Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies

Primary immunodeficiencies are the result of biological defects associated with functional immune abnormalities. It consists of a group of disorders showing a higher incidence and severity of infections, expression of immunological dysregulation such as inflammation and lymphoproliferation. The immunophenotyping and in vitro functional characterization of immunodeficient patients contribute, together with the clinical aspects, to define the underlying immune defect particularities. Flow cytometry applications in primary immunodeficiency assessment are multiple and include the study of a wide range of specific cell lymphocyte subpopulations. This chapter describes the main techniques used in the diagnosis of a wide variety of primary immunodeficiencies, in which intracellular proteins or activation markers involved in immunity are evaluated, as well as functional proliferation, cytokine production, phosphorylation of transcription factors, cytotoxic and degranulation capacity. Flow cytometry is a tool that allows rapid and accurate evaluation of multiple lymphocyte populations and immunological function, and this information is essential for the diagnosis and evaluation of patients with primary immunodeficiencies

El uso de la metodología Art Thinking para trabajar las emociones en el aula a través del color en Educación Secundaria: una propuesta de intervención didáctica

La comunicación presenta una propuesta de intervención cuyo objetivo es trabajar, a través del color, las emociones y la conciencia emocional a través del Art Thinking en el primer curso de la E.S.O. en la materia de Educación Plástica, Visual y Audiovisual. En esta propuesta se apuesta por unir el arte con las temáticas relevantes a nivel social haciendo uso de la educación emocional como contenido transversal y fomentando la implicación y la reflexión del alumnado sobre las problemáticas de la sociedad actual. Además de las temáticas trasversales previamente comentadas -guerra, violencia de género y cuidado del medio ambiente, entre otras- se ha partido de los contenidos que establece el curriculum para primer ciclo de la E.S.O. según la Orden del 15 de enero de 2021. Estos forman parte del bloque I (Expresión plástica) y del bloque II (Comunicación audiovisual), específicamente el color y su naturaleza y el lenguaje visual y audiovisual. La propuesta se lleva a cabo en seis sesiones en las que se ha hecho uso del Art Thinking (Acaso & Megías, 2017) como metodología didáctica, favoreciendo el protagonismo del alumnado en su propio aprendizaje. En cada una de las sesiones se trabaja un color y una emoción diferente asociada al mismo y el alumno podrá expresar sus emociones y relacionarlas con los colores que les evoquen las distintas situaciones planteadas a través del dibujo. La evaluación de los aprendizajes se realizará a través del portafolios en el que el alumnado recogerá sus vivencias y este será calificado haciendo uso de una rúbrica. También se incluye el diseño de la evaluación de la propuesta para conocer el nivel de satisfacción del alumnado.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

International retrospective study of allogeneic hematopoietic cell transplantation for activated PI3K-delta syndrome

Immunodeficiència primària; Limfoproliferació; Inhibidor de mTORInmunodeficiencia primaria; Linfoproliferación; Inhibidor de mTORPrimary immunodeficiency; Lymphoproliferation; MTOR inhibitorBackground Activated phosphoinositide 3-kinase delta syndrome (APDS) is a combined immunodeficiency with a heterogeneous phenotype considered reversible by allogeneic hematopoietic cell transplantation (HCT). Objectives This study sought to characterize HCT outcomes in APDS. Methods Retrospective data were collected on 57 patients with APDS1/2 (median age, 13 years; range, 2-66 years) who underwent HCT. Results Pre-HCT comorbidities such as lung, gastrointestinal, and liver pathology were common, with hematologic malignancy in 26%. With median follow-up of 2.3 years, 2-year overall and graft failure–free survival probabilities were 86% and 68%, respectively, and did not differ significantly by APDS1 versus APDS2, donor type, or conditioning intensity. The 2-year cumulative incidence of graft failure following first HCT was 17% overall but 42% if mammalian target of rapamycin inhibitor(s) (mTORi) were used in the first year post-HCT, compared with 9% without mTORi. Similarly, 2-year cumulative incidence of unplanned donor cell infusion was overall 28%, but 65% in the context of mTORi receipt and 23% without. Phenotype reversal occurred in 96% of evaluable patients, of whom 17% had mixed chimerism. Vulnerability to renal complications continued post-HCT, adding new insights into potential nonimmunologic roles of phosphoinositide 3-kinase not correctable through HCT. Conclusions Graft failure, graft instability, and poor graft function requiring unplanned donor cell infusion were major barriers to successful HCT. Post-HCT mTORi use may confer an advantage to residual host cells, promoting graft instability. Longer-term post-HCT follow-up of more patients is needed to elucidate the kinetics of immune reconstitution and donor chimerism, establish approaches that reduce graft instability, and assess the completeness of phenotype reversal over time.This research was funded in part from the Intramural Program of the National Cancer Institute, National Institutes of Health. The funding source had no involvement in study design; collection, analysis, and interpretation of data; writing of the report; or in the decision to submit the article for publication

Biological brain age prediction using machine learning on structural neuroimaging data: Multi-cohort validation against biomarkers of Alzheimer’s disease and neurodegeneration stratified by sex

Brain-age can be inferred from structural neuroimaging and compared to chronological age (brain-age delta) as a marker of biological brain aging. Accelerated aging has been found in neurodegenerative disorders like Alzheimer’s disease (AD), but its validation against markers of neurodegeneration and AD is lacking. Here, imaging-derived measures from the UK Biobank dataset (N=22,661) were used to predict brain-age in 2,314 cognitively unimpaired (CU) individuals at higher risk of AD and mild cognitive impaired (MCI) patients from four independent cohorts with available biomarker data: ALFA+, ADNI, EPAD and OASIS. Brain-age delta was associated with abnormal amyloid-b, more advanced stages (AT) of AD pathology and APOE-e4 status. Brain-age delta was positively associated with plasma neurofilament light, a marker of neurodegeneration, and sex differences in the brain effects of this marker were found. These results validate brain-age delta as a non-invasive marker of biological brain aging related to markers of AD and neurodegeneration.The project leading to these results has received funding from “la Caixa” Foundation (ID 100010434), under agreement LCF/PR/GN17/50300004 and the Alzheimer’s Association and an international anonymous charity foundation through the TriBEKa Imaging Platform project (TriBEKa-17-519007). Additional support has been received from the Universities and Research Secretariat, Ministry of Business and Knowledge of the Catalan Government under the grant no. 2017-SGR-892 and the Spanish Research Agency (AEI) under project PID2020-116907RB-I00 of the call MCIN/ AEI /10.13039/501100011033. FB is supported by the NIHR biomedical research center at UCLH. MSC receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 948677), the Instituto de Salud Carlos III (PI19/00155), and from a fellowship from ”la Caixa” Foundation (ID 100010434) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 847648 (LCF/BQ/PR21/11840004).Report de recerca signat per 27 autors/es: Irene Cumplido-Mayoral 1,2; Marina García-Prat 1; Grégory Operto 1,3,4; Carles Falcon 1,3,5; Mahnaz Shekari 1,2,3; Raffaele Cacciaglia 1,3,4; Marta Milà-Alomà 1,2,3,4; Luigi Lorenzini 6; Silvia Ingala 6; Alle Meije Wink 6; Henk JMM Mutsaerts 6; Carolina Minguillón 1,3,4; Karine Fauria 1,4; José Luis Molinuevo 1; Sven Haller 7; Gael Chetelat 8,10; Adam Waldman 9; Adam Schwarz 10; Frederik Barkhof 6,11; Ivonne Suridjan 12, 11; Gwendlyn Kollmorgen 13; Anna Bayfield 13; Henrik Zetterberg 14,15,16,17,18; Kaj Blennow 14,15 12; Marc Suárez-Calvet 1,3,4,19; Verónica Vilaplana 20; Juan Domingo Gispert 1,3,5; ALFA study; EPAD study; ADNI study; OASIS study // 1) Barcelonaβeta Brain Research Center (BBRC), Pasqual Maragall Foundation, Barcelona, Spain; 2) Universitat Pompeu Fabra, Barcelona, Spain; 3) IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain; 4) CIBER Fragilidad y Envejecimiento Saludable (CIBERFES), Madrid, Spain; 5) Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain; 6) Department of Radiology and Nuclear Medicine, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands; 7) CIRD Centre d'Imagerie Rive Droite, Geneva, Switzerland; 8) Normandie Univ, UNICAEN, INSERM, U1237, PhIND "Physiopathology and Imaging of Neurological Disorders", Institut Blood and Brain @ Caen-Normandie, Cyceron, Caen, France; 9) Centre for Dementia Prevention, Edinburgh Imaging, and UK Dementia Research Institute at The University of Edinburgh, Edinburgh, UK; 10) Takeda Pharmaceutical Company Ltd, Cambridge, MA, USA; 11) Institutes of Neurology and Healthcare Engineering, University College London, London, UK; 12) Roche Diagnostics International Ltd, Rotkreuz, Switzerland; 13) Roche Diagnostics GmbH, Penzberg, Germany; 14) Institute of Neuroscience and Physiology, University of Gothenburg, Mölndal, Sweden; 15) Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden; 16) Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, United Kingdom; 17) UK Dementia Research Institute at UCL, London, United Kingdom; 18) Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China; 19) Servei de Neurologia, Hospital del Mar, Barcelona, Spain; 20) Department of Signal Theory and Communications, Universitat Politècnica de Catalunya, Barcelona, Spain.Preprin

Newborn Screening for SCID: Experience in Spain (Catalonia)

Linfocitos T; Cribado de recién nacidos; Inmunodeficiencia combinada severaLimfòcits T; Cribratge de nounats; Immunodeficiència combinada severaT-lymphocytes; Newborn screening; Severe combined immunodeficiencyNewborn screening (NBS) for severe combined immunodeficiency (SCID) started in Catalonia in January-2017, being the first Spanish and European region to universally include this testing. In Spain, a pilot study with 5000 samples was carried out in Seville in 2014; also, a research project with about 35,000 newborns will be carried out in 2021–2022 in the NBS laboratory of Eastern Andalusia. At present, the inclusion of SCID is being evaluated in Spain. The results obtained in the first three and a half years of experience in Catalonia are presented here. All babies born between January-2017 and June-2020 were screened through TREC-quantification in DBS with the Enlite Neonatal TREC-kit from PerkinElmer. A total of 222,857 newborns were screened, of which 48 tested positive. During the study period, three patients were diagnosed with SCID: an incidence of 1 in 74,187 newborns; 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns who also benefited from the NBS program. The results obtained provide further evidence of the benefits of early diagnosis and curative treatment to justify the inclusion of this disease in NBS programs. A national NBS program is needed, also to define the exact SCID incidence in Spain

First universal newborn screening program for severe combined immunodeficiency in Europe: two-years' experience in Catalonia (Spain)

Newborn screening; Severe combined immunodeficiency; T-cell receptor excision circlesCribratge de nadons; Immunodeficiència combinada greu; Cercles d'excisió de receptors de cèl·lules TCribado de recién nacidos; Inmunodeficiencia combinada grave; Círculos de escisión de receptores de células TSevere combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first 2 years of experience are evaluated here. All babies born between January 2017 and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In 2018, the retest cutoff in the detection algorithm was updated based on the experience gained in the first year, and changed from 34 to 24 copies/μL. This decreased the retest rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αβ and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 tested positive, 15 of which were male. During the study period, one patient was diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 newborns (43% of positive detections). Nine patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TRECs between 3 and 6 months of life, four infants had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia

Early Diagnosis and Treatment of Purine Nucleoside Phosphorylase (PNP) Deficiency through TREC-Based Newborn Screening

Newborn screening; Severe combined immunodeficiencyCribatge nounat; Immunodeficiència combinada severaCribado neonato; Inmunodeficiencia combinada gravePurine nucleoside phosphorylase (PNP) deficiency is a rare inherited disorder, resulting in severe combined immunodeficiency. To date, PNP deficiency has been detected in newborn screening only through the use of liquid chromatography tandem mass spectrometry. We report the first case in which PNP deficiency was detected by TREC analysis.This research was funded by Jeffrey Modell Foundation

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report

Chromosomal rearrangements; Primary immunodeficiencies; Syndromic immunodeficienciesReordenacions cromosòmiques; Immunodeficiències primàries; Immunodeficiències sindròmiquesReordenamientos cromosómicos; Inmunodeficiencias primarias; Inmunodeficiencias sindrómicasSyndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient’s CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient’s complex clinical phenotype.This study was funded by Instituto de Salud Carlos III, grants PI17/00660 and PI20/00761, cofinanced by the European Regional Development Fund (ERDF). This study was also funded by the Jeffrey Modell Foundation. This work is supported by the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases Network (ERN-RITA)

Expanding the clinical and genetic spectra of primary immunodeficiency-related disorders with clinical exome sequencing: expected and unexpected findings

Inmunodeficiencias primarias; Secuenciación de próxima generación; Secuenciación clínica del exomaImmunodeficiències primàries; Seqüenciació de propera generació; Seqüenciació clínica d’exomesPrimary immunodeficiencies; Next generation sequencing; Clinical exome sequencingPrimary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically heterogeneous group of over 350 disorders affecting development or function of the immune system. The increasing use of next-generation sequencing (NGS) technology has greatly facilitated identification of genetic defects in PID patients in daily clinical practice. Several NGS approaches are available, from the unbiased whole exome sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases provided interesting and somewhat unexpected findings that expand the clinical and genetic spectra of PID-related disorders, and are useful to consider in the differential diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders.This study was funded by Instituto de Salud Carlos III, grants PI14/00405 and PI17/00660, cofinanced by the European Regional Development Fund (ERDF)

Early Diagnosis and Treatment of Purine Nucleoside Phosphorylase (PNP) Deficiency through TREC-Based Newborn Screening

Purine nucleoside phosphorylase (PNP) deficiency is a rare inherited disorder, resulting in severe combined immunodeficiency. To date, PNP deficiency has been detected in newborn screening only through the use of liquid chromatography tandem mass spectrometry. We report the first case in which PNP deficiency was detected by TREC analysis