Martin Perez-Andres

• University of Salamanca

Objectives The development of External Quality Assessment Schemes (EQAS) for clinical flow cytometry (FCM) is challenging in the context of rare (immunological) diseases. Here, we introduce a novel EQAS monitoring the primary immunodeficiency Orientation Tube (PIDOT), developed by EuroFlow, in both a ‘wet’ and ‘dry’ format. This EQAS provides feedb...

Introduction Good syndrome (GS) is a rare adult-onset immunodeficiency first described in 1954. It is characterized by the coexistence of a thymoma and hypogammaglobulinemia, associated with an increased susceptibility to infections and autoimmunity. The classification and management of GS has been long hampered by the lack of data about the underl...

Background Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Obj...

Low‐count monoclonal B‐cell lymphocytosis (MBLlo) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID‐19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID‐19 of MBLlo versus non‐MBL patients. For this study, we analyzed the kinetics of i...

New lignohydroquinone conjugates (L-HQs) were designed and synthesized using the hybridization strategy, and evaluated as cytotoxics against several cancer cell lines. The L-HQs were obtained from the natural product podophyllotoxin and some semisynthetic terpenylnaphthohydroquinones, prepared from natural terpenoids. Both entities of the conjugate...

Introduction Multiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can ide...

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole-cell to acellular pertussis vaccines, although other factors may also contribute. Here, we applied high-dimensional flow cytometry t...

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole cell to acellular pertussis vaccines, although other factors may also contribute. To develop future vaccines and improve current vac...

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput mu...

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells...

Introduction Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memo...

Background Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informat...

Natural products are the ideal basis for the design of novel efficient molecular entities. Podophyllotoxin, a naturally occurring cyclolignan, is an example of natural product which displays a high versatility from a biological activity point of view. Based on its unique chemical structure, different derivatives have been synthesized presenting the...

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell s...

Cancer dissemination and distant metastasis most frequently require the release of tumor cells into the blood circulation, both in solid tumors and most hematological malignancies, including plasma cell neoplasms. However, detection of blood circulating tumor cells in solid tumors and some hematological malignancies, such as the majority of mature/...

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discus...

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sens...

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with sur...

Monozygotic twins provide a unique opportunity to better understand complex genetic diseases and the relative contribution of heritable factors in shaping the immune system throughout life. Common Variable Immunodeficiency Disorders (CVID) are primary antibody defects displaying wide phenotypic and genetic heterogeneity, with monogenic transmission...

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first...

Idiopathic T-CD4 lymphocytopenia (ICL) is a rare and heterogeneous syndrome characterized by opportunistic infections due to reduced CD4 T-lymphocytes (<300 cells/μl or <20% T-cells) in the absence of HIV infection and other primary causes of lymphopenia. Molecular testing of ICL has revealed defects in genes not specific to CD4 T-cells, with pleio...

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapp...

Flow cytometry immunophenotyping is essential for diagnosis, classification and monitoring of clonal hematopoietic diseases, particularly of hematological malignancies and primary immunodeficiencies. Optimal use of immunophenotyping for these purposes requires detailed knowledge about the phenotypic patterns of normal hematopoietic cells. In the pa...

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to...

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the flu...

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for...

Currently, it is well established that monocytes are a heterogeneous type of cell consisting of phenotypically and functionally distinct subpopulations found to be numerically altered in blood in patients with a wide variety of disease conditions, such as infection, autoimmunity, respiratory and cardiovascular diseases, and inflammatory disorders.1...

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression o...

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the d...

Despite the recent progress in treatment of multiple myeloma (MM), it is still an incurable malignant disease, and we are therefore in need of new risk stratification tools that can help us to understand the disease and optimize therapy. Here we propose a new subtyping of myeloma plasma cells (PCs) from diagnostic samples, assigned by normal B-cell...

A new family of molecular hybrids, between cyclolignans related to podophyllic aldehyde and several diterpenylnaphthohydroquinones (DNHQ), was prepared and its biological activity evaluated in several human solid tumor cell lines, which are representatives of the most prevalence solid tumors in Western world. Both, cyclolignan and quinone fragments...

Background: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity and neoplasia. Immunoglobulin heavy chain (IgH)-isotype serum levels can partly explain such age-related differences, but their relationship with the IgH-isotype distribution within memory...

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG a...

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orien...

Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive disease resulting from variants in the WAS gene, characterized by a triad of immunodeficiency, eczema, and thrombocytopenia. Despite the fact that WAS is traditionally differentiated from immune thrombocytopenia (ITP) by small size of WAS platelets, in practice, microthrombocytopenia may o...

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19–/CD45low/–/CD38high/CD1...

Resumen Concepto Las inmunodeficiencias primarias (IDP) son enfermedades hereditarias muy heterogéneas, producidas en su mayoría por defectos en un gen que condiciona una alteración cuantitativa y/o cualitativa del sistema inmune y, en consecuencia, mayor riesgo de infecciones por gérmenes encapsulados u oportunistas, fenómenos autoinmunes o alérg...

Waldenstrom’s macroglobulinaemia (WM) is an MYD88L265P-mutated lymphoplasmacytic lymphoma that invades bone marrow and secretes monoclonal immunoglobulin M (IgM). WM cells are usually unable to undergo class switch recombination, and have mutated IGHV, with a typical immunophenotype CD19⁺/CD22low+/CD23–/CD25⁺/CD27⁺/CD45⁺/CD38low+/SmIgM⁺ (negative f...

Although major steps have been recently made in understanding the role of the distinct subsets of dendritic cells (DC)/antigen-presenting cells (APC), further studies are required to unravel their precise role, including in-depth immunophenotypic characterisation of these cells. Here, we used eight-colour flow cytometry to investigate the reactivit...

Background: Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings. Methods: Here we evaluate the efficiency of consensu...

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO...

A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a...

Breast cancer is a major cause of mortality in women. The transcription factor SNAI2 has been implicated in the pathogenesis of several types of cancer, including breast cancer of basal origin. Here we show that SNAI2 is also important in the development of breast cancer of luminal origin in MMTV-ErbB2 mice. SNAI2 deficiency leads to longer latency...

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The B-cell compartment where the multiple myeloma (MM) stem cell resides remains unclear. For more than a decade, it has been generally accepted that a less differentiated B-cell is responsible for the outgrowth of the more differentiated monoclonal plasma cell (M-PC) compartment. In line with this hypothesis, circulating peripheral blood (PB) CD19...

This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. N...

The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with...

Introduction It is currently well-known that B-cell chronic lymphocytic leukemia (CLL) patients have an impaired immune function -particularly in advanced disease-, which significantly contributes to a higher risk of infections. The introduction of new effective therapeutic agents, such as the purine analogues (or alkylating agents with concomitant...

Introduction Management of B-cell chronic lymphocytic leukemia (CLL) is currently undergoing profound changes. Accordingly, new treatment options with an expected less toxicity than standard regimens are been explored. Recent results show that chemoimmunotherapy may improve the life expectancy of CLLpatients and has proven to be more efficient than...

Sos1 and Sos2 are ubiquitously expressed, universal Ras-GEFs acting in multiple signal transduction pathways activated by upstream cellular kinases. The embryonic lethality of Sos1 null mutants has hampered ascertaining the specific in vivo contributions of Sos1 and Sos2 to processes controlling adult organism survival or development of hematopoiet...

3945 Background Recurrent immunoglobulin heavy chain (IGH) translocations - t(14q32) - such as t(4;14) or t(11;14) are considered to be primary cytogenetic events which play an important role in the pathogenesis of clonal plasma cell (PC) disorders. However, previous cytogenetic studies in these diseases have mainly been done in whole bone marrow...

Background. Cytogenetic studies in clonal plasma cell diseases have mainly been done in whole bone marrow or CD138+ microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) - are primary oncogenetic events. The aim of this study is to determine cytogenetic patterns of highly purified aberra...

Background. Multiple myeloma remains largely incurable. However, a few patients experience ≥10-years relapse-free survival and can be considered as operationally cured. Interestingly, long-term disease control in multiple myeloma is not restricted to patients with complete response, since some revert into an MGUS profile. Design and Methods. We com...

Analysis of the immune system of multiple myeloma patients achieving long-term disease control, by multidimensional flow cytometry

Background. Multiple myeloma remains largely incurable. However, a few patients experience ≥10-years relapse-free survival and can be considered as operationally cured. Interestingly, long-term disease control in multiple myeloma is not restricted to patients with complete response, since some revert into an MGUS profile. Design and Methods. We com...

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 nu...

To the Editor: We recently read with great interest an article in The Journal of Experimental Medicine from the [laboratory of Thomas Rothstein][1] ([Griffin et al., 2011][2]). This paper described a new B cell population present in human cord blood and adult peripheral blood that displays

The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory...