Re: Flow--Counting anything

  
It is probably hard to tell. Even if you put the suspension under microscope there will be both "sticking" and coincidence looking the same, although the coincidence will be different - in the volume of the hemocytometer chamber, as opposed to linear coincidence in the flow. 
 
Perhaps, what can help is diluting a sample and running again. Dilution should decrease coincidence, but the number of stuck particles should not change. This is if you dilute after first mixing cells and beads. If you just prepare different dilutions from the beginning, the chances of sticking will also be lower in lower concentrations.
 
In theory, the mathematical probability of coincidence can be calculated knowing the particle diameter (length), event rate, flow speed (linear) and cytometer resolution.
 
I guess, if the flow rate is not extremely high the coincidence should not be high enough to be worried about. Otherwise, we would have odd double positivity in any antibody stained sample (apart from flow count tests) and determination of low frequency cells identified by co-staining of two (or more) antibodies would not be possible.
 
Regards.
 
Vladislav Rozenkov, MD, PhD
 
-----Original Message-----
From: Nebe-Von-Caron, G <g.nebe-von-caron@unipath.com>
To: cyto-inbox
Sent: Fri, 24 Feb 2006 11:57:54 -0000
Subject: RE: Flow--Counting anything


Interesting problem. Do you know how much of this is coincidence and how much is sticking?
 
Regards
 
Gerhard
-----Original Message-----
From: ckohler@bi-vetmedica.com [mailto:ckohler@bi-vetmedica.com] 
Sent: 22 February 2006 14:16
To: cyto-inbox
Subject: RE: Flow--Counting anything


Dear Vladislav and Samuele,
 
On the subject of volume and flow beads. With the problems I had where beads altered my readings due to bacteria sticking to them, I ran my bead dilutions separately at the same flow speed as I ran my bacteria samples. I currently run 3 dilutions, beginning, middle and end of the sample run, to verify my flow rate. Prior this, I ran my beads in multiple dilutions to determine my variability. With 9 sets of dilutions, each ran 3 times over two different days--5%RSD, 3.6% if I throw out one really low number. The instrument I have continues to operate at consistent flow rates. 
 
Just some food for thought...
Caroline
 
-----Original Message-----
From: rozenkov@netscape.net [mailto:rozenkov@netscape.net] 
Sent: Saturday, February 18, 2006 11:24 PM
To: cyto-inbox
Subject: Re: Flow--Counting anything


Dear Samuele, 
 
The reason for your doubt is, I think, that you consider that all flow cytometers run the flow in a volumetric fashion, i.e. that the precise volumes of suspension run through the machine are known.
 
In fact, most flow cytometers do not measure the volume of suspension, but only give in the manuals approximate velocities in ml/min for high, medium and low speed, for instance. Therefore, the volume of sample that contains some counted number of cells is unknown, so the concentration can not be calculated. That is why we need to use reference beads of known concentration and, therefore, knowing the number of beads and cells (events) of interest mixed in the sample and the bead concentration we can calculate the cell concentration.
 
I knew only one brand of flow cytometers, Partek that measured precise volume of the sample. They have a range of models all with volumetric counting. Now I learnt about the second one, Guava from Roland?s very comprehensive and non-biased posting.
 
Regards.
 
Vladislav Rozenkov, MD, PhD
 
-----Original Message-----
From: Samuele Secchiero <strikeiron13@yahoo.com>
To: cyto-inbox
Cc: cytometry@flowcyt.cyto.purdue.edu
Sent: Tue, 14 Feb 2006 10:35:14 +0100 (CET)
Subject: Re: Flow--Counting anything


Hello,
Sorry but reading your answers I realized that I have a doubt in this subject.
If you have cells in a known concentration and you count them with a cytofluorimeter and your results are corresponding, then may you assume that cytofluorimeter counts are correct, without using an internal reference (beads)?
Thanks in advance
 
Samuele Secchiero
University of Trieste, Department of general pathology

rozenkov@netscape.net ha scritto: 
Dear Myron,
 
Thank you for the correction. They had to be reversed.
 
Vladislav
 
-----Original Message-----
From: Waxdal, Myron <M.Waxdal@intracel.com>
To: cyto-inbox
rdue.edu>
Sent: Thu, 9 Feb 2006 08:31:39 -0500
Subject: RE: Flow--Counting anything


Dear All
 
Dr Rozenkov's response to Caroline is a nice summary of why the flow cytometer provides good data on the concentration of single cells (or other particles) in a sample. However it contains one paragraph which may lead to confusion.
 
Impreciseness of pipettes does not affect counting with beads, differently from manual count. This is because the same volumes of cells and beads are taken, so their impreciseness does not matter. Of course, by the state of the art methodology, you have to use the same pipette for cells and beads. Pipette inaccuracy still influence both methods
 
In this paragraph the terms imprecision and accuracy are reversed. Precision/imprecision is the ability of an instrument/technique (in this case a pipette) to be reproducible and is essential for the flow counting method.  The pipette must deliver the same volume of	beads and sample each time (such as 101 uL). The pipette does not have to be really accurate, that is to deliver exactly 100 uL.
 
Waxy
-----Original Message-----
From: rozenkov@netscape.net [mailto:rozenkov@netscape.net] 
Sent: Wednesday, February 08, 2006 12:34 AM
To: cyto-inbox
Subject: Re: Flow--Counting anything


Hello Caroline, 
 
Definitely, flow counting is a good, reliable and widely used method. That is why a number of companies make flourospheres for flow count, to mention just Coulter, BD, Spherotex, Caltex, Molecular probes, etc. Probably, representatives of these companies will be able to supply you with technical data and references if you ask them.
 
We are now using Flow-count beads from Coulter and the kit's insert leaflet has quite a bit of information and a reference list of six papers. I think this insert is not available on the web-site, so if you are interested, but can not obtain it from the company, I can fax or mail it to you.
 
The data there shows that flow count results were comparable to manual count. It is really hard to do worse than manual count with hemocytometer. You mentioned about 20% accuracy... Yes, it is known and fully accepted in scientific and clinical applications that if you count a standard minimum of 200 cells in a hemocytometer then error can be up to +/-20%. This means that if your count the same sample two times and your second count is 50% higher than the first one, this is still within 20% around the mean!
 
So, the first reason why flow count is more accurate than manual is that you can count thousands of events or hundreds of thousands in seconds. It is impossible to count so many events manually.
 
If you collect 5000 cells in a minute, probably your concentrations are relatively not high and, therefore can not be counted in a hemocytometer without concentrating by spinning down and reducing supernatant. Some amount of cells or bacter


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