CSMLS Histotechnology and Cytology Questions and Answers — Questions and Answers
Question 1: A histotechnologist is cutting paraffin sections of a kidney block and observes regularly spaced parallel lines, or 'chatter', across the tissue. What is the most likely cause of this microtomy artifact?
- The block was too warm during sectioning.
- The clearance angle of the knife was too small.
- The block or knife was not securely clamped in the microtome. (Correct answer)
- The paraffin wax was contaminated with clearing agent.
Correct answer: The block or knife was not securely clamped in the microtome.
Chatter is a vibration artifact characterized by thick and thin zones in the section. It is most frequently caused by a lack of rigidity in the system, such as a loosely clamped block or knife holder, which allows for vibration during the cutting stroke. [2] Other less common causes can include cutting too fast or a block that is overly hard.
Question 2: A gynecological smear is stained using the Papanicolaou method. Which cellular component is specifically highlighted by the Orange G (OG-6) solution?
- Nuclei of immature parabasal cells.
- Cytoplasm of metabolically active cells like intermediate cells.
- Cilia and red blood cells.
- Cytoplasm of mature, keratinized squamous cells. (Correct answer)
Correct answer: Cytoplasm of mature, keratinized squamous cells.
In the Papanicolaou stain, Orange G (OG-6) is an acidic counterstain that has a high affinity for keratin. It specifically stains the cytoplasm of mature, superficial squamous cells, which are often keratinized, a distinct orange-yellow color. [9, 13] Hematoxylin stains the nuclei, while the Eosin Azure (EA) component provides differential staining for the cytoplasm of less mature cells. [13]
Question 3: A lymph node biopsy is submitted for routine H&E staining and may also require future immunohistochemistry (IHC). Which of the following is the most appropriate and widely used fixative for this purpose?
- Zenker's fluid
- 10% Neutral Buffered Formalin (NBF) (Correct answer)
- Bouin's solution
- Carnoy's fixative
Correct answer: 10% Neutral Buffered Formalin (NBF)
10% Neutral Buffered Formalin (NBF) is the gold standard for routine histology because it provides excellent preservation of tissue morphology and is compatible with the vast majority of immunohistochemical stains. [10, 24, 25] While other fixatives have specific uses, NBF offers the best balance of morphological detail and antigen preservation for both H&E and subsequent IHC studies. [25, 27]
Question 4: After automated tissue processing, a batch of cassettes feels soft and the tissue is greasy to the touch. During microtomy, the tissue crumbles and fails to form a ribbon. This is most likely due to incomplete action by which reagent?
- Clearing agent (e.g., xylene) (Correct answer)
- Fixative (e.g., formalin)
- Dehydrating alcohol
- Infiltrating paraffin
Correct answer: Clearing agent (e.g., xylene)
The clearing agent's purpose is to remove the dehydrating alcohol and make the tissue miscible with the molten paraffin wax. If the clearing step is incomplete, residual alcohol will prevent the paraffin from fully infiltrating the tissue. [28] This results in a soft, greasy block where the tissue is not properly supported, leading to crumbling upon sectioning. [19, 20, 23]
Question 5: The Periodic acid-Schiff (PAS) stain is fundamentally based on the oxidation of which chemical group to form aldehydes, which then react with the Schiff reagent?
- Amino groups
- Sulfhydryl groups
- 1,2-Glycols (Correct answer)
- Phosphate groups
Correct answer: 1,2-Glycols
The principle of the PAS stain involves the use of periodic acid to selectively oxidize 1,2-glycol groups (vicinal diols) present in carbohydrates. [5] This oxidation breaks the carbon-carbon bond and creates two aldehyde groups. These aldehydes then react with the colorless Schiff reagent to produce a distinctive magenta color, highlighting structures rich in glycogen, mucins, and basement membranes. [4, 5, 11]
Question 6: A cervical cytology report describes squamous cells with significant nuclear enlargement, perinuclear halos, and nuclear membrane irregularities (koilocytotic atypia). According to the Bethesda System, this finding is characteristic of which interpretation?
- Low-grade Squamous Intraepithelial Lesion (LSIL) (Correct answer)
- High-grade Squamous Intraepithelial Lesion (HSIL)
- Negative for Intraepithelial Lesion or Malignancy (NILM)
- Atypical Squamous Cells of Undetermined Significance (ASC-US)
Correct answer: Low-grade Squamous Intraepithelial Lesion (LSIL)
Koilocytes are the hallmark cellular changes associated with the cytopathic effects of Human Papillomavirus (HPV). [6, 34] The Bethesda System for Reporting Cervical Cytology classifies changes consistent with HPV infection, including koilocytotic atypia, as a Low-grade Squamous Intraepithelial Lesion (LSIL). [6, 21]
CSMLS Histotechnology and Cytology Questions and Answers
A histotechnologist is cutting paraffin sections of a kidney block and observes regularly spaced parallel lines, or 'chatter', across the tissue.
What is the most likely cause of this microtomy artifact?