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1.
Transfusion ; 60(5): 1032-1041, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32237236

RESUMO

BACKGROUND: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. STUDY DESIGN AND METHODS: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. RESULTS: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. CONCLUSIONS: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.


Assuntos
Deformação Eritrocítica/fisiologia , Eritrócitos/citologia , Eritrócitos/fisiologia , Dispositivos Lab-On-A-Chip/normas , Técnicas Analíticas Microfluídicas , Bancos de Sangue/métodos , Bancos de Sangue/normas , Velocidade do Fluxo Sanguíneo/fisiologia , Preservação de Sangue/efeitos adversos , Preservação de Sangue/métodos , Preservação de Sangue/normas , Criopreservação , Contagem de Eritrócitos/instrumentação , Contagem de Eritrócitos/métodos , Contagem de Eritrócitos/normas , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Hemólise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/normas , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Fatores de Tempo
2.
Nat Commun ; 11(1): 1162, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139684

RESUMO

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells s-1 without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.


Assuntos
Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Aprendizado Profundo , Euglena gracilis , Estudos de Viabilidade , Citometria de Fluxo/instrumentação , Hematologia/instrumentação , Hematologia/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Células Jurkat , Técnicas Microbiológicas/instrumentação , Microscopia de Fluorescência/instrumentação , Sensibilidade e Especificidade
3.
J Clin Pathol ; 73(10): 676-677, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32034055

RESUMO

AIM: The aim was to assess the flagging performance of Sysmex XN-10 haematology analyser for malaria detection through the parasitic red blood cell ('pRBC') alarm. METHODS: We retrospectively studied 584 blood samples performed on the Sysmex XN-10 analyser that were tested for malaria. Sensitivity, specificity, positive and negative predictive values, and prevalence were established for the pRBC alarm. RESULTS: Sensitivity, specificity, and positive and negative predictive values for the pRBC flag were 7.8%, 100%, 100% and 87.7%, respectively. The prevalence of pRBC flag of 0.026% in the overall population was significantly different from the prevalence of 1.027% in the population tested for malaria. CONCLUSIONS: Considering the excellent specificity and the low prevalence of the flag in the overall population, we suggest, in case of the presence of pRBC flag, the implementation of a rapid review of the blood smear looking for Plasmodium, mostly if the patient had fever and had not been tested for malaria.


Assuntos
Contagem de Células Sanguíneas/instrumentação , Citometria de Fluxo/instrumentação , Hematologia/instrumentação , Malária/diagnóstico , Automação Laboratorial/instrumentação , Contagem de Células Sanguíneas/métodos , Eritrócitos/parasitologia , Citometria de Fluxo/métodos , Hematologia/métodos , Humanos , Malária/sangue , Sensibilidade e Especificidade
6.
Brasília; CONITEC; nov. 2019.
Não convencional em Português | BRISA/RedTESA | ID: biblio-1120899

RESUMO

CONTEXTO: No dia 14 de dezembro de 2018, o Secretário da Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde tornou pública, por meio da Portaria nº77/2018, a decisão de incorporar o medicamento eculizumabe para tratamento de pacientes com hemoglobinúria paroxística noturna (HPN) no âmbito do Sistema Único de Saúde (SUS). Tal incorporação, entretanto, foi atrelada às medidas condicionantes, dentre elas a elaboração do Protocolo de Uso do medicamento. Dentre tais critérios a serem definidos, o diagnóstico acurado dos pacientes a serem tratados é de extrema importância, dada a gravidade da doença, os possíveis riscos associados ao tratamento e o alto custo do medicamento ofertado. Para tal, esta nota técnica visa esclarecer as evidências disponíveis sobre a citometria de fluxo - método atualmente considerado por especialistas como padrão ouro no diagnóstico da HPN - e avaliar a sua ampliação de uso, uma vez que a tecnologia já está incorporada no SUS. TECNOLOGIA: Citometria de fluxo. EVIDÊNCIAS CIENTÍFICAS: A busca de evidências foi realizada no Pubmed, limitado para estudos em humanos, e recuperou 437 artigos. Para a melhor tomada de decisão foram selecionados os estudos mais relevantes na investigação da acurácia, sensibilidade e especificidade da citometria de fluxo como método diagnóstico da HPN, como protocolos clínicos bem fundamentados e estudos de metodologia robusta. A citometria de fluxo é recomendada para diagnóstico de HPN pelas principais organizações de interesse (International Clinical Cytometry Society - ICCS, European Society for Clinical Cell Analysis - ESCCA, International PNH Interest Group - IPIG). AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO (AIO): Foi realizada uma análise simplificada de impacto orçamentário. Os custos assumidos nessa análise foram restritos aos de realização do procedimento, de acordo com o Sistema de Gerenciamento da Tabela de Procedimentos, Medicamentos e OPM do SUS (SIGTAP). Estima-se um impacto orçamentário de R$ 5,8 milhões ao final de cinco anos com a realização de citometria de fluxo para diagnóstico de HPN e acompanhamento de pacientes com alta atividade. CONSIDERAÇÕES FINAIS: Apesar do consenso sobre a utilização da citometria de fluxo, a ausência de outros métodos de diagnóstico específicos para a HPN inviabilizou a elaboração dos estudos iniciais de acurácia, sensibilidade e especificidade. Entretanto, vários estudos comparando sensibilidade e especificidade entre diferentes metodologias para citometria de fluxo foram encontrados, como comparações entre a CF convencional e de alta sensitividade, diferentes reagentes, linhagens celulares e aparelhos. No contexto proposto neste documento, a citometria de fluxo demonstrou-se um método de diagnóstico adequado, por isso o seu uso deve ser considerado no sistema público de saúde. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Pelo exposto, a CONITEC, em sua 81ª reunião ordinária, nos dias 04 e 05 de setembro de 2019, recomendou a ampliação do uso da citometria de fluxo no SUS, para o diagnóstico de Hemoglobinúria Paroxística Noturna. A matéria foi disponibilizada em consulta pública. CONSULTA PÚBLICA: A Consulta Pública nº 56 foi realizada entre os dias 18/09/2019 e 27/09/2019. Foram recebidas 17 contribuições, sendo quatro pelo formulário para contribuições técnicocientíficas e 13 pelo formulário para contribuições sobre experiência ou opinião. Todas as 13 contribuições sobre experiência e opinião concordaram com a recomendação da Nota Técnica para ampliação do uso da CF para diagnóstico de pacientes com HPN, sendo 12 pessoas físicas representadas por familiares, amigos ou cuidadores de paciente (50%) e interessados no tema (25%), e uma pessoa jurídica (Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular). Já o formulário para contribuições técnicas contou com quatro participações, todas de acordo com a recomendação, sendo duas destas enviadas pela fabricante do medicamento eculizumabe, Alexion Pharmaceuticals. Após apreciação das contribuições encaminhadas pela Consulta Pública, o plenário da Conitec entendeu que não houve argumentação suficiente para alterar a recomendação inicial. RECOMENDAÇÃO FINAL: Os membros da CONITEC presentes na reunião do plenário realizada no dia 10 de outubro de 2019, deliberaram, por unanimidade, recomendar a ampliação do uso da citometria de fluxo para diagnóstico de hemoglobinúria paroxística noturna. Foi assinado o Registro de Deliberação nº 482/2019. Decisão: Ampliar o uso de citometria de fluxo para diagnóstico de hemoglobinúria paroxística noturna, no âmbito do Sistema Único de Saúde ­ SUS, conforme Portaria nº 61, publicada no Diário Oficial da União nº 226, seção 1, página 151, em 21 de novembro de 2019.


Assuntos
Humanos , Citometria de Fluxo/instrumentação , Hemoglobinúria Paroxística/diagnóstico , Avaliação da Tecnologia Biomédica , Sistema Único de Saúde , Brasil , Análise Custo-Benefício/economia
7.
J Immunol Methods ; 475: 112680, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655051

RESUMO

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.


Assuntos
Citometria de Fluxo/instrumentação , Citometria de Fluxo/normas , Imunofenotipagem/instrumentação , Imunofenotipagem/normas , Humanos , Padrões de Referência
8.
J Immunol Methods ; 474: 112646, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31419409

RESUMO

INTRODUCTION: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. METHODS: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. RESULTS: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) p = .058, CD11b: 2.13× (1.6×-2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. CONCLUSION: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15-20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


Assuntos
Antígeno CD11b/sangue , Citometria de Fluxo , Imunofenotipagem/métodos , Leucócitos/metabolismo , Neprilisina/sangue , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Receptores de Complemento 3b/sangue , Adolescente , Adulto , Idoso , Automação Laboratorial , Biomarcadores/sangue , Feminino , Citometria de Fluxo/instrumentação , Voluntários Saudáveis , Humanos , Imunofenotipagem/instrumentação , Leucócitos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho , Adulto Jovem
9.
Zhongguo Yi Liao Qi Xie Za Zhi ; 43(4): 270-274, 2019 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-31460719

RESUMO

In order to meet the needs of the flow cytometry for the simultaneous analysis of multiple fluorescence wavelengths and small volume, the design method of flow cytometry spectrum analysis system is presented by analyzing the characteristics of Dyson structure. And according to the method, a flow cytometry spectrum analysis system is disigned with Dyson type.The system's spectral range is 400 nm to 800 nm, the defocused spot size is less than the pixel size 24µ mm, the ransfer function value is above 0.8 at the Nyquist cut-off frequency 21 lp/mm,the spectral resolution is less than 3 nm, and the overall size is 83.54 mm×85.60 mm.The system has good optical performance and small volume, which meets the needs of the flow cytometry fluorescence spectral analysis. The outstanding innovation of this system is the application of Dyson light splitting structure and EMCCD detector which is high speed and high sensitivity.


Assuntos
Desenho de Equipamento , Citometria de Fluxo , Citometria de Fluxo/instrumentação
10.
Biosens Bioelectron ; 142: 111526, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31362203

RESUMO

Efficient manipulation of micro biological cells has always been a very important task in healthcare sector for which a Micro Electro Mechanical System (MEMS) based impedance flow cytometry has been proven to be a promising technique. This technique utilise the advantage of dielectrophoresis (DEP) force which is generated by non-uniform electric field in a microfluidic channel using an appropriate external AC supply at certain frequency range. The DEP forces generated in micro-channel depend upon various biological and physical parameters of cell and suspending medium. Apart from that design parameters of microfluidic channel and dimension of electrodes used for generating DEP action also plays major role in micro cell/bead manipulation. This article give remarks on the operating parameters which affects the cell manipulation and interrogates the currently accepted various electrode orientations in microfluidic MEMS flow cytometer technologies for effective manipulation of micro entities like healthy human cells (T-lymphocytes, B- lymphocytes, Monocytes, Leukocytes erythrocytes and human kidney cells HEK293), animal cells (neuroblastoma N115 and sheep red blood cells), cancer cells (MCF-7, MDA-435 and CD34+), yeast cells (saccharomyces cerevisiae, listeria innocua and E. coli) and micro particles (polystyrene beads) based on their dielectric properties using DEP action. Article focuses on the key electrode orientations for generation of non-uniform electric field in microfluidic flow cytometer like tapered electrodes, trapezoidal electrode arrays, Interdigitated electrodes, curved microelectrode and 3D electrode orientations and give remarks on their advantages and limitations. The cell manipulation with current MEMS impedance flow cytometry orientations targeting possibilities of implementation of the lab-on-chip devices has been discussed.


Assuntos
Citometria de Fluxo/instrumentação , Sistemas Microeletromecânicos/instrumentação , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Impedância Elétrica , Desenho de Equipamento , Citometria de Fluxo/métodos , Humanos , Sistemas Microeletromecânicos/métodos , Micromanipulação/instrumentação , Micromanipulação/métodos
11.
Sensors (Basel) ; 19(12)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248214

RESUMO

Miniaturizing flow cytometry requires a comprehensive approach to redesigning the conventional fluidic and optical systems to have a small footprint and simple usage and to enable rapid cell analysis. Microfluidic methods have addressed some challenges in limiting the realization of microflow cytometry, but most microfluidics-based flow cytometry techniques still rely on bulky equipment (e.g., high-precision syringe pumps and bench-top microscopes). Here, we describe a comprehensive approach that achieves high-throughput white blood cell (WBC) counting in a portable and handheld manner, thereby allowing the complete miniaturization of flow cytometry. Our approach integrates three major components: a motorized smart pipette for accurate volume metering and controllable liquid pumping, a microfluidic cell concentrator for target cell enrichment, and a miniaturized fluorescence microscope for portable flow cytometric analysis. We first validated the capability of each component by precisely metering various fluid samples and controlling flow rates in a range from 219.5 to 840.5 µL/min, achieving high sample-volume reduction via on-chip WBC enrichment, and successfully counting single WBCs flowing through a region of interrogation. We synergistically combined the three major components to create a handheld, integrated microflow cytometer and operated it with a simple protocol of drawing up a blood sample via pipetting and injecting the sample into the microfluidic concentrator by powering the motorized smart pipette. We then demonstrated the utility of the microflow cytometer as a quality control means for leukoreduced blood products, quantitatively analyzing residual WBCs (rWBCs) in blood samples present at concentrations as low as 0.1 rWBCs/µL. These portable, controllable, high-throughput, and quantitative microflow cytometric technologies provide promising ways of miniaturizing flow cytometry.


Assuntos
Citometria de Fluxo/instrumentação , Microfluídica/instrumentação , Microscopia de Fluorescência/instrumentação , Miniaturização/instrumentação , Animais , Cães , Leucócitos/metabolismo , Microfluídica/métodos , Pressão , Reologia , Vibração
12.
IEEE Trans Nanobioscience ; 18(3): 369-372, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31180894

RESUMO

Fine needle aspirate sampling of tumors requires acquisition of sufficient cells to complete a diagnosis. Aspirates through such fine needles are typically composed of small cell clusters in suspension, making them readily amenable to microfluidic analysis. Here we show a microfluidic device with integrated electrodes capable of interrogating and identifying cellular components in a patient-derived sample of dissociated tumor cells using micro-electrical impedance spectroscopy ( µ EIS). We show that the µ EIS system can distinguish dissociated tumor cells in a sample consisting of red blood cell (RBCs) and peripheral blood mononucleated cells (PBMCs). Our µ EIS system can also distinguish dissociated tumor cells from normal cells and we show results for five major cancer types, specifically, lung, thyroid, breast, ovarian, and kidney cancer. Moreover, our µ EIS system can make these distinctions in a label-free manner, thereby opening the possibility of integration into standard clinical workflows at the point of care.


Assuntos
Espectroscopia Dielétrica , Neoplasias/patologia , Análise de Célula Única , Células Tumorais Cultivadas/citologia , Biópsia por Agulha Fina , Espectroscopia Dielétrica/instrumentação , Espectroscopia Dielétrica/métodos , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
13.
Int J Lab Hematol ; 41(5): 607-614, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31162830

RESUMO

BACKGROUND: The MRD status detected using multiparameter flow cytometry (MFC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT) has crucial prognostic value for patients with acute myeloid leukemia (AML) in morphologic complete remission (CR). We designed a novel panel of antibodies to identify aberrant differentiation/maturation profiles of residual leukemia cells in patients who were not diagnosed at our institution, relapsed with an antigenic shift, or lack leukemia-associated immunophenotypes. METHODS: We compared the MRD status detected using MFC and real-time quantitative polymerase chain reaction (RT-qPCR) in the same 158 bone marrow samples collected from 44 AML patients carrying leukemia-specific fusion genes. The clinical performance of the MFC-based MRD status was analyzed in 168 AML patients who exhibited morphologic CR (135) or active disease (33) before HSCT. RESULTS: Strong concordance was found between MFC-based and RT-qPCR-based MRD status (κ = 0.868). Among the patients displaying CR, the positive MRD status detected using MFC was correlated with a worse prognosis [HRs (P values) for relapse, event-free survival, and overall survival: 4.83 (<0.001), 2.23 (0.003), and 1.79 (0.049), respectively]; the prognosis was similar to patients with an active disease before HSCT. Patients with a positive MRD before HSCT might experience a benefit from developing chronic graft-vs-host disease. CONCLUSIONS: The assessment of MRD using our self-built different-from-normal AML-MRD detection panel exhibited reliable sensitivity, specificity, and accuracy. In addition, patients with a positive MRD status before transplantation may have higher risk of relapse and worse survival.


Assuntos
Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mieloide/terapia , Neoplasia Residual/diagnóstico , Doença Aguda , Adolescente , Adulto , Criança , Intervalo Livre de Doença , Feminino , Citometria de Fluxo/instrumentação , Humanos , Imunofenotipagem/métodos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/genética , Proteínas de Fusão Oncogênica/genética , Prognóstico , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Homólogo , Adulto Jovem
14.
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
15.
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
16.
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
17.
PLoS One ; 14(5): e0217298, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125378

RESUMO

OBJECTIVE: The aim of this study is to evaluate the performance of different platelet counting methods (optical, impedance, fluorescence and hand counting) applied in different analysers by comparing with the international flow cytometric reference method (IRM). METHODS: A total of 333 blood samples from different subgroups (168 cases with thrombocytopenia, 136 cases with normal platelet counts and 29 cases with thrombocytosis) were tested. Regarding IRM as the gold standard, we compared the accuracy and precision of different platelet count methods; i.e. LH780 (impedance), BC-6000 Plus (optical (O) and impedance (I)), Sysmex XN-9000 (optical (O), impedance (I), fluorescence (F)), and hand counting. RESULTS: Sysmex XN-9000-F (r = 0.988) had the best correlation with IRM for thrombocytopenic samples; BC-6000 Plus-I (r = 0.966) was more relevant to IRM than any other method for samples with normal platelet counts. Correlation between Sysmex XN-9000-I (r = 0.960) and IRM was the highest among these methods for samples with thrombocytosis. For bias evaluation, the average bias of Sysmex XN-9000-F was -1.5 × 109/L (95% LA = -9.4 to +6.4) for samples with thrombocytopenia, compared with IRM. BC-6000 Plus-I had a small mean difference with IRM for samples with normal platelet counts or thrombocytosis. Moreover, all evaluated methods had acceptable sensitivity, specificity, and concordance rates as compared with IRM in the diagnosis of thrombocytopenia and thrombocytosis. CONCLUSIONS: Platelet counting by Sysmex XN-9000-F is more accurate than other methods for thrombocytopenic samples. BC-6000 Plus-I has superior association and consistency for normal platelet counts. As for thrombocytosis patients, Sysmex XN-9000-I has the highest correlation with IRM while Sysmex XN-9000-O has the highest diagnosis efficacy.


Assuntos
Citometria de Fluxo/instrumentação , Contagem de Plaquetas/instrumentação , Trombocitose/sangue , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Contagem de Plaquetas/métodos , Contagem de Plaquetas/normas , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trombocitopenia/sangue
18.
Int J Lab Hematol ; 41(4): 542-549, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102331

RESUMO

INTRODUCTION: Diagnosis of myelodysplastic syndromes (MDS) is usually challenging. In this context, we have attempted to employ data derived from automated analysis of bone marrow (BM) samples as an ancillary tool for the discrimination between reactive marrow and MDS. METHODS: A total of 101 BM anticoagulated samples referred for flow cytometry (FCM) analysis on the clinical suspicion of MDS had been previously counted in a Mindray BC-6800 hematology analyzer (testing set). Among them, 22/101 randomly selected BM samples (comparison set) had been also simultaneously counted by an Advia 2120 and a CELL-DYN Sapphire hematology analyzer. Selected parameters obtained by Mindray BC-6800 were retrospectively evaluated with ROC and regression analysis in an attempt to formulate a discriminative scoring system (SS) for MDS. This system was further evaluated in the comparison set. RESULTS: The diagnosis of MDS was established in 37/101 patients assessed ("MDS" group). Three patients were diagnosed with myelodysplastic/myeloproliferative neoplasm (MDS/MPN), while 61 revealed a "reactive" bone marrow ("RBM" group). Statistical analysis revealed significant differences in Hb, RDW-CV%, NRBC%, and RET% values between the "MDS" and the "RBM" group. Specific cutoff values were then indicated and employed for the formulation of a SS of high sensitivity (86.84%) and specificity (86.89%). The encouraging performance characteristics of the proposed SS were also confirmed in the BM comparison set. CONCLUSION: Automated BM counts on hematology analyzers contributed to the formulation of a SS for the screening discrimination between reactive and MDS BM fluids, which seems to be applicable and informative, regardless of the analyzer used.


Assuntos
Células da Medula Óssea/patologia , Citometria de Fluxo/instrumentação , Síndromes Mielodisplásicas , Doenças Mieloproliferativas-Mielodisplásicas , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/patologia , Doenças Mieloproliferativas-Mielodisplásicas/diagnóstico , Doenças Mieloproliferativas-Mielodisplásicas/patologia
19.
Ann Hematol ; 98(6): 1413-1420, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30830246

RESUMO

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.


Assuntos
Apresentação de Dados , Detecção Precoce de Câncer/métodos , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leucemia Promielocítica Aguda/diagnóstico , Adulto , Idoso , Antígenos CD/análise , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Medula Óssea/patologia , Bandeamento Cromossômico , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 15/ultraestrutura , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 17/ultraestrutura , Fator XIII/análise , Feminino , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Humanos , Imunofenotipagem/instrumentação , Hibridização in Situ Fluorescente , Leucemia Promielocítica Aguda/sangue , Leucemia Promielocítica Aguda/genética , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Estudos Retrospectivos , Translocação Genética
20.
Eur Biophys J ; 48(3): 267-275, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30903263

RESUMO

We introduce a simple, label-free cytometry technique, based on the spatio-temporal fluctuation analysis of pixel gray levels of a cell image utilizing the Gray Level Information Entropy (GLIE) function. In this study, the difference in GLIE random fluctuations and its biophysical etiology in a comparison cell model of leukemic Jurkat cells and human healthy donor lymphocytes was explored. A combination of common bright field microscopy and a unique imaging dish wherein cells are individually held untethered in a picoliter volume matrix of optical chambers was used. Random GLIE fluctuations were found to be greater in malignant Jurkat cells than in benign lymphocytes, while these fluctuations correlate with intracellular vesicle Mean Square Displacement (MSD) values and are inhibited by myosin-2 and adenosine triphosphate (ATP) inhibitors. These results suggest that the incoherent active forces acting on the cytoskeleton which cause mechanical dissipative fluctuation of the cytoskeletal and related intracellular content are the biophysical cellular mechanism behind the GLIE random fluctuation results. Analysis of the results in Jurkat cells and normal lymphocytes suggests the possible potential of this simple and automated label-free cytometry to identify malignancy, particularly in a diagnostic setup of multiple cell examination.


Assuntos
Citometria de Fluxo/instrumentação , Leucemia/patologia , Linfócitos/citologia , Trifosfato de Adenosina/metabolismo , Humanos , Células Jurkat
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