Histotechniques

1. Histotechniques Dr Mulazim Hussain Bukhari MBBS, DCP, MPhil, FCPS, PhD Associate Prof Pathology King Edward Medical University, Lahore CME,DEpartment of Pathology,King Edward Medical University,Lahore

2. Tissue Processing • Specimen Accessioning • Gross Examination • Fixation • Tissue Processing • Sectioning • Frozen Sections • Staining • H and E staining • Cover slipping • Decalcification • Artifacts in Histologic Sections • Problems in Tissue Processing

3. Safety in the Lab • The lab should be well illuminated and well-ventilated. • Rules and Regulations governing • formalin and • hydrocarbonds • such as xylene • and toluene. • Limits set by the Occupational Safety and Health Administration (OSHA) that should not be exceeded. • These limits should be revised and revived to reduced any mishap

4. Cont. • Every chemical compound used in the laboratory should have a materials safety data sheet on file • that specifies the nature, • toxicity, • and safety precautions to be taken when handling the compound. • The laboratory must have a method for disposal of hazardous wastes. • Health care facilities processing tissues often contract this to a waste management company. • Tissues that are collected should be stored in formalin • and may be disposed by incineration • or by putting them through a "tissue grinder" attached to a large sink (similar to a large garbage disposal unit).

5. Cont. • Check the sharpness of scalpel, scissors and quality of other ones like ruler, probes weighing machines • Every instrument used in the laboratory should meet electrical safety specifications and have written instructions regarding its use. • Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose.

6. Cont. • Fire safety procedures are to be posted. • Safety equipment including fire extinguishers, • fire blankets, • and fire alarms should be within easy access. • A shower and eyewash should be readily available. • No smoking, eating or movements in the labs • Use disposable gloves

7. Cont. • Laboratory accidents must be documented and investigated with incident reports and industrial accident reports. • Specific hazards that you should know about include: • Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive.

8. Sodium azide • Many reagent kits have sodium azide as a preservative. • You are supposed to flush solutions containing sodium azide down the drain • with lots of water, or • there is a tendency for the azide to form metal azides in the plumbing. • These are also explosive.

9. Drainage • Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used. • Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. • Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed. • Hazards of usually used formalin

10. Objective • Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. • The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy • The persons who do the tissue processing and make the glass microscopic slides are histotechnologists

11. Specimen Accessioning • 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. • The specimens are accessioned by giving them a number that will identify each specimen for each patient

13. Grossing • Describing the specimen • Placing all or parts of it into a small plastic cassette • When a malignancy is suspected • Inking a gross specimen for margins

15. Fixation Types of fixatives (AMAPO) • Aldehydes • Mercurials • Alcohols • Picrates • Oxidizing agents

16. Fixation - factors affecting fixation There are a number of factors that will affect the fixation process: • Buffering • Penetration • Volume • Temperature • Concentration • Time interval • Position of tissue

17. Buffering • Fixation is best carried out close to neutral pH, in the range of 6-8. • Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. • Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. • Common buffers include phosphate, bicarbonate, cacodylate, and veronal. • Commercial formalin is buffered with phosphate at a pH of 7.

18. Penetration • Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. • Formalin and alcohol penetrate the best, and glutaraldehyde the worst. • Mercurials and others are somewhere in between. • One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). • Penetration into a thin section will occur more rapidly than for a thick section

19. Volume • The volume of fixative is important. • There should be a 10:1 ratio of fixative to tissue. • Obviously, we often get away with less than this, but may not get ideal fixation. • One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. • Agitation of the specimen in the fixative will also enhance fixation.

20. Temperature • Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. • Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor.

21. Concentration of fixative • Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. • Formalin is best at 10%; • glutaraldehyde is generally made up at 0.25% to 4%. • Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat.

22. Time interval • Also very important is time interval from of removal of tissues to fixation. • The faster you can get the tissue and fix it, the better. • Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline. • The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur

23. Kaiserling formula for preservation for surgical specimens for museum • Formalin pure 5 liter • Distilled water 22.5 liter • Potassium acetate(CH3COOK ) 250gm • Chloral hydrate 50 gm • 27 liter • Potassium acetate is used in mixtures applied for tissue preservation, fixation, and mummification. Most museums today use the formaldehyde-based method recommended by Kaiserling in 1897 and containing potassium acetate. • For example, Lenin's mummy was soaked in a bath containing potassium acetate

25. Gough Sections: • Whole organs may be sectioned on paper by the methods of Gough and Wentworth. • These sections provide valuable information on whole organ structure and serve as links between mounted museum specimens and histologic sections.

26. Colour restoration • Small amount of sodium hydrosulphite to preserve the colour. • If the container is properly sealed, the colour restoration is then permanent. • For photography, the procedure is to first wash and clean the specimen. • It is then soaked in an excess of 60% ethanol until the colour has been restored satisfactorily

27. Characteristics of Fixatives Chemical Fixatives Freeze Substitution Microwave Fixation

28. Ideal Fixative • Penetrate cells or tissue rapidly • Preserve cellular structure before cell can react to produce structural artifacts • Not cause autofluorescence, and act as an antifade reagent

29. Chemical Fixation • Coagulating Fixatives • Crosslinking Fixatives

30. Coagulating Fixatives • Ethanol • Methanol • Acetone

31. Coagulating Fixatives Advantages • Fix specimens by rapidly changing hydration state of cellular components • Proteins are either coagulated or extracted • Preserve antigen recognition often Disadvantages • Cause significant shrinkage of specimens • Difficult to do accurate 3D confocal images • Can shrink cells to 50% size (height) • Commercial preparations of formaldehyde contain methanol as a stabilizing agent

32. Crosslinking Fixatives • Glutaraldehyde • Formaldehyde • Ethelene glycol-bis-succinimidyl succinate (EGS)

33. Cross-linking Fixatives • Form covalent crosslinks that are determined by the active groups of each compound

34. Principles of Fixation • Once tissues are removed from the body, they undergo a process of self-destruction or autolysis • which is initiated soon after cell death by the action of intracellular enzymes causing the breakdown of protein and eventual liquefaction of the cell.

35. Principles of Fixation • Autolysis is independent of any bacterial action, • retarded by cold, • greatly accelerated at temperatures of about 30°C and • almost inhibited by heating to 50°C

36. Cont. • Autolysis is more severe in tissues which are rich in enzymes, • such as the liver, • brain and kidney, • and is less rapid in tissues such as elastic fibre and collagen.

37. Cont. • By light microscopy, autolysed tissue presents a `washed-out' appearance with swelling of cytoplasm, • eventually converting to a granular, homogeneous mass which fails to take up stains.

38. How Autolysed tissue looks like • The nuclei of autolytic cells may show some of the changes of necrosis • including condensation (pyknosis), • fragmentation (karyorrhexis) and • lysis (karyolysis) • D/D these are not accompanied by an inflammatory or cellular response.

39. How Autolysed tissue looks like • There may be diffusion of intracellular substances of diagnostic significance, such as glycogen which is lost from the cells in the absence of prompt and suitable fixation. • Autolysis also causes desquamation of epithelium which separates from its basement membranes.

40. Bacterial Action on dead tissue • Bacterial decomposition can also produce changes in tissues that mimic those of autolysis and is brought about by bacterial proliferation in the dead tissue.

41. Bacterial Action on dead tissue • Such bacteria may normally be present in the body during life such as the non-pathogenetic organisms present in the bowel, or may be present in diseased tissues at the time of death such as in septicaemia.

42. The objective of fixation • is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. • Fixation arrests autolysis and bacterial decomposition and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. • Aside from these requirements for the production of tissue sections, increasing interest in cell constituents and the extensive use of immunohistochemistry to augment histological diagnosis has imposed additional requirements.

43. Cont. • Fixation should also provide for the preservation of tissue substances and proteins. • Fixation is, therefore, the first step and the foundation in a sequence of events that culminates in the final examination of a tissue section.

44. Common pitfalls of fixation • It is relevant to point out that fixation in itself constitutes a major artifact. • The living cell is fluid or in a semi-fluid state, Whereas fixation produces coagulation of tissue proteins and constituents, a necessary event to prevent their loss or diffusion during tissue processing; the passage through hypertonic and hypotonic solutions during tissue processing would otherwise disrupt the cells. • For example, if fresh unfixed tissues were washed for prolonged periods in running water, severe and irreparable damage and cell lysis would result. • In contrast, if the tissues were first fixed in formalin, subsequent immersion in water is generally harmless.

45. Summary of objective • Fixation: • Confers chemical stability on the tissue • Hardens the tissue (helps further handling) • Halts enzyme autolysis • Halts bacterial putrefaction • May enhance later staining techniques • Introduces a 'consistent artifact'

46. Aldehydes • include formaldehyde (formalin) and glutaraldehyde. • Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. • This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost.

47. Cont. • Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow. • The standard solution is 10% neutral buffered formalin. • A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues.

48. Formaldehyde • Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections. • It is a gas with a very pungent odor, soluble in water to a maximum extent of 40% by weight and is sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid).

49. Formaldehyde • Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde.