Towards the development of a direct method for the detection of autologous blood transfusions in sport doping: targeting the red blood cells “storage lesions”

doi:10.21203/rs.3.rs-3000420/v1

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Research Article

Towards the development of a direct method for the detection of autologous blood transfusions in sport doping: targeting the red blood cells “storage lesions”

https://doi.org/10.21203/rs.3.rs-3000420/v1

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published 01 Dec, 2023

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Background and objective: Autologous blood transfusion is a form of “blood doping” banned by the World Anti-Doping Agency. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions” that occur to red blood cells between the withdrawal and the re-infusion.

Methods: We specifically focused on (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2 (PRDX2), (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles. All of the above were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh and blood samples mixed “ex vivo” to simulate an autotransfusion.

Results: Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to the reduced concentration of Band 3 protein and glycophorin A in the erythrocyte membrane, to the externalization of phosphatidyl serine and to the reduced concentration of CD47. In parallel, increasing levels of erythrocyte microparticles are produced during storage.

Conclusions: The most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

Doping in sport

Blood doping

Autologous blood transfusions

Glycophorin-A

Band 3 complex

Peroxiredoxin-2

CD47

Phosphatidylserine

Red blood cell microparticles.

Autologous blood transfusions (ABT) are a form of "blood doping" banned in sport.

ABT are not detectable by direct methods, but only by the individual, longitudinal monitoring of hematological parameters. The efficacy of this approach can be reduced by the effects of several confounding factors and/or masking strategies.

The multiparametric procedure here proposed considers different biomarkers of "blood storage", with special focus on the diagnostic markers of the so called "storage lesions" of the red blood cells, namely:

changes regarding erythrocytes membrane proteins, i.e., Band 3 single protein and macro-complex and Glycophorin-A (GYPA);
alteration of Peroxiredoxin-2 (PRDX2), classified as a biomarker of oxidative stress due to its antioxidant function;
biomarkers of erythrocyte senescence, such as phosphatidylserine (PS) and Cluster of Differentiation 47( CD47), also known as Integrin Associated Protein (IAP);
micro-vesiculation phenomena, with formation and release of red blood cell microparticles (RBM).

The term “blood doping” indicates the misuse of substances or techniques aimed to increase the number of red blood cells (RBCs) in the bloodstream, improving oxygen delivery to muscles and enhancing athletic performance, especially in endurance sport disciplines. A wide group of banned practices are considered blood doping, including homologous (HBT) or autologous (ABT) blood transfusion of whole blood or erythrocytes concentrates (reviewed in [1]). Any manipulation of blood and/or blood is components considered doping and prohibited by the World Anti-Doping Agency (WADA) [2]. Officially approved and validated direct tests are available for the majority of the blood doping strategies and for HBT (reviewed in [3]), but one of the greatest challenges for anti-doping scientists is the development of a direct method to detect the recourse to ABT.

In details, ABT consist of the withdrawal of a certain amount of blood (typically, in the range 400–500 mL), its storage for a sufficient period (maximum of 40 days) to allow the full replacement of total blood volume, and its reinfusion, usually a short time before the competition. Although the risks related to immunoreactions is extremely low in the case of ABT compared to HBT, the risk of cardiovascular diseases is still high. Moreover, inappropriate conditions of storage may lead to infections and other complications.

Currently, the only officially validated method for ABT detection is indirect: it is based on the individual, longitudinal monitoring of specific hematological parameters carried out in the framework of the hematological module of the Athletes’ Biological Passport (ABP) [4]. Deviations from each athlete’s individual reference values can indicate the recourse to substances and/or methods aimed to obtain a “blood doping” effect. However, ABP has some limitations [5], such as the impossibility to detect micro-dosage transfusions of RBC, the recourse to masking agents (e.g. plasma volume expanders), and the generation of confounding profiles due to specific physical and/or environmental conditions (e.g. altitude and/or high-intensity training) [6].

During storage, RBCs go through numerous irreversible changes, that involve membrane markers, cytoskeletal proteins, and enzymes, resulting in morphological, biochemical, and functional modifications, collectively defined as “RBC storage lesions” [7]. More specifically, erythrocytes undergo oxidative lesions, loss of ATP, changes in the metabolome and proteome [8] that cause modifications of shape and volume, and membrane integrity. It is widely known that during storage, the morphology of RBCs can be significantly modified: erythrocytes lose their characteristic discoidal shape and develop progressive spiculation on their surface (echinocytes), accruing spherical form (spherocytes) [9, 10]. The most probable sites of damage will be the cytoskeletal and membrane proteins [11]. Moreover, these membrane modifications lead to increased RBCs osmotic fragility and changes in electrolyte imbalance, resulting in augmented oxidative stress and damages of those enzymes responsible for the oxidant-antioxidant balance. Due to their altered shape and compromised functionality, RBCs experience early senescence and begin the apoptotic process. Furthermore, senescent erythrocytes undergo a reduction of their dimension, due to the phenomena of micro-vesiculation, with the parallel formation of RBCs microparticles (RMPs) [12, 13].

These damages involve structural, morphological, metabolic, and functional irreversible modifications.

2.1 Aim, design and setting of the study

In this study we focused on the RBCs “storage lesions” to develop a direct method for the detection of ABT, complementing the current indirect approach based on the hematological module of the Athlete Biological Passport, that, as anticipated above, is centered on the individual, longitudinal monitoring of some key hematological parameters. More specifically, our research focused on four different aspects of storage damages, detailed below:

Changes regarding RBCs membrane proteins, such as Band 3 single protein and macro-complex and Glycophorin-A (GYPA), were investigated over storage time. Band 3 macro-complex represents the main erythrocytes transmembrane protein, and it was demonstrated to bind Rh complex [14] and various cytoskeletal proteins [15], having an important role in the maintaining of membrane integrity, cytoskeletal organization [11, 16] and in maintaining electrolyte imbalance [17]. The surface biomarker GYPA represents the major sialoglycoprotein of RBCs membrane and it is generally considered to be specific to erythroid cells [18].

Alteration of Peroxiredoxin-2 (PRDX2), the third most abundant protein of RBCs, classified as biomarker of oxidative stress due to its antioxidant function. RBCs are constantly exposed to oxidative stress and PRDX2 function to reduce peroxides is very important for their survival [19]. During storage oxidative stress increases and PRDX2 function is impaired, due to the impossibility of the enzyme to return to its reduced form [20].

Biomarkers of RBCs senescence, such as Phosphatidylserine (PS) and CD47. PS is a phospholipid of RBCs membrane. In the standard condition, it is in the inner leaflet of the plasmatic membrane, but it migrates to the outer side in case of senescent or damaged erythrocytes [21]. PS exposure on RBCs determines the beginning of the apoptotic phase and represents a clear signal for the macrophage for phagocytosis [22]. On the contrary, CD47 is a transmembrane protein involved in the regulation of cell survival [23]. A decline of cell surface CD47 expression during RBCs aging in vivo promotes the clearance of senescent RBCs [24]. During storage, in an ex-vivo context, the mechanism of clearance is impaired. For this reason, we selected PS and CD47 as biomarkers of RBCs senescence since they cannot be removed in simulated storage conditions.

Evaluation of the micro-vesiculation phenomena in RBCs over storage time. Among the irreversible “RBC storage lesions” there is a severe reduction of erythrocytes size, due to the formation of RMPs [25]. RMPs are membrane particles measuring around 1 nm, produced by stimulated or apoptotic erythrocytes through modulation or membrane lipid organization and cytoskeleton reorganization. RMPs express antigens characteristic of their original cell type [12]. In vivo conditions, they are usually eliminated by the clearance mechanism, but during storage, this process is impaired, still being traceable after the reinfusion. In the present study, we investigated the variation of RBCs parameters described above, before, and after storage of blood samples under standard blood banking conditions.

Finally, we simulated ex vivo blood transfusion experiments, assessing the relevance of the selected parameters as potential biomarkers of ABT.

2.2. Sample collection and storage

Samples from elite athletes, tested negative to antidoping screening tests, were collected in EDTA-treated tubes and enriched with 14% of citrate phosphate dextrose with adenine (CPDA, Sigma-Aldrich, USA) and stored under standard blood banking conditions at 2–8°C for 20 (T1) or 40 days (T2). In compliance with the WADA International Standards for Laboratories [26], all samples selected for this study were from athletes who had priorly given consent to the use of their samples for research purposes, according to the information reported on the doping control form. Samples were analyzed within 24-hours from collection (T0) and/or after a fixed period of storage. To simulate an autologous blood transfusion, a group of samples was obtained by mixing freshly collected whole blood samples with 10% of stored blood (T2), following a procedure already described by our group [13]. To evaluate age-related differences, erythrocytes were separated by density, using Percoll (Sigma-Aldrich, USA) discontinuous gradients, according to the procedure described by Bosch et al [27].

All biomarkers considered in this study are listed in Table 1, that reports also the corresponding analytical techniques for their determination and the different storage times.

Table 1

Summary of the biomarkers, biological matrices and analytical techniques considered in the present study.
Biomarkers	Matrix	Storage time (days)	Analytical technique
Glycophorin-A	Whole blood	0; 20; 40	Flow Cytofluorimetry
Band 3	Whole blood; erythrocytes fractions	0; 20; 40	Flow Cytofluorimetry; Western Blot
Peroxiredoxin-2	Whole blood; erythrocytes fractions	0; 20; 40	Western Blot
CD47	Whole Blood	0; 20; 40	Flow Cytofluorimetry
Phosphatidylserine	Serum; mixed	0; 20; 40; Mixed (90% t0 + 10% t40)	ELISA assay
Red Blood Cells Microparticles	Whole blood; mixed	0; 20; 40; Mixed (90% t0 + 10% t40)	Flow Cytofluorimetry

2.3. Flow Cytofluorimetry analysis

Whole blood samples were diluted in PBS to reach the concentration of 1 million RBCs/100 µL. Aliquots of 100 µL were stained with 5 µL of specific primary antibodies: anti-Glycophorin-A-PE (Beckman Coulter Srl, Italy), anti-Band3-FITC (ThermoFisher, USA), anti-CD47-PE (ThermoFisher, USA) or eosin-5’-maleimide dye (1:5 E5M, ThermoFisher, USA). Samples were incubated for 45 minutes at RT, washed, and assayed by the flow cytofluorimeter (Cytomics FC500, Beckman Coulter, USA). The settings used were the following: Forward Scatter (FS) discriminator set at the detector channel 20; 50000 events/sample. To analyze RMPs, it was developed a time-saving approach that allows RMPs identification without extraction [25, 28]. Proper gates were set on CXP Analysis Software (Beckman Coulter, USA), according to cell morphology and surface protein expression. Fluorescent microspheres (Beckman Coulter, USA) were used to include in the analysis only events of 1µm or lower, which is the conventional diameter of RMPs [29] (Fig. 1a). The surface expression GYPA was also considered (Fig. 1b) and three gates were defined to identify RBCs (Fig. 1c, gate C), transition events (TE) (Fig. 1c, gate H), and RMPs (Fig. 1c, gate I).

2.4. Western Immunoblotting assay

Ghost membrane extraction was performed according to Low et al. [30]. Proteins, separated by SDS-PAGE, were transferred to a PVDF membrane (Immobilon, Italy) and stained with primary antibodies against PRDX2 (1:1600, R&D Systems, USA) and Band 3 protein (1:100000, ThermoFisher, USA). Membranes were then incubated with secondary antibody (Gt anti-MS IgG, HRP-conjugate, ThermoFisher, USA). After the substrate addition, images were acquired with ImageQuant LAS400 (GE-Healthcare, USA). Bands of interest were quantified by densitometry utilizing the GasEPO software (WADA).

2.5. ELISA assay

PS levels were measured through an enzyme-linked immunosorbent assay (ELISA) sandwich kit (LifeSpan Biosciences, USA). Plates were read on a Victor³ Multilabel Plate Reader (Perkin Elmer, USA) at 450 nm.

2.6. Data analysis

Basic statistics was performed using Statistica 12.0 (Statsoft, USA) and GraphPad Prism (GraphPad Software Inc, USA) softwares. To compare the mean values of the RBCs parameters within different experimental group, repeated measures analysis of variance (ANOVA) was used. Tukey HSD test was used as a post-hoc analysis. Statistical significance was set at p < 0.05.

3.1. RBCs surface markers and membrane proteins

3.1.1. Band-3

The results of Band 3 concentration over storage time, are reported in Fig. 2a (flow cytofluorimetric assays) and in Figs. 2b and c (western immunoblotting assay). FC analysis demonstrated that Band 3 expression levels decrease over storage by 19% after 20 days and 39% after 40 days, with statistical significance from T0 to T2 (p < 0.05). No significance differences were found after 20 days of storage. Levels of Band 3 through western immunoblotting assay, were performed on extracted erythrocytes ghost membranes [30] of freshly collected and stored samples, aliquoted at T1 (20 days of storage) and T2 (40 days of storage) (n = 7). It was also evaluated whether the presence of CPDA reduces the progressive decrease of the selected biomarkers over storage time. Results are shown in Fig. 2b and c and confirmed the previous data of FC, confirming a progressive and statistically significant reduction of Band 3 protein over storage time, expressed as the mean band intensity normalized with β-actin. Besides, Band 3 protein is a component of the Band 3 macro-complex, which binds various other membrane and cytoskeletal proteins. We decided to further explore changes on Band 3 macro-complex in addition to the single protein, to have a better framework of the cytoskeletal damage due to storage. In this case, we did not use a specific antibody for the Band 3 protein, but the E5M dye, which binds to a ξ-NH2 group of Lys-430 on Band 3 macrocomplex and a deficiency in Band 3 results in a decrease of fluorescence intensity [31]. As shown in Fig. 2d, our results indicate that the presence of CPDA protect the integrity of the RBCs over 20 days of storage, but it is not sufficient to prevent RBCs membrane damages after 40 days. Band 3 levels decrease with statistical significance with a p-value lower than 0.5 after 40 days both with and without the addition of CPDA solution (Fig. 2d). Interestingly, while Band 3 single protein levels have shown a trend of decrease after 20 days of storage without CPDA solution (Fig. 2a), this phenomenon is reverted when the whole Band 3 macro-complex is analyzed (Fig. 2d). Furthermore, Band 3 concentration was estimated in young, old, and middle-aged erythrocytes, to assess whether the above-mentioned changes were correlated with an accumulation of senescent RBCs, caused by impaired clearance in ex-vivo conditions. Results are shown in Fig. 2e. As it can be seen, there is a trend of decreasing in Band 3 levels comparing old RBCs to younger ones, consistently with our hypothesis of the reproducibility of Band 3 expression pattern in stored samples and aged erythrocytes. Moreover, our results show that Band 3 levels in young erythrocytes after 20 days of storage are lower than freshly collected samples, with statistical significance and a p-value lower than 0.05. Unfortunately, results after 40 days are not available due to the fragility of the cells, showing severe age-related degradation phenomena.

3.1.2. GYPA

GYPA levels were analyzed by flow cytofluorimetry in freshly collected blood samples and stored samples, respectively after 20 days (T1) and 40 days (T2). The results are expressed as the average value of the Mean Fluorescence Intensity (MFI) of seven different blood samples. GYPA resulted in a progressive decrease during storage (47% after 20 days and 63% after 40 days). Results from both T0 to T1 and T2 showed statistical significance with a p-value lower than 0.05. GYPA levels decreased significantly also between 20 and 40 days of storage (Fig. 2f).

3.1.3. GYPA/Band 3 ratio

We also analyzed how the ratio between GYPA levels and Band 3 protein, measured as described above, changes during storage. GYPA/Band 3 ratio showed a reduction by 32% and 37%, respectively, after 20 and 40 days of storage, with statistical significance both between T0 and T1 and T0 to T2 (Fig. 2g).

3.2. Biomarkers of oxidative stress: PRDX2

Since PRDX2 is located in the cytosol, to allow the PRDX2 specific antibody to cross the membrane and bind to the target protein, a preliminary fixation and permeabilization pretreatment of the RBC membrane would be necessary before the flow cytofluorimetric assay [32]. Unfortunately, due to the increased fragility of RBCs over storage time, this process did not allow to obtain repeatable results, and the differential sensitivity was overall unsatisfying. Therefore, the levels of PRDX2 in the RBCs before, during, and after storage were assessed by western immunoblotting assay, which is widely used for its analysis [33–35], on erythrocytes ghost membranes. PRDX2 showed an inverted pattern of expression over storage time: for indeed, PRDX2 levels were shown to increase after 20 days of storage and this phenomenon is even more evident without CPDA solution. Results showed in Fig. 3a indicate that there is an augmentation in PRDX2 levels immediately after 20 days, with statistical significance and a p-value lower than 0.05. This increase is maintained after 40 days, but without a progressive trend of growth. These results can be explained by an accumulation of PRDX2 due to the increased storage-dependent oxidative stress. In these conditions the reduction of oxidized PRDX2 is impaired, resulting in the loss of the protein antioxidant function. PRDX2, then, accumulates in its oxidized form, migrating from the cytosol to the membrane, becoming more detectable by the analysis of RBCs ghost membranes through western blot. Figures 3a and 3b show respectively the progressive increase of PRDX2 concentration of individual samples over storage time. PRDX2 levels were also analyzed in RBCs fraction isolated by density, to assess whether PRDX2 concentration was higher in older RBCs as well as in stored ones. Figure 3c describes our results on PRDX2 levels on freshly collected and stored erythrocytes fractions. No differences were found between aged and young RBCs membranes.

3.3. RBCs markers of senescence

3.3.1. CD47

Figure 4a show the trend pf CD47 concentration in whole blood samples (n = 9) as a function of the storage time. As it can be seen, CD47 concentration on RBCs surface decreases over storage time. In the presence of the CPDA solution the extent of “RBCs storage lesions” is less pronounced after 20 days of storage, but membrane damages still occur after 40 days. CD47 levels decrease with statistical significance with a p-value lower than 0.5 after 40 days both with and without the addition of CPDA solution (Fig. 4a). No differences were found after 20 days of storage compared to freshly collected samples.

3.3.2. Phosphatidylserine

PS levels were evaluated in freshly collected (n = 9), stored (T1 n = 13, T2 n = 13) and in mixed samples (n = 8) by a sandwich ELISA assay. Our data indicate the progressive formation of apoptotic cells over storage time. Figure 4b shows that the expression of PS is higher in stored samples already after 20 days of storage. PS levels become even higher after 40 days of storage and this increase is still traceable in mixed samples (90% fresh blood + 10% stored blood). The mean population values of PS concentration were calculated and reported in Table 2, with the related standard error and the sum of the mean ± 95% of the standard error. For both experiments, Tukey HSD post-hoc analysis highlighted a statistically significant difference between PS concentration between T0 samples and T1, T2, and mixed (90% fresh + 10% stored) samples.

Table 2

Mean values of phosphatidylserine (PS) concentration in the different experimental groups, with related standard error and the sum of the mean ± 95% of the standard error.
Time	PS mean	PS std error	PS -95%	PS + 95%	N
T0	0,48	0,43	-0,39	1,36	9
T1	2,51	0,36	1,78	3,23	13
T2	5,01	0,36	4,28	5,74	13
Mixed	3,91	0,46	2,98	4,84	8

To simulate the condition of the ABT, values of PS in serum were analyzed focusing on freshly collected blood samples (T0) and ex vivo transfused samples (“Mixed 10%”). As shown above, PS values are always higher in mixed samples compared to freshly collected ones. Therefore, a threshold of PS concentration in serum can be selected to discriminate between transfused and un-transfused samples

3.4. RMPs formation

RMPs formation in freshly collected (n = 30), stored for 20 (T1, N = 16) and 40 days (T2, n = 16), and “Mixed 10%” (n = 24) whole blood samples was analyzed without extraction, with a gating setting through FC. The result is expressed by the mean of the morphological events counted within the gate (< 1 micrometer). Figure 5 shows the RMPs events expressed as a percentage of RMPs/RBCs (a) and RMPs/All events (b) ratios in freshly collected, stored with CPDA solution and mixed samples. Our results show the increase in RMPs concentration in stored (T2) or mixed samples. These data suggest that RMPs might be still traceable in transfused samples, although their concentration is higher in T2 samples. On the contrary, no differences in RMPs concentrations are detectable between freshly collected samples and samples analyzed after 20 days of storage with the addition of CPDA, resulting in a sudden increase in RBCs micro-vesiculation phenomena between 20 and 40 days.

Further information can be obtained also by following the transitional events (TE). TE/RBCs and TE/All events ratios remain stable over storage time. Flow cytofluorimeter settings allowed to count a maximum of 100000 events in each gate and the total amount of events (“All” events). RMPs/RBCs, RMPs/All events, TE/RBCs, TE/All events, and relative percentages were analyzed. The mean values of the population regarding percentages RMPs/RBCs and RMPs/All events ratios were calculated and reported in Table 3, with the related standard error. For both experiments, Tukey HSD post-hoc analysis highlighted a statistically significant difference between RMPs concentration between T0 samples and both T2 samples stored with CPDA solution and Mixed 10%, while no differences were found between T0 and T1 samples.

Table 3

Mean values of the concentration percentage of RMPs/RBC and RMPs/All events in the different experimental groups, with related standard error.
Time	RMPs/RBC100 mean*	RMPs/RBC100 std error*	RMPs/All100 mean*	RMPs/All100 std error*	N
T0	0,78	0,19	0,77	0,17	30
T1	1,05	0,37	1,04	0,34	8
T2	5,16	0,40	4,90	0,36	7
Mixed	4,23	0,21	4,02	0,19	24

In addition to the above, we also measured the levels of RMPs in freshly collected blood samples and in stored samples without CPDA solution. As expected, RMPs formation over storage time is higher without the addition of CPDA solution and it is detectable even after 20 days, but with clearer and statistically significant results after 40 days (Fig. 6a and b). Figure 6c and d show the box plot of the percentage of the mean value of RMPs/RBCs ratio, respectively after 20 days (Fig. 6c) and 40 days of storage (Fig. 6d), with and without CPDA. RMPs formation is significatively higher without the addition of CPDA solution, with higher values after 40 days, demonstrating a reduction of the percentage of RBC showing “storage lesions” in presence of a blood preservative.

Finally, the role of preservatives (in our case, CPDA), was also assessed: while no differences in TE/RBCs ratio percentage were highlighted between groups when samples were stored with CPDA solution, it was demonstrated that this ratio increases significantly (p < 0.05) when samples were stored without CPDA solution (Fig. 7).

Based on the above results, we focused our analysis on freshly collected samples (T0) and mixed samples (90% fresh and 10% stored), to better understand the possible detectability of RMPs in transfused samples. RMPs/RBCs*100 values were calculated for T0 and Mixed 10% samples. As we reported in Fig. 5, the concentration of detected RMPs is always higher in the artificially transfused samples rather than in fresh un-transfused ones. Thus, RMPs resulted as a more secure index to identify transfused samples from the un-transfused ones. Moreover, a detection strategy based on the RMPs concentration threshold can be applied to both autologous and homologous transfusion practice in presence of a storage period, since micro-vesiculation phenomena are storage-dependent and independent from the transfusion method. Based on those data, specific values can be calculated to identify a threshold of positivity for anti-doping tests.

The development of innovative method to detect the recourse to HBT and ABT has always represented a challenge in antidoping analysis. In the present study, a multi-parametric approach, based on the “RBC storage lesions”, was suggested as a strategy to detect ABT for doping purposes, that might complement the effectiveness of the indirect testing procedures based on the hematological module of the ABP. “Storage lesions” originate from biochemical events that lead to irreversible changes and alterations in long-term stored RBCs after blood banking. The evaluation of the biochemical effects of the transfusion using RBCs stored for a sufficiently long time can therefore represent an effective tool to detect the illicit recourse to blood transfusions. In this context, the results obtained in this study offer a broad framework of the changes that occur to RBCs after long-term storage, allowing to hypothesize the possibility of detecting those changes after their reinfusion. From the data we obtained, the following conclusions can be drawn:

The irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, are closely related to the reduced concentration of Band 3 protein and GYPA in the erythrocyte membrane over storage time, highlighting the suitability of those two proteins as biomarker of “RBC storage lesions”.

Erythrocytes undergo a fastened senescence and apoptotic process due to progressive, storage-dependent oxidative stress, revealed by the increase of PS-exposing RBCs and the reduced concentration of CD47. The combined analysis of those two biomarkers may help anti-doping authorities to detect the presence of transfused blood in whole blood samples.

We applied a novel method to evaluate RMPs formation, through a system of gating by flow cytofluorimetry. This approach resulted to be efficient and timesaving, avoiding the complex procedure of RMPs extraction and increasing the repeatability of the method. The efficacy of this approach allowed us to define a threshold of RMPs concentration to identify transfused samples, with an accuracy of 75%. The actual applicability of this approach should be implemented with further experiments, but it may already be used by the anti-doping laboratories as a fast-screening strategy, activating the more complex procedure for the extraction of RMPs only in case of suspicious results.

Based on the above, the most promising method to discriminate freshly collected samples from stored or transfused ones should be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

Although of utility for the development of a multiparametric strategy for the detection of autologous blood transfusions, the present pilot study has two major limitations, outlined below:

The biomarkers of storage investigated in this study were analyzed in freshly collected, stored, and “artificially transfused” blood samples (obtained, as previously described, through an ex-vivo mixture constituted by 90% freshly collected blood sample and 10% stored blood sample). Our results need therefore to be confirmed also on samples collected after a real transfusion, to assess the suitability of the selected biomarkers for the development of a novel method to detect the illicit recourse to ABT.

The pool of the tested samples in this study was limited to those samples belonging to athletes that resulted negative in the context of the anti-doping analysis and who gave their preliminary consent for their samples to be used for research purposes.

Increasing the pool of the tested samples will allow to reduce the above drawback, also allowing to highlight more pronounced and statistically significant differences between freshly collected and transfused samples.

ABT

autologous blood transfusion

CD47

cluster of differentiation 47

CPDA

citrate-phosphate-dextrose anticoagulant solution

flow cytofluorimetry

GYPA

glycophorin A

HBT

homologous blood transfusion

IAP

Integrin Associated Protein

PRDX2

peroxiredoxin-2

phosphatidylserine

RBC

red blood cell

RMP

red blood cell microparticle

transition event

Ethics approval and consent to participate

Selection of samples was carried out in compliance with the WADA Ethical Code: all samples selected for this study were collected for routine anti-doping tests and were from athletes who had priorly given written consent to the use of their samples for research purposes, according to the information reported on the doping control form.

Consent for publication

Not applicable.

Availability of data and material

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Competing interests

Giorgia M. Biasini, Francesco Botrè, Xavier de la Torre, and Francesco Donati declare that declare that they have no competing interests.

Funding

The study was supported in part by a research grant from the Section of Vigilance on Doping of the Italian Department of Health (Grant “SVD_2022_2” to F Botrè).

Authors Contribution

FD preliminarily designed the original research plan, processed the results and revised the paper; GMB finalized the research plan, performed the experimental part of the study, collected and processed the results and drafted the paper; XdT discussed the results and contributed to writing the paper; FB obtained the funding for the study, supervised the experimental plan, discussed the results, fully reviewed, revised and finalized the paper and submitted it; all authors approved the submission.

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Towards the development of a direct method for the detection of autologous blood transfusions in sport doping: targeting the red blood cells “storage lesions”

Status:

Journal Publication

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Abstract

Figures

Key Points

1. Background

2. Methods

2.1 Aim, design and setting of the study

2.2. Sample collection and storage

2.3. Flow Cytofluorimetry analysis

2.4. Western Immunoblotting assay

2.5. ELISA assay

2.6. Data analysis

3. Results

3.1. RBCs surface markers and membrane proteins

3.1.1. Band-3

3.1.2. GYPA

3.1.3. GYPA/Band 3 ratio

3.2. Biomarkers of oxidative stress: PRDX2

3.3. RBCs markers of senescence

3.3.1. CD47

3.3.2. Phosphatidylserine

3.4. RMPs formation

4. Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1