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
Interference in thyroid function tests – problems and solutions
, /in Featured Articles /by 3wmediaInterference in immunoassay is a well described phenomenon and all clinical immunoassays, including thyroid function tests, are potentially at risk. Spurious results can lead to over investigation or mismanagement if not detected, but a proactive approach by the laboratory will help to identify and resolve these problems.
by Dr Olivia Bacon and Dr David J. Halsall
Background
Thyroid disorders are relatively common, and are associated with long-term morbidity and mortality. Clinical signs and symptoms are often non-specific, so reliable laboratory tests are critical for diagnosis. Therefore, thyroid function tests (TFTs) are frequently requested immunoassays with around 10 million results being reported each year by UK laboratories. In the UK, TFTs typically include a high sensitivity immunoassay for thyroid stimulating hormone (TSH) with an immunoassay estimation of non-protein bound thyroxine (fT4), either run simultaneously or added if the TSH value is outside the reference interval [1].
For the majority of tests, both results will be within the reference interval and thyroid disease can be excluded. In some patients TFTs support the diagnosis of hypothyroidism (raised TSH with fT4 low, or lownormal) or hyperthyroidism (TSH undetectable, and fT4 elevated), and these results will confirm clinical findings. However, due to the high volume of TFTs performed, it is not unusual for the laboratorian to be faced with a set of TFTs that are either internally inconsistent, or incompatible with the clinical details provided. Many medications can affect the thyroid axis, as can other non-thyroidal pathologies; these are often transient, but can cause unusual patterns of TFT. Much rarer genetic or pituitary conditions can also cause discordant TFTs [2]. However, if drug effects are excluded, it is necessary at this stage for the laboratorian to consider that one of the TFT results is incorrect, as analytic error is at least as common as these rare thyroid conditions. As spurious TFT results can lead to over investigation, or even inappropriate treatment, it is critical, but not trivial, for the laboratory to confirm the analytical validity of the TFT results.
In one study of more than 5000 samples received for TSH analysis, assay interference with the potential to adversely affect clinical care was detected in approximately 0.5% of patients [3]. This equates to a rather alarming 50,000 tests per annum in the UK.
Although assay design is continually improving, no routine immunoassay is currently robust to interference. Technical errors with many routine chemistry methods caused by inappropriate sample collection or handling, chemical or spectral interference can be detected during result validation. However, detection of spurious TFT immunoassay results is more challenging as there is no automatic ‘flag’ from the analyser, and there is usually a wide range of plausible values for these analytes, making it difficult to question those which are ‘suspicious’. Consequently clinical validation, where results are checked for discordance with the clinical correlates and other laboratory tests, is used to detect potentially incorrect results before reporting. For TFTs this is aided by the characteristic reciprocal relationship between TSH and fT4 in patients with an intact pituitary–thyroid axis.
Mechanisms of interference in TSH assays
Endogenous interfering antibodies are a well described cause of immunoassay interference [4]. In TSH assays these antibodies can have affinity for TSH itself or towards assay components. Anti-reagent antibodies can be ‘anti-animal’ antibodies, specific to the species in which the reagent antibody was raised, or weak, polyspecific ‘heterophilic’ antibodies, which may be part of the natural process of the generation of antibody diversity [5]. Anti-animal antibodies are more prevalent in animal handlers or patients treated with therapeutics based on animal immunoglobulins.
Anti-reagent antibodies can interact with either the capture or detection antibodies in two-site assays, blocking the generation of signal in the presence of analyte (false negative result) or by causing antibody cross-linking in the absence of analyte (false positive result) [Fig. 1].
Anti-TSH antibodies can generate high molecular weight TSH : antibody complexes (‘macro-TSH’). Depending on the exact site of the antibody–analyte interaction, false positive TSH results may occur as the macro-TSH is unlikely to be biologically active [6].
Detection of interference in TSH assays
Once suspected, a robust laboratory strategy is required for confirming or excluding assay interference. Method comparison using an alternative method is often used as the first step. Most laboratories use two-site immunoassays for TSH, but assay formulations, antibody species and incubation times vary between manufacturers. Varying amounts of blocking agents, designed to prevent non-specific binding of heterophile antibodies, may be included. Significant disagreement between two TSH methods is a strong indicator of assay interference.
Dilution studies are a simple but effective tool to investigate the analytical validity of an immunoassay. Non-linearity to dilution suggests a result is unreliable. However, although a good ‘rule in’ test, linearity to dilution alone cannot be used to exclude interference [3,7].
Immunosubtraction is a useful method to confirm the presence of antibody interference. This can be done crudely using polyethylene glycol (PEG) precipitation or more specifically using anti-immunoglobulin agaroses. Proprietary heterophile blocking tubes can also be used to confirm the presence of this class of interferent [3,4].
Once assay interference is established it can still be difficult to determine the correct TSH value, as there is no ‘gold standard’ method for TSH. However, an alternative immunoassay result which gives the expected responses to dilution and immunosubtraction, and correlates with fT4 results plus clinical findings, can be used with a reasonable degree of confidence.
Mechanisms of interference in fT4 assays
fT4 assays present a particular analytical challenge as >99.9% of T4 in the serum is protein bound, and the unbound T4 fraction must be measured without upsetting the equilibrium between the two fractions [8]. Therefore, an abnormal T4 binding protein, or agent which affects binding protein affinity in vitro, has the potential to generate incorrect results. Most commercial fT4 assays are one-site immunoassays based on competitive principles, using either labelled T4 analogue or anti-T4 antibodies for detection. Both heterophile and anti-T4 antibodies therefore also have the potential to interfere with these methods [4].
Non-esterified fatty acids (NEFAs) are a common T4 displacing agent as they can release T4 from the low affinity, high capacity T4 binding site on albumin. NEFAs can be generated in vitro, usually as a consequence of heparin therapy, which stimulates the action of lipoprotein lipase on triglyceride. Although the measured fT4 result is genuinely high, it does not reflect the in vivo situation [9].
Familial dysalbuminaemic hyperthyroxinaemia (FDH) is a benign genetic condition where the affinity of albumin for T4 is increased, such that circulating albumin-bound T4 is elevated. Despite the high total T4 (tT4), concentrations of free hormone in vivo are unaffected due to the homeostatic regulation of the thyroid axis. However, FDH is often associated with falsely high fT4 measurements using commercial immunoassays [10] [Fig. 2]. Both the increased affinity of the variant albumin for some labelled T4 analogues, as well as potential disruption of the T4 : albumin equilibrium during the assay, are likely mechanisms. The presence of the FDH mutation can be confirmed using molecular genetic approaches.
Detecting interference in fT4 assays
Despite the greater analytical challenge, confirming interference in fT4 assays can be easier than for TSH due to the availability of physical separation methods, such as equilibrium dialysis, as ‘gold standard’ assays [8]. However, these methods are technically difficult and not available in most clinical biochemistry laboratories. Also, these methods do not solve the in vitro problems of hormone displacement.
Again a first approach is often method comparison, using a different immunoassay architecture. Dilution and immunosubtraction studies can also be informative, although some fT4 methods are not robust to matrix effects so careful control experiments are required.
Measurement of total rather than free T4 can be useful in situations where there is a suspicion of abnormal T4 binding proteins. For example, total T4 will be elevated in the presence of anti-T4 antibodies and in FDH.
Clinical causes of aberrant TFTs
As mentioned above there are well described pharmacological and pathological causes of unusual TFTs; an increased awareness of analytical artefacts should not detract from the detection of these conditions. For example thyroxine treatment, a TSH secreting pituitary tumour (TSHoma), the genetic condition thyroid hormone resistance, FDH or TFT antibody interference can give elevated fT4 results with a TSH within the reference interval. Attempts by the laboratory to exclude assay interference should complement both the diagnosis of transient and genetic thyroid conditions as well as the more common drug related effects.
Conclusions and future directions
Immunoassay manufacturers have invested considerable resources into reducing the potential for antibody-mediated assay interference, for example by including blocking agents, or using antibody fragments rather than intact antibodies as assay reagents. Although these measures are effective, it is worth bearing in mind that changes to assay formulations may introduce novel types of interference. We have observed negative interference in one fT4 assay which appears related to the presence of a blocking agent introduced to reduce the risk of positive interference in this method [11]. Mass spectrometric methods have been introduced to eliminate antibody interference in both fT4 and tT4 methods, but unfortunately the fT4 methods still require careful optimization to avoid interference caused by binding proteins and displacing agents.
As current TFT methods remain prone to analytical interference the clinical laboratory must remain vigilant to the potential for assay interference, promote effective communication with requesting clinicians, and have procedures in place for investigation of discordant results.
References
1. Association for Clinical Biochemistry (ACB), British Thyroid Association (BTA), British Thyroid Foundation (BTF). UK guidelines for the use of thyroid function tests.2006; www.acb.org.uk/docs/TFTguidelinefinal.pdf.
2. Gurnell M, Halsall DJ, Chatterjee VK. What should be done when thyroid function tests do not make sense? Clin Endocrinol. (Oxf) 2011; 74(6): 673–678.
3. Ismail AA, Walker PL, Barth JH, Lewandowski KC, Jones R, Burr WA. Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid-stimulating hormone and gonadotropin immunoassay results. Clin Chem. 2002; 48(11): 2023–2029.
4. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998; 44: 440–454.
5. Kaplan IV, Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem. 1999; 45: 616–618.
6. Halsall DJ, Fahie-Wilson MN, Hall SK, Barker P, Anderson J, Gama R, Chatterjee VK. Macro thyrotropin-IgG complex causes factitious increases in thyroid-stimulating hormone screening tests in a neonate and mother. Clin Chem. 2006; 52: 1968–1969.
7. Ross HA, Menheere PP, Thomas CM, Mudde AH, Kouwenberg M, Wolffenbuttel BH. Interference from heterophilic antibodies in seven current TSH assays. Ann Clin Biochem. 2008; 45: 616.
8. Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best Pract Res Clin Endocrinol Metab. 2013; in press.
9. Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab. 2009; 23(6): 753–767.
10. Cartwright D, O’Shea P, Rajanayagam O, Agostini M, Barker P, Moran C, Macchia E, Pinchera A, John R, Agha A, Ross HA, Chatterjee VK, Halsall DJ. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009; 55(5): 1044–1046.
11. Bacon O, Gillespie S, Koulouri O, Bradbury S, O’Toole A, Stuart-Thompson D, Taylor K, Pearce S, Gurnell M, Halsall DJ. A patient with multiple Roche serum immunoassay interferences including false negative serum fT4. Ann Clin Biochem. 2013; 50(Suppl 1): T50.
The authors
Olivia Bacon PhD and David Halsall* PhD, FRCPath, CSci
Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, Cambridge, UK
*Corresponding author
E-mail: djh44@cam.ac.uk
Trace elements and clinical chemistry
, /in Featured Articles /by 3wmediaPatients are routinely monitored for levels of trace elements to investigate situations of deficiency or toxicity. This article covers some of the reasons why trace elements are investigated in the clinical setting and discusses, with examples, how the measurements are carried out using advanced analytical instrumentation. It then goes on to suggest some important new developments in the field of inorganic mass spectrometry, which could have an important impact on future clinical assays.
by Dr Chris Harrington
Trace elements are defined as having a concentration of less than 100 µg/g or 100 mg/L and traditionally there are two main reasons for their measurement in a clinical setting: for the determination of deficiency or toxicity.
There are about 10 inorganic micronutrients essential for human health which include the transition elements Cu, Co (as vitamin B12), Cr, Fe, Mn, Mo and Zn, the metalloid Se and the halogen elements F and I. The human body also contains As, B, Ni, Si, Sn and V, but there is no firm evidence any of these are essential for health. These elements have a number of biochemical roles, e.g. co-factors for different enzymes; constituents of important molecules, such as the thyroid hormones; and electron transport, due to their redox chemistry. The toxic effect of any trace element is dose-dependent, but there are a number which exert toxicity at low concentration and examples of these include: Hg, Ti, Pb, Cd and As. The degree of harmful toxicity will not only depend on the concentration, but also on the actual chemical form and exposure time. In the case of an element such as As, it is highly toxic when present as arsine (AsH3) because it is a gas, but when exposure is via a more complex organometallic compound such as arsenobetaine (C5H11AsO2) which is common in fish and seafood, an equivalent dose of As would be harmless. Commonly exposure to a toxic trace element is determined by analysis of a urine sample normalized to the creatinine concentration and comparison to an established guidance value such as a biological exposure index (BEI). However, clearly in the case of As, a measurement of total As in the urine will not differentiate between different chemical forms. To achieve this aim each of the separate As-containing species will need to be determined using methods based on elemental speciation, whereby a chromatographic separation is coupled to a suitable atomic spectroscopic detector, for example HPLC-ICP-MS (inductively coupled plasma mass spectrometry in combination with HPLC).
A more recent development in the clinical measurement of trace elements relates to the orthopedic area and the increasing use of metal alloys containing Cr, Co, Mo and Ti as the components of metal-on-metal (MoM) hip replacements. As a result of complications with the use of such implants and the potential for failure requiring revision surgery, all patients in the UK with MoM replacements are now monitored on an annual basis. The guidelines issued by the UK Medicines and Healthcare products Regulatory Agency (MHRA) in 2010 [MDA/2010/033] and subsequently updated in 2012 [MDA/2012/008 and 036] provide advice to healthcare professionals involved in the management of patients implanted with MoM hip replacements. The initial alert recommended that all patients should be followed up regularly by measurement of Co and Cr in whole blood samples and that this should be carried out most frequently on patients with symptoms consistent with high rates of failure. The medical device alert stipulates that if either element was elevated above a concentration of 7 µg/L (134 and 119 nmol/L for Cr and Co respectively), then further tests should be performed including imaging, to identify patients with potentially failing MoM hip joints. Whereas there are already action limits for these elements relating to occupational exposure, the concentration of 7 µg/L was chosen after consultation with orthopedic clinicians and using information from the National Joint Registry for England and Wales, as a level at which the joint was not showing optimum performance. It was not set as an indication of toxicity but rather as an indicator of joint performance and is thus interpreted with this in mind.
Internal quality control and external quality assurance are important prerequisites for measuring trace elements and making appropriate diagnosis or treatment decisions. A good example of this is the routine annual follow-up of patients with MoM hip-replacements, where clinicians need to make sure that their decisions are based on well controlled analytical measurements. How, for instance, can a clinician decide if an increase in concentration of Co or Cr results from a change in the particular joint and does not arise from a change in the laboratories measurement performance? We recently looked at data [1] from the UK National External Quality Assessment Scheme for trace elements (TEQAS). This supplies whole blood specimens which are spiked with known amounts of a number of trace elements including Co and Cr. The mean recovery over the samples measured in the 2011–12 scheme year was 96.4% (SD 2.23, CV 2.3%) for Co and 96.1% (SD 3.19, CV 3.3%) for Cr. The excellent agreement between the amounts in the specimens and the mean value indicates the results are accurate, and agreement between the pools distributed on different occasions shows they are reproducible over time. This should provide the necessary confidence to the clinical decision maker that the laboratories providing the Co and Cr results are competent and the results are suitably accurate.
Analytical instrumentation
The instrumental mainstay of clinical laboratories which specialise in the measurement of trace elements is inductively coupled plasma mass spectrometry (ICP-MS), which is a form of inorganic MS measuring elemental ions rather than molecular ions. Developed as a commercial analytical technique in the early 1980s it was initially used in environmental and geological laboratories, but after instrumental improvements it is now gaining popularity in the clinical area. This is mainly because it is multi-elemental in nature.
A review of new research and instrumental approaches in the elemental analysis of clinical and biological materials, foods and beverages is published annually as an Atomic Spectrometry Update [2].
The instrumentation itself will not be discussed as many texts [3] deal with the fundamentals of ICP-MS. However, the significant strengths of the technique include: multi-elemental detection in a single run; wide elemental coverage up to m/z 254 (UO+); high sensitivity with low limits of detection, down to sub ng/L levels (limited by purity of the reagents); fast analysis times as a result of the scanning speed of the quadrupole analyser; wide linear working range, up to 9 orders of magnitude in the same run; isotopic information, making high accuracy calibration via isotope dilution mass spectrometry available; and it can be used for specialist applications such as speciation analysis, where it is used as a chromatographic detector for HPLC, GC, CE or GE separations. The main weaknesses of the technique are: the presence of isobaric interferences on some elements, which mean the isotope of one element is at the same m/z ratio for the analyte of interest, for instance Ca has an isotope at m/z 48 which is the same as the most abundant isotope for Ti, making the measurement of Ti in clinical samples problematic; the formation of polyatomic ions from sample matrix and atmospheric ions can impinge on the m/z for the analyte of interest, an example would be the measurement of Cr at its most abundant isotope at m/z 52 which has a major interference from the formation of ArC; and the formation of doubly charged ions, for instance Gd2+ can interfere with Se at m/z 78. Luckily instrumental developments based on the use of a reaction/collision cells containing a suitable gas have been introduced to overcome the problems due to polyatomics and doubly charged ions. These work by using a reactive gas, e.g. H2 in the cell and removing the interference by reactively neutralising it, which we have recently demonstrated for the removal of the Gd2+ interference from the measurement of Se [4], or using a collision gas, e.g. He to remove the larger polyatomic ions by collision induced kinetic energy discrimination. These newer instruments are extremely robust and can rapidly deliver highly accurate measurements for multi-elements at low concentrations in difficult matrices such as whole blood, serum or urine.
Future trends and developments
Over the last 5–10 years the capabilities of ICP-MS for the detection of molecules that do not contain a trace element have been investigated. By using a reagent with specificity for the analyte and which carries a metal or nanoparticle tag, the molecule of interest becomes visible for detection by ICP-MS. The reagents used are often antibodies, so the protocols often mimic those developed for immunochemical assays and, in theory, can be applied to the determination of the same analytes, including peptides, proteins and other specific biomarkers. So why would this be advantageous compared to conventional immunochemical assays? Most importantly this approach has a greater potential for multiplexing than spectroscopic methods; there are a large number of elemental tags to choose from and no overlap between them. As illustrated in Figure 1 this is not the case with fluorescence signals.
Other advantages include: analyte quantification with high precision; low detection limits; large dynamic range; low matrix effects from other components of the biological sample (i.e. contaminating proteins in the sample have no effect on elemental analysis); low background from plastic plates (i.e. plastic containers do not cause interference on elemental detection as it can with fluorescence), and superior spectral resolution. As can be seen in Figure 1 this has generated a new approach to flow-cytometry based on ICP-MS and it’s very likely that other new approaches to other biochemical analytes will shortly become commercially available.
Acknowledgements
Scott Tanner, University of Toronto for permission to use the figures from the CyTOF flow cytometer based on ICP-ToF-MS (DVS Sciences Inc).
References
1. Harrington CF, Taylor A. BMJ 2012; 344: e4017.
2. Taylor A, Day MP, Hill S, Marshall J, Patriarca M, White M. J Anal At Spectrom. 2013; 28: 425–459.
3. Inductively Coupled Plasma Mass Spectrometry Handbook. Ed. Nelms S, Blackwell 2005.
4. Harrington CF, Walter A, Nelms S, Taylor A. Ann Clin Biochem. 2013; submitted.
The author
Chris Harrington PhD, MRSC
SAS Trace Element Laboratory, Faculty of Health and Science, University of Surrey, Guildford, Surrey, GU2 7XH, UK
E-mail: Chris.harrington1@nhs.net
Managing and diagnosing diabetes at the point-of-care
, /in Featured Articles /by 3wmediaClinical decisions need to be made at the earliest possible time to facilitate the administration of quick and accurate treatment plans. Point of care testing (POCT) enables tests to be convenient and fast, making them suitable for use with a broad range of patients, including diabetics. Glycated hemoglobin (HbA1c) is commonly tested in diabetics as it provides a reliable measure of glycemic control (Figure 1). However, the role of HbA1c in the diagnosis of diabetes has only more recently been documented. HbA1c levels reflect average circulating glucose levels over the lifespan of red blood cells (2-3 months). Once hemoglobin molecules have been glycated, they become highly stable, enabling a greater level of clinical information to be obtained from them than a single glucose measurement taken at a particular point in time.
by Gavin Jones, Diabetes Product Manager, EKF Diagnostics
By taking serial HbA1c measurements, an individual’s control over their glucose levels can be assessed in response to changes in management strategies. Measurements should be taken every 2-6 months with target HbA1c levels set individually and therapy adjusted accordingly to provide the most effective treatment (1). The target ranges of HbA1c for diabetic patients, depending on their risk of severe hypoglycemia, cardiovascular status and co-morbidities, should be set between 6.5 – 7.5% DCCT (48 – 58 mmol/mol), with the non-diabetic reference range being 4.0 – 6.0% DCCT (20 – 42 mmol/mol). One point for consideration is that HbA1c results may be affected by any condition that leads to a change in red blood cell survival. But even then, HbA1c can be used to detect trends in a patient’s glycemic control.
HbA1c in POCT-based diabetes monitoring
HbA1c determination was originally based on methods such as ion exchange and affinity chromatography with alternative affinity and immunological methods following later, taking HbA1c into the POC environment.
Typically, when using laboratory-based testing, patients with existing diabetes are monitored for HbA1c every 2-6 months, requiring a visit to a nurse or phlebotomist and a follow-up appointment 1 to 2 weeks later to discuss the results. Use of POCT would mean that after just one visit, patients can leave with their results, eliminating the need for a follow-up appointment. By enabling an earlier therapeutic decision, diabetes control can be improved whilst also providing economic benefits in terms of cost and time.
Diabetes diagnosis
The benefits of HbA1c in the management of diabetes can also be directly applied to the diagnosis of diabetes. Unlike glucose levels, which are affected by what has been eaten and drunk in the previous 2-3 hours, the measurement of HbA1c levels does not require fasting. As a simple and immediate test for diabetes, POC HbA1c can support the early identification of at-risk individuals. This would rapidly enable them to make small changes to their lifestyle to significantly reduce the risk of developing type 2 diabetes.
Patients diagnosed with diabetes who are able to maintain low blood HbA1c levels also have a significantly reduced chance of complications after diagnosis (2); early detection by POCT can reduce this risk even further. The ability to rapidly assess and change these risk outcomes has significant health benefits and reduces the costs associated with recurrent leg ulcers, blindness, heart disease and stroke, for example, all of which are conditions and complications commonly associated with type 2 diabetes.
The World Health Organization (WHO) has recommended the use of HbA1c for the diagnosis of diabetes (3). In the UK for example, the National Institute for Clinical Excellence (NICE) has published guidelines for diabetes prevention which aim to identify people at high risk of type 2 diabetes and offer cost-effective, appropriate interventions to prevent or delay onset (4). Used in conjunction with a lifestyle health risk assessment, these guidelines advocate the monitoring of HbA1c levels to allow healthcare providers to advise individuals on treatment regimens, depending on their classification as low, moderate or high risk. Current guidance, therefore, supports the use of HbA1c in screening for type 2 diabetes, and in the management of patients with diabetes. The use of POCT could improve the management of patients with established diabetes in both primary and secondary care settings and enable earlier type 2 diabetes diagnosis.
What to look for in a POC HbA1c analyser
Most POC HbA1c analysers use a single drop of blood (4-10 µL), which is applied to a reagent cartridge. The cartridge is then directly inserted into a desktop device for analysis. Time to results is generally between 3 to 10 minutes, depending on the analyzer. This quick turn-around time, in combination with simple operation, is key to maintaining effective POC testing.
Simplicity
To minimize human error and the subsequent need for repeat testing, a POC analyser should be as easy as possible to use. Also the analyser should be highly intuitive, requiring little user training. Features that support ease-of-use include ready-to-use reagent cartridges which can be inserted straight into the analyser. The blood sample can then be added directly, without the need for premixing or pipetting. Minimizing the number of steps in the procedure not only reduces the potential for user error, but also helps to standardize results by eliminating variation from different users.
Audit trails
For patient safety purposes, audit trails must be readily available. Use of barcode scanning for patient and user identification, as well as confirmation of the batch of reagents and controls used, ensures an analyser can provide such information in a timely manner. Two levels of quality controls that are recorded and held within the analyser’s memory are also ideal for auditing purposes.
Certification
Certification of the analyser in order to confirm delivery of accurate, standardized results should also be a key consideration. In an effort to standardize HbA1c results, the AACC set up the ’National Glycohemoglobin Standardization Program’ (NGSP) in 1996. In parallel, the International Federation of Clinical Chemistry (IFCC) developed reference methods for glycated hemoglobin. In 2006 and 2007, an international consensus between IFCC and AACC was agreed upon (5).
The calibration and certification of laboratories and manufacturers to the same standards has improved the conformity of results. However, in practice, differences can still be observed among technologies and between individual systems. These observed differences arise because of heterogeneity of hemoglobins, underlying differences in technologies (e.g. ion exchange, boronate affinity, immunoassay), calibration drifts or lot to lot variability. Providing the manufacturer follows the recommendations of the IFCC and NGSP to ensure instruments and reagents are accurately aligned and traceable to the reference method, this should not be a problem.
Methodology
There are POC HbA1c analysers available (e.g., the Quo-Lab, EKF Diagnostics, Cardiff, UK) (Figure 2) where results are not affected by hemoglobin variants (which do not result in reduced erythrocyte life span), labile glycated hemoglobin or hematocrit levels. Such analysers use Boronate Fluorescence Quenching Technology (BFQT) (6) (Figure 3) which is associated with simple, yet powerful multiple optical measurements. This is based on well-documented boronate affinity chromatography systems used in reference laboratories. However, as BFQT does not require chromatographic separation, the methodology allows for fast, simple and accurate POC measurement of HbA1c to deliver comparable results to chromatography-based techniques.
Summary
Type 2 diabetes can be managed easily and effectively through the monitoring of HbA1c levels, as opposed to blood glucose. POC diagnosis enables early detection in higher risk patients, before any additional complications arise. POCT therefore not only improves patient access to testing, but provides accurate diagnoses there and then. Treatment strategies can be determined immediately, eliminating the need for a follow-up visit to discuss the results. The ability for diagnosis to occur near to the patient provides greater convenience, thus increasing the likelihood of compliance.
When selecting an analyser for use at the POC, users need to bear in mind that it needs to be a convenient and appropriate option. The focus should be on meeting regulatory requirements, as well as ease of use in order to ensure rapid testing, with accurate, standardized resulting data.
References:
1. Diabetes UK. HbA1c Standardization: Information for Clinical Healthcare Professionals. 2009. http://www.diabetes.org.uk/Guide-to-diabetes/Monitoring/Blood_glucose/Glycated_haemoglobin_HbA1c_and_fructosamine/HbA1c_Standardisation_Information_for_Clinical_Healthcare_Professional.
2. Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86.
3. World Health Organization. Use of glycated hemoglobin (HbA1c) in the diagnosis of diabetes mellitus. 2011. www.who.int/diabetes/publications/report-hba1c_2011.pdf.
4. National Institute for Health and Clinical Excellence. Preventing type 2 diabetes: risk identification and interventions for individuals at high risk. 2012. www.nice.org.uk/nicemedia/live/13791/59951/59951.pdf.
5. Geistanger A, Arends S, Berding C, Hoshino T, Jeppsson JO, Little R, Siebelder C, Weykamp C; on behalf of the IFCC Working Group on Standardization of Hemoglobin A1c. Statistical Methods for Monitoring the Relationship between the IFCC Reference Measurement Procedure for Hemoglobin A1c and the Designated Comparison Methods in the United States, Japan, and Sweden. Clin Chem. 2008 Aug;54(8):1379-85.
6. Wilson DH, Bogacz JP, Forsythe CM, Turk PJ, Lane TL, Gates RC and Brandt DR. Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection. Clinical Chemistry October 1993 vol. 39 no. 10 2090-2097
Research findings presented at AACC hold promise for improving patient care
, /in Featured Articles /by 3wmediaLeaders from the medical diagnostics, laboratory medicine, and healthcare fields convened in Houston, Texas, July 28 – August 1 for the AACC annual meeting, the world’s largest diagnostics conference and expo. Over 17,000 attendees took part in the event and the exhibit totalled more than 625 companies. A selection of research papers presented in Houston are summarized below.
New biomarkers for prostate cancer
Dimitra Georganopoulou, PhD, Ohmx Corporation, presented results of a pilot study to find a new biomarker for prostate cancer aggressiveness. The researchers measured the enzyme activity of prostate-specific antigen (PSA), termed the “aPSA”, in patient specimens that had been removed by radical prostatectomy. They wanted to determine if this activity level could be a clue to how aggressive the cancer was. The team found that there was a significant negative correlation between prostate cancer progression and the aPSA in prostatic fluid. Patients with the least amount of aPSA (PSA activity) had the most aggressive prostate cancer. Tests for an “aggressiveness biomarker” would provide critical information for making decisions about when clinical treatment should occur or when it could be postponed. Many men might be able to avoid radical treatments if their cancer was known to be non-aggressive. Likewise, men whose cancer was too aggressive to employ the “active surveillance” or “watchful waiting” approach would have more information to help them make meaningful personal decisions with the help of their doctors about what level of treatment was right for them. The findings from this study could lead to the development of a new tool to use along with existing screening tests.
PSA Enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness.
Dimitra Georganopoulou, PhD, OHMX Corporation, Evanston, Ill., U.S.A.
Diagnosing cystic fibrosis at the point of care
Xuan Mu from Peking Union Medical College presented test results from cystic fibrosis patients using an exciting new point-of-care method. Microfluidics and colour changes within a Band-Aid type of adhesive strip on the skin allow the new device to rapidly, accurately, and quantitatively diagnose cystic fibrosis in a small amount of sweat. Detecting sweat chloride has been the gold standard in diagnosing cystic fibrosis for more than 50 years. The new test detects increased chloride in sweat using a colour change in paper on an adhesive strip when a very small amount of sweat is absorbed. The intensity of changed colour is recorded with a cell phone camera, and is then measured against a colour model. Cystic fibrosis in an inherited disease of the body’s mucus glands. Technically a rare disease, the incidence of cystic fibrosis varies around the world and by ethnic group. Different mutations in the CFTR gene cause the severity and symptoms of CF to vary considerably. Respiratory and digestive systems are affected, as well as sweat glands and reproductive systems. The new point-of-care test device can distinguish healthy people from cystic fibrosis samples and conveniently integrates the many separate steps of current sweat chloride tests whose results take several hours to obtain. Treatment advances have increased the life expectancy of cystic fibrosis patients over the past several decades from the mid-teens in the 1970s to more than 36 years today in the U.S. An early diagnosis and a comprehensive treatment plan can improve both survival and quality of life of patients. This new method demonstrates a fast and cost-effective opportunity in diagnosing cystic fibrosis.
On-site colorimetric detection of sweat chloride ion for diagnosing cystic fibrosis.
Xuan Mu, Peking Union Medical College, Beijing, China
Determining the safety of olanzapine for schizophrenia and bipolar disorder
AACC member Werner Steimer from Munich, Germany presented the results of research showing that study patients who carried a specific genetic variation in an antipsychotic-metabolizing enzyme developed significantly higher serum concentrations of the drug olanzapine. The increased drug concentrations were still noteworthy even when researchers accounted for differences in the patient’s age, sex, weight, and other medications that they used. This is the first study to demonstrate that this polymorphism influences serum levels of olanzapine, and the study is extremely timely in the context of the recent FDA safety alert on the injectable form of olanzapine, an “atypical” or second generation antipsychotic medication. Under investigation are two unexplained deaths of patients who received an intramuscular injection of Zyprexa Relprevv (olanzapine pamoate) and showed very high blood levels of the drug, although they had received appropriate doses. They died 3-4 days after injection. Olanzapine is approved by the U.S. FDA for treating schizophrenia and bipolar disorder in adults and children older than 13, and is one of the most widely prescribed of the atypical antipsychotics. Olanzapine is available in tablet, injectable, and long-acting “depot” formulations. Long acting medications can be more tolerable to some patients and help them adhere to treatment. Olanzapine is metabolized in the liver by specific cytochrome P450 enzymes. Some individuals have genetic variations – polymorphisms – of cytochrome enzymes. These can impact the way that drugs are broken down and distributed throughout the body and sometimes even the strength or effectiveness of treatment.
The CYP1A2*1D Polymorphism has a significant impact on Olanzapine serum concentrations.
Werner Steimer, MD, Klinikum Rechts der Isar – Technische Universität München, Munich, Germany.
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