The DEHP/Plasticizer Test

Well I decided to make a new thread instead, setting it free of the Contador debate.

This is more for the future of the test, and whether we will see this test coming to fruition and being implemented (and reason alone for a 2 year sanction).

The two key articles are:

January 2010: Monfort et al. Urinary di-(2-ethylhexyl)phthalate metabolites in athletes as screening measure for illicit blood doping: a comparison study with patients receiving blood transfusion. Transfusion

December 2010: Solymos et al. Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing. Analytical and Bioanalytical Chemistry

From Monfort et al:

the concentrations of DEHP metabolites in the urine of transfused subjects was significantly higher than in the control and nontransfused patients groups.
DEHP metabolite concentrations for these subjects were not significantly different from those obtained in the control group
What this means is that not only are concentrations much higher in the tranfused than the control, but also the "nontransfused" group who were in contact with medical products (catheters, IVs etc). This is good news as it means there could be a threshold which clearly draws the line between environmental exposure and a transfusion.

Among athletes, especially high concentrations of DEHP metabolites were found in four samples: one from aquatics, one from cycling, and the other two from soccer (Subjects A, B, C, and D in Table 2). In those samples, concentrations of DEHP metabolites overlapped with some of those obtained in actual patients being transfused.
Cycling n = 46, Rowing n = 22, Swimming n = 18, Soccer n = 41.

Not just cycling!

Recent studies also reflect a steady decrease over recent years of the common exposure to phthalates and this fact further supports that high unexpected concentrations of DEHP metabolites can be easily detected compared with usual urinary background.
The discussion goes on, it mentions that a current problem is that transfusions (homologous) can only be detected in a blood sample (not urine). I think this is an irrelevant point as autologous transfusions are probably a lot more common and they are undetectable in anything - hence that is the bigger problem, not the lack of a urine test.

The appearance of some outliers among athletes (Subjects A, B, C, and D in Table 2) may indicate either a particularly higher environmental exposure than usual or a sudden excretion as a consequence of blood transfusion processes.
"Higher environmental exposure" would have to be very high it seems, when all these concentrations were above the control group. Such environmental exposure doesn't seem like the thing which would happen in the middle of an Olympics or Grand Tour.

-Detection window of 24 to 48 hours.

In our study,medical treatments with PVC devices did not increase the day-long concentrations of DEHP metabolites and, thus, they would not probably explain positive results. Nevertheless, the possibility of other sources of substantial DEHP exposure cannot be completely excluded. For this reason, a close follow-up of any athlete would be needed after any suspicious result of urinary DEHP metabolites concentrations. This follow-up
would include a confirmatory test for allogeneic (or autologous, if the test is available) blood transfusion, and it should also include longitudinal studies to evaluate the normal basal exposure to DEHP of each athlete. The basal
levels of DEHP metabolites may be incorporated as a part of the individual athlete biologic passport, to be able to detect sudden and unexplained increases in DEHP metabolites concentrations in urine that should allow sports authorities to suspect on the misuse of blood transfusions.
Now this is worrying, it's saying that this test alone shouldn't be grounds for a sanction. The whole reason this test sounds promising is because it could be something in transfusions which is easily detectable, with no microdosing or easy to get around thresholds. If you have to fall back on other tests (like EPO, or biopassport) then it's just not going to be major breakthrough.

Compared to other tests that are performed for blood doping detection, such as flow cytometry, the method proposed here is cheaper, less time-consuming, and easy to perform because it is based on liquid chromatography coupled to tandem mass spectrometry technology, nowadays available in all antidoping control laboratories.
From Solymos et al:

100 person control group, 5 person transfusion group (two samples each) and a large doping test athlete group (468).

In comparison to the control group, significantly higher concentrations of the secondary DEHP metabolites were observed in urine samples received after blood transfusion (Fig. 3). The maximum concentrations of 5oxo-MEHP and 5OH-MEHP in the control samples were 18- and 13-fold lower as the minimum concentrations determined after transfusion (Table 4). The 99.9% upper reference values of the control group were determined at 54.4 ng/mL for 5oxo-MEHP and 94.8 ng/mL for 5OHMEHP which are 12- and 9-fold lower than the lowest value measured after blood transfusion.
Note in this study, the 5 patients gave two samples between 0 to 24 hours.

Three out of four outliers were taken out-of competition at the same time originating from one cycling team.
Now I find this confusing. Isn't the idea to use transfusions "in competition" - how many days before or after a race is classified as "out of competition"? Did they go on a training camp near a DEHP factory?

This study also comes to the conclusion that the DEHP test alone is not proof of blood doping.

It also cites other studies which show higher variability amongst the general population - but at most levels they are still below the transfusion concentrations. Also, there is quite a clear distinction between the median concentration for the athletes and the four in the "transfusion zone".

Interesting bit in the conclusion:

Currently, the presented assay covers only DEHP. However, an implementation of alternatively used plasticizers in blood bags or other medical devices seems to be promising, assuming comparable physical properties of used substances. As a future prospect, the applicability of the method on different plasticizers has to be investigated
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So whilst the results seem promising, both studies paint a gloomy picture by suggesting that the tests could not be standalone proof.

To the cynical eye it would seem quite clearly that a high concentration in a cyclist only has one real explanation and that there's no doubt a positive for DEHP metabolites should be enough to sanction.

I wonder what the process is with WADA and how they work out a legally viable implementation of a new test. i.e. if they heed the advice of these studies there's no way it's good enough, but how hard will WADA work to do their own research and legal work to try and perfect the implementation.

Optimistically, this could actually strengthen the bio passport. The passport is a dead duck as far as getting a sanction is concerned, but maybe this is the final piece of the jigsaw which could make the passport hold up in the courts. Any positive for plasticizer in the middle of a GT would be highly suspicious and if it coincided with a high shift in blood values it could just be enough.
 
Aug 13, 2009
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I would doubt the test will ever be stand alone proof, but as part of the Biopassport it is a real tool.
 
Pending seeing the whole Solymos article I can’t comment too much. One would obviously like to see a larger transfusion group than just five subjects, but the control group is substantial, and the maximum and 99.9% values seem to be in rough agreement with those in other studies, IIRC. Bert’s values for one of these metabolites peaked at 480 ng/ml, and a second metabolite peaked at a value of 210 ng/ml., I believe. That is a little lower than the lowest value apparently reported for a transfused subject in this study (about 650 and 900, from the Abstract), which again points to the need for more subjects.

Added later: Going back to the Segura study (the first one in the OP, Monfort is first author), they obtained the values shown below. (ng/ml). The first set is controls at 90th percentile, and agrees reasonably well with the 99.9% values in the Solymos study. However, these values were not cleanly separated from those of all transfused patients. This is shown in the second and third sets,the 25th percentile values for patients 24 hrs and 48 hours after transfusion. That is, one-fourth of these patients had values this low or lower. These data suggest finding a value that eliminates most false positives will result in a large number of false negatives, i.e., transfused subjects whose values are not high enough to separate them clearly from controls.

In the Segura study there were 25 transfused subjects, so this again suggests a larger number of transfused patients is needed to establish a criterion. In fact, the Segura data show that there is enormous variability in the metabolite levels in transfused patients, and another study I posted here last summer reported an equally large variability in these values over time in controls. The Solymos study claims the variability in controls is not that large, but I haven't seen their full data yet. OTOH, they don't have enough transfused subjects to make any claims about the variability in them.

The Seguara study also demonstrates the critical point that the test has to be applied within a couple of days or so of transfusion, as the metabolites are cleared and rapidly approach control values.

Controls (90%)
MEHP 27.1
MEHHP 64.6
MEOHP 75.8

24 hr transfused (25%)
MEHP 51.8
MEHHP 93.7
MEOHP 98.1

48 hr transfused (25%)
MEHP 34.3
MEHHP 26.6
MEOHP 26.9
 
Oct 7, 2010
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I find this very interesting, I like the data and when looking at catching offenders, the more evidence in total that you can amass is far better in my estimation. I think we fail in certain ways because we want a litmus test, a happy blue face if we are pregnant or something. The sophistication of the drugs, masking and the like will rely on multiple factors of detection in the future.

The mere mention of DEHP also brings to mind the ongoing debate about plasticizers in the products we have around the house, and with the BPA scares I searched a bit on a whim.

http://www.livestrong.com/article/166500-side-effects-and-dangers-of-refilling-plastic-bottles/

This link is entirely tongue in cheek, sorry couldnt resist.

http://www.ehhi.org/plastics/avoiding_exposures.shtml

My overall curiosity is with the wide array of products that can raise DEHP levels, how skewed can results be? After all, cyclists are constantly in contact every day with on the bike waterbottles, water containers, plastic wraps, and tupperware type containers for our leftovers. These DEHP particles are more likely to be in urine and show regularly there, rather than in blood tests. Supposedly microwaving these plastics help release the chemicals further. I love science, technology and the like, but I have my doubts that a DEHP test itself is going to be a smoking gun, but more like the gun powder residue left on an assailants hands.
 
Good thread. Thanks for doing so much work Ferminal.

I too think it's going to be a while before this test is used to determine blood doping. But with any luck it won't be long until it's at least used as part of the bio passport.

There's also long-term hope in that at some point in the future the test will be accepted and validated through enough peer review that retroactive testing can take place to catch cheats of the past.
 
Oct 16, 2010
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drummond said:
The WADA validated method for detecting blood transfusions through plasticizers.

http://www.elpais.com/articulo/deportes/plastico/prueba/dopaje/elpepidep/20110110elpepidep_23/Tes
Thanks Drummond,
If I'm not mistaken, this is quite sensational news.
It suggests that the plasticizers in AC's blood are no longer just a rumor, but a fact, and that they might still be used against him in the currently running case.

In any case, great article by El Pais, for breaking the news, and showing that the Spanish press are objectively observing AC's case.
 
Mar 19, 2010
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Last friday I was "sealing" some chicken in the frying pan. The frying pan was new. The Spatula was a black plastic. The spatula actually left stains on the frying pan where it had melted; I ate the chicken and it was delicious.

Also, -this might be genius- wont blood dopers just find some other recipient for the blood, or way of masking the plasticisers?

They could do worse than squander another 7m$ on a test and catch no one like they did with growth hormone; rather than spend 7m$ testing for cardiac defects, bone mineral density and what have you.

I'm afraid I can't offer a solution to catching autologous blood doping, which makes my argument invalid.
 
Dec 18, 2009
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Unless you can find something positive in the blood its virtually impossible to ban someone.

The only way the doping culture will change is when there is a change in culture and whilst that its perpetuated through dodgy DS's and Prat McQuaid nothing will change.
 
Sep 25, 2009
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wait a minute…

didn’t we already establish that the test is too little to late and for all intents and purposes is almost useless...i mean it's of very limited use - like retro testing or catching really stupid ?

I recall reading in this very forum (many months ago in fact !) that as soon as the dopers learn about the plasticizer test they can easily switch to a different type of blood storage containers.

this very forum contained pictures and descriptions of those containers - in existence for decades since the early days of blood banking...

may be we’re losing the fight against dopers because we think they are stupid ?

or is it us who're naive and stupid ?
 
drummond said:
The WADA validated method for detecting blood transfusions through plasticizers.

http://www.elpais.com/articulo/deportes/plastico/prueba/dopaje/elpepidep/20110110elpepidep_23/Tes
Another very interesting link. I'm not sure publication was accelerated for Bert's case, because at the time of submission, the middle of October, or soon after, there was talk of a quick resolution of the case. In any case, once the paper has been accepted and is in press, it's official and can be cited just as any published article can. So from this point of view, there was no need to rush publication once the article was accepted--unless someone wanted to make sure the cycling media became aware of it.

I'm interested, though, in the juxtaposition of this publication with the recent REFC announcement inviting UCI's cooperation on Bert's case. Several here have speculated that REFC wouldn't have put out this invitation if they hadn't thought an acquittal or light sentence was forthcoming. But this publication throws a little more support to the transfusion scenario. I would think if REFC had known about it, they wouldn't have invited UCI in?

I hadn't realized that "mistress" in Spanish is "wada". LOL, I have known mistresses with a WADA-like interest in ascertaining my whereabouts at random times not available to me in advance.
 
Oct 16, 2010
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python said:
wait a minute…

didn’t we already establish that the test is too little to late and for all intents and purposes is almost useless...i mean it's of very limited use - like retro testing or catching really stupid ?

I recall reading in this very forum (many months ago in fact !) that as soon as the dopers learn about the plasticizer test they can easily switch to a different type of blood storage containers.

this very forum contained pictures and descriptions of those containers - in existence for decades since the early days of blood banking...

may be we’re losing the fight against dopers because we think they are stupid ?

or is it us who're naive and stupid ?
+1
indeed, all too often it is necessary to point out, or recall to mind, the obvious.
Indeed, to even think that the BP or this new plasticizer test is gonna change anything is naive.
Though if only it helps to nail down AC, I'll be more than satisfied.
 
python said:
wait a minute…

didn’t we already establish that the test is too little to late and for all intents and purposes is almost useless...i mean it's of very limited use - like retro testing or catching really stupid ?

I recall reading in this very forum (many months ago in fact !) that as soon as the dopers learn about the plasticizer test they can easily switch to a different type of blood storage containers.

this very forum contained pictures and descriptions of those containers - in existence for decades since the early days of blood banking...

may be we’re losing the fight against dopers because we think they are stupid ?

or is it us who're naive and stupid ?
Currently, the presented assay covers only DEHP. However, an implementation of alternatively used plasticizers in blood bags or other medical devices seems to be promising, assuming comparable physical properties of used substances. As a future prospect, the applicability of the method on different plasticizers has to be investigated
Or would they use glass?

If it makes them carry something other than a plastic bag which they can cut up and flush then it's a win.

PotentialPro said:
My overall curiosity is with the wide array of products that can raise DEHP levels, how skewed can results be? After all, cyclists are constantly in contact every day with on the bike waterbottles, water containers, plastic wraps, and tupperware type containers for our leftovers. These DEHP particles are more likely to be in urine and show regularly there, rather than in blood tests. Supposedly microwaving these plastics help release the chemicals further. I love science, technology and the like, but I have my doubts that a DEHP test itself is going to be a smoking gun, but more like the gun powder residue left on an assailants hands.
Well no doubt the control groups were exposed to these sorts of environments, one group even used DEHP containing medical products, but still the concentrations were well below that of a transfusion.

Chances of a cyclist in the middle of a stage race somehow getting a monster (transfusion range) dose of DEHP accidentally? Close to nil.
 
Mar 13, 2009
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I find this to be quite interesting:

The researchers studied three groups of people; a control group comprising 100 individuals with no special exposure to the plasticizer DEHP, ten hospitalised patients receiving transfusion and 468 athletes. Researchers concluded that “significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.”

There were also indications that four of the athletes had shown clear signs of transfusions, including three riders from the same team. They had been targeted in out of competition controls.

Read more: http://www.velonation.com/News/ID/7038/Plasticizer-test-moving-closer-to-official-implementation-Contador-case-linked.aspx#ixzz1AeEocoD6
 
I wonder if Velonation read the paper, it didn't exactly say it was "clear".

"Since information regarding the medical treatment of these athletes was not available, increased levels of secondary DEHP metabolites may indicate an unusual increased environmental exposure or blood transfusion. Three out of four outliers were taken out-of competition at the same time originating from one cycling team."

Of course we shouldn't be too critical when cycling media are actually calling a spade a spade (or a doper a doper in this case)...

What would it take to say, retrospectively test a certain rider's 2009 TdF samples? One rider in particular had a very suspicious passport and a high DEHP reading would be the nail in the coffin. Although said samples have probably been misplaced or destroyed.
 
May 26, 2010
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Ferminal said:
I wonder if Velonation read the paper, it didn't exactly say it was "clear".

"Since information regarding the medical treatment of these athletes was not available, increased levels of secondary DEHP metabolites may indicate an unusual increased environmental exposure or blood transfusion. Three out of four outliers were taken out-of competition at the same time originating from one cycling team."

Of course we shouldn't be too critical when cycling media are actually calling a spade a spade (or a doper a doper in this case)...
DEHP is a plastizer, if found can it not indicate other transfusions or just blood???
 
May 26, 2010
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Ferminal said:
What else would you be transfusing? Remember that any IV is banned in competition.
for sure that all IV's are banned but lots of other stuff is IV'd for recovery and just like Clen they'll talk about how it was not blood but something else and its not possible to gain anything from Saline IVs etc.......
 
Benotti69 said:
for sure that all IV's are banned but lots of other stuff is IV'd for recovery and just like Clen they'll talk about how it was not blood but something else and its not possible to gain anything from Saline IVs etc.......
The medical group (with catheters, IVs etc) still didn't go near the concentrations of the tranfusion group.

There's no way your average cynic like us would conclude that it was anything other than a blood transfusion, but that doesn't make it definitive and legally certifiable.
 
Jan 15, 2010
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Fester said:
Last friday I was "sealing" some chicken in the frying pan. The frying pan was new. The Spatula was a black plastic. The spatula actually left stains on the frying pan where it had melted; I ate the chicken and it was delicious. .
Well, end of story!
 
Jan 6, 2011
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Important to remember that the plasticizers test is only a test to "prove" their existence in a sample and the possible use of a medical procedure but not the exact nature of the procedure and not with certainty whether the subject blood doped. For example an athlete would now be smart to insist his doctor use glass transfusion bottles rather than the flexible bags.
Excuses will arise why certain athletes in the past may have had plasticizers in their sample but the future dopers will simply adjust their method to bypass the flexible bags.
 
Jul 2, 2009
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Fester said:
The test must be bullet proof before it's applied. If anything has come out of the Contador enquiry is that it isn't.
Don't you think that:

a) the creators of the test will have have considered the possibility other methods of ingestion

b) the creators know considerably more about the issues involved than you (or anyone on here)?
 
Galmozzi said:
... For example an athlete would now be smart to insist his doctor use glass transfusion bottles rather than the flexible bags.

Excuses will arise why certain athletes in the past may have had plasticizers in their sample but the future dopers will simply adjust their method to bypass the flexible bags.
I am not an expert on blood handling, storage, and preparation procedures, but it may well be that the use of bottles will require equipment that is 'non-standard' to a degree that makes shifting quite difficult.

Money still talks, of course. But, if you are counting on the Spanish health care system to store your products for you it could be difficult if the plastic bags are de rigeur. Let alone the fact that it will draw attention to you.

Why does this patient need blood to be treated differently, again?

Anyone know anything about the blood equipment?

Dave.
 

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