Cd138 pc5

Circulating tumor plasma cells in the peripheral blood (PB) were detected using flow cytometry. Briefly, PB samples were collected in tubes containing EDTA and processed using a bulk-lysis procedure (Versalyse, Beckman Coulter, Brea, CA, USA) within 24 h of sample collection. Labeling was performed using a single 8-colors antibody combination: CD45-KrOr, CD138-PC5.5, CD56-AA700, CD27-PC7, CD19-ECD, CD200-AA750 (Beckman Coulter), Kappa-APC, and Lambda FITC (BD Biosciences, Franklin Lakes, NJ, USA), and CD38-BV421 (Biolegend, San Diego, CA, USA). First, membrane staining was performed and this was followed after a PBS washing procedure by intracytoplasmic labeling (anti-Kappa and anti-Lambda) using a Fix & Perm Cell Permeabilization Kit (Beckman Coulter, Brea, CA, USA). After the final PBS wash, the final cell pellet was resuspended in 500 µL of PBS before analysis using the flow cytometer (Navios, Beckman Coulter, Brea, CA, USA). From 200,000 to 1 million leucocytes were acquired for each patient to ensure the quality of the analysis and to allow a limit of detection to be set at ≥20 tumor plasma cells (0.01% to 0.002%, according to the number of leucocytes). Data were analyzed using Kaluza 2.1 software (Beckman Coulter, Brea, CA, USA).

Laboratory analyses included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), total serum IgE, total serum immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 subclasses. Flow cytometry was performed using a Navios cytometer (Beckman Coulter, Brea, CA, USA) on fresh peripheral blood collected in ethylenediaminetetraacetic acid tubes using a lyse-no-wash technique (ammonium chloride) and the following panel of directly conjugated antibodies: CD3-fluorescein isothiocyanate, CD4-ECD, CD8-Pacific Blue, CD19-A700, CD20-allophycocyanin, CD27-phycoerythrin-cyanine 7, CD38-A750, CD45-Krome Orange, CD56-phycoerythrin, CD138-PC5.5 (Beckman Coulter). Naive B cells, memory B cells, plasmablasts, and plasma cells were identified within the CD19⁺ gate as CD19⁺CD20⁺CD27⁻CD38⁺ cells, CD19⁺CD20⁺CD27⁺CD38⁻ cells, CD19⁺CD20⁻CD27⁺CD38^+bright cells, and CD19⁺CD20⁻CD38⁺CD138⁺ cells, respectively. Total B cells were identified both as CD19⁺ cells (CD19⁺/side scatter [SSC] within the leukogate) and CD20⁺ cells (CD20⁺/SSC within the leukogate).