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Thermo Fisher Scientific and Fluid Management Systems, Inc. Food Safety and POPs Seminar

Use of Sample Pooling as a Strategy to Reduce Analytical Costs and Improve Detection Frequency of PCDDs/PCDFs/cPCBs and other POPs in National Biomonitoring Surveys. Wayman E. Turner, Samuel P. Caudill, Troy P. Cash, Wanda E. Whitfield , Emily S. DiPietro, P. Cheryl McClure, and Andreas Sjodin.

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Thermo Fisher Scientific and Fluid Management Systems, Inc. Food Safety and POPs Seminar

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  1. Use of Sample Pooling as a Strategy to Reduce Analytical Costs and Improve Detection Frequency of PCDDs/PCDFs/cPCBs and other POPs in National Biomonitoring Surveys Wayman E. Turner, Samuel P. Caudill, Troy P. Cash, Wanda E. Whitfield , Emily S. DiPietro, P. Cheryl McClure, and Andreas Sjodin Thermo Fisher Scientific and Fluid Management Systems, Inc. Food Safety and POPs Seminar Sunday August 26, 2012 Cairns, Queensland, Australia National Center for Environmental Health Division of Laboratory Science

  2. Median serum TCDD levels in selected populations Ground troop veterans in AOVS Controls Low Medium High Ranch Hand veterans Controls Officers - non-flying Officers - flying (navigators) Officers - flying (pilots) Enlisted men - flying Enlisted men - non-flying Occupational exposures NIOSH workers - quintile 1 quintile 2 quintile 3 quintile 4 157 quintile 5 355 German plant workers New Zealand sprayers Seveso, Italy population 4540 Without chloracne 16,600 With chloracne 0 20 40 60 80 100 1000 10,000 Serum dioxin pg/g lipid (ppt)

  3. Objectives Determine whether military records can be used to identify US Army veterans likely exposed and not exposed to agent orange (dioxin) If so, determine how well these levels correlate with the exposure indices. Participants 646 Vietnam of III Corps (X = 300 Days, 1966-69) 97 Comparisons Agent Orange Validation Study

  4. In the Agent Orange Validation Study, Vietnam ground troop veterans and non-Vietnam veterans had similar TCDD levels Serum TCDD distribution in these Vietnam Veterans same as in comparison group. No association between current serum dioxin levels and exposure indices.

  5. POPs levels in human milk,Sweden 1972-2007 POPs in Human Milk Fact Sheet 4.3 ENHIS (Dec, 2009)

  6. Time tends for TCDD levels “Birth Cohort Effect” - observed level related to birth year. Source: DioxinFacts.org

  7. Estimated Average TEQ - Time Trends Chlorine Chemistry Division of the American Chemistry Council modeled the future body levels (from 2002 to 2020) starting with the mid-point measurement for the average dioxin ppt-TEQ for each reported age group (20 years and older) reported in the Third National Exposure Report (2005). Chlorine Chemistry Division of the American Chemistry Council based projections for the 15 year old age group on the 2001-2002 pooled blood samples (Needham, 2005). Source: DioxinFacts.org

  8. New Zealand StudyOur first encounter with sample pooling Collection of serum samples was carried out in conjunction with the National Nutrition Survey (NNS) which was itself linked to the New Zealand Health Survey (NZHS) (Ministry of Health, 1999). The target population of the NZHS was all New Zealanders aged 15 or older. To increase the number of Maori (the indigenous people of New Zealand) and Pacific Island people in the NZHS, additional households were selected from areas in which there was a high proportion of Maori or Pacific Island people. Bates et al. Chemosphere 58 (2005) 943-951

  9. New Zealand - Pooling Samples • In the late 1990s our laboratory was contracted to analyze the samples for PCDDs/PCDFs/cPCBs, PCBs and OC pesticides, but because of the extreme expense associated with the measurements and the need for large sample volumes to detect very low concentrations, the researchers asked if it might be possible to pool samples. • Pooling samples before making analytical measurements can reduce costs by reducing the number of analyses and increase the number of detectable results (lower LODs).

  10. New Zealand - Pooling Samples • We subsequently prepared 60 25-50 g pools from 80 possible strata (5 age, 2 ethnicity, sex and 4 regions) to characterize PCDDs/PCDFs/cPCBs, PCBs, and organochlorine pesticides in the New Zealand samples. Individual samples available were ~2 mL. • This was the first study to obtain representative measures of these compounds in the adult population of an entire country.

  11. New Zealand Study Bates et al. Chemosphere 54 (2004) 1431-1443

  12. NHANES National Health and Nutrition Examination Survey • The NHANES sampling plan is designed to select a representative sample of the civilian, non-institutionalized population in the United States based on age, gender, and race/ethnicity. • The sampling plan follows a complex, stratified, multistage, probability-cluster design. • NHANES uses oversampling of certain population subgroups to increase the reliability and precision of health status indicator estimates for these groups. • So NHANES data are not obtained using a simple random sample.

  13. NHANES Stratified, multistage, probability-cluster design.

  14. Pooling Individual Samples • If the distribution of a population is Gaussian (Normal), the distribution of the averages of samples of size N will also be normally distributed with identical mean but with variance reduced proportional to the size of N. If X ~ N(µ, σ) then ΣXi/N ~ N(µ, σ/√N)

  15. Distribution of Averages of Samples of Size NGreen: Individual Samples Blue/Red: Averages N=5/25

  16. Distribution of Averages of Samples of Size NGreen: Individual Samples Blue/Red: Averages N=5/25

  17. Pooling Samples • Biological averaging assumption (BAA) from microarray and proteomics literature: The measured value of a pooled sample is comparable to an arithmetic average of the levels in the individual samples making up the pool.

  18. Pooling Samples • So for log-normal data the biological averaging assumption suggests that pooled-sample measurements will behave like arithmetic averages. • Based on the Central Limit Theorem, as N increases, the distribution of the pooled-sample measurements from a log-normal distribution will be normally distributed. • And the mean of a group of pooled-samples will be larger than the geometric mean of the individual samples from the original distribution.

  19. Disadvantages Associated with Pooling • Individual level information is lost - possibly limiting analyses to descriptive and ecologic studies. • Association studies will be difficult, if not impossible. • May not be possible to include all minority demographic groups. • May be difficult, if not impossible, to incorporate sampling weights, which could lead to biased estimates.

  20. Disadvantages Associated with Pooling • Maximum and minimum values not known.

  21. Disadvantages Associated with Pooling • Individuals in a cluster (i.e., county, school, city, census block) are more correlated to one another than to those in other clusters. • Because NHANES uses cluster sampling, the data cannot be treated as a simple random sample where all values are statistically independent.

  22. Disadvantages Associated with Pooling • The impact of the complex sample design on variance estimates is measured by the design effect (DEFF). • DEFF = Var(θ)cluster / Var(θ)simple random sample • With pooled samples, estimating DEFF may be impossible or difficult leading to biased standard error estimates, which in turn will result in biased confidence limits and incorrect significance levels.

  23. Is it time to get out of the pool(ing)?

  24. “Any data is better than no data!”Larry Needham

  25. NHANES 2001-2002 Pools

  26. 34 People per pool (Total 1,734 people; 51 pools) Unweighted: used for estimates of the “means” 0.75mL Serum per person 25.5 g Serum per pool 2 g BFRs/PCBs/Persistent Pesticides 22 g PCDDs/PCDFs/cPCBs; PCNs 0.5 g Total Lipids 0.4 g PFCs Serum Pools: NHANES 2001-2002

  27. Geometric mean TEQ NHANES 2001-2002 Pools Patterson et al. Chemosphere 73 (2008) S262-S277 Caudill et al. Chemosphere 69 (2007) 371-380 (Geometric mean estimation from pooled samples)

  28. Simulation Study using PooledSamples for BiomonitoringFurther refinements in pooling strategy • Considered 15 pooled-sample designs. Samples per pool (minimum, maximum # pools) 2(18,72) 3(12,48) 4( 9,36) 5( 7,29) 6( 6,24) 7( 5,20) 8( 4,18) 9( 4,16) 10( 3,14) 11( 3,13) 12( 3,12) 13( 2,11) 14( 2,10) 16( 2, 9) 18( 2, 8)

  29. Simulation Study using PooledSamples for Biomonitoring Solid line closed circles: median percent bias in estimates of geometric mean & Cis. Dashed line open circles: median percent bias in estimates of 95th percentile & CIs.

  30. Black Curve: Distribution of Individual SamplesBlueCurve: Distribution of Means of 8 Samples

  31. Pooled-Sample Design for Dioxins and PCBsUsing NHANES 2005-2006 Samples • Theoretically, sample weighting can also be accomplished with pooled-samples by using sample volumes proportional to the sampling weights. Vij = Wij/Wi. where Vij is the volume of the ith sample in the jth pool, Wij is the sampling weight of the ith sample in the jth pool, and Wi. is the sum of the weights of all samples in the jth pool.

  32. Pooled-Sample Design for Dioxins and PCBsUsing NHANES 2005-2006 Samples • DLS statistician assigns individual samples to pools in groups of 8 samples per pool. • Merge NCHS sub-sampling weight files and demographic information files. • Select only samples from subjects in major race/ethnicity categories: NHW NHB and MA. Group samples into 4 age groups: (12-19; 20-39; 40-59; 60+). • Sort data by demographic group and by subsample weight within each demographic group.

  33. Pooled-Sample Design for Dioxins and PCBsUsing NHANES 2005-2006 Samples • Assign 8 samples to each pool to create several 8-sample pool sets in each demographic group. • Create a variable called "factor" which is equal to the subsample weight of each sample in a pool divided by the sum of subsample weights of all samples in the same pool. • The desired volume of each sample is equal to 40*factor.

  34. Pooled-Sample Design for Dioxins and PCBsUsing NHANES 2005-2006 Samples8 Samples per Pool

  35. Pooling NHANES 2005-2006 SamplesWas it worth all the trouble? • The samples in the NHANES 2005-2006 survey were pooled with 8 samples per pool and analyzed for 74 PCDDs/PCDFs/cPCBs, PCBs PCNs and OC pesticides. The number of samples to be analyzed was reduced from 2041 to only 247 at a savings of ~$2.8 million. • Pooling samples also resulted in lower limits of detection (LODs) because of the larger sample volumes are available for analysis.

  36. PCDD/PCDF/cPCBs: Unadjusted Geometric Means and Selected Percentiles (95% Confidence Intervals) of Serum Concentrations (pg/g lipid) by Age, Sex, Race for the NHANES 2003-2004

  37. PCDD/PCDF/cPCBs: Unadjusted Geometric Means and Selected Percentiles (95% Confidence Intervals) of Serum Concentrations (pg/g lipid) by Age, Sex, Race for the NHANES 2003-2004

  38. Comparison of 50th Percentile for 12378D from NHANES 2003-2004 and 2005-2006

  39. Comparison of 50th Percentile for 2378D from NHANES 2003-2004 and 2005-2006

  40. NHANES 2005-2006 Pools50th, 75th, 90th & 95th Percentiles 12378Dby Age, Race & Sex

  41. NHANES 2005-2006 Pools50th, 75th, 90th & 95th Percentiles 2378Dby Age, Race & Sex

  42. Mean & Range TEQ NHANES 2005-2006 Pools by Race and Age Group

  43. Recent References on Pooling • Caudill S.P. Use of pooled samples from the national health and nutritional examination survey. Statistics in Medicine (2012) DOI: 10.1002/sim.5341. • Caudill S.P. Important issues related to using pooled samples for environmental chemical biomonitoring. Statistics in Medicine 30 (2010) 515-521. • Caudill S.P. Characterizing populations of individuals using pooled samples. Journal of Exposure Science and Environmental Epidemiology 20 (2010) 29-37. • Caudill S.P., Turner W.E., Patterson Jr. D.G. Geometric mean estimation from pooled samples. Chemosphere 69 (2007) 371-380.

  44. What is the most frequently asked question? • How much blooddo you want?

  45. The standard answer… “It depends.”

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