The determination of autoantibodies is an important component in the diagnosis and differentiation of glomerular disease. Key analyses include antibodies against phospholipase A2 receptors (anti-PLA2R), the glomerular basement membrane (anti-GBM), neutrophil granulocyte cytoplasm (ANCA), double-stranded DNA (anti-dsDNA) and nucleosomes (ANuA). With these tests autoimmune reactions can be identified as causative factors of renal disease.
by Dr Jacqueline Gosink
Glomerulonephritis (GN) is an inflammation of the blood-filtering structures of the kidneys (glomeruli) which can lead to kidney failure if left untreated. The disease is associated with the symptom complexes nephritic syndrome and nephrotic syndrome. Nephritic syndrome is characterised by hematuria, mild to moderate proteinuria and hypertension and is observedain diseases such as post-infectious GN, lupus nephritis, rapid progressive GN and IgA nephropathy. Nephrotic syndrome combines heavy proteinuria, hypoalbuminemia, hyperlipidemia and edema and is typical of membranous GN, minimal change GN and focal segmental glomerulosclerosis.
Because of the wide range of potential causes, the diagnosis of GN can be difficult. The diagnostic process is based on clinical examination, biopsy, and laboratory tests on urine and blood. The serological analysis of specific autoantibodies allows autoimmune forms of GN to be identified and distinguished from nephropathies of other origins, for example hereditary conditions, infections, drug intoxication, electrolyte or acid-base disturbances, diabetes and hypertension.
Autoantibodies in GN may be directed against specific renal targets, such as PLA2R or the GBM, resulting in diseases that predominantly injure the kidneys. Or they may be non-organ-specific, for example ANCA, anti-dsDNA or ANuA. Non-organ-specific autoantibodies cause damage to a wide variety of organs. Thus, GN may represent just one manifestation of a complex systemic autoimmune disease, for example systemic lupus erythematosus (SLE) or ANCA-associated vasculitis (AAV).
Anti-PLA2R antibodies
Autoantibodies against PLA2R are a new and highly specific marker for primary membranous glomerulonephritis (MGN), also known as idiopathic membranous nephropathy. Primary MGN is a chronic inflammatory autoimmune disease of the glomeruli and is one of the leading causes of nephrotic syndrome in adults. It is distinguished from secondary MGN, which is triggered by an underlying disease such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as SLE. Primary MGN accounts for 70-80% of cases of MGN, while the secondary form comprises around 20-30%. Clinical differentiation of the two forms is crucial since primary MGN is treated with immunosuppressants, whereas therapy for secondary MGN focuses on the causal disease.
The immune reactions leading to primary MGN, which were first described in 2009 [1], stem from autoantibodies binding to PLA2R (transmembrane glycoproteins, [Figure 1]) on the surface of the podocytes [Figure 2]. PLA2R of type M have been identified as the major target antigen of the autoantibodies. The antigen-antibody complexes are deposited in the GBM, triggering complement activation with overproduction of collagen IV and laminin. This damages the podocytes, resulting in protein entering the primary urine. With increasing proteinuria there is a higher long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications.
Primary MGN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to detect immunoglobulin-containing deposits in the GBM. Serological determination of anti-PLA2R antibodies supports the diagnostic procedure and has the advantage of being less time-consuming and less stressful for patients. Anti-PLA2R antibody analysis is, moreover, suitable for monitoring the activity of primary MGN and the response to therapy.
Until recently there was no reliable test to detect anti-PLA2R antibodies. A new recombinant-cell anti-PLA2R indirect immunofluorescence test (IIFT) developed to address this deficit has rapidly established itself as the gold standard for the serological diagnosis of primary MGN. The assay utilizes transfected human cells expressing recombinant PLA2R as the antigenic substrate [Figure 3] to provide monospecific antibody detection [2, 3]. The sensitivity of the test for primary MGN amounts to around 50-80% depending on the characteristics of cohort individuals, for example their disease activity or therapy status. In a retrospective clinical study [2] the Anti-PLA2R IIFT demonstrated a sensitivity of 52% in a cohort of 100 patients with biopsy-proven primary MGN and a specificity of 100% with respect to control subjects. In the first prospective study [4] the sensitivity amounted to 82% in patients with biopsy-proven MGN where no secondary cause could be found. An ELISA based on purified recombinant PLA2R has also been developed. It demonstrates >98% correlation with the IIFT and is particularly useful for quantification of antibody levels in therapy monitoring.
Anti-GBM antibodies
Autoantibodies against GBM are a highly specific and sensitive marker for Goodpasture’s syndrome, a rare, but potentially fatal autoimmune disease which is characterized by rapidly progressive GN and lung haemosiderosis. Diagnosis of this disease is challenging because of the speed of progression to organ failure and the initially unspecific symptoms. Serological parameters such as anti-GBM play a crucial role in obtaining an early diagnosis.
The primary target antigen of anti-GBM antibodies is the NC1 domain of the alpha chain of type IV collagen. The antibodies target the alveolar basement membrane or the GBM. In cases without lung involvement they are detected in more than 60% of patients and in cases with lung involvement in over 90%. Clinical progression of the disease correlates with antibody concentration, with high-titre circulating anti-GBM antibodies indicating an unfavourable prognosis.
Anti-GBM antibodies can be detected serologically by IIFT using sections of primate kidney as the antigenic substrate. Inclusion of a second substrate comprising microdots of purified GBM allows results to be confirmed at a glance. The substrates are positioned side by side as BIOCHIP Mosaics in the test fields of a microscope slide [Figure 4] and incubated in parallel. Further substrates for differential diagnostics, for example HEp-2 cells, granulocytes or other microdot substrates, can also be included in the BIOCHIP Mosaics, yielding a detailed patient antibody profile following a single incubation. Serum anti-GBM antibodies can alternatively be detected or confirmed quantitatively using the Anti-GBM ELISA.
ANCA
ANCA determination is a well-established tool for serological diagnosis and differentiation of different types of AAV, which often present as a rapidly progressive GN among other symptoms. The most important ANCA parameters include antibodies against proteinase 3, which are sensitive and specific markers for Wegener’s granulomatosis, and antibodies against myeloperoxidase (MPO), which occur in microscopic polyangiitis and other forms of AAV.
The standard method for detecting ANCA is IIFT using granulocytes to identify the typical staining patterns of anti-PR3 antibodies (cytoplasmic, cANCA) and anti-MPO antibodies (perinuclear, pANCA). BIOCHIP Mosaics are particularly useful for this application as they allow different substrates to be combined and analysed in parallel [Figure 5]. Recently, several new substrates have been developed to improve the ease and reliability of ANCA analysis still further. HEp-2 cells coated with granulocytes allow immediate differentiation between ANCA and anti-nuclear antibodies, while BIOCHIPs containing microdots of purified MPO or PR3 enable monospecific antibody characterization at the same time as the ANCA screening [5, 6].
Monospecific enzyme immunoassays such as ELISA or immunoblot are used to characterize the specificity of the target antigen. A recent major advance in ANCA ELISA is the development of a novel PR3 diagnostic antigen comprising an optimized mixture of native human (hn) PR3 and designer recombinant PR3 expressed authentically in human cells (hr). An ELISA based on this combined antigen provides unsurpassed sensitivity for the detection of anti-PR3 antibodies – 14% higher than even a capture ELISA (7). The Anti-PR3-hn-hr ELISA thus enhances ANCA diagnostics and is also suitable for long-term evaluation of patients.
Anti-dsDNA and anti-nucleosome antibodies
Anti-dsDNA and ANuA are among the immunological parameters used to diagnose SLE, which counts nephritis among its many and variable manifestations. These two markers provide the highest specificity and sensitivity in the serological diagnosis of SLE.
Anti-dsDNA antibodies are found in 60-90% of patients and represent the most established marker for SLE. A recently developed ELISA provides an exceptionally high sensitivity and specificity for detection of these antibodies owing to the use of a novel coating technology based on highly adhesive nucleosomes. The unspecific reactions that typically occur with traditionally used coating materials are thus avoided, and the clear presentation of the major DNA epitopes ensures a remarkably high sensitivity. In a published clinical comparison study using a large cohort of patients with SLE and other diseases [8], the Anti-dsDNA-NcX ELISA demonstrated the highest sensitivity for SLE (60.8%), exceeding that of conventional ELISA (35.4%), Crithidia luciliae IIFT (27.4%) and even Farr-RIA (53.1%) [Figure 6].
ANuA [Figure 7] are specific for SLE and are a prognostic indicator for SLE with renal involvement. The frequency of ANuA is especially high in severe cases requiring transplantation (79%), compared to less severe lupus nephritis (18%) and SLE without nephritis (9%) [9]. The relevance of ANuA is, however, highly dependent on the assay used to detect them. If insufficiently purified nucleosomes are used in ELISA, then sera from patients with scleroderma or other diseases also frequently react, resulting in an unacceptably low specificity. The 2nd generation Anti-Nucleosome ELISA, in contrast, is based on a patented preparation of highly purified mononucleosomes, which are free of contaminating histone H1, non-histone proteins such as Scl-70, and chromatin DNA fragments. This ELISA provides an SLE specificity of close to 100% and a sensitivity of around 54%. Significantly, with this highly specific test ANuA have been shown to be present in 16-18% of SLE sera that are negative for anti-dsDNA antibodies [Table 1] [10, 11]. Thus, the determination of ANuA substantially enriches the serological diagnosis of SLE. When both ANuA and anti-dsDNA antibodies are analysed in parallel as first-line serological tests, the detection rate for SLE can be increased to 87%.
Conclusions
Recent developments in autoantibody diagnostics for nephrology include the groundbreaking anti-PLA2R IIFT for identifying primary MGN, as well as considerable improvements in the sensitivity, specificity and convenience of tests for ANCA, anti-GBM, anti-dsDNA and ANuA. These advances have boosted the ease, reliability and relevance of autoantibody testing, aiding the diagnosis of autoimmune forms of GN, especially in their early stages. This is crucial to allow the implementation of interventional therapy and prevent the nephropathy progressing to a fatal end stage.
References
1. Beck et al. N. Engl. J. Med. 2009: 361: 11.21
2. Hoxha et al. Nephrology Diagnosis Transplantation 2011: 26 (8): 2526-32.
3. Debiec et al. Nat. Rev. Nephrol. 2011: 7(9): 496-8
4. Hoxha et al. Kidney International. 2012: 82: 797-804
5. Buschtez et al. Zeitschrift für Rheumatologie 2007: Band 66: 43, 10942-10.
6. Damoiseaux et al. JIM 2009: 348: 67-73
7. Damoiseaux J. et al. Ann. Rheum. Dis. 2009; 68: 228-233.
8. Biesen et al. Lupus 2008; 17(5): 506-507.
9. Stinton et al. Lupus 2007; 15: 394-400.
10. Suer et al. J. Autoimmunity 2004: 22: 325-334.
11. Schluter et al. J. Lab Med. 2002; 26: 516-517.
The author
Jacqueline Gosink, PhD
Euroimmun AG
Luebeck, Germany
Patients 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
Despite some limitations, current molecular diagnostic methods have a great potential to include targets useful in the rapid identification of microorganisms and antimicrobial resistance, to analyse directly unprocessed samples and to obtain quantitative results in pneumonia, an entity of complex microbiological diagnosis due to the features of the pathogens commonly implicated.
by Dr A. Camporese
A change in culture without culture?
Developing accurate methods for diagnosing respiratory tract infections has long been a challenge for the clinical microbiology laboratory [1].
The current semi-quantitative agar-plate based culture method used in most clinical microbiology laboratories for analysing specimens from patients with suspected community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), or ventilator-associated pneumonia (VAP), although adequate for recovering and identifying a wide variety of bacterial species from respiratory specimens, is slow, and cannot differentiate between colonization and infection. Moreover, results may be misleading, particularly if a Gram stain is not performed in parallel to ascertain the adequacy of expectorated sputum samples or endotracheal aspirates [2].
As the Infectious Diseases Society of America (IDSA) and American Thoracic Society (ATS) CAP guideline notes, one of the problems with diagnostic tests for respiratory tract infections “is driven by the poor quality of most sputum microbiological samples and the low yield of positive culture results” [3]. Moreover, the highest predictive value of a culture occurs only when Gram stain shows a predominant morphotype, and the culture yields predominant growth of a single recognized respiratory pathogen of that morphotype (e.g., Streptococcus pneumoniae) [2].
Unfortunately, such concordance decreases rapidly when specimens are collected after the initiation of antimicrobial therapy or when their arrival at the microbiology laboratory is significantly delayed.
One approach that may improve the diagnosis of respiratory tract infections and shorten the time necessary to place patients on appropriate therapy is the use of nucleic acid amplification methods.
Straight ahead toward molecular assays
Today clinical microbiologists appear to be on the threshold of a potentially important transition, with a substantial increase in the use of molecular diagnostic tests to replace or augment the century-old methods of culture, as many experts now view traditional microbiology as slow and antiquated, especially when compared with newer technologies used in other areas of laboratory medicine [4].
Traditional methods demonstrated poor sensitivity and specificity for detecting specific pathogens, particularly when the specimen being cultured is from a non-sterile anatomical compartment, such as the respiratory tract.
For this reason, molecular methods are becoming more widely used also for the detection of respiratory pathogens, in part because of their superior sensitivity, relatively rapid turnaround time, and ability to identify pathogens that are slow growing or difficult to culture.
However, to have a positive impact on patient management, molecular tests will need to be easy to use, and provide clear, definitive results that will give physicians the data necessary to start, or in some cases withhold, antimicrobial agents [5].
Further, to be really successful, industry must determine which combination of molecular targets [Table 1] and clinical specimens will produce results that will effectively guide anti-infective therapy regimens for patients with pneumonia or other respiratory tract diseases.
Another key challenge for industry will be to develop assays that are not only rapid, but also readily accessible, because development of an assay that is rapid, but unavailable on evening or night shifts, or at weekends, because of its technical complexity, limits the clinical value of the test.
Moreover, to be successful, molecular assays will need to be perceived by health care systems as cost-effective, but cost-effectiveness should be determined not only by comparison to the costs of performing slower, conventional methods in the laboratory, but also by consideration of the cost savings achieved from optimized antimicrobial therapy, decreased use of additional diagnostic tests, and shorter hospital stays [2].
To address issues on these topics, the IDSA and the Food and Drug Administration (FDA) co-sponsored a workshop on molecular diagnostic testing for respiratory tract infections in November 2009, with the participation of the FDA, industry, authorities in microbiology, statisticians and others. Respiratory tract infections were selected because this is the site of most infections treated with antibiotics in paediatric and adult practice, and they also represent a group of infections in which an etiologic agent is seldom identified in non-research settings [4].
The IDSA believes that patient care could be improved by accurate and rapid identification of pathogens, which would promote more judicious use of antibiotics, permit pathogen directed therapy, and provide potentially important
epidemiologic information.
Thus, the IDSA strongly desires development and implementation of molecular diagnostic tests that are easy, rapid, technically uncomplicated, applicable to specimens that are readily obtained, reasonably priced, sensitive and specific, because such tests will greatly improve antimicrobial stewardship, thereby helping to reduce the spread and impact of antibiotic resistance. Such tests will also facilitate conduct of clinical trials supporting the approval of new antibacterial agents [4].
Respiratory samples suitable for molecular assays
A variety of respiratory samples are amenable to molecular testing, including expectorated sputum, bronchoalveolar lavages (BALs), protected bronchial brushes, and endotracheal aspirates [2, 5, 6]. Of these, expectorated sputum samples are by far the most common respiratory samples submitted to the clinical microbiology laboratory, but are also the poorest in overall quality.
Endotracheal aspirates from ventilated patients are often of better quality than that of expectorated sputum obtained from patients with CAP/HAP, but may still be contaminated with upper respiratory tract flora.
Therefore, obtaining specimens from the site of infection that are not contaminated with upper respiratory tract flora remains to date a real and constant problem. BALs and protected brush samples seem more likely to yield samples from the site of infection, but require significantly more effort to obtain, and thus offer a much smaller market for a new molecular test.
Moreover, there is a significant gap in our knowledge as to how well molecular tests for bacterial pathogens would perform on expectorated sputum samples, compared with performance on BALs or protected brush samples from the same patient collected within a similar period [2].
This knowledge gap is also a barrier to test development, because a molecular test that cannot be performed on expectorated sputum (given all the problems with specimen quality) may not have broad enough appeal among physicians to make it a financially viable product (from the industry perspective).
Technology perspectives
There are a wide array of emerging technologies for the detection and quantification of respiratory pathogens directly from clinical specimens. Some of these technologies, such as real-time PCR, have potential for high-throughput testing, and others will allow rapid near patient testing, but more studies are needed to fully determine their performance characteristics and determine their ideal clinical application [6,7].
Molecular assays may target either a single pathogen or multiple respiratory pathogens in a single assay. There are merits to both single-pathogen and multiplex approaches. Certain bacterial respiratory pathogens cause such distinct clinical syndromes that assays that target them individually still have clinical utility. These include already many organisms, such as Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, or Bordetella pertussis [Table 1].
Moreover, some multiplex assays for respiratory tract disease already include many targets for a rapid diagnosis of CAP, HAP, and VAP, but, in designing new assays, it will be critical to understand whether an assay for a determined number of bacterial pathogens will meet physicians’ needs and provide adequate data for initiating or altering anti-infective therapy [7, 8].
Potential and currently available targets for multiplex or individual molecular assays for respiratory tract samples in immunocompetent and/or immunocompromised patients with CAP, HAP, or VAP are presented in Table 1 [7].
Further, in this age of multidrug resistance, expanding the target selection to include key antimicrobial resistance genes that would alter existing therapy or guide empirical therapy, should also be considered [Table 1].
Lastly, if molecular-based diagnostic methods currently available are helpful in detecting single and multiple bacterial pathogens simultaneously, including the most frequent cause of CAP/HAP/VAP, the real-time PCR is also well known for its ability to quantify targets.
Where available, the application of quantitative molecular tests for the detection of key pathogens, such as S. pneumoniae, both in sputum and in blood, defining a threshold for classification, such as a colonizer or as an invasive pathogen, might be relevant in CAP patients, mainly in whom antibiotic therapy has been initiated, and might be a useful tool for severity assessment [9, 10].
Conclusion
Significant progress exists on the development and improvement of molecular-based methods feasible to be applied to the diagnosis of lower respiratory tract infection.
Multiplex assays, user-friendly formats, results in a few hours, high sensitivity and specificity in pathogen identification, detection of antibiotic resistance genes and target quantification, among others, are some of the contributions of novel molecular-based diagnosis approaches.
Developing new molecular tests for other bacterial respiratory pathogens, particularly microorganisms that can be both asymptomatic colonizers and overt pathogens of the respiratory tract, detection of pathogens and new key antimicrobial resistance genes in unprocessed samples, and determination of the microbial load by quantitative multi-pathogen tests will be some of the future challenges of molecular diagnosis in CAP/HAP/VAP.
References
1. Bartlett JG. Decline in microbial studies for patients with pulmonary infections. Clin Infect Dis 2004; 39: 170–172.
2. Tenover FC. Developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens. Clin Infect Dis 2011; 52(S4): S338–S345.
3. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007; 44(S2): S27–S72.
4. Infectious Disease Society of America (IDSA). An unmet medical need: rapid molecular diagnostics tests for respiratory tract infections. Clin Infect Dis 2011; 52(S4): S384–S395.
5. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011; 52(S4): S326–S330.
6. Lung M and Codina G. Molecular diagnosis in HAP/VAP. Curr Opin Crit Care 2012; 18: 487–494.
7. Camporese A. Impact of recent advances in molecular techniques on diagnosing lower respiratory tract infections (LRTIs). Infez Med 2012; 4: 237–244.
8. Johansson N, Kalin M, Tiveljung-Lindell A, et al. Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods. Clin Infect Dis 2010; 50: 202–209.
9. Werno AM, Anderson TP, Murdoch DR. Association between pneumococcal load and disease severity in adults with pneumonia. J Med Microbiol 2012; 61: 1129–1135.
10. Woodhead M, Blasi F, Ewig S, et al. Guidelines for the management of adult lower respiratory tract infections-Full version. Clin Microbiol Infect 2011; 17(S6): E1–E59.
The author
Alessandro Camporese MD
Clinical Microbiology and Virology Department
S. Maria degli Angeli Regional Hospital, Pordenone, Italy
E-mail: alessandro.camporese@aopn.fvg.it
Currently, the diagnosis of bowel diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) relies on invasive and expensive procedures. Identification of biomarker-based tests to aid diagnosis is an important area of research. Here we review the use of mass spectrometry in this search and discuss recent findings.
by Dr B. De Lacy Costello, Professor N. M. Ratcliffe and S. Shepherd
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disease caused by an inappropriate response of the immune system to commensal gut microbes [1]. There are two types of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). UC affects the large bowel only, affecting variable lengths of the colon continuously from the rectum, primarily affecting the mucosa [Fig. 1]. CD can affect any part of the GI tract, and is a transmural disease [2]. Common symptoms of IBD are severe abdominal pain, defecation urgency and diarrhoea, which can contain blood.
Irritable bowel syndrome (IBS) is a functional disorder of the digestive tract. It is characterized by its symptoms, with no physiological changes in the GI tract. IBS can be diarrhoea predominant (IBS-D), constipation predominant (IBS-C) or symptoms can alternate between the two (IBS-A). Common symptoms include abdominal pain and cramps, bloating and flatulence, and unusual bowel habit. IBS has, as yet, no known cause. People with IBS show abnormal gut motility and hypersensitivity to pain in the GI tract. Stress and anxiety are known to cause changes in gut motility [3] with stress and anxiety being common symptoms of IBS. When under physical or psychological stress IBS patients showed increased gastro-intestinal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut microbiota in patients with IBS, the evidence being that IBS symptoms often occur after infective gastroenteritis or in patients in remission from IBD or diverticulitis. SIBO (small intestinal bowel overgrowth) has also been implicated in IBS and other function bowel disorders. One current hypothesis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial permeability, causing dysregulation of the enteric nervous system [5]. Genome-wide association studies have successfully identified many genetic loci involved in susceptibility to IBD, and it is thought that genetic factors may also play a role in IBS [1].
Diagnosis of GI disease
IBS-D can present with symptoms similar to IBD and other non-functional bowel conditions. The diagnosis of IBS is often one of exclusion, where more serious bowel diseases, such as IBD or colon cancer which present with common symptoms, are ruled out. The current gold standard for diagnosis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have associated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with functional bowel disease are significant, with healthcare costs for IBS patients being significantly higher than non IBS controls [7].
There are currently no known biomarkers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastrointestinal disease, but there is still a need to identify biomarkers and to develop quicker, lower cost and less invasive testing for diagnosis of gastro-intestinal disease.
Biomarkers such as lactoferrin, calprotectin, c-reactive protein (CRP) and erythrocyte sedimentation rate (ECR) have all been used to help distinguish functional from inflammatory bowel disorders and to diagnose IBD. Serological markers such as antibodies to bacterial and fungal antigens that can indicate an abnormal response to commensal microbes can also be useful in identifying IBD.
Fecal calprotectin and lactoferrin are protein biomarkers of inflammation. In 2010 a meta-analysis of six studies (n=670) in adults by Van Rheenen et al. [8] found that screening patients by testing fecal calprotectin levels would have reduced the number of endoscopies performed by 67%, although its diagnosis would have been delayed in 6% of patients. When taking a weighted mean of 19 studies including 1001 patients, where IBD patients were compared with controls of IBS and other colonic diseases, fecal lactoferrin has a sensitivity and specificity of 80% and 82%, respectively [9].
Although these biomarkers can be useful as part of the screening process when establishing a diagnosis [6, 8], there is currently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry techniques are at the forefront of research for biomarker prospecting for IBS/IBD.
Mass spectrometry
Mass spectrometry (MS) has the ability to identify numerous compounds in a single sample. It is also high throughput allowing rapid analysis of many samples, which is especially useful for large studies or for the diagnosis of many samples. The ability to obtain results quickly, usually in less than 1 hour makes it attractive for clinical use.
Proteomic approach
Although MS (with associated sample vaporisation methods) was originally limited to low molecular weight volatile compounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, changing the way proteins are analysed. The soft ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of proteins and other macromolecules [10]. The identification of proteins through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequencing and data can be analysed automatically. MS can also be used to determine the abundance of a molecule in a sample [10].
Differential protein expression can identify different diseases, and can indicate the degree of the disease state, or be used to assess the effects of treatment – for example the response of IBD patients to anti-TNF alpha antibodies (infliximab) [11]. It also has applications in the identification of protein biomarkers.
In 2011 MALDI-MS was used by M’koma et al. for tissue analysis; through profiling of the proteome of the colonic submucosa they were able to distinguish UC from CD by comparing proteomic spectra. Definitive diagnosis of either UC or CD is important as people with UC also have an increased risk of colon cancer [12].
Goo et al. have investigated protein biomarkers for IBS. ESI with LC-MS was used on protein fragments from the urine of women with IBS. They found differences in some specific components of the urinary proteome, and demonstrated that there is a possibility for future biomarker studies for IBS [13].
There are still limitations to mass spectrometric protein analysis, for example the difficulty in detecting hydrophobic membrane proteins. However, it seems promising that, with the advances in mass spectrometry technology, there will be an increase in the discovery of protein biomarkers and key pathogenic factors of gastro–intestinal disease, and improved diagnosis and therapy.
Metabolomic approach
The metabolome is the set of small molecule metabolites found in a biological sample. Unlike proteomics, metabolomics can be a direct measure of production of compounds and activity of cells or systems in an organism. This can be especially useful when looking for disease biomarkers in IBS and other bowel diseases as it can be used to understand the environment of the GI tract, as well as factors such as digestion and absorption of dietary products and gut microbial activity [14], which are implicated in IBS pathogenesis.
Researchers have explored the use of various techniques incorporating MS on breath [15], urine [16] and stool [17] samples in search of metabolic biomarkers of bowel disease for non-invasive testing and many possible candidates have been identified.
The commonly used analytical techniques in metabolomics are GC-MS (gas chromatography-mass spectrometry) or LC-MS (liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) spectrometry. NMR has the advantage that there is no need to have the compounds in the vapour phase, although the limit of detection using NMR is much poorer than MS.
LC-MS metabolomic studies have been recently undertaken using urine to identify putative colon inflammation biomarkers [18]. The authors note that urinary biomarkers would be preferable to sampling intestinal tissue or blood as the collection of urine samples is non-invasive and multiple samples are more
readily obtained.
The analysis of volatile organic compounds (VOCs) or metabolites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other commonly used methods of VOC detection are selected ion flow tube mass spectrometry (SIFT-MS) [Fig. 2], and the similar technique of PTR-MS (proton transfer MS).
There are already several FDA approved tests using volatiles from breath. These include testing for heart transplant rejection, hemoglobin breakdown in children and measurement of hydrogen or methane to diagnose GI lactose or fructose malabsorption. The measurement of breath hydrogen has also been used to diagnose SIBO. Recent work by Španĕl et al. using SIFT-MS quantified the breath pentane concentration of study subjects using the reaction of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].
Testing for fecal biomarkers of bowel disease is facile as samples are easily obtained and have been in contact with the gastro intestinal tract. The changes in the odour of feces and flatus reported in many bowel conditions are due to changes in the VOC profile. This altered VOC profile could lead to identification of biomarkers of disease state. A recent pilot study carried out by Ahmed et al. using GC-MS on fecal samples from IBD and IBS patients identified a key set of VOMs which were able to distinguish IBS-D from Active IBD with a sensitivity of 96% and a specificity of 80% [19].
Conclusions
MS techniques show promise for the identification of biomarkers of various GI disease states, which have the potential to reduce invasive testing, improve patient care and reduce healthcare costs.
Instrumentation is still expensive and relatively large, limiting its use in hospital settings and particularly limiting its use for near-patient testing. Also biomarker discovery is still in its infancy and much remains to be clarified in relation to the significance of markers to disease and the underlying metabolic pathways.
However, work to reduce the size and cost of mass spectrometers is well advanced and would open up the possibility of instruments being deployed for point-of-care detection and monitoring of diseases including IBS and IBD.
References
1. Khor B, Gardet A, Xavier RJ. Nature 2011; 474(7351): 307–317.
2. Geboes K. Churchill Livingstone Elsevier 2003; 255–276.
3. Drossman DA, Camilleri M, Mayer EA, Whitehead WE. Gastroenterology 2002; 123(6): 2108–2131.
4. Murray CD, Flynn J, Ratcliffe L, Jacyna MR, et al. Gastroenterology, 2004; 127(6): 1695–1703.
5. Simrén M, Barbara G, Flint HJ, Spiegel BM, Spiller RC, et al. Gut 2013; 62(1): 159–176.
6. Kok L, Elias SG, Witteman BJ, Goedhard JG, Muris JW, et al. Clinical chemistry 2012; 58(6): 989–998.
7. Maxion-Bergemann S, Thielecke F, Abel F, Bergemann R. Pharmacoeconomics 2006; 24: 21–37.
8. Van Rheenen PF, Van de Vijver E, Fidler V. BMJ 2010; 341: doi 10.1136/bmj.c3369.
9. Gisbert JP, McNicholl AG, Gomollon F. Inflammatory bowel diseases 2009; 15(11): 746–1754.
10. Alberici RM, Simas RC, Sanvido GB, Romão W, Lalli PM, Benassi M, Eberlin MN. Analytical and bioanalytical chemistry 2010; 398(1): 265–294.
11. Han NY, Kim EH, Choi J, Lee H, Hahm KB. Journal of Digestive Diseases 2012; 13(10): 497–503.
12. M’Koma AE, Seeleyv EH, Washington MK, Schwartz DA, Muldoon RL, Herline A, Caprioli RM. Inflammatory bowel diseases 2011; 17(4): 875–883.
13. Goo YA, Cain K, Jarrett M, Smith L, et al. Journal of Proteome Research 2012; 11(12): 5650–5662.
14. Collino S, Martin FPJ, Rezzi S. British journal of clinical pharmacology 2013; 75(3): 619–629.
15. Hrdlicka L, Dryahina K, Spanel P, Bortlik M, et al. Gastroenterology 2012; 142(5): S-784.
16. Rao AS, Camilleri M, Eckert DJ, Busciglio I, Burton DD, Ryks M, Zinsmeister AR. Am J Physiol Gastrointest Liver Physiol 2011; 301(5): G919–G928.
17. Garner CE, Smith S, de Lacy Costello B, White P, Spencer R, Probert C, Ratcliffe NM. FASEB J. 2007; 21(8): 1675–1688.
18. Otter D, Cao M, Lin H-M, Fraser F, Edmunds S, et al. J Biomed Biotechnol. 2011; 2011: 974701
19. Ahmed I, Greenwood R, de Lacy Costello B, Ratcliffe NM, Probert CS. PloS one, 2013; 8(3): e58204.
The authors
Ben De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc
Institute of Bio-Sensing Technology, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY
*Corresponding author
E-mail: Norman.Ratcliffe@uwe.ac.uk
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
November 2025
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.