A study protocol for a randomisedcontrolled trial evaluating clinicaleffects of platelet transfusion products:the Pathogen Reduction Evaluation andPredictive Analytical Rating Score(PREPAReS) trial

Paula F Ypma,1,2 Pieter F van der Meer,2 Nancy M Heddle,3 Joost A van Hilten,2

Theo Stijnen,4 Rutger A Middelburg,2,5 Tor Hervig,6 Johanna G van der Bom,2,5

Anneke Brand,2 Jean-Louis H Kerkhoffs,1,2 for the PREPAReS Study Group

To cite: Ypma PF, van derMeer PF, Heddle NM, et al. Astudy protocol for arandomised controlled trialevaluating clinical effects ofplatelet transfusion products:the Pathogen ReductionEvaluation and PredictiveAnalytical Rating Score(PREPAReS) trial. BMJ Open2016;6:e010156.doi:10.1136/bmjopen-2015-010156

▸ Prepublication history forthis paper is available online.To view these files pleasevisit the journal online(http://dx.doi.org/10.1136/bmjopen-2015-010156).

Received 8 October 2015Revised 23 December 2015Accepted 5 January 2016

For numbered affiliations seeend of article.

Correspondence toDr Paula F Ypma;p.ypma@sanquin.nl

ABSTRACTIntroduction: Patients with chemotherapy-inducedthrombocytopaenia frequently experience minor andsometimes severe bleeding complications. Unrestrictiveavailability of safe and effective blood products ispresumed by treating physicians as well as patients.Pathogen reduction technology potentially offers theopportunity to enhance safety by reducing bacterialand viral contamination of platelet products along witha potential reduction of alloimmunisation in patientsreceiving multiple platelet transfusions.Methods and analysis: To test efficacy, arandomised, single-blinded, multicentre controlled trialwas designed to evaluate clinical non-inferiority ofpathogen-reduced platelet concentrates treated by theMirasol system, compared with standard plasma-storedplatelet concentrates using the percentage of patientswith WHO grade ≥2 bleeding complications as theprimary endpoint. The upper limit of the 95% CI of thenon-inferiority margin was chosen to be a ≤12.5%increase in this percentage. Bleeding symptoms areactively monitored on a daily basis. The adjudication ofthe bleeding grade is performed by 3 adjudicators,blinded to the platelet product randomisation as well asby an automated computer algorithm. Interim analysesevaluating bleeding complications as well as seriousadverse events are performed after each batch of60 patients. The study started in 2010 and patientswill be enrolled up to a maximum of 618 patients,depending on the results of consecutive interimanalyses. A flexible stopping rule was designedallowing stopping for non-inferiority or futility.Besides analysing effects of pathogen reduction onclinical efficacy, the Pathogen Reduction Evaluation andPredictive Analytical Rating Score (PREPAReS) isdesigned to answer several other pending questionsand translational issues related to bleeding andalloimmunisation, formulated as secondaryand tertiary endpoints.Ethics and dissemination: Ethics approval wasobtained in all 3 participating countries. Results of the

main trial and each of the secondary endpoints will besubmitted for publication in a peer-reviewed journal.Trial registration number: NTR2106; Pre-results.

INTRODUCTIONPatients with thrombocytopaenia due to mye-loablative chemotherapy in the setting ofserious and acute bone marrow disease are atrisk of haemorrhagic complications.1 Platelettransfusions are extensively used for treatmentand prophylaxis of bleeding in these patients.Bleeding and refractoriness to platelet transfu-sions are prominent issues in patient

Strengths and limitations of this study

▪ This study will be conducted as a large rando-mised controlled trial concerning Mirasol patho-gen reduction technology; it will provideclinicians and blood banks with important infor-mation about this pathogen-reduction technique.

▪ The secondary outcomes of this study will offeropportunity to better predict bleeding episodesin thrombocytopaenic patients and insight intoimmunological refractoriness to platelettransfusions.

▪ The clinical relevance of WHO grade 2 bleedingis debated; however, WHO grade=2 bleeding isincorporated in the primary bleeding endpoint(grades 3 and 4 bleeding are obvious importantbleeding complications).

▪ The control group of platelet concentrates con-tains different platelet doses due to country-specific guidelines; subanalysis based on plateletcontent is feasible.

▪ The lack of blinding of the assessors collectingdata on bleeding symptoms is a limitation to thestudy design.

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management, and clinicians face the challenge of choos-ing the most appropriate transfusion strategy. Meeting thedemands of 24 h availability of safe platelet products is alogistic challenge, since the maximum storage time of pla-telets in the blood bank is 5 days.2 Platelets are stored atroom temperature, which places them at higher risk ofbacterial contamination as compared with other bloodcomponents.3 Extended storage of platelet concentratesfor up to 7 days has been approved in Europe for clinicaluse, but requires a post-marketing surveillance phase and100% bacterial testing.4 5 Bacterial outgrowth to hightitres, associated with longer storage times, has prompted afurther search for techniques that warrant safe plateletproducts. Currently, in the Netherlands, 0.44% of all plate-let products are found to be contaminated.6 Transfusion-transmitted bacterial infections with clinical consequencesare rare, with estimated incidence of 1 in 70 000 to 1 in118 000 cases.7 Pathogen-reduction strategies aim toreduce the risk of bacterial contamination of stored plate-let products.8 A recent review assessing the effectiveness ofpathogen-reduced platelet products in general concludedthat pathogen-reduced platelets did not differ from stand-ard platelets with regard to clinically significant or severebleeding complications or adverse reactions.9 Althoughthe use of pathogen-reduced platelet products(PR-platelet concentrates) is not formally accepted asstandard of care everywhere, gradually these products arebeing implemented in transfusion centres worldwide.Experts in the field of transfusion recently elucidatedreasons for either implementing pathogen-reduction tech-niques or postponing decision-making on implementa-tion.10 These reasons consist of the absence of regulatoryapproval, and concerns on safety and increment ofpathogen-reduced platelets.10 Mirasol pathogen reductiontechnology (Mirasol PRT) is a treatment system designedfor plasma and platelet products. This technology usesriboflavin, a naturally occurring vitamin B2, which interactswith nucleic acids leading to a chemical reaction whenexposed to ultraviolet (UV) light.11 Riboflavin-basedphotochemical treatment has been shown to be effectiveagainst a broad range of pathogens.12 13 In addition toreducing the infectious risk of viruses and bacteria forwhich blood is currently tested, this technology also offersa safety benefit by protecting against unknown and/oremerging pathogens, or pathogens that cannot bedetected otherwise in an early phase of infection (windowphase). Until now, only one randomised study evaluatingMirasol PRT, comparing corrected count increments(CCIs) of treated versus untreated apheresis or buffy coat-derived plasma–platelet concentrates, has been per-formed.14 This trial showed acceptable 1 h CCIs for bothmethods of preparation and no difference in platelet orred cell usage, suggesting no increase in bleeding ten-dency. Besides effective pathogen reduction, anotherimportant (pre)clinical premise concerning Mirasol treat-ment is potential effectiveness in reducing alloantibodyformation. Indeed, the ‘trial to reduce alloimmunisationto platelets study group’ showed evidence in patients

suffering from acute myeloid leukaemia that UV-B illumin-ation of platelets is equally effective as leukofiltration inpreventing generation of alloantibodies, as well asalloantibody-mediated refractoriness to platelets.15

RationalePolicymakers, product providers and investigators agreethat substantial changes in platelet production, such aspathogen reduction technologies, should also be vali-dated for their clinical efficacy, including an assessmentof bleeding. The Pathogen Reduction Evaluation andPredictive Analytical Rating Score (PREPAReS) study wasinitiated to investigate Mirasol-treated platelet productsfor clinical efficacy. The research question that isaddressed is summarised in figure 1. The aim is todescribe here the design of the PREPAReS trial andprovide rationale for the design elements that wereincorporated into this study.

ObjectivesPrimary objectiveThe primary aim of this study is to assess the non-inferiority of PR-plasma-platelet concentrates (PCs)

Figure 1 Pathogen Reduction Evaluation and Predictive

Analytical Rating Score (PREPAReS) research question

(PICOT format).

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compared with plasma-PCs in terms of WHO bleedingcomplications ≥grade 2 (expressed as percentage ofpatients) in haemato-oncological patients with thrombo-cytopaenia during one transfusion episode.

Secondary objectives1. To assess the transfusion failures defined as 1 h CCI

<7.5 and 24 h <4.5, 1 and 24 h CI and CCI of allplatelet transfusions.

2. To assess the percentage of days that bleedings>WHO grade 2 occur.

3. To evaluate whether clinical factors interact with thedifferent study products leading to a difference inplatelet refractoriness.

4. To assess the safety (incidence of adverse reactions).5. To assess the transfusion requirement (red cells and

platelets).6. To assess the transfusion interval.7. To assess the rate of human leucocyte antigen

(HLA) alloimmunisation.8. To evaluate whether in vitro measures relate to in

vivo outcome measures as the 1 h and the 24 h CCI.9. To establish the relation between albuminuria and

bleeding tendency.10. To establish whether transmission of commensal

viruses can be prevented by the Mirasol treatment.

METHODS AND ANALYSISPREPAReS trial designThe PREPAReS study is a randomised multicentre trialusing a parallel arm design. Three countries involving10 centres are participating in PREPAReS. In theNetherlands, there are three academic centres and onenon-academic hospital participating. In Norway, one aca-demic centre participates and in Canada, one academiccentre and four general hospitals participate. In all ofthese hospitals, there are facilities for haematologicalintensive care for patients with acute leukaemia, andautologous and/or allogeneic stem cell transplantationfor haemato-oncological diseases. Sanquin Blood Supply,the Dutch Blood Bank, is the study sponsor. Patientenrolment started in 2010 and is expected to end in2016. The study was approved by a central ethical com-mittee as well as the local Research Ethics Boards(REBs) of the participating sites. Figure 2 summarisesthe design of the trial and each of the trials aspects isdescribed in detail below.

Eligibility criteriaPatients aged 18 years or older are eligible for inclusionin the study; they are expected to require at least twoplatelet transfusions during a period of profoundthrombocytopaenia. This may be following intensivechemotherapy or a conditioning regimen prior to haem-atopoietic stem cell transplantation, due to aplasticanaemia or a result of the bone marrow disease crowd-ing out megakaryopoiesis. Exclusion criteria include

microangiopathic thrombocytopaenia and idiopathicthrombocytopenic purpura (ITP); bleeding grade >2 atrandomisation; known immunological refractoriness toplatelet transfusions; HLA and/or HPA alloimmunisa-tion or clinical relevant autoantibodies; pregnancy; indi-cations to use hyperconcentrated platelets; priortreatment with pathogen-reduced blood products;known allergy to riboflavin or its photoactive products.Sites that routinely perform HLA/platelet antibodyscreening when immunological refractoriness is sus-pected will base this decision on a positive screening testresult. Sites that do not routinely carry out this screeningwill base this decision on clinical judgement. Withregard to patients presenting with a bleeding conditionof grade >2 before enrolment, a template was designedto support decision-making, primarily enabling theenrolment of patients with old bruises (figure 3).Patients previously treated with pathogen-reduced plate-let products using other technologies are also excluded.

RandomisationPatients who meet the eligibility criteria are randomised toreceive either standard plasma-stored, or Mirasol-treatedplatelet products, during a transfusion period (for defin-ition of ‘transfusion period’: figure 1). Randomisationtakes place using a web-based tool, as part of ProMISe(Project Manager Internet Server). The ProMISe system,developed at the Leiden University Medical Center, pro-vides custom-made databases for scientific medicalresearch as well as an application for online data entry,quality checks and reporting. The treatment allocationscheme is created within ProMISe and is generated by thecomputer, ensuring concealment of treatment allocation.The randomisation method uses permuted blocks of vari-able size, randomly varying from 2 to 6 participants.Stratification factors in the randomisation are: centre,diagnosis (acute myeloid leukemia (AML) vs no AML)and treatment phase (transplant vs no transplant).16

Patients are randomly assigned to the treatment allocationat the time that they are expected to require their firstplatelet transfusion. All eligibility criteria will be checkedwith an electronic checklist during randomisation.Randomisation will be requested by the staff memberresponsible for recruitment. Each patient will be given aunique patient study number, which will be given immedi-ately by the randomisation provider (ProMISe) and con-firmed by email. This email is sent to the staff memberrequiring randomisation as well as to the generalPREPAReS email address, controlled by the principalinvestigator. Patients may be re-randomised in a subse-quent transfusion period (see figure 1) following a nextintensive chemotherapy course or a pre-transplant condi-tioning regimen and the statistical analysis will account formultiple randomisations per individual.17

Treatment armsAll platelet products are produced by local blood suppli-ers (Sanquin Blood Bank in the Netherlands,

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Haukeland University Blood Bank in Norway andCanadian Blood Services in Canada). Platelet concen-trates are all prepared from pooled buffy coats and arere-suspended in plasma. The pooled platelet concen-trates are leukoreduced by filtration. All platelet pro-ducts are stored with gentle agitation at 20–24°C up to7 days in the Netherlands and Norway, and for amaximum of 5 days in Canada. Patients randomised tothe standard platelet product arm of the study receiveplatelet products prepared in this manner.The platelet products transfused to patients in the

pathogen-reduced arm of the study are prepared asfollows: 500 μM riboflavin is added to the leukoreducedplasma–platelet concentrates within 28 h of platelet col-lection, mixed and exposed to UV-B light (wavelength280–315 nm) for 5–10 min (depending on the volumeof the platelet concentrate) with constant agitation at120 rpm, giving a total dose of 6.2 J/mL. Following treat-ment, the platelets are stored in the same way as theuntreated platelets, making sure that the products arenot exposed to direct daylight. In all participatingcentres, a standardised automated culture method isused to detect bacterial contamination of the plateletproduct.18

Products are γ-irradiated if indicated by the requestingcentre, even though the Mirasol procedure is licensedfor white cell inactivation to prevent graft versus hostdisease, making γ-irradiation unnecessary. The rationalefor γ-irradiation in this trial reflects a logistical concernthat two different policies of irradiation based on thetreatment arms of the study could increase the risk oferror. It could result in patients in the standard treat-ment arm incorrectly receiving non-irradiated products,or patients in the treatment arm receiving non-irradiated products in case of an off protocoltransfusion.

Table 1 shows characteristics of the platelet productsin the participating countries. In Canada, platelet con-centrates are routinely prepared from four buffy coats,with on average 300×109 platelets per unit. The SPRINTtrial had shown that pathogen-reduced platelet unitswith fewer than 300×109 per unit were associated with asignificantly shorter transfusion interval and lower CCIs(though not an increased bleeding risk) compared withuntreated platelet concentrates with <300×109 plateletsper unit.19 20 Since there was a chance that the plateletcontent in a four buffy coat pool would be below thisthreshold, the Canadian sites provided a five buffy coatplatelet pool for patients allocated to the PR arm,increasing the number of platelets by 25%. TheCanadian hospitals wanted to maintain four buffy coatsin the control arm, as this is the current standard ofpractice to which the PR arms should be compared.Differences in platelet numbers could potentially lead toa bias in the study. Therefore, before the final decisionwas made, quality control data from Canada and theNetherlands for the year 2011 were compared. TheDutch untreated units from five buffy coats (n=5068)contained, on average, 380±55×109 platelets. Treatedunits from five buffy coats (n=347) contained 366±55×109, that is, about 5% lower than untreated ones.Canadian untreated platelet concentrates from fourbuffy coats (n=1302) had an average of 312±54×109 pla-telets. Therefore, five buffy coats would theoretically give390×109 platelets, but with the 5% decrease due to thehandling associated with Mirasol treatment, theexpected average would come to 371±64×109 platelets.Assuming that Canada would include one-third of thepatients, and the Netherlands and Norway two-thirds,the overall distribution of platelet numbers per concen-trate was estimated. This estimation was based on the fol-lowing facts: platelet concentrates in the untreated

Figure 2 PREPAReS trial

design. The flow chart

summarises the design of the

PREPAReS trial; eligible patients

may be enrolled more than once,

for example, when they receive

consolidation chemotherapy.

*See also figure 3. PR,

pathogen-reduced; PREPAReS,

Pathogen Reduction Evaluation

and Predictive Analytical Rating

Score.

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group were derived from four buffy coats for Canadaand Norway, and five buffy coats for the Dutch sites; inthe Mirasol group, they were four buffy coats for Norwayand five buffy coats for all other sites. Under these con-ditions, it was calculated that the platelet counts wouldlargely overlap, although in the untreated group, about23% would contain <300×109 vs 7% in the Mirasolgroup. In the SPRINT trial, 12% of the platelet concen-trates in the control group contained <300×109 plateletsversus 20% in the pathogen-inactivated group.19

Therefore, we accepted that, in Canada, untreated plate-let concentrates were made from four buffy coats, whilein the Mirasol-treated group, they were made from five

buffy coats. Subanalysis based on platelet content is feas-ible. Moreover, a higher platelet dose may offset part ofthe lower platelet increments seen with Mirasol-treatedplatelet concentrates.14

Indications for platelet transfusionsIndications for platelet transfusions are distinguishedinto platelet count-related prophylaxis (PP),intervention-related prophylaxis (IP) and treatment ofbleeding (TB). For each transfusion, the indication isrecorded. The Dutch transfusion guidelines are used asguidance for the indication of platelet transfusions,these imply: trigger for PP 10×109/L; for IP 50×109/L;and for TB until bleeding stops or up to a platelet countof 100×109/L. When bleeding complications occur intra-cranially, in the retina or in any other vital organ, theadvised platelet count is >100×109/L.21 The treatingphysician determines if or when a transfusion isordered.

Outcomes (primary and secondary)The primary outcome for the study is bleeding greaterthan or equal to WHO grade 2 (WHO). Earlier com-parative platelet transfusion studies concerning plateletadditive solutions, and even the Mirasol system forpathogen inactivation, used CCIs as primary outcomemeasure.14 The CCI, however, is inadequate as a surro-gate marker for the occurrence of bleeding symp-toms.22 23 There is general agreement that substantialchanges in platelet production and processing shouldnot only be validated by CCI measurement, but, also,clinical validation for their haemostatic effectiveness isneeded. Haemostatic assessment ideally is performedusing a consistent and well-described manner.24 Severallarge randomised controlled trials (RCTs) that were pub-lished in recent years used the WHO bleeding scale.25–28

The WHO bleeding scale can be regarded as a gradingmethod that classifies the seriousness of bleeding. In the

Figure 3 Inclusion in the study

in case of a bleeding

complication. Template for

decision-making in case of a

bleeding complication at

presentation before enrolment of

the Pathogen Reduction

Evaluation and Predictive

Analytical Rating Score

(PREPAReS) trial. Bleeding

grade is based on WHO bleeding

scale.

Table 1 Characteristics of platelet products in three

participating countries in the PREPAReS trial

Country Participating

in PREPAReS

The

Netherlands Norway Canada

Blood supplier Sanquin

Blood Bank

Haukeland

University

Blood

Bank

Canadian

Blood

Services

Anticoagulant used

for whole blood

collection

CPD CPD CPD

Method of platelet

preparation

Buffy coat Buffy coat Buffy coat

Standard pool size of

Plasma platelets 5 4 4

Pathogen-reduced

platelets

5 4 5

Bacterial testing Yes Yes Yes

Storage solution Plasma Plasma Plasma

Duration of storage 7 days 7 days 5 days

CPD, citrate-phosphate-dextrose; PREPAReS, Pathogen ReductionEvaluation and Predictive Analytical Rating Score.

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WHO bleeding scale, grade 0 is absence of bleeding;grade 1 bleeding generally is considered not to be clinic-ally relevant, in the grade 2 bleeding category, bleedingof variable clinical relevance is encountered (skin,mucosal membranes, bleeding at invasive sites, etc),whereas grades 3 (bleeding requiring red cell transfu-sions) and 4 bleeding (central nervous system bleedingand fatal bleeds) are the most serious. In a pilot studywhere daily bleeding was assessed in the Dutch centresthat currently participate in the PREPAReS, we foundthat 54% of patients present with grade >2 bleeding atsome point in their clinical course.29 As this is compar-able to the STOP trial, published in 2009, we used thesepilot data for the power calculation of the PREPAReS.25

Secondary outcomes contribute to the evaluation ofnon-inferiority of pathogen-reduced platelet products inthis patient population: the frequency of 1 h and 24 hpost-transfusion platelet increment failures (defined as1 h CCI <7.5 and 24 h CCI <4.5), the percentage of daysthat bleedings ≥WHO grade 2 occur, the incidence ofadverse reactions, the transfusion requirement of redcells and platelets, and the platelet concentrate transfu-sion interval, and the incidence of HLA alloimmunisa-tion. The evaluation of whether in vitro measures relateto in vivo outcome measures as the 1 h and the 24 hCCI is also defined as secondary outcome, and is incor-porated in several tertiary outcomes as explained ahead.

Tertiary outcome measures; linking in vitro quality tests toin vivo outcomesThe large cohort of patients randomised in thePREPAReS being closely watched for bleeding symp-toms, creates possibilities for additional research ques-tions. Outcome measures, focusing on the connectionof preclinical and clinical outcomes with respect tosafety issues (decrease of microbial transmission andHLA antibody formation) as well as other translationalaspects, are addressed in the PREPAReS trial. Predictionof individual bleeding episodes on clinical grounds, orwith the aid of laboratory measurements, remains ablind spot in the aim of maximal prevention strategies.

In vitro platelet quality testing and relation with CCICurrently, provided the pH is above the threshold forproduct release (pH22◦ >6.2), there are no in vitro mea-surements available that accurately predict platelet recov-ery, survival or haemostatic function in plateletconcentrates.30–32 A combination of individually well-established metabolic, activation and functional para-meters of platelet concentrates, combined into one‘rating score’, may predict either the 1 h and/or the24 h CCI. For such an in vitro rating value, we selectedassays that can be performed shortly before transfusionof the platelet concentrates. We selected three para-meters for this rating system, each of which could playan independent role in predicting in vivo effectivenessof stored platelet concentrates, for reasons discussedbelow.33 First, we considered CD62P expression, an

activation marker, on the platelet surface. A higherCD62P expression has been associated with enhancedplatelet clearance from the circulation.34–37 Second,impaired platelet survival in animals was found to beassociated with the apoptosis marker annexin A5binding.38 39 The third assay is the lactate concentration(as surrogate for lactate production) as a low lactate pro-duction rate is considered a good indicator of mitochon-drial function.31 40 41 The in vitro outcomes of each ofthese three parameters are scored from 0 to 2, where 0points would indicate a poor quality and 2 points a goodquality, rated on an arbitrarily chosen linear basis. Thecombined rating then results in a value between 0 (poorquality) and 6 (optimal quality; table 2). By relating thein vitro rating scores with CCI values and haemostaticassessment data of the transfusion day, and the day after,the relation between this combination of metabolic(lactate concentration), activation (CD62P) and apop-tosis (annexin A5) parameters of transfused platelet con-centrates, and the usefulness of combining these intoone ‘rating’ value, will be evaluated. Other laboratorytests for platelet responsiveness of the product will beperformed and linked to clinical outcome, includingthromboelastography and dynamic light scattering(ThromboLUX, LightIntegra, Canada).42 We apply thesame tests also on patient samples prior to and aftertransfusion, to investigate if there is an improvement inthese platelet function assays and how that relates to thetests performed on that platelet concentrate. Theresearch staff performing daily bleeding assessments isblinded for the outcomes of the in vitro tests of theproduct and of patient samples.

HLA antibodiesThe Mirasol treatment not only inactivates proliferationof residual contaminating white blood cells in plateletproducts preventing graft versus host disease, but alsoimpairs the antigen-presenting potential of treated leu-kocytes. Animal studies contribute to the hypothesis thatthe Mirasol procedure could play a role in reducing thealloimmunisation and induce tolerance to a subsequentallograft.43–47 To further investigate these assumptions inthe current study, samples are obtained from patientswho are negative for HLA antibodies prior to transfu-sions, and analysed to investigate de novo or boosteredformation of HLA antibodies during a follow-up period.These samples are collected weekly during hospitalisa-tion up until day 28, and then on day 56, and tested in

Table 2 Rating score for quality testing platelet

concentrates

Score in vitro test 2 points 1 point 0 points

CD62P expression <20% 20–30% >30%

Annexin A5 binding <10% 10–20% >20%

Lactate concentration <10 mM 10–20 mM >20 mM

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the Luminex-based assay for presence of HLA anti-bodies.48 In a selected cohort of primed and unprimedpatients, following the first on-protocol transfusion, the1 h samples are analysed for induction of HLA class IImolecules on T-cells of the donor (if detectable) andrecipient, and at day 56, blood is collected and frozen-processed for evaluation of persisting donor cells.

Endothelial damageCytotoxic therapy for haemato-oncological disease playsa central role in the occurrence of a bleeding event.49–51

Maintenance of vascular integrity is important toprevent microvascular and macrovascular bleeding symp-toms. Endothelial damage is a presumed mechanism forincreased (transfused) platelet consumption.52 A subsetof patients included in the PREPAReS trial (thisdepends on hospital participation) undergo serum Creactive protein and urine testing for albumin twiceweekly. These laboratory markers for endothelialdamage53 54 will be analysed in relation to the extent ofthe bleeding tendency patients develop during the plate-let transfusion period.

Tests for commensal virusesThe pathogen reduction technology applied in thePREPAReS study aims to reduce the transmission ofpathogens. The actual risk of viral transmission is in theorder of 1 in 1 million or less, and for clinically relevanttransmission of bacteria about 1 in 70 000 to 1 in118 000.7 55 Hence, the PREPAReS trial is not poweredto detect prevention of transmission of pathogens.Commensal viruses in the blood supply are not screenedfor, as they are present in healthy blood donors, and arethought to be non-pathogenic, albeit this may not bethe case for (premature) infants and immunocomprom-ised patients. Because of their universal presence inhumans, commensal viruses can therefore serve as surro-gate markers for pathogen-reduction efficacy.Transmission of commensal viruses during transfusioncan be most efficiently tracked using the highly genetic-ally diverse commensal anelloviruses, of which numerousstrains can be readily distinguished by their distinct DNAsequences. Genetic comparisons of the strains of anello-viruses in donor and recipient samples, therefore, offera sensitive system to confirm viral transmission duringtransfusion and to detect its elimination in patients whoreceive Mirasol-treated platelet units.56

Assessment of bleedingDuring the transfusion period, patients are screened forbleeding every day by a member of the study team.Every assessor is trained by an instruction slide set onthe study website, which discusses usual and unusual ordebatable bleeding sites and presentations, followed by atest. The assessments are performed by physical examin-ation and patient interviews, as well as checking hospitalmedical and nursing charts. In Canada, a validatedpatient self-assessment form is used on weekends,57 with

additional follow-up at the beginning of the week byreviewing the patient’s medical record and communicat-ing with the care givers who were on service over theweekend period, to ascertain if bleeding has occurred.

BlindingBecause the Mirasol-treated platelet products show acolour difference that does not allow proper blinding tothe treatment arm, the study is not blinded for researchpersonnel evaluating bleeding signs and symptoms.Nurses involved in giving the transfusion or assistingwith data collection other than bleeding may be awareof the randomisation results. The actual allocation mustnot be disclosed to the staff members responsible forthe adjudication process, nor to the monitors and thesponsor. In none of the corresponding patient docu-ments is the allocation disclosed.

Grading the bleedingThe grade of bleeding is assigned later by three inde-pendent adjudicators blinded to the patient’s treatmentallocation, based on the specified WHO bleedingscale.26 If discrepancies occur, a consensus meeting isorganised to make a final decision on the bleedinggrade. In addition, an automated algorithm for com-puter adjudication on consecutive haemostatic assess-ment data sets was developed, and showed an excellentrelationship with ‘human’ adjudication.58 Human andcomputerised adjudication is performed independentlyonce the data manager has given permission to adjudi-cate a subsequent series of 60 participants with completedata. In the event that discrepancies occur between themanual and computerised adjudication, agreementarrives through consensus. The blinded adjudicatorsalso answer any questions or issues concerning unclearbleeding symptoms from participating sites through thestudy by email.

Adverse eventsAdverse events are, according to the definitions, anyunfavourable or unintended event affecting patients onstudy. In cases of prolongation of hospitalisation, deathor significant clinical sequelae, these events are definedas serious adverse events (SAEs), the occurrence ofwhich the study sponsor (Sanquin) and the Data SafetyMonitoring Board (DSMB) will be informed at shortnotice. During protocol treatment, all deaths, all SAEsthat are life-threatening and any unexpected SAE mustbe reported to the Sanquin data manager using the SAEweb form within 48 h of the initial observation of theevent. The protocol treatment period is defined as theperiod from the first study-related platelet transfusionuntil 30 days after the last study-related platelet transfu-sion, or until the start of another systemic anticancertreatment cycle, if that occurs earlier. All details shouldbe documented on the SAE and death report. Reportswill also be sent to the local Canadian REBs and toHealth Canada, according to pre-established criteria.

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Further, any death (regardless of cause) that has cometo the attention of the study staff, occurring more than30 days after the last study-related platelet transfusion,must be reported to the Sanquin data manager usingthe death report web form.Safety aspects of the study are closely watched by the

DSMB, which receives unblinded data for its judgement.In the case of a suspected relation of the SAE to thepathogen-reduction technique, Terumo blood compo-nent technology (Terumo BCT) is notified.

Data managementIn the PREPAReS trial, data are collected during a trans-fusion episode, as defined (figure 1). Data collection isperformed by trained local research staff and data entryin the ProMISe database is completed in a standardisedfashion from two central research locations in Canadaand the Netherlands. Other participating centres in theNetherlands and Canada as well as the Norwegiancentre send their paper case report forms (CRFs) to thedata entry site. A central data manager performs andmonitors data entry, and looks after timely CRF delivery.Data on the CRF will include at least: scores of bleeding,baseline observations at entry, follow-up observations,observations before, during and after platelet and redcell transfusions, adverse transfusion reactions, and theparameters needed for calculating 1 and 24 h CCIs. Anyother parameters necessary to evaluate the study end-points and reason for end of protocol treatment are alsodocumented.

End of protocol treatmentReasons for going off protocol are:▸ Transfusion independency >7 days or hospital dis-

charge, whichever occurs first.▸ Patient non-compliance (especially refusal to con-

tinue; the reason for discontinuation must berecorded in the source documentation and the StudyExit CRF).

▸ Intercurrent death.▸ Reasons for going off protocol treatment, but with

full study monitoring, including daily scoring ofbleeding, are:

▸ Serious adverse transfusion reactions necessitatingother products;

▸ Immunological refractoriness.

Sample size and rationale for non-inferiorityThe PREPAReS study is designed on a hypothesis ofnon-inferiority. Apart from the superior safety profile ofthe product to date,59 the effectiveness of the product toprevent bleeding may be less than that of the controlproduct, to some extent. Previous studies25 27 29 indi-cated that, in this specific patient group, on average,50% of patients with thrombocytopaenia exhibit bleed-ing complications ≥grade 2 at any time while on study.Based on discussions with clinicians and on the basis ofearlier published studies,26 the investigators agreed that

up to a 12.5% increase in grade 2 or greater bleedingwould be acceptable to still claim non-inferiority of thePR platelet product. Thus, non-inferiority will be con-cluded when the upper limit of the two-sided 95% CIfor the difference between the risk of a ≥grade 2 bleed-ing in the Mirasol group compared with the controlgroup is below 12.5%. Under the assumption of 50%risk in both groups, the required number of patients todemonstrate non-inferiority with 80% power is 618 (309per arm), taking into account the stopping rule as wellas a 7% drop out of patients. For safety reasons, interimanalyses are planned in this study after every 60th inclu-sion. A flexible stopping rule based on α and β spendingfunctions was designed, allowing stopping for non-inferiority or futility. The (unblinded) DSMB will, ateach interim analysis, either give the green light for con-tinuation or not; the study team remains blinded.

Statistical analysisPrimary outcomeThe primary effect parameter is the difference in theprobability of a WHO bleeding complication ≥grade 2during one transfusion episode for pathogen-reducedplatelet concentrates compared with standardplasma-stored platelet concentrates. Patients can contrib-ute more transfusion episodes to the study and the out-comes of different transfusion periods may be correlatedwithin patients. Furthermore, the probability of a≥grade 2 bleeding might depend on the number of theepisode within a patient. The primary effect parameteris estimated according to a generalised estimating equa-tion (GEE) approach using a generalised linear modelwith identity link and independence working correl-ation. Covariates in the model will be the treatmentgroup, the episode number (dichotomised as first orlater) and the interaction between these two covariates.The primary treatment effect is then calculated as aweighted mean of the effect in first episodes and in laterepisodes, where the weights are proportional to thenumbers of first and later episodes in the study. Ifthe upper bound of the two-sided 95% CI is <12.5%, thereduced platelet treatment will be concluded to be non-inferior to standard plasma-stored platelet concentrates.

Secondary outcomesWe only briefly describe the analysis of the most import-ant secondary outcomes. Transfusion failures aredefined as 1 h CCI <7.5 and 24 h CCI <4.5, where the1 h and 24 h CCI are defined as:

CCI1=24h ¼post- -- pretransfusion count (x109=L)

� body surface area (m2)transfused number of platelets (x1011=L)

Per episode, the failure rates are calculated as thenumber of failed transfusions as proportion of the total

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number of transfusions. Next, the failure rates are ana-lysed by a GEE approach analogous to the primaryoutcome analysis.The 1 h and 24 h CCI will be analysed with a fully spe-

cified linear mixed model, taking into account the cor-relations between transfusions within episodes as well asbetween episodes within patients. The percentage ofdays that bleedings ≥WHO grade 2 occur per episodewill be analysed following the GEE approach, as for theprimary outcome. The rate of alloimmunisation will beanalysed using the log-rank test.

MonitoringThe study will have monitoring for quality and regula-tory compliance. In the Netherlands and Norway, moni-toring will be performed by Sanquin. In Canada,monitoring of the participating sites and McMasterTransfusion Research Program (MTRP) (acting as theCanadian Clinical Research Officer (CRO)) will be per-formed through a contract with an external companythat specialises in monitoring of clinical trials. The fre-quency depends on inclusion rates, questions andpending issues from earlier audits: once or twice a year.

Ethics and disseminationEthics approval was first obtained in the Netherlands atthe Medical Ethics Committee South West Holland (TheHague, The Netherlands). They judged the study design,ethics, interim analyses, the allowed level of non-inferiorityand safety rules. On approval, the study was started in theHAGA Teaching Hospital in The Hague. Additionalregional approval was obtained for the remaining threeDutch hospitals. In Norway, ethics approval was obtainedfrom the Regional Committee for Medicine and HealthResearch Ethics (region West, Bergen, Norway). InCanada, ethics approval was obtained at each of the fivesites from the local REB. The trial is registered with theNetherlands Trial Register NTR2106.A manuscript with the results of the primary study will

be published in a peer-reviewed journal. Separate manu-scripts will be written on each of the secondary aims,and these will also be submitted for publication in peer-reviewed journals.On completion of the trial, and after publication of

the primary manuscript, data requests can be submittedto the researchers at the Center for Clinical TransfusionResearch, Sanquin Research, Leiden, The Netherlands.

DISCUSSIONSupportive care of patients with life-threateninghaemato-oncological disease is an important issue.Photochemical pathogen reduction contributes to therisk reduction of transfusion with regard to transmittedinfections.12 It remains, however, unclear whether thisimproved safety is not outweighed by the potentiallyreduced clinical efficacy of PRT platelets, that is,

decreased post-transfusion increments, increased plateletproduct consumption and even increased bleeding risk.9

With regard to Mirasol-treated platelets, a recent RCTshowed decreased post-transfusion increments but didnot find an increase in bleeding complications.14 Themain goal of the PREPAReS study is to demonstrate non-inferiority of Mirasol-treated platelets as compared withstandard untreated platelets, using the percentage ofpatients with ≥ WHO grade 2 bleeding as primaryoutcome. Alloimmunisation as well as in vitro plateletquality testing related to clinical outcome are two otherimportant cornerstones of this study. This study willcollect day-to-day data of over 600 patients, 2000 platelettransfusions and 24 000 days of follow-up, allowing forseveral other important issues, for instance the capacityof the current WHO system to grade bleeding complica-tions in predicting major bleeding complications, thepossibility of testing other bleeding scales and serving asa platform for several site studies including albuminuriaas a predictor for vascular damage, increased plateletconsumption and, perhaps, bleeding.60–62

The potential reduction of alloimmunisation throughthe use of Mirasol-treated platelets would be of additivebeneficial value, potentially even more clinically significantin comparison to the reduction of transfusion transmittedinfectious diseases. Last but not least, linking in vitro plate-let product quality markers potentially enables us toimprove the preclinical testing of novel platelet products.

Author affiliations1Department of Hematology, HAGA Teaching Hospital Den Haag, TheNetherlands2Center for Clinical Transfusion Research, Sanquin Research, Leiden, TheNetherlands3Faculty of Health Sciences, Department of Medicine, Canadian BloodServices, McMaster University, and Centre for Innovation, Hamilton, Ontario,Canada4Leiden University Medical Centre, Leiden, The Netherlands5Department of Clinical Epidemiology, Leiden University Medical Center,Leiden, The Netherlands6Department of Immunology and Transfusion Medicine, and Department ofClinical Science, Haukeland University Hospital, University of Bergen, Bergen,Norway

Acknowledgements The authors thank Ray Goodrich, David Cox and RonaldBrand, for their role in the design of the PREPAReS study. This trial is beingcoordinated by Sanquin Research, Center for Clinical Transfusion Research andthe trial is funded by Terumo BCT. The McMaster Transfusion ResearchProgram, which is coordinating the study in the Canadian centres, receivespartial infrastructure funding from Canadian Blood Services and Health Canada.

Contributors J-LHK, PFvdM, AB, JAvH and PFY were involved in conceptionand trial design. PFY, PFvdM and J-LHK were involved in drafting of thearticle. NMH, PFvdM, AB, JAvH, RAM, TS, TH and JGvdB were involved incritical revision of the article for important intellectual content. All the authorswere involved in final approval of the article. TS provided statistical expertise.Preparing study design, collection, management, analysis and interpretationof data; writing of the report; and the decision to submit the report forpublication is the responsibility of Sanquin, the study sponsor. The studyfunder, Terumo BCT, has an advisory role. Although strictly from a regulatoryperspective for the Canadian part of the study, Terumo BCT is the sponsor ofthe trial; also, in Canada, by contract, all study related activities as mentionedare under the authority of Sanquin.

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Funding The PREPAReS study is investigator initiated; Sanquin Blood Supplyis the sponsor of the study. The PREPAReS study is funded by Terumo BCT.

Competing interests None declared.

Ethics approval Central Ethics Committee: METC Zuidwest Holland, theNetherlands.

Provenance and peer review Not commissioned; externally peer reviewed.

Open Access This is an Open Access article distributed in accordance withthe Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license,which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, providedthe original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

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