17 Quality Assurance and Quality Control in the In Vitro Fertilization Laboratory Karen L Martin Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, South Yorkshire, U.K INTRODUCTION The main objective of any in vitro fertilization (IVF) unit is to provide good service for their patients Although this can be interpreted in several ways, it is usually perceived as achieving a live birth (1) One of the most important factors contributing to this outcome is the performance of the IVF laboratory responsible for the creation of embryos from patients’ gametes The viability of the embryos created and transferred into the woman’s uterus is a major determinant in establishing a successful pregnancy As the development and viability of gametes and embryos in vitro is compromised by contaminants and relatively small fluctuations in the environment, such as temperature and pH (2,3), it is imperative that optimal culture conditions for gametes and embryos are attained and maintained in the IVF laboratory to ensure the best care for patients The purpose of employing quality assurance (QA) and quality control (QC) schemes is to ensure reproducibility in all methods and performance within the IVF laboratory (4), and thus provide consistent, optimal conditions for gamete and embryo culture Moreover, the diagnostic as well as the therapeutic role of the IVF laboratory can benefit from employing QA and QC programs (5) 365 366 Martin WHAT ARE QA AND QC? QC involves measures and activities undertaken to control the quality of products, methods, equipment, and environment to ensure that the laboratory is functioning correctly, and is carried out to prevent undetected problems leading to a compromise in service to the patient Internal quality control (IQC) refers to those measures undertaken on site QA is a comprehensive program that includes all activities and programs intended to ensure or improve the performance of the laboratory and thus the quality of care to patients QA includes measures such as record keeping, evaluation and education of laboratory staff, results reporting, treatment auditing, incident reporting, etc., as well as QC methodology Both QA and IQC are systems that look within a particular testing site In comparison, external quality assessment (EQA) looks at differences between different sites testing the same analyte to ensure continuity of patient care between institutions and/or to ensure standards meet those of accredited/regulatory bodies WHY EMPLOY QA AND QC SCHEMES IN THE IVF LABORATORY? Until relatively recently, QA and QC programs were little employed within IVF laboratories despite their obligatory use and proven benefits in other types of clinical facilities Although this was originally attributed to IVF being a new field of medicine which did not fall under the auspices of any accreditation/regulatory authorities, 25 years later, changing legislation in some countries and/or the need to raise standards to compete with other units is leading to greater numbers of IVF laboratories employing QA and QC schemes Furthermore, QA and QC programs have not only been shown to benefit the running of an existing IVF laboratory (4,5), but are also invaluable when setting up new facilities (6) The benefits and application of QA and QC schemes will therefore be discussed in relation to both the new and the established IVF unit Benefits of Employing QA and QC Schemes When Setting Up a New IVF Laboratory When setting up a new IVF laboratory, or even making alterations to an existing laboratory, there are usually a large number of new and untested variables which should be validated before use in clinical practice This may apply to equipment, methodology, environment, consumables, and / or culture media The use of QA and QC to test these new variables will ensure that optimal standards of practice are attained from the outset in the new IVF laboratory, thus ensuring that patient treatment is not compromised In a prospective analysis, Cutting et al.(6) demonstrated that Quality Assurance and Quality Control in the IVF Laboratory 367 employment of a QC scheme identified problems with equipment before treatment commenced when setting up the Assisted Conception Unit (ACU), Center for Reproductive and Developmental medicine (CRMF), Sheffield Teaching Hospitals Foundation Trust, Jessop Wing, Sheffield, U.K These problems are likely to have led to a compromise in pregnancy rates if they had gone unchecked As such, QC can give confidence to the scientific staff and thus the whole IVF team that gamete and embryo development are not compromised by new laboratory facilities and procedures The use of EQA where possible is also important to ensure that laboratory practices and personnel in the new IVF laboratory are comparable to acceptable standards of other laboratories or preferably accredited/ regulatory bodies Again this ensures that patient treatment is not compromised at the expense of a learning curve and gives confidence to the IVF team from the outset Benefits of Employing QA and QC Schemes in an Established IVF Laboratory There are several advantages of employing QA and QC schemes in the established IVF laboratory These include ensuring optimal laboratory performance is consistent and maintained Their use also allows rapid and effective troubleshooting should there be a problem such as a decrease in fertilization rate As such, QC especially allows the staff to determine whether the cause lies within the laboratory, and if so, where the problem resides (4) Identifying problems through QC should therefore lead to an improvement in work processes and protocols More importantly, a QC scheme should detect problems before they become significant and impact on the care of patients In addition to the above, QA and QC are also useful when incorporating new technologies and/or methodologies into the established IVF laboratory and thus for improving laboratory performance This is particularly applicable to the field of assisted reproductive technology as new developments are frequently emerging Validation and monitoring of new methods and techniques is important as the introduction of new procedures can compromise the quality of service to patients The effectiveness of new techniques and protocols can be readily assessed with QA and QC measures giving confidence to the team upon implementation of the new procedures In turn, moving forward confidently with new developments educates and thus motivates the team as well as improving patient care The use of EQA in the existing laboratory is important for evaluating personnel performance as part of their career development, thus highlighting training and educational needs, which in turn should also motivate staff Employing EQA to check whether laboratory performance meets specified requirements also increases customer confidence and the credibility of the IVF unit placing it in a more competitive position with its counterparts 368 Martin WHAT COMPRISES A QC PROGRAM? A QC program involves the monitoring of: (i) equipment; (ii) culture media and consumables; (iii) the environment; and (iv) protocols When setting up a new IVF unit or introducing new changes to an already established laboratory, validation of any new variables is required, which once established to be satisfactory should then be regularly monitored All testing, whether it is the initial validation or routine monitoring, should take place at an adequate frequency using appropriate, accurate, reliable, and preferably accredited testing methods Furthermore, all aspects of the testing should be accurately documented Equipment Validation All new and major items of equipment (e.g., flow hoods, incubators, etc.) should be installed by the manufacturer and properly commissioned with certification Even then, internal validation using QC measures of the equipment before its use in clinical practice is highly recommended to ensure that it meets the manufacturer’s specifications (6) All new major items of equipment should be switched on monitored regularly allowing sufficient time for performance validation to ensure that problems are clearly identified and rectified before use in clinical treatment The time required to validate an item of equipment will vary depending on its function In setting up the ACU, CRMF in Sheffield, a period of months was allowed to run and test new major items of equipment after their installation before employing them in clinical practice (6) As a result, problems were identified and solved with major items of equipment that otherwise would not have been detected if testing had lasted only a relatively short period of time Table summarizes the types of equipment tested in situ within the ACU, CRMF, the type of test used and the frequency of testing In addition to the tests listed, a mouse embryo bioassay was also used to assess the incubators before use in clinical treatment for their ability to support mammalian embryo development Although the mouse embryo bioassay is generally considered a biologically relevant bioassay (7), it has been criticized for being insufficiently sensitive (8) However, its sensitivity can be manipulated by taking into account the strain of mouse used, the stage of embryo retrieval and culture, and the type of culture medium used (4,9) To test the three incubators in the ACU, CRMF, mouse 2-cell embryos of the MF-1 strain were randomly divided between these and a fourth (control) incubator in which successful mouse embryo development had previously been established The control incubator was located in a separate research laboratory in the hospital within the Academic Unit of Reproductive and Developmental Medicine, University of Sheffield Groups of 50 embryos were cultured in 50 mL microdroplets of KM3 medium (10) overlaid with liquid paraffin oil at 5% CO2 in air and 37 C (11) Embryo Heraeus Vivendi LEC MATa Huntera Hunter Grant Gallenkamp Planar Incubator Water system Refrigerator Heated stages Cryopreservation equipment Integral to class II f low hoods Source: From Ref a Hot blocks Hot oven Manufacturer Equipment Kryo 360 LR500 BioMAT2 Hunter N24 200AIN QBT2 OVB-307 Purelab Maxima BB6220 Model Function Every use Daily Monthly Monthly Monthly Monthly Every use Weekly Daily As required Weekly CO2 (%) Temperature Environment Endotoxin Temperature Temperature Temperature Temperature Temperature Temperature Frequency of testing Test Unit, Centre for Reproductive and Developmental Medicine, Sheffield, U.K Fyrite gauge Max/min thermometer Mouse bioassay Limulus Amoebocyte Lysate test Max/min thermometer Surface thermometer Surface thermometer Surface thermometer Mercury thermometer Temperature indicator strips Test run with medium only Method n/a 10 C to ỵ50ặ 1 C 50 C to ỵ150ặ 0.1 C As above As above 20 C to ỵ150ặ 5 C 121 C to 160ặ 2% 5ặ 0.5% 10 C to ỵ50ặ 1 C n/a 0.03 EU/mL Rang accuracy Table Internal Quality Control Measures Used to Assess the Performance of New Equipment Installed in the Assisted Conception Quality Assurance and Quality Control in the IVF Laboratory 369 370 Martin Figure The percentage of 2-cell mouse embryos of the MF-1 strain reaching the hatched blastocyst stage in three new incubators newly installed in the Assisted Conception Unit, Centre for Reproductive Medicine and Fertility, Sheffield, U.K and a fourth, control incubator located in the Academic Unit of Reproductive Medicine and Fertility, University of Sheffield Ãp < 0.05 Fisher’s exact test Source: From Ref morphology and cleavage rates were monitored daily over a period of days and the percentage of embryos reaching the blastocyst stage on day postfertilization compared using Fisher’s exact test (Fig 1) Using the QC measures outlined in Table and the mouse embryo bioassay, several problems were identified with the new equipment installed and commissioned by the manufacturers in the ACU, CRMF, including the incubators, refrigerators, and the water purification system (6) The temperature of two of the incubators took more than ten weeks to stabilize at 37 C, whereas the third incubator exhibited unacceptable fluctuations even after this period of time (Fig 2) Furthermore, the CO2 levels in the third incubator also failed to stabilize satisfactorily (Fig 3) The sub-optimal performance of the third incubator was confirmed by the mouse embryo bioassay The proportion of mouse embryos reaching the hatched blastocyst stage using this instrument was significantly lower than that achieved by the control incubator (p < 0.05, Fisher’s exact test; Fig 1) The QC measures also identified a problem with two of the refrigerators which both failed to maintain the correct temperature for storing embryo culture medium (i.e., 2–8 C; Medicult, U.K.) The temperature of both refrigerators was consistently between to 10 C The ultra-pure water tested positive for endotoxins (> 0.03 EU/mL) on two separate occasions Sanitization was carried out immediately according to the manufacturer’s instructions and the equipment re-tested On both occasions, the water tested negative following sanitization Quality Assurance and Quality Control in the IVF Laboratory 371 Figure Temperature recordings from three incubators newly installed in the Assisted Conception Unit, Centre for Reproductive Medicine and Fertility, Sheffield, U.K All readings were carried out daily using a using a maximum /minimum thermometer (À10 C to ỵ50 ặ 1 C) Source: From Ref One of the main outcomes from this study was that independent double-checking and performance validation of new equipment before use in clinical treatment, even after installation and commissioning by the manufacturer, is very important Figure CO2 recordings from three incubators newly installed in the Assisted Conception Unit, Centre for Reproductive Medicine and Fertility, Sheffield, U.K All readings were carried out weekly using a fyrite gauge (10 C to ỵ50 Æ 1 C) Source: From Ref 372 Martin Equipment Monitoring Following installation and validation, continuous and regular monitoring of the equipment once in clinical use is imperative to ensure its performance is maintained and that problems are detected as they occur In the ACU, CRMF, independent temperature recordings of the refrigerators and incubators continued to be tested daily, the hot blocks and heated stages monthly, and the hot oven every time it was used; the CO2 levels in the incubators were monitored weekly The cryopreservation equipment was also assessed after each freezing run by checking that the printed readout detailing the embryo cryopreservation program followed the specified program details (6) Taking into account the manufacturers’ recommendations on frequency of servicing, all equipment was also placed on a service contract (6) Regular servicing of items of equipment minimizes the risk of equipment malfunction and, although seemingly expensive at the outset, is usually costeffective in the long-term Culture Media and Consumables Validation Certification of product QC testing by the manufacturer should be obtained for all consumables used in the laboratory Given the increasing wide choice and availability of culture media and other chemicals/solutions for human IVF, it is becoming less acceptable to make such consumables in-house unless they are rigorously assessed using QC However, even when purchasing company-manufactured media, detailed inspection and critical evaluation of a company’s QC program is essential to ensure that it meets appropriate standards for IVF For example, some manufacturers lay claim to using the mouse embryo bioassay to test their products However, their definition of cytotoxicity may be