Clinical Follow-up Demonstrates Differential Accuracy of Flow Cytometry and Cytomorphology for the Detection of Hematologic Malignancy in Hypercellular Body Cavity Fluids.
Authors: Cesana Clara , Klersy Catherine , Scarpati Barbara , Brando Bruno , Volpato Elisabetta , Bertani Giambattista , Faleri Maurizio , Nosari Annamaria , Cantoni Silvia , Ferri Ursula , Scampini Linda , Barba Claudia , Lando Giuliana , Morra Enrica , Cairoli Roberto 
Published: October 2019

Abstract
Abstract 5018
Flow cytometry (FCM) and cytomorphology (CM) evaluation of hematologic malignancy in hypercellular body cavity fluids (BCF) needs clinical validation and standardized approaches for definitive sample assessment.
A total of 1,011 hypercellular BCF analyzed by FCM between 2002 and 2009 were retrospectively examined. As a prerequisite for inclusion in the study, each of the following was needed: suspected or known hematologic malignancy at withdrawal, CM performed on the same sample, and availability of follow-up data for reliable retrospective clinical outcome (RCO). Ninety-two BCF [61 effusions, 31 bronchoalveolar lavage fluids (BALF)] were selected for the analysis.

Results
obtained by the two methods were matched with RCO. A combined method assessment (CMA) was also tested, accounting as negative those samples not assessed as frankly positive by at least one method.
Thirty-six percent of BCF resulted RCO-positive. By applying each method to RCO, concordant results between FCM and CM were observed in 69 cases (75%). Fifty-six cases (61%) were double true negative (TN), 11 cases (12%) were double true positive (TP), and 2 cases (2%) were double false negative (FN). The latter were BALF samples from patients with primary pulmonary lymphomatoid granulomathosis in which FCM revealed a prominent inflammatory background of CD3+ T-cells; clonality could not be assessed due to the small number of B-cells. Mismatched results between FCM and CM were observed in 23 cases (25%). FCM gave TP results in 19 cases (20.5%) with FN or uncertain CM, TN results in 3 cases (3%) with uncertain CM, and FN results in 1 case (1%) with TP CM. This was a pleural effusion in which, in the presence of anaplastic cell features, primary effusion lymphoma was definitively diagnosed by immunocytochemical staining for ORF73/latent nuclear antigen-1 of HHV-8 on B-cell marker negative neoplastic cells. By applying the CMA to RCO, 59 TN results (64%), 31 TP results (33.5%) and 2 (double) FN results (2%) were obtained. Overall, 100% specificity and positive predictive value were detected for each method. FCM/CMA sensitivity was significantly higher than CM sensitivity (p<.0001). The highest sensitivity for each method was displayed in the analysis of samples from B/T-cell precursor lymphoma/leukemia. FCM sensitivity was significantly higher than that of CM in the evaluation of effusions (p<.005) and of samples from B/T-cell differentiated lymphoma (p<.0001). CMA sensitivity was the highest in the latter, and reached 100% in the former. In the BALF setting, FCM/CMA displayed 75% sensitivity, while CM being non-diagnostic in each of RCO-positive samples.
In conclusion, clinical follow-up demonstrates FCM to retain significantly higher accuracy than CM in detecting hematologic malignancy contamination in both effusions and BALF, especially in the subset of differentiated lymphomas. Combining methods improves diagnostic performance almost completely by means of FCM.

Disclosures
No relevant conflicts of interest to declare.