Commonly blood is collected into citrate-dextrose-phosphate solution and RBC concentrates are prepared by the removal of plasma and leukocytes 11 , Polyvinyl chloride PVC bags plasticized with di 2-ethylhexyl phthalate DEHP are typically used for blood collection and storage 6.

The influence of DEHP and two alternative plasticizers, 1,2-cyclohexane-dicarboxylic acid diisononyl ester DINCH and n-butyryl-tri-n-hexyl citrate BTHC , was studied on RBCs stored in PVC bags for 42 days 6. DEHP and DINCH bags offered protection against vesiculation, osmotic stress and loss of Hb, whereas RBCs in BTHC bags stored rather poorly 6.

When RBC concentrates in mannitol-adenine-phosphate solution were stored for 6 wks in PVC bags containing DEHP, DINCH and di 2-ethylhexyl 4-cyclohexene-1,2-dicarboxylate DOTH , or 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester DL9TH and DOTH, there were no significant differences in the total amount of eluted plasticizer, hemolysis and osmotic fragility between the cells in DEHP and non-DEHP blood containers The cells lose their deformability and change their phenotype e.

occurrence of spherical cells and echinocytes. As the intraerythrocytic concentrations of adenosine triphosphate ATP and 2,3-bisphosphoglycerate 2,3-BPG decrease, the O2affinity increases these changes are reversible and restored in vivo within a few days.

The function of cation pumps is impaired resulting in the loss of potassium and accumulation of sodium. There is irreversible loss of parts of the membrane through vesiculation Because the collected blood contains RBCs differing in age, the question arises as to the life span of transfused RBCs.

Luten et al. The degree of hemolysis and the extracellular bicarbonate, potassium, lactate and lactate dehydrogenase levels of SS RBCs were lower than those of LS RBCs. The mean hr post-transfusion recovery of SS RBCs was After the first day, the life spans of the remaining SS and LS RBCs did not differ days vs.

Hod et al. Contrasting fresh RBCs, re-infusion of the older RBCs caused a mean increase in serum total bilirubin by 0.

The higher concentrations of non-transferrin-bound iron correlated with enhanced proliferation in vitro of a pathogenic strain of Escherichia coli.

Therefore, circulating free iron derived from the rapid clearance of transfused, older stored RBCs may promote bacterial infections. Furthermore, major polyunsaturated fatty acids and their oxidation products oxylipins were detected in RBC concentrates stored for 42 days Extracellular vesicles in stored RBC concentrates may cause an inflammatory response in the recipients of older blood 2.

Another negative sign is the occurrence of hemolysis or the leakage of Hb from RBCs Along these lines, measurements of O2max and performance on a cycle ergometer following the re-infusion of autologous RBCs to healthy volunteers have indicated that day RBCs are inferior to 7-day RBCs at delivering O2to tissues 5.

Therapeutically, cryopreserved RBCs play primary roles in military operations and as a reservoir of RBCs with rare phenotypes Scanning electron microscopic investigations of cryopreserved RBCs have yielded increased RBC volumes and shape alterations Although glycerol is a non-toxic substance, its high intracellular concentration and slow rate of osmosis relative to water makes removal of excess glycerol after thawing necessary to prevent osmotic lysis upon transfusion.

The process of glycerol removal can be equally damaging to RBCs as the cooling process. Alternative cryoprotectants include betaine 53 , trehalose 3 , hydroxyethyl starch, and the ice recrystallization inhibitor poly vinyl alcohol In contrast to the cooling, the warming process itself is usually benign and can be done rapidly 9.

Basically, cryopreservation is technically more difficult than refrigeration. However, a high throughput has been made possible by commercial semi-automated cell washing instruments. Severe immune reactions can occur on mismatched transfusion 1.

Whereas for allogeneic transfusions testing of the recipient suffices, for autologous transfusion additionally the packed RBCs must be tested.

The transfusion of RBC concentrates should always be carried out using a standard transfusion set with a filter pore diameter — μm. The RBCs should be given via an exclusive venous port; drugs or other agents should not be mixed with the RBCs or administered simultaneously through the same port Potential risks include transfusion-associated transmissions of bacteria, viruses, parasites or prions and non-immunologically mediated adverse reactions such as transfusion associated circulatory overload TACO , thrombovascular diseases, hyperkalemia, citrate overload and transfusion hemosiderosis Covin et al.

Namely, the authors have described a case of transfusion-related acute lung injury TRALI and hypotension following ABT in a surgical patient Doping-prone athletes probably turned back to RBC transfusion after reliable detection methods for rhEpo were established 23 , Donated cryopreserved RBCs may be stored for up to 30 years, while refrigerated RBCs must be re-infused within 42 days due to increasing storage lesions.

Thus, refrigeration is a less probable strategy for ABT doping because it takes at least a month for full [Hb] restoration after blood donation 24 , 43 , The stored RBCs are probably re-infused to the athletes one to four days before competition Leigh-Smith 26 has earlier provided a brief review of initial work into the performance-modulating effects of RBC transfusion.

Accordingly, the increase in total RBC mass is most important, while the transient increases in blood volume and cardiac output are too short-lived to be of real importance. In a very recent review, Solheim et al.

The authors noted that only four of 28 studies were of very high quality, i. placebo-controlled, double-blinded crossover studies. A linear relationship has been demonstrated between the volume of donated RBCs and the change in O2max.

RBC re-infusion increases endurance performance by elevating the arterial O2content and by reducing lactate concentrations, i. reduced anaerobic energy contribution at submaximal intensities Re-infusion of autologous blood acutely increases blood volume, central venous pressure and cardiac stroke volume However, experimental evidence indicates that the blood volume has usually returned to the pre-transfusion value 24 hrs after re-infusion due to a rapid compensatory reduction in plasma volume For the detection of allogeneic RBCs, flow cytometry is useful because of the differences in blood group antigens 17 , However, there is still no direct method for the detection of re-infused autologous RBCs, despite long-standing research 37 , 46 , As an alternative, for chemical detection of ABTs, measurements of metabolites of DEHP in urine have been performed As noted above, however, alternatives to DEHP for RBC storage are available 44 , including DINCH, DOTH, and DL9TH.

However, there are limitations of the ABP approach with respect to efficacy, sensitivity rate of detection of correct positives and specificity lack of false positives. Mørkeberg et al. Hbmassproved the only tenable prospect to detect acute transfusions. However, the measurement of Hbmassis based on the inhalation of CO, which is inappropriate in the routine testing for blood doping In a recent study, Malm et al.

In total, blood samples were included. A majority of samples remained within limits of normal individual variation at all times Leuenberger et al.

Plasma iron levels increased up to fold 6 hrs after blood re-infusion and remained fold elevated one day after the procedure. However, the intake of oral or parenteral iron is a confounding factor for the method.

Quantification of hepcidin may be another supportive way to detect ABTs In an experimental trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was re-infused 36 days later Hepcidin concentrations increased 12 hrs and 1 day after blood re-infusion Other investigators have reported Hb profile changes e.

reduced HbA1C level after ABT In addition, altered circulating 29 and erythroid-related 16 microRNAs have been proposed as potential novel biomarkers for detection of ABT misuse in sports.

These findings deserve scientific merit, at present. However, the combination of omics-based technologies with classic hematological variables may eventually provide tools for the detection of ABT and other blood doping procedures ABTs following PABDs in moderately anemic patients scheduled for elective surgery are medically favorable compared to allogeneic blood transfusions The procedure is particularly useful for patients with rare blood group types, irregular antibodies or blood matching problems However, PABD has fallen into disuse in clinical practice, partly due to the large portion of pre-donated blood that is withdrawn.

In contrast, illegal ABT with cryopreserved RBCs appears to be a method of choice in doping athletes, because RBC transfusions can enhance performances, and no valid laboratory method exists to detect autologous RBCs after re-transfusion Interestingly, these cases first came to light through admissions by athletes or support personnel.

Conflict of Interest The authors have no conflict of interest. Home Archive Archive Issue 3 Autologous Red Blood Cell Transfusions in Clinics and their Misuse in Sports.

DOI: accepted: January published online: March Jelkmann W. Autologous red blood cell transfusions in clinics and their misuse in sports. Olympic Committee regarding a young orthopedic surgeon named Tom Dickson, who had worked with the USCF National Team and was interested in sports medicine.

Dickson had complained to the USOC about a blood boosting incident involving U. cyclists that he said he witnessed during the Olympics. Lea quickly set up a three-person inquiry panel to investigate the incident. He notably did not include Executive Director Dave Prouty for reasons that soon became clear.

His plan was to use this incident as a basis for ousting Dave Prouty, Eddie B. I pointed out that there was no evidence linking Prouty to the conspiracy, so it looked like a far reach to try to get him. Lea sounded disappointed that I felt that way.

Inquiring minds want to know. An inquiry into the blood boosting incident was held at the U. Olympic Training Center in Colorado Springs on November 17, USCF Executive Director Dave Prouty had requested that he be included in the investigative panel. President Rob Lea realized that his plot to get rid of Prouty was too obvious, so he decided at the last moment to include him in the panel.

When Lea and I were by ourselves at one point just before the inquiry began, I smilingly asked him if he thought Prouty would recommend that the Executive Director be fired.

Lea looked at me blankly, as if he didn't understand what I was talking about. In the morning we listened to Dr. Tom Dickson's account of his observations. He reported on which athletes he had seen getting transfusions and which others he had heard about.

He didn't mention taking part in any of this, but we later learned that he had apparently assisted willingly in some of the transfusions.

I was curious about why it had taken Dickson three months to report his observations. He remarked that it had taken him awhile to get to talk to a hematologist and learn that what he had seen was medically unethical.

I later learned that what really happened was that after he returned home to Allentown , Pennsylvania , a national-level woman rider who knew about the transfusions at the Olympics asked Dickson if he would do the same thing for her in preparation for a forthcoming major race.

He agreed and did a preliminary test of blood compatibility with her sister. The rider had a severe allergic reaction, causing her arm to swell up. This apparently scared Dickson and made him realize that he didn't know what he was doing.

At that point he decided to complain to the USOC about the earlier transfusion incidents. Troika tango. The afternoon session of our inquiry was scheduled to be an interview of the organizers of the transfusion scheme except for Dr. Falsetti , who declined to talk. Dave Prouty had correctly perceived that he was on Lea's hit list and, in typical style, had brought his lawyer into the proceedings.

Since his lawyer was also the Federation's lawyer, they decided to bring in another lawyer to specifically look after the Federation's interests, namely Bart Enoch. When those lawyers started threatening the accused conspirators, the latter went out and hired a real badass lawyer.

This lawyer proliferation was a direct result of Lea's political posturing. It was a giant waste of everyone's time and money. The only thing that the three lawyers accomplished was to inhibit the inquiry and run up the bill.

Before the hearing convened, the USCF lawyer and Prouty's lawyer had gotten together to decide just what questions could be asked. They developed a short list of things they wanted to know and told the rest of us that we couldn't ask anything.

Prouty clucked a bit but didn't press the issue. He knew from past experience that I could get much more outrageous, given provocation. The inquiry began with Coaches Eddie B. Ed Burke, three riders, and their mean-looking lawyer on one side of a wide table and the four inquisitors and our two lawyers on the other side.

From the outset, the three lawyers discussed only procedural issues while posturing and emitting content-free lawyer talk. There were a couple of times when the lawyers felt that they needed to confer in private with each other and went into the hall.

This gave the rest of us a chance to have some friendly conversation and actually begin to communicate a bit until they returned.

After awhile, Eddie B. I do not understand why we must speak through lawyers. I said that I was sorry that we could not discuss what happened in a more straightforward way.

The lawyers all scowled but didn't respond verbally. After using up most of the available time discussing procedural matters, their lawyer finally answered the six questions that our lawyers had asked. Actually, he didn't so much answer them as evade them.

The lawyers then seemed to run out of gas and quieted down a bit, so I went ahead and asked a number of questions that I had been forbidden to bring up and received what seemed like straight answers. In the weeks following the inquiry I communicated with the U.

Olympic Committee about their policies, did some private interviews of riders, and developed recommendations for disciplinary action and legislation to inhibit future undertakings of this type. When I attended the next Executive Committee meeting in Chicago in mid-December , I noticed that Rob Lea made no specific proposals for dealing with the vampire project.

It also appeared that he had given up on his plan to get rid of Prouty. He was getting heat from me over his conduct and some of the other officers joined the attack.

At that point, he seemed to give up on everything. I was not too surprised when he resigned a week or so later.

As a result of his bizarre performance, Lea holds the record for shortest term in the office of USCF President. He was there just two months, during which time he demonstrated that he was unable to cope with the first problem that came along.

He tried to turn this problem into grounds for a political purge and, when that failed, he lost interest in the job. I was not displeased that Lea had resigned. Like former U. President Jimmy Carter, Lea seemed to be at his best in running for office but, once elected, was not prepared to do the job.

Lea still had a parting shot left. He passed some inside information and a lot of nonsense to a national magazine in an attempt to get his enemies. Meanwhile, the U. Olympic Committee covered itself with a thick layer of whitewash.

Rolling Stoned. We had hoped to keep a lid on the vampirism incident until the USCF Board of Directors meeting scheduled for January 18, That would permit the Board to review the results of our investigation, adopt disciplinary and remedial measures, then make public both what had happened and what was being done about it.

This was not to be, however. Early in the morning of January 4, I received a phone call from a writer for Rolling Stone magazine.

He asked me a series of questions based on certain memos from our investigation that he said he possessed. It did not take a lot of analysis to figure out where he had gotten them -- only two copies had been made of some of the memos I wrote.

One copy was still in my filing cabinet and the other one I had sent to Rob Lea. I promptly called Rob and asked him why he was telling the press about these incidents. He denied doing it, apparently not realizing that I knew for sure that he had.

This dishonest response seemed to me to be tragically consistent with his general conduct. The newspapers and television people had the story within a day or two and my telephone was ringing a lot.

I decided to confirm all information that was in the memos that Rolling Stone had and to not comment on information that was not in those memos. By following this policy, I aimed to remove the insider's advantage that Rob Lea's friend at Rolling Stone enjoyed.

I saw in the press that Dr. Tom Dickson was talking freely about what others had done, but still was not mentioning his role in the transfusion incidents. He reportedly offered various explanations for these incidents, such as an alleged fear of failure that drove the U.

cyclists to take these risks. Olympic whitewash. Officials of the U. Olympic Committee began trying to rewrite history so as to divert attention from their failed responsibility to set policy. In an article that appeared January 12, Dr. Irving Dardik , Chairman of the U. blood boosting.

In fact, that ban was so informal that it did not appear in any of their regulations or guidance documentation. In other words, the bureaucrats were trying to cover their blank slate, to avoid blame. Don Miller, the Executive Director of the USOC, was quoted the next day as saying that the use of blood transfusions to improve performance is unethical.

That was certainly correct. He went on to say that the International Olympic Committee has opposed such practices since Unfortunately, there seems to be no documentary support for that claim -- they kept their alleged opposition secret.

More whitewash. The lead article in the Sports Illustrated edition of January 21, had a rather accurate and comprehensive review of the incident. Tom Dickson as saying he was opposed to the project from the beginning.

That was clearly a lie. The USOC's Dr. All [this discussion of] questionable legality to me [is] immaterial. I was pleased to see Dr. Dardik get kicked out of the USOC a year or so after this incident.

He had apparently played a few too many political games for even that political organization to swallow. An amusing series of sports cartoons appeared in newspapers carrying Tank McNamara. They showed cyclists circling a track with bottles of blood connected to one arm, hanging bat-like from shower bars and traveling in vampire-style coffins.

The worst major article was the last one to appear and the one based on the extensive documentation that had been provided by Rob Lea. The Rolling Stone issue of February 14 had a headline in 32 point type on the cover, just below Mick Jagger's sardonic face, reading:. medalists were doped to win.

cycling team. I wrote a letter to Rolling Stone refuting some of the more flagrant assertions in their article and followed it up with phone calls to the editors.

My answer to that was unprintable. Vampirism -- Driving the Stake. The USCF Board of Directors disciplined the organizers of the vampire project and took steps to prevent a recurrence of blood boosting.

When the Board met on January 18, , the first order of business was to elect a new president to replace the defunct Rob Lea. Phil Voxland returned to power.

As it turned out, Lea did Voxland a great favor -- under the USCF Constitution, a president many serve for at most four consecutive years, but by taking a two month vacation, the clock was reset and Voxland was eventually able to extend his tenure to about five years. No discipline of the athletes involved in blood boosting was contemplated at any time because they had not violated any rules and had, for the most part, followed the guidance of the coaches and other officials.

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Pathological features observed in both human and experimental cerebral malaria ECM are endothelial dysfunction and changes in blood components. Blood transfusion has been routinely used in patients with severe malarial anemia and can also benefit comatose and acidotic malaria patients.

ECM mice showed severe thrombocytopenia and decreases in hematocrit. Artemether treatment markedly aggravated anemia within 24 h. Whole blood administration significantly prevented further drop in hematocrit and partially restored the platelet count.

Increased levels of plasma angiopoietin-2 Ang-2 remained high 24 h after artemether treatment but returned to normal levels 24 h after blood transfusion, indicating reversal to quiescence. Ang-1 was depleted in ECM mice and levels were not restored by any treatment.

Blood transfusion prevented the aggravation of the breakdown of blood brain barrier after artemether treatment and decreased spleen congestion without affecting splenic lymphocyte populations.

Critically, blood transfusion resulted in markedly improved survival of mice with ECM These findings indicate that whole blood transfusion can be an effective adjuvant therapy for cerebral malaria. Changes in blood and blood vessels are a pathological hallmark of Plasmodium falciparum infection, and are particularly intense in its deadly complication, cerebral malaria CM.

Mechanical obstruction of cerebral blood vessels by sequestration of parasitized red blood cells pRBCs reduces cerebral blood flow and oxygen consumption 1 , 2 , 3. Anemia and loss of RBC deformability impair the perfusion of various organs 4. Severe malaria also leads to alterations of biochemical characteristics of the plasma, with depletion of plasmatic factors related with vascular health such as l -arginine 5 , haptoglobin 6 and angiopoietin-1 Ang-1 , a critical regulator of endothelial integrity 7.

Ang-1 maintains vascular quiescence by signaling through the Tie-2 receptor 8 , whereas Ang-2, stored in Weibel-Palade bodies, can be rapidly released upon endothelial activation and displace Ang-1, sensitizing the endothelium to low concentrations of inflammatory cytokines such as TNF 8.

Indeed, Ang-2 levels are elevated in severe malaria 9 and have been associated with CM retinopathy 10 , and blood-retinal breakdown associated with death or sequelae in pediatric CM Together with coagulation disorders like platelet activation and thrombocytopenia 12 , 13 , these changes disturb endothelial quiescence, leading to vascular dysfunction, impaired cerebral perfusion, acidosis 14 and breakdown of the blood—brain barrier BBB Whole blood transfusion is a practice already adopted in the adjuvant treatment of patients with severe malarial anemia Recently, Ackerman and colleagues have shown that whole blood transfusion was associated with improved survival in children with severe falciparum malaria, and patients with impaired consciousness and hyperlactatemia benefited from transfusion even at moderate levels of anemia 19 , berghei ANKA PbA 21 , 22 , was used to investigate the effects of whole blood transfusion as adjunctive therapy to artemether in the late stages of the disease.

This experimental model shows a number of similarities with human CM as well as some differences. The pros and cons of this model have been discussed 21 , 22 , 23 , A major difference is that a hallmark of human CM is sequestration of Plasmodium falciparum -infected erythrocytes in the brain post-capillary venules, resulting in vascular blockage and impaired perfusion 25 , In ECM, leukocyte adhesion with vascular plugging of cerebral vessels is the common pathological feature of the neurological syndrome, but P.

berghei -infected erythrocyte accumulation in the brain has been documented and a recent study showed that P. berghei -infected erythrocytes are trapped in brain capillaries and contribute to impaired cerebral blood flow 27 , 28 , In both human and experimental CM, cerebrovascular blockage and severe vasculopathy occur, making this model appropriate to investigate interventions intended to restore vascular function, cerebral blood flow and cerebral oxygenation.

Indeed, in this study whole blood transfusion showed a marked benefit on survival and on parameters associated with vascular integrity in ECM.

Hypothermia rectal temperature between 31 and 36 °C was used for defining late-stage ECM and as the objective criterion for treatment on day 6 post-infection Thermocouple probe Oakton® Acorn TM; Oakton Instruments, IL, USA was used to measure rectal temperature of mice on day 6 post-infection.

The ARRIVE guidelines were taken in consideration while designing and performing experiments. Two preliminary experiments were performed to determine the conditions for the main experiments. First, an experiment was performed to establish the effect of artemether and artemether plus whole blood transfusion on hematocrit in mice with late-stage ECM.

Transfusion of whole blood in mice poses a substantial challenge. Transferring this amount of blood to mice, in particular mice with late-stage ECM, which present with vasoconstriction and vascular plugging by leukocyte, is even more challenging.

Therefore, we followed a protocol for whole blood transfusion by means of IP injection, as previously described Artemether only showed the best outcome and was used therefore for the main experiments see Supplementary Data.

For the main experiments, on day 6 post infection, hypothermic mice 31—36 °C were equally and randomly distributed in two groups ECM treated with artemether only and ECM treated with artemether plus μL of whole blood transfusion.

The time elapsed between blood collection, pooling and administration to sick mice was kept below 30 min. Uninfected mice and mice with ECM, untreated, were used as controls. In the survival experiments, mice with late-stage ECM were treated with artemether with or without μL of whole blood and, in the subsequent days, they received artemether only daily for another 4 days.

After last artemether dose, mice were followed for 7 days before being euthanized with pentobarbital. Blood from uninfected, healthy mice and from mice with ECM, untreated, were used as controls. Blood was drawn by cardiac puncture and μL were transferred to EDTA-coated microtubes and analyzed for hematological components, including hematocrit and platelet counts, using a pocHi automated hematology analyzer Sysmex at the Institute of Science and Technology in Biomodels ICTB-Fiocruz.

For plasma components analysis, blood was collected in heparinized tubes and centrifuged for 6 min at rpm. Spleen tissues were removed, weighed, and then processed for further analysis. Plasma samples were thawed and diluted to measure concentration of Ang-1 and Ang-2, using mouse Ang-1 and Ang-2 Picokine ELISA kits Boster , or to measure mouse haptoglobin Duoset.

The procedure was performed as previously described After 1 h of dye circulation animals were euthanized and perfused transcardially with 10 mL of ice-cold saline. Later, the brain was harvested and incubated for 48 h at 37 °C in 3 mL of The amount of Evans blue extracted was calculated using a standard curve ranging from to 1.

Animals were submitted to cardiac perfusion with 10 mL of cold PBS. Spleens were removed, weighed and mechanically dissociated, single cell suspensions were treated with lysis buffer Sigma and splenocytes counted with a hematocytometer.

Samples were collected using a FACS CANTO II flow cytometer BD Biosciences. Data analysis was performed using the FlowJo All experiments were repeated at least once.

Data were analyzed using a statistical software package GraphPad Prism 7. Shapiro—Wilk test was used to check distribution among the tested groups. Comparisons between 2 groups were performed using Student t-test and Mann—Whitney test, and multiple groups were compared using One-way ANOVA.

For survival analysis, the Mantel-Cox log-rank test was used. Preliminary experiments were performed in order to define the feasibility of blood transfusion via intraperitoneal injection, as described 31 , and to define a suitable treatment to compare with the performance of artemether plus whole blood transfusion in late-stage ECM.

As shown in Supplemental Fig. Supplemental Fig. Indeed, addition of saline actually led to a worse outcome, with all mice dying in 24 h. Mice with ECM showed a drop in hematocrit ECM mice treated with artemether alone showed a post-treatment decrease in hematocrit, reaching In contrast, ECM mice that received artemether plus μL of whole blood showed a preservation of hematocrit levels at 6 h Effect of artemether treatment with and without μL of whole blood on hematocrit level and platelet counts.

Treatment with ARM led to further decreases in hematocrit after 6 and 24 h Treatment with ARM only did not change platelet levels within 6 h and 24 h.

Artemether treatment did not improve the platelet count at 6 h The combination, however, of artemether treatment plus whole blood raised platelet counts by twofold at 6 h Ang-1 levels did not recover after artemether treatment whether combined with blood transfusion or not.

Despite resolution of elevated Ang-2 levels after blood transfusion, the Ang-1 to Ang-2 ratios remained low Fig. Plasma levels of Angiopoietin-1 and Ang-2 in mice with ECM treated with artemether with or without blood transfusion.

Plasma haptoglobin levels, blood—brain barrier permeability and spleen weight in mice with ECM treated with artemether with or without blood transfusion. The levels of haptoglobin were not affected by either treatment at 6 h. B Permeability of blood brain barrier BBB was quantified by Evans blue assay.

Treatment with ARM only did not change spleen weight within 6 or 24 h. Mice with ECM showed increased Evans blue dye leakage BBB integrity worsened 6 h after treatment with artemether alone Twenty-four hours after treatment, BBB integrity recovered in both treatment groups compared to untreated ECM mice or at 6 h post-treatment.

Mice with ECM showed increased spleen weight, which did not improve 6 or 24 h after artemether-only treatment, but mice treated with artemether plus whole blood had decreased spleen weight at 6 and 24 h after treatment Fig. Data regarding splenocyte populations are shown in Fig.

The increase in spleen weight in mice with ECM was paralleled by an increase in total number of splenocytes, which was not affected by the antimalarial treatment.

Mice with ECM showed increased numbers of splenic B cells, which return to normal 24 h after artemether treatment. In all cases, blood transfusion had no effect on the spleen cell populations compared to artemether alone.

Effect of artemether treatment with and without blood transfusion on splenocyte populations. Adjuvant therapy with blood transfusion resulted in a marked improvement of survival There was a sharp decrease of parasitemia 24 h after treatment, and at this timepoint parasitemia was lower in mice treated with artemether plus whole blood 1.

This difference was not observed in the subsequent timepoints. Mice with ECM showed decreased body weight Even after 5 days of artemether-only treatment the body weight did not fully recover Effect of artemether treatment with and without blood transfusion on survival, parasitemia and body weight of mice with ECM.

A Survival: mice with ECM treated with ARM only showed a survival rate of Whole blood given as adjunctive therapy to ARM resulted in significant increase in survival to Four survival experiments where conducted and results pooled.

B Parasitemia: treatment with artemether, with or without whole blood transfusion, led to a marked decrease in parasitemia in 24 h. For survival, four separate studies were performed, the results combined and log-rank test was performed for statistical analysis.

The high lethality and post treatment neurological sequalae in patients with cerebral malaria demand new adjuvant therapies. The main finding of the present study is that whole blood transfusion resulted in substantial improvement of survival of mice with late-stage ECM treated with artemether.

However, artemether treatment resulted in further marked drops in hematocrit after 6 and 24 h, a phenomenon also reported in human malaria 34 , especially in non-immune travelers with hyperparasitemia Transfusion of μL of whole blood to mice with ECM resulted in improved hematocrit at both 6 and 24 h, whereas saline had the opposite effect, and all mice died in 24 h.

This worse outcome may reflect similar findings in human severe malaria, where fluid resuscitation did not improve and actually worsened patient outcomes 55 , 56 , Although the findings in the experimental CM model cannot be directly extrapolated for the human disease, it is expected that maintenance of the hematocrit strengthens the oxygen-carrying capacity of the blood, which in a setting of cerebral ischemia may be a critical advantage for the patient.

Fresh RBCs also improve the hemorheological properties, which is known to be deteriorated in severe malaria infections 36 , And finally, lower hematocrit results in decreased vascular wall shear stress 38 , with decreased eNOS activity, which leads to worsened endothelial function Therefore, increasing hematocrit through whole blood transfusion should help restore endothelial function 40 , 41 , help clear vessels blocked by parasitized erythrocytes and inflammatory cells, increasing tissue perfusion and decreasing acidosis 19 , 42 , 43 as well as immune cell-mediated endothelial damage.

The effects on endothelial function are supported by the observation that blood transfusion prevented worsening of BBB breakdown and restored Ang-2 levels. Indeed, decreased levels of Ang-1 and increased levels of Ang-2, disturbing endothelial quiescence with loss of vascular health 7 , 8 , have been associated with pediatric severe malaria 10 , 44 , Since Ang-2 has been proposed as a risk factor for cognitive injury in pediatric CM, this finding is of critical importance, indicating that fresh blood counteracts the ECM-related inflammation and vascular insult.

These findings are in line with data showing that interventions that counteract vascular dysfunction and inflammation are beneficial in ECM 32 , 40 , 41 , 46 , 47 , It is noteworthy, however, that the improvement in Ang-2 levels by transfusion was observed at 24 but not at 6 h, indicating that the intervention takes time to show benefit on this parameter.

Since in human CM most deaths occur in the first 24—48 h after hospitalization, the relevance of this finding for clinical disease still needs to be established. Thrombocytopenia is also one of the risk factors for mortality in African children with falciparum malaria Whole blood transfusion induced significant upturn in circulating platelets in mice with ECM.

The availability of fresh, quiescent platelets, could help restoring the normality of the coagulation system without the deleterious inflammatory actions of activated platelets This effect of partially restoring the platelet counts in 24 h cannot be ascribed only to a passive, repository effect due to the platelets present in the limited amount of transfused blood.

Therefore, it is apparent that blood transfusion stimulates the body to actively respond, increasing platelet production. Other effects such as improved BBB response and decreased splenic congestion also support an active modulatory, rather than just repository, effect of blood transfusion.

Acute and severe hemolysis usually leads to a consumption of haptoglobin, as seen in severe malaria In mice with ECM, anemia was only mild to moderate whereas inflammation is overwhelming, and this might help to explain why haptoglobin levels were high, since haptoglobin is an acute phase protein that increases with conditions such as inflammation and infection On the other hand, a hemolytic event might also help to explain the decrease in haptoglobin levels following artemether treatment.

Blood transfusion did not seem to interfere with haptoglobin levels following artemether treatment. Interestingly, blood transfusion actually helped to decrease the weight of enlarged spleen in mice with ECM, suggesting that it helped to decrease congestion. It is possible that fresh red blood cells improved blood flow throughout the body, improving overall hemodynamics and decreasing the burden at the spleen.

Indeed, in sickle cell disease acute splenic sequestration is treated by RBC transfusion The increase in spleen weight in mice with ECM was paralleled by an increase in the total splenocyte population.

The vast majority of the increase in splenocyte numbers was in the B cell compartment, in line with previous findings Although there was a change in the dynamics of different splenocyte populations in mice with ECM and after treatment with artemether, blood transfusion had no effect on the outcomes of each cell population.

The benefit of blood transfusion, however, on hematological and vascular parameters in mice with ECM treated with artemether was associated with a marked improvement in survival.

These findings are in line with a recent prospective multicenter observational study showing that blood transfusion improved survival of children hospitalized with severe falciparum malaria 19 , However, the authors showed that when signs of vital organ hypoperfusion are present, blood transfusion can benefit patients even at higher hemoglobin thresholds 7.

The effect of whole blood transfusion on survival in pediatric severe malaria must be evaluated in randomized controlled trials that would balance the potential benefits of transfusion against the potential risks such as infection transmission, hemolytic reactions, and circulatory overload.

For patients with higher hemoglobin levels, our study in mice indicates that even the transfusion of half the usual blood volume can be of great benefit, an approach that could reduce the risk of circulatory overload.

In conclusion, the transfusion of whole blood as an adjuvant therapy in ECM showed promising results and identified an unexpected interaction between transfusion and vascular inflammation.

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Rénia, L. Cerebral malaria: in praise of epistemes. An ultrastructural study of the brain in fatal Plasmodium falciparum malaria. Blood doping is the practice of misusing certain techniques and substances to increase the number of circulating red blood cells and hemoglobin mass in the body.

Since hemoglobin within the red blood cells carry oxygen to the muscles, this lets the body transport more oxygen to working muscles and can increase their aerobic capacity and endurance, as well as improve recovery. Blood doping involves using pharmaceuticals, such as EPO and other biosimilars, to stimulate the production of more red blood cells, or the infusion of additional red blood cell volume.

The manipulation of blood and blood components to enhance performance is prohibited at all times under the World Anti-Doping Agency WADA Prohibited List.

EPO is part of a class of substances called Erythropoiesis-Stimulating Agents ESAs. In a clinical setting, EPO is primarily used for kidney failure, chemotherapy, and other medical conditions involving red blood cell loss and anemia.

EPO is prohibited at all times under the WADA Prohibited List and is the most commonly used non-Specified Substance in the class of Peptide Hormones, Growth Factors, and Related Substances in category S2. EPO has been on the WADA Prohibited List since its inception in , and prior to that, it was on the International Olympic Committee IOC List of Banned Substances and Methods.

EPO has a long history of abuse in endurance sports. EPO has significant clinical utility and therapeutic benefit when used appropriately, but its misuse to gain a performance benefit can result in serious health consequences.

It depends on many factors, including dose, frequency, route of administration, and type of ESA administered. Some newer generation ESAs are designed to remain active in the blood for weeks rather than days to make it easier for less frequent administration when used by clinically ill patients; thus, the detection window of these substances is much longer.

A test for EPO was first presented at the Olympic Games in Sydney, Australia that was based on a complementary analysis using blood and urine matrix. With this test, a blood screening took place first, followed by a urine test to confirm possible use of EPO.

Atypical ABP profiles are used to facilitate target testing and guide further ESA analyses. The EPO detection method is widely accepted by the scientific community and has gone through an extensive scientific validation process.

Accredited anti-doping laboratories worldwide have also successfully used it for years. In September , the WADA Laboratory Committee reaffirmed its support of the method when applied appropriately. According to WADA, the Court of Arbitration for Sport CAS has also supported the validity of the EPO detection method in all its decisions relating to EPO.

Blood Doping and EPO: An Anti-Doping FAQ Blood doping, which may include the use of erythropoietin EPO , is among the most well-known methods of doping in sport. Learn more about the prohibited method, as well as the prohibited substance EPO, in this anti-doping FAQ: What is blood doping?

What is EPO? Is EPO prohibited in sport?