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
After the 3rd volume devoted to parental anticoagulants, it stands to reason that the Clinical Development Department had to turn its attention to the other side of anticoagulant treatment – the oral anticoagulants – in this series launched in 2014 with the objective of publishing a Practical Manual every year.
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Background
Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are minor ethanol metabolites that can be used to detect recent alcohol consumption [1, 2]. Following the ingestion of alcohol, over 95% is metabolized by alcohol dehydrogenase to acetaldehyde. Up to 5% of ethanol is excreted unchanged in breath, sweat and urine. A small amount of ethanol (<0.1%) is metabolized in the liver by conjugation of glucuronic acid or sulphate to form EtG and EtS (Fig. 1). Following alcohol consumption, ethanol itself can only be detected in breath or urine for up to 6 or 12 hours, respectively (depending on the amount of alcohol consumed) [3]. In comparison, it has been demonstrated that EtG and EtS can be detected in urine for at least 24 hours and over 48 hours with heavy alcohol consumption [4].
The ability of these markers to detect alcohol intake over a longer time period means that they can be useful to identify alcohol relapses in alcohol-dependent individuals in treatment programmes [5]. In the UK, alcohol treatment programmes rely on breath ethanol and self-reporting to detect recent alcohol intake. However, this will only detect a proportion of individuals who are continuing to drink alcohol; this has been a low as 7% in one study comparing breathalyser/self-reported alcohol intake to urine EtG measurement [6]. Therefore, EtG and EtS can be helpful to detect those in alcohol treatment who are continuing to drink alcohol but deny it and have a negative breath ethanol test [7]. This allows additional interventions in individuals who are continuing to drink, which may ultimately improve outcomes. During 2016–17, 80 454 individuals entered alcohol treatment in England; of those 61% were free of alcohol dependence following the standard 12-week programme [8]. Therefore, improved detection of continuing alcohol consumption could lead to initiation of earlier intervention and altered strategies to increase the numbers successfully completing treatment.
Measurement of ethyl glucuronide and ethyl sulphate
Liquid chromatography (LC) to separate analytes with detection using mass spectrometry (MS) is now routinely used in clinical laboratories for an increasing number of tests. It is routine practice in urine toxicology testing for results to be confirmed by either LC or gas chromatography with detection using MS and it has been recommended by the United States Substance Abuse and Mental Health Services Administration (SAMHSA) that MS confirmation should be used for the measurement of EtG and EtS [9].
In tandem MS, two mass spectrometers are arranged sequentially with a ‘collision cell’ placed between the two instruments (Fig. 2). Using selective reaction monitoring, the first mass spectrometer (MS1) selects the ion with the mass/charge (m/z) ratio of interest. The selected ion (parent ion) is fragmented into small ions that enter the second mass spectrometer (MS2) where an ion with a specific m/z ratio is selected (daughter). Detection of analytes using an m/z ratio is very specific and sensitive allowing detection of very small amounts of EtG and EtS.
A number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for EtG and EtS have been published and a reference method has been proposed for EtG using solid phase extraction followed by LC-MS/MS [10]. Deuterated standards (EtG-d5 and EtS-d5) are readily available to purchase for use as internal standards ensuring reproducibility and reliability; an internal standard must mimic the analyte of interest but have a different mass to allow the MS detector to differentiate between the analyte of interest and the internal standard.
Sample preparation in published methods ranges from solid phase extraction to protein precipitation to dilution of urine in mobile phase. Solid phase extraction or protein precipitation of urine samples prior to LC-MS/MS can reduce the presence of potentially interfering substances which may cause ion suppression. It may also help to increase the lifespan of the column. For chromatographic separation of EtG and EtS, the mobile phases are usually formic acid in HPLC grade water and acetonitrile. Published methods have used both isocratic and gradients of mobile phase A and B to achieve separation of EtG and EtS; this is dependent on the sample preparation, the exact composition of the mobile phases and the column chosen. A rapid sample preparation of diluting urine samples in mobile phase A and then adding internal standard has been shown to be effective with no ion suppression or enhancement at or near the retention times for EtG and EtS [11]. Our experience has been to use an increasing gradient of mobile phase B (acetonitrile) from 1% to 10% over the first 2 minutes and then 10% to 100% from 2 minutes to 2.5 minutes. The increase from 1% to 10% acetonitrile elutes EtS/EtS-d5 at 1.27 minutes and the increase from 10% to 100% elutes EtG/EtG-d5 at 2.03 minutes. Figure 3 shows an example chromatogram for a urine sample collected from an individual attending the community based alcohol treatment programme; the high EtG and EtS results demonstrate that this person was continuing to drink alcohol.
Using MS to measure EtG and EtS requires the availability of LC-MS/MS equipment within the laboratory, the technical expertise required to set up an LC-MS/MS method and a dedicated member of staff to perform the analysis. In laboratories already using LC-MS/MS for other assays, there should be no difficulty in setting up a method to measure urine EtG and EtS.
An enzyme immunoassay method is also available to measure EtG and may be adapted for use on many automated laboratory analysers. This method has been shown to compare well to an LC-MS method [12]. For routine use, an immunoassay for EtG on an automated analyser has a number of advantages including rapid turnaround times, availability of EtG analysis out of routine working hours and the same staff members performing the analyses of multiple tests at the same time. However, there is no requirement for urine EtG and EtS analysis to be performed 24/7 as they would not be required in an acute setting. Generally, clients in a community treatment programme attend weekly, so once or twice weekly analysis using LC-MS/MS should be adequate for feedback of results to clients at their next visit. Not requiring a dedicated member of staff (as would be required for LC-MS/MS) is advantageous but according to SAMHSA guidelines, immunoassay results will require confirmation using a MS method. In addition, there is currently no immunoassay method available to measure EtS. This is important as there are a number of scenarios that can cause a false positive EtG result with a negative EtS result. For example, ‘positive’ EtG results (but not EtS results) have been demonstrated after the consumption of non-alcoholic beers (alcohol content 0.5%) [13]. EtG could also be formed in subjects with glycosuria and E.coli infection. If ethanol was formed due to the fermentation of sugars in the urine, this could be converted to EtG by bacteria present in the urine [14]. EtS would not be produced so again EtS can verify whether the EtG result is a true positive. Both EtG and EtS have been detected in individuals who used ethanol-based mouthwash or hand gel; however, the mouthwash was gargled 4 times/day which is much higher than the recommended frequency of use [15]. Owing to these factors, it is advisable to measure both EtG and EtS, which is currently only possible if using LC-MS/MS.
Cut-off values for EtG and EtS
There has been a lot of debate in the literature about suitable cut-off values to use for EtG and EtS. Some authors have suggested using the lower limit of detection (LLOD) or lower limit of quantitation (LLOQ) for the method so that any detectable EtG and EtS is a ‘positive’ result. However, the LLOD and LLOQ in LC-MS/MS methods will be variable between laboratories depending on a number of factors including sample preparation, column choice, chromatography and the tandem MS optimization. For EtG and EtS, the published LLOQs range from 0.05–0.20 mg/L and 0.04–0.10 mg/L respectively. New Clinical & Laboratory Standards Institute (CLSI) guidelines were published in 2016 and these should help to improve standardization between LC-MS/MS methods [16]. Alternatively, cut-off values could be defined by measuring EtG and EtS in a non-drinking population and incorporating measurement uncertainty (0.26 mg/L and 0.22 mg/L for EtG and EtS respectively) [11]. For EtG, a cut-off of 0.50 mg/L has been proposed to reduce the risk of false positive results. The disadvantage of a higher EtG cut-off is a reduction in sensitivity. Jatlow et al. demonstrated that using a 0.50 mg/L cut-off would only detect the intake of a low dose of alcohol 12 hours earlier (estimated blood alcohol 20 mg/dL) in 50% of participants. However, all participants had results above 0.10 mg/L and 0.20 mg/L after the same low alcohol dose 12 hours earlier [4]. SAMHSA have suggested separating EtG results into ‘high’ positive (>1.00 mg/L), ‘low’ positive (0.50–1.00 mg/L) and ‘very low’ positive (0.10–0.50 mg/L). They suggest that a ‘very low’ positive result may indicate previous heavy drinking (1–3 days ago), previous light drinking (12–36 hours ago) or ‘extraneous’ exposure [9].
Another consideration for urine EtG and EtS analysis is the dilution of urine samples; in urine toxicology testing, it is standard practice to measure creatinine to check the validity of a urine sample. There is limited data on the utility of EtG and EtS creatinine ratios. However, it is good practice to measure creatinine and question the validity of the EtG and EtS results if the creatinine is ≤2.0 mmol/L [17].
Conclusion
Urine EtG and EtS are valuable additional tools to detect recent alcohol intake in individuals undergoing treatment for alcohol dependence to ensure continued abstinence. Owing to the risk of false positive EtG results from unintentional exposure (e.g. non-alcoholic beer, urine infection with glycosuria, ethanol-based hand gel/mouthwash), the measurement of EtS in addition to EtG is recommended. An immunoassay is available for EtG but only MS allows the detection of both EtG and EtS to confidently confirm recent alcohol intake. There are a number of published methods for LC-MS/MS for EtG and EtS which are applicable for routine use in a clinical laboratory.
References
1. Dahl H, Stephanson N, Beck O, Helander A. Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide. J Anal Toxicol 2002; 26: 201–204.
2. Helander A, Beck O. Ethyl Sulphate – a metabolite of ethanol in humans and a potential biomarker of acute alcohol intake. J Anal Toxicol 2005; 29: 270–274.
3. Helander A, Beck O, Jones W. Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol and 5-hydroxytryptophol. Clin Chem 1996; 42: 618–624.
4. Jatlow P, Agro A, Wu R, Nadim H, Toll BA, Ralevski E, Nogueira C, Shi J, Dziura JD, et al. Ethylglucuronide and ethyl sulfate assays in clinical trials, interpretation and limitations: results of a dose ranging alcohol challenge study and two clinical trials. Alcohol Clin Exp Res. 2014; 38: 2056–2065.
5. Dahl H, Voltaire Carlsson A, Hillgren K, Helander A. Urinary ethyl glucuronide and ethyl sulphate for detection of recent drinking in an outpatient treatment program for alcohol and drug dependence. Alcohol Alcohol 2011; 46: 278–282.
6. Wetterling T, Dibbelt L, Wetterling G, Göder R, Wurst F, Margraf M, Junghanns K. Ethyl glucuronide (EtG): better than breathalyser or self-reports to detect covert short-term relapses into drinking. Alcohol Alcohol 2014; 49: 51–54.
7. Armer J, Gunawardana L, Allcock R. The performance of alcohol markers including ethyl glucuronide and ethyl sulphate to detect alcohol use in clients in a community alcohol treatment programme. Alcohol Alcohol 2017; 52: 29–34.
8. Knight J, Brand P, Willey P, van der Merwe J. Adult substance misuse statistics from the National Drug Treatment Monitoring System (NDTMS): 01 April 2016 – 31 March 2017. Public Health England 2017
(https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/658056/Adult-statistics-from-the-national-drug-treatment-monitoring-system-2016-2017.pdf).
9. The role of biomarkers in the treatment of alcohol use disorders. Substance Abuse and Mental Health Services Administration (SAMHSA) Advisory 2012; 11(2) (https://store.samhsa.gov/shin/content/SMA12-4686/SMA12-4686.pdf).
10. Helander A, Kenan N, Beck O. Comparison of analytical approaches for liquid chromatography/mass spectrometric determination of the alcohol biomarker ethyl glucuronide in urine. Rapid Commun Mass Spectrom 2010: 24: 1737–1743.
11. Armer J, Allcock R. Urine ethyl glucuronide and ethyl sulphate using liquid chromatography-tandem mass spectrometry in a routine clinical laboratory. Ann Clin Biochem 2017; 54: 60–68.
12. Bottcher M, Beck O, Helander A. Evaluation of a new immunoassay for urine ethyl glucuronide testing. Alcohol Alcohol 2008; 43: 46–48.
13. Thierauf A, Gnann H, Wohlfarth A, Auwärter V, Perdekamp MG, Buttler KJ, Wurst FM, Weinmann W. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of “non-alcoholic” beer. Forensic Sci Int 2010; 202: 82–85.
14. Helander A, Ollson I, Dahl H. Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consumption. Clin Chem 2007; 53: 1855–1857.
15. Reisfield G, Goldberger B, Pesce A, Crews BO, Wilson GR, Teitelbaum SA, Bertholf RL. Ethyl glucuronide, ethyl sulfate, and ethanol in urine after intensive exposure to high ethanol content mouthwash. J Anal Toxicol 2011; 35: 264–268.
16. Lynch K. CLSI C62-A: a new standard for clinical mass spectrometry. Clin Chem 2016; 62(1): 24–29.
17. European guidelines for workplace drug testing in urine. European Workplace Drug Testing Society 2015 (http://www.ewdts.org/data/uploads/documents/ewdts-urine-guideline-2015-11-01-v2.0.pdf).
The authors
Jane Armer*1 BA MSc FRCPath and Rebecca Allcock2 BSc MSc FRCPath
1Department of Blood Sciences, East Lancashire Hospitals NHS Trust, Blackburn, UK
2Department of Clinical Biochemistry, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, UK
*Corresponding author
E-mail: jane.oakey@elht.nhs.uk
Sepsis is a medical emergency that needs rapid identification and treatment to create the best possible outcomes. However, in the early stages it can be very difficult to distinguish sepsis from uncomplicated infection. This article summarizes recent developments in sepsis nomenclature and definitions as well as providing an insight into the role that biomarkers might play in diagnosis and prognosis.
Background
Sepsis is a life-threatening condition associated with high morbidity and mortality, with the risk of death ranging from 30% to 80% depending on the severity of the disease. The World Health Organization estimates that more than 30 million people are affected by sepsis worldwide every year [1], although for reasons discussed by Candel et al., the actual epidemiology of sepsis is difficult to ascertain [2]. In the UK and USA it is thought that sepsis is the cause of around 37 000 and nearly 270 000 deaths per year, respectively [3, 4]. Outcomes of sepsis are better if it is detected and treated early, but despite the large numbers of people affected by it, public awareness of it is still low. In recent years, awareness campaigns have been launched and this year several popular TV and radio programmes in the UK have featured sepsis storylines (Call the Midwife, Coronation Street and The Archers).
Definitions
The difficulties experienced in studying the epidemiology of sepsis are likely to reflect the problems of characterization and diagnosis of the disease, which is in turn a reflection of the complex nature of the condition. Original definitions of sepsis date back to 1991, with the idea that sepsis was caused by systemic inflammatory response syndrome (SIRS) in resulting from infection. In 2001 the definitions were re-examined but left largely unchanged. In 2016, a task force re-evaluated and updated definitions of sepsis and septic shock (Box 1), taking into account improved understanding of the pathobiology of sepsis, which is now recognized to involve early activation of both pro- and anti-inflammatory responses, along with major modifications in non-immunologic pathways such as cardiovascular, neuronal, autonomic, hormonal, bioenergetic, metabolic, and coagulation [5]. A lay definition of sepsis published in 2011 [6] was also accepted by the 2016 task force (Box 1). The definitions created in 1991, 2001 and 2016 have been designated Sepsis-1, Sepsis-2 and Sepsis-3, respectively, to indicate the need for ongoing refinement.
Diagnosis of sepsis
Early diagnosis and treatment of sepsis is associated with improved outcomes, but the difficulty lies in distinguishing sepsis from uncomplicated infection. Identification of patients with sepsis is largely achieved through the use of the Sequential (or Sepsis-Related) Organ Failure Assessment (SOFA) score (Table 1) in the hospital setting or the quick SOFA (qSOFA) score (See Figure 1 “Operationalization of Clinical Criteria Identifying Patients With Sepsis and Septic Shock” in Singer et al. [5]). Commencement of treatment should occur within the first hour of admission and should not be delayed by waiting for results from the lab, as the SOFA score can be applied retrospectively. Management of sepsis also requires (amongst other things) that blood samples are taken before broad spectrum antibiotics are administered and that once the pathogen has been identified antibiotic usage can be refined to aid antimicrobial stewardship (See the Surviving Sepsis Campaign [7] and NICE guidelines [8] for full details of early sepsis management). Sepsis is most commonly caused by bacterial infection, but can also be due to fungal, viral or parasitic infection. However, identification of the pathogen and its antibiotic susceptibility and/or resistance by classic culture techniques is slow and molecular- and proteomic-based approaches, such as matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) spectroscopy, may improve turnaround times [9].
Biomarkers
The difficulty of distinguishing sepsis from uncomplicated infection has long driven the search for suitable biomarkers to aid sepsis diagnosis. An ideal biomarker would be able to distinguish sepsis from non-infectious causes of critical illness, having a fast and specific increase in sepsis and a rapid decrease after effective therapy. A number of potential biomarkers have been identified, although none are specific enough to be used alone.
Procalcitonin and C-reactive protein
The most-studied biomarkers are procalcitonin and C-reactive protein (CRP). CRP is an acute-phase protein that is secreted from the liver in the response to inflammatory processes and is therefore sensitive but not specific for sepsis. Procalcitonin, again is produced in response to inflammation and infection, and is so far the only biomarker to be used clinically, as it differentiates better than CRP between infectious and non-infectious causes of critical illness. A meta-analysis found that procalcitonin had a mean sensitivity and specificity of around 70% and an area under receiver operator characteristic curve of less than 0.80 [10]. However as levels of procalcitonin are known to be raised after surgery, trauma and viral infection, the Surviving Sepsis Campaign concluded that procalcitonin levels are not adequate to distinguish sepsis from other causes of inflammation [11], although it may be useful for indicating when treatment with antibiotics can end [12].
Interleukin 6 (IL-6)
IL-6 was initially a biomarker of interest for rapid sepsis diagnosis as it has a fast kinetic profile – the concentration increases within 2 hours of onset of sepsis and decreases within 6 hours. However, the results from studies have been mixed, with some suggesting that it was able to discriminate between sepsis and non-infectious illness, whereas others found that procalcitonin was better, hence it has not been added to current guidelines [11].
Promising biomarkers
A number of other biomarkers have been identified that show promise include soluble urokinase-type plasminogen activator receptor, presepsin and proadrenomedullin [2, 13]. Additionally, recently, reduced serum levels of fetuin-A (a major hepatokine) were found to be independently associated with predicting progression to septic shock and higher rates of mortality [14].
Biomarker panels
Even today, no single biomarker has the diagnostic strength to identify patients suffering from sepsis and it is likely that assessing panels of biomarkers will increase the sensitivity and accuracy of diagnosis of sepsis, compared to any individual biomarker (for example, see the study by Kofoed et al. [15]). More recently, the power of mass spectrometry and “-omics studies” is being investigated with some promise, although still suffering from limitations [13].
References
1. Sepsis. World Health Organization 2018; http://www.who.int/news-room/fact-sheets/detail/sepsis.
2. Candel FJ, et al. Current aspects in sepsis approach. Turning things around. Rev Esp Quimioter 2018; 31(4): 298–315.
3. Improving outcomes for patients with sepsis: a cross-system action plan. NHS England 2015; https://www.england.nhs.uk/wp-content/uploads/2015/08/Sepsis-Action-Plan-23.12.15-v1.pdf.
4. Sepsis. Centers for Disease Control and Prevention 2018; https://www.cdc.gov/sepsis/datareports/index.html.
5. Singer M, et al. The Third International Consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 2016; 315(8): 801–810.
6. Czura CJ. Merinoff symposium 2010: Sepsis – speaking with one voice. Mol Med 2011; 17(1-2): 2–3.
7. Surviving Sepsis Campaign: International guidelines for management of sepsis and septic shock: 2016. Surviving Sepsis Campaign 2016; http://www.survivingsepsis.org/Guidelines/Pages/default.aspx.
8. Sepsis: recognition, diagnosis and early management; NICE guideline [NG51]. National Institutes for Health and Care Excellence 2017; https://www.nice.org.uk/guidance/NG51/chapter/Recommendations#identifying-people-with-suspected-sepsis.
9. Ward KM, Harris R. Sepsis: earlier organism identification using MALDI-TOF. Clin Lab Int 2015; Nov: 14–18.
10. Wacker C, et al. Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. Lancet Infect Dis 2013; 13: 426–435.
11. Dellinger RP, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013; 41(2): 580–637.
12. Sager R, et al. Procalcitonin-guided diagnosis and antibiotic stewardship revisited. BMC Med 2017; 15: 15.
13. Ludwig KR, Hummon AB. Mass spectrometry for the discovery of biomarkers of sepsis. Mol Biosyst 2017; 13(4): 648–664.
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