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High-quality cell block preparation from scraping of conventional cytology slide: a technical report on a modified cytoscrape cell block technique

Yeon Il CHOI, Mirzarakhimov JAKHONGIR*, Suk Jin CHOI**, Lucia KIM**, In Suh PARK**, Jee Young HAN**, Joon Mee KIM** and Young Chae CHU**

Department of Pathology, Inha University Hospital, Incheon, Korea, *Department of Histology and Medical Biology, Tashkent Medical Academy, Uzbekistan and **Department of Pathology, Inha University College of Medicine, Incheon, Korea

Abstract

Background: Immunocytochemistry (ICC) on formalin-fixed paraffin embedded cell blocks is an ancillary tool commonly recruited for differential diagnoses of fine needle aspiration cytology (FNAC) samples. However, the quality of conventional cell blocks in terms of adequate cellularity and evenness of distribution of cytologic material is not always satisfactory for ICC. We introduce a modified agarose-based cytoscrape cell block (CCB) technique that can be effectively used for the preparation of cell blocks from scrapings of conventional FNAC slides. Methods: A decoverslipped FNAC slide was mounted with a small amount of water. The cytological material was scraped off the slide into a tissue mold by scraping with a cell scraper. The cytoscrape material was pelleted by centrifugation and pre-embedded in ultra-low gelling temperature agarose and then re-embedded in conventional agarose. The final agarose gel disk was processed and embedded in paraffin. Results: The quality of the ICC on the CCB sections was identical to that of the immunohistochemical stains on histological sections. By scrapping and harvesting the entirety of the cytological material off the cytology slide into a compact agarose cell button, we could avoid the risk of losing diagnostic material during the CCB preparation. Conclusion: This modified CCB technique enables concentration and focusing of minute material while maintaining the entire amount of the cytoscrape material on the viewing spot of the CCB sections. We believe this technique can be effectively used to improve the level of confidence in diagnosis of FNAC especially when the FNAC slides are the only sample available.

Keywords: Fine needle aspiration cytology, cell block, immunocytochemistry

Address for correspondence: Suk Jin Choi, M.D. Department of Pathology, Inha University Hospital, 27, Inhang-ro, Jung-gu, Incheon, 22332, Rep. of KOREA. Tel: +82-32-890-3972, Fax: +82-32-890-3464. E-mail: 204058@inha.ac.kr

TECHNICAL REPORT

INTRODUCTION

Cytological diagnosis of fine needle aspiration cytology (FNAC) samples is usually based on microscopical examinations of Pap- or H&E-stained smears. However, diagnosing based on cytomorphology alone, without the backup of ancillary tests, could be less straightforward when histoarchitectural features are not available or if there are severe air-drying artifacts obscuring cytomorphological findings. Because immunocytochemistry (ICC) on formalin-fixed paraffin-embedded (FFPE) cell block sections is an important ancillary test for differential diagnoses of difficult FNAC cases, the preparation of the cell block is prerequisite

for optimal interpretation of the ICC.1 However, the sample for the preparation of a cell block is not always available. Furthermore, the quality of the cell block is not always satisfactory for proper interpretation of ICC due to suboptimal cellularity or uneven distribution of diagnostic cells through different levels.2 Alternatively, when the previously stained conventional cytology smears are the only clinical sample available, the cytoscrape cell block (CCB) technique can be resorted to in order to prepare the cell blocks from scraping of FNAC slides.3-6

However, it is not easy to procure a high-quality CCB in terms of adequate cellularity and even distribution of diagnostic material throughout the

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different levels in spite of sufficient cellularity on the conventional FNA slide. This is mainly because of incomplete recovery of the minute amount of cytological material into the cell pellet.7 In this report, by presenting cytology cases that were confidentially diagnosed with the aid of ICC on CCB sections, we intend to contribute a technical report on a simple and inexpensive CCB technique that can be effectively used for the preparation of compact agarose-based high-quality CCBs.

MATERIALs AND METHODs

In order to illustrate the diagnostic utility of the high-quality CCBs, we recruited 10 consecutive cases including a few challenging cases of lymph node FNAC that were successfully diagnosed with the aid of ancillary tests on CCB sections using the same protocols for ICC and bright-field dual in situ hybridization tests standardized for FFPE tissue sections (Table 1).

Scraping and harvesting of cytoscrape material from the Papanicolaou-stained FNAC slideThe schematic flow chart of CCB preparation is represented in Fig. 1. A representative conventional Papanicolaou (Pap)-stained FNAC slide with adequate cellularity from each case was decoverslipped in xylene and then rehydrated through graded alcohols. A cell scraper (Corning cell® scrapers, Sigma-Aldrich) that is commonly used in the tissue culture laboratory was used to scrape off the cytological materials entirely from the decoverslipped slide (Fig. 2A). Two or three sets of scrapings were performed for each slide: for each set of scraping, 200~300 µl of water was mounted on the decoverslipped slide prior to scraping so that the cytological materials smeared on the slide could remain wet while being scraped off the slide. Then, the suspension with the cytoscrape material was then transferred into a small tissue mold and finally transferred into a 1.5 ml microcentrifuge tube (Figs. 2B-2C).

Preparation of compact cytoscrape agarose cell blockThe cell block of the cytoscrape material was prepared in the same way as described previously.8 In brief, the suspension of the cytoscrape material was pelleted by centrifugation (Fig. 2D) and the pellet was resuspended in a microcentrifuge tube with a small amount of 2% (w/v) ultra-low gelling temperature (ULGT)

agarose (Agarose Type IX-A, Sigma-Aldrich, St. Louis, MO, USA). After re-pelleting by centrifugation and discarding the surplus ULGT agarose, the cell pellet was refrigerated at 4℃ for 5 to 10 minutes. The resulting solidified ULGT agarose cell button was put into the cap of the microcentrifuge. Then, the cell button was filled with conventional agarose so that the cell button could be re-embedded in it (Fig. 2E). Finally, the agarose gel disk with the cell button at the bottom was removed from the cap by the aid of a needle (Fig. 2F) and put in a tissue cassette (Fig. 2G) for routine tissue processing for the preparation of a paraffinized CCB.

Immunocytochemistry and in situ hybridizationSerial sections cut from the CCBs were routinely processed for H&E staining and immunostaining. An automated slide stainer (Ventana BenchMark XT, Ventana Medical Systems Inc., Tucson, AZ, USA) and the Ventana OptiView DAB detection kit were used to analyze the result of ICC and bright-field dual in situ hybridization tests using the markers and protocols standardized for FFPE tissue sections.

Results

Preparation of cytoscrape cell blocksBy scraping the cytological material off the slide while wet with a small amount of water mounted on it, it was easy to transfer cytoscrape material entirely into a small volume of water (less than 1 ml). This procedure helped us to prevent any loss of diagnostically important cells or minute tissue fragments during the preparation of the cell pellet. By pre-embedding the cell pellet and re-embedding it in conventional agarose, it was possible to have the cytoscrape material concentrated on the viewing spots of the CCB section. Accordingly, the quality of the CCBs in terms of cellularity and distribution of cells through different levels was satisfactory for a cytomorphological evaluation and ancillary tests including ICC and bright-field dual in situ hybridization tests.

Case description

Case 1A 36-year-old previously healthy Korean man presented with a 10-day history of a huge painless mass in the neck. Routine bloods tests including a CBC and chemistry were all normal. He was referred to the Department of Pathology for FNA cytology. The Pap-stained FNAC

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CYTOSCRAPE CELL BLOCK TECHNIQUE

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FIG. 1: A schematic flow chart of the cytoscrape cell block preparation

FIG. 2: Actual practice of the technique. The cytoscrape material is scraped by using a cell scraper while the slide remains wet (A). The cytoscrape material harvested in a small volume (B) is pelleted (C), pre-embedded in ultra-low gelling temperature agarose (D) and re-embedded in conventional agarose (E). Finally, the solidified agarose disk (F) is removed into a tissue cassette (G)

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slides were highly cellular, disclosing singles and loose clusters of malignant round cells characterized by scant cytoplasm and relatively monomorphic nuclei with pale open chromatin, suggesting lymphoid malignancy (Fig. 3A). The cytoscrape material from a Pap-stained slide was pelleted in ULGT agarose and re-embedded in conventional agarose (Fig. 3B) and processed for the preparation of a CCB (Figs. 3C-3D). On ICC with a panel of antibodies performed on the CCB sections, the tumour cells were negative for CD3, CD5, CD20, and cyclin D. However, they were positive for TdT (Fig. 3E) and PAX5. The cytomorphological and immunocytochemical

profiles were sufficient for rendering a diagnosis of B-lymphoblastic lymphoma/leukemia. The bone marrow biopsy that followed confirmed the diagnosis.

Case 2A 55-year-old Korean man presented with an intra-abdominal peri-gastric mass clinically assumed to be a malignant lymphoma. An endoscopic ultrasound-guided FNAC was performed and the Pap-stained slides revealed malignant large epithelioid cells with conspicuous mitotic/apoptotic figures predominantly arranged in cohesive sheets, clusters or anastomosing cords

FIG. 3: Findings of fine needle aspiration cytology and immunocytochemistry of case 1 diagnosed as lympho-blastic lymphoma/leukemia. The scraped material from slide with malignant round tumor cells (A, Pap, ×10) was pelleted and pre-embedded in an ultra-low gelling temperature agarose and re-embedded in conventional agarose gel disk (B). The quality of the cytoscrape cell block (C, H&E, ×400; D, ×400) was satisfactory for ancillary tests including immunocytochemistry for TdT (E, ×400)

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(Figs. 4A-4B). For the differential diagnosis between high grade carcinoma and malignant lymphoma, ancillary tests were performed on sections of the CCB (Figs. 4C-4D) obtained from a redundant smear. A diagnosis of aggressive B-cell lymphoma consistent with diffuse large B-cell lymphoma was rendered based on ICC with panels of antibodies, including CD3 (Fig. 4E), CD20 (Fig. 4F), MUM1 (Fig. 4G) and PAX5. The result of an in situ hybridization with an Epstein-Barr virus-encoded RNA probe was negative. The FFPE tissue sections of a separately submitted tiny piece of tissue sample revealed the same cytoarchitectural and immunophenotypic features (Fig. 4H).

Case 3A 35-year-old previously healthy Vietnamese woman presented with a 2-weak history of intermittent thoracic back pain accompanied by epigastric pain and discomfort. The upper gastrointestinal endoscopic examination showed multiple nodular erosions in the gastric mucosa, which were initially interpreted as “probably acute infectious gastritis”. Because the patient’s symptom was aggravated despite symptomatic treatment over a week, a further workup including a chest CT scan and abdominopelvic CT scans disclosed multiple nodules in both lungs and pathological factures in the thoracic spine accompanied by significant generalized lymphadenopathies in the whole body. An image-guided FNAC of the pelvic lymph node was performed. The Pap-stained slides disclosed loosely cohesive malignant epithelioid cells arranged in syncytial tissue fragments (Figs. 5A-5C). One of the redundant cellular slides was decoverslipped and the cytological material was entirely incorporated into a compact CCB (Figs. 5D-5F) as described above. ICC showed that the tumour cells were negative for CD45 (Fig. 5G), CD3 (Fig. 5H), and CD20 (Fig. 5I), while they were reactive for CD30 (Fig. 5J) and ALK (Fig. 5K). The cytomorphological and ICC profile were sufficient to render a diagnosis of ALK-positive anaplastic large cell lymphoma of the small cell variant, which was histologically confirmed by a needle biopsy of the lesion (Fig. 5L).

Case 4A 74-year-old Korean woman, who had been free of disease for 14 years since she received a subtotal gastrectomy for signet ring cell carcinoma confined to the gastric mucosa,

presented with a recently detected 2 cm-sized palpable mass in her left axilla. The gastric mucosa was free of disease recurrence, except a tiny polyp that showed a focal infiltration of atypical cells that were negative for cytokeratin. The patient was referred for FNAC to a pathologist in the department of pathology. The FNAC characterized by many singles and some papillary clusters of malignant epithelioid cells with prominent nucleoli and moderate amounts of cytoplasm was initially diagnosed as metastatic carcinoma morphologically suggestive of ductal carcinoma of the breast. However, further clinical evaluation via a mammography and ultrasonography disclosed multiple 1-1.5 cm-sized nodules in both breasts and as well as in both axillae. Because her clinical presentation was unusual for carcinoma of the breast, one of the cellular smears (Fig. 6A) was processed as described above to create a CCB (Fig. 6B) for ICC using a panel of antibodies. The tumour cells were negative for cytokeratins (MNF116 and Cam5.2), oestrogen receptor, progesterone receptor, c-erbB2, and epithelial membrane antigen. Finally, a revised diagnosis of metastatic epithelioid malignant melanoma was rendered based on diffuse strong immunoreactivity for HMB45 (Fig. 6C) and Melan A. Immunohistochemistry using the antibodies disclosed a minute focus of infiltration of malignant melanoma in the tiny polyp of the gastric mucosa (Fig. 6D).

DisCussion

The result of ICC applied on an FNAC slide is not necessarily identical to that applied on sections of a FFPE cell block, and hence not always reliable.9-13 This is because of a substantial difference in method of sample preparation between the FNAC samples and tissue samples. Although ICC on FFPE cell block sections can augment diagnostic accuracy in daily practice of diagnostic cytopathology, the quality of the conventional cell blocks in daily cytopathology practice is not always satisfactory because of poor cellularity and uneven distribution of cytologic material through different levels.14

To overcome these problems, a CCB technique has been be recruited for the preparation of a cell block from cytoscrape material.4-6 However, the conventional CCB technique has not been generally adopted in routine cytology practice because this technique does not always warrant preparation of high-quality CCBs. Previously, we have shown that even a minute amount of

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FIG. 4: Findings of fine needle aspiration cytology and immunocytochemistry of case 2 diagnosed as diffuse large B-cell lymphoma. The scraped material from a slide with malignant epithelioid tumor (A, Pap, ×100; B, ×400) processed for the preparation of a cytoscrape cell block (C, H&E, ×40; D, ×400). The diagnosis was rendered with the aids of immunocytochemistry for CD3 (E, ×100) and CD20 (F, ×100), and MUM1 (G, ×100), which was confirmed by additional biopsy (H&E, H, ×400; inset, CD20)

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cellular material can be entirely incorporated into a compact cell button by using a modified cell block technique in which two different agaroses with different gelling temperatures are used for the preparation of cell blocks.8 Since we have applied this technique to the preparation of CCBs, we were able to avoid the loss of the cytoscrape material during the CCB preparation. By concentrating the minute but entire amount of the cytoscrape material in a viewing spot with relatively even distribution of cytological

FIG. 5: Findings of fine needle aspiration cytology and immunocytochemistry of case 3 diagnosed as ALK-positive anaplastic large cell lymphoma of small cell variant. The scraped material from a slide with malignant epithelioid tumor cells arranged in syncytial fragments (A, Pap, ×40; B, ×100; C, ×400) was processed and embedded in a cytoscrape cell block (D, HE, ×40; E, ×100; F, ×400). Based on the result of ICC on the cell block sections using a panel of antibodies including CD45 (G), CD3 (H), and CD20 (I), CD30 (J) and ALK (K), a diagnosis of ALK-positive anaplastic large cell lymphoma of small cell variant. The diagnosis was confirmed by a needle biopsy of the lesion (L, HE, ×100; inset, ×400)

material through different levels of the CCB sections, we were able to prepare high-quality CCBs that were suitable not only for ICC but also for bright-field dual in situ hybridization using the same protocols standardized for of FPPE tissue. In our modified CCB technique, the decoverslipped slide was mounted with a small amount (200~300 µl) of water so that the cytological material on the slide could be kept wet while being scraped off. In addition, instead

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FIG. 6: Findings of fine needle aspiration cytology and immunocytochemistry of case 4 diagnosed as metastatic malignant melanoma. The scraped material from a slide (A, ×10; left inset, ×100; right inset, ×400) was processed and blocked in a cytoscrape cell block (B, HE, ×40; inset, ×400) for ICC using a panel of antibodies. The result of ICC for HMB45 (C, ×100) on the cell block section was suggestive of metastatic melanoma, which was confirmed by an incidentally identified tiny polyp of the gastric mucosa (D, H&E; left inset, HMB45, ×400; right inset, S100, ×400)

of using a surgical blade or microtome blade for scraping off the cytological material from the slide, we used a cell scraper with a wide blade that is commonly used in any tissue culture laboratory. We assume that these modifications can not only minimize the damage to the cytological material caused by the procedure itself but also help to recover the cytoscrape material as entirely and as rapidly as possible. When the only sample for the pathology report is an FNAC slide that can only be successfully diagnosed with the help of ancillary tests using diagnostic markers on CCB sections, consideration should be given to obtaining photographic records of the smear appearance for purposes of documentation and future review if necessary. However, when the smear on the FNAC slide has sufficient cellularity, only a part of the slide with thick smear can be scraped off the slide and the rest of the smear can be left intact so that the valuable diagnostic material can be preserved

in its original form on the same slide. This is possible because the decoverslipped slide does not have to be destained as it has already been demonstrated by a couple of previous works.5,6 We assume that the residual cytological material on the same slide may also be effectively used for in situ cytogenetic tests using fluorescent in situ hybridization (FISH) probes especially when optimal interpretation FISH signal on cell block sections is difficult due to artifactual cellular aggregation and nuclear truncation.15

We believe our modified CCB technique will markedly improve the confidence in an ICC-based differential diagnosis of difficult FNAC cases, especially when the slides are the only sample available for ancillary tests and repeat FNACs are not possible. Further studies are warranted to confirm its utility in in situ analysis of prognostic markers and markers for target therapy.

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ACKNOWLEDGEMENT

This work was supported by Inha University Research Grant (52036-1). There is no potential conflict of interest relevant to this article.

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