Gynecologic cytology preparation and the Pap stain

7 DOH Abu Dhabi (HAAD) lab licensing practice questions on Gynecologic cytology preparation and the Pap stain, part of Histopathology and Cytology. The first 7 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   One clinic's conventional cervical smears show enlarged squamous cells with pale, smudged nuclei, poor chromatin detail and orange-red cytoplasm. Smears from other clinics stained in the same run look normal. The most likely cause is:

   1. AToo long in 95% alcohol after the EA
   2. BExhausted hematoxylin in the shared staining line
   3. CSmears air-dried before reaching the fixative
   4. DToo long a time in the OG-6 bath

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   Answer: C · Smears air-dried before reaching the fixative

   Drying before fixation lets cells flatten and swell, so they look larger, with pale, poorly defined nuclei and cytoplasm that takes eosin and orange G intensely. Because other clinics' smears in the same run are normal, the reagents are not at fault. The clinic should fix each smear within seconds of spreading, in 95% ethanol or with a spray fixative. Severe air-drying artifact can make a smear impossible to interpret.

   • A: Alcohol rinses affect every slide in the run and do not change cell size.
   • B: Exhausted hematoxylin would give pale nuclei on every smear in the run.
   • D: OG-6 time is the same for all smears in the run and does not enlarge cells.

2. Question 2 · Medium

   After fixation, which sequence of main steps is correct for a Pap stain?

   1. AHematoxylin, bluing, OG-6, EA, dehydrate, clear
   2. BEA, OG-6, hematoxylin, bluing, clear, dehydrate
   3. CHematoxylin, EA, OG-6, dehydrate, bluing, clear
   4. DOG-6, hematoxylin, EA, bluing, dehydrate, clear

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   Answer: A · Hematoxylin, bluing, OG-6, EA, dehydrate, clear

   Smears are hydrated, stained in hematoxylin, differentiated if a regressive method is used, and blued. They then pass through 95% alcohol into OG-6 and then EA, with alcohol rinses in between, since both counterstains are made up in alcohol. Final absolute alcohol removes the water, and xylene clears the cells before mounting. Clearing before full dehydration leaves water in the cells and makes them hazy.

   • B: EA follows OG-6, and clearing must come after dehydration.
   • C: OG-6 comes before EA, and bluing belongs right after the hematoxylin.
   • D: Nuclei are stained and blued before the cytoplasmic dyes.

3. Question 3 · Medium

   In the Pap stain, which component of the EA counterstain gives intermediate and parabasal squamous cells their blue-green cytoplasm?

   1. AEosin Y
   2. BBismarck brown Y
   3. CLight green SF yellowish
   4. DOrange G

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   Answer: C · Light green SF yellowish

   EA contains eosin Y and light green SF yellowish, usually with phosphotungstic acid, and some formulas still include Bismarck brown. Light green binds the cytoplasm of non-keratinized cells such as intermediate and parabasal cells, which appear blue-green (cyanophilic). Eosin Y stains superficial cell cytoplasm, nucleoli and cilia pink. Orange G is applied separately, as OG-6, and stains keratin.

   • A: Eosin Y gives superficial cells their pink cytoplasm.
   • B: Bismarck brown adds little color and is left out of many EA formulas.
   • D: Orange G, in OG-6, stains keratinized cells orange.

4. Question 4 · Medium

   Compared with a conventional smear, a liquid-based cervical preparation:

   1. ANeeds no fixation, as the vial fluid already stains cells
   2. BCannot be used for HPV testing from the same vial
   3. CGives a thin, even cell layer with less blood and mucus
   4. DMust be air-dried before it is placed in the vial

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   Answer: C · Gives a thin, even cell layer with less blood and mucus

   The collection device is rinsed into a vial of alcohol-based preservative, so the cells are fixed at once instead of drying on a slide. The processor removes much of the blood, mucus and inflammatory debris and deposits a thin layer of cells in a defined circle. Residual fluid can be used for HPV and other molecular tests. Screening is quicker, and fewer slides are unsatisfactory because of obscuring material.

   • A: The vial fluid fixes cells but does not stain them; the slide is Pap stained later.
   • B: Residual vial fluid is routinely used for HPV testing.
   • D: The cells are fixed in the vial's preservative; air-drying would damage them.

5. Question 5 · Hard

   A cervical smear from a 35-year-old woman shows one small cluster of malignant-looking cells lying in a different focal plane from the rest of the sample, with no similar cells elsewhere. A malignant pleural fluid was stained in the same dishes just before it. What is the most likely explanation?

   1. AFloater carried over through the shared stains
   2. BMislabeling of the smear at the clinic
   3. CContamination from the clinic's speculum
   4. DTumor cells shed from the patient's own cervix

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   Answer: A · Floater carried over through the shared stains

   Cells can detach from heavily cellular slides into the staining solutions and settle on later slides. Such a floater typically lies in a different focal plane and does not match the rest of the smear. To prevent this, labs filter or change stains on a schedule and stain non-gynecologic and highly cellular specimens separately from gynecologic slides. The pathologist should review the finding together with the earlier slide.

   • B: Mislabeling would swap the whole sample, not add one small cluster.
   • C: A speculum cannot carry cells from another patient's pleural fluid.
   • D: Cells from the patient would lie in the same plane as the rest of the smear.

6. Question 6 · Easy

   A conventional cervical smear is routinely wet-fixed before Pap staining in:

   1. AAbsolute methanol after air-drying
   2. B95% ethanol
   3. CBouin's solution
   4. D10% neutral buffered formalin

   Answer and explanation in LearnFree account

7. Question 7 · Easy

   In the Papanicolaou stain, cell nuclei are stained by:

   1. AHematoxylin
   2. BEosin azure (EA)
   3. COrange G (OG-6)
   4. DLight green SF

   Answer and explanation in LearnFree account