Featured Articles

Making standardization simple

, 26 August 2020/in Featured Articles /by 3wmedia

Mass spectrometry (MS) is a well-known and broadly used analytical technique, and one that is particularly effective when coupled with liquid chromatography (LC). LC-MS/MS operates by analyte separation, ionization, mass analysis and detection, and lends itself as an ideal technique to meet the needs of a range of laboratory types. Over the past decade, LC-MS/MS has been applied across several different fields of clinical diagnostics and has become commonplace for forensic and clinical toxicology. However, until now it has only been used across a limited number of specialities, including endocrinology and therapeutic drug monitoring.

By Professor Brian Keevil and Dr Sarah Robinson

Such a powerful technique has the potential to bring significant advantages to the clinical setting, and would enable clinicians to analyse multiple analytes at greater specificities than immunoassay-based methods. It has the potential to supersede alternative methods since it avoids the issues surrounding interferences and the subsequent generation of unreliable data. Even with such advantages, LC-MS/MS has not yet been further adopted by the clinical community. The lack of an automated system has limited its suitability to routine clinical use, while also presenting challenges to laboratories under pressures to standardize and harmonize their practices. Current LC-MS/MS systems involve multiple and complex manual stages that are open to human error while being both time- and labour-intensive. Furthermore, the lack of standardization of LC-MS/MS methods is deterring clinical labs from benefiting from their advantages.

Standardization is critical in clinical laboratories since it is necessary to ensure the correct results are obtained and they are in accordance with results from other labs, especially for therapeutic drug monitoring and endocrine applications.

The challenge of standardization
One of the barriers to more widespread LC-MS/MS use is the lack of properly standardized methods and different laboratories will often use a wide range of techniques, equipment and internal standards. Together, these factors may mean that different results are generated from the same sample.

This level of variation makes it challenging to obtain proper standardization of LC-MS/MS results and is highly problematic. Not only does it become difficult to control results within a lab and ensure they remain comparable year on year, but it can create discrepancies between labs. This could ultimately lead to incorrect patient diagnoses and clinicians recommending the wrong treatment programmes.

The drive for change
Until now, LC-MS/MS systems have been designed with the research laboratory in mind and, as such, are highly configurable making them great for developing methods. However, the needs of the clinical lab are different from those of the research community. The clinical setting requires a dedicated system that not only promotes, but also facilitates standardization. Studies have shown that, through careful use of the same instrument, column and methods, it is possible to generate consistent and reliable resulting data from LC-MS/MS systems based at different laboratories. There is currently a drive from organizations, such as the International Federation of Clinical Chemistry (IFCC), the Centers for Disease Control and Prevention (CDC), and the Endocrine Society, to harmonize assays across laboratories to improve levels of quality. The adoption of one dedicated system among an entire network of laboratories would not only satisfy this organizational drive, but also help clinicians be confident that the data across their entire network is standardized, and thus comparable and repeatable.

The availability of a dedicated system with standardized methods and procedures would make this process significantly easier and remove one of the primary barriers to uptake of this gold standard technique. A dedicated system would need to be optimized for the specific methods run by each laboratory, and available with columns, reagents, calibrators and controls that are consistent and designed specifically for the system. This would help to ensure all data generated is both reproducible and accurate – paramount to patient diagnosis and care. In addition, a clinical LC-MS/MS system would need to be automated and easy to use. Clinical labs are extremely busy so even the most junior members of the staff must be able to operate the instrument and walk away with the confidence that samples are being analysed without error or the need for manual intervention. A system such as this would help to ensure patients were properly diagnosed and appropriate treatment plans devised.

Breaking through the barrier
If a network of laboratories decided to start using a dedicated clinical analyser, it would be able to adopt common reference ranges and reagents, which would provide much greater confidence in the consistency of results. For example, if a patient was transferred to a different hospital mid-way through treatment then there would be a level of assurance that the test results would be the same from both facilities. The data would therefore be directly comparable as long as both labs were using the same dedicated LC-MS/MS system.

Proper standardization is extremely important, yet challenging, and is a key consideration when deciding on an analytical method for implementation. An automated, dedicated clinical LC-MS/MS system would enable inter-laboratory standardization, while allowing interference-prone immunoassay-based tests to be phased out and replaced by clinical LC-MS/MS analysers. The results obtained from one laboratory would then be consistent over many years, and match those results generated from the same patient samples in other labs using the same system. Furthermore, such a system could be operated by the entire laboratory team, removing the need for in-depth and specialist training. This ease of use would decrease the investment required in training, while freeing up more experienced team members to focus on their research.

Conclusion
Analytical techniques are a core component to clinical workflow to ensure accurate patient diagnosis and treatment. LC-MS/MS has clear advantages over alternative immunoassay-based methods, with the ability to analyse multiple analytes at greater specificities. However, its uptake across the clinical community has been slow. This is because LC-MS/MS systems to date have been developed for use in research laboratories, and although the data have been demonstrated to be of high quality, the technology does not simply translate to the needs of the clinical lab.

With analytical needs that directly correlate to patient treatment plans, analytical methods within the clinical lab need to be automated, standardized, reliable and provide walk-away capabilities. This clear need for a dedicated analytical technique has driven the development of the new Thermo Scientific™ Cascadion™ SM Clinical Analyzer*. This dedicated clinical LC-MS/MS system is accurate, easy to use, and has been designed specifically for the clinical laboratory, facilitating standardization both on an inter- and intra-laboratory level to enable clinicians to fully leverage the power of this technique. The impact of this system would help laboratories and laboratory networks to meet their clinical needs.

To find out more, visit www.thermofisher.com/cascadion
*This product is in development and not available for sale. This product is not CE marked or FDA 510(k) cleared.

The authors
Professor Brian Keevil¹ and Dr Sarah Robinson²
¹Consultant Clinical Scientist and Head of the Clinical Biochemistry Department, University Hospital of South Manchester
²Market Development Specialist, Thermo Fisher Scientific

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Rapid and reliable detection of medulloblastoma-associated DNA methylation patterns: MS-MIMIC

, 26 August 2020/in Featured Articles /by 3wmedia

Disease-associated variations in DNA methylation profiles hold significant potential for diagnostic and research applications. Unfortunately, scant and degraded samples often limit the analyses which can be performed. To overcome these issues, we developed Mass Spectrometry Minimal Methylation Classifier (MS-MIMIC) to identify then reliably analyse disease-specific DNA methylation profiles. The technique has now been validated in a cohort of pediatric medulloblastoma cases.

by Dr Ben Chaffey, Dr Debbie Hicks, Dr Edward Schwalbe and Prof. Steve Clifford

Background
Altered DNA methylation patterns have emerged as valuable biomarkers of disease pathogenesis, showing clear potential in diagnostics, sub-classification and prediction of therapeutic response/ disease course [1–7]. However, clinical assessment of these altered patterns can be problematic, with sample materials often being degraded/scant, such as formalin-fixed paraffin-embedded (FFPE) tissue and core biopsies, and certain platforms, such as DNA methylation microarrays, having a requirement for batched assessments of relatively large numbers of samples. This compromises generation of data from real-world samples within clinically meaningful timeframes, hampering translation of research findings into routine practice.

In this project, we focused on developing a new DNA methylation state assay to provide molecular subgrouping of cases of medulloblastoma (Fig. 1), the most frequently occurring malignant brain tumour in children. This disease has an approximate incidence of 1.5 cases per million, rising to 6 per million in children aged 1–9 years. It also occurs in adults, although in this group it is around ten times less common [8].

Although rates of survival to 5 years and beyond following diagnosis are around 65–70%, medulloblastoma still causes around 10% of all childhood cancer deaths. Initial treatment generally consists of complete or near complete surgical resection followed by adjuvant treatment with both post-operative radiotherapy and chemotherapy. Despite the fact that survival rates have improved over past decades, the delivery of individualized therapies based on patient-specific disease-risk profiles remains a major goal; intensified treatment for poor-risk disease, while reducing therapy for favourable-risk cases, with the overall aim of maximizing survival while minimizing late effects [9].

Medulloblastomas can be placed into one of four distinct subgroups, which are defined by specific methylomic, transcriptomic and genomic features. These are WNT, SHH, Group 3 and Group 4 [10]. Each group displays characteristic clinical and pathological features, drug targets and outcomes, and contributes significantly to the 2016 World Health Organization (WHO) classification of brain tumours [11]. Molecular subgrouping is, therefore, an important step in determining the most appropriate course of treatment and follow-up for individual patients [12].
Mass spectrometry minimal methylation classifier (MS-MIMIC) assay
The assay we have developed and validated, MS-MIMIC, is a novel polymerase chain reaction (PCR)-based assay for the multiplexed assessment of multiple signature CpG loci. We first identified a DNA methylation signature of 17 CpG loci using genome-scale Illumina 450k DNA methylation microarray data from 220 medulloblastoma cases. The 50 most discriminatory CpG loci for each molecular subgroup (200 loci in total) were considered as candidates for inclusion in the signature set. These were triaged using a 10-fold cross validated classification fusion algorithm, followed by a reiterative primer design process where amenability to primer design and multiplex bisulfite PCR was assessed in silico before finally undergoing in vitro PCR validation.

Candidate signature CpG loci were then analysed by a specific custom iPLEX assay [13] (Agena Bioscience). In this method (displayed schematically in Fig. 2), methylation-dependent SNPs representative of CpG methylation status are induced by initial treatment of DNA with sodium bisulfite [14] followed by multiplexed target-region amplification PCR, then single base extension and termination of target-specific probe oligonucleotides. The products of this reaction are analysed using MALDI-ToF (matrix-assisted laser desorption and ionisation – time of flight) mass spectrometry (MassARRAY System, Agena Bioscience). Each potential CpG locus variant yields a product with a unique and characteristic mass, enabling their rapid and unambiguous identification. MALDI-ToF analysis of single base variants is widely used to provide clinical DNA diagnostics in related genotyping applications [15], and is the key technical innovation which enables the robust assessment of medulloblastoma molecular subgroup, especially for samples which are refractory to analysis using conventional DNA methylation-array based methods.

Using these techniques, we generated an optimal, multiply-redundant 17-CpG locus signature and a robust assay for its detection.
A Support Vector Machine (SVM) classifier for the signature was then developed, using the existing 450k DNA methylation array data as a training set. SVM is a supervised machine learning technique commonly used in multiple areas of data analysis, including analysis of microarray data [16], making it well-suited to this application. Crucially, it returns a probability of group membership, enabling the assessment of confidence of subgroup assignment.

Next, we assessed MS-MIMIC performance against Illumina 450k methylation microarrays using an independent validation cohort of 106 medulloblastoma DNA samples which contained examples of all four medulloblastoma subgroups. These samples were also derived from tissue which reflected different clinical fixation methods commonly in use; fresh-frozen biopsies (n=40), FFPE tumour section (n=39), or FFPE-derived nuclear preparations [17] (n=27) produced by cytospin, a pre-analytical method that uses centrifugation to create a monolayer of cells for analysis on a slide from a low-concentration cellular suspension sample [18]. In this validation cohort, MS-MIMIC faithfully recapitulated DNA methylation array molecular subgroup assignments.

Quality control measures for CpG locus-specific assay failure were established; up to six failed CpG loci per sample were tolerated within the multiply-redundant signature/classifier, without impacting performance. Forty-three out of 106 validation cohort samples were affected by at least one locus failure, reflecting the damaged nature of DNA generally obtained from some of these samples. Five out of 106 samples had more than seven failed CpGs and were deemed not classifiable (NC). Molecular subgroup classifications were then compared, with MS-MIMIC classifications showed complete concordance with the reference subgroup, as determined by DNA methylation array [10]. Furthermore, CpG-level methylation estimates (β-values) were equivalent between methods (R² = 0.79). As anticipated, fresh-frozen derivatives performed best (n=39/40; 98% successfully subgrouped), with 91% success (n=56/61) using FFPE-derived DNA from tumour sections and cytospin preparations (Fig. 3a–c).

Application of MS-MIMIC to the HIT-SIOP-PNET4 clinical trials cohort
Following successful assay development and validation, we next wished to test MS-MIMIC methylation signature detection in limited, poor quality, archival, clinical biopsies. Analysis of remnant material from the HIT-SIOP-PNET4 cohort [17] offered the first opportunity to determine the potential utility of molecular subgroup status to predict disease outcome in a clinical trial of risk-factor negative, ‘standard risk’ (SR) medulloblastoma. Only FFPE sections (n=42/153 available tumour samples) and cytospin nuclear preparations (approximately 30 000 nuclei isolated and centrifuged onto microscope slides; n=111/153) remained from this study archive and all DNA preparations fell below quality and quantity thresholds (>200 ng double-stranded DNA (dsDNA)) required for methylation profiling using conventional research methods (Illumina 450k and MethylationEpic arrays [16]). Using MS-MIMIC, 70% (107/153) of samples were successfully subgrouped, and subgroup assignments and β-value estimations were consistent across duplicate determinations. Assay performance was equivalent across the input DNA range (<2 ng (limit of detection) to 100 ng dsDNA (41.4 ng median DNA input).

Reasons for assay failure included unsuccessful bisulfite conversion/PCR (6%; 9/153), and inability to classify due to assay QC failure (24%; 37/153). These findings from HIT-SIOP-PNET4 reveal important subgroup-dependent molecular pathology in SR medulloblastoma. Group 4 was most common (n=62; 58%), with approximately equivalent numbers of WNT (18/170; 16%), SHH (17/107; 16%) and Group 3 (10/107; 9%) tumours observed. The majority (11/13) of events (defined as disease recurrence or progression following treatment) affected Group 4 patients [82% 5-year progression-free survival (PFS)], with >95% PFS in non-Group 4 patients. Subgroup assignment will thus be essential to inform future clinical and research studies in SR medulloblastoma.

Discussion
Detection of disease-specific variations in DNA methylation patterns has great potential for both supporting biomedical research and improving the quality of care that is delivered to patients. MS MIMIC has so far only been applied to medulloblastoma but this approach has clear potential for use in other cancers [7] and in other diverse settings, for example smoking [1], obesity [2], human fetal alcohol spectrum disorder [3] and aging [4].

Key resources which must be available for development of an MS-MIMIC assay for a given condition are a suitable collection of data concerning disease-state specific methylation patterns obtained using an array system such as those mentioned above, samples with which to perform assay validation, bioinformatics knowledge and support to create, optimize and operate the disease-specific SVM classifier system, plus access to a MassARRAY System for analysis. MS-MIMIC is discussed in greater detail in Schwalbe et al., 2017 [19].

References
1. Besingi W, Johansson A. Smoke-related DNA methylation changes in the etiology of human disease. Hum Mol Genet 2014; 23: 2290–2297.
2. Wahl S, Drong A, Lehne B, Loh M, Scott WR, Kunze S, Tsai PC, Ried JS, Zhang W et al. Epigenome-wide association study of body mass index, and the adverse outcomes of adiposity. Nature 2017; 541: 81–86.
3. Portales-Casamar E, Lussier AA, Jones MJ, MacIsaac JL, Edgar RD, Mah SM, Barhdadi A, Provost S, Lemieux-Perreault LP et al. DNA methylation signature of human fetal alcohol spectrum disorder. Epigenetics Chromatin 2016; 9: 1–20.
4. Ong ML, Holbrook JD. Novel region discovery method for Infinium 450 K DNA methylation data reveals changes associated with aging in muscle and neuronal pathways. Aging Cell 2014; 13: 142–155.
5. Mehta D, Klengel T, Conneely KN, Smith AK, Altmann A, Pace TW, Rex-Haffner M, Loeschner A, Gonik M et al. Childhood maltreatment is associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder. Proc Natl Acad Sci USA 2013; 110: 8302–8307.
6. Bacalini MG, Gentilini D, Boattini A, Giampieri E, Pirazzini C, Giuliani C, Fontanesi E, Scurti M, Remondini D et al. Identification of a DNA methylation signature in blood cells from persons with Down Syndrome. Aging 2015; 7: 82–96.
7. Sturm D, Witt H, Hovestadt V, Khuong-Quang DA, Jones DT, Konermann C, Pfaff E, Tönjes M, Sill M et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 2012; 22: 425–437.
8. Smoll NR, Drummond KJ. The incidence of medulloblastomas and primitive neurectodermal tumours in adults and children. J Clin Neurosci 2012; 19: 1541–1544.
9. Pizer BL, Clifford SC. The potential impact of tumour biology on improved clinical practice for medulloblastoma: progress towards biologically driven clinical trials. British Journal Of Neurosurgery 2009; 23: 364–375.
10. Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S et al. Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 2012; 123, 465–472.
11. Louis DN, Cavenee WK, Ohgaki H, Wiestler OD. WHO classification of tumours of the central nervous system, 4th edn. pp.184–200. IARC Press, 2016.
12. Schwalbe EC, Williamson D, Lindsey JC, Hamilton D, Ryan SL, Megahed H, Garami M, Hauser P, Dembowska-Baginska B et al. DNA methylation profiling of medulloblastoma allows robust subclassification and improved outcome prediction using formalin-fixed biopsies. Acta Neuropathol 2013; 125: 359–371.
13. Gabriel S, Ziaugra L, Tabbaa D. SNP genotyping using the Sequenom MassARRAY iPLEX platform. Current Protocols in Human Genetics, Chapter 2: Unit 2.12. Wiley & Sons 2009.
14. Wang RY, Gehrke CW, Ehrlich M. Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res 1980; 8: 4777–4790.
15. Griffin TJ, Smith LM. Single-nucleotide polymorphism analysis by MALDI-ToF mass spectrometry. Trends Biotechnol 2000; 18: 77–84.
16. Hovestadt V, Remke M, Kool M, Pietsch T, Northcott PA, Fischer R, Cavalli FM, Ramaswamy V, Zapatka M et al. Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumour material using high-density DNA methylation arrays. Acta Neuropathol 2013; 125: 913–916.
17. Clifford SC, Lannering B, Schwalbe EC, Hicks D, O’Toole K, Nicholson SL, Goschzik T, Zur Mühlen A, Figarella-Branger D et al. Biomarker-driven stratification of disease-risk in non-metastatic medulloblastoma: Results from the multicentre HIT-SIOP-PNET4 clinical trial. Oncotarget 2015; 6: 38827–38839.
18. Koh CM. Preparation of cells for microscopy using cytospin. Meth Enzymol 2013; 533: 235–240.
19. Schwalbe EC, Hicks D, Rafiee G, Bashton M, Gohlke H, Enshaei A, Potluri S, Matthiesen J, Mather M et al. Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures. Sci Rep 2017; 7: 13421.

The authors
Ben Chaffey¹ PhD, Debbie Hicks² PhD, Edward Schwalbe³ PhD, Steve Clifford²* PhD
¹NewGene Ltd, International Centre for Life, Newcastle-upon-Tyne, UK
²Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle-upon-Tyne, UK
³Northumbria University, Newcastle-upon-Tyne, UK

*Corresponding author
E-mail: steve.clifford@ncl.ac.uk

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Genomic tracking of MRSA outbreaks could help infection control

, 26 August 2020/in Featured Articles /by 3wmedia

Hospital-acquired infections (HAIs) today rank among the major causes of death and morbidity in hospitalized patients and are estimated to be responsible for 175,000 deaths per year in industrialized countries. HAIs have been growing exponentially worldwide since the 1980s primarily because of the indiscriminate use of antibiotics which have triggered the growth of multidrug resistant bacterial strains – also known as superbugs – and the transmission of such strains between patients, as well as between patients and hospital staff and vice versa. Methicillin-resistant Staphylococcus aureus (MRSA) is a superbug that is resistant to several widely used antibiotics. In the general community, MRSA mostly causes skin infection, is spread by skin-to-skin contact and, if left untreated, can also get deeper into the body, causing potentially life-threatening infections. It is generally estimated that about 3 percent of the population chronically carries MRSA. However, in a healthcare setting such as a hospital or nursing home, MRSA infection is more frequent and often more severe, leading to pneumonia, surgical site infections, bloodstream infections and possibly sepsis. The risk factors are indeed much higher in hospitals because of the increased vulnerability of some patients (the elderly and those with weakened immune systems) and because of the multiple potential pathways for MRSA entry into the body provided by wounds (including surgical wounds), burns as well as feeding tubes, intravenous lines or urinary catheters. MRSA is also prevalent in nursing homes where healthy carriers have the opportunity to spread it among the resident population and staff.
A very recent study, published in the October 25 edition of Science Translational Medicine, used genomic sequencing technology for the genomic surveillance of MRSA in the East of England. A team at the Wellcome Trust Sanger Institute sequenced the genetic code of every single MRSA-positive sample processed over a 12-month period by a routine clinical microbiology lab receiving samples from three hospitals and 75 general practitioner practices. Samples from 1465 people were analysed, revealing a total of 173 transmission clusters involving 598 people and ranging from outbreaks affecting two patients up to 44. These findings shed some new light on MRSA transmission within and between hospitals and the community and could pave the way for more targeted, efficient and effective infection control practices. While genomic surveillance of MRSA cannot by itself prevent an outbreak from occurring, it can certainly help to reduce the numbers of infected people. The cost-effectiveness of implementing this strategy needs to be carefully evaluated. Although the whole genome of a bacterium can now be sequenced for around 140€, this might still prove too much for many healthcare systems.

LC-MS/MS measurement of serum steroids in the clinical laboratory

, 26 August 2020/in Featured Articles /by 3wmedia

In recent decades liquid chromatography–tandem mass spectrometry (LC-MS/MS) has become more widespread in the clinical laboratory, bridging the analytical gap between high-throughput (but interference prone) immunoassays and the highly specific (but labour intensive) technique of gas chromatography–mass spectrometry (GC-MS). This article discusses serum steroid measurement by LC-MS/MS and describes a multiplexed LC-MS/MS steroid panel recently launched at Imperial College Healthcare NHS Trust.

by Dr Emma L. Williams

Introduction
Historically steroid hormones have been measured, primarily in urine, by GC-MS and in serum and plasma by radio-immunoassay. Both techniques require sample extraction prior to analysis and for the former there is a need for derivatization to form volatile derivatives. Thus the assays are laborious and time consuming and have been the preserve of research and specialist laboratories. More recently automated immunoassays have been used in routine clinical laboratories, but these are notorious for being highly prone to interference as a result of their inherent specificity problems [1]. In recent decades LC-MS/MS has come to the fore, offering a promising alternative to immunoassays for high-throughput, specific measurement of serum steroids and it is now the method of choice in many clinical laboratories. LC-MS/MS measurement of serum steroids is informative in the clinical investigation of conditions such as hirsutism, polycystic ovarian syndrome (PCOS) and infertility. In addition LC-MS/MS steroid measurement forms part of a diagnostic triad, along with urine steroid profiling by GC-MS and whole gene sequencing of genomic DNA, for inherited steroidogenic defects including the congenital adrenal hyperplasias (CAH) and disorders of sexual differentiation.

LC-MS/MS measurement
Significant advances in LC-MS/MS technology have enabled the development of high-throughput, sensitive and precise assays for steroid measurement. Figure 1 depicts the biosynthetic pathways of steroidogenesis. LC-MS/MS assays have now been published for all of the steroids in this pathway, using a variety of approaches for sample preparation prior to analysis. Protein precipitation, liquid–liquid extraction, solid phase extraction and supported liquid extraction have all been used for the preparation step. In my laboratory, semi-automated off-line solid phase extraction has been implemented in order to achieve higher throughput. This extraction approach is used to prepare samples prior to ultra-performance (UP)LC-MS/MS analysis using electrospray ionization with detection by multiple reaction monitoring (MRM). The majority of steroids are measured in positive ionization mode, although we use negative ionization mode for aldosterone and dehydroepiandrosterone sulphate (DHEAS).

For accurate LC-MS/MS quantitation, stable isotope internal standards (IS) are required. Addition of IS to all samples, calibrators and quality controls (QCs) is carried out prior to extraction and LC-MS/MS analysis. The ratios of analyte to IS signals are determined to correct for effects of the matrix upon signal intensity, which may be due to ion suppression or enhancement. Typically in LC-MS/MS assays the IS will have two or more hydrogens replaced by deuterium atoms. The IS has a different mass and ion transition to the analyte, while retaining its chemical and physical properties and thus behaves the same way as the analyte throughout the analytical procedure. Carbon-13 labelled IS are increasingly being used as they have become more available. These co-elute more completely with the non-labelled analyte and are, therefore, more effective at correcting for matrix effects compared to deuterium labelling, which alters polarity and increases the possibility of non-co-elution.

An important factor to consider in steroid LC-MS/MS assays is that of specificity, given the similarities in structures of the various steroid intermediates in the steroidogenic pathway.
There are several examples of steroids that have the same molecular weight and are, therefore, isobaric. It is vital that these isobaric steroids are chromatographically resolved as they will undergo the same ion transitions in the mass spectrometer. If not resolved, they would be measured as if they were the same steroid and, therefore, be a cause of positive interference. For example 11-deoxycortisol and 21-deoxycortisol have the same molecular weight (Fig. 2) and undergo the same ion transitions, but can be chromatographically resolved using the selectivity of the mobile phase. It can be seen in Figure 3 that these steroids are successfully resolved in our laboratory method, which uses reverse phase T3 chromatography.

LC-MS/MS steroid assays
In the clinical laboratory, testosterone is the serum steroid most frequently measured by LC-MS/MS analysis. In the external quality assessment scheme offered by the United Kingdom National External Quality Assessment Service (UK NEQAS), 43 (21%) participating labs use LC-MS/MS, with the remainder relying upon automated immunoassays. In my laboratory, both measurement techniques are used, whereby all female samples with elevated immunoassay testosterone results >2.0 nmol/L are reflexed for LC-MS/MS confirmation. In a recent audit of over 5000 female samples in which testosterone was measured we found that of over 800 elevated samples reflexed for confirmation, 23% of these are subsequently found to have normal LC-MS/MS results within the reference range. It is, therefore, essential that elevated female immunoassay results are confirmed by LC-MS/MS to avoid falsely elevated results being reported. Norethisterone, a synthetic form of progesterone used in hormonal contraceptives, is a commonly encountered cause of positive interference in immunoassays for testosterone in female samples [2].

Advantages of multiplexed assays
Testosterone is measured in the investigation of females presenting with clinical signs of hyperandrogenism, e.g. acne and hirsutism and in the investigation of infertility and PCOS. Following the introduction of LC-MS/MS assays into the clinical laboratory for the combined measurement of testosterone and androstenedione it became clear that androstenedione is the cause of hyperandrogenism in a subgroup of patients with PCOS [3]. These cases previously may have been undiagnosed when the testosterone measured in isolation was found to be normal. This observation highlights the benefits of being able to measure two or more steroids simultaneously, which is not possible with radio-immunoassays or in routine automated immunoassays.

17-Hydroxyprogesterone (17-OHP) measurement is used to screen for 21-hydroxylase deficiency; the most common cause of CAH, accounting for ~85% of cases. 17-OHP sits at a branch point for either cortisol or androgen synthesis (Fig. 1) and accumulates when 21-hydroxylase is deficient. However, it can also be raised in normal newborns, particularly in premature neonates, and is influenced by birth weight and stress. In 21-hydroxylase deficiency, 21-deoxycortisol is formed as a side product from the accumulated 17-OHP in a reaction catalysed by 11-beta hydroxylase. The LC-MS/MS measurement of 21-deoxycortisol for the diagnosis of CAH was first described by Cristoni et al. [4] and it allows accurate diagnosis of 21-hydroxylase deficiency in newborns independent of prematurity, birth weight and stress [5]. Shackleton has proposed that a second tier panel comprising 17-OHP, cortisol, 21-deoxycortisol and androstenedione is used in newborn screening for 21-hydroxylase deficiency with a third tier of urinary GC-MS analysis to clinch the final diagnosis [6]. The addition of 11-deoxycortisol to this panel permits the diagnosis of 11-beta-hydroxylase deficiency, the second most common form of CAH. Such a panel has been applied to second tier testing for CAH [7].

In my laboratory a semi-automated solid phase extraction (SPE) LC-MS/MS method for the simultaneous measurement of androstenedione, testosterone and 17-OHP has been in use since April 2016. The SPE uses Waters Oasis PRiME HLB, 96 well, μ-elution plates and is performed using a Tecan Freedom Evo automated Liquid Handler. One hundred microlitres of sample is mixed with IS and proteins are precipitated with methanol and water. Supernatants are applied to the wells of the SPE plate and drawn through under vacuum. Following washing with 0.1% formic acid in 35% methanol, steroids are eluted with methanol and water enabling direct LC-MS/MS analysis of the eluates.
Using a Waters Acquity UPLC system, samples are injected onto a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm) and separated by water/methanol/ammonium acetate/formic acid gradient elution. The analysis is performed using a Waters Acquity-TQD mass spectrometer in electrospray positive ionization mode. The analytes and their co-eluting isotopic ISs are detected using MRM. Quantifier transitions (m/z) monitored are 287>97 for androstenedione, 289>97 for testosterone and 331>97 for 17-OHP.

The method underwent full validation prior to implementation according to Clinical and Laboratory Standards Institute (CLSI) guidelines and as recommended by Honour [8] and demonstrated excellent linearity over the analytical range, with all r2 values ≥0.99. Overall process efficiency was 100–108.3%, demonstrating excellent recovery and minimal ion suppression/enhancement. Intra-assay precision was 2.6–8.1% for all analytes across the measurement range, and inter-assay precision varied from 4.9 to 10.8%. Analysis of UK NEQAS samples revealed minimal negative bias and the high specificity of the assay was confirmed by spiking and interference studies. The newly developed assay compared favourably with the stand-alone LC-MS/MS methods in use previously in our laboratory, with no requirement to re-derive reference intervals. This supra-regional assay service (SAS) accredited steroid panel assay has been in routine use in our LC-MS/MS laboratory since April 2016, streamlining the analytical service. The assay is carried out two or three times a week, with each full plate accommodating around 80 patient samples, plus standards and controls, with automated sample extraction completed in ~ 90 minutes and the LC-MS/MS sample to sample injection time is 5 minutes.

We have recently evaluated a seven steroid LC-MS/MS assay with the addition of cortisol, DHEAS, 11-deoxycortisol and 21-deoxycortisol into the panel. Figure 3 shows the total ion chromatogram of the steroids quantified by this assay. Using a Waters Acquity-TQD mass spectrometer and a slightly modified experimental set-up, the lower limits of quantification obtained were 16.5 nmol/L for cortisol, 2nmol/L for DHEAS, 7nmol/L for 11-deoxycortisol and 2nmol/L for 21-deoxycortisol.
In conclusion, LC-MS/MS steroid panels are a valuable addition to the diagnostic work up of patients being investigated for hyperandrogenism and in the investigation of steroidogenic defects. The increased availability of semi-automated, high-throughput LC-MS/MS assays for multiplexed steroid measurement has opened the door for their future application in targeted metabolomic research. Finally, in the clinical laboratory setting the future continues to look bright for the role of accurate and robust measurement by LC-MS/MS in place of immunoassays as the method of choice for routine serum steroid measurement.

References
1. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol Oxf 2006; 64: 234–244.
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The author
Emma L. Williams PhD, FRCPath
North West London Pathology, Imperial College Healthcare NHS Trust, London
W6 8RF, UK

E-mail: emma.walker15@nhs.net

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, 26 August 2020/in Featured Articles /by 3wmedia

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