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ASCP H practice questions

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Question 1Fixation and Tissue Processing

Which fixative is the standard for routine surgical pathology specimens?

  1. Bouin's solution
  2. 10% neutral buffered formalin Correct answer.
  3. Absolute ethanol
  4. 2.5% glutaraldehyde

Explanation

Neutral buffered formalin is buffered to pH 7.0-7.6, preserves morphology well, and is compatible with most routine stains and immunohistochemistry. Glutaraldehyde is reserved mainly for electron microscopy, Bouin's for selected special stains, and ethanol is a coagulant dehydrant.

Question 2Fixation and Tissue Processing

What ratio of fixative volume to tissue volume is recommended for adequate formalin fixation?

  1. 10:1 Correct answer.
  2. 50:1
  3. 5:1
  4. 1:1

Explanation

At least ten volumes of fixative per volume of tissue provides enough reagent to compensate for dilution by tissue fluids and to maintain fixation. Smaller volumes may be exhausted before fixation is complete.

Question 3Fixation and Tissue Processing

Formalin fixes tissue mainly by:

  1. chelating calcium ions
  2. precipitating nucleic acids
  3. dissolving membrane lipids
  4. cross-linking proteins through methylene bridges Correct answer.

Explanation

Formaldehyde reacts with amino and amide groups on proteins, creating methylene bridges that stabilize tissue structure. This cross-linking can mask antigens but does not fix lipids.

Question 4Fixation and Tissue Processing

Which fixative is preferred for transmission electron microscopy?

  1. Bouin's solution
  2. 10% neutral buffered formalin
  3. 2.5% glutaraldehyde Correct answer.
  4. Carnoy's solution

Explanation

Glutaraldehyde produces stronger protein cross-linking and preserves fine ultrastructure, often followed by osmium tetroxide post-fixation. Formalin alone gives poorer electron microscopic detail, and Bouin's and Carnoy's are coagulant light microscopy fixatives.

Question 5Fixation and Tissue Processing

Which clearing agent is most widely used in routine paraffin processing?

  1. Xylene Correct answer.
  2. 10% formalin
  3. Absolute ethanol
  4. Acetone

Explanation

Xylene is miscible with both alcohol and paraffin, making it the standard clearing agent in routine processing. Ethanol is a dehydrant, formalin is a fixative, and acetone is not the routine clearing agent.

Question 6Fixation and Tissue Processing

Dehydration of tissue during processing is usually performed with:

  1. neutral buffered formalin
  2. graded ethanol solutions Correct answer.
  3. xylene
  4. molten paraffin

Explanation

Graded ethanol removes water gradually, limiting shrinkage before the tissue is cleared. Xylene clears tissue, formalin fixes it, and paraffin infiltrates it.

Question 7Fixation and Tissue Processing

What is the usual melting point of paraffin wax used for routine tissue embedding?

  1. 56-58°C Correct answer.
  2. 70-75°C
  3. 45-50°C
  4. 37-40°C

Explanation

Paraffin with a melting point of 56-58°C is firm enough for room-temperature sectioning while remaining liquid at embedding temperatures. Much softer waxes fail to support the tissue, and much harder waxes require excessive heat.

Question 8Fixation and Tissue Processing

Which fixative best preserves glycogen in tissue?

  1. 10% neutral buffered formalin
  2. Carnoy's solution Correct answer.
  3. Zenker's fluid
  4. Bouin's solution

Explanation

Carnoy's solution contains absolute ethanol, chloroform, and acetic acid and rapidly preserves glycogen, whereas aqueous formalin tends to dissolve it. Zenker's and Bouin's are not preferred for glycogen preservation.

Question 9Fixation and Tissue Processing

Yellow-brown granular pigment seen in blood-rich tissue fixed in unbuffered formalin is:

  1. melanin
  2. hemosiderin
  3. lipofuscin
  4. formalin pigment Correct answer.

Explanation

Acid formalin reacts with hemoglobin to form acid hematin, also called formalin pigment. It can be removed with saturated alcoholic picric acid or ammonia.

Question 10Fixation and Tissue Processing

Zenker's fixative contains which metal salt whose pigment is removed with iodine?

  1. zinc chloride
  2. lead nitrate
  3. osmium tetroxide
  4. mercuric chloride Correct answer.

Explanation

Zenker's fluid contains mercuric chloride, which deposits brown mercury pigment in tissue. Iodine in Lugol's solution converts the pigment to soluble mercuric iodide so it can be washed out.

Question 11Fixation and Tissue Processing

Tissue fixed in Bouin's solution has a yellow color that is removed by:

  1. treatment with iodine
  2. washing in 70% alcohol Correct answer.
  3. rinsing in distilled water
  4. soaking in xylene

Explanation

Bouin's solution contains picric acid, which imparts a yellow color that is washed out in 70% alcohol. Iodine removes mercury pigment rather than picric acid.

Question 12Fixation and Tissue Processing

Why is neutral buffered formalin preferred over unbuffered formalin?

  1. It fixes tissue more rapidly
  2. It preserves lipids better
  3. It decalcifies bone
  4. It prevents formation of formalin pigment Correct answer.

Explanation

Buffering to pH 7.0-7.6 prevents the acidic conditions that produce acid hematin pigment. Buffering does not substantially speed fixation, preserve lipids, or decalcify bone.

Question 13Fixation and Tissue Processing

Prolonged fixation in formalin can cause:

  1. tissue swelling and protein loss
  2. spontaneous decalcification
  3. increased cross-linking and antigen masking Correct answer.
  4. dissolution of paraffin

Explanation

Overfixation adds protein cross-links that can mask epitopes and necessitate antigen retrieval. It does not dissolve paraffin or decalcify bone.

Question 14Fixation and Tissue Processing

Inadequate fixation of a thick tissue slice most often produces:

  1. central autolysis and poor staining Correct answer.
  2. yellow discoloration
  3. excessive hardening of the center
  4. mercury pigment

Explanation

Formalin penetrates at roughly 1 mm per hour, so thick slices remain unfixed in the center and undergo autolysis. Yellow color indicates picric acid, and mercury pigment comes from Zenker's fixative.

Question 15Fixation and Tissue Processing

What is the purpose of applying vacuum during tissue processing?

  1. to speed dehydration by evaporating alcohol
  2. to decalcify bone
  3. to sterilize the tissue
  4. to remove trapped air and improve paraffin infiltration Correct answer.

Explanation

Vacuum removes air and volatile clearing agent from tissue spaces, allowing paraffin to infiltrate more completely. It is not a sterilizing or decalcifying step.

Question 16Fixation and Tissue Processing

Which decalcifying agent is a chelator that gently preserves antigens and nucleic acids?

  1. nitric acid
  2. formic acid
  3. EDTA Correct answer.
  4. hydrochloric acid

Explanation

EDTA chelates calcium ions slowly and is gentler on antigens and DNA than mineral acids. Nitric, formic, and hydrochloric acids decalcify faster but can damage tissue components.

Question 17Fixation and Tissue Processing

The endpoint of decalcification is commonly assessed by:

  1. staining a section with eosin
  2. testing with iodine
  3. measuring tissue pH
  4. the ammonium oxalate test Correct answer.

Explanation

Residual calcium in the decalcifying solution reacts with ammonium oxalate to form a precipitate; absence of precipitate indicates that decalcification is complete. Iodine testing detects mercury pigment, not calcium.

Question 18Fixation and Tissue Processing

Which fixative is preferred for preserving uric acid crystals?

  1. Zenker's fluid
  2. 10% neutral buffered formalin
  3. alcoholic formalin Correct answer.
  4. Bouin's solution

Explanation

Uric acid crystals dissolve in aqueous fixatives, so absolute alcohol with formalin is used to preserve them. Aqueous neutral buffered formalin, Bouin's, and Zenker's would dissolve or distort the crystals.

Question 19Fixation and Tissue Processing

Osmium tetroxide is used in electron microscopy because it:

  1. stains glycogen
  2. preserves and stains lipids Correct answer.
  3. decalcifies bone
  4. removes formalin pigment

Explanation

Osmium tetroxide binds unsaturated lipids in membranes, providing both preservation and contrast for electron microscopy. It is not a decalcifier or a glycogen stain.

Question 20Fixation and Tissue Processing

Raising the temperature of formalin fixation generally:

  1. prevents protein cross-linking
  2. has no effect on the rate of fixation
  3. slows fixative penetration
  4. accelerates fixation but increases autolysis and heat artifact Correct answer.

Explanation

Chemical fixation speeds up as temperature rises, but heat also accelerates autolysis and can produce heat artifact, so routine fixation is performed at room temperature.

Question 21Fixation and Tissue Processing

Prolonged exposure of tissue to xylene during processing tends to make it:

  1. yellow and pigmented
  2. swollen and edematous
  3. soft and gelatinous
  4. hard and brittle Correct answer.

Explanation

Extended clearing removes residual lipids and over-dehydrates tissue, producing hard, brittle blocks that section poorly. Xylene does not swell or pigment tissue.

Question 22Fixation and Tissue Processing

Which preanalytical factor most directly increases autolytic damage before fixation?

  1. delay between excision and fixation Correct answer.
  2. use of a 10:1 fixative ratio
  3. section thickness at microtomy
  4. paraffin melting point

Explanation

Autolysis begins as soon as the blood supply stops and worsens with time and warm temperature before fixative penetrates. Fixative ratio, paraffin melting point, and section thickness do not act before fixation.

Question 23Fixation and Tissue Processing

Surface decalcification of a paraffin block is performed by:

  1. immersing the entire block in nitric acid
  2. applying acid to the exposed tissue surface Correct answer.
  3. heating the block to 60°C
  4. soaking the block in EDTA for a week

Explanation

Surface decalcification applies a small amount of acid to the cut face for a short time to soften a focal calcium deposit without decalcifying the whole specimen. Immersing the block or prolonged EDTA would damage the entire specimen.

Question 24Fixation and Tissue Processing

Microwave-assisted processing is used mainly to:

  1. replace formalin fixation entirely
  2. improve antigen preservation indefinitely
  3. eliminate the need for dehydration
  4. shorten processing time by heating and agitating reagents Correct answer.

Explanation

Microwaves heat reagents and tissue, accelerating fixation, dehydration, and infiltration so processing time is shortened. Processing steps are not eliminated, and excessive heat can damage antigens.

Question 25Fixation and Tissue Processing

What tissue slice thickness is recommended to allow adequate formalin penetration?

  1. 3-5 mm Correct answer.
  2. 1-2 mm
  3. 10-15 mm
  4. 20-25 mm

Explanation

Slices 3-5 mm thick allow formalin to penetrate within a reasonable time; thicker slices remain unfixed centrally. Very thin slices are unnecessary and difficult to orient.

Question 26Fixation and Tissue Processing

What is the primary purpose of tissue fixation before processing?

  1. To soften bone so that sections can be cut
  2. To dissolve lipids and permit clearing to occur
  3. To remove water so paraffin can enter the tissue
  4. To preserve cellular structure and block autolysis Correct answer.

Explanation

Fixation stabilizes proteins and other macromolecules, inactivates enzymes, and kills microorganisms, thereby preventing autolysis and putrefaction. Dehydration, clearing, and decalcification are separate steps performed later in the workflow.

Question 27Fixation and Tissue Processing

What is the usual pH of neutral buffered formalin (NBF)?

  1. 5.0 to 5.5
  2. 3.0 to 4.0
  3. 7.0 to 7.4 Correct answer.
  4. 8.5 to 9.0

Explanation

NBF is buffered to approximately physiological pH, about 7.0 to 7.4, which minimizes acid-related artifacts such as formalin pigment. Unbuffered formalin becomes acidic as formic acid forms during storage.

Question 28Fixation and Tissue Processing

What is the recommended minimum ratio of 10% formalin volume to tissue volume?

  1. 1:1
  2. 5:1
  3. 2:1
  4. 10:1 Correct answer.

Explanation

A 10:1 ratio of fixative to tissue ensures sufficient reagent to penetrate the specimen and dilute tissue fluids. Lower ratios exhaust the fixative and slow or prevent complete fixation.

Question 29Fixation and Tissue Processing

Why is tissue dehydrated through a graded series of alcohols rather than placed directly into absolute alcohol?

  1. Graded alcohols neutralize residual formalin before clearing
  2. Graded alcohols reduce sudden shrinkage and hardening Correct answer.
  3. Graded alcohols remove tissue lipids more completely
  4. Graded alcohols keep paraffin from dissolving during processing

Explanation

Moving gradually from lower to higher alcohol concentrations limits abrupt water loss and reduces shrinkage and distortion. Direct transfer to absolute alcohol can harden the surface and impede penetration.

Question 30Fixation and Tissue Processing

Formalin fixes tissue mainly by which mechanism?

  1. Dissolving lipid membranes and stabilizing them physically
  2. Cross-linking proteins through methylene bridges Correct answer.
  3. Precipitating proteins by lowering the pH below 4
  4. Chelating calcium ions and preventing coagulation

Explanation

Formaldehyde reacts with amino groups, especially on lysine residues, forming methylene bridges that stabilize protein structure. It does not act primarily by acid precipitation or calcium chelation.

Question 31Fixation and Tissue Processing

After several cassettes have been processed, the first xylene station is noticeably cloudy. What is the most likely cause?

  1. The xylene was too warm
  2. The tissue was over-fixed in formalin
  3. The paraffin bath was contaminated with water
  4. The tissue was incompletely dehydrated Correct answer.

Explanation

Cloudiness in xylene usually indicates carried-over water from incomplete dehydration. Ensuring fresh alcohols and adequate dehydration steps prevents this artifact and allows proper clearing.

Question 32Fixation and Tissue Processing

Which clearing agent is preferred for delicate tissue because it causes less shrinkage and hardening than xylene, although it acts more slowly?

  1. Methanol
  2. Toluene
  3. Chloroform Correct answer.
  4. Acetone

Explanation

Chloroform is a slower, gentler clearing agent that produces less hardening of delicate specimens. Acetone and methanol are primarily dehydrants, and toluene behaves similarly to xylene.

Question 33Fixation and Tissue Processing

At room temperature, 10% formalin penetrates solid tissue at approximately what rate?

  1. 1 mm per hour Correct answer.
  2. 0.5 mm per hour
  3. 5 mm per hour
  4. 0.1 mm per hour

Explanation

The practical rule is about 1 mm of penetration per hour at room temperature, so a 4-mm-thick slice needs roughly 2 hours for central fixation. Denser tissue or thicker blocks require longer fixation.

Question 34Fixation and Tissue Processing

Why does adipose tissue usually require longer processing times than densely cellular tissue?

  1. Adipocytes hold more water and need longer dehydration
  2. Lipid absorbs paraffin and blocks sectioning
  3. Fat cells resist formalin and remain unfixed
  4. Lipid must be dissolved and replaced by clearing agent Correct answer.

Explanation

Adipose tissue consists largely of lipid, which is not fixed and must be extracted by clearing agents such as xylene. The large volume of lipid requires extended clearing before paraffin can adequately infiltrate the tissue.

Question 35Embedding and Microtomy

What is the standard section thickness for routine paraffin-embedded H&E sections?

  1. 10 µm
  2. 4 µm Correct answer.
  3. 20 µm
  4. 1 µm

Explanation

Routine paraffin sections for H&E are cut at approximately 3–5 µm, with 4 µm being typical; this thickness gives a single cell layer suitable for nuclear detail and is reproducible on a rotary microtome. Thicker sections overlap cells and obscure detail.

Question 36Embedding and Microtomy

Paraffin ribbons are typically floated on a water bath held at which temperature?

  1. 60–65 °C
  2. 20–25 °C
  3. 40–45 °C Correct answer.
  4. 80–85 °C

Explanation

The flotation bath is kept roughly 5–10 °C below the melting point of the paraffin (about 56–58 °C), so 40–45 °C flattens the ribbon without melting it. Water that is too cool leaves wrinkles and too warm water distorts or dissolves the section.

Question 37Embedding and Microtomy

A cryostat chamber is usually set to which temperature range for routine frozen sections?

  1. −5 to 0 °C
  2. −60 to −70 °C
  3. −20 to −25 °C Correct answer.
  4. 10 to 15 °C

Explanation

Most fresh tissue cuts well when the chamber and specimen are held near −20 to −25 °C, where the tissue is firm but not brittle. Warmer temperatures leave the block soft and smeared, while much colder temperatures cause fracturing.

Question 38Embedding and Microtomy

Which medium is used to embed tissue for sectioning in a cryostat?

  1. OCT compound Correct answer.
  2. Paraffin wax
  3. Celloidin
  4. Agar

Explanation

OCT (optimal cutting temperature) compound is a water-based medium that freezes hard at cryostat temperatures and supports the tissue during cutting. Paraffin and celloidin require processing at temperatures incompatible with frozen sectioning.

Question 39Embedding and Microtomy

The first step in microtomy, in which excess paraffin is cut away until the full tissue face is exposed, is called:

  1. Flotation
  2. Decalcification
  3. Ribboning
  4. Trimming (facing) Correct answer.

Explanation

Facing or trimming removes surplus wax until a complete, representative plane of tissue is reached, ensuring the first collected sections contain the full specimen. Only after facing are intact sections taken for staining.

Question 40Embedding and Microtomy

Which slide type is preferred for sections that may detach during intense pretreatment such as heat-induced epitope retrieval?

  1. Frosted-end slides
  2. Cover-glass slips
  3. Charged (positively coated) slides Correct answer.
  4. Plain glass slides

Explanation

Charged slides carry a positively charged surface (for example aminosilane or poly-L-lysine) that binds the negatively charged tissue section and resists detachment during heat and enzyme pretreatment. Plain glass provides no such adhesion.

Question 41Embedding and Microtomy

Very small or fragmented biopsies are at risk of being lost during processing; which approach helps keep them together?

  1. Agar pre-embedding Correct answer.
  2. Staining before processing
  3. Double embedding
  4. Sectioning at 20 µm

Explanation

Wrapping tiny fragments in agar, or placing them in a biopsy bag or mesh cassette insert, keeps them together and prevents loss during processing and embedding. Thin fragments otherwise pass through cassette pores or become scattered in the wax.

Question 42Embedding and Microtomy

How should paraffin blocks be stored after sectioning?

  1. At room temperature Correct answer.
  2. Incubated at 60 °C
  3. Immersed in formalin
  4. Frozen at −20 °C

Explanation

Paraffin blocks are archival and are stored dry at controlled room temperature, away from heat, direct light and pests, so that additional sections can be cut later. Extreme temperatures damage the wax and the tissue.

Question 43Embedding and Microtomy

The clearance angle between the blade facet and the block face is normally set at about:

  1. 20–25 degrees
  2. 35–40 degrees
  3. 5–10 degrees Correct answer.
  4. 45–50 degrees

Explanation

A clearance angle of roughly 5–10 degrees lets the blade edge cut cleanly with minimal contact between the facet and the block. Too little clearance causes rubbing and compression, while too much produces vibration and chatter.

Question 44Embedding and Microtomy

What is the purpose of the anti-roll plate on a cryostat?

  1. To clamp the block onto the chuck
  2. To collect sections from the bath
  3. To prevent curling of sections Correct answer.
  4. To cool the specimen before cutting

Explanation

The anti-roll plate sits parallel to the blade and guides the newly cut frozen section so it stays flat instead of curling as it leaves the knife. It does not cool or secure the specimen.

Question 45Embedding and Microtomy

A technologist sees evenly spaced parallel chatter lines across sections of a dense specimen. Which is the most likely cause?

  1. A loose block or hard tissue Correct answer.
  2. Tissue that was over-decalcified
  3. Clearance angle adjusted too low
  4. Water bath temperature set too high

Explanation

Regular, evenly spaced lines (chatter or venetian-blind artifact) arise from vibration during cutting, typically from a loose block or chuck, a very hard or dense specimen, or a poorly supported tissue. Tightening the block and softening dense tissue reduce the artifact.

Question 46Embedding and Microtomy

Sections show compression with thinning and parallel creases. Which is the most likely cause?

  1. Prolonged hydration of tissue
  2. Too much trimming of the block
  3. A dull blade Correct answer.
  4. Water bath temperature too cool

Explanation

A dull or damaged edge pushes and compresses tissue rather than slicing it cleanly, so the section thins and creases. A fresh portion of blade or a new blade corrects the problem.

Question 47Embedding and Microtomy

Sections show numerous small holes and torn areas throughout the tissue. Which is the most likely cause?

  1. Water bath contaminated with dust
  2. Blade clearance angle too steep
  3. Very rapid cooling of the block
  4. Poor paraffin infiltration Correct answer.

Explanation

Incomplete replacement of tissue fluid by paraffin leaves unsupported regions that crumble and tear as the blade passes, producing holes throughout the section. Ensuring complete dehydration, clearing and vacuum infiltration prevents this.

Question 48Embedding and Microtomy

Fine, regular vertical lines running the full length of every section most often indicate:

  1. Incomplete dehydration
  2. Static electricity
  3. A nick in the blade edge Correct answer.
  4. Excess paraffin in the bath

Explanation

A small defect or nick in the blade edge leaves a continuous stripe that repeats in every section because the same damaged point contacts the block each pass. Moving to an unused portion of the blade or replacing it clears the lines.

Question 49Embedding and Microtomy

How should a skin punch biopsy be embedded?

  1. Epidermis down, cut tangentially
  2. On its side at a 45-degree angle
  3. Epidermis up, cut perpendicular Correct answer.
  4. Dermis down, cut parallel to surface

Explanation

Skin is oriented with the epidermal surface facing up and the block cut perpendicular to the surface, so the section shows the full epidermis and dermis in a single plane. Parallel or oblique cuts give tangential sections that miss the complete epidermal profile.

Question 50Embedding and Microtomy

Skeletal muscle is best embedded so that it is cut:

  1. Tangentially
  2. Without deliberate orientation
  3. Obliquely at 45 degrees
  4. Longitudinally Correct answer.

Explanation

Longitudinal orientation aligns the muscle fibers with the plane of section, allowing fiber architecture, striations and nuclei to be assessed consistently. Random or oblique orientation produces inconsistent fiber profiles.