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<TITLE>Re: [RASMB] standard tests; should ABRF be involved?</TITLE>
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<BLOCKQUOTE>John -<BR>
<BR>
You raise some very sound points here. The idea was that we would all be using the same procedure, right down to temperature equilibration times, software used*, mode of use of software (e.g. we all float the meniscus position, within common limits, same resolution levels etc), optical system (probably absorption optics) - and anything else which is capable of being fixed.<BR>
<BR>
As regards the problem labelled 'lastly', Neil Errington and I {N Errington, A J Rowe (2003) "Probing conformation and conformational change in proteins is optimally undertaken in relative mode" European Biophysics Journal 32 (5) 511-517} <I>have </I> actually done a series of experiments over some years, with the same sample, triplicated cells each time (a commercial QA job, obviously!). The sample was far from ideal - an engineered vaccine with a significant spread of size values ore or less normally distributed around a mean (EM studies). Even so, an average precision of ±0.37% precision was attained (SD around a normalised mean). Less extensive trials with better defined systems suggest lower values can be achieved. As Neil and I also noted (loc cit) operator to operator consistency is also an issue, but the uncertainty which we gave there (±0.3%) was based upon non-standardised operator usage - interesting enough in its own right, but things ought to be much better if everyone follows the same procedure.<BR>
<BR>
Arthur<BR>
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<BLOCKQUOTE><FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">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.<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">The problem is to distinguish real differences in temperature from other effects that can change the values of the sedimentation coefficient:<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">(1) systematic errors due to differences in the software used to evaluate the data;<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">(2) systematic errors due to how the meniscus position is defined (this is a bigger problem than most people realize);<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">(3) potential systematic differences between absorbance and interference data (has anyone really looked at this carefully?);<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">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!).<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">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. <BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">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.<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">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.<BR>
</FONT></FONT></FONT> <BR>
<FONT COLOR="#0000FF"><FONT SIZE="2"><FONT FACE="Arial">John<BR>
</FONT></FONT></FONT><BLOCKQUOTE><FONT SIZE="2">-----Original Message-----<BR>
<B>From:</B> rasmb-admin@server1.bbri.org [mailto:rasmb-admin@server1.bbri.org] <B>On Behalf Of </B>Arthur Rowe<BR>
<B>Sent:</B> Monday, December 13, 2004 7:47 AM<BR>
<B>To:</B> John Correia; mchien@beckman.com; rasmb@server1.bbri.org<BR>
<B>Subject:</B> Re: [RASMB] Re: XL-A/I temp control<BR>
<BR>
</FONT>Hi Everyone<BR>
<BR>
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. <BR>
<BR>
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 <I>precision </I>(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.<BR>
<BR>
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 . . . .<BR>
<BR>
Regards to all<BR>
<BR>
Arthur<BR>
<BR>
-- <BR>
*************************<BR>
Arthur Rowe<BR>
Lab at Sutton Bonington<BR>
tel: +44 115 951 6156<BR>
fax: +44 115 951 6157<BR>
*************************<BR>
<BLOCKQUOTE><BR>
<B>From: </B>"John Correia" <jcorreia@biochem.umsmed.edu><BR>
<B>Date: </B>Fri, 10 Dec 2004 11:16:43 -0600<BR>
<B>To: </B><mchien@beckman.com>, <arthur.rowe@nottingham.ac.uk>, <rasmb@server1.bbri.org><BR>
<B>Subject: </B>Re: [RASMB] Re: XL-A/I temp control<BR>
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<BLOCKQUOTE><FONT SIZE="2">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. <BR>
</FONT><BR>
<FONT SIZE="2">What matters is not the set point but knowing the actual temperature.<BR>
</FONT><BR>
<FONT SIZE="2">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.<BR>
</FONT><BR>
<FONT SIZE="2">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.) <BR>
</FONT><BR>
<FONT SIZE="2">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. <BR>
</FONT><BR>
<FONT SIZE="2">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".<BR>
</FONT><BR>
<FONT SIZE="2">PS - why Beckman has never joined up with Anton Paar to bundle Density Meters into XLA/XLI quotes amazes me.<BR>
</FONT><BR>
<FONT SIZE="2">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.<BR>
</FONT><BR>
<BR>
<BR>
-------------------------------------------------------------------<BR>
Dr. John J. "Jack" Correia<BR>
Department of Biochemistry<BR>
University of Mississippi Medical Center<BR>
2500 North State Street<BR>
Jackson, MS 39216<BR>
(601) 984-1522 <BR>
fax (601) 984-1501 <BR>
email address: jcorreia@biochem.umsmed.edu <BR>
homepage location: http://biochemistry.umc.edu/correia.html<BR>
dept homepage location: http://biochemistry.umc.edu/<BR>
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>>> Arthur Rowe <arthur.rowe@nottingham.ac.uk> 12/10/04 08:17AM >>><BR>
Hi Everyone <FONT COLOR="#000080">{this is a second (</FONT><FONT COLOR="#FF00FF">now 3rd!</FONT><FONT COLOR="#000080">) try at getting this mail out - first attempt got lost in cyber-space, it seems}<BR>
</FONT><BR>
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 <I>is</I> when it is going round in the rotor at speed.<BR>
<BR>
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 "<FONT COLOR="#FF0000"><TT>to ensure their operation within the published specification".</TT></FONT> . I am trying to get the <U>accuracy</U> of the temperature read-out to be close to the <U>precision</U> 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.<BR>
<BR>
Even the method mentioned (equilibrate for 3 hours - under vacuum - and then check "<FONT COLOR="#FF0000"><TT>with a calibrated external temperature sensing device to verify accuracy"</TT></FONT> 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? <BR>
<BR>
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 <U>as measured by the defined procedure</U>. 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. <BR>
<BR>
<FONT COLOR="#008000"><I>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.<BR>
</I></FONT><BR>
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 <BR>
<BR>
Regards to all (and many thanks to Mei-Ling Chien)<BR>
<BR>
Arthur<BR>
<BR>
-- <BR>
*************************<BR>
Arthur Rowe<BR>
Lab at Sutton Bonington<BR>
tel: +44 115 951 6156<BR>
fax: +44 115 951 6157<BR>
*************************<BR>
<BLOCKQUOTE><BR>
<B>From: </B>mchien@beckman.com<BR>
<B>Date: </B>Fri, 3 Dec 2004 10:11:46 -0800<BR>
<B>To: </B>"'rasmb@rasmb-email.bbri.org'" <rasmb@server1.bbri.org><BR>
<B>Subject: </B>[RASMB] Re: XL-A/I temp control<BR>
<BR>
</BLOCKQUOTE><BR>
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Hi All,<BR>
<BR>
Below is response regarding XL-A/I temperature control from our Technical<BR>
Support Department.<BR>
<BR>
******************************************************<BR>
Mei-Ling Chien PhD<BR>
Staff Development Scientist, Centrifugation<BR>
Platform & Automation Business Center<BR>
Beckman Coulter Inc.<BR>
<BR>
mchien@beckman.com<BR>
(650) 859-1948<BR>
******************************************************<BR>
<BR>
<BR>
The basis for temperature control specifications were instrument design<BR>
specifications for temperature control and dynamic system testing during the<BR>
prototype phase of the product.<BR>
<BR>
If there is a discrepancy in temperature control and measurement between<BR>
instruments of the same design then a dynamic calibration check should be<BR>
performed on both instruments <FONT COLOR="#FF0000">to ensure their operation within the published<BR>
specification.<BR>
</FONT><BR>
First the physical condition of components within the temperature control and<BR>
vacuum system should be verified through inspection. Then an electronic<BR>
calibration for temperature control and vacuum can be performed. Lastly a<BR>
dynamic test or rotor dunk test is performed (rotor should be precooled or<BR>
preheated to avoid testing delay). The rotor and its contents must be allowed<BR>
to equilibrate for up to 3 hours or more. When set temperature equals indicated<BR>
temperature at the instrument interface, the rotor temperature is then checked<BR>
<FONT COLOR="#FF0000">with a calibrated external temperature sensing device to verify accuracy</FONT>.<BR>
<BR>
If the checks fall out of specification then appropriate troubleshooting is<BR>
required to isolate the electronic or mechanical fault in the temperature<BR>
control or vacuum system. Once the fault is corrected the temperature control<BR>
checks are performed again.<BR>
<BR>
Quote from Bob Giebeler, Analytical Ultracentrifugation in Biochemistry<BR>
and Polymer Science, 1992,16-25 for the Optima XLA/I.<BR>
"Temperature control is considerably more stable, provides more rapid cool-down<BR>
and heat-up rates, is thermally more uniform, and has equivalent accuracy as<BR>
compared to previous models including the Model E. This control system uses an<BR>
isothermal radiometer temperature-sensing system to sense the temperature of the<BR>
rotor that is emissivity-independent ad view factor-corrected in software.<BR>
Heating and cooling of the rotor are accomplished by the refrigeration can that<BR>
surrounds the rotor, which is in turn heated and cooled by thermoelectric<BR>
modules. This environment is very isothermal, and at equilibrium, irrespective<BR>
of speed or temperature, rotor temperature is within about one degree of the<BR>
refrigeration can temperature.<BR>
<BR>
<BR>
The control system that regulates rotor temperature, as monitored by the<BR>
radiometer, is highly software-intensive. This software encompasses triple<BR>
proportional-integral-differential control algorithms and proportional-integral<BR>
smoothing algorithms. In addition, radiometer view factors are measured during<BR>
rotor cool-down to allow more rapid rotor cool-down and more accurate<BR>
temperature monitoring during cool-down. While at equilibrium, refrigeration<BR>
can temperature fluctuation does not typically exceed +0.5C, and the<BR>
corresponding rotor temperature fluctuation is less than +0.2C.<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
<BR>
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