Skip to main content

Advertisement

Log in

Comprehensive examination of conventional and innovative body fluid identification approaches and DNA profiling of laundered blood- and saliva-stained pieces of cloths

  • Original Article
  • Published:
International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

Body fluids like blood and saliva are commonly encountered during investigations of high volume crimes like homicides. The identification of the cellular origin and the composition of the trace can link suspects or victims to a certain crime scene and provide a probative value for criminal investigations. To erase all traces from the crime scene, perpetrators often wash away their traces. Characteristically, items that show exposed stains like blood are commonly cleaned or laundered to free them from potential visible leftovers. Mostly, investigators do not delegate the DNA analysis of laundered items. However, some studies have already revealed that items can still be used for DNA analysis even after they have been laundered. Nonetheless, a systematical evaluation of laundered blood and saliva traces that provides a comparison of different established and newly developed methods for body fluid identification (BFI) is still missing. Herein, we present the results of a comprehensive study of laundered blood- and saliva-stained pieces of cloths that were applied to a broad range of methods for BFI including conventional approaches as well as molecular mRNA profiling. The study included the evaluation of cellular origin as well as DNA profiling of blood- and saliva-stained (synthetic fiber and cotton) pieces of cloths, which have been washed at various washing temperatures for one or multiple times. Our experiments demonstrate that, while STR profiling seems to be sufficiently sensitive for the individualization of laundered items, there is a lack of approaches for BFI with the same sensitivity and specificity allowing to characterize the cellular origin of challenging, particularly laundered, blood and saliva samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Bond JW, Hammond C (2008) The value of DNA material recovered from crime scenes. J Forensic Sci 53:797–801. https://doi.org/10.1111/j.1556-4029.2008.00746.x

    PubMed  Google Scholar 

  2. Virkler K, Lednev IK (2009) Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 188:1–17. https://doi.org/10.1016/j.forsciint.2009.02.013

    CAS  PubMed  Google Scholar 

  3. Harbison SA, Fleming RI (2016) Forensic body fluid identification: state of the art. Res Reports Forensic Med Sci 6:11–23. https://doi.org/10.2147/RRFMS.S57994

    Google Scholar 

  4. Muro CK, Doty KC, de Souza FL, Lednev IK (2016) Forensic body fluid identification and differentiation by Raman spectroscopy. Forensic Chem 1:31–38. https://doi.org/10.1016/j.forc.2016.06.003

    CAS  Google Scholar 

  5. Li R (2015) Forensic serology. CRC Press, Boca Raton

    Google Scholar 

  6. Cox M (1991) A study of the sensitivity and specificity of four presumptive tests for blood. J Forensic Sci:1503–1511

  7. Schweers BA, Old J, Boonlayangoor PW, Reich KA (2008) Developmental validation of a novel lateral flow strip test for rapid identification of human blood (rapid stain identification blood). Forensic Sci Int Genet 2:243–247. https://doi.org/10.1016/j.fsigen.2007.12.006

    PubMed  Google Scholar 

  8. Bauer M, Patzelt D (2003) Protamine RNA as molecular marker for spermatozoa in semen stains. Int J Legal Med 117:175–179. https://doi.org/10.1007/s00414-002-0347-2

    CAS  PubMed  Google Scholar 

  9. Juusola J, Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 152:1–12. https://doi.org/10.1016/j.forsciint.2005.02.020

    CAS  PubMed  Google Scholar 

  10. Haas C, Klesser B, Maake C, Bär W, Kratzer A (2009) mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet 3:80–88. https://doi.org/10.1016/j.fsigen.2008.11.003

    CAS  PubMed  Google Scholar 

  11. Hanson EK, Lubenow H, Ballantyne J (2009) Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 387:303–314. https://doi.org/10.1016/j.ab.2009.01.037

    CAS  PubMed  Google Scholar 

  12. Courts C, Madea B (2011) Specific microRNA signatures for the detection of saliva and blood in forensic bodyfluid identification. J Forensic Sci 56:1464–1470. https://doi.org/10.1111/j.1556-4029.2011.01894.x

    CAS  PubMed  Google Scholar 

  13. Wang Z, Luo H, Pan X, Liao M, Hou Y (2012) A model for data analysis of microRNA expression in forensic body fluid identification. Forensic Sci Int Genet 6:419–423. https://doi.org/10.1016/j.fsigen.2011.08.008

    CAS  PubMed  Google Scholar 

  14. Wang Z, Zhang J, Luo H, Ye Y, Yan J, Hou Y (2013) Screening and confirmation of microRNA markers for forensic body fluid identification. Forensic Sci Int Genet 7:116–123. https://doi.org/10.1016/j.fsigen.2012.07.006

    CAS  PubMed  Google Scholar 

  15. Zubakov D, Boersma AWM, Choi Y, van Kuijk PF, Wiemer EA, Kayser M (2010) microRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med 124:217–226. https://doi.org/10.1007/s00414-009-0402-3

    PubMed  PubMed Central  Google Scholar 

  16. Park JL, Park AM, Kwon OH, Lee HC, Kim JY, Seok HH, Lee WS, Lee SH, Kim YS, Woo KM, Kim SY (2014) Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification. Electrophoresis 35:362–3068. https://doi.org/10.1002/elps.201400075

    Google Scholar 

  17. Frumkin A, Wasserstrom A, Budowle B, Davidson A (2011) DNA methylation-based forensic tissue identification. Forensic Sci Int Genet 5:517–524. https://doi.org/10.1016/j.fsigen.2010.12.001

    CAS  PubMed  Google Scholar 

  18. Lee HY, Jung SE, Lee EH, Yang WI, Shin KJ (2016) DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood. Forensic Sci Int Genet 24:75–82. https://doi.org/10.1016/j.fsigen.2016.06.007

    CAS  PubMed  Google Scholar 

  19. Poon H, Elliott J, Modler J, Frégeau C (2009) The use of Hemastix® and the subsequent lack of DNA recovery using the Promega DNA IQTM system. J Forensic Sci 54:1278–1286. https://doi.org/10.1111/j.1556-4029.2009.01173.x

    CAS  PubMed  Google Scholar 

  20. Miranda GE, Prado FB, Delwing F, Darunge E Jr (2014) Analysis of the fluorescence of body fluids on different surfaces and times. Sci Justice 54:427–431. https://doi.org/10.1016/j.scijus.2014.10.002

    PubMed  Google Scholar 

  21. Nelson DG, Santucci KA (2002) An alternate light source to detect semen. Acad Emerg Med 9:1045–1048

    PubMed  Google Scholar 

  22. Vandenberg N, Orschoot RAH (2006) The use of polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests. J Forensic Sci 51:361–370. https://doi.org/10.1111/j.1556-4029.2006.00065.x

    CAS  PubMed  Google Scholar 

  23. Lindenbergh A, de Pagter M, Ramdayal G, Visser M, Zubakov D, Kayser M, Sijen T (2012) A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces. Forensic Sci Int Genet 6:565–577. https://doi.org/10.1016/j.fsigen.2012.01.009

    CAS  PubMed  Google Scholar 

  24. Lindenbergh A, Maaskant P, Sijen T (2013) Implementation of RNA profiling in forensic casework. Forensic Sci Int Genet 7:159–166. https://doi.org/10.1016/j.fsigen.2012.09.003

    CAS  PubMed  Google Scholar 

  25. van den Berge M, Bhoelai B, Harteveld J, Matai A, Sijen T (2016) Advancing forensic RNA typing: on non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling. Forensic Sci Int Genet 20:19–129. https://doi.org/10.1016/j.fsigen.2015.10.011

    Google Scholar 

  26. Andrews C, Coquoz R (1994) PCR DNA typing of washed stains. In: Bär W, Fiori A, Rossi U (eds) Advances in forensic haemogenetics 5. Springer Verlag, Heidelberg, pp 343–345. https://doi.org/10.1007/978-3-642-78782-9_90

    Chapter  Google Scholar 

  27. Edler C, Gehl A, Kohwagner J, Walther M, Krebs O, Augustin C, Klein A (2017) Blood trace evidence on washed textiles—a systematic approach. Int J Legal Med 131:1179–1189. https://doi.org/10.1007/s00414-017-1549-y

    PubMed  Google Scholar 

  28. Kamphausen T, Fandel SB, Gutmann JS, Bajanowski T, Poetsch M (2015) Everything clean? Transfer of DNA traces between textiles in the washtub. Int J Legal Med 129:709–714. https://doi.org/10.1007/s00414-015-1203-5

    PubMed  Google Scholar 

  29. Radacher M, Dunkelmann B, Höckner G, Neuhuber F, Pölzgutter E, Breksler E, Baderer D, Steinletzberger N (2011) Luminol im Vergleich mit Fluorescein und Blue Star, Blue Star Forensic Magnum im Vergleich mit Lumiscene. Kriminalistik 3:180–184

    Google Scholar 

  30. Adair TW, Shaw RL (2005) Enhancement of bloodstains on washed clothing using luminol and LVC reagents. I.A.B.P.A, News

    Google Scholar 

  31. Horjan I, Barbaric L, Mrsic G (2016) Applicability of three commercially available kits for forensic identification of blood stains. J Forensic Leg Med 38:101–105. https://doi.org/10.1016/j.jflm.2015.11.021

    PubMed  Google Scholar 

  32. Mushtaq S, Rasool N, Firiyal S (2016) Detection of dry bloodstains on different fabrics after washing with commercially available detergents. Aust J Forensic Sci 48:87–94. https://doi.org/10.1080/00450618.2015.1029971

    Google Scholar 

  33. Tobe SS, Watson N, Daeid NN (2007) Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA. J Forensic Sci 52:102–109. https://doi.org/10.1111/j.1556-4029.2006.00324.x

    CAS  PubMed  Google Scholar 

  34. Zubakov D, Hanekamp E, Kokshoorn M, van Ijcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med 122:135–142. https://doi.org/10.1007/s00414-007-0182-6

    PubMed  Google Scholar 

  35. An JH, Shin KJ, Yang WI, Lee HY (2012) Body fluid identification in forensics. BMB Rep 45:545–553

    CAS  PubMed  Google Scholar 

  36. Pang BCM, Cheung BKK (2008) Applicability of two commercially available kits for forensic identification of saliva stains. J Forensic Sci 53:1117–1122. https://doi.org/10.1111/j.1556-4029.2008.00814.x

    PubMed  Google Scholar 

  37. Haas C, Hanson E, Kratzer A, Bär W, Ballantyne J (2011) Selection of highly specific and sensitive mRNA biomarkers for the identification of blood. Forensic Sci Int Genet 5:449–458. https://doi.org/10.1016/j.fsigen.2010.09.006

    CAS  PubMed  Google Scholar 

  38. Haas C, Hanson E, Anjos MJ, Banemann R, Berti A, Borges E (2013) RNA/DNA co-analysis from human saliva and semen stains—results of a third collaborative EDNAP exercise. Forensic Sci Int Genet 7:230–239. https://doi.org/10.1016/j.fsigen.2012.10.011

    CAS  PubMed  Google Scholar 

  39. Kohlmeier F, Schneider PM (2012) Successful mRNA profiling of 23 years old blood stains. Forensic Sci Int Genet 6:274–276. https://doi.org/10.1016/j.fsigen.2011.04.007

    CAS  PubMed  Google Scholar 

  40. Raj PA, Edgerton M, Levine MJ (1990) Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265:3898–3905

    CAS  PubMed  Google Scholar 

  41. Sabatini LM, Carlock LR, Johnson GW, Azen EA (1987) cDNA cloning and chromosomal localization (4q1 1-13) of a gene for statherin, a regulator of calcium in saliva. Am J Hum Genet 41:1048–1060

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Juusola J, Ballantyne J (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int 135:85–96

    CAS  PubMed  Google Scholar 

  43. Sakurada K, Ikegaya H, Fukushima H, Akutsu T, Watanabe K, Yoshino M (2009) Evaluation of mRNA-based approach for identification of saliva and semen. Legal Med 11:125–128. https://doi.org/10.1016/j.legalmed.2008.10.002

    CAS  PubMed  Google Scholar 

  44. Setzer M, Juusola J, Ballantyne J (2008) Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J Forensic Sci 53:296–305. https://doi.org/10.1111/j.1556-4029.2007.00652.x

    CAS  PubMed  Google Scholar 

  45. Sirker M, Schneider PM, Gomes I (2016) A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions. Int J Legal Med 130:1431–1438. https://doi.org/10.1007/s00414-016-1373-9

    PubMed  Google Scholar 

  46. Zubakov D, Kokshoorn M, Kloosterman A, Kayser M (2009) New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains). Int J Legal Med 123:71–74. https://doi.org/10.1007/s00414-008-0249-z

    PubMed  Google Scholar 

  47. van den Berge M, Ozcanhan G, Zijlstra SA, Lindenbergh A, Sijen T (2016) Prevalence of human cell material: DNA and RNA profiling of public and private objects and after activity scenarios. Forensic Sci Int Genet 21:81–89. doi:https://doi.org/10.1016/j.fsigen.2015.12.012

  48. Nakanishi H, Hara M, Takahashi S, Takada A, Saito K (2014) Evaluation of forensic examination of extremely aged seminal stains. Legal Med 16:303–307. https://doi.org/10.1016/j.legalmed.2014.04.002

    CAS  PubMed  Google Scholar 

  49. van Steendam K, de Ceuleneer M, Dhaenens M, van Hoofstat D, Deforce D (2013) Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic sciences. Int J Legal Med 127:287–298. https://doi.org/10.1007/s00414-012-0747-x

    PubMed  Google Scholar 

  50. Dammeier S, Nahnsen S, Veit J, Wehner F, Ueffing M, Kohlbach O (2016) Mass-spectrometry-based proteomics reveals organ-specific expression patterns to be used as forensic evidence. J Proteome Res 15:182–192. https://doi.org/10.1021/acs.jproteome.5b0070

    CAS  PubMed  Google Scholar 

  51. Hanson E, Ingold S, Haas C, Ballantyne J (2015) Targeted multiplexed next generation RNA sequencing assay for tissue source determination of forensic samples. Forensic Sci Int Genet Supplement Series 5:e441–e443. https://doi.org/10.1016/j.fsigss.2015.09.175

    Google Scholar 

  52. Zubakov D, Kokmeijer I, Ralf A, Rajagopalan N, Calandro L, Wootton S, Langit R, Chang C, Lagace R, Kayser M (2015) Towards simultaneous individual and tissue identification: a proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the ion torrent PGM. Forensic Sci Int Genet 14:122–128. https://doi.org/10.1016/j.fsigen.2015.04.002

    Google Scholar 

  53. Lin MH, Albani PP, Fleming R (2016) Degraded RNA transcript stable regions (StaRs) as targets for enhanced forensic RNA body fluid identification. Forensic Sci Int Genet 20:61–70. https://doi.org/10.1016/j.fsigen.2015.09.012

    PubMed  Google Scholar 

  54. Winter J, Diederichs S (2011) Argonaute proteins regulate microRNA stability: increased microRNA abundance by Argonaute proteins is due to microRNA stabilization. RNA Biol 8:1149–1157. https://doi.org/10.4161/rna.8.6.17665

    CAS  PubMed  Google Scholar 

  55. Hutvagner G, Simard MJ (2008) Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 9:22–32. https://doi.org/10.1038/nrm2321

    CAS  PubMed  Google Scholar 

  56. Goray M, Mitchell JR, van Oorschot RAH (2012) Evaluation of multiple transfer of DNA using mock case scenarios. Legal Med 14:40–46. https://doi.org/10.1016/j.legalmed.2011.09.006

    CAS  PubMed  Google Scholar 

  57. Wiegand P, Heimbold C, Klein R, Immel U, Stiller D, Klintschar M (2010) Transfer of biological stains from different surfaces. Int J Legal Med 125:727–731. https://doi.org/10.1007/s00414-010-0424-x

    PubMed  Google Scholar 

  58. Goray M, Eken E, Robert J, Mitchell RJ, van Oorschot RAH (2010) Secondary DNA transfer of biological substances under varying test conditions. Forensic Sci Int Genet 4:62–67. https://doi.org/10.1016/j.fsigen.2009.05.001

    CAS  PubMed  Google Scholar 

  59. Phipps M, Petricevic S (2007) The tendency of individuals to transfer DNA to handled items. Forensic Sci Int 168:162–168. https://doi.org/10.1016/j.forsciint.2006.07.010

    CAS  PubMed  Google Scholar 

  60. Brayley-Morris H, Sorrell A, Revoir AP, Meakin GE, Courts DS, Morgan RM (2015) Persistence of DNA from laundered semen stains: implications for child sex trafficking cases. Forensic Sci Int Genet 19:165–171. https://doi.org/10.1016/j.fsigen.2015.07.016

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We kindly thank our blood and saliva donors for supplying samples. We also want to acknowledge Titia Sijen and Margreet van den Berge from the NFI for their support and helping hand with the establishment of mRNA analysis in our laboratory.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to G. Kulstein.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Supplementary Fig. 1

RNA profile of 100 μl volume sample on cotton washed at 40 °C (GIF 441 kb)

High Resolution Image (TIFF 507 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kulstein, G., Wiegand, P. Comprehensive examination of conventional and innovative body fluid identification approaches and DNA profiling of laundered blood- and saliva-stained pieces of cloths. Int J Legal Med 132, 67–81 (2018). https://doi.org/10.1007/s00414-017-1691-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00414-017-1691-6

Keywords

Navigation