Throw away your 8-peak beads...now.

Why is it that each booth with a new piece of hardware I walked up to at CYTO had the same set of plots 'demonstrating' how 'sensitive' their instrument is?  I can't tell you how disheartening it is after years of clamoring for a re-definition of instrument 'sensitivity' to see marketing materials littered with histograms proudly displaying 8 peaks.  What does that actually mean?  and Why do instrument manufacturers design instruments around this 'standard'?
Now, I can't totally claim innocence here.  As you can see here, and here, I do use 8-peak beads as part of my panel of instrumentation tests.  However, it's not the only test I run, and I use it more so to dismiss potential problems than single out an instrument that is performing particularly well.  One of the best things 8-peaks can tell you about an instrument is the presence of background due to laser light bleed-through, or possibly a bad filter.  8-peaks are also pretty useful as an all-around alignment bead.  Beyond that, there's not much you can infer from the resolution of 8-peak beads as to the ability of said instrument to resolve dimly stained cells labeled with a particular fluorochrome from unstained cells.  For example, the ability to resolve 8-peaks in the FITC channel doesn't mean a whole lot as to its ability to resolve dim GFP signal from negative cells.  Likewise, the inability to resolve 8-peaks doesn't necessarily mean that channel will perform poorly for a dim fluorescence signal.  Let's look at an example.
I recently had the pleasure of evaluating the 8HT from EMD-Millipore (check out the full review here).  For comparison's sake, I ran the same set of tubes on our Beckman Coulter Gallios.  After collecting the data and doing the requisite comparisons, I noticed how closely the 8-peak data matched between the two, especially in the far red channel where we'd usually detect PECy7.  The first slide below shows the 8-peak data for the PECy7 channel.  The last 3 or 4 peaks are pretty much overlapping with one another.  If we were using the 8-peaks as a metric of 'sensitivity' we'd probably conclude that these two instruments are pretty similar in their ability to resolve dimly fluorescent PECy7 stained cells from unstained cells, right?...right?

Not so fast.  If we stain capture beads with a PECy7 antibody and look at the ability to resolve the 4 peaks that represent different levels of antigen density from each other as well as a blank bead, we can better assess (emphasis on the second syllable, please) the true 'sensitivity' of the two instruments.  Looking below at this figure we can easily see that the Gallios is able to resolve PECy7 much better than the 8HT.  This conclusion matches perfectly with real-world staining examples run on these instruments.  It's obvious that if PECy7 was a pretty darn important conjugate for my panels, you know which instrument I'd be buying (if I could afford it, that is).

So, what's the take-home message here?  Well, it's simple, 8-peak data cannot be used as a surrogate for how well an instrument will detect your panel of fluorochromes.  What should you do?  Again, simple:  make sure YOU run YOUR favorite flavors of fluors on the instrument you're evaluating to give you an idea of how well it's set up for YOUR experiments.  You can certainly do this by staining your cell samples of choice, or you could use a multi-peak capture bead to look at resolution.  And if you want to quantitate this a bit more, you could extrapolate the peaks down to an area just above the blank bead to determine precisely how dim of a population you'd be able to resolve.

EMD-Millipore 8HT Review

In my quest to find a mid-range cytometer to replace my ailing FACSCantos, I've come upon the 8HT from EMD-Millipore (whom I'll probably just call Millipore for now, or maybe even Guava at times). The 8HT is a 2 laser, 8-parameter cytometer (2 Scatter and 6 Fluorescence, setup in a 4-2 configuration).  It represents the "top-of-the-line" instrument in the field of 8 cytometers that vary in their number of lasers, detectors and the absence/presence of a microtiter plate loader.  The 8HT can accept either a 96 well plate or 10, 1.5mL microfuge tubes.  There are also a set of 6 tube slots for various washing/rinsing purposes.  There are a few unique features to this instrument which I'll briefly describe below before getting into the data.

All of the legacy Guava instruments and the new Millipore additions share the same microcapillary fluidics system.  Whereas in flow-cell containing instruments, a sheath fluid is funneled through a conical shaped flow-cell where it creates the hydrodynamic force used to align the sample core stream through the laser interrogation point, here there is no sheath fluid.  The microcapillary system is basically a clear straw through which the sample is drawn, and the microcapillary walls provide the physical barrier to align the sample, essentially replacing the sheath fluid.  This basically means there's no PBS tanks to fill, and no giant waste container to dump down the sink.  Also, since the sample core cannot expand in diameter to increase event rate like it would in a hydrodynamically focused system, the way in which you increase event rate is by literally increasing the speed at which the fluid is drawn through the capillary.  With the capillary system, you do get a bonus in absolute counts for everything.

A second unique feature of the system is the modulated laser setup they've implemented to get around the need for separate pinholes and laser delays.  Basically, the 488nm and 640nm laser lines are modulated out-of-phase with each other at a high frequency so that red fluorescence emitting from the cell while the blue laser is exciting is sensed separately from red fluorescence emitting from the cell while the red laser is exciting.  I was pretty skeptical at first, but this works surprisingly well.  For example, APC fluorescence was pretty well excluded from the Red1 channel (Blue excitation/Red emission).  What makes this remarkable is that Red1 and Red2 are actually the same detector, but because of the modulation, the emission is able to be separated out cleanly.  

The software, InCyte was pretty good.  The thing that bugs me a little is the mere presence of the old Guava modules.  The 8HT software, in general seems a bit schizophrenic.  You can jump back and forth between a green background Guava software to a grey background InCyte.  I think it would be less confusing if there were just one platform to use.  The InCyte software is pretty capable on its own, so I can't see what's the use of the Guava software.  I'm not one to use canned application-specific templates, so that makes anything with a green background pretty much useless.  There are a few unique analysis tricks built-in as well, which show your data in a heat map-like graphic.  However, probably my most favorite feature of the software is something fairly minor.  To adjust the threshold on FSC, you can simply drag a red dotted line up and down the scale.  There is no confusion on where the threshold is set.

So far, so good, right?  Well, here's where things fall apart - Data.  Like many of the other units I've tested in this range, their fluorescence resolution seems to be lacking.  I'd put the 8HT pretty much on-par with the rest, but let's take a look at some figures.  I ran my standard battery of tests including, Single Peak UltraRainbows (to get a glimpse at alignment via the CV), 8-peak rainbows (to assure a certain level of dynamic range and resolution), PI stained CENs (to look at linearity as well as well-to-well carryover), and my 'gold-standard' dim population resolution (using antibody stained capture beads).  This time, to mix things up a bit, I chose to run the EXACT same samples on our Gallios, and just to make things absolutely fair, the samples were run on the 8HT first and then on the Gallios (just in case they started to deteriorate over time).

Single peak URFP, showing CVs of the fluorescence channels on the 8HT versus the Gallios.

8HT URFP Bead CV

Gallios URFP Bead CV

Next up, the ubiquitous 8-peak Rainbow Beads.

8HT 8-peak Rainbow Bead Resolution

Gallios 8-peak Rainbow Bead Resolution

PI Stained CENs:  Here we notice a problem with the 8HT.  My guess is that the unbound PI in solution is too much for the system to handle, and the detector is being swamped by light.  Obviously, this could be alleviated by titrating the PI out, but as you can see below, the Gallios' baseline restoration has no problem with the unbound PI.  If you think about it, in a capillary system, the amount of sample fluid that is illuminated along with the cell is much higher than in a hydrodynamically focused system running at a narrow core stream, so unbound fluorochrome in solution surrounding the cells has a huge impact on background.  Keep this in mind when looking at the Dim population resolution data.  Also, for carryover, I'm simply looking for cells in the subsequent well to have been stained by PI carried-over from the prior well.  In this case, carryover appears to be less than 1:10,000.

For the Dim Population test, antibody binding beads stained with different fluorescently labelled CD4 antibodies were run on the 8HT at the 'very low' flow rate and the 'medium' flow rate.  There are suppose to be 4 stained peaks (blue) and an unstained peak (grey).  The lowest stained peak represents about 2500 bound antibodies (CD4 on Human PBMCs is about 50,000 binding sites).  Here, we get a really great picture of the background issues.  When looking at a blank bead by itself, the background is pretty low.  However, once you have some fluorescence present, the background peaks merge together and offer no resolution.  This effect is enhanced when you increase the flow rate.  Again, the exact same samples are run on the Gallios.

So, as I've said many times, the convenience that these systems offer may be nice, but I'm not sure it outweighs the lack of fluorescence resolution.  I'm sure things could be optimized so that this system would perform better, but I'll always come back to the fact that we don't run into these problems on our LSRII, Fortessa, Gallios, or even our Cantos, Caliburs and Scans.  What's the difference?  Hydrodyanmically focused streams with gel-coupled collection optics, high-quality/high-powered lasers, and bit-dense/fast sampling electronics.  The quest continues...