Interference in immunoassay is a well described phenomenon and all clinical immunoassays, including thyroid function tests, are potentially at risk. Spurious results can lead to over investigation or mismanagement if not detected, but a proactive approach by the laboratory will help to identify and resolve these problems.
by Dr Olivia Bacon and Dr David J. Halsall
Background
Thyroid disorders are relatively common, and are associated with long-term morbidity and mortality. Clinical signs and symptoms are often non-specific, so reliable laboratory tests are critical for diagnosis. Therefore, thyroid function tests (TFTs) are frequently requested immunoassays with around 10 million results being reported each year by UK laboratories. In the UK, TFTs typically include a high sensitivity immunoassay for thyroid stimulating hormone (TSH) with an immunoassay estimation of non-protein bound thyroxine (fT4), either run simultaneously or added if the TSH value is outside the reference interval [1].
For the majority of tests, both results will be within the reference interval and thyroid disease can be excluded. In some patients TFTs support the diagnosis of hypothyroidism (raised TSH with fT4 low, or lownormal) or hyperthyroidism (TSH undetectable, and fT4 elevated), and these results will confirm clinical findings. However, due to the high volume of TFTs performed, it is not unusual for the laboratorian to be faced with a set of TFTs that are either internally inconsistent, or incompatible with the clinical details provided. Many medications can affect the thyroid axis, as can other non-thyroidal pathologies; these are often transient, but can cause unusual patterns of TFT. Much rarer genetic or pituitary conditions can also cause discordant TFTs [2]. However, if drug effects are excluded, it is necessary at this stage for the laboratorian to consider that one of the TFT results is incorrect, as analytic error is at least as common as these rare thyroid conditions. As spurious TFT results can lead to over investigation, or even inappropriate treatment, it is critical, but not trivial, for the laboratory to confirm the analytical validity of the TFT results.
In one study of more than 5000 samples received for TSH analysis, assay interference with the potential to adversely affect clinical care was detected in approximately 0.5% of patients [3]. This equates to a rather alarming 50,000 tests per annum in the UK.
Although assay design is continually improving, no routine immunoassay is currently robust to interference. Technical errors with many routine chemistry methods caused by inappropriate sample collection or handling, chemical or spectral interference can be detected during result validation. However, detection of spurious TFT immunoassay results is more challenging as there is no automatic ‘flag’ from the analyser, and there is usually a wide range of plausible values for these analytes, making it difficult to question those which are ‘suspicious’. Consequently clinical validation, where results are checked for discordance with the clinical correlates and other laboratory tests, is used to detect potentially incorrect results before reporting. For TFTs this is aided by the characteristic reciprocal relationship between TSH and fT4 in patients with an intact pituitary–thyroid axis.
Mechanisms of interference in TSH assays
Endogenous interfering antibodies are a well described cause of immunoassay interference [4]. In TSH assays these antibodies can have affinity for TSH itself or towards assay components. Anti-reagent antibodies can be ‘anti-animal’ antibodies, specific to the species in which the reagent antibody was raised, or weak, polyspecific ‘heterophilic’ antibodies, which may be part of the natural process of the generation of antibody diversity [5]. Anti-animal antibodies are more prevalent in animal handlers or patients treated with therapeutics based on animal immunoglobulins.
Anti-reagent antibodies can interact with either the capture or detection antibodies in two-site assays, blocking the generation of signal in the presence of analyte (false negative result) or by causing antibody cross-linking in the absence of analyte (false positive result) [Fig. 1].
Anti-TSH antibodies can generate high molecular weight TSH : antibody complexes (‘macro-TSH’). Depending on the exact site of the antibody–analyte interaction, false positive TSH results may occur as the macro-TSH is unlikely to be biologically active [6].
Detection of interference in TSH assays
Once suspected, a robust laboratory strategy is required for confirming or excluding assay interference. Method comparison using an alternative method is often used as the first step. Most laboratories use two-site immunoassays for TSH, but assay formulations, antibody species and incubation times vary between manufacturers. Varying amounts of blocking agents, designed to prevent non-specific binding of heterophile antibodies, may be included. Significant disagreement between two TSH methods is a strong indicator of assay interference.
Dilution studies are a simple but effective tool to investigate the analytical validity of an immunoassay. Non-linearity to dilution suggests a result is unreliable. However, although a good ‘rule in’ test, linearity to dilution alone cannot be used to exclude interference [3,7].
Immunosubtraction is a useful method to confirm the presence of antibody interference. This can be done crudely using polyethylene glycol (PEG) precipitation or more specifically using anti-immunoglobulin agaroses. Proprietary heterophile blocking tubes can also be used to confirm the presence of this class of interferent [3,4].
Once assay interference is established it can still be difficult to determine the correct TSH value, as there is no ‘gold standard’ method for TSH. However, an alternative immunoassay result which gives the expected responses to dilution and immunosubtraction, and correlates with fT4 results plus clinical findings, can be used with a reasonable degree of confidence.
Mechanisms of interference in fT4 assays
fT4 assays present a particular analytical challenge as >99.9% of T4 in the serum is protein bound, and the unbound T4 fraction must be measured without upsetting the equilibrium between the two fractions [8]. Therefore, an abnormal T4 binding protein, or agent which affects binding protein affinity in vitro, has the potential to generate incorrect results. Most commercial fT4 assays are one-site immunoassays based on competitive principles, using either labelled T4 analogue or anti-T4 antibodies for detection. Both heterophile and anti-T4 antibodies therefore also have the potential to interfere with these methods [4].
Non-esterified fatty acids (NEFAs) are a common T4 displacing agent as they can release T4 from the low affinity, high capacity T4 binding site on albumin. NEFAs can be generated in vitro, usually as a consequence of heparin therapy, which stimulates the action of lipoprotein lipase on triglyceride. Although the measured fT4 result is genuinely high, it does not reflect the in vivo situation [9].
Familial dysalbuminaemic hyperthyroxinaemia (FDH) is a benign genetic condition where the affinity of albumin for T4 is increased, such that circulating albumin-bound T4 is elevated. Despite the high total T4 (tT4), concentrations of free hormone in vivo are unaffected due to the homeostatic regulation of the thyroid axis. However, FDH is often associated with falsely high fT4 measurements using commercial immunoassays [10] [Fig. 2]. Both the increased affinity of the variant albumin for some labelled T4 analogues, as well as potential disruption of the T4 : albumin equilibrium during the assay, are likely mechanisms. The presence of the FDH mutation can be confirmed using molecular genetic approaches.
Detecting interference in fT4 assays
Despite the greater analytical challenge, confirming interference in fT4 assays can be easier than for TSH due to the availability of physical separation methods, such as equilibrium dialysis, as ‘gold standard’ assays [8]. However, these methods are technically difficult and not available in most clinical biochemistry laboratories. Also, these methods do not solve the in vitro problems of hormone displacement.
Again a first approach is often method comparison, using a different immunoassay architecture. Dilution and immunosubtraction studies can also be informative, although some fT4 methods are not robust to matrix effects so careful control experiments are required.
Measurement of total rather than free T4 can be useful in situations where there is a suspicion of abnormal T4 binding proteins. For example, total T4 will be elevated in the presence of anti-T4 antibodies and in FDH.
Clinical causes of aberrant TFTs
As mentioned above there are well described pharmacological and pathological causes of unusual TFTs; an increased awareness of analytical artefacts should not detract from the detection of these conditions. For example thyroxine treatment, a TSH secreting pituitary tumour (TSHoma), the genetic condition thyroid hormone resistance, FDH or TFT antibody interference can give elevated fT4 results with a TSH within the reference interval. Attempts by the laboratory to exclude assay interference should complement both the diagnosis of transient and genetic thyroid conditions as well as the more common drug related effects.
Conclusions and future directions
Immunoassay manufacturers have invested considerable resources into reducing the potential for antibody-mediated assay interference, for example by including blocking agents, or using antibody fragments rather than intact antibodies as assay reagents. Although these measures are effective, it is worth bearing in mind that changes to assay formulations may introduce novel types of interference. We have observed negative interference in one fT4 assay which appears related to the presence of a blocking agent introduced to reduce the risk of positive interference in this method [11]. Mass spectrometric methods have been introduced to eliminate antibody interference in both fT4 and tT4 methods, but unfortunately the fT4 methods still require careful optimization to avoid interference caused by binding proteins and displacing agents.
As current TFT methods remain prone to analytical interference the clinical laboratory must remain vigilant to the potential for assay interference, promote effective communication with requesting clinicians, and have procedures in place for investigation of discordant results.
References
1. Association for Clinical Biochemistry (ACB), British Thyroid Association (BTA), British Thyroid Foundation (BTF). UK guidelines for the use of thyroid function tests.2006; www.acb.org.uk/docs/TFTguidelinefinal.pdf.
2. Gurnell M, Halsall DJ, Chatterjee VK. What should be done when thyroid function tests do not make sense? Clin Endocrinol. (Oxf) 2011; 74(6): 673–678.
3. Ismail AA, Walker PL, Barth JH, Lewandowski KC, Jones R, Burr WA. Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid-stimulating hormone and gonadotropin immunoassay results. Clin Chem. 2002; 48(11): 2023–2029.
4. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998; 44: 440–454.
5. 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.
6. Halsall DJ, Fahie-Wilson MN, Hall SK, Barker P, Anderson J, Gama R, Chatterjee VK. Macro thyrotropin-IgG complex causes factitious increases in thyroid-stimulating hormone screening tests in a neonate and mother. Clin Chem. 2006; 52: 1968–1969.
7. Ross HA, Menheere PP, Thomas CM, Mudde AH, Kouwenberg M, Wolffenbuttel BH. Interference from heterophilic antibodies in seven current TSH assays. Ann Clin Biochem. 2008; 45: 616.
8. Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best Pract Res Clin Endocrinol Metab. 2013; in press.
9. Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab. 2009; 23(6): 753–767.
10. Cartwright D, O’Shea P, Rajanayagam O, Agostini M, Barker P, Moran C, Macchia E, Pinchera A, John R, Agha A, Ross HA, Chatterjee VK, Halsall DJ. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009; 55(5): 1044–1046.
11. Bacon O, Gillespie S, Koulouri O, Bradbury S, O’Toole A, Stuart-Thompson D, Taylor K, Pearce S, Gurnell M, Halsall DJ. A patient with multiple Roche serum immunoassay interferences including false negative serum fT4. Ann Clin Biochem. 2013; 50(Suppl 1): T50.
The authors
Olivia Bacon PhD and David Halsall* PhD, FRCPath, CSci
Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, Cambridge, UK
*Corresponding author
E-mail: djh44@cam.ac.uk
Sleeping sickness elimination: are we dreaming?
, /in Featured Articles /by 3wmediaRecent sleeping sickness epidemics killed over 400,000 people in less than 20 sub-Saharan African countries. Serological screening of populations at risk and treatment of confirmed patients have drastically reduced the annually reported cases. Elimination seems feasible but only with new control tools and strategies adapted to the new epidemiological situation.
by Dr Philippe Büscher, Quentin Gilleman and Dr Pascal Mertens
Sleeping sickness, also called human African trypanosomiasis (HAT), is caused by two subspecies of the protozoan parasite Trypanosoma brucei (T.b.). The disease is transmitted by blood sucking tsetse flies that only occur in sub-Saharan Africa. T.b. gambiense causes a rather chronic disease and is found in West and Central Africa. T.b. rhodesiense causes a more fulminant form of the disease in Eastern Africa. Other Trypanosoma species cause diseases in animals, including cattle and small ruminants [Figure 1].
Infection and pathology
After inoculation with the saliva of an infective tsetse fly, the parasites invade lymph, blood and all peripheral organs where they multiply and survive the immune response of the host by a biological mechanism called antigenic variation. Eventually, the parasites invade the brain causing intrathecal inflammation associated with neurological disorders such as altered sleep-wake rhythm, behavioural changes, motor disabilities etc.
Except for some very rare cases, the disease is always lethal and even after successful treatment, many patients, especially children, never recover completely and remain disabled for the rest of their life. Sleeping sickness is a rural disease affecting poor populations living in the forests and wooded savannah where tsetse flies breed. Today, T.b. rhodesiense is mainly found in wild animals in game parks and natural reserves where it is often transmitted to rangers and visiting tourists.
Epidemiological background
At the turn of the 20th century, both gambiense and rhodesiense sleeping sickness caused devastating epidemics killing about one million people within two decades. By sustained implementation of vector control (including habitat destruction and insecticide spraying), culling of wild animals, and systematic screening of the population and treatment of patients by specialized teams, the colonial governments gained control over the epidemics and reduced the annual number of cases to less than 5000 cases around 1960. However, around 1990, a new epidemic of gambiense HAT was rampant in many countries with several tens of thousands of annually reported patients [1]. Countries most affected were typically poor and socio-politically unstable such as Angola, Central African Republic, D.R. of the Congo, Rep. of Congo, Sudan and Uganda to name a few.
Current situation
Today, about 20 years later, the number of reported cases has fallen again to about 7000 in 2012 of which 85% were diagnosed and treated in one single country, the D.R. of the Congo [2]. This achievement was made possible by a combination of different factors among which the availability of performing diagnostic tests and effective treatment, the recognition of sleeping sickness as Neglected Tropical Disease (NTD), thus attracting attention by donor agencies, humanitarian organizations and the private sector, and the combined effort of the World Health Organization, national HAT control programmes, bilateral cooperations and Non Governmental Organizations to organize large scale active case finding in the affected regions.
Active case finding is typically done by mobile teams that consist of up to seven persons trained in diagnosis and treatment of HAT. They go out in the field for several weeks, carrying all necessary equipment, diagnostics and drugs to screen the population at risk with a serological antibody detection test, to examine seropositive suspects by microscopy and to treat parasitologically confirmed patients in their villages or to refer them to the nearest specialized treatment centre. Since more than 20 years now, the recommended screening test is the Card Agglutination Test for Trypanosomiasis (CATT), a rapid test that detects gambiense specific antibodies [3].
Neglected Tropical Disease (NTD)
The recent success in HAT control has led to the inclusion of gambiense HAT in the WHO’s list of NTD’s that could be eliminated as a public health problem in Africa by 2020 with zero transmission in 2030 [2]. However, with the currently available tools for HAT control, elimination may remain an elusive target. Indeed, eradication of the tsetse flies, although proven to be feasible in some isolated foci with only one species transmitting trypanosomes, probably will never be achieved in endemic countries with dense forests and with large protected zones. As a consequence, tsetse flies will continue to transmit the disease, not only from man to man but also from the domestic and wild animal reservoir to man.
Diagnosis and treatment of infection
Today, treatment of sleeping sickness patients relies on toxic drugs and most often requires several weeks of hospitalization. Therefore, treatment is
administered only to patients in which the parasites have been detected in the blood, lymph or cerebrospinal fluid. Given that even the most sensitive parasite detection tests remain negative in 10% to 20% of actually infected patients, untreated patients may continue to act as a parasite reservoir, sometimes for years before they are treated or die. With the venue of molecular diagnostics, it was believed that such tests would sooner or later replace microscopic parasite detection. However, HAT patients have to be diagnosed in rural environments that are not compatible with today’s DNA- or RNA-based diagnostics and molecular test do not perform better than parasitology. Therefore, it is questionable if the individual patient will ever benefit from molecular diagnostics for sleeping sickness [4].
New control tools
Should we then despair about sleeping sickness elimination? Not at all, at least not for gambiense HAT. History shows that in countries that are socio-politically stable, where the rural population has access to functional primary healthcare facilities and where changing land use has suppressed the tsetse fly population, sleeping sickness has disappeared as is the case in Benin, Burkina Faso, Ghana and Togo [5]. For countries where these conditions cannot be met in the near future, newly developed HAT control tools may play a major role in disease elimination. For example, GIS technology allows to combine the GPS coordinates of all villages where HAT patients are reported with demographic and environmental data, and to precisely map the populations at risk [6].
New rapid test
Also, a new rapid diagnostic test for gambiense HAT serodiagnosis has been developed (HAT Sero-K-SeT, Coris BioConcept, Belgium). The HAT Sero-K-SeT is individually packed, thermostable, equipment-free , robust and has shown excellent diagnostic performance in a phase I evaluation [7]. Its target product profile, and especially its very high specificity, makes it fully compatible for use in foci with very low prevalence and in fixed health centres with minimal infrastructure [Figure 2]. In addition, strategies involving newly developed small size (0,25 x 0,25 m) insecticide-treated targets to kill the riverine tsetse fly are more cost effective than former models [8].
New drugs in the pipeline
Finally, the search for new drugs has identified a new class of compounds of which one, the SCYX-7158 has been selected for the development of a safe, one-dose oral treatment of both stages of sleeping sickness [9]. Once such a drug becomes available, parasite detection and stage determination that can only be accurately performed by expert medical staff, may become dispensable and decision to treat might be taken on the serodiagnostic evidence of infection.
Conclusion
Elimination of at least one form of sleeping sickness seems possible but only with the long-term commitment of donor agencies and ministries of health in endemic countries and with the cost efficient deployment of the newly developed control tools in rationally designed elimination strategies adapted to the local epidemiological situation.
References
1. World Health Organization. Control and surveillance of African trypanosomiasis. WHO Technical Report Series 1998; 881: 1-113.
2. World Health Organization. Report of a WHO meeting on elimination of African trypanosomiasis (Trypanosoma brucei gambiense), 3-5 December 2012, Geneva, Switzerland. WHO/HTM/NTD/IDM 2013.4 http://apps.who.int/iris/bitstream/10665/79689/1/WHO_HTM_NTD_IDM_2013.4_eng.pdf (accessed 27 May 2013)
3. Chappuis F, Loutan L, Simarro P, Lejon V and Büscher P. Options for the field diagnosis of human African trypanosomiasis. Clinical Microbiology Reviews 2005; 18: 133-146.
4. Deborggraeve S and Büscher P. Molecular diagnostics for sleeping sickness: where’s the benefit for the patient? The Lancet Infectious Diseases 2010; 10: 433-439.
5. Simarro PP, Diarra A, Ruiz Postigo JA, Franco JR, and Jannin JG. The human african trypanosomiasis control and surveillance programme of the world health organization 2000-2009: the way forward. PLoS Neglected Tropical Diseases 2011; 5: e1007.
6. Simarro PP, Cecchi G, Franco JR et al. Estimating and mapping the population at risk of sleeping sickness. PLoS Neglected Tropical Diseases 2012; 6: e1859.
7. Büscher P, Gilleman Q and Lejon V. Novel rapid diagnostic tests for sleeping sickness. New England Journal of Medicine 2013; 368: 1069-1070.
8. Esterhuizen J, Rayaisse JB, Tirados I et al. Improving the cost-effectiveness of visual devices for the control of riverine tsetse flies, the major vectors of human African trypanosomiasis 3. PLoS Neglected Tropical Diseases 2011; 5: e1257.
9. Jacobs RT, Nare B, Wring SA et al. SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis. PLoS Neglected Tropical Diseases 2011; 5: e1151.
The authors
Philippe Büscher1* PhD, Quentin Gilleman2 MSc, and Pascal Mertens2 PhD
1 Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-2000 Antwerp, Belgium
2 Coris BioConcept, Crealys Park, Rue Jean Sonet 4a, B-5032 Gembloux, Belgium
*Corresponding author
E-mail: pbuscher@itg.be
Tel. +32 3247 6371
Women and heart disease
, /in Featured Articles /by 3wmediaCardiovascular disease (CVD) is still widely considered as a middle-aged man’s disease and this is clearly a misconception. In actual fact, CVD is the number one cause of death for women worldwide. Also, compared with men, women have a number of additional risk factors that are specific to them and should not be ignored by medical professionals. Laboratory testing has a key role to play in the diagnosis and follow up of women with CVD. CLI talked to Jean Onofrio, Senior Director, Global Assay Marketing, Siemens Healthcare Diagnostics, about this important health issue for women.
Q.What impact does cardiovascular disease have on women?
Cardiovascular disease, or CVD, is a significant health concern for women. In fact, it’s the number one killer of women globally, [1] and according to the World Health Organization (WHO), accounts for one-third of deaths in women.
CVD also is the main cause of death for older women. Women generally develop CVD about 10 years later in life than men, likely due to the protective, anti-oxidant effects of estrogen prior to menopause.
Unfortunately, the misperception that CVD is a middle-aged man’s disease still persists. Understanding CVD’s global impact on women is one positive step toward battling the disease.
Q. What are the risk factors for CVD in women? How do these compare to risk factors in men?
While many CVD risk factors, such as age, family history and high blood pressure, are similar in both genders, there are some, including diabetes, tobacco use and high triglyceride levels, that put women at higher risk. Other risk factors, like obesity and depression, are more prevalent in women. There are also some risk factors unique to women, including pregnancy complications, oral contraceptive use, hormone replacement therapy and polycystic ovary syndrome. It’s important for women to understand their CVD risk factors and discuss their concerns with their physician.
Q. How does the mortality rate of women with CVD compare to the mortality rate of CVD in men?
While the mortality rate is high for older women, a heart attack can occur at any age. For younger women, heart attacks are actually more deadly than for men. According to the American Heart Association (AHA), among adults aged 45-62, women are twice as likely as men to die within the first year after a heart attack.
Also, more than twice as many women will develop heart failure within five years of surviving a heart attack compared to men, and three times more women than men will suffer a stroke after surviving a heart attack.
Q. What are some of the challenges associated with diagnosing CVD in women?
Women having a heart attack commonly present with symptoms other than chest pain, which makes diagnosis challenging. Rather, women often experience such less common symptoms as fatigue, indigestion, appetite loss and
“heart flutters.”
Even though these symptoms may not be severe, they may still lead to deadly consequences. Unfortunately, many women, and often clinicians, disregard their symptoms, attributing them to other non-life-threatening conditions.
Adding to this challenge, women with CVD aren’t as likely as men to receive aggressive diagnosis and treatment. Consider that women receive only about 34 percent of interventional treatments, with and witout the placements of stents.
Q. What role does laboratory testing play in the diagnosis and management of women with CVD? What about biomarkers?
CVD is largely preventable, and simple laboratory tests can help assess a person’s risk.
Laboratory professionals play an increasingly important role in providing access to both traditional and novel cardiac biomarkers that are available throughout the disease continuum. Also, whether conducted in the central lab or at the point-of-care, cardiac tests, such as high-sensitivity troponin, are key diagnosis tools.
By leveraging the appropriate use of laboratory diagnostic testing, clinicians can help enhance the assessment, diagnosis and follow-up care for women with CVD.
Reference
1.http://gamapserver.who.int/gho/interactive_charts/women_and_health/causes_death/ chart.html; accessed 11/27/12
Use of global hemostatic markers for risk stratification and personalized treatment of coronary artery disease
, /in Featured Articles /by 3wmediaCoronary artery disease has been linked to a hypercoagulable state of the blood, and the use of global hemostatic assays such as thromboelastography, thrombin generation or the overall hemostatic assay may allow for prediction of adverse events in these patients as well as targeted, individualized treatment.
by Dr C. Reddel, Dr J. Curnow and Professor D. Brieger
Global hemostatic markers in coronary artery disease
Hemostasis is the process by which bleeding is stopped, involving blood coagulation and platelet aggregation. This process depends on the delicate balance of many pro- and anti-coagulant factors, and when hemostatic balance is disrupted, pathological clot formation may occur leading to potentially fatal venous or arterial thrombosis. Appropriate fibrinolysis, the breakdown of blood clots, is also essential to the process of hemostasis.
Coronary artery disease is considered an inflammatory disease in which patients are predisposed to arterial thrombosis, which can lead to myocardial infarction. Additionally, the presence of coronary artery disease can increase the risk of venous thrombosis [1]. This points to an overall hypercoagulable state of the blood in this disease. Although the use of antiplatelet and anticoagulant therapies is a common and necessary method of reducing this risk, this may unnecessarily expose patients to a risk of bleeding. There is a need to risk stratify patients and individually tailor thromboprophylaxis.
Imbalances in the hemostatic system can be assessed in citrated plasma samples from patients either by measuring individual coagulation and fibrinolytic factors, or by global coagulation assays. Such imbalances have been found to be associated with various pro-thrombotic states, such as cancer, pregnancy or trauma. In stable and acute coronary artery disease, there is evidence for links between prognosis and markers of coagulation and fibrinolysis, including prothrombin fragment 1+2, fibrinopeptide A, thrombin–antithrombin and plasmin–antiplasmin complexes, D-dimer, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator [2, 3]. However, measuring single factors does not reflect the overall hemostatic balance as other pro- or anti-coagulant, and pro- and anti-fibrinolytic factors may compensate for the deficient or elevated factor. Therefore measurement of the overall coagulable state of the blood may provide a more relevant picture.
Standard laboratory coagulation tests, such as prothrombin time (PT) or activated partial thromboplastin time (APTT), can be useful for patients with bleeding disorders, but do not reliably detect hypercoagulability in this context. Recently, there has been interest in global assays of coagulation and fibrinolysis as methods of assessing the overall potential of a patient’s blood to form or lyse a clot. These include assays of thrombin generation, thromboelastography and the overall hemostatic potential assay.
Thromboelastography
Thromboelastography is a method measuring clot formation and lysis in whole blood. A pin is suspended into a cuvette of whole blood heated to 37°C, and the cup and pin move relative to each other, so that when the clot forms the interference is detected by the pin. Thromboelastography (TEG, Haemonetics, Braintree, Massachusetts, USA) and Thromboelastometry (ROTEM, Tem International GmbH, Munich, Germany) are two commercial variants of the assay. The assay measures not only time to clot, but speed of clot formation, clot strength and elasticity, and can be modified to assess platelet function, fibrinogen, hyperfibrinolysis and effect of anticoagulant treatment. The use of whole blood means the role of the cell is incorporated into the assay, although this necessitates immediate use of the sample.
Thromboelastography is a point-of-care assay which is used to measure and characterize peri-operative bleeding. It may additionally be useful in monitoring antiplatelet therapy such as aspirin or clopidogrel. Recently, it has also been used to detect hypercoagulability in patients with coronary artery disease, and further, has been demonstrated to predict thrombotic events in patients who have undergone coronary stenting or coronary artery bypass grafting [4, 5].
Thrombin generation assay
The thrombin generation assay was first described in 1953, but has more recently been simplified, standardized and commercialized, including in the form of the Calibrated Automated Thrombogram (Thrombinoscope BV, Maastricht, The Netherlands) and Technothrombin (TGA, Technoclone, Vienna, Austria) [6]. In this assay, ex vivo potential for thrombin generation is measured in platelet-rich or platelet-poor plasma. In a 96-well plate, thrombin generation is triggered by addition of tissue factor, phospholipids and calcium at 37°C, and conversion of a substrate for thrombin measured over an hour by fluorescence.
Thrombin is central to the process of hemostasis, and various pro-thrombotic states have been associated with variations in plasma potential to generate thrombin. Patients with stable coronary artery disease have elevated thrombin generation [Fig. 1] [7], and patients with acute coronary syndrome have still higher thrombin potential [8]. Antiplatelet therapies most likely do not affect the thrombin generation assay in platelet-poor plasma, but it may be possible to monitor the effect of anticoagulant drugs (including novel oral anticoagulants) using the assay, and preliminary assessment has suggested the assay can predict bleeding and ischemic events in patients with coronary artery disease [9].
Overall Hemostatic Potential (OHP) assay
The Overall Hemostatic Potential (OHP) assay is a test of fibrin generation and fibrinolysis first described in 1999 [10]. Similar to the thrombin generation assay, it is performed in citrated plasma in 96-well plates and triggered by tissue factor or thrombin and calcium at 37°C. It is a turbidometric assay, measuring the change in absorbance over an hour at 405nm, which allows for a kinetic analysis of fibrin clot formation. Tissue plasminogen activator is also added to half the wells, which triggers fibrinolysis. The assay measures coagulation potential and fibrinolytic potential, and is carried out on stored plasma samples.
A limitation of the plasma-based thrombin generation and OHP assays is the absence of cells. These assays have nonetheless identified differences between patients with pro-thrombotic states and healthy controls, and the use of plasma allows for samples to be stored and batch-tested, which is an advantage for screening large numbers of patients. The OHP assay additionally requires no specialized equipment, apart from a standard plate reader, and although not standardized, it is inexpensive. Unlike thromboelastography which is relatively insensitive to hypofibrinolysis, the OHP assay can detect and quantify hypofibrinolysis as well as hyperfibrinolysis.
Very recently the OHP assay has been used to show hypercoagulability and hypofibrinolysis in patients with acute and stable coronary artery disease [Fig. 2] [7, 11]. The observations in this latter population suggest the potential for this assay to predict future events, and prospective studies are required to determine its utility in this context.
Future trends and requirements
There is a growing body of evidence that ex vivo hypercoagulability of patients’ blood or plasma has prognostic value in arterial or venous thrombotic events. Global markers of hemostasis, including results of thromboelastography, the thrombin generation and OHP assays, may prove clinically relevant in identifying individual patients at risk of adverse event, and thus allow the tailoring of thromboprophylaxis. Further large-scale prospective trials are needed to directly address this.
References
1. Anandasundaram B, Lane DA, Apostolakis S, Lip GY. The impact of atherosclerotic vascular disease in predicting a stroke, thromboembolism and mortality in atrial fibrillation patients: a systematic review. J Thromb Haemost. 2013; 11: 975–987.
2. Stegnar M, Vene N, Bozic M. Do haemostasis activation markers that predict cardiovascular disease exist? Pathophysiol Haemost Thromb. 2003; 33: 302–308.
3. Gorog DA. Prognostic value of plasma fibrinolysis activation markers in cardiovascular disease. J Am Coll Cardiol. 2010; 55:2 701–709.
4. Hobson AR, Agarwala RA, Swallow RA, Dawkins KD, Curzen NP. Thrombelastography: current clinical applications and its potential role in interventional cardiology. Platelets 2006; 17: 509–518.
5. McCrath DJ, Cerboni E, Frumento RJ, Hirsh AL, Bennett-Guerrero E. Thromboelastography maximum amplitude predicts postoperative thrombotic complications including myocardial infarction. Anesth Analg. 2005; 100: 1576–1583.
6. Hemker HC, Giesen P, AlDieri R, Regnault V, de Smed E, Wagenvoord R, et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002; 32: 249–253.
7. Reddel CJ, Curnow JL, Voitl J, Rosenov A, Pennings GJ, Morel-Kopp MC, et al. Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay. Thromb Res. 2013; 131: 457–462.
8. Orbe J, Zudaire M, Serrano R, Coma-Canella I, Martinez de Sizarrondo S, Rodriguez JA, et al. Increased thrombin generation after acute versus chronic coronary disease as assessed by the thrombin generation test. Thromb Haemost. 2008; 99: 382–327.
9. Campo G, Pavasini R, Pollina A, Fileti L, Marchesini J, Tebaldi M, et al. Thrombin generation assay: a new tool to predict and optimize clinical outcome in cardiovascular patients? Blood Coag Fibrinolysis 2012; 23: 680-687.
10. He S, Bremme K, Blomback M. A laboratory method for determination of overall haemostatic potential in plasma. I. Method design and preliminary results. Thromb Res. 1999; 96: 145–156.
11. Leander K, Blomback M, Wallen H, He S. Impaired fibrinolytic capacity and increased fibrin formation associate with myocardial infarction. Thromb Haemost. 2012; 107: 1092–1099.
The authors
Caroline Reddel* PhD; Jennifer Curnow MBBS, PhD, FRACP, FRCPA; David Brieger MBBS, PhD, FRACP, FACC
ANZAC Research Institute, Concord Repatriation General Hospital, Concord NSW, 2139, Australia
*Corresponding author
E-mail: creddel@anzac.edu.au
YKL-40: a new prognostic biomarker in patients with coronary artery disease
, /in Featured Articles /by 3wmediaInflammation is of importance for the progression of coronary artery disease. Until now, there has been no biomarker to monitor the effect of treatment regimes. YKL-40 is a new biomarker of inflammation, which if highly elevated in the disease, is a strong prognostic predictor of death and potentially can be used to monitor disease activity.
by Prof. J. Kastrup, Dr M. Harutyunyan-Bønsager and Dr N. D. Mygind
Clinical background
The number of patients with coronary artery disease (CAD) is increasing worldwide, and CAD is the most common cause of death in western countries. Although the prognosis and quality of life for patients has improved due to more aggressive and invasive treatment regimes, in the US someone will have a coronary event approximately every 25 seconds, and someone will die of one approximately every minute. Therefore CAD is an increasing economic burden and the total estimated direct and indirect costs of CAD in the US in 2010 were $503.2 billion [1].
Currently, there is a lack of new biomarkers for monitoring the effect of the patients’ treatment and for predicting their risk of a heart attack, heart failure and cardiac death.
Coronary artery disease and inflammation
It has been well established that inflammation plays an important role in development and progression of atherosclerosis in the coronary arteries [2]. Moreover, inflammation is also involved in the inflammatory pathways inducing extracellular matrix remodelling and heart failure progression [3]. The inflammatory biomarker high-sensitivity C-reactive protein (hs-CRP) is associated with atherosclerosis and the incidence of coronary events [4], but its association with the extent and severity of atherosclerosis remains controversial. Therefore, it is not very useful for continuous monitoring of treatment effects and progression of the disease.
The inflammatory biomarker YKL-40
YKL-40 is a glycoprotein mainly produced by macrophages and neutrophils, which are important for the development of atherosclerosis, and is stimulated by hypoxia [5]. Serum YKL-40 is suggested to be a biomarker of diseases characterized by inflammation [5] and its plasma concentration has been shown to increase reversibly in patients by more than 25% following an inflammatory stimulus.
YKL-40 is not a disease specific biomarker, but plays a role in cell migration and adhesion, angiogenesis, remodelling of the extracellular matrix, cell proliferation and differentiation [5]. Macrophages in atherosclerotic plaques, especially those located more deeply in the atherosclerotic lesion, express YKL-40 [6], and macrophages in early atherosclerotic lesions express the highest amount of YKL-40 mRNA. As Hs-CRP is mainly produced in the liver, it is likely that biomarkers such as YKL-40 (secreted from inflammatory cells within the atherosclerotic plaque) could be superior for monitoring CAD.
YKL-40 in healthy subjects
The normal YKL-40 value in a healthy subject from the general population has recently been published [7]. In 3130 subjects the median YKL-40 value was 40 µg/L and increased exponentially with age.
YKL-40 in coronary artery disease
Serum YKL-40 has been found to be increased in both acute and coronary artery disease [8]. Serum YKL-40 levels were also significantly increased in patients with acute ST-elevation myocardial infarction and thereafter consistently decreased from a maximum value just after the myocardial infarction and during a 360 day follow-up period towards its normal levels. Plasma YKL-40 levels were found to correlate inversely with left ventricular ejection fraction (LVEF) recovery, but not with infarct size in patients with STEMI [9, 10].
Although highly increased in patients with stable CAD, it has not been possible to detect any relationship between serum YKL-40 level and the degree of CAD as evaluated by the number of vessels involved or the degree of artery stenosis [11]. In patients with stable CAD, revascularization with balloon angioplasty of significant stable coronary artery lesions has no effect on YKL-40 levels within a 6 month follow-up period (unpublished data).
This indicates that YKL-40 not is a measurement of the amount of ischemia within the myocardium. Serum YKL-40 seems to be more a measurement of ongoing inflammatory activity rather than the presence of stabilized chronic lesions.
Therefore, it is very interesting that serum YKL-40 was a very strong prognostic biomarker for death within a 2.6 and 6 year follow-up period in patients with stable CAD [12, 13] [Fig. 1].
YKL-40 and heart failure
The consequence of CAD is often the development of severe heart failure. It has recently been demonstrated that serum YKL-40 is increased in heart failure and that YKL-40 is an independent significant prognostic biomarker for death [15]. It is interesting that serum YKL-40 measured in all-comers at acute hospital admission is a very strong predictor of death, especially within the first year, in patients with heart disease [16]. Of patients admitted with disease of the heart, those with elevated YKL-40 had a hazard ratio of death within the first year after discharge from the hospital at 2.5 compared to heart patients with normal serum YKL-40 levels. YKL-40 remained an independent biomarker of mortality, even after adjusting for other known risk factors such as age, hs-CRP and NT-proBNP [16].
YKL-40 for monitoring CAD activity
Statin treatment is used in CAD for lowering cholesterol levels. However, it also has an anti-inflammatory action. Therefore, it is very interesting that serum YKL-40 is significantly lower in patients with stable CAD on statin treatment compared to patients without [14] [Fig. 2].
This difference seems to be independent of the effect that statins have on lowering cholesterol levels, indicating that the YKL-40 level can be regulated by the direct anti-inflammatory action of statins [14]. This is unlike the situation with the inflammatory biomarker hs-CRP, which has been shown to correlate to cholesterol levels in statin-treated CAD patients [14].
Moreover, the mortality is also lower in stable CAD on statins compared to non-statins [12, 13]. This indicates that YKL-40 could be used to monitor the anti-inflammatory effect of statin treatment. Whether YKL-40 is also useful for
monitoring the effects of other anti-angina medications remains to be investigated.
Conclusion and future perspective
YKL-40 is a new inflammatory biomarker in ischemic heart disease. It is increased in both acute and chronic coronary artery disease and is a very strong diagnostic biomarker for death. It is suggested to be a mirror of the active inflammatory atherosclerotic processes in CAD, more than a measurement of degree of myocardial ischemia induced by stable coronary lesions. Since YKL-40 is lower in patients on statin treatment, it can potentially be used to monitor disease activity and the effect of anti-inflammatory or stabilizing treatment regimes.
Conflict of interest
A patent application (WO 2009/092382) is published and pending.
References
1. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, et al. Circulation 2012; 125(1): e2–e220.
2. Hansson GK. J Thromb Haemost 2009; 7 Suppl 1: 328–331.
3. Radauceanu A, Ducki C, Virion JM, Rossignol P, Mallat Z, McMurray J, et al. J Card Fail 2008; 14(6): 467–474.
4. Corrado E, Rizzo M, Coppola G, Fattouch K, Novo G, Marturana I, et al. J Atheroscler Thromb 2010; 17(1): 1–11.
5. Kastrup J. Immunobiology 2012; 217(5): 483–491.
6. Boot RG, van Achterberg TA, van Aken BE, Renkema GH, Jacobs MJ, Aerts JM, et al. Arterioscler Thromb Vasc Biol 1999; 19(3): 687–694.
7. Bojesen SE, Johansen JS, Nordestgaard BG. Clin Chim Acta 2011; 412: 709–712.
8. Wang Y, Ripa RS, Johansen JS, Gabrielsen A, Steinbruchel DA, Friis T, et al. Scand Cardiovasc J 2008; 42(5): 295–302.
9. Nojgaard C, Host NB, Christensen IJ, Poulsen SH, Egstrup K, Price PA, et al. Coron Artery Dis 2008; 19(4): 257–263.
10. Hedegaard A, Ripa RS, Johansen JS, Jorgensen E, Kastrup J. Scand J Clin Lab Invest 2010; 70(2): 80–86.
11. Mathiasen AB, Harutyunyan MJ, Jorgensen E, Helqvist S, Ripa R, Gotze JP, et al. Scand J Clin Lab Invest 2011; 71(5): 439–447.
12. Kastrup J, Johansen JS, Winkel P, Hansen JF, Hildebrandt P, Jensen GB, et al. Eur Heart J 2009; 30(9): 1066–1072.
13. Harutyunyan M, Gotze JP, Winkel P, Johansen JS, Hansen JF, Jensen GB, Hilden J, Kjøller E, Kolmos HJ, Gluud C, Kastrup J. Immunobiology 2013; 218(7): 945–951.
14. Mygind ND, Harutyunyan MJ, Mathiasen AB, Ripa RS, Thune JJ, Gotze JP, et al. Inflamm Res 2011; 60(3): 281–287.
15. Harutyunyan M, Christiansen M, Johansen JS, Køber L, Torp-Petersen C, Kastrup J. Immunobiology. 2012; 217(6): 652–656.
16. Mygind ND, Iversen K, Køber L, Goetze JP, Nielsen H, Boesgaard S, Bay M, Johansen JS, Nielsen OW, Kirk V, Kastrup J. J Intern Med 2013; 273(2): 205–216.
The authors
Jens Kastrup* MD, DMSc; Marina Harutyunyan-Bønsager MD; and Naja Dam Mygind MD
Department of Cardiology B, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark
*Corresponding author
E-mail: jens.kastrup@regionh.dk
A breakthrough in timely ovarian cancer diagnosis?
, /in Featured Articles /by 3wmediaWhile globally ovarian cancer is the eighth most common cancer in women, in the developed countries (with the exception of Japan) the disease is much more prevalent. In Europe it is the fifth most frequently diagnosed cancer in women, with an average lifetime risk of 1 in 70, and in both Europe and North America the disease accounts for over 5% of all female cancer deaths. In addition, unlike with most other cancers, the five year survival rate of only 45% has barely improved in the last 30 years. This poor prognosis is largely due to the non-specific symptoms, resulting in diagnosis at Stage III or IV when the tumour has already metastasized. But if ovarian cancer is diagnosed early, the five year survival rate exceeds 90%.
Much work in recent decades has concentrated on finding a simple screening method that would allow more timely diagnosis; so far none has had a significant effect on mortality. An assay for the most frequently used biomarker, CA125, was developed around 30 years ago. Normally elevated in the serum of patients diagnosed with symptomatic ovarian cancer, CA125 is ideal in disease management, but its use to enable early disease detection has remained controversial. Specificity is very limited as the serum level is raised in several benign conditions (such as endometriosis) as well as in other cancers. In addition sensitivity is only about 50% in patients with Stage I or II disease. More recently human epididymis protein 4 (HE4) has been advocated as a useful marker for ovarian cancer detection. Its level is not elevated as a result of benign pelvic disease so its specificity is higher than CA125, but levels of HE4 are also raised in some other cancers. Recent work on ovarian cancer screening has suggested that screening utilizing a combination of these two biomarkers may be the best approach for early disease detection.
Now exciting preliminary data from the Anderson Cancer Center have just been published. Over 4,000 women, healthy at the start of the study, were classified into three risk groups based on a mathematical model- the ROCA- incorporating their age and CA125 serum level. Follow-up over eleven years was dependent on the evolving perceived risk. The US researchers were ‘cautiously optimistic’ about this approach, but await results from a similar trial in the UK, involving more than 200,000 women, which will be available within two years. Hopefully, though, screening using the ROCA will lead to more timely diagnosis and thus a better survival rate for ovarian cancer patients.
Phage-displayed peptides as novel reagents for norovirus detection
, /in Featured Articles /by 3wmediaCurrent methods for detecting noroviruses (NoVs) have significant limitations in sensitivity and feasibility for use in remote locations. Our group recently identified phage-displayed peptides with specific binding to NoVs and sensitivity comparable to that of existing antibodies. These reagents can be easily optimized by mutagenesis and represent promising diagnostic tools.
by Amy M. Hurwitz, Prof. Robert L. Atmar and Prof. Timothy G. Palzkill
Norovirus infection and diagnosis
Each year, norovirus (NoV) infections cause approximately 267 million new cases of gastroenteritis and 200,000 deaths worldwide [1]. Infection spreads rapidly in areas of close human contact, such as cruise ships and hospitals, and is treated only by rehydration, as no antiviral therapy currently exists. An infectious dose estimated to be as low as 18 virions and high environmental stability contributed to classification of NoVs as a category B biodefense agent in the U.S. Therefore, rapid, accurate and highly sensitive diagnosis is important for outbreak recognition and control, and also to guide physicians in patient management. The potential health and economic consequences that may be ameliorated by early NoV detection have led to a high demand for optimized detection reagents that can be used to develop reliable diagnostic assays with minimal requirements for expensive, bulky equipment or technical training.
NoVs are divided into six different genogroups (GI–GVI) based on the amino acid sequence of the major capsid protein (VP1). These are organized further into more than 30 genotypes, and finally into numerous strains or variants [2]. The VP1 protein assembles to form an icosahedral shell with an inner shell (S) domain and outer protruding (P) domain. The P domain is on the virus surface and is the most accessible, while the S domain has the highest sequence conservation across different strains. Given the ability of NoVs to evolve rapidly to result in novel or recombinant strains, continual optimization of detection reagents may be necessary in order to recognize the majority of human-infecting strains. Strains classified into GI and GII are most relevant for human infections, and thus the focus for diagnostic assay development efforts.
Current diagnostic methods and their limitations
Methods used currently for the diagnosis of norovirus infection are far from ideal as they exhibit several limitations that hinder their use for individual patient diagnoses or in rural and developing locations. The gold standard for diagnosis is reverse transcriptase (RT)-PCR, which requires multiple sets of primers to detect about 90% of human-infecting strains [3]. This method has significant equipment and expertise requirements, which are often not available outside of large institutions. Further, the expense of running multiple samples and the need for timely instrument accessibility limit the feasibility of applying RT-PCR as point-of-care applications or for preventing the rapid spread of an outbreak.
Other existing methods include immune electron microscopy (IEM) and enzyme immunoassays. IEM was the first method described for identifying NoVs and was used originally to classify viruses based on structural appearance. This method has limited sensitivity, and also requires expensive equipment and skilled expertise. Enzyme immunoassays, developed after the discovery of type-specific antibody epitopes on the NoV capsid, detect viral particles in human stool samples [4]. This method offers increased specificity and has led to the development of commercially available ELISA and lateral flow assays.
Currently, the only FDA-approved antigen detection assay is an ELISA called RIDASCREEN® (3rd Generation) produced by R-Biopharm, which uses an antibody cocktail with specificity for GI and GII NoVs [Fig. 1]. Due to limitations in sensitivity, this assay is only approved for use during outbreaks and takes several hours to produce results. Several companies, including R-Biopharm, have developed rapid diagnostic assays that use lateral flow technology and have also demonstrated strong specificity for NoV GI and GII strains. However, these have similar limitations with sensitivity and thus are only recommended for preliminary screening to be confirmed by RT-PCR, and are distributed primarily outside of the United States [5]. Overall, there is a clear need for improved diagnostic methods to detect norovirus rapidly with strong specificity, high sensitivity, and with minimal equipment and expertise requirements.
Novel diagnostic phage reagents
Recent studies in our laboratory have identified short, 12-mer peptide reagents with specific binding to the GI.1 NoV genotype [6]. The small size of these peptides displayed on phages offers the ability to access epitopes that may be buried in the capsid protein and not accessible to antibodies, and the potential for increased avidity through multiple linked peptide molecules. To identify peptides with specific binding to NoV, we used phage display technology to screen commercially available, large-scale libraries of randomized peptides that are fused to the gene III protein and expressed in five copies on one end of the phage. Rounds of biopanning were performed in which filamentous phage libraries were screened for phages displaying peptides that bind immobilized Norwalk (NV) GI.1 virus-like particles (VLPs). The phage libraries were added to VLPs and, after washing away non-binding phages, the phages displaying VLP-binding peptides were eluted with low pH [Fig. 2A]. Two to four subsequent rounds of biopanning using the resulting phage populations enriched for phages displaying peptides with the highest binding affinity for NV. DNA sequencing of individual phage clones recovered after multiple rounds of biopanning revealed three peptides, named NV-O-R5-3, NV-O-R5-6, and NV-N-R5-1, that occurred most commonly, and the phage clones displaying the peptides were further characterized for their NoV binding properties [6].
Phage-based ELISAs confirmed the binding specificity of phage-displayed peptides to NV VLPs. These affinity-binding assays used NV VLP captured by immobilized rabbit polyclonal anti-NV antibody in order to maintain the structural integrity of VLPs. Single phage clones were added to the captured VLPs and binding was detected using anti-M13 phage antibody that was conjugated to horseradish peroxidase to provide a signal for bound antibody [Fig. 2B]. Of the three peptide-displaying phage clones analysed, NV-N-R5-1 exhibited a dose-dependent response with decreasing NV VLP concentration and the highest sensitivity with a limit of detection at 1.56 ng NV VLP. Additional phage ELISAs indicated that NV-N-R5-1 binds to the P domain of the capsid protein, which extends the furthest out from the virus, and has comparable sensitivity for NV as existing antibodies used for diagnostics [6]. These results provide proof-of-concept and a strong lead reagent for developing novel phages displaying peptides as effective detection reagents for NoV. Further, the methods described establish a platform methodology for using phage display to identify antigen-specific binding reagents that may be applied to any pathogen with distinct surface epitopes.
Current status
To develop our lead phage-displayed peptide into a commercially viable tool, we are currently optimizing its binding affinity for other genogroups of NoV in order to broaden its diagnostic applications. Phage display technology provides a simple platform for constructing collections of new mutations in a lead peptide that can be used for additional rounds of biopanning to screen for variants with optimal affinity properties [Fig. 2C]. The three phage-displayed peptides discussed above share conserved amino acid sequence motifs that likely confer binding specificity for particular epitopes on the NV capsid protein. Directed evolution through mutagenesis of amino acids surrounding these consensus sequences can enable us to improve binding affinity to NV and alter binding specificities starting with the lead phage peptide, NV-N-R5-1. In particular, developing phage-displayed peptides with optimized binding affinity for the NoV GII.4 genotype, which accounts for >80% of NoV infections worldwide [1], and other GI and GII NoV genotypes will have the greatest relevance for diagnostic applications.
Future development of bacteriophage reagents
For decades, phages have been used to identify their target bacterial strains and species in order to diagnose the cause of infections by phage typing. More recent applications have begun to leverage synthetic biology and genomic engineering strategies to customize phage specificity and reporter signals to enable ‘near-real-time’ detection of a broader range of human pathogens [7]. Our recent work has established a methodology for the identification, characterization, and development of phage-based affinity reagents that may be applied to different pathogens and translated into diagnostic applications. The process outlined in Figure 2 demonstrates the progression from (A) identifying lead reagents against a target of interest, (B) characterizing binding affinity for the antigenic target, (C) optimizing leads through directed evolution or genomic engineering strategies, and finally (D) producing scalable quantities of reagent for commercial diagnostic applications. Zou and colleagues, for example, used a similar method to identify a phage-displayed peptide reagent with specific binding to transmittable gastroenteritis virus (TGEV) that also showed potential antiviral activity [8]. Several groups have also developed phage-based reagents to detect bacterial pathogens, such as Salmonella enterica and Escherichia coli [9, 10].
In summary, the use of phage-based reagents for microbial diagnostics offers many advantages in comparison to more commonly used detection reagents, such as antibodies. Phage display technology enables rapid identification and validation of candidate phage reagents with specificity for new or evolved pathogens through biopanning of commercial or custom made phage libraries (Fig. 2A, B). Phage manipulation through directed evolution facilitates development of reagents with optimized binding affinity and specificity to a target of interest (Fig. 2B). Finally, production of large quantities of phages is accomplished rapidly and inexpensively, as simple preparation methods can produce sufficient phage for hundreds of assays (Fig. 2D). As viral pathogens such as NoV continually evolve, the flexibility provided by phage-based reagents will be essential for developing next generation diagnostics for effective containment of outbreaks. A cocktail of phages, each of which binds to a specific target NoV genotype, may ultimately be the ideal strategy for producing an assay to detect the broadest possible range of NoVs without sacrificing specificity. Overall, phages have an enormous potential for use as detection reagents in clinical, agricultural, food, and environmental settings, and represent an underutilized resource for diagnostic development.
References
1. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunological Reviews 2008; 225(1): 190–211.
2. Kroneman A, Vega E, Vennema H, Vinjé J, White P, Hansman G, Green K, Martella V, Katayama K, Koopmans M. Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology 2013; doi:10.1007/s00705-013-1708-5.
3. Atmar RL, Estes MK. The epidemiologic and clinical importance of norovirus infection. Gastroenterology Clinics of North America 2006; 35(2): 275–290.
4. Parker TD, Kitamoto N, Tanaka T, Hutson AM, Estes, MK. Identification of Genogroup I and Genogroup II broadly reactive epitopes on the norovirus capsid. Journal of Virology 2005; 79(12): 7402–7409.
5. Ambert-Balay K, Pothier P. Evaluation of 4 immunochromatographic tests for rapid detection of norovirus in faecal samples. Journal of Clinical Virology 2013; 56(3): 194–198.
6. Rogers JD, Ajami NJ, Fryszczyn BG, Estes MK, Atmar RL, Palzkill TG. Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples. Journal of Clinical Microbiology 2013; 51(6): 1803–1808.
7. Lu TK, Bowers J, Koeris MS. Advancing bacteriophage-based microbial diagnostics with synthetic biology. Trends in Biotechnology 2013; 31(6): 325–327.
8. Zou H, Zarlenga DS, Sestak K, Suo S, Ren X. Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential. Antiviral Research 99(3): 383–390.
9. Schofield DA, Sharp NJ, Westwater C. Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage 2012; 2(2): 105–283.
10. Galikowska E, Kunikowska D, Tokarska-Pietrzak E, Dziadziuszko H, Loś JM, Golec P, Węgrzyn G, Loś M. Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents. European Journal of Clinical microbiology & Infectious Diseases 2011; 30(9): 1067–1073.
The authors
Amy M. Hurwitz1 BS, Robert L. Atmar2,3 MD, Timothy G. Palzkill*2,4 PhD
1 Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA
2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
3 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
4 Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
*Corresponding author
E-mail: timothyp@bcm.edu
Systematic multiplex PCR for the diagnosis of infectious gastroenteritis
, /in Featured Articles /by 3wmediaCurrent methods for the detection of gastroenteric pathogens are insensitive, slow, and laborious. Testing directed at specific organisms misses important infections. Systematically testing all fecal samples using multiplex PCR for common viral, bacterial and parasitic pathogens allows laboratories to increase diagnostic yield, improve workflow, reduce waste and turn-around times.
by Dr Gary McAuliffe
Introduction
Within a single diagnostic laboratory, multiple methods are used to detect gastroenteric pathogens. Selective agar plates differentiate bacterial pathogens, whereas immunoassays are used to detect viruses and parasites such as Giardia lamblia and Cryptosporidium spp.. Laboratories perform microscopy with special stains for the detection of Entamoeba histolytica and Dientamoeba fragilis. PCR has generally been restricted to the detection of norovirus, but many studies have demonstrated its potential for the detection of other enteric pathogens.
Multiplex PCR (M-PCR) panels have been shown to enhance detection of gastroenteric organisms. These panels combine several enteric pathogen targets in one or more PCR reaction vessel(s). O’ Leary et al. demonstrated 100% sensitivity compared with culture for the detection of four bacterial pathogens [1]. Wolffs et al. used a panel targeting seven viral pathogens and detected a pathogen in 97% of samples compared with 49% by conventional methods [2]. Stark et al. showed 100% sensitivity and specificity of a M-PCR panel targeting four parasites, which also reliably differentiates E. histolytica from non-pathogenic E. dispar and E. moshkovskii [3]. de Boer et al. successfully replaced bacterial culture at their institution with a molecular screening approach targeting four bacteria and G. lamblia [4].
Several commercial M-PCR fecal panels are available [Table 1]. These may be directed against parasites, viruses or bacteria, or contain targets from all three groups of organisms, allowing systematic testing of stool samples for all common gastrointestinal pathogens.
In the author’s study, 1758 samples from community and hospital patients were tested using the Fast-Track Diagnostics bacterial, viral and parasite M-PCR panels. Pathogens were detected in 30% of samples by this systematic M-PCR approach compared with 18% using conventional testing as directed by clinician request [5][Table 2].
Advantages of a systematic M-PCR approach
Studies have demonstrated enhanced detection of entero-hemorrhagic E. coli (EHEC), Clostridium difficile, G. lamblia, E. histolytica, norovirus, adenovirus and rotavirus by PCR compared with immunoassays and other conventional assays [2–4, 6, 7]. Bacterial PCR has generally performed comparably with culture, with the exception of Salmonella spp., where reduced detection by PCR has been demonstrated in several, but not all, studies [4, 5, 8]. An advantage of increased sensitivity is that smaller amounts of feces are required for testing, and multiple samples are not required for the detection of common parasites. Clinicians should be aware that organisms such as adenovirus and norovirus can be shed in feces for several weeks following infection and that PCR detects low levels of these organisms which may not be clinically relevant. Selective bacterial media lack specificity, requiring time and further testing to discount commensal organisms. E. histolytica cysts cannot be reliably differentiated from other members of the Entamoeba complex by microscopy and staining. M-PCR generally overcomes these issues, though specificity depends on the target sequence chosen. For adenovirus, some panels target the hexon gene which does not differentiate between enteric and non-enteric serotypes, whereas others target sequences specific to enteric sub-types.
Currently laboratories restrict their testing to a limited range of pathogens for which they have sensitive and affordable assays available. A number of organisms that laboratories do not commonly test for, such as astrovirus, sapovirus, Vibrio spp., and non-O157 EHEC, can be missed. M-PCR allows a wider range of organisms to be targeted. In the author’s study astrovirus was found to be the second most common cause of gastroenteritis, and non-O157 EHECs were detected in sixteen samples by targeting the stx genes. Our laboratories did not have assays for these organisms prior to the study. Laboratories may design their own panels to reflect locally and internationally important pathogens, or buy commercial panels which reflect these requirements. Rare or imported infections not targeted by the panels will not be detected, and laboratories need to decide when additional tests are required. Up to five targets may be tested in a single real-time PCR reaction vessel; therefore increasing the number of targets reduces the number of samples on a given PCR amplification run. The xTAG system (Luminex Corp.) overcomes this limitation by post-amplification analysis using microspheres with up to 100 differing spectra. Eleven targets are currently included in the xTAG gastroenteritis panel within a single PCR reaction vessel [9]. TaqMan array cards (Life technologies) also overcome this restriction by allowing simultaneous real-time PCR in 384 PCR reaction wells. This platform allows up to eight samples to be run in parallel [10].
Fecal samples are usually tested for a limited range of viruses, bacteria, or parasites dependent upon clinician request and laboratory algorithms. Studies have shown that important pathogens such as G. lamblia and EHEC are missed using this approach. Testing for enteric viruses is not widely employed outside hospitals despite their prevalence. The M-PCR approach tests every sample for every target in the panels. It is less reliant on clinicians’ knowledge of the organisms that the patient has likely been exposed to, or those that may be causing the patients clinical syndrome. This systematic approach accounted for the majority of the increased diagnostic yield in the author’s study.
In our laboratory it can take 3–4 days for the identification of Salmonella spp. by culture. Time to generation of results is significantly reduced with the M-PCR approach. In the author’s study, samples collected were batch tested the following day, giving results for eleven pathogens simultaneously within 24 hours of collection. Hands-on time is also reduced. The preparation and reading of a trichrome stain can take up to 40 minutes by an experienced operator, whereas the hands-on time for testing a sample by PCR is a quarter of this.
The workflow created by the systematic M-PCR approach fits well with current laboratory systems; testing is performed in a single pathway rather than by several laboratory departments. Conventional fecal testing employs multiple diagnostic kits and selective media, some of which come with significant cost, short expiry times, and extensive quality control requirements. In our laboratory stool samples for bacterial culture are inoculated onto seven agar plates which need to be stored, processed, and incubated, generating a significant amount of waste. O’ Leary et al. reported that M-PCR significantly reduced this wastage [1]. M-PCR does not obviate the need for bacterial culture as it does not offer an antibiogram, but it allows focused testing of samples found to be positive for these bacterial targets.
Pathogens such as Shigella spp, G. lamblia and D. fragilis are labile in stool. Delays may occur in transportation, inoculation or examination which can compromise yield. For M-PCR less initial processing is required. Specimens are inoculated into tubes containing Stool Transport and Recovery buffer (STAR) (Roche Diagnostics) which binds inhibitors, and stabilizes nucleic acids for later processing. PCR is also able to detect organisms rendered non-viable by inadequate transport, or the use of antibiotics. Feces contains high levels of bilirubin and bile salts, which can lead to inhibition of PCR amplification. Inoculating samples into STAR buffer on arrival, and using extraction methods such as the EasyMag platform (Biomerieux) can help overcome this issue. In the author’s study 1.7% of samples exhibited inhibition, but all gave adequate internal control amplification following dilution and repeat testing.
Cost is a major factor preventing systematic testing of fecal samples by conventional techniques in diagnostic laboratories. The costs of PCR are reducing relative to conventional tests, and batch testing of samples using the systematic M-PCR approach is becoming a viable option. In the author’s study testing a sample for bacteria, viruses and parasites was significantly cheaper by M-PCR than using equivalent conventional tests [NZ$152 (£75) versus NZ$280 (£140)]. Laboratories have successfully reported replacing their conventional methods with a molecular screening approach for bacteria [1, 4] or viruses [2]. Where the cost of replacing all traditional diagnostics with systematic testing may currently remain restrictive, laboratories may choose to replace testing for organism groups, e.g. viruses, and institute systematic testing at a later date.
Conclusion
Current conventional methods for the detection of enteric pathogens are labour intensive, insensitive, slow, and applied piecemeal to submitted fecal samples. Testing stool samples using multiplex PCR panels which simultaneously detect all common bacteria, viruses and parasites increases the detection of gastroenteric pathogens. This approach improves turn-around time, workflow, reduces labour and waste. Costs are reducing, making systematic M-PCR testing an attractive alternative to currently used techniques.
References
1. O’Leary J, et al. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol. 2009; 47: 3449–3453.
2. Wolffs PF, et al. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. J Clin Microbiol. 2011; 49: 1926–1931.
3. Stark D, et al. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol. 2011; 49: 257–262.
4. de Boer RF, et al. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol. 2010; 48: 4140–4146.
5. McAuliffe GN, et al. Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples. J Infect. 2013; 67(2): 122–129.
6. Costantini V, et al. Diagnostic accuracy and analytical sensitivity of IDEIA norovirus assay for routine screening of human norovirus. J Clin Microbiol. 2010; 48: 2770–2778.
7. Luna RA, et al. Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children’s hospital. J Clin Microbiol. 2011; 49: 851–857.
8. Cunningham SA, et al. Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture. J Clin Microbiol. 2010; 48:2929–2933.
9. Coste JF, et al. Microbiological diagnosis of severe diarrhea in kidney transplant recipients by use of multiplex PCR assays. J Clin Microbiol. 2013; 51(6): 1841–1849.
10. Liu J, et al. A laboratory developed TaqMan array card for simultaneous detection of nineteen enteropathogens. J Clin Microbiol. 2013; 51(2): 472–480.
The author
Gary McAuliffe MBBS
Microbiology Department, LabPlus Laboratory, Auckland, New Zealand
E-mail: GMcAuliffe@adhb.govt.nz
A blood test for checking stomach health
, /in Featured Articles /by 3wmediaHelicobacter pylori (Hp) -infection and atrophic gastritis (AG) are the most important risk conditions preceding gastric cancer (GC). Following extensive research and development, a Finnish biotechnology company, Biohit Oyj, has launched the GastroPanel test, a panel of four stomach-specific biomarkers that give accurate information on both the structure and function of gastric mucosa.
by Dr Kari Syrjänen
Since the risk of GC and peptic ulcer disease among individuals with healthy stomach is very low, it is essential to distinguish between subjects with healthy stomach and those with gastric disorders. With GastroPanel – a simple blood test – it is now possible to detect the patients who are at high risk for GC because they harbour either Hp -infection, AG or both in their stomach mucosa. Hp -infection alone increases the risk of GC several-fold, and this risk is over 90-fold among patients with Hp -related severe AG of both the corpus and antrum (pangastritis)[1, 3].
Another area of use for the GP test are the dyspeptic complaints, which in western countries appear in 20-40% of the population. According to most current medical practices, the assessment of these complaints should invariably include a gastroscopic examination for which the existing resources are clearly insufficient and which is actually not really necessary. The same applies to the costly and risky “test” medications with proton-pump inhibitors (PPIs), since it is now possible to screen the patients at true risk and for whom gastroscopy is indicated by using the GastroPanel test. With this approach, approximately 40-70% of the limited and expensive endoscopy capacity can be released for colonoscopies, i.e. for screening and early detection of colorectal cancer. Because of the fact that, particularly among the elderly, dyspeptic complaints are frequently of large intestinal origin, it is cost-effective to supplement the examinations of these dyspeptic patients with colorectal screening methods like the ColonView test, a stool based detection of Hb and Hb/Hp complex, or colonoscopy.
The safe and cost-effective GastroPanel test enables early detection of many different disorders, and thus helps avoiding the majority of subsequent health problems. Besides gastric and esophageal cancer, undetected AG of the corpus (acid-free stomach) can eventually also lead to malabsorption of vitamin B12, iron, magnesium, calcium, and some drugs. AG of the antrum, in turn, increases the risk of peptic ulcer disease and GC.
Concomitant AG of the antrum and corpus (pangastritis) is the single most important risk condition for GC. A minority of GCs can develop directly from HP-induced gastritis, without recognizable stages of mucosal atrophy. It is well known that vitamin B12 deficiency can lead to pernicious anemia (PA), dementia, depression, and injuries of the peripheral nervous system. Calcium deficiency, in turn, leads to osteoporosis. The absorption of many drugs is impaired in acid-free stomach. The risk of serious intestinal infections (giardiasis, malaria, Clostridium difficile and E. coli EHEC) can be increased particularly among senior citizens with AG.
Within public healthcare, it is possible to achieve substantial cost savings by replacing the systematic use of gastroscopy with a simple and inexpensive first-line diagnostic tool like the GastroPanel test for all patients with dyspeptic symptoms.
References
1. Suovaniemi O. GastroPanel-tutkimus osaksi dyspepsian hoitokäytäntöä. Yleislääkäri 2007; 4:104-106.
2. Malfertheiner P, Mégraud F, O’Morain C ym. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007; 56:772-781.
3. Agreus L, Kuipers EJ, Kupcinskas L, Malfertheiner P, Di Mario F, Leja M, Mahachai V, Yaron N, van Oijen M, Perez Perez G, Rugge M, Ronkainen J, Salaspuro M, Sipponen P, Sugano K, Sung J. Rationale in diagnosis and screening of atrophic gastritis with stomach-specific plasma biomarkers. Scand J Gastroenterol 2012; 47:136-147.
The authors
Prof Kari Syrjänen,* MD, PhD, FIAC,
Chief Medical Director, Biohit Oyj.
Lea Paloheimo PhD
Director of Business Development and Quality, EurClinChem,
*Corresponding author
E-mail: kari.syrjanen@biohit.fi
Gene testing gets primed for the mass market
, /in Featured Articles /by 3wmediaQuestions about gene testing were highlighted dramatically this summer after Hollywood superstar Angelina Jolie announced she had undergone a preventive double mastectomy. The reason: gene tests showed she carried the breast cancer-linked BRCA1 mutation. In an Op-Ed piece in the ‘New York Times’, the actress encouraged other women, who believed they were also at risk, to also get tested.
Ms. Jolie’s decision has been hailed by some, criticized by others. However, it may well mark a watershed, when gene testing began a paradigm shift to the mass market. Her announcement, for example, led to a doubling of cancer checks at top clinics in London.
US Patent ruling will bring costs down
Such trends are likely to be reinforced, strongly, by a US Supreme Court ruling in June 2013 (shortly after Ms. Jolie’s announcement) that human genes cannot be patented. The decision reversed three decades of US intellectual property case law, and within days, several US labs announced they would be offering BRCA tests. The latter could previously only be tested for by a single company, Myriad Genetics. Though patent laws are national matters, it is likely that the US court ruling will make an impact elsewhere. In Europe, the EU Biotech Directive allows patenting of gene tests, while Myriad itself recently won a Federal Court ruling in Australia upholding its BRCA patents.
Revenues from genetic screening were $5.9 billion in the US in 2011, according to a study by the respected Battelle Institute. To put the figure in perspective, this is about 10% of the total US clinical testing market. Globally, sales of genetic tests could be conservatively estimated at $10-$15 billion. Scores of vendors already offer a range of tests – from selective screening for some hundred-odd major disease genes to complete sequencing of a person’s genome.
The once-prohibitive costs of gene tests have seen downwards pressure over the past decade. As with other consumer technology cycles, lower prices are expected to drive an expansion in affordability, in users and revenues, in a virtuous cycle. One of the key market catalysts has been direct-to-consumer testing (DTC) companies. US DTC leader 23AndMe has seen its gene tests used by about 200,000 consumers. For just $99, the company provides information on 50 carrier traits, 20 drug classes and disease risk information. 23AndMe is currently seeking FDA certification. European firms are less visible. A leading vendor, deCode Genetics, shut down its DTC service after being acquired by Amgen in late 2012. The Iceland-based firm had been offering its deCodeme personal genomic scanning service for just under $1,000, as well as screening for cardiovascular diseases and common cancers – in a package for $350. Other major DTC players in Europe are also from the US, among them Navigenics, DNADirect and Genelex.
Price falls are now almost certain to accelerate after the US Supreme Court decision on gene patents. Myriad, for example, was using its monopoly on BRAC to charge $3,000 and more for a test. After the Court ruling, the test is projected to see a steep fall in its price to just $100.
Drivers of consumer tests
The key reason for the growth of DTC is that genetic testing has so far largely been restricted to specialist labs and top academic medical centres. In spite of a sharp rise in the number of registered tests to over 7,500, most have yet to be translated into clinical applications.
A study by United Health, the US managed health group, found 63% of physicians saying that screening provided them “the ability to diagnose conditions that would otherwise be unknown.” However, a larger number, about three of four, also noted there were patients in their practices “who would benefit from a genetic test but have not yet had one.” United Health estimates that the US testing market alone would reach about $15 to $21 billion by 2021. In Europe too, an increase in formal healthcare settings for gene testing is likely to be welcomed, given growing concerns about DTC. A recent survey of clinical geneticists found 84% of respondents expressing concern about “replacing face-to-face supervision by a medical doctor with supervision via telephone” through DTC testing firms. A little less than half the respondents said they had at least one patient make contact with them after they had undergone a DTC genetic test, and 86% said they would provide post-test counselling to such patients. The survey posed the likelihood of a ‘cascade effect’ in the future, particularly should physicians spend more time on patients with DTC test results that are not medical priorities. As a result, it seems market growth will be accompanied by the encouragement of general hospitals and physician practices to do gene testing.
The emergence of personal medicine
The impact of mass gene testing will clearly be enormous. One new frontier is personal medicine, where medicine choice and dosages would be prescribed according to a patient’s specific genetic profile. Further down the horizon may be an end to several inherited diseases. In January 2009, the UK saw the birth of the first baby “tested preconceptionally for a genetic form of breast cancer.” The baby was born at University College London (UCL) Hospital, using Preimplantation Genetic Diagnosis, which involves undertaking an in vitro fertilization treatment cycle to have several embryos available for genetic tests.
More recently, UCL announced that its scientists had developed a microchip test to analyse 35 different genetic mutations linked to cancer, and enable doctors to identify and target specific genes from a small sample of tissue. UCL Professor Charles Swanton said the test marked the beginning of tailored cancer care in the NHS.
Ethical questions remain
Nevertheless, there is some way to go. One barrier consists of still-lingering questions about the ethical implications of gene tests.
Here, the first issue is uncertainty. Even now, gene testing (including that for the high-profile BRCA 1 and 2) only predicts an increase in risk, not certainty of disease. This transfers the choice and responsibility for an irreversible prophylactic intervention to a patient, and to his or her best guess. It also rules out the possibility of effective, new and less-invasive surgical interventions emerging in the future.
Such technology evolution challenges – of better choices becoming available – apply broadly to all genetic testing. Some tests do not (as yet) identify all possible gene mutations which lead to a particular disease, or have only limited predictive value. Finally, it remains unclear whether a mutation is not just a symptom of a disease, rather than being a cause.
For example, in cystic fibrosis (CF), there is still no way to predict disease severity, even when a fetus has inherited two mutations. Parents thus face the dilemma of deciding whether to continue or end a pregnancy without full knowledge. In the meanwhile, even as data on CF mutations grows steadily by the year, promising new drug therapies are becoming available. For example, Ivacaftor (Vertex Pharmaceuticals), which addresses the G551D mutation affecting 4% of CF patients, is now being evaluated for the more prevalent F508del mutation.
The above dilemmas are aggravated by the question of false positives and false negatives. In spite of being at the cutting edge of mass screening techniques for Down’s syndrome and neural tube defects, Quad tests for pregnant women still retain a 5% false positive and 20% false negative rate. Elsewhere, while metabolic genetic disorders such as phenylketonuria can be identified by fetal gene tests and then addressed by dietary changes, many others lack treatment options.
The broader debate on gene tests and its ethics is unlikely to go away soon, but policy makers are broadly swinging to accept its inevitability. The Human Genetics Commission in Britain stated in April 2011 that there were “no ethical barriers preventing the use of genetic testing in couples before they conceive.” Within months, the German parliament enacted a law to allow testing fertilized embryos for possible life-threatening genetic defects, via Preimplantation Genetic Diagnosis (like that launched by University College London in early 2009). Critics in Germany have been especially vociferous, calling the move “a step toward designer babies.”
One of the biggest concerns about genetic testing is the emergence of ‘a la carte’ health insurance, providing choice of cover and premium based on a person’s particular disease risks and (eventual) treatment requirements, rather than loading the highest-risk beneficiaries atop the lower-risk ones.
In the US, resulting concerns about discrimination due to genetic testing led to the 2008 Genetic Information Nondiscrimination Act (GINA), which bars denial of health insurance or employment because of a genetic predisposition to a particular disease.
In Europe, different laws and regulations in the Member States seek to address ethical questions. A major hurdle here is the lack of an “approved definition of a genetic test,” in spite of the EU-funded project EuroGentest. One of the latter’s goals was to “try to develop at least some key elements for a working definition” of a gene test.
Interference in thyroid function tests – problems and solutions
, /in Featured Articles /by 3wmediaInterference in immunoassay is a well described phenomenon and all clinical immunoassays, including thyroid function tests, are potentially at risk. Spurious results can lead to over investigation or mismanagement if not detected, but a proactive approach by the laboratory will help to identify and resolve these problems.
by Dr Olivia Bacon and Dr David J. Halsall
Background
Thyroid disorders are relatively common, and are associated with long-term morbidity and mortality. Clinical signs and symptoms are often non-specific, so reliable laboratory tests are critical for diagnosis. Therefore, thyroid function tests (TFTs) are frequently requested immunoassays with around 10 million results being reported each year by UK laboratories. In the UK, TFTs typically include a high sensitivity immunoassay for thyroid stimulating hormone (TSH) with an immunoassay estimation of non-protein bound thyroxine (fT4), either run simultaneously or added if the TSH value is outside the reference interval [1].
For the majority of tests, both results will be within the reference interval and thyroid disease can be excluded. In some patients TFTs support the diagnosis of hypothyroidism (raised TSH with fT4 low, or lownormal) or hyperthyroidism (TSH undetectable, and fT4 elevated), and these results will confirm clinical findings. However, due to the high volume of TFTs performed, it is not unusual for the laboratorian to be faced with a set of TFTs that are either internally inconsistent, or incompatible with the clinical details provided. Many medications can affect the thyroid axis, as can other non-thyroidal pathologies; these are often transient, but can cause unusual patterns of TFT. Much rarer genetic or pituitary conditions can also cause discordant TFTs [2]. However, if drug effects are excluded, it is necessary at this stage for the laboratorian to consider that one of the TFT results is incorrect, as analytic error is at least as common as these rare thyroid conditions. As spurious TFT results can lead to over investigation, or even inappropriate treatment, it is critical, but not trivial, for the laboratory to confirm the analytical validity of the TFT results.
In one study of more than 5000 samples received for TSH analysis, assay interference with the potential to adversely affect clinical care was detected in approximately 0.5% of patients [3]. This equates to a rather alarming 50,000 tests per annum in the UK.
Although assay design is continually improving, no routine immunoassay is currently robust to interference. Technical errors with many routine chemistry methods caused by inappropriate sample collection or handling, chemical or spectral interference can be detected during result validation. However, detection of spurious TFT immunoassay results is more challenging as there is no automatic ‘flag’ from the analyser, and there is usually a wide range of plausible values for these analytes, making it difficult to question those which are ‘suspicious’. Consequently clinical validation, where results are checked for discordance with the clinical correlates and other laboratory tests, is used to detect potentially incorrect results before reporting. For TFTs this is aided by the characteristic reciprocal relationship between TSH and fT4 in patients with an intact pituitary–thyroid axis.
Mechanisms of interference in TSH assays
Endogenous interfering antibodies are a well described cause of immunoassay interference [4]. In TSH assays these antibodies can have affinity for TSH itself or towards assay components. Anti-reagent antibodies can be ‘anti-animal’ antibodies, specific to the species in which the reagent antibody was raised, or weak, polyspecific ‘heterophilic’ antibodies, which may be part of the natural process of the generation of antibody diversity [5]. Anti-animal antibodies are more prevalent in animal handlers or patients treated with therapeutics based on animal immunoglobulins.
Anti-reagent antibodies can interact with either the capture or detection antibodies in two-site assays, blocking the generation of signal in the presence of analyte (false negative result) or by causing antibody cross-linking in the absence of analyte (false positive result) [Fig. 1].
Anti-TSH antibodies can generate high molecular weight TSH : antibody complexes (‘macro-TSH’). Depending on the exact site of the antibody–analyte interaction, false positive TSH results may occur as the macro-TSH is unlikely to be biologically active [6].
Detection of interference in TSH assays
Once suspected, a robust laboratory strategy is required for confirming or excluding assay interference. Method comparison using an alternative method is often used as the first step. Most laboratories use two-site immunoassays for TSH, but assay formulations, antibody species and incubation times vary between manufacturers. Varying amounts of blocking agents, designed to prevent non-specific binding of heterophile antibodies, may be included. Significant disagreement between two TSH methods is a strong indicator of assay interference.
Dilution studies are a simple but effective tool to investigate the analytical validity of an immunoassay. Non-linearity to dilution suggests a result is unreliable. However, although a good ‘rule in’ test, linearity to dilution alone cannot be used to exclude interference [3,7].
Immunosubtraction is a useful method to confirm the presence of antibody interference. This can be done crudely using polyethylene glycol (PEG) precipitation or more specifically using anti-immunoglobulin agaroses. Proprietary heterophile blocking tubes can also be used to confirm the presence of this class of interferent [3,4].
Once assay interference is established it can still be difficult to determine the correct TSH value, as there is no ‘gold standard’ method for TSH. However, an alternative immunoassay result which gives the expected responses to dilution and immunosubtraction, and correlates with fT4 results plus clinical findings, can be used with a reasonable degree of confidence.
Mechanisms of interference in fT4 assays
fT4 assays present a particular analytical challenge as >99.9% of T4 in the serum is protein bound, and the unbound T4 fraction must be measured without upsetting the equilibrium between the two fractions [8]. Therefore, an abnormal T4 binding protein, or agent which affects binding protein affinity in vitro, has the potential to generate incorrect results. Most commercial fT4 assays are one-site immunoassays based on competitive principles, using either labelled T4 analogue or anti-T4 antibodies for detection. Both heterophile and anti-T4 antibodies therefore also have the potential to interfere with these methods [4].
Non-esterified fatty acids (NEFAs) are a common T4 displacing agent as they can release T4 from the low affinity, high capacity T4 binding site on albumin. NEFAs can be generated in vitro, usually as a consequence of heparin therapy, which stimulates the action of lipoprotein lipase on triglyceride. Although the measured fT4 result is genuinely high, it does not reflect the in vivo situation [9].
Familial dysalbuminaemic hyperthyroxinaemia (FDH) is a benign genetic condition where the affinity of albumin for T4 is increased, such that circulating albumin-bound T4 is elevated. Despite the high total T4 (tT4), concentrations of free hormone in vivo are unaffected due to the homeostatic regulation of the thyroid axis. However, FDH is often associated with falsely high fT4 measurements using commercial immunoassays [10] [Fig. 2]. Both the increased affinity of the variant albumin for some labelled T4 analogues, as well as potential disruption of the T4 : albumin equilibrium during the assay, are likely mechanisms. The presence of the FDH mutation can be confirmed using molecular genetic approaches.
Detecting interference in fT4 assays
Despite the greater analytical challenge, confirming interference in fT4 assays can be easier than for TSH due to the availability of physical separation methods, such as equilibrium dialysis, as ‘gold standard’ assays [8]. However, these methods are technically difficult and not available in most clinical biochemistry laboratories. Also, these methods do not solve the in vitro problems of hormone displacement.
Again a first approach is often method comparison, using a different immunoassay architecture. Dilution and immunosubtraction studies can also be informative, although some fT4 methods are not robust to matrix effects so careful control experiments are required.
Measurement of total rather than free T4 can be useful in situations where there is a suspicion of abnormal T4 binding proteins. For example, total T4 will be elevated in the presence of anti-T4 antibodies and in FDH.
Clinical causes of aberrant TFTs
As mentioned above there are well described pharmacological and pathological causes of unusual TFTs; an increased awareness of analytical artefacts should not detract from the detection of these conditions. For example thyroxine treatment, a TSH secreting pituitary tumour (TSHoma), the genetic condition thyroid hormone resistance, FDH or TFT antibody interference can give elevated fT4 results with a TSH within the reference interval. Attempts by the laboratory to exclude assay interference should complement both the diagnosis of transient and genetic thyroid conditions as well as the more common drug related effects.
Conclusions and future directions
Immunoassay manufacturers have invested considerable resources into reducing the potential for antibody-mediated assay interference, for example by including blocking agents, or using antibody fragments rather than intact antibodies as assay reagents. Although these measures are effective, it is worth bearing in mind that changes to assay formulations may introduce novel types of interference. We have observed negative interference in one fT4 assay which appears related to the presence of a blocking agent introduced to reduce the risk of positive interference in this method [11]. Mass spectrometric methods have been introduced to eliminate antibody interference in both fT4 and tT4 methods, but unfortunately the fT4 methods still require careful optimization to avoid interference caused by binding proteins and displacing agents.
As current TFT methods remain prone to analytical interference the clinical laboratory must remain vigilant to the potential for assay interference, promote effective communication with requesting clinicians, and have procedures in place for investigation of discordant results.
References
1. Association for Clinical Biochemistry (ACB), British Thyroid Association (BTA), British Thyroid Foundation (BTF). UK guidelines for the use of thyroid function tests.2006; www.acb.org.uk/docs/TFTguidelinefinal.pdf.
2. Gurnell M, Halsall DJ, Chatterjee VK. What should be done when thyroid function tests do not make sense? Clin Endocrinol. (Oxf) 2011; 74(6): 673–678.
3. Ismail AA, Walker PL, Barth JH, Lewandowski KC, Jones R, Burr WA. Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid-stimulating hormone and gonadotropin immunoassay results. Clin Chem. 2002; 48(11): 2023–2029.
4. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998; 44: 440–454.
5. 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.
6. Halsall DJ, Fahie-Wilson MN, Hall SK, Barker P, Anderson J, Gama R, Chatterjee VK. Macro thyrotropin-IgG complex causes factitious increases in thyroid-stimulating hormone screening tests in a neonate and mother. Clin Chem. 2006; 52: 1968–1969.
7. Ross HA, Menheere PP, Thomas CM, Mudde AH, Kouwenberg M, Wolffenbuttel BH. Interference from heterophilic antibodies in seven current TSH assays. Ann Clin Biochem. 2008; 45: 616.
8. Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best Pract Res Clin Endocrinol Metab. 2013; in press.
9. Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab. 2009; 23(6): 753–767.
10. Cartwright D, O’Shea P, Rajanayagam O, Agostini M, Barker P, Moran C, Macchia E, Pinchera A, John R, Agha A, Ross HA, Chatterjee VK, Halsall DJ. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009; 55(5): 1044–1046.
11. Bacon O, Gillespie S, Koulouri O, Bradbury S, O’Toole A, Stuart-Thompson D, Taylor K, Pearce S, Gurnell M, Halsall DJ. A patient with multiple Roche serum immunoassay interferences including false negative serum fT4. Ann Clin Biochem. 2013; 50(Suppl 1): T50.
The authors
Olivia Bacon PhD and David Halsall* PhD, FRCPath, CSci
Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, Cambridge, UK
*Corresponding author
E-mail: djh44@cam.ac.uk