Affecting around one in twenty pregnancies, pre-eclampsia is a leading cause of fetal morbidity and mortality globally. Around half a million babies die as a result of the condition annually. Severe pre-eclampsia, leading to eclampsia characterized by seizures, is also the second leading cause of maternal mortality (after hemorrhage) in most countries: an estimated 76,000 women die from it each year. A diagnosis of this multisystemic disorder has classically been made if hypertension and proteinuria are present. Pre-eclampsia can only be resolved by delivery of the placenta, thus management must weigh the severity of the condition against the risk to the fetus of an induced, premature delivery.
The launch of a rapid test measuring the plasma level of placental growth factor (PLGF), a biomarker of placental function, four years ago offered the possibility of a more timely diagnosis of pre-eclampsia and its severity that could facilitate optimal management for both mother and baby, including the administration of corticosteroids to accelerate fetal lung development prior to premature delivery. The level of PLGF normally rises during pregnancy up to 26 to 30 weeks’ gestation, and then falls until full-term, but its level is abnormally low in women with pre-term pre-eclampsia. Recently the published results of a large multicentre study using this rapid test made very encouraging reading. During the study, PLGF was measured in 625 pregnant women between 20 and 35 weeks gestation with suspected pre-eclampsia. The condition was confirmed in 55% of these women, with outcome being the delivery of the fetus within 14 days. The authors concluded that the test had high sensitivity in women presenting with suspected pre-eclampsia before 35 weeks’ gestation, and indicated need for delivery better than other diagnostic methods.
Although this research is good news for pregnant women and their babies, another aspect of pre-eclampsia has largely been ignored and is not generally known by either health-workers or women themselves, namely the subsequent increased health risk in older women who suffered from pre-eclampsia in pregnancy. A robust meta-analysis has linked the condition with a fourfold increased risk of hypertension, and a twofold increased risk of ischemic heart disease, stroke and venous thromboembolism, later in life. A recent study from Australia found that the endothelial dysfunction associated with pre-eclampsia persists, causing the increased risk. At the very least previous pre-eclampsia should be flagged as important in an older woman’s medical history!
Bee 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
Eosinophilic 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
February | March 2025
Beukenlaan 137
5616 VD Eindhoven
The Netherlands
+31 85064 55 82
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.