Combining surface-enhanced Raman scattering (SERS) of saliva and two-dimensional shear wave elastography (2D-SWE) of the parotid glands in the diagnosis of Sjögren's syndrome
Graphical abstract
Introduction
Primary Sjögren's syndrome (SjS) is an immune cell mediated disease with typical presentation involving dryness of the mouth and eyes, fatigue and polyarthralgia [1]. SjS represents the second most common systemic autoimmune disease after Hashimoto's thyroiditis [2], with incidence estimates ranging between 3 and 11 cases per 100,000 individuals [2,3].
According to the most widely accepted theory termed ‘autoimmune epithelitis’, SjS is determined by an aberrant inflammatory response mediated by both innate and acquired immunity, primed by the epithelial cells that act as atypical antigen presenting cells and then further expanded by circulating pro-inflammatory cytokines [1]. Consequently, SjS is characterized by a diffuse lymphocytic infiltration of the parotid glands as well as by circulating antibodies against Ro (also called Sjögren's syndrome-associated antigen A (anti-SSA)) and/or La (Sjögren's syndrome-associated antigen B (anti-SSB) [4]. Although much has been discovered about the molecular mechanisms behind SjS, druggable pharmacological targets that could result in disease modifying therapies are still awaited [5].
Besides the classic triad, SjS also presents with general symptoms such as fever and weight loss as well as organ specific complications of the cutaneous, articular, pulmonary, cardiovascular, nephro-urological, nervous and haematological systems, which are present in around 50–60% of cases [6,7]. Interestingly, systemic symptoms can precede the characteristic syndrome, a condition termed occult SjS or non-sicca onset SjS.
SjS is sometimes diagnosed concomitantly with other systemic autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis, in which case the SjS is referred to as secondary SjS [8]. An important and potentially life-threatening complication of SjS is represented by lymphoma, which develops predominantly in the major salivary glands, the most common variant being the low-grade extranodal marginal zone B-cell non-Hodgkin lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma) [9].
Despite extensive evidence demonstrating an impaired quality of life in patients with SjS [[10], [11], [12]], treatment options are scarce and mainly based on clinical experience with other systemic autoimmune diseases and expert opinions [1]. The ocular and salivary manifestations of SjS (the sicca syndrome) can usually be managed with education and environment modifications and with medications applied topically, whereas other manifestations of SjS require systemic medication such as corticoids, methotrexate, hydroxychloroquine or biologicals, the use of which is unfortunately based on low-level evidence.
For many years, in research and clinical practice, the diagnosis of SjS has been based on the classification criteria elaborated by the American-European Consensus Group (AECG) [13]. The new classification criteria endorsed by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) are based on minor salivary gland biopsy, anti-SSA/Ro antibodies, ocular staining and salivary and tear flow [14]. Concurrently, patients must meet inclusion criteria based on a standardized set of questions related to ocular and oral dryness. Importantly, these symptoms must not be explained by a history of head and neck radiation, active hepatitis C infection, AIDS, sarcoidosis, amyloidosis, graft-versus-host disease or IgG4-related disease. These exclusion criteria underscore an important shortcoming of the current guidelines, which are intended for classification and not for diagnostic purposes. Moreover, the current guidelines rely on salivary gland biopsy for the definite diagnosis of SjS, an invasive procedure which can be associated with complications such as bleeding or infection and which requires highly trained medical personnel. Therefore, there is a need for novel non-invasive techniques that could aid the diagnosis of SjS.
Ultrasound elastography (USE) is a non-invasive, readily available and cost-effective technique which employs ultrasound waves for enquiring the perturbed tissue stiffness that accompanies both diffuse parenchymal diseases and space occupying lesions [15,16]. USE has already gained wide acceptance for detecting liver fibrosis [17] and it becomes increasingly important for other organs as well, including the breast [18,19] and the thyroid and salivary glands [20,21].
Depending on whether the stiffness is calculated based on longitudinal or transversal sound waves, USE is classified in strain elastography and the more modern shear wave elastography (SWE), respectively. SWE can be further subdivided in transient elastography (TE), point shear wave elastography/acoustic radiation force impulse quantification (pSWE/ARFI-SWE), and 2-dimensional 2D-SWE [15]. The technique of 2D-SWE has the advantage that it provides quantitative information and therefore it is less operator-dependent [16].
In the context of SjS, several lines of evidence suggest that USE along with classical ultrasonography of parotid glands has a valuable input in regard to the differential diagnoses of SjS and there is an ongoing effort to include ultrasound imaging in the guidelines on SjS [22,23]. Accomplishing this undertaking rests upon improving the inter-observer variability of the USE, which was shown to be a significant issue, especially in the case of inexperienced users [24]. Newer types of quantitative elastography such as 2D-SWE was shown to reliably assess perturbations in the stiffness of the parotid glands, but this method has not been yet evaluated in the case of SjS [25].
Raman spectroscopy is a type of vibrational spectroscopy that has attracted much attention due to its potential application in life sciences [26]. The physical effect behind Raman spectroscopy is represented by the inelastic scattering of the incoming photons, which excite the vibrational energy structure of the molecules within the sample, thus providing molecular specific information. In the realm of musculoskeletal diseases such as rheumatoid arthritis, osteoarthritis, gout or calcium pyrophosphate deposition disease, promising results were reported on a wide range of biological samples such as bone, cartilage, synovial fluid or chondrocytes (reviewed in [27,28]). However, the use of Raman spectroscopy is often limited by the low yield of the method, since on average only one in 107 photons is scattered inelastically and thus caries information about the molecular structure of the sample.
This shortcoming can however be overcome by a wide array of methods, including surface-enhanced Raman scattering (SERS) [29]. Other amplification methods are stimulated Raman scattering (SRS) [30], coherent anti-Stokes Raman scattering (CARS) [31] or resonant Raman scattering (RRS) [32].
The amplification of the Raman scattering in SERS is based on the presence of nanometer-sized metal structures, which display characteristic collective oscillations of electrons termed plasmons [33]. One of the most convenient and widely used SERS substrates are represented by metal nanoparticles in suspension, which are particularly well suited for analyzing liquid samples (including biofluids) [34]. Although the exact mechanism of SERS is still debated, the method is increasingly used as an analytic method in diverse areas of science, including life sciences. For instance, promising results were reported on the use of SERS for assessing the molecular perturbations in biofluids such as serum, urine, saliva or cerebrospinal fluid [34,35].
In this study, we employed SERS spectroscopy on saliva from patients with SjS and controls, with the aim of using the molecular spectral information gained via SERS for complementing the findings of 2D-SWE, a type of elastography which has not been yet evaluated in the case of SjS. By employing multivariate statistical analysis, we combined the input of 2D-SWE with that of SERS and used hybrid 2D-SWE -SERS models for discriminating between SjS and controls. To the best of our knowledge, this is the first study which describes the use of SERS on saliva for diagnosing SjS as well as the possibility of combining SERS with state-of-the-art quantitative ultrasound techniques such as 2D-SWE.
Section snippets
Materials and methods
In this nonblinded preliminary study, we included n = 31 patients diagnosed with primary SjS based on the ACR/EULAR classification criteria (13), while the controls were represented by n = 22 healthy volunteers with similar age and sex distribution to the control group. At the time when the study was performed, all SjS patients were aware of their disease. The study was approved by the Ethics Committee of the Iuliu Hatieganu University of Medicine and Pharmacy and all participants provided
Results
2D-SWE is an advanced elastography techniques which can display qualitatively and quantitatively the shear modulus of the scanned tissues. A typical example of a 2D-SWE scan of the parotid gland is shown in Fig. 1. Using 2D-SWE, we have quantified the average shear modulus of the parotid glands.
Among the 31 SjS patients in our study, 29 were female, while in the case of the control group, 21 out of the 22 control subjects were females. The age of the subjects had a parametric distribution in
Discussion
In line with previous studies on the epidemiology of SjS showing a female-to-male ratio as high as 20:1 [38,39], among the 31 SjS patients in our study, 29 were female. In the case of the control group, 21 out of the 22 control subjects were females, such that both groups had similar female-to-male ratios.
In this proof-of-principle study, we sought to combine 2D-SWE of parotid glands with SERS of saliva samples in order to devise a novel, non-invasive strategy of diagnosing SjS. 2D-SWE is a
Conclusion
The results of this study suggest that SERS could contribute to the diagnosis of SjS by complementing morphological information related to the stiffness of the glands with information regarding the molecular composition of saliva. Thus, PCA-LDA models combining data from 2D-SWE and SERS of saliva yielded an overall accuracy of 81%, which is higher than the overall accuracy corresponding to just SERS (overall accuracy 75%) or just 2D-SWE data (overall accuracy 71%). This study is the first
CRediT authorship contribution statement
Vlad Moisoiu: Methodology, Writing - original draft, Writing - review & editing. Maria Badarinza: Methodology, Writing - original draft, Writing - review & editing. Andrei Stefancu: Methodology, Writing - review & editing. Stefania D. Iancu: Methodology, Writing - review & editing. Oana Serban: Methodology, Writing - review & editing. Nicolae Leopold: Conceptualization, Writing - review & editing. Daniela Fodor: Conceptualization, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
A. Stefancu and S.D. Iancu acknowledge support from the College for Advanced Performance Studies, Babeș-Bolyai University. S.D. Iancu also acknowledges the performance fellowship from the Babeș-Bolyai University contract nr. 35996/28.11.2018.
Funding
Financial support from the Competitiveness Operational Programme 2014–2020 (POC-A1-A1.1.4-E-2015), financed under the European Regional Development Fund, project number P_37_765 is highly acknowledged. Also, a grant from the Romanian Ministry of Research
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These authors contributed equally to this work, being considered co-first authors.