Парадигм Смена в Моноклональном Обнаружении Протеина: От электрофореза до Массовых Спектрометрических методов.

INTRODUCTION

Monoclonal protein (M-protein): Definition and clinical significance

Monoclonal gammopathy (MG) is a condition characterized by the overproduction of a monoclonal immunoglobulin or fragment because of the clonal proliferation of cells in the B lymphocyte lineage [ 1 ]. The abnormal Ig, known as M-protein, can be detected in serum and/or urine [ 2 ]. M-protein may consist of intact Ig and/or monoclonal fragments, or it may consist solely of monoclonal free light chains (FLCs) or heavy chains [ 3 ]. M-protein is essential for diagnosing disease, monitoring treatment responses, and developing a prognosis in various hematologic disorders [ 4 ].

M-protein is a key biomarker of plasma cell disorders (PCDs) and is related to various diseases associated with MG [ 5 ]. The spectrum of hematologic conditions related to MG includes multiple myeloma (MM), smoldering multiple myeloma, MG of undetermined significance (MGUS), plasmacytoma, Waldenström macroglobulinemia, plasma cell leukemia (PCL), light chain deposition disease (LCDD), chronic lymphocytic lymphoma, and other low-grade lymphomas [ 6 ]. In addition to these hematological conditions, MG has been associated with various systemic disorders such as nephropathy, neuropathy, oculopathy, dermopathies, autoimmune diseases, and coagulopathies [ 1 ]. Furthermore, MG can manifest as multi-systemic diseases, such as amyloid light-chain (AL) amyloidosis and polyneuropathy, organomegaly, endocrinopathy, MG, and skin changes syndrome [ 7 ].

Traditional methods for M-protein detection

Conventional M-protein diagnostics rely on electrophoresis (EP)-based techniques, such as protein electrophoresis (PEP) for screening and quantification [ 8 ], along with immunosubtraction electrophoresis (IEP) or immunofixation electrophoresis (IFE) for isotype characterization [ 8 ]. Additionally, turbidimetric and nephelometric assays are used to quantify Ig isotypes (IgG, IgA, and IgM) and FLCs (kappa, κ, and lambda, λ) [ 9 ]. No single test provides comprehensive information on M-protein types and concentrations; therefore, these methods must be used in combination. Performing one of these tests is insufficient for accurate isotype identification and M-protein quantification, as each technique provides complementary diagnostic insights.

EP-based methods have critical limitations, particularly in detecting low levels of M-protein (<10 g/L) [ 10 ]. Another challenge arises with the increasing use of therapeutic monoclonal antibodies (t-mAbs), as these antibodies can mimic endogenous M-protein, leading to false-positive results in PEP, IEP, and IFE tests [ 11 ]. As novel therapies continue to improve patient outcomes, a growing need exists for highly sensitive and specific analytical tools, particularly for assessing minimal residual disease (MRD), wherein detecting trace amounts of M-protein is critical for prognosis and disease monitoring [ 12 ].

Emergence of mass spectrometry (MS) in M-protein analysis

MS has emerged as a superior alternative to traditional methods, offering enhanced sensitivity and specificity in M-protein detection [ 13 ]. MS-based methods, including matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS), liquid chromatography–electrospray ionization–quadrupole time-of-flight MS (LC–ESI-qTOF MS), and LC–ESI-Orbitrap MS (LC–ESI-Orbitrap MS), enable the detection of low-abundance M-protein, t-mAb differentiation from endogenous M-protein, and improved assessment of disease progression [ 14 ].

Various MS platforms offer distinct advantages in terms of ionization efficiency, mass resolution, and analytical performance, which determine their usability for analyzing specific types of proteins [ 15 ]. MALDI-TOF employs laser-induced desorption and produces predominantly single- or double-charged ions, enabling rapid screening with minimal sample preparation [ 16 ]. In contrast, LC–ESI-based systems generate multiply charged ions through electrospray ionization, enhancing the detection of larger and more complex molecules [ 17 ]. The qTOF MS combination offers accurate, high-resolution mass detection suitable for intact protein analysis [ 18 ]. The Orbitrap analyzer provides ultra-high mass accuracy and resolving power, which are particularly advantageous for peptide-level workflows and quantitative applications [ 19 ].

Sample preparation strategies are typically categorized into top-down, middle-down, and bottom-up approaches [ 18 ]. Top-down methods are used to analyze intact protein chains without enzymatic digestion, preserving complete molecular weight information. Middle-down strategies involve selective reduction or limited proteolysis, typically targeting the disulfide bond between heavy and light chains. Bottom-up approaches digest proteins into peptides prior to MS analysis, enabling highly sensitive detection of specific amino acid sequences through tandem MS.

Since the introduction of MS-based methods, research on and interest in their clinical applications have increased markedly ( Fig. 1 ). Over the past decade, clinical data have demonstrated that MS-based approaches offer greater sensitivity and accuracy for M-protein identification and quantification, positioning them as a robust alternative to conventional techniques. In this review, we provide a comprehensive overview of MS-based methods for M-protein analysis, highlighting their clinical applications, advantages, and future perspectives in laboratory diagnostics.

CURRENT DIAGNOSTIC METHODS FOR MG

Diagnostic testing for MG

EP-based methods, including PEP, IEP, and IFE, are commonly used for detecting, quantifying, and characterizing M-protein in serum and urine in clinical practice. Serum PEP is used particularly for patients with intact Ig multiple myeloma. However, its sensitivity is limited, especially in light chain multiple myeloma (LCMM), non-secretory multiple myeloma (NSMM), and LCDD. Serum IFE and urine IFE have higher sensitivity and are therefore used to confirm the presence of M-protein [ 20 ]. IFE is also essential for identifying the M-protein isotype. Urine PEP detects monoclonal FLCs, commonly known as Bence Jones proteins. However, collecting 24 hr urine samples is challenging, and urine PEP has lower sensitivity than serum-based tests. In early-stage disease or in patients secreting small amounts of FLCs, urine PEP may fail to detect M-protein. Therefore, urine IFE is recommended only for patients suspected of having AL amyloidosis based on serum IFE and FLC tests [ 21 ].

An FLC analyzer is widely used for high-sensitivity screening and detection of FLCs [ 22 ]. Combining serum PEP and FLC testing substantially improves diagnostic sensitivity for MM and related disorders. The International Myeloma Working Group (IMWG) recommends using serum FLC testing alongside serum PEP and IFE to rule out MM [ 9 , 23 ]. For other MG-related diseases, a combination of serum PEP, IFE, and FLC assays is recommended for enhanced M-protein and/or FLC detection [ 20 ]. FLC assays use either polyclonal antibodies (e.g., Freelite) or monoclonal antibodies (e.g., N-latex) to detect FLCs and offer higher sensitivity than EP-based methods. Luminex, a recent highly sensitive instrument capable of detecting FLC levels <0.001 g/L in serum [ 24 ], is particularly useful for detecting small amounts of monoclonal FLCs, especially when serum PEP and urine PEP fail. It is especially effective for diagnosing LCMM, NSMM, LCDD, and AL amyloidosis. However, Luminex FLC assays are affected by limitations, including lot-to-lot reagent variability, extreme polymerization, unrecognized epitopes, indirect determination of monoclonality via the κ: λ ratio, antigen excess, poor inter-assay agreement, and low concordance across laboratories using the same reagents [ 22 ].

Monitoring disease progression and treatment responses in patients with MM

MM is a hematologic malignancy characterized by the proliferation of clonal plasma cells in the bone marrow (BM), which can cause a range of systemic complications [ 25 ]. MM is a major concern in clinical practice as early treatment and continuous monitoring for relapse are essential for improving patient prognosis. Effective treatment strategies aim to improve patient outcomes and survival rates, with advanced therapeutic approaches, personalized treatment plans, and supportive care enhancing patients’ quality of life and survival.

Recent advancements in treatment combined with t-mAbs have significantly improved MM treatment outcomes [ 25 ]. The IMWG response criteria employ M-protein as a follow-up biomarker, defining a complete response (CR), very good partial response, and partial response based on serum and urine M-protein levels. Achieving MRD negativity is associated with extended overall survival (OS) and progression-free survival (PFS) [ 25 , 26 ]. Therefore, advanced monitoring methods with high sensitivity for screening small amounts of M-protein in serum are needed.

Detection of MRD

MRD negativity is defined by the absence of clonal plasma cells in BM aspirates, as assessed using highly sensitive methods that can detect one malignant cell among ≥10 5 normal nucleated cells. MRD is a critical prognostic factor for patients with MM that is strongly associated with PFS and OS [ 26 ]. Several techniques are currently available for MRD assessment in BM, including multiparameter flow cytometry and molecular methods such as allele-specific oligonucleotide quantitative PCR and next-generation sequencing [ 27 ]. These methods offer significantly greater sensitivity than traditional M-protein diagnostics, achieving limits of detection (LoDs) of 1 malignant cell among 10 –5 normal nucleated cells [ 18 ].

Historically, MRD assessments have primarily relied on BM samples because of the limited sensitivity of traditional methods with peripheral blood (PB) samples. Next-generation sequencing and next-generation flow cytometry are primarily used to analyze BM samples, which are generally associated with higher sensitivity than PB samples [ 28 ]. However, novel techniques are emerging to utilize PB samples, including high-resolution MS for detecting M-protein, offering a non-invasive alternative with enhanced sensitivity [ 29 ].

Limitations of EP-based methods

The presence of co-migrating polyclonal proteins, observed as a background peak in serum PEP, can limit the sensitivity for low M-protein levels [ 18 ], often requiring subjective interpretation. Interferences such as hemolysis, the presence of fibrinogen, and elevated C-reactive protein can create pseudo-M-protein peaks, complicating the diagnostic process. Pre-analytic variables, such as sample handling and the use of radio-opaque agents, can also introduce artifacts that mimic M-protein, thus compromising the accuracy of these conventional methods [ 10 ]. In addition, t-mAbs, such as daratumumab, elotuzumab, and isatuximab, have emerged as new interferences for serum PEP and IFE [ 12 ]. t-mAbs can be confused with endogenous M-protein during PEP or IFE interpretation when a large amount of IgG or κ-type M-protein co-migrates with the t-mAb. This co-migration can mask the clearance of M-protein by IFE, which is necessary for classifying CR based on the IMWG criteria [ 2 ]. To confirm a CR in patients with MM, the absence of M-protein in serum and urine must be demonstrated using IFE. However, t-mAbs can interfere with IFE results, complicating the confirmation of CR. To overcome this situation, the daratumumab IFE reflex assay (DIRA), which can distinguish daratumumab from endogenous M-protein, was developed [ 30 ]. However, DIRA can only detect daratumumab, not other t-mAbs, making EP-based methods difficult to interpret when t-mAbs other than daratumumab are administered. A more comprehensive and advanced approach is needed to accurately distinguish t-mAbs from endogenous M-protein and improve the reliability of M-protein monitoring. Table 1 compares EP-based and MS-based methods in terms of their sensitivity and the ability to differentiate t-mAbs.

CLINICAL REVIEW OF MS-BASED METHODS

MALDI-TOF MS combined with the reduction in M-protein levels

The middle-down approach, which involves the reduction of antibody levels after Ig enrichment, improves the efficiency of sample preparation. Table 2 summarizes the clinical applications including method development, diagnosis, risk factor identification, treatment response monitoring, and prognostic evaluation of middle-down analysis using MALDI-TOF MS with reduction [ 31 ]. MALDI-TOF MS has evolved from a laboratory-developed test into a clinically applicable tool for detecting and monitoring M-protein in PCDs such as MM, MGUS, and AL amyloidosis [ 18 ]. MALDI-TOF MS combined with reduction enables the rapid and cost-effective processing of large sample volumes.

MASS-FIX, developed at Mayo Clinic Laboratories (Rochester, MN, USA) as an advancement of the initial qualitative MASS-SCREEN method, enables the detection, isotyping, and quantification of M-protein using nanobody-coupled bead resins [ 32 ]. To improve sensitivity and facilitate automation, MALDI-TOF has been coupled with reduction techniques such as MASS-SCREEN and MASS-FIX, making MALDI-TOF a viable alternative to conventional methods such as serum PEP and IFE. The EXENT system (The Binding Site, Birmingham, UK), recently commercialized in Europe, further enhances analytical sensitivity in M-protein detection by integrating immuno-enrichment and paramagnetic bead–based purification [ 33 ].

Automated MALDI-TOF MS demonstrated a detection limit of 0.1 g/L, comparable to that of serum PEP [ 34 ]. MASS-FIX has been validated for both serum and urine samples; it identified the same M-protein isotype as IFE did in 98% of serum samples and 95% of urine samples [ 18 ]. Compared with conventional methods such as IFE, MALDI-TOF significantly reduces the processing time and labor involved while maintaining high sensitivity [ 35 ]. In addition, MALDI-TOF MS effectively distinguishes t-mAbs, such as daratumumab, from endogenous M-protein, reducing misinterpretation for patients with MM undergoing treatment [ 35 ]. Beyond M-protein detection, MALDI-TOF MS has been used to analyze light chain glycosylation, a risk factor for progression to MM and AL amyloidosis [ 36 ]. These findings suggest that MALDI-TOF MS might play a role in early diagnosis and disease monitoring, particularly for patients with MGUS at risk of progression to symptomatic disease [ 37 ].

LC–ESI-qTOF MS combined with M-protein reduction

Using identical reduction techniques for sample preparation, LC–ESI-qTOF MS demonstrates superior sensitivity and accuracy over MALDI-TOF MS reflecting its higher instrumental performance [ 38 ]. The monoclonal Ig rapid accurate mass measurement (miRAMM) method, based on LC–ESI-qTOF MS, provides higher sensitivity and specificity for M-protein detection than does MALDI-TOF MS [ 39 ]. miRAMM has proven highly effective for detecting M-protein in patients with MM, AL amyloidosis, and other PCDs ( Table 3 ).

Barnidge et al . [ 40 ] demonstrated that miRAMM consistently identified monoclonal peaks with stable molecular weights in patients with MM over a 7 yr period and effectively detected M-protein in 157 urine samples with a sensitivity comparable to that of IFE. Moreover, the results of a later study demonstrated that the molecular masses of M-protein light chains were consistent across both BM and PB samples [ 41 ].

Barnidge et al . [ 42 ] validated the use of miRAMM for detecting serum FLC in patients with AL amyloidosis, accurately identifying both monomeric and dimeric forms with high sensitivity, with an LoD of 0.006 g/L for monomers. Compared with MALDI-TOF, miRAMM demonstrated superior performance in distinguishing t-mAbs such as daratumumab, elotuzumab, and isatuximab; miRAMM detected molecular weight differences within ±0.5 Da [ 43 ]. miRAMM can detect t-mAb concentrations as low as 0.005 g/L, which is below the LoD of traditional EP-based methods. miRAMM confirmed t-mAbs (daratumumab, elotuzumab, and isatuximab) in 100% of cases (192/192 samples) [ 44 ], whereas MALDI-TOF only differentiated daratumumab and IgG /κ type M-protein in 84% of cases (26/31 samples) [ 45 ].

LC–ESI-Orbitrap MS combined with M-protein digestion

Bottom-up MS methods, particularly LC–ESI-Orbitrap MS combined with digestion, have demonstrated significant advancements in MM and MRD monitoring ( Table 4 ). Unique peptides derived from the variable regions of M-proteins are used for disease monitoring. The LC–ESI combination enables the effective separation of peptides in complex serum samples, whereas high-resolution Orbitrap detection facilitates highly accurate identification and quantification of disease-specific peptides. Techniques such as parallel reaction monitoring and the use of stable isotope-labeled peptides enable high sensitivity, reaching detection limits as low as 0.001 g/L for M-protein, even in the presence of t-mAbs [ 46 ]. Previous findings demonstrated that this method significantly enhanced the sensitivity of MRD detection in patients with MM [ 47 ].

Bergen et al . [ 48 ] introduced an MS-based method to monitor MRD via de novo light-chain sequencing. Among 67 patients with immunohistochemistry (IHC)-negative (N=57) or 6-color flow cytometry (FC)-negative (N=10) results in BM samples, 62 (93%) were found to have detectable disease by LC-MS/MS using serum samples, suggesting LC-MS/MS as a feasible alternative to BM-based IHC or FC for MRD detection. The limit of quantification (LoQ) was 0.001 g/L, indicating 2,000 times higher sensitivity than that of serum PEP.

Serum-based MS approaches are considered reliable, non-invasive alternatives for MRD monitoring and offer the additional advantage of detecting relapse months earlier than conventional methods [ 49 ]. LC–ESI-Orbitrap MS combined with M-protein digestion has achieved remarkable sensitivity, particularly for MRD detection, with or without the need for BM sampling [ 49 ]. However, serum-based MS methods provide an alternative to invasive BM sampling, which may sometimes yield non-representative results because of the heterogeneous dispersion of MM cells.

Comparison of MS-based methods

MALDI-TOF is a high-throughput, cost-effective technique capable of detecting M-proteins at moderate concentrations. Recent advancements, such as MASS-FIX and EXENT, which are commercially available in both the United States and Europe, have significantly improved its capabilities by introducing automated solutions. However, MALDI-TOF still has limited sensitivity compared with more advanced techniques such as LC–ESI-qTOF and LC–ESI-Orbitrap. Furthermore, because of its relatively low resolution, MALDI-TOF has difficulty in effectively differentiating t-mAbs from endogenous proteins, particularly when molecular mass differences are <10 Da [ 50 ].

LC–ESI-qTOF offers significantly higher sensitivity than MALDI-TOF, making it highly suitable for detecting low-abundance M-protein and assessing MRD. Additionally, it can differentiate t-mAbs with high specificity, offering detailed molecular insights crucial for clinical diagnostics. LC–ESI-qTOF analysis following MALDI-TOF covers cases missed by MALDI-TOF [ 51 ]. However, this technique is not yet commercially available for all clinical applications, limiting its widespread use, and requires more complex and costly instrumentation and management, limiting its routine use in some laboratories [ 23 ].

Among MS-based methods, LC–ESI-Orbitrap exhibits the highest sensitivity and specificity, making it ideal for detecting trace levels of M-protein and conducting detailed MRD assessments. This method also excels in molecular characterization, offering a detailed analysis of protein structures, which is invaluable in clinical settings. The major limitations of LC–ESI-Orbitrap are its high cost and restricted availability. Because of the complexity of operation and data interpretation involved, which require specialized expertise and advanced training to ensure accurate results, its accessibility for routine clinical use is limited.

ADVANCES AND CLINICAL UTILITY OF MS-BASED METHODS

Comparison of sensitivity

The LoD for serum PEP is 0.5–2 g/L [ 52 ]. Serum IFE, with an LoD of 0.1–0.2 g/L, is 5–10 times more sensitive than serum PEP [ 53 ]. Although serum PEP methods are capable of quantifying M-protein, IFE lacks this capability. Despite advances in automation through capillary electrophoresis (CE), its analytical sensitivity remains limited. The LoQ for both serum PEP and CE is approximately 2 g/L [ 18 ], with accuracy significantly decreasing for M-protein concentrations <10 g/L [ 54 ]. At concentrations of 10 g/L, the precision and accuracy of serum PEP decline because of its limited resolving power and the increasing influence of the polyclonal background on measurements [ 55 ].

MS-based techniques, including MALDI-TOF, LC–ESI-qTOF, and LC–ESI-Orbitrap, exhibit significantly higher sensitivity and specificity than do EP-based methods. MALDI-TOF, which previously showed an LoD of approximately 0.5 g/L, has recently undergone major advancements that have heightened its sensitivity [ 18 ]. MASS-FIX and EXENT have improved the detection capabilities of MALDI-TOF MS. MASS-FIX improved sensitivity by lowering the LoD to <0.1 g/L [ 55 ], and EXENT has further enhanced it, achieving an LoD of <0.015 g/L [ 56 ]. LC–ESI-qTOF demonstrated even higher sensitivity, with an LoD of 0.005 g/L [ 39 ]. LC–ESI-qTOF has higher sensitivity and specificity than traditional EP-based methods, making it suitable for more precise detection of low-abundance M-protein and t-mAbs. LC–ESI-Orbitrap provided the highest sensitivity among MS-based methods, with an LoD <0.001 g/L [ 57 ]. With its extremely high specificity, LC–ESI-Orbitrap is especially powerful for detecting trace levels of M-protein in MRD assessments.

Overall, MS-based methods are superior to EP-based methods in both sensitivity and specificity for detecting and monitoring low-abundance M-protein.

Time and cost efficiency using automation

MS-based methods have significant advantages over EP-based methods in terms of both time and cost efficiency. When compared with PEP and IFE, the automated systems using MALDI-TOF have shown significantly less time and labor required to process large sample volumes, making it a more cost-effective solution for clinical laboratories. Because of the automation of tasks such as sample handling and data analysis, MALDI-TOF MS is more efficient in detecting M-protein. Notably, MASS-SCREEN can process 96 patient samples in just 20 mins, highlighting its effectiveness in high-throughput testing [ 32 ]. The results of a study using 19,523 serum samples from patients with PCDs confirmed that MASS-FIX is more cost-effective for high-volume testing, significantly reducing both time and labor costs [ 35 ]. MASS-FIX outperformed traditional IFE in terms of sensitivity, specificity, and automation, enabling a single technician to perform 30% more tests in an 8 hr shift than could be performed with IFE [ 35 ].

Additional benefits of using MS

Beyond quantitative data for M-protein quantification, MS-based methods provide mass to charge ( m/z ) ratios, allowing differentiation between a patient’s medication history and t-mAbs [ 58 ]. Table 5 summarizes reported t-mAb detection data and the corresponding mass values obtained using MS. The m/z ratios can provide structural details, such as distinguishing between monoclonal and biclonal samples or between monomeric and dimeric forms.

Furthermore, MALDI-TOF can detect light chain N-glycosylation, which serves as an important prognostic indicator [ 36 ]. This capability is particularly valuable for identifying risk factors associated with progression to MM and for assessing MRD in patients with MM, aiding in OS and PFS predictions after treatment [ 28 ].

MALDI-TOF automation further enhances the speed and accuracy of M-protein detection, thus enhancing MRD assessment and patient outcome prediction [ 58 ]. Integrating automated MS-based approaches into clinical workflows enables more efficient disease monitoring and risk stratification, ultimately improving treatment decision-making and long-term prognosis [ 28 ].

INSIGHTS FROM THE PAST AND FUTURE DIRECTIONS OF MS-BASED METHODS

Over the past decade, MS-based methods have accumulated sufficient data to enable high-throughput automated testing with reliable detection and long-term monitoring of M-protein. These methods have demonstrated superior performance in detecting and monitoring low-abundance M-protein, particularly at concentrations of <10 g/L, offering significantly enhanced sensitivity and specificity over EP-based methods. Additionally, MS-based techniques enable t-mAb differentiation and the identification of new risk factors, such as light chain glycosylation, reinforcing their potential as a stable and reliable diagnostic platform.

The resolution of MS techniques increases in the order of MALDI-TOF, LC–ESI-qTOF, and LC–ESI-Orbitrap, and corresponding improvements in sensitivity and specificity for clinical M-protein detection have been observed [ 16 – 19 ]. However, these advancements come with higher instrumental costs and increased complexity in system operation, data processing, and maintenance. Therefore, platform selection should be guided by available resources, laboratory infrastructure, and specific clinical objectives.

Looking ahead, MS-based methods have the potential to replace EP-based techniques in routine clinical practice, particularly for detecting and quantifying low-abundance M-protein. To facilitate this transition, standardized guidelines must be developed to seamlessly integrate MS technologies into clinical workflows. Furthermore, ongoing research and validation studies are necessary to establish assay performance metrics, enhance automation, and refine data interpretation for regulatory approval. As technological advancements continue, addressing current limitations and financial feasibility will be crucial for improving accessibility and enabling the widespread adoption of MS-based methods in clinical diagnostics.

CONCLUSIONS

MS-based methods have emerged as an alternative to EP-based methods in clinical laboratories. Studies over the past decade have demonstrated the effectiveness and utility of MS-based methods. Advanced MS-based methods, including MALDI-TOF, LC–ESI-qTOF and LC–ESI-Orbitrap, have demonstrated significant improvements over traditional EP-based methods in terms of sensitivity, specificity, and comprehensive M-protein analysis. MS-based methods provide highly sensitive detection of small amounts of M-protein, improved accuracy in quantification, detailed molecular characterization, and reliable monitoring of treatment efficacy. Consequently, MS-based methods enable earlier detection of M-protein, aid in assessing disease progression risk, and effective monitoring and differentiation of t-mAbs.

ACKNOWLEDGEMENTS

None.

Footnotes

AUTHOR CONTRIBUTIONS

Lee J conceptualized the overall scope of the review and drafted the manuscript with a focus on the clinical perspectives. Yoo S assisted in collecting and structuring the background information. Yang S contributed expertise in mass spectrometry, reviewed the methodological descriptions, and ensured the technical details. Song SH provided overall supervision and offered insightful guidance and revisions throughout the process.

CONFLICTS OF INTEREST

None declared.

RESEARCH FUNDING

None declared.

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