internal validation of rapidhit™ id system in casework …...eugenio alladio. 1. 1 - reparto...
TRANSCRIPT
Internal Validation of RapidHIT™ ID System in
Casework Samples
Dr. Andrea BERTI Ph.D
The Applied Biosystems™ RapidHIT™ IDSystem is a fully-integrated system,simple-to-use, and capable of producinglab-quality forensic DNA profiles in aslittle as 90 minutes. The system isproposed for the analysis of referencesamples or highly concentrated samples.
Laboratory backlog analysis
Crime scene analysis on site
DVI Scenario
( need ISO17025 for NDBDNA)
Remote analysis on local PoliceOffice
Published data review Validation plan set up◦ Genemapper IDX Panels and bin customization◦ AT and ST calculation◦ Reproducibility◦ Repeatability◦ Accuracy◦ Sensitivity◦ Real casework sample analysis
• Review data• Submit to accreditation board
2019 2018
ST= ~200 RFU**
**(99% confidence; < 1%Drop out)
Global.AT7 Global.B.AT7 Global.G.AT7 Global.Y.AT7 Global.R.AT7 Global.P.AT736,09 50,28 31,11 40,02 12,21 35,86
*STR-validator v. 2.1.0
*
AT= ~ 50 RFU
7 ladders
Size Mean (bp)
Size Standard Deviation (bp)
Max Size Standard Deviation
86,26714286 0,067753053 0,163794296
91,39142857 0,070339314
VS
SALIVA BLOOD
Intercolor balancewas expressed as aratio of the meanpeak height of a dyeover the meansample peak height.
SALIVA BLOOD
Intracolor balance was calculated byaveraging the RFU values for each allele ofheterozygous genotypes of polymorphicloci and by dividing the RFU of eachhomozygous peak RFU by 2 for a dye Thelowest normalized RFU value was thendivided by the highest normalized RFUvalue and expressed as a percentage
HB table locusMarker Hb.n Hb.Min Hb.Mean Hb.Sd Hb.Max Hb.Perc.5D22S1045 30 0,567027 0,779104 0,118667 0,947189 0,589225SE33 42 0,442879 0,786169 0,117042 0,993612 0,628775D12S391 43 0,601351 0,842294 0,085471 0,998267 0,681055vWA 40 0,557707 0,861825 0,099651 0,997996 0,684042D3S1358 42 0,660959 0,892522 0,090561 0,994178 0,700961D2S1338 47 0,546135 0,859863 0,09567 1 0,711495TH01 38 0,471074 0,875607 0,10064 0,996055 0,715481D8S1179 28 0,64497 0,860583 0,095907 1 0,718903D16S539 33 0,702087 0,861638 0,086152 0,995872 0,739497D2S441 29 0,674079 0,903379 0,080344 0,995558 0,753353D21S11 46 0,615646 0,871093 0,07437 1 0,754002D19S433 37 0,64064 0,880842 0,078923 0,989099 0,763044D1S1656 47 0,637931 0,884426 0,077689 0,995676 0,767734D10S1248 29 0,663126 0,887557 0,077535 0,992629 0,787076FGA 47 0,701613 0,908109 0,073624 0,998324 0,793303D18S51 43 0,74554 0,89439 0,064811 0,99191 0,793548
We started an internal validation inorder to accreditate RapidHIT™ IDSystem for ISO/IEC 17025. Over100 samples (mock and realcasework) were collected in theLab. The sample set includedblood, saliva, semen spotted ondifferent matrices. The sampleswere processed either with rapidDNA or standard CE internalprocedure.
The profiles were compared in termsof accuracy, completeness andstochastic effects, such as drop outor drop in.
SALIVA 100ul, 50 ul, 20 ul X3 BLOOD 20ul, 4 ul, 2 ul X3 SEMEN 20ul, 10 ul, 4 ul X 3
# 25- Reference saliva sample (different matrix) # 24 –Reference blood sample (different matrix) Negative Controls
# 2-Standard NIST- # 2-CTS* proficiency test
#55- Real Casework Samples
*(http://cts-forensics.com/)
X3
DILUTION SERIES
N 3 3 3Mean 92,794 608,21 1160,5StDev 28,857 225,85 244,83Minimum 64,292 380,77 878,05Maximum 121,99 832,44 1311,1
Statistics 20 50 100
1005020
1200
1000
800
600
400
200
0
ul fluido
med
ia He
ig
Distribution of Data by GroupCompare the center and the variability across samples. Identify any outliers.
Boxplot of media Heig by ul fluidoSummary Report
Saliva
Semen
Blood
Matrice/ supporto
Tampone (oral swabb) FTA card Denim
Saliva
2 drop-out di allele su 288 alleli
% drop-out = 0.7%
15 drop-out di allele su 512 alleli
% drop-out = 2.9%
n.a.
Sangue
0 drop-out di allele su 256 alleli
% drop-out = 0%
0 drop-out di allele su 256 alleli
% drop-out = 0%
0 drop-out di allele su 256 alleli
% drop-out = 0%
Blood
Saliva
Bone 3 Months in Formalin
Blood >1 YearBlood >10 Years
Blood >10 Years
Bone 3 Monthsin Formaline
tracciaabbondante
traccia esigua
Negative 8% 100%
positive 92% 0%
0%10%20%30%40%50%60%70%80%90%
100%
Saliva
High Concentrate
LowConcentrate
tracciaabbondante
traccia esigua
Negative 25% 73%
positive 75% 27%
0%
20%
40%
60%
80%
100%
120%
Blood
High Concentrate
LowConcentrate
The results of our validation studyconclude that the Applied BiosystemsRapidHIT ID System is a robust solutionfor the generation of DNA profiles fromhighly concentrated saliva and bloodsamples from reference and crimecaseworks.
Marco Santacroce1
Elena Salata1
Ciro Trapani1Martina Botti2Cesare Rapone1
Eugenio Alladio1
1 - Reparto Carabinieri Investigazioni Scientifiche di Roma – Sezione di Biologia, Viale Tor di Quinto 119, 00191 - Rome, Italy.2- Comando Compagnia CC Pomezia-Roma, Italy
Acknowledgments
-Thermo Fisher Scientific
Speaker was provided travel and hotel support by ThermoFisher Scientific for this presentation, but no remuneration.
When used for purposes other than Human Identification orPaternity Testing the instruments and software modules citedare for Research Use Only. Not for use in diagnosticprocedures.Thermo Fisher Scientific and its affiliates are not endorsing,recommending, or promoting any use or application ofThermo Fisher Scientific products presented by third partiesduring this seminar.Information and materials presented or provided by thirdparties are provided as-is and without warranty of any kind,including regarding intellectual property rights and reportedresults.Parties presenting images, text and material represent theyhave the rights
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