Flow rates can subsequently be calculated by reading the volume of saliva in the cylinder in milliliters and dividing by the number of minutes of saliva collection. Alternatively, saliva can be aspirated into a syringe and measured, or the cup of saliva can be weighed and after subtracting the weight of the container, salivary ßow rate is calculated as g/min, considered equivalent to mL/min.

Unstimulated and stimulated whole saliva measurements are considered positive, i.e., decreased, when 1.5 mL/15 min or less, and 3.5 mL/5 min or less whole saliva is collected, unstimulated and stimulated saliva, respectively. Unstimulated saliva >0.1 mL/min was found to be sufÞcient to avoid the sensation of oral dryness [28]. A positive test for unstimulated saliva in patients with primary SS was found to be highly reproducible when repeated over a 1-year interval under standardized conditions [33].

The cut-off value for unstimulated whole salivary ßow rate is ≤0.1 mL/min and that of parafÞn-chewing stimulated whole saliva is ≤0.7 mL/min. These ßow rates are signiÞcantly lower than Ògenerally acceptedÓ low levels; 0.3 and 1.5 mL/min, unstimulated and stimulated, respectively [34].

14.2Saliva as a Diagnostic Fluid

Reliable diagnosis, assessment of a patientÕs individual risk, and the ability to monitor disease progression and treatment outcome are highly desirable goals in health care [35, 36]. In this context, oral ßuids meet the need of an easy, non-invasive and inexpensive sampling method, which involves minimal discomfort for the patient [37, 38]. The use of saliva as a suggested diagnostic ßuid allows repeated sample collection from large cohorts including risk groups such as aging individuals and young children. In most situations saliva can be collected in sufÞcient amounts, and such sampling does not expose patients or clinical personnel to additional health risks [37, 38]. Minimal sample processing, requirements before being able to analyze saliva for its constituents, further improves the

applicability of saliva-based analyses in research and clinics [38, 39].

Saliva can be collected from an individual gland by using, for example, a Lashley cup, which adheres via mild suction to the duct oriÞce [38, 40]. Alternatively saliva can be sampled as whole saliva, which contains, compared to gland speciÞc, more components that do not originate from the salivary glands speciÞcally [38, 40Ð42]. Gland-speciÞc saliva is primarily useful for the detection of pathological changes related to a speciÞc gland, whereas whole saliva, due to its composition, is more prone to reßect both the state of all salivary glands and further the condition of an individualÕs systemic health [38, 40].

As already alluded to, whole saliva is a complex mixture that also includes, depending on the patientÕs oral status, a non-inßammatory serum transudate or inßammatory exudate derived from crevicular junctures around the teeth (i.e., gingival crevicular ßuid), blood derivatives from oral ulcerations, and desquamated epithelial cells. Furthermore, exogenous components such as microorganisms, microbial products, and food debris are components of whole saliva [38, 40].

Gland speciÞc or whole saliva can be collected with or without help of masticatory action, e.g., chewing on parafÞn, or gustatory, e.g., application of citric acid, stimulation [38, 40]. For SS patient classiÞcation, the use of saliva to assess an individualÕs salivary gland involvement mandates the determination of the unstimulated whole salivary ßow rate [18]. Importantly, stimulation of salivary ßow does not only change the amount of saliva being secreted but also alters the relative volume of ßuid contributed by the different types of glands to whole saliva. Furthermore, upon stimulation the molecular composition of saliva also changes according to the secreted proteinsÕ biochemical properties [38, 43].

The quantity of saliva, however, is not only solely affected by the sampling method, but is also affected by other parameters such as hydration status, body position, exposure to light, circadian rhythms, circannual rhythms, food intake, smoking, and drugs [3]. Reliable measurement of secretion rate and valid diagnostic results critically depend on strict compliance with

protocols for saliva collection controlling these factors [40, 43]. The most common methods for collecting whole saliva are the dripping/drooling method, in which saliva is allowed to passively drip/drain from the lower lip, and the spitting method, in which the individual actively disgorges the ßuid into a collection tube [38, 40]; see also Sialometry. Handheld, microchip-based devices combining saliva collection and immediate analyses of multiple salivary protein or nucleic acid targets are further being explored for possible point-of-care diagnosis [44].

14.2.1 Biomarker Analyses in Saliva

Realization of the potential of saliva as a bioßuid for health and disease discrimination and disease surveillance depends on three mainly factors: Þrst, the ability of monitoring tissue-related changes in saliva relays on the paradigm that a speciÞc tissue state is reßected in the spectrum and quantity of speciÞc proteins liberated into certain bioßuids [45]; second, the identiÞcation of speciÞc biomarkers, which reliably indicate a speciÞc condition [35, 45, 46]; and thirdly, the applicability of the proposed testing procedure in a clinical setting [35, 44].

In practice, a validated biomarker reduces the often ungraspable complexity behind the process it indicates to a rather simple measurement. In parallel, a high concentration of a certain protein in serum combines complexities related to factors, such as genetic background, metabolism, inßammation, and state of blood vessels walls, to an indicator of disease [47].

Today, most researchers anticipate that saliva principally reßects the entire spectrum of system states related with a patientÕs condition of health, irrespective of whether the disease involves the salivary gland or not [37Ð39, 45]. In addition, saliva is likely to reßect diseases involving the salivary glands more adequately than serum due to enrichment or unique presence of locally synthesized proteins in association with the disease process [37]. In this context, comprehensive efforts to catalogue the salivary transcriptome [42] and proteome [41] of healthy individuals are

crucial to expand our general understanding of saliva and its composition.

One particular challenge when using saliva as a diagnostic ßuid regards the notion that informative analytes are often present in signiÞcantly lower amounts in saliva compared to serum (see supplementary Table 2 of Ref. [48] for a selective overview). Indeed, molecules that are not produced by the glandular epithelium require to be translocated from the local vasculature into the salivary glandÕs interstitial space [39]. Subsequently, these constituents, together with molecules produced by tissues associated with the glandular epithelium, are transported by either the transcellular route (passive diffusion or active transport) or the paracellular route (ultraÞltration) into the lumen of the salivary glands where they become part of Þrst, primary saliva and later, after traversing the ducts, Þnal saliva [39].

The serumÐsaliva recovery rate depends on the biochemical properties of the molecule of interest [38]. This rather stable rate can, however, to a variable extent, be contaminated by serum components present in the gingival crevicular ßuid or derived from oral ulcers [38]. At present, a few saliva-based diagnostic tests have been approved by the health authorities and are commercially available for screening purposes. Most notably, based on speciÞc antibodies, saliva can be used to detect an HIV infection [49]. Considered as accurate as serological tests, these tests offer clinicians and patients a novel, simple, safe, and well-tolerated diagnostic method, which, in addition, improves the possibilities for epidemiological surveys. Interestingly, oral lesions did not seem to affect the results obtained by this method [49]. However, compared to exogenous factors, where this very factor, e.g., microorganism or drug component, largely deÞnes the strategy for patient diagnosis and follow-up, the classiÞcation of other disease categories, such as cancer, cardiovascular, metabolic, neurological, and autoimmune disorders, often lacks such clear rational. The diagnosis of the latter is still largely based on clinical examination, often combined with histopathological evaluation of tissue biopsies and/or serological parameters.

The recent development of sophisticated, large-scale, and high-throughput genomic and proteomic platforms has created unprecedented possibilities to identify novel biomarker signatures for autoimmune diseases in a wide range of bioßuids [35, 36, 45, 46, 50]. Improved sensitivity and the possibility of detecting multiple molecules simultaneously greatly aided saliva to become a bioßuid recognized for its potential in biomarker discovery and subsequent use in a clinical setting. Technologies applied to biomarker discovery are either, at least theoretically, unbiased platforms such as microarrays covering the whole genome and mass spectrometry or biased technologies such as bead-based multiplex immunoassays, antigen arrays, or antibody arrays [35]. The inherent bias in the latter category is due to the use of existing capturing agents, e.g., puriÞed proteins, peptides, or antibodies [35]. Which technology platform most accurately mirrors the true situation is often difÞcult to estimate, emphasizing the importance of appropriate and uniform guidelines for quality assurance and quality control [35]. One important task in the future also consists in the creation of bioinformatics tools for the analysis of disparate datasets such as mRNA proÞles, protein proÞles, and cell surface phenotypes [35, 51].

14.2.2Exploration of Saliva as a Diagnostic Fluid in Sjögren’s Syndrome

Compared with other systemic diseases that may secondarily affect the salivary glands, SS is a rheumatic disease, in which the exocrine glands are the principal target of an autoimmune reaction [52]. Histopathologically characterized by focal mononuclear cell inÞltration in the salivary glands in the majority of patients, the disease signiÞcantly reduces salivary glandÕs secretory capacity through mechanisms that are not yet fully understood [52]. The degree of functional impairment, similar to the degree of inßammation, varies greatly among patients with SS and a direct correlation between these two parameters is not always obvious [53].

The actual diagnostic parameters, unfortunately, do not allow conclusions regarding underlying pathological processes, and a single marker or parameter speciÞcally associated with all cases of SS has not been identiÞed [18]. Also, the demand for an analytical test allowing detection of SS at an early stage has not yet been met.

Representing a less complex bioßuid compared to blood, saliva has been suggested to contain disease-related molecules that reßect salivary gland pathology and conditions involving the oral cavity [38, 39, 41, 45]. Supporting such a notion, transcriptome-based diagnosis of oral cancers by analyzing saliva was shown to be slightly more accurate compared to blood-based analyses [54].

Salivary ßow rates vary among patients with SS and salivary constituents have been subject to several biochemical studies in the Þeld of SS since the early 1970s [39]. Sialochemistry consistently showed increased concentrations of sodium and chloride in saliva from patients with SS, whereas the levels of potassium and calcium appeared to be comparable with measurements obtained in saliva from healthy controls [15]. Other constituents altered in saliva from patients with SS included proteases such as lysozyme, kallikreins, MMP-2, and MMP-9 and members of the serine protease inhibitor family, e.g., cystatin C and cystatin S [39].

Elevated levels of total IgA and IgG together with the presence of autoantibodies against Ro/SSA and La/SSB have also been reported. Anti-La/SSB antibodies in saliva were primarily found in patients with especially low salivary ßow rates and in some patients the antibody was detectable in saliva but not in serum or vice versa [55, 56]. Although anti-Ro/SSA and anti-La/SSB autoantibody levels in saliva and serum correlate signiÞcantly [56] and the number of antigen-speciÞc antibody-producing cells in the salivary glands appears to be closely associated with Ro/SSA and La/SSB serology [57], the diagnostic value of these autoantibodies when measured in saliva remains to be determined.

By applying methods such as immunoßuorescence and in situ hybridization, the presence and local production of several inßammatory mediators and autoantibodies in the salivary glands

have been described (see supplementary Table 1 of Ref. [48] for a selective overview). The recent application of modern technology platforms to the Þeld of SS allows for more comprehensive analyses of saliva collected from patients with SS and experimental animal models of SS [48, 58Ð60]. Although both frequencies of certain gene transcripts and speciÞc proteins may represent valuable biomarkers, research based on speciÞc proteins can rule out certain factors of uncertainty such as mRNA stability and correlation between mRNA copy numbers and levels of the corresponding protein. In addition, direct detection of proteins may facilitate conclusions regarding underlying molecular processes implicated in the pathogenesis of SS [60]. Indeed, a study combining global gene expression analyses with quantitative 2D gel electrophoresis-based mass spectrometry in pooled saliva obtained from SS patients revealed poor correlations between genomic and protein markers [58]. Interestingly, regarding the proteomic analyses, the panel of candidate peptide/protein markers for SS was clearly distinct from the panel described for oral cancer [54, 58], suggesting that the reported alterations in the salivary proteome may reßect rather speciÞc conditions.

Using surface-enhanced laser desorption/ionization time-of-ßight mass spectrometry and 2D gel electrophoresis-based mass spectrometry, Ryu et al. proposed to further explore lactoferrin, β(beta)-2-microglobulin, polymeric Ig receptor, lysozyme C, and carbonic anhydrase IV as potential diagnostic markers for SS [61]. These proteins, identiÞed independently by both methods, were present in signiÞcantly different quantities in pooled parotid saliva from patients with SS when compared to non-SS controls with complaints of xerostomia. Subsequent mass spectrometry-based studies identiÞed 24 and 42 proteins, which were signiÞcantly altered in pooled whole saliva from primary SS patients compared with healthy controls [58, 62]. Furthermore, pilocarpine administration partially restored levels or qualitative presence of several proteins identiÞable by mass spectrometry to values comparable with measurements obtained in saliva from healthy controls [63]. Consistent in

all studies, salivary proteomic proÞles generated by mass spectrometry of primary SS saliva compared with proÞles obtained from healthy controls indicated increased levels of proteins related to inßammation whereas amounts of acinar proteins were decreased. Interestingly, the proÞle associated with secondary SS resembled to some extent that of primary SS while in other aspects, being more similar to the proÞle of healthy subjects [59].

Nonetheless, mass spectrometry-based identiÞcation and quantiÞcation of immunological regulators, which operate at nanomolar to picomolar levels, still represent a great challenge, as abundant proteins might often mask relevant immunological modulators [50]. However, the technology is evolving at a fast pace and methods such as stable isotope protein tagging and subtractive proteomics may improve the number of less abundant and/or immune system-related proteins identiÞed by mass spectrometry [50]. The gold standard for quantitative measurements is, however, still immunoassays due to the unmatched sensitivity provided by antibodies [36].

Applying comprehensive bead and antibodybased multiplex assays with the purpose to discriminate between mice presenting with SS-like disease and an SS unrelated strain, revealed besides 18 biomarkers in serum, 3 chemokines measured in saliva that had the potential to individually and reliably predict strain [48]. In a subsequent study applying the same technology, analyses revealed speciÞc biomarker signatures, which, largely based on salivary proteins, could reliably predict treatment success regarding prevention of onset of hyposalivation as a result of immunization with heat shock protein 60 kDa (Hsp60) or Hsp60-derived peptide aa437Ð460 (Fig. 14.2a) [60]. Based on the same dataset, normal and impaired salivary secretion capacity, irrespective of strain and treatment group membership, could also accurately be predicted (Fig. 14.2b) [60].

Nevertheless, despite or due to signiÞcant technological advances and substantial research initiatives over the recent years, the reality of today is more than ever marked by an obvious gap between the large number of potential