Multicoloured flow cytometry to detect metastatic carcinoma in lymph node by epithelial cell adhesion molecule.

BACKGROUND: The metastatic carcinoma in the lymph node may be missed in routine fine needle aspiration cytology (FNAC). There are limited studies on the role of epithelial cell adhesion molecule (EpCAM) in detecting metastatic carcinoma in the FNAC of lymph nodes by flow cytometry (FCM). AIMS: To evaluate the role of EpCAM in flow cytometry detecting metastatic carcinoma in the fine needle aspiration cytology (FNAC) of the lymph node. MATERIALS AND METHODS: In this prospective study, successive 42 cases of lymph nodes were subjected to FNAC followed by flow cytometry to detect the EpCAM positive cell population. The sample was used for cytology and FCM (CD45, CD14, and EpCAM antibodies tagged with fluorochromes). The percentage of EpCAM positive cell population in each case was calculated and compared in the metastatic carcinomas and reactive lymphoid hyperplasia (RLH) cases. RESULT: There were 29 cases of metastatic carcinoma and 13 non-neoplastic cases (12 RLH and one granulomatous inflammation). The average percentages of EpCAM in metastatic carcinoma and reactive lymphoid cells were 11.37 and 1.24, respectively. The independent sample t-test showed a significant difference (0.001) in the percentage of EpCAM in the two groups. The cut of value of 3% EpCAM in FCM showed 97% sensitivity and 92% specificity to detect metastatic carcinoma in FNAC of the lymph node. CONCLUSION: The percentage of EpCAM in FCM may be helpful in detecting metastatic carcinoma in the lymph node. The FCM is a rapid and quantitative test with high sensitivity and specificity.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis.

BACKGROUND: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation - plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. DESIGN: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ2 test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. RESULTS: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). CONCLUSION: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Application of multicolour flow cytometry in the detection of metastatic carcinoma in serous effusions: Special emphasis in atypical cytology.

INTRODUCTION: We evaluated the role of simultaneous use of multiple antibodies in flow cytometry (FCM) to detect metastatic carcinomas in effusion samples. METHODS: Cytological examination of 75 successive cases of effusion samples was performed. There were 48 peritoneal, 26 pleural and one pericardial fluid. Multi-coloured FCM examination was undertaken using a cocktail of CD45, CD14 and epithelial cell adhesion molecule (EpCAM), antibodies tagged with different fluorochromes. The percentage of EpCAM positivity was calculated in the CD45 and CD14 dual negative population by selective gating. The EpCAM value was correlated with the cytological findings, follow-up data and MOC-31 immunostaining. RESULTS: There were 20 benign, 35 malignant and 20 atypical cases diagnosed on cytomorphology. The primary sources of carcinomas were mainly from the ovary, followed by lung, gall bladder, intestine and other areas. Out of 20 cytologically benign cases, there were two malignant cases on the final follow-up, and EpCAM on FCM picked up all 18 benign cases and one malignant case. Out of 35 cytologically detected malignant cases, EpCAM picked up 32 malignant cases. The EpCAM detected 15/18 malignant and both benign cases out of 20 cytological atypical cases. EpCAM antibody by FCM showed 87% sensitivity, 100% specificity, 100% positive predictive value and 74% negative predictive value. CONCLUSION: This comprehensive study highlights the potential use of multi-coloured FCM along with cytological examination to diagnose metastatic carcinoma in effusion samples. Multi-coloured FCM is rapid and quantitative and is helpful in atypical cases.

Micronucleus assay in buccal smears of breast carcinoma patients.

The aim of this study is to compare micronucleus assay in buccal smear of breast carcinoma patients versus normal benign cases as control group. In this prospective study, we selected a total 32 patients of carcinoma of breast and 49 patients of benign breast lesions diagnosed in fine needle aspiration cytology (FNAC). Acridine orange stain was done on buccal smears of these cases and micronucleus (MN) scoring was performed in 40× 0bjective in a fluorescent microscope. The MN score was expressed as positivity per 1,000 cells. The MN scoring in buccal smear was compared in malignant and benign breast cases. In fluorescence microscope, the micronucleus was detected as round orange shaped small intracytoplasmic structure around the nucleus. The mean MN scores in buccal smears of benign and carcinoma cases were 0.5014 ± 0.45768 and 2.1938 ± 1.08656 cases respectively. Independent sample Student's t test showed significantly high MN score in buccal smear of the cancer patients (P < 0.001). Micronucleated cells are significantly increased in buccal cells of the breast carcinoma cases. The increased number of MN in buccal smears raises the possibility that the genetic damage in breast cancer patients is generalized. In future, MN scoring could be used as biomonitoring of DNA damage and in early detection of high risk cases of carcinoma of breast.

Artificial neural network in diagnosis of metastatic carcinoma in effusion cytology.

AIMS AND OBJECTIVES: To build an artificial neural network (ANN) model for the detection of carcinoma in effusion cytology. MATERIALS AND METHODS: We selected a total of 114 effusion cytology cases consisting of 57 each benign and malignant case. In all these cases, detailed cytological features, image morphometric data, densitometric data, and chromatin textural data were collected. Based on these data, we made a back propagation ANN model for diagnosing malignancy in effusion cytology. This network was designed as 25-2-1 (input nodes-hidden nodes-output node). Online back propagation method was applied for training the network. The training of the network was continued until the network error was reduced to 0.000654. Simultaneously, we also performed logistic regression (LR) analysis test to compare with ANN model performance. RESULT: ANN model worked excellent after adequate training. With the help of this model, it was possible to identify correctly all the malignant cases in validation and test set. The result of the multivariate LR analysis was also similar as that of ANN model and all the cases were also classified correctly. CONCLUSIONS: In this study, we successfully constructed an ANN model to diagnose metastatic carcinoma in effusion cytology. ANN is very promising in the diagnosis of metastatic carcinoma in effusion cytology. In future, ANN model may help the cytopathologist to diagnose the difficult cases in effusion fluid.