英文文献.docx

英文文献.docx

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英文文献

ABObloodgroupingfromhardandsofttissuesofteethbymodifiedabsorption-elutiontechnique

BKRamnarayan,MManjunath,1andAnaghaAnanthJoshi2

DepartmentsofOralMedicine,Diagnosis,andRadiology,DayanandaSagarCollegeofDentalSciencesandHospital,ShavigeMalleswaraHills,Bangalore,Karnataka,India

1DepartmentofOralMedicineandRadiology,V.S.DentalCollegeandHospital,VVPuram,Bangalore,Karnataka,India

2DepartmentofPathology,KempegowdaInstituteofMedicalSciencesandResearchCentre,Banashankari,Bangalore,Karnataka,India

Addressforcorrespondence:

Dr.B.K.Ramnarayan,DepartmentsofOralMedicine,Diagnosis,andRadiology,DayanandaSagarCollegeofDentalSciencesandHospital,ShavigeMalleswaraHills,Bangalore-560078,Karnataka,India.E-mail:

ramnarayanbk@

Authorinformation►CopyrightandLicenseinformation►

Copyright:

©JournalofForensicDentalSciences

Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Noncommercial-ShareAlike3.0Unported,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.

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Abstract

Background:

Teethhavealwaysbeenknownasstabletissuethatcanbepreservedbothphysicallyandchemicallyforlongperiodsoftime.Bloodgroupsubstanceshavebeenknowntobepresentinboththehardandsofttissuesoftheteeth.

Objectives:

ThisstudyaimedatdetectionofABObloodgroupsubstancesfromsoftandhardtissuesofteethandalsotoevaluatethereliabilityofteethstoredforarelativelylongperiodasasourceofbloodgroupsubstancesbyabsorption–elutiontechniquewithsomemodifications.

Results:

Bloodgroupobtainedfromtheteethwascomparedwiththoseobtainedfromthebloodsample.Pulpshowedaverylargecorrelationinbothfreshandlong-standingteeththoughitdecreasedslightlyinthelatter.Hardtissueshowedalargecorrelationinboththegroupsindicatingthathardtissueisquitereliabletodetectbloodgroupandthatthereisnomuchdifferenceinthereliabilityinboththegroups.However,combiningpulpandhardtissue,correlationismoderate.Correlationofbloodgroupingwiththeage,sex,andjawdistributionwascarriedout.

Conclusion:

Bloodgroupidentificationfromhardandsofttissuesofteethaidsintheidentificationofanindividual.

Keywords:

ABO,absorption–elution,bloodgrouping,dentin,forensicodontology,pulp,teeth

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Introduction

Bloodgroupinghasbeenoneofthecornerstonesofidentificationofbiologicalmaterial.Theterm“Bloodgroup”isappliedtoinheritedantigensdetectedontheredcellsurfacebyspecificantibodies.[1]TheABObloodgroupsystemwasfirstdescribedbyKarlLandsteinerin1900andremainsthebulwarkofforensicbloodgroupinvestigation.Thereasonsforthisaremanifold.Itistheprimary,mostcommon,conspicuous,andeasilydetectablegroups.[2]ThoughadvanceshavebeenmadeintermsofDeoxyribonucleicacid（DNA）analysis,fingerprinting,etc.,bloodgroupingstillhasamajorroleinforensicscienceinthefieldofpersonidentification,paternitydispute,andotherscenarios.Thisisattributedtothefactthatgeneticandantigenicconstituentsofanindividualarenotaffectedbyenvironmentalconditions.[3]Latteshasaptlysaid“Thefactthatbelongingtoadefinitebloodgroupisafixedcharacterofeveryhumanbeingandcanbealteredneitherbythelapseoftimenorbyintercurrentdisease”.Bloodgrouplikefingerprintisanunalterableprimarycharacter.[4]

Teethbeingthehardestofalltissuescanbepreservedintactforlongperiodsoftimeafterdeath.Theyarestablechemicallyandtheyretaintheircharacteristiceveninthemostadversecondition.[5]Thepresenceofbloodgroupsubstancesandothergeneticmarkerssuchasenzymesinsoftandharddentaltissuesmakesitpossibletoassistintheidentificationofhighlydecomposedbodies.[6]Pulpaltissuebeingcontainedwithindentalhardtissues.Post-mortemchangesareseenverylate.Sincetoothpulpishighlyvascular,bloodgroupantigensaremostcertainlyboundtobepresent.Indentin,itispresumedthatthesesubstancesarelocatedinthedentinaltubules.ThepossibledistributionofABOsubstancesfromthepulpalcavitywalltothedentinedgeandtotheenamelgraduallydecreasesbecauseoffewerpossibilitiesofdiffusionofantigensfrombothbloodandsaliva.[7]

Absorption–elution（AE）techniquewasdevisedbySiracusa[8]in1923andhasbeenrefinedbyKind.[9]Variousmodificationshavetakenplacesincethentoimproveitssensitivity.Thistechniquehasbeenusedextensivelytodeterminebloodgroupfromdriedstains,tissues,secretions,andteethinvariousforensiclaboratories.Thismethodisverysensitive,highlyspecific,andleastinterferedwiththenatureofthesubstrata.Ithasalsobeenfoundthatthematerialonceusedisavailableforre-usewithpracticallynolossinitsantigenicproperty.[10]Recenttoothspecimencouldbeexpectedtoprovidegoodsourcesfordeterminationofbloodgroups.However,theeffectofautolysis,dehydration,lossofpulpantigens,orhighnumberoferrorsduetoforeignantigenbornebybacteriaincariousteethmayleadtovariationinthestudy.Therefore,itwasthoughtjustifiabletoconductthisstudytodetermineABObloodgroupsubstancesfrombothsoftandhardtissuesofnotonlyrelativelyfreshteethbutalsofromteethkeptstandingforarelativelylongperiodof6-10monthsbytheAEtechniquewithcertainmodifications.

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MaterialsandMethods

Sixtyclinicallysoundhumanpermanentteethwerecollectedrandomlyalongwiththerespectivebloodsamplesfrompatientswhoreportedforextractionduetoorthodonticandtherapeuticpurposes.Cariousteeth,grosslyattrited,andabradedteethwereexcludedfromthestudy.Thestudywasdividedintotwogroups:

GroupI（freshteeth）comprisedof30teethwhichwereanalyzedwithinaperiodof6weeks.GroupII（teethkeptforlongperiodoftime）comprisedof30teeththatwerecollectedandkeptforaperiodof6-10monthsandthenanalyzed.

Atthetimeofextractionofteeth,subsequenttoawrittenconsent,1mlofvenousbloodwasdrawnunderasepticconditionandstoredinlabeledEthyleneDiamineTetraAceticAcid（EDTA）bottles.Thisbloodsamplewasthensubjectedtobloodgroupingusingtheforwardgroupingslidemethodatroomtemperature.Thebloodgroupsthusdeterminedweretabulatedandusedasthegoldstandard.Theextractedteethwerewashedunderrunningwatertoremoveblood,saliva,anddebrisattachedtoit.Theteethweredriedwithgauzeandplacedinlabeledplasticrecipientsuntilusedindrystate.

Fromeachtooth,pulpwasextirpatedandtheremainingtoothstructurewaspulverizedinthefollowingway.Thetoothissplithorizontallyatthecervicalmarginwithacarborundumdiscusingamicromotor.Adropofsalinewasputintothepulpchamberandrootcanaltowetthepulp,whichwasextirpatedusingabarbedbroach.Theremainingtoothispulverizedwithstraightfissureburstartingfromthepulpalend.Theextirpatedpulpandpowderarestoredinsterilelabeledrecipients.Inablindstudy,pulpandpulverizedtoothpowderwerethensubjectedtoAEtest.Toothpulpwasdividedintotwoequalpartsandputintosterilelabeledtesttubes.Similarly,10mgofpowdereachwasplacedinlabeledtesttubes.Twodropsofanti-Aoranti-Bserawereadded,respectively,toeachtesttubecontainingpulpsamples.Similarly,antiserawasaddedtothepowdersamplealsoandmixed.Confirmingthetestsamplesbeingsufficientlysoakedwithantiserum,thetesttubeswerepluggedwithcottonandwereplacedat4°Covernighttoallowabsorption.

Afterremovingantisera,eachsamplewaswashed6timeswith10mlicecoldsalinesolutioninthefollowingway.Afteradditionofsaline,thetestsampleswereagitatedandthencentrifugedfor5minat4,000rpm.ThesupernatantwasdecantedandsuckedawaywithaPasteurpipette.Anyexcesssalinewasremoved.Thentwodropsof10%bovineserumalbumin（BSA）insalinewasaddedtothetestsamplesandthetesttubeswereplacedinapre-heatedwaterbathat56°Cfor10mintoelutetheantibodies.ThepowdertestsamplesinBSAwerecentrifugedat1,500-2,000rpmfor1-2min.thesupernatantwaspipettedandusedforagglutinationreaction.Adropof0.5%AandBredbloodcell（RBC）suspensionsisimmediatelyputintotherespectivetesttubes;thesamplesweregentlyshakenandincubatedat37°Cfor30mintoenhanceagglutination.Later,onedropofthesolutionwasplacedonamicroscopicslide,thencoverslipwasplaced,andfinallyagglutinationwasreadmicroscopicallyatamagnificationof×10and×40[Figures​[Figures11–3].

Figure1

Photomicrograph（×10）showingagglutinationofRBCs

Figure3

Photomicrograph（×40）showingnoagglutinationofRBCs

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Results

Sixtyteethwerecollectedfrompatientsintheagegroupof10-60years.Theage,sex,andjawdistributionofthesamplesinboththegroupsareasshowninTable1.Whenbloodgroupingfromthepulpandhardtissuecorrelatedwithbloodgroupofpersonwhichwasidentifiedfromthereference,thetestresultwasrecordedas“positive”andiftheydidnotmatch,thenitwasrecordedas“negative”andtabulated.

Table1

Age,sex,andjawdistributionoftwogroups

Thecomparisonofbloodgroupsbetweenreference,pulp,andhardtissueweredoneusingonlineJava-Scripttests.TwogroupswereconsideredtobestatisticallysimilarifP>0.05.

InGroupI（n=30）,whenpulpwascomparedwiththereference,28teethshowedpositiveresults,whiletwowerenegative.Thesensitivityofpulpincomparisontobloodwasfoundtobe93%.