We applied Y-STRs (DYS385/DYS19/YCAII) to an adhesive plaster left at a crime scene. This plaster may have included body fluids from more than one person. Firstly, we performed preliminary examinations, ABO-blood type examinations, and commonly used DNA examinations (D1S80, HLADQα, TH01, and PM) on these specimens. As a result of these examinations, we could evidence that suspect A did not contacted with the plaster, but could not confirm the presence of perspiration from suspect B. As the next step, we applied Y-STR examination to the plaster. Using this examination, we detected alleles that coincided to those of suspect B. We also concluded that the fluid from an unidentified person was vaginal fluid based on crime scene investigation. Y-STRs examination data obtained from 124 persons in Tokushima prefecture showed that 1.613% of individuals demonstrated haplotypes 10-18/ 15/19-23, which was detected from the plaster and from suspect B. Therefore, we considered that there was a high probability that the persiration detected in the plaster was that of suspect B. Based on these studies, we concluded that Y-STR examination of trace evidence was very useful to screen suspects using materials that contained body fluid from more than one person

Fujita, Yoshihiko

Kubo, Shin-ichi

Yoshida, Yasushi

Tokushima University Institutional Repository

INTRODUCTIONY-chromosome short tandem repeat (STR) poly-morphisms are male-specific and inherited from fatherto son, and are potential tools for personal identificationin sexual assaults and paternity tests between fatherand sons (1-4).Wehadstudied thedistributionof haplo-types on three Y-chromosome STRs including DYS385, DYS19 and YCAII in a Japanese population fromTokushima prefecture.In this paper, we report the identification of a suspectby DNA analysis from doubtful material containingmixed body fluids frommore than one person. In thiscase, the suspect was not identified by conventionalautosomal DNA typing, such as D1S80, HLADQA1,TH01, and PM system. Y-STR examination establishedthe personal identification of the suspect.CASE REPORTTwo suspects, A and B, conspired to break into thevictim’s house. They suddenly covered the mouth ofthe victim while she slept, blindfolded her and threat-ened her to further suppress her resistance. SuspectA tried to forcibly rape the victim. These men weresuspected of two other rape incidents, in addition tothis one, and therewere few items left at the crime scenes.In this case, an adhesive plaster was considered toCASE REPORTForensic casework of personal identification using amixtureof body fluids frommore thanonepersonbyY-STRsanalysisYasushi Yoshida1,2, Yoshihiko Fujita1,2, and Shin-ichi Kubo11Department of Forensic Medicine, Institute of Health Biosciences, The University of Tokushima GraduateSchool, Tokushima,Japan ; and 2Scientific Investigation Research Laboratory, Tokushima Prefectural PoliceHeadquarters, Tokushima, Japan.Abstract : We applied Y-STRs (DYS385/DYS19/YCAII) to an adhesive plaster left at a crimescene. This plaster may have included body fluids from more than one person. Firstly, weperformed preliminary examinations, ABO-blood type examinations, and commonly usedDNA examinations (D1S80, HLADQα, TH01, and PM) on these specimens. As a result of theseexaminations, we could evidence that suspect A did not contacted with the plaster, but couldnot confirm the presence of perspiration from suspect B. As the next step, we applied Y-STRexamination to the plaster. Using this examination, we detected alleles that coincided tothose of suspect B. We also concluded that the fluid from an unidentified person was vaginalfluid based on crime scene investigation. Y-STRs examination data obtained from 124 personsin Tokushima prefecture showed that 1.613% of individuals demonstrated haplotypes 10-18/15/19-23, which was detected from the plaster and from suspect B. Therefore, we consideredthat there was a high probability that the persiration detected in the plaster was that of suspectB. Based on these studies, we concluded that Y-STR examination of trace evidence was veryuseful to screen suspects using materials that contained body fluid from morethan one person.J. Med. Invest. 51 : 238-242, August, 2004Keywords : forensic casework, mixed body fluid, Y-STRsReceived for publication June 10, 2004 ; accepted July 16, 2004.Address correspondence and reprint requests to Shin-ichi Kubo,Department of ForensicMedicine, Institute of Health Biosciences,The University of Tokushima Graduate School, Tokushima 770-8503, Japan and Fax : +81-88-633-7084.The Journal of Medical Investigation Vol. 51 2004２３８have been left behind by suspect B.MATERIALS AND METHODS1) SamplesThe evidentiary materials were an adhesive plasterthough to have been left behind by the suspect. Bloodsamples were collected from suspects A and B, andfrom one of the victim.2) Preliminary examination for body fluids and ABO-Blood type examinationWe tested the plaster for the presence of variousbodyfluids using the following methods. LeucomalachiteGreen method was used as a screening test for blood-stain (5). For the detection of urea, p-dimethyl aminocinnam aldehyde (DAC) reagent and urease-bromothymol blue (BTB) methods were applied (6, 7). ABOBlood type antigens were detected by the absorption-inhibition method (8) andmodified absorption-elutionmethod (9) for body fluids, and by the slide methodfor each blood group (10).3) DNA-extract method and conventional autosomalDNA typingDNAwas extracted from eachmaterial by the modi-fied phenol-chloroform method (11). D1S80 typingwas carried out as described in a previous report (12)using D1S80 Primer Mix (LifeCodes, USA). HLADQA typing and PM system were carried out accordingto themanufacturer’s instructions using an AmpliTypePM＋DQA1 PCR Amplification and Typing kit (Perkin-Elmer, USA). TH01 typing was carried out accordingto the manufacturer’s instructions using a GenePrintSTR System-TH 01(Promega, USA).4) Y-STRs examinationThe primers (DYS385, DYS19 and YCAⅡ) usedfor PCR amplification were designed as shown inTable 1 (http://ystr.charite.de./). The PCR reactionsamples contained : 2 ng DNA template, 1.6 x PCRbuffer Ⅱ (Applied Biosystems), dNTP’s200µM,BSA1.6 µg/ml, AmpliTaq Gold (Applied Biosystems) 2U,MgCl2 1.5 mM. The total reaction volume was 20 µl.The PCR cycle conditions were as follows:10min at94℃(soak) 30cycles:1min at 94℃(denaturation), 2min at 54℃ (primer annealing), 2 min at 68℃(primerextension) 60 min at 68℃(extension). PCR productswere separated by electrophoresis on4%acrylamidedenaturing gel. Band patternswere visualized by silverstaining (Fig.1).RESULTS AND DISCUSSIONResult of preliminary examination for body fluidsand ABO-Blood type examination, and conventionalautosomal DNA typing Y-STRs are shown in Table 2.1) Preliminary examination for body fluidsThe plaster showed a weakly positive-reaction byDAC reagent and urease-BTB method. Generally itis known that sweat contains a slight amount ofurea (13). Therefore, it was considered that the sweat,containing urea, soaked into the bandage.Moreover, the Leucomalachite Green method didnot show any reaction, indicating that blood had notadhered to the plaster.Fig 1. D1S80 typing results obtained from adhesive plaster, thesuspect A, suspect B and the VictimLane 2 is the adhesive plaster. Lane 8 is suspectA. Lane 4 is suspectB. Lane9 is victim. Lane6and lane11are positive controls. Lane1,lane 3, lane 5, lane 7 and lane 10 are allelic ladder. White dot :main band.Table 1. Primer sequences for PCR amplification of loci inthe Y-STRSequence (f : forward, r : reverse)DYS 385f : 5’-GTGACAGAGCTAGACACCATGC-3’r : 5’-CCAATTACATAGTCCTCCTTTC-3’DYS 19f : 5’-CTACTGAGTTTCTGTTATAGT-3’r : 5’-ATGGCATGTAGTGAGGACA-3’YCA IIf : 5’-TATATTAAATAGAAGTAGTGA-3’r : 5’-TATCGATGTAATGTTATATTA-3’The Journal of Medical Investigation Vol. 51 August 2004 ２３９2) ABO-Blood type examinationsBlood type Bwas detected from theplaster.Thebloodtype of suspect A was type A, while that of suspect Bwas type B. The victim’s ABO-blood type was type O.Thus, it was considered that sweat from suspect Bhad soaked into the bandage. However, the possibilitythat other person’s fluids (BorO type) existed togetherwas left.3) Conventional autosomal DNA type examinationElectrophoresis showed four bands of D1S80 allelefrom the plaster (Fig.1). And those bands includedthe alleles of suspect B, but did not contain the allelesof suspect A nor the victim.Allele 16 and allele 32 were observed besides alleles ofsuspect B. Therefore, it was considered that someone’sbody fluid excluding suspect B and victim soakedinto the bandage.TH01 from the plaster showed 3 bands. And thosethree bands contained alleles from suspect B and avictim, but there were no alleles from suspect A.Regarding the HLADQA1type, many alleles fromtwo or more persons were observed from the plaster.Table 2. Summary for the results of preliminary examination, ABO blood and DNA typingMaterials Adhesive plaster Suspect A Suspect B The VictimBlood Leuco* -Sweat and/or urine DAC +/-Urease-BTB +/-Blood type ABO B A B ODNA typeD1S80 16, 21, 24, 32 30-30 21-24 24-30HLADQA1 1.3, 3, 4.2/4.3, (1.2) 3-4.1 1.3-3 1.3-3TH01 6, 8, 9 7-9 6-9 9-9PM LDLR BB BB BB BBGYPA AB AB BB BBHBGG BB BB BB BBD7S8 AB AB BB AAGC A,B,C AC BC BCY-STRs DYS 385 10-18 12-15 10-18 -DYS 19 15 13 15 -YCA II 19-23 19-19 19-23 -- : negative-reaction or no detection. +/- : weak positive-reaction. * : Leucomalachite GreenFig 2. TH01 typing results obtained from the adhesive plaster, suspect A, suspect B and the VictimLane 2 is the adhesive plaster. Lane 8 is suspect A. Lane 4 is suspect B. Lane 9 is the victim. Lane 6 and lane11 are positive controls. Lane 1, lane 3, lane 5, lane 7 and lane 10 are allelic ladders. Black dot : main band.Y. Yoshida et al. Personal identification from a body flulds mixture by Y-STRs２４０The bands contained the alleles of suspect B and avictim, but not those of suspect A. In GC of the PMsystem, the alleles from the plaster showedmany spotsfrom two ormore persons. In other types of PM, therewas no inconsistency in the alleles from suspects A andB or victim.One overall judgment, it was considered that plastercontained the sweat from suspect B, and body fluidfrom someone other than the victim tested. However,the existence of the suspect’s sweat had not been de-finitively proven as also shown by the research resultsof Fujita et al. (14) Furthermore, we found that similaramounts of DNAswere intermixed basedon the reportsof Budowle et al.(15), de Pancorbo et al. (16) and SusanC. et al . (17).4) Y-STRs examinationIn Fig.3, it can be seen that the alleles from the plasterwere identical to those of suspect B, but different fromthose of suspect A. There were no male alleles otherthan those of suspect B included among alleles obtainedfrom the bandage. In Y-STR, if homogeneousDNAfromtwo or more persons were present in fairly equivalentamounts and are not the same types, these allelesmustsurely be detectable (18,19).As the result of DNA typing, we found that the sweatof suspect B and fluid other than that of the victim testedhad adhered to the plaster. And we concluded that theother fluid was vaginal fluid. Vaginal fluid may some-times contain sufficient urea for cells in the containedfluid, to sink into the plaster. Proofof the fluid’s presenceusually is obtained by microscopic test and serologytest. However, when the sample involves an extremelysmall quantity, testingbecomesextremelydifficultquan-titatively. In this case investigating the unknownsampleusing Y-STR yielded significant forensic findings.Finally, we found that the material had a mixture ofmale sweat and vaginal fluid adhering to it, and it wasconsidered that Y-STR examinationwas useful for thepersonal identification of mixed body fluids from per-sons of different gender.5) Individual identificationWe studied the frequencyof threeY-STRs ina Japanesepopulation fromTokushima prefecture (datanot shown).Aler et al. (20), Tracqui et al. (21) andHonda et al. (22)reported that the potential for personal identificationusing Y-STRs is very high.Wewere also able to establishhighly personal identification in124 Japanese, as80haplotypes was observed, yielding a HD (Haplotypediversity) value of 0.97. According to our study de-scribed above, the frequency was 1.613% (2/124 in ex-amined cases). Suspect B was specifiedwith high like-lihood as one of the criminals.In summary, combination ofDNA analysis, autosomaland Y-chromosomalDNApolymorphism, demonstratedthat the body fluids that had soaked into the bandagewere from suspect B and another unidentified female.REFERENCES1. Betz A, Baessler G, Dietl G, Steil X, WeyermannG, PflugW : DYS STR analysis with epithelial cellsin a rape case. Forensic Sci Int 118 : 126-130, 20012. Corach D, Filgueira R L, MarinoM, PenacinoG,Sala A : Routine Y-STR typing in forensic case-work. Forensic Sci Int 118 : 131-135, 20013. Prinz M, Ishii A, Coleman A, BaumH J, ShalerR C : Validation and casework application of a Ychromosome specific STR multiplex. ForensicSci Int 120 : 177-188, 20014. Koyama H, IwasaM, Tsuchimochi T, Maeno Y,Isobe I,Matsumoto T, NagaoM : Utility of Y-STRhaplotype andmtDNA sequence in personal iden-tification of human remains. Am J Forensic MedFig 3. Y-STR (DYS385/DYS19/YCAII) typing results obtainedfrom adhesive plaster, suspect A, suspect B and the VictimLane 2 is the adhesive plaster [10-18/15/19-23]. Lane 3 is suspectA [12-15/13/19-19]. Lane 4 is suspect B [10-18/15/19-23]. Lane 5is the victim. Lane 1 and lane 6 are molecular weight standards.Black dot : main band.The Journal of Medical Investigation Vol. 51 August 2004 ２４１Pathol 23 : 181-185, 20025. Hunt A C, Corby C, DoddBE : The identificationof human stains-a critical survey. Journal of Fo-rensic Medicine 7 : 112-130, 19606. Cook J W : Test paper of urease and acid-baseindicator for detection of urea. J AssocOff AgricChem 31 : 797-799, 19487. Rhodes E F, Thornton J I : DMAC test for urinestains. J. Police Sci. Admin. 4 : 88-89, 19768. Gaensslen R E, ed. : BLOODGROUPS, Nationalinstitute of Justice, Sourcebook in forensic serology,immunology, and biochemistry.Washington, 1983,pp.285-2869. 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Gaensslen R E, ed. : Identification of urine, Na-tional institute of Justice, Sourcebook in forensicserology, immunology, and biochemistry.Wash-ington, 1983, pp.191-19414. Fujita Y, Tokunaga I, Kubo S : Forensic Identifi-cation of a vaginal fluid and salivamixture throughDNA Analysis. ACTA Crim Japon 69 : 48-52, 200315. Budowle B, Lindsey J A, DeCou J A, Koons BW,Giusti AM, ComeyCT : Validation and populationstudies of the loci LDLR, GYPA, HBGG, D7S8,and Gc (PM loci), and HLA-DQ alpha using amultiplex amplification and typing procedure.J Forensic Sci 40 : 45-54, 199516. de PancorboMM,Castro A, Fernandez-FernandezI, Garcia-Orad : A Population genetics and foren-sic applications using multiplex PCR (CSF1PO,TPOX, and TH 01)loci in the Basque Country.J Forensic Sci 43 : 45-54, 199817. Susan C, Rebecca R : Validation of the AmpliFLPTMD1S80 PCR amplification kit for forensic case-work analysis according toTWGDAMGuidelines.J Forensic Sci 40 : 424-434, 199518. Zehner R, Bohrer U :DYS19and amelogenin inartifical blood stains with defined amounts ofmale and female cell. Int J Legal Med 111 : 340-342, 199819. PrinzM, Boll K, BaumH, Shaler B :Multiplexingof Y chromosome specific STRs and performancefor mixed samples. Forensic Sci Int 85:209-218,199720. Aler M, Salas A, Murcia E, Gisbert-GrifoM, Car-racedo A : Population study of eight novel Y-chromosome STRs (DYS 460, DYS 461, GATA-A10, GATA-C4, GATA-H4, DYS434, DYS437, DYS439) in a southeast Iberian population : lookingfor highly informative Y-chromosome haploptypes.Int J Legal Med 117 : 127-131, 200321. Keyser-Tracqui C, Ricaut F X, Blandin P, LudesB : French allel frequencies and haplotypes of nineY-specific STRs. J Forensic Sci 48 : 242-244, 200322. Honda K, Tun Z, YonngD, Terao T: Examinationof Y-STR mutations in sex chromosomal abnor-mality in forensic cases. Forensic Sci Int 118 :136-140, 2001Y. Yoshida et al. Personal identification from a body flulds mixture by Y-STRs２４２

Forensic casework of personal identification using a mixture of body fluids from more than one person by Y-STRs analysis

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Forensic casework of personal identification using a mixture of body fluids from more than one person by Y-STRs analysis

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