Evaluation of the automated LIAISON® SARS-CoV-2 TrimericS IgG assay for the detection of circulating antibodies.

OBJECTIVES: COVID-19 has brought about tests from many manufacturers. While molecular and rapid antigen tests are targeted for early diagnosis, immunoassays have a larger role in epidemiological studies, understanding longitudinal immunity, and in vaccine development and response. METHODS: The performance of the LIAISON® SARS-CoV-2 TrimericS IgG assay was evaluated against the Beckman ACCESS SARS-CoV-2 IgG assay in New Mexico, and against the Siemens ADVIA Centaur COV2G assay in New York. Discordant samples were parsed using a microneutralization assay. RESULTS: A SARS-CoV-2 antibody positivity rate of 23.8% was observed in the samples tested in New York (September 2020), while in the same month the positivity rate was 1.5% in New Mexico. Positive and negative agreement were 67.6% (95% CI 49.5-82.6%) and 99.8% (95% CI 99.5-99.9%), respectively, with the Beckman test, and 98.0% (95% CI 95.7-99.3%) and 94.8% (95% CI 93.4-96.0%), respectively, with the Siemens test. Receiver operating characteristic analysis for the detection of SARS-CoV-2 antibodies discloses an AUC, area under the curve, of 0.996 (95% CI 0.992-0.999) for the LIAISON® SARS-CoV-2 TrimericS IgG assay. The criterion associated to the Youden Index was determined to be >12.9 kAU/L with a sensitivity of 99.44% and a specificity of 99.82%. CONCLUSIONS: The LIAISON® SARS-CoV-2 TrimericS IgG assay is highly sensitive and specific. The balance of these parameters, without emphasis on high specificity alone, is particularly important when applied to high prevalence populations, where a highly sensitive assay will result in reporting a lower number of false negative subjects.

Clinical and Analytical Performance of an Automated Serological Test That Identifies S1/S2-Neutralizing IgG in COVID-19 Patients Semiquantitatively.

In the coronavirus (CoV) disease 2019 (COVID-19) pandemic, highly selective serological testing is essential to define exposure to severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Many tests have been developed, yet with variable speeds to first results, and are of unknown quality, particularly when considering the prediction of neutralizing capacity. The LIAISON SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescence assay. The clinical and analytical performances of the test were validated in an observational study using residual samples (>1,500) with a positive or negative COVID-19 diagnosis. The LIAISON SARS-CoV-2 S1/S2 IgG assay proved to be highly selective and specific and offered semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The assay's diagnostic sensitivities were 91.3% and 95.7% at >5 or ≥15 days from diagnosis, respectively, and 100% when assessed against a neutralizing assay. The assay's specificity ranged between 97% and 98.5%. The average imprecision of the assay was a <5% coefficient of variation. Assay performance at 2 different cutoffs was evaluated to optimize predictive values. The automated LIAISON SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day; first results are available within 35 min, with a throughput of 170 tests/hour. The semiquantitative results provided by the test also associate with the presence of neutralizing antibodies and may provide a useful tool for the large-scale screening of convalescent-phase plasma for safe therapeutic use.

Trueness, precision and stability of the LIAISON 1-84 parathyroid hormone (PTH) third-generation assay: comparison to existing intact PTH assays.

BACKGROUND: Over the past few decades, parathyroid hormone (PTH) immunoassays have progressed through successive generations resulting in increased specificity and accuracy for detecting circulating PTH. With the introduction of third-generation assays, in which the biologically active PTH(1-84) is specifically targeted, the PTH(7-84) and other fragments are not detected. The specific recognition of only PTH(1-84) whole molecule allows for more reliable standardization and calibration than with the existing assays. METHODS: Samples from patients on hemodialysis or with primary hyperparathyroidism and apparently healthy subjects were examined in different collection matrices (EDTA plasma, unspun EDTA plasma and SST) stored for 0, 24 or 72 h at room temperature to reflect the prevailing sample collection methods, shipping and processing conditions of centralized labs in the United States. Samples were analyzed by the LIAISON 1-84 PTH and N-TACT assays, and by three additional commercially available intact PTH assays. RESULTS: Defined samples, prepared using two different standards (WHO 95/646 international standard and the synthetic Bachem PTH(1-84)), show little bias with the LIAISON 1-84 PTH assay, but not with the other intact PTH assays. Furthermore, PTH is stable for up to 72 h in plasma, but less stable in serum beyond 24 h. CONCLUSIONS: The FDA-approved LIAISON 1-84 PTH assay is accurate and reliably measures the biologically active PTH molecule in plasma or serum stored at room temperature for up 72 and 24 h, respectively.

Modulation of inflammatory and immune responses by vitamin D.

Vitamin D (VitD) is a prohormone most noted for the regulation of calcium and phosphate levels in circulation, and thus of bone metabolism. Inflammatory and immune cells not only convert inactive VitD metabolites into calcitriol, the active form of VitD, but also express the nuclear receptor of VitD that modulates differentiation, activation and proliferation of these cells. In vitro, calcitriol upregulates different anti-inflammatory pathways and downregulates molecules that activate immune and inflammatory cells. Administration of VitD has beneficial effects in a number of experimental models of autoimmune disease. Epidemiologic studies have indicated that VitD insufficiency is frequently associated with immune disorders and infectious diseases, exacerbated by increasing evidence of suboptimal VitD status in populations worldwide. To date, however, most interventional studies in human inflammatory and immune diseases with VitD supplementation have proven to be inconclusive. One of the reasons could be that the main VitD metabolite measured in these studies was the 25-hydroxyVitD (25OHD) rather than its active form calcitriol. Although our knowledge of calcitriol as modulator of immune and inflammatory reactions has dramatically increased in the past decades, further in vivo and clinical studies are needed to confirm the potential benefits of VitD in the control of immune and inflammatory conditions.

LIAISON® Calprotectin for the prediction of relapse in quiescent ulcerative colitis: The EuReCa study.

INTRODUCTION: Fecal calprotectin (FC) is established as a diagnostic marker to differentiate between inflammatory bowel diseases and non-inflammatory conditions. Furthermore, it may be effective in monitoring response to treatment, and to predict relapse during maintenance therapy. DESIGN: This was a prospective longitudinal study carried out in Italy, France and Spain. The primary objective was to correlate the LIAISON® Calprotectin assay measurements to quiescent ulcerative colitis (UC) or relapse as assessed by clinical data. Patients were assessed every 3 months for 12 months, and at 18 months. RESULTS: The last FC measured prior to relapse was the variable that predicted relapse in a statistically significant manner. With a 62.3 µg/g cut-off the area under the curve was 0.619, and the sensitivity was 62.9% (95% Confidence Interval [CI] 44.9%-78.5%) and specificity 63.0% (95% CI 53.1%-72.1%). Using machine learning methods, the last FC measurement was shown to have the largest impact in predicting relapse. An algorithm was developed that included other variables available following a clinician's visit, which resulted in an area under the curve of 0.754 for predicting relapse. CONCLUSION: In the present study FC measured by the LIAISON® Calprotectin assay on the visit before relapse is predictive of relapse in patients with quiescent UC. In a proof of concept, the accuracy of prediction can further be improved including other variables in an algorithm developed by machine learning. TRIAL REGISTRATION: The trial is registered at clinicaltrials.gov with reference number NCT05168917.

mRNA COVID-19 vaccine booster fosters B- and T-cell responses in immunocompromised patients.

SARS-CoV-2 vaccination has proven effective in inducing an immune response in healthy individuals and is progressively us allowing to overcome the pandemic. Recent evidence has shown that response to vaccination in some vulnerable patients may be diminished, and it has been proposed a booster dose. We tested the kinetic of development of serum antibodies to the SARS-CoV-2 Spike protein, their neutralizing capacity, the CD4 and CD8 IFN-γ T-cell response in 328 subjects, including 131 immunocompromised individuals (cancer, rheumatologic, and hemodialysis patients), 160 health-care workers (HCW) and 37 subjects older than 75 yr, after vaccination with two or three doses of mRNA vaccines. We stratified the patients according to the type of treatment. We found that immunocompromised patients, depending on the type of treatment, poorly respond to SARS-CoV-2 mRNA vaccines. However, an additional booster dose of vaccine induced a good immune response in almost all of the patients except those receiving anti-CD20 antibody. Similarly to HCW, previously infected and vaccinated immunocompromised individuals demonstrate a stronger SARS-CoV-2-specific immune response than those who are vaccinated without prior infection.

1,25-dihydroxyvitamin D as Predictor of Renal Worsening Function in Chronic Kidney Disease. Results From the PASCaL-1,25D Study.

Background: Heterogeneous progression of chronic kidney disease (CKD) toward dialysis advocates improving in renal care management. Diagnosis and staging of CKD relies on estimated glomerular filtration rate (eGFR) and albuminuria. Tubular biomarkers emerged as new predictors of worsening renal function (WRF), due to partial inaccuracy of eGFR and existing WRF in non-proteinuric patients. Active vitamin D is synthesized in renal tubules and participates to mineral adaptation in CKD. Circulating 1,25-dihydroxyvitamin D [1,25(OH)2D] was poorly investigated as a biomarker of endocrine tubular function and predictor of WRF. Objective: Investigate capability of 1,25(OH)2D to predict parathormone (PTH) increase and WRF in CKD stage 3-4. Methods: PASCaL-1,25D was an observational, prospective, monocentric study. Primary outcomes were absolute and 20% increase in PTH, and WRF defined as 20% reduction in eGFR or dialysis initiation at 6 months. Results: Seventy-one patients completed follow up. Absolute increase in PTH (1-84) was independently predicted by lower 1,25(OH)2D levels (p = 0.0134). No association was detected between 1,25(OH)2D and iPTH increase. Higher 1,25(OH)2D was associated with reduced risk of WRF at univariate analysis [OR 0.89 (95% CI 0.86-0.93), p = 0.006]. The 1,25(OH)2D/PTH (1-84) ratio was associated with non-significant 84% risk reduction for WRF [OR 0.16 (95% CI 0.06-0.41), p = 0.05]. Low 1,25(OH)2D reached 100% sensitivity in predicting WRF in CKD stage 3 (AUC 9.909, p < 0.0001) and non-elderly patients (AUC 0.883, p < 0.0001). Machine learning models retained 1,25(OH)2D/PTH (1-84) as relevant predictor of WRF together with eGFR and albuminuria. Age influenced interaction between renal and mineral biomarkers. Conclusion: 1,25(OH)2D deserves attention as biomarker of tubular health, and sensible predictor of WRF on the short run among non-elderly patients affected by stage 3 CKD. The 1,25(OH)2D/PTH (1-84) ratio may represent a composite biomarker of tubular reserve/endocrine response to the transition from adaptive to maladaptive equilibrium in CKD-MBD.

Comparative Cost and Effectiveness of a New Algorithm for Early Lyme Disease Diagnosis: Evaluation in US, Germany, and Italy.

PURPOSE: This Lyme disease early detection economic model, for patients with suspected Lyme disease without erythema migrans (EM), compares outcomes of standard two-tier testing (sTTT), modified two-tier testing (mTTT) and the DiaSorin Lyme Detection Algorithm (LDA), a combination of both serology tests and Interferon-ɤ Release Assay. PATIENTS AND METHODS: A patient-level simulation model was built to incorporate effectiveness estimation from a structured focused literature review, and health-care cost inputs for the United States, Germany, and Italy. Simulated clinical outcomes were 1) percent of patients with timely and correct diagnosis, 2) patients appropriately treated and exposed to antibiotics therapy, and 3) patients with late Lyme disease manifestations. Expected health outcomes were expressed in terms of differences in quality-adjusted life years (QALYs) due to disseminated Lyme disease and persisting symptoms, and economic outcomes were analyzed from a third-party payer perspective. RESULTS: The DiaSorin LDA resulted in a better sensitivity compared to sTTT and mTTT, 84% vs 49% and 45%, respectively, in the base case (13% of infected patients in the tested population). Due to the improved diagnostic performance, the LDA-based strategy is expected to be more effective, providing mean incremental 0.024 QALYs per tested patient, or 0.19 per infected patient. Furthermore, from a third-party payer perspective, the adoption of the LDA-based strategy would reduce the expected health-care cost for suspected and confirmed Lyme disease by roughly 40%, ie about $410, €130, and €170 per tested patient in the United States, Germany, and Italy, respectively, compared to sTTT. The results are most sensitive to the infection rate in the tested population, with LDA maintaining a cost advantage for Lyme disease active infection rates ≥0.8-2.5%. CONCLUSION: LDA early diagnostic testing and subsequent treatment of subjects with early Lyme disease without EM are expected to outperform traditional management strategies both clinically and economically in the US, Germany, and Italy.

Serum active 1,25(OH)2D, but not inactive 25(OH)D vitamin D levels are associated with cardiometabolic and cardiovascular disease risk in psoriasis.

BACKGROUND AND AIMS: Vitamin D exists as an inactive 25-hydroxyvitamin D (25(OH)D) in the bloodstream, which is converted to active 1,25-dihydroxyvitaminD (1,25(OH)2D) in target tissues. Cohort studies reporting cardiovascular disease among individuals with low vitamin D are inconsistent and solely measure 25(OH)D. Psoriasis, a chronic inflammatory disease, is a vitamin D deficient state and is associated with increased cardiovascular disease risk. While serum 25(OH)D is routinely measured, we hypothesized that measurement of 1,25(OH)2D in psoriasis may perform better than 25(OH)D in capturing cardiovascular risk. METHODS: Consecutive psoriasis patients (N = 122) at baseline underwent FDG PET/CT and CCTA scans to measure visceral adipose volume, aortic vascular uptake of FDG, and coronary plaque burden respectively. Blood levels of both 1,25(OH)2D and 25(OH)D were measured by chemiluminescence (LIAISON XL DIaSorin, Stillwater, MN). RESULTS: The psoriasis cohort was middle-aged (mean ±â€¯SD: 49.6 ±â€¯13.0), predominantly male (n = 71, 58%), in majority Caucasians (n = 98, 80%), and had moderate-to-severe skin disease [psoriasis area severity index score, PASI score, med. (IQR): 5.5 (3.2-10.7)], with almost one-fourth of the cohort on biologic psoriasis therapy for skin disease management (n = 32, 27%) at baseline. Interestingly, serum levels of 1,25(OH)2D but not 25(OH)D were found to be inversely associated with visceral adipose, a marker of cardiometabolic risk in fully adjusted models (ß = - 0.43, p = 0.026 and ß = -0.26 p = 0.13). Similarly, we found an inverse relationship between 1,25(OH)2D, but not 25(OH)D, and aortic vascular uptake of FDG independent of traditional risk factors (ß = -0.19, p = 0.01). Finally, we found that serum 1,25(OH)2D, but not 25(OH)D, was inversely associated with non-calcified coronary plaque burden, as measured by CCTA independent of traditional risk factors (ß = -0.18, p = 0.03). CONCLUSIONS: In conclusion, we demonstrate that low 1,25(OH)2D levels were associated with visceral adipose volume, vascular uptake of FDG and coronary plaque burden independent of traditional risk factors, suggesting that 1,25(OH)2D may better capture the cardiometabolic risk associated with vitamin D deficient states.

Anchor-based fluorescent amplicon generation assays (FLAG) for real-time measurement of human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus viral loads.

BACKGROUND: Monitoring the human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes. METHODS: The fluorescent amplicon generation (FLAG) technology uses the PspGI restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing an accessory oligonucleotide "anchor" that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated an internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay. RESULTS: The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1-10(7) copies/microL), analytical sensitivity (0.420 copies/microL), and intra- and interassay imprecision. CONCLUSIONS: The a-FLAG assay is an alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity.

Development of automated assays for anticardiolipin antibodies determination: addressing antigen and standardization issues.

The lack of reliable standardization tools as well as the poorly defined nature of the "Cardiolipin antigen" makes the development of the anticardiolipin antibody (ACA) assays (for anti-IgG and IgM detection) highly challenging. This article describes how several issues have been solved during the development of an automated ACA immunoassays, based on a technology that includes paramagnetic microbeads as solid-phase reagents and chemiluminescence as a signal. The technology is adapted to an automatic immunoanalyzer, called LIAISON, which performs, in an automatic manner, the whole assay, starting from the primary tube of the bleeding to the display of the assay result. Briefly, the magnetic microbeads were coated with an ethanolic solution of cardiolipin (CL) followed by an affinity-purified, cross-linked human beta2-glycoprotein I. CL-coated paramagnetic microbeads, after incubation with an ACA-positive sera plus addition of immunogold-protein A, were visualized by SEM, showing the presence of well-defined protein clusters on the microbeads surface as an indication of the successful occurrence of the "antigen" coating. The assay standardization was achieved on the basis of human samples containing various amount of ACA, which were previously classified according to consensus doses. The evaluation of the optimized LIAISON Cardiolipin assays (IgG and IgM) was conducted by using clinically characterized APS sera. The results of the evaluation showed that the LIAISON assays perform at least similar to certain well-established ACA enzyme-linked immunosorbent assay (ELISA) products.

A novel, fully-automated, chemiluminescent assay for the detection of 1,25-dihydroxyvitamin D in biological samples.

BACKGROUND: 1,25-Dihydroxyvitamin D (1,25-(OH)2D), the hormonal form of vitamin D, is difficult to measure because of its low circulating levels (pg/mL), and similarity to more abundant metabolites. Here a fully-automated chemiluminescent assay that accurately and precisely measures 1,25-(OH)2D is described. METHOD: The novel 1,25-(OH)2D assay was conceived based on four pillars: (1) the VDR's ligand binding domain (LBD) as a capture molecule; (2) reaction conditions wherein 1,25-(OH)2D favors binding to LBD vs. the vitamin D binding protein; (3) exploitation of liganded-LBD's conformational change; (4) a monoclonal antibody specific to liganded-LBD. This specific, conformational, sandwich approach, unique for automated measurement of haptens, is superior to more cumbersome, conventional competitive formats. RESULTS: Accuracy of the 1,25-(OH)2D assay was corroborated by its alignment against LC-MS/MS with fit Deming regression equations of y=0.98x + 1.93 (r=0.92), and y=1.07x+3.77 (r=0.94) for different methods from Endocrine Sciences, Laboratory Corporation of America® and the University of Washington, respectively. Good analytical precision was manifested by its low estimated limit of quantitation (1.57pg/mL), average intra-assay imprecision (3.5%CV; range 1.1-4.7%), and average inter-assay imprecision (4.5%CV; range 3.4-7.2%). Expected and measured recovery values were congruent (93.4% mean). CONCLUSIONS: The novel 1,25-(OH)2D method exhibited excellent correlation with well validated LC-MS/MS assays from two laboratories. Significantly, its 65min turn-around time is quicker, and sample volume smaller (75µl) than current methods.

A low plasma 1,25(OH)2 vitamin D/PTH (1-84) ratio predicts worsening of renal function in patients with chronic heart failure.

BACKGROUND: Dysregulation of the vitamin D system promotes renal dysfunction and has direct detrimental effects on the heart. Progressive deterioration of renal function is common in patients with chronic heart failure (HF) and is invariably associated with unfavorable outcomes which can be improved by early identification and timely interventions. We examined the relation between two plasma markers of vitamin D metabolism and worsening of renal function (WRF) in a large cohort of patients with chronic HF. METHODS: Plasma levels of 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone PTH (1-84) were measured in 1237 patients with clinical evidence of chronic and stable HF enrolled in the multicentre GISSI-HF trial and followed for 3.9years. We examined the relation of 1,25(OH)2D, PTH(1-84), and their ratio with WRF, defined as first increase in serum creatinine concentration ≥0.3mg/dL and ≥25% at two consecutive measurements at any time during the study. RESULTS: Lower 1,25(OH)2D/PTH(1-84) ratio was associated with a higher baseline serum concentration of creatinine, winter season, female sex and older age; 335 patients (29.6%) experienced an episode of WRF. After adjustment, a lower 1,25(OH)2D/PTH(1-84) ratio remained significantly associated with a higher risk of WRF (HR=0.75 [0.62-0.90], p=0.002) and correctly reclassified events. This ratio also independently predicted mortality and admission to hospital for cardiovascular reasons. CONCLUSIONS: The plasma 1,25(OH)2D/PTH(1-84) ratio is a promising indicator of future risk of deterioration of renal function in patients with chronic HF and mild renal impairment, that may serve to optimize therapies and improve outcomes.

1,25-Dihydroxyvitamin D to PTH(1-84) Ratios Strongly Predict Cardiovascular Death in Heart Failure.

OBJECTIVES: Vitamin D deficiency and hyperparathyroidism are common in patients with heart failure (HF). There is a growing body of evidence supporting the role of vitamin D and parathyroid hormone (PTH) in cardiac remodeling and worsening of HF. Lack of reliable automated testing of 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active metabolite of vitamin D, has limited its contribution to the prognostic assessment of HF. Here, the association of 1,25(OH)2D and PTH(1-84) levels was evaluated for prediction of cardiovascular death in chronic HF patients. METHODS: We conducted a single center prospective cohort including 170 chronic HF patients (females n = 36; males n = 134; NYHA II-IV; mean age: 67 years; etiology: ischemic n = 119, dilated cardiomyopathy n = 51; mean LVEF: 23%). The primary outcome was cardiovascular death. RESULTS: Serum levels of 1,25(OH)2D decreased markedly with increased HF severity. Medians were 33.3 pg/mL for NYHA-II patients, 23.4 pg/mL for NYHA-III, and 14.0 pg/mL for NYHA-IV patients (p<0.001). Most patients had levels of 25(OH)D below 30ng/mL, and stratification by NYHA functional class did not show significant differences (p = 0.249). The 1,25(OH)2D to PTH(1-84) ratio and the (1,25(OH)2D)2 to PTH(1-84) ratio were found to be the most significantly related to HF severity. After a median follow-up of 4.1 years, 106 out of 170 patients reached the primary endpoint. Cox proportional hazard modeling revealed 1,25(OH)2D and the 1,25(OH)2D to PTH(1-84) ratios to be strongly predictive of outcomes. CONCLUSIONS: 1,25(OH)2D and its ratios to PTH(1-84) strongly and independently predict cardiovascular mortality in chronic HF.

Development of recombinant human IgA for anticardiolipin antibodies assay standardization.

Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents.