Re: Compensation in Clinical Lab

If the machine behaves reproducibly, and your cells and reagents never vary 
either, then indeed you're right, but also maybe lucky.  One of the best 
ways for a cytometrist (good or not) to assess the quality of their data is 
to feed the machine with compensation controls (no stain then each stain on 
its own), that way there's no need to "study the entrails" of smears, you 
can get straight to the meat of how the machine presents what one dye alone 
does unequivocally.

Using those controls also allows one to demonstrate that one's machine can 
differentiate the dyes one is actually using, rather than the (not 
necessarily related) dyes used in one's (not so cheap) QC beads,

Cheers

Ray


----- Original Message ----- 
From: "Mark Simmerson" <Mark.Simmerson@sch.nhs.uk>
To: cyto-inbox
Sent: Tuesday, December 11, 2007 3:35 PM
Subject: RE: Compensation in Clinical Lab


> Hi Flowers
> I'm a bit puzzled by the advocating of doing compensation as frequently as 
> once a day.
> As long as one can demonstrate that your flow cytometer is stable via 
> regular QC to
> ascertain optimum laser alignment, instrument standardisation of light 
> scatter intensity,
> fluorescence intensity and hydrodynamic focussing, plus instrument 
> linearity, then why do
> you need to perform time consuming, costly (and unnecessary) compensation 
> experiments.
> I would suggest that if you keep the same panels, instrument settings, 
> etc, then the only
> time one need recheck compensation is when the instrument may have been 
> altered - eg
> after a service.
> We installed a FC500 about 18 months ago and once the initial compensation 
> settings were
> ascertained, we have not needed to alter them. I checked the settings 
> after our recent
> service and found that the settings were so similar to the original that 
> no action was
> required.
> This doesn't mean that one can safely ignore compensation, but surely a 
> good flow
> cytometrist always examines the data to ensure that data is not - for 
> example - smeared
> along either the x or y axis.
> Cheers
> Mark Simmerson
> Lead BMS Flow Cytometry
> Sheffield Children's Hospital
>
>>>> "Irina Grigorieva PhD" <Irina.Grigoriena@northside.com> 11/29/07 9:29 
>>>> pm >>>
> Hello Richard,
> Our lab is using FACSCanto. We have 2 six-color instruments and we like
> them. When using Canto software (for lymphocyte typing pane or CD34
> enumeration), you should not even have problems with compensation, just
> run 7-color beads setup and do the automated compensation.
> For immunophenotyping you use CST beads for daily setup (Diva 6
> software), so you have to perform the compensation exercise. But unlike
> CST setup, you do not have to do this exercise daily	(I am not even
> talking about every run). First, you establish your compensation values
> and we do it by using CD8 in every color for a normal donor. Then you
> compare your results for every antibody you use in your panels with CD8
> (ex. compare CD8-PEcy7 with CD33-PECy7, if you use CD33 in this color).
> Then you run your cocktails (or whatever format you use for your test
> antibodies) and verify compensation settings. When initially
> establishing compensation values we always additionally check them on CD
> Chex, just to confirm the values. We call this procedure 'Establishing
> and validating compensation settings'.
> Now you have a different task of monitoring your compensation settings
> and this procedure does not necessary include running compensation
> exercise. We run daily normal controls and we have established our
> normal ranges for each antibody. Therefore, we can use these data to
> overall assess our daily compensation, comparing the patterns and
> values. And then each lab is deciding for themselves when the patterns
> do not look consistent or numbers do not match exactly. One day prior to
> that happening is date of your compensation exercise. Depending on the
> lab it may be 1 day, 1 week, 1 month.
> We have started doing it every day 2 years ago, and then changed our
> schedule according to the patterns we saw overtime. The instrument is
> very reliable, settings do not require a lot of adjustments even at the
> time of scheduled compensation exercise. Therefore, my lab policy
> states: After initial establishing and validating compensation settings
> the verification of the use of correct settings is performed weekly.
> Additionally the same compensation exercise is performed in a case of
> any questionable results, changing the reagents, and also when laser,
> PMT optical filter components or settings are modified.
> From my personal experience  there are a lot of myths created around
> flow cytometry: danger of using tandems, risk of using stored samples
> for acquisition, constant failure of PMT and compensation settings, etc.
> All this forces clinical flow people to perform a lot of excessive work,
> not necessary needed. Since we run ASR tests (beauty and trouble of
> clinical flow), we have to do an extra work to create the system with
> proper parameters, normal ranges, controls and standards. Then you can
> use your own system to validate all your procedures, from storing
> conditions to number of compensation runs per unit of your time.
> Overall I truly believe that with modern equipment, very capable
> software and reliable reagents our clinical flow life should become less
> complicated, although Current Protocols recommendations do not support
> my theory.
> Regards. Irina
>
> Irina Grigorieva, PhD
> Director, Flow Cytometry Laboratory
> Northside Hospital, Atlanta, GA
> (404)- 851-6541
> e-mail: irina.grigorieva@northside.com
>
>
>
> ________________________________
>
> From: Richard D. Schretzenmair [mailto:rds@mail.med.upenn.edu]
> Sent: Tuesday, November 27, 2007 7:10 PM
> To: cyto-inbox
> Subject: Compensation in Clinical Lab
>
>
> Hello,
>
> Currently, we are validating a FACSCanto for clinical use. In trying to
> develop standard protocols for the operation of this instrument, a
> question arose about how often compensation is required? We had intended
> to compensate daily, but Current Protocols says that compensation is
> required for every run (in the compensation myths section). When you are
> compensating 6 colors, then include each antibody conjugated to a tandem
> dye, your could easily exceed a dozen tubes for each compensation run.
> In the era of tight hospital budgets, the cost of antibodies,
> compensation beads (or compensation samples) and labor needs to be
> considered.
>
> It is my understanding that compensation is dictated by the instrument
> setup (filters, mirrors, PMT Voltages, lasers) and the specific
> fluorochromes used. This machine is QC'ed using DiVa 6's CST software,
> which does a maniacally thorough job at QC. So, if the instrument setup
> is not changed, CST QC is passed and PMT voltages held constant, how
> often does compensation really have to be performed? Samples are run
> immediately after staining. It seems that doing compensation with each
> run (several times a day), would be more likely to introduce human
> error, than to correct for any true changes in compensation.
>
> Considering the factors effecting compensation, is it really required
> for every run, or even daily, given the above circumstances?
>
> Rich
>
>
>
>
>
> Richard D. Schretzenmair
>
>
>
>
> Flow Cytometry and Cell Sorting Shared Resource
>
> Abramson Cancer Center
>
> University of Pennsylvania School of Medicine
>
> 297 John Morgan Bldg.
>
> 3620 Hamilton Walk
>
> Philadelphia, PA  19104-6082
>
>
>
>
> Phone: 215-898-3528
>
> FAX: 215-898-4227
>
>
>
>
> rds@mail.med.upenn.edu
>
>
>
>
>
>
>
>
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