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1.
Isr Med Assoc J ; 21(7): 503, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31507132

RESUMO

BACKGROUND: Tumor treating fields (TTFields) are low-intensity, intermediate frequency electric fields that affect proliferating cells. TTFields are FDA approved for treatment of newly diagnosed and recurrent glioblastoma. Combining TTFields with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to the ability of TTFields to induce immunogenic cell death. Conversely, TTFields may interfere with immune functions critical for effective T-cell responses. OBJECTIVES: To evaluate the effects of TTFields on pivotal antitumoral T-cell functions. METHODS: T-cells from healthy donor peripheral blood (PB) or from viably dissociated human glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen stimulation. Multiparametric flow cytometry (8-color) was used to assess T-cell responses by monitoring select pivotal functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), and activation/exhaustion (PD-1). Cellular viability was assessed in a dedicated assay. A chimeric antigen receptor (CAR) T-cell-based assay directly evaluated cellular cytotoxicity. RESULTS: Activated PB T-cells and tumor-infiltrating T-cells (TILs) preserved all monitored anti- tumoral functions under TTFields, apart from proliferation. This finding also applied specifically to PD-1 + TILs, comprised predominantly of tumor antigen-specific cells. Activated T-cells that attempted to proliferate under TTFields demonstrated decreased viability, in line with TTField MOA. Small or no reduction in viability was found in T-cells that did not attempt to proliferate, whether activated or resting. CONCLUSIONS: All monitored anti-tumoral T cell functions, except for proliferation, were unhindered by TTFields. Our results support further investigation into combinations of TTFields with T-cell based immunotherapeutic approaches.


Assuntos
Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Terapia por Estimulação Elétrica/métodos , Glioblastoma/terapia , Citometria de Fluxo/métodos , Glioblastoma/patologia , Humanos , Recidiva Local de Neoplasia , Linfócitos T/citologia
2.
J Cancer Res Clin Oncol ; 145(11): 2803-2811, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31463716

RESUMO

BACKGROUND: Flow cytometry (FCM) plays a crucial role in the differential diagnosis of Burkitt lymphoma/leukemia (BL) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The presence of surface IgM (sIgM) alone or with light chain restriction indicates a mature blast phenotype (BIV by EGIL) and is usually observed in BL. However, sIgM expression could also be detected in transitional BCP-ALL cases. These similarities in immunophenotype and ambiguous correspondence with other laboratory findings may challenge the correct BL diagnostics. METHODS: We retrospectively reviewed the available data from immunophenotypic, morphological, cytogenetic, and molecular genetic studies of 146 children (85 boys and 61 girls) with a median age of 10 years (range 0-18 years) who were diagnosed with BL and BCP-ALL. The blasts' immunophenotype was studied by multicolor FCM. The conventional cytogenetic analysis included G-banded karyotyping and fluorescence in situ hybridization (FISH). RESULTS: In 54 children classified as BIV-ALL according to the EGIL, it was demonstrated that sIgM in a minority of cases can be associated with various types of BCP-ALL. Analysis of the antigen expression profile of 105 patients with verified BL (n = 21) and BCP-ALL (n = 84) showed significant differences in BL and the sIgM(+) vs BCP-ALL immunophenotype. Thus, even in cases of ambiguous sIgM expression, these two diseases could be reliably discriminated by complex immunophenotyping. Moreover, 10 patients (7 boys and 3 girls) with BL leukemic cells did not express sIgM, and they were diagnosed with BL on the basis of other laboratory and clinical signs. CONCLUSIONS: In conclusion, our study shows that BIV subtype is heterogeneous group of leukemia including not only the BL, but also BCP-ALL. In ambiguous cases, only a combination of multiple immunophenotypic, cytomorphologic, and genetic diagnostic technologies can allow the precise discrimination of BL and BCP-ALL and selection of the appropriate treatment scheme.


Assuntos
Linfócitos B/patologia , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Cariotipagem/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/classificação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Estudos Retrospectivos
3.
Nat Protoc ; 14(8): 2370-2415, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31278398

RESUMO

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in ~3 months.


Assuntos
Citometria de Fluxo/métodos , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais (Computação) , Análise de Célula Única/métodos , Células Cultivadas , Humanos , Dispositivos Lab-On-A-Chip , Microalgas/citologia , Processamento de Sinais Assistido por Computador , Software
4.
Anal Bioanal Chem ; 411(23): 6039-6047, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31304564

RESUMO

Circulating tumor DNA (ctDNA) is a tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It has been greatly focused in the recent decade because of its potential clinical utility for liquid biopsies. Development of ctDNA analytical techniques with high sensitivity and cost-efficiency will undoubtedly promote the clinical spread of ctDNA testing. In this paper, we propose a novel flow cytometry-based ctDNA sensing strategy which combines enzyme-free amplification and magnetic separation. The target DNA is capable of triggering a hybridization chain reaction, producing a fluorescent long linear assembly of DNA, which can be further captured by magnetic beads to present fluorescent signals using flow cytometry. In comparison with some conventional methods, our strategy has the advantages of easy operation and cost-efficiency, and thereby shows a promising application in clinical diagnosis. Graphical abstract.


Assuntos
DNA Tumoral Circulante/sangue , Citometria de Fluxo/métodos , Imãs/química , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Tumoral Circulante/análise , Humanos , Limite de Detecção , Biópsia Líquida/métodos , Hibridização de Ácido Nucleico/métodos
5.
Fish Shellfish Immunol ; 92: 871-880, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299464

RESUMO

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.


Assuntos
Bass/fisiologia , Citometria de Fluxo/veterinária , Imunidade Inata , Neutrófilos/fisiologia , Explosão Respiratória/fisiologia , Animais , Citometria de Fluxo/métodos , Oxirredução , Rodaminas/química
6.
Hematol Oncol ; 37(4): 401-408, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31291481

RESUMO

Multiple myeloma (MM) is malignant tumor with abnormal proliferation of bone marrow plasma cells. The existing clinical tools used to determine treatment response and tumor relapse are limited in sensitivity. We investigated the CD138+ microparticles (MPs) of MM patients to find out whether MPs could provide a novel means to monitor the malignant cells in MM patients. Our study showed that the levels of MPs were significantly elevated in MM patients. The MP counts in peripheral blood from new diagnosed MM patients were significantly higher than patients in CR and HD. Consist with the total MPs, the number of the PC-derived MPs (CD138+) increased in BM from MM patients compared with CR and HD. The ratio of the PC-derived MPs (CD138+) in BM increased in MM patients compared with CR and HD. The correlation test revealed that the CD138+ MPs in BM and PB were all positively correlated with the plasmacyte ratio in bone marrow (BMPC) and the ß2 -MG. New diagnosed MM patients and controls were compared, and ROC curves were used to identify cutoff points with optimal sensitivity and specificity concerning the ratios and counts of CD138+ MPs in BM and PB. The AUC of the CD138+ MP counts in BM was 0.767, and in PB was 0.680. The AUC of the CD138+ MP ratios in BM was 0.714, and in PB was 0.666. According to this, the counts of CD138+ MPs in BM showed to be a powerful marker of diagnosis. We demonstrated that CD138+ MPs from the plasma provide support for a potential monitoring biomarker of MM.


Assuntos
Células da Medula Óssea/química , Medula Óssea/patologia , Micropartículas Derivadas de Células/química , Mieloma Múltiplo/sangue , Proteínas de Neoplasias/sangue , Sindecana-1/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores Tumorais/sangue , Separação Celular/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Curva ROC , Sensibilidade e Especificidade , Sindecana-1/análise , Microglobulina beta-2/análise
7.
Analyst ; 144(17): 5232-5244, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31360935

RESUMO

Leishmaniasis comprises a group of infectious diseases with worldwide distribution, of which both the visceral and cutaneous forms are caused by Leishmania parasites. In the absence of vaccines, efficacious chemotherapy remains the basis for leishmaniasis control. The available drugs are expensive and associated with several secondary adverse effects. Due to these limitations, the development of new antileishmanial compounds is imperative, and plants offer various perspectives in this regard. The present study evaluated the in vitro leishmanicidal activity of flavonoids isolated from Solanum paludosum Moric. and investigated the mechanisms of cell death induced by them. These compounds were evaluated in vitro for their antileishmanial activity against Leishmania amazonensis promastigotes and they showed prominent leishmanicidal activity. The EtOAc fraction, gossypetin 3,7,8,4'-tetra-O-methyl ether (1), and kaempferol 3,7-di-O-methyl ether (3) were selected to be used in an in vitro assay against L. amazonensis amastigotes and cell death assays. The flavonoids (1) and (3) presented significant activity against L. amazonensis amastigotes, exhibiting the IC50 values of 23.3 ± 4.5 µM, 34.0 ± 9.6 µM, and 10.5 ± 2.5 µM for the EtOAc fraction, (1), and (3), respectively, without toxic effects to the host cells. Moreover, (1) and (3) induced blocked cell cycle progression at the G1/S transition, ultimately leading to G1/G0 arrest. Flavonoid (3) also induced autophagy. Using Raman spectroscopy in conjunction with principal component analysis, the biochemical changes in the cellular components induced by flavonoids (1) and (3) were presented. The obtained results indicated that the mechanisms of action of (1) and (3) occurred through different routes. The results support that the flavonoids derived from S. paludosum can become lead molecules for the design of antileishmanial prototypes.


Assuntos
Antiprotozoários/farmacologia , Morte Celular/efeitos dos fármacos , Flavonoides/farmacologia , Citometria de Fluxo/métodos , Leishmania/efeitos dos fármacos , Animais , Antiprotozoários/química , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Flavonoides/química , Quempferóis/química , Quempferóis/farmacologia , Leishmania/citologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Análise Espectral Raman , Estreptófitas/química
8.
Anal Chim Acta ; 1076: 154-161, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203960

RESUMO

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein.


Assuntos
Antígenos CD/imunologia , Biomarcadores Tumorais/imunologia , Células Neoplásicas Circulantes/imunologia , Receptores da Transferrina/imunologia , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Ligantes , Biópsia Líquida , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
9.
Nat Commun ; 10(1): 2770, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235780

RESUMO

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.


Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo/métodos , Microbiota , Microbiologia do Solo , Aminoácidos/análise , Aminoácidos/química , Bactérias/genética , Bactérias/metabolismo , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Coloração e Rotulagem/métodos
11.
Nat Protoc ; 14(7): 1946-1969, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160786

RESUMO

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis.


Assuntos
Anticorpos Monoclonais , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Análise de Célula Única/métodos , Algoritmos , Biomarcadores , Tampões (Química) , Corantes , Biologia Computacional/métodos , Fatores de Transcrição Forkhead/imunologia , Humanos , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia
12.
Eur J Histochem ; 63(2)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243942

RESUMO

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.


Assuntos
Autofagia , Citometria de Fluxo/métodos , Proteínas Associadas aos Microtúbulos/análise , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Cloroquina/farmacologia , Fluorescência , Humanos , Leucócitos Mononucleares/microbiologia , Mycobacterium bovis/química , Coloração e Rotulagem , Células THP-1
13.
Nat Commun ; 10(1): 2686, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217423

RESUMO

Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis. Each disease is characterized by a diverse set of potential manifestations, which determine patients' disease phenotype. Current understanding of phenotype determinants is limited, despite increasing prevalence and healthcare costs. Diagnosis and monitoring of disease requires invasive procedures, such as endoscopy and tissue biopsy. Here we report signatures of heterogeneity between disease diagnoses and phenotypes. Using mass cytometry, we analyze leukocyte subsets, characterize their function(s), and examine gut-homing molecule expression in blood and intestinal tissue from healthy and/or IBD subjects. Some signatures persist in IBD despite remission, and many signatures are highly represented by leukocytes that express gut trafficking molecules. Moreover, distinct systemic and local immune signatures suggest patterns of cell localization in disease. Our findings highlight the importance of gut tropic leukocytes in circulation and reveal that blood-based immune signatures differentiate clinically relevant subsets of IBD.


Assuntos
Citometria de Fluxo/métodos , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Leucócitos/imunologia , Espectrometria de Massas/métodos , Adulto , Idoso , Biópsia , Separação Celular , Colonoscopia , Feminino , Humanos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Intestinos/imunologia , Intestinos/patologia , Masculino , Pessoa de Meia-Idade , Exacerbação dos Sintomas , Adulto Jovem
14.
Int J Lab Hematol ; 41 Suppl 1: 56-62, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31069980

RESUMO

Flow cytometry (FCM) allows scientists to rapidly quantify up to 50 parameters for millions of cells per sample. The bottleneck in the application of the technology is data analysis, and the high number of parameters measured by the current generation of instruments requires the use of advanced computational algorithms to make full use of their capabilities. This review summarizes the main steps of FCM data analysis, focusing on the use of the most recent bioinformatic tools developed for an R-based programming environment. In particular, for each stage of the data analysis, libraries and packages currently available are listed, and a brief description of their functioning is included.


Assuntos
Algoritmos , Biologia Computacional/métodos , Citometria de Fluxo , Software , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos
15.
Int J Lab Hematol ; 41 Suppl 1: 73-81, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31069981

RESUMO

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidyl-inositol glycan complementation Class A (PIG-A) gene. Depending on the severity of the mutation in the PIG-A gene, there is a partial or absolute inability to make glycosylphosphatidyl-inositol (GPI)-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Flow cytometric detection of PNH clones has become the gold standard and has played an increasingly important role in the diagnosis, monitoring, and clinical management of patients with PNH. Recently, a 4-part set of Consensus Guidelines have been published by flow experts in the field to address the key assay-specific considerations for the identification of PNH clones in RBC and WBC, how to report such data and a full validation document for the assays described. Below, we have summarized the most significant aspects of this International effort.


Assuntos
Antígenos CD55/sangue , Antígenos CD59/sangue , Citometria de Fluxo/métodos , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/líquido cefalorraquidiano , Proteínas de Membrana/sangue , Antígenos CD55/genética , Antígenos CD59/genética , Consenso , Citometria de Fluxo/normas , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/genética , Humanos , Proteínas de Membrana/genética , Guias de Prática Clínica como Assunto
16.
Int J Lab Hematol ; 41 Suppl 1: 63-72, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31069989

RESUMO

Primary immunodeficiencies (PID) or inborn errors of immunity affect phenotype and/or function of one or more components of the immune system. The exponential growth of genetic analysis has enabled identification of known and novel mutations. However, genetic analysis continues to be expensive and is not easily accessible. Flow cytometry, on the other hand, has been well established for many years in clinical laboratories and its use for the analysis of immunodeficiencies is expanding. Surface, intracellular, and intranuclear proteins can easily be evaluated by flow cytometry, enabling both phenotypic and functional identification of specific cell populations and therefore facilitating the identification of a variety of PIDs. While genetic analysis provides a definitive diagnosis for PIDs, flow cytometry is necessary to confirm or establish the immune phenotype of a gene mutation. Furthermore, flow cytometry provides a rapid means to identify an immunological defect at a relatively low cost.


Assuntos
Citometria de Fluxo/métodos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Testes Genéticos , Humanos , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/genética , Mutação , Fenótipo
17.
Transplant Proc ; 51(4): 1016-1020, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31101162

RESUMO

AIM: Antibody assessment during pretransplantation term is important to detect donor specific antibodies. These donor-specific antibodies are determined by various crossmatch methods (flow cytometric [FCXM], complement-dependent cytotoxic [CDCXM], and Luminex [LMXM]). Recently, single antigen bead (SAB) assays have been used for the assessment of hypersensitized patients. The aim of this study was to compare sensitivity and specificity of the 3 crossmatch methods in reference to the SAB method. METHOD: In this study, 69 hypersensitized patients with high class I and/or class II panel reactive antibodies were tested using the flow cytometric SAB method. Serum samples were cross-matched by 3 crossmatch methods with the cells of a volunteer healthy individual, and the results were evaluated according to HLA and cross-reactive epitope groups (CREGs). RESULTS: Sensitivity was found to be better with T FCXM (0.91) and class I LMXM (0.87). Specificity of peripheral blood lymphocyte CDCXM method (1.0) was found to be better than the other 2 methods (0.33 and 0.57, respectively). Sensitivity of class II LMXM (0.88) was found to be better than the others (0.42 for B CDCXM and 0.82 for B FCXM, respectively). The specificity of the B CDCXM, B FCXM, and class II LMXM was similar (0.44, 0.44, and 0.33, respectively). CREGs results were similar to HLA results. CONCLUSION: Although CDCXM has high specificity for the detection of anti-HLA antibodies, it has low sensitivity. To increase sensitivity, FCXM or LMXM methods may be used with the CDCXM test. These results will be beneficial for laboratories and clinicians during graft survival and patient health assessment.


Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , Isoanticorpos/análise , Adulto , Feminino , Citometria de Fluxo/métodos , Antígenos HLA/imunologia , Humanos , Linfócitos/imunologia , Pessoa de Meia-Idade , Sensibilidade e Especificidade
18.
Transplant Proc ; 51(4): 1021-1023, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31101163

RESUMO

Cytotoxic flow cytometric crossmatch (cFCXM), identified by detecting complement-mediated cytotoxic cell death in addition to the capability of showing the alloantibodies binding onto lymphocytes at the same time, can reduce the necessary time and workload in evaluating alloantibodies. More data from clinical samples are needed for cFCXM to be accepted by tissue typing laboratories. In this study, we compared cFCXM with complement-dependent lymphocytotoxicity and standard flow cytometric crossmatch in 41 renal pretransplant patients. A comparison of the obtained data was performed using Spearman's correlation test. We found that cFCXM showed no statistically significant differences with complement-dependent lymphocytotoxicity and flow cytometric crossmatch. We believe that cFCXM can be used in clinical laboratories in the near future following intra-laboratory validation.


Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Teste de Histocompatibilidade/métodos , Transplante de Rim , Feminino , Humanos , Isoanticorpos/análise , Isoanticorpos/imunologia , Masculino
19.
Transplant Proc ; 51(4): 1027-1028, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31101165

RESUMO

AIM: The cell-based flow cytometric and bead-based Luminex crossmatch methods have been used alongside the standard complement-dependent cytotoxic crossmatch (CDCXM) test to detect donor specific anti-HLA antibodies. In this study, it was aimed to compare flow cytometric crossmatch (FCXM), CDCXM, and Luminex donor-specific crossmatch (LM-XM) tests for pre-transplant assessment of patients who applied to Tepecik Education and Research Hospital for kidney transplantation from related or deceased donors. METHOD: HLA tissue typing of 1120 patients were tested using the sequence specific oligonucleotide probe method with low resolution. FCXM and LM-XM were performed according to the manufacturer's instructions. The CDCXM test was performed according to the standard procedure. The results were analyzed using SPSS version 21.0 software (IBM, Armonk, NY, United States). P < .05 was accepted as statistically significant. RESULTS: FCXM, CDCXM, and LM-XM tests were performed on 58.2% (n = 652), 91% (n = 1019), and 55.4% (n = 620) of 1120 patients. There were statistically significant differences between FCXM/CDCXM, LM-XM/CDCXM, and FCXM/LM-XM (P < .0001), although there was also a moderate correlation between them (for class I, r = .599, r = .693, and r = .507; for class II, r = .546, r = .471, and r = .495, respectively). The results obtained according to donor type were compatible with the total study group. CONCLUSION: The utilization of FCXM and/or LM-XM tests together with the CDCXM method before kidney transplantations from related and/or deceased donor may facilitate the determination of target cells of donor-specific antibodies or their antibody class, which may increase the success of transplantation.


Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , Isoanticorpos/administração & dosagem , Adulto , Feminino , Citometria de Fluxo/métodos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade
20.
Pan Afr Med J ; 32: 2, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31068996

RESUMO

Introduction: This study aimed to evaluate the performances of the MUSE® flow cytometer compared with the reference GUAVA® flow cytometer. Methods: We conducted an experimental study on HIV-infected patient samples. Venous blood samples, collected in a K3 EDTA tube, were analyzed within 24-48 hours by MUSE® and GUAVA® cytometers at the International Center for medical diagnosis (Centre International de Diagnostic médical) in Yaoundé. Results: In total, 227 samples were analyzed. There was a strong intraclass correlation (p<0.0001) between MUSE® and GUAVA® cytometers with a correlation coefficient 0.998 (95% CI: 0,998-0,999) for the absolute values and 0,992 (95% CI: 0,989-0,994) for the percentages. A strong positive linear correlation (p=0.0001) was found between MUSE® and GUAVA® cytometers with linear regression slope r2 = 0.98 (95% CI=0,97-0,99) for the absolute values and r2= 0.98 (95% CI= 0.96-1,00) for the percentages. The biases were -4,80 cells/µl (-101.31-91.71) for the absolute values and -0.89% (IC: -6,08-4.3) for the percentages. The percentage of data points outside the limits of agreement was 12/227 (5.29%) and 10/227 (4.41%) respectively for the absolute values and percentages. Cohen's kappa coefficient was 0.92 and the area under the curve was 0,9975 (CI 95%: 0.99-1). Conclusion: MUSE®AUTO CD4/CD4% cytometer is a powerful instrument because its results are consistent with those obtained by the reference cytometer. It can enable tracking of patients infected with HIV, in particular in the developing countries.


Assuntos
Contagem de Linfócito CD4/métodos , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Infecções por HIV/diagnóstico , Adulto , Contagem de Linfócito CD4/instrumentação , Camarões , Feminino , Citometria de Fluxo/instrumentação , Infecções por HIV/imunologia , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade
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