- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence
摘要： Background: The introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis may allow for more harmonized ANA IIF reporting, provided that a thorough quality assurance program controls this process. The aim of this study was to evaluate various quality indicators used for ANA IIF analysis with the final goal of optimizing the iQC program. Methods: In an experimental setup, we introduced artificial errors, mimicking plausible problems during routine practice on a QUANTA-Lyser-NOVA View? system (Inova Diagnostics, San Diego, CA, USA). Predetermined quality indicators were evaluated against predefined acceptance criteria. In addition, we retrospectively investigated the applicability of the selected quality indicators in the daily routine practice during three pre-defined periods. Results: Both the experimental as the retrospective study revealed that pre-analytical, analytical and post-analytical errors were not highlighted by company internal quality control (iQC) materials. The use of patient derived iQC samples, median fluorescence intensity results per run and the percentage of positive ANA IIF results as additional quality indicators ensured a more adequate ANA IIF quality assurance. Furthermore, negative and moderate positive sample iQC materials merit clinical validation, as titer changes of >1 correspond to clinically important shifts. Traditional Westgard rules, including a clinically defined stop limit, revealed to be useful in monitoring of the supplemental quality indicators. Conclusions: A thorough ANA IIF quality assurance for daily routine practice necessitates the addition of supplemental quality indicators in combination with well-defined acceptance criteria.
关键词： automation,indirect immunofluorescence,antinuclear antibodies,quality control
更新于2025-09-23 15:23:52
-
Validation de l’immunofluorescence indirecte en mosa?que versus technique de référence IFI et Elisa dans les maladies bulleuses auto-immunes
摘要： L’immunofluorescence indirecte (IFI) permet de mieux typer la dermatose bulleuse auto-immune (DBAI). L’étude décrit une technique d’IFI en mosa?que. Il s’agit d’une IFI sur mosa?que de six substrats (BIOCHIP Mosa?csTM) : ?sophage de primate, peau humaine traitée par une solution de NaCl, cellules exprimant les desmogléines 1 et 3, la BP230 et la BP180. Chaque substrat est dans une biopuce pour une analyse simultanée. Elle consiste à mettre en contact le sérum des patients avec les substrats. Si une réaction positive est obtenue, les autoanticorps de classes IgA, IgG et IgM présents se fixent au déterminant antigénique de la protéine visée et ensuite le complexe antigène—anticorps est révélé par un anticorps (IgG) marqué au fluorochrome, et est visualisé à l’aide d’un microscope à fluorescence conventionnel. Il s’agit d’une étude diagnostique monocentrique réalisée en 2015 avec des sérums de 2012 à 2015. Quatre-vingt sérums au total testés sur 67 patients (47 pemphigo?des bulleuses [PB] et 2 pemphigo?des cicatricielles, 16 pemphigus vulgaires, 9 pemphigus superficiels, 2 épidermolyses bulleuses acquises). Quatre témoins négatifs ont été utilisés. Chaque élément de la mosa?que est comparé avec la technique de référence (IFI sur peau humaines provenant de reliquats chirurgicaux avec peau splitée). Soixante-dix-neuf sérums ont été analysés, pour 40 sérums, il est réalisé une triple lecture et pour 39 une double lecture. En utilisant l’IFI en mosa?que par rapport à la technique de référence, les sensibilités toutes maladies confondues de la desmogléine 1, desmogléine 3, BP180 NC16A étaient respectivement de : 100 %, 92 %, 85 %. L’antigène BP 230 était reconnu dans 59 % des PB. Pour tous les substrats sauf la peau séparée, la spécificité variait de 84 à 100 %. Pour la peau séparée, elle était de 60 %. Parmi les 40 sérums ayant eu une triple lecture pour 31 patients les résultats étaient identiques pour les 3 lecteurs (78 %). Parmi les 39 sérums ayant eu une double lecture pour 35 les résultats étaient identiques pour les deux lectures (90 %). Les résultats de notre étude sont en accord avec ceux rapportés dans les études retrouvées dans la littérature. L’IFI en mosa?que permet d’avoir un spectre antigénique complet disponible en une seule analyse (un seul test qui permet de différencier les principales DBAI) ; de différencier le pemphigus superficiel et vulgaire. Elle permet une interprétation simple sans spectrophotométrie, les résultats sont rapides en une journée. Elle est facilement reproductible. L’IFI en mosa?que a une bonne spécificité et sensibilité pour le diagnostic des DBAI. C’est une méthode standardisée, plus rapide, pour faciliter le diagnostic de ces maladies. Cette technique s’inscrit dans le cadre de la certification des laboratoires dans la mesure ou l’utilisation de substrats humains ou animaux n’est plus autorisée.
关键词： Immunofluorescence indirecte,BIOCHIP technologie,auto-immune,Dermatose bulleuse
更新于2025-09-23 15:22:29
-
A computer-aided diagnosis system for HEp-2 fluorescence intensity classification
摘要： Background and objective: The indirect immunofluorescence (IIF) on HEp-2 cells is the recommended technique for the detection of antinuclear antibodies. However, it is burdened by some limitations, as it is time consuming and subjective, and it requires trained personnel. In other fields the adoption of deep neural networks has provided an effective high-level abstraction of the raw data, resulting in the ability to automatically generate optimized high-level features. Methods: To alleviate IIF limitations, this paper presents a computer-aided diagnosis (CAD) system classifying HEp-2 fluorescence intensity: it represents each image using an Invariant Scattering Convolutional Network (Scatnet), which is locally translation invariant and stable to deformations, a characteristic useful in case of HEp-2 samples. To cope with the inter-observer discrepancies found in the dataset, we also introduce a method for gold standard computation that assigns a label and a reliability score to each HEp-2 sample on the basis of annotations provided by expert physicians. Features by Scatnet and gold standard information are then used to train a Support Vector Machine. Results: The proposed CAD is tested on a new dataset of 1771 images annotated by three independent medical centers. The performances achieved by our CAD in recognizing positive, weak positive and negative samples are also compared against those obtained by other two approaches presented so far in the literature. The same system trained on this new dataset is then tested on two public datasets, namely MIVIA and I3Asel. Conclusions: The results confirm the effectiveness of our proposal, also revealing that it achieves the same performance as medical experts.
关键词： HEp-2 samples,Deep learning,Invariant Scattering Convolutional Networks,Computer-aided diagnosis,Indirect immunofluorescence
更新于2025-09-23 15:21:21
-
Detection of u-serrated patterns in direct immunofluorescence images of autoimmune bullous diseases by inhibition-augmented COSFIRE filters
摘要： Background and objective: Direct immunofluorescence (DIF) microscopy of a skin biopsy is used by physicians and pathologists to diagnose autoimmune bullous dermatoses (AIBD). This technique is the reference standard for diagnosis of AIBD, which is used worldwide in medical laboratories. For diagnosis of subepidermal AIBD (sAIBD), two different types of serrated pattern of immunodepositions can be recognized from DIF images, namely n- and u-serrated patterns. The n-serrated pattern is typically found in the most common sAIBD bullous pemphigoid. Presence of the u-serrated pattern indicates the sAIBD subtype epidermolysis bullosa acquisita (EBA), which has a different prognosis and requires a different treatment. The manual identification of these serrated patterns is learnable but challenging. Methods and Materials: We propose an automatic technique that is able to localize u-serrated patterns for automated computer-assisted diagnosis of EBA. The distinctive feature of u-serrated patterns as compared to n-serrated patterns is the presence of ridge-endings. We introduce a novel ridge-ending detector which uses inhibition-augmented trainable COSFIRE filters. Then, we apply a hierarchical clustering approach to detect the suspicious u-serrated patterns from the detected ridge-endings. For each detected u-serrated pattern we provide a score that indicates the reliability of its detection. In order to evaluate the proposed approach, we created a data set with 180 DIF images for serration pattern analysis. This data set consists of seven subsets which were obtained from various biopsy samples under different conditions. Results and conclusion: We achieve an average recognition rate of 82.2% of the u-serrated pattern on these 180 DIF images, which is comparable to the recognition rate achieved by experienced medical doctors and pathologists.
关键词： Autoimmune bullous diseases,serration pattern analysis,COSFIRE filter,direct immunofluorescence image,inhibition,ridge-ending detection
更新于2025-09-23 15:21:01
-
Detailed process analysis for glomerular capillary formation by immunofluorescence on ultra-thick sections
摘要： Glomerular capillary formation is one of the fundamental mysteries in renal developmental biology. However, there are still debates on this issue, and its detailed formation process has not been clarified. To resolve this problem, we performed antibody staining with ultra-thick section on embryonic and postnatal mouse kidneys. We obtained the expression patterns of several genes that play an important role in the development of glomerular capillaries. We found that blood vessel of the fetal kidneys expanded through proliferation and sprouting. During the comma-stage and S-shaped stage, 3–4 capillaries began to bud and migrate into the glomerular cleft, forming a capillary bed in the Bowman's capsule. Then, the capillary bed expanded into mature glomerular capillary by intussusceptive angiogenesis. The afferent and efferent arterioles were formed through pruning. The distribution of VEGFA in the nephron epithelial cells but not only in podocytes, induced multiple capillaries sprouted into the glomerular cleft. And CXCR4 played an important role in the differentiation and expansion of capillary bed into glomerular capillary. Immunofluorescence performed with ultra-thick section allowed us to investigate the development of complex structure tissues systematically and comprehensively.
关键词： Ultra-thick sections,Glomerular capillary,Immunofluorescence
更新于2025-09-23 15:19:57
-
[Methods in Molecular Biology] Autoantibodies Volume 1901 (Methods and Protocols) || Determination of Anti-aquaporin 5 Autoantibodies by Immunofluorescence Cytochemistry
摘要： Cell-based assay by immunofluorescence cytochemistry (CBA-IFC) has been shown to be the most accurate method to detect anti-aquaporin (AQP) 4 autoantibodies. Detection of anti-AQP5 autoantibodies is delicate, which depends on the proper expression of AQP5 on the plasma membrane. Here, we describe methods to determine anti-AQP5 autoantibodies by CBA-IFC. Both anti-AQP5 IgG and IgA can be detected by this method.
关键词： Plasma membrane,Autoantibodies,Cell-based assay,Aquaporin 5,Immunofluorescence cytochemistry
更新于2025-09-23 15:19:57
-
Quenching autofluorescence in the alimentary canal tissues of <i>Bactericera cockerelli</i> (Hemiptera: Triozidae) for immunofluorescence labeling
摘要： Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli ?ulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, 'Candidatus Liberibacter solanacearum' (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H2O2) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above-mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H2O2, or SBB treatments. The compatibility assays indicated that irradiation and H2O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.
关键词： gut,endosymbionts,pathogen,Liberibacter,psyllid,immunofluorescence,vector
更新于2025-09-19 17:15:36
-
Detection of <i>Escherichia coli</i> O157:H7 with Antibody Conjugated Amino-Functionalized Graphene Quantum Dots as Immunofluorescence Probes
摘要： Escherichia coli O157: H7 (E. coli O157: H7) is a foodborne pathogenic bacterium which can cause fever, diarrhoea and vomiting in humans. Thus, a rapid, simple, and specific bioprobe for pathogen detection in contaminated foods has been attracted more and more attention. In this work, the strong fluorescent amino-functionalized graphene quantum dots (af-GQDs) were prepared by hydrothermal method. The microtopographic height, surface morphology and spectroscopic properties of af-GQDs are characterized by the high resolution transmission electron microscope (HRTEM), atomic force microscope (AFM), UV-vis, fluorescence, Raman spectroscopic techniques. All the results showed that the af-GQDs can be effectively applied in the preparation of biocompatible immunofluorescence probe and in the detection of E. coli O157: H7. The minimum detection limit is 100 cfu/mL. It is a simple, rapid, sensitive, low-cost and easy to be popularized method, which provides a feasible way to monitor E. coli O157: H7 in food safety.
关键词： E. coli O157: H7,Bioprobe,Amino-functionalized graphene quantum dots,Foodborne bacteria,Immunofluorescence
更新于2025-09-12 10:27:22
-
Rapid immunofluorescence assay for staphylococcal enterotoxin A using magnetic nanoparticles
摘要： A competitive immunoassay for staphylococcal enterotoxin A (SEA) detection in milk was developed, using immobilised antibody onto magnetic nanoparticles (MNPs). MNPs were prepared and then modified to introduce amino groups on them. The morphology and size of the obtained both unmodified and modified MNPs were characterized using TEM analyses. Monoclonal anti-SEA antibody was immobilised onto the modified MNPs (MNP-Ab). Staphylococcal enterotoxin A was conjugated with fluorescent dye ATTO620NHS. The characteristics of fluorescence conjugate were examined. The amount of MNP-Ab and concentration of the fluorescent conjugate used for competitive immunoassay were optimized: 0.25 mg and 53 lg mL(cid:1)1, respectively. The detection limit of developed immunoassay was determined – 0.23 ng mL(cid:1)1 SEA in spiked milk samples. The immunoassay takes only 30 min, the magnetic separation is fast (<10 s) and the volume of the sample for analysis is very small (200 lL).
关键词： fluorescent conjugate,staphylococcal enterotoxin,milk,Antibody immobilisation,magnetic nanoparticles,immunofluorescence
更新于2025-09-09 09:28:46
-
Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings
摘要： Introduction: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. Methods: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. Conclusions: DIF is not very sensitive under clinical laboratory settings.
关键词： qRT-PCR,Direct immunofluorescence,Human metapneumovirus
更新于2025-09-04 15:30:14