Publication summaries on the Troponin T hs Gen 6 assay and TSIX study program

Speaker: Konstantin Schmidt, PhD. Scientific Communications Lead, Roche Diagnostics International

Today, we are reviewing the results of the REF-TSIX global reference study published by Daniels LB et al in Clinical Chemistry. This publication summarises the primary read-out of this study and establishes the global reference range values for the next-generation high-sensitivity cardiac Troponin T Gen 6 assay. High-sensitivity cardiac troponin—or hs-cTn—is currently the gold standard for the early diagnosis and risk stratification of patients with suspected acute myocardial infarction, or AMI. The 4th universal definition of myocardial infarction defines any cardiac troponin elevation above the 99th percentile upper reference limit (URL) as the recommended reference standard for identifying myocardial injury and is a prerequisite to the diagnosis of myocardial infarction. Nevertheless, various factors, such as age, sex, kidney function, and comorbidities, influence cardiac Troponin values, which, in turn, influence the 99th percentile URL. Therefore, establishing a robust reference range requires a strictly defined, "apparently healthy" population. For this reason, the International Federation of Clinical Chemistry, IFCC, recommends stringent exclusion criteria for the recruitment of a healthy reference population, defining the 99th percentile URL. Furthermore, the IFCC defined cardiac Troponin assays as “high-sensitive” if they i) are able to measure cTn with an analytical precision coefficient of variation (CV) of ≤10% at the 99th percentile URL, and ii) for cTn to be measurable above the assay’s lower limit of detection (LoD) in equal to or more than 50% of healthy female and male participants. 

The REF-TSIX study was a prospective, multicenter, observational study designed to meet this challenge. It recruited participants from 34 sites across the United States of America, Europe, China, and Japan. The objective was to determine sex-specific and uniform 99th percentile URLs for the Gen 6 assay and determine whether the assay fulfills the IFCC “high-sensitivity” criteria. To ensure the reference population was truly healthy, the investigators applied rigorous exclusion criteria. Over 7,900 participants were initially recruited. Per study protocol the following exclusion criteria were applied: symptoms suggestive of cardiovascular disease, a history of cardiovascular disease, NT-proBNP concentrations ≥125 pg/mL (in those aged <75 years) or ≥450 pg/mL (in those aged ≥75 years), chest trauma within the last 14 days, uncontrolled hypertension requiring immediate antihypertensive drug treatment, chronic kidney disease, known diabetes mellitus or glycated hemoglobin (HbA1c) ≥48 mmol/mol, or a history of recreational substance use. Additionally, the exclusion criteria recommended by the IFCC published during the conduct of the study were applied pos-hoc. Therefore, participants were additionally excluded if they were on treatment for any cardiovascular condition or risk factor, including hypertension or hyperlipidemia, had NT-proBNP concentrations ≥125 pg/mL (age-independent), an abnormal body mass index (BMI; <18 kg/m2 or >35 kg/m2), any chronic condition that could affect the heart, recent hospitalization within the last 3 months, or were smokers at the time of enrollment. This resulted in a final analysis cohort of 4,147 individuals, of which 52% were female. This ensured that the derived values represent a physiology free from silent cardiac or renal dysfunction. It was recently reported that the Troponin T hs Gen 6 assay features a formulation change and higher-affinity antibodies compared to the previous generation. The assay showed improved analytical sensitivity and precision, particularly at the lower end of the measuring range. This improved sensitivity is evident in the study results. In this healthy reference cohort, Troponin T was measurable above the limit of detection (LoD 1.5 ng/L) in 81.0% of female and 99.2% of male participants. This is a critical finding as the IFCC requires that a high-sensitivity cardiac Troponin assay measures troponin in at least 50% of healthy female and male individuals. For the primary endpoint the study established the following 99th percentile URLs: For females, the limit is 18 ng/L. For males, the limit is 32 ng/L. The uniform, or overall, limit is 27 ng/L. Consistent with other hs-cTn assays, the values for females are significantly lower than for males. As recommended by the UDMI, sex-specific thresholds should be used to avoid under-diagnosis of cardiac conditions in women. The coefficient of variation at the female, male and uniform 99th percentile URL in plasma was 3.3%, 2.9%, and 3.0%, respectively. This shows that the Troponin T hs Gen 6 assay meets the IFCC high-sensitivity criteria. The study also evaluated the impact of sample type. Reassuringly, the results showed that the upper reference limits were comparable between lithium-heparin plasma and serum. This consistency offers flexibility in clinical laboratory workflows. The advantage of the large, global, multi-center REF-TSIX study is being able to analyse the cohort stratified by region and age. Interestingly, the distribution of Troponin T hs Gen 6 values was consistent across regions in males and females. This underlines the use of a global 99th percentile URL. When stratifying Troponin T hs Gen 6 values by age, cTnT increased with age, with a greater increase observed in females compared with males. This observation has been made in previous studies. However, it should be noted that age-specific 99th percentile URLs are not currently recommended for the diagnosis of myocardial injury or infarction and need to be studied further in future studies. 

In summary, the REF-TSIX study successfully defined robust, global reference ranges for the Troponin T hs Gen 6 assay using a large, strictly defined healthy population. It is important to note that while these reference values are established, the diagnostic performance of these new cut-offs is currently being validated in the PERFORM-TSIX study, involving patients presenting with symptoms of acute coronary syndrome. We will summarise the results of this study in one of our future episodes. Ultimately, these data confirm that the Gen 6 assay meets the stringent IFCC criteria for high-sensitivity designation, providing precise tools for the next generation of cardiac care. Thank you for your attention. for high-sensitivity designation, providing precise tools for the next generation of cardiac care.

Speaker: Konstantin Schmidt, PhD. Scientific Communications Lead, Roche Diagnostics International

High-sensitivity cardiac troponin—or hs-cTn—is currently the gold standard for the early diagnosis and risk stratification of patients with suspected acute myocardial infarction, or AMI. International guidelines emphasize the need for rapid diagnostic pathways to accurately ensure prompt treatment and efficient patient disposition. However, as clinical demands evolve, so must diagnostic tools. Today, we are discussing the study design of the PERFORM-TSIX study design by Daniels LB et al. recently published in Clinical Research in Cardiology. This is a large-scale, global initiative evaluating the next-generation Troponin T high-sensitivity Gen 6 assay. This new assay is designed to provide a rapid, quantitative determination of cardiac Troponin T in both serum and plasma. Compared to previous assay generations, the Gen 6 assay provides higher analytical sensitivity and accuracy, especially at very low concentrations, alongside improved interference tolerance, particularly regarding hemolysis. PERFORM-TSIX is a prospective, observational, longitudinal cohort study. What makes this study unique is the global coverage, the sample size and the diversity of patients and healthcare settings included. Previous troponin studies focus on a single country or healthcare system. 

PERFORM-TSIX enrolled 5,631participants across 50 sites globally from institutions ranging from local community hospitals to academic centers. The enrollment included over 3,200 patients in the USA. Nearly 1,400 patients in Europe. Over 800 patients in China and 125 patients in Japan. This is an "all-comers" cohort consisting of adults aged 20 and older presenting to the Emergency Department with signs or symptoms of possible acute coronary syndrome. The inclusion criteria were set broadly, extending beyond chest pain to include dyspnea, syncope, and generalized fatigue. There are no exclusion criteria to ensure that the study results could reflect real-world clinical practice. The primary objective of PERFORM-TSIX is to determine the sensitivity of the Gen 6 assay for a centrally adjudicated AMI diagnosis at 3 hours post-presentation. The final diagnosis for every patient is adjudicated by an independent Clinical Events Committee. These clinicians are blinded to the Gen 6 assay results and use the Fourth Universal Definition of Myocardial Infarction as their reference. Beyond the primary 3-hour endpoint, the study evaluates clinical performance at multiple other time points as well, namely: 0, 1, 5, and 6 hours. Furthermore, a major component of the study is the validation of rapid diagnostic algorithms. This includes: Validation of thresholds for the 0/1-hour algorithm to rule in or rule out AMI. Validation of a 0/2-hour algorithm for rule-in and rule-out of AMI. By validating these accelerated pathways, the study aims to support faster clinical decision-making, which can significantly reduce the total length of stay in the emergency department. PERFORM-TSIX will also explore the prognostic value of the Gen 6 assay. Patients are followed at 30 and 180 days post-presentation to track Major Adverse Cardiovascular Events, or MACE and all-cause death. This includes monitoring for subsequent MI, all-cause mortality, and further hospitalizations. By linking the initial troponin results and algorithmic categories—Rule-Out, Observe, or Rule-In—to long-term outcomes, the study will provide a comprehensive look at the assay’s utility in long-term risk stratification. 

In summary, the PERFORM-TSIX study is positioned to provide a robust, global validation of the next-generation Troponin T hs Gen 6 assay. Its large sample size and geographically diverse population ensure that its findings will be applicable across a wide range of healthcare settings —from community hospitals to tertiary care centres. Preliminary data presented at international congresses exhibited a preview of the excellent clinical performance, underlining that the assay is ready for clinical practice. All of these studies will be summarised in upcoming episodes. Thank you for watching. For more details on the PERFORM-TSIX study design, please refer to the full publication in Clinical Research in Cardiology.

Speaker: Konstantin Schmidt, PhD. Scientific Communications Lead, Roche Diagnostics International

Welcome. Today, we are reviewing the results of the analytical performance of the next-generation high-sensitivity cardiac troponin T Gen 6 assay recently published by Knoll et al in the Journal of Applied Laboratory Medicine. Clinical practice guidelines worldwide recommend high-sensitivity cardiac troponin assays for the evaluation of myocardial injury and the diagnosis of acute myocardial infarction, or AMI. As per universal definition of myocardial infarction, AMI diagnosis requires: a rise and/or fall in cTn concentrations, with ≥1 value above the sex-specific 99th percentile upper reference limit (URL), along with clinical evidence of ischemia. Cardiac troponin assays that are high sensitive with increased sensitivity and precision especially at the lower end of the measuring range may facilitate more accurate and efficient exclusion of AMI and may further improve cardiovascular risk prediction in the general population where overt cTn elevations are less common. In this publication, the analytical sensitivity, precision, lot-to-lot, matrix, platform-to-platform variation, and interference resistance of the Troponin T hs Gen 6 assay were analyzed. The new Troponin T hs Gen 6 assay has received various updates compared to its predecessor. The original M11.7 antibody was improved by affinity maturation of the monoclonal antibody used in the previous assay version. Thereby, the antibodies used in the Troponin T hs Gen 6 assay recognize the same epitopes, amino acid positions 125–131 and 136–147, located in the stable center region of the cTnT protein. To further improve the performance of the TnT hs Gen 6 assay, the assay formulation was optimized as well. Lastly, a new three-level quality control system was introduced. Compared with the previous assay generation’s two-level quality control, the new has one sample between the LoQ and the lowest sex-specific 99th percentile (~4 nanograms per liter); One with a concentration around the highest sex-specific 99th percentile URL (~30 nanograms per liter); and lastly one sample with multiples of the 99th percentile URL (~220 nanograms per liter) as recommended by the IFCC. To comply with ISO requirements, the metrological traceability of the Gen 6 assay was standardized against recombinant human cardiac troponin T that is traceable to the National Institute of Standards and Technology (NIST) reference materials. This is an important update from the fifth-generation assay, which was standardized to its predecessor, the Generation 4 assay, where the original standardization was against bovine troponin T. Because of this restandardization, values measured by the Gen 6 assay are not numerically equivalent to those from the fifth-generation assay. In general, Troponin T hs Gen 6 values were higher than those for the fifth-generation cTnT hs assay for a given sample. It should also be noted that a conventional conversion factor cannot be applied to convert Gen 5 to Gen 6 values. However, the two assays display a positive, monotonic, non-linear relationship and are highly correlated to each other. The analytical sensitivity and precision of the assay were evaluated using lithium-heparin plasma and serum samples on the cobas® e 801, e 402, and Pro analyzers. The Gen 6 assay demonstrated excellent sensitivity, comfortably meeting its pre-specified targets: the Limit of Blank (LoB) was 1.0 nanograms per liter, the Limit of Detection (LoD): 1.5 nanograms per liter and the Limit of Quantitation (LoQ) at a 10% coefficient of variation (CV) was 3 nanograms per liter and at 20% CV was 1.5 nanograms per liter. The Troponin T hs Gen 6 assay demonstrated high sensitivity and high precision at the low end of the measuring range. Across three reagent lots the repeatability CVs were ranging from 1.0% to 5.8% and the intermediate precision CVs were 2.0–9.0% for mean troponin T concentrations between 2.6 and 9230 nanograms per liter in LiHep plasma samples. Excellent linearity was demonstrated across the entire measuring range of 1.5 to 9500 nanograms per liter. Lastly, high lot-to-lot comparability and sample matrix comparability were observed for LiHep plasma and serum samples. A common challenge in laboratory medicine is that as an assay's sensitivity increases, so too can its susceptibility to analytical interferences. The Gen 6 assay overcomes this through the optimized formulation. The assay demonstrated increased resistance against hemoglobin interference up to 1000 milligrams per deciliter, which is a ten-fold improvement over the previous assay generation. It also improved resistance to bilirubin interference by two-fold, tolerating up to 50 milligrams per deciliter. This means that even in markedly hemolyzed or icteric samples, the impact on assay measurements is minimized. Additionally, the assay maintains robust state-of-the-art resistance to biotin up to 1200 nanograms per milliliter, consistent with the predecessor assay. As mentioned earlier numerical values of Gen 5 and Gen 6 are not directly comparable over the complete measuring range due to the new standardization. However, a detailed analysis showed that at the very low end of the measuring range, meaning 1.5–5 nanograms per liter, values for the fifth-generation and Troponin T hs Gen 6 assays are comparable and demonstrate a somewhat linear behavior. As values below 3 nanograms per liter would not be measurable with the fifth-generation assay and would be unreadable in clinical practice, this shows that Troponin T hs Gen 6 assay has a higher analytical sensitivity compared to the fifth generation. In conclusion, the Troponin T hs Gen 6 assay delivers high analytical sensitivity, excellent precision at the low end of the measuring range, and robust resistance to endogenous interferences. Clinically, this improved analytical sensitivity will enable better differentiation of low troponin values at and even below the 99th percentile upper reference limit and give the opportunity to even allow measurement of small delta changes. This capability holds significant potential to improve the performance of optimized rapid algorithms and risk stratification thresholds, thereby facilitating earlier and safer rule-out of patients with suspected acute myocardial infarction. Thank you for watching this summary of the analytical performance of the Troponin T hs Gen 6 assay, and for more details, please refer to the full publication by Knoll et al published in the Journal of Applied Laboratory Medicine.