Effusion is an abnormal fluid accumulation in one or more body cavities, which reflects some pathology and requires rapid diagnosis.^[1]Adenocarcinomas afflicting the lung, ovary and breast frequently metastasize to the serosal cavities.^[2]Many patients neoplastic disease present with accumulation of fluid in one of the serosal cavities.

Currently, cytomorphology is gold standard for diagnosing malignant effusion. Errors in sampling, screening and interpretation confer a moderate false-negative rate to cytology reporting.^[3]Some specimens make it very difficult to distinguish the malignant cells, mesothelial cells and inflammatory cells like macrophages on cytology.^[2]Cytology shows an average 97.0% specificity and 58.2% sensitivity with multiple specimens increasing sensitivity.^[4]

The diagnostic efficacy of cytology can be increased by performing immunohistochemistry on the cell block sections. The results of immunohistochemistry are largely dependent on the cellularity of the cell block sections.The complexity of immunostaining process is not time-efficient to make diagnosis promptly. Hence, an adjunct technique needs to be introduced that increases the diagnostic efficacy of malignant effusion samples.^[4]

Over the last few decades flow cytometry is an emerging technology. DNA flow cytometry requires nuclei or single cells in a suspension of fluid. DNA specific dye is used for staining dissociated cells. DNA stoichiometry helps in binding these dyes. The stained cells are passed singly through a laser beam. Photomultiplier tube is used in flow cytometry to measure the fluorescence emitted by the cells. DNA flow cytometry is studied by many researchers to detect malignant effusions which showed variation in specificity and sensitivity. Studies carried out by Green, Griffin and Laurini et al show that this is a rapid procedure which has a high specificity (96.8%) and sensitivity (100%) for malignant effusion diagnosis due to metastatic adenocarcinoma.^[5]

A large population of cells can be evaluated accurately in a small period by flow cytometry. It is a method which is applied to fresh specimens which have viable cells hence avoiding any artifacts of fixation and it could be complimentary to cytopathology.^[6],^[7]Measurement of content of DNA is the most accepted application which allows identifying aneuploidy of cells and gives information on cell proliferation by analyzing the various cell cycle phases.^[8]

Recently flow cytometry analysis of DNA aneuploidy is used to increase cytological accuracy of effusion analysis^[9] and to recognize abnormal cells not identified by cytology.^[10]Flow cytometry is a rapid, reproducible and sensitive method of cellular antigen detection.

In solid tumours there is good correlation between aneuploidy and neoplastic cells.^[11]Flow cytometry gives positive results on basis of aneuploidy whereas cytology gives positive results by detection of abnormal morphology.^[11]

Tribukait proposed a principle by which DNA ploidy determination and sample classification as diploid and aneuploidy was done.^[12] DNA Index is the ratio of abnormal peak position to the diploid peak of normal lymphocytes + or - 10%. Diploid nuclear value was 1.

Immunohistochemistry is an ancillary method which is used widely in cytopathology to increase diagnostic accuracy. Epithelial Membrane Antigen (EMA) is useful in identification of carcinoma cells in serous effusion samples. Flow cytometric immunophenotyping applying an antibody panel is used to determine patient specific data in this personalised medicine era.^[13] Few studies have reported that flow cytometry is useful in cell detection with stem cell marker expression.^[14]

DNA flow cytometry is a rapid and easy procedure, which provides a histogram which can be easily interpreted, and used as a supplement for the diagnosis of pleural effusions.^[15]Several studies have reported DNA aneuploidy provides a diagnosis of malignant effusion.^[16]

This study is conducted to identify the utility of flow cytometry in the diagnosis of malignant cells in clinical effusion specimens using DNA ploidy analysis by flow cytometry and compare the results with immunohistochemistry using EMA taking cytology as bench mark.

Materials and Methods

Serous effusions from 31 patients were prospectively analyzed over a two year period (July 2016 to Aug 2018). The study was conducted in Dr.D.Y.Patil Medical College, Hospital and Research Centre. The hospitalized patients with pleural or peritoneal effusions were included in this study. Approval for the study was obtained from the Institute ethics committee.

All the samples were received fresh, without anticoagulant or fixatives and were processed within 24 hours. Centrifugation of the fluid was done at 3000 rpms for 5 minutes. Five to six smears were made from the sediment and stained with Leishman stain, hematoxylin and eosin stain (H&E) and Papanicolaou stain. After further centrifugation, cell block for paraffin sections was made from the cell button obtained and slides stained with H&E and IHC stain was done using anti-EMA antigen (BioGenex). Appropriate positive and negative controls were used. The cytological examination was performed on all fluid specimens and histologic examination of biopsy tissues was performed using standard hematoxylin and eosin stain (H & E), whenever possible.

Flow cytometric analysis of the effusion samples was done using the procedure given in the BD Cycle TEST PLUS DNA Reagent Kit (Becton, Dickinson & Co.; BD Biosciences). The technique of DNA analysis by flow cytometry has been previously reported.^[11]

In this, the fluid samples were centrifuged and 50 microlitre pellet was transferred in tube with addition of 1ml buffer solution followed by vortex and centrifugation. The supernatant was discarded with addition of 1 ml buffer solution followed by vortex and centrifugation. The supernatant was discarded with addition of 1 ml buffer solution. The cell count was obtained with a hemocytometer using standard laboratory methods and the concentration was adjusted to 1.0 x 10^[6] cells/mL with buffer solution. Staining was done by adding 250 microlitre of solution A and incubated for 10 mins at room temperature. 200 microlitre of solution B (trypsin inhibitor and RNase buffer) was added and incubated at room temperature for 10 mins. To this 200 microlitre of cold solution C (PI stain solution) was added and incubated in the dark or in fridge (2 -8 degrees Celsius). The sample was filtered through nylon mesh prior to FCM analysis. Peripheral blood Lymphocytes of healthy individuals were used as diploid reference control. Effusion specimens were analysed on multi parameter six colour flow cytometer (FACS-Jazz; Becton, Dickinson & Co.; USA) and DNA index (DI) was calculated as ratio of DNA quantity of testing G0/G1 cells peak/DNA quantity of control G0/G1 cells peak.

The tumour cells ploidy was calculated by ratio of the average DNA quantity of the neoplastic cells that are at the G0/G1 phase to a normal quantity of a similarly processed control sample.^[17]

The results of cytological examination of the stained smears, immunohistological examination of cell blocks and FCM DNA index were divided in 3 groups. Group A consisted of malignant effusions. Group B had benign effusions. Group C consisted of effusions associated with malignancy where effusion sample was negative for malignancy, but the patient had malignancy. Cytology was considered as the gold standard for effusion diagnosis in our study.

Results

31 patients underwent cytological examination and DNA analysis of the effusion samples. Out of 31 samples, 4 were peritoneal fluid and remaining were pleural effusions. The study group comprised of almost equal sex distribution with 16 females (51.6%) and 15 males (48.4%).

There were five malignant pleural effusions. In three cases, there was concordance between cytologic diagnosis and DNA analysis. Four pleural effusions were malignant on cytology and three of them had an abnormal DI. One of these five was initially positive for malignancy on cytology and turned out to be negative on repeat cytologic examination but expressed abnormal DNA histogram with a DI of 1.5. One pleural effusion sample was positive for malignancy on cytology, but the DI was found to be normal (Table 1).

23 patients had effusion because of benign disease. All benign effusions except five had a DNA histogram which was normal. Hypodiploid cells were found in 4 patients having pleural effusion. The DI was 0.8 in two patients while it was 0.6 in three patients. One patient having peritoneal effusion was a case of Nephrotic syndrome showing hypodiploid cells had a DI of 0.6. Cytologic examination of group B effusions including these five hypodiploid effusions was negative for malignancy (Table 2).

There were three patients who had malignancy associated effusions. These patients were diagnosed with primary malignancy in other sites, but the effusion cytological report was negative. DNA analysis result was also negative for these effusions (Table3).

Considering cytology as the gold standard, the specificity of DNA analysis was 0.96 (95% confidence values, 0.81-0.99) and the sensitivity was 0.75 (95% confidence values, 0.19-0.99). The predictive value was 0.75 (95% confidence limits, 0.29-0.96). As the sample size is small the confidence limits are wide (Table 4).

Immunohistochemistry results with EMA

Among the thirty-one effusions, five cases showed anti EMA positivity (16.1 %) and twenty-six were anti EMA negative (83.9 %). Of the 5 cases, 3 agreed with cytology and 2 agreed with abnormal DI.

Among the benign effusions and effusions associated with malignancy all except 2 were negative for anti EMA. These two cases consisted of a patient operated for benign ovarian tumor and the other was a case of renal cell carcinoma, both were negative for malignancy on cytological examination.

The specificity, sensitivity and diagnostic accuracy of EMA was 92.6%, 75% and 90.3% respectively. (Table 5)

In this study, by DNA flow cytometric analysis, a total of 4 aneuploid patterns were detected and three of these could be shown to contain malignant cells by cytologic examination and 2 also showed positive for anti EMA antibody by immunohistochemistry.

Table 1: Results of group a patient data: Diagnosis, Cytology, EMA and DNA index

Table 2: Results of Group B patient data: Diagnosis, Cytology, EMA and DNA index

*Peritoneal effusion

Table 3: Results of Group C patient data: Diagnosis, Cytology, EMA and DNA index

Table 4: Comparative results of Cytology and Flow Cytometry

Table 5: Comparative results of Cytology and EMA

Discussion