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Purdue s J. Paul Robinson Discusses Flow Cytometry and His Plan for ISAC

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At A Glance

Name: J. Paul Robinson

Position: Professor, Immunopharmacology and Biomedical Engineering, Purdue University; Director, Purdue University Cytometry Laboratories

Background: Postdoc and Research Scientist, University of Michigan Medical School — 1984-1988; Postdoc and PhD, University of New South Wales — 1981-1984

J. Paul Robinson has been involved with flow cytometry for nearly three decades, and has authored several instructional manuals on the subject. Fresh off of a trip to Budapest for the Hungarian Biophysical Society Cell Analysis Section's annual meeting, and in the midst of preparing for a trip to next week's International Society of Analytical Cytology's International Congress in Montpellier, France (where he will be named president-elect), Robinson found some time to chat with Inside Bioassays.

You've written several books on flow cytometry methods? How did you originally become involved in the field?

In the late 1970's, when I was in Australia in the immunology department [at the University of New South Wales], we did all of our immunology basically using a microscope. And my boss, who used to go to experimental biology meetings every year came back one day, when I was a graduate student, and said "We're going to buy a flow cytometer," and I didn't have a clue what he was talking about. And I said "What is it?" And he said "It's a machine that looks at cells with a laser and it phenotypes them, and we don't have to look under a microscope." And I thought, "My glory!" And I didn't know anything about it.

I had to review a couple of papers at our journal club, and he gave me some papers on flow cytometry and said "Tell everybody about flow cytometry." I remember sitting there trying to explain this, and I had no idea what I was talking about. But that's where it started. We bought one and set it up. And I finished my PhD and went to Michigan to do a postdoc for two years in 1984 — I had gone to the World Immunology Conference in Kyoto in 1983, and there I met Peter Ward from the University of Michigan, and he invited me to come and give talks, and so I did. He offered me a postdoc straight away, so I came for two years in 1984.

What was some of the early research that you conducted using flow cytometry?

Well, I was interested in neutrophil function, and our lab discovered that we could study neutrophils — one of the nice things about flow cytometry is that I had spent a lot of my time isolating and purifying populations of cells, so you can actually study cells in a heterogeneous environment. You don't need to separate them and have pure populations of cells. So this was very powerful. We were interested in several other areas, but I followed through in this area over many years. In the early 80's, when I was at Michigan, we would talk with our dental colleagues, and they'd say: "We'd like to look at crevicular neutrophils." We asked how many neutrophils can you get out of a crevicular space, and they would say, "Oh, about 10. But you guys keep telling us that you can run very few cells." So we did a study, and amazingly we could get enough cells out of a crevicular space to study the cell function. And so, when you work hard at those things, it turns out that you can study a very few cells. The fact that you can actually study the function of cells, I think, is the real key thing about flow cytometry. But you can also phenotype them at the same time. And if you want to, you can recover the cells. So the various studies that we did were dependent on the fact that it was easier to study a mixed population of cells — particularly in blood, where it takes a long time to purify a population. You don't really know what you're doing to the cells as you purify them over six to eight hours sometimes before you do an assay.

So are you still studying neutrophils today?

Yes, as a matter of fact we're doing work studying mitochondria and oxidative metabolism, basically, because neutrophils are a great source of cells that convert oxygen through the oxygen metabolism pathway, through superoxide, hydrogen peroxide, through to water. And those oxygen radical pathways are very important in many other areas: Apoptosis, DNA damage, et cetera. They're important in just about everything. Inflammation is one of the key aspects of physiology.

And the other thing we've done is sort of focused on the fact that nobody standardized the methods. When I moved to Purdue in 1988, I was setting up my lab — and when you move to a new institution, you don't have anything there, you walk into an empty lab — and it took me three or four months to buy all the stuff that I needed. And I wrote the Handbook of Flow Cytometry Methods, because I knew that I would have to train technicians and students, and I wondered how we would do that, because the methods are very specific and very delicate. We published that, and lots of copies went out and actually now that has been converted to Current Protocols in Cytometry. I wrote [Handbook of Flow Cytometry Methods] in 1988, and it's still on the market — people still buy that book, but I don't know why because it's out of date.

So what are the most important advances that have occurred since that first handbook, and how do these translate into future advances?

I think the future advances have got to be in truly looking at high-content and high-throughput, and what I'll call spectral classification. This is moving a little bit away from traditional flow cytometry technology that uses a photomultiplier tube and an optical filter and a laser, and shoots cells past it. We will have much more sophisticated technology that [has] very comprehensive multi-anode detectors that, instead of using filters to separate the signals, we'll simply collect a broad spectrum of wavelengths — 30 or 40 channels of wavelengths. And then we will use linear unmixing and deconvolution to extract wavelength spectra out of this information. It will be heavily mathematical, and it will be classification based. We'll do what the chemists have been doing for years. This is the next major leap in the technology, because what that will do is force us into the informatics age, where we collect an enormous amount of information, and then we allow the computers to help us extract the information. Now we follow a very simplistic pathway — which can become complicated — but it's a simplistic pathway. People like Mario Roederer [of the NIH] are collecting 10, 12, or 14 colors using single PMTs and filters, but the compensation issues are very complex, and I think that spectral cytometry as I'm proposing will reduce our dependence on some of those complex problems. I'm really looking forward to being able to do diagnostic classification on a flow cytometer, which I think we should have been able to do 10 years ago, but with the computation power that we now have, it's very doable.

What are your thoughts on the commercial offerings in flow cytometry?

I think all of them are good. I never put myself in a position where I upset any of [the providers]. As incoming president of ISAC I've got to walk a very fine line. But I do believe that all the companies have some unique advantages in their technologies. We're very fortunate that we have several dedicated companies. For example, there are big companies in the US, such as Becton Dickinson, Beckman Coulter, and DakoCytomation. But there are several other small companies — Guava, for example, [and] companies like Partec in Germany. And there are some technologies that I have not yet verified, but it's pretty clear that the next generation of technology is going to have features that are not on current systems. I can predict what some of those are going to be; in fact, I know what some of them are going to be, but I can't say what they are. But I can say that many companies are going to move into areas of flow and image. I think that's going to be a very interesting opportunity. If this were two weeks later I could say some more, because I know some interesting stuff is going to be released at the ISAC meeting.

So tell me what's next in your position as president-elect of ISAC? Do you have any general plans or ideas at this point?

I do. I can point you to a website (http://www.cyto.purdue.edu/vote/index.htm) that talks about what our plan was to really advance the organization. The first thing we're going to implement is an ISAC fellows program, where we hope to raise the number of students and postdocs interested in our field. We have very few — an insignificant number. And I think we need 150 students and postdocs in our society. So, I'm proposing to go and bring the pharmaceutical companies into our society, because we have mostly instrumentation companies. But I believe the application areas are very important — and next-generation applications will absolutely be moving into drug discovery. We cannot avoid that. So moving into the high-content, high-throughput screening areas is really a goal of ours. Along with the current incoming president, Maria Pallavicini, we will start the program with the goal to bring 150 students into the organization within the next three years. This is a huge undertaking, because that's a lot of students. But we want to do that at the same time as we're bringing the pharmaceutical companies in to show this is where we're going.

And I think the society needs to take a higher role in public policy. We forget that we have to influence public policy in terms of our very high-cost technology. We'll improve our role in education and training, which I think will endear us more to the pharmaceutical companies that find this important, and also this will be important for students and graduate members. We need to keep ourselves in line with the current and changing technologies, because we are truly a technology society — we work with the most leading-edge technology, and we have to be totally aware of how those technologies change and how the impact of what we do will also change.

And one more thing, which is a hard one to get publicity for. The problem with our technology is that it's very high-cost. It's a problem because in third-world countries, AIDS is probably one of the most significant problems. So I'm working with a colleague, Frank Mandy, from [Health Canada in] Ottawa. We are very keen to make sure that there is low-cost technology available for HIV testing. And this is an issue because we work in a group of people that depends upon expensive instruments. If we don't have the highest cost, most expensive instruments, then the things we want to do at the leading edge can't be done. But the conundrum is: How do you deal with this unbelievable problem in Africa of AIDS testing? So this becomes standardization and low-cost testing. These are issues that [ISAC] has to deal with. If we don't deal with them, we can't expect the politicians to deal with them either. As a technology society, we have to address that. I think the work that Frank Mandy is doing around the world is extremely important and we are going to focus on that in this and next year's meeting. We are in the center of this measurement technology, so we're obligated to try to make sure it's not only in the most expensive environments; we have to make sure that it's accurate, standardized, and available in environments that simply don't have the money that we have, in the United States, particularly.

 

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