T Arbuckle, F McNeill, M Inskip , D Chettle, WI Manton, M Fisher, N Healey, R Bray, L. Marro

1. All ages study of blood-lead and bone-lead in urban Toronto: lessons learned from a collaborative study T Arbuckle, F McNeill, M Inskip, D Chettle, WI Manton, M Fisher, N Healey, R Bray, L. Marro Main funding: Health Canada, [Chemicals Surveillance Bureau, Environmental and Radiation Health Sciences Directorate]

2. Co-investigators and groups Health Canada Tye Arbuckle (Co -PI) Mandy Fisher Mike Inskip (now with McMaster University) Norm Healey (now with Azimuth Consulting Group) Leonora Marro Department of Health Physics, McMaster Univ David Chettle Fiona McNeill (Co-PI) SepidehBehinaein (Ph.D student) University of Texas at Dallas William Manton Hospital coordinating centre– St Joseph’s Hospital, Toronto Jessica Tyrwhitt Toronto Public Health Riina Bray

3. Talk outline • Goals, study design and context • Techniques used • Results so far ….. • Lessons learned • Discussion / summary

4. Study goals The first Canadian study that we are aware of which attempts to measure bone Pb in young children. The pilot study goals were to demonstrate: • the feasibility of including in vivo XRF measurements of Pb in bone for young children in future surveys [like the ‘Canadian Health Measures Survey’] • the feasibility of 1) remote bone Pb measurements; • 2) partnership with a local community health centre for recruitment and subject measurement; • 3) quantification of serum Pb concentrations in environmentally exposed subjects • 4) recruitment of childhood subjects for venous blood sampling; • 5) development and delivery of appropriate risk communication materials.

5. Testing and recruitment successTOTAL: 263/272 Participants Age ExpectedTested Males FemalesMales Females 1 to 5 10 10 10 6 6 to 10 23 20 23 16 11 to 19 23 20 23 20 20 to 35 21 25 21 25 36 to 50 25 29 25 29 51 to 64 21 24 21 24 65 to 85 10 11 9 11 Total 133 139132 131 • 1 participant cancelled; 2 participants did not assent to participate in the study; • 5 appointments were cancelled - equipment malfunction (participants rescheduled)

6. Methods / techniques for success:plans and reality #1 • Very effective (and persistent) hospital coordinator for reaching potential volunteers / scheduling visits • Patient-friendly & very skilled child phlebotomist and measuring staff and well-designed procedures for family groups being measured + questionnaire time allowed. • Financial ($50) incentive • YES • YES – but early-on suspected a brief contamination event; Used TV games and books & adult distractions to keep children engaged • YES – but poorer participation rate by younggirls vs the boys

7. Methods / techniques for success:plans and reality # 2 • Training for obtaining serum samples (Class-100 clean room, gloves etc., centrifuge on site) and pre-cleaned sample tubes, sample dispatch processes. • Choice of experienced analytical lab for Pb • Communications with volunteers about health risk by Toronto Public Health personnel very familiar with PbB exposure routes in homes. • YES – but Couriers lost one batch of samples • YES: …although challenges. • Low blood lead values did not necessitate many home visits.

8. 1st blood sample - venipuncture for:whole blood and hematocrit Bluetop Vacutainer tube Place a drop of blood sample onto slide and draw into a capillary tube.  The capillary tube is then spun to separate the red blood cells from the plasma at which time a Hematocrit value can then be measured. 

9. 2nd sample : Venipuncture for:blood serum sample to measure a person’s Hematocrit is by • Use butterfly +syringe to slowly collect • 5ml blood. Remove butterfly tip, leave sample stand 20 minutes. • NB The following are done in CLEAN BOX • Slowly transfer blood to centrifuge tube* • Spin down using bench centrifuge • Decant plasma (Special tip*) into Teflon jar* • Record patient I.D. against jar number • Tighten lid & freeze sample • * provided

10. Why is use of a HEPA Clean Box (Class-100 air) important? • Differences: at least 100 times (!) less Pb is present in Plasma vs. whole blood • E.g., If PbB = 1 ug/dL, plasma Pb = 0.01 ug/dL or 100 pg/ml • Where things can get contaminated • Pb on airborne particles • Pb on hands or any surfaces. • One speck of dust enough to contaminate sample • Pb can also leach-out from plastics

11. Blood collection protocol • Blood volume: the phlebotomist must do their best to obtain as much as possible - up to the desired amount of 5mL. • For the serum sample, the tube is left to stand for 20 minutes • After centrifuging, a dedicated pipeter is used to transfer plasma; when not in use, it is kept in the clean box as the photo shows (hanging). • The rack of pre-cleaned 15 mL teflon jars and the centrifuge tubes must never leave the clean box. • The transfer of the supernatant plasma to the tubes must be done in the box.

12. Treatment of sample when taken

13. Benchtop centrifuges for plasma / hematocrit

14. Results: Blood lead vs age (*male / *female)

15. Systematic representation of vascular organisation in the diaphysis of a long-bone (From Mayo Foundation for Medical Education and Research)

16. Summary • Blood lead levels were lower than expected …but in the range found for the CHMS survey; Lead levels in serum required special attention for analysis • Serum lead levels were so low that the Texas laboratory had to develop a new technique to measure them. • Special attention to training of collection staff was a good investment (and critically important for obtaining reliable data). • Bone lead data were successfully obtained for young children by in vivo XRF; Themethod is being further evaluated as a tool for measuring children’s skeletal burden. • A multi-disciplinary team approach was considered essential for this study.

17. Summary (2) • Analysis of data continues: e.g.: • Bone/PbB/serum data • Questionnaire results related to values • Additional information on isotope ratios in blood samples & possible source of environmental Pb