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.
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.
Study Type
INTERVENTIONAL
Allocation
NON_RANDOMIZED
Purpose
OTHER
Masking
NONE
Enrollment
35
Collection of 2 samples of 1.5 to 2mL of saliva by passive salivation in healthy volunteers
Collection of vaginal secretions with 2 dry swabs in women
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.
PEYRON
Montpellier, France
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
Time frame: 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.
Time frame: 3 months
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.
Time frame: 3 months
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, κ = 1. If they totally disagree (or agree due solely to chance), κ ≤ 0.
Time frame: 3 months
Analytical Sensibility
Lowest saliva concentration (µL) detected by the method on saliva samples and on vaginal samples soaked with saliva in controlled condition
Time frame: 3 months
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
Time frame: 3 months
Diagnostic specificity
Analysis of vaginal samples and dry samples in controlled condition, to determine the rate of false positives and true negatives.
Time frame: 3 months
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