Anti-Human FMC7 FITC/CD23 PE/CD19 PerCP-Cy™5.5

Protocol tips

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	The lymph node samples were cut, mechanically disintegrated, and then filtered. A specimen of 25–50 µL (<20 × 109 cells/L) was pipetted into five tubes and labelled with the Ab. Following a 15‐minutes incubation, red blood cells were automatically lysed and washed with Cell Wash solution (BD Biosciences) using BD FACS Lyse/Wash Assistant (BD Biosciences), followed by the automatic fixation of the specimen with Cell FIX solution (BD Biosciences).	Flow cytometry analysis of the specimens was performed with Becton Dickinson FACSCanto II. At least 20,000 cells were acquired for analysis.

Protocol tips
The lymph node samples were cut, mechanically disintegrated, and then filtered. A specimen of 25–50 µL (<20 × 109 cells/L) was pipetted into five tubes and labelled with the Ab. Following a 15‐minutes incubation, red blood cells were automatically lysed and washed with Cell Wash solution (BD Biosciences) using BD FACS Lyse/Wash Assistant (BD Biosciences), followed by the automatic fixation of the specimen with Cell FIX solution (BD Biosciences).
Downstream tips
Flow cytometry analysis of the specimens was performed with Becton Dickinson FACSCanto II. At least 20,000 cells were acquired for analysis.

Publication protocol

"The specimens were collected into plastic tubes with EDTA anticoagulant. The bone marrow and pleural fluid specimens were filtered. The lymph node samples were cut, mechanically disintegrated, and then filtered. A specimen of 25–50 µL (<20 × 109 cells/L) was pipetted into five tubes and labelled with the following antibodies: 1) TBNK tube (CD45‐PerCP‐Cy5.5/CD3‐FITC/CD4‐PE‐Cy7/CD8‐APC‐Cy7/CD19‐APC/CD16 + 56‐PE); 2) CD19‐PerCp‐Cy5.5/kappa‐FITC/lambda‐PE + CD200‐PE‐Cy7 + CD95‐AP C; 3) CD19‐PerCp‐Cy5.5/CD23‐PE/FMC7‐FITC + CD79b‐APC + CD5‐PE‐Cy7; 4) CD20‐APC‐Cy7 + CD10‐PE + CD43‐APC + CD35‐FITC + CD38‐PE‐Cy7; 5) CD20‐PerCp‐Cy5.5/CD22‐PE/CD103‐FITC + CD25‐PE‐Cy7 + CD11c‐APC (all BD Biosciences, San José, CA, USA). Following a 15‐minutes incubation, red blood cells were automatically lysed and washed with Cell Wash solution (BD Biosciences) using BD FACS Lyse/Wash Assistant (BD Biosciences), followed by the automatic fixation of the specimen with Cell FIX solution (BD Biosciences). In the case of the second test tube, red blood cell lysis and washing in BD FACS Lyse/Wash Assistant were followed by pipetting of the antibody; after incubation, the specimen was automatically washed and fixed by means of Cell FIX solution (BD Biosciences).

Flow cytometry analysis of the specimens was performed with Becton Dickinson FACSCanto II. At least 20,000 cells were acquired for analysis. The technical parameters of the flow cytometry (voltage, spectral overlap compensation) were set automatically using BD Cytometer Setup and Tracking Beads (Becton Dickinson). Daily internal quality control was performed with IMMUNO‐TROL Cells, manufactured by Beckman Coulter.

The data was recorded with Becton Dickinson FACSDiva software. For all markers, their surface expression in a population of B‐cells was analyzed. The neoplastic population was selected using a conventional gating strategy with FSC, SSC, and the expression of CD19/CD20. The intensity of expression was assessed as positive or negative (a cut‐off of 30%) or quantitatively with the MFI on a logarithmic scale. In markers with a bimodal expression pattern (bimodality being considered if two undoubted distinct neoplastic subpopulations with the different marker expression were visible on dot‐plot diagram), the MFI of the positive cell population was recorded. Kappa MFI and Lambda MFI were compared separately to minimize the bias caused by the use of different antibodies/fluorochromes. The analyzed populations were selected and the MFI values recorded by a single technician (DS). For CLL cases, the CLL scores were determined on the basis of the expression of CD5, CD23, CD79b, FMC7, and immunoglobulin (Ig) light chains."

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