ImmunologyInForensicScience

Dr.J.R.Gaur

Introduction

Thebasictaskoftheforensicserologistandimmunologististoanswer
certainspecificquestionsrelatedtotheexaminationofblood,otherbody
fluidsandtheirstains.Sometimes,othertissuematerialslikebones,hair,
skinandfleshetc.arealsorequiredtobeexaminedfromforensicaspects.
Themajorquestions,whichhavetobeansweredareusualy:

 Isthestainofbloodorofaspecificbodyfluidlikesaliva,semenor
vaginalsecretionetc.?
 Ifitisofblood,semenorsalivastain,isitofahumanbeing.?
 Ifitisofhumanorigin,towhichgrouportypeitbelongs.?
 Isitpossibletoobtainfurtherinformationtowardsindividualizationof
stainsorbiologicalmaterialssentforexamination?

Suchquestionsaforensicserologisthastoanswerafterforensic, Chemical,
Biological, Serological and immunological laboratory
investigationsineverycaseofmurder,assault,rape,dacoitorhitandrun
accidentetc.,wheretherehasbeenbloodshedorsomeexhibitsstained
withbloodorbodyfluidshavebeenrecoveredbythepoliceduring
investigationofdifferentcasesandaresenttoaforensiclaboratoryfor
examinations.

Sometimes,the evidence ofbody fluid’s typing is an excelent

coroborativeevidencetolinkorde-linktheaccusedandtheweaponof
offencewiththecrime.Insomecases,wheretherearenoeye-witnesses,
thebloodgroupingorbodyfluidtypingevidencemaybeconcluding
physicalevidenceinfavourof,oragainsttheaccused.

Theaforesaidfourquestionscanbeansweredbytheforensicserologist
afterperformingchemical,microscopic,immunological,enzymeand
serumproteintypingandDNAprofilingtests.However,afterthediscovery
ofABObloodgroupsystem byLandSteiner,K.(1901),therehasbeen
almostaninvasioninthetwentiethcenturyforintroducingimmunological
methodsinForensicScience.Thesemethodsarestilofutmostuseinthe
laboratories devoid ofDNA typing facilities orin routine tests.
Immunologicalmethodsareprevalentinforensicscienceeventodayas
thesetestsareverysensitiveandspecific.Abriefaccountofsome
immunologicalmethods used in Forensic Science is given in the
succeedingparagraphsofthischapter.

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WhatisImmunology?

Immunologyisthestudyofantigenandantibodyreactionsandthe
immuneresponseproducedbytheantigensinalivingbeing.Antigensare
thesubstances,whichilicitanimmuneresponseinthehost.Antigenmay
beofseveraltypes,solubleandparticulateproteins,viruses,sub-celular
particulatesandentirecomplexcelssuchastumorcelsandbacteria,
whichareforeigntothehost.

SpeciesofOrigintest

Aftertheidentificationofbiologicalstainsandothertissuematerial,a
forensicscientisthastofindoutspeciesoforiginofthesame,whichcan
bedonebythevariousmethodsthroughimmunologicalantigenand
antibodyreactions.Thestainortissuematerialinthetestprovide
antigenicmaterialforthepurposeandspeciesspecificantibodiesraised
areusedasantiserainthesetests.Thetestcanbeperformedasringtest
intheprecipitintubesorbyimmunodiffusioninAgarorAgarosegel.
Furtherthistestcanbecariedoutbycross-overelectrophoresis,immuno
electrophoresisandrocketimmunoelectrophoresisetc.Theappearance
ofmilkybandsandatthemeetingjunctionofantigenandantibodydueto
theformationofantigenandantibodycomplexesindicativeofpositive
reaction.In1946Qudindevelopedthesingletubediffusionmethodandin
1949Ouchterlonyprescribedthemethodofantigenantibodyreactionsfor
thedetectionoforiginofspeciesingels.Variousmethodsweredevised
bythescientistsinthesucceedingyears.

Raisingofspecificspecies

Strictimmunizationscheduleisamustforgoodtitreofantisera.The
amountofantigen,typeofanimalandtherouteofantigenadministration
playavitalroleintheimmuneresponse.Minuteamountsofantigenis
desirableforimmunization.Thelargerdosesofantigeninducetolerance
andhencearenotadvisable.Rabbitistheconvenientandidealanimalfor the
experimentalimmunization.Best route of administration for
particulateantigensisperitonealand forthesolubleantigensthe
intradermalorintramuscularrouteisrecommended.

Haptenconjugationtocarriermacromolecules

Haptensarelowweightmolecules,whichcannotproduceanimmune
response.Thesesmalermoleculeshavetobeconjugatedtoacarried
molecule,usualyaproteinlikeBovineSerum Albuminbeforetheyare
usedasimmunogensfortheproductionofantisera.Severalcompounds
inforensicinvestigations,whichfalinthecategoryofhaptens.

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Immunobloting

Oneofthetechniqueslargelyusedtodetectantigen-antibodyreactionsis
“Immunoblotingtechnique”.Thisinvolvedimmobilizationoftheantigen
onamatrixsuchasnitrocelulose(NC)paperandprobingwithan
antibody.Immunoblotingenhancesthedetectionofantigensbecause, the
antigensaftertheyare transferred from the gelinto the NC
membranes,theyarereadilyavailablefortheantibodyprobesfortheir
detection.Antigens,aftertheirtransfercanbestoredforlongertimeat –
0
20 Cforfutureuse.Thevisualizationofantigenandantibodycomplex
isaccomplishedbyusingasecond-antibodyspecifictothefirstantibody.
Thesecondantibodyisradioactivelylabeledorcoupledtoenzymelike horse-
radish peroxidase.Thelaterkind usesa colour-reaction for
visualization.IntheImmunoblotingtechniquetheconditionsareprovided
suchthatonlythespecificexperimentalconditionsaresuchthatonly
interactionsofhighspecificityareretainedbeforevisualization,Kashyap (1989).

MicrobeadBasedELISAforthequantificationofL-fetoprotein
(AFT)

In1956BergstranddiscoveredL-fetoprotein(AFP)forthefirsttimein
humanfoetalserum.AFPisasinglepolypeptidewithamolecularweight
ofapproximately 70,000 da.The physicochemicalproperties and
aminoacidcompositionofAFParesimilartothoseofalbumin.Itis
secretedintothefoetalserumandreachesapeaklevelatabout13weeks
ofgestationperiodandgradualydeclinesthereafter.Fromforensicpoint
ofviewAFPisusefulfordiscriminatingthefoetalbloodstainsfromthe
adultbloodstains.ItcanbedonewithmicrobeadbasedELISAtechnique.

BloodGroups

KarlLandsteineronthebasisofhisobservationsonbloodclassified
humanbloodintofourgroupsA,B,AB,andOin1901anddiscoveredABO
bloodgroupsystemwhichhasbeenwidelyinuseforbloodtransfusions,
populationgeneticsstudiesandForensicAnalysisthroughouttheworld.

SincethediscoveryofABObloodgroupsystembyKarlLandsteiner,the
knowledgeinForensicSerologyhasexpandedtremendously.Today,more
than160antigens,150serum proteinsand250celularenzymeshave
beenfoundinhumanblood.Threeclassesofbloodconstituentshave
beenchosenbyserologistsfortheanalysisofbloodsamples.

1.TheGroupspecificantigens.

2.TheCelularenzymesandproteins.

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 3.Theserumenzymesandproteins.

ThebasicABO bloodgroupsystem ispolymorphicsystem inwhich

severalotherrareformsofA,B&Hantigenshavebeenaddeduplateron
andareverymuchusefulinforensicsciencesfortheestablishmentof
identityofaperson.

MNSystem

Itisatwoalelesystem discoveredbyLandsteinerandLevinein1927.
M N
TheycaledtheantigensofthissystemtobeMandNandalelesasL andL
.LateronlikeABOseveralsubgroupsofMandNwerediscovered.
Amongtheseisoneconsistingofpersonswhohavetherare,weak
N2
antigenN2,whichdependsonthepresenceofthealeleL anotherof
g
thosewhohavetheantigenM ,transmitedbytheveryrarealele
mg g
designatedasL .TheantigenM isremarkableinthatitdoesnotreact
Mg M Mg
witheitherantibodyanti–Moranti–N.OnlyheterozygousL L andL
N
L individualshavebeenfoundsofar.AhomozygousL Mg LMgperson –
whomustbeexcessivelyrare-testedwithanti–M andanti–Nsera
wouldbeneitherMnorNnorMN.DistributionofMNtypesinEuropeans
isM=28%,N=22%andMN=50%.

TheSsAntigens

In1947,twentyyearsafterthediscoveryofM andNantigens,anew
antigens,S,wasfound.Lateronrecessiveantigen‘s’wasdiscoveredand
thegenotypes,SS,Ssandsswerealsorecognised.TheseantigensSand
swerefoundassociatedwithalMNtypes.PercentageofSstypesin
EuropeansisSS=11%,Ss=44%andss=45%.

LweisTypes

Mourantin1946foundthatanunknownantibodybelievedtobenaturaly
occuringwasfoundinthebloodoftwodifferentpersons.Thisantibody
recognisedanantigenpresentinabout22%oftheEnglishpopulation.The system
wascaledasLewisafteroneofthetwoindividualsinwhichit
wasfound.LateronLepositivetobesecretorsofABHsubstancesin
saliva.CommonphenotypesofLe(a+b-)werefoundtooccureinmostof
theworldpopulations.TherarephenotypeLe(a+b+)isfoundininfants
butonlyuptotheageofoneyearorso,itishoweververyrareinadults.Le (a-
b-)veryuncommoninIndianpopulationsisashighas46%inWest
x
AfricanNegros.Le antibodyisfoundtoreactwithLe(a-b-)blood
c d
samples.TwomoreantigensLe andLe whichareveryrarehavealso
beenidentifiedinsomehumanpopulations.

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RhSystem

LevineandStetson(1939)haddescribedanatypicalantibodywhichlater
wasshowntobeantiRhinspecificity.TherealimportanceofRhfactor
washoweverrealizedin1940whenWeinerandPetersreportedevenafter
ABOcompatibilityintransfusionsseraofsomepersonsshowedstil
crossreactivity.Levineandhisco-workersin1941publishedseriesof
paperswhentheyfoundthatRhnegativemothergotimmunizedfroma
Rh+vefoetuswhichgaverisetoerythroblastosisfoetalisorHDNB.

TheeightmainRhhaplotypeshadbeenidentified.
-
FisherNotation WeinerNotation Frequency in
English
Populations.
-
CDe R1 0.421
cDE R2 0.141
cDe Ro 0.026
CDE Rz 0.002
Cde r‘ 0.010
cdE r“ 0.012
CdE ry Verylow
cde r 0.389

w x v u u
Rhsystem isfurthercomplicatedbyC C C C g,vandD etc.genes.
WhicharequiterarebutDuiscommonthantheotherandisaNegroid trait.

TheKelsystem

Thisantibodywasfoundintheserumofamotherwhoseinfantsuffered from
thehaemolyticdiseaseofthenewborn(HDNB).Kantigenwas
foundtooccurin10%oftheBritishpopulationanditwaspostulatedthat
thesystemwasgovernedbyapairofalelicgenescaledKandkwhich
controledtheproductionofcorespondingantigensKandk.Thegroups being:

Phenotype Genotype
KelPositive KK=0.2%

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 Kk=10.0%
KelNegative kk=90.0%
Lateron‘k’genewascaledtobecelanofactorandanotherraretypein
— — 0 0
thissystem,i.e.K k orK k wasdiscovered.Tiltoday21Kelrelated
antigenshavebeendescribed,butalhavenotbeenfoundtobeKel aleles.

TheDuffySystem

ItwasdiscoveredbyCutbushandChanarinin1950(BoormanandDodd,
1957).
Phenotypeasdefined Genotype Frequency%
a b
byAntiFyandFy
Fy(a+b-) a a 17
FyFy
Fy(a+b+) a b 49
FyFy
Fy(a-b+) b b 34
FyFy
3 4 5
Lateron,threeantigensFy Fy andFy werediscoveredandaddedtothis
systemwhichwererareinnature.

TheKiddbloodgroupsystem

TheKiddbloodgroupsystemwasdiscoveredin1951,theantibodybeing
foundintheserumofwoman,Mrs.Kidd,whosesixthchildsufferedfrom
haemolyticdiseaseofthenewborn.Theroleoftheantibodyinconnection
withthisdiseasecouldnotbeassessedbecausethenaterualserumalso
containedAnti-Kel.TheKiddantibodyhasbeenfoundtobestimulated
eitherbytransfusionorbypregnancyorboth.Theantibodywascaled anti-
Jklateranit-Jkwasdiscovered

Phenotype Genotype Approx.Occurrence

Jk(a+b-) a a 26%
Jk Jk
Jk(a+b+) a b 50%
Jk Jk
Jk(a-b+) b b 24%
Jk Jk
Thissystemhasnotfurtherbeenexpandedsofar.

LutheranSystem

In1946itwasfoundthatanunknownantibodyinthebloodofapatient

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whohadreceivedmanybloodtransfusions.Theantibodywasimmune,
b
theLu wasdiscoveredbyCutbushandChanarinin1956in“Originof
Man”(BuetnerandJanusch,1966).Noneoftheantibodiesofthissystem
hadanyclinicalsignificance.

Phenotypes Genotype Grequency%

a b
Lu(a+b+) Lu Lu 8%
a a
Lu(a+b-) Lu Lu 0.2%
b b
Lu(a-b+) Lu Lu 92%
3, 4 5
Lu Lu andLu antibodieshavebeendiscoveredlaterinthissystem.

PSystem

System discoveredin1927,howeverin1949anti-Pwasdiscoveredby
0
makingitactiveat4 C.

Phenotype Europeanfrequency
P1 79%
P2 21%
Pk1 Veryrare
Pk2 Veryrare
p Veryrare

TheIbloodGroupSystem

In1956onenewantibodywasfoundintheserumofapatientsuffering from
haemolyticanemiaofthecoldantibodytype,itwascaled-I.Of 22000 donors
tested,onlyfive were negative and were therefore designatedas‘i’fals.Later,I1
wasfoundonlyinwhitesandi2inNegros.
FurtherseveralotherbloodgroupsystemswereaddedinSerologyviz.SID,
Wright(Wr),Dombrock(DO),Diego(Di)andColtonetc.Buttheantigens
arenotverystableinstainsandthusareofforensicsignificanceonlyin
paternitydisputesifthecorespondingantiseraareavailable.Detectionof
theseantigensinstainsisnotpossibleastheantigensbeingweak.

TheHLASystem

HighlypolymorphicsystemisofutmosthelpinForensicScienceforthe
typingofbloodsamplesincrimecasesandpaternitydisputes.Though,
therehasbeenproblem ofcrossreactivityinstain’stesting.TheHLA

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antigensatA&Blocushavebeentriedfortestingvariousstains.

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ApplicationofrarebloodgroupsinForensicScience

Rareantigenswheneverdetectedinbloodorbloodstainsareofutmost
importanceandevidentialvalueintheindividualizationofbloodandin
paternitydisputes.TheHLAantigensmakeHLAsystemsverymuch
polymorphicandhelpedalotinorgantransplantsandalsosolving
paternitydisputes.However,theprobabilityofmatchingonthebasisofal
bloodgroupsystemsSerumproteins&enzymesneverreachesbeyond94
to95%.Buttherarebloodgroupantigenshavealwaysbeenofutmost
value,whenever,foundasitbringstheprobabilitynearing100%.

LimitationsofalrarebloodgroupsinForensicanalysis.

AlrarebloodgroupssystemshadnotbeenofForensicutilitywhenthe
testshavetobecarriedfromthebloodstainsandotherbodyfluidstains.
Becausetheputreficationathightemperatureandhumidityandwiththe
elapseoftimeinreachingtotheexpertsforexaminationintheLaboratory.
Genefrequencyofsomerareantigensandtheredetectibilityperiodsafter
preserving blood stainsatroom temperatureand deep frozenare
presentedthroughthetransparenceswhichindicatethatthemaximum
detectableperiodoftheMN antigenstobesixmonthsandLewis
antigensuptothreedaysonly.Andotherareantigenswhichare
sometimeslessresistanttothehightemperatureandhumiditycannotbe
detectedfromthestains.

IdentificationofSpeciesofOriginandgroupingtestsin
Forensicanalysis.

Scientistalovertheworldhaveworkedextensivelyfordevisingvarious
th
methodsofanalysisandmodifyingthemextensivelyinthe20 Century.
ThereadersarereferedtotheimportantcompilationslikeBiology Methods
Manual(1978)ofMetropolitan Police,Forensic Science
Laboratory,LondonandtheworksofChowdhuri,S.(1979),Kashyap,V.K.
(1989)andGaur,J.R.(1989)inIndia,whohavepresentedimportant
methodologyanddataonthesubject.ItmayalsobementionedthatGaur,
J.R.andBhala,V.(1993)presented“Aserologicalprofileofthepeopleof
Haryana(India)”.Thebloodgroupgenotypeandphenotypefrequency
datacontainedinthisstudygivesrecentdataofbloodgroupsinthatpart
ofIndiaandcanbeusedforforensicpurposesintheIndiancontest.Gaur, J.R.
(1989)alsostudiedandpresenteddataonthedetectibilitystudiesof
variousbloodgroupantigensintheenvironmentalconditionsofHaryana
andincontroledlaboratoryconditions.

Thus,immunologicalmethodsareofutmostimportanceinForensic
Serology.Theyhaveplayedasignificantroleincrimeinvestigationand

9
theadministrationofJusticeinthepastandpresentandshalcontinueto
dosoinfuture.

10
References:-

1.Bergstrand,C.G.,(1956),Demonstrationofnewproteinfractioninserum
fromhumanfetus,ScandJ.Clin&Lab.Invest.,8-174.
2.BiologyMethodsManual,(1978),MetropolitanPoliceForensicScience
Laboratory109,LambethRoad,LondonSE17LP,England.
3.Boorman,K.E.andB.E.Dood,(1957),Bloodgroupserology.,Litle,Brown, Boston.

4.Buetner,J.andJanusch,(1969),Originsofman,WileyEasternPrivate
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5.Chowdhuri.,S.,(1979),Examinationofbiologicalstains,BPR&D,ND,p.p.1-
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bloodonageing,Ph.D.thesis,PunjabUniversity,Chandigarh(India),p.p.1-
191(Unpublished)
7.Gaur,J.R.andBhala,V.(1993),Aserologicalprofileofthepeopleof
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Publications,NewDelhi,p.p.70-83.
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differentiatingindividual,495.
11.Mourant.,A.E.,(1954),Thedistributionofhumanbloodgroups,BlackWel,
Oxford,London.
12.Ouchterlony,(1949),“Antigenantibodyreactionsingels”,ActaPathol.
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th
14.Race,R.R.,andSanger(1975),Bloodgroupsinman,6 ed.(Oxford:Black
WelScientificPublishers.

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