T R A N S F U S I O N P R A C T I C E

Plasticizers excreted in urine: indication of autologous bloodtransfusion in sports_3331 647..657

Núria Monfort,* Rosa Ventura,* Petra Platen,* Timo Hinrichs, Klara Brixius, Wilhelm Schänzer,

Mario Thevis, Hans Geyer, and Jordi Segura

BACKGROUND: Misuse of autologous blood transfu-sions in sports remains undetectable. The metabolitesof the plasticizer di-(2-ethylhexyl)phthalate (DEHP) wererecently proposed as markers of blood transfusion,based on high urinary concentrations of these com-pounds observed in patients subjected to blood transfu-sion. This study evaluates DEHP metabolites in urinefor detecting autologous blood transfusion.STUDY DESIGN AND METHODS: One blood bag wasdrawn from moderately trained subjects and the redblood cells (RBCs) were reinfused after differentstorage periods. Group 1 (12 subjects) was reinfusedafter 14 days, and Group 2 (13 subjects), after28 days of storage. Urine samples were collectedbefore and after reinfusion for determination ofthe concentrations of three DEHP metabolites,mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, and mono-(2-ethyl-5-oxohexyl)phthalate.RESULTS: Concentrations of DEHP metabolites on thedays before reinfusion were in agreement with thosedescribed after common environmental exposure. A fewhours after the reinfusion a significant increase wasobserved for all metabolites in all volunteers. Concen-trations 1 day later were still higher (p < 0.05) thanbefore reinfusion. Variations in urine dilution supportednormalization by specific gravity. Concentrations ofDEHP metabolites tended to be higher after longerstorage times of RBCs.CONCLUSION: Autologous transfusion with RBCsstored in plastic bags provokes an acute increase in theurinary concentrations of DEHP metabolites, allowingthe detection of this doping malpractice. The window ofdetection is approximately 2 days. The method mightbe applied to urine samples submitted for antidopingtesting.

The increase of oxygen delivery to muscles is oneof the most effective performance-enhancingpossibilities in sports. To obtain such advantage,the initially preferred administration of recom-

binant erythropoietin or the use of blood transfusion fromanother donor (known as allogeneic or homologous trans-fusion) are nowadays detected by antidoping laboratoriesby means of urine and blood analysis, respectively.1,2 As aconsequence, the possibility of using the athlete’s ownblood or red blood cells (RBC; known as autologous trans-fusion) has appeared as an alternative for increasingoxygen availability to muscles for doping purposes. Infact, some police raids have identified networks ofmedical or paramedical people helping athletes to storeand reinfuse their own stored blood.3 In that case, thetransfused RBCs bear the same surface antigens than theRBCs of the recipient and, thus, the analytical approachused for the detection of allogeneic transfusion2 is notuseful.

ABBREVIATIONS: DEHP = Di-(2-ethylhexyl)phthalate;

MEHHP = mono-(2-ethyl-5-hydroxyhexyl)phthalate; MEHP =mono-(2-ethylhexyl)phthalate; MEOHP = mono-(2-ethyl-5-

oxohexyl)phthalate.

From the Bioanalysis Research Group, IMIM Hospital del Mar

Research Institute and the Universitat Pompeu Fabra, Barce-

lona, Spain; Department of Sports Medicine and Sports Nutri-

tion, Ruhr University Bochum, Bochum, Germany; and the

Institute of Cardiology and Sports Medicine and the Center for

Preventive Doping Research, German Sport University, Cologne,

Germany.

Address reprint requests to: Jordi Segura, Bioanalysis

Research Group, IMIM Hospital del Mar Research Institute,

IMIM-Hospital del Mar, carrer Dr Aiguader 88, 08003 Barcelona,

Spain; e-mail jsegura@imim.es.

*These authors are first co-authors as they contributed

equally to the manuscript.

Received for publication January 20, 2011; revision

received July 21, 2011, and accepted July 21, 2011.

doi: 10.1111/j.1537-2995.2011.03331.x

TRANSFUSION 2012;52:647-657.

Volume 52, March 2012 TRANSFUSION 647

Several possibilities have been proposed in the litera-ture to detect the misuse of autologous blood trans-fusion,4-6 although none of them have yet received officialapproval. In addition, all of them are based on blood vari-ables, but blood is a material not always available fordoping control. In contrast, the use of a method based onurine analysis would offer new possibilities for this so farunsolved testing situation, because it could be applied toall doping control tests, where urine is always collected.

Most bags for blood or RBC storage are made frompolyvinyl chloride (PVC) containing plasticizers toassure appropriate flexibility.7 Di-(2-ethylhexyl)phthalate(DEHP) is the most used plasticizer, being in some coun-tries the only one authorized for such purpose. An acuteexposition to DEHP is expected after a transfusion proce-dure. However, DEHP, being part of some other plasticmaterials, is widespread in the environment and allpeople are exposed to it. Thus, basal concentrations ofDEHP metabolites can be detected in urines of generalpopulation. The main metabolites of DEHP are theproducts of hydrolysis to mono-(2-ethylhexyl)phthalate(MEHP) and two subsequently oxidized compoundsmono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP) andmono-(2-ethyl-5-oxoyhexyl)phthalate (MEOHP).8 Thefact that DEHP has endocrine disruptive properties hasinitiated the study of markers for any undue overexposure.DEHP itself is not usually used as marker because it iswidely present and it is quickly metabolized in the body;thus, DEHP metabolites have been routinely used tomeasure DEHP exposure for epidemiologic purposes.9-11 Itis well known, for instance, that when children are sub-jected to blood transfusions, the huge urinary excretionof the metabolites indicate such an acute directexposure.12-14 In a recent study performed in our group,15

the excretion of DEHP metabolites in urine of hospitalizedadult patients receiving RBC transfusion was much higherthan in a normal population and much higher than in eliteathletes. According to these results, DEHP metabolites inurine were proposed as markers for the detection of pro-hibited blood transfusion in sports.

The objective of this study was to measure the con-centrations of DEHP metabolites in urine of healthymoderately trained individuals receiving autologousRBC transfusions, to evaluate the potential use of DEHPmetabolites measurements as indicators of the misuse ofblood transfusion in sports.

MATERIALS AND METHODS

SubjectsAn experiment of autologous RBC trans-fusion was performed with 25 moder-ately trained subjects. The study wasapproved by the Ethics Committee of

the Ruhr-University Bochum (Reg. No. 3200-08). Beforethe beginning of the study, subjects gave their writteninformed consent for their participation.

Five hundred milliliters of blood was collected fromall the participants in the study. The procedures of bloodcollection, preparation of the RBCs, storage, and reinfu-sion were carried out in a specialized laboratory accordingto usual clinical practice using blood bags (Macopharma,Tourcoing, France) and leukoreduction filtration systems(Fenwal, Inc., Lake Zurich, IL). The blood preparationprocedure involved a centrifugation step (4068 ¥ g for18.5 min at 22°C). A quadruple blood pack unit was used.The system consists of a primary blood bag containingcitrate-phosphate-dextrose adenine (CPDA) solutionand a satellite bag containing saline-adenine-glucose-mannitol to preserve RBCs. Leukoreduction was carriedout at 18 to 26°C for 15 to 45 minutes. Up to 60 minutesafter blood donation, subjects were asked to drink at least500 mL of mineral water or juice to avoid circulatory regu-lation disturbances.

The subjects were randomly allocated to two transfu-sion protocols: For a group of 12 subjects, RBCs were rein-fused 14 days after storage (Group 1), and for 13 subjects,RBCs were reinfused 28 days after storage (Group 2).Anthropometric data of the subjects participating in thestudy are described in Table 1. For Group 1, spontaneousurine samples were collected on Days -15, -14 (blood col-lection), -13, -7, -1, 0 (reinfusion), 1, 2, 3, 5, 7, 10, 14, and21. For Group 2, the equivalent days were Days -32, -29,-28 (blood collection), -27, -21, -14, -7, -1, 0 (reinfusion),1, 2, 3, 5, 7, 10, 14, and 21. Urine samples were collected assoon as subjects were able to spontaneously produce, atleast, 100 mL of urine. On the days of the bleeding andreinfusion, urine samples were obtained along the periodfrom 1 to 3 hours thereafter. On the other days, subjectscame to the laboratory at nearly the same time as on thedays of the bleeding and reinfusion. Samples were col-lected in glass bottles (free of DEHP), coded, stored at-20°C, transported frozen to laboratory, and restored at-20°C until analysis.

Sample analysesSamples were analyzed for DEHP metabolites at IMIM-Hospital del Mar Research Institute (LAB 1) by a method

TABLE 1. Anthropometric data of the subjects participating inthe study*

Group Sex Number Age (years) Weight (kg) Height (cm)

1 Male 7 24.7 � 2.4 77.4 � 19.7 174.6 � 6.2Female 5 27.6 � 12.0 68.5 � 6.6 170.8 � 5.4

2 Male 8 24.8 � 3.2 77.7 � 9.9 179.5 � 6.9Female 5 25.0 � 1.9 63.4 � 8.1 166.6 � 4.5

* Data are reported as mean � SD.

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previously described.15 Samples were blinded to the ana-lysts. After being thawed at room temperature, centri-fuged urine samples had their specific gravity measuredby means of a refractometer. When appropriate (seebelow), concentrations were corrected for specific gravity.This mathematical correction process, usually carried outin doping control for some analytes,16 was carried out byapplying the formula

Concentration adjusted Concentration1.020 1 Specific gra

= ×−( ) vvity of the Sample 1 .−( )[ ]

Analytical results were confirmed by an independentblind analysis carried out by another laboratory (Centerfor Preventive Doping Research, German Sport Univer-sity; LAB 2). The method used in LAB 2 was previouslydescribed and it was different regarding sample pre-paration and instrumental analysis.17 Concentrationsused for the results presented in this paper were those ofLAB 1.

Statistical analysisCorrelations of results obtained between metabolites andbetween the two different laboratories were calculated byPearson’s coefficient. Other statistical comparisons werecalculated with the paired or unpaired t test, consideringp-values lower than 0.05. Descriptive statistics and statis-tical comparisons were carried out in computer software(Microsoft Office Excel 2003, Microsoft Corp., Redmond,WA; and SPSS 12.0, SPSS, Inc., Cary, NC). Data were

processed untransformed except when mentioned in thetext (log transformation).

RESULTS

Concentrations of DEHP metabolitesConcentrations of DEHP metabolites obtained in all sub-jects participating in the study are described in Tables 2and 3. Samples corresponding to the same period of timearound blood collection were grouped together forpercentile stratification (Table 2). Some days includedsamples from all 25 volunteers (out of some missing non-collected samples) while other periods (-4, +14, and +21days after blood collection) only involved the subjects ofGroup 2. Concentrations corresponding to percentiles10th, 25th, 50th, 75th, and 90th are indicated for eachperiod.

Concentrations of DEHP metabolites on the periodsaround the reinfusion day are presented in Table 3. Hugeconcentrations were found on the day of the reinfusion forall metabolites in all volunteers, with the 90th percentilebeing 864.1, 1187.5, and 1063.5 ng/mL for MEHP, MEHHP,and MEOHP, respectively. A decrease was observed the dayafter reinfusion (Day 1 in Table 3), but the concentrationswere still higher than for the other days at all percentiles.

Concentrations of MEHHP were usually slightlyhigher than those of MEOHP and substantially higherthan those of MEHP. Correlations in concentrations of thedifferent metabolites are shown in Fig. 1A. Pearson corre-lation coefficients were 0.99 for MEHHP versus MEOHPand 0.92 for MEHP versus MEOHP.

TABLE 2. Distribution of absolute concentrations of DEHP metabolites in urine, taking all subjects studied alongthe days before and after blood collection (Day 0)

Concentration (ng/mL)

MetaboliteDays around

blood collection Number 10th percentile 25th percentile 50th percentile 75th percentile 90th percentile Maximum

MEHP -4 13 1.4 2.9 5.7 11.2 11.8 15.3-1 21 1.2 2.1 5.7 11.6 14.4 17.00 25 1.0 1.3 2.2 4.8 8.2 11.91 25 1.0 1.3 3.5 6.4 8.2 22.47 23 1.1 1.7 4.1 5.8 9.6 26.0

14 13 1.3 1.8 8.0 11.8 15.9 18.721 13 1.3 3.0 5.2 8.3 20.9 24.7

MEHHP -4 13 1.4 8.7 9.9 21.4 27.4 29.9-1 21 1.7 2.8 10.7 28.6 54.5 91.30 25 0.4 1.3 4.4 8.2 16.8 35.71 25 1.4 2.7 6.1 18.2 22.6 121.77 23 0.9 2.1 5.9 10.4 21.6 75.6

14 13 1.4 3.7 17.7 27.8 43.3 43.621 13 4.0 5.4 15.8 25.3 45.3 93.6

MEOHP -4 13 0.6 4.9 8.0 14.0 15.3 26.1-1 21 1.2 2.3 9.8 15.6 22.8 41.90 25 0.4 0.6 2.2 4.9 10.5 29.11 25 0.6 2.0 3.9 12.2 17.2 76.47 23 0.8 2.0 4.9 8.0 14.5 40.0

14 13 1.4 2.6 12.2 16.2 24.0 37.121 13 3.1 3.5 10.6 19.9 24.2 27.5

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Volume 52, March 2012 TRANSFUSION 649

Correlation of concentrations between LAB 1 and LAB2 is shown in Fig. 1B. A Pearson’s coefficient of 0.95 wasobtained.

The mean concentrations of DEHP metabolites onthe reinfusion day of subjects of Group 1 (RBC stored for14 days) and subjects of Group 2 (RBC stored for 28 days)are compared in Fig. 1C. Normal distribution for log trans-formed concentrations on the reinfusion day was verified.Measured concentrations tended to be higher for Group 2with longer RBC storage time: log MEHP (significant,p < 0.05), log MEHHP (not significant, p = 0.06), and logMEOHP (not significant, p = 0.13).

Results adjusted for specific gravitySome of the studied urines appeared highly diluted whileothers were of normal color and appearance. Measure-ments of specific gravity were carried out, and the resultsindicated that there were 2 days in which the dilution ofthe urines was especially relevant (Fig. 2). The first day

was the day of the blood collection (Day 0 in Fig. 2A), forwhich a mean specific gravity of 1.006 was observed com-pared with 1.016 the day before. The other day was thereinfusion day (Day 0 in Fig. 2B), although in this day, thedilution was smaller (mean value of 1.010 compared with1.014 the day before reinfusion).

The range of specific gravities varied from 1.001 to1.029 in the whole period with a wide span between sub-jects (Fig. 2) and a general trend for lower specific gravitiesin females. Taking into account those variations, togetherwith the clear decrease in specific gravity in the days ofblood collection and reinfusion, the need of harmoniza-tion of results based on a similar dilution status of thedifferent samples was obvious.

The whole set of concentrations was adjusted towarda theoretical specific gravity of 1.020 for all samples. Thegraphical representation of specific gravity–adjustedresults obtained for subjects of Group 1 (RBC stored for 14days) is displayed in Fig. 3. The day of blood collection wasDay -14 in this group and day of reinfusion was Day 0. A

TABLE 3. Distribution of absolute concentrations of DEHP metabolites in urine, taking all subjects studied alongthe days before and after reinfusion (Day 0)*

Concentration (ng/mL)

MetaboliteDays around

reinfusion Number 10th percentile 25th percentile 50th percentile 75th percentile 90th percentile Maximum

MEHP -7 24 1.2 2.8 4.1 7.6 20.9 26.0-1 25 1.2 2.1 4.1 5.8 10.2 51.30 25 28.3 91.0 212.2 309.3 864.1 1207.51 25 2.1 4.8 8.3 16.6 26.9 31.62 25 1.7 3.0 6.1 9.5 17.9 22.33 25 0.8 1.9 3.3 7.8 10.8 13.85 25 1.0 1.7 5.1 9.3 11.6 32.47 25 1.0 1.8 3.7 5.1 6.7 13.1

10 24 1.1 2.3 4.1 7.0 13.6 17.614 24 1.3 2.4 3.7 7.0 21.0 27.621 25 1.4 2.2 3.8 5.9 9.1 36.2

MEHHP -7 24 1.6 4.0 6.3 21.6 45.3 93.6-1 25 2.2 3.7 7.6 12.5 27.9 145.10 25 121.8 216.0 440.8 676.8 1187.5 2786.31 25 7.6 14.2 21.7 53.8 99.2 151.32 25 1.5 8.1 12.5 31.9 37.2 64.43 25 1.4 3.9 7.9 17.3 24.3 148.65 25 0.9 3.2 9.0 21.4 25.9 53.97 25 1.0 2.3 4.7 16.2 21.6 49.5

10 24 1.6 6.4 8.0 18.7 40.2 46.514 24 1.2 3.0 9.8 15.6 38.5 50.421 25 2.3 3.8 6.7 11.4 26.2 56.4

MEOHP -7 24 1.0 3.1 5.7 11.5 24.2 40.0-1 25 1.6 2.1 5.5 7.9 36.2 44.50 25 113.3 211.3 348.6 582.9 1063.5 2807.01 25 6.8 12.3 15.8 39.6 84.9 97.02 25 1.8 5.0 10.7 18.8 24.4 37.23 25 1.4 2.7 5.7 11.8 16.4 27.35 25 0.9 1.7 8.3 15.4 18.6 19.07 25 0.6 1.2 3.4 8.6 13.0 42.6

10 24 0.9 3.5 6.8 13.5 27.8 31.214 24 0.7 2.3 6.3 8.6 24.4 34.021 25 1.5 3.0 5.0 8.3 19.1 38.2

* Thirteen of the subjects included in Day -7 (Table 3) are also included in Day 21 after blood collection (Table 2), which corresponds to thesame samples.

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650 TRANSFUSION Volume 52, March 2012

similar figure is presented for the subjects of Group 2(Fig. 4; blood collection is Day -28 in this group).

To obtain information on the (un)stability of urinaryconcentrations of DEHP metabolites in the sameindividual in different days in absence of known acuteexposures, coefficients of variation of concentrationsof DEHP metabolites were calculated for each oneof the volunteers using samples collected in differentdays before the reinfusion process. As it is presentedin Table 4, the mean percent intraindividual coeffi-cients of variation (CVs; 95% confidence interval[CI]) were 53.6 (40.9-66.3), 64.0 (51.3-76.7), and58.7 (45.4-72.0) for MEHP, MEHHP, and MEOHP,respectively.

Cluster of similar situationsTo compare different situations, the results were arrangedfor all volunteers in the following groups:

• Basal concentrations (n = 13 samples; 4 days beforeblood collection, only available for the subjects ofGroup 2).

0,1

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Fig. 1. Relationships involving absolute concentrations (Conc)

of DEHP metabolites in urine. (A) Correlation between the

three studied metabolites (MEHP vs. MEOHP, �; MEHHP vs.

MEOHP, ); (B) correlation between the sum of the three

metabolites obtained for this study (LAB 1) and the same

samples analyzed by another independent laboratory with a

different method (LAB 2); and (C) mean and SEM of concen-

trations obtained during the reinfusion day corresponding to

Group 1 (14 days of RBC storage, ) and Group 2 (28 days of

RBC storage, ) RBCs.

Specific gravity around reinfusion

1,0000

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Fig. 2. Specific gravity measurements for all samples collected

in the study (A) on the previous days and up to 2 weeks after

blood collection and (B) on the previous days and up to 1

week after blood reinfusion.

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• Day of blood collection (n = 25 samples, all subjects).• One day after blood collection (n = 25 samples, all

subjects).• Day of reinfusion (n = 25 samples, all subjects).• One day after reinfusion (n = 25 samples, all subjects).• Two days after reinfusion (n = 25 samples, all

subjects).

Figure 5 displays the graphical representation ofthese groups. Apart from the huge difference between thereinfusion day and all other periods (Fig. 5, left panels), it

is noteworthy that concentrations cor-responding to the day after blood rein-fusion (Fig. 5, Column E, right panels)were still significantly higher (p < 0.05)than the remaining other concentra-tions for the three metabolites.

DISCUSSION

The most striking finding of thisresearch was the extremely high excre-tion of DEHP metabolites on the dayof blood reinfusion. Urinary DEHPmetabolites had been suggested asmarkers of the misuse of blood transfu-sions in sports.15 The results of this studyconfirm that DEHP metabolites areuseful for detecting such malpractice. Inurine samples collected on the day ofthe reinfusion, the excretion of themetabolites increases markedly com-pared to the day before, thus indicatingbeyond reasonable doubt that an acutehuge exposure to DEHP has taken place.

To detect abnormal concentrationsof metabolites of the plasticizer DEHPin urine it is of utmost importance thecomparison with the common excretionrates and concentrations arising in anysubject from the unavoidable exposureto such a widely used plasticizer inmodern society. The urinary concentra-tions of DEHP metabolites in environ-mentally exposed population havebeen largely investigated.18-20 Reportedconcentrations of DEHP metabolitesin general population may be foundin several publications (see Table 7 inFromme et al.21). In summary, themedian of the usual concentrationsreported for MEHP, MEHHP, andMEOHP range approximately from 0.9 to10.3, 8.0 to 52.1, and 14.0 to 41.4 ng/mL,respectively. Nevertheless, it is recog-

nized that rare acute exposures (occupational exposure,certain diet or lifestyle) may result in medium to severeexcretion of such products in the urine of subjects exposedto not well-defined sources.22-24

The analytical method used in our approach15

involved the analysis of urine samples for the content ofrelevant DEHP metabolites, MEHP, MEHHP, and MEOHP.The basal concentrations of DEHP metabolites in ourpopulation of healthy medium-trained subjects were atthe low side of the usual range, probably because it is amore homogeneous population than those studied else-

0200400600800

1000120014001600

-20 -15 -10 -5 0 5 10 15 20 25

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MEHHP

MEOHP

MEHP

Fig. 3. Graphical representation of specific gravity–adjusted concentrations (Conc) of

DEHP metabolites of Group 1 (12 subjects where RBCs were stored for 14 days).

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652 TRANSFUSION Volume 52, March 2012

where. It is important to notice that the populationstudied belongs to a German cohort, which may reflect acommon European lifestyle. Also, a trend toward a slightdecrease of basal DEHP metabolites has been described inrecent years for subjects living in this country.25 The resultswere confirmed by an independent laboratory using dif-ferent method and instrumentation, thus giving addi-tional strength to the findings reported. In fact, previousanalysis of basal concentrations of DEHP metabolites inelite athletes has also shown concentrations slightly lowerthan those observed in general population.15

The comparison of concentrationsof xenobiotics in urine always suffersfrom the different hydration status ofthe subjects studied, especially in spotsamples. The measurements of specificgravity confirm the wide scope of urinedilutions being registered among thevolunteers (Fig. 2). Also the mere inter-vention of blood collection (accompa-nied by additional drinking) or RBCreinfusion led to a dilution of the urinecollected those days, as shown in Fig. 2.Additionally, when considering theworld of sport, it seems rational to takeinto account the well-known differencesin urinary specific gravity between ath-letes playing different sports and disci-plines. As urines may be excreted indifferent hydration states, one way ofharmonizing findings between subjectsis to adjust concentrations by specificgravity or creatinine content. The latteris less convenient in sport when muscleexercise and the precursor creatine maybe altered and, therefore, in the sportsarea adjustments by specific gravity arepreferred. In fact, adjustment of con-centrations to a theoretical standardurine with a specific gravity of 1.020 isbeing routinely used in sports drugtesting for other purposes.16 The specificgravity–adjusted concentrations allowthe results obtained to be studied in amore uniform way, in spite of the factthat concentrations in extremely dilutedurine samples may be overemphasized.

Specific gravity–adjusted concen-trations for the two groups of volunteersare presented in Fig. 3 (Group 1) andFig. 4 (Group 2). The concentrations ofDEHP metabolites observed for the dayof reinfusion are so high that all otherconcentrations in those figures appearclose to the X-axis. Elevated adjusted

concentrations also appear on the day after reinfusion(Day 1) in both groups of subjects (see below). Our resultssuggest that it is possible to establish a threshold valueabove which to be fully certain of an acute high exposureto DEHP (blood transfusion being one potential explana-tion) or even another threshold value to suspect for someacute exposure on the day before. Additionally, if thenormal basal exposure to DEHP of each athlete is evalu-ated and incorporated to the biologic passport (biologicpassport concept in urine26), the possibilities of easilydetecting minor blood transfusion processes will increase.

MEHHP

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-35 -30 -25 -20 -15 -10 -5 0 5 10 252025

-35 -30 -25 -20 -15 -10 -5 0 5 10 252025

Fig. 4. Graphical representation of specific gravity–adjusted concentrations (Conc) of

DEHP metabolites of Group 2 (13 subjects where RBCs were stored for 28 days).

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Volume 52, March 2012 TRANSFUSION 653

In this regard, the intraindividual reproducibility inconcentrations of DEHP metabolites along differentdays before reinfusion was evaluated (Table 4). The CVobtained showed, however, low reproducibility, probablydue to small uncontrolled occasional DEHP exposuresthat increased DEHP metabolites in the spot urinesamples.

The distinction among different situations (basal,blood collection, reinfusion) based on DEHP metabolitesexcreted in urine is presented in Fig. 5. Specific gravity–adjusted concentrations for the day of the reinfusion andthe next day are significantly different from all other situ-ations. Even 2 days after the reinfusion day there is a trendto still higher concentrations, which is, however, not sig-nificant. Moreover, no differences were observed in thespecific gravity–adjusted concentrations between menand women. In fact, up to three categories of results can beconsidered: 1) common concentrations expected in non-doped subjects, 2) high concentrations on the day of thereinfusion process, and 3) slightly elevated concentrationsappearing beyond 24 hours from RBC reinfusion.

The ratio between MEHP and the sum of the threemetabolites (%MEHP3)27,28 was evaluated to explore othermarkers to increase the discriminatory power of themethod proposed. The results obtained indicate that theratio is rather constant under common exposure (mean,

24%) but it decreases to 19% the day of reinfusion and to15% (significant) 1 day afterward. It is interesting to notethat the acute infusion of DEHP seems to promote eitheran increase in the rate of oxidative metabolism to MEHHPand MEOHP or a more rapid elimination of MEHP. Theuse of %MEHP3 could help to better interpret suspiciousanalytical results.

Our protocol implied urine sampling in the period upto 3 hours after reinfusion and at 24-hour intervals forsome days thereafter. In the period in between the first 3hours and 24 hours after reinfusion, our protocol did notallow us to define for how many hours these extreme con-centrations of DEHP metabolites remained in the urine ofstudied subjects. Some future studies with more samplesobtained in that time interval should clarify the issue.

Interestingly, adjusted concentrations of DEHPmetabolites obtained in subjects of Group 2 had a ten-dency to be higher than those observed for subjects ofGroup 1 (Fig. 1C). This result is fully in agreement withprevious data showing that DEHP leaking into the bloodbags increases along the storage period.29 Accordingly,storage periods longer than 28 days may result in evenhigher differences than those found in this study for thethree metabolites. In fact, a good correlation existsbetween the concentrations of MEHP, MEHHP, andMEOHP (Fig. 1A) confirming that the kinetic profile is

TABLE 4. Individual distribution of adjusted concentrations of DEHP metabolites*

Group Sex

Specific gravity–adjusted concentations (ng/mL) before reinfusion day

MEHP MEHHP MEOHP

Mean CV (%) Range Mean CV (%) Range Mean CV (%) Range

1 Male 4.9 43.9 2.5-7.5 6.4 55.6 2.6-10.2 5.4 54.7 2.2-9.3Female 12.5 147.3 3.5-45.6 6.5 40.4 3.7-9.9 2.7 29.4 1.8-3.5Male 5.8 37.5 4.2-9.3 9.7 39.8 5.0-13.8 5.5 38.4 2.9-7.6Male 5.6 51.6 2.2-9.1 10.5 41.3 5.6-15.5 6.8 31.4 4.7-9.7Female 6.0 27.1 4.1-8.2 11.6 84.2 3.6-27.9 7.0 74.4 2.6-15.6Male 10.2 26.9 6.3-12.4 13.1 46.6 6.1-22.6 8.0 35.4 5.3-12.2Female 8.6 57.6 2.5-15.8 13.6 103.0 1.7-37.4 9.9 91.7 3.6-25.7Male 16.1 98.0 6.7-44.0 15.1 36.2 10.9-22.7 7.9 25.9 6.1-11.0Male 7.6 83.3 2.8-18.5 17.1 122.0 3.8-53.6 9.2 121.9 1.6-28.4Female 15.0 77.3 7.7-32.2 25.5 81.6 11.4-56.5 10.4 87.2 3.9-23.8Male 10.1 87.2 3.7-22.9 41.8 131.5 10.4-124.2 25.5 138.0 7.0-78.3Female 11.0 22.6 8.8-13.7 45.8 38.4 26.1-60.0 39.0 33.6 24.2-49.1

2 Female 4.4 34.2 1.6-6.2 8.4 84.5 1.2-24.9 6.4 73.3 0.8-16.9Female 10.4 42.0 3.9-18.0 9.2 64.8 2.7-19.3 10.0 79.2 2.1-24.8Male 7.9 25.7 5.6-11.9 11.0 24.9 7.0-14.5 10.1 24.3 7.7-14.7Male 9.7 42.0 5.9-18.5 12.2 29.2 6.9-17.1 8.2 25.7 5.1-10.2Female 6.6 50.5 3.0-13.3 15.5 34.0 4.8-21.3 11.2 30.8 5.2-16.8Male 5.0 18.8 3.6-6.0 18.4 58.4 8.7-40.1 9.4 46.5 6.1-17.0Female 8.4 50.9 4.0-15.6 21.7 67.8 8.5-49.3 17.0 70.7 6.2-38.1Male 10.6 41.3 5.5-19.4 24.0 37.8 12.4-41.8 13.0 44.6 6.9-24.9Male 8.2 23.8 5.8-11.5 24.6 42.0 15.9-44.0 13.9 30.9 8.4-23.5Male 10.0 36.3 5.6-14.6 27.4 79.7 12.3-78.4 15.2 43.5 9.1-29.2Female 7.0 39.1 2.8-11.2 30.3 47.3 5.2-54.2 23.3 46.8 5.0-42.3Male 20.9 98.9 7.9-68.5 37.5 108.5 1.9-112.3 22.6 108.1 1.1-70.3Male 14.7 76.3 4.2-36.8 50.6 99.7 9.2-142.3 18.1 80.1 4.3-43.8

Mean CV (%) 53.6 64.0 58.795% CI (%) 40.9-66.3 51.3-76.7 45.4-72.0

* Data are sorted according to increasing values of mean MEHHP concentrations.

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654 TRANSFUSION Volume 52, March 2012

relatively similar whatever metabolite is considered.30 Thecorrelation between DEHP metabolites was observed inprevious studies.19,21,31,32

Up to now, all attempts to propose methods to detectautologous blood transfusions had been addressed toblood measurements. The method proposed in this articleis the first experimental approach to screen for bloodtransfusion misuse (either homologous or autologous) bymeans of urine analyses. Urine samples are easier tocollect than blood samples and, moreover, the existingmethod in blood only allows homologous blood transfu-sion detection. The method here developed is proposed asa fast and low-cost screening test. In cases where potentialexposure to DEHP not linked to blood transfusion mightbe an explanation for the observed findings, otherapproaches described to detect autologous blood transfu-sion through blood measurements might be used ascomplementary or alternative evidences, if so considered.

PVC plasticized with DEHP is themost common material used for storageof RBC-containing blood products.33,34

Testing for DEHP may speed the adop-tion of alternatives (e.g., materials freeof DEHP) among doping athletes, asnormally occurs for other doping sub-stances and methods. Further studiesare needed to address blood transfusionpractices where materials free of DEHPcould be used. Similarly, evaluation ofthe DEHP content on the reinfusedmaterials when frozen glycerolatedRBCs are used for transfusion should beinvestigated. Frozen glycerolated RBCsneed washing before reinfusion, and theremaining content of DEHP in the rein-fusion materials should be investigated.These frozen RBCs are believed to beintensively used for doping purposes

In summary, this study confirmsthat DEHP metabolites in urine can beused as markers to detect most of themisuse of either autologous or alloge-neic (homologous) blood transfusions.The accumulation of further data andthe potential inclusion of the testamong the battery of methods in use indoping control might allow testing forblood transfusion all subjects for whichurine (e.g., 277,934 samples in year200935) is the most common biologicfluid available for doping control.

ACKNOWLEDGMENTS

The authors thank G. Balcells, A. Fabregat, J.

Franke, E. Marek, M. Rau, H. Uschkureit, W. Bloch, and S. Guddat

for the participation in different steps of the project. We also

thank Ruhr-Plasma-Center Bochum (Dr C. Hellermann) for their

support in the transfusion procedure and blood storage. This work

was supported by a grant from WADA (08B05PP), by grants from

DIUE Generalitat de Catalunya (2009 SGR 492), by the Fundació

IMIM (Barcelona, Spain), and by the Center for Preventive Doping

Research (Cologne, Germany). Background support to IMIM-

Hospital del Mar by Consell Català de l’Esport and to the Center for

Preventive Doping Research by the FRG Federal Ministry of Inte-

rior is acknowledged. The present work was recognized with the

award Premio Nacional de Investigación en Medicina del Deporte

2010, Universidad de Oviedo (Oviedo, Spain).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest rel-

evant to the manuscript submitted to TRANSFUSION.

Fig. 5. Mean and SEM of specific gravity adjusted concentrations of DEHP metabo-

lites in samples clustered according to six different situations (A = basal; B = day of

blood collection; C = 1 day after blood collection; D = day of reinfusion; E = 1 day

after reinfusion; F = 2 days after reinfusion). Right panel is a Y-axis–expanded insert

of left panel.

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Volume 52, March 2012 TRANSFUSION 655

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