The Paracelsus 10,000 study is a population based, prospective cohort study. A formative evaluation of required resources was completed in the years 2012 and 2013 and the project structure was established. Recruitment of participants started in April 2013 and was finished in March 2020, while examinations were performed throughout the whole study period from Monday to Friday (except during school holidays). Trained staff with a background in medicine, biology or sports science conducted data collection. Facilities for the study were provided by the University Hospital Salzburg (Landeskrankenhaus and Christian Doppler Klinik), who is the sponsor of the Paracelsus 10,000 study. Facilities on site include examination rooms, office space, computer and waiting rooms for the participants, storage rooms and biobank facilities.
2.1. Recruitment and characteristics of the study population
People living in the city of Salzburg and surrounding areas were randomly selected from the local population registry and invited by letter to participate in the study. We aimed to recruit equal numbers of men and women. The population was stratified by age with the goal to recruit about 50% of subjects in the age range between 50 and 59, 25% of subjects in the age ranges between 40 and 49 years and 25% of subjects in the age range between 60 and 69 years. Participation was voluntary and without financial reward, while participants benefited from a preventive medical check-up. All in all 60,000 invitation letters were distributed, yet a certain ratio did not reach the intended recipients due to changed address or death. Due to data security guidelines and an unmanageable effort, it was not possible to conduct statistics about unanswered invitations. In total, we examined 10,062 participants, of whom 286 were not randomly selected, but they had proactively approached the study and were included when they fulfilled entry criteria. Participants were stratified into two groups: 1) participants aged 23- 77 years who received the basic examination program (basic program) and 2) participants aged 50-59 years who received the basic program and additional examinations (extended program). These subjects were randomly chosen from the pool of 50-59 years old participants. Shortly after the start of the first recruitment period, we stopped inviting participants younger than 40 due to the small response rate in this age group. In the end we investigated 18 participants younger than 40. Due to the long retention of the first study phase, some participants exceeded their 70th birthday. In total, 323 participants of the study cohort were older than 69 years. Both of these marginal age groups were excluded from the calculations of the results section due to better comparability with the data of Statistics Austria. For reasons of optimal capacity usage, 610 participants received additional examinations on a voluntary basis, without being randomly selected for the examination program. For return rate and distribution of age, gender and program see figure 1, for the list of basic and extended examination, see table 1. Even though single investigations had to be omitted in case of physical inability, there were no predefined exclusion criteria for the invited participants.
All participants signed an informed consent and the study protocol was approved by the ethics committee of the State of Salzburg (415-E/1521/3-2012).
In order to get an overview of characteristics of our study cohort, we conducted comparisons with the population of Austria and the State of Salzburg. Variables compared were sex, age, migration background, educational attainment, type of employment, degree of urbanization, smoking habits and physical activity. The information was extracted from a questionnaire answered by the participants. We used national official statistics by Statistics Austria as reference [23] to compare the data of our cohort. Whenever possible we excluded all age groups from the reference data that did not overlap with the age range of 40 to 69 years. Detailed information about the reference data used and the matching of answer options can be found in the supplementary material.
2.2. Investigations
All examinations of an individual participant were carried out on the same day and each examination was conducted at the same time of the day, to control for circadian rhythmicity. In addition to a complete blood count and urinalysis, the following examinations were included in the study program:
Anthropometry and body composition
- Body height
- Body weight
- Abdominal circumference
- Body composition: Multiple frequency bioelectric impedance Analysis was performed while participants adopted a supine position with arms spread apart from the body and legs separated. Signal input and output electrodes were placed on the dorsum of the right hand and foot. Recording electrodes were placed at standard positions at wrist and ankle, the measurement was repeated three times, while participants were not necessarily fasting (Body Impedance Analyzer™, Nutri Plus Data Input, Germany).
- Dexa-Scan X-ray densitometry for measuring bone mineral density and body composition (Hologic Discovery A™, Hologic APEX, USA).
Laboratory parameters:
Red blood cells (RBC), white blood cells (WBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, reticulocytes measured with Sysmex XN-1000™ (Sysmex Austria GmbH).
Thrombin time (TT), partial thromboplastin time (PTT), prothrombin time (PT)/international normalized ratio (INR), fibrinogen, antithrombin III measured with BCS XP™ (Siemens Healthcare Diagnostics GmbH).
Red blood cells (RBC), white blood cells (WBC) measured with Sysmex XN-1000™ (Sysmex Austria GmbH)
Total cholesterol, Low-density Lipid (LDL)-cholesterol, High-density Lipid (HDL)-cholesterol, triglycerides, apolipoprotein-B, apolipoprotein-AI, lipoprotein (a) measured with Cobas 6000™ (Roche Diagnostics GmbH).
Fasting blood glucose measured with Cobas 6000 (Roche Diagnostics GmbH), HbA1c measured with Cobas Integra 400 Plus Analyzer™ (Roche Diagnostics GmbH) and fasting insulin.
Serum creatinine, glomerular filtration rate (eGFR), serum urea, serum electrolytes (Na, K, Cl, Ca, Mg, P), serum uric acid, urine chemistry (albumin, creatinine, albumin/creatinine ratio, total protein, glucose), urine sediment (specific weight, pH value, red blood cells (RBC), white blood cells (WBC), nitrite, total protein, glucose, ketone bodies, urobilinogen, bilirubin), , electrolytes (Na, K, Cl, Ca, Mg, P) in urine measured with Cobas 6000™ (Roche Diagnostics GmbH) while urine sediment was measured with Cobas U 601™ (Roche Diagnostics GmbH).
Aspartate transaminase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (gGT), alkaline phosphatase (AP), cholinesterase (CHE) measured with Cobas 6000™ (Roche Diagnostics GmbH).
P-amylase, lipase measured with Cobas 6000™ (Roche Diagnostics GmbH).
Iron, transferrin, transferrin saturation, ferritin measured with Cobas 6000™ (Roche Diagnostics GmbH).
- high sensitivity c-reactive protein (hsCRP), thyroid-stimulating hormone (TSH), prostate specific antigen (PSA, for men), total serum protein measured with Cobas 6000™ (Roche Diagnostics GmbH).
Cardiovascular parameters:
- Blood pressure was measured bilaterally in a sitting position. The measurement was repeated three times with a previous resting period of each 60s (Watch BP office AFIB™ from the company Microlife AG Swiss Corporation, Switzerland).
- Electrocardiography (ECG) 12-lead, in a supine position (Schiller Cardiovit AT-102 Plus 12-Kanal-EKG™, Germany).
- Ankle–brachial pressure index was measured three times in a supine position (Boso ABI-system 100 PWV™, Bosch + Sohn, Germany).
- 24 h blood pressure: A portable device was taken home by participants and measured blood pressure, pulse and pulse wave velocity in 15-minute intervals from 8 a.m. to 10 p.m. and in 30-minute intervals during night (Mobile-O-Graph PWA™, Industrielle Entwicklung Medizintechnik und Vertriebsgesellschaft mbH (I.E.M.), Germany).
- Coronary Artery Calcium Score (Agatston-Score) using Computed Tomography (Siemens Somatom Definition AS+™, Erlangen, Germany) [24].
Spirometry, measurement of liver stiffness, oral glucose tolerance test:
- Pulmonary function test was carried out, determining forced vital capacity and forced expiratory volume over 1s using a portable spirometer (Easy OneTM Spirometer™, ndd Medical Technologies, Zurich, Switzerland). The tests were performed in sitting position in triplicate with values being accepted when consecutive maneuvers yielded values within 10% of each other [25]. Exclusion criteria were recent operations, cardiovascular and acute pulmonary diseases.
- Liver stiffness measurement and semi-quantitative estimation of fat content via continuation attenuated parameter using Fibroscan 530 Compact™ (Echosens Paris, France).
- An oral glucose tolerance test (OGTT) was performed following the standard protocol of the American Diabetes Association. Before and at 30min, 60min, 90min and 120min after oral intake of 75g glucose venous blood was drawn for measurement of plasma glucose and insulin levels.
Medical history
Trained staff conducted a face-to-face interview recorded as audio file that included medical history, family history and medication. Further inventories were merged to one computer-based questionnaire that was answered discretely by participants either in the study rooms (possibly with assistance by the staff) or at home. Participants who were not familiar with computer work or who needed translation answered the questionnaire manually and the study staff digitalized the data. The questionnaire included:
- ODQ-D (Oswestry Low Back Pain Disability) [26]
- WOMAC (Western Ontario and McMaster Osteoarthritis Index) about cox arthrosis [27]
- Screening questionnaire of the Austrian Social Insurance [28].
During the face-to-face interview, neurological diseases were surveyed [29] and the Salzburg Dementia Prediction Test was conducted [12]. The questionnaire filled out by the participant included further questions on epilepsy.
The interview also included questions on major depressive disorder, bulimia and anorexia nervosa, generalized anxiety disorder, manic and hypomanic episodes, panic disorder, agoraphobia, social phobia, obsessive-compulsive disorder, posttraumatic stress disorder, substance and alcohol abuse. The participants filled in additional inventories using a custom made digital questionnaire of a secure computer at the hospital:
- BDI (Beck Depression Inventory) slightly modified [29].
- Barrat Impulsiveness Scale (BIS-15) [30; 31].
Ultrasound of the carotid arteries
- Ultrasound examination of the carotid artery bilaterally in a supine position (Panasonic Healthcare Diagnostics US). Intima media thickness and total plaque area were measured.
Assessment of lifestyle factors
Further inventories on lifestyle that were answered discretely by participants were included:
- AUDIT (Alcohol Use Disorders Identification Test) [32]
- Inventory on diet according to the EPIC study (European Prospective Investigation into Cancer and Nutrition) [33]. The German version of the EPIC questionnaire was kindly provided by the DIFE (Deutsches Institut für Ernährungsforschung, Potsdam, Germany).
- Quality of life Assessment (QoL) in F 12 short form including twelve [34] [35].
- A seven-day diet survey including a program that calculates nutritional value (DGE –PC professional, version 5.1) by the German Nutrition Society [36].
- ESS (Epworth-Sleepiness-Scale) [37]
Additionally, questions about origin and family background, religion, employment, education, domestic circumstances, smoking habits, menopause, Mediterranean diet, allergies and sexuality were included. Further, questions were added on usage of mobile phone, sensitivity towards chemical smells, electromagnetic pollution, air and noise pollution.
Health related physical activity
- The objective measurement of physical activity was calculated from a triaxial accelerometer (3D-Activity Sensor Move II™, movisens GmbH, Karlsruhe, Germany), which collected raw data at a sampling rate of 64 Hz. The accelerometer was placed on the right hip during seven consecutive days (24h) in a typical working week, but was removed for activities involving water (i.e. showering, swimming). The accelerometer data-set per participant were analyzed, when valid 10 h/d recording on at least five days per week were available using software from Movisens GmbH (DataAnalyzer™, movisens GmbH, Karlsruhe, Germany), extracting the bouted and non-bouted amount of physical activity in minutes, stratified into light, moderate and vigorous physical activity.
- International Physical Activity Questionnaire (IPAQ) was competed after accelerometer recording to display the last seven days [38] [39]. We extended the inventory slightly by adding questions on electrically assisted bicycle and the times in standing or sitting position, as well as sleeping times.
- Questionnaire about regular exercise in different phases of life were recorded in the self-assessed inventory.
Physical Fitness Testing
- Incremental cardiopulmonary exercise testing (CPET) was conducted on a stationary bicycle ergometer (ergo select 200P™, ergo line GmbH, Blitz, Germany) including measures of heart rate (12-lead ECG, blood pressure, blood oxygen level (pulsoximeter) and spirometry gases. A gas-exchanger analyzer (Master Screen CPX-Jaeger™, VIASYS Healthcare – Respiratory Technologies, LabManager V.5.32.0.) was used for continuous respiratory gas analysis and volume measurements. Before, during and after the CPET the expired air was sampled by using a facemask (Hans Rudolph, Kansas, USA), a volume sensor (Triple-V™) and a gas analyser (Master Screen CPX™), which was connected to a semipermeable sampling line (Twin Tube, all products are manufactured by Jaeger™, Höchberg, Germany). The exercise protocols were adapted to reach volitional exhaustion after 8-12 min of test duration, starting with different workloads and increments regarding sex and body mass-range (American College of Sports Medicine, 2013; Pühringer, 2020) [40] [41]. Exclusion criteria were cardiovascular disease, anemia and musculoskeletal disorders.
- Six-meter gait speed test (6GST): maximum and habitual walking speed was recorded via a wireless photoelectric beamer over a distance of 10 m, where the walking speed in m/s over 6 m was recorded (TC™, Brower Timing System, Draper, USA).
- Isometric grip strength of the right and left hand was measured in a sitting position via a hydraulic hand dynamometer by recording the best of three repetitions for right and left hand grip strength in kg (5030J1™, Jamar, Patterson Medical, Dallas, USA).
We used the internet based software EvaSys™ to collect parts of the data (questionnaires, interview, anthropometry, parts of coronary artery calcium score, Dexa-scan and carotids parameter). By means of individual codes, the study staff or the participants themselves could fill in data into pre-programmed masks. The data was then stored in the system and was ready for download at any time. Even though the program is internet based, it was in cooperated into the intern hospital IT system, protected by the associated firewall.
2.5. Biobank sample collection
Blood was drawn in sitting position between 7.30 and 10.30 a.m. after at least 10 hours of fasting. The whole blood was collected in special Vacutainer tubes (Greiner Bio-One™) for preparation of different types of biological materials.
Blood (EDTA, citrate and heparin), fating serum and spontaneous urine were centrifuged at 2.000g for 15 minutes in a refrigerated centrifuge (CF5804R™, Eppendorf). The supernatant was aliquoted into 2D Data-Matrix coded Screw Cap tubes, that were placed in barcoded Loborack-96™ from Micronic™ for long time storage at -80°C. The tubes were closed with pierceable TPE Capcluster using electric Capper (Micronic™). The barcodes on the tubes and racks were scanned using Traxer Code Reader (Micronic™). Information about the type of biological material and location in the freezers was managed using Track-it sample management (Micronic™) and LabCollector (AgileBio™) softwares.
We collected 24 aliquots of fasting serum, 24 aliquots of fasting plasma EDTA, 8 aliquots of fasting citrate plasma, four aliquots of fasting heparin plasma, 8 aliquots of urine, 12 ml whole EDTA blood, two aliquots of buffy coat and approximately 50gr of stool from each participant. All biological materials are stored at -80°C in Ultra-Low Temperature Freezers from Thermo Fischer Scientific™.
2.6. Quality control
During the course of the study, several measures of quality control were implemented. In the first phase of the study, all data manually entered in EvaSys was checked for accuracy. With rising numbers of participants and data sets, only randomly selected data of single participants were checked in detail. The first major quality control was conducted after recruitment of 6,000 subjects. Here, data was checked for plausibility via determination of threshold values. If data exceeded defined thresholds, the raw-data were consulted again. Additionally, duplicate observations were deleted and missing values were completed if possible. Of all the values (blood pressure and anthropometry parameters) of the 307 participants that were randomly checked for mistakes, 0.54 % were found to be faulty. In the next and for now last phase of quality control, the statistical software R [42] was used to check for internally inconsistent answers (e.g. medication without known diseases or differences in self-report and interview). In case of doubt, data values were excluded.