ROLE OF ARGYROPHYLIC NUCLEOLAR ORGANIZER REGION STAINING IN IDENTIFICATION OF MALIGNANT CELLS IN EFFUSION

Gill M, Singh U, Mahapatra QS, Gehlot S, Gupta V, Sen R. J Cytol 2011;28:191-5

INTRODUCTION

Pleural, peritoneal and pericardial cavities have a common embryologic mesenchymal origin forming a double layer, namely visceral and parietal layers. Presence of fluid in these cavities constitutes an effusion which can be transudate or exudate. Exudative effusion can be caused by a variety of diseases which could be inflammatory or neoplastic. Malignant tumors give rise to effusion by secondary inflammatory reaction due to circulatory disturbance or direct involvement of serous membranes by tumor invasion. The detection of malignant cells in effusion is important for clinical staging of tumor and deciding the line of treatment.

   . and moreover. but fails to allow a definitive diagnosis in a considerable number of cases which are classified as atypical. Recurrence of malignant tumor after treatment of primary tumor is an early indication of residual or resurgent malignancy. but such methods are time consuming and costly. Cytological examination of effusions helps to differentiate between benign and malignant effusions. electron microscopy and immunocytochemical studies supplement cytological evaluation of effusions. One of the common problems in effusion cytology is to distinguish reactive mesothelial cells from neoplastic cells Histochemical stains. some antigens suitable for this purpose are expressed by normal as well as reactively proliferated mesothelial cells.

21 and 22. irregularly distributed throughout the nucleus and had heterogeneity in size. 15. Quantitative and qualitative AgNOR study is more accurate in cytological smears because the whole nucleus can be assessed rather than only part of it as occurs with tissue sections. reactive or benign neoplastic cells. which appear as intranuclear black spots. Their frequency within the nuclei is significantly higher in malignant cells than in normal. Derenzini et al. They can be visualised by silver staining technique that recognizes argyrophilia-associated proteins (AgNORs).Argyrophilic nucleolar organizer regions (AgNORs)      Argyrophilic nucleolar organizer regions (AgNORs) are loops of DNA that transcribe ribosomal RNA and are located in the short arm of the acrocentric chromosomes 13. studied AgNORs in cytologic preparations of human serous effusions and found that interphasic NORs in neoplastic cells were more numerous. . 14.

Identification of NORs is a reliable method of supplementing conventional cytologic methods to differentiate malignant cells from benign.    Inflammatory reactive cells had only a few regularly clustered NORs. . Reactive mesothelial cells had regularly clustered distribution of NORs which were less in number and rather uniform in size.15 by the manual and automated method. in serous effusion showed that the AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases.09 ± 1. the mean number of AgNOR dots per nucleus was 2. Another study done by Mohanty et al. In benign reactive effusion cases. Thus. respectively.51 and 8. respectively.69 by the manual and automated method.83 ± 1.71 and 2. whereas that for malignant effusion cases was 7.48 ± 2.33 ± 0.

Samples were centrifuged at a speed of 2000 rpm for 5 minutes. Smear for Leishman staining was air dried. Cases in which long-term follow-up or histopathological correlation was not possible were excluded. malignant and atypical. Smears to be stained with H and E and AgNOR staining were quickly immersed in 95% ethanol before any drying occurred. . one each for Leishman and hematoxylin and eosin (H and E) staining and two for AgNOR staining. The cases were classified as benign.MATERIALS AND METHOD       A total of 100 cases of effusion samples were taken up for the present study which was conducted over a period of 2 years. The effusion samples of both indoor and outdoor patients of all age groups were included in the study. Four smears were prepared from sediment.

Working solution was prepared by mixing solution A and B in a proportion of 1: 2 volumes and was poured over the smears and left for 60 minutes at room temperature. Smears were again washed with distilled water and dehydrated through ascending grades of alcohol. Fifty percent aqueous silver nitrate solution was prepared by dissolving 5 g of silver nitrate in distilled water to make 10 ml solution (solution B).     AgNOR staining solution was prepared by dissolving 2 g of gelatin in 1% aqueous formic acid to make 100 ml solution at a concentration of 2% (solution A).5%. cleared with xylene and . The silver colloid formed was washed off with distilled water and the smears were counter stained with neutral red of safranin 0.

 . the cells that could be identified as benign mesothelial were omitted and only the atypical cells in each field were studied.  AgNORs were counted in 100 such cells from each sample.  In cytologically atypical effusion. malignant and atypical cases and the AgNOR counting was done.  The mean number of AgNORs per nuclei was calculated for each sample. The pattern of AgNOR dispersion and their shape was compared in benign.AgNORs were counted as black dots in the nuclei of all unequivocal malignant cells in cytologically positive effusion and proliferated mesothelial cells in cytologically negative samples.

. confidence interval and degree of freedom were calculated. P value.STATISTICAL ANALYSIS   Unpaired t-test was applied to analyze the statistical significance of difference of mean AgNOR counts in different cytomorphological categories.

A total of 100 cases of pleural. 28 malignant and 15 samples were labeled as atypical.  AgNORs were counted in 100 cells in each case and results were compared among the three cytomorphological categories (benign. 57 effusion samples were benign. malignant and atypical).  Cytomorphologically.  Fifty cases of pleural effusion. H and E and AgNOR staining. peritoneal and pericardial effusion samples were subjected to conventional Leishman.  . 47 cases of peritoneal effusion and three cases of pericardial effusion were examined.

10 were malignant and 11 were atypical. one was benign and two were malignant . 16 were malignant and four were atypical. Among the 47 peritoneal effusion samples. 27 were benign. Out of 50 pleural effusion samples. Out of three cases of pericardial effusion samples. 29 were benign.

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prominent nucleoli and multinucleation  .  Some of mesothelial cells showed features of activation like nuclear enlargement. mesothelial cells and macrophages.Cytological findings were non-specific and smears showed varying admixtures of inflammatory cells. coarsely stippled chromatin.

A giant cell is also seen . macrophages and mesothelial cells.Benign effusion-smear showing lymphocytes.

indicating that serous membranes had not been involved by malignancy and effusions were only reactive effusions revealing mesothelial cells with some of them showing features of activation. there was an underlying malignancy. along with macrophages and lymphocytes only. . but no malignant or atypical cell could be detected in Leishman and H and Estained smears. In the 16 cases.

Smears showing a group of mesothelial cells in a benign effusion .

AgNOR staining in benign effusion showing 1-2 dots/nucleus in all cell types .

1 to 2 AgNOR dots/nucleus with regular borders in activated mesothelial cells .

20 were macroscopically hemorrhagic and were proved malignant on FNAC or biopsy.MALIGNANT EFFUSIONS  Out of these 28 effusions categorized as malignant cytologically. The remaining eight cases were later found to be malignant either by pleural biopsy. . FNAC of lungs and FNAC or histopathology of the relevant lymph nodes.

Malignant effusion-smears showing malignant cells Smear showing irregular 4-5 AgNOR dots/ nucleus in malignant cells .

moreover.ATYPICAL CASES  These cases were investigated further and were proved to be malignant. the AgNORs were variably sized. distributed irregularly and had irregular borders . Number of AgNOR dots in malignant and atypical cases was higher.

Smear showing 4-5 dots/nucleus in atypical cells .Smear showing atypical cells.

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95% confidence interval of the difference of two means was from 2. it was considered to be extremely statistically significant. The degree of freedom was 70. Unpaired t-test was also applied to analyze the statistical significance of difference of mean AgNOR count in atypical and benign effusions. Unpaired t-test was applied to analyze the statistical significance of difference of mean AgNOR count in malignant and benign effusions.6144.0001.0313. 95% confidence interval of the difference of two means was from 1. The 'P' value here was also less than 0. Here.0001.0001 in the different cytomorphological categories.6887 to 2. .3856 to 2. the 'P' value was less than 0. As the P value was less than 0.

Interphasic NOR distribution can be used as a parameter to detect nucleolar abnormalities that characterize malignant cells.DISCUSSION  Diagnosis of malignancy is often difficult on pure morphologic grounds and malignant cells cannot always be differentiated from macrophages and particularly reactive mesothelial cells.  .

especially in case where a definite cytological diagnosis was difficult to make on Leishman and H and E smears.  In this study.SUMMARY The present study was done to assess the utility of AgNORs in diagnosis of benign and malignant effusion. malignant effusions and a third group consisting of atypical cases which could not be classified with certainty as to whether they were reactive mesothelial cells or malignant cells. 100 cases of serous effusion were studied comprising of benign effusions. In malignant group. the benign group consisted of cells showing 1 to 2 dots which were regular in size and shape. 3 to 5 dots were observed per cell distributed within the nucleus  .  All effusions were subjected to AgNOR staining.

 A similar study done by Palaoro et al. showed that the assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid . shape and irregular contours. the reactive mesothelial cells showed 1 to 2 dots and malignant cells showed 3 to 4 irregular dots.  In this way.The dots had variable size. clear separation could be achieved between activated mesothelial cells and malignant cells.  In atypical group.  Another study done by Asotra and Sharma substantiated the fact that AgNOR counting in fine needle aspiration smears is a simple. sensitive and cost-effective method for  .

 .  Another advantage of this technique is that it can even be done on Papanicolaou and May-GrunwaldGiemsa-stained smears after destaining.Conclusion AgNOR can be considered as an extremely useful additional diagnostic tool for cytodiagnosis of effusions. so this technique can be used for cytodiagnosis even when the extra unstained smears are not available.