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

Question 1: Which type of microtome is best suited for cutting undecalcified bone sections?

Cryostat
Vibratome
Rotary microtome
Sliding microtome (Correct answer)

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

Green nuclear staining
Deep blue cytoplasm
Excessive eosin uptake
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 3: Which fixative is preferred for electron microscopy due to superior ultrastructural preservation?

10% neutral buffered formalin
Bouin's solution
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 4: 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 5: What is the primary advantage of frozen sections over paraffin sections during intraoperative consultation?

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

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 6: Which of the following describes the avidin-biotin complex (ABC) method in IHC?

Indirect amplification system using avidin-biotin affinity (Correct answer)
Direct antibody-chromogen system
Fluorescent antibody method
Enzyme-only detection 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 7: What action should be taken if a microtome blade injury occurs in the histology lab?

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

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

To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs) (Correct answer)
To prevent antibody aggregation
To improve antigen retrieval
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 9: 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 10: 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)
Eosin rinse
Differentiation agent

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

To remove surface paraffin and expose full tissue depth (Correct answer)
To calibrate section thickness
To test staining quality
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 12: In the Gram stain used in histology (Brown and Brenn technique), how do gram-positive bacteria appear?

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

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 13: 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 14: What temperature is paraffin wax typically maintained at during infiltration?

60°C (Correct answer)
75°C
45°C
37°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 15: What is the standard section thickness used for routine paraffin H&E sections?

20–25 µm
1–2 µm
4–5 µm (Correct answer)
10–12 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 16: What is the term for the process of surrounding embedded tissue with paraffin wax in a mold?

Casting
Infiltration
Blocking
Embedding (Correct answer)

Correct answer: Embedding

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

Question 17: What is the difference between the direct and indirect IHC methods?

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

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

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

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

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

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

Red
Yellow
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 21: Which decalcifying agent is most commonly used for routine bone processing in histology?

Nitric acid
Hydrochloric acid (HCl) (Correct answer)
Acetic 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 22: What is the standard recommended storage temperature for most unstained paraffin sections awaiting IHC testing?

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

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

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

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 24: What color does reticulin stain (Gordon and Sweet silver method) produce for reticular fibers?

Green
Black (Correct answer)
Blue
Red

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

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

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

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

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 27: 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 28: What is the ideal pH range for neutral buffered formalin to prevent artifact formation?

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

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

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

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 30: 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.

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%

To Pass

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