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July 11, 2019 - The benefits and perils of orthogonal measurements

One of the many reasons Spectradyne's customers get excited about our MRPSTM technology is that it is inherently "orthogonal" to the measurement techniques most of them use. Most scientists working with micron or submicron particles have some form of optical instrumentation at their disposal: either Dynamic Light Scattering (DLS), Multi-Angle Light Scattering (MALS), or Nanoparticle Tracking Analysis (NTA). These are standard measurement technologies in the pharma industry. Spectradyne's MRPS technology is, by contrast, a 21st century version of an electrical sensing method (the Coulter principle) that, at its core, has been around for over 60 years. Although it's been around a long time and is the gold standard method of counting blood cells, the challenges of applying the method to deep submicron particles have only recently been solved, in a robust manner, by Spectradyne's MRPS technology.

In this blog post, we'd like to review why orthogonal measurements are important, and why customers want them. This will then lead us to discuss why, in fact, some customers don't REALLY want an orthogonal measurement after all, though they may not want to admit it!

First, let's review why our customers want orthogonal measurements. Just about all of Spectradyne's customers are scientists (or managers who once were scientists!), and all scientists know that a measurement result is more powerful if the result is obtained more than once, better if obtained by different researchers/labs, and even better if obtained by a different type of instrument. Especially if one has an exciting result, a natural scientific mind-set tries to confirm that result with another method. If you don't, someone else certainly will! So, researchers want that orthogonal result because it provides confirmation and validation and is just plain good scientific practice.

But of course, therein lies a problem: what if the second method, orthogonal as it is, doesn't confirm the first result? What if Spectradyne's MRPS measurement in fact indicates that those extracellular vesicles or liposomes aren't quite as purified as you thought, based on NTA measurements? It's been known to happen, as detailed in earlier blog posts about exosomes and protein aggregates!

In fact, we can take this scenario a step further. If a new measurement method is actually independent/orthogonal to a first method, meaning that it is probing the sample through a different interaction mechanism, it's actually quite remarkable if the same result is obtained. A simple example is the sizing difference obtained from DLS and Electron Microscopy (EM) measurements. DLS probes the size of particles through their Brownian motion in liquid, thereby extracting the hydrodynamic radii of the particles. Electron Microsocopy (EM) probes a completely different aspect of a material: the material's re-emission and scattering of high energy electrons, and therefore giving information related to the core physical dimensions of particles. In this example, different information is obtained from each method; there's not one method that's right and one that's wrong. Of course, in this case the fact that the absolute sizing is different between the two methods is less important than determining if results are correlated: when DLS reports that sample A has larger particles than sample B, a similar trend in sizes should also be reported by EM.

In the case that orthogonal results are correlated, and therefore consistent with each other, a researcher is in a happy place because her measurement result has been confirmed by two methods. The results may not agree in an absolute sense, but that's to be expected. If they are at least consistent with each other, then that's confirmation enough. However, what if a first result, perhaps a desirable one, is NOT confirmed by the second, orthogonal, method?

Unfortunately, this will definitely happen sometimes! The power of a result increases when a second method confirms it exactly because there is a possibility that the second method will expose something new or different that wasn't picked up by the first method. One can't have the benefit of the affirmative measurement without the possibility of a negative result!

This is the reality that this blog post wants to illuminate: An orthogonal measurement is an independent method that may confirm a result, but it may also NOT confirm a result. And this alternate method is powerful exactly because of this possibility. It means that, in striving for an orthogonal result, a researcher may re-live the uncomfortable part of the old saying "be careful what you wish for, because you just might get it." At Spectradyne, we encounter this directly, every week, because our MRPS technology is orthogonal to most of the measurements our customers already have. Most significantly, our technology has exposed the "false peak" phenomenon that researchers commonly encounter in their NTA measurements. Many an on-site demo has become uncomfortable when we realize that a researcher didn't REALLY want an orthogonal measurement but rather just a confirmation from a second method.

If you are a scientist reading this post, ask yourself: how will you respond if confronted by an uncomfortable result that doesn't agree with your expectations? Will you admit the possibility that your understanding is incomplete and a deeper investigation is warranted? Or will you ignore the new measurement and search for another method, perhaps one not so independent, that "confirms" the first one?

Do you really want an orthogonal measurement, or just a second one? We'd advocate for courage, that more data is always better, so that having an orthogonal method is indeed the way to go.

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