Accreditation of flow cytometry in Europe†
How to cite this article: Sack U, Barnett D, Demirel GD, Fossat C, Fricke S, Kafassi N, Nebe T, Psarra K, Steinmann J, Lambert C. Accreditation of flow cytometry in Europe. Cytometry Part B 2013; 84B: 135–142.
ISO 15189 has been introduced to enable any clinical laboratory, irrespective of geographic location, to be accredited against internationally recognized standards and therefore facilitate direct international comparison of laboratories. Together with increasing use of ISO 15189 for standardization and competition purposes, often triggered by demands of patients and clinicians, clinical flow cytometry laboratories are becoming increasingly challenged to introduce compliant quality management systems. Whilst in most countries, ISO 15189 accreditation is not yet compulsory, there is increasing evidence to suggest that the implementation of this standard is growing. As a result, the European Society of Clinical Cell Analysis (ESCCA) has analysed the impact of accreditation in clinical flow cytometry laboratories. It found, through a discussion forum, that staff qualification, adaptation of multicolour antibody panels, and implementation of a comprehensive quality system (including quality assessment) have been identified as major challenges. © 2013 International Clinical Cytometry Society
Flow cytometry is now considered part of the routine repertoire in most clinical diagnostic laboratories for the analysis of peripheral blood cells in variety of disease states. However, in contrast to other laboratory-based procedures, flow cytometry, due to its high complexity, have not yet been standardized to the same level as many other laboratory diagnostics. Twenty years ago, the United Kingdom introduced a voluntary program of accreditation through Clinical Pathology Accreditation (UK) Ltd. with similar programs being introduced in North America. While these efforts to introduce accreditation were embraced in the relevant geographical regions, there was little standardization between the processes involved. However, in recent years several ISO standards have been introduced, ISO 15189 (developed and for medical laboratories from the standard ISO/IEC 17025) and ISO/IEC 17043 that is specific for providers of external quality assessment (EQA or proficiency testing (PT)) programs with the aim of facilitating parity in accreditation processes and thus ensuring that laboratories in different geographical regions that achieve ISO 15189 accreditation operate in a similar manner and provide similar levels of service. However, passing an accreditation process is still a real challenge and involves an enormous amount of work and effort by all staff involved.
WHAT IS ACCREDITATION?
Accreditation, “a third-party recognition of competence to perform specific tasks,” attests that a laboratory has been successful in meeting the requirements of international accreditation standards (1). For medical laboratories, confirms the successful implementation of elements of a comprehensive quality management system according to ISO 15189:2007. The standard ISO 15189 “Medical laboratories—Particular requirements for quality and competence” was first published in 2003 by the International Organization of Standardization and is the first internationally accepted set of requirements specifically designed for the medical laboratory (2, 3).
ISO 15189 is specific to clinical laboratories and was adopted to fit conditions in clinical laboratories. In contrast, ISO 9001 is a certification standard providing generic criteria for quality management systems, originally and most often applied as manufacturing standard (for industry). When used for certification purposes, the formal attestation of compliance with ISO 9001 does not imply any attestation of competence.
Clinical flow cytometry is usually placed within an accredited clinical sector, for example in immunology or hematology. However, ISO 15189 does not include any specific flow cytometry standards or criteria.
Accreditation should demonstrate that the methods and techniques used are used in the proper application, respond to the requirements for patient care, and that the laboratory is fully competent in the specific method. However, accreditation also helps to improve laboratory management by providing a better understanding of the laboratory structures and processes to achieve a consistently high level of quality every day. It helps reduce the risk of failure and enhances confidence from patients, physicians, hospitals, and contract partners. It is assumed that accredited laboratories produce quality assessment (QA) reproducible results. Furthermore, by clarifying and formalizing its procedures, the laboratory will also improve its governance, process transparency, and diagnostic support, but in the main, diagnostic quality will be improved (4). So, all risks and incertitude due to the daily practice of a diagnostic laboratory can be better controlled and routine can be improved by the accreditation process. Accreditation impels the tracking of samples, reagents, and processes and keeps a record for possible retrospective enquiry from the preanalytical to the postanalytical phases.
It should be stressed at this point that accreditation is different from certification; accreditation is awarded directly by the national accreditation body, not by accredited certification sites. The European Cooperation for Accreditation (EA) is a nonprofit organization that networks national accreditation bodies located in Europe. The members of the EA have signed a multilateral recognition agreement to recognize equivalence and reliability. Therefore, there is an agreement on accreditation across Europe. The International Laboratory Accreditation Cooperation (ILAC) is the international umbrella organization that covers all national and regional accreditation organizations in the field of laboratories and inspection bodies.
Accreditation is also not standardization. Standardization of methods aims to homogenize processes, instruments, and reagents to get highly similar results. This may imply the choice of common tools or parameters and is of course of very high interest for sharing data especially in interlaboratory trials. In contrast, accreditation means that the lab has established processes that lead to robust and comparable data generation, regardless of the process used to generate that data. Accreditation does not dictate that all laboratories should work in the exactly same way, but ensures that quality procedures are in place to monitor the processes and identify and address any untoward instances to improve their services and prevent errors. Therefore, laboratories intending to obtain and retain accreditation must formalise all procedures relevant to reaching such a quality level and should maintain records with reference to international standards. Accreditation is the confirmation by a national accreditation body that these processes are in use.
However, ISO 15189 requirements go far beyond the typical quality assessment (QA) measures in the analytical phase of medical laboratory analysis. The standard includes criteria for the examination of management competence and impartiality, review of contracts, competency of personnel as result of qualification, training, compliance evaluation, competency assessment and professional development, environmental conditions, laboratory equipment, the preanalytical phase, analytical procedures, and the postanalytical phase including reporting of results. The aim of accreditation according to ISO 15189 is to provide assurance, reliability, and reproducibility of test results for patients and laboratory staff independent of location and thus, in turn, drive up quality. It is a generic standard designed to be applied to any clinical laboratory irrespective of the offered medical laboratory disciplines or tests.
Essentially, ISO 15189 accreditation is divided into two distinct areas: management and technical. The former details the general principles that need to be applied by laboratory management with specific emphasis on the quality management system, education, and training. The technical section covers every stage of laboratory work ranging from system implementation and selection to participation in EQA programs, particularly emphasizing that participation in ISO/IEC 17043 accredited EQA programmes is recommended.
For each test undertaken, the laboratory should have a written policy to identify and define potential errors and risks so that these are dealt with in an appropriate and timely fashion. Therefore, these standards are designed to ensure that every clinical laboratory, irrespective of geographical location, has a prescribed and agreed set of protocols. Exactly how these standards are implemented is the responsibility of each laboratory providing that there is documentary and auditable evidence for standard compliance. For example, ISO 15189 does not state how to implement or write a Quality Management System (QMS) or Standard Operating Procedures (SOP), but it does define the minimum criteria expected for such documents and that they must be in place to achieve accreditation: in essence, write down exactly what you do, do what is written, and document that you have done it (5). The accreditation procedure therefore helps formalise the process.
Although ISO 15189 is not compulsory across the EU, countries such as France, Germany, Italy, Spain, and the United Kingdom, have driven the adoption of accreditation, via professional bodies, societies, and customers. Table 1 details the agencies charged with granting accreditation. As stated earlier, ISO 15189 is not test or technique specific and therefore flow cytometry is usually, for example in the UK, accredited within the process of the department where it resides. Today, more and more laboratories working on flow cytometry are in the process of accreditation.
Table 1. National Accreditation Bodies in Europe
|Austria||BMWFJ: Federal Ministry for Economy, Family and Youth|| |
|Belgium||BELAC: Belgian Accreditation Structure|| |
|Bulgaria||BAS: Bulgarian Accreditation Service|| |
|Croatia||HAA: Croatian Accreditation Agency|| |
|Czech Republik||CAI: Ceskâ Institut pro Akreditaci, o.p.s.|| |
|Cyprus||CYS-CYSAB: Cyprus Organization for the Promotion of Quality|| |
|Denmark||DANAK: The Danish Accreditation and Metrology Fund|| |
|Estonia||EAK: Estonian Accreditation Centre|| |
|Finland||FINAS: Finnish Accreditation Service|| |
|France||COFRAC: Comite Francais d'Accreditation||GBEA Good biological practices guidelines|
|Germany||DAkkS: Deutsche Akkreditierungsstelle||Guidelines by the German Medical Association, compulsory|
|Greece||ESYD: Hellenic Accreditation System|| |
|Hungary||NAT: Hungarian Accreditation Board|| |
|Iceland||ISAC-Accreditation: Icelandic Patent Office|| |
|Ireland||INAB: Irish National Accreditation Board|| |
|Italy||ACCREDIA: Italian Accreditation Body; Regions|| |
|Kosovo||DAK: Kosovo Accreditation Directorate|| |
|Latvia||LATAK: Latvian National Accreditation Bureau|| |
|Lithuania||State Health Care Accreditation Agency under the Ministry of Health||Good laboratory practices|
|Luxembourg||OLAS: Luxembourg Office of Accreditation|| |
|FYR of Macedonia||IARM: Institute for Accreditation|| |
|Malta||NAB-Malta: National accreditation board|| |
|Montenegro||ATCG: Accreditation Body of Montenegro|| |
|Norway||NA: Norwegian Accreditation|| |
|Poland||PCA: Polish Centre for Accreditation|| |
|Portugal||IPAC: Portuguese Institute for Accreditation||Good biological practices guidelines|
|Romania||RENAR: Romanian Accreditation Association (Asociatia de Acreditare din Romania)|| |
|Russian Federation||STC-IS: Scientific Technical Centre on Industrial Safety|| |
|Serbia||ATS: Accreditation Body of Serbia|| |
|Slovakia||SNAS: Slovak National Accreditation Service|| |
|Slovenia||Ministry of health: SA: Slovenska Akreditacija|| |
|Spain||ENAC: Entidad Nacional de Acreditacion (Spain)|| |
|Sweden||SWEDAC: Swedish Board for Accreditation and Conformity Assessment|| |
|Switzerland||SAS: State Secretariat for Economic Affairs (SECO), Swiss Accreditation Service|| |
|Turkey||TURKAK: Turkish Accreditation Agency||Minimal requirements for clinical laboratories published by Ministry of Health|
|UK||UKAS: United Kingdom Accreditation Service|| |
Besides accreditation, various systems have been established with the same purpose, but under different basic conditions. These approaches do not replace accreditation and are independent of it, despite parallels in the underlying quality management approaches.
For example, in transfusion medicine, flow cytometry processes for quality control are subject to appropriate Good Manufacturing Practice (GMP) regulations. Similarly, anti-doping laboratories must follow WADA standards (6). The European Federation for Immunogenetics (EFI) has established standards (currently version 5.3) for histocompatibility testing or for cross match by flow cytometry (7). Furthermore, the British Association of Research Quality Assurance (BARQA) and the NIH/NIAID/DAIDS have published guidelines for Good Clinical Laboratory Practice (GCLP) for laboratory investigation within clinical trials (8, 9).
Some pathology disciplines have established expert groups to identify areas where guidelines are required and recommend the application of relevant national/international guidelines. There are also many peer reviewed guidelines and professional development courses in this field. They help clinical cytometrists by giving advice, explaining guidelines, and offering blueprints. This will contribute to harmonisation in quality improvement in cytometric labs (Clinical and Laboratory Standards Institute (CLSI), formerly National Committee for Clinical Laboratory Standards (NCCLS); MiFlowCyt) (10–12).
The European system of accreditation resembles in several points “good laboratory practice” (GLP) (13) as known from the United States, but includes more aspects than GLP. Interestingly, GLP is used in Europe for safety testing, e.g., during preclinical drug development, but has not been adapted for use in Europe and is not a recognized international standard.
The College of American Pathologists (CAP) accreditation program (14) also differs mainly from ISO 15189 requirements.
For U.S.-driven clinical trials applying for FDA approval, requirements should be fulfilled in those laboratories that have achieved ISO 15189. A comparison between 15189 accreditation and FDA rules can be found here (15).
STEPS TO FOLLOW WHEN UNDERGOING ACCREDITATION
Accreditation is a continuous process, starting after adequate structures and processes have been established and ongoing after the first assessment. Accreditation bodies offer support and information such as interview forms, checklists, or recommendations to smooth the process; and, many consensus documents and guidelines do exist specifically for flow cytometry, such as the global consensus protocols of the NCCLS (Global Consensus Standardization for Health Technologies) (16).
Following the decision to apply for accreditation, there are several tasks that need to be accomplished by the laboratory management. For a successful outcome, dedicated personnel are critical and there must be a quality manager in place who oversees the whole process and should be independent from the technical staff so that objectivity can be obtained. All levels of staff should be encouraged to embrace the process.
The key to success is accurate record keeping and documentation. Everything undertaken in the laboratory needs to be documented and traceable. Although it is laborious, once started and understood by the personnel, it becomes a natural component of the daily work and is beneficial for all related parties. Responsibilities for each and every position in the laboratory must be defined in detail.
The central document to the whole process is the quality manual. This must describe in detail the structure and the processes undertaken within the laboratory. Furthermore, SOPs and additional documents will describe and explain how a procedure is to be performed. SOPs may also include related activities such as how quality control is undertaken how and who interprets the results, how to recognize spurious results, and what actions to take in the case of nonconformity. It is worth establishing checklists and undertaking regular audits (vertical, horizontal, and examination) to help in structuring this paperwork and identifying gaps in procedures.
REQUIREMENTS FOR THE STAFF
The employment and training of qualified staff is a crucial requirement of accreditation and in most countries basic flow cytometry training is provided through general training of medical laboratory scientists. However, in most countries there is no specific and formal flow cytometrists training or certification. Mostly, qualification must be supported by approved certificates and maintained regularly over years of practice with each country having its own qualification system for maintaining medical laboratory scientist certification (Table 2).
Table 2. Qualification of Flow Cytometrists in Accredited Labs
|France||Medical biologist (MD, Pharma D)||French health ministry/National university boards|
|Engineers||French teaching ministry|
|Biology technician||No specific accreditation for cytometry|
|Germany||Immunologist DGfI||German Society for Immunology|
|Laboratory physician||German Medical Association|
|Greece||Medical biopathologist||Greek health ministry|
|Medical hematologist||Greek health ministry|
|Laboratory scientist||Greek registry of clinical chemists (not officially recognized) (also IFCC, EurClinChem)|
| ||No specific accreditation for cytometry|
|Italy||Not yet in place||Prospectively: SiBioc (Italian Soc. for clinical chemistry and molecular biology)|
|Lithuania||Laboratory medicine physician||Licensed by State Health Care Accreditation Agency under the Ministry of Health|
|Laboratory technician||Certification system in progress|
|Portugal||Not yet in place||No specific certification for flow cytometry|
|Slovenia||Clinical chemist||Licensed on behalf of Ministry of Health by|
|Medical biologist||Slovenian Chamber of Laboratory Medicine or Medical Chamber of Slovenia|
|Laboratory medicine physician|| |
|Laboratory technician|| |
|Medical pathologist|| |
|United Kingdom||Clinical scientist and/or biomedical scientist||Health Professions Council, but no specific qualification for flow cytometry, usually discipline driven|
Every method applied in the laboratory must be validated or verified, especially if “in-house” methods are being used. Like any diagnostic laboratory, flow cytometry laboratories must run validated tests. In contrast to most tests in clinical chemistry, only a minority of diagnostic panels can be introduced to flow cytometric laboratories without adaptations. Many tests have been developed and scientifically validated for flow cytometry without any gold standard references (Table 3). In early 2000, an initiative was established to develop reference standards for flow cytometry (EuroStandards) (17), but concluded that as the techniques were not fully validated then reference standards could not be created but rather controls, were at this stage, more likely.
Table 3. Recent Clinical Cytometric Tests
| a. Leukocyte differential (immuno diff)|
| b. Lymphocyte subsets (secondary immunodeficiencies, HIV…)|
| c. Primary immunodeficiencies (SCID, CVID…)|
| d. Lymphoma|
| e. Acute leukemia|
| f. Multiple myeloma|
| g. Stem cells (CD34)|
| h. PNH|
| i. HLA-B27|
| j. Monocyte immunoparalysis (HLA-DR on monocytes)|
|2. Nucleic acid analysis|
| a. DNA content (aneuploidy)|
| b. Cell cycle analysis|
| c. RNA content (retics, reticulated platelets)|
|3. Autoantibodies against…|
| a. Platelets (screening for platelet associated immunoglobulin or specific SASPA)|
| b. Neutrophils (IgG)|
| c. Lymphocytes (HIV)|
| d. Donor cells (flow crossmatch in transplantation)|
|4. Cellular function|
| a. Lymphocyte transformation (Scatter, CD69, BrdU…)|
| b. Cytokine secretion tests (intracellular cytokines)|
| c. NK-Assay (natural killer assay eg. with tumor target cell line K652)|
| d. Phagocytosis|
| e. Oxidative burst|
| f. Chemotaxis|
| g. Basophil degranulation (allergy test)|
| h. Serotonin release (gray platelet syndrome)|
|5. Dye binding|
| a. EMA-Test for spherocytosis|
|6. Suspension bead arrays|
Any deviation from the manufacturers' instructions must be documented and fully validated and these records must be retained. A flow cytometry laboratory applying for ISO 15189 accreditation must validate four areas: instrument validation (ISO 15189 4.6.2, 5.3.2), method validation (ISO 15189 5.5.2), Laboratory Information System validation, and validation of results (ISO 15189 5.8.13). Method validation is potentially the most difficult and time consuming. In Section 5.5.3, linearity, precision, accuracy, limit of detection, sensitivity, and specificity are listed as performance parameters for validation. Therefore, each flow cytometry laboratory needs to design, plan, and perform these validations for each test they report for patients. Once implemented and become routine for the users, methods should be reviewed on a periodic basis as dictated by the quality management system (i.e., quality control/assessment and/or audits or repeated untoward incidents) and should be monitored by the quality manager.
Furthermore, often, the object of flow cytometric analysis is to measure the diversity and variability of a widely heterogeneous cell population; for example, leukemia/lymphoma testing is especially characterized by examining the profile of antigen expression and comparing this to normal populations. The complexity of leukaemia diagnostics is dynamic and the technical approaches to undertaking this test are constantly being reviewed in accordance with the latest opinion/data, particularly with the introduction of new instruments that are capable of multiparametric analysis. There are many working groups attempting to establish international consensus documents, but the challenges are numerous and include: validation of novel tests, generation of normal values, establishing quality control mechanisms (both internal and external), development of international standards, and the harmonization of reporting results.
Instruments have to be calibrated and standardized in clearly defined terms. Calibrated results obtained from standardised instruments can be compared objectively and quantitatively with those from other laboratories. Synthetic or biological calibration particles can be used for calibration. Synthetic particles are manufactured from polymers (beads, plastic-beads, latex-beads, micro-beads). Biological particles can be stained with the same fluorochromes used for cell staining. For standardization of cytometers, both synthetic and biological particles are applicable according to the IVD specification given by manufacturers with all results plotted on a Levy-Jennings type plots to allow the identification of early problems.
INTERNAL AND EXTERNAL QUALITY CONTROL
Internal quality control by daily calibration and external control by following external assessment schemes (the latter designed to comply with ISO/IEC 17043) are essential for accreditation. ISO 15189 is a best practice laboratory standard and is not a standardization itself; standardization is driven by EQA. Thereby, cross-site conformity is ensured. In European countries, there are a variety of EQA schemes fulfilling this norm (Table 4). EQA participation creates a network for communication and can be a good tool for enhancing a national laboratory network. Samples received for EQA testing, as well as the information shared by the EQA provider, are also useful for conducting professional development activities.
Table 4. EQA Scheme Providers
|Denmark||Immunophenotyping||UK NEQAS via DEKS|
| ||Leukemia/lymphoma|| |
| ||CD34|| |
|FRANCE||Immunophenotyping||STATUS-FLOW Euro Cell diag (R&D systems)|
| ||Leukemia/lymphoma||AFSAP National agency|
| ||CD34||Task forces|
| ||PNH||Commercial EQC with interlaboratory data management UK NEQAS|
| ||Leukemia/lymphoma||INSTAND (this program offers EQA services to most European countries as an independent supplier to individual laboratories)|
| ||CD34||UK NEQAS|
| ||PNH|| |
|Greece||Leukemia/lymphoma||Helenic Cytometric Society|
| ||Immunophenotyping||UK NEQAS|
| ||CD34|| |
|Italy||No regional schemes for FCM||Small, local, non-accredited schemes but UK NEQAS delivers EQA services to 168 laboratories|
| ||CD34|| |
| ||PNH|| |
|Netherlands||No regional schemes for FCM||Participation in foreign external quality schemes (INSTAND and UK NEQAS)|
|Portugal||No regional schemes for FCM||Participation in foreign external quality schemes (INSTAND and UK NEQAS)|
|Slovenia||No regional schemes for FCM||Participation in foreign external quality schemes (INSTAND and UK NEQAS)|
|Turkey||Immunophenotyping||Yeditepe/Centro FC PT ILC|
|UK||Immunophenotyping||UK NEQAS (this program delivers EQA services in all European countries either as official programs such as in Denmark, The Netherlands, Italy, and Portugal or as an independent supplier to individual laboratories)|
| ||Leukemia/lymphoma, MRD|| |
| ||CD34|| |
| ||PNH|| |
| ||HLA crossmatch|| |
|USA||Immunophenotyping||CAP and UK NEQAS|
| ||Leukemia/lymphoma|| |
| ||CD34|| |
| ||PNH|| |
| ||DANN cell cycle|| |
| ||Fetal blood|| |
| ||ZAP70|| |
External quality control schemes usually use stabilised whole blood material to reduce the degradation of cells encountered when fresh material is used. With the introduction of ISO/IEC 17043, EQA programmes are now facing the same challenges posed by ISO 15189 in clinical laboratories and have to have fully documented operating procedures including sample stability verification, data handling and reporting, and statistically validated mechanisms to ensure identification of participants who are consistently out of consensus. EQA programmes themselves do not provide certification of either competence to undertake flow cytometry or approve certification. The EQA programs accredited to ISO/IEC 17043 are applied by approved EQA providers and independent assessments take place accordingly.
EQA programs (proficiency testing) can only cover these requirements in part, particularly as reference methods (with the exception of immunoplatelet counting) do not exist. Although the task is not straightforward, fulfilling the requirements of accreditation is the best way to improve quality in diagnostic labs. Accreditation forces a quality-driven competition between laboratories and raises standards.
WHICH PROBLEMS ARE KNOWN REGARDING FLOW CYTOMETRY ACCREDITATION?
In addition to the specific challenges with regard to accreditation in clinical flow cytometry as discussed earlier, there are other challenges that cross all laboratory disciplines. The accreditation process is expensive and time consuming. However, rules for accreditation have been established for highly standardised test systems, for example in clinical chemistry. To apply them to cytometry laboratories, interpretation of ISO 15189 should be adapted to specific points in flow cytometry, such as instrument settings, sample preparation, or performance follow-up. Thus, validation of novel tests, generation of normal values, daily quality control in the absence of international standards, activities to guarantee day-to-day stability of running tests, and interpretation of test results raise lots of challenges for cytometric laboratories. Besides checklists of accreditation bodies, a lot of reference books and articles are very helpful (16, 18–28).
WHAT IS THE OPINION OF FLOW CYTOMETRISTS ABOUT ACCREDITATION?
In 2010, ESCCA made a survey of flow cytometry specialists about accreditation. One hundred fifty-seven participants replied to the questionnaire. Interestingly, 98% agreed with the need for accreditation, although only 19% of them had to undergo accreditation by national law. However, 76% were included in at least one national or international regulatory scheme.
WHAT SHOULD BE DONE TO SUPPORT ACCREDITATION IN FLOW CYTOMETRY?
The role of scientific communities such as ESCCA is restricted to providing assistance in the form of education in how to achieve and meet these standards. On the basis of this knowledge, ESCCA should provide recommendations that support accreditation bodies when assessing competencies in the field of flow cytometry. These efforts for laboratories and accreditation bodies could include:
Recommendation for daily cytometer operation
Offering comparative calibration procedures between laboratories
Validation of novel panels and advice on how to validate cytometric protocols
Recommendation of harmonised validated and well documented antibody panels with known reference values and clear indications
Critical review of data analysis, expert systems, and gating strategies
Organization of interlaboratory comparisons of best practices
Providing an open access library of “reference cases” and variants (images and FCS files)
Furthermore, ESCCA should support EQA providers in offering relevant samples and supporting clinically relevant reporting in EQA schemes. Finally, ESCCA should support continuous certification schemes for individuals showing that they are up-to-date as well as competent to perform and understand clinical flow cytometry. However, there are many challenges to developing a specific clinical cytometry syllabus. The certificate (or exam) would have to be at a high level to attract individuals and ensure that national registration bodies could and would recognize the certificate.
We are grateful to Audrey Braun for proofreading and profoundly improving the manuscript.