Chromogenic ELISA Research Articles

Cost-Effectiveness of Rapid Hemosil Versus in-House and Send-out ADAMTS13 Activity Testing in the Care of Patients with Immune Thrombotic Thrombocytopenic Purpura

INTRODUCTION Immune thrombotic thrombocytopenic purpura (iTTP) is a thrombotic microangiopathy requiring immediate treatment when the pretest probability is intermediate or high, a probability determined with the use of the PLASMIC and/or French clinical scores. Ruling out iTTP with a biomarker ADAMTS13 activity level can take several days at most hospital systems in the United States, all the while empiric treatment for iTTP accrues risks and costs. The newly developed rapid turnaround and fully automated, chemiluminescent immunoassay (HemosIL) reveals high concordance with chromogenic ELISA and FRET-based assays, the diagnostic standard-of-care. Unlike rapid in-house testing requiring technical expertise, the HemosIL assay can abrogate the cost and risk exposure associated with treating patients for iTTP whose biomarker activity level later rules out this diagnosis. Despite existing concerns of testing cost, we hypothesized that rapid and fully automated diagnostic testing would be cost-saving with similar quality-adjusted life-expectancy, as compared to both in-house FRET-based evaluation and send-out FRET-based evaluation. We further posited that this effect would be greater in the context of 1) intermediate- versus high-probability PLASMIC scores, and 2) patients treated with caplacizumab versus without. We sought to fill these gaps by conducting the first cost-effectiveness evaluation of the HemosIL assay in patients treated with and without caplacizumab. METHODS For this independent analysis free of industry influence, we built a Markov simulation of patients with intermediate- (PLASMIC score =5) or high- (PLASMIC score 6-7) pretest probability for iTTP empirically treated while awaiting results under three testing strategies: 1) HemosIL rapid turnaround (<1 hour), 2) 24-hour in-house, FRET-based evaluation, and 3) 72-hour send-out, FRET-based evaluation. For transition probabilities, our model was informed by extensive literature published on test characteristics, as well as derivation and validation cohorts for the PLASMIC score. Adverse events for therapeutic plasma exchange (TPE) and caplacizumab were informed from clinical trial data and real-world data, with costs in 2023 US dollars sourced from the Centers for Medicare and Medicaid Services. Utilities were informed by literature on critically ill adults hospitalized in the medical intensive care unit and were originally derived with validated EuroQol methodology. Age- and sex-adjusted background mortality were employed. The primary outcome was the incremental cost-effectiveness ratio (ICER) or, the incremental net monetary benefit (iNMB) if the intervention (HemosIL) was found to be cost-saving, reported over a 3-day time-horizon and across accepted willingness-to-pay thresholds in USD per quality-adjusted life-year (QALY). Four scenario analyses were examined: 1) PLASMIC 5 with caplacizumab, 2) PLASMIC 5 without caplacizumab, 3) PLASMIC 6-7 with caplacizumab, 4) 6-7 PLASMIC without caplacizumab. We concluded by executing deterministic and probabilistic sensitivity analyses, varying all parameter estimates +/-50% and capturing uncertainty in all parameters simultaneously over 10,000 Monte Carlo simulations. RESULTS Across all scenario analyses, testing with HemosIL was cost saving and accrued the same amount of QALYs (Table 1). These savings with HemosIL ranged from $2,900 (PLASMIC 6-7, without caplacizumab) through $44,700 (PLASMIC 5, with caplacizumab) per-patient-tested versus send-out, and from $800 to $19,800 versus in-house testing. No parameter variation changed the outcome of any scenario to favoring a non-HemosIL testing strategy in deterministic sensitivity analysis. In probabilistic sensitivity analyses across accepted willingness-to-pay thresholds in the United States, HemosIL was favored in 100% of iterations in the PLASMIC 5 context (with and without caplacizumab), and 99.9% and 98.6% in the PLASMIC 6-7 context with and without caplacizumab use, respectively (Table 2). CONCLUSION Despite cost concerns, HemosIL testing for patients with intermediate- or high-risk PLASMIC score produces significant cost savings for each patient tested and empirically treated. This is achieved by reducing the costs associated with unnecessary therapeutic plasma exchange and caplacizumab therapy.

ADAMTS13 levels in a plasma-derived FVIII concentrate: A potential therapeutic option for patients with congenital thrombotic thrombocytopenic purpura

IntroductionTreatment of congenital thrombotic thrombocytopenic purpura (cTTP), a disease characterized by the congenital deficiency of ADAMTS13, remains a challenge as there are no specific treatments available yet, other than therapy based on the use of fresh frozen plasma (FFP). Since cTTP is caused by low levels of ADAMTS13 protein, commercially available coagulation factor concentrates have been considered as potential ADAMTS13 source in place of FFP. The study aimed to validate the therapeutic potential of a plasma-derived factor VIII (FVIII) product as a source of ADAMTS13. MethodsThe quantitation of ADAMTS13 activity levels in eight lots of a plasma-derived FVIII product, (Koāte®) was carried out with three different methodologies: a Fluorescence Resonance Energy Transfer (FRET) assay, a chemiluminescence assay, and a chromogenic ELISA. ADAMTS13 protein antigen levels were measured by the FRET technique as well. In addition, von Willebrand factor (VWF) activity (VWF ristocetin cofactor, VWF:RCo, and VWF collagen binding, VWF:CB) and antigen (VWF:Ag) were measured using chemiluminescence assays. Qualification protocols were applied to the methods used. ResultsThe results showed high levels of ADAMTS13 in all eight Koāte® lots analyzed, with antigen and activity levels respectively of 10.72 IU/ml ± 3.94 and 5.62 IU/ml ± 1.39. Despite the significant content of ADAMTS13, VWF integrity seems not to be affected (0.81 ± 0.15 VWF:RCo/VWF:Ag and 0.75 ± 0.15 VWF:CB/VWF:Ag ratios). ConclusionsThese findings suggest that Koāte® could be a potential candidate for the treatment of cTTP, warranting evaluation in a clinical trial.

International Council for Standardization in Haematology (ICSH) recommendations for laboratory measurement of ADAMTS13.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune-mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l−1 and 4.2 nmol l−1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml−1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol l−1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C18 columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg−1, and 200 μg·kg−1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg−1.