Image of rhEPO


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Image of rhEPO

Postby Snation » Mon Sep 11, 2006 6:48 pm

If I can get this to work, I have an image of the electrophoresis of rhEPO. To be honest I can't tell some of what the hell they are talking about in the caption.

This is from a series of reports just days old in the journal Blood. The first report suggests that there are false positives in the rhEPO prcedure. Two groups oppose the view, and one is Don Catlin's group. The other is Lasne's group, the group that first published the test. You are looking at an image from the lab that devised the rhEPO test.

I suppose if we took a couple of hours we all could figure it out. However, my point is this: they are not looking at numbers, they are reading the results of a pharmacological lab procedure.


Image

Figure 1. Epo profiles obtained by double-blotting. Panel A is reproduced from Beullens et al.1(Fig1A), and panels B and C are results from our laboratory. (A) Lane 1 shows epoetin beta; lane 2, darbepoetin {alpha}; lane 3, urine sample considered as "false positive" (the arrow shows a white hole corresponding to ineffective transfer of proteins in this area); and lane 4, the same sample as in lane 3 1 hour later. (B) Lane 5 shows a mixture of epoetin beta and darbepoetin {alpha}; lane 6, natural urinary Epo from a sample taken after strenuous exercise (note some shift toward the cathode of the banding pattern); lane 7, natural urinary Epo; lane 8, urine sample showing the protein (P) unrelated to Epo outside the window of integration (dotted box) used for interpretation of an antidoping control result; and lane 9, urine sample in case of epoetin administration (note the difference with the natural urinary Epo pattern even in the case of the post-strenuous exercise sample). (C) Two-dimensional electrophoresis of a urine sample showing both Epo and protein P.
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Postby 26mi235 » Mon Sep 11, 2006 7:28 pm

Here are the comments of Floyd Landis' attorney. [My understanding of the roles of this board are to keep the doping issues to those of track and field. However, since this is a question about a test that, like the EPO test, is not completely cut and dried, it seems relevant]

http://www.cyclingnews.com/news.php?id= ... /sep12news

"WADA's own protocols require that all testosterone metabolite differentials provide clear evidence of testosterone usage to find an athlete positive. Given the data, three of the four testosterone metabolite differentials tested in Landis' sample are reported as negative considering the margin of error.

The only testosterone metabolite that can be argued as positive under the WADA Positivity Criteria resulted from an unknown laboratory error and is not the result of testosterone usage.

The one metabolite that has been identified by WADA-accredited laboratories as the best, and longest-term indicator, of exogenous testosterone usage was reported as negative in Landis' urine samples."
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Postby Snation » Mon Sep 11, 2006 8:05 pm

When you read the lawyer's statements, what do they sound like? A defense attorney.

That's what the deal is now - forensic labs. So the attorneys are going to question every step and fight every measure.

Jacobs is talking about lab errorsand 'wrongful death' suits. Stuff of litigation lawyers.

This is the same Howard Jacobs who helped Jones. He also defended Tim Montgomery, and Tyler Hamilton.

Jacobs is a one man wrecking crew right now. He is a dfense attorney, but he is on a crusade to blow up every lab test that adversly affects an athlete.
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Postby JRM » Tue Sep 12, 2006 7:27 am

Is this test similar to DNA (gel) electrophoresis, or is it a mass spectrometer readout? Or is it something different?
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Postby Snation » Tue Sep 12, 2006 7:50 am

Here is one description: The adopted test is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and recombinant human Epo.

And again:

"The electrophoretic technique is based on the observation that endogenous EPO generally has a greater negative charge than rhEPO"

I am not an expert on this test, but I will double check it with lab buddies. It is part electrophoresis (the focusing part). It is not a mass spect readout.

EPO and rhEPO are migrated using electrophoresis. Then antibodies to proteins are used to create images (the bands you see above which is the immunoblotting). Because the glycoproteins differ between rhEPO and EPO, there is seperation.

A link on doubt of the test: http://blogs.nature.com/news/blog/2006/ ... blood.html
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Postby Daisy » Tue Sep 12, 2006 2:13 pm

JRM wrote:Is this test similar to DNA (gel) electrophoresis, or is it a mass spectrometer readout? Or is it something different?

Something different.

There are two different ways (actually more, but let's keep it simple) to separate proteins using electrophoresis.

Isoelectric focusing (IEF) separates based on charge.

SDS-PAGE electrophoresis separates based on size.

DNA electrophoresis uses size based separation, however, the EPO test described here is using IEF.

As simply as possible, the IEF is achieved by setting up a pH gradient along an electrical field. All proteins have a charge that is dependant on the pH (acidity) of the solution. Consequently, in an electric field the protein will have a tendency to move towards the anode or cathode depending on its charge.

The trick in the IEF experiment is that as the protein moves towards the cathode or anode the pH is variable. At a certain point between the cathode and anode the charge of the protein becomes zero. At this point the protein stops moving in the electric field (i.e. it becomes focused at one point as a band).

Each band seen in the samples represent a subtle variant of the EPO that can be distinguished by the difference in charge (detected and visualised with an antibody). The synthetic EPO (recombinant) has a different charge compared to the natural EPO (endogenous). This is due to differences in the patterns of sugars that decorate the protein (known as glycosylation).

I have taken the figure from the original paper by Lanse and de Ceaurriz that was published in Nature (405, 8 June 2000, page 635) and labelled it to make it easier to interpret.
Image
Last edited by Daisy on Tue Sep 12, 2006 6:54 pm, edited 1 time in total.
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Postby JRM » Tue Sep 12, 2006 6:05 pm

Ah, excellent description. I wasn't aware of the process, but now you've given me a new example to use in my "Electromagnetism for Pre-Med" course! Thanks, Daisy!
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Postby El Toro » Tue Sep 12, 2006 8:14 pm

JRM, Daisy's invoice for the consultancy fee will be in the mail tomorrow.....
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Postby Snation » Tue Sep 12, 2006 9:11 pm

Nice post Daisy!

Can you comment on the double blot part of the rhEPO test?
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Postby Daisy » Tue Sep 12, 2006 10:14 pm

Snation wrote:This is from a series of reports just days old in the journal Blood. The first report suggests that there are false positives in the rhEPO prcedure. Two groups oppose the view, and one is Don Catlin's group. The other is Lasne's group, the group that first published the test. You are looking at an image from the lab that devised the rhEPO test.Image


After reading a few papers in this area it appears you have stumbled across an interesting case. The papers you found in Blood represent the responses to a paper published in February.
This paper was a direct result of research done on the Belgian triathlete, Rutger Beke, after he failed an EPO test, both A and B sample. Yet, the data in this paper caused his ban to be overturned. Their claim is that Beke's body can produce a 'false'-positive test after intense activity (phenomenon known as post-exercise proteinuria).

What you found in Blood, that is hot off the press, are scathing (almost sarcastic) criticism from both Catlin (who runs a testing lab) and Lasne (who developed the test as published in Nature (2000), see my post above) of the Beullens et al. paper. The figure above is from Lasne comparing the quality of Beullens results (panel A) compared with an example from a standard test (panel B). Lasne clearly states that since Beullens results had no controls and the quality was so bad their conclusions were invalid. I have read the paper and I have to agree with Lasne's criticism.

I had not realised but this case actually caused WADA to review their procedure carefully and release a statement confirming that their tests were valid.
I don't know the details of why this athlete managed to overturn his ban. However, I suspect it is due to this scientific study having some weight among lawyers and suits who did not fully understand the data. According to reports below they did get an outside opinion from a Professor Bogaerts, of the University of Leuven, but I do not know his area of expertise. Judging from the quality of the experiments and data presented, I am surprised this paper made it through peer review. It is no wonder that Catlins response was so sarcastic.

The precedent set here is quite interesting from two perspectives. First, lawyers will now be able to challenge a positive test for EPO using the "Beke exception". But more interesting, if Beke's physiology really is prone to giving a positive to EPO he should test positive after every competition. Time will tell if he is really an anomaly or whether he was saved by scientific 'experts' that were not that good at doing an EPO test.

If I have time I might be able to put together my commentary of the data set as I have read in the papers concerned. For sources with respect to the Beke story read the three below.






    Rutger Beke's Positive EPO Test
    Eric Schwartz (duathlon) on October 26, 2004
    http://www.duathlon.com/articles/3675

    Rutger Beke tested positive for EPO. He was reported to have tested positive for EPO with his A and B samples at a triathlon in Belgium on September 1.

    Beke maintains his innocence and vows to fight the case. According to McCrary, Beke is “absolutely devastated by this. He is confused. He is depressed. He knows for a fact that he has not used any banned substance.”

    [Scientists]..... will study Rutgers biological makeup to help determine how he could have produced a positive EPO test when in fact he has never used any such product. In fact, Rutger has voluntarily agreed to be followed and tested for as long as it takes to understand how his body could have produced this type of result.

    RUTGER BEKE CLEARED OF DOPING CHARGE
    8/10/2005,
    http://www.katalystmultisport.com/news.php?p=20

    Over the last 10 months Beke compiled a team of scientists and medical professionals to help understand why he would have tested positive for EPO when in fact he had never used any performance enhancing substances. During the appeals process, the scientific evidence was presented and eventually led the Commissions independent scientific expert, professor Bogaerts of the University of Leuven, to state that there was no scientific evidence to prove that Rutger Beke had ever taken any banned substances. With that finding, the Commission made a final ruling of not-guilty and Beke can once again pursue his dreams of winning the Ironman World Championships.

    “With the help of scientists, I have proven that I can test positive on the existing WADA EPO-test without ever taking any EPO. This is what happened in Knokke and explains why I have been falsely accused. Even the director of the French Wada-lab (the lab that invented the EPO test) has admitted that I am innocent.

I'm not sure about this claim about the French Wada-lab director admitting he is innocent. It sounds at odds with the WADA statement that came out in Sept last year.

    Testing Times: EPO Examined
    Thursday, March 23, 2006 by James Hewitt
    http://www.pezcyclingnews.com/?pg=fullstory&id=3874

    A number of athletes have questioned the validity of the EPO test but in August last year, Belgian triathlete Rutger Beke had his conviction for EPO use overturned on the basis of a scientific investigation that concluded the he was found to excrete molecules that would yield a positive test due to ‘post-exercise proteinuria'.

    His body excreted more of these proteins naturally and would push the percentage of such proteins over the 80% limit. The next problem is that the antibodies used to identify the EPO proteins are not specific to EPO. They may react with other unrelated proteins in the urine. These questions were raised with WADA who conducted a review of the IOC’s urine test for EPO. They concluded that it “has undergone an extensive scientific validation..." and that "it is a well-established procedure widely accepted by the scientific community.”

    However, in a pre-published study in ‘Blood‚ the Journal of the American Society of Haematology’ on February 21st this year it claims, “these earlier publications missed critical control experiments and were not designed to exclude non-specific false-positive misidentification of other non-EPO urine components”. This is a specific reference to the reaction of EPO antibodies with proteins unrelated to EPO.

    In January 2005, WADA issued a directive to make testing more qualitative. This based the criteria on the bands which form on the special paper onto which the gel is transferred following the reaction with the antibodies. Rather than simply declaring a minimum percentage, at least three bands are identified with the two most intense bands being identified to declare the result. This is determined visually or by a statistical technique called densitometry. This validity of this method is contested by some but research is ongoing into the refinement of the EPO testing protocol.
Last edited by Daisy on Wed Sep 13, 2006 7:20 am, edited 1 time in total.
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Postby Daisy » Tue Sep 12, 2006 10:36 pm

Snation wrote:Nice post Daisy!

Can you comment on the double blot part of the rhEPO test?


I have not had time to find the publication that describes it in detail.
    Double blotting: a solution to the problem of non specific binding of a of a secondary antibodies in immunoblotting procedures.
    Lasne, F. J. Immunol Methods 2003; 276 223-226


However, i think i can guess what they are doing.

After the isoelectric focusing they blot the proteins on to a nylon membrane (western blot). They then probe this with the antibody that is specific for the EPO. It is quite common to then use a so-called secondary antibody to bind to the first antibody (primary antibody) to amplify the signal. It would seem that in this case the secondary antibody is reacting non-specifically with proteins in the urine sample and this makes the test much harder to interpret.

For this reason they have added a second blot (quite an unusual procedure).

1) They do it by western blotting the primary antibodies from the first blot to a second nylon membrane. The conditions allow the movement of the antibodies but the original urine proteins remain on the first blot.

2) In this way the second nylon membrane has the primary antibodies ONLY and their spatial distribution is intact.

3) Now this second blot is probed with the secondary antibodies and since there are no urine protein there should be no non specific binding.


This sounds like a real pain. I expect they are developing a new test, with new EPO antibodies, so they can use a different secondary antibody that does not have non-specific binding.
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Postby EPelle » Tue Sep 12, 2006 10:42 pm

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Search parameter: 2003; 276 223-226 yielded only:

    Lasne F.
    Laboratoire National de Depistage du Dopage, 143 avenue Roger Salengro, 92290, Chatenay-Malabry, France. f.lasne@lndd.com

    "Double-blotting" (DB) has been developed to overcome the problem of nonspecific binding of secondary antibodies in immunoblotting (IB). After it has been probed by the primary antibody, the membrane with the blotted proteins is assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules are thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first one. The second membrane can then be probed by the secondary antibodies without the risk of nonspecific binding. This method has been developed for the study of erythropoietin (EPO) in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical IB protocols. However, its concept makes it usable in other applications that come up against this kind of problem.

    PMID: 12738375 [PubMed - indexed for MEDLINE]


However, can:t seem to open the publication. There are linked references to the right of the article as well.
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Postby Daisy » Tue Sep 12, 2006 10:47 pm

Thanks. i have not tried to find it yet so that helps. I think I should be able to get a hard copyu or even digitial copy tomorrow. 9afternnon for you).

There is a new test deing developed using 2D electrophoresis. You can see an example of that in panel C above.



That will separate on the basis of size and charge. Possibly this will solve the problem of the non-specific binding of the secondary antibody. The non-specific binding would still be present, but would not interfere with the interpretation of the EPO bands.
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Postby eldrick » Wed Sep 13, 2006 12:54 am

EPelle wrote:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed

Search parameter: 2003; 276 223-226 yielded only:

    Lasne F.
    Laboratoire National de Depistage du Dopage, 143 avenue Roger Salengro, 92290, Chatenay-Malabry, France. f.lasne@lndd.com

    "Double-blotting" (DB) has been developed to overcome the problem of nonspecific binding of secondary antibodies in immunoblotting (IB). After it has been probed by the primary antibody, the membrane with the blotted proteins is assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules are thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first one. The second membrane can then be probed by the secondary antibodies without the risk of nonspecific binding. This method has been developed for the study of erythropoietin (EPO) in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical IB protocols However, its concept makes it usable in other applications that come up against this kind of problem.

    PMID: 12738375 [PubMed - indexed for MEDLINE]


this is an admission by the wada team that the monoblot test was non-specific & assumedly unreliable

so the queston is : when was the double-blot test introduced ?

any +ve tests done with the monoblot prior to this therefore have grounds for a challenge

i'm stating the obvious so forgive me ( but it may simplify things for posters not overfamiliar with this ) :

it appears that the belgians are saying that the wada admitted ( lasne guy ) problems with the monoblot haven't been 100% corrected with the double-blot
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Postby Daisy » Wed Sep 13, 2006 1:10 am

eldrick wrote:so the queston is : when was the double-blot test introduced ?

any +ve tests done with the monoblot prior to this therefore have grounds for a challenge

i don't know the timeline but the first paper of the test dates back to 2000. the full protocol was published later. it seems that they never tested with mono blots and may never have been able to get one to work with enough reliability.

There are some interesting stats from catlin with respect to reliability in his Blood response.

He has detected 9 positives; 3 confessed, three accepted bans, 2 maintained their innocence but lost on appeal and one had a doctor who has been indicted for distribution of rhEPO.

These 9 tests are from 6800 samples that have been processed in his lab and 2600 of those were doping control samples from athletes. With respect to the controls he does not say if these were standards or blind.

eldrick wrote:it appears that the belgians are saying that the wada admitted ( lasne guy ) problems with the monoblot haven't been 100% corrected with the double-blot

The non-specific binding of the secondary antibodies is not the problem discussed by the Belgians. They are saying there is non-specific binding of the primary antibody. This lights up another band on the IEF western blot. However, it is quiet convincing from Lasne's reply that the so-called P protein does not interphere with the EPO bands on the IEF. In fact, its pI is not even in the pH range used by the WADA tests. If you look at the 2D gel (panel C, 5 posts above) you can notice the P protein off to the far right. The EPO ladder is to the left.
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Postby Snation » Wed Sep 13, 2006 5:29 am

He said 'stumbled'. I am greviously injured :-)

Yes it is an interesting case. The false positive was reported in Blood as above. The original lab in Belgium (?) and Catlin proteted in this month's journal

I tried reading the reports. Very dense. Very technical.

I even asked my lab buddies; it was tough for them to explain.

So, it is interesting a lawer like Dick Pound is so confident, when medical people struggle with the evaluation of this test.

Daisy what do you think of the overall validity and reliability of the rhEPO test?
Last edited by Snation on Wed Sep 13, 2006 12:23 pm, edited 1 time in total.
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Postby Snation » Wed Sep 13, 2006 5:33 am

The responses from Lasne and Catlin tried to be polite, but they were pointing out perceived deficiencies in the report of the false +.

I would love to be in attendance in a conference where these results are discussed.

I see an anti-doping conference is scheduled for Europe at the end of the month. Anyone going?
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Postby Snation » Wed Sep 13, 2006 5:35 am

May I applaud Daisy again!
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Postby Daisy » Wed Sep 13, 2006 9:48 am

Snation wrote:He said 'stumbled'. I am greviously injured :-)

I meant stumbled into Beke not the EPO data ;) But maybe you had him in your sights too?

Snation wrote:Daisy what do you think of the overall validity and reliability of the rhEPO test?

I think the validity and reliability is probably pretty good. I have summarised all these basic data from Lasne with regard to the non specific binding. (See the compilation figure below).

Remember, you can not compare directly between different experiments, in this case labelled A, B and C. It is possible that comparison within data set C is not good idea too, since I think the lane with the P protein is from a different experiment. This might explain the lower exposure for the EPO bands, nevertheless, the reproducability is quite good between the different publications.

Looking at these data with respect to non-specific binding there are two things to observe.

1) From data set B, the secondary antibodies do bind to the urine proteins causing a real smearing problem. The single blot is completely useless for analysis. The double blot seems to work effectlively at getting getting rid of this background.

2) From data set C, the non specific binding of the EPO antibody (primary) to the P protein does not seem to interfere with the EPO specific bands since it focuses outside the area of analysis (bounded by the two red lines in the figure below).

Even if there were problems with non specific binding the very first acceptance crieteria laid out by WADA for a positive result is:
    "Spots smears areas of excessive background or absent signal in a lane that significantly interferes with application of the identification criteria shall invalidate the lane."
Obviously this is subjective, but, it shows they are trying to run a tight ship. An i assume an athletes lawyer can review the test results so they will be under very close scrutiny if there is a positive. It would be very interesting to see the quality of a typical high throughput test versus these publication quality IEF/DB's.

I'll get back to you with respect to discussing the specific criticisms raised by Beullens et al. Specifically, they discuss the possability of a false positive from two angles; the problem of post-exercise proteinuria and the P protein contaminant.

Image
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Postby Snation » Wed Sep 13, 2006 12:47 pm

Daisy, I corrected my pronoun, man. Sorry.

In relationship to 'stumbled', indeed to did stumble upon this case. I was doing a search on Pubmed for the rhEPO test. I saw that this controversy was pretty fresh (I believe the letters are from the Sept jounral issue).

I was struck with a couple things:
- a medical journal like Blood was publishing papers on rhEPO. I am not a hematologist, but I thought this journal would be looking at anemia, leukemia, etc. etc. Never did I think blood doping articles would be published here.

I also note a fellow like Don Catlin is an endocrinologist, as I recall. He was asked to establish a lab for the Olympics in the USA. He is a solid man, endowed with alot of talent. I bet he never envisioned his academic career would entail chasing drug cheats.

- I was surprised at the repartee between the groups. I am all too well aware of academic politics :-( However I noted in these reports there was a hostile tone.

Why wouldn't the lab with the false positive simply call up Lasne's lab or Catlin's shop, rather than try to publish it in a journal?

I have seen academics get irritated with other; however in this case it is potentially very high profile.

- The reports are very dense to read. One needs a pretty good lab background to understand them.

How do you think a legal challenge is going to go on the rhEPO test?
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Postby eldrick » Wed Sep 13, 2006 1:49 pm

i doubt haematology journals get offered more month-on-month than articles along the lines of :

"effect of varying vincristine doses on aml regression"

or

"new gastric antibodies postulated in aetiology of pernicious anaemia"

controversial articles related to epo must come as a godsend !
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Postby Daisy » Wed Sep 13, 2006 2:16 pm

Snation wrote:Why wouldn't the lab with the false positive simply call up Lasne's lab or Catlin's shop, rather than try to publish it in a journal?

If their data was solid, I would have expected them to submit blind samples from Beke for official testing. Ideally this should include mulitple samples from Beke, pre and post exercise, as well as samples from other athletes taken during the same physical tests. To date, Beke is a complete anomaly, from a physiological perspective, you'd think they would want to prove this beyond doubt. Confirmation of their data from another lab would give them huge credance and be a huge blow for WADA.

Will Beke volunteer for such a study?
Snation wrote:How do you think a legal challenge is going to go on the rhEPO test?

Now that's a question for the Law Dude, assuming he has not already tuned out of this thread?
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Postby eldrick » Wed Sep 13, 2006 2:37 pm

it's worth looking into the:

"exercise proteinuria" angle

i haven't checked this, but normal kidney function doesn't allow proteinuria - usually a sign of nephropathy

if it's an accepted "norm" with exercise ( but even at worst, low molecular weight ones woud be involved - not epo ), then i apologise

otherwise, the belgian tri-ahlete had more serious worries than just an epo test !
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Postby gh » Wed Sep 13, 2006 2:40 pm

Daisy wrote:...Now that's a question for the Law Dude, assuming he has not already tuned out of this thread?


I believe only Dr. Fronkensteen and Eye-gore are left viewing the proceedings.
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Postby eldrick » Wed Sep 13, 2006 2:52 pm

gh wrote:
Daisy wrote:...Now that's a question for the Law Dude, assuming he has not already tuned out of this thread?


I believe only Dr. Fronkensteen and Eye-gore are left viewing the proceedings.


you don't escape that easily !

you qualified as, & will always be a bloody scientist !
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Postby Snation » Wed Sep 13, 2006 4:34 pm

Eldrick, good point. In fact in his letter to the journal, Don Catlin suggested the triathlete has renal problems and should get an eval!!!

Good pickup!
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Postby gh » Wed Sep 13, 2006 5:03 pm

eldrick wrote:
gh wrote:
Daisy wrote:...Now that's a question for the Law Dude, assuming he has not already tuned out of this thread?


I believe only Dr. Fronkensteen and Eye-gore are left viewing the proceedings.


you don't escape that easily !

you qualified as, & will always be a bloody scientist !


Hump, what hump?
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Postby eldrick » Wed Sep 13, 2006 5:11 pm

---
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Postby EPelle » Wed Sep 13, 2006 9:25 pm

Snation wrote:How do you think a legal challenge is going to go on the rhEPO test?


Sometimes an athlete is right on that line between being acquitted or banned for life, and it's up to a scientist to determine whether it's a positive. As Don Catlin, the head of the UCLA lab that analyzed Jones' test, said during a visit to his lab earlier this year, they don't call a test positive when they think the athlete is dirty, they call it positive when they believe they can defend it in court.


http://www.sunherald.com/mld/sunherald/ ... ald_sports
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