Detection of Saliva by Immunoaffinity and Mass Spectrometry

NCT06183385 | Unknown

• 1 other identifier: RECHMPL23_0098
• Study Type: interventional
• Target: 35
• Locations: 1 country
• Sites: 1

Brief 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.

Conditions

Sexual Assault

Keywords

saliva; proteins; biomarkers; mass spectrometry

Outcome Measures

Primary Outcomes (2)

• 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

• 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, κ = 1. If they totally disagree (or agree due solely to chance), κ ≤ 0.

  3 months

Secondary Outcomes (5)

• Reproducibility between vaginal samples soaked with saliva

  3 months

• Intermediate fidelity between technicians on vaginal samples soaked with saliva

  3 months

• Analytical Sensibility

  3 months

• Diagnostic sensibility

  3 months

• Diagnostic specificity

  3 months

Study Arms (2)

Saliva

OTHER

Collection of two saliva samples (approx. 1.5 to 2 mL each) by passive drooling in healthy volunteers These samples will be centrifuged and the supernatant collected.

Other: Saliva collection; Other: IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry

Vaginal secretions

OTHER

In order to be as close as possible to real life, two vaginal swabs (introduced through the vaginal orifice over a length of 2 to 3 cm) will be taken with a sterile dry swab during the gynecological examination (prior to endovaginal ultrasound or any other endovaginal procedure) of patients included in the Reproductive Medicine Service.

Other: Vaginal secretion collection; Other: IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry

Interventions

Saliva collection OTHER

Collection of 2 samples of 1.5 to 2mL of saliva by passive salivation in healthy volunteers

Saliva

Vaginal secretion collection OTHER

Collection of vaginal secretions with 2 dry swabs in women

Vaginal secretions

IP-LC-MS/MS : immunoprecipitation enrichment with liquid chromatography coupled to tandem mass spectrometry OTHER

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.

Saliva; Vaginal secretions

Eligibility Criteria

• Age: 18 Years+
• Sex: all
• Healthy Volunteers: Yes
• Age Groups: Adult (18-64), Older Adult (65+)

You may qualify if:

• 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)

You may not qualify if:

• 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

Sponsors & Collaborators

• University Hospital, Montpellier: lead

Study Sites (1)

PEYRON

Montpellier, 34295, France

Location

Related Publications (13)

• 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

  BACKGROUND

• 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.

  PMID: 29734053 BACKGROUND

• 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.

  PMID: 16566772 BACKGROUND

• 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.

  BACKGROUND

• 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.

  PMID: 35023573 BACKGROUND

• 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.

  PMID: 35446574 BACKGROUND

• 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.

  PMID: 23525663 BACKGROUND

• 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.

  PMID: 33289932 BACKGROUND

• 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.

  PMID: 25070728 BACKGROUND

• 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.

  PMID: 22843116 BACKGROUND

• 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.

  PMID: 29579379 BACKGROUND

• 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.

  PMID: 27943336 BACKGROUND

• 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.

  PMID: 30071450 BACKGROUND

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Study Officials

• Pierre-Antoine PEYRON, PI

  University Hospital, Montpellier

  STUDY DIRECTOR

Central Study Contacts

Pierre-Antoine PEYRON, PI

CONTACT

04 67 33 85 86; pa-peyron@chu-montpellier.fr

Laëtitia LEVEQUE

CONTACT

04 67 33 85 86; l-leveque@chu-montpellier.fr

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: NON RANDOMIZED
• Masking: NONE
• Purpose: OTHER
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: August 18, 2023
• First Posted: December 27, 2023
• Study Start: January 15, 2024
• Primary Completion: July 15, 2024
• Study Completion: December 31, 2024
• Last Updated: December 27, 2023

Record last verified: 2023-12

Locations

FR (1)