Population cohorts

MOLI-SANI Study

Neuromed IRCCS, Pozzilli (IS) , Italy

Study design and endpoints

The Moli-sani study participants underwent anthropometric, biological and instrumental screening:

·         Trained research personnel took BP and anthropometric measurements using methods standardized beforehand during preliminary training sessions. BP was measured by an automatic device (OMRON-HEM-705CP) 3 times on the not dominant arm and the last 2 values were taken as the BP. Measurements were made in a quiet room with comfortable temperature with the participants lying down for at least 5 min. Body weight and height were measured on a standard beam balance scale with an attached ruler with subjects wearing no shoes and only light indoor clothing. BMI was calculated as kg/m2. Waist circumferences were measured according to the NIH, Heart, Lung, and Blood guidelines.

·         Subjects underwent electrocardiogram and spirometric test aimed to evaluate pulmonary diffusion capacity, gas diffusion and pulmonary volumes through plethysmography.

·         Venous blood samples were obtained by clean venipuncture between 07:00 and 09:00 a.m. from participants who had fasted overnight and had refrained from smoking for at least 6 h. Biochemical analyses were performed in the centralized Moli-sani laboratory.

Glycaemia, cholesterol, HDL, LDL, C-reactive protein, D-Dimers and blood cell count were measured on fresh samples in the centralized Moli-sani clinical chemistry laboratories in the two recruiting centers. D-Dimer levels were measured in fresh citrate plasma, utilizing HemosIL, an automated latex immunoassay on IL coagulation System ACL9000. Quality control was maintained using internal laboratory standard in-house plasma pool. Inter and intra-day CV was 5.4% and 7.6%, respectively.

High sensitivity CRP was measured in fresh serum sample, using quantex CRP ultra sensitive immuno turbidimetric assay on ILab350.

Serum lipids and blood glucose were assayed by enzymatic reaction methods using an automatic analyzer (ILab 350). LDL-cholesterol was calculated according to Friedewald.

Blood cell counts including platelets, white and red blood cells, mean corpuscular volume (MCV) and haemoglobin (Hb) were performed by a Coulter LH Hematology analyzer (Beckman-Coulter).

Moreover, in the central laboratory of the European project BiomarCaRE the following biomarkers were measured on frozen samples: Apoliprotein A, Apoliprotein B, C-peptide, Creatinine, C-reactive protein, Cystatin C, Glucose, HDL, Insulin, LDL, Lipoprotein(a), N-terminal-pro B-type natriuretic peptide, Testosteron, Triglycerides, hs Troponin I, Vitamin D.

·         Citrated plasma, EDTA plasma, serum, cellular pellet and urinary spots are stored in liquid nitrogen in a specific Biobank. DNA is going to be extracted and stored at -80°C

Each subject was asked to answer to three different types of questionnaires: anamnestic, food frequency and quality of life (FS36) questionnaires and underwent psychometric tests to evaluate any depressive status, stress response and attitude to suicide:

·         The first questionnaire, beyond personal data, collected information on social status and childhood, physical activity, smoking habits, hormonal status in women, body weight history, inflammatory disease in the past year, antibiotics and influenza vaccine, hypertension, diabetes and dyslipidemia and relative dietary and pharmacological treatment, ischemic cardiovascular disease and relative treatment (type of medication, dosage and compliance) and possible hospitalization, pulmonary diseases (relative treatment and possible hospitalization), depressive status or anxiety with relative previous and current treatment, haematological disease, kidney disease, gastrointestinal disease, endocrinological disease, Alzheimer disease, tumours (malignant and benign), surgical intervention and radiological tests in the past five years (with indication of the Centres where they were performed).

Finally, the questionnaire presented a detailed pharmacological analysis registering each chronic or acute medication in the past 15 days, with indication of commercial name, type of packaging, dosage, frequency, compliance and clinical indication (people are asked to bring with them the GP prescription or the drug packaging of the medication they use to take).

·         Dietary assessment. The European Prospective Investigation into Cancer and Nutrition FFQ was used to determine daily nutritional intakes consumed in the past year. This questionnaire was designed and validated in a multicenter study performed in 10 European countries, with the aim to evaluate the relation between diet and cancer. The questionnaire, computerized with tailor-made software, allowed researchers to interview participants in a fully interactive way, including illustrations of sample dishes of definite sizes or by reference to standard portion sizes. All the answers provided by the participants were registered in a database in real time. The NAF software (Nutritional Analysis of Food Frequency Questionnaires, National Cancer Institute, Milan, Italy) (17) was used to transform information about food composition into daily intake of food items (g/d), energy (kcal or kJ/d), and macro- and micronutrients (g or mg/d). Nutrient data for specific foods were obtained from the food composition database for epidemiological studies in Italy.

On the basis of the recorded data, each subject was provided with his own cardiovascular risk according to the cuore.exe algorithm elaborated within the Cuore Project by ISS.

A follow-up based on linkage with hospital discharge records, clinical records and mortality regional registry was performed at December 2011 (FUP1) with a median of 4.3 years.

·         Validated end-points followed up: Deaths (with specific causes of death), fatal or Non-fatal AMI, cardiac revascularization, fatal and non-fatal stroke events, heart failure, atrial fibrillation, diabetes, cancer, neurodegenerative disease (ongoing).

·         A second follow-up (at December 2019) is ongoing.

·         At June 2015 re-examination at the cohort has stated.

MOLI_SANI Study