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Sample Preparation and Specimen Quality for NGS assays and Molecular Diagnostics

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Amid Zand:

Hi everyone. Welcome to this podcast from Cambridge Health Tech Institute for the Knowledge Foundation and CHI's ninth International Sample Prepped Technologies Conference which runs from June 25-26th in Bethesda, Maryland. I am Amid Zand, associate [inaudible 00:00:15] producer at Cambridge Heath Tech Institute.

We have with us today one of our speakers at the conference, Dr. Patrick Hurban, vice-president of Research and Development at Expression Analysis which is recently acquired by Quintiles. Dr. Hurban joined Quintiles in 2012 as a global head of genomic research and development. Upon the acquisition of Expression Analysis by Quintiles. He is responsible for the identification and implementation of new genomic capabilities for consulting and special projects and for the development and validation of genomic assays.

These include broad-based screening assays such as exome and RNA sequencing as well as microarray based expression profiling and genotyping. Dr. Hurban has over 25 years of experience in molecular genetics including over 15 years in positions of increasing responsibility in high profile genomics focused organizations. His research interests have focused on the genetic control of gene expression and have spanned diverse fields such as toxicology, developmental and cancer biology. Dr. Hurban, thank you for joining us.

Patrick Hurban:

Absolutely. Thank you for having me.

Amid Zand:

At the Sample Prepped Technologies Conference is designed to cover and discuss the best practices in pre-analytical processing for molecular diagnostics and biodetection applications. How do you think pre-analytical variables can impact the sensitivity of NGS assays?

Patrick Hurban:

They can have a tremendous impact. Here at DA Quintiles we do a great deal of testing. It's all genomic testing. We develop and validate a number of different assays. The pre-analytical workflows ... really, what happens to the sample before it comes to our facility have a lot to do with how we're going to design the assay, what we're going to expect of the assay and the type of specific test cases that we'll include in our validation. Perhaps the most well-known example is the difference between frozen samples and formal and fixed parafin embedded samples.

With fresh frozen samples, whether we're looking at RNA or DNA, we have a certain kind of presumption about the quality of the material provided that that tissue or other type of sample was flash frozen and stored well prior to it coming to our facility. For a formal and fixed sample on the other hand, we know that not only are the conditions of fixation going to be pretty rough on those nucleic acids and can lead to certain kinds of degradation, and as a result we may, certain kinds of assays that we might use on fresh frozen samples just simply aren't going to work very well on those formal and fixed samples.

In addition, we can see that the exact conditions that are used for fixation and storage of those samples, or even if we have a parafin block, if we cut the sample immediately or we cut the sample and then wait for a long time before we actually analyze the types of results that we get ultimately, whether that results is an accurate reflection of the biology that we're really after in trying to sample. We need to make sure that when we are developing an assay we keep all of that in mind and if we're going to be getting different kinds of samples or the intended use of an assay encompasses all of those different types of samples than it's really important that we include those parameters in our validation.

Amid Zand:

Speaking of the NGS assays and based on your experience at Quintiles, how important are biospecimen quality requirements in the process of NGS validation?

Patrick Hurban:

You know, they're tremendously important but I kind of look at it, really, from two perspectives. One, is sort of the ideal perspective. The other is the more practical perspective. I'll talk about the ideal perspective first and that is that you know, of course, for any sample that we analyze we want it to be as high quality and as pristine as possible. If we're looking at genomic DNA, we want it to be free of contaminants. We want it to be nice, high molecular weight. If it meets those kind of requirements, almost any type of assay that we perform is going to be suitable with that type of material.

As we have more say, contaminants, that may be in that sample or as we get higher levels of degradation or cross-linking or even base changes, the sorts of things that happen when you fix the sample in [inaudible 00:04:30]. Well, then we run into certain types of issues that we have to ... that cause us to be careful about how we interpret the data. Usually when we're developing an assay, we will certainly do some initial feasibility work using nice intact pristine samples that we have a lot of control over. Not only from a sample quality standpoint but also understanding the genomic content of that sample so we can understand whether our assay is returning a result that we would consider to be true or not.

But, that's where the practical side comes in because we know that if you're collecting samples as part of a clinical trial or if you're accessing biobanks that have large collections of samples, the reality is that these samples are not going to be in pristine condition. What we have to understand is, for a given assay and how we're going to use it, if we're going to use it for example for a retrospective analysis of a large collection of formal and fixed tissues, well then one of the things we need to do is make sure that we, specifically, test formal and fixed tissues as part of our validation and development process. There, you know, I had an interesting discussion with a vendor recently who provides us with a lot of formal and fixed tissues that we can use for these types of testing purposes.

They talked quite a bit about how wonderful and uniform their processes were in all of their different pathology labs so that they were able to provide us with very high quality formal and fixed samples. A question that I had for this gentleman was, "Well, can you provide me with some samples that have much lower quality because if I validate my assay using only your high quality samples than I'm not going to understand how well that assay performs in the real world lower quality samples that I know I'm going to encounter when I actually use the test?"

We have to think of quality from a number of different perspectives. How it's going to impact your assay? But, we always have to be grounded in something very practical and understand that no matter how much we want perfect, pristine samples that's just not what we're going to get when we actually go to testing. That has to be part of your assay development and validation planning.

Amid Zand:

We are excited to have you speak at the conference in June. Why are you planning to attend and present at the conference? Can you tell us a little bit about your expectations and what you're excited about the most at the upcoming conference?

Patrick Hurban:

Well, first of all, thank you for inviting me. I mean, I'm really honored to be a part of the conference itself not only as a participant but also as a speaker. I attended last year. It was a fabulous conference. I really got to meet some folks who were having the same ... they're encountering the same types of issues we were. Very early in this discussion here today you used a phrase that I think is emblematic of why I want to attend this conference and why I'm so excited about it. That is, best practices.

All of us engaged in these types of assays and that are part of this field, we really want to utilize best practices because we want to be able to get the most out of the samples that we have. All of these assays that we run, many of them can be kind of expensive, but really, the most expensive thing is not the assay itself but the idea that you could be wrong. Learning from your colleagues how best to go about this and just exchanging ideas with the experts in the field, people who have encountered the same issues and who have developed their own creative solutions, just that kind of cross-fertilization you can get by gathering people who are really active in the field. I think that's what I'm really looking forward to. I got a great deal not only out of the podium presentations but also just out of the ideas that can be exchanged during breaks between sessions and just sort of the social interaction that I have with a certain amount of my colleagues.

I'm really looking forward not only to the formal talks but just the chance to network with some folks who are really working at the same sets of issues in the field. I really like being able to exchange ideas with them.

Amid Zand:

That was Dr. Patrick Hurban, vice-president of research and development at Expression Analysis. He will be speaking at the upcoming ninth annual prep technologies conference taking place on June 25th and 26th in Bethesda, Maryland. If you would like to hear him in person, go to for registration information and enter the key code 'podcast'. I am Amid Zand, associate conference producer. Thank you for listening.

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