June 2019 Medical Mass Spectrometry Vol. 3 No. 1 Review Applications of mass spectrometry in clinical chemistry Mamoru Satoh, Fumio Nomura* Division of Clinical Mass Spectrometry, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan Abstract Mass spectrometry (MS) is now an essential technology for laboratory medicine. The most successful applica- tion of MS in laboratory medicine is the rapid identification of microorganisms using matrix-assisted laser desorption/ion- ization time-of-flight (MALDI-TOF) MS. However, the adoption of MS-based assays for routine testing in clinical chemis- try is very slow in Japan. In this review, we summarize the current status of clinical mass spectrometry in the US, Europe, and Japan, and discuss the possible reasons why Japan is behind in this regard. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a highly accurate and reproducible analytical tech- nique, but a number of challenges and pitfalls remain for routine use in clinical laboratories, such as the effects of various pre-analytical factors. Indeed, we observed a significant noise peak during routine measurements of vitamin D metabolites, most likely due to a separating gel in blood collection tubes used for particular specimens sent by another hospital. Ion sup- pression due to matrix effects and problems associated with stable isotope labeled internal standards should also be consid- ered. MS assays are typically laboratory developed tests at present. As LC/MS/MS procedures become more automated and more MS-related in vitro diagnostics become commercially available, the application of LC/MS/MS to laboratory medicine will be significantly accelerated. Key words: LC/MS/MS, MALDI-TOF MS, clinical chemistry, matrix effect, pre-analytical factors Introduction ics”. For example, it was recently reported that the BoxCar Advances in mass spectrometer have been remarkable, acquisition method enables single-shot proteomics analysis and increasing numbers of highly sensitive, user-friendly of 10,000 proteins in 100 minutes1). Over recent years, novel instruments are released by instrument manufacturers sophisticated instruments have been developed not only for every year. Mass spectrometry (MS) is now an essential basic research but also for specialized clinical applications. technology for basic biological research and clinical medi- In the latter cases, sample preparation and LC-MS are inte- cine. Rapid progress in MS-related technologies, including grated into the system. Moreover, advanced ionization pro- sample preparation methods, labeling reagents, stable iso- cess, operating system, and the required reagents including tope labeling reagents and peptide synthesis technologies, calibrators, quality control samples and ready-to-use sol- has led to the evolution of“ Proteomics” and“ Metabolom- vents are now commercially available. Several ionization methods, such as Desorption Electrospray Ionization *Corresponding author 2) 3) (DESI) , Probe Electro Spray Ionization (PESI) and Fumio Nomura, MD, PhD. Rapid Evaporative Ionization Mass Spectrometry Division of Clinical Mass Spectrometry, Chiba University (REIMS)4) have been used for intraoperative rapid patho- Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒ logical diagnosis and real-time analysis of biological sam- 8670, Japan Tel: ＋81‒43‒226‒2627, Fax: ＋81‒43‒226‒2627 ples. Mass cytometry is a novel technique similar to flow E-mail: [email protected] cytometry and is widely used in hematological examina- 5) Received March 18, 2019. Accepted April 26, 2019. tions . Clinical applications of mass spectrometry will Epub June 18, 2019. become more widespread as technological innovations DOI: 10.24508/mms.2019.06.005 advance. 2 June 2019 Medical Mass Spectrometry Vol. 3 No. 1 MS is used in various fields in Japan, including newborn stances, antibodies are not specific enough and may lead to screening for inborn errors of metabolism, forensic toxicol- cross-reactivity between closely related compounds. LC/ ogy, and therapeutic drug monitoring. The most successful MS/MS can overcome this limitation and is increasingly application of MS in laboratory medicine is the rapid identi- used for clinical chemistry, mainly in Europe and the fication of microorganisms using matrix-assisted laser United States. For example, approximately 15% of the desorption/ionization time-of-flight (MALDI-TOF) MS. papers presented at the scientific poster session of the Indeed, there has been a revolutionary shift in clinical diag- American Association for Clinical Chemistry (AACC) over nostic microbiology6). Since an early report of the applica- the past few years dealt with“ Mass Spectrometry Applica- tion of MALDI-TOF MS-based bacterial identification in tions.” At AACC, a number of MS manufacturers display clinical laboratory on a routine basis7), this technology is and introduce their new instruments for laboratory person- increasingly used globally. In Japan, as of August 2018, nel. The Association for Mass Spectrometry Applications to there were around 200 instruments (either MALDI Bio- the Clinical Labs (MSACL) has the mission to accelerate typer: Bruker, USA or VITEK® MS: bioMérieux, France) the implementation of mass spectrometry in the clinical lab- in operation for routine clinical use. MALDI-TOF MS is oratory with the goal of contributing to the improvement of particularly useful for rapid bacterial identification in blood patient standard-of-care as well as reducing health care culture specimens, providing analyses 1.5 to 2 days faster costs. Their annual meetings are held twice a year, one each than conventional methods and thus helping shorten hospi- in the United States of America and Europe. tal stays and improve overall prognosis. Moreover, a 2018 The Clinical Laboratory Improvement Amendments revision of the Japanese health insurance medical fee sys- (CLIA) in the United States is a body of United States fed- tem allows additional charges for microbial identification eral regulatory standards. CLIA certified laboratories can by MS-based methods. This development will accelerate conduct laboratory developed tests (LDT), including the implementation of MALDI-TOF MS in major hospitals LC-MS/MS, in the absence of officially certified IVDs, and in Japan. health insurance reimbursement is generally possible. More In contrast, the adoption of liquid chromatography-tan- recently, FDA has decided to extend their regulations to dem mass spectrometry (LC-MS/MS)-based assays for rou- LDTs as well. The details of this policy have remained tine clinical chemistry testing is very slow in Japan and unknown. A provocative editorial was published in the remains limited to large institutions and reference laborato- Journal of Clinical Endocrinology and Metabolism (JCEM) ries. Many challenges and limitations prevent LC-MS/MS in 20138) indicating that effective January 1, 2015, manu- from being implemented routinely. First, LC/MS/MS scripts reporting sex steroid assays as important endpoints involves complex, labor-intensive workflow compared to must be MS-based assays instead of immunoassays, and the fully automated immunoassay platforms. Another bottle- methods used must be reported or cited in sufficient detail. neck is the absence of officially approved in vitro diagnos- Although this policy has been suspended because of the tic reagents (IVD), including calibrators and preparation complexity of the issues, we believe that it will be eventu- reagents, although there is an FDA-cleared test kit (SCIEX ally effective in the very near future. Vitamin D 200M Assay for the TopazTM System). As more The situation in Japan: Immunoassays are also widely IVD reagents and kits are officially approved and become used in Japan because of the minimal sample preparation commercially available, more LC/MS/MS platforms will be required and rapid turnaround time (TAT). Most laboratory widely used in clinical chemistry laboratories. tests for outpatients are conducted as a same-day test in hospitals that have their clinical laboratories, which Current Status of Mass Spectrometry in Clini- demands short TAT of less than one hour. This requirement cal Chemistry for rapid TAT is one reason preventing the widespread use The situation in the US and Europe: Currently, immuno- of LC-MS/MS in the clinical chemistry field in Japan. assays are widely used for testing proteins, peptides and LC/MS/MS can serve as a gold standard for evaluating hormones in clinical laboratories. However, immunoassays the accuracy of other methods. Serum 25-hydroxyvitamin have several limitations and drawbacks, such as being D [25(OH)D] is an indicator of vitamin D status and is gen- dependent on antibody specificity. Under many circum- erally quantified by immunoassays in Japan. The Japanese 3 June 2019 Medical Mass Spectrometry Vol. 3 No. 1 Society of Clinical Chemistry recently evaluated the stan- between the antibody and other low-abundance metabolites dardization of six automated immunoassays available in with similar structures10). We speculate that a similar expla- Japan9) using the LC/MS/MS method10) developed at Chiba nation will be possible for the discordant results of E2, but University Hospital as a reference method. exact reasons remain to be clarified. The range of correlation coefficients between the values obtained by LC/MS/MS and each immunoassay was 0.73 Challenges and pitfalls in clinical applications to 0.97. The results obtained using six immunoassay kits of LC-MS/MS deviated greatly from the MS assay results