Histotechnology D2L questions fully solved & updated.

What thickness are paraffin sections routinely cut? - ANSWER-4-6 um Why should the temperature of the flotation bath be set specifically at 5-10 °C below the melting point of the paraffin wax? - ANSWER-water bath that is too hot may distort tissue morphology Warm enough to relieve compression Cool enough so the wax does not melt What thickness are paraffin sections cut for the Luxol Fast Blue procedure? - ANSWER-10 - 15 um Why are paraffin blocks put on ice prior to cutting? - ANSWER-to soften the tissue; harden the wax What thickness are paraffin sections cut for the Congo Red procedure? - ANSWER-8-10 um What epithelium occurs only in the female reproductive tract? - ANSWER-Simple columnar ciliated n the gross room, what is the maximum thickness to which a tissue should be dissected? - ANSWER-3mm What is the primary fixative used for electron microscopy? - ANSWER-Glutaraldehyde What will happen when there is too much gelatin in the floatation bath? - ANSWER-The whole slide will turn pink List (3) things that a satisfactory decalcifying agent should ensure. - ANSWER-Complete removal of calcium without damaging tissue, Rapid removal of calcium, No effect on staining Name (2) strong and (1) weak acid decalcifying agents. - ANSWER-Strong: HCl, Nitric acid, Weak: Formic, Picric, Acetic State (4) factors that affect the rate of decalcification. - ANSWER-Size of tissue, Density of tissue, Volume of fluid, Type of fluid., Agitation can speed up the rate of decalcification ist (6) situations in which coated slides are particularly useful. - ANSWER-any hard or tough tissue (ex. Prostate with calcification), brain, blood clot, silver impregnation stains, immunohistochemistry, frozen sections List two causes for each of the following problems which may be encountered when paraffin sectioning. State the way to alleviate each problem. Washboard Alternate thick & thin sections Scoring Sections will not ribbon Compression Sections disintegrate on flotation bath Curved ribbon - ANSWER-a. Washboard: Cause: Solution: blade or block is loose tighten clearance angle is too steep adjust tissue is hard or too cold warm b. Alternate thick & thin sections Cause: Solution: Blade / block loose tighten Clearance angle too small adjust Tissue too hard warm c. Scoring: Cause: Solution: Scoring / nick on blade clean / replace knife Hard particles in tissue surface decalcify d. Section will not ribbon: Cause: Solution: Block edges not parallel to knife position block correctly in chuck Cutting too fast or too slow change speed Wax too hard warm Debris on knife edge clean / replace knife e. Compression: Cause: Solution: Dull blade replace blade Wax too warm cool on ice f. Sections disintegrate on flotation bath: Cause: Solution: Poor processing reprocess / new tissue if possible Flotation bath too warm cool g. Curved ribbon: Cause: Solution: Block edges not parallel to knife adjust block in the chuck One side of knife dull replace knife Tissue varying in consistency cut slower than usual. No real fix. State the difficulties and / or problems that may be encountered in / or following paraffin sectioning if the tissues were improperly fixed or processed in the following ways: Incomplete fixation Incomplete dehydration Over dehydration Incomplete clearing Excessive time in the clearing agent Incomplete filtration Excessive infiltration time and / or wax too hot - ANSWER-Incomplete fixation Tissue components can separate easily on the flotation bath during cutting. Tissue morphology is not well maintained and nuclei can appear smeared. Improper fixation is irreversible. b. Incomplete dehydration Tissue will be mushy and softer than usual. It may show properly processed edges, but a lighter or whiter colour in the centre and thicker areas. Will likely smell like formalin. Water may be visible inside the tissue cassettes and on the bottom of the wax bath. c. Over dehydration Tissue will be very brittle. Cutting will be difficult if even possible. d. Incomplete clearing Tissues will appear opaque or cloudy due to two immiscible substances present together in the tissue - alcohol and paraffin. Tissues will have a soft consistency. Tissue will eventually dry up and shrink. e. Excessive time in the clearing agent Too much time in the xylene can produce brittle tissue, making it difficult or even impossible to cut. f. Incomplete filtration This means that the wax will not completely replace the xylene from the clearing step. Wax mixed with xylene will not properly harden to support the tissue. Cutting will be very difficult. The block will still smell like xylene. Over time during storage, the clearing agent will evaporate from the face of the block and the tissue will contract. g. Excessive infiltration time and / or wax too hot Prolonged time in the paraffin wax causes shrinkage and hardening of the tissue. Wax that has been overheated breaks down, making sectioning and pick-up onto slides very difficult. List two techniques or procedures that make use of cryostat sections. - ANSWER-Quick section for rapid diagnosis (frozen section), Oil Red O, Enzymes for immunohistochemistry, Immunofluorescence, Muscle biopsy What is the antiroll plate made of? - ANSWER-Plastic or plastic coated glass At what thickness are frozen sections cut for lipid demonstration? - ANSWER-10 - 18 um At what thickness are frozen sections cut for rapid H&E staining? - ANSWER-6 - 8 um State (3) advantages and (2) disadvantages of frozen sectioning. - ANSWER-a. Advantages: No lipid or enzyme loss No processing artifact Rapid diagnosis b. Disadvantages: Ribbons are impossible Storage of frozen tissue is difficult, if not impossible (need -70° C freezer or liquid nitrogen tank; can thaw and store in NBF) Thin sections, such as 3-4um, are difficult Cutting artifacts more likely and detail distorted Name (2) chemicals that can be used to disinfect a cryostat. - ANSWER-Formaldehyde fumes, Glutaraldehyde, Germicidal spray, alcohol Explain the meaning of the term "resonance system" with regard to dye chemistry. - ANSWER-A benzene ring and a chromophore giving a change in energy level with the formation of a closed system. Now, the light will absorb in the visible spectrum Define the term auxochrome. Name the (4) auxochromes in relation to dye classification. - ANSWER-Ionizing radical that gives the molecule the property of electrolytic dissociations (salt forming). Ex. Gives the dye a positive or negative charge and making it colour-fast. i. OH - hydroxyl ii. COOH - carboxyl iii. NH2 - amino iv. SO3H - sulphonic Name and define (4) physical mechanisms of staining. - ANSWER-a. Adsorption - attraction of dye particles to tissue surface b. Porosity - the relationship of the size of the dye molecules to tissue permeability c. Absorption - ability of dye to penetrate tissue and bind with its positive and negative components d. Selective Solubility - dye is less Explain the principle and state a staining method for each of the chemical theories of staining listed below. Salt linkage Hydrogen bonding Histochemical reaction Metachromasia Metallic impregnation Argyrophyllic reaction - ANSWER-a. Salt linkage - electrostatic attraction: tissue components usually have charged groups in protein . Example: H&E, AlBl, b. Hydrogen bonding - 2 atoms linked by H (covalent bond) O-H-N Example: GAF, Congo Red, Verhoeff c. Histochemical reaction - formation of an insoluble coloured compound at a freaction site in tissue. Example: PAS, Perl's Prussian Blue d. Metachromasia - one dye colours certain tissue components (chromotropes), a different colour than the original dye Example: Toluidine Blue e. Metallic impregnation - disposition of salts of heavy metals over selective cell and tissue structures...usually a black result, can be opaque also. f. Argyrophyllic reaction - local reduction and selective precipitate of Ag by the aldehyde groups of the carbohydrate of the reticulin. These groups reduce the ammoniacal Ag solution to dark brown silver oxide on the fibres. This is thenreduced to a black metallic silver by an extraneous reducer Example: formalin in Gordon & Sweets g. Argentaffin reaction - Tissue binds Ag, reducing it directly to visible metallic silver. These tissues contain phenolic groups and tyrosine derivatives as well as large quantities of aldehydes. Example: GMS, Melanin, PAMS h. Metallic substitution - replacement of calcium salts by Ag and then reduced to a visible brown or black colour. Example: VonKossa Define progressive and regressive staining. Give (2) examples of each - ANSWER-Progressive - specific intensity, no differentiation step, Regressive - tissue is overstained and then differentiated Explain how the following differentiating fluids work. State one application for each. Acids and acid alcohols Simple solvents Mordants - ANSWER-Acids and acid alcohol - mass action, breaks tissue mordant bond Example: routine hematoxylin b. Simple solvents - excess dye dissolved by solubility Example: routine eosin, water washes c. Mordants - present in excess compared to the amount in the tissue. Mass action- the dye leaves the tissue to combine with the free mordant in the solution Define a mordant and explain its use in Histology. List (2) staining solutions which contain mordants. - ANSWER-Mordant - is the salt of a divalent or trivalent metal. It supplies the link between the dye and the tissue. Example: hematoxylins such as Harris', Mayer's, Verhoeff's Explain the purpose of accentuators and list two staining solutions which contain accentuators. What is an accelerator? - ANSWER-a. Accentuator - act as a catalyst to speed up the reaction or increase the intensity Example: acetic acid in eosin, light green; phenol in carbol fuchsin b. Accelerator - special application of accentuator when impregnation involves nervous tissue Example: chloral hydrate in some Metallic impregnation for nerve fibres If mounting media is too thin when coverslipping, what will happen? - ANSWER-Air around the edges can introduce bubbles. The tissue can be distorted if not completely covered. You may be tempted to 'press down' on the coverslip to disperse the mounting media if there is not enough, but this can crush and distort the tissue also. Explain briefly how horseradish peroxidase takes part in the demonstration of the antigen under investigation. - ANSWER-HRP splits H2O2 into H2O + O2 which oxidizes the chromogen and produces a coloured compound DAB (3-diaminobenzidine) and AEC (3-amino-9-ethylcarbazole) are chromogens used to demonstrate the antigen under investigation. What are the primary differences between them? - ANSWER-a. DAB Insoluble in alcohol Uses permanent resin to coverslip Result = brown b. AEC Soluble in alcohol Aqueous mounting media Result = red List (8) causes of non-specific staining. - ANSWER-a. Thick sections, bubbles b. Normal serum not applied before primary c. Concentration of normal serum or buffer salt too low d. Paraffin not completely removed On microscopic examination of an H&E tissue section, unstained patches are seen. What could be the cause? - ANSWER-Incomplete dewaxing, slides stuck together, solutions too low After the ABC immunohistochemical procedure, the test section expected to be positive, was much paler than the positive control. Why? - ANSWER-Different fixatives used on each tissue, different tissues stain differently Why would slides turn milky when placed in distilled water after de-waxing and hydrating? - ANSWER-Xylene is still present When Hematoxylin is either over-oxidized or over-differentiated, what, if anything will be wrong with the appearance of the tissue in an H&E preparation? - ANSWER-Nuclei will be pale In an H&E stain, if the ammonia water is not properly rinsed out before the eosin, what, if anything will be wrong with the appearance of the tissue? - ANSWER-Cytoplasm will appear pale When checking the Gram stain control, all bacteria appear purple. Why would this happen? - ANSWER-The tissue was under differentiated by alcohol/acetone On microscopic examination, what causes an H&E stained tissue section to have bright pink stained areas throughout? - ANSWER-Air bubbles under the tissue section On microscopic examination of a Masson's Trichrome stained section, why are the muscle and collagen both blue? - ANSWER-Differentiation was too long Why would there be a microscopic appearance of precipitate on a tissue section stained with Oil Red O? - ANSWER-Working solution not filtered, contamination from containser, working solution not made fresh What could cause a tissue section stained with Masson's Trichrome to shows no nuclear staining? - ANSWER-use of wrong hemotoxylin, over-oxidized hemotoxylin, iron-mordanted hemotoxylin needed When comparing two sections stained on different occasions with the Von Kossa method, the positively stained area in one appears much blacker than in the other. Which light source gives the stronger result? - ANSWER-Sunlight On a tissue section stained by the Verhoeff method, why would elastic fibres appear very light? - ANSWER-Solution is over-oxidized Over-differentiation Overstaining in the Van Gieson's solution In the Grocott's Methenamine Silver method, why would fungi appear completely black on microscopic examination? - ANSWER-Working solution was too hot Too long in the working silver solution Working solution was made too strong After blueing sections in ammonia water, what could cause all the tissues to lift off the slides? - ANSWER-Ammonia water is too strong There was no slide adhesive Slides were not dried long enough in the oven What could potentially cause the silver solution to explode during a staining method using silver nitrate? - ANSWER-Ammoniacal silver solution + silvered glassware can be explosive Why would tissue fixed in formal-alcohol not freeze properly? - ANSWER-Alcohol inhibits freezing On an H&E stained section of skin, why would only the dermis be showing under microscopic examination? - ANSWER-incorrect orientation during embedding the block was not trimmed full face On microscopic examination of a control slide for reticulin fibres, why might the reticulin fibres appear dark black and both collagen and elastic fibres appear dark brown? - ANSWER-Too long in the silver solution When a specimen arrives in the Histology Laboratory without a requisition, the technologist should: - ANSWER-Alert the sender, wait for the requisition and then accession the specimen Why might a precipitate be observed on a section stained with Perl's Prussian Blue? - ANSWER-A red precipitate indicates the nuclear fast red was not filtered A blue precipitate indicates a reaction with dirty glassware Contamination of the solution during the procedure When cutting a block of breast tissue, the centre of the block is mushy. The H&E stained sections reveal a large hole in the middle of the section. What happened? - ANSWER-Improper fixation Improper dehydration Improper clearing Improper infiltration Which of the following problems during tissue processing cannot be remedied? - ANSWER-Improper fixation Why could a section stained by the reticulin fibre method show a fine granular precipitate microscopically? - ANSWER-Metal forceps touched the silver solution Silver solution not filtered Improper rinsing Why would mast cells appear blue on microscopic examination of a metachromatic stain? - ANSWER-dehydrated with alcohol incorrect Ph When examining a Congo Red stained section under a polarizing microscope, why could there be no apple-green birefringence observed? - ANSWER-Section was cut too thin What is it called when a tissue section of liver stained with PAS, shows magenta colored areas which are visible only in one half of the cytoplasm of the cells? - ANSWER-Glycogen streaming Two sections from the same block of live, one treated with diastase, and both stained with PAS, are both PAS positive. What is the probable cause? - ANSWER-Diastase was old or missed A positive control and a test section stained with the Avidin-Biotin method shows non-specific staining. What could cause this? - ANSWER-Endogenous peroxidase or biotin not blocked Antibody concentration was too high Dilution was too low What problem may have occurred if the basement membrane of the tubules in a section of kidney stained by the Periodic Acid Methenamine Silver method is dark brown and the glomerular basement membrane is unstained? - ANSWER-Not in the silver solution long enough If tissue has been fixed in B5, with what must slides be treated before taking them through staining? - ANSWER-Iodine and hypo What chemical(s) can be used to remove acid formaldehyde hematin? - ANSWER-Alcoholic picric acid How can the formation of formalin pigment be prevented? - ANSWER-Using 10% neutral buffered formalin