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

Question 1: In the context of laboratory safety, what is the purpose of a Safety Data Sheet (SDS)?

To document equipment maintenance
To track reagent expiration dates
To provide comprehensive hazard, handling, storage, and emergency information for chemical substances (Correct answer)
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 2: 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 3: What color does reticulin stain (Gordon and Sweet silver method) produce for reticular fibers?

Blue
Red
Green
Black (Correct answer)

Correct answer: Black

The silver impregnation method deposits metallic silver on reticulin (type III collagen) fibers, staining them black against a golden-brown background.

Question 4: Which decalcifying agent is most commonly used for routine bone processing in histology?

Nitric acid
Hydrochloric acid (HCl) (Correct answer)
Sulfuric acid
Acetic 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 5: Zenker's fixative contains which hazardous heavy metal component?

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

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 6: What processing artifact results from inadequate fixation before processing?

Autolysis (Correct answer)
Chatter artifact
Retraction artifact
Folding artifact

Correct answer: Autolysis

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

Question 7: What preparatory step should be performed on tissue BEFORE beginning acid decalcification?

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

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

To remove excess paraffin
To filter contaminants from reagents
To prevent overheating of tissue
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 9: Which fixative is considered the standard routine fixative used in most histopathology laboratories?

10% neutral buffered formalin (Correct answer)
Glutaraldehyde 2.5%
Bouin's solution
Carnoy's fixative

Correct answer: 10% neutral buffered formalin

10% neutral buffered formalin (NBF) is the universal standard fixative because it preserves tissue morphology well and is compatible with most staining and molecular applications.

Question 10: In the Prussian blue stain, what positive control tissue is typically used?

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

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

Embedding (Correct answer)
Blocking
Infiltration
Casting

Correct answer: Embedding

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

Question 12: What is the purpose of rehydration after deparaffinization before H&E staining?

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

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

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

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 14: What is the role of internal quality controls in an H&E staining batch?

To replace the need for external proficiency testing
To verify that each staining run is performing within acceptable standards before patient slides are released (Correct answer)
To calibrate the automated stainer software
To document reagent purchases

Correct answer: To verify that each staining run is performing within acceptable standards before patient slides are released

Internal controls run with each batch confirm that staining reagents, timing, and technique are working acceptably, ensuring patient results are reliable.

Question 15: What is the standard recommended storage temperature for most unstained paraffin sections awaiting IHC testing?

Freezer (-20°C) always
Incubator at 37°C
Room temperature for indefinite periods
Room temperature for up to a limited time; refrigeration (4°C) for longer storage (Correct answer)

Correct answer: Room temperature for up to a limited time; refrigeration (4°C) for longer storage

Unstained sections are best stored at room temperature short-term or at 4°C for extended periods, as oxidation degrades antigenic epitopes over time.

Question 16: What does CAP checklist item require regarding the turnaround time (TAT) for surgical pathology specimens?

Laboratories must establish, monitor, and document TAT goals for different specimen types (Correct answer)
There is no TAT requirement
TAT only applies to cytology specimens
TAT must be under 24 hours for all 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 17: What is the purpose of the anti-roll plate in a cryostat?

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

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

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

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

Delafield's hematoxylin
Weigert's hematoxylin
Harris hematoxylin (Correct answer)
Mayer'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 20: What is the primary purpose of tissue fixation in histology?

To harden tissue for paraffin infiltration
To dehydrate tissue before embedding
To prevent autolysis and putrefaction (Correct answer)
To stain cellular components for visibility

Correct answer: To prevent autolysis and putrefaction

Fixation prevents autolysis (self-digestion by intracellular enzymes) and putrefaction (bacterial decomposition) by stabilizing tissue proteins and halting enzymatic activity.

Question 21: Which fixative is preferred when preservation of glycogen in tissue is the primary concern?

10% neutral buffered formalin
Absolute alcohol (ethanol) (Correct answer)
Bouin's solution
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 22: In a Masson's trichrome stain, what color does collagen appear?

Blue or green (Correct answer)
Black
Red
Yellow

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

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

Correct answer: Glutaraldehyde

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

Question 24: Which reagent is used for decalcification of bone specimens?

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

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

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

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

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

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

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

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

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

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

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

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

Glycogen
Mucin
Neutral lipids and triglycerides (Correct answer)
DNA

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 31: What is the purpose of using a diastase control with a PAS stain?

To differentiate neutral from acid mucins
To confirm that PAS-positive material is glycogen by removing it with diastase pretreatment (Correct answer)
To enhance mucin staining
To block non-specific background

Correct answer: To confirm that PAS-positive material is glycogen by removing it with diastase pretreatment

Diastase digests glycogen; if PAS positivity disappears after diastase treatment, the material was glycogen rather than other PAS-positive substances.

Question 32: What temperature is paraffin wax typically maintained at during infiltration?

60°C (Correct answer)
37°C
45°C
75°C

Correct answer: 60°C

Paraffin wax is maintained at approximately 60°C during infiltration to keep it in its liquid state for proper tissue impregnation.

Question 33: What is the primary purpose of fixation in tissue processing?

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

Correct answer: To prevent autolysis and preserve tissue morphology

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

Question 34: What does diffuse cytoplasmic staining for CK7 suggest in a carcinoma workup?

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

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

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

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 36: In a well-stained H&E section, red blood cells (RBCs) should appear what color?

Blue-purple
Bright red/orange (Correct answer)
Colorless
Green

Correct answer: Bright red/orange

RBCs lack nuclei and stain brightly eosinophilic (bright red to orange) due to their hemoglobin content.

Question 37: What is the purpose of the mordant in the hematoxylin staining process?

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

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

Non-additive coagulant fixative (Correct answer)
Metallic fixative
Additive cross-linking fixative
Oxidizing 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 39: What is the angle of the blade (clearance angle) typically set to on a rotary microtome?

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

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

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

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

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

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 42: What chromogen is most commonly used with HRP-based IHC and produces a brown reaction product?

DAB (3,3'-diaminobenzidine) (Correct answer)
BCIP/NBT (blue)
AEC (red)
Fast Red

Correct answer: DAB (3,3'-diaminobenzidine)

DAB (3,3'-diaminobenzidine) is oxidized by HRP in the presence of hydrogen peroxide to produce an insoluble brown precipitate at antigen sites.

Question 43: What does 'over-differentiation' of hematoxylin produce in an H&E section?

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

Correct answer: Pale or unstained nuclei

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

Question 44: 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 45: Which stain differentiates hyaline cartilage from fibrocartilage in musculoskeletal histology?

H&E only
Safranin O/Fast Green (Correct answer)
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 46: What action should be taken if a microtome blade injury occurs in the histology lab?

Only report if bleeding is severe
Apply a bandage and document later
Immediately wash the wound, report to supervisor, and follow the exposure control plan (Correct answer)
Continue working and report at the end of shift

Correct answer: Immediately wash the wound, report to supervisor, and follow the exposure control plan

Any sharps injury must be immediately addressed by washing with soap and water, reporting to a supervisor, and following the facility's bloodborne pathogen exposure control plan.

Question 47: Why is endogenous peroxidase activity blocked in IHC protocols using HRP-based detection?

To improve antigen retrieval
To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs) (Correct answer)
To prevent antibody aggregation
To prevent tissue damage

Correct answer: To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs)

Endogenous peroxidases in hemoglobin and other cellular components will react with the DAB chromogen, creating false-positive background staining if not blocked.

Question 48: Under CLIA regulations, who is responsible for the overall operation and administration of a histology laboratory?

Each individual technician equally
The senior histotechnician
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 49: Which of the following describes the avidin-biotin complex (ABC) method in IHC?

Enzyme-only detection system
Indirect amplification system using avidin-biotin affinity (Correct answer)
Fluorescent antibody method
Direct antibody-chromogen system

Correct answer: Indirect amplification system using avidin-biotin affinity

The ABC method uses a biotinylated secondary antibody that binds to a preformed avidin-biotin-enzyme complex, amplifying signal through the high-affinity avidin-biotin interaction.

Question 50: When is formic acid decalcification preferred over hydrochloric acid?

When nuclear morphology and DNA preservation for molecular studies are important (Correct answer)
When cost reduction is the primary consideration
When speed of decalcification is the highest priority
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 51: What is the recommended tissue-to-fixative volume ratio to ensure adequate fixation?

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

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 52: What is the embedding medium used for frozen sections in a cryostat?

Paraffin wax
Agarose gel
Glycerin
OCT (Optimal Cutting Temperature) compound (Correct answer)

Correct answer: OCT (Optimal Cutting Temperature) compound

OCT compound supports tissue during cryosectioning by providing a firm matrix without ice crystal artifact formation.

Question 53: What is the purpose of antigen retrieval in immunohistochemistry (IHC)?

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

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

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

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

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

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

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

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

3.7–4% formaldehyde (Correct answer)
37–40% formaldehyde
1% formaldehyde
10% 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 58: What is the purpose of the dehydration step in tissue processing?

To remove fixative residue
To remove water before infiltration with paraffin (Correct answer)
To clear pigments from tissue
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 59: What is 'chatter' in microtomy?

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

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

Removal of membrane lipids to expose proteins
Formation of methylene bridges that cross-link proteins (Correct answer)
Coagulation of proteins through dehydration
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 61: What is the purpose of the differentiation step using acid-alcohol in H&E staining?

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

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 62: Which fixative type is most likely to mask antigenic epitopes, necessitating antigen retrieval before immunohistochemistry?

10% neutral buffered formalin (Correct answer)
Absolute ethanol
Methanol
Acetone

Correct answer: 10% neutral buffered formalin

Formalin cross-links proteins and masks antigenic epitopes; heat-induced or proteolytic antigen retrieval must be performed to unmask these epitopes before IHC staining.

Question 63: In the Ziehl-Neelsen (acid-fast) stain, what color do acid-fast organisms (e.g., Mycobacterium tuberculosis) appear?

Green
Blue
Black
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 64: What is the recommended tissue thickness for routine formalin fixation to ensure adequate penetration?

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

Correct answer: 5–10 mm

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

Question 65: 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 section without primary antibody to check background
A tissue treated with buffer instead of antibody

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

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

Correct answer: 70%

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

Question 67: What is the standard section thickness used for routine paraffin H&E sections?

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

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 68: A tissue block shows a grainy, washed-out appearance after H&E staining. What processing artifact is most likely responsible?

Over-dehydration
Paraffin over-infiltration
Inadequate clearing
Under-fixation (Correct answer)

Correct answer: Under-fixation

Under-fixation allows continued autolysis, resulting in poorly preserved, washed-out tissue morphology after staining.

Question 69: What temperature range is a cryostat chamber maintained at for optimal sectioning of most tissues?

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

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 70: 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 71: Which type of microtome is best suited for cutting undecalcified bone sections?

Sliding microtome (Correct answer)
Cryostat
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 72: Which stain is most commonly used for rapid intraoperative frozen section evaluation?

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

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

Methanol
Acetone
Ethanol
Xylene (Correct answer)

Correct answer: Xylene

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

Question 74: Ki-67 IHC is used to assess what cellular property?

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

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

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

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

Red fluorescence only
Negative staining
Blue birefringence under polarized light
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 77: What is the purpose of the mucicarmine stain in histopathology?

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

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

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

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

Water
Acetone
Xylene (Correct answer)
Ethanol

Correct answer: Xylene

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

Question 80: What is the function of Scott's tap water (or ammonia water) in the H&E staining process?

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

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

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

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 82: 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 83: Which of the following is NOT a standard step in automated tissue processing?

Dehydration
Clearing
Fixation
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 84: What is the role of the mordant in a silver impregnation stain like GMS?

To dehydrate sections before silver application
To reduce silver ions to metallic silver
To sensitize tissue so it adsorbs silver ions (Correct answer)
To remove background silver deposits

Correct answer: To sensitize tissue so it adsorbs silver ions

The mordanting step (chromic acid oxidation in GMS) sensitizes the fungal cell wall by creating aldehyde groups that subsequently reduce silver.

Question 85: The Alcian blue stain at pH 2.5 is used to identify what type of tissue component?

Glycogen
Neutral mucins
Elastic fibers
Acid mucopolysaccharides (acid mucins) (Correct answer)

Correct answer: Acid mucopolysaccharides (acid mucins)

Alcian blue at pH 2.5 stains both sulfated and carboxylated (acid) mucopolysaccharides blue, distinguishing them from neutral mucins.

Question 86: What does a reagent QC failure on a negative control indicate?

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

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

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

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 88: What is the purpose of a Levey-Jennings chart in histology laboratory quality control?

Schedule preventive maintenance
Visually monitor control values over time to detect trends and shifts (Correct answer)
Record patient results
Track reagent inventory

Correct answer: Visually monitor control values over time to detect trends and shifts

Levey-Jennings charts plot control values over time against mean ± standard deviation limits, allowing visual detection of systematic errors, trends, and out-of-control situations.

Question 89: What is the recommended water bath temperature for floating paraffin sections before mounting?

55–60°C
30–35°C
40–45°C (Correct answer)
20–25°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 90: According to CAP guidelines, what is the recommended fixation time range for standard surgical resection specimens in 10% NBF?

1–2 weeks
6–72 hours (Correct answer)
1–2 hours
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 91: Which fixative is specifically required for optimal ultrastructural preservation in electron microscopy?

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

Correct answer: Glutaraldehyde

Glutaraldehyde (2–4%) is the fixative of choice for electron microscopy because its bifunctional aldehyde groups provide superior cross-linking and ultrastructural preservation.

Question 92: Which IHC marker is used to identify neural/neuroendocrine differentiation in tumors?

MNF116
Synaptophysin or chromogranin (Correct answer)
CK20
CD3

Correct answer: Synaptophysin or chromogranin

Synaptophysin and chromogranin A are neuroendocrine markers expressed in dense-core secretory granules of neuroendocrine cells and their neoplasms.

Question 93: Which reagent is used to mount coverslips on H&E sections stained using an alcohol-based final dehydration?

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

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

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

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

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

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 96: Which personal protective equipment (PPE) is required when handling formalin-fixed specimens in the histology laboratory?

Gloves, lab coat, and eye protection at minimum (Correct answer)
No PPE required for fixed specimens
Only a face shield
Gloves only

Correct answer: Gloves, lab coat, and eye protection at minimum

Formalin is a known carcinogen and respiratory irritant; minimum PPE includes nitrile gloves, lab coat, and eye protection when handling formalin-fixed specimens.

Question 97: Which regulatory body sets standards for quality systems in US clinical laboratories, including histology?

FDA only
OSHA only
CLIA (administered by CMS), with accreditation from CAP or Joint Commission (Correct answer)
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 98: What is formalin pigment and how is it prevented?

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

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 microtomy artifact is caused by a nick or defect in the cutting blade?

Score lines (linear streaks through the section) (Correct answer)
Chatter
Venetian blind effect
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 100: Which endpoint test is used to determine if decalcification is complete?

X-ray examination or chemical endpoint test (Correct answer)
Gram stain
Congo red staining
pH indicator strips only

Correct answer: X-ray examination or chemical endpoint test

X-ray examination verifies calcium removal radiographically, while the chemical test detects calcium ions in the decalcifying solution.

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.

Questions

35 min

Time Limit

70%

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