Diagnostic testing for Graves' or non-Graves' hyperthyroidism: A comparison of two thyrotropin receptor antibody immunoassays with thyroid scintigraphy and ultrasonography.

OBJECTIVE: Graves' disease (GD) is the most common cause of hyperthyroidism. In many cases, when the aetiological diagnosis of GD is not evident based on the clinical evaluation and thyroid function testing, it may become challenging to distinguish Graves' hyperthyroidism from other forms of thyrotoxicosis. The current study was primarly carried out to compare the diagnostic effectiveness of two TSH receptor antibody immunoassays (IMAs), ultrasonography and thyroid scintigraphy in hyperthyroidism scenario. METHODS: We retrospectively analysed consecutive patients with newly diagnosed and untreated thyrotoxicosis who underwent thyroid functional tests, both TRAb and TSI measurements, thyroid scintigraphy and ultrasonography. TRAb assessment was carried out by Kryptor® compact PLUS, while TSI by Immulite® . Echo pattern 3 corresponded to 'thyroid inferno', and the final diagnosis of GD vs non-Graves' hyperthyroidism was made according to the thyroid scan (qualitative scintigraphy). Receiver operating characteristic (ROC) curves were drawn using the final diagnosis as reference. Clinical sensitivity and specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were calculated for all the tests. RESULTS: A total of 124 untreated hyperthyroid patients were included in our study (GD, n = 86 vs non-Graves' hyperthyroidism, n = 38). ROC curves showed that the optimal cut-off values associated with the highest diagnostic sensitivity and specificity was 0.7 IU/L for TRAb Kryptor® (93 [85.4-97.4] and 86.8 [71.9-95.5]) and 0.1 IU/L for TSI Immulite® (94.2 [86.9-98.1] and 84.2 [68.7-93.9]), respectively. For the echo pattern 3, we found a good sensitivity (92.1%) and a high PPV (95.2%) but a quite low specificity value (69.8%) and a relative low NPV (57.5%). For thyroid scintigraphy, the TcTU cut-off value of 1.3% corresponded to the best limit for sensitivity and specificity in our patients (95.3 [88.5-98.7] and 96.4 [81.6-99.4]). The Passing-Bablok regression equation and the Bland-Altman test showed a great degree of correlation and agreement existed between TRAb Kryptor® and Immulite® TSI results. CONCLUSIONS: Thyroid scintigraphy remains the most accurate method to differentiate causes of thyrotoxicosis. However, TRAb assays can be alternatively adopted in this setting, limiting the use of thyroid scintigraphy (TcTU evaluation) to TRAb-negative patients. Thyoid US is less accurate than both TRAb/TSI and thyroid scintigraphy, but the 'thyroid inferno' pattern provides a high PPV for GD.

Frequency of serological non-responders and false-negative RT-PCR results in SARS-CoV-2 testing: a population-based study.

Objectives The sensitivity of molecular and serological methods for COVID-19 testing in an epidemiological setting is not well described. The aim of the study was to determine the frequency of negative RT-PCR results at first clinical presentation as well as negative serological results after a follow-up of at least 3 weeks. Methods Among all patients seen for suspected COVID-19 in Liechtenstein (n=1921), we included initially RT-PCR positive index patients (n=85) as well as initially RT-PCR negative (n=66) for follow-up with SARS-CoV-2 antibody testing. Antibodies were detected with seven different commercially available immunoassays. Frequencies of negative RT-PCR and serology results in individuals with COVID-19 were determined and compared to those observed in a validation cohort of Swiss patients (n=211). Results Among COVID-19 patients in Liechtenstein, false-negative RT-PCR at initial presentation was seen in 18% (12/66), whereas negative serology in COVID-19 patients was 4% (3/85). The validation cohort showed similar frequencies: 2/66 (3%) for negative serology, and 16/155 (10%) for false negative RT-PCR. COVID-19 patients with negative follow-up serology tended to have a longer disease duration (p=0.05) and more clinical symptoms than other patients with COVID-19 (p<0.05). The antibody titer from quantitative immunoassays was positively associated with the number of disease symptoms and disease duration (p<0.001). Conclusions RT-PCR at initial presentation in patients with suspected COVID-19 can miss infected patients. Antibody titers of SARS-CoV-2 assays are linked to the number of disease symptoms and the duration of disease. One in 25 patients with RT-PCR-positive COVID-19 does not develop antibodies detectable with frequently employed and commercially available immunoassays.

Measuring thyroglobulin in patients with thyroglobulin autoantibodies: evaluation of the clinical impact of BRAHMS Kryptor® Tg-minirecovery test in a large series of patients with differentiated thyroid carcinoma.

Background The present study was undertaken to evaluate the clinical impact of a thyroglobulin (Tg) minirecovery test (Tg-mRec) in a large series of differentiated thyroid carcinoma (DTC) patients treated and monitored homogeneously in a tertiary referral center. Methods Included were 1120 serum samples from 798 DTC patients. Tg, Tg autoantibodies (TgAb) and Tg-mrec measurements were performed on the automated Kryptor® platform and results compared to the corresponding clinical status. Results Among included samples 228 (20%) were TgAb-positive (TgAb+) and 892 (80%) TgAb-negative (TgAb-), respectively. Tg cutoff points were settled at 0.31 µg/L and 0.15 µg/L for TgAb- and TgAb+ patients, respectively, by ROC curve analysis. The diagnostic performance of serum Tg was reduced in TgAb+ compared to TgAb- patients, however, 87% of TgAb+ patients with recurrent disease and, particularly, all patients with distant metastases were correctly detected by adopting an optimized Tg cutoff for TgAb+ patients. A disturbed recovery was found in only 1% of TgAb- patients and in these cases no clinically relevant information was added by the Tg-mRec. Among TgAb+ patients with undetectable Tg and undisturbed Tg-mRec, no one had recurrent disease. However, a falsely undetectable Tg was demonstrated in two patients with recurrent disease who next to increased TgAb also had a disturbed Tg-mRec test. Conclusions There is no additional clinical benefit from performing Tg-mRec in most patients. It can however be considered in TgAb+ patients with undetectable Tg levels as it may help differentiate between patients with true negative and false negative Tg levels in the presence of such antibodies.

Procalcitonin measurement to screen medullary thyroid carcinoma: A prospective evaluation in a series of 2705 patients with thyroid nodules.

BACKGROUND: To prospectively evaluate the role of procalcitonin (PCT) in screening of patients with thyroid nodules for medullary thyroid carcinoma (MTC). MATERIALS AND METHODS: We measured PCT in 2705 patients with thyroid nodules referred to our centre between January 2011 and December 2017. Those with a positive PCT were operated after positive confirmatory tests such as fine-needle aspiration, measurement of calcitonin (CT) in serum and fine-needle aspiration washouts or CT stimulation testing. Patients with a negative PCT were operated based on the results of further diagnostics. The diagnostic performance of PCT was evaluated, and the best cut-off level was selected by ROC curve analysis. RESULTS: Among 2705 patients, 9 with positive serum PCT (ie, above 0.1 µg/L) and 370 with negative PCT underwent thyroid surgery. MTC was histologically confirmed in all patients with positive PCT but not found in patients with negative PCT. Serum PCT levels were significantly higher in patients with MTC (median 0.64 µg/L, range 0.16-12.9 µg/L) than in those without (median 0.075 µg/L, range 0.075-0.16 µg/L; P < .0001). ROC curves were plotted to calculate the optimal PCT value separating patients with MTC from those without. The best cut-off was 0.155 µg/L with sensitivity, specificity, positive and negative predictive values as well as accuracy of 100%, 99.7%, 91.7%, 100% and 99.7%, respectively. Positive and negative likelihood ratios were 329 and zero, respectively. CONCLUSIONS: Measurement of PCT is a sensitive and accurate method for detecting MTC in patients with thyroid nodules and can thus be a reliable alternative to CT measurement.

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.

Thyroglobulin autoantibodies before radioiodine ablation predict differentiated thyroid cancer outcome.

BACKGROUND: Serum thyroglobulin (Tg) is essential to manage differentiated thyroid carcinoma (DTC). However, Tg determination is affected by circulating Tg antibodies (TgAb), and a role of TgAb as surrogate biomarker has been proposed. Here we evaluated the role of TgAb measured before and after radioiodine ablation (RRA) as potential predictors of prognosis. METHODS: Patients treated since 2006 were screened. Cancers with structural relapse were defined as recurrent. Both Tg and TgAb were measured by immunoassays on the fully automated Kryptor® platform (BRAHMS Gmbh, Henningsdorf, Germany). RESULTS: A series of 215 DTC patients was enrolled, of whom 28.8% had positive preablation TgAb. Overall, 2.8% patients died by DTC and 11% recurred. High-risk class (p=0.004) and cancer relapse (p=0.007) occurred more frequently in positive TgAb, whereas better disease-free survival was observed in negative group (hazard ratio 2.59, p=0.01). Having positive preablation TgAb was significantly associated with risk to develop recurrence (odds ratio 3.57, p=0.004). Among positive TgAb subgroup, higher levels were recorded in recurrent cases (p=0.0001), and the most accurate preablation TgAb threshold was 107.5 IU/mL. When TgAb were measured at first follow-up, recurrence rate was significantly (p<0.0001) higher in persistently TgAb-positive patients (75%) than normalized ones (2.4%). At that time, the highest negative predictive value could be obtained when considering TgAb normalization (<33 IU/mL) or reduction by ≥36.4%. CONCLUSIONS: Positive TgAb before RRA indicates higher risk of poor prognosis, but their significant drop 6-12 months later could be considered a favorable factor.

Evaluation of the BRAHMS Kryptor(®) thyroglobulin minirecovery test in patients with differentiated thyroid carcinoma.

BACKGROUND: The present study was undertaken to evaluate a new sensitive thyroglobulin (Tg) mini-recovery test (Tg-mrec) for the detection of potential interferences in sera from patients with differentiated thyroid carcinoma (DTC) and low Tg levels. METHODS: 167 DTC patients with serum Tg <2 µg/L were enrolled. Both TgAb and Tg-mrec measurements were performed on the automated Kryptor(®) platform. Serum pretreatment in proprietory blocking tubes was perfomed to screen for heterophile antibody interferences. The concordance rates between tests were evaluated. RESULTS: One case of over-recovery occurred in a patient with discordant Tg results in different immunoassays. The prevalences of a positive TgAb test and a reduced recovery rate were 15%-12%, respectively. Serum TgAb and Tg-mrec tests were both positive in 16 patients, both negative in 138 patients and discordant in 13 patients, respectively. The concordance rate between the Tg-mrec test and the TgAb assay was 92% (Cohens' kappa 0.894; 95% confidence interval: 0.82-0.93, p<0.001). In all, among seven clinically relevant interferences, three were found by TgAb, five by Tg-mrec and six by using both tests. CONCLUSIONS: The automated Kryptor(®) Tg-mrec test has a complimentary value to TgAb immunoassay testing in the detection of potential interferences in Tg measurements in patients with DTC and low Tg values (i.e., <2 µg/L). Tg-mrec may detect interferences from TgAb not measured directly by an individual TgAb assay or from other interfering substances, such as heterophile antibodies.

Comparison of serum calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients with thyroid nodules.

BACKGROUND: To prospectively evaluate the role of procalcitonin (PCT) in detecting or excluding medullary thyroid carcinoma (MTC) among patients with thyroid nodules and increased calcitonin (CT) levels. METHODS: Fourteen of 1236 patients referred for thyroid nodules had increased serum CT >10 pg/mL. A stimulation test with pentagastrin was done and both CT and PCT were measured after stimulation. All patients underwent thyroid ultrasound, fine-needle cytology and, if indicated, surgery with histological and immunohistochemical examination of the surgical specimens. RESULTS: After follow-up, two MTCs were found. These two patients had basal CT >100 pg/mL and detectable (>0.1 ng/mL) PCT, with 100% sensitivity. Pentagastrin stimulated CT achieved values above 100 pg/mL in two MTCs and in other two cases with no MTC outcome (50% PPV and 83% NPV). On the contrary, all patients with no MTC had both basal and stimulated undetectable PCT (100% PPV and 100% NPV). CONCLUSIONS: The addition of basal PCT measurement in patients with thyroid nodule(s) and increased CT may significantly improve accuracy of CT measurement without needing a PG stimulation test.

Serum thyroglobulin reference values according to NACB criteria in healthy subjects with normal thyroid ultrasound.

BACKGROUND: The present study was undertaken to establish serum thyroglobulin (Tg) normal reference values in a large group of healthy subjects. METHODS: Four hundred and thirty-eight non-smoking healthy subjects were selected to assess the Tg reference values (209 males, 229 non-pregnant females, age 34.7±13.1 years). Inclusion criteria were: no personal or familial history of thyroid disease, thyrotropin levels from 0.5 to 2.00 mUI/L, negative thyroperoxidase and thyroglobulin antibodies. In addition, the patients had a normal size thyroid (females ≤18 mL, males ≤25 mL) without nodules on the thyroid ultrasound (TUS). According to National Academy of Clinical Biochemistry (NACB) criteria the Tg results were transformed to a logarithmic scale and reference ranges were calculated as mean±2 SD. RESULTS: Serum Tg was measured on the Beckman Coulter UniCel DxI 800 automated platform by the simultaneous 1-step immunoenzymatic Access Thyroglobulin assay (Beckmann-Coulter SA, Nyon, Switzerland). Serum Tg levels were higher in females than in males (p=0.0022). Accordingly, gender-specific reference values were calculated (i.e., males: 1.40-29.2 ng/mL; females: 1.50-38.5 ng/mL). CONCLUSIONS: To the best of the authors' knowledge, the first reference interval study for Tg that integrates NACB criteria and TUS assessment for the selection of the reference population is provided here.

Thyroid volume influences serum calcitonin levels in a thyroid-healthy population: results of a 3-assay, 519 subjects study.

BACKGROUND: We aimed to assess determinants for serum calcitonin (CT) levels and to define reference ranges for different CT immunoassays integrating thyroid ultrasonography (TUS) and conventional clinical and biochemical criteria to select the reference population. METHODS: Five hundred and nineteen thyroid-healthy subjects were included in this prospective cross-sectional population study. Thyroid volume was measured by TUS, while serum CT levels were measured by three different immunoassays. RESULTS: Significant interassay differences were found and the agreement between assays was poor. CT levels were higher in males than in females in all immunoassays. Using the first two assays, both gender and thyroid volume were independent determinants for CT levels. While using the third assay, one thyroid volume was the only determinant for CT levels. CONCLUSIONS: Thyroid volume is a relevant determinant for CT levels. In the clinical practice, the difference of the thyroid sizes in males and females warrants gender-specific reference ranges.

Multicenter Technical Validation of 30 Rapid Antigen Tests for the Detection of SARS-CoV-2 (VALIDATE).

During COVID19 pandemic, SARS-CoV-2 rapid antigen tests (RATs) were marketed with minimal or no performance data. We aimed at closing this gap by determining technical sensitivities and specificities of 30 RATs prior to market release. We developed a standardized technical validation protocol and assessed 30 RATs across four diagnostic laboratories. RATs were tested in parallel using the Standard Q® (SD Biosensor/Roche) assay as internal reference. We used left-over universal transport/optimum media from nasopharyngeal swabs of 200 SARS-CoV-2 PCR-negative and 100 PCR-positive tested patients. Transport media was mixed with assay buffer and applied to RATs according to manufacturer instructions. Sensitivities were determined according to viral loads. Specificity of at least 99% and sensitivity of 95%, 90%, and 80% had to be reached for 107, 106, 105 virus copies/mL, respectively. Sensitivities ranged from 43.5% to 98.6%, 62.3% to 100%, and 66.7% to 100% at 105, 106, 107 copies/mL, respectively. Automated assay readers such as ExDia or LumiraDx showed higher performances. Specificities ranged from 88.8% to 100%. Only 15 of 30 (50%) RATs passed our technical validation. Due to the high failure rate of 50%, mainly caused by lack of sensitivity, we recommend a thorough validation of RATs prior to market release.

Characteristics of Three Different Chemiluminescence Assays for Testing for SARS-CoV-2 Antibodies.

Several tests based on chemiluminescence immunoassay techniques have become available to test for SARS-CoV-2 antibodies. There is currently insufficient data on serology assay performance beyond 35 days after symptoms onset. We aimed to evaluate SARS-CoV-2 antibody tests on three widely used platforms. A chemiluminescent microparticle immunoassay (CMIA; Abbott Diagnostics, USA), a luminescence immunoassay (LIA; Diasorin, Italy), and an electrochemiluminescence immunoassay (ECLIA; Roche Diagnostics, Switzerland) were investigated. In a multigroup study, sensitivity was assessed in a group of participants with confirmed SARS-CoV-2 (n = 145), whereas specificity was determined in two groups of participants without evidence of COVID-19 (i.e., healthy blood donors, n = 191, and healthcare workers, n = 1002). Receiver operating characteristic (ROC) curves, multilevel likelihood ratios (LR), and positive (PPV) and negative (NPV) predictive values were characterized. Finally, analytical specificity was characterized in samples with evidence of the Epstein-Barr virus (EBV) (n = 9), cytomegalovirus (CMV) (n = 7), and endemic common-cold coronavirus infections (n = 12) taken prior to the current SARS-CoV-2 pandemic. The diagnostic accuracy was comparable in all three assays (AUC 0.98). Using the manufacturers' cut-offs, the sensitivities were 90%, 95% confidence interval [84,94] (LIA), 93% [88,96] (CMIA), and 96% [91,98] (ECLIA). The specificities were 99.5% [98.9,99.8] (CMIA), 99.7% [99.3,99.9] (LIA), and 99.9% [99.5,99.98] (ECLIA). The LR at half of the manufacturers' cut-offs were 60 (CMIA), 82 (LIA), and 575 (ECLIA) for positive and 0.043 (CMIA) and 0.035 (LIA, ECLIA) for negative results. ECLIA had higher PPV at low pretest probabilities than CMIA and LIA. No interference with EBV or CMV infection was observed, whereas endemic coronavirus in some cases provided signals in LIA and/or CMIA. Although the diagnostic accuracy of the three investigated assays is comparable, their performance in low-prevalence settings is different. Introducing gray zones at half of the manufacturers' cut-offs is suggested, especially for orthogonal testing approaches that use a second assay for confirmation.

Interference in thyroid-stimulating hormone determination.

BACKGROUND: Thyroid-stimulating hormone (TSH) measurement plays a major role in the diagnosis of thyroid disorders. Despite the good quality of immunochemical tests measuring TSH levels, the presence of interfering substances can sometimes alter the TSH results. DESIGN: We reported the case of a 79-year-old man affected by primary autoimmune hypothyroidism hospitalized for pneumonia. A TSH value > 100 mIU L(-1) (reference: 0.44 mIU L(-1)) was found at admission. No signs and symptoms of hypothyroidism were found upon clinical examination and serum concentration of the free thyroxine (FT4) was normal. RESULTS: Serum treatment in heterophile antibody blocking tubes did not change the TSH result in our assay, while normal levels were found in a different immunoassay method. An abnormal pattern was found in protein electrophoresis at admission, with IgG / j and IgM / k monoclonal bands proved in immunofixation. Interestingly, the disappearance of monoclonal bands was paralleled with a normalization of the TSH value. CONCLUSIONS: We suggest in this study that the TSH determination might be influenced by the presence of transient paraproteins.

Reference Intervals for Platelet Counts in the Elderly: Results from the Prospective SENIORLAB Study.

Currently, age- and sex-independent reference limits (RLs) are frequently used to interpret platelet counts in seniors. We aimed to define and validate reference intervals (RIs) for platelet counts within the framework of the prospective SENIORLAB study. Subjectively healthy Swiss individuals aged 60 years and older were prospectively included and followed for morbidity and mortality. Participants who had circumstances known to affect platelet counts were excluded. The obtained RIs were validated with indirect statistical methods. Frequencies of abnormal platelet counts in a population-based setting, including 41.5% of the entire age-specific population of the Principality of Liechtenstein, were compared by using age- and sex-independent RIs and the RLs obtained in the present study. For males (n = 542), 95% RIs for platelet counts were defined as follows: 150-300 × 109/L (60-69 years); 130-300 × 109/L (70-79 years); and 120-300 × 109/L (80 years and above). For females (n = 661), the consolidated age-independent 95% RI was 165-355 × 109/L. These RI values were validated by indirect RI determination of 51,687 (30,392 female/21,295 male) patients of the same age. Age- and sex-independent RIs exhibited imbalanced frequencies of abnormal platelet counts between the two sexes, which were corrected by introducing age- and sex-specific RLs. In conclusion, females have higher platelet counts than males. Whereas the upper RL for males remains constant, the lower RL decreases with age. We propose to abandon the practice of employing sex- and age-independent RL for platelet counts in the elderly.

EDTA-Anticoagulated Whole Blood for SARS-CoV-2 Antibody Testing by Electrochemiluminescence Immunoassay (ECLIA) and Enzyme-Linked Immunosorbent Assay (ELISA).

While lateral flow test formats can be utilized with whole blood and low sample volumes, their diagnostic characteristics are inferior to immunoassays based on chemiluminescence immunoassay (CLIA) or enzyme-linked immunosorbent assay (ELISA) technology. CLIAs and ELISAs can be automated to a high degree but commonly require larger serum or plasma volumes for sample processing. We addressed the suitability of EDTA-anticoagulated whole blood as an alternative sample material for antibody testing against SARS-CoV-2 by electro-CLIA (ECLIA; Roche, Rotkreuz, Switzerland) and ELISA (IgG and IgA; Euroimmun, Germany). Simultaneously drawn venous serum and EDTA-anticoagulated whole blood samples from 223 individuals were included. Correction of the whole blood results for hematocrit led to a good agreement with the serum results for weakly to moderately positive antibody signals. In receiver-operating characteristic curve analysis, all three assays displayed comparable diagnostic accuracy (area under the curve (AUC)) using corrected whole blood and serum (AUCs: 0.97 for ECLIA and IgG ELISA; 0.84 for IgA ELISA). In conclusion, our results suggest that the investigated assays can reliably detect antibodies against SARS-CoV-2 in hemolyzed whole blood anticoagulated with EDTA. Correction of these results for hematocrit is suggested. This study demonstrates that the automated processing of whole blood for identification of SARS-CoV-2 antibodies with common ECLIA and ELISA methods is accurate and feasible.

Temporal Course of SARS-CoV-2 Antibody Positivity in Patients with COVID-19 following the First Clinical Presentation.

Knowledge of the sensitivities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests beyond 35 days after the clinical onset of COVID-19 is insufficient. We aimed to describe positivity rate of SARS-CoV-2 assays employing three different measurement principles over a prolonged period. Two hundred sixty-eight samples from 180 symptomatic patients with COVID-19 and a reverse transcription polymerase chain reaction (RT-PCR) test followed by serological investigation of SARS-CoV-2 antibodies were included. We conducted three chemiluminescence (including electrochemiluminescence assay (ECLIA)), four enzyme-linked immunosorbent assay (ELISA), and one lateral flow immunoassay (LFIA) test formats. Positivity rates, as well as positive (PPVs) and negative predictive values (NPVs), were calculated for each week after the first clinical presentation for COVID-19. Furthermore, combinations of tests were assessed within an orthogonal testing approach employing two independent assays and predictive values were calculated. Heat maps were constructed to graphically illustrate operational test characteristics. During a follow-up period of more than 9 weeks, chemiluminescence assays and one ELISA IgG test showed stable positivity rates after the third week. With the exception of ECLIA, the PPVs of the other chemiluminescence assays were ≥95% for COVID-19 only after the second week. ELISA and LFIA had somewhat lower PPVs. IgM exhibited insufficient predictive characteristics. An orthogonal testing approach provided PPVs ≥ 95% for patients with a moderate pretest probability (e.g., symptomatic patients), even for tests with a low single test performance. After the second week, NPVs of all but IgM assays were ≥95% for patients with low to moderate pretest probability. The confirmation of negative results using an orthogonal algorithm with another assay provided lower NPVs than the single assays. When interpreting results from SARS-CoV-2 tests, the pretest probability, time of blood draw, and assay characteristics must be carefully considered. An orthogonal testing approach increases the accuracy of positive, but not negative, predictions.

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study.

Pan-immunoglobulin assays can simultaneously detect IgG, IgM and IgA directed against the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 S1-RBD Ig). In this work, we aim to evaluate a quantitative SARS-CoV-2 S1-RBD Ig electrochemiluminescence immunoassay (ECLIA) regarding analytical, diagnostic, operational and clinical characteristics. Our work takes the form of a population-based study in the principality of Liechtenstein, including 125 cases with clinically well-described and laboratory confirmed SARS-CoV-2 infection and 1159 individuals without evidence of coronavirus disease 2019 (COVID-19). SARS-CoV-2 cases were tested for antibodies in sera taken with a median of 48 days (interquartile range, IQR, 43-52) and 139 days (IQR, 129-144) after symptom onset. Sera were also tested with other assays targeting antibodies against non-RBD-S1 and -S1/S2 epitopes. Sensitivity was 97.6% (95% confidence interval, CI, 93.2-99.1), whereas specificity was 99.8% (95% CI, 99.4-99.9). Antibody levels linearly decreased from hospitalized patients to symptomatic outpatients and SARS-CoV-2 infection without symptoms (p < 0.001). Among cases with SARS-CoV-2 infection, smokers had lower antibody levels than non-smokers (p = 0.04), and patients with fever had higher antibody levels than patients without fever (p = 0.001). Pan-SARS-CoV-2 S1-RBD Ig in SARS-CoV-2 infection cases significantly increased from first to second follow-up (p < 0.001). A substantial proportion of individuals without evidence of past SARS-CoV-2 infection displayed non-S1-RBD antibody reactivities (248/1159, i.e., 21.4%, 95% CI, 19.1-23.4). In conclusion, a quantitative SARS-CoV-2 S1-RBD Ig assay offers favorable and sustained assay characteristics allowing the determination of quantitative associations between clinical characteristics (e.g., disease severity, smoking or fever) and antibody levels. The assay could also help to identify individuals with antibodies of non-S1-RBD specificity with potential clinical cross-reactivity to SARS-CoV-2.

Early post-treatment risk stratification of differentiated thyroid cancer: comparison of three high-sensitive Tg assays.

OBJECTIVE: To assess the diagnostic performance of three high-sensitive assays in a cohort of TgAb-negative and TgAb-positive differentiated thyroid cancer (DTC) patients. DESIGN: Retrospective study on prospectively selected DTC patients. METHODS: Serum samples from 154 DTC patients were obtained 6-12 months after radioiodine ablation and tested by Beckman, Roche, BRAHMS Tg and TgAb assays, respectively. Receiver operating characteristics curves for Tg were plotted using outcome over time as benchmark and assay-specific Tg thresholds were obtained for TgAb-negative and TgAb-positive patients. RESULTS: The frequency of positive TgAb was 21, 20 and 20% for Beckman, Roche and BRAHMS, respectively. In TgAb-negative patients, clinical sensitivities and specificities of 100% and 85-95%, respectively, were observed across all assays. In TgAb-positive patients, clinical sensitivities and specificities of 80-100% and 92-96%, respectively, were observed using lower thresholds than in patients without TgAb. CONCLUSIONS: Adopting appropriate thresholds, lower than those for TgAb-negative patients, is possible to reliably follow TgAb-positive patients using highly sensitive Tg assays.