DEPARTMENT OF HEALTH SCIENCES

Northern Finland Birth Cohort 1966 46-year follow-up study

Protocol

300312 / JA

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Contents 1 BACKGROUND ...... 6 2 SUBJECTS AND AREAS OF RESEARCH ...... 6 3 TIMETABLE ...... 6 4 STUDY COMPONENTS ...... 7 4.1 QUESTIONNAIRES ...... 7 4.2 CLINICAL EXAMINATIONS ...... 8 4.3 EXAMINATION FACILITIES ...... 9 4.4 APPOINTMENT SCHEDULING ...... 9 4.5 STAFF, RESPONSIBILITIES AND CONTACT DETAILS ...... 10 4.5.1 Staff ...... 10 4.5.2 Substitute arrangements...... 12 4.6 EXECUTION ...... 13 4.6.1 INVITATIONS ...... 13 4.6.2 FIELDWORK ...... 14 4.6.2.1 SAMPLES AND ANALYSES ...... 14 4.6.2.1.1 Questionnaires ...... 14 4.6.2.1.2 Health examinations in Oulu ...... 14 4.6.2.1.3 Health examinations in Oulainen and Kemi ...... 14 4.6.2.1.4 Eye examinations ...... 14 4.6.2.1.5 Glucose tolerance tests ...... 15 4.6.2.1.6 White blood cell isolation ...... 15 4.6.2.1.7 Saliva samples ...... 15 4.6.2.2 CLINICAL EXAMINATIONS, SUBJECTS’ ROUTE BETWEEN EXAMINATION STATIONS AND FIELD STAFF’S RESPONSIBILITIES...... 15 4.6.2.2.1 Oulu examination stations, Day 1 (Appendix 21) ...... 15 4.6.2.2.2 Oulu examination stations, Day 2 (Appendix 21) ...... 26 4.6.2.2.3 Oulainen and Kemi with the same equipment and staff as Oulu (flow chart in Appendix 22) ...... 28 4.6.2.2.4 Northern Finland and Southern Finland teams (flow chart in Appendix 23) ...... 28 4.6.2.3 CLINICAL EXAMINATION PROTOCOLS ...... 35 4.6.2.3.1 Registration, identity checks and consent for the use of data ...... 35 4.6.2.3.2 Biological samples ...... 35 4.6.2.3.3 Upper arm blood pressure and heart rate measurements ...... 41 4.6.2.3.4 Central blood pressure measurements ...... 42 4.6.2.3.5 Waist and hip measurements ...... 43 4.6.2.3.6 Height and weight measurements ...... 43 4.6.2.3.7 Bioimpedance measurements ...... 44 4.6.2.3.8 Measures the subject’s hand grip strength ...... 45 4.6.2.3.9 Back strength tests ...... 46 4.6.2.3.10 Skin analyses ...... 46 4.6.2.3.11 Spirometry ...... 47 4.6.2.3.12 Activity tracker and sleep tracker data ...... 50 4.6.2.3.13 Panoramic radiography and X-ray images of ankles, feet and knees ...... 50 4.6.2.3.14 Dental and oral examination, photography and 3D imaging ...... 54 4.6.2.3.15 Physiotherapy examinations ...... 54 4.6.2.3.16 Step tests and autonomic nervous system measurements ...... 54 4.6.2.3.17 Skin prick testing ...... 59 4.6.2.3.18 Pressure pain sensitivity, thermal sensitivity and heat pain threshold tests ...... 63 4.6.2.3.19 Carotid ultrasonography and echocardiography ...... 65 4.6.2.3.20 Eye examinations ...... 65 5 DATA STORAGE, TRANSMISSION AND MANAGEMENT ...... 66 5.1 DATA STORAGE AND TRANSMISSION ...... 66 5.2 DATA PROCESSING, CLEANING AND MANAGEMENT ...... 66 5.3 COMMUNICATION OF RESULTS TO SUBJECTS ...... 67 5.3.1 Results to be communicated on the day ...... 67 5.3.2 Results to be sent by post ...... 67 5.3.3 Procedure in the event of abnormalities ...... 68 5.3.3.1 Blood pressure ...... 68 5.3.3.2 ECG ...... 68 5.3.3.3 Blood tests ...... 68 3

5.3.3.4 Skin health ...... 68 5.3.3.5 Radiography ...... 69 5.3.3.6 Oral and dental health ...... 69 5.3.3.7 Heart and jugular vein ultrasound ...... 69 5.3.3.8 Eye examinations ...... 69 5.4 EXCLUSION CRITERIA FOR CLINICAL EXAMINATIONS ...... 69 5.5 FIRST AID ...... 69 5.6 COMMUNICATION WITH THE MEDIA ...... 69

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APPENDICES*

1. Map of Finland, teams and regions 2. Background, lifestyle and health questionnaire 3. Economic, work and resources questionnaire 4. Supplementary questions 5. Supplementary dental health questionnaire 6. Opinions and experiences questionnaire 7. Research locations and subject numbers 8. Mailing list plan 9. Cover letter and information about the questionnaires 10. Cover letter, information about the study and clinical consent form for subjects in and around Helsinki 11. Cover letter, information about the study and clinical consent form for subjects in and around Kajaani 12. Cover letter, information about the study and clinical consent form for subjects in and around Oulu, excluding eye examination 13. Cover letter, information about the study and clinical consent form for subjects in and around Oulu, including eye examination 14. Cover letter, information about the study and clinical consent form for subjects in and around Kemi 15. Map for study participant in Oulu, including eye examination 16. Map for study participant in Oulu, excluding eye examination 17. Clinical examination rooms in the Department of Health Sciences 18. Examination rooms in the Department of Dentistry 19. Schedule and checklist for subjects in Oulu, excluding eye examination 20. Schedule and checklist for subjects in Oulu, including eye examination 21. Flow chart of Day-1 and Day-2 examinations in Oulu 22. Flow chart of examinations to be performed by the Oulu team in Oulainen and Kemi 23. Flow chart of examinations to be performed by the Southern and Northern Finland teams 24. Flow chart of blood sample collection and processing in Oulu 25. Flow chart of blood sample collection and processing by the Southern and Northern Finland teams 26. Urine and stool sample collection instructions for subjects 27. White blood cell isolation protocol 28. Saliva sample collection and processing protocol 29. Activity tracker manual for subjects 30. Activity tracking log 31. Physical activity tracking protocol 32. Activity tracker manual for staff 33. Physiotherapy examinations 34. Skin prick testing template 35. Nutrition poster for supplementary questions 36. Mailing list plan 37. Shipping list for blood samples to be sent to Oulu University Hospital 38. Temperature monitoring form 39. Weight/height template for information sharing between examination stations of the Southern and Northern Finland teams 40. Autonomic nervous system measurement form 5

41. BP/height/weight template for information sharing between examination stations of the Oulu team 42. Sugar stress tests (outside Oulu) 43. Sugar stress test flow chart for Oulu 44. Heart and jugular vein ultrasound examination protocol 45. Information and feedback form for heart and jugular vein examinations 46. Examination certificate for subjects 47. Blood sample separation and separator tubes 48. Sugar stress test flow chart (outside Oulu) 49. Blood sample data sheet 50. Blood sugar meter control form 51. Sticker sheet for the Southern and Northern Finland teams 52. Sticker sheet for the Oulu team 53. Clinical consent forms

*Appendixes are not available in English or contain personal information and are thus not included in the version to be appear on the NFBC webpage.

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1 BACKGROUND The purpose of this study is to track longitudinally, from the foetal period onwards, the prevalence of certain medical conditions (such as cardiovascular diseases, lung diseases, skin diseases, musculoskeletal disorders, mental disorders, oral and dental disorders), social and financial welfare, health behaviours (alcohol, tobacco and nutrition) and mortality. This 46-year follow-up also includes a particular focus on work. The objective is to identify high-risk groups and potential genetic and environmental predictors to enable early intervention and prevention.

2 SUBJECTS AND AREAS OF RESEARCH The Northern Finland Birth Cohort includes all those individuals due to be born between 1 January 1966 and 31 December 1966 in the regions of Oulu and Lapland – 12,231 births in total. According to the Population Register Centre, approximately 10,300 of these individuals were alive in early 2012 and around 7,000 still lived in Oulu or Lapland (Appendix 1). Previous follow-up studies covering the entire cohort were conducted at ages 1, 14 and 31 years. The follow-up study to be conducted in 2012–2013 covers the entire cohort at the age of 46–47 years.

3 TIMETABLE o Staff training from 1 to 17 February 2012 o Invitations to the second pilot by 20 February 2012 o First pilot from 20 to 24 February 2012 o Invitations to the official study by 7 March 2012 o Second pilot from 12 to 16 March 2012 o Piloting of the Southern and Northern Finland field protocols from 26 to 28 March 2012 o Launch of the study on Tuesday, 10 April 2012 (the Oulu team in Oulu, the Northern Finland team in Kajaani and the Southern Finland team in Helsinki) o Northern and Southern Finland field teams (Kajaani and Helsinki) off on 30 April 2012 and 18 May 2012 o Oulu field team at work on 30 April 2012 and off on 18 May 2012 o Summer break from Monday, 2 July to Friday, 27 July 2012 (4 weeks) o Autumn break from 22 to 26 October 2012 o Christmas break from Monday, 24 December 2012 to Wednesday, 2 January 2012 o Winter break from 4 to 8 March 2013 o The aim is to complete the study in Oulu in April 2012, after which some of the Oulu team will relocate to Oulainen and Kemi (MLT1 and CRN1–5) o Summer break from Monday, 1 July to Friday, 26 July 2013 (4 weeks) o Autumn break from 21 to 25 October 2013 o The aim is to complete the study by the end of 2013 (realistically in early 2014) o Data pooling in 2014 7

4 STUDY COMPONENTS

4.1 QUESTIONNAIRES The study will include a set of questionnaires distributed to all surviving cohort members. Login details for the questionnaires, which are to be completed online (Webropol) before the clinical examinations, will be sent out with the invitation to the 46-year follow-up (Appendices 9 and 36). Subjects will also be able to request paper versions of the questionnaires by telephone. Invitations to complete the questionnaires and general information about the 46-year follow-up will be sent to all surviving cohort members whose addresses are known and who live in Finland. The invitations will be sent out in three batches at two-week intervals in April 2012 so that the first batch will arrive by post on 2 April 2012 (Appendices 8 and 36). The subjects will be asked to complete the questionnaires within one month of the invitation. New Webropol login details and paper versions of the questionnaires will be sent to any subjects who do not complete the questionnaires by the deadline. If another month passes without a subject completing the questionnaires, the Webropol login details and paper versions will be sent to them one more time. The first two questionnaires will include a consent form in which subjects are asked to give their consent to having their answers included in the study and combined with previous research data and register data as well as to the research team accessing their patient records and sharing the research data with businesses involved in the study. Subjects who complete their questionnaires online using the Webropol tool will be asked to give their consent electronically, and subjects who complete their questionnaires on paper will be asked to fill in and sign a paper form. Subjects will also be asked to answer supplementary questions on a computer in connection with their clinical examinations. Each subject attending clinical examinations will be given a questionnaire to fill in either while they wait or afterwards at home. Subjects will be asked to return their paper questionnaires by post to the University of Oulu Department of Health Sciences. Any subjects who have missed questions will be contacted by telephone and asked to provide the missing answers. Both the questionnaires returned by post and the data retrieved from Webropol will be checked at the Department of Health Sciences. Any minor omissions in subjects’ answers will be rectified by telephoning the subjects concerned. Subjects will be asked for their telephone number in the consent form. Subjects who have left all or most of the questions unanswered will be added to the mailing list for reminders and sent new questionnaires to complete. Questionnaires and timetable Name Timetable Format Background, lifestyle and health Before the clinical examinations: Online using the Webropol tool questionnaire (Appendix 2) or on paper; paper versions of the - Login details to Webropol questionnaires can be requested and sent out with invitations to by telephone the 46-year follow-up in three Finances, work and resources batches at two-week intervals questionnaire (Appendix 3) - Reminder with paper versions of the questionnaires and new Webropol login details sent by post one month later - Second reminder with paper versions of the questionnaires and new Webropol login 8

details sent by post one month after the first reminder Supplementary questions In connection with clinical Online questionnaire (Infopath) (Appendix 4 and nutrition poster examinations at all examination and BDI-II on paper; the entire / Appendix 35) stations between April 2012 and questionnaire can be completed December 2013 on paper in case of computer problems Supplementary dental health In connection with clinical Computerised questionnaire questionnaire (Appendix 5) examinations in Oulu between April 2012 and April 2013 Opinions and experiences Handed out in connection with Paper questionnaire handed out in questionnaire (Appendix 6) clinical examinations between connection with clinical April 2012 and January 2014 to examinations; subjects who have be filled in while waiting or not attended clinical afterwards at home and returned examinations by the end of 2013 by post will be sent the questionnaire by post, along with the BDI-II questionnaire

4.2 CLINICAL EXAMINATIONS The clinical examinations will be designed to collect information by means of a range of tests and blood sampling. The examinations will be carried out by three field teams (Appendix 1). The field teams will meet with subjects in the subjects’ hometown or in a location no further than 100 km from their hometown (Appendix 7). 1. The Oulu field team will be based in Oulu for the first year (from April 2012 to April 2013). The examinations will take place at the University of Oulu Faculty of Medicine and in Oulu University Hospital, and the pool of subjects will include all those residing within between 80 and 100 km of Oulu (n = 3,050). Once the examinations in Oulu have been completed, some of the Oulu team (CRN1–5 and MLT1) will relocate to Kemi (n = 716) and Oulainen (n = 590) to examine subjects residing in these areas. 2. The Northern Finland field team will rotate between local-authority health centres and other similar facilities around the Oulu Region and Lapland between April 2012 and December 2013, examining subjects residing in each local authority (n = 2,853). 3. The Southern Finland field team will rotate between the largest towns and cities of Western, Central, Eastern and Southern Finland from April 2012 to December 2013, examining subjects residing in these areas (n = 3,281). Subjects will be invited to the clinical examinations by post (Appendices 8 and 10–14) between two and four weeks before examinations are due to begin in each location. Invitations to subjects residing in large towns and cities will be sent out in batches if the total number of subjects exceeds 400. The invitations will include login details and a link to an online appointment booking tool that subjects can use to book a suitable time for their examinations. Those who do not take advantage of the tool will be contacted by telephone to arrange an appointment. Subjects will also be sent a text- message reminder in the days preceding their appointment to reduce the no-show rate. The appointment booking tool will give the subjects invited by the Oulu team a choice of 10 appointments a day from Monday to Friday (9 per day in April/May 2012) and the subjects invited by the Northern and Southern Finland teams a choice of seven appointments per day from Monday to Friday (6 per day in April 2012). There will be no appointments in July, over Christmas or during 9 the teams’ autumn and winter breaks. Subjects will be told to arrive at their designated examination station 10 minutes before their scheduled appointment to allow for their blood pressure to stabilise.

4.3 EXAMINATION FACILITIES Subjects residing in and around Oulu will be first examined in the clinical examination rooms of the Faculty of Medicine’s Department of Health Sciences (Appendix 17), followed by dental examinations at the Department of Dentistry (Appendix 18) and eye examinations at the eye clinic of Oulu University Hospital. Clear signage will be provided between the examination stations, and subjects will be given maps and a simplified flow chart (Appendices 15–16) as well as a checklist of examination stations (Appendices 19–20), which they will be asked to tick off after visiting each one. Receptionists will be on hand to guide subjects from one examination station to the next. The Northern Finland field team will examine subjects in local-authority facilities around the Oulu Region and Lapland. Arrangements will be made separately with each local authority. The first choice will always be the local health centre. If the local health centre is unable to provide examination rooms, other facilities (schools, colleges, etc.) will be contacted. The Northern Finland field team will need three examination rooms and one room for questionnaires and cognitive tests. The Southern Finland field team will begin its work in Helsinki by examining subjects living in and around Helsinki. The team will then rotate between the largest towns and cities of Western, Eastern, Central and Southern Finland, examining subjects residing in these areas. The Southern Finland field team will need three examination rooms and one room for questionnaires and cognitive tests. The rooms must be reserved exclusively for this study. The aim will be to have the examination rooms furnished in advance with the required chairs, desks and an examination table for sample- taking and ECG. The field staff will bring their own instruments. For telecommunications, the staff will use either the examination station’s local area network or, in the absence thereof, the 3G network.

4.4 APPOINTMENT SCHEDULING The invitations sent to subjects will include an internet address, a username and a password to enable subjects to book their appointments online. The internet address will take the subjects to the login page of an appointment booking tool (http://nfbc.oulu.fi/mrbs). Once logged in, subjects will be asked to find their hometown from a drop-down menu to see the appointment calendar of their local examination station. Once a subject has clicked on their preferred appointment time in the calendar, a page will open containing a box in which the subject can enter their name and telephone number if they want a text-message reminder about their appointment. The subject will then be asked to click ‘Save’ to return to the calendar to view the appointment reserved for them. Appointments that have been booked by other subjects will appear as ‘Reserved’ without the subjects’ names. The field staff and Project Headquarters will be able to access a list showing subjects’ names. Subjects who are due to be examined in Oulu will be asked to also book an appointment for a sugar stress test after they have booked their actual examination. Subjects who fail to book an appointment using the tool will be contacted by telephone to make arrangements. The calls will be made by receptionists, secretaries and Clinical Research Nurses, who will have a slot in their daily schedule set aside for this purpose. The plan is to also assign a time slot for making calls in the evenings. An Excel spreadsheet will be used to keep track of calls. Staff members will use the same appointment booking tool as subjects. Subjects will also be able to call in themselves to book or reschedule an appointment, to ask questions or to request paper versions of questionnaires. Two contact telephone numbers will be provided in the invitation. Calls will be transferred between the two numbers if one number is busy or not answering. 10

Subjects who want to attend the more comprehensive examination available in Oulu will be given an opportunity to do so. If none of the appointments available at a subject’s local examination station suits them, they will be given a choice of appointments at the next nearest examination station. Subjects will be sent an automated text-message reminder two or three days before their scheduled clinical examination. Field staff will be able to print out daily appointment lists from a designated folder. Subjects will also be provided with an ID sticker sheet (Appendices 51–52) at the first examination station they visit, which they will carry in a document wallet throughout the day and which staff can use to identify them.

4.5 STAFF, RESPONSIBILITIES AND CONTACT DETAILS

4.5.1 STAFF Project Headquarters • Scientific Director of Northern Finland Birth Cohort (NFBC) Studies: Marjo-Riitta Järvelin, • Executive Director of Northern Finland Birth Cohort (NFBC) Studies: Sirkka Keinänen-Kiukaanniemi, • Deputy Director of Northern Finland Birth Cohort (NFBC) Studies: Anja Taanila, • Field Manager: Juha Auvinen, • Project Secretary: Tuula Ylitalo, • Senior Clinical Research Nurse: Anu Outinen-Tuuponen, • Senior Medical Laboratory Technologist: Kirsi Krum, • Information System Developer / Data Manager: Arto Muhli, • Developer / Data Manager: Ville Huikari, • Software Developer: Markku Koiranen, • Information System Developer: Jari Jokelainen, Field staff Oulu field team (MLT1 and CRN1–5 to relocate to Oulainen and Kemi once examinations in Oulu are completed) • Medical Laboratory Technologist (MLT) 1: Anni Ruotsalainen • Clinical Research Nurse (CRN) 1: Hanna-Leena Grönroos, • Clinical Research Nurse (CRN) 2: Titta Korteniemi, • Clinical Research Nurse (CRN) 3: Anu Outinen-Tuuponen, • Clinical Research Nurse (CRN) 4: Riitta Alaraatikka • Clinical Research Nurse (CRN) 5: Eila Ylitalo • Receptionist, 8.00 am – 1.00 pm / Appointment Scheduler, 1.45–3.30 pm: Liisa Puurunen • Dermatologists: o First pilot: Päivi Hägg, o Second pilot: Kaisa Tasanen-Määttä, o Examinations between April and June 2012: Laura Huilaja, o and March/April 2013: Suvi-Päivikki Sinikumpu • Radiographer: Anna Laulumaa, • Dental Receptionists: Tanja Korhonen and Paula Pesonen, • Dentist 1: Eelis Rytkönen, 11

• Dentist 2: o Linnea Närhi, o Heikki Kiviahde, o Marja-Liisa Laitala, o In reserve: Pirkko Jaskari, • Dental Nurse 1: Aili Jurvakainen, • Dental Nurse 2: Arja Moilanen, • Saliva sample processing staff: o Henna Niskala, o Tanja Kuusisto, o Eeva-Maija Kiljander, o Sanna Juntunen, • Physiotherapists: (three in rotation) o Pekka Antila (Mon, Wed and Fri) o Sirpa Kortesalmi (Tue/Thu), o Tuula Tarvainen (Tue/Thu), • Cardiologist: Kari Kaikkonen • Optician / Eye Nurse: Anna Autio • Field Nurse, Eye Examinations: o Nina Yrjänäinen, o Anne Kurikka, • Ophthalmic Photographer: Jukka Veijola,

Northern Finland field team • Medical Laboratory Technologist (MLT) 1: Leila Pehkonen, • Clinical Research Nurse (CRN) 1: Sari Väärälä • Clinical Research Nurse (CRN) 2 (team leader): Rauni Seppälä

Southern Finland field team • Medical Laboratory Technologist (MLT) 1: Saija Glader, • Clinical Research Nurse (CRN) 1 (team leader): Pirjo Rähmönen, • Clinical Research Nurse (CRN) 2: Satu Lahtela-Kupiainen,

Consultants/Analysts Questionnaires Juha Auvinen, Upper arm blood pressure Juha Auvinen, Central blood pressure Karl-Heinz Herzig, Bioimpedance, height, weight and waist and hip measurements Juha Auvinen, Blood samples Aimo Ruokonen, ECG Kari Kaikkonen, Tuomas Kenttä, Activity trackers Riikka Ahola, 12

Anna Jauho, Dermatology Kaisa Tasanen-Määttä, PAL tests Juha Veijola, Skin prick testing Juha Pekkanen, Spirometry Juha Pekkanen Juha Auvinen, Marjo-Riitta Järvelin, Radiography Jaakko Niinimäki, Saliva tests Tuula Salo, Dental examinations Raija Lähdesmäki, Leena Niskanen, Physiotherapy examinations Jaro Karppinen, Juha Auvinen, Tero Klemola, Step test, heart rate variability and baroreflex Mikko Tulppo, Antti Kiviniemi, Juha Auvinen, Back strength Juha Auvinen, Hand grip strength Juha Auvinen, Echocardiography and carotid ultrasonography Kari Kaikkonen, Eye examinations Ville Saarela,

4.5.2 SUBSTITUTE ARRANGEMENTS Field staff are being trained to substitute for each other in the event of sickness absences and holidays. Substitute arrangements for the Oulu field team (CRN1–5 and MLT1) will be as follows: 1. MLT1’s role will be covered by Kirsi Krum. 2. CRN1’s role will be covered by CRN4, with Kirsi Krum covering sugar stress tests. If CRN4 is also unavailable, CRN1’s role will be covered by Kirsi Krum. 3. CRN2’s role will be covered by Riikka Siiro-Virtanen or, in her absence, CRN5, with Kirsi Krum covering sugar stress tests. 4. CRN3’s role will be covered by CRN1, with CRN1’s role covered as per point 1. 5. CRN4’s role will be covered by CRN2, with CRN2’s role covered as per point 2. 13

6. CRN5’s role will be covered by CRN3, with Kirsi Krum covering sugar stress tests in the morning. If CRN3 is also unavailable, CRN5’s role will be covered by Juha Auvinen. Staff shortages in the Northern and Southern Finland field teams (CRN1–2 and MLT1) will be covered by a member of the Oulu team, subject to the Oulu team’s internal substitute arrangements as shown above. The aim will be to send a volunteer and, if there are no volunteers, there will be a rota according to which staff shortages in the Northern and Southern Finland field teams will be covered. 1. The MLTs’ roles will be covered by the Oulu team’s MLT or, if the Oulu team’s MLT is unavailable, by Kirsi Krum. 2. CRN1s’ roles will be covered by the Oulu team’s CRN3, CRN5 and CRN1. 3. CRN2s’ roles will be covered by the Oulu team’s CRN2 and CRN4. 4. The field teams’ CRN1s and CRN2s will substitute for each other if the Oulu team is unable to provide cover. Substitute arrangements for the dental examination team. Substitute arrangements for the dermatology team. No heart and jugular vein ultrasound examinations will be performed in the absence of the cardiologist. These subjects will be examined either after their sugar stress test in the morning or at a later date by appointment. The eye examination team includes two field analysts competent to cover each examination station. The musculoskeletal examination team includes three physiotherapists who will be able to substitute for each other. All team members will be expected to contribute to the general management of the study and to prioritise as necessary.

4.6 EXECUTION

4.6.1 INVITATIONS Addresses for the cohort members will be requested from the Population Register Centre in February 2012. The first invitation to the 46-year follow-up (Appendix 9) will be sent to cohort members by post in March/April 2012 (in three batches at two-week intervals). The invitation will include login details for the Webropol tool. Invitations to clinical examinations will be sent to subjects between two and four weeks before their local examination station is due to open. Invitations to busy examination stations will be sent out in batches (one batch per month). These letters will include a detailed description of the clinical examination process, the feedback that will be given to subjects and two consent forms for subjects to familiarise themselves with and bring with them to their examination (Appendices 10–14). 14

4.6.2 FIELDWORK

4.6.2.1 SAMPLES AND ANALYSES

4.6.2.1.1 Questionnaires All surviving cohort members whose address has been successfully retrieved from the Population Register Centre and who live in Finland will be asked to fill in two questionnaires before their clinical examinations in April 2012. The first questionnaire will focus on background, lifestyle and health and the second on finances, work and resources, and the subjects will be able to complete the questionnaires online or on paper. All subjects who attend clinical examinations will be given supplementary questions to answer on a computer and a BDI-II questionnaire to fill in on paper while they wait. Subjects examined in Oulu will be asked to complete a supplementary dental health questionnaire. In addition, all subjects who attend clinical examinations will be handed an opinions and experiences questionnaire to be filled in while they wait or afterwards at home and returned by post. The opinions and experiences questionnaire and the BDI-II questionnaire will also be sent by post to cohort members who fail to attend their clinical examinations after the study has been completed at the beginning of 2014.

4.6.2.1.2 Health examinations in Oulu Subjects living within between approximately 80 and 90 km of Oulu will be invited to a more comprehensive battery of clinical examinations in Oulu. These examinations will cover all the same components as the examinations performed in other areas, plus the following: − Skin health − Comprehensive oral and dental examination − Radiography − Physiotherapy examinations − Heart and jugular vein ultrasound examination (some subjects)

4.6.2.1.3 Health examinations in Oulainen and Kemi Subjects living within approximately 70 km of Oulainen or Kemi will be invited to a more comprehensive battery of clinical examinations in Oulainen or Kemi. These examinations, although more comprehensive than the examinations performed by the Northern and Southern Finland field teams, will not be as comprehensive as those performed in Oulu. In addition to the components included in the examinations performed by the Northern and Southern Finland field teams, the examinations in Oulainen and Kemi will also cover the following: − Autonomic nervous system measurements − Thermal sensitivity and heat pain threshold tests

4.6.2.1.4 Eye examinations Half of the cohort members (approximately 5,100 subjects) will be randomly selected for eye examinations. Subjects living in and around Oulu will have their eye examination in Oulu after their other examinations on the same day. The plan is for the eye examination team to disperse to other examination stations once the examinations in Oulu are completed to examine the other cohort members selected for the eye examination sample. 15

4.6.2.1.5 Glucose tolerance tests Subjects living in and around Oulu will be invited to attend a separate oral glucose tolerance test at the University of Oulu Department of Dentistry ideally between two and six weeks after their other examinations. Medical directors of health centres in Northern Finland will also be asked to assign medical laboratory technologists to perform glucose tolerance tests in health centres after the clinical examinations. The participating health centres will be provided with all the equipment they need (tubes, needles, glucose solution, etc.) and, if necessary, compensated for their medical laboratory technologists’ time. The cost of shipping cryogenic tubes will also be reimbursed. The samples will be frozen and analysed in Oulu at a later date.

4.6.2.1.6 White blood cell isolation Subjects living in and around Oulu will be asked to provide a blood sample (10 ml CPT tube) for the purposes of white blood cell isolation (peripheral white blood mononuclear cells). Medical laboratory technologists from the Department of Microbiology will collect tubes from the Faculty of Medicine once a day. The samples will be processed at the Department of Microbiology, frozen by degrees and stored in a liquid nitrogen tank in the Department of Health Sciences’ walk-in freezer.

4.6.2.1.7 Saliva samples Subjects living in and around Oulu will be asked to give a resting saliva sample and a paraffin- stimulated saliva sample on the morning of their sugar stress test.

4.6.2.2 CLINICAL EXAMINATIONS, SUBJECTS’ ROUTE BETWEEN EXAMINATION STATIONS AND FIELD STAFF’S RESPONSIBILITIES

4.6.2.2.1 Oulu examination stations, Day 1 (Appendix 21) Clinical Research Nurse (CRN) 1: Equipment and instruments - Computer - Upper arm blood pressure monitor - Central blood pressure monitor - ECG machine Data collection - Filing of consent forms - Entries in the examination template o CRN’s initials o Receipt of consent form o BP x 3, PR x 3 o Central BP measurement successful Y/N o ECG taken Y/N o Notes, where applicable - Automatically migrated instrument data o Central BP data o ECG - Average of the BP measurements entered into a separate BP/height/weight template for the cardiologist as shown on the BP monitor screen Procedures 16

- Checks the subject’s identity and personal details from the appointment list - Checks the worksheet to see whether the subject has completed the questionnaires and consent forms sent by post and, if not, gives the forms to the subject and asks the subject to fill them in while they wait - Explains the purpose and objectives of the study and describes the examinations on a general level, emphasising the confidential and voluntary nature of the study. Tells the subject about the feedback they will be given (immediate feedback / Appendix X and results to be reported afterwards / Appendix X). - Asks for the consent forms completed by the subject or, if the subject has not completed the forms or brought the forms with them, gives a new consent form to the subject to fill in. This form asks for the subject’s consent to the use of information collected during their health examination. CRN1 enters the date and their name and signature to confirm receipt of the forms. The consent form will be completed in two identical copies, one of which will be given to the subject and the other filed at the examination station / in the project folder / in the analyst’s folder. - Gives the subject a copy of the opinions and experiences questionnaire / login details for the Webropol tool. The subject will also be able to fill in the questionnaire while they wait and hand it in at the end of the day. - Places an ID sticker on the copy of the consent form that will remain at the examination station, all the questionnaires completed by the subject and the BP/height/weight template (Appendix 41) - Provides the subject with a document wallet that they can use to keep their questionnaires and documents in order and to write on - The document wallet will also contain a lunch voucher, a map of examination stations and a sticker sheet for other CRNs - Asks the last four subjects of the day whether they are happy with the first option on the Pruxis canteen’s menu - Measures the subject’s blood pressure and pulse three times at one-minute intervals - Measures the subject’s central blood pressure twice - Records the ECG Feedback - Reads out the subject’s upper arm blood pressure measurements Preparations and other tasks - Communicates the subjects’ food choices to the Pruxis canteen by telephone - Makes back-up copies of the collected data - Cleans the instruments - Checks whether more forms and instruments are needed - Prepares document wallets for the following day’s subjects - Prints out ID and laboratory stickers for the following day - Centrifuges and separates remaining sugar stress test samples and delivers them to MLT1 - Helps MLT1 to process samples as required - Delivers saliva samples collected by the dental examination team in connection with delivering blood samples - Prepares document wallets for the following day’s subjects

Medical Laboratory Technologist (MLT) 1: Equipment and instruments - Computer - Freezer 17

- Refrigerator - Examination table - Sample shaker Data collection - Entries in the examination template o CRN’s initials o Subject’s ID o Date o Confirmation of the subject’s having fasted for at least 12 hours o If the subject has not fasted for 12 hours, record of what the subject has eaten and drunk o Time when blood samples were taken o Confirmation as to whether all blood samples were successful o Confirmation as to whether centrifugation and separation were performed on time o Confirmation as to whether all separator tubes contain sample o CRNs track sample-taking/processing using a separate template and enter the data into the system at the end of the day. The template will be filed in the project folder. Procedures o See separate protocol Feedback - None to the subject on the site. Preparations and other tasks o Gets instruments and tubes ready for the following day o Delivers fresh samples and sugar stress test samples to Oulu University Hospital o Orders and prints out laboratory referral stickers from Oulu University Hospital for the following day’s subjects o Prints out stickers for the Northern and Southern Finland teams’ fresh samples and delivers to CRN at Oulu University Hospital’s laboratory

Clinical Research Nurse (CRN) 2: Equipment and instruments - Computer - InBody machine - Digital scale - Stadiometer - Tape measure - Roman chair - Hand grip dynamometer and calibration tools Data collection - Entries in the examination template o CRN’s initials o Subject’s height o Subject’s weight o Waist and hip measurements o Hand grip strength o Back strength test result o Notes, where applicable 18

- Automatically migrated instrument data o InBody machine - Subject’s height and weight entered into a separate height/weight template (for CRN3) Procedures - Asks to see the subject’s study folder and height/weight template - Briefly explains the measurements to be taken - Asks the subject to strip down to their underwear and take off any jewellery and hair accessories - Asks the questions included in the InBody checklist - Measures the subject’s height o Subject’s height x 2 o Subject to stand up straight facing forward o Subject's heels, buttocks, shoulders and head to rest against the stadiometer o The outer corner of the subject’s eye to align with the top of the ear canal o Height to be recorded to the nearest 0.1 cm - Measures the subject’s weight o Subject’s weight x 1 o Subject to stand with their feet slightly apart, resting on both feet and facing forward o Weight to be recorded to the nearest 0.1 kg - Measures the subject’s waist o Circumference x 1 o CRN2 to be seated o Subject to stand in front of CRN2 with their feet slightly apart o Measurement to be taken at the midway point between the iliac crest and the lowest ribs o Tape measure to run parallel to the floor and over any abdominal fat o Tape measure to be snug but not compress the skin o Subject to breathe calmly in and out o Measurement to be recorded to the nearest 0.5 cm - Measures the subject’s hips o Circumference x 1 o Hips to be measured at the widest point approximately two finger widths above the pubic bone o Measurement to be taken at the midway point between the top of the pelvis and the inguinal crease o Measurement to be recorded to the nearest 0.5 cm - Performs a bioimpedance analysis (InBody) o Analysis to be performed using Lookin'Body software o See InBody protocol - Measures the subject’s hand grip strength o Dynamometer switched ‘On’ and set to ‘Continuous’ o ‘Channel A’ selected and ‘Zero Level’ at 0.00–0.01 o CRN2 to check the subject’s grip of the handle and adjust if necessary o Dynamometer setting from ‘Continuous’ to ‘Peak’ o ‘Reset’ before each measurement o Subject to stand with their feet slightly apart and arms by their sides o Maximal grip strength x 3 with both hands 19

o Contraction to be maintained for four seconds and a gap of 15 seconds to be left between measurements - Measures the subject’s back strength o Subject to lie on their front holding their body in a horizontal line o CRN2 to sit on the subject’s ankles o Plumb bob to be suspended above the subject’s back o Subject to tense their back muscles with their arms by their sides and keep their body horizontal o Position to be adjusted as necessary (so that the plumb bob touches the subject’s back) o Test to be timed using a stopwatch o Subject to place their hands on the floor when they are no longer able to hold the position to indicate to CRN2 to stop the clock Feedback - Reads out the subject’s body fat percentage, height, weight, waist and hip measurements, back strength test result and hand grip test result Preparations and other tasks - Restarts the InBody machine after each weekend to let it warm up for 10 minutes - Inserts flash drive 11 and checks that the InBody stick is in place - Calibrates instruments and checks accuracy - Makes back-up copies of the collected data - Cleans the instruments

Supplementary questions in a separate room (2 computers): Receptionist Equipment and instruments - Computer - Box for completed BDI forms Data collection - Subject’s answers to the supplementary questions - BDI forms Procedures - Enters the subject’s ID on the computer and helps them with the questionnaire as necessary - Subject to fill in the BDI form and place in a sealed box next to the computer - Subject to be permitted snacks while filling in the questionnaire - If the 40 minutes reserved for filling in the questionnaire is not enough and several questions remain unanswered, the receptionist will save the questionnaire and provide the subject with a paper version indicating how far the subject got on the computer. The subject will be asked to finish filling in the questionnaire on paper and to return the form at the end of the day. Remember to place an ID sticker on the form! Feedback - None to the subject

Dermatologist: Equipment and instruments - Computer Data collection - Entries in the examination template o 20

Procedures - Feedback - Preparations and other tasks -

Clinical Research Nurse (CRN) 3: Equipment and instruments - Computer (Windows XP) - Spirometer - Ventolin Evohaler and Volumatic - Stopwatch - Polar Active and Hookie trackers - Polar FlowLink for data transmission - USB divider Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Confirmation as to whether a bronchodilator was administered (yes/no) and time of administration o Time when the subject began wearing the Hookie and Polar trackers - Automatically migrated instrument data o Spirometry results - ID sticker on the diary Procedures Spirometry o Asks the subject to enter the room o Performs the first spirometry test o Administers a bronchodilator (all subjects) o Performs a second spirometry test 10 minutes later Distribution of activity trackers o Attaches a Hookie tracker to the subject’s belt before they arrive. Enters the serial numbers of both trackers (Hookie and Polar Active) in the measurement log and the diary. Places a sticker on the diary. o Explains to the subject how to use the Hookie tracker (pilot subjects’ belts will also include an ActiGraph tracker) and how to use and return their Polar Active tracker o Explains to the subject how to use their diary and return it with the trackers o Creates an account for the subject in the Polar GoFit system o Enters the subject’s details (height and weight measurements from CRN2) o Configures the subject’s activity trackers using Polar GoFit and WebSync o Anonymises the subject’s Polar Active data o Fits the trackers on the subject and hands out user manuals, diary and return envelope o Asks whether the subject has any questions about how to use the trackers or the diary. Explains to the subject that they will receive their activity data by post with their other results. Appointment scheduling 21

o Checks that the subject has booked a sugar stress test appointment and, if they have not, enquires politely whether the subject has forgotten or does not wish to take part. Suggests that the subject return their activity trackers and bring their urine/stool samples to their sugar stress test if the test is scheduled for between two and four weeks after the clinical examinations. Final steps o Shows the subject to an iPad in the waiting room to take a PAL test. The receptionist will open the program, give the subject a set of headphones and tell them to follow the instructions. Feedback - Spirometry results and activity tracker data to be sent to the subject by post Preparations and other tasks - Resets the Hookie trackers first thing in the morning and sets them to charge in a USB charging dock - Calibrates the spirometer and checks accuracy before subjects arrive - Checks whether more spirometer mouthpieces are needed - Cleans the instruments - Helps with glucose tolerance tests until 9.10 am

PAL tests in the waiting room: Equipment and instruments - iPad (1 + 1 spare) - Lockable iPad stand Data collection - PAL test to be completed independently Procedures - CRN3 will lead the subject to the room where the receptionist will open the program, enter the subject’s ID according to what is shown on their forms, give the subject a set of headphones and tell them to follow the instructions. - At the end of the test, the receptionist will show the results to the subject and close the program. Feedback - Results to be shown to be subject on the screen at the end of the test Preparations and other tasks - iPad charging - USB data migration at the end of the day - Back-up copies

Radiographer: Equipment and instruments - Computer - Mobile X-ray machine - Image viewer - Imaging plates - Grids - Stand Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable 22

- Automatically migrated instrument data o X-ray images Procedures o Panoramic radiography (to be shared with dentists) o Weight-bearing AP and lateral views of feet o Knees Feedback - Images to be sent to the subject on a CD by post Preparations and other tasks - Pre-population of the imaging software with subjects’ IDs

Dentist reception room: Equipment and instruments - Computer Data collection - Supplementary dental health questionnaire on a computer Procedures - Subject to complete the questionnaire on a computer with the dental hygienist on hand to help if necessary Feedback - None to the subject Preparations and other tasks - Back-up copies at the end of the day

Dental Hygienist: Equipment and instruments - Computer - iTero intraoral scanner - Digital camera Data collection - Images uploaded to iTero to be sent for analysis in Holland and returned between 20 and 40 minutes later to the NFBC server in a cleaned-up format - Digital photography of the subject’s mouth and teeth Feedback - None to the subject Preparations and other tasks - Back-up copies at the end of the day

Dentist + Dental Nurse (2 examination stations side by side): Equipment and instruments - Computer - Oral care unit Data collection - Entries in the examination template o Dental examination template Procedures - See separate protocol Feedback - Subject to be provided feedback on paper at the end of the examination Preparations and other tasks 23

- Back-up copies on the NFBC server at the end of the day

Physiotherapist: Equipment and instruments - Computer - Spinal Mouse - Camera, stand and foot-pedal shutter remote - Calibration stick - SLR protractor - Caliper - Paediatric blood pressure cuff - Examination table - Disinfectant, antiseptic wipes, ID stickers Data collection - Entries in the examination template o Physiotherapist’s initials o Status information - Automatically migrated instrument data o Spinal Mouse files to be saved and later opened using the same software to export relevant variables Procedures o Calls the subject into the room o Explains the spinal and foot mobility assessment process o Asks the subject to strip down to their underwear o Measures spinal mobility using Spinal Mouse (starts the program and the device and configures settings) o Lumbar motor control impairment tests o Photography of the subject’s feet and the soles of their shoes o Photography of the soles of the subject’s feet and passive calf tension measurement o Two-point discrimination test on the subject’s back o Sciatic neurology based on the subject’s sciatic anamnesis o Observation Feedback - Running commentary of the results of mobility tests Preparations and other tasks - Places ID stickers on picture tags - Transfers pictures from the camera’s memory card to the computer, distributes between folders and makes back-up copies for the server

Clinical Research Nurse (CRN) 4: Equipment and instruments - Computer - AD converter - Bridge amplifier - Plethysmograph – ear sensor (1 + 1 spare) - Nasal sensor (1 + 1 spare) - Nexfin unit - BP finger clip (S, M, L) - Upper arm blood pressure monitor 24

- Manual defibrillator with ECG signal - Polar RX800 heart rate monitor with an infrared link for migrating data to the computer at the end of the day - Step board and podium (for women) - Juice to be offered to the subject after the test Data collection - Entries in the examination template o BP sitting down and standing o Step test time o Pulse at end of the step test / after four minutes o Notes explaining why the test had to be abandoned, where applicable - Automatically migrated instrument data o All electronic data o Heart rate monitor data transfer to the computer via the infrared link at the end of the day Procedures - See separate protocol Feedback - Verbal description of the subject’s fitness index as in the 31-year follow-up Preparations and other tasks - Sugar stress tests for three subjects in the morning (Day 2)

Clinical Research Nurse (CRN) 5: Equipment and instruments - Computer - Skin prick testing solutions, lancets, cellulose, protective table cloth, skin tape and pen - Refrigerator (for skin prick testing solutions) - Pressure and heat pain threshold meters Data collection - Skin prick test results and notes on a paper form - Entries in the examination template o Pressure pain threshold test notes (right/left side, etc.) - Automatically migrated instrument data o Pressure and heat pain threshold measurements Procedures - See skin prick testing protocol - See pressure and heat pain threshold test protocol Feedback - Skin prick test results to be sent to the subject by post Preparations and other tasks - Skin prick testing solutions to be taken out to warm up in the morning and returned to the refrigerator in the afternoon - SenseBox calibration, subjects’ IDs to be entered into the software - Sugar stress tests for three subjects in the morning (Day 2) Feedback - Skin prick test results to be sent to the subject by post

------Cardiologist to perform echocardiography and carotid ultrasonography on the first, third, fifth and seventh subject of the day ------25

Cardiologist: Equipment and instruments - Computer - GE Healthcare Vivid E9 ultrasound system - Heart sensor - Jugular sensor (11 MHz, IMT measurements) - Image processing software (AFI, IMT) - External hard drive for back-up copies and data processing - Examination table - Height-adjustable chair for the cardiologist - Pillow for the subject - Wedge pillow for the subject - Ultrasound gel - Ultrasonic sensor cleaner - ECG electrodes - Paper towels for the subject - Medical gloves for the cardiologist - Leaflets and feedback form for the subject Data collection - Automatically migrated instrument data o Ultrasound images and measurements Procedures - See separate protocol Feedback - Results of the ultrasound examination to be explained to the subject verbally - If abnormalities that require further investigation or follow-up are found, the subject will be given their diagnosis on paper to show to their doctor (Appendix X).

------50% of subjects randomly selected for eye examinations to proceed to the eye clinic ------

Optician / Eye Nurse: Equipment and instruments - Computer - Equipment provided by the eye clinic Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Visual acuity o Refraction o Intraocular pressure o Corneal thickness Procedures - Feedback - Preparations and other tasks -

26

Field Nurse, Eye Examinations: Equipment and instruments - Computer - Equipment provided by the eye clinic Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable - Automatically migrated instrument data? o Visual field Procedures - Feedback - Preparations and other tasks -

Ophthalmic Photographer: Equipment and instruments - Computer - OCT machine Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable - Automatically migrated instrument data o OCT images Procedures - Feedback - Preparations and other tasks -

4.6.2.2.2 Oulu examination stations, Day 2 (Appendix 21) Saliva tests (Dental Hygienist and Dental Nurse): Equipment and instruments - Computer Data collection - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Resting saliva sample, volume (ml) o Stimulated saliva sample, volume (ml) Procedures - Feedback - Preparations and other tasks 27

- Freezing of samples - Dental nurses and hygienist to collect Day-2 saliva samples in the morning and perform Day-1 dental examinations in the afternoon

Clinical Research Nurse (CRN) 4: Equipment and instruments - Computer - Ascensia Contour 15-second blood sugar meter + test strips and normal control solution Data collection - Entries in the sampling/processing template o Subject’s ID o Date o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Time when the baseline sample was taken o Start time of glucose solution ingestion o Time when the 30-minute sample was taken o Time when the 60-minute sample was taken o Time when the 120-minute sample was taken o Any nausea/vomiting during the test Procedures - Signs in subjects 1, 2 and 3 - See separate protocol Feedback - Measurements to be sent to the subject by post Preparations and other tasks - Blood sugar meter controls - Restocking and refrigeration of glucose solutions - Restocking of the trolley - Distribution of stickers - CRN4 and CRN5 to collect Day-1 data in the afternoon and perform Day-2 glucose tolerance tests in the morning - Subject to be sent off to lunch once the last sample has been taken and to prepare for the afternoon’s examinations - CRN1 to centrifuge and separate remaining samples and deliver to MLT1 in the main building of the Faculty of Medicine

Clinical Research Nurse (CRN) 5: Equipment and instruments - Computer - Ascensia Contour 15-second blood sugar meter + test strips and normal control solution Data collection - Entries in the sampling/processing template o Subject’s ID o Date o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Time when the baseline sample was taken 28

o Start time of glucose solution ingestion o Time when the 30-minute sample was taken o Time when the 60-minute sample was taken o Time when the 120-minute sample was taken o Any nausea/vomiting during the test Procedures - Signs in subjects 4, 5 and 6 - See separate protocol Feedback - Measurements to be sent to the subject by post Preparations and other tasks - Blood sugar meter controls - Restocking and refrigeration of glucose solutions - Restocking of the trolley - Distribution of stickers - CRN4 and CRN5 to collect Day-1 data in the afternoon and perform Day-2 glucose tolerance tests in the morning - Subject to be sent off to lunch once the last sample has been taken and to prepare for the afternoon’s examinations - CRN1 to centrifuge and separate remaining samples and deliver to MLT1 in the main building of the Faculty of Medicine

4.6.2.2.3 Oulainen and Kemi with the same equipment and staff as Oulu (flow chart in Appendix 22) Clinical Research Nurse (CRN) 1: see above Medical Laboratory Technologist (MLT) 1: see above + below: - Blood samples to be picked up at the end of the day. Fresh samples to be delivered to Oulu University Hospital for analysis and samples to be frozen at the Department of Health Sciences to be placed in the designated -80 ⁰C chest freezer in the walk-in freezer. The Senior Medical Laboratory Technologist to take the samples from the rack and place them in the correct containers the following morning and to record the samples in Excel / the sample management system. - Samples destined for Oulu University Hospital to be stored and transported in a +4 ⁰C cooler. - Samples due to be frozen to be placed in an expanded polystyrene box with multiple ice packs. - Fills out shipping lists by container and packs them in the cooler with the day’s samples. Clinical Research Nurse (CRN) 2: see above Supplementary questions in the waiting room: see above Clinical Research Nurse (CRN) 3: see above PAL tests in the waiting room: see above Clinical Research Nurse (CRN) 4: see above Clinical Research Nurse (CRN) 5: see above

4.6.2.2.4 Northern Finland and Southern Finland teams (flow chart in Appendix 23) Medical Laboratory Technologist (MLT) 1: Equipment and instruments - Computer 29

- Upper arm blood pressure monitor - InBody machine - Digital scale - Stadiometer - Spirit level - Freezer - Refrigerator - Examination table - Sample shaker - Centrifuge - Laboratory supplies, tubes and shipping materials - Printer - Questionnaires, consent forms, BDI-II forms, ID stickers and document wallets for subjects Data collection - Filing of consent forms - Entries in the examination template o CRN’s initials o Subject’s ID o Date o Receipt of consent form o BP x 3, PR x 3 o Subject’s height o Subject’s weight o Confirmation of the subject’s having fasted for at least 12 hours o If the subject has not fasted for 12 hours, record of what the subject has eaten and drunk - Hand-written notes (template to be saved in the folder) to be entered into the examination template at the end of the day o Time when blood samples were taken o Confirmation as to whether all blood samples were successful o Confirmation as to whether centrifugation and separation were performed on time o Confirmation as to whether all separator tubes contain sample o Notes, where applicable - Automatically migrated instrument data o InBody machine - Subject’s height and weight entered into a separate height/weight template for CRN1 (Appendix 39) Procedures - Checks the subject’s identity and personal details from the appointment list - Asks the subject whether they have completed the questionnaires and consent forms sent by post and, if they have not, gives the forms to the subject and asks the subject to fill them in while they wait or afterwards at home and return by post (provides an envelope). - Explains the purpose and objectives of the study and describes the examinations on a general level, emphasising the confidential and voluntary nature of the study. Tells the subject about the feedback they will be given. - Asks for the consent forms completed by the subject or, if the subject has not completed the forms or brought the forms with them, gives a new consent form to 30

the subject to fill in. This form asks for the subject’s consent to the use of information collected during their health examination. CRN1 enters the date and their name and signature to confirm receipt of the forms. The consent form will be completed in two identical copies, one of which will be given to the subject and the other filed at the examination station. - Gives the subject a copy of the opinions and experiences questionnaire. The subject will also be able to fill in the questionnaire while they wait and hand it in at the end of the day. - Places an ID sticker on the consent form that will be filed at the examination station, all the questionnaires completed by the subject and the height/weight template - Provides the subject with a document wallet that they can use to keep their questionnaires and documents in order and to write on - The document wallet will also contain a sticker sheet for other staff - Measures the subject’s blood pressure and pulse three times at one-minute intervals using an automatic blood pressure monitor - Asks the subject to strip down to their underwear and take off any jewellery and hair accessories - Asks the questions included in the InBody checklist - Measures the subject’s height o Subject’s height x 2 o Subject to stand up straight facing forward o Subject's heels, buttocks, shoulders and head to rest against the stadiometer o The outer corner of the subject’s eye to align with the top of the ear canal o Height to be recorded to the nearest 0.1 cm - Measures the subject’s weight o Subject’s weight x 1 o Subject to stand with their feet slightly apart, resting on both feet and facing forward o Weight to be recorded to the nearest 0.1 kg - Performs a bioimpedance analysis (InBody) o Analysis to be performed using Lookin'Body software o See InBody protocol - Takes and processes blood samples (see separate protocol) - Instructs the subject to have a snack while answering supplementary questions on the computer (enters the subject’s ID to get them started and shows them the nutrition poster) and completing the BDI-II form, and places an ID sticker on the form Feedback - Reads out the subject’s blood pressure and pulse, body fat percentage, height, weight and waist and hip measurements Preparations and other tasks - Makes back-up copies of the collected data o By clicking ‘Next Patient’ in the examination template after each subject and ‘Close’ at the end of the day o By clicking the desktop icon to back up InBody data at the end of the day - Cleans the instruments - Checks whether more forms and instruments are needed - Prepares document wallets for the following day’s subjects - Prints out ID and laboratory stickers for the following day - Restarts the InBody machine after each weekend to let it warm up for 10 minutes - Checks that the InBody stick is in place 31

- Calibrates instruments (scale once a week, stadiometer with a spirit level once a day) and checks accuracy (CRN’s measurements once a week) - Fills out shipping lists by container and packs them with the day’s samples to be shipped to Oulu University Hospital

Supplementary questions in a separate room: Equipment and instruments - Computer - Nutrition poster on the wall Data collection - Subject’s answers to the supplementary questions - BDI forms Procedures - MLT1 to enter the subject’s ID on the computer to get them started with the questionnaire - Subject to fill in the BDI form and place in a sealed box for CRN2 - Subject to be permitted snacks while filling in the questionnaire - If the 40 minutes reserved for filling in the questionnaire is not enough and several questions remain unanswered, the receptionist will save the questionnaire and provide the subject with a paper version indicating how far the subject got on the computer. The subject will be asked to finish filling in the questionnaire on paper and to return the form to CRN2 at the end of the day. Remember to place an ID sticker on the form! Feedback - None to the subject Preparations and other tasks - Makes back-up copies of the collected data o By clicking ‘Save and Send’ to automatically save a copy of the questionnaire on the server and then ‘Close’ to save a PDF version on the server

Clinical Research Nurse (CRN) 1: Equipment and instruments - Computer (Windows XP) - Spirometer - Ventolin Evohaler and Volumatic - Skin prick testing solutions, lancets, cellulose, protective table cloth, Bemetson cream, skin tape, pen and EpiPen in case of anaphylaxis - Stopwatch - Polar Active and Hookie trackers - Polar FlowLink for data transmission - USB divider Data collection - Skin prick test results and notes on a paper form - Entries in the examination template o CRN’s name o Reasons why procedures were unsuccessful, where applicable o Confirmation as to whether a bronchodilator was administered (yes/no) and time of administration o Time when the subject began wearing the Hookie and Polar trackers 32

- Automatically migrated instrument data o Spirometry results - ID sticker on the diary Procedures - Skin prick testing o See separate protocol - Spirometry o Asks the subject to enter the room o Performs the first spirometry test o Administers a bronchodilator (all subjects) o Performs a second spirometry test 10 minutes later - Asks the subject whether they took any medication in the morning and whether they take any medication regularly - Distribution of activity trackers o Attaches a Hookie tracker to the subject’s belt before they arrive. Enters the serial numbers of both trackers (Hookie and Polar Active) in the measurement log and the diary. Places a sticker on the diary. o Explains to the subject how to use the Hookie tracker (pilot subjects’ belts will also include an ActiGraph tracker) and how to use and return their Polar Active tracker o Explains to the subject how to use their diary and return it with the trackers o Creates an account for the subject in the Polar GoFit system o Enters the subject’s details (height and weight measurements from CRN2) o Configures the subject’s activity trackers using Polar GoFit and WebSync o Anonymises the subject’s Polar Active data o Fits the trackers on the subject and hands out user manuals, diary and return envelope o Asks whether the subject has any questions about how to use the trackers or the diary. Explains to the subject that they will receive their activity data by post with their other results. - Measures the subject’s hand grip strength o Dynamometer switched ‘On’ and set to ‘Continuous’ o ‘Channel A’ selected and ‘Zero Level’ at 0.00–0.01 o CRN2 to check the subject’s grip of the handle and adjust if necessary o Dynamometer setting from ‘Continuous’ to ‘Peak’ o ‘Reset’ before each measurement o Subject to stand with their feet slightly apart and arms by their sides o Maximal grip strength x 3 with both hands o Contraction to be maintained for four seconds and a gap of 15 seconds to be left between measurements Feedback - Spirometry and skin prick test results and activity tracker data to be sent to the subject by post Preparations and other tasks - Resets the Hookie trackers first thing in the morning and sets them to charge in a USB charging dock - Checks whether more Polar and Hookie trackers are needed and contacts Oulu if necessary - Calibrates the spirometer and checks accuracy before subjects arrive - Calibrates the hand grip dynamometer 33

- Checks whether more spirometer mouthpieces are needed - Cleans the instruments

Clinical Research Nurse (CRN) 2: Equipment and instruments - Computer - ECG machine and electrodes - Razors, skin sanitiser, exfoliating tape - Polar RX800 heart rate monitor, belt and infrared link - Pressure pain threshold meter - Boxes, plumb bob and non-slip mat for step tests and back strength tests - Upper arm blood pressure monitor - Stopwatch - Metronome - Hair sample kit (bags, scissors, ID stickers) - Tape measure - Box for completed BDI forms Data collection - Entries in the examination template o CRN’s initials o Subject’s ID o Back strength test time (minutes and seconds) o Hand grip strength reading o Waist and hip measurements o Whether pressure pain threshold test was performed on the left side and why o Step test time and heart rate at the end of the test o Whether the subject was given a urine and stool sample kit (Y/N) o Whether a hair sample was taken (Y/N) o Notes, where applicable (whether any tests were abandoned and why, potential anomalies and reasons, whether the memory test was abandoned and why) - Automatically migrated instrument data o Pressure pain threshold o Heart rate monitor data transfer to the computer via the infrared link at the end of the day o ECG data Procedures - Records the ECG - Performs heart rate variability and step tests o See separate protocol - Measures the subject’s waist o Circumference x 1 o CRN2 to be seated o Subject to stand in front of CRN2 with their feet slightly apart o Measurement to be taken at the midway point between the iliac crest and the lowest ribs o Tape measure to run parallel to the floor and over any abdominal fat o Tape measure to be snug but not compress the skin o Subject to breathe calmly in and out o Measurement to be recorded to the nearest 0.5 cm 34

- Measures the subject’s hips o Circumference x 1 o Hips to be measured at the widest point approximately two finger widths above the pubic bone o Measurement to be taken at the midway point between the top of the pelvis and the inguinal crease o Measurement to be recorded to the nearest 0.5 cm - Performs the pressure pain threshold test o See separate protocol - Measures the subject’s back strength o Subject to lie on their front holding their body in a horizontal line o CRN2 to sit on the subject’s ankles o Plumb bob to be suspended above the subject’s back o Subject to tense their back muscles with their arms by their sides and keep their body horizontal o Position to be adjusted as necessary (so that the plumb bob touches the subject’s back) o Test to be timed using a stopwatch o Subject to place their hands on the floor when they are no longer able to hold the position to indicate to CRN2 to stop the clock - Explains the urine and stool sample processes o See separate protocol - Takes a hair sample o See separate protocol - Final steps o Shows the subject to an iPad in the waiting room to take a PAL test, enters the subject’s ID, gives them a set of headphones and tells them to follow the instructions and to place the headphones on the desk when the results appear on the screen at the end of the test Feedback - Reads out the subject’s waist and hip measurements, step test fitness index and back strength index Preparations and other tasks

PAL tests in the waiting room: Equipment and instruments - iPad (1 + 1 spare) - Lockable iPad stand Data collection - PAL test to be completed independently Procedures - CRN2 will lead the subject to the room where the receptionist will open the program, enter the subject’s ID according to what is shown on their forms, give the subject a set of headphones and tell them to follow the instructions. - At the end of the test, the receptionist will show the results to the subject and close the program. Feedback - Results to be shown to be subject on the screen at the end of the test Preparations and other tasks - iPad charging 35

- USB data migration at the end of the day - Back-up copies

4.6.2.3 CLINICAL EXAMINATION PROTOCOLS

4.6.2.3.1 Registration, identity checks and consent for the use of data Each subject will be asked for their name and date of birth at the first examination station. They will then be signed in on the appointment list. ID stickers will be placed on the front page of consent forms. The subject will be given an overview of the examinations to be performed during the day. They will then be asked to sign the consent forms. One copy will be given to the subject and the other will be filed in the NFBC archives. The subject will be given a document wallet already containing an ID sticker sheet and a flow chart of the examination stations to be visited during the day. A copy of the opinions and experiences questionnaire and any questionnaires that the subject has not already returned by post or online will also be placed in the document wallet. ID stickers will also be placed on these forms. The subject will be instructed to complete the forms while they wait during the day and to return the forms to any examination station to be delivered to the Department of Health Sciences by a member of staff.

4.6.2.3.2 Biological samples A wide range of samples will be taken for laboratory analyses. The samples will be stored at the University of Oulu Department of Health Sciences and sent out to various laboratories for analysis as required.

4.6.2.3.2.1 Blood samples The aim will be to take blood samples from all subjects who attend clinical examinations. The maximum total volume of blood to be taken will be approximately 90 ml. The MLT will check the subject’s identity and ask them about fasting. If the subject has fasted for less than 12 hours, the MLT will ask them about the foods and drinks consumed and make a note of the actual fasting time. Blood will be drawn from a vein in the crook of the elbow into a vacuum- sealed test tube with the subject lying down. The MLT will be provided with a rack pre-filled with the tubes to be used. Each subject’s samples will remain in the same rack throughout the study, and an ID sticker will be placed on the rack when the samples are taken. The MLT will use a tray containing two racks: one pre-filled with blood collection tubes and one with cryogenic tubes and tubes to be sent to Oulu University Hospital. Each subject’s samples will remain in their designated racks on the same tray until ready for freezing/refrigeration. The subject will be asked to expose the crook of their elbow, and a tourniquet will be placed around their left upper arm. The skin will be cleaned with A12 T 80% denatured alcohol. The tourniquet will be released as soon as the needle enters the vein. A 21G safety butterfly needle will be used to collect the samples. The samples will be placed in a sample shaker to mix after first shaking them a few times manually. Once the samples have been collected, the MLT will place ID stickers on the sample tubes and write the time when the samples were taken in the first sample processing template. Blood samples will be taken in the following order (Appendix 24 for Oulu and Appendix 25 for Southern and Northern Finland): o Serum tube (1 x 10 ml) 36

o Serum gel tube (3 x 10 ml) o Serum gel tube (1 x 5 ml) o Trace element tube (1 x 7 ml) o Lithium heparin tube (1 x 5 ml) o (Lithium heparin tube (1 x 10 ml) in Oulu only) o EDTA tube (6 x 5 ml) o EDTA tube (1 x 10 ml) o PAXgene tube (1 x 10 ml) o FC mixture tube (1 x 5 ml)

4.6.2.3.2.1.1 Blood sample processing Serum and plasma tubes will be placed in a centrifuge with a 13.5-cm rotor and centrifuged at 3,800 rpm (2,200 G) for 11 minutes. The rotor radius is measured from the centre of the rotor to the base of the tube. If the radius is not 13.5 cm, the rotor speed can be calculated as follows: Rotor speed (rpm): 푅퐶퐹 푅푃푀 = √ ∗ 1000 푟 ∗ 1.118

Samples due to be centrifuged (Appendices 24 and 25) must be centrifuged within between 30 and 60 minutes of collection. Serum samples should be allowed to clot for 30 minutes after collection. Plasma tubes can in theory be centrifuged immediately after collection, but centrifuging all blood samples together is easier for practical reasons. It will be the MLT’s responsibility to ensure that samples are processed within the allocated window. If processing is delayed, a note will be made in the sample processing template. Samples will be separated by order of priority as shown in Appendices 24 and 25 into cryogenic tubes and tubes to be sent to Oulu University Hospital. After the separation process, fresh samples due to be analysed in Oulu University Hospital will be placed in a refrigerator and cryogenic tubes frozen at -20 ⁰C at all field examination stations. The order in which blood samples are to be collected and separated is set out in Appendix 47. The serum collected in 10-ml serum and serum gel tubes will be separated into 12 cryogenic tubes and three tubes to be sent to Oulu University Hospital. Plasma from the 5-ml lithium heparin tube will be separated into two cryogenic tubes and red blood cells into two micro cryogenic tubes. Plasma from the FC mixture tube will be separated into one tube to be sent to Oulu University Hospital. One of the 10-ml serum gel tubes will need to be protected from light immediately after collection and before centrifugation by wrapping it in a piece of foil. After centrifugation, 1 ml of the light- shielded serum gel sample will be separated into a brown cryogenic tube for a vitamin-D analysis and the rest of the serum can be used in other cryogenic tubes. The 5-ml serum gel tube will also need to be protected from light after centrifugation, as the sample will be used to analyse, for example, bilirubin production. For the purposes of bilirubin analysis, only serum, not whole blood, must be protected from light. The 7-ml trace element tube, six 5-ml EDTA tubes and the 10-ml PAXgene tube will not be centrifuged at all but will instead be stored as whole blood, as shown in the flow charts of blood sample collection and processing (Appendices 24 and 25). The whole blood tubes will be allowed to cool in room temperature after collection before storing them at the correct temperature. Two of the 5-ml EDTA tubes will be refrigerated and taken to Oulu University Hospital for analysis at that 37 temperature. Four of the 5-ml EDTA tubes, the 7-ml trace element tube and the 10-ml PAXgene tube will be frozen at -20 ⁰C after collection. The PAXgene tube will be kept at room temperature for a few hours prior to freezing at -20 ⁰C.

4.6.2.3.2.1.2 Sample logistics The blood samples that will be taken in the course of the 46-year follow-up of the 1966 Northern Finland Birth Cohort Study can be divided roughly into fresh samples that will be analysed in Oulu University Hospital and frozen samples that will be initially stored at examination stations, and logistics for the two types of samples will be slightly different. The location of field teams at any given time will also affect sample logistics. As a rule, fresh samples due to be analysed in Oulu University Hospital will be shipped from examination stations once a day or at least four times a week (on Tuesdays, Wednesdays, Thursdays and Fridays). Fresh samples will be shipped refrigerated at +4 °C. The samples will be placed in thermos flasks each containing two refrigerated gel packs. The flasks will be placed in an expanded polystyrene box with one or two frozen ice packs. The tubes will be inside resealable plastic bags with absorbent tissue paper in case they break in transit. An itemised shipping list (Appendix 37) will accompany each shipment. The list will show if any samples are missing from a subject. A sticker showing the address of Oulu University Hospital’s Customer Service will be placed on the box: Oulu University Hospital, Laboratory, Customer Service PO Box 500 90029 Oulu University Hospital Fresh samples collected at regional examination stations will be given to Matkahuolto or Itella to deliver, depending on the location. The main criterion in the choice of service provider will be schedule. Project Headquarters will research the alternatives in each location and pick the best way to ship samples. A thermometer will be used to check shipment temperatures once a week. Fresh samples from the Oulu examination stations will be hand-delivered to Oulu University Hospital for analysis by the MLT or the Senior Medical Laboratory Technologist. Once the Oulu field team has relocated to Oulainen and Kemi, fresh samples can be brought back to Oulu to be analysed in Oulu University Hospital at the end of each day, in which case the samples must be placed in a cooler with a sufficient number of refrigerated ice packs. Frozen samples from regional examination stations will be shipped once a week or once a fortnight. The samples will be packed in dry ice and delivered by Matkahuolto. Project Headquarters will supply regional examination stations with the dry ice needed for shipping whenever a shipment is due. The samples will be in freezer containers and placed inside a large expanded polystyrene box filled with dry ice. A sticker showing the address of the Project Headquarters will be placed on the box: University of Oulu Aapistie 5A 90220 Oulu ATTN: Kirsi Krum / Department of Health Sciences Project Headquarters will need to be notified of each shipment of frozen samples in advance. If any field team members are due in Oulu for the weekend, they can also bring frozen samples with them. In such cases the samples will need to be placed in a cooler with a sufficient number of frozen 38 ice packs to keep the samples frozen during transit. If a field team moves to a new examination station in the middle of a week, frozen samples can be transported in the chest freezer where they we originally placed. The chest freezer must contain a sufficient number of ice packs at all times to ensure that samples remain frozen while the freezer is switched off during transit. The chest freezer must be plugged in as soon as the team arrives at the new examination station. Samples collected at field examination stations will be frozen in cardboard containers with 10 x 10 slots for 2-ml cryogenic tubes and 8 x 8 slots for 5-ml cryogenic tubes. All samples (blood and urine) should ideally be placed in designated slots inside the containers to facilitate further storage at the Department of Health Sciences. All sugar stress test samples collected from the same subject at different times will need to be placed next to each other in the same container.

4.6.2.3.2.2 Urine and stool samples Each subject will be given a stool and urine sample kit to take home and instructed to drop off their samples at a later date or to bring the samples with them when to their sugar stress test. In other words, urine and stool samples from subjects in Oulu can be collected in connection with the sugar stress test, and subjects examined elsewhere (by the Northern and Southern Finland teams) will need to bring their samples separately either the day after or a few days later. The kit (containing a kidney dish, gloves, an expanded polystyrene box, an ice pack, two stool sample containers, a urine sample container and a urine sample tube) along with instructions and a sample data sheet (Appendix 26) will be given to the subjects when they attend their clinical examination. The CRN on duty will already have placed ID stickers on the sample data sheet, the urine sample tube and the stool sample containers. Subjects will be instructed to return their samples to any CRN, who must check that all the fields in the sample data sheet have been filled in and then place the urine sample in a refrigerator and the stool samples in a freezer.

4.6.2.3.2.2.1 Urine sample processing Subjects will be asked to produce their urine sample in the morning and place it in a 10-ml urine sample tube without preservatives. The sample must be brought to the examination station in an expanded polystyrene box containing a frozen ice pack. Urine samples must be processed as soon as possible, albeit giving priority to timely blood sample processing. The MLT will enter the time when the subject produced their urine sample and the time when the sample was processed into the examination template. Recording the times will be extremely important especially if processing needs to be postponed due to more urgent blood sample processing. Urine samples will be centrifuged at 3,640 rpm (2,000 G) for five minutes. After centrifugation, each sample will be separated into to cryogenic tubes and frozen at -20 ⁰C. Stickers will be placed on the tubes as shown in Appendices 51 and 52. The samples will be delivered to the Department of Health Sciences together with frozen blood samples.

4.6.2.3.2.2.2 Stool sample processing Each subject will return two 20-ml stool sample containers. A sticker will be placed on each container and the date on which the sample was provided will be recorded in the examination template. Subjects will be instructed to freeze their stool samples. Stool samples stored at field examination stations will be frozen at -20 ⁰C without doing anything to them first.

4.6.2.3.2.3 Special samples taken in Oulu Subjects living in and around Oulu will be asked to provide more laboratory samples than subjects examined in other locations. In addition to the standard battery of tests, the Oulu team will also take a blood sample for white blood cell isolation purposes, perform a modified blood sugar stress test 39 and collect saliva samples from subjects. The study only has enough resources to collect these samples from subjects in Oulu.

4.6.2.3.2.3.1 Isolated white blood cells Subjects examined in Oulu will provide an additional blood sample to fill a 10 ml CPT tube for the purposes of white blood cell (white blood mononuclear cell) isolation. The additional sample will be collected in accordance with the flow chart of blood sample collection and processing. The sample must be mixed carefully at the end of the collection process and left in room temperature. The sample must NOT be placed in a refrigerator. Staff from the Department of Microbiology will pick up CPT blood samples several times a day for further processing in the laboratory. The samples will be processed as shown in Appendix 27. Three or four cryogenic tubes of white blood cells per subject will be frozen for further use. ID stickers will be supplied to the microbiology laboratory with the samples. The samples will ultimately be stored at -195 ⁰C.

4.6.2.3.2.3.2 Saliva samples Saliva samples will be collected at the Department of Dentistry in Oulu. A receptionist at the Department of Dentistry will explain the saliva sample collection process to the subjects (Appendix 28). Both a resting saliva sample and a stimulated saliva sample will be collected. The receptionist will deliver the saliva samples in crushed ice to the Department of Dentistry’s Chief Laboratory Technician (CLT). Subjects will bring the stickers to be placed on the cryogenic tubes containing their saliva samples. The CLT will process saliva samples as and when they are collected. The CLT will first measure the volume of each sample using a medication cup with a millilitre scale and record the volume on a computer/form (resting: X ml; stimulated: X ml). Once the samples have been measured, the saliva will be transferred into an Eppendorf tube using a pipette (1.5 ml) and centrifuged at 1,200 rpm for 10 minutes. After centrifugation, the supernatant will be transferred into two cryogenic tubes (0.5 ml each) for freezing. The solid residue left in the Eppendorf tube will also be frozen. The samples will be labelled as shown in Appendix 52. Six saliva samples per subject will be frozen for further use. The CLT will place the processed saliva samples in the designated rack inside a freezer kept at -20 ⁰C in the sample processing room. CRN1 will collect the saliva samples in connection with the processing of sugar stress test samples and place them in the clinical examination station’s freezer, from where the Senior Medical Laboratory Technologist will move them to the Department of Health Sciences’ walk-in freezer for deep freezing at -80 ⁰C.

4.6.2.3.2.4 Oral glucose tolerance tests Subjects living in and around Oulu and other subjects as agreed will be invited to come back another day for a modified blood sugar stress test. Sugar stress tests in Oulu will be performed by CRN4 and CRN5 at the Department of Dentistry, with CRN1 assisting with sample processing. In other locations, sugar stress tests will be performed by health centre staff, with all equipment and instruments supplied in advance by the NFBC team (Appendices 42 and 48). Contrary to standard protocol, these sugar stress tests will involve taking samples at four different times (a baseline sample plus samples at 30 minutes, 60 minutes and 120 minutes), and a serum sample for insulin and metabolomics analyses will be collected in addition to blood sugar samples at each point. Subjects who have been diagnosed with diabetes and are taking diabetes medication will be excluded from the sugar stress test population. Each subject will be asked about fasting before the test. Subjects must have fasted for at least 12 hours and, if they have not, they will not be able to be tested. If a subject has fasted since the previous evening, albeit for less than 12 hours, a sugar stress 40 test may be performed but a note about the fasting time will need to be entered in the subject’s records. If a subject has had breakfast, the test cannot be performed. Each subject’s baseline blood sugar level will be measured from their fingertip using a blood sugar meter (Ascensia Contour) before baseline samples are taken and the glucose solution administered. If the baseline blood sugar reading is above 8.0, the sugar stress test cannot go ahead. Baseline samples will nevertheless be taken. The subject will be told to contact their local health centre / occupational health care provider for follow-up. If a subject’s baseline blood sugar reading is within the acceptable margin, baseline samples will be taken in the following order: o Serum gel tube (1 x 5 ml) o FC mixture tube (1 x 5 ml) The accuracy of blood sugar meters will be tested daily using a special control solution (Appendix 50). Venous blood samples will be collected by drawing blood from a vein in the crook of the elbow into a vacuum-sealed test tube. The subject will be asked to expose the crook of their elbow, and a tourniquet will be placed around their left upper arm. The skin will be cleaned with A12 T 80% denatured alcohol. The tourniquet will be released as soon as the needle enters the vein. A 21G vacuum needle will be used to collect the samples. Once the baseline samples have been taken, the subject will be asked to start ingesting the glucose solution and to empty the entire container within five minutes. The CRN on duty will record the time when the subject began to ingest the solution / when the baseline samples were taken in the examination template. New samples will be taken 30 minutes, 60 minutes and 120 minutes after the administration of the glucose solution. The blood sample tubes will be used and the samples taken in the same order as the baseline samples. Blood can also be drawn from the other arm or from a different vein to avoid stressing one vein too much. Blood sugar stress test samples will be processed in the same way as other blood samples (see 4.6.2.3.2.1.1). The samples will be separated as shown in Appendix 24 (Appendix 43 for regional sugar stress tests). The CRN on duty will instruct subjects to avoid physical strain and to refrain from smoking, eating and drinking during the sugar stress test. Subjects will be advised to sit in the waiting room and pass the time by reading magazines or watching the television.

4.6.2.3.2.5 ECG recordings A 15-lead resting ECG will be recorded digitally from all subjects who attend clinical examinations. ECGs will be recorded using a GE CAM-14 digital ECG machine and CardioSoft software.

ECG protocol: 1. Open CardioSoft from the desktop 2. Click the ‘New Test’ button in the top left corner 3. Check that ‘Resting ECG’ is selected 4. Click on ‘New Patient’ 5. Enter the subject’s details (initials of their first name and surname, date of birth as 1 January 1966, gender and estimated height and weight) in the fields at the bottom of the screen 6. Prepare the subject: o Ask them to remove their top and expose their ankles and wrists; jewellery does not need to be removed unless it would obstruct the electrodes o Shave the subject and remove any grease with alcohol o Use exfoliating tape to roughen the skin 41

o Attach the electrodes according to the agreed protocol and connect the leads to the correct electrodes as instructed o Ask the subject to relax and refrain from moving or talking during the recording process 7. Click on the blue ‘New Test’ button on the screen 8. Check the connections at the top of the pop-up window: green indicates that a connection is working, yellow that there is a problem with a connection, white that a connection is inactive, and red that an electrode has come loose or that the connection has been lost 9. When you are ready to register the result, press the ‘Record ECG’ button to record the result from the previous 10 seconds 10. The program saves the graph and a summary opens on the screen 11. To finish or switch to a new subject, click the ‘Home’ button on the left to return to the start page

Electrode placement (as indicated on the leads): 1. Limbs: o red R on the right wrist o yellow L on the left wrist o green F on the left ankle o black N on the right ankle 2. Chest: o C1 on the right sternal border in the fourth intercostal space o C2 on the left sternal border in the fourth intercostal space o C4 on the mid-clavicular line in the fifth intercostal space o C3 midway between C2 and C4 o C5 on the anterior axillary line horizontally in line with C4 o C6 on the midaxillary line horizontally in line with C4 3. Extra connections: o I on the upper sternal border o E in the centre horizontally in line with C5 and H o H on the right in line with C5 o M on the back in line with E

4.6.2.3.3 Upper arm blood pressure and heart rate measurements All subjects who attend clinical examinations will have their blood pressure and heart rate measured from their upper arm. Subjects will be asked to sit in the waiting room for 10 minutes before their first examination in the morning. Moreover, it will take the CRN in charge of the first examination station five minutes to explain what will happen during the day and deal with consent forms before the subject’s blood pressure is measured. Blood pressure will be measured using an Omron M10-IT automatic blood pressure monitor. The manufacturer’s instructions for use are provided in Appendix XX. Subjects will be seated while their blood pressure is measured. Once the subject has sat down by the examination table, a cuff will be fitted around their right upper arm. The size of the cuff will be 15.2 mm x 60 cm and fit upper arms with a circumference of between 22 and 42 cm. If the subject’s upper arm measures less than 22 cm, a smaller cuff will be provided. The cuff will be wrapped tightly around the subject’s upper arm, however without applying any additional pressure. Enough space can be left for an index finger to fit between the cuff and the subject’s upper arm on the axillary side. Prior to fitting the cuff, the subject will be asked to remove any tight-fitting clothing to prevent applying extra pressure on their right upper arm and so as to not leave any clothing underneath the cuff. The cuff will be placed around the subject’s right upper arm with the tubing to the side. The bottom of the cuff should be one or two centimetres above the crook of the 42 elbow. The machine will be programmed to take three measurements in a row with a 60-second break in between. The CRN will press ‘Start’ to begin the process and wait until the machine has taken all three measurements. The average of the three measurements will be shown on the screen. Individual measurements can be viewed by pressing ‘Set’. The CRN will enter the individual readings into the examination template. It is important for the subject to keep their arm approximately at the height of their heart (level with the fourth intercostal space at an angle of no more than 40 degrees relative to their body) and to not speak during the procedure. If the procedure is unsuccessful, the machine will repeat the process if it is set on automatic. If the procedure fails three times, the machine will stop the process. If only two of the three measurements are successful, the screen will show the average of the two readings. If only one measurement is successful, the reading on the screen will be based on that measurement alone.

4.6.2.3.4 Central blood pressure measurements In addition to the standard upper arm blood pressure measurement process, subjects in Oulu, Oulainen and Kemi will also have their central blood pressure measured from their radial artery using a SphygmoCor device at the start of the day.

Measuring central blood pressure using a SphygmoCor device: • The device switches on as you turn on the computer • Central blood pressure is measured from the subject’s left wrist • Two readings per subject will be taken • It is important for both the CRN on duty and the subject to be sitting comfortably

Opening the software and creating a new file: 1. Open SphygmoCor from the icon on the desktop 2. Click on ‘Create New’ in the top right corner 3. Click ‘Yes’ when the program asks ‘Create New Patient?’ 4. Enter the subject’s details (ID, initials of their first name and surname, date of birth as 1 January 1966 and gender) 5. Click on ‘Update’ in the top right corner 6. Click ‘Yes’ to confirm changes 7. Click the ‘Study’ button in the top left corner 8. Enter the subject’s lowest upper arm BP reading (systolic) and your own initials under ‘Operator’ 9. Click on ‘Capture Data’ in the top right corner

Measuring process: • Place the subject’s left wrist on top of the support in as relaxed a position as possible • Use two fingers to find the point on the subject’s wrist where their arterial pulse is the strongest and place the pulse pen perpendicularly against the radial artery. Move the pen around until you find a spot that produces a strong, steady pulse on the screen, wait until you have seen at least two lines from the bottom and then press the pedal to save the reading. • Try to have the ‘Signal Strength’ on the left of the screen showing at least 400 and the ‘Operator Index’ at least 80 43

• To take a second reading from the same subject, click on the ‘Study’ button in the top right corner again. This resets the subject’s upper arm blood pressure reading and you can proceed straight to clicking the ‘Capture Data’ button in the top right corner.

To switch to a new subject, click the ‘Patient’ button in the top left corner. This saves the previous subject’s data in the patient list and you can start the process again by clicking ‘New Patient’.

4.6.2.3.5 Waist and hip measurements All subjects who attend clinical examinations will have the circumference of their waist and hips measured. The accuracy of tape measures will be checked once a week using a metallic metre stick at the 60-cm and 100-cm points. A log will be kept of the control measurements. If there is a discrepancy of 0.5 cm or more between the tape measure and the metre stick, a new tape measure will be introduced. The new tape measure will be checked against the metallic metre stick before use. The subject will be asked to strip down to their underwear. The subject’s waist and hips will be measured with the subject standing with their feet slightly apart and with their weight evenly on both feet. The subject’s feet should be approximately 15 cm apart. The CRN on duty will stay seated to make it easier to read the measurements. The waist measurement will be taken from the midway point between the iliac crest and the lowest ribs. The tape measure must be snug but not compress the skin. The tape measure should run over any abdominal fat. It is important that the tape measure runs parallel to the floor. The subject can be asked to rotate 90 degrees for the CRN to be able to check that the tape measure also runs parallel to the floor along the subject’s back. The subject will be told to breathe calmly in and out and the result at the point when the subject is breathing out will be recorded. The reading will be rounded to the nearest 0.5 cm in the examination template. No measurement will be taken from pregnant women. The subject’s hips will be measured at the widest point with the tape measure running approximately two finger widths above the pubic bone. The tape measure must not run level to the inguinal crease or above the hip bones. The reading will be rounded to the nearest 0.5 cm in the examination template.

4.6.2.3.6 Height and weight measurements Staff will bring a stadiometer to the examination station. The stadiometer will be assembled and stood up against a wall with both of the support blocks touching the wall. The support blocks will be attached to the wall using, for example, adhesive putty. The straightness of the stadiometer will be checked using a spirit level before use each day, and a log will be kept of the control measurements. A visual inspection of the stadiometer will also be performed and the built-in spirit level checked before each measurement and the position of the stadiometer adjusted if necessary. A CRN will check the accuracy of the stadiometer once a week by measuring their own height and recording the result in the control log. If a stadiometer appears to be inaccurate, accuracy can be checked with the metallic measure provided. A control check with the metallic measure will also be performed whenever a new stadiometer is assembled and once a week in larger towns and cities. The subject will be asked to take off their shoes and outer garments. The subject’s height will be measured twice while the subject is undressed (underwear and socks may be worn). The subject will be asked to remove any hair accessories that could obstruct the process and to undo their hair. The subject will be asked to stand on the stadiometer platform in a natural position with their feet together, their back straight and facing forward. The subject’s heels, buttocks, shoulders and head should rest against the stadiometer. The CRN will check the positioning of the subject’s head: they 44 should be facing forward with the outer corner of the eye horizontally in line with the top of the ear canal. The sliding horizontal headpiece will then be lowered until it touches the top of the subject’s head. The subject’s height will need to be read at eye level. If the CRN is significantly shorter than the subject, they must step onto a podium to read the result. If the subject is taller than the stadiometer, the subject will be asked for their height and a note will be entered in the examination template to indicate that the height recorded is not based on a measurement. The subject’s height will be recorded in the examination template to the nearest 0.1 cm. The subject will be asked to step off the stadiometer platform briefly before their height is measured a second time in the same way as before. Any deviations from the aforementioned procedure will be recorded in the examination template. Staff will bring a digital scale to the examination station. The scale can be left on between appointments but should be switched off at the end of the day. The CRN on duty will check the accuracy of the scale once a week with 20-kg and 3.5-kg weights and record the results in a control log. The subject’s weight will be measured while the subject is undressed (underwear and socks may be worn). The subject will be asked to stand in the centre of the scale with their feet slightly apart and their weight evenly on both feet. The subject must stay still and facing forward while the CRN reads the result. The CRN will wait until the reading has stabilised and then record the reading in the examination template. The subject’s weight will be recorded to the nearest 0.1 kg. Any deviations from the aforementioned procedure will be recorded in the examination template. Please note that if a subject is in a wheelchair, the CRN will need to ask them for their height and/or weight and record the result(s) in the examination template with a note indicating that measurements could not be taken. If a subject refuses to have measurements taken and to disclose their height and/or weight, the height and weight fields in the examination template will be left empty and a note will be added to explain why. If a subject is pregnant, their weight will not be measured and instead the subject will be asked for their pre-pregnancy weight and that figure will be entered into the examination template with an explanatory note.

4.6.2.3.7 Bioimpedance measurements Make sure that the InBody machine is on and connected to the computer. Launch the Lookin'Body 3.0 software from the icon. 1. Click on ‘Profile’ in the top left corner to create a new profile. 2. Enter the subject’s details in the fields in the ‘Personal Profile’ tab. If the fields already contain, for example, the previous subject’s data, click ‘New’ at the bottom of the screen to clear the fields. 3. Press ‘Save’ to save the new subject’s details. Click ‘OK’ when ‘ID input done’ pops up on the screen. 4. Press the ‘InBody Test’ button. 5. Prepare the subject by wetting their hands and the soles of their feet and explain how they are to stand on the machine. Tell the subject to step onto the InBody platform. 6. Click ‘Yes’ when ‘Do you want to start InBody analysis?’ appears on the computer screen. 7. A Lookin'Body S window will open in the bottom right corner of the computer screen and a message will appear reading ‘InBody analysis is in progress’ with a percentage for tracking progress. 45

8. Check the screen on the InBody display to make sure that the subject’s details are showing correctly and tell the subject to grab the handles and assume the correct position when prompted by the machine. 9. When ‘Completed’ appears in the top right corner of the InBody display, tell the subject to step off the platform. Make sure that the results are showing on the computer. 10. The analysis is now complete and the machine ready to start again in step 1 for the next subject. InBody checklist o Do not disconnect cables at the back of the computer or the InBody machine unnecessarily or switch the InBody machine off to get around an error message. o Check that the subject has removed their watch, jewellery and items of clothing that could get in the way. o Make sure that the subject does not have a pacemaker, other implanted electronic medical devices or metallic implants. o No InBody analysis will be performed on pregnant women. o Ensure a good contact with the skin by wiping the subject’s hands and the soles of their feet with, for example, antibacterial electrolyte tissue. o If in step 7 the Lookin'Body S window reads ‘Checking the status of InBody measurement’, →the machine is unable to detect the subject’s weight→ and the subject must adjust their position on the InBody platform. To achieve maximum accuracy, o the analysis must be performed on an empty stomach after 12 hours of fasting, o the subject can be asked to go to the toilet before the analysis to ensure that the subject’s bladder is as empty as possible, o the subject should remove any heavy clothing, jewellery and similar for the machine to record an accurate weight, o the subject should be on their feet for at least five minutes before the analysis, o the subject’s height must be entered accurately, and o the room temperature must be between 20 and 25 °C. Technical support: o Mika Fiskari, (Project Team Member, Oulu University of Applied Sciences) o Pasi Viitanen, (Project Team Member, Oulu University of Applied Sciences) o Iida Saksi, (Project Team Member, Oulu University of Applied Sciences) o Kaisa Orajärvi, (Medical Laboratory Engineer, Oulu University of Applied Sciences)

4.6.2.3.8 Measures the subject’s hand grip strength The dynamometer will be calibrated weekly using 20-kg and 3.5-kg weights. In addition to the weights, two velcro straps and a calibration beam are needed for the process. Switch the dynamometer ‘On’ and set it to ‘Continuous’. Insert a small screwdriver into the ‘Zero Level’ slot and adjust until the screen reads to 0.00. Tie one of the straps to the beam and hang the dynamometer handle from the strap. Tie the other strap with the 20-kg hanging off it to the 46 dynamometer handle. Insert a small screwdriver into the ‘G’ slot along the bottom of the front of the dynamometer and adjust until the screen reads 20 kg. Repeat the process with the 3.5-kg weight. The dynamometer needs to be prepared before each subject by switching it on, setting it to ‘Continuous’ and placing the handle on the desk. Check that ‘Channel A’ is selected and the ‘Zero Level’ reads 0.00–0.01. Change the setting from ‘Continuous’ to ‘Peak’. The dynamometer is now ready for use. Press ‘Reset’ before each new measurement. The hand grip strength test measures the subject’s grip strength. The subject will be asked to stand with their feet a shoulder width apart holding the dynamometer handle in their right hand. The subject’s grip of the handle should be light at this point and their arm should be relaxed and by their side. The second phalanx of the subject’s index finger should be perpendicular to the handle. The subject will be asked to grip the handle as hard as they can for four seconds while keeping their body upright. The subject’s arm must not be bent or supported against their body. Three measurements will be taken at 15-second intervals first with the subject’s right hand and then with their left. All the readings will be entered into the examination template and the best result will be communicated to the subject. The meter reading represents the subject’s maximal grip strength. The subject will be asked to identify their dominant hand and a note of this will be made in the examination template. Please note that if, for any reason, a subject is unable to perform the test (due to e.g. amputation, surgery or pain), the relevant fields in the examination template will be left empty and a note will be added to explain why. If a subject has had one or more fingers amputated but still performs the test, their results will be recorded as normal and an explanatory note will be added.

4.6.2.3.9 Back strength tests Staff will need a stopwatch, a podium, a non-slip mat and a lever arm with a weight (plumb bob) hanging off it for static back strength tests. The subject will be asked to lie face down on the podium with the mat underneath them for comfort. The subject’s upper body from the crista iliaca anterior superior up should hang over the edge of the podium. The subject will first be told to have their hands on the floor for support until a horizontal line is established. Once the subject is in the correct position, the CRN will suspend the plumb bob above the subject so that it just touches the subject’s back and so that their body position can be monitored during the test. The CRN will sit lightly on the subject’s ankles for support during the test. The subject will be told to bring their arms level with their body, tense their back muscles and hold their body in a position where the plumb bob touches their back. If contact between the subject’s back and the plumb bob is lost at any point during the test, the subject will be told to immediately correct their position. If the subject is able to resume the correct position, the test can continue. Otherwise, the test will be over. The CRN will time how long the subject is able to maintain the position, up to a maximum of four minutes. The subject will be told to place their hands on the floor when they are no longer able to hold the position to indicate to the CRN to stop the clock. The test result will be communicated to the subject and entered into the examination template.

4.6.2.3.10 Skin analyses Enclosed (to be added later) 47

4.6.2.3.11 Spirometry All subjects who attend clinical examinations will undergo spirometry and a bronchodilator test with inhaled salbutamol. The test will be performed using a Spira MasterScreen Pneumo spirometer and two pneumotachographs. Staff will also need a three-litre calibration pump, a nasal clip, personal mouthpieces with bacterial/viral filters, a thermometer and a humidity gauge (to account for ambient conditions) and a salbutamol inhaler (Ventolin Evohaler cum Volumatic 0.1 mg/dos; 200 dos). Instructions for starting up the spirometry system: 1. Turn on the power unit from the switch at the back and open the spirometry software on the desktop. 2. The apparatus performs a self-test and heats the pneumotachograph for approximately five minutes. 3. ‘System check active’ will appear on the screen. The system is now ready for calibration. Click ‘OK’ to confirm ‘90 days information’. Instructions for calibrating the spirometer: The apparatus will need to be calibrated every morning and whenever ambient conditions change (temperature by +/- 4 °C and as explained in more detail in the spirometry folder). 1. Check the ambient conditions from the barometer, thermometer and humidity gauge. 2. Click on the ‘Ambient conditions’ icon: o Temperature o Rel. humidity o Baro. pressure 3. Do not change the ‘Altitude’ setting from 300. 4. Save by pressing the ‘F12’ key. Volume calibration: 1. Click on the ‘Volume Calibration’ icon. 2. Fit a grey spacer and a filter onto the manual calibration pump and attach the pump to the side of the pneumotachograph with a human symbol. 3. Press the ‘F1’ key in the top left corner to begin the calibration process. 4. Click ‘OK’ when prompted with ‘Hold the handle still and upright’. 5. Start moving the piston fully in and out at a steady pace. 6. Continue pumping at a steady pace inside the smallest volume calibration range. The apparatus resets the pump curve display automatically. Once this happens, pump two curves inside each of the three ranges. 7. ‘Calibration ended successfully’ will appear in the bottom left corner when calibration is complete. 8. Press the ‘F12’ key to save and detach the calibration pump from the pneumotachograph. Instructions for performing spirometry: 1. Prepare the subject. 48

2. Explain the basics of spirometry: o Spirometry measures lung volume and flow (ventilation capacity). o The bronchodilator test is a method for studying the reversibility of bronchoconstriction. 3. Ask the subject about contraindications: o Respiratory infection with fever (a gap of two weeks is recommended) o Recent (within the last three months) heart attack o Unstable angina pectoris o Chest pain that makes the test uncomfortable for the subject o Severe arrhythmia o Pulmonary tuberculosis (positive sputum test) o Pneumothorax o Post-operative pulmonary complications o Dementia or confusion o Pain in the mouth or face that makes the test uncomfortable for the subject o Urge incontinence o Risk of premature labour towards the end of pregnancy o Recent surgery 4. Enter the subject’s details: o Click on the ‘Patient Data’ icon to create a new profile o Last name → enter the first letter of the subject’s last name o First name → enter the first letter of the subject’s first name o Identification → enter the subject’s ID, e.g. 65554 o Press ‘Enter’ o Date of birth → enter 1 January 1966 (for all subjects) o Gender → enter male/female o Enter the subject’s height to the nearest millimetre o Enter the subject’s weight to the nearest 100 g o Save by pressing the ‘F12’ key 5. Attach an additional filter (mouthpiece) to the side of the pneumotachograph marked with a human symbol. Make sure to use a new, clean bacterial/viral filter for each subject. 6. Start the test by launching the ‘Spirometry Flow–Volume’ program. 7. Click ‘OK’ when prompted with ‘Hold the handle still’. 8. Instructions for the subject: o Explain the test to the subject. 49

o Tell the subject to sit up straight and to keep a good posture and their neck steady during the test. The subject’s jaw must be at a 90-degree angle to their neck. The subject must not lean forward during the test. 9. Place the nasal clip on the subject’s nose. 10. Place the sensor in the subject’s mouth and ask them to clamp their teeth so that their lips are tightly sealed around the mouthpiece. 11. Click on the ‘Flow Volume F3’ icon to start the test. 12. The test always begins with the subject breathing normally, after which they will be told to fill their lungs with a long, deep breath and then immediately (without holding their breath) empty their lungs with a forceful, long breath lasting at least until the indicator (‘Time ex’) switches to green, followed by quickly filling their lungs again. 13. The subject must not remove the sensor until told to do so. 14. Correctly instructing and motivating the subject (“keep blowing!”) is extremely important. 15. Always click ‘No’ if the program asks ‘Shall this trial be ignored for the evaluation?’. 16. The ‘Type of quality’ window can be closed. 17. Press ‘F7’ at the end of the test. 18. Start a new test by clicking on the ‘Flow Volume F3’ icon. 19. Try to produce three consistent flow–volume loops. 20. Give the subject a moment to rest between measurements. 21. Press ‘F12’ to save. 22. Click on the ‘Printer Report Pre Measurements’ icon. 23. The ‘File name’ window can be closed. Instructions for performing the bronchodilator test: - Contraindications for bronchodilator testing: o Chest pain while exhaling o Severe arrhythmia Please note o Asthma medication taken earlier in the day does NOT prevent bronchodilator testing o Pregnancy does not prevent the administration of a bronchodilator - The bronchodilator test will be performed using a salbutamol inhaler (Ventolin Evohaler cum Volumatic 0.1 mg/dos; 200 dos). Two doses of Ventolin will be administered, amounting to 0.4 mg in total. - Bronchodilator administration: 1. Shake the inhalation aerosol container. 2. Ask the subject to exhale calmly for as long as they can. 3. Place the dosing chamber mouthpiece firmly in the subject’s mouth. 4. Spray two doses of the aerosol into the dosing chamber. 50

5. Ask the subject to immediately fill their lungs with the aerosol-laden air from the dosing chamber and then hold their breath for 10 seconds. (The Volumatic spacer must be removed from the subject’s mouth before they begin to hold their breath.) 6. Inhalation must occur within five seconds of spraying the drug. 7. Repeat the process between 30 and 60 seconds later. - Spirometry will be performed 10 (to 15) minutes after the administration of the drug. - USE A STOPWATCH TO TIME THE INTERVAL. - Reopen the spirometry program. - Click on the ‘Flow Volume F3’ icon to start the test. - Repeat the steps from the pre-bronchodilator stage. - Try to produce two consistent flow–volume loops. At the end of the day: - Press ‘F12’ to save and exit the program. - Click on the ‘Printer Report Post Measurements’ icon. - Dispose of the mouthpieces. - Wash the nasal clips. Notes: - Start the process for each new subject by entering the subject’s details. - Use the entries in the examination template to populate the program. - If a test is unsuccessful, make a note of the reason (e.g. coughing, fatigue, back pain) in the examination template. Instructions for cleaning pneumotachographs: (to be added later)

4.6.2.3.12 Activity tracker and sleep tracker data Appendices 29 to 32

4.6.2.3.13 Panoramic radiography and X-ray images of ankles, feet and knees X-rays will be taken of subjects attending clinical examinations in Oulu at the Department of Dentistry where a special X-ray examination room will be set up. The images will be archived in digital format.

Panoramic radiography Dental panoramic radiography will be performed using Planmeca 2002 CC Proline and Dimax2 panoramic X-ray machines. Staff will also need the following: o Mouth prop with replaceable bite blocks o Jaw prop to be used instead of a mouth prop for subjects with no teeth and for subjects who cannot wear a mouth prop otherwise Contraindications: 51

o Inability to sit up or stand up o Inability to stay still (tremor) o Large body size and abnormal upper back (short neck or severe kyphosis) Protocol: 1. Ask the subject to remove all removable items and devices (partial or complete dentures, orthodontic devices, etc.) from the head and neck area. 2. Put on a radiation apron. 3. Select the subject’s profile in the Dimaxis software and get the machine ready for panoramic radiography before positioning the subject. 4. Position the subject: o Ask the subject to stand in front of the machine (with their neck straight and their shoulders relaxed). o Adjust the height of the chin rest and ask the subject to position their chin on the rest and to grab hold of the handles (subjects with a short neck should keep their hands crossed in front of them with their arms straight). o Position the subject’s upper and lower front teeth in the bite block grooves (with the bite block lying horizontally). If a jaw prop is used, position the subject so that the bottom edge of their lower lip rests on the top of the prop. o Align the horizontal beam with the lower margins of the orbits and the ear canal (Frankfort plane). o Align the image receptor (vertical beam) with the mesial side of the upper canine. o Check the subject’s midsaggital plane, make sure that the subject is facing forward and then close the temple support. o Once the subject is in position, ask them to gently close their lips over the bite block, to swallow and stick their tongue against the roof of their mouth (the shadow of the tongue prevents the overexposure of the upper teeth), to stay still and breathe normally. 5. Press the ‘Ready’ button and step behind a shielded screen while constantly holding down on the exposure button and monitoring the subject through the window throughout the procedure. Once the procedure is completed, the temple support opens automatically and the subject can step out of the machine. 6. Check the image quality: o The contrast ratio must be sufficiently high. o The upper and lower jaws and the mandibular joints must be clearly visible. o The roots of the teeth and the bone around them (lamina dura) must be clearly visible also in the front. 7. Clean the machine carefully after use. Technical properties: Panoramic exposure values Subject kV mA 52

Female, small 62 6 Female, normal 64 6 Female, large 64 9 Male, small 64 6 Male, normal 66 6 Male, large 68 9 - Focus-film distance: 480 mm - Filtering: 2.5 mm A1 - Enlargement: 1.2 - Radiation dose: The Finnish Radiation and Nuclear Safety Authority’s DAP reference value for adults is 120 mGy/cm2.

ANKLES, FEET AND KNEES Subjects’ ankles, feet and knees will be X-rayed using a mobile Simax X-ray machine. There are no contraindications. The images will be read using Fuji FCR XG-1 software. - The optimal sensitivity value (S value) with Fuji is 200–400. - The radiographer must wear a gonad apron. - The subject will be asked to remove their shoes and trousers. Protocol: 1. Feet o Exposure: 50 kV, 8 mAs; lateral 52 kV, 10 mAs o FFD: 115 cm, small focus o No additional filtering o Aids: podium and sandbag o Imaging plate: 24 x 30 AP images: - Ask the subject to stand on the X-ray box and position the tube at a craniocaudal angle of 15°. - Weight-bearing radiographs are taken of both feet simultaneously. - The subject’s feet must lie flat against the imaging plate. - Check the image quality: • The subject’s toes must be straight. • The metatarsal bones must be visible. • Soft tissue must be visible. Lateral views: - Ask the subject to stand on the podium with the cassette between their legs. 53

- Tell the subject to put their weight on the foot being X-rayed. - Make sure that the whole foot (including the ankle) is visible. - Check the image quality: • The whole foot (from the toes to the heel) must be visible. • The ankle joint must be straight. • Soft tissue must be visible. 2. Knees o Directional X-ray (D image) o Imaging plate: 35 x 43 o 60 kV, 20–32 mAs or automatic centre o FFD: 150 cm, small focus o Additional filtering at 2 mm Al AP standing up: - Ask the subject to stand with their calves resting against the frame with their knees straight. - Weight evenly on both feet - Inward rotation of feet at 5–10° with the outer edge of the foot straight - X-ray both knees even if just one has been requested. - Stand the cassette up and use a straight tube. - Check the image quality: • The subject’s knees must be clearly visible. • Enough of the subject’s legs below and above the knees must be visible (for measuring the varus/valgus angle). • Patella along the central line with the intra-articular space clearly visible • Proximal heads of the tibia and fibula partially overlapping each other in the projection • Epicondyles of the femurs along the same plane Semiflexion (of knees with the subject standing up): - Ask the subject to bend their knees with their knees, toes and thighs against the frame. - Weight evenly on both feet - Outward rotation of feet at 10° - Position the tube at a craniocaudal angle of 10° along the intra-articular space. - Stand the cassette up. - Check the image quality: • The subject’s knees must be clearly visible. 54

• Enough of the subject’s legs below and above the knees must be visible (for measuring the varus/valgus angle).

4.6.2.3.14 Dental and oral examination, photography and 3D imaging Enclosed (to be added later)

4.6.2.3.15 Physiotherapy examinations Appendix 33

4.6.2.3.16 Step tests and autonomic nervous system measurements All subjects who attend clinical examinations will have their heart rate variability and blood pressure measured from their upper arm first while seated and then while standing up. This will be followed by the same step test that was performed in connection with the 31-year follow-up. Subjects examined in Oulu, Oulainen and Kemi will also have their blood pressure monitored from their fingertip while seated and standing up, variation in peripheral blood circulation measured from the auricle using a plethysmograph, nasal breathing frequency measured with a thermometer and a three-lead ECG taken from the chest. Measurement protocol for Oulu, Oulainen and Kemi: Explain the process to the subject: “This test is designed to measure the performance of your respiratory and circulatory system. Your blood pressure will be measured from your fingertip and upper arm, your breathing frequency from your nose, your circulation from your auricle, your heart beat using a heart rate monitor and your heart function with an ECG. I would now like you to remove some of your clothing (T-shirt?).” Start placing the electrodes and sensors on the subject. Carry on with the explanation: “I will first ask you to stay seated for a couple of minutes and then to stand up for a couple of minutes when I tell you. I will then remove the leads and tell you to take a four-minute step test paced by a metronome. Always place your feet completely flat on the step. You can practise first before you sit down (to check the rhythm, your position and that you straighten your knees and hips fully and to get used to switching feet at one-minute intervals, i.e. one minute leading with your left foot, followed by one minute leading with your right foot, etc.). I will tell you when to STAND UP, when I am about to REMOVE THE LEADS, when you need to START THE STEP TEST and when to STOP.” Ask the following questions before you start the test: Yes No 1 Do you have a heart condition? 1 2 2 Do you suffer from joint pain or do you have any 1 2 inflammation or swelling in your joints? 3 Do you get chest pain or feel dizzy when walking? 1 2 4 Do you feel abnormally tired? 1 2 5 Do you have a temperature? 1 2 6 Have you been told to avoid unnecessary stress by 1 2 your doctor? 55

If the subject answers ‘No’ to all the questions, proceed with the step test. If the subject answers ‘Yes’ to one or more questions, consult the test coordinator. Yes No 7 Are you on any medication at the moment? 1 2 “Certain medications can affect your heart rate during physical exercise, and your results may not be completely accurate.” Explain the circumstances in which the test will be abandoned: “If you start to feel any chest pain, nausea or signs of arrhythmia during the test, let me know and we will stop the test.” - Make a note of anything abnormal (such as if the subject is unable to perform the test or refuses) in the examination template. If the subject is unable to keep going for four minutes or struggles to keep pace with the metronome, make a note in the examination template. Remember to rewind the metronome before each test. - Check that the subject has taken off their shoes. - Set the correct height of the step: 33 cm for women (podium on the floor) and 40 cm for men. - Ask the subject to step onto the platform at a rate of 23 times per minute (set the metronome at 46 beats per minute). - Attach the leads and sensors. - Wet the electrodes for the heart rate monitor belt with water and place on the subject’s chest (clean with water). - Fit the heart rate monitor watch onto the subject’s wrist (or place on the desk?). - Attach the ECG electrodes to the subject (placement?). - Fit the nasal sensor onto the subject. - Fit the ear sensor onto the subject. - Fit a finger clip onto the subject’s right index finger (?). - Fit a cuff onto the subject’s left upper arm (?). - Time the stages of the test: 1. 4 minutes for undressing, fitting leads and sensors and explaining the procedure 2. 3 minutes sitting down 3. 3 minutes standing up 4. 2-minute break: removal of leads and sensors and rehearsal of the step test procedure 5. 4-minute step test 6. 2 minutes for recovery 7. 2 minutes for removing the monitors and for the subject to dress 2. Keep the heart rate monitor on for the entire duration of the test. Other leads and sensors should only be attached while the subject is seated or standing up and removed for the duration of the break. - Monitor the subject’s rhythm during the step test, help them to adjust if necessary and tell them when to switch feet at one-minute intervals (the subject should be able to find the right rhythm within a minute).

Nexfin quick guide:

Quick guide for using the software:

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Measurement protocol for the Southern and Northern Finland teams: Instruments: - Polar RX800RC heart rate monitor watch - Polar WearLink heart rate monitor belt (XS/S, M–XXL, XXXL) - Omron M10 blood pressure monitor - Metronome - 40-cm step test box, 7-cm podium for women and non-slip mat Performing the test: 1. Ask the subject about contraindications for the step test: o Do you have a heart condition? o Do you suffer from joint pain or do you have any inflammation or swelling in your joints? o Do you get chest pain when walking? o Do you have a temperature? o Have you been told to avoid unnecessary stress by your doctor? o If the subject answers ‘No’ to all the questions, proceed with the step test. If the subject answers ‘Yes’ to one or more questions, assess whether the test should be abandoned. Musculoskeletal pain is not an absolute contraindication, and if the subject wants to take the test, let them to try for as long as they are able. Recent heart-related chest pain during physical activity, a temperature and rest prescribed by a doctor, however, are always contraindications. Heart rate variability and blood pressure while seated and standing up will nevertheless be measured from all subjects even if they are unable to do the step test. 2. Explain the circumstances in which the test will be abandoned: o “If you start to feel any chest pain, nausea or signs of arrhythmia during the test, let me know and we will stop the test.” o The CRN on duty will also monitor the subject’s condition during the test. 3. Wet the electrodes of the heart rate monitor belt with warm water and fit the belt to the subject’s chest (making sure that both the belt and the text on the transmitter are the right way up), making sure to use the correct size and adjust as necessary. 4. Ask the subject to sit down on a chair. 5. Fit the blood pressure monitor cuff around the subject’s left upper arm. If the cuff cannot be fitted onto the subject’s left arm, fit it onto their right arm instead and make a note of the reason in the examination template. 6. Explain the procedure to the subject: “I will first ask you to sit for three minutes while I measure your blood pressure from your upper arm. I will tell you when the three minutes are up and you can stand up. I will then ask you to stand still for three minutes while I measure your blood pressure. I will take off the blood pressure monitor after you have been standing for three minutes and will ask you to start the four-minute step test.” 7. Measure the subject’s blood pressure from their left upper arm. 8. Press the red button on the heart rate monitor watch once to find the subject’s heart rate signal. Place the watch onto the desk no further than two metres away from the subject (keep checking the watch to make sure that the signal has not been lost). The monitor must always be in front of the subject or off to their side (not behind them) to ensure a good signal. The subject’s heart rate should appear on the monitor. if it does not, check the tightness and moisture levels of the transmitter belt. 9. Press the red button again to start measuring the subject’s heart rate. 57

10. Let the heart rate monitor run for 90 seconds and then take the subject’s blood pressure from their upper arm. Record the subject’s blood pressure in the examination template. 11. Ask the subject to stand up when the timer gets to three minutes. 12. Take the subject’s blood pressure from their upper arm when the timer gets to four minutes and 30 seconds (when the subject has been standing for 90 seconds). Record the subject’s blood pressure in the examination template. 13. Stop the heart rate monitor after six minutes (when the subject has been standing for another 90 seconds) by pressing the bottom left button on the watch twice. 14. Remove the blood pressure monitor cuff from the subject’s arm. 15. Ask the subject to step in front of the step test platform. Place a podium on the floor for women or a non-slip mat for men. 16. Check that the subject has taken off their shoes. 17. Turn on the metronome at 46 beats per minute. 18. Press the red button on the heart rate monitor watch once to find the subject’s heart rate signal. 19. Explain the step test to the subject: “I now want you to start stepping onto the platform in time with the metronome. Switch feet at one-minute intervals, pushing off with your left foot first. Always place your feet completely flat on the step. You can practise stepping onto the platform once.” 20. Press the red button again to start timing the test and measuring the subject’s heart rate when the subject begins the test. 21. Make sure that the subject starts with their left foot and switches feet at one-minute intervals. 22. Check the subject’s maximum heart rate at the end of the four-minute test, record it in the examination template and use it to find the fitness index to communicate to the subject. 23. Monitor the subject’s rhythm during the step test and help them to adjust by demonstrating the correct rhythm yourself if necessary. Make sure that the subject straightens their knees and hips fully when they step onto the box. 24. Cheer the subject on to complete the full four minutes. If the subject is unable to keep going for the full four minutes, record their time in the examination template and add a note explaining why the test was abandoned (e.g. due to pain in the left knee). 25. Ask the subject to sit down on a chair for one minute after the test. 26. Stop the heart rate monitor timer after one minute by pressing the bottom left button twice. 27. Explain the results of the fitness test to the subject. 28. Enter the subject’s step test time in the examination template. Make a note of any issues with timekeeping or loss of signal. 29. Take the heart rate monitor belt off the subject.

Heart rate monitor quick guide: - Make sure that the heart rate monitor is measuring the subject’s RR variability (heart rate variability). Find the buttons on the right side of the monitor and use the red button to select ‘Settings’. Check that ‘RR Data’ under ‘Features’ is selected. Keep pressing the ‘Stop’ button to return to the home screen. Always check the settings after replacing the batteries. (‘GPS’, ‘Shoes’ and ‘Altitude’ should be ‘Off’ or set to ‘None’). - Press the red button once before starting the procedure. The subject’s heart rate 58

should appear on the monitor. if it does not, check the tightness and moisture levels of the transmitter belt. - Once the subject’s heart rate is displayed on the screen, press the red button to start recording. - Stop the recording by pressing the ‘Stop’ button twice. The monitor returns to the clock mode and the measurement is saved. - Troubleshooting: o If there is no signal, check that tightness and moisture levels of the belt. o If this does not help, switch to a different belt (without changing the transmitter unit). o If this does not work either, switch to a different monitor and transmitter unit. o The monitor should give an alert when the batteries need replacing, but dying batteries can also make the screen dimmer when recording begins. o The heart rate monitor uses 2032 batteries and the transmitter 2025 batteries. Remember to check the settings on the heart rate monitor after changing the batteries. - The batteries in both the watch and the transmitter belt should be replaced once a month. - If you are having problems with the watch or the transmitter unit and you decide to switch, always switch both! Migrating heart rate monitor data to a computer: 1. Start the Polar ProTrainer software on the computer 2. Select ‘Settings’ → ‘Change User’ → Team ‘LaptopXX’ (= computer name) → User ‘LaptopXX-1’ → OK 3. Use the buttons on the right side of the watch to select ‘Connect’ 4. Position the heart rate monitor against the infrared reader (with the screen facing the desk and the top of the monitor facing the reader) 5. Select data migration in the top left corner of the Polar ProTrainer window (a monitor icon with a yellow cogwheel) 6. The computer will now connect to the monitor 7. Select ‘Session Files’ in the pop-up window 8. Select all unsaved sessions (files without a blue book symbol next to them). You can select up to 10 session files from the same day at a time. Select the files you want to save (CTRL + left mouse button OR press SHIFT and click on the first file and then the 10th file to select files 1–10) 9. Click ‘Save’ to save the files onto the computer 10. The blue book symbol next to a file means that the data have been saved in the selected Polar ProTrainer profile 11. Select the files you want to remove (CTRL + left mouse button) and click ‘Delete’ 12. Click ‘Close* twice to disconnect the monitor from the computer o If there are more than 10 sessions to save for the day, save the first 10 in one Polar ProTrainer profile (LaptopXX-1) and the rest in a second profile (LaptopXX-2) and, if there are more than 20 files, in a third profile (LaptopXX-3) o Select ‘Settings’ → ‘Change User’ → ‘LaptopXX-2’ (or ‘LaptopXX-3’) o Reconnect to the heart rate monitor and save the rest of the files as explained above o Please note that if you have not deleted data saved in another profile from the monitor, these files will also show up as unsaved! 13. The files are saved in the selected profile under the correct date in the calendar 59

14. Double-click on a date to view the day’s sessions 15. Name each session using the subject’s ID and the name of the test (e.g. ID0001-ortho = stand-up test, ID0001-step = step test). Use the start time of each test as a guide for selecting the correct name. 16. Once you have named the files, click on the disk icon to save them. 17. Remember to check that the date and time are correct on the monitor (standard time / summer time)! Changing the time on the heart rate monitor: 1. Use the buttons on the right side of the heart rate monitor to select ‘Connect’ 2. Position the heart rate monitor against the infrared reader (with the screen facing the desk and the top of the monitor facing the reader) 3. Select data migration in the top left corner of the Polar ProTrainer window (a monitor icon with a yellow cogwheel) 4. The computer will now connect to the monitor 5. Select ‘Clock’ in the pop-up window 6. Synchronise the heart rate monitor with the computer clock by pressing the icon in the bottom right corner (a picture of a monitor with a red arrow pointing at it) 7. Wait for the hourglass cursor to return to an arrow and then select ‘Close’. The time and date will now have been updated.

4.6.2.3.17 Skin prick testing All subjects who attend clinical examinations will undergo skin prick testing. Skin prick testing produces a positive reaction when an allergen introduced to the skin of an allergic person triggers the release of mast cell mediators and causes an itchy weal at the test site within approximately 15 minutes. Interpreting the results requires both a positive control (histamine) and a negative control (the diluent used to preserve the allergen extract). The first shows how the person’s skin reacts to histamine and the second how the skin reacts to just the diluent and a mechanical puncture. The reliability of skin prick testing depends on a range of variables that can be controlled. These include, among others, many drugs, the test solutions used, the distances between test sites, the puncture technique and measurement accuracy. Read the following instructions carefully beforehand and ask if anything is unclear. It is important to follow the instructions exactly and to only perform the test as shown. The results should be carefully recorded in the examination template as instructed. These instructions are designed for research purposes and may not be suitable for clinical practice with patients. Exclusion criteria: o If a subject has an acute rash (e.g. eczema) around the test site that causes their skin to be inflamed or broken, a different test site should be used. o Subjects who have previously had an anaphylactic reaction to skin prick testing should be excluded (ask the subject about previous reactions). o Pregnant women should be excluded. Preparations: 1. Take the solutions out of the refrigerator half an hour before testing is due to begin. If, for some reason, testing needs to begin before the solutions have had half an hour to warm up (e.g. first thing in the morning), use your hands to warm each bottle briefly before use. Only use the latter technique if you absolutely have to. The 60

solutions should, as a rule, be allowed to warm up on their own. Do not allow the solutions to freeze. 2. Check that you have enough solution bottles and that they are correctly numbered and in the right order in the rack. 3. Arrange the test kit and prick test templates (Appendix 34) on a well-lit desk under a spotlight lamp. 4. Cover the desk with a protective cloth. 5. Make sure that you have enough lancets, numbered stickers and wide clear tape, a sharp-tipped pen, a see-through ruler, a plastic cup containing A12t cleaner for disinfecting the pen, cellulose and cleansing skin wipes. 6. The solution bottles keep for six months once opened; mark the date of opening next to the number on each bottle. 7. Remember to put the solutions back into the refrigerator at the end of the day and to regularly check the temperature inside the refrigerator. 8. Each skin prick testing kit contains two EpiPen adrenaline injectors in case of anaphylaxis. If a subject begins to complain about nausea and goes pale or faints, lay them down with their feet up. Once the subject comes to, give them a sugary drink, if possible. Anaphylaxis is a sudden and life-threatening IgE-mediated reaction triggered within one and 15 minutes of exposure. The first symptoms include feeling hot, redness, tingling of the skin and mouth, itching, heart palpitations, globus sensation, heaviness in the chest, blocked nose and nausea. These are followed by swelling, hives, hoarseness, coughing fits, wheezing, abdominal pain, diarrhoea, paleness, drop in blood pressure and ultimately unconsciousness. START TREATMENT IMMEDIATELY! Administer adrenaline by injecting the contents of the EpiPen into the subject’s thigh muscle. Clear the subject’s airway and help them to breathe if necessary. Keep checking their pulse and breathing. A subject who has been administered adrenaline must always be taken to the nearest health centre / hospital for follow-up. Also contact the physician in charge of the study. Familiarise yourself with the medication and the injection technique in advance so that you do not need to refer to the instructions in a real-life emergency. Performing the test: o Place an ID sticker on the form, complete the first section of the template and go through the questions with the subject. Consult the antihistamine list if the subject cannot remember the name of an antihistamine they are taking. Make a note of whether the subject has taken an antihistamine in the previous 48 hours. Antihistamine use is not an obstacle to testing, however. o If possible, use the subject’s left forearm. Check the condition of the skin and mark the test area with numbered tape. Alternatively, you can use the subject’s right forearm or back. Make a note if you are not using the usual test area. o If the subject’s skin is dirty or greasy (e.g. if they have used a lotion, a moisturiser or hand cream earlier in the day), ask the subject to wash their arm with lukewarm water and pat dry with a hand towel without rubbing the skin. This will give the subject’s skin a chance to dry and recover before the test. o Place droplets of solutions 1 to 7 for the first subject of each day and solutions 1 to 6 for the others to the left of the tape by the numbered spots. 61

o Positive control (position 1) closest to the crook of the elbow o Negative control (position 2) o Birch pollen (position 3) o Timothy grass (position 4) o Cat (position 5) o Dust mite (Dermatophagoides pteronyssinus) (position 6) closest to wrist o Repeat of the positive control for the first subject of the day only o Double-testing will be used with the first subject of the day by repeating the positive control with a histamine solution in position 7 on the same (left) forearm after the other solutions. The first subject of the day will therefore have two control weals. The weals are traced and marked in the same template after the last test allergen (No 7). In double-testing, the standard deviation between the results of the two positive controls should be under 20% overall. o Press a lancet perpendicularly against the skin in the middle of each droplet and hold the tip in place for one or two seconds to allow the test solution to react with the cells underneath the skin. o Use a new, sterile lancet for each droplet and be careful not to mix the droplets. Pierce the skin with an ALK lancet perpendicularly (at a 90-degree angle) to the skin through each droplet, hold the lancet in place for approximately two seconds and then remove. The puncture should not bleed but can appear as a small red dot. Lift the lancet straight up off the droplet and place immediately in a designated waste container (e.g. an empty plastic bottle). Make sure to always prick the droplets in the same order and to pierce each droplet. o Use sufficiently thick cellulose wadding to dry the skin and ensure that the droplets are absorbed and do not bleed into each other. Pay attention to the absorption process to make tracing the reactions easier. Enter the time when the test was performed in the template. o Set a timer for 14 minutes. Read the results after 15 minutes by tracing the weals with a sharp-tipped fountain pen (PILOT BP-S FINE or similar). Lighting the skin from the side makes the weals easier to see. Trace the outer edge of each weal (NOT the area of redness) with the pen. This ensures that the inside edge of the pen line runs exactly along the outer edge of the weal. Always draw the circles in the same order as you placed the solutions. The weals should be circled cutting off any pseudopods so that only the outline of the weals, excluding pseudopods, is traced. Once you have finished tracing the weals, place a strip of clear tape on top of the pen marks to lift the marks and then transfer to the template in the columns reserved for each allergen. Make a note of any pseudopods in the designated column (ps) in the template (+) and exclude them from the maximum diameters. Mark the injection sites with a dot. Immediately press a strip of wide, clear tape (Scotch Magic 19 mm x 33 m) firmly over the pen marks to transfer the marks to the tape. Then place the tape in the correct place in the template. Make a note of when the results were read in the template. o Measure the inside diameter of the circles drawn around each weal (i.e. from the edge of the pen line closest to the weal). Measure the maximum diameter of each 62

weal in millimetres and the perpendicular (90-degree) diameter from the centre of the weal at its widest point. Round the result to the nearest full millimetre (e.g. 1.0– 1.4 mm => 1 mm, 1.5–-1.9 mm => 2 mm, etc.). Record a negative result for test sites that show the puncture mark but no weal or a weal of less than 0.5 mm. Negative results are indicated as dots on the tape and should be entered into the template as 0+0. Calculate the subject’s results by adding up the numbers and dividing the sum by two. The calculations can also be performed later or by another CRN. The person performing the calculations should enter their ID at the bottom of the template. o Remove the tape and give the subject a cleansing wipe for wiping off the pen marks and to disinfect the skin and relieve itching. o If the subject’s skin is particularly itchy, apply some Bemetson solution to the affected area and, if necessary, give them 10 mg of antihistamine to be taken orally. o Make a note of any mistakes and other problems with the test at the bottom of the template. Examples of mistakes and problems: o You notice that you have pricked the same droplet twice. This can cause a double reaction and make the result unreliable → start the whole test again on the subject’s other arm. o You are unable to see a puncture mark or the test is unsuccessful in some other way → start the whole test again on the subject’s other arm. o You get a negative result from the positive control (histamine). Possible reasons include the following: o The subject has taken an antihistamine → abandon the test. o Motion sickness medication and antidepressants, internal cortisone therapy and cortisone cream applied to the test site within three days of the test can also cause a negative reaction → abandon the test. o You have failed to follow the protocol (the order of solutions) → start the whole test again on the subject’s other arm. o A solution has lost its potency → this is likely to be the reason if you get a negative result from the positive control on multiple subjects and there appears to be no other reason. o You get a positive result from the negative control and all the other weals are consequently also at least the size of the negative control weal. Possible reasons include the following: o Dermatographism – the subject’s mast cells are triggered into releasing histamine by the mechanical irritation alone (this occurs to some degree with 5–20% of the population) → abandon the test. o The subject reacts to the diluent → abandon the test. o You have failed to follow the protocol (the order of solutions) → start the whole test again on the subject’s other arm. Enter the results of the second test in the template and make a note of the fact that the test had to be restarted and why. Also record the reasons for any abnormal results (see above) if known. 63

4.6.2.3.18 Pressure pain sensitivity, thermal sensitivity and heat pain threshold tests

4.6.2.3.18.1.1 Pressure pain sensitivity tests The pressure pain sensitivity and pressure pain threshold of all subjects who attend clinical examinations will be measured using a Somedic algometer (Somedic AB, Hörby, Sweden). The test will be performed by pressing a 10-mm tip perpendicularly against the skin. The test will begin at 0 kPa with the pressure increasing at 50 kPa/s. The subject will be told to press down a button when the pressure applied becomes uncomfortable and to release the button as soon as it becomes unbearable. The test will in any case end when a light comes on to indicate that the pressure applied has reached 1,200 kPa. The measurement will be taken from the right side of the subject’s body if the subject is not experiencing any pain at the time of the test or on the painful side if they are in pain at the time of the test. Protocol: 1. Explain the test to the subject. Avoid using the word ‘pain’: “I will now measure your sensitivity to pressure”. Start the test by opening the SenseApp software from the desktop. 2. Enter the subject’s details: ‘Subjects’ → ‘New’: Enter the ID number in the activated ‘Enter subject’ field. ‘Name’: Enter the initials of the subject’s first name and surname. ‘Gender’: Enter male/female → SAVE. Follow the same steps to enter the details of all the subjects to be examined during the day. Press ‘Close’ to finish. 3. Select the subject to be examined: ‘Groups’ → ‘Oulu’ → tick the box next to the correct ID and clear any previous selections. Press ‘Save’ and then ‘Close’ to finish. 4. Select ‘AlgoDuoLimit / CohortOulu1966’. 5. Check that the settings are correct (50/10/1,200). 6. Unplug the computer power supply. → ‘Start registration’. 7. This is where you can calibrate the algometer, which should be done once a day. To skip the calibration process, click on ‘Skip all’. Calibration and testing: o Click on ‘Adjust 0’. Hold the sensor steady pointing straight at the ceiling and wait for the reading to reset at 0. Place the gold sensor on top, remove your hand and wait for the reading to stop at 10. o Check the LED lights in the handle by navigating to the ‘Off’ button with the arrows on the screen. o Check the subject’s button by pressing it and making sure that the ‘Button pressed’ light starts flashing. 8. Give the button to the subject and tell them to press it down when the pressure becomes uncomfortable and to release the button when it becomes unbearable and the subject wants to stop the test. 9. Find the correct spot and place a folded cleansing wipe between the subject’s skin and the pressure sensor. 10. Press perpendicularly down on the skin, gradually increasing the amount of pressure until the green light comes on. A blue light indicates that you have to press harder, and the amber and red lights are a sign that you are using too much force. Follow the sensor lights. The software alerts you with a red light (this can be a very brief flash) once you have reached the maximum pressure → end the test. 64

11. Press down on each test site twice. Wait 5 to 10 seconds between measurements. If the subject is not experiencing any pain at the time of the test, perform the test on the right side of their body. However, if the subject is in pain, perform the test on the sore side. Make a note in the examination template if you perform the test on the left side. Press ‘Save’ after each successful measurement. If a measurement fails, press ‘Reset’ to do the test again. Test sites: o SHOULDER: Begin with the subject seated on a chair and the CRN standing on a podium. Find the centre of the subject’s trapezius muscle half-way between the lateral tip of the clavicle and the spine, approximately 4 cm dorsad of the supraclavicular fossa at the tallest point of the shoulder. Press down perpendicularly. Press ‘Save’ to keep the measurement. o WRIST: Ask the subject to lie down on the examination table on their back. Place a hard A4-size base under the subject’s wrist. Make sure that the subject’s wrist rests firmly against the base. Find the wrist joint (on the back of the hand in the middle of the wrist between the heads of the ulna and the radius). Press ‘Save’ to keep the measurement. o LEG: Estimate the midway point between the patella and the ankle. The test site is a couple of centimetres off the tibia on the outer side of the leg muscle half-way between the midway point of the leg and the patella (approximately ¼ of the leg). Pressing down perpendicularly is easier with the subject’s hip (foot) turned inward. This position also helps to keep the leg still. Press ‘Save’ to keep the measurement. o BACK: Ask the subject to turn onto their front. Find the lowest intervertebral disc in the subject’s lower back and press down on the soft tissue in the centre (not the bone). Press ‘Save’ to keep the measurement. 12. Once you have measured all the test sites, press ‘Quit’ (wait for the blue light first to activate). The software reverts to the home screen. 13. Go to ‘Groups’ to select the next subject, then to ‘AlgoLimit’ to select the correct program (CohortOulu1966), click ‘Skip all’ to skip the calibration process, etc. 14. Reconnect the computer power supply to ensure that you have enough battery for the remaining measurements.

4.6.2.3.18.1.2 Thermal sensitivity and heat pain threshold tests Subjects in Oulu, Oulainen and Kemi will also undergo thermal sensitivity and heat/cold pain threshold testing. The tests will be performed using Somedic’s modular sensory analyser (Somedic AB, Hörby, Sweden), which has a metallic thermode surface of 12.5 cm2. Place the thermode on the desk and ask the subject to place the side of their palm (hypothenar eminence) against the thermode with as much of their skin as possible touching the metal plate. Explain the test to the subject: 1. “I am now going to measure the thermal sensitivity of your skin. Place the side of your right palm against the metal plate. Press the button as soon as you can feel a change in the temperature of the metal.” Test 1/4: ‘Prepare seg.’ / ‘Start’. The temperature of the plate drops and returns to +32 °C automatically five times, after which the program asks you to confirm a switch to warm. Click ‘OK’ to repeat an automated heat sensitivity test five times. 2. “I will now measure your left hand. Place the side of your left palm against the metal plate. Press the button as soon as you can feel a change in the temperature of the metal.” 65

Test 2/4: Use the arrow keys to navigate to the second test, which is the same as the first one but for the left palm. Press ‘Start’ to begin the test. 3. “I will now measure your sensitivity to cold from your right palm. The temperature of the plate will drop down to +10 °C and then return back to body temperature. Press the button if the temperature starts to feel unbearably cold.” Use the arrow keys to navigate to ‘Test 3/4’ and press ‘Start’ to begin the test. Take one measurement only. 4. “I would now like you to put your right palm against the metal plate again. The metal will get gradually hotter. Press the button when the temperature becomes unbearably hot. If you do not press the button at any point, the plate will heat up to 50 °C and then cool back down to body temperature. This temperature is not hot enough to damage your skin.” Use the arrow keys to navigate to ‘Test 4/4’ and press ‘Start’ to begin the test. You can set the measurement interval yourself by pressing ‘OK’ whenever you want to start a new test. The interval can vary but should nevertheless always be around 10 seconds. Repeat the measurement three times. 5. Press ‘End sequence’ to stop the test and save the results.

4.6.2.3.19 Carotid ultrasonography and echocardiography Appendix 44

4.6.2.3.20 Eye examinations Enclosed (to be added later).

66

5 DATA STORAGE, TRANSMISSION AND MANAGEMENT

5.1 DATA STORAGE AND TRANSMISSION All the data produced in the course of the study will be saved in the NFBC database. The CRN on duty will enter the results of clinical examinations into the examination template on a laptop, and the template is automatically converted into an Excel spreadsheet. Back-up copies will be made as instructed and saved in the NFBC database managed by the Department of Health Sciences at the end of each day.

Examination/test Data processing Bioimpedance Central blood pressure ECG Polar Active Hookie Supplementary questions Supplementary dental health questionnaire Spirometry PAL Radiography (panoramic Retrospective analysis radiography and knee and foot X-rays) Intraoral scan (iTero) Raw image files sent to Straumann to be compiled into a single 3D image file Spinal Mouse Export to Excel Lumbar motor control Retrospective anonymised impairment (video) analysis by an independent researcher Heart rate variability and baroreflex Pressure pain sensitivity Export to Excel? Carotid ultrasonography and Image files and metrics to be echocardiography saved Visual field OCT Retrospective analysis

The results of blood sample analyses carried out in Oulu University Hospital will be imported manually into the NFBC database once a month in Excel format.

5.2 DATA PROCESSING, CLEANING AND MANAGEMENT The data produced in the course of the study will need to be cleaned before they can be analysed. The subjects’ answers to Webpropol questionnaires will need to be checked. Answers given on paper will need to be entered into the template. 67

5.3 COMMUNICATION OF RESULTS TO SUBJECTS

5.3.1 RESULTS TO BE COMMUNICATED ON THE DAY The following results will be communicated to subjects verbally in connection with their clinical examinations. Subjects will also be told that all results that can be given (= that can be compared against a baseline) will be sent to them by post in the next couple of months. - Blood pressure and pulse - Body fat percentage - Height and weight - Waist and hip measurements - Hand grip strength index - Back strength index - Skin health and potential changes - Memory test results on screen, relative to 50-year-olds - Potential dental and oral changes that require follow-up - Calf tightness - Step test fitness index Any dental or oral changes that require follow-up or treatment will be marked in a special feedback form given to subjects at the end of their dental examination (Appendix X). The cardiologist’s ultrasound findings will be explained to subjects verbally, in addition to which they will be provided a written record of any abnormalities that require follow-up or further investigation.

5.3.2 RESULTS TO BE SENT BY POST In addition to the aforementioned results, the following results will be sent to the subjects by post in the next couple of months. - Upper arm blood pressure and pulse - Height and weight - Waist and hip measurements - Hand grip strength index - Back strength index - Step test fitness index - Spirometry results - Skin prick test results - On a CD: o Panoramic radiography o Knee X-rays o Weight-bearing AP and lateral foot X-rays 68

- Blood test results (CMP, CRP, CREA, ALB, BIL, ALT, ALP, AMYL, AST, CK, GT, CHOL, HDL, LDL, TRIGLY, NA, K) - Bioimpedance analysis - Polar Active data - Visual acuity, intraocular pressure and refractive error, where applicable - Any other abnormalities that require follow-up or treatment identified in the course of the examinations Both an Excel spreadsheet and a written report will be compiled of the results. These will be printed out and sent to the subjects in batches at two-week intervals by post. The letter will be accompanied by a CD containing each subject’s X-rays.

5.3.3 PROCEDURE IN THE EVENT OF ABNORMALITIES

5.3.3.1 BLOOD PRESSURE A subject’s blood pressure will be deemed to be elevated if their diastolic reading is 90 mmHg or higher every time their blood pressure is measured during the morning of their clinical examinations. The subject will be asked whether they are under observation or receiving treatment for high blood pressure. If the subject is already under observation or being treated, they will be told to keep following their doctor’s orders. Otherwise, the subject will be told to book a follow-up appointment with a nurse at their local health centre in the near future. If a subject’s diastolic reading is 100 mmHg or higher, they will be urged to book an appointment with a doctor in the next few days. If the subject is also exhibiting nausea, dizziness, headache or other similar symptoms, they will be told to see a doctor immediately.

5.3.3.2 ECG The following abnormalities detected by ECG will be shared with a doctor: o First-degree or second-degree AV block o Silent atrial fibrillation o Resting heart rate > 120 beats/min (SVT, slow VT) Other potential abnormalities (bundle branch block, QT interval, etc.) will not be communicated to the subject in connection with the examination.

5.3.3.3 BLOOD TESTS Blood test results will be printed out once a week from Oulu University Hospital’s Weblab software and shared with the Field Manager, who can consult Professor Aimo Ruokonen about any abnormalities if necessary. Any abnormalities that require acute (< 1 month) management, treatment or follow-up will be reported to the subject in question by telephone, urging them to contact their local health centre / occupational health care provider.

5.3.3.4 SKIN HEALTH If a dermatologist identifies skin changes on a subject that require treatment, further investigation or follow-up, they will tell the subject to contact their local health centre or occupational health care 69 provider and give the subject a brief note to take with them. If necessary, the dermatologist can also refer the subject directly to the dermatology clinic of Oulu University Hospital.

5.3.3.5 RADIOGRAPHY A radiologist will perform a retrospective analysis of the subjects’ X-rays. Any abnormalities that require acute management or treatment will be communicated to the subject in question, urging them to book a follow-up appointment.

5.3.3.6 ORAL AND DENTAL HEALTH Any dental or oral changes that require follow-up or treatment will be marked in a special feedback form given to subjects at the end of their dental examination (Appendix X). Subjects will be told verbally about any abnormalities that require acute treatment or management and urged to book an appointment.

5.3.3.7 HEART AND JUGULAR VEIN ULTRASOUND The cardiologist’s ultrasound findings will be explained to subjects verbally, in addition to which they will be provided a written record of any abnormalities that require follow-up or further investigation. Subjects will be told verbally about any abnormalities that require acute treatment or management and urged to book an appointment.

5.3.3.8 EYE EXAMINATIONS Any abnormalities detected in the course of eye examinations that clearly require treatment will be communicated to the subject in question either on the day or afterwards by telephone.

5.4 EXCLUSION CRITERIA FOR CLINICAL EXAMINATIONS All subjects who wish to attend clinical examinations will be able to do so. The exclusion criteria for each examination are listed in the relevant protocol, in addition to which the CRN on duty will exercise judgement in borderline cases.

5.5 FIRST AID All CRNs involved in the study will have practised basic CPR with a CPR doll. Examination stations set up in health centres or hospitals will also have access to emergency staff in acute situations. In the event of an emergency at an examination station not located in a health centre or a hospital, staff will call the national emergency number (112) and proceed with basic first aid. Basic CPR will be administered if necessary. Staff will not have access to emergency medication or other resuscitation equipment. Staff will aim to prevent emergencies by following the exclusion criteria and continuously monitoring the subjects. The skin prick testing protocol includes instructions for dealing with anaphylaxis.

5.6 COMMUNICATION WITH THE MEDIA The media have been told about the study at BusinessOulu’s press conference in November 2011 and in a press release issued by the University of Oulu towards the end of March 2012. Long-term communication plans for the NFBC project are being drawn up at the moment.