Comparative Performance Assessment of Novel Fluorescence Immunoassay POCTs for Measuring Circulating Levels of Vitamin-D.

Vitamin D (Vit D) is a fat-soluble molecule acting like a hormone, and it is involved in several biological mechanisms such as gene expression, calcium homeostasis, bone metabolism, immune modulation, viral protection, and neuromuscular functions. Vit D deficiency can lead to chronic hypocalcemia, hyperparathyroidism, and many other pathological conditions; in this context, low and very low levels of 25-hydroxy-vitamin D (25-OH-D) were found to be associated with an increased risk of COVID-19 infection and the likelihood of many severe diseases. For all these reasons, it is important to quantify and monitor 25-OH-D levels to ensure that the serum/blood concentrations are not clinically suboptimal. Serum concentration of 25-OH-D is currently the main indicator of Vit D status, and it is currently performed by different assays, but the most common quantitation techniques involve immunometric methods or chromatography. Nevertheless, other quantitation techniques and instruments are now emerging, such as AFIAS-1® and AFIAS-10® (Boditech and Menarini) based on the immunofluorescence analyzer, that guarantee an automated system with cartridges able to give quick and reliable results as a point-of-care test (POCT). This work aims to compare AFIAS-1® and AFIAS-10® (Boditech and Menarini) Vit D quantitation with Ultra High-Performance Liquid Chromatography coupled with tandem mass spectrometry that currently represents the gold standard technique for Vit D quantitation. The analyses were performed in parallel on 56 samples and in different conditions (from fresh and frozen plasma) to assess the reliability of the results. Any statistically significant differences in methods, the fixed error, and the error proportional to concentration were reported. Results obtained in all conditions showed a good correlation between both AFIAS® instruments and LC-MS/MS, and we can affirm that AFIAS-1® and AFIAS-10® are reliable instruments for measuring 25-OH-D with accuracy and in a fast manner.

Immunohistochemistry and immunofluorescence utilization audit by subspecialty in an academic setting: A step toward stewardship.

There are immunohistochemistry (IHC) and immunofluorescence (IF) panels described in the literature and established by personal and institutional experiences that are in common use by pathologists in their daily practice. Stewardship is a difficult discussion because IHC utilization is influenced by many factors including the pathologist's experience, background, practice setting, personal bias, and medicolegal culture. We developed the methodology to audit the IHC/IF utilization in our academic subspecialty practice. We aim to share this methodology and to provide our data that can be used for consideration by other subspecialized academic practices. This analysis included a total of 63,157 specimens that were accessioned during 2022, representing 38,612 cases. The likelihood of ordering IHC/IF ranged from 1 % (in genitourinary pathology) to 59 % (in renal pathology). The average percentage of specimens with IHC/IF was 21 % for the entire practice. In cases where IHC/IF was ordered, the number of stained slides averaged 4.9 per specimen for the entire practice. The number of IHC/IF slides per specimen ranged from 1.9 (in gastrointestinal pathology) to 12.2 (in renal pathology). The highest number of antibodies ordered for a single specimen by subspecialty ranged from 11 (in cardiac pathology) to 63 (in dermatopathology). Renal pathology was the only subspecialty that had an average number of IHC/IF slides that was statistically significantly different from all other subspecialties. We described the various patterns of utilization by subspecialty and rationalized their subtle differences. We also analyzed the types of cases that exceeded the reimbursement limits set by the Centers for Medicare and Medicaid Services (CMS).

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers. We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types. This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

A Low-Cost Modular Imaging System for Rapid, Multiplexed Immunofluorescence Detection in Clinical Tissues.

To image 4-plex immunofluorescence-stained tissue samples at a low cost with cellular level resolution and sensitivity and dynamic range required to detect lowly and highly abundant targets, here we describe a robust, inexpensive (<$9000), 3D printable portable imaging device (Tissue Imager). The Tissue Imager can immediately be deployed on benchtops for in situ protein detection in tissue samples. Applications for this device are broad, ranging from answering basic biological questions to clinical pathology, where immunofluorescence can detect a larger number of markers than the standard H&E or chromogenic immunohistochemistry (CIH) staining, while the low cost also allows usage in classrooms. After characterizing our platform's specificity and sensitivity, we demonstrate imaging of a 4-plex immunology panel in human cutaneous T-cell lymphoma (CTCL) formalin-fixed paraffin-embedded (FFPE) tissue samples. From those images, positive cells were detected using CellProfiler, a popular open-source software package, for tumor marker profiling. We achieved a performance on par with commercial epifluorescence microscopes that are >10 times more expensive than our Tissue Imager. This device enables rapid immunofluorescence detection in tissue sections at a low cost for scientists and clinicians and can provide students with a hands-on experience to understand engineering and instrumentation. We note that for using the Tissue Imager as a medical device in clinical settings, a comprehensive review and approval processes would be required.

Assessment of Growth Plate Chondrocytes Proliferative Activity in Embryonic Endochondral Ossification via Ki-67 Immunofluorescence.

Cell proliferation is one of the key events that regulates organism development. In the limb, chondrocytes differentiate into a multi-layered cellular template called the growth plate. Chondrocyte proliferation is essential to provide the necessary cells that allow growth of a bone. Deregulated cell proliferation will lead to truncated bone elements. Immunofluorescence is a biological technique that uses specific antibodies to detect the subcellular localization of a proliferative marker within cellular or tissue context. In this chapter, we illustrate how to perform immunofluorescence to detect the localization of Ki-67 (a marker of actively growing/proliferating chondrocytes) in order to assess the growth fraction of the columnar chondrocytes in the growth plate in paraffin-embedded mouse tissue limb.

Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence.

We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.

Validation of multiplex immunofluorescence and digital image analysis for programmed death-ligand 1 expression and immune cell assessment in non-small cell lung cancer: comparison with conventional immunohistochemistry.

AIMS: This study aimed to validate the application of combined multiplex immunofluorescence (mIF) and digital image analysis (DIA) in formalin-fixed and paraffin-embedded tissues for the quantitative assessment of programmed death-ligand 1(PD-L1) and immune cells (ICs) in non-small cell lung cancer (NSCLC). METHODS: Fifty resected samples of NSCLC were sequentially stained with a DNA-tagged mIF (panel including PD-L1, CKpan, CD8, CD68 and 4',6-diamidino-2-phenylindole (DAPI)) and conventional immunohistochemistry (cIHC). The assessment of cell density and consistency of tumour proportion score (TPS) via DIA were compared with those by pathologists. RESULTS: A strong correlation in the cell population of immune markers was obtained between mIF and cIHC (for PD-L1: R=0.9304, CKpan: R=0.8231, CD8: R=0.9314 and CD68: R=0.8366) within 95% limits of agreement. The continuous TPS calculated using mIF was highly consistent with the IHC staining results which were evaluated by pathologists (R=0.9362). However, in the comparison of TPS using interval variables, a poor agreement was obtained at a cut-off of 1% (κ=0.197), whereas excellent agreement was achieved at cut-offs of 50% (κ=0.908) and 5% (κ=0.823). DIA on mIF showed that PD-L1 commonly colocalised with CD68+ macrophages and CD8+ cytotoxic cells were closer to PD-L1-/CK+ tumour cells (TCs) than to PD-L1+/CK+ TCs in spatial distribution. CONCLUSIONS: A combination of mIF and DIA is useful for the quantification of PD-L1 expression and IC populations in NSCLC. Further validation of TPS at a cut-off of 1% and assay harmonisation is essential for translating this method in a diagnostic setting.

TGF-ß-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes using In Vivo Inhibition, Immunofluorescence and Flow Cytometry.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Isolation and Assessment of Pancreatic Islets Versus Dispersed Beta Cells: A Straightforward Approach to Examine Cell-Cell Communication.

Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.

Quantitative Assessment of the Immune Microenvironment in Patients With Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE: Iatrogenic laryngotracheal stenosis (iLTS) is characterized by fibroinflammatory narrowing of the upper airway and is most commonly caused by intubation injury. Evidence suggests a key role for CD4 T cells in its pathogenesis. The objective of this study is to validate emerging multiplex immunofluorescence (mIF) technology for use in the larynx and trachea while quantitatively characterizing the immune cell infiltrate in iLTS. In addition to analyzing previously unstudied immune cell subsets, this study aims to validate previously observed elevations in the immune checkpoint PD-1 and its ligand PD-L1 while exploring their spatial and cellular distributions in the iLTS microenvironment. STUDY DESIGN: Controlled ex vivo cohort study. SETTING: Tertiary care center. METHODS: mIF staining was performed with formalin-fixed, paraffin-embedded slides from 10 patients with iLTS who underwent cricotracheal resection and 10 control specimens derived from rapid autopsy for CD4, CD8, CD20, FoxP3, PD-1, PD-L1, and cytokeratin. RESULTS: There was greater infiltration of CD4+ T cells, CD8+ T cells, CD20+ B cells, FoxP3+CD4+ Tregs, and FoxP3+CD8+ early effector T cells in the submucosa of iLTS specimens as compared with controls (P < .05 for all). PD-1 was primarily expressed on T cells and PD-L1 predominantly on CD4+ cells and "other" cells. CONCLUSION: This study leverages the power of mIF to quantify the iLTS immune infiltrate in greater detail. It confirms the highly inflammatory nature of iLTS, with CD4+ cells dominating the immune cell infiltrate; it further characterizes the cellular and spatial distribution of PD-1 and PD-L1; and it identifies novel immunologic targets in iLTS.

Immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in lichen planus.

Ex vivo confocal laser scanning microscopy (CLSM) provides rapid, high-resolution imaging, fluorescence detection and digital haematoxylin-eosin (H&E)-like staining. We aimed to assess the performance of ex vivo CLSM in identifying histomorphology and immunoreactivity in lichen planus (LP) and comparing its accuracy with conventional histopathology and direct immunofluorescence (DIF). Thirty-three sections of 17 LP patients stained with acridine orange (AO) and FITC-labelled anti-fibrinogen antibody and 21 control samples stained with AO were examined using ex vivo CLSM. Ex vivo CLSM was in perfect agreement with conventional histopathology in identifying interface dermatitis, vacuolar degeneration and band-like infiltration. ROC analysis showed that the presence of vacuolar degeneration, interface dermatitis and band-like infiltration was useful to distinguish LP sections from controls (p < .0001). The detection rates of fibrinogen deposition using DIF and in conclusion ex vivo CLSM were 93.8% and 62.5%, respectively. ex vivo CLSM enables histopathological and immunofluorescence examination in LP with the advantage of digital H&E-like staining.

Quantitative assessment of neural elements in a rat model using nerve growth factor after remnant-preserving anterior cruciate ligament reconstruction: a histological and immunofluorescence pilot study.

BACKGROUND: Immunofluorescence analyses of anterior cruciate ligament (ACL) allografts following remnant-preserving ACL reconstruction using Achilles tendon allografts have provided evidence for the presence of neural elements. In this study, we aimed to examine the expression of neural elements and quantify the presence of neural cells in ACL remnants and Achilles allografts using nerve growth factor (NGF) therapy after remnant-preserving ACL reconstruction. METHODS: Experiments were conducted on 5 pairs of rats (approximately 8 weeks old and weighing 320 g at the time of surgery). Longitudinally, split Achilles tendons from the paired rats were freshly frozen and later defrosted with warm saline and allografted onto the right ACL of the other rat that was partially detached at the femoral attachment site. A sham operation was conducted on the left knee to be used as a control. NGF was injected into both knee joints every week for 6 weeks after surgery. The presence of neural cells in the ACL of the sham-operated knee, allografted Achilles tendon, and ACL remnant was examined 6 weeks post-surgery using H and E and immunofluorescent staining. RESULTS: H and E staining did not reveal neural cells in any of the three groups. However, immunofluorescence analysis showed the presence of nestin-positive neural elements in the normal ACL tissues as well as ACL remnants. Additionally, neural elements were examined in 7 of the 8 (87.5%) allograft tissues. Quantitative analysis showed no difference in the number or area of nuclei among the three groups. However, the number and area of neural cells in the Achilles allografts were significantly lower than those in the other two groups (p = 0.000 and p = 0.001, respectively). CONCLUSION: Our observations indicate that ACL remnants promote the new ingrowth and persistence of neural cells. We suggest that the ingrowth of neural elements can support the persistence and new ingrowth of mechanoreceptors, thereby enhancing the functional stability of knee joints. Moreover, the expression of neural cells in the Achilles allografts was lower than that in normal ACL tissues and ACL remnants in the quantitative evaluation, thereby confirming the essential role of ACL remnants in knee joint functionalization.

Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19 / Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19

INTRODUÇÃO: O coronavírus da Síndrome Respiratória Aguda Grave 2 (abreviado para SARSCoV-2, do inglês Severe Acute Respiratory Syndrome Coronavirus 2), anteriormente conhecido como novo coronavírus (2019-nCoV), é um agente zoonótico recémemergente que surgiu em dezembro de 2019, em Wuhan, China, causando manifestações respiratórias, digestivas e sistêmicas, que se articulam no quadro clínico da doença denominada COVID-19 (do inglês Coronavirus Disease 2019). Ainda não há informações robustas sobre a história natural da doença, tampouco sobre as medidas de efetividade para o manejo clínico dos casos de infecção pelo COVID19, restando ainda muitos detalhes a serem esclarecidos. No entanto, sabe-se que o vírus tem alta transmissibilidade e provoca uma síndrome respiratória aguda que varia de casos leves ­ cerca de 80% ­ a casos muito graves com insuficiência respiratória - entre 5% e 10% dos casos ­, os quais requerem tratamento especializado em unidades de terapia intensiva (UTI). Sua letalidade varia, principalmente, conforme a faixa etária. TECNOLOGIA: Os testes de diagnóstico para a COVID-19 se destacaram na pandemia de coronavírus em andamento como uma ferramenta essencial para rastrear a propagação da doença. Uma ampla gama de testes diagnósticos para o SARS-CoV-2 está disponível comercialmente, alguns dos quais receberam autorizações para uso por várias agências reguladoras nacionais. Com as informações da sequência genética devidamente identificadas, os testes de diagnóstico baseados na detecção da sequência viral por reação em cadeia da polimerase com transcriptase reversa (RT-PCR) ou plataformas de sequenciamento logo se tornaram disponíveis. Isso permitiu a confirmação do diagnóstico e melhores estimativas da atividade da infecção, que vêm aumentando em velocidades alarmantes. Para a detecção mais sensível de SARS-CoV, MERS-CoV e SARS-CoV-2, recomendavam-se a coleta e o teste de amostras respiratórias superiores e inferiores. O diagnóstico de casos suspeitos era confirmado por testes de RNA com RT-PCR em tempo real ou sequenciamento de próxima geração. Foi demonstrado que o RNA viral poderia ser detectado a partir do swab nasal e faríngeo, lavagem broncoalveolar e plasma sanguíneo usando RT-PCR direcionado ao gene do vírus (5). O padrão-ouro para diagnóstico laboratorial da COVID-19 é a reação da transcriptase reversa, seguida de reação em cadeia da polimerase (RT-PCR) para amostras coletadas no trato respiratório superior ou inferior. OBJETIVO: O objetivo deste relatório é analisar a acurácia dos testes diagnósticos registrados na Agência Nacional de Vigilância Sanitária (ANVISA) até a presente data. METODOLOGIA: foi realizada uma busca por diagnósticos para COVID-19 com registros vigentes na ANVISA. Para tal, foram utilizados os termos "COVID 19", SARS e coronavírus no campo de consulta de registro de produtos para saúde no site da Agência (https://consultas.anvisa.gov.br/#/saude/). Os passos para acesso ao banco de dados de produtos diagnósticos na ANVISA são: 1) consulta produtos; 2) consulta a banco de dados; 3) produtos para a saúde e 4) pesquisa de produtos para a saúde registrados. CONCLUSÕES: A ANVISA já avaliou mais de 120 pedidos de registro de produtos para testagens relacionadas à COVID-19 desde o dia 18 de março. A maior parte das petições aguarda complementação de informações por parte das empresas e outras estão sendo analisadas com prioridade. O tempo médio para avaliação dos registros na ANVISA gira em torno de 15 dias. Atualmente, mais da metade dos registros concedidos diz respeito a testes rápidos para anticorpos. Até a presente data, foram registrados 64 testes para diagnóstico da COVID-19, sendo 15 deles moleculares. O teste de polymerase chain reaction em tempo real (RT-PCR) para identificação de SARS-CoV-2 é um teste de elevada sensibilidade e especificidade, ainda que os doentes com maior carga viral possam ter maior probabilidade de um teste positivo. Os testes moleculares baseados em RNA exigem instalações laboratoriais específicas com níveis restritos de biossegurança e técnica. A sensibilidade e especificidade dos testes sorológicos variaram entre os fabricantes. É importante destacar que uma baixa sensibilidade do teste diagnóstico pode resultar em uma maior probabilidade de detectar falsos-negativos, o que poderia interferir principalmente em casos de indivíduos assintomáticos. Em geral, a sensibilidade dos testes foi superior a 85% e a especificidade, superior a 94%. Os testes sorológicos medem a quantidade de dois anticorpos (IgG e IgM) que o organismo produz quando entra em contato com um invasor. Contudo, o desenvolvimento da resposta de um anticorpo à infecção pode ser dependente do hospedeiro e levar tempo. No caso de SARS-CoV-2, estudos iniciais sugerem que a maioria dos pacientes se converte entre 7 e 11 dias após a exposição ao vírus, embora alguns pacientes possam desenvolver anticorpos mais cedo. Devido a esse atraso natural, o teste de anticorpos pode não ser útil no cenário de uma doença aguda (11). Os testes de anticorpos para SARS-CoV-2 podem facilitar (i) o rastreamento de contatos (os testes baseados em RNA também podem ajudar); (ii) a vigilância sorológica nos níveis local, regional, estadual e nacional; e (iii) a identificação de quem já teve contato com o vírus e, portanto, pode (se houver imunidade protetora) ser imune (11,12). Alguns conjuntos de reagentes para testes sorológicos foram autorizados pela ANVISA em caráter emergencial devido à gravidade da situação e à necessidade de ampliar a testagem da população, mas a validação desses reagentes pelos laboratórios é fundamental, uma vez que poucos trabalhos conseguiram ser publicados até o momento. As aprovações estão de acordo com a Resolução da Diretoria Colegiada (RDC) 348/2020, que define os critérios e os procedimentos extraordinários e temporários para tratamento de petições de registro de medicamentos, produtos biológicos e produtos para diagnóstico in vitro, e mudança pós-registro de medicamentos e produtos biológicos em virtude da emergência de saúde pública internacional decorrente do novo coronavírus. Na RDC, para registro de testes diagnósticos, a ausência de qualquer estudo de desempenho ou restrição de dados deve ser justificada por motivações técnicas que permitam a avalição da confiabilidade dos resultados e da efetividade diagnóstica do produto. Os registros concedidos nas condições dessa Resolução terão a validade de um ano, exceto para situações em que a avaliação da estabilidade seja apresentada por comparação com produtos similares e os demais critérios descritos no Regulamento sejam atendidos. Nesse caso, poderão ter a concessão regular de validade de registro de produtos para saúde por um período de 10 anos. Em resumo, as duas categorias de testes para SARS-CoV-2 podem ser úteis nesse surto, pois, eventualmente, a coleta de múltiplas amostras, regiões e em tempos diferentes durante a evolução da doença pode ser necessária para o diagnóstico da COVID-19.

Diagnostic evaluation of IgM ELISA and IgM Immunofluorescence assay for the diagnosis of Acute Scrub Typhus in central Nepal.

BACKGROUND: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium, Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. METHOD: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody to Orientia tsusugamushi was detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. RESULT: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73-87.68%), specificity 94.82% (95% CI: 93.43-95.99%), positive likelihood ratio 16.21% (95% CI: 12.71-20.67%), negative likelihood ratio 0.17% (95% CI: 0.13-0.21%), disease prevalence 22.08% (95% CI: 20.06 -24.21%), positive predictive value 82.12% (95% CI: 78.28-85.42%) and negative predictive value 95.44% (95% CI: 94.27-96.38%) respectively. CONCLUSION: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

3D Cell-Culture Models for the Assessment of Anticoagulant and Anti-Inflammatory Properties of Endothelial Cells.

Endothelial cells (EC) play a crucial role in the pathophysiology of cardiovascular diseases, ischemia/reperfusion injury, and graft rejection in (xeno-)transplantation. In such nonphysiological conditions, EC are known to lose their quiescent phenotype and switch into an actively pro-inflammatory, procoagulant, and anti-fibrinolytic state. This case happens essentially because the endothelial glycocalyx-a layer of proteoglycans and glycoproteins covering the luminal surface of the endothelium-is shed. Heparan sulfate, one of the main components of the endothelial glycocalyx, contributes to its negative charge. In addition, many plasma proteins such as antithrombin III, superoxide dismutase, C1 inhibitor, and growth factors and cytokines bind to heparan sulfate and by this scenario contribute to the establishment of an anticoagulant and anti-inflammatory endothelial surface. Shedding of the glycocalyx results in a loss of plasma proteins from the endothelial surface, and this phenomenon causes the switch in phenotype. Particularly in xenotransplantation, both hyperacute and acute vascular rejection are characterized by coagulation dysregulation, a situation in which EC are the main players.Since many years, EC have been used in vitro in 2D flatbed cell culture models, with or without the application of shear stress. Such models have also been used to assess the effect of human transgenes on complement- and coagulation-mediated damage of porcine EC in the context of xenotransplantation. The methods described in this chapter include the analysis of endothelial cell-blood interactions without the necessity of using anticoagulants as the increased EC surface-to-volume ratio allows for natural anticoagulation of blood. Furthermore, this chapter contains the description of a novel microfluidic in vitro model carrying important features of small blood vessels, such as a 3D round-section geometry, shear stress, and pulsatile flow-all this in a closed circuit, recirculating system aiming at reproducing closely the in vivo situation in small vessels.

Assessment of DNA damage by 53PB1 and pKu70 detection in peripheral blood lymphocytes by immunofluorescence and high-resolution transmission electron microscopy.

PURPOSE: 53BP1 foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA double-strand break (DSB) marker. In addition, high-resolution transmission electron microscopy (TEM) with immunogold labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA repair process. To establish this TEM method in the PBLs of patients with cancer, we analyzed and characterized whether different modes of irradiation influence the formation of DSBs, and whether accompanying chemotherapy influences DSB formation. METHODS: We obtained 86 blood samples before and 0.1, 0.5, and 24 h after irradiation from patients (n = 9) with head and neck or rectal cancers receiving radiotherapy (RT; n = 4) or radiochemotherapy (RCT; n = 5). 53BP1 foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70 dimers and 53BP1 clusters within euchromatin and heterochromatin of PBLs. RESULTS: IFM analyses showed that during radiation therapy, persistent 53BP1 foci in PBLs accumulated with increasing numbers of administered RT fractions. This 53BP1 foci accumulation was not influenced by the irradiation technique applied (3D conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1 foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT patients than in PBLs of RT only patients. Moreover, not every residual 53BP1 focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1 clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion or, possibly, defective repair. CONCLUSION: IFM 53BP1 foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1 focus but not every 53BP1 focus represents a DSB.

Lateral flow fluorescent immunoassay based on isothermal amplification for rapid quantitative detection of Salmonella spp.

Salmonella spp. are zoonotic pathogens of substantial public health concern. To enable detection in the field or under instrument-free conditions, we developed a rapid and robust lateral flow fluorescent immunoassay based on strand exchange amplification (SEA-LFIA) for the quantitative detection of Salmonella spp. As far as we know, this work is the first report regarding the use of Bst DNA polymerase-assisted SEA for fluorescence sensing to detect Salmonella spp. The SEA method was further confirmed by enzymatic digestion and Sanger dideoxy sequencing. The specificity of SEA-LFIA assay was verified by 89 Salmonella strains (18 Salmonella reference strains and 71 clinical isolates) and 15 non-Salmonella reference strains (different genera). The sensitivity of SEA-LFIA assay was 6 × 100 CFU mL-1 of Salmonella pure culture or 3 × 104 CFU 25 g-1 of artificially spiked raw chicken meat. Using this assay, it was found that 37 (16%) of the 236 samples collected were positive, which was consistent with the results of conventional PCR. The cutoff value is 15 and SEA-LFIA assay only takes ∼30 min without high equipment and reagent cost. In addition, the proposed strategy can be easily extended by redesigning the corresponding amplification primers to detect target analytes. In conclusion, the optimized SEA-LFIA assay is an efficient and specific method for the detection of Salmonella spp., and can potentially serve as a new on-site diagnostic tool in life sciences.

Flow cytometry-based isolation of tumor-associated regulatory T cells and assessment of their suppressive potential.

Regulatory T cells (Tregs) play a major role in establishing an immunosuppressive tumor microenvironment. In order to fully uncover their role and molecular regulation in tumor-bearing hosts, it is critical to combine phenotypical characterization with functional analyses. A standard method to determine the suppressive potential of Tregs is with an in vitro suppression assay, in which the impact of freshly isolated Tregs on T cell proliferation is assessed. The assay requires the isolation of substantial numbers of Tregs from tissues and tumors, which can be challenging due to low yield or cell damage during sample preparation. In this chapter, we discuss a flexible suppression assay which can be used to assess the suppressive potential of low numbers of murine Tregs, directly isolated from tumors. We describe methods for tissue preparation, flow cytometry-based sorting of Tregs and optimal conditions to perform a suppression assay, to obtain reliable and reproducible results.

Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.

Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.