Fertility Unit, Department of Obstetrics and Gynaecology, Queen's Medical Centre, B Floor, East Block, Nottingham, NG7 2UH & Assisted Conception Unit, Birmingham Women's Hospital, Edgbaston, Birmingham, B15 2TG, UK
* CorrespondenceEmail: mathew.tomlinson{at}gmc.nhs.uk
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Abstract |
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Key words: cryopreservation/embryo storage/risk management/sperm storage
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Introduction |
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The risk management of storage services is therefore becoming an increasingly sensitive and important issue, particularly with regard to: injury to personnel, sample loss, premature sample thaw and the possibility of transmission of infectious disease between samples (Tedder et al., 1995). It is of course not within the scope of this article to discuss the entire risk management process nor is it within the author's area of expertise. Suffice it to say that centres should at least begin the process by carrying out formal risk assessments for all of the various components, which contribute to the cryostorage service.
A risk assessment is defined as a method for the early identification of adverse events (hazards), which precedes a management phase, which would then include the identification and implementation of specific control measures to deal with each potential hazard. Dividing the assessment by the area of impact and the nature of the risk may help the exercise. For example, the area of impact may be the cryostorage facility and the nature of the risk could be one of the following: financial (risk to the business); natural event (flood/fire); human resources (training/staffing levels); infection control; health and safety; compliance with regulation and patient or user satisfaction (quality assurance). Figure 1 shows a working model of this. Each potential risk is quantified with a basic scoring system and ranked in order of priority. A commonly used scoring system is one based on an Australian/New Zealand model (AZ/NZ54360:1999) where the risk score is a product of the consequences and the chance of it occurring; i.e. risk=consequence x likelihood. A score is given and an indication of the adequacy of current controls. Obviously, with adequate controls in place, a low risk score will be obtained. An example might be staff injury (or worse) due to the failure of an oxygen depletion monitor, which potentially could have severe consequences possibly even leading to a fatality, so is given a relatively high score. However, with adequate controls in place and the relatively small likelihood, the overall risk score is low. Once this exercise has been performed for all conceivable hazards and adequate controls are recognized, the necessary resources can be identified and incorporated into business planning.
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This article is not intending to review of all the current literature and neither is it a consensus view. It is however based on the available evidence, experience of the author and shared experience with professional colleagues in the field. It is aimed at the professional involved in the day to day running of and/or management of a cryostorage facility for fertility preservation. By increasing general awareness of each of these important points, it is hoped that the article will help individual centres in assessing risk within their own storage facility.
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Physical security of vessels and specimens |
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Liquid nitrogen supply and staff safety |
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Alongside such measures, management and staff must not become complacent of the hazards posed by liquid nitrogen itself. A recent death in Edinburgh, Scotland, of a technician who entered a cryofacility depleted of room oxygen by a discharging nitrogen vessel has recently refocused our attention on the dangers of nitrogen (see http://www.news.bbc.co.uk/1/hi/scotland/484813.stm).
Injury to staff, or indeed members of the public, can occur if nitrogen is not treated with respect. Supply vessels should be regularly checked and serviced and their movement throughout the building should be carefully controlled. Neither delivery/laboratory staff nor members of the public should accompany a vessel being transported to upper floors of a building in an elevator. An efficient nitrogen extraction system and a suitable oxygen monitoring system should be mandatory and staff should have some training in the use of cryogenic vessels and all available personal protective equipment (PPE).
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Relative safety of the containment system (vials or straws) |
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PVC straws are fragile after immersion at ultra-cold temperature and have a tendency to break: anyone experienced in carrying out an audit of a cryovessel will have found, and possibly been forced to discard, a number of broken PVC straws. Furthermore, commonly used PVA powder plugs or indeed solid plastic plugs used to seal these straws (and sometimes for identification purposes) are known to be unreliable. As such, they not only represent a potential cross infection risk but could also could lead to mis-identification.
Polypropylene cryovials allow ingress of liquid nitrogen due to an ineffective seal. Manufacturers clearly state that they are for use in vapour only, yet centres continue to use them in the liquid phase despite a theoretical infection and explosion risk. Nunc recommend their use in liquid nitrogen, only if enclosed in Cryoflex, which is a polymer that can be heat-wrapped around vials using a Bunsen or heat gun, which itself poses another risk to the samples. Understandably, individuals are reluctant to use this routinely for precious sperm and embryos (see www.nuncbrand.com/page.asp?id302langgb)>www.nuncbrand.com/page.asp? ID=302=GB).
Ionomeric resin (CBS) straws have a more effective sealing method than PVC straws and are less likely to break.
Glass ampoules are rarely used but represent a considerable hazard if they explode or break.
Based on this, the ionomeric resin straw would appear to be the inventory option with the lowest associated risk. However, data regarding their long-term incident-free use are not available, and over-reliance on any of them to solve our containment problems would be risky in itself.
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Storage in the gaseous phase of nitrogen (vapour storage) |
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A major advantage with liquid storage is that as long as nitrogen is in intimate contact with the inventory, it will remain at a stable 196°C. With vapour however, temperature gradients will exist from top to bottom with conventional vapour freezers, e.g. Taylor Wharton 10K, or even from the edge to the middle with modern nitrogen jacketed freezers, e.g. CBS 1500A. This will vary according to: (i) how full the freezer is; (ii) how big the freezer is; and (iii) what racking material is used. Plastic inventory/racking has been used by some manufacturers but is wholly unsuitable for low temperature storage, due to over-insulation of the samples furthest away from the nitrogen reservoir. Using plastic canisters during testing of our own system, temperatures lower than 100°C could not be achieved at the top of the inventory. As mentioned earlier, long term storage can only take place at lower than 135°C. In vapour, packing the freezer both with samples and with as much highly conductive material as possible is therefore essential to ensure temperature stabilization. Aluminium or steel containers should therefore be used to house the samples, and to improve conduction further, either cryocanes (often used with cryovials) or aluminium canister/goblet dividers can also be used to lower the temperature and provide a necessary safety margin (see Figure 2).
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Autofilling systems |
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The problem appeared to be a failure of the pressure sensing system (which measures the liquid nitrogen level in the freezer) leading to a failure to fill, and consequently warming of the samples. Failure of autofill sensing devices is not common but can occur, and lead to either under-filling as above or even over-filling, particularly if there are few fail-safe devices built into the freezer design. Over-filling, in which the freezer fails to detect the maximum permitted liquid level and continues to fill, bathing the inventory in liquid nitrogen, may not only lead to sample losses but it is also extremely hazardous. Firstly, it defeats the object of having vapour storage in the first instance, as without an extremely robust and leak-free containment system, this can lead to the very cross-contamination incidents we are trying to avoid. Secondly, it can cause severe problems in the cryoroom as the complete emptying of the supply vessel will spill onto the floor, displace oxygen from the room and reduce or completely interrupt the supply to other vessels. Manufacturers are already looking at fail-safe mechanisms in the event of over-filling, such as back-up sensors or even overflow devices to decant off excess liquid nitrogen. Laboratories are still required to be extremely vigilant whatever storage system is in place, as automation is clearly no excuse for leaving freezers un-monitored or unattended. A simple alarm and monitoring device with an appropriate manual fill operating procedure may have saved the lost samples associated with the MHRA alert, and we owe it to the patients we serve to put suitable measures in place.
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Using suitable equipment and materials to do the job |
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Bearing in mind that vessel trauma is probably the most common cause of new vessel failure, centres should be extremely mindful when vessels are moved or transported. Even single patient shipments using dry shippers should be performed with caution. Patients should be advised against transporting all of their sperm or embryos in one shipment and couriers only used as a last resort or only for donor samples. Those centres having to move an entire sperm or embryo bank should perhaps consider the purchase of spare vessels and consider taking the opportunity to decommission/decontaminate existing ones.
By complying with current tissue banking code of practice (UK Department of Health, 2001) or likely changes in EU law, risks associated with processing and storage of gametes and embryos should be reduced as a matter of course. Validation of materials, equipment and procedures used during storage and demonstration of their continued hazard-free use will all serve to protect the samples and patient recipients and can only be observed as a positive result of changes in regulation.
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Witnessing and security of labelling |
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Labels on straws or vials must withstand immersion in liquid nitrogen and/or extreme cold. Anecdotal reports of poor practice such as paper tags for labelling straws and inaccurate and misaligned markings on straws have led to a number of transcription incidents. The consequences could be (and no doubt have been) disastrous. Labelling must be clear and accurate, using appropriate labelling pens and avoiding poor handwriting, which can also lead to transcription errors. At least three patient identifiers should be given, one of which may be a unique code or number. Automated labellers or barcode generators may certainly reduce operator time and error and help to provide clear and accurate labels.
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Screening of patients for infectious diseases prior to storage |
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Methods of sperm cryopreservation/sample processing to reduce transmission risk |
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Early warning and monitoring systems for the cryoroom |
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At the British Andrology Society (BAS) workshop (2002) on sperm banking for cancer patients, the need for improvements in alarm and early warning systems was highlighted. A survey carried out and presented at the meeting suggested that in the UK, only 50% of clinics had alarms of any description on their sperm and embryo freezers. Furthermore, <25% of centres had alarms linked to external warning systems such as auto-diallers or fire alarm panels to deal with out of hours emergencies (Tomlinson and Pacey, 2003). Obvious problems with funding and being over-burdened with false alarms were cited as reasons for not having suitable systems in place. Since this workshop, many units have begun to address the problem, particularly after a recent directive from the Human Fertilisation and Embryology Authority (HFEA) made alarming the cryobank mandatory in the UK. There are a number of methods of alarming a system, the simplest being a nitrogen level float gauge placed in a dewar lid and linked to an audible alarm. Obviously local alarms are of some use during the working day but useless outside of normal working hours. It is therefore imperative that these are linked to some form of early warning system. These may take the form of a warning beacon on a hospital or university fire panel or more often than not may be autodiallers, which automatically telephone on-call personnel or senior laboratory staff.
Ideally, laboratories should aim to install a comprehensive system which not only provides early warning in emergency situations but also provides continuous monitoring of vital equipment, serving as important quality assurance for our service users. A number of advanced laboratories are now installing such systems with additional features such as remote call-in facility for interrogating and diagnosing any fault or incident, often circumventing the need for immediate on-site visit. Theoretically, monitoring can then take place from almost anywhere, providing that the software is installed onto a portable PC and an appropriate mobile phone link-up is available. The huge advantage that this provides is that although false alarms can and do occur, proper management of the system during the week and in normal working hours keeps them to a minimum and out of hours visits to the cryoroom are generally unnecessary. As a safety net, there has to be a well-structured staff on-call system throughout the week and sufficient staff should be available for rotation to prevent this being over-burdensome.
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Concluding remarks |
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A combination of risk reduction strategies should be implemented to keep cells and tissues in optimum condition, backed up by an early warning system to prevent premature thaw. Centres should begin the risk management process and identify areas of risk within their own service: priority should obviously be given to the areas of highest risk. An early warning system should be mandatory as it is now in the UK. This has to be affordable, manageable, easy to use and implemented alongside other risk reduction strategies.
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Acknowledgements |
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References |
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Human Fertilisation and Embryology Authority (HFEA) 6th Code of Practice, 2004.
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Medical Devices Agency (2001) A code of practice for the production of human derived therapeutic products. UK Department of Health.
Meryman HT (1963) Preservation of living cells. Fed Proc 22, 8189.[ISI][Medline]
Mortimer D (2004) Symposium: Cryopreservation and assisted human conception. Current and future concepts and practices in human sperm cryobanking. RBM Online 9 no. 2.
NHS Executive (1997) Guidance notes on the processing, storage and issue of bone marrow and blood stem cells. UK Department of Health.
Tedder RS, Zuckerman MA, Goldstone AH et al. (1995) Hepatitis B transmission from contaminated cryopreservation tank. Lancet 15, 137140.
Tomlinson MJ and Sakkas D (2000) Safe and effective cryopreservationshould sperm banks and fertility centres move toward storage in nitrogen vapour? Hum Reprod 15, 24602463.
Tomlinson MJ and Pacey AA (2003) Practical Aspects of Sperm Banking for Cancer Patients. Hum Fertil 6, 100105.
Submitted on August 18, 2004; accepted on November 19, 2004.
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