[Cytometry] Compensation Frequency Question

D. Robert Sutherland rob.sutherland at utoronto.ca
Thu Mar 22 01:55:49 EDT 2012

We virtually never redo the comp on this assay except if major service has occurred. The FC500 instruments are very stable otherwise. We do lot-to-lot testing before deploying new Stem-Kits, and we run high and low range 'control' stabilized cells from R&D Systems every day we run the assay; in other words 5-6 days per week. We have the protocols set up on more than one instrument and we regularly perform instrument-to-instrument  comparisons on the same samples on the same day.
If we see the comp going off a bit over a period of time (and we monitor every plot in the ISHAGE protocol) we tweak the comp post-acquisition, then apply the slightly altered matrix to the next sample, and save the new settings.
Setting the comp with stabilized cells eg from Streck, R&D Systems or NEQAS virtually guarantees inappropriate settings for fresh samples (totally unsurprisingly). 
We use an extra plot that displays 7-AAD versus SSC gated on ALL CD34+ cells, not just the viable ones (this plot is not available to users of the Stem-CXP software) to see that our viable CD34+ cells are 'on-scale' in the first decade or so (and not crushed on the axis).  In this manner, ALL CD34+ cells (dead or alive) are visible and not masked behind other cell types (that are present in far superior numbers). It is then easy to see if the CD34+ cells gated from the first three gating regions are comped properly, and that dead CD34+ cells are visible in the 7-AAD+ region and the viable 7-AAD-negative CD34+ cells are visible and properly on-scale in the 7-AAD-negative. This is the key plot in the entire assay to show the comp is optimal, such that Viable CD34+ cels do not appear in the '7-AAD+ region, or that 7-AAD+ CD34+ cells do not appear in the viable CD34+ cell region.

Regarding Stem-Trol, there was once a time when this stuff was useful, since it was all that was available to prove that the CD34 and CD45 antibodies and 7-AAD had been added to the assay and were staining as expected. However, with the ready availability of commercially manufactured, stabilized whole blood-derived CD34+ cell-containing products from the organisations listed above and at appropriate CD34+ cell concentrations, it is time to move on from Stem-Trol. 
Some folks claim that by using Stem-Trol adequately, it somehow demonstrates that they know how to deploy the ISHAGE protocol correctly; This is BS! 
Stem-Trol's assayed cell concentration ranges from about 1200-1400 cells per microlitre and is very tricky to use properly, as Kanchan and many others have discovered over the years.
More recently some folks have taken to diluting this stuff to more 'useful' concentrations so that they can make their own 'high' and 'low' controls (typically in the 10/microlitre and 35/microlitre range). Given the problems associated with using this product as it was intended (ie undiluted), this seems like a very bad idea to me, regardless of all the extra verification/validation issues that raise their ugly heads with such an approach.  
In addition, the light scatter of these cells (derived as they are from the KG1 cell line) is totally different to that of normal CD34+ cells (fresh OR stabilised). I have witnessed all manner of issues with data files from folks relying on this stuff to prove they know how to do this assay properly. This material is in fact dangerous because folks open up their light scatter and fluorescence parameter gates much wider than was intended when we designed this methodology. In other words, the use of such products can teach the unwary how NOT to use the protocol optimally, and before too long, the protocol ends up bearing very little resemblance to that intended when it was developed. 
When manufacturers of CD34 enumeration kits similarly disregard the intent behind the original protocols and change gates and/or alter the gating strategies, we end up having to deal with the consequences.


Rob Sutherland
University Health Network/Toronto General Hospital

On 2012-03-20, at 4:51 PM, KANCHAN JESWANI wrote:

> Hi thereWell, we do Autosetup for every kit opened and also once a month as a regular policy.But the voltages and compensation values changes are very very minimal .However,stemtrol values do give us a headache sometimes...many a times it is out of range..... ThanksKanchan JeswaniClinical scientistDubai cord blood bank n research centre
>> From: aheisserer at BBAK.ORG
>> To: Cytometry at lists.purdue.edu
>> Date: Mon, 19 Mar 2012 09:28:24 -0800
>> Subject: [Cytometry] Compensation Frequency Question
>> Hello everyone out there.
>> For the Beckman Coulter Stem Kit, we were wondering how often people are performing compensation.
>> The company told us that the frequencies they have seen are once ever, once per lot of Stem Kit, once per box of Stem Kit, once a month, once a week, and daily.
>> This is a huge range and since the standards do not tell us how often, just that it needs to be done, we thought we would poll people who use it and find out how often they compensate.
>> So, all our fellow flow users who use the Stem Kit, how often do you compensate?
>> Sincerely,
>> A. Darlene Heisserer, MLS, ASCP
>> Stem Cell Laboratory Technologist
>> Blood Bank of Alaska, Inc.
>> 907-222-5671
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