Qtifiti fifl t tki iQuantification of inflammatory ...
Transcript of Qtifiti fifl t tki iQuantification of inflammatory ...
Q tifi ti f i fl t t ki iQuantification of inflammatory cytokines in dried blood spot (DBS) samples
Thom McDade, PhDDepartment of AnthropologyDepartment of Anthropology
Laboratory for Human Biology ResearchCells to Society (C2S):
The Center on Social Disparities and HealthThe Center on Social Disparities and Health,Institute for Policy Research
Why inflammatory cytokines?
Pathophysiology of Pathophysiology of diseases of aging
Sensitive to social and Sensitive to social and developmental contexts
Beyond CRP: upstream regulation of inflammationy p g
Pro- and anti-inflammatory inputs
Cytokines cross the blood-brain barrier Cytokines cross the blood brain barrier
Measuring inflammation in DBS
CRPL t b t 1• Low cost, robust assay1
• Widespread application
IL-6• Low concentrations in unstimulated• Low concentrations in unstimulated
samples is a major challenge• Recently validated ELISA2
1McDade et al (2004) Clin Chem 50:652 41McDade et al. (2004) Clin Chem 50:652-4.2Miller and McDade (2012) Am J Hum Bio 24:863-5.
Highly sensitive IL-6 ELISA for DBS
Protocol highlights Assay performance Adv/disadv
Platform: R&D Systems Quantikine HS IL-6 ELISA
Calibrators: DBS material manufactured in lab with Calibrators: DBS material manufactured in lab with serum calibrators provided in kit
Sample quantity: 4 x 3.2 mm discs per wellSample 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
Highly sensitive IL-6 ELISA for DBS
Protocol highlights Assay performance Adv/disadv
Lower limit of detection: 0.67 pg/mL
Linearity of dilution: 0 80 1 25 mean = 1 05 Linearity of dilution: 0.80-1.25, mean = 1.05
Variability high med low Variability high med low
Intra-assay (%CV) 6.3 8.9 14.6
Inter assay (%CV) 9 4 9 5 15 4Inter-assay (%CV) 9.4 9.5 15.4
Highly sensitive IL-6 ELISA for DBS
Protocol highlights Assay performance Adv/disadv
N=46
Highly sensitive IL-6 ELISA for DBS
Protocol highlights Assay performance Adv/disadv
Advantages
Tool for measuring IL 6 at very low concentrations Tool for measuring IL-6 at very low concentrations
Relies on gold-standard R&D protocol
Can be implemented with standard EIA equipment Can be implemented with standard EIA equipment
Disadvantages
R i 1 2 d f h l bl d (f d li t ) Requires 1-2 drops of whole blood (for duplicates)
Limited assay range (<10 pg/mL)
Expensive, particularly in comparison with CRP
Advantages of multiplexingg p g
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 Analyze up to 10 biomarkers in single volume of sample
Lower cost per biomarker (but higher overall cost)
Multiplex analysis of inflammatory cytokines in DBS: Luminex platformcytokines in DBS: Luminex platform
Sample: 4 x 3.2mm discs
LLD 1 5 / LLLD: 1-5 pg/mL
Reliability: Not great
Multiplex analysis of inflammatory cytokines in DBS: MesoScale Discoverycytokines in DBS: MesoScale Discovery
Electrochemiluminescent immunoassayimmunoassay
Microtiter plate-based (96-well)( )
Quantify up to 10 biomarkers
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
Platform: MSD Pro-Inflammatory 7-plex, Ultrasensitive
Calibrators: DBS material manufactured in lab with Calibrators: DBS material manufactured in lab with serum calibrators provided in kit
Sample quantity: 2 x 3/16 in discs per wellSample 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
Multiplex assay of cytokines in DBS
Multiplex assay of cytokines in DBS
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
Multiplex assay of cytokines in DBS
IL6 IL10 TNFα
Lower limits of detection (pg/mL) 0.15 0.36 0.70(pg )
Linearity of dilution (mean) 1.03 1.03 1.04
Intra-assay variability (%CV) 5.6 4.0 7.5
Inter-assay variability (%CV) -- tbd --
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
IL6
N=72 25
30Scatter Plot with Passing & Bablok Fit
N=72
15
20
6 (p
g/m
L)
10
15
Plas
ma
IL6
Plasma =1.80 x DBS – 0.86
0
5
0 5 10 15 20DBS IL6 (pg/mL)
R = 0.98
DBS IL6 (pg/mL)
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
IL6
N=704
4.5Scatter Plot with Passing & Bablok Fit
N=70
2.5
3
3.5
6 (p
g/m
L)
1
1.5
2
Plas
ma
IL6
Plasma =1.84 x DBS – 0.88
0
0.5
0 0.5 1 1.5 2 2.5 3DBS IL6 (pg/mL)
R = 0.71
DBS IL6 (pg/mL)
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
IL6
N=71 10
12Scatter Plot with Passing & Bablok Fit
N=71
6
8
ma
(pg/
mL)
4
6
R&
D p
lasm
Plasma =1.95 x DBS – 0.88
0
2
0 1 2 3 4 5 6 7DBS IL6 (pg/mL)
R = 0.89
DBS IL6 (pg/mL)
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
IL10
N=72 25
30Scatter Plot with Passing & Bablok Fit
N=72
15
20
10 (p
g/m
L)
10
15
Plas
ma
IL1
Plasma = 4.94 x DBS – 2.13
0
5
0 2 4 6 8 10 12DBS IL10 (pg/mL)
R = 0.71
DBS IL10 (pg/mL)
Multiplex assay of cytokines in DBS
Protocol highlights Assay performance Adv/disadv
Advantages
Tool for measuring several cytokines at very low Tool for measuring several cytokines at very low concentrations
Wide dynamic range: <0.5 pg/mL to 5,000 pg/mLde dy a c a ge 0 5 pg/ o 5,000 pg/
Reduced cost per biomarker/per sample
DisadvantagesDisadvantages
Requires 1-2 drops of whole blood (for duplicates)
Big up front instrumentation costs Big up-front instrumentation costs
Acknowledgements
Fundingg
NIA Network on Biological Risk (R24AG037898)
USC/UCLA Center on Biodemography and PopulationUSC/UCLA Center on Biodemography and Population Health (P30AG017265)
National Science Foundation, Physical Anthropology (BCS-1027687)
Colleagues in the Laboratory for Human Biology Research
Elizabeth Miller
Bill Funk
Adam Leigh
American Journal of Human Biology
Impact factor: 2.3Indexed: EverywhereIndexed: EverywhereTime to decision: 27 days
Toolkit: Methods in Human Biology• Laboratory and field methods for
measuring human energy expenditure
Fi ld d l b t th d i• Field and laboratory methods in human milk research
• Essential anthropometric methods• Essential anthropometric methods