Anti-doping organisations may need to compensate for a hematocrit (HCT) variability and HCT concentration bias before Dried Blood Spot (DBS) tests can be used reliably for anti-doping tests, a new Study has highlighted. DBS tests involve various devices that an athlete can place on their finger, heel, or arm. This draws out a small amount of blood that is then deposited onto a DBS index card, which is then sealed using a methodology that is apparently tamper proof, and posted to a Laboratory accredited by the World Anti-Doping Agency (WADA) for analysis.

The DBS method was trialled by the Ultimate Fighting Championships (UFC) in 2018, and was trialled with seven anti-doping organisations (ADOs) last year. In June this year, WADA announced a Request for Funding Applications (RFA) for research into the development of DBS for use in anti-doping, and its use was discussed at the Institute of National Anti-Doping Organisations’ (iNADO) recent Annual General Meeting (AGM).

ADOs want to use DBS as a complementary anti-doping test to the existing urine and blood tests. DBS has been heralded as enabling detection of a wide range of substances and biomarkers whilst allowing easier transport, longer storage and greater opportunities for reanalysis. It’s also cheaper than blood or urine analysis.

Blood tests can prove prohibitively expensive for many ADOs because it costs money to send a trained blood collection officer and phlebotomist to process a sample; and because a vial of blood needs to be refrigerated when sent to a WADA Laboratory within three days, without being frozen. This also requires temperature logging at every stage of the journey to the Laboratory. 

Game changer

DBS doesn’t require any of this. It been presented as potentially enabling athletes to conduct anti-doping tests at home, which would understandably be a game changer in anti-doping. 

"I think it could be a game changer." -USADA Emeritus Chair Edwin Moses shares his thoughts on the virtual testing pilot program and dried blood spot testing. ???????? #cleansport #innovation pic.twitter.com/08L2wd7oDc

— USADA (@usantidoping) July 2, 2020

“DBS for anti-doping has a huge potential”, explains Marc Luginbühl of the CAMAG DBS Laboratory in Muttenz, Switzerland, who authored ‘Dried blood spots for anti‐doping: Why just going volumetric may not be sufficient’ with his colleague Stefan Gaugler. “Novel applications and new developments are published weekly and knowledge is constantly growing. However, for a reliable, quantitative DBS strategy, some extra efforts will be required.”

WADA’s approach to DBS has focused on devices that ensure the collection of a known volume of blood to allow quantitative analysis. A minimum volume of 20 microlitres (μL) per spot was set as a benchmark. 

“Reliable quantitative DBS analytics in an athlete population can only be performed if the hematocrit [HCT] concentration is taken into consideration”, says Luginbühl. “If the hematocrit is not accounted for during quantitative analysis, an underestimation or an overestimation of the drug concentrations is likely. For threshold substances, where a certain concentration has to be exceeded to report an adverse analytical finding [AAF], false positives and false negatives could be the case if no hematocrit correction is taken into consideration. A proper DBS method validation should therefore evaluate the hematocrit impact and suggest strategies to compensate for potential effects.”

It is understood that DBS sampling introduces an HCT area bias, because the viscosity of the blood changes as the number of red blood cells increases, leading the total area of a DBS from the same volume of blood to vary. WADA’s approach aims to compensate for this by normalising sample volumes. However, as Luginbühl and Gaugler’s Study explains, HCT can vary by up to 15% in normal adults based on whether a sample is taken in Summer or Winter. In athletes, variations are even more pronounced.

Previous studies have shown that HCT varies during exercise due to issues such as (de)hydration, oxygen uptake, altitude, and more. WADA’s Blood Sample Collection Guidelines account for this, preventing blood samples from being collected within two hours after exercise and requiring athletes to declare their movements in the previous two weeks. However, ‘a general DBS strategy to measure and compensate for these variations within the anti-doping laboratory has not been presented yet’, the Study notes.

The lack of HCT correction in the DBS analysis proposed by WADA could also cause problems at the analytical stage, the Study notes. This is firstly because the binding of substances to plasma proteins within the blood can vary, and the HCT can influence the amount of a drug being detected within a fixed volume of blood. Secondly, the Study argues that an extraction bias needs to be assessed for each analytical procedure as variations are possible due to an increase or decrease in HCT.  

As perhaps might be expected from scientists that work in a Laboratory that specialises in DBS analysis and equipment, Luginbühl and Gaugler have solutions that allow HCT assessment during DBS tests. “The HCT extraction bias is a DBS-specific parameter, which is dependent on the choice of sampling device (the material on which the blood is sampled, such as cellulose membrane or a hydrophilic porous material), the analyte, and the extraction process”, explains Luginbühl. “Methods such as prolonged DBS extraction, the use of sonication, or an introduction of the internal standard prior extraction (spraying before extraction) can help to compensate for the bias”.

Assessing HCT is, of course, possible during the sampling process, however that would compromise the simplicity of the DBS process, which is the main attraction for ADOs. Therefore, a logical solution appears to be post sample assessment of HCT. Again, Luginbühl and Gaugler have a solution. 

“The automated hematocrit analyzer has a fibre-coupled LED light source that generates an excitation light, with a nominal wavelength at 589nm. The light is transmitted towards the centre of the DBS and the reflected light is transmitted to a spectrometer. The resulting value can then be set into relation to the HCT or further processed.”

The future of DBS in anti-doping

The potential of DBS is huge. The current reliance on ‘wet’ samples places refrigeration and storage requirements on ADOs. WADA’s Blood Collection Guidelines specify that samples must arrive at the Laboratory within 72 hours (less, for some forms of analysis) and as mentioned, they must remain refrigerated throughout the process and not be allowed to freeze.

There are 30 accredited Laboratories listed on WADA’s internet site. Four are currently suspended, and two warn that activities may be temporarily disrupted due to Covid-19. As the Bankgok and New Delhi Laboratories are currently suspended, any Southeast Asian ADO must ship blood samples to Beijing, Doha, Seoul or Tokyo. There is only one accredited Laboratory in Africa (South Africa).

Given the expenses and complicated logistics, it is perhaps a miracle that any blood samples from the Global South are collected and analysed at all. DBS has the potential to change that, allowing blood testing to take place in remote locations, where it has long been suspected that doping is taking place unhindered.

Luginbühl and Gaugler’s Study merely means that the DBS process may need to be refined before being used effectively in anti-doping. WADA is likely to already be aware of the issues it outlines, as the Editor of Drug Testing and Analysis – where the Study was published – is Mario Thevis. He is a prominent anti-doping scientist and a Member of WADA’s Prohibited List Expert Group. If the issues in the Study are not addressed, then the danger is that anti-doping rule violations (ADRV) revealed though DBS will be far too easy for anti-doping’s many expert lawyers to challenge.