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Background Alpha-1-Antitrypsin (AAT) deficiency (AATD) is a hereditary disorder that manifests primarily as pulmonary emphysema and liver cirrhosis. The clinically most relevant mutation causing AATD is a single nucleotide polymorphism Glu342Lys (Z-mutation). Despite the recommendation to test every COPD patient, the condition remains severely underdiagnosed with a delay of several years between first symptoms and diagnosis. The Grifols’ AlphaKit® QuickScreen is a novel qualitative point-of-care (POC) in vitro screening test developed for the detection of the Z AAT protein in capillary whole blood. The objective of this prospective, international, multi-center, diagnostic, interventional real-world study was to assess the performance of this device for the detection of AATD in test-naïve COPD patients. Methods 1044 test-naïve COPD patients were recruited from 9 centers in Spain and 10 centers in Germany, ranging from primary to tertiary care. To evaluate the performance of the test, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated compared with the gold standard (genotyping). Results Genotyping and phenotyping of all 1019 evaluable samples revealed 4.12% of patients as carriers of at least one Z-allele, while 0.29% carried the homozygous genotype Pi*ZZ. The evaluation of the test’s ability to detect the PiZ protein yielded the following results: specificity 97.8%, sensitivity 73.8%, negative predictive value 98.9%, and positive predictive value 58.5%. All false negatives ( n  = 11) were heterozygote Pi*MZ samples. Conclusions The tested device can be used as an appropriate tool to exclude AATD in primary care and in the overall COPD population, except in patients with a high a-priori- probability of AATD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in 2020. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA. We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, 2014, and Oct 4, 2016) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least 20 days post symptom onset (collected between Feb 1, 2020, and May 31, 2020). Receiver operating characteristic (ROC) curves were used to assess assay thresholds. At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2–94·8) and specificity was 99·9% (99·4–100%); for the DiaSorin assay sensitivity was 96·2% (94·2–97·7) and specificity was 98·9% (98·0–99·4); for the Oxford immunoassay sensitivity was 99·1% (97·8–99·7) and specificity was 99·0% (98·1–99·5); for the Roche assay sensitivity was 97·2% (95·4–98·4) and specificity was 99·8% (99·3–100); and for the Siemens assay sensitivity was 98·1% (96·6–99·1) and specificity was 99·9% (99·4–100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset. Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health. Public Health England and UK National Institute for Health Research.

Protein reference materials play a vital role in generating standard curves for numerical output of immunoassays. However, quality control (QC) of protein reference materials to ensure accurate quantitation and lot-to-lot consistency remains a significant challenge. Current approaches in the field depend on relative quantitation against certified standards or spectroscopic methods without a true understanding of the actual quantity of material present. Here we present a novel approach termed digital Proximity Ligation Assay (dPLA) combining Proximity Ligation Assay (PLA) with Digital PCR (dPCR) for QC of protein reference materials, leveraging the absolute quantification nature of dPCR for the best-in-class quantification precision and accuracy. This approach follows the conventional PLA method to conjugate unique oligonucleotide sequences to the antibody pair. The antibody pair is then incubated with the target reference material. After the formation of an immunocomplex of two antibodies and the target, the ligated sequence is heat extracted and quantified using dPCR (Figure 1). In addition, dPCR allows the background signal to be directly subtracted to calculate the actual number of intended binding events, potentially improving linearity and sensitivity. To prove both concepts, pTau181 is used due to its wide commercial availability. The pTau181 standard materials were obtained from Thermo Fisher Scientific, quantified by dPLA, and compared to conventional qPCR readout. In comparison to the conventional PLA, dPLA can differentiate small changes in signal (Figure 2). The same pTau181 standards at baseline, 80% dilution, and 60% dilution were quantified in three different runs. The dPLA showed within 3% CV for each standard whereas the conventional readout cannot differentiate the diluted concentrations. We then performed 5x serial dilution of the baseline standard. With background subtraction, the linearity is improved, with the potential to quantify the least concentrated standard (Figure 3). Using dPLA, reference materials are quantified in an absolute manner, enhancing reproducibility across different lots and ensuring consistent and reliable measurements. This method can also be used to minimize instrument-to-instrument variation caused by standard materials variations, potentially allowing diagnostic platforms to be deployed widely with superior lab-to-lab consistency.

The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring type 1 diabetes (T1D)-associated autoantibodies and the concordance of results among laboratories. IASP organizes international interlaboratory assay comparison studies in which blinded serum samples are distributed to participating laboratories, followed by centralized collection and analysis of results, providing participants with an unbiased comparative assessment. In this report, we describe the results of glutamic acid decarboxylase autoantibody (GADA) assays presented in the IASP 2018 workshop. In May 2018, IASP distributed to participants uniquely coded sera from 43 new-onset T1D patients, 7 multiple autoantibody-positive nondiabetic individuals, and 90 blood donors. Results were analyzed for the following metrics: sensitivity, specificity, accuracy, area under the ROC curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95), and concordance of qualitative and quantitative results. Thirty-seven laboratories submitted results from a total of 48 different GADA assays adopting 9 different formats. The median ROC-AUC and pAUC95 of all assays were 0.87 [interquartile range (IQR), 0.83-0.89] and 0.036 (IQR, 0.032-0.039), respectively. Large differences in pAUC95 (range, 0.001-0.0411) were observed across assays. Of formats widely adopted, bridge ELISAs showed the best median pAUC95 (0.039; range, 0.036-0.041). Several novel assay formats submitted to this study showed heterogeneous performance. In 2018, the majority of the best performing GADA immunoassays consisted of novel or established nonradioactive tests that proved on a par or superior to the radiobinding assay, the previous gold standard assay format for GADA measurement.

Abstract Background Treponemal immunoassays are increasingly used for syphilis screening with the reverse sequence algorithm. There are few data describing performance of treponemal immunoassays compared to traditional treponemal tests in patients with and without syphilis. Methods We calculated sensitivity and specificity of 7 treponemal assays: (1) ADVIA Centaur (chemiluminescence immunoassay [CIA]); (2) Bioplex 2200 (microbead immunoassay); (3) fluorescent treponemal antibody absorption test (FTA-ABS); (4) INNO-LIA (line immunoassay); (5) LIAISON CIA; (6) Treponema pallidum particle agglutination assay (TPPA); and (7) Trep-Sure (enzyme immunoassay [EIA]), using a reference standard combining clinical diagnosis and serology results. Sera were collected between May 2012-January 2013. Cases were characterized as: (1) current clinical diagnosis of syphilis: primary, secondary, early latent, late latent; (2) prior treated syphilis only; (3) no evidence of current syphilis, no prior history of syphilis, and at least 4 of 7 treponemal tests negative. Results Among 959 participants, 262 had current syphilis, 294 had prior syphilis, and 403 did not have syphilis. FTA-ABS was less sensitive for primary syphilis (78.2%) than the immunoassays or TPPA (94.5%-96.4%) (all P ≤ .01). All immunoassays were 100% sensitive for secondary syphilis, 95.2%-100% sensitive for early latent disease, and 86.8%-98.5% sensitive in late latent disease. TPPA had 100% specificity. Conclusions Treponemal immunoassays demonstrated excellent sensitivity for secondary, early latent, and seropositive primary syphilis. Sensitivity of FTA-ABS in primary syphilis was poor. Given its high specificity and superior sensitivity, TPPA is preferred to adjudicate discordant results with the reverse sequence algorithm over the FTA-ABS. We compared performance of 5 treponemal immunoassays, the Treponema pallidum particle agglutination assay (TPPA), and the fluorescent treponemal antibody absorption test (FTA-ABS). FTA-ABS was less sensitive for primary syphilis (78%) than the immunoassays or TPPA (94%-96% sensitivity). TPPA was 100% specific.

Many different cystatin C-based equations exist for estimating glomerular filtration rate. Major reasons for this are the previous lack of an international cystatin C calibrator and the nonequivalence of results from different cystatin C assays. Use of the recently introduced certified reference material, ERM-DA471/IFCC, and further work to achieve high agreement and equivalence of 7 commercially available cystatin C assays allowed a substantial decrease of the CV of the assays, as defined by their performance in an external quality assessment for clinical laboratory investigations. By use of 2 of these assays and a population of 4690 subjects, with large subpopulations of children and Asian and Caucasian adults, with their GFR determined by either renal or plasma inulin clearance or plasma iohexol clearance, we attempted to produce a virtually assay-independent simple cystatin C-based equation for estimation of GFR. We developed a simple cystatin C-based equation for estimation of GFR comprising only 2 variables, cystatin C concentration and age. No terms for race and sex are required for optimal diagnostic performance. The equation, [Formula: see text] is also biologically oriented, with 1 term for the theoretical renal clearance of small molecules and 1 constant for extrarenal clearance of cystatin C. A virtually assay-independent simple cystatin C-based and biologically oriented equation for estimation of GFR, without terms for sex and race, was produced.

The IFCC Committee for Standardization of Thyroid Function Tests developed a global harmonization approach for thyroid-stimulating hormone measurements. It is based on a multiassay method comparison study with clinical serum samples and target setting with a robust factor analysis method. Here we describe the Phase IV method comparison and reference interval (RI) studies conducted with the objective to recalibrate the participating assays and demonstrate the proof-of-concept. Fourteen manufacturers measured the harmonization and RI panel; 4 of them quantified the harmonization and first follow-up panel in parallel. All recalibrated their assays to the statistically inferred targets. For validation, we used desirable specifications from the biological variation for the bias and total error (TE). The RI measurements were done with the assays' current calibrators, but data were also reported after transformation to the new calibration status. We estimated the pre- and postrecalibration RIs with a nonparametric bootstrap procedure. After recalibration, 14 of 15 assays met the bias specification with 95% confidence; 8 assays complied with the TE specification. The CV of the assay means for the harmonization panel was reduced from 9.5% to 4.2%. The RI study showed improved uniformity after recalibration: the ranges (i.e., maximum differences) exhibited by the assay-specific 2.5th, 50th, and 97.5th percentile estimates were reduced from 0.27, 0.89, and 2.13 mIU/L to 0.12, 0.29, and 0.77 mIU/L. We showed that harmonization increased the agreement of results from the participating immunoassays, and may allow them to adopt a more uniform RI in the future.

Background: We report the development of a novel high-sensitivity cardiac troponin T (hs-cTnT) assay, a modification of the Roche fourth-generation cTnT assay, and validation of the analytical performance of this assay. Methods: Validation included testing of analytical sensitivity, specificity, interferences, and precision. We established the 99th percentile cutoff from healthy reference populations (n = 616). In addition, we studied differences in time to a positive result when using serial measurements of hs-cTnT vs cTnT in patients with a confirmed diagnosis of non-ST elevation myocardial infarction (non-STEMI). Results: The hs-cTnT assay had an analytical range from 3 to 10 000 ng/L. At the 99th percentile value of 13.5 ng/L, the CV was 9% using the Elecsys® 2010 analyzer. The assay was specific for cTnT without interferences from human cTnI or cTnC, skeletal muscle TnT, or hemoglobin concentrations up to 1000 mg/L, above which falsely lower values would be expected. When the assay was evaluated clinically, a hs-cTnT higher than the 99th percentile concentration identified a significantly higher number of patients with non-STEMI on presentation (45 vs 20 patients, P = 0.0004) compared with cTnT, and a final diagnosis of non-STEMI was made in 9 additional patients (55 vs 46 patients, P = 0.23) after serial sampling. Time to diagnosis was significantly shorter using hs-cTnT compared with cTnT [mean 71.5 (SD 108.7) min vs 246.9 (82.0) min, respectively; P < 0.01]. Conclusions: The analytical performance of hs-cTnT complies with the ESC-ACCF-AHA-WHF Global Task Force recommendations for use in the diagnosis of MI.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Today, biomedical researchers still collect tales of antibody woe faster than country-music labels spin out sad songs. The most common grumble is the cheating reagent: the antibody purchased to detect protein X surreptitiously binds protein Y (and perhaps ignores X altogether). Another complaint is 'lost treasure': a run of promising experiments that stalls when a new batch of antibodies fails to reproduce previous findings (see 'A market in a bind'). But technological advances and shifts in the scientific community now promise to cut through this antibody quagmire.