Resources & FAQs

Salimetrics Lab Resources: FAQ’s, Resources, and Salivary Bioscience Guidance

The most common questions (and even some of the more obscure ones) are answered here in our saliva lab services resources forum.  No time to read through these answers? Ask An Expert for help.

 

Testing FAQ's

Are there any recommended pipetting techniques?

​The key to accuracy in pipetting is a consistent technique.  Consistent speed and smoothness while pipetting is necessary.  Avoid any sudden motions when drawing or dispensing fluids.

Always pre-wet the pipette tips – see; Pre-wetting the pipette tip(s) below. (Always change pipette tips between each standard, control and sample!)

Pre-wetting the pipette tip(s) will result in more accurate and consistent pipetting of solutions which is very important for pipetting duplicates onto an immunoassay plate.  Pre-wetting tips also applies to multichannel pipettes.

a.Firmly place the correct size pipette tip onto the end of the pipetter.

b.Depress the plunger button at the top of the pipetter with your thumb until you feel the first stop point.

c.Immerse the pipette tip under the surface of the liquid to be dispensed to a depth of 2-5 cm, staying clear of the container walls and bottom.  Release the plunger button slowly to draw liquid up into the tip. 

d.Pause with the tip in the liquid for one or two seconds after aspirating.  The liquid in the tip “bounces” slightly when the plunger is released.  A slow, consistent pause helps minimize errors due to this effect.

e.Withdraw the tip from the liquid & dispense back into the container by gently pressing the plunger all the way down.  This “pre-wets” the tip. 

 

What should I do if my saliva samples are viscous?

​The key to accuracy in pipetting is a consistent technique.

Consistent speed and smoothness while pipetting is necessary. Avoid any sudden motions when drawing or dispensing fluids.

Always pre-wet the pipette tips – see; Pre-wetting the pipette tip(s) below. (Always change pipette tips between each standard, control, and sample!)

Pre-wetting the pipette tip(s) will result in more accurate and consistent pipetting of solutions which is very important for pipetting duplicates onto an immunoassay plate. Pre-wetting tips also applies to multichannel pipettes.

a. Firmly place the correct size pipette tip onto the end of the pipetter.

b. Depress the plunger button at the top of the pipetter with your thumb until you feel the first stop point.

c. Immerse the pipette tip under the surface of the liquid to be dispensed to a depth of 2-5 cm, staying clear of the container walls and bottom. Release the plunger button slowly to draw liquid up into the tip. 

d. Pause with the tip in the liquid for one or two seconds after aspirating. The liquid in the tip “bounces” slightly when the plunger is released. A slow, consistent pause helps minimize errors due to this effect.

e. Withdraw the tip from the liquid & dispense back into the container by gently pressing the plunger all the way down. This “pre-wets” the tip.

 

How should I label my samples to send to Salimetrics for testing?

​For samples being sent to Salimetrics, label the exterior of the tube using pre-printed, bar-coded labels provided, or write the ID number with a waterproof pen. Further details can be found in the Saliva Collection Handbook on page 4.

 

How long can my sample stay on the plate before adding conjugate/substrate?

​To ensure highest quality assay results, pipetting of samples and reagents must be done as quickly as possible (without interruption) across the plate. Ideally, the process should be completed within 20 minutes or less.

 

Is it cost effective to send samples to Salimetrics for testing or should we do the testing ourselves using Salimetrics kits?

​Utilizing a high quality lab will improve the quality of your data. If your immunoassay lab has highly experienced technicians, the latest equipment, and is up-to-date on current saliva collection, handling, storage, and testing protocols, it can cost less to test small studies yourself. For large projects, the SalivaLab is often the most cost-effective option when high-quality data is desired. If you would like to learn how to get high-quality data in your lab, we would welcome you at “Spit Camp” to teach you how to do this. Many who come to Spit Camp decide very quickly if it’s worth it to send samples to Salimetrics. There are other options as well. Salimetrics has a network of laboratories we call “Centers of Excellence (COE) Labs”. We monitor their performance on a regular basis. If there is a COE in your region, this is a very good alternative to consider so you will get guaranteed, reliable results using our laboratory products.

 

How do I ship samples to Salimetrics for testing?

​Complete instructions on how to send your samples to Salimetrics can be found under the Send Samples link at the SalivaLab.

 

Why do I have to freeze and centrifuge saliva samples before testing them?

​Whole saliva contains glycoproteins know as mucins, which form mucus when dissolved. This thick and sticky substance can cause problems in transferring small volumes by pipette. Freezing saliva samples will precipitate mucins. Centrifuging removes mucins and other particulate matter which may interfere with antibody binding, leading to falsely elevated results.

 

Do I need to keep samples cold for saliva testing?

​Salimetrics only accepts samples that are shipped on dry ice to the SalivaLab.

 

Can I send/ship samples from outside the United States to Salimetrics for assay?

Yes.  To ship samples to Salimetrics from outside the United States:• Contact Salimetrics for specific instructions regarding documentation that must accompany your samples. A deviation from the proper procedure will cause a delay in getting samples through United States Customs.• Transit times may be more than one day. Please take this into account when packaging your samples.• Shipping regulations may differ outside of the United States. Please investigate this before shipping. Visit the SalivaLab site at; http://www.salimetrics.com/submit-samplesfor more information.

 

 

Collection FAQ's

What is serum/saliva correlation for all Salimetrics kits?

​For salivary analytes that move into oral fluid from the circulation, serum-saliva correlations are often available in the literature. Many analytes of interest in oral fluid are produced by cells which have migrated into the oral mucosal compartment or are secreted by the salivary glands. In this case, the serum-saliva correlation is a moot point. Moreover, we are beginning to question the importance of the serum-saliva correlation for many markers. The levels of analytes in saliva may be meaningful regardless of whether the serum-saliva association is weak or strong. 

 

Do you have screening questionnaires available?

​We do have an example of a Pre-Screening Health Questionnaire. You can Click Here to download it.

What things should I be screening for when collecting saliva?

​Generally speaking, screening questions need to be customized to fit the research questions being asked and the unique characteristics of the participants being studied. Thus, there is not a universal screening questionnaire that is appropriate for every study. You can this information in the Saliva Collection Handbook under “Before Saliva Collection”.

 

Is there an accepted procedure I can use to stimulate saliva flow without interfering with the results?

​The key word here is “accepted,” which is highly subjective. Chewing on unflavored paraffin/wax or rolling a marble around under the tongue has been used by some, but not recommended for flow-dependent analytes. Salimetrics does not recommend using any stimulants when collecting saliva.

 

What is the best time to collect saliva samples pre/post stimulus/experimental manipulation my analyte of interest?

​In general, the time of day for collection can play a part in the level detected; in most cases, the time of day should be standardized for all study participants.  Most steroid hormone levels are higher in the am.  Please make an appointment to talk with one of our experts by phone.  The paper below presents a very good overview:
Granger, D. A., Fortunato, C. K., Beltzer, E. B., & Virag, M.., Bright., M. & Out, D. (2012). Salivary Bioscience and research on adolescence: A integrated perspective.  Journal of Adolescence., 32, 1081-1095 PMID 22401843

 

Why did you stop recommending salivettes?

​Salimetrics had several reasons for discontinuing the recommendation for Salivette usage. The main reason is that Salimetrics observed unacceptable lot to lot variation in the % recovery of analytes from Salivettes. The second reason is that the manufacture did not make any claims about what samples collected using the device could be assayed for. The third reason is that the manufacturer is not engaged in salivary bioscience research—we wanted a better more consistent product to use in our own research.

 

Why do you recommend avoiding alcohol comsumption for 12 hours before sample collection?

​Salimetrics recommends documenting alcohol consumption as a precaution. Alcohol stimulates saliva flow and is respirated out of the body, thus increased ETOH will be in saliva samples after consumption. High levels of ETOH in saliva samples interfere with the binding of antibodies in enzyme immunoassays. This is really only an issue if the research participant has been drinking heavily within 12 hours or is “hung over”. One or 2 alcoholic beverages within the prior 12 hours is not a problem.

 

Can/should I use protease inhibitors for saliva collection with Salimetrics swabs?

​Salimetrics does not recommend the use of protease inhibitors with our kits.  The kits are designed and validated for use without inhibitors.  

 

Can I use a stimulant to collect my saliva?

​Since contemporary salivary immunoassays have been designed to resolve the need for stimulating saliva flow to collect sufficient sample volume, Salimetrics does not recommend the use of stimulants to collect saliva samples.  The use of stimulants can yield falsely high or low values in immunoassays. Our goal is to minimize these sources of variation in saliva test results. If an oral stimulant must be used, then careful application is needed to apply that material consistently between sampling occasions, and pilot studies must be conducted to ensure that the use of the material does not cause interference in the assays to be used.

 

What is the difference between a ‘research’ kit and a ‘diagnostic’ kit?

​Diagnostic kits have specialized labeling requirements dictated by the US FDA. If you intend to use the result from testing to make a clinical or diagnostic decision, then you are required to use aDiagnostic Kit.  If you are using the testing to answer a basic or applied research question then the “research use” version of the kits are for you.

 

Can I use your kits (or any Salimetrics product) after the expiration date?

​Salimetrics does/can not guarantee results for expired kits and we generally do not retain replacement components for expired kit components. You can, at your own discretion, still run the assay and if the control values are in range then the assay results should be valid. 

 

What collection device would you recommend for my study?

​The choice about which sample collection tool you use is key to high quality results. Generally speaking, you need to consider multiple variables in this choice. You can view a list of the approved collection devices for each analyte by viewing the “Collection Protocol” on the analytes page on our website. However, we strongly suggest that you use our on-line service, or Ask An Expert, to get custom advise.  You will want to specify who your research participants are, how many samples you’ll collect, whether they will be self-collecting, and which analytes you want to assay now (and possibly later).

 

Can I collect samples for testing analytes using cotton?

Salimetrics does not recommend the use of cotton for collecting saliva samples. Passing saliva through an organic, cotton based material causes interference with many analytes, and when sample volumes are small, it can be very difficult to recover that volume by compression or centrifugation.  SalivaBio swabs have been designed specifically to resolve these issues. 

 

 

Can I store Saliva in SalivaBio Swabs?

Saliva can be stored frozen in SalivaBio swabs for short periods of time (weeks, months). However, generally speaking, the sooner samples can be expressed or centrifuged to remove saliva from the swabs, the better. This is mainly due to the fact that saliva storage tubes are not designed for long term storage at ultra-cold temperatures. Often the air space in these vials is large relative to the volume of the sample. Also, when saliva is distributed in the swab, the area of surface exposure to air inside the tube is increased. This creates opportunity for evaporation by sublimation which reduces the sample volume. This process has the potential to change the concentration per volume measurements for analytes, and especially so when the levels expected are very low and the volumes collected are very small.

 

 

How do I get saliva out of the swabs?

Specimens can be removed from SalivaBio swabs by centrifugation or compression and transferred to a vial appropriate for testing or cyrostorage.​ Visit the Saliva Collection Handbook for more information.

 

Is it necessary to have subjects rinse their mouth before taking the samples?

Salimetrics recommends rinsing the mouth with water before collecting saliva samples. Rinsing removes food particles and mucous.Be sure to wait 10 minutes after rinsing before collecting to make sure samples won’t be diluted. The Saliva Collection Handbook has more detailed instructions for collecting saliva. Of course, you can always call Salimetrics customer support to discuss your particular situation as well at 800-790-2258.

 

 

How many analytes can you test from one sample?

Generally Salimetrics recommends a maximum of 3 tests per sample. You can test all analytes from one sample if there is enough volume to run each test twice. Another alternative is to collect a large sample and aliquot the saliva into smaller vials/tubes to avoid freeze thaw when for multiple analyte testing. 

 

 

How do I measure flow rate when collecting saliva?

​See the Effects of Flow Rate and Mouth Location on Salivary Analytes section of the Saliva Collection Handbook

 

Can I thaw my saliva samples the day before use?

Salimetrics recommends thawing the saliva samples completely, vortexing, and centrifuging at 1500 x g (@3000 rpm) for 15 minutes on the day the assay is to be preformed. We have not compiled data on overnight stability of analytes.  

 

 

DNA FAQ's

Can I use saliva collected for DNA with the Oragene device to test for other analytes?

​No, the fluid in the Oragene device will dilute the saliva by an unknown amount and also has the potential to interfere with EIA, thus impeding the accurate measurement of analytes.

 

Can I use saliva collected for DNA to test for other analytes?

​Yes, saliva collected for DNA analysis using SalivaBio or Salimetrics collection devices can be used for other analytes as well. DNA in whole saliva is obtained from the cell pellet after centrifugation.  The supernatant can be used for testing other analytes.  DNA is collected from the actual SalivaBio swabs after centrifugation, so the saliva in the swab storage tube can be used testing other analytes.  Salimetrics recommends collecting the DNA material before testing for other analytes but it still possible to get the DNA sample after testing for other analytes provided that care is taken to prevent cross contamination during the testing phase. See this publication for more information;

Zsofia Nemoda, Maria Horvat-Gordon, Christine K Fortunato, Emilie K Beltzer, Jessica L Scholl and Douglas A Granger;Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples;BMC Medical Research Methodology 2011, 11:170 doi:10.1186/1471-2288-11-170.

 

How do I use your saliva collection devices to collect samples for DNA testing?

​Information on this topic was published online in the following paper:

Nemoda, Z.., Horvat-Gordon, M., Fortunato, C. K., Beltzer, E., Scholl, J. L., Granger, D. A. (2011). Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples.  BMC Medical Research Methodology, 11, 170. PMID 22182470

 

Handling and Storage FAQ's

Is it ok to have saliva frozen in the home and mailed overnight to a central collection location?

​For home saliva collection, if shipping on dry ice is not possible/practical, then they should be frozen and shipped frozen using 3 ice paks in a u-line insulated cooler for 36 hour delivery.  We advise including a temperature tracking device in your shippers. Salimetrics recommends FedEx overnight shipping. 

 

Can saliva samples be shipped by individuals in the regular mail?

​Choices of how samples will be shipped depends on several variables including budget constraints, shipping distance, domestic or international source of origin, and ability to control the cold chain. Ideally, samples once frozen after collection would stay frozen. Salimetrics website has advise about how to pack and ship samples to maintain the cold chain. Generally speaking, samples coming from individuals should be sent to a project coordination site locally, batched and then shipped on dry ice to Salimetrics. We advise including a temperature tracking device in your shippers, and these devices can be obtained via Salimetrics. Salimetrics only accepts samples that are shipped on dry ice to the Salimetrics SalivaLab.

 

What is Salimetrics recommendation for saliva storage? Whole saliva (passive drool)?

​Saliva handling and storage after collection is critical to the process of generating high quality results. Some analytes are unstable when handled at room temperature. Peptides (e.g., oxytocin) are so unstable that we recommend the samples be flash frozen within minutes of collection. Most analytes in saliva do not require flash freezing to maintain sample integrity. One of the major differences between saliva and traditional biospecimens is bacteria load.  Saliva has many different types of bacteria, some types are present in very high levels, and the types and levels vary from person to person. To protect unstable analytes and to prevent bacterial growth, we advise that all samples should be frozen at -20 C or below as soon as possible. If freezing is not practical, then it is appropriate to hold samples temporarily (for hours) at 4ºC. Samples stored for more than 4-5 months should be frozen at -40ºC or below.

You can find more detailed information in the Salimetrics’Saliva Collection Handbookon page 5.

 

How long can saliva for each analyte be stored frozen at -60C or below?

Once frozen at – 40 C or below, the stability analytes in saliva is no different than the long term stability of analytes in other traditional biological specimens. Theoretically, storage at -40 C or below is the best option and most economic option for the storage of saliva samples in biorepositories. 

 

How do I aliquot samples?

​In this video we demonstrate how to split saliva samples via aliquoting. We demonstrate proper technique and also review workspace prep and sample organization.