Journal of Cytology
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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 25  |  Issue : 1  |  Page : 18-22
Role of AgNORs in thyroid lesions on fine needle aspiration cytology smears

Department of Pathology, Indira Gandhi Medical College, Shimla, India

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Background: Fine needle aspiration has an important role in diagnosis of thyroid neoplasm. However, it is difficult to differentiate between follicular adenoma and follicular carcinoma by cytology alone. Recently, silver staining has been performed for nucleolar organizer regions (AgNORs) to differentiate various tumors.
Aims: The present study was undertaken to see if the AgNOR technique could distinguish between benign and malignant lesions, particularly, follicular neoplasm.
Materials and Methods: One hundred forty cases of thyroid lesions were examined, which included colloid goiter (n = 36), multinodular goiter (n = 38), subacute thyroiditis (n = 6), Hashimoto's thyroiditis (n = 17), lymphocytic thyroiditis (n = 3), follicular neoplasm (n = 18), Hurthle cell neoplasm (n = 3), papillary carcinoma (n = 16), and medullary carcinoma (n = 3). Diagnosis was confirmed by histopathology in 80 cases. The usual one-step silver colloidal reaction was performed at room temperature for 35 minutes and intranuclear dots of silver deposits were counted in 100 cells.
Results: AgNOR counts of benign and malignant lesions were compared and were found to be statistically significant (P < 0.001). The mean AgNOR counts were higher in neoplastic lesions.
Conclusions: AgNOR counting in fine needle aspiration smears is a simple, sensitive, and cost-effective method for differentiating benign from malignant thyroid follicular neoplasms.

Keywords: AgNORs; fine needle aspiration cytology; thyroid.

How to cite this article:
Asotra S, Sharma J. Role of AgNORs in thyroid lesions on fine needle aspiration cytology smears. J Cytol 2008;25:18-22

How to cite this URL:
Asotra S, Sharma J. Role of AgNORs in thyroid lesions on fine needle aspiration cytology smears. J Cytol [serial online] 2008 [cited 2022 Dec 1];25:18-22. Available from:

   Introduction Top

Nodular thyroid diseases indicated by the presence of single or multiple nodules within the thyroid gland, remain a common clinical problem. The vast majority of nodules are nonneoplastic. To avoid unnecessary surgery, a thyroid scan, ultrasonography, and fine needle aspiration cytology (FNAC) are used as diagnostic tools to differentiate malignant nodules from a benign lesion. [1],[2]

The routine use of FNAC in the assessment of thyroid nodules has reduced the number of patients subjected to thyroidectomy for benign diseases of the thyroid. [3] The sensitivity, specificity, and accuracy of FNAC for malignancy detection have eclipsed the diagnostic utility of other diagnostic methods. [4],[5]

Various attempts have been made to improve the diagnostic accuracy of FNAC, including morphometric studies, DNA measurement, immunohistochemical, and enzyme techniques for thyroid cancer with varying degrees of success. [6] It is well known that the silver staining technique for nucleolar organizer regions (AgNORs) has been successfully applied to a wide variety of neoplastic lesions on pathological materials in order to distinguish benign from malignant lesions. [2],[6],[7] In the human karyotype, NORs are located on each of the short arms of the acrocentric chromosomes 13,14,15,21 and 22. [8],[9]

NORs can be located by staining with silver nitrate under prescribed conditions, the structures then demonstrated are termed AgNORs. In cytopreparation, the mean value and the mean standard deviation [SD] of the area per cell of the AgNORs seem highly sensitive parameters with high specificity to distinguish between benign and malignant thyroid lesions. [6],[10] In smears, these parameters are more accurate because the whole nucleus can be assessed.

   Materials and Methods Top

This study is based on the analysis of patients presenting with thyroid swelling in the Departments of Surgery and Pathology of the Indira Gandhi Medical College, Shimla during the period of July 2003 to August 2004. Utility of AgNOR staining was studied in FNAC of the thyroid lesions along with the cytomorphological features, and results were confirmed by histopathology.

One hundred forty FNAC cases of the thyroid were included in the present study. The clinical findings, history, and laboratory investigations were noted in all the cases. The thyroid lesions were aspirated with a 23-gauge needle attached to a 20 mL disposable syringe fixed to a syringe holder. The smears were air-dried and stained with May-Grünwald-Giemsa (MGG). Cytomorphological features including cellularity, architectural pattern, amount and type of colloid, cellular-nuclear pleomorphism, chromatin pattern, intranuclear inclusion, intranuclear grooves, oncocytic cells, metaplastic cells, foam cells, multinucleated giant cells, Psammoma body, and siderophages were assessed. Other smears were subjected to silver colloidal staining for AgNORs to find out the number of AgNORs in different lesions.

The argyrophilic proteins associated with nucleolar organizer regions, appeared as black dots both within the nucleolus and elsewhere in the nucleoplasm. AgNORs were counted in randomly selected 100 nuclei in each case under an oil immersion lens and the mean number of AgNORs per nucleus was calculated.

All discrete variables were expressed as percentages and continuous variables as mean ± SD. Significance of continuous variables was analyzed using one-way analysis of variance [ANOVA] and P < 0.05 was considered to be statistically significant (Confidence interval = 95%).

   Results Top

Out of 140 patients that had undergone FNAC, there were 100 nonneoplastic and 40 neoplastic cases. Of the latter, 21 were benign and 19 malignant. Females (84.28%) formed the major group with the male to female ratio being 1:5.4. Age ranged from 15 to 82 years with the maximum number of patients being in the 20-40 years' age group; the mean age was 42.08 years. AgNOR counts were evaluated in 140 FNA smears, colloid goiter cases were taken as control. The numbers of dots were recorded for 100 follicular cells, and the mean number was calculated [Table - 1]. The cytology diagnosis was confirmed histopathologically in 80 cases. [Table - 2] shows the AgNOR counts in the cases where histopathology was available.

Colloid goiter: Thirty-six cases were studied. The smears were hypercellular and characterized by plenty of colloid material. The cells were of variable morphology in the background of the smears, ranging from cuboidal follicular cells to large cells with abundant cytoplasm and lipofuscin pigment. These were present either in loose aggregates or dissociated forms. A fair number of bare nuclei and inflammatory cells including foamy macrophages were also seen. The mean number of AgNOR dots was 2.94 ± 0.852 (2.01-6.02). These were taken as normal controls for comparison with other lesions.

Multinodular goiter: Thirty-eight cases of FNAC were studied. The smears were hypercellular consisting of aggregates and sheets of follicular cells with abundant cytoplasm showing marginal vaculations in MGG-stained smears. Bare nuclei were common and there was moderate anisokaryosis in some cells. These lesions had a mean AgNOR count of 2.82 ± 0.088 in the range of 2.02-3.9 and significantly exceeded ( P = 0.01) the normal values.

Subacute thyroiditis: Six cases were included in cytology. The thyroditis cases showed a fair number of inflammatory exudates consisting of neutrophils, few lymphocytes, macrophages, epithelioid cells, and multinucleated giant cells. The mean AgNOR count was 2.61 ± 0.281 (2.02-3.82), which was highly significant ( P < 0.005) when compared to normal values.

Lymphocytic thyroiditis: Three cases were diagnosed by FNAC. The mean AgNOR count was 3.59 ± 0.04 (2.82-4.88); this was higher than normal values, but was not statistically significant ( P > 0.1).

Hashimoto's thyroiditis: Seventeen cases were included. They showed moderate numbers to abundant lymphocytes, plasma cells, multinucleated giant cells, variable number of oxyphilic epithelial cells, and scanty colloid. The mean AgNOR counts was 2.76 ± 0.082 (2.2-3.6); it was highly significant when compared to the normal values ( P = 0.005).

Follicular neoplasm: AgNOR staining was performed in 18 cases of follicular neoplasm and dots were counted in 100 consecutive cells by standard methods. The mean AgNOR count was 3.15 ± 0.80 (2.01-5.2).

Hurthle cell neoplasm: Three cases were studied by FNAC. Smears were composed predominately of Hurthle cells, which were present in sheets and in dyshesive form. The cells were polyhedral or plasmacytoid. These cells had distinct borders and abundant granular acidophilic cytoplasm. These tumors had a mean AgNOR counts of 3.48 ± 0.554 (2.4-4.22). All nuclei had clear black dots with no cluster arrangement.

Papillary carcinoma: Sixteen cases were studied. All aspirates were highly cellular and were distinguished by papillary clusters. The cells showed mild to moderate cellular-nuclear pleomorphism, nuclear grooves, and nuclear inclusions. In certain areas, multinucleated giant cells and hemosiderin containing macrophages were also present. The mean AgNOR count was 5.08 ± 0.278 (2.4-6.2). All dots present in the nuclei were distinct with no cluster formation.

Medullary carcinoma: Three FNAC cases were studied which showed high cellularity with mainly dispersed cells, plasmacytoid cells with moderate anisokaryosis, and fragments of amorphous pink, background material. The mean AgNOR counts was 3.47 ± 0.54 (2.4-4).

The mean AgNOR count in malignant neoplastic thyroid lesions was found to be 4.27 ± 0.41 by FNAC, which was of high statistical significance when compared to normal values ( P < 0.001).

Statistical analysis showed that cytological comparisons of AgNOR counts of benign and malignant lesions were statistically significant ( P < 0.01), FNAC comparisons showed overlapping AgNOR counts between nonneoplastic and neoplastic lesions.

There was no cut-off point for AgNOR counts among various groups, although mean counts were higher in neoplastic lesions.

Analysis of variance on AgNORs

In all ten diagnostic groups, statistical analysis was done by one-way analysis of variance. All benign and malignant groups showed significant differences in AgNOR counts.

   Discussion Top

Nodular thyroid disease is a common clinical problem. A vast majority of nodules are nonneoplastic. The ease and safety with which material can be obtained and the low cost of the technique ensure a place for FNAC in the existing diagnostic armamentarium. The majority of thyroid nodules have a characteristic cytological pattern, allowing specific diagnosis. [11] However, FNAC has certain limitations, which restrict its use as a simple diagnostic test, especially in cases of thyroid neoplasm. In the present study, AgNOR staining was used along with assessment of cytomorphological features in FNAC of the thyroid to differentiate between benign and follicular neoplasms of the thyroid.

Gharib et al. , [3] had reviewed fine needle aspiration biopsy of the thyroid, based on pooled data of seven series. They found that 69% lesions were benign, 10% were suspicious lesions, 4% were malignant lesions and 4% were nondiagnostic. The sensitivity and specificity of FNAC were 65-98 and 72-100%, respectively. The false negative and positive rates for FNAC were 1-11 and 1-8%, respectively. Our study found the sensitivity and specificity of FNAC to be 88.23 and 94.6%, respectively, and determined that 72.4% lesions had benign etiology, 15% were suspicious and 14% lesions were malignant.

Mehrotra et al. , [12] studied cytomorphological features and the value of the silver colloidal staining method in distinguishing nonneoplastic, benign and malignant neoplasms in 140 cases. Lower AgNOR counts were recorded in cases of thyroiditis (1.375 ± 0.414) whereas follicular carcinoma had higher AgNOR counts (5.04 ± 0.52). In our study, the mean AgNOR count in colloid goiters was 3.11 ± 1.03. A lower AgNOR count was recorded in subacute thyroiditis (2.61 ± 0.82) and Hashimoto's thyroiditis (2.97 ± 0.99) similar to the results reported by Mehrotra et al. [12]

The AgNOR count of colloidal goiters was found to be statistically significant ( P = 0.01) compared to that of multinodular goiters. However, the AgNOR counts of colloid goiters was not statistically significant ( P > 0.1) compared to that of benign neoplasms, although the number was higher than the control values.

The AgNOR count of colloid goiters (4.27 ± 0.41) was found to be highly significant ( P < 0.001) compared to that of malignant lesions of the thyroid [Table - 3]. The AgNOR counts showed no cut-off point among various groups, although mean counts were higher in neoplastic lesions. Therefore, this method can be used as an additional diagnostic measure along with a combination of cytological features to categorize various lesions of the thyroid in cytology in the absence of histological studies.

   Conclusions Top

AgNOR counting in fine needle aspiration smears is a simple, sensitive and cost-effective method for differentiating benign from malignant thyroid follicular neoplasms.

   References Top

1.Crocker J, Nar P. Nucleolar organizer regions in lymphoma. J Pathol 1987;151:111-8.  Back to cited text no. 1  [PUBMED]  
2.Montironi R, Braseiseki A, Scerpelli M, Matera G, Albeste R. Value of quantitative necleolar features vis preoperative cytological diagnosis of follicular neoplasia of the thyroid, J Clin Pathol 1991;44:509-14.  Back to cited text no. 2    
3.Gharib H, Goellner JR. Fine-needle aspiration biopsy of the thyroid: an appraisal. Ann Intern Med 1993;118:282-9.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Leong AS. Editoral. Diagn Cytopathol 1995;12:142-51.  Back to cited text no. 4    
5.Karmakar T, Dey P. Role of AgNORs in diagnosis of thyroid follicular neoplasms on fine needle aspirations smears. Diagn Cytopathol 1995;2:148-9.  Back to cited text no. 5    
6.Solymosi T, Toth V, Sapi Z, Bodo M, Gal I, Csanate L. Diagnostic value of AgNORs method in thyroid cytopathology: correlation with morphometric measurements. Diagn Cytopathol 1996;14:140-4.  Back to cited text no. 6    
7.Slowinska-Klencka D, Klencki M, Popowicz B, Sporny S, Lewinski A. Multiparameters analysis of AgNOR in thyroid lesions: comparison with PCNA expression. Histopathol 2004;19:785-92.  Back to cited text no. 7    
8.Derlin J, Hsuch C, Chiehchao T, Weng H, Hung B. Thyroid follicular neoplasms diagnosed by high resolution ultrasonography with fine needle aspiration cytology. Acta Cytol 1997;41:487-91.  Back to cited text no. 8    
9.Deshpande V, Kapila K, Siva Sai K, Verma K. Follicular neoplasms of the thyroid. Acta Cytol 1997;43:369-76.  Back to cited text no. 9    
10.Augustynowicz A, Dzieciol J, Barwijecek-Maehata M, Duden J, Puchalaski Z, Suekowski S. Assessment of proliferative activity of thyroid hurthle cell tumors using PCNA, Ki-67 and AgNOR methods. Folia Histochem Cytobiol 2004;42:165-8.  Back to cited text no. 10    
11.Hashemi F, Nasseroleslamic. Comparison of argyrophillic nucleolar organizer regions in normal thyroid, nodular thyroid, nodular goiter and thyroid neoplasms. J Iran Univ Med Sci 1999;6:246-52.  Back to cited text no. 11    
12.Mehrotra A, AgarwaL PK, Chandra T. Cytopathology and AgNORs counts in fine needle aspiration cytology smears of thyroid lesion. Diagn Cytopathol 1998;19:238-43.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]

Correspondence Address:
Sarita Asotra
Flat No-5, Block No-5, Phase-III, New Shimla - 171 009, Himachal Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9371.40653

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  [Figure - 1], [Figure - 2], [Figure - 3]

  [Table - 1], [Table - 2], [Table - 3]

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