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Accreditation of a forensic genetics laboratory in Italy
Forensic Science International: Genetics Supplement Series 4 (2013) e294–e295
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Accreditation of a forensic genetics laboratory in Italy U. Ricci *, C. De Sanzo, I. Carboni, S. Iozzi, A.L. Nutini, F. Torricelli AOU Careggi, SOD Diagnostica Genetica, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 August 2013 Accepted 2 October 2013
Although in Italy DNA analysis is used since the beginnings, the introduction of a specific legislation had a very important impact for the quality of the results. Only authorized laboratories can now implement the database with genetic profiles; these labs have to follow ISO/IEC standard and according to the Council Framework Decision 2009/905/JHA of 30 November 2009 and the Recommendation 7 of ENFSI this standard is the ISO17025/2005 accreditation. Since December 22th 2009, ACCREDIA is the only appointed Italian Accreditation authority can issue the accreditation certificate. There is no rule to define the ISO test method for the study of DNA polymorphisms in forensics, thus each laboratory should build their own analytical system, reliable and reproducible, through its own internal validation. Here, we focus on some points of the accreditation process developed in our laboratory concerning internal validation protocol. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Accreditation ISO/IEC 17025 Forensic genetics DNA database
1. Introduction In 1997 the Council of the European Union invited Member States to consider the establishment of national DNA databases [1]. The Pru¨m Convention (sometimes known as Schengen III Agreement [2]) is a treaty which was signed on May 27th 2005 by Austria, Belgium, France, Germany, Luxembourg, Netherlands and Spain in the town of Pru¨m in Germany in order to exchange data regarding DNA, fingerprints and vehicle registrations and to cooperate against terrorism. Italy has acceded to the Treaty with the law 85/2009 published on the13th July 2009 – Suppl. Ordinary n8 108 [3]. In the article 11 entitled ‘‘Methodology for the analysis of artifacts and biological samples for the profiles typing to include in the national DNA database’’ is specified that ‘‘DNA profiles can be included in the national DNA database only if they has been obtained in certified laboratories in accordance with ISO/IEC’’; in accordance with ENFSI Recommendation 7 [4] this standard is ISO/IEC 17025. Since the 22nd December 2009, Accredia [5] is the only appointed Italian Accreditation authority can issue the accreditation certificate. ACCREDIA evaluates the technical competence and professional integrity of the operators, through compliance with mandatory regulations and voluntary norms in order to ensure the value and credibility of the attestations they release. Here we highlight some important aspects of the ISO17025/2005 test method, concerning the
* Corresponding author at: AOU Careggi SOD Diagnostica Genetica, Largo Brambilla 3, I-50134 Florence, Italy. Tel.: +39 055 7946204; fax: +39 055 7946200. E-mail address: [email protected] (U. Ricci). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.150
internal validation process adopted to interpret DNA forensic samples. The purpose of this report is to show the general characteristic of the internal method and the validation process used, aware that it is a dynamic method that has to be continuously implemented. 2. Organization and standards adopted 2.1. General The laboratory shall use appropriate methods and procedures for all tests and/or calibrations within its scope (5.4.1 General). An appropriate internal method was described in the procedure, according to section 5.4.2 (Selection of methods), Laboratorydeveloped methods or methods adopted by the laboratory may also be used if they are appropriate for the intended use and if they are validated. This method was based on the German protocol described by Schneider et al. (unpublished and [6]) and it consists in the evaluation of both quality and quantity of DNA quantified in Real-Time. Thus a ‘‘Standard protocol’’ or a ‘‘Low template protocol’’ was adopted depending on the quantification results. 2.2. DNA standard Commercial control DNA were used for validation considering that these cell lines were part of NIST component. For example, component 9 of the NIST SRM2391b is the commonly used 9947A genomic DNA and component 10 is the commonly used control 9948 (Coble M, personal communication). Also previously typed DNA from known donors were used for some applications.
U. Ricci et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e294–e295
2.3. Quantification A NanoDrop 1000 Spectrophotometer (Thermoscientific, USA) and an Applied Biosystems 7900HT Fast Real-Time PCR System (Life Technologies, USA), in association with Quantifiler1 Duo DNA Quantification Kit, were used for DNA quantification. Experimental design includes:
e295
(b) Measuring the reproducibility and concordance of the method by analysing twenty control DNA and ladders for all kits, by evaluating the DNA profiling and standard deviation for each injection; (c) A specific control chart was prepared on the basis of the migration sizes of the positive samples. 3. Conclusion
(a) comparison of two methods for DNA extracted from largevolume samples like saliva swabs or blood; (b) evaluation of results’ repeatability in standard DNA included in kits and artificial dilution of genomic DNA; (c) evaluation of performance in female-male mixtures with different ratio. 2.4. PCR amplification and DNA electrophoresis AmpFlSTR1 Identifiler1 Plus, AmpFlSTR1 NGMTM, AmpFlSTR1 MiniFilerTM, AmpFlSTR1 Yfiler1 PCR Amplification Kits (Life Technologies, USA) and PowerPlex1 ESI 17 (Promega, USA) were used. 26plex described by Hill et al. [7] was also included in the validation protocol. Experimental design includes: (a) evaluation of robustness and repeatability using half quantity of reagents for PCR. DNA profile, heterozygous balance, signal intensity and standard deviation were evaluated; (b) modification of injection parameters: ‘‘light’’, ‘‘normal’’ and ‘‘heavy’’ injection protocols were adopted; (c) elimination of primers excess in electrophoretic pattern; (d) evaluation of robustness and repeatability using 100 pg/PCR input DNA; (e) evaluation of the performance using 10 pg/PCR input DNA: % of drop-out and drop-in were verified; (f) evaluation of the performance using 10 pg/PCR input DNA with +1 and +3 PCR cycles suggested by instruction manual. Interpretation process was suggested by Caragine et al. [8]; (g) evaluation of mixture by using female/male and male/female in ratio of 1:1, 1:2, 1:4, 1:6, 1:8, 1:10.
2.5. Validation of the electrophoretic system A 3500 Genetic Analyzer (Life Technologies) was used for electrophoresis [9,10]. Experimental validation with this instrument includes: (a) Measuring the robustness of the method determining LOD (limit of detection) and LOQ (limit of quantification) for all above indicated kits. These stochastic parameters were determined using thirty negative controls in association with ‘‘light’’, ‘‘normal’’ and ‘‘heavy’’ injection protocols indicated above;
Here we show the developmental process of the internal validation for DNA polymorphisms adopted by forensic genetics laboratory of the AOU Careggi, Florence (Italy). The lab is now accredited to ISO 17025 by Accredia as Lab.1268 and the denomination of the test is ‘‘DNA typing for human identification, paternity and kinship testing’’ [11]. We believe that the spread of the adopted methods by all who adopt ISO17025 standards is necessary for a possible future sharing of common protocols in the forensic geneticists community. This is the paper nr. 1 after the accreditation ISO/IEC17015 (laboratory nr. 1268 by the Italian Accreditation Body Accredia). Conflict of interest None. Acknowledgements We thank all colleagues who have before obtained in the laboratory the objectives of the standards ISO9001 and ISO15189, laying the foundation for achieving accreditation 17025. Moreover, we are grateful to Life Technologies for contribution to participate at this meeting. References [1] http://europa.eu/legislation_summaries/other/l33097_en.htm. [2] Christopher Walsch (July 2009), Europeanization and Democracy: Negotiating the Pru¨m Treaty and the Schengen III Agreement. Croatian Political Science Review 45(5), Retrieved 29 August 2010. [3] http://www.governo.it/biotecnologie/documenti/LEGGE_30_giugno_2009_n_85.pdf. [4] DNA-database management review and recommendations ENFSI DNA Working Group; April 2012. [5] http://www.accredia.it/context.jsp?ID_LINK=76&area=6. [6] P.M. Schneider, R. Fimmers, W. Keil, et al., The German Stain Commission: recommendations for the interpretation of mixed stains, Int. J. Legal Med. 123 (January (1)) (2009) 1–5. [7] C.R. Hill, J.M. Butler, P.M. Vallone, A 26plex autosomal STR assay to aid human identity testing, J. Forensic Sci. 54 (September (5)) (2009) 100. [8] T. Caragine, R. Mikulasovich, J. Tamariz, et al., Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler, Croat. Med. J. 50 (June (3)) (2009) 250–267. [9] E.L.R. Butts, M.C. Kline, D.L. Duewer, et al., NIST validation studies on the 3500 Genetic Analyser, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e184–e185. [10] J.R. Gilder, T.E. Doom, K. Inman, et al., Run-specific limits of detection and quantitation for STR-based DNA testing, J. Forensic Sci. 52 (January (1)) (2007) 97–101. [11] http://www.accredia.it/accredia_labsearch.jsp?ID_LINK=293&area=7&dipartimento=L%2CS&.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Accreditation of a forensic genetics laboratory in Italy U. Ricci *, C. De Sanzo, I. Carboni, S. Iozzi, A.L. Nutini, F. Torricelli AOU Careggi, SOD Diagnostica Genetica, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 August 2013 Accepted 2 October 2013
Although in Italy DNA analysis is used since the beginnings, the introduction of a specific legislation had a very important impact for the quality of the results. Only authorized laboratories can now implement the database with genetic profiles; these labs have to follow ISO/IEC standard and according to the Council Framework Decision 2009/905/JHA of 30 November 2009 and the Recommendation 7 of ENFSI this standard is the ISO17025/2005 accreditation. Since December 22th 2009, ACCREDIA is the only appointed Italian Accreditation authority can issue the accreditation certificate. There is no rule to define the ISO test method for the study of DNA polymorphisms in forensics, thus each laboratory should build their own analytical system, reliable and reproducible, through its own internal validation. Here, we focus on some points of the accreditation process developed in our laboratory concerning internal validation protocol. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Accreditation ISO/IEC 17025 Forensic genetics DNA database
1. Introduction In 1997 the Council of the European Union invited Member States to consider the establishment of national DNA databases [1]. The Pru¨m Convention (sometimes known as Schengen III Agreement [2]) is a treaty which was signed on May 27th 2005 by Austria, Belgium, France, Germany, Luxembourg, Netherlands and Spain in the town of Pru¨m in Germany in order to exchange data regarding DNA, fingerprints and vehicle registrations and to cooperate against terrorism. Italy has acceded to the Treaty with the law 85/2009 published on the13th July 2009 – Suppl. Ordinary n8 108 [3]. In the article 11 entitled ‘‘Methodology for the analysis of artifacts and biological samples for the profiles typing to include in the national DNA database’’ is specified that ‘‘DNA profiles can be included in the national DNA database only if they has been obtained in certified laboratories in accordance with ISO/IEC’’; in accordance with ENFSI Recommendation 7 [4] this standard is ISO/IEC 17025. Since the 22nd December 2009, Accredia [5] is the only appointed Italian Accreditation authority can issue the accreditation certificate. ACCREDIA evaluates the technical competence and professional integrity of the operators, through compliance with mandatory regulations and voluntary norms in order to ensure the value and credibility of the attestations they release. Here we highlight some important aspects of the ISO17025/2005 test method, concerning the
* Corresponding author at: AOU Careggi SOD Diagnostica Genetica, Largo Brambilla 3, I-50134 Florence, Italy. Tel.: +39 055 7946204; fax: +39 055 7946200. E-mail address: [email protected] (U. Ricci). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.150
internal validation process adopted to interpret DNA forensic samples. The purpose of this report is to show the general characteristic of the internal method and the validation process used, aware that it is a dynamic method that has to be continuously implemented. 2. Organization and standards adopted 2.1. General The laboratory shall use appropriate methods and procedures for all tests and/or calibrations within its scope (5.4.1 General). An appropriate internal method was described in the procedure, according to section 5.4.2 (Selection of methods), Laboratorydeveloped methods or methods adopted by the laboratory may also be used if they are appropriate for the intended use and if they are validated. This method was based on the German protocol described by Schneider et al. (unpublished and [6]) and it consists in the evaluation of both quality and quantity of DNA quantified in Real-Time. Thus a ‘‘Standard protocol’’ or a ‘‘Low template protocol’’ was adopted depending on the quantification results. 2.2. DNA standard Commercial control DNA were used for validation considering that these cell lines were part of NIST component. For example, component 9 of the NIST SRM2391b is the commonly used 9947A genomic DNA and component 10 is the commonly used control 9948 (Coble M, personal communication). Also previously typed DNA from known donors were used for some applications.
U. Ricci et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e294–e295
2.3. Quantification A NanoDrop 1000 Spectrophotometer (Thermoscientific, USA) and an Applied Biosystems 7900HT Fast Real-Time PCR System (Life Technologies, USA), in association with Quantifiler1 Duo DNA Quantification Kit, were used for DNA quantification. Experimental design includes:
e295
(b) Measuring the reproducibility and concordance of the method by analysing twenty control DNA and ladders for all kits, by evaluating the DNA profiling and standard deviation for each injection; (c) A specific control chart was prepared on the basis of the migration sizes of the positive samples. 3. Conclusion
(a) comparison of two methods for DNA extracted from largevolume samples like saliva swabs or blood; (b) evaluation of results’ repeatability in standard DNA included in kits and artificial dilution of genomic DNA; (c) evaluation of performance in female-male mixtures with different ratio. 2.4. PCR amplification and DNA electrophoresis AmpFlSTR1 Identifiler1 Plus, AmpFlSTR1 NGMTM, AmpFlSTR1 MiniFilerTM, AmpFlSTR1 Yfiler1 PCR Amplification Kits (Life Technologies, USA) and PowerPlex1 ESI 17 (Promega, USA) were used. 26plex described by Hill et al. [7] was also included in the validation protocol. Experimental design includes: (a) evaluation of robustness and repeatability using half quantity of reagents for PCR. DNA profile, heterozygous balance, signal intensity and standard deviation were evaluated; (b) modification of injection parameters: ‘‘light’’, ‘‘normal’’ and ‘‘heavy’’ injection protocols were adopted; (c) elimination of primers excess in electrophoretic pattern; (d) evaluation of robustness and repeatability using 100 pg/PCR input DNA; (e) evaluation of the performance using 10 pg/PCR input DNA: % of drop-out and drop-in were verified; (f) evaluation of the performance using 10 pg/PCR input DNA with +1 and +3 PCR cycles suggested by instruction manual. Interpretation process was suggested by Caragine et al. [8]; (g) evaluation of mixture by using female/male and male/female in ratio of 1:1, 1:2, 1:4, 1:6, 1:8, 1:10.
2.5. Validation of the electrophoretic system A 3500 Genetic Analyzer (Life Technologies) was used for electrophoresis [9,10]. Experimental validation with this instrument includes: (a) Measuring the robustness of the method determining LOD (limit of detection) and LOQ (limit of quantification) for all above indicated kits. These stochastic parameters were determined using thirty negative controls in association with ‘‘light’’, ‘‘normal’’ and ‘‘heavy’’ injection protocols indicated above;
Here we show the developmental process of the internal validation for DNA polymorphisms adopted by forensic genetics laboratory of the AOU Careggi, Florence (Italy). The lab is now accredited to ISO 17025 by Accredia as Lab.1268 and the denomination of the test is ‘‘DNA typing for human identification, paternity and kinship testing’’ [11]. We believe that the spread of the adopted methods by all who adopt ISO17025 standards is necessary for a possible future sharing of common protocols in the forensic geneticists community. This is the paper nr. 1 after the accreditation ISO/IEC17015 (laboratory nr. 1268 by the Italian Accreditation Body Accredia). Conflict of interest None. Acknowledgements We thank all colleagues who have before obtained in the laboratory the objectives of the standards ISO9001 and ISO15189, laying the foundation for achieving accreditation 17025. Moreover, we are grateful to Life Technologies for contribution to participate at this meeting. References [1] http://europa.eu/legislation_summaries/other/l33097_en.htm. [2] Christopher Walsch (July 2009), Europeanization and Democracy: Negotiating the Pru¨m Treaty and the Schengen III Agreement. Croatian Political Science Review 45(5), Retrieved 29 August 2010. [3] http://www.governo.it/biotecnologie/documenti/LEGGE_30_giugno_2009_n_85.pdf. [4] DNA-database management review and recommendations ENFSI DNA Working Group; April 2012. [5] http://www.accredia.it/context.jsp?ID_LINK=76&area=6. [6] P.M. Schneider, R. Fimmers, W. Keil, et al., The German Stain Commission: recommendations for the interpretation of mixed stains, Int. J. Legal Med. 123 (January (1)) (2009) 1–5. [7] C.R. Hill, J.M. Butler, P.M. Vallone, A 26plex autosomal STR assay to aid human identity testing, J. Forensic Sci. 54 (September (5)) (2009) 100. [8] T. Caragine, R. Mikulasovich, J. Tamariz, et al., Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler, Croat. Med. J. 50 (June (3)) (2009) 250–267. [9] E.L.R. Butts, M.C. Kline, D.L. Duewer, et al., NIST validation studies on the 3500 Genetic Analyser, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e184–e185. [10] J.R. Gilder, T.E. Doom, K. Inman, et al., Run-specific limits of detection and quantitation for STR-based DNA testing, J. Forensic Sci. 52 (January (1)) (2007) 97–101. [11] http://www.accredia.it/accredia_labsearch.jsp?ID_LINK=293&area=7&dipartimento=L%2CS&.