Tuesday, January 31, 2012

Introducing the Cytometry and Antibody Technology Facility (CAT)

I can never tell if and when things like this become "official."  It's not like administrators are going to put out a letter to the University announcing such things, so I thought I'd mark the occasion with a post about it.  What is "it" you say?  For years the Flow Cytometry Facility and Fitch Monoclonal Antibody Facility have been working together in a cooperative spirit.  These efforts have mostly surrounded the creation of custom conjugated antibodies for use on our systems.  They will now be fostered more deliberately in the formation of a new facility.  And so it is with great pleasure that I announce that these two titans of technology shall become one - introducing the Cytometry and Antibody Technology Facility, CAT for short.  Now, I must admit, I'm not crazy about the name either, but give it a chance, it'll grow on you.  Just keep thinking to yourself, co-STAN-za!  Don't worry, UCFlow is not going away.  The two subcores will retain their identities and remain pretty much intact, however we'll be able to leverage each other's resources to create some interesting new services for you.

The birth of a mAb:  Two cell divisions shortly after a subclone
If you've read entries here before, then you're probably familiar with UCFlow, so let me tell you a bit about our new partners, the Fitch Monoclonal Antibody Facility.  For starters, the 'Fitch' part of the name is referring to cellular immunologist Frank W. Fitch, MD’53, SM’57, PhD’60 (University of Chicago), the Albert D. Lasker professor emeritus of pathology and the Ben May Institute, who joined Chicago’s faculty in 1957. As the institute’s third director, Fitch oversaw its growth into a collection of laboratories working in multiple areas of cancer research. A teacher and a mentor who encouraged students to think creatively while still abiding by scientific rigor, Fitch has also served as the editor in chief of the Journal of Immunology and as president of the American Association of Immunologists.  He was a pioneer of antibody technology and was the founder of the Monoclonal Antibody Facility.  Today, the facility's technical director is Ms. Carol McShan who has over 30 years of experience with tissue culture techniques, and novel monoclonal production.  A major thrust of the facility over the years has been the production of novel monoclonal antibodies.  This involves immunizing mice (or rats) with the specific antigen, screening the mice for a response, fusing the mouse's splenocytes with an immortalized fusion partner to create hybridomas, and finally subcloning and screening the hybridomas according to the end use of the antibody (flow cytometry, immunofluorescence, western blotting, etc...).  In addition to novel monoclonal production, the facility also produces high-titer antibody supernatant, purified antibodies, and fluorescently coupled antibodies.  A complete list of services can be found on their web site:  fitchantibodies.uchicago.edu.

Hollow Fiber Bioreactors, utilized to produce high titer mAb.
Before we talk about what's in store for our joined future, let's discuss why I think this makes sense.  I'm going to throw out a couple of economics terms to try and make a correlation to how things are done in the for-profit business world and then tie them back to what we're doing here.  What we're really talking about here is a transition from lateral expansion to vertical expansion (or integration).  My buddy wikipedia tells me that lateral expansion is the growth of a business enterprise through the acquisition of similar companies, in the hope of achieving economies of scale.  Think of BD purchasing Cytopeia or Accuri.  They gain scale due to the significant install base of these instruments.  In a lot of ways this is pretty typical of successful companies.  It can be a quick way to really expand your business and can lead to even greater growth.  This lateral expansion sums up the past few years of the flow cytometry facility.  We've added lots of pieces of equipment, grew our business, and expanded considerably.  All of this expansion was pretty much of the same stuff - that is, cytometers.  No, we weren't out 'buying up' other core facilities or things like that, but in our own way, we experienced a sort of lateral expansion.  But there comes a point when you've pretty much maxed out this horizontally-directed growth and you need to pivot.  So, what's the next logical step?  You guessed it; vertical integration.

Vertical expansion therefore, is the growth of a business enterprise through the acquisition of companies that produce the intermediate goods needed by the business or help market and distribute its product.  This type of vertical pipeline integration is another very common strategy employed by companies.  When BD purchased Pharmingen, that's exactly what was going on.  If you are making the reagents that will be used on your instruments, you can control the entire package and make sure everything fits together nicely.  This also works in reverse too.  I'll remind you of the acquisition of Guava (and later, Amnis) by Millipore, or Invitrogen purchasing Applied Biosystems (becoming Life Technologies) and getting the supply of hardware to complement their reagents.  This is sort of how I see the "merger" of UCFlow and the Fitch Monoclonal Facility.  We can leverage the expertise in reagent development and production from the Monoclonal Facility so that our users have affordable and efficient access to commonly used reagents and will therefore be able to do more experiments with their limited funding (cf. more recharge revenue).  Hopefully the synergy of expert reagent production and state-of-the-art technology will create a positive feedback loop for both sub-cores.

So what can you expect in the future?  Our main focus at the start will be an expansion of the current Hybridoma Bank.  Right now, the facility can produce purified antibody from 21 hybridomas including such favorites as anti-CD4 (GK1.5), anti-CD8 (2.43), the FC blocking antibody 2.4G2, anti-CD3 (2C11),  NK1.1, CD19, anti-GFP, and many more.  If we can get the hybridomas for an antibody, we can add it to our list.  The next step to this is creating a plan to quickly couple these antibodies to a wide range of fluorochromes on-demand.  We've spent some time with the Lightning Link technology from Innova Biosciences and it looks promising.  This system allows for coupling to a good range of colors in as little as 20 minutes.  We're still exploring many options, so if you have a favorite setup, let us know.  We're excited about the prospects for this new phase of growth in the facility and will keep you posted about new developments as they happen.

Tuesday, January 10, 2012

What to do with aging equipment: Upgrade or Replace? And a Mini MoFlo XDP Review.

I started writing this post specifically to follow through on a comment I made when talking about our upgrade of our aging MoFlo to the XDP platform (thanks for the reminder Carol).  But then, I started thinking about all the equipment we've held onto and decided to upgrade and asked myself, was it really worth it?  Before I answer that, let's lay out a bit of discussion on the matter, and then I'll finish up with my thoughts on our upgrade to the XDP.

When I think about equipment, I like to put things into 3 categories, namely Cutting Edge, Mid-Cycle, and End-of-life (EOL).  I take all my equipment and shuffle them into these categories and move them around every so often as needed.  This way, I can put things like service contracts, maintenance budgets, capital investment, and upgrades into perspective according to pre-determined criteria.  For example, I'll stick instruments like our 4-laser Fortessa, or ImageStreamX into our Cutting Edge category.  This means they probably won't require a huge maintenance budget since things aren't likely to break yet.  However, they may need more personnel time because the applications performed on them are likely to be complex.  I'll shuffle staff and training resources to those instruments.  Mid-Cycle equipment are things like our 4 year old LSRIIs.  Things are likely to start breaking and so they may eat up some service budget, but they are the workhorses and need to be running full-time.  The applications are probably fairly routine, so they may not require as much custom tech time.  Lastly, our EOL instruments are things like our ancient FACScan and FACSCantos, and it's these instruments on which we need to make decisions.  Depending on the maintenance of these EOL'ed instruments, they may require varying amounts of service and since they may not be as desirable to use as the cutting edge cytometers, you'll need to determine how much money you're willing to invest to keep them limping along.

These EOL instruments can be a pretty decent consumer of budget and may or may not return all of their costs from recharge.  It is with these instruments that we must decide; replace or upgrade (or I guess you could just let them die a slow death).  You'll need to first determine if there is an upgrade path for your instrument.  In the case of the MoFlo, this was a whole-hearted YES, thanks to the good folks at Propel Labs.  Other instruments where this may be a possibility include FACScans and FACSCaliburs, which can be transformed into completely new instruments courtesy of Cytek Development.  If there is not a path to upgrade, then the decision is an easy one.  However, if you're looking into an upgrade, you'll need to weigh the costs against the benefits and definitely compare it to simply purchasing a brand new instrument.  If you're going to shell out a bunch of money on an upgrade, it may make more sense to look into getting a new cytometer.  Sure, you may have to settle with something a bit less powerful, but it'll be nice and shiny and (hopefully) problem-free for a few years.

So, let's put this all into practice with a retrospective look at our decision to upgrade our MoFlo.  We had our MoFlo originally installed in 2000, and approaching 2008, it was definitely showing its age.  Many of the buttons on the "rack" had fallen off, and it seemed like a waste of money to replace entire electronic bays on a rack to simply fix a button.  In addition, parts to fix the MoFlo were somewhat scarce, and it looked like many of the components were approaching their demise.  At the time, options for a new sorter were limited to the FACSAria, the inFlux (both from BD), the Reflection (from iCyt), and the MoFlo XDP (from Beckman Coulter).  All of these instruments easily approached the $0.5Million mark, so buying a brand new sorter without an SIG or a generous donor was pretty much a long shot.  Seeing as our MoFlo was still humming along just fine we decided to look into an upgrade.  The goal going into this thought process was to have a sorter that would handle a lot of the cell line type sorts using GFP or other RFPs and perhaps a few phenotyping experiments.  We did not have the expectation that it would rival our FACSAria and start performing multicolor phenotyping sorts as well as the Aria does.  We also noted that our "GFP" sorts accounted for about 30% of all our sorts and guess what?  We had 3 sorters; a perfect match.  We were able to upgrade our MoFlo for about 1/3rd the cost of a new sorter and get a few more years of life out of it while we waited for the next big thing!

MoFlo-XDP Mini Review:

I can say that the XDP upgrade pretty much met our expectations.  It handles most of our GFP/RFP sorts just fine, and is able to do a few more sorts on markers that are relatively bright.  It by no means can resolve populations as cleanly as our Arias, but it does well enough for many things.  The single best feature of the XDP is zero coincidence aborts.  You may be thinking to yourself, well isn't the Aria marketed as having very low abort rates as well?  It is, but when i say zero aborts, I really mean zero aborts, even when you have 30,000 - 40,000 events going through per second.  The place this comes in handy are rare event sorts at high throughput rate.  We can sort very rare populations and have a really good yield when compared to our Arias. What this really means, however, is that you have absolute control over your yield.  If you need every single cell possible, you can run fast, have confidence that you'll be able to make a sort decision on every single cell, and using a yield sort mode, sort out every single cell.  Sure, it won't be very pure, but at least you have them all and can decide to resort again if you need purity.  Our specification for sorting yield is a 1% population with 70% yield using the purify sort mode (to achieve 98% purity or better).  The max event rate able achieve this on the XDP is about 30,000 eps.  The max rate able to achieve this on our Arias is slightly less (~22,000 eps).  The touch screen is a bit annoying at first, but I've gotten use to it.  The biggest problem with it is the implementation of the slider and up and down arrows.  The slide is way too sensitive, and the up and down arrows are way too slow.   This interface is used for adjusting things like frequency and amplitude and plate voltages.  The new and "improved" Sort Master, dubbed Intellisort, works intermittently for us.  It took a lot of playing around, but we can get it to hold onto a node pretty well these days, but for a while we completely ignored it.  I still think this can be done way better, and apparently Intellisort II delivers, but I'm not going to hold my breath for that one.

So, am I happy with my decision?  Absolutely!  Would I do it again today?  Not too sure. What it boils down to is, I spent a good chunk of change for a sorter that has 4 lasers and about 4 usable detectors at any given time.  The need for the 4 lasers is pretty low, so I could get by with a 2-laser 4-color sorter and be able to do everything I'm currently doing on my 4-laser 10-color MoFlo-XDP.  If I were given the option today, knowing what I planned to use the sorter for, I might check out the possibility of getting a brand new sorter that was stripped down to the basics for cell line transfection sorting.  I'm thinking something like this perhaps might do the trick.