19 June 2017
Forensic reference samples
A complete solution for the automated laboratory process for DNA profiling Alexander Kneppers Program Manager Technology and Innovation Division of Biological Traces
17 May 2017 HIDS
DNA profiling – past & present
Anno 2000 Anno 2017 Number of requests
1000 45.000
Number of reference samples
1000 35.000
Number of employees 25 >100
Amount of DNA needed for profile
1 ng (170 cells)
0,006ng (1 cell)
Number of loci
10 (SGM Plus)
16 > 24 Plus (NGM > Global STR)
2
17 May 2017 HIDS
DNA Databank of the Netherlands
• > 250.000 persons • > 70.000 traces • > 57.000 matches (person/trace) • > 5.400 matches (trace/trace)
• 57 % matches (person/trace)
3
17 May 2017 HIDS
Workflow: Following a case
Report to clientReport by
primary ROReview by second RO
ClientDNA labTrace recoveryClient PlanningFrontdesk
Reporting Officer(RO)
Rework
Rework
R&D
Dutch DNA database
4
Reference samples
17 May 2017 HIDS
LabAutomation... .. is a necessity as a consequence of:
5
Time
Quantity of samples
Time
Sensitivity of DNA tests
Time
Delivery time
Time
Quality of DNA profiling
17 May 2017 HIDS
Goals of NFI Optimization in DNA profiling
6
Profiling of reference and trace samples to: • reduce costs and speed up the process • eliminate the possibility of sample misplacements • reduce the occurrence of contamination to a bare minimum
By: • whenever possible replace manual processes with automated processes • improve the remaining manual processes
17 May 2017 HIDS
Automated process allows for: • Efficient sample collection and transport • Full track and trace of sample handling during its life time • Complete audit trail for sample storage • Automation of ‘Business rules’ • Immediate automated storage of samples waiting for processing • Release for profiling after official request for profiling granted • Demonstrable retrieval and destruction of samples • Less manual handling of samples by logistic and laboratory personnel
7
17 May 2017 HIDS
Automated storage of reference samples
Goal: Store reference samples in such a way that: • Risk of losing and/or misplacing samples is eliminated; • Quality of the sample is maintained (sample integrity); • Audit trail is correct and complete (data integrity).
Points of interest: • The NFI is obliged by Dutch law to store certain samples up to 80 years; • Initial capacity: 250.000 samples • Fully automated storage solution • During first few years of storage most interaction/transfers of sample • Nucleic Cards at 20 °C • DNA extracts/lysates at 4 °C • Automated tube picking and delivery at 4 °C and 20 °C
8
17 May 2017 HIDS
2D-coded tubes/cards for laboratory samples At the basis of automation are 2D-coded tubes
and cards. Used for • High quality storage (low evaporation) • Automated delivery from storage of samples
based on work lists for different steps in the DNA-profiling process: • DNA extraction • Quantification • (Direct) PCR
• Manual labelling of tubes is eliminated • Visual control of correct sample order or
sample transfer is eliminated
9
17 May 2017 HIDS
Automation and Innovation Program • Automation of reference samples for DNA profiling: o Sample selection and long term storage o Direct PCR using card punching and PCR setup o Direct PCR buffer for swabs o Post PCR setup
o Applied Biosystems™3500XL Genetic Analyzer o Softgenetics GeneMarker® HID o New (Y-)STR kits
o Full integration and support by LIMS system o For all tendering procedures for purchasing new equipment and/or
labware/kits
10
17 May 2017 HIDS
DNA Profiling of samples: automated process
11
Implemented 2014
Implement Q1 2018
17 May 2017 HIDS
Current process references • Based on FSS automated process • 4 saliva swabs • StoreStar sample placement • DNA Extraction • Picogreen quantification
• Instrumentation is old and must be replaced
12
17 May 2017 HIDS
Tender for collection kit based on DNA cards
• Under responsibility of the National Police • Based on Buccal Swabs • Suitable for direct PCR • User tests by users of kit (National Police, Immigration
Service) • Technical requirements and assessment of cards by NFI • Field test with approx. 200 samples to determine first pass
success rate > 90 %
13
17 May 2017 HIDS 14
Subject Supplier A Supplier B Copan/ Thermo Fisher
Direct sampling No, transfer to collection card yes No, transfer to
collection card
Indicator yes no yes
Treated cards yes
no, pretreatment
necessary before PCR
yes
Treated Cards: Collection devices that are treated with a chemical intended to protect the collected samples. Such cards are not safe for putting directly in the subject’s mouth and therefore require a transfer step. These cards consists of a special paper treated with chemicals that lyse cell membranes and denaturate proteins to immobilize and preserve the DNA.
Untreated Cards: Collection devices with the collection paper made of 100% cotton paper. These devices allow for direct collection and can collect a sample without having to transfer to another substrate.
Three responses to tender
17 May 2017 HIDS
User tests of collection kit
15
• Packaging of kit and components. • Ease of use of kit
• Number of steps in sample collection • Instruction for usage
• Control steps for sampling (colour change of cards)
• Prevention of contamination (for kit user and donor) • Packaging/sealing of samples for transport/storage
• Experience of donor of the sample
17 May 2017 HIDS
Technical tests NFI
16
• 20 donors
• 4 punches per card
• Typing with Promega PowerPlex® Fusion 6C
• Parameters
• Concordance
• Full profile
• Profile quality (peak height/intra- and inter locus balance)
17 May 2017 HIDS
Results technical tests NFI
Parameter Supplier
A Supplier
B
Copan/ Thermo Fisher
Scientific Peak height 10 6 10 Full Profile 9 4 10 Success rate 10 X 10 Interlocus balance
9 5 8
Intralocus balance
10 7 9
Total score 48 X 47
17
Based on quality and price tender won by Copan/Thermo Fisher Scientific
17 May 2017 HIDS 18
Profiling process
Preferred route • Fully automated procedure • EasyPunch
Alternative route • CPA200™ • Direct PCR on swab • DNA extraction of punches
17 May 2017 HIDS
• Automated system with Punch device and liquid
handling • Punch head 1,2 mm • Card imaging (saliva on indicator cards, recognition of
punches) • Track and trace of samples through barcodes • monitoring and reporting of punch in PCR plate wells • Cleaning function of puncher • No manual interaction in setup of DNA profiling • Fully integrated with LIMS System • Validation on parameters
• Sample tracking • Sample and punch detection • Punching • Contamination prevention • Robustness
19
Hamilton® easyPunch STARlet
17 May 2017 HIDS
CPA 200™ • Semi automatic punching
system • Manual punching for • cards that cannot be
processed on the automated system
• low quality samples for visual selection of punch area
• Validation on parameters • sample tracking • punching • contamination prevention
20
17 May 2017 HIDS
Field test buccal samples
• As part of tender process for new collection kit • To determine first pass success rate > 90 % • Data to be used for internal validation • Double collection of samples in one Police district (East
Netherlands) • DNA profiling using easyPunch system and CPA200 with PPF6C kit • Reduced PCR volume 6,3 µl • 3500XL electrophoresis • GeneMarker HID analysis • Police Academy involved for changing course material for
certification of personnel and retraining of certified personnel • 223 samples
21
17 May 2017 HIDS 22
0
10
20
30
40
50
60
70
80
++ + +/- - --
nu
mb
er
of
collect
ion
card
s
colour change
Visual score on colour change
17 May 2017 HIDS 23
0
10
20
30
40
50
60
70
full partial no
# c
ard
s
++
+
+/-
-
--
0
10
20
30
40
50
60
70
80
full partial no
# c
ard
s
++
+
+/-
-
--
Colour change vs profile outcome
easyPunch
CPA200
17 May 2017 HIDS 24
11
43 17
29
0
Peak height: 5000 -‐ 10.000 RFU
++
+
+/-‐
-‐
-‐-‐
28
44
12
4 12
Peak height: > 10.000 RFU
++
+
+/-‐
-‐
-‐-‐
11
20
42
20 3
Peak height: 0 -‐ 1000 RFU
++
+
+/-‐
-‐
-‐-‐
13
20
35
26
4
Peak height: 1000 -‐ 5000 RFU
++
+
+/-‐
-‐
-‐-‐
Peak height vs colour change (Easypunch fp)
17 May 2017 HIDS
Profile quality
25
0.00
0.20
0.40
0.60
0.80
1.00
1
6
11
16
21
26
31
36
41
46
51
56
61
66
71
76
81
86
91
96
101
106
111
116
121
126
131
136
141
146
151
156
161
166
171
176
181
186
191
196
201
206
Intra locus balance
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Inter locus balance
17 May 2017 HIDS 26
Process Full DNA profiles Partial DNA
profiles No DNA profile
Easypunch 212 8 3 CPA 200 218 2 3
Process Number of cards Full Profiles First Pass
Succes rate
Easypunch 223 205 92%
CPA200 223 214 96%
Results Field tests
Concordancy test: 121 profiles full concordant based on NGM
17 May 2017 HIDS 27
20 Copan Floq swabs, direct in “swab solution”
• First pass succes rate: 100%
20 GE Healthcare swabs , direct in “swab solution”
• First pass succes rate: 100%
Swab extracts 6 weeks at +4°C stored
• First pass succes rate: 100%
Alternative DNA Profiling
17 May 2017 HIDS
Overall conclusions
28
• Overall solution for DNA typing process of reference samples
• Reduction of time and costs
• Covering complete life span of reference sample
• Reduced risks on sample misplacement/switching, protocol errors, cross-contamination, operator contamination
• Complete sample tracking, audit trail on storage
• Tender procedures
• Time consuming procedure
• Quality based tests during selection process is of added value
• Select products based on right balance between quality and price
17 May 2017 HIDS
Acknowledgements • Direct PCR • Zulaiga Habiboellah • Angelique Hardon • Anne-Wil van der Zwan • Dico van Meerten
29
• IT • Martin Slagter • Mirjana Chamberlain • Rudi de Smit
• PPF6C • Titia Sijen • Francisca Duijs • Linda van de Merwe • Laura Willemstein
• 3500/GeneMarker HID • Krista Smeele • Jelle van der Zwaag • Ellen Vollebregt
17 May 2017 HIDS
Disclaimer Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration When used for purposes other than Human Identification or Paternity Testing the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so
30
