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
Accurate quantitative targeted analysis of low molecular weight compounds is one of the most important needs in clinical diagnostic laboratories. The enhanced analytical specificity and sensitivity of modern liquid chromatography–tandem mass spectrometry based methods satisfy this requirement for screening of endocrine related disorders, including those affecting steroidogenic systems or overproduction of catecholamines.
by Dr M. Peitzsch and Professor G. Eisenhofer
Liquid chromatography – tandem mass spectrometry (LC-MS/MS)
The development of electrospray ionization (ESI) enabled the introduction of aqueous chromatographic eluates into mass spectrometers, an advance for which John Bennett Fenn was awarded the Nobel Prize in chemistry in 2002. Subsequent refinements in liquid chromatography coupled with ESI mass spectrometry led to analytical applications directed at a broad range of macromolecules from peptides, proteins, glycoproteins and glycolipids to lower molecular weight polar and non-polar compounds, including fatty acids, vitamins, nucleic acids, steroids, amino acids and biogenic amines.
Introduction of tandem mass spectrometry (MS/MS) represented a further breakthrough enabling analyses of relationships between ‘parent or precursor ions’ in the first stage and ‘daughter or product ions’ in the second stage of the instrument [1]. For targeted quantitative analyses, the filtering capabilities and the multiple reaction monitoring (MRM) possible through MS/MS triple quadrupole instruments provide not only high selectivity, but also improved signal-to-noise ratios. In recent years, the increasing commercial availability of stable isotope labelled substances, used as internal standards, has facilitated the application of stable isotope dilution internal standardization as the gold standard for accurate quantitative analyses. Since the physicochemical properties of the target analyte and the stable-isotope-labelled internal standard are similar, this approach compensates for all variations which occur during sample extraction, injection, chromatography, ionization, and ion detection with dow stream improvements in analytical precision and accuracy [2].
The high analytical specificity of LC-MS/MS allows less rigorous sample purification and chromatographic resolution than for standard high performance liquid chromatographic (HPLC) procedures employing ultraviolet, electrochemical or fluorimetric detection. This, and other developments in column chemistry, such as those allowing ultra-high performance liquid chromatography (UPLC), in turn enables higher sample throughput than offered by conventional HPLC procedures. Fusion of LC-MS/MS with other technologies, such as multiplexing parallel LC systems and turbo-flow technology, provides additional advantages for efficient and accurate high-throughput quantitative analyses. Further, automated online sample extraction systems minimize time spent on sample preparation and allow multiple applications to be efficiently handled by one instrument.
All the above possibilities for extending sample throughput, combined with the versatility of a single LC-MS/MS system to take over the jobs of multiple standard HPLC systems, provide advantages that justify the initial high cost of the instrument. Recognized impediments to implementing LC-MS/MS technology include the complexity of the instrumentation associated with the necessity for highly skilled personnel, especially for method development. A lack of standardization combined with a shortage of inter-laboratory comparison programs for quality assurance represent other limitations to acceptance by clinical laboratories.
LC-MS/MS in clinical diagnostics laboratories
The improvements in precision and accuracy offered by LC-MS/MS are now well recognized as offering critical advances over standard HPLC and immunoassay procedures, which are subject to analytical interferences or do not allow precise and accurate identification of structurally-related compounds, such as steroid hormones. Such advances are important to the fields of endocrinology and clinical laboratory medicine where accurate quantitative analysis is crucial for diagnostic purposes [2].
LC-MS/MS applications are now used in clinical and forensic toxicology, such as for drugs-of-abuse testing. In clinical laboratory medicine, LC-MS/MS is used for measurements of endocrine hormones such as steroids, biogenic amines and thyroid hormones, as well as for therapeutic drug monitoring and in new-born screening for assessment of inborn errors of metabolism.
In contrast to commonly used immunoassays, LC-MS/MS enables measurements of multiple analytes for each sample processed. Such determination of analyte profiles includes those for the various thyroid hormones, different vitamin D metabolites and steroid profiles, all available in single analytical runs. Profiles of steroid hormones, although mainly used in research applications, hold considerable promise for the routine clinical assessment of a wide range of
steroidogenic disorders.
LC-MS/MS based screening for catecholamine producing tumours
Pheochromocytomas and paragangliomas (PPGLs) are tumours arising respectively in adrenal and extra-adrenal chromaffin cells that are characterized by an overproduction of catecholamines. Without diagnosis and an appropriate treatment, the excessive secretion of catecholamines by PPGLs can lead to disastrous consequences.
For initial biochemical screening different tests are available, including plasma or urinary measurements of the catecholamines – norepinephrine, epinephrine and dopamine – and their respective O-methylated metabolites – normetanephrine, metanephrine and 3-methoxytyramine. Whereas the free metabolites are usually measured in plasma, analyses in urine are commonly performed after acid hydrolysis in which free metabolites are liberated from sulfate conjugates.
In 2002, Taylor and Singh presented an LC-MS/MS method for the analysis of deconjugated urinary fractionated metanephrines [3]. The outlined advantages of this method over other methods, such as immunoassay and HPLC-ECD (electrochemical detection), included relative freedom from drug interferences, high sample throughput and short chromatographic run times. Subsequently, there has been a plethora of related methods published, including many that enable detection of the much lower concentrations of plasma free metanephrines than urinary deconjugated metanephrines.
Development of new sample preparation procedures, either offline or online to the LC-MS/MS system, have been particularly useful for automated high-throughput procedures [4, 5]. More recent improvements in LC-MS/MS instrumentation have led to improved analytical sensitivity, now even enabling accurate and precise measurements of picomolar plasma concentrations of 3-methoxytyramine, the O-methylated metabolite of dopamine [5–7]. This valuable biomarker not only allows detection of dopamine producing PPGLs, but can also be used to detect malignancy [9]. Using LC-MS/MS, the diagnostic performance of 3-methoxytyramine as a marker of malignancy was characterized by an enhanced diagnostic sensitivity of 86% and specificity of 96% [8] [Fig. 1].
Problems with drug interferences in HPLC-ECD and immunoassay-based methods are largely overcome using LC-MS/MS. For example, problems of acetaminophen (paracetamol) interferences in HPLC-ECD procedures are not a problem for LC-MS/MS [8, 10]. Chromatographic disruptions associated with certain disorders, such as renal insufficiency, are also less of a problem by LC-MS/MS than by HPLC-ECD [8].
Use of plasma free normetanephrine, metanephrine and methoxytyramine for reliable diagnosis of PPGLs requires collection of blood samples after 30 minutes of supine rest and an overnight fast. These conditions pose difficulties for many clinicians, which can result in excessive false-positive results or worse, missed diagnoses when inappropriately high upper cut-offs have been derived from seated sampling. Measurements of urinary metanephrines provide a reasonable alternative test for those situations where blood samples cannot be collected appropriately.
As mentioned above, urinary metanephrines are commonly measured after an acid-hydrolysis deconjugation step. This procedure is based mainly on historical convention, where initially less sensitive instruments did not allow measurements of the much lower urinary concentrations of free rather than deconjugated metanephrines. Improvements in analytical sensitivity now, however, allow analysis of urinary free metanephrines, [11, 12]. Unlike the sulfate-conjugated derivatives, which are produced by a sulfotransferase enzyme located in the gastrointestinal tract, the free metabolites are produced within chromaffin cells. This provides a potential advantage for measurements of the free metabolites. Another advantage is that there are no suitable quality controls or calibrators for measurements of urinary deconjugated metanephrines [12, 13]. Those that are available are almost entirely in the free form so that procedures will always pass quality control even if the deconjugation step is missed and values for patient samples are grossly under-estimated. Measurements of urine free metanephrines avoid this potential pitfall in quality assurance.
Finally, with measurements of urinary free metanephrines it is possible to combine the measurements with urinary catecholamines in a single run [12;14]. This also provides an advantage over measurements of urinary deconjugated metanephrines, where the deconjugation step does not allow measurements of free catecholamines.
The difficulties in applying LC-MS/MS in the clinical chemistry laboratory, such as associated with high initial instrument costs and need for expertise, are easily overshadowed by the analytical advantages. High sample throughput and the analytical versatility offered by LC-MS/MS, which enables rapid method switching, in particular represent important advantages over standard HPLC methods. Nevertheless, such advantages are not easily realized by the small hospital-based laboratory where high sample throughput is not an important consideration. In the US the highly competitive nature of the heath care system is an incentive for centralized testing where efficiency and low operating costs associated with high sample throughput (economy of scale) are more easily realized. In the US the switch from immunoassays or HPLC-based methodology to superior LC-MS/MS technology is therefore likely to remain more advanced than in Europe.
Summary and conclusion
Modern LC-MS/MS systems provide well-recognized accuracy for quantitative targeted measurements of analytes used for clinical diagnostics. The high-throughput capabilities and versatility of LC-MS/MS instrumentation enable multiple applications for rare diseases to be handled by a single instrument. Furthermore, single analyte assays can be extended to accurate profiling by LC-MS/MS assays, providing deeper insight into endocrine metabolic disorders. This, however, remains largely a research-based application and for LC-MS/MS to be readily adapted for routine use in the clinical laboratories, other advantages such as those associated with economy of scale must be appreciated and realized.
References
1. Glish GL, Vachet RW. The basics of mass spectrometry in the twenty-first century. Nature Reviews Drug Discovery 2003; 2: 140–150.
2. Vogeser M, Parhofer KG. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) – Technique and applications in endocrinology. Exp Clin Endocrinol Diabetes 2007; 115: 559–570.
3. Taylor RL, Singh RJ. Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clinical Chemistry 2002; 48: 533–539.
4. Lagerstedt SA, O’Kane DJ, et al. Measurement of plasma free metanephrine and normetanephrine by liquid chromatographym-tandem mass spectrometry for diagnosis of pheochromocytoma. Clinical Chemistry 2004; 50: 603–611.
5. Peaston RT, Graham KS, et al. Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma. Clinica Chimica Acta 2010; 411: 546–552.
6. Eisenhofer G, Goldstein DS, et al. Biochemical and clinical manifestations of dopamine-producing paragangliomas: Utility of plasma methoxytyramine. J Clin Endocrinol Metab 2005; 90: 2068–2075.
7. Eisenhofer G, Lenders JW, et al. Plasma methoxytyramine: A n ovel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status. European Journal of Cancer 2012; 48(11): 1739–1749.
8. Peitzsch M, Prejbisz A, et al. Analysis of plasma 3-methoxytyramine, normetanephrine and metanephrine by ultra performance liquid chromatography – tandem mass spectrometry: utility for diagnosis of dopamine-producing metastatic phaeochromocytoma. Ann Clin Biochem 2013; 50: 147–155.
9. Eisenhofer G, Tischler AS, et al. Diagnostic Tests and Biomarkers for Pheochromocytoma and Extra-adrenal Paraganglioma: From Routine Laboratory Methods to Disease Stratification. Endocrine Pathology 2012; 23: 4–14.
10. Petteys JB, Graham KS, et al. Performance characteristics of an LC–MS/MS method for the determination of plasma metanephrines. Clinica Chimica Acta 2012; 413: 1459–1465.
11. Boyle JG, Davidson DF, et al. Comparison of diagnostic accuracy of urinary free metanephrines, vanillyl mandelic acid, and catecholamines and plasma catecholamines for diagnosis of pheochromocytoma. J Clin Endocrinol Metab 2007; 92: 4602–4608.
12. Peitzsch M, Pelzel D, et al. Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites. Clinica Chimica Acta 2013; 418: 50–58.
13. Simonin J, Gerber-Lemaire S, et al. Synthetic calibrators for the analysis of total metanephrines in urine: Revisiting the conditions of hydrolysis. Clinica Chimica Acta 2012; 413: 998–1003.
14. Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem 2009; 46: 129–136.
The authors
Mirko Peitzsch*1 PhD and
Graeme Eisenhofer1,2 PhD
1 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus at the Technical University Dresden, Dresden, Germany
2 Department of Medicine III, University of Dresden, Dresden, Germany
*Corresponding author
E-mail: Mirko.Peitzsch@uniklinikum-dresden.de
The aim of this study was to assess the practicability and evaluate the analytical characteristics of the Mindray BS-2000M, a new automatic chemistry analyser, manufactured by Shenzhen Mindray Bio-Medical Electronics Co., Ltd (Mindray Shenzhen, China).
The evaluation involved 21 clinical chemistry parameters measured using indirect potentiometry, spectrophotometric and immunoturbidimetric assays.
Spectrophotometric assays
Alanine aminotransferase (ALT, according to IFCC with-out pyridoxal phosphate), aspartate aminotransferase (AST, according to IFCC method), gamma-glutamyltransferasa (GGT, according to Szasz), total bilirubin (TBIL, Diazotized sulfanilic acid method), calcium (Ca, Arsenazo III method), creatine kinase (CK, IFCC method), creatinine (Crea, modified Jaffe method), glucose (Glu, Hexokinase method), high density lipoprotein-cholesterol (HDL-C, direct method), magnesium (Mg, Xylidyl blue method), phosphorus (P, Phosphomolybdate method), total cholesterol (TC, Cholesterol oxidase – Peroxidase method), triglycerides (TG, Glycerokinase Peroxidase – Peroxidase method), total protein (TP, Biuret method), uric acid (UA, Uricase-Peroxidase method), iron (Fe, Colorimetric assay-Ferrozine), α-amylase [α-AMY, substrate: 4, 6- ethylidene-(G7)-1, 4-nitrophenyl-(G1) –α, D-maltoheptaoside (EPS-G7), enzymatic colorimetric assay according to IFCC method] and urea (Urea, Urease-glutamate dehydrogenase).
Immunoturbidimetric assays
Apolipoprotein A1 (ApoA1).
Indirect potentiometry assays (ISE)
Sodium (Na), potassium (k) and chloride (Cl).
Analytical evaluation
Among all the available parameters to verify the Mindray BS-2000M analytical performance, those which are more frequently requested in routine clinical practice were selected (e.g., glucose, creatinine, total protein).
An imprecision study was carried out according to the CLSI EP5-A2 guideline [1]. The within run imprecision was evaluated using two control materials. Final results were expressed as coefficient of variation (CV%). We checked that these CV satisfied the allowable maximum imprecision based on biological variability [2]. These data were taken from the listing of biological variation by Ricos et al [3], recently updated in 2012.
The inaccuracy study was done according to the CLSI EP9-A2 guideline [4], measuring at least 40 patient samples for the two analysers (Mindray BS-2000M and ADVIA 2400 Siemens Healthcare Diagnostics, USA) for 5 days.
In the inaccuracy study the mean bias and 95% confidence interval (CI) was calculated with the Bland-Altman plot analysis, and the linear regression was assessed using Passing-Bablok regression method [5-6].
The results of the imprecision study [Table 1] showed that for all the parameters imprecision was lower than the maximal allowable applying to biological variability based criteria, with the exception of sodium (0.7%) and chloride (0,8%) in control 1 and total proteins (1,7%) in control 2. Nevertheless, these three parameters fulfill the commonly used “State of the art” criterion. According to this criterion, the maximal allowable imprecision for physiological concentrations must be less than the 0.20 fractile of the between-run imprecision (CV) of the laboratories in a external quality assessment scheme (7). The CV% limit for these three parameters following this approach would be 0.9% for Na, 1.6% for Cl and 1.7% for TP.
In the comparison study, a close correlation was observed for all parameters studied (r range: 0.92 – 1.00) [Table 2]. It is noted that there were no significant differences for 11 of the 21 parameters studied. For the other parameters statistically significant differences were found but, except for creatinine, those differences were not considered to have a clinical significance. The constant and proportional differences may be due to different standardization of both procedures. Traceable calibration materials should be used related to the reference method and also switchable materials that reveal the degree of deviation of multiple methods with respect to the true value should be used [8].
References
1. Clinical and Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline -second edition. CLSI document EP5-A2. Wayne, PA:CLSI, 2004.
2. Fraser CG, et al. Proposed quality specifications for the imprecision and inaccuracy of analytical systems for clinical chemistry. Eur J Clin Chem Clin Biochem 1992; 30: 311.
3. Ricos C, et al. Desirable quality specifications for total error, imprecision, and bias, derived from biological variation. http://www.Westgard.com/biodatabase1.htm. Accessed on February 15, 2013.
4. Clinical and Laboratory Standards Institute. Method comparison and bias estimation using patient samples; approved guideline – second edition. CLSI document EP9-A2. Wayne, PA: CLSI, 2002.
5. Bland JM, et al. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1: 307.
6. Passing H, et al. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry, Part I. J Clin Chem Clin Biochem 1983; 21: 709.
7. Sebastian-Gámbaro, et al. An improvement on the criterion of the state of the art to estimate the maximal allowable imprecision. Eur J Clin Chem Biochem. 1996; 34: 445.
8. Documento de consenso. Sociedad Española de Bioquímica Clínica (SEQC) y Sociedad Española de Nefrología (SEN). Recomendaciones sobre la utilización de ecuaciones para la estimación del filtrado glomerular en adultos. Química Clínica 2006; 25: 423.
The author
Dr. Jose Luis Bedini,
Hospital Clinic I Provincial De Barcelona,
Barcelona,
Spain
MINDRAY
Questions 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.
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is rapidly emerging as the technology of choice for measuring steroid hormones. This review will focus on the utility of clinical mass spectrometry for the assessment of endocrine disorders.
by Dr P. Monaghan, L. Owen, Prof. P. Trainer and B. Keevil
Mass spectrometry or immunoassay?
The technological armamentarium of the modern day clinical laboratory has been greatly enhanced by the introduction and continued evolution of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This technique is almost universally applicable to the measurement of small molecule compounds such as steroid hormones and is proving to be invaluable for the endocrinologist towards diagnosing and managing complex endocrine disorders [1]. Furthermore, LC-MS/MS is rapidly expanding for the application of quantitative peptide hormone and protein measurement. LC-MS/MS offers a number of considerable advantages over conventional immunoassay (IA) technology: greater analytical sensitivity and specificity, lack of susceptibility to interference from anti-reagent antibodies and cross-reacting compounds, multiplexing capability for steroid profiles, and low running costs for consumables in comparison to antibody-based reagents. However, like IA, LC-MS/MS is also vulnerable to interference that can compromise the analytical integrity of the method. The potential sources of analytical interference and inaccuracy to consider for IA and LC-MS/MS methodologies are summarized in Table 1.
Improved specificity: safer medical management of Cushing’s syndrome
The use of the 11β-hydroxylase inhibitor metyrapone generally has an adjunctive role in the medical management of Cushing’s syndrome with the aim of improving the medical status of patients prior to surgery or radiotherapy. Patients receiving adrenal-directed anti-steroidogenic drugs such as metyrapone require frequent clinical and biochemical monitoring to minimize the risk of treatment-induced hypoadrenalism.
Current clinical guidance advocates that metyrapone dose is titrated against serum cortisol concentration and some centres, including our own, assess normalization of cortisol production via the measurement a day curve with a mean serum cortisol target between 150–300 nmol/L. The monitoring of metyrapone therapy relies on the measurement of serum cortisol that by the vast majority of laboratories is performed by routine IA. However, metyrapone treatment causes altered steroid metabolism and therefore serum cortisol measurement is susceptible to positive interference when performed by IA due to cross-reactivity with precursor steroids such as 11-deoxycortisol (11DOC) that build up in the circulation as a result of the metyrapone blockade of the adrenal steroidogenic pathway.
Our group has recently quantified the level of positive interference in serum cortisol IA for patients receiving metyrapone therapy by employing a direct quantitative comparison with LC-MS/MS [2]. A modest correlation between plasma adrenocorticotropic hormone (ACTH) concentration and the extent of positive interference in the IA for serum cortisol was also observed as 90% of patients in our study had ACTH-driven Cushing’s syndrome [3]. Our study concluded that for patients receiving metyrapone therapy, cortisol analysis by LC-MS/MS mitigates the potential for erroneous clinical decisions concerning dose titration [Figure 1] and is likely to reduce the risk of unrecognized hypoadrenalism which may result in symptoms that mimic the side-effects of metyrapone treatment, or at worst be fatal.
Improved sensitivity: estradiol measurement
Progress in both LC-MS/MS and online sample preparation technology (pre-analytics) has advanced the analytical sensitivity of this methodology to the extent that for the measurement of many steroid hormones, modern MS applications have now transcended conventional IA methods in this regard. An example of this is the high sensitivity measurement of serum estradiol. External quality assurance data reveals that a wide range of concentrations can be obtained by immunoassay when measuring samples for estradiol at lower concentrations. Furthermore, a recent position statement from the Endocrine Society has stressed the need for better analytical methods to address the current poor performance of assays for measuring low concentrations of estradiol [4]. To this end, our group has developed a novel direct assay that is applicable to routine clinical use for the measurement of estradiol and estrone (therefore permitting calculation of total estrogen status) in male patients and patients on aromatase inhibitors [5]. This high sensitivity assay uses ammonium fluoride in the mobile phase to facilitate more efficient ionization and thereby increase analytical sensitivity. Additionally, an on-line solid phase extraction (OSM) system [Figure 2 (Waters, Manchester, UK)] allows a large volume of extract to be loaded and this coupled with a XEVO™TQS tandem mass spectrometer enables unprecedented analytical sensitivity to be achieved.
Conclusions and future prospects
LC-MS/MS is a very powerful tool which is enabling substantial innovations in the endocrine laboratory. Indeed, it is likely that the majority of emerging small molecules will be addressed by LC-MS/MS analysis. There are two keys areas in which future research and development for LC-MS/MS ought to be directed. Firstly, the utility of LC-MS/MS for the quantification of peptide hormones and proteins is already becoming a reality with published methods available for measurement of renin activity [6], parathyroid hormone [7] and insulin-like growth factor-1 [8] amongst others. These current methods require the skills of highly trained personnel in order to develop and run these assays, and it is hoped that continued innovation in this area will culminate in the development of rapid protein assays that are applicable to routine clinical use. Secondly, it seems feasible with existing technology to develop fully automated random-access LC-MS/MS analysers that will enable greater ease of use in non-specialist laboratory settings. However, the automation of mass spectrometry will not be achieved without a concerted effort from the in vitro diagnostics industry to fully realize the potential of LC-MS/MS across clinical medicine.
References
1. Monaghan PJ, Keevil BG, Trainer PJ. The use of mass spectrometry to improve the diagnosis and the management of the HPA axis. Rev Endocr Metab Disord 2013 Mar 15. [Epub ahead of print].
2. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone. Ann Clin Biochem 2011; 48: 441–446.
3. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Response to ‘Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone’ by Halsall DJ and Gurnell M. Ann Clin Biochem 2012; 49: 204–205.
4. Rosner W, et al. Challenges to the measurement of estradiol: An Endocrine Society Position Statement. J Clin Endocrinol Metab 2013; 98: 1376–1387.
5. Owen LJ, Wu FC, Labrie F, Keevil BG. A rapid direct assay for the routine measurement of oestradiol and oestrone by LC-MS/MS. Ann Clin Biochem [In press].
6. Carter S, Owen LJ, Kerstens MN, Dullaart RP, Keevil BG. A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-phase extraction. Ann Clin Biochem 2012; 49: 570–579.
7. Kumar V, Barnidge DR, Chen LS, Twentyman JM, Cradic KW, Grebe SK, Singh RJ. Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry. Clin Chem 2010; 56: 306–313.
8. Kay R, Halsall DJ, Annamalai AK, et al. A novel mass spectrometry-based method for determining insulin-like growth factor 1: assessment in a cohort of subjects with newly diagnosed acromegaly. Clin Endocrinol 2013; 78: 424–430.
9. Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: Minimizing risk. Ann Clin Biochem 2011; 48: 418–432.
10. Vogeser M, et al. Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem 2010; 56: 1234–1244.
11. Duxbury K, Owen LJ, Gillingwater S, Keevil BG. Naturally occurring isotopes of an analyte can interfere with doubly deuterated internal standard measurement. Ann Clin Biochem 2008; 45: 210–212.
12. Davison AS, Milan AM, Dutton JJ. Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2013; 50: 274.
13. Twentyman JM, Cradic KW, Singh RJ, Grebe SK. Ionic cross talk can lead to overestimation of 3-methoxytyramine during quantification of metanephrines by mass spectrometry. Clin Chem 2012; 58: 1156–1158.
The authors
Phillip J. Monaghan*1 PhD, Laura J. Owen2 MSc, Peter J Trainer3 MD, and Brian G Keevil2 MSc
1Department of Clinical Biochemistry, 3Department of Endocrinology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK.
2Department of Clinical Biochemistry, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK.
*Corresponding author
E-mail: Phillip.Monaghan@nhs.net
January 2025
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