Cytometry and Antibody Technology

Propel Labs Avalon: A first look.

by | Aug 28, 2012 | Archives, Instrument Demo | 0 comments

This archived post was originally written by Ryan Duggan when he was the Technical Director. Ryan has since moved to a position outside of the university. 

N.B.  The Avalon cell sorter is now the S3 cell sorter distributed by Bio-Rad.  It’s the same hardware/software, with a new color scheme to match the Bio-Rad colors.  You can find the product’s new page here:

I recently had the opportunity to check out the highly anticipated Avalon cell sorter from little-known Propel Labs (Ft. Collins, CO).  Little known, that is, until this year’s CYTO conference where they unveiled the new 2-laser, 4-color bench-top cell sorter.  This made such a huge splash at CYTO even competitor vendors couldn’t help but check it out.  After the dust settled from the whirlwind tour, would the Avalon prove to be a true, easy-to-use, bench-top cell sorter?  Read on for the answer.

Do you remember when the original FACSAria was announced?  It was billed as so easy to use, you could train all your users to run the sorter themselves.  BD was forced to quickly retract those marketing talking points as things like “Nozzle-gate” came to light.  Since then, there have been a few attempts at “user-friendly” sorters, but all them required too much hardware manipulation by the user.  So, you could imagine I was pretty skeptical when the new Avalon was touted to be easy-to-use.  As such, my evaluation of the Avalon focused primarily around this notion that I could train any of my users, who possessed a certain level of flow cytometry know-how, to sort their cells on this instrument with minimal instruction.  I was NOT disappointed.  Secondarily, I wanted to test the basics, like sorting speed, yield, purity, and to a lesser degree fluorescence resolution.  Let’s begin.

The Propel Labs Avalon Cell Sorter

First, I will say the instrument is pretty nice looking.  At 70 cm wide x 65 cm tall x 65 cm deep, it’s bigger than a breadbox, but not much bigger.  And the best part is, what you see here in the image to the right is the ENTIRE instrument, no wet cart, no external power supplies, no nothing.  Everything is contained within that footprint.  Behind the larger grey door on the front houses the sheath (more on that in a sec), waste tank, and DI water tank (used for shutdowns).  And here’s the kicker;  You can load a tank of 8X sheath fluid, and it dilutes your concentrate to 1X on-board using the DI water.  Now, there’s even less of a chance of introducing contamination when making up your 1X PBS for your cell sorters.

To test the system’s sorting ability, I ran a sample using my normal cell sorting specification.  The spec we use at UCFlow is:  Event Rate = Frequency/4, and sorting a 5% population, the efficiency should be >80% and the purity should be >98%.  This spec is mostly governed by physics and poisson statistics, so if an instrument cannot pass this spec, then there is something else going on (too high of a hard abort rate, too slow of an electronics processing system, etc…).  Below are the results (analyzed in FlowJo, of course).  As you can see, the Avalon easily handled this test.  N.B. The sample was fixed/no-wash peripheral blood stained with CD3, CD4, and CD56, which is why the background is pretty high and the separation is not spectacular.  This is also why the post-sort populations actually look better; we’ve essentially ‘washed’ the sample by diluting it in the collection media.  You’ll also note here the somewhat standard droplet formation values:  30 PSI, 100um tip, 38KHz.  There are 3 modes for sorting, Purify, Enrich, and Single Cell, and in Purify or Enrich, the sort envelope is 1/2 droplets.

Sorting test using 3-color staining of fixed/nowash Peripheral Blood.

The 100mW 488nm and 561nm lasers are plenty powerful to resolve most populations.  This, combined with a careful selection of Semrock filters, and an in-house developed collection lens makes the Avalon no slouch when it comes to fluorescence resolution.  The requisite 8-peak bead data is presented below.  Use this information however you’d like, but use it with caution.

8-peak data collected by UCFlow during Demo
So, we know it can sort, and we know it can resolve fluorescence adequately, but what about user-friendliness.  Well, you’ve reached the best part!  There’s a single power button on the system; everything else is controlled through the software.  There’s a one-click startup sequence that primes the fluidics, and turns the stream on.  There’s a one-click, one bead alignement QC AND Drop-Delay assessment, and voila, you’re ready-to-sort.  There are a few details that occur behind the scenes here that are worth mentioning, but for the average user, they won’t need to know any of this.  It just works! So, what happens during the start up is a coarse alignment of the stream to the light collection pinhole.  This is completely automated, and happens via pico-motor controllers on the x/y stage of the nozzle.  The laser lines are fixed in place.  Also, at this point, the system scans through the droplet frequency and amplitude, picks the optimal node, and optimizes the drop charge phase.  After the coarse alignment, the QC/DropDelay beads are put on and the alignment is peaked.  Lastly, it does a drop-delay test by sorting these same beads and counting the beads left in the waste trough at each adjustment in delay.  In the waste trough is a small laser which excites the beads, and a fiber cable that carries any fluorescence to one of the systems PMTs.  When the PMT fluorescence drops down to zero, the drop-delay is set.  Again, all of this happens without any user intervention.  Once set, the delay is monitored and adjusted as needed, and sorts are terminated in the case of a clog.  The software, although a bit buggy yet, had a nice familiar feel taking a page from the MS Office suite and utilizing the Ribbon feature.
Look and feel of Avalon Sorter Software
The sample tube holder and collection receptacles are temperature controlled from 4C – 37C, and completely contained for excellent aerosol management.  The sample tube holder has this really cool loading action.  There’s a front and rear position; the front position is where your sample is loaded, and the rear position is where the sample line is backflushed and rinsed, inside and out.  You can see the loading and unloading action in the video below.  Here, the Avalon covers are off for easy viewing.


Sort collection is equally unique.  The system sorts 2-way, with a 5 postion tube holder (each side).  So, you could load 10 collection tubes on the system, and once they fill the tubes in position 1, it’ll automagically slide the next (empty) tubes into place.  No more having to stop the sort, pull out full collection tubes, and put in new empty ones.  The only complaint here is the lack of 4-way sorting and true, 96 well plate sorting.  I say ‘true’ because there is actually an attachment that allows you to place two 8-well strips on the collection receptacle allowing you to sort 16 wells at a time.  It certainly slows down your high-throughput sorting, but it works.
Looking into the sort chamber on the Avalon.
5-position collection receptacle in foreground

Final thoughts.  The Avalon seems to capitalize on all the little things that so many companies got wrong in the past.  Simplifying the design, minimizing the steps required to go from ‘power-up’ to ‘ready-to-sort’, and making a seemingly solid instrument, all add up to a very attractive package.  To top things off, it comes at a bargain price, starting at just 99,950.00 US$ for a 1-laser, 2-color system.  Sure, it won’t replace the need for a 5-laser FACSAria III, but it can surely supplement your sorting needs by offloading those simple 2-4 color sorts clogging up your schedule.  The only question then is, How soon can you scrounge up the funds?


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