This chapter covers the quality assurance aspects of cfDNA testing on the level of the performing laboratory. Standards, accreditation procedures, and among others European Directives and Regulations for in vitro diagnostics (IVD) have been developed in order to maximize patient safety. ISO 15189 specifies the requirements for medical laboratories, and recently agreed on CE-IVD Regulations in Europe will become effective in the coming years and translated into national rules and legislation.
KeywordsAccreditation, Cell-free DNA-based prenatal testing, External quality assessment, ISO 15189, Noninvasive prenatal testing, Patient safety, Quality assurance, Quality, Standardization, Validation
Cell-Free DNA-Based Prenatal Testing as a Genetic Test in Laboratories
Cell-free DNA-based prenatal testing is a screening test that estimates the risk of fetal aneuploidy (trisomy 21, 18, and 13) by analyzing cell-free DNA (cfDNA) circulating in the maternal plasma . The requirements for cfDNA tests are similar as for other screening and diagnostic tests in the medical domain . This includes all steps from blood collection, transport and storage to DNA extraction and analysis, and interpretation of results .
Harmonization of procedures is needed to obtain reproducible and comparable results and minimize the risk of individual deviations and unexpected events. The term “standardization” refers to “standards.” Standards are a published set of minimum requirements for the execution of processes, designed for harmonization and to ensure safety . They are developed, written, and subsumed by experts representing all relevant parties. As standards are very often international, referral to national regulations is not made. A list of harmonized standards used to demonstrate compliance with relevant EU legislations has been published at ec.europa.eu/growth/single-market/european-standards/harmonised-standards_en .
ISO 15189 is an international standard that specifies the quality management system requirements particular to medical laboratories. The standard is provided by the International Organization for Standardization and covers all steps relevant for laboratory tests done to obtain information about the health of a patient in relation to diagnosis, treatment, or prevention of disease.
Laboratories that implement a quality management system according to ISO 15189 must fulfill management as well as technical requirements. Management requirements include, for instance, specifications for personnel, facilities, and equipment. Technical requirements include preanalytical steps, analytical procedures, and postanalytical steps. Furthermore, ISO 15189 identifies the needs and expectations of customers. Through corrective and preventive actions, it focuses on continuous quality improvement and addresses prevention of errors and nonconformities. Some of the most important parts of ISO 15189 are discussed later.
Documents, Document Control, and Documentation
Written “standard operating procedures” (SOP) have to be in place and accessible for each staff member for their respective field. They have to be updated or reconfirmed at agreed points in time (“lifecycle” check; “document control”). Laboratories have to guarantee that the content of the SOP is correct during their development, implementation, and revision.
All working steps, from sample to report, must be documented.
A laboratory is required to perform internal audits in each section at least once a year. An audit aims at controlling compliance to the ISO guidelines and to the specified procedures, and at providing information about eventual actions needed to improve processes and quality.
The validation process ensures that the results a laboratory issues are compared to a “standard” and are thus trustworthy. A framework for validation of clinical molecular genetic tests has been described by Mattocks et al. . There are different possibilities for validating a method/process. The most common ways are to compare the results with an already validated method, to perform a cross-validation of results with another laboratory or reference institute, and to participate in an external quality assessment scheme ( Chapter 11 ).
Validation studies should comprise all stages of a method or process and should especially focus on critical steps. In cfDNA-based testing preanalytical steps, which are operator dependent, and evaluation of results with bioinformatics programs, are part of the validation process.
Almost all methods in laboratories require special instruments. These instruments must be qualified, which means that their functioning has been proven.
Personnel and Training
Skilled staff has to be available. The laboratory is in charge of identifying the need for staff and to train the staff both in performing tests and in complying to and contributing to the quality environment of the laboratory.
It is impossible to fully prevent nonconformities, deviations from instructions, and other adverse events in a laboratory. Therefore part of the ISO focuses on appropriate plans and options to correct errors and prevent them from reoccurring. To achieve this goal, detailed root-cause analyses have to be performed and appropriate corrective and preventive actions must be defined afterward. Individuals should not be blamed for errors and adverse events should be addressed as an opportunity to improve processes and procedures.
Internal Quality Control
Laboratories should implement internal quality control procedures that detect errors occurring during handling and check proper functioning of instruments. For example, including negative controls enables to detect contamination and including positive controls ascertains successful handling of samples.
In general, internal quality controls should provide information about potential nonconformities before a result is reported to a patient.
External Quality Assessment
The last aspect of ISO is EQA, also known as proficiency testing (PT). EQA is discussed in more detail in Chapter 11 . In ISO 17043 EQA has been defined as “evaluation of participant performance against preestablished criteria by means of inter-laboratory comparison” . In general, participation in EQA schemes represents the last major step in the entire validation process. It is educational and allows long-term, retrospective analysis of laboratory performance. It is a key strategy for comparing analytical test performance between laboratories , permits laboratories to compare results with their peers, and can reveal intermethod variability . Regular participation in EQA schemes enables laboratories to improve their test performance and correlates with higher quality assurance scores . A review by Hoeltge et al. on three rounds of PT results with data from 6300 accredited laboratories revealed that an increase in number of participations reduced the error rate . Haselmann et al. reported that the error rate within a molecular genetic EQA scheme for genotyping significantly decreased over the years, demonstrating that the performance of laboratories increased. Additionally, the ability of EQA schemes to improve the quality of diagnostics was proven by the decreased error rate of samples that were provided twice .
In principle, every laboratory is allowed to participate in EQA schemes and the choice of a suitable EQA provider lies with the laboratory, unless otherwise dictated by legislation. Lists of EQA providers and their available schemes are available to help laboratories to identify an appropriate EQA provider. To the best of the author’s knowledge, the listing on the Eurogentest and IFCC websites cover all European and globally active providers for molecular genetic diagnostics and their representative Schemes .
With respect to PT for cell-free DNA-based prenatal testing, ISO 15189 requests that an EQA covers the entire process from preanalytical sample handling to sample analysis and interpretation and reporting .
The International Society for Prenatal Diagnosis has recommended that “providers of cfDNA testing utilize laboratory services that meet national guidelines for quality control and proficiency testing” . Implementation of a quality management system according to ISO 15189, although voluntary, is the gold standard for medical laboratories and has been recommended for cfDNA testing . It represents a “formal recognition by an authoritative body that a laboratory has the competence to carry out specific tasks” .
Each country has a relevant authoritative body that provides a list of all accredited laboratories. This authoritative body is responsible for the entire accreditation process including surveys by specialized auditors and correction of potential nonconformities. The specialized auditors have the expertise to authenticate the executed test validation and the reliability of results. Laboratories performing successfully receive a certificate that can be presented to the users.
Accreditation offers many benefits, not restricted to benefits to the laboratories. The most important is that the person requesting a test can trust the validity of the results because accreditation focuses on the use of validated methods and on the appropriate use of procedures and test reagents. The test-requesting person can be assured that proper procedures are in place to continuously guarantee and improve quality. On the side of the laboratory, accredited laboratories gain competitive advantages over nonaccredited laboratories by giving objective proof of quality and competence. In addition, accreditation can prevent costs associated with multiple assessments, because the authoritative bodies of other countries accept the national accreditation, hence leading to international recognition of the accredited laboratory .
Once a laboratory has been successfully accredited, it is subject to regular reassessments in order to ensure the retention of compliance to the ISO.
Quality management systems of medical laboratories are subject to regulations, standards, and accreditation. National and international regulations set the obligatory requirements. Standards, although usually voluntary, provide specific instructions. The implementation of standards leads to accreditation, which testifies of conformity assessed by an impartial party ( Fig. 1 ).
Quality Assurance of Cell-Free DNA-Based Prenatal Testing Laboratory Procedures
In the following section, we take a closer look at the single steps of the cfDNA test laboratory process and the criteria to be fulfilled to assure quality according to ISO 15189 for cell-free DNA-based prenatal testing. The general requirements outlined in the previous sections also apply to cell-free DNA-based prenatal testing ( Fig. 2 ).