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.
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).
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.
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.