1- Histology and Histo-technique

1. 1- Histology and Histo-technique BY Dr. TAREK ATIA

2. Histology is a science concerned with the microscopic structure of tissues and organs in relation to their function. Also called microanatomy or microscopic anatomy.

3. MICROSCOPY - Light microscope - Electron microscope: Scanning / Transmission - Fluorescence microscope - Inverted microscope - Phase contrast microscope

4. Light microscope Electron microscope

7. Tissue Preparation for Light Microscope Tissue specimens received in the surgical pathology laboratory have a request form that lists the patient information and history along with a description of the site of origin.

9. 1- Fixation • Fixation should be carried out as soon as possible after removal of the tissues to prevent autolysis. • Actually, there is no perfect fixative, though formaldehyde comes the closest. • Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.

10. The aim of fixation • Prevent autolysis by inhibiting the hydrolytic enzymes of lysosomes. • Stop bacterial effect, so it prevent putrefaction. • Preserve the fine structure of the tissues. • Harden the tissues due to protein coagulation. • Increase the affinity of tissues to staining effect.

11. Types of fixatives • There are five major groups of fixatives, classified according to mechanism of action: • Aldehydes • Mercurials • Oxidizing agents • Picrates • Alcohols

12. Aldehydes: Includeformaldehyde (formalin) and glutaraldehyde. • It is good for immuno-histochemistry techniques. • Formalin penetrates tissue well, but is relatively slow. • The standard solution is 10% neutral buffered formalin.

13. Mercurials: • They contain mercuric chloride and include such well-known fixatives as Zenker's solution. • These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail.

14. Oxidizing agents: Include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. • Picrates: • include fixatives with picric acid. • Foremost among these is Bouin's solution.

15. Alcohols: • Including methyl alcohol (methanol) and ethyl alcohol (ethanol). • However, they are very good for cytological smears because they act quickly and give good nuclear detail.

16. 2-Factors affecting fixation There are a number of factors that will affect the fixation process: • Buffering: Fixation is best carried out close to neutral pH, in the range of 6-8. • Penetrationof tissues depends upon the diffusability of each individual fixative, which is a constant.

17. The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. • Increasing the temperature, as with all chemical reactions, will increase the speed of fixation. • Concentrationof fixative should be adjusted down to the lowest level possible. • Time interval: Also very important is time interval from removal of the tissues to the fixation.

18. Tissue Processing • The technique of getting fixed tissue into paraffin is called tissue processing. The main steps in this process are Dehydration, Clearingand Embedding.

19. 1- Dehydration • Gradual removal of water from the tissue using ascending grads of ethyl alcohol to prevent tissue shrinking. • The specimens are dehydrated in an ascending graded of ethanol; from water through 10%-20%-50%-95%-100% ethanol.

20. Duration of dehydration should be kept to the minimum consistent with the tissues being processed. • Tissue blocks 1mm 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 • The dehydrating agents includeEthanol, Methanol, Acetone.

21. 2. Clearing: Replacement of alcohol in tissue by clearing fluid like Xylene, Toluene, Chloroform, or Benzene 4. Cutting: - Paraffin block are cut by microtomeusing a sharp metal knife, into thin sections ~ 6µ 3. Impregnation and Embedding: - Tissues are impregnated in paraffin wax to replace xylene • Other embedding medium include Agar, Gelatin, or Paraplast.

22. Orientation Of Tissue In The Block 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.

23. 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 • Tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally) • Multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

24. 6. Mounting: - Sections spread on the hot plate and mounted on glass slides. 7. Staining: - Variable stains are used for specific tissues.

25. Automated tissue processor

27. Sectioning • Once the tissues have been embedded, they must be cut into very thin sections (4 to 6 microns) that can be placed on a slide. • This is done with a microtome. The important thing for proper sectioning is a very sharp knife.

30. Frozen Sections • Frozen sections are performed with an instrument called a cryostat. • The cryostat is just a refrigerated box containing a microtome. • The temperature inside the cryostat is about -20 to -30 C. • The tissue sections are cut and picked up on a glass slide. • The sections are dried and then stained.

31. Automated stainer Staining • The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. • Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylene then, to alcohols and lastly to water. • There are no stains that can be done on tissues containing paraffin. Few slides or Frozen sections are better stained manually

32. Coverslipping The stained section on the slide must be covered with a thin piece glass to protect the tissue from being scratched, and to preserve the tissue section for years to come.

33. Decalcification Bone specimens as well as calcified tissues are the most type here. The calcium must be removed before embedding to allow sectioning. A variety of reagentshave been used to decalcify tissue such as mineral acids, organic acids, EDTA, and electrolysis.

34. Strong mineral acidssuch as nitric and hydrochloric acids • Strong acids will remove large quantities of calcium at a rapid rate, butthey will cause damage of cellular morphology. • Organic acids such as acetic and formic acid. • However, they act more slowly on dense cortical bone. • EDTA can remove calcium safely. • It works slowly, and penetrates tissue poorly.