Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

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.

Hybridoma Screening by Antigen Capture: Immunoprecipitation.

Immunoprecipitation is rarely used for screening hybridoma fusions because the assays are tedious and time-consuming. However, it can be useful when working with complex antigens because the precipitated antigen is normally detected after sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis and thus it is simple to discriminate between true and false positives. Furthermore, the assay provides information regarding the molecular weight of the antigen.

Contemporary Challenges in Clinical Flow Cytometry: Small Samples, Big Data, Little Time.

BACKGROUND: Immunophenotypic analysis of cell populations by flow cytometry has an established role in primary diagnosis and disease monitoring of many hematologic diseases. A persistent problem in evaluation of specimens is suboptimal cell counts and low cell viability, which results in an undesirable rate of analysis failure. In addition, the increased amount of data generated in flow cytometry challenges existing data analysis and reporting paradigms. CONTENT: We describe current and emerging technological improvements in cell analysis that allow the clinical laboratory to perform multiparameter analysis of specimens, including those with low cell counts and other quality issues. These technologies include conventional multicolor flow cytometry and new high-dimensional technologies, such as spectral flow cytometry and mass cytometry that enable detection of over 40 antigens simultaneously. The advantages and disadvantages of each approach are discussed. We also describe new innovations in flow cytometry data analysis, including artificial intelligence-aided techniques. SUMMARY: Improvements in analytical technology, in tandem with innovations in data analysis, data storage, and reporting mechanisms, help to optimize the quality of clinical flow cytometry. These improvements are essential because of the expanding role of flow cytometry in patient care.

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)

Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library.

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

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).

Antigen-detection in the diagnosis of SARS-CoV-2 infection: interim guidance, 6 October 2021

Diagnostic testing for SARS-CoV-2 is a critical component to the overall prevention and control strategy for COVID-19. Countries should have a national testing strategy in place with clear objectives that can be adapted according to changes in the epidemiological situation, available resources and tools, and country-specific context. It is critical that all SARSCoV-2 testing is linked to public health actions to ensure appropriate clinical care and support and to carry out contact tracing to break chains of transmission. Since the early days of the SARS-CoV-2 pandemic, laboratories have been using nucleic acid amplification tests (NAATs), such as real time reverse transcription polymerase chain reaction (rRT-PCR) assays, to detect SARS-CoV-2, the virus that causes COVID-19. Since mid-2020, less expensive and faster diagnostic tests that detect antigens specific for SARS-CoV-2 infection have become commercially available, and several have achieved WHO Emergency use listing. Antigen-detecting diagnostic tests are designed to directly identify SARS-CoV-2 proteins produced by replicating virus in respiratory secretions (or oral fluid/saliva) and have been developed as both laboratory-based tests and rapid diagnostic tests (RDTs) intended for near-patient use. The diagnostic development landscape is dynamic, with over two hundred tests for SARS-CoV-2 antigen detection on the market, of which 85% can be delivered at the point of care and the other 15% for use on high throughput machines in laboratory-based settings.

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage.

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time-dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins.

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.

APRANK: Computational Prioritization of Antigenic Proteins and Peptides From Complete Pathogen Proteomes.

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize potentially antigenic proteins and peptides in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We trained and tested this method with a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have evaluated this integrative method using non-parametric ROC-curves and made an unbiased validation using Onchocerca volvulus as an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

Barriers to antigen detection and avoidance in chronic hypersensitivity pneumonitis in the United States.

BACKGROUND: Chronic hypersensitivity pneumonitis (CHP) is an interstitial lung disease (ILD) caused by long term exposure to an offending antigen. Antigen avoidance is associated with improved outcomes. We are unable to identify the antigen source in approximately half of patients. When an antigen is successfully identified, patients have difficulty with avoidance. METHODS: We conducted three structured group discussions with US based ILD specialists utilizing the nominal group technique (NGT). Participants listed barriers to antigen detection and avoidance in CHP. Each participant ranked what they perceived to be the top three barriers in the list in terms of importance. The master list of barriers was consolidated across the three groups into themes that were prioritized based on receiving the highest rankings by participants. RESULTS: Twenty-five physicians participated; 56% had experience caring for CHP patients for ≥ 16 years. Sixty barriers to antigen detection were categorized into seven themes of which the top three were: 1. unclear significance of identified exposures; 2. gaps in clinical knowledge and testing capabilities; 3. there are many unknown and undiscovered antigens. Twenty-eight barriers to antigen avoidance were categorized into five themes of which the top three were: 1. patient limitations, financial barriers and lack of resources; 2. individual patient beliefs, emotions and attachments to the antigen source; and 3. gaps in clinical knowledge and testing capabilities. CONCLUSIONS: This study uncovered challenges at the individual patient, organizational, and societal levels and ranked them in terms of level of importance. These findings provide information to guide development and validation of multidisciplinary support and interventions geared towards antigen identification and avoidance in CHP.

Flow cytometry detection of surface and intracellular antigens in pancreas from a single mouse embryo.

We here report a flow-cytometry-based protocol to measure single-cell protein expression in small samples. The protocol is optimized for simultaneous detection of fluorescent proteins and intracellular and surface antigens in the embryonic pancreas from the mouse. Owing to low cell numbers, current protocols for flow cytometric analysis of embryonic tissues rely on tissue pooling. Our protocol enables analysis of one pancreas per sample, thereby facilitating detection of biological variation and minimizing the number of experimental animals needed. For complete details on the use and execution of this protocol, please refer to Nyeng et al (2019).