Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections.

BACKGROUND: Porcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera. RESULTS: ICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen's κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃. CONCLUSIONS: In summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

Detection of effusion tumor cells under different storage and processing conditions.

BACKGROUND: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. METHODS: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. RESULTS: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. CONCLUSIONS: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Statistical Analysis of Multiplex Immunofluorescence and Immunohistochemistry Imaging Data.

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue.

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

Monitoring the interactions between alpha-synuclein and Tau in vitro and in vivo using bimolecular fluorescence complementation.

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-ß (Aß) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

Spectrum of renal diseases in the elderly, above age 50.

BACKGROUND: The spectrum of kidney diseases varies in the elderly population with frequent inconsistencies between clinical presentation and histopathological diagnosis. The immunofluorescence (IF) may provide additional information in such situations. AIMS: The purpose was to study the spectrum of kidney diseases in patients above 50 years undergoing renal biopsy and utility of light chain (LC) IF in the diagnosis. SETTINGS AND DESIGN: This was a retrospective, crosssectional, singlecenter-based study. MATERIAL AND METHODS: The clinical details, histopathological findings, and LC IF pattern in native renal biopsy of patients above 50 years were noted. STATISTICAL ANALYSIS: Continuous variables were presented as mean ± standard deviation (SD). Categorical variables were expressed as frequencies and percentages. RESULTS: A total of 205 patients were included in the study. The most common clinical presentation was acute kidney injury/rapidly progressive glomerulonephritis (AKI/RPGN) (49%). Glomerular diseases (72%) were more common. Crescentic glomerulonephritis (21%) and membranous nephropathy (MN) (19%) were the most common glomerulopathy. LC restriction was observed in LC cast nephropathy (LCCN), primary amyloidosis, and LC also helped in classifying the cases of MPGN-type morphology. CONCLUSIONS: AKI/RPGN was the most common indication for renal biopsy in patients above 50 years. Crescentic GN and MN were the frequent glomerular pathology. LC IF is a useful adjunctive tool to classify various renal diseases.

Evaluation of DNA double-strand break repair capacity in human cells: Critical overview of current functional methods.

DNA double-strand breaks (DSBs) are highly deleterious lesions, responsible for mutagenesis, chromosomal translocation or cell death. DSB repair (DSBR) is therefore a critical part of the DNA damage response (DDR) to restore molecular and genomic integrity. In humans, this process is achieved through different pathways with various outcomes. The balance between DSB repair activities varies depending on cell types, tissues or individuals. Over the years, several methods have been developed to study variations in DSBR capacity. Here, we mainly focus on functional techniques, which provide dynamic information regarding global DSB repair proficiency or the activity of specific pathways. These methods rely on two kinds of approaches. Indirect techniques, such as pulse field gel electrophoresis (PFGE), the comet assay and immunofluorescence (IF), measure DSB repair capacity by quantifying the time-dependent decrease in DSB levels after exposure to a DNA-damaging agent. On the other hand, cell-free assays and reporter-based methods directly track the repair of an artificial DNA substrate. Each approach has intrinsic advantages and limitations and despite considerable efforts, there is currently no ideal method to quantify DSBR capacity. All techniques provide different information and can be regarded as complementary, but some studies report conflicting results. Parameters such as the type of biological material, the required equipment or the cost of analysis may also limit available options. Improving currently available methods measuring DSBR capacity would be a major step forward and we present direct applications in mechanistic studies, drug development, human biomonitoring and personalized medicine, where DSBR analysis may improve the identification of patients eligible for chemo- and radiotherapy.

Oligonucleotide conjugated antibody strategies for cyclic immunostaining.

A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach.

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

Increased Accuracy to c-Fos-Positive Neuron Counting.

There is not a described method to count the core label of c-Fos-positive neurons, avoiding false-positive and false-negative results. The aim of this manuscript is to provide guidelines for a secure and accurate method to calculate a threshold to select which core of c-Fos-positive neurons marked by immunofluorescence has to be scored. A background percentage was calculated by dividing the intensity value (0 to 255) of the core of c-Fos-positive neurons by its surrounding background from the 8-bit images obtained in a previous study. Using the background percentage from 20% up to 98%, raising 2% once for each score, as threshold to choose which core has to be counted, a script was written for the R program to count the number of the c-Fos-positive neurons and the comparison between control and experimental groups. The differences of the average number of the core counted c-Fos-positive neurons between control and experimental groups, at all thresholds studied, showed a rising value related to an increase of the background percentage threshold as well as a decrease of its p value related to an increase of the threshold of background percentage. For the smallest thresholds (high intensity of label), the differences between groups are suppressed (false negative). However, for the biggest thresholds (nonspecific label), these differences are always the same (false positive). Therefore, to avoid the false-negative and the false-positive values, it was chosen as the threshold of 62% the inflection point of the linear regression, which is equally different from the biggest and smallest values of the differences between groups.

Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ macrophages as a predictor of improved survival in triple negative breast cancer.

Triple negative breast cancer (TNBC) comprises 10-15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33-0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25-0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.

Anti-Proliferative Effects of E2F1 Suppression in Glioblastoma Cells.

Glioblastoma (GBM) is an aggressive malignant brain tumor; surgery, radiation, and temozolomide still remain the main treatments. There is evidence that E2F1 is overexpressed in various types of cancer, including GBM. E2F1 is a transcription factor that controls the cell cycle progression and regulates DNA damage responses and the proliferation of pluripotent and neural stem cells. To test the potentiality of E2F1 as molecular target for GBM treatment, we suppressed the E2F1 gene (siRNA) in the U87MG cell line, aiming to inhibit cellular proliferation and modulate the radioresistance of these cells. Following E2F1 suppression, associated or not with gamma-irradiation, several assays (cell proliferation, cell cycle analysis, neurosphere counting, and protein expression) were performed in U87MG cells grown as monolayer or neurospheres. We found that siE2F1-suppressed cells showed reduced cell proliferation and increased cell death (sub-G1 fraction) in monolayer cultures, and also a significant reduction in the number of neurospheres. In addition, in irradiated cells, E2F1 suppression caused similar effects, with reduction of the number of neurospheres and neurosphere cell numbers relative to controls; these results suggest that E2F1 plays a role in the maintenance of GBM stem cells, and our results obtained in neurospheres are relevant within the context of radiation resistance. Furthermore, E2F1 suppression inhibited or delayed GBM cell differentiation by maintaining a reasonable proportion of CD133+ cells when grown at differentiation condition. Therefore, E2F1 proved to be an interesting molecular target for therapeutic intervention in U87MG cells.

Application of Immunoprofiling Using Multiplexed Immunofluorescence Staining Identifies the Prognosis of Patients with High-Grade Serous Ovarian Cancer.

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420-0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Patient derived organoids in prostate cancer: improving therapeutic efficacy in precision medicine.

With advances in the discovery of the clinical and molecular landscapes of prostate cancer (PCa), implementation of precision medicine-guided therapeutic testing in the clinic has become a priority. Patient derived organoids (PDOs) are three-dimensional (3D) tissue cultures that promise to enable the validation of preclinical drug testing in precision medicine and coclinical trials by modeling PCa for predicting therapeutic responses with a reliable efficacy. We evaluate the advances in 3D culture and PDO use to model clonal heterogeneity and screen for effective targeted therapies, with a focus on the technological advances in generating PDOs. Recent innovations include the utilization of PDOs both in original research and/or correlative studies in clinical trials to examine drug effects within the PCa tumor microenvironment (TME). There has also been a significant improvement with the utilization of various extracellular matrices and single cell assays for the generation and long-term propagation of PDOs. Single cell derived PDOs could faithfully recapitulate the original tumor and reflect the heterogeneity features. While most PDO use for precision medicine understandably involved tissues derived from metastatic patients, we envision that the generation of PDOs from localized PCa along with the incorporation of cells of the TME in tissue models would fulfill the great potential of PDOs in predicting drug clinical benefits. We conclude that single cell derived PDOs reiterate the molecular features of the original tumor and represent a reliable pre-clinical PCa model to understand individual tumors and design tailored targeted therapies.

Biomolecular imaging of colorectal tumor lesions using a FITC-labeled scFv-Cκ fragment antibody.

For the sensitive diagnosis of colorectal cancer lesions, advanced molecular imaging techniques using cancer-specific targets have emerged. However, issues regarding the clearance of unbound probes and immunogenicity remain unresolved. To overcome these limitations, we developed a small-sized scFv antibody fragment conjugated with FITC for the real-time detection of colorectal cancer by in vivo molecular endoscopy imaging. A small-sized scFv fragment can target colon cancer secreted protein-2 (CCSP-2), highly expressed in colorectal adenocarcinoma tissues; moreover, its full-length IgG probe has been used for molecular imaging previously. To assess the efficacy of anti-CCSP-2 scFv-FITC, surgical specimens were obtained from 21 patients with colorectal cancer for ex vivo molecular fluorescence analysis, histology, and immunohistochemistry. Orthotopic mice were administered with anti-CCSP-2 scFv-FITC topically and intravenously, and distinct tumor lesions were observed by real-time fluorescence colonoscopy. The fluorescence imaging of human colon cancer specimens allowed the differentiation of malignant tissues from non-malignant tissues (p < 0.05), and the CCSP-2 expression level was found to be correlated with the fluorescence intensity. Here, we demonstrated the feasibility and safety of anti-CCSP-2 scFv-FITC for molecular imaging as well as its potential in real-time fluorescence colonoscopy for the differential diagnosis of tumor lesions.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects.

BACKGROUND: Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy. METHODS: Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models. RESULTS: We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity. CONCLUSIONS: Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

A tale of tripartite: An experience from a tertiary care center of Eastern India.

BACKGROUND: Crescentic glomerulonephritis (Cr GN) is pattern of glomerular injury resulting from wide range of diseases sharing a common pathogenesis. OBJECTIVES: The objective of our study was to analyze the clinicopathological spectrum and outcome of Cr GN with special reference to its immunopathological subtypes using a panel of immunofluorescence stains. MATERIALS AND METHODS: Native renal biopsies with crescentic pattern of injury were included. Detailed Clinical and laboratory variables were analyzed along with the treatment protocol and renal outcome, wherever available. Renal biopsy slides were evaluated for various glomerular and extraglomerular features. Both qualitative and quantitative data were analyzed. RESULTS: A total of 57 cases of Cr GN were included; majority (47.36%) of cases were pauci-immune in nature. Among clinical features, ranges of proteinuria and creatinine level were significantly different between subgroups. The various light microscopic parameters, including proportion of cellular crescents and capillary wall necrosis were different. Presence of arteriolar changes also showed association with unfavorable outcome. Three unusual associations, including IgA nephropathy, membranous glomerulonephritis and Hepatitis B infection were detected. Adequate follow-up information was available in 35 of the patients. Of these, 14 were dialysis-dependent at the last follow-up. CONCLUSIONS: Type III Cr GN (pauci-immune Cr GN) was the commonest cause of Cr GN in our population. Adult patients required renal replacement therapy more frequently than pediatric cases those are chiefly infection associated. Critical appraisal of clinical, histopathological and immunofluorescence finding help to identify individual subtypes as treatment and outcome varies accordingly.

Genetic and acquired blistering disorders of pediatric age group: An experience from Eastern India.

INTRODUCTION: Blistering or vesiculobullous disorders in pediatric population are either immunobullous or mechanobullous. Spectrum was analyzed using demographic details, clinical features, histopathology, direct immunofluorescence (DIF) and Immunofluorescence mapping (IFM). METHODOLOGY: This was a single institution based observational study in children below 18 years. The demographic details were collected using proforma containing particulars of the patient, history, complaints, and other parameters. Punch biopsy of the skin lesion was done. Biopsy samples were examined under light microscope followed by DIF using fluorescent conjugated polyclonal antibody against immunoglobulins IgG, IgM, IgA, and complement C3. The salt-split technique was also used in particular cases. IFM was done using anticytokeratin (CK) 5 & 14, antilaminin 332, anticollagen VII, and anticollagen IV antibodies. RESULTS: Out of total 50 cases, linear IgA bullous dermatosis (LABD) was the commonest. The average concordance between clinical and final diagnosis (histopathological examination + DIF) was 87.5% and discordance was 12.5%. The agreement between histopathological examination and DIF was found to be substantially significant (κ = 0.6892). IFM depicted epidermolysis bullosa simplex with reduced CK 14 expression, dystrophic epidermolysis bullosa with reduced Collagen VII expression and junctional epidermolysis bullosa with absent laminin 5 expression. CONCLUSION: The spectrum of bullous lesions in childhood was properly delineated and subcategorization of EB was done. Histopathological examination showed the hallmarks that were conclusive in most of the cases except in LABD and EB. DIF and IFM proved indispensable in those cases. Thus, DIF is not a substitute for histopathology but complementary to it.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.