1. Histological Techniques By DR ANYANWU GE

2. Introduction Histological technique deals with the preparation of tissue for microscopic examination. The aim of good histological technique to preserve microscopic anatomy of tissue. Make them hard so that very thin section (4 to 5 micron) can be made.

3. Introduction Good staining should be possible. After staining, the section should represent the anatomy of the tissue as close to as possible to their structure in life. This is achieved by passing the total as selected part of the tissue through a series of process.

4. Techniques These techniques are: 1. Fixation 2. Dehydration 3. Cleaning 4. Embedding 5. Cutting 6. Staining

5. Fixation This is the process by which the constituents of cells and tissue are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture.This is achieved by exposing the tissue to chemical compounds, call fixatives.

6. TISSUE FIXATION Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. The aim of fixation: The aim of fixation: 1- To prevent autolysis and bacterial attack. 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 2- To fix the tissues so they will not change their volume and shape during processing. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 4- To leave tissue as close as their living state as possible, and no small molecules should be lost. 4- To leave tissue as close as their living state as possible, and no small molecules should be lost. Fixation is coming by reaction between the fixative and protein which form a gel, so keeping every thing as their in vivo relation to each other. Fixation is coming by reaction between the fixative and protein which form a gel, so keeping every thing as their in vivo relation to each other.

7. Mechanism of action of fixatives Most fixatives act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents.

8. Contin….. Good fixative is most important factors in the production of satisfactory results in histopathology. Following factors are important: Fresh tissue Proper penetration of tissue by fixatives Correct choice of fixatives

9. Contin…. No fixative will penetrate a piece of tissue thicker than 1 cm. For dealing with specimen thicker than this, following methods are recommended: 1.Solid organ: Cut slices as necessary as but not thicker than 5 mm..

10. Continu…. 2.Hollow organ: Either open or fill with fixative or pack lightly with wool soaked in fixative. 3.Large specimen: It requires dissection, Inject fixative along the vessels or bronchi as in case of lung so that it reaches all parts of the organs.

11. Properties of an Ideal Fixative Prevents autolysis and bacterial decomposition. Preserves tissue in their natural state and fix all components. Make the cellular components insoluble to reagent used in tissue processing. Preserves tissue volume.

12. Properties of an Ideal Fixative Avoid excessive hardness of tissue. Allows enhanced staining of tissue. Should be non-toxic and non-allergic for user. Should not be very expensive.

13. Temperature The fixation can be carried out at room temperature. Tissue should not be frozen once it has been placed in the fixative solution, for a peculiar ice crystals distortion will result.

14. Speed of fixation The speed of fixation of most fixative is almost 1 mm/hour. Therefore, a fixation time of several hours is needed for most specimens.

15. Amount of fixative fluid This should be approximately 10-20 times the volume of the specimen. Fixative should surround the specimen on all sides.

16. Factor affecting fixation Size and thickness of piece of tissue. Tissue covered by large amount of mucous fix slowly. Tissue covered by blood or organ containing very large amount of blood also fix slowly. Fatty and lipomatous tissue fix slowly. Fixation is accelerated by agitation. Fixation is accelerated by maintaining temperature around 60oc.

17. Classification of Fixatives Classified into three categories. Classified into three categories. 1. 1. Tissue fixatives 2. Cytological fixatives 3. 3. Histochemical fixatives

18. Tissue fixatives There are many tissue fixatives i.e Buffered formalin Buffered gluteraldehyde Zenker’s formal saline Bowen’s fluid

19. Cytological fixatives Cytological fixatives are Ethanol Methanol Ether

20. Histochemical fixatives These are Formal saline Cold acetone Absolute alcohol

21. TISSUE PROCESSING the aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut, and yet soft enough not to damage the knife or tissue. Stages of processing: 1- Dehydration. 2- Clearing. 3- Embedding.

22. Dehydration to remove fixative and water from the tissue and replace them with dehydrating fluid. There are a variety of compounds many of which are alcohols. several are hydrophilic so attract water from tissue. To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol. To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol. In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage. In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage.

23. Types of dehydrating agents : Types of dehydrating agents : Ethanol, Methanol, Acetone. Ethanol, Methanol, Acetone. Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm

24. Clearing replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon the following: Choice of a clearing agent depends upon the following: - The type of tissues to be processed, and the type of processing to be undertaken. - The type of tissues to be processed, and the type of processing to be undertaken. - The processor system to be used. - The processor system to be used. - Intended processing conditions such as temperature, vacuum and pressure. - Intended processing conditions such as temperature, vacuum and pressure. - Safety factors. - Safety factors. - Cost and convenience. - Cost and convenience. - Speedy removal of dehydrating agent. - Speedy removal of dehydrating agent. - Ease of removal by molten paraffin wax. - Ease of removal by molten paraffin wax. - Minimal tissue damage. - Minimal tissue damage.

25. Some clearing agents: Some clearing agents: - Zylene. - Toluene. - Chloroform. - Benzene. - Petrol.

26. Embedding is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. Paraffin wax properties : Paraffin wax properties : Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: - improve ribboning. - improve ribboning. - increase hardness. - increase hardness. - decrease melting point - decrease melting point - improve adhesion between specimen and wax - improve adhesion between specimen and wax

27. Precaution while embedding in wax The wax is clear of clearing agent. The wax is clear of clearing agent. No dust particles must be present. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size.

28. There are four main mould systems and associated embedding protocols presently in use : There are four main mould systems and associated embedding protocols presently in use : 1- traditional methods using paper boats 1- traditional methods using paper boats 2- Leuckart or Dimmock embedding irons or metal containers 2- Leuckart or Dimmock embedding irons or metal containers 3- the Peel-a-way system using disposable plastic moulds and 3- the Peel-a-way system using disposable plastic moulds and 4- systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome. 4- systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome.

29. Tissue processing Embedding moulds: (A) paper boat; (B) metal bot mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring ( F) or cassette bases (G)

30. General Embedding Procedure 1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. General Embedding Procedure 1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. 2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. 3- Fill the mould with paraffin wax. 4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. 5- Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 5- Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 6- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould. 6- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould. 7- Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 7- Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 8- Remove the block from the mould. 8- Remove the block from the mould. 9- Cross check block, label and worksheet. 9- Cross check block, label and worksheet.

32. Processing methods and routine schedules Machine processing Machine processing manual processing manual processing

33. CUTTING using the microtome using the microtome

34. A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. The most common applications of microtomes are A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. The most common applications of microtomes arehistology

35. 1- Traditional histological technique : tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 25 micrometers thick. From there the tissue can be mounted on a microscope slide, stained and examined using a light microscopeparaffin

36. Microtome knives STEEL KNIVES STEEL KNIVES NON-CORROSIVE KNIVES FOR CRYOSTATS NON-CORROSIVE KNIVES FOR CRYOSTATS DISPOSABLE BLADES DISPOSABLE BLADES GLASS KNIVES GLASS KNIVES DIAMOND KNIVES DIAMOND KNIVES

37. STAINING

38. H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. Hematoxylin and Eosin (H & E)

39. Staining machine

40. Staining There are hundreds of stains available. Classification of Stains: Acid stains Basic stains Neutral stains

41. Acid Dyes In an acid dye the basic component is colored and the acid component is colorless. Acid dyes stain basic components e.g. eosin stains cytoplasm. The color imparted is shade of red.

42. Basic Dyes In a basic dye the acid component is colored and the basic component is colorless. Basic dyes stain acidic components e.g. basic fuchsin stains nucleus. The color imparted is shade of blue.

43. Neutral Dyes When an acid dye is combined with a basic dye a neutral dye is formed. As it contains both colored radicals, it gives different colors to cytoplasm and nucleus simultaneously. This is the basis of Leishman stain.

44. Special stains When a specific components of tissue e.g. fibrous tissue, elastic tissue, nuclear material is to be stained, certain special stains are used which specifically stain that component tissue.

45. Procedure of staining: There are two types of staining, There are two types of staining, Manual Staining Automatic staining

46. Manual Staining In a small laboratory when a few slides are stained daily, this is the method of choice. Although it is time consuming it is economical. Different reagent containers are placed in a special sequence and the slides are removed from one container to another manually.

47. Automatic staining In this procedure an automatic stainer is required. It has a timer, which controls the time. It has a mechanical device which shifts the slides from one container to next after the specified time. Advantages of automated stainer are: It reduces the man power It controls the timing of staining accurately Large number of slides can be stained simultaneously Less reagents are used Note: Slides stained either manually or by automatic stainer, pass through same sequences.

48. And KEEP SMILING