The DIV-AD study aims to find out whether levels of Alzheimer's disease markers in blood differ among the main ethnical groups living in central Barcelona.

It will also examine how factors such as age, income, education, and health conditions may affect these levels.

In addition, the study will identify barriers that may make it difficult to use these blood tests in primary care.

The final goal is to help ensure that everyone in Barcelona has fair access to early prevention strategies for Alzheimer's disease.

Background and Rationale Alzheimer's disease (AD) is the leading cause of dementia worldwide and represents a major public health challenge. Blood-based biomarkers (BBBs) for AD - including phosphorylated tau isoforms (p-tau181, p-tau217, p-tau231), amyloid-beta peptides (Aβ40, Aβ42), brain-derived tau (BD-tau), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) - have emerged as accessible, scalable, and minimally invasive tools for the early and accurate detection of AD pathology. These biomarkers are increasingly being integrated into research studies, clinical trials, and prevention programs. Their accessibility makes them particularly promising for community-level screening and recruitment into prevention studies.

However, the vast majority of research on BBBs has been conducted in highly educated, non-Hispanic White (NHW) populations from the United States and Europe. Ethnic minorities and migrant communities remain substantially underrepresented in this body of literature, raising serious concerns about the generalizability and equity of current biomarker thresholds, reference ranges, and clinical applications. A growing number of studies suggest that BBB levels may differ across ethnic groups, potentially due to disparities in demographic, socioeconomic, lifestyle, and biological factors. Without a clear understanding of these differences, the implementation of BBBs in routine clinical care risks perpetuating or even widening existing health disparities.

Spain, and Barcelona in particular, occupies a leading position in the development and validation of BBBs. Yet, to date, no study has systematically examined the performance of BBBs in a diverse, multi-ethnic cohort within the Spanish or European context. The Primary Care Team Raval Sud (EAP Raval Sud) in Barcelona provides a unique real-world setting to address this gap: it serves a highly diverse population, with over 60% of its approximately 32,000 registered patients being foreign-born, representing a broad spectrum of ethnic backgrounds including South Asian (Pakistani, Indian, Bangladeshi), Filipino, Latin American, and Maghrebi communities.

Study Overview The DIV-AD BARCELONA study is a prospective, observational, cross-sectional investigation that will enroll 250 cognitively unimpaired (CU) adults aged 50-75 years from five self-identified ethnic groups, recruited at EAP Raval Sud. The study pursues two overarching goals: (1) to characterize and compare BBB levels across diverse ethnic groups and identify the demographic, socioeconomic, medical, and biological determinants of any observed differences; and (2) to assess the frequency of biomarker-defined AD pathology across these groups, validate a cross-cultural cognitive screening tool (RUDAS), evaluate dementia knowledge and stigma, and develop culturally tailored educational and healthcare interventions to promote equitable access to AD prevention.

Study Procedures

Recruitment will occur during routine primary care visits at EAP Raval Sud. Potential participants will be identified and approached exclusively by their own primary care physician, in keeping with standard confidentiality protocols. Following informed consent, each participant will complete two study visits:

• Visit 1 (Primary Care Physician, ~20 min): Eligibility verification, informed consent, collection of sociodemographic and clinical data, self-reported ethnicity and ancestry questionnaire, and administration of the Rowland Universal Dementia Assessment Scale (RUDAS) as a cross-cultural cognitive screening instrument.
• Visit 2 (Nurse + Neurologist + Optional Sociologist/Anthropologist, ~3 hours): Blood extraction and capillary dried plasma spot collection via finger prick. The nurse will also record vital signs, biometric data, and administer lifestyle questionnaires (diet, physical activity, sleep, alcohol). The neurologist will perform a structured neurological and cognitive assessment including CDR, MMSE, SCD questionnaire, HADS, and DSM-V criteria for mood disorders, and will administer the Dementia Knowledge Assessment Scale (DKAS). An optional qualitative interview with sociologists/anthropologists will explore social determinants of health, healthcare access barriers, stigma about dementia, and culturally specific illness narratives.

Biological Sample Analysis Plasma BBBs (p-tau181, p-tau217, p-tau231, Aβ40, Aβ42, BD-tau, GFAP, NfL, and derived ratios) will be measured at the BBRC laboratory using Simoa HD-X and Lumipulse G1200 technologies. Routine biochemistry (renal and hepatic function, lipid and glycemic profiles, complete blood count, ionogram, vitamin B12, folate, thyroid function, inflammatory profile) will also be analyzed. Genetic ancestry will be determined using the Axiom Genome-Wide Human Origins Array. Pre-analytical variables (fasting state, time of blood draw, processing time, hours of sleep) will be systematically recorded.

Statistical Approach Primary analyses will compare BBB levels across the five self-identified ethnic groups using unadjusted non-parametric tests (Mann-Whitney U, ANOVA) and multiple linear regression models adjusting for age, sex/gender, education, socioeconomic indicators, medical comorbidities, concomitant medications, and APOE status. Multiple comparison corrections (FDR, Bonferroni) will be applied. Secondary analyses will use a two-threshold approach for plasma p-tau217 (0.22 and 0.34 pg/mL) to stratify participants into high, intermediate, and low AD pathology risk, with group comparisons by chi-squared test. RUDAS validation against MMSE will assess internal consistency (Cronbach's alpha ≥0.70) and convergent validity (Spearman's rho ≥0.70). Qualitative interview data will be analyzed using content analysis with Atlas.ti software.

Ethical Considerations and Disclosure The study has been approved by the Parc de Salut Mar Research Ethics Committee (CEIm code: 2025/12083/I) and will be conducted in accordance with the Declaration of Helsinki (2024), Spanish Law 14/2007 on Biomedical Research, and GDPR. Results of routine blood analyses and any clinically relevant findings from neurological assessments will be communicated to participants and/or their primary care team as appropriate.

INCLUSION CRITERIA

• Cognitively Unimpaired individuals aged between 50 and 75 years at the time of signing the informed consent
• Individuals interested in participating, able to follow study procedures, and who understand that all the tests are carried out in a research context.
• Explicit desire to participate in the tests and procedures of the study, including: collection of study variables, cognitive and neurologic assessment, blood sampling.
• Signing the informed consent, agreeing not to receive the research results that will not be of no clinical interest for the participant.

EXCLUSION CRITERIA

• Cognitive decline defined by CDR>0.
• Individuals with cognitive complaints and who have sought for medical help for cognitive impairment at any time in their life
• Patients with relevant medical illnesses that may significantly interfere with the interpretation of results. This includes auditory and visual perception disorders, active oncological disease under treatment (localized tumors are excluded) and any condition that, in the investigator's opinion, may interfere with the correct performance/interpretation of the study procedures and/or with future permanence in the study.
• Major psychiatric disorders (according to the DSM-V manual) or diseases that affect cognitive abilities (major depressive episode, bipolar disorder, schizophrenia, etc).
• Acquired brain damage: history of head trauma with macroscopic parenchymal or non-axial lesion, large-vessel ischemic or hemorrhagic stroke, brain tumours and any other etiologies that can cause acquired brain damage (i.e., chemotherapy or brain radiotherapy).
• Parkinson's disease, epilepsy under treatment and with frequent seizures (> 1/month) in the last year, multiple sclerosis or other neurodegenerative diseases.

The study will enroll 250 cognitively unimpaired adults aged 50-75 years, recruited from the patient registry of the Primary Care Team Raval Sud (EAP Raval Sud) in Barcelona, Spain. This primary care center is embedded in the Raval neighborhood of the Ciutat Vella district, one of the most ethnically diverse areas of Barcelona, characterized by a high proportion of foreign-born residents (over 63%)