Mass Spectrometry: An Emerging Path for Clinical Diagnostics

While many products enable clinicians and medical professionals to provide patient care quickly, reliably, and cost-effectively, there are aspects of clinical diagnostics that need a deeper investigation of the disease markers that in turn amount to early detection of fatal or near-fatal diseases. These evaluations are being conducted by focusing on protein profiling, bioinformatics, and protein-ligand assays for diagnostics in specific cancers such as prostate cancer. There has been a growing need for techniques for ultrasensitive detection tests with clinical diagnostics as the final goal.

In a Clinical Chemistry editorial in 2003, Petricoin and Liotta from the FDA and NCI proteomics programs suggested that advances in mass-spectrometry could “ignite a revolution in the field of molecular medicine.”¹ The idea behind that statement is that while traditional ELISA-based technologies or immunoassay-based diagnostics have been the norm in disease detection for decades, the editors claim that these methods are low throughput, costly, and low sensitivity.

For example, a reliable and sensitive test that’s widely available for cancer detection is the protein-ligand (ELISA) assay—considered to be robust, linear, and reliable. But it also requires tests for a very stringently validated analyte for detection, in addition to an extremely high-affinity antibody for analyte detection. A traditional lab test that searches for cancer-related markers has been the single-marker test with a one-at-a-time approach. Not only is this laborious and time-consuming with an added factor of human error, the protein profiling is extremely complicated and interpretation can be very subjective.

The Techniques

In clinics where monitoring of multiple disease markers for a quick and convincing result to detect early onset of disease such as colorectal or ovarian cancer is paramount, mass spectrometry-based proteomic pattern diagnostics has now provided a rapid and reliable solution. With mass spectrometry-based clinical diagnostics, the use of a drop of blood to characterize multiple proteins in a MALDI-TOF platform is now commonplace. The patterns obtained from this technology coupled with bioinformatics profiling can lead to a very specific, extremely sensitive method to detect and monitor diseases.

Mass spectrometry-based diagnostics using proteomics has been used for ovarian, breast, and prostate cancer detection.² The distinctive tumor-host environment yields amplification cascades specific to the disease process. Even in early stages, these protein-protein interactions, abundances, or 3D conformation changes can indicate the onset of tumor cells and growth via MS-protein profiles. The technique combined with screening bioinformatics to reduce nonspecific and background noise makes for a unique protein marker test that yields a tremendous amount of genetic information for disease diagnostics.

The Controversies

In a 2005 Clinical Chemistry paper by Semmes et al focusing on cancer diagnostics, the controversies of using mass spectrometry as a clear diagnostic tool were put forth.³ Commentaries between Diamandis⁴ and the analysis of data by Petricoin et al1 questioned the reproducibility of SELDI-based approaches.

Diamandis listed a few open questions related to diagnostics SELDI-TOF technology to include the nonlinear relationship between peak height and molecular abundance, validated serum markers, analytical sensitivities, and the unknown relationship between distinguishing molecules and cancer biology. Other scientists (Sorace & Zahn and Baggerly et al) addressed the study-design bias of such evaluations.

The questions included whether such proteomics techniques would improve early detection of cancers such as prostate and ovarian cancers. In fact, data has shown that when lung, colorectal, and breast cancer tissues are detected at an early stage, the survival rate increases 85%.⁵

Addressing Issues and Implementing Better Technology

A lot of progress has been made in the last 5 years to address the questions above. QC reference standards have been established (see “The Need for Control(s)” below). Cleaner algorithms for peak selection for specific markers have been implemented. Swifter analysis and results have also been determined.

Currently, FDA regulations for medical devices list that cross-reactivity and background signals from ELISA or ELISA-like assays need to be addressed in medical devices for clinical labs. MS diagnostics now employ a near-perfect solution to signal-to-noise ratios by implementing stringent protein profiling conditions, least residual contamination, and a clear signal for disease detection.

A key player in mass spectrometry clinical diagnostics is Milford, Mass-based Waters Corp. Waters’ MassTrak system ensures increased sensitivity and reproducibility, and eliminates cross-reactivity with accuracy and precision of therapeutic drug monitoring and biomarkers with improved sensitivity. With more than 5 decades of experience in HPLC, mass spectrometry, software, and chemistry, Waters has recognized the need for lowering cross-reactivity and increasing signal-to-noise ratios in any method for disease markers and detection.

According to Pat Martell, its director of clinical marketing, “Waters MassTrak LC-MS/MS-based solutions are built upon our work with clinical laboratories, helping them address the need to provide accurate and precise quantification of new drugs or disease biomarkers.” They are FDA Class I medical devices that are on an MS-based platform addressing interference and cross-reactivity issues. He adds, “We provide a cleared MassTrak solution for the therapeutic drug monitoring of Tacrolimus (an immunosuppressant drug for kidney and liver transplant patients).”

One of Waters’ first FDA-cleared products screened for PKU, and its second screened for Tacrolimus. Waters is the only LC-MS/MS platform solution provider that has gained regulatory clearance for its solution.

Other companies focused on proteomics, or biomarker profiling, are now moving into the field of MS-clinical diagnostics. A May 2010 press release from Shimadzu and bioMérieux, Durham, NC, announced a collaborative effort between the two to use mass spectrometry in microbiology, combining their experience in infectious diseases organisms and Shimadzu’s MALDI-TOF technology for integration into the microbiology lab workflow.⁶

In a similar release (May 2010), Illumina announced the FDA approval of its BeadXpress system. According to Illumina, this FDA approval allows the company to now pursue diagnostics development in a commercial platform for genotyping, protein profiling, and gene expression.

Half a decade back when the raging question of whether mass-spectrometric protein profiling would meet the desired clinical laboratory practice, a few guidelines were recommended (See Figure 1 below).

Figure 1. Stages of mass spec analyses

The above observations were put forth primarily to address the fact that mass spectrometric protein profiling allowed for multiple new markers for cancer and other diseases.⁷ This obviously was cause for celebration, which then led to frustration due to these methods not being quickly adopted into clinical diagnostic practice. But the main reason for this delay is the fact that translating a discovery method platform into clinical practice requires the transfer of many intricate steps, including the reproducibility of data between labs and within labs.

The Need for Control(s)

As evident from the table above, most of the analysis in MS-diagnostics is based on reference standards, followed by a comparative peak analysis for ideal biomarker recognition. In early 2010, San Jose, Calif-based Cerilliant Corp announced an agreement with Thermo Fisher Scientific Inc, Middletown, Va, to develop and manufacture certified solution standards for the Thermo Scientific ToxSpec Analyzer. While not clinical diagnostics, but rather in clinical toxicology, these solutions can be used readily in LC-MS (ToxSpec Analyzer) for detection of drugs of abuse.

This combined, cost-effective solution addresses the need for standardization of controls and assays in testing laboratories. According to Mitzi Rettinger, Cerilliant VP, Sales and Marketing, “We offer Certified Spiking Solutions™ for clinical applications, including forensics, diagnostics, and toxicology. Our standards provide critical information data points for patient care, NTIs (narrow therapeutic index), hormone testing, and others.”

Cerilliant offers excellent sample-handling options.

According to her, many in the industry are moving toward LC-MS for certain types of clinical diagnostic testing. Whether it is GC-MS or LC-MS, the idea is to make sample handling easier, and this is where Cerilliant can play a major role.

“Thorough neat material characterization, precision balances and weighing technique, and rigorous testing ensures accuracy and shelf life,” she adds, as she describes the validated process for the production of Certified Spiking Solutions that Cerilliant is known for. Cerilliant solution standards are manufactured under ISO Guide 34, ISO/IEC 17025, and ISO 9001:2008, and incorporate FDA’s GMP and GLP requirements. Cerilliant’s shelf-stable, Snap-N-Spike™ solutions reduce variability in testing results so that clinical technicians do not run the risk of reporting errors on patient samples.

Last year, Cerilliant also introduced its Certified Spiking Solutions for Tacrolimus, Cyclosporin A, Mycophenolic acid, and Sirolimus immunosuppressant drugs, critical for therapeutic drug monitoring. The company also prides itself in its Snap-N-Spike format eliminating weighing errors of hazardous chemicals reducing occupational hazards, and allowing for easy spiking into a laboratory’s matrix of choice.

Collaborative Efforts

Multiple collaborations have been initiated between MS-centric companies and companies with biomarker discovery capabilities in the past few years.

In a press release last year, Thermo Fisher announced its agreement with NextGen Sciences, Ann Arbor, Mich, a biomarker company providing discovery and assay services. The collaborative agreement since last year is to allow Thermo Fisher Scientific’s Biomarker Research Initiatives in Mass Spectrometry (BRIMS) center to work with NextGen Sciences to apply its technologies to the company’s biomarkerexpress™ platform, and develop SRM assays for peptides and proteins in biological fluids and tissues.

The biomarkerexpress workflow will include Thermo Scientific LTQ Orbitrap XL mass spectrometer, Thermo Scientific SIEVE software for label-free differential analysis, and Thermo Scientific Proteome Discoverer software for the discovery phase.

Based on its press release statement, Michael Pisano, PhD, CEO of NextGen Sciences, says, “While we were putting in place the infrastructure for our biomarker services we looked for reliable and accurate instruments, and Thermo Scientific technology was found to be just that.”

In May 2010, Rockville, Md-based OriGene Technologies and Seattle-based nonprofit organization Institute for Systems Biology (ISB) announced its collaboration in creating an SRM/MRM (single/multiple reaction molecule) mass-spectrometric database for 5,000 human proteins. The idea behind this collaboration is to facilitate quantitative protein analysis as well as address lab-to-lab validation that has been questioned in the early years of MS-clinical diagnostics.

According to its press release, ISB President and Cofounder Leroy Hood, MD, cautioned that while proteomics combined with systems biology will push discovery further, all discoveries need to be “validated … with a large-scale mass spectrometry standard database.”

Most new and emerging technologies evolve through intense and progressive collaborations among groups with complementary technologies and ideas. Such is the case with MS-clinical diagnostics too, as is evident with increasing collaborative efforts every year to reduce the variation of results for disease detection.

Research in the Right Direction: Academic Innovations for the Future

In a February 2010 article in Medical Devices Today, it was noted that mass spectrometry techniques can be used to detect cancer during surgery.⁸ This would be a revolution in real-time clinical diagnostics should these techniques move out of academic labs into viable products.

Waters’ MassTrak LC-MS/MS

Researchers from Justus Liebeg University Giessen, Germany, use a scalpel in development, to be used in surgery that will distinguish between cancerous and normal tissue. Current technologies include preoperative scans and microscope reads that are error prone and time-consuming.

Comparing tissue mass (and protein profiling) may ease physician decision-making during critical presurgery times. This modified scalpel uses gaseous ions compatible with MS techniques that are released as harmful by-products and collected to reduce harm to lungs during surgery. These by-products are collected by the scalpel and directed into the mass spectrometer, and data is compared to references available on the database in real time.

Purdue University mass spectrometry researchers are coating tissue with charged particles, similar to their German counterparts, but are extending this to include a larger amount of tissue characteristics.

The drawback of this research is obviously the need to obtain cheaper methods to get the same techniques to market. However, elegant methods to improve sensitivity, decrease noise, and improve diagnostics are now being employed in both academic and industrial applied research. Once a commercially viable technology transfer takes place, MS-clinical diagnostics can then move into a relatively inexpensive direction focused on disease diagnostics and greater patient care.

According to Martell, “The clinical laboratory is keen to optimize their work processes in a cost-effective manner while at the same time improving their assay turnaround time.” He also suggests that many clinical laboratories are interested in cleared kits that are precise and sensitive, which in turn reduces the lab’s compliance and validation issues in quality systems. Waters and other GC-MS, LC-MS companies understand the need behind that statement and support the labs by ensuring maximum information through minimum manipulation and analysis time.

To compare chemistry control products, search our buyer’s guide

The use of mass spectrometry in clinical diagnostics is a burgeoning field and is growing rapidly. Most instrument companies are collaborating with either biomarker discovery groups, protein profiling teams, or clinical diagnostics companies to release products for the clinical industry.

Most of the research now is to ease the use of these products, reduce the sample-to-answer time, increase the stringency of results, and employ automated liquid handling systems to reduce operator times. In addition, the future of clinical diagnostics with mass spectrometry focuses on more sensitive bioinformatics tools, better resolution, and higher throughput in sample preparation, leading to statistically significant analytical results.

Madhushree Ghosh, PhD, is a San Diego-based science and health writer.

References

Petricoin EF, Liotta LA. Mass spectrometry-based diagnostics: The upcoming revolution in disease detection. Clin Chem. 2003;49:533-554.
Adam BL, Qu Y, Davis JW, et al. Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res. 62(13):3609-3614.
Semmes OJ, Feng Z, Adam BL, et al. Evaluation of serum protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry for the detection of prostate cancer: 1. Assessment of platform reproducibility. Clin Chem. 2005;51(1):102-112.
Diamandis EP. Proteomic patterns in biological fluids: Do they represent the future of cancer diagnostics? Clin Chem. 2003;49(8):1272-1278.
Etzioni R, Urban N, Ramsey S, et al. The case for early detection. Nat Rev Cancer. 2003;3(4):243-252.
Shimadzu and bioMérieux enter into partnership for mass spectrometry applications in microbiology. Available at: www.biomerieux.com/servlet/srt/bio/portail/dynPage…. Accessed June 1, 2010.
Hortin GL. Can mass spectrometric protein profiling meet desired standards of clinical laboratory practice? Clin Chem. 2005;51(1):3-5.
Clinical Edge: New tools to detect cancer. Medical Devices Today, February 2010. Available at: www.medicaldevicestoday.com/2010/02/clinical-edge.html. Accessed June 1, 2010.