Simple and rapid flow cytometry assay for the detection of malignant epithelial cells in body fluids.

INTRODUCTION: Morphology is routinely used for detecting malignant cells in body fluids, but it has limitations. Recently, flow cytometry (FCM) is used as an effective tool for studying non-haematological malignancies. The main objective of this study is to standardize a simple and rapid FCM test for the detection of malignant epithelial cells in body fluids. MATERIALS AND METHODS: Body fluids that had been processed for cytology/cytology and FCM were enrolled in this prospective study. We developed a fluorescent-labelled, monoclonal antibody panel composed of cell surface markers for this FCM assay. We compared the results of cytology/cell block and FCM. RESULTS: A total of 121 fluid samples were studied. Comparing the diagnostic performance of cytology/cell block and FCM, 52 (43%) cases were positive and 60 (49.5%) cases were negative for carcinoma cells by both techniques. Nine cases showed discordant results between the two techniques. Six cases were cytology+ but FCM- and three cases were FCM+ cytology-. Clustered Epithelial Cell Adhesion Molecule (EpCAM)-positive events with high scatter properties were definitive for positive diagnosis by FCM. We studied PD-L1 expression in 13 cases by FCM. Six cases were reported as false negative by this FCM assay due to hypocellularity and lack of EpCAM expression in malignant cells. CONCLUSIONS: This FCM assay is simple, easier and cost-effective yielding sensitive results with no inter-observer variability. FCM would become a valuable tool to complement routine diagnostic cytology and reduces misdiagnosis.

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Role of high acquisition flow cytometry in the detection of marrow involvement in patients with extramedullary B cell non-Hodgkins lymphoma: a comparison with marrow aspirate cytology, trephine biopsy, and PET.

Detection of marrow infiltration is crucial in extramedullary B cell non-Hodgkin lymphoma (B-NHL). We studied the efficiency of high acquisition flow cytometry (FCM) in detecting marrow involvement in B-NHL patients and compared its performance with marrow aspiration (BMA) cytology, marrow biopsy (BMB), and positron emission tomography (PET). No case with marrow infiltration was found to be BMB positive and FCM negative. BMA cytology showed poor sensitivity and specificity. Only 50% of FCM positive cases showed evidence of marrow involvement by PET. Neither the nature of lymphoma nor the burden of the marrow disease showed a correlation with PET positivity. Four cases that were positive only by PET had findings in areas other than the iliac region. We found more cogent and convincing results with high event acquisition in FCM. This study shows that a combination of PET and FCM with high event acquisition is the best way for assessing marrow involvement in B-NHL cases.

Independent diagnostic utility of CD20, CD200, CD43 and CD45 in chronic lymphocytic leukaemia.

Immunophenotyping plays a major role and is essential for establishing the diagnosis of chronic lymphocytic leukemia (CLL). Though CLL has a characteristic phenotype, diagnosis may be challenging due to immunophenotypic overlap with other B cell non-Hodgkin's lymphomas (B-NHL). Markers like CD200, CD43, CD20 and CD45 were found valuable in CLL and we investigated their diagnostic efficiency and accuracy in 174 patients with leukemic B-NHL. On the integration of four markers by a scoring system, 96% (49/51) of CLL cases showed a score of 3 or 4 and 90% (36/40) of non-CLL cases had a score of 0 or 1. This scoring system for CLL diagnosis showed a sensitivity of 98.2% and 96% in the analytical cohort and validation cohort respectively, which was significantly higher than the classical Matutes score. Hence we strongly suggest considering the expression of CD200, CD20, CD43 and CD45 in the diagnosis of B-NHL cases.