All you really need to get started is some FCS data. There are
only a few user-selectable parameters that govern QTube's
behavior (see “Setting Parameters”), and the default
parameters are appropriate for many applications. So we encourage
you to just try it out!
Remember that QTube wants to compare fingerprints for parameters
that are common to two or more tubes, where the data for each tube is
stored in its own FCS file.
At the top of QTube there are the familiar MenuBar and ToolBar.
Hovering over a button in the ToolBar will display the
of the button. At the very bottom is a giant button that will
compose an email to Cira from you to discuss licensing for use outside
your organization, if you wish. In between, the screen is
divided into two
sections by an adjustable divider. In the upper one will be a
list of parameters that
QTube finds in your FCS files. You will use the table that
appears here to select parameters you want included in the
fingerprints. The results will appear in the lower one.
So, after launching the QTube application, click on “File ->
FCS Files...” or on the left-most button in the Toolbar [ ].
A File selection dialog will appear. Browse to the directory that
contains the files you want to analyze (QTube will remember this
location for subsequent runs, making it convenient to come back to the
same or nearby location later).
You can select multiple files in several ways. If the files are
listed contiguously you can click on the first file of a group, and
then shift-click on the last one to select the entire group. Or,
you can CTRL-click on each file to add it to the list. Regardless
of which method you choose, once all of the files that you want
analyzed have been selected, click on the “Open”
button. This returns you to the QTube main panel.
Note that the “Run” button [ ] is disabled.
That's because Qtube doesn't yet know which parameters you
wish to be compared. QTube at this point has analyzed the FCS
headers and determined which parameters are common across all of the
tubes based on the FCS Standard $PnN and $PnS parameter names assigned
by your cytometer. These parameters are shown in black text
– the parameters that differ across the tubes are shown, but are
disabled to avoid inadvertent use of parameters that should not be
compared because different reagents are used for the same parameter in
So, select the parameters you wish compared by clicking in the
In the example above, we have selected two of the three available
parameters: 2 (Side Scatter) and 5 (CD45-PerCP).
Now just click the Run [ ] button. In a few
seconds (depending on the
speed of your computer) the results will be displayed in the panel
QTube presents the results for several “metrics” in a color-coded
colors mean that the tube was within normal limits as compared with the
average of all of your tubes, and a “good” sample will be
green across the board.
colors mean that there is some deviation. You should examine the data
for this tube (or tubes) to find out why, although very likely you will
still be able to successfully analyze these data.
that a large deviation has occurred for this tube. There could be
several explanations. Perhaps a bubble interrupted the
flow. Re-acquiring this tube should clear up this problem.
If for some reason the cytometer calibration(s) changed, you may need
to re-calibrate and then re-acquire the entire panel. If
there was a sample or reagent mixup, simply re-acquiring the data may
not help. This should be investigated carefully and addressed
using your lab's procedures.
Note on Memory
If the total number of
events in the
set of files you've chosen is large, QTube will sample at random an
equal number of events from each file. This is to enable
run within a small memory footprint on your computer. If this
happens, QTube will inform you, and tell you how many events from each
file it's using.
We've included two different metrics for measuring similarity,
each with somewhat different statistical properties. You can be
quite sure that if both metrics for all tubes are “in the
green” that your data are highly self-consistent (that is, unless
you've gotten threshold parameters badly boluxed up!).
Each metric is computed from the same fingerprint representation. The
fingerprint is expressed as an array of values, each of which
corresponds to a subregion in the space you specified by your choice of
fingerprinting parameters. Each value is the ratio of the
observed probability in the subregion divided by the expected (uniform)
- The max metric is computed by first taking the log2 of the
relative densities. Thus, if there are twice as many events than
expected in a subregion, the value will be +1.0. If there are
half as many as the expected number it will be -1.0. The max metric is
then the maximum absolute value of these transformed densities.
This best represents the extreme deviation from the norm. It is
probably the most sensitive of the metrics in that it is sensitive to a
- This metric is simply the standard deviation of the relative
densities in the fingerprint. It provides information regarding the
values around the expected value. Like the max metric, values
close to zero mean that the data are very consistent. Unlike the
max metric, a value of 1 means something a little more complicated, and
a precise interpretation would require examining how the values are
distributed. In general these values should be considerably
smaller than the max metric values.
The resolution parameter governs the scale to which Cytometric
Fingerprints are taken. Essentially, we sub-divide events into
relatively smaller subregions of multi-parameter space at the higher
resolutions. If you only have two or three common parameters, low
or medium resolution should be adequate. If you are comparing
multiple tubes on more than three parameters (an unusual circumstance)
you may want to consider using High resolution. Higher resolution
will take a few seconds longer to run, depending on the speed of your
computer and the size of your FCS files.