MSACL 2016 EU Abstract

Best Practices for Long-term Quality Assurance of Quantitative Clinical Chemistry Proteomics Tests

Renee Ruhaak (Presenter)
Leiden University Medical Center

Authorship: L. Renee Ruhaak, Yuri E.M. van der Burgt, Nico P.M. Smit, Fred P.H.T.M. Romijn, Mervin Pieterse, Arnoud van der Laarse, Christa M. Cobbaert
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands

Short Abstract

Mass spectrometry (MS) is a promising technology that experiences an increased interest for its application in quantitative clinical chemistry proteomics (qCCP), due to its high specificity and its potential for multiplexing. However, MS is a complex technology, which requires expanded quality assurance procedures and predefined quality criteria to guarantee accurate and reliable results in the clinical laboratory setting. Here, we review our best practices for quality assurance of MS-based qCCP tests in time and space. We are confident that our findings can be generalized and transferred to other applications and labs, enabling ISO 15189:2012 accreditation of qCCP tests.

Long Abstract

Clinicians make medical decisions based on test results that are assumed to be accurate and interchangeable among labs, no matter in which laboratory and on which day the test results were obtained. Therefore, quality assurance and standardization of tests is of utmost importance both during test development and routine application. The use of mass spectrometry (MS) for absolute quantification of protein biomarkers has become a promising alternative for clinical immunoassays that are either flawed or do not recognize disease specific proteoforms [1]. Mass spectrometry has high specificity for detecting and quantifying defined measurands such as specific protein(s) or proteoforms and matrix effects are accounted for by using isotopically labelled internal standards. MS has the potential to provide highly accurate and robust results, and is considered to be a disruptive technology enabling rapid translation of promising protein biomarkers into medical tests.

Mass spectrometry is anno 2016 far more complex than immunoassay-based technologies used in (semi-)automated medical laboratories. To bring qCCP tests into medical laboratories, metrological traceability of test results is key. Firstly, during sample preparation the proteins of interest are digested by trypsin into proteotypic peptides, which means that the measurand is changed across the traceability chain. The quality and type of trypsin used are highly relevant and co-determine the enzymatic digestion efficiency. The digestion conditions are relevant too and should be optimized in such a way that equimolarity between protein and quantification peptide is guaranteed The challenge is that the concentration of signature peptide(s) should reflect the concentration of intact protein(s), taking into account peptide instabilities, and the presence of genetic variants, polymorphisms or PTMs. Secondly, to get accurate results, at least two peptides should be monitored per protein, and each peptide is monitored with at least one quantifying and one qualifying transition, without the risk of product ion cross-talk. Thirdly, internal standards (peptide, intact protein, or other) should be representative for the protein measurand(s), at an appropriate concentration and with accurate purity. Fourthly, calibrators are human, value-assigned specimens that behave like patient samples and undergo the entire MS-workflow, just like the unknowns. To ensure accuracy of test results in qCCP tests, commutability of internal standards and external calibrators is of high importance.

After qCCP development, quality procedures and predefined quality criteria are essential to guarantee constant performance of qCCP tests in time. Recently, the Clinical and Laboratory Standards Institute (CLSI) guideline C62-A on “Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline” [2] was released that provides a good starting point but does not foresee in specific quality assurance recommendations for qCCP tests [3]. Procedures are needed for preventive instrument maintenance, both for liquid handling equipment and the LC/MS on a weekly, quarterly and yearly basis to evade instrument contamination and avoid a gradual decline in instrument performance. Furthermore procedures and specifications should be determined to establish the instrument starting conditions, which include intervals and acceptance criteria for instrument calibration and the incidence and number of system suitability checks together with their acceptance criteria (e.g. similar to [4]). The latter checks should provide an accurate indication of the suitability of the systems for the clinical test at any given time.

Besides such preventative measures, quality assurance should also be incorporated in the sample run, both by inclusion of internal QC samples and by monitoring of test specific parameters such as retention time, ion ratio, peak shape and absolute abundance of the internal standard. Criteria for these variables should be predefined and could either be minimal and/or maximal performance criteria, or maximal allowable deviation (%).

We summarize our best practices for long-term quality assurance of lab developed qCCP tests. Application of predefined quality criteria enables constant performance of qCCP tests, which is a prerequisite for ISO 15189 accreditation of these lab-developed tests.


References & Acknowledgements:

1. Hoofnagle, A.N. and M.H. Wener, The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J Immunol Methods, 2009. 347(1-2): p. 3-11.

2. Clinical and Laboratory Standards Institute, C62-A. Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline. 2014.

3. Vogeser, M., Mass spectrometry in the clinical laboratory - Challenges for quality assurance, in Current trends in Mass Spectrometry. 2015. p. 14-19.

4. Abbatiello, S.E., et al., Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS). Mol Cell Proteomics, 2013. 12(9): p. 2623-39.


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