*2.4. Whole Blood Cytometry*

To assess different lymphocyte subpopulations and perform the B-cell immunophenotyping, we used fresh whole blood. Both analyses were performed using the BD FACSCanto II Flow Cytometer (BD Biosciences, San Diego, CA, USA).

For the lymphocyte subpopulations, we used BD Trucount tubes and the BD Multitest 6 Color BTNK kit (BD Biosciences, San Diego, CA, USA), which included the following mixtures of fluorophore-conjugated monoclonal antibodies (mAb): anti-CD45- PerCP-Cy5.5, anti-CD3-FITC, anti-CD8-APC-Cy7, anti-CD4-PECy7, anti-CD19-APC, and anti-CD16+CD56-PE. Pre-sample preparation included the following steps: 20 μL of the antibody mix was added to a Trucount tube with 50 μL of patient blood. The tube was vortexed and incubated for 15 min at room temperature. Subsequently, 450 μL of FACS lysing solution (BD Biosciences) was added and left to act for 10 min. Finally, the cells were acquired on the flow cytometer and analyzed with the BD FACSCanto Clinical software (BD Biosciences, San Diego, CA, USA).

For B-cell immunophenotyping, we use an eight-color panel of the following mAb: anti-CD45-APH-7, anti-CD19-V500, anti-CD10-V450, anti-CD38-PECy7, anti-CD21-PE, anti-CD27-PerCP-Cy5, anti-IgD-FITC, and anti-IgM-APC (BD Biosciences, San Diego, CA, USA). The blood sample was stained with the mAb mixture in a test tube for 20 min at room temperature in the dark. After incubation, red cells were lysed with 1 mL of lysing solution (BD Pharm Lyse) for 10 min. Cells were then washed twice with PBA (1% bovine serum albumin in PBS) and fixed using 1% formalin in PBS. Finally, the cells were acquired on the flow cytometer, analyzed with the BD FACSDiva software (BD Biosciences, San Diego, CA, USA), and read with Infinicyt software (Cytognos, Salamanca, Spain).
