thatasocalledgenomicstormatthelevelofthe leukocytetranscriptomeoccurredaftertraumaticinjury addingfurtherhumantranslationalinvestigativesupport tothefactthatthesystemicinflammatoryresponsesyn drome ID: 456520
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leukocytegeneexpressionofthesepatientsillustrated thataso-calledgenomicstormatthelevelofthe leukocytetranscriptomeoccurredaftertraumaticinjury, addingfurtherhumantranslationalinvestigativesupport tothefactthatthesystemicinflammatoryresponsesyn- drome(SIRS)andcompensatoryanti-inflammatoryre- sponses(CARS)occurredsimultaneouslyratherthan sequentially[6,7].Patientswhoexhibitedacomplicated clinicaltrajectory,definedasgreaterthanfourteendays ofpersistentorgandysfunctionordeath,hadexacerba- tionandprolongationoftheirtranscriptomicresponse, andfailuretoreturntobaselineexpressionpatterns[6]. Inaddition,arapidgenomiccompositescorewasdevel- oped,using63selectgenes,whichdeterminewithin12 to24hoursofinjurythosepatientswhoaredestinedto haveacomplicatedclinicaltrajectory[8,9]. Interestingly,recentlypublisheddatabyourgroup utilizingmurinemodelsofinfectionandtraumadonot completelysupportthisseverelyexacerbatedgeneex- pressionpatterninmiceofadvancedage,althoughres- torationofgenomichomeostasisiscertainlydelayed [10,11].Althoughmurineandhumanresponsestoin- flammationarecertainlynotequivalentatthelevelof thetranscriptome[12],genomicexpressionpatternsin someindividualpathways,suchasinnateimmunity,can bewell-replicatedinmice[13].Inaddition,researchers areperformingtranslationdatainhumansthatsupports thesespecificdifferencesininflammatoryresponsesto injuryorinfectionintheelderly[14]. Todate,genomicanalysesinthisseverelyinjuredpa- tientcohorthavebeencarriedoutprimarilyontotal leukocytepopulations,ratherthanonisolatedperipheral polymorphonuclearneutrophils(PMNs),whicharethe predominantcirculatingleukocytesaftersevereinjury [6].Inaddition,thecohortsfromtheseanalysescon- tainedonlypatientsyearsold.Therefore,thegoalof thisstudywasthree-fold:(1)determinewhetherad- vancedageisassociatedwithincreasedmorbidityand poorclinicaloutcomesbothwithstandardmeasuresof outcome(thatis,28-daymortality),aswellasmorere- centlyproposedmeasuresoflong-termdisposition;(2) characterizethePMNgenomicresponseaftersevereblunt traumaticinjurywithhemorrhagicshock,and;(3)deter- mineifthegenomicstormidentifiedinyoungercohortsis alsoseeninPMNsfromtheagedaftertrauma.Wehy- pothesizedthatadvancedagewouldbeassociatedwith worsenedoutcomes,andauniquegenomicresponsein severelyinjuredpatientswithhemorrhagicshock. Methods ApprovalwasobtainedfromtheUniversityofFlorida InstitutionalReviewBoardtoanalyzede-identifiedhu- mandataobtainedfromtheGGTraumaRelatedData- base(TRDB)priortoinitiationofthisstudy[15].The clinicalprotocolandconsentformswerereviewedand approvedbythecentraladministrationsiteatMassachu- settsGeneralHospital(InstitutionalReviewBoard(IRB) MGHProtocol#2002P001743).Inaddition,theclinical protocolwasreviewedandapprovedbyeachofthe sevenparticipatingclinicalsites.Ineverycase,signedin- formedconsentwasobtainedfromtheindividualpatient ortheirdesignatedlegalrepresentative.Ifinformedcon- sentwasobtainedfromthelegalrepresentative,thepa- tientwasre-consentedaftertheyhadachievedaclinical statewheretheycouldprovideinformedconsent.Based onindividualIRBs,thetimeperiodrequiredtoobtain informedconsentfromthepatientorlegalrepresenta- tivevariedfrom24hourstowithinhospitalizationdue tothevulnerablenatureofthepatientandtheirlegal representatives.Inallcases,theIRBsacceptedtheargu- mentthatpatientsandtheirfamiliesareoftenvulnerable intheearlypost-traumaperiod,andrequiresometime periodtoadjusttotheseverityofthepatient sinjury, andprovideinformedconsent. Datasourceandcohortselection TheTRDBcontainsauditedandde-identifieddataob- tainedfromseverelyinjuredtraumapatientsenrolled fromsevenlevel-1traumacentersbetween2001and 2011[16].Inclusioncriteriaincludedadultpatients(age 16yearsold),whohadbeenseverelyinjured(injuryse- verityscore(ISS)�15)havingundergoneblunttrauma withoutsevereTBI,andwithevidenceofhemorrhagic shock(systolicbloodpressure(SBP)mmHgorbase deficit 6mEq/L,andrequiringbloodtransfusion).In ordertoaddressconfoundingbyvariationsintreatment strategiesbetweencenters,clinicalstandardoperating procedures(SOPs)wereestablishedandappliedtoall enrolledpatients.Compliancewithapplicationofthese SOPswassubjectedtoauditoverthecourseofthestudy [15,17]. AsofOctober2013,theTRDBcontaineddetailed, prospectivelycollecteddemographic,clinical,andout- comesdataon1,928patientswithblunttraumaandin hemorrhagicshock,whometthecriteriaforthisana- lysis.Thesepatientswereseparatedintotwomainco- horts,eitheradvancedage( 55yearsold)oryoung( yearsold)forepidemiologicanalysis.Theageof55years wasusedasthecutoffforseveralreasons.First,theini- tialphaseoftheGGlimitedenrollmenttothose years,whichwaslaterextendedtoincludeallpatients overtheageof16andwasusedastheoriginalagecut- offbytheGGtodefinetheagedpopulation[18].Sec- ond,studiesbyDemetriadesandcolleaguesanalyzing thevalidityofthetraumaandinjuryseverityscore (TRISS)methodologyandbySauaia etal .lookingat earlypredictorsofpostinjuryMOFfurthersupported thiscutoff.Bothillustratedthatbeingage55yearsor Vanzant etal.CriticalCare (2015) 19:77 Page2of15 olderaftertraumawasassociatedwithworseoutcomes thanpredicted,evenaftercontrollingforotherinjury factors[19,20].Usingthesedefinitionsforyoungand aged,therewere1,395and533patientsintheyoung andagedcohorts,respectively. Inadditiontothetraumapatientsenrolledforepide- miologicdatacollectionintheTRDB,asubsetof244se- verelyinjuredtraumapatients,16to90yearsold,and anadditional21healthycontrolswereenrolledforblood samplingforenrichedPMNgenomicanalysis.When separatedbasedonage,therewere67agedand177 youngtraumapatients.ThepatientsintheGGwerealso classifiedbasedonclinicaloutcomesintocomplicated, intermediateoruncomplicatedcohorts.Inthisregard, complicatedoutcomesweredefinedaseitherICU hospitalizationlongerthan14dayswithevidenceofon- goingorgandysfunction,ordeath[6,15].Uncomplicated outcomesweredefinedasthosewithorganrecoveryand ICUhospitalizationfordays.Thosewhofellbetween thesetwoclassificationswereconsideredintermediate. Therefore,thepatientsweredividedintofourdistinct groupsbasedonclinicaloutcomes(eithercomplicated oruncomplicated)andageasfollows:complicatedaged (n=25),uncomplicatedaged(n=8),complicatedyoung (n=42)anduncomplicatedyoung(n=55). Next,weattemptedtoremoveconfoundinginthese cohortsduetogenderorinjuryseveritybycreating matchedpairsbasedongender,abbreviateinjuryscores (AIS),aswellas,thenumberandtimingofthesamples obtainedorthedeathofthepatient.Thegenderof healthycontrolswasmatchedtothecohortascloselyas possible.Unfortunately,nohealthycontrolsolderthan 55yearswereavailable.However,thetranscriptomic analysisofuncomplicatedagedandyoungpatientsde- terminedthattherewasnodetectablegenomicdiffer- encebetweenthetwogroups.Regardless,thisresulted inthecreationofamuchsmallersubsetfortranscrip- tomicanalysis,whichincluded4matcheduncompli- catedagedandyoungtraumapairs,aswellas17 matchedcomplicatedagedandyoungtraumapairs. Thesewerecomparedto4and17healthycontrolpa- tients,respectively. Clinicaloutcomesandmultivariablelogisticregression analysis Baselinepatientdemographics,injuryseverity,fluidand bloodproductresuscitationparameters,seriallaboratory valuesandmultipleclinicaloutcomes,includingcompli- catedrecoveryand28-daymortality,wereobtainedfrom theTRDB.Ventilator-associatedpneumonia(VAP)was usedratherthanventilatorassociatedevents(VAE),as VAEhadnotbeendefinedbytheCDCatthetimeof studyinitiationandwasthereforenottrackedinthe database.Univariateanalyseswereperformedbetween youngandagedcohortsusingFisher sexacttestandthe Wilcoxontwo-sampletestasappropriate. Todeterminetheroleofageasanindependentpre- dictorofpooroutcome,multivariatestepwiselogisticre- gressionmodelswerecreatedusingpriorknownand suspectedconfoundingriskfactors,aswellasanysig- nificantpredictivefactorsidentifiedbyunivariateana- lysis.Patientswhodiedwithin48hoursofinjurywere excludedfromthecomplicatedoutcomesmodelinorder toremoveconfoundingeffectsofdeathfromirreversible hemorrhagicshockornon-survivableinjuries.Allpa- tientswereincludedfor28-daymortalitymodeling.All significancetestsweretwo-sided,witha0.05alphalevel. StatisticalanalyseswereperformedwithSAS(v.9.3, Cary,NC,USA). Geneexpressionprofileanalysis Ofthe1,928traumapatients enrolled,244underwent isolatedPMNbloodsampling, includingaged(n=67)and young(n=177)patients.Inadd ition,therewere17healthy controlsamplesfrompatientsyearsold.Patients enrolledintheleukocytesub populationsamplingportion ofthestudyhadblooddrawnwithin12hoursofinjuryand thensubsequentlyat1,4,7,14,21and28daysafterinjury whilehospitalized.Circulati ngPMNswereisolatedbyposi- tiveselectionusingmicrofluidiccassettes[21].Ingenuity SystemAnalysis(IPA®)wasusedtoperformgenomicana- lysisamongthetraumapatientsandhealthycontrolsafter RNAextractionandhybridizat ionontoproprietaryHH1/2/3 GeneChips [22],manufacturedbyAffymetrix(SantaClara, CA,USA)specificallyfortheGGprogram[21,23]. Threeseparateanalyseswereperformedasfollows:(1) allagedandyoungtraumapatientscomparedtohealthy controls;(2)matcheduncomplicatedyounganduncom- plicatedagedtrauma-patientpairscomparedtomatched controls;and(3)matchedcomplicatedyoungandcom- plicatedagedtrauma-patientpairscomparedtomatched controls.Matchingforthelatterinvolvedidentifying complicatedpatientsfromeachagegroupwhowerethe samesex,hadthesameAIS,hadthesamenumberand timepointofsampleisolation,andhadbotheithersur- vivedordiedby28days.Foreachofthesesets,weiden- tifiedsignificanttrauma-responsivegenesandthe differenceinthegenome-wideexpressionpatternsover- all,aswellasdifferencesatdays0.5,1.0,and4.0days afterinjury( P 1, F -test).Leaveoneoutcross- validationwasperformedtocomputethemisclassifica- tionrate,andaMonteCarlosimulationwasconducted totestifthemiscalculationratewassignificantlybetter thanpredictedbychance. Inaddition,thedatasetswereanalyzedforindividual geneexpressiondifferences(magnitudeoffoldchange, P 001),aswellasforindividualpathway(Gene OntologyandBiocarta,distancefromreference(DFR), Vanzant etal.CriticalCare (2015) 19:77 Page3of15 P )(22),andfunctionalpathwaydifferences(IPA, Z -score(&#x-2,-;Җ.;â¦é2)).A Z -scoreor&#x-2,-;Ҕ.;â¦é2,representsa significantchangeata95%CI[24]. AsecondaryanalysisofisolatedPMNexpressionusing the63trauma-responsivegenesthathadpreviouslybeen foundtobepredictiveofcomplicatedoutcomesafterse- veretrauma[8]wasperformedandcomparedinasimi- larfashionastoabove. Toquantifytheoverallperturbationingeneexpres- sion,amodifiedDFRmetricwascalculatedbasedon previouslyreportedmethodologyusingtheequation.[9]. TheDFRisasinglenaturallogarithmicgenomicscore thatsummatesalltheindividualgeneexpressionalter- ationsfrombaseline,whetherup-ordownregulated, where e i isthepatient sexpressionlevel,and M i and V i arethemeanandvarianceofthecontrolgroupforthe i th probeset.Thus,eachpatient soverallalteredtran- scriptomicresponsecanberepresentedbyasinglenat- urallog-transformedmetric,althoughitcanalsobe calculatedwithoutthenaturallogtransformationas well.TheDFRvaluesforanalyzedpatientswerecalcu- latedwithouttakingthenaturallogofthesumsandthe resultsreportedarereflectiveofthis. One-wayanalysisofvariance(ANOVA),Newman- Keul,Kruskal-Wallis,orHolm-Sidakmultiplecompari- sontestswereperformedwhenappropriate,witha 0.05alphalevelunlessindicateddifferentlyabove.Gen- omicstatisticalanalyseswereperformedusingGraphPad Prism6.00(LaJolla,CA,USA). Plasmacytokineandchemokineanalysis Patientswhowereenrolledintheleukocytesubpopula- tionsamplingalsohadplasmasamplesthatwereob- tainedduringthestudyperiod.Plasmasampleswere collectedwithin12hoursoftraumaonset,andat1,4,7, 14,21and28daysafterinjury,oruptothetimeofhos- pitaldischargeifthepatientwasaccessible.Analysiswas performedonthe17matchedyoungandagedtrauma- patientpairswithcomplicatedoutcomes,alongwith controls,whowereusedforthegeneexpressionanalysis mentionedpreviously. Plasmasamplesweretestedforthefollowinganalytes: IL-6,IL-8,IL-10,IL-1 ,Interferongamma-inducedprotein (IP)-10,Monocytechemoattractantprotein(MCP)-1and TNF- ,accordingtothemanufacturer sprotocol,onthe LuminexMAGPIX®xMapsystem(Luminex,Austin,TX, USA)usingMilliplex®MAPmultiplexkits(Millipore,Billerica, MA,USA).Thesampleconcentrationswerethengenerated withtheMilliplex®Analyst5.1softwareusingbest-fitcurves. Two-wayANOVAandgenerationofgenerallinear models(GLMs)wereusedforcomparison.Becausethe numberofsamplesinthetwocohortsvariedovertime, whetherduetodischargeordeathoverthestudyperiod, GLMsweredeterminedtobetheappropriatemethod forcomparison.Theywereusedtoexaminewhetherthe significantdifferencesseeninplasmacytokine/chemo- kineconcentrationswereduetoageand/ortimeafter injuryandtheresultsreportedarefromthisanalysis ( P 95%CI).Statisticalanalysiswasperformed usingSAS(v.9.3,Cary,NC,USA). Thedataobtainedinthemethodsanddiscussedinthis publicationhavebeendepositedinapublicrepository,the NationalCenterforBiotechnologyInformation s(NCBI) GeneExpressionOmnibus[25]andareaccessible [GEO:GSE64711](http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE64711). Results Patientclinicalcharacteristics,outcomesandmultivariate logisticregressionmodelanalysis Theoverallcohortconsistedof1,928severelyinjuredpa- tientsinhemorrhagicshock.Afterdividingthepopulation intoyoung(age5years)andaged(age 55years)co- horts,therewasnosignificantdifferencebetweenthe groupsininjuryseverityortotalamountofbloodtrans- fused(Table1).Agedpatientshadanincreasednumberof comorbidconditionsatadmission,aswellasevidenceof moresevereshockwithlowerinitialSBP,increasedlactate levelsandhigheracutephysiologyandchronichealth evaluation(APACHE)IIscoresonadmissionascompared totheiryoungercounterparts(Table1). Whencomparingclinicaloutcomesamongotherrisk factors,age�55yearswasassociatedwithsignificantly higherMOFscores,longerICUlengthofstay(LOS),in- creasedventilatordays,andhigherratesofbothnon- infectiousandinfectiouscomplications(Table1).The incidenceofVAPandtracheostomyplacementwassig- nificantlyelevatedinpatientsofadvancedage.In addition,amongotherriskfactors,age�55yearswasas- sociatedwithsignificantlyhigherratesofcomplicated outcomeand28-daymortality(Table1).Amongthose patientswhosurvived,theagedcohortwasmorelikely tobedischargedtoskillednursingfacilitiesorlong-term acutecarefacilities,ratherthanhomeorrehabilitation facilities(Table2). Multivariatelogisticregressionanalysisrevealedthat age55yearsorolderwasastrongindependentriskfac- torforthedevelopmentofacomplicatedclinicalout- come(definedaseitherICUhospitalization�14days withevidenceofongoingorgandysfunction,ordeath) aftercontrollingforinjuryseverity,shockseverity,blood transfusionandcomorbidities(Table3).Similarly,ad- vancedagewasastrongindependentriskfactorfor28- daymortality(Table3). Neutrophilmicroarraygenomicanalysis Initialgenomicanalysisdemonstratedthattherewasno significantage-baseddifferenceinthetranscriptomic Vanzant etal.CriticalCare (2015) 19:77 Page4of15 Table1Patientdemographicsandoutcomesofpatientswithsevereblunttraumainjuryandhemorrhagicshock Patientdemographicsandoutcomes Young(ageyears)(n=1,395)Aged(age 55years)(n=533) Number(%)ormedian(IQR) P -value Demographics Age,years34(24to45)66(59to75) Gender,male954(68%)331(62%)0.010 a NISS36(27to48)34(27to48)0.10 Bodymassindex,kg/m 2 27(24to31)28(25to32) a MaxApacheIIscore0to24hours28(24to32)32(27to37) a InjurytoEDarrival,hours1.3(0.7to2.3)1.4(0.8to2.9)0.001 a LowestEDSBP,mmHg84(72to98)78(66to88) a Maxlactate12to24hours,mmol/L2.8(2.0to4.3)3.0(2.1to4.5)0.035 a Totalblood0to12hours,U4.6(2.1to9.1)5.0(2.6to9.0)0.83 Totalcrystalloid0to12hours,L9.9(7.0to14.0)9.0(6.1to12.7) a Majoracutesurgicalprocedures1,312(94%)475(89%) a Pre-existingcomorbidities Preexistingmedications563(40%)412(77%) a Majormedicalcomorbidity, 1813(62.4%)419(87.1%) a Hypertension99(7%)214(40%) a Congestiveheartfailure0(0%)0(0%)- Atrialarrhythmia7(0.5%)31(5.6%) a Ventriculararrhythmia1(0.1%)4(0.8%)0.009 a Peripheralvasculardisease7(0.5%)16(3.0%) a Cerebrovasculardisease9(0.7%)40(7.5%) a Dementia0(0%)20(3.8%) a Obstructivepulmonarydisease64(4.6%)30(5.6%)0.35 Malignancy23(1.7%)47(8.8%) a Smoking423(30%)84(15.8%) a Alcoholism187(13%)54(10%)0.054 Psychiatricdisease147(10%)45(8%)0.17 Solidorgantransplant0(0%)6(1.1%) a HIV8(0.6%)1(0.2%)0.46 Outcomes MaxMarshallMOFscore4.7(3.2to6.7)5.3(3.8to7.2) a MaxDenver2MOFscore2(0to3)2(1to4) a Onventilator,days6(2to13)8(3to16) a Tracheostomy308(22%)145(27%)0.019 a ICUreadmission137(10%)51(10%)0.93 Non-infectiouscomplications592(42%)277(53%) a Surgicalsiteinfections202(15%)61(11%)0.09 VAP363(26%)159(30%)0.023 a ICUlengthofstay,days9(4to17)11(5to20) a Hospitallengthofstay,days19(10to31)18(9to30)0.07 Complicatedoutcome391(30%)226(47%) a Mortalityat28days54(6.7%)76(15.8%) a P -valueconsideredsignificantatdesignatedbysuperscripta.Complicatedoutcomeisdefinedas�.05;&#x-304;&#x.200;Ä14daysofpersistentorgandysfunctionorde ath.NISS, newinjuryseverityscale;ED,emergencydepartment;SBP,systolicbloodpressure;MOF,multiple-organfailure;VAP,ventilator-associatedpneu monia. Vanzant etal.CriticalCare (2015) 19:77 Page5of15 expressionpatternsintheuncomplicatedcohortsat 12hoursandonedayafterinjury( resultsnotshown ). Therefore,furtheranalysisofthesegroupswasnot performed. Asourepidemiologicalanalysisillustratedthataged patientshadaworseoutcometoseveretrauma,we wishedtodetermineifthereweredifferencesduetoage inthetranscriptomicresponsetoinjuryandshock, whiletryingtoexcludetheconfoundersofthemagni- tudeoftheinjury,gender,thehospitalcourse,andtreat- ment.Thus,wecomparedthegenome-wideexpression patternsofmatched(forthevariablesof:complicated outcome;gender;AIS;numberofsamplesisolatedatthe sametimepoints;andwhetherthepatientdiedduring hospitalization)patientswithcomplicatedoutcomes. Thisrevealed3,121probesets(2,095genes)thatwere differentiallyexpressedbetweenthetwoagegroupsat 12hoursaftertrauma( F -test, P 1).Leaveoneout cross-validationwasusedtoconfirmthatthedifferences inPMNgeneexpressionbetweenyoungandagedpatients couldnotbeexplainedbychancealone(Figure1A). WhenexaminingtheDFRscalculatedforeachpa- tient sPMNgeneexpressionpatternsovertime,itwas observedthatboththeagedandyoungcohortsexperi- enceddifferenttranscriptomicresponsesfromcontrols atallmeasuredtimepoints.However,theagedcohort hadsignificantlymoreaberrantexpression4daysafter hospitaladmissionthantheyoungercomplicatedtrauma cohort(DFR(×10 3 ):116±54versus88±29(SD); P 05)(Figure1B). Subsequently,secondaryanalysiswasconductedonthe 63totalleukocytegeneswhosedysregulationisknownto predictcomplicatedoutcomesafterseveretrauma[8]. Thisdemonstratedthatonly51ofthe63previouslyiden- tifiedgenesweresignificantlyexpressedinthePMNsof boththeyoungandagedtraumacohortswithcomplicated outcomes.Therefore,onlythese51geneswereusedfor subsequentcomparison.Analysisdemonstrateddifferen- tialexpressionovertimebetweentheagedandyoung complicatedtraumapatients.TheDFRsofthese51genes intheagedcohortwithcomplicatedcourses,revealedthat theirgeneexpressionpatternsweresignificantlylessper- turbedat12hoursand1dayafterinjury(DFR(×10 3 ): 1.031±0.364versus0.776±390and1.741±0.705versus 0.984±0.506; P .05).Bydayfourafterinjury,theaged patientshadsignificantlyincreasedalterationsintheir geneexpressionpatterns(DFR(×10 3 ):1.549±0.680ver- sus1.024±0.673; P .05)whencomparedtocontrols andtheiryoungercounterparts(Figure2). Focusingonindividualgenefold-changesat12hours and1dayafterinjury,theyoungcohortwasnotedto havesignificantalterationsingeneexpressioninvolved inneutrophilchemotaxis(thatis, CCR3,IL-8 ),increased Table2Dischargedispositionofyoungandagedtraumapatients Patientdischargedisposition Young(ageyears)(n=1,395)Aged(age 55years)(n=533) Number(%) P -value Home480(34.4%)66(12.4%) a Inpatientrehabilitation357(25.5%)96(18.0%) a LTAC52(3.7%)38(7.1%)0.002 a Skillednursingfacility283(20.3%)193(36.2%) a Other43(3.1%)12(2.3%)0.36 Death(asinpatient)180(12.9%)128(24.0%) a A P -valuewasconsideredsignificantdesignatedbysuperscripta.LTAC,long-termacutecarefacility. Table3Multivariateanalysisexaminingtheassociation betweenageandcomplicatedoutcomeandmortality aftersevereblunttrauma RiskfactorOddsratio(95%CI) P -value Complicatedoutcome 1 Newinjuryseverityscore�342.82(2.26to3.51) Totalblood�9.5U,0to12hours2.72(2.06to3.59) Age 55,years2.25(1.76to2.89) Lactate�6,mmol/L,0to6hours1.82(1.41to2.36) Bodymassindex�28,kg/m 2 1.56(1.26to1.94) EDSBPmmHg1.46(1.16to1.85) Crystalloid�12.5,L,0to12hours1.43(1.12to1.83)0.004 Majormedicalcomorbidity, 11.34(1.05to1.72)0.019 Mortalityat28days 2 TotalBlood�9.5(U)0to12hours3.91(2.93to5.22) Lactate�6,mmol/L,0to6hours2.22(1.67to2.96) Age 55,years2.12(1.59to2.82) Newinjuryseverityscore�341.74(1.32to2.29) EDSBPmmHg1.66(1.20to2.30)0.002 1 Modelfitstatistics:areaunderthecurve(AUC), c =0.748;Akaikeinformation criterion(AIC)=1982;likelihoodratiotest, P 01). 2 Modelfitstatistics: AUC,c=0.775;AIC=1433;likelihoodratiotest, P )Allriskfactorswere foundtobesignificant,definedasa P -valueComplicatedoutcomeis definedas�14daysofpersistentorgandysfunctionordeath.U,units;ED, emergencydepartment;SBP,systolicbloodpressure. Vanzant etal.CriticalCare (2015) 19:77 Page6of15 inflammation(thatis, HP,MMP8,HMGB1 )andin- creasedresponsesoftheinnateandadaptiveimmune system(thatis, CD24,CD44,IL4R )whencomparedto controls.Inaddition,theyoungcohorthadanincreased magnitudeofgenomicchanges,ascomparedtothoseof theagedcohort(Table4).Byday4postinjury,the gene-foldchangesseenintheyoungbegantotrendback towardcontrolbaselineexpression,withdecreasedex- pressionofpro-inflammatorygenesandincreasedim- muneresponse(antigenpresentationandco-stimulatory moleculegenes).Theagedcohort,however,continued todemonstratesignificantalterationsingenesinvolved indecreasedchemotaxisofneutrophils,upregulationof myeloidderivedsuppressorcells(MDSC),increasedin- flammation,anddecreasedresponseoftheinnateand adaptiveimmunesystem,ascomparedtocontrols,4 daysafterinjury(Table4). GeneontologyandBiocartaDFRpathwayanalysis demonstratedthatcomplicatedyoungtraumapatients hadsignificantlymoreaberrantexpressionpatterns(fold changeversuscontrolbaselineexpression)forPMN pathwaysinvolvedininnateimmunityandneutrophil Figure1 Heatmapandcalculateddifferencefromdistancefromreference(DFR)forpolymorphonuclearneutrophil(PMN)genome-wide expression. Usingafalsediscoveryadjustedprobabilityof.001andatwo-folddifferenceinexpression,thetemporalpatternoftheexpression ofthetraumaresponsivegenesthatdifferedbetweenthematchedaged( 55years)andyoung(55yearsold)traumacohortswithcomplicated outcomes,aswellashealthycontrols,ispresented. (A) Clusteranalysisofthecohorts0.5daysafterinjuryshowedthattherewere3,121probesets (2,095genes)withexpressionthatwassignificantexpressedamonggroups( F -test, P .001).Inaddition,theoverallpatternofgeneexpressionwas significantlydifferentineachcohort,asdeterminedbyleaveoneoutcross-validation. (B) SummaryoftheDFRscorecalculatedforeachpatientinthe complicatedagedandyoungcohortsatdays0.5,oneandfourdaysafterinjury.AnalysisrevealedsignificantdifferencesintheDFRsatallthepost traumatimepointsbetweenthetwocohortswhencomparedtocontrols.Inaddition,theadvancedagecohorthadsignificantlymoreaberrantgene expressionascomparedtotheyoungpatientsonday4(Newman-Keulsmultiplecomparisontest, * P .05). Vanzant etal.CriticalCare (2015) 19:77 Page7of15