Quality Assurance and Quality Control in the In Vitro Fertilization Laboratory pdf

The purpose of employing quality assur-ance QA and quality control QC schemes is to ensure reproducibility in all methods and performance within the IVF laboratory 4, and thus provide co

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 labora-tory 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 devel-opment 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 assur-ance (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 More-over, the diagnostic as well as the therapeutic role of the IVF laboratory can benefit from employing QA and QC programs (5)

365

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 lab-oratory 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 pro-grams intended to ensure or improve the performance of the laboratory and thus the quality of care to patients QA includes measures such as rec-ord 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 appli-cation 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 com-promised In a prospective analysis, Cutting et al.(6) demonstrated that

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 lab-oratory practices and personnel in the new IVF lablab-oratory are comparable

to acceptable standards of other laboratories or preferably accredited/ regulatory bodies Again this ensures that patient treatment is not compro-mised 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 estab-lished IVF laboratory These include ensuring optimal laboratory performance

is consistent and maintained Their use also allows rapid and effective trouble-shooting 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 incor-porating new technologies and/or methodologies into the established IVF laboratory and thus for improving laboratory performance This is partic-ularly 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 proce-dures 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 highlight-ing trainhighlight-ing 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

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 labora-tory, 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 equip-ment before its use in clinical practice is highly recommended to ensure that

it meets the manufacturer’s specifications (6) All new major items of equip-ment 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 2 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 1 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 mam-malian embryo development Although the mouse embryo bioassay is generally considered a biologically relevant bioassay (7), it has been criti-cized 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 pre-viously been established The control incubator was located in a separate research laboratory in the hospital within the Academic Unit of Repro-ductive and Developmental Medicine, University of Sheffield Groups of

50 embryos were cultured in 50 mL microdroplets of KM3 medium (10)

Ct

C

Ct

C

Ct

C

Ct

C

C

morphology and cleavage rates were monitored daily over a period of 5 days and the percentage of embryos reaching the blastocyst stage on day 5 post-fertilization compared using Fisher’s exact test (Fig 1)

Using the QC measures outlined in Table 1 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

whereas the third incubator exhibited unacceptable fluctuations even after

incuba-tor also failed to stabilize satisfacincuba-torily (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 incu-bator (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

U.K.) The temperature of both refrigerators was consistently between 8 to

on two separate occasions Sanitization was carried out immediately accord-ing to the manufacturer’s instructions and the equipment re-tested On both occasions, the water tested negative following sanitization

Figure 1 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 Concep-tion 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,

One of the main outcomes from this study was that independent dou-ble-checking and performance validation of new equipment before use in clinical treatment, even after installation and commissioning by the manu-facturer, is very important

Figure 2 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

Con-ception Unit, Centre for Reproductive Medicine and Fertility, Sheffield, U.K All

From Ref 6

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 incu-bators continued to be tested daily, the hot blocks and heated stages

incu-bators were monitored weekly The cryopreservation equipment was also assessed after each freezing run by checking that the printed readout detail-ing 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 cost-effective 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 pur-chasing 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 <40% blastocyst development using an F1 hybrid or inbred strain This is a very low cut-off point which may not identify all embryo toxic products Furthermore, proof of testing upon delivery of each new batch of consumables/culture media is not always forthcoming (4) Once again, QC of new consumables, even those already QC-tested by the manufacturer, is recommended before their use in the IVF laboratory to ensure their suitability This is especially applicable to those consumables not specifically designed for use in IVF (e.g., gloves, some cell culture flasks, and dishes) which may be suitable for culturing other types of mammalian cells but may prove to be embryo toxic (12)

A useful method for testing consumables is the human sperm survival assay (13) This is an inexpensive and convenient bioassay, not involving the use of animals, which monitors sperm survival over hours/days in culture while in contact with the products As with the mouse embryo bioassay, consideration must be given to the conditions under which the assay is per-formed to ensure appropriate sensitivity to detect cytotoxicity of clinical significance Claassens et al (14) found that optimum sensitivity was

achieved when sperm were cultured in the absence of serum A calculated sperm motility index value of < 0.75 was used to indicate sperm toxicity, and items identified as sperm-toxic within eight hours were considered to

be of clinical significance due to close agreement with the mouse embryo bioassay (14) Validation of consumables before use in clinical treatment

is important to ensure that an alternative product can be sourced should they prove to be unsuitable If an alternative product needs to be found then sperm survival tests should be repeated as appropriate

Culture Media and Consumables Monitoring

Despite QC testing by the manufacturer and initial validation of a product,

QC testing of all new batches of culture media is important because trans-port and incorrect storage conditions can affect media quality (4) The

QC measures should include testing for endotoxin levels because their presence in embryo culture medium can cause a significant decrease in preg-nancy rates (15) The limulus amoebocyte lysate test is a simple and rapid method for checking endotoxin levels in company-manufactured media as well as the water of any in-house water purification system (6) The osmolar-ity of all new batches of culture medium should also be checked to ensure that

it is within the acceptable range based on the manufacturer’s information Similarly, all new batches of plastics and other consumables should be subject to a sperm survival test because seemingly minor changes in the manufacturing process can affect the quality of the product rendering it embryo-toxic Moreover, the manufacturers sometimes fail to report changes in specifications/manufacturing process to end users

The batch / lot numbers for all consumables and media to come into contact with gametes and embryos should be documented, preferably in each patients’ records, to ensure comprehensive tracing of every product should a problem arise (4)

Environment Validation

Involvement in the planning and design of a new IVF unit, or building/ decorating alterations to an existing unit, can save a lot of trouble further down the line At the first instance, consideration should be given to possible sources of indoor air contaminants because volatile organic compounds (VOCs) and chemical airborne contaminants (CACs) can significantly affect mouse embryo culture (2) Factors affecting the levels of internal air pollutants include the air quality outside the building, equipment, heating, ventilation and air-conditioning systems, human activities, building compo-nents and furnishings, and other temporary sources such as redecorating and repair activities When possible, consideration should therefore be given

to the site and location of the laboratories The use of air purification units

should also be considered, if necessary, to reduce the levels of airborne con-taminants To minimize the levels of VOCs and CACs when setting up the ACU, CRMF, sealants and toxic glues were avoided, and low odor specia-lized paint was used for decorating the laboratories (6) The quality of the gas supplying the incubators and the type of pipework used should also be carefully considered Class II biological safety cabinets with High efficiency particulate air-filtered air, which provide personnel as well as environmental and product protection, were also installed for all gamete and embryo hand-ling The positioning of air conditioning or purification units should be carefully considered so that the airflow of the class II biological cabinets is not compromised

Laboratory lighting should also be taken into account at the planning stage because ultraviolet (UV) light may have detrimental effects on mam-malian oocyte maturation and embryo development (16,17) Tungsten light bulbs controlled by dimmer switches can be installed to provide low-level lighting in the laboratories (6) Alternatively, UV light filtration units can

be used to filter out harmful UV wavelengths on fluorescent lights Once building works/decorating is completed, microbiology testing of the air quality by an accredited laboratory is recommended At the ACU, CRMF, swabs and settle plates were used to test the presence of fungus

or colony-forming units (CFUs) on laboratory surfaces and in the incuba-tors and flow hoods (6) Acceptable limits of air quality for a clean area were considered to be <100 CFUs and no fungus (Department of Microbiology, Royal Hallamshire Hospital, Sheffield, U.K.) and less than one CFU and no fungus within the class II f low cabinets (18) Initial validation of the air quality within the new laboratories before treatment commenced was found

to be satisfactory (Fig 4—August) Similarly, no bacterial growth was

Figure 4 Microbiological testing of air quality in the in vitro fertilization laboratories

of the Assisted Conception Unit, Centre for Reproductive Medicine and Fertility, Sheffield, U.K Abbreviation: CFUs, colony-forming units Source: From Ref 6