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Symposium presentation: Development of a Dried Blood Spot Method for Leptin
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Transcript of Symposium presentation: Development of a Dried Blood Spot Method for Leptin
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%
18
Dried Blood Spot (DBS) Dilution Study
19
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.
29
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