Wednesday, May 30, 2012

Laboratory plasticware – damaging to our experiments?

On one of my kitchen shelves sits a plastic food container discolored a reddish-orange from previously storing leftover spaghetti, a stain which no amount of washing can seem to fade it the slightest. From this, it is evident that plastics do interact with food products and the evidence is clearly visible in every kitchen around the nation.  Those of us in the lab use plastic tubes, flasks, and plates to store reagents or grow cells; even though the interaction between the plastic and our samples may not leave such a noticeable mark, it is still reasonable to think that some interaction occurs, potentially leaving contaminants in our samples and affecting our experimental results.  In fact, researchers have studied the effects that laboratory plasticware have, and the findings though troubling is extremely eye opening.

In a 2008 Science paper, researchers found diHEMDA and oleamide in water and DMSO/methanol, respectively, after these fluids were used to rinse plastic tubes.  Both were then shown to have an inhibitory effect on an enzyme, human monoamine oxidase-B. And, DMSO rinsed through plastic tubes could even inhibit GABAA receptor-ligand binding due to the ability of oleamide to bind to the receptor1.  In an even more recent story, Nature News reported in 2010 that plastic tubes release compounds that increase absorbance readings of the samples.  Mass spectrometry confirmed an increase of chemicals from the plastic leaching into the samples after tubes were heated (which can occur from innocuous activities such as centrifuging for longer periods of time) or when inorganic solvents were used2.

Remarks made in a Nature News article and readers' comments on the website indicate just how prevalent the problem of contaminants from plastics is3. For instance, from the Nature News website, Yarek Rivers comments, "We had this very problem in our own lab, when chemicals leaching from plastic tubes increased the UV absorbance of our samples, and confounding nucleic acid quantitation.  Even very simple assays can be significantly impacted by these effects."  Additionally, it may not be safe to assume that experiments are equally designed and executed as long as both control and experimental samples undergo the same treatment or growth conditions in the plastic because the interaction with plastics can be variable.

A Nature reader, William Wustenberg suggested there is "wide variation in quantity and character of the leachables in supposedly identical materials" and "exposure of plastics to other compounds from processing, packaging, handling and storage can make a profound difference in the leachable profiles."  To prevent confounding or non-reproducible results, an idea would be to research what kind of chemicals could leach out of certain products, with information made widely available to researchers of course.  In support of this idea, Andrew Holt's lab, which authored the Science paper discussed above, found that colored microcentrifuge tubes seemed to be a source of contaminants affecting his experiments, with clear tubes eliminating the problem. 

Leading Tissue Culture plastic manufacturer TPP is fully aware of the interaction between cells in culture and their plastic environments that potentially dictate growth, propagation and senescence. They firmly declare the absence of any additives into the polypropylene manufacturing process and the use of only virgin plastics in the resin4. Other TC manufacturers also detail the effects of laboratory reagents on different types of plastic (polystyrene, polycarbonate, etc.) that could be very helpful for researchers5.  For instance, it cautions researchers that oxidizing acids attack polystyrene plastic whereas there is no effect on polytetrafluorethylene products.  A survey of additional laboratory plasticware providers reveals that companies are generally aware of this issue, and some have taken measures to provide more information about this issue.  One of the leaders in plastic bottle manufacturing informs customers on their website that "common additives [in the plastic] include stabilizers like BHT; lubricants like calcium or zinc stearates, colorants."  Additionally, "there may also be some monomer of the plastic available for extraction in the final molded product."  However, they reassure customers by saying that the "extractables typically occur in very low concentrations (ppm or ppb)," and that "even though a plastic contains an additive, it may not be extractable in a particular fluid" due to the requirements of being soluble in the fluid and being present on the surface of the plastic. 

Based on the information provided on the companies' websites, there are also plasticware manufacturers that go further, making an effort to decrease the interference of plastic with our samples.  TPP does this by "using ultrapure raw-material that is certified to be free of chemical softeners and additives."  Laboratory tips manufactured by Sorenson Bioscience also do not contain additives such as silicone, which had been used in the past to prevent DNA and protein binding to the plastic but was later shown to denature DNA. Rather, Sorenson’s method of preventing sample binding is to use a well studied and proven method called Low-Binding Surface Technology that involves bonding a proprietary polymer to the inside of the tip to create a hydrophobic surface, thereby decreasing the surface tension that promotes the sticking of DNA and proteins6. The polymer is not affected by chemicals or solvents present in samples, and there is no leaching of contaminants into the sample and interference with the experiment.  Perfect for quantitative assays, these low-binding surface products would solve problems of inaccurate measurements of protein or nucleic acid concentration, as mentioned by Nature reader Yarek Rivers. In blinded tests performed by an independent laboratory, these Sorenson Low Binding Tips as well as other manufacturers' conventional tips were used to pipet DNA or protein solutions and then washed with water.  The water, containing DNA or protein that had bound to the tip, was analyzed by spectrophotometry. Results from these tests confirmed that the tips work as advertised; the averaged absorbance values from 8 experiments revealed that Sorenson's Low Binding Tips have significantly decreased DNA or protein binding.  As a final example, Axygen's Maxymum Recovery products use an ultra-smooth mold and a modified polypropylene resin, with no chemical additives involved as well, to achieve a smooth pipette tip surface devoid of occlusions and cavities, thereby eliminating samples from sticking to the tips7

Current approaches to prevent the interaction of plastics with nucleic acids and proteins rely on unique and well researched technologies to aid something as simple as liquid handling in a laboratory as well as something as complex as growing primary cells isolated from adult tissue. Thus, there needs to be ongoing communication between the scientist community and the plastic manufacturing industry to aid efficient dissemination of information about which plastic products are most appropriate for certain types of samples.  Both scientists and manufacturers are ultimately working towards a common goal – ensuring the validity of ground-breaking findings and improving the reproducibility of data which must be repeated by different individuals and labs – and therefore ought to work together to achieve it.



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