Examination of Proposed Manufacturing Standards and Implementation of ISO 18385

Transcription

1 Examination of Proposed Manufacturing Standards and Implementation of ISO Promega Corporation April 30,

2 Phantom of Heilbronn Promega Corporation 2

3 Crime scene Crime lab Potential Sources of Human DNA Contamination Environmental events Law enforcement personnel Sample collection devices Lab staff Other samples in lab Lab consumables Promega Corporation 3

4 ENFSI, SWGDAM, & BSAG Recommendations Promega Corporation 4

5 ENFSI, SWGDAM, & BSAG Recommendations Follow Good Manufacturing Processes Minimize interaction of staff with products When contact is necessary, ensure products are protected Perform Post-Production Treatment Ethylene oxide gas treatment UV cross-linking Perform Continual QC Checks Use a sensitive method of detection Test an adequate number of samples Maintain Elimination Database Check in the event of suspected contamination Promega Corporation 5

6 Certification What Does it Mean? Test methods using a sensitive STR profiling assay using a sensitive real-time PCR assay analyzed on a 2% agarose gel stained with EtBr Pass criteria Test sensitivity: 32 pg 2 pg of genomic DNA, equivalent to less than one human cell Limit of quantitation: 0.2 pg/µl Promega Corporation 6

7 UK Guidelines (June 2012) Promega Corporation 7

8 International Effort to Standardize Promega Corporation 8

9 ISO Draft: Some Definition Promega Corporation 9

10 Partial Profile from ~ 6.7pg DNA (24sec, 90RFU) Y-axis: 1,400RFU Promega Corporation 10

11 What is Forensic Grade? What detection method is acceptable to crime labs? What limit of detection is acceptable to crime labs? What level of undetectable is achievable by manufacturers? What level of undetectable is practical? Promega Corporation 11

12 Factors Influencing STR Analysis Sensitivity STR Kit PCR Cycle Number CE Instrument CE Run Parameters Data Analysis Promega Corporation 12

13 Promega Corporation Sensitivity: CE Instrument

14 Instrument Sensitivity Without stochastic influence of amplification 500pg DNA amplified with PowerPlex Fusion System Serially dilute amplification product Condition Default Enhanced 3500 CE 15sec, 1.2kV, 175RFU 24sec, 1.2kV, 90RFU Total the number of alleles called in all 3 replicates Divide by 135 (total possible alleles) Promega Corporation 14

15 Instrument Sensitivity Without stochastic influence of amplification Dilution Calculated DNA Amount (pg) 15sec 175RFU % Alleles Called on 3500 CE 15sec 90RFU 24sec 175RFU 24sec 90RFU 1: : : : : : : : : Default condition: no alleles called at/below ~1.67pg Enhanced condition: no alleles called at/below ~0.1pg Promega Corporation 15

16 One Allele Called with 0.5pg (24sec, 90RFU) Y-axis: 100RFU Promega Corporation 16

17 No CE Instrument Baseline Noise No amplification mix: 1X PowerPlex Fusion Primer Pair Mix 1X PowerPlex Fusion Master Mix 1µL no amp mix 10µL Hi-Di formamide 1µL ILS 500 Condition Default Enhanced 3500 CE 15sec, 1.2kV, 175RFU 24sec, 1.2kV, 90RFU No allelic peaks observed in 94 no-amplification injections Promega Corporation 17

18 Promega Corporation Sensitivity: Low Template Condition

19 Sensitivity with Consensus Allele Calling Serially dilute DNA Amplify with PowerPlex Fusion System; 3 replicates Condition Default Enhanced 3500 CE 15sec, 1.2kV, 175RFU 24sec, 1.2kV, 90RFU Count number of alleles called in at least 2 of 3 replicates Promega Corporation 19

20 Number of Alleles Number of Alleles STR Analysis is Sensitive Down to 1 pg sec, 175RFU sec, 90RFU pg 21 Limit of Detection Limit of Detection pg Rep 1 Rep 2 Rep 3 Consensus Rep 1 Rep 2 Rep 3 Consensus sec, 175RFU pg 45 24sec, 90RFU Limit of Detection Limit of Detection pg Rep 1 Rep 2 Rep 3 Consensus Rep 1 Rep 2 Rep 3 Consensus Promega Corporation 20

21 qpcr: Suitable Option for Human DNA Detection? Practical advantages for non-str kits: Straightforward data interpretation Can test more samples Faster More economical More sensitive with mtdna primers mtdna present at higher copies (~ 500 copies per cell) Promega Corporation 21

22 qpcr Analysis Serially dilute DNA Plexor qpcr System; 45 cycles; 3 replicates Determine Cq value Promega Corporation 22

23 qpcr is Sensitive Down to 0.25pg Limit of Detection Promega Corporation 23

24 Summary CE instruments are very sensitive No alleles called from ~1.67 pg under default conditions No alleles called from ~0.1 pg under enhanced conditions STR kits are very sensitive Full profile from 50 pg DNA Partial profile from one cell One allele called from 1pg under low template conditions qpcr analyses can detect 0.25pg DNA Promega Corporation 24

25 Promega Corporation Towards ISO 18385

26 ISO Develops International Standards Influence Observe Keep up-to-date Promega Corporation 26

27 Relevant ISO Standards Forensic Labs: ISO/IEC 17025:2005 specifies the general requirements for the competence to carry out tests and/or calibrations, including sampling. It covers testing and calibration performed using standard methods, non-standard methods, and laboratory-developed methods. Forensic Manufacturers: ISO 9001:2008 specifies requirements for a quality management system where an organization needs to demonstrate its ability to consistently provide product that meets customer and applicable statutory and regulatory requirements, and aims to enhance customer satisfaction through the effective application of the system, including processes for continual improvement of the system and the assurance of conformity to customer and applicable statutory and regulatory requirements. Promega Corporation 27

28 How/Why US Became a Participating Country To provide input on achievable standard Requirements for Switching ANSI (American National Standards Institute) needed to create a TAG (Technical Advisory Group) TAG requires: Administration by non-biased entity ASCLD Monetary support of administrative cost Promega Corporation 28

29 Developing a New ISO Standard ISO/PC 272: Forensic Sciences ISO Participating Countries Australia Belgium France Germany Japan Korea Netherlands Singapore Sweden Switzerland Thailand United Kingdom United States 12 Observing Countries Argentina Austria Bulgaria Denmark Ecuador Finland Iran New Zealand Poland Romania Slovakia Spain Promega Corporation 29

30 Recent ISO Activities Month March 2014 September 2014 December 2014 April 2015 Activity First draft of standard released (DIS 18385) ISO Committee meeting in Berlin, Germany; vote on first draft Decision: send for second draft round Second draft of standard released (DIS ) ISO Committee meeting in Washington, D.C. to vote on second draft Promega Corporation 30

31 Purpose of ISO For forensic manufacturers Purpose (and title): Minimizing the risk of human DNA contamination in products used to collect, store and analyze biological material for forensic purposes Scope: products used in sample collection through STR amplification Does not include post-amplification products (e.g., Allelic Ladder, CE consumables) ISO compliant companies will label products as ISO Forensic DNA Grade Promega Corporation 31

32 What does ISO mean to you? Assurance that products with the ISO Forensic DNA Grade label were manufactured using control measures specifically designed to minimize the risk of human DNA contamination Forensic labs should ask questions about ISO certification from their manufacturers. Especially since the standard will be self-certified, at least initially. Promega Corporation 32

33 General overview of draft requirements Have a Quality System in place (such as ISO 9001) Have a policy for a staff contamination detection system Left vague due to differing country laws Perform a risk assessment for potential human DNA contamination Implement risk mitigations (e.g., manufacturing controls) where necessary Have Cleaning and Environmental Monitoring Procedures Post-production treat products, where possible Per a validated treatment process Treatment must not negatively impact product performance Batch-release products which cannot be post-production treated Test for human DNA contamination by STR, qpcr, or other equivalent or more sensitive test method Promega Corporation 33

34 How Promega is preparing for ISO ISO 9001-certified since 1998 Has an established staff contamination detection system Reviewing existing risk assessment Process mapping each manufacturing step/process Will update assessment where necessary Reviewing existing manufacturing control measures Will implement additional control measures where necessary Reviewing existing cleaning and environmental monitoring procedures Will implement additional procedures where necessary Promega Corporation 34

35 How Promega is preparing for ISO cont. Post-production treat products, where possible Evaluating post-production treatment efficacies Performing stability studies to ensure no negative product impact Will validate process once the post-production treatment is determined Batch-release products which cannot be post-production treated Reviewing existing QC testing for human DNA contamination Will implement additional QC testing where necessary Promega Corporation 35

36 Thank You! Kristina Pearson Kara Raymond Jenni Setlak Charlie Stollberg Sarah Bettinger Ginger Goiffon Rohaizah James Doug Storts Susan Wigdal Andy Hopwood Susan Frackman Ann MacPhetridge Lotte Downey Promega Corporation 36