European Journal of Cardiovascular Medicine

Cytological analysis of serous effusion fluids is a valuable diagnostic tool that aids in the evaluation of a wide range of pathological conditions, including infections, systemic diseases, and malignancies (1). Due to its minimally invasive nature and relatively high diagnostic yield, effusion cytology has become an essential first-line investigation, especially in cases with suspected neoplastic etiology (2). However, considerable variability in reporting practices across institutions has historically contributed to interpretational inconsistencies, affecting clinical decision-making and communication among healthcare providers (3).

To address these challenges, the International System for Reporting Serous Fluid Cytopathology (ISRSFC) was developed, offering a five-tiered framework aimed at standardizing effusion cytology reporting globally (4). This classification comprises the following categories: non-diagnostic (ND), negative for malignancy (NFM), atypia of uncertain significance (AUS), suspicious for malignancy (SFM), and malignant (MAL). Each tier is linked with a corresponding risk of malignancy (ROM), which serves as an evidence-based guide for subsequent clinical management (5). The Indian Academy of Cytologists (IAC) has also endorsed a similar approach, emphasizing uniformity in reporting and promoting cytohistological correlation (6).

Preliminary studies applying the ISRSFC have demonstrated its practicality and diagnostic utility in various clinical settings, with reported ROMs aligning reasonably well across different institutions (7,8). Nonetheless, further institutional experiences are essential to validate its applicability and assess category-specific diagnostic accuracy in diverse patient populations.

This study aims to evaluate the feasibility of implementing the ISRSFC and IAC guidelines in the routine cytological assessment of serous effusion samples at a tertiary care center. Additionally, we aim to determine the ROM associated with each diagnostic category and to explore the spectrum of underlying malignancies through clinico-pathological correlation.

A total of 784 effusion samples from pleural, peritoneal, pericardial, cranium- spine and joint cavities were included from a period of January 2022 to December 2023 Cases were selected based on the availability of complete clinical data and adequate cytological smears. Samples with insufficient cellularity or lacking relevant patient information were excluded. All specimens were received fresh and processed on the same day of collection.

Cytological Processing

Fluid samples were subjected to centrifugation at 2500 rpm for 10 minutes. The resultant cell pellet was used to prepare conventional smears, which were stained using Diff-Quik and Hematoxyllin- Eosin(H-E). In selected cases, cell blocks were prepared using plasma-thrombin or formalin-based techniques to enhance diagnostic yield. Immunohistochemistry (IHC) was performed when required to confirm the primary site of malignancy.

Classification and Diagnostic Categorization

All cases were retrospectively reviewed and categorized according to the five-tier diagnostic framework proposed by the International System for Reporting Serous Fluid Cytopathology (ISRSFC), which includes:

1. Non-diagnostic (ND)
2. Negative for malignancy (NFM)
3. Atypia of uncertain significance (AUS)
4. Suspicious for malignancy (SFM)
5. Malignant (MAL)

Two experienced cytopathologists independently reviewed the slides to reduce interobserver variability. In case of discrepancy, a consensus diagnosis was reached after joint re-evaluation.

Clinicopathological Correlation

Clinical history, radiological imaging findings, and, where available, histopathological diagnoses were reviewed for correlation. These parameters were used to assess the final diagnosis and confirm the presence or absence of malignancy.

Risk of Malignancy (ROM) Assessment

ROM was calculated for each ISRSFC category by determining the proportion of cases within that category that had histologically or clinically confirmed malignancy.

• (b)

Figure 1. CATEGORY 1  Non Diagnostic shows (a) haemorrhage  (HE 40)X (b) degenerated cells Diff-Quik 40X

(a)                                                                                     (b)

Figure 2. CATEGORY 2 Negative for malignancy shows (a) lymphocytes  (HE 40X) (b) reactive mesothelial cells Diff-Quik 40X

(a )                                                                                        ( b)

Figure 3. CATEGORY 3 Atypia of undetermined significance (a) & (b) show occasional cluster of atypical cells (Diff-Quik 40X)

                  (a)                                                                                   (b)

Figure 4. CATEGORY 4 Suspicious for malignancy shows (a) &(b)Atypical cells with enlarged, hyperchromatic nucleus with irregular nuclear membrane  (HE 40X)

(a)                                                                                                          (b)

Figure 5. CATEGORY  5 Malignant (mal) shows (a) &(b) tumour cells in three dimensional clusters (Diff-Quik 40X)and in sheets (HE 40X)

Statistical Analysis

Descriptive statistics were used to summarize demographic and clinical data. The ROM for each diagnostic category was expressed as a percentage. Data were analyzed using Microsoft Excel and SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Inter-category comparison was performed where applicable, and p-values <0.05 were considered statistically significant.

A total of 784 serous effusion samples were analyzed, consisting of 395 (50.38%) were pleural, 289 (36.8%) peritoneal, 39 (4.9%) pericardial, 37 (4.6%) CSF and 24 (3.06%) synovial fluid (Table 3). The patients included 416 females (53.1%) and 368 males (46.9%), with a mean age of 52.8 ± 15.4 years, ranging from 1 to 88 years.

Distribution According to ISRSFC Categories

When classified according to the ISRSFC guidelines, the majority of cases (n = 532, 67.9%) were reported as negative for malignancy (NFM), followed by malignant cases (n = 156, 19.9%). Other categories included non-diagnostic (ND) in 21 cases (2.7%), atypia of uncertain significance (AUS) in 28 cases (3.6%), and suspicious for malignancy (SFM) in 47 cases (6.0%) (Table 1).

Table 1. Distribution of effusion cases according to ISRSFC diagnostic categories

Risk of Malignancy (ROM)

Histopathological or clinico-radiological follow-up was available for 368 cases. The calculated ROM for each diagnostic category was as follows: ND – 14.2% (3/21), NFM – 18.6% (44/237), AUS – 51.4% (18/35), SFM – 75.9% (22/29), and MAL – 93.5% (43/46) (Table 2). These values indicate an increasing trend of ROM with higher cytological suspicion categories, validating the utility of the ISRSFC system in malignancy risk stratification (Table 2).

Table 2. Risk of malignancy (ROM) across ISRSFC categories

Table 3. Distribution of different body fluid

Malignancy Patterns

Among the malignant cases, lung carcinoma was the predominant primary tumor identified in pleural effusions (n = 66), while ovarian carcinoma accounted for most peritoneal malignancies (n = 38). Other identified primaries included breast, gastrointestinal, and pancreatic cancers. Cell block preparations were performed in 84 cases and contributed to definitive diagnosis in 72 (85.7%) of them.

These findings highlight the diagnostic reliability and clinical relevance of the ISRSFC classification in routine effusion cytology practice (Tables 1 and 2).

Effusion cytology remains a cornerstone in the initial diagnostic work-up of patients presenting with serous fluid accumulation, particularly in malignancy evaluation due to its minimally invasive nature and rapid turnaround time (1). However, inconsistent reporting terminology across institutions has posed challenges in ensuring diagnostic reproducibility and guiding appropriate clinical management (2,3). The introduction of the International System for Reporting Serous Fluid Cytopathology (ISRSFC) has addressed this gap by offering a standardized five-tier classification system with defined cytological criteria and risk of malignancy (ROM) for each category (4).

In our study, the most frequently reported category was “Negative for Malignancy” (67.9%), followed by “Malignant” (19.9%). This distribution is consistent with previous studies by Lobo C et al. and Sun T et al., where NFM and MAL constituted the bulk of effusion diagnoses (5,6). The non-diagnostic category in our cohort accounted for 2.7%, which falls within the acceptable range reported in the literature (1–5%) and reflects good sample quality and smear preparation techniques (7).

The ROM observed across categories followed the expected ascending trend: 14.2% in ND, 18.6% in NFM, 51.4% in AUS, 75.9% in SFM, and 93.5% in MAL. These values closely align with those reported by Kala C et al. and Bharti S et al., thereby supporting the clinical reliability of the ISRSFC risk stratification framework (8,9). The high ROM in the malignant category underscores the robustness of cytological criteria for identifying frank malignancy in serous fluids (10).

The atypia of uncertain significance (AUS) and suspicious for malignancy (SFM) categories remain areas of diagnostic challenge due to overlapping morphological features and paucicellular samples. In our study, the ROM for AUS was 51.4%, suggesting that this category warrants careful follow-up and adjunctive testing such as cell block and immunocytochemistry (11). The suspicious category demonstrated a ROM of 75.9%, which is consistent with findings by Mandava H et al., indicating that most SFM cases eventually turn out to be malignant upon histological confirmation (12).

Lung carcinoma was the most common primary site associated with pleural effusion, while ovarian carcinoma predominated in peritoneal fluid, which aligns with the findings of several institutional studies conducted in both oncological and general hospital settings (13,14). The utility of ancillary techniques such as cell block and immunohistochemistry was evident in our study, as 85.7% of cell block preparations contributed to a definitive diagnosis, supporting previous reports advocating their use in challenging cases (15).

Despite the strengths of the ISRSFC system, challenges remain in cases with low cellularity, mixed inflammatory backgrounds, or poorly differentiated malignancies. Integration of clinical and radiologic findings, in conjunction with cytopathological features, remains vital for comprehensive diagnosis. The reclassification of archived effusion samples in our study revealed improved clarity in reporting and enhanced communication with treating clinicians.

In conclusion, our findings affirm the applicability and diagnostic value of the ISRSFC guidelines in routine effusion cytology. The risk-stratified approach provided by the five-tier system not only enhances interobserver consistency but also aids in predicting the likelihood of malignancy, thereby informing clinical decision-making.

1. Kolte S, Zaheer S, Aden D, Ranga S. Application of the international system for reporting serous fluid cytopathology on reporting various body fluids; experience of a tertiary care hospital. Cytojournal. 2022 Sep 5;19:52. doi: 10.25259/Cytojournal_49_2021. PMID: 36128470.
2. Rajeswaran PK, Srinivasan V, Mahesh SSV, Ashok AU. A retrospective analysis of the application of the newly proposed International System for Reporting Serous Fluid Cytopathology on serous effusion specimens: An institutional experience. Acta Cytol. 2023;67(1):70–9. doi: 10.1159/000527398. PMID: 36476509.
3. Rodriguez EF, Jones R, Gabrielson M, Santos D, Pastorello RG, Maleki Z. Application of the International System for Reporting Serous Fluid Cytopathology (ISRSFC) on reporting pericardial effusion cytology. Acta Cytol. 2020;64(5):477–85. doi: 10.1159/000507311. PMID: 32422631.
4. Ahuja S, Malviya A. Categorisation of serous effusions using the International System for Reporting Serous Fluid Cytopathology and assessment of risk of malignancy with diagnostic accuracy. Cytopathology. 2022 Mar;33(2):176–84. doi: 10.1111/cyt.13089. PMID: 34913541.
5. Lobo 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. 2021 May;49(5):596–605. doi: 10.1002/dc.24440. PMID: 32339444.
6. Sun T, Wang M, Wang H. Risk of malignancy assessment of the International System for Reporting Serous Fluid Cytopathology: Experience in a community hospital setting and comparison with other studies. Cancer Cytopathol. 2022 Dec;130(12):964–73. doi: 10.1002/cncy.22638. PMID: 35994357.
7. Sachan R, Gupta A, Awasthi PN, Singh P, Anand N, Chandra S, et al. Application of international system for reporting serous fluid cytology (ISRSFC) in effusion samples-a prospective study in an oncology setting. J Am Soc Cytopathol. 2023 Sep-Oct;12(5):351–61. doi: 10.1016/j.jasc.2023.04.005. PMID: 37244848.
8. Kala C, Kala S, Singh A, Jauhari RK, Bajpai A, Khan L. The International System for Reporting Serous Fluid Cytopathology: An institutional experience on its implication and assessment of risk of malignancy in effusion cytology. J Cytol. 2023 Oct-Dec;40(4):159–64. doi: 10.4103/joc.joc_111_22. PMID: 38058672.
9. Bharti S, Nalwa A, Elhence PA, Rao M, Bharti JN, Khera S, et al. Risk stratification of pleural fluid cytology based on the International System for Reporting Serous Fluid Cytology in a tertiary care centre. Acta Cytol. 2022;66(5):449–56. doi: 10.1159/000524144. PMID: 35413720.
10. Kundu R, Srinivasan R, Dey P, Gupta N, Gupta P, Rohilla M, et al. Application of Indian Academy of Cytologists guidelines for reporting serous effusions: An institutional experience. J Cytol. 2021 Jan-Mar;38(1):1–7. doi: 10.4103/JOC.JOC_224_20. PMID: 33935385.
11. Alashetty S, Sadasivan B, Dharmalingam P, Rajagopal N, Kavya L, Pai MM. The role of novel tiered reporting system in serous fluid cytology and risk of malignancy assessment: A retrospective study in a tertiary care center. J Cytol. 2023 Jul-Sep;40(3):107–13. doi: 10.4103/joc.joc_107_22. PMID: 37745807.
12. Mandava H, Venkata Renuka I, Potti R, Mounica B, Kalla I. Deciphering serous effusions using the new International System for Reporting Serous Fluid Cytopathology. Cureus. 2024 May 10;16(5):e60042. doi: 10.7759/cureus.60042. PMID: 38774462.
13. Wang M, Sun T, Jiao J, Wang H. Application of the International System for Reporting Serous Fluid Cytopathology to pericardial fluid: Root cause analysis of indeterminate diagnoses, cytohistological correlation, and assessment of malignancy risk. Cancer Cytopathol. 2023 Jul;131(7):433–41. doi: 10.1002/cncy.22696. PMID: 36973963.
14. Jha S, Sethy M, Adhya AK. Application of the International System for Reporting Serous Fluid Cytopathology in routine reporting of pleural effusion and assessment of the risk of malignancy. Diagn Cytopathol. 2021 Oct;49(10):1089–98. doi: 10.1002/dc.24837. PMID: 34289263.
15. Yang H, Zhu J, Wang P. Application of the International System for Reporting Serous Fluid Cytopathology (ISRSFC) in reporting serous effusion: A retrospective study. Medicine (Baltimore). 2023 Oct 27;102(43):e35707. doi: 10.1097/MD.0000000000035707. PMID: 37904355.