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Reference ranges of blood-circulating leukocyte populations by, e.g., age and sex, are required for monitoring immune-cell kinetics. Most previous reports in which flow cytometry has been used to define the reference ranges for leukocyte counts included a limited number of donors and/or cell populations and/or did not consider age and sex simultaneously. Moreover, other factors not previously considered in the definition of normal ranges, such as the presence of chronic-lymphocytic-leukemia (CLL)-like low-count monoclonal B-cell lymphocytosis (MBLlo), might also be associated with an altered distribution of leukocytes in blood in association with an immunodeficiency and increased risk of infection and cancer. Here, we established reference cell-count ranges for the major populations of leukocytes in blood of non-MBL and MBLlo adult Caucasians matched by age and sex using the EuroFlow Lymphocyte Screening Tube (LST). A total of 706 Caucasian adult donors—622 non-MBL and 84 MBLlo—were recruited from the general population. Among non-MBL donors, the total leukocyte, neutrophil, basophil dendritic cell and monocyte counts remained stable through adulthood, while the absolute numbers of T- and B-cell populations and plasma cells decreased with age. The number of eosinophils and NK-cell increased over time, with clear differences according to sex for certain age ranges. In MBLlo subjects, few differences in the absolute cell counts by age (vs. non-MBL) were observed, and MBLlo men and women showed similar trends to non-MBL subjects except for the B-cell count drop observed in >70 y-men, which was more pronounced in MBLlo vs. non-MBL controls. Building robust age- and sex-matched reference ranges for the most relevant immune-cell populations in the blood of non-MBL donors is essential to appropriately identify an altered immune status in different clinical settings and highlight the altered immune-cell profiles of MBLlo subjects.

Lysosomal storage disorders (LSDs) are a group of rare and genetic diseases produced by mutations in genes coding for proteins involved in lysosome functioning. Protein defect leads to the lysosomal accumulation of undegraded macromolecules including glycoproteins, glycosaminoglycans, lipids, and glycogen. Depending on the stored substrate, several pathogenic cascades may be activated leading to multisystemic and progressive disorders affecting the brain, eye, ear, lungs, heart, liver, spleen, kidney, skin, or bone. In addition, for some of these disorders, hematological findings have been also reported. In this paper, we review the major hematological alterations in LSDs based on 56 case reports published between 2010 and 2020. Hematological alterations were reported in sphingolipidosis, mucopolysaccharidoses, mucolipidoses, neuronal ceroid lipofuscinosis, glycogenosis, glycoproteinosis, cystinosis, and cholesteryl ester storage disease. They were reported alterations in red cell linage and leukocytes, such as anemia and morphology changes in eosinophils, neutrophils, monocytes, and lymphocytes. In addition, changes in platelet counts (thrombocytopenia) and leukocyte abnormalities on non-peripheral blood samples were also reported for some LSDs. Although in most of the cases these hematological alterations are not pathognomonic of a specific disease or group of LSDs, since they can be easily identified in general clinical laboratories, their identification may contribute to the diagnosis of these disorders. In this sense, we hope that this review contributes to the awareness of the importance of hematological alterations in the diagnosis of LSDs.

Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females., This work was supported by the RD06/0020/0035 and RD12/0036/0048 grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER); CB16/12/00400 grant (CIBERONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER); the FIS PI06/0824-FEDER, PS09/02430-FEDER, PI12/00905- FEDER, DTS15/00119-FEDER, PI16/00787-FEDER and PI17/00399-FEDER grants, from the Fondo de Investigación Sanitaria of Instituto de Salud Carlos III; the GRS206/A/08 grant, (Ayuda al Grupo GR37 de Excelencia, SAN/1778/2009) from the Gerencia Regional de Salud (Consejería de Educación and Consejería de Sanidad of Castilla y León, Valladolid, Spain) and the SA079U14 grant (Consejería de Educación and Consejería de Sanidad of Castilla y León, Valladolid, Spain). ML Gutiérrez is supported by grant PTA2014-09963-I from the Instituto de Salud Carlos III.

Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes., We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114 and FIS PI14/01538), Fondos FEDER (EU), Junta Castilla-Leon SA198A12-2 and BIO/SA03/15. The proteomics analysis was performed in the Proteomics facility of Centro de Investigacion del Cancer (Salamanca, Spain) that belongs to ProteoRed, PRB2- ISCIII, supported by grant PT13/0001. P.D. is supported by a JCYL-EDU/346/2013 PhD scholarship.

Primary Health Care Group of Salamanca for the study of MBL: list of members (alphabetical order): Alonso Martín, María Monserrat (C.S. Fuentes de Oñoro); Asensio Oliva, María Carmen (C.S. Santa Marta de Tormes), Bárez Hernández, Pilar (C.S. Garrido Sur); Cabo Sastre, Luis (C.S. Ledesma); Carreño Luengo, María Teresa (C.S. Ledesma); Casado Romo, José María (C.S. Alba de Tormes); Cubino Luis, Rocio (C.S. Sancti Spiritus); De Vega Parra, José (C.S. Peñaranda); Franco Esteban, Eloy (C.S. Pizarrales-Vidal); García García, María Concepción (C.S. Guijuelo); García Rodríguez, Bernardo Lucio (C.S. La Alberca); Garzón Martín, Agustín (C.S. Peñaranda); Goenaga Andrés, Rosario (C.S. Ledesma); Gómez Cabrera, Rosalia (C.S. Garrido Sur); Gómez Sánchez, Francisco (C.S. Periurbana Norte); González Moreno, Josefa (C.S. Guijuelo); González Vicente, Ángel Carlos (C.S. Aldeadávila de la Ribera); Guarido Mateos, José Manuel (C.S. Vitigudino); Hernández Sánchez, María Jesús (C.S. Vitigudino); Herraes Martín, Ricardo (C.S. La Alberca); Herrero Sánchez, Amparo (C.S. Fuentes de Oñoro); Jiménez Ruano, María Josefa (C.S. Garrido Norte); Jimeno Cascón, Teresa Basa (C.S. Elena Ginel Díez); Macías Kuhn, Francisco (C.S. Ledesma); Mateos Rubio, Pablo (C.S. Ledesma); Márquez Velasco, María Salud (C.S. Sancti Spiritus); Merino Palazuelo, Miguel (C.S. Fuentes de Oñoro); Miguel Lozano, Rubén (C.S. Garrido Norte); Montero Luengo, Juan (C.S. San Juan); Muriel Díaz, María Paz (C.S. Miguel Armijo); Pablos Regueiro, Araceli (C.S. Vitigudino); Pascual Martín, J. Antonio (C.S. Fuentes de Oñoro); Pastor Alcalá, Luis (C.S. Vitigudino); Pedraza García, Jesús (C.S. Vitigudino); Pérez Díaz, Manuel (C.S. Pizarrales-Vidal); Pérez García, Manuel (C.S. Alba de Tormes); Prieto Gutiérrez, María Teresa (C.S. Peñaranda); Ramos Arranz, Manuel (C.S. Ledesma); Ramos Mongue, Aurora Esther (C.S. Ledesma); Rodríguez Medina, Ana María (C.S. Alba de Tormes); Rodríguez Vegas, Margarita (C.S. Ledesma); Romo Cortina, Javier (C.S. Elena Ginel Díez); Roselló Carmen, Elena (C.S. Vitigudino); Sánchez Alonso, Begoña (C.S. Aldeadávila de la Ribera); Sánchez Bazo, Begoña (C.S. Aldeadávila de la Ribera), Sánchez White, Nicolás (C.S. Sancti Spiritus); Sandín Pérez, Rafael (C.S. San José); Sanz Santa-Cruz; Fernando (C.S. Capuchinos); Soto Jiménez, Francisco (C.S. Santa Marta de Tormes); Velasco Marcos, María Auxiliadora (C.S. Elena Ginel Díez); Vicente López, Horacio Marcos (C.S. Aldeadávila de la Ribera); Vicente Santos, M. Sebastián (C.S. Aldeadávila de la Ribera)., Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal Bcell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL., This work was supported by the: RD06/0020/0035 and RD12/0036/0048 grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER); CB16/12/00400 grant (CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER); the FIS PI06/0824-FEDER, PS09/02430-FEDER, PI12/00905-FEDER and DTS15/00119-FEDER grants, from the Fondo de Investigación Sanitaria of Instituto de Salud Carlos III; the GRS206/A/08 grant, (Ayuda al Grupo GR37 de Excelencia, SAN/1778/2009) from the Gerencia Regional de Salud, (Consejería de Educación and Consejería de Sanidad of Castilla y León, Valladolid, Spain); FS/1-2010 and FS/19-2013 grants, from the Fundación Memoria D. Samuel Solórzano, (University of Salamanca, Salamanca, Spain).

A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next- Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig. Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches. For this purpose, an in-house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS., We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI11/02114 and FIS PI114/01538. We also acknowledge Fondos FEDER (EU) and Junta Castilla León (grant BIO/SA07/15). This work has been also sponsored by Fundación Solórzano (FS/23-2015). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I+D+I 2013-2016, funded by ISCIII and FEDER. The authors would like to thank all the clinicians and technicians in the Cytometry and Cell Purification Services of the University of Salamanca, the Spanish National DNA Bank (Banco Nacional de DNA Carlos III, University of Salamanca) and the Genomic Unit of Cancer Research Centre (IBMCC, USAL-CSIC) for their support in the data collection for the preparation of this manuscript. P.D. is supported by a JCYL-EDU/346/2013 Ph.D. scholarship.

Primary Health Care Group of Salamanca for Study of MBL: et al., Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets. Little is known about the distribution of normal lymphoid cells and their subsets in the peripheral blood (PB) of subjects with monoclonal Bcell lymphocytosis (MBL). In our study, we compared the absolute number of PB lymphoid cells and their subpopulations in 95 MBL cases with normal lymphocyte counts vs. 617 age-/sex-matched non-MBL healthy subjects (controls), using highly sensitive flow cytometry. MBL cases showed significantly reduced numbers of normal circulating B-cells, at the expense of immature and naïve B-cells; in addition, CD41CD81 double-positive T-cells and CD81 T-cells were significantly lower and higher vs. controls, respectively. Moreover, most normal B-cell subsets were significantly decreased in PB at ≥1% MBL-counts, vs. ''low-count'' MBL cases, and lower amounts of immature/naïve B-cells were detected in biclonal (particularly in cases with coexisting CLL-like- and non-CLL-like B-cell clones) vs. monoclonal MBL subjects. In summary, our results show imbalanced (reduced) absolute numbers of recently produced normal circulating B-cells (e.g., immature and naïve B-cells) in MBL, which becomes more pronounced as the MBL cell count increases. © 2012 Wiley Periodicals, Inc., Contract grant sponsor: Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich (FWF), Austria; Contract grant number: J 2906-B13. Contract grant sponsor: Austrian Society of Hematology and Oncology; Grant numbers: RTICC RD06/0020/0035-FEDER, PI06/0824-FEDER and PS09/02430-FEDER. Contract grant sponsor: Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain; Contract grant numbers: GRS206/A/08 and GREX-GR37. Contract grant sponsors: Gerencia Regional de Salud de Castilla y León and Ayuda al Grupo GR37 de Excelencia de Castilla y León, Consejería de Educación, Junta de Castilla y León, Valladolid, Spain; Contract grant number: SAN/1778/2009. Contract grant sponsor: Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain; Contract grant number: FS/ 16.2008. Contract grant sponsor: Fundación Samuel Solórzano, University of Salamanca, Salamanca, Spain, A.R-C. was supported by Fundación Científica de la Asociación Española contra el Cáncer., The Primary Health Care Group of Salamanca for Study of MBL: et al.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western world. The disease is typically diagnosed in adults >40 years old, who show an expansion (>5 × 109 cells per l) of clonal B-cells with a unique CD5+, CD23+, B-cell receptor (BCR)low immunophenotype in peripheral blood (PB) and bone marrow, frequently in association with involvement of other lymphoid tissues, disease symptoms and a heterogeneous clinical outcome.

Monoclonal B-cell lymphocytosis (MBL) indicates the presence of less than 5 × 109/L circulating monoclonal B cells in otherwise healthy subjects. Recently, it has been reported that circulating chronic lymphocytic leukemia (CLL)–like B cells can be detected using 4- or 5-multicolor flow cytometry in 5% to 7% of adults with normal lymphocyte counts. We investigated the frequency of circulating monoclonal B cells in 608 healthy subjects older than 40 years with normal blood counts, using a highly sensitive 8-color flow cytometry approach and systematic screening for total PB leukocyte count higher than 5 × 106. We show that the frequency of PB monoclonal B cells is markedly higher than previously reported (12% for CLL-like B cells, found at frequencies of 0.17 ± 0.13 × 109 cells/L), the incidence progressively increasing with age. Most cases (62%) showed clonal B-cell levels below the maximum sensitivity of the techniques described by others (< 0.01%), supporting the notion that detection of MBL may largely depend on the sensitivity of the flow cytometry approach used.

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL-like monoclonal B-cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL-like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non-stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL-like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non-stereotyped clones. Whilst the overall size of the stereotyped B-cell clones in peripheral blood did not appear to be associated with the CLL-related cytogenetic profile of B-cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)-associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis., Funded by: Red Temática de Investigación Cooperativa en Cáncer (RTICC) of Instituto de Salud Carlos III – FONDOS FEDER. Grant Numbers: RD06/0020/0035, RD12/0036/0048, FIS PI06/0824-FEDER, PS09/02430-FEDER; FIS PI12/00905-FEDER Fondo de Investigación Sanitaria of Instituto de Salud Carlos III. Grant Numbers: GRS206/A/08, SAN/1778/2009; Gerencia Regional de Salud, Consejería de Educación and Consejería de Sanidad of Castilla y León. Grant Numbers: FS/1-2010, FS/19-2013; Fundación Memoria D. Samuel Solórzano, Universidad de Salamanca; Fundação para a Ciência e Tecnologia of Portugal. Grant Number: SFRH/BD/31609/2006; Fundación Científica de la Asociación Española contra el Cáncer. Grant Number: AECC-2008.

This is an open-access paper.-- et al., Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes., AH was supported by a grant from the Fundação Para a Ciência e Tecnologia of Portugal (SFRH/BD/31609/2006); ARC was partly supported by a grant from Fundación Científica de la Asociación Española contra el Cáncer (AECC-2008) and by a grant from Red Temática de Investigación Cooperativa en Cáncer del Instituto de Salud Carlos III - FEDER (RD12/0036/0048). The research was supported by the following grants: Red Temática de Investigación Cooperativa en Cáncer (RTICC) del Instituto de Salud Carlos III - FONDOS FEDER (RD06/0020/0035 and RD12/0036/0048); FIS PI06/0824-FEDER, PS09/02430-FEDER and FIS PI12/00905-FEDER, from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain; GRS206/A/08 from the Gerencia Regional de Salud de Castilla y León and Ayuda al Grupo GR37 de Excelencia de Castilla y León, Consejería de Educación; SAN/1778/2009, Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain and FS/1-2010 Fundación Memoria D. Samuel Solórzano, Universidad de Salamanca, Salamanca, Spain.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]:Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown. [Methodology/Principal Findings]:For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLlo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+ clonal B-cells. [Conclusions/Significance]: These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLlo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution., AH was supported by a grant from the Fundação para a Ciência e Tecnologia of Portugal (SFRH/BD/31609/2006), ARC was supported by a grant from Fundación Científica de la Asociación Española contra el Cáncer (AECC-2008). This work was financially supported by the following grants: Red Temática de Investigación Cooperativa en Cáncer del Instituto de Salud Carlos III - FONDOS FEDER (RD06/0020/0035); FIS PI06/0824-FEDER, PS09/02430-FEDER and FIS PI12/00905, from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain; GRS206/A/08 from the Gerencia Regional de Salud de Castilla y León and Ayuda al Grupo GR37 de Excelencia de Castilla y León, Consejería de Educación; SAN/1778/2009, Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain and FS/1-2010 Fundación Memoria D. Samuel Solórzano, Universidad de Salamanca, Salamanca, Spain.

The Primary Health Care Group of Salamanca for the Study of MBL2: et al., [Background]: Monoclonal B-cell lymphocytosis (MBL) indicates 40 years old (62 ± 13years) with normal lymphocyte counts (2.1 ± 0.7 × 109/L) were immunophenotyped using high-sensitive flow cytometry, based on 8-color stainings and the screening for >5 × 106 total PB leukocytes. [Results]: Thirteen subjects (2.0%; 9 males/4 females, aged 73 ± 10 years; absolute lymphocyte count: 2.4 ± 0.8 × 109/L) showed a non-CLL-like clonal B-cell population, whose frequency clearly increased with age: 0.4%, 3%, and 5.4% of subjects aged 40-59, 60-79, and ≥80 years, respectively. One single B-cell clone was detected in 9/13 cases, while two B-cell clones were found in 4/13 (n = 17 MBL populations). Nine MBL cell populations showed a CD5- phenotype (usually overlapping with marginal zone-derived (MZL) or lymphoplasmacytic (LPL) non-Hodgkin lymphoma (NHL) B-cells, or an unclassifiable NHL), but CD5-/+d (n = 3) and CD5+ (n = 3 non-CLL-like MBL, consistent with a mantle-cell lymphoma (MCL)-like phenotype, and n = 2 CLL-like) MBL were also identified; iFISH supported the diagnosis in most cases. No preferential IGHV usage of B-cell receptor could be found. Twelve cases reevaluated at month +12 showed circulating clonal B-cells, at mean levels significantly higher than those initially detected. [Conclusions]: Non-CLL-like MBL cases frequently show biclonality, in association with MZL-, LPL-, MCL-like, or unclassifiable phenotypic profiles. As with CLL-like MBL, the frequency of non-CLL-like MBL increases with age, with a clear predominance of males. © 2010 International Clinical Cytometry Society., Grant sponsor: Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain; Grant numbers: RTICC RD06/0020/0035-FEDER, PI06/0824, PS09/02430; Grant sponsor: Gerencia Regional de Salud de Castilla y León; Grant number: GRS206/A/08; Grant sponsor: Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain; Grant number: SAN/1778/2009; Grant sponsors: Fundación Samuel Solórzano, University of Salamanca, Salamanca, Spain; Grant number: FS/16-2008; Grant sponsor: Consejeria de Educación, Junta de Castilla y León, Valladolid, Spain; Grant number: Ayuda al grupo GR37 de Excelencia de Castilla y León. A.R-C. is supported by Fundación Científica de la Asociación Española contra el Cáncer.

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be. Published 2010 Wiley-Liss, Inc.

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) na?̈ve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL). © 2010 International Clinical Cytometry Society., Grant sponsor: MSCNet European strep; Grant number: N_ E06005FF; Grant sponsor: Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain; Grant numbers: RTICC RD06/0020/0035, PI06/0824, PS09/02430; Grant sponsor: Gerencia Regional de Salud de castilla y León; Grant number: GRS206/A/08; Grant sponsor: Ayuda GR37 de Excelencia de Castilla y León, Consejeria de Educación, Junta de Castilla y León, Valladolid, Spain; Fundación Samuél Solorzano, Salamanca, Spain; Grant number: FS/16-2008.