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Peter, et al.-<br>
I think this is great idea! It is the best way to estimate accuracy
versus precision in AUC. There was similar work done by the ABRF, but
if I am not mistaken, it was for comparison of different techniques.<br>
The AUC2005 meeting will provide a good forum for discussing this more.<br>
Best to all-<br>
Tom<br>
<br>
<a class="moz-txt-link-abbreviated" href="mailto:pschuck@helix.nih.gov">pschuck@helix.nih.gov</a> wrote:<br>
<blockquote
cite="mid34382.69.140.182.115.1103395858.squirrel@69.140.182.115"
type="cite">
<pre wrap="">----------------------------------------------------------------------------------
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Regarding a standardized test, I believe it is a good idea, and I totally
agree with John's concerns about analysis and that we either should not do
it or spend enough effort and thought to make it worthwhile. For the
latter, my suggestions would be
1) run the sample in triplicate on each machine (in the same run) to get a
feeling for the variance caused by things other than temperature (e.g.
analysis, centerpiece imperfections, imperfections in matching the buffer
volume, etc.) In addition, each participant could optionally do
repetitions of the run to assess run-to-run variability. Could also
invovle two categories of data - absorbance/interference detection.
2) we should establish a very detailed protocol for the experiment,
standardizing rotor speed, sample volume, temperature equilibration
periods, etc.
3) Following John's idea, the data analysis should be performed centrally
by one or more persons, blind to the identity of each run, each analysis
person analyzing the complete set of all runs performed. (I'd volunteer
for the analysis.) We could put the data anonymously on a website, and
each participant, knowing the code of his/her own machines, could judge
him/herself if a centrifuge is an outlier and decide whether to introduce
a temperature correction factor or not.
4) I propose we should all do the spectroscopic temperature calibration
method, as well. That would address if deviations from the mean of the
experimental s-values for a machine does actually correlate with a
detected calibration error (addressing the concern about statistical
accuracy of the calibration). I wonder if that, too, could be anlayzed by
a single person, without access to the results of the s-values and blind
to the origin of the data sets. I'm not familiar with the method, but
would it be possible and advantageous to share the same cell (if it's
sealed and stable enough) and mail it from one site to the other?
5) the choice of sample and sample stability is probably critical factor.
I would suggest something large, maximizing the sharpness of the boundary
and the possibility to calculate the meniscus position in the data
analysis. Also, if a large molecule aggregates it would be easier to
identify the monomer peak and confine the analysis to that. How about an
antibody (perhaps donation)? We could make solutions and reference buffer
in a single lab, testing for sufficient stability over a couple of weeks,
and then mailing them out to each participant who would agree to run it
within a certain period of time?
In this proposed scenario, we would need
* a donation of protein sample
* a lab to do the preliminary protein stability test and mail the sample out
* enough participating labs (machines) to get a good statistics
* a site to gather the data, encode it, put it on a website, and send it
out for the data analysis
* data analysis sites for calibration spectra
* data analysis sites for velocity runs
* a site to do the statistical analysis of all results and the data subsets
* a coordinator of these tasks
In my view, ideally these tasks should all be done by different labs, and
there's probably several missing aspects.
Regarding the organization of such a study, although in my view ABRF
involvement could be very valuable, I don't think we necessarily need a
formal structure or entity for this particular study. It appears to me it
could probably be handled very well by our RASMB group, since we already
have an excellent communication and discussion forum established.
In my view, we would only need a volunteer to collect all suggestions from
this discussion, to put up for discussion all additional points of
concern, consolidate it in a detailed proposal, solicit participants and
volunteers for the different tasks.
Overall, it would certainly be a lot of work, but if we can divide it up
it may be feasible.
Seasonal Greetings,
Peter Schuck
</pre>
<blockquote type="cite">
<pre wrap="">Arthur's suggestion of running the same sample in multiple labs may be a
good one, but I'm not so certain the results will clearly indicate
differences in temperature calibration, at least not unless it is all
thought through carefully in advance.
The problem is to distinguish real differences in temperature from other
effects that can change the values of the sedimentation coefficient:
(1) systematic errors due to differences in the software used to evaluate
the data;
(2) systematic errors due to how the meniscus position is defined (this is
a
bigger problem than most people realize);
(3) potential systematic differences between absorbance and interference
data (has anyone really looked at this carefully?);
The obvious way to deal with the first two would be to have one person do
all the data analysis, but that could be a burden (no, I'm NOT
volunteering!).
Lastly, there is simply the problem of precision of the values from any
one
lab. How many labs have really evaluated their reproducibility, for
example
for running the same sample in triplicate in the same run, and from
run-to-run? If the run-to-run precision is say 1%, then that translates
into
a 0.4 degree temperature uncertainty.
Overall then you really need VERY high quality data to evaluate the
temperature calibration issue. A casual effort is potentially a waste of
everyone's time.
Lastly, I might suggest that this may be something that should be handled
under the auspices of the Molecular Interactions Research Group of the
Association of Biotechnology Resource Facilities (ABRF), who have already
run (and published) tests involving both BIAcore and AUC studies of the
same
protein in multiple labs. Preston Hensley and Ed Eisenstein are, I
believe,
currently part of that group so perhaps one of them could comment on this.
John
-----Original Message-----
From: <a class="moz-txt-link-abbreviated" href="mailto:rasmb-admin@server1.bbri.org">rasmb-admin@server1.bbri.org</a> [<a class="moz-txt-link-freetext" href="mailto:rasmb-admin@server1.bbri.org">mailto:rasmb-admin@server1.bbri.org</a>]
On
Behalf Of Arthur Rowe
Sent: Monday, December 13, 2004 7:47 AM
To: John Correia; <a class="moz-txt-link-abbreviated" href="mailto:mchien@beckman.com">mchien@beckman.com</a>; <a class="moz-txt-link-abbreviated" href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a>
Subject: Re: [RASMB] Re: XL-A/I temp control
Hi Everyone
I entirely agree that the temperature on machines needs to be calibrated.
Equally, I agree with Jack's statement that "most users . . . never
calibrate the temperature". As to whether it is actually easy to do same
using the Stafford & Liu method, opinions might differ. We ourselves find
that the integration required to get the area under the curve is easy
enough
to estimate with data from the spec, but less easy (baseline definition,
noisier values) with data from the XL-A.
The suggestion that a quick set of standard runs be undertaken on multiple
machines would serve to clarify as to whether it all really matters, at
the
level of doing careful calibration - if one is talking at the precision
(not
accuracy) level of the instrument. After all, with a (presumably
standardised) production line, one might have supposed that any errors at
a
given temperature might have been pretty much the same from one instrument
to another. The sketchy data which we have to hand suggests that that is
not
the case.
Measuring an s value of a given solute via an agreed procedure is about as
objective a procedure as one can define. Variations are easily transformed
into temperature uncertainties. If that is what we are looking for, then
this is the easiest way to find it. The numbers of instruments volunteered
to date (10) looks enough to give a pretty good idea of what the variation
level actually is - but any more would bolster the stats nicely . . . .
Regards to all
Arthur
--
*************************
Arthur Rowe
Lab at Sutton Bonington
tel: +44 115 951 6156
fax: +44 115 951 6157
*************************
From: "John Correia" <a class="moz-txt-link-rfc2396E" href="mailto:jcorreia@biochem.umsmed.edu"><jcorreia@biochem.umsmed.edu></a>
Date: Fri, 10 Dec 2004 11:16:43 -0600
To: <a class="moz-txt-link-rfc2396E" href="mailto:mchien@beckman.com"><mchien@beckman.com></a>, <a class="moz-txt-link-rfc2396E" href="mailto:arthur.rowe@nottingham.ac.uk"><arthur.rowe@nottingham.ac.uk></a>,
<a class="moz-txt-link-rfc2396E" href="mailto:rasmb@server1.bbri.org"><rasmb@server1.bbri.org></a>
Subject: Re: [RASMB] Re: XL-A/I temp control
I suspect other will give technical responses about the ability of these
temperature controllers to function at the +/- 0.1 C level. My take is
slightly different.
What matters is not the set point but knowing the actual temperature.
To my knowledge most users never use the Stafford & LUS method to
calibrate
the temperature on their machine. (can we vote on the RASMB in some way?)
It is no more tedious than waiting three hours before starting a run. I
have checked the calibration on my machine twice, once in '93 when I got
it,
and again in 1999 when a result caused me to question the temperature
accuracy. A series of measurements up to 40 C and back down to 4 C (with
parallel measurements in a spec) takes about two days. It does not take
three hours to go from 20 to 25 C, as verified by the stability of the
area
under the CoCl2/ethanol curve. At a setting of 4 C I get 3.6, at 20 I get
19.7, at 40 I get 39.8. The values drifted by 0.1 C in 7 years. So if we
mean by accuracy +/- 0.5 C of setting, my machine is within spec. When I
fit data I use the actual temperature, not the set point. & I trust the
setting on the screen at low vacuum and hit start when it reports the set
temperature.
Comparisons between uncalibrated machines honestly make no sense to me.
Calibrate & be done with it. (Arthur, if you do that in Nottingham &
still
get different values of S between machines I would be concerned, &
amazed.)
I also use the calibrated temperature values when I measure density in an
Anton Paar DMA 5000. It has a peltier cell good between 0 - 80 C, so I
dial
in 19.7 instead of 20 C and measure away. I suspect the error from a
calculated density (viscosity & Vbar) is larger than (albeit coupled to)
an
assumed temperature.
At this point the inquiring "student" should assume errors in various
parameters and propagate them into S or MW by the appropriate equations -
my
favorite book for teaching this is Bevington, "Data Reduction and Error
Analysis for the Physical Sciences".
PS - why Beckman has never joined up with Anton Paar to bundle Density
Meters into XLA/XLI quotes amazes me.
PPSS - Walter claims to keep the original solution around, in a cell, from
the CoCl2 calibration work, and years later it still gives the same
results.
Quick checking may not be as difficult as one might think.
-------------------------------------------------------------------
Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-1522
fax (601) 984-1501
email address: <a class="moz-txt-link-abbreviated" href="mailto:jcorreia@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</a>
homepage location: <a class="moz-txt-link-freetext" href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</a>
dept homepage location: <a class="moz-txt-link-freetext" href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</a>
-------------------------------------------------------------------
</pre>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<pre wrap="">Arthur Rowe <a class="moz-txt-link-rfc2396E" href="mailto:arthur.rowe@nottingham.ac.uk"><arthur.rowe@nottingham.ac.uk></a> 12/10/04 08:17AM >>>
</pre>
</blockquote>
</blockquote>
</blockquote>
<pre wrap="">Hi Everyone {this is a second (now 3rd!) try at getting this mail out -
first attempt got lost in cyber-space, it seems}
Mei-Ling Chien gives us a very useful review of the nature of the
temperature measurement and control system in the XL-I/A instrument.
However, I do not think that this fully addresses the problems which one
has
in determining what the absolute temperature of one's sample actually is
when it is going round in the rotor at speed.
It is, of course, only a worry to those (very limited) number of people
for
whom an absolute s value is of importance, normally for hydrodynamic
modelling purposes (although formulation issues should not be forgotten).
When I raised this issue on RASMB a week or so back, my concern was not
"to
ensure their operation within the published specification". . I am
trying
to get the accuracy of the temperature read-out to be close to the
precision
of which the system is capable. I have no evidence at all to suggest that
the accuracy is outside the quoted spec of 0.5º. It is just that I - in my
greedy way - want 0.1º.
Even the method mentioned (equilibrate for 3 hours - under vacuum - and
then
check "with a calibrated external temperature sensing device to verify
accuracy" is not unambiguous in what it will yield. Quite apart from
matters such as adiabatic effects when one releases the vacuum to use an
"external temperature sensing device", can one be sure that the thermal
emissivity of a spinning rotor surface, averaged over everything that is
passing by, is equal to that of a piece of the rotor surface 'seen' in a
stationary rotor?
None of these are new concerns, and I certainly lay no claim to the IPRs!
I
imagine, from what Mei-Ling Chien has communicated, that we at least know
clearly that the ±0.5º refers to the accuracy of the temperature as
measured
by the defined procedure. Walter Stafford's colorimetric method (Stafford
&
Liu) did not suggest the presence of errors outside the stated accuracy
limit, and is surely a valid way to approach the absolute temperature
issue.
But is is pretty tedious to use as a procedure, and certainly as a routine
QA method is not feasible.
As an approach to the size of the problem, would there be support for
Borries Demeler's suggestion (a single sample to be circulated and
multiple
users on multiple machines to report an s value under defined conditions)?
After all, the NCMH + Borries's Lab gives us 6 machines for starters.
Any way, we here keep trying here to locate the holy grail - a simple,
cheap, effective method for determining the in-cell temperature to ±0.1º
Regards to all (and many thanks to Mei-Ling Chien)
Arthur
--
*************************
Arthur Rowe
Lab at Sutton Bonington
tel: +44 115 951 6156
fax: +44 115 951 6157
*************************
From: <a class="moz-txt-link-abbreviated" href="mailto:mchien@beckman.com">mchien@beckman.com</a>
Date: Fri, 3 Dec 2004 10:11:46 -0800
To: <a class="moz-txt-link-rfc2396E" href="mailto:'rasmb@rasmb-email.bbri.org'">"'rasmb@rasmb-email.bbri.org'"</a> <a class="moz-txt-link-rfc2396E" href="mailto:rasmb@server1.bbri.org"><rasmb@server1.bbri.org></a>
Subject: [RASMB] Re: XL-A/I temp control
----------------------------------------------------------------------------
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----------------------------------------------------------------------------
------
Hi All,
Below is response regarding XL-A/I temperature control from our Technical
Support Department.
******************************************************
Mei-Ling Chien PhD
Staff Development Scientist, Centrifugation
Platform & Automation Business Center
Beckman Coulter Inc.
<a class="moz-txt-link-abbreviated" href="mailto:mchien@beckman.com">mchien@beckman.com</a>
(650) 859-1948
******************************************************
The basis for temperature control specifications were instrument design
specifications for temperature control and dynamic system testing during
the
prototype phase of the product.
If there is a discrepancy in temperature control and measurement between
instruments of the same design then a dynamic calibration check should be
performed on both instruments to ensure their operation within the
published
specification.
First the physical condition of components within the temperature control
and
vacuum system should be verified through inspection. Then an electronic
calibration for temperature control and vacuum can be performed. Lastly a
dynamic test or rotor dunk test is performed (rotor should be precooled or
preheated to avoid testing delay). The rotor and its contents must be
allowed
to equilibrate for up to 3 hours or more. When set temperature equals
indicated
temperature at the instrument interface, the rotor temperature is then
checked
with a calibrated external temperature sensing device to verify accuracy.
If the checks fall out of specification then appropriate troubleshooting
is
required to isolate the electronic or mechanical fault in the temperature
control or vacuum system. Once the fault is corrected the temperature
control
checks are performed again.
Quote from Bob Giebeler, Analytical Ultracentrifugation in Biochemistry
and Polymer Science, 1992,16-25 for the Optima XLA/I.
"Temperature control is considerably more stable, provides more rapid
cool-down
and heat-up rates, is thermally more uniform, and has equivalent
accuracy as
compared to previous models including the Model E. This control system
uses an
isothermal radiometer temperature-sensing system to sense the temperature
of
the
rotor that is emissivity-independent ad view factor-corrected in
software.
Heating and cooling of the rotor are accomplished by the refrigeration
can
that
surrounds the rotor, which is in turn heated and cooled by
thermoelectric
modules. This environment is very isothermal, and at equilibrium,
irrespective
of speed or temperature, rotor temperature is within about one degree
of
the
refrigeration can temperature.
The control system that regulates rotor temperature, as monitored
by
the
radiometer, is highly software-intensive. This software encompasses
triple
proportional-integral-differential control algorithms and
proportional-integral
smoothing algorithms. In addition, radiometer view factors are measured
during
rotor cool-down to allow more rapid rotor cool-down and more
accurate
temperature monitoring during cool-down. While at equilibrium,
refrigeration
can temperature fluctuation does not typically exceed +0.5C,
and
the
corresponding rotor temperature fluctuation is less than +0.2C.
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</pre>
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<pre wrap=""><!---->
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<pre class="moz-signature" cols="72">--
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX: 603-862-0031
E-mail: <a class="moz-txt-link-abbreviated" href="mailto:Tom.Laue@unh.edu">Tom.Laue@unh.edu</a>
<a class="moz-txt-link-abbreviated" href="http://www.bitc.unh.edu">www.bitc.unh.edu</a>
<a class="moz-txt-link-abbreviated" href="http://www.camis.unh.edu">www.camis.unh.edu</a></pre>
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