Thursday, September 4, 2014

A First Look at the Beckman Coulter CytoFLEX - Strong Performance in a Small Box

Over the past few years, we've been inundated with small, inexpensive cytometers with the promise that they can perform as well as the big boys. Up until now, I would have told you not to waste your time... up until now.

In 2013, an unknown company called Xitogen set up a booth at the annual CYTO conference. Before long, there was a buzz racing through the exhibit floor aisles of a flow cytometer starting at ~US$25,000 (1 laser, 2 colors). The Chinese company, headquartered in the Suzhou Industrial Park, set out to provide an alternative for Chinese researchers to acquire affordable flow cytometry instrumentation without having to deal with overpriced imported hardware from the big players. With U.S. zero install base, and zero user-generated data, CYTO 2013 came and went, and the buzz surrounding Xitogen died out. It was pretty obvious the better known cytometer manufacturers would be taking a look at the company for a possible acquisition, and in April of 2014, Beckman Coulter announced they would purchase Xitogen for an undisclosed amount of money.  The acquisition was finalized in June 2014. At CYTO2014, Beckman Coulter revealed the re-branded instrument now called CytoFLEX.

I had the chance to spend about a month with the CytoFLEX and what follows are some of my thoughts about the key features, successes and failures of this instrument.

General Technical Specs:

The CytoFLEX came to me as a 3 laser system including a 50mW 488nm laser, a 55mW 640nm
Beckman Coulter CytoFLEX Analyzer
laser, and a 93mW 405nm laser. The system also has 9 fluorescence channels in a 4-3-2 configuration, respectively. In addition, there are 3 light scatter parameters, the typical blue laser scatter yielding forward and side scatter, and an additional side scatter parameter off the 405nm laser. Pulse height and area are collected for all parameters, and a width signal can be selected for any one of the parameters. The fluidics system is controlled through peristaltic pumps for both the sheath and sample lines, and the sample volume flow rates can range from 10ul/min up to 240ul/min with 10, 30, and 60 ul/min presets (referred to as Low, Med, and Hi, respectively). A single tube holder with built-in backflush loads samples into the instrument one-at-a-time, and the hardware is controlled by the bare bones, but highly functional CytExpert acquisition software.

The system that is suppose to ship some time in October will be configurable with 3 spatially separated lasers (with a 4th coming soon?), with a variety of laser options and colors available.  The base configuration should include 3 lasers, and 13-colors in a 5-5-3 config (violet, blue, red, respectively).

Look and feel:

A look inside the CytoFLEX revealing lots of unused space.
The instrument itself is quite small, fitting roughly into a 40cm cube, but even the box itself seems to be too big for whats being housed inside.  A peak under the hood reveals a ton of unused space (multiwell autosampler, perhaps!!!).  Pretty much every component on this instrument looks like a fraction of its counterpart on more common cytometers. However, it's quite clear that every penny possible was pinched in the manufacturing of this instrument.  Everything about it screams cheap.  That's not necessarily a bad thing per se, but as soon as you start opening up lids and doors and see some of the components inside, it becomes clear how they were able to create a functional instrument at bargain prices.  Beckman Coulter has said that part of what they will do to the CytoFLEX is to add some polish to the components without adding cost.  A final product and price point has yet to be revealed, but we expect to see it in the wild this fall.

Fluidics:

The Sheath and Waste tanks sit beside the instrument and have a single output/input line, respectively. They hold about 5 liters, which should last most of a day with moderate use and reasonable amounts of backflushing. The preferred sheath for this system is some high quality H2O (Insert Waterboy reference here). Beckman Coulter will likely sell you a box of water at a premium and call it "Coulter Sheath" but you'll be just fine grabbing some DI from your MilliQ system.

Again the system moves fluid throughout using a pair of peristaltic pumps. The non-fluid movement of peristaltic pumps tend to make them not ideal for a system that requires stable fluid flow, but in testing the CytoFLEX, I saw no fluctuations in any of the channels over long runs with beads (plotting bead intensity vs. time). This type of instability due to peristaltic pump oscillation had been reported in some iterations of the Accuri C6 when it first came out. In the CytoFLEX, special attention was paid to create a pulseless peristaltic pump, which definitely holds true in my testing.

Although the sample volume flow rate has a custom setting that allows it to go up to 240ul/min, in
Close-up of the sample tube loading arm.
my tests, I saw dramatic declines in scatter profiles and less obvious, but still present, losses in resolution of fluorescence profiles beyond 100ul/min. I think the 240 setting would be great for cleaning the sample line out, or maybe forcing through a stubborn clog, but not for collecting data.  This type of flow rate is pretty much on par with other hydrodynamically focused fluidics system (unlike, for example the Attune that uses acoustic focusing and can easily go up to 1000ul/min with minimal degradation of profiles). Although the 80um wide beam spots may insulate the wide sample core stream from really poor resolution.

The sample loading stage is a bit funky at this point.  The loading stage moves in and out with the smoothness of 20 grit sandpaper sliding across berber carpeting (i.e. not smooth at all). This loading/unloading operation slows down the process just enough to be annoying, but you get use to it after a while. No plate loader (yet), No multi-tube loader (yet).

Optics: 

The optical system on the CytoFLEX is the biggest departure from any other instrument developed.  A lot of the technology is proprietary, and as much information as I was able to deduce I'll share here, but I could be flat wrong on some things, so take what I say with a grain of salt. 

Custom made Laser modules
Don't expect to see the familiar Coherent Laser Cubes on this instrument.  In fact, these lasers are custom made, in-house in the Chinese facilities (where the entire instrument is manufactured).  When you take off the laser compartment cover, you're greeted with non-descript tiny black boxes with a sticker on them telling you which laser it is.  Here is where they can save a lot of money.  Without being beholden to the Coherent behemouth, they're not locked into Coherent prices.  And, since they are making the lasers themselves, they can customize everything about them according to this specific instrument.  Worried about the quality?  I was too, until I saw the performance.  Of course, what I'm not able to test is long-term laser life on these.  The stated spec on Xitogen's web site for lifetime is 20,000 hours, but this hasn't been tested in the field, as far as I know.  The air launched beams go through the typical steering and shaping optics and terminate at the flow cell in front of one of a 7 "pinholes" on the instrument.  Beam sizes and power efflux are restricted to a 5um x 80um gaussian profile courtesy of the beam shaping optic and its large 1.3 NA, which means most of the laser power gets focused to the "pinhole" in a slit (N.B. I say "pinhole" since it's unclear if there are actual pinholes in the traditional sense or some other sort of voodoo magic).  This should allow for maximal excitation of fluors and minimal crosstalk between laser lines.
A look at the laser path with the covers off and interlock defeated
(Don't try this at home kids!)

Emitted light is collected by fiber optic bundles which carry the light to the detector blocks. The detector blocks, referred to as Fiber Array Photo Detector Modules or FAPDs is where all the innovation takes place.  The first thing you'll notice when looking inside the FAPDs is the small size of the filter sticks.  Pulling one out reveals a tiny piece of glass no more than a few millimeters square. However, the rest of the components inside the FAPD are completely foreign to someone who's looked at dichroics, bandpasses, and PMTs his whole cytometry life.  The light exiting the fiber passes through a wavelength division multiplexer, which acts like conventional dichroics to partially
Looking into the FAPD with a filter stick removed.
split the light into distinct ranges, and then the light is further refined by the bandpass filters before hitting the photodiode.  Photodiode? Don't you mean PMT?  No, you read that right, this system uses Avalanche Photodiodes (APDs).  These semiconductor detectors are well-known for their high sensitivity, and silicon based APDs have good quantum efficiency in the visible and near-IR range as well as low noise.  If they're so sensitive why haven't they been used before?  Good question, and as far as I can tell, the problem has always been the amount of voltage that needs to be applied to achieve high sensitivity and this high voltage causing breakdown of the APD.  Somehow, this has been circumvented in the CytoFLEX. The other interesting thing about the detectors is that the response of the APDs across the entire range is absolutely linear. They stand by this fact so much so that if you set up compensation on FITC vs. PE at one set of voltages, and then change the voltages, the system will automatically adjust the compensation values to take into account the new voltage settings.  This can only happen if the response is linear from end-to-end and therefore compensation is merely a mathematical equation with voltages as one of the variables.

Electronics:

The system uses 16-bit A/D converters and boasts of 7-decades of dynamic range.  Normally 16-bits doesn't get you that much range, but by oversampling the pulses at 40MHz, and adding up all the samples, a full 7-log scale can be achieved.  However, like most of these large scales, the first decade tends to exhibit poor resolution and is "hidden" by default.  So the scale goes from 10^2 up to 10^7. Qualitatively, I will say that I was able to resolve all 8-peaks of the 8-peak rainbow bead set with some room on both sides of peaks 1 and 8 - that doesn't always happen.

One of the only complaints I had about this instrument was the loss of data at moderate to high event rates. This has to be due to the pulse processing speed of the electronics system and its inability to process the signals fast enough.  It's likely influenced by sample concentration and the system's dynamic integration window - not unlike the FACSDiVa window extension setting.  If the window is reduced, % abort would likely decrease.  Also, increasing the threshold would also have the effect of better resolution between pulses and thereby decrease the abort rate. I did not explore either of these options when running and just used the default window extension and threshold. As you can see from the chart, even going at a moderate rate of 10,000 events per second yields an alarming abort rate. Going even faster results in a recovery of 50% or less. It's important to separate your ideas about % aborts on analyzers from high-speed sorters.  Cell sorters have the advantage of pushing the cells through at very high velocities resulting is narrower pulses and an easier time resolving two closely related pulses.  But, on analyzers, the cell velocity is much slower, resulting is broad pulses and more difficulty resolving closely related pulses. Therefore, the abort rates are typically going to be higher on slow flow analyzers, however we're not as aware of these abort rates on analyzers because we are
always only concerned with frequencies of populations and not absolute yield of populations (like on cell sorters).  So, 10% abort over 20,000 events per second might be reasonable, however, 10% abort rates over 10,000 eps is probably not.


For this test, I created a concentrated sample using a suspension cell line, which, at 60ul/min should yield 50,000 events per second.  I then created serial dilutions from there all the way down until an expected 2,500 events per second.  I ran each tube on the instrument and recorded the event rate displayed by the system's counters. 

Software/Interface:

If you've used CellQuest and FACSDiVa in your cytometry lifetime, you'll feel right at home here. There's not much to say about the software except that it works.  It was super easy for me to pick up.  I was shown nothing as far as how to operate the instrument, do compensation, etc... and I was able to figure it out with minimal struggle. The CytExpert software does one thing really well and that is it gives you a large, unobstructed view of your data, and just enough controls in a thin side panel to allow you to acquire data.  I'm sure there are some analysis tools built in, but I don't care, I just want to acquire data, dump it into FlowJo and worry about analysis later. It gives you the ability to do automated compensation, use biexponential display, perform gating, and show stats windows. It would be interesting to see if something like Kaluza-G would ever make it onto something like this. But then again, Beckman Coulter already has 900 acquisition softwares already, what's one more!

Performance:

Finally the good stuff. What can I say, this thing rocks. In terms of fluorescence sensitivity, it beat the pants off of anything I've ever tested full stop. I've put a range of values together for fluorescence resolution that shows the spread of instruments I've tested.  The value (called qNORM) represents the lowest number of antibodies bound that can be resolved from unstained lymphocytes.  The lower the value, the better, and as you can see, the CytoFLEX, with its APD detectors and DIY lasers easily beats the average across the board.  Of course I ran all the other common bead sets on this instrument.  Everything I threw at it, it handled with ease. 

8-Peak Resolution at low and high flow rates:

As you can see, resolving 8-peak beads is a cinch on the CytoFLEX pretty much across all channels. Even at the highest flow rate (240ul/min) the fluorescence resolution remains relatively unchanged, however the light scatter experiences some funky spread at the high flow rate.

APD Voltage Optimization:

Using a blank bead, the voltage was moved up and down the scale at appropriate intervals.  The rCV was calculated on the single bead peak in each of the fluorescence channels. Using a similar test as PMT optimization, I wanted to see if the APDs behaved in a similar way. It does appear that there is a sweet spot for APD voltages that vary across parameters.  This mimics PMT optimization profiles commonly seen before.


Linearity:

A pretty simple linearity test using PI stained CENs, and everything checks out as expected.  However, like I mentioned earlier, linearity on this instrument has a bigger role on this instrument than others.  With the CytExpert software, you can setup compensation at one set of voltages, change voltages (because it's a different cell line, or the sample is too bright, or other reason), and it will recalculate compensation based on the new voltages.  This may have been (or may still be) part of the FACSVerse software, but I've never used one of those, so I'm not sure about that.  Theoretically, then, you could create a set of comp tubes using non-tandem antibodies once, and then recall those comps each time, even if you've changed voltages. Anywho, linearity is great, it deviates from the theoretical line by less than 1% across the board.
PI Stained CENs comparing the theoretical line and the actual data

qNORM Resolution Comparison:

Without going to much into the methodology (because I've done it so many times before), what follows is a comparison of pretty much every instrument I've ever tested (grey boxes with quartile whiskers) with the CytoFLEX (blue circles) overlain.  This metric measures the number of bound antibodies that can be resolved from unstained lymphocytes. So, lower numbers equals better low-end resolution.  As you can see the CytoFLEX compares very well with all the best instruments out there. It definitely beats every instrument I own in the FITC, PE, PECy7, and APC channels.  In the PacBlue channel, it's about average.  I'm pretty sure this system can resolve pretty much any dim population you can throw at it.


Final Thoughts:

It's evident to me that the CytoFLEX would suit the needs of many demanding applications.  There's really no questioning its performance in terms of fluorescence detection. Light scatter resolution of cell populations wasn't as good as some of my better instruments. However, small particle detection, especially using 405nm side scatter is reported to give <200nm resolution.  I typically don't test small particle stuff since it's not really my thing. The fluidics seemed stable and robust for the time I had it.  I ran as many cell samples as I could to see if I could clog it up or make things look bad, and other than the high abort rates at high event rates, I saw no issues from a fluidics standpoint.  The software is fine for what it needs to do.  I'm so entrenched in doing analysis in FlowJo that I couldn't care less if there are histogram overlays or other fancy analysis-only plots in my acquisition software.  I just want it to be fast and simple to use. 

We also don't really know about the long term reliability of the hardware components. Sure everything works fine over the course of a month, but what about a couple years or more.  Will it have the staying power and uptime of a FACScan? This, I'm afraid, only time will tell.

But, I think the most important take home message here is that this instrument proves that flow cytometry hardware is absolutely a commodity in the eye of the consumer. As fancy as one wants to make hardware these days, no one is going to be impressed.  And the fact that hardware can be made cheaply reinforces this fact.  What this means is that we'll finally see a shift in focus away from over-engineered hardware to hardware that just works, but this time with a super slick user interface that people are attracted to.  The future is all about software and services, and I, for one, couldn't be happier!

Postscript: At the time of publishing, Beckman Coulter launched a new splash page with specs and a glimpse of the new exterior of the CytoFLEX.  You can reach that page here

Wednesday, January 29, 2014

21st Century Learning using an Ancient Model Applied to Flow Cytometry Training

There are so many things I'd love to learn.  I always imagined myself playing the guitar, or executing a no-hands backflip, or even writing a mobile application, but so far, I still cannot do any of those things. Of course, each of these things are certainly in the realm of possibilities for me.  I'm actually somewhat musically inclined, I can do many gymnastic-type flips, and I know a thing or two about the languages of the coding world, and yet, I can't amend my CV with any of these goals.  So, how might I go about learning these skills that envelope both knowledge of abstract concepts (like coding) and physical moves (like playing a guitar)?

I don't know about you, but whenever I'm trying to figure something out, I default to YouTube. YouTube is great for things like this, in that you can pretty much find a video demonstrating something on any topic you're interested in.  However, where things tend to fall apart is the one-way nature of YouTube.  The demonstrator is broadcasting out a message that I may stumble upon years after it was uploaded and there's not a great way for me to interact with the original creator.  Sure, I could leave a comment in the hopes that it'll be answered, but I'm just as likely to get an unhelpful snide remark.

In fact, many models of learning these days follow a similar strategy.  E-Learning is all the craze these days.  Popularized by online e-learning houses such as Kahn Academy or Coursera or even on a larger scale, institutions such as University of Phoenix, e-learning promises all the bang for little to no buck, all taking place in the confines of your oversized easy-chair. But, questions remain as to how effective these programs are. I know I've signed up for courses a few times only to stop going after the second or third lecture. In some ways the information is presented as little more than a canned powerpoint presentation with some voice-over description.  A step up from here is the possibility to interact, real-time with the presenter via chat or video conference.  Even, with the best implementation of these technologies, remote e-learning is difficult.

Let's flip this conversation completely on its head for a moment.  For millennia, the way in which people learned a trade or skill or gained any sort of knowledge was through a Master/Apprentice process.  The elders of the group who had the necessary experience and expertise took a young apprentice under his wing and taught him the way.  If it helps, I always conjure up images of Qui-Gon Jinn teaching his Padawan Obi-Wan Kenobi in the ways of the force.  You could imagine the education the apprentice received was really good, but the process was somewhat inefficient since a Master may only have a limited number of apprentices. Contrast this with e-learning and the dichotomy should be clear.  E-learning may provide a highly efficient means of disseminating information, but the actual learning may be inadequate whereas the Master/Apprentice model may provide for world-class learning but is inefficient in terms of disseminating information to a large group of eager learners.

To bring this conversation closer to home, how do we go about teaching the art of flow cytometry to the next generation of scientists?  I would say, up until this point, the passing on of flow cytometry knowledge has favored the Master/Apprentice model.  This is certainly the way I learned, and probably the way I would prefer to learn just about anything.  In recent years, however, many core facilities, companies, and professional organizations have tested out the e-learning model of teaching flow cytometry.  Like the e-learning trailblazers, these differ in quality from powerpoint slideshows to interactive, well-produced, and highly animated videos.  We've tossed around the idea of moving towards an e-learning model at UCFlow, and what's held us back (aside from the technical complexities involved in producing something worth putting your brand on) is a core belief that we can produce better cytometrists with the more intimate master/apprentice model.

The question then becomes, can we leverage modern communications technologies to make the master/apprentice model work more efficiently?  Well, of course the answer is yes, otherwise I wouldn't have bothered writing this post.  But, instead of describing a fictional method in overly verbose prose, I want to point you to what I think is the ultimate model of learning PERIOD.

It pairs the master/apprentice model with new technologies like video conferencing, chat, hangouts, google glass, wearable tech, etc...  It also gamifies the process to promote better engagement.  Imagine this scenario.  I know a little flow, but I'm faced with this new application.  I'd really like to start doing microparticle analysis.  I log into the cytometry masters portal, and search microparticles.  Up pops a list of microparticle experts with various specialities and levels.  For example Jane is a level 50 Endothelial MicroParticle Master, and can accept a new apprentice for the next month.  She prefers to communicate via Google+ Hangouts and is in the Pacific Time Zone.  I connect with Jane, learn all her tricks and tips, and then I can level-up in my knowledge of microparticle detection, bringing me to a level 10 master.

Would you like to see how this works?  Luckily, this has already been launched using a different, but I'd dare to say very similar, technology - photography.  The super awesome photographer, Trey Ratcliff (stuckincustoms.com) launched a brand new site, The Arcanum (thearcanum.com) that uses this exact model.  Master/Apprentice, Modern Communications Technologies, Gamification.  Watch the video below to see what it's all about.  What I love about this is that it's visual; you're learning directly from an expert of whom you can ask all the nuanced questions you like; it uses all the latest gadgetry; and the gamification of the process makes it way more engaging.

The next question I have is, Who wants to build the flow cytometry version of this with me????


Monday, November 18, 2013

He said, She said, PEBCAC?

In flow cytometry core facilities, scenarios such as the one that follows are commonplace.
The first thought that comes to me
after a user reports a problem.
An end-user is attempting to collect data, for some reason there's an issue, the end-user requests assistance from the core facility staff, some resolution is achieved, lather, rinse, repeat.
But, the interesting thing is the back and forth between facility personnel and the user.  Each party is trying to figure out in what way the other party messed up the experiment.  A veritable "he said/she said" ensues and eventually a resolution is achieved.  The way in which the resolution comes about can take many forms depending on the level-headedness of the parties involved. However, core facility personnel are typically about as protective of their instruments and services as a mama grizzly is towards her newborn cubs.  Similarly, a precocious grad student, who has spent umpteen hours preparing her samples, couldn't imagine a situation where she could have made an error.  To celebrate this perennial back and forth, I present to you the 10 most common phrases (5 from each side) overheard between core facility personnel and end-users during the initial throws of an experimental/instrument mishap.

5 Most common statements from core facility personnel when presented with a problem by an end-user

  1. Did you try and reboot the instrument (software)?
  2. Hmph, my QC beads look fine.
  3. Did you filter your samples before bringing them here?
  4. I don't know... everything looks pretty dead/negative to me.
  5. No one else has had any problems on here today.
5 Most common statements from an end user when they encounter a problem at the core facility

  1. Why does this thing break every time I try and use it?
  2. I had X million cells, so why did the instrument only run X/5 cells?
  3. The instrument is clogged or something.  The person before me didn't clean it well enough.
  4. Well, will the problem be fixed soon? This data is for a grant proposal due tomorrow.
  5. I hope you're not going to charge me for this.
Of course, I'm a bit biased when it comes to this scenario, so you may have your own favorite anecdotes to share.  You can do so in the comments. Flame on!




Tuesday, July 23, 2013

10 Tips for purchasing your next cytometer

So you've got some money to spend and you figure, heck, I do so much flow, maybe I'll just buy a(nother) cytometer.  Presented here are some tips to avoid buyer's remorse, see though the marketing spin, and make an educated decision on which instrument to purchase.  But, before we get into that, let me first state that I'm NOT going to make this decision for you and tell you which instrument to buy.  I am merely going to provide you with the tools to make as good a decision as you can.  In fact, these are the very same steps I go through whenever I'm in the market.  And so, I present to you, 10 tips for purchasing your next cytometer.

#1.  Define your needs.  What are the applications you will run on this system?  How many parameters (realistically) do you run on average?  How many parameters will you run in the near-future?  Are there any specialty dyes you run?  Do you prepare samples in tubes or plates?  At what event rate do you run your samples?

Example:  I have some projects in mind which require 8 - 10 fluorescence parameters.  At 3 parameters per laser, I probably need a 3-laser system minimally.  I like to stain/run my cells in a 96 well plate, so a plate sampler option is needed.  My experiments typically involve immunophenotyping rare subsets, so I collect 10^6 cells at rates of about 15,000/second.  I don't need to sort.

#2.  Query the end-users.  If others in the lab or core facility will use the instrument, ask them the same questions as in #1.

Example:  Another user in the lab does a lot of screening of her mCherry transfected cell lines.  She doesn't collect a lot of cells, but screens many samples.  She would require a yellow/green laser for excitation, and fast 96-well sampling capabilities.

#3.  Refine your needs.  Combining the information you learn from steps 1 and 2, you should be able to refine the needs for this instrument.  Annotating this list, and possibly triaging needs and wants will be very helpful at this point.  Of course you are working within a budget, so you'll certainly want to keep that in mind as you survey the market.

#4.  Survey the market.  You probably already have an idea of the "big" players in the market, but even if you didn't, simply typing the query "flow cytometer" in your browser brings up 9 different instrument manufacturers within the first two pages of a Google search.  You can follow these links, collecting information on the various instruments.  For something like this, I like to use an electronic note taking application like Evernote to keep everything together, and make notes as I go through the process.  For those web sites where information and materials are not easily accessible, sending an email to a local sales representative will get you all the marketing materials you could ever ask for.

#5.  Learn how to read marketing materials.  Speaking of marketing materials, there are a few things you should be aware of.  Beyond the very basics (lasers available, number of detectors, etc...), much of what you see in your average cytometer specification sheet is useless information.  It's basically a list of values for meaningless metrics that MUST be put into the materials to match what the competition is stating.  For example, the ever-present detection threshold of FITC and PE.  Most all spec sheets will include something like an MESF Detection Threshold of <150 for FITC and <100 for PE.  This means absolutely nothing in terms of how well the system will work for your applications let alone how other colors will fair.  You'll also see outrageous specifications for event rates, like 100,000 events per second!  Lastly, and probably my favorite, is the panel of histograms showing the resolution of multi-intensity hard-dyed beads (e.g. 8-peak Spherotech beads).  You can pretty much ignore all this information, and focus on the things that matter.  How many lasers?  How many detectors?  Can you upgrade the system in the field with more lasers/detectors? Is there a multi-well sampler? etc...

#6.  Create the matrix.  By now, you have a list of needs/wants, and you have a bunch of marketing materials.  Put it all together in tabular format.

Fictitious Instrument Comparison Chart, with the 3 contenders.

#7.  Gain hands-on experience for the top 3 contenders - make sure the OEM knows the fate of the sale hinges on the success of the hands-on demo. Run your battery of tests that matter to you, evaluating the results, as well as ease-of-use, software, UI, hardware.  Simply staining your favorite panel of antibodies and running it on the instrument will give you a TON of information as to how these cytometers stack up.  Running real samples (not just beads) is an absolute must.

#8.  Take to the social network (and take everyone's opinion with a grain of salt).  Useful things that can come back from the community include; recurring hardware/software failures, maintenance issues, service issues, responsiveness, etc...  For any negative responses that come up, make sure to bring these to the attention of the manufacturer (respecting people's confidentiality, of course) and ask for a response.  Get everything in writing.  No phone conversations!

#9.  Negotiate the purchasing terms with multiple companies.  Make sure the sales representative is aware of their competition.  Aside from asking for the best possible price, discuss other value added options.  For example, an extension of the warranty, free training slots, discounted multi-year service agreement, free shipping, free upgrades (higher powered lasers, multiwell samplers, extra emission filters, next version of software).  Get everything in writing, no phone conversations (did I mention that already)!

#10.  Take advantage of year-end discounts.  If possible, time your negotiations and purchase with the end of the company's fiscal year.  You'd be surprised what sort of deal you can get if the company is close to reaching their target for the year.

BONUS Tip:  Don't be afraid of venturing away from the "big companies."  When dealing with newer companies and newer technologies, getting cutting-edge hardware can be a double-edged sword. Although you may be able to get a deal on price, make sure there are some protections in place that allow you to get future upgrades or revisions to problematic hardware for free.  At the very least, you should be able to get a percentage of your money back if it's a total failure.  Again, get it in writing up front.

So there you have it.  I think if you keep these common sense tips in mind when purchasing your next cytometer you won't be disappointed.  Got any other tips that have helped you make the right purchasing decision?  Why not leave a comment below.






Thursday, June 6, 2013

A Cell Sorter in Every Lab. Can Core Facilities Survive?

 It happens in every industry across all times - What initially requires sophistication and expertise becomes simpler and more accessible to the masses and the former "experts" feel threatened and rail against the advancements.  For many years, Flow Cytometry core facilities cornered the market on ALL cytometry taking place at an institution.  As instruments became easier to use, some facilities allowed their users to begin operating the analyzers unassisted.  Affordable flow cytometers first came to market in the form of the Guava with some success, and then, in late 2006, Accuri exploded on the scene with it's affordable, easy-to-use C6 analyzer.  Undoubtedly, these instruments were marketed to individual investigators seemingly bypassing core facilities altogether.

The last stronghold of core facilities seemed to be cell sorting.  Alas, these instruments are sufficiently complicated as to assure even the most skittish of core facility technologist. That was, until easy-to-use sorters became more available.  The FACSAria (BD) was marketed as the first bench-top cell sorter capable of doing everything its more complicated predecessors could do.  That didn't pan out so well, much to the chagrin of BD.  However, after multiple iterations of the FACSAria, as well as other, easy-to-use cell sorters, we're on the cusp of a turning point in cell sorting, much like that day in 2006 when Accuri launched its C6.  In fact, here at the University of Chicago, we've jumped on this bandwagon with both feet, adding the BioRad S3 Cell Sorter to our group of cell sorters.  In addition to training users to operate the FACSAria's, we now can spend much less time training users to sort on the S3.  For a user who is familiar with the general operation of a flow cytometer, we can get them proficient on the S3 in less than a half hour.  Now, roughly one third of simple sort clogging up the FACSAria schedules can be done on the S3 with no increase in facility personnel and minimal increase in facility operating expenses.  Win/Win/Win!

However, some of us in the field may feel like this is yet another assault on our job security.  "If I don't control the sorting, what will I do all day."  I think this view is extremely myopic.  From an economic standpoint, it's always better to get something for nothing than to have to put real resources into doing it.  When you think about it more closely, if we don't have to expend resources into operating cell sorters, we can focus those resources on other important things.  I mean, it's sort of silly to have a trained professional sitting in front of a cell sorter drawing regions around GFP+ cells.  These types of sorts can easily be transferred back to the user once she is trained to operate a basic cell sorter.  This frees up time for the experts to focus on the cutting-edge stuff or development work.

This whole discussion then turns to a more philosophical discourse on what the role of resource core facilities will be in the future.  It's time to pivot, folks!  Gone are the days of a facility with 1.25 FTE's per cell sorter, staring at dots popping up on the screen all afternoon.  The business side of me wants to focus less on hardware recharge and more on services.  Why be a warehouse of hardware, when you can be the (more lucrative) service center.  Let's assume, for a moment, that cell sorters like the S3 or Sony's SH800 become so solid and affordable that many labs decide to buy their own.  Facilities focused solely on hardware recharge revenue will quickly spiral towards obsolescence. Facilities focused on services will already have other revenue streams to compensate for the lost hardware recharge.  Allow me to illustrate with a few examples.

Sample processing as a service (SPaaS):  An investigator has an idea for an experiment on a cohort of patients seen in the clinic.  We, as a core facility can coordinate pick-up of blood tubes from the phlebotomist, process the blood, bank the plasma and cells, and then stain the cells using standard panels and perform cytokine bead assays, microparticle analysis, or other assay on the plasma.  Translational applications like this are not only becoming more and more common, but many times those investigators in the best position to do these studies do not have large research labs set up to do this themselves.  Since we're not sitting in front of the FACSAria 8 hours a day, we now have time to develop these panels, and market this service.

Internal Instrument Service and Maintenance (IISM):  Investigators start purchasing their own analyzers/sorters, however, they may not have the skill, time, or resources to perform routine service and maintenance.  We, in the core facility already have the know-how to fix many of the issues on these instruments, and we have the SOPs in place to perform the necessary maintenance and quality assurance.  Instead of paying a service contract fee to BD AND having to do all the routine maintenance themselves, the investigators could contract the flow core facility to perform maintenance and service.  The core facility can, in turn, take out a self-insurance policy to maintain the instrument and recover actual costs.  The more instruments the facility can perform this service on, the better insulated you can be against catastrophic incidents.  This is essentially the model I've been using in my own lab; Budget 50% of the cost of a service contract, and pay for service calls as needed.  In the 15+ years I've been around, we've never lost money.  This works only because the number of instruments is sufficiently large.  The use, operation, and initial investment of the hardware took place completely outside the core facility.  The investigators' staff and students need training?  We can do that!  They want to assure the instrument is performing optimally?  We can do that!  They need someone to come fix the instrument because it's not working?  We can do that!  Or, if we can't, we call the OEM and pay for a service call.

Of course, there will still be other technologies associated with a traditional core facility that will still follow the normal hardware use recharge model.  High-end cytometers/sorters, imaging cytometers (a la the ImageStream), mass cytometry (CyTOF), etc...  However, we can anticipate these technologies following suit.  Soon, the CyTOF will be super easy to run, and imaging technologies will be simpler and simpler (e.g. the FlowSight).  As these technologies become commoditized like analyzers and now cell sorters, it's going to be service-focused facilities that will stand the test of time.




Monday, March 11, 2013

ABRF 2013 Recap - A flow cytometrist's take.

On the ABRF about page, it states, "The Association of Biomolecular Resource Facilities is an international society dedicated to advancing core and research biotechnology laboratories through research, communication, and education."  But, if you know anything about ABRF, you're likely involved in a molecular biology-based core facility, namely a genomics, proteomics, or related facility.  I guess I always knew about ABRF, their meetings, and what they were about, but somehow it never really interested me much.  I mean, anything with the word "biomolecular" in it gives me not-so-pleasant flashbacks to biochemistry classes.  So you can see why a guy who spends all his time working with whole cells might not take a second look at marketing materials from ABRF.  However, it seems as if the tide has shifted.

What piqued my interest this year was some interesting movement in two of the ABRF research groups that had formed in recent years.  You see, unlike other societies which may only focus on annual meetings, ABRF has interest groups that form with the intent of doing research projects.  A core group of ABRF members with common interests (e.g. flow cytometry) may come together and propose research projects to work on.  ABRF supports these efforts by providing the necessary sponsorship. A newly formed Flow Cytometry Research Group (FCRG), and a recently revived Antibody Technology Research Group (ARG) were working on some projects that seemed really interesting and very pertinent to what I do.  Seeing as I now share my time between our antibody production and flow cytometry cores you can probably guess why I'm excited by these two research groups.  Combining this with the general core facility management stuff that's always happened at ABRF pretty much made up my mind about attending this year...and I'm glad I did!

Sure there were some interesting talks about exome sequencing and insanely parallel westerns, and even the need to foster convergent technologies in order to make inroads into cancer research, but the real highlights came at the FCRG and ARG meetings.  The ARG group had been working on a modified immunization strategy to both increase the initial immune response as well as prolong that response in order to trick the immune system into making antigen specific, antibody secreting B cells.  One might not think of cutting edge technology when talking about novel monoclonal antibody production, but they had some interesting ideas.  For example, using CpG's in combination with a standard adjuvant (e.g. Freunds) when an antigen doesn't seem to be eliciting a good response.  This would be hugely important information for our Antibody facility.  The FCRG also had some interesting data surrounding the ill effects on cell function after cell sorting.  Everyone has their anecdotes about sorting at high pressure vs. low pressure, or on a jet-in-air sorter vs. cuvette sorter, but there's not much data out there in a well-controlled experiment.  Again, hugely important information.  
2013 ABRF President, David Friedman presenting Lee & Len Herzenberg with the ABRF Award

Another interesting draw for me was the fact that Drs. Lee and Len Herzenberg were being honored with the ABRF Award, and anyone hanging around with any involvement with flow cytometry got to get in a group picture with the Herzenbergs.  I have to admit being a little star-struck around them.  I really wanted to ask them for an autograph, but I didn't.  We were treated to some behind-the-scenes photos of the early days of flow cytometry.  And, it was also interesting to note how this development coincided with the development of the first personal computers.  In fact, Lee spoke about some of the first computer programs built by Wayne Moore and Dave Parks.  Probably the best part were the pictures of the 1970s versions of Wayne Moore and Dave Parks... Yeah, they look pretty much the same.
Member of the Flow Cytometry Research Group posing with the Herzenberg's following their tandem ABRF lecture

I have to say, I was quite impressed with the meeting in general.  The company that assists ABRF in putting on this show does a stellar job, and since I'm sort of involved in putting on shows on a much smaller scale, I definitely pick up on those things.  It definitely had the grandeur of a CYTO meeting, but you didn't feel completely lost in the crowd.  The biggest let down for me was the exhibitor area.  Not that it was poorly set up or anything like that, it's just none of my people were there.  One measly flow cytometry exhibitor was there, and only because they're brand new in flow cytometry and also do some stuff in the molecular biology area.  Thanks Bio-Rad. Other than that very minor demerit, it was a great conference, even for a flow guy.  I'll predict that the next time CYTO is in Europe, the US cytometry contingent will flock to the ABRF meeting.  You heard it here first, folks!  Actually, I heard it first from someone else at the meeting and am just shamelessly taking credit for the idea.

Thursday, December 13, 2012

Long live the Listserv; Death to the Listserv.

Good ole listservs (or listserves or list serves, if you will).  A staple in the academic's toolbox of communication technologies.  But the question is, how useful are they in today's hyper-connected, socially-networked, always-on environment?  Here we'll touch on a few pros/cons as well as get you plugged in to cytometry networking hubs online.  

Why I love Listservs:

  • Let's face it, we all live in our inboxes, right?  So, what better place to funnel all your networking than via e-mail?  It's fast, easy-to-use, and is easily accessible, especially with today's smart phone install base.  
  • It's a pretty good networking tool.  Regarding the listserv's involvement in Cytometry, there has been no better platform to allow end-users to interact with field experts.  Of course, we cannot mention the words 'cytometry' and 'listserv' in the same breath without pointing you to the preeminent spot for networking, the Purdue Cytometry List. For years (over 20, now) the Purdue List (as it's commonly referred to) has allowed cytometry professionals to interact and network.
  • Being an active Listserv participant also gains you exposure, which can lead to new opportunities.  


Why I wish Listservs would crawl up into a little ball and be subjected to a slow painful demise:

  • Poor search leads to lazy researchers.  The fact that it can be difficult to search listservs inevitably results in people asking the same questions that were answered already on the list.  Long-time list participants may respond with not-so-pleasant remarks to such a query.  This sometimes even leads to a discussion on the merits of re-visiting previously answered questions, when we should be discussing the original question itself.  
  • "Out-of-office" replies...need I say more?
  • The "Good Samaritan Effect."  People who may know the answer to a posted question may pass by without helping because they assume someone else will help them out.   
  • Too broad or too diverse.  Listservs typically cannot be broken down into categories, allowing people to focus in on a specific area of the listserv's main topic.
  • No inline rich media.  Depending on the listserv, you may not be able to attach documents, pictures, movies, etc...  Try and explain your gating strategy only using words...It's not fun.
  • Wikipedia tells me that LISTSERV was developed in 1986.  Nineteen Hundred and Eight Six!!!!!  Do you know how long ago that was?  It was the last time the Bears won the Superbowl (OUCH!).
  • Do we really need to be shuffling around emails to 4000 people on a listserv?  Answer: No.
21st century tools for a 21st century technology

Obviously, I wouldn't bring you here unless I had some alternatives to offer.  And, you can probably already guess a few of the punchlines, but allow me to state the obvious.  SOCIAL NETWORKING.  LinkedIn, Facebook, Google+, and Twitter have demonstrated the power of social networking platforms.  Already, there is a substantive presence of cytometry on each of these platforms; some of them are actually quite fruitful.  These tools allow for:
  • Sharing rich media inline with text to create better communication of ideas.
  • A better sense of interactivity of the group instead of a one-to-one interaction.
  • Fantastically good search tools for finding exactly the information you need
  • Using email notification settings, you have the ability to interact as much or as little as you'd like.
  • Speaking of email. Many of these services allow you to fully interact with the group using the email interface, if that's more your style.
  • Strong sense of community - Via avatars and in-depth profiles, you're able to build better relationships with colleagues.
  • Expand your interaction with people on the fringe since they're already using these networks for other (personal and professional) purposes.
So, what's out there?  

Well, if you just do some searches, you're bound to find groups online.  To point out one near and dear to me, I'll plug the fledgling Google+ Cytometry Community (for which I'm one of the moderators).  Google+ as a platform, is becoming quite full-featured in this regard, and the potential to have a very interactive online community is strong.  Of course, the fact that it's backed by powerful search and a host of integrated tools (Drive, Gmail, Picasa, etc...) makes it, de facto, a force to be reckoned with.  It is becoming more and more clear that tools like Google+ are most definitely the future, and antiquated platforms like listservs are (slowly) in decline.  My advice to you?  Get out there and start interacting.  Why not hop over to Google+ and grab yourself an account (if you don't have one yet) and then stop by the Cytometry Community to say hi!