Technologies based on mass spectrometry have already been adopted in various biomedical research fields and healthcare sectors, including toxicology, immunology, analytical chemistry, and endocrinology. More recently, mass spectrometry-based assays have gained increasing popularity amongst clinical diagnostic laboratories and emerged as a promising tool for modern pathology owing to their multiplexing abilities and exceptional analytical performance.¹

Principles of mass spectrometry

Mass spectrometry analysis involves the generation of positively or negatively charged ions from analytes in a sample (ionization step), followed by the detection of selected charged ions of interest. By measuring the ratio of the molecular mass to the charge (m/z) of molecules and creating a mass spectrum of relative abundance of ions against m/z (also known as a chromatogram), mass spectrometry can provide an accurate quantitative insight into the molecular composition of samples.^1–3 The peak areas of analytes in chromatograms are compared to those of an internal standard with known concentration, allowing for the precise determination of the concentration of different analytes.⁴

Additionally, this powerful analytical technique can also be used to characterize the structure and chemistry of molecules within samples.⁵ As the analytes pass through the mass spectrometer, fragmentation may occur depending on specific bonds. As the chemical nature and strength of bonds affect fragmentation, mass spectrometry can be used to generate a mass spectral fingerprint, which is characteristic for each molecule and dependent on its molecular structure.⁶

There are many variations of mass spectrometry using different spectrometer configurations. For instance, in tandem mass spectrometry (MS/MS), multiple mass spectrometers with different fragmentation techniques are combined to detect molecular ions with similar m/z ratios.⁷ Mass spectrometry imaging (MSI) is another variation of mass spectrometry with several promising applications in pathology and clinical diagnostics.^8,9

Current and emerging applications of mass spectrometry in anatomical pathology

The value of mass spectrometry in the discovery and detection of biomarkers in various types of specimens, including biopsy tissues, is becoming increasingly recognized. With the rapid decrease in the costs of omics technologies over the past few years, whole-proteome profiling from tissue slides has become more accessible to diagnostic labs as a means of characterization of global protein expression patterns to evaluate the pathophysiology of diseases.¹ Mass spectrometry has proved particularly useful for the pathological diagnosis of renal diseases and amyloidosis. Specifically, assays combining laser microdissection with mass spectrometry (LMD/MS) have been developed to detect and characterize renal protein deposits and amyloid fibrils in clinical biopsy specimens. Validation studies have shown that these LMD/MS-assays offer nearly 100% specificity and sensitivity.^10–13

MSI is perhaps one of the most attractive mass spectrometry-based assays in pathology. MSI entails the use of mass spectrometry to evaluate the spatial distribution of various molecules (ranging from proteins to lipids, metabolites, and glycans) in fresh/frozen or formalin-fixed paraffin-embedded (FFPE) biopsy tissue sections.¹⁴ Therefore, MSI can shed light on the relationship between the distribution of molecules, morphological alterations in tissues, and disease pathology.¹⁵

MSI is particularly useful in oncology. Applications of MSI in pathology include cancer diagnosis; molecular characterization, classification, and staging of tumors; evaluation of tumor surgical margins; and discovery of cancer biomarkers.^1,16

“Mass spectrometry imaging is the main segue into molecular pathology for rapid tissue-based diagnostics,” said Professor Ron Heeren, co-chair of the Maastricht Multimodal Molecular Imaging Institute.⁹ “Whether it is disease staging, tumor margin assessment, therapy prediction, or cellular phenotyping, it is evident that MS imaging is making a stronger and stronger impact on the clinical decision-making process.”

Advantages of mass spectrometry in tissue pathology

One of the main advantages of mass spectrometry making it a powerful tool in anatomical pathology and diagnostics is its ability to not only identify but also quantify biomolecules in a wide variety of biological specimens.¹ In contrast with other established analytical assays, mass spectrometry offers multiplexing opportunities as it can detect and quantify multiple analytes in a high-throughput manner. The multiplexing ability of mass spectrometry makes it an ideal tool for the analysis of complex samples, such as pathology specimens.^17,18

As MSI can be used to analyze tissues directly without the need for multiple, time-consuming staining steps, it can significantly reduce the time to diagnosis or even be used to guide intraoperative tumor excision.¹⁹ In addition, mass spectrometry offers high analytical specificity and sensitivity; thus, the adoption of mass spectrometry for the analysis of biopsy tissues can improve diagnostic accuracy.^1,3

Examples of commercially available MSI technologies for molecular pathology

Hyperion Imaging System is a commercially available MSI system provided by Fluidigm. The system offers the potential for high-multiplex imaging and single-cell protein analysis in FFPE or fresh/frozen tissue sections (≤ 7 μm thickness). It can be used to characterize the tissue microenvironment at the single-cell level, spatially characterize numerous cell types in tissues, discover novel biomarkers, and predict treatment response.^20,21

Similarly, MIBIscope developed by IONpath is the first commercially available multiplexed ion beam imaging system. MIBIscope is compatible with standard tissue processing techniques and allows the simultaneous, high-definition visualization of more than 40 biomarkers. By doing so, the platform helps clinicians monitor diseases, evaluate treatment response, and elucidate the pathology of diseases.

Remaining challenges and future perspectives

As ionization methods, instrumentations, and technologies continue to advance, novel clinical applications based on mass spectrometry are expected to become the mainstay of anatomical pathology and diagnostics, among other clinical fields. Nonetheless, several challenges remain to be addressed to allow for the implementation of mass spectrometry-based tissue analysis methods in routine diagnosis.

As with all emerging and rapidly evolving methods, the advantages and limitations of mass spectrometry-based tissue analysis methods over traditional histology methods need to be established and communicated among pathologists, clinical laboratory technicians, researchers, and other potential users of these novel methods.^8,22 Furthermore, the acquisition of images with high spatial resolution may considerably reduce the pixel-by-pixel sampling speed, increasing the overall image acquisition and analysis time.¹

Other limitations of mass spectrometry that need to be addressed by manufacturers and the clinical mass spectrometry community are the lack of automation in instrumentation and data analysis, the requirement of skilled personnel, the need for validated biomarkers, the relatively high cost (compared with standard H&E), the lack of standardized workflows, and the lack of well-established regulatory guidelines.^1,23,24 Tackling issues related to turnaround time, standardized workflows, and biocomputational data analysis and storage is also required to help facilitate the smooth integration of mass spectrometry in routine pathology and modern diagnostics.

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References

1. Fung AWS, Sugumar V, Ren AH, Kulasingam V. Emerging role of clinical mass spectrometry in pathology. J Clin Pathol. 2020;73(2):61-69. doi:10.1136/jclinpath-2019-206269
2. Paital B. Mass Spectrophotometry: An Advanced Technique in Biomedical Sciences. Adv Tech Biol Med. 2015;4(3). doi:10.4172/2379-1764.1000182
3. Banerjee S. Empowering Clinical Diagnostics with Mass Spectrometry. ACS Omega. 2020;5(5):2041-2048. doi:10.1021/acsomega.9b03764
4. Johnsen E, Leknes S, Wilson SR, Lundanes E. Liquid chromatography-mass spectrometry platform for both small neurotransmitters and neuropeptides in blood, with automatic and robust solid phase extraction. Sci Rep. 2015;5(1):9308. doi:10.1038/srep09308
5. Biemann K. Structure Determination of Natural Products by Mass Spectrometry. Annu Rev Anal Chem (Palo Alto Calif). 2015;8:1-19. doi:10.1146/annurev-anchem-071114-040110
6. Konermann L, Vahidi S, Sowole MA. Mass Spectrometry Methods for Studying Structure and Dynamics of Biological Macromolecules. Anal Chem. 2014;86(1):213-232. doi:10.1021/ac4039306
7. Mittal RD. Tandem Mass Spectroscopy in Diagnosis and Clinical Research. Indian J Clin Biochem. 2015;30(2):121-123. doi:10.1007/s12291-015-0498-9
8. Schwamborn K. The Importance of Histology and Pathology in Mass Spectrometry Imaging. Adv Cancer Res. 2017;134:1-26. doi:10.1016/bs.acr.2016.11.001
9. Leung F, Eberlin LS, Schwamborn K, Heeren RMA, Winograd N, Graham R. Mass spectrometry-based tissue imaging: The next frontier in clinical diagnostics? Clin Chem. 2019;65(4):510-513. doi:10.1373/clinchem.2018.289694
10. Sethi S, Vrana JA, Theis JD, Dogan A. Mass spectrometry based proteomics in the diagnosis of kidney disease. Curr Opin Nephrol Hypertens. 2013;22(3):273-280. doi:10.1097/MNH.0b013e32835fe37c
11. Vrana JA, Gamez JD, Madden BJ, Theis JD, Bergen HR 3rd, Dogan A. Classification of amyloidosis by laser microdissection and mass spectrometry-based proteomic analysis in clinical biopsy specimens. Blood. 2009;114(24):4957-4959. doi:10.1182/blood-2009-07-230722
12. Mollee P, Boros S, Loo D, et al. Implementation and evaluation of amyloidosis subtyping by laser-capture microdissection and tandem mass spectrometry. Clin Proteomics. 2016;13:30. doi:10.1186/s12014-016-9133-x
13. Park J, Lee GY, Choi J-O, et al. Development and Validation of Mass Spectrometry-Based Targeted Analysis for Amyloid Proteins. Proteomics Clin Appl. 2018;12(3):e1700106. doi:10.1002/prca.201700106
14. Buchberger AR, DeLaney K, Johnson J, Li L. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Anal Chem. 2018;90(1):240-265. doi:10.1021/acs.analchem.7b04733
15. Chughtai K, Heeren RMA. Mass Spectrometric Imaging for Biomedical Tissue Analysis – Chemical Reviews (ACS Publications). Chem Rev. 2011;110(5):3237-3277. doi:10.1021/cr100012c.Mass
16. Kriegsmann J, Casadonte R, Kriegsmann K, Longuespée R, Kriegsmann M. Mass spectrometry in pathology – Vision for a future workflow. Pathol Res Pract. 2018;214(8):1057-1063. doi:10.1016/j.prp.2018.05.009
17. Longuespée R, Casadonte R, Kriegsmann M, et al. MALDI mass spectrometry imaging: A cutting-edge tool for fundamental and clinical histopathology. Proteomics – Clin Appl. 2016;10(7):701-719. doi:10.1002/prca.201500140
18. Kriegsmann J, Kriegsmann M, Casadonte R. MALDI TOF imaging mass spectrometry in clinical pathology: A valuable tool for cancer diagnostics (review). Int J Oncol. 2015;46(3):893-906. doi:10.3892/ijo.2014.2788
19. Jannetto PJ, Fitzgerald RL. Effective use of mass spectrometry in the clinical laboratory. Clin Chem. 2016;62(1):92-98. doi:10.1373/clinchem.2015.248146
20. Flint LE, Hamm G, Ready JD, et al. Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging. Anal Chem. 2020;92(18):12538-12547. doi:10.1021/acs.analchem.0c02389
21. Porta Siegel T, Hamm G, Bunch J, Cappell J, Fletcher JS, Schwamborn K. Mass Spectrometry Imaging and Integration with Other Imaging Modalities for Greater Molecular Understanding of Biological Tissues. Mol Imaging Biol. 2018;20(6):888-901. doi:10.1007/s11307-018-1267-y
22. Chaurand P, Schwartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM. Integrating Histology and Imaging Mass Spectrometry. Anal Chem. 2004;76(4):1145-1155. doi:10.1021/ac0351264
23. Lynch KL. CLSI C62-A: A New Standard for Clinical Mass Spectrometry. Clin Chem. 2016;62(1):24-29. doi:10.1373/clinchem.2015.238626
24. Lynch KL. Accreditation and Quality Assurance for Clinical Liquid Chromatography-Mass Spectrometry Laboratories. Clin Lab Med. 2018;38(3):515-526. doi:10.1016/j.cll.2018.05.002