PS1_Histo_Lect3_part 2_Power Point Presentation.pptx

PATHOLOGICAL SCIENCES 1 TISSUE PROCESSING LECTURE 3 Miss Yulia Humrye, BSc (Hon)[email protected] SM 3.28b Processing artefacts:  Artefacts arise due to incomplete processing - poor sectioning, especially fatty or hard tissues  Insufficient tissue fixation / formalin penetration – resul...

PATHOLOGICAL SCIENCES 1 TISSUE PROCESSING LECTURE 3 Miss Yulia Humrye, BSc (Hon)[email protected] SM 3.28b Processing artefacts:  Artefacts arise due to incomplete processing - poor sectioning, especially fatty or hard tissues  Insufficient tissue fixation / formalin penetration – results in incomplete processing, which affects overall morphology, chromatin detail, tissue integrity.  Excessive time in formalin produces pigment = formalin pigment A formalin-fixed paraffin section of kidney showing the typical deposition of acid formaldehyde hematin (formalin pigment) associated with red blood cells. The pigment is brown to black in colour and is birefringent under polarized light. In this case the specimen remained in fixative for an extended period before processing. FURTHER CONSIDERATIONS ABOUT TISSUE PROCESSING  In recent years the processing is more tailored to the specimen under investigation  Xylene-free processing with advanced processing machines can be implemented. Isopropyl alcohol can be used to replace xylene either in combination with ethanol, or as both dehydrating and clearing agent.  Processing of fatty tissues requires extended time.  Large blocks (mega blocks) require longer time but processing equipment has improved and in return the number of larger blocks has increased to meet reporting demands.  Tissue reprocessing – the process of tissue processing is reversible and where processing has been unsuccessful e.g. all of the water has not been removed from the tissue , then material can be rehydrated and the process recommenced.  Hard tissue – a pre-treatment can be used either a softener solution or a de-calcification agent (e.g. formic acid). TISSUE EMBEDDING PROCESS Tissue embedder 1. Tissues removed from molten wax 2. Placed and orientated in mould 3. Cooled on cold plate 4. Cassette is placed on top of the 5. Cold blocks are removed from the 6. Blocks Paratrimmed MICROTOMY  Blocks trimmed  Cooled on wet ice before sectioning  Sectioned on a rotary Manual Leica microtome Manual Sakura Manual Thermo Scientific Manual Pfm Semi-automated Leica Automated Pfm The Accu-Cut® SRM™ 300 LT Manual Microtome is the first and only American made manual microtome with a patented multi-colored LED backlit chuck to illuminate paraffin embedded tissue samples making them much easier to see and safer to section. https://www.sakuraus.com/Products/Microtomy/Accu-Cut-SRM-300-LT-Manual- MICROTOMY Routine section thicknes s 3-4 microns STAINING EQUIPMENT Leica Automated Stainer And coverslipper Dako Automated Stainer and coverslipper Sakura Automated Stainer and COVERSLIPS Automated + Manual Or HISTOLOGY RESULTS H&E stained tissue: microscopic view Mega slide demonstrates prostate gland tissue stained with H&E H&E microscopic view: pipelle endometrial biopsy a b H&E microscopic view: MyoSure Routine H&E slide: a. Pipelle endometrial biopsy endometrial biopsy; b. MyoSure endometrial QUALITY ASSURANCE: INTERNAL QUALITY CONTROL Block checking  After cut-up before blocks are put for processing, the case number and the number of blocks are recorded, either photographed or written in order. Microscope checking  Each slide or a selection of slides  After embedding all blocks (case number and number of blocks) are recorded too.  After sections cut and stained the blocks are checked against the slides cut so that the shape of the sections are checked and the patient name and the case number are checked. Block – slide check from a case, is/are checked under the microscope for staining quality and for quality of microtomy.  Sections that are of poor quality are rejected and a replacements are cut. Examples of microtomy defects 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