*5.2. Sicca Syndrome and Glandular Assessment*

The investigation for objective dysfunction of the salivary and lacrimal glands is useful for the diagnosis and symptomatic management of the patient. Anatomical or functional imaging can be used to assess changes in the major SG during pSS.

The evaluation of dry eyes requires a simple ophthalmological examination. The Schirmer test consists of positioning a small strip of filter paper inside the inferior fornix of each eye. The eyes are then closed for 5 min. After this time, the strips are removed, and the amount of tears absorbed by capillarity is measured in millimetres from the edge of the strip in contact with the ocular surface. Dryness is significant if ≤5 mm/5 min. The evaluation then continues with the evaluation of the stability of the tear film by the Break-up Time (BUT) and the search for conjunctival or corneal lesions linked to dryness (keratoconjunctivitis sicca). These various tests use the slit lamp and the ocular instillation of dyes. BUT is measured by placing a drop of fluorescein in each eye and measuring the time during which the coloured tear film uniformly covers the ocular surface, before the appearance of dry spots. A tear BUT test of less than 10 s (averaged over 3 testings') is considered pathological but is

not specific of pSS manifestations. Finally, damage to the conjunctiva and cornea is highlighted by ocular surface staining techniques (fluorescein and lissamine green) [253]. The anomalies are scored using standardized scores: van Bijsterveld scale or the SICCA Ocular Staining Score (OSS). Respective cut-offs of ≥4 and ≥5 correspond to pathological situations suggestive of pSS. Those tests are more specific of pSS than Schirmer and Break-up time tests. Rose Bengal dye is no longer used because of its poor tolerance and local toxicity.

The evaluation of hyposalivation can be easily performed by sialometry. In its simplest form, sialometry consists of measuring the Unstimulated Whole Salivary Flow rate (UWSF) and the Stimulated Whole Salivary Flow rate (SWSF). UWSF is performed by asking the patient—fasted for minimum 2 h—to passively drain all the saliva produced in a tared jar for 15 min. The jar is then weighed and the saliva volume estimated. UWSF less than 0.1 mL/min is considered pathological (normal range 0.3–0.4 mL/min). UWSF represents a minor classification criterion. SWSF is measured in the presence of mechanical stimulation. SWSF can be measured using the Saxon test or Gum test protocols. Saxon test is performed by asking the patient to chew for 2 min a tared compress which will then be weighed. Gum test is performed as USWF, but in this case, the patient chews chewing gum and then spits saliva in a container. A diagnosis of hyposalivation is made if SWSF is ≤0.5–0.7 mL/min (normal range 1.5–2.0 mL/min). It is also possible to measure the salivary flow specific to each major SG by aspiration or cannulation. However, these techniques are of little use to the rheumatologist and especially uncomfortable for the patient.

Radiosialography is an X-ray imaging technique requiring the retrograde injection of a contrast solution into the excretory ducts of the major SG. This technique indirectly highlights glandular damage by studying changes in the "tree structure" of the excretory ducts [254]. Given the invasive nature and the complications of this technique, it has been abandoned in favour of other non-invasive techniques.

SG scintigraphy (SGS) studies the uptake, the concentration and the basal or stimulated secretion of a radioactive tracer by the parotid and submandibular glands following an infusion of Technethium-99 pertechnetate. SGS interpretation is mainly based on Schall's classification [255], a qualitative score classifying anomalies in 4 grades—from grade 1 (normal) to grade 4 (the total absence of uptake and mouth activity). With ≥3 as cut-off, sensitivity and specificity are 54–87% and 78–98%, respectively [256]. Salivary scintigraphy is one of the classification criteria of 2002 for pSS but has disappeared from the most recent classification criteria of 2016. An abnormal scintigraphy makes it possible to objectify a dysfunction of the SG but does not allow etiological diagnosis as no image is specific of pSS. However, it may be of interest for treatment: if the examination shows SG with normal uptake but with a major dysfunction of excretion (possibly due to an autonomic disorder), the patient could benefit from a sialagogue treatment. In case of a scintigraphy demonstrating no uptake of the tracer, the parenchyma is probably totally destroyed and a sialagogue treatment will be useless.

Ultrasound is a simple, non-invasive way to assess the parenchyma of parotid and submandibular glands for diagnostic and prognostic evidence for pSS. Mode-B ultrasound using a high frequency linear probe allows characterization of size, homogeneity, presence of hypo-/anechoic areas, hyperechoic bands and clearness of SG borders. These different items were included in several diagnostic scores [257]. The OMERACT group, in an attempt to standardize, developed in 2019 a semi-quantitative scoring (0–3) based on the presence of hypoechoic/anechoic zones within the parenchyma of the parotid and submandibular glands [258]. A score ≥ 2 is abnormal and suggestive of pSS. At present, SG ultrasound (SGUS) is not part of classification criteria but may well be in the future [259]. Unfortunately, correlations between histological abnormalities (lymphocytic infiltration, diseased parenchyma or ductal ectasia/cysts) and SGUS lesions have not been corroborated [254]. SGUS scores improvement after treatment with Rituximab prove that part of the abnormalities are correlated with the disease activity and not only damage accrual [260,261]. To date, there is currently insufficient evidence to use SGUS as a prognostic or treatment response factor. Thanks to its high spatial and contrast resolution, low cost and accessibility, SGUS has replaced MRI in the diagnosis of the pSS patient.

#### *5.3. Labial Minor SG Biopsy*

The minor SG biopsy (MSGB) is a simple procedure that can be performed with little equipment. Several biopsy techniques have been described in the literature [262,263]. After disinfection, the reappearance of small drops of saliva makes it possible to identify the accessory SG at the level of the lateral third of the lower lip. The mucosa above these glands is anesthetized with an injection of lidocaine. The mucosa is then opened with a scalpel over 5–10 mm and the glands removed with forceps. The individualization and extraction of the glands is made easier by the hydrodissection that occurs during local anaesthesia and by the eversion of the lip. Lobules are herniated towards the surface of the wound by the application of pressure—digital or instrumental—on the external part of the lip. For quality concerns, the removal of 4–6 glands—allowing the study of minimum 8 mm<sup>2</sup> of glands—is recommended [264]. A parotid biopsy is only exceptionally performed because technically more complex with a theoretical risk of damage to the facial nerve, for a diagnostic contribution identical to MSGB based on focus-score. On the other hand, the detection of lymphoepithelial lesions and early stage lymphomas—having a prognostic value—is more frequent/easier to detect on parotid biopsies [263].

The central element of MSGB pathology is the presence of clusters of more than 50 mononuclear cells (mainly lymphocytes) called foci. These foci in periductal or perivascular areas adjacent to normal acini are counted, reported to the area investigated and expressed as a Chisholm–Mason score [265] or a Focus-score [266]. Compared to the initial descriptions of those scores, some experts recommend counting all foci, including those associated with areas of fibrosis or atrophy, for fear of changing the Focus-score [264]. The Focus-score corresponds to the average number of foci per 4 mm<sup>2</sup> of gland. It goes from 0 to 12, 12 corresponding by convention to the coalescence of the foci. The Chisholm score ranks chronic sialadenitis from 0 to 4. Grade 0 corresponds in the absence of infiltration; grade 1 corresponds to a slight infiltration of mononuclear cells, however not forming a focus; grade 2 corresponds to the presence of an infiltrate of mononuclear cells organizing in foci but whose density is <1 focus per 4 mm2; grades 3 and 4 correspond to the presence of 1 or > 1 focus per 4 mm2, respectively. The presence of focal sialadenitis characterized by a Focus-score ≥ 1 (Chisholm grade ≥ 3) is a major diagnostic argument for pSS and is included in the different classification criteria. Due to its sensitivity and specificity >80% and its significant positive predictive value [267], the presence of a chronic focal sialadenitis (Focus-score ≥ 1) is particularly useful in the diagnosis of early pSS, even with specific manifestations and autoantibodies negativity [249].

Although not part of the classification criteria, other anomalies can be described: fibrosis, acinar atrophy, ectasia or metaplasia of the excretory ducts, histiocytic granulomas, presence of germinal centre-like structures, lymphoepithelial or myoepithelial sialadenitis (LESA/MESA) [268,269]. LESA/MESA are characterized by lymphocytic infiltration of ducts and basal cell hyperplasia, resulting in a multilayered epithelium. In addition, pathology allows differential diagnosis with sarcoidosis, IgG4-related disease, amyloidosis and lymphoma. Finally, MSGB provides information on the patient's prognosis: a Focus-score ≥3 and the presence of germinal centre-like structures or LESA/MESA are associated with more severe disease and an increased frequency of local and systemic manifestations, including lymphoma. For this reason, we recommend doing MSGB even if the diagnosis can be made based on anti-Ro/SSA positivity with objective sicca syndrome.

The parotid biopsy has fallen somewhat into disuse due to the ease of performing a minor SG biopsy with equivalent diagnostic performance. On the other hand, the possible discrepancies with MSGB [270,271], the possibility of early detection of lesions associated with a poor prognosis, the possibility of biopsying the same gland again to monitor the disease and the possibility of correlating it with SGUS semiology make parotid biopsy a tool that would need to be reassessed in the future [263].

#### *5.4. Antinuclear Antibodies (ANA) Profile*

The other major element in the diagnosis of pSS is the presence of anti-Ro/SSA and/or anti-La/SSB autoantibodies. The Ro/La system is a heterogeneous antigenic complex, composed by three different proteins (52kDa Ro, 60kDa Ro and La) and four small RNAs particles [272]. The search for antinuclear antibodies (ANA) by Immunofluorescence (IF) on HEp-2/HeLa cells is therefore an important element in the diagnosis of pSS. ANA is positive in 70% of pSS patients, usually with a fine speckled fluorescence [273]. Anti-Ro/SSA and/or anti-La/SSB autoantibodies are identified in 50–90% and 25–60% of patients, respectively [274]. It should be borne in mind that the Hep-2 cells do not sufficiently express Ro/SSA antigen, explaining the fact that 10% of patients anti-Ro/SSA-positive in ELISA have negative ANA in IF on HEp-2 cells [274]. Therefore, in case of suspicion of pSS, it is necessary to request the anti-Ro/SSA antibodies identification by ELISA, even in the presence of a negative ANA IF screening. Two types of anti-Ro/SSA autoantibodies can be differentiated: anti-Ro52 and anti-Ro60 [272]. Anti-Ro52/SSA have no specific ANA fluorescence staining pattern (might even exhibit a cytoplasmic pattern [274]), is precipitin negative and is not detected by ELISAs based on natural SSA/Ro. Ro52+ Ro60+ patients are likely to have pSS while Ro52+ Ro60- patients are not [275]. Isolated anti-Ro52/SSA positivity is statistically linked to primary myositis and systemic sclerosis. On the other hand, anti-Ro52/SSA and anti-La/SSB have the highest relative risks of congenital heart block in offspring from anti-Ro/SSA positive patients because these two antigens are expressed in foetal cardiac tissue from the 18th to 24th week [272]. Anti-La/SSB is mainly found in the presence of an anti-Ro/SSA, evoking a mechanism of epitope spreading. In only 2–3% of cases, pSS patients present with an isolated Anti-La/SSB antibody [276,277]. The presence of another ANA pattern or the identification of "atypical" ANAs can allow the identification of a secondary SS, an overlap with another systemic disease or a specific pSS subgroup [159]. The prognostic implication of these antibodies is discussed in the prognosis section.

## *5.5. Blood Workup*

In addition to ANA testing, the initial blood workup for suspected autoimmune systemic disease includes a complete blood count; a coagulation profile with antiphospholipid panel; urea/creatinine dosage and urine sediment and 24-h urine protein or urine protein/creatinine levels; Na+/K+/HCO3 −/Cl−/Uric Acid levels to investigate renal tubulopathy; hepatic enzymes levels; creatine phosphokinase (CPK) to investigate myositis; C3/C4/CH50 levels, Rheumatoid Factor (RF), Cyclic Citrullinated Peptide (CCP) antibodies, Coombs test; serum protein electrophoresis and total IgG, IgM and IgA levels to investigate presence of polyclonal hypergammaglobulinemia and/or monoclonal gammapathy; HCV serology; VDRL/TPHA; free T4 levels, TSH, anti-thyroid peroxidase, anti-thyroglobulin, anti-mitochondrial, anti-smooth muscle, anti-gastric parietal cell antibodies in case of associated auto-immune diseases. Hypergammaglobulinemia and lymphopenia are classically described during pSS. Their presence may be an additional argument, but their diagnostic performance is not known.
