Embedding processes-pptx

Histopathology Lecture slides about tissue embedding for histopathology and histopathology university students

1. By Geoffrey Mutale (MMLS-MUST) 1 Histopathology lecture slide Embedding processes MUTALE GEOFFREY Masters in -Advanced Histopathology & Diagnostic Cytology

2. OBJECTIVES At the end of this lesson, you will be able to : Describe the process of embedding Different embedding media Tissue orientation By Geoffrey Mutale (MMLS-MUST) 2

3. INTRODUCTION Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mould . Since the tissue blocks are very thin in thickness they need a supporting medium in which the tissue blocks are embedded. This supporting medium is called embedding medium. By Geoffrey Mutale (MMLS-MUST) 3

4. Cont’ Various embedding substances are paraffin wax, celloidin , synthetic resins, gelatine , etc. The choice of embedding media depends upon Type of microscope ,Type of microtome & Type of tissue eg . hard tissue like bone or soft tissue like liver biopsy By Geoffrey Mutale (MMLS-MUST) 4

5. Examples of impregnating media Paraffin wax Agar Gelatin Low viscosity nitrocellulose( L.V.N) Water soluble wax ( carbo wax) Celloidin Epoxy Resin By Geoffrey Mutale (MMLS-MUST) 5

6. CONT’ Paraffin wax – Paraffin is solid at room temperature. The melting point of paraffin ranges from 40-60°C. For tropical countries hard wax having a melting point of 58-60°C is suitable. This is the most commonly used tissue embedding medium . By Geoffrey Mutale (MMLS-MUST) 6

7. Cont’ Carbowax : It is a water soluble wax. Therefore tissues are directly transferred to water soluble wax after fixation and washing. By Geoffrey Mutale (MMLS-MUST) 7

8. Cont’ Paraplast This is a mixture of purified paraffin and plastic. It is easy to make the serial sections of 4 microns with this embedding medium due to its elasticity . By Geoffrey Mutale (MMLS-MUST) 8

9. Cont’ Ester wax It is harder than paraffin but has a low melting point of 46-48°C. It is soluble in alcohol so there is no need for the  clearing step when using this tissue embedding medium By Geoffrey Mutale (MMLS-MUST) 9

10. Cont’ Epoxy Resin Epoxy resins are used for electron microscopy. Epoxy polymers of araldite differ from methcrylate in that they are crosslinked causing the cured solid block of araldite to be insoluble in any solvent. Longer filtration is required because the viscosity of resin is greater than methacrylate. By Geoffrey Mutale (MMLS-MUST) 10

11. Cont ’ Agar embedding: It is mainly used in double embedding. Multiple fragments and friable tissue may be impregnated in one block when sectioning on the cryostat. Another use of agar embedding is for FNAC specimens. By Geoffrey Mutale (MMLS-MUST) 11

12. Cont’ Celloidin media : Celloidin ( colloidin ) is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo By Geoffrey Mutale (MMLS-MUST) 12

13. CONT ’ Advantages: Its rubbery consistency allows tissue blocks that are either very hard or of varying consistency, to be cut without undue distortion . Dense tissues which are hard to infiltrate (e.g . bones and brains) and specimen which tend to collapse easily due to air spaces (e.g. eyes) are supported better, thereby avoiding the crumbling of tissues during sectioning. By Geoffrey Mutale (MMLS-MUST) 13

14. CONT’ It permits cutting of tissue sections which are thicker than paraffin wax, and is therefore recommended for processing of neurological tissues. It does not require heat during processing; hence, producing minimum shrinkage and tissue distortion especially for cutting large bone sections. By Geoffrey Mutale (MMLS-MUST) 14

15. Cont’   It is, therefore, recommended in cases when minimum shrinkage is required and frozen section technique cannot be done. By Geoffrey Mutale (MMLS-MUST) 15

16. Disadvantage Celloidin impregnation is very slow (lasting for several days or weeks ) Very thin sections (less than 10u) are difficult to cut . Photomicrographs are difficult to obtain . It is very volatile and therefore must be kept in bottles with ground – glass stoppers to prevent evaporation . By Geoffrey Mutale (MMLS-MUST) 16

17. Cont’ Gelatin : Gelatin impregnation is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies. It is used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut. By Geoffrey Mutale (MMLS-MUST) 17

18. PROPERTIES OF AN IDEAL EMBEDDING MEDIUM The embedding medium is considered as ideal if it bears the following qualities: The melting point of the embedding medium in the pure state should be below 65°C to avoid damage to the tissues by heating during impregnation. The embedding medium must be solidifying at room temperature . By Geoffrey Mutale (MMLS-MUST) 18

19. Cont’ The embedding medium must penetrate the tissues replacing the fluid in which it is saturated, usually a clearing agent. By Geoffrey Mutale (MMLS-MUST) 19

20. DOUBLE EMBEDDING OF TISSUES As the name suggests, in this type of embedding two types of embedding medium are used. This term particularly refers to the embedding in which the tissues are first penetrated with nitrocellulose and then embedded in the Paraffin wax. The goal of this is to produce a block that can be sectioned with a matrix of nitrocellulose in addition to paraffin. By Geoffrey Mutale (MMLS-MUST) 20

21. Double embedding By Geoffrey Mutale (MMLS-MUST) 21

22. Cont’ Tissue embedding machine All the blocking steps can be performed with the help of tissue embedding machine. The embedding machine contains the following parts; Mould warmer, cassette bath, working surface warmer with a nozzle for pouring the wax, forceps well and cold plate . By Geoffrey Mutale (MMLS-MUST) 22

23. Tissue embedding machine By Geoffrey Mutale (MMLS-MUST) 23

24. Cont’ The cold plate is of high efficiency refrigeration system having temperature control ranging from different freezing points to 4 or 5 degree C. It can occupy about 50-60 blocks. Large 3-5 litre capacity paraffin reservoir with adjustable temperature Optional vacuum lids, which allows for vacuum infiltration of tissues. By Geoffrey Mutale (MMLS-MUST) 24

25. Method of Embedding Open the tissue cassette, check requisition form entry to ensure the correct number of tissue pieces is present. Select the mould ; there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. By Geoffrey Mutale (MMLS-MUST) 25

26. Cont’ Simple glossed wall or floor tiles may also be used in place of glass plate . Fill the mould with paraffin wax. Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. By Geoffrey Mutale (MMLS-MUST) 26

27. Cont’ Glycerine may be applied to the L pieces and also to the metal or glass plate on which the moulds are placed for embedding. By Geoffrey Mutale (MMLS-MUST) 27

28. Cont’ Place the tissue in the mould according to the side to be sectioned. This side should be facing down against the mould . A small amount of pressure may be used in order to have more even embedding. Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. By Geoffrey Mutale (MMLS-MUST) 28

29. Cont’ This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. Insert the identifying label or place the labelled embedding ring or cassette base onto the mould Add more paraffin into the mould to fill the cassette and mould . By Geoffrey Mutale (MMLS-MUST) 29

30. Cont’ Cool the block on the cold plate. Remove the block from the mould . Cross check block, label and requisition form By Geoffrey Mutale (MMLS-MUST) 30

31. Types of moulds By Geoffrey Mutale (MMLS-MUST) 31 This is the traditional embedding method. The “L moulds ( Leuckhart ) are adjusted according to the shape and size of the tissue

32. Cont’ Reusable metallic moulds Most used moulds PVC disposable moulds By Geoffrey Mutale (MMLS-MUST) 32

33. Orientation of different tissue During embedding the orientation of tissue is important. Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. By Geoffrey Mutale (MMLS-MUST) 33

34. Tissue orientation By Geoffrey Mutale (MMLS-MUST) 34

35. Cont’ During embedding it is important to orient the tissue in a way that will provide the best information to the pathologist. Embedding should be done according to the type of tissue. The requisition form should always be read during embedding for proper orientation . By Geoffrey Mutale (MMLS-MUST) 35

36. general considerations Elongate tissues are placed diagonally across the block. Tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers. By Geoffrey Mutale (MMLS-MUST) 36

37. Cont’ Tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinized epithelia are oriented to face the knife diagonally). Usually tissues are embedded with the surface to be cut facing down in the mould . By Geoffrey Mutale (MMLS-MUST) 37

38. Cont’ Multiple tissue pieces are aligned across the long axis of the mould , and not placed at random. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. In circumstances where precise orientation is essential, tissue should be marked or agar double embedded. By Geoffrey Mutale (MMLS-MUST) 38

39. Points to Note Embedding is the process in which tissues or specimens are enclosed in a mass of the embedding medium using a mould . Embedding medium are supporting medium into which the tissue block are embedded. Various embedding substances such as paraffin wax, celloidin , synthetic resins, gellatine are used depending the type of microscope, type of microtome, type of tissue. By Geoffrey Mutale (MMLS-MUST) 39

40. Points to Note….. Epoxy resin is used for electron microscopy Agar embedding is used in double embedding and FNAC specimens Celloidin media is used for cutting hard tissues Gelatin is used when frozen sections are required on friable tissues A variety of moulds are used for embedding. There may be L moulds or plastic moulds By Geoffrey Mutale (MMLS-MUST) 40

41. cont Various substances can be added to paraffin wax in order to modify its consistency and melting point to improve efficiency during microtomy Additives increase the hardness of block Correct orientation of tissue in a mould is the most important steps in embedding By Geoffrey Mutale (MMLS-MUST) 41

42. The end Thank you By Geoffrey Mutale (MMLS-MUST) 42

43. References Bancroft, J.D. and Stevens, A.: theory and practice of histological techniques ed.3, Churchill livingstone inc. 1990. Edinburgh. London, Melbourne and New York. 2. Lillie, R.D.: Histopathologic technique and practice histochemistry ed. 3, New York, 1965 McGraw Hill Book co. 3. Manual of histologic and special staining techniques ed. 2, New York, 1960, The Blakiston Division McGraw Hill Book Co. By Geoffrey Mutale (MMLS-MUST) 43