A Spectrum of Views on Clinical Mass Spectrometry

The June 2009 issue of Clinical Chemistry contained our very first Q&A, which has since become a monthly feature in the journal. In that Q&A we asked 5 experts about mass spectrometry (MS)9 in the clinical laboratory. We wanted to find out where we stood and where we needed to be. Not only has it been nearly 7 years since we first asked about clinical MS, but we have devoted the entire January 2016 issue of Clinical Chemistry to this important technology. In this Q&A we ask 6 experts representing instrument design, research, and the clinical laboratory for their perspectives on where we stand in 2016. We were particularly interested in the challenges instrument manufacturers face in meeting the needs of customers and regulatory agencies, the potential of MS moving toward point-of-care (POC) testing, whether there was a next “big thing” in MS on the horizon, and whether MS had matured to the point that it was becoming a true clinical instrument. As scientists involved in instrument development, what demands are manufacturers facing with new applications or instrument designs? How about regulatory hurdles? Reza Javahery: Increased analytical sensitivity, reproducibility, durability (uptime), and ease of use all continue to be features demanded by users. Thus, we cannot focus on just one of these areas. Serviceability is also a major concern. As far as regulatory hurdles, we are still in an environment where there are no clear guidelines. Bradley Hart: As manufacturers, we are tasked with challenges that include improving ease of use and connectivity to automation and laboratory information systems/laboratory information management systems (LIS/LIMS), handling smaller sample sizes and spot samples, improving sensitivity for challenging applications, translating and enabling clinical omics assessment panels, and ultimately providing solutions that enable customers to deliver personalized and precision medicine. In addition, manufacturers of equipment used as components of laboratory-developed tests must employ quality management systems to ensure the equipment is satisfactory for the measurement of patient samples per the requirements of applicable regulatory agencies. As such, manufacturers have invested in quality systems that meet the ISO 13485 standard and related requirements. Additional investment is also required to register products around the world. Those investments require continued funding to maintain system effectiveness and to rigorously monitor and resolve product quality or performance issues. As MS moves into “intended use” or “closed” diagnostics platforms, further regulatory requirements are imposed. Provision of associated collection devices, reagent kits, and methods, and the requirement to assure clinical efficacy in addition to analytical performance, leads to additional process control and investment. The time needed to meet regulatory requirements may increase uncertainty in terms of time to market. Some risks can be minimized by assembling solutions across companies who have broad clinical capabilities, and through corporate partnering. Regulatory investment can also siphon funds away from research, so critical strategic business decisions must be made to drive successful ventures that advance science and meet all regulatory requirements. Do you see a near-future push toward POC, miniaturized, or “sold for purpose” instruments? If so, what is going to get us there, and how long it is going to take us? Richard Smith: I believe this will become an important area, with both decreasing cost of such platforms as well as increasing effectiveness. Much of this will be driven by advances in the ‘front end’ portions of such platforms, and to a lesser extent by the MS itself. The big challenges for the short term involve decreasing platform cost, while also continuing to advance performance, enhance throughput, improve robustness of operation, and implementing effective automation. I think we need some substantial departures from present platforms to really address the combination of these challenges. Bradley Hart: Yes, but with caveats. A primary value of MS is that it can easily measure multiple analytes with very high sensitivity and selectivity. If you ask a mass spectrometer to perform a single measurement, it devalues the core capability that a mass spectrometer provides. Assuming that the “for purpose” use includes multivariate analysis such as large toxicology panels or measurement of multiple proteins and their isoforms and modifications, it's easy to imagine that MS will be optimized towards “for purpose” systems. Miniaturization of MS has been achieved on a variety of fronts, specifically ion traps, but innovations in manufacturing technology will continue to drive miniaturization of systems in all aspects, including sample prep and vacuum systems. It will take the combination of a specific use that fits the performance characteristics of a miniaturized system and a clear business opportunity for these types of systems to reach the routine market. If a market develops that drives a “for purpose, killer application,” and miniaturization and technology development continues at its current pace, then it's not a big step for MS to be employed as a POC technology. MS could be employed for a system today as a POC analyzer, but the industry still lacks a compelling application and business opportunity to drive investment. LC-MS lends itself to a wide variety of clinical applications, including large and small molecules. A nexus of activities by the industry and manufacturers could deliver an LC-MS–based “clinical analyzer” with a wide menu of intended use tests. If those efforts are successful, then we could reasonably expect POC systems to enter the market in 5–10 years. Reza Javahery: Yes, depending on requirements for POC. We are almost in a position of replacing mass spectrometers in central hospital laboratories, similar to how x-ray and other scanning equipment is going to the patient rather than the patient coming to the equipment. Sample preparation is a hurdle in this respect, but there are many other industries heavily moving forward by producing robotics so that sample preparation can be done in real time. Are the improvements in MS instrument sensitivity going to be in the ionization, ion transfer, or ion detection stage? Richard Smith: The answer depends on the platform and the manner in which it is applied. In many cases the key challenge is having the sensitivity needed to routinely detect and quantify low-concentration analytes, and here all 3 of these aspects are important and somewhat intertwined. The ion transfer challenge has been essentially solved using well-implemented ion inlets and ion funnel designs with electrospray ionization (ESI) sources, since it is possible to achieve almost perfect ion focusing and transfer. Improved interface designs are still needed that can handle much larger ion currents from more intense ESI sources without the need for expensive pumping arrangements, but in general the source of the issues and how to address them is understood, and the solutions are beginning to become broadly available. The ionization step is actually the greatest challenge, particularly using on-line liquid chromatography (LC)-ESI-MS, which has become the real workhorse of modern MS. The efficiency of ESI increases as the flow rate from the LC decreases (and where the electrosprayed droplet size decreases and the charge available per analyte molecule increases) and for some species can approach 100% at very low flow rates <10–50 nL/min. Effective LC separations at such low flow rates presently require the use of nanobore LC columns that are both more difficult to prepare and use, and also readily plugged. The use of these columns also creates significant challenges in maintaining stable LC flow rates, reproducible mobile phase gradients, and ESI performance. At present I would not want to risk valuable samples to achieve the most sensitive measurements possible, which might require, for example, the use of 15-micron inner diameter packed columns to achieve very low flow rates and optimal ESI efficiency. Rather, I would probably use 50-micron- or even 75-micron-diameter columns, which work best with somewhat higher flow rates and provide much more robust performance, but result in somewhat reduced sensitivities. But this might not make any significant difference, depending on the platform and the specific application. For example, an orbitrap or Fourier transform ion cyclotron resonance (FTICR) analyzer is constrained by both the charge capacity of the trap and the time it takes to make a measurement, and thus benefits of nanoflow LC would generally only be significant when sample sizes are very small. For other MS platforms (e.g., TOF or triple quadrupole) that do not have such constraints, much greater gains can be achieved. Bradley Hart: Ionization, or more specifically the generation of ions and efficient transfer from atmospheric pressure to the first vacuum region, is still a major challenge for manufacturers. This area is fraught with intellectual property challenges and is one of the major areas of differential performance amongst the vendors. Today, each vendor's design may have preferential performance towards specific analytes, but vendors are working on designs to ionize the widest variety of compounds with minimal ion suppression effects. It is possible with “for purpose” systems that the ion source designs would be simplified and optimized for specific analytes and specific sample introduction flow rates or techniques. There are continuous incremental improvements in ion transfer efficiencies, and these again may be specific to certain types of mass analyzers, including relatively new mass analyzers like the orbitraps. There are also specific transmission techniques that are related to transmission of large molecules vs small molecules. Efficient transmission of large proteins or antibodies requires specific settings that may be invoked as “modes” of operation or specifically optimized in “for purpose” designs. Ion detection technologies also continue to improve incrementally, but new digital technologies offer promise for substantive leaps in sensitivity, selectivity, and unique ways to detect ions. Reza Javahery: Sample extraction from a complex mixture is most challenging. If the ionization process occurs at atmosphere pressure, then ion introduction into the mass spectrometer remains the critical issue that needs to be addressed. It is highly important to be able to transfer most, if not all, ions of interest to the mass analyzer. In modern MS, ion transportation has become a key factor for the production of high-sensitivity instrumentation. We are seeing an increasing number of reports on new protein biomarkers detected by MS, but few have made it into routine analysis. What are the bottlenecks? What will it take for MS protein marker assays to be ready for regular use? Samir Hanash: There are several major bottlenecks. A consequence of an increasing number of reports on new biomarkers is a tendency to confuse which report(s) and which biomarker(s) to pursue further by, say, a diagnostics company, particularly when the evidence is relatively weak with respect to the extent to which the discovery and initial validation are relevant to the intended clinical application. Assuming that the evidence is strong for particular MS-based marker(s), the next challenge stems from whether markers that result from other discovery strategies for the same application(s), e.g., nucleic acid–based markers instead of proteins, may be equally strong and may be more amenable to clinical grade assays. Assuming the latter is not the case, the next decision to be made is likely going to be the nature of the assay to be developed, the standard approach being an immunoassay. For MS marker assays to be ready for clinical use requires a substantial investment on the part of MS instrumentation companies to develop suitable front-end sample preparation methodologies with the necessary analytical reproducibility together with MS instrumentation that yields the prerequisite sensitivity (typically below ng/mL) for assays of biospecimens like plasma and urine. Clearly much progress has been made and it is likely that in the near future, based on informal feedback from reliable industry sources, effort in this direction will intensify. Ravinder Singh: I agree that few biomarkers have made it into routine use. MS has helped in the detection of new protein biomarkers in well-controlled studies; however, not many biomarkers have proven to be clinically useful in confirmation studies and used in large clinical studies. Biomarker detection either by MS or any other technology is essential in generating a proper hypothesis, but ultimately confirming the biomarker in circulation for improving patient care is what makes the final product for patients and clinicians. The identification and confirmation of the biomarker, as well as elucidating its pathophysiology, are critical before assigning an analyte as novel biomarker. Because of constraints, where are clinical and tests are being in the For example, the clinical use of which was a biomarker at one is being diagnostics is by using and It is critical for the development and validation of novel biomarkers that clinical with and are of current and thus can new biomarkers that can improve clinical or patient MS of instrument when use nanoflow vs liquid What is of this technology becoming more and laboratory not employ nanoflow or LC techniques. we have with LC robustness even at the more standard flow rates used for measurement of small molecules. The quality of the LC depends on such as how the LC is how the are and how system are for these of requires and highly The of LC systems has a to of MS assays in our which is and to with to maintain the systems. in robustness and ease of the step are needed for of LC-MS systems into routine Increased robustness can be by of the LC system design with the of decreasing system A in the number of moving and a more in flow and a in the number of needed to achieve optimal performance would a long toward decreasing the number of and improving the to Richard Smith: This is not a a of us have been with this challenge for well a by the needed to achieve more robust performance. As you can see from to an I not that we will have robust nanoflow platforms that achieve optimized sensitivity in the near The system needed would have robust performance, high and reproducible and highly efficient with MS, with this high I believe that the key is to away from the use of nanoflow Bradley Hart: from that the of time in LC-MS is to the LC The mass analyzer itself has very high is a particular challenge for routine operation, and while improvements have been made to the of in this the need to sensitivity for the mass spectrometer has been flow sensitivity to and It is expensive to nanoflow LC vs vacuum pumping systems. In the discovery phase or work where proteins and were of sample were very and using an issue with sample If a marker was in nanoflow were or to since it was just much work to spectrometers are sensitivity years. have that flow rates or higher could be used in of nanoflow with systems. As the discovery phase and we focus on specific proteins and we can a larger sample thus instrument sensitivity requirements. As specific proteins for will away from and columns are being more and more with MS applications in (e.g., reduced for when the systems (e.g., flow and systems work and provide and and again the mass spectrometer sensitivity requirements. systems and columns for and will the required in and will improve reproducibility vs nanoflow for routine into in 5 years where do you see MS new in the and of Do you see a next “big thing” in We will likely see increasing use of MS technology for measurement of clinically important and protein the increasing focus on the benefits of personalized the next “big thing” might be a in the number of MS applications for of current is use of MS to measure for the of and of For of MS or in routine clinical laboratories, probably the important in technology needed is A routine analyzer can perform more than almost the mass spectrometer itself is of measurements with a high of the step requires that samples be in a where one sample must be before the next is the chromatography with the of systems that can multiple columns with that are each in LC still remains the major step to MS systems will likely not be able to with standard analyzers in terms of and time can be in a nearly manner without increasing the number of system Samir Hanash: The next big is a to MS-based assays with sample or of for assay by and analysis. Bradley Hart: The next “big thing” in the application of MS to clinical is of MS and measurements to enable our customers to MS will provide more of a that for of that are specific to certain patient and validation of new and will drive significant industry cost Reza Javahery: MS will continue to provide more and more valuable information that is to many current diagnostics of its and The analytical sensitivity of MS a major in many specifically in providing necessary information that we could not with other techniques. I that many potential advances in MS become since MS technology is relatively and has Ravinder Singh: The will from the use of a single biomarker for confirmation of a single MS has the potential of multiple and will in complex reports for clinicians. The validation of multiple biomarkers in like and for small molecules and proteins will be the need of the time. The of analyte by MS, e.g., and and and of proteins the of the and will clinical value to the and novel Richard Smith: I think detection of what we think of as being modern MS platforms are and while there will be I think are going to be incremental and I believe the combination of ion with MS is going to be in broad areas of application. can be of than is with much smaller sample sizes than and since it not involve a phase it all the issues associated with It is also robust and most major MS vendors offer to the based on one approach or short in terms of either their or sensitivity and very I see as the next big for MS, and that will enhance its performance and robustness for the broad of all its possible in the and of is that in 5 to years from you will see to most MS the I also see major coming from MS platforms in which substantial of analyzers work in in gains in throughput, But I think this is also likely a and clinical applications have heavily (e.g., methods, but there is and MS for protein analysis. there any evidence that MS is on some Ravinder Singh: Not technologies will in use in clinical since both have MS has the potential of and novel biomarkers but may be the best for the performance like and an and sensitive assays for proteins is very for manufacturers as a to get and assays are more likely to be used in the scientists that MS can provide to other laboratory techniques. our are beginning to the analytical performance benefits by MS and are specifically that measurements be made using MS technology. For example, our have by LC-MS to that are when using for measurement of We have also used LC-MS to resolve several cases of that were not by or techniques. In addition to proteins, our interest in small molecules has of our laboratory for from and and a large of the are from to of in these patient our have that be using MS. We also on MS techniques to resolve by and to routinely monitor and Samir Hanash: I would not as but we are all of the challenges in antibodies or with the necessary and We have many markers that are on the for which in coming with a has It would be more to develop an MS-based assay if we could achieve the prerequisite sensitivity and with the MS that MS involve Are there improvements or new that you have used to improve MS Ravinder Singh: is important for low-concentration there has not been much For of and toxicology where the of analytes are much higher than the there is minimal sample extraction and required and rather and is The use of extraction has but the extent of and has not been cost effective for low is becoming again in the clinical for detection of proteins in and has the work flow of our laboratory would from quality and throughput, we have done to our current we with LC The requirement for substantial for development, and of is the most challenging issue we face for of additional MS We would develop a larger number of MS assays if available could be What do you want to see in MS to it a true clinical MS instrumentation was for science and Clinical their work flow around of rather than from technology specifically for clinical For of MS in routine laboratories, a major in approach equipment and process design is MS systems of instrument sample reagent and application of quality For MS to the needs of the clinical all components and need to be and The current of together components in robustness and process sample use a and no of any process be automation of sample reagent and and will improve efficiency. must be instrument components such that feedback from any point in the process will a quality be to each result and in real time. types of and would enable of MS instrumentation out of by specifically and into the highly routine clinical laboratory. Ravinder Singh: Not many MS for clinical are and are It is very challenging to have the liquid MS, and laboratory information systems. LC-MS methods, equipment is very complex and requires expensive from vendors and for multiple applications to be all these are no will be able to get an for the final product to be useful and to the instrument. mass spectrometry point of care laboratory information system laboratory information management system electrospray ionization liquid chromatography Fourier transform ion cyclotron resonance ion and

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