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INTRODUCTION: A highly sensitive and specific nucleic acid test (NAT) for the blood-borne viruses human immunodeficiency virus (HIV), hepatitis C (HCV), and hepatitis B (HBV) is essential for the safety of blood components. Since more than 2 decades, NAT screening of blood donations has become standard in developed countries that have implemented the individual-donation (ID-NAT) and mini-pool NAT (MP-NAT) approaches. With this powerful technique, confirmation of initial reactive (IR) NAT samples becomes a challenge. Different algorithms are currently in use to eliminate false reactive results. To show that the algorithm implemented in 2007, that uses repeat testing of IR samples in duplicate runs, is a safe strategy, especially in low endemic countries, data from a 10-year experience of ID-NAT were extensively analyzed when follow-up data were available. METHODS: From July 2007 to December 2014, the Procleix Ultrio assay on a Procleix Tigris system, and from January 2015 to December 2017, the cobas MPX on a cobas 8800 platform, were used for ID-NAT screening. All IR samples were subjected to repeat testing in duplicate independent runs. Only when both tests remained negative were the products released. Donor data from the last 10 years were investigated retrospectively, looking for the reoccurrence of a reactive result in a follow-up sample. Only those donors with at least an x + 1 donation result were included for the confirmation of a false reactive result. RESULTS: From the 1,830,657 donations tested, 2,450 samples were IR (0.13%); only 228 were repeat reactive ([RR], 18 HIV, 61 HCV, and 149 HBV samples), and 2,222 were non-RR (0.12%). Follow-up data were available from 1,267 donors (57%) for further analysis. All except one of these donors were ID-NAT-negative in all follow-up samples. The one exception was from a donor who acquired a fresh HBV infection 10 years after the IR donation (in the x + 28 donation) and subsequently seroconverted. Subsequent serological tests from all succeeding donations (x + 1, x + 2, etc.) were negative in all the other cases, proving that no seroconversion took place after the IR ID-NAT result. CONCLUSIONS: The algorithm to deal with IR ID-NAT donations using duplicate repeat testing is very safe and cost-effective in low-prevalence countries. There is no unnecessary destruction of blood products, no counseling of false reactive donors, and also no need to add further complexity to the screening algorithm.
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BACKGROUND: Pathogen reduction methods for blood components are effective for a large number of viruses though less against small, non-enveloped viruses such as Parvovirus B19 (B19V). This article describes the passive transmission by transfusion of two B19V-contaminated pooled platelet concentrates (PCs) which were treated with the Intercept® blood pathogen reduction system. CASE REPORTS: Two transfusion cases of B19V-contaminated Intercept-treated pooled PCs were described. Due to the analysis delay, the PCs were already transfused. The viral content of each donation was 4.87 × 10(10) IU/ml in case 1and 1.46 × 10(8) IU/ml in case 2. B19V (52 IU/ml) was detected in the recipient of the case 1 PC, whereas no virus could be detected in the case 2 PC recipient. A B19V IgM response and a transient boost of the underlying B19V IgG immune status and was observed in recipient 1. Recipient of the case 2 PC remained B19V IgG- and IgM-negative. B19V DNA sequence and phylogenetic analysis revealed a 100% homology between donor and recipient. CONCLUSION: This report describes passive B19V transmission by a PC with very high B19 viral load which elicited a transient boost of the B19V immunity, but not by a PC with a lower B19V content, suggesting that there is a B19 viral load threshold value at which B19V inactivation is exceeded.
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BACKGROUND: Nucleic acid test (NAT) hepatitis B virus (HBV) screening for all blood donations with a sensitivity limit of 25 IU/mL in the individual donation is mandatory in Switzerland since 2009. The aims of the two studies were to define the percentage of antibody to hepatitis B core antigen (anti-HBc) or anti-HBc and antibody to hepatitis B surface antigen (anti-HBs)-positive donors bearing HBV DNA and to gather HBV viral load data on HBV NAT yields during the routine screening since the introduction of the HBV NAT. STUDY DESIGN AND METHODS: Archive samples from anti-HBc-positive donors (Group I) were analyzed with a quantitative HBV DNA test and further with anti-HBc and anti-HBs assays. In addition, all the HBV NAT-only-yield samples (Group II) from the routine donor screening performed between July 2007 and May 2013 were included in the study. RESULTS: From the 667 samples investigated (131 donors), three donors (2.3%) had donated eight samples (1.2%) with detectable HBV DNA; however, all had very low viral loads (≤ 10 IU/mL). From the 1,160,426 donations screened with the routine HBV NAT assay, 16 HBV NAT yields were detected: two window period (WP) and 14 occult hepatitis B infection (OBI) cases. In eight of these positive donations (two WP and six OBI), the HBV viral loads were not more than 10 IU/mL, in three cases between 10 and 25 IU/mL, and in the remaining five donations between 37 and 166 IU/mL. CONCLUSION: The highly sensitive HBV NAT assay with a threshold significantly below 10 IU/mL is a valuable alternative to anti-HBc and a less sensitive HBV NAT screening in blood donor screening.
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Doadores de Sangue , DNA Viral/sangue , Seleção do Doador , Anticorpos Anti-Hepatite B/sangue , Vírus da Hepatite B , Carga Viral , Feminino , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico , Estudos Retrospectivos , SuíçaRESUMO
Starting in 2013, blood donors must be tested at least using: (1) one monoclonal anti-D and one anti-CDE (alternatively full RhCcEe phenotyping), and (2) all RhD negative donors must be tested for RHD exons 5 and 10 plus one further exonic, or intronic RHD specificity, according to the guidelines of the Blood Transfusion Service of the Swiss Red Cross (BTS SRC). In 2012 an adequate stock of RHD screened donors was built. Of all 25,370 RhD negative Swiss donors tested in 2012, 20,015 tested at BTS Berne and 5355 at BTS Zürich, showed 120 (0.47%) RHD positivity. Thirty-seven (0.15%) had to be redefined as RhD positive. Routine molecular RHD screening is reliable, rapid and cost-effective and provides safer RBC units in Switzerland.
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Doadores de Sangue/legislação & jurisprudência , Tipagem e Reações Cruzadas Sanguíneas , Seleção do Doador , Sistema do Grupo Sanguíneo Rh-Hr/genética , Tipagem e Reações Cruzadas Sanguíneas/métodos , Tipagem e Reações Cruzadas Sanguíneas/normas , Seleção do Doador/legislação & jurisprudência , Seleção do Doador/normas , Feminino , Humanos , Masculino , SuíçaRESUMO
This work aims to assess the value of a generalized molecular RHD screening strategy which could replace routine serological screening of weak D by indirect antiglobulin test. Three independent studies were performed at the two Blood Transfusion Services Berne and Zurich. Donors investigated were 652 RhD negative, but RhC and/or RhE positive, 17,391 mainly Rhccee, and 8200 with normal RhCcEe phenotype distribution. In study I single samples, in studies II and III minipools of 24 and 20 donor samples were tested, respectively. Among 26,243 phenotypically RhD negative blood donors, 65 carriers of RHD alleles were identified. Thirty-one of them were redefined as RhD positive.
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Alelos , Tipagem e Reações Cruzadas Sanguíneas/métodos , Seleção do Doador/métodos , Técnicas de Genotipagem/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Feminino , Humanos , Masculino , FenótipoRESUMO
We aim to develop new portable desktop tissue analysers (DTAs) to provide fast, low-cost, and precise test results for fast nanomechanical profiling of tumours. This paper will explain the reasoning for choosing indentation-type atomic force microscopy (IT-AFM) to reveal the functional details of cancer. Determining the subtype, cancer stage, and prognosis will be possible, which aids in choosing the best treatment. DTAs are based on fast IT-AFM at the size of a small box that can be made for a low budget compared to other clinical imaging tools. The DTAs can work in remote areas and all parts of the world. There are a number of direct benefits: First, it is no longer needed to wait a week for the pathology report as the test will only take 10 min. Second, it avoids the complicated steps of making histopathology slides and saves costs of labour. Third, computers and robots are more consistent, more reliable, and more economical than human workers which may result in fewer diagnostic errors. Fourth, the IT-AFM analysis is capable of distinguishing between various cancer subtypes. Fifth, the IT-AFM analysis could reveal new insights about why immunotherapy fails. Sixth, IT-AFM may provide new insights into the neoadjuvant treatment response. Seventh, the healthcare system saves money by reducing diagnostic backlogs. Eighth, the results are stored on a central server and can be accessed to develop strategies to prevent cancer. To bring the IT-AFM technology from the bench to the operation theatre, a fast IT-AFM sensor needs to be developed and integrated into the DTAs.
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BACKGROUND & AIMS: Clinical cases of viral infections possibly involving the transfusion of blood components are systematically investigated. METHODS: Serological and molecular markers of hepatitis B virus were used including HBsAg, anti-HBc, anti-HBs, HBV DNA, and viral load. Full genome sequencing and phylogenetic analyses were performed. RESULTS: An acute HBV infection was diagnosed in the mother of a 16-month-old daughter who had been transfused at age three weeks with one quarter of a regular red cell concentrate (RCC). The repeat donor of the index donation was free of HBV markers in two previous donations but seroconverted to anti-HBc and anti-HBs 3 months post-donation of a unit containing only low level of HBV DNA. One other newborn recipient of the same RCC was asymptomatically HBV infected. A third newborn recipient whose mother had been HBV vaccinated and carried moderate level of anti-HBs was not infected. Full length nucleotide sequence identity between HBV strains from the mother and the two infected transfusion recipients provided evidence of the transfusion origin of all three infections in the absence of donor sequence. CONCLUSIONS: Reverse vertical HBV transmission was likely the result of casual mother contact with a baby carrying extremely high viral load. The blood products intended to immunodeficient newborn should be submitted to more thorough viral testing considering their increased susceptibility to infections.
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Vírus da Hepatite B/isolamento & purificação , Hepatite B/transmissão , Transmissão Vertical de Doenças Infecciosas , Reação Transfusional , Doença Aguda , Adulto , Feminino , Genoma Viral , Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Lactente , Recém-Nascido , Filogenia , Carga ViralRESUMO
During the last few decades, efforts to increase the safety of blood and blood products have mainly focused on preventing the viral infections HCV, HIV, HBV and Treponema pallidum. The evolution of these approaches and the achieved increase in safety is shown for the last 25 years in Switzerland. In detail, the prevalences and incidences of the infection disease and the theoretical estimated residual risks (RR) of these blood-borne infections are presented. Prevalences, incidences and, in particular, the RR have decreased considerably over the last 25 years. This was achieved primarily by the adoption of strict criteria for the selection of blood donors, refined questionnaires, the introduction of increasingly sensitive serological screening tests and the implementation of nucleic acid testing (NAT) for these blood-borne pathogens. These NAT assays have significantly shortened the window period between infection and the first detection of the infectious agent in the blood of an infected individual. A form of "real life" comparison or confirmation is provided by the reported lookback procedures (LBP) and the haemovigilance data of the Swiss competent authority, Swissmedic. These data are in agreement, and thus support the very low prevalences, incidences and RR.
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Infecções por HIV , Hepatite C , Humanos , Vírus da Hepatite B/genética , Segurança do Sangue , Treponema pallidum/genética , Suíça/epidemiologia , Programas de Rastreamento/métodos , Infecções por HIV/prevenção & controle , Hepatite C/epidemiologia , Doadores de Sangue , Técnicas de Amplificação de Ácido NucleicoRESUMO
Towards optimizing the growth of extracellular matrix to produce repair cartilage for healing articular cartilage (AC) defects in joints, scaffold-based tissue engineering approaches have recently become a focus of clinical research. Scaffold-based approaches by electrospinning aim to support the differentiation of chondrocytes by providing an ultrastructure similar to the fibrillar meshwork in native cartilage. In a first step, we demonstrate how the blending of chitosan with poly(ethylene oxide) (PEO) allows concentrated chitosan solution to become electrospinnable. The chitosan-based scaffolds share the chemical structure and characteristics of glycosaminoglycans, which are important structural components of the cartilage extracellular matrix. Electrospinning produced nanofibrils of â¼100 nm thickness that are closely mimicking the size of collagen fibrils in human AC. The polymer scaffolds were stabilized in physiological conditions and their stiffness was tuned by introducing the biocompatible natural crosslinker genipin. We produced scaffolds that were crosslinked with 1.0% genipin to obtain values of stiffness that were in between the stiffness of the superficial zone human AC of 600 ± 150 kPa and deep zone AC of 1854 ± 483 kPa, whereas the stiffness of 1.5% genipin crosslinked scaffold was similar to the stiffness of deep zone AC. The scaffolds were degradable, which was indicated by changes in the fibril structure and a decrease in the scaffold stiffness after seven months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes (HACs) showed a cell viability of over 90% on the scaffolds and new extracellular matrix deposited on the scaffolds.
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Cartilagem Articular/fisiologia , Quitosana/química , Reagentes de Ligações Cruzadas/química , Iridoides/química , Polietilenoglicóis/química , Materiais Biocompatíveis/química , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/citologia , Humanos , Nanofibras/química , Regeneração , Alicerces Teciduais/químicaRESUMO
As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.
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Cartilagem Articular/química , Microscopia de Força Atômica/métodos , Microtecnologia/métodos , Nanotecnologia/métodos , Animais , Materiais Biomiméticos/química , Sulfatos de Condroitina/química , Colágeno/química , Elasticidade , Géis/química , Técnicas In Vitro , Teste de Materiais/instrumentação , Teste de Materiais/métodos , Microscopia de Força Atômica/instrumentação , Microtecnologia/instrumentação , Modelos Biológicos , Nanotecnologia/instrumentação , Poliésteres/química , Polietilenoglicóis/química , Polietilenotereftalatos/química , Proteoglicanas/química , Sefarose/química , Cloreto de Sódio/química , Suínos , Água/químicaRESUMO
A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs' nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.
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Microscopia de Força Atômica/métodos , Poro Nuclear/metabolismo , Animais , Transporte Biológico/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oócitos/metabolismo , Xenopus laevis/metabolismoRESUMO
BACKGROUND: The risk of transfusion-transmitted hepatitis B virus (HBV) in Switzerland by testing blood donors for hepatitis B surface antigen (HBsAg) alone has been historically estimated at 1:160,000 transfusions. The Swiss health authorities decided not to introduce mandatory antibody to hepatitis B core antigen (anti-HBc) testing but to evaluate the investigation of HBV nucleic acid testing (NAT). STUDY DESIGN AND METHODS: Between June 2007 and February 2009, a total of 306,000 donations were screened routinely for HBsAg and HBV DNA by triplex individual-donation (ID)-NAT (Ultrio assay on Tigris system, Gen-Probe/Novartis Diagnostics). ID-NAT repeatedly reactive donors were further characterized for HBV serologic markers and viral load by quantitative polymerase chain reaction. The relative sensitivity of screening for HBsAg, anti-HBc, and HBV DNA was assessed. The residual HBV transmission risk of NAT with or without anti-HBc and HBsAg was retrospectively estimated in a mathematical model. RESULTS: From the 306,000 blood donations, 31 were repeatedly Ultrio test reactive and confirmed HBV infected, of which 24 (77%) and 27 (87%) were HBsAg and anti-HBc positive, respectively. Seven HBV-NAT yields were identified (1:44,000), two pre-HBsAg window period (WP) donations (1:153,000) and five occult HBV infections (1:61,000). Introduction of ID-NAT reduced the risk of HBV WP transmission in repeat donors from 1:95,000 to 1:296,000. CONCLUSIONS: Triplex NAT screening reduced the HBV WP transmission risk approximately threefold. NAT alone was more efficacious than the combined use of HBsAg and anti-HBc. The data from this study led to the decision to introduce sensitive HBV-NAT screening in Switzerland. Our findings may be useful in designing more efficient and cost-effective HBV screening strategies in low-prevalence countries.
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Doenças Endêmicas/prevenção & controle , Vírus da Hepatite B/genética , Hepatite B/diagnóstico , Hepatite B/prevenção & controle , Hepatite B/transmissão , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação Transfusional , Adulto , Idoso , Algoritmos , Transfusão de Sangue/estatística & dados numéricos , Análise Custo-Benefício , DNA Viral/análise , DNA Viral/genética , Eficiência Organizacional , Doenças Endêmicas/estatística & dados numéricos , Feminino , Testes Genéticos/economia , Testes Genéticos/métodos , Testes Genéticos/normas , Geografia , Hepatite B/epidemiologia , Antígenos de Superfície da Hepatite B/análise , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/normas , Prevalência , Fatores de Risco , Suíça/epidemiologiaRESUMO
We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.
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Cartilagem Articular/fisiologia , Cartilagem Articular/ultraestrutura , Microscopia de Força Atômica/métodos , Animais , Artroscopia/métodos , Fenômenos Biomecânicos/fisiologia , Osteoartrite/patologia , Estresse Mecânico , SuínosRESUMO
QUESTIONS UNDER STUDY: The risk of transfusion-transmitted HBV remains significant in Switzerland, where routine screening for hepatitis B virus (HBV) in blood donations relies solely on serological hepatitis B surface antigen (HBsAg) testing. This study was designed to determine the prevalence of anti-hepatitis B core (anti-HBc) and HBV nucleic acid testing (NAT) positive donations in two different Swiss donor populations, to help in deciding whether supplemental testing may bring additional safety to blood products. METHODS: In a first population of donors, 18143 consecutive donations were screened initially for HBsAg, anti-HBc (with one EIA assay) and with HBV NAT in minipools of 24 donations. The screening repeatedly reactive anti-HBc donations were then "confirmed" with two supplemental anti-HBc assays, an anti-hepatitis B surface assay (anti-HBs) and with single donation HBV NAT. In a second population of donors, 4186 consecutive donations were screened initially with two different anti-HBc assays in addition to the mandatory HBsAg screening test. The screening repeatedly reactive donations with at least one anti-HBc assay were tested for anti-HBs. RESULTS: In the first subset of 18143 donations, 17593 (97.0%) were negative for HBsAg, anti-HBc and HBV NAT in minipools. 549 (3.0%) were HBsAg and HBV NAT negative, but repeatedly reactive for anti-HBc. Of these 549 donations, 287 could not be "confirmed" with two additional anti-HBc assays and were negative with an anti-HBs assay, as well as with single donation HBV NAT. Only 211 (1.2% of the total screened donations) were "confirmed" positive with at least one of two supplemental anti-HBc assays. One repeatedly reactive HBsAg donation, from a first-time donor, was confirmed positive for HBsAg and anti-HBc, as well as with single donation HBV NAT. In the second subset of 4186 donations, 4014 (95.9%) were screened negative for HBsAg and for anti-HBc, tested with two independent anti-HBc assays. 172 donations (4.1%) were HBsAg negative but repeatedly reactive with at least one of the two anti-HBc assays. Of these 172 samples, 86 were reactive with the first anti-HBc assay only, 13 were reactive with the second anti-HBc assay only and 73 (1.7% of the total screened donations) were "confirmed" positive with both anti-HBc assays. CONCLUSION: The prevalence of anti-HBc "confirmed" positive donations in the two Swiss blood donor populations studied was low (<2%) and we found only one HBV NAT positive (HBsAg positive) donation among more than 18000. Concerning blood product safety, an increase in the deferral rate of less than 2% of anti-HBc positive, potentially infectious donors, would in our opinion make routine anti-HBc testing of blood donations cost-effective. There is however still a need for more specific assays to avoid an unacceptably high deferral rate of "false" positive donors. In contrast, the introduction of HBV NAT in minipools gives minimal benefit due to the inadequate sensitivity of the assay. It remains to evaluate more extensively the value of individual donation NAT, alone or in addition to anti-HBc, as supplemental testing in the context of several Swiss blood donor populations.
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Doadores de Sangue , Hepatite B/transmissão , Algoritmos , DNA Viral/análise , Hepatite B/prevenção & controle , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Humanos , Imunoensaio , Sensibilidade e Especificidade , Reação TransfusionalRESUMO
Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.
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Fenômenos Mecânicos , Microscopia de Força Atômica/métodos , Calibragem , Coloides , Módulo de ElasticidadeRESUMO
This report focuses on nanotools based on the scanning force microscope (SFM) for imaging, measuring, and manipulating biological matter at the sub-micron scale. Because pathophysiological processes often occur at the (sub-) cellular scale, the SFM has opened the exciting possibility to spot diseases at a stage before they become symptomatic and cause functional impairments in the affected part of the body. Such presymptomatic detection will be key to developing effective therapies to slow or halt disease progression.
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Equipamentos para Diagnóstico/tendências , Microscopia de Varredura por Sonda/métodos , Nanotecnologia/métodos , Instrumentos Cirúrgicos/tendências , Cartilagem Articular/patologia , Cartilagem Articular/ultraestrutura , Vasos Coronários/patologia , Vasos Coronários/ultraestrutura , Humanos , Microscopia de Varredura por Sonda/instrumentação , Nanotecnologia/instrumentaçãoRESUMO
The aim of this study was to demonstrate that differences in the local composition of bi-zonal fibrocartilaginous tissues result in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF-11) and cultured for 4 weeks in mixed flask, a rotary Cell Culture System (RCCS), or statically. Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings, respectively. Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2 microm diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues. In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.
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Cartilagem Articular , Engenharia Tecidual , Animais , Cartilagem Articular/citologia , Cartilagem Articular/ultraestrutura , Bovinos , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Resistência à TraçãoRESUMO
Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a 'guiding' structure to support the body's self-healing process.
Assuntos
Implantes Absorvíveis , Cartilagem Articular/crescimento & desenvolvimento , Condrócitos/fisiologia , Regeneração Tecidual Guiada/instrumentação , Poliésteres/química , Alicerces Teciduais , Materiais Biocompatíveis/química , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/citologia , Condrogênese/fisiologia , Módulo de Elasticidade/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Engenharia Tecidual/instrumentaçãoRESUMO
Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.
Assuntos
Cartilagem Articular/ultraestrutura , Colágenos Fibrilares/química , Osteoartrite do Quadril/patologia , Osteoartrite do Joelho/patologia , Cartilagem Articular/patologia , Articulação do Quadril/metabolismo , Articulação do Quadril/patologia , Humanos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Microscopia Eletrônica de Varredura , Osteoartrite do Quadril/metabolismo , Osteoartrite do Joelho/metabolismoRESUMO
The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense.