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P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specificmonoclonal antibody. The gene fragment of P32D (GP32D) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32D was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32D. Utilizing rP32D, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32D were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with kappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32D, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method. [ABSTRACT FROM AUTHOR]

Background: Older patients who are predisposed to bullous pemphigoid (BP) may exhibit reluctance to undergo skin biopsy due to potential complications. Objectives: This study aimed to conduct a comparative evaluation among histology, direct immunofluorescence (DIF), and indirect immunofluorescence (IIF) to determine the optimal diagnostic tool in elderly patients. Methods: A retrospective study was conducted on 841 patients suspected of having BP. All cases were initially classified as BP and non-BP in accordance with the diagnostic criteria. Student's t -test and chi-squared test examined differences between the 2 groups. We evaluated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio detected by the 3 tools. We stratified the analysis by age to compare the performance of the diagnostic tools and examined the risk factors associated with BP using logistic regression. Results: Overall, histology exhibited the highest sensitivity (89.4%), while DIF demonstrated the highest specificity (67.1%). In the elderly, the IIF test exhibited the highest specificity (57.5%), the highest positive likelihood ratio (2.047), and the lowest negative likelihood ratio (0.226). Among patients taking Dipeptidyl Peptidase-4 (DPP-4) inhibitors, IIF demonstrated the highest positive likelihood ratio (3.194) and the second-lowest negative likelihood ratio (0.235). Conclusions: In cases that elderly patients suspected of having BP are reluctant to undergo skin biopsy, IIF demonstrates the optimal diagnostic method due to its highest positive likelihood ratio, the lowest negative likelihood ratio among the 3 diagnostic measures. Moreover, IIF is found to be a more effective tool for detecting BP in patients using DPP-4 inhibitors. [ABSTRACT FROM AUTHOR]

Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney tissue, is restricted by operator-dependent interpretation. A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal aorta vascular smooth muscle (VSM) cell line-based assay with those of the rodent tissue-based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis, 40 primary sclerosing cholangitis, 50 chronic viral hepatitis, 20 alcohol-related liver disease, 40 steatotic liver disease, and 40 healthy controls) were assayed for MF-SMA and SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96 (70%) and 87 (63%) AIH-1 patients, and 2 controls (P < 0.0001). Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%, P = ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870, [0.789–0.952]). MF-SMA were associated with higher serum γ‐globulin [26 (12–55) vs 20 g/l (13–34), P < 0.005] and immunoglobulin G (IgG) levels [3155 (1296–7344) vs 2050 mg/dl (1377–3357), P < 0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA. (i) The search for smooth muscle antibodies (SMA) is a cornerstone of hepatic autoimmune serology, being one of the diagnostic criteria of autoimmune hepatitis (AIH). (ii) SMA are directed against structures of the cytoskeleton such as microfilaments, intermediate filaments, and microtubules. Although they may be detected in sera of patients with viral hepatitis, drug-induced hepatitis, and alcohol-related and non-alcohol-related fatty liver disease, SMA directed against actin microfilaments (MF-SMA) are highly AIH-specific. (iii) The recommended rodent tissue-based IFL for detecting MF-SMA is somewhat complicated, and the interpretation of the results depends on the operator's experience. The use of an embryonal aorta vascular smooth muscle cell line-based IFL assay provides high accuracy and easy detection of MF-SMA, the most AIH-specific SMA. Graphical Abstract [ABSTRACT FROM AUTHOR]

Introduction: The presence of anti-nuclear autoantibodies (ANA) is closely associated with immune complex-mediated autoimmune conditions. Accurate detection and measurement of these autoantibodies play a pivotal role in both diagnosing and managing such diseases. The indirect immunofluorescence method has emerged as the gold standard for detecting ANA, facilitated by advancements in serological testing for autoimmune disorders. ANA screening, while non-specific, offers a reliable, rapid, and comprehensive assessment, revealing diverse patterns indicative of various autoimmune disorders. Materials and Methods: Between January 2023 and June 2023, a total of 179 serum samples were subjected to ANA screening using the indirect immunofluorescence (IIF) method on Hep-2000 cells. Observations and Results: Among the 179 serum samples analyzed, 40 (22.35%) tested positive for antinuclear antibodies (ANA). Among the positive samples, speckled pattern was the most prevalent, observed in 20 cases (50%), followed by homogeneous pattern in 8 cases (20%), nucleolar pattern in 6 cases (15%), mixed pattern in 3 cases (7.5%), cytoplasmic pattern in 2 cases (5%) and Centromere in 1 case(2.5%). Notably, higher positivity rates were observed in the age groups of 20-40 years (35%) and 40-60 years (32.5%), with females showing a predominance. Conclusion: The speckled pattern emerged as the most prevalent among positive cases, closely followed by homogenous patterns. This underscores the importance of conducting observational studies to comprehensively grasp the epidemiology of autoimmune disorders and ascertain the predictive value of ANA indirect immunofluorescence (IIF) testing in clinical settings. Furthermore, exploring the feasibility of integrating ANA IIF as a screening tool for autoimmune disorders warrants consideration. [ABSTRACT FROM AUTHOR]

Background and Objectives: Age-related macular degeneration (AMD) is one of the leading causes of central vision loss among elderly patients, and its dry form accounts for the majority of cases. Although several causes and mechanisms for the development and progression of AMD have previously been identified, the pathogenesis of this complex disease is still not entirely understood. As inflammation and immune system involvement are strongly suggested to play a central role in promoting the degenerative process and stimulating the onset of complications, we aimed to analyze the frequency of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (AECAs) in patients with dry AMD and to determine their relationship with the clinical features of the disease, notably the area of geographic atrophy (GA). Materials and Methods: This study included 41 patients with advanced-stage dry AMD and 50 healthy controls without AMD, matched for gender and age. ARAs were detected by indirect immunofluorescence using monkey retina as an antigen substrate, and the presence of AECAs was determined using cultivated human umbilical vein endothelial cells and primate skeletal muscle. Results: ARAs were detected in 36 (87.8%) AMD patients (titers ranged from 1:20 to 1:320) and in 16 (39.0%) (titers ranged from 1:10 to 1:40) controls (p = 0.0000). Twenty of the forty-one patients (48.8%) were positive for AECAs, while in the control group, AECAs were present only in five sera (10.0%). The titers of AECAs in AMD patients ranged from 1:100 to 1:1000, and in the control group, the AECA titers were 1:100 (p = 0.0001). There were no significant correlations between the presence of AECAs and disease activity. Conclusions: This study demonstrates a higher prevalence of circulating AECAs in patients with dry AMD; however, no correlation was found between the serum levels of these autoantibodies and the area of GA. [ABSTRACT FROM AUTHOR]

Introduction: Anti-nuclear antibody (ANA) testing is among the most common immunological test requested in the diagnostic immunology laboratory. The main purpose of this test is to screen for the underlying systemic autoimmune rheumatic diseases (SARDs). The gold standard laboratory method for ANA detection is by the indirect immunofluorescence (IIF) assay. In most laboratories, positive ANA-IIF is reported in terms of titration and pattern. Objective: This study was conducted with the aim of determining the correlation between ANA-IIF titration and pattern for the diagnosis of SARDs. Materials and methods: A retrospective study was conducted whereby the positive ANA-IIF samples from 1st July 2018 until 31st December 2019 and 1st January 2021 until 31st March 2021 were included in this study. The duplicate samples were excluded. ANA-IIF titration and pattern were recorded for all patients. The demographic, clinical, and final diagnosis data were retrieved from each patient's clinical note. Results: A total of 179 patients were included for analysis. The majority of the patients were female (79.9%) and from Malay ethnicity (66.5%). Sixty-five patients (36.3%) had ANA-IIF positive at 1:80 titration followed by 45 patients (25.1%) positive at titration of equal or more than 1:160. Speckled was the predominant pattern visualised in 90 patients (50.3%) followed by homogeneous in 76 patients (42.5%). Forty-five patients (25.1%) were finally diagnosed with SARDs with 41 of them diagnosed as SLE. ANA titration was significantly associated with the final diagnosis of SARDs at all titres (p<0.001) but the best cut-off was noted at a titre of equal or more than 1:320 with the sensitivity and specificity of 86.7% and 77.6% respectively. The homogeneous pattern was also significantly associated with SARDs (p=0.04). The final diagnosis of SARDs were significantly higher in female (p=0.03) and their age was significantly younger (p<0.001). Conclusion: ANA-IIF titration of equal or more than 1:320 can be used as the best titration for differentiating between SARDs and non-SARDs in a positive ANA sample. Patients with homogeneous pattern were more likely to be diagnosed with SARDs than other ANA-IIF patterns. [ABSTRACT FROM AUTHOR]

Background: Autoimmune encephalitis (AIE) has been ranked as the third most common cause of encephalitis after viral encephalitis and acute disseminated encephalomyelitis. The estimated incidence is 5-8 cases per 100,000 population. The objective of this study was to develop a costeffective test for detection of anti NMDA R antibodies by using in house prepared rodent brain tissue sections which could facilitate timely diagnosis and management of anti NMDA R Encephalitis, which if left undiagnosed may prove fatal. Methods: A total of 500 samples sent for autoimmune encephalitis related antibody testing were included in this cross-sectional study from April 2019 to March 2021 at department of Immunology, Shifa International Hospital (SIH), Islamabad. Rodent brain was dissected to prepare tissue sections on which samples were tested by Indirect Immunofluorescence. Simultaneously samples were tested on cell-based assay (CBA) which is gold standard for testing anti NMDA R antibodies. Sensitivity, specificity, positive and negative predictive values were calculated. Results: Median age of patients who tested positive for anti NMDA encephalitis was 19 years (range: 1 to 57). Out these 76% were female and 24% males. 5% patients tested positive for anti NMDA antibodies out of those suspected to be suffering from autoimmune encephalitis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of rodent brain IF for anti-NMDR antibodies taking CBA as gold standard was 92.6%, 98.5%, 78.1% and 99.6% respectively. The accuracy of the procedure was 98.2%. Conclusion: Indirect immunofluorescence (IF) on rodent brain tissue sections can be useful as a cost-effective alternate for resource constrained laboratories for timely detection of anti NMDA R antibodies facilitating timely diagnosis and management of anti-NMDA receptor encephalitis patients. [ABSTRACT FROM AUTHOR]

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist's performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Methodology: Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. Results: The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25 kDa, 27–31 kDa and 45–55 kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Conclusion: Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins. [ABSTRACT FROM AUTHOR]

Introduction: Antinuclear Antibodies (ANA) detection by Human Epithelial Substrate (Hep2) is the recommended screening test for the diagnosis of ANA-Associated Rheumatic Diseases (AARD). ANA is ordered by various specialists in a tertiary care hospital, and a positive ANA result is followed by testing for specific autoantibodies. High pretest probability, pattern type with intensity, and referral departments are key factors determining the autoimmune diagnosis. Aim: To analyse the ANA referral patterns among the various departments and also to estimate the prevalence and clinical significance of rare ANA patterns. Materials and Methods: A retrospective observational study was conducted at Nizam’s Institute of Medical Sciences, Hyderabad, a tertiary care hospital, in Hyderabad, Telangana, India where ANA test reports (n=16,994) from various departments over three years (November 2017 to October 2020) were evaluated between November 2022 to December 2022. ANA tests were performed on Hep2 substrate at a 1:100 dilution, and ANA patterns were reported according to the International Consensus on ANA Patterns (ICAP) nomenclature. Statistical analysis of department-wise ANA positivity and fluorescence intensity was conducted, and the final diagnoses of patients with rare ANA patterns (

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results. [ABSTRACT FROM AUTHOR]

Introduction: The combination of patterns is a frequent and challenging situation in the daily laboratory routine of autoantibodies testing using HEp-2 cells indirect immunofluorescence assay (HEp-2-IFA). Recently, the Brazilian Consensus on Autoantibodies (BCA) named these combinations as complex patterns (CPs) and organized them into 3 subtypes: multiple, mixed, and composite. This study aimed to describe the most frequent combinations of HEp-2-IIF patterns according to this new nomenclature. Methods: Routine HEp-2-IFA results reported in January and June 2017 were reviewed using the new BCA classification. Visual pattern recognition was performed by experts on HEp-2-IFA readings, using the International Consensus on Antinuclear Antibodies (ANA) Patterns (ICAP) and BCA recommendations. Results: 54,990 serum samples from different patients were tested for ANAHEp-2, and 11,478 (20.9%) were positive at a titer ≥ 1/80. Among these positive samples, 1,111 (9.7%) displayed CPs, divided into 95 different combinations. A higher proportion of CPs was observed in the pediatric age group. Multiple, mixed, and composite patterns were present in 85.3, 5.4, and 9.5% of the samples, respectively. In the multiple/mixed pattern group (n=1,005), double, triple, and quadruple combinations (ICAP/BCA codes) were observed in 97.7%, 2.2%, and 0.1%, respectively. The double nuclear pattern was the most prevalent combination observed (67.6%). The most common CPs registered were AC-4 (nuclear fine speckled) + AC-6,7 (nuclear discrete dots) (n=264); AC-2 (nuclear dense fine speckled) + AC-6,7 (n=201); AC-4+AC-8,9,10 (nucleolar) (n=129); and AC-3 (centromere)+AC-4 (n=124). All of these combinations were in the multiple subgroup. Conclusion: Almost 10% of positive results in the HEp-2 procedure displayed CPs. Among the 3 subtypes of CPs proposed, the multiple pattern was the most prevalent, especially in the pediatric population. The AC-4, AC-2, and AC-6,7 were the most prevalent single patterns observed in the combinations described in this study. There was a significant association between age and the prevalence of most combined patterns. The AC-4+AC-6,7 combination was the most prevalent complex pattern detected regardless of the age group. The AC-2 +AC-6,7 was more prevalent in younger individuals. The concepts involved in the CPs definition should add value to the reading and interpretation of the HEp-2-IIF assay. [ABSTRACT FROM AUTHOR]

Mucous membrane pemphigoid, formerly known as cicatricial pemphigoid, is a rare and difficult-to-treat bullous disorder that occurs most commonly in older adults. We describe a 32-year-old woman who was diagnosed with anti-laminin 332 mucous membrane pemphigoid through indirect immunofluorescence for laminin 332 following nonspecific histologic and direct immunofluorescence findings. At 16 weeks following completion of her first cycle of with rituximab 375mg/m2 weekly for four weeks, her mucosal erosions had resolved. Although not widely available, this case highlights the utility of anti-laminin 332 immunofluorescence for diagnostic confirmation of this entity and the efficacy of rituximab in obtaining disease control.

Immunofluorescence on human epithelial type 2 cells is the standard screening assay for the detection of antinuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding. However, the less commonly reported ones include the cytoplasmic fibrillar patterns on indirect immunofluorescence technique (IIFT). The cytoplasmic fibrillar patterns include the cytoplasmic linear (AC-15), cytoplasmic filamentous (AC-16), and cytoplasmic segmental (AC-17). We report a case of cytoplasmic linear (F-actin) detected through IIFT during ANA screening in a 77-year-old man and later reconfirmed on liver mosaic biochip through IIFT on vascular smooth muscle substrate (VSM-47) without features suggestive of anti-smooth muscle antibody involvement post-complementary and alternative medicine therapy initiation.

IntroductionThe combination of patterns is a frequent and challenging situation in the daily laboratory routine of autoantibodies testing using HEp-2 cells indirect immunofluorescence assay (HEp-2-IFA). Recently, the Brazilian Consensus on Autoantibodies (BCA) named these combinations as complex patterns (CPs) and organized them into 3 subtypes: multiple, mixed, and composite. This study aimed to describe the most frequent combinations of HEp-2-IIF patterns according to this new nomenclature.MethodsRoutine HEp-2-IFA results reported in January and June 2017 were reviewed using the new BCA classification. Visual pattern recognition was performed by experts on HEp-2-IFA readings, using the International Consensus on Antinuclear Antibodies (ANA) Patterns (ICAP) and BCA recommendations.Results54,990 serum samples from different patients were tested for ANA-HEp-2, and 11,478 (20.9%) were positive at a titer ≥ 1/80. Among these positive samples, 1,111 (9.7%) displayed CPs, divided into 95 different combinations. A higher proportion of CPs was observed in the pediatric age group. Multiple, mixed, and composite patterns were present in 85.3, 5.4, and 9.5% of the samples, respectively. In the multiple/mixed pattern group (n=1,005), double, triple, and quadruple combinations (ICAP/BCA codes) were observed in 97.7%, 2.2%, and 0.1%, respectively. The double nuclear pattern was the most prevalent combination observed (67.6%). The most common CPs registered were AC-4 (nuclear fine speckled) + AC-6,7 (nuclear discrete dots) (n=264); AC-2 (nuclear dense fine speckled) + AC-6,7 (n=201); AC-4+AC-8,9,10 (nucleolar) (n=129); and AC-3 (centromere)+AC-4 (n=124). All of these combinations were in the multiple subgroup.ConclusionAlmost 10% of positive results in the HEp-2 procedure displayed CPs. Among the 3 subtypes of CPs proposed, the multiple pattern was the most prevalent, especially in the pediatric population. The AC-4, AC-2, and AC-6,7 were the most prevalent single patterns observed in the combinations described in this study. There was a significant association between age and the prevalence of most combined patterns. The AC-4+AC-6,7 combination was the most prevalent complex pattern detected regardless of the age group. The AC-2+AC-6,7 was more prevalent in younger individuals. The concepts involved in the CPs definition should add value to the reading and interpretation of the HEp-2-IIF assay.

The aim was to investigate the effect of particles released during grinding of dental composites on human gingival keratinocytes (HGK). Specimens from Filtek™ Supreme XTE and ceram.x® universal were prepared and ground to dust. The dust was filtered (≤ 5 µm) and the particle size distribution was examined using NANO-flex®-180° dynamic light scattering (DLS). Suspensions at five concentrations (3, 10, 30, 100 and 300 µg/mL) were prepared using keratinocyte growth medium (KGM). These suspensions, as well as a positive (CuO) and a negative control (KGM) were added to HGK. The cells treated with Filtek™ Supreme XTE suspensions were analyzed by real-time monitoring using RTCA iCELLigence™. In addition, light and scanning electron microscopic images of the exposed cells were taken. Indirect immunofluorescence staining was performed to detect the extracellular matrix protein fibronectin. In distilled water, DLS showed similar particles' range (171.9 nm- 2.7 µm) for both composites. In saliva, larger particles were detected (Filtek™ Supreme XTE: 243 nm-6,5 µm; ceram.x® universal: 204 nm- 4,6 µm). iCELLigence™ revealed similar results of cell growth parameters for HGK incubated with composite dust (≤ 5 µm) at different concentrations. The microscopic images indicated unaltered cell structures and formation of large agglomerates with high particle concentration (> 100 µg/mL). Exposure to composite dust resulted in upregulation of fibronectin expression. Grinding of dental composite materials generates dust particles of different sizes. The particle size distribution seems to be more influenced by the suspending medium than the material itself. While cell growth of HGK seem not to be affected by the particles, an upregulation of fibronectin in the intercellular space concomitant by increasing particle concentration may indicate an increase of cell migration/mobility. • Grinding of composites can generate dust particles in the nanoscale range. • The surrounding medium influences the size of composite dust particles. • HGK exposure to composite dust results in upregulation of fibronectin in the intercellular space. [ABSTRACT FROM AUTHOR]

Anti-neutrophil cytoplasmic antibodies (ANCA) are mainly associated with medium and small vessel vasculitis. Two main methodologies currently available for detection of these antibodies are indirect immunofluorescence (IIF) and monospecific proteinase 3 (PR3) and myeloperoxidase (MPO) based immunoassays. However, well-defined guidelines regarding mode of testing for ANCA in laboratories still don't exist, leading to problems in diagnosis and further patient management. Anti-neutrophil cytoplasmic antibodies testing by IIF and enzyme linked immunosorbent assay (ELISA) often pose a significant challenge in diseases other than vasculitis and in overlapping autoimmune conditions. Anti-neutrophil cytoplasmic antibodies reporting by IIF can be challenging in certain circumstances. This case series aims to discuss four cases with probable interference of anti-nuclear antibodies (ANA) during ANCA testing by IIF resulting in ANCA false positivity. All four cases on subsequent reflex testing by line immunoassay (LIA) for PR3, MPO and glomerular basement membrane (GBM) antigens proved otherwise. While analysing for the presence of ANCA by IIF, the possible interference of ANA leading to a false positive ANCA result should be kept in mind and alternative methods of testing like ELISA, extended granulocyte based IIF assays with MPO and PR3 coated beads, etc., should also be advised. Probability of atypical ANCA in diseases other than vasculitis should also be considered in case of ambiguous results. [ABSTRACT FROM AUTHOR]

The article explores the use of indirect immunofluorescence on salt-split skin sections to diagnose bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) in patients. The study compares the sensitivity and specificity of human and monkey skin samples in detecting autoantibodies from BP and MMP patients. While human skin proved more sensitive in detecting BP autoantibodies, monkey skin showed higher sensitivity for MMP autoantibodies due to differences in antigen expression. The study suggests carefully selecting the appropriate substrate to enhance the diagnostic performance of the immunofluorescence technique. [Extracted from the article]

Background and Objectives: Age-related macular degeneration (AMD) is one of the leading causes of central vision loss among elderly patients, and its dry form accounts for the majority of cases. Although several causes and mechanisms for the development and progression of AMD have previously been identified, the pathogenesis of this complex disease is still not entirely understood. As inflammation and immune system involvement are strongly suggested to play a central role in promoting the degenerative process and stimulating the onset of complications, we aimed to analyze the frequency of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (AECAs) in patients with dry AMD and to determine their relationship with the clinical features of the disease, notably the area of geographic atrophy (GA). Materials and Methods: This study included 41 patients with advanced-stage dry AMD and 50 healthy controls without AMD, matched for gender and age. ARAs were detected by indirect immunofluorescence using monkey retina as an antigen substrate, and the presence of AECAs was determined using cultivated human umbilical vein endothelial cells and primate skeletal muscle. Results: ARAs were detected in 36 (87.8%) AMD patients (titers ranged from 1:20 to 1:320) and in 16 (39.0%) (titers ranged from 1:10 to 1:40) controls (p = 0.0000). Twenty of the forty-one patients (48.8%) were positive for AECAs, while in the control group, AECAs were present only in five sera (10.0%). The titers of AECAs in AMD patients ranged from 1:100 to 1:1000, and in the control group, the AECA titers were 1:100 (p = 0.0001). There were no significant correlations between the presence of AECAs and disease activity. Conclusions: This study demonstrates a higher prevalence of circulating AECAs in patients with dry AMD; however, no correlation was found between the serum levels of these autoantibodies and the area of GA.

Introduction: Recognition of Dense Fine Speckled (DFS) Antinuclear Antibody (ANA) pattern is challenging as it is frequently confused with speckled or homogeneous patterns. Identification and confirmation of DFS pattern is important as it is more often seen in healthy individuals, routine laboratory referrals with relatively lesser prevalence in Systemic Autoimmune Rheumatic Diseases (SARD). As there is limited data on the prevalence of DFS pattern in Indian population, present study determined the prevalence and clinical significance of DFS pattern followed by anti-DFS 70 Enzyme Linked Immunosorbent Assay (ELISA) for confirmation. Aim: To estimate the prevalence of anti-DFS 70 antibodies in ANA screening tests, SARD, and healthy individuals by Indirect Immunofluorescence (IIF) screening and confirmation by anti-DFS 70 ELISA. Materials and Methods: A cross-sectional study was conducted over a period of 15 months from January 2017 to April 2018 at Department of Rheumatology, Nizams Institute of Medical Sciences, a Tertiary Care Institute in Hyderabad, Telangana, India. A total of 7,550 serum samples were tested by IIF during the study duration. Sera displaying DFS pattern was tested for anti-DFS antibodies and anti-Extractable Nuclear Antigen (ENA) antibodies by ELISA. A total of 49 patients from rheumatology department satisfying SARD criteria and 184 healthy patients were also tested for anti-DFS antibodies. Results: Anti-DFS 70 antibodies were seen in 0.47%, 16.3%, 39.6% among routine ANA screening referrals, SARD patients and healthy individuals, respectively. DFS 70 antibodies were positive in 60% of DFS IIF positive samples with none of them showing anti-ENA positivity or clinical evidence of SARD. Anti-DFS 70 positivity was more commonly seen in younger patients. Anti-DFS antibodies were significantly more common in healthy individuals compared to SARD patients (p-value=0.0022). Conclusion: It is important to confirm DFS IIF pattern with a specific ELISA as isolated DFS antibodies are frequently associated with non SARD conditions.

Since the outbreak of coronavirus (COVID-19) in 2019, various rare movement disorders and cognitive changes have been recognized as potential neurological complications. The early treatment of some of these allows rapid recovery; therefore, we must diagnose these manifestations in a timely way. We describe the case of a 76-year-old man infected with severe acute respiratory syndrome coronavirus-2 who presented with confusion and hallucinations and was admitted to our hospital 14 days after the onset of symptoms. One day later, he developed generalized myoclonus, dysarthria and ataxia, and tonic clonic seizures and was admitted to the intensive care unit. A diagnosis of COVID-19-associated autoimmune encephalitis with characteristics of limbic encephalitis and immune-mediated acute cerebellar ataxia and myoclonus syndrome was supported by alterations in the limbic system shown in magnetic resonance imaging, lateralized discharges shown in electroencephalography, a slightly elevated protein level in the cerebrospinal fluid (CSF), and indirect immunofluorescence in the CSF with autoantibody binding to anatomical structures of the cerebellum and hippocampus. The patient improved with 2 weeks of corticosteroid treatment and four sessions of plasmapheresis. Our current case study describes a rare case of COVID-19-related limbic encephalitis with immune-mediated acute cerebellar ataxia and myoclonus syndrome (ACAM syndrome) and strengthens the need for tissue-based assays (TBAs) to screen the serum and/or CSF of patients highly suspected to have autoimmune encephalitis. We believe that the timely diagnosis and targeted aggressive immunotherapy were mainly responsible for the patient's total recovery. [ABSTRACT FROM AUTHOR]

Immunofluorescence on human epithelial type 2 cells is the standard screening assay for the detection of antinuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding. However, the less commonly reported ones include the cytoplasmic fibrillar patterns on indirect immunofluorescence technique (IIFT). The cytoplasmic fibrillar patterns include the cytoplasmic linear (AC-15), cytoplasmic filamentous (AC-16), and cytoplasmic segmental (AC-17). We report a case of cytoplasmic linear (F-actin) detected through IIFT during ANA screening in a 77-year-old man and later reconfirmed on liver mosaic biochip through IIFT on vascular smooth muscle substrate (VSM-47) without features suggestive of anti-smooth muscle antibody involvement post-complementary and alternative medicine therapy initiation. [ABSTRACT FROM AUTHOR]

Background: M2-type anti-mitochondrial autoantibodies are considered the hallmark of primary biliary cholangitis and are directed mainly against the E2 subunits of the 2-oxo acid dehydrogenase complex enzymes (PDC, BCOADC and OGDC). The aim of this study was to determine whether a Dot-blot that includes these E2 subunits separately could confirm the results of methods with non-separated subunits in patients with low positive or discordant results between techniques. Methods: Sera of 24 patients with low positive or discordant results and of 10 patients with clear positive results by non-separated subunits methods were analyzed by Dot-blot with separated subunits. Results: Autoantibodies against E2 subunits of PDC, BCOADC or OGDC were detected in all patients, except in one case from the low positive or discordant results group, by Dot-blot with separated subunits. Conclusions: It would be advisable to use methods that include the three E2 subunits, and a Dot-blot with separated subunits could confirm doubtful cases by non-separated assays. [ABSTRACT FROM AUTHOR]

Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization. A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper). Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents. Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization. [ABSTRACT FROM AUTHOR]

Background: The autoimmune response may be one of the causative factors for oral submucous fibrosis (OSMF). Serum levels of antinuclear antibodies (ANAs), anti-smooth muscle antibodies (ASMAs), and antithyroid microsomal antibodies (ATMAs) were evaluated in patients with and without OSMF. Objectives: This study aimed to evaluate the presence of autoantibodies in patients suffering from OSMF and those without it and compare both groups for ANAs, ATMAs, and ASMAs. Materials and Methods: The participants were 70 subjects with an age group of 18–80 years. Thirty-five patients with the diagnosis of OSMF were assigned to the case group, while 35 subjects without any OSMF were assigned to the control group. Serum samples were collected from the subjects and screened for serum autoantibodies (ANAs, ASMAs, and ATMAs) by indirect immune fluorescence. Results: In the case group, most of the patients were positive for ANAs and none were positive for ASMAs. ANA positivity was found to be more significant (P < 0.001) than ATMAs and ASMAs in both groups. While 10% of patients in the control group were positive for ATMAs, the control group showed negative status for both ANAs and ASMAs. There was a male predominance with the prevalence of ANAs. Conclusion: The study suggested a possible role of autoimmune response in OSMF.

Systemic lupus erythematosus (SLE) is an autoimmune connective disorder characterized by the presence of autoantibodies. Indirect immunofluorescence is considered as the gold standard for the detection of antinuclear antibodies (ANA). False-negative ANA results have resulted in misclassification of some patients as ANA-negative SLE. There are many possible explanations for this negative ANA status of SLE patients. Current evidence in literature suggests that ANA-negative lupus is a questionable entity.

Pneumocystis jirovecii pneumonia (PJP) in immunocompromised patients entails high mortality and requires adequate laboratory diagnosis. We compared the performance of a real time-PCR assay against the immunofluorescence assay (IFA) in the routine of a large microbiology laboratory. Different respiratory samples from HIV and non-HIV-infected patients were included. The retrospective analysis used data from September 2015 to April 2018, which included all samples for which a P. jirovecii test was requested. A total of 299 respiratory samples were tested (bronchoalveolar lavage fluid (n = 181), tracheal aspirate (n = 53) and sputum (n = 65)). Forty-eight (16.1%) patients fulfilled the criteria for PJP. Five positive samples (10%) had only colonization. The PCR test was found to have a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 96%, 98%, 90% and 99%, compared to 27%, 100%, 100% and 87%, for the IFA, respectively. PJ-PCR sensitivity and specificity were >80% and >90% for all tested respiratory samples. Median cycle threshold values in definite PJP cases were 30 versus 37 in colonized cases (p < 0.05). Thus, the PCR assay is a robust and reliable test for the diagnosis PJP in all respiratory sample types. Ct values of ≥36 could help to exclude PJP diagnosis. [ABSTRACT FROM AUTHOR]

Pemphigus herpetiformis (PH) is an autoimmune intraepithelial bullous skin disorder. A 61-year-old female presented with history of multiple pruritic erosions, ulcers all over body, and diffuse loss of hair over scalp. Oral and genital mucosas were uninvolved. Subcorneal separation with suprapapillary thinning of epidermis, neutrophilic spongiosis, and elongation of rete ridges were seen on histopathology. Direct immunofluorescence (DIF) revealed IgG deposits in intercellular zone in fish net like pattern and focal linear IgA deposits along basement zone. Indirect immunofluorescence (IIF) revealed antibodies to desmoglein1 (Dsg-1) positive. A final diagnosis of PH was given. The patient responded well to treatment with dapsone and steroids.

Abstract Background The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. Methods A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. Results From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively (p

Due to the rapid mutation of porcine epidemic diarrhea virus (PEDV), existing vaccines cannot provide sufficient immune protection for pigs. Therefore, it is urgent to design the affinity peptides for the prevention and control of this disease. In this study, we made use of a molecular docking technology for virtual screening of affinity peptides that specifically recognized the PEDV S1 C-terminal domain (CTD) protein for the first time. Experimentally, the affinity, cross-reactivity and sensitivity of the peptides were identified by an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) test, separately. Subsequently, Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), Western blot and indirect immunofluorescence were used to further study the antiviral effect of different concentrations of peptide 110766 in PEDV. Our results showed that the P/N value of peptide 110766 at 450 nm reached 167, with a KD value of 216 nM. The cytotoxic test indicated that peptide 110766 was not toxic to vero cells. Results of the absolute quantitative PCR revealed that different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of peptide 110766 could significantly reduce the viral load of PEDV compared with the virus group (p < 0.0001). Similarly, results of Western blot and indirect immunofluorescence also suggested that the antiviral effect of peptide 110766 at 3.125 is still significant. Based on the above research, high-affinity peptide 110766 binding to the PEDV S1-CTD protein was attained by a molecular docking technology. Therefore, designing, screening, and identifying affinity peptides can provide a new method for the development of antiviral drugs for PEDV. [ABSTRACT FROM AUTHOR]

Human visceral leishmaniasis (HVL) is a neglected disease that occurs in 98 countries on five continents, and it is endemic in tropical and subtropical regions. In South America, the etiological agent of HVL is Leishmania infantum (syn. Leishmania chagasi), mainly transmitted through the bite of an infected sandfly female from the genus Lutzomyia. In American HVL endemic areas, the occurrence of asymptomatic infection is common, which contributes to the possibility of L. infantum transmission during a blood transfusion. To know the prevalence of L. infantum asymptomatic infection in blood donors from the microregion of Adamantina, we investigated 324 peripheral blood samples from donors through immunofluorescence (IFAT) and PCR–RFLP techniques. Seven blood samples (2.16%) tested positive for Leishmania by IFAT, and from those, six presented positive results by PCR (85.71%), which were later identified as L. infantum by RFLP. The presence of L. infantum in the peripheral blood of blood donors supported the hypothesis of transmission by blood transfusion and points to the need to include tests for visceral leishmaniasis in blood bank screening tests and pre-storage measures, especially in endemic areas to prevent the exponential increase of HVL by blood transfusion. [ABSTRACT FROM AUTHOR]

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To investigate the prevalence and clinical significance of the dense fine speckled (DFS) pattern in a large‐scale cohort of Chinese patients. Data on the antinuclear antibody (ANA) and extractable nuclear antigen (ENA) autoantibody obtained from 165 498 patients who attended Peking Union Medical College Hospital from January 2019 to December 2021 were retrospectively analysed. The prevalence of the DFS pattern was 1.14%, and it mainly appeared in young patients (≤24 years old). A higher positive rate of the DFS pattern was observed in patients with dermatosis (18.12%) and systemic autoimmune rheumatic diseases (SARDs) (13.53%). The DFS pattern titre was mostly low or medium (≤1:160). A higher titre was associated with an increased risk of SARDs (p <.001), dermatosis (p =.015) and pulmonary disease (p =.016). In 37 patients with ENA autoantibody positivity, anti‐SSA antibody was the most common (2.55%). If the low titre (<1:160) or the DFS pattern with negative ENA autoantibody were to be used as exclusion criteria for SARDs, the diagnosis would have been missed in 42 or 77 patients, respectively. The prevalence of the DFS pattern was low in ANA test samples and was more common in patients with dermatosis and SARDs, but the titre was usually higher in patients with SARDs. There was no evidence that the DFS pattern could be used as an exclusion criterion for SARDs diagnosis. The DFS pattern was associated with certain pathological states, which may inform the clinical significance of the DFS pattern. [ABSTRACT FROM AUTHOR]

Bovine parainfluenza virus 3 (BPIV3) is one of several viruses that contribute to bovine respiratory disease complex (BRDC). During this study, isolation of BPIV3 was attempted from 20 PCR-positive swabs by Madin-Darby Bovine Kidney (MDBK) cells. Nine samples showed obvious cytopathic lesions identified as BPIV3 by reverse-transcription polymerase chain reaction amplification and sequencing. The genomes of isolates XJ21032-1 and XJ20055-3 were sequenced using Illumina sequencing technology and determined to have lengths of 15,512 bp and 15,479 bp, respectively. Phylogenetic analysis revealed that isolate XJ21032-1 was genotype B, and isolate XJ20055-3 was genotype C. In addition, the two isolates had multiple amino acid changes in nucleocapsid protein, fusion protein, and hemagglutinin/neuraminidase, major antigenic proteins. This allows the further recognition of the presence of BPIV3 type B in Chinese cattle herds. We hope this will help trace the origin of BPIV3, improve the understanding of differences between genotypes, and provide data support for vaccine development. [ABSTRACT FROM AUTHOR]

Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued by the International Autoimmune Hepatitis Group. They also allow the distinction between type 1 AIH, characterized by positive anti-nuclear and/or anti-smooth muscle antibody, and type 2 AIH, characterized by positive anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. Anti-soluble liver antigen is the only AIH-specific autoantibody, and is found in 20–30% of both type 1 and type 2 AIH. Anti-neutrophil cytoplasmic antibody is frequently positive in type 1 AIH, being associated also with inflammatory bowel disease and with primary/autoimmune sclerosing cholangitis. The reference method for autoantibody testing remains indirect immunofluorescence on triple tissue (rodent liver, kidney and stomach), allowing both the detection of the majority of liver-relevant reactivities, including those autoantibodies whose molecular target antigens are unknown. Of note, the current knowledge of the clinical significance of autoantibodies relies on studies based on this technique. However, immunofluorescence requires trained laboratory personnel, is observer-dependent, and lacks standardization, leading to ongoing attempts at replacing this method with automated assays, the sensitivity, and specificity of which, however, require further studies before they can be used as a reliable alternative to immunofluorescence; currently, they may be used as complementary to immunofluorescence. [ABSTRACT FROM AUTHOR]

The laboratory diagnostics of primary biliary cholangitis (PBC) have substantially improved, thanks to innovative analytical opportunities, such as enzyme-linked immunosorbent assays (ELISA) and multiple immunodot liver profile tests, based on recombinant or purified antigens. This study aimed to identify the best diagnostic test combination to optimize PBC diagnosis. Between January 2014 and March 2017, 164 PBC patients were recruited at the hospitals of Parma, Modena, Reggio-Emilia, and Piacenza. Antinuclear antibodies (ANA) and anti-mitochondrial antibodies (AMA) were assayed by indirect immunofluorescence (IIF), ELISA, and immunodot assays (PBC Screen, MIT3, M2, gp210, and sp100). AMA-IIF resulted in 89.6% positive cases. Using multiple immunodot liver profiles, AMA-M2 sensitivity was 94.5%, while anti-gp210 and anti-sp100 antibodies were positive in 16.5% and 17.7% of patients, respectively. PBC screening yielded positive results in 94.5% of cases; MIT3, sp100, and gp210 were detected by individual ELISA test in 89.0%, 17.1%, and 18.9% of patients, respectively. The association of PBC screening with IIF-AMA improved the diagnostic sensitivity from 89.6% to 98.2% (p < 0.01). When multiple immunodot liver profile testing was integrated with AMA-IIF, the diagnostic sensitivity increased from 89.1% to 98.8% (p < 0.01). The combination of IIF with solid-phase methods significantly improved diagnostic efficacy in PBC patients. [ABSTRACT FROM AUTHOR]

Objective: The primary aim of this study was to conduct a meta‐analysis to compare the diagnostic accuracy of automated indirect immunofluorescence (automated‐IIF) and fully automated solid‐phase immunoassays (solid‐phase assays), compared with gold standard conventional manual indirect immunofluorescence (manual‐IIF) for antinuclear antibody (ANA) detection. Methods: Indirect meta‐comparison was performed using prospective studies reporting comparative data between automated‐IIF and fully automated solid‐phase assays individually to conventional manual‐IIF. Diagnostic tests regarding different automated solid‐phase assays and automated‐IIF for ANA detection were retrieved from the Cochrane Library, PubMed, Embase, Web of Science, Chinese Biological Medicine Database (CBM), China National Knowledge Infrastructure (CNKI), and WANFANG electronic databases from their inception to January 2022. Assessment of the quality of the studies was undertaken using a second version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS‐2) tool. The investigated diagnostic indices including pooled sensitivity, pooled specificity, pooled positive likelihood ratio (PLR), pooled negative likelihood ratio (NLR), pooled diagnostic odds ratio (DOR), area under the summary receiver operating characteristic (AUC) of automated‐IIF and solid‐phase assays, respectively. Relative diagnostic odds ratio (RDOR) was calculated to indirectly compare the diagnostic accuracy of automated‐IIF and solid‐phase assays. To visualize results, we provide forest plots showing the RDOR with 95% confidence intervals (CI) of the 2 methods against the "gold standard" manual‐IIF by R software. Deeks' funnel was used to investigate the publication bias. Results: A total of 16 studies involving 6111 subjects were included in the analysis. The pooled sensitivity, pooled specificity, pooled PLR, pooled NLR, pooled DOR and the AUC were 0.85 (95% CI: 0.84‐0.86), 0.82 (95% CI 0.81‐0.84), 14.22 (95% CI 8.55‐23.65), 0.06 (95% CI 0.03‐0.12), 287.0 (95% CI 124.30‐662.68) and 0.983 for automated‐IIF respectively, and as for solid‐phase assays those were 0.73 (95% CI 0.70‐0.75), 0.87 (95% CI 0.85‐0.89), 5.66 (95% CI 3.33‐9.62), 0.30 (95% CI 0.20‐0.47), 19.14 (95% CI 8.00‐45.79) and 0.894. The results of indirect comparison indicated that automated‐IIF had statistically significant higher accuracy for the detection of ANA. Conclusion: This meta‐analysis and indirect comparison suggest that automated‐IIF should be recommended as an alternative assay for ANA screening under the condition of increased demand for ANA testing in clinical immunology laboratories. [ABSTRACT FROM AUTHOR]

Les profils d'immunofluorescence indirecte sur cellules HEp2 évocateurs d'anticorps anti-vimentine sont considérés comme non pertinents d'un point de vue clinique. Des études datant des années 1980 avaient en effet démontré qu'ils s'observaient dans divers contextes cliniques, pathologiques ou non. Leur relevance clinique mérite cependant une réévaluation contemporaine, eu égard aux dilutions sériques initiales et aux conjugués qui ont évolué au fil des décennies. Sur 5 083 recherches d'anticorps anti-nucléaires effectuées de septembre à décembre 2019 au centre hospitalier universitaire Lyon-sud, 35 sérums (0,69 %) ont donné un aspect de fluorescence évocateur d'anticorps anti-vimentine. Les données cliniques étaient disponibles pour 34 patients, 21 hommes (62 %) et 13 femmes (38 %), d'âge moyen 59 ans (25-80 ans). Quinze patients (44 %) présentaient un cancer (6 sur 15) et/ou une hémopathie maligne (9 sur 15) à type de lymphome (4 cas), de myélofibrose (2 cas), de thrombocytémie essentielle (2 cas), ou de leucémie myéloïde chronique (1 cas). Treize patients (38 %) présentaient une pathologie cardiovasculaire (4 cas de thrombose veineuse profonde/embolie pulmonaire, 5 cas d'atteinte cardiaque avec élévation de la troponine, 3 cas d'accident vasculaire cérébral, 1 cas d'artériopathie oblitérante des membres inférieurs). Ces résultats préliminaires suggèrent une association entre ces aspects de fluorescence et deux entités nosologiques : hémopathies malignes/cancers et pathologies cardiovasculaires graves. Indirect immunofluorescence patterns on HEp2 cells suggestive of anti-vimentin antibodies are considered as irrelevant clinically. Indeed, initial studies demonstrated that they were observed in various clinical contexts, pathological or not. Their clinical relevance, however, should be reevaluated, given the initial serum dilutions and conjugates that have evolved over the decades. Out of 5083 anti-nuclear antibodies analyzes carried out from September to December 2019 at the CHU Lyon-Sud, 35 sera (0,69 %) gave a fluorescence staining suggestive of anti-vimentin antibodies. Clinical data were available for 34 patients, 21 men (62 %) and 13 women (38 %), mean age 59 years (25-80 years). Fifteen patients (44 %) had cancer (6 out of 15) and/or hematological malignancy (9 out of 15) such as lymphoma (4 cases), myelofibrosis (2 cases), essential thrombocythemia (2 cases), or chronic myeloid leukemia (1 case). Thirteen patients (38 %) had cardiovascular disease (4 cases of deep vein thrombosis/pulmonary embolism, 5 cases of cardiac disease with elevated troponin, 3 cases of stroke, 1 case of obliterating arteriopathy of the lower limbs). These preliminary results suggest an association between these aspects of fluorescence and two nosological entities: hematological malignancies/cancers and severe cardiovascular diseases. [ABSTRACT FROM AUTHOR]

P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Wang, Chen, Ma, Yin, Yang, Liu and Du.)

Background Antineutrophil cytoplasmic antibodies (ANCA) are important biomarkers in the diagnosis of ANCA-associated vasculitis, and indirect immunofluorescence (IIF) had been the method of choice for its detection from the very beginning. However, international consensus on ANCA testing (2017) advocates the use of high-quality immunoassays as the primary screening method. The purpose of this study was to evaluate the diagnostic performance of enzyme-linked immunosorbent assay (ELISA) compared to IIF in detecting ANCA. Methods One-hundred eighty-nine serum samples of suspected or known cases of systemic vasculitis were screened for ANCA by IIF and proteinase-3- and myeloperoxidase-ELISA. In IIF, positive results were further divided into cytoplasmic pattern of ANCA and perinuclear pattern of ANCA, depending upon the pattern of fluorescence. McNemar’s chi-squared test was applied to check the equality of proportions of positive results, and Kappa statistics was used to measure the agreement between the two methods. Diagnostic performance of ELISA was evaluated taking IIF as reference. Results IIF detected ANCA in 17.5% cases and ELISA detected it in 11.6% cases. A good agreement between the overall performance of ELISA and IIF was observed (K-value: 6.8, p-value: < 001). However, a significant difference in the proportion of positive results by the two methods was observed in McNemar’s test (two-sided p-value: 0.007). Taking IIF as standard, ELISA showed 60.6% sensitivity, 98.7% specificity, and predictive value of positive and negative results of 90.9 and 92.2%, respectively. Conclusion The new generation antigen-specific ELISAs had high specificity but the chances of missing cases in primary screening due to the low sensitivity and high false negativity (39.4%) need to be dealt with.

Parasites of the genus Sarcocystis can infect several species of animals and cause multiple diseases such as equine protozoal myeloencephalitis. Felines are considered hosts of this protozoa; therefore, the present study aimed to detect anti-Sarcocystis spp.–specific antibodies in domestic cats that were under clinical evaluation, using the indirect immunofluorescence antibody test. Anti-Sarcocystis-specific immunoglobulin Gs were detected in 24 out of 497 (4.82%) cat serum samples. These findings support the fact that natural Sarcocystis infections do occur in cats. Furthermore, it highlights the importance of domestic cats as both intermediate and definitive hosts in the Sarcocystis life cycle, maintaining the parasite and serving as a source of infection for various other animals. To the best of our knowledge, this is the first study to identify antibodies against the genus Sarcocystis in cats from a region in southern Brazil. [ABSTRACT FROM AUTHOR]

Autoimmune diseases are pathological conditions in which the immune system mistakenly attacks its own tissues. This study evaluates the performance of two techniques, which are identifiers of autoantibody specifics: immunoblot and immunodot. This study was conducted in 300 patients of whom 62 were tested positive for antinuclear antibodies. The patients were initially screened for antinuclear antibodies using indirect immunofluorescence. Then, the identification of specific autoantibodies such as anti-extractable nuclear antigens (ENAs) was carried out using the immunoblot and immunodot techniques. The results showed that immunoblot and immunodot did not present a significant difference in their sensitivity against anti-SSA/52, SSB, CENP-B, PCNA, U1-snRNP, Jo-1, Pm-scl, and Mi-2 (p > 0.05). However, the two techniques showed a significant difference in their sensitivity toward autoantibodies anti-DNAn, anti-histone, anti-SmD1, and anti-ds-DNA (p < 0.05). The immunoblot data were in complete accordance with the immunodot data (100%) regarding the detection of autoantibodies such as anti SSA/52, SSB, CENP-B, PCNA, U1-snRP, Jo-1, Pm-scl, and Mi-2, 80% regarding SmD1, and 75% concerning ds-DNA. We should certainly pay closer attention to the efficiency of the techniques used in the diagnosis of autoimmune diseases.

QseC is a membrane sensor kinase that enables bacteria to perceive autoinducers −3, adrenaline, and norepinephrine to initiate downstream gene transcription. In this study, we found that the QseC protein of Glaesserella parasuis can serve as an effective antigen to activate the host's immune response. Therefore, we investigated the immunogenicity and host protective effect of this protein. ELISA and indirect immunofluorescence results showed that QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. We used MTS reagents to detect lymphocyte proliferation levels and found that QseC protein can cause splenic lymphocyte proliferation with memory and specificity. Further immunological analysis of the spleen cell supernatant revealed significant upregulation of levels of IL-1β, IL-4 and IFN-γ in the QseC + adjuvant group. In the mouse challenge experiment, it was found that QseC + adjuvant can provide effective protection. The results of this study demonstrate that QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research. • QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. • QseC activated splenic lymphocyte proliferation and exhibit memory and specificity. • QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research. [ABSTRACT FROM AUTHOR]