Cell Viability Multiplexing: Quantification of Cellular Viability by Barcode Flow Cytometry and Computational Analysis.

Fluorescent cell barcoding (FCB) is a useful flow cytometric technique for high-throughput multiplexed analyses and can minimize technical variations after preliminary optimization and validation of protocols. To date, FCB is widely used for measurement of phosphorylation status of certain proteins, while it can be also employed for cellular viability assessment. In this chapter, we describe the protocol to perform FCB combined with viability assessment on lymphocytes and monocytes using manual and computational analysis. We also provide recommendations for FCB protocol optimization and validation for clinical sample analysis.

Linguagem e comunicação na perspectiva do sujeito com esquizofrenia / Language and communication from the perspective of people with schizophrenia / Lenguaje y comunicación desde la perspectiva del sujeto con esquizofrenia

Introdução: a esquizofrenia apresenta um conjunto de sintomas cognitivos, psicóticos, afetivos e emocionais que influenciam a linguagem e o processo comunicativo. Objetivo: refletir sobre o impacto das manifestações linguístico-discursivas da esquizofrenia na comunicação, a partir da perspectiva de sujeitos com esse transtorno mental, além de descrever as principais manifestações linguísticas presentes no discurso desses indivíduos. Método: estudo transversal, descritivo e qualitativo, com modelo de amostragem não probabilística por conveniência, realizado em um Centro de Atendimento Psicossocial. Realizou-se análise de prontuários e entrevista semiestruturada com usuários do serviço de saúde diagnosticados com esquizofrenia. Os resultados foram submetidos à análise estatística descritiva, sendo que as entrevistas foram exploradas de forma qualitativa por meio da "Análise Temática do Conteúdo", após transcrição das falas na íntegra. Resultados: metade dos participantes relatou satisfação e conforto comunicativo, enquanto a outra metade informou dificuldade de comunicação associada a sentimentos de frustração, má disposição, nervosismo e receio de falar em público. Tais sentimentos parecem estar relacionados à postura de desvalorização e estigma do interlocutor perante o discurso de pessoas com esquizofrenia. Observaram-se as seguintes manifestações linguístico-discursivas: descarrilamento e alogia. Não é possível afirmar que uma das manifestações linguístico-discursivas observadas nos participantes, a alogia, esteja associada, exclusivamente, à sintomatologia do transtorno mental, visto que a condição de segregação e estigma social pode ser um fator determinante na redução da produção do discurso. Conclusão: os sintomas de linguagem característicos da esquizofrenia impactam as relações de comunicação dos sujeitos diagnosticados com esse transtorno mental.

Introduction: schizophrenia has a set of cognitive, psychotic, affective and emotional symptoms that influence language and the communicative process. Objective: to reflect on the impact of linguistic-discursive manifestations of schizophrenia on communication, from the perspective of individuals with this mental disorder, in addition to describing the main manifestations present in the speech of these individuals. Method: cross-sectional, descriptive and qualitative study, with a non-probabilistic sampling model for convenience, carried out in a Psychosocial Service Center. Analyses of medical records and semi-structured interviews with users of the health service, diagnosed with schizophrenia, were performed. The results were subjected to descriptive statistical analysis, and the interviews were explored in a qualitative way through the "Thematic Content Analysis", after transcribing the speeches. Results: half of the participants reported satisfaction and communicative comfort, while the other half reported communication difficulties, associated with feelings of frustration, nervousness and fear of speaking in public. Such feelings seem to be related to the attitude of devaluation and stigma of the interlocutor regarding the speech of people with schizophrenia. The following discursive linguistic manifestations were observed: derailment and allogy. It is not possible to state that one of the linguistic-discursive manifestations observed in the participants, the allogy, is associated exclusively with the symptoms of mental disorder, since the condition of segregation and social stigma can be a determining factor in reducing the production of speech. Conclusion: the language symptoms characteristic of schizophrenia impact the communication relationships of subjects diagnosed with this mental disorder.

Introducción: la esquizofrenia tiene un conjunto de síntomas cognitivos, psicóticos, afectivos y emocionales que influyen en el lenguaje y el proceso comunicativo. Objetivo: Reflexionar sobre el impacto de las manifestaciones lingüístico-discursivas de la esquizofrenia en la comunicación, desde la perspectiva de sujetos con este trastorno mental, además de describir las principales manifestaciones presentes en el discurso de estos individuos. Método: estudio transversal, descriptivo y cualitativo, con un modelo de muestreo no probabilístico por conveniencia, realizado en un Centro de Servicios Psicosociales. Se realizaron análisis de historias clínicas y entrevistas semiestructuradas a usuarios del servicio de salud, diagnosticados de esquizofrenia. Los resultados fueron sometidos a análisis estadístico descriptivo, y las entrevistas fueron exploradas de manera cualitativa a través del "Análisis de contenido temático", luego de transcribir los discursos en forma íntegra. Resultados: la mitad de los participantes reportó satisfacción y comodidad comunicativa, mientras que la otra mitad reportó dificultades de comunicación, asociadas con sentimientos de frustración, nerviosismo y miedo a hablar en público.Tales sentimientos parecen estar relacionados con la actitud de desvalorización y estigma del interlocutor con respecto al discurso de las personas con esquizofrenia. Se observaron las siguientes manifestaciones lingüísticas discursivas: descarrilamiento y alogía. No es posible afirmar que una de las manifestaciones lingüístico-discursivas observadas en los participantes, la alología, esté asociada exclusivamente a los síntomas del trastorno mental, ya que la condición de segregación y estigma social puede ser un factor determinante en la reducción de la producción del habla. Conclusión: los síntomas del lenguaje característicos de la esquizofrenia impactan las relaciones de comunicación de los sujetos diagnosticados con este trastorno mental.

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis.

Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique because of inter-operator variability and differences in data analysis. FCB was implemented by combining two-dye barcoding (DyLight 350 plus Pacific Orange) with five-color surface marker antibody and intracellular staining for phosphoprotein signaling analysis. We proposed a robust method to measure intra- and inter-assay variability of FCB in T/B cells and monocytes by combining range and ratio of variability to standard statistical analyses. Data analysis was carried out by conventional and semi-automated workflows and built with R software. Results obtained from both analyses were compared to assess feasibility and reproducibility of FCB data analysis by machine-learning methods. Our results showed efficient FCB using DyLight 350 and Pacific Orange at concentrations of 0, 15 or 30, and 250 µg/mL, and a high reproducibility of FCB in combination with surface marker and intracellular antibodies. Inter-operator variability was minimized by adding an internal control bridged across matrices used as rejection criterion if significant differences were present between runs. Computational workflows showed comparable results to conventional gating strategies. FCB can be used to study phosphoprotein signaling in T/B cells and monocytes with high reproducibility across operators, and the addition of bridge internal controls can further minimize inter-operator variability. This FCB protocol, which has high throughput analysis and low intra- and inter-assay variability, can be a powerful tool for clinical trial studies. Moreover, FCB data can be reliably analyzed using computational software.