Buyers' guide: Automated nucleic acid extraction ...

Buyers’ guide Automated nucleic acid extraction systems CEP09015

June 2009

Contents

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Introduction ............................................................................................. 3 Technical considerations ......................................................................... 5 Operational considerations...................................................................... 8 Economic considerations ...................................................................... 10 Purchasing ............................................................................................ 11 Market review........................................................................................ 13 Acknowledgements ............................................................................... 47 Glossary ................................................................................................ 48

References............................................................................................ 51 Appendix 1: Supplier contact details ..................................................... 53 Appendix 2: EU procurement procedure ............................................... 56 Author and report information................................................................ 59

CEP 09015: June 2009

Introduction

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Molecular biological techniques are increasingly being incorporated into routine clinical microbiology laboratories. Their uses include the detection, identification or typing of pathogens, prediction of drug resistance or molecular genetics [1]. Such techniques can offer significant advantages over more traditional methods. A recent review of some of the applications, advantages and disadvantages of molecular methods used in the clinical laboratory is given by Morshed et al [2]. Benefits offered by the use of molecular methods in the clinical microbiology laboratory are: • • • • • •

Increased sensitivity (eg through the use of amplification techniques such as polymerase chain reaction, PCR) High specificity (as a result of the precise base-pairing of DNA) More rapid results leading to decreased turnaround times (eg detection of noncultivable or slow-growing pathogens) Increased laboratory safety (nucleic acid extraction frequently renders specimens non-infectious) Facilitated epidemiological studies (eg strain identification during outbreaks or transmissions which can then be controlled or prevented in future) Improved treatment monitoring (eg prediction of drug resistance or efficacy, virulence factors or host factors)

Extraction of nucleic acid (NA) from a range of specimens is an integral and essential step in many molecular biological techniques, most notably those for NA amplification, including polymerase chain reaction (PCR), strand displacement amplification (SDA), and ligase chain reaction (LCR). While some commercially available nucleic acid amplification tests (NAATs) incorporate their own nucleic acid extraction system (for example, the COBAS system from Roche Diagnostics links a dedicated extraction machine, the Ampliprep, with a TaqMan Analyzer real-time PCR amplification machine), many NAATs are available which are less prescriptive as to which extraction method is used. Extraction systems can be expensive and need to match the requirements of each specific laboratory. There are two main methodologies involved in NA extraction - both use a solid phase either in the form of (i) particles (often magnetically charged); (ii) membranes (generally silica). The basic principle is the same in both of these - to capture, wash away unwanted molecules and then recover the purified nucleic acid into elution buffer. There are many factors which are of importance when considering which automated extraction platform should be adopted. One of the most important ones is the required throughput. Automated systems can extract from a single sample up to 96 CEP 09015: June 2009

Introduction

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samples in one run. For some applications, such as molecular genetic testing, relatively small batches of samples are processed at any time (up to 16) [3]. For other purposes such as HIV viral load testing, the batches can be considerably higher. This Buyers Guide will concentrate on those systems with a ‘high’ throughput, which is defined as at least 96 samples per run, and includes some more generic liquid handling platforms. Another factor of high importance is the level of flexibility associated with the extractor, since with a greater flexibility comes versatility which can prolong the useful life of a machine, but may be associated with an increased risk of suboptimal usage or performance [4]. It is beyond the scope of this Buyers Guide to assess the performance of the systems.

National guidance The use of molecular techniques has become the ‘gold standard’ for the diagnosis or characterisation of microorganisms, primarily in virological testing, but increasingly so for bacteriological targets as well, particularly those which are noncultivable or slow growing such as Treponema pallidum (syphilis) or Mycobacterium tuberculosis (TB) [2;5;6]. For example, current testing guidelines from the British Human Immunodeficiency Virus Association (BHIVA) recommend drug resistance testing for all newly diagnosed persons, and for monitoring genotypic resistance following treatment failures in order to select appropriate therapy [7]. Similarly, blood DNA levels are used to decide when to begin therapy for Hepatitis B virus (HBV) infection, and circulating viral loads are used to guide decisions on whether HBV and Hepatitis C Virus (HCV)-infected healthcare workers can practice [8;9]. Testing for viral RNA is recommended as part of the testing algorithm for HCV [5] and knowledge of the HCV genotype can indicate the likely success of the most common treatment regime. In his 2006 Review of NHS Pathology, Lord Carter of Coles considers whether England has been slower to adopt new automation technology than other countries, but concludes that it is catching up [8]. He recognises that increasing use of automation, including molecular testing systems in microbiology (ie automated extractors and related equipment), can provide many benefits including a reduction in lost samples, sampling errors, use of qualified staff time, and test turnaround times. Problems with the adoption of new technology, however, include the lack of a national framework for evaluation, and adoption up to this point has been inconsistent across the NHS. There is a need to ensure that where multiple platforms exist with similar roles but different specifications (sometimes dictated by requirements or practice in other countries, and intellectual property rights) there is reassurance that test results are standardised.

CEP 09015: June 2009

Technical considerations

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Quality control Quality of extraction As part of good laboratory practice, and to conform with laboratory accreditation schemes, it is essential to assess any automated extractor with the sample type(s) and targets used within the testing laboratory. Factors which should be considered as performance measures are the yield, purity and integrity of the extracted NA [1;4]. Inferior extraction can result in the persistence of substances inhibitory to downstream applications (eg PCR, sequencing or cloning) or nucleic acid of a poor quality such as that caused by shearing of longer nucleic acid strands [9]. Ongoing monitoring of performance is important, either through co-extraction of a quantified internal quality control (IQC) added to every sample, or by inclusion of an extraction or run control with each batch of samples (external quality control; EQC) where the former is not practical. Including samples from external quality assessment (EQA) schemes, where available, will also form an important part of ensuring appropriate performance of equipment [10]. Liquid level detection or clot detection are sometimes available – these can indicate a poor quality specimen (eg the presence of particulate matter or bubbles in a sample, or an insufficient volume) but have the disadvantage of additional time added to the extraction process. 21 CFR Part II 21 CFR Part II is an FDA regulation pertaining to the keeping of complete records of events during manufacturing. Some NA platforms offer compliance with this regulation, meaning that a complete audit trail of each specimen and extraction run is available.

Limitations Contamination or carryover Since the products of NA extraction are often amplified (eg by PCR), it is essential that there is no cross-contamination of samples. Dispensers with disposable tips (incorporating filters) are therefore preferable to reusable probes; in some instances these may be used more than once for a single sample to minimise waste. Simple dispensing of reagents, however, may be compatible with the use of reusable probes allowing increased speed and reduced consumable costs. Some robotic systems automatically (or can be programmed to) choose routes of movement for the arm(s) which minimise contamination risks (‘no fly zones’). Ideally, automated NA systems will be located in a dedicated ‘clean’ area without possible exposure to postamplification products [1]. Completely enclosed systems can minimise any environmental contamination issues, and some systems incorporate HEPA filters, can be operated within laminar flow cabinets, or use in-built UV irradiation to decrease this risk further.

CEP 09015: June 2009

Technical considerations

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Specimen volume For some specimen types received in a clinical microbiology laboratory only a very small volume is obtained (eg blood samples from neonates) and it is important, therefore, to know the minimum addition volume that the instrument can handle. It may be necessary to dilute specimens prior to extraction which will add to the extraction time and/or necessitating an additional manual preparation step. For target NA at low concentrations, the ability to extract a large volume (where available) is desirable. Elution The elution volume must be sufficiently low to enable detection of targets at low concentration where necessary, without compromising the elution efficiency. While on the extraction platform, the elution tubes or plates may not be stored below ambient temperature meaning that longer term storage here should be avoided. Following removal from the extractor, the eluted material may require transfer from the elution vessel into a more appropriate one for downstream processing or longerterm storage. Specimen type/problematical specimens Some specimens (eg serum or plasma) are homogeneous and hence relatively straightforward to extract. Other types such as faecal or respiratory samples are less homogeneous and may require additional steps. It is important to remove all enzyme inhibitors, incompatible salts and other competing materials before use of the NA in downstream applications (such PCR, sequencing or cloning) [1;3]. Particulate matter present in samples such as faecal material can prove problematic for filter-based extraction systems where the filter may become blocked or have reduced flowthrough efficiency. Some systems incorporate clot or clog detection which can indicate potential problems early in the extraction run. Extraction run failures How the automated NA system deals with problems occurring during a run (for example a blocked filter in a vacuum-based system, or a power failure) is important, particularly for processing samples of limited volume. In some systems, the complete run may need to be repeated leading to sample or reagent wastage or even loss of the sample if irreplaceable. Programming equipment Some dedicated NA extraction systems come complete with all required software programs (protocols) and no user intervention is required. For those systems which are more flexible, consideration should be given to whether trained individuals are able to generate new protocols, or whether the manufacturer needs to be involved; how user-friendly this process is may vary considerably. A few or even a single universal protocol that works for all specimens and extraction targets is CEP 09015: June 2009

Technical considerations advantageous, not least where a single extraction run may comprise of mixed specimen types. In some instances, copies of the software for protocol design or downstream analysis of results may be made available for use on PCs not directly connected to the NA extraction platform. Maintenance of equipment Maintenance requirements need to be factored in to any estimations of machine throughput. Operators need to undertake daily, weekly and monthly maintenance schedules, and manufacturers need to fulfil service requirements.

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Operational considerations

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Training Training is required for operation of any automated equipment. The extent of this, how many staff members are included in the initial training provided by the company and whether there is an ongoing training commitment by the company should be established, as should any associated costs.

Logistical issues Choice of automated equipment may be limited by logistical issues. These include: •

the amount of space required (footprint plus any additional area, for example for lid opening, air circulation or waste disposal)

•

whether an instrument is benchtop or floor-standing (benches will need to be able to support the weight of the extraction platform for benchtop equipment)

•

power requirements, including UPS (uninterruptible power supply)

•

operating temperature and humidity

•

heat generated by the equipment

•

connectivity (eg to Laboratory Information Management Systems (LIMS) or other pieces of laboratory equipment such as liquid handlers).

Waste disposal Significant levels of plastic waste may be associated with each extractor run, especially when disposable tips are used; some companies may offer collection and recycling of plastic packaging waste. Liquid waste such as guanidinium thiocyanate may also require specialist disposal as per local regulations.

Customer support The level of customer support provided by the company should be considered, especially for high-throughput laboratories, which require a rapid response to address faults and other operational issues. As laboratories move away from traditional office hour working (9am to 5pm), access to customer services outside of these times may be required.

CEP 09015: June 2009

Operational considerations Servicing Service contracts vary considerably between companies, although the majority will offer a one-year warranty either from date or delivery or installation. An annual preventative maintenance visit is recommended by most manufacturers, and purchasers should negotiate for the most appropriate service agreement to match their requirements.

CEP 09015: June 2009

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Economic considerations

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Economic considerations Aside from staffing, the costs associated with automated extraction machines can be broadly divided into three: the machine itself (and any associated hardware such as a computer), the consumables required (comprising of the disposable plastic ware and the extraction kit reagents), and the after care or support offered by the company. Automated extraction system cost The list price of a system is often open to negotiation with the manufacturer or supplier, and discounts linked to minimum purchase of associated kits or consumables are possible. Given the progression of technology, purchasers could consider rental schemes. While there may not always be a saving in the time taken to perform NA extraction using an automated system compared to manual, a proportion of the operator time is freed up for other tasks. It is beyond the scope of this Buyers’ Guide to discuss more general aspects of the economic impact of the introduction of laboratory automation, but an analysis of this was performed by Gurevitch [11]. Futureproofing Laboratory requirements generally change over time, resulting in a piece of equipment becoming obsolete. Some instruments, particularly the more generic liquid handling platforms, allow for future modifications to the format and the interaction with other pieces of equipment. Consumables The majority of automated NA extraction systems require proprietary consumables such as plasticware, and use of such items produced by other manufacturers which could have cost benefits may invalidate the equipment warranty. However, items such as microtitre plates are often manufactured to ANSI/SBS standards meaning that they should be suitable for all systems. Re-use of disposable tips (for the same sample) within an extraction run may be possible for some platforms to reduce tip requirements. For the use of the liquid level detection feature (where available), conductive tips are required which incur an additional cost compared to nonconductive tips. Liquid level sensing, which calculates levels based on the known liquid volume and containment vessel dimensions does not require conductive tips. Some systems have their own or recommended extraction kits or are able to provide information on the performance of different kits. It is unlikely, however, that performance information will be available for all possible extraction kits and, in any case, these must be validated within the laboratory hosting the system [10].

CEP 09015: June 2009

Purchasing

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Purchasing procedures The Trust Operational Purchasing Procedures Manual provides details of the procurement process [12]. European Union procurement rules apply to public bodies, including the NHS, for all contracts worth more than £90,319 (from January 1st 2008) [13] (Appendix 2). The purpose of these rules is to open up the public procurement market and ensure the free movement of goods and services within the EU. In the majority of cases, a competition is required and decisions should be based on best value. NHS Supply Chain (NHS SC) offers national contracts or framework agreements for some products, goods and services. Use of these agreements is not compulsory and NHS organisations may opt to follow local procedures.

Sustainable procurement This section identifies relevant sustainability issues and provides some guidance on how these can be incorporated into procurement decision making processes. The UK Government launched its current strategy for sustainable development, Securing the Future [14] in March 2005. The strategy describes four priorities in progressing sustainable development: • sustainable production and consumption – working towards achieving more with less • natural resource protection and environmental enhancement – protecting the natural resources and habitats upon which we depend • sustainable communities – creating places where people want to live and work, now and in the future • climate change and energy – confronting a significant global threat. The strategy highlights the key role of public procurement in delivering sustainability. Power supply and energy consumption Energy costs will need to be factored in to the whole life cost calculation for the equipment; some indication of these are given in the following section. Any guidance provided by the manufacturer or supplier on using the extractors in the most energy efficient manner should be assessed, and it may be of value to ask whether they have set any targets for improving this. Some extractors may incorporate features to reduce energy consumption when idle, for example. CEP 09015: June 2009

Purchasing

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End of life disposal Consideration should be given to the likely financial and environmental costs of disposal at the end of the product’s life. Where appropriate, suppliers of equipment placed on the market after the 13th August 2005 should be able to demonstrate compliance with the UK Waste Electrical and Electronic Equipment (WEEE) regulations (2006) [15]. The WEEE regulations place responsibility for financing the cost of collection and disposal on the producer. Electrical and electronic equipment is exempt from the WEEE regulations where it is deemed to be contaminated at the point at which the equipment is scheduled for disposal by the final user. However, if it is subsequently decontaminated such that it no longer poses an infection risk, it is again covered by the WEEE regulations, and there may be potential to dispose of the unit through the normal WEEE recovery channels.

CEP 09015: June 2009

Market review

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Market review The following tables provide a comparative summary of automated nucleic acid extraction platforms. They are listed as the dedicated nucleic acid platforms first, followed by liquid handling platforms with a nucleic acid extraction format, and finally more generic liquid handling platforms. Initial tables comprise of physical characteristics (size, weight, power requirements etc); these are followed by details of some of the features (extraction chemistry, pipetting precision, throughput etc) and finally there are single page summaries for each platform, with details of costs, warranty and servicing. Where platforms are more generic, costs or timings for extraction runs are not available due to the variety in possible chemistry, reagent and platform conformation combinations. Maintenance Where provided, information on the maintenance of NA extraction platforms indicated that between five and twenty minutes are required daily for maintenance tasks (typically visually checking, cleaning surfaces, flushing the system and loading or unloading reagents or samples). Weekly tasks typically took between fifteen and thirty minutes, and monthly maintenance was often not required but could take up to 40 minutes. Yearly preventative maintenance checks by representatives of the companies involved were generally recommended and would be included in warranty/service agreements.

CEP 09015: June 2009

Market review

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Characteristics of dedicated NA extraction platforms (1) Instrument name

m 2000sp

chemagic Magnetic MagMAX™ Express-96 Deep Well Magnetic Particle Separation Module I (MSM1) Processor

BioRobot Universal (Universal); BioRobot MDx(MDx)

Floor standing

Benchtop

Benchtop

Benchtop

C

C

C

Universal: O; MDx: C

Size: WxDxH (to nearest cm)

145x78x175

68x60x38

194x65x143

145x81x82

Actual space required: WxDxH (cm) (allowing for air circulation, lid opening etc)a

155x88x185

78x70x38

194x65x193 (for lid opening)

165x91x82

282

28

85

140

P

NR

P

P

15-32

10-40 Max 80 ≤ 31˚C (linear dec to 50% RH at 40˚C)

10-35

15-32

10-80

15-85 at 30˚C

100-240 ± 10%

100-240

90-240; 16A

249 ±10%

70 W

150 W max (55 W standby)

1.5kVA max

800 W

N

Y

Y

Y

Configuration (floor, benchtop) Open/closed platform (open= flexible format ; closed= fixed platform setup)

Weight (nearest kg) (shipping weight will be higher) PC required (R)/not required (NR)/provided (P) Operating ambient temp (°C) Operating relative humidity (%, non condensing) Power requirements (V; all at 50/60Hz) Power consumption Linking more than one system through a single interface a

30-80 ≤ 30˚C

Even where not declared by the company, it is likely that the actual space required will be greater than the footprint to allow for air circulation etc

CEP 09015: June 2009

Market review

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Characteristics of dedicated NA extraction platforms (2) Instrument name

QIAsymphony SP

QIAxtractor (formerly XTractor-Gene)

LabTurbo 96

Thermo KingFisher Flex

Benchtop

Benchtop (vacuum/waste station under bench)

Benchtop

Benchtop

C

O

O

C

Size: WxDxH (to nearest cm)

130x72x103

49x64x77 (v/w station: 40x51x55)

86x79x90

68x60x38

Actual space required: WxDxH (cm) (allowing for air circulation, lid opening etc)a

150x125x163

As above

As above

As above

175

Workstation = 31 (v/w station = 18)

150

28

NR (touch screen operation)

R - provided at additional cost

NR (touch screen operation)

NR

15-32

15-35

15-35

4-40