CO rebreathing method to measure tHbmass

[I Watch Cycling In July]

Sorry off topic, but I want kerbs cycle to find it.

With altitude ... if you get the stimulous right, tHbmass will gradually start to rise after 10-14 days... As tHbmass increases, plasma volume begins to normalise, so both retics and [Hb] are normal. When you return from altitude, retics do not go below normal

Just want to clarify this further. The relationship of "Usually a low normal Retic indicates that the body has a high normal total number of RBCs" has been asserted in a number of places. It's really the possibility of a sustained high-ish tHb/low-ish retic scenario that interests me, rather than altitude effect. I have yet to see the relationship articulated as clearly in any highly credible source, but it seems to contradict your answer to a previous post of mine.

Is it simply wrong?...Or did you mean that after returning from (effective) altitude training, tHbmass might be at the higher end of normal, retics at the lower end, but both still within normal range?...Or is a return from altitude training a bad example, because it doesn't lead to a high-ish tHb/low-ish retic scenario, but other considerations could?

 

[Krebs cycle]

to detect the transfusion of one blood bag, the uncertainty in the measurement of the mass of hemoglobin should be no more than 1 or 2%. Now, imagine the scenario in which you administer 1 ml of a substance, this substance diffuses in a few liters of blood, a blood sample of 5 ml is withdrawn and the substance quantified. A simple calculation shows that the requirement in the precision of the overall process (administration, dilution, sample collection and analysis) is too high to make it interesting for anti-doping purposes.

Radioactive tracers and other dilution procedures are nevertheless used by the US army.

The radiotracer method used by the USARIAM group is fraught with danger (in terms of potentional sources of error) because there are many more steps required. The CO rebreathing method is far superior. I don't know why those guys even bother with the red cell labelling technique. They have argued in the past that CO is taken up by myoglobin in the muscle however, there is clearcut evidence that this does not occur when the PaO2 is high. The rebreathing pipe is filled with 100% O2 prior to injection of the CO dose.

The precision of measurement of the CO rebreathing method is <2% so we are talking 20g out of 1000g. Changes to tHbmass resulting from transfusions are WELL in excess of this value.

Search for "Schmidt and Prommer" in pubmed and ye shall find what you are looking for. The stability of the variable of interest and the accuracy of the test is not a problem. It is logistics that need to be addressed.

 

[Escarabajo]

...
However, the more I think about the legal issues, the more practical problems appear...

I agree. I think this is the biggest hurdle for this type of testing.
Thanks for the information.

 

[Krebs cycle]

Just want to clarify this further. The relationship of "Usually a low normal Retic indicates that the body has a high normal total number of RBCs" has been asserted in a number of places. It's really the possibility of a sustained high-ish tHb/low-ish retic scenario that interests me, rather than altitude effect. I have yet to see the relationship articulated as clearly in any highly credible source, but it seems to contradict your answer to a previous post of mine.

Is it simply wrong?...Or did you mean that after returning from (effective) altitude training, tHbmass might be at the higher end of normal, retics at the lower end, but both still within normal range?...Or is a return from altitude training a bad example, because it doesn't lead to a high-ish tHb/low-ish retic scenario, but other considerations could?

I haven't read extensively on the following but I suspect that time plays a factor. If you increase your tHbmass gradually, the volume regulatory mechanisms adapt simultaneously, hence the reason why retics do not go below the normal range. If you suddenly whack in a transfusion, the lack of adaption in other volume regulatory pathways is not present and therefore, you elicit a clear homeostatic response which is to suppress erythropoiesis. This is speculation at present, but it makes sense from a theoretical point of view.

We need to be searching the literature firstly for what is know about the regulatory pathways involved in erythropoiesis (there are many steps in the pathway which have both inhibitory and excitatory influences) and secondly, for studies that closely examine these pathways in response to either rapid or gradual changes in red cell volume.

The comments from Jakob Morkberg in the other thread are highly relevant to this discussion ie: it will still be possible to beat this test and indeed, the biological passport, but what it does is begin to place limits on the range that changes can occur. Those boundries will begin to place limitations on artificial performance enhancement, so then it becomes a case of whether or not it is even worth the risk to dope when it only offers a minimal advantage.

 

[Krebs cycle]

Hm, 24 h later? What would BigBoat say to that? "They drain blood into coke cans" Could be dodgy.

pffft, non-issue. you only need around 500 ul whole blood per test. You would loose more than that with a paper cut.

 

[Escarabajo]

pffft, non-issue. you only need around 500 ul whole blood per test. You would loose more than that with a paper cut.

I don't think he meant that. What I understood is that if the rider is tested and he new that he had a transfusion done then his numbers would look dodgy. So he would perform a "Big Boat" operation overnight of draining some of the blood to alter the following test. Even if the numbers align back to normal (which I understand would be very difficult), it is enough erratic information for the rider to claim that there is something wrong with the test.

 

[Krebs cycle]

True.

snip

With the CO method, I see the following problem:
1) Sure you can draw 2 blood samples after breathing CO. The problem is that (i) we don't know how stable HbCO is in the sample and how it depends on handling, temperature etc. (probably that could be figured out) and (ii) the rider could complain that something went wrong with the breathing of CO in the first place. Since no lawyer of the rider in question was present during that part, I don't know whether any sample taken could actually count legally as a B sample. That's how CO testing is different from traditional testing; it has the added component of introducing foreign chemicals before taking samples!

Yes the problem is that if a positive test occurs, teh lawyers will jump onto the leak issue.

2) If you test again after 24 h, the rider could have extracted some blood in the meantime and so produce an innocent result. They should know how much they've put in, so they should know how much they should take out again to appear normal. And even 24 h is very short notice to get the rider's lawyer and expert on board.

But how will they know they need to take blood out if you don't give them results? You just knock on the door of the team bus 24hrs later and say come with me. They could anticipate and take the blood out anyway, but if say, the athlete is supervised at all times in between finishing a stage and when the test is conducted, then it would be rather counterproductive to take blood out the night before a TT or mountaintop finish for example.

I think the science of the CO method is fine and I hope the test will be deployed rather sooner than later. However, the more I think about the legal issues, the more practical problems appear.

I agree, there are significant logistical hurdles to be overcome, and I suspect these hurdles are the reason that it has not already been introduced. However, all we can hope is that the strict set of rules is drawn up in order to get the most meaningful data from the testing if it is introduced.

 

[Alpe d'Huez]

Really good thread. Glad to see this topic brought up again. Thanks Krebs!

Correct me if I'm wrong, but didn't Michael Ashenden suggest a test that didn't involve re-breathing, but merely blood analysis from a pin *** was possible to implement?

Is there any way your test could be administered under water Krebs? That would prevent leakage, or detect it.

The big problem here though is still the UCI. They won't do this because they think the peloton is clean already. Well, I don't know if they honestly think that, but that's what they say. The other "legal" problem is they have no testes. If they did, they'd start to put their foot down, and would draw up a new rules and testing guideline saying these are the race rules, and it includes this kind of testing. If you don't like it, find another vocation. Instead they are always going to defer to WADA, or someone else, and want to be selective in their testing, that way nothing happens beyond their control, and nothing happens that causes them any problems.

 

[Cobblestones]

But how will they know they need to take blood out if you don't give them results? You just knock on the door of the team bus 24hrs later and say come with me. They could anticipate and take the blood out anyway, but if say, the athlete is supervised at all times in between finishing a stage and when the test is conducted, then it would be rather counterproductive to take blood out the night before a TT or mountaintop finish for example.

You have to tell the rider in advance before you analyze a B sample such that he can get lawyers and experts of his own in place to observe the analysis.

For normal blood or urine samples, there's no problem. It can take months until all meet and break the seal of the sample.

For the CO test, I would argue that using the re-breather is part of the sample analysis process (that's different from urine and blood sample taking) and therefore the rider should have the chance to have a lawyer and expert of his choice observe it. In that case, you'd have to inform the rider well ahead before you take the B sample. If you follow that train of thought, then you cannot surprise the rider.

I agree with you that draining blood before taking a B sample in a GT would be very counterproductive to performance. Now, if a rider has to chose between a bad GT performance and a 2 year doping ban, then I think we know what most of them will do.

 

[Krebs cycle]

We use a hand held CO monitor near the mouth to detect the leak, so we know when it occurs. The problem is that the athletes will discover pretty much immediately that the test result is rendered useless if they break the seal on purpose.

A more full (fool) proof method would be a method via which a small amount of blood is withdrawn, then the CO bolus added to this blood externally via some sort of dialysis device, and then reinserted. This would be a technical challenge indeed, but it would remove the need for rebreathing.

Dopingectomy alluded to a method which uses radiolabelled red cells, this method is considered (by some) to be the gold standard, however it is much more technically difficult and would take a lot longer to complete the test.

The B sample issue is certainly a problem too (damn lawyers!!). This is very much a BIG sticking point then unless the rules on that can be altered.

Alpe d' huez, the original CO rebreathe method used venous samples. Asho wrote a paper showing that capillary samples could be used instead.


Great comments everyone. The red tape that exists illustrates part of the reason why it is obviously so difficult to make prosecutions. Why can't cyclists or any athletes just not cheat in the first place? I find the world a strange place sometimes.... eg: a new therapeutic method is discovered and developed which has the potential to alleviate suffering of millions of people, but A) the product is too expensive for those who really need it and drug companies are not humanitarian agencies and B) the new product allows cheating athletes to gain unfair advantages over thei competitors.

Advances in medical technology are supposed to be about improving the lives of humans and lifting quality of life, but in some cases there exists this ethical darkside as well.

 

[Bala Verde]

That's why I brought up the B-sample or legal issues. A test can work perfectly, but when they don't accomodate/guarantee the rights of the suspect, they are near worthless.

Maybe one way out of 'breaking the seal' could be that when a subject purposely fails the test, he gets a doping infraction similar to missing a test due to incorrect whereabouts.

I noticed Basso already uses the rebreathing method:

Seasonal variations of Ivan Basso’s hemoglobin mass. Hemoglobin mass was determined by the CO rebreathing method, optimized by Schmidt and Prommer (Eur J Appl Physiol 2005). In the figure each measurement value is expressed as percentage of the all measurements average.

If you were apply the blood passport model - collect data over time and consolidate them in a graph - it might work. When values are off, I haven't heard anyone run to his lawyer to dispute that the test(s) to determine Hb/Hemtocrit/Ret% at certain dates were compromised.

 

[Cobber]

That's why I brought up the B-sample or legal issues. A test can work perfectly, but when they don't accomodate/guarantee the rights of the suspect, they are near worthless.

Maybe one way out of 'breaking the seal' could be that when a subject purposely fails the test, he gets a doping infraction similar to missing a test due to incorrect whereabouts.

I noticed Basso already uses the rebreathing method:

If you were apply the blood passport model - collect data over time and consolidate them in a graph - it might work. When values are off, I haven't heard anyone run to his lawyer to dispute that the test(s) to determine Hb/Hemtocrit/Ret% at certain dates were compromised.

This is a very interesting graph. Basically, Basso's Hb mass varies by no more than +/- 4% from his yearly average. What value, or %
change would constitute a positive? Any ideas what his other values were for these tests (Hb/crit/retic%)?

 

[Bala Verde]

I used the data from his latest .pdf with his blood values, publicly available at http://www.mapeisport.it/IvanBasso/?LNG=EN. The Hb mass tests (CO rebreathing) did not seem to overlap with the dates the other blood tests for the blood passport were taken.

I can't guarantee that all the values have been accurately transfered to the excel sheet, so let me know if something is wrong.

Since the Off score graphs didn't display exact values, the only way to determine them is to either A) calculate them or B) guestimate them using the graph. I am not a statician, so I used the following formulas to determine the off scores.

1)z score= (observation-mean)/standard deviation
2)OFF score = Hb (g/L) - sixty times the square root of the percentage of RBCs identified as reticulocytes.

I hope someone can confirm that this is correct.

IVAN BASSO: BLOOD PASSPORT DATA – DATI PASSAPORTO EMATICO UPDATING 25.08.09

2/5/2008 UCI Blood Passport data; 25/9/2008 UCI Blood Passport data; 7/10/2008 UCI Blood Passport data; 4/11/2008 UCI Blood Passport data; 30/1/2009 CEDAL lab; 11/02/2009 UCI Blood Passport data; 6/3/2009 CEDAL lab; 20/3/2009 UCI Blood Passport; 3/4/2009 CEDAL lab; 15/4/2009 UCI Blood Passport (prelievo effettuato in altura, dopo 12 giorni di allenamento in quota /blood sample taken in altitude, after 12 days of altitude training); 20/4/2009 CEDAL lab; 29/4/2009 UCI Blood Passport; 30/4/2009 CEDAL lab (dopo allenamento/after training session); 7/5/2009 UCI Blood Passport (Prima/before Giro d’Italia); 18/5/2009 UCI Blood Passport (metà/middle Giro d’Italia); 31/5/2009 UCI Blood Passport; 29/06/2009 UCI Blood Passport; 11/7/2009 CEDAL Lab; 24/7/2009 UCI Blood Passport; 27/7/2009 CEDAL Lab.; 9/8/2009 UCI Blood Passport; 15/8/2009 UCI Blood Passport; 24/08/2009 CEDAL Lab.

 

[Oldman]

is completely safe for any athlete (including riders with all kinds of allergies ). I'm sure people have been thinking about it. The problem I see is that since IV's are generally banned to start with, it would be quite ironic to require an IV injection for a doping test. Also, I could imagine a number of reasons why a rider would reject an IV or why it would create trouble in many possible ways. For instance, I don't think you'd have to wait very long for the first rider to claim "the French spiked my IV".

I've had the test administered by and allergist. You hit the nail on the head plus the liability of administration: imagine a rider or team claiming illness or performance problems afterward? It also takes some time to conduct. This sounds like another credible idea that would require refinement.

 

[Cobblestones]

This is a very interesting graph. Basically, Basso's Hb mass varies by no more than +/- 4% from his yearly average. What value, or % change would constitute a positive? Any ideas what his other values were for these tests (Hb/crit/retic%)?

Yes, it's wonderful. That's why Hb mass is such a nice indicator. There must be one or maybe 2 points during the Giro this year on that graph. A pint of blood should stand out like a sore thumb. I also see from the table that Hb concentration goes down during the Giro (as it should due to plasma expansion) and the retic count is not suppressed. It doesn't look like he got a refill in the Giro. Now he didn't get a stage win or a podium either, but that belongs on the other side of the forum.

 

[Bala Verde]

Yes, it's wonderful. That's why Hb mass is such a nice indicator. There must be one or maybe 2 points during the Giro this year on that graph. A pint of blood should stand out like a sore thumb. I also see from the table that Hb concentration goes down during the Giro (as it should due to plasma expansion) and the retic count is not suppressed. It doesn't look like he got a refill in the Giro. Now he didn't get a stage win or a podium either, but that belongs on the other side of the forum.

I am eagerly waiting for Basso's mid/post vuelta values. I'll add them to the sheet to see what they look like.

Unfortunately, he only had 2 tests during a GT/or published only 2...

 

[Cobber]

Yes, it's wonderful. That's why Hb mass is such a nice indicator. There must be one or maybe 2 points during the Giro this year on that graph. A pint of blood should stand out like a sore thumb. I also see from the table that Hb concentration goes down during the Giro (as it should due to plasma expansion) and the retic count is not suppressed. It doesn't look like he got a refill in the Giro. Now he didn't get a stage win or a podium either, but that belongs on the other side of the forum.

It sure is! I did some quick calculations.... if you assume an average adult blood volume of 5 litres, a 4% change in total Hb would constitute the addition or removal of 200 ml of blood. This is still a significant amount, and so abrupt changes might be something of concern. A pint would cause slightly more than 10% change, which would be extremely obvious. Maybe we will see athletes "micro-dosing" transfusions on a few years?!

 

[Cobber]

Getting back to what I was suggesting above.... I found a link to this instrument that can measure blood volume to 98% accuracy. Combining this with Hg conc should also give an accurate measure of total Hg. (Unfortunately) the half-life if the isotope they use (I-131) is only 8 days, so again this would make B sample analysis tricky. However, I think accurate B sample analysis could be conducted within a week of the original test.

http://www.bva100.com/

 

[Escarabajo]

It sure is! I did some quick calculations.... if you assume an average adult blood volume of 5 litres, a 4% change in total Hb would constitute the addition or removal of 200 ml of blood. This is still a significant amount, and so abrupt changes might be something of concern. A pint would cause slightly more than 10% change, which would be extremely obvious. Maybe we will see athletes "micro-dosing" transfusions on a few years?!

Nice info.

The Big Boat theory of doing it more than 2 times during a Grand Tour might become a reality.

 

[US Patent Exploding Cyclist]

The remaining CO left in the lung is measured (in ppm) by conducting a complete forced expiration,

What method is used for the "expiration"? Is the subject just blowing out in a different tube or are you using a nebulizer? It seems there might be room for argument in this aspect of the test as well as the "leaking" already mentioned. Especially for those who have asthma and those others who have "asthma".

 

[mitochondrion]

CO rebreathing practicalities

Carbon monoxide binds to haemoglobin more avidly than oxygen and as a result random testing on the day of an important grand tour stage would result in considerable performance loss on that day.

Breathing high flow oxygen through a reservoir mask would decrease the time to excretion of CO to probably less than 2 hours in practice. Unfortunately loading oxygen prior to a race is illegal, although its effects would be negligible if not detrimental as a race progressed.

I routinely use transfusion to improve oxygen delivery in my clinical practice, but I would postulate that the only practical way of determining whether an athlete has undergone autologous transfusion would be to examine red cell size , shape and reticulocyte count daily during a tour. Any changes could then be considered abnormal, not the current fudge factor seen in the sampling only 5 times in 3 weeks.

 

[Escarabajo]

...

I routinely use transfusion to improve oxygen delivery in my clinical practice, but I would postulate that the only practical way of determining whether an athlete has undergone autologous transfusion would be to examine red cell size , shape and reticulocyte count daily during a tour. Any changes could then be considered abnormal, not the current fudge factor seen in the sampling only 5 times in 3 weeks.

Don't you think Lance's values were clear doping during the Tour? It looks clear to me. The passport criteria just need to be revised. The off score is not enough.

 

[mitochondrion]

An eloquent idea but calibration may prove impossible.

Lance and his medical advisors know that his published values are entirely within explainable limits. Even if future grand tours test more often, perhaps the quantity of transfusion and performance enhancement will drop but the best programmes will still produce winners or even podium places for 37 year olds.

 

[Krebs cycle]

What method is used for the "expiration"? Is the subject just blowing out in a different tube or are you using a nebulizer? It seems there might be room for argument in this aspect of the test as well as the "leaking" already mentioned. Especially for those who have asthma and those others who have "asthma".

its just a gradual exhalation until you hit residual volume. not a problem for asthmatics, they should be well practices at doing FVCs anyway!

 

[Krebs cycle]

Getting back to what I was suggesting above.... I found a link to this instrument that can measure blood volume to 98% accuracy. Combining this with Hg conc should also give an accurate measure of total Hg. (Unfortunately) the half-life if the isotope they use (I-131) is only 8 days, so again this would make B sample analysis tricky. However, I think accurate B sample analysis could be conducted within a week of the original test.

http://www.bva100.com/

this looks better than radio labelling the red cell itself. There was a previous method which used evan's blue dye which attaches to albumin but the only company in the world who made it stopped production so the method went the way of the dinosaur.