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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