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Spiking Armstrong's '99 Samples

Mon, 04/06/2009 - 2:32am by Andy Shen

In my interview with Michael Ashenden we discussed the improbability of Lance Armstrong's '99 Tour samples being spiked. In an effort to cross all our t's and dot our i's I followed up with him to completely eliminate the possibility of foul play. First we have to assume that a lab worker has somehow accessed the UCI's files and figured out which samples belong to Armstrong. This saboteur now has to spike these samples so they fit a profile of an athlete who is injecting EPO every few days, where the % of isoforms spike up and then tail off gradually. I asked Dr. Ashenden how much urine was in each test sample, and how much EPO would be needed to spike those samples to a precise %. Here's his answer:

That's an interesting angle - I'll do my best to explain.

Based on the doping control forms, which report the volume of urine collected, all his samples were 120-125 ml except for Prologue (75ml) and Stage 1 (115ml). From that, the lab takes 20ml for the EPO test.

The urine is placed in a cup that has a special filter attached. The cup is placed in a centrifuge and high molecular weight material is retained on the filter. This step is repeated a second time, and the final retentate (it is a liquid!) left on the filter will contain the EPO.

The concentration of EPO in the urine is measured by immunoassay. The concentration of EPO in the retentate is adjusted to an optimal value (no adjustment if the concentration is low, but adjustments made if the concentration is too high). Twenty MICROLITRES of the retentate are applied to the gel (1 litre = 1 million microlitres).

I've reattached the spreadsheet with an additional column, that shows the concentration of EPO in the retentate corresponding with each sample. You can see the value is quite variable, and does NOT correspond with the percentage of basic isoforms. (See table below)

To answer your question - how much EPO would you spike to vary between 100% and 89.7%? I honestly don't know how you could even attempt such a process...but I will give it a shot just to illustrate.

For the Prologue sample with 100% isoforms, the easy explanation would be that you would 'drown' the sample in EPO to ensure that it was 100%. Note - this is not failsafe, because compare sample 16 July (906 UI/L and 95.2%) against Prologue (600UI/L and 100%) and you see there is no correlation between EPO concentration and % isoforms!!!

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 (so 16 July says that 95.2% of the EPO in that sample was in the basic region and only 4.8% in the 'natural' region). So when you leave the realm of getting 100% basic isoforms, the amount of synthetic you would have to add must be in relation to the amount of natural EPO already present.

So let's use 4 July as a test case: you know you have 20 uL of retentate with an EPO concentration of 210 UI/L. So in 20uL of that sample there is 210 divided by 50,000 = 0.0042 UI of EPO in that 20 uL. Let's approximate and say that we need 89.7% of that to be synthetic - which is 0.0037674 UI. (Just to explain: UI stands for 'units international' which is the standard way to report EPO levels).

The concentration of commercial EPO is around 20,000 UI per 0.6ml of injection. So if you wanted to add 0.0037674 UI you would add 0.000000113 ml (i.e., 0.6ml divided by 20,000/0.0037674 = 0.000000113 ml) of the synthetic EPO solution. This is 0.113 microlitres.

Let's now try and manipulate that same sample to arrive at 95% basic isoforms. You would need .95 x 0.0042 UI = 0.00399 UI. At a concentration of 20,000UI per 0.6ml you would need 0.000000126 ml. This is 0.126 microlitres.

So 0.000000113 ml gives you 89.7% and 0.000000126 ml gives you 95%. Good luck finding a pipette able to get down to that level of accuracy so you could inject the corresponding amount.... Remember 1 microlitre is one millionth of a litre, so you want to get your accuracy down to less than 1 ten millionth of a litre.

And perhaps most importantly - remember that the smallest overdose in this context and the synthetic EPO will drown out the endogenous and you will get 100% basic isoforms (because literally their intensity will drown out any of the endogenous EPO appearing).

Andy I am honestly doing my best here - but I am not confident I am correct! I hate very low numbers and the quantities we are talking here are minute. But I am pretty confident my logic is correct, if not my maths. Do you know a chemist or pharmacologist who you could run the figures past???

So to summarize, this lab technician would have to centrifuge the sample and run a test to measure the EPO concentration. Then he'd have to calculate the percentage he was shooting for and spike the sample with that amount. From one sample to the next he'd need to be accurate to approximately .126-.113uL, or .013uL. Just for reference, a drop of water is 50 uL, so this is .013x.02=.00026 of a drop of water. So unless he has a pipette that can spike one sample with .013uL more of EPO from one sample to the next, he won't be able to replicate that pattern of EPO leaving the body from one day to the next. Dr. Ashenden is not aware of any pipettes with that degree of accuracy. If there are any readers out there with any expertise on this matter, we'd love to hear from you.

 

Stage

Vial #

Visual

Interpretation

% Isoforms

EPO rententate

UI/L

Prologue

160297

+

100

600

1

157372

+

89.7

210

2-7

 

 

 

 

8

186584

+

To be reanalyzed

1470

Rest day

 

 

 

 

9

185557

+

96.6

265

10

185479

+

88.7

268

11

185476

 

Sample missing

 

12

185475

+

95.2

906

13

185895

+

Weak intensity, no % recorded

<125

14

186397

+

89.4

?

Rest day

 

 

 

 

15-20

 

 

Undetectable, insufficient EPO in urine

 

 

 

 

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Silly
By: Matteo Pulley
Sat, 08/25/2012 - 12:09am

The idea that it would be difficult to add a minute amount is silly, if not misleading. It doesn't take a lot of creativity to see that one could add one drop to a large container of water, then extract one drop from that weakened solution to achieve the levels of concentration that you're looking for.

I've taken one year of college level chemistry and I have a BS in aerospace engineering.

Serial dilutions
By: Ex lab tech
Thu, 06/11/2009 - 1:40pm

"I'm neither an exercise physiologist nor a pharmacologist nor a chemist, but this looks pretty easy to fake. Couldn't you just dilute with 10L-20L Normal Saline?"

You're right, serial dilutions to get something into a range where you can pipette accurately is a very common technique, which you learn as an undergrad in just about any lab science pretty much on day 1.

C1V1=C2V2 :)

It'd be technically possible to spike samples in this way. It still seems pretty far fetched though, they'd have to be exceptionally underhand and devious for the whole lab not to figure something is up.

simpler explanation?
By: Wheelsucker
Fri, 04/10/2009 - 12:45am

Dude, I'm pretty sure I just spent a lot of time trying to explain that showing a decrease in isoforms over time really wouldn't be that complicated. Especially uncomplicated if you're not looking to hit specific percentages of isoforms... just lower in general.

-Peter

You're correct that the
By: Wheelsucker
Fri, 04/10/2009 - 12:44am

You're correct that the pipetting might get too difficult if you're looking to hit these exact figures. However, the issue is not that you're looking to get to exactly 87% isoforms (or whatever the number is).. It's getting less isoforms than before to simulate a decrease in isoforms over time. All you have to do is pipette a little less, and that's not that hard.

I do think the 20 minute shower was odd. He probably should have waited to shower because of the payout matrix:

The guy is legit, and he doesn't shower: all good (unless he's doped)
The guy is legit, and he goes to take a shower: violation
The guy is not legit, and he doesn't shower: he's smelly
The guy is not legit, and he goes to shower: he's not smelly

It's not hard to see that you probably shouldn't shower if you have nothing to hide. However, we all know the feeling of being pestered by someone at the end of a hard 5 hour ride. The brain doesn't always work right. Very odd indeed.

Haha. Who am I? I did sign my name at the bottom of my posts...

-Peter

Lance's Response in regard to 18:07
By: Wheelsucker
Thu, 04/09/2009 - 10:02pm
old thread but simpler explanation...
By: Wheelsucker
Thu, 04/09/2009 - 2:11pm

...so this states that spiking Lance's samples would have been too tough to accomplish. What if the cuplrit, rather than spiking samples, simply swapped the labels with that of another cyclist who is a known doper. If the labels are coded to keep things anonymous then this would have been easier as they would have only had to swap the codes in the database or wherever they are listed.
Do they (Can they) DNA test the urine to confirm that it came from Lance?

glad we're back on track now
By: Wheelsucker
Wed, 04/08/2009 - 2:07pm

glad we're back on track now

Gimme Paw
By: Wheelsucker
Wed, 04/08/2009 - 1:17pm

We've gone plaid!

I see your Schmaltz is as
By: Wheelsucker
Wed, 04/08/2009 - 1:14pm

I see your Schmaltz is as big as mine. Let's see how well you handle it.

Prepare ship for ludicrous
By: Wheelsucker
Wed, 04/08/2009 - 1:13pm

Prepare ship for ludicrous speed! Fasten all seatbelts, seal all entrances and exits, close all shops in the mall, cancel the three ring circus, secure all animals in the zoo!

schwartz?
By: Wheelsucker
Wed, 04/08/2009 - 1:12pm

may the schmalz be with you!
also, may the schlitz be with you, too.

may the schwartz be with
By: Wheelsucker
Wed, 04/08/2009 - 1:11pm

may the schwartz be with you.

spaceballs quote, real
By: Wheelsucker
Wed, 04/08/2009 - 12:51pm

spaceballs quote, real classy bro

I am your father's brother's
By: Wheelsucker
Wed, 04/08/2009 - 12:50pm

I am your father's brother's nephew's cousin's former roommate.

"I'm neither an exercise
By: Wheelsucker
Wed, 04/08/2009 - 12:43pm

"I'm neither an exercise physiologist nor a pharmacologist nor a chemist, but this looks pretty easy to fake. Couldn't you just dilute with 10L-20L Normal Saline?"

Then who are you?

Activia does a body good.
By: Wheelsucker
Tue, 04/07/2009 - 11:32pm

Activia does a body good.

You wouldn't even have to
By: Wheelsucker
Tue, 04/07/2009 - 11:09pm

You wouldn't even have to dilute by 10 or 20L. Just take out a 1µl of stock EPO and dilute x1000 with PBS and now you are within range of using a p2 or even a p20 pipettor. I still think that the error in pipetting would still be significant enough to make this 'spiking' difficult.

"you don't think"
By: Wheelsucker
Tue, 04/07/2009 - 4:17pm

You're entitled to your opinion, and I'm not disagreeing that there is a high-ish probability that he doped. However, it doesn't seem like there's ponderously more against Armstrong than say... DiLuca, Simoni, Moreau, or even LeMond.

They were "probably still secret." There's no way to know. That's like saying positives are never known to the press after the A-sample is tested. Oh wait... the press always gets that info. It doesn't seem that secrecy is a very high priority.

There are many explanations. Without a positive or a bag of blood sitting in a dope-doctors refrigerator... it's tough to get anywhere in these discussions.

-Peter

If by cracking the UCI code
By: Andy Shen
Tue, 04/07/2009 - 2:10am

If by cracking the UCI code you mean Ressiot getting the numbers, Armstrong himself had to agree to release those, so unless he released them more than once they were probably still a secret at the time of the testing. This doesn't rule out some other foul play, of course.

I wasn't cautious enough while writing this. Ashenden in the message above and subsequent follow up emails was more careful, I should've followed his example.

I still don't think it was tampering, though.

The simplest solution
By: Wheelsucker
Tue, 04/07/2009 - 1:34am

Andy, it is true that oftentimes the simplest solution is the right one, and I'm also not saying that Lance was clean. However, I am chuckling at the dismissal of "spiking" as either reasonably beyond the capabilities of a saboteur or requiring such an intense utilization of resources that it would be prohibitively expensive to undertake such an operation. It's actually quite easy. Apparently, cracking the UCI code is also easy.

Sometimes things are complicated.

Anyway, non-repeatable results are always viewed very skeptically in science. There isn't enough urine to retest it, right? If he's doping, you just have to trust that he'll be caught given the intensity of the efforts to catch him. We can all cry about how he was "probably" doped (1999-2005), but there's nothing we can do about it now. It's in the past. Let's move forward and try to catch the dopers racing right now (including Lance if he's doped).

Look what happens if we start digging in the past:
I'm guessing LeMond was on anabolic steroids, insulin, cortisone, and homologous transfusions... possibly EPO. After all, he was on the infamous PDM team. On top of that, the 58 second beating he handed to Fignon in a 25k TT (an almost-admitted doper) was a little too miraculous. For Lemond to win against dope machines while clean would be the (2nd) greatest comeback in history or the greatest fraud. In that Tour, 7 of the top 10 were related in some way to some kind of doping scandal. Are we sure he has mitochondrial myopathy? Are we sure it isn't... steroid myopathy... or nothing at all?

-Peter

Nice. Believe it or not, one
By: Andy Shen
Mon, 04/06/2009 - 11:49pm

Nice. Believe it or not, one of MA's follow up emails mentioned the possibility of diluting, but I didn't include it as I thought I was already going too far afield. I also received another scenario today, where you just use someone else's samples, or even inject yourself in that pattern and substitute the urine. But as always, go for the simple explanation, which points back to LA.

All this of course, still assumes that you've cracked the UCI code.

Are you sure it would be that difficult?
By: Wheelsucker
Mon, 04/06/2009 - 11:19pm

I'm neither an exercise physiologist nor a pharmacologist nor a chemist, but this looks pretty easy to fake. Couldn't you just dilute with 10L-20L Normal Saline?

Math:
20,000 UI per 0.6 microliter injection.

Dilute with 20L NS. 20,000 UI per 20L NS (0.6 microliters of original fluid is negligible). Don't forget to stir to homogenize the mixture.

The resultant mixture now has a concentration of 1,000 UI/L instead of 33,333 UI/L (original concentration). Interesting, eh? Given that you work in a lab if you're doing this, you would have no problem testing the concentration to make sure it's good. After all, that is your job. You toss it all out except for 1L making sure that it's the right concentration and then start pipetting:

(0.0042 UI) / (1000 UI/L) = 4.2 microliters
(0.0040 UI) / (1000 UI/L) = 4.0 microliters (~95% isoforms)
(0.0038 UI) / (1000 UI/L) = 3.8 microliters (~89.7% isoforms)

Micropipettes regularly measure 0.2-2 microliters.

I imagine a lab tech would have no trouble doing this. Normal Saline isn't exactly rare.

Armstrong tests positive for bad attitude
By: Wheelsucker
Mon, 04/06/2009 - 10:07pm

PARIS (AFP) — A report has been compiled on the behaviour of American cycling legend Lance Armstrong during a recent out of competition drug test, the French Anti-doping Agency (AFLD) announced on Monday.

AFLD president Pierre Bordry revealed that he had sent the report to the International Cycling Union (UCI) and the World Anti-Doping Agency (WADA) on March 30. He did not reveal the report's contents.

The AFLD sent a sample-taker to test Armstrong after a training session at Saint-Jean-Cap-Ferrat on the Cote d'Azur on March 17. The official took a sample of urine, blood and other bodily matter.

According to the AFLD, the sample-taker warned Armstrong that he would compile a report about his attitude.

"The UCI does not have jurisdiction to judge this case," said UCI press officer Enrico Carpani, referring to articles nine and 13 of the organisation's anti-doping legislation.

"As it concerns a test carried out by a national agency that happened outside of competition, it's the agency which has the authority."

The AFLD will await a response from the UCI before deciding whether or not Armstrong's behaviour constitutes an infringement of the world anti-doping code.

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