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CO rebreathing method to measure tHbmass

Thought I'd make a post about this for interested peeps as it seems a few people are aware of the test but not sure how it works.

I have the equipment to do the test in my lab and I have administered the test on at least 100 occasions. I've also done the test myself around 10 times.

It works on the principle of dilution. If you measure the saturation of CO bound to Hb (%HbCO) in the blood before and after a precise dose of CO is given, then the smaller the change in %HbCO, the larger the tHbmass.

%HbCO is measured from a capillary blood sample first. The subject then rebreathes through a glass pipe that looks rather like a big crack pipe or something, which has attachments for a syringe to administer the CO dose and a 3L rebreathing bag. You insert a CO2 scrubber inside the pipe so that you don't end up hyperventilating like a psychopath with a brown paper bag. Upon inserting the CO dose into the rebreathing pipe, the subject breathes for exactly 2min. The remaining CO left in the lung is measured (in ppm) by conducting a complete forced expiration, and the remaining CO in the rebreathing bag is also measured by sucking it out with a hand held piston. Further capillary blood samples are taken after about 5min.

It really is quite a simple test to conduct and is over in about 10-15min. The measurement of %HbCO in the blood samples is rapid and would always be performed immediately to ensure stability of the blood is not compromised by longterm storage.

I don't think the test could ever be conducted in the morning before racing, because you can notice some sluggishness in performance if you train immediately after the test, but after about 3hr, you don't feel any different to normal. The amount of CO administered is so small that within 6-8hrs you are back to baseline values. I don't believe the test can be administered twice in a row, but it most certainly can be done on a daily basis without any long term effect on aerobic performance.

The main source of error is if there is a leak of CO from the rebreathing pipe, but if it is conducted properly, the variation in test results is actually lower than many standard full blood count variables used in the bio-passport. The lower the inherent variability in a measure, the better its use from an anti-doping perspective, because it means the thresholds can be tighter.

The beauty of the test really lies in the fact that it zeros in on the criterion variable. The object of doping manipulation IS to increase tHbmass, so why not measure it directly instead of relying on a host of indirect markers? The only way to beat the test is to know exactly what your previous test result was and know exactly when you are going to be tested, because you could of course remove the appropriate amount of blood, do the test, then just put it back afterwards. Not such an easy thing to do though, if the actual results don't get released to the team doctor. Remember that a big decrease in tHbmass is also evidence of tampering.

The big problem is the leak issue. I think there will be significant legal hurdles to overcome in the event that an athlete continually sabotages the test by causing a leak on purpose ie: not keeping a seal around the mouthpiece through which they must breathe. If they can fix that problem somehow, then we have a winner!
 
Jul 8, 2009
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BroDeal said:
The duct tape might not go ever well, but I'm sure no one will mind a mouthpiece on their testes.


LMAO :D!



I don't think the test will be implemented as long as it worsens performance... It doesn't matter if it only worsens it for a short time, or that you do the test after the race - the racers will always use that argument why it shouldn't be used. Unless they are clean of course.
 
Jun 18, 2009
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Great post, Krebs cycle. Thanks!

Maybe they could control for leakage from the pipe by using a dilute detergent solution around anywhere a leak could occur. Bubbles would be evidence of the need for a retest.

I also wonder if there would be a way to administer the CO directly to the blood stream in a safe way, say really dilute in a saline IV. That way, there would be no leakage, and no need to measure residual CO in the testing apparatus.

Edit: Another idea...... CO rebreathing is really used to measure the volume of blood, right? That is, you can measure the amount of hemoglobin in a ml of blood, but unless you know the total volume of blood the total mass of hemoglobin is not known. So, rather than using CO rebreathing, why not just administer iv a known amount of a substance (say a drug with a very long serum half life or a small amount of homologous blood), allow it to normalize in the blood for a period of time, then draw 5 ml of blood and measure hemoglobin and the drug concentration. This would be an indirect way to measure total hemoglobin concentration (hemoglobin concentration X dilution factor of added substance).
 
Mar 10, 2009
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One of the advantages of the test is its capacity to instantly produce a value. However couldn't this also be one of the main drawbacks?

How can a rider request a 'B sample' or retest, when his 'first test' resulted in an 'abnormal' value. Storage of samples serves the purpose of having concrete proof for accuser as well as suspect, ie blood or urine can be retested for exonegenous substances. That, however, takes a while due to procedures.

How can the CO rebreathing method be used, so that it can accomodate the rights of the suspect? Or would the values be used in a blood passport type of fashion in which multiple results are consolidated in a chart/graph, whereby parameters are gradually set to determine an individual's bandwidth?
 
May 13, 2009
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We actually had a thread on this in the other section (there wasn't a clinic at that time). It got spammed up a bit, but some posts there are still worth reading.

Here it is.

Anyway, good to bring the subject up again.
 
May 13, 2009
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BroDeal said:
The duct tape might not go ever well, but I'm sure no one will mind a mouthpiece on their testes.

Make that 'testis' for a certain rider.

Cobber: The test will actually measure the total Hb mass, not a concentration. So there is no reason to measure total blood volume (with plasma) or anything like that. CO only binds to Hb, so any kind of variations in plasma volume do not matter at all.

About introducing CO by IV, I don't know how that would work. You wouldn't put it in as a gas obviously. If you introduce it bound to some carrier, that's another can of worms. I can see the riders complain big time.

Bala Verde: What you could do is to draw two samples. One is tested on the spot, if that turns out suspicious, the other might be tested as well. But I agree, there's definitely a storage problem. It is unclear how long CO stays bound to the Hb.
 
Jun 18, 2009
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Cobblestones said:
Make that 'testis' for a certain rider.

Cobber: The test will actually measure the total Hb mass, not a concentration. So there is no reason to measure total blood volume (with plasma) or anything like that. CO only binds to Hb, so any kind of variations in plasma volume do not matter at all.

About introducing CO by IV, I don't know how that would work. You wouldn't put it in as a gas obviously. If you introduce it bound to some carrier, that's another can of worms. I can see the riders complain big time.

Bala Verde: What you could do is to draw two samples. One is tested on the spot, if that turns out suspicious, the other might be tested as well. But I agree, there's definitely a storage problem. It is unclear how long CO stays bound to the Hb.

The test does measure total Hb mass, but it does so by measuring the concentration of CO in the blood after inputting a known amount. Thus, it estimates the volume of the blood as part of the test. The equation is:

Total mass (grams) = concentration (grams/litre) X volume (litres)

The test I suggested does the same thing, but in a different way. Adding a known amount of a substance to the blood then measuring its concentration will give the volume of the blood. Multiplying this number by the concentration of hemoglobin in the blood will give the total mass of hemoglobin, without having to worry about CO toxicity, or athletes sealing on testes! :eek:

As far as direct CO infusion is concerned, the gas will dissolve in water (and presumably saline) in small quantities, as shown here (http://www.engineeringtoolbox.com/gases-solubility-water-d_1148.html). Since CO has an extremely high affinity for hemoglobin, almost all of what is infused should be bound to hemoglobin after a few minutes, and thus the CO rebreathing test could be replicated without the problems suggested above.
 
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Cobber said:
The test does measure total Hb mass, but it does so by measuring the concentration of CO in the blood after inputting a known amount. Thus, it estimates the volume of the blood as part of the test. The equation is:

Total mass (grams) = concentration (grams/litre) X volume (litres)

The test I suggested does the same thing, but in a different way. Adding a known amount of a substance to the blood then measuring its concentration will give the volume of the blood. Multiplying this number by the concentration of hemoglobin in the blood will give the total mass of hemoglobin, without having to worry about CO toxicity, or athletes sealing on testes! :eek:

As far as direct CO infusion is concerned, the gas will dissolve in water (and presumably saline) in small quantities, as shown here (http://www.engineeringtoolbox.com/gases-solubility-water-d_1148.html). Since CO has an extremely high affinity for hemoglobin, almost all of what is infused should be bound to hemoglobin after a few minutes, and thus the CO rebreathing test could be replicated without the problems suggested above.

I think you're wrong here. The CO will almost exclusively bind to Hb (very little will be dissolved in the plasma or anything). So, what one measures is the amount of HbCO in percent of Hb. If one assumes that all CO which is no longer in the rebreathing apparatus has been bound to Hb to form HbCO, by measuring the percentage of HbCO (relative to all Hb), you can calculate the total amount of Hb (in number of molecules for instance).

The formula would be something like this:

(#CO molecules which the test subject has breathed in)/(percent of HbCO molecules with respect to all Hb)=#of Hb molecules in total

Nowhere enters the total blood volume or plasma volume.

Here is a graph of CO solubility in water. At body temperature (~40 degree Celsius), you can dissolve about 20 mg in 1 kg of water. How much gas is needed again? Let's assume solubility is the same in saline, and maybe you want to administer no more than a few ml of saline, then the most CO you can administer would be about 20 microgram. Is that enough? The CO content of a cigarette is about 10-20 mg which is 1000 times higher. Upthread, we compared the necessary amount of CO for this test to the CO content in the smoke of a cigarette.
 
Jun 18, 2009
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Cobblestones said:
I think you're wrong here. The CO will almost exclusively bind to Hb (very little will be dissolved in the plasma or anything). So, what one measures is the amount of HbCO in percent of Hb. If one assumes that all CO which is no longer in the rebreathing apparatus has been bound to Hb to form HbCO, by measuring the percentage of HbCO (relative to all Hb), you can calculate the total amount of Hb (in number of molecules for instance).

The formula would be something like this:

(#CO molecules which the test subject has breathed in)/(percent of HbCO molecules with respect to all Hb)=#of Hb molecules in total

Nowhere enters the total blood volume or plasma volume.

Hmmmm.... I think we are both right. Decision, REF?!

The CO rebreathing method measures the % HbCo relative to all Hb. Because the amount of HbCO is dependent on a known amount of CO being administered, you know the total amount of HbCo in the body. This amount is then multiplied by the ratio of total Hb to HbCo, giving the total amount of Hb. Correct?

I agree that there is no direct measure (or estimation) of blood volume in this procedure. However, if the concentration of hemoglobin in the blood is known, this would be very easy to calculate.

ie. if total Hb (from CO rebreathing) = 10g, and concentration (from standard blood test) of Hb = 1g/L, then total blood volume would be 10L.

The method I suggested would use Hb concentration (from standard blood test) and total blood volume (from test I suggested) to generate total Hb concentration. While my method would require total blood volume to be determined, the output is the same as the CO rebreathing method. ie. total amount of Hb in the blood.
 
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Cobber said:
Hmmmm.... I think we are both right. Decision, REF?!

The CO rebreathing method measures the % HbCo relative to all Hb. Because the amount of HbCO is dependent on a known amount of CO being administered, you know the total amount of HbCo in the body. This amount is then multiplied by the ratio of total Hb to HbCo, giving the total amount of Hb. Correct?

I agree that there is no direct measure (or estimation) of blood volume in this procedure. However, if the concentration of hemoglobin in the blood is known, this would be very easy to calculate.

ie. if total Hb (from CO rebreathing) = 10g, and concentration (from standard blood test) of Hb = 1g/L, then total blood volume would be 10L.

The method I suggested would use Hb concentration (from standard blood test) and total blood volume (from test I suggested) to generate total Hb concentration. While my method would require total blood volume to be determined, the output is the same as the CO rebreathing method. ie. total amount of Hb in the blood.

Oh, I agree, your method would also work. I think for your method you need to enter something into the bloodstream which (i) isn't there before (or if it is, you'd have to measure before and after concentrations), (ii) stays sufficiently long to completely dilute without disappearing, and (iii) is completely safe for any athlete (including riders with all kinds of allergies :eek:). 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". ;)
 
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Cobber said:
Edit: Another idea...... CO rebreathing is really used to measure the volume of blood, right? That is, you can measure the amount of hemoglobin in a ml of blood, but unless you know the total volume of blood the total mass of hemoglobin is not known. So, rather than using CO rebreathing, why not just administer iv a known amount of a substance (say a drug with a very long serum half life or a small amount of homologous blood), allow it to normalize in the blood for a period of time, then draw 5 ml of blood and measure hemoglobin and the drug concentration. This would be an indirect way to measure total hemoglobin concentration (hemoglobin concentration X dilution factor of added substance).

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.
 
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dopingectomy said:
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.

Well, if you want to measure down to 1-2%, then what you have to do is measure the amount of substance you give down to 0.7-1.5% and the final concentration (in the 5ml blood which is drawn) also down to 0.7-1.5%. Simple error propagation rules dictate that the result will be 1-2% uncertain. Here, I assume that the dilution is perfect (it probably is after waiting 5-10 minutes). It does not matter whether the sample is always 5ml, because the sample size does not enter the calculation (only the concentration). I agree, it's tough and it's easier with CO. In particular it's easier to control the amount of substance given (which is probably the hard part) with the CO method.

ETA: adding a pint of blood to the otherwise ~5 l of blood in the average human is a 10% effect. If the pint isn't just blood, but one pint of packed cells, the effect is even bigger. I agree you might want to measure a bit better than 10%, but 3-5% might actually suffice.
 
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dopingectomy said:
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.

That really depends on the accuracy of the test for the hypothetical compound being injected. It should be incredibly easy to standardize any test along these lines using a series of volunteers. A compound would just need to be found that is safe, has a long serum half life, and can be detected in the blood with suitable sensitivity to make the test work (and, I guess, have no influence on and blood parameters or any performance-enhancing effects). There must be hundreds of drugs/compounds that fit these criteria.
 
Thanks Krebs for the information.

Thanks Cobber and Cobbles for the discussion. I am a little lost, but hopefully I picked up something.

Maybe there is a different reason why they don’t do it by infusion (I noticed Cobbles mentioned a good explanation about the use of IV within the race). It would be the easier way.

Bala have a point about having samples frozen for later testing. I just don't think that using the samples right on the spot without a lawyer or third party will hold in a court later.
 
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Cobber said:
why not just administer iv a known amount of a substance, then draw 5 ml of blood and measure hemoglobin and the drug concentration.

Why not just give a big saline IV after a tough day and measure the before/after concentration of ANY blood variable that is easy to measure accurately, to get to the total blood volume, then calculate tHbmass....I guess the whole plasma shift malarkey could be technical issue though, and then there is the ethical issue below...

Cobblestones said:
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.

+1 The real problem I see with the whole CO re-breathing method is the irony of testing for foreign substances by the introduction of foreign substances. 'Fark off you hypocrites' would be a reasonable reaction IMHO.
 
Bala Verde said:
One of the advantages of the test is its capacity to instantly produce a value. However couldn't this also be one of the main drawbacks?

How can a rider request a 'B sample' or retest, when his 'first test' resulted in an 'abnormal' value. Storage of samples serves the purpose of having concrete proof for accuser as well as suspect, ie blood or urine can be retested for exonegenous substances. That, however, takes a while due to procedures.

How can the CO rebreathing method be used, so that it can accomodate the rights of the suspect? Or would the values be used in a blood passport type of fashion in which multiple results are consolidated in a chart/graph, whereby parameters are gradually set to determine an individual's bandwidth?
Simple, you do the test again 24hr later. The second test IS your "B sample".

I think the test on its own is is good enough, however, a bio-passport style analysis will help you to determine more precisely the difference between doping and exposure to hypoxia. eg: autologous transfusions = acute change in tHbmass = not possible with exposure to hypoxia.
 
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Krebs cycle said:
Simple, you do the test again 24hr later. The second test IS your "B sample".

I think the test on its own is is good enough, however, a bio-passport style analysis will help you to determine more precisely the difference between doping and exposure to hypoxia. eg: autologous transfusions = acute change in tHbmass = not possible with exposure to hypoxia.

Hm, 24 h later? What would BigBoat say to that? "They drain blood into coke cans" Could be dodgy.
 
Cobblestones said:
Hm, 24 h later? What would BigBoat say to that? "They drain blood into coke cans" Could be dodgy.
And they always will claim that the test was wrong, that they needed an expert or third party during the testing. The fact is that you don't know that the second test is going to be your B sample. So the riders will always say that they can not defend themselves, that they were set up, blah, blah, blah.
 
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Escarabajo said:
And they always will claim that the test was wrong, that they needed an expert or third party during the testing. The fact is that you don't know that the second test is going to be your B sample. So the riders will always say that they can not defend themselves, that they were set up, blah, blah, blah.

True.

As far as I understand the legal part of testing (honestly, not very well) the point with the B sample is not that you get a second opinion. The point is that the rider can have lawyers and experts of his choice to monitor the testing procedure. The A sample is entirely worthless when it comes to sanctioning. It's only the B sample which counts. Normally, when the A sample shows a non-negative, the rider is informed that the B sample will be tested. They all meet (with lawyers and possibly experts) to break the seal of the B sample and monitor the testing. Whatever comes out of that is going to be accepted as the result by both parties.

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!
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.

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. Maybe one has to change the legal framework a bit for that particular test. Maybe instead of a full-blown sanction, a ban from riding for 2 weeks could be given just as for violating the 50% rule. Of course, the 50% rule is due to 'health concerns'. A suddenly increased total Hb mass is not unhealthy. It's strange for sure, and indicative of doping, but it's not a health concern in itself. So maybe that's not the way to go either.