Compared to the current standard technologies in clinical diagnosis, MS-based technologies are far more complex in both instrumentation and data analysis, whose performance is influenced by many factors in sample matrix and handling, stability of infrastructure and technician skills. The establishment of a clinical laboratory involves a coordinated effort encompassing various aspects such as space allocation, workflow design, equipment selection, ventilation, lighting, plumbing, electrical systems, and data management. It is important to note that mass spectrometers are not always classified as in vitro diagnostic (IVD) instruments.Furthermore, the choice of method and mass spectrometry not only depends on the expense and maintenance of instrumentation but also requires thorough considerations for the return on investment. The evaluation list to establish a mass spectrometer is detailed in Table 1.

Table 1 Evaluation list to set up LC-MS/MS in a clinical laboratory

When implementing technically complex mass spectrometry (MS) or liquid chromatography-mass spectrometry (LC-MS) procedures, a well-thought-out plan is essential. This process should align with Clinical Laboratory Improvement Amendments (CLIA) compliance, as depicted in Fig. 1, which provides a structured list of major milestones and expected outcomes for transitioning a research laboratory into a clinical laboratory. The initial step involves preparing the laboratory space and infrastructure for conducting assays. To successfully transfer and implement a fully developed MS assay at a different location, the development and validation of the assay, along with the establishment of standard operating procedures (SOPs), may require several months to a year to ensure comprehensive analytical accuracy.

Fig. 1

Preparation, timeline and milestone to establish an immuno-multiple reaction monitoring (iMRM) mass spectrometry-based thyroglobulin assay meeting the Clinical Laboratory Improvement Amendments (CLIA) guideline

Staff training and competency Ensuring accurate, reliable, and timely testing is the next phase, which includes staff training and competency assessment adhering to CLIA standards. Compared to the operation of a research laboratory, this is a unique step to be part of quality management In a clinical laboratory, all the laboratory personnel training and competence assessment need to be planned, documented and evaluated. On a biannual or annual basis, staff competency needs to be evaluated and documented to demonstrate the necessary knowledge, skill and behaviors to perform their respective duties. Given the complexity of MS technology, proficient LC-MS/MS technical specialists are crucial. While certified laboratory technicians with a general clinical laboratory background can handle routine tasks and result reporting, mass spectrometry technical specialists are often needed for troubleshooting related to LC-MS/MS instrumentation. In this study, two certified staff members were trained for tasks like sample preparation, instrumentation operation, data analysis, and report generation. Service contracts were also acquired from the vendor for LC-MS/MS instrument troubleshooting and maintenance.

Quality control The implementation of a quality management plan is a unique procedure that is not common practice in a research environment. It is crucial to establish guidelines and frameworks for maintaining quality throughout the project. Finally, proficiency testing is conducted through interlaboratory comparison testing to assess individual laboratory performance, ensuring competence and quality assurance. For the thyroglobulin (Tg) assay developed in this study, proficiency testing involved comparing quality control (QC) and clinical samples with the Clinical Chemistry Laboratory overseen by Dr. Andrew Hoofnagle at the University of Washington Medical Center in the USA.

Planning for Laboratory infrastructure (Fig. 2)Fig. 2

Laboratory layout for sample reception room (A), sample preparation station (B), and mass spectrometry analysis room (C)

While targeted protein mass spectrometry assays have gained significant popularity in biomarker discovery and validation, additional prerequisites must be met to make them practically applicable within the constraints of medical laboratory infrastructure. These prerequisites encompass various factors that can be controlled, including the laboratory environment, technician proficiency, instrument calibration, storage conditions, reagent and sample handling, and the quality of assay reagents (as cited in [13]). In contrast to research laboratories, where the primary focus often revolves around advancing technology, in a medical laboratory context, the emphasis is on maintaining strict consistency within the workspace and hardware instrumentation. This consistency is essential to ensure the robustness of operation, the quality of data generation, and adherence to stringent standards.

To transform a traditional biomedical research laboratory, a significant effort was made to plan and fully renovate the laboratory space. This newly designed space was carefully arranged to accommodate three main areas: the sample reception room, the sample preparation station, and the mass spectrometry analysis room, as depicted in Fig. 2.

Sample reception room (Fig. 2A): This area serves as the initial point of entry for samples. It includes a designated space for inspecting incoming sample packages and containers to check for any damage. Additionally, the quality of the samples is assessed against established recruitment criteria. Following this, samples are registered, labeled with barcodes for proper tracking, and safely stored. The room layout allows for potential expansion to handle increased sample throughput if required.

Sample preparation station (Fig. 2B): The sample preparation station is equipped with essential laboratory equipment, including a fume hood, clinical centrifuge, pipettes, thermomixer, and more. It is specifically designed for the transformation of thyroglobulin (Tg) proteins into peptides, which is a critical step in the analysis. The process involves several key steps. Initially, immunoaffinity (IA) beads are prepared by attaching Tg-peptide antibodies to magnetic beads. Next, serum samples undergo denaturation, alkylation, and trypsin digestion to produce peptides. These Tg peptides are subsequently enriched using the IA beads and are stored at a temperature of 4℃ in preparation for later LC-MS/MS analysis.

Mass spectrometry analysis room (Fig. 2C): This room is equipped to accommodate the necessary components for mass spectrometry analysis. It houses the high-performance liquid chromatography (HPLC) equipment, the mass spectrometer, a computer for data acquisition and analysis, as well as auxiliary components such as a Liquid nitrogen tank, a nitrogen generator, a noise reduction device, and a waste venting line. The design and setup of this room follow the guidelines outlined in the “Site Planning Guide” provided by the mass spectrometer manufacturer. While this guide offers valuable information about minimum space requirements, electrical power needs, gas supplies, ventilation, computer connectivity, and operational conditions, it may not cover all the specific operational requirements for running a clinical mass spectrometry laboratory. Nonetheless, it serves as a fundamental reference point for the planning and setupprocess.

Case study: building clinical laboratory infrastructure in Taiwan under CLIA requirement

As per the outlined plan, we started to establish the CLIA-equivalent laboratory, i.e. to convert a research laboratory to operate the Tg-MS assay under CLIA requirement, in the research space of the pharmacogenomics laboratory under the Centers of Genomic and Precision Medicine, National Taiwan University. This laboratory holds certification from the Taiwan FDA as a medical laboratory (equivalent to ISO15189 certification) for gene testing (Fig. 3). Given that the Tg-MS assay is intended for clinical diagnostics, it is imperative to validate the method’s quality and competence. In Taiwan, adherence to specific requirements for quality management in clinical assays is guided by the Laboratory Developed Tests and Services (LDTS) guidelines put forth by the Taiwan Food and Drug Administration (TFDA) and biomedical molecular testing method validation guidelines (TAF-CN-G38) monitored by Taiwan Accreditation Foundation (TAF), both literally following the ISO 15,189 international standards. For the assay development and validation, we followed the guideline outlined in the quantitative measurement of proteins and peptides by mass spectrometry (CLSI-C64) the Clinical & Laboratory Standards Institute (CLSI) guideline, a framework initiated by CPTAC and collaboratively developed by the research and clinical laboratory community, including academia, regulatory bodies, and industry, and published by the Clinical & Laboratory Standards Institute. At our site, we meticulously assessed the analytical merits of the Tg-MS assay, encompassing accuracy, precision, reportable range, cut-off value, specificity, and stability of the Tg-MS assay. The summary and comparison of regulatory requirements between the TFDA-LDTS and TAF-CNLA-G38 guidelines are presented in Table 2. Additionally, specific chapters in CLSI C64 were adopted for evaluating assay development and validation, and these are enumerated for comparative purposes.

Fig. 3

The CLIA-equivalent laboratory was established within the pharmacogenomics laboratory under the Centers of Genomic and Precision Medicine, National Taiwan University (A). The laboratory comprised three distinct areas: (B) the sample reception room for barcode labeling and scanning and sample storage; (C) the sample preparation station for the transformation of Tg proteins into peptides; and (D) the mass spectrometry analysis room

Table 2 Comparison of regulatory requirement among CLSI, LDTS, and TAF for analytical performance in developing a LC-MS assay. TFDA-LDTS: Taiwan Food and Drug Administration; TAF: Taiwan Accreditation Foundation; CLSI: Clinical & Laboratory Standards Institute

In the initial phase of setting up the laboratory facility, the primary tasks involve ensuring a dedicated electricity supply and the installation of a safe venting system for the disposal of waste generated from the use of organic solutions in the mass spectrometry (MS) process. As illustrated in Fig. 3, this photo depicts the CLIA-equivalent laboratory established within the Centers for Genomics and Precision Medicine at the National Taiwan University.

The repeatability and reproducibility of an assay depend significantly on the condition of the laboratory infrastructure. To ensure the generation of trustworthy and traceable measurement data, certain critical requirements must be met. These include a consistent and reliable supply of electricity, preferably backed up with an uninterruptible power supply (UPS), and well-controlled environmental conditions in terms of temperature and humidity within an air-conditioned room. These conditions should be both moderate and uniform.

Converting a conventional biochemical laboratory into a dedicated mass spectrometry analysis room requires a series of crucial modifications. Specifically, new electrical outlets, designed with specifications of 220 volts and 45-amp circuits, were installed. Additionally, a fume hood equipped with acid-base cabinets was incorporated into the lab. The inclusion of this fume hood necessitated the installation of new ducts to efficiently accommodate it and facilitate the proper ventilation of the mass spectrometry instrument. This process entailed various steps, including applying for departmental approvals and ensuring compliance with safety and regulatory requirements for power distribution. It’s worth noting that this entire transformation, from the approval stage to the completion of the renovation, could span several months due to the comprehensive nature of the modifications and the need for meticulous compliance with established standards and regulations.

Maintaining the appropriate temperature for mass spectrometry required the installation of an air conditioner with precise temperature control, along with an air compressor located in a separate room. To meet the nitrogen gas requirements of LC/MS applications, a nitrogen generator was put in place. To minimize noise within the lab, a well-placed nitrogen generator bench and the separation of the air compressor were practical solutions.

In summary, it is highly advisable to plan well in advance for such a lengthy and intricate process of transforming a conventional research laboratory into a CLIA-equivalent laboratory infrastructure. Moreover, it is crucial to recognize that, in contrast to a standard research laboratory, securing service contracts for equipment maintenance is paramount. These contracts encompass not only technological support but also the capability to troubleshoot issues, ensuring the sustained reliability of LC-MS/MS instruments over the long term.

Assay development pipeline and demonstration of laboratory proficiency

Following the regulation and guidelines of LDTS and CLSI C64, we developed and validated the Tg-MS assay from the latest version established by Hoofnagle et al. [11]. in the medical laboratory at National Taiwan University, Taiwan. In brief, the unique peptide of Tg, FSPDDSAGASALLR (abbreviated as FSP), and its two dominant fragment ions (y7 and y12) are selected as target transitions for LC-MRM analysis to ensure the specific detection of Tg protein. Pairs of synthetic internal standards (SIS) of light and heavy isotope-labeling (IS) of the same sequence of FSP peptides are prepared to optimize the LC-MS/MS and validate the retention time of the FSP peptide in the LC-MRM. Further optimizations on the instrument settings such as collision energy and LC gradient are required for enhancing the transition signals of both target and SIS peptides. In our case, the two transitions have different optimal collision energies; 33.2% and 38.2% for y7 and y12 transitions, respectively. Compared to the common practice for a research assay, the additional key steps for a clinical assay development and validation of performance meeting regulatory guideline are summarized as follows:

Quality control standards To develop the Tg-MS assay, we adopted a commercially available human serum as Tg standard to prepare calibrators and quality control samples (QCs). The initial concentration of Tg in the commercial human serum was determined as 13.12 ng/mL by LC-MRM assay from the certified clinical laboratory in the UW Medical Center. With this human serum, we prepare 5 calibrators with Tg concentrations ranging from 0.23 to 13.12 ng/mL. Three QCs of different Tg concentration, including QC-high (10.99 ng/mL), QC-Mid (3.3 ng/mL) and QC-low (0.99 ng/mL), are designed for evaluation of the assay quality. It is recommended to prepare a large batch of calibrators and QCs to support at least half- or one-year use to ensure the consistency and stability of the assay.

Quantification performance For every batch of Tg-MS assay, 400 µL of 5 calibrators, 3 QCs and a number of clinical samples (with IRB approval and informed consent) are aliquoted to undergo denaturation, reduction, and alkylation processes, following by the addition of SIS peptide, tryptic digestion, and the affinity purification of FSP and SIS peptides using peptide immunoaffinity enrichment with a well-characterized monoclonal antibody against FSP peptide. All the samples are then analyzed by LC-MRM. Then, we construct the calibration curve by using these 5 calibrators. A linearity with r2 higher than 0.95 in the calibration curve is accepted for measuring the Tg levels in three QCs and other clinical samples. For acceptance of the assay performance, the measured Tg levels of three QCs should be less than 20% deviation from the expected values. Under these two conditions, the measurement of Tg in the clinical samples demonstrated the quality of LC-MRM assay and the reliability of the results.

Evaluation of analytical merits In order to validate the performance of Tg-MS assay at our site, we performed a series of measurements of QCs to evaluate the analytical merits that adhere to the LDTS and CLSI C64 guideline, including reportable range, accuracy, precision, sensitivity, specificity, interference, and stability of the assay (Table 3). Specifically, we performed 4 replicate analyses of 2–3 QCs per day for at least 3 —5 days to evaluate the within-day repeatability as well as 1 replicate analysis of 2–3 QCs for at least 7–10 days to evaluate the between-day reproducibility. Our data shows that the coefficient of variation (CV) of all three QCs in within-day and between-day experiments were 5 – 10% and < 10% CV, respectively. Furthermore, collecting a long-term result of QCs showed that the deviation between the measured and expected values of Tg in QC-mid and QC-high are 7.8% and 8%, respectively. These results demonstrated a precise and accurate measurement of serum Tg levels by the Tg-MS assay at our site. We also evaluated the interference from hemolysis by mixing up one hemolyzed serum (250–500 mg/dL) and non-hemolyzed serum in specific ratios to prepare 0%, 25%, 50%, 75%, and 100% hemolyzed serum samples. The data indicated that a serum sample with up to 25% hemolysis (62.5–125 mg/dL) is tolerated with Tg-MS assay.

Table 3 The overview of analytical performance of Tg-iMRM-MS established in Taiwan

Proficiency test Finally, proficiency testing was performed to confirm harmonization or standardization of the established Tg-iMRM MS assay between our laboratory and the University of Washington Medical Center. Proficiency testing (PT) is a very important concept for clinical assays which is very foreign to research laboratories. PT is a long-term evaluation of the accuracy and reliability of the assay. CLIA requires proficiency testing for all assays in a clinical laboratory on a routine basis and these results will be graded and evaluated. Consecutive failures of PT results for an assay will result in discontinuation of the assay in the lab. The PT can be part of a program such as CAP or self-arranged with other laboratories. In our particular case, there is no official CAP program for Tg PT. We established a PT process with the Washington University Medical Center.

In our protocol, only the Tg antibody and affinity bead were the same with the University of Washington Medical Center, most of the chemicals and reagents as well as the LC-MS system were different. Thus, proficiency testing with a routine exchange of QC and clinical samples between multiple clinical laboratories is necessary to provide reliable results using distinct vendors, models, platforms or institutions [14]. As shown in Table 3, we exchanged 35 QCs, clinical samples, and diluted clinical samples with UW Medical Center to compare the measured Tg levels between two sites. The Tg measurements between two sites are linear and highly correlated (r2 = 0.996 and slope = 0.9287). The concordant results across a small set of patients with and without TgAb demonstrated the standardization between the two laboratories and quality assurance of the established CLIA-equivalent Tg-iMRM MS assay in Taiwan.