Percentile transformation and recalibration functions allow harmonization of thyroid-stimulating hormone (TSH) immunoassay results.

Background The comparability of thyroid-stimulating hormone (TSH) results cannot be easily obtained using SI-traceable reference measurement procedures (RPMs) or reference materials, whilst harmonization is more feasible. The aim of this study was to identify and validate a new approach for the harmonization of TSH results. Methods Percentile normalization was applied to 125,419 TSH results, obtained from seven laboratories using three immunoassays (Access 3rd IS Thyrotropin, Beckman Coulter Diagnostics; Architect System, Abbott Diagnostics and Elecsys, Roche Diagnostics). Recalibration equations (RCAL) were derived by robust regressions using bootstrapped distribution. Two datasets, the first of 119 EQAs, the second of 610, 638 and 639 results from Access, Architect and Elecsys TSH results, respectively, were used to validate RCAL. A dataset of 142,821 TSH values was used to derive reference intervals (RIs) after applying RCAL. Results Access, Abbott and Elecsys TSH distributions were significantly different (p < 0.001). RCAL intercepts and slopes were -0.003 and 0.984 for Access, 0.032 and 1.041 for Architect, -0.031 and 1.003 for Elecsys, respectively. Validation using EQAs showed that before and after RCAL, the coefficients of variation (CVs) or among-assay results decreased from 10.72% to 8.16%. The second validation dataset was used to test RCALs. The median of between-assay differences ranged from -0.0053 to 0.1955 mIU/L of TSH. Elecsys recalibrated to Access (and vice-versa) showed non-significant difference. TSH RI after RCAL resulted in 0.37-5.11 mIU/L overall, 0.49-4.96 mIU/L for females and 0.40-4.92 mIU/L for males. A significant difference across age classes was identified. Conclusions Percentile normalization and robust regression are valuable tools for deriving RCALs and harmonizing TSH values.

Establishing normal values of total testosterone in adult healthy men by the use of four immunometric methods and liquid chromatography-mass spectrometry.

BACKGROUND: The total testosterone (T) cutoffs clinically adopted to define late-onset hypogonadism (LOH) do not consider the differences that exist between different analytical platforms, nor do they consider the body mass index (BMI) or age of the patient. We aimed at providing method, age and BMI-specific normal values for total T in European healthy men. METHODS: A total of 351 eugonadal healthy men were recruited, and total T was measured with four automated immunometric assays (IMAs): ARCHITECT i1000SR (Abbott), UniCel DxI800 (Beckman Coulter), Cobas e601 (Roche), IMMULITE 2000 (Siemens) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reference ranges (RRs) were calculated for each method. RESULTS: Passing and Bablok regression analysis and Bland-Altman plot showed an acceptable agreement between Abbott and LC-MS/MS, but a poor one between LC-MS/MS and the other IMAs. Age-specific T concentrations in non-obese (BMI <29.9 kg/m2) men were greater than in all men. The total T normal range, in non-obese men aged 18-39 years, measured with LC-MS/MS was 9.038-41.310 nmol/L. RRs calculated with LC-MS/MS statistically differed from the ones calculated with all individual IMAs, except Abbott and among all IMAs. Statistically significant differences for both upper and lower reference limits between our RRs and the ones provided by the manufacturers were also noticed. CONCLUSIONS: We calculated normal ranges in a non-obese cohort of European men, aged 18-39 years, with four commercially available IMAs and LC-MS/MS and found statistically significant differences according to the analytical method used. Method-specific reference values can increase the accuracy of LOH diagnosis and should be standardly used.

A multicenter study for the evaluation of the reference interval for TSH in Italy (ELAS TSH Italian Study).

Background The aims of this study were: (1) to calculate reliable thyroid stimulating hormone (TSH) reference intervals using laboratory databases; (2) to evaluate the relationship between TSH, sex and age values in different large Italian populations. Methods The TSH values stored in the laboratory information system of clinical laboratories of four Italian city hospitals, including 146,801 TSH measurements (with the respective age and sex data of individuals) were taken in consideration. Assuming a log-normal distribution, to log-transformed TSH values were applied the Dixon's iterative principle in order to exclude the outliers. At the end of this iterative process 142,821 log-transformed TSH results remained. The four clinical laboratories measured serum TSH concentrations using the same TSH immunoassay method (Access TSH 3rd IS, using UniCel DxI platform). Results The TSH reference interval calculated in the present study (0.362-5.280 mIU/L) is similar to that suggested by the manufacturer for the Access TSH 3rd IS assay (0.45-5.33 mIU/L). TSH values in females were significantly higher than in males (females: mean=2.06 mIU/L; standard deviation [SD]=1.26 mIU/L; n=101,243; males: mean=1.92 mIU/L; SD=1.19 mIU/L; n=41,578; p<0.0001). Moreover, a negative linear relationship was observed between TSH throughout all interval age values (from 0 to 105 years). Conclusions The results of the present multicenter study confirm that data mining techniques can be used to calculate clinically useful reference intervals for TSH. From a pathophysiological point of view, our results suggest that some Northern populations of Italy might still suffer some harmful effects on the thyroid gland due to mild to moderate iodine intake deficiency. Specific clinical trials are needed to confirm these results.

Evaluation of the first fully automated immunoassay method for the measurement of stimulating TSH receptor autoantibodies in Graves' disease.

BACKGROUND: Thyroid stimulating hormone (TSH) receptor autoantibodies (TRAb) are pathogenetic and diagnostic hallmarks of Graves' disease (GD). Three varieties of TRAb have been described: stimulating (S-TRAb), blocking (B-TRAb), and apoptotic (A-TRAb) autoantibodies. Very recently, the first immunoassay method (Immulite TSI assay, Siemens Healthcare Diagnostics) declared to measure serum S-TRAb concentration, has been made available in an automated commercial platform. The aim of the study was to evaluate the ability of this new test to identify patients suffering from GD, in comparison with two current IMA methods for total TSH receptor autoantibodies (T-TRAb) measurement. METHODS: Sera of 383 subjects [72 patients with untreated GD, 55 patients with autoimmune thyroiditis (AIT), 36 patients with multinodular non-toxic goiter, 100 patients with other non-thyroid autoimmune diseases (NTAD) and 120 healthy subjects (HS)] were evaluated. RESULTS: The threshold obtained by receiver operating characteristic (ROC) analysis was 0.54 IU/L, very similar to that proposed by the manufacturer (0.55 IU/L). Thyroid-stimulating immunoglobulins (TSI) were positive in all GD patients and negative in all but three controls (clinical sensitivity and specificity of 100% and 98.7%, respectively). Passing and Bablok regression analysis and Bland-Altman plot showed an acceptable agreement between TSI Immulite assay and other two immunoassay methods (Cobas/Elecsys, Roche and TRAK RIA, BRAHMS Thermo Scientific). CONCLUSIONS: The diagnostic performance of fully automated Immulite TSI assay in GD patients is at least comparable to that of current TRAb immunoassays (IMAs) suggesting the possibility of including such assay in rapid and cost-saving diagnostic and monitoring algorithms. However, our results do not provide full evidence that this assay is specific for S-TRAb only, and future studies comparing Immulite TSI assay to stimulating activity are required.

Measurement of thyroglobulin, calcitonin, and PTH in FNA washout fluids.

Different imaging tools, circulating endocrine markers, and fine-needle aspiration (FNA) cytology are of great importance in the diagnosis and follow-up of different thyroid and parathyroid diseases. Sometimes, however, they are conflicting or inconclusive: interestingly, measuring endocrine markers (i.e. thyroglobulin, calcitonin, parathyroid hormone) in fluids from FNA proved to be a very useful complementary diagnostic tool in such cases. The determination of endocrine markers in fluids other than serum/plasma has been developed in the last years. Although studies have reported overall satisfactory results, a good standardization of procedures has not yet been reached, and further efforts should be made in order to better define pre-analytical, analytical, and post-analytical aspects. Here we reviewed critically the literature on the measurement of FNA endocrine markers, focusing on laboratory issues, such as preparation of the sample, choice of solution, and technical features of determination of these markers. Indeed, information for use of FNA-Tg, FNA-CT, and FNA-PTH in clinical practice was also provided.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Immunoassay of thyroid peroxidase autoantibodies: diagnostic performance in automated third generation methods. A multicentre evaluation.

BACKGROUND: The use of automated immunometric methods for the detection of anti-thyroid peroxidase antibodies (TPOAb), the main serological marker of autoimmune thyroid diseases (AITD), has expanded in recent years. However, it is not known whether these new automated platforms have improved the diagnostic performance of TPOAb assays. The aim of this study was to evaluate the potential improvement of the inter-method agreement of current automated third generation systems, 12 years after a previous study, which had assessed the analytical variability between semi-automated second generation methods of TPOAb detection. METHODS: Eight pools of sera from patients with chronic lymphocytic thyroiditis, exhibiting different TPOAb concentrations, were collected from routine laboratory diagnostics and distributed to seven companies throughout Italy. All automated third generation methods were calibrated against the Medical Research Council (MRC) reference preparation 66/387. RESULTS: The overall mean variability (CV) was 93.6% when results were expressed in part as arbitrary Units (U/mL) and in part as International Units (IU/mL). The conversion of all values in IU/mL resulted in a significant decrease of CV (49.8%). The CV expressed as COM (cut-off concentration multiples) was 64.0%. Agreement of qualitative results was 95.3% with a pronounced difference in the threshold values proposed by manufacturers (range 3.2-35.0 IU/mL). CONCLUSIONS: These findings confirm the improvement of harmonisation between different methods of automated third generation TPOAb assays. Nevertheless, further efforts should be made in the definition of the positive cut-off concentration to avoid misclassification of AITD patients as well as in a new international reference preparation and in the autoantigen purification modality.

Current state of diagnostic technologies in the autoimmunology laboratory.

The methods for detecting and measuring autoantibodies have evolved markedly in recent years, encompassing three generations of analytical technologies. Many different immunoassay methods have been developed and used for research and laboratory practice purposes, from the early conventional (or monoplex) analytical methods able to detect single autoantibodies to the more recent multiplex platforms that can quantify tens of molecules. Although it has been in use for over 50 years, indirect immunofluorescence remains the standard method for research on many types of autoantibodies, due to its characteristics of diagnostic sensitivity and also to recent technological innovations which permit it a greater level of automation and standardization. The recent multiplex immunometric methods, with varying levels of automation, present characteristics of higher diagnostic accuracy, but are not yet widely diffused in autoimmunology laboratories due to the limited number of autoantibodies that are detectable, and due to the high cost of reagents and systems. Technological advancement in autoimmunology continues to evolve rapidly, and in the coming years new proteomic techniques will be able to radically change the approach to diagnostics and possibly also clinical treatment of autoimmune diseases. The scope of this review is to update the state of the art of technologies and methods for the measurement of autoantibodies, with special reference to innovations in indirect immunofluorescence and in multiple proteomic methods.

Preliminary evaluation of the first international reference preparation for anticitrullinated peptide antibodies.

BACKGROUND: A lyophilised reference serum from one patient with rheumatoid arthritis (RA) diluted with serum samples from healthy subjects was evaluated as a possible first international standard for anticitrullinated peptide antibodies (ACPAs). METHODS: The authors used 12 commercial ELISAs for ACPA detection in the reference serum and for testing the linearity of the assays by studying twofold serial dilutions. To test the effectiveness of the standardisation, sera from 20 RA patients with variable antibody concentrations were analysed, and the relative concentrations were calculated using both the kit's own curve and the six dilutions of the reference serum as a calibration curve. Fifty sera from normal healthy subjects were used to calculate cut-off values for the reference serum using each commercial kit. RESULTS: The calibration curve obtained for each of the 12 methods using the reference sample dilutions as calibrator allowed harmonisation of the ACPA concentration of the 20 RA serum samples, significantly reducing the dispersion of the values. The mean coefficient of variation (CV) was reduced from 76.4% to 27.9% (p=0.018) and from 85.9% to 33.5% (p=0.028) for the medium/high and negative samples, respectively. Low positive sera CV was also reduced, but to a smaller degree, from 82.5% to 55.5% (p=0.043). CONCLUSION: This first evaluation of the behaviour of the ACPA reference serum demonstrated that it tested positive in all the assays and that it may be used as a reference standard for establishing calibration curves, reducing the dispersion of antibody values and better comparing results obtained from different methods/laboratories.

Clinical usefulness of the serological gastric biopsy for the diagnosis of chronic autoimmune gastritis.

AIM: To assess the predictive value for chronic autoimmune gastritis (AIG) of the combined assay of anti-parietal-cell antibodies (PCA), anti-intrinsic-factor antibodies (IFA), anti-Helicobacter pylori (Hp) antibodies, and measurement of blood gastrin. METHODS: We studied 181 consecutive patients with anemia, due to iron deficiency resistant to oral replacement therapy or to vitamin B12 deficiency. RESULTS: 83 patients (45.8%) tested positive for PCA and underwent gastroscopy with multiple gastric biopsies. On the basis of the histological diagnosis, PCA-positive patients were divided into 4 groups: (1) 30 patients with chronic atrophic gastritis; they had high concentrations of PCA and gastrin and no detectable IFA; (2) 14 subjects with metaplastic gastric atrophy; they had high PCA, IFA, and gastrin; (3) 18 patients with nonspecific lymphocytic inflammation with increased PCA, normal gastrin levels, and absence of IFA; (4) 21 patients with multifocal atrophic gastritis with "borderline" PCA, normal gastrin, absence of IFA and presence of anti-Hp in 100% of the cases. CONCLUSIONS: The assay of four serological markers proved particularly effective in the diagnostic classification of gastritis and highly correlated with the histological profile. As such, this laboratory diagnostic profile may be considered an authentic "serological biopsy."

The old and new tests for celiac disease: which is the best test combination to diagnose celiac disease in pediatric patients?

BACKGROUND: In the diagnosis of celiac disease (CD), serum assays for anti-endomysium (EMA) and anti-transglutaminase (anti-tTG) antibodies have excellent diagnostic accuracy. However, these assays are less sensitive in young pediatric patients. Recently, a new ELISA test using deamidated gliadin peptides (DGP) as antigen has proved to be very sensitive and specific even in pediatric patients. In addition, anti-actin IgA antibodies (AAA) is another test that can be used in CD patients because antibody concentrations correlate with the degree of villous atrophy. This study evaluated the clinical accuracy of anti-tTG, EMA, AGA, anti-DGP and AAA and the effectiveness of these in different combinations for diagnosing CD in a large cohort of pediatric patients. METHODS: Sera of 150 children under 6 years of age were tested: 95 patients had a diagnosis of CD, while 55 patients who did not suffer from CD were used as controls. Anti-DGP IgA/IgG and AAA were assayed with ELISA kits, while anti-tTG IgA/IgG and AGA IgG/IgA were assayed using a quantitative fluoroimmunoassay. The EMA test was conducted by indirect immunofluorescence. RESULTS: Seventy-six of 95 (80%) CD patients were positive for DGP IgA and/or tTG IgA. Eighty of 95 (84.2%) patients were positive for DGP IgG and/or tTG IgA. None of the controls were positive for these antibodies. Eighty-four of 95 (88.4%) patients and 8/55 (14.5%) controls were positive for AAA and/or anti-tTG IgA. CONCLUSIONS: In very young children, association of anti-tTG IgA with anti-DGP IgG is the best test combination for diagnosing CD, yielding a cumulative sensitivity of 84.2% and a specificity of 100%.

SARS-COV-2-related immune-inflammatory thyroid disorders: facts and perspectives.

Introduction: During the COVID-19 pandemic thyroid gland alteration/dysfunction has been emerged as a possible endocrine complication. The present review is focused on inflammatory and autoimmune thyroid complications triggered by SARS-CoV-2 infection by searching through databases like MEDLINE and Scopus up to April 2021.Areas covered: Beside the occurrence of 'non-thyroidal illness' in severe clinical conditions, alterations of thyroid function and structure may occur during COVID-19 as a consequence of either direct or indirect effects of SARS-CoV-2 infection on the gland. On the one hand, SARS-CoV-2 uses ACE2 as a receptor to infect the host cells and ACE2 is highly expressed by follicular thyroid cells. On the other hand, COVID-19 is associated with a systemic inflammatory and immune response, involving Th1/Th17/Th2 lymphocytes and proinflammatory cytokines, which resembles the immune activation that occurs in immune-mediated thyroid diseases. COVID-19-related thyroid disorders include destructive thyroiditis and onset or relapse of autoimmune thyroid disorders, leading to a broad spectrum of thyroid dysfunction ranging from thyrotoxicosis to hypothyroidism, that may worsen COVID-19 clinical course and affect prognosis.Expert opinion: Physicians should be aware of the possible occurrence of thyroid dysfunction during and after SARS-CoV-2 infection. Further longitudinal studies are warranted to evaluate potential long-term sequelae.

SARS-CoV-2 infection as a trigger of autoimmune response.

Currently, few evidences have shown the possible involvement of autoimmunity in patients affected by coronavirus disease 2019 (COVID-19). In this study, we elucidate whether severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) stimulates autoantibody production and contributes to autoimmunity activation. We enrolled 40 adult patients (66.8 years mean age) admitted to Alessandria Hospital between March and April 2020. All the patients had a confirmed COVID-19 diagnosis and no previously clinical record of autoimmune disease. Forty blood donors were analyzed for the same markers and considered as healthy controls. Our patients had high levels of common inflammatory markers, such as C reactive protein, lactate dehydrogenase, ferritin, and creatinine. Interleukin-6 concentrations were also increased, supporting the major role of this interleukin during COVID-19 infection. Lymphocyte numbers were generally lower compared with healthy individuals. All the patients were also screened for the most common autoantibodies. We found a significant prevalence of antinuclear antibodies, antineutrophil cytoplasmic antibodies, and ASCA immunoglobulin A antibodies. We observed that patients having a de novo autoimmune response had the worst acute viral disease prognosis and outcome. Our results sustain the hypothesis that COVID-19 infection correlates with the autoimmunity markers. Our study might help clinicians to: (a) better understand the heterogeneity of this pathology and (b) correctly evaluate COVID-19 clinical manifestations. Our data explained why drugs used to treat autoimmune diseases may also be useful for SARS-CoV-2 infection. In addition, we highly recommend checking patients with COVID-19 for autoimmunity markers, mainly when deciding on whether to treat them with plasma transfer therapy. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Recent data sustain the idea that autoimmune phenomena exist in patients with coronavirus disease 2019 (COVID-19), but other investigations are necessary to define the possible link between severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) infection and autoimmune disease onset. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ In this monocentric study, we demonstrated how SARS-CoV-2 infection could be associated with an autoimmune response and development of autoantibodies. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ Patients with COVID-19 having an increased level of inflammatory markers and strong autoantibodies positivity (i.e., antinuclear antibodies and antineutrophil cytoplasmic antibodies) presented the worst clinical outcome. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ These results suggest that the drugs normally used to treat autoimmune diseases should also be considered during SARS-CoV-2, improving public health. In addition, before starting a transfer plasma therapy, it is important to also evaluate the autoimmunity conditions of the patients with COVID-19. Transferring antibodies or trying to neutralize them should be done with precaution. It is possible that the risk of developing or increasing the autoimmune response may enhance.

Receptor autoimmunity: diagnostic and therapeutic implications.

Receptor autoimmunity is one of the ways in which autoimmune diseases appear in humans. Graves' disease, myasthenia gravis, idiopathic membranous nephropathy, and autoimmune acute encephalitis are the major autoimmune diseases belonging to this particular group. Receptor autoimmune disease are dependent on the presence of autoantibodies directed against cell-surface antigens, namely TSH receptor in thyrocytes, acetylcholine receptor in neuromuscular junction, phospholipase 2 receptor in podocytes, and NMDA receptor in cortical neurons. In this article we outline the distinctive features of receptor autoimmunity and the specific relationship between the autoimmunology laboratory and the presence/concentration of autoantibodies. Some immunological features distinguish receptor autoimmunity. Anti-receptor autoantibody pathologies are considered T cell-dependent, B-cell-mediated autoimmune disorders: the knowledge about the presence of circulating and/or localized autoantibodies to target organs and identification of autoantigens involved in the autoimmune reaction is of paramount importance. Due to the close correlation between the concentration of anti-receptor autoantibodies, the autoimmune target of some cell-surface receptors and the intensity of symptoms, the measurement of these immunoglobulins has become central to diagnose autoimmune diseases in all affected patients, not just in clinically dubious cases. The measurement of autoantibodies is also relevant for differential diagnosis of autoimmune and non-autoimmune forms with similar symptoms. From the methodological point of view, quantitative immunoassay methods of measurement should be preferred over semi-quantitative ones, for the capacity of the first class of methods to define precisely the reference ranges and decision levels overcoming the measurement uncertainty of semi-quantitative methods.

Heterophile antibodies may falsely increase or decrease thyroglobulin measurement in patients with differentiated thyroid carcinoma.

BACKGROUND: To examine the prevalence of significant interference from heterophile antibodies (HAb) in the measurement of serum thyroglobulin (Tg), we evaluated a large cohort of samples from patients with differentiated thyroid carcinoma (DTC). METHODS: Serum Tg measurements were performed in 406 serum samples before and after incubation of each serum sample in heterophile-blocking tubes (HBT) at room temperature for 1 h. We calculated the difference between the original Tg value and the value obtained after HBT treatment. We considered any sample showing an absolute percent difference >3 SD from the mean percent difference as being affected by HAb interference. RESULTS: We identified five patients (1%) as showing interference from HAb. Of these, three (60%) showed a false positive or falsely increased Tg concentration without any recurrence following clinical work-up; two (40%) showed a false negative or falsely reduced Tg levels, and metastases were detected in both cases by imaging procedures. CONCLUSIONS: HAb may increase as well as reduce the measured Tg in a significant number of patients. A positive HAb interference should be suspected if Tg elevation does not fit the clinical pictures. A negative interference is a more challenging problem because increases in Tg generally occur as the first sign of recurrence of DTC. Therefore, treatment using HBT tubes of all sera referred for Tg measurement should be considered in order to prevent both unwarranted investigations or therapy, and delayed diagnosis of recurrence in patients affected by DTC.

Diagnostic efficacy of the ELISA test for the detection of deamidated anti-gliadin peptide antibodies in the diagnosis and monitoring of celiac disease.

BACKGROUND AND AIM: We evaluated the diagnostic performance of an ELISA test for anti-gliadin IgA and IgG antibodies, which uses synthetic deamidated gliadin peptides (anti-gliadin antibodies, AGAs) as coating; the results were compared with a test that uses extracted gliadin (AGAe). METHODS: The study was conducted on the sera of 144 patients suffering from celiac disease (CD), including 20 patients with IgA deficiency and 9 who were following a gluten-free diet (GFD), and 129 controls. RESULTS: In the 115 CD patients (without IgA deficiency), the sensitivity of AGAe IgA and IgG was 32.2 and 60.9%, whereas that of AGAs IgA and IgG was 59.1 and 72.2%. The specificity for AGAe IgA and IgG, and AGAs IgA and IgG was 93.8 and 89.9%, and 96.9% and 99.2%, respectively. Of the 20 patients with CD and IgA deficiency, 7 tested positive for AGAe IgG and 14 for AGAs IgG. The test using deamidated gliadin peptides performed better in terms of sensitivity and specificity than the AGA tests with extracted antigen. CONCLUSIONS: The very high specificity of the AGAs IgG test (99.2%) also suggests that patients who test positive with this assay require a thorough followup, even if the anti-tissue transglutaminase antibodies (anti-tTG) and anti-endomysial autoantibodies (EMA) assays are negative.

Diagnostic accuracy of a new fluoroenzyme immunoassay for the detection of TSH receptor autoantibodies in Graves' disease.

PURPOSE: Thyrotropin receptor (TSHR) autoantibodies (TRAbs) are a hallmark of Graves' disease (GD). The aim of this study was to evaluate the diagnostic accuracy of a new third generation automatic fluorescence enzyme immunoassay for TRAb measurement in GD, in comparison with two current IMAs. METHODS: Sera of 439 subjects (57 patients with untreated GD, 34 with treated GD, 15 with GD and Graves' orbitopathy, 52 with multinodular non-toxic goiter, 86 with Hashimoto's thyroiditis, 20 with toxic adenoma or toxic multinodular goiter, 55 with non-thyroid autoimmune diseases and 120 normal controls) were tested for TRAbs with the ELiA™ anti-TSH-R assay (ThermoFischer Scientific, Uppsala, Sweden), the TRAK™ RIA, Brahms (Thermo Scientific, Hennigsdorf, Germany) and the Immulite™ TSI assay (Siemens Healthcare, Llanberis, UK). RESULTS: Sensitivity and specificity of the ELiA™ anti-TSH-R assay, TRAK™ RIA and Immulite™ TSI assay were 94.7% and 99.6, 100 and 98.2%, 100 and 98.2%, respectively. Spearman's coefficient and Passing-Bablok regression showed a satisfactory correlation between EliA™ and TRAK™ [rho: 0.925; 95% CI: 0.883-0-953. Intercept: - 0.875 (95% CI: - 2.411 to 0.194); slope: 1.086 (95% CI: 0.941 to 1.248)], and between ELiA™ and TSI™ [rho: 0.947; 95% CI: 0.912 0.969. intercept: 1.085 (95% CI: 0.665 to 2.116); slope 1.315 (95% CI:1.116 to 1.700)]. CONCLUSIONS: The diagnostic performance of ELiA™-TSH-R assay is comparable to that of some current TRAb assays. It may be adopted into clinical practice for the differential diagnosis of hyperthyroidism, to screen for transient hyperthyroidism, and to monitor disease activity and treatment effects.

Recent advances in diagnostic technologies and their impact in autoimmune diseases.

Conventional immunological methods for the detection of serum autoantibodies have been an essential tool for the diagnosis of autoimmune diseases for 40 years: in the last decade autoantibody tests have become accepted criteria for the diagnosis and classification of the main systemic and organ-specific autoimmune diseases. The high degree of purification reached by the autoantigens used in these methods has allowed high diagnostic sensitivity and specificity, especially in the case of some new autoantibodies of particular clinical significance, such as anti-nucleosome, anti-transglutaminase, anti-TSH receptor and anti-citrullinated protein autoantibodies. In the last 5 years the advent of proteomic technology, which allows the simultaneous measurement of a number of autoantibodies (multiplexing), has opened up new horizons in the diagnosis of autoimmune diseases. Multiplexing is particularly interesting for clinical laboratories, for organisational, logistical/managerial, physiopathological and research reasons. The emerging technologies are represented by systems based on planar or non-planar (suspension) arrays: the latter include methods which use addressable microbeads or nanobarcoded particles. Within a few years, the new methods will allow testing of individual autoantibody profiles, which will probably improve understanding of the physiopathology of autoimmunity, allow early diagnosis (due to the predictive value of autoantibodies), and drive the diffusion of antigen-specific therapies in autoimmune diseases.

The laboratory approach to the diagnosis of autoimmune diseases: is it time to change?

Array technology and proteomics are about to launch the era of multiplexed analysis, which allows simultaneous detection of numerous autoantibody specificities and the possibility of defining broad autoantibody profiles. This will probably improve disease staging, risk stratification, prognosis and treatment. However, although these technologies are very promising, they are still in their infancy, and therefore need to undergo strict analytical and clinical validation processes. The latter should involve clinicians and pathologists in prospective, multicentric studies conducted on large numbers of patients to define the specific significance of the various autoantibody profiles. Establishing common standards for the publication and sharing of microarray-generated data will be important for this purpose. Only when these studies have been completed will these new technologies find a place in clinical laboratories. Although we are entering a decade which will probably see a radical change in the diagnostic approach to autoimmune diseases, we do not yet have sufficient knowledge to apply proteomic technologies on a large scale. For the time being, therefore, it is advisable to continue using well-established approaches and diagnostic algorithms such as those reported in the international guidelines, which have been prepared in accordance with the principles of appropriateness and evidence-based medicine.