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



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



N=46



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


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




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 --



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)



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


0

0.5

0 0.5 1 1.5 2 2.5 3DBS IL6 (pg/mL)

R = 0.71

DBS IL6 (pg/mL)



IL6

N=71 10


N=71

6

8

ma

(pg/

mL)

4

6

R&

D p

lasm


0

2

0 1 2 3 4 5 6 7DBS IL6 (pg/mL)

R = 0.89

DBS IL6 (pg/mL)



IL10

N=72 25


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)



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

qtifiti fifl t tki iquantification of inflammatory

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