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BACKGROUND: Mechanisms underlying neurodegeneration in multiple sclerosis (MS) remain poorly understood but mostly implicate molecular pathways that are not unique to MS. Recently detected tau seeding activity in MS brain tissues corroborates previous neuropathological reports of hyperphosphorylated tau (p-tau) accumulation in secondary and primary progressive MS (PPMS). We aimed to investigate whether aberrant tau phosphorylation can be detected in the cerebrospinal fluid (CSF) of MS patients by using novel ultrasensitive immunoassays for different p-tau biomarkers. METHODS: CSF samples of patients with MS (n = 55) and non-inflammatory neurological disorders (NIND, n = 31) were analysed with in-house Single molecule array (Simoa) assays targeting different tau phosphorylation sites (p-tau181, p-tau212, p-tau217 and p-tau231). Additionally, neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) were measured with a multiplexed Simoa assay. Patients were diagnosed with clinically isolated syndrome (CIS, n = 10), relapsing-remitting MS (RRMS, n = 21) and PPMS (n = 24) according to the 2017 McDonald criteria and had MRI, EDSS and basic CSF analysis performed at the time of diagnosis. RESULTS: Patients with progressive disease course had between 1.4-fold (p-tau217) and 2.2-fold (p-tau212) higher p-tau levels than relapsing MS patients (PPMS compared with CIS + RRMS, p < 0.001 for p-tau181, p-tau212, p-tau231 and p = 0.042 for p-tau217). P-tau biomarkers were associated with disease duration (ρ=0.466-0.622, p < 0.0001), age (ρ=0.318-0.485, p < 0.02, all but p-tau217) and EDSS at diagnosis and follow-up (ρ=0.309-0.440, p < 0.02). In addition, p-tau biomarkers correlated with GFAP (ρ=0.517-0.719, p ≤ 0.0001) but not with the albumin quotient, CSF cell count or NFL. Patients with higher MRI lesion load also had higher p-tau levels p ≤ 0.01 (<10 vs. ≥ 10 lesions, all p ≤ 0.01). CONCLUSION: CSF concentrations of novel p-tau biomarkers point to a higher degree of tau phosphorylation in PPMS than in RRMS. Associations with age, disease duration and EDSS suggest this process increases with disease severity; however, replication of these results in larger cohorts is needed to further clarify the relevance of altered tau phosphorylation throughout the disease course in MS.
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OBJECTIVES: Recently published 2023 ACR/EULAR APS classification criteria emphasize the importance of quantifying single-, double-, and triple-antiphospholipid antibody positivity, distinguishing between IgG and IgM isotypes, and delineating moderate/high levels of anticardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies. We aimed to establish clinically important moderate/high thresholds for aCL and anti-ß2GPI IgG/IgM chemiluminescent immunoassays (CLIA), in particular QUANTA Flash, comparable to our in-house ELISAs used for over two decades, and to evaluate their diagnostic performance. METHODS: QUANTA Flash CLIA and in-house ELISAs were used to measure aCL and anti-ß2GPI IgG/IgM. Moderate thresholds for QUANTA Flash CLIA were determined using a non-parametric approach, calculating a 99th percentile on serum samples from 139 blood donors, and by mirroring the diagnostic performance of in-house ELISA on 159 patient samples. RESULTS: Thresholds for QUANTA Flash CLIA achieving diagnostic performance equivalent to in-house ELISAs were 40â¯CU for moderate and 80â¯CU for high levels for aCL and anti-ß2GPI IgG and IgM. The assays showed good qualitative agreement, ranging from 76.10 to 91.19â¯%. When considering in-house ELISA results, 14 out of 80 (17.5â¯%) patients did not fulfill the new ACR/EULAR laboratory classification criteria, while 27 out of 80 (33.8â¯%) did not when considering QUANTA Flash CLIA results. CONCLUSIONS: We determined moderate and high thresholds for aCL and anti-ß2GPI IgG and IgM detected with QUANTA Flash CLIA, aligning with long-established in-house ELISA thresholds. These thresholds are crucial for seamlessly integrating of the new 2023 ACR/EULAR classification criteria into future observational clinical studies and trials.
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INTRODUCTION: The established cerebrospinal fluid (CSF) phosphorylated tau181 (p-tau181) may not reliably reflect concomitant Alzheimer's disease (AD) and primary age-related tauopathy (PART) found in Creutzfeldt-Jakob disease (CJD) at autopsy. METHODS: We investigated CSF N-terminal p-tau181, p-tau217, and p-tau231 with in-house Simoa assays in definite CJD (n = 29), AD dementia (n = 75), mild cognitive impairment (MCI) due to AD (n = 65), and subjective cognitive decline (SCD, n = 28). Post-mortem examination performed in patients with CJD 1.3 (0.3-14.3) months after CSF collection revealed no co-pathology in 10, concomitant AD in 8, PART in 8, and other co-pathologies in 3 patients. RESULTS: N-terminal p-tau was increased in CJD versus SCD (p < 0.0001) and correlated with total tau (t-tau) in the presence of AD and PART co-pathology (rho = 0.758-0.952, p ≤ 001). Concentrations in CJD+AD were indistinguishable from AD dementia, with the largest fold-change in p-tau217 (11.6), followed by p-tau231 and p-tau181 (3.2-4.5). DISCUSSION: Variable fold-changes and correlation with t-tau suggest that p-tau closely associates with neurodegeneration and concomitant AD in CJD. HIGHLIGHTS: N-terminal phosphorylated tau (p-tau) biomarkers are increased in Creutzfeldt-Jakob disease (CJD) with and without concomitant AD. P-tau217, p-tau231, and p-tau181 correlate with total tau (t-tau) and increase in the presence of amyloid beta (Aß) co-pathology. N-terminal p-tau181 and p-tau231 in Aß-negative CJD show variation among PRNP genotypes. Compared to mid-region-targeting p-tau181, cerebrospinal fluid (CSF) N-terminal p-tau has greater potential to reflect post-mortem neuropathology in the CJD brain.
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In this study, we optimized the dissociation of synovial tissue biopsies for single-cell omics studies and created a single-cell atlas of human synovium in inflammatory arthritis. The optimized protocol allowed consistent isolation of highly viable cells from tiny fresh synovial biopsies, minimizing the synovial biopsy drop-out rate. The synovium scRNA-seq atlas contained over 100,000 unsorted synovial cells from 25 synovial tissues affected by inflammatory arthritis, including 16 structural, 11 lymphoid, and 15 myeloid cell clusters. This synovial cell map expanded the diversity of synovial cell types/states, detected synovial neutrophils, and broadened synovial endothelial cell classification. We revealed tissue-resident macrophage subsets with proposed matrix-sensing (FOLR2+COLEC12high) and iron-recycling (LYVE1+SLC40A1+) activities and identified fibroblast subsets with proposed functions in cartilage breakdown (SOD2highSAA1+SAA2+SDC4+) and extracellular matrix remodeling (SERPINE1+COL5A3+LOXL2+). Our study offers an efficient synovium dissociation method and a reference scRNA-seq resource, that advances the current understanding of synovial cell heterogeneity in inflammatory arthritis.
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Blood phosphorylated tau (p-tau) biomarkers, including p-tau217, show high associations with Alzheimer's disease (AD) neuropathologic change and clinical stage. Certain plasma p-tau217 assays recognize tau forms phosphorylated additionally at threonine-212, but the contribution of p-tau212 alone to AD is unknown. We developed a blood-based immunoassay that is specific to p-tau212 without cross-reactivity to p-tau217. Here, we examined the diagnostic utility of plasma p-tau212. In five cohorts (n = 388 participants), plasma p-tau212 showed high performances for AD diagnosis and for the detection of both amyloid and tau pathology, including at autopsy as well as in memory clinic populations. The diagnostic accuracy and fold changes of plasma p-tau212 were similar to those for p-tau217 but higher than p-tau181 and p-tau231. Immunofluorescent staining of brain tissue slices showed prominent p-tau212 reactivity in neurofibrillary tangles that co-localized with p-tau217 and p-tau202/205. These findings support plasma p-tau212 as a peripherally accessible biomarker of AD pathophysiology.
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Doença de Alzheimer , Humanos , Doença de Alzheimer/diagnóstico , Neuropatologia , Plasma , Emaranhados Neurofibrilares , Autopsia , Proteínas tau , Biomarcadores , Peptídeos beta-AmiloidesRESUMO
OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. METHODS: An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. RESULTS: The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8â¯% and Cohen's kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7â¯%, 95.0â¯%, and 0.937; capture ELISA 92.3â¯%, 98.3â¯%, and 0.939; and QUANTA Flash 89.7â¯%, 95.9â¯%, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0-8 CU had LR 0.08, 8-29 CU had LR 1.03, 29-121 CU had LR 7.76, 121-191 CU had LR 12.4, and >191 CU had LR ∞. CONCLUSIONS: The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results.
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Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Humanos , Mieloblastina , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , PeroxidaseRESUMO
The detection of cryoglobulins (CG) used to diagnose cryoglobulinemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in patients` serum and a decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians.
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Antiphospholipid antibodies (aPLA) are a laboratory criterion for the classification of antiphospholipid syndrome (APS) and are known to cause clinical symptoms such as vascular thrombosis or obstetric complications. It is suggested that aPLA may be associated with thromboembolism in severe COVID-19 cases. Therefore, we aimed to combine clinical data with laboratory findings of aPLA at four time points (admission, worsening, discharge, and 3-month follow-up) in patients hospitalized with COVID-19 pneumonia. In 111 patients with COVID-19 pneumonia, current and past history of thrombosis and pregnancy complications were recorded. Nine types of aPLA were determined at four time points: anticardiolipin (aCL), anti-ß2-glycoprotein I (anti- ß2GPI), and antiphosphatidylserine/prothrombin (aPS/PT) of the IgM, IgG, or IgA isotypes. During hospitalization, seven patients died, three of them due to pulmonary artery thromboembolism (none were aPLA positive). Only one of the five who developed pulmonary artery thrombosis was aPLA positive. Out of 9/101 patients with a history of thrombosis, five had arterial thrombosis and none were aPLA positive at admission and follow-up; four had venous thrombosis, and one was aPLA positive at all time points (newly diagnosed APS). Of these 9/101 patients, 55.6% were transiently aPLA positive at discharge only, compared to 26.1% without a history of thrombosis (p = 0.041). Patients with severe forms of COVID-19 and positive aPLA should receive the same dose and anticoagulant medication regimen as those with negative aPLA because those antibodies are mostly transiently positive and not linked to thrombosis and fatal outcomes.
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Alzheimer's disease (AD) is the most common neurodegenerative disease, with a complex genetic background. Apart from rare, familial cases, a combination of multiple risk loci contributes to the susceptibility of the disease. Genome-wide association studies (GWAS) have identified numerous AD risk loci. Changes in cerebrospinal fluid (CSF) biomarkers and imaging techniques can detect AD-related brain changes before the onset of clinical symptoms, even in the presence of preclinical mild cognitive impairment. In this study, we aimed to assess the associations between SNPs in well-established GWAS AD risk loci and CSF biomarker levels or cognitive test results in Slovenian patients with cognitive decline. The study included 82 AD patients, 28 MCI patients with pathological CSF biomarker levels and 35 MCI patients with normal CSF biomarker levels. Carriers of at least one polymorphic TOMM40 rs157581 C allele had lower Aß42 (p = 0.033) and higher total tau (p = 0.032) and p-tau181 levels (p = 0.034). Carriers of at least one polymorphic T allele in SORCS1 rs1358030 had lower total tau (p = 0.019), while polymorphic SORCS1 rs1416406 allele was associated with lower total tau (p = 0.013) and p-tau181 (p = 0.036). In addition, carriers of at least one polymorphic T allele in BCHE rs1803274 had lower cognitive test scores (p = 0.029). The study findings may contribute to the identification of genetic markers associated with AD and MCI and provide insights into early disease diagnostics.
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Doença de Alzheimer , Disfunção Cognitiva , Doenças Neurodegenerativas , Humanos , Estudo de Associação Genômica Ampla , Biomarcadores , Disfunção Cognitiva/genética , Alelos , Doença de Alzheimer/genéticaRESUMO
The correct balance between reactive oxygen species and antioxidant defense in an organism is disturbed in oxidative stress. To assess oxidative balance in 36 SSc patients and 26 healthy controls (HCs), we measured reactive oxidative metabolites (ROMs), total antioxidant capacity (TAC), lipid peroxidation (measuring 4-HNE), and DNA oxidative damage (measuring 8-OHdG) in serum. Furthermore, DNA breaks in leukocytes of 35 SSc patients and 32 HCs were evaluated using COMET. While we report high ROMs for both SSc patients and age/sex matched HC samples, there was a significant increase in TAC in SSc patients as compared to HCs, and thus also a significantly higher oxidative stress index in SSc patients. TAC was significantly higher in SSc patients with ILD and gastrointestinal involvement, as well as in patients with anti-topoisomerase antibodies. We observe no difference in serum lipid peroxidation status or oxidative DNA damage. However, SSc patients had significantly more leukocyte DNA breaks than HCs; the most damage was observed in patients treated with immunosuppressives. Thus, our study confirms presence of oxidative stress and increased DNA damage in leukocytes of SSc patients; however, it points toward increased antioxidant capacity, which needs to be further studied.
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Studies on the serum biomarkers of granulomatous inflammation and pulmonary interstitial disease in intrathoracic sarcoidosis have shown conflicting results. We postulated that differences in the concentrations of serum biomarkers can be explained by the heterogenous patterns of sarcoidosis seen on thoracic HRCT. Serum biomarker levels in 79 consecutive patients, newly diagnosed with intrathoracic sarcoidosis, were compared to our control group of 56 healthy blood donors. An analysis was performed with respect to HRCT characteristics (the presence of lymph node enlargement, perilymphatic or peribronchovascular infiltrates, ground-glass lesions, or fibrosis), CXR, and disease extent. Serum levels of CXCL9, CXCL10, CTO, and CCL18 were statistically significantly increased in all patients compared to controls. Serum levels of CA15.3 were statistically significantly increased in all patients with parenchymal involvement. SAA was increased in patients with ground-glass lesions while SP-D levels were statistically significantly increased in patients with lung fibrosis. Only SP-D and CA15.3 showed a significant correlation to interstitial disease extent. In conclusion, we found that sarcoidosis patients with different HRCT patterns of intrathoracic sarcoidosis have underlying biochemical differences in their serum biomarkers transcending Scadding stages. The stratification of patients based on both radiologic and biochemical characteristics could enable more homogenous patient selection for further prognostic studies.
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Doenças Pulmonares Intersticiais , Sarcoidose , Humanos , Proteína D Associada a Surfactante Pulmonar , Doenças Pulmonares Intersticiais/patologia , Sarcoidose/diagnóstico por imagem , Pulmão/patologia , Tomografia Computadorizada por Raios X , BiomarcadoresRESUMO
Oxidative stress and neuroinflammation are important processes involved in Alzheimer's disease (AD) and mild cognitive impairment (MCI). Numerous risk factors, including genetic background, can affect the complex interplay between those mechanisms in the aging brain and can also affect typical AD hallmarks: amyloid plaques and neurofibrillary tangles. Our aim was to evaluate the association of polymorphisms in oxidative stress- and inflammation-related genes with cerebrospinal fluid (CSF) biomarker levels and cognitive test results. The study included 54 AD patients, 14 MCI patients with pathological CSF biomarker levels, 20 MCI patients with normal CSF biomarker levels and 62 controls. Carriers of two polymorphic IL1B rs16944 alleles had higher CSF Aß1-42 levels (p = 0.025), while carriers of at least one polymorphic NFE2L2 rs35652124 allele had lower CSF Aß1-42 levels (p = 0.040). Association with IL1B rs16944 remained significant in the AD group (p = 0.029). Additionally, MIR146A rs2910164 was associated with Aß42/40 ratio (p = 0.043) in AD. Significant associations with cognitive test scores were observed for CAT rs1001179 (p = 0.022), GSTP1 rs1138272 (p = 0.005), KEAP1 rs1048290 and rs9676881 (both p = 0.019), as well as NFE2L2 rs35652124 (p = 0.030). In the AD group, IL1B rs1071676 (p = 0.004), KEAP1 rs1048290 and rs9676881 (both p = 0.035) remained associated with cognitive scores. Polymorphisms in antioxidative and inflammation genes might be associated with CSF biomarkers and cognitive test scores and could serve as additional biomarkers contributing to early diagnosis of dementia.
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OBJECTIVE: To develop EULAR points-to-consider for therapeutic drug monitoring (TDM) of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases (RMDs). METHODS: The points-to-consider were developed in accordance with EULAR standardised operation procedures by a multidisciplinary task force from eight European countries, based on a systematic literature review and expert consensus. Level of evidence and strength of the points-to-consider were determined, and mean levels of agreement among the task force were calculated using a 10-point rating scale. RESULTS: Six overarching principles and 13 points-to-consider were formulated. The level of agreement among the task force for the overarching principles and points-to-consider ranged from 8.4 to 9.9.The overarching principles define TDM and its subtypes, and reinforce the underlying pharmacokinetic/pharmacodynamic principles, which are relevant to all biopharmaceutical classes. The points-to-consider highlight the clinical utility of the measurement and interpretation of biopharmaceutical blood concentrations and antidrug antibodies in specific clinical scenarios, including factors that influence these parameters. In general, proactive use of TDM is not recommended but reactive TDM could be considered in certain clinical situations. An important factor limiting wider adoption of TDM is the lack of both high quality trials addressing effectiveness and safety of TDM and robust economic evaluation in patients with RMDs. Future research should focus on providing this evidence, as well as on further understanding of pharmacokinetic and pharmacodynamic characteristics of biopharmaceuticals. CONCLUSION: These points-to-consider are evidence-based and consensus-based statements for the use of TDM of biopharmaceuticals in inflammatory RMDs, addressing the clinical utility of TDM.
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Produtos Biológicos , Doenças Musculoesqueléticas , Doenças Reumáticas , Humanos , Produtos Biológicos/uso terapêutico , Monitoramento de Medicamentos , Doenças Musculoesqueléticas/tratamento farmacológico , Anticorpos , Europa (Continente) , Doenças Reumáticas/tratamento farmacológicoRESUMO
Blood phosphorylated tau (p-tau) biomarkers, including p-tau217, show high associations with Alzheimer's disease (AD) neuropathologic change and clinical stage. Certain plasma p-tau217 assays recognize tau forms phosphorylated additionally at threonine-212, but the contribution of p-tau212 alone to AD is unknown. We developed a blood-based immunoassay that is specific to p-tau212 without cross-reactivity to p-tau217. Thereafter, we examined the diagnostic utility of plasma p-tau212. In five cohorts (n=388 participants), plasma p-tau212 showed high performances for AD diagnosis and for the detection of both amyloid and tau pathology, including at autopsy as well as in memory clinic populations. The diagnostic accuracy and fold changes of plasma p-tau212 were similar to those for p-tau217 but higher than p-tau181 and p-tau231. Immunofluorescent staining of brain tissue slices showed prominent p-tau212 reactivity in neurofibrillary tangles that co-localized with p-tau217 and p-tau202/205. These findings support plasma p-tau212 as a novel peripherally accessible biomarker of AD pathophysiology.
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Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering.
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Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis and/or obstetric complications in the presence of antiphospholipid antibodies (aPL). Catastrophic APS (CAPS) is the most severe form of the disease, in which microvascular thromboses develop rapidly, leading to multiorgan failure. Monocytes, along with endothelial cells, are critical players in the pathogenesis of APS. Recruitment of these cells to the site of injury/inflammation involves a series of events, including capture, rolling, adhesion enhancement, and transmigration, which are controlled by surface adhesion molecules. The aim of our study was to investigate the surface adhesion profile of monocytes from APS patients and monocytes stimulated in vitro with aPL from a CAPS patient. The surface expression of the adhesion molecules LFA1, L-selectin, MAC1, PSGL1, and VLA4 was analyzed by flow cytometry. To our knowledge, this preliminary study was the first to show that VLA4 was significantly increased on the surface of monocytes from APS patients. Moreover, in vitro stimulations mimicking CAPS showed an even greater increase in VLA4. Our data suggest that the surface adhesion profile on monocytes is altered in APS and CAPS and may be involved in the thrombotic pathophysiology of the disease by enhancing monocyte adhesion.
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OBJECTIVES: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context. METHODS: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available. RESULTS: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies. CONCLUSIONS: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity.
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Artrite Reumatoide , Imunoglobulina A , Imunoglobulina M , Fator Reumatoide , Artrite Reumatoide/diagnóstico , Humanos , Imunoglobulina A/química , Imunoglobulina M/química , Peptídeos Cíclicos , Fator Reumatoide/metabolismo , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Commercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays. MATERIALS AND METHODS: This is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays. RESULTS: For both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays. CONCLUSION: Adoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.
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Background: Safe and effective vaccines against COVID-19 are critical for preventing the spread of SARS-CoV-2, but little is known about the humoral immune response more than 9 months after vaccination. We aimed to assess the humoral immune response after the first, second, and third (booster) doses of BNT162b2 vaccine in SARS-CoV-2 naïve and previously infected healthcare professionals (HCP) and the humoral immune response after infection in vaccinated HCP. Methods: We measured anti-spike (anti-S) and anti-nucleocapsid antibodies at different time points up to 12 months in the sera of 300 HCP who had received two or three doses of BNT162b2 vaccine. Mixed-model analyses were used to assess anti-S antibody dynamics and to determine their predictors (age, sex, BMI, and previous infection). Results: Naïve individuals had statistically lower anti-S antibody concentrations after the first dose (median 253 BAU/ml) than previously infected individuals (median 3648 BAU/ml). After the second dose, anti-S antibody concentrations increased in naïve individuals (median 3216 BAU/ml), whereas the second dose did not significantly increase concentrations in previously infected individuals (median 4503 BAU/ml). The third dose resulted in an additional increase in concentrations (median 4844 BAU/ml in naïve and median 5845 BAU/ml in previously infected individuals). Anti-S antibody concentrations steadily decreased after the second dose and after the third dose in naïve and previously infected individuals. In addition, we found that age had an effect on the humoral immune response. Younger individuals had higher anti-S antibody concentrations after the first and second doses. After infection with the new variant Omicron, a further increase in anti-S antibody concentrations to a median value of 4794 BAU/ml was observed in three times vaccinated HCP whose anti-S antibody concentrations were relatively high before infection (median 2141 BAU/ml). Our study also showed that individuals with systemic adverse events achieved higher anti-S antibody concentrations. Conclusion: In this study, significant differences in humoral immune responses to BNT162b2 vaccine were observed between naïve and previously infected individuals, with age playing an important role, suggesting that a modified vaccination schedule should be practiced in previously infected individuals. In addition, we showed that the high anti-S antibodies were not protective against new variants of SARS-CoV-2.
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COVID-19 , Vacinas , Anticorpos Antivirais , Formação de Anticorpos , Vacina BNT162 , Vacinas contra COVID-19 , Atenção à Saúde , Humanos , SARS-CoV-2RESUMO
Brain-derived tau secreted into CSF and blood consists of different N-terminal and mid-domain fragments, which may have a differential temporal course and thus, biomarker potential across the Alzheimer's disease continuum or in other neurological diseases. While current clinically validated total tau assays target mid-domain epitopes, comparison of these assays with new biomarkers targeting N-terminal epitopes using the same analytical platform may be important to increase the understanding of tau pathophysiology. We developed three total tau immunoassays targeting specific N-terminal (NTA and NTB total tau) or mid-region (MR total tau) epitopes, using single molecule array technology. After analytical validation, the diagnostic performance of these biomarkers was evaluated in CSF and compared with the Innotest total tau (and as proof of concept, with N-p-tau181 and N-p-tau217) in three clinical cohorts (n = 342 total). The cohorts included participants across the Alzheimer's disease continuum (n = 276), other dementias (n = 22), Creutzfeldt-Jakob disease (n = 24), acute neurological disorders (n = 18) and progressive supranuclear palsy (n = 22). Furthermore, we evaluated all three new total tau biomarkers in plasma (n = 44) and replicated promising findings with NTA total tau in another clinical cohort (n = 50). In CSF, all total tau biomarkers were increased in Alzheimer's disease compared with controls (P < 0.0001) and correlated with each other (rs = 0.53-0.95). NTA and NTB total tau, but not other total tau assays, distinguished amyloid-positive and amyloid-negative mild cognitive impairment with high accuracies (AUCs 84% and 82%, P < 0.001) matching N-p-tau217 (AUC 83%; DeLong test P = 0.93 and 0.88). All total tau assays were excellent in differentiating Alzheimer's disease from other dementias (P < 0.001, AUCs 89-100%). In Creutzfeldt-Jakob disease and acute neurological disorders, N-terminal total tau biomarkers had significantly higher fold changes versus controls in CSF (45-133-fold increase) than Innotest or MR total tau (11-42-fold increase, P < 0.0001 for all). In progressive supranuclear palsy, CSF concentrations of all total tau biomarkers were similar to those in controls. Plasma NTA total tau concentrations were increased in Alzheimer's disease compared with controls in two independent cohorts (P = 0.0056 and 0.0033), while Quanterix total tau performed poorly (P = 0.55 and 0.44). Taken together, N-terminal-directed CSF total tau biomarkers increase ahead of standard total tau alternatives in the Alzheimer's disease continuum, increase to higher degrees in Creutzfeldt-Jakob disease and acute neurological diseases and show better potential than Quanterix total tau as Alzheimer's disease blood biomarkers. For progressive supranuclear palsy, other tau biomarkers should continue to be investigated.