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biology
Review
Relation between Exercise Performance and Blood Storage
Condition and Storage Time in Autologous Blood Doping
Benedikt Seeger and Marijke Grau *
Citation: Seeger, B.; Grau, M.
Relation between Exercise
Performance and Blood Storage
Condition and Storage Time in
Autologous Blood Doping. Biology
2021,10, 14. https://doi.org/
10.3390/biology10010014
Received: 15 November 2020
Accepted: 25 December 2020
Published: 29 December 2020
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censeeMDPI, Basel, Switzerland. This
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Creative CommonsAttribution (CCBY)
license(https://creativecommons.org/
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Molecular and Cellular Sports Medicine, German Sport University Cologne, 50677 Cologne, Germany;
benediktseeger@web.de
*Correspondence: m.grau@dshs-koeln.de; Tel.: +49-221-4982-6116
Simple Summary:
Autologous blood doping (ABD) refers to sampling, storage, and re-infusion of
one’s own blood to improve circulating red blood cell (RBC) mass and thus the oxygen transport
and finally the performance capacity. This illegal technique employed by some athletes is still
difficult to detect. Hence knowledge of the main effects of ABD is needed to develop valid detection
methods. Performance enhancement related to ABD seems to be well documented in the literature,
but applied study designs might affect the outcome that was analyzed herein. The majority of
recent studies investigated the effect of cold blood storage at 4
◦
C, and only few studies focused on
cryopreservation, although it might be suspected that cryopreservation is above all applied in sport.
The storage duration—the time between blood sampling and re-infusion—varied in the reported
literature. In most studies, storage duration might be too short to fully restore the RBC mass. It is
thus concluded that most reported studies did not display common practice and that the reported
performance outcome might be affected by these two variables. Thus, knowledge of the real effects of
ABD, as applied in sport, on performance and associated parameters are needed to develop reliable
detection techniques.
Abstract:
Professional athletes are expected to continuously improve their performance, and some
might also use illegal methods—e.g., autologous blood doping (ABD)—to achieve improvements.
This article applies a systematic literature review to investigate differences in the ABD meth-
ods and the related performance and blood parameters owing to different storage conditions—
cryopreservation (CP) and cold storage (CS)—and different storage durations. The literature research
resulted in 34 original articles. The majority of currently published studies employed CS during ABD.
This contrasts to the applied storage technique in professional sports, which was mainly reported
to be CP. The second outcome of the literature research revealed large differences in the storage
durations applied, which were in the range of one day to 17 weeks between blood sampling and
re-infusion, which might affect recovery of the red blood cell mass and thus performance outcome
related to ABD. Data revealed that performance parameters were positively affected by ABD when
a minimal storage duration of four weeks was adhered. This article identified a need for further
research that reflect common ABD practice and its real effects on performance parameters, but also
on related blood parameters in order to develop valid and reliable ABD detection methods.
Keywords:
autologous blood doping; exercise performance; storage conditions; storage duration;
red blood cells
1. Introduction
International world class athletes are expected to continuously improve their per-
formances [
1
,
2
] and some athletes not only use legal but also illegal methods to achieve
this improvement [
2
,
3
]. Autologous blood doping (ABD) appears to be a widely applied
illegal method to enhance an athlete’s performance [
4
,
5
]. The reason why ABD represents
a preferred method might be due to the finding that the muscular oxygen (O
2
) supplying
Biology 2021,10, 14. https://doi.org/10.3390/biology10010014 https://www.mdpi.com/journal/biology
Biology 2021,10, 14 2 of 14
system is a major limiting factor in endurance sports [
6
] and ABD is able to increase this
system without being detectable so far [
7
,
8
]. This assumption is further supported by the
recent disclosures and the ongoing doping investigation and trial in Germany referred to
as “Operation Aderlass” [
9
,
10
]. However, since doping does not only harm the sport’s
reputation [
11
], but also possibly the athlete’s health [
12
,
13
], it is important to develop a
reliable and valid ABD detection method. This systematic review aims to analyze pub-
lished protocols of applied ABD techniques and the related effects on tested performance
parameters in order to evaluate the effectiveness of the methods applied, especially related
to changes of the
in vivo
red blood cell (RBC) system, and to compare the applied methods
with actual doping practice. This knowledge will be needed to assess the real effects of
published ABD-related protocols in order to understand the related changes in the RBC
system necessary for the development of ABD detection methods.
The ABD procedure consists of three major steps: (1) the blood sampling from a
subject, (2) the processing and storage of the blood or blood components, and (3) the
re-transfusion of whole blood or a red blood cell (RBC) concentrate to the initial donor [
4
].
Each of these steps can be implemented with some variations, which in turn might affect
the effect size of ABD [
4
]. First, the donated blood volume (BV) can vary [
12
]. Second, two
different storage techniques, cryopreservation (CP) or cold storage (CS), are applied [
14
].
Thereby, CP refers to blood storage between
−
65–
−
140
◦
C, while CS refers to blood storage
at 4
◦
C, which is equivalent to refrigerator level [
14
,
15
]. The storage techniques will be
explained in more detail in Section 3.1, when they are discussed. The storage duration
might differ between the storage techniques since CS stored blood is durable for 35–42 days,
while CP allows a blood storage for up to ten years [12,16].
The blood preparation and storage methods of the blood have not been compared so
far. This appears to be a research gap, because the restoration of RBC mass after sampling
is an important factor for the effect size of ABD. In addition to that, the storage technique
might influence the quality of the re-infused RBC. It is widely agreed that an increase in
RBC and a corresponding increase of the hemoglobin mass (Hb) is the major determinant
of ABD-related performance enhancements [
3
,
12
]. ABD might affect RBC structure and/or
function, which might affect exercise performance [
17
,
18
], but the precise mechanisms
related to the different ABD methods remain to be investigated Third, according to current
literature, the amount of re-infused blood after storage varies between 135 and >900 mL,
but a possible dose response relation of re-transfused blood and performance parameters
was not described so far [19,20].
The aim of this review was to analyze and compare the current literature regarding
reported differences in the storage conditions and the storage duration applied during
ABD and to assess the provided changes in exercise performance in relation to the study
design. This review also aimed to direct the focus on RBC changes caused by the ABD
techniques. This information may provide new starting points for anti-doping advocates
in the development of ABD detection techniques.
2. Method
A systematic search protocol was constructed that followed the PRISMA guidelines
for systematic reviews to ensure the reproducibility of this systematic literature search [
21
].
This procedure is also recommended by the MDPI [22]. The research process is presented
in a flow diagram (Figure 1) inspired by the PRISMA 2009 Flow-Diagram [
21
]. However,
since no meta-analysis was conducted, the final step of the PRISMA 2009 Flow Diagram
“Studies included in quantitative synthesis” was not carried out.
The primary aim, and thus end point, of this review was to evaluate described effects
of blood re-transfusion on exercise performance with a special focus on the described
storage technique and storage duration and to relate these changes to possible changes in
the red blood cell system.
Biology 2021,10, 14 3 of 14
Biology 2021, 10, x 3 of 14
Figure 1. Research process. Flow diagram inspired by PRISMA 2009 Flow Diagram.[21].
To select articles, that are eligible for the reviewing process, a full text search was
conducted. Applied keywords included “autologous blood doping” and “autologous
blood transfusion” combined with either “sport”, “exercise”, or “performance”—using
the fixed Boolean operators of each database. The used databases included PubMed, Bio-
Medsearch, Spolit, Web of Science, EBSCOhost, and ProQuest. The inclusion criteria for
articles of these databases were: (1) articles in peer reviewed journals, (2) the reference to
a sport context, (3) articles investigating blood parameters and/or exercise performance,
and (4) the accessibility of the full text. No restrictions concerning the publication dates
were made. Articles that fulfilled the inclusion criteria in title and abstract were taken into
the record. The first step in the research process led to n = 588 articles. Additionally, a
forward and a backward search was conducted for the identified articles. The search en-
gine Google Scholar (scholar.google.com) was used for these searches. The forward back-
ward search resulted in additional n = 27 articles, thus a total amount of n = 615 articles
was identified for the screening process. During the screening process, n = 527 articles
were removed as they did not meet the review’s criteria. The screening resulted in n = 88
articles for further research. After removing duplicates, a full text examination of the re-
maining n = 48 articles was conducted to identify the articles qualified for the qualitative
analysis. During this step, review articles were removed too, since they do not present
primary data (they were included in the search as they are part of the literature and are
eligible sources to identify applicable articles). The final search process resulted in n = 34
articles.
The analysis of the selected articles focused on two major aspects. First, the blood
storage technique applied (CS or CP) were described and compared regarding possible
differing effects of CS and CP on changes of performance and related blood parameters.
Second, the time interval between blood donation and re-infusion was analyzed. The ef-
fect of improved performance after re-transfusion of stored blood relates to the restoration
Figure 1. Research process. Flow diagram inspired by PRISMA 2009 Flow Diagram [21].
To select articles, that are eligible for the reviewing process, a full text search was
conducted. Applied keywords included “autologous blood doping” and “autologous blood
transfusion” combined with either “sport”, “exercise”, or “performance”—using the fixed
Boolean operators of each database. The used databases included PubMed, BioMedsearch,
Spolit, Web of Science, EBSCOhost, and ProQuest. The inclusion criteria for articles of
these databases were: (1) articles in peer reviewed journals, (2) the reference to a sport
context, (3) articles investigating blood parameters and/or exercise performance, and (4)
the accessibility of the full text. No restrictions concerning the publication dates were made.
Articles that fulfilled the inclusion criteria in title and abstract were taken into the record.
The first step in the research process led to n= 588 articles. Additionally, a forward and
a backward search was conducted for the identified articles. The search engine Google
Scholar (scholar.google.com) was used for these searches. The forward backward search
resulted in additional n= 27 articles, thus a total amount of n= 615 articles was identified
for the screening process. During the screening process, n= 527 articles were removed as
they did not meet the review’s criteria. The screening resulted in n= 88 articles for further
research. After removing duplicates, a full text examination of the remaining n= 48 articles
was conducted to identify the articles qualified for the qualitative analysis. During this
step, review articles were removed too, since they do not present primary data (they were
included in the search as they are part of the literature and are eligible sources to identify
applicable articles). The final search process resulted in n= 34 articles.
The analysis of the selected articles focused on two major aspects. First, the blood
storage technique applied (CS or CP) were described and compared regarding possible
differing effects of CS and CP on changes of performance and related blood parameters.
Second, the time interval between blood donation and re-infusion was analyzed. The effect
of improved performance after re-transfusion of stored blood relates to the restoration of
initial RBC mass and the additional benefit of extra blood volume after the re-transfusion.
Biology 2021,10, 14 4 of 14
The articles were analyzed whether or not a full recovery of RBCs might be reasonable, the
minimal period between donation and re-infusion to gain an effect on exercise performance
and if the length of the period affects the ABD outcome.
3. Results and Discussion
ABD is applied in order to increase RBC mass and thus oxygen transport capacity of
the blood and to improve exercise performance [
23
]. ABD not only increases oxygen deliv-
ery to the working muscle but also increases the heat tolerance of the athletes [
24
]. The In-
ternational Olympic Committee (IOC) banned blood boosting after the 1984 Olympics [
25
].
Flow cytometry methods for membrane surface double population of antigens may reveal
the homologous blood transfusion approach [
26
–
28
] but autologous blood transfusion
is not detected by this method. Thus, new approaches are designed as indirect markers
for blood doping, including total Hb mass measurements, or to test for the excretion of
metabolites of bag plasticizers in the urine. Another attempt to detect autologous trans-
fusions is the Athlete Biological Passport (ABP) [
29
,
30
] which contains the longitudinal
monitoring of biologic measures to identify patterns that might be related to ABD and the
evaluation of such abnormal patterns by a panel of experts. These parameters might be
affected by the storage duration and storage technique via an effect on RBC. Parameters
include RBC count, Hb concentration, hematocrit, mean cellular Hb concentration, mean
cellular volume, and the reticulocyte percentage [
31
,
32
]. Further, OFF-Hr score ([Hb]—60
√
Ret%, normal range: 85–95 [
7
]) and abnormal blood profile score (ABPS)—which are cal-
culated parameters and are also mainly dependent on RBC related parameters—are also
monitored in the ABP [
33
,
34
]. This strategy might involve several drawbacks, including
differences in threshold values between the different sports associations and the difficult in-
terpretation of hematological parameters because of wide inter-individual differences [
35
].
Recent data suggest that the RBC function itself might be affected by the ABD process
and that the measurements of certain RBC parameters, for example RBC deformability,
might in the future provide a promising attempt to detect ABD [
17
,
36
]. Still, valid detection
methods are unavailable at present, but might benefit from the knowledge on the real
effects of performance improvements after ABD and the relation to changes in RBC or Hb
concentration.
As mentioned earlier, ABD is capable of improving the muscular O
2
supply system
and thus endurance exercise performance. Several studies analyzed within this review
reported either improvements in the VO
2max
or VO
2peak
, or augmentation of the time to
exhaustion test (TTE) or the time trial test (TT). VO
2max
refers to the maximum possible
O
2
availability (VO
2
) for the working muscles. VO
2
peak is provided if the monitored
oxygen uptake values do not flatten during the exercise test but still increase at time of
termination of the exercise test [
37
,
38
]. Thus, both tests are considered to be the most
adequate to examine a subject’s endurance capacity [
37
,
39
]. Because VO
2max
and VO
2peak
are often used incorrectly as synonyms [
40
], this review will not differentiate between
theses parameters. The TTE measures the time a subject is able to perform a standardized
exercise protocol [
41
]. The TT quantifies the time a subject needs to finish a standardized
task, or the standardized work a subject is able to complete within a fixed time frame [
42
].
The findings of improvements in endurance exercise due to ABD were in line with the
current literature [3,12,19].
The wide range of exercise improvements is probably related to differences in the
implied ABD method. Different storage technique and varying storage durations appear
to be a moderator to the ABD outcome. Therefore, the following section aims to outline
differences in the applied ABD—concerning storage duration and storage technique—
and how these affect performance and performance related blood parameters. Therefore,
Sections 3.1 and 3.2
each discuss a part of the ABD method. To gain an optimal under-
standing, Table 1serves as an overview of the applied methods and important parameters
of the selected articles.
Biology 2021,10, 14 5 of 14
Table 1. Overview of analyzed original articles related to effects of blood storage techniques and storage duration on performance outcome in autologous blood doping (ABD).
Article nFitness Donated Stored Infused TTE/TT VO2m/p RBC Hb
Bejder et al. [20]9M ht 450 mL CS 4 wk 136 mL TT + 4.4% * p/ + 3.3% * + 2.9% *
CS 4 wk 369 mL TT + 5.1% * p/ + 9.8% * + 8.9% *
Bennett-Guerrero et al. [43]4M t 900 mL CS 1 wk 900 mL TTE + 8.4% * m + 8.7% *
4M t 900 mL CS 1 wk 900 mL TTE −2.6% * m + 1.9% *
Berglund et al. [44]
8M+4F
t 3 ×450 mL CP ~14 wk n/a F + 18% *
M + 12% *
Berglund & Hemmingson [45] 6M/F ht 1 ×900 mL + 1 ×450 mL CS 4 wk 1350 mL TT + 5.3% * + *
Berglund et al. [46] 6M/F ht 1 ×900 mL + 1 ×450 mL CS 4 wk 1350 mL TT n/a + 7.9% *
Brien et al. [47] 6M t 2 ×450 mL CP 400 mL TT + *
Buick et al. [48] 11M ht 1000 mL CP 900 mL TTE + 33% * m + 5% * + 8% *
Celsing et al. [49] 9M t 5 ×450 mL CS + CP 1–9 wk 1800 mL m/
Celsing et al. [50] 8M t 8 ×450 mL 5–7 wk n/a m/
Ekblom et al. [51]3M ut 3 ×400 mL 6 wk n/a TTE + 23% * m + 9% * + 18% * + 13% *
4M ut 800 mL 4 wk n/a TTE + * m + * + 4.9% *
Ekblom et al. [52] 5M t 800 mL CS 360 mL m + 8% * + 4.9% *
Goforth et al. [53] 6M ht 2 ×450 mL CP 330 mL TT + 2% * m + 11.9% * + 10% *
Gullbring et al. [54] 6M ut 530–689 mL CS 1 wk n/a PWC + 3.9% * /
Kanstrup & Ekblom [55] M t 900 mL + 500/750 mL CS 500/750 mL TTE + 24% * m + 2–11% * + 4% *
Kots et al. [56] 10M ht/ut 500 mL 3 wk 500 mL TTE + 40% * m/ + 14.8% * + 14.3% *
Lamberti et al. [57] 24M t 450 mL CS/CP 5 wk n/a + * + *
Malm et al. [58]10M t 2 ×450 mL CP 15 wk n/a TTE + 15% * m + 17% * + * + *
30M/F
t 1 or 2 ×450 mL CP 2 wk n/a / /
Mørkeberg et al. [16]23M t3×450 mL CS 4 wk n/a + 3.6%*
CP 10 wk + 6.5% *
Muza et al. [59]. 12M SF 2 ×450 mL CP ~12 wk 600 mL m + 11% * + 11% * + 10% *
Pottgiesser et al. [60] 10M n/a 550 mL CS 7 wk 280–350 mL + * + 5–8% *
Pottgiesser et al. [61] 11M n/a 550 mL/1000 mL CS 1 d 330–550 mL −*
Robertson et al. [62] 9F n/a 2 ×450 mL CP ~9–17 wk 475 mL m + * + * + *
Robinson et al. [63] 6M n/a 1000–1200 mL ~2 wk 1000–1200 mL TTE + * m/
Sallet et al. [64] 7M ht 450 mL CS ~3 wk 450 mL + /
Biology 2021,10, 14 6 of 14
Table 1. Cont.
Article nFitness Donated Stored Infused TTE/TT VO2m/p RBC Hb
Sawka et al. [65] 30M n/a 450 mL CP 600 mL m + * + 10% *
Sawka et al. [66] 9M t 2 ×450 mL CP 6 wk 600 mL m + 11% * +11% * + 10% *
Spriet et al. [67]
4M ht
1×900 mL + 1 ×450 mL CP ~10 wk
450 mL m/ + *
4M ht 900 mL m + 7.5–10.7% * + *
4M ht 1350 mL m + 10–13.3% * + *
Thomson et al. [68] 4M t 2 ×500 mL CP ~12 wk n/a TT + 10% * m + * + *
Thomson et al. [69] 4M ut 2 ×500 mL CP n/a TT + * m + * + *
Turner et al. [70] 7M n/a 2 ×450 mL CP n/a TT + 5.3% * m + * + 3.9% *
Williams et al. [71]5M ht 500 mL CS 3 wk 500 mL TTE/
5M ht 275 mL TTE/
Williams et al. [72] 16M ht 460 mL CP 3 wk 460 mL TTE/ / /
Williams et al. [73] 12M ht 2 ×460 mL CP ~9 wk 920 mL TT + * + * + *
Ziegler et al. [74] 8M n/a 450 mL ~4 wk 245 mL TT + 4.6% * p + 4.8% * + * + *
Legend: VO
2m/p
= VO
2max
/VO
2peak
; m = max; p = peak; RBC = red blood cell; Hb = hemoglobin; CS = cold storage at (4
◦
C); CP = cryopreserved; wk = week(s); d = day(s); M = male; F = female; ht = highly
trained; t = active/moderately trained; ut = untrained; SF = special forces; TT = time trial test; TTE = time to exhaustion test; PWC = physical working capacity test; + = increase; / = no change;
−
= decrease;
* = significant; X.X% = change in %; n/a = not available.
Biology 2021,10, 14 7 of 14
3.1. Blood Storage Techniques
The performed literature analysis suggested two different storage techniques applied
during ABD: CP and CS.
ABD involves sampling of 1–4 units of blood (1 unit corresponds to 450 mL blood)
8–12 weeks before competition to allow recovery of the RBC mass back to pre-donation
level. The blood is centrifuged, the plasma is usually transferred back to the donor or
discarded, and the RBC are stored and re-infused into the donor/athlete 1–7 days before
a competition [
12
]. During CS, RBC were stored in the storage solution SAGM (saline,
adenine, glucose, mannitol) at 4
◦
C, but several published articles suggest that the quality of
RBC decreases during storage at 4
◦
C and that re-infusion of long term stored RBC might be
deleterious [
75
,
76
]. These deleterious effects are reduced when RBC are cryopreserved [
36
].
During CP, blood is sampled in common blood bags and anticoagulated using CPDA-1
solution (citrate, phosphate, dextrose, and adenine). The blood bags are automatically
processed to separate the RBC. Glycerol is added as cryoprotectant [
15
,
77
] and the RBC are
either fast frozen (>100
◦
C/min) and stored below
−
140
◦
C or slowly frozen (~1–3
◦
C/min)
and stored between
−
65 and
−
80
◦
C [
15
,
77
]. The hypothermia of the CP stops the aging of
RBC and thereby prevents the maturity of the cells [
36
], while the 4
◦
C of CS only slows
the aging but does not prevent the cells death [
78
,
79
]. Cryopreserved RBC are thawed
when needed by an automated cell processor device [
12
]. Differences between CS and CP
include the allowed storage time and the occurrence of the so-called storage lesions [
12
].
Maximum storage time of CS ranges between 35 and 42 days, while cryopreserved RBC
are allowed to be stored up to 10 years [
12
,
14
]. Further aspects of the storage durations will
be addressed in Section 3.2. Blood storage is associated with the loss of RBC. According to
recent literature, the RBC loss in CS is about 6% per week, while the loss of RBC during CP
is stated to be about 15% in total due to the processing for the CP storage [14].
From the 34 selected articles, 11 studies used CS while 15 articles used CP, another
three articles used both techniques and five articles did not mention their storage technique.
Improved performance parameters and/or increased associated blood parameters were
presented by nine out of 11 (9/11) CS studies, 14/15 CP studies, and 2/3 CS+CP studies.
Thus, it seems plausible that improvements of the performance might be achieved by
both storage techniques [
20
,
48
,
53
]. It was not possible to argue whether there is a clear
detectable relation of the performance increases and a specific storage technique because
the selected studies showed high variations in their study designs. Only Celsing and
co-workers [
49
], Lamberti and colleagues [
57
], and Mørkeberg and associates [
16
] applied
both storage techniques at the same time. But the study by Celsing et al. [
49
] mixed both
storage techniques within one experiment and thus a comparison between the techniques
was not possible. Furthermore, Lamberti and co-workers did not differentiate between CP
and CS in their results and did not present any data to compare the storage techniques. In
contrast, Mørkeberg and colleagues differentiated between Hb level changes in CP and CS.
The authors detected a 3.6% increase in Hb content after re-infusion of blood stored under
CS conditions and an increase in Hb level of 6.5% after the re-infusion of blood stored
under CP conditions (both groups were re-infused with “all three bags” [
16
] (p. 287). This
might suggest a higher increase in Hb concentration by CP. However, the study design
implemented a time frame of 4 weeks between blood sampling and re-infusion for the CS
group and a period of 10 weeks for the CP group. Because this difference might affect the
recovery of RBC amount and Hb mass
in vivo
, the results were not suitable to compare
both storage techniques.
The data suggested that possible differences between CS and CP in performance and
related blood parameters appeared to be less understood but relevant because the storage
condition and the known quality changes potentially affected the results described. Further,
the quality of the stored RBC was affected by so called storage lesion, which differs between
CS and CP. The precise findings of storage lesions will not be addressed in this review
and were described in detail elsewhere [
80
,
81
]. However, some aspects will be described
Biology 2021,10, 14 8 of 14
herein. RBC lose their deformability, an important parameter to ensure nutrient and oxygen
supply in the microcirculation, during CS [
82
], while RBC deformability of cryopreserved
RBC appears not to be negatively affected [
36
]. The decrease in RBC deformability in
CS might be associated to RBC senescence, which includes not only a reduction in cell
volume, externalization of phosphatidylserine, and reduction in CD47 [
83
], but also a loss
in 2,-3-DPG and ATP and reduction in nitric oxide bioavailability. During senescence in
CS, cell volume loss stiffens the RBC and leads to reduction in RBC deformability [
84
].
Thus, old RBC are prone to hemolysis or phagocytosis during the circulation and might
reduce the outcome during re-infusion [
75
]. Stopped or highly reduced RBC ageing
during CP storage is thus of advantage to preserve the quality of the cells. Further, the
deglycerolization procedure applied during the thawing of CP cells and mainly responsible
for the RBC loss during CP is suggested to destroy mainly old RBC [
85
]. Hence, the
average RBC population age is reduced in CP in comparison to CS. This further positively
affects the RBC deformability, density, size, and O
2
affinity [
18
,
84
], which leads to an
increased diffusion capacity and an improved muscular O
2
supply [
18
,
86
]. Since there
appear to be several differences in the RBC count, Hb content concerning the different
storage techniques, it appears to be relevant to investigate these differences directly.
Furthermore, it is noteworthy that the majority of the articles published in the last 20
years either implied CS [
19
,
20
,
43
,
60
,
61
,
64
] or do not comment on the storage technique [
87
].
This appears to be contradictory to the actual applied ABD techniques in professional
sports [
11
,
88
]. Because the different storage techniques in ABD probably lead to different
blood adaptations and possibly different enhancements of the endurance performance,
there is a need for future studies. These should focus on the effects of CP on functional
blood parameters in relation to endurance capacity. This knowledge will allow to identify
parameters affected by this procedure in order to develop new detection methods.
3.2. Blood Storage Duration
The second variable affecting the outcome of the ABD method is the storage duration.
This refers to the time period between the blood donation from a subject and the blood
re-infusion back to the initial donor. The importance of this phase during ABD is due to the
fact that the increase in RBC mass after re-infusion evolves not from the re-infusion itself,
but from the interplay between the recovery of RBC mass
in vivo
after sampling [
89
,
90
]—
caused by an artificially created momentary anemia [
91
–
93
]—and the following re-infusion
of additional RBC [
3
,
12
]. A decrease in circulating RBC after blood sampling increases ery-
thropoietin (EPO) expression in the kidney and release, which stimulates erythropoiesis in
the bone marrow [
91
]. The production of RBC starts with a pluripotent hematopoietic stem
cell [
89
,
90
]. Among others, EPO is the key driver for of the differentiation of the progenitor
cells into colony forming units-erythroid, proerythroblasts, and erythroblasts
[94,95].
Ery-
thropoiesis lasts approximately five to nine days. Reticulocytes are then released into the
circulation to mature into RBC [90].
The RBC restoration of the RBC mass to pre-donation level [
96
] is essential for the
performance enhancement caused by ABD. An additional aspect of the increase in erythro-
poiesis rate after blood sampling is a probable change in the RBC quality. The neoformation
of young RBC reduces the average age of the circulating RBC pool [
17
]. This reduction
in RBC age results from the interplay of an increased RBC production accompanied by a
normal RBC mortality rate [
17
,
97
]. Young RBC are more deformable compared to other
RBC and are suggested to show increased oxygen transport capacity [
84
,
86
]. Thus, rejuve-
nation of circulating RBC might improve ABD effect size [
18
]. However, further studies are
needed to support this relation.
Although the restoration period appears to be a central element in ABD, only two
articles tested the effect of different storage durations on exercise performance [
51
,
58
].
However, one of the two studies [
51
] tested differences in sampled blood volume in parallel
and thus, the results of this article were less suitable for a comparison of storage durations.
The second study [
58
] tested the exercise performance of the candidates after re-transfusion
Biology 2021,10, 14 9 of 14
of the blood after a storage duration of 15 weeks and 2 weeks, respectively. After a storage
duration of 15 weeks, TTE increased by 15%, VO
2max
by 17%, and also RBC count and Hb
were significantly increased. In contrast, after 2 weeks of storage, Hb level and RBC count
were not significantly altered. A performance test was not carried out [
58
]. These data
indicate that the storage duration should be long enough to allow the blood parameters to
reach the initial level prior to re-transfusion of the stored blood in order to significantly
affect exercise performance [58].
According to current literature, recovery of the RBC content after donation of 500 mL
of whole blood is estimated to last 59 days [
60
,
74
,
98
]. Referring to the selected articles in
this review, only Robertson and co-workers [
62
] respect this recovery period. However,
studies reporting a shorter period between blood donation and re-infusion present im-
provements in endurance performance and related blood parameters [
20
,
51
,
74
]. In general,
the selected articles present a wide range of time frames between blood donation and
re-infusion of 1 day up to 17 weeks [
61
,
62
]. The analysis of these articles revealed that the
minimal recovery period needed to establish significant improvements in the endurance
performance or relevant blood parameters appeared to be 4 weeks. Nine out of thirty-four
articles described a storage time of less than 4 weeks (1 day to 3 weeks). However, only
four of these articles detected significant improvements in TTE of up to 40% [
43
,
56
,
63
],
physical working capacity (PWC) of 3.9% [
54
], Hb level of 14.3%, and RBC of 14.8% [
56
].
These findings suggest that this rather short recovery period might be sufficient for the
tested subjects to at least recover a part of their RBC mass after sampling and that this
might be sufficient to improve the performance. However, the recovery process of RBC
is rather complex and mainly depends on an increased erythropoiesis, which might take
one week to produce additional RBC [
99
]. Therefore, full recovery of the whole RBC mass
during a recovery time of three weeks seems unlikely. Further, three of the four mentioned
articles [
43
,
54
,
63
] sampled a blood volume >500 mL, which might need even more time to
recover than the reported 59 days. The three articles that reported improvements in exercise
performance after a recovery time of less than three weeks showed some limitation in the
study design. The authors scheduled the baseline test after the blood sampling, shortly
before the re-infusion [
43
,
56
,
63
]. Thus, baseline values of the performance parameters, but
also RBC count and level of Hb, might be artificially reduced because of the blood loss.
The increase in performance parameters likely express the effect of a restoration rather
than an “on top” effect that is intended. Thus, a central element of the ABD mechanism—
the need of RBC mass recovery—was disregarded. In contrast to that, Gullbring and
co-workers [
54
]—the fourth article detecting an increase in endurance performance with
storage durations less than four weeks—performed the baseline measurements before the
blood sampling. PWC performance significantly increased by 3.9%. in the post re-infusion
test. However, it cannot be ruled out that this result might also be related to an adaptation
to the test protocol because the authors scheduled several tests between sampling and
re-transfusion as well.
Furthermore, the test performed one day pre re-infusion presents a similar PWC
performance compared to the test 1h post re-infusion. In addition, no significant increase
in the Hb level was detected by the authors. This missing Hb increase presents a further
indicator that the performance increase might be rather related to muscular adaptation than
to changes in the subjects’ blood parameters. In contrast to the aforementioned articles, five
of the articles analyzed herein with a storage duration of less than 3 weeks did not report
any significant improvements in the tested performance—or relevant blood parameters.
Furthermore, all articles with a storage duration of 4 weeks or more present significant
changes in either the endurance performance, relevant blood parameters, or in both.
In summary, articles that described improvements in performance parameters after
blood re-infusion but had storage durations less than four weeks also presented methodical
weaknesses. Articles with storage durations less than four weeks and a reasonable study
design regarding the exercise tests presented no significant improvements, but all articles
with a storage time of 4 weeks or more, which might be needed to fully recover RBC mass,
Biology 2021,10, 14 10 of 14
report improvements in performance and/or in related blood parameters including RBC
or Hb. Thus, it was concluded that a minimum of four weeks is needed as RBC restoration
time for any ABD effect on performance parameters. Future anti-blood doping studies
should consider this information during the development of detection methods.
4. Limitations
The present article provides a better understanding of two key factors affecting the
exercise performance related to ABD. Nevertheless, the small number of key words com-
prised in the applied search string might represent a limitation of the present review. For
instance, in some articles the re-transfusion of RBC is also referred to as induced erythro-
cythemia. However, this term was not included in the conducted search, because it would
also lead to articles not associated to autologous blood doping. Also, the literature search
of this review only focused on peer reviewed original articles and thus excludes alternative
sources such as proceedings, conference papers, and books [100].
5. Conclusions
This review revealed that the storage technique applied, and the storage duration
adhered are two major factors that affect the endurance performance capacity in the context
of autologous blood doping (ABD). The results of the literature review indicated that
cold storage (CS) shows disadvantages compared to cryopreservation (CP) with regard to
possible storage time. Also, the quality of RBC highly reduced during CS, which might
lead to adverse effects for the exercise capacity. The review indicated that CP might be
favored in the ABD procedure. The literature analyzed herein further revealed that a large
proportion of published articles reported storage durations that might be insufficient to
allow the restoration of initial RBC mass. This might in part explain the absence of positive
effects of re-infused RBC on exercise parameters, but might also lead to the assumption
that the reported positive effects of ABD on exercise performance might at least in part
result from other influencing variables.
In order to understand the physiological changes in the RBC system during ABD,
which is indispensable to develop ABD detection techniques, it might be of relevance to
focus on three topics in future research: (1) investigation of the influence of CP on the ABD
outcome because this might be the most used in current doping cases, (2) investigation of
the influence of a proper restoration time on ABD outcome, (3) investigation of the changes
in functional and structural RBC parameters during ABD.
Author Contributions:
Conceptualization, B.S. and M.G.; methodology, B.S.; investigation, B.S.;
writing—original draft preparation, B.S. and M.G.; visualization, B.S.; supervision, M.G.; project
administration, M.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research received funding by the HIFF of the German Sports University Cologne.
Data Availability Statement: Not Applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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