Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples.

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy.

Quality assurance practices in Europe: a survey of molecular genetic testing laboratories.

In the 2000s, a number of initiatives were taken internationally to improve quality in genetic testing services. To contribute to and update the limited literature available related to this topic, we surveyed 910 human molecular genetic testing laboratories, of which 291 (32%) from 29 European countries responded. The majority of laboratories were in the public sector (81%), affiliated with a university hospital (60%). Only a minority of laboratories was accredited (23%), and 26% was certified. A total of 22% of laboratories did not participate in external quality assessment (EQA) and 28% did not use reference materials (RMs). The main motivations given for accreditation were to improve laboratory profile (85%) and national recognition (84%). Nearly all respondents (95%) would prefer working in an accredited laboratory. In accredited laboratories, participation in EQA (P<0.0001), use of RMs (P=0.0014) and availability of continuous education (CE) on medical/scientific subjects (P=0.023), specific tasks (P=0.0018), and quality assurance (P<0.0001) were significantly higher than in non-accredited laboratories. Non-accredited laboratories expect higher restriction of development of new techniques (P=0.023) and improvement of work satisfaction (P=0.0002) than accredited laboratories. By using a quality implementation score (QIS), we showed that accredited laboratories (average score 92) comply better than certified laboratories (average score 69, P<0.001), and certified laboratories better than other laboratories (average score 44, P<0.001), with regard to the implementation of quality indicators. We conclude that quality practices vary widely in European genetic testing laboratories. This leads to a potentially dangerous situation in which the quality of genetic testing is not consistently assured.

Improvement of interpretation in cystic fibrosis clinical laboratory reports: longitudinal analysis of external quality assessment data.

Participation in external quality assessment (EQA) is a key element of quality assurance in medical laboratories. In genetics EQA, both genotyping and interpretation are assessed. We aimed to analyse changes in the completeness of interpretation in clinical laboratory reports of the European cystic fibrosis EQA scheme and to investigate the effect of the number of previous participations, laboratory accreditation/certification status, setting and test volume. We distributed similar versions of mock clinical cases to eliminate the influence of the difficulty of the clinical question on interpretation performance: a cystic fibrosis patient (case 1) and a cystic fibrosis carrier (case 2). We then performed a retrospective longitudinal study of reports over a 6-year period from 298 participants for case 1 (2004, 2008, 2009) and from 263 participants for case 2 (2006, 2008, 2009). The number of previous participations had a positive effect on the interpretation score (P<0.0001), whereas the laboratory accreditation/certification status, setting and test volume had no effect. Completeness of interpretation improved over time. The presence of the interpretation element 'requirement for studying the parents to qualify the genotype' increased most (from 49% in 2004 to 93% in 2009). We still observed room for improvement for elements that concerned offering testing for familial mutations in relatives and prenatal/preimplantation diagnosis (16% and 24% omission, respectively, for case 1 in 2009). Overall, regular participation in external quality assessment contributes to improved interpretation in reports, with potential value for quality of care for patients and families by healthcare professionals involved in genetic testing.

Mutation nomenclature in practice: findings and recommendations from the cystic fibrosis external quality assessment scheme.

Currently, two nomenclature systems are in use to describe sequence variants for cystic fibrosis: the established traditional nomenclature system and the more recent Human Genome Variation Society (HGVS) nomenclature system. We have evaluated the use of both systems in the laboratory reports of 217 participants in the cystic fibrosis external quality assessment scheme of 2009. The mutation c.1521_1523delCTT (p.Phe508del, F508del) was described by traditional and HGVS nomenclature by 32 of 216 (15%) laboratories that correctly identified the mutation, whereas 171 (79%) laboratories used traditional nomenclature only and 13 (6%) laboratories used HGVS nomenclature only. Overall, 29 of 631 (5%) reports used nomenclature that was evaluated as being seriously incorrect and/or misleading and 136 (22%) reports contained attempts at HGVS coding, of which 104 (76%) contained no coding errors; just 33 (24%) mentioned the correct cDNA name and cited the nucleotide reference sequence. We recognized an urgent need for more consistent and correct usage of nomenclature. We recommended that cystic fibrosis transmembrane conductance regulator testing reports should include a description of the identified sequence variants in both HGVS and traditional nomenclature and provided basic recommendations and other guidance.

Customer satisfaction survey to improve the European cystic fibrosis external quality assessment scheme.

BACKGROUND: The Cystic Fibrosis European Network, coordinated from within the Katholieke Universiteit Leuven, is the provider of the European cystic fibrosis external quality assessment (EQA) scheme. The network aimed to seek feedback from laboratories that participated in the cystic fibrosis scheme in order to improve services offered. In this study we analysed responses to an on-line customer satisfaction survey conducted between September and November 2009. METHODS: The survey was sent to 213 laboratories that participated in the cystic fibrosis EQA scheme of 2008; 69 laboratories (32%) responded. Scores for importance and satisfaction were obtained from a five-point Likert scale for 24 attributes. A score of one corresponded to very dissatisfied/very unimportant and five corresponded to very satisfied/very important. Means were calculated and placed in a two-dimensional grid (importance-satisfaction analysis). Means were subtracted from each other to obtain gap values (gap-analysis). RESULTS: No attribute had a mean score below 3.63. The overall mean of satisfaction was 4.35. Opportunities for improvement enclosed clarity, usefulness and completeness of the general report and individual comments, and user-friendliness of the electronic datasheet. CONCLUSIONS: This type of customer satisfaction survey was a valuable instrument to identify opportunities to improve the cystic fibrosis EQA scheme. It should be conducted on a regular basis to reveal new opportunities in the future and to assess effectiveness of actions taken. Moreover, it could be a model for other EQA providers seeking feedback from participants. Overall, the customer satisfaction survey provided a powerful quality of care improvement tool.

Multiallelic synthetic quality control material: lessons learned from the cystic fibrosis external quality assessment scheme.

AIM: With the arrival of increasingly complex molecular tests, we are obliged to create new ways to monitor and troubleshoot the underperformance of these multiplex assays. A synthetic multiallelic quality control material has been designed to augment genomic DNA controls. We aimed to evaluate the control on a large scale, testing it on a wide variety of oligonucleotide ligation assays, test protocols, and analysis software. In addition, we investigated how laboratories treat untried and complex materials. METHODS: The synthetic control monitored 32 cystic fibrosis transmembrane conductance regulator mutations and polymorphisms simultaneously. Participants of a cystic fibrosis external quality assessment scheme were invited to analyze the quality control. RESULTS: In total, 58 laboratories participated in this study. Twenty-seven (47%) laboratories detected 32 variants; another 27 laboratories (47%) detected from 31 to 4 variants and 4 participants reported no variants (6%). The main observations included administrative errors when indicating variants on a checklist, errors caused by misreading the instructions for use of the control or assay, and technical problems related to the assay used. CONCLUSION: Synthetic quality control materials proved to be valuable in troubleshooting underperforming assays and complement existing genomic controls. The study also revealed a strong need for increased quality control in the postanalytical phase of testing.

Approaches to quality management and accreditation in a genetic testing laboratory.

Medical laboratories, and specifically genetic testing laboratories, provide vital medical services to different clients: clinicians requesting a test, patients from whom the sample was collected, public health and medical-legal instances, referral laboratories and authoritative bodies. All expect results that are accurate and obtained in an efficient and effective manner, within a suitable time frame and at acceptable cost. There are different ways of achieving the end results, but compliance with International Organization for Standardization (ISO) 15189, the international standard for the accreditation of medical laboratories, is becoming progressively accepted as the optimal approach to assuring quality in medical testing. We present recommendations and strategies designed to aid genetic testing laboratories with the implementation of a quality management system, including key aspects such as document control, external quality assessment, internal quality control, internal audit, management review, validation, as well as managing the human side of change. The focus is on pragmatic approaches to attain the levels of quality management and quality assurance required for accreditation according to ISO 15189, within the context of genetic testing. Attention is also given to implementing efficient and effective quality improvement.

Variability in the use of CE-marked assays for in vitro diagnostics of CFTR gene mutations in European genetic testing laboratories.

DNA diagnostics of genetic diseases increasingly shifts towards utilization of commercial assays. Cystic fibrosis (CF)-related DNA diagnostics were used as a model for a pilot survey of the variability in the utilization of qualitative CE-marked in vitro diagnostic (IVD) assays and the scale of their modification by end users. A structured questionnaire, developed in the context of the EuroGentest project, was distributed within the frame of the 2005 annual CF external quality assessment (EQA) scheme. Its aim was to evaluate the variability in the use of different CE-marked IVD assays in routine CF DNA diagnostics. Survey results were analysed and sequentially discussed with respective users and/or manufacturers. In total, 125 responses from EQA scheme participants were received. Almost half of the respondents modified manufacturer-recommended protocols. They also reported sporadic and/or recurrent problems with assay performance and genotyping of particular alleles. Nonetheless, only half of the respondents performed in-house verification before the implementation of the assay in clinical diagnostics and/or after modification of the recommended protocol. Results of this survey substantiate the importance of guidelines for proper verification of CE-marked IVD assays in DNA diagnostics, using CF as a model.

Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis.

Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material.