Multiplex Spatial Protein Detection by Combining Immunofluorescence with Immunohistochemistry.

Technologies for staining and imaging multiple antigens in single tissue sections are developing rapidly due to their potential to uncover spatial relationships between proteins with cellular resolution. Detections are performed simultaneously or sequentially depending on the approach. However, several technologies can detect limited numbers of antigens or require expensive equipment and reagents. Another serious concern is the lack of flexibility. Most commercialized reagents are validated for defined antibody panels, and introducing any changes is laborious and costly. In this chapter, we describe a method where we combine, for the first time, multiplexed IF followed by sequential immunohistochemistry (IHC) with AEC chromogen on Leica Bond staining processors with paraffin tissue sections. We present data for successful detection of 10 antigens in a single tissue section with preserved tissue integrity. Our method is designed for use with any combination of antibodies of interest, with images collected using whole slide scanners. We include an image viewing and image analysis workflow using nonlinear warping to combine all staining passes in a single full-resolution image of the entire tissue section, aligned at the single cell level.

Uma análise dos principais diagnósticos de Sars-cov-2 disponíveis no Brasil: uma revisão de literatura / Diagnosis and main types of coronavirus in humans: a narrative review / Diagnóstico y principales tipos de coronavirus en humanos: una revisión narrativa

Introdução: Com a emergência do SARS-CoV-2 foi disponibilizado uma grande quantidade de ferramentas de diagnóstico. Neste contexto, a falta de vacina, de tratamento e o grande número de casos graves e morte, possibilitou a aprovação emergencial de diversos testes, que ainda necessitam de estudos populacionais para seu registro definitivo. Objetivo: Realizar uma revisão de literatura para avaliar as metodologias de diagnóstico disponíveis no Brasil, de acordo com a realidade local de saúde, explorando o momento epidemiológico a complexidade do teste e a finalidade da sua aplicação. Metodologia: Trata-se de um estudo bibliográfico, descritivo do tipo revisão de literatura. Foram utilizadas as seguintes bases de dados científicos para buscas: PUBMED, MEDLINE, LILACS E COCHRANE LIBRARY, através de descritores selecionados na plataforma DECS. Resultados: O cenário de diversos ensaios, baseados em diferentes metodologias, como os testes baseados em RNA viral, em detecção de antígenos virais ou de anticorpos, associados ao conhecimento da história natural do vírus, possibilita uma análise crítica do melhor diagnóstico de acordo com a clínica do paciente, os epidemiológicos, o objetivo do diagnóstico e a acurácia do ensaio. Atualmente, há mudança no padrão imunológico da população e a descrição de tipos e subtipos de SARS-CoV-2 com mudanças gênicas, que podem levar a mudanças na acurácia diagnóstica ou a re-emergência em surtos de doença grave. Conclusão: Ainda é incerto o caminho evolutivo da história natural da Covid-19 e os ensaios diagnósticos estão em diferentes estágios de desenvolvimento, validação e produção e cada tipo de teste tem suas próprias vantagens e desvantagens distintas inerentes a plataforma tecnológica de origem e uma combinação de tipos de testes usados em momentos diferentes pode ser útil para a condução clínica dos pacientes e no controle da pandemia por SARS-CoV-2.

Introduction: With the emergence of SARS-CoV-2, a large number of diagnostic tools were made available. In this context, the lack of vaccine, treatment and the large number of severe cases and death, allowed the emergency approval of several tests, which still require population studies for their definitive registration. Objective: To carry out a literature review to evaluate the diagnostic methodologies available in Brazil, according to the local health reality, exploring the epidemiological moment, the complexity of the test and the purpose of its application. Methodology: This is a bibliographic, descriptive study of the literature review type. The following scientific databases were used for searches: PUBMED, MEDLINE, LILACS AND COCHRANE LIBRARY, through selected descriptors on the DECS platform. Results: The scenario of several tests, based on different methodologies, such as tests based on viral RNA, on detection of viral antigens or antibodies, associated with knowledge of the natural history of the virus, allows a critical analysis of the best diagnosis according to the patient's clinical, epidemiological, diagnostic objective and assay accuracy. Currently, there is a change in the immune pattern of the population and the description of types and subtypes of SARS-CoV-2 with genetic changes, which can lead to changes in diagnostic accuracy or the re-emergence in outbreaks of severe disease. Conclusion: The evolutionary path of the natural history of Covid-19 is still uncertain and diagnostic assays are at different stages of development, validation and production and each type of test has its own distinct advantages and disadvantages inherent in the technology platform of origin and a combination of types of tests used at different times can be useful for the clinical management of patients and in the control of the SARS-CoV-2 pandemic.

Introducción: Con la aparición del SARS-CoV-2, se dispuso de un gran número de herramientas diagnósticas. En este contexto, la falta de vacuna, tratamiento y el gran número de casos graves y muerte, permitieron la aprobación de urgencia de varias pruebas, que aún requieren estudios poblacionales para su registro definitivo. Objetivo: Realizar una revisión bibliográfica para evaluar las metodologías diagnósticas disponibles en Brasil, de acuerdo con la realidad sanitaria local, explorando el momento epidemiológico, la complejidad de la prueba y la finalidad de su aplicación. Metodología: Se trata de un estudio bibliográfico, descriptivo, del tipo revisión de literatura. Para las búsquedas se utilizaron las siguientes bases de datos científicas PUBMED, MEDLINE, LILACS Y COCHRANE LIBRARY, a través de descriptores seleccionados en la plataforma DECS. Resultados: El escenario de varias pruebas, basadas en diferentes metodologías, como pruebas basadas en el ARN viral, en la detección de antígenos virales o anticuerpos, asociado al conocimiento de la historia natural del virus, permite un análisis crítico del mejor diagnóstico de acuerdo con la clínica del paciente, epidemiológica, objetivo diagnóstico y precisión de la prueba. Actualmente, hay un cambio en el patrón inmunológico de la población y la descripción de tipos y subtipos de SARS-CoV-2 con cambios genéticos, que pueden conducir a cambios en la precisión diagnóstica o la reaparición en brotes de enfermedad grave. Conclusiones: El camino evolutivo de la historia natural del Covid-19 es aún incierto y los ensayos de diagnóstico se encuentran en diferentes etapas de desarrollo, validación y producción y cada tipo de prueba tiene sus propias ventajas y desventajas distintas inherentes a la plataforma tecnológica de origen y una combinación de tipos de pruebas utilizadas en diferentes momentos puede ser útil para el manejo clínico de los pacientes y en el control de la pandemia de SARS- CoV-2.

Diagnostic applications of microsphere-based flow cytometry: A review.

Microsphere-based flow cytometry is a highly sensitive emerging technology for specific detection and clinical analysis of antigens, antibodies, and nucleic acids of interest. In this review, studies that focused on the application of flow cytometry as a viable alternative for the investigation of infectious diseases were analyzed. Many of the studies involve research aimed at epidemiological surveillance, vaccine candidates and early diagnosis, non-infectious diseases, specifically cancer, and emphasize the simultaneous detection of biomarkers for early diagnosis, with accurate results in a non-invasive approach. The possibility of carrying out multiplexed assays affords this technique high versatility and performance, which is evidenced in a series of clinical studies that have verified the ability to detect several molecules in low concentrations and with minimal sample volume. As such, we demonstrate that microsphere-based flow cytometry presents itself as a promising technique that can be adopted as a fundamental element in the development of new diagnostic methods for a number of diseases.

Enhancing Antigen Retrieval to Unmask Signaling Phosphoproteins in Formalin-fixed Archival Tissues.

The introduction of targeted therapy has revolutionized cancer treatment. Nonetheless, for this approach to succeed, it is crucial to identify the targets, particularly when activated, in tumor tissues. Phosphorylation is a posttranslational modification that causes activation of numerous oncogenic protein kinases and transcription regulators. Hence, phosphoproteins is a class of biomarkers that has therapeutic and prognostic implications directly relevant to cancer patients' management. Despite the progress in histopathology methodology, analysis of the expression of phosphoproteins in tumor tissues still represents a challenge owing to preanalytical and analytical factors that include antigen retrieval strategies. In this study, we tested the hypothesis that optimizing antigen retrieval methods will improve phosphoproteins unmasking and enhance their immunohistochemical staining signal. We screened 4 antigen retrieval methods by using antibodies specific for 3 oncogenic phosphoproteins to stain human lymphoma tumors that were developed in severe combined immunodeficiency mice and subsequently fixed in formalin for 2 years. Then, we used antibodies specific for 15 survival phosphoproteins to compare the most effective method identified from our screening experiment to the antigen retrieval method that is most commonly utilized. Using the antigen retrieval buffer Tris-EDTA at pH 9.0 and heating for 45 minutes at 97°C unmasked and significantly enhanced the staining of 9 of the 15 phosphoproteins (P<0.0001). Our antigen retrieval approach is cost effective and feasible for clinical and research settings. We anticipate that combining this approach with the newly proposed methods to improve tissue fixation will further improve unmasking of phosphoproteins in human and animal tissues.

Persistent SARS-CoV-2 antigen presence in multiple organs of a naturally infected cat from Brazil

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the disease coronavirus 2019 (COVID-19) in humans. SARS-CoV-2 has been identified in cats with or without clinical signs. Case presentation: We describe the pathological and molecular findings in a six-month-old asymptomatic cat with SARS-CoV-2 infection from Brazil, belonging to a human family with COVID-19 cases. The pool of nasopharynx and oropharynx swabs at day zero tested positive by RT-qPCR for SARS-CoV-2. No amplification resulted from molecular testing performed on days 7 and 14. The cat was hit by a car and died 43 days after the molecular diagnosis. Immunohistochemistry at post-mortem examination demonstrated nucleocapsid protein in samples from the lungs, kidneys, nasal conchae, trachea, intestine, brain and spleen. Conclusion: The present study has highlighted the possibility that viral antigens can be detected by immunohistochemistry in multiple organs six weeks after infection, although the same tissues tested negative by RT-PCR.(AU)

Estudio del desempeño de dos métodos de detección de antígenos rápidos en hisopados combinados y en hisopados nasofaríngeos para la detección de SARS-CoV-2 / Performance study of two rapid antigen detection methods in combined swabs and nasopharyngeal swabs for detection of SARS-CoV-2

Resumen Se realizó una comparación del desempeño de los métodos rápidos de detección de antígenos para el diagnóstico de SARS-CoV-2 Veritor System de Becton Dickinson y Panbio de Abbott versus una reacción en cadena de la polimerasa con retrotranscripción en tiempo real (RT-PCR) de Roche en un triage de demanda espontánea de pacientes febriles de un hospital público, para la detección de COVID-19. Se procesaron 36 hisopados de pacientes sospechosos por los tres métodos. La concordancia entre ambos métodos con la RT-PCR fue del 97%. La sensibilidad de los métodos de detección de antígenos versus la RT-PCR fue del 83% y la especificidad fue del 100%. El valor predictivo positivo (VPP) fue del 100% y el valor predictivo negativo (VPN) fue del 97%. La muestra que resultó discordante presentó un ciclo umbral (Ct) de 29,8. El método para detección de antígenos tuvo un desempeño aceptable, incluso con resultados de sensibilidad mayores que los declarados por los fabricantes (84% para el Veritor System y 93,3% para el Panbio).

Abstract A comparison of the performance of the rapid antigen detection methods for the diagnosis of SARS-CoV-2 Veritor System from Becton Dickinson and Panbio from Abbott versus a real-time polymerase chain reaction with reverse transcription (RT-PCR) Roche in a spontaneous demand triage of febrile patients of a public hospital was made, for the detection of COVID-19. Thirty six swabs from suspected patients were processed by the three methods. The concordance between both methods with RT-PCR real time was 97%. The sensitivity of the antigen detection methods versus RT-PCR real time was 83% and specificity was 100%. The positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 97%. The sample that was discordant presented a threshold point (Ct) of 29.8. The method for antigen detection resulted in an acceptable performance, even with S results higher than those declared by the manufacturers (84% for the Veritor System and 93.3% for the Panbio).

Resumo Uma comparação do desempenho dos métodos rápidos de detecção de antígenos para o diagnóstico deSARS-CoV-2 Veritor System de Becton Dickinson e Panbio de Abbott versus uma reação em cadeia da polimerasecom transcrição reversa em tempo real (RT-PCR) da Roche em uma triagem de demanda espontâneade pacientes febris de um hospital público, para a detecção de COVID-19. Foram processadas 36 amostrasde esfregaços de pacientes suspeitos pelos três métodos e a concordância entre os dois métodos com aRT-PCR foi de 97%. A sensibilidade dos métodos de detecção de antigenos versus a RT-PCR foi de 83% ea especificidade de 100%. O valor preditivo positivo (VPP) foi de 100% e o valor preditivo negativo (VPN)foi de 97%. A amostra que resultou discordante apresentou um ciclo limiar (Ct) de 29,8. O método paradetecção de antígenos teve um desempenho aceitável, mesmo com resultados de sensibilidade superioresaos declarados pelos fabricantes (84% para o Veritor System e 93,3% para o Panbio).

Efficacy of the Antigenicity-Retaining Ability of Fixative Solutions for Liquid-Based Cytology: Immunocytochemistry of Long-Term Storage.

INTRODUCTION/OBJECTIVE: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. MATERIALS AND METHODS: Sediments of cultured RAJI cells (derived from Burkitt's lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. RESULTS: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. CONCLUSIONS: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

Multicolor Immunofluorescence Staining of Paraffin-Embedded Human Bone Marrow Sections.

Immunofluorescence is an indispensable method for the identification, localization and study of the expression of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections of human bone marrow. However, the procedure shows technical limitations because of the chemical and physical treatments required for sample processing before imaging. Here we describe a revisited protocol to obtain high-resolution images of human bone marrow trephine biopsies, improving the antigen-antibody recognition and preserving the morphology and the architecture of the bone marrow microenvironment.

Many roles for oligodendrocyte precursor cells in physiology and pathology.

Oligodendrocyte precursor cells (OPCs) are a fourth resident glial cell population in the mammalian central nervous system. They are evenly distributed throughout the gray and white matter and continue to proliferate and generate new oligodendrocytes (OLs) throughout life. They were understudied until a few decades ago when immunolabeling for NG2 and platelet-derived growth factor receptor alpha revealed cells that are distinct from mature OLs, astrocytes, neurons, and microglia. In this review, we provide a summary of the known properties of OPCs with some historical background, followed by highlights from recent studies that suggest new roles for OPCs in certain pathological conditions.

Real-Time and Sensitive Immunosensor for Label-Free Detection of Specific Antigen with a Comb of Microchannel Long-Period Fiber Grating.

This study aimed to develop a comb of microchannel and immunosensor based on long-period fiber grating using the process of Lithographie Galvanoformung Abformung-like micro-electromechanical systems (LIGA-like MEMS) for real-time and label-free detection of specific antigen. The coupling between propagating core and cladding modes was conducted from the comb of microchannel long-period fiber grating (CM-LPFG). The CM-LPFG-based immunosensor consisted of a microchannel structure through photoresist stacking processes and was sandwiched with an optical fiber to obtain a long-period structure. Specific immunoglobulin against protein antigen was immobilized onto an optical fiber surface and produced a real-time resonance effect on sensing specific protein antigen from the extracted protein mixtures of the cancer cell lines. The variable transmission loss was -14.07 dB, and the resonant wavelength shift was 11.239 nm. The low limit of detection for total protein concentration was 1.363 ng/µL. Our results revealed that the CM-LPFG-based immnosensor for real-time detection of label-free protein antigen is feasible and sensitive based on the diversification of a transmission loss and achieves specific immunosensing purposes for lab-on-fiber technology.

Identification and Immunophenotypic Characterization of Normal and Pathological Mast Cells.

Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 106 unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to studying MCs from other tissues and species.

Critical assessment of staining properties of a new visualization technology: a novel, rapid and powerful immunohistochemical detection approach.

Immunohistochemical staining of tissue sections is a vital technique in pathological diagnostics and theranostics. Several kinds of detection systems are available-each of them with their advantages and disadvantages. Here we present the results of a study assessing a prototype immunohistochemical detection technology (PIDT) for visualization of antigens in tissue sections. Different tumor tissues (n = 11) were stained with selected antibodies (n = 30) and a subset of these under different fixation conditions. The staining properties were assessed according to six staining quality parameters (signal distribution, intensity, tissue and slide background, acutance, clarity of details, and subcellular morphological details), and the results were compared with those of a well-established detection system (EnVision FLEX). Overall, both detection methods revealed good to optimal results regarding the evaluated parameters even under unfavorable fixation conditions. However, with the prototype detection technology a quicker turnaround time was reached primarily due to shorter primary antibody incubation times. Moreover, PIDT-stained tissues showed higher signal intensity and a uniform signal distribution over the tissue slide, still, with well-preserved tissue morphology and without impairing the gradation of staining intensity of different cell types. In particular, the prototype detection technology performed better in poorly or delayed fixed tissue. In situations where rapid and profound results are in demand, and particularly in the context of a small laboratory setting, this prototype detection technology could be a useful addition to the established detection systems.

Sistema Rh-Hr y variantes del antígeno D en tres poblaciones afroecuatorianas del Valle del Chota / Rh-Hr system and D antigen variants in three Afro-Ecuadorian populations in the Chota Valley / Sistema Rh-Hr e variantes do antígeno D em três populações afro-equatorianas no vale do Chota

Resumen La identificación inequívoca del antígeno D en medicina transfusional es de vital importancia para evitar reacciones postransfusionales y la enfermedad hemolítica del recién nacido. Es común el uso de reactivos serológicos monoclonales o tarjetas de gel y su interpretación está definida por cruces, de acuerdo con la reacción serológica. El propósito de este estudio fue determinar la frecuencia del factor Rh y las variantes del antígeno D en una población afroecuatoriana. Se trató de un estudio descriptivo, transversal con muestreo aleatorio simple de 541 pobladores. Para la tipificación del factor Rh se utilizó la metodología en tubo con antisueros monoclonales y para la detección de las variantes de D se utilizaron tarjetas de gel IDCoombs Anti-IgG. Las lecturas se verificaron mediante el análisis del índice kappa. Se aplicó estadística descriptiva y el análisis de Chi cuadrado para establecer la relación de las variables y su significación. Se identificó una frecuencia del 92% de individuos Rh(D) positivo y un 8% Rh(D) negativo. El 4,80% de los individuos presentaban la variante D débil y el 79% reacciones serológicas entre 2 y 3(+) indicativas de otras variantes del antígeno D. El fenotipo más común fue el R0/R0. Estos datos demuestran la necesidad de confirmar la existencia de variantes del antígeno D en esta población para un mejor manejo de la sangre. Una limitante constituye la disponibilidad de técnicas moleculares para la genotipificación de D; sin embargo, se podría implementar la fenotipificación RHCE como estrategia pretransfusional.

Abstract The unequivocal identification of D antigen in transfusion medicine is of vital importance to avoid post-transfusion reactions and hemolytic disease of the newborn. The use of monoclonal serological reagents or gel cards is common and their interpretation is defined according to the serological reaction by crosses. The purpose of this study was to determine the frequency of Rh factor and D antigen variants in the Afro-Ecuadorian population. This was a descriptive, cross-sectional study with simple random sampling of 541 residents. Tube typing with monoclonal antisera was used to typify Rh factor and ID-Coombs Anti-IgG gel cards were used to detect D variants, and the readings were verified by analysis of the kappa index. Descriptive statistics and Chi-square analysis were applied for the relationship of the variables and their significance. A frequency of 92% of Rh(D) positive individuals and 8% Rh(D) negative individuals were identified. Almost 5% (4.80%) of the individuals presented the weak D variant and 79% serological reactions between 2-3(+) indicative of other D antigen variants, the most common phenotype being R0/R0. These data demonstrate the need to confirm the existence of D antigen variants in this population for better management and availability of blood. A limitation is the availability of molecular techniques for D genotyping, however, RHCE phenotyping could be implemented as a pretransfusion strategy.

Resumo A identificação inequívoca do antígeno D na medicina transfusional é de vital importância para evitar reações pós-transfusionais e a doença hemolítica do recém-nascido. É comum o uso de reagentes sorológicos monoclonais ou cartões de gel e sua interpretação é definida por cruzamentos de acordo com a reação sorológica. O objetivo deste estudo foi determinar a frequência do fator Rh e as variantes do antígeno D numa população afro-equatoriana. Foi um estudo descritivo, transversal, com amostragem aleatória simples de 541 residentes. Para a tipagem do fator Rh foi utilizada a metodologia em tubo com anti-soros monoclonais e para a detecção das variantes de D, os cartões de gel ID-Coombs Anti-IgG. As leituras foram verificadas por análise do índice kappa. Foi aplicada estatística descritiva e para estabelecer a relação das variáveis e sua significação se utilizou a análise do qui-quadrado. Identificando uma frequência de 92% dos indivíduos Rh (D) positivos e 8% Rh (D) negativos. 4,80% dos indivíduos apresentavam a variante D fraca e 79% reações sorológicas entre 2 e 3(+) indicativas de outras variantes do antígeno D, sendo o fenótipo mais comum o R0/R0. Esses dados demonstram a necessidade de confirmar a existência de variantes do antígeno D nessa população para melhor gerenciamento e disponibilidade de sangue. Uma limitação é a disponibilidade de técnicas moleculares para a genotipagem de D, no entanto, a fenotipagem de RHCE poderia ser implementada como uma estratégia de pré-transfusão.

Massively parallel interrogation and mining of natively paired human TCRαß repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Diagnóstico de la Tricomonas vaginalis en la mujer / Diagnosis of Trichomonas vaginalis in women

OBJETIVO: revisar los diferentes métodos de diagnóstico de la tricomoniasis vaginal disponibles hasta el presente. MATERIALES Y MÉTODOS: se revisó la bibliografía latinoamericano e internacional a través de los sitios electrónicos de Pub-Med y Scielo. RESULTADOS: la Tricomonas vaginalis es considera como la enfermedad de transmisión sexual no viral, curable más frecuente y prevalente en el mundo. Se revisan los diferentes de métodos para diagnosticar la presencia de la tricomonas vaginalis en pacientes femeninos con síntomas y signos de la infección producida por el protozoario flagelado. CONCLUSIONES: se revisaron los diferentes métodos de diagnostico de la infección producida por la Tricomonas vaginalis en pacientes femeninas, desde los clásicos hasta los más actuales que emplean alta tecnología.

OBJECTIVE: to review the different diagnostic methods of Trichomonas vaginal available at the present time. MATERIAL AND METHOD: it was reviewed the Latin-American and international bibliography using the Pub-Med and Scielo web sites. RESULTS: Trichomonas vaginalis is considered the most common and prevalent sexual transmitted disease curable and non-viral worldwide. It was reviewed the different methods to diagnose the presence of Trichomonas vaginalis in female patients with symptoms and signs of infection produces by the flagellate protozoa. CONCLUSION: Different methods of diagnosis of the infection produced by Trichomonas vaginalis, since the classics to the most current methods that use high technology, were reviewed.

Prevalencia del antígeno D débil y su clasificación fenotípica en donantes voluntarios de sangre / Prevalence of "weak" D antigen and its phenotypic classification in voluntary blood donours / Prevalência do antígeno D "fraco" e sua classificação fenotípica em doadores voluntários de sangue

En el campo de la medicina transfusional la correcta identificación de los fenotipos del sistema Rh y en especial del antígeno D debe ser de manera inequívoca por su relevancia clínica. El antígeno D tiene variantes denominadas D parcial, D débil y DEL, las que se producen por mutaciones de los alelos RHD/RHCE o por una supresión en la expresión fenotípica. Se trató de un estudio descriptivo, retrospectivo de corte transversal en el que se realizó una revisión de registros primarios durante el período 2011-2014 validados de acuerdo con el protocolo de Hernández-Sampieri R. Se utilizó estadística descriptiva mediante la aplicación del software informático SPSS versión 22.0 y se estableció la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado. Se determinó una prevalencia de donantes RhD negativos de 1,8 a 2,5% y RhD débil de 1,79 a 2,28%. La fenotipificación serológica permitió identificar que los tipos 2 y 5 eran los más frecuentes. También se estableció la existencia de aloinmunización por anti-D, anti-C y anti-E. Se estableció de esta manera la existencia de D débil y una importante aloinmunización en la población de donantes de sangre tipificados como D negativo y D débil, por lo que se recomienda implementar un algoritmo de identificación del antígeno D en servicios de medicina transfusional.

In the field of transfusion medicine, the correct identification of the phenotypes of the Rh system and especially of the D antigen must be unequivocal for clinical relevance. The D antigen has variants called partial D, weak D and DEL. These are produced by mutations of the RHD/RHCE alleles or a suppression in phenotypic expression. The objective of this study was to establish the frequency of weak D antigen in the population of blood donours from 17 Ecuadorian states and their phenotypic combinations. It was a descriptive, retrospective cross-sectional study performed during the 2011-2014 period and validated with primary records in accordance with the Hernández-Sampieri R protocol. A descriptive statistics through the application of SPSS computer software version 22.0 was used and the relationship between independent variables through the Chi-square statistic method was established. A prevalence of RhD negative donours from 1.8 to 2.5% and weak D 1.79 to 2.28% was observed The serological phenotyping made it possible to identify that type 2 and 5 were the most frequent. The presence of alloimmunization by anti-D, anti-C and anti-E was also established. Besides, the presence of weak D types and significant alloimmunization in the donour population of blood typed as D negative and weak was established, so it is recommended to implement an algorithm for the identification of D antigen in transfusional medicine services.

No campo da medicina transfusional, a correta identificação dos fenótipos do sistema Rh e especialmente do antígeno D deve ser inequívoca devido a sua relevância clínica. O antígeno D tem variantes chamadas de D parcial, D fraca e DEL, as quais são produzidos por mutações dos alelos RHD/RHCE ou por uma supressão na expressão fenotípica. O objetivo deste estudo foi estabelecer a frequência do antígeno D fraco em uma população de doadores de sangue de 17 províncias equatorianas e suas combinações fenotípicas. Foi uma estudo descritivo, retrospectivo de corte transversal em que se realizou uma revisão dos registros primários validados de acordo com o Protocolo Hernández-Sampieri R durante o período 2011-2014. Utilizou-se estatísticas descritivas através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes através da análise estatística de qui-quadrado. Foi determinada uma prevalência de doadores RhD negativos de 1,8 a 2,5% e RhD fraco de 1,79 a 2,28%. A genotipagem serológica permitiu identificar que os tipos 2 e 5 são os mais frequente. A existência de alo imunização por anti-D, anti-C e anti-E também foi estabelecida. A existência de D fraco e uma alo imunização significativa na população de doadores de sangue tipificados como D negativo e fraco, por isso é recomendado implementar um algoritmo de identificação do antígeno D em serviços de medicina transfusional.

Mural Cell SDF1 Signaling Is Associated with the Pathogenesis of Pulmonary Arterial Hypertension.

Pulmonary artery smooth muscle cells (PASMCs) and pericytes are NG2+ mural cells that provide structural support to pulmonary arteries and capillaries. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization, but whether similar mechanisms are responsible for their behavior is unknown. RNA-seq was used to compare the gene profile of pericytes and PASMCs from PAH and healthy lungs. NG2-Cre-ER mice were used to generate NG2-selective reporter mice (NG2tdT) for cell lineage identification and tamoxifen-inducible mice for NG2-selective SDF1 knockout (SDF1NG2-KO). Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is highly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that, similar to PAH, tdT+ cells accumulate in muscularized microvessels and demonstrate significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared with control mice, SDF1NG2-KO mice in chronic hypoxia had reduced muscularization and lower abundance of NG2+ cells around microvessels. SDF1 stimulation in healthy pericytes induced greater contractility and impaired their capacity to establish endothelial-pericyte communications. In contrast, SDF1 knockdown reduced PAH pericyte contractility and improved their capacity to associate with vascular tubes in coculture. SDF1 is upregulated in NG2+ mural cells and is associated with PA muscularization. Targeting SDF1 could help prevent and/or reverse muscularization in PAH.

Fluorescence Signal Amplification by Using ß-Galactosidase for Flow Cytometry; Advantages of an Endogenous Activity-Free Enzyme.

We previously proposed using a hydrolysis enzyme for fluorescent signal amplification in flow cytometric detection of antigen proteins, which was named the catalyzed reporter penetration (CARP) method. In this method, antigen proteins are labeled with enzyme-modified antibodies, and then fluorophore-modified substrates stain cells by penetrating the cell membrane upon hydrolysis of the substrate. We proved the concept by using alkaline phosphatase (AP) as the hydrolysis enzyme. However, a required prior inactivation process of endogenous AP activity on the cell surface risked disrupting recognition of antigen proteins by antibodies. In this report, the CARP method was extended to ß-galactosidase (ß-gal) as an amplification enzyme, which circumvented the requirement of an initial inactivation process because endogenous ß-gal activity on the surface of examined cells was found to be negligible. The substrate structure for ß-gal was optimized and used for the CARP method. The CARP method showed significantly higher fluorescent signals than a conventional method using fluorophore-modified antibodies. Moreover, the degree of amplification of the fluorescence signal was higher for antigens with low expression levels, showing that the CARP method is a suitable signal amplification method over current conventional approaches.