Abstract
Clinical manifestations, as distinct from thrombotic and obstetric morbidity, were recently included in the update of classification criteria of the antiphospholipid syndrome (APS). However, the existence of several patients with clinical manifestations suggestive of APS, but negative for criteria antiphospholipid antibodies (aPLs) [anti-cardiolipin antibodies (aCL), anti-β2-glycoprotein I antibodies (aβ2-GPI), and lupus anticoagulant] may suggest an update of diagnostic criteria. In this study, we analysed the prevalence of six non-criteria aPLs in a large monocentric cohort of patients with seronegative APS (SN-APS), to investigate their possible diagnostic role. aCL IgA, aβ2-GPI IgA, and aβ2-GPI Domain 1 antibodies were detected by chemiluminescence, anti-phosphatidylserine/prothrombin (aPS/PT) IgG, anti-vimentin/cardiolipin (aVim/CL) IgG, and anti-carbamylated-β2-glycoprotein I (aCarb-β2-GPI) IgG by ELISA in sera from 144 SN-APS patients. In SN-APS patients, aCL IgA was detected in 4/144 (2.77%), aβ2-GPI IgA in 2/144 (1.39%), aβ2-GPI-Domain 1 in 1/144 (0.69%), aPS/PT in 16/144 (11.11%), aVim/CL in 37/144 (25.69%), and aCarb-β2-GPI in 43/144 patients (29.86%). Patients negative for all non-criteria aPL assays were 77/144 (53.47%). Notably, the Venn diagram showed that aCarb-β2-GPI together with aVim/CL represented the prevalent combination of positive antibodies. In SN-APS patients, aCL IgA were associated with recurrent thrombosis (OR 11.48; P = 0.03); in obstetric SN-APS patients, aPS/PT were significantly associated with foetal deaths (OR 4.84; P = 0.01), aVim/CL with spontaneous abortions (OR 2.71; P = 0.016). This study indicates that aPS/PT, aVim/CL and aCarb-β2-GPI antibodies may represent useful tools to identify ‘seronegative’ APS patients, who are negative for criteria aPLs, supporting the need to make testing for non-criteria aPLs more accessible in patients with SN-APS.
Keywords: antiphospholipid syndrome, seronegative APS, anti-β2-GPI, anti-vimentin/cardiolipin, anti-phosphatidylserine/prothrombin, carbamylation
This study demonstrates the presence of six non-criteria antiphospholipid antibodies, anti-cardiolipin antibodies IgA, aβ2-GPI IgA, aβ2-GPI Domain 1, aPS/PT, aVim/CL, aCarb-β2-GPI antibodies in a large cohort of ‘seronegative’ antiphospholipid syndrome (SN-APS) patients. They revealed potential usefulness in the identification of a significant proportion of SN-APS. Patients tested positive showed a higher prevalence of recurrent thrombosis, foetal deaths, or spontaneous abortions, suggesting that this test may be considered a suitable approach in the evaluation of SN-APS patients.
Graphical Abstract
Graphical Abstract.
Introduction
Forty years after the first description of antiphospholipid syndrome (APS) [1], clinical manifestations, as distinct from thrombotic and obstetric morbidity, were included in the 2023 update of classification criteria [2]. The call for an update derives from different aspects surrounding the disease.
Regarding laboratory diagnosis, APS depicts a mosaic, in which antiphospholipid antibodies (aPLs) are a heterogeneous family of antibodies, including more than 30 different antibody specificities that have been described in patients with the syndrome. The need to update these diagnostic criteria derives from the existence of a patient subgroup that corresponds to the so-called seronegative APS (SN-APS) [3–5].
Despite the growing number of national and international studies on SN-APS and non-criteria aPLs, patients with clinical manifestations suggestive of APS, but doggedly negative for criteria aPLs [anti-cardiolipin (aCL), anti-β2-glycoprotein I (aβ2-GPI), and lupus anticoagulant (LA)], no unconventional antibodies were included in the new criteria [6–12]. Beyond classification criteria, SN-APS represents a diagnostic problem in which ‘non-criteria aPLs’ could help physicians to identify those patients who needs specific treatment [13]. The clinical scenario of SN-APS was described for the first time in 2003 from Hughes and Khamashta and seronegativity predisposing factors were evaluated [14]. SN-APS patients, just like classic APS patients, mainly manifest an increased risk for thrombotic events and pregnancy morbidities. The diagnostic identification of patients with SN-APS, providing therapeutic or preventative medication, remains a remarkable challenge. The diagnostic problem could be linked, first, to a misdiagnosis or to a previous positive aPL test turned negative.
Furthermore, but more likely, seronegativity is due to the methodological approach, regarding the type, sensitivity and specificity of the assay employed to detect aPLs, or yet, the existence of unknown antibodies playing a role in the APS pathogenesis and diagnosis [15–17]. Based on different results of SN-APS patient series, it may be speculated that the prevalence of SN-APS patients will decrease over time paralleling the discovery and identification of new autoantibodies associated with the disease. Discoveries of new autoantibodies have helped to detect potential SN-APS patients as well as provide additional insight into the mechanisms of the disease.
To date, several ‘new’ tests have been proposed and used to better identify SN-APS, and it was possible to demonstrate the presence of non-criteria aPLs or new laboratory methods have been proposed to detect aPLs in SN-APS patients. Autoantigen specificity of these non-criteria aPLs may include different phospholipids, protein/phospholipid complexes, proteins, and post-translationally modified proteins [18–21]. Among these, anti-phosphatidylserine/prothrombin (aPS/PT) and anti-vimentin/cardiolipin (aVim/CL) antibodies represent the main tool for diagnosis of SN-APS patients [6, 11, 22–24]. Moreover, aβ2-GPI Domain 1 antibodies [25–27], aβ2-GPI IgA, aCL IgA [28–30], and anti-carbamylated-β2-glycoprotein I antibodies (aCarb-β2-GPI) [31] were also suggested for the identification of APS patients and have been the object of previous investigations.
Interestingly, several data highlighted a role for these non-criteria aPLs, not only as serological markers but also in the signal transduction pathway(s) involved in the immunopathogenesis of thrombosis and/or APS clinical manifestations.
In this study, we analyzed the prevalence of six non-criteria aPLs, also evaluating their association, in a large monocentric cohort of SN-APS patients, to investigate their possible diagnostic role in these patients.
Materials and methods
Patients
The study included consecutive patients referred to the Lupus Clinic, Rheumatology Unit of Sapienza University of Rome from January 2014 to May 2023 before the publication of ACR/EULAR Antiphospholipid Syndrome Classification Criteria [2]. They presented clinical features consistent with APS diagnosis due to thrombotic and obstetrical manifestations, but completely negative for conventional aPLs (aCL, aβ2-GPI, and LA tests) in at least two consecutive occasions 12 weeks apart between tests. Enrolled patients must have at least one of the following criteria: age < 50 years, recurrent thrombotic events, extra criteria manifestations, or another autoimmune disease. In SN-APS patients, other possible causes of thrombosis or obstetric morbidities were ruled out by a multidisciplinary team. All patients were tested for common inherited thrombophilic defects (no homozygous mutation of Factor II, Factor V; normal range of antithrombin III, homocysteine, protein S, and protein C); in addition, patients with a history of neoplasia and sepsis were excluded. Regarding obstetrical manifestations, patients with pregnancy morbidity due to chromosomal diseases, infections or pregnancy complications attributable to uterine malformation or other anatomical reasons were also excluded.
As control groups, we analyzed sera from 50 healthy donors (HD), without medical history of thrombosis, obstetrical morbidity, and autoimmune diseases.
Sera were collected from two blood samples at least 12 weeks apart and stored at −20 °C until use. This study was approved by ethics committees of Sapienza University of Rome (protocol 0215/2021), and participants gave written informed consent.
Detection of criteria aPL antibodies
aCL and aβ2-GPI antibodies (IgG, IgM) were detected using an immune-enzymatic assay by the QUANTA Lite detection kit using a QUANTA-Lyser 3000 system (Inova Diagnostic Inc., San Diego, CA, USA). All the obtained results were confirmed by chemiluminescence assay by QUANTA Flash detection kit using a BIO-FLASH system (Inova Diagnostic Inc.). In addition, LA was evaluated by two coagulation systems, a dilute sensitized activated partial thromboplastin time (aPTT) and a dilute Russell’s viper venom time (dRVVT), and then performing a confirmation test (Hemoliance Instrumentation Laboratory, Lexington, MA, USA).
Detection of anti-cardiolipin IgA, anti-β2-glycoprotein IgA, and anti-β2-glycoprotein I Domain 1 antibodies by chemiluminescence
Antibodies of IgA class against CL and β2-GPI and antibodies specific for Domain 1 of β2-GPI were detected by chemiluminescence assay by QUANTA Flash detection kit, using a BIO-FLASH system (INOVA Diagnostic Inc.).
Detection of anti-phosphatidylserine/prothrombin antibodies by ELISA
Antibodies (IgG) against PS/PT complex were detected by ELISA using a QUANTA Lite detection kit, using a QUANTA-Lyser 3000 system (INOVA Diagnostic Inc.), according to manufacturer’s instructions.
Detection of anti-vimentin/cardiolipin and anti-Carbamylated-β2-glycoprotein I antibodies by ELISA
To detect aVim/CL and aCarb-β2-GPI antibodies (IgG), sera of all patients and HD were tested by ELISA, as previously reported [12, 31].
In particular, Cardiolipin (50 μg/ml in methanol) (from bovine heart, Sigma–Aldrich, Milan, Italy) and human recombinant vimentin (5 μg/ml in 0.05 mM NaHCO3 buffer, pH 9.5) (R&D System, Minneapolis, MN, USA), in the amount of 100 μl/well, were used to coat a 96-well polystyrene plate (Thermo Fisher Scientific, Waltham, MA, USA).
In parallel experiments, β2-GPI was carbamylated as previously described [31] and a 96-well polystyrene plate was coated with 100 μl/well of this Carb-β2-GPI (1 μg/well, diluted in 0.05 M NaHCO3 buffer, pH 9.5).
In both assays, coated plates were incubated overnight at 4 °C and then washed three times with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T), and the ELISA was performed as described below. After washing, plates were incubated for 2 h at room temperature (RT) with a blocking buffer, composed of 1% bovine serum albumin (BSA) in PBS (100 μl/well). Then, plates were washed as above and incubated with 100 μl/well of patient sera (diluted 1:100 in the blocking buffer) for 1 h at RT. Goat polyclonal anti-vimentin antibodies (R&D Systems) and rabbit anti-carbamyl-lysine polyclonal antibodies (CliniSciences, Nanterre, France) were used as positive controls in the ELISA. To visualize the antibody reaction, after washing, the plates were incubated (1 h at RT) with 100 μl/well of horseradish peroxidase-conjugated antibodies, anti-human IgG or anti-rabbit IgG or anti-goat IgG (Sigma–Aldrich) diluted in the blocking buffer. Then, after washing as above, 100 μl/well of O-phenylenediamine dihydrochloride buffer was added to the wells to reveal the bound peroxidase. Finally, color development was stopped with 100 μl/well of H2SO4 0.2 M for 5 min. The absorbance of the wells was measured with a microplate reader at 492 nm. Each sample was analyzed in triplicate.
Furthermore, all the sera were analyzed with the same procedure but without coated Vim/CL or Carb-β2-GPI. Data were analyzed as the mean optical density corrected for background (wells without coated antigens). For both assays, cut-off values were calculated using the mean of optical density ± 2 SD of 50 HD.
Statistical analysis
The distribution of continuous variables was analyzed by the Shapiro–Wilk test. Data are expressed as mean ± standard deviation (SD) or median (interquartile range, IQR) according to the variable distribution. Chi square test or Fisher’s exact test was used to compare categorical variables. The Spearman rank correlation was performed for aPLs OD. P-values < 0.05 were considered statistically significant. Data analysis was performed using SPSS version 20 (SPSS Inc., Chicago, IL, USA).
Results
Clinical characteristics of SN-APS
We enrolled 144 SN-APS patients with a median age of 40.5 years (IQR 18) and a female-to-male ratio of 7:1. The clinical and demographic characteristics of patients are reported in Table 1.
Table 1:
clinical and demographic characteristics of SN-APS patients
| Feature | n = 144 (%) |
|---|---|
| Male/female | 18/126 |
| Median age in years (IQR) | 40.5 (18) |
| PAPS | 95 (66.0) |
| SLE | 31 (21.5) |
| Other autoimmune diseases | 22 (15.3) |
| Pregnancy morbidity | 62/126 (49.2) |
| Spontaneous abortions | 40/126 (31.7) |
| Normal foetus deaths | 24 (19) |
| Premature births | 9 (7.1) |
| Thrombosis | 92 (63.9) |
| Arterial thrombosis | 46 (31.9) |
| Venous thrombosis | 59 (41) |
| Recurrent thrombosis | 32 (22.2) |
| Thrombosis + pregnancy morbidity | 11/126 (8.7) |
| Non-criteria APS features | 52 (36.1) |
| Livedo reticularis | 25 (17.4) |
| Thrombocytopenia | 12 (8.3) |
| Migraine | 23 (16) |
| Seizures | 4 (2.8) |
| Cognitive | 1 (0.7) |
| Cardiovascular risk factors | 62 (56.9) |
| Hypercholesterolemia | 14 (9.7) |
| Smoking | 27 (18.8) |
| Hypertension | 27 (18.8) |
| OC/HRT | 11/126 (8.7) |
| Diabetes | 5 (3.5) |
| Hyperhomocysteinemia | 7 (4.9) |
SN-APS: seronegative antiphospholipid syndrome; PAPS: primary antiphospholipid syndrome; OC: oral contraceptive; HRT: hormone replacement therapy.
All SN-APS patients had clinical features strongly suggestive of APS. Moreover, 34% had another systemic autoimmune disease, 22% reported recurrent thrombosis, and 36% had manifestations not included in the 2006 classification criteria [32].
The control group of 50 HD, with no medical history of thrombosis, obstetric morbidity, and autoimmune diseases, was matched for gender and age to the SN-APS group.
Occurrence of non-criteria aPLs in SN-APS and control group
Sixty-seven of 144 (46.53%) patients tested positive for at least one non-criteria aPL, in 54 patients the assays were repeated on a second occasion, at least 12 weeks apart, and 43 out 54 (79.63%) confirmed the positivity. Thirteen patients were not tested on a second occasion due to loss at follow-up. However, 77/144 (53.47%) patients were negative for all non-criteria aPL test used.
In SN-APS patients, aCL IgA was detected in 4/144 (2.77%), aβ2-GPI IgA in 2/144 (1.39%) and aβ2-GPI-Domain 1 antibodies in 1/144 (0.69%). Reactivity against PS/PT was observed in 16/144 (11.11%). Moreover, 37/144 patients (25.69%) were positive for aVim/CL antibodies. Finally, our results showed the presence of aCarb-β2-GPI antibodies in 43/144 patients (29.86%) (Fig. 1).
Figure 1:
occurrence of the non-criteria aPLs in SN-APS patients. Anti-β2-glycoprotein I Domain 1 (aβ2-GPI Domain 1), anti-β2-glycoprotein IgA (aβ2-GPI IgA), anti-cardiolipin IgA (aCL IgA), anti-phosphatidylserine/prothrombin (aPS/PT), anti-vimentin/cardiolipin (aVim/CL), and anti-carbamylated-β2-glycoprotein I (aCarb-β2-GPI)
Figure 2 shows the distribution of the autoantibodies in the group of positive patients to these tests, also showing the percentage of patients displaying multiple positivity for different autoantibodies. Notably, the aCarb-β2-GPI test detected autoantibodies in most of SN-APS patients and the Venn diagram showed that aCarb-β2-GPI together with aVim/CL antibodies represented the prevalent combination of positive antibodies (Fig. 3). All HD samples were negative for all the non-criteria aPLs under test. The receiver operating characteristic (ROC) analysis for aVim/CL and aCarb-β2-GPI test in SN-APS patients is shown in Fig. 4.
Figure 2:
antibody profiles of SN-APS patients. Anti-β2-glycoprotein I Domain 1 (aβ2-GPI Domain 1), anti-β2-glycoprotein IgA (aβ2-GPI IgA), anti-cardiolipin IgA (aCL IgA), anti-phosphatidylserine/prothrombin (aPS/PT), anti-vimentin/cardiolipin (aVim/CL), and anti-carbamylated-β2-glycoprotein I (aCarb-β2-GPI). The aCarb-β2-GPI assay detected autoantibodies in the majority of the patients
Figure 3:
Venn diagrams of the six non-criteria aPLs in SN-APS patients. The distribution of anti-β2-glycoprotein I Domain 1 (aβ2-GPI Domain 1), anti-β2-glycoprotein IgA (aβ2-GPI IgA), anti-cardiolipin IgA (aCL IgA), anti-phosphatidylserine/prothrombin (aPS/PT), anti-vimentin/cardiolipin (aVim/CL), and anti-carbamylated-β2-glycoprotein I (aCarb-β2-GPI) was shown. Positivity for all autoantibodies was determined by the respective cut-off values
Figure 4:
receiver operating characteristic (ROC) analysis of aVim/CL and aCarb-β2-GPI in SN-APS. The variation in antibody levels presented an area under the curve (AUC) of 0.773 and 0.708, respectively
Clinical association of non-criteria aPL in SN-APS patients
In SN-APS patients, the presence of aCL IgA was associated with recurrent thrombosis [OR 11.48 (95% CI 1.15–114.51); P = 0.03]. In obstetric SN-APS patients, aPS/PT was significantly associated with foetal deaths [OR 4.84 (95% CI 1.55–15.1); P = 0.01], while aVim/CL was associated with spontaneous abortions [OR 2.71 (95% CI 1.19–6.18); P = 0.016]. Multiple positives were not significantly associated with any specific disease phenotype.
Correlation of non-criteria aPL titers each other
Subsequentially, we examined whether aPL titers were correlated with each other. aCarb-β2-GPI correlated with aVim/CL (r 0.475 [CI 95% 0.33-0.60], P < 0.0001), aCL IgA (r 0.203 [CI 95% 0.04-0.36], P = 0.015), and aβ2-GPI-Domain 1 (r 0.218 [CI 95% 0.05-0.37], P = 0.009); aVim/CL correlated with aPS/PT (r 0.202 [CI 95% 0.04-0.36], P = 0.015) and aCL IgA (r 0.193 [CI 95% 0.03-0.35], P = 0.020); aPS/PT correlated with aβ2-GPI-Domain 1 (r 0.207 [CI 95% 0.04-0.36], P = 0.013)]; aβ2-GPI-Domain 1 correlated with aβ2-GPI IgA (r 0.167 [CI 95% −0,001 to 0.33], P = 0.046).
Follow-up of SN-APS patients tested positive for non-criteria aPL
Among SN-APS patients who tested positive for at least one non-criteria, four cases of recurrent thrombosis were observed [three cases of arterial thrombosis, including two during treatment with antiaggregant therapy and one during vitamin K antagonist (VKA), and one case of venous thrombosis in treatment with new oral anticoagulant therapy], one patient presented with recurrent transverse myelitis during VKA and hydroxychloroquine (HCQ) therapy and another one reported a severe thrombocytopenia (VKA plus HCQ treatment) after a median follow-up of 45 months (IQR 43.5).
Twenty-three pregnancies were recorded in 20 patients. Fourteen (61%) pregnancies were successful under conventional treatment for criteria APS.
Discussion
In this study, we tested a large cohort of SN-APS patients, with clinical features strongly suggestive of APS, showing that in almost half of the patients, it was detectable the presence of at least one extra-criteria aPL.
It is not surprising that 22% of patients had recurrent thrombotic events, given the greater complexity in diagnosing and treating seronegative autoimmune diseases. Consequently, patients often receive treatment later, leading to a more severe disease. Therefore, it is crucial to explore additional extra-criteria aPLs, the positivity of which can aid clinicians in both diagnosis and treatment management.
Recently, a systematic review compared non-criteria APS (NC-APS), mainly SN-APS, with definite APS according to Sapporo criteria [5, 32]. The included studies, published between 2012 and 2021, evaluated 3798 participants and found no difference in the prevalence of thrombosis and pregnancy morbidity between NC-APS and definite APS. Regarding recurrent thrombosis, two studies that analyzed patients with SN-APS found no difference compared with definite APS. Of the 10 studies that reported a statistical comparison of pregnancy outcomes between NC-APS and definite APS, 7 found similar good outcomes [5].
Non-criteria aPLs have the potential not only to assist clinicians in diagnosing and making treatment management but also to serve as biomarkers capable of identifying clinical phenotypes. Although aCL IgA prevalence in SN-APS patients was quite low, according to with previous analyses [7, 28, 29, 33], this study indicates that this test in some cases can be useful. Indeed, our results demonstrate that aCL IgA can identify those patients with recurrent thrombosis, whereas aPS/PT and aVim/CL were more associated with pregnancy events, foetal death, and miscarriages, respectively. Therefore, these non-criteria aPLs could be useful tools.
Interestingly, the most prevalent antibodies – aCarb-β2-GPI and aVim/CL – are strongly correlated with each other. In addition, aCarb-β2-GPI correlates with aβ2-GPI-Domain 1, known for its specificity being directed towards the pathogenic domain of β2GPI [34]. Furthermore, aVim/CL correlates with aCL IgA, just as aβ2-GPI-Domain 1 correlates with aβ2-GPI IgA. It could be assumed that the Vim/CL and β2-GPI-Domain 1 antigens are also detected by the IgA isotopes of aCL and aβ2-GPI, respectively.
In conclusion, the present analysis confirms and extends previous studies, which revealed that aPS/PT [22, 23] and aVim/CL [12, 24] may represent useful tools to identify ‘seronegative’ APS patients, who are negative for criteria aPLs. The major novelty of this investigation is detection of aCarb-β2-GPI antibodies in a significant proportion of these patients and, mainly, identification of the association aCarb-β2-GPI – aVim/CL as the most prominent biomarker of this group of patients. The role of aCarb-β2-GPI antibodies is not surprising; recently, post-translational modification of target autoantigens has been described as a trigger for autoimmune response [31]. The potential pathological consequences of this process are related to the structural changes of the proteins with exposure of cryptic or neo-epitopes, triggering of an autoantibody response [35–38]. In particular, in SN-APS patients’ inflammation, NETosis and/or oxidative stress may represent key mechanisms for inducing β2-GPI carbamylation [39–41].
However, despite this analysis which analyzes a wide spectrum of non-criteria antibodies, a proportion of patients remains negative to all tests, suggesting that other unidentified specificities should be still found.
While much progress has been made in these 40 years, it is necessary to expand knowledge for those patients who still pose a challenge in clinical management, such as patients with SN-APS.
Given that, the present study, along with others in the scientific literature, supports the need to make testing for non-criteria aPLs more accessible in patients with SN-APS.
Acknowledgements
We thank Alessandro Lisci for technical support.
Contributor Information
Simona Truglia, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Gloria Riitano, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Silvia Mancuso, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Serena Recalchi, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Luca Rapino, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Cristina Garufi, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Valeria Manganelli, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Tina Garofalo, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Roberta Misasi, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Cristiano Alessandri, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Maurizio Sorice, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Agostina Longo, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Fabrizio Conti, Reumatologia, Dipartimento di Scienze Cliniche Internistiche, Anestesiologiche Cardiovascolari, Sapienza University, Rome, Italy.
Antonella Capozzi, Dipartimento di Medicina Sperimentale, Sapienza University, Rome, Italy.
Ethical approval
The present study received approval from the Institutional ethics committee (Sapienza University of Rome protocol 0215/2021), and the research was performed according to the 1964 Declaration of Helsinki and its updated versions. All study participants gave written informed consent.
Conflict of interest
None declared.
Funding
This work was supported by a grant from Project Rome Technopole, Flagship 7, Spoke 1 Sapienza University of Rome (Cod.: Rome_Tech_Spoke_1_DMS).
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author Prof M. Sorice on reasonable request.
Author contributions
Simona Truglia (Conceptualization, Investigation), Gloria Riitano (Investigation, Methodology), Silvia Mancuso (Data curation), Serena Recalchi (Formal analysis), Luca Rapino (Investigation), Cristina Garufi (Methodology), Valeria Manganelli (Software), Tina Garofalo (Visualization), Roberta Misasi (Validation), Cristiano Alessandri (Validation), Maurizio Sorice (Conceptualization, Writing—original draft, Writing—review & editing), Agostina Longo (Project administration), Fabrizio Conti (Conceptualization, Writing – original draft, Writing—review and editing), and Antonella Capozzi (Conceptualization, Writing—original draft, Writing—review and editing)
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author Prof M. Sorice on reasonable request.