89 research outputs found
EuroFlow-based flowcytometric diagnostic screening and classification of primary immunodeficiencies of the lymphoid system
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 innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naive pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed >= 8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide
The EuroFlow PID orientation tube for flow cytometric diagnostic screening of primary immunodeficiencies of the lymphoid system
Copyright © 2019 van der Burg, Kalina, Perez-Andres, Vlkova, Lopez-Granados, Blanco, Bonroy, Sousa, Kienzler, Wentink, Mejstríková, Šinkorova, Stuchly, van Zelm, Orfao and van Dongen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.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 all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.The coordination and innovation processes of this study were supported by the EuroFlow Consortium (Chairmen: MvdB and AO). MvZ is supported by Senior Research Fellowship GNT1117687 from the Australian National Health and Medical Research Council. TK and EM were supported by projects 15-28541A from Ministry of Health, LO1604 from Ministry of Education, Youth and Sports and GBP302/12/G101 from Grant Agency of the Czech Republic. MP-A, EB, and AO were supported by a grant from the Junta de Castilla y León (Fondo Social Europeo, ORDEN EDU/346/2013, Valladolid, Spain) and the CB16/12/00400 grant (CIBER/ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, - Madrid, Spain- and FONDOS FEDER), the FIS PI12/00905-FEDER grant (Fondo de Investigación Sanitaria of Instituto de Salud Carlos III, Madrid, Spain) and AP119882013 grant (Fundación Mutua Madrileña, Madrid, Spain). Publishing costs for this article were covered by the International Union of Immunological Societies (IUIS).info:eu-repo/semantics/publishedVersio
ФЕКАЛЬНЫЙ КАЛЬПРОТЕКТИН В ДИФФЕРЕНЦИАЛЬНОЙ ДИАГНОСТИКЕ ОСТРЫХ КИШЕЧНЫХ ИНФЕКЦИЙ
Infectious diarrhea takes a leading position in all infectious diseases worldwide. As a result of the pathogens diversity, patient-specific diagnosis and treatments are needed. Additional laboratory tests are crucial for differentiation among pathogens, as routine bacteriological and virological methods are time-consuming. One of them is fecal calprotectin.Aim: examination of the potential role of fecal calprotectin in infectious diarrhea.Materials and methods: the national and international medical literature sources were analyzed.Results: the data regarding fecal calprotectins’ structure, functional activity and diagnostic value are presented.Conclusions: the study of fecal calprotectin, as a correlative biomarker of bacterial gastroenteritis, can be helpful for clinicians with diagnostic approach to the patient.Введение: острые кишечные инфекции с диарейным синдромом занимают лидирующее место в инфекционной патологии в мировом масштабе. Разнообразие возбудителей заставляет искать индивидуальный диагностический и терапевтический подход к пациентам. Рутинные бактериологические и вирусологические методы занимают продолжительное время до получения результатов, поэтому введение дополнительных лабораторных показателей, помогающих клиницистам при дифференциальной диагностике, было бы практически важным. Один из таких маркеров – фекальный кальпротектин.Цель: изучение роли фекального кальпротектина в диагностике острых кишечных инфекций с диарейным синдромом.Материалы и методы: проанализированы различные литературные источники по указанной проблеме, доступные в национальной и международной медицинской литературе.Результаты: представлены данные относительно структуры, функциональной активности и диагностической роли фекального кальпротектина при кишечных инфекциях.Заключение: исследование фекального кальпротектина помогает дифференциально- диагностическому процессу у пациентов с острыми кишечными инфекциями, коррелируя с бактериальной этиологией заболевания
СИНДРОМ ЭЙДИ КАК ОСЛОЖНЕНИЕ ВЕТРЯНОЙ ОСПЫ
Adie syndrome is a rare complication of chickenpox, which is seen in female mainly up to 6 years of age, one week after vesicular rash appearance.Aim: to introduce a case report of Adie syndrome as a complication of chickenpox.Material and methods: a case report of 8 years old female is introduced, who was hospitalized in Infectious Diseases Department — Varna, because of chickenpox. Anisocoria was diagnosed while the disease. We used epidemiologic and documental methods. Analyzes was made in the relevant departments of the St. Marina Hospital. The international literature data associated with the problem was studied.Results: we describe a patient with chickenpox. On the 9th day of the disease, Adie syndrome was diagnosed on the base of next symptoms: left mydriasis, accommodation paralysis, absent light reaction.Conclusion: Adie syndrome is a rare complication of chickenpox, with self-limitation tendency in the period of a year, but when early diagnosis is made together with specific treatment, normalization of eye functions occurs earlier. Синдром Эйди, редкое осложнение ветряной оспы, наблюдается, главным образом, у девочек до 6 лет через неделю после появления высыпаний.Цель: представить клинический случай пациента с синдромом Эйди как осложнения ветряной оспы.Материалы и методы: наблюдалась 8-летняя девочка, госпитализированная в Инфекционную больницу г. Варны, Болгария, по поводу пустулёзной формы ветряной оспы, осложнённой анизокорией. Использованы клинико-эпидемиологические и документальные методы. Все исследования проведены в соответствующих структурных звеньях стационара. Проанализированы данные по вышеупомянутой проблематике, доступные в международной периодической литературе.Результаты: на 9-ый день с момента появления сыпи были установлены клинические признаки синдрома Эйди: левосторонний мидриаз, паралич аккомодации и замедленная реакция зрачков на свет.Заключение: синдром Эйди — редкое осложнение ветряной оспы, проявляющее тенденцию к саморазрешению в течение одного года, но при ранней постановке диагноза и адекватном лечении нормализация зрительной функции наступает гораздо раньше.
Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum
BACKGROUND: Antibody responses to sand fly saliva have been suggested to be a useful marker of exposure to sand fly bites and Leishmania infection and a potential tool to monitor the effectiveness of entomological interventions. Exposure to sand fly bites before infection has also been suggested to modulate the severity of the infection. Here, we test these hypotheses by quantifying the anti-saliva IgG response in a cohort study of dogs exposed to natural infection with Leishmania infantum in Brazil. METHODS: IgG responses to crude salivary antigens of the sand fly Lutzomyia longipalpis were measured by ELISA in longitudinal serum samples from 47 previously unexposed sentinel dogs and 11 initially uninfected resident dogs for up to 2 years. Antibody responses were compared to the intensity of transmission, assessed by variation in the incidence of infection between seasons and between dogs. Antibody responses before patent infection were then compared with the severity of infection, assessed using tissue parasite loads and clinical symptoms. RESULTS: Previously unexposed dogs acquired anti-saliva antibody responses within 2 months, and the rate of acquisition increased with the intensity of seasonal transmission. Over the following 2 years, antibody responses varied with seasonal transmission and sand fly numbers, declining rapidly in periods of low transmission. Antibody responses varied greatly between dogs and correlated with the intensity of transmission experienced by individual dogs, measured by the number of days in the field before patent infection. After infection, anti-saliva antibody responses were positively correlated with anti-parasite antibody responses. However, there was no evidence that the degree of exposure to sand fly bites before infection affected the severity of the infection. CONCLUSIONS: Anti-saliva antibody responses are a marker of current transmission intensity in dogs exposed to natural infection with Leishmania infantum, but are not associated with the outcome of infection
EuroFlow-based flowcytometric diagnostic screening and classification of primary immunodeficiencies of the lymphoid system
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 innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the Eu
The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System
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 all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID
(without a need for specificity to diagnose particular PID) and to guide and prioritize
further diagnostic modalities and clinical management. The tube composition has been
defined in a stepwise manner through several cycles of design-testing-evaluationredesign in a multicenter setting. Equally important appeared to be the standardized preanalytical procedures (sample preparation and instrument setup), analytical procedures
(immunostaining and data acquisition), the software analysis (a multidimensional view
based on a reference database in Infinicyt software), and data interpretation. This
standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID
patients with defined genetic defects. In addition, an application of new EuroFlow
software tools with multidimensional pattern recognition was designed with inclusion of
maturation pathways in multidimensional patterns (APS plots). The major advantage of
the EuroFlow approach is that data can be fully exchanged between different laboratories
in any country of the world, which is especially of interest for the PID field, with generally
low numbers of cases per center
Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance
OBJECTIVES: The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. METHODS: Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. RESULTS: Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly-p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. CONCLUSIONS: Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide
Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis
Phlebotomine female sand flies require a blood meal for egg development, and it is during the blood feeding that pathogens can be transmitted to a host. Leishmania parasites are among these pathogens and can cause disfiguring cutaneous or even possibly fatal visceral disease. The Leishmania parasites are deposited into the bite wound along with the sand fly saliva. The components of the saliva have many pharmacologic and immune functions important in blood feeding and disease establishment. In this article, the authors identify and investigate the protein components of saliva of two important vectors of leishmaniasis, Phlebotomus tobbi and P. sergenti, by sequencing the transcriptomes of the salivary glands. We then compared the predicted protein sequences of these salivary proteins to those of other bloodsucking insects to elucidate the similarity in composition, structure, and enzymatic activity. Finally, this descriptive analysis of P. tobbi and P. sergenti transcriptomes can aid future research in identifying molecules for epidemiologic assays and in investigating sand fly-host interactions
The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System
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 all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center
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