certified reference materials for prenatal and …...certified reference materials for prenatal and...
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Certified Reference Materials for Prenatal and Neonatal Testing
Korea Research Institute of Standards and Science (KRISS)
Center for Bioanalysis
Ji-Seon Jeong, PhD
2017 JCTLM M&S meeting
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Background
News from The hankyoreh, 2016-08-28
Charts on delayed motherhood (1995 and 2014)
Korea Poland
Sweden UK
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U.S. Non-Invasive prenatal testing market, 2014 - 2025 (USD Million)
• Possibility of fetus abnormalities ↑ = Prenatal testing ↑
• High false-positive rate in Quad screening vs. Higher risk of miscarriage after amniocentesis
• Demand of prenatal testing with high accuracy !
• The global Non-Invasive Prenatal Testing (NIPT) market size was valued at USD 1.1 billion in
2016. Korean NIPT market size was developed 1000% for 1-year (2016)Figures from
www.carolguze.com
Source: Persistence Market Research (Nasdaq newswire, July 01, 2016)
Why NIPT?
The strategy of KRISS CRM for NIPT
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Subject Target Technical issue
Matrix matched CRM Serum
- Inactivation of DNAse
- Elimination of endogenous cfDNA
- Nucleosomal DNA
** Exact matching to cfDNA in human serum
Full ID-MS of DNA
quantificationTrace level
- SIL-DNA production for compensation of
sample loss or digestion imperfection
- High-sensitivity detection method
- Traceability of DNA quantity for CRM
preparation
Development of an LC-MS/MS Method using an Isotopically Labeled
DNA as an Internal Standard for the Quantification of Cell Free DNA
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MRM scans of dNMP and SIL-dNMP
Compensation of extraction loss
Linearity
Patent pending : Production of SIL-DNA
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• Preparation of DNA-free serum
- Comparable with blank (water) after treatment
- Similar properties but only ‘DNA-free’ after treatment
• First batch of KRISS CRM for NIPT: it used for test materials for proficiency test by
Korean Institute of Genetic Testing Evaluation.
Preparation of DNA-free serum
Confirmation of DNA-clearance in serum
Proficiency tests for NIPT
Patent pending : Elimination of endogenous cfDNA
Neonatal/Newborn screening
7Reference from www.babysfirsttest.org, and the homepage
of Ministry of Health and Welfare, Korea
Simple !
Less-invasive!
Easy!
Economic !
Dried blood spot (DBS)
Things to think for DBS as CRM
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0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 5 10 15
Peak a
rea r
atio (
unla
bele
d/labe
led P
he)
nominal value (Phe; mg/dL)
J_50uL spotted
J_100uL spotted
company B-1
company B-2
company S
company P
• Diversity of samples: sensitive to blood conditions, and manufacturing conditions
(Viscosity, Hematocrit, Adsorbed volume, Paper type, etc)
• Extremely small size sample: slight differences different results
• Sample homogeneity: within spot and between spot
JS Jeong et al, Anal Bioanal Chem, 405:25(2013)8063-8072
Different Results !
1 2 3 4 5 6 7
0.8
1.1
1.2
1.3
1.4
1.5
Phe
Tyr
pe
ak a
rea
ra
tio
(u
nla
be
led
/la
be
led
AA
)
position
1 2
6 34
75
< 2% RSDs within spot
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• Whole spot vs. partial spot (area = sample volumn)
• No extraction or measurement issue
• Revise calculation of disc area : underestimation of sampling volume
• Whole spot sampling for certification
Paper in preparation: Sampling error in DBS
After adjustment of disc area
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JS Jeong et al, Anal Bioanal Chem, 405:25(2013)8063-8072
• Black box in sampling step: Lack of traceability chain to final stage
• Metrological challenge: KRISS is developing SI-traceable DBS CRMs for neonatal
screening
0.0%
4.0%
8.0%
12.0%
16.0%
20.0%
(a) control (b) pre-mixed (c) pre-layered(d) post-layered
CV (
%)
Phe Tyr
Blood
Blood Spot
Disc
Extract
Sample for analysis
Internal standard
?
?
Beyond screening!
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ID-MS/NGS: NIPT
ID-MS: DBS
NIPT CRMs
DBS CRMs
• Nucleic Acids
• NGS
• Clinical analysis
• Serum CRMs
Measurement
capability
• Clinical labs/industry
• 430,000 births/yr, Korea
• Non-invasive prenatal test
• New born screening
New CRM needs
• Matrix matched NIPT CRM
• SI-Traceable DBS CRM
• Expanding applications based
on NIPT and DBS
Outcomes
2017
NA/NGS
ID-MS
