Presentation at CDC conference 2009 of successful clinical trials Emory University Hospital & Georgia Department of Human Services Children Lead Poison Prevention.

1. Accuracy of Oral Fluid Lead Testing Lynn Gardner, MD, Emory University Atlanta Georgia; Robert Geller, MD, Emory University Atlanta Georgia; Robyn Hannigan, Ph.D. University of Massachusetts; Yu Sun, MD, MPH Ga. Department of Community Health; Anil Mangla, M.S.,Ph.D., MPH. FRIPH, Ga. Department of Community Health

2. Disclaimer Drs. Gardner, Geller, and Mangla are independent evaluators of this lab method and have no conflicts of interest with this lab methodology or with any vendor of testing services.

5. Behavioral abnormalities

7. Difficulty in obtaining blood samples in children

9. Limitations The prevalence of lead poisoning in the study cohort was less than the national average of 1.5% Due to the small number of elevated blood lead levels, the performance characteristics of this test at higher blood lead levels was not established.

10. This study compares oral fluid lead levels and blood lead levels in a clinical setting.

11. Study Site

12. Methods Oral fluid samples collected on 500 children aged 6 months old to 5 years old Children having venous blood lead levels drawn for routine screening purposes were eligible.

13. Methods The blood lead levels were measured using standard methodology at a CLIA certified laboratory. The oral fluid lead levels were measured using an ICP-MS (DRC II, PerkinElmer Sciex). Oral fluid samples from 50 children were gathered twice to provide internal controls, but were only counted once.

14. Methods The mean absolute difference between the sample groups was determined to test the hypothesis that group means are equal.

15. Results Five hundred patients agreed to enroll. No eligible patient declined to participate. 474 patients had both blood and oral fluid samples available for analysis. 26 did not have blood available.

17. # with blood lead level <4mcg/dL = 455

18. % correlation = 92%

19. # with oral fluid lead level ≥4mcg/dL = 19

20. # with blood lead level ≥4mcg/dL = 19

22. Data Results 9/474 patients were identified as false positives (1.9%) 0/474 patients were missed as false negatives (0%)

23. Determining Cut-Off values A sensitive test is positive when a disease is present A specific test is usually negative when a patient is disease free Higher sensitivity of a test is associated with fewer false negatives To establish a cut-off value, determine the test characteristics to assess highest sensitivity with highest specificity

24. Determining Cut-Off values

25. Conclusions Oral fluid may be a reliable medium to use when screening children for lead exposure for levels < 4mcg/dL. Oral fluid lead levels ≥ 4mcg/dL should be confirmed by a venous blood sample. The sample size in the ≥ 4mcg/dL group (n=19) was inadequate to draw conclusions on accuracy at higher lead levels. Further studies are being conducted.

27. Eliminating inability to obtain an adequate sample.

28. Decreasing sample collection times.

30. Preliminary Laboratory Validation

32. Samples are weighed (triplicate masses taken)

33. Samples are acid digested (ultrapure HNO3) by microwave digestion

34. Samples, once digested, are brought to volume and spiked with internal standard (Indium, final concentration 20 ppb)

35. Samples are analyzed by ICP-MS (PerkinElmer DRCII)

36. Follow EPA 6020 with modification

37. Pb isotopes monitored (204, 206, 207, 208)

38. Concentrations based on external calibration (208Pb) and internal standardization (115In)

39. Calibration every 10 samples

40. Required R2 > 0.999 to proceed

41. Check standards run every 5 samples

43. Analytical concentrations corrected for preparation dilution and blank concentration

44. “Oral Fluid” concentration reported as mg/dL

45. Method Detection Limit performed for each analytical run

46. Within and Between-Day variation assessed using spiked swab samples

48. Inter-Run Precision (CV) and Accuracy

49. Oral Fluid Lead Proficiency Correlation 27 pairs of samples were analyzed by an ICP-MS for saliva and blood lead for each individual patient.

50. Method Detection Limit Performed at the start ofeach daily analytical run 7 individual standards comprised of a “spiked” swabcontaining 0.30 ug/dL Pb Standard Deviation of 7measurements * Student-t for n=6 at 99% confidence 15 MDL determinationsMDL = 0.006 +/- 0.002 ug/dL Pb(0.06 ng Pb per mL fluid) Calibration data using external calibration standards (n=6) and blank, Internal standard (20 ppb In), instrument configuration (EPA 6020, DRC II ICP-MS, no cell gas).

51. METHODS Standard Operating Procedure – EPA 6020 for ICP-MS with the following modifications Indium as single internal standard, 6 calibration standards (0.1 to 10 ug/dL Pb), 2 QA/QC standards (0.2, 7.0 ug/dL), run allowed to proceed if R2 > 0.999, QA/QC stds within 95% of known. Concentration of standards (2% ultrapure HNO3) entered in software as ug/dL to allow for direct reporting of results in appropriate units, blank subtracted prior to quantification, all Pb counts per second normalized to In counts per second prior to quantification by the software. Matrix suppression study revealed logarithmic response of detector to higher matrix concentrations, correction factor applied for dilutions during sample preparation and matrix effect (total dissolved solids) = 1.639log(counts per second Pb/In) Digestion blanks also analyzed every 10 samples (see outline of methods) = below method detection limit All data processing, calibrations, and QA/QC handled by PerkinElmer software (EPA 6020 methods adapted for use in this project)