Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

OBJECTIVE: Antidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly sensitive methods, such as solid-phase immunoassays, and highly specific methods, such as the Crithidia luciliae indirect immunofluorescence test (CLIFT). Even so, discordant results are common, thus hindering the diagnostic process. Therefore, this study aimed to characterise a cohort of patients with discrepant results for a dsDNA fluorescence enzyme immunoassay (FEIA) and CLIFT during 2016-2018 and to follow patients up until December 2021. METHODS: We performed an observational, longitudinal and retrospective study on 417 samples from 257 patients who had been referred for suspected connective tissue diseases or followed up after diagnosis. All of them were positive for antinuclear antibodies (ANAs) using an indirect immunofluorescence assay (IFA) on Hep-2 cells, the entry criterion in our laboratory, and positive for FEIA dsDNA. Samples were then tested with CLIFT according to our routine protocol, which includes CLIFT testing after FEIA dsDNA results ≥10 UI/ml. After the assessment of data quality, the final analysis was based on 222 patients. RESULTS: Eighty-three patients (37.4%) had positive results in both tests and met the diagnostic criteria for SLE. However, 139 patients (62.6%) had discrepant results (FEIA+, CLIFT-). Of these, 58 patients (41.7%) had a diagnosis of SLE, with 47 (33.8%) having been previously diagnosed and under treatment. The remaining 11 patients (7.9%) had a new diagnosis of SLE, which was made up within 4 years of the initial screening. A total of 81 of the 139 patients (57.5%) with discrepant results did not meet lupus criteria during the follow-up period. CONCLUSIONS: The study showed that CLIFT could be negative in both treated and newly diagnosed SLE, thus underlining the importance of follow-up of dsDNA-positive results using solid-phase tests. Therefore, quantitative tests such as FEIA could add value to the diagnosis and management of patients with suspected SLE.

A Straightforward Cytometry-Based Protocol for the Comprehensive Analysis of the Inflammatory Valve Infiltrate in Aortic Stenosis.

Aortic stenosis (AS) is a frequent cardiac disease in old individuals, characterized by valvular calcification, fibrosis, and inflammation. Recent studies suggest that AS is an active inflammatory atherosclerotic-like process. Particularly, it has been suggested that several immune cell types, present in the valve infiltrate, contribute to its degeneration and to the progression toward stenosis. Furthermore, the infiltrating T cell subpopulations mainly consist of oligoclonal expansions, probably specific for persistent antigens. Thus, the characterization of the cells implicated in the aortic valve calcification and the analysis of the antigens to which those cells respond to is of utmost importance to develop new therapies alternative to the replacement of the valve itself. However, calcified aortic valves have been only studied so far by histological and immunohistochemical methods, unable to render an in-depth phenotypical and functional cell profiling. Here we present, for the first time, a simple and efficient cytometry-based protocol that allows the identification and quantification of infiltrating inflammatory leukocytes in aortic valve explants. Our cytometry protocol saves time and facilitates the simultaneous analysis of numerous surface and intracellular cell markers and may well be also applied to the study of other cardiac diseases with an inflammatory component.

Case Report: Successful Response to Intravenous Immunoglobulin and Steroid Pulses in a Renal Transplant Recipient With Severe Covid-19 Disease and Associated Acute Allograft Failure.

The impact of Covid-19 pneumonia caused by SARS-CoV-2 on transplanted populations under chronic immunosuppression seems to be greater than in normal population. Clinical management of the disease, particularly in those patients worsening after a cytokine storm, with or without allograft impairment and using available therapeutic approaches in the absence of specific drugs to fight against the virus, involves a major challenge for physicians. We herein provide evidence of the usefulness of high-dose intravenous immunoglobulin (IVIG) combined with steroid pulses to successfully treat a case of Covid-19 pneumonia in a single-kidney transplanted patient with mechanical ventilation and hemodialysis requirements in the setting of a cytokine storm. A rapid decrease in the serum level of inflammatory cytokines, particularly IL-6, IL-8, TNF-α, MCP-1 and IL-10, as well as of acute-phase reactants such as ferritin, D-dimer and C-reactive protein was observed after the IVIG infusion and methylprednisolone bolus administration with a parallel clinical improvement and progressive allograft function recovery, allowing the patient's final discharge 40 days after the treatment onset. The immunomodulatory effect of IVIG together with the anti-inflammatory and immunosuppressive potential of steroids could be an alternative strategy to treat severe cases of Covid-19 pneumonia associated with an uncontrolled inflammatory response in transplanted populations.