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
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
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
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).
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.
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
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.
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.
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
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
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
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
Fixation Types of fixatives (AMAPO) • Aldehydes • Mercurials • Alcohols • Picrates • Oxidizing agents
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
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.
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
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.
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.
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.
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
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
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.
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
Characteristics of Fixatives Chemical Fixatives Freeze Substitution Microwave Fixation
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
Chemical Fixation • Coagulating Fixatives • Crosslinking Fixatives
Coagulating Fixatives • Ethanol • Methanol • Acetone
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
Crosslinking Fixatives • Glutaraldehyde • Formaldehyde • Ethelene glycol-bis-succinimidyl succinate (EGS)
Cross-linking Fixatives • Form covalent crosslinks that are determined by the active groups of each compound
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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'
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.
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.
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).
Formaldehyde • Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde.