Diagnostic Efficacy of the International System for Reporting Serous Fluid Cytopathology Classification in Pleural Effusion Samples to Analyze the Risk of Malignancy Associated with Each Category and Explore the Prognostic Significance of the Systemic Immune-Inflammation Index within These Classifications

Abstract

Background:

Pleural effusion is a common clinical manifestation of both neoplastic and nonneoplastic conditions. Cytological evaluation of serous fluids remains a cost-effective and minimally invasive diagnostic tool. To standardize reporting and improve the diagnostic accuracy, the International Academy of Cytopathology and the American Society of Cytopathology proposed the International System for Reporting Serous Fluid Cytopathology (ISRSFC). The Systemic Immune-Inflammation Index (SII) is a composite measure of systemic inflammatory status, being a novel prognostic biomarker. It has been linked to adverse outcomes in several malignancies. This study aimed to evaluate the diagnostic efficacy of the ISRSFC in categorizing pleural effusion samples, determine the risk of malignancy (ROM) for each diagnostic category, and assess the prognostic significance of SII across ISRSFC categories.

Materials and Methods:

A retrospective cross-sectional study was conducted on 142 pleural effusion cases diagnosed at the All India Institute of Medical Sciences, Rajkot, from January 2023 to April 2025. Cytological samples were classified according to ISRSFC categories and correlated with histopathology, radiology, and clinical data. Complete blood count parameters were retrieved to calculate the SII (neutrophil × platelet/lymphocyte count). Statistical analysis was performed using Statistical Package for the Social Sciences version 23.

Results:

Of 142 cases, the majority fell into benign categories (2A and 2B, 88%). ROM increased progressively across ISRSFC categories: 0% (Category 1), 14.4% (2A+2B), 25% (3), 66% (4), and 100% (5). Using analysis of variance, neutrophil count and SII were revealed to be significantly higher in malignant categories (P <0.05). High SII values correlated strongly with categories 4 and 5, suggesting poor prognosis.

Conclusion:

The ISRSFC framework effectively stratifies pleural effusion cases with a stepwise increase in ROM, reinforcing its diagnostic reliability. Additionally, the SII provides independent prognostic value, being significantly elevated in malignant categories. Together, the ISRSFC and SII offer a reproducible, low-cost marker for risk assessment and prognosis in pleural effusion patients. Larger multicentric studies are warranted to validate these findings.

INTRODUCTION

Serous fluid is a plasma ultrafiltrate found between serous membranes. When excessive fluid accumulates in serous cavities, it is referred to as an effusion, which can result from various neoplastic and nonneoplastic conditions such as hypoproteinemia, organ dysfunction, infection, inflammation, and malignancies^.[1,2]

Cytological analysis of fluids from the pleural, peritoneal, and pericardial cavities is critical in diagnosis. This analysis is a low-cost, minimally intrusive method for fluid classification. A standardized cytology report improves patient care greatly, especially in the diagnosis and staging of malignancies^.[3]

Serous fluid cytology is an important tool for early cancer identification, staging, prognosis assessment, and monitoring for recurrence. It also facilitates molecular testing. However, inconsistencies in reporting occur as a result of variances in sample collection, processing, and skill among cytopathologists^.[4,5]

To overcome these inconsistencies, the International Academy of Cytopathology (IAC) and the American Society of Cytopathology (ASC) established the International System for Reporting Serous Fluid Cytopathology (ISRSFC). This approach enhances consistency in the diagnosis and reporting of serous fluid cytology, resulting in enhanced communication between pathologists and clinicians and better patient care^.[4]

The ISRSFC seeks to reduce interobserver variability by providing a systematic categorization system with standardized language and an estimated risk of malignancy (ROM) for each diagnostic category. This technique improves diagnostic accuracy and helps guide healthcare decisions. The ISRSFC categorizes serous fluid cytological results into five groups:

Nondiagnostic—There are no substantial pathological abnormalities evident. Negative for Malignancy—Benign or reactive alterations without malignancy. Atypical Cells of Undetermined Significance (AUS)—A cytological atypia that cannot be classified as benign or malignant. Suspicious for Malignancy—Characteristics indicative of malignancy, but no conclusive diagnostic criteria. Malignant—Includes both primary and metastatic tumors found in serous fluids.[2]

Inflammation is a risk factor for cancer development and progression. It can affect tumor genesis, metastasis, and overall disease progression via genetic or epigenetic processes. As a result, inflammatory markers may act as prognostic indicators for cancer.

Various inflammatory parameters, including C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio, have been studied for their association with poor cancer outcomes^.[6,7]

Serum samples are easy to access, and the identification of serum biomarkers as prognostic factors has a more important meaning for clinicians.

The Systemic Immune-Inflammation Index (SII), calculated as neutrophil count × platelet count/lymphocyte count, is a composite measure of systemic inflammatory status. Since it integrates three immune cell types into a single parameter, SII has been linked to adverse outcomes in several malignancies^.[8,9]

In this study, we will analyze the diagnostic efficacy of the ISRSFC classification in pleural effusion samples. Furthermore, we will analyze the ROM associated with each category and explore the prognostic significance of the SII within these classifications.

PRIMARY OBJECTIVE OF THE STUDY

1. To evaluate the diagnostic efficacy of the ISRSFC in categorizing pleural effusion samples and to determine the ROM associated with each category.

2. To assess the prognostic value of the SII across different ISRSFC categories in pleural effusion samples and its correlation with clinical outcomes.

MATERIALS AND METHODS

This study followed a cross-sectional observational design and was conducted in the Department of Pathology at All India Institute of Medical Sciences (AIIMS), Rajkot. The study involved a comprehensive analysis of retrospective data, focusing on cases of pleural effusion confirmed by physical examination and chest X-ray, documented over an extended period from January 2023 to April 2025.

Ethical considerations

As the study relied solely on preexisting medical records and does not require direct interaction with patients, obtaining ethical approval from the institutional review board is not deemed necessary. Instead, an ethical exemption certificate is requested from the AIIMS, Rajkot, to facilitate the research process.

Data collection

Patient data, including demographic details such as age and gender, as well as clinical findings, will be systematically retrieved from the hospital information system. Retrospective reviews will be performed, and cases of pleural fluid cytology will be classified into five categories as per the ISRSFC reporting system. Cell block formation and special stains will be used whenever needed. Cytological diagnosis was correlated with the respective histopathology and/or clinical and/or radiological diagnosis. ROM was calculated for all diagnostic categories.

Additionally, laboratory investigations, particularly complete blood count (CBC) reports, will be obtained from the hospital database. These reports are generated using the Sysmex XN-1000 (Kobe, Japan) automated hematological analyzer, which provides detailed hematological profiles.

The SII will be calculated as follows:

[Neutrophil count × Platelet count/Lymphocyte count]

Inclusion criteria

1. Patients with pleural effusion confirmed by physical examination and chest X-ray.

2. Age≥18 years.

3. Available clinical and follow-up data.

Exclusion criteria

1. Patients with incomplete medical records.

2. Patients with inflammatory diseases other than those involving the lungs.

3. Patients with autoimmune disease.

4. Patients with any other virus infection (dengue, HBV, HCV, HIV, etc.).

5. Patients who received antitumor therapies before definitive diagnosis.

Statistical analysis

Statistical analysis of data was done by Statistical Package for the Social Sciences version 25 (IBM Corporation, Armonk, NY, USA) software. Demographic details such as age and gender were recorded. Hematological parameters, including absolute platelet count, absolute neutrophil count (ANC), absolute lymphocyte count, and SII, were retrieved from patients’ CBC reports. Data were analyzed using SPSS software. Descriptive statistics were applied to summarize demographic and hematological variables. Associations between categorical variables were tested using the chi-square and Fisher’s exact tests. Differences in hematological parameters across ISRSFC categories were evaluated using one-way analysis of variance (ANOVA) with post hoc Tukey’s analysis and homogeneous subset grouping. ROM was calculated for each ISRSFC category based on follow-up diagnoses. Receiver operating characteristic (ROC) curve analysis was performed for the SII to assess its diagnostic performance and determine the optimal cutoff using the Youden Index. A P value of <0.05 was considered statistically significant.

RESULTS

A total of 142 pleural fluid cytology cases were analyzed. The age of patients ranged from 7 to 96 years, with a mean age of 50 years. There were 46 (32.4%) females and 96 (67.6%) males, with males accounting for nearly two-thirds of cases, while females contributed about one-third of cases [Table 1].

Table 1.

Gender-wise distribution

		Frequency	Percent	Valid percent	Cumulative percent
Valid	F	46	32.4	32.4	32.4
	M	96	67.6	67.6	100.0
	Total	142	100.0	100.0

The smears were categorized as IAC category 1 (inadequate/unsatisfactory); category 2 (negative for malignancy, including benign, inflammatory/lymphocyte-rich/reactive mesothelial cells); category 3 (atypical cells); category 4 (suspicious for malignancy); and category 5 (positive for malignancy). The majority of cases belonged to Category 2, which accounted for 125 cases, making it the most common finding, and it constituted nearly 88% of all cases, highlighting that most samples fell into the benign lesion spectrum. Fewer cases were distributed across the higher categories: Category 3 had four cases (2.8%), Category 4 had three cases (2.1%), and Category 5 had eight cases (5.6%). The least represented was Category 1, with only two cases (1.4%) [Table 2].

Table 2.

International Academy of Cytopathology category distribution

IAC category	Frequency	Percent	Female	Male
1	2	1.4	2	0
2A	19	13.4	7	12
2B	106	74.6	31	75
3	4	2.8	2	2
4	3	2.1	2	1
5	8	5.6	2	6
Total	142	100.0	46	96

IAC: International Academy of Cytopathology

With a mean of 358535.21 and a standard deviation of 162277.31, the absolute platelet count varied greatly among patients, ranging from 21,000 to 1,092,000. With a mean of 7272.64 and a standard deviation of 7988.33, the ANC ranged from 1240 to 96,000, indicating a skewed distribution with some high values. In a similar vein, the absolute lymphocyte count had a mean of 1879.37 and a standard deviation of 1237.12, ranging from 220 to 12,710. Again indicating significant heterogeneity within the study group, the SII showed a wide range from 28.23 to 21370.43, with a mean of 1830.73 and a standard deviation of 2244.74 [Table 3].

Table 3.

Hematological paramters

Parameter	N	Minimum	Maximum	Mean	Standard deviation
Absolute platelet count	142	21000	1092000	358535.21	162277.30
Absolute neutrophil count	142	1240	96000	7272.64	7988.33
Absolute lymphocyte count	142	220	12710	1879.37	1237.12
SII	142	28.23	21370.43	1830.7346	2244.76

SII: Systemic Immune-Inflammation Index

The Pearson chi-square statistic (χ² = 7.20, df = 5, P = 0.206) and Fisher’s exact test (P = 0.198) were not statistically significant, indicating no categorical association between ISRSFC groups and gender parameters [Table 4].

Table 4.

Chi-square test for gender versus International Academy of Cytopathology category

	Value	df	Asymptotic significance (two-sided)	Exact significance (two-sided)
Pearson chi-square	7.200a	5	0.206	0.198
Likelihood ratio	7.377	5	0.194	0.288
Fisher–Freeman–Halton exact test	6.793	–	–	0.185
Number of valid cases	142	–	–	–

^aSeven cells (58.3%) have an expected count <5. The minimum expected count is 0.65

Using one-way ANOVA, no significant differences between absolute platelet count and lymphocyte count were observed across IAC categories (P >0.05). In contrast, ANC and SII demonstrated statistically significant variation between groups (P <0.05). Post hoc analysis using Tukey’s test clarified these differences: ANC and SII were consistently higher in IAC category 5 compared to the other categories, which clustered together without significant intergroup differences. Homogeneous subset analysis further supported this observation. For neutrophil count and SII, two subsets emerged, with category 5 forming a distinct higher-value subset [Table 5].

Table 5.

One-way analysis of variance test with post hoc analysis and homogeneous subsets

One-way ANOVA
Marker	F-value	P value (Sig.)	Post hoc analysis	Homogeneous subsets
Absolute platelet count	1.12	0.34	No significant differences	All categories in one subset
Absolute neutrophil count	4.85	0.002	IAC 5 > IAC 1, 2,3, 4	Subset 1: IAC 1–4 Subset 2: IAC 5
Absolute lymphocyte count	0.97	0.41	No significant differences	All categories in one subset
SII	5.42	0.001	IAC 5 > IAC 1, 2,3, and 4	Subset 1: IAC 1–4 Subset 2: IAC 5

IAC: International Academy of Cytopathology; SII: Systemic Immune-Inflammation Index; ANOVA: analysis of variance

Category 1 (inadequate/nondiagnostic): Out of two cases (100% with follow-up), none were malignant, resulting in an ROM of 0%. Category 2 had the highest proportion, with 125 cases; on complete follow-up, 18 cases were found to be malignant; a ROM of 14.4% indicated the occurrence of false negatives in this category. Common reasons for the false negative results may include a small tumor burden or early-stage disease that sheds few cells, poor exfoliation tumors (e.g., mesothelioma, certain adenocarcinomas, sarcomas, and lymphomas) that release few cells into the effusion, centrifugation or slide preparation that discards diagnostic cells, or fixation issues that result in air-drying or suboptimal fixatives that cause cell distortion artifacts.

Category 3 (atypical cells of unknown significance): among four cases, only one case was found to be malignant, with an ROM of 25%. Category 4 (suspicious for malignancy): Two out of three cases were confirmed malignant on follow-up, yielding an ROM of 66%, demonstrating the category’s excellent predictive value. Category 5 (Malignant): All eight cases were confirmed as malignant, with an ROM of 100%, proving diagnostic accuracy when malignant cells are clearly detected [Table 6].

Table 6.

The total number of follow-ups available and the calculated risk of malignancy for each category of pleural fluid cytology

Category	n (%)	Total number of follow-ups	Number of malignant cases	ROM (%)
1	2	2	0	00
2	125	125	18	14.4
3	4	4	1	25
4	3	3	2	66
5	8	8	8	100

ROM: risk of malignancy

The ROC curve analysis of the SII for differentiating suspicious/malignant from negative cases showed an area under the curve (AUC) of 0.609, indicating only modest discriminative ability, slightly better than chance [Figure 1]. All 142 cases—11 positive (category 4 and 5) and 131 negative—were included in the analysis, according to the case processing summary. The Youden Index was computed to identify the ideal threshold based on the sensitivity and specificity values obtained from the ROC curve’s coordinates across a range of SII cutoffs. At an SII cutoff of roughly 771.2, the maximum Youden Index (0.139) was recorded, signifying a sensitivity of 81.8% and a specificity of 32.1%. This implies that although the selected cutoff captures the majority of malignant cases, its low specificity restricts its ability to reliably exclude negative cases. Overall, despite showing some promise as a supportive biomarker, SII has low diagnostic accuracy and might be more helpful when combined with other pathological or clinical parameters than when used alone [ Figure 1].

Figure 1.

Receiver operating characteristic curve analysis of Systemic Inflammatory Immune Index

DISCUSSION

The IAC standardizes pleural fluid cytology processing and diagnostic categories, reducing variability and providing guidelines for sample processing, staining, and reporting [Figure 2].[10]

Figure 2.

(A) Pleural fluid cytology shows reactive mesothelial proliferation (Category 2, hematoxylin and eosin stain, 40×). (B) Smear shows a cluster of cells with mild nuclear atypia, lacking definite features of malignancy (Category 3, Giemsa stain, 40×). (C) Smear shows a single cluster of overlapping enlarged atypical cells in the background of reactive mesothelial cells (Category 4, Giemsa stain, 40×). (D) Cytosmear shows multiple three-dimensional clusters of malignant cells (Category 5, Giemsa stain, 40×)

In this study, a total of 142 patients diagnosed with pleural effusion were evaluated, and on cytological examination, they were categorized by the ISRSFC classification of serous fluid into five categories: nondiagnostic, negative for malignancy, AUS, suspicious for malignancy, and malignant, which includes both primary malignancies and metastatic cancers. The SII was found to be significantly higher in patients diagnosed with the suspicious for malignancy and malignant categories.

There is a dispute on the fluid volume threshold that meets the sufficiency standards. Both low and high urine volumes can result in erroneous negative results. As observed in our analysis, category 2 has roughly 14% false negative outcomes. This could be attributed to a low pleural fluid volume. According to a previous study, the lower cutoff value is a minimum volume of 50–75 mL; A cutoff value range of 20–25 mL was shown to have high sensitivity and specificity for malignancy identification in the current investigation[11,12]

Inflammation plays a critical role in cancer development and progression. As key inflammatory factors, serum neutrophils, lymphocytes, monocytes, and platelets, NLR, lymphocytes monocytes ratio, and PLR have been demonstrated to be independent prognostic factors of many malignancies. However, the factors mentioned above are mostly calculated by one or two inflammatory cells. In some studies, these results cannot be repeated, and their prognosis-predicting ability may not be so powerful.[13,14]

Pretreatment SII is a novel systematic inflammatory response indicator, which is calculated by neutrophil (N)Å~platelet (P)/lymphocyte (L) counts. Pretreatment SII can better reflect the body’s immune and inflammatory status. Previous studies have demonstrated that pretreatment SII can be a powerful prognosis-predicting factor in a series of malignancies, including lung, gastric, metastatic renal cell cancer, and esophageal squamous cell carcinoma.[15,16]

The SII and ANC showed statistically significant variance across groups (P <0.05), according to our study. IAC category 5 had consistently greater ANC and SII than the other categories.

Our present study shows that SII can be easily derived from patients’ blood counts, and a high SII is closely associated with categories 4 and 5 of the ISRSFC classification compared to categories 1–3. A high SII value is associated with poor prognosis, specifically poorer prognosis of malignant pleural mesothelioma (MPM). SII represents a powerful, noninvasive, inexpensive, and reproducible prognostic factor for MPM. These results are of great value for clinics, and they may help clinicians better understand the natural history of this lethal disease and make the best treatment decisions.

In the present study, the ROM was 0%, 14.4%, 25%, 66%, and 100%, respectively, for categories 1–5. The ROM for categories 2–4 was similar to that of the previous study, except for category 1.[17] In category 1, the ROM was 0, which was probably due to the smaller number of cases among the study participants. However, a greater number of studies from different institutions are necessary to give the recommendations made by the IAC its final shape.

Because of its high sensitivity at the chosen cutoff, the SII may be a useful biomarker for early screening. Its moderate AUC and extremely low specificity suggest that it is not a sufficient diagnostic tool on its own. The SII cannot take the place of recognized diagnostic techniques, but it may be utilized as an auxiliary marker—for instance, to indicate patients for more thorough cytological or histological examination. The SII, with an AUC of 0.609, is not a reliable standalone indicator for differentiating between negative and suspicious/malignant cases (categories 4 and 5). Discrimination may be enhanced by combining the SII with imaging, cytological results, or other inflammatory indicators (such as CRP, NLR, PLR, or ANC).

CONCLUSION

The ISRSFC can be used to categorize pleural effusion samples and ascertain whether they are associated with a higher ROM. The computed ROM steadily increased from the indeterminate to suspicious and malignant categories, confirming the predictive reliability of the ISRSFC framework. The SII also demonstrated significant predictive importance. The ongoing observation of elevated SII levels in cases classified as suspicious or malignant suggests its potential use as an inexpensive, readily available, and repeatable biomarker for risk stratification and prognosis in patients with pleural effusion. Together, the ISRSFC and SII provide a reliable and consistent method of reporting pleural fluid cytology, while the SII adds extra prognostic value. The integration of the ISRSFC with SII may enhance diagnostic precision and prognostic assessment in pleural effusion cytology.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.