Letter to the Editor: “CLL-like B-lymphocytesare systematically present at very low numbers in peripheral blood of healthy adults”
SUPPLEMENTARY MATERIAL
Material and Methods
Subjects of study. A total of 639healthy subjects (292 males -46%- and 347 females -54%-; median age of 62±13 years, ranging from 40 to 97 years) randomly selected across the Primary Health Care areaof Salamanca(Spain), with an overall population of 353,287 inhabitants, were recruited into this study.Absolute peripheral blood (PB) leukocyte and lymphocyte counts were of 6·3±1·6 x109/L and 2·1±0·7 x109/L, respectively, and the mean absolute B-cell number was of 0·16±0·1 x109/L. The frequency of positive CLL-like cases in the first 608 individuals has been previously reported.1 The research protocol was approved by the Ethics Committee of the Cancer Research Centre of Salamanca;prior to entering this study, informed written consent was given by each participant, in accordance with the Declaration of Helsinki.
Immunophenotypic Studies. A total amount of between 0·9 and 1·2 mL/tube EDTA-anticoagulated PB was immunophenotyped using a direct immunofluorescence stain-and-then-lyse technique,2 with the following multicolor antibody stainings: 1) CD20-pacific blue (PB)/ CD45-pacific orange (PO)/ CD8-fluorescein isothiocyanate (FITC)+anti-sIgλ-FITC / CD56-phycoerythrin (PE) +anti-sIgκ-PE/ CD4-peridinin chlorophyll protein-cyanin 5.5 (PerCPCy5.5)/ CD19-PE–cyanin 7 (PE-Cy7)/ CD3-allophycocyanin (APC)/ CD38-Alexa Fluor 700 (AF700); 2) CD20-PB/ CD45-PO/ Cybcl2-FITC/ CD23-PE/ CD19-PerCPCy5.5/ CD10-PE-Cy7/ CD5-APC/ CD38-AF700 and 3) CD20-PB/ anti-sIgλ-FITC/ anti-sIgκ-PE/ CD19-PerCPCy5.5/ CD10-PE-Cy7/ CD5-APC. Reagents were purchased from Becton/Dickinson Biosciences (San Jose, CA, USA) except for CD19-PECy7 (Beckman/Coulter, Miami, FL), CD20-PB (e-Biosciences, San Diego, CA, USA), CD38-AF700 (Exbio, Prague, Czech Republic), CD45-PO (Invitrogen, Carlsbad, CA, USA) and bcl2-FITC, anti-sIgλ-FITC and anti-sIgκ-PE (Dako, Glostrup, Denmark). For the staining of Cybcl2, the Fix & Perm reagent kit (Invitrogen) was used, following the recommendations of the manufacturer. Data acquisition was performed on a FACSCanto II flow cytometer (BDB) using the FACSDiva software (v6.1; BDB) for >5x106 leukocytes/tube, as previously described1. Instrument setup and calibration were performed according to well-established protocols,3 and a daily quality control program was followed, using the Cytometer Setup and Tracking (CST) Beads and CST Module (BDB); in addition a tube containing phosphate buffered saline (PBS) was run between samples to avoid carryover of cells from a sample into the next one to be measured. Data analysis was performed using the INFINICITYTMsoftware program (Cytognos SL, Salamanca, Spain). The minimum number of cellular events required to define the presence of a CLL-like B-cell cluster was of 50 cells with the most informative staining from those described above, after excluding dead cells and cell doublets according to scatter. In each case, the precise volume of blood screened was recorded; besides, in all CLL-like MBL cases, the actual volume of PB containing the first 50 CLL-like B-cell events identified, was also calculated.
In a subgroup of 9 donors aged >70 years who did not show evidence of CLL-like B-cells in the screening phase, a second 50 mL PB sample was obtained in EDTA vacutainer tubes after informed consent was given by each subject. CD19+ B lymphocytes contained in 50 mL PB were purified using the AutoMACS device and CD19 monoclonal antibodies directly bound to iron microspheres (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the recommendations of the manufacturer.4Purified B-cells (purity of: 85%±8%) were then stained as described above, with the following multicolor stainings: CD20-PB/ CD45-PO/ anti-sIgλ-FITC/ anti-sIgκ-PE/ CD79b-PerCPCy5.5/ CD19-PE-Cy7/ CD5-APC/ CD3-APC-H7. CD3-APC-H7 and CD79b-PerCPCy5.5 were purchased from BDB. In addition in a subgroup of 50 MBL cases with, the persistence of the CLL-like clonal B-cells was systematically confirmed through the measurement of a second independent PB sample.
Assessment of the clonal nature of CLL-like B cells. Assessment of clonalitywas performed on FACSorted CLL-like B cells (purity: 98%0.8%)through analysis of IGH gene rearrangements (n=11 cases) and multicolor interphase fluorescence in situ hybridization (iFISH) studies directed against the detection of the most common genetic abnormalities associated with CLL (n= 35), as previously described in detail.5,6These studies could not be performed in any of those 9 cases in which 50mL of PB were screened for the presence of CLL-like MBL B-cells, since all sample was used for the flow cytometry immunophenotyping studies. In all 11 cases analyzed, sorted CLL-like MBL populations showed clonal IGH gene rearrangements.
Statistical methods. Descriptive and comparative statistics (either the Pearson 2 test or the T-student / Mann-Whitney U tests) were performed using the SPSS software program (SPSS 15·0 Inc. Chicago, IL).P-values of <0·05 were considered to be associated with statistical significance.
A statistical predictive model (power regression) was built, in order to estimate the percentage of subjects showing CLL-like cells for the whole cohort of individuals, as well as for each group of subjects defined according to age (40-59, 60-69; and>70 years),in the hypothetical case that higher volumes of PB would be stained and analyzed for each subject.Power regression analysis was performed using MATLAB software (MathWorks, Inc., Natick, MA, USA) after determining the percentage of cases who had detectable CLL-like B-cells in PB, among all subjects included in the study, for increasingly higher volumes of PB (and number of CLL-like cells) measured (from 1μL to 1200μL). Parameters were adjusted by a Nonlinear Least Square algorithm and confidence bounds (95%) were systematically calculated for all groups of cases. This regression model allowed the estimation of the percentage of MBL cases that would had been detected, for each age group,for blood volumes larger thanthoseactuallymeasured.
ACKNOWLEDGMENTS
The authors thank members of the Primary Health Care Group of Salamanca for the Study of MBL (listed below), who were the direct responsible for collection of samples and data from the cohort of healthy individuals recruited.
The authors thank R Goenaga, F Macías, M Pérez-García and M Rodríguez-Vegas, also for their collaboration in collecting the additional 50 mL of PB from healthy adult donors.
Members of The Primary Health Care Group of Salamanca for the Study of MBL:
ASENSIO OLIVA MARIA CARMENC.S. SANTA MARTA DE TORMES
BAREZ HERNANDEZ PILARC.S. GARRIDO SUR
CARREÑO LUENGO MARIA TERESAC.S. LEDESMA
CASADO ROMO JOSE MARIAC.S. ALBA DE TORMES
CUBINO LUIS ROCIOC.S. F. VILLALOBOS
DE VEGA PARRA JOSEC.S. PEÑARANDA
FRANCO ESTEBAN ELOYC.S. PIZARRALES-VIDAL
GARCIA RODRIGUEZ BERNARDO LUCIOC.S. LA ALBERCA
GARZON MARTIN AGUSTINC.S. PEÑARANDA
GOENAGA ANDRÉS ROSARIOC.S. LEDESMA
GOMEZ SANCHEZ FRANCISCOC.S. PERIURBANA NORTE
GONZALEZ MORENO JOSEFAC.S. GUIJUELO
GUARIDO MATEOS JOSE MANUELC.S. VITIGUDINO
HERNANDEZ SANCHEZ MARIA JESUSC.S. VITIGUDINO
JIMENEZ RUANO MARIA JOSEFAC.S. GARRIDO NORTE
JIMENO CASCON TERESA BASAC.S. ELENA GINEL DIEZ
MACIAS KUHN FRANCISCOC.S. LEDESMA
MERINO PALAZUELO MIGUELC.S. FUENTES DE OÑORO
MIGUEL LOZANO RUBENC.S. GARRIDO NORTE
MONTERO LUENGO JUANC.S. SAN JUAN
MURIEL DIAZ Mª PAZC.S. MIGUEL ARMIJO
PABLOS REGUEIRO ARACELIC.S. VITIGUDINO
PÉREZ GARCÍA MANUELC.S. ALBA DE TORMES
RAMOS MONGUE AURORA ESTHERC.S. LEDESMA
RODRIGUEZ MEDINA ANA MARIAC.S. ALBA DE TORMES
RODRIGUEZ VEGAS MARGARITAC.S. LEDESMA
SANCHEZ ALONSO BEGOÑAC.S. ALDEADAVILA DE LA RIBERA
SANCHEZ BAZO BEGOÑAC.S. ALDEADAVILA DE LA RIBERA
SANCHEZ SANCHEZ TERESAC.S. ALDEADAVILA DE LA RIBERA
SANCHEZ WHITE NICOLASC.S. F.VILLALOBOS
SANDIN PEREZ RAFAELC.S. SAN JOSE
SANZ SANTA-CRUZ FERNANDOC.S. PIZARRALES-VIDAL
VELASCO MARCOS MARIA AUXILIADORAC.S. ELENA GINEL DIEZ
SUPPLEMENTARY REFERENCES
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