MLT GlobeHisto & Cyto Techniques: Fixation, processing & staining – page 4
94 Histo & Cyto Techniques MCQs on Fixation, processing & staining with answers and explanations.
In immunohistochemistry on formalin-fixed tissue, heat-induced epitope retrieval (e.g., citrate buffer pH 6) is performed mainly to:
Formalin cross-links hide epitopes. Heating in buffer breaks these links and unmasks antigens so the primary antibody can bind.
In an HRP–DAB immunohistochemistry method, sections are treated with dilute hydrogen peroxide before the primary antibody in order to:
Red cells and granulocytes contain peroxidase activity that would convert DAB to brown product and give false positives. Hydrogen peroxide inactivates it beforehand.
In immunohistochemistry, a negative reagent control is prepared by:
Replacing the primary antibody shows whether staining is due to the detection system or nonspecific binding. A tissue known to express the antigen is a positive control.
Which fixative contains absolute ethanol, chloroform and glacial acetic acid, acts very quickly and preserves glycogen and nucleic acids well?
Carnoy's is a rapid alcohol-based fixative that preserves glycogen and nucleic acids but dissolves lipids and causes shrinkage. Zenker's contains mercuric chloride and dichromate.
Osmium tetroxide is used as a secondary fixative in electron microscopy mainly because it:
Osmium tetroxide reacts with unsaturated lipids, fixing membranes and adding contrast (it turns black). It penetrates poorly, which is why glutaraldehyde is used first.
For breast carcinoma specimens tested for ER, PR and HER2, ASCO/CAP guidelines recommend fixation in 10% neutral buffered formalin for:
Under-fixation (less than 6 hours) and over-fixation (more than 72 hours) can both give false results for hormone receptors and HER2. The fixation time should be recorded.
ASCO/CAP guidelines state that a breast resection for ER, PR and HER2 testing should be placed in fixative within:
Cold ischemia time should be as short as possible and no more than 1 hour, because antigens and nucleic acids degrade before fixation. The time to fixation should be documented.
Formaldehyde penetrates tissue at roughly 1 mm per hour. To allow even fixation, tissue slices placed in cassettes should be about:
Slow penetration means thick pieces fix poorly in the center, so tissue should be thinly sliced (about 3–5 mm) before fixation. A large fixative volume does not overcome poor penetration.
Tissue from a lymph node that may later be used for PCR-based molecular testing should NOT be fixed in:
Bouin's contains picric and acetic acids, which break down DNA and RNA, making the tissue poor for molecular tests. Buffered formalin and alcohol-based fixatives are acceptable.
While sectioning a paraffin block, the knife meets a small calcified area and the sections tear. The quickest remedy is to:
Surface decalcification of the exposed block face for a short time softens small calcium deposits so good sections can be cut. Reprocessing does not remove calcium.
Applying vacuum during paraffin infiltration mainly:
Reduced pressure draws out air bubbles and volatile clearing agent, so paraffin enters dense or fatty tissue faster and more completely. It does not change the wax melting point.
Mayer's hematoxylin is chemically 'ripened' (hematoxylin oxidized to hematein) by adding:
Sodium iodate oxidizes hematoxylin to hematein, the actual dye. Potassium alum is the mordant, and lithium carbonate is used for bluing.
Mucicarmine staining is most useful to show:
Mucicarmine stains epithelial mucins red and also stains the mucopolysaccharide capsule of Cryptococcus neoformans. Elastic fibers need Verhoeff or orcein stains.
Spirochetes such as Treponema pallidum in a tissue section are best demonstrated with:
Silver impregnation methods such as Warthin–Starry blacken thin spirochetes that are hard to see with other stains. Ziehl–Neelsen is for acid-fast bacilli.
Van Gieson solution, used to stain collagen red, contains:
Van Gieson combines saturated picric acid (yellow for muscle and cytoplasm) with acid fuchsin (red for collagen). Aniline blue with phosphomolybdic acid is part of the Masson trichrome.
Phosphotungstic acid–hematoxylin (PTAH) is used mainly to show:
PTAH stains muscle cross-striations, fibrin and nuclei blue, with collagen red-brown. It is useful for skeletal muscle tumors and fibrin deposits.
In suspected Wilson disease, copper in a liver biopsy is best demonstrated with:
Rhodanine binds copper and shows it as orange-red granules. Perls stains ferric iron and von Kossa shows calcium salts.
In a combined Alcian blue (pH 2.5)–PAS stain of intestinal mucosa, neutral mucins appear:
Alcian blue stains acid mucins blue, then PAS stains the remaining neutral mucins magenta; cells with both appear purple. This helps detect intestinal metaplasia.
An immunohistochemistry slide developed with AEC chromogen must be mounted with an aqueous medium because:
AEC gives a red product that is soluble in organic solvents, so dehydration and resinous mounting would remove it. DAB forms a brown product that is insoluble and can be dehydrated.
A small piece of unrelated tissue appears on the slide of another patient's biopsy, lying on top of the section. The most likely source and prevention are:
Fragments left on the flotation bath can stick to the next patient's slide. The water surface should be skimmed with tissue paper between blocks to prevent these 'floaters'.