SPIDIA-DNA: An External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses

Abstract

a b s t r a c t a r t i c l e i n f oBackground: 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.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: The SPIDIA experience

“…Details of the adopted statistical procedure are reported by Malentacchi et al [20]. Briefly, a two-step statistical procedure based on a distribution-free approach was adopted in order to process the quality parameter variables.…”

“…The analysis of DNA integrity has been performed by analyzing PFGE gel with the ImageJ imaging software (www.rsbweb.nih.gov/ij), as described by Malentacchi et al [20]. The ImageJ peak corresponding to the size of DNA fragments that are most represented in the sample, was used for the evaluation of gDNA integrity.…”

“…Evaluation of High Molecular Weight DNA integrity by ImageJ software 157 SPIDIA DNA samples have been analyzed using ImageJ software in order to obtain numerical data from gel electrophoresis pictures. The procedure has been already reported by Malentacchi et al [20]. The distribution of the ImageJ peak (kb) in the SPIDIA DNA samples is reported in Fig.…”

“…We have already reported the description and the results of 1st SPIDIA DNA EQA [20], a large, Pan European trial, involving 197 laboratories from 29 countries. The aim of this trial was to investigate the influence of blood storage on gDNA quality and quantity.…”

“…The aim of this paper is to deeply investigate the role of gDNA fragmentation measured in the 1st SPIDIA DNA EQA samples on some considered pre-analytical factors and its influence on the results of an analytical multiplex PCR test based on semi-quantitative long range PCRs to detect and characterize the repertoire recombination of human T lymphocyte receptor [21,22] [20]. Blood was collected from two consented, adult donors (tested negative for HIV, HBV and HCV) after approval by the Institutional Committee of Azienda Ospedaliero-Universitaria Careggi (Florence, Italy).…”