ASCP Board of Certification Histotechnician (HT) Examination — Questions and Answers

Question 1: What does basophilic staining indicate in H&E histology?

Affinity for eosin (acidic dye)
Affinity for hematoxylin (basic dye) (Correct answer)
Affinity for both dyes equally
Lack of staining

Correct answer: Affinity for hematoxylin (basic dye)

Basophilic structures have a negative charge that attracts the basic dye hematoxylin, staining them blue-purple.

Question 2: What type of hematoxylin is most commonly used in routine histology laboratories in the US?

Harris hematoxylin (Correct answer)
Delafield's hematoxylin
Mayer's hematoxylin
Weigert's hematoxylin

Correct answer: Harris hematoxylin

Harris hematoxylin is the most widely used progressive/regressive hematoxylin in US histology labs for routine H&E staining.

Question 3: What IHC marker is used to confirm the presence of muscle differentiation in a tumor?

CD20
CK7
TTF-1
Desmin or smooth muscle actin (SMA) (Correct answer)

Correct answer: Desmin or smooth muscle actin (SMA)

Desmin and smooth muscle actin are cytoskeletal proteins expressed in muscle cells; their IHC positivity confirms muscle differentiation in tumor cells.

Question 4: What color does eosin stain the cytoplasm and collagen in a routine H&E preparation?

Green
Purple
Blue
Pink/red (Correct answer)

Correct answer: Pink/red

Eosin is an acidic dye that stains positively charged proteins in the cytoplasm and extracellular matrix pink to red.

Question 5: What is the recommended tissue thickness for routine formalin fixation to ensure adequate penetration?

5–10 mm (Correct answer)
20–25 mm
15–20 mm
1–3 mm

Correct answer: 5–10 mm

Tissue sections of 5–10 mm allow adequate formalin penetration within the recommended fixation time.

Question 6: In the Ziehl-Neelsen (acid-fast) stain, what color do acid-fast organisms (e.g., Mycobacterium tuberculosis) appear?

Black
Green
Blue
Bright red/carmine (Correct answer)

Correct answer: Bright red/carmine

Acid-fast organisms retain the carbol-fuchsin primary stain after acid-alcohol decolorization and appear bright red against a blue background.

Question 7: What type of blade is used for cutting frozen sections on a cryostat?

Carbide-tipped blade
Diamond blade
Low-profile disposable blade (Correct answer)
High-profile disposable blade

Correct answer: Low-profile disposable blade

Low-profile disposable blades are used in cryostats because they fit the cryostat microtome head and are designed for frozen tissue.

Question 8: What microtomy artifact is caused by a nick or defect in the cutting blade?

Venetian blind effect
Score lines (linear streaks through the section) (Correct answer)
Chatter
Tissue folding

Correct answer: Score lines (linear streaks through the section)

A nicked or damaged blade creates linear streaks or score lines through the tissue section in the direction of cutting.

Question 9: What is the purpose of antigen retrieval in immunohistochemistry (IHC)?

To unmask epitopes altered by formalin fixation (Correct answer)
To deparaffinize sections
To apply the secondary antibody
To block endogenous peroxidase

Correct answer: To unmask epitopes altered by formalin fixation

Formalin cross-links proteins and masks antigenic epitopes; antigen retrieval (heat or enzyme-based) reverses these cross-links to restore antibody binding.

Question 10: According to OSHA's Hazard Communication Standard, what must be present on all chemical containers in the histology laboratory?

A barcode and lot number
Technician initials only
The manufacturer's name only
GHS-compliant labels with hazard pictograms, signal word, and hazard statements (Correct answer)

Correct answer: GHS-compliant labels with hazard pictograms, signal word, and hazard statements

OSHA HazCom requires GHS-compliant labeling including pictograms, signal word (Danger/Warning), hazard statements, and precautionary statements on all chemical containers.

Question 11: What is the primary purpose of decalcification in histological tissue preparation?

To remove lipids so tissue can be infiltrated with paraffin
To improve the staining intensity of soft tissue components
To harden soft tissue before microtomy
To remove calcium mineral deposits so calcified tissue can be sectioned (Correct answer)

Correct answer: To remove calcium mineral deposits so calcified tissue can be sectioned

Decalcification removes calcium salts from hard tissues such as bone and teeth, making them soft enough to be sectioned on a standard microtome without damaging the blade.

Question 12: Melanoma cells are typically IHC positive for which set of markers?

CK7, CK20, CEA
CD45, CD20, CD3
S100, HMB-45, Melan-A (MART-1) (Correct answer)
SMA, desmin, CD34

Correct answer: S100, HMB-45, Melan-A (MART-1)

S100, HMB-45, and Melan-A are melanocytic differentiation markers used in combination to confirm melanoma diagnosis.

Question 13: What is the primary advantage of using EDTA for decalcification compared to acid methods?

It requires no prior fixation step before use
It is more effective for large, dense cortical bone specimens
It is significantly faster than acid decalcification
It preserves tissue morphology and antigenicity better, enabling IHC and molecular studies (Correct answer)

Correct answer: It preserves tissue morphology and antigenicity better, enabling IHC and molecular studies

EDTA chelates calcium ions slowly and gently, preserving cellular morphology, antigenic epitopes for immunohistochemistry, and nucleic acids for molecular pathology testing.

Question 14: What is the purpose of the differentiation step using acid-alcohol in H&E staining?

To selectively remove excess hematoxylin from non-nuclear elements (Correct answer)
To remove eosin background
To increase nuclear stain intensity
To deparaffinize tissue

Correct answer: To selectively remove excess hematoxylin from non-nuclear elements

Acid-alcohol removes excess hematoxylin from cytoplasm and connective tissue, leaving stain only in the nuclei.

Question 15: According to CAP guidelines, what is the recommended fixation time range for standard surgical resection specimens in 10% NBF?

1–2 hours
6–72 hours (Correct answer)
1–2 weeks
3–5 hours

Correct answer: 6–72 hours

CAP guidelines recommend a minimum of 6 hours and a maximum of 72 hours of fixation in 10% NBF to ensure adequate protein cross-linking, particularly important for breast cancer biomarker testing.

Question 16: What is 'chatter' in microtomy?

Incomplete paraffin removal
Tissue folding during cutting
Tissue tearing at edges
Parallel lines or waves across a section due to vibration (Correct answer)

Correct answer: Parallel lines or waves across a section due to vibration

Chatter produces parallel lines across the section caused by vibration between the blade and the block during cutting.

Question 17: Ethanol (absolute alcohol) is classified as which type of fixative based on its mechanism of action?

Non-additive coagulant fixative (Correct answer)
Additive cross-linking fixative
Oxidizing fixative
Metallic fixative

Correct answer: Non-additive coagulant fixative

Ethanol is a non-additive coagulant fixative that fixes tissue by precipitating and denaturing proteins through dehydration, without forming covalent cross-links.

Question 18: Over-retrieval in heat-induced antigen retrieval (HIER) can cause which artifact?

Section detachment from the slide (Correct answer)
Absence of all staining
Blue background staining
Increased specific staining only

Correct answer: Section detachment from the slide

Excessive heat or prolonged retrieval times can cause sections to detach from slides, especially if adhesion is suboptimal.

Question 19: What is the trimming step in paraffin block preparation used for?

Final fixation
Removing the tissue from the cassette
Staining the block surface
Removing excess paraffin to expose the tissue face (Correct answer)

Correct answer: Removing excess paraffin to expose the tissue face

Trimming removes excess paraffin and exposes the full tissue face so diagnostic-quality sections can be cut.

Question 20: What preparatory step should be performed on tissue BEFORE beginning acid decalcification?

No prior treatment is needed; decalcification alone is sufficient
Fix in 70% alcohol for 24 hours before decalcification
Process through a graded alcohol series before decalcification
Fix in 10% neutral buffered formalin before decalcification (Correct answer)

Correct answer: Fix in 10% neutral buffered formalin before decalcification

Tissue must be adequately fixed in 10% NBF before acid decalcification to preserve the protein components of the tissue, since acid solutions alone do not adequately fix proteins.

Question 21: Which of the following actions helps prevent tissue sections from folding during microtomy?

Warming the paraffin block
Chilling the block face on ice before cutting (Correct answer)
Using a dull blade
Increasing section thickness

Correct answer: Chilling the block face on ice before cutting

Chilling the block face hardens the paraffin slightly, reducing the tendency for thin sections to fold or wrinkle during cutting.

Question 22: What is the proper disposal method for used xylene in the histology laboratory?

Neutralize with baking soda and discard
Pour down the drain with water
Collect in labeled hazardous waste containers for licensed disposal (Correct answer)
Evaporate in a fume hood

Correct answer: Collect in labeled hazardous waste containers for licensed disposal

Xylene is a flammable, toxic solvent regulated as hazardous waste; it must be collected in properly labeled containers and disposed of by a licensed hazardous waste contractor.

Question 23: What is the purpose of the post-fixation step using osmium tetroxide in electron microscopy preparation?

Dehydration of tissue
Clearing of aldehydes
Decalcification of bone
Fixation of lipids and enhancement of contrast (Correct answer)

Correct answer: Fixation of lipids and enhancement of contrast

Osmium tetroxide fixes lipids and adds electron density to membranes, enhancing ultrastructural contrast for electron microscopy.

Question 24: What is the function of Scott's tap water (or ammonia water) in the H&E staining process?

Differentiation agent
Bluing agent to convert hematoxylin complex to blue (Correct answer)
Eosin rinse
Dehydration step

Correct answer: Bluing agent to convert hematoxylin complex to blue

Scott's tap water (magnesium sulfate and sodium bicarbonate) raises the pH, converting the brownish-red hematoxylin complex to its characteristic blue color.

Question 25: What is the consequence of over-decalcification on tissue?

Improved section quality
Increased tissue thickness
Loss of nuclear staining and tissue damage (Correct answer)
Better paraffin infiltration

Correct answer: Loss of nuclear staining and tissue damage

Over-decalcification damages nucleic acids and proteins, resulting in poor nuclear staining and compromised tissue morphology.

Question 26: Which special stain is used to identify iron (hemosiderin) deposits in tissue?

Congo red
Prussian blue (Perl's stain) (Correct answer)
Oil Red O
Alcian blue

Correct answer: Prussian blue (Perl's stain)

Perl's Prussian blue reacts ferric iron with potassium ferrocyanide to produce a bright blue precipitate at iron deposition sites.

Question 27: What does a reagent QC failure on a negative control indicate?

Non-specific or background staining is present, suggesting a reagent or protocol problem (Correct answer)
The positive control worked correctly
Tissue autolysis occurred
The staining run can be reported as valid

Correct answer: Non-specific or background staining is present, suggesting a reagent or protocol problem

A positive signal on a negative control means non-specific staining is occurring, which could lead to false-positive results in patient samples.

Question 28: Ki-67 IHC is used to assess what cellular property?

Cellular proliferation index (Correct answer)
Apoptosis rate
Hormone receptor status
Lymphoid lineage

Correct answer: Cellular proliferation index

Ki-67 is a nuclear protein expressed only in proliferating cells; IHC for Ki-67 measures the proportion of actively dividing cells (proliferation index).

Question 29: What is the difference between the direct and indirect IHC methods?

Direct uses two antibodies; indirect uses one
Direct is more sensitive than indirect
There is no practical difference
Direct uses enzyme-labeled primary antibody; indirect uses unlabeled primary and enzyme-labeled secondary antibody (Correct answer)

Correct answer: Direct uses enzyme-labeled primary antibody; indirect uses unlabeled primary and enzyme-labeled secondary antibody

In the direct method, the primary antibody carries the detection label; in the indirect method, an unlabeled primary antibody is detected by a labeled secondary antibody for greater sensitivity.

Question 30: What does CAP (College of American Pathologists) accreditation require regarding staining controls?

Each staining run must include appropriate positive and negative controls (Correct answer)
Controls are only required for IHC, not special stains
Controls are optional if the lab has experienced staff
Controls must be run weekly, not on each batch

Correct answer: Each staining run must include appropriate positive and negative controls

CAP accreditation mandates that each staining run includes appropriate positive and negative controls to verify stain performance before patient results are reported.

Question 31: What processing artifact results from inadequate fixation before processing?

Chatter artifact
Folding artifact
Retraction artifact
Autolysis (Correct answer)

Correct answer: Autolysis

Inadequate fixation allows continued enzymatic activity, leading to autolysis and degraded tissue morphology.

Question 32: What is the recommended water bath temperature for floating paraffin sections before mounting?

30–35°C
40–45°C (Correct answer)
20–25°C
55–60°C

Correct answer: 40–45°C

A water bath at 40–45°C is warm enough to relax and flatten paraffin sections without melting the wax.

Question 33: What is the purpose of the water bath used during paraffin sectioning?

To remove paraffin from sections
To fix sections before mounting
To stain sections
To float and flatten sections by removing compression wrinkles (Correct answer)

Correct answer: To float and flatten sections by removing compression wrinkles

The water bath (typically 40–45°C) allows compressed sections to expand and flatten before being picked up on slides.

Question 34: What is the primary purpose of a tissue processor's vacuum function?

To prevent overheating of tissue
To remove excess paraffin
To filter contaminants from reagents
To speed reagent infiltration into dense tissues (Correct answer)

Correct answer: To speed reagent infiltration into dense tissues

Vacuum accelerates the infiltration of reagents, especially paraffin, into dense or fatty tissues by reducing air pockets.

Question 35: Which fixative is preferred for electron microscopy due to superior ultrastructural preservation?

Bouin's solution
10% neutral buffered formalin
Glutaraldehyde (Correct answer)
Zenker's fixative

Correct answer: Glutaraldehyde

Glutaraldehyde provides superior ultrastructural preservation by cross-linking proteins more effectively than formaldehyde for electron microscopy.

Question 36: Which regulatory body sets standards for quality systems in US clinical laboratories, including histology?

OSHA only
CLIA (administered by CMS), with accreditation from CAP or Joint Commission (Correct answer)
FDA only
CDC only

Correct answer: CLIA (administered by CMS), with accreditation from CAP or Joint Commission

CLIA (Clinical Laboratory Improvement Amendments), administered by CMS, sets federal quality standards for all US clinical labs, with CAP and The Joint Commission as authorized accreditation organizations.

Question 37: Which decalcifying agent is most commonly used for routine bone processing in histology?

Hydrochloric acid (HCl) (Correct answer)
Acetic acid
Nitric acid
Sulfuric acid

Correct answer: Hydrochloric acid (HCl)

Hydrochloric acid (5–10% HCl) is widely used for routine decalcification because it is effective, relatively fast, and provides acceptable tissue preservation compared to other strong acids.

Question 38: What is the 'bluing' step in H&E staining intended to accomplish?

Remove excess eosin
Convert soluble hematoxylin to an insoluble blue-black complex by raising pH (Correct answer)
Increase eosin intensity
Deparaffinize sections

Correct answer: Convert soluble hematoxylin to an insoluble blue-black complex by raising pH

Exposure to a slightly alkaline solution (Scott's water or ammonia water) shifts the hematoxylin complex from red-brown to a permanent blue-black.

Question 39: What cellular component is primarily stained by hematoxylin in an H&E stain?

Nuclei (DNA/RNA) (Correct answer)
Lipids
Cytoplasm
Collagen

Correct answer: Nuclei (DNA/RNA)

Hematoxylin is a basic dye that binds to negatively charged nucleic acids in the nucleus, staining it blue-purple.

Question 40: What is the primary purpose of fixation in tissue processing?

To remove lipids from tissue
To dehydrate tissue for embedding
To decalcify bone specimens
To prevent autolysis and preserve tissue morphology (Correct answer)

Correct answer: To prevent autolysis and preserve tissue morphology

Fixation prevents autolysis and microbial decomposition by cross-linking proteins to preserve tissue architecture.

Question 41: What is the function of the heat plate or slide warmer after mounting sections?

To stain the sections
To fix the section
To deparaffinize the section
To melt and drive out water and adhere the section to the slide (Correct answer)

Correct answer: To melt and drive out water and adhere the section to the slide

The slide warmer (55–60°C) melts paraffin slightly and drives out water, ensuring the section adheres firmly to the slide.

Question 42: Which reagent is used to mount coverslips on H&E sections stained using an alcohol-based final dehydration?

Glycerin jelly
Xylene-based mounting medium (e.g., Permount) (Correct answer)
Aqueous mounting medium
Canada balsam only

Correct answer: Xylene-based mounting medium (e.g., Permount)

Xylene-based mounting media like Permount are miscible with the dehydrated, xylene-cleared sections and provide permanent, clear mounts.

Question 43: What is the recommended tissue-to-fixative volume ratio to ensure adequate fixation?

1:10 (Correct answer)
1:5
1:1
1:20

Correct answer: 1:10

A 1:10 tissue-to-fixative ratio ensures sufficient fixative volume to penetrate the specimen completely and maintain fixative concentration throughout the process.

Question 44: What is the purpose of the mucicarmine stain in histopathology?

Identify iron deposits
Highlight acid and neutral mucins, especially useful for Cryptococcus (Correct answer)
Stain collagen fibers
Identify amyloid

Correct answer: Highlight acid and neutral mucins, especially useful for Cryptococcus

Mucicarmine stains mucin deep rose-red and is particularly useful for identifying the mucoid capsule of Cryptococcus neoformans.

Question 45: What is the ideal pH range for neutral buffered formalin to prevent artifact formation?

6.8–7.4 (Correct answer)
6.0–6.8
5.0–6.0
7.4–8.0

Correct answer: 6.8–7.4

NBF should be maintained between pH 6.8 and 7.4 to prevent the formation of formalin pigment (acid formaldehyde hematin) and optimize tissue preservation.

Question 46: What is the purpose of the dehydration step in tissue processing?

To clear pigments from tissue
To remove water before infiltration with paraffin (Correct answer)
To remove fixative residue
To enhance nuclear staining

Correct answer: To remove water before infiltration with paraffin

Dehydration removes water from tissue using ascending grades of alcohol so the tissue can be infiltrated with paraffin wax.

Question 47: What is the significance of a negative IHC result when the positive control is also negative?

The staining run failed — results are invalid and the test must be repeated (Correct answer)
The tumor is truly antigen-negative
Background inhibited staining
The antibody worked but the tumor lacked the antigen

Correct answer: The staining run failed — results are invalid and the test must be repeated

A failed positive control means the staining protocol did not work; any negative results from that run are technically invalid and the assay must be repeated.

Question 48: What staining result indicates that sections were not completely deparaffinized before H&E staining?

Overly pale nuclei
Unstained or poorly stained areas with paraffin residue (Correct answer)
Excessive eosin background
Green cytoplasm

Correct answer: Unstained or poorly stained areas with paraffin residue

Residual paraffin acts as a barrier to aqueous dyes, producing unstained patches or areas with poor dye penetration.

Question 49: What does the term 'cross-reactivity' mean in the context of IHC antibody specificity?

The antibody binds to unintended antigens structurally similar to the target (Correct answer)
The primary antibody reacts with the secondary antibody
The antibody stains both frozen and paraffin sections
The antibody works with multiple chromogens

Correct answer: The antibody binds to unintended antigens structurally similar to the target

Cross-reactivity occurs when an antibody binds antigens other than its intended target due to shared epitopes, leading to false-positive staining.

Question 50: During dehydration, tissues are passed through graded alcohols starting from what concentration?

95%
50%
70% (Correct answer)
100%

Correct answer: 70%

Graded dehydration typically begins at 70% alcohol to avoid osmotic shock and cell distortion from sudden water removal.

Question 51: In IHC, what is the function of the blocking step with normal serum?

To enhance primary antibody penetration
To fix the tissue before antibody application
To amplify signal detection
To prevent non-specific binding of the secondary antibody to tissue proteins (Correct answer)

Correct answer: To prevent non-specific binding of the secondary antibody to tissue proteins

Normal serum (from the species used to raise the secondary antibody) saturates non-specific protein binding sites, reducing background staining.

Question 52: What does a negative IHC result on a poorly fixed specimen most likely represent?

False negative due to antigen destruction by inadequate fixation (Correct answer)
Successful antibody staining with no target present
Over-retrieval artifact
True absence of the antigen

Correct answer: False negative due to antigen destruction by inadequate fixation

Inadequate fixation causes protein degradation that destroys antigenic epitopes, resulting in false-negative IHC results even when the antigen is present.

Question 53: What tissue processing problem is indicated by a crumbly, brittle paraffin block?

Under-fixation
Insufficient paraffin infiltration
Over-processing (over-dehydration or excessive paraffin heat) (Correct answer)
Incomplete dehydration

Correct answer: Over-processing (over-dehydration or excessive paraffin heat)

Over-processing desiccates tissue, making it brittle and causing the block to crumble during sectioning.

Question 54: Which stain is most commonly used for rapid intraoperative frozen section evaluation?

PAS stain
Masson's trichrome
Gram stain
Hematoxylin and eosin (H&E) (Correct answer)

Correct answer: Hematoxylin and eosin (H&E)

H&E is the standard rapid stain for frozen sections because it provides quick nuclear and cytoplasmic contrast for diagnostic evaluation.

Question 55: What is the approximate formaldehyde concentration in a solution labeled '10% neutral buffered formalin'?

10% formaldehyde
3.7–4% formaldehyde (Correct answer)
1% formaldehyde
37–40% formaldehyde

Correct answer: 3.7–4% formaldehyde

10% formalin is a 1:10 dilution of the 37–40% formaldehyde stock solution, yielding an actual formaldehyde concentration of approximately 3.7–4%.

Question 56: In a Masson's trichrome stain, what color does collagen appear?

Yellow
Red
Blue or green (Correct answer)
Black

Correct answer: Blue or green

Masson's trichrome stains collagen blue (or green in the Goldner variant), muscle red, and nuclei dark brown-black.

Question 57: What does 'over-differentiation' of hematoxylin produce in an H&E section?

Pale or unstained nuclei (Correct answer)
Deep blue cytoplasm
Green nuclear staining
Excessive eosin uptake

Correct answer: Pale or unstained nuclei

Over-differentiation removes too much hematoxylin, resulting in pale or ghost nuclei that are difficult to visualize.

Question 58: What is the permissible exposure limit (PEL) for formaldehyde established by OSHA?

2 ppm (8-hour TWA)
5 ppm (8-hour TWA)
0.1 ppm (8-hour TWA)
0.75 ppm (8-hour TWA) (Correct answer)

Correct answer: 0.75 ppm (8-hour TWA)

OSHA sets the PEL for formaldehyde at 0.75 ppm as an 8-hour time-weighted average, with a 2 ppm short-term exposure limit.

Question 59: What is the purpose of the anti-roll plate in a cryostat?

To hold the tissue block in place
To clean the blade edge
To prevent the cut section from rolling up (Correct answer)
To cool the blade

Correct answer: To prevent the cut section from rolling up

The anti-roll plate guides and holds the cut section flat so it can be easily transferred to a glass slide.

Question 60: The Warthin-Starry stain is used to identify which organisms in tissue?

Acid-fast bacteria
Spirochetes and Helicobacter pylori (Correct answer)
Gram-negative rods only
Fungi

Correct answer: Spirochetes and Helicobacter pylori

Warthin-Starry is a silver-based stain that demonstrates spirochetes (e.g., Treponema) and Helicobacter pylori as black organisms on a yellow background.

Question 61: What is the primary advantage of frozen sections over paraffin sections during intraoperative consultation?

Better antigen preservation for IHC
Rapid turnaround time for immediate surgical decisions (Correct answer)
Superior morphological detail
Easier to cut thin sections

Correct answer: Rapid turnaround time for immediate surgical decisions

Frozen sections can be prepared in 10–15 minutes, allowing surgeons to receive immediate pathological guidance during an operation.

Question 62: What is the angle of the blade (clearance angle) typically set to on a rotary microtome?

0–2°
25–30°
15–20°
3–8° (Correct answer)

Correct answer: 3–8°

A clearance angle of 3–8° prevents the blade back from dragging on the tissue block while still allowing clean sectioning.

Question 63: In the Prussian blue stain, what positive control tissue is typically used?

Normal kidney
Liver with hemosiderosis or spleen with iron deposits (Correct answer)
Lung with no pathology
Normal colon

Correct answer: Liver with hemosiderosis or spleen with iron deposits

Liver or spleen from a case with known hemosiderosis or iron overload serves as a reliable positive control for Perl's Prussian blue stain.

Question 64: Which of the following is NOT a standard step in automated tissue processing?

Clearing
Fixation
Dehydration
Decalcification (Correct answer)

Correct answer: Decalcification

Decalcification is a separate pre-processing step performed on calcified tissues and is not part of the standard automated processing cycle.

Question 65: What temperature range is a cryostat chamber maintained at for optimal sectioning of most tissues?

-15°C to -25°C (Correct answer)
0°C to -5°C
-5°C to -10°C
-30°C to -40°C

Correct answer: -15°C to -25°C

A cryostat chamber maintained at -15°C to -25°C provides the optimal firmness for most tissues without making them too brittle.

Question 66: What is the primary chemical mechanism by which formalin fixes tissue?

Coagulation of proteins through dehydration
Removal of membrane lipids to expose proteins
Formation of methylene bridges that cross-link proteins (Correct answer)
Precipitation of nucleic acids by pH change

Correct answer: Formation of methylene bridges that cross-link proteins

Formalin reacts with amino groups of proteins to form methylene bridges, creating a stable cross-linked protein network that preserves tissue architecture.

Question 67: When should an autoclave (steam sterilization) be used in the histology laboratory?

To sterilize paraffin blocks before sectioning
To clear sections in preparation for staining
To dry processed tissue sections
To decontaminate biohazardous waste and instruments (Correct answer)

Correct answer: To decontaminate biohazardous waste and instruments

Autoclaving uses pressurized steam to sterilize biohazardous waste, contaminated instruments, and materials before disposal or reuse.

Question 68: Which special stain is used to highlight elastic fibers in tissue sections?

Congo red
Mucicarmine
Verhoeff's iron hematoxylin (VVG) or Weigert's resorcinol-fuchsin (Correct answer)
Oil Red O

Correct answer: Verhoeff's iron hematoxylin (VVG) or Weigert's resorcinol-fuchsin

Verhoeff-Van Gieson (VVG) stain colors elastic fibers blue-black, collagen red, and other tissue elements yellow.

Question 69: What is the purpose of using the polymer-based detection system (e.g., EnVision) over ABC in IHC?

Lower cost
Requires fewer wash steps
Eliminates endogenous biotin interference and provides consistent amplification (Correct answer)
Uses a different chromogen

Correct answer: Eliminates endogenous biotin interference and provides consistent amplification

Polymer systems eliminate false positives from endogenous biotin (a problem in liver, kidney, and adipose tissue) while providing high sensitivity without biotin-streptavidin interference.

Question 70: When is formic acid decalcification preferred over hydrochloric acid?

When speed of decalcification is the highest priority
When cost reduction is the primary consideration
When nuclear morphology and DNA preservation for molecular studies are important (Correct answer)
When decalcifying large, dense cortical bone specimens

Correct answer: When nuclear morphology and DNA preservation for molecular studies are important

Formic acid (5–10%) is a weaker organic acid that decalcifies more slowly but causes less tissue damage, better preserving nuclear morphology and DNA integrity for molecular pathology.

Question 71: What does the PAS (Periodic Acid-Schiff) stain primarily identify?

Nuclei and DNA
Glycogen, neutral mucins, and basement membranes (Correct answer)
Amyloid deposits
Elastic fibers

Correct answer: Glycogen, neutral mucins, and basement membranes

PAS stains 1,2-glycol groups in polysaccharides, glycoproteins, and mucins magenta-red by oxidizing them with periodic acid and then applying Schiff reagent.

Question 72: What is the purpose of proficiency testing (PT) in clinical histology laboratories?

To train new staff on staining techniques
To verify reagent shelf life
To externally validate laboratory performance by testing unknown samples against peer laboratories (Correct answer)
To calibrate microtome blade angles

Correct answer: To externally validate laboratory performance by testing unknown samples against peer laboratories

Proficiency testing uses blind samples distributed to multiple laboratories; results are compared to peer performance and expected values to confirm laboratory accuracy.

Question 73: Which reagent is used for decalcification of bone specimens?

Hydrochloric acid only
Xylene
EDTA or nitric acid (Correct answer)
Acetic acid

Correct answer: EDTA or nitric acid

EDTA (chelating agent) and nitric acid are common decalcifying agents that remove calcium from bone to allow sectioning.

Question 74: What does CAP checklist item require regarding the turnaround time (TAT) for surgical pathology specimens?

TAT must be under 24 hours for all specimens
There is no TAT requirement
Laboratories must establish, monitor, and document TAT goals for different specimen types (Correct answer)
TAT only applies to cytology specimens

Correct answer: Laboratories must establish, monitor, and document TAT goals for different specimen types

CAP requires laboratories to define TAT goals appropriate for specimen type and urgency, and to monitor and document performance against those goals.

Question 75: What is the characteristic staining result of amyloid with Congo red?

Blue birefringence under polarized light
Red fluorescence only
Negative staining
Apple-green birefringence under polarized light (Correct answer)

Correct answer: Apple-green birefringence under polarized light

Amyloid stained with Congo red shows salmon-pink color in brightfield and apple-green birefringence under polarized light.

Question 76: What causes the 'venetian blind' artifact in paraffin sections?

Dull microtome blade
Insufficient water bath temperature
Under-fixation of tissue
Alternating thick and thin areas due to incomplete ribbon advancement (Correct answer)

Correct answer: Alternating thick and thin areas due to incomplete ribbon advancement

The venetian blind effect results from uneven section thickness caused by inconsistent block advancement between cuts.

Question 77: In the Gram stain used in histology (Brown and Brenn technique), how do gram-positive bacteria appear?

Yellow
Red/pink
Blue-black (Correct answer)
Green

Correct answer: Blue-black

Gram-positive bacteria retain the crystal violet-iodine complex after decolorization and appear blue-black in the Brown and Brenn tissue Gram stain.

Question 78: What is the purpose of rehydration after deparaffinization before H&E staining?

To fix tissue before staining
To remove excess eosin
To improve section adherence
To remove residual xylene and prepare tissue for aqueous dyes (Correct answer)

Correct answer: To remove residual xylene and prepare tissue for aqueous dyes

After xylene deparaffinization, tissue must be rehydrated through descending grades of alcohol to prepare it for aqueous hematoxylin and eosin solutions.

Question 79: Which reagent is commonly used as a clearing agent to remove alcohol before paraffin infiltration?

Methanol
Acetone
Xylene (Correct answer)
Ethanol

Correct answer: Xylene

Xylene is miscible with both alcohol and paraffin, making it the standard clearing agent in tissue processing.

Question 80: Which of the following is a quality control indicator for an H&E stained section?

Crisp blue nuclei with pink cytoplasm and no background staining (Correct answer)
All cell types stain uniformly one color
Nuclei stain red and cytoplasm stains blue
Collagen stains blue

Correct answer: Crisp blue nuclei with pink cytoplasm and no background staining

A well-stained H&E section shows sharp blue-purple nuclei, pink cytoplasm, and clean background without precipitate or uneven staining.

Question 81: Which fixative is preferred when preservation of glycogen in tissue is the primary concern?

Absolute alcohol (ethanol) (Correct answer)
Bouin's solution
10% neutral buffered formalin
Carnoy's fixative

Correct answer: Absolute alcohol (ethanol)

Absolute alcohol preserves glycogen by precipitating surrounding proteins through dehydration without dissolving the water-soluble glycogen molecules.

Question 82: What is a positive control in IHC and why is it required?

A section stained only with the chromogen
A tissue known to contain the target antigen, run to confirm the staining protocol works (Correct answer)
A tissue treated with buffer instead of antibody
A tissue section without primary antibody to check background

Correct answer: A tissue known to contain the target antigen, run to confirm the staining protocol works

A positive control (tissue with known antigen expression) confirms that antibody, detection reagents, and staining conditions are functioning correctly.

Question 83: What is the minimum required documentation for each H&E staining run in an accredited laboratory?

Reagent lot numbers, dates, technician ID, and control results (Correct answer)
No documentation required
Staining time and temperature only
Patient name only

Correct answer: Reagent lot numbers, dates, technician ID, and control results

Accreditation standards require each staining run to be documented with reagent lot numbers, preparation/expiration dates, technician identification, and control staining results.

Question 84: Before H&E staining, paraffin sections must be deparaffinized using which reagent?

Ethanol
Acetone
Xylene (Correct answer)
Water

Correct answer: Xylene

Xylene dissolves paraffin wax before the section is rehydrated through graded alcohols for aqueous staining.

Question 85: Zenker's fixative contains which hazardous heavy metal component?

Mercuric chloride (Correct answer)
Cadmium sulfate
Lead acetate
Arsenic trioxide

Correct answer: Mercuric chloride

Zenker's fixative contains mercuric chloride, which is highly toxic and requires special handling and disposal procedures, contributing to its decline in modern laboratories.

Question 86: What is the purpose of the mordant in the hematoxylin staining process?

To remove excess hematoxylin
To prevent fading
To differentiate background from nuclei
To link the hematoxylin dye to tissue components (Correct answer)

Correct answer: To link the hematoxylin dye to tissue components

A mordant (typically alum) acts as a bridge between the dye and tissue, forming a stable dye-mordant-tissue complex.

Question 87: What does diffuse cytoplasmic staining for CK7 suggest in a carcinoma workup?

Adenocarcinoma or urothelial carcinoma origin (Correct answer)
Squamous cell carcinoma
Melanoma
Neuroendocrine differentiation

Correct answer: Adenocarcinoma or urothelial carcinoma origin

CK7 (cytokeratin 7) positivity indicates glandular epithelial origin, and combined with other markers, helps narrow the primary site of adenocarcinomas.

Question 88: What is the purpose of sectioning a 'waste' ribbon before collecting diagnostic sections?

To clean the blade
To test staining quality
To remove surface paraffin and expose full tissue depth (Correct answer)
To calibrate section thickness

Correct answer: To remove surface paraffin and expose full tissue depth

Cutting and discarding initial waste sections removes the trimmed surface layer and reveals intact tissue at the proper depth.

Question 89: What does Oil Red O stain identify in tissue sections?

Mucin
DNA
Glycogen
Neutral lipids and triglycerides (Correct answer)

Correct answer: Neutral lipids and triglycerides

Oil Red O is a lysochrome dye that stains neutral fats and lipids bright red; it requires frozen sections as lipids are removed during paraffin processing.

Question 90: Which fixative is considered the gold standard for routine histological processing?

10% neutral buffered formalin (Correct answer)
Bouin's solution
Zenker's fixative
Glutaraldehyde

Correct answer: 10% neutral buffered formalin

10% neutral buffered formalin is the standard fixative because it preserves morphology well and is compatible with most downstream staining techniques.

Question 91: In the context of laboratory safety, what is the purpose of a Safety Data Sheet (SDS)?

To provide comprehensive hazard, handling, storage, and emergency information for chemical substances (Correct answer)
To track reagent expiration dates
To document equipment maintenance
To record patient demographics

Correct answer: To provide comprehensive hazard, handling, storage, and emergency information for chemical substances

SDS (formerly MSDS) documents provide detailed chemical hazard information including toxicity, flammability, PPE requirements, and spill/exposure procedures.

Question 92: Which heat-based antigen retrieval method is most commonly used in routine IHC?

Pressure cooker in acetone
Microwave in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) (Correct answer)
Autoclave in distilled water
Water bath in ethanol

Correct answer: Microwave in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

Heat-induced epitope retrieval (HIER) using citrate buffer at pH 6 or EDTA at pH 9 in a microwave or steamer is the standard method for most antibodies.

Question 93: What is the standard section thickness used for routine paraffin H&E sections?

20–25 µm
10–12 µm
1–2 µm
4–5 µm (Correct answer)

Correct answer: 4–5 µm

Routine paraffin sections are cut at 4–5 µm to allow adequate light transmission and single-cell-layer evaluation.

Question 94: Under CLIA regulations, who is responsible for the overall operation and administration of a histology laboratory?

The senior histotechnician
Each individual technician equally
The hospital administrator
The laboratory director (Correct answer)

Correct answer: The laboratory director

CLIA designates the laboratory director as ultimately responsible for all laboratory operations, test performance, quality, and regulatory compliance.

Question 95: Which type of microtome is best suited for cutting undecalcified bone sections?

Cryostat
Sliding microtome (Correct answer)
Vibratome
Rotary microtome

Correct answer: Sliding microtome

A sliding (sledge) microtome with a heavy-duty blade is suited for hard tissues like undecalcified bone because it exerts more force.

Question 96: Which stain differentiates hyaline cartilage from fibrocartilage in musculoskeletal histology?

Safranin O/Fast Green (Correct answer)
H&E only
PAS stain
Congo red

Correct answer: Safranin O/Fast Green

Safranin O stains proteoglycans (found in hyaline cartilage matrix) red-orange, while fibrocartilage with less proteoglycan appears green with Fast Green counterstain.

Question 97: Which decalcification agent is the fastest but causes the most tissue damage, reserved for cases where speed is critical?

Nitric acid (5–10%) (Correct answer)
Electrolytic decalcification
EDTA chelation
Formic acid (5–10%)

Correct answer: Nitric acid (5–10%)

Nitric acid is the most rapid decalcifying agent but causes significant tissue damage including poor nuclear staining and reduced antigenicity, limiting its use to situations where speed outweighs quality concerns.

Question 98: What is formalin pigment and how is it prevented?

A yellow discoloration from picric acid; prevented by washing in tap water
A black deposit from osmium tetroxide; prevented by shortening fixation time
A blue artifact from alkaline formalin; prevented by using unbuffered formalin
A brown artifact from acidic formalin reacting with hemoglobin; prevented by using buffered formalin (Correct answer)

Correct answer: A brown artifact from acidic formalin reacting with hemoglobin; prevented by using buffered formalin

Formalin pigment (acid formaldehyde hematin) is a brown refractile artifact that forms when acidic, unbuffered formalin reacts with hemoglobin in bloody tissue; buffering the formalin to neutral pH prevents its formation.

Question 99: What is a common cause of eosin staining appearing too pale in H&E sections?

Over-dehydration during processing
Over-fixation in formalin
Insufficient eosin concentration or exhausted eosin solution (Correct answer)
Excessive differentiation with acid-alcohol

Correct answer: Insufficient eosin concentration or exhausted eosin solution

Diluted or exhausted eosin solution lacks sufficient dye concentration to adequately stain cytoplasmic proteins.

Question 100: What is the term for the process of surrounding embedded tissue with paraffin wax in a mold?

Embedding (Correct answer)
Infiltration
Blocking
Casting

Correct answer: Embedding

Embedding is the process of orienting tissue in molten paraffin within a mold to create a block for sectioning.

ASCP Board of Certification Histotechnician (HT) Examination

The ASCP HT certification exam tests competency in histotechnology laboratory techniques including tissue fixation, processing, microtomy, and staining procedures. It validates the knowledge and skills required to prepare high-quality tissue sections for pathological diagnosis.

100

Questions

150 min

Time Limit

70%

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