Tissue Processing of Histopathology.pptx

A tissue processor is used to prepare tissue samples for analysis by fixing, staining, dehydrating or decalcifying them.
The techniques for processing the tissue, whether biopsies, larger specimen removed at surgery

1. Tissue Processing

2. Introduction Fresh Tissues are soft hence cannot be cut on microtome. In order to get thin sections of good quality for final diagnosis , the tissue needs firm solid medium which gives sufficient rigidity to ensure thin sectioning. Because water & wax are not miscible, tissues must be gradually dehydrated with alcohol.

3. Important maintenance tips • Any spillage or overflow should be cleaned immediately. • Accumulation of wax on any surface should be removed. • The temperature of the paraffin wax bath should be set to 3°C above the melting point of the paraffin wax and monitored daily • Timings should be checked when placing tissue cassettes in the processor, especially when delayed schedules are selected • Warm water flushes should be incorporated, keeping the lines free of salts, protein and debris

4. Principle The Principle of tissue processing is to remove all extractable water from the tissue replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without damage or distortion. The steps involved are:- Dehydration :- removal of water & fixative from the tissue. Clearing:- Removal of dehydrating solution , thus making the tissue components receptive to infiltration medium. Impregnation:- removal of clearing agent & infiltration of paraffin wax in order to give internal support to tissue.

5. Dehydration Removes free or unbound water molecule of the tissue as the supporting medium (paraffin) is not miscible with water. • Sharp difference of concentration gradient of the dehydrating fluid may damage the delicate tissue. Gradual dehydration is necessary. Too much time in the dehydrating fluid: the tissue becomes hard and brittle. • Routine laboratory: 70, 90 and 100% alcohol for 2 h each.

6. Common dehydrating agents: – Ethyl alcohol – Methylated spirit – Methanol – Butyl alcohol – Isopropyl alcohol – Dehydrating agents other than alcohol: dioxane, ethylene glycol and acetone

7. Comparison of different dehydrating agents Dehydrating agents Advantages Disadvantages Ethyl alcohol • Rapid and efficient dehydrating agent • Needs licence from the government • Inflammable • Hard and brittle tissue if kept for long time Methanol Equally effective as ethanol • Volatile • High cost Isopropyl alcohol • Relatively rapid action • Non-toxic • Minimal tissue shrinkage • Not possible to use in celloidin technique Dioxane • Rapid action • No shrinkage of tissue • Highly toxic gas is generated Ethylene glycol Rapid • No graded solution is needed • Tissue can be kept in it for long time Very expensive • Clearing agent is needed Acetone • Rapid action • Cheaper than ethanol • Good for fatty tissue processing Quickly evaporates • Inflammable • Prolonged use may cause shrinkage and brittleness of tissue

8. Clearing Aims of clearing: – Removal of dehydrating agent (e.g. alcohol) to facilitate impregnation of paraffin wax – To make the tissue clear and improve the microscopic examination • Ideal clearing agent: – Low viscosity and high penetration rate – Low melting point – Miscible with both alcohol and molten wax – No tissue damage – Less toxic – Less inflammable – Cheap

9. Selection of appropriate clearing agent: Type of tissue, type of processor, processing condition (such as heat, vacuum) safety factors and cost Volume of clearing agent: 40 times the volume of the specimen • Total duration: – Smaller biopsy: 1 h – Larger tissue: Three changes in xylene or toluene 60 min each • End point detection: Tissue becomes transparent • Prolonged exposure to clearing agent: The brittle and more friable tissue • Different clearing agents: Xylene, toluene, chloroform, amyl nitrate, cedarwood oil and limonene

10. Comparison of clearing agents Properties Xylene Toluene Chloroform Esters Tissue shrinkage Yes Yes Minimum No Tissue hardening Yes No No No Inflammable Yes Yes No Yes Harmful effect Irritant but less harmful Irritant Dangerous toxic gas liberates in heating Safe Cost Cheap Cheap Very expensive High cost

11. Aims: To provide support to the tissue. Principle: Clearing agent is removed by the process of diffusion, and the tissue space is now infiltrated with the embedding media. Ideal impregnating medium: • Miscible with clearing agent • Liquid in higher temperature and solid in room temperature • Homogenous and stable • Non-toxic and cheap • Transparent • Fit for sectioning the tissue

12. Infiltration and Embedding 1. Size of tissue: Thicker large tissue takes more time to impregnate with the embedding medium. It also contains more clearing agent to remove. 2. Type of tissue: Hard tissue such as bone and cartilage takes more time for embedding than soft tissue. 3. The type of clearing agent: Certain clearing agents are easy to remove than others. Such as xylene and toluene are easy to remove than cedarwood oil. 4. Type of processing: Vacuum embedding enhances impregnation. Ester wax, polyester wax, resins, agar, gelatin, celloidin.

13. Equipments & Reagents Tissue cassettes Automatic Tissue Processor Glass jars Incubator Reagents Required:- Ethanol Xylene Paraffin Isopropenol

14. Influencing Factors of Tissue Processing Size of the tissue: – The smaller the size, the better the processing. Agitation: – Agitation facilitates the contact of tissue with fresh solution. Heat: – Increases the better penetration of flui Viscosity: – The higher the viscosity of the medium, lower the penetration. Negative pressure: – Negative pressure removes trapped air in the tissue. – Removal of clearing agent by increasing volatility.

15. Dehydration In paraffin tissue processing various dehydrating agents used are alcohol ethanol, methanol, Isopropenol. butyal alcohol & acetone. Before taking the tissue for processing it is necessary to remove fixative from the tissue. After proper fixation tissue should be washed under running tap water & then kept in 70% alcohol for minimum 30 minutes which helps to remove fixative from the tissue. It is also called as “resting stage” in processing. If tissue will be directly introduced to high grade of alcohol after fixation it will usually show high grade of shrinkage due to rapid removal of water. After this tissue is introduced to ascending grades of alcohol & final 2-3 changes of absolute alcohol. Between transfer of graded alcohol tissue should be allowed to drain the previous solution.

16. Dehydration Procedure 1. automatic tissue processor a. overnight 2. Baths: water, 70,95,100,100 % alcohol 3. Clearing agent: 2 baths of xylene

17. Clearing Various clearing agents used are :- chloroform, Benzene, Xylene, Cedar wood oil. Clearing is done to remove the dehydrating solution & to make the passage clear for impregnating medium to infiltrate in the tissue. The clearing agent should be miscible with both the dehydrating agent & impregnating agent. Many of clearing agent have a similar refractive index to that of protein constituents making the tissue translucent . The end point of clearing can be noted by the transparent appearance of the tissue after xylene treatment.

18. Impregnation with wax Once clearing is over the tissues are transferred to melted paraffin wax bath for impregnation replacing the clearing agent with the embedding medium. We use paraffin wax of melting point 60-62 O C, so it will remove bubbles & replace xylene with paraffin. Two baths of melted paraffin each of 1.5 hrs & 2 hours are given.

19. Factors affecting the tissue processing:- Heat:- Instead of processing the tissue at RT the processing of tissue done at 37 C will help for rapid dehydration which in turn will help to reduce to total processing time . Agitation:- Continuous agitation will help for rapid dehydration. Vacuum:- These will increase the speed of penetration of the reagent which in terms will help for rapid & good quality processing. Size of tissue & volume of reagent should be appropriate. Overcrowding of tissues in the container will results in insufficient processing.

20. Procedure for loading the tissue Processor All the cases from gross room are tide in cassettes, plastic metal bought into machine room where they are kept under running tap water for 5 minutes. Then these cassettes are dipped in 70% Isopropenol till the machines are switched on. Then according to order of utilization of machines cases are put into corresponding tissue baskets. These tissue baskets are then loaded into respective machines. Entry of this is maintained in processing register , machines are switched on at around ______ The temperature of paraffin bath _____? is noted in register. The processed tissue is embedded in next morning.

21. Tissue Embedding

22. Introduction The processed & & impregnated tissues need firm supporting embedding medium . This enables us to have thin sections on the microtome. Criteria for Good Embedding medium It should offers the advantages of fast embedding It should give good consistency of serial sections It should have wide range of section thickness It should produce blocks which are durable for long time.

23. Embedding Medium Paraffin wax is a most commonly & widely using embedding medium It is a hydrocarbon produced in cracking mineral oils , the melting point ranging between 40 to 70 c. We use paraffin wax, of a melting point 58 to 60 o C which is commercially added with resins. Praplast is highly purified paraffin wax which produces thin sections & is mainly to cut lymph nodes.

24. Principle The tissue is blocked by transferring it from the final wax bath to a moulds filled with molten paraffin wax & oriented on the mold in such away that the surface to be cut rests on the base of the mould. The block is then quickly cooled.

25. Materials required Stainless steel metal moulds of different sizes- many small lab. Uses “L” (Leukhartz) mould – mace up of brass Plastic embedding rings. Pointed forceps. Lead pencils Register Pair of tongues Gas Burner Cardboard boxes Small plane labels scissors. Rough knives, Enamel tray REAGENTS Required:- Embedding machines. Hot plate Paraffin Dispenser Cooling Plate

26. Procedure L mould is placed on a metal sheet. Molten wax is poured with the help of metal kettle. Individual tissue bit is placed at bottom & pressed with help of forcep. The corresponding label is attached at the side the mould. This is allowed to cool. Moulds are taken out & the slab is cut around bits. Many large laboratories having large work load are using Tissue embedding system. This system comprise of three . i.e. wax dispenser cold plate & heating storage area for moulds. Many large laboratories use metal moulds & plastic cassettes are made in the same plastic cassette used for processing. The label with appropriate pathology number is affixed on the plastic rings

27. Orientation of Tissues Skin :- must be oriented in such a way that the epidermal edge , sub cutaneous tissue & deeper layers of all should be seen in a finished slide. Epithelial edge should be paralleled to the cutting edge at the upper side of the block while cutting so that cutting knife comes first in contact with the soft part & then hard part. This gives minimum tension on knife & we get a uniform section. Bone Marrow :- is placed horizontally ; parallel to the cutting edge so that it is easy to get whole section. Stomach :- it should embedded in such a way that all layers i.e. mucosa , submucosa , muscularis & serosa should seen in sectioning. Cervix :- the flat surface of cervix should be at bottom of the mould so that ectocervix , endocervix & transformation zone should come first in final slide. Lymphnode :- should placed at the same level, only a few mm appear with parallel placement. If the mucosa is not seen microscopically the block should be melted and tissue should be rotated through 90 & then re-embedded.

28. Points to remember Care is taken that the blocks are not over cooled or else they will crack & the sectioning becomes difficult. The use of over heated forceps should as it burns the contact area of the tissue. Over heated paraffin also should not used. Do not overheat rough knife as it will burn the label & make it unreadable.

29. Trimming & Cutting

30. Introduction Microtome is a mechanical device for cutting thin uniform slices of tissues , thin enough for examinations under a microscope. Prior to sectioning paraffin block should be trimmed so that the tissue is fully exposed. The requirement for section cutting are a well maintained microtome sharp knife & properly processed tissue.

31. Different types of Microtomes:-

32. Sledge Microtome In this type, knife is fixed & tissue block moves. These are generally used to cut sections of larger blocks. These microtome are generally heavy.

33. The Rocking Microtome:- The name of this type of microtome is based on it’s rocking action of the cross arm. In this type knife is fixed at one place & block moves. This is generally used to cut small tissues.

34. Sliding Microtome This type of microtome is generally used to cut block of celloidin material. In sliding microtome , knife moves horizontally & blocks remain fixed at one place.

35. Rotary Microtome The name of this microtome is based on it’s rotary action of hand wheel. These microtomes are heavier & stable. This type of microtome is used for cutting all types of blocks. & is commonly used in histopathology laboratories.

36. Freezing Microtome:- This is used to cut Frozen section specifically

37. Microtome Knife Microtome knives are used to cut thin & serial sections of paraffin block containing tissue Bite. Types of knives:- Plano concave i.e profile ’A’ Biconcave ( Profile B) Wedge (Profile C):- As Tool Edge (Profile D) Disposable blades:- They are most popular, are of two types – low profile & high profile. Advantages of disposable blades:- Sharpening time & abrasive coast is saved. Very thin sections are possible with this blades.

38. Knife Angles:- For cutting thin section it is important to know what a knife angle is ! Bevel Angle :- It can varied between 18 to 35 degree. It is formed by convergence of two planes. Angle of Clearance :- This angle is formed between knife edge & block surface. It is around 2 to 5 degrees.

39. Knife sharpening Old method of sharpening is manual sharpening. It involves two process – Honing & Stropping. Honing :- It is the process in which all the nicks & irregularities in the cutting edge of the knife are removed to make the cutting edge straight & sharp. After prolonged use or after cutting very hard tissue the cutting edge will have become so damaged that stropping will not re sharpen it ; small pieces of metal may have been removed which will give a jagged edge, causing lines or tears in section cutting. A straight cutting edge & the correct bevel must be restored by grinding the knife on a hone. Types of hones are :- Belgian black vein/Belgian yellow , Arkansas , Carborundum Grads of honing material :- 1. Coarse :- This type of particles are used for quick sharpening of badly damaged knives, but we should careful as it causes nicks in knife. 2. Fine

40. Lubricants They are used in sharpening knives. They acts as coolant & protects the temper of extreme edge. They allow the flow away of fine metal particles from the knife edge & movement of fresh abrasive particles towards the knife edge. They reduces the tendency of stones pores to become blocked with finely divided metal particles. Material used are aqueous lubricants 1% liquid soap , 50% glycerol & 10-30% soluble oil etc.

41. Precaution taken during honing Lubricants must use. Blades must kept perfectly flat while honing because if the knife edge be raised even the slightest , the edge will be turned. After honing is complete the knife should wiped with piece of soft cloth moistened with Xylene. The edge may be view with low power objective microscope to ascertain removal of nicks.

42. Stropping It is the process of polishing the already fairly sharpened edge after honing Stropping removes the buffs formed during honing. The knife edge cannot be sufficiently sharp to cut good sections directly from honing or sharpening. Stropping must follow sharpening. This is the final technique for sharpening the knife before cutting sections.

43. Types of strops: 1. Rigid / fixed – Rigid type is preferred by many histotechnologists as it is easy to manipulate and rounding of the knife edges is minimized. 2. Flexible / hanging Strop is made from rump of horse and is fixed on wooden piece with handle of the size of Length - 12 inch, Breadth - 2 inch, Height - 2 inch. Back of strop is made of canvass to give support to leather during stropping. Small quantity of vegetable oil is applied beneath the leather to keep the strops soft

44. Section Cutting C l inical diagnosis need to be confirmed by laboratory investigations for a proper patient management and therapy. For Histopathological diagnosis, a good thin tissue section is very important. Well fixed paraffin embedded block are pre requisites for getting thin sections. To get thin good quality tissue section for staining by histo technologist and histopathological examination by the histopathologist. Instruments required: 1. Good microtone 2. Sharp microtone knife 3. Properly processed tissue 4. Block holders 5. Water bath 6. Slide warmer 7. Forceps 8. Number 0 brush 9. Albuminized slides 10. Experience

45. Trimming of Tissue Blocks When everything has gone well in fixing, dehydrating, infiltrating the specimen and embedding it, you should have obtained a paraffin block containing the specimen surrounded with –generally- a too generous amount of paraffin wax. The surplus paraffin needs to be removed and the paraffin block trimmed in its final form prior to sectioning. Trimming removes excess paraffin from tissue & tissue surface is exposed properly o get complete section of tissue.

46. PROCEDURE After trimming, blocks as well as knife are cooled on ice tray for 10-15 minutes. The block is clamped firmly to the microtome block holder. Stage, blade or knife holder & block holder clamps all should be enough tight. Adjust microtome knob as per desired microns.

47. Procedure 5) Rotate the wheel slowly with rhythmic movement till the ribbon of sections is obtained. 6) Select section in cut from the ribbon & taken in a jar containing water & diluted alcohol. Major folds are removed from the section. 7) The section is now taken on albuminized slide & it is dipped in water bath to remove microscopic folds. This is called spreading of sections. Temperature of water bath is maintained at 57-58 . 8) The section is blotted on filter papers & pathology number is scratched on the glass slide. These unstained slides are kept in an oven for heating at 60 C.

48. Points to Remember Use knife guards to prevent knife injury. Use Needle for Scalpel to lift ribbon from blade. Knives should be handled carefully. When not in use, it should be kept in a knife box. Knife should be cleaned with xylene after every use. Do not allow the edge of knife to touch any surface. Separate knife should be used to cut bony tissue.