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
The threat of allergies, which affect about one in five people in the US and Europe is emerging as a major public health challenge. The problem is also fast becoming severe in the developing world.
Very much an enigma
In spite of these trends, the World Allergy Organisation (WAO) notes that “services for patients with allergic diseases are fragmented and far from ideal,” and that this is true even in the developed world. The key reason is that allergies still remain little understood.
In Europe, for example, the EU Commission acknowledges that the epidemiology of allergies remains “very much an enigma.” In spite of “relatively homogeneous lifestyles” across the region, allergy rates vary from 3.7% among 13-14 year olds in Greece to 32.2% for the same age group in the United Kingdom.
Children hit hardest
As hinted by the EU Commission figures above, the impact of allergies is especially pronounced in a vulnerable demographic, namely children.
Indeed, the Florence, Italy-based European Academy of Allergy and Clinical Immunology (EAACI) reports that “the number of children with allergies has doubled in the last ten years, and visits to A&E have increased seven-fold.” The situation is no different in the US, where a recent study by the Centers for Disease Control and Prevention (CDC) finds allergy to be among the most common medical conditions affecting children aged below 17.
The allergy challenge has been confounded by the fact that its origins now include a bewildering (and growing) range of food products. In Europe, the InformAll Database (developed with funding from the European Union) currently contains information about the “more than 120 foods” reported to be associated with allergy.
The burden of this, once again, is disproportionately high on children. Globally, an estimated 220 to 250 million people could be suffering from food allergy, according to the WAO.
In Britain, the respected National Institute for Clinical Excellence (NICE) zeroes down on food allergy as being “among the most common of the allergic disorders” and “a major pediatric health problem” because of “the potential severity of reactions and a dramatic increase in prevalence over the past recent decades.”
Food allergies a specific challenge
Though the CDC study mentioned above found the biggest challenge for US children to be respiratory allergies, their share – at 17% – has remained constant since the late 1990s. The fastest growth, on the other hand, was shown with skin allergies, up from 7.4% in 1997–1999 to 12.5% in 2009–2011.
In contrast, the prevalence of food allergies in US children is not only smaller than either respiratory or skin allergies, but also showed a slower increase than the latter, from 3.4% to 5.1%.
However, the US figures conceal more than they reveal.
Firstly, managing (or even) identifying food allergies is not straightforward. Unlike respiratory allergies (which have a long-established intervention modus), or skin allergies (which are easier to pinpoint), the diagnosis of food allergies is far more problematic. This is because “nonallergic food reactions, such as food intolerance, are frequently confused” with food allergies.
The allergy continuum
Making things worse is the allergy continuum.
According to a review of two million patient visits in the US (the largest ever of its kind), food allergies in childhood are instrumental in the so-called ‘allergy march’, a medical condition by which there is an escalation in the risk “for the development of additional and more severe allergy-related conditions, including asthma, later in life.”
In other words, tackling food allergies effectively may hold the key to reducing the burden of other allergies in later life.
Profiling allergies: differences between children and adults
Food allergies in children are most commonly caused by eggs, milk, peanuts, tree nuts and wheat; in adults, milk and wheat are excluded as typical allergens, and instead replaced by fish and shellfish.
However, the EU Commission’s observation about the ‘enigma’ of allergies applies to food too. “In continental Europe, the most common food allergies are to fresh fruit and vegetables, whilst in Anglo-Saxon countries hazelnuts, peanuts and walnuts are the most problematic. Allergy to fish and shellfish prevails in Scandinavia and Northern Europe.”
Tracking the severity of allergies
An allergic reaction to food usually occurs quite quickly (in some cases, within minutes of eating a particular food, and in others, 2-3 hours afterwards). Typical symptoms include an abnormal swelling of the tongue, diarrhea, and hives.
In severe cases, the reaction (as with other allergies) is anaphylaxis, which can be life-threatening.
A study by Mayo Clinic covering a period of 10 years (1990 to 2000) found an age-specific rate for anaphylaxis highest in the under-19 year population (at 70 per 100,000 person-years, compared to an overall age- and sex-adjusted rate of just under 50). The Mayo clinic study also found that ingested foods accounted for one-third of all cases (33.2%), significantly ahead of the second- and third-ranked causes: insect stings with 18.5% and medication with 13.7%.
As troubling is the growth in the incidence of anaphylaxis, again in children. Hospital data from New York State shows that hospitalization for anaphylaxis among patients younger than 20 increased more than 4-fold between 1990 and 2006.
Growing costs
The economic impact of food allergy is significant. In the US, children’s food allergies are estimated to cost as much as $24.8 billion per year.
It is also growing. In the UK, hospitalization for food allergies has increased by as much as 500% since 1990.
Food allergies cannot be cured, but they can be managed by dietary control – in other words through avoidance of allergen-inducing foods. However, there is sometimes little room for a learning curve. In certain people, even tiny amounts of a food allergen (for example, 1/44,000 of a peanut kernel) can prompt an allergic reaction.
Currently, aside from avoidance, the standard of care for food allergies remains “ready access to self-injectable epinephrine.”
Both the US National Institutes of Health (NIH) and Britain’s NICE have drawn up recommendations for the diagnosis and management of food allergy. At the European level, the European Academy of Allergy and Clinical Immunology (EAACI) published its first guidelines on the subject in summer 2013.
An ‘allergy epidemic’: the institutional response
However, the challenge of food allergies is likely to continue.
One key gap is an institutional network of qualified specialists, which link in seamlessly into the wider public health system. In 2006, a subcommittee at Britain’s House of Lords concluded that allergy services were insufficient to deal with it and described the growing incidence of allergic conditions as an ‘allergy epidemic’. Their recommendations urged setting up “at least one allergy centre, led by a full time allergy specialist” in each Strategic Health Authority, supported by “a chest physician, dermatologist, ENT specialist, clinical immunologist, gastroenterologist, occupational health practitioner and pediatrician,” and assisted by “specialist nurses and dieticians trained in allergy.”
The House of Lords subcommittee also strongly called for “diagnostic facilities necessary to investigate complex allergies” staffed by personnel who have received “accredited allergy training.” In other words, such a system will only be meaningful if laboratories are harnessed to address the exploding allergy challenge, and provided with sufficient funds for equipment and staff. Until such time, the response will mean little more than using best practices guidelines (from bodies such as the NIH, NICE and the EAACI) to streamline what essentially remains an ad-hoc infrastructure.
The need for support by clinical labs is implicit in the NICE guidelines on food allergies, which stress that “skin prick tests and blood tests are equally cost-effective” and that “blood tests are cost-effective independent of number of individuals tested.” On the other hand, the guidelines also highlight the need for “valid test results” “to reduce incidence of adverse reactions and improve quality of life,” and prevent the (yet unquantified cost of) anxiety and worry, as well as the “avoidance of food that is actually safe to eat.”
The future: no cures in sight, yet
As of now, there is no cure for food allergies.
A seemingly-promising Phase II, randomized, double-blind study on the anti-asthmatic omalizumab against peanut allergy (one of the most dangerous food allergies) was stopped in 2011, with most subjects not reaching the endpoints. The investigators concluded that “no firm conclusions can be drawn” from their effort, but said it deserved “further investigation.”
The omalizumab research actually reached the same deadlock as another similar anti-IgE preparation, HU-9015, in 2003. This study was, ironically, stopped after its sponsors found the prospects for omalizumab to be more promising.
Nevertheless, researchers continue with their efforts, especially with regard to peanut allergy. As of this date, according to a communication from the National Institutes of Health, 14 studies and trials on peanut hypersensitivity alone are recruiting volunteers, one more than for asthma.
March 2026
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