FINE NEEDLE ASPIRATION. Use of a Thin-Layer Technique in Thyroid Fine Needle Aspiration DO NOT DUPLICATE

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1 FINE NEEDLE ASPIRATION Use of a Thin-Layer Technique in Thyroid Fine Needle Aspiration Despoina Malle, M.D., Ph.D., Rosalia-Maria Valeri, M.D., Ph.D., Kalliopi Pazaitou-Panajiotou, M.D., Ph.D., Anastasia Kiziridou, M.D., Ph.D., Iraklis Vainas, M.D., Ph.D., and Charicleia Destouni, M.D., Ph.D. Objective To investigate the efficacy of the ThinPrep Processor (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) in fine needle aspiration (FNA) of thyroid gland lesions. Study Design This study included 459 thyroid FNA specimens obtained from patients who came to our endocrinology department with various thyroid disorders over 3 years. The cytologic material was prepared using both the conventional and ThinPrep method in the first 2 years (285 cases), while in the last one only the ThinPrep method was used (174 cases). The smears were stained using a modified Papanicolaou procedure and May-Grünwald-Giemsa stain. Immunocytochemistry was performed on thin-layer slides using specific monoclonal antibodies when needed. Thin-layer and direct smear diagnoses were compared with the final cytologic or histologic diagnoses, when available. Results Our cases included 279 adenomatoid nodules, 15 cases of Hashimoto thyroiditis, 45 follicular neoplasms, 14 Hürthle cell tumors, 58 papillary carcinomas and 15 anaplastic carcinomas. Thin-layer preparations showed a trend toward a Our experience suggests that thin-layer cytology is a promising technique for investigating thyroid lesions... lower proportion of inadequate specimens and a lower false negative rate. Cytomorphologic features showed some differences between the 2 methods. Colloid was less frequently observed on ThinPrep slides, while nuclear detail and micronucleoli were more easily detected with this technique. Moreover, ThinPrep appeared to be the appropriate method for the use of ancillary techniques in suspicious cases. Conclusion Thin-layer cytology improves the diagnostic accuracy of thyroid FNA and offers the possibility of performing new techniques, such as immunocytochemistry, on the same sample in order to detect malignancy as well as the type and origin of thyroid gland neoplasms. (Acta Cytol 2006;50:23 27) Keywords: thyroid cancer; thyroid neoplasms; aspiration biopsy, fine-needle; thin-layer cytology. Fine needle aspiration (FNA) cytology has greatly improved the clinical management of thyroid nodules 1-3 but has some inherent limitations related to inadequate sampling and cytologic features overlapping between benign and malignant follicular lesions. 4-8 In recent years liquid-based, thin-layer techniques be- From the Departments of Cytopathology, Endocrinology and Histopathology, Theagenion Anticancer Hospital, Thessaloniki, Greece. Drs. Malle and Valeri are Cytopathologists, Department of Cytopathology. Dr. Pazaitou-Panajiotou is Endocrinologist, Department of Endocrinology. Dr. Kiziridou is Pathologist, Department of Histopathology. Dr. Vainas is Endocrinologist and Head, Department of Endocrinology. Dr. Destouni is Cytopathologist and Head, Department of Cytopathology. Address correspondence to: Despoina Malle, M.D., Ph.D., Pontou 47-49, Kalamaria, Thessaloniki, Greece (pepimalle@hotmail.com). Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. Received for publication June 21, Accepted for publication July 19, /06/ /$19.00/0 The International Academy of Cytology ACTA CYTOLOGICA 23

2 Malle et al came available as alternatives to conventional cytopreparatory methods to surmount the diagnostic difficulties. Although thyroid FNA is widely accepted as the primary diagnostic procedure for thyroid nodules, very few articles have been published on liquid-based techniques The aim of this study was to investigate the efficacy of the ThinPrep Processor (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.), an Awareness of the cytomorphologic features associated with [thin-layer preparations] is essential to avoiding misleading diagnostic pitfalls in daily practice. automated, thin-layer method, in FNA of thyroid lesions. Materials and Methods This study included 459 thyroid FNA specimens obtained from unselected patients referred to our hospital for evaluation of a palpable thyroid lesion. Each nodule was aspirated using a standard technique. Multiple aspirations (2 or 3 per nodule) were performed until an adequate specimen was obtained. The cytologic material was prepared using both the conventional and the ThinPrep method in the first 2 years (285 cases), while in the last one only the ThinPrep method was used (174 cases). At first, direct smears were prepared using the conventional method, and the remaining aspirate in the needle was rinsed immediately in a vial containing 20 ml of CytoLyt preservative solution (Cytyc) and then processed according to the manufacturer s instructions. 13 The vial was placed in the ThinPrep 2000 automated slide processor (Cytyc), and a dispersion cycle homogenized the cell suspension. The cells were automatically collected on a polycarbonate filter membrane. A thin, evenly dispersed monolayer of cells was deposited from the filter onto the slide in a 20-mmdiameter circle. The smears were stained using a modified Papanicolaou procedure and May-Grünwald-Giemsa stain. Immunocytochemistry was performed on thin-layer slides, mainly for differentiation between benign and malignant lesions, using specific monoclonal antibodies for thyreoglobulin, CK19, galectin 3 and CD44. The NexES Automated Slide Stainer (Ventana, Tucson, Arizona, U.S.A.) and related Ventana reagents were used. The samples were immersed in a citrate buffer solution (ph 7.3) and heated for 20 minutes at 350 W. The slides were rinsed in tap water for 5 minutes. Then they were incubated with 3% H 2 O 2 for 4 minutes to quench the endogenous peroxidase activity. We used the primary antibodies diluted in an appropriate dilution with Ventana antibody diluent in a Ventana User Fillable Dispenser. A standard immunocytochemical avidin-biotin method was applied. The primary antibody was bound by a biotin-conjugated mouse secondary antibody formulation. This step was followed by an avidin enzyme conjugate, which bound to the biotin present on the secondary antibody. The primary antibody secondary antibody avidin enzyme complex was then visualized utilizing a precipitating enzyme product. Each step was performed for a precise time and and at a precise temperature. At the end of each incubation step, the Ventana ES instruments washed the slides to stop the reaction and remove unbound material that would hinder the desired reaction in subsequent steps and applied a liquid coverslip, which minimized evaporation of the aqueous reagents from the specimen-containing slide. Diaminobenzidine was used as a chromogen for detection of the antigens. Incubation with copper sulfate was performed for enhancement of the color reaction. The smears were finally counterstained with hematoxylin and coverslipped for examination. The whole staining process lasted about 90 minutes at 41 C with the slides at 37 C. The results were compared with the corresponding histologic diagnoses and clinical follow-up. Results The microscopic examination of all cases revealed mainly benign lesions and, in a smaller percentage of cases, neoplastic ones. The cytologic results were classified as benign lesions (adenomatoid nodules, with or without cystic change, and Hashimoto s thyroiditis), follicular neoplasms, Hürthle cell neoplasms, suspicious for malignancy, positive for malignancy and unsatisfactory. At least 6 groups of 10 follicular cells were required for specimen adequacy (Table I). Histologic correlation was available in all suspicious and malig- Table I Final Cytologic Diagnoses in All Cases Cytologic diagnosis No. of cases Unsatisfactory 18 Benign lesions (adenomatoid nodules) 279 Hashimoto s thyroiditis 15 Follicular neoplasms 45 Suspicious for malignancy 15 Hürthle cell tumors 14 Papillary carcinomas 58 Anaplastic carcinomas 15 Total ACTA CYTOLOGICA Volume 50 Number 1 January February 2006

3 Thin-Layer Technique Table II Comparison of Direct and ThinPrep Smears in Cases with the Split Sample Protocol nant cases and in one third of benign lesions. During the first 2 years a correlation between the conventional and ThinPrep methods could not be made for accuracy because a split sample protocol was used (Table II). Nevertheless, in most of the cases, the thin-layer diagnoses agreed with the direct smear diagnoses. In some cases of adenomatoid nodules with cystic change the diagnosis was based on ThinPrep smears since there was not diagnostically sufficient material by the conventional method. The rate of unsatisfactory samples for the ThinPrep method was 3.92% and for the conventional smears, 8.93%. All the cases with a final diagnosis of a malignant lesion were diagnosed with both preparations. In the evaluation of cases, the cytomorphology of thin-layer slides and direct smears showed some differences. Colloid was observed less frequently on thinlayer slides and tended to be present as droplets rather than a diffuse layer or film. Tissue fragments and spherules appeared less frequently on thin-layer slides. However, the ThinPrep smears showed a cleaner background and greater cellularity. Moreover, nuclear detail and micronucleoli were more easily detected. Other architectural features, such as microfollicles, honeycomb arrangement and papillae, occurred with equal frequency in both preparations. The determination of malignancy was much easier with the Thin- Prep technique, as nuclear and cytoplasmic detail were more easily detected. Some artifacts seen in conventional smears were not present in ThinPrep materials. In contrast, chronic lymphocytic thyroiditis was diagnosed more frequently on direct smears than on thin-layer slides. Additionally, ThinPrep smears were more suitable for immunoperoxidase staining. In suspicious and all malignant cases immunocytochemistry was performed using specific monoclonal antibodies. The suspicious cases were 11 atypical adenomas, 1 papil- ThinPrep diagnosis Adeno- Hashi- Follicu- Suspi- Hürthle Papil- Conventional Unsatis- matoid moto s lar neo- cious cell lary car- Anaplastic smear diagnosis factory nodules thyroiditis plasms cases tumors cinoma carcinoma Total Unsatisfactory Adenomatoid nodules Hashimoto s thyroiditis Follicular neoplasms Suspicious cases 9 9 Hürthle cell tumors Papillary carcinoma Anaplastic carcinoma 9 9 Total lary carcinoma, 1 Hürthle cell tumor and 2 follicular cell carcinomas. Positive thyroglobulin staining was noted in about 49% of benign nodular goiters, Hashimoto s thyroiditis and papillary carcinomas. Negative or weaker expression was found in oncocytic tumors as well as in anaplastic thyroid carcinomas. Galectin-3 positive expression was noted in almost all malignant cases while not detected in benign lesions. Overexpression of CK19 was observed in 54 of 58 papillary carcinomas and in 1 suspicious case, which turned out to be an atypical adenoma. Overexpression of CD44 was observed in 51 of 58 papillary carcinomas and in 1 case of Hürthle cell tumor. Discussion FNA has become an accepted diagnostic modality in evaluating thyroid nodules, 1-3 but the preoperative characterization of follicular lesions is very difficult and remains a challenge. Maximizing diagnostic accuracy requires good clinical information, close attention to aspiration and preparation technique, and experience in cytodiagnosis. The ThinPrep Processor is a slide preparation device that recently has become widely available and has gained popularity as a collection and preparation technique. 14,15 However, the results from previous studies on thyroid FNA are controversial. Similar to our observations, Biscotti et al 9 noted less colloid in thin-layer slides than direct smears, and Biscotti et al 9 and Mesonero et al 16 reported that the colloid occurred in droplets or was fragmented rather than appearing as a diffuse film or watery substance. Both groups of authors found that cell type and cellular arrangement were well preserved in ThinPrep slides. Furthermore, Biscotti et al 9 noted superior nuclear detail in thin-layer slides, while Mesonero et al 16 found no differences in nuclear or cytoplasmic fea- Volume 50 Number 1 January February 2006 ACTA CYTOLOGICA 25

4 Malle et al tures in thin-layer slides and direct smears. According to a Scurry and Duggan 17 study, there are no significant differences between the 2 methods in any quality indicators. They reported that benign lesions were more easily diagnosed by the thin-layer technique (24 31%). Additionally, a lower proportion of inadequate specimens (41 50%) and, most important, a lower false negative rate (3 9%) was observed by ThinPrep. However, in other studies 10 of thyroid FNA in which the direct smear and thin-layer method were compared, slightly reduced diagnostic accuracy was found using the thin-layer technique. In our experience, immunocytochemistry was easier to apply using the ThinPrep technique than the conventional method because of the high quality of archival cytologic material available and smaller quantity of monoclonal antibody needed. Positive staining of thyroglobulin was helpful in defining the origin of cells in metastatic lesions because follicular cells of the thyroid are the only cells that have the ability to synthesize thyroglobulin. Galectin-3 serves as a differential diagnostic marker between benign and malignant thyroid neoplasms and may be a useful adjunct to the currently available diagnostic tools for preoperative diagnosis of malignant thyroid tumors. 18,19 According to a Bartolazzi study, 20 the immunodetection of galectin-3 on thyroid cells is a highly specific and sensitive strategy for identifying malignant lesions of the thyroid on conventional histologic and cytologic preparations. Those authors found that the sensitivity and specificity of galectin-3 immunodetection alone in discriminating benign from malignant thyroid lesions were > 99% and 98%, respectively, and that the positive predictive value and diagnostic accuracy were 92% and 99% as well. Positive expression of CD44 and CK19 maybe of value in the differential diagnosis of thyroid papillary carcinomas from its mimics. Nasser et al 21 concluded as well that CK19 is an effective, highly sensitive (92%) and specific (97%) ancillary tool for the diagnosis of papillary carcinoma, in thyroid FNA. Also, Chhieng et al 22 demonstrated that CD44 immunostaining applied to the thyroid FNA specimens maybe a useful adjunct in the difficult differential diagnosis of thyroid papillary carcinoma from its mimics. ThinPrep is of greater value concerning sample quality and increases diagnostic accuracy, mainly because of the better cytomorphologic picture and cleaner background. It is easier and less time consuming to screen and interpret the slides, as the cells are limited to a smaller area on a clear background with excellent cellular preservation. However, the Thin- Prep method requires some experience for a correct interpretation in order to avoid diagnostic pitfalls.the method is simple and not expensive and permits faster and better evaluation of thyroid FNA, using a smaller number of cells and slides. Also, it offers the possibility of creating archival material and applying new techniques, such as immunocytochemistry, 23 in the same sample. An additional advantage is that in geographic locations where well-equipped cytology laboratories are not available, material can be collected and transported in ThinPrep vials. Our experience suggests that thin-layer cytology is a promising technique for investigating thyroid lesions, but awareness of the cytomorphologic features associated with ThinPrep processing is essential to avoiding misleading diagnostic pitfalls in daily practice. 12 Our experience suggests that thin-layer cytology is a promising technique for investigating thyroid lesions. Awareness of the cytomorphologic features associated with [thin-layer preparations] is essential to avoiding misleading diagnostic pitfalls in daily practice. References 1. Silverman JF, West RL, Larkin EW, Park HK, Finley JL, Swanson MS, Fore WW: The role of fine-needle aspiration biopsy in the rapid diagnosis and management of thyroid neoplasm. Cancer 1986;47: Nunez C, Mendelsohn G: Fine-needle aspiration and needle biopsy of the thyroid gland. Pathol Annu 1989;24: Kini SR: Thyroid. In Guide to Clinical Aspiration Biopsy. Third volume. Edited by TS Kline. New York, Igaku-Shoin, Hall TL, Layfield LJ, Phillippe A, Rosenthal DL: Sources of diagnostic error in fine needle aspiration of the thyroid. Cancer 1989;63: Caraway NP, Sneige N, Samaan NA: Diagnostic pitfalls in thyroid fine-needle aspiration: A review of 394 cases. Diagn Cytopathol 1993;9: Sidawy MK, Del Vecchio DM, Knoll SM: Fine needle aspiration of thyroid nodules: Correlation between cytology and histology and evaluation of discrepant cases. Cancer (Cancer Cytopathol) 1997;81: Baloch ZW, Sack MJ, Yu GH, Livolsi VA, Gupta PK: Fineneedle aspiration of thyroid: An institutional experience. Thyroid 1998;8: Bartolazzi A: Improving accuracy of cytology for nodular thyroid lesions. Lancet 2000;355: Biscotti CV, Hollow JA, Toddy SM, Easley KA: ThinPrep versus conventional smear cytologic preparations in the analysis of thyroid fine needle aspiration specimens. Am J Clin Pathol 1995;104: Frost AR, Sidawy MK, Ferfelli M, Tabarra SO, Bronner NA, Brosky KR, Sherman ME: Utility of thin-layer preparations in thyroid fine needle aspiration: Diagnostic accuracy, cytomorphology and optimal sample preparation. Cancer (Cancer Cytopathol) 1998;84: ACTA CYTOLOGICA Volume 50 Number 1 January February 2006

5 Thin-Layer Technique 11. Zhang Y, Fraser JL, Wang HH: Morphologic predictors of papillary carcinoma on fine needle aspiration of thyroid with ThinPrep preparations. Diagn Cytopathol 2001;24: Afify AM, Liu J, Al- Khafaji BM: Cytologic artifacts and pitfalls of thyroid fine needle aspiration using Thin Prep: A comparative retrospective review. Cancer (Cancer Cytopathol) 2001;93: ThinPrep 2000 Operator s Manual. Boxborough, Massachusetts, Cytyc Corporation, Rosenthal DL, Manjikian V: Techniques in the preparation of a monolayer of gynecologic cells for automated cytology: An overview. Anal Quant Cytol Histol 1987;9: Linder J: Automation in cytopathology. Am J Clin Pathol (suppl 1) 1992;98:S47 S Mesonero CE, Sickel J: Thyroid fine needle aspiration: A comparison of thin-layer slide preparation with conventional smears. Cytology 1993;37: Scurry JP, Duggan MA: Thin layer compared to direct smear in thyroid fine needle aspiration. Cytopathology 2000;11: Gasbarri A, Martegani MP, Del Prete F, Lucante T, Natali PG, Bartolazzi A: Galectin-3 and CD44v6 isoforms in the preoperative evaluation of thyroid nodules. J Clin Oncol 1999;17: Saggiorato E, Cappia S, De Giuli P, Mussa A, Pancani G, Caraci P, Angeli A, Orlandi F: Galectin-3 as a presurgical immunocytodiagnostic marker of minimally invasive follicular thyroid carcinoma. J Clin Endocrinol Metab 2001;86: Bartolazzi A, Gasbarri A, Papotti M, Bussolati G, Lucante T, Khan A, Inohara H, Marandino F, Orlandi F, Nardi F, Vecchione A, Tecce R, Larsson O, Thyroid Cancer Study Group: Application of an immunodiagnostic method for improving preoperative diagnosis of nodular thyroid lesions. Lancet 2001;357: Nasser SM, Pitman MB, Pilch BZ, Faquin WC: Fine-needle aspiration biopsy of papillary thyroid carcinoma: Diagnostic utility of cytokeratin 19 immunostaining. Cancer 2000;90: Chhieng DC, Ross JS, McKenna BJ: CD44 immunostaining of thyroid fine-needle aspirates differentiates thyroid papillary carcinoma from other lesions with nuclear grooves and inclusions. Cancer 1997;81: Cochand-Priollet B, Prat J-J, Polivka M, Thienpont L, Dahan H, Wassef M, Guillausseau P-J: Thyroid fine needle aspiration: The morphological features on ThinPrep slide preparations: Eighty cases with histological control. Cytopathology 2003;14: Volume 50 Number 1 January February 2006 ACTA CYTOLOGICA 27