Quantification of inflammatory cytokines in dried blood spot (DBS) samples

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1 Quantification of inflammatory cytokines in dried blood spot (DBS) samples Thom McDade, PhD Department of Anthropology Laboratory for Human Biology Research Cells to Society (C2S): The Center on Social Disparities and Health, Institute for Policy Research

2 Why inflammatory cytokines? Pathophysiology of diseases of aging Sensitive to social and developmental contexts Beyond CRP: upstream regulation of inflammation Pro- and anti-inflammatory inputs Cytokines cross the blood-brain brain barrier

3 Measuring inflammation in DBS CRP Low cost, robust assay 1 Widespread application IL-6 Low concentrations in unstimulated samples is a major challenge Recently validated ELISA 2 1 McDade et al. (2004) Clin Chem 50: Miller and McDade (2012) Am J Hum Bio 24:863-5.

4 Highly sensitive IL-6 ELISA for DBS Platform: R&D Systems Quantikine HS IL-6 ELISA Calibrators: DBS material manufactured in lab with serum calibrators provided in kit Sample quantity: 4 x 3.2 mm discs per well 8 discs for duplicate measures Samples and calibrators eluted in filter plates to Samples and calibrators eluted in filter plates to maximize recovery

5 Highly sensitive IL-6 ELISA for DBS Lower limit of detection: 0.67 pg/ml Linearity of dilution: , mean = 1.05 Variability high med low Intra-assay (%CV) Inter-assay (%CV)

6 Highly sensitive IL-6 ELISA for DBS N=46

7 Highly sensitive IL-6 ELISA for DBS Advantages Tool for measuring IL-6 at very low concentrations Relies on gold-standard R&D protocol Can be implemented with standard EIA equipment Disadvantages Requires 1-2drops of whole blood d(for duplicates) Limited assay range (<10 pg/ml) Expensive, particularly in comparison with CRP

8 Advantages of multiplexing Assay multiple inflammatory cytokines at once Pro- and anti-inflammatory signaling Patterns of cytokine response (e.g., Th1 vs. Th2) Conserves sample Analyze up to 10 biomarkers in single volume of sample Lower cost per biomarker (but higher overall cost)

9 Multiplex analysis of inflammatory cytokines in DBS: Luminex platform Sample: 4 x 3.2mm discs LLD: 1-5 pg/ml Reliability: Not great

10 Multiplex analysis of inflammatory cytokines in DBS: MesoScale Discovery Electrochemiluminescent immunoassay Microtiter plate-based (96-well) Quantify up to 10 biomarkers

11 Multiplex assay of cytokines in DBS Platform: MSD Pro-Inflammatory 7-plex, Ultrasensitive Calibrators: DBS material manufactured in lab with serum calibrators provided in kit Sample quantity: 2 x 3/16 in discs per well 4 discs for duplicate measures (1 large drop) Samples and calibrators eluted in filter plates to Samples and calibrators eluted in filter plates to maximize recovery

12 Multiplex assay of cytokines in DBS

13 Multiplex assay of cytokines in DBS

14 Multiplex assay of cytokines in DBS IL6 IL10 TNFα Lower limits of detection (pg/ml) Linearity of dilution (mean) Intra-assay variability (%CV) Inter-assay variability (%CV) -- tbd --

15 Multiplex assay of cytokines in DBS IL6 N= Scatter Plot with Passing & Bablok Fit Plasma IL6 (pg/ml) Plasma =1.80 x DBS 0.86 R = DBS IL6 (pg/ml)

16 Multiplex assay of cytokines in DBS IL6 N= Scatter Plot with Passing & Bablok Fit Plasma IL6 (pg/ml) Plasma =1.84 x DBS 0.88 R = DBS IL6 (pg/ml)

17 Multiplex assay of cytokines in DBS IL6 N= Scatter Plot with Passing & Bablok Fit R&D plasm ma (pg/ml) Plasma =1.95 x DBS 0.88 R = DBS IL6 (pg/ml)

18 Multiplex assay of cytokines in DBS IL10 N= Scatter Plot with Passing & Bablok Fit Plasma IL1 10 (pg/ml) Plasma = 4.94 x DBS 2.13 R = DBS IL10 (pg/ml)

19 Multiplex assay of cytokines in DBS Advantages Tool for measuring several cytokines at very low concentrations Wide dynamic range: <0.5 pg/ml to 5,000 pg/ml Reduced cost per biomarker/per sample Disadvantages Requires 1-2 drops of whole blood (for duplicates) Big up-front instrumentation costs

20 Acknowledgements Funding NIA Network on Biological Risk (R24AG037898) USC/UCLA Center on Biodemography and Population Health (P30AG017265) National Science Foundation, Physical Anthropology (BCS ) Colleagues in the Laboratory for Human Biology Research Elizabeth Miller Bill Funk Adam Leigh

21 American Journal of Human Biology Impact factor: 2.3 Indexed: Everywhere Time to decision: 27 days Toolkit: Methods in Human Biology Laboratory and field methods for measuring human energy expenditure Field and laboratory methods in human milk research Essential anthropometric methods