathological Sciences 1 Surgical cut-up and Fixation Lecture 2 Miss Yulia Humrye, BSc (Hon) [email protected] SM 3.28b Contents           Cellular injury, necrosis, apoptosis Tissue and cells preservation and fixation Characteristics of fixatives Standard methods of fixation Formalin ideal anti-bacterial treatment Tissue selection and description Types of samples Sampling endometrium Sampling skin Examples of surgical specimens and slides Cellular injury … Cellular Injury Numerous causes Physical and chemical agents  Various mechanisms Disruption, membrane failure, metabolic interference  May be reversible, or end in cell death  Cell replication in human body at a rate of 10,000 new cells per second  Cell dearth is a normal physiological process as well as reaction to injury  Similarly, failure or poor regulation of death processes may underlie some diseases  Lethal Cell Injury Necrosis = accidental cell death A pathological term referring to areas of dead cells within a tissue or organ. This is an outcome rather than process.  Apoptosis = programmed cell death Is a process of physiological cell deletion that has an opposite role to mitosis in the regulation of cell populations.  To fix or not to fix … To Fix Probably from Old French verb “fixer” from fixe - "fixed” From Latin fixus "fixed” - immovable; established, settled "position from which it is difficult to move" Fix Why fixation is needed?      Cessation of normal life functions in the tissue (killing) Stabilization of the structure of the tissue (preservation) The goal of fixation is to preserve structure as faithfully as possible compared to the living state. Post-mortem change is a summation of: Ischaemia Autolysis Putrefaction These changes are minimised/prevented by the process called - fixation Fixation = the process of preserving cells Ischaemia – restriction in blood supply to tissues, causing a shortage of oxygen and food (glucose), and build up of waste.  Autolysis – uncontrolled release of intra-cellular enzymes from lysosomes (proteases, nucleases) – autolysed cells stained with H&E show enhanced cytoplasmic staining (more pink = eosinophilia) and reduced nuclear staining (less purple).  Putrefaction – action of micro-organisms: bacteria, fungi with associated smell = production of gases. Together = post-mortem changes If the shape and structure of cells are allowed to deteriorate then diagnosis becomes impossible.  Prevention of cellular deterioration is done by one of two processes: fixing or freezing. Features of necrosis and apoptosis Tissue and cell preservation … Fixed? Fixation aims to preserve cells and tissue constituents in as close to a lifelike state if possible.  Stabilise proteins  Arrest autolysis  Prevent bacterial decomposition  Allows further preparative procedures Appearance of the tissue after fixation is artefactual Cellular components when examined alter with the effect of different pathological conditions Factors affecting the rate of fixation  Temperature  Size of specimen and penetration of fixative  Volume  pH and buffers  Osmolarity  Concentration  Duration Characteristics of fixation … Characteristics of fixation Fixation is largely irreversible  Fixation prevents post mortem changes  Fixation preserves cells/organelles/tissues  Fixation modifies proteins  Hardening of the specimen  Fixation helps processing, sectioning, staining  Choice of fixative NB ALL are hazardous (toxic vapours, highly irritant)  Chemically active (react with proteins)  Penetrate tissue rapidly  Retain tissue and cellular structure  Safe to use (including disposal)  Cost effective Types of fixative  Simple – only one active ingredient  Compound - 2 or more active ingredients NB many formulations include buffers: these do not act as fixatives, but maintain physiological pH, osmolarity, retain shape.  Coagulative/precipitant –  Denature proteins e.g. alcohol Non-coagulative/non-precipitant – Formalin also described as cross-linking as bridges are made between proteins. Can be partially washed out with running water (single additions). Coagulant fixatives      Both organic and non-organic solutions may coagulate proteins making them insoluble Cellular architecture in-vivo is maintained primarily by lipoproteins and fibrous proteins such as collagen Coagulating these proteins maintains tissue histomorphology at the light microscopy level Coagulant fixatives result in cytoplasmic flocculation and poor preservation of mitochondria and secretory granules These fixative are not useful in ultrastructural analysis Non-coagulant cross-linking fixatives    Fixatives forming cross-links both within and between proteins and nucleic acids Examples formaldehyde, glutaraldehyde and other aldehydes Aldehyde groups are chemically and biologically recitative and are responsible for many histochemical reactions The side chains of peptides or proteins which are most reactive with methylene hydrate have the highest affinity for formaldehyde – lysine, cysteine, histidine, arginine, tyrosine, and reactive hydroxyl groups of serine and threonine