Infinicyt software v 2

FCS files from 553 BM aspirates from newly diagnosed MM patients were analyzed using the semi-automated algorithm named “FlowCT” v.0.0.9^{51 (link)}, which is based on the analysis of multiple files by automated cell clustering. Briefly, FCS files were merged, underwent quality control, were normalized through batch removal steps and clustered using FlowSOM. After the computational clustering, the Infinicyt software v.2.0 (Cytognos SL, Salamanca, Spain) was used for the identification of each cluster. Statistical analysis was then performed based on the output of the software.

Bone marrow samples were processed following the general recommendations of the EuroFlow group (Kalina et al., 2012 (link)) and stained with at least an eight-color panel including monoclonal antibodies against the following antigens combined in several tubes: surface immunoglobulin-M (SIgM), CD5, CD19, CD20, CD22, CD23 (FCER2), CD25 (IL2RA), CD27, CD38, CD45 (PTPRC), CD56 (NCAM1), CD79B, CD81, CD117 (KIT), CD138, and intracytoplasmic IgM (CyIgM), kappa (CyК) and lambda (Cyλ). A minimum of 1 million cells were acquired per tube in a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) using BD FACSDiva™ software v6.1 (Becton Dickinson Biosciences). Data were analyzed using Infinicyt™ software v2.0 (Cytognos, Salamanca, Spain). Light chain-restricted clonal lymphocytes (CD19⁺) and plasma cells (CD38⁺ or strong CD138⁺) were quantified (Paiva et al., 2014 (link); Puig et al., 2017 ).