Images used with permission, ASSAY and Drug Development Technologies.
Awareness of leachates received a boost in 2008, when Science published a paper showing that bioactive contaminants leaching from plasticware "demonstrate potent effects on enzyme and receptor proteins." Shortly thereafter, an article in the Journal of Biomolecular Screening identified a bioactive substance leaching from automated compound-handling plastic tips. "We need to do more to address this problem," says Lynn Rasmussen, HTS supervisor at Southern Research Institute in Birmingham, AL, and chair of the SLAS Labware Leachables Special Interest Group (SIG).
The presence of leachates in labware was documented in the literature as early as 1980, when Bunge et al. reported that neurites in four-day cultures retracted and disintegrated or failed to form when the cultures were started in plastic, according to Walter D. (Lane) Niles, Ph.D., chief scientific officer and cofounder, Etaluma, Carlsbad, CA. A number of other journal articles documenting problems from leachates were published since then, including the two mentioned above. Therefore, Niles refers to leachates as the "last variable," urging researchers to acknowledge the problem and to consider the material composition of their work vessels as a factor in experimental results.
Most people are aware of reports of BPA [bisphenol A] leaching into food and beverages from plastic containers, can liners, soda bottles and other consumer products, says Rasmussen. "BPA and other substances are put into plastic to give a product specific characteristics—for example, to make it flexible or hydrophilic or hydrophobic—but they can also leach out of the plastic. The fact that these substances can leach out into our experiments and lead to erroneous results was a major topic of discussion in the Labware Leachates SIG at SLAS2014." [Editor's note: SLAS member Michael Mancini, Ph.D. and colleagues have developed a test that can help identify replacements for BPA].
"Leachates may result in false positive or false negative data that misleads researchers in a drug discovery program," Rasmussen stresses. "False positives usually can be sorted out because the investigators use various assays with different endpoints to confirm a compound's activity. But false negatives are more problematic because you'll never know what you didn't see. If a compound falls off the radar screen, you forget about it and focus on the hits. That means if the perfect lead candidate generated false negative results because of a leachate problem, you'll never know that you missed it.
"Arguably, the biggest impact of a leachate problem is cost," Rasmussen continues. "I heard a lot of frustration at our SLAS2014 SIG meeting from end users who were having leachate problems. They clearly were getting bad data, so they had to stop their high-throughput screening (HTS) campaign and spend time and money troubleshooting the problem, then spend more time and money repeating the process in order to get good data from the part of the run that was impacted by the artifact." In a large HTS center where many samples are run in a short period of time, researchers are likely to detect something that is impacting data quality, whatever it is," Rasmussen says. "But if you're a principal investigator in a small lab where you run a few plates a week, you're not running enough samples to determine if a problem is being caused by a leachate or something else. Identifying and resolving assay artifacts is important because false results may lead you down the wrong path, wasting time and money."
Niles has found in his work with neural stem cells that "there seems to be something in the plastics that profoundly affects cell growth. This means when we're trying to scale up cell cultures, we take a hit in yield and in doubling times (the mitosis rates of the cells)," he says. "I don't find plastic surfaces and biological cells to be all that compatible. When they work together, it's because many people do very cursory experiments using tumor cells that are hard to kill and typically grow for 24 hours. In the stem cell world, we grow cells for several weeks, and sometimes, for reasons we may not understand, they just die."
Niles worked at Aurora Biosciences in the 1990s, when the company developed a 3456-well plate. "The first thing we discovered was that if we fabricated that plate out of polystyrene, we couldn't get any cells to grow in it," he recalls. "It turned out that the mass of plastic used for those plates was much greater than for a standard 96-well or 384-well plate. So if there were anything in the plastic that could leach out and cause cell growth difficulties, it would most likely manifest in those plates." Serendipitously, while looking for an alternative to polystyrene, which affects experiments because it absorbs ultraviolet light and has autofluorescence characteristics, "we fortuitously stumbled upon cyclic olefin polymer (COP)—and we found we could grow cells in plates made of COP and the cells would thrive." Several companies now manufacture devices for implant and high-density multiwall plates using COP as a "cleaner" alternative to polystyrene because it is considered "medical grade," whereas polystyrene is not, he notes. "For example, COP intrinsically contains about two orders of magnitude less residual heavy metal polymerization and hydrogenation catalysts. Labware manufacturers have yet to produce standard tissue cultures wares such as six-well plates and T-series flasks in COP, however."
Other companies have been trying to reduce potential leachates in their products. "We avoid critical additives introduced during manufacturing that are known to interfere with bioassays, including plasticizers, biocides and slip agents (which make resins easier to mold)," says Nils Gerke, Ph.D., global product manager, consumables at Eppendorf in Hamburg, Germany. "Of course, it's not possible to say anything is 100% leachate-free. But by using raw materials, such as certified virgin polypropylene, to make our pipette tips and microfuge tubes, we can minimize the possibility that bioactive substances in those products will compromise a user's experiment."
Some companies use special methods for cleaning resins and some manufacture their own resins, according to Niles. "They may use cleaner resins for high density multi-well plates, but not for general work articles, and that's probably because of cost," he says. "Cleaner resins require different parameters for injection molding because they have different melt viscosities. There also are other technical requirements. For example, if a company wanted to make T25 flasks out of a cleaner material, it would have to reengineer the sprue and runner configuration of the mold and make other changes. The bottom line is, if we as end users want labware that is less likely to leach, we are at the mercy of vendors. Washing plasticware in the lab with ethanol or a solvent won't do it."
Rasmussen's own work was affected by leachates. In a poster presented at SLAS2012, she discussed a situation that occurred when she and her colleagues at Southern Research Institute were participating in the Molecular Libraries Probe Production Centers Network. "We were running a fluorescent assay and realized something was interfering with our fluorescent endpoint," she recalls. "If we took the compounds that were giving us problems in a fluorescent assay and ran them in a luminescent assay, we did not see the same problem."
It turned out that the artifact that produced false positive results in two of the three assays tested appeared to have been caused by a contaminant from a single lot of cyclic olefin copolymer microplates [Ed note: These are similar to, but not the same as, the COP plates mentioned above]. "This lot of plates had been used to store chemical libraries that were solubilized in DMSO, which is more likely to leach contaminants from plastic than are aqueous-based tissue culture media," Rasmussen says. "In this case, the leachate produced an effect that was dramatic and observable, which brought it to our attention—and because we had multiple copies of the compounds in an assortment of plate types, we could further narrow the artifact down to a specific lot of plates. However, if we had only run those compounds in luminescent assays, we would never have seen the problem. The point is, what is leached from plastic depends on the combination of plastic and solvent, and you will not see the effect of the leachate in all assays,"
The efforts of some manufacturers to offer "cleaner" labware notwithstanding, users still need to acknowledge the potential for leachates to undermine their work and take steps to control it, Rasmussen advises. "It is a problem and it is real," she emphasizes. "I've heard people say, 'My buffer sat in a plastic container over the weekend and on Monday I got bad data. I made new buffer, ran it again on Tuesday, and I got good data. I guess the buffer was bad, so I poured it down the drain. The new buffer works and I really don't care why it didn't work on Monday.' But leachates from the container the buffer sat in over the weekend could have been why it failed; there might have been nothing wrong with the buffer itself."
Once researchers accept that contaminants can leach into their reaction solutions or assays, "they need to take this fact into consideration when planning new measurements or assays," says Eppendorf's Gerke. "It's important to standardize your processes and use the same consumables during an investigation to minimize the possibility of variability. It's also important to keep the possibility in mind when analyzing results at the end." In addition, when authors publish, "they should include in the methods section of an article which products they use, so a reader has a chance to reproduce the complete experiment with the same materials, reagents and consumables."
Gerke would like to see some "basic standards similar to the MIQE guidelines," which provide minimum information for publication of quantitative real-time PCR experiments. However, he acknowledges that for consumables, "it might not be easy to define a group. The qPCR community can set up their own rules and then act in accordance with them. But consumables are used in every lab, so it may not be easy to focus it."
Other factors confound efforts to reduce or eliminate leachates. While it makes sense to try to use the cleanest possible consumables, "some things are beyond the control of the manufacturers who mold our products," Rasmussen observes. "For example, they have minimal control over their source supply of plastic. They have a spec sheet that says it's 95% this and 3% that and 2% something else. But they don't have the parts per million of this or that, and they don't test for these things. What I have learned is that in the world of industrial plastic manufacture, there is less scientific rigor than we researchers are accustomed to. To us, a lot is a batch of something that was made all at once and is homogeneous. By contrast, plastic is made before it is changed into products such as labware. The process can span months and the feed stock can vary over the span of a production run. Therefore, what is being manufactured at the beginning is not necessarily what is being made at the end. At that point, all the plastic in the run meets the specifications, but the potential leachable contaminants may not be known—and even if they are known, they probably are not listed in the specification document."
On the user side, Rasmussen adds, "even if we wanted to test for leachates, what would we test for and how would we do it? Would we take microtiter plates and grind them into little pieces and put them in a shaker with DMSO for 48 hours?"
Lane would like to see an ANSI standard for testing for chemical leachates in consumables. "We need to know specific things related to materials, like how many biocides will leach out? How many oleamides will leach out?" he says.
"The whole issue of leachates is just getting onto people's radar, and we're only now beginning to discuss what's practical and possible to do versus what's impractical or not possible. It's all a work in progress," says Rasmussen. She urges readers to join the SLAS Labware Leachables SIG and LinkedIn group associated with it, and to participate in the SIG meeting at SLAS2015. "This is comparable to the process that ultimately achieved the ANSI/SLAS microplate standards. It's not something end users alone can fix. It's something that end users and manufacturers have to work on together to solve."
October 6, 2014