Multimodal assessment of orbital immune cell infiltration and tissue remodeling during development of graves disease by 1 H19 F MRI.

PURPOSE: To evaluate key molecular and cellular features of Graves orbitopathy (GO) by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling. METHODS: To this end, we utilized a murine model of GO induced by immunization with a human thyroid-stimulating hormone receptor A-subunit plasmid. Altogether, 52 mice were used: 27 GOs and 25 controls (Ctrl) immunized with ß-galactasidose plasmid. From these, 17 GO and 12 Ctrl mice were subjected to multimodal MRI at 9.4T, whereas 23 mice only underwent histology. Beyond anatomical hydrogen-1 (1 H) MRI, we employed transverse relaxation time (T2 ) mapping for visualization of edema, chemical exchange saturation transfer (CEST) for detection of hyaluronan, and fluorine-19 (19 F) MRI for tracking of in situ-labeled immune cells after intravenous injection of perfluorcarbons (PFCs). RESULTS: 1 H/19 F MRI demonstrated substantial infiltration of PFC-loaded immune cells in peri and retro-orbital regions of GO mice, whereas healthy Ctrls showed only minor 19 F signals. In parallel, T2 mapping indicated onset of edema in periorbital tissue and adjacent ocular glands (P = 0.038/0.017), which were associated with enhanced orbital CEST signals in GO mice (P = 0.031). Concomitantly, a moderate expansion of retrobulbar fat (P = 0.029) was apparent; however, no signs for extraocular myopathy were detectable. 19 F MRI-based visualization of orbital inflammation exhibited the highest significance level to discriminate between GO and Ctrl mice (P = 0.006) and showed the best correlation with the clinical score (P = 0.0007). CONCLUSION: The present approach permits the comprehensive characterization of orbital tissue and holds the potential for accurate GO diagnosis in the clinical setting. Magn Reson Med 80:711-718, 2018. © 2018 International Society for Magnetic Resonance in Medicine.

Challenges in the Standardization of Autoantibody Testing: a Comprehensive Review.

Standardization and harmonization are complementary tools to achieve higher testing quality in laboratory medicine. Both are of great relevance and are strongly needed in autoimmune diagnostics, due to the impressive advance in basic research and technological development observed in this diagnostic field in recent years that has led to the introduction of many new tests and new analytical methods. It is, therefore, essential that this strong innovative thrust is translated into clinical practice in a coordinated way to avoid confusion and the risk of potentially harmful errors for the patient. However, while standardization of antibody assays is a very complex task, harmonization of procedures and behaviors is a more feasible target and should necessarily include all the phases of the total testing process-in the pre-analytical phase, appropriateness of test requests, harmonization of autoantibody terminology, and adoption of uniform nomenclature for laboratory tests; in the analytical phase, harmonization of measurements, and sharing of test profiles and diagnostic algorithms; and in the post-analytical phase, harmonization of data reporting, and criteria for interpreting immunoserological results, especially harmonization of units, reference intervals, decision limits, and definition and notification of critical values. We here provide and discuss some examples of harmonization initiatives related to anti-nuclear antibodies, TSH receptor, and anti-thyroid peroxidase antibodies and to antibodies associated with autoimmune hepatitis and with celiac disease. These initiatives could be the starting steps to achieve a wider consensus and a closer interaction among stakeholders in the path of autoimmune diagnostics harmonization to enhance clinical effectiveness and provide greater patient safety.

Comparative Assessment of Female Mouse Model of Graves' Orbitopathy Under Different Environments, Accompanied by Proinflammatory Cytokine and T-Cell Responses to Thyrotropin Hormone Receptor Antigen.

We recently described a preclinical model of Graves' orbitopathy (GO), induced by genetic immunization of eukaryotic expression plasmid encoding human TSH receptor (TSHR) A-subunit by muscle electroporation in female BALB/c mice. The onset of orbital pathology is characterized by muscle inflammation, adipogenesis, and fibrosis. Animal models of autoimmunity are influenced by their environmental exposures. This follow-up study was undertaken to investigate the development of experimental GO in 2 different locations, run in parallel under comparable housing conditions. Functional antibodies to TSHR were induced in TSHR A-subunit plasmid-immunized animals, and antibodies to IGF-1 receptor α-subunit were also present, whereas control animals were negative in both locations. Splenic T cells from TSHR A-subunit primed animals undergoing GO in both locations showed proliferative responses to purified TSHR antigen and secreted interferon-γ, IL-10, IL-6, and TNF-α cytokines. Histopathological evaluation showed orbital tissue damage in mice undergoing GO, manifest by adipogenesis, fibrosis, and muscle damage with classic signs of myopathy. Although no inflammatory infiltrate was observed in orbital tissue in either location, the appearances were consistent with a "hit-and-run" immune-mediated inflammatory event. A statistically significant increase of cumulative incidence of orbital pathology when compared with control animals was shown for both locations, confirming onset of orbital dysimmune myopathy. Our findings confirm expansion of the model in different environments, accompanied with increased prevalence of T cell-derived proinflammatory cytokines, with relevance for pathogenesis. Wider availability of the model makes it suitable for mechanistic studies into pathogenesis and undertaking of novel therapeutic approaches.

Sample commutability in external quality assessment surveys (EQAS) for thyroid-related antibodies--state of the art.

External quality assessment surveys for thyroid-related antibodies have been offered by INSTAND for 20 years. During this time, some problems have remained, especially those between the similarity of samples sent and routine patient samples. Here the questions of "matrix effects" and "commutability of results" are topics discussed at most EQA-meetings. This short communication deals with the effects of lyophilization on results for thyroid-associated antibodies in an EQA-survey carried out by over 230 participants in October 2005 by INSTAND in Düsseldorf, Germany. The results show that there are small but statistically significant differences (at the level p < 0.05) in results from liquid and lyophilized samples from the same source, the tendency to higher results in the lyophilized samples for anti-TPO and anti-Tg and to lower results for TRAb. The precision in measurement was significantly better for anti-TPO and TRAb in the lyophilized samples, there being no significant difference for anti-Tg. The differences in results between liquid and lyophilized samples were minimal when compared with the numerical results for anti-TPO and anti-Tg, despite the fact that all kits were calibrated with the NIBSC reference materials, although the latter are now both 40 years old.

[National external quality assessment in auto-immunity: auto-antibodies against thyroid constituents]. / Contrôle national de qualité en auto-immunité: anticorps anti-thyroïdiens.

The French Health Products Safety Agency organized in 2005, for the scheme of the national external quality assessment, a survey on antibodies against thyroid constituents which included for the first time the quantitative assay. The purpose of this survey was to assess the quality of the different methods of these assays. The overall qualitative results are satisfactory. However, this survey pointed out a lower performance for immunodot which appeared to have been misused. Concerning the titer of antibodies, results show a broad dispersion between reagents. This confirms the lack of a real standardisation despite of the existence of the international MRC standards.

Comparability of method results and performance in a national external quality assessment scheme between 1993 and 2003 using thyroid associated antibodies as examples.

Four thyroid antibodies (antibodies to microsomes [MAb], thyroid peroxidase [anti-TPO], thyroglobulin [anti-Tg] and TSH-receptor [TRAB, THYBIA]) have been followed up over a 10-year period in a national external quality assessment scheme (EQAS) organised by the Institute for Standardisation and Documentation in the Medical Laboratory (INSTAND e.V.). The following points were observed: I. The introduction of samples with properties similar to patient serum (filtered, recalcified defibrinated plasma without stripping) improved performance and inter-method comparability for thyroid antibodies. II. Regular statistical analysis of EQAS data allows adjustment of target ranges to be made when necessary. III. There are large inter-method variations in reporting, both on qualitative and quantitative results. IV. The samples often gave rise to different constellations of antibodies, which were kit-dependent. V. Despite use of international reference preparations, there was no numerical comparability between quantitative methods for the same analyte. In general, the performance in EQAS for thyroid antibodies has improved over the past decade. There is still a real need for standardisation in the field of thyroid antibody analysis.

Soluble CD antigen (cytokine) expression in various hyperthyroid states and use in the assessment of propylthiouracil treatment.

The soluble CD antigens sCD8, sCD23, and sCD25 are increased in untreated Graves' hyperthyroidism. These levels remain elevated when euthyroidism is established in response to propylthiouracil (PTU) therapy but decrease to control values after PTU treatment is discontinued, when euthyroidism has been established and maintained. Neither sCD8 nor sCD23 are elevated in patients with euthyroid Graves' ophthalmopathy nor in the hyperthyroid phase of subacute thyroiditis. sCD25 is increased to an intermediate degree in these disorders. Soluble CD8 > or = 450 U/ml is sensitive, specific, and predictive of PTU success as sole therapy or need for definitive therapy in untreated and PTU-treated Graves' hyperthyroidism, exceeding the predictive values of thyroid-stimulating hormone receptor antibody, thyroid peroxidase antibody, and T3 radioimmunoassay.