Chest pain is a common presentation in Emergency Medicine and can cause a diagnostic dilemma. Does the patient need urgent admission for percutaneous coronary intervention following a myocardial infarction? Or will discharge with reassurance and a bottle of antacids sort out their indigestion? Measurement of cardiac troponin has become an essential part of emergency assessment of chest pain. Despite this, clinicians and laboratory professionals should be conscious that elevated cardiac troponin is not inevitably indicative of cardiac pathology – and in some cases, may be deceptive…
by Ceri Parfitt and Dr Christopher Duff
Troponin and assessment of acute chest pain
The Universal Definition of Myocardial Infarction and Injury has incorporated measurement of cardiac troponin (cTn) since 2000, with the most recent revision in 2012 [1]. In the UK, the National Institute of Health and Care Excellence (NICE) the ‘Chest pain of recent onset: assessment and diagnosis’ pathway [2] is well established in clinical practice, and Emergency Department clinicians are confident in the use of cTn assays to identify patients with acute coronary syndrome (ACS).
The troponin complex comprises three regulatory proteins – troponin C, troponin T and troponin I – essential for skeletal and cardiac muscle contraction, through regulation of actin/myosin filaments. Cardiac-specific isoforms of both troponin T (cTnT) and troponin I (cTnI) have been identified. cTn complexes can be detected in the blood within 2–3 hours of myocardial damage, peak within 24 hours and persist for 1–2 weeks [3, 4]. This characteristic pattern of release and specificity for cardiac injury has cemented cTn as the biomarker of choice for diagnosis of ACS, replacing creatinine kinase, aspartate transaminase and lactate dehydrogenase. Measurement of cTn is a standard assay available on an urgent basis in the majority of modern clinical laboratories. Both cTnT and cTnI can be measured by two-site ‘sandwich’ immunoassay, based on formation of complexes of cTn, a ‘capture’ anti-cTn antibody and a ‘label’ anti-cTn antibody (Fig. 1a). Owing to patent regulations, Roche Diagnostics is the sole distributor of cTnT immunoassays, whereas various companies offer cTnI immunoassays on several different platforms. Although results cannot be directly compared, the two assays largely provide the same diagnostic information.
A patient presents with chest pain and raised troponin
Despite the widespread use of cTn in Emergency Departments worldwide, clinicians may often be unaware of spurious causes of raised cTn. Application of the pathway without considering patient history and previous presentations can potentially lead to invasive, unnecessary (and expensive) investigations. We recently encountered an example of this in our local Emergency Department. A 34-year-old male with no significant cardiac risk factors repeatedly presented to the Emergency Department complaining of chest pain over a period of 3 years, resulting in more than 40 individual analyses of cTnI (using the Siemens TnI-Ultra assay on ADVIA Centaur XP). The results were variable, but always elevated (48–4030 ng/L, decision limit 40 ng/L). The patient underwent thorough investigation, but no cardiac pathology was ever identified. The Cardiology Department contacted the laboratory to discuss the apparent mismatch between patient presentation and biochemical findings.
Has there been a laboratory error?
Spurious results can arise for a number of reasons, including mislabelled samples, sample contamination, poor sample quality, analyser malfunction, calibration/quality control errors and transcription errors [5]. Where possible, these factors are minimized by stringent sampling techniques and robust laboratory procedures, or can be identified through the technical and clinical validation processes, and highlighted to the clinical team by laboratory staff. In many cases, a simple repeat sample is sufficient to confirm/rule out error. However, clinicians should always be vigilant for results that are not consistent with patient presentation. The persistent nature of the elevated cTnI in this case – over a period of 3 years – rules out sporadic errors, such as mislabelled samples or analyser malfunction. Furthermore, no other patients with unexpectedly high cTnI concentrations were discussed with the laboratory. These factors strongly suggested that the solution to the mystery lay within the patient, rather than in management of their samples.
Alternative causes of elevated troponin: cardiac and non-cardiac
Although elevated cTn is typically associated with myocardial infarction, other cardiac pathologies also cause cTn release into the circulation through direct or hypoxic myocyte damage. These include aortic dissection, pericarditis, myocarditis, acute heart failure and cardiac contusion following trauma [4, 6]. Unsurprisingly, cardiac interventions such as defibrillator shocks, coronary angioplasty, percutaneous coronary intervention and open heart surgery are also associated with variable increase in cTn [6]. Chemotherapy agents, including anthracyclines, alkylating agents, anti-metabolites and anti-microtubules are all associated with toxic damage to myocytes [4].
In addition to cardiac sources of cTn, non-cardiac sources must also be considered. A significant proportion (estimated at 36–85 %) of patients admitted to critical care units for sepsis/systemic inflammatory response syndromes (SIRS) have elevated cTn concentrations. In these patients, cTn release is thought to be a consequence of oxygen supply-demand mismatch in the myocardium: although oxygen demand is increased, due to fever and tachycardia, systemic hypoxemia reduces oxygen supply due to respiratory failure, circulatory dysfunction and hypotension [7, 8]. Patients with end-stage renal disease (ESRD) are often noted to have chronically elevated cTn. The underlying cause for this is unclear, but various hypotheses have been presented, including reduced renal clearance of cTn microfragments, sub-clinical cardiac disease, and metabolic abnormalities [9]. cTn concentrations are also found to be elevated in patients with acute pulmonary embolism. Again, the cause has not been conclusively established, but it is believed that abrupt increases in pulmonary artery pressure leads to dilatation of the right ventricle, with associated myocyte injury [4].
For most patients, any of these situations can be identified by examining the patient records, or through further cardiac investigations. In the case presented here, after thorough analysis of the patient’s history, and given the resolutely normal cardiac investigations, we concluded that it was unlikely that the elevation in cTn was due to pathological factors, whether cardiac or non-cardiac. This led us to examine the local cTnI assay – and the patient’s samples – in more detail, to ascertain the cause of the spurious results.
Could immunoassay interference be involved?
Non-pathological causes of false positives in cTn analysis include human anti-mouse antibodies (HAMA), rheumatoid factor (RF) and heterophilic antibodies [10–12]. HAMA are characterized by strong avidity to well-defined antigens. They are thought to develop following exposure to mouse immunoglobulins, which may occur through monoclonal antibody therapy, vaccination, infection, blood transfusion, or simply through animal handling [10]. In contrast, heterophilic antibodies are endogenous antibodies present in serum/plasma produced against poorly defined antigens. They often have weak affinity, but multiple specificities. Heterophilic antibodies may develop following infection and are thought to affect up to 30% of the population [10, 12, 13]. RFs are endogenous human antibodies with heterophilic activity, often arising following autoimmune disease [12].
Genuine assay interference is less commonly encountered these days, with the development of highly specialized commercial assays. Most modern immunoassays contain agents able to block low concentrations of interfering proteins. However, the fact that interference is less frequently observed may lead laboratory staff members to believe that interference is less prevalent in the general population. This is not the case. In fact, the prevalence of assay interference may be increasing, as immunotherapy and the use of radiolabelled antibodies are established in routine practice [13]. Although heterophilic antibodies usually have little clinical significance in vivo, their ability to interfere with two-site immunoassays can have major effects on patient management. The magnitude of interference varies between samples and may even vary within a patient over time [13]. The mechanism for antibody interference is complex and has not been fully elucidated, but is likely to involve ‘bridge’ formation between the capture antibody and label antibody (Fig. 1b) [13, 14]. Several standard techniques are available to investigate assay interference [12, 13]. In this case, we used dilution studies, polyethylene glycol precipitation, heterophilic antibody blocking tubes and an alternative assay method. In all cases, samples from the patient were compared with a control sample with a similar cTnI concentration, from a patient with confirmed ACS.
Investigations into immunoassay interference
Serial dilution of samples should produce a linear decrease in concentration if interfering species are absent. However, if interfering substances are present, then non-linear recovery following dilution is commonly observed, as the interferent is ‘diluted out’. Both the patient’s serum and control serum were diluted with saline, and cTnI was measured in each dilution. As expected, the cTnI concentration in the control sample decreased proportionally to the dilution factor. In contrast, any dilution of the patient sample resulted in undetectable cTnI, consistent with presence of an interfering species (Fig. 2a). Immunoglobulins can be removed by treatment with polyethylene glycol (PEG). PEG forms poorly soluble complexes with immunoglobulins, which can be precipitated then removed by centrifugation, before the sample is re-analysed. In the patient sample, PEG precipitation resulted in undetectable levels of cTnI. Levels were also reduced in a control sample (62% recovery), though it is not uncommon to see at least some reduction in recovery using this approach, regardless of whether interfering immunoglobulins are present or not (Fig. 2b). Heterophilic blocking reagents prevent non-analyte mediated bridging of antibodies by heterophilic interference. Incubation of a patient sample in a heterophilic blocking tube (Scantibodies Laboratory Inc.) before cTnI analysis resulted in 4% recovery of cTnI, suggesting analytical interference. In the control sample, 94% of cTnI was recovered (Fig. 2c). Finally, a patient sample was sent to a referral laboratory for analysis of cTnT, as opposed to cTnI. A cTnT concentration below the decision limit (99th percentile) was obtained (cTnI result: 398 ng/L; cTnT result: 6 ng/L) (Fig. 3).
As initial investigations strongly suggested the presence of an assay interferent, further work was conducted to identify the precise nature of the interfering species. Two patient samples with elevated cTnI were analysed for presence of HAMAs, which was negative in both cases. This was not unexpected, given that the patient had no history of exposure to animal proteins in either a therapeutic or a social setting. The same sample was tested for RFs, using the local assay (Orgentec RF IgM), which returned a slightly elevated result. This slightly elevated concentration is not considered clinically significant, but may contribute to interference in the assay. Thus, heterophilic antibody interference remains the most likely cause of cTnI elevation in this patient. Further investigations have been limited, however, by recently decreasing concentrations of cTnI in this patient. Unpredictable variation in heterophilic antibody activity is a known phenomenon however, and the laboratory continues to oversee samples received from the patient.
Outcome and conclusions
The patient continues to regularly attend the Emergency Department with chest pain. No cardiac pathology has ever been identified in this patient and his chest pain has been attributed to a combination of gastro-oesophageal reflux disease and health anxiety. Senior review is now required in the Emergency Department before the patient is investigated or admitted. If ACS is ever suspected in this patient, the laboratory is able to pre-treat samples with heterophilic blocking tubes to provide an estimate of cTnI concentration, with referral for cTnT analysis required for a definitive result. This alternate pathway ensures that the patient is protected in case of a genuine cardiac event.
This case demonstrates that clinicians must be aware of assay interference, and highlights the benefit of discussing patients with the laboratory when test results do not correlate with clinical presentation. Laboratory staff members are rarely able to visit patients and, thus, are not in a position to suspect interference without input from the clinical team. In this case, failure to identify the underlying cause of elevated cTnI at an early stage resulted in a number of unnecessary and invasive investigations, significant costs to the NHS, and continued anxiety to the patient.
References
1. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, et al. Third universal definition of myocardial infarction. Eur Heart J 2012; 33: 2551–2567.
2. National Institute of Health and Care Excellence (NICE). Chest pain of recent onset: assessment and diagnosis. NICE clinical guideline 95, 2010 (https://www.nice.org.uk/guidance/cg95).
3. Wu AHB. Analytical issues for clinical use of cardiac troponin. In: Morrow DA (ed.) Contemporary cardiology: cardiovascular biomarkers: pathophysiology and disease management. Humana 2006, pp 27–40.
4. Korff S, Katus HA, Giannitsis E. Differential diagnosis of elevated troponins. Heart 2006; 92: 987–993.
5. Plebani M. The detection and prevention of errors in laboratory medicine. Ann Clin Biochem 2010; 47: 101–110.
6. Sara JDS, Holmes DR, Jaffe AS. Fundamental concepts of effective troponin use: important principles for internists. Am J Med 2015; 128: 111–119.
7. Ver Elst KM, Spapen HD, Nguyen DN, Garbar C, Huyghens LP, Gorus FK. Cardiac troponins I and T are biological markers of left ventricular dysfunction in septic shock 2000; 46: 650–657.
8. Spies C, Haude V, Fitzner R, Schroder K, Overbeck M, Runkel N, Schaffartzik W. Serum cardiac troponin T as a prognostic marker in early sepsis. Chest 1998; 113: 1055–1063.
9. Jaffe AS. The 10 commandments of troponin with special reference to high sensitivity assays. Heart 2011; 97: 940–946.
10. Lippi G, Aloe R, Meschi T, Borghi L, Cervellin G. Interference from heterophilic antibodies in troponin testing. Case report and systematic review of the literature. Clin Chim Acta 2013; 426: 79–84.
11. Makaryus AN, Markaryus MN, Hassid B. Falsely elevated cardiac troponin I levels. Clin Cardiol 2007; 30: 92–94.
12. Zaidi A, Cowell R. False positive cardiac troponin elevation due to heterophile antibodies: more common than we recognise? BMJ Case Rep 2010; 2010: bcr1120092477.
13. Ismail AAA, Walker PL, Cawood ML, Barth JH. Interference in immunoassay is an underestimated problem. Ann Clin Biochem 2002; 39: 266–373.
14. Kaplan IV, Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem 1999; 45: 616–618.
The authors
Ceri Parfitt MSc, Christopher Duff* PhD
Department of Clinical Biochemistry, Pathology Directorate, Royal Stoke University Hospital, University Hospitals of North Midlands NHS Trust, Staffordshire, UK
*Corresponding author
E-mail: Chris.duff@uhnm.nhs.uk
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, /in Featured Articles /by 3wmediaHospital-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.
Is a celiac screen in a ‘tired all the time’ test profile of any value?
, /in Autoimmunity & Allergy, Featured Articles /by 3wmediaby L. Hughes, Dr A. Ballantyne, Dr C. Ford, Dr A. Ekbote and Prof. R. Gama Celiac disease (CD) is a common autoimmune gastrointestinal disease. Several serological tests are available to screen for CD. Since CD can present with fatigue, serological screening was incorporated into a ‘tired all the time’ testing profile available to general […]
Evaluation of a highly automated fecal calprotectin assay for the differential diagnosis of IBD or IBS
, /in Featured Articles /by 3wmediaFecal calprotectin is an effective biomarker in the differential diagnosis of inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS). Since the National Institute for Health and Care Excellence (NICE) recommended its use there has been a significant increase in demand for analysis. New methods on mainline chemistry analysers can be implemented in response to the increase in workload.
by Sally Willett, Pamela Bowe, Frankie Leslie and Wayne Bradbury
Introduction
Chronic abdominal pain with diarrhea or constipation are common presenting symptoms in general practice. The differential diagnosis in this patient population is varied, but includes irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).
IBS is a chronic, relapsing and often lifelong disorder associated with disordered defecation and abdominal distention. It is not associated with any distinctive pathology and although it is troublesome for the patient it is not associated with any serious comorbidity. IBS is a relatively common diagnosis with a prevalence of 10–20% in the general population [1].
IBD is a much more serious condition, associated with a high morbidity. The term IBD includes Crohn’s disease and ulcerative colitis, conditions in which gastrointestinal inflammation can lead to major complications. Patients may require surgery and are at increased risk of colorectal cancer. Evolving treatment options, including novel drugs and surgery, aim to secure and maintain remission [2].
It is important to distinguish IBD from non-IBD, such as IBS, so that conditions can be appropriately managed and monitored. Endoscopy with histological examination of biopsy samples remains the gold standard in differentiating IBD and IBS, but is very expensive, time consuming and invasive. Conventional diagnostic testing included markers of inflammation including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). However, these markers cannot localize inflammation to the gut. There has been intensive research into fecal biomarkers, specific for gastrointestinal inflammation over the last decade.
Calprotectin is a small calcium binding protein which contributes ~60% of the protein content of the cytosol in neutrophils [3]. During the intestinal inflammation observed in patients with IBD neutrophils migrate to the intestinal mucosa. As the inflammatory process damages the mucosal architecture the neutrophils are shed into the lumen and calprotectin is detectable in the feces. A raised fecal calprotectin concentration (>50 µg/g) has been shown to have a good diagnostic sensitivity and specificity for the detection of IBD [4].
Analytical methods for the detection of calprotectin in feces have evolved since the original enzyme-linked immunosorbent assay (ELISA) method was described in 1992 [5]. Commercial immunoassays are now available and quantitative lateral flow immunochomatographic point-of-care tests have been marketed to generate rapid results in the clinic setting. Many laboratories still use ELISA technology to analyse fecal samples for calprotectin. Such analysis is relatively labour intensive and often fecal extracts are run in duplicate at increased cost.
Since the National Institute for Health and Care Excellence (NICE) recommended the use of fecal calprotectin in primary care [2], there has been a significant increase in demand for this test. We investigated the performance of the new BÜHLMANN fCALTM turbo method which is CE marked for use on a number of mainline chemistry analysers. Implementation of this method has the potential to streamline analysis, relieving staff time and reducing cost.
Method
The BÜHLMANN fCALTM turbo particle enhanced turbidimetric immunoassay (PETIA) method on the Roche Cobas 6000 (c501) was compared to the BÜHLMANN Calprotectin ELISA method on the Dynex DS2. The study was performed within the Blood Sciences Department at North Cumbria University Hospitals.
The PETIA method uses polystyrene nanoparticles coated with specific antibodies to bind calprotectin in fecal extracts. Calprotectin in the sample mediates immune-particle agglutination and the resultant increase in turbidity is quantified by optical density.
Fecal samples were extracted using the BÜHLMANN CALEX® extraction device prior to analysis on both methods. Fifty-eight patient samples were analysed and results compared using regression analysis. Intra-assay precision was determined using 10 replicates of patient samples and inter-assay precision was calculated using 17 replicates of internal quality control material. NEQAS samples were analysed and bias relative to the all laboratory trimmed mean (ALTM) was assessed.
Results and discussion
Comparison of patient results showed good correlation (R2=0.97) consistent with previous studies [6, 7]. Regression analysis produced the following equation:
fCALTM turbo = (1.14×DS2 result)−23.42
The fCALTM turbo method demonstrated a negative bias at concentrations <100 µg/g and a positive bias at higher concentrations when compared with the ELISA method (Fig. 1), which has also been observed by De Sloovere et al. [6]. The positive bias observed at higher concentrations is accounted for in local guidelines. Since the initial evaluation a field safety notice (FSN) was distributed informing users that a positive bias of 15.6% was observed using the BÜHLMANN CALEX® extraction devices. This has subsequently been corrected with the CALEX® Cap “N” devices. After the introduction of the revised extraction devices external quality assurance (EQA) results have improved, and local results show a mean bias of 54 µg/g from the NEQAS ALTM (Fig. 2). A commutable reference material for calprotectin is required to define analytical accuracy in the future.
Intra-assay precision, as determined by percent coefficient of variation (%CV), was 3.1% and 1.3% at concentrations of 48 µg/g and 247 µg/g respectively (n=10). Inter-assay precision was 3.3% at 73 µg/g and 1.1% at 247 µg/g (n=17). This is consistent with De Sloovere et al. who demonstrated %CVs of ~3% using the fCAL turbo method [6]. Since running the method routinely the internal quality control data shows a running %CV of 4.5% at 75 µg/g and 2.6% at 245 µg/g (n=23).
Historically, fecal samples required weighing and diluting in extraction buffer before analysis, which was very labour intensive and prone to error. The introduction of extraction devices has simplified the pre-analytical steps significantly. The introduction of the PETIA method into our laboratory has further simplified analysis and reduced staff time, as the fecal extracts are loaded directly onto the Cobas 6000 in barcoded CALEX tubes. The PETIA method has a large analytical range (20–1800 µg/g feces) reducing the requirement for costly repeat analysis on dilution. Although the ELISA method favours batch analysis, the PETIA method is suitable for random access testing, improving assay turnaround times. An additional wash step is implemented to eliminate carry over between fecal and blood samples.
Conclusion
It is important to accurately differentiate IBD from IBS so that appropriate patient care pathways can be instigated. The methodologies available for the quantification of fecal calprotectin have evolved significantly over the last decade. The BÜHLMANN fCALTM turbo PETIA method on the Roche Cobas 6000 (c501) demonstrated acceptable performance and is suitable for routine use within a diagnostic laboratory.
References
1. National Institute for Health and Clinical Excellence (NICE). Irritable bowel syndrome in adults: diagnosis and management. NICE clinical guideline 61, 2008.
2. NICE. Faecal calprotectin diagnostic tests for inflammatory diseases of the bowel. NICE diagnostic guideline 11, 2013.
3. Fagerhol M, Dale I, Andersson T. A radioimmunoassay for a granulocyte protein as a marker in studies on the turnover of such cells. Bull Eur Physiopathol Respir 1980; 16(Suppl): 273–282.
4. Walsham N and Sherwood R. Fecal calprotectin in inflammatory bowel disease. Clin Exp Gastroenterol 2016; 9: 21–29.
5. Roseth AG, Fagerhol MK, Aadland E, Schiønsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992; 27: 793–798.
6. De Sloovere M, De Smet D, Baert F, Debrabandere J, Vanpoucke HJM. Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of IBD. Clin Chem Lab Med 2017; 28: 1435–1446.
7. Nilsen T, Sunde K, Hansson L, Havelka AM, Larsson A. A novel turbidimetric immunoassay for fecal calprotectin optimized for routine chemistry analysers. J Clin Lab Anal Analysis 2017; 31: 1–6.
The authors
Sally Willett FRCPath, Pamela Bowe* MSc, Frankie Leslie BSc, Wayne Bradbury FRCPath
Blood Sciences, North Cumbria University Hospitals NHS Trust, Cumberland Infirmary, Carlisle, UK
*Corresponding author
E-mail: Pamela.Bowe@ncuh.nhs.uk
Calprotectin use in primary care. Are testing criteria being followed?
, /in Featured Articles /by 3wmediaIn 2013, the National Institute for Health and Care Excellence (NICE) published guidelines recommending the use of fecal calprotectin as a screening test for differentiating between inflammatory bowel disease and irritable bowel syndrome. Since then, despite a relatively slow uptake with only a few laboratories offering the test, fecal calprotectin has garnered much interest with more hospitals incorporating it into their pathology services. Therefore, the question of how well primary care is using the service arises. This, along with a brief historical context of inflammatory bowel disease and its diagnosis together with implications of calprotectin testing are discussed here.
by Dr Benjamin Palmer, Dr Wisam Jafar and Steven McCann
Historical context
Inflammatory bowel disease (IBD) is a term used to describe two relapsing chronic gastrointestinal disorders: ulcerative colitis (UC) and Crohn’s disease (CD). Both cause considerable morbidity amongst young patients in whom they occur more frequently [1]. With an estimated prevalence of 2.5–3.0 million people affected in Europe the incidence of IBD is increasing [1]. Although the etiology of IBD is unknown several theories have been put forward the most important and frequently cited elements being: gut microbiota; genetic pre-disposition; environment and immune dysregulation [2]. The two main types of IBD seen in the clinical setting are UC and CD: definitive diagnosis and confirmation is provided by endoscopy and histology. However, in 5% of cases a definitive diagnosis cannot be established: in such cases patients are diagnosed with inflammatory bowel disease unclassified (IBDU) [3, 4].
The gastrointestinal tract is colonized by a large number of diverse bacteria living in symbiosis with their host (microbiota) [5]. Disruption to the diversity and quality of the gut microbiota (dysbiosis) is now implicated in the pathogenesis of both UC and CD [2, 5, 6]. Other important participants are cytokines: as their release causes intestinal inflammation they have been implicated in some of the clinical symptoms such as diarrhea [7–9].
Historically, the two types of IBD were differentiated by the different cytokines and mature CD4+ T helper (Th) cells found elevated in tissue biopsies. UC was characterized by elevated levels of Th2 cells and interleukin (IL)-4, -5, -10 and -13 cytokines, whereas CD was defined by high levels of Th1 cells and the interferon gamma (IFNγ) cytokine [3]. However, new emerging information suggests that it is not as cut and dried as this: emphasis has now shifted towards such pathways as IL-23/IL-17 activation of more pathogenic Th17 cells playing a more significant role in the pathophysiology of not just IBD but other inflammatory diseases [10].
Complicating the clinical scenario is the high prevalence (around 10−20% of the UK population) of an unrelated functional bowel disorder called irritable bowel syndrome (IBS): the true prevalence is thought to be higher as it is suspected that many people may not seek medical advice [11]. Although it does not cause serious morbidities, IBS manifests with symptoms similar to those of IBD, making diagnosis difficult. Unlike IBD, it is not associated with inflammation and, hence, this provides a means of making a differential diagnosis.
Until recently, the diagnosis of IBD was made by clinical evaluation and a combination of biochemical and mainly endoscopic investigations that often involved repeat consultations and testing [3]. This, taken together with an ageing population, an increased public awareness of bowel cancer and the introduction of national screening campaigns has led to increased service demands in endoscopy. The fact that fecal markers, such as calprotectin, are secreted by inflamed intestinal mucosa has led to the development of laboratory assays that can detect gastrointestinal inflammation. It was because of these challenges and recent developments that led the National Institute for Health and Care Excellence (NICE) to publish guidelines on the use of fecal calprotectin in differentiating IBD from IBS in adults provided cancer is not suspected and that local reporting guidelines along with appropriate quality assurance procedures are in place [12]. A cut-off of 50 μg/g is recommended in which all patients with a calprotectin level ≥50 μg/g may be suggestive of IBD and should be referred to Gastroenterology, whereas a calprotectin level <50 μg/g is unlikely to be caused by IBD and should, therefore, be managed with a presumptive diagnosis of IBS [12]. One key study used by NICE in recommending this threshold was carried out by Tibble et al. in which 602 outpatients with lower gastrointestinal tract symptom were included [13]. In this study the authors clearly demonstrated that the IBD group of patients differed significantly (P=0.001–<0.0001) from the IBS group of patients and that a cut-off level of 10 mg/L (equivalent to 50 μg/g) provided optimal diagnostic performance [13]. However, the cut-off level of any test is dependent on the method used and, therefore, each laboratory should establish their method-specific cut-off level [12].
IBD pathway and audit outcome
Following the recommendations of NICE, fecal calprotectin testing was incorporated into the pathology services at Stepping Hill hospital on 1 November 2014. The test is available for patients from primary care between 20 and 40 years of age who are presenting with abdominal pain or discomfort, bloating or altered bowel habits for 6 months or longer. Patients with red flag symptoms (anemia, abdominal mass, gastrointestinal infection, rectal bleeding, unexplained weight loss or a family history of ovarian or bowel cancer) should be referred directly to Gastroenterology (Fig. 1).
If the calprotectin test is negative the patient should be managed by primary care with a presumptive diagnosis of IBS; if the test is positive the patient should be referred to Gastroenterology. Then in 2016 a 1-year retrospective audit was carried out with the aim of determining how well primary care was adhering to the clinical pathway [14]. In order to achieve this, a questionnaire was designed and sent to all primary care surgeries in the catchment area for each patient who received calprotectin testing (n=587): the responses (n=217) to these questionnaires were then compared to the IBD pathway [14].
The outcome of this audit revealed that most areas of the IBD pathway were not being adhered to and, therefore, GP re-education and training was needed; a similar finding to another hospital [10]. The worst area of non-conformance was to ensure that patients had had signs/symptoms for at least 6 months: 69% of requests were not compliant [14]. Exclusion of gastrointestinal infection was second (63%), followed by ensuring age was 20–40 years (48%) [14]. Of the 216 questionnaires returned, 35% of patients had had red flag signs/symptoms at the time of the request [14]. Alarmingly, rectal bleeding was the most frequently encountered, followed by anemia, unexplained weight loss and a family history of bowel/ovarian cancer [14]. None of the patients had abdominal mass [14]. Conversely, high compliance was observed for the withdrawal of non-steroidal inflammatory drugs (NSAIDs) and antibiotics before testing [14]. Overall, only 24 requests (11%) were fully compliant for all criteria of the IBD clinical pathway [14].
Patient/clinician considerations
Unlike other fecal markers such as elastase, calprotectin is much less stable (up to 3 days at room temperature) and, therefore, it is important that patient samples are sent to the laboratory within 72 hours of collection [16]. It is still the case that samples are received by the laboratory with no date or time of collection and these are consequently rejected for analysis. Considering that calprotectin is elevated in gastrointestinal infection, in order for the test to be of clinical use, it is imperative that this is excluded.
The age restriction imposed on calprotectin testing is important and is based on the fact that IBS is more common in younger adults, that the prevalence of IBS decreases with increasing age and that new onset of symptoms after 50 years is uncommon [11, 17]. By focusing on this age group a large number of patients who do not require diagnostic testing will be excluded and, therefore, they will not add to the delay that current IBD sufferers face in receiving a colonoscopy.
Patients >40 years of age should be referred to Gastroenterology without delay, as the risk of developing colorectal cancer increases with age: colorectal cancer is the third most commonly diagnosed cancer in the UK and one of the major complications of IBD [18].
Since IBD is a relapsing disease the clinician should be aware that screening results from some patients with IBD may be negative, particularly if the disease is in a period of remittance and, therefore, the presentation of the patient, not the test result, should be the ultimate deciding factor over whether to refer. Finally, underpinning this is the need for local laboratories to determine their own method-specific cut-off values using evidence-based medicine. As with all screening tests the aim should be to optimize the test’s ability to exclude disease (IBS) so that fewer patients without disease are referred.
References
1. Trbojevic Akmacic I, Ventham NT, Theodoratou E, Vuckovic F, Kennedy NA, Krištic J, Nimmo ER, Kalla R, Drummond H, et al. Inflammatory bowel disease associates with proinflammatory potenial of the immunoglobulin G glycome. Inflamm Bowel Dis 2015; 21: 1237–1247.
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The authors
Benjamin Palmer*1 PhD, MRSC; Wisam Jafar2 MBChB, MRCP(gastro), MSc, MA, FRCP; Steven McCann2 MSc, FRCPath
1Betsi Cadwaladr Health Board, Glan Clwyd Hospital, Rhyl, Denbighshire, Wales, UK
2Stockport NHS Foundation Trust, Stockport, Cheshire, UK
*Corresponding author
E-mail: Ben.palmer@wales.nhs.uk
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by Ceri Parfitt and Dr Christopher Duff
Troponin and assessment of acute chest pain
The Universal Definition of Myocardial Infarction and Injury has incorporated measurement of cardiac troponin (cTn) since 2000, with the most recent revision in 2012 [1]. In the UK, the National Institute of Health and Care Excellence (NICE) the ‘Chest pain of recent onset: assessment and diagnosis’ pathway [2] is well established in clinical practice, and Emergency Department clinicians are confident in the use of cTn assays to identify patients with acute coronary syndrome (ACS).
The troponin complex comprises three regulatory proteins – troponin C, troponin T and troponin I – essential for skeletal and cardiac muscle contraction, through regulation of actin/myosin filaments. Cardiac-specific isoforms of both troponin T (cTnT) and troponin I (cTnI) have been identified. cTn complexes can be detected in the blood within 2–3 hours of myocardial damage, peak within 24 hours and persist for 1–2 weeks [3, 4]. This characteristic pattern of release and specificity for cardiac injury has cemented cTn as the biomarker of choice for diagnosis of ACS, replacing creatinine kinase, aspartate transaminase and lactate dehydrogenase. Measurement of cTn is a standard assay available on an urgent basis in the majority of modern clinical laboratories. Both cTnT and cTnI can be measured by two-site ‘sandwich’ immunoassay, based on formation of complexes of cTn, a ‘capture’ anti-cTn antibody and a ‘label’ anti-cTn antibody (Fig. 1a). Owing to patent regulations, Roche Diagnostics is the sole distributor of cTnT immunoassays, whereas various companies offer cTnI immunoassays on several different platforms. Although results cannot be directly compared, the two assays largely provide the same diagnostic information.
A patient presents with chest pain and raised troponin
Despite the widespread use of cTn in Emergency Departments worldwide, clinicians may often be unaware of spurious causes of raised cTn. Application of the pathway without considering patient history and previous presentations can potentially lead to invasive, unnecessary (and expensive) investigations. We recently encountered an example of this in our local Emergency Department. A 34-year-old male with no significant cardiac risk factors repeatedly presented to the Emergency Department complaining of chest pain over a period of 3 years, resulting in more than 40 individual analyses of cTnI (using the Siemens TnI-Ultra assay on ADVIA Centaur XP). The results were variable, but always elevated (48–4030 ng/L, decision limit 40 ng/L). The patient underwent thorough investigation, but no cardiac pathology was ever identified. The Cardiology Department contacted the laboratory to discuss the apparent mismatch between patient presentation and biochemical findings.
Has there been a laboratory error?
Spurious results can arise for a number of reasons, including mislabelled samples, sample contamination, poor sample quality, analyser malfunction, calibration/quality control errors and transcription errors [5]. Where possible, these factors are minimized by stringent sampling techniques and robust laboratory procedures, or can be identified through the technical and clinical validation processes, and highlighted to the clinical team by laboratory staff. In many cases, a simple repeat sample is sufficient to confirm/rule out error. However, clinicians should always be vigilant for results that are not consistent with patient presentation. The persistent nature of the elevated cTnI in this case – over a period of 3 years – rules out sporadic errors, such as mislabelled samples or analyser malfunction. Furthermore, no other patients with unexpectedly high cTnI concentrations were discussed with the laboratory. These factors strongly suggested that the solution to the mystery lay within the patient, rather than in management of their samples.
Alternative causes of elevated troponin: cardiac and non-cardiac
Although elevated cTn is typically associated with myocardial infarction, other cardiac pathologies also cause cTn release into the circulation through direct or hypoxic myocyte damage. These include aortic dissection, pericarditis, myocarditis, acute heart failure and cardiac contusion following trauma [4, 6]. Unsurprisingly, cardiac interventions such as defibrillator shocks, coronary angioplasty, percutaneous coronary intervention and open heart surgery are also associated with variable increase in cTn [6]. Chemotherapy agents, including anthracyclines, alkylating agents, anti-metabolites and anti-microtubules are all associated with toxic damage to myocytes [4].
In addition to cardiac sources of cTn, non-cardiac sources must also be considered. A significant proportion (estimated at 36–85 %) of patients admitted to critical care units for sepsis/systemic inflammatory response syndromes (SIRS) have elevated cTn concentrations. In these patients, cTn release is thought to be a consequence of oxygen supply-demand mismatch in the myocardium: although oxygen demand is increased, due to fever and tachycardia, systemic hypoxemia reduces oxygen supply due to respiratory failure, circulatory dysfunction and hypotension [7, 8]. Patients with end-stage renal disease (ESRD) are often noted to have chronically elevated cTn. The underlying cause for this is unclear, but various hypotheses have been presented, including reduced renal clearance of cTn microfragments, sub-clinical cardiac disease, and metabolic abnormalities [9]. cTn concentrations are also found to be elevated in patients with acute pulmonary embolism. Again, the cause has not been conclusively established, but it is believed that abrupt increases in pulmonary artery pressure leads to dilatation of the right ventricle, with associated myocyte injury [4].
For most patients, any of these situations can be identified by examining the patient records, or through further cardiac investigations. In the case presented here, after thorough analysis of the patient’s history, and given the resolutely normal cardiac investigations, we concluded that it was unlikely that the elevation in cTn was due to pathological factors, whether cardiac or non-cardiac. This led us to examine the local cTnI assay – and the patient’s samples – in more detail, to ascertain the cause of the spurious results.
Could immunoassay interference be involved?
Non-pathological causes of false positives in cTn analysis include human anti-mouse antibodies (HAMA), rheumatoid factor (RF) and heterophilic antibodies [10–12]. HAMA are characterized by strong avidity to well-defined antigens. They are thought to develop following exposure to mouse immunoglobulins, which may occur through monoclonal antibody therapy, vaccination, infection, blood transfusion, or simply through animal handling [10]. In contrast, heterophilic antibodies are endogenous antibodies present in serum/plasma produced against poorly defined antigens. They often have weak affinity, but multiple specificities. Heterophilic antibodies may develop following infection and are thought to affect up to 30% of the population [10, 12, 13]. RFs are endogenous human antibodies with heterophilic activity, often arising following autoimmune disease [12].
Genuine assay interference is less commonly encountered these days, with the development of highly specialized commercial assays. Most modern immunoassays contain agents able to block low concentrations of interfering proteins. However, the fact that interference is less frequently observed may lead laboratory staff members to believe that interference is less prevalent in the general population. This is not the case. In fact, the prevalence of assay interference may be increasing, as immunotherapy and the use of radiolabelled antibodies are established in routine practice [13]. Although heterophilic antibodies usually have little clinical significance in vivo, their ability to interfere with two-site immunoassays can have major effects on patient management. The magnitude of interference varies between samples and may even vary within a patient over time [13]. The mechanism for antibody interference is complex and has not been fully elucidated, but is likely to involve ‘bridge’ formation between the capture antibody and label antibody (Fig. 1b) [13, 14]. Several standard techniques are available to investigate assay interference [12, 13]. In this case, we used dilution studies, polyethylene glycol precipitation, heterophilic antibody blocking tubes and an alternative assay method. In all cases, samples from the patient were compared with a control sample with a similar cTnI concentration, from a patient with confirmed ACS.
Investigations into immunoassay interference
Serial dilution of samples should produce a linear decrease in concentration if interfering species are absent. However, if interfering substances are present, then non-linear recovery following dilution is commonly observed, as the interferent is ‘diluted out’. Both the patient’s serum and control serum were diluted with saline, and cTnI was measured in each dilution. As expected, the cTnI concentration in the control sample decreased proportionally to the dilution factor. In contrast, any dilution of the patient sample resulted in undetectable cTnI, consistent with presence of an interfering species (Fig. 2a). Immunoglobulins can be removed by treatment with polyethylene glycol (PEG). PEG forms poorly soluble complexes with immunoglobulins, which can be precipitated then removed by centrifugation, before the sample is re-analysed. In the patient sample, PEG precipitation resulted in undetectable levels of cTnI. Levels were also reduced in a control sample (62% recovery), though it is not uncommon to see at least some reduction in recovery using this approach, regardless of whether interfering immunoglobulins are present or not (Fig. 2b). Heterophilic blocking reagents prevent non-analyte mediated bridging of antibodies by heterophilic interference. Incubation of a patient sample in a heterophilic blocking tube (Scantibodies Laboratory Inc.) before cTnI analysis resulted in 4% recovery of cTnI, suggesting analytical interference. In the control sample, 94% of cTnI was recovered (Fig. 2c). Finally, a patient sample was sent to a referral laboratory for analysis of cTnT, as opposed to cTnI. A cTnT concentration below the decision limit (99th percentile) was obtained (cTnI result: 398 ng/L; cTnT result: 6 ng/L) (Fig. 3).
As initial investigations strongly suggested the presence of an assay interferent, further work was conducted to identify the precise nature of the interfering species. Two patient samples with elevated cTnI were analysed for presence of HAMAs, which was negative in both cases. This was not unexpected, given that the patient had no history of exposure to animal proteins in either a therapeutic or a social setting. The same sample was tested for RFs, using the local assay (Orgentec RF IgM), which returned a slightly elevated result. This slightly elevated concentration is not considered clinically significant, but may contribute to interference in the assay. Thus, heterophilic antibody interference remains the most likely cause of cTnI elevation in this patient. Further investigations have been limited, however, by recently decreasing concentrations of cTnI in this patient. Unpredictable variation in heterophilic antibody activity is a known phenomenon however, and the laboratory continues to oversee samples received from the patient.
Outcome and conclusions
The patient continues to regularly attend the Emergency Department with chest pain. No cardiac pathology has ever been identified in this patient and his chest pain has been attributed to a combination of gastro-oesophageal reflux disease and health anxiety. Senior review is now required in the Emergency Department before the patient is investigated or admitted. If ACS is ever suspected in this patient, the laboratory is able to pre-treat samples with heterophilic blocking tubes to provide an estimate of cTnI concentration, with referral for cTnT analysis required for a definitive result. This alternate pathway ensures that the patient is protected in case of a genuine cardiac event.
This case demonstrates that clinicians must be aware of assay interference, and highlights the benefit of discussing patients with the laboratory when test results do not correlate with clinical presentation. Laboratory staff members are rarely able to visit patients and, thus, are not in a position to suspect interference without input from the clinical team. In this case, failure to identify the underlying cause of elevated cTnI at an early stage resulted in a number of unnecessary and invasive investigations, significant costs to the NHS, and continued anxiety to the patient.
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The authors
Ceri Parfitt MSc, Christopher Duff* PhD
Department of Clinical Biochemistry, Pathology Directorate, Royal Stoke University Hospital, University Hospitals of North Midlands NHS Trust, Staffordshire, UK
*Corresponding author
E-mail: Chris.duff@uhnm.nhs.uk