Morphometric Analysis and Immunocytochemical Staining on Cytospin Preparation in Effusion Cytology: A Study

Abstract

Introduction: Morphological differentiation between reactive mesothelial proliferation and metastatic carcinoma cells may be extremely difficult in conventional centrifuge deposit smears stained with papanicolaou and romanowsky dyes. In the present study immunocytochemistry and morphometric analysis were performed on cytospin preparation of effusion categorized as atypical/suspicious on toluidine blue stained wet films. Materials and methods: A total of 100 cases comprising 26 benign (control group) and 74 malignant (study group) effusions were included in the study. Samples showing atypical /suspicious cells on preliminary conventional centrifuged wet film stained with toluidine blue; 63 were aspirated from pleural, 36 from peritoneal and 1 from pericardial effusions. These samples were processed for 6 cytospin preparations, 1 air dried stained with giemsa and others fixed in ethanol stained with papnicolaou stain, Cytokeratin 8/18, Epithelial Membrane Antigen (EMA) and Calretinin. Morphometric analysis was performed using software Image Pro Plus Version 6.3 on a minimum of 20 positive and negative stained cells on IHC stained smears. Results: The sensitivity and specificity of CK 8/18 in diagnosing benign, atypical and malignant cases were 91.89% and 89.5% respectively. The sensitivity and specificity of EMA was 90.5% and 86.6% respectively. Calretinin had 97% sensitivity for mesothelial cells. The 3 negative cases did not express CK 8/18 and EMA also. Thus, Calretinin can be accepted as a technique control to decide that immunocytochemical staining is working in a given case because mesothelial cells are generally present in benign as well as malignant effusions as native exfoliated cells. Nuclear area, cytoplasmic area and N:C ratio of mesothelial cells in benign effusions were 56.2 ± 2.03 um2, 182.41 ± 4.61 um2 and 0.31 ± 0.01 um2 and in malignant effusions were 63.15 ± 2.44 um2, 185.67 ± 2.15 um2 and 0.34 ± 0.01 um2 respectively. Using ROC (Receiver Operating Characteristic) curve nuclear area 88.30 μm2 area [sensitivity (98.6%) and specificity (94%)] cytoplasmic area 200.55 μm2 area [sensitivity (100%) and specificity (90%)] and N:C ratio 0.345 [sensitivity (93%) and specificity (94%)] considered as cut off values. So, we can use this value to discriminate between reactive mesothelial proliferations from malignant cells. Conclusion: IHC can be easily performed on cytospin preparation without requiring antigen retrieval and is extremely useful in differentiating metastasis from reactive mesothelial proliferation. The results of morphometric analysis were useful adjunct.