Doping inspector backs Armstrong

[I Watch Cycling In July]

I've been trying to determine what the analyte is.... ....If the samples were spiked for a recovery study, the point would be to determine how much was detected per the method. In other word, I know how much I put in, so, how much did I get back.

When an athletes sample is being tested they compare the signal strength in the relative bands with the signal strength with 'spiked' reference samples prepared by the above method...don't they? Rather than going through the whole process of adding different known amounts to several aliquots from the athletes original 120ml, to calculate the concentration?

Don't know what % is. Usually, in analytical testing, it means it's compared to a standard with a known concentration.... .... That's why looking at the results as % is a little frustrating to me, as it could masks what may be going if this were a recovery study.... ....The results for the rEPO (isoforms) are reported as IU/L, but the nEPO is not reported as a concentration.

“The problem is that the IEF test contrasts the amount of synthetic EPO with the amount of endogenous EPO - and expresses the former as a percentage of total EPO in the sample.....” Ashenden

The significance (or otherwise) of rEPO being expressed as a % of total EPO concentration is the somewhat obscure point I have been trying to find out about. Just because it's expressed as a % of total concentration, does not automatically mean that the measurement errors in concentration will carry through to the % rEPO. Superficially that sounds ***. However, if the different isoforms in the sample maintain the same ratios with respect to each other, regardless of the overall proportion of isoforms recovered versus isoforms lost (and why wouldn't they if they're very similar molecules) then the error in % rEPO could be much less than the error in the absolute concentrations. At which point, even trying to calculate how much to spike you sample with becomes tricky....but I'm not sure about the if above, and whether I have the gist of the method though?

Not that this would make spiking impossible of course; a few iterations on any random samples handy would do the job, but might be fairly time consuming and therefore risky ....once the blind had been broken that is.

Based on the comment regarding the adjustment for optimal value, one would expect that at least 40ul of retentate is produced, as the test would need to be re-run......he doesn't mention how he would adjust the sample.

Isn't that just a simple dilution before processing or re-processing? So the total isoforms in bands wont be so high, that individual bands are so dense their relative concentrations can't be measured quantitatively?

 

[RTMcFadden]

When an athletes sample is being tested they compare the signal strength in the relative bands with the signal strength with 'spiked' reference samples prepared by the above method...don't they? Rather than going through the whole process of adding different known amounts to several aliquots from the athletes original 120ml, to calculate the concentration?

For simplicities sake, if you want to continue this discussion, let’s agree on some terminology. Let’s equate signal strength with absorbance, and refer to “spiked” and “unspiked” as reference or control samples (positive and negative). Also, instead of the term ”tester,” let’s call the people who collect the samples “collectors” and the people to run the analytical tests “analysts.”

Don’t’ they? Maybe, maybe not. I don’t know exactly how they obtain their positive and negative reference samples (external standards). As previously pointed out by Python, in the medical labs world, standards are normally supplied by 3rd parties and can be obtained from natural sources (i.e. patients) or manufactured. In the Pharma production world, standards can be supplied by 3rd parties (USP or the like) or internally. In the research world, standards can be supplied by 3rd parties or internally, but the earlier you are in the R&D process, the more likely they are internally supplied. If you’re interested some insight into how reference standards are established, specifically for EPO, take a look at Collaborative study for the establishment of erythropoietin BRP batch 2 (http://lib.bioinfo.pl/pmid:15659283). To give you fair warning, the reference provided is only a entryway into a whole world of discussion, not it’s end point. (It's a hole.)

Comparison? Again, don’t know. The comparison may be a gel thing, or may also be a concentration thing. Remember, in gel electrophoresis (GE), the bands (compounds) move across the gel in relation to the amount of current applied, a function of intensity and time. As such, the reference standards may simply be used to identify where the requisite bands are located, in relation to the unknowns. Of this I have not doubt, it’s standard practice. The use of the standard for concentration I’m less sure of. You have issues of limits of detection (LoD) and limits of quantitation (LoQ). The data indicates that the lower LoQ is 125 UI/L and that the upper LoQ is less than 1470 UI/L but more than 906 UI/L for rEPO. No data is supplied for nEPO.. Without more detail regarding the method (procedure), I can’t really even guess. If anyone is interested in learning more about GE, you can start here http://learn.genetics.utah.edu/content/labs/gel/.

 

[RTMcFadden]

The significance (or otherwise) of rEPO being expressed as a % of total EPO concentration is the somewhat obscure point I have been trying to find out about. Just because it's expressed as a % of total concentration, does not automatically mean that the measurement errors in concentration will carry through to the % rEPO. Superficially that sounds ***. However, if the different isoforms in the sample maintain the same ratios with respect to each other, regardless of the overall proportion of isoforms recovered versus isoforms lost (and why wouldn't they if they're very similar molecules) then the error in % rEPO could be much less than the error in the absolute concentrations. At which point, even trying to calculate how much to spike you sample with becomes tricky....but I'm not sure about the if above, and whether I have the gist of the method though?

Some information regarding the relationship between nEPO and rEPO can be found in The effects of microdose recombinant human erythropoietin regimens in athletes haematologica/the hematology journal | 2006; 91(8) | 1143 | (http://www.haematologica.org/cgi/reprint/91/8/1143.pdf). Ashenden indicated that a paradigm exists “which holds that endogenous erythropoietin production is suppressed when the red cell mass has been increased beyond the homeostatic set point.” To me, this means that there is an inverse relationship between the concentration of nEPO and hemoglobin (Hb). So, the addition of rEPO would raise Hb concentration, thereby decreasing nEPO concentration, which makes physiological sense.

Superficially… Yes it does. I think the whole rEPO:tEPO thing is a their way of establishing criteria in such a way as to provide a very high degree of assurance that the results are true/real. To put it another way, to negate the possibility of a rider being sanctioned based upon false positive. What bothers me is when the results are 100%, was there no nEPO, was it below the detection limit, what’s going on. This is why I’m more interested in the concentration.

 

[RTMcFadden]

Isn't that just a simple dilution before processing or re-processing? So the total isoforms in bands wont be so high, that individual bands are so dense their relative concentrations can't be measured quantitatively?

I would think. It's either because it would wash out the detector or because the band would be so big as to run (bleed) into other bands.

 

[Bag_O_Wallet]

Is this still theorizing about how it might be possible to spike samples?

Again, any suggestion that this actually happend?

 

[RTMcFadden]

Not that this would make spiking impossible of course; a few iterations on any random samples handy would do the job, but might be fairly time consuming and therefore risky ....once the blind had been broken that is.

It’s not simple, but it’s not impossible either. Here’s what I have a problem with. In the discussion regarding the probability of correctly selecting LA’s samples, it was noted by somebody that the testing took place over 20 days. It’s also been established that there were 87 samples. So, 87/20 = 4.35 or 5 samples per day. Digger indicated that there were 4 samples (I think) tested each day. These facts roughly correlate because gels usually have between 8 and 10 slots and two were used for control samples. In addition, I read somewhere that the procedure takes 2.5 days to perform. However, what I glean from this information is that only one gel was prepared each day. So, to me, based upon the procedure, it’s entirely possible that one person could have performed all of this testing with the identified time frame.

 

[Race Radio]

Is this still theorizing about how it might be possible to spike samples?

Again, any suggestion that this actually happend?

None.

It appears a groupie is confusing word count with content again.

 

[RTMcFadden]

Is this still theorizing about how it might be possible to spike samples?

Again, any suggestion that this actually happend?

I think we've moved beyond theorizing about the possibility. I think we've established that it's technically possible and even speculated that it may be done a routine basis (positive reference sample preparation).

Any suggestion is what is left up to each of us. I don't think anyone in this forum has the requisite information to defnitively prove this either way.

 

[RTMcFadden]

None.

It appears a groupie is confusing word count with content again.

There's nothing for you to learn here. Move along.

 

[Bag_O_Wallet]

I think we've moved beyond theorizing about the possibility. I think we've established that it's technically possible and even speculated that it may be done a routine basis (positive reference sample preparation).

Any suggestion is what is left up to each of us. I don't think anyone in this forum has the requisite information to defnitively prove this either way.

Then what's the end game if you can't prove or disprove?

 

[RTMcFadden]

Then what's the end game if you can't prove or disprove?

Information. The conversations has basically dwindled to IWCIJ and myself. He seems interested in the science and asks some good questions. Everyone else seems to have made their mind up.

 

[Bag_O_Wallet]

Information. The conversations has basically dwindled to IWCIJ and myself. He seems interested in the science and asks some good questions. Everyone else seems to have made their mind up.

Cool.

obligatory additional words.

 

[Digger]

Information. The conversations has basically dwindled to IWCIJ and myself. He seems interested in the science and asks some good questions. Everyone else seems to have made their mind up.

'Everyone else' has been asking you questions which you obviously feel afraid to answer. I especially like the question you've repeatedly failed to answer, the one that says if you have any evidence, whatsoever, that the samples were spiked?

 

[Race Radio]

There's nothing for you to learn here. Move along.

You are correct, you bring nothing new to the discussion...... although I am looking forward to your exploration of the space Alien theory.

 

[red_flanders]

I think we've moved beyond theorizing about the possibility. I think we've established that it's technically possible and even speculated that it may be done a routine basis (positive reference sample preparation).

Since the expert in the field completely rejects it's possiblity, I'd say you've got a long, long way to go before you've "established that it's technically possible". You may think it's technically possible, but since no one who actually knows anything about how the labs run the testing or who has developed the test, or any other small pieces of germane information has commented, and in fact their comments on the record totally contradict your claim...I'd say you've got bupkis.

Any suggestion is what is left up to each of us. I don't think anyone in this forum has the requisite information to defnitively prove this either way.

I would agree. So unless someone who actually has the information needed can provide a reason why it's possible and some evidence that it happened, you're just working really, really hard to create a red herring.

"Why?" seems to be the only relevant question.

 

[Mellow Velo]

Information. The conversations has basically dwindled to IWCIJ and myself. He seems interested in the science and asks some good questions. Everyone else seems to have made their mind up.

Maybe it's because.....

There's nothing for you to learn here. Move along.

I guess everyone has taken your advice.
Either that, or a lot of posters have you on ignore.

Or maybe folks can appreciate just how much you like proving your theories to yourself.
Just don't play with yourself, too much.

 

[Anonymous]

I think we've moved beyond theorizing about the possibility. I think we've established that it's technically possible and even speculated that it may be done a routine basis (positive reference sample preparation).

Any suggestion is what is left up to each of us. I don't think anyone in this forum has the requisite information to defnitively prove this either way.

Again, simple point of argument in this instance: In this case it is impossible to prove a negative. It is therefore your responsibility to prove otherwise. But that is not the point of your post. You are here producing propaganda. The stupid thing is that it fools nobody, and your efforts are transparent and fruitless. But by all means, keep up the bad work!

 

[Anonymous]

There's nothing for you to learn here. Move along.

There is nothing for you to teach here. Take your own advice.

 

[Anonymous]

You are correct, you bring nothing new to the discussion...... although I am looking forward to your exploration of the space Alien theory.

It was Nazi Frogmen, get your facts straight!

 

[Dr. Maserati]

Information. The conversations has basically dwindled to IWCIJ and myself. He seems interested in the science and asks some good questions. Everyone else seems to have made their mind up.

The difficulty is that you are not basing your information on facts but theories.
The Vrijman report clearly mentions that spiking was done on some samples contributed by volunteers and staff. The 98 & 99 samples were used to help meet the numbers needed to have an effective database.

I will happily admit I know little about gels - except that i like the Gu Strawberry and Banana.

 

[I Watch Cycling In July]

Then what's the end game if you can't prove or disprove?

Understanding the testing processes better.

 

[CentralCaliBike]

There is nothing for you to teach here. Take your own advice.

The only thing I wonder about when I see something like this is it seems like there is only value is giving information that would conclude LA is, and has always doped, any information that disagrees with that position seems to have no value on this forum to a majority of the posters. The only problem with that is science and law tend to want to look at both sides and not be stuck with one. While I personally doubt the 1999 samples were spiked it is interesting to see how it could be done and that has some value to me (even if it is not a likely scenario).

 

[red_flanders]

The only thing I wonder about when I see something like this is it seems like there is only value is giving information that would conclude LA is, and has always doped, any information that disagrees with that position seems to have no value on this forum to a majority of the posters. The only problem with that is science and law tend to want to look at both sides and not be stuck with one. While I personally doubt the 1999 samples were spiked it is interesting to see how it could be done and that has some value to me (even if it is not a likely scenario).

Most people on this forum have spent years if not decades looking at both sides. At some point, one reaches inescapable conclusions based on overwhelming, repetitive evidence.

Because many have come to a conclusion does not mean they were pre-disposed to do so. I've been watching cycling since the mid-seventies on my uncle's knee spending summers in Beligium. I've seen a LOT. Even so, I was initially predisposed to think Lance clean in the first year or two of his return.

An avalanche of evidence and common sense ended that a long time ago. It does NOT mean I didn't look at both sides for a long time. Give your fellow posters some credit. They object because you are hammering on red herrings. It helps perpetuate the myth if left unchallenged. It becomes tiresome as an intellectual exercise with no evidence to back it up.

 

[CentralCaliBike]

Most people on this forum have spent years if not decades looking at both sides. At some point, one reaches inescapable conclusions based on overwhelming, repetitive evidence.

Because many have come to a conclusion does not mean they were pre-disposed to do so. I've been watching cycling since the mid-seventies on my uncle's knee spending summers in Beligium. I've seen a LOT. Even so, I was initially predisposed to think Lance clean in the first year or two of his return.

An avalanche of evidence and common sense ended that a long time ago. It does NOT mean I didn't look at both sides for a long time. Give your fellow posters some credit.

So, since you looked at both sides and have decided on one - any others coming in should not bother?

 

[python]

So, since you looked at both sides and have decided on one - any others coming in should not bother?

this is a cheap red herring mister. for some one pandering about justice and never picking up a single factual argument despite the multiple opportunities it's actually a low blow. you deliberately misinterpreted a rather simple statement. that makes you in my book a bs artist. you continue to disregard the evidence and instead of discussing it you essentially obfuscating the issue.