[Cytometry] Using FITC threshold to analyze high density specimens

Dorothea Torous DTorous at LitronLabs.com
Thu Feb 5 09:56:43 EST 2009

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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