Introduction

Serous fluids from the pleural, peritoneal, and pericardial cavities constitute majority of the non-gynaecological samples being submitted to any cytology laboratory. [1] They reflect a wide array of aetiologies such as infection, injury/trauma, mechanical obstruction, metabolic diseases, and malignancy. [2] Around 10% to 25% of these effusions are caused by malignancy. [3]

Serous effusions usually are the first clinical manifestation of malignancy and cytology not only detects malignancy but is also useful in determining the primary site of origin, staging, recurrence status and prognosis of the tumors. [4,5] It is a cost effective, minimally invasive, simple and safe procedure which when used in conjunction with ancillary techniques such as cell block, flow cytometry, immunocytochemistry and molecular analysis provides a definitive diagnosis and has a profound impact on the clinical management. [3] The sensitivity of cytomorphological evaluation of malignant effusions ranges from 40% to 97% and the specificity ranges from 90% to 100%. [2,3] The diagnostic efficacy of effusion cytology is varied for reasons such as sample volume, cell content, different collection/preparation techniques in different laboratories, overlap between benign and malignant entities and the experience of the cytopathologist. Until now, there was no standardized reporting system for fluid cytology that could strictly define the diagnostic terminologies and establish standards for the sample adequacy to improve interobserver agreement and communication, influencing further management based on the risk of malignancy (ROM) assessment for each category. [5,6]

Hence, The International System of Reporting Serous Fluid Cytopathology (TIS) has been developed in collaboration with the International Academy of Cytology (IAC) and American Society of Cytopathology (ASC) to address these issues and provide a consistent and tiered reporting for these specimens. The five proposed diagnostic categories are Non-Diagnostic (ND), Negative for Malignancy (NFM), Atypia of Undetermined Significance (AUS), Suspicious for Malignancy (SFM) and Malignant (MAL) – primary and secondary. [7]

Currently, only a few studies have been done based on the above proposed new system. Therefore, the present study was conducted with the aim to reclassify serous fluids into the categories as per TIS, estimate the risk of malignancy for each category and determine the diagnostic efficacy of serous fluid cytology when TIS was applied.

Materials and Methods:

The approval for conducting the study was obtained from the institutional ethical committee and the number is 19/2021 dated 20/01/2021 board. This was a cross-sectional study conducted over a duration of two years from 1^st April 2021 upto 30^th March 2023. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 2000. Purposive sampling technique was followed. The study sample included all pleural, pericardial, or peritoneal effusion samples submitted to the cytopathology department during the study period. The exclusion criteria included those slides that were returned to the patients and slides that were broken and unavailable for reviewing. Total 235 cases were included in the study.Complete clinical data were recorded. The fluid was centrifuged and slides from the sediment were prepared. Haematoxylin-eosin,Pap and Giemsa staining was done.

Slides of all the cases were reviewed by two experienced cytopathologist (AK and SM) with more than 10 years) experience. All the cases were classified as per The International System of Reporting Serous Fluid Cytopathology (TIS) into one of the following categories. Non-Diagnostic (ND), Negative for Malignancy (NFM), Atypia of Undetermined Significance (AUS), Suspicious for Malignancy (SFM) and Malignant (MAL) – primary and secondary. [7]

Final diagnosis was confirmed by follow-up, clinical history, radiology, and cell block in 74 cases.

Risk of malignancy was calculated for individual diagnostic category. The sensitivity, specificity, positive predictive value, and negative predictive value depicting the diagnostic efficacy of TIS were calculated under three circumstances. In first case, malignant cases were considered as positives, while SFM and AUS were taken as negatives. In second case, malignant and SFM cases were considered positives while AUS were taken as negatives. Lastly, malignant, SFM and AUS cases were considered as positives.

Statistics:

Continuous variables were expressed as mean and standard deviation. Categorical variables were expressed as percentages and proportions. Sensitivity, specificity, positive predictive value, and negative predictive value were determined to assess the diagnostic efficacy of TIS for fluid cytology. Microsoft excel was used for data compilation and analysis.

Results

Demographics and general charecteristics of fluid:

In the present study, a total of 235 fluid aspirates were included. Final confirmatory diagnosis was obtained in 74 cases based on thorough clinical history, cell block or radiological findings. Out of the total 235 cases, maximum cases were from the peritoneal cavity 127 (54.04%), followed by pleural cavity 108 (45.95%). There was no case of pericardial fluid. Mean age of presentation was 54 ± 2.5 years. Male to female ratio was 1.97:1. Quantity of fluid sent to the cytopathology ranged from 5 ml to 20 ml. In maximum number of cases, fluid was clear or straw coloured 167 (71.06%) followed by blood-tinged fluid in 50 cases (21.27%). Fluid was turbid in 15 cases (6.38%), and milky in 3 cases (1.28%). Demographics and general characteristics of fluid is shown in Table 1.

Cyto-diagnostic categories:

Out of a total of 235 cases of fluid examined, distribution of cases in the diagnostic categories were as follows, 11 (4.6%) in non-diagnostic (ND), 208 (88.51%) in negative for malignancy (NFM) (Figure 1), 5 (2.12%) in atypia of undetermined significance (AUS) (Figure 2), 2 (0.85%) in suspicious for malignancy (SFM) (Figure 3) and 9 (3.82%) in malignant (MAL) category (Figure 4). (Table 2)

Figure 1: A: Sediment smears showed only neutrophils. Acute inflammatory process. B: Shows only lymphocytes. CBNAAT was negative. A and B: Negative for malignancy. (Hematoxylin and Eosin, × 400)
Figure 2: Atypical cell in an acute inflammatory background. -Atypia of undetermined significance. (Hematoxylin and Eosin, × 400)	Figure 3: Atypical cell clusters in an acute inflammatory background- Suspicious for malignancy. (Hematoxylin and Eosin, × 400)
Figure 4: A: Tumor cells forming 3D balls in malignant effusion. B: Two population of cells suggestive of malignancy. C: Cell block from pleural fluid showing signet ring cells of adenocarcinoma stomach. (Hematoxylin and Eosin, × 400)

Predominant cell pattern in benign and malignant cases:

Among 208 cases in whom benign diagnosis was made, maximum cases showed lymphocyte predominance (140/208, 67.3%). Only neutrophils (30/208, 14.4%) and mesothelial cells (5/208, 2.4%) were also noted. Rest of the cases showed mixed pattern of cells. Malignancy was detected most commonly in pleural fluid. All the positive cases in pleural fluid were metastatic, with the most common site of primary being from lung followed by breast carcinoma. In peritonel fluid, metastasis from gastrointestinal tract tumours were seen. Site wise distribution of malignant cases and predominant non-cancerous cell pattern in each case is depicted in Table 3.

Correlation between cytology and final diagnosis:

Correlation between cytology and final diagnosis was performed on the basis of histopathology, clinical history or radiology findings.(Table 4) Final diagnosis could be affirmed in 74 cases (3.18%). Concordance between cytology and final diagnosis was obtained in 71 cases whilst discordant diagnosis was obtained in three cases. All the three cases in which discordant diagnosis was made were placed in the atypical category of TIS. Amongst these three discordant diagnoses, two were false positive and one was a case of false negative diagnosis.

Diagnostic efficacy:

The sensitivity, specificity, positive predictive value, and negative predictive value under various circumstances is shown in Table 5. We calculated the values depicting the diagnostic efficacy of TIS under three circumstances. In first case, malignant cases were considered as positives, while SFM and AUS were taken as negatives. In second case, malignant and SFM cases were considered positives while AUS were taken as negatives. Lastly, malignant, SFM and AUS cases were considered as positives. In all the scenarios, the sensitivity and specificity was high.

Risk of malignancy in individual diagnostic categories:

Risk of malignancy was obtained for each category by dividing the number of cases diagnosed as malignant by the total number of cases in that category. Risk of malignancy was 100% each in SFM and malignant categories and 40% in AUS category.

Discussion

Serous fluids offer rich cytopathological as well as molecular information on benign and malignant conditions. Being a simple, cost-effective, and minimally invasive diagnostic tool, it seems timely that a unified approach to reporting these specimens has been proposed. [3,8] The International System for Reporting Serous Fluid Cytopathology (ISRFC) consists of 5 categories namely – non-diagnostic (ND), negative for malignancy (NFM), atypia of undetermined significance (AUS), suspicious of malignancy (SFM) and malignant (MAL). [8]

The present study was a retrospective, cross sectional analysis of 235 pleural and peritoneal samples based on the “The International System for Reporting Serous Fluid Cytopathology (ISRFC)”, recategorizing them into the five categories and calculating the diagnostic accuracy and ROM for each category. Overall, most of the specimens were peritoneal samples (54.04%, n = 127) with a mean age of 51 years.

Non-Diagnostic (ND)

The ND category includes those specimens that are acellular or nearly acellular, without atypia, compromised by degeneration, poor preservation, obscuring blood, etc. making them uninterpretable. Eleven of the 235 cases (4.6%) were reclassified to this category. Confirmatory diagnosis was available for 3 of the 11 cases and all 3 were concordant. Therefore, it is recommended that enough specimen is collected (75ml) for accurate reporting. [8] However, in this study, volume of sample ranged from 5-20ml with maximum number of specimens falling in the 5-10ml range.

Negative for Malignancy (NFM)

208 cases fell into the NFM category as they contained only benign or reactive cellular components with no morphological, phenotypic, or molecular evidence of mesothelial or non-mesothelial malignancy. Concordance between cytology and final diagnoses was obtained in 55 of the cases.

Atypic of Undetermined Significance (AUS)

In the present study, in AUS category, 5 cases were indeterminate for mesothelial and non-mesothelial malignancy and contained cells (macrophages, mesothelial cells, or cells from cytologically bland malignant tumors) of uncertain nature, either because of qualitative or quantitative factors. 2 of the 5 cases were concordant while the rest were discordant. Amongst these 3 discordant diagnoses, 2 were false positive and one was a case of false negative diagnosis. The false-positive rate reported in literature is 0.1-1%. [11,12] The low cellularity, measurement errors, screening errors and failure to recognise cytopathological features could explain these findings. [9] The ROM was 40% which is in consensus with ROM in various literatures ranging from 20-70% with an average of 45%. [2,3,5-7,10,11]

Suspicious for Malignancy (SFM)

The suspicious for malignancy category includes specimens showing cytologic features usually found in malignant lesions but insufficient either in quality or quantity for a definitive diagnosis of malignancy and 2 concordant cases were assigned this category. The ROM was 100% which is higher than most of the literature ranging from 57-88%. [2,3,5-7,10,11] The lack of standardized diagnostic criteria thus far and an intrinsic bias of knowing patients’ history could be potential reasons for the difference in ROM values.[2,11]

Malignancy (M)

A total of 9 out of 235 cases (3.82%) were categorized into the malignant category as the specimens included those with definitive findings and/or supportive studies indicating mesothelial or non-mesothelial malignancies. This percentage reported in lower than that reported in literature which ranges from 10.4-20.5%. [12] This may be attributed to the small sample size and lack of ancillary testing. All nine cases were concordant on final diagnoses.

Eight of the 9 cases (88.89%) were pleural fluids malignancies with the primary being predominantly lungs (75%) followed by breast (25%). Previous reports show that pleural fluid malignancies account for 10-30% only and of these 50-70% are due to lung, breast, and ovarian cancer, as well as lymphoma. [13,14] This discrepancy between the results of present study and previous reports could be due to different selection criteria, study methodology and ancillary techniques employed. The peritoneal fluid malignancy was from a primary gastrointestinal adenocarcinoma which has been reported as the most common cause of malignant ascites in both sexes. [15] Literature has shown that ROM for this category ranges from 89-100% with a mean ROM of 99% (±0.1%). [11] In this study, ROM for this category was 100%.

Taking only malignant cases as positive, Lobo et. al., in their study of 1496 pleural effusion samples and 763 peritoneal effusion samples had a sensitivity of 61.4%. specificity of 100%, PPV of 100% and NPV of 71.7%. [3] Another study by Pinto D et. al, showed similar results with 52.21% sensitivity, 100% specificity, 100% PPV and 76.28 NPV. [5] In the present study, although the specificity closely matched their results, the sensitivity and NPV were 100% and PPV for 75%. These results support the role of cyto-analysis in confirming malignancy. The comparison of diagnostic efficacy of TIS with that of the existing literature is shown in Table 7.

Considering malignant and suspicious for malignancy cases as positives and AUS as negatives, the present study showed a sensitivity of 91.67%, specificity of 100%, PPV of 100% and NPV of 98.21%. This was in stark contrast to the findings of other similar studies having sensitivity values ranging from 60-67% and NPV ranging 74-79%. [3,7] However, a study by Pergaris et. al. involving 1028 cases in total showed similar specificity, PPV and NPV of 99.5%, 98.62% and 91.6% respectively. [6]

If malignant, suspicious for malignancy and AUS are considered as positives, the present study showed a higher sensitivity value of 92.85%, a higher NPV value of 98.21% and a lower PPV of 86.67% compared to Lobo et. al. and Pinto D et. al. [3,5]

Comparison of diagnostic efficacy of TIS in the present study with that reported in the literature is depicted in Table 7.

Limitations

In this study, all samples evaluated were conventional preparations. Many studies have reported higher accuracy with liquid-based preparations, despite some controversy. [16] The absence of pericardial samples reflects the clinical management of the situation. The criteria for drainage are strict and patients presenting with malignant pericardial effusion often present with concomitant malignant pleural effusion which is easier to tap. Follow-up confirmatory diagnosis was not available for majority of the cases.

It is a known fact that the combination of clinicopathological findings as well as immunocytochemistry play a crucial role in diagnosis of malignant serous effusions. Immunocytochemistry aside, serous effusions also serve as an ideal material for molecular analysis. [17,18] Due to technical limitations, ancillary studies were not possible in our set-up. These techniques would have allowed to reassign more cases to malignant category and identify the primary.

The IRSSF will eliminate many inconsistencies in reporting effusions and the nonspecific terms such as “suggestive of”, “suspicious for” etc, thereby providing a common language for all cytopathologists, clinicians, and institutions to be utilised in patient management.

Conclusion:

ISRSFC is easy to apply and has high diagnostic efficacy. Most of the serous effusions fall in negative for malignancy category. Mesothelial cells are not important for specimen adequacy.

References

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