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2.
Mutat Res ; 854-855: 503200, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660824

RESUMO

Germ cell tumour (GCT) patients who fail to respond to chemotherapy or who relapse have a poor prognosis. Timely and accurately stratifying such patients could optimise their therapy. We identified endogenous DNA damage levels as a prognostic marker for progression-free (PFS) and overall (OS) survival in chemotherapy-naïve GCT patients. In the present study, we have extended our previous results and reviewed the prognostic power of DNA damage level in GCTs. Endogenous DNA damage levels were measured with the comet assay. Receiver operator characteristic analysis was applied to determine the optimal cut-off value and to evaluate its prognostic accuracy. PFS and OS were estimated by the Kaplan-Meier method and compared using the log-rank test. Hazard ratio (HR) estimates were calculated by Cox regression analysis. A cut-off value of 6.34 provided the highest sensitivity and specificity, with area under curve values of 0.813 and 0.814 for disease progression and mortality, respectively. A % DNA in tail > 6.34 was significantly associated with shorter PFS (HR = 9.54, 95 % confidence interval [CI]: 3.43-26.55, p < 0.001) and OS (HR = 14.62, 95 % CI: 3.14-67.95, p = 0.001) by univariate analysis. The prognostic value of DNA damage measurement was confirmed by multivariate models (HR = 6.45, 95 % CI: 2.22-18.75, p = 0.001 for PFS and HR = 9.40, 95 % CI: 1.70-52.09, p = 0.010 for OS), when HR was adjusted for relevant clinical categories. The added prognostic value of DNA damage in combination with International Germ Cell Cancer Collaborative Group (IGCCCG) risk groups has been revealed. Endogenous DNA damage is an independent prognosticator for PFS and OS in GCT patients and its clinical use, particularly in combination with IGCCCG risk groups, may help in stratifying these patients.


Assuntos
Células Sanguíneas/patologia , Dano ao DNA/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Adulto , Células Cultivadas , Ensaio Cometa/métodos , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Fatores de Risco
4.
Sci Rep ; 9(1): 14670, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31605010

RESUMO

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.


Assuntos
Linhagem da Célula/genética , Perfilação da Expressão Gênica/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Circular/genética , Células Sanguíneas/patologia , Linhagem Celular Tumoral , Criança , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pediatria , Leucemia-Linfoma Linfoblástico de Células Precursoras/classificação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RNA Circular/classificação
5.
Clin Nucl Med ; 44(11): e624-e626, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31584492

RESUMO

F-Fluorothymidine-positron emission tomography with CT fusion ([F]FLT-PET/CT) offers a unique and non-invasive method for three-dimensional localization and quantification of functional bone marrow. [F]FLT-PET/CT has potential application in radiotherapy planning when risk of marrow toxicity is a significant clinical concern. In this patient with chemo-refractory, transformed lymphoma and treatment-induced cytopenias, [F]FLT-PET/CT was a novel and useful adjunct to (1) image the distribution of functional bone marrow reserve, (2) guide "marrow-sparing" radiotherapy planning, and (3) quantify the effects of radiotherapy-induced bone marrow suppression both in-field and out-of-field.


Assuntos
Células Sanguíneas/efeitos da radiação , Medula Óssea/fisiopatologia , Medula Óssea/efeitos da radiação , Didesoxinucleosídeos , Tratamentos com Preservação do Órgão , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Idoso , Células Sanguíneas/patologia , Medula Óssea/diagnóstico por imagem , Contagem de Células , Feminino , Humanos , Linfoma/radioterapia
6.
Comput Biol Med ; 113: 103385, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31437626

RESUMO

Identification of genes whose regulation of expression is functionally similar in both brain tissue and blood cells could in principle enable monitoring of significant neurological traits and disorders by analysis of blood samples. We thus employed transcriptional analysis of pathologically affected tissues, using agnostic approaches to identify overlapping gene functions and integrating this transcriptomic information with expression quantitative trait loci (eQTL) data. Here, we estimate the correlation of gene expression in the top-associated cis-eQTLs of brain tissue and blood cells in Parkinson's Disease (PD). We introduced quantitative frameworks to reveal the complex relationship of various biasing genetic factors in PD, a neurodegenerative disease. We examined gene expression microarray and RNA-Seq datasets from human brain and blood tissues from PD-affected and control individuals. Differentially expressed genes (DEG) were identified for both brain and blood cells to determine common DEG overlaps. Based on neighborhood-based benchmarking and multilayer network topology approaches we then developed genetic associations of factors with PD. Overlapping DEG sets underwent gene enrichment using pathway analysis and gene ontology methods, which identified candidate common genes and pathways. We identified 12 significantly dysregulated genes shared by brain and blood cells, which were validated using dbGaP (gene SNP-disease linkage) database for gold-standard benchmarking of their significance in disease processes. Ontological and pathway analyses identified significant gene ontology and molecular pathways that indicate PD progression. In sum, we found possible novel links between pathological processes in brain tissue and blood cells by examining cell pathway commonalities, corroborating these associations using well validated datasets. This demonstrates that for brain-related pathologies combining gene expression analysis and blood cell cis-eQTL is a potentially powerful analytical approach. Thus, our methodologies facilitate data-driven approaches that can advance knowledge of disease mechanisms and may, with clinical validation, enable prediction of neurological dysfunction using blood cell transcript profiling.


Assuntos
Células Sanguíneas/metabolismo , Encéfalo/metabolismo , Simulação por Computador , Bases de Dados de Ácidos Nucleicos , Regulação da Expressão Gênica , Doença de Parkinson/metabolismo , Biomarcadores/metabolismo , Células Sanguíneas/patologia , Encéfalo/patologia , Estudo de Associação Genômica Ampla , Humanos , Doença de Parkinson/patologia
7.
J Thromb Thrombolysis ; 48(2): 187-194, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31177487

RESUMO

Relatively little information is known about the definitive role of phosphatidylserine (PS) in the hypercoagulability of heart failure (HF). Our objectives were to assess the levels of PS exposure on microparticles (MPs) and blood cells (BCs) in each group of HF patients and to evaluate their procoagulant activity (PCA). HF patients in each NYHA functional class II-IV (II n = 30, III n = 30, IV n = 30) and healthy controls (n = 25) were enrolled in the present study. PS exposure on MPs, BCs was analyzed with flow cytometry. MPs were classified based on their cellular origin: platelets (CD41a+), neutrophils (CD66b+), endothelial cells (CD31+CD41a-), erythrocytes (CD235a+), monocytes (CD14+), T lymphocytes (CD3+), and B lymphocytes (CD19+). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+ BCs and MPs were performed by lactadherin. There was no significant difference in MP cellular origin between healthy and HF subjects. However, the total number of PS+ MPs was significantly increased in HF patients compared with healthy controls. In addition, circulating PS+ BCs cooperated with PS+ MPs to markedly shorten coagulation time and dramatically increase FXa/thrombin generation and fibrin formation in each HF group. Moreover, blockade of exposed PS on BCs and MPs with lactadherin inhibited PCA by approximately 80%. Our results lead us to believe that exposing PS on the injured BCs and MPs played a pivotal role in the hypercoagulability state in HF patients.


Assuntos
Micropartículas Derivadas de Células/fisiologia , Células Endoteliais/fisiologia , Insuficiência Cardíaca/sangue , Fosfatidilserinas/metabolismo , Trombofilia/etiologia , Adulto , Idoso , Células Sanguíneas/patologia , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade
8.
Hormones (Athens) ; 18(2): 173-178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31154656

RESUMO

PURPOSE: To investigate DNA methylation changes in peripheral blood from patients with gestational diabetes mellitus (GDM) and preeclampsia (PE) due to poorly treated GDM. METHODS: Eighteen pregnant women participated in the study: 6 with GDM, 6 with PE, and 6 healthy controls. The promoter methylation status of genes was profiled using the Human Inflammatory Response and Autoimmunity EpiTect Methyl II Signature PCR Array profiles. The results were validated with quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Fewer inflammation-related genes were significantly hypomethylated in PE cases compared to healthy subjects than in GDM cases. Some of the examined genes show different methylation patterns between GDM and PE. CONCLUSIONS: The epigenetic changes observed in this study indicate that GDM and PE exhibit specific DNA methylation profiles, with possible clinical applications.


Assuntos
Células Sanguíneas/metabolismo , Metilação de DNA , Diabetes Gestacional/genética , Inflamação/genética , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/genética , Transcriptoma , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Células Sanguíneas/patologia , Estudos de Casos e Controles , Diabetes Gestacional/sangue , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/complicações , Análise em Microsséries , Pré-Eclâmpsia/diagnóstico , Gravidez , Prognóstico , Regiões Promotoras Genéticas
9.
Lab Chip ; 19(15): 2500-2511, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31246211

RESUMO

Development of therapeutic approaches to treat vascular dysfunction and thrombosis at disease- and patient-specific levels is an exciting proposed direction in biomedical research. However, this cannot be achieved with animal preclinical models alone, and new in vitro techniques, like human organ-on-chips, currently lack inclusion of easily obtainable and phenotypically-similar human cell sources. Therefore, there is an unmet need to identify sources of patient primary cells and apply them in organ-on-chips to increase personalized mechanistic understanding of diseases and to assess drugs. In this study, we provide a proof-of-feasibility of utilizing blood outgrowth endothelial cells (BOECs) as a disease-specific primary cell source to analyze vascular inflammation and thrombosis in vascular organ-chips or "vessel-chips". These blood-derived BOECs express several factors that confirm their endothelial identity. The vessel-chips are cultured with BOECs from healthy or diabetic patients and form an intact 3D endothelial lumen. Inflammation of the BOEC endothelium with exogenous cytokines reveals vascular dysfunction and thrombosis in vitro similar to in vivo observations. Interestingly, our study with vessel-chips also reveals that unstimulated BOECs of type 1 diabetic pigs show phenotypic behavior of the disease - high vascular dysfunction and thrombogenicity - when compared to control BOECs or normal primary endothelial cells. These results demonstrate the potential of organ-on-chips made from autologous endothelial cells obtained from blood in modeling vascular pathologies and therapeutic outcomes at a disease and patient-specific level.


Assuntos
Células Sanguíneas/patologia , Células Progenitoras Endoteliais/patologia , Dispositivos Lab-On-A-Chip , Trombose/patologia , Adulto , Proliferação de Células , Estudos de Viabilidade , Humanos , Estresse Oxidativo , Trombose/sangue
10.
J Cell Biochem ; 120(10): 16307-16315, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31127656

RESUMO

Leukemia is a cancer, which is derived from leukocytes and precursors of leukocytes in the bone marrow. A large number of pivotal biological processes are linked to leukemia pathogenesis. More insights into these mechanisms can provide a better developing pharmacological platform for patients with leukemia. Among the different players in leukemia pathogenesis, exosomes have appeared as a new biological vehicle, which can transfer oncogenic signals to blood cells. Exosomes are nano-carriers, which enable transferring numerous cargos such as DNA fragments, RNAs, messenger RNAs, microRNAs, long noncoding RNA, and proteins. Targeting the contents of exosomes leads to the alteration of host cell behavior. Increasing evidence has indicated that leukemia-derived exosomes could be utilized as prognostic, diagnostic, and therapeutic biomarkers for individuals suffering from leukemia. In this regard, the importance of exosomes in terms of initiation and progression of leukemia was underlined in this study.


Assuntos
Biomarcadores Tumorais/sangue , Células Sanguíneas/metabolismo , Exossomos/metabolismo , Leucemia/sangue , Células Sanguíneas/patologia , DNA de Neoplasias/sangue , Exossomos/patologia , Humanos , Leucemia/diagnóstico , MicroRNAs/sangue , Proteínas de Neoplasias/sangue , RNA Neoplásico/sangue
11.
J Allergy Clin Immunol Pract ; 7(8): 2624-2633.e2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100552

RESUMO

BACKGROUND: Noninvasive markers of type 2 inflammation are needed to identify children and adolescents who might benefit from personalized biologic therapy. OBJECTIVE: We hypothesized that blood eosinophil counts would predict 1 or more acute visits for asthma and that prediction could be improved with the addition of a second, noninvasive type 2 inflammatory biomarker. METHODS: Children and adolescents 5 to 21 years (N = 589) with an asthma exacerbation necessitating systemic corticosteroid treatment in the previous year completed a characterization visit and telephone calls at 6 and 12 months. The primary outcome was an acute visit for asthma with receipt of systemic corticosteroids. Acute visits were verified by medical record review. Exploratory outcomes included time to first acute visit and hospitalization. RESULTS: Acute visits occurred in 106 (35.5%) children and 72 (24.8%) adolescents. Elevated blood eosinophils were associated with increased odds and shorter time to first acute visit, but optimal cut-points differed by age (≥150 vs ≥300 cells/µL for children vs adolescents, respectively). The addition of a second marker of type 2 inflammation did not improve prediction in children, but increased the odds and hazard of an acute visit up to 16.2% and 11.9%, respectively, in adolescents. Similar trends were noted for hospitalizations. CONCLUSIONS: Blood eosinophils and other noninvasive markers of type 2 inflammation may be useful in the clinical assessment of children and adolescents with asthma. However, features of type 2 inflammation vary by age. Whether children and adolescents also respond differently to management of type 2 inflammation is unclear and warrants further evaluation.


Assuntos
Asma/diagnóstico , Células Sanguíneas/patologia , Eosinófilos/patologia , Inflamação/diagnóstico , Medicina de Precisão/métodos , Adolescente , Adulto , Biomarcadores/metabolismo , Criança , Pré-Escolar , Citocinas/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Prognóstico , Células Th2/imunologia , Adulto Jovem
12.
J Neuroimmunol ; 332: 49-56, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30933850

RESUMO

The experimental autoimmune encephalomyelitis (EAE) model is indispensable for autoimmunity research, but model-specific T cell dynamics are sparsely studied. We used next-generation immunosequencing across lymphoid organs, blood and spinal cord in response to immunization with myelin basic protein (MBP) to study T cell repertoires and migration patterns. Surprisingly, most spinal cord T cells were unique to the individual animal despite the existence of shared MBP-specific clones, suggesting a previously underestimated T cell diversity. Almost complete emigration of pathogenic clones from blood to spinal cord indicates that blood is not a suitable compartment to study EAE-mediating T cells.


Assuntos
Seleção Clonal Mediada por Antígeno/genética , Encefalomielite Autoimune Experimental/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Subpopulações de Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Células Sanguíneas/imunologia , Células Sanguíneas/patologia , Movimento Celular , Células Clonais/imunologia , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Tecido Linfoide/imunologia , Tecido Linfoide/patologia , Camundongos , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Organismos Livres de Patógenos Específicos , Medula Espinal/imunologia , Medula Espinal/patologia
16.
Cytometry A ; 95(5): 510-520, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31012276

RESUMO

Cellular biophysical properties are the effective label-free phenotypes indicative of differences in cell types, states, and functions. However, current biophysical phenotyping methods largely lack the throughput and specificity required in the majority of cell-based assays that involve large-scale single-cell characterization for inquiring the inherently complex heterogeneity in many biological systems. Further confounded by the lack of reported robust reproducibility and quality control, widespread adoption of single-cell biophysical phenotyping in mainstream cytometry remains elusive. To address this challenge, here we present a label-free imaging flow cytometer built upon a recently developed ultrafast quantitative phase imaging (QPI) technique, coined multi-ATOM, that enables label-free single-cell QPI, from which a multitude of subcellularly resolvable biophysical phenotypes can be parametrized, at an experimentally recorded throughput of >10,000 cells/s-a capability that is otherwise inaccessible in current QPI. With the aim to translate multi-ATOM into mainstream cytometry, we report robust system calibration and validation (from image acquisition to phenotyping reproducibility) and thus demonstrate its ability to establish high-dimensional single-cell biophysical phenotypic profiles at ultra-large-scale (>1,000,000 cells). Such a combination of throughput and content offers sufficiently high label-free statistical power to classify multiple human leukemic cell types at high accuracy (~92-97%). This system could substantiate the significance of high-throughput QPI flow cytometry in enabling next frontier in large-scale image-derived single-cell analysis applied in biological discovery and cost-effective clinical diagnostics. © 2019 International Society for Advancement of Cytometry.


Assuntos
Fenômenos Biofísicos , Citometria de Fluxo/métodos , Processamento de Imagem Assistida por Computador , Análise de Célula Única , Células Sanguíneas/patologia , Calibragem , Linhagem Celular Tumoral , Humanos , Leucemia/patologia , Análise Multivariada , Fenótipo , Reprodutibilidade dos Testes
17.
Sci Rep ; 9(1): 2922, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814612

RESUMO

The recent advances in myeloma treatment result in significantly better outcomes, defined as increased progression free survival (PFS) and overall survival (OS). Since there is a proven correlation between the extend of response and prolonged survival, there is an urgent need for highly sensitive assays for the detection of minimal residual disease (MRD). Next generation flow cytometry has become a valuable approach for sensitive evaluation of the depth of complete response (CR). Here, we report the diagnostic performance and validation results of a single-tube 9-color panel assay. The validation design included intra-assay analysis measuring accuracy, inter-assay analysis estimating method's linearity and precision and inter-assay analysis evaluating repeatability. Furthermore, in inter-operator analysis assessed the comparability of the result analysis of different operators. Staining stability was evaluated in age-of-stain experiments. Our validation results show that a reliable detection of residual myeloma cells is feasible to a detection level of 10-5 with a single-tube assay for a variety of materials (peripheral blood, bone marrow and stem cell apheresis). This study establishes highly sensitive, fully standardized approach for MRD detection in myeloma that is ready for implementation in routine diagnostic laboratories.


Assuntos
Células Sanguíneas/patologia , Células da Medula Óssea/patologia , Citometria de Fluxo/métodos , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Remoção de Componentes Sanguíneos , Linhagem Celular Tumoral , Estudos de Viabilidade , Humanos , Mieloma Múltiplo/terapia , Variações Dependentes do Observador , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do Tratamento
18.
Anal Chem ; 91(7): 4413-4420, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30816698

RESUMO

In this work, we developed a simple electrochemical method for ultrasensitive and label-free detection of circulating tumor cells (CTCs) based on direct plasmon-enhanced electrochemistry (DPEE). After plasmonic gold nanostars (AuNSs) were modified on the glassy carbon (GC) electrode, the aptamer probe was immobilized on the AuNSs surface, which can selectively capture the CTCs in samples. Upon localized surface plasmon resonance (LSPR) excitation, the electrochemical current response can be enhanced remarkably due to efficient hot electrons transport from AuNSs to the external circuit. The captured cells on the AuNSs surface will influence the hot electrons transport efficiency, leading to a decreased current response. Using ascorbic acid (AA) as the electroactive probe, it was found that the current responses of the AuNSs/GC electrode upon light irradiation decrease with the cell concentration. Due to the special molecular recognition of the aptamer and enhanced electrochemical performance of the plasmon, the proposed method enables an ultrasensitive and label-free detection of CTCs with excellent selectivity. The experimental results show that CCRF-CEM cell concentrations as low as 5 cells/mL can be successfully detected, which is superior to most reported work up to now. Using the present method, MCF-7 cells as low as 10 cells/mL can be also successfully detected, indicating the universality of the proposed method for CTCs detection. Furthermore, the cytosensor can successfully distinguish CTCs from normal cells in blood samples. The as-proposed strategy provides a promising application of DPEE in the development of novel biosensors for nondestructive analysis of biological samples.


Assuntos
Células Sanguíneas/patologia , Separação Celular/métodos , Técnicas Eletroquímicas/métodos , Células Neoplásicas Circulantes/patologia , Ressonância de Plasmônio de Superfície/métodos , Aptâmeros de Nucleotídeos/química , Ácido Ascórbico/química , Sequência de Bases , Carbono/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro , Humanos , Limite de Detecção , Células MCF-7 , Nanopartículas Metálicas/química
19.
Theranostics ; 9(5): 1417-1425, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867841

RESUMO

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications. Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI). Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM+CK+CD45-DAPI+) and 70.8% of patient blood samples were positive for CSCs (CD133+CK+CD45-DAPI+), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM+CK+CD45-DAPI+CD133+ and EpCAM+CK+CD45-DAPI+CD133-) and CSC subtypes (CD133+CK+CD45-DAPI+EpCAM+ and CD133+CK+CD45-DAPI+EpCAM-) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation. Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.


Assuntos
Adenocarcinoma/patologia , Células Sanguíneas/patologia , Carcinoma Ductal Pancreático/patologia , Separação Celular/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologia , Humanos
20.
Histol Histopathol ; 34(8): 857-873, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30779051

RESUMO

The diagnosis of myelodysplastic syndromes is based on the presence of cytopenias, dysplastic morphological features on peripheral blood (PB) and bone marrow (BM), cytogenetic abnormalities and requires to rule out other diseases resembling these conditions. Optical cytomorphology is the cornerstone of diagnosis of MDS. The recognition of cytological myelodysplasia has a crucial value in diagnosis and prognosis of MDS. Assessment of cytological morphology requires, like other diagnostic techniques (flow cytometry, cytogenetics, histological morphology), experienced observers and the availability of high quality and properly stained samples. The morphological analysis has shown moderate reproducibility among hematopathologists. The better characterization and standardization of morphological features has improved the reliability and reproducibility of MDS diagnosis. Maintaining the competence in morphology assessment requires experience and continuous training. For the correct assessment of cytologic myelodysplasia it is essential to keep in mind the morphology of normal myelopoiesis. To the extent of our knowledge there are no studies describing together morphological data on normal and dysplastic myelopoiesis in the framework of MDS. Therefore, by combining these data, this manuscript could serve as a useful tool for quotidian process of diagnosis.


Assuntos
Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/fisiopatologia , Mielopoese , Células Sanguíneas/patologia , Medula Óssea/patologia , Citogenética , Citometria de Fluxo , Humanos , Microscopia
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