Mass spectrometry: exciting perspectives for clinical labs

Mass spectrometry is poised for a new era, as clinical labs and researchers, hospital managers and industry prepare themselves for expansion in its use. Fuelling growth are trends towards personalized healthcare, the identification of novel biomarkers for translational medicine, large-scale epidemiological screenings as well as everyday clinical chemistry tests beyond just toxicology and endocrinology. There is room for such growth. At present, clinical lab applications of mass spectrometers account for only about 5% of the market.

Superior sensitivity and specificity, samples reusable
Mass spectrometry identifies a molecule by its unique mass-to-charge ratio, and is both highly sensitive and specific. In spite of concerns about cost and steep learning curves, the superiority of mass spectrometry versus immunoassays has never been disputed. Indeed, a study by the US National Cancer Institute (NCI) in 2008 focused on using mass spectrometry to distinguish between breath samples from patients with ovarian epithelial cancer versus those with polycystic ovarian syndrome or endometriosis.

Another advantage of mass spectrometry is its ability to use the same serum for multiple analyte profiling. This makes it useful in large-scale clinical studies, where samples have often been archived. Another NCI study, for instance, used mass spectrometry to identify biomarkers in blood from patients with acute myeloid leukemia; some of the samples were almost 10 years old. Dated samples have also been used for a range of other biomarkers, including malignant melanoma, soft tissue sarcomas and non-small cell lung cancer.

Gas chromatography and liquid chromatography
As a technology, mass spectrometry is not new in a lab setting. Gas chromatograph MS (GC-MS) has been used for ages in the diagnosis of organic metabolic disorders.  More recently, liquid chromatograph mass spectrometry (LC-MS) has become a recommended resource for screening newborns.

The longer use of GC-MS means a bigger user base, as well as a more extensive legacy database, richer software libraries and advanced algorithms. Although GC-MS requires more complex processes for sample preparation (discussed below), it is relatively inexpensive compared to LC-MS systems, and has been considered effective enough for the bulk of applications.

The challenge of standardization
However, there is still some way to go before mass spectrometry attains wider use. One key barrier is a lack of standardization, above all in the preparation of samples. Clinical labs have different approaches to this issue, especially in terms of purification. This leads to sometimes-significant differences in results. Confounding the problem are continuing changes in the methods used for sample preparation, over time even within individual laboratories.

In the US, the Clinical and Laboratory Standards Institute has published two sets of recommendations on the use of MS. However, these leave quite a bit of room for interpretation and are considered no more than broad guidelines.

Preparation of samples for mass spectrometry
Typically, two steps are involved in preparing a sample: the concentrating of analytes, followed by ionization. The sample itself consists of two parts: the analytes of interest, and other components which are collectively known as the sample matrix. Sample preparation is considered the most difficult when whole blood or fractions are involved, given a relatively low density of analytes. Urine lies at the the other end of the spectrum, since the kidneys have already done most of the job of concentrating analytes.

Techniques for preparing samples include solid-phase extraction (SPE), immunoextraction (or immunoaffinity purification) and so-called ‘dilute-and- shoot’. In SPE, analytes and other matrix compounds are separated on the basis of their physical and chemical properties, among them charge and polarity. SPE systems consist of a liquid, mobile phase and a solid stationary phase (usually disposable cartridge-based). The liquid phase uses two different solvents, one for binding and washing, and another for elution.

Immunoextraction separates antibodies bound to the analytes from ‘free’ matrix components, by immobilizing them to a chromatographic column or polystyrene beads. After incubation with an immobilized antibody, unwanted components are washed away, and the enriched analyte is then eluted; another method is to concentrate the sample by drying, followed by re-suspension and injection into the chromatography system.

The third mechanism for preparing MS samples, dilute-and-shoot, is generally used in samples with a relatively high concentrations of analytes (e.g. urine). Here, dilution is usually effective enough to reduce matrix components to a
manageable level.

Successful ionization essential
The process of analysis relies wholly on successful ionization, as mass spectrometers can only detect charged analytes in a gaseous phase. Ionization can be either positive (cationic) or negative (anionic). The most common techniques for ionization in a clinical lab consist of chemical ionization and electrospray.

Chemical ionization generates ions by combining heat and plasma (produced by high-voltage electricity), at atmospheric pressure. While high temperatures vaporize the sample, the plasma (also known as a corona discharge) ionizes the evaporated solvent. Following this, mechanical interaction of the sample components (including analytes of interest) leads to the formation of negative or positive ions.

On its part, electrospray ionization uses electricity, heat and air to successively reduce the size of droplets that elute off the chromatographic column and sharply increase their charge. Ions (above all, proteins) desorb from the liquid droplet surface into a gas phase and then enter the mass spectrometer.

Challenges for vendors
Until recently, industry has focused on process improvements, while researchers have concentrated on improving the specificity and sensitivity of mass spectroscopy. Innovations from vendors have aimed at increasing the efficiency of ionization and of ion transfer, and accelerating discovery of biomarkers by combining size exclusion and affinity capture to enrich low molecular weight proteins, and more quickly separate diseased from clear samples. Some companies have also coupled reference databases of micro-organisms to their mass spectroscopy systems.

The greatest challenge for industry, however, has been to increase user acceptability. Research scientists rather than clinical lab technologists have been the traditional target for mass spectrometry manufacturers. The former, typically, have more interest in top-of-the-line technical specifications and performance than user-friendliness. The potential demand from clinical labs is forcing vendors to change approach. As a result, several are now beginning to package equipment sales with training and support.

Industry is also paying attention to systems integration, to bundle sample preparation instrumentation into a mass spectrometry suite and control its findings. Indeed, software has so far proved to be one of the biggest impediments to the growth of mass spectrometry, once again given the delicate balance between enabling new users to operate a system on the one side, while permitting complex adjustment of performance parameters on the other. OEMs have sought to plug this gap with bespoke add-ons but, as all IT systems designers know, this adds to system cost.

Researchers aim for more precision, ease of use
On the R&D side, a potentially promising area consists of so-called time-of-flight (TOF) mass spectrometers. TOF provides accuracy of 1 part per million by accelerating gas phase ions toward a detector via an electric field. Other initiatives are focused on robotic assistance, turbulent-flow chromatography and ion mobility – with considerable potential seen in linear ion traps. Scientists are also exploring the use of nanospray interfaces as well as microfluidics, though most successes to date have been at bench scale. In the future, such improvements will permit a reduction of detection thresholds, along with greater precision, ease of use and efficiency.

Some trade-offs inevitable
For both researchers and industry, the Holy Grail is to devise adequate user-configurability for trade-offs between high throughput on one side (required, for example, in epidemiological studies or newborn screening), and sensitivity and specificity on the other. Even now, detection of steroids such as cortisol, estradiol and testosterone remain a challenge at the lower end of their reference range, but require high precision in certain categories of patients, for example elderly female patients.

Lab use of mass spectrometry still minor, room for growth
No one doubts that the market for mass spectrometry is potentially huge. Globally, sales have been rising briskly, after falling due to the recession. A study from Los Angeles-based Strategic Directions International estimates the 2011 mass spectrometer market at USD 3.9 billion, with projections of USD 4.8 billion by 2014. The US and Canada hold the largest share of the market (38%) followed by Europe (31%) and Japan (13%), with other countries accounting for the remainder. Leaders in the mass spectrometer market include AB Sciex, Thermo Fisher Scientific, Waters and Agilent Technologies (all from the US), along with Hitachi and Shimadzu. European companies have a smaller presence, and include Germany’s GSG, Spectromat and Thermolinear.

As mentioned before, the clinical lab segment accounts for a very small share of total sales. The biggest users are pharmaceutical companies (a share of 20% of sales, with mass spectrometers increasingly used for metabolomic screening and drug discovery). Government follows closely (with an 18.5% share), universities (12.6%) and environmental/general testing services (9.4%). Electronics, the food and chemical industries also buy more mass spectrometers than clinical laboratories or hospitals.
 
However, the hope is that continuing growth in this entrenched base of other users will drive down unit costs of mass spectrometers, just as clinical labs get ready to increase their own requirements.