MARK-AGE standard operating procedures (SOPs) : A successful effort

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MechanismsofAgeingandDevelopment151(2015)18–25

ContentslistsavailableatScienceDirect

Mechanisms of Ageing and Development

j o ur na l h o me p a g e:w w w . e l s e v i e r . c o m / l o c a te / m e c h a g e d e v

MARK-AGE standard operating procedures (SOPs): A successful effort

María Moreno-Villanueva

^a,∗,1

, Miriam Capri

^b,1

, Nicolle Breusing

^c

, Anne Siepelmeyer

^d

, Federica Sevini

^b

, Alessandro Ghezzo

^h

, Anton J.M.de Craen

^e

, Antti Hervonen

^f

,

Mikko Hurme

^f

, Christiane Schön

^d

, Tilman Grune

^c,g

, Claudio Franceschi

^b

, Alexander Bürkle

^a

aMolecularToxicologyGroup,DepartmentofBiology,UniversityofKonstanz,78457Konstanz,Germany

bDIMES-DepartmentofExperimental,DiagnosticandSpecialtyMedicine,CIG-InterdepartmentallCentre“L.Galvani”,AlmaMaterStudiorum,Universityof Bologna,40126Bologna,Italy

cInstituteofNutritionalMedicine,UniversityofHohenheim,70599Stuttgart,Germany

dBioTeSysGmbH,73728Esslingen,Germany

eDepartmentofGerontologyandGeriatrics,LeidenUniversityMedicalCenter,Leiden,TheNetherlands

fSchoolofMedicine,UniversityofTampere,33014Tampere,Finland

gGermanInstituteofHumanNutrition,14558Nuthetal,Germany

hNationalAssociation“FamigliediPersoneconDisabilitaAffettivae/oRelazionale”(ANFFAS)Onlus,Macerata,Italy

a r t i c l e i n f o

Articlehistory:

Received29January2015

Receivedinrevisedform17March2015 Accepted23March2015

Availableonline26March2015

Keywords:

Standardoperatingprocedures Biobank

Humanstudies

a b s t r a c t

WithintheMARK-AGEproject,apopulationstudy(3337subjects)wasconductedtoidentifyasetof biomarkersofageingwhich,asacombinationofparameterswithappropriateweighting,wouldmeasure biologicalagebetterthananysinglemarker.TheMARK-AGEprojectinvolves14Europeancountriesand atotalof26researchcentres.Insuchastudy,standardoperatingprocedures(SOPs)areanessentialtask, whicharebindingforallMARK-AGEBeneﬁciaries.TheSOPscoverallaspectsofsubject’srecruitment, collection,shipmentanddistributionofbiologicalsamples(bloodanditscomponents,buccalmucosa cellsorBMCandurine)aswellastheanthropometricmeasurementsandquestionnaires.

1. Introduction

AcrossallEuropeancountries,thenumberofelderlypeopleis increasingsteadily.Therefore,itisbecomingmoreandmoreimpor- tanttodeﬁneareliablemethodofassessmentofthestateofageing, whichhasnotbeenpossiblewiththeavailabletechniques.The MARK-AGEstrategytosolvethisproblemistheidentiﬁcationof anage-relatedchangeinbodyfunctionorcompositionthatcould serveasameasureof“biologicalage”andwhichpredicttheriskof onsetofage-relateddiseasesmoreaccuratelythanchronological agedoes.Suchparametersaretermed“biomarkersofageing”.

TheMARK-AGEprojectinvolvesresearchersfrom14European countries:Austria,Belgium,Denmark,Finland,France,Germany, Greece,Italy,theNetherlands,Poland,Romania,Spain,Switzerland and the United Kingdom. A total of 26 “Beneﬁciaries” (i.e.the

∗Correspondingauthor.Tel.:+497531884414;fax:+497531884033.

E-mailaddress:maria.moreno-villanueva@uni-konstanz.de (M.Moreno-Villanueva).

1 Theseauthorscontributedequallytothiswork.

participatinginstitutionsorcompanies)collaboratedwiththeaim ofidentifyingpowerful“biomarkersofageing”(Table1).Therange ofcandidatebiomarkerstobetestedincludes(a)“classical”ones forwhichdatafromseveralsmallerstudieshavebeenpublished;

(b)“new”ones,basedonrecentpreliminarydata,aswellas(c)

“novel”ones,basedonrecentresearchonmechanisticaspectsof ageing,conductedbyprojectparticipants(Vanhoorenetal.,2007;

Garagnanietal.,2012;Dall’Olioetal.,2013;Collinoetal.,2013).

Inconsiderationofthehugeamountsofsamplestobecollected SOPsneedtobedefinedconcerningprobandrecruitment,sampling andprocessingofbodyfluids.ASOPisasetofwritteninstructions thatdocumenthowtheinvolvedrecruitingandstudystaffexecutes thetasksandwhichmaterialsareused.Thesedetailedinstructions includestep-by-stepdetailsoftheprocessesandprovidesinstruc- tionsinordertoperformthetaskinaconsistentmanner.SOPsare anessentialcomponentassuringconsistencyinthequalityofdata collectionasitprovidesthestaffwiththeinformationtoperform theirjobproperlyandinastandardizedmannerfollowingstudy requirements.Thecredibilityofhumanstudiesthatarenotcon- ductedinaccordancewithSOPmightbecompromised.Therefore theMARK-AGEConsortiumdedicatedthefirstyeartoelaborate

http://dx.doi.org/10.1016/j.mad.2015.03.007

Erschienen in: Mechanisms of Ageing and Development ; 151 (2015). - S. 18-25 https://dx.doi.org/10.1016/j.mad.2015.03.007

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-312478

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Table1

ListofMARK-AGEpartnerswithashortdescriptionoftheirtasks.

Partnernumber Beneﬁciaryname Country Task

1 UniversitaetKonstanz Germany Co-ordinatorandanalysingcentre(PARPactivityandDNArepair)

2 BioTeSysGmbH Germany Recruitingandanalysingcentre(VitC,VitE,Gluthatione)

3 FundaciónCentroNacionaldeInvestigaciones OncológicasCarlosIII

Spain Analysingcentre(telomerelength)

4 DNageB.V. TheNetherlands Recruitingcentre

5 ErasmusUniversitairMedischCentrum Rotterdam

TheNetherlands Prematurelyageingmousemodels 6 FacultésUniversitairesNotre-DamedelaPaix

deNamur

Belgium Recruitingcentre 7 ImperialCollegeofScience,Technologyand

Medicine

UK ChangedtoCranﬁeld(Nr.27) 8 OesterreichischeAkademieder

Wissenschaften

Austria Recruitingandanalysingcentre(measles,inﬂuenzaAandB,tetanus IgGantibodiesandnumberofcellsproducingINFafterInﬂuenzaand CMVstimulation)

9 IstitutoNazionaleRiposoeCuraperAnziani Italy Analysingcentre(intracellularandextracellulartraceelements)

10 NESTECSA Switzerland Analysingcentre(identiﬁcationofmetabolitesusingMagnetic

ResonanceSpectroscopy)

11 NationalHellenicResearchFoundation Greece Recruitingandanalysingcentre(serumlevelsofpolipoproteinJ) 12 InstytutBiologiiDo´swiadczalnejim.M.

NenckiegoPAN

Poland Recruitingandanalysingcentre(Activation-andDNA Damage-inducedcelldeathand

13 InstitutulNationaldeGerontologiesiGeriatrie AnaAslan

Romania Analysingcentre(LDLoxandNOx) 14 RijksinstituutvoorVolksgezondheidenMilieu TheNetherlands Analysingcentre(clinicalchemistry) 15 StratiCELLScreeningTechnologiesSA/NV Belgium Analysingcentre(cytokines)

16 AarhusUniversitet Denmark Analysingcentre(intermediateﬁlamentproteinvimentin)

17 AstonUniversity UK Analysingcentre(enolaseandthioredoxin1onCD4+cellsand

transferrinresidues) 18 VlaamsInstituutvoorBiotechnolgievzw Belgium Analysingcentre(N-glycans)

19 UniversitaetHohenheim Germany Biobankandanalysingcentre(lycopene,carotinoide,cysteine,gamma tocopherol,VitA,VitC,uricacid,VitE,totalglutathione,

malondialdehyde,proteincarbonyls)

20 Martin-LutherUniversitaetHalle-Wittenberg Germany Analysingcentre(advancedglycationendproducts(AGEs)and micro-RNA(miRNA)pattern)

21 AlmaMaterStudiorum–UniversitàdiBologna Italy Recruitingandanalysingcentre(DNAmethylationstatus,APOE genotypeandlevelsofheteroplasmyinmtDNA)

22 UnileverUKCentralResourcesLimited UK Analysingcentre(urinaryandplasma8-isoprostane,urinary creatinineandserumadiponectin)

23 UniversitàdegliStudidiRoma“LaSapienza” Italy Analysingcentre(DNMT,PARP1and2expressionandmethylation) 24 UniversitéPierreetMarieCurie–Paris6 France Analysingcentre(proteasomeactivity)

25 AcademischZiekenhuisLeiden–Leids UniversitairMedischCentrum

TheNetherlands Recruitingandanalysingcentre(cholesterolandtriglyceridesinHDL, HDL1,HDL2,LDL,LDL1,LDL2,VLDL,VLDL1,VLDL2)

26 TampereenYliopisto Finland Recruitingandanalysingcentre(cellfreeDNAinserum)

27 CranﬁeldUniversity UK Analysingcentre(Tcellreceptorexcisioncircles(TRECs)asbiomarker

ofthymusoutput)

standardizedprotocolsandprocedures.Further,theuseofSOPs wasreviewedandre-enforcedbythecoordinatorregularly,The MARK-AGEprojectisdescribedindetailinaseparatemanuscript (seeBürkleandco-workers,thisissue).

2. Materialandmethods 2.1. Recruitmentofsubjects

Twolargegroupsofsubjectswererecruited,i.e.(1)randomly recruited age-stratiﬁedindividuals fromthe generalpopulation covering theage range 35–74 years (“RASIG”)and (2) subjects bornfromalong-livingparentbelongingtoafamilywithlongliv- ingsibling(s)alreadyrecruitedintheframeworkoftheEUGEHA project(Genetics of HealthyAgeing http://www.geha.unibo.it/).

For genetic reasons such individuals (“GEHA offspring”) are expectedtoageat a slowerrate.Theywere recruitedtogether withtheirspouses(“SGO”ascontrolsofthesharedenvironment).

(3)Asmallnumberofpatientswithprogeroidsyndromes(Cock- ayne, WernerandDown syndromes)werealso includedinthe study.PriortorecruitmentofsubjectseachBeneﬁciaryinvolved obtainedthelocalEthics Committeeapproval.Exclusioncriteria were foreseen: (i) self-reportedseropositivity for HIV, for HBV (except seropositivity by vaccination) and HCV; (ii) measured

seropositivityforHBVandHCV(iii)presenceofadiagnosedcan- cerdiseaseandcurrentuseofanti-cancerdrugsorglucocorticoids (chronictreatment);(iv)lessthan50%oflifetimespentincountry ofresidence;or(v)inabilitytogiveInformedConsentor(vi)any acuteillness(e.g.commoncold)withinsevendaysprecedingblood collection(seeBuerkleetal.;CapriandMoreno-Villanuevaetal., thisissue)

2.1.1. Ethicalapproval

TheMARK-AGEstudywascarriedoutinaccordancewiththe declarationofHelsinki,whichistheacceptedbasisforclinicalstudy ethics,andmustbefullyfollowedandrespectedbyallengaged inresearchonhumanbeings.Duringtheﬁrstfundingperiod,one ofthetop-prioritytasksfortheBeneﬁciariesinvolvedinrecruit- mentofMARK-AGEsubjectswastoobtainethicalapprovalfrom thecompetentauthoritiesintherespectivecountries.Astheeth- icalrequirementsdiffer betweencountries, it wasnecessary to makeappropriateadaptations.Inordertoobtainethicalapproval, thefollowingdocumentswerecreated:“Informedconsent”,“Par- ticipantInformationSheet”and“SynopsisofMARK-AGEproject”

(Supplementarymaterials).Thesedocumentswereoriginallycre- atedinEnglishandtheBeneﬁciariesinvolvedintherecruitment thentranslatedthemintotheirrespectivenationallanguage,i.e.

Dutch,Finnish,French,German,Greek,ItalianandPolish.Before

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startingtheinterviewandtheexaminationofthesubjects,each potentialsubjectwasinformedindetailaboutthestudyprocedures andwrittenInformedConsentwasobtained.

2.1.2. Questionnaires

Thequestionnaires,whichwereﬁlledoutbythesubjectand atrainedinterviewer,allowstandardiseddocumentationofbio- graphicinformationandhealthstatusoftheMARK-AGEsubjects.

Thecognitivetestsincorporated inthe questionnaireprovide a scorethatiscommonlyacceptedinthescientificcommunity,asis documentedbymanypublications.Thesequestionnaireswerefirst writteninEnglishandthentranslatedtotherespectivenational languages.Thequestionnairesconsistoftwoparts,onetobefilled outbythesubjectathomebeforetheinterview,andtheotherto becompletedbytheintervieweratthetimeoftheexamination.

Theextensivequestionnairescapturedemographic information, lifestyle,cognitivestatus,mood,healthstatus,andanthropometricmeasurements(bodymassindex,waistandhipcircumference, bloodpressureatrest,heartrateatrest,lungcapacity,nearvision, five-timeschair standingand handgripstrength among others) (Supplementarymaterials).Peopleover65yearsofagewerealso testedwithstandardizedmini-mentalstateexamination(SMMSE) (Molloyetal.,1991)andactivitiesofdailyliving(ADL)(Kempen etal.,1996).Further,aspecificquestionnairewasdevelopedby beneficiary#21forindividualsofdifferentagesaffectedbyDown Syndrome and enrolled in this project as “accelerated ageing cohort”(seeCapriandco-workers,thisissue).Inparticular,spe- cificcognitivetestsincludedinthegeneralquestionnairearebriefly describedbelow:

2.1.3. Cognitivetests

2.1.3.1. 15-Picturelearningtest.Immediateanddelayedmemory functionwasassessed bythe 15-picture learningtest (15-PLT).

Subjectswereshown15picturesofwell-knownitemsandthen askedtorecallasmanyaspossible.Thetestwasrepeatedthree consecutivetimesandafter20min.Outcomeparameterswerethe numberofcorrectpicturesaftereachtrialandafter20min(delayed recall).Thetotalnumberofcorrectanswersafterthreetrialswas deﬁnedastheimmediaterecall.Furthermore,thenumberofincor- rectpictureswasreportedforeachtrial.Alowscoreindicatesworse cognitiveperformance(BrandandJolles,1985).

2.1.3.2. Stroop-colour-word-test. The Stroop-colour-word-test (Stroop)was used totest selective attention.The test involves threepartsthatdisplayed40stimulieach,whichthesubjectwas askedtoreadornameasquicklyaspossible:(1)colournames,(2) colouredpatches,and(3)colournamesprintedinincongruously colouredink;forexample,“green”printedinblueletters,where thesubjectistosay“blue”.Performanceonpart3isdetermined foralargepartbythetimeneededtodiscardirrelevantbutvery salient information (verbal), in favour of a less obviousaspect (colournaming),alsoknownascognitiveinterference.Themain outcomevariables warethetimesneededfor eachof thethree testparts,ahigherscorethereforeindicatesworseperformance (Stroop,1935).

2.1.3.3. Digit-symbolsubstitutiontask. Thedigit-symbolsubstitutiontask(DSST)wasusedtoassessprocessingspeed.IntheDSST, digitswerepresentedand thesubjectswereaskedtowritethe corresponding symbols in a blank space according to a given key.Outcomeparameterwasthenumberofcorrectdigit-symbol combinationswithin90s.Alow scoreindicatesworsecognitive performance(Lezaketal.,2004).

2.1.3.4. Zung self-rating depression scale.The Zung self-rating depressionscaleisashortself-administeredsurveytoquantifythe

depressedstatusofapatient.Thereare20itemsonthescalethat ratetheratingaffective,psychologicalandsomaticsymptomsasso- ciatedwithdepression.Therearetenpositivelywordedandten negativelywordedquestions.Eachquestionisscoredonascaleof 1through4(basedonthesereplies:“alittleofthetime”,“someof thetime”,“goodpartofthetime”,“mostofthetime”).Scoreson thetestrangefrom20through80.Ahigherscorerepresentamore depressedstatus(Zung,1965).

2.2. Questionnairefordownsyndrome

Thequestionnaire comprises two parts: partIequal tothat adoptedonotherMARKAGEpopulations(demographicinforma- tion,lifestyle,healthstatus,andanthropometricmeasurements);

partIIcontaininga standardisedbatteryof15 teststoevaluate differentdomainssuchasbehaviourandneuropsychologicalfea- tures,memoryandlanguage.AberrantBehaviourCheckList(Aman etal.,1985);AdaptiveSkillsVinelandScale(Sparrowetal.,1986), VisualObjectSpatialPerception(Rapportetal.,1998);Weschler IntelligenceScaleforChildrenIIIsubtests(Wechsler,1991)were partoftestbatteryamongothersandalsoincludedinaprevious workwhereage-relatedchangesofadaptiveandneuropsycological featuresinpersonswithDSwereassessed(Ghezzoetal.,2014).

2.3. Biobank

TheMARK-AGEBiobankwasembeddedattheInstituteofBio- logicalChemistryandNutritionattheUniversityofHohenheim, Germany,withinthefacilitiesofoneMARK-AGEBeneﬁciary.The Biobankwasaimedtoprovidetherecruitmentcentreswithstan- dardizedmaterialforsamplescollection,managesamplesincome from the recruitment centres, sample outcome to the analytic partners,samplere-labeling,samplesplitting,samplestorageand organizationofaninternaldatabaseforsampletracking.Thecen- tralroleoftheBiobankwasimplementedintheMark-AgeSOPs.

TheBiobankhardwareconsistedofthreeultra-lowtemperature freezers(−80^◦C,NewBrunswickScientific,Enfield,USA)andfour nitrogentanks(−196^◦C,AirLiquide,Paris,France),whichassured thepermanentfreezingconditionsforthesamplesandthestruc- turedstorageofseveralthousandsofsamples.Inallfreezersand tankstemperaturefluctuationswerepermanentlyrecorded.Fur- thermore,thefillinglevelofthenitrogentankswasdocumented.

FreezerswereadditionallyequippedwithaCO₂ backup system holdingconditionsforabout2days.Acentralcurrentgenerator atthefacilityguaranteedthemaintenanceofthefreezerfunction afteralocalpowerfailure.Allfreezerswereequippedwithanalarm system,automaticallyinformingmaintenancestaffandscientists (viamobilephone)aboutpotentialtroubles.Theincomingandout- goingsamples,aswellasthestorageplace,wereregisteredina centralBiobankcomputer.Thedataweredailyuploadedtoacen- tralserverinadifferentbuildinginordertoavoidanyfataldata hazard.

2.4. Recruitingcentres

Therecruitingcentresweresuppliedwithcollectionmaterialby theBiobank:eightbloodmonovettes(Sarstedt,Germany)ranging from2.7to9mlcontaininglithium-heparine,EDTAornoneofthese asanti-coagulants,29cryotubesrangingfrom0.5to15ml(Greiner Bio-one,Germany)andoneBMCkit(BioTeSysGermany)consist- ingofaspecialtoothbrush,stabilizersolutionandemptytubes.

Alltubeswerealreadylabelledwithaprimarysubjectcode(PSC) consistingof7numbers(thefirsttwonumbersdefiningtheMARK- AGErecruiter centrefollowed bya fivedigit number).In order toalleviatesampleprocessingintherecruitingcentres,cryotubes werecolouredaccordingtothedestinedsampletype(red:EDTA

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samples,blue:lithium-heparinesamples,white:serumsamples, yellow:urinesamples,green:BMCsandwholebloodsamples).

2.5. Bloodcollection

All subjects were asked to donate blood (50ml) by phle- botomyafterovernightfasting.EDTA,lithiumheparinandserum monovetteswere used.One EDTAmonovette containing 2.7ml wholebloodwassent tothelocalclinicalchemistry laboratory for blood counts.Haemoglobin (Hb),hematocrit (Hct),erythro- cytes,mean cellvolume(MCV), meancorpuscularhaemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocytes,thrombocytes,eosinophilgranulocytes,basophilgran- ulocytes, neutrophil granulocytes, lymphocytes and monocytes were assessed. Allother monovettes were processed to obtain plasma,serum,andperipheralbloodmononuclearcells(PBMC).

2.6. Buccalmucosacellscollection

Priortoharvesting,volunteershadtorinsetheirmouththor- oughlywithtapwatertoclearfoodresiduefromtheoralcavity.

Buccalmucosacells wereharvestedby brushingeachcheek 25 timesfromupsidedownwithmedium pressureusinga special toothbrush(TEPEspecial care,TePeMundhygieneprodukteVer- triebsGmbH, Hamburg, Germany). The cells were collectedby washing outthe toothbrushin a 50ml test tube containingan aliquot of4,5ml stabilizersolution.Ifthe cellsolutionwasnot turbidenough the brushing of cheeks wasrepeated (10 times eachcheek)usinganewtoothbrushandrinsedinthesametest tubeagain.Afterwardstheentirestabilizer-BMCsuspensionwas

pipettedintoa5mlcryotubeandfrozenat−80^◦Cuntilshipment totheBiobank.

2.7. Urinecollection

Theurinecollectiontookalsoplaceattherecruitingcentres.

MARK-AGEsubjectswereaskedtocollectatleast19mlurineina sterilescrew-topcontainer.Theobtainedvolumewassplitin14ml urine+140␮l0.1MSodiumazide(NaN₃)aspreservativeand5ml justurinefollowingtherequirementsofanalyticlaboratories.

2.8. Database

TheMARK-AGEdatabaseservedasacentralsiteforelectronic storage of questionnaire information, anthropometric data and analyticaldataonblood,urineandbuccalmucosalcellsfromeach MARK-AGEsubject.Inatestphasetheprocedureofenteringdata inthedatabasewas“rehearsed”repeatedlybytheBeneﬁciaries,in ordertopreventanyproblemsduringthephaseofactiverecruit- ment.Theestablishmentofthedatabaseisdescribedindetailina separatemanuscript(seeMoreno-Villanuevaandco-workers,this issue).

2.9. Samplesshipment

Aliquots of biological material werestored at −80^◦C (BMC, wholeblood,serum,plasmaandurine)orat-196^◦Cliquidnitro- gen(PBMCs)attherecruitmentcentresuntiltheirshipmenttothe Biobank.Atthebeginningoftheprojectsizeofthepacketsand theamountofdryicenecessaryforkeepingsamplesfrozenduring 3dayshadbeenestimated.Alsoaﬁctitiousshipment,including

Fig.1. SamplesshipmentfromrecruitingcentrestotheBiobank(Hohenheim,Germany).

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shakingthepacketsinregulartime intervals(astheywouldbe movedbymeansoftransport)hadbeenperformedatthecoor- dinatorcentre. Allsamplesfromoneprobandwerepackedinto aPSC-labelledplastic bag.Packetscontainingseveralbagswere shippedondryicetotheBiobanktoastipulatedtimepointina regularmanner(Fig.1).

AttheBiobanksampleswerefurtheraliquoted,re-labelledand transferredtothecorrespondingstoragedevices.Theyremained temporarystored untiltheirshipmenttotheanalyzingcentres.

Justbeforeshipmenttubesweretransferredfromstoragedevices intolaboratory-speciﬁccryo-safestorageboxes(formandsizedif- feredbetweenanalysingcentres).Theseboxescontainingthetubes wereshippedondryicetotheanalyticlaboratoriesinregularinter- valsdependingontheirstorecapacity.Theanalysingcentresstored theboxesinthecorrespondingstoragedevicesuntilanalysiswere performed.

3. Results

3.1. Recruitmentstrategy

Theprincipalstrategywastogettheattentionofthepopulation usingthecommunicationmedia,severalarticlesandinterviews inlocal andnational newspapersand TVprogrammes. Recruit- mentcentresgaveanextendedreportontheMARK-AGEproject and encouragedthepopulationtoparticipate leaving a contact telephonenumber.TherecruitmentstrategiesandMARK-AGEpop- ulationsaredescribedindetailinaseparatemanuscript(seeCapri andco-workers,thisissue).A greateffortwasalsogiveninthe recruitmentofDSindividualsandtheirfamilies.Thefirstmeeting withthemwasathomeinordertogetacquaintedwithvolunteers, togettheinformedconsent,andtoanswerthefirstpartofthe questionnairetogetherwiththeirparentsorcaregivers.Thesec- ondpartofthequestionnairewiththespecificbatteryoftestswas performedwithasecondappointment(sometimesalsoathird)to completealltheinterviews.

3.2. SOPsforsamplecollectionandpreparationattherecruiting centres

TheﬁrstimportantelementoftheMARK-AGESOPprotocolwas theusageofidenticalmaterialforsamplecollection,preparation andstorage.Asdescribedearlierrecruitingcentresweresupplied withthesamesamplecollectionmaterial(Sarstedt,Germany)and cryotubesforsamplestorage(Greinerbio-one,Germany)bythe Biobank.Duetotheextremelysensitivitytofreeze-thawingcycles, PBMCsandwholebloodsampleswerealreadysplitfortheana- lyticcentresattherecruitmentcentresandonlyre-labelledatthe Biobank.

All recruitment centres ordered the same reagents and material for PBMC isolation: Percoll (Amersham Biosciences), RPMI(Invitrogen/Gibco),DMSO (SigmaAldrich,Germany,Peni- cillin/Streptomycin (Invitrogen, Germany), sterile sodium azide (NaN₃)(Merck,Germany).BioTeSysprovidedastabilizersolution forBMCs.Inordertoavoidvariabilitybetweendifferentfoetalcalf serum(FCS)charges,thesameFCSchargewasusedforallsamples (MerckMillipore,Germany).Phosphatebufferedsaline(PBS)was orderedseparatelyineachrecruitingcentre.

Allbiological sampleswereprocessed within2hand 10min followingamasterprotocolelaboratedforMARK-AGEpurposes (Fig.2).Bytheuseoftwocentrifuges(1×atroomtemperature, 1× at 4^◦C) the different preparation tasks were able to man- agein parallelin orderto shortenthetotal time untilfreezing toaminimum.Thecollectedurinewassplitbyattherecruiting centreintotwoaliquotsof5mland 14mlurineasindicated in

chapter2.3.Urinealiquotswerefrozenat−80^◦Cuntilshipment totheBiobank.Insummary,wholeblood,serumandEDTA/LiHep plasmaaliquotswerepreparedfromthebloodmonovettes,and stored at −80^◦C immediately after cryotubes were ﬁlled. Fur- ther,PBMCswereisolatedbydensitygradientcentrifugationusing Percoll(70%Percoll(GEHealthcare,Germany)+10%1.5MNaCl) andstoredovernightat−80^◦Cinfreezingcontainers(Mr.Frosty, Nalgene®,VWR,Germany)withfreezingmediumcontaining20%

RPMI 1640(Thermo Fisher Scientiﬁc, Germany) (with 1%Peni- cillin/Streptomycin)+70%FCS (Merck Millipore,Germany)+10%

DMSO(Sigma,Germany).ThenextdayPBMCsweretransferredto liquidnitrogen(−196^◦C)untilshipmenttotheBiobank.Foreach proband,samplecollectiontime,samplepreparationstartingtime andsamplefreezingtimeweredocumentedbytherecruitingstaff.

Abnormalitieslikeerythrocytecontaminationinanysampleswere alsorecorded.

In order totrain the MARK-AGE teams involved in recruitment,samplecollectionandpreparation,datacollectionandentry aworkshopaboutstandardoperatingprocedureswasorganized.

Afterwardsthewholeprocedureofrecruitment,samplescollec- tionanddataenteringwasmimickedinatestphasebeforethereal recruitmentstarted.

3.3. Biobank

Thealiquotsobtainedattherecruitingcentreswereregularly shippedondryicetothecentralBiobankforstorageanddistribu- tiontotheBeneﬁciarieswhoperformedtherespectiveanalyses.A shipmentplanwasmadeinordertodeﬁnetheoptimalpacketsize, volumeandweightfortheshipmentstoand fromtheBiobank.

Packetsfromrecruitingcentreswithsamplesfroma maximum of 40 MARK-AGE probands each (about 1160 cryotubes) were acceptedbytheBiobankperweek.Uponrequirementoftheana- lyzingcentresbatchesofsampleswereretrievedfromthestorage andsentoutforanalysis.Thisroutinewasperformedviaanindi- vidualagreementwitheachBeneﬁciarydependingof analytical requirements,e.g.peripheralstoragecapacity,analysednumbers etc.OnaveragetheBiobankorganized4shipmentsconsistingof 4–12packetseachperyear.Inotherwordsevery2–3monthsthe Biobanksuppliedanalyticpartnerswithpacketscontainingsam- plesofabout100–300probands.

Urine,plasmaandwholebloodsamplesweresplitintoseveral smalleraliquotsattheBiobankandthenre-labelledwiththesec- ondarysubjectcode(SSC)bytheBiobankstaffteam.Afterwards,all 58aliquotsofeachprobandwerestoredinthecorrespondingfreez- ersortanksuntilshipmenttotheanalyticcentres.However,before allsamplesweredistributedwithintheConsortiumonemonovette ofthelithium-heparine plasmawassentforhepatitistestingto Beneficiary#14(RIVM).Thepositivesampleswerenotdistributed totheBeneficiariesbuteliminatedaftersterilisation.Insummary, basedon3169classifiedassuccessfullyrecruitedvolunteersinthe study(seeCapriandMoreno-Villanuevaetal.,this issue),more than158,400sampleswerere-labelledandtemporarilystoredat theBiobank(Fig.3).

As indicated above all tubes provided by the Biobank were labelledwithaprimarysubjectcode(PSC).Duetotheriskofsam- pleslosing theirlabelling duringshipment orhandlings special heavy-dutylabels(CILS,Worthing,UK)wereusedforcodingsam- ples.

3.4. SOPsforanalyticcentres

IntheanalyticcentresPBMCcryovialswereremovedfromliq- uidnitrogentoa37^◦Cwaterbath.Avolumeof0.5ml37^◦Cwarm thawingmedium(90%RPMI+10%FCS)wasaddeddropwise.After oneminutecellsuspensionwastransferredintoapolypropylene

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Fig.2.Laboratoryproceduresforpreparationofbiologicalmaterial.Elaborationofaverydetailedmasterprotocolforobtainingserum,plasma,wholebloodandPBMCfrom thelimitedamountofblood(50ml)tobetakenfromMARK-AGEsubjects.

15mltubeandthawingmediumwasaddedstepwise(1.0ml,wait oneminute,2.0ml,waitoneminute,4.0ml,waitoneminute).After centrifugation(250×gfor10min)cellswereresuspendedinRPMI cellculturemediumorbufferaccordingwiththecorresponding analyticprocedures.

4. Discussion

Appropriate and optimised SOPs are the best basement of the success of a project especially when a large volunteers’

recruitmentisforeseenaswellasMARK-AGE.Manybeneﬁciaries werepreviouslyinvolved inEUGEHAproject(Franceschietal., 2007)thushavingexpertiseinSOPstasksincludingrecruitment, samplescollectionandBiobanksetup.

4.1. Recruitment

Thepreparationandlaunchofrecruitmentactivityencountered somedifﬁcultiesrelatedtologistics.Insomecasestheinterviews andblood drawneededtotakeplaceatthesubjects’domiciles,

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Fig.3. SampleﬂowononeMARK-AGEsubject.

especiallyincaseofGOandSGOsubjects.Unfortunatelyseveral ofthemwerelivinginremoteplacesthereforethetimeneeded toprocessbiologicalsampleswascriticalforsomeoftheintended biomarkers.Especiallybiomarkersrelatedtooxidativestresscould beaffectedduetotheinstabilityofthosemolecules.Theinﬂuence ofthetransporttimeonbiologicalmaterialcouldnotbechecked forallbiomarkers.Therefore,theConsortiumdecidedtodocument thetimeofsamplingcollection,sampleprocessing,samplestor- ageandsampleshipmentinordertoexcludethosemeasurements inﬂuencedbytimeforlogisticreasons.

Anothertricky taskregarded ethicalissues,which hadtobe inagreementwithcurrentEUdirectives(Seppetetal.,2011).The elaborationofallnecessarydocumentsinastandardisedmanner appearedtobecomplicatedduetodifferencesincountrieslegisla- tions.TheGreekEthicCommitteedidnotallowtakingmorethan 50mlbloodfromoldvolunteers.Thislimitedtheamountofserum, plasma,PBMCandwholebloodandforcedtheConsortiumtoelab- orateaprotocol,whichmaximalexploitstheavailablebiological material.

4.2. Samplescollection

Rightattheonsetoftherecruitmentactivity,extensivequal- itycontrolanalyseswerecarriedoutbyBeneﬁciary#1duringthe testphaseinordertoensurethequalityofthebiologicalmaterial comingfromtherecruitmentcentres.Theanalyseswerefocused onthequalityofPBMCsand includedcellcounts, DNAdamage andrepair measurements andanalysesof celldeath(apoptosis andnecrosis).Thedatashowedthatin manycasesthenumber ofviablecellsobtainedwasinacceptable.Consequentlythecryop- reservationandthawingproceduresforPBMCs,samplestransport ondryice andsamplelabelling attheBiobank wereoptimised.

AfterwardstheviabilityofthePBMCwasimprovedfrom30%to 80–90%.Beneﬁciary#21repeatedtheanalysesandtheresultswere conﬁrmed.

Anotherchallengewastoprovidetheanalyticlaboratorieswith sufficientbiologicalmaterialforrunningtheirmeasurements.This wasespecially thecaseforPBMCaliquots,unfortunatelyahigh percentageofthesamplesdidnotcontainthenecessaryamount of cells. Therefore thesample distribution was rearranged and theaffectedBeneficiariescouldbeprovidedwithadditionalPBMC aliquots.Inaddition,Beneficiariesalsoinvestedtimeandresources intheoptimizationoftheirassaysinordertomakethemmore

sensitiveandtobeabletodetecttheirpotentialbiomarkersinlower amountsofmaterial.

Theseoptimizationswerecarriedoutusingsamplesfromaddi- tionalvolunteers(notenrolledintheMARK-AGEpopulation)and whichwere,inpart,providedbyBeneﬁciary#1.

The effect of temperature on biological samples is known (Pasella etal.,2013)thereforethequalityofserum andplasma sampleswasmonitored.Beneﬁciary#14checkedtheincidenceof hemolytic,ictericorlipemicsamplessinceinsuchsamplesamean- ingfulanalysisofclinicalchemistryparametersmaynotbepossible.

Theincidencereportedwasbelow0.7%,thusattestingtothevery highqualitystandardsoftheMARK-AGEsamples.

4.3. Biobank

Oneoftheproblemsforstandardisedsamplecollectionwasthe differenceinexistinglaboratorymaterialattherecruitmentcen- tres.Inordertoequipallrecruiterswithstandardisedmaterialthe Biobankorderedthosefromonecentralsource.Anotherproblem, relativelyuniquefortheMARK-AGEBiobank,istherequirementof storageofdifferentsamples(blood,cells,plasma,serum,urine)in differentvolumes.IncontrasttootherBiobanks,e.g.storingonly DNAsamples,eachofthesampleshasitsspecialrequirements,as tubesizeandstoragecondition.Whilstthereisnoperfectsolution, processingand storage ofbiological samplesis doneas a com- promiseandwithintheconstraintsofadeﬁnitebudget.Alimited numberofsamplesremainintheBiobank,forfutureusagebythe Consortium.

5. Conclusions

BycarefulpreparingtheSOPstheMARK-AGEConsortiumwas abletohandlethesamplesfrom3169probands.Standardizationof bloodcollectionmaterialandprocedureallowedamaximalcom- parisonofsamplesfromdifferentrecruitmentcentresandallowed alsoamaximalsecurityofsamplehandling.Thecarefulmonitoring ofsampleﬂowbytheBiobankandtheexchangeofsubjectcodes bythecentraldatabaseminimizedsampleloss,ensuredsample qualityandguaranteedthedouble-blinddesignofthestudy.In generalitcanbeconcludedthatrestrictingSOPsarerequiredto ensurequalityofalargecohorttrial,especiallyifvariousmetabolic samplesarepartofthestudy.

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Acknowledgments

WewishtothanktheEuropeanCommissionforﬁnancialsup- port through the FP7large-scale integrating project“European Study to Establish Biomarkers of Human Ageing” (MARK-AGE;

grantagreementno.:200880).Wewouldalsoliketothankallstudy participantswhovoluntarilyofferedtheirtimeandsupportforthe beneﬁtoftheMARK-AGEproject.WewishtothankBarbaraBausch, GudrunvonScheven,MonikaSchulz,Andrea Flaccus,Christiane Hallwachs,NadineGrebenstein,StephanieAllenfortandBiotesys stafffortheirtechnicalsupport.WearealsogratefultoDr.Elisa Cevenini,Dr.LauraCelani,Dr.MariaScurti,Dr.ElisaPinitogether withalltheUNIBOteamfortheiressentialcontributiononSOPs optimization.GratefullyalsotoDr.DanielaFollo,Dr.LauraLami,Dr.

ClaudiaPizzoli,Dr.MariaCaterinaSolimandoandDr.AlicePalmieri fortheireffortinDSquestionnaireoptimization.(Bologna,Italy).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.mad.2015.03.007.

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