PS1_Histo_Lect3_part 1_Power Point Presentation.pptx

PATHOLOGICAL SCIENCES 1 TISSUE PROCESSING LECTURE 3 Miss Yulia Humrye, BSc (Hon)[email protected] SM 3.28b CONTENTS 1. Tissue processing 2. Tissue embedding 3. Microtomy (cutting sections) 4. Staining and mounting 5. Quality assurance LEARNING OBJECTIVES  Discuss the principles and pract...

PATHOLOGICAL SCIENCES 1 TISSUE PROCESSING LECTURE 3 Miss Yulia Humrye, BSc (Hon)[email protected] SM 3.28b CONTENTS 1. Tissue processing 2. Tissue embedding 3. Microtomy (cutting sections) 4. Staining and mounting 5. Quality assurance LEARNING OBJECTIVES  Discuss the principles and practices of tissue processing  Discuss the reagents used in tissue processing  Discuss principles of embedding  Discuss principles of cutting blocks using a microtome  Discuss systems to maintain the quality of work within the laboratory STEPS FROM SPECIMEN TO REPORT 1. Fixation   2. Specimen collection, transportation and receipt  3. Tissue selection and description  4. Routine Tissue processing  5. Tissue embedding  6. Microtomy  7. Staining and mounting  8. Quality assurance  9. Reporting  10. Specimen disposal  11. Block & Slide archiving Specimen (in fixative) and request form Specimen description and selection of tissue blocks Processing Fixation Dehydration Clearing Impregnation Tissue embedding Examination of H&E stained slides under the light microscope (digital pad is used for Section mounting on a slide Microtome cutting of FFPE blocks Formalin-fixed Paraffinembedded (FFPE) blocks ROUTINE TISSUE PROCESSING: THE PARAFFIN TECHNIQUE  The aim is to prepare tissues in a supporting medium ready to be cut into thin sections.  To achieve the aim firstly is to remove water from tissues  Thereafter, to saturate tissues with molten paraffin wax  Wax is immiscible with water, the tissue must transfer through a number of intermediary steps util it is able to be impregnated with wax – paraffin processing.  The tissue blocks prepared during specimen dissection are immersed in a number of different chemical solutions.  Paraffin wax is the embedding medium of choice for routine specimens – it is cheap, easy to handle, non-toxic, hard enough to support the cutting of thin sections, when solid paraffin was has consistency very similar to that of the tissue itself. STAGES OF TISSUE PROCESSING  Fixation – routine fixation in 10% Neutral buffered formalin  Dehydration removes water with graded series of ethanol *can also use methanol, propanol, acetone.  Clearing reagent acts as a link between the dehydrator and wax. Xylene is the most widely used. *safe natural extracts/substitutes are available.  Impregnation is to infuse all parts of the tissue structure with the embedding medium (most commonly wax) *resin is an alternative if thin sections are needed e.g. EM REAGENTS USED IN TISSUE PROCESSING  Dehydrating solutions – the most popular are industrial methylated spirit (IMS) (primary ethanol) and isopropyl alcohol (isopropanol) (also can act as dehydrating and clearing agent).  Clearing solutions – in routine practice Xylene (di-methylbenzene).  Impregnation agents – in routine practice Paraffin wax (crystalline hydrocarbon). Pure paraffin wax is not used, but with additives such as: *dimethyl sulphoxide (to assist with even good penetration of tissues) *microcrystalline (has good ribboning properties) *ceresin, beeswax, rubber or asphalt (provides good adhesion between the tissue and supporting medium) HEALTH & SAFETY Industrial Methylated Spirit Xylene  Highly flammable  Flammable  Harmful by inhalation and if  Harmful by inhalation and in contact swallowed  Requires specialist disposal  If comes in contact with eyes or skin it needs to be washed well in running water with skin, is irritating to eyes and skin  Carcinogen  Xylene waste must be disposed of as specialist disposal  If skin becomes splashed – wash well under running water.  If it is splashed in eyes – following rinsing under running water, medical advice should be sought. ROUTINE PROCESSING Tissue processing schedules are validated by using surplus clinical material not required for diagnosis. The manufacturers will offer guidance and set of programs suitable for different tissues e.g. types fatty, large, small. Time of processing schedule may vary depending on the brand of equipment and reagents used. TISSUE PROCESSORS TISSUE PROCESSORS: SHANDON THERMO FISHER VS LEICA ASP300 Leica ASP300 Shandon Thermo Fisher