Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

No abstract

Primary cutaneous B-cell lymphomas are a heterogeneous group of mature B-cells neoplasms with tropism for the skin, whose biology and clinical course differ significantly from the equivalent nodal lymphomas. The most indolent forms comprise the primary cutaneous marginal zone and follicle center B-cell lymphomas that despite the excellent prognosis have cutaneous recurrences very commonly. The most aggressive forms include the primary cutaneous large B-cell lymphomas, consisting in two major groups: the leg type, with poor prognosis, and others, the latter representing a heterogeneous group of lymphomas from which specific entities are supposed to be individualized over time, such as intravascular large B-cell lymphomas. Treatment may include surgical excision, radiotherapy, antibiotics, corticosteroids, interferon, monoclonal antibodies and chemotherapy, depending on the type of lymphoma and on the type and location of the skin lesions. In subtypes with good prognosis is contraindicated overtreatment and in those associated with a worse prognosis the recommended therapy relies on CHOP-like regimens associated with rituximab, assisted or not with local radiotherapy. We review the primary cutaneous B-cell lymphomas, remembering the diagnostic criteria, differential diagnosis, classification, and prognostic factors and presenting the available therapies.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of leukemia/lymphoma, whose diagnosis can be difficult to achieve due to its clinical and biological heterogeneity, as well as its overlapping features with other hematologic malignancies. In this study we investigated whether the association between the maturational stage of tumor cells and the clinico-biological and prognostic features of the disease, based on the analysis of 46 BPDCN cases classified into three maturation-associated subgroups on immunophenotypic grounds. Our results show that blasts from cases with an immature plasmacytoid dendritic cell (pDC) phenotype exhibit an uncommon CD56− phenotype, coexisting with CD34+ non-pDC tumor cells, typically in the absence of extramedullary (e.g. skin) disease at presentation. Conversely, patients with a more mature blast cell phenotype more frequently displayed skin/extramedullary involvement and spread into secondary lymphoid tissues. Despite the dismal outcome, acute lymphoblastic leukemia-type therapy (with central nervous system prophylaxis) and/or allogeneic stem cell transplantation appeared to be the only effective therapies. Overall, our findings indicate that the maturational profile of pDC blasts in BPDCN is highly heterogeneous and translates into a wide clinical spectrum -from acute leukemia to mature lymphoma-like behavior-, which may also lead to variable diagnosis and treatment.

Background Resistance to recombinant human erythropoietin (rhEPO) occurs in some chronic kidney disease (CKD) patients, which may be due to enhanced systemic inflammatory response and to the erythropoiesis-suppressing effect of pro-inflammatory cytokines, some of which are produced by T cells. Aim of study The aim of this study was to investigate the relationship between resistance to rhEPO therapy in hemodialysis CKD patients and inflammatory markers [Creactive protein (CRP), soluble interleukin (IL)-2 receptor (sIL2R), and serum albumin levels], blood cell counts, Tcell phenotype, cytokine production by T cells, and serum cytokine levels. Materials and Methods We studied 50 hemodialysis CKD patients, 25 responders and 25 nonresponders to rhEPO, and compared them to each other and with 25 healthy controls. When compared to controls, CKD patients showed increased serum levels of CRP, IL-6, and sIL2R and a T-cell lymphopenia, due to decreased numbers of both CD4 + and CD8 + T cells. T cells from CKD patients had an immunophenotype compatible with chronic T-cell stimulation as shown by the increased percentage of CD28

Mucosal T lymphocytes from patients with ulcerative colitis (UC) were previously shown to display a deficiency in branched N-glycosylation associated with disease severity. However, whether this glycosylation pathway shapes the course of the T cell response constituting a targeted-specific mechanism in UC remains largely unknown. In this study, we demonstrated that metabolic supplementation of ex vivo mucosal T cells from patients with active UC with -acetylglucosamine (GlcNAc) resulted in enhancement of branched N-glycosylation in the T cell receptor (TCR), leading to suppression of T cell growth, inhibition of the T helper 1 (Th1)/Th17 immune response, and controlled T cell activity. We further demonstrated that mouse models displaying a deficiency in the branched N-glycosylation pathway (, ) exhibited increased susceptibility to severe forms of colitis and early-onset disease. Importantly, the treatment of these mice with GlcNAc reduced disease severity and suppressed disease progression due to a controlled T cell-mediated immune response at the intestinal mucosa. In conclusion, our human ex vivo and preclinical results demonstrate the targeted-specific immunomodulatory properties of this simple glycan, proposing a therapeutic approach for patients with UC.

Monoclonal TCR␣␤ ؉ /CD4 ؉ T-large granular lymphocyte (T-LGL) lymphocytosis is a T-cell disorder with a restricted TCR-V␤ repertoire. In the present study we explored the potential association between the expanded TCR-V␤ families, the CDR3 sequences of the TCR-V␤ gene, and the HLA genotype of patients with monoclonal TCR␣␤ ؉ /CD4 ؉ T-LGL lymphocytosis.

Indolent natural killer (NK) cell lymphoproliferative disorders include a heterogeneous group of patients in whom persistent expansions of mature, typically CD56 ؉ , NK cells in the absence of any clonal marker are present in the peripheral blood. In the present study we report on the clinical, hematological, immunophenotypic, serological, and molecular features of a series of 26 patients with chronic large granular NK cell lymphocytosis, whose NK cells were either CD56 ؊ or expressed very low levels of CD56 (CD56 ؊/؉dim NK cells), in the context of an aberrant activation-related mature phenotype and proved to be monoclonal using the human androgen receptor gene polymerase chain reaction-based assay. As normal CD56 ؉ NK cells, CD56 ؊/؉dim NK cells were granzyme B ؉ , CD3 ؊ , TCR␣␤/ ␥␦ ؊ , CD5 ؊ , CD28 ؊ , CD11a ؉bright , CD45RA ؉bright , CD122 ؉ , and CD25 ؊ and they showed variable and heterogeneous expression of both CD8 and CD57. Nevertheless, they displayed several unusual immunophenotypic features. Accordingly, besides being CD56 ؊/؉dim , they were CD11b ؊/؉dim (heterogeneous), CD7 ؊/؉dim (heterogeneous), CD2 ؉ (homogeneous), CD11c ؉bright (homogeneous), and CD38 ؊/؉dim (heterogeneous). Moreover, CD56 ؊/؉dim NK cells heterogeneously expressed HLA-DR. In that concerning the expression of killer receptors, CD56 ؊/؉dim NK cells showed bright and homogeneous CD94 expression, and dim and heterogeneous reactivity for CD161, whereas CD158a and NKB1 expression was variable. From the functional point of view, CD56 ؊/؉dim showed a typical Th1 pattern of cytokine production (interferon-␥ ؉ , tumor necrosis factor-␣ ؉ ). From the clinical point of view, these patients usually had an indolent clinical course, progression into a massive lymphocytosis with lung infiltration leading to death being observed in only one case. Despite this, they frequently had associated cytopenias as well as neoplastic diseases and/or viral infections. In summary, we describe a unique and homogeneous group of monoclonal chronic large granular NK cell lymphocytosis with an aberrant activation-related CD56 ؊/؉dim /CD11b ؊/؉dim phenotype and an indolent clinical course, whose main clinical features are related to concomitant diseases. (Am J