Managing risk associated with cryopreservation

Mathew Tomlinson*

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


    Abstract
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Patients who consent to the frozen storage of sperm or embryos quite rightly expect the storing centre to do everything reasonably possible to keep them in optimum conditions. Both the process of cryopreservation and the cryofacility are loaded with risk, from patient/sample processing, through to the eventual utilization or disposal of specimens. The risk management process should focus on minimizing losses, including staff injury, premature warming of cells and tissues, mistaken identity, and transmission of infection. Early warning and monitoring systems should be in place for quality assurance and to prevent incidents involving cryovessels turning critical. Centres must ensure that every reasonable practical measure that can be put in place is done so, and that resourcing of the service adequately reflects the liability it represents.

Key words: cryopreservation/embryo storage/risk management/sperm storage


    Introduction
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The cryopreservation of sperm, embryos and reproductive tissues is an essential element of any assisted conception (or indeed cancer) service. To those patients who consent to storage, their frozen sperm or embryos are absolutely priceless and quite rightly they expect them to be kept in the best possible condition. It is therefore imperative that patients and other users of the services such as oncologists, gynaecologists and general practitioners are reassured that quality systems are in place to look after their samples for the entire storage period, which, of course may be many years.

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., 1995Go). 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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Figure 1. Risk assessments demonstrating risk scoring system.

 
In terms of cryostorage, specific attention should focus on the following areas:
physical security of vessels and specimens;
liquid nitrogen supply and staff safety;
the relative safety of the containment system (vials or straws);
the type of nitrogen storage (liquid vs vapour phase);
the suitability of equipment to do the job;
witnessing and security of labelling;
screening of patients for infectious diseases prior to storage;
sample processing in order to lessen the risk of transmission;
early warning and monitoring systems;

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.


    Physical security of vessels and specimens
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
It is vital that all clinics keep specimens secure within the individual cryovessels and the vessels themselves are secure within the premises. In many countries, including the UK, the locking of cryovessels is a regulatory requirement (HFEA, 2004Go). The store itself should ideally be located on a ground floor location (to reduce unnecessary liquid nitrogen transport) and provide easy of access for the delivery vehicle. This should not however be adjacent to a busy road where there may a small but real risk of damage from a road traffic accident. Overall security should be assessed and, if necessary, systems installed such as swipe card access and closed-circuit television (CCTV) cameras. Access should also be limited to a few authorized and qualified members of staff. Multiple dewars should never be kept in small/poorly ventilated laboratories, as, should vessel integrity become compromized and the vacuum fail, there is a significant risk of oxygen depletion. Risk registers should perhaps include reference to less likely occurrences such as fire, theft or even a major accident such as a heavy vehicle impacting on the building.


    Liquid nitrogen supply and staff safety
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Storage within any freezer that warms much above –135°C, the so-called ‘glassy transformation temperature’ (Merryman, 1963Go) is likely to result in significant damage to the frozen cells or tissues. Maintenance of ultralow temperatures and hence a continued supply of liquid nitrogen must therefore be ensured for the safekeeping of specimens. Centres must ensure that if the nitrogen supply fails for whatever reason, there are fail-safe mechanisms in place to cope. Supply failure can occur for a number of reasons, for example, if the pressurized vessel vacuum fails or discharges its contents; the automated refrigerator over-fills (see later); the supply company fails to deliver. Such events could be survived, providing contingency measures are put in place, such as extra capacity in terms of nitrogen delivery vessels and appropriate written procedures and training for all relevant staff for dealing with supply problems. Centres are often highly dependant on delivery from the nitrogen supply companies for regular filling of pressurized storage vessels. Controls should also therefore anticipate potential delivery vehicle problems, for example heavy road traffic, accident or breakdown and contingency may include having spare capacity, either by keeping a surplus in spare pressurized vessels or by keeping an external bulk tank e.g. >1000 litres.

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).


    Relative safety of the containment system (vials or straws)
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
In response to the now infamous hepatitis B cross-contamination incident involving ruptured blood bags in liquid nitrogen (Tedder et al., 1995Go), the NHS executive recommended several risk reduction steps, one of which was the use of secondary containment or ‘double bagging’ of the samples. Unfortunately, the reproductive field is not ‘over-blessed’ with choice when deciding upon containment for sperm and embryos. Double bagging is not currently a realistic option (apart from perhaps cryovials; see below) and one over-riding consideration is the suitability of the container to allow uniform cooling of the sample (discussed at length by Mortimer, 2004Go). There are basically four options: (i) PVC straws; (ii) cryovials; (iii) ionomeric resin (CBS) straws; and (iv) glass ampoules. The relative merits of each could be discussed in detail but this is not the purpose of this article. What seems reasonably indisputable in terms of relative risk includes the following:

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.


    Storage in the gaseous phase of nitrogen (vapour storage)
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Discussions surrounding the relative merits of liquid vs vapour storage have provided an ongoing debate (see Tomlinson and Sakkas, 2000Go). Although storage in vapour itself is not without risk—certainly pathogenic growth, particularly fungal growth, can occur (Fountain et al., 1997Go)—it would appear that without the presence of a liquid medium, transport of pathogens around a vessel is less likely. As part of an overall risk reduction strategy, aimed in particular at reducing the chance of cross infection within a freezer, the UK National Blood Service and accredited tissue banks have essentially moved entirely over to storage in the vapour phase. It is therefore highly possible that the reproductive field may eventually follow suit, especially if it forms part of the recommendations put forward in the EU directive on the storage of cells and tissues (see http://www.europa.eu.int/comm/health/ph_threats/human_substance/tissues_en.htm). This may either require investment in automated (intelligent) freezers or adaptation of existing liquid dewars (see Clarke, 1999Go).

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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Figure 2. Vapour phase storage with aluminium canister-style racking and cryocanes to improve thermal conductivity.

 
Large towers with drawers for cryovials also provide suitable conductivity, although they are cumbersome and do not easily allow the storage of different containment systems i.e. they are limited to vials. A relatively full freezer, with an appropriately designed racking and inventory will achieve temperatures as low as –190°C just below the freezer lid with a temperature range of between only –175°C to –190°C (Table I).


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Table I. Data logging of computer controlled vapour freezers and adapted liquid nitrogen dewar containing a 10 cm nitrogen reservoir

 
Adaptation of existing liquid nitrogen dewars as suggested by Clarke (1999)Go is an alternative solution to storage in vapour without the need for large-scale investment. However, although more than satisfactory temperatures can be obtained in a dewar with only a few cm of nitrogen in the bottom (Table I), centres must be aware of potential pitfalls. There are obvious implications for dewar capacity, as the lower layer of the dewar is used as the nitrogen reservoir. For the average sized dewar, this leaves enough canister height for only a single layer of straws and insufficient height for standard cryocanes, although these may be ‘cut down’. Furthermore, there is little point keeping the inventory free from immersion in liquid in storage, then filling the dewar from the top, as nitrogen itself is a known source of contamination (Fountain et al., 1997Go). Therefore, filling hoses should be placed in the dewar bottom and nitrogen level carefully checked until it reaches the desired level. The dewar will stay sufficiently cold even with 2–3 cm nitrogen in the bottom, but once this evaporates it will warm rapidly (Table I). Therefore, careful temperature, and in particular, liquid level monitoring is required to reduce significant operator anxiety and to avoid a potential disaster.


    Autofilling systems
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The risk associated with autofilling systems was well highlighted by a recent Medicines and Healthcare Regulatory Agency (MHRA) incident alert to a failure with a CBS (CBS, USA) vapour freezer (see http://www.devices.mhra.gov.uk/mda/mdawebsitev2.nsf/webvwSearchResults/182F98F2F29B8A9F80256DBA00472658?OPEN).

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.


    Using suitable equipment and materials to do the job
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
All equipment has a finite lifespan and as nitrogen vessels age their vacuum will slowly diminish until eventually the vessel will hold nitrogen for too short a period for it to be of economic use. There is usually warning of this (frost on outside of vessel, shortened fill intervals) and plenty of time to provide a replacement. Situations such as this should be pre-empted, however, by having appropriate capital equipment or 15 year replacement programs, e.g. vessels on a 10 year cycle. Vacuum failure can also be an acute event, for example if a joint fails (vessels are particularly susceptible to trauma at the point where the neck core is fixed), in which case the vessel will lose its nitrogen and begin warming within hours. This is largely unpredictable and is as likely to occur in new vessels as it is in an old one. Clearly in this instance there needs to be in place a further risk strategy to minimize losses. These would certainly include avoidance of trauma, e.g. smooth flooring for roller bases; an early warning system (as mentioned later in this article), providing spare vessels and/or perhaps spreading the risk by splitting the samples over two (or more) vessels.

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.


    Witnessing and security of labelling
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Witnessing is now an established part of the assisted conception service, especially in the UK where it is mandatory for a second person to verify all processes involving transfer of gametes or embryos, e.g. transfer of sperm/cryoprotectant to straws or vials. It may also be prudent in the first place to have verification of the initial analysis, i.e. to check that the specimen pot, request, consent and report forms all correspond to the same patient. A mix up of two specimens at the processing/storage phase is a well-documented potential risk and requires all reasonable preventative measures to be in place.

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.


    Screening of patients for infectious diseases prior to storage
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The screening of all patients for HIV and hepatitis B and C must be implemented by law in the UK by December 2004. This strategy, designed to reduce the risk of cross contamination has also been implemented by the blood and tissue banking services and will no doubt be of some benefit. Despite being extremely unlikely, transmission, both in sperm and especially embryo storage tanks remains a possibility. Patient perception is paramount and screening certainly helps provide them with some re-assurance. Centres should not become complacent and over-reliant on screening, however, particularly as quarantine to cover the ‘window of seroconversion’ may not be practical and other, as yet undiscovered, pathogens will no doubt cause us a problem in the future. Moreover, there is also the possibility of rare false negative results from the virology lab, which would again cause significant anxiety if no fail-safe system were in place.


    Methods of sperm cryopreservation/sample processing to reduce transmission risk
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The risks associated with storing sperm and embryos tend to be very different. By the time an embryo is created in vitro, a large number of washing/processing steps have already taken place, each one reducing potentially contaminating pathogens and therefore transmission risk. On the other hand most sperm storage is relatively crude and is undertaken using raw semen with very little processing other than the addition of cryoprotectant. Potentially, this raw semen could harbour a relatively high pathogenic load, and as such, the risk of transmission within a nitrogen dewar is considerably higher. Reductions in seminal viral load to almost undetectable levels have been clearly demonstrated by groups treating HIV discordant couples, simply by sperm washing and density gradient centrifugation which removes contaminating leukocytes and seminal plasma (Kim et al., 1999Go). Most laboratories aim to freeze the sample in its raw state as soon as it is physically possible, which immediately puts stored sperm in a higher risk category than embryos. There is no disadvantage however, by delaying storage and preparing sperm beforehand; in fact data in our own centre suggest that post-thaw sperm recovery can be enhanced by sperm preparation and further incubation (see Table II). There are other benefits apart from the reduction of the pathogenic load of the sample, including: reduced workload for the embryology service using the sample post-thaw; improved sperm quality post-thaw; and it allows the storage of more concentrated sperm pellets, a particular advantage to those with poorer sperm quality in whom cryosurvival is notoriously low, e.g. sperm banking cancer patients.


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Table II. Motile sperm concentration (x106/ml; assessed manually and by CASA) in samples frozen in seminal plasma, thawed and prepared (freeze–prep) compared with those prepared prior to freezing (prep–freeze)

 

    Early warning and monitoring systems for the cryoroom
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Well-publicized incidents (both storage and non-storage) in the last 3 years have all done little to increase public confidence in our particular branch of medicine. Indeed, recent storage incidents affecting cancer patient samples served as a reminder to us all that the samples we handle are extremely precious, and we owe it to our service users to do everything possible to keep them in good order (see http://www.news.bbc.co.uk/1/hi/scotland/1876306.stm)

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, 2003Go). 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.


    Concluding remarks
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The cryoroom represents an enormous responsibility, and even liability, particularly for scientists involved in reproductive medicine. As responsible keepers, we owe it to the users of our service (clinicians and patients) to protect stored sperm, embryos, eggs, tissues etc. by whatever means we can. It is clear that in many centres, the storage facility is inadequately resourced and few of the measures discussed here have been implemented or even considered. Centres must look at their resourcing in a risk management context and ensure that pricing adequately reflects the true cost of the service. To believe that patients are unaware of such risk is irresponsible; indeed, patient expectations are constantly on the increase and they more or less expect such measures to be in place already. For example, well-informed patients have even asked for their stored sperm to be placed in two separate geographic locations to reduce the risk of losses due to fire or other natural disaster.

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
The author would like to thank the Medical Physics team of Birmingham Women's Hospital for their tremendous help over the past 2 years.


    References
 Top
 Abstract
 Introduction
 Physical security of vessels...
 Liquid nitrogen supply and...
 Relative safety of the...
 Storage in the gaseous...
 Autofilling systems
 Using suitable equipment and...
 Witnessing and security of...
 Screening of patients for...
 Methods of sperm...
 Early warning and monitoring...
 Concluding remarks
 Acknowledgements
 References
 
Clarke G (1999) Sperm cryopreservation: is there a significant risk of cross-contamination? Hum Reprod 14, 2941–2943.[Free Full Text]

Fountain D, Ralston M, Higgins N et al. (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37, 585–591.[CrossRef][ISI][Medline]

Human Fertilisation and Embryology Authority (HFEA) 6th Code of Practice, 2004.

Kim LU, Johnson MR, Barton S, Nelson MR, Sontag G, Smith JR, Gotch FM and Gilmour JW (1999) Evaluation of sperm washing as a potential method of reducing HIV transmission in HIV-discordant couples wishing to have children. AIDS 16, 645–651.[CrossRef]

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, 81–89.[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, 137–140.

Tomlinson MJ and Sakkas D (2000) Safe and effective cryopreservation–should sperm banks and fertility centres move toward storage in nitrogen vapour? Hum Reprod 15, 2460–2463.[Abstract/Free Full Text]

Tomlinson MJ and Pacey AA (2003) Practical Aspects of Sperm Banking for Cancer Patients. Hum Fertil 6, 100–105.

Submitted on August 18, 2004; accepted on November 19, 2004.





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