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Find video protocols related to scientific articles indexed in Pubmed.
The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis.
J. Leukoc. Biol.
PUBLISHED: 11-07-2014
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SM comprises a heterogeneous group of disorders, characterized by an abnormal accumulation of clonal MCs in 1 or more tissues, frequently involving the skin and BM. Despite the fact that most adult patients (>90%) carry the same genetic lesion (D816V KIT mutation), the disease presents with multiple variants with very distinct clinical and biologic features, a diverse prognosis, and different therapeutic requirements. Recent advances in the standardization of the study of BM MC by MFC allowed reproducible identification and characterization of normal/reactive MCs and their precursors, as well as the establishment of the normal MC maturational profiles. Analysis of large groups of patients versus normal/reactive samples has highlighted the existence of aberrant MC phenotypes in SM, which are essential for the diagnosis of the disease. In turn, 3 clearly distinct and altered maturation-associated immunophenotypic profiles have been reported recently in SM, which provide criteria for the distinction between ISM patients with MC-restricted and multilineage KIT mutation; thus, immunphenotyping also contributes to prognostic stratification of ISM, particularly when analysis of the KIT mutation on highly purified BM cells is not routinely available in the diagnostic work-up of the disease.
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Identification and immunophenotypic characterization of normal and pathological mast cells.
Methods Mol. Biol.
PUBLISHED: 08-24-2014
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Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 10(6) unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single-cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to study MCs from other tissues and species.
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Flow cytometry in mastocytosis: utility as a diagnostic and prognostic tool.
Immunol Allergy Clin North Am
PUBLISHED: 04-22-2014
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This article presents information for the identification and characterization of mast cells from bone marrow and other tissues using multiparametric flow cytometry. In addition, it provides guidelines for the application of this technique in the subclassification of systemic mastocytosis and assessment of the long-term prognosis of patients individually.
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The protein expression profile of meningioma cells is associated with distinct cytogenetic tumour subgroups.
Neuropathol. Appl. Neurobiol.
PUBLISHED: 02-05-2014
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Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinico-histopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour.
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Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM.
J. Allergy Clin. Immunol.
PUBLISHED: 03-19-2013
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Indolent systemic mastocytosis (ISM) without skin lesions (ISMs(-)) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs(+)). Interestingly, in almost one-half of ISMs(-) patients, MC-mediator release episodes are triggered exclusively by insects.
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CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis.
Histopathology
PUBLISHED: 02-20-2013
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CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry.
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Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis.
J. Allergy Clin. Immunol.
PUBLISHED: 02-10-2013
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Despite the fact that a great majority (>90%) of patients with systemic mastocytosis (SM) carry a common genetic lesion, the D816V KIT mutation, little is known regarding the molecular and biological pathways underlying the clinical heterogeneity of the disease.
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Serum tryptase monitoring in indolent systemic mastocytosis: association with disease features and patient outcome.
PLoS ONE
PUBLISHED: 01-01-2013
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Serum baseline tryptase (sBT) is a minor diagnostic criterion for systemic mastocytosis (SM) of undetermined prognostic impact. We monitored sBT levels in indolent SM (ISM) patients and investigated its utility for predicting disease behaviour and outcome.
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Association between inflammatory infiltrates and isolated monosomy 22/del(22q) in meningiomas.
PLoS ONE
PUBLISHED: 01-01-2013
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Meningiomas contain highly variable levels of infiltrating tissue macrophages (TiMa) and other immune cells. In this study we investigated the potential association between the number and immunophenotype of inflammatory and other immune cells infiltrating the tumor as evaluated by multiparameter flow cytometry, and the clinico-biological, cytogenetic and gene expression profile (GEP) of 75 meningioma patients. Overall, our results showed a close association between the amount and cellular composition of the inflammatory and other immune cell infiltrates and the cytogenetic profile of the tumors. Notably, tumors with isolated monosomy 22/del(22q) showed greater numbers of TiMa, NK cells and (recently)-activated CD69(+) lymphocytes versus meningiomas with diploid and complex karyotypes. In addition, in the former cytogenetic subgroup of meningiomas, tumor-infiltrating TiMa also showed a more activated and functionally mature phenotype, as reflected by a greater fraction of CD69(+), CD63(+), CD16(+) and CD33(+) cells. GEP at the mRNA level showed a unique GEP among meningiomas with an isolated monosomy 22/del(22q) versus all other cases, which consisted of increased expression of genes involved in inflammatory/immune response, associated with an M1 TiMa phenotype. Altogether, these results suggest that loss of expression of specific genes coded in chromosome 22 (e.g. MIF) is closely associated with an increased homing and potentially also anti-tumoral effect of TiMa, which could contribute to explain the better outcome of this specific good-prognosis cytogenetic subgroup of meningiomas.
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Immunophenotypic characterization of bone marrow mast cells in mastocytosis and other mast cell disorders.
Methods Cell Biol.
PUBLISHED: 07-05-2011
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Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained.
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Heme-oxygenases during erythropoiesis in K562 and human bone marrow cells.
PLoS ONE
PUBLISHED: 05-30-2011
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In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.
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Evaluation of the WHO criteria for the classification of patients with mastocytosis.
Mod. Pathol.
PUBLISHED: 05-06-2011
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Diagnosis and classification of mastocytosis is currently based on the World Health Organization (WHO) criteria. Here, we evaluate the utility of the WHO criteria for the diagnosis and classification of a large series of mastocytosis patients (n=133), and propose a new algorithm that could be routinely applied for refined diagnosis and classification of the disease. Our results confirm the utility of the WHO criteria and provide evidence for the need of additional information for (1) a more precise diagnosis of mastocytosis, (2) specific identification of new forms of the disease, (3) the differential diagnosis between cutaneous mastocytosis vs systemic mastocytosis, and (4) improved distinction between indolent systemic mastocytosis and aggressive systemic mastocytosis. Based on our results, a new algorithm is proposed for a better diagnostic definition and prognostic classification of mastocytosis, as confirmed prospectively in an independent validation series of 117 mastocytosis patients.
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Cell-cycle distribution of different cell compartments in normal versus reactive bone marrow: a frame of reference for the study of dysplastic hematopoiesis.
Cytometry B Clin Cytom
PUBLISHED: 03-04-2011
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Limited information is currently available about the proliferation activity and cell-cycle distribution of different bone marrow (BM) cell subsets defined according to their lineage and maturation stage in normal versus cytopenia-associated reactive BM samples. Here, we report a three-color flow cytometry approach to investigate the cell-cycle distribution of different BM cell compartments-CD34(+) hematopoietic progenitor and precursor cells (HPC), maturing neutrophils and monocytic cells, mature lymphocytes, eosinophils, and nucleated red blood cell precursors (NRBC)-from normal (n = 47) versus cytopenia-associated reactive (n = 47) BM samples. Highly similar proliferation profiles were detected in normal versus reactive BM, with a higher proliferation index (PI) for the more immature CD34(+) HPC, CD11b(-) maturing neutrophils and NRBC versus other BM cell compartments. The only differences observed between normal and reactive BM were restricted to the more mature (CD13(hi) /CD11b(+) ) bands/neutrophils and to monocytic cells, which showed an increased PI (0.9% ± 0.8% vs. 0.6% ± 0.5% and 6 ± 3.6 vs. 4.6 ± 4.5, respectively) at the expense of a lower PI of CD34(+) HPC in reactive conditions. Of note, bands/mature neutrophils and mature lymphocytes showed either residual numbers or absence of S + G? /M-phase cells in both normal and reactive BM. Our results suggest that a slight shift of proliferation from the early precursors to the more mature granulomonocytic compartment occurs in reactive BM, which could reflect an attempt of the hematopoietic system to rapidly produce functional neutrophils and monocytes, at the expense of a lower expansion of the minor compartments of CD34(+) HPC.
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Commentary: Comparison of current flow cytometry methods for monoclonal B cell lymphocytosis detection.
Cytometry B Clin Cytom
PUBLISHED: 09-15-2010
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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-Hodgkins 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 Lukes 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.
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Non-CLL-like monoclonal B-cell lymphocytosis in the general population: prevalence and phenotypic/genetic characteristics.
Cytometry B Clin Cytom
PUBLISHED: 09-15-2010
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Monoclonal B-cell lymphocytosis (MBL) indicates <5 × 10(9) peripheral blood (PB) clonal B-cells/L in healthy individuals. In most cases, MBL cells show similar phenotypic/genetic features to chronic lymphocytic leukemia cells-CLL-like MBL-but little is known about non-CLL-like MBL.
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Mast cells from different molecular and prognostic subtypes of systemic mastocytosis display distinct immunophenotypes.
J. Allergy Clin. Immunol.
PUBLISHED: 01-12-2010
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Systemic mastocytosis (SM) is a heterogeneous group of disorders with distinct clinical and biological behavior. Despite this, little is known about the immunophenotypic features of the distinct diagnostic categories of SM.
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Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach.
Blood
PUBLISHED: 05-06-2009
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Monoclonal B-cell lymphocytosis (MBL) indicates the presence of less than 5 x 10(9)/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 x 10(6). 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 x 10(9) 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.
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Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients.
J. Allergy Clin. Immunol.
PUBLISHED: 01-09-2009
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Indolent systemic mastocytosis is a group of rare diseases for which reliable predictors of progression and outcome are still lacking.
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Immunophenotypic identification and characterization of tumor cells and infiltrating cell populations in meningiomas.
Am. J. Pathol.
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Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.
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The proliferation index of specific bone marrow cell compartments from myelodysplastic syndromes is associated with the diagnostic and patient outcome.
PLoS ONE
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Myelodysplastic syndromes (MDS) are clonal stem cell disorders which frequently show a hypercellular dysplastic bone marrow (BM) associated with inefficient hematopoiesis and peripheral cytopenias due to increased apoptosis and maturation blockades. Currently, little is known about the role of cell proliferation in compensating for the BM failure syndrome and in determining patient outcome. Here, we analyzed the proliferation index (PI) of different compartments of BM hematopoietic cells in 106 MDS patients compared to both normal/reactive BM (n = 94) and acute myeloid leukemia (AML; n = 30 cases) using multiparameter flow cytometry. Our results show abnormally increased overall BM proliferation profiles in MDS which significantly differ between early/low-risk and advanced/high-risk cases. Early/low-risk patients showed increased proliferation of non-lymphoid CD34(+) precursors, maturing neutrophils and nucleated red blood cells (NRBC), while the PI of these compartments of BM precursors progressively fell below normal values towards AML levels in advanced/high-risk MDS. Decreased proliferation of non-lymphoid CD34(+) and NRBC precursors was significantly associated with adverse disease features, shorter overall survival (OS) and transformation to AML, both in the whole series and when low- and high-risk MDS patients were separately considered, the PI of NRBC emerging as the most powerful independent predictor for OS and progression to AML. In conclusion, assessment of the PI of NRBC, and potentially also of other compartments of BM precursors (e.g.: myeloid CD34(+) HPC), could significantly contribute to a better management of MDS.
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Prognostic Impact of del(17p) and del(22q) as assessed by interphase FISH in sporadic colorectal carcinomas.
PLoS ONE
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Most sporadic colorectal cancer (sCRC) deaths are caused by metastatic dissemination of the primary tumor. New advances in genetic profiling of sCRC suggest that the primary tumor may contain a cell population with metastatic potential. Here we compare the cytogenetic profile of primary tumors from liver metastatic versus non-metastatic sCRC.
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Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.
Am. J. Hematol.
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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 B-cell 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 naive B-cells; in addition, CD4+CD8+ double-positive T-cells and CD8+ 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/naive 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 na?ve B-cells) in MBL, which becomes more pronounced as the MBL cell count increases.
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Immunophenotyping in systemic mastocytosis diagnosis: CD25 positive alone is more informative than the CD25 and/or CD2 WHO criterion.
Mod. Pathol.
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Aberrant expression of CD2 and/or CD25 by bone marrow, peripheral blood or other extracutaneous tissue mast cells is currently used as a minor World Health Organization diagnostic criterion for systemic mastocytosis. However, the diagnostic utility of CD2 versus CD25 expression by mast cells has not been prospectively evaluated in a large series of systemic mastocytosis. Here we evaluate the sensitivity and specificity of CD2 versus CD25 expression in the diagnosis of systemic mastocytosis. Mast cells from a total of 886 bone marrow and 153 other non-bone marrow extracutaneous tissue samples were analysed by multiparameter flow cytometry following the guidelines of the Spanish Network on Mastocytosis at two different laboratories. The CD25+ and/or CD2+ bone marrow mast cells World Health Organization criterion showed an overall sensitivity of 100% with 99.0% specificity for the diagnosis of systemic mastocytosis whereas CD25 expression alone presented a similar sensitivity (100%) with a slightly higher specificity (99.2%). Inclusion of CD2 did not improve the sensitivity of the test and it decreased its specificity. In tissues other than bone marrow, the mast cell phenotypic criterion revealed to be less sensitive. In summary, CD2 expression does not contribute to improve the diagnosis of systemic mastocytosis when compared with aberrant CD25 expression alone, which supports the need to update and replace the minor World Health Organization CD25+ and/or CD2+ mast cell phenotypic diagnostic criterion by a major criterion based exclusively on CD25 expression.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.