Journal of Cytology

: 2021  |  Volume : 38  |  Issue : 1  |  Page : 1--7

Application of Indian Academy of cytologists guidelines for reporting serous effusions: An institutional experience

Reetu Kundu1, Radhika Srinivasan1, Pranab Dey1, Nalini Gupta1, Parikshaa Gupta1, Manish Rohilla1, Shruti Gupta1, Amanjit Bal2, Arvind Rajwanshi1,  
1 Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Dr. Radhika Srinivasan
Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012


Background: Recently, the Indian Academy of Cytologists (IAC) has published the guidelines for interpretation and reporting of serous effusions. Till date, there are no studies on its applicability. Aims: The present study was carried out to assess the feasibility of applying the IAC reporting categories to effusions, determine the frequency, and provide an estimate of the risk of malignancy (ROM) for individual diagnostic categories. Materials and Methods: All cases of serous effusion fluids reported in the year 2019 were retrieved from the archives and reassigned as per the IAC diagnostic categories. The clinical and histopathological follow-up information was obtained wherever possible. Results: A total of 1340 effusion samples were received from 1085 patients. There were 561 (51.7%) males and 524 (48.3%) females. Majority were pleural (1066, 79.5%), followed by peritoneal (187, 14%) and pericardial (87, 6.5%) effusions. The age ranged from 7 months to 92 years. There were 35 (2.6%) samples in category 1 (non-diagnostic), 954 (71.2%) in category 2 (benign), 17 (1.3%) in category 3 (atypical), 59 (4.4%) in category 4 (suspicious for malignancy) and 275 (20.5%) in category 5 (malignant). The estimated ROM in serous effusion samples was 20% for category 1, 16.7% for category 2, 50% for category 3, 94.4% for category 4 and 100% for category 5. Conclusions: The categorization of serous effusion cytology samples as per the IAC diagnostic categories and as per the reporting format developed by the IAC is feasible and the management recommendations are mostly appropriate.

How to cite this article:
Kundu R, Srinivasan R, Dey P, Gupta N, Gupta P, Rohilla M, Gupta S, Bal A, Rajwanshi A. Application of Indian Academy of cytologists guidelines for reporting serous effusions: An institutional experience.J Cytol 2021;38:1-7

How to cite this URL:
Kundu R, Srinivasan R, Dey P, Gupta N, Gupta P, Rohilla M, Gupta S, Bal A, Rajwanshi A. Application of Indian Academy of cytologists guidelines for reporting serous effusions: An institutional experience. J Cytol [serial online] 2021 [cited 2021 Feb 27 ];38:1-7
Available from:

Full Text


Cytological evaluation is often the first line of investigation in the clinical setting of serous effusions.[1] Effusions may be caused by a wide variety of stimuli and not uncommonly are harbingers of an underlying malignancy.[2] Accurate identification and typing of tumor cells in effusion samples serves as a guide to patient management. On the other hand, exclusion of malignancy allows for appropriate management in case of non-malignant effusions.

Established international reporting terminology systems are already in place for cervical,[3] urinary tract,[4] thyroid,[5] salivary gland,[6] pancreatico-biliary,[7] and pulmonary cytology.[8] As of now, there is absence of a globally accepted and widely used reporting terminology system for serous effusion cytology samples. Not long ago, the international system for reporting serous fluid cytopathology was announced.[9] In the beginning of this year, 2020, the Indian Academy of Cytologists (IAC) published guidelines for collection, preparation, interpretation, and reporting of serous effusion fluid samples with the vision of attaining uniformity across all laboratories, ensuring good cytopathology practice and implementing a standard reporting format with same recommendations in similar context.[10] After cytological evaluation the case is placed into any of the five recommended categories (Category 1, Unsatisfactory for evaluation; Category 2, No malignant cells detected/benign cellular changes; Category 3, Atypical cells, NOS; Category 4, Atypical cells, suspicious for malignancy; Category 5, Malignant cells seen).

The present study was carried out to assess the feasibility and utility of application of categorization of effusion cytology samples into the various diagnostic categories recommended by the IAC.

 Materials and Methods

All cases of serous cavity effusion fluids received in the Department of Cytology from January- December 2019 were retrieved. The study was carried out in accordance with the standards laid down in the Helsinki Declaration. Relevant details were noted from the sample requisition forms. Medical records were searched for the clinical, radiological, and histopathological follow-up. Cases with no hospital record of malignancy in one year were presumed to have a benign course. An attempt was made to categorize cases as per the recent IAC Guidelines for interpretation and reporting of serous effusion samples.[10]

As per local practice, most effusion samples were received already anticoagulated using 1% ammonium oxalate or heparin. The sample was processed by centrifugation and sediment smears prepared, one of which was stained by May-Grünwald Giemsa (MGG) and second smear was kept unstained for subsequent special staining such as Ziehl-Neelsen and periodic acid Schiff staining if required. The remaining sediment was fixed in Cytorich red for liquid based cytology (LBC) (Surepath®, Becton-Dickinson, USA) processing and one smear was made and stained by the Papanicolaou method. Thus, in each case, 2 smears, 1 air-dried MGG stained, and 1 Papanicolaou stained LBC smear was examined. After processing, the left-over sample was stored in the refrigerator at 2–8°C until the case was reported by the cytopathologist. Following the initial reporting, cell blocks (CB) were prepared the next day by centrifugation of more fluid (minimum 30 mL with maximum 100 mL) wherever available and obtaining cell sediment. In cases, with lesser amounts of initial sample, the residual material, if any, after preparation of LBC smear was converted into a CB. CB were made by the plasma-thromboplastin clot method and immunocytochemistry (ICC) for tumor typing was done wherever required.

Upon request from the treating clinician in cases of lung cancer, testing for epidermal growth factor receptor (EGFR) driver mutations in exons 18, 19, 20, and 21 was done by real-time polymerase chain reaction (PCR)-based method. Testing for anaplastic lymphoma kinase (ALK) and rat osteosarcoma (ROS1) rearrangements by ICC on CBs was done using D5F3 and clone D4D6 Ventana automated immunohistochemistry assay, respectively.

Routinely, the initial cytomorphological evaluation was carried out by light microscopy and based on the interpretation and sign-out, each case was categorized into any of the 5 recommended diagnostic IAC categories.


A total of 1340 serous effusion cytology samples were received from 1085 patients over a period of one year. Overall, there were 561 (51.7%) male and 524 (48.3%) female patients. Majority of the effusions were pleural effusions (1066, 79.5%), followed by peritoneal (187, 14%) and pericardial (87, 6.5%) effusions. The ages ranged from 7 months to 92 years with mean age of 49.07 ± 19.07 years. Based on the cytodiagnosis, all cases were assigned to the IAC diagnostic categories [Table 1].{Table 1}

IAC effusion Diagnostic categories

Category 1, Non-diagnostic

Out of 35 total cases in category 1, majority (30/35) were pleural effusions. Clinical/histological follow up information was available in 5 cases. Precursor T-cell lymphoblastic lymphoma was diagnosed in 1 pleural effusion case whereas the outcome in the remaining 4 unsatisfactory peritoneal effusions was either benign or no evidence of malignancy.

Category 2, Benign

A total of 954 (71.2%) cases were placed in category 2, making it the most common cause of effusion irrespective of the site of effusion. They were further sub-categorized as reactive mesothelial proliferation (467, 49%), acute inflammation (82, 8.6%), chronic inflammation (230, 24.1%), lymphocyte rich effusion (171, 17.9%), and specific infections (4, 0.4%). Specific infections identified included tuberculosis [Figure 1], aspergillosis and nocardiosis in a small number of cases. In 9 cases with the first sample reported as negative for malignant cells, subsequent samples evaluated from the same patient were assigned to category 4 (2 cases) and 5 (7 cases). All these cases had a malignant outcome on follow-up. The remaining 45 cases in category 2 had benign outcome.{Figure 1}

Category 3, Atypical

A total of 17 (1.3%) cases were placed in category 3 [Figure 2] and 15/17 cases were pleural effusions. Follow up was available in 4 cases, 2 of which had malignant outcome. Pleural biopsy revealed adenocarcinoma in one case while the second showed ovarian high grade serous carcinoma with omental metastasis.{Figure 2}

Category 4, Suspicious of malignancy

A total of 59 (4.4%) cases were in category 4. Follow-up was available in 18 cases with 17 having a malignant outcome while one was benign. Of these 17 cases, 9 were pleural effusions with primary in breast (3 cases), unknown primary (2 cases), and 1 case each of squamous cell carcinoma lung, small cell carcinoma lung, and head and neck squamous cell carcinoma and Non-Hodgkin lymphoma of duodenum. Pleural biopsy done in one case did not reveal malignancy. Peritoneal effusion in 5/17 cases showed malignancy on follow-up (ovary, serous carcinoma high grade, 3 cases; Krukenberg tumor, 1; uterine carcinosarcoma, 1). Again, 3 cases with pericardial effusion revealed metastatic adenocarcinoma from unknown primary in 2 cases while biopsy from mediastinal mass showed, T- lymphoblastic lymphoma in one case.

Category 5, Malignant

A definite diagnosis of malignancy in effusions was made in 275 (20.5%) serous effusions with 225 pleural, 17 pericardial, and 33 peritoneal effusions. The type of malignancy detected is shown in [Table 2]. Clinical and/or histopathological follow up was available in 72 cases of pleural effusion. The most common primary site of origin was the lung primary seen in 45 cases (adenocarcinoma, 36; squamous cell carcinoma, 3; small cell carcinoma, 4; non-small cell carcinoma, 2). Testing for EGFR driver mutations was performed by RT-PCR in 27/36 using the direct fluid cell sediment or residual material after LBC processing. A total of 21/27 (77.8%) cases showed positivity for EGFR mutations with exon 19 deletion in 12, L858R exon 21 mutation in 4, dual exon 19 deletion/exon 20 T790M mutation in 2, dual exon 21 L858R/exon 20 insertion in 1 and exon 20 T790M mutation and exon 20 insertion in 1 case, respectively. A representative case with EGFR exon 19 deletion in the pleural fluid metastases of lung adenocarcinoma is illustrated in [Figure 3]. Six out of 27 cases were negative for all the known mutations in exons 18 to 21 of EGFR gene. Immunocytochemistry for ALK (D5F3 clone) and ROS1 ICC was done in 21 cases, while ALK ICC alone was done in 4 cases. Only one case showed 3 + staining on ALK ICC. In 6 cases of pleural effusion, ovarian origin of the primary was confirmed.{Figure 3}{Table 2}

In peritoneal effusions, metastatic adenocarcinoma (25/33 cases) was most common and ovary the most common primary site detected on follow-up (20/25) [Table 2]. In these cases, the panel for immunocytochemistry consisted of PAX8, WT1, p53, CK7, and CK20. Positivity for PAX8, WT 1 and mutant p53 staining pattern confirmed high grade serous carcinoma of tubal/ovarian origin in 17/20 cases and a representative case is illustrated in [Figure 4]. The remaining cases represented peritoneal involvement in borderline serous tumour ovary in 1 case and low-grade serous carcinoma ovary in 2 cases, respectively. Pericardial effusions showed both metastatic adenocarcinoma and lymphoma infiltration. All the lymphomas were of thymic origin T-lymphoblastic lymphomas.{Figure 4}

Repeat cytology evaluation

Out of 1340 serous effusion cytology samples received, 265 were repeat samples. Upon cytological evaluation of the repeat samples a shift in category was seen in 31 cases (11.7%). Out of 7 pleural effusions initially reported as unsatisfactory (category 1), repeat cytological evaluation was diagnostic and 6 cases were put in category 2 and 1 proved to be a case of Precursor T-cell lymphoblastic lymphoma. In 9 cases, where the initial sample was reported as category 2 (benign effusion), repeat samples evaluated showed upgradation to category 4 in 2 cases and to category 5 in 7 cases. For cases placed in category 3, atypical cells, NOS a repeat sample was obtained in 6 cases (5 pleural and 1 pericardial). 3 cases were downgraded to category 2, while 3 cases were upgraded to category 5. In category 4, suspicious for malignancy a repeat effusion sample for cytology was received in 9 cases (8 pleural and 1 pericardial) and all the cases shifted to category 5. Representative cases are illustrated in [Figure 5]. Out of 265 repeat samples, no change in category was observed in 234 samples (category 2, 196 cases; category 4, 2 cases; category 5, 36 cases).{Figure 5}

Discordant case- “False” false positive case

Pleural effusion received from a 52-year-old female was reported as suspicious for malignancy (Category 4). The smears were highly cellular and showed papillaroid clusters of cells with moderate cytoplasm, coarse vesicular chromatin, prominent nucleoli, and many mitotic figures [Figure 6]a and [Figure 6]b. Many showed windows and cytoplasmic blebbing. Cell block- ICC on the pleural fluid sediment revealed tumour cells negative for MOC31 [Figure 6]c (excluding adenocarcinoma), CK7, CK20, TTF1, napsin A, p63 and WT1 and calretinin [Figure 6d] (excluding mesothelial origin). Possibility of carcinoma or mesothelioma could not be confirmed on ICC and the morphological diagnosis was retained upon review. Pleural biopsy showed a few reactive mesothelial cells and was reported as negative for malignancy [Figure 6f]. Follow-up revealed that the patient died of disease after 4 months. Hence, case was reviewed again and at this time, D2-40 ICC performed revealed strong diffuse membranous positivity [Figure 6e] confirming a diagnosis of mesothelioma.{Figure 6}

Estimated risk of malignancy

Based on the follow-up information available as per clinical record or histopathology, the estimated risk of malignancy (ROM) in serous effusion samples was calculated and results shown in [Table 3]. The upper bound estimate of ROM was 20% for category 1 (non-diagnostic), 16.7% for category 2 (benign), 50% for category 3 (atypical), 94.4% for category 4 (suspicious for malignancy), and 100% for category 5 (malignant). It is pertinent here to note that the ROM for category 2 is 0 if based on the initial category 2 report alone and 16.7% based on the initial category 2 report and which was subsequently upgraded to category 4/5.{Table 3}


Cytological evaluation of body cavity fluids is an integral part of day-to-day cytopathology practice. In the present study, categorization of effusion samples into 5 diagnostic categories was done as per the recommendations of IAC.[10]

Thirty-five effusion samples (2.6%) were non diagnostic (category 1). These samples were either less in volume (<10 ml), showed bacterial contamination, or were not optimally anticoagulated and clotted or had excess of anticoagulant leading to formation of crystals. Repeat evaluation was advised in all these cases along with proper instructions for sample collection and transportation.[10]

Majority (954/1340, 71%) of the effusions were placed in category 2. The etiologic factors for benign serous effusions are organ failure, autoimmune diseases, infectious diseases, cirrhosis, hypoalbuminemia, peritoneal dialysis, etc.[11] Serous effusions in category 2 were further sub-categorized into reactive mesothelial proliferation (467, 49%), acute inflammation (82, 8.6%), chronic inflammation (230, 24.1%), lymphocyte rich effusion (171, 17.9%), and specific infections (4, 0.4%). Sub-categorization often provides a lead to the etiology. Reactive mesothelial proliferation was seen in nearly half of the benign effusions. A wide variety of stimuli cause proliferation, marked morphological alterations in mesothelial cells and that may pose diagnostic difficulties.[12] Worldwide tuberculosis is an important cause of lymphocyte predominant pleural effusion.[13] This represents a delayed hypersensitivity reaction to the tubercular antigen. However, rarely, there is direct extension of pulmonary disease into the pleural cavity as was seen in 2 cases in this series with AFB positivity demonstrated by the Ziehl-Neelsen stain. One case each of aspergillosis and nocardiosis was also seen which again represents spread of pulmonary disease into the pleural space. In all these cases, there was necrotizing inflammation and hence, the learning practice point is that special stains for identification of organisms should be requested in effusion smears with necrotizing inflammation. The upper bound limit of ROM of category 2 was 16.7% based on malignant outcome in 9 cases which were reported benign (category 2) initially but upon repeat evaluation were upgraded to category 4/5. Hence, the recommendation for this category which currently reads as “corelate clinically, and to imaging and microbiological studies” may be modified and also include a repeat cytology evaluation if there is a suspicion of malignancy on clinical/imaging grounds.

Repeat cytological evaluation was very useful in other categories as well. Thus, the 265 (19.7%) repeat samples were present across all categories and mostly carried out in cases with a high clinical suspicion for malignancy or when a second sample was requested for cell block and immunocytochemistry. A shift in category was seen in 31 (11.6%) cases. Category 3- atypical cells, NOS and category 4- atypical cells, suspicious for malignancy described as preliminary “holding” categories have a potential to move into clear cut benign and malignant categories on repeat sampling and/or ancillary testing.[14] Thus the recommendation of a repeat cytology in category 1, 3, and 4 seems very justified.

Any category 4 report should be viewed seriously by the clinician and taken as malignant until proven otherwise as highlighted in our study by the discordant “False” false positive case wherein the cytology report was suspicious for malignancy and pleural biopsy was reported as negative for malignancy but subsequently proven to be malignant mesothelioma by D2-40 immunocytochemistry is highlighted [Figure 5]. Thus, in all category 4 reports, a good correlation to the imaging findings is advocated and if the initial small biopsy and histopathology is reported benign, a repeat biopsy is strongly indicated. The importance of using an extended panel of immunocytochemistry for confirming mesothelioma cannot be overstated.

An unequivocal malignant diagnosis was rendered in 275 (21%) cases which were category 5. Amongst these malignant effusions, pleural effusions constituted 21.1%, followed by pericardial (19.5%) and peritoneal (17.6%) effusions. Our results are at variance with the malignancy rates observed in effusions in a recent study wherein malignant pericardial effusions constituted 56.2% followed by peritoneal samples (33%) and pleural effusions (31.9%).[14] It is possible that infections like tuberculosis in our country could have led to higher rates of non-malignant effusions. The most common malignancies in pleural effusion were from lung and breast as observed in other studies.[15],[16] Malignant pleural/peritoneal effusion from an ovarian primary were confirmed as high grade serous carcinoma by cell-block immunocytochemistry using a panel comprising CK7, WT1, PAX8 and p53. A minimum panel of PAX8, WT1, and p53 allowing for specific pre-neoadjuvant chemotherapy diagnoses of ovarian high grade serous carcinoma in effusions is advocated by Bansal et al.[17] All our cases received neoadjuvant chemotherapy following immunophenotyping as high grade serous carcinoma of ovarian origin.

Testing for EGFR, ROS1, and ALK mutations in all patients with advanced non-small cell lung cancer with an adenocarcinoma component is now essential as per the latest guidelines from the International Association for the Study of Lung Cancer.[18] Testing for EGFR driver mutations in effusion sediments is feasible and was very rewarding as 77.8% cases had targetable molecular alterations.

The present study emphasizes the need for classifying effusion cytology samples into various diagnostic categories using standardized terminologies which allow for uniformity across various laboratories. For certain organs like the thyroid, the cytological diagnosis can be compared with the subsequent histopathology, available in many cases. However, in case of effusion cytology, histopathology is not necessarily available in majority of the cases and hence the difficulty in cytohistological correlation making the evaluation of performance parameters problematic. Even when the histopathology is available, the primary tumor site may still remain unproven. Hence, clinical, radiological, and histopathological follow-up should all be taken into account for determination of accuracy of cytological diagnosis in effusion samples. Obtaining follow-up information can be very challenging in our setup; however, this was taken into account to calculate the estimated ROM. Thus, the upper bound limit of ROM was 20% for category 1, 16.7% for category 2, 50% for category 3, 94.4% for category 4 and 100% for category 5. In a recent meta-analysis on serous effusion cytology, the mean ROM for cytology diagnoses of nondiagnostic, negative for malignancy, atypia of uncertain significance, suspicious for malignancy and malignant were 17.4%, 20.7%, 65.9%, 81.8%, and 98.9%, respectively.[19] Recently, the international system for reporting serous fluid cytology (TIS) was developed with the efforts of International Academy of Cytology and the American Society of Cytopathology which is identical with the IAC diagnostic categories of reporting.[20] It is hoped that there will be more such studies from across the country to enable a meta-analysis which can refine the management strategy in these IAC/International diagnostic categories.

To conclude, the categorization of serous effusion cytology samples as per the reporting format developed by the IAC which is in line with the international system is feasible and recommendations appropriate for the different diagnostic categories. Standardization in interpretation and reporting terminologies with negligible interobserver variation also ensures an accurate cytological diagnosis, which opens the door for appropriate clinical patient care and management.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Gupta S, Sodhani P, Jain S. Cytomorphological profile of neoplastic effusions: An audit of 10 years with emphasis on uncommonly encountered malignancies. J Cancer Res Ther 2012;8:602-9.
2Hanselaar AGJM. Additional techniques in serous effusions. Anal Cell Pathol 2002;24:1-4.
3Nayar R, Wilbur DC. The Bethesda system for reporting cervical cytology: A historical perspective. Acta Cytol 2017;61:359-72.
4Barkan GA, Wojcik EM, Nayar R, Savic-Prince S, Quek ML, Kurtycz DF, et al. The Paris system for reporting urinary cytology: The quest to develop a standardized terminology. Acta Cytol 2016;60:185-97.
5Cibas ES, Ali SZ. The 2017 Bethesda system for reporting thyroid cytopathology. Thyroid 2017;27:1341-6.
6Rossi ED, Faquin WC, Baloch Z, Barkan GA, Foschini MP, Pusztaszeri M, et al. The Milan system for reporting salivary gland cytopathology: Analysis and suggestions of initial survey. Cancer Cytopathol 2017;125:757-66.
7Pitman MB, Layfield LJ. Guidelines for pancreaticobiliary cytology from the Papanicolaou society of cytopathology: A review. Cancer Cytopathol 2014;122:399-411.
8Layfield LJ, Baloch Z, Elsheikh T, Litzky L, Rekhtman N, Travis WD, et al. Standardized terminology and nomenclature for respiratory cytology: The Papanicolaou society of cytopathology guidelines. Diagn Cytopathol 2016;44:399-409.
9Chandra A, Crothers B, Kurtycz D, Schmitt F. Announcement: The international system for reporting serous fluid cytopathology. Acta Cytol 2019;63:349-51.
10Effusion guidelines committee of IAC guidelines drafting and finalization committee, Srinivasan R, Rekhi B, Rajwanshi A, Pathuthara S, Mathur S, et al. Indian academy of cytologists guidelines for collection, preparation, interpretation, and reporting of serous effusion fluid samples. J Cytol 2020;37:1-11.
11Mercer RM, Corcoran JP, Porcel JM, Rahman NM, Psallidas I. Interpreting pleural fluid results. Clin Med (Lond) 2019;19:213-7.
12Davidson B. Malignant effusions: From diagnosis to biology. Diagn Cytopathol 2004;31:246-54.
13Vorster MJ, Allwood BW, Diacon AH, Koegelenberg CFN. Tuberculous pleural effusions: Advances and controversies. J Thorac Dis 2015;7:981-91.
14Lobo C, Costa J, Petronilho S, Monteiro P, Leça L, Schmitt F. Cytohistological correlation in serous effusions using the newly proposed international system for reporting serous fluid cytopathology: Experience of an oncological center. Diagn Cytopathol 2020. doi: 10.1002/dc. 24440.
15 Froudarakis ME, Plojoux J, Kaspi E, Anevlavis S, Laroumagne S, Karpathiou G, et al. Positive pleural cytology is an indicator for visceral pleural invasion in metastatic pleural effusions. Clin Respir J 2018;12:1011-6.
16Awadallah SF, Bowling MR, Sharma N, Mohan A. Malignant pleural effusion and cancer of unknown primary site: A review of literature. Ann Transl Med 2019;7:353.
17Bansal A, Srinivasan R, Rohilla M, Sundaram A, Rai B, Rajwanshi A, et al. Morphologic and immunocytochemical features of high-grade serous carcinoma of ovary in ascitic fluid effusion and fine-needle aspiration cytology. Am J Clin Pathol 2020;154:103-14.
18Jain D, Roy-Chowdhuri S. Molecular pathology of lung cancer cytology specimens: A concise review. Arch Pathol Lab Med 2018;142:1127-33.
19Farahani SJ, Baloch Z. Are we ready to develop a tiered scheme for the effusion cytology? A comprehensive review and analysis of the literature. Diagn Cytopathol 2019;47:1145-59.
20Pinto D, Chandra A, Crothers BA, Kurtycz DFI, Schmitt F. The international system for reporting serous fluid cytopathology-diagnostic categories and clinical management. J Am Soc Cytopathol 2020;9:469-77.