Diagnostic and Prognostic Biomarkers for IgG4-related Disease.

1. Project design The project will consist of a first diagnostic study (objectives 1-2) of cross-sectional analytical design to assess the validity of the biomarkers. Subsequently, a second prospective cohort study (objective 3) will be carried out to evaluate whether these biomarkers are prognostic factors for relapse in patients with IgG4-related disease after treatment.
2. Scope of the project The project will be carried out at the Hospital Sant Pau which is a tertiary referral center of the AIS Dreta (Área Integral de Salud) of Barcelona. The hospital covers a population of 407,550 inhabitants. IgG4-Related Disease is a rare disease that requires diagnostic tests and treatments for hospital use, so almost all cases are referred and followed up at the hospital. At the Functional Autoimmune Unit of the Hospital de Sant Pau the investigators perform monographic consultations on systemic vasculitis, the investigators are an active part of the Aortic Pathology Unit (source of patients with aortitis and retroperitoneal fibrosis), and the investigators maintain a close relationship with the other services involved in this pathology (otorhinolaryngology, digestive and nephrology).
3. Period of performance and recruitment procedure Patients will be selected by sequential sampling between April 2023 and December 2023 in the Systemic Autoimmune Diseases monographic consultations.
4. Sample size IgG4-related disease is a rare disease and has therefore been defined under a pragmatic and realistic approach. A total of 50 participants will be recruited at Hospital Sant Pau in Barcelona. It is planned to recruit 50 patients with IgG4 disease which would be the totality of newly diagnosed patients during 12 months in our center and 50 patients with osteoarthritis or soft tissue pathology. In a previous study (doi: 10.1097/MD.00000000000000003785) the correlation of serum IgG4 concentration with active disease was r=0.17 while the correlation of IgG4+ plasmablasts (10.1136/annrheumdis-2014-205233) with active disease was r=0.47. The expected size to detect a difference (r1-r) of 0.28 is 84 patients with a beta of 20% and alpha of 5%.
5. Data collection Data will be collected from the electronic medical record of the hospital database.

• Demographic and disease assessment: Collection of demographic (age, sex) and disease information (onset, duration of symptoms, treatments). Disease onset will be defined as the time at which ER- IGg4 diagnostic counseling is performed according to the Umehara criteria. The status of the outcome event (disease relapse) and the date on which it occurs will be collected. The start date of follow-up, date of outcome event, last follow-up visit or last contact with the patient in the case of lost subjects will be collected.
• Clinical evaluation: The clinical evaluation will be carried out according to medical criteria, taking into account the combination of the patient's clinical status and the complementary radiological and serological examinations, classifying the patient as active or inactive.
• Radiological studies: Data from radiological examinations in the form of CT and/or FDG18F-PET/CT performed in the diagnosis and/or evolutionary control of the patients will be collected. The organs affected and the increase of 18F FDG in case of PET-CT will be taken into account. In those patients who have scans at diagnosis and control visits, a qualitative comparative study of the images will be performed (no change, improvement, worsening).
• Laboratory studies: Plasmablasts are one of the differentiation stages of B lymphocytes. At the peripheral blood level, the investigators can identify them by labeling against molecules on their surface. Plasmablasts are defined as CD45+ CD19+ IgM- IgD- CD27+ CD38high cells. The use of these markers also allows us to identify other differentiation stages such as naïve B lymphocytes (CD45+ CD19+ IgD+ CD27-), marginal zone (CD45+ CD19+ IgD+ CD27+), class non-switched (CD45+ CD19+ IgM+ IgD+ IgD+ CD27+ CD38-), class switched memory (CD45+ CD19+ IgM- IgD- CD27+ CD38-), transitional (CD45+ CD19+ IgM+ IgD+ CD27- CD38high CD24high), CD19low (CD45+ CD19low CD27+) or CD21low (CD45+ CD19+ CD21low CD38-).

To analyze the differentiation stages of B lymphocytes, the investigators will perform a flow cytometry analysis. Briefly, from peripheral blood in EDTA, the investigators will perform a wash and subsequent staining with the different monoclonal antibodies directed against the surface molecules of interest labeled with fluorochromes. After a 15-minute incubation, the red blood cells are lysed, washed again and analyzed in the BD FACSLyric cytometer.

For the determination of follicular T helper (Thf) lymphocytes and Thf1 and Thf2 subpopulations the investigators will also perform cytometry analysis with monoclonal antibody staining, defining Tfh1 as CD4+ CD45RA- CXCR5+ CCR6- CXCR3+ T lymphocytes and Tfh2 as CD4+ CD45RA- CXCR5+ CCR6- CXCR3- T lymphocytes.

The analysis of interleukins IL-4, IL-5, IL-10 and IL-13 will be performed, from our serum, by individualized ELISAs for each of them.