Detection of Saliva by Immunoaffinity and Mass Spectrometry

Sexual Assault Clinical Trial

— SIMS  

Official title:

Detection of Specific Salivary Proteins by Immunoaffinity and Mass Spectrometry

Clinical Trial Details — Status: Not yet recruiting

Administrative data

• NCT number: NCT06183385
• Other study ID #: RECHMPL23_0098
• Status: Not yet recruiting
• Phase: N/A
• Start date: January 15, 2024
• Est. completion date: December 31, 2024

Study information

• Verified date: December 2023
• Source: University Hospital, Montpellier
• Contact: Pierre-Antoine PEYRON, PI
• Phone: 04 67 33 85 86
• Email: pa-peyron[@]chu-montpellier.fr
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

The identification of saliva in genital area during a criminal investigation can be a critical component in the prosecution of a sexual assault in France, as non-consensual oral-genital intercourses have been considered as crimes since 2021.The development of highly specific methods for saliva detection is therefore crucial as the commonly employed screening methods lack specificity. Protein mass spectrometry has proven to be a sensitive and specific method but is particularly time consuming. A faster and more sensitive hybrid approach using automated immunoaffinity mass spectrometry (IP-LC-MS/MS) has been recently developed and has been found to be particularly performant for the detection of a seminal fluid protein (semenogelin), allowing a high-throughput seminal fluid identification in semen samples. Like semenogelin, specific salivary proteins such as histatin type 1, cystatin D or proline-rich proteins (PRPs) could be detected using this promising approach, which has never been tested on saliva samples. In collaboration with the Clinical Proteomics Platform and the Department of Reproductive Medicine of the University Hospital of Montpellier, we aim to develop a protocol for the detection of specific saliva proteins by IP-LC-MS/MS in sexual assault-type samples.

Description:

Each participant will be contacted by a phone call the day before the visit, to present for a presentation of the study.The day of the visit, two types of samples will be collected :

• Saliva samples: on healthy volunteers in the Department of Legal Medicine (2 samples of 1.5 - 2 mL are collected for each volunteer).

• Vaginal samples : on women consulting in the Department of Reproductive Medicine (2 dry swabs on each patient). 2 groups, One group with vaginal secretion fluid samples only, and one group with vaginal secretion fluid + sperm samples.

Recruitment information / eligibility

• Status: Not yet recruiting
• Enrollment: 35
• Est. completion date: December 31, 2024
• Est. primary completion date: July 15, 2024
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years and older

Eligibility

Inclusion Criteria:

Saliva Samples

• Men and women aged 18 or more

Vaginal samples :

• Women aged 18 or more

• No unprotected vaginal sexual intercourse during the 10 days prior to the visit (group 1)

• Unprotected vaginal sexual intercourse with ejaculation in the 24 hours prior to the visit (group 2)

Exclusion Criteria:

Saliva Samples :

• Active pathology of the saliva glands (infection, tumor)

• Unprotected oral-genital sexual intercourse in the 24 hours prior to the visit

• Failure to obtain written informed consent after a reflection period

• Pregnant or breast feeding women

• Adult protected by law or under guardianship or curatorship

• No affiliation to the French Social Security System or no benefit from such a system

Vaginal Samples :

• Oral-genital sexual intercourse (cunnilingus) in the 24 hours prior to the visit

• Failure to obtain written informed consent after a reflection period

• Pregnant or Breastfeeding women

• Adult protected by law or under guardianship or curatorship

• No affiliation to the French Social Security System or no benefit from such a system

Related Conditions & MeSH terms

• Sexual Assault

Intervention

Other:
• Saliva collection
Collection of 2 samples of 1.5 to 2mL of saliva by passive salivation in healthy volunteers
• Vaginal secretion collection
Collection of vaginal secretions with 2 dry swabs in women
• IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry
sample preparation and analysis : Impregnation of the tips (absorbent cotton) of sterile dry swabs and vaginal swabs with controlled quantities of saliva. Immunocapture of salivary proteins of interest (histatin type 1, cystatin D, PRPs) by protein A affinity purification LC-MS/MS analysis of eluted purified peptides (Multiple Reaction Monitoring mode) Dilutions will be made from 10 to 10 (1/10, 1/100, 1/1000...), then after reaching a detectability threshold, specified by a second more precise analysis (e.g. if no signal at 1/1000, analysis at 1/500 then 1/250 etc.). Analytical sensitivity will be tested on three samples of each type (salivary, vaginal, vaginal + semen) to ensure reproducibility of results. Operators, unaware of the presence of saliva in the samples, will then carry out saliva-specific protein detection analysis using the IP-LC-MS/MS method.

Locations

Country	Name	City	State
France	PEYRON	Montpellier

Sponsors (1)

Lead Sponsor	Collaborator
University Hospital, Montpellier

Country where clinical trial is conducted

France, 

References & Publications (13)

Blanchard Muller M., Baccino E., Peyron P.A. Détection de traces salivaires dans le cadre d'agressions sexuelles : intérêt de l'utilisation d'une méthode immuno-chromatographique rapide (RSID-SalivaTest®) selon un protocole simplifié. Rev Médecine Légale 2023.

Brown CO, Robbins BL, McKiernan HE, Danielson PB, Legg KM. Direct seminal fluid identification by protease-free high-resolution mass spectrometry. J Forensic Sci. 2021 May;66(3):1017-1023. doi: 10.1111/1556-4029.14646. Epub 2020 Dec 8. — View Citation

Davidovics R, Saw YL, Brown CO, Prinz M, McKiernan HE, Danielson PB, Legg KM. High-throughput seminal fluid identification by automated immunoaffinity mass spectrometry. J Forensic Sci. 2022 May;67(3):1184-1190. doi: 10.1111/1556-4029.14975. Epub 2022 Jan 13. — View Citation

de Beijer RP, de Graaf C, van Weert A, van Leeuwen TG, Aalders MCG, van Dam A. Identification and detection of protein markers to differentiate between forensically relevant body fluids. Forensic Sci Int. 2018 Sep;290:196-206. doi: 10.1016/j.forsciint.2018.07.013. Epub 2018 Jul 24. — View Citation

Illiano A, Arpino V, Pinto G, Berti A, Verdoliva V, Peluso G, Pucci P, Amoresano A. Multiple Reaction Monitoring Tandem Mass Spectrometry Approach for the Identification of Biological Fluids at Crime Scene Investigations. Anal Chem. 2018 May 1;90(9):5627-5636. doi: 10.1021/acs.analchem.7b04742. Epub 2018 Apr 13. — View Citation

Legg KM, Powell R, Reisdorph N, Reisdorph R, Danielson PB. Discovery of highly specific protein markers for the identification of biological stains. Electrophoresis. 2014 Nov;35(21-22):3069-78. doi: 10.1002/elps.201400125. Epub 2014 Oct 1. — View Citation

Legg KM, Powell R, Reisdorph N, Reisdorph R, Danielson PB. Verification of protein biomarker specificity for the identification of biological stains by quadrupole time-of-flight mass spectrometry. Electrophoresis. 2017 Mar;38(6):833-845. doi: 10.1002/elps.201600352. Epub 2017 Jan 30. — View Citation

LOI n°2021-478 du 21 avril 2021 visant à protéger les mineurs des crimes et délits sexuels et de l'inceste. JORF n°0095 du 22 avril 2021. Texte n°4

Sato H, Nakajima D, Ishikawa M, Konno R, Nakamura R, Ohara O, Kawashima Y. Evaluation of the Suitability of Dried Saliva Spots for In-Depth Proteome Analyses for Clinical Applications. J Proteome Res. 2022 May 6;21(5):1340-1348. doi: 10.1021/acs.jproteome.2c00099. Epub 2022 Apr 21. — View Citation

Van Steendam K, De Ceuleneer M, Dhaenens M, Van Hoofstat D, Deforce D. Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science. Int J Legal Med. 2013 Mar;127(2):287-98. doi: 10.1007/s00414-012-0747-x. Epub 2012 Jul 29. — View Citation

Vandenberg N, van Oorschot RA. The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests. J Forensic Sci. 2006 Mar;51(2):361-70. doi: 10.1111/j.1556-4029.2006.00065.x. — View Citation

Wornes DJ, Speers SJ, Murakami JA. The evaluation and validation of Phadebas(R) paper as a presumptive screening tool for saliva on forensic exhibits. Forensic Sci Int. 2018 Jul;288:81-88. doi: 10.1016/j.forsciint.2018.03.049. Epub 2018 Apr 20. — View Citation

Yang H, Zhou B, Deng H, Prinz M, Siegel D. Body fluid identification by mass spectrometry. Int J Legal Med. 2013 Nov;127(6):1065-77. doi: 10.1007/s00414-013-0848-1. Epub 2013 Mar 24. — View Citation

* Note: There are 13 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Reproducibility between saliva samples	Intra-assessor agreement on saliva samples, which involves testing the first sample and retesting the second sample; The study's primary endpoint will be to measure the reliability of the protocol, consisting of reproducibility or intra-assessor agreement,for the detection of specific salivary proteins (histatin type 1, PRPs, cystatin D) using the IP-LC-MS/MS method on adult saliva samples.; Intra-assessor agreement (test/retest) will be measured by their means +/- standard deviation.; Several analyses (between 3 or 4) on the same sample will be carried out to determine whether the results are identical	3 months
Primary	Intermediate fidelity between technicians on saliva samples	Inter-assessor agreement(Cohen's kappa coefficient) on saliva samples, which involves testing several samples by at least 2 different technicians; The study's primary endpoint will be to measure the reliability of the protocol, consisting of intermediate fidelity or inter-assessor agreement (Cohen's kappa coefficient) for the detection of specific salivary proteins (histatin type 1, PRPs, cystatin D) using the IP-LC-MS/MS method on adult saliva samples.; The kappa coefficient gives a score ranging from 0 to 1. If the coders totally agree, ? = 1. If they totally disagree (or agree due solely to chance), ? = 0.	3 months
Secondary	Reproducibility between vaginal samples soaked with saliva	Intra-assessor agreement on vaginal samples soaked with saliva; The secondary endpoints, will consist of repeating the primary endpoints (i.e. reproducibility and intermediate fidelity) on the vaginal samples impregnated with sperm or not.; Again, the same sample will be tested several times (between 3 or 4) to see if the results come back similar.	3 months
Secondary	Intermediate fidelity between technicians on vaginal samples soaked with saliva	: Inter-assessor agreement (Cohen's kappa coefficient) on vaginal samples soaked with saliva; The secondary endpoints, will consist of repeating the primary endpoints (i.e. reproducibility and intermediate fidelity) on the vaginal samples impregnated with sperm or not.; The kappa coefficient gives a score ranging from 0 to 1. If the coders totally agree, ? = 1. If they totally disagree (or agree due solely to chance), ? = 0.	3 months
Secondary	Analytical Sensibility	Lowest saliva concentration (µL) detected by the method on saliva samples and on vaginal samples soaked with saliva in controlled condition	3 months
Secondary	Diagnostic sensibility	Analysis of saliva samples and vaginal samples soaked with saliva in controlled condition, to determine the rate of detection of true positives and false negatives	3 months
Secondary	Diagnostic specificity	Analysis of vaginal samples and dry samples in controlled condition, to determine the rate of false positives and true negatives.	3 months

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