Development of Reference Methods and Reference Materials for the Determination of Cardiac Troponin I in Human Serum

Summary

Cardiac troponin I (cTnI) is considered the 'gold standard' for the diagnosis of myocardial infarction and the assessment of cardiac damage. Yet this clinical measurement is plagued with significant inter-assay comparability problems. Through collaboration with a variety of professional organizations, research laboratories, and commercial manufacturers world-wide, NIST is developing the measurement infrastructure needed to improve the quality of clinical cTnI measurement. The NIST effort involves the development of new reference measurement procedures utilizing both enzyme-linked immunoassays and quantitative mass spectrometry to quantify cTnI in serum at levels as low as 50 ng/L. Serum pools with multiple levels of cTnI will be used to produce certified reference materials.

Description

The clinical measurement of serum cardiac troponin I (cTnI) has become one of the most important tools in the diagnosis of acute myocardial infarction and myocardial damage. Unfortunately, considerable variability in clinical cTnI assay results is observed when comparing assays from different manufacturers. With so much variation between assays, physicians and clinical laboratory staff are required to establish their own decision points for cTnI based on the assay used. Standardization of clinical cTnI measurements is needed to provide more reliability in the use of cTnI assays.

Although in 2004 NIST issued SRM 2921 (Human Cardiac Troponin Complex), the use of this certified reference material by assay manufacturers has not led to the desired level of inter-assay comparability. A higher level of troponin I assay standardization has not been achieved because NIST SRM 2921 alone does not provide the complete reference measurement system necessary to improve cTnI measurement quality. What is still needed are matrix-based reference materials and the reference measurement procedures through which certified concentrations of cTnI in matrix reference materials can be determined. Through collaboration with the International Federation of Clinical Chemistry (IFCC), other national metrology institutes, clinical researchers, and cTnI assay manufacturers, reference measurement procedure and serum-based reference materials for cTnI are being developed. Once these materials and methods are established, the components of complete reference measurement system for cTnI will be in place. The impact of this reference measurement system will be a significant improvement in the accuracy and comparability of clinical cTnI assays allowing this important clinical measurement to be used with a higher level of confidence.

Additional Technical Details

The first goal of this project is the development of an ELISA-based reference measurement procedure to measured cTnI in serum. This task is being accomplished through collaboration between NIST and the National Physical Laboratory (NPL) of the United Kingdom. To support this effort, NIST has developed a variety of analytical techniques to evaluate the quality of commercial anti-cTnI monoclonal antibody reagents. These techniques include multiplexed bead-based assays and quantitative mass spectrometry of both antibodies and antigen. The result of the analytical characterization of the monoclonal antibodies was used to guide the design of the ELISA-based reference material. This analytical 'toolbox' for antibody evaluation also enables the affinity reagents used in the ELISA to be characterized at the molecular level, producing an ELISA whose performance has been characterized analytically more thoroughly than commercial ELISAs. This results in an ELISA that can be considered to be a higher order method as compared to routine cTnI assays. To complement the ELISA reference measurement procedure, a mass spectrometry-based assay for serum cTnI is also being developed.

After establishing the ELISA reference measurement procedure, a multi-level cTnI serum reference material will be developed through collaboration with the IFCC. To produce a serum pool that is commutable with the majority of current routine cTnI assays, a series of different pooling strategies will be evaluated through a pilot project involving assay manufacturers. The outcome of this pilot project will be an optimized protocol for the production of the large serum pools needed for the cTnI reference materials.

Major Accomplishments:

• An ELISA-based reference measurement procedure has been developed for measuring human cardiac troponin I in serum and will be evaluated in a pilot project to assess commutability
• To support the development of the ELISA-based troponin I reference measurement procedure, a variety of techniques (including bead-based multiplexed assays and mass spectrometry measurements) have been developed to evaluate commercial anti-troponin I antibody performance with regards to relative binding strengths and specificity.
• A mass spectrometric method to quantify human cardiac troponin I in serum is being developed to complement the ELISA-based reference measurement procedure for the future certification of a serum matrix reference material.

Associated Product(s)

SRM 2921 Human Cardiac Troponin Complex

Associated Publications:

Panteghini, M., Bunk, D.M., Christenson, R.H., Katrukha, A., Porter, R.A., Schimmel, H., Wang, L., Tate, J.R., "Standardization of troponin I measurements: an update," Clin. Chem. Lab. Med. 2008;46(11):1501-6.