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!



Wednesday, October 17, 2012

Life Technologies Attune Cytometer... Deja Vu

It wasn't so long ago that I blogged my impressions of the newly released Attune Cytometer from Life Technologies.  Although overall, the system seemed pretty good, I did find some disheartening issues with both the hardware and software.  There's no reason why this instrument shouldn't be one of the best pieces of hardware on the market, regardless of price-point.  The entire premise of acoustic focusing should lead to excellent fluorescence resolution.  So, when Life Technologies approached me again to give the Attune another look, I obliged.  The reason for the poor performance the first go around?  Poor alignment and a very generous install specification.  With some of those issues taken care of, I was able to spend a few months with both the Blue/Red laser configured system and the Blue/Violet system.  I'll reference back to the original article in various places, and highlight some of the improvements I've seen with the systems.

Fluidics:

Much of the fluidics system has remained unchanged for the most part.  A few of the biggest issues from the prior testing have been addressed specifically point #8 where there seemed to be some fluctuation of the fluorescence signal after a re-draw of the sample syringe.  That no longer happens and is not an issue.  The other issue is the return of unused sampled once you've acquired enough events.  Now, at the end of a run, you can enable a script to return the unused portion of the sample to your tube.  I'll also mention the startup and shutdown procedures here as well.  They've really done it right on this system.  You can start and shutdown the fluidics without having to log in (useful for usage tracking in a core facility).  Also, the system turns itself off after the shutdown is complete, which is nice at the end of the day.  Lastly, their performance tracking QC module works well and is very informative, yielding Levy-Jennings-type plots for all your parameters as well as giving you an overall pass/fail (similar to CS&T in DiVa).

Software:

If you've used DiVa, you've used the Attune software.  The software was pretty snappy overall, and handled large data files (>100,000 events) without hesitation.  There's still a lag switching between tubes while you're waiting for the fluidics to finish its process.  It's long enough for you to notice it, but not so long that it becomes annoying.  It's times like this however, that I wish I would have had the 96 well loader on the system.  Also, the plot scale needs to be changed.  Right now, they're displaying a 7-log scale of which the first 2 logs are pretty much useless.  In setting up an unstained control, you end up putting your cells between the 2nd and 3rd decade anyway, so there's no reason to have empty white space at the bottom of your plots.  This only adds to a user's confusion on how to optimally set up their voltages.

There is no noticeable differences in Optics or Electronic other than the introduction of a Blue/Red laser configured instrument, which is built as a 4-2 configuration.

Data:

As far as testing goes, I focused on resolution tests I had run previously on the system, which utilizes stained capture beads.  I was working on a further development of this technique which has resulted in a quantitative metric called qNORM.  The qNORM can be defined as the minimum number of antibodies bound to a cell that can be resolved from unstained lymphocytes.  For example a qNORM value of 1000 would mean that this channel on this instrument could resolve a cell type that was stained with as few as 1000 antibodies from the unstained cells.  In testing the Attune systems, I used a few other instruments in and around the flow facility for comparison's sake. The first table below summarizes the instruments used (including the laser powers and the vintage/health of the specific instrument tested).  The second bar graph shows the qNORM values for the different instruments for 5 fluorochromes.  Please note:  lower qNORM values = better resolution.

Instruments at UCFlow tested for the qNORM characterization

qNORM values for 5 channels on the respective instruments.  Error bars are calculated from 4 separate experiments.
What we can take home as far as the Attune's performance on these tests is that it falls in line with its cohort on some of the channels (similar in FITC to a FACSCanto and MACSQuant), but does surprisingly well in other channels (as good as the Gallios or Fortessa in the PECy7 and APC Channels).  You'll notice an anomaly in the PECy7 performance of the BV Attune compared with the BR Attune.  This is due to a suboptimal filter on the BV for PECy7 (a 650 LP instead of a specific PECy7 filter such as a 780/60 BP) and a non-red sensitive PMT in that channel.  Both of these deficits can be easily fixed by swapping in a appropriate filter when using that color or using a more red-sensitive PMT.

Final Thoughts:  The units I evaluated this time around performed much better on all the tests run.  The most remarkable feature of the Attune is its ability to perform on par with the more expensive cytometers in some of the channels AND retain that level of resolution at much higher flow rates.  I said it before, and I'll say it again, "There's no reason why this instrument shouldn't be one of the best pieces of hardware on the market regardless of price-point", and if they continue to develop the platform, specifically working on the software issues highlighted above, I think it could easily take that crown.