If your research study requires the analysis of salivary biomarkers such as Cortisol, decisions need to be taken early to ensure that no issues arise that will affect the result and ultimately your peer reviewed paper.
The initial planning process should answer questions such as: how to collect the samples, collection method, at what time of day, how many samples would be appropriate, how should they be stored?
All of these issues are thoroughly covered by Salimetrics within the section Spit Tips
Next where do we test the samples?
To process the samples within your own laboratory you will need to check that all facilities are available to you. The required Laboratory Equipment and methods are covered in the final section of this post.
HOWEVER should you decide to send them to a laboratory within say Europe, these are the considerations:
In order to publish accurate results we suggest the following is considered:
1. If the Lab is within the UK, do they have an HTA Licence?
2. For laboratories offering saliva testing, do they use the best commercially available Assays that have been manufactured to GMP, give full reproducible results, been fully validated, contain standards and controls, have been quoted in past research papers and as such are regarded as the highest standard available?
3. Does the laboratory offering to test your samples use an in-house assay that does not go through the same QA/QC as the commercial assays, open you to criticism, raise concerns that the quote was too cheap? Can the laboratory offer customer service advice right though the project? Do they manufacture and validate their assays / collection devices ?
What do I need to set up my own Laboratory?
Basic Laboratory
You will need to have a general laboratory lab space within which to conduct your research. For example.....
Laboratory benching, that meets the current specifications for work with saliva
Adequate ventilation
Precision balances (For weighing saliva samples to determine flow rate and weekly checks on your pipettes)
Vortex mixers (useful for mixing reagents or making saliva dilutions)
Heater/ stirrers (useful for mixing wash buffers)
Single and multichannel pipettes (essential - these are used for accurately dispensing saliva and reagents into your assay kit. The quality of the final assay results is greatly influenced by pipetting - both in terms of operator experience and well maintained pipettes)
Air conditioning (to keep lab temperature to around 21C) Most Salimetrics assays are conducted at room temperature but if the lab is much colder or warmer than 21C the assay results may be unreliable
The assays most commonly use aspirate/ dispenses of:
Single pipette:
Multichannel: 100µl, 200µl and 320µl (for salivary Alpha Amylase)
High Quality Water
As a minimum you need grade 3 distilled water but ideally ultra-pure water (18 MΩ water) would be used for all buffers in ELISA and salivary Alpha Amylase. Essential to use: Grade 3 distilled a minimum specification. We currently use a Purite Neptune for ultra pure water (18.3 MΩ water) and a Purite Select for the grade 3 water.
Centrifuges:
The rate-limiting step for our assays is often how quickly we can get samples centrifuged. Most samples require 1500RCF for 15 minutes and do not require temperature control. As the saliva is being centrifuged it is important to use sealed adaptor lids so in the event that a tube breaks in the centrifuge, saliva is not passed into laboratory air.
Class 11 Cabinets
The class II cabinets are used to protect both the operator and contents of the cabinet from bacterial/ viral infection. Whether or not a class II is required depends on the institutional view on health & safety and the nature of the samples being analysed. Not essential but the decision to install depends on the Health and Safety policy of your institution. Given that the Salimetrics laboratory processes large and continuous numbers of samples from a wide number of European customers and participants, we chose to use class11 cabinets for all our testing. We use cabinets by Bioquell and Faster
We use twin HEPA filtration but given the work we do there is little need for ways to sterilize the cabinet or filters (for example UV Lights) As you may be operating equipment such as washers, inside cabinets it is useful to have electrical sockets installed in the cabinet
Refrigeration
Many reagents need to be stored at 2-8ºC (fridge) and saliva samples, ideally, at -80C or below. Fridges should be high quality laboratory specifications that can maintain a stable temperature. (‘Domestic’ fridges often show large temperature fluctuations between 2 -8ºC), this is essential
We largely use Gramm Fridges and -20C freezers and New Brunswick -86ºC freezers
Ambient fridges can maintain 22ºC, which is very useful if your lab temperature is not always well controlled. not essential but we use an LMK ambient incubator
Wireless Temperature Monitoring
It is very useful to get 24/7 warnings of when fridges/ freezers fail, for example during power cuts. Not essential but if you have to consider the costs of replacing kits or unique saliva samples it may be well worth the cost of monitoring. There are a number of solutions depending on the size of your lab and the how and when you wish to be contacted. We are currently using a system from Tutela medical (http://www.tutelamedical.com)
Liquid CO2 Injection for -80c Freezers
If the temperature of the -80ºC freezer falls below a certain predefined temperature liquid C02 is injected into the freezer to keep the contents cold
Not essential but provides an additional amount of time before you start to risk damage to unique samples. Currently we use a single C02 bottle can maintain our primary -80ºC freezer at -70C for around 6 hours and additional bottles can extend this. This system provides some ‘peace of mind’ during power cuts or system failures.
We currently use a New Brunswick C02 backup system.
If you are using C02 liquid you will also need a C02 sensor and alarm as the CO2 Gas can be fatal at We use Quantam sensor and alarm system
Microplate Reader (Essential)
Simple absorbance reader required with most common filters being 405nm (for salivary Alpha Amylase)), 450nm (for most ELISA) and 620nm (for reference read) we currently use a Tecan Sunrise and a Tecan Infinite F200. The Sunrise is an entry-level system that is more than adequate for Salimetrics ELISA.
It is important to get some form of data processing software with the reader, for example Tecan Magellan software.
If you are considering salivary Alpha Amylase assays then an incubator option is desirable.
Plate Washer (Essential)
8 or 16 channel head will be sufficient, we currently use the Tecan Hydroflex with 18 channel head and 3 reagent bottles. If you are doing more than a few ELISA plates the washer is all but essential.
Plate Shaker Incubator
An orbital shaker that can ideally shake at least two plates at the same time. If you were considering non Salimetrics ELISA or salivary Alpha Amylase then an incubator unit would be very useful. Not essential but relatively inexpensive and whilst you can tap plates to mix a shaker provides far more standardised and efficient shaking
LIMS (Laboratory Information Management System)
LIMS databases store, analyse and report assay data. They are useful if you are processing large numbers of samples, The database also helps to ensure you have detailed information on the location and history of each sample that is required for storage under the Human Tissue Act. We use a custom LIMS from AIS LIMS ltd (www.ais-lims.com)
Automated Liquid Handler
Automated Liquid Handlers can partially or totally automate the ELISA process, but far from essential. All the Salimetrics assays were developed to be used in laboratories without automated equipment. However if you are looking to test large numbers of samples there are advantages in using an automated system. We use a Tecan Freedom Evo with 8 fixed tips, fast wash system, robotic manipulator, 2 x incubator units (MIOs), Tecan Sunrise and Tecan Hydroflex controlled using TecanEvoWare and Tecan EvoWare PLUS software with Liebert UPS power backup. The unit is enclosed in a Bigneat HEPA filter cabinet. These installations has just been replaced so we now have some 10 years experience of this assay and have just invested in the latest automated liquid handler from Tecan Robot available (There are other manufacturers but this was our first choice
Autoclave (Essential)
The autoclave disinfects all lab waste and saliva by sterilizing at 131ºC for 30 minutes. Normal lab waste, once sterilized, goes into the normal waste system and saliva is then incinerated. We use a benchtop 40L LTE Osprey autoclave
Human Tissue Act License
The handling and storage of Saliva falls within the Human Tissue Act. We therefore have invested in an HTA License costing some £6,000 per annum and have been audited by representatives from the HTA and received full approval for our documentation and systems
Salimetrics Saliva Testing Service
As you can see the Salimetrics testing laboratory in Cambridge has state of the art equipment and staffed by colleagues with over a decade of experience in saliva testing. We pride ourselves by offering a fast, efficient and accurate service to researchers across Europe. Testing, dependent upon the number of samples, can be as low as £8 per duplicate sample and in addition to the published range of Assays we now offer Cytokine testing, so if you have a need ask us if we can assist you!
We also offer guidance on Saliva Collection (Talk to us prior to collecting your samples) assistance with project planning and continued customer service during and after we have completed the process
Finally we have the contact details of experts who can assist with the supply of equipment and installation advice, so contact us for details
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T: +44 (0) 1638 782619
Comprehensive support at www.salimetrics.com
Blogsite: www.salimetricseurope.blogspot.com
Twitter: @Salimetrics
Anglia Ruskin University:
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