Only a decade ago, a mass spectrometer may have looked out of place in a clinical laboratory. The technology, once mostly confined to chemical research facilities, debuted on the clinical scene around ten years ago with the release of commercial FDA-cleared microbial identification mass spectrometry (MS) platforms. But even before that, MS was recognized as a more specific alternative to immunoassays, with fewer false positives, in illicit drug testing. Since then, the breadth of analytes reliably evaluated by MS in laboratory tests has increased tremendously, ranging from small molecules and metal analytes to biomolecules. Applications abound in identification of infectious diseases, inborn errors of metabolism, and cancer diagnostics.
For clinical applications, the most frequently performed MS techniques are gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), tandem MS (MS/MS, GC-MS/MS, LC-MS/MS), and matrix-assisted laser desorption/ ionization time of flight MS (MALDI-TOF MS).
As a clinical tool, MS boasts high analytical specificity and sensitivity, multiplexing capability, and low sample cost. Runs can be staggered into the machine to maximize instrument time. MS is also versatile since protocols can be developed for a new biomarker without waiting for an FDA-approved kit or assay.
Despite its benefits, there are some hurdles to widespread adoption of MS in clinical labs. The instruments come with high price tags. Skilled operators are required to develop and validate protocols for applications that lack an FDA-cleared, end user–friendly MS platform. MS is also labor intensive, since the process is not fully automated. The regulatory landscape for the majority of clinical applications is still uncertain; laboratory-developed tests, (i.e., protocols developed in-house), will eventually need to conform to FDA guidelines, but when the final guidelines will be released is at present unclear.
Analyte or biomarker quantification in laboratory tests
The high sensitivity and specificity achievable by MS make it superior to immunoassays for analysis of several drug types, (e.g., steroids, illicit drugs, abused prescription drugs, and therapeutic prescription drugs). For analysis of immunosuppressant prescription drugs, such as tacrolimus, immunoassays fail to yield accurate results due to cross-reactivity with patient autoantibodies. Accurate quantification of tacrolimus is essential as too-low levels run the risk of tissue rejection while high levels run the risk of overdose. In some cases, MS also outstrips immunoassays in the determination of levels of serum thyroglobulin (Tg), a thyroid glycoprotein and biomarker for recurrent thyroid cancer. The immunoassay is unreliable in patients who have serum antithyroglobulin autoantibodies or heterophile antibodies because of cross-reactivity with the Tg immunoassay antibody, which leads to false-positive test results. In such instances, LC-MS/MS is more specific than a Tg immunoassay. Assays for proteins, including monoclonal antibodies, are also emerging, but are still in the early stages of development. Finally, inductively coupled mass spectrometry can be employed to analyze metal content in biosamples, (e.g., blood, plasma, and urine, to evaluate acute or chronic metal exposure).
The most renowned application for clinical MS is microbial identification by MALDI-TOF MS. This technique has revolutionized microbiology laboratories since the introduction of FDA-cleared instruments for diagnostic identification of patient infections caused by bacteria and yeasts. The process begins by culturing patient-derived biosamples and selecting a microbial colony for evaluation by the MALDI-TOF MS. The spectrum produced by analysis of the microbial colony is matched to the spectrum of a known organism in an approved, FDA-cleared database. At present, analysis of mycobacteria and filamentous fungi is still challenging and not yet approved for clinical diagnostics by the FDA, but research is ongoing. Rare and exotic microbes can be analyzed against a custom-built, research-use-only database, but that process has not received regulatory approval as a diagnostic tool. Additional novel clinical applications on the horizon include antimicrobial susceptibility testing, which would identify the ideal antibiotic treatment, and direct MALDI-TOF MS analysis from blood culture and biofilms, which could substantially shorten the time to identification.
Inborn errors of metabolism
A very early clinical application of MS was in the analysis of acylcarnitine for identifying newborns with inborn errors of fatty acid oxidation. Defects in twenty-five enzymes of this metabolic pathway leads to eighteen conditions associated with human disease, the most frequent of which is medium-chain acyl-CoA dehydrogenase deficiency. Additionally, MS can be used to screen neonates with cystinosis, glutaric aciduria type I, and lysosomal disorders (LSDs). LSDs encompass a broad spectrum of diseases, including Fabry disease; Gaucher disease; Krabbe disease; Mucopolysaccharidoses types I and II; Niemann-Pick disease types A, B, and C; and Pompe disease. In these applications, MS, generally GCor LC-MS/MS, is applied in a targeted manner to identify key biomarker metabolites associated with the conditions. Alternatively, metabolomics, a systems biology approach that considers the entire metabolome, could be used to identify aberrant metabolic patterns that may be linked to defects in certain biochemical pathways and hence to deactivation of specific genes. Though still in the nascent stages of development, the potential applications of metabolomics for inborn errors of metabolism are vast.
Cancer diagnostics and surgery
MALDI imaging mass spectrometry (MALDI IMS) couples the sensitivity of MS with spatial information. A raster of the MALDI laser beam collects MS data at sampling spots evenly spaced across a matrix-embedded tissue specimen, which are combined to reconstruct a spatial MS map of the histological sample. MALDI IMS has been used predominantly in the analysis of tumor tissue pathology, either in a diagnostic capacity, (i.e., to distinguish tumor from healthy tissue, or in a prognostic capacity, i.e., to identify patients who will benefit from a certain treatment or to predict their survival). Although not yet available in an FDA-cleared platform, the technology is rapidly advancing and could be amenable to automated, user-friendly protocols that would translate well to the clinic.
Another MS technique even promises to deliver real-time monitoring of tumor tissue during surgical resection, aiding the surgeon in complete tumor removal. The invention, called the intelligent knife or iKnife, uses rapid evaporative ionization MS to ablate tumor tissue into a mass spectrometer. An algorithm developed using a training set of cancerous and normal tissues allows the iKnife to differentiate tumor from healthy tissue, defining the margin along the tumor mass.
Like many clinical tools, MS is also set to join the ’omics revolution through proteomics, e.g., in patient plasma, metabolomics, and lipidomics analysis and metabolomics flux studies, both for diagnostics and biomarker discovery. Capillary electrophoresis interfaced with MS can offer better analyte separation and hence resolution for biomarker identification. Other technological advances include movement toward miniaturization of MS instruments, which could eventually be applied to diagnostic point-of-care tests.