We think of plastics as a problem due to the waste. However, it seems some also bias our experiments.
I couldn’t really believe my eyes as I went through the literature, but it seems quite clear that some plastics affect your data.
Let’s discover in how far the contents of your tubes, tips, and plates leach into your samples.
Today's Lesson: Leachables from Plastics
To what extend do our lab items affect our results
Number of the Day
Whether Bachelor’s student or postdoc, we measure DNA/RNA concentration by measuring UV absorbance at 260 nm. However, it might be that the largest contributor to the peak was actually leachates from the plastic items you used. In fact, Lewis et al. showed that, for example, benzaldehyde compounds from microcentrifuge tubes can severely affect our readouts. Let’s therefore take a closer look at what people have found and in how far you might be affected:
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How Plastics Can Affect Your Data
Almost all plastics that we use contain so-called additives.
These are molecules added to make manufacturing easier, make them more resistant to UV light, or enhance their properties in other ways.
Whether it is (2Z)-1,4-diphenyl-2-butene or bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), plastic-related leachables come in all sorts of shapes and forms. Some additives help release plastic parts from the mould cavities in which they are manufactured, while others improve the material’s physical properties after production.
A few years ago, I did some research and arrived at the conclusion that it is safe to turn on the UV light in your cell culture hood because plastic degradation is too weak to lead to significant leaching.
This is why it was so unsettling to realise that while this might be true, there is a whole range of additives that can affect your data:
Effects on Enzymes and Assays
One of the first groups to notice these effects was McDonald et al.
The group was at some point confused because, although their monoamine oxidase B assay had always worked well, they now observed variability.
They state: 10% DMSO or water in 40% of tube volume, 1 hour, 20°C, from Fisherbrand (clear bars) or Sarstedt (hatched bars) plastic tubes or glass vials (C). x axis labels indicate tube volumes (ml). C indicates control; SZ, siliconized 0.5-ml tube. What compound caused the higher activity in the DMSO case couldn’t be resolved but it was an effect seen repeatedly.
They searched for the reason, and indeed, what they found was that it was the tips they used for pipetting.
They leached DiHEMDA and oleamide, which inhibit MAO-B.
In case you are interested: to identify these leachates, you simply pipette or, in other cases, when it is about tubes or plates, you incubate these items with solvents like water, methanol, or DMSO. Then you normally run GC-MS or LC-MS analysis, sometimes even NMR, to see which compounds are present.
Another group found that a coloring agent called NP10 inhibits mitochondrial complex I, which was a problem for their human sample analysis.
Upon checking, they saw that NP10 stops cell division when cells are grown in restrictive media but not in normal medium.
Interestingly, the publication identifying the coloring agent as having an effect on mitochondrial complexes was published as a letter to the editor, in contrast to the Scientific Report on the right.
Still in 2020, a collection of case studies, essentially showed that when analyzing extracts, a range of interfering leachables could be identified in common lab items and membranes.
Effects on Analyses
Almost 20 years ago, a group found that a compound called Tinuvin 770, which is a UV stabiliser, would essentially mask other signals in their LC-MS.
Click to enlarge. The original graphs from Schauer et al.
That makes it almost impossible to carry out a normal analysis.
In more recent studies, the group around Canez et al. found several hundred contaminants in materials used for lipid extraction analysis.
While some overlay weak signals from actual analytes, they also found that some lipids from these plastics are the same as those you analyse in human serum samples.
Effects on Cells in Culture
We also find that these leachables can affect cells in culture.
Hammond et al. unraveled that the degradation product of a plasticizer causes cell death.
Comparison of (a) HPLC-UV chromatograms of water extracts (48 h incubation at 50°C) with (B) viable cell density measured in cell-based assay experiments for the various bags tested. The peak positions of degradants of TBPP are indicated in (A). Please note the effect in F-1. The point is that these bags contain bDtBPP but Hammond et al. weren’t able to detect it in the water, only when the medium was stored directly in the bags.
What is remarkable here is that it was just the bags in which the water that would later be used to create medium was stored that caused the effect.
However, other groups also saw differences in neuronal morphology when growing their cells, although they were unable to actually identify the compound that caused the effect.
A recent study then concluded that while most extractables did not show a significant effect, a few of them do, which can be seen across a broad range of cellular activities in cell painting assays.
Pahl and colleagues tell us that during cell painting assay evaluation the cellular compartments are imaged in five different channels. What you see above is the percentage of changed imaged channels for CPA-active compounds. ER: Endoplasmic Reticulum; Hoechst: Nuclei; Mito: Mitochondria; Ph_golgi: Golgi/Cell Membrane/Cytoskeleton; Syto: Cytoplasmic RNA/Nucleoli.
Even More Problems
This is not all the literature that exists.
Some studies essentially suggest that certain leachables interfere, for example, with acetylcholine receptors.
It was even shown that a leachable could bind to the aforementioned monoamine oxidase B, which would later be seen when people conducted X-ray crystallography and wondered what the unknown inhibitor was. Also, here is an interesting review if you want more.
Also, the paper from the very beginning demonstrates that whether it is sonication, multiple PCR cycles, or centrifugation, these can lead to leachables causing peaks at 220 and 260 nanometres.
Lewis et al. identify two main “types” of absorption patterns after brief heating (30 min, 100°C) of microtubes with water. They state that most commercially available tubes are type 1. You see Microtubes: type 1, VWR, Cat. no. 20170 – 038; type 2, Ambion, Cat. no. 12400.
In fact, even Eppendorf has run some tests that clearly show you can falsely identify amounts of DNA of up to 3 to 5 micrograms per millilitre, although all the tube contains is water.
Even when running PCR cycles with plain water controls, for some items, you will see SYBR signals.
Applying the Knowledge
As always, it is essential to keep a cool head.
It does not necessarily mean that your experiments are affected, but testing this might make sense.
Many papers have shown that there are big differences among manufacturers.
Click to enlarge, or better, visit the white papers. The data on the left come from an Eppendorf white paper showing fluorescence signals without baseline subtraction, measured for water samples incubated in PCR plates. And yes, while nobody is storing their DNA at 40 or 95 °C, as indicated in this white paper, you certainly reach higher temperatures during PCR.
Unfortunately, even variation among batches is noticeable.
Sometimes this can be due to the supplier changing the manufacturer that actually produces the items.
Of course, many of these effects also depend on concentration, volume, and the contact time with the plastics.
For the future, it makes sense to ask your supplier whether they disclose information about additives.
For instance, Eppendorf does (unsurprisingly given their later efforts, although early papers had shown effects when using their items too.)
That means if you now see questionable data, you will have another idea of what to check for.
Moreover, you have a new argument for switching to glass, although it has some leachables too.
How We Feel Today
References
Lewis, L.K., et al., 2010. Interference with spectrophotometric analysis of nucleic acids and proteins by leaching of chemicals from plastic tubes. BioTechniques, 48(4), pp.297–302. doi:10.2144/000113387.
McDonald, G.R., et al., 2008. Bioactive contaminants leach from disposable laboratory plasticware. Science, 322(5903), p.917. doi:10.1126/science.1162395.
Jug, U., et al., 2020. Interference of oleamide with analytical and bioassay results. Scientific Reports, 10, 2163. doi:10.1038/s41598-020-59093-1.
Schauer, K.L., et al., 2013. Mass spectrometry contamination from Tinuvin 770, a common additive in laboratory plastics. Journal of Biomolecular Techniques, 24(2), pp.57–61. doi:10.7171/jbt.13-2402-004.
Canez, C.R., et al., 2024. Studies of labware contamination during lipid extraction in mass spectrometry-based lipidome analysis. Analytical Chemistry, 96(8), pp.3544–3552. doi:10.1021/acs.analchem.3c05431.
Canez, C.R., et al., 2024. Investigation of the effects of labware contamination on mass spectrometry-based human serum lipidome analysis. Analytical Chemistry, 96(21), pp.8373–8380. doi:10.1021/acs.analchem.3c05433.
Hammond, M., et al., 2014. A cytotoxic leachable compound from single-use bioprocess equipment that causes poor cell growth performance. Biotechnology Progress, 30(2), pp.332–337. doi:10.1002/btpr.1869.
Lee, T.W., et al., 2015. Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons. Journal of Neurochemistry, 133(1), pp.53–65. doi:10.1111/jnc.13009.
Pahl, I., et al., 2024. Assessing biologic/toxicologic effects of extractables from plastic contact materials for advanced therapy manufacturing using cell painting assay and cytotoxicity screening. Scientific Reports, 14, 5933. doi:10.1038/s41598-024-55952-3.
Reuhl, T.O., et al., 1990. Tissue culture tube contaminant inhibits excitatory synaptic channels. Brain Research Bulletin, 25(3), pp.433–435. doi:10.1016/0361-9230(90)90234-q.
Hubálek, F., et al., 2003. Polystyrene microbridges used in sitting-drop crystallization release 1,4-diphenyl-2-butene, a novel inhibitor of human MAO B. Acta Crystallographica Section D: Biological Crystallography, 59(10), pp.1874–1876. doi:10.1107/s0907444903016883.
Olivieri, A., et al., 2012. On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware. Canadian Journal of Physiology and Pharmacology, 90(6), pp.697–703. doi:10.1139/y2012-049.
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