This document outlines the key methods and techniques used in pathology, focusing on histopathology. It describes the process of fixing, processing, embedding and sectioning tissue samples, then staining them with hematoxylin and eosin for examination under a microscope. Tissue samples are fixed in formalin, dehydrated, cleared, infiltrated with paraffin wax, embedded and sectioned thinly before being mounted on slides and stained with H&E to identify structures. This staining method allows examination of cell and tissue morphology.
This document outlines the key methods and techniques used in pathology, focusing on histopathology. It describes the process of fixing, processing, embedding and sectioning tissue samples, then staining them with hematoxylin and eosin for examination under a microscope. Tissue samples are fixed in formalin, dehydrated, cleared, infiltrated with paraffin wax, embedded and sectioned thinly before being mounted on slides and stained with H&E to identify structures. This staining method allows examination of cell and tissue morphology.
This document outlines the key methods and techniques used in pathology, focusing on histopathology. It describes the process of fixing, processing, embedding and sectioning tissue samples, then staining them with hematoxylin and eosin for examination under a microscope. Tissue samples are fixed in formalin, dehydrated, cleared, infiltrated with paraffin wax, embedded and sectioned thinly before being mounted on slides and stained with H&E to identify structures. This staining method allows examination of cell and tissue morphology.
TECHNIQUES EMPLOYED IN PATHOLOGY, SO THAT SUBSEQUENT ADVANCED KNOWLEDGE OF PATHOLOGY MAY BE LEARNED SWIFTLY. FOLLOWING ARE SOME OF THE IMPORTANT AND COMMON METHODS, TECHNIQUES AND TOOLS USED IN THE STUDY OF PATHOLOGY I) HISTOPATHOLOGY (SURGICAL PATHOLOGY) HISTOPATHOLOGY DEALS WITH THE GROSS AND MICROSCOPIC EXAMINATION OF TISSUES. TISSUES ARE RETRIEVED SURGICALLY FROM THE BODY AND SENT TO THE HISTOPATHOLOGY LABORATORY FOR THE PATHOLOGICAL EXAMINATION OF THE SPECIMEN. THE REPRESENTATIVE TISSUE SECTIONS SHOULD NOT BE MORE THAN 3-4 MM IN THICKNESS. THE SECTIONS ARE PLACED IN STEEL OR PLASTIC CAPSULES, OR CASSETTES AND ARE SUBJECTED TO THE FOLLOWING STEPS OF PROCESSING, BEFORE THEY CAN BE EXAMINED UNDER A MICROSCOPE. 1. FIXATION: THE MAIN PURPOSE OF FIXATION IS TO PRESERVE TISSUES, SO AS TO MAINTAIN THEIR NATURAL STRUCTURE AS CLOSELY AS POSSIBLE FOR MICROSCOPIC EXAMINATION, AS WELL AS PRESERVATION FOR MUSEUM DISPLAY. FORMALINE IS THE MOST COMMONLY USED FIXATIVE FOR LIGHT MICROSCOPY THE OTHER FIXATIVES USED IN PATHOLOGY ARE GLUTARALDEHYDE (FOR ELECTRON MICROSCOPY) 2. TISSUE PROCESSING:
THE FIXED TISSUE MUST BE PROCESSED INTO A
FORM IN WHICH IT CAN BE CUT INTO THIN MICROSCOPIC SECTIONS. THE MAIN PURPOSE OF PROCESSING IS TO REPLACE THE WATER IN THE TISSUES WITH AN EMBEDDING MEDIUM (USUALLY PARAFFIN WAX), WHICH GIVES IT A FIRM CONSISTENCY, SO AS TO ENABLE THIN SECTIONING FROM THE TISSUE. TISSUE PROCESSING INVOLVES THE FOLLOWING STEPS: A. DEHYDRATION
THE PURPOSE OF THIS STEP IS TO REMOVE WATER
AND FIXATIVES FROM THE TISSUES (DEHYDRATE), AND REPLACE IT WITH A DEHYDRATING FLUID (MOST COMMONLY ETHYL ALCOHOL), WHICH IS SUBSEQUENTLY REPLACED PERMANENTLY BY WAX. OTHER DEHYDRATING AGENTS ARE ACETONE, METHYL ALCOHOL, AND ISOPROPYL ALCOHOL. B. CLEARING
THE NEXT STEP IS CLEARING IN WHICH THE
DEHYDRATING AGENT (ETHYL ALCOHOL) IS REPLACED BY A MEDIUM IN WHICH PARAFFIN WAX IS COMPLETELY SOLUBLE. THIS STEP ALSO MAKES THE TISSUE TRANSPARENT, HENCE CALLED CLEARING. THE MOST COMMONLY USED CLEARING AGENT IS XYLENE. OTHER CLEARING AGENTS INCLUDE TOLUENE, BENZENE, CHLOROFORM. C. IMPREGNATION
IN THE STAGE IN WHICH THE CLEARING AGENT
(XYLENE) IS FINALLY REPLACED BY MOLTEN PARAFFIN WAX. WAX IMPREGNATION HARDENS THE TISSUES, MAKING THEIR HANDLING AND SECTIONING EASIER. D. TISSUE EMBEDDING AND BLOCKING: BLOCKS ARE CONVENTIONALLY PREPARED BY USING METALLIC LEUCKHART’S L MOLDS. TISSUE EMDEDING AND BLOCKING CAN ALSO BE PERFORMED IN SPECIALIZED EQUIPMENT CALLED TISSUE EMBEDDING STATION. 3. MICROTOMY ONCE THE TISSUES HAVE BEEN EMBEDDED INTO BLOCKS, THEY MUST BE CUT INTO THIN SECTIONS THAT CAN BE PLACED ON THE SLIDE. ROTARY MICROTOME IS THE MOST COMMONLY USED MICROTOME, IN WHICH THE KNIFE IS FIXED AND THE TISSUE BLOCK IS MOVABLE, AND CAN BE ADVANCED TOWARD THE BLOCK THE IDEAL HISTOPATHOLOGICAL SECTIONS SHOULD BE UNIFORM AND 3-4 MICRONS IN THICKNESS. THE THINNER THE SECTIONS, THE BETTER AND CRISPER ARE THE MORPHOLOGY AFTER STAINING. THE SECTIONS ARE FLOATED IN A WATER BATH MAINTAINED AT A TEMPERATURE OF 5˚C -10˚C LESS THAN THE MELTING POINT OF THE WAX (54˚C - 62˚C). THESE SECTIONS ARE NOW GENTLY LIFTED ONTO GLASS SLIDES PRECOATED WITH EGG ALBUMIN, WHICH ENHANCE THE ADHERENCE OF THE SECTION TO THE SLIDE. 4. HEMATOXYLIN AND EOSIN STAINING BEFORE STAINING, THE PARAFFIN IS WAX IS REMOVED FROM TISSUE BY RUNNING THE SLIDES THROUGH XYLENE TO ALCOHOL FOLLOWED BY WATER. HEMATOXYLIN AND EOSIN (H&E) IS THE UNIVERSALLY USED STAIN FOR ROUTINE MICROSCOPY. HEMATOXYLIN IS A NUCLEAR STAIN, WHICH IS EXTRACTED FROM THE LOGWOOD OF A TREE – HAEMATOXYLUM CAMPECHIANUM. HEMATIN, WHICH IS THE ACTIVE INGREDIENT OF THE STRAIN, WHICH IMPARTS THE BLUE COLOR, IS PRODUCED BY OXIDATION. EOSIN IS A XANTHENE DYE, WHICH STAINS THE CYTOPLASM PINK. 5. MOUNTING AND COVER SLIPPING BEFORE STAINTING, THE PARAFFIN WAX IS REMOVED FROM TISSUE BY RUNNING THE SLIDES THROUGH XYLENE TO ALCOHOL FOLLED BY WATER. THE STAINED SECTION ON THE SLIDE IS MOUNTED IN A MOUNTING MEDIUM(DISTRENE, PLASTICISE, XYLENE), AND THEN COVERED WITH A FINE PIECE OF GLASS, COVER SLIP. MOUNTING PROTECTS THE TISSUE FROM BEING SCRATCHED, PROVIDES A BETTER OPTICAL QUALITY FOR VIEWING UNDER THE MICROSCOPE AND PRESERVES THE TISSUE SECTION FOR YEARS.