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Mario Pazzagli - One of the best experts on this subject based on the ideXlab platform.

  • THE Pre-Analytical Phase OF THE LIQUID BIOPSY
    New Biotechnology, 2019
    Co-Authors: Francesca Salvianti, Stefania Gelmini, Filomena Costanza, Irene Mancini, Gemma Sonnati, Lisa Simi, Mario Pazzagli, Pamela Pinzani
    Abstract:

    Abstract The term ‘liquid biopsy’, introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes. The analysis of the liquid biopsy relies on a plethora of methods whose standardisation cannot be accomplished without disclosing criticisms related to the Pre-Analytical Phase. Thus, Pre-Analytical factors potentially influencing downstream cellular and molecular analyses must be considered in order to translate the liquid biopsy approach into clinical practice. The present review summarises the most recent reports in this field, discussing the main Pre-Analytical aspects related to CTCs, cfNAs and exosomes in blood samples for liquid biopsy analysis. A short discussion on non-blood liquid biopsy samples is also included.

  • How standardization of the Pre-Analytical Phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics.
    New biotechnology, 2019
    Co-Authors: Peter H.j. Riegman, Mario Pazzagli, Karl-friedrich Becker, Kurt Zatloukal, U. Schröder, Uwe Oelmüller
    Abstract:

    Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample Pre-Analytical Phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of ‘not fit for purpose’ samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.

  • Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.
    Data in brief, 2016
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses – [1]; Influence of Pre-Analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

  • Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality
    Clinica chimica acta; international journal of clinical chemistry, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Abstract Background In order to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on Pre-Analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent Pre-Analytical conditions.

  • influence of storage conditions and extraction methods on the quantity and quality of circulating cell free dna ccfdna the spidia dnaplas external quality assessment experience
    Clinical Chemistry and Laboratory Medicine, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Claudio Orlando, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Stefania Gelmini
    Abstract:

    BACKGROUND Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic Pre-Analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the Pre-Analytical Phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for Pre-Analytical Phase studies. METHODS Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

Francesca Malentacchi - One of the best experts on this subject based on the ideXlab platform.

  • Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.
    Data in brief, 2016
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses – [1]; Influence of Pre-Analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

  • Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality
    Clinica chimica acta; international journal of clinical chemistry, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Abstract Background In order to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on Pre-Analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent Pre-Analytical conditions.

  • influence of storage conditions and extraction methods on the quantity and quality of circulating cell free dna ccfdna the spidia dnaplas external quality assessment experience
    Clinical Chemistry and Laboratory Medicine, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Claudio Orlando, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Stefania Gelmini
    Abstract:

    BACKGROUND Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic Pre-Analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the Pre-Analytical Phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for Pre-Analytical Phase studies. METHODS Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

  • SPIDIA-RNA: second external quality assessment for the Pre-Analytical Phase of blood samples used for RNA based analyses.
    PloS one, 2014
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Hui Zhang
    Abstract:

    One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the Pre-Analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and Pre-Analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  • SPIDIA-DNA: An External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses
    Clinica chimica acta; international journal of clinical chemistry, 2013
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, C.c. Hartmann, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Ales Tichopad
    Abstract:

    Abstract Background The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. Methods SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. Results 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. Conclusions The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the Pre-Analytical Phase of DNA analysis of blood samples.

Chiara Maura Ciniselli - One of the best experts on this subject based on the ideXlab platform.

  • Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.
    Data in brief, 2016
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses – [1]; Influence of Pre-Analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

  • Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality
    Clinica chimica acta; international journal of clinical chemistry, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Abstract Background In order to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on Pre-Analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent Pre-Analytical conditions.

  • influence of storage conditions and extraction methods on the quantity and quality of circulating cell free dna ccfdna the spidia dnaplas external quality assessment experience
    Clinical Chemistry and Laboratory Medicine, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Claudio Orlando, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Stefania Gelmini
    Abstract:

    BACKGROUND Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic Pre-Analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the Pre-Analytical Phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for Pre-Analytical Phase studies. METHODS Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

  • SPIDIA-RNA: second external quality assessment for the Pre-Analytical Phase of blood samples used for RNA based analyses.
    PloS one, 2014
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Hui Zhang
    Abstract:

    One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the Pre-Analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and Pre-Analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  • SPIDIA-DNA: An External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses
    Clinica chimica acta; international journal of clinical chemistry, 2013
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, C.c. Hartmann, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Ales Tichopad
    Abstract:

    Abstract Background The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. Methods SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. Results 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. Conclusions The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the Pre-Analytical Phase of DNA analysis of blood samples.

Sara Pizzamiglio - One of the best experts on this subject based on the ideXlab platform.

  • Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.
    Data in brief, 2016
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses – [1]; Influence of Pre-Analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

  • Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality
    Clinica chimica acta; international journal of clinical chemistry, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Abstract Background In order to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on Pre-Analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent Pre-Analytical conditions.

  • influence of storage conditions and extraction methods on the quantity and quality of circulating cell free dna ccfdna the spidia dnaplas external quality assessment experience
    Clinical Chemistry and Laboratory Medicine, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Claudio Orlando, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Stefania Gelmini
    Abstract:

    BACKGROUND Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic Pre-Analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the Pre-Analytical Phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for Pre-Analytical Phase studies. METHODS Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

  • SPIDIA-RNA: second external quality assessment for the Pre-Analytical Phase of blood samples used for RNA based analyses.
    PloS one, 2014
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Hui Zhang
    Abstract:

    One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the Pre-Analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and Pre-Analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  • SPIDIA-DNA: An External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses
    Clinica chimica acta; international journal of clinical chemistry, 2013
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, C.c. Hartmann, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Ales Tichopad
    Abstract:

    Abstract Background The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. Methods SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. Results 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. Conclusions The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the Pre-Analytical Phase of DNA analysis of blood samples.

Paolo Verderio - One of the best experts on this subject based on the ideXlab platform.

  • Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.
    Data in brief, 2016
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses – [1]; Influence of Pre-Analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

  • Second SPIDIA-DNA External Quality Assessment (EQA): Influence of Pre-Analytical Phase of blood samples on genomic DNA quality
    Clinica chimica acta; international journal of clinical chemistry, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Hady Ibrahim-gawel, Stefania Gelmini
    Abstract:

    Abstract Background In order to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on Pre-Analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent Pre-Analytical conditions.

  • influence of storage conditions and extraction methods on the quantity and quality of circulating cell free dna ccfdna the spidia dnaplas external quality assessment experience
    Clinical Chemistry and Laboratory Medicine, 2015
    Co-Authors: Francesca Malentacchi, Mario Pazzagli, Claudio Orlando, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Stefania Gelmini
    Abstract:

    BACKGROUND Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic Pre-Analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the Pre-Analytical Phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for Pre-Analytical Phase studies. METHODS Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

  • SPIDIA-RNA: second external quality assessment for the Pre-Analytical Phase of blood samples used for RNA based analyses.
    PloS one, 2014
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Kalle Günther, Hui Zhang
    Abstract:

    One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the Pre-Analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and Pre-Analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  • SPIDIA-DNA: An External Quality Assessment for the Pre-Analytical Phase of blood samples used for DNA-based analyses
    Clinica chimica acta; international journal of clinical chemistry, 2013
    Co-Authors: Francesca Malentacchi, Lisa Simi, Mario Pazzagli, Claudio Orlando, Ralf Wyrich, C.c. Hartmann, Paolo Verderio, Sara Pizzamiglio, Chiara Maura Ciniselli, Ales Tichopad
    Abstract:

    Abstract Background The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the Pre-Analytical Phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. Methods SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. Results 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. Conclusions The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the Pre-Analytical Phase of DNA analysis of blood samples.