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ASCP HT practice questions

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Question 1Specimen Procurement, Handling, and Fixation

Which fixative is the standard of care for routine surgical pathology specimens?

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

Explanation

Neutral buffered formalin is isotonic, buffered to pH 7.0-7.4, and preserves morphology and antigenicity well for routine processing. The other solutions are specialized fixatives that produce color changes or require additional steps.

Question 2Specimen Procurement, Handling, and Fixation

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

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

Explanation

At least 10 volumes of fixative per volume of tissue ensures enough formaldehyde to penetrate and fix the specimen adequately. A 20:1 ratio is often used for cellular specimens.

Question 3Specimen Procurement, Handling, and Fixation

At approximately what rate does formalin penetrate and fix tissue at room temperature?

  1. 1 mm per hour Correct answer.
  2. 5 mm per hour
  3. 0.1 mm per hour
  4. 10 mm per hour

Explanation

Formalin penetrates tissue at roughly 1 mm per hour, so thick specimens must be sliced so fixative reaches the center. Larger intact specimens can undergo central autolysis despite surface fixation.

Question 4Specimen Procurement, Handling, and Fixation

What is the acceptable pH range for 10% neutral buffered formalin?

  1. 3.5 to 4.0
  2. 7.0 to 7.4 Correct answer.
  3. 5.0 to 5.5
  4. 8.5 to 9.0

Explanation

Phosphate buffer maintains neutral buffered formalin near physiologic pH, which minimizes formalin pigment and preserves staining. Acidic formalin causes acid hematin deposition and poorer nuclear detail.

Question 5Specimen Procurement, Handling, and Fixation

Which treatment removes brown-black formalin pigment from tissue sections?

  1. Saturated alcoholic picric acid Correct answer.
  2. Iodine followed by sodium thiosulfate
  3. 1% hydrochloric acid in 70% alcohol
  4. Saturated lithium carbonate

Explanation

Formalin pigment, or acid hematin, is removed by saturated alcoholic picric acid. Iodine and thiosulfate remove mercury pigment, while lithium carbonate removes picric acid yellowing.

Question 6Specimen Procurement, Handling, and Fixation

Mercury pigment from tissues fixed in B5 or Zenker's solution is removed by treating sections with:

  1. Lithium carbonate
  2. Periodic acid
  3. Iodine followed by sodium thiosulfate Correct answer.
  4. Saturated alcoholic picric acid

Explanation

Iodine converts mercury deposits to soluble mercuric iodide, and sodium thiosulfate clears the iodine and removes the pigment. This step is required after mercury-containing fixatives.

Question 7Specimen Procurement, Handling, and Fixation

Bouin's fixative imparts a yellow color to tissue; which solution is used to remove this yellow discoloration?

  1. Saturated lithium carbonate in 70% alcohol Correct answer.
  2. Iodine solution
  3. Ammonia water
  4. Sodium thiosulfate

Explanation

Picric acid in Bouin's solution stains tissue yellow, and the color is removed by saturated lithium carbonate in 70% alcohol or by prolonged washing in 70% alcohol. Iodine and thiosulfate are used for mercury pigment.

Question 8Specimen Procurement, Handling, and Fixation

Which embedding medium is commonly used to support tissue for frozen sections?

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

Explanation

OCT compound is a water-soluble embedding medium that freezes to support tissue in a cryostat. Paraffin requires dehydration and clearing, so it cannot be used for fresh frozen sections.

Question 9Specimen Procurement, Handling, and Fixation

A specimen is left at room temperature for several hours before fixation. Which change is most likely to occur?

  1. Increased tissue hardness
  2. Improved antigen preservation
  3. Complete fixation
  4. Autolysis and loss of cellular detail Correct answer.

Explanation

Without fixative, lysosomal enzymes digest tissue and bacteria cause putrefaction, leading to autolysis and loss of morphology. Cold ischemia time should be minimized for all surgical specimens.

Question 10Specimen Procurement, Handling, and Fixation

Which decalcifying agent is preferred when preservation of DNA and antigenicity is critical?

  1. 20% formic acid
  2. 5% nitric acid
  3. Hydrochloric acid
  4. EDTA Correct answer.

Explanation

EDTA is a chelating agent that removes calcium slowly at neutral pH and causes minimal damage to nucleic acids and antigens. Strong acids decalcify rapidly but degrade DNA and mask antigens.

Question 11Specimen Procurement, Handling, and Fixation

Which decalcification method is most appropriate for a bone marrow core biopsy that will undergo molecular testing?

  1. Boiling in nitric acid
  2. 24 hours in 10% hydrochloric acid
  3. Rapid decalcification with 5% nitric acid
  4. EDTA at neutral pH Correct answer.

Explanation

EDTA chelates calcium at neutral pH and preserves DNA and RNA for molecular studies. Strong acids such as nitric or hydrochloric acid degrade nucleic acids and are avoided for molecular testing.

Question 12Specimen Procurement, Handling, and Fixation

Which fixative is best for preserving glycogen in tissue sections?

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

Explanation

Glycogen is water-soluble and leaches out in aqueous fixatives; alcohol-based fixatives such as absolute ethanol or Carnoy's preserve it. Aqueous formalin and Bouin's solution dissolve glycogen.

Question 13Specimen Procurement, Handling, and Fixation

For electron microscopy, tissues are routinely fixed in which solution?

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

Explanation

Glutaraldehyde cross-links proteins more extensively than formalin and preserves ultrastructure for electron microscopy. Formalin is inadequate for fine structural detail.

Question 14Specimen Procurement, Handling, and Fixation

Which fixative is used specifically to preserve lipids for electron microscopy?

  1. Bouin's solution
  2. Osmium tetroxide Correct answer.
  3. Glutaraldehyde
  4. Formalin

Explanation

Osmium tetroxide reacts with unsaturated lipids and stabilizes membranes, and it is used as a post-fixative for electron microscopy. Formalin and glutaraldehyde do not retain lipids well.

Question 15Specimen Procurement, Handling, and Fixation

A sputum specimen for cytology is best collected in which fixative?

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

Explanation

Saccomanno's fixative contains ethanol and polyethylene glycol and preserves cells and mucoid material for sputum cytology. Formalin is not optimal for cytologic detail, and Zenker's or Bouin's are special histology fixatives.

Question 16Specimen Procurement, Handling, and Fixation

What is the OSHA permissible exposure limit (PEL) for formaldehyde as an 8-hour time-weighted average?

  1. 5 ppm
  2. 2 ppm
  3. 15 ppm
  4. 0.75 ppm Correct answer.

Explanation

OSHA sets the formaldehyde PEL at 0.75 ppm as an 8-hour time-weighted average, with a 2 ppm short-term exposure limit. Formalin should be handled in ventilated areas to stay below these limits.

Question 17Specimen Procurement, Handling, and Fixation

Which practice best ensures adequate fixation of a large surgical specimen?

  1. Slicing the specimen at 3-5 mm intervals before fixation Correct answer.
  2. Placing the intact specimen in formalin
  3. Using a smaller volume of fixative to increase concentration
  4. Fixing at 4°C for 24 hours

Explanation

Formalin penetrates at about 1 mm per hour, so serial slicing allows fixative to reach the center of large specimens. An intact large specimen may autolyze centrally despite surface fixation.

Question 18Specimen Procurement, Handling, and Fixation

Which specimen should be transported fresh (not in formalin) to the surgical pathology laboratory?

  1. A skin excision for routine histology
  2. A lymph node for flow cytometry Correct answer.
  3. A routine colon resection
  4. A product of conception

Explanation

Flow cytometry requires viable cells, so lymph nodes are transported in saline or tissue culture medium rather than formalin. Formalin kills cells and destroys surface markers needed for the assay.

Question 19Specimen Procurement, Handling, and Fixation

What is the effect of overfixation in formalin on immunohistochemistry?

  1. Complete loss of tissue morphology
  2. Masking of antigenic sites requiring antigen retrieval Correct answer.
  3. Enhanced antigen staining
  4. Precipitation of formalin pigment

Explanation

Prolonged formalin fixation creates excessive protein cross-links that mask epitopes and reduce antibody binding. Antigen retrieval can partially restore staining, but overfixation is avoided when possible.

Question 20Specimen Procurement, Handling, and Fixation

A tissue section for frozen section is best frozen at which temperature range in a cryostat?

  1. -20 to -30°C Correct answer.
  2. -40 to -50°C
  3. -10 to -15°C
  4. -60 to -70°C

Explanation

Most tissues section well at -20 to -30°C, where they are firm but not brittle. Too warm a temperature causes smearing, while too cold a temperature causes shattering.

Question 21Specimen Procurement, Handling, and Fixation

Formalin fixes tissue primarily by which chemical mechanism?

  1. Chelation of calcium ions
  2. Oxidation of lipid membranes
  3. Dehydration and precipitation of proteins
  4. Cross-linking of proteins through methylene bridges Correct answer.

Explanation

Formaldehyde reacts with amino and amide groups on proteins to form methylene bridges, stabilizing tissue structure. Dehydration and precipitation describe alcohol fixatives, and chelation describes EDTA decalcification.

Question 22Specimen Procurement, Handling, and Fixation

Acid hematin (formalin pigment) forms under which condition?

  1. Prolonged fixation in glutaraldehyde
  2. Alcohol dehydration after fixation
  3. Neutral pH with phosphate buffer
  4. Acidic formalin in blood-rich tissue Correct answer.

Explanation

Formalin pigment forms when formaldehyde reacts with hemoglobin in an acidic environment. Buffering formalin to pH 7.0-7.4 prevents acid hematin deposition.

Question 23Specimen Procurement, Handling, and Fixation

Which statement about Carnoy's fixative is correct?

  1. It is an aqueous fixative that preserves lipids
  2. It requires post-fixation with iodine
  3. It is a rapid alcohol-chloroform-acetic acid fixative that causes tissue shrinkage Correct answer.
  4. It is buffered to pH 7.4 and used for routine processing

Explanation

Carnoy's solution contains ethanol, chloroform, and acetic acid; it penetrates rapidly and preserves nucleic acids and glycogen but causes marked tissue shrinkage. Zenker's fixative, not Carnoy's, requires iodine treatment.

Question 24Specimen Procurement, Handling, and Fixation

Zenker's fixative requires which post-fixation step to remove mercury pigment?

  1. Immersion in osmium tetroxide
  2. Washing in lithium carbonate
  3. Treatment with iodine and sodium thiosulfate Correct answer.
  4. Treatment with picric acid

Explanation

Zenker's solution contains mercuric chloride, which deposits mercury pigment in tissue. Iodine converts the pigment to soluble mercuric iodide, and sodium thiosulfate clears it.

Question 25Specimen Procurement, Handling, and Fixation

Which fixative composition is correct for Bouin's solution?

  1. Ethanol, chloroform, and acetic acid
  2. Glutaraldehyde, buffer, and sucrose
  3. Picric acid, formalin, and acetic acid Correct answer.
  4. Formalin, potassium dichromate, and mercuric chloride

Explanation

Bouin's solution is a mixture of picric acid, formalin, and acetic acid and is used for selected specimens such as testis and gastrointestinal biopsies. Zenker's contains dichromate and mercuric chloride, while Carnoy's contains alcohol, chloroform, and acetic acid.

Question 26Specimen Procurement, Handling, and Fixation

What is the minimum recommended volume ratio of 10% neutral buffered formalin to tissue for adequate fixation?

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

Explanation

A minimum of 10 volumes of formalin per volume of tissue is required to maintain the concentration of the fixative and permit adequate diffusion into the specimen. With less fluid, tissue fluids and blood rapidly dilute the formalin and fixation becomes incomplete.

Question 27Specimen Procurement, Handling, and Fixation

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

  1. 5 mm per hour
  2. 1 mm per hour Correct answer.
  3. 1 mm per day
  4. 1 cm per hour

Explanation

Formalin advances through tissue at roughly 1 mm per hour at room temperature, so fixation of the center of a thick slice lags well behind the surface. This is why large specimens must be sliced thinly and why fixation time is estimated from tissue thickness.

Question 28Specimen Procurement, Handling, and Fixation

A bottle of 10% neutral buffered formalin contains approximately what concentration of formaldehyde?

  1. 0.4%
  2. 10%
  3. 4% Correct answer.
  4. 1%

Explanation

The term 10% formalin refers to 10 g of formaldehyde gas dissolved in 100 mL of water, which yields a working concentration of about 4% formaldehyde. Neutral buffered formalin is therefore described as a 4% formaldehyde solution.

Question 29Specimen Procurement, Handling, and Fixation

Which reagent sequence correctly dehydrates a tissue specimen during routine processing?

  1. Water, 50% ethanol, xylene, then 100% ethanol
  2. 70%, 80%, 95%, then 100% ethanol Correct answer.
  3. 100%, 95%, 70%, then water
  4. Xylene, 100% ethanol, 95% ethanol, then water

Explanation

Dehydration must proceed from lower to higher alcohol concentrations so that water is removed gradually, limiting osmotic shock and tissue shrinkage. Starting in absolute alcohol or reversing the gradient causes abrupt water loss and hard, shrunken tissue.

Question 30Specimen Procurement, Handling, and Fixation

The pH of properly prepared 10% neutral buffered formalin is approximately:

  1. 7.2 Correct answer.
  2. 5.5
  3. 9.0
  4. 4.0

Explanation

Neutral buffered formalin is adjusted to pH 7.0–7.4 with phosphate buffers to prevent the acidity that promotes formalin pigment and degrades nucleic acids and antigens. Unbuffered formalin drifts toward an acidic pH as formaldehyde oxidizes to formic acid.

Question 31Specimen Procurement, Handling, and Fixation

A needle core biopsy of kidney must be evaluated by direct immunofluorescence for immune complex deposition. Which transport medium is appropriate for this specimen?

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

Explanation

Michel's solution is an ammonium sulfate-based holding medium that preserves immunoglobulins and complement for immunofluorescence when the specimen cannot be frozen promptly. Cross-linking and dehydrating fixatives alter or destroy the antigens and immune deposits being demonstrated.

Question 32Specimen Procurement, Handling, and Fixation

A direct smear of a fine needle aspirate must be wet-fixed immediately after preparation. Which fixative is standard for this purpose?

  1. Normal saline
  2. 95% ethanol Correct answer.
  3. 10% neutral buffered formalin
  4. Xylene

Explanation

Cytology smears are wet-fixed in 95% ethanol to prevent air-drying artifact, which enlarges cells and distorts nuclear chromatin. Immediate fixation while the smear is still wet preserves nuclear detail for Papanicolaou staining.

Question 33Specimen Procurement, Handling, and Fixation

Which is the correct order of the main steps in routine processing of a formalin-fixed tissue specimen?

  1. Clearing, dehydration, paraffin infiltration
  2. Paraffin infiltration, dehydration, clearing
  3. Dehydration, clearing, paraffin infiltration Correct answer.
  4. Dehydration, paraffin infiltration, clearing

Explanation

Processing removes water with graded alcohols, replaces the alcohol with a solvent such as xylene that is miscible with wax, and then infiltrates the tissue with melted paraffin. Each step must follow the previous one because the reagents are only mutually miscible in that order.

Question 34Specimen Procurement, Handling, and Fixation

For transmission electron microscopy, tissue should be minced into cubes no larger than approximately:

  1. 5 mm³
  2. 1 cm³
  3. 3 mm³
  4. 1 mm³ Correct answer.

Explanation

Glutaraldehyde-based EM fixatives penetrate only a fraction of a millimeter per hour, so blocks thicker than about 1 mm develop poorly fixed, artifact-ridden centers. Mincing tissue into 1 mm cubes at the time of collection is essential for ultrastructural preservation.

Question 35Specimen Procurement, Handling, and Fixation

Routine paraffin wax used for tissue embedding melts at approximately:

  1. 45–48 °C
  2. 56–58 °C Correct answer.
  3. 40–42 °C
  4. 50–52 °C

Explanation

Routine histological paraffin has a melting point of about 56–58 °C, hot enough to flow into and support the tissue yet cool enough to limit thermal damage. Harder waxes with higher melting points are used for thin sectioning, and softer waxes for friable tissue.

Question 36Specimen Procurement, Handling, and Fixation

Autolysis in an unfixed tissue specimen is best described as:

  1. Shrinkage produced by graded alcohol dehydration
  2. Bacterial decomposition of tissue by contaminating organisms
  3. Denaturation of proteins caused by formalin cross-linking
  4. Self-digestion of cells by their own lysosomal enzymes Correct answer.

Explanation

Autolysis is enzymatic self-digestion that begins when the blood supply ceases and lysosomal enzymes are released into the cytoplasm. It is distinguished from putrefaction, which is decomposition by bacteria, and from fixation and processing artifacts.

Question 37Specimen Procurement, Handling, and Fixation

Which test is used to confirm that decalcification of a bone specimen is complete?

  1. Measurement of the osmolality of the decalcifying fluid
  2. Silver nitrate test on a fragment of the tissue
  3. Periodic acid–Schiff staining of the decalcifying fluid
  4. Ammonium hydroxide and ammonium oxalate test on the decalcifying fluid Correct answer.

Explanation

The decalcifying fluid is mixed with ammonium hydroxide and ammonium oxalate; a cloudy precipitate of calcium oxalate indicates that calcium is still being removed. A clear result, confirmed on a fresh portion of fluid, indicates that decalcification is complete.

Question 38Specimen Procurement, Handling, and Fixation

Tissue frozen slowly for cryotomy shows prominent ice crystal artifact. Which practice best minimizes this artifact?

  1. Holding the tissue in a −20 °C freezer overnight
  2. Immersing the tissue in formalin before freezing
  3. Snap-freezing the tissue in chilled isopentane Correct answer.
  4. Equilibrating the tissue at room temperature before freezing

Explanation

Rapid freezing produces many small ice crystals, whereas slow freezing allows large extracellular crystals to grow and disrupt morphology. Snap-freezing with a chilled, high-conductivity medium such as isopentane therefore gives the best preservation for frozen sections.

Question 39Specimen Procurement, Handling, and Fixation

A bone specimen that was inadequately decalcified is brought to the microtome. What is the most likely result?

  1. Torn, chattered sections and a nicked blade Correct answer.
  2. Complete dissolution of the tissue in the flotation bath
  3. Excessive nuclear staining with hematoxylin
  4. Loss of the section from the slide during staining

Explanation

Residual calcium hardens the tissue, dulls the blade, and produces chatter, tearing, and holes in the section. Decalcification must be complete, or the block face softened with a surface decalcifying agent, before sectioning.

Question 40Specimen Procurement, Handling, and Fixation

Uric acid crystals in a tissue specimen are best preserved by fixing the tissue in:

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

Explanation

Urate crystals are water soluble and dissolve in aqueous fixatives such as formalin, leaving empty spaces in the section. Non-aqueous fixation in absolute alcohol preserves the crystals so they can be demonstrated in the finished slide.

Question 41Processing, Embedding, and Microtomy

What is the primary purpose of passing tissue through a graded series of increasing ethanol concentrations during processing?

  1. To neutralize residual fixative
  2. To dissolve paraffin within the tissue
  3. To cross-link proteins in the tissue
  4. To remove water from the tissue Correct answer.

Explanation

Graded ethanol dehydrates the specimen by replacing tissue water with alcohol in stepwise increments. The gradual change limits osmotic shock and shrinkage. Water must be removed before a clearing agent, and later paraffin, can enter the tissue.

Question 42Processing, Embedding, and Microtomy

Which clearing agent is most commonly used in routine histology processing?

  1. Liquid paraffin
  2. Absolute ethanol
  3. Xylene Correct answer.
  4. Formalin

Explanation

Xylene is miscible with both alcohol and paraffin and is the standard clearing agent for routine schedules. Clearing replaces the dehydrant so that wax can infiltrate the tissue.

Question 43Processing, Embedding, and Microtomy

Routine paraffin sections for hematoxylin and eosin staining are typically cut at which thickness?

  1. 3–5 µm Correct answer.
  2. 10–15 µm
  3. 0.5–1 µm
  4. 20–30 µm

Explanation

Paraffin sections for routine light microscopy are cut at 3–5 µm, which gives a single layer of cells suitable for H&E evaluation. Thinner sections are used for specialized techniques, and thicker sections are used for specific purposes such as research or whole-mount work.

Question 44Processing, Embedding, and Microtomy

What is the typical temperature range for a tissue flotation bath used to flatten paraffin ribbons?

  1. 70–75 °C
  2. 55–60 °C
  3. 20–25 °C
  4. 40–45 °C Correct answer.

Explanation

A flotation bath is kept a few degrees below the melting point of the paraffin, typically 40–45 °C, so the ribbon relaxes without melting. Overheating causes the section to spread excessively and can damage morphology.

Question 45Processing, Embedding, and Microtomy

When embedding a mucosal biopsy in paraffin, the block should be oriented so that the cut surface lies:

  1. Vertical and parallel to the knife edge
  2. At a 45-degree angle within the mold
  3. Facing the side wall of the cassette
  4. Flat against the bottom of the mold Correct answer.

Explanation

The surface of interest must face the bottom of the mold so that it is the first plane reached by the knife. This ensures the diagnostic epithelium or lesion appears in the initial sections.

Question 46Processing, Embedding, and Microtomy

Frozen sections cut in a cryostat for routine intraoperative evaluation are usually cut at which thickness?

  1. 4–6 µm Correct answer.
  2. 20–25 µm
  3. 1–2 µm
  4. 10–12 µm

Explanation

Cryostat sections for frozen diagnosis are typically 4–6 µm, which balances cellular detail with the need to cut rapidly. Thinner sections are difficult to obtain consistently from frozen tissue.

Question 47Processing, Embedding, and Microtomy

The primary function of a tissue cassette during processing is to:

  1. Seal the specimen from processing fluids
  2. Prevent shrinkage during dehydration
  3. Provide a mold for paraffin embedding
  4. Hold the specimen during processing Correct answer.

Explanation

The cassette houses the specimen through fixation, dehydration, clearing, and infiltration while allowing free exchange of reagents. It also carries the identifying number that links the tissue to the case.

Question 48Processing, Embedding, and Microtomy

Clearing agents render tissue translucent because they:

  1. Remove the fixative pigment
  2. Match the refractive index of protein Correct answer.
  3. Dissolve the cell membranes
  4. Increase the water content of the tissue

Explanation

After dehydration, the clearing agent fills the tissue spaces and has a refractive index close to that of protein, so light passes through without scattering. This produces the characteristic translucent appearance.

Question 49Processing, Embedding, and Microtomy

Why are graded ethanol solutions used instead of moving tissue directly into absolute ethanol?

  1. Graded steps reduce tissue shrinkage Correct answer.
  2. Absolute ethanol cannot penetrate tissue
  3. Graded steps fix the tissue before processing
  4. Graded steps remove paraffin more effectively

Explanation

Moving tissue abruptly from water to absolute alcohol causes sudden osmotic shifts and marked shrinkage and distortion. Stepwise increases in alcohol concentration allow water to leave gradually, preserving morphology.

Question 50Processing, Embedding, and Microtomy

Tissue left in absolute ethanol too long during processing is likely to:

  1. Become soft and mushy
  2. Become hard and brittle Correct answer.
  3. Show excessive eosinophilia
  4. Retain water and fail to clear

Explanation

Prolonged exposure to absolute alcohol removes bound water and over-hardens the tissue, producing brittle blocks that crumble or chatter during microtomy. Processing schedules therefore limit time in the highest alcohol concentrations.