Application of the Indian Academy of Cytologists Recommendations for Reporting Serous Fluid Cytopathology in Routine Reporting of Ascitic Fluid Specimen and Assessment of the Risk of Malignancy.

Background: A five-tiered reporting system for effusion fluid cytology has been published by the Indian Academy of Cytologists (IAC). Only a single study has evaluated the applicability of this system in routine reporting. Aims: We intend to evaluate the practical utility of this system in routine reporting of ascitic fluid cytology. Materials And
Methods: Nine hundred and sixty-one cases of ascitic fluid cytology were included in this study. The clinical, radiological, cytomorphological, and follow-up data of these cases were reviewed. All cases were recategorized according to the proposed IAC system, and the risk of malignancy (ROM) for each category was estimated.
Results: Age of the patients ranged from 1 to 92 years, and fluid volume ranged from 10 ml to 3 l. The number of cases included in each category and their respective ROM were as follows: category 1: 41, 21.42%; category 2: 805, 14.9%; category 3: 5, 33.3%; category 4: 31, 90%; and category 5:79, 96.4%. Conclusions: The new IAC guidelines for the serous fluid is representative, informative, and could be easily applied at our institutional level. We used the recommended diagnostic categories for reclassifying the ascitic fluid samples based on their cytosmear findings and conclude that the system has enormous utility at each level starting from the collection of fluid samples till the delivery of the report. Copyright:

INTRODUCTION

Peritoneal effusion is the result of a reactive, infective, or malignant pathology.[1] Peritoneal fluid cytosmear examination is an important part of initial assessment in understanding the etiology of the disease process. However, over the years, there is no consistent and standardized reporting protocol. This led to varied cytological diagnosis that did not aid the treating clinicians to make therapeutic decisions in further management of the patients. In an attempt to standardize the reporting protocol for serous effusions, a group of experts from the Indian Academy of Cytologists (IAC) proposed national guidelines that can be applied at various laboratories with different set-ups. As per the recommendations, every step from the beginning of sample collection to the final drafting of the report is divided into three broad categories, namely, essential, optimal, and optional. This system classifies the reporting of the effusion cytosmears into five categories and strikes clarity in the reporting protocol across the laboratories.[2]

We implemented these reporting guidelines and recategorized the ascitic fluid cytosmear findings into the five proposed categories and calculated the risk of malignancy (ROM) for each category.

MATERIAL AND METHODS

After receiving approval from the institutional ethics committee (Ref. No. T/IM-F/18-19/13), a retrospective study was performed using the data on the ascitic fluid samples retrieved from departmental archives. The study was conducted over a period of 1½ years from January 2019 to June 2020, and a total of 961 ascitic fluid samples were included. All the incomplete requisition forms not bearing appropriate name, age, registration number, diagnosis, and clinical details were rejected as per the IAC guidelines.

The samples were collected in an anticoagulant-free container and processed within 4 h of receipt. The fluid specimens were centrifuged at 1500 revolutions per minute for 10 min. Further, the supernatant was decanted and the sediment pellets were placed in cytospin chambers after resuspension. Two cytospin slides for each sample were prepared, one of which was air-dried and the other was fixed in absolute alcohol. The air-dried smear was stained with May Grunwald–Giemsa (MGG) stain and the alcohol-fixed one was stained with Papanicolaou stain (PAP stain). Rest of the sample was used for the preparation of cell block by mixing 1 ml of sediment with four drops of plasma and thrombin each. The cell blocks were subjected to routine processing and hematoxylin and eosin-stained slides were prepared. Immunohistochemistry was performed on the cell blocks whenever necessary.

The slides were reviewed and reclassified by two experienced cytopathologists (AA and MS) based on the morphology of cells and the background of the cytosmears. The recommended IAC guidelines were used to recategorize the cytosmear findings of the ascitic fluid. Data entry was done using the Microsoft Excel 2016 version.[2]

The associated ROM for each category was calculated by dividing the number of malignant cases in each category by the total number of cases where follow-up data was available either by histopathology or radiology or clinical records.

RESULTS

In total, 961 ascitic fluid specimens were reviewed with the volume ranging from 10 to 3000 ml. The study included 326 females and 635 males with age ranging from 1 to 92 years and the mean being 47 years. Cell blocks were available for 906 cases, out of which immunohistochemical analysis was done on 42 cell blocks. Immunohistochemistry on cell blocks was done only in cases where adequate number of cells was present and if any atypical cells were found on the sections. Table 1 shows a comparison of the number of cases in each category before and after recategorization by the IAC criteria. Before recategorization, various terminologies were used by various reporting faculty members (consultants). Although no definite criteria were proposed for the unsatisfactory category, a case was reported as inadequate/blood only/paucicellular smear/inconclusive whenever there were insufficient cells or there was blood only. Clubbing all these diagnoses yielded 26 cases (2.7%). Maximum cases were reported as benign effusion (n = 823, 85.63%), which included all the cases variously reported as negative for malignancy/no malignant cells seen/suppurative effusion/lymphocyte-rich effusion/reactive effusion. Five cases (5.2%) were reported as atypical cells present, and 38 cases (3.95%) were found to be reported as “suspicious for malignancy.” The number of “positive for malignancy” cases accounted for 7.18% (n = 69). Recategorization led to change in the number of cases of these categories.

Table 1

Comparison of cytodiagnosis before and after recategorization

Initial cytodiagnosis	n (%)	IAC categories	n (%)
Inadequate/blood only/paucicellular effusion	26 (2.7)	Nondiagnostic	41 (4.26)
Benign effusion/negative for malignancy/no malignant cells seen/suppurative effusion/lymphocyte-rich effusion/reactive mesothelial cells only	823 (85.64)	No malignant cells detected	805 (83.77)
Atypical cell present	5 (5.2)	Atypical cells, NOS	5 (5.2)
Suspicious of malignancy	38 (3.95)	Atypical cells, suspicious of malignancy	31 (3.25)
Positive for malignancy/metastatic adenocarcinoma/infiltration by lymphoma/leukemia	69 (7.18)	Malignant cells seen	79 (8.22)
Total	961		961

IAC=Indian Academy of Cytologists, NOS=not otherwise specified

Table 2 shows the data after recategorization of cases according to the IAC criteria. It also represents the total number of follow-ups available and the calculated ROM for each category. Representative cases belonging to each category are illustrated in [Figures 1-5]. Evaluating the clinical and follow-up data of these cases revealed that the most common causes of non-neoplastic effusions were chronic liver disease, tuberculosis, subacute bacterial peritonitis, acute pancreatitis, noncirrhotic portal hypertension, and congestive heart failure. The most common etiologies of neoplastic effusions were metastasis from primary adenocarcinoma in the lower gastrointestinal tract, stomach, ovary, lungs, breast, liver, pancreaticobiliary, neuroendocrine carcinoma, and hematolymphoid malignancies such as non-Hodgkin lymphoma infiltration and acute leukemias. Mesothelioma was diagnosed in only one case.

Table 2

Risk of malignancy of the IAC categories

Categorie	n (%)	Total follow-up	No. of malignant cases on follow-up	ROM (%)
1	41 (15.7)	14	3	21.42
2	805 (83.76)	268	40	14.9
3	5 (0.52)	3	1	33.3
4	31 (3.22)	20	18	90.0
5	79 (8.22)	56	53	96.4

IAC=Indian Academy of Cytologists, ROM=risk of malignancy. ROM: of cases found to be malignant/total no. of cases with follow-up

Figure 1

Two cases categorized as “no malignant cells detected.” (a) Cytosmear showing clusters of benign mesothelial cells (PAP stain 200×). (b) Section of cell block of the case showing clusters of benign mesothelial cells (H & E stain, 200×). (c) Cytosmear of another case showing sheets of neutrophils, macrophages, and occasional reactive mesothelial cells. There were no malignant cells (PAP stain, 200×). (d) Section of cell block of the case showing sheets of neutrophils (H & E stain, 200×). H & E = hematoxylin and eosin, PAP = Papanicolaou stain

Figure 5

Another case categorized as “malignant cells seen.” (a) Cytosmear shows high cellularity with sheets of lymphoid cells. The lymphoid cells are twice the size of mature lymphocytes and have opened up chromatin and prominent nucleoli (MGG-Geimsa stain, 200×). (b) Sheets of atypical lymphoid cells (PAP stain, 200×). (c) Section of cell block shows sheets of atypical lymphoid cells (H & E stain, 200×). (d and e) The cells are immunopositive for CD20 and Tdt, suggesting a precursor B lymphoblastic lymphoma. H & E = hematoxylin and eosin, MGG = May Grunwald–Giemsa, PAP = Papanicolaou stain. IAC = Indian Academy of Cytologists, NOS = not otherwise specified

A case categorized as “atypical cells, not otherwise specified.” (a) Cytosmear showing singly scattered cells with enlarged hyperchromatic nuclei among reactive mesothelial cells and lymphocytes. The patient had chronic liver disease. (b) Section of the cell block of this case showing moderate cellularity with clusters of cells with morphology of reactive mesothelial cells. Background shows many inflammatory cells and blood. No atypical cells could be found in these sections (H & E, 400×). (c–e) The cell clusters show strong positivity for calretinin and WT1, whereas they are negative for CD15. The inflammatory cells in the background show positivity for CD15. H & E = hematoxylin and eosin

A case categorized as “atypical cells, suspicious of malignancy.” (a) Cytosmears show many cells with enlarged, hyperchromatic nuclei and few clusters of reactive mesothelial cells. The patient had radiological evidence of omental thickening. (b) Section from the cell block reveals papillary clusters of cells with atypical nuclei. (c-e) The cells show strong positivity for Epithelial membrane antigen (EMA) and CD15 and are negative for calretinin, confirming the epithelial nature of the cells

A case categorized as “malignant cells seen.” (a) Cytosmear is highly cellular and shows mostly discohesive large cells with eccentric, hyperchromatic nuclei in a hemorrhagic background (MGG stain, 400×). (b) Section of cell block showing scattered malignant cells in a hemorrhagic background (H & E stain, 400×). (c–e) The tumor cells are immunopositive for EMA and PAX8 and immunonegative for calretinin, confirming their epithelial nature and ovarian origin. H & E = hematoxylin and eosin, MGG = May Grunwald–Giemsa

DISCUSSION

The IAC guidelines for reporting serous fluid cytology[2] provide a base for standardizing the processing and diagnostic categories for better communication and reduction in the variability of fluid cytosmear results. The guideline also provides a detailed standard operating procedure for fluid sample processing, staining, and use of ancillary techniques for proper reporting. We conducted a retrospective study and recategorized the cytosmear results of the ascitic fluid and present our experience with this recent guideline. To the best of our knowledge, this is the second study evaluating the usage of the proposed IAC guidelines.

The fluid volume threshold that addresses the adequacy criteria has been debatable. As stated in the earlier reports,[3456] minimum volume of 50–75 ml is the lower cut-off value, and a volume above this increases the chances of detecting malignancy in a pleural fluid sample. In the present study, a cut-off value range of 13.5–22.5 ml was determined to have a good sensitivity and specificity for detection of malignancy. However, it is recommended that the criteria should be set at an individual center level and a uniformity may be attained.

The heterogenous diagnostic terminologies like “inadequate,” “blood only,” “hemorrhagic,” “degenerated cells,” and “paucicellular” were cataloged into category 1 and were designated a common cytopathology diagnosis of “unsatisfactory.” This was applicable only when the representative amount of ascitic fluid was submitted. Fifteen cases that initially were rendered with a diagnosis of “negative for malignancy” were reclassified under category 1 in view of the scant cellularity that consisted of mostly inflammatory cells from the blood. It is wise to deem these fluid cytosmears as inadequate to avoid false-positive results. ROM associated with this group is high (21%). The cases should have mandatory clinical and radiological correlation, and biopsies should be sent for histopathologic examination wherever necessary.

The number of benign diagnoses summed up to 823 before the classification, and after recategorization, this number reduced to 805 (83.76%). Many cases that were labeled as benign were reclassified as “unsatisfactory” when the criteria laid down by the IAC system were applied. ROM for this category is low (14.9%).

The cytosmears showing “atypical cells” have been allocated to two categories in the IAC guidelines: category 3 (atypical cells, not otherwise specified [NOS]) and category 4 (atypical cells, suspicious for malignancy). In the indexed study, the associated ROM for the latter is significantly higher (33.33% for category 3 and 90% for category 4). The use of ancillary techniques benefits in these cases and extreme caution must be observed while categorizing them. Repeat sampling may help to unmask the malignant cells in such situations.[7]

The cytosmears with distinct malignant cells were ascribed category 5 and the associated ROM is 96.4% in the present study. Out of 56 available follow-ups, three were benign. All these three cases were found to be reactive mesothelial proliferations. The reactive changes in the proliferating mesothelial cells could lead to a false-positive diagnosis of malignancy. Cell block preparation with immunohistochemistry with mesothelial markers (calretinin, D2-40, WT1, desmin) and epithelial markers (EMA, CD15, CK7, and CK20) could help in the proper diagnosis.

Only one previous study by Kundu et al.[7] has evaluated the utility of this newly formulated IAC guidelines. Our findings are in concordance with theirs. They found a ROM of 20%, 16.7%, 50%, 94.4%, and 100% for the categories 1–5, respectively, which is very similar to our findings. However, a greater number of studies from different institutions are necessary to give the recommendation made by IAC its final shape. Recently, the International Society of Cytologists has published guidelines for reporting fluid cytology.[8] It is also a five-tiered system, encompassing similar diagnostic categories, for example, nondiagnostic, negative for malignancy, atypia of undetermined significance, suspicious of malignancy, and malignant with estimated ROM of 17.4% ± 8.9%, 21% ± 0.3%, 66% ± 10.6%, 82% ± 4.8%, and 99% ± 0.1%, respectively, for the categories. Many studies have evaluated these recommendations and have found them to be useful in routine reporting of fluid cytology.[910]

The new IAC guidelines for the serous fluid is representative, informative, and could be easily applied at our institutional level. We used the recommended diagnostic categories for reclassifying the ascitic fluid samples based on their cytosmear findings and concluded that the system has enormous utility at each level starting from the collection of fluid samples till the delivery of the report. For the laboratories that are not equipped for performing the ancillary techniques, the samples can be transported as per the guidelines to the centers having such facilities for proper handling of the specimen and rendering an informative report that could channelize the clinicians in the right direction and patient care.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.