[Cytometry] Using FITC threshold to analyze high density spec imens

Roberts, Daniel Daniel.Roberts at covance.com
Thu Feb 5 14:45:48 EST 2009

Dr. Dertinger,

I have seen something similar to this when analyzing cells stained with PI
and labeled with Alexa488 (FACSCanto II, Diva v6.1.1). Our samples were not
super concentrated, but were in fact very dilute. So much so that I didn't
want to spend 20 minutes acquiring each sample on low. However, after
increasing the flow rate from low to medium, I noticed the same type of
spectral drifts. In some instances the CV's more than doubled. 

I image that someone can comment on how or if the digital processing plays a
part in this, but our solution was to adjust the stock flow rate as
indicated below (for details please send me an email).

FACSCanto II Stock Settings
  Stock / Flow Rate / New Setting / New Flow Rate
L 0.45, 10 ul/min, Did not change
M 1.15, 60 ul/min, 0.6, 21 ul/min
H 2.06, 120 ul/min, 0.9, 41 ul/min

My new flow rate settings have eliminated spectral shifting, and I can now
even analyze on high. Comparatively, the stock settings on a Calibur are (in
ul/min): 12, 35 and 60 for L, M, and High, respectively.

Thus, Canto Med = Caliburs High (stock settings).

If you have the HTS module take caution, as I'm not sure how adjusting the
main flow rate would affect those fluidics (though I think they have
separate controls). Another lab confirmed this phenomenon, which initially
lead me to take action. I recommend anyone performing DNA analyses on a
Canto to either stay on Low or adjust the settings down. 

Best of Luck,
PIG-a for life, 
Technical Resource Specialist
Genetic Toxicology Department
Covance Laboratories, Vienna, VA
703-245-2200 x5136 (Office)
x5589 (Lab)

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Dorothea Torous
Sent: Thursday, February 05, 2009 9:57 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Using FITC threshold to analyze high density specimens

I'm submitting this for a colleague, so please respond directly to him.


We have two flow applications that involve interrogating millions of rodent
RBCs for an aberrant phenotype. This is not difficult on our Calibur
(CellQuest Pro) or our Canto II  (DiVa) using FSC thresholding. However,
there are other times when we want to restrict analysis to reticulocytes,
yet still interrogate one million of these rarer cells for the aberrant
phenotype (e.g. CD59-negative phenotype).

We have a strategy that works well on the Calibur...rather than physically
enriching for retics, we bring very high density blood specimens to the
flow. Each tube has the equivalent of approximately 25 microliters of blood
per 1 mL! The specimens have been stained with a green fluorescent nucleic
acid dye to resolve mature RBCs from retics from leukocytes. We then use a
FL1 threshold set sufficiently high to eliminate the mature RBCs from
consideration. As this eliminates upwards of 97% of all events, the
instrument is able to deal with the high density, even when we move from a
low to a medium fluidics rate setting. In this manner, we can efficiently
evaluate one million retics within 8 minutes or so on average.

We have been struggling with the Canto though. When we bring the high
density specimens to the instrument and use a FITC threshold, all we can do
is run them on the low fluidics rate. At this rate, our one million retic
analysis would take 40 minutes or more. Unlike the Calibur, we are unable to
go to a higher fluidics rate. The light scatter profiles change, as well as
fluorescence. This is especially true for the PE-low/negative events as
opposed to the PE-bright events. What is overwhelming the processor? What is
this new digital instrument doing that doesn't allow it to keep up with our
10+ year old instrument?! Any thoughts/suggestions would be greatly

Thanks in advance!


Stephen D. Dertinger, Ph.D.
Director of Research
Litron Laboratories
200 Canal View Blvd.
Rochester, NY 14623
tele: 585-442-0930
fax: 585-442-0934
email: sdertinger at litronlabs.com
web: www.litronlabs.com <http://www.litronlabs.com> 

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