MT 5: HISTOPATHOLOGY HISTOPATHOLOGY (LABORATORY) METHODS OF FRESH TISSUE EXAMINATION Histopathologic techniques Involves different procedures that have been adopted for the preparation of materials and tissues for microscopic investigation whether they are normal or abnormal Includes examination of smears, preservation and processing of tissue sections prior to actual evaluation of tissue details Importance A well processed tissue can help in the confirmation and proper evaluation of disease entity leading to a proper mode of treatment Main concern of a Medical Technologist See to it that the tissues collected have been properly preserved and adequately prepared for microscopic study Central Dogma of Tissue Processing Prepare the section of the solvent of the next reagent Tissue Processing 1. Logging 2. Fixation 3. Decalcification 4. Dehydration 5. Clearing 6. Impregnation 7. Embedding 8. Trimming 9. Sectioning 10. Staining 11. Mounting 12. Reading 13. Reporting Examination of fresh tissues Teasing or dissociation Squash preparation (crushing) Smear preparation (streaking, spreading, pullapart)
TEASING/ DISSOCIATION DISSOCIATION A selected tissue specimen is immersed in a watch glass containing NSS, carefully dissected or separated and examined SQUASH PREPARATION Small pieces of tissue are placed on a microscopic slide and forcibly compressed with another slide and coverslip SMEAR PREPARATION PREPARATION Cellular materials are spread lightly over a slide a. STREAKING - With an applicator stick or platinum loops, the material is rapidly but gently applied in a straight line or zigzag line throughout the slide attempting to obtain a relatively uniform distribution of secretion b. SPREADING - A selected portion of the specimen is transferred to a clean slide - Spread moderately into thick film c.
PULL APART - A drop of secretion or sediment is placed at the center of a clean slide - Another slide is placed over it and the specimen is allowed to disperse evenly between the two slides - Slight movement of the two slides are then pulled apart with a single uninterrupted motion - The specimen is then examoined under the microscope in the unstained state/ applied with vital stain - Thick secretion/ serous fluids: concentrated sputum, enzymatic lavage, samples from gastrointestinal tract and blood
d. TOUCH PREPARATION/ IMPRINT/ IMPRESSION MTD
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This is done by bringing into contact the surface of a freshly cut piece of a tissue onto the surface of a clean slide The tissue is pressed lightly to the side allowing to be transferred directly to the slide for examination using a phase contrast microscope or bright filed microscope after application of vital stain Has the added advantage in that cells may be examined without destroying their actual intercellular relationship and without separating them from their normal surrounding
FROZEN SECTION Utilized when rapid diagnosis of tissue is required If lipids and nervous tissue elements are to be demonstrated -25° C (-13° F) Rapid diagnosis (guide for intra-operative patient management) To optimally process tissues for special studies for diagnosis, treatment, or research To confirm that lesional tissue is present for diagnosis on permanent sections (sample adequacy) Limitations: Limited section sampling Ice crystal or freezing artefact Inferior quality compared to paraffin sections Lack of special studies (time constraint) - Special stains, immunohistochemistry, culture Lack of consultation for difficult cases
Tissue Processing Fixation Decalcification (for boen tissues) Dehydration Clearing Impregnation Embedding Trimming Sectioning Staining Mounting
CLEARING/ DEALCOHOLIZATION Replacing of alcohol in tissues Prepares tissue for infiltration because most clearing agents are miscible with the infiltrating agent 10% Neutral Buffered Formalin
FIXATION Kills, hardens, preserves tissues for the next histopath steps “Life-like” appearance – prevention of degeneration, putrefaction, decomposition, distortion – protein stabilization (cross links formed between fixative and proteins) Reduce risk of infection Promotes staining Inhibit bacterial decomposition Tissue size: 1-2cm long x 1-2cm wide x 4-5 mm thick TimeL 24-48 hrs Amount: must not be less than 10x the volume of tissue being fixed Specimen Color Texture Consistency Size Type of fixative: 10% formalin, Bouin’s fluid, Regaud’s fluid
Zenker’s Fluid (Mercuric Chloride)
Regaud’s Fluid (Chromate)
Running water Alcohol,
3-12 hrs
1-12 hrs
1-6 hrs
1-6 hrs
Alcohol,
1-6 hrs
1-6 hrs
Alcohol,
3-12 hrs
1-6 hrs
1-6 hrs
3-12 hrs
Alcohol,
3-12 hrs
1-6 hrs
1-6 hrs
3-12 hrs
Alcohol,
3-12 hrs
1-6 hrs
1-6 hrs
3-12 hrs
Alcohol,
3-12 hrs
1-6 hrs
1-6 hrs
3-12 hrs
Alcohol,
3-12 hrs
1-6 hrs
1-6 hrs
3-12 hrs
Xylol I
1-2 hrs 1 hr
1-2 hrs 1 hr
1-2 hrs 1 hr
1-2 hrs 1 hr
Xylol II
INFILTRATION/ IMPREGNATION Is the process whereby the clearing agent is completely removed from the tissue and replaced with a medium that will completely fill all the tissue spaces and cavities Oven is set at 2-5 degrees above the melting point of wax used Paraffin wax 1
Specimen
Color
Texture
Consistency
Size
Fixative 10% Formalin Bouin’s fluid Regaud’s fluid
DEHYDRATION Process of removal of water in tissues Uses ascending concentration of agent to gradually and completely remove water from tissues
Bouin’s Fluid (Picric Acid)
Paraffin wax 2
Paraffin wax 3
