Coincidence on Facscan

From: Arnold Pizzey (a.pizzey@ucl.ac.uk)
Date: Wed May 15 2002 - 00:36:39 EST


>I am just scanning back on my flow list messages and I found your question,
>but did not see any replies. I have been wondering about this as well. I f
>you received any replies, I would appreciate if you could forward them to
>me.
>
>Thank you for your time

Hello Artur,

I have to say that overall, the replies were not that useful. Most talked
about coincidence in sorting rather than in analysis. I did however carry
out some preliminary experiments to assess the scale of the problem on one
of my elites. Two aliquots of human RBCs were separately stained with
fluorescein and Phycoerythrin conjugated anti Glycophorin-A, washed and
recombined. I then ran them through the flow and analysed the data obtained
on a dot plot, of course you would expect to obtain two separate clouds and
no double positives. At low concentration this was essentially the case
however, at data rates above ~1000 events/sec a distinct double staining
population appears. At 2000 events/sec 13% were scored as double positives
-this equates to a 'real' value of 39% as the possible combinations of
coincidence are: green/green, yellow/yellow, green/yellow (only the latter
is scored as double positive). Incidentally, this data was obtained after
pre-processing FS peak Vs FS Integral signals and excluding obvious
doublets that fall away from the diagonal. I suspect that  pulse
width/height analysis is inefective on 'true' coincident cells (those that
are exactly coincident).  I used RBCs as they were my targets for analysis,
I would imagine that you could do the same thing with CD45 for leukocytes.

It would I think, be a good idea for those of us who have not already done
so, to assess the degree of coincidence on our machines before undertaking
any high-speed multicolour analysis -it would have certainly saved me a lot
of time!

As I am reposting, I should also mention that I had a quick look at the
second question. I had been assessing the expression level of a multiple
myeloma marker and relating the values obtained to calibration beads of a
known MESF. My worry was that there was a difference in saturation of the
fluorochrome on the cells as compared to that on the beads. Therefore I ran
both at diferent laser powers from 10mW up to 1000mW, the results I
obtained sugest that, on the Beckman-Coulter elite (100uM flow cell,
16x125uM beam shaper) that there is a departure from input/output linearity
 for fluoroceine above ~100mW. It should be borne in mind that jet in air
instruments would see this effect at far higher laser powers due to the
poorer coupling of the laser to the stream

Best regards,

Arnold





>Hello all,
>
>
>
>1) I was on a visit to another department the other day where a FACScan was
>being used to diagnose MRD using a multi-colour protocol at flow rates
>around 2000/sec.
>On my machines (Beckman-Coulter Elites) I would expect to see a
>considerable amount of coincidence at these rates. My question is, are
>FACScans less susceptible to this effect than elites at these rates and if
>not, what data rate would be best to minimize this effect.
>
>
>2) I have been assaying the level of expression of a cell surface marker by
>comparing the fluorescence obtained against 'Spherobead' calibration beads,
>the fluorescence obtained are then expressed as MESF levels -so far, so
>good. I have upgraded the laser on the machine (an epics elite) from the
>old Cyonics 15mW unit to an Innova 90 -I now use 100mW excitation. My
>concern is that the fluorescein on the cells may have reached saturation
>whilst the proprietary fluorescent compound on the calibration beads has
>not -thus giving a low reading for the cells. I intend to try different
>levels of excitation when I get a minute, in the mean time, I would
>appreciate any thoughts
>
>
>Best regards,
>
>
>Arnold
>

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	Arnold Richard Pizzey
	Department of Haematology
	Royal Free and University College London Medical School
	98 Chenies Mews
	London WC1E 6HX
	U.K

	voice:	+44 020-7679-6234
	Fax:	+44 020-7679-6222
	email:	a.pizzey@ucl.ac.uk
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