To define and characterize a novel pre-Descemet's layer in the human cornea.
Clinical and experimental study.
We included 31 human donor sclerocorneal discs, including 6 controls (mean age, 77.7 ...years).
Air was injected into the stroma of donor whole globes (n = 4) and sclerocorneal discs (n = 21) as in the clinical deep anterior lamellar keratoplasty procedure with the big bubble (BB) technique. The following experiments were performed: (1) creation of BB followed by peeling of the Descemet's membrane (DM); (2) peeling off of the DM followed by creation of the BB, and (3) creation of the BB and continued inflation until the bubble popped to measure the popping pressure. Tissue obtained from these experiments was subjected to histologic examination.
Demonstration of a novel pre-Descemet's layer (Dua's layer) in the human cornea.
Three types of BB were obtained. Type-1, is a well-circumscribed, central dome-shaped elevation up to 8.5 mm in diameter (n = 14). Type-2, is a thin-walled, large BB of maximum 10.5 mm diameter, which always started at the periphery, enlarging centrally to form a large BB (n = 5), and a mixed type (n = 3). With type-1 BB, unlike type-2 BB, it was possible to peel off DM completely without deflating the BB, indicating the presence of an additional layer of tissue. A type-1 BB could be created after first peeling off the DM (n = 5), confirming that DM was not essential to create a type-1 BB. The popping pressure was 1.45 bar and 0.6 bar for type-1 BB and type-2 BB, respectively. Histology confirmed that the cleavage occurred beyond the last row of keratocytes. This layer was acellular, measured 10.15 ± 3.6 microns composed of 5 to 8 lamellae of predominantly type-1 collagen bundles arranged in transverse, longitudinal, and oblique directions.
There exists a novel, well-defined, acellular, strong layer in the pre-Descemet's cornea. This separates along the last row of keratocytes in most cases performed with the BB technique. Its recognition will have considerable impact on posterior corneal surgery and the understanding of corneal biomechanics and posterior corneal pathology such as acute hydrops, Descematocele and pre-Descemet's dystrophies.
The authors have no proprietary or commercial interest in any materials discussed in this article.
Efficient Human Epithelial-2 cell image classification can facilitate the diagnosis of many autoimmune diseases. This paper proposes an automatic framework for this classification task, by utilizing ...the deep convolutional neural networks (CNNs) which have recently attracted intensive attention in visual recognition. In addition to describing the proposed classification framework, this paper elaborates several interesting observations and findings obtained by our investigation. They include the important factors that impact network design and training, the role of rotation-based data augmentation for cell images, the effectiveness of cell image masks for classification, and the adaptability of the CNN-based classification system across different datasets. Extensive experimental study is conducted to verify the above findings and compares the proposed framework with the well-established image classification models in the literature. The results on benchmark datasets demonstrate that 1) the proposed framework can effectively outperform existing models by properly applying data augmentation, 2) our CNN-based framework has excellent adaptability across different datasets, which is highly desirable for cell image classification under varying laboratory settings. Our system is ranked high in the cell image classification competition hosted by ICPR 2014.
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.
Autoimmune phenomena and clinically apparent autoimmune diseases, including autoimmune hepatitis, are increasingly been reported not only after natural infection with the SARS-CoV-2 virus, but also ...after vaccination against it. We report the case of a 63-year old man without a history of autoimmunity or SARS-CoV-2 natural infection who experienced acute severe autoimmune-like hepatitis seven days after the first dose of the mRNA-1273 SARS-CoV-2 vaccine. Liver histology showed inflammatory portal infiltrate with interface hepatitis, lobular and centrilobular inflammation with centrilobular necrosis, in absence of fibrosis and steatosis. Serum immunoglobulin G was slightly elevated. Autoimmune liver serology showed an indirect immunofluorescence pattern on triple rodent tissue compatible with anti-mitochondrial antibody (AMA), but, unexpectedly, this pattern was not mirrored by positivity for primary biliary cholangitis (PBC)-specific molecular tests, indicating that this antibody is different from classical AMA. Anti-nuclear antibody (ANA) was also positive with a rim-like indirect immunofluorescence pattern on liver and HEp2 cell substrates, similar to PBC-specific ANA; however, anti-gp210 and a large panel of molecular-based assays for nuclear antigens were negative, suggesting a unique ANA in our patient. He carries the HLA DRB1*11:01 allele, which is protective against PBC. Response to prednisone treatment was satisfactory. The clinical significance of these novel specificities needs to be further evaluated in this emerging condition.
•A case of acute severe autoimmune-like hepatitis after mRNA-1273 SARS-CoV-2 vaccine.•Indirect immunofluorescence compatible with anti-mitochondrial antibody.•Negative primary biliary cholangitis-specific molecular tests.•Positive anti-nuclear antibody with a rim-like and speckled patterns on HEp2 cells.•Negative anti-gp210 and multiple molecular-based assays for nuclear antigens.
•Performance of Abbott ARCHITECT SARS-CoV-2 IgG assay was retrospectively evaluated.•The assay showed a very high specificity compared to IFA.•Sensitivity was 100 % for samples collected after 14 ...days post-symptoms onset.•Sensitivity vs. microneutralization was 94.4 %.
Serological tests for anti-SARS-CoV-2 antibodies are becoming of great interest to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic.
We evaluated the diagnostic performance of the Abbott ARCHITECT SARS-CoV-2 IgG test, a fully automated indirect immunoassay that detects antibodies directed to a recombinant SARS-CoV-2 Nucleocapsid antigen.
Abbott ARCHITECT SARS-CoV-2 IgG immunoassay was compared to an indirect immunofluorescence assay (IFA) on sera from patients with COVID-19 collected at different days after symptoms onset or infected by other human coronaviruses. Comparison with neutralization test was also performed.
After 7, 14 and >14 days after onset ARCHITECT was positive on 8.3 %; 61.9 % and 100 % of the tested samples compared to 58.3 %; 85.7 % and 100 % by IFA. The sensitivity was 72 % vs. IFA and 66.7 % vs. a real-time PCR, the specificity was 100 %. On 18 samples with neutralizing activity, 17 were positive by Abbott ARCHITECT SARS-CoV-2 IgG.
In our study, Abbott ARCHITECT SARS-CoV-2 IgG assay showed a satisfactory performance, with a very high specificity. IgG reactivity against SARSCoV-2 N antigen was detectable in all patients by two weeks after symptoms onset. In addition, concordance between this serological response and viral neutralization suggests that a strong humoral response may be predictive of a neutralization activity, regardless of the target antigens. This finding supports the use of this automated serological assay in diagnostic algorithm and public health intervention, especially for high loads of testing.
Objective
The spectrum of antinuclear antibodies (ANAs) is changing to include both nuclear staining as well as cytoplasmic and mitotic cell patterns (CMPs) and accordingly a change is occurring in ...terminology to anticellular antibodies. This study examined the prevalence of indirect immunofluorescence (IIF) anticellular antibody staining using the Systemic Lupus International Collaborating Clinics inception cohort.
Methods
Anticellular antibodies were detected by IIF on HEp‐2000 substrate using the baseline serum. Three serologic subsets were examined: ANA positive (presence of either nuclear or mixed nuclear/CMP staining), anticellular antibody negative (absence of any intracellular staining), and isolated CMP staining. The odds of being anticellular antibody negative versus ANA or isolated CMP positive was assessed by multivariable analysis.
Results
A total of 1,137 patients were included; 1,049 (92.3%) were ANA positive, 71 (6.2%) were anticellular antibody negative, and 17 (1.5%) had an isolated CMP. The isolated CMP–positive group did not differ from the ANA‐positive or anticellular antibody–negative groups in clinical, demographic, or serologic features. Patients who were older (odds ratio OR 1.02 95% confidence interval (95% CI) 1.00, 1.04), of white race/ethnicity (OR 3.53 95% CI 1.77, 7.03), or receiving high‐dose glucocorticoids at or prior to enrollment (OR 2.39 95% CI 1.39, 4.12) were more likely to be anticellular antibody negative. Patients on immunosuppressants (OR 0.35 95% CI 0.19, 0.64) or with anti‐SSA/Ro 60 (OR 0.41 95% CI 0.23, 0.74) or anti–U1 RNP (OR 0.43 95% CI 0.20, 0.93) were less likely to be anticellular antibody negative.
Conclusion
In newly diagnosed systemic lupus erythematosus, 6.2% of patients were anticellular antibody negative, and 1.5% had an isolated CMP. The prevalence of anticellular antibody–negative systemic lupus erythematosus will likely decrease as emerging nomenclature guidelines recommend that non‐nuclear patterns should also be reported as a positive ANA.
Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of ...PCa‐derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time‐resolved fluorescence immunoassay (TR‐FIA) for capture/detection of PCa‐derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin‐coated wells. After application of exosomes, Europium‐labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR‐FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR‐FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR‐FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR‐FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease‐specific detection assays.
What's new?
The vesicles cast off by cancer cells could serve as billboards advertising the cancer's presence – if we knew how to read them. Detecting markers in prostate cancer vesicles is currently labor intensive. These authors set out to change that, by developing an immunoaffinity technique to expose these cellular markers more easily. Using the assay, they detected two cell surface proteins, CD9 and CD63, in the urine of men who had prostate cancer. They found far less of the markers in men without cancer, men without prostates, and women, suggesting CD9 and CD63 could be useful prostate cancer markers.
Abstract Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) remains the hallmark of diagnosing autoimmune diseases despite the introduction of multiplex techniques. Non-organ ...specific AAB are screened in routine diagnostics by IIF on HEp-2 cells. However, IIF results vary due to objective (e.g., cell fixation) and subjective factors (e.g., expert knowledge). Therefore, inter- and intralaboratory variance is relatively high. Standardisation of AAB testing by IIF remains a critical issue in and between routine laboratories and may be improved by automated interpretation systems. An overview of existing interpretation techniques will be given taking into account own data of the first fully automated reading system AKLIDES. The novel system provides fully automated reading of IIF images and software algorithms for the mathematical description of IIF AAB patterns. It can be used for screening and preclassification of non-organ specific AAB in routine diagnostics regarding systemic autoimmune and autoimmune liver diseases. Furthermore, this system paves the way for economic data processing of cell-based IIF assays and can contribute to the reduction of interlaboratory variance of AAB testing. More sophisticated pattern recognition algorithms and novel calibration systems will improve standardised quantifications of IIF image interpretation.
Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, ...spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.
Objective
Growing evidence suggests increasing frequencies of autoimmunity and autoimmune diseases, but findings are limited by the lack of systematic data and evolving approaches and definitions. ...This study was undertaken to investigate whether the prevalence of antinuclear antibodies (ANA), the most common biomarker of autoimmunity, changed over a recent 25‐year span in the US.
Methods
Serum ANA were measured by standard indirect immunofluorescence assays on HEp‐2 cells in 13,519 participants age ≥12 years from the National Health and Nutrition Examination Survey, with approximately one‐third from each of 3 time periods: 1988–1991, 1999–2004, and 2011–2012. We used logistic regression adjusted for sex, age, race/ethnicity, and survey design variables to estimate changes in ANA prevalence across the time periods.
Results
The prevalence of ANA was 11.0% (95% confidence interval 95% CI 9.7–12.6%) in 1988–1991, 11.4% (95% CI 10.2–12.8%) in 1999–2004, and 16.1% (95% CI 14.4–18.0%) in 2011–2012 (P for trend <0.0001), corresponding to ~22.3 million, ~26.6 million, and ~41.5 million affected individuals, respectively. Among adolescents age 12–19 years, ANA prevalence increased substantially, with odds ratios of 2.07 (95% CI 1.18–3.64) and 2.77 (95% CI 1.56–4.91) in the second and third time periods relative to the first (P for trend = 0.0004). ANA prevalence increased in both sexes (especially in men), older adults (age ≥50 years), and non‐Hispanic white individuals. These increases in ANA prevalence were not explained by concurrent trends in weight (obesity/overweight), smoking exposure, or alcohol consumption.
Conclusion
The prevalence of ANA in the US has increased considerably in recent years. Additional studies to determine factors underlying these increases in ANA prevalence could elucidate causes of autoimmunity and enable the development of preventative measures.
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