Autoantibody detection is a powerful laboratory tool for clinical diagnosis in the autoimmune diseases field. Among the techniques most widely used worldwide, indirect immunfluorescence (IFA) plays a particularly important role not only in the diagnosis but in the follow up of many diseases and remains the hallmark despite the introduction of new techniques in the routine of clinical laboratories. Witness to this is the renaissance of the antinuclear antibodies (ANA) screening on HEp2 cells by this techique or the renewal of the detection of anti-endomysium antibodies on monkey esophagus as the gold standard serological test for celiac disease. Therefore, IFA is a technique in full validity and requires a level of standardization that unfortunately is far from being achieved.
by Petraki Munujos, PhD
The efforts to improve standardization of indirect immunofluorescence as a diagnostic tool are numerous worldwide. Traditionally, the players involved in standardization have been clinical laboratories, clinicians, regulators, and to a lesser degree, diagnostic reagents manufacturers. Energy has been concentrated basically in aspects like the control of laboratory procedures, unification of nomenclatures and classifications, guidelines on how to report the results, preparation of recommendations, definition of diagnostic criteria and diagnostic algorithms and development of external quality control programs. In these iniatives, laboratory staff, clinicians and regulators are mainly involved. Nevertheless, those aspects regarding the design, development and manufacturing of the reagents, which involve manufacturers, are basically ignored. And this is probably due to the fact that the evolution of the technology has led to a truncated view of the test procedure resulting in a misconception of what needs to be standardized. In other words, the execution of many procedures is nowadays being shared between the manufacturer, who actually initiates the assay, and the laboratory, where the test is finalized. In old scientific articles related to ANA, the Material and Methods section usually started with the cell culture, the preparation of the slides and the fixation among others, and the sample incubation was only one more step of the whole procedure. Currently, the Material and Methods section starts with the sample preparation and instead of describing all the preliminary steps, one can find the name and references of the manufacturer. Figure 1 illustrates what would be the whole test procedure, showing the part performed in the clinical laboratory, actually the only part which is taken into consideration when dealing with standardization. So, to ensure appropriate use of indirect immunofluorescence testing, clinicians, diagnostic laboratories, regulators and reagents manufacturers should be involved and share the tasks of identifying and managing the key points leading to proper results.
Evidences of disparity
At the level of the manufacturer, the potential variability in the performance of the kits lies in features like the reagents and materials that are purchased or manufactured to become components of the kit, the procedures and conditions of manufacturing (fixatives, temperatures, formulations), the reliability of the serum samples used to set up the calibration of the determination (basically, the sample dilution which actuallly acts as the cut-off point), and the stability of the final product (1).
When approaching the participation of the manufacturer in the standardization of antibody testing, it is observed that what basically matters for industry is the standardization of the manufacturing processes. This normally occurs in an environment of Quality System Certifications, like GMP, ISO-9001 or ISO-13485 and under the requirements of the European Directive on In Vitro Medical Devices, and it is strengthened by the manufacturer’s own interest in having robust and reliable processes. Nevertheless, despite regulatory compliant and well implemented standardized processes, there are several aspects that make final reagents differ from one manufacturer to another. Below are reviewed some examples of variation on the results depending on the manufacturer source.
Dense fine speckles 70 (DFS70) antigen
As with other fluorescence patterns, the typical DFS pattern (lens epithelium-derived growth factor) can vary depending on the manufacturer source of the HEp2 slides used. The variations consist basically in different sensitivities and even in positive and negative results for the same sample run in different slide brands. Inconsistencies are also observed when comparing fluorescence with the results obtained by means of ELISA (2,3).
Ribosomal P protein (Rib P)
In studies performed by Mahler et al. (4) to determine the sentitivity of the immunofluorescence technique to detect antibodies against ribosomal P protein, several different HEp2 slides manufacturers were used, resulting in significant differences in patterns of staining for monospecific anti-Rib-P sera. Differing patterns were observed for the same sample, from a fine speckled nucleoplasmic pattern, to a diffuse cytoplasmic staining, or a fine speckled cytoplasmic pattern.
CDC/AF Reference Human Sera
When running reference sera on HEp2 slides coming from different manufacturers, variations of unknown origin can be observed. While most brands produce the expected specific pattern, there are often differences among brands like the ones shown in Figure 2.
Labile nuclear antigens
Most of the patterns observed when analysing the presence of ANA in patients sera by IFA on HEp2 cells slides are suitably detected in most slides brands. However, there are some antigens for which expression may significantly vary from one manufacturer to another like Jo1, PCNA or SSA/Ro (5). These antigens are not always well preserved in the substrates and they can be extremely sensitive to handling, to certain fixatives and in some cases, they can be just washed out during the manufacturing process, resulting in a poor presence or a total lack of antigenic molecules available to capture the antibody being analysed.
Antineutrophil cytoplasmic antibodies (ANCA)
The neutrophil substrates used in the detection of ANCA may vary in their ability to give the typical immunofluorescence patterns described and established by consensus groups, i.e. a diffuse granular cytoplasmic staining with higher interlobular intensity (C-ANCA), a compact staining of the perinuclear zone of the cytoplasm (P-ANCA) and a broad non homogeneous perinuclear staining, eventually accompanied by a diffuse cytoplasmic pattern with no accentuation of the interlobular zone (X-ANCA). In general, substrates differ in their ability to distinguish between a C-ANCA and X-ANCA. In a study by Pollock et al. (6), it was observed that although all commercial neutrophil substrates consistently demonstrated nuclear extension of perinuclear fluorescence with sera containing P-ANCA with MPO specificity, there were more problems in P-ANCA testing than in C-ANCA, due basically to the eventual presence of additional cytoplasmic fluorescence.
Crithidia luciliae
In a similar way as observed in HEp2 cells immunofluorescence patterns, the anti-nDNA test on Crithidia luciliae slides may show significant differences among manufacturers. The variety of strains available in cell banks contribute to the heterogeneity of results. Apart from the kinetoplast, other organelles can be stained by antibodies from the sample, like the nucleus, the basal body and the flagellum. Depending on the conditions of preparation of C. luciliae substrates and on the nature of the sample analysed, different patterns of stained organelles can be observed. Nevertheless, the only specific staining to be considered as a positive result is the kinetoplast staining. In addition to anti-nDNA antibodies, there are other antibodies in the serum of lupus patients that can react with the substrate. The so called anti-nucleosome antibodies are antibodies that react with histones exposed in the nucleosome. It is well known that treating C. luciliae substrate with HCL eliminates histone from the kinetoplast (7). This could be another point of possible discrepancy among manufacturing processes if some include the histone removal procedure and some others do not. Furthermore, the cell cycle of C. luciliae may influence histone appearance in the kinetoplast. Therefore, the manufacturing process of C. luciliae slides, including culture, harvest, fixation and drying, can cause variation in the results.
Aspects providing variablity
Among the players participating in autoimmune diagnostics, there is no doubt that manufacturers hold the know-how of preparing diagnostic kits and are the true experts in the development of test methods. However, despite the standardized manufacturing processes and the CE-certifications or FDA approvals, there are several aspects that are found to be sources of variabilty. These aspects should be addressed and recommendations on key points should be created by specialized committees with the participation of laboratory experts, clinicians and manufacturers. The definition and control of the raw materials incorporated in the kit production is a common and regulated practice in any kind of manufacturing process. But recommendations on nature, compostion or quality grades of key materials, including culture media, cell type and strain or fluorescent conjugates is still lacking. In the case of tests based on cellular substrates, extracellular matrix (ECM) proteins are commonly used to aid the spreading and growth of cells on the slide glass surface. Many ECM proteins contain defined amino acid sequences to which cell surface integrin receptors bind specifically. ECM, together with growth factors in the culture medium, work to produce an appropriate in vitro proliferative response, promoting cell growth and spreading. Altering cell-ECM contacts results in coordinated changes in cell, cytoskeletal, and nuclear form. Thus, the choice of the right ECM to coat the glass slides used as growing surface deserves our attention since it might have a direct effect on the fluorescent pattern finally observed (8). It is also common to use synchronization agents to achieve a greater rate of mitotic cells. Due to the fact that these compounds may be toxic for the cell, some cell disturbances may occur that can impact the morphology or the behaviour of the final cell preparation.
Diagnosis by means of tissue sections remains very important in liver autoimmune diseases like autoimmune hepatitis (AIH) or primary billiary cirrhosis (PBC). In particular, the detection of anti-smooth muscle antibodies (ASMA), antibodies to liver-kidney microsomes (LKM antibodies) and anti-mitochondrial antibodies (AMA) are considered important diagnostic tools. Only a few guidelines have been published on the obtention of tissue sections (9), while the variations in the preparation of tissue blocks regarding orientation, preservation conditions, and sectioning keep on contributing to the heterogeneity of results, especially in the case of tissues that are not morphologically homogeneous. For instance, the LKM antibodies can only be well defined if the kidney section has the proper orientation that allows the distinction between proximal and distal renal tubules and, thus, between LKM and AMA.
Considering that the expression and topographical distribution of autoantigens is under the direct influence of the HEp-2 fixation method, some immunofluorescence patterns are not adequately expressed due to the way that the antigenic substrate is prepared. This aspect equally affects tissue and cell substrates. As for the sensitivity of the tests, differences among manufacturers are due to the use of fixatives to prolong shelf-life. The use of slides without fixation seems to be the best choice for most autoantibody patterns. Nevertheless, there are several staining patterns that need the substrate to be fixed (figure 3), like anti-islet cells antibodies or anti-adrenal cortex antibodies.
A less frequent but significant source of variability in the immunofluorescence on tissue sections can be found in the origin of the animal used (Figure 4). Definition of suitable species and strains should be addressed in some cases in which the levels of antigen expression may differ. This affects the sensitivity of the test, especially in samples with moderate or low titers of antibody.
Considering the complexity and diversity of manufacturing processes and subprocesses and their impact on the final test performance, it is important to combine the efforts of laboratory experts, clinicians and manufacturers in the task of standardizing those key aspects that could otherwise keep on undermining the successful harmonization of the results obtained in the clinical laboratory.
References
1. Fritzler MJ, Wiik A, Fritzler ML, Barr SG. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 2003, 5:192-201
2. N.Bizzaro, E.Tonuttiand D.Villalta, «Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al,» Arthritis Rheum, vol. 63, no. 12, pp. 4036-4037, 2011
3. Mahler M. The clinical significance of anti-DFS70 antibodies as part of ANA testing. In: K. Conrad, E.K.L. Chan, M.J. Fritzler, R.L. Humbel, P.L. Meroni, G. Steiner, Y. Shoenfeld (Eds.). Infection, Tumors and Autoimmunity, AUTOANTIGENS, AUTOANTIBODIES, AUTOIMMUNITY, Volume 9, p.342-350. PABST, 2013.
4. Mahler M, Ngo JT, Schulte-Pelkum J, Luettich T, Fritzler MJ. Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies. Arthritis Research & Therapy 2008, 10:R131
5. Dellavance A, de Melo Cruvinel W, Carvalho Francescantonio PL, Pitangueira Mangueira CL, Drugowick IC, RodriguesSE; Coelho Andrade LE. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides J Bras Patol Med Lab 2013;49( 3):182-190.
6. Pollock W, Clarke K, Gallagher K, Hall J, Luckhurst E, McEvoy R, Melny J, Neil J, Nikoloutsopoulos A, Thompson T, Trevisin M, Savige J. Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate. J Clin Pathol 2002;55:680–683
7. Kobkitjaroen J, Jaiyen J, Kongkriengdach S, Potprasart S, Viriyataveekul R. Comparison of Three Commercial Crithidia luciliae Immunofluorescence Test (CLIFT) Kits for Anti-dsDNA Detection. Siriraj Med J 2013;65:9-11
8. (Integrin Binding and Cell Spreading on Extracellular Matrix Act at Different Points in the Cell Cycle to Promote Hepatocyte Growth Hansen LK,. Mooney DJ, Vacanti JP, Ingber DE. Molecular Biology of the Cell 1994;5:967-975
9. Vergani D, Alvarez F, Bianchi FB, Cançado ELR, Mackay IR, Manns MP, Nishioka M, Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. Journal of Hepatology 2004;41: 677–683
OmniLab CAL 8000 Hematology System / SAL 8000 Molecular System
, /in Featured Articles /by 3wmediaTowards early diagnosis of AD
, /in Featured Articles /by 3wmediaAlzheimer’s disease (AD), a progressive and eventually fatal neurodegenerative condition, was first described over a century ago. The prevalence of the disease has greatly increased since then: indeed the World Health Organization estimates that around 36 million people are living with dementia, the majority of whom are suffering from AD. This number is expected to double by 2030 and triple by 2050, mostly due to increased human longevity: the incidence of AD increases exponentially after the age of 65, with nearly 50% of people over 85 affected. Very early diagnosis and timely and effective therapy are urgently needed if health and social services are not to be totally overwhelmed catering for the needs of both patients and their frequently elderly carers.
Changes in the brains of AD patients may commence up to two decades before clinical symptoms become apparent. The two major abnormalities, beta-amyloid plaques (Aβ) and neurofibrillary tangles (NFT), are very visible at autopsy and continued improvements in medical imaging technologies may allow eventual visualization in the brains of living patients. A definitive diagnosis of AD, though, is usually still based on neuropsychological testing and MRI and/or CT scans to rule out other causes of cognitive decline at a stage of the disease when the drugs currently available, which regulate neurotransmitters, are no longer very effective.
Ongoing research to allow earlier diagnosis has found that gradually increasing concentrations of both Aβ and NFT can be detected in the cerebrospinal fluid of AD patients. And two very recently published studies give additional cause for optimism. The first, published in Nature Genetics, was a large international study that scanned the DNA from more than 74,000 AD patients and healthy controls from 15 different countries to find novel genetic risk factors. As well as the genes already implicated in the disease, such as APOE4, which is strongly linked to late-onset AD, eleven new genes were discovered that had previously not been linked to the condition. This work could facilitate very early diagnosis in individuals at risk. And a smaller British Medical Research Council study discovered a compound that actually prevents further neurodegeneration in animal models.
It has been recognized, however, that an international approach would be most effective in reducing the impact of AD and other types of dementia. To this end health ministers from the G8 countries will be meeting in London in December to develop a coordinated plan of action. It is to be hoped that the result of their deliberations will be global cooperation between companies, researchers and clinicians, and ultimately timely diagnosis and therapy for this appalling condition.
TDM of levetiracetam and pregabalin: the need and the method
, /in Featured Articles /by 3wmediaTherapeutic drug monitoring of anti-epileptic drugs has greatly advanced since the development of colorimetric assays for the measurement of phenytoin and phenobarbital in the mid-1950s. Today, not only have laboratory technology and assay development advanced, but so have the pharmaceutical agents available for the treatment of epilepsy disorders. However, under UK National Institute for Health and Clinical Excellence (NICE) Guidelines, therapeutic drug monitoring is still justified for newer anti-epileptic drugs like levetiracetam and pregabalin, for which we have developed quick and robust LC-MS/MS assays.
by Jonathan C. Clayton, Katherine Birch and Carrie A. Chadwick
Background
Therapeutic drug monitoring (TDM) is an important consideration in the treatment of epilepsy. It has long been known that a dose of a given drug may be effective in one patient but not in another [1]. This is of particular importance when too high a concentration of drug can have toxic effects, and too low a concentration has no therapeutic effect. Problems arise when, in different patients, a specific dosage leads to a therapeutically significant concentration in one, but could be ineffective or even toxic in another. Understanding the relationship between dosage and the concentration of the active drug at receptor sites has long been a topic for research [2], which has led to the development of assays to measure the plasma concentration of anti-epileptic drugs (AEDs). TDM of AEDs has advanced since colorimetric assays for phenytoin and phenobarbital were developed in the mid-1950s [3]. Older AEDs such as phenytoin and valproate have narrow therapeutic ranges (the plasma drug concentration range below which the drug may be ineffective and above which the patient may experience toxic effects). However, even the plasma concentration at which a given drug is effective may vary from individual to individual, depending on a number of factors known as pharmacokinetics [4]. Many newer AEDs, such as lamotrigine and topiramate do not have the narrow therapeutic range as seen with the older AEDs, however, TDM is still applicable [5]. Today both older AEDs such as phenytoin, phenobarbital and sodium valproate as well as newer AEDs such as lamotrigine and topiramate are subject to TDM [4]. This has led to the development of new assays for monitoring the serum concentration of these drugs. Methods include immunoassays such as enzyme multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA), kinetic interaction of microparticles (KIMS) and chemiluminescent assays (CLIA) [6]. However, more liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are being developed for newer AEDs, which can detect a number of AEDs in a single assay [7].
Best Practice Guidelines for TDM published in 2008 [1], along with a review discussing TDM of the newer AEDs [8] have provided a rationale for developing methods for two second generation AEDs, levetiracetam and pregabalin. These drugs are becoming increasingly popular with levetiracetam being used as an adjunct for partial and generalized tonic–clonic seizures, and pregabalin used as an adjunct for partial seizures [9]. Pregabalin, and to a lesser extent levetiracetam, is also used in the treatment of non-epileptic disorders such as neuropathic pain [9]. The increasing popularity of these drugs with clinicians has led to an increasing demand for determination of plasma concentrations of these drugs. TDM is justified for determining compliance with treatment with either drug, but also for determining overdosing, and dosing in renal failure, of levetiracetam.
Here, we describe methods for the detection and quantification of levetiracetam or pregabalin in serum using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The methodology is identical for both levetiracetam and pregabalin and so, should demand for TDM of these drugs increase in the future, there is scope for them to be combined into one assay.
Materials and methods
Levetiracetam (1 mg/mL in MeOH) and pregabalin (1 mg/mL in MeOH) stock solutions, levetiracetam-D6 (100 µg/mL in MeOH) and pregabalin-D6 (100 µg/mL in MeOH) were purchased from Cerilliant (distributed by LGC Standards, Middlesex, UK). EQA materials used for accuracy assessment were kindly supplied by the LGC Heathcontrol EQA scheme. HPLC grade water and methanol were purchased from Sigma-Aldrich Ltd (Poole, Dorset, UK). All other chemicals were purchased from Sigma-Aldrich Ltd or VWR Ltd. ClinChek® Control Levels 1 and 2 were purchased from RECIPE (Munich, Germany).
Standards
Standard solutions were made by preparing serial dilutions of stock solution in PBS/BSA (phosphate buffered saline containing 0.5% bovine serum albumin) (137 mmol/L NaCl, 2.7 mmol/L KCl, 5.4 mmol/L Na2HPO4•7H2O, 1.8 mmol/L KH2PO4, 0.5% BSA). The standards were stored at –20°C until use.
Internal standards
Each internal standard was prepared to a final concentration of 10 mg/L in HPLC grade methanol containing 50 mmol/L ZnSO4∙7H2O. The internal standards were stored at room temperature until use.
Sample preparation
For assay purposes, standards, quality control (QC) and serum samples were prepared in an identical fashion. In a 96-well plate, 80 μL internal standard solution (in ZnSO4 in MeOH) are added to 20 μL sample followed by agitation and centrifugation. Eighty microlitres of H2O was then added to each well, the plate heat sealed, agitated and centrifuged.
Chromatography and mass spectrometry
Chromatography was performed on a Waters Acquity UPLC system equipped with a Waters Acquity UPLC BEH C18 1.7 μm 2.1 x 50 mm column. Mobile phase A consisted of 10 mmol/L ammonium acetate and mobile phase B consisted of MeOH.
A flow rate of 0.5 mL/min was maintained for the run time of 2.5 minutes. A linear gradient of mobile phase B from 2% to 50% was run between 0 and 1 minutes, followed by a constant concentration of 50% mobile phase B. Ninety-eight per cent mobile phase B was run from 1.75 to 2.5 minutes. The injection volume was 5 μL.
Mass spectrometric determination was carried out using a Waters TQD in ESI+ mode. The source temperature was 130 °C, desolvation temperature was 400 °C, cone gas flow was 50 L/hr and the desolvation gas flow was 800 L/hr. Targetlynx™ software was used to process the data and quantify the drugs in the standards, controls and patient samples.
Method validation
Validation of the assays was carried out according to Honour [10]. Precision and bias were determined by measuring QC samples over 5 batches with 5 samples in each batch. The coefficients of variance (CVs) were calculated for intra-batch and inter-batch precision. Bias was calculated from the nominal target values for each of the QC materials.
Accuracy was assessed using EQA materials from the LGC Heathcontrol AE1 Anti-epileptic drug EQA scheme.
Matrix effects were determined by running a water blank, extracted water and extracted drug-free serum against a background infusion of each drug.
The limit of blank (LOB) was determined by running 10 extracted water samples and was quantified as the highest concentration measured in the absence of analyte.
The lower limit of quantitation (LLOQ) was determined by spiking drug-free serum with known quantities of each drug, and was quantified as the lowest detectable concentration whose CV was <15% and bias <20%.
Specificity was determined by spiking PBS/BSA with high concentrations of six more commonly used AEDs (carbamazepine, carbamazepine epoxide, phenobarbital, phenytoin, primidone and sodium valproate.
Carry-over was determined by spiking drug-free serum with high concentrations of each drug, and analysing followed by drug-free serum.
Results
Chromatography and mass spectrometry
Levetiracetam and levetiracetam-D6 had a retention time of 0.88 minutes and the cycle time from injection to injection was 3 minutes. Pregabalin and pregabalin-D6 had a retention time of 0.82 minutes and the cycle time from injection to injection was 3 minutes. The chromatography profile is identical for both of the drugs. The profile produced clean, sharp peaks with no co-eluting elements. The quantification transition for levetiracetam was m/z 170.90>69.16 and the confirmation transition was m/z 170.90>98.17. For pregabalin, the quantification transition was m/z 159.90>55.12 and the confirmation transition was m/z 159.90>83.08. For the internal standards, levetiracetam-D6 had the transition m/z 177.00>132.00 and pregabalin-D6 had the transition m/z 166.10>102.90.
Method validation
The intra- and inter-assay CVs are <8% for both drugs suggesting good precision of the assay. The inter- and intra-assay bias for levetiracetam was acceptable at <6%, while for pregabalin the inter- and intra-assay bias was <10% apart from the inter-assay bias at 10 mg/L (Table 1). External quality assessment materials were analysed as per patient samples. The results (Table 2) were compared with the target value supplied by LGC Heathcontrol, and with the returns of other laboratories using similar methods (LC-MS and LC-MS/MS) in order to determine the accuracy of the assay. Matrix effects were investigated using injections of drug-free serum, extracted water and blank water against a constant background infusion of each drug in methanol (50 mg/L levetiracetam, 25 mg/L pregabalin). No matrix effects are seen around the relevant retention times for either drug (Fig. 1). The LOB was quantified as the highest apparent analyte concentration in the absence of analyte. The LLOQ was quantified as the lowest level of analyte detectable whose CV was <15% and whose bias was <20% (Table 3). The methods for both levetiracetam and pregabalin showed no interference from any other commonly prescribed AEDs, with responses of ‘0’ to the interference samples from both methods. Blank serum samples and extracted water samples run immediately after samples containing either ~200 mg/L levetiracetam or 100 mg/L pregabalin gave responses of ‘0’, indicating no problems with carry-over. Discussion
We have developed and validated LC-MS/MS assays for the quantification of levetiracetam and pregabalin in serum.
Two optimal transitions were identified for both drugs, thus providing a ‘quantifier’ transition and a ‘confirmation’ transition in order to increase confidence of identification owing to the risk of misidentification of analytes with the same molecular weights as the drugs of interest.
The chromatography method is identical for both levetiracetam and pregabalin, and with the two drugs having different retention times (0.88 and 0.82 minutes respectively), should there ever be a wish to combine these assays into one single run, this should be straightforward. Additionally, should assays for any other AEDs be developed, this chromatography method would be an appropriate starting point. Serum proteins are precipitated by the addition of ZnSO4 in methanol, which also aids the retained solubility of the drug. Following centrifugation, an equal volume of H2O is added so the drug is in 50 : 50 methanol/water. Following a further centrifugation, 5 µl of supernatant is injected onto the column. The method is quick and robust. The assay has acceptable precision and bias. All the EQA materials ran well within their acceptable ranges, close to the target value.
Other LC-MS/MS methods for the detection of levetiracetam [11, 12] and pregabalin [13] have been described, all of which have longer cycle times between injections, larger sample volume requirements, and, in some cases, have more complex sample preparation. The method described here benefits from being quick, with a simple sample preparation procedure.
Methods for the measurement of levetiracetam in saliva have been described [11] and it has been shown that there is good correlation between saliva, plasma and serum, meaning saliva would be a suitable alternative to serum [14]. To date, no such method has been described for pregabalin, but cases of pregabalin toxicity have been described which would advocate the development of further methods for the TDM of pregabalin [14].
The monitoring of levetiracetam and pregabalin is justified [1, 5] to monitor compliance and overdosing, and quick and robust methods for their measurement in serum have been described here. Further work could include development of assays for the measurement of these drugs in saliva, with comparison studies required.
References
1. Patsalos PN, Berry DJ, Bourgeois BF, Cloyd JC, Glauser TA, et al. Antiepileptic drugs—best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission of therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008; 49: 1239–1276.
2. Eadie MJ. Therapeutic drug monitoring—antiepileptic drugs. Br J Clin Pharmacol. 1998; 46: 185–193.
3. Theodore WH. Rational use of antiepileptic drug levels. Pharmac Ther. 1992; 54: 297–305.
4. Glauser TA, Pippenger CE. Controversies in blood-level monitoring: reexamining its role in the treatment of epilepsy. Epilepsia 2000; 41(Suppl. 8): S6–S15.
5. National Institute for Health and Clinical Excellence. The epilepsies: the diagnosis and management of the epilepsies in adults and children in primary and secondary care. Clinical guidelines 137. NICE 2012; http://guidance.nice.org.uk/CG137 (accessed 15 October 2013).
6. Aldaz A, Ferriols R, Aumente D, Calvo MV, Farre MR, et al. Pharmacokinetic monitoring of antiepileptic drugs. Farm Hosp. 2011; 35: 326–329.
7. Shibata M, Hashi S, Nakanishi H, Masuda S, Katsura T, Yano I. Detection of 22 antiepileptic drugs by ultra-performance liquid chromatography coupled with tandem mass spectrometry applicable to routine therapeutic drug monitoring. Biomed Chromatogr. 2012; 26: 1519–1528.
8. Krasowski MD. Therapeutic drug monitoring of the newer anti-epilepsy medications. Pharmaceuticals 2010; 3: 1909–1935.
9. Wahab, A. Difficulties in treatment and management of epilepsy and challenges in new drug development. Pharmaceuticals 2010; 3: 2090–2110.
10. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem. 2011; 48: 97–111.
11. Guo T, Oswald LM, Mendu DR, Soldin SJ. Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry. Clin Chim Acta 2007; 375: 115–118.
12. Blonk MI, van der Nagel BC, Smit LS, Mathot RA. Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2010; 878: 675–681.
13 Nirogi R, Kandikere V, Mudigonda K, Komarneni P, Aleti R. Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma. J Chromatogr B. 2009; 877: 3899–3906.
14. Patsalos PN, Berry DJ. Therapeutic drug monitoring of antiepileptic drugs by use of saliva. Ther Drug Monit. 2013; 35: 4–29.
The authors
Jonathan Clayton* MPhil, MSc; Katherine Birch DipRCPath; and Carrie Chadwick FRCPath
The Buxton Laboratories, The Walton Centre NHS Foundation Trust, Liverpool, UK
*Corresponding author
E-mail: Jonathan.clayton@nhs.net
Therapeutic drug monitoring of methadone
, /in Featured Articles /by 3wmediaMethadone maintenance therapy is central to the treatment of opiate dependence. Assessment of adherence is essential to ensure success and to prevent misuse of prescribed medications. A variety of specimen types can be tested for methadone and its main metabolite using a number of different analytical methods. The benefits and limitation associated with each are discussed.
by Dr Elizabeth Fox and Dr Deepak Chandrajay
Introduction
Opiate dependence is an important problem worldwide. In the UK, individuals seeking help with their addiction are referred to substance misuse services where they are usually offered methadone or buprenorphine substitution therapy [1]. Methadone is a synthetic opioid with pharmacological actions similar to opiates mediated through the mu receptor. Treatment is initiated at a dose of 10–40 mg daily and gradually increased by 10–20 mg weekly. The usual maintenance dose is 60–120 mg daily, but some clients require a higher dose for symptomatic relief [2]. Its long half-life allows for a once-daily dosing schedule and the accumulation in the body means that steady-state plasma concentrations are easily achieved after repeated administration.
Methadone reduces or eliminates withdrawal symptoms and helps the subject reach a drug-free state in a controlled way. There is evidence of reduced illicit opiate misuse, criminal activity and mortality when patients are on maintenance therapy [2, 3]. Injecting behaviours and incidence of HIV infection are also reduced [4].
Methadone is a lipid soluble drug with an oral bioavailability of approximately 95%. It is metabolized by cytochrome P-450 (CYP) enzymes and demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Both parent drug and metabolite are excreted in the urine and can also be detected in blood, oral fluid, sweat and hair. Co-administration of CYP enzyme inducing drugs such as rifampicin, phenytoin, and zidovudine can precipitate opiate withdrawal symptoms. Fluoxetine and fluvoxamine can inhibit CYP enzymes and have an opposite effect on methadone metabolism [5].
In contrast to most other forms of therapeutic drug monitoring where blood concentration is maintained within a narrow therapeutic window, methadone is monitored almost exclusively to confirm adherence with the treatment regimen. The client may seek to falsify the drug test to feign adherence when the drug is actually being sold to others, or simply to mask illicit drug use. Such individuals will submit a specimen spiked with methadone mixture, therefore effective methods for methadone testing should use matrices which are resistant to tampering and/or include measures to detect falsified samples. Absence of EDDP from a methadone-positive urine sample strongly suggests that it has been spiked with medication. Measurement of urine creatinine will identify samples which have been diluted or substituted, for example with tea. Methadone mixture is green so a green tinge to urine should raise suspicions of sample spiking. The temperature and pH of fresh urine specimens can be recorded to assess reliability. Salivary IgG is useful to confirm integrity of oral fluid specimens.
Analytical methods used for methadone testing
The key analytical methods used to measure methadone and EDDP are immunoassay, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). The benefits and limitations of each are summarized in Table 1. Immunoassay is rapid, high throughput when automated and low cost. Sensitivity is determined by the detection cut-off concentration of the test kit and specificity by the specific antibody used in the kit. Point-of-care test (POCT) kits are available for use with urine and oral fluid in the clinic and require little expertise or training. POCT offers the significant advantage of producing instant results that can be discussed during the consultation. Laboratory-based immunoassays can be run on multichannel clinical chemistry analysers and do not require additional staff training. All immunoassay-based techniques are prone to interference from unrelated compounds due to cross-reaction with the specific antibody. Cross reactivity data are available from the manufacturer and should be borne in mind when interpreting results. False-positive methadone results have been documented with diphenhydramine, doxylamine and phenothiazines [6]. Immunoassay-based tests, whether designed for POCT or laboratory use, are sold as screening tests. The manufacturers recommend the confirmation of positive results using an alternative methodology such as LC-MS/MS or GC-MS. That said, not all laboratories and substance misuse clinics routinely confirm methadone and EDDP positive results. UK Department of Health guidance on adherence testing recommends only that positive screen tests are confirmed ‘if appropriate’[1].
Mass spectrometric techniques offer the best possible sensitivity and specificity and are considered the ‘gold standard’. Test menus are user-defined which allows simultaneous detection of methadone and EDDP and any other drug as required. Disadvantages of these techniques are that they require expensive specialist instrumentation, labour intensive sample preparation, and complex data interpretation. Turnaround times can be lengthy and they are not amenable to POCT. LC-MS/MS methods require considerably less sample preparation than GC-MS and are now used in many clinical laboratories. A few labs including our own have adopted LC-MS/MS for first-line drug screening of urine and oral fluid specimens.
Specimen types for methadone testing
The main sample types are summarized in Table 2.
Urine
Regular urine testing is the most commonly used means of confirming adherence with methadone prescription. The advantages of urine are that both methadone and EDDP are readily detected and collection is easy and non-invasive. Presence of EDDP provides proof that methadone has been ingested and not spiked into the sample. A disadvantage of urine is that it is easy to manipulate. A sample of donor urine, if the donor is taking methadone, submitted in place of the patient’s own is difficult to recognize. Supervised collection is not always desirable as it is an invasion of privacy and subjects may suffer from ‘shy bladder’. The concentrations of methadone and EDDP do not correlate well with dose (because of the variability of untimed urine samples), so qualitative urine analysis is only suitable for confirming use.
Commercially available methadone and EDDP immunoassays typically have a fixed cut-off or detection threshold of between 100 and 300 µg/L. Multidrug panel tests usually include either methadone or EDDP. Choosing a test which specifically detects EDDP will minimize the chance that a spiked urine is passed off as positive. An estimated 4% of methadone-positive samples submitted to our laboratory lack detectable EDDP; a methadone-only assay would not identify these specimens (unpublished observation). Our approach to urine testing is to measure both methadone and EDDP by LC-MS/MS in all samples. The testing strategy in an increasing number of substance misuse clinics is to use POCT as a first-line test, then to refer suspicious or disputed samples to the lab for confirmation. A laboratory immunoassay would offer no further information for samples that have already been tested at the point of care and could theoretically suffer the same interference.
Oral fluid
An alternative matrix for methadone testing is oral fluid. The advantage of oral fluid is that collection is simple, easily observable and can be done in the consultation room. POCT devices are available for instant results or samples can be sent for laboratory analysis. Both immunoassay and mass spectrometric analytical methods are available. The amount of methadone and EDDP present in oral fluid is dependent upon salivary pH. Methadone is a basic drug and under acidic conditions it becomes ionized and ‘trapped’ in the saliva. Unstimulated saliva is more acidic than stimulated saliva so false negatives can be avoided by asking the subject to abstain from eating, drinking or chewing for 10 minutes prior to collection. A recent study involving subjects on daily methadone doses found that the concentration of methadone in saliva correlated poorly with dose and that EDDP was below detection in 12% of samples [7]. However, methadone was readily detectable in all samples suggesting that oral fluid is a useful specimen for confirming adherence. Oral fluid methadone does not reflect the plasma concentration so would not be useful for assessing dose adequacy. Contamination with methadone from the oral cavity is a problem and absence of EDDP, if measured, should be interpreted with caution as it does not necessarily equate to sample adulteration.
Blood
The main advantage of using blood to monitor methadone therapy is that it’s virtually impossible to falsify the sample. Plasma concentration correlates with methadone dose but the concentrations at which therapeutic effect is achieved have not been well defined. Several studies have suggested target concentrations; other studies have found no correlation between plasma concentration and either heroin use or opiate withdrawal symptoms [8]. A further study suggested that the pharmacodynamics of methadone can be altered by the presence of other drugs therefore altering the relationship between plasma methadone and effect [9]. There is debate in the literature as to whether plasma concentration is any more useful than daily dose for predicting response to treatment [10]. Given the polypharmacy present in the majority of subjects receiving methadone, routine use of plasma methadone to titrate dose is likely to need further evaluation. Intravenous drug users tend to have poor venous access so collecting samples may be challenging. Methods using dried blood-spot samples to circumvent this problem have been described but skin contamination with methadone is likely to be an issue [11]. Blood is not the ideal specimen to assess use of other substances because of the very short detection window, so additional testing may be required. In conclusion, blood testing is best reserved for difficult cases where knowledge of the plasma concentration may be helpful.
Other matrices
Monitoring of methadone therapy using sweat analysis has been evaluated. Patches are typically worn for up to 7 days then dispatched to the laboratory for analysis. They are tamper-evident and claim to be difficult to adulterate. Large inter- and intra-individual variations in sweat methadone concentration have been observed and there is only a weak correlation between patch concentration and dose. Sweat testing is, however, useful for detecting exposure to other substances so may be applicable to some cases. Hair analysis can be used to retrospectively confirm adherence with methadone treatment but is not useful for real-time assessment.
Concluding remarks
The current trend is for substance misuse services to perform methadone adherence testing in the clinic and refer samples to the laboratory for confirmation where necessary. Substance misuse clinic personnel are not laboratory scientists, therefore a key role of the laboratory that performs confirmatory testing is to develop a good working relationship and ensure all aspects of testing are fully understood.
References
1. Department of Health (England) and the devolved administrations. Drug Misuse and Dependence: UK Guidelines on Clinical Management. London: Department of Health (England), the Scottish Government, Welsh Assembly Government and Northern Ireland Executive. 2007; www.nta.nhs.uk/uploads/clinical_guidelines_2007.pdf.
2. National Institute for Health and Clinical Excellence. Methadone and buprenorphine for the management of opioid dependence. Technology appraisal guidance 114. NICE 2007; http://guidance.nice.org.uk/TA114.
3. Advisory Council on the Misuse of Drugs. Reducing drug-related deaths: a report by the Advisory Council on the Misuse of Drugs. ACMD, Home Office 2000; ISBN 0-11-341239-8.
4. NTORS, The National Treatment Outcome Research study. 2001; http://webarchive.nationalarchives.gov.uk/+/www.dh.gov.uk/en/publicationsandstatistics/publications/publicationspolicyandguidance/dh_4084908.
5. McCance-Katz EF, Sullivan L and Nallani S. Drug interactions of clinical importance among the opioids, methadone and buprenorphine and other frequently prescribed medications: a review. Am J Addict. 2010; 19(1): 4–16.
6. Lancelin F, Kraoul L, Flatischler N, Brovedani-Rousset S, Piketty ML. False-positive results in the detection of methadone in urines of patients treated with psychotropic substances. Clin Chem. 2005; 51(11): 2176–2177.
7. Gray TR, Dams R, Choo RE, Jones HE, Heustis MA. Methadone disposition in oral fluid during pharmacotherapy for opioid-dependence. Forensic Sci Int. 2011; 206 (1–3): 98–102.
8. Shiu JR, Ensom MHH. Dosing and monitoring of methadone in pregnancy: literature review. Can J Hosp Pharm. 2012; 65(5): 380–386.
9. Kharasch ED, Walker A, Whittington D, Hoffer C, Sheffels Beynek P. Methadone metabolism and clearance are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A) activity. Drug Alcohol Depend. 2009; 101(3): 158–168.
10. Hallinan R, Ray J, Byrne A, Agho K, Attia J. Therapeutic thresholds in methadone maintenance treatment: a receiver operating characteristic analysis. Drug Alcohol Depend. 2006; 81(2): 129–136.
11. Saracino MA, Marcheselli C, Somaini L, Pieri MC, Gerra G, et al. A novel test using dried blood spots for the chromatographic assay of methadone. Anal Bioanal Chem. 2012; 404(2): 503–511.
The authors
Liz Fox* PhD, FRCPath and Deepak Chandrajay MBBS, MRCP
Specialist Laboratory Medicine, St James’s University Hospital, Leeds, UK
*Corresponding author
E-mail: Elizabeth.fox@leedsth.nhs.uk
The challenge of food allergies
, /in Featured Articles /by 3wmediaThe threat of allergies, which affect about one in five people in the US and Europe is emerging as a major public health challenge. The problem is also fast becoming severe in the developing world.
Very much an enigma
In spite of these trends, the World Allergy Organisation (WAO) notes that “services for patients with allergic diseases are fragmented and far from ideal,” and that this is true even in the developed world. The key reason is that allergies still remain little understood.
In Europe, for example, the EU Commission acknowledges that the epidemiology of allergies remains “very much an enigma.” In spite of “relatively homogeneous lifestyles” across the region, allergy rates vary from 3.7% among 13-14 year olds in Greece to 32.2% for the same age group in the United Kingdom.
Children hit hardest
As hinted by the EU Commission figures above, the impact of allergies is especially pronounced in a vulnerable demographic, namely children.
Indeed, the Florence, Italy-based European Academy of Allergy and Clinical Immunology (EAACI) reports that “the number of children with allergies has doubled in the last ten years, and visits to A&E have increased seven-fold.” The situation is no different in the US, where a recent study by the Centers for Disease Control and Prevention (CDC) finds allergy to be among the most common medical conditions affecting children aged below 17.
The allergy challenge has been confounded by the fact that its origins now include a bewildering (and growing) range of food products. In Europe, the InformAll Database (developed with funding from the European Union) currently contains information about the “more than 120 foods” reported to be associated with allergy.
The burden of this, once again, is disproportionately high on children. Globally, an estimated 220 to 250 million people could be suffering from food allergy, according to the WAO.
In Britain, the respected National Institute for Clinical Excellence (NICE) zeroes down on food allergy as being “among the most common of the allergic disorders” and “a major pediatric health problem” because of “the potential severity of reactions and a dramatic increase in prevalence over the past recent decades.”
Food allergies a specific challenge
Though the CDC study mentioned above found the biggest challenge for US children to be respiratory allergies, their share – at 17% – has remained constant since the late 1990s. The fastest growth, on the other hand, was shown with skin allergies, up from 7.4% in 1997–1999 to 12.5% in 2009–2011.
In contrast, the prevalence of food allergies in US children is not only smaller than either respiratory or skin allergies, but also showed a slower increase than the latter, from 3.4% to 5.1%.
However, the US figures conceal more than they reveal.
Firstly, managing (or even) identifying food allergies is not straightforward. Unlike respiratory allergies (which have a long-established intervention modus), or skin allergies (which are easier to pinpoint), the diagnosis of food allergies is far more problematic. This is because “nonallergic food reactions, such as food intolerance, are frequently confused” with food allergies.
The allergy continuum
Making things worse is the allergy continuum.
According to a review of two million patient visits in the US (the largest ever of its kind), food allergies in childhood are instrumental in the so-called ‘allergy march’, a medical condition by which there is an escalation in the risk “for the development of additional and more severe allergy-related conditions, including asthma, later in life.”
In other words, tackling food allergies effectively may hold the key to reducing the burden of other allergies in later life.
Profiling allergies: differences between children and adults
Food allergies in children are most commonly caused by eggs, milk, peanuts, tree nuts and wheat; in adults, milk and wheat are excluded as typical allergens, and instead replaced by fish and shellfish.
However, the EU Commission’s observation about the ‘enigma’ of allergies applies to food too. “In continental Europe, the most common food allergies are to fresh fruit and vegetables, whilst in Anglo-Saxon countries hazelnuts, peanuts and walnuts are the most problematic. Allergy to fish and shellfish prevails in Scandinavia and Northern Europe.”
Tracking the severity of allergies
An allergic reaction to food usually occurs quite quickly (in some cases, within minutes of eating a particular food, and in others, 2-3 hours afterwards). Typical symptoms include an abnormal swelling of the tongue, diarrhea, and hives.
In severe cases, the reaction (as with other allergies) is anaphylaxis, which can be life-threatening.
A study by Mayo Clinic covering a period of 10 years (1990 to 2000) found an age-specific rate for anaphylaxis highest in the under-19 year population (at 70 per 100,000 person-years, compared to an overall age- and sex-adjusted rate of just under 50). The Mayo clinic study also found that ingested foods accounted for one-third of all cases (33.2%), significantly ahead of the second- and third-ranked causes: insect stings with 18.5% and medication with 13.7%.
As troubling is the growth in the incidence of anaphylaxis, again in children. Hospital data from New York State shows that hospitalization for anaphylaxis among patients younger than 20 increased more than 4-fold between 1990 and 2006.
Growing costs
The economic impact of food allergy is significant. In the US, children’s food allergies are estimated to cost as much as $24.8 billion per year.
It is also growing. In the UK, hospitalization for food allergies has increased by as much as 500% since 1990.
Food allergies cannot be cured, but they can be managed by dietary control – in other words through avoidance of allergen-inducing foods. However, there is sometimes little room for a learning curve. In certain people, even tiny amounts of a food allergen (for example, 1/44,000 of a peanut kernel) can prompt an allergic reaction.
Currently, aside from avoidance, the standard of care for food allergies remains “ready access to self-injectable epinephrine.”
Both the US National Institutes of Health (NIH) and Britain’s NICE have drawn up recommendations for the diagnosis and management of food allergy. At the European level, the European Academy of Allergy and Clinical Immunology (EAACI) published its first guidelines on the subject in summer 2013.
An ‘allergy epidemic’: the institutional response
However, the challenge of food allergies is likely to continue.
One key gap is an institutional network of qualified specialists, which link in seamlessly into the wider public health system. In 2006, a subcommittee at Britain’s House of Lords concluded that allergy services were insufficient to deal with it and described the growing incidence of allergic conditions as an ‘allergy epidemic’. Their recommendations urged setting up “at least one allergy centre, led by a full time allergy specialist” in each Strategic Health Authority, supported by “a chest physician, dermatologist, ENT specialist, clinical immunologist, gastroenterologist, occupational health practitioner and pediatrician,” and assisted by “specialist nurses and dieticians trained in allergy.”
The House of Lords subcommittee also strongly called for “diagnostic facilities necessary to investigate complex allergies” staffed by personnel who have received “accredited allergy training.” In other words, such a system will only be meaningful if laboratories are harnessed to address the exploding allergy challenge, and provided with sufficient funds for equipment and staff. Until such time, the response will mean little more than using best practices guidelines (from bodies such as the NIH, NICE and the EAACI) to streamline what essentially remains an ad-hoc infrastructure.
The need for support by clinical labs is implicit in the NICE guidelines on food allergies, which stress that “skin prick tests and blood tests are equally cost-effective” and that “blood tests are cost-effective independent of number of individuals tested.” On the other hand, the guidelines also highlight the need for “valid test results” “to reduce incidence of adverse reactions and improve quality of life,” and prevent the (yet unquantified cost of) anxiety and worry, as well as the “avoidance of food that is actually safe to eat.”
The future: no cures in sight, yet
As of now, there is no cure for food allergies.
A seemingly-promising Phase II, randomized, double-blind study on the anti-asthmatic omalizumab against peanut allergy (one of the most dangerous food allergies) was stopped in 2011, with most subjects not reaching the endpoints. The investigators concluded that “no firm conclusions can be drawn” from their effort, but said it deserved “further investigation.”
The omalizumab research actually reached the same deadlock as another similar anti-IgE preparation, HU-9015, in 2003. This study was, ironically, stopped after its sponsors found the prospects for omalizumab to be more promising.
Nevertheless, researchers continue with their efforts, especially with regard to peanut allergy. As of this date, according to a communication from the National Institutes of Health, 14 studies and trials on peanut hypersensitivity alone are recruiting volunteers, one more than for asthma.
Molecular diagnosis for eosinophilic esophagitis: a next-generation technique with pathogenic insight
, /in Featured Articles /by 3wmediaEosinophilic esophagitis (EoE) is a clinical disorder induced by food allergy with its current diagnosis based on histological examination of esophageal biopsies and clinical symptoms. A next-generation molecular diagnostic panel based on a 96-gene qPCR array was recently introduced for a definitive and objective diagnosis of EoE and demonstrated high diagnostic merit relative to the current method. This test provides insight into the pathological processes of EoE in a cost effective manner and will likely bring a personalized medicine approach to the field of esophagitis.
by Dr Ting Wen and Professor Marc E. Rothenberg
Eosinophilic esophagitis and current form of diagnosis
The tide of technical advances in the new century has sparked a molecular revolution in the fields of clinical diagnostics and predictive medicine, and is crucial for the provision of personalized medicine. Thus far, molecular diagnosis of diseases has been largely confined to cancer, autoimmunity and genetic disorders, while areas such as gastrointestinal (GI) disorders and allergic inflammation have been underexplored.
Eosinophilic esophagitis (EoE) is a type of immunological food allergy mediated by allergic hyper-responses to food, typically the six most common food allergens (milk, egg, wheat, soy, nuts, fish). Dietary elimination and steroidal intervention are two of the most effective therapies, and are often used together. At the cellular level, the inflammation is a well concerted process caused by local lymphocytes (primarily Th2 cells), mast cells and eosinophils within the esophagus that likely contributes to most of the clinical symptoms. Clinically, EoE is characterized by esophageal dysfunction (e.g. dysphagia) and is historically defined by a tissue biopsy exhibiting ≥15 eosinophils per high-power field (EOS/HPF), a cut-off agreed by a panel of field experts based on case discussions 5 years ago and referred to as the consensus recommendation (CR) 2007 [1, 2]. Both histological and clinical symptoms are necessary for diagnosis [ideally with proton pump inhibitor (PPI) trial confirmation] and form the basis of CR2007. Thus far, histological examination is the only widely accepted EoE diagnostic test [2]. However, this method is subjective, time-consuming and subject to variability (patchy sampling and inter-pathologist variability), as well as expensive. In addition, it is non-specific to a certain extent, as there are a number of other diseases sharing esophageal eosinophilia [3], and therefore the histological method cannot identify specific exposure to medications (such as glucocorticoid) nor differentiate patients in remission from EoE from non-EoE patients (neither exhibit eosinophilia). Therefore, the current ‘gold standard’ method has limitations and its results may be questionable. [4].
EoE transcriptome, qPCR array and algorithm development
The EoE transcriptome was identified by Blanchard et al. in 2006 [5] and subsequent studies identified ~1000 genes that were bi-directionally dysregulated at different magnitudes; thus serving as the foundation for molecular differentiation. We adopted a low density array in which four identical custom arrays are embedded on a 384-well fluidic card (Fig. 1), which allows cost-efficient Taqman-based PCR to be performed on potentially informative genes that are part of the EoE transcriptome. With this design, a maximum of 95 genes (plus one housekeeping control) can be assayed to map molecular pathogenic signatures and four samples can be processed in a given batch thus improving the turn-around time. During a recent study that involved a large cohort of 194 unique samples and used the qPCR-based array described above (termed the EoE diagnostic panel, or EDP), the test performed at nearly 100% accuracy in terms of revealing the bi-directional EoE signature, at much lower cost and with faster turn-around (same-day) compared with the standard microscopic analysis [6]. Notably, the EDP is designed to work with both fresh tissue and formalin-fixed, paraffin-embedded (FFPE) samples, with major steps involving RNA isolation, reverse transcription, qPCR amplification, raw data rendering, a dual algorithm application and diagnostic report generation (Fig. 1) (Table 1).
The test is highly reproducible between batches and samples, as results from samples tested months apart still correlated well because of the accuracy and specificity of Taqman qPCR. Having generated the raw cycle threshold (Ct) values of qPCR, the critical next step was to develop a method of interpreting the results in a way that every physician and patient was able to comprehend. One of the unique features of the EDP is the novel dual algorithm associated with the panel, which provides additional assurance. Briefly, the first algorithm is a clustering analysis based on the Pearson correlation of 77 genes followed by dimensionality reduction. With 50 upregulated genes and 27 downregulated genes, the bi-directional dysregulation provides a pronounced contrast for signature recognition. A dendrogram (hierarchical tree) is then derived from the inter-sample distance metrics aided by commercial analysis softwares such as GeneSpring (Agilent Inc.). The first branch of the dendrogram serves as a diagnostic bifurcation point. The second algorithm performed in parallel is essentially a mathematical summing-up of the change in Ct value for each gene relative to the housekeeping gene (GADPH) and takes into account the bi-directional changes (+ and – vectors). The end read-out of this algorithm is an absolute integer that provides a definitive EoE diagnosis and which correlates linearly with disease severity. This direct output allows the physician to readily assess the disease status and potential therapy. These types of dual algorithms are not mathematically challenging, so it is expected that the same algorithm may be expanded for broader use in the diagnosis of other inflammatory diseases, especially those with a diagnostic ‘grey zone’ or other clinical dilemma.
Data management, FFPE compatibility and commercialization potential
Compared to other more advanced gene-expression platforms, the Taqman qPCR array-based EDP has the advantage of cost effectiveness, minimal data size and straightforward experimental analysis. Basically, the raw Ct data are exported from the qPCR machine (ABI 7900HT) in text (.txt) or Excel format (typically in seconds), and are then further processed by software such as GeneSpring or simply by formula calculation in Excel. The goal is to generate an ‘EoE score’ and a clustered heat diagram leading to definitive diagnosis and disease evaluation on the basis of patient specific profiles of the 95-gene signature, therefore enabling a form of personalized medicine.
Of note, the EDP offers good compatibility with FFPE samples, as shown by a sub-study with 45 randomly selected FFPE samples, reaching 96% sensitivity and 100% specificity using histology as the ‘gold standard’. While FFPE RNA is known to be susceptible to considerable degradation with time (also shown in this study by Agilent QC assay), the EDP signature acquisition does not seem to be compromised over the reported archiving duration of 3 years. One can only imagine how many clinical questions could be answered using vast archived pools of FFPE samples and the associated amount of clinical outcome information.
With the simple qPCR-based procedures and the easily accessible computational algorithm, as well as the demonstrated clinical utility, the commercialization potential of the EDP is promising. The future use of the EDP in clinical practice is not reliant on complicated techniques, advanced hardware, professional expertise or significant start-up funds. Together with the rapid turn-around time and minimal demands on lab personnel, the initial barrier for adoption of the EDP in practice is low. It is also worth mentioning that such novel qPCR arrays combined with novel scoring algorithms could be readily applied to the diagnosis of other allergic inflammatory diseases with minimal modifications.
Conclusions and future expectations
Given the growing interest in “next-gen” molecular techniques, there seems to be ample justification for the development of this novel diagnostic method. Compared to the classic histology-based EoE diagnosis, there are several aspects that the molecular method is uniquely capable of providing. First, the molecular panel is able to distinguish remission patients despite normal histology, which is clinically important and not achievable using standard histology. The EDP is essentially based on reading gene expression in 96 channels (96-D), providing multi-dimensional pathogenic clues and personalized medical information compared with histology and other reported immunohistochemistry (IHC)-based approaches. Recently, a 4-protein panel was reported as having high diagnostic merit [7]. Although having a greater variety of diagnostic methods for EoE is beneficial, the lengthy IHC workload, subjective interpretation and insufficient pathogenic resolution make the molecular method advantageous. Preliminary results also suggest that the EDP can be used to predict the likelihood of EoE relapse prospectively, indicating a predictive medicine component of this technique. The EDP also provides a means to assess steroid exposure based on steroid responding gene elements, which could be used to evaluate patient compliance. Finally, regarding sensitivity and specificity, there is currently no option but to continue to use histology as the ‘gold standard’, because it is not yet clear which method more objectively reflects the disease status. Further research is needed to clarify this, especially in the light of the debatable specificity of histology [3, 4]. Although the EDP doesn’t provide all the answers, for example there is no predictive component for identifying which type of treatment will be more successful (diet versus steroid) and the sample source is still deemed invasive, it nevertheless represents a substantial advance. The conventional method has its merits, namely directly visualizing infiltrating eosinophils (for which EoE was named) and cellular topology, and will continue to be used in parallel. However, the advantages of the new platform are likely to be appreciated by researchers and physicians in an era where disease definition and pathogenic understanding are increasingly at the molecular level.
References
1. Furuta GT, Liacouras CA, Collins MH, Gupta SK, et al. Eosinophilic esophagitis in children and adults: a systematic review and consensus recommendations for diagnosis and treatment. Gastroenterology 2007; 133(4): 1342–1363.
2. Liacouras CA, Furuta GT, Hirano I, Atkins D, et al. Eosinophilic esophagitis: updated consensus recommendations for children and adults. J Allergy Clin Immunol. 2011; 128(1): 3–20 e6; quiz 21–22.
3. Dellon ES, Gonsalves N, Hirano I, Furuta GT, et al. ACG clinical guideline: Evidenced based approach to the diagnosis and management of esophageal eosinophilia and eosinophilic esophagitis (EoE). Am J Gastroenterol. 2013; 108(5): 679–692; quiz 693.
4. Rodrigo S, Abboud G, Oh D, DeMeester SR, et al. High intraepithelial eosinophil counts in esophageal squamous epithelium are not specific for eosinophilic esophagitis in adults. Am J Gastroenterol. 2008; 103(2): 435–442.
5. Blanchard C, Wang N, Stringer KF, Mishra A, et al. Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis. J Clin Invest. 2006 116(2): 536–547.
6. Wen T, Stucke EM, Grotjan TM, Kemme KA, et al. Molecular diagnosis of eosinophilic esophagitis by gene expression profiling. Gastroenterology 2013; doi:10.1053/j.gastro.2013.08.046.
7. Dellon ES, Chen X, Miller CR, Woosley JT, Shaheen NJ. Diagnostic utility of major basic protein, eotaxin-3, and leukotriene enzyme staining in eosinophilic esophagitis. Am J Gastroenterol. 2012 107(10): 1503–1511.
The authors
Ting Wen PhD and Marc Rothenberg* MD, PhD
Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
*Corresponding author
E-mail: Rothenberg@cchmc.org
Improved tools to diagnose venom allergies
, /in Featured Articles /by 3wmediaBee and wasp venom allergy is a potentially life-threatening condition and diagnostic errors can therefore have serious consequences. Currently, the diagnosis of allergy to stinging insects relies on patient case history and quantification of specific IgE antibodies and skin prick testing to identify the responsible insect. However, the diagnosis can sometimes be problematic as patients may have very low levels of specific IgE and also because many patients show positive test results to several venom species. Moreover it is often difficult for the patient to identify the offending insect. Component based specific IgE testing helps to increase the sensitivity of testing as well as to resolve which stinging insect species the patient is sensitized to. By applying these new component-specific IgE tests and including testing for serum tryptase, the certainty in identifying patients that will benefit from relevant and safe venom immunotherapy treatment increases greatly.
by Magnus Borres, MD, PhD, MPH
Background
Venoms from stinging insects such as bees and wasps (Hymenoptera) can induce anaphylaxis in susceptible people, and stinging insects are the second most common cause of anaphylaxis in Europe and USA (prevalence of 0.3 to 7.5% in Europe). Most of the severe and fatal reactions to insect stings in Europe are caused by members of the Vespidae family – commonly known as wasps. In contrast to many other IgE mediated allergic reactions, venom allergies may arise very unexpectedly as they can affect also individuals that do not have a genetic pre-disposition to make IgE antibodies.
The reactions elicited by a bee or wasp sting can range from mild/local immediate reactions, to larger often late local reactions up to immediate systemic reactions, eventually leading into life threatening conditions requiring emergency treatment.
Markers and risk factors in venom allergies
The presence of specific IgE antibodies to venoms supports the diagnosis of an allergic reaction. In many patients however, the levels are low and there is no direct correlation between the levels of specific IgE antibodies and the risk for reactions. In fact, it is not uncommon that severe reactions occur in patients with very low or sometimes even undetectable venom-specific IgE levels. This exemplifies the need for highly sensitive diagnostic tests that can detect and quantify very low specific IgE levels.
The risk of developing severe reactions after a Hymenoptera sting is dependent on several factors, such as the patient’s history of previous reactions, serum tryptase levels, age and specific IgE-sensitization. People who have already suffered from severe systemic reactions due to stings are predisposed for future reactions – up to 80% will develop severe reactions following a subsequent sting. However, in 50% of the fatal cases no previous systemic reaction has occurred. Serum tryptase is an important marker for evaluating the risk for systemic reactions, where elevated baseline tryptase levels indicate a higher risk for severe anaphylactic reactions. Approximately a fourth of patients who experience severe venom reactions have elevated baseline levels of this marker. The risk for severe reaction to venom stings also increases with age, and is higher in adults than in children and adolescents. This may be explained by an increased number of mast cells in addition to other contributing clinical conditions in older people.
Identify the little beast!
For patients who are highly allergic to insect stings the treatment option is to undergo venom immunotherapy (VIT) aiming at inducing tolerance. For selecting the most effective treatment, correct identification of the Hymenoptera species that causes reactions in the patient is crucial. This is however not trivial as many patients do not know what insect stung them, and as approximately 60% of the patients show up positive to both bee and wasp in venom extract-based tests.
Diagnostic in vitro tests in venom allergies
Patient history forms the basis in diagnosing a venom allergy and specific IgE antibody test results can support the doctor in the diagnosis and in choosing the appropriate treatment. Whether the reaction in a patient is IgE mediated or not needs to be established. This is usually done by in vitro testing for specific IgE and/or skin prick testing, but as many as 10-20% who seek medical care for sting-induced reactions are negative in these extract-based tests. The reason may be that the reaction was due to another pathogenic mechanism than an allergic reaction, or it could have been caused by an underlying mast cell disease. When using conventional extract based test, which due to the preparation procedure may be low in content of certain allergenic proteins, the sensitivity may not be high enough to pick up certain sensitizations. In addition patients reacting for the first time to a sting may initially have levels of venom-specific IgE below the detection limit.
On the other hand, it is common that patients appear to be sensitized to both bee and wasp venoms when using extractbased specific IgE tests, even in cases when proven non-reactive to one of the species. Diagnostic tests capable of discriminating between clinically relevant and irrelevant sensitizations, while reliably detecting true co-sensitization to both species greatly improves proper diagnosis and selection of therapeutic interventions.
There has thus been a need to increase both the sensitivity to detect low levels of IgE antibodies and the specificity to distinguish between sensitization to different Hymenoptera species. Recently this has become possible through the introduction of component-resolved diagnostics, or molecular allergology.
What is molecular allergology?
Molecular allergology allows the measurement of specific IgE antibodies to single, pure allergen molecules, thereby helping to identify the exact allergenic molecule (component) that a patient is sensitized to. All allergen sources contain several allergenic molecules, and the ability to produce these by recombinant means and assay them individually greatly increases the precision of specific IgE measurements. Using this component-resolved testing it is possible to discriminate between species-specific sensitizations, where the patient is genuinely sensitized to the allergen source, and sensitizations due to cross-reactivity. Cross-reactivity occurs when antibodies directed against one molecule cross-recognize a very similar but yet distinct protein. Such cross-reactivity may arise due to high similarity between some components in bees and wasps, but may also be caused by carbohydrate structures (CCDs) on proteins in plants and invertebrates. CCD antibodies do not cause symptoms and are thus clinically irrelevant, but may confuse test results greatly. Recombinant components used in molecular allergology are free of CCD structures and are therefore very specific. In addition, tests that identify antibodies to CCD are available to further increase diagnostic accuracy.
Molecular allergology improves the allergy diagnosis
Results from extract-based tests give the first, although crude answers that guide the diagnosis, while further analyses using component-based testing take the diagnosis to completely new levels by offering improved test sensitivity, resolving ambiguous extract test results and guiding the selection of optimal treatment. In cases where the patient has a convincing history of bee or wasp allergy, but extract -based tests turn out negative, allergen component testing offers increased sensitivity to detect relevant sensitizations. These tests contain only one single pure allergen component therefore the sensitivity to detect antibodies directed to this unique protein is increased as compared to extract-based tests. However the strength of the extract-based test is that they do contain all relevant allergenic proteins in the allergen source, including minor allergens.
Component-based testing enables the discrimination between true co-sensitization and sensitization due to cross-reactivity. Extract-based test results may indicate sensitization to both bee and wasp venoms, but using component testing it is possible to investigate if these sensitizations are clinically irrelevant or truly suggest allergy to both species. The recombinant markers for bee (Api m 1), common wasp (Ves v 1 and Ves v5) and/or paper wasp (Pol d 5) should be used to determine unambiguously if the sensitization is species-specific or not. CCD-antibodies can also give rise to double positive test results in the absence of specific Hymenoptera venom sensitization since these antibodies often are induced by grass sensitization.
When extract-based test turn out positive to either bee or wasp only, there is little questioning about what species that patient reacts to. Even though this indicates a true Hymenoptera venom sensitization, additional testing with component-based tests can confirm if the patient is sensitized to a major allergen in the relevant species. Venom immunotherapy treatment may be more effective in patients who are sensitized to these major allergens.
New diagnostic tools in Hymenoptera venom allergy are now available for clinicians
The recent development of IgE tests against species-specific allergen components in Hymenoptera venom allergy offers diagnostic tools that greatly improve the ability to differentiate between sensitization to bees and wasp, and helps in discriminating between clinically relevant and irrelevant sensitizations.
Identification of which molecules that triggers the severe reaction is of vital importance for the clinician when considering venom immunotherapy. The combined use of venom components and Tryptase optimize the diagnosis and management of patients with a suspicion of venom allergy. Currently only Thermo Fisher Scientific, formerly known as Phadia, Uppsala, Sweden, have both tryptase and allergen component test available on the same technology platform.
The author
By Magnus Borres, MD, PhD, MPH
Pediatric Allergist, Uppsala University Hospital, Uppsala, Sweden
Medical Director, ImmunoDiagnostics, Thermo Fisher Scientific, Uppsala, Sweden
Molecular allergology – probing deeper into the triggers of allergies
, /in Featured Articles /by 3wmediaMolecular allergology is a cutting edge technology that enables the triggers of allergies to be characterized to a new level of detail. Two new component-resolved immunoblot test systems provide in-depth profiling of allergic reactions against birch and grass pollens and against bee and wasp venoms. The molecular tests supplement the established Euroline allergy range, which comprises a comprehensive spectrum of application-oriented profiles designed for use in any diagnostic laboratory.
by Dr Jacqueline Gosink
Advanced diagnostic approach
Molecular allergology or component-resolved diagnostics is a novel approach to allergy diagnostics, whereby single purified allergen components (SPAC) are used for specific IgE detection in place of the usual whole extracts. This powerful technology introduces a new dimension to differential allergy diagnostics.
Precise, in-depth profiling
The raw allergen preparations of substances such as pollen that are traditionally used for in vitro allergy diagnostics are generally not well characterized and are thus difficult to standardize. In contrast, the allergenic targets used in molecular allergology tests are defined recombinant proteins, which are capable of delivering precise information about the source of sensitization.
The in-depth profiling enables allergologists to:
Multiple pollen sensitizations
Pollen allergies are the most frequently occurring inhalation allergies, with sensitizations to birch and grass pollen as the most common ones. Typically, patients with multiple pollen sensitizations suffer from rhinitis, conjunctivitis and allergic asthma. The allergen extract-based determination of specific IgE antibodies encompasses sensitizations against major allergens and cross reacting minor allergens.
The Euroline SPAC Pollen 1 profile (Figure 1) combines the major and minor allergens of birch (Bet v1, Bet v2, Bet v4, Bet v6) and timothy grass (Phl p1, Phl p5, Phl p7, Phl p12), allowing the differentiation of pollen cross reactions from true multiple pollen sensitizations.
The efficacy of the assay has been confirmed by clinical studies. In one study the test successfully confirmed sensitizations to birch or grass pollen in 77 patients with clinically and anamnestically diagnosed allergies (1), and in a further study the test verified allergic reactions in 44 patients with birch and grass pollen double sensitizations (2). Furthermore, the test system correlated well with comparable commercial assays, demonstrating an EAST class correlation of 95-100% for each of the allergen components.
Bee and wasp venom allergies
Bee and wasp venom stings can pose a problem in the summer months. Whereas a normal reaction to a sting involves local swelling, itching and reddening, persons with an allergy can develop severe systemic reactions, including anaphylactic shock. Bee and wasp venom reactions can be identified using the single allergen components i208 (bee venom) and i209 (wasp venom). i208 represents the main bee venom marker rApi m1 from the honey bee (Apis mellifera) and i209 is the main allergen rVes v5 from the common wasp (Vespula vulgaris). Both preparations are free of cross-reactive carbohydrate determinant (CCD), providing higher reliability in result interpretation. The SPAC analysis allows true double sensitization to be distinguished from cross reactions between insect venoms. The Euroline SPAC Insect Venoms 1 profile (Figure 1) provides the recombinant antigens i208 and i209 together with the corresponding extracts i1 (bee venom) and i3 (wasp venom), allowing an efficient and comprehensive investigation of bee and wasp venom sensitizations with one test.
Fast and easy test procedure
The molecular allergology immunoblot tests are fast and simple to perform and are suitable for use in any diagnostic laboratory. The test procedure is based on established Euroline technology and consists of three basic steps: serum incubation (60 min), conjugate incubation (60 min) and chromogen substrate incubation (10 min). The in-between washing steps are short, and the entire procedure can be completed in 2.5 to 3 hours. All reagents are ready to use, saving time and reducing the risk of errors.
Only small amounts of sample material, typically 400 μl, are required per test. In a special volume-optimized version of the protocol the test can be performed with as little as 100 μl of patient sample, making it ideal for use in pediatrics.
Since the allergens are configured as a line blot with related allergens grouped together, the evaluation of profiles is effortless. Results are classified according to the RAST/EAST system. All profiles additionally include an indicator band of CCD to aid interpretation of the relevance of specific IgE results, for example in cases where positive IgE reactions are inconsistent with the clinical picture.
Fully automated processing
The standardized design of Euroline test strips allows automated processing using immunoblot incubators such as the EUROBlotOne (Figure 2). This advanced system automates the entire Euroline procedure from sample entry to report release. The compact, tabletop device has a high walkaway capacity: up to 44 strips can be incubated per run, and different tests can be combined in one run. All dilution, incubation and washing steps are performed automatically, and the integrated barcode scanner ensures that the correct samples are pipetted. User-friendly menus provide easy navigation, and error-detection features ensure high reliability. Test strips are subsequently digitalized using a special camera module.
Results are then automatically evaluated and archived using the worldwide-established and user-friendly EUROLineScan software. The software automatically identifies, quantifies and assigns bands, and a full results report is available within minutes of completing the incubation (Figure 3). The extensive individual data is administered and documented by the system, and all images and data are electronically archived, eliminating the need to store potentially infectious blot strips. The software can be easily integrated into LIS software, for example the EUROLabOffice system, for a smooth daily laboratory routine.
Comprehensive Euroline allergy range
The new molecular allergy tests are part of the established Euroline allergy range, which provides efficient multiparameter analysis of IgE antibodies against up to 36 different allergens in parallel. The immunoblots are composed from a wide portfolio of allergens, comprising both SPAC and native extracts which have been extensively purified and carefully quality controlled to ensure consistency. All profiles are application-oriented, each one being designed to address a particular diagnostic inquiry.
The Euroline system offers a very competitive price per allergen, making this system the ideal choice for laboratories wanting to perform state-of-the art allergy diagnostics on a small budget.
Perspectives
The advent of molecular allergology technology represents a quantum leap for allergy diagnostics. Component-resolved allergy test systems are unrivalled in the depth of diagnostic information they deliver and hence the level of support they provide for therapeutic decision-making. The Euroline SPAC range will soon be expanded to include further test systems based on this cutting-edge technology.
References
1. Weimann et al. 30th Annual Congress of the EAACI, Istanbul, Turkey, June 2011.
2. Weimann et al. 20th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine (EuroMedLab), Milan, Italy, May 2013.
The author
Jacqueline Gosink PhD
Euroimmun AG
Luebeck, Germany
Autoantibodies against MDA-5: very important serological markers in amyopathic dermatomyositis with rapidly progressive interstitial lung disease
, /in Featured Articles, Pathology & Histology /by 3wmediaby Assoc. Prof. Y. Muro, Assoc. Prof. K. Sugiura and Prof. M. Akiyama Autoantibodies against MDA-5 are serologically important biomarkers because they are mainly detected in patients with amyopathic dermatomyositis complicated with rapidly progressive interstitial lung disease (ILD). Anti-MDA-5 antibodies are useful not only for diagnosis but possibly also for monitoring disease activity in ILD.
The relevance of the manufacturer in indirect immunofluorescence standardization
, /in Featured Articles /by 3wmediaAutoantibody detection is a powerful laboratory tool for clinical diagnosis in the autoimmune diseases field. Among the techniques most widely used worldwide, indirect immunfluorescence (IFA) plays a particularly important role not only in the diagnosis but in the follow up of many diseases and remains the hallmark despite the introduction of new techniques in the routine of clinical laboratories. Witness to this is the renaissance of the antinuclear antibodies (ANA) screening on HEp2 cells by this techique or the renewal of the detection of anti-endomysium antibodies on monkey esophagus as the gold standard serological test for celiac disease. Therefore, IFA is a technique in full validity and requires a level of standardization that unfortunately is far from being achieved.
by Petraki Munujos, PhD
The efforts to improve standardization of indirect immunofluorescence as a diagnostic tool are numerous worldwide. Traditionally, the players involved in standardization have been clinical laboratories, clinicians, regulators, and to a lesser degree, diagnostic reagents manufacturers. Energy has been concentrated basically in aspects like the control of laboratory procedures, unification of nomenclatures and classifications, guidelines on how to report the results, preparation of recommendations, definition of diagnostic criteria and diagnostic algorithms and development of external quality control programs. In these iniatives, laboratory staff, clinicians and regulators are mainly involved. Nevertheless, those aspects regarding the design, development and manufacturing of the reagents, which involve manufacturers, are basically ignored. And this is probably due to the fact that the evolution of the technology has led to a truncated view of the test procedure resulting in a misconception of what needs to be standardized. In other words, the execution of many procedures is nowadays being shared between the manufacturer, who actually initiates the assay, and the laboratory, where the test is finalized. In old scientific articles related to ANA, the Material and Methods section usually started with the cell culture, the preparation of the slides and the fixation among others, and the sample incubation was only one more step of the whole procedure. Currently, the Material and Methods section starts with the sample preparation and instead of describing all the preliminary steps, one can find the name and references of the manufacturer. Figure 1 illustrates what would be the whole test procedure, showing the part performed in the clinical laboratory, actually the only part which is taken into consideration when dealing with standardization. So, to ensure appropriate use of indirect immunofluorescence testing, clinicians, diagnostic laboratories, regulators and reagents manufacturers should be involved and share the tasks of identifying and managing the key points leading to proper results.
Evidences of disparity
At the level of the manufacturer, the potential variability in the performance of the kits lies in features like the reagents and materials that are purchased or manufactured to become components of the kit, the procedures and conditions of manufacturing (fixatives, temperatures, formulations), the reliability of the serum samples used to set up the calibration of the determination (basically, the sample dilution which actuallly acts as the cut-off point), and the stability of the final product (1).
When approaching the participation of the manufacturer in the standardization of antibody testing, it is observed that what basically matters for industry is the standardization of the manufacturing processes. This normally occurs in an environment of Quality System Certifications, like GMP, ISO-9001 or ISO-13485 and under the requirements of the European Directive on In Vitro Medical Devices, and it is strengthened by the manufacturer’s own interest in having robust and reliable processes. Nevertheless, despite regulatory compliant and well implemented standardized processes, there are several aspects that make final reagents differ from one manufacturer to another. Below are reviewed some examples of variation on the results depending on the manufacturer source.
Dense fine speckles 70 (DFS70) antigen
As with other fluorescence patterns, the typical DFS pattern (lens epithelium-derived growth factor) can vary depending on the manufacturer source of the HEp2 slides used. The variations consist basically in different sensitivities and even in positive and negative results for the same sample run in different slide brands. Inconsistencies are also observed when comparing fluorescence with the results obtained by means of ELISA (2,3).
Ribosomal P protein (Rib P)
In studies performed by Mahler et al. (4) to determine the sentitivity of the immunofluorescence technique to detect antibodies against ribosomal P protein, several different HEp2 slides manufacturers were used, resulting in significant differences in patterns of staining for monospecific anti-Rib-P sera. Differing patterns were observed for the same sample, from a fine speckled nucleoplasmic pattern, to a diffuse cytoplasmic staining, or a fine speckled cytoplasmic pattern.
CDC/AF Reference Human Sera
When running reference sera on HEp2 slides coming from different manufacturers, variations of unknown origin can be observed. While most brands produce the expected specific pattern, there are often differences among brands like the ones shown in Figure 2.
Labile nuclear antigens
Most of the patterns observed when analysing the presence of ANA in patients sera by IFA on HEp2 cells slides are suitably detected in most slides brands. However, there are some antigens for which expression may significantly vary from one manufacturer to another like Jo1, PCNA or SSA/Ro (5). These antigens are not always well preserved in the substrates and they can be extremely sensitive to handling, to certain fixatives and in some cases, they can be just washed out during the manufacturing process, resulting in a poor presence or a total lack of antigenic molecules available to capture the antibody being analysed.
Antineutrophil cytoplasmic antibodies (ANCA)
The neutrophil substrates used in the detection of ANCA may vary in their ability to give the typical immunofluorescence patterns described and established by consensus groups, i.e. a diffuse granular cytoplasmic staining with higher interlobular intensity (C-ANCA), a compact staining of the perinuclear zone of the cytoplasm (P-ANCA) and a broad non homogeneous perinuclear staining, eventually accompanied by a diffuse cytoplasmic pattern with no accentuation of the interlobular zone (X-ANCA). In general, substrates differ in their ability to distinguish between a C-ANCA and X-ANCA. In a study by Pollock et al. (6), it was observed that although all commercial neutrophil substrates consistently demonstrated nuclear extension of perinuclear fluorescence with sera containing P-ANCA with MPO specificity, there were more problems in P-ANCA testing than in C-ANCA, due basically to the eventual presence of additional cytoplasmic fluorescence.
Crithidia luciliae
In a similar way as observed in HEp2 cells immunofluorescence patterns, the anti-nDNA test on Crithidia luciliae slides may show significant differences among manufacturers. The variety of strains available in cell banks contribute to the heterogeneity of results. Apart from the kinetoplast, other organelles can be stained by antibodies from the sample, like the nucleus, the basal body and the flagellum. Depending on the conditions of preparation of C. luciliae substrates and on the nature of the sample analysed, different patterns of stained organelles can be observed. Nevertheless, the only specific staining to be considered as a positive result is the kinetoplast staining. In addition to anti-nDNA antibodies, there are other antibodies in the serum of lupus patients that can react with the substrate. The so called anti-nucleosome antibodies are antibodies that react with histones exposed in the nucleosome. It is well known that treating C. luciliae substrate with HCL eliminates histone from the kinetoplast (7). This could be another point of possible discrepancy among manufacturing processes if some include the histone removal procedure and some others do not. Furthermore, the cell cycle of C. luciliae may influence histone appearance in the kinetoplast. Therefore, the manufacturing process of C. luciliae slides, including culture, harvest, fixation and drying, can cause variation in the results.
Aspects providing variablity
Among the players participating in autoimmune diagnostics, there is no doubt that manufacturers hold the know-how of preparing diagnostic kits and are the true experts in the development of test methods. However, despite the standardized manufacturing processes and the CE-certifications or FDA approvals, there are several aspects that are found to be sources of variabilty. These aspects should be addressed and recommendations on key points should be created by specialized committees with the participation of laboratory experts, clinicians and manufacturers. The definition and control of the raw materials incorporated in the kit production is a common and regulated practice in any kind of manufacturing process. But recommendations on nature, compostion or quality grades of key materials, including culture media, cell type and strain or fluorescent conjugates is still lacking. In the case of tests based on cellular substrates, extracellular matrix (ECM) proteins are commonly used to aid the spreading and growth of cells on the slide glass surface. Many ECM proteins contain defined amino acid sequences to which cell surface integrin receptors bind specifically. ECM, together with growth factors in the culture medium, work to produce an appropriate in vitro proliferative response, promoting cell growth and spreading. Altering cell-ECM contacts results in coordinated changes in cell, cytoskeletal, and nuclear form. Thus, the choice of the right ECM to coat the glass slides used as growing surface deserves our attention since it might have a direct effect on the fluorescent pattern finally observed (8). It is also common to use synchronization agents to achieve a greater rate of mitotic cells. Due to the fact that these compounds may be toxic for the cell, some cell disturbances may occur that can impact the morphology or the behaviour of the final cell preparation.
Diagnosis by means of tissue sections remains very important in liver autoimmune diseases like autoimmune hepatitis (AIH) or primary billiary cirrhosis (PBC). In particular, the detection of anti-smooth muscle antibodies (ASMA), antibodies to liver-kidney microsomes (LKM antibodies) and anti-mitochondrial antibodies (AMA) are considered important diagnostic tools. Only a few guidelines have been published on the obtention of tissue sections (9), while the variations in the preparation of tissue blocks regarding orientation, preservation conditions, and sectioning keep on contributing to the heterogeneity of results, especially in the case of tissues that are not morphologically homogeneous. For instance, the LKM antibodies can only be well defined if the kidney section has the proper orientation that allows the distinction between proximal and distal renal tubules and, thus, between LKM and AMA.
Considering that the expression and topographical distribution of autoantigens is under the direct influence of the HEp-2 fixation method, some immunofluorescence patterns are not adequately expressed due to the way that the antigenic substrate is prepared. This aspect equally affects tissue and cell substrates. As for the sensitivity of the tests, differences among manufacturers are due to the use of fixatives to prolong shelf-life. The use of slides without fixation seems to be the best choice for most autoantibody patterns. Nevertheless, there are several staining patterns that need the substrate to be fixed (figure 3), like anti-islet cells antibodies or anti-adrenal cortex antibodies.
A less frequent but significant source of variability in the immunofluorescence on tissue sections can be found in the origin of the animal used (Figure 4). Definition of suitable species and strains should be addressed in some cases in which the levels of antigen expression may differ. This affects the sensitivity of the test, especially in samples with moderate or low titers of antibody.
Considering the complexity and diversity of manufacturing processes and subprocesses and their impact on the final test performance, it is important to combine the efforts of laboratory experts, clinicians and manufacturers in the task of standardizing those key aspects that could otherwise keep on undermining the successful harmonization of the results obtained in the clinical laboratory.
References
1. Fritzler MJ, Wiik A, Fritzler ML, Barr SG. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 2003, 5:192-201
2. N.Bizzaro, E.Tonuttiand D.Villalta, «Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al,» Arthritis Rheum, vol. 63, no. 12, pp. 4036-4037, 2011
3. Mahler M. The clinical significance of anti-DFS70 antibodies as part of ANA testing. In: K. Conrad, E.K.L. Chan, M.J. Fritzler, R.L. Humbel, P.L. Meroni, G. Steiner, Y. Shoenfeld (Eds.). Infection, Tumors and Autoimmunity, AUTOANTIGENS, AUTOANTIBODIES, AUTOIMMUNITY, Volume 9, p.342-350. PABST, 2013.
4. Mahler M, Ngo JT, Schulte-Pelkum J, Luettich T, Fritzler MJ. Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies. Arthritis Research & Therapy 2008, 10:R131
5. Dellavance A, de Melo Cruvinel W, Carvalho Francescantonio PL, Pitangueira Mangueira CL, Drugowick IC, RodriguesSE; Coelho Andrade LE. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides J Bras Patol Med Lab 2013;49( 3):182-190.
6. Pollock W, Clarke K, Gallagher K, Hall J, Luckhurst E, McEvoy R, Melny J, Neil J, Nikoloutsopoulos A, Thompson T, Trevisin M, Savige J. Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate. J Clin Pathol 2002;55:680–683
7. Kobkitjaroen J, Jaiyen J, Kongkriengdach S, Potprasart S, Viriyataveekul R. Comparison of Three Commercial Crithidia luciliae Immunofluorescence Test (CLIFT) Kits for Anti-dsDNA Detection. Siriraj Med J 2013;65:9-11
8. (Integrin Binding and Cell Spreading on Extracellular Matrix Act at Different Points in the Cell Cycle to Promote Hepatocyte Growth Hansen LK,. Mooney DJ, Vacanti JP, Ingber DE. Molecular Biology of the Cell 1994;5:967-975
9. Vergani D, Alvarez F, Bianchi FB, Cançado ELR, Mackay IR, Manns MP, Nishioka M, Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. Journal of Hepatology 2004;41: 677–683