MLT GlobeHisto & Cyto Techniques: Fixation, processing & staining – page 3
94 Histo & Cyto Techniques MCQs on Fixation, processing & staining with answers and explanations.
Unstained Pap smears that must be mailed should be:
Fixation before drying prevents air-drying artifact. A coating agent or glycol-containing fixative protects the smear during transport.
Which is a drawback of ethanol-based cytology fixatives?
Ethanol fixes by dehydration and protein precipitation, giving crisp nuclei but some shrinkage. It is used at room temperature.
A Pap slide shows golden-brown refractile deposits over cells ('cornflake' artefact). What is the likely cause?
If xylene evaporates before mountant is applied, air is trapped over cells, giving brown refractile artefact; overstaining only darkens nuclei.
Air-drying artefact in cytocentrifuge slides for Pap staining is best reduced by:
Cytospin spots dry quickly; immediate wet fixation preserves Pap morphology. Distilled water lyses cells, and heat worsens drying.
Which decalcifying agent is slowest but best preserves tissue antigens and nucleic acids?
EDTA removes calcium by chelation, gently but slowly (days to weeks). Strong acids are fast but damage staining, antigens and DNA, which matters for immunohistochemistry and molecular tests.
10% neutral buffered formalin is prepared from concentrated (37–40%) formaldehyde. The actual formaldehyde concentration in the working fixative is about:
'10% formalin' means 10% of the stock 37–40% solution, which gives about 3.7–4% formaldehyde. The 10% refers to formalin, not formaldehyde.
Brown-black, birefringent granules are seen across a bloody spleen section fixed in unbuffered formalin. How could this artifact have been prevented?
This is acid formaldehyde hematin (formalin pigment), which forms when acidic formalin reacts with hemoglobin. Buffering to neutral pH prevents it; alcoholic picric acid removes it.
Sections fixed in a mercury-containing fixative show black crystalline deposits. Before staining, the deposits are removed by:
Mercury pigment is converted by iodine (e.g., Lugol's) to mercuric iodide, and the iodine is then removed with sodium thiosulfate. Alcoholic picric acid removes formalin pigment.
Tissue placed in xylene during processing turns milky and cloudy. What is the most likely cause?
Xylene does not mix with water. Residual water from incomplete dehydration gives a milky appearance, so the tissue should be returned to absolute alcohol.
Paraffin infiltration baths are kept far above the wax melting point for many hours. The most likely effect on the tissue is:
Paraffin should be kept only about 2–4 °C above its melting point. Overheating makes tissue hard, brittle and shrunken, and damages antigens.
How should a skin biopsy be oriented in the embedding mold?
Skin is embedded on edge (perpendicular to the cutting face) so every layer appears in the section. Flat embedding would give sections of only one layer.
Sections show alternating thick and thin bands running parallel to the knife edge. This artifact is called:
Chatter results from vibration, often due to a loose blade or block, very hard or over-dehydrated tissue, or an incorrect clearance angle. Scoring runs perpendicular to the knife edge.
Every section from a block shows a straight tear running perpendicular to the knife edge at the same position. The most likely cause is:
A nick in the blade or a hard deposit such as calcium drags through the tissue and splits every section at the same place. Move to a new area of the blade.
For paraffin with a melting point of 56–58 °C, the flotation water bath is usually kept at about:
The water bath is kept about 10 °C below the paraffin melting point so the ribbon flattens without melting. A bath at the melting point would disperse the section.
Which method gives the most accurate determination of the end point of decalcification?
Radiography shows remaining calcium most accurately. The chemical ammonium oxalate test is also acceptable, while physical probing damages tissue and is unreliable.
A bone biopsy left in strong acid decalcifier over the weekend is sectioned. What staining problem is most expected?
Over-decalcification in strong acid damages nucleic acids, so nuclei stain weakly and poorly with hematoxylin. Timely endpoint testing prevents this.
In Harris or Mayer hematoxylin, the mordant that links hematein to tissue is:
Alum hematoxylins use aluminum salts as the mordant, forming a positively charged dye-lake. Sodium iodate is an oxidizing agent; iron is the mordant in Weigert's hematoxylin.
In regressive hematoxylin staining, tissue is:
Regressive staining deliberately overstains, then removes excess dye with acid alcohol. Progressive staining stains to the desired endpoint without differentiation.
A liver biopsy shows magenta granules with PAS that disappear on a serial section treated with diastase before PAS. The granules are:
Diastase digests glycogen, so PAS positivity that is lost after diastase indicates glycogen. Neutral mucins and fungi remain PAS positive after digestion.
The Perls Prussian blue reaction demonstrates hemosiderin because potassium ferrocyanide in hydrochloric acid reacts with:
Acid releases ferric iron from hemosiderin, which combines with ferrocyanide to form blue ferric ferrocyanide. Iron tightly bound in hemoglobin does not react.