MHCY112 Embedding PDF
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Michener Institute of Education at UHN
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Summary
This document provides an overview of embedding procedures, including learning objectives, equipment descriptions, and various steps in the process. It includes diagrams and descriptions of different types of tissues and embedding molds.
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Embedding Learning Objectives 1. Define “Embedding". 2. State the general principle of embedding. 3. Label the following parts on a diagram of the Tissue-Tek II embedding center, and state their function(s): a)Wax reservoir b)Storage area c)Forceps warmer d)Wax dispensing nozzle...
Embedding Learning Objectives 1. Define “Embedding". 2. State the general principle of embedding. 3. Label the following parts on a diagram of the Tissue-Tek II embedding center, and state their function(s): a)Wax reservoir b)Storage area c)Forceps warmer d)Wax dispensing nozzle e)Hot orientation plate f)Hot working platform g)Cold plate h)Main cooling plate i) Cold drawer 2 Learning Objectives j. Heat switch k.Cold switch l. Thermostat 4. Describe Tissue-Tek II and III processing cassettes and the labeling procedure appropriate for each. 5. Outline the procedure for embedding a piece of tissue. 6. Compare the differences between properly and poorly oriented tissues, with particular reference to the following: a)Rectangular piece of tissue b)Collagen capsule c)Layered tissue d)Fragments (curetting) e)Tubular structures f)Multiple pieces. 3 Learning Objectives 7. Given a diagram or an embedded block, recognize the following embedding errors/faults and state their consequences: a)Mold too small for tissue b)Mold too large for tissue c)Tissue floating d)Error in identification e)Incorrect processing f)Contaminated wax g)Air bubbles or water droplets h)Wax stratification i) Tissue not centered j) Ring not filled adequately k)Incorrect orientation 4 Embedding Embedding involves enclosing the tissue in the infiltration medium used for processing and then allowing the medium to solidify 9/21/2023 5 Principle of Embedding Choice of Paraffin with appropriate melting point (affects tissue support and ribboning) Proper specimen orientation Light pressure be applied over the entire specimen during orientation Open and embed one cassette of tissue at a time to avoid tissue mix up Number of tissue pieces submitted in each cassette should be recorded 6 9/21/2023 7 Leica Embedding Machine 9/21/2023 8 Parts & Functions: 1.The wax reservoir stores and heats the paraffin wax. 2.The storage area keeps the metal molds warm for embedding. 3.The forceps warmer holds forceps and prevents wax accumulation. 4.The wax dispensing nozzle dispenses paraffin from the reservoir and is controlled by a switch just behind the nozzle. Pushing the switch starts the wax flow – releasing it stops the wax flow. The nozzle contains a filter. 5.The hot orientation plate allows for orientation of the tissue specimen in hot liquid wax. 6.The cold plate allows the tissue to be set to the base of the embedding mold, thus maintaining its orientation. 9/21/2023 9 Parts & Functions: 7. The main cooling plate is a large refrigerated plate to solidify blocks. 8. The cold drawer collects the overflow of excess paraffin wax and must be emptied periodically. 9. The heat switch controls the heater for the wax reservoir and the hot orientation plate. 10. The cold switch controls the cold plate and the main cooling plate. 11. A squeegee is supplied, to wipe excess paraffin wax from the hot orientation plate to the grooved right-hand part of the working platform & into the cold drawer. 9/21/2023 10 Cassettes The TISSUE-TEK II system uses the plastic bottoms of Tissue-Tek II or Tissue-Tek III processing cassettes They are filled with wax through the perforations. A great advantage of this system is that the specimen identification number is written on the cassette when the specimen is grossed, and therefore does not require further transposing from one place to another. This lessens the possibility of transcription errors. These cassettes also fit standard base molds. 9/21/2023 11 9/21/2023 12 Embedding Molds 1 9/21/2023 3 Embedding Procedure 1 9/21/2023 4 Embedding Procedure 1. Select correct size mold 2. Pick up tissue specimen from the opened cassette with heated forceps and look for the flat or marked side of the tissue. 3. Partially fill the well of the mold with molten paraffin 4. Place tissue against the base of the mold with the flat or marked side down. 5. Orient the tissue diagonally (on a slight angle) then introduce the “initial chilling” on the cold plate 1 9/21/2023 5 Embedding cont. 6. Apply gentle pressure on the tissue so that it will be embedded flat. 7. Place cassette on top and add more wax through the perforations. 8. Transfer the mold-cassette “system” on to the cooling plate (temp: -4° C to - 6° C) 9. When cooled, remove blocks from molds 10. File and sort in ascending order 1 9/21/2023 6 Embedding A newer embedding technology: https://www.youtube.com/watch?v=f2Di a7kgf0Q 1 9/21/2023 7 Specimen Orientation When embedding, placement and orientation of tissue in the base mold is very important. The following slides give examples of correct and incorrect embedding. Different types of tissues are illustrated by line drawings; the drawings show the face of the block to be cut. 9/21/2023 18 Rectangular Piece of Tissue A slight angle is recommended. Orient the tissue specimen on an angle (~45). 9/21/2023 19 Specimen With An Obvious Collagen Capsule Imagine the blue bold line is the cutting blade A Figure A shows ok embedding, except it would be nice to orient the tissue at an angle Figures B show good embedding with the capsule not getting in touch with the B blade first. The tissue is at an angle too that optimizes cutting Figure C illustrates poor embedding; cutting collagen first is difficult and may C cause tissue compression. 9/21/2023 20 Layered Tissue (Skin) Figure A shows good embedding. All layers are flat against the base. Figure B is unacceptable because only one layer (the epidermis) can be seen on the face of the block. 9/21/2023 21 Fragments (Curettings) These often arrive wrapped in lens paper or gauze. Each individual piece must be seen as much as possible Do not pile them up All of the tissue is represented on the slide when cut. 9/21/2023 22 Tubes (Appendix, Veins) Tubes should be embedded so that a cross-section is cut. Such specimens are more difficult to cut, but all layers and the lumen (the space within) must be visible. 9/21/2023 23 Multiple-Piece Block Tissues are embedded side-by-side, and therefore won't be dragged through one another during microtomy. 9/21/2023 24 Error in Identification Error in identification may increase the possibility of a wrong diagnosis. 9/21/2023 25 Soft Tissues Incorrect processing. Difficult or impossible to cut. Tissues described as “mushy” or “soft” 9/21/2023 26 Difficult to cut Contaminated wax (e.g., suture or dirt) Difficult to cut. Suture must be removed. 9/21/2023 27 Air bubbles/ water Air bubbles or water droplets. Loss of internal support makes it difficult to cut 9/21/2023 28 Wax Stratification Wax stratification. Block may fall apart and damage tissue. Do not allow lower layer to harden completely before filling cassette. 9/21/2023 29 Peripheral Tissue Tissue not centered. Difficult to cut, especially if the tissue is touching the side or edge. 9/21/2023 30 Poor block support Cassette not filled in adequately. Poor support for the block. More wax required. 9/21/2023 31 Incorrect orientation Incorrect orientation Wrong side of tissue will be cut (not the side the pathologist wants to see) Tumour or area of interest may be lost in cutting 9/21/2023 32 Final Product…. Paraffin block and corresponding stained slide 3 9/21/2023 3 References 1. Armed Forces Institute of Technology. Laboratory methods in histotechnology, 1992. Washington DC: American Registry of Pathology; 1992. 2. Carson FL. Histotechnology: A Self-Instructional Text. ASCP Press. 2020 Edition: 5th. 9/21/2023 34