Júlia Almeida

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

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) naïve, 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).

Although the immunophenotype of plasma cells (PCs) from multiple myeloma (MM) patients has been extensively explored, information on the phenotypic characteristics of PCs in monoclonal gammopathy of undetermined significance (MGUS) patients is scanty and frequently controversial. Thus, the question of whether or not PCs are phenotypically different in the two disorders and whether this criteria could be useful for the differential diagnosis between MGUS and MM remains to be explored. In the present study, the immunophenotypic profile of bone marrow PCs (BMPCs) from a group of 76 MGUS patients has been analyzed by flow cytometry and compared with that of BMPCs present in both MM patients (n = 65) and control subjects (n = 10). For that purpose, a large panel of monoclonal antibodies against PC-related antigens was used together with a sensitive methodology in which a minimum of 10(3) PCs were studied. In all MGUS cases studied, two clearly defined and distinct PC subpopulations could be identified. One PC subpopulation, population A (33 +/- 31% of total PCs), constantly displayed a high CD38 expression with low forward light scatter (FSC)/side light scatter (SSC) and was positive for CD19 and negative for CD56 (only a small proportion of these PCs were weakly positive for CD56). The other PC subpopulation, population B (67 +/- 31% of total PCs), showed the opposite pattern; the antigen CD56 was strongly positive and CD19 was constantly negative, and it showed a lower CD38 expression and higher FSC/SSC values than population A. Clonality studies (cytoplasmic light chain restriction, DNA content studies, and polymerase chain reaction assessment) confirmed the clonal nature of PCs from population B and the polyclonal origin of PCs from population A. Moreover, the polyclonal PCs from MGUS displayed a phenotypic profile identical to that found in PCs from healthy individuals. By contrast, clonal PCs from all MGUS patients displayed a similar antigenic profile to myelomatous PCs, with clear phenotypic differences with respect to normal PCs: lower intensity of CD38 expression and a variable reactivity for markers that were not expressed in normal PCs, such as CD28, CD117, and sIg. Although the presence of residual polyclonal PCs was a constant finding in MGUS patients, it was a rare event in MM and, when present (only 22% of MM cases), its frequency was significantly lower than that observed in MGUS (0.25% versus 32.9%, respectively; P < 0.0001). Only 1.5% of patients with MM had more than 3% of normal PCs, whereas 98% of patients with MGUS had more than 3%. Moreover, as shown by multivariate analysis, the number of residual polyclonal PCs was the most powerful single parameter for the discrimination between MGUS and MM patients at diagnosis, even when only stage I MM cases were considered.