blood stain typing by electrophoresis

C310/09(6)

Blood Stain Typing by Electrophoresis

An educational service of:

PO Box 752Beaumont, TX USA 77704-0752

28 1

Handbook for Rapid Blood Stain Typing Using Electrophoresis for the Crime Laboratory

Prepared by:

R.C. Briner, Ph.D.

C. R. Longwell, M.S.

SEMO Regional Crime LaboratorySoutheast Missouri State University

Cape Girardeau, MO 63701

Section 4 Final Notes

Titan®IIIandtheSuperZApplicatorSystemarefast,easyandpracticalforforensicinvestigationofmanydifferentproteinsandenzymes.Theexam-plesinthisbookletwillgivetheinvestigativelaboratorianspecifictechniquesforsomeassaysand theequipmentandreagentsareeasilymodified forotheruses.

PleasecontacttheauthorsatSEMORegionalCrimeLaboratoryorHelenaLaboratoriesifyouneedassistanceorhaveinformationtoshare.

For more information, call Helena Laboratories.Toll free 800-231-5663.

2 27

AcknowledgementsThefollowingstudentshaveprovidedvaluableassistanceinthedevelopmentofthesemethods:Retha(Mat-thews)Edens1,AliceAbbot2,RandallWebster3,Yvonne(Moll)Matthews4andRalphWillis5.Weespeciallyap-preciateMs.DonnaPruneau6forreviewingandupdatingthismanual(February,1989).TheseproceduresareinuseincourseFS552(BloodandBodyFluids),anelectiveintheScienceDivisionatSoutheastMissouriStateUniversity,CapeGirardeau,Missouri.TheauthorswouldliketothankHelenaLaboratories,Beaumont,Texas,forsupportofthisproject.PortionsofthisworkweresupportedbytheResearchCouncil,SoutheastMissouriStateUniversity,CapeGirardeau,Missouri.WewouldalsoliketothankMs.PattyButler7fortypingtheoriginaldraftofthishandbook.

1.No.11WestAdams,Cahokia,IL. 2.St.John’sMercyHospitalClinicalLaboratory,St.Louis,MO. 3.HannibalPoliceDepartment,Hannibal,MO. 4.P.O.Box945,CollegeStation,TX. 5.CrimeLaboratory,MissouriSouthernCollege,Joplin,MO. 6.SEMORegionalCrimeLaboratory,SoutheastMissouriStateUniversity,CapeGirardeau,MO. 7.Secretary,CollegeofScienceandTechnology,SoutheastMissouriStateUniversity,CapeGirardeau,MO.

Peptidase A (Pep A)(For Negroids)

1. TankBuffer:SameasforGLO-IandEsD

2. GelBuffer:SameasforGLO-IandEsD

3. Starch/AgarGel:SameasforGLO-IandEsD

4. SamplePreparationandApplication:Soakdriedbloodstainsforabout15minutesindistilledwaterandapplytogelinthesamemannerasGLO-IandEsD.Runaknowntype2-1asastandard.

5. Electrophoresis:250voltsfor45minutes.Amperageatthestartabout10mA,attheendabout6mA.

6. ReactionBuffer(SameasPGMreactionbuffer) 3.64gmTris,pH8.0 Dissolvein500mLofdistilledwater.AdjusttopH8.0withconcentratedHCl.

7. ReactionMixture 40mgL-valyl-L-leucine 20mgL-aminoacidoxidase(crudesnakevenom) 10mgMTT(lightsensitive) 4.0mgPMS(lightsensitive) 10mLreactionbuffer

8. Visualization:Overlay0.2gmagarose(SigmaTypeII)dissolvedin10mLofreactionbuffer.

Heattheagaroseuntildissolved.Allowtheagarosetocoolto55°Cbeforeaddingthereactionmixture.This isquicklypouredover thegel. Incubate thegel for1hourat37°C.

Thephenotypesappearasbluebandsonalightyellowbackground.

Figure 13 – Peptidase A (PepA)

Anode(+)

Applicationpoint

Cathode(–) Samplenumber 1 2 3 4 5 6 7 8

Electrophoretic patterns of three types of Pep A: Type 1-1: Samples 1, 2, 3, 7, 8 and 9 Type 2-1: Samples 4 and 6

Sample 5 is a suspected 1 + 2-1 mix from a vaginal/seminal sample

26 3

Table of Contents

Section 1 General Information I. Introduction 4

II. FactorsInfluencingResults 5

III. SampleExtractionandStorage 5

IV. Materials 5

V. ElectrophoresisTechniques 6

VI. ResultsandDiscussion 9

VII. References 9

Section 2 Typing of Dried Blood Stains With Cellulose Acetate Part I: Isoenzymes Phosphoglucomutase(PGM) 10

Erythrocyte/Vaginal/SeminalAcidPhosphatase (EAP/VAP/SAP) 12

AdenylateKinase(AK) 14

AdenosineDeaminase(ADA) 16

CarbonicAnhydraseII(CA-II)andHemoglobin 18

LactateDehydrogenase(LDH) 21

Glucose-6-PhosphateDehydrogenase(G-6-PD) 22

Part II: Proteins GroupSpecificComponent(Gc) 23

Haptoglobin 23

Section 3 Typing Dried Blood Stains Using Agar Starch Gels

Isoenzymes EsteraseD(EsD)andGlyoxylase1(GLO1) 24

PeptidaseA(PepA) 27


Figure 11 – Glyoxylase-I (GLO-I)

Anode(+)

Applicationpoint


Electrophoretic patterns of three types of GLO-1: Type 1-1: Samples 5 Type 2-1: Samples 1, 3, 4, 6 and 7 Type 2-2: Samples 2 and 8

Figure 12 – Esterase D (EsD)

Anode(+)

Applicationpoint


Electrophoretic patterns of two types of EsD: Type 1-1: Samples 1, 3, 4, 5 and 6 Type 2-2: Samples 2, 7 and 8

4 25

Section 1 General Information I. Introduction

Inthepastdecade,progressindevelopingmethodologiesfortypingbloodfactorsusingisoenzymestudieshasmadebloodstainevidenceoneof themost important investiga-tive toolsavailable to forensicscientists.Originally,allprocedures for isoenzyme typinginvolved electrophoresis on starch gels.At the present time, four major electrophoreticsupportmediaorutilized:starchgel,agarose,polyacrylamidegelandcelluloseacetate.Thispresentationisaresultoftheresearchperformedinourlaboratoriesdirectedtowardadaptationofisoenzymetypingofbloodfactorsbyelectrophoresisonsupportedcelluloseacetatemembranes(CAM).

Traditionally,theonlymethodforidentifyinghumanbloodsamplesinthecrimelaboratorywastypingbytheABOsystem.UsingtheABOsystem,themostrarebloodtypeencoun-teredisAB,whichisfoundinonly3%ofthepopulation.WhilethepercentageofpersonshavingbloodtypeABisverylow,almost50%ofthehumanpopulationhastypeO.Itisap-parentfromthesestatisticsthatasystemformoredefiniteidentificationofbloodsystemsismandatoryifbloodtypingistobeusedinacrimelaboratory.

Intheearly1970s,researchwasinitiatedtoimprovethesystemfordeterminingthetypeofhumanbloodfoundondriedbloodstains.InadditiontotheABOsystem,othersystemswereutilized.TheerythrocyteMNandRh(primarilyRhD)antigensystemswereused.Pro-teinstudiesinvolvedhemoglobinandhaptoglobin.Severalenzymesystemswereutilized;themostusefulofthesebeingphosphoglucomutase(PGM),adenylatekinase(AK),eryth-rocyteacidphosphatase(EAP),esteraseD(EsD),andadenosinedeaminase(ADA).InitialworkwasperformedbyBryanJ.CullifordoftheMetropolitanPoliceForensicLaboratoryofLondon,England.1Determiningbloodtypesusingacombinationofthesystemscited,greatlydiminishestheoccurrenceofaparticulartypeinthepopulation.Althoughelectro-phoresiswasthemethodofchoiceindeterminingtheproteinspresentinabloodstain,thetimeinvolvedbecameaprimeconcernsincestarchgel(theonlymediausedatthattime)electrophoresisproceduresoftenrequired15to30hoursforcompletion.

Inthemid1970s,workersattheUniversityofPittsburghproposedamethodformultipleenzymeseparationonasingleelectrophoresisplate.ThisworkwascompletedunderthedirectionofB.Wraxall.2Bycoveringaparticularportionofthestarchgelanddevelopingeachportionspecificallyforthedesiredenzyme,itwaspossibletoidentifyseveralenzymesystemsatthesametime.AdditionalworkbyWraxalldevelopedamultiplesystemsusingastarch/agarosegel.

Since that time, the research of Dr. B. Grunbaum and P.L. Zajac using the BeckmanMicro-zone®SystemandadaptingthestarchgelprocedurestoSartorious®celluloseace-tatemembraneshasdrasticallyreducedthetimerequired.TheTitan®IIICelluloseAcetateElectrophoresisSystemfromHelenaLaboratoriesisnowbeingusedsuccessfullybytheSoutheastMissouriRegionalCrimeLaboratory.Methods for identifyinggeneticmarkersareperformedonsupportedcelluloseacetateelectrophoresisplates.Theresultsaredis-cernableandreadilyreproducible.Studiesonalltheenzymesystemscanbecompletedinlessthaneighthours.

TheEsDbandsappearaswhitefluorescentbandsonadarkbackground.UseanEsD2-1asacontrol.AftervisualizingEsD,markthepositionofthenumber2bandoftheEsDpattern.ThegeliscoveredwithGLOIreactionmixturebeginningwiththispositionandincubatedagain.

b. GLOI:20mgreducedGlutathione 50µL40%Methylglyoxal 7.5mLGLOIreactionbuffer

SoakmixtureintoWhatmanpapercuttofitthesizeofthegelfromthesecondbandoftheEsDpatterntotheedgeoftheplate(approximately6.5cmx10cm).Thefilterpaperisplacedonthegelbeginningatthecathodeside,gentlyrollingdowntherestofthepaper,beingsurenoairbubblesremainunderthepaper.Incubate30to40minutesat37°C.

Heat0.2gmsagarose(SigmaTypeII)in20mLdistilledwateruntildissolved.Allowtheagarosetocoolto60°Candthenadd2dropsofiodinesolution.(Iodinesolutioncontains1.65gmsofKI,2.94gmsofI2dissolvedin30mLsofwater.)Mixtheaga-rose/iodinemixture.Thismixturewillbealightorangecolor.Aftertheelectrophoresisgelhasbeenincubatedasabove,theagarose/iodinemixtureisquicklypouredontothegelstartingatonecornerofthegelallowingittoflowovertheentiregelsurface.Thephenotypeappearsasbluebandsonalightyellowbackground.Theheterozy-gote(2-1)hasthreebluebandspresentandshouldbeusedasacontrol.Asecondagarose/iodinemixturemaybeappliedtothegelifthebluebandsappearweakinthefirstgel.Thisisdoneafterremovingthefirstagarose/iodinegel.

24 5

II. Factors Influencing Results Severalvariablesinfluencetheresultsobtainedwithanelectrophoreticprocedure.These

includesamplepreparationandapplication,celluloseacetateplatepreparation,pHandionicstrengthofthebuffers,voltage,amperage,andcolordevelopmenttechniques.Thecelluloseacetateplatesmustbesoakedintheindividualsystemplatebufferpriortosam-pleapplication.Thesoakingbufferisusuallyadilutionofthetankbuffer.Thecorrectdilu-tionofplatebufferisessentialforgoodresolutionandforpreventionofhighamperageandexcessheat.Theplatemustbesoakedgraduallyfromthebottomuptoensuresaturationwithoutairbubbles.

Airbubblesinthecelluloseacetateimpedethemovementoftheelectriccurrentthroughtheplate.Oncetrappedintheplate,airbubblescannotberemovedandtheplatemustbediscarded.

ThepHofthetank,plateandreactionbuffersisextremelyimportant.AllenzymeshaveanoptimalpHforactivity.Denaturationoftheenzymesmayoccurifreagentswiththein-correctpHareused.Improperreactiontemperatureshavethesameeffect.Correctionicstrengthofthebuffersisalsoveryimportant.

Propervoltageandamperageareextremelyimportanttogoodelectrophoreticresolution.Voltageshouldremainconstant,allowingtheamperagetostartlow(about2milliamperesperplate)inordertoavoidoverheatingthesystem.Thetemperatureoftheelectrophore-sisplaterisesastheamperagerises.Ifthetemperatureexceedstheoptimalrange,theenzymewilldenature.

The amount of time required for electrophoresis varies with the enzyme system understudy.Noneofourproceduresrequiremorethanonehour.

Thetechniqueusedfordevelopingthecelluloseacetateplateaftercompletionofelectro-phoresisiscritical.Thetwobasictypesofdevelopmentarecolorimetricandfluorometric.Afterdevelopment,resultsmustberecordedandfiled.AllplatesarephotographedwithblackandwhitePolaroidfilm.

III. Sample Extraction and Storage Topreserveenzymaticactivity,anymaterialscontainingdriedbloodstainsshouldbefro-

zen.Atthetimeoftesting,cuta0.5cmx0.5cmpieceofstainedmaterialfromtheoriginalsampleandplaceinawellwith2to3dropsofsolvent.Distilledwater isroutinelyusedas thesolvent;however,Cleland’s reagentmustbeused forphosphoglucomutaseanderythrocyte acid phosphatase assays. Samples extracted with purified water may besoakedovernightinahumiditychamberat4°C.SamplesextractedinCleland’sreagent(dithioerythritol;availablefromSigma)arestablefor3to5hoursandshouldnotbestoredovernight.Theprocedure frommakingCleland’s reagent is included in thePGMproce-dure.


Isoenzymes Esterase D (EsD) and Glyoxylase I (GLO I) Thisprocedureshouldbeperformedwithintwodaysofreceivingsample.Maximumsam-

pleageis10days.After10daysthesamplemaybetoodeterioratedtoproduceareadableresult.

1. TankBuffer:HEPES,pH7.5 5.95gmHEPES0.05M(N-2hydroxyethylpiperazine-N-2ethanesulfonicacid) Dissolvein400mLdistilledwater.AdjusttopH7.5with40%NaOH.Q.S.tofinalvol-

umeof500mLwithdistilledwater.

2. GelBuffer:1:14dilutionoftankbuffer,pH7.4. Dilute10mLoftankbufferwith140mLdistilledwater.

3. Starch/AgarGel 1%Agarose,SigmaTypeV(lowEEO) 2%Hydrolyzedstarch(ConnaughtLabs,Willowdale,Ontario,Canada). AvailablefromFisher,Cat.No.S-676. Dissolve0.3gmagaroseand0.6gmstarchin30mLgelbuffer.Afterdissolving,de-gas

thesolutionpriortopouringinto8cmx10cmx2mmdeepplasticplates.Gelmustsitovernightbeforeuse.

4. SamplePreparationandApplication a. Soakdriedbloodstains for15minutes in0.1NClelend’s reagent,pH8.0.Runa

knowntype2-1asastandard(GLOandEsD). b. Eightwells(1mmx4mm,3mmapart)arecutinthegel2cmfromthecathodicend

withametaltemplate.Apply5µLofbloodineachwellusingamicropipet.

5. Electrophoresis:250voltsfor30minutes. Itisveryimportanttokeeptheelectrophoresischamberverycoolduringtherun.The

tankbuffermustbekeptcolduntilreadyforuse.Tokeepthechamberatalowtem-perature,anotherlidormetalpanisfilledwithiceandplacedontopoftheclosedelec-trophoresistank.Plateisrunfacedownonspongewicks.Amperageatstart8to9mA;attheend,about10mA.

6. ReactionBuffer a. EsD: 0.41 gm sodium acetate, anhydrous (0.05M). Dissolve in 100 mL distilled

water.AdjusttopH6.5with1%aceticacid. b. GLOI:2.42gmNaH2PO4,anhydrous(0.2M) 1.31gmNa2HPO2,anhydrous(0.072M) Dissolvein100mLdistilledwater,adjusttopH6.2withphosphoricacid.

7. ReactionMixtureandVisualization a. EsD:4mgMU-Acetate(4-methylumbelliferylacetate) 1.0mLacetone(spectralgrade) 5mLreactionbuffer DissolveMU-acetateinabout1.0mLofacetoneandimmediatelyadd10.0mLofthe

reactionbuffer.SoakintoWhatman3MMpaper4cmx10cm.Laythispaperoveraportionofthegelfromtheorigintowardanode.Leaveatroomtemperatureforabout5minutes.RemovethepaperafterincubationatroomtemperatureandreadEsDbyusingUVlight.

6 23

IV. Materials

TitanIIICelluloseAcetateSystemavailablefromHelenaLaboratories. Hardware Cat. No. TITAN®plusPowerSupply(110V) 1504 ZipZone®Chamber 1283 SuperZApplicatorKit 4088 DevelopmentWeight 5014 TitanPhotoVuBoxCamera 5048 Bufferizer 5093 IODIncubator,OvenDryer(110V) 5116 Micro-Hood 8009 Consumables Titan®III-LipoCelluloseAcetate(60x76mm) 3900 Electra®HRBuffer 5085 Supre-HemeBuffer 5802 Electra®B1Buffer 5016 LDVisIsoenzymeReagent 5909 HemolysateReagent 5125 Blotters 5034 ZipZone®ChamberWicks 5081 DevelopmentSlides 5008 TitanPlasticEnvelope 5052 TitanIdentificationLabels 5006 HelenaMarker 5000

V. Electrophoresis Techniques

A. Preparation of Titan III-Lipo Plates

1. ProperlycodetherequirednumberofTitanIII-Lipoplatesbymarkingontheglossy,hardsidewithamagicmarker.Itissuggestedthatthemarkbeplacedinonecorneroftheplatesothatitisalwaysalignedwithsample#1.

2. Soaktheplatesfor20minutes in the individualsystemplatebuffer.Thesoakingbufferisusuallyadilutionofthetank buffer.The plates must be soaked gradually fromthebottomup toensuresaturationwithout trappingairbubblesinthecelluloseacetate.

The plates may be soaked in the Bufferizer (availablefrom Helena Laboratories, Cat. No. 5093) or they maybesoaked ina laboratorybeakerusing theprincipleoftheBufferizer.Placetheplatesinthebeakerinaverticalpositionandallowtheplatebuffertoslowlyflowdowntheside of the beaker from a separatory funnel or directlyfrom the buffer container.Alternately, fill a beaker withbufferandslowlyandsteadilyimmersetheplates(verti-calposition)intothebuffer.

Part II. Proteins Group Specific Component (Gc)

1. TankBuffer:Tris/Glycine,pH8.4 21.8gmGlycine 4.5gmTris Dissolvein900mLwater.AdjusttopH8.4withNaOH.Q.S.toafinalvolume

of1000mLwithdistilledwater.

2. PlateBuffer:Sameastankbuffer.

3. SamplePreparationandApplication:Extractbloodstainswith3dropsof6Murea(3.6gm:10mLH2O).Placeallofextractinasmallplasticcentrifugetube.Fillthetubehalfwaywithchloroform.Vortextomix.Centrifugethemixtureinamicrocentrifugeathighspeed.Thechloroformwashesdownthedebrisinthestain.

ExtractionofstainswithHemolysateReagent(HelenaCat.No.5125)willelim-inateagingbandswhichappearabovetheGcbands.ThiswillallowBCtypingtobeperformedonoldstains.*

4. Electrophoresis:500voltsfor10minutes;Initialamperage=2to4mA/plate.

5. Visualization(Immunofixation) a. Afterelectrophoresis,theplatesarecoveredwithhumanGcantiserumdi-

luted1:4withphysiologicalsaline(2mLserum+6mLsaline).Allowplatetosoakfor20minutes.

Thedilutedantiserumcanbereusedandisstableabout1monthafterdilu-tion,dependingonthenumberoftimesused.Storeintherefrigerator.

b. Removeexcessserumfromplateandsoakovernightinphysiologicalsa-line(0.85%).

c. Stainfor5minutesin0.2%PonceauS(v/vindistilledwater). d. Todestain,placeplatesinasolutionof5%aceticacid,agitatefor15min-

utes.Pouroutthissolutionandaddfreshaceticacid,agitateforanother15minutesuntil thebackgroundbecomeswhite.Platesmaybewashedovernight.Theplatesare thendried; thebandsaredarkpinkonawhitebackground.

TwosuggestedsourcesforGcantiseraare: Atlantic Antibodies In Vitro Research Sources, Inc. 10NonesuchRoad P.O.Box110 P.O.Box1032 Benson,MD21018 Scarborough,ME04074 (301)877-7110 (207)883-4154 Figures9and10onpage22illustratetheelectrophoreticpatternsofGc.

Haptoglobin (Hp) The stain extract prepared for the Gc procedure may be used for haptoglobin

determinationonpolyacrylamidegradientgel.However,beforeusingtheextract,preparea1:1mixturewith40%sucrose.

*PersonalCommunication:Willard(Bud)Stuver,Supervisor,Serology/BiologySection,Metro-DadePoliceCrimeLaboratory,Miami,FL33125.

22 7

B. Preparation of the Electrophoresis Tank (Zip Zone Chamber)

1. Prior to using the tank, fill the inner chamber halfway withwaterandkeepfrozenuntilthetankisused.

2. Whenpreparingforelectrophoresis,pour50mLoftankbufferintothecathodechamberanda1:1dilution(25mLtankbuf-fer/25mLH2O)intheanodechamber.(Exceptwhenstatedotherwise;checkspecificprocedures.)

3. Wet two Zip Zone® Chamber Wicks in the tank buffer anddrape one over each support bridge. Be sure they makecontactwiththebufferandthattherearenoairbubblesun-derthewicks.Donotallowthewickstotouchtheiceintheinnerchambers.Coverthetankuntilused.

C. Sample Application

1. Fillthewellsofthesamplewellplatewithbloodstainextractusingasmallcapillarypipette.

2. Primetheapplicatorbydepressingthetipsintothesamplewells 3 or 4 times.Apply this loading to a piece of blotterpaper.Primingtheapplicatormakesthesecondloadingmuchmoreuniform.Donotloadtheapplicatoragainatthispoint,butproceedquicklytothenextstep.

3. Remove the wettedTitan III plate from the buffer and blot

oncefirmlybetweentwoblotters.Placetheplateinthealign-ingbase, celluloseacetatesideup,aligning the topof theplate with the black scribe line marked “cathodic applica-tion”.Theidentificationmarkshouldbealignedwithsample#1.Mostproceduresrequireacathodicapplicationwiththeexceptionofphosphoglucomutase(PGM)whichrequiresanextremecathodicapplication.

HELENA

1 2 3 4 5 6 7 8

HELENA

1 2 3 4 5 6 7 8

Buffer

Ice

Buffer

CATHODE APPLICATION

CENTER APPLICATION

HELENA LABORATORIES

Placementofplateforextremecathodicapplication

Figure 9 – Group Specific Component (Gc)

Anode(+)

Applicationpoint


Electrophoretic patterns of two types of Gc are demonstrated as follows:

Type 2-1: Samples 1, 3, 5, 6, 7 and 8 Type 2-2: Samples 2 and 4


Anode(+)

Applicationpoint


Electrophoretic patterns of three types of Gc are demonstrated as follows: Type 1-1: Samples 3, 6 and 7 Type 2-2: Samples 1 and 8 Type 2-1: Samples 2, 4 and 5

4. Apply thesampleto theplatebydepressingtheapplicatortipsintothesamplewells3or4timesand promptly transferring the applicator to thealigning base. Press the button down and holdfor5seconds.Makeasecondor thirdsuperim-posed application by repeating this step. Threeapplicationsof .25µLeach issufficient formostisoenzymestudies.

D. Electrophoresis of the Sample Plate

1. Quickly place the plate, cellulose acetate sidedown,intheelectrophoresistank.Placeaweight(plasticrod,glassslide,etc.)ontheplate(s)toin-surecontactwith thewicks.Cover thechambersecurely.

2. Electrophorese according to the instructions oftheappropriateprocedure.

E. Development

1. Foreachsampleplateused,areactionplatemustalsobesoakedaccordingtothesoakingmethoddescribedinStepA.Thereactionplateisusuallysoakedinthereactionbuffer.Seeindividualproce-dures,Section2.

2. Remove each plate from the reaction soaking buffer 5 minutes before completion of electrophoresis and blot gently. Place theplate, celluloseacetatesideup,ontoaglassdevelopmentslideandplaceonalevelcountertoporalevelingtable.

3. Pourapproximately2.0to5.0mLofreactionmix-tureontotheplate.Tilttheslideuntilthereactionmixturecovers theplate.Covereachplatewithasmallbox(ifthereactionmixtureislightsensi-tive)andallowtostandforfiveminutes.

4. Aftercompletionofelectrophoresis, remove thesample plate from the electrophoresis tank andblotlightlytoremoveexcessbuffer.

5. Carefullylayerthesampleplate,celluloseacetatesidedown,ontothereactionplate.Placeablotteroverthesandwichedplates.Removeexcesssub-stratefromthesandwichbydrawingtheedgeofthealigningbaseacrosstheplateseveraltimes.Blotawayexcesssubstrate.

6. PlacetheplatesbetweenmoistenedblottersandplaceonthetopofapreheatedMicro-HoodorinaHelenaIODorotherlaboratoryincubatorat37°C.Placeapreheateddevelopmentweightontopoftheblotters.

8 21

TITANPOWER SUPPLY

VOLTAGE CONTROL

II

II

II

II

IIII

II

II

II

II

+ _

Glucose-6-Phosphate Dehydrogenase (G-6-PD)

1. TankBuffer:Supre-HemeBuffer(HelenaCat.No.5802),pH8.4.Tris-EDTABoratebuffer.Diluteto1000mLwithdistilledwater.


3. Preheatincubatoranddevelopmentweightto37°C.

4. SamplePreparationandApplication:Onecathodicapplicationofdistilledwaterextractofstain.

5. Electrophoresis:350voltsfor20minutes.

6. ReactionBuffer:Undilutedtankbuffer.

7. G-6-PDReactionMixture a.G-6-PDReagent,HelenaCat.No.5620or b.10.0mgGlucose-6-Phosphate 6.0mgNAD(NicotinamideAdenineDinucleotide) 7.0mgMTT(lightsensitive) 7.0mgPMS(lightsensititve) Reconstitutereagentwith3to5mLofundilutedtankbuffer.Thismakessufficient

reagentfor3to4reactionplates.

8. Incubation:20minutesat37°C.

9. Visualization:Separateplatesandsoakin7.5%trichloroaceticacid(TCA)for2to3minutes.Rinsein5%aceticacidfor5minutes.Blot,dryplatesandreadundertransmittedlight.

20 9

7. Aftertheappropriateincubationperiod,preparetheplatesforevalu-ationasdescribedinindividualprocedures(Section2).Afterdevel-opment,allplatesmustbedriedbeforereadingeitherbyairdryingorwithalightstreamofwarmforcedair.Bothplates,thesampleplateandreactionplate(negative),canberead.Inmostinstances,driedplatesmaybestoredindefinitelyforfuturereferenceifplacedinaTitanPlasticEnvelopeorsmallplasticstoragebag.

VI. Results and Discussion Atthepresenttime,thefollowingproceduresarebeingperformedrou-

tinely inSEMORegionalCrimeLaboratoryusing thecelluloseacetatemembrane(CAM)system:

Phosphoglucomutase(PGM) Erythrocyte/Vaginal/SeminalAcidPhosphatase(EAP/VAP/SAP) AdenylateKinase(AK) GroupSpecificComponent(Gc) Other procedures which are currently performed on CAM are: Glucose-6-PhosphateDehydrogenase(G-6-PD) LactateAcidDehydrogenase(LDH) CarbonicAnhydraseII(CA-II)5

Isoenzymetypingofbloodusingcelluloseacetatemembranesasasup-portmediumforelectrophoreticseparationhasproventobearelativelyin-expensiveandrapidmethodforidentifyingbloodstainsaccordingtopoly-morphicproteinsandenzymes.Usingtheseprocedures,determinationscanbeperformedusingmicrolitervolumesofspecimen(stainextract).

EsteraseD(EsD),Glyoxylase-I(GLO-I)andPeptidaseA(PepA)determi-nationsareperformedwithstarch-agarosegelduetosuperiorseparationandvisualization.Haptoglobindeterminationsareperformedutilizingagradientpolyacrylamidesystem(overnight).Ifthehaptoglobinprocedureis runat300volts, itcanbecompleted in fourhours. It ispossible foratrainedanalysttorunallsystems, includingspecies,ABOtypingandhaptoglobinineighthours.

VII. References 1. Culliford,B.J.,TheExaminationandTypingofBloodstainsintheCrime

Laboratory,LawEnforcementAssistanceAdministration,Washington,D.C.,20402,Stock#2700-0083.

2. Wraxall, Bryan G.D., Ed., Bloodstain Analysis System Procedures Manual,SerologicalResearchInstitute,Emeryville,California,1977.

3. Grunbaum,B.W.,Selvin,S.,Pace,N.,Black,D.M.,JofForensicSci,23,577,1979.

4. Grunbaum, B.W., Ed., Handbook for Individualization of Forensic Blood and Bloodstains,UniversityofCalifornia,Berkeley,California,1980.

5. Noppinger, K.E., Morrison, R.D., J of Forensic Sci, 26:1, 176-180,1981.

Lactate Dehydrogenase (LDH) 1. TankBuffer:ElectraHRBuffer(HelenaCat.No.5805),pH8.8. Trisbarbital-sodiumbarbitalbuffer.Diluteto1750mLwithdistilledwater.



4. SamplePreparationandApplication:Applytwocathodicapplicationsofdistilledwaterextractofstain.Seminalstainsmayhavetobeseriallydiluteduntilareadableresultisachieved.

5. Electrophoresis:180voltsfor30minutes;Initialamperage=8mAeach.

6. ReactionPlates:SoakplatesinPGMreactionbufferabout15minutesbeforecompletionofelectrophoresis.

7. LDHReactionMixture 5mLPGMreactionbuffer 25mgCalciumLactate 2mgNAD(NicotinamideAdenineDinucleotide) 2.5mgMTT(lightsensitive) 2.0mgPMS(lightsensititve) OruseLDVISIsoenzymeReagent,Cat.No.5909,HelenaLaboratories

8. Incubation:15minutesat37°Cwithdevelopmentweightontop.

9. Visualization:LDHpatternappearsasbluebands.Viewundertransmittedlight.

Figure 8 – Lactic Acid Dehydrogenase (LDH) Anode(+)

Applicationpoint


LDH electrophoretic patterns from various sources are illustrated as follows: Liver: Samples 1 and 2 Heart: Samples 3 and 4

Mixture of menstrual blood and seminal fluid: Samples 5, 6, 7, and 8

10 19

Section 2 Typing of Dried Blood Stains With Cellulose Acetate

Part 1: Isoenzymes Phosphoglucomutase (PGM)

1. TankBuffer:Tris/maleicacidbuffer0.075M;pH7.4 6.05gmTris:Tris(hydroxymethyl)aminomethane 5.81gmMaleicacid 1.46gmEDTA(FreeAcid):EthylenediaminetetraaceticAcid 3.47gmMgCl2•6H2O Dissolvein900mLofdistilledwaterandadjusttopH7.4with40%NaOH.

Q.S.tofinalvolumeof1000mLwithdistilledwater. CathodeBuffer:50mLoftankbuffer AnodeBuffer:15mLtankbuffer+35mLdistilledH2O

2. PlateBuffer:30mLtankbuffer+270mLdistilledH2O


4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sreagent.Postmortemblood

mayhavetobeseriallydilutedwithCleland’sreagentuntilareadableresultcanbeachieved.Runknowntype2-1standardoneachplate.Makeoneortwoapplications,extremelycathodic.

NOTE:TheextractmadeinCleland’sreagentisstablefor3to5hours.

5. Electrophoresis:250voltsfor30minutes;Initialamperage=2mA/plate.

6. ReactionBuffer:pH8.0 3.64gmTris Dissolvein500mLdistilledwater.AdjusttopH8.0withconcentratedHCl.

7. ReactionPlates:Soakplatesinreactionbufferapproximately15minutespriortocompletionofelectrophoresis.

8. PGMReactionMixture a)PreparedPGMmixturein3.0mLofreactionbufferor b)30.0mgGlucose-1-Phosphate* 0.3mgGlucose-1,6-Diphosphate* 20.0unitsGlucose-6-PhosphateDehydrogenase 2.5mgPMS:PhenazineMethosulfate(Lightsensitive) 2.5mgMTT:ThiazoylBlue(Lightsensitive) 2.0mgNADP:NicotinamideAdenineDinucleotidePhosphate 5.0mLreactionbuffer Stirmixtureuntildissolved.Pouronreactionplatesabout5minutes

beforecompletionofelectrophoresis. *AvailabletogetherfromSigmaChemicalCompany;-D-Glucose1-PhosphateDisodium

Salt,cat.no.G-1259

9. Incubation:15minutesat37°C,withweightontoporuntilbluebandsappear.

10.Visualization:Separateplatesandsoak5minutesin5%aceticacid.Blot, dryandreadusingtransmittedlight.

Figure 7 – Carbonic Anhydrase (CA-II)

Anode(+)

Applicationpoint


Electrophoretic patterns for two types of CA-II are demonstrated as follows: Type 1-1: Samples 1, 2, 4, 5 and 6 Type 2-1: Samples 3, 7, and 8

18 11

Figure 1 – Phosphoglucomutase (PGM) Anode(+)

Applicationpoint


Electrophoretic patterns for two types of PGM are demonstrated as follows: Type 1-1: Samples 2, 3, 4, 6, 7 and 8 Type 2-2: Samples 1 and 5


Applicationpoint


Electrophoretic patterns for three types of PGM are demonstrated as follows: Type 1-1: Samples 1, 6, 7 and 8 Type 2-2: Samples 3 and 4 Type 2-1: Samples 2 and 5

Carbonic Anhydrase II (CA-II) and Hemoglobin (For Negroids)

1. TankBuffer:TAPS,0.075M,pH8.4 9.12gmTAPS(Tris(Hydroxymethyl)MethylaminopropaneSulfonicAcid) Dissolvein500mLdistilledwater.AdjusttopH8.4with40%NaOH(sodiumhydroxide).

MonitorpHclosely.



4. SamplePreparationandApplication:Prepareadistilledwaterextractofthestain.Runaknowntype2-1asstandard.Make5-6centerapplications.

5. Electrophoresis:Constantamperageof9.0mAperplatefor45minutes.Voltageisapproximately400.

Itisveryimportanttokeeptheelectrophoresischamberverycoolduringtherun.Thetankbuffermustbekeptcolduntilreadyforuse.Tokeepthechamberatalowtemperature,anotherlidormetalpanisfilledwithiceandplacedontopoftheclosedelectrophoresistank.

NOTE:Thehemoglobinbandsareeasilyseenasdistinctredbandsontheplatewithin10-15minutesafterstartingelectrophoresis.HemoglobinvariantscanbedeterminedwhiletheplateisstillinthetankandaremoredistinctbeforestainingforCA-II.

6. ReactionBuffer 3.60gmNa2HPO4,dibasic 0.05gmMgCl2•6H2O Dissolvein250mLdistilledwater.AdjusttopH7.5withHCl(1.0N).

7. ReactionMixture a.8.0mgFluoresceindiacetate 1.0mLAcetone 20mLreactionbuffer b.0.4gmagarose,SigmaTypeII 20mLdistilledwater c.Heatagaroseuntilitisdissolved.Coolto60°C.Dissolvefluoresceindiacetatein

1.0mLacetoneandimmediatelyadd20mLofreactionbuffer.Addthismixturetothecooledagarose.Pourtheagarose/fluoresceindiacetatemixtureintoflatPetridishes;makes3to4plates.Storeinrefrigerator.

d.Placethecelluloseacetateplateontotheagarose/fluoresceindiacetategel,celluloseacetatesidedown.

8. Incubation:20to30minutesat37°Corlongerifnecessary.

9. Visualization:CA-IIbandsarebrightyellowfluorescentbandsonadarkbackground.

12 17

Erythrocyte/Vaginal/Seminal Acid Phosphatase (EAP/VAP/SAP)

1. TankBuffer:CitratePhosphatepH5.5 4.42gmSodiumCitrate 3.36gmDibasicSodiumPhosphate(Na2HPO4•H2O)(2.98gmanhydrous) 0.372gmEDTA(FreeAcid) Dissolvein900mLofdistilledwaterandadjusttopH5.5withphosphoricacid.




4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sReagent.Runknowntype

BAasstandard.Makethreecathodicapplications.



6. ReactionBuffer 5.25gmCitricAcid(anhydrous) 2.0gmSodiumHydroxide Dissolvein500mLofdistilledwater.AdjusttopH5.0withsodiumhydroxide

orcitricaciddependingonpH.


8. EAPReactionMixture 2.0mg4-methylumbelliferylphosphate 5.0mLreactionbuffer 0.75mLglycerol(finalsolutionis15%glycerolvol/vol) Pouronreactionplatesabout5minutesbeforecompletionofelectrophoresis.

9. Incubation:15minutesat37°C,withdevelopmentweightontop.

10. Visualization(Fluorescence):Separateanddryplates.Mistlightlywith0.1NNaOH.ReadunderU.V.lightbox.Storeplatesinplasticbags.Bandscanberegeneratedbymistingwith0.1NNaOH.

Figure 6 – Adenosine Deaminase (ADA)

Anode(+)

Applicationpoint


Electrophoretic patterns for two types of ADA are demonstrated as follows: Type 1-1: Samples 1, 2, 3,4, 5 and 8 Type 2-1: Samples 6 and 7

16 13

Figure 3 – Erythrocyte Acid Phosphatase (EAP)

Anode(+)

Applicationpoint


Electrophoretic patterns for six types of EAP are demonstrated as follows: Type BB: Samples 1 and 3 Type BA: Samples 2 and 8 Type DB: Samples 4 Type CB: Samples 5 Type CA: Samples 6 Type AA: Samples 7

Figure 4 – Vaginal/Seminal Acid Phosphatase

Anode(+)

Applicationpoint

Cathode(–) Samplenumber 1 2 3 4

Electrophoretic patterns for two types of vaginal/seminal fluid acid phosphatase are demonstrated as follows:

Type 1-1: Samples 1 and 3, seminal stain Type 2-2: Samples 2 and 4, vaginal stain

Adenosine Deaminase (ADA)

1.TankBuffer:CitratePhosphatepH5.5 4.42gmSodiumCitrate 3.36gmDibasicSodiumPhosphate(Na2HPO4•H2O)(2.98gmanhydrous) 0.372gmEDTA(FreeAcid) Dissolvein900mLofdistilledwaterandadjusttopH5.5withphosphoricacid.




4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sReagent.RunaknownType2-1

asastandard.Makefourorfivecathodicapplications. NOTE:TheextractmadeinCleland’sreagentisstablefor3to5hours.

5. Electrophoresis:200voltsfor60minutes;Initialamperage=2mA/plate

6. ReactionBuffer:pH7.9 9.0gmTris 8.92gmMgCl2•6H2O Dissolvein500mLdistilledwater.AdjusttopH7.9with6MHCl.

7. ReactionMixture 10.0mgAdenosine 0.1units(4µL)XanthineOxidase 0.5units(0.05µL)NucleosidePhosphorylase 5.0mgPMS(lightsensitive) 5.0mgMTT(lightsensitive) 3mLReactionbuffer

NOTE:AddMTTandPMSjustbeforepouringonreactionplateandworkinthedarkbecausebothchemicalsarelightsensitive.Filterreactionmixtureontoreactionplate.

8. Incubation:15minutesat37°Coruntilbluebandsappear.

9. Visualization:Separateplatesandsoak5minutesin5%aceticacid.Blotwell,dryandreadresultsundertransmittedlight.

NOTE:Ifupperbandisdarker,thereisapossibilityofoxidation.UsemercaptoethanolratherthanCleland’sreagenttoextractthedriedstain.

14 15

Adenylate Kinase (AK)





4. SamplePreparationandApplication:Twotothreecathodicapplicationsofdistilledwaterextractofstain.Ifpossible,runaknown2-1AKonthetestplatetohelpclarifyreadings.

5. Electrophoresis:200voltsfor60minutes;Initialamperage=2mA/plate. ADAandAKmayberuninsametank,utilizingseparateplatesforeachsystem.


7. ReactionMixture 150.0mgGlucose 10.0mgADP(Adenosine-5’diphosphate) 10.0mgNADP 5.0mgPMS(lightsensitive) 5.0mgMTT(lightsensitive) 14IUHexokinase* 6IUGlucose-6-PhosphateDehydrogenase* 5mLReactionbuffer NOTE:AddMTTandPMSjustbeforepouringonreactionplateandworkinthedark

becausebothchemicalsarelightsensitive.Filterreactionmixtureontothereactionplate. *AvailabletogetherfromSigmaChemicalCompany;Hexokinase-Glucose-6-PhosphateDehydrogenase,

cat.no.H-8629.


9. Visualization:Separateplatesandsoakfor5minutesin5%aceticacid.Blotwell,dryplatesandreadbluebandsundertransmittedlight.

Figure 5 – Adenylate Kinase (AK)

Anode(+)

Applicationpoint


Electrophoretic patterns for two types of AK are demonstrated as follows: Type 1-1: Samples 1, 2, 3, 4, 5, 7 and 8 Type 2-1: Sample 6

16 13

Figure 3 – Erythrocyte Acid Phosphatase (EAP)

Anode(+)

Applicationpoint


Electrophoretic patterns for six types of EAP are demonstrated as follows: Type BB: Samples 1 and 3 Type BA: Samples 2 and 8 Type DB: Samples 4 Type CB: Samples 5 Type CA: Samples 6 Type AA: Samples 7

Figure 4 – Vaginal/Seminal Acid Phosphatase

Anode(+)

Applicationpoint

Cathode(–) Samplenumber 1 2 3 4

Electrophoretic patterns for two types of vaginal/seminal fluid acid phosphatase are demonstrated as follows:

Type 1-1: Samples 1 and 3, seminal stain Type 2-2: Samples 2 and 4, vaginal stain

Adenosine Deaminase (ADA)

1.TankBuffer:CitratePhosphatepH5.5 4.42gmSodiumCitrate 3.36gmDibasicSodiumPhosphate(Na2HPO4•H2O)(2.98gmanhydrous) 0.372gmEDTA(FreeAcid) Dissolvein900mLofdistilledwaterandadjusttopH5.5withphosphoricacid.




4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sReagent.RunaknownType2-1

asastandard.Makefourorfivecathodicapplications. NOTE:TheextractmadeinCleland’sreagentisstablefor3to5hours.

5. Electrophoresis:200voltsfor60minutes;Initialamperage=2mA/plate


7. ReactionMixture 10.0mgAdenosine 0.1units(4µL)XanthineOxidase 0.5units(0.05µL)NucleosidePhosphorylase 5.0mgPMS(lightsensitive) 5.0mgMTT(lightsensitive) 3mLReactionbuffer

NOTE:AddMTTandPMSjustbeforepouringonreactionplateandworkinthedarkbecausebothchemicalsarelightsensitive.Filterreactionmixtureontoreactionplate.


9. Visualization:Separateplatesandsoak5minutesin5%aceticacid.Blotwell,dryandreadresultsundertransmittedlight.

NOTE:Ifupperbandisdarker,thereisapossibilityofoxidation.UsemercaptoethanolratherthanCleland’sreagenttoextractthedriedstain.

12 17

Erythrocyte/Vaginal/Seminal Acid Phosphatase (EAP/VAP/SAP)





4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sReagent.Runknowntype

BAasstandard.Makethreecathodicapplications.



6. ReactionBuffer 5.25gmCitricAcid(anhydrous) 2.0gmSodiumHydroxide Dissolvein500mLofdistilledwater.AdjusttopH5.0withsodiumhydroxide

orcitricaciddependingonpH.


8. EAPReactionMixture 2.0mg4-methylumbelliferylphosphate 5.0mLreactionbuffer 0.75mLglycerol(finalsolutionis15%glycerolvol/vol) Pouronreactionplatesabout5minutesbeforecompletionofelectrophoresis.

9. Incubation:15minutesat37°C,withdevelopmentweightontop.

10. Visualization(Fluorescence):Separateanddryplates.Mistlightlywith0.1NNaOH.ReadunderU.V.lightbox.Storeplatesinplasticbags.Bandscanberegeneratedbymistingwith0.1NNaOH.

Figure 6 – Adenosine Deaminase (ADA)

Anode(+)

Applicationpoint


Electrophoretic patterns for two types of ADA are demonstrated as follows: Type 1-1: Samples 1, 2, 3,4, 5 and 8 Type 2-1: Samples 6 and 7

18 11


Applicationpoint


Electrophoretic patterns for two types of PGM are demonstrated as follows: Type 1-1: Samples 2, 3, 4, 6, 7 and 8 Type 2-2: Samples 1 and 5


Applicationpoint


Electrophoretic patterns for three types of PGM are demonstrated as follows: Type 1-1: Samples 1, 6, 7 and 8 Type 2-2: Samples 3 and 4 Type 2-1: Samples 2 and 5

Carbonic Anhydrase II (CA-II) and Hemoglobin (For Negroids)

1. TankBuffer:TAPS,0.075M,pH8.4 9.12gmTAPS(Tris(Hydroxymethyl)MethylaminopropaneSulfonicAcid) Dissolvein500mLdistilledwater.AdjusttopH8.4with40%NaOH(sodiumhydroxide).

MonitorpHclosely.



4. SamplePreparationandApplication:Prepareadistilledwaterextractofthestain.Runaknowntype2-1asstandard.Make5-6centerapplications.

5. Electrophoresis:Constantamperageof9.0mAperplatefor45minutes.Voltageisapproximately400.

Itisveryimportanttokeeptheelectrophoresischamberverycoolduringtherun.Thetankbuffermustbekeptcolduntilreadyforuse.Tokeepthechamberatalowtemperature,anotherlidormetalpanisfilledwithiceandplacedontopoftheclosedelectrophoresistank.

NOTE:Thehemoglobinbandsareeasilyseenasdistinctredbandsontheplatewithin10-15minutesafterstartingelectrophoresis.HemoglobinvariantscanbedeterminedwhiletheplateisstillinthetankandaremoredistinctbeforestainingforCA-II.

6. ReactionBuffer 3.60gmNa2HPO4,dibasic 0.05gmMgCl2•6H2O Dissolvein250mLdistilledwater.AdjusttopH7.5withHCl(1.0N).

7. ReactionMixture a.8.0mgFluoresceindiacetate 1.0mLAcetone 20mLreactionbuffer b.0.4gmagarose,SigmaTypeII 20mLdistilledwater c.Heatagaroseuntilitisdissolved.Coolto60°C.Dissolvefluoresceindiacetatein

1.0mLacetoneandimmediatelyadd20mLofreactionbuffer.Addthismixturetothecooledagarose.Pourtheagarose/fluoresceindiacetatemixtureintoflatPetridishes;makes3to4plates.Storeinrefrigerator.

d.Placethecelluloseacetateplateontotheagarose/fluoresceindiacetategel,celluloseacetatesidedown.

8. Incubation:20to30minutesat37°Corlongerifnecessary.

9. Visualization:CA-IIbandsarebrightyellowfluorescentbandsonadarkbackground.

10 19

Section 2 Typing of Dried Blood Stains With Cellulose Acetate

Part 1: Isoenzymes Phosphoglucomutase (PGM)

1. TankBuffer:Tris/maleicacidbuffer0.075M;pH7.4 6.05gmTris:Tris(hydroxymethyl)aminomethane 5.81gmMaleicacid 1.46gmEDTA(FreeAcid):EthylenediaminetetraaceticAcid 3.47gmMgCl2•6H2O Dissolvein900mLofdistilledwaterandadjusttopH7.4with40%NaOH.




4. SamplePreparationandApplication Cleland’sReagent(0.01N) 154gm/dLDithiothreitol+100mLH2O.AdjustpHto8.0with6MNaOH. Soakbloodstainsfor15minutesinfreshCleland’sreagent.Postmortemblood

mayhavetobeseriallydilutedwithCleland’sreagentuntilareadableresultcanbeachieved.Runknowntype2-1standardoneachplate.Makeoneortwoapplications,extremelycathodic.



6. ReactionBuffer:pH8.0 3.64gmTris Dissolvein500mLdistilledwater.AdjusttopH8.0withconcentratedHCl.


8. PGMReactionMixture a)PreparedPGMmixturein3.0mLofreactionbufferor b)30.0mgGlucose-1-Phosphate* 0.3mgGlucose-1,6-Diphosphate* 20.0unitsGlucose-6-PhosphateDehydrogenase 2.5mgPMS:PhenazineMethosulfate(Lightsensitive) 2.5mgMTT:ThiazoylBlue(Lightsensitive) 2.0mgNADP:NicotinamideAdenineDinucleotidePhosphate 5.0mLreactionbuffer Stirmixtureuntildissolved.Pouronreactionplatesabout5minutes

beforecompletionofelectrophoresis. *AvailabletogetherfromSigmaChemicalCompany;-D-Glucose1-PhosphateDisodium

Salt,cat.no.G-1259

9. Incubation:15minutesat37°C,withweightontoporuntilbluebandsappear.

10.Visualization:Separateplatesandsoak5minutesin5%aceticacid.Blot, dryandreadusingtransmittedlight.

Figure 7 – Carbonic Anhydrase (CA-II)

Anode(+)

Applicationpoint


Electrophoretic patterns for two types of CA-II are demonstrated as follows: Type 1-1: Samples 1, 2, 4, 5 and 6 Type 2-1: Samples 3, 7, and 8

20 9

7. Aftertheappropriateincubationperiod,preparetheplatesforevalu-ationasdescribedinindividualprocedures(Section2).Afterdevel-opment,allplatesmustbedriedbeforereadingeitherbyairdryingorwithalightstreamofwarmforcedair.Bothplates,thesampleplateandreactionplate(negative),canberead.Inmostinstances,driedplatesmaybestoredindefinitelyforfuturereferenceifplacedinaTitanPlasticEnvelopeorsmallplasticstoragebag.

VI. Results and Discussion Atthepresenttime,thefollowingproceduresarebeingperformedrou-

tinely inSEMORegionalCrimeLaboratoryusing thecelluloseacetatemembrane(CAM)system:

Phosphoglucomutase(PGM) Erythrocyte/Vaginal/SeminalAcidPhosphatase(EAP/VAP/SAP) AdenylateKinase(AK) GroupSpecificComponent(Gc) Other procedures which are currently performed on CAM are: Glucose-6-PhosphateDehydrogenase(G-6-PD) LactateAcidDehydrogenase(LDH) CarbonicAnhydraseII(CA-II)5

Isoenzymetypingofbloodusingcelluloseacetatemembranesasasup-portmediumforelectrophoreticseparationhasproventobearelativelyin-expensiveandrapidmethodforidentifyingbloodstainsaccordingtopoly-morphicproteinsandenzymes.Usingtheseprocedures,determinationscanbeperformedusingmicrolitervolumesofspecimen(stainextract).

EsteraseD(EsD),Glyoxylase-I(GLO-I)andPeptidaseA(PepA)determi-nationsareperformedwithstarch-agarosegelduetosuperiorseparationandvisualization.Haptoglobindeterminationsareperformedutilizingagradientpolyacrylamidesystem(overnight).Ifthehaptoglobinprocedureis runat300volts, itcanbecompleted in fourhours. It ispossible foratrainedanalysttorunallsystems, includingspecies,ABOtypingandhaptoglobinineighthours.

VII. References 1. Culliford,B.J.,TheExaminationandTypingofBloodstainsintheCrime

Laboratory,LawEnforcementAssistanceAdministration,Washington,D.C.,20402,Stock#2700-0083.

2. Wraxall, Bryan G.D., Ed., Bloodstain Analysis System Procedures Manual,SerologicalResearchInstitute,Emeryville,California,1977.

3. Grunbaum,B.W.,Selvin,S.,Pace,N.,Black,D.M.,JofForensicSci,23,577,1979.

4. Grunbaum, B.W., Ed., Handbook for Individualization of Forensic Blood and Bloodstains,UniversityofCalifornia,Berkeley,California,1980.

5. Noppinger, K.E., Morrison, R.D., J of Forensic Sci, 26:1, 176-180,1981.

Lactate Dehydrogenase (LDH) 1. TankBuffer:ElectraHRBuffer(HelenaCat.No.5805),pH8.8. Trisbarbital-sodiumbarbitalbuffer.Diluteto1750mLwithdistilledwater.



4. SamplePreparationandApplication:Applytwocathodicapplicationsofdistilledwaterextractofstain.Seminalstainsmayhavetobeseriallydiluteduntilareadableresultisachieved.

5. Electrophoresis:180voltsfor30minutes;Initialamperage=8mAeach.

6. ReactionPlates:SoakplatesinPGMreactionbufferabout15minutesbeforecompletionofelectrophoresis.

7. LDHReactionMixture 5mLPGMreactionbuffer 25mgCalciumLactate 2mgNAD(NicotinamideAdenineDinucleotide) 2.5mgMTT(lightsensitive) 2.0mgPMS(lightsensititve) OruseLDVISIsoenzymeReagent,Cat.No.5909,HelenaLaboratories

8. Incubation:15minutesat37°Cwithdevelopmentweightontop.

9. Visualization:LDHpatternappearsasbluebands.Viewundertransmittedlight.

Figure 8 – Lactic Acid Dehydrogenase (LDH) Anode(+)

Applicationpoint


LDH electrophoretic patterns from various sources are illustrated as follows: Liver: Samples 1 and 2 Heart: Samples 3 and 4

Mixture of menstrual blood and seminal fluid: Samples 5, 6, 7, and 8

4. Apply thesampleto theplatebydepressingtheapplicatortipsintothesamplewells3or4timesand promptly transferring the applicator to thealigning base. Press the button down and holdfor5seconds.Makeasecondor thirdsuperim-posed application by repeating this step. Threeapplicationsof .25µLeach issufficient formostisoenzymestudies.

D. Electrophoresis of the Sample Plate

1. Quickly place the plate, cellulose acetate sidedown,intheelectrophoresistank.Placeaweight(plasticrod,glassslide,etc.)ontheplate(s)toin-surecontactwith thewicks.Cover thechambersecurely.

2. Electrophorese according to the instructions oftheappropriateprocedure.

E. Development

1. Foreachsampleplateused,areactionplatemustalsobesoakedaccordingtothesoakingmethoddescribedinStepA.Thereactionplateisusuallysoakedinthereactionbuffer.Seeindividualproce-dures,Section2.

2. Remove each plate from the reaction soaking buffer 5 minutes before completion of electrophoresis and blot gently. Place theplate, celluloseacetatesideup,ontoaglassdevelopmentslideandplaceonalevelcountertoporalevelingtable.

3. Pourapproximately2.0to5.0mLofreactionmix-tureontotheplate.Tilttheslideuntilthereactionmixturecovers theplate.Covereachplatewithasmallbox(ifthereactionmixtureislightsensi-tive)andallowtostandforfiveminutes.

4. Aftercompletionofelectrophoresis, remove thesample plate from the electrophoresis tank andblotlightlytoremoveexcessbuffer.

5. Carefullylayerthesampleplate,celluloseacetatesidedown,ontothereactionplate.Placeablotteroverthesandwichedplates.Removeexcesssub-stratefromthesandwichbydrawingtheedgeofthealigningbaseacrosstheplateseveraltimes.Blotawayexcesssubstrate.

6. PlacetheplatesbetweenmoistenedblottersandplaceonthetopofapreheatedMicro-HoodorinaHelenaIODorotherlaboratoryincubatorat37°C.Placeapreheateddevelopmentweightontopoftheblotters.

8 21

TITANPOWER SUPPLY

VOLTAGE CONTROL

II

II

II

II

IIII

II

II

II

II

+ _

Glucose-6-Phosphate Dehydrogenase (G-6-PD)

1. TankBuffer:Supre-HemeBuffer(HelenaCat.No.5802),pH8.4.Tris-EDTABoratebuffer.Diluteto1000mLwithdistilledwater.



4. SamplePreparationandApplication:Onecathodicapplicationofdistilledwaterextractofstain.

5. Electrophoresis:350voltsfor20minutes.

6. ReactionBuffer:Undilutedtankbuffer.

7. G-6-PDReactionMixture a.G-6-PDReagent,HelenaCat.No.5620or b.10.0mgGlucose-6-Phosphate 6.0mgNAD(NicotinamideAdenineDinucleotide) 7.0mgMTT(lightsensitive) 7.0mgPMS(lightsensititve) Reconstitutereagentwith3to5mLofundilutedtankbuffer.Thismakessufficient

reagentfor3to4reactionplates.

8. Incubation:20minutesat37°C.

9. Visualization:Separateplatesandsoakin7.5%trichloroaceticacid(TCA)for2to3minutes.Rinsein5%aceticacidfor5minutes.Blot,dryplatesandreadundertransmittedlight.

22 7

B. Preparation of the Electrophoresis Tank (Zip Zone Chamber)

1. Prior to using the tank, fill the inner chamber halfway withwaterandkeepfrozenuntilthetankisused.

2. Whenpreparingforelectrophoresis,pour50mLoftankbufferintothecathodechamberanda1:1dilution(25mLtankbuf-fer/25mLH2O)intheanodechamber.(Exceptwhenstatedotherwise;checkspecificprocedures.)

3. Wet two Zip Zone® Chamber Wicks in the tank buffer anddrape one over each support bridge. Be sure they makecontactwiththebufferandthattherearenoairbubblesun-derthewicks.Donotallowthewickstotouchtheiceintheinnerchambers.Coverthetankuntilused.

C. Sample Application

1. Fillthewellsofthesamplewellplatewithbloodstainextractusingasmallcapillarypipette.

2. Primetheapplicatorbydepressingthetipsintothesamplewells 3 or 4 times.Apply this loading to a piece of blotterpaper.Primingtheapplicatormakesthesecondloadingmuchmoreuniform.Donotloadtheapplicatoragainatthispoint,butproceedquicklytothenextstep.

3. Remove the wettedTitan III plate from the buffer and blot

oncefirmlybetweentwoblotters.Placetheplateinthealign-ingbase, celluloseacetatesideup,aligning the topof theplate with the black scribe line marked “cathodic applica-tion”.Theidentificationmarkshouldbealignedwithsample#1.Mostproceduresrequireacathodicapplicationwiththeexceptionofphosphoglucomutase(PGM)whichrequiresanextremecathodicapplication.

HELENA

1 2 3 4 5 6 7 8

HELENA

1 2 3 4 5 6 7 8

Buffer

Ice

Buffer

CATHODE APPLICATION

CENTER APPLICATION

HELENA LABORATORIES

Placementofplateforextremecathodicapplication


Anode(+)

Applicationpoint


Electrophoretic patterns of two types of Gc are demonstrated as follows:

Type 2-1: Samples 1, 3, 5, 6, 7 and 8 Type 2-2: Samples 2 and 4


Anode(+)

Applicationpoint


Electrophoretic patterns of three types of Gc are demonstrated as follows: Type 1-1: Samples 3, 6 and 7 Type 2-2: Samples 1 and 8 Type 2-1: Samples 2, 4 and 5

6 23

IV. Materials

TitanIIICelluloseAcetateSystemavailablefromHelenaLaboratories. Hardware Cat. No. TITAN®plusPowerSupply(110V) 1504 ZipZone®Chamber 1283 SuperZApplicatorKit 4088 DevelopmentWeight 5014 TitanPhotoVuBoxCamera 5048 Bufferizer 5093 IODIncubator,OvenDryer(110V) 5116 Micro-Hood 8009 Consumables Titan®III-LipoCelluloseAcetate(60x76mm) 3900 Electra®HRBuffer 5085 Supre-HemeBuffer 5802 Electra®B1Buffer 5016 LDVisIsoenzymeReagent 5909 HemolysateReagent 5125 Blotters 5034 ZipZone®ChamberWicks 5081 DevelopmentSlides 5008 TitanPlasticEnvelope 5052 TitanIdentificationLabels 5006 HelenaMarker 5000

V. Electrophoresis Techniques

A. Preparation of Titan III-Lipo Plates

1. ProperlycodetherequirednumberofTitanIII-Lipoplatesbymarkingontheglossy,hardsidewithamagicmarker.Itissuggestedthatthemarkbeplacedinonecorneroftheplatesothatitisalwaysalignedwithsample#1.

2. Soaktheplatesfor20minutes in the individualsystemplatebuffer.Thesoakingbufferisusuallyadilutionofthetank buffer.The plates must be soaked gradually fromthebottomup toensuresaturationwithout trappingairbubblesinthecelluloseacetate.

The plates may be soaked in the Bufferizer (availablefrom Helena Laboratories, Cat. No. 5093) or they maybesoaked ina laboratorybeakerusing theprincipleoftheBufferizer.Placetheplatesinthebeakerinaverticalpositionandallowtheplatebuffertoslowlyflowdowntheside of the beaker from a separatory funnel or directlyfrom the buffer container.Alternately, fill a beaker withbufferandslowlyandsteadilyimmersetheplates(verti-calposition)intothebuffer.

Part II. Proteins Group Specific Component (Gc)

1. TankBuffer:Tris/Glycine,pH8.4 21.8gmGlycine 4.5gmTris Dissolvein900mLwater.AdjusttopH8.4withNaOH.Q.S.toafinalvolume

of1000mLwithdistilledwater.


3. SamplePreparationandApplication:Extractbloodstainswith3dropsof6Murea(3.6gm:10mLH2O).Placeallofextractinasmallplasticcentrifugetube.Fillthetubehalfwaywithchloroform.Vortextomix.Centrifugethemixtureinamicrocentrifugeathighspeed.Thechloroformwashesdownthedebrisinthestain.

ExtractionofstainswithHemolysateReagent(HelenaCat.No.5125)willelim-inateagingbandswhichappearabovetheGcbands.ThiswillallowBCtypingtobeperformedonoldstains.*

4. Electrophoresis:500voltsfor10minutes;Initialamperage=2to4mA/plate.

5. Visualization(Immunofixation) a. Afterelectrophoresis,theplatesarecoveredwithhumanGcantiserumdi-

luted1:4withphysiologicalsaline(2mLserum+6mLsaline).Allowplatetosoakfor20minutes.

Thedilutedantiserumcanbereusedandisstableabout1monthafterdilu-tion,dependingonthenumberoftimesused.Storeintherefrigerator.

b. Removeexcessserumfromplateandsoakovernightinphysiologicalsa-line(0.85%).

c. Stainfor5minutesin0.2%PonceauS(v/vindistilledwater). d. Todestain,placeplatesinasolutionof5%aceticacid,agitatefor15min-

utes.Pouroutthissolutionandaddfreshaceticacid,agitateforanother15minutesuntil thebackgroundbecomeswhite.Platesmaybewashedovernight.Theplatesare thendried; thebandsaredarkpinkonawhitebackground.

TwosuggestedsourcesforGcantiseraare: Atlantic Antibodies In Vitro Research Sources, Inc. 10NonesuchRoad P.O.Box110 P.O.Box1032 Benson,MD21018 Scarborough,ME04074 (301)877-7110 (207)883-4154 Figures9and10onpage22illustratetheelectrophoreticpatternsofGc.

Haptoglobin (Hp) The stain extract prepared for the Gc procedure may be used for haptoglobin

determinationonpolyacrylamidegradientgel.However,beforeusingtheextract,preparea1:1mixturewith40%sucrose.

*PersonalCommunication:Willard(Bud)Stuver,Supervisor,Serology/BiologySection,Metro-DadePoliceCrimeLaboratory,Miami,FL33125.

24 5

II. Factors Influencing Results Severalvariablesinfluencetheresultsobtainedwithanelectrophoreticprocedure.These

includesamplepreparationandapplication,celluloseacetateplatepreparation,pHandionicstrengthofthebuffers,voltage,amperage,andcolordevelopmenttechniques.Thecelluloseacetateplatesmustbesoakedintheindividualsystemplatebufferpriortosam-pleapplication.Thesoakingbufferisusuallyadilutionofthetankbuffer.Thecorrectdilu-tionofplatebufferisessentialforgoodresolutionandforpreventionofhighamperageandexcessheat.Theplatemustbesoakedgraduallyfromthebottomuptoensuresaturationwithoutairbubbles.

Airbubblesinthecelluloseacetateimpedethemovementoftheelectriccurrentthroughtheplate.Oncetrappedintheplate,airbubblescannotberemovedandtheplatemustbediscarded.

ThepHofthetank,plateandreactionbuffersisextremelyimportant.AllenzymeshaveanoptimalpHforactivity.Denaturationoftheenzymesmayoccurifreagentswiththein-correctpHareused.Improperreactiontemperatureshavethesameeffect.Correctionicstrengthofthebuffersisalsoveryimportant.

Propervoltageandamperageareextremelyimportanttogoodelectrophoreticresolution.Voltageshouldremainconstant,allowingtheamperagetostartlow(about2milliamperesperplate)inordertoavoidoverheatingthesystem.Thetemperatureoftheelectrophore-sisplaterisesastheamperagerises.Ifthetemperatureexceedstheoptimalrange,theenzymewilldenature.

The amount of time required for electrophoresis varies with the enzyme system understudy.Noneofourproceduresrequiremorethanonehour.

Thetechniqueusedfordevelopingthecelluloseacetateplateaftercompletionofelectro-phoresisiscritical.Thetwobasictypesofdevelopmentarecolorimetricandfluorometric.Afterdevelopment,resultsmustberecordedandfiled.AllplatesarephotographedwithblackandwhitePolaroidfilm.

III. Sample Extraction and Storage Topreserveenzymaticactivity,anymaterialscontainingdriedbloodstainsshouldbefro-

zen.Atthetimeoftesting,cuta0.5cmx0.5cmpieceofstainedmaterialfromtheoriginalsampleandplaceinawellwith2to3dropsofsolvent.Distilledwater isroutinelyusedas thesolvent;however,Cleland’s reagentmustbeused forphosphoglucomutaseanderythrocyte acid phosphatase assays. Samples extracted with purified water may besoakedovernightinahumiditychamberat4°C.SamplesextractedinCleland’sreagent(dithioerythritol;availablefromSigma)arestablefor3to5hoursandshouldnotbestoredovernight.Theprocedure frommakingCleland’s reagent is included in thePGMproce-dure.


Isoenzymes Esterase D (EsD) and Glyoxylase I (GLO I) Thisprocedureshouldbeperformedwithintwodaysofreceivingsample.Maximumsam-

pleageis10days.After10daysthesamplemaybetoodeterioratedtoproduceareadableresult.

1. TankBuffer:HEPES,pH7.5 5.95gmHEPES0.05M(N-2hydroxyethylpiperazine-N-2ethanesulfonicacid) Dissolvein400mLdistilledwater.AdjusttopH7.5with40%NaOH.Q.S.tofinalvol-

umeof500mLwithdistilledwater.

2. GelBuffer:1:14dilutionoftankbuffer,pH7.4. Dilute10mLoftankbufferwith140mLdistilledwater.

3. Starch/AgarGel 1%Agarose,SigmaTypeV(lowEEO) 2%Hydrolyzedstarch(ConnaughtLabs,Willowdale,Ontario,Canada). AvailablefromFisher,Cat.No.S-676. Dissolve0.3gmagaroseand0.6gmstarchin30mLgelbuffer.Afterdissolving,de-gas

thesolutionpriortopouringinto8cmx10cmx2mmdeepplasticplates.Gelmustsitovernightbeforeuse.

4. SamplePreparationandApplication a. Soakdriedbloodstains for15minutes in0.1NClelend’s reagent,pH8.0.Runa

knowntype2-1asastandard(GLOandEsD). b. Eightwells(1mmx4mm,3mmapart)arecutinthegel2cmfromthecathodicend

withametaltemplate.Apply5µLofbloodineachwellusingamicropipet.

5. Electrophoresis:250voltsfor30minutes. Itisveryimportanttokeeptheelectrophoresischamberverycoolduringtherun.The

tankbuffermustbekeptcolduntilreadyforuse.Tokeepthechamberatalowtem-perature,anotherlidormetalpanisfilledwithiceandplacedontopoftheclosedelec-trophoresistank.Plateisrunfacedownonspongewicks.Amperageatstart8to9mA;attheend,about10mA.

6. ReactionBuffer a. EsD: 0.41 gm sodium acetate, anhydrous (0.05M). Dissolve in 100 mL distilled

water.AdjusttopH6.5with1%aceticacid. b. GLOI:2.42gmNaH2PO4,anhydrous(0.2M) 1.31gmNa2HPO2,anhydrous(0.072M) Dissolvein100mLdistilledwater,adjusttopH6.2withphosphoricacid.

7. ReactionMixtureandVisualization a. EsD:4mgMU-Acetate(4-methylumbelliferylacetate) 1.0mLacetone(spectralgrade) 5mLreactionbuffer DissolveMU-acetateinabout1.0mLofacetoneandimmediatelyadd10.0mLofthe

reactionbuffer.SoakintoWhatman3MMpaper4cmx10cm.Laythispaperoveraportionofthegelfromtheorigintowardanode.Leaveatroomtemperatureforabout5minutes.RemovethepaperafterincubationatroomtemperatureandreadEsDbyusingUVlight.

4 25

Section 1 General Information I. Introduction

Inthepastdecade,progressindevelopingmethodologiesfortypingbloodfactorsusingisoenzymestudieshasmadebloodstainevidenceoneof themost important investiga-tive toolsavailable to forensicscientists.Originally,allprocedures for isoenzyme typinginvolved electrophoresis on starch gels.At the present time, four major electrophoreticsupportmediaorutilized:starchgel,agarose,polyacrylamidegelandcelluloseacetate.Thispresentationisaresultoftheresearchperformedinourlaboratoriesdirectedtowardadaptationofisoenzymetypingofbloodfactorsbyelectrophoresisonsupportedcelluloseacetatemembranes(CAM).

Traditionally,theonlymethodforidentifyinghumanbloodsamplesinthecrimelaboratorywastypingbytheABOsystem.UsingtheABOsystem,themostrarebloodtypeencoun-teredisAB,whichisfoundinonly3%ofthepopulation.WhilethepercentageofpersonshavingbloodtypeABisverylow,almost50%ofthehumanpopulationhastypeO.Itisap-parentfromthesestatisticsthatasystemformoredefiniteidentificationofbloodsystemsismandatoryifbloodtypingistobeusedinacrimelaboratory.

Intheearly1970s,researchwasinitiatedtoimprovethesystemfordeterminingthetypeofhumanbloodfoundondriedbloodstains.InadditiontotheABOsystem,othersystemswereutilized.TheerythrocyteMNandRh(primarilyRhD)antigensystemswereused.Pro-teinstudiesinvolvedhemoglobinandhaptoglobin.Severalenzymesystemswereutilized;themostusefulofthesebeingphosphoglucomutase(PGM),adenylatekinase(AK),eryth-rocyteacidphosphatase(EAP),esteraseD(EsD),andadenosinedeaminase(ADA).InitialworkwasperformedbyBryanJ.CullifordoftheMetropolitanPoliceForensicLaboratoryofLondon,England.1Determiningbloodtypesusingacombinationofthesystemscited,greatlydiminishestheoccurrenceofaparticulartypeinthepopulation.Althoughelectro-phoresiswasthemethodofchoiceindeterminingtheproteinspresentinabloodstain,thetimeinvolvedbecameaprimeconcernsincestarchgel(theonlymediausedatthattime)electrophoresisproceduresoftenrequired15to30hoursforcompletion.

Inthemid1970s,workersattheUniversityofPittsburghproposedamethodformultipleenzymeseparationonasingleelectrophoresisplate.ThisworkwascompletedunderthedirectionofB.Wraxall.2Bycoveringaparticularportionofthestarchgelanddevelopingeachportionspecificallyforthedesiredenzyme,itwaspossibletoidentifyseveralenzymesystemsatthesametime.AdditionalworkbyWraxalldevelopedamultiplesystemsusingastarch/agarosegel.

Since that time, the research of Dr. B. Grunbaum and P.L. Zajac using the BeckmanMicro-zone®SystemandadaptingthestarchgelprocedurestoSartorious®celluloseace-tatemembraneshasdrasticallyreducedthetimerequired.TheTitan®IIICelluloseAcetateElectrophoresisSystemfromHelenaLaboratoriesisnowbeingusedsuccessfullybytheSoutheastMissouriRegionalCrimeLaboratory.Methods for identifyinggeneticmarkersareperformedonsupportedcelluloseacetateelectrophoresisplates.Theresultsaredis-cernableandreadilyreproducible.Studiesonalltheenzymesystemscanbecompletedinlessthaneighthours.

TheEsDbandsappearaswhitefluorescentbandsonadarkbackground.UseanEsD2-1asacontrol.AftervisualizingEsD,markthepositionofthenumber2bandoftheEsDpattern.ThegeliscoveredwithGLOIreactionmixturebeginningwiththispositionandincubatedagain.

b. GLOI:20mgreducedGlutathione 50µL40%Methylglyoxal 7.5mLGLOIreactionbuffer

SoakmixtureintoWhatmanpapercuttofitthesizeofthegelfromthesecondbandoftheEsDpatterntotheedgeoftheplate(approximately6.5cmx10cm).Thefilterpaperisplacedonthegelbeginningatthecathodeside,gentlyrollingdowntherestofthepaper,beingsurenoairbubblesremainunderthepaper.Incubate30to40minutesat37°C.

Heat0.2gmsagarose(SigmaTypeII)in20mLdistilledwateruntildissolved.Allowtheagarosetocoolto60°Candthenadd2dropsofiodinesolution.(Iodinesolutioncontains1.65gmsofKI,2.94gmsofI2dissolvedin30mLsofwater.)Mixtheaga-rose/iodinemixture.Thismixturewillbealightorangecolor.Aftertheelectrophoresisgelhasbeenincubatedasabove,theagarose/iodinemixtureisquicklypouredontothegelstartingatonecornerofthegelallowingittoflowovertheentiregelsurface.Thephenotypeappearsasbluebandsonalightyellowbackground.Theheterozy-gote(2-1)hasthreebluebandspresentandshouldbeusedasacontrol.Asecondagarose/iodinemixturemaybeappliedtothegelifthebluebandsappearweakinthefirstgel.Thisisdoneafterremovingthefirstagarose/iodinegel.

26 3

Table of Contents

Section 1 General Information I. Introduction 4

II. FactorsInfluencingResults 5

III. SampleExtractionandStorage 5

IV. Materials 5

V. ElectrophoresisTechniques 6

VI. ResultsandDiscussion 9

VII. References 9

Section 2 Typing of Dried Blood Stains With Cellulose Acetate Part I: Isoenzymes Phosphoglucomutase(PGM) 10

Erythrocyte/Vaginal/SeminalAcidPhosphatase (EAP/VAP/SAP) 12

AdenylateKinase(AK) 14

AdenosineDeaminase(ADA) 16

CarbonicAnhydraseII(CA-II)andHemoglobin 18

LactateDehydrogenase(LDH) 21

Glucose-6-PhosphateDehydrogenase(G-6-PD) 22

Part II: Proteins GroupSpecificComponent(Gc) 23

Haptoglobin 23


Isoenzymes EsteraseD(EsD)andGlyoxylase1(GLO1) 24

PeptidaseA(PepA) 27


Figure 11 – Glyoxylase-I (GLO-I)

Anode(+)

Applicationpoint


Electrophoretic patterns of three types of GLO-1: Type 1-1: Samples 5 Type 2-1: Samples 1, 3, 4, 6 and 7 Type 2-2: Samples 2 and 8

Figure 12 – Esterase D (EsD)

Anode(+)

Applicationpoint


Electrophoretic patterns of two types of EsD: Type 1-1: Samples 1, 3, 4, 5 and 6 Type 2-2: Samples 2, 7 and 8

2 27

AcknowledgementsThefollowingstudentshaveprovidedvaluableassistanceinthedevelopmentofthesemethods:Retha(Mat-thews)Edens1,AliceAbbot2,RandallWebster3,Yvonne(Moll)Matthews4andRalphWillis5.Weespeciallyap-preciateMs.DonnaPruneau6forreviewingandupdatingthismanual(February,1989).TheseproceduresareinuseincourseFS552(BloodandBodyFluids),anelectiveintheScienceDivisionatSoutheastMissouriStateUniversity,CapeGirardeau,Missouri.TheauthorswouldliketothankHelenaLaboratories,Beaumont,Texas,forsupportofthisproject.PortionsofthisworkweresupportedbytheResearchCouncil,SoutheastMissouriStateUniversity,CapeGirardeau,Missouri.WewouldalsoliketothankMs.PattyButler7fortypingtheoriginaldraftofthishandbook.

1.No.11WestAdams,Cahokia,IL. 2.St.John’sMercyHospitalClinicalLaboratory,St.Louis,MO. 3.HannibalPoliceDepartment,Hannibal,MO. 4.P.O.Box945,CollegeStation,TX. 5.CrimeLaboratory,MissouriSouthernCollege,Joplin,MO. 6.SEMORegionalCrimeLaboratory,SoutheastMissouriStateUniversity,CapeGirardeau,MO. 7.Secretary,CollegeofScienceandTechnology,SoutheastMissouriStateUniversity,CapeGirardeau,MO.

Peptidase A (Pep A)(For Negroids)

1. TankBuffer:SameasforGLO-IandEsD

2. GelBuffer:SameasforGLO-IandEsD

3. Starch/AgarGel:SameasforGLO-IandEsD

4. SamplePreparationandApplication:Soakdriedbloodstainsforabout15minutesindistilledwaterandapplytogelinthesamemannerasGLO-IandEsD.Runaknowntype2-1asastandard.

5. Electrophoresis:250voltsfor45minutes.Amperageatthestartabout10mA,attheendabout6mA.

6. ReactionBuffer(SameasPGMreactionbuffer) 3.64gmTris,pH8.0 Dissolvein500mLofdistilledwater.AdjusttopH8.0withconcentratedHCl.

7. ReactionMixture 40mgL-valyl-L-leucine 20mgL-aminoacidoxidase(crudesnakevenom) 10mgMTT(lightsensitive) 4.0mgPMS(lightsensitive) 10mLreactionbuffer

8. Visualization:Overlay0.2gmagarose(SigmaTypeII)dissolvedin10mLofreactionbuffer.

Heattheagaroseuntildissolved.Allowtheagarosetocoolto55°Cbeforeaddingthereactionmixture.This isquicklypouredover thegel. Incubate thegel for1hourat37°C.

Thephenotypesappearasbluebandsonalightyellowbackground.

Figure 13 – Peptidase A (PepA)

Anode(+)

Applicationpoint


Electrophoretic patterns of three types of Pep A: Type 1-1: Samples 1, 2, 3, 7, 8 and 9 Type 2-1: Samples 4 and 6

Sample 5 is a suspected 1 + 2-1 mix from a vaginal/seminal sample

28 1

Handbook for Rapid Blood Stain Typing Using Electrophoresis for the Crime Laboratory

Prepared by:

R.C. Briner, Ph.D.

C. R. Longwell, M.S.

SEMO Regional Crime LaboratorySoutheast Missouri State University

Cape Girardeau, MO 63701


Titan®IIIandtheSuperZApplicatorSystemarefast,easyandpracticalforforensicinvestigationofmanydifferentproteinsandenzymes.Theexam-plesinthisbookletwillgivetheinvestigativelaboratorianspecifictechniquesforsomeassaysand theequipmentandreagentsareeasilymodified forotheruses.

PleasecontacttheauthorsatSEMORegionalCrimeLaboratoryorHelenaLaboratoriesifyouneedassistanceorhaveinformationtoshare.

For more information, call Helena Laboratories.Toll free 800-231-5663.

C310/09(6)

Blood Stain Typing by Electrophoresis

An educational service of:

PO Box 752Beaumont, TX USA 77704-0752

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