According to the World Health Organization, depression affects more than 300 million people and is the leading cause of ill health and disability worldwide. Currently, diagnosis of depression involves the use of questionnaires about the patient’s general health, the way they are feeling and how this is affecting them. Blood tests are carried out during diagnosis, but are for the purpose of excluding other conditions, such as thyroid disease or vitamin D deficiency, that can give rise to symptoms similar to depression. There is no physical test for depression per se. Treatment for depression ranges from ‘wait and see’ and exercise for very mild forms through to self-help groups, talking therapies, such as counselling and cognitive behavioural therapy, for mild to moderate depression, as well as antidepressant medication for the more severe end of the spectrum. There are several classes of antidepressant drugs and treatment is largely through a process of trial and error in order to determine what does or does not work for certain patients, as it is recognized that there is a large variation in the way individuals respond to the different medications. Additionally, although numbers vary, conservative estimates suggest at least 30% of patients do not respond to antidepressant medication, and suffer from what is termed treatment-resistant depression.
However, recently, a line of research about one cause of depression has been gaining traction: the role of inflammation. Recent work suggests that an overactive immune system causing higher levels of inflammation results in an increased risk of depression and that these patients are less likely to respond to antidepressants; perhaps, therefore, the cause of treatment-resistant depression. It has also been noticed that patients taking anti-inflammatory medication for rheumatoid arthritis experience improvements in mood that are more profound than just feeling happier because of reduced pain; changes that have been confirmed by brain scans. Professor Ed Bullmore, Head of the Department of Psychiatry at the University of Cambridge, is certain that inflammation can cause depression and his new book, The Inflamed Mind: A radical new approach to depression, is about to bring these ideas to the attention of a much more general audience. The exciting relevance of this research for clinical lab diagnostics is the thought that a blood test for biomarkers of inflammation will help in an objective diagnosis of a certain type of depression and that treatment will be much better tailored to the individual – perhaps the individuals who fail to respond to current antidepressants. Even if this benefits only a small proportion of people with depression, because of the prevalence of the condition a large number of people will benefit.
Steroid metabolism
All steroids share a cyclopentanoperhydrophenanthrene nucleus, with individual species varying according to the presence of different functional groups attached to this four-ring structure, as well as by the oxidation state of the rings. Cortisol structure is given as an example in Figure 1. In humans, the major sites of steroid hormone production are the adrenal gland and the gonads. Steroids are synthesized from cholesterol via a series of enzyme-catalysed steps (Fig. 2), which are under tight regulation in healthy individuals by feedback mechanisms involving the hypothalamus and anterior pituitary. Steroids have a wide range of physiological functions which are summarized in Table 1.
Adrenocortical carcinoma – a diagnostic challenge
There are many endocrine disorders that result in the improper synthesis of steroids, and one of the rarest and most severe is adrenocortical carcinoma (ACC). ACC is a malignancy of the adrenal cortex with an annual incidence of 1 or 2 cases per million [1]. The majority of ACC cases are sporadic and occur in the fifth or sixth decade of life and more commonly in women; although ACC can be associated with several familial syndromes including Li-Fraumeni, Beckwith-Wiedemann, Lynch syndrome and multiple endocrine neoplasia type 1 [2]. Functional steroid hormone-producing tumours occur in around two-thirds of cases [3], presenting with varied signs and symptoms of steroid overproduction, most commonly Cushing’s syndrome (cortisol excess) and hyperandrogenism. ACC can progress rapidly in some patients, therefore it is vital that it is distinguished from benign adrenal adenomas, as ACC has a 5-year survival rate of <50% [2]. A surgical cure is only possible if the carcinoma is detected in its localized stage, otherwise the median survival period is <15 months [4].
The diagnosis of ACC is challenging as there is no single diagnostic tool that is able to distinguish ACC from other adrenal masses, including benign adenomas with glucocorticoid or mineralocorticoid excess, phaeochromocytoma and non-functioning adenomas. Imaging alone is insufficient for diagnosis, as although patients with ACC almost always present with tumours ≥4 cm, the presence of a large mass only has a clinical specificity of 61% [5]. Additionally, whereas up to two-thirds of tumours are functional, less than half of ACC cases present with clinical signs of steroid overproduction [3], with a further proportion presenting with other symptoms including abdominal pain. However, a significant proportion are discovered incidentally [2].
The European Network for the Study of Adrenal Tumours (ENSAT) currently recommends that the initial biochemical work-up for suspected ACC includes measurement of serum cortisol (both basal and assessment of suppression after dexamethasone), dehydroepiandrostenedione sulphate (DHEAS), androstenedione, testosterone, 17-hydroxyprogesterone, estradiol and aldosterone (if the patient is hypokalemic or hypertensive). An alternative approach is to measure steroid metabolites in urine using gas chromatography-mass spectrometry (GC-MS); increases in the excretion of metabolites of the steroid precursors 11-deoxycortisol, 17-hydroxypregnenolone and pregnenolone have been shown to provide particularly high diagnostic utility in ACC. Unfortunately, urine steroid profiling is not commonly available in clinical laboratories owing to lengthy sample preparation and complex result interpretation. Further, serum 11-deoxycortisol, 17-hydroxypregnenolone or pregnenolone measurements are rarely performed either because of lack of demand, or specificity of the available immunoassays which may be subject to significant levels of cross-reactivity.
As a result of these limitations, the use of LC-MS/MS is increasingly being adopted to provide more specific steroid hormone measurements. An approach we have taken in our laboratory is to develop and fully evaluate a multiplexed LC-MS/MS method panelling 13 steroids in serum [6] to include many of the steroid synthetic pathway intermediates currently not available for ACC work-up.
Use of a serum steroid panel
The steroids included in our serum panel are highlighted in Figure 2 and are as follows:
Samples are prepared for analysis by an initial protein precipitation step to remove steroids from their binding proteins, followed by liquid-liquid extraction in order to cleanly extract the steroids from remaining matrix components. Prepared extracts are then analysed by LC-MS/MS in which steroids are first resolved on a reverse phase C18 column by gradient elution followed by MS/MS detection using positive atmospheric pressure chemical ionization (APCI) operated in multiple reaction monitoring mode. Chromatographic separation of several isobaric (same mass to charge ratio) steroids is essential, as is the use of deuterated internal standards for all steroids in the method.
When we applied our method to adrenal tumour samples [6], we were able to show that between 4 and 7 steroids were elevated in all ACC cases in comparison to non-ACC adrenal tumours where a maximum of 1–2 steroids were abnormal. The cortisol precursor 11-deoxycortisol was most useful in the discrimination between ACC and non-ACC adrenal lesions, whereas other steroids markedly elevated in ACC included 17-hydroxypregnenolone and pregnenolone. Indeed, all steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC. This validates the use of a panelling approach when investigating adrenal masses.
Our findings compare well with urine steroid profiling studies. Although urine steroid profiling using 24-hour collections may offer greater clinical sensitivity compared to a single blood measurement owing to diurnal rhythms of steroid production, urine measurements rely on accurately timed collections that are often performed incorrectly and are inconvenient to the patient. Advantages of our LC-MS/MS serum panel compared to urine steroid profiling by GC-MS include a less labour intensive sample preparation, as well as less expertise required for the interpretation of complex profiles, as the serum method only targets selected steroids rather than the large number of their metabolites in urine.
Use of our LC-MS/MS serum steroid panel in ACC patients has further demonstrated the limitations of assessing serum steroids by immunoassay. We observed evidence of notable interference in ACC patients in the cortisol, progesterone, 17-hydroxyprogesterone and androstenedione immunoassays, inferred to be due to elevated concentrations of structurally related steroid precursors.
Future work
Currently, our 13-steroid serum panel has been used to study a relatively small number of ACC patients (because of the rarity of the disease), and clearly larger prospective studies are required to more fully determine the diagnostic utility of our panel in ACC. Further work is also required to clarify the effects of age, sex and diurnal variation on serum steroid panelling; nonetheless the most useful markers of ACC are markedly elevated above variation attributable to these biological factors. In addition to the complexity of interpreting biomarker panels, it is not only important to consider specific reference ranges, but to also consider the patterns in results which require an omics-based analysis approach to interpretation. The challenge surrounding this, as well as the requirement for clear presentation and reporting of results to clinicians requires close involvement of clinical colleagues for the development and introduction of such testing strategies.
The analysis of steroid panels by LC-MS/MS can also undoubtedly be used in other conditions including inborn errors of steroid metabolism such as congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome (PCOS).
Although we have demonstrated the advantages of our LC-MS/MS steroid panel compared to routine immunoassays, there are undoubtedly disadvantages of using LC-MS/MS. These include the initial cost of instrument purchase, the increased expertise required and often a more laborious sample preparation. Additionally, the specificity of mass spectrometry should not be readily assumed; careful selection of multiple reaction monitoring (MRM) transitions and chromatography conditions are essential to separate isobaric steroids and other interfering compounds. However, in the context of improving the biochemical tools available to us to aid the diagnosis of ACC, the advantages of LC-MS/MS far outweigh these limitations.
Summary
In summary, LC-MS/MS serum steroid panelling offers an additional tool for the challenge that is the diagnosis of ACC. Our method combines measurement of both common and rarely measured steroids in a single sample, which we have shown provides useful data to aid the discrimination of ACC from benign adrenal tumours. Use of LC-MS/MS gives several advantages over the immunoassay and GC-MS-based methods currently used to assess steroid overproduction, but further work is required to demonstrate the full potential of its use in the diagnosis of ACC.
References
1. Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endocrinol Metab 2013; 98: 4551–4564.
2. Else T, Kim AC, Sabolch A, Ramond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD. Adrenocortical carcinoma. Endocr Rev 2014; 35: 282–326.
3. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, Schneider P, Smith DJ, Stiekema H, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumours. J Clin Endocrinol Metab 2011; 96: 3775–3784.
4. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012;366:2189–2197.
5. Hamrahian AH, Ioachimescu AG, Remer EM, Motta-Ramirez G, Bogabathina H, Levin HS, Reddy S, Gill IS, Siperstein A, Bravo EL. Clinical utility of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadenomas: Cleveland Clinical experience. J Clin Endocrinol Metab 2005; 90: 871–877.
6. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, Diaz-Cano S, Galata G, Schulte KM, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocortical carcinoma. Clin Chem 2017; 63: 1836–1846.
The authors
Victoria Treasure* MSc and Dr David Taylor PhD
Department of Clinical Biochemistry
(Viapath), King’s College Hospital NHS Foundation Trust, London, UK
*Corresponding author
E-mail: Victoria.treasure@nhs.net
The increasing global burden of sepsis in healthcare calls for better diagnostic tests that allow earlier detection of sepsis and infections that could lead to sepsis. The major problem for patients at risk for sepsis is an immunological imbalance. Cells of the innate immune system, such as monocytes and neutrophils, are the first-line of defence against infections. In the presence of sepsis, these cells produce a flood of inflammatory cytokines, causing widespread inflammation that can lead to death. Monocytes perform multiple immunological functions, and play a role in the development of sepsis-induced inflammation and immunosuppression. Monocyte subpopulations with different functions and morphologies vary in number over the course of the inflammatory response. The monocyte distribution width (MDW) is a novel cellular marker of monocyte anisocytosis that can add significant value to the white blood cell (WBC) count and help detect sepsis in patients entering the emergency department (ED).
by Elena A. Sukhacheva
Sepsis epidemiology and definitions
Sepsis is a major healthcare burden and, despite progress in diagnostic and treatment options, mortality from sepsis remains unacceptably high. The number of septic patients in the U.S., UK and EU is increasing [1–4]. Clearly, there is an unmet need for better diagnostic tests that can provide both the early detection of sepsis and the detection of severe infections that may progress to sepsis, if not diagnosed early enough. Global increases in sepsis frequency may be related to the aging population, as the incidence of sepsis is disproportionately increased in elderly adults, and age is an independent predictor of mortality [5]. Furthermore, immunosuppressive drugs, which are increasingly being used for diverse conditions, may result in more severe infections and increased sepsis frequency [6].
The definition of sepsis has recently been changed from the previous Sepsis-2 definition of a systemic inflammatory response (SIRS) in the presence of an infection [7], to the current Sepsis-3 definition of a life-threatening organ dysfunction caused by a dysregulated host response to infection [8]. The new Sepsis-3 definition reflects newfound understanding that the immune response in sepsis is more complex than previously thought, comprising both pro- and anti-inflammatory mechanisms.
Immune response in sepsis
It is now clear that the major problem for patients with sepsis, or at high risk of developing sepsis, is immunological imbalance, and dysregulation of the mechanisms of innate and adaptive immunity. Sepsis occurs when the immune system begins, in one way or another, to lose the battle against severe infection. After sepsis onset, the production of pro-inflammatory cytokines (IL-1β, IL-6, and tumour necrosis factor [TNFα]) by the cells of the innate immune system (neutrophils and monocytes) may result in a “cytokine storm” that produces overwhelming inflammation, which can lead to blood pressure collapse, coagulation abnormalities and, ultimately, organ failure and death. In the later stages of disease, patients who survive the cytokine storm may die from sepsis-related immunosuppression and an inability of the immune system to combat infection efficiently [9]. Inflammatory and immunosuppressive processes may overlap in sepsis [10,11], further complicating the biology of this fatal condition whose mechanisms are still poorly understood by scientists. Figure 1 shows the current understanding of immune imbalance in sepsis [12]. While all immune cells are involved in the immune response in sepsis [13–16] (Figure 2), this document is mainly focused on changes in monocytes, with other cell populations discussed only briefly.
Under normal conditions, neutrophils usually stay in the circulation for only a few hours and undergo apoptosis within 24 hours of release from the bone marrow. In sepsis, the delay in neutrophil apoptosis [17,18], combined with the increased neutrophil production in the bone marrow, results in neutrophilia. The function of these neutrophils, however, is impaired [19], with decreased chemotactic activity [20,21], decreased antibacterial function and increased production of anti-inflammatory cytokine interleukin 10 (IL-10) [22].
Sepsis also has a profound effect on all the main lymphocyte subpopulations [14]: CD4+ T-cells, CD8+ T-cells and B-cells undergo increased apoptosis; T-regulatory cells are more resistant to sepsis-induced apoptosis, leading to an increased proportion of T-regulatory cells and an immunosupressive phenotype. T-helper cell polarization from a pro-inflammatory Th1 phenotype towards an anti-inflammatory Th2 phenotype also contributes to increased immunosuppression in sepsis.
Monocytes also undergo multiple changes in sepsis, but before discussing these phenomena, it is important to discuss some basic information about the biology and classification of monocytes.
Monocytes’ biology and classification
Monocytes are cells of the innate immune system, the body’s first-line of defence against infection. Other cells of this system include neutrophils, basophils, eosinophils, mast cells, as well as certain types of lymphocytes such as γδ-T-cells and natural killer cells. The innate immune response develops during the first hours and days after pathogen invasion, and the majority of pathogens entering the human body usually are inactivated by this response and do not require adaptive mechanisms with lymphocyte involvement.
Myeloid precursors in the bone marrow differentiate into promonocytes and then into mature monocytes that enter the peripheral blood. These monocytes stay in the circulation for one to three days, after which they migrate into tissues and organs, where they turn into macrophages and dendritic cells. Morphologically, monocytes are large cells measuring 10 to 18 µm in diameter, with convoluted nuclei and azurophilic granules in their cytoplasm.
Monocytes and dendritic cells perform multiple immunological functions that include phagocytosis, antigen presentation and cytokine production. The function of these cells is regulated by a number of cell surface receptors:
Three subpopulations of monocytes have been characterized in peripheral blood [23–25]. Classical monocytes make up the main monocyte population. Expressing high level CD14 and no CD16 (CD14++CD16-), they represent 80–90% of monocytes in peripheral blood. “Intermediate” monocytes expressing CD16 (CD14++CD16+) are normally found at low numbers, but increase with cytokine stimulation and inflammation. Nonclassical monocytes display decreased expression of CD14 and increased expression of CD16 (CD14+CD16++), and comprise 9%+/-5% of all monocytes, with an average count in healthy donors of approximately 45+/-22 cells/µL [26].
In the literature, nonclassical monocytes are sometimes referred to as inflammatory or pro-inflammatory monocytes; however, published recommendations for the nomenclature of monocytes and dendritic cells in the blood clearly advocate avoiding functional terminology, “because this leads to confusion as the label ‘inflammatory’ has been used for different subpopulations in humans and mice [24].” Also, “these terms may prematurely ascribe functional attributes to cells based on ex vivo studies while they largely remain to be functionally characterized in vivo [24].” Subsets of nonclassical monocytes are expanded dramatically in several pathological conditions including sepsis [26–28], HIV-1 infection [29–33], diabetes [34–35], tuberculosis [36] and other disease states [37].
The recent detailed analysis performed by Mukherjee et al. [28] revealed the functions of monocyte subsets as follows: classical monocytes are phagocytic with no inflammatory attributes, nonclassical subtypes display inflammatory characteristics on activation and display properties for antigen presentation, and intermediate subtypes appear to have both phagocytic and inflammatory functions [28]. In 2017, research based on single-cell RNA sequencing discovered even more subtypes, describing six subpopulations of dendritic cells and four monocyte subpopulations [39]. This classification was based solely on transcriptional activity, and further studies will be needed to understand function and describe the phenotype of all cell subpopulations. Nonetheless, it is clear that morphologically similar cells that we call monocytes may actually have very different functions in human immunity.
Monocytes in sepsis
Monocytes, as cells of first-line defence against infection, are involved in the immune response from very early stages. Abundant literature exists on monocytes and the changes they undergo in sepsis.
A recent study on the dynamics of monocyte subpopulations in peripheral blood at the onset of infection has demonstrated a decrease in the number of peripheral blood monocytes during the early stages of lipopolysaccharide (LPS)-induced acute inflammation in humans. This loss may be due to the migration of monocytes from the blood into tissues, where they differentiate into macrophages and dendritic cells, or it may reflect an increase in monocytes residing in the marginal pool or rolling on the vessel walls [40]. For all three subpopulations of monocytes, the number of cells was decreased at one to two hours after LPS injection. This decrease was followed by a return to the baseline count, but with differences in timing for the three monocyte subsets. This difference in timing means that the early stages of infection, before the appearance of any clinical symptoms, are characterized by differences in the proportions of monocyte subpopulations relative to baseline pre-infection proportions.
Functional changes in monocytes and, in parallel, changes in their cellular morphology, have been demonstrated in the past for a human THP-1 monocytic cell line infected with viable C. pneumonia bacteria [41]. The differentiation of infected cells into macrophages was accompanied by a change to an ameboidor diffused morphology as assessed by microscopy after Giemsa staining.
Multiple studies have demonstrated the importance of HLA-DR expression on monocytes as a prognostic marker in septic patients. A decreased level of HLA-DR expression on monocytes has been found to be a negative prognostic indicator [42–44] and may be used to evaluate the functional activity of the immune system [45,46]. Decreased HLA-DR, as a marker of monocyte anergy, correlates with decreased antigen presentation capacity and decreased pro-inflammatory cytokine release. This has been analyzed mainly by flow cytometry, but, recently, new methods based on real-time PCR have emerged [47,48].
Another monocyte marker, CD16, plays an important role in orchestrating the response of monocytes to Gram-negative sepsis. It has been demonstrated that CD16 on human monocytes is a key regulator of the TRIF-dependent TLR4 signalling pathway, and this pathway is preferentially activated in the CD16+ monocyte subset [49]. Recent publications suggest the variability of monocyte properties in sepsis. Detailed analysis of gene expression in patient monocytes during sepsis and after recovery demonstrated plasticity of monocytes in the course of disease [50]. The significant up-regulation of pro-inflammatory cytokines (IL-1b, IL-6) and chemokines (CCL3 and CCL5) has been demonstrated in sepsis monocytes compared to monocytes after recovery. Transcriptional factor NF-kB, a central transcriptional regulator of the inflammatory response, was also activated in sepsis monocytes, supporting their involvement in severe inflammation. At the same time, anti-inflammatory cytokine IL-10 was found to be up-regulated in sepsis monocytes. These studies once again highlight the diversity of monocytes’ function in sepsis pathogenesis, and their key role in disease progression, with the possible polarization from a pro-inflammatory state to an immunosuppressive state.
More recently, Crouser et al. demonstrated that the morphological variability that occurs during monocyte activation in the early inflammatory response can be captured by measuring the monocyte distribution width (MDW), an indicator of monocyte anisocytosis. Investigators showed that MDW could be a novel cellular marker that may help detect sepsis early in patients admitted to the emergency department (ED) [51]. Multiple morphometric characteristics of monocytes were obtained using a DxH 800 cellular analysis system, which employs physical measurement of cell volume, conductivity and multiple angles of laser scatter to classify leukocytes into five sub-populations and detect the presence of abnormal cells. This study showed that anisocytosis of circulating monocytes provides significant added value to WBC count for the detection of sepsis in the ED population.
Conclusion
In summary, monocytes are a very heterogeneous population of cells that differ in phenotype, size, nuclear morphology, gene profile and function [52]. In sepsis, this diversity is even more pronounced due to functional changes of monocyte subsets, and is accompanied by a variation in monocyte morphology.
Morphological variability is just the tip of the iceberg of the underlying biological heterogeneity, and may be an important early marker of sepsis or severe infections with a high risk of progressing to sepsis. A recent publication from Crouser [51], together with previous research on sepsis using cellular morphometric parameters gathered using a DxH 800 analyser [53–56], may build the foundation for practical usage of MDW in combination with currently-used sepsis markers (WBC, PCT, CRP, IL-6) for early sepsis screening and diagnosis, leading to early initiation of appropriate therapy.
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The author
Elena A. Sukhacheva, Ph.D.
Senior Manager,
Global Scientific Affairs, Hematology, Beckman Coulter Diagnostics,
Miami, FL, USA
Despite significant inherent advantages of liquid chromatography-tandem mass spectrometry (LC-MS/MS) over immunoassay techniques in clinical laboratory applications, its adoption into routine practice has been slower than might have been expected. The barriers to more widespread uptake are a function of issues in the laboratory workflow. This article analyses those issues and discusses how they can be overcome by improved automation and integration with the laboratory information management system, drawing on examples from the North West London Pathology (NWLP) clinical laboratories at Imperial College Healthcare NHS Trust.
by Dr Emma L. Williams
Introduction
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen over two decades of use in specialist clinical laboratories in the UK, offering a number of significant advantages over immunoassay techniques. These advantages include increased specificity, sensitivity and accuracy, as well as the detection of multiple analytes within a single assay. There is no need for an antibody for analyte detection and the method is not susceptible to the antibody-based interferences that plague immunoassays [1]. LC-MS/MS is suitable for multiple sample matrices and avoids the need for radioactive tracers. LC-MS/MS assays also have a wider dynamic measurement range and have improved between-method bias when compared to immunoassays.
LC-MS/MS initially played a role in specialist clinical laboratories in areas such as newborn screening, inborn errors of metabolism, toxicology and in immunosuppressant and therapeutic drug monitoring. More recently LC-MS/MS has established a role in diagnostic endocrinology, with the first appearance of LC/MS-MS for the measurement of vitamin D in the international vitamin D external quality assurance scheme (DEQAS) in 2005. There are now over 150 labs registered in this scheme using LC/MS-MS for the measurement of vitamin D. However, automated immunoassay still dominates and represents 69% of participants registered in the DEQAS scheme. Why has there not been more widespread adoption?
A number of issues have inhibited wider adoption and routine use of LC/MS-MS in the clinical laboratory. First among these is the use of labour-intensive manual workflows, which result in lower throughput, decreased productivity and longer turnaround time. Furthermore, a high level of technical expertise is needed, not only for method development, but also for troubleshooting assay and equipment failures. In addition to the high initial capital costs of purchasing the equipment, ongoing personnel costs are higher because of the need for more technically competent staff. With a clear understanding of where the bottlenecks in the process arise, these barriers can be overcome.
Figure 1 depicts the six main steps of a typical LC/MS-MS workflow, from sample receipt and extraction, separation in the LC, MS/MS analysis, data review and reporting of the results [2]. Of these steps it is the pre- and post-analytical stages that are the most time consuming and therefore if there is a focus on streamlining these, maximum benefit can be achieved. A number of steps can be taken to streamline the workflow, and these come under three broad headings of reduced manual processes, increased throughput and improved integration. Dependence on manual processes can be reduced by the automation of liquid handling and extraction, use of barcode reading for worklist generation and implementation of automated data analysis. Throughput can be increased with strategic column and sample management and by analyte multiplexing. Integration can be improved by bi-directional interfacing of the LC/MS-MS system to the laboratory information management system (LIMS) allowing automatic worklist upload and results download. These three strategic areas will be discussed in more detail below.
Reduced manual processes
Unlike the case with immunoassay, samples for LC-MS/MS usually require extraction prior to analysis. Historically this extraction step utilized liquid–liquid extraction or protein precipitation, these being carried out after the addition of internal standard to the calibrators, quality controls and patient samples. All of these steps involved manual pipetting and were very slow and time consuming. Use of an automated liquid-handling platform for the pipetting of samples and addition of internal standard allows some of the steps of liquid–liquid extraction and protein-precipitation methods to be automated. These liquid-handling platforms are available from a number of suppliers including Hamilton and Tecan.
With the advent of 96-well plate technology it became possible to carry out fully automated off-line solid phase extraction (SPE) using platforms such as the Freedom Evo (Tecan) and the Biomek NX (Beckman Coulter). More recently, supported liquid extraction (SLE), which allows solvent extraction to occur on a diatomaceous earth inert support, has also become available in a 96-well plate format. The Extrahera system (Biotage) enables automation of SLE by carrying out all of the pipetting and extraction steps required. In the NWLP laboratory, this system is used for the extraction of patient samples for vitamin D measurement by LC-MS/MS. A sample throughput of up to 50,000 samples per annum is achieved with capacity remaining for additional extractions for use in other LC-MS/MS applications. The system is robust and reliable with good pipetting precision and uses disposable pipette tips, thus avoiding sample carry over. Figure 2 depicts the Tecan Freedom Evo 200 and Biotage Extrahera liquid handlers in use in the NWLP laboratory.
In some manufacturers’ LC-MS/MS systems, on-line sample preparation and extraction is enabled by use of turbo flow or 2D chromatography. On-line protein precipitation and SPE is also now available using the Clinical Laboratory Automated sample preparation Module (CLAM)-2000 (Shimadzu Corporation) [3] and the Rapidfire 365 MS system (Agilent) [4] respectively. These latter examples most closely resemble the immunoassay workflow, whereby samples are introduced into the analytical system without any sample preparation or pre-treatment.
Increased throughput
Increased throughput can be achieved through the use of column and sample managers, allowing multiple assay batches to be queued up for overnight analysis of different LC-MS/MS assays. LC multiplexing enables multiple columns to be coupled to one tandem mass spectrometry system, maximizing the MS detection capability. In this approach, the use of quaternary solvent pumps in the LC enables column switching between different columns using different mobile phases. Finally there is analyte multiplexing, which can use manufacturers’ kits or in-house laboratory developed tests (LDTs). This approach enables multiple analytes to be detected in a single chromatographic separation by the use of multiple reaction monitoring for MS/MS detection. Perkin Elmer and Chromsystems both provide kits enabling the simultaneous measurement of multiple steroid hormones within a single assay panel. In the NWLP laboratory an in-house LDT steroid panel for the simultaneous measurement of androstenedione, 17-hydroxyprogesterone and testosterone has been implemented. This multiplexed assay has replaced the previous stand-alone assays for these analytes, thus increasing throughput and offering faster turnaround time. The assay utilizes off-line SPE using Waters Oasis PRiME HLB 96-well plates and the Tecan Freedom Evo 200 automated liquid handler [5].
Improved integration
Improved integration can be achieved by the use of bi-directional interfacing between the LIMS and the LC-MS/MS instrument software. Nowadays, manufacturers of LC-MS/MS systems offer customer support to allow their systems to be interfaced to the LIMS. One example is the MassLynx LIMS interface (Waters), which enables both worklist download and results upload. The MassLynx LIMS interface is accessed via the LC-MS/MS system software allowing sample worklists, created by barcode scanning of the patient samples, to be imported directly. Following peak integration and analyte quantitation the results are directly transmitted from the LC-MS/MS to the LIMS via an HL7 interface. This avoids the need for manual transcription thus saving a great deal of staff time and eliminating transcription errors.
The ultimate aim of LC-MS/MS integration is to achieve complete integration of LC-MS/MS instruments into the automated workflow of high-throughput routine clinical laboratories. With the recent introduction of the Cascadion LC-MS/MS analyser (Thermo Fisher Scientific) this ultimate aim has now been achieved [6]. This analyser offers a complete LC-MS/MS solution including primary blood tube sampling, on-board sample extraction, LIMS connectivity and a random access workflow enabling the provision of a 24/7 service. Traceable manufacturer’s kits are offered for the measurement of a panel of immunosuppressant drugs, testosterone and vitamin D with further assay kits in the development pipeline. The Cascadion analyser is shown in Figure 3.
Summary
LC/MS-MS automation and integration is now a reality, allowing faster sample processing and improved turnaround time, as well as offering increased staff productivity, improved quality and reduced error rate. Staff time is liberated for further service development, allowing the more rapid introduction of validated in-house LDTs into the assay repertoire. Finally there is the possibility of complete analyser integration allowing routine, high-throughput analysis, as is already the standard approach for the common immunoassay platforms. This exciting development will support the more widespread adoption of LC-MS/MS in the routine clinical laboratory by offering complete automation and integration, overcoming the barriers discussed in this article and enabling the inherent advantages of LC/MS-MS in clinical laboratory practice to be more fully realized.
References
1. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol Oxf 2006; 64: 234–244.
2. Zhang YV, Rockwood A. Impact of automation on mass spectrometry. Clin Chim Acta 2015; 450: 298–303.
3. Shimadzu. CLAM-2000. Fully automated sample preparation module for LCMS. (https://www.shimadzu.com/an/lcms/clam/index.html).
4. Jannetto PJ, Langman LJ. High-throughput online solid-phase extraction tandem mass spectrometry: Is it right for your clinical laboratory? Clin Biochem 2016; 49: 1032–1034.
5. Williams EL. LC-MS/MS measurement of serum steroids in the clinical laboratory. Clinical Laboratory International 2017; Sept: 18–20.
6. ThermoFisher Scientific. Cascadion SM Clinical Analyzer (www.thermofisher.com/cascadion).
The author
Emma L. Williams PhD, FRCPath
North West London Pathology, Imperial College Healthcare NHS Trust, London, UK
E-mail: emma.walker15@nhs.net
January 2026
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
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