DEVELOPMENT OF A DRIED BLOOD SPOT

METHOD FOR LEPTIN

AACC Annual Meeting Oral Abstract Session

Cardiovascular Disease, Chronic Kidney Disease and

Obesity: Diagnostic Paradigms

August 1, 2016

Jack A. Maggiore, PhD, MT(ASCP)

Assistant Laboratory Director

Doctor’s Data, Inc.

St. Charles, Illinois

CURRENT DISCLOSURES

Doctor’s Data, Inc. – Primary employer and

laboratory service provider for this validated test

MediaLab, Inc. – Continuing education contract

provider, not relevant to this presentation

2

LEPTIN OVERVIEW

Satiety hormone produced by abdominal

adipose tissue

Leptin is opposed by the actions of the

hormone Ghrelin

Both hormones act on receptors in the

hypothalamus to regulate appetite to achieve

energy homeostasis

In obesity, a decreased sensitivity to leptin

occurs, resulting in an inability to detect

satiety despite high energy stores

Leptin resistance as an early predictor of

Metabolic Syndrome? 3

BLOOD SPOT TESTING

History

1960’s - Dr. Robert Guthrie Inborn Errors of Metabolism - PKU test

1990’s – Commercial laboratory testing HIV Screening

Quantitative Testing of Metabolic Biomarkers- Lipids, HbA1c

Advantages

Smaller blood volume

Facilitates self-collection

At-home testing in fasting state

Minimized sample transit costs to laboratory

4

DRIED BLOOD SPOT METHOD DEVELOPMENT

Pre-Analytical Considerations

Sample matrix compatibility

Sample volume requirements

Sample integrity

Analyte stability

5

PRE-ANALYTICAL VARIABLES

Blood Collection

Collection Media

Sample Transport

Sample Integrity and Measurand Stability

6

BLOOD COLLECTION

Lancets

Single-Use

Self-Retracting

Pressure Triggered

Incision, NOT puncture

Free-Flowing Capillary Blood

Yielding 50μL Blood Drops (x3)

7

Image Courtesy of

SurgiLance™

COLLECTION MEDIA Standardized Collection Card

8

SAMPLE TRANSPORT

Desiccation

Protection from weather elements

9

SAMPLE INTEGRITY AND

MEASURAND STABILITY

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

Day 0 Day 4 Day 7 Day 10 Day 14 Day 19 Day 24 Day 30

Lep

tin

(n

g/m

L)

Leptin in Blood Spots - Stability

Room Temperature Incubated, 37°C Refrigerated, 2-8°C Frozen, -20°C

10

DRIED BLOOD SPOT METHOD DEVELOPMENT

Pre-Analytical Consideration

Analytical Considerations

Well-characterized Leptin standards?

Lack of accepted reference method

Lack of automated high-throughput options

11

ANALYTICAL VARIABLES

Analytical Methods

Principles

Extracted Sample Homogeneity

Precision

Matrix Compatibility

Linearity and Recovery

Sensitivity

Accuracy: Comparability DBS vs Serum

12

ANALYTICAL METHODS

Leptin by LC-MS/MS

Immunoassay Methods

13

ALPCO ULTRASENSITIVE LEPTIN ELISA

14

Image courtesy of ALPCO

15 Schematic Courtesy of ALPCO Diagnostics

SAMPLE PROCESSING

Dilution Factor

Assay requires dilution of serum sample prior to

analysis

Account for sample dilution as part of blood spot

extraction

⑩ 3-mm punched spots in required diluent volume

Extraction Buffer Selection

Homogeneity

Incubation Time

Incubation Temp

Rotator Speed

16

DRIED BLOOD SPOT (DBS)

PRECISION TESTING

(n = 20)

Low DBS Control High DBS Control

Mean

ng/mL

Total

%CV

Mean

ng/mL

Total

%CV

DBS

Leptin 8.9 5.4% 12.4 6.3%

17

LINEARITY AND RECOVERY

Graph and Summary

y = 0.9981x + 0.0116 R² = 0.9999

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0

Ob

serv

ed

Lep

tin

(n

g/m

L)

Expected Leptin (ng/mL)

DBS Leptin Leptin (ng/mL) %

Recovery Expected Observed

70.00 70.00 100.0%

35.00 34.50 98.6%

17.50 17.80 101.7%

8.75 9.00 102.9%

4.38 4.20 96.0%

2.19 2.10 96.0%

1.09 1.03 94.3%

0.55 0.65 117.9%

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Dried Blood Spot (DBS) Dilution Study

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LINEARITY AND RECOVERY

DBS Volume

(3mm spots) in

extraction buffer

Expected

Leptin

(ng/mL)

Observed

Leptin

(ng/mL)

% Recovery

⑩ 10.0 10.0 100%

⑨ 9.0 9.2 102%

⑧ 8.0 8.4 105%

⑦ 7.0 7.3 104%

⑥ 6.0 6.2 103%

⑤ 5.0 5.1 102%

④ 4.0 4.2 105%

③ 3.0 3.2 107%

② 2.0 2.2 110%

① 1.0 0.9 90%

ANALYTICAL AND FUNCTIONAL SENSITIVITY

The ability to differentiate a measurable

concentration from a true value of zero

Determined to be 0.0056 ng/mL

Manufacturer’s Limit of Blank (LoB) claim = 0.01

ng/mL

Functional Sensitivity

Concentration at which interassay CV% ≤20

Determined to be 0.55 ng/mL

20

COMPARISON TESTING

y = 0.932x + 0.365 R² = 0.9721

r = 0.986 S(y/x) = 1.76

n = 45

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0

Blo

od

Sp

ot

Le

pti

n (

ng

/mL

)

Serum Leptin (ng/mL)

Leptin - Serum vs Blood Spot

21

DRIED BLOOD SPOT METHOD DEVELOPMENT

Pre-Analytical Consideration

Analytical Considerations

Post-Analytical Considerations

22

POST-ANALYTICAL VARIABLES

Result Reporting

Reference Intervals

Collection Success Rate

23

LAB REPORT

24

REFERENCE INTERVALS Validated Using Healthy Adult Donors (n = 40)

Males: 1.8 – 20.0 mg/mL

Females: 4.7 – 39.0 mg/mL

25

COLLECTION SUCCESS RATE

>99% Overall sample acceptance rate for

Blood Spot Assays at Doctor’s Data

Vitamin D

Celiac Disease and Non-Celiac Gluten

Sensitivity

Metabolomic Profile

Hemoglobin A1c

Insulin

hs-C-Reactive Protein

Leptin

26

OTHER DBS METABOLOMIC ASSAYS

27

y = 0.9994x + 1.6398 R² = 0.9802 r = 0.9901

n = 42

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0

DB

S I

ns

uli

n (µIU/m

L)

Serum Insulin (µIU/mL)

Insulin Serum vs Dried Blood Spot

y = 0.9483x + 0.2345 R² = 0.9514 r = 0.9754

n = 38

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

DB

S h

s-C

RP

(m

g/L

)

Serum hs-CRP (mg/L)

hs-CRP Serum vs Dried Blood Spot

CONCLUSIONS • Fully validated laboratory developed test

• Requires three 50 µL drops of capillary whole blood

• Blood spot extracts run and read from standard ELISA assay

• Excellent blood spot correlation with serum

• 19-day sample stability at ambient temperatures

• Intra-assay imprecision <4.0%; Total imprecision 5.4 – 6.3%

• Blood spot leptin precision and sensitivity equivalent to serum

• Recent interest in the analysis of blood leptin has increased

among the medical community related to its association with the

development of the metabolic syndrome and pre-diabetes

• Elevated leptin levels have been associated with acute

cardiovascular events and stroke

• Leptin levels may be altered through lifestyle change and may

provide a useful biomarker to evaluate the effects of exercise

and dietary changes 28

REFERENCES

1. Koh KK, Park SM, Quon MJ. Leptin and cardio-vascular

disease. Circulation. 117(25):3238-49;2008.

2. Könner AC, Brüning JC. Selective insulin and leptin

resistance in metabolic disorders. Cell Metabolism. 16(2):144-

152;2012.

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ACKNOWLEDGMENTS Doctor’s Data

Darrell Hickok

David Quig, PhD

Andrea Gruszecki, ND

Erlo Roth, MD

Karen Urek, MT(ASCP)

Carol Cruzan, C(ASCP)

Bonnie Reynolds

ALPCO Diagnostics

Terance Fisher

Jennifer Mayes

Christopher Wisherd

Bethany Bell

Sean Conley 30

QUESTIONS

31