American Association for Clinical Chemistry
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Expo Issue 2011: On the Road to cTnI Standardization

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Expo Issue 2011

On the Road to cTnI Standardization
Session Highlights Challenges in Standardization, Harmonization

By Genna Rollins

Cardiac troponin I (cTnI) has become the bedrock biomarker for assessing suspected acute coronary syndrome, and research based on new high-sensitivity cTnI assays not yet commercially available in the U.S. point to its potential role in new avenues of risk assessment, diagnosis, and, eventually, treatment of cardiac diseases. Yet, as solid a place as cTnI has in clinical cardiology, there are some challenges related to measurement of this important analyte. Today’s morning symposium, “Cardiac Troponin I Standardization—A Model for Harmonization,” will explore the analytical issues related to cTnI measurement and update attendees on a substantial international effort to achieve standardization and harmonization for cTnI.

The symposium, which starts at 9:30 a.m. in Room B314 of the Georgia World Congress Center, is being moderated by Heinz Schimmel, PhD, a scientific officer at the European Commission’s Institute for Reference Materials and Measurements Joint Research Center in Geel, Belgium. “Cardiac troponin I standardization is needed because it is a very important marker for myocardial infarction, and because there are very discrepant results on the same samples using the different commercially available assays,” he explained. “This poses challenges that clinical laboratorians need to be aware of, and we hope to educate the audience about all the issues involved as well as the process for achieving standardization.” At least 18 different commercial cTnI assays are available in the U.S.

Schimmel is one of nine members of the International Federation for Clinical Chemistry and Laboratory Medicine’s (IFCC) Working Group on cTnI standardization (See Box, below). Two other Working Group members will speak at the symposium, including chair Jillian Tate, BSc, MSc, a senior scientist in the department of clinical pathology at Pathology Queensland in Brisbane, Australia, and David Bunk, PhD, a research chemist at the National Institute of Standards and Technology (NIST) in Gaithersburg, Md. The Working Group met earlier this week in Atlanta, so symposium panelists will be able to provide attendees with the latest update on their progress and deliberations.

Members of the IFCC Working Group on cTnI Standardization

Jillian Tate, BSc, MSc, Chair, Australia
Julian Barth, MD, U. K.
David Bunk, PhD, U.S.
Robert Christenson, PhD, U. S.
Alexei Katrukha, PhD, Finland
Mauro Panteghini, MD, Italy
Robert Porter, PhD, U. K.
Heinz Schimmel, PhD, Belgium
Lili Wang, PhD, U. S.

The Working Group also has five corresponding members representing clinical chemistry societies from France, Germany, Greece, Japan, and Portugal, and 10 representing IVD manufacturers.

The Challenge of Heterogeneous Proteins

Already, considerable effort has gone into standardizing cTnI, but as with other heterogeneous and low-concentration proteins, the process has been fraught with challenges, according to Schimmel. When the IFCC Working Group started, there was up to a 100-fold discrepancy between the various assays in measuring the same sample. NIST led development of a standard reference material (SRM 2921) from an isoform of cTnI with a value assigned by mass spectrometry, but this proved not to be a complete standardization solution. “Against expectations, this material is only leading to a harmonization effect in 50 percent of the commercial assays, meaning that only about half of them came together with more comparable results after using the material. The rest remained with scattering to the same extent as before,” Schimmel recalled.

Some commercial assays were recalibrated using the reference material, which reduced the interassay discrepancy for those assays to a much-improved, but still undesirable, 5% difference. “A five-fold difference is still an awful lot if you have to make decisions on the basis of such deviating measurement results,” said Schimmel. Studies verifying this continued variability led the Working Group to determine that the reference material was not commutable, meaning that it does not show properties like those of human samples.

Creating a Secondary Reference Material

The Working Group then realized that a commutable secondary reference material with value assignment by a higher order reference measurement procedure would be needed. Panelist Tate will update the audience on the round robin study aimed at developing a serum-based secondary reference material for cTnI. This complex project involves collecting at least 90 blood samples from patients diagnosed with myocardial infarction in order to develop a matrix serum material.

Separately, the U.K.’s National Physical Laboratory (NPL), working collaboratively with NIST, is developing a standardized immunoassay procedure to achieve a value transfer from the purified SRM 2921 to the matrix (secondary reference) material, activities that panelist James Noble, PhD, a scientist in NPL’s Analytical Science Division, will describe during his presentation. An immunoassay is being used because mass spectrometry methods are too insensitive, according to Schimmel. “The concentration of troponin in the matrix material will be so low that it will be below the limits of quantitation for mass spectrometry methods,” he explained. The Working Group already has reported that its proposed reference measurement procedure showed no bias when tested on samples containing various troponin complexes (Clin Chem Lab Med 2010;48:1603–10).

Changing Assay Targets

Another step in narrowing inter-assay variability came with a proposal made by the Working Group and endorsed by the IFCC that called for cTnI assays to target the stable region of troponin. Specifically, IFCC recommended that antibodies used in cTnI assays should selectively recognize epitopes in the central part of the cTnI molecule that are not affected by troponin IC or ITC complex formation. Schimmel explained that while this had proven helpful, it too had not adequately reduced the discrepancy between assays.

“Although the assays should now measure the stable region of troponin I, this hasn’t lead to a sufficient agreement between the assays,” he said. “There are more issues to be sorted out, and what the group is currently looking at is the interaction of the target molecule with other troponin components. There may be interaction with other compounds in the serum, which may have an impact on assay performance. Another issue to be considered by the IFCC Working Group is the definition of performance goals for assays at low levels of troponin I.”

Can cTnI be Standardized?

Given the twists and turns the Working Group has encountered so far, is standardization of cTnI even possible? Schimmel believes it is. “We think troponin can be standardized,” he said. “We have to be careful in how we do it, taking into consideration all the parameters which have an influence on our measurements. If they can be controlled—and that’s what the Working Group is trying to do—we think it will be possible to standardize at least the majority of the assays.”

With so many efforts underway on so many fronts, Schimmel hesitated to predict when cTnI standardization would be considered fait accompli. “All the different steps are linked together. The greatest mistake you can make if you want to standardize complex proteins like cardiac troponin is that you try to separate the issues,” he said. “The collection of samples for the serum matrix material will really be a challenge, but once we can demonstrate that this candidate reference material is commutable, then the value assignment should be completed rather rapidly.”

The Working Group’s considerable progress despite myriad challenges should encourage attendees about standardization not only of cTnI, but also other analytes, according to Schimmel. “The basic message is that people really should understand that standardization of heterogeneous proteins is not a simple thing to do,” he observed. “However, on the other hand, if you try to understand the issues and do the right studies, there is a good chance that you can standardize even complex proteins.”