Forensic Reliability of Body Fluids in Sexual Assault Investigations: A Systematic Review

Abstract

The forensic reliability of biological fluids in sexual assault investigations depends on substrate type, environmental exposure, time since deposition, and analytical methodology. This systematic review evaluates the forensic reliability of major biological fluids, semen, blood, saliva, and vaginal secretions by comparing detectability and persistence on porous and non-porous substrates, assessing environmental and temporal effects on DNA integrity, and examining the performance of identification methods. A systematic search of PubMed, Scopus, and Web of Science (2001–2025) was conducted following PRISMA 2020 guidelines. Eligible studies investigated fluid persistence, degradation, or identification reliability under controlled or casework-relevant conditions. A weighted scoring framework categorised relative reliability. Twenty-seven studies met inclusion criteria. Semen and blood demonstrated higher reliability across substrates, particularly when collected within recommended timeframes. Porous substrates reduced surface detectability but occasionally preserved DNA from rapid degradation. Elevated temperature, humidity, and prolonged intervals consistently reduced DNA quality and detection success. Molecular approaches, including mRNA profiling, showed enhanced specificity in degraded or mixed samples, though methodological variability limited direct comparability across studies. The forensic reliability of biological fluids is context-dependent, shaped by complex interactions between substrate characteristics, environmental exposure, and analytical technique. Semen and blood remain robust DNA sources, while emerging technologies offer improved specificity in challenging scenarios. Standardised evaluation frameworks and timely evidence collection remain essential to enhance evidential value and minimise misinterpretation in sexual assault investigations.

1. Introduction

Forensic science relies heavily on the accurate identification and interpretation of bodily fluids such as blood, semen, and saliva, which frequently play a decisive role in sexual assault investigations [1]. These biological materials can provide critical information for reconstructing events, establishing contact between individuals, and corroborating or challenging testimonial evidence, particularly in cases where witnesses are absent or accounts are disputed. However, the detection and interpretation of body fluid evidence remain inherently challenging. Many biological stains are invisible to the naked eye, present only in trace quantities, or occur as complex mixtures, while others may visually resemble non-biological substances, increasing the risk of misinterpretation at both the crime scene and during laboratory analysis [1,2].

A wide range of biological materials may be encountered in forensic contexts, including blood, menstrual blood, semen, saliva, vaginal secretions, skin cells, sweat, and urine [2,3]. Among these, blood, semen, saliva, vaginal fluid, sweat, and urine are most frequently recovered in cases involving sexual violence [3]. Accurate identification of these fluids is essential for establishing timelines, demonstrating victim–perpetrator contact, and supporting investigative and prosecutorial decision-making. In particular, DNA recovered from semen, saliva, or epithelial skin cells may represent the only objective means of confirming or excluding physical contact, thereby substantially influencing arrest and charging outcomes [4].

The forensic reliability of body fluid evidence is strongly influenced by multiple interacting factors, including the time elapsed since deposition, the type of surface on which the fluid is deposited, environmental exposure, and sample handling procedures. Although biological fluids degrade rapidly within the human body, they may persist for longer periods on external substrates such as clothing or bedding. Nevertheless, DNA integrity is highly susceptible to degradation caused by heat, humidity, microbial activity, ultraviolet radiation, and inappropriate storage conditions, all of which can compromise analytical outcomes and reduce the likelihood of successful DNA profiling [5]. Consequently, the preservation of evidential value depends on timely collection, appropriate drying, controlled storage conditions, and strict avoidance of contamination.

In sexual assault investigations, the timing of evidence collection is particularly critical. Delayed reporting, often associated with trauma, fear, or social stigma, substantially reduces the probability of recovering intact biological material. The Sexual Assault Medical Forensic Examination (SAMFE) plays a pivotal role in mitigating this risk by enabling systematic collection of biological evidence, documentation of injuries, and acquisition of toxicological samples when conducted within the recommended post-assault window [6]. However, even when collection occurs promptly, the nature of the substrate remains a key determinant of detectability and persistence. Porous materials such as cotton and denim tend to absorb biological fluids into their fibres, reducing surface-level detectability and hindering recovery, particularly following washing or prolonged exposure. In contrast, non-porous surfaces retain fluids on the surface, facilitating visual detection but potentially increasing susceptibility to removal or environmental degradation [7].

Advances in forensic science have led to the development of increasingly sensitive techniques for body fluid detection and interpretation. Semen remains one of the most valuable forensic fluids due to its high DNA content and strong discriminatory potential, while blood demonstrates superior environmental resilience and long-term molecular stability [5,8]. Immunochromatographic assays, alternative light sources (ALS), and molecular approaches such as mRNA profiling have enhanced the ability to detect and differentiate body fluids, even in aged or environmentally exposed samples [2,9]. Nevertheless, the performance of these methods varies substantially depending on fluid type, surface characteristics, environmental conditions, and time since deposition. As a result, interpretation of body fluid evidence increasingly requires a probabilistic, context-driven approach that accounts for transfer, persistence, and degradation processes [10].

Despite substantial methodological progress, a comprehensive synthesis comparing the persistence, detectability, and degradation patterns of major forensic body fluids across varying substrates and environmental conditions remains limited. In particular, the combined effects of surface porosity, environmental exposure, and post-deposition time on fluid-specific forensic reliability have not been systematically evaluated.

The primary objective of this study is to systematically evaluate existing evidence on the forensic reliability of body fluid identification, preservation, and interpretation in sexual assault investigations. Specifically, this review aims to: (i) compare the detectability and persistence of major body fluids on porous and non-porous substrates; (ii) evaluate the effects of time since deposition on DNA quality and quantity across different biological fluids; and (iii) determine which body fluids demonstrate the highest reliability under varying environmental and evidence collection conditions.

2. Materials and Methods

This study was conducted as a systematic review in accordance with the PRISMA guidelines, with the study selection process summarised in the PRISMA flow diagram (Figure 1). The review protocol was registered with PROSPERO (Registration ID: CRD420251239570) to ensure methodological transparency and consistency. A total of 160 records were identified through database searches and register-based sources. Following the removal of duplicate records and those excluded for predefined reasons, 105 articles were screened based on titles and abstracts. Of these, 39 full-text articles were assessed for eligibility, and 27 studies met the inclusion criteria and were included in the final qualitative assessment.

Peer-reviewed literature relevant to forensic body fluid analysis in sexual assault investigations was retrieved from multiple electronic databases, including PubMed, ScienceDirect, and Forensic Science International. Google Scholar was used as a supplementary source, alongside selected forensic science repositories, with ResearchGate and Mendeley utilised to identify additional relevant publications. The search strategy employed combinations of relevant keywords and a specific Boolean string: (“sexual assault” OR rape OR “sexual violence”)

AND

(“body fluid” OR “biological fluid” OR semen OR blood OR saliva OR “vaginal secretion*” OR “vaginal fluid” OR “menstrual blood”)

AND

(persistence OR degradation OR “DNA degradation” OR “time since deposition” OR detectability OR stability)

AND

(substrate OR surface OR porous OR “non-porous” OR fabric OR textile OR clothing)

AND

(“forensic identification” OR “body fluid identification” OR mRNA OR microRNA OR “DNA methylation” OR proteomic* OR “LC-MS/MS” OR “mass spectrometry” OR immunochromatographic OR “alternative light source” OR ALS)

Searches were limited to English-language publications published between 2001 and 2025. Reference lists of all included studies were manually screened to identify additional eligible articles.

All retrieved records were imported into Mendeley for reference management, and duplicate entries were removed prior to screening. Title and abstract screening was independently conducted by two reviewers to assess relevance against predefined inclusion and exclusion criteria. Full-text screening was subsequently performed by both reviewers, with any discrepancies resolved through discussion and consensus. Only human studies directly relevant to the forensic detection, persistence, and interpretation of body fluids in the context of sexual assault investigations were included.

Data extraction focused on five key variables: (i) type of body fluid, (ii) detection method (such as alternative light sources [ALS], prostate-specific antigen [PSA]), (iii) surface porosity, (iv) time since deposition, and (v) environmental conditions. Study quality was assessed using a structured narrative approach, considering methodological transparency, relevance to forensic practice, and consistency of reported outcomes. Extracted data were organised using Microsoft Excel, while manuscript preparation and formatting were undertaken using Google Docs.

Due to substantial heterogeneity across study designs, analytical techniques, and outcome measures, a meta-analysis was not performed. Instead, findings were evaluated using descriptive statistical summaries, comparative tables, weighted reliability scoring, and narrative analysis. To support comparative interpretation, a weighted reliability scoring framework was applied based on three predefined criteria: detectability, persistence, and environmental robustness. Each criterion was scored on a scale from 0 to 3, where 0 indicated non-detectability and 3 indicated high forensic reliability. Total reliability scores were calculated by summing the three component scores. Subgroup analyses were conducted where appropriate to explore variations in performance across surface types, environmental exposures, and detection methods. This structured approach enabled a comprehensive evaluation of the forensic reliability of biological body fluids under conditions relevant to sexual assault investigations.

To facilitate structured comparison across heterogeneous studies, a qualitative reliability grading framework was developed. Body fluids and detection approaches were categorised as demonstrating high reliability when evidence indicated consistently high sensitivity and specificity (>90% where reported), low cross-reactivity, demonstrable persistence under varied environmental conditions, and reproducible performance across multiple studies. Moderate reliability was assigned where performance remained acceptable but was affected by substrate type, degradation, or occasional cross-reactivity. Low reliability was attributed where substantial sensitivity reduction, high false-positive rates, rapid degradation, or inconsistent reproducibility were reported. These classifications were based on analysed findings from included studies rather than single-study outcomes [5,8,11].

3. Results and Discussion

3.1. Detectability and Persistence of Major Body Fluids on Porous and Non-Porous Substrates

The collective findings of the included studies demonstrate that surface type exerts a substantial influence on both the detectability and persistence of biological fluids, with non-porous substrates consistently outperforming porous materials. Non-porous surfaces retain fluids at the surface rather than absorbing them, thereby facilitating stronger fluorescence under alternative light sources (ALS) and more efficient interaction with biochemical and molecular detection techniques. As summarised in

Table 1. Frequency, detectability, and persistence of major body fluids on porous and non-porous substrates as reported in previous studies.

Study ID	Study Design	Surface Type	Surface Detail	Body Fluid	Detection Method	Detectability	Persistence
Miranda et al. 2014 [12]	Experimental laboratory	Porous	Cloth, wood	Blood	ALS	High when dry; weak when wet	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Porous	Cloth, wood	Semen	ALS	Strong fluorescence	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Porous	Cloth, wood	Saliva	ALS	Moderate	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Porous	Cloth, wood	Urine	ALS	Detectable but weaker	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Non-porous	Ceramic tile	Blood	ALS	Strong detection	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Non-porous	Ceramic tile	Semen	ALS	High fluorescence	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Non-porous	Ceramic tile	Saliva	ALS	Good detection	Up to 60 days
Miranda et al. 2014 [12]	Experimental laboratory	Non-porous	Ceramic tile	Urine	ALS	Detectable	Up to 60 days
Spiker et al. 2014 [13]	Controlled laboratory	Non-porous	Smooth ceramic tile	Spermatozoa	Microscopy	Highest recovery (polyester swab best)	Short-term
Spiker et al. 2014 [13]	Controlled laboratory	Semi-porous	Rough tile	Spermatozoa	Microscopy	Moderate recovery	Short-term
Spiker et al. 2014 [13]	Controlled laboratory	Semi- porous	Quarry tile	Spermatozoa	Microscopy	Lowest recovery	Short-term
Gregório et al. 2017 [14]	Experimental	Porous	Pads, diapers, panty liners	Semen	ATR-FTIR	Clear spectral peaks	Not Reported
Gregório et al. 2017 [14]	Experimental	Porous	Pads, diapers, panty liners	Vaginal fluid	ATR-FTIR	Strong vs. markers	Not Reported
Gregório et al. 2017 [14]	Experimental	Porous	Pads, diapers, panty liners	Urine	ATR-FTIR	Detectable but weaker	Not Reported
Rodriguez et al. 2019 [15]	Experimental	Porous	Underwear (cotton)	Semen	ALS, Sg test	High detectability	Stains visible for days
Rodriguez et al. 2019 [15]	Experimental	Non-porous	Condom (inner and outer)	Semen	ALS, Sg test	Internal positive; external weak–negative	Several days; degradation when stored 55–69 days
Burnier et al. 2021 [16]	Proficiency + casework	Non-porous	Latex condom	Blood	Py-GC-MS/GC-MS	Blood detectable on condom surface	Not Reported
Clarke & Hassan 2025 [17]	Experimental	Non-porous	Plastic/Glass	Semen	PSA, AP, microscopy	High	PSA decreases after 8–24 h
Clarke & Hassan 2025 [17]	Experimental	Porous	Cotton fabric	Semen	PSA, AP, microscopy	Moderate	Reduced after 24–48 h
Newton 2013 [18]	Experimental	Non-porous	Plastic & Metal	Vaginal fluid	Fluorescent markers	Positive	Fluorescence fades with time
Astrup et al. 2012 [19]	Experimental	Non-porous	Slide	Spermatozoa	Microscopy, staining	Strong detection	Detectable several hours
Maynard et al. 2001 [20]	Experimental	Non-porous	Condom	Semen	GC-MS, Py-GC-MS	Often negative (condom barrier	Very low transfer; dependent on brand
Laffan et al. 2011 [21]	Laboratory evaluation	Porous	Cotton fabric, swabs	Semen	RSID-Semen, PSA	High sensitivity; RSID more specific	Short-term (post-coital)
Rankin-Turner 2020 [22]	Experimental	Non- porous	Glass, metal, plastic, ceramic tile	Blood	In situ mass spectrometry	High (strong and distinct chemical profiles)	Moderate–High (ageing signatures detectable longer due to surface retention)
Rankin-Turner 2020 [22]	Experimental	Porous	Fabric	Blood	In situ mass spectrometry	Moderate–Low (reduced signal due to absorption)	Low–Moderate (faster chemical degradation)
Rankin-Turner 2020 [22]	Experimental	Non-porous	Condom	Semen	In situ mass spectrometry	High (clear semen-specific chemical signatures)	Moderate (persistence enhanced by impermeable substrates)
Rankin-Turner 2020 [22]	Experimental	Porous	fabric	Semen	In situ mass spectrometry	Low–Moderate (signal loss due to absorption)	Low (reduced persistence compared with non-porous surfaces)

ALS → Alternative Light Sources (ALS), PSA → Prostate-Specific Antigen (PSA), GC–MS → Gas Chromatography–Mass Spectrometry (GC–MS), ATR-FTIR → Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy (ATR-FTIR), RSID → Rapid Stain Identification (RSID™), SG test → Specific Gravity test, AP → Acid Phosphatase.

, blood, semen, saliva, and urine exhibited brighter fluorescence signals and prolonged detectability on non-porous ceramic tiles compared with porous substrates such as fabric or wood, where absorption diminished surface-level visibility and recovery [7,12].

Non-porous materials, including ceramic tiles, glass, plastic, metal, and latex condoms, consistently enhance forensic recovery because biological residues remain exposed on the surface. This surface retention improves the performance of detection approaches such as ALS screening, presumptive chemical tests, swabbing, and microscopic examination [23]. Miranda et al. [12] reported that blood and semen produced the most intense ALS fluorescence on ceramic tiles, attributable to surface-level preservation and minimal substrate interference. Similarly, Burnier et al. [16] demonstrated that blood deposited on latex condoms remained chemically intact over time, enabling reliable identification by gas chromatography–mass spectrometry (GC-MS), as latex prevents fluid absorption and protects biomolecular integrity. Using in situ mass spectrometry, Rankin-Turner [22] further showed that blood and semen deposited on non-porous surfaces produced strong, distinctive chemical and metabolite profiles, with enhanced signal stability due to unrestricted surface accessibility.

In contrast, porous substrates such as fabrics, cloth, and wood absorb biological fluids into their fibres, reducing the quantity of recoverable material and weakening surface-based detection signals. Medina-Paz et al. [24] observed that fluid diffusion into fabrics resulted in diminished detection intensity and lower analytical signals, particularly on materials such as leather, where surface recovery was poor and biomolecular degradation was accelerated. Laffan et al. [21] further reported that porous substrates significantly reduce DNA recovery efficiency due to fibre entrapment and uneven fluid distribution, producing increased variability in DNA yield and profile quality, especially in aged or environmentally exposed samples. Semi-porous materials may present additional challenges: Spiker [13] reported that the rough surface structure of quarry tiles impeded effective swabbing and entrapped cellular material, leading to markedly reduced spermatozoa recovery. Consistent with these observations, Rankin-Turner [22] found reduced chemical signal intensity and faster loss of detectable biomarkers when blood and semen were deposited on absorbent materials, reflecting limited surface accessibility and enhanced degradation.

Surface type also plays a critical role in determining the persistence of biological fluids over time. On non-porous substrates, fluids typically dry as thin surface films, limiting moisture retention and reducing microbial growth, oxidation, and enzymatic degradation [25]. Miranda et al. [12] found that biological fluids deposited on ceramic tiles remained detectable for up to 60 days, displaying substantially greater fluorescence stability than those on porous materials, where signal loss occurred more rapidly. Similarly, Burnier et al. [16] showed that blood on latex condoms retained its chemical profile over extended periods, as the substrate shielded the stain from enzymatic and microbial deterioration.

By contrast, porous materials promote faster degradation due to moisture retention and increased exposure to substrate-bound enzymes and microorganisms. Cano-Trujillo et al. [23] noted that absorbed fluids are trapped within microenvironments that accelerate chemical and biological breakdown. Gregório et al. [14] demonstrated that ATR-FTIR signals for semen, vaginal fluid, and urine were primarily confined to the superficial absorbent layers of pads and diapers, with deeper layers exhibiting rapid signal loss due to molecular degradation. Similarly, Hanson and Ballantyne [26] reported rapid degradation of vaginal fluid mRNA when absorbed into fabric or tissue, highlighting the vulnerability of nucleic acids on porous substrates. Collectively, these findings indicate that biological fluids exhibit a markedly shorter forensic lifespan on porous materials.

From a mechanistic perspective, the influence of substrate type on biological degradation may be partly explained by differences in effective surface area and oxygen exposure. Porous materials such as cotton and denim possess complex fibrous matrices that increase internal surface area relative to smooth non-porous substrates. This expanded microenvironment facilitates greater diffusion of oxygen and moisture into deposited biological stains, potentially accelerating oxidative degradation of proteins and nucleic acids. Oxidative processes contribute to molecular fragmentation and structural modification, thereby reducing detectability over time [5]. In contrast, non-porous surfaces such as plastic or metal typically restrict absorption and may limit internal oxygen penetration, although they remain vulnerable to environmental exposure at the surface level [27]. These substrate-dependent differences highlight that degradation is not solely time-dependent but is influenced by physicochemical interactions between biological material and the deposition matrix.

Conventional swabbing techniques typically employ cotton-based applicators, which are themselves porous substrates. While swabs are designed to maximise biological recovery through absorption, prolonged retention of moisture within fibrous matrices may theoretically promote oxidative and microbial degradation if drying and storage are delayed. However, standard forensic protocols mitigate this risk through prompt air-drying, controlled storage conditions, and timely laboratory processing [5]. Furthermore, swabbing represents a transfer mechanism rather than a long-term storage environment; once dried, the risk of ongoing oxidative acceleration is substantially reduced. Emerging collection technologies, including flocked swabs and alternative absorbent materials, may offer improved recovery efficiency while minimising retention-associated degradation. These considerations underscore that substrate-related degradation is influenced not only by deposition surface but also by post-collection handling procedures.

The effectiveness of detection methods is further modulated by substrate characteristics. Presumptive tests such as luminol, Kastle–Meyer, acid phosphatase, and amylase assays offer rapid screening but frequently lose sensitivity on porous surfaces, where absorption limits reagent contact and increases the likelihood of substrate interference and false-positive reactions [28]. The limited specificity of amylase-based saliva tests is particularly problematic, as α-amylase is present in multiple body fluids and may be retained within porous materials along with environmental contaminants.

Confirmatory techniques, including PSA and semenogelin immunoassays, sperm microscopy, and molecular markers, offer greater specificity but remain highly dependent on efficient sample recovery, which is more consistently achieved on non-porous substrates. As reflected in Table 1, spermatozoa are more readily identified by microscopy on smooth surfaces than on textured materials [13]; ATR-FTIR enables differentiation of body fluids but yields weakened signals within deeper absorbent layers [14]; mRNA markers degrade rapidly on porous substrates [26]; and GC-MS provides stable chemical profiles when residues remain accessible on non-porous condoms [16]. Advanced mass spectrometric approaches have further demonstrated the ability to identify semen components in aged samples, particularly when deposited on non-porous surfaces that preserve residue accessibility [22].

Taken together, these findings indicate that non-porous surfaces such as ceramic tiles, glass, plastic, and latex condoms should be prioritised during forensic evidence collection, as they retain biological fluids at the surface and preserve detectable chemical and molecular signatures [12,16]. Although porous materials may still retain evidential value, they often require more sensitive and targeted confirmatory techniques, including RSID assays, ATR-FTIR, mRNA profiling, or microscopic examination, to compensate for reduced fluorescence, limited recovery, and accelerated degradation [15,26]. These results underscore the importance of considering substrate type when selecting detection strategies and interpreting body fluid evidence in sexual assault investigations.

3.2. Effects of Time Since Deposition on DNA Quality and Quantity Across Biological Fluids

The assessment of DNA quality and persistence across biological fluids is inherently dependent upon both the analytical methodology employed and the characteristics of the deposition substrate. DNA degradation has typically been evaluated using short tandem repeat (STR) profiling, quantitative PCR (qPCR) analysis, and electrophoretic peak height assessment, which serve as proxies for molecular integrity and amplifiable template quantity [5]. Importantly, the performance of these analytical techniques may vary depending on whether samples are recovered from porous or non-porous substrates, as substrate-mediated retention, moisture exposure, and oxidative processes influence DNA stability [27,29]. Accordingly, interpretation of DNA persistence data must be contextualised within both analytical and environmental parameters.

Table 2. Changes in DNA quality and quantity of blood, saliva, and semen over time, based on Al-Kandari et al. [30].

Time (Days)	Blood: DNA Quantity	Blood: DNA Quality	Saliva: DNA Quantity	Saliva: DNA Quality	Semen: DNA Quantity	Semen: DNA Quality
1	High	High	High	High	High	High
3	High	High	High	High	High	High
5	Moderate	Moderate	Moderate	Moderate	High	High
7	Moderate	Moderate	Moderate	Moderate	Moderate	Moderate
10	Low	Low	Low	Low	Moderate	Moderate

Reliability categories defined according to criteria outlined in Materials and Methods section, paragraph 7.

,

Table 3. Changes in DNA quality and quantity of blood, saliva, and semen over time, based on Sirker et al. [31].

Time (Weeks)	Saliva (DNA Quality)	Blood (DNA Quality)	Semen (DNA Quality)
1	Moderate-High	High	High
2	Moderate	High	High—Moderate
4	Low	High-Moderate	Moderate
8	Very low	Moderate	Moderate
12	Very low	Moderate-Low	Low
20	None	Moderate-Low	Moderate-Low
24	None	Low	Low
33	None	Low	Very low
>40	None	Very low	None

Reliability categories defined according to criteria outlined in Materials and Methods section, paragraph 7.

and

Table 4. Changes in DNA quality and quantity of blood, saliva, and semen over time, based on Zhang et al. [32].

Time (Days)	Blood	Semen	Saliva	Vaginal Secretion	Menstrual Blood
7	High	Moderate—High	Moderate	High	High
14	Moderate	Moderate	Low	Moderate	High—Moderate
21	Low—Moderate	Low	Very Low	Low	Moderate
30	Low	Very low	None	Very low—None	Moderate—Low

Reliability categories defined according to criteria outlined in Materials and Methods section, paragraph 7.

and Figure 2, Figure 3 and Figure 4 illustrate changes in DNA quality and quantity in blood, saliva, and semen over time.

Graph Legend for Figure 2, Figure 3 and Figure 4:

• High: 6
• Moderate—High: 5
• Moderate: 4
• Moderate—Low: 3
• Low: 2
• Very Low: 1
• None: 0

The trends illustrated in Figure 2, Figure 3 and Figure 4 and summarised in Table 2, Table 3 and Table 4 consistently demonstrate a time-dependent decline in both DNA quantity and quality across all examined biological fluids, although the rate and extent of degradation vary markedly by fluid type. In the short term, semen exhibits the greatest stability, maintaining high DNA quality and quantity up to Day 5 and moderate levels by Day 10, as shown in Figure 2. During this period, saliva undergoes the most rapid deterioration, declining from high to low DNA quality by Day 10, while blood displays a more gradual reduction.

These fluid-specific differences become more pronounced over longer observation periods. Figure 3, which extends the analysis to 40 weeks, indicates that blood remains the most stable fluid over time, retaining moderate DNA quality for several months. Semen demonstrates a progressive decline from high to moderate levels before eventually reaching low detectability during extended storage. In contrast, saliva shows a steep reduction in DNA integrity, becoming low by Week 4 and undetectable after approximately Week 12. This long-term pattern aligns with existing evidence indicating that blood consistently outperforms other biological fluids in preserving nucleic acid integrity over prolonged periods [33].

Figure 4 further supports these observations by comparing five biological fluids over a 30-day period. Blood and menstrual blood emerge as the most persistent fluids, maintaining detectable DNA throughout the observation window. Semen shows moderate stability during the initial two weeks but undergoes a pronounced decline thereafter. Saliva becomes undetectable by the end of the monitoring period, confirming its limited forensic longevity. Vaginal secretions exhibit particularly rapid degradation, reflecting the fast breakdown of mRNA and nucleic acids reported in previous studies [32].

When the findings across all datasets are generated, a clear hierarchy of DNA stability emerges. Blood and menstrual blood demonstrate the highest long-term persistence, semen exhibits intermediate stability, and saliva and vaginal secretions degrade most rapidly. These trends are supported by microbial-based time-since-deposition studies, which report faster compositional shifts in saliva and vaginal fluids compared with menstrual blood and semen [32,34]. Díez López et al. [35] employed 16S ribosomal RNA (rRNA) sequencing to characterise the salivary microbiome rather than analysing human messenger RNA (mRNA). Accordingly, the findings relate to microbial community profiling rather than host RNA-based body fluid identification.

The observed differences in DNA stability across body fluids can be largely attributed to their underlying cellular composition and biochemical environments. Blood contains a high concentration of nucleated cells, resulting in consistently high DNA yields and superior stability, even under extended storage or environmental exposure. Semen similarly contains abundant DNA due to densely packed spermatozoa, conferring moderate to high stability despite some susceptibility to enzymatic degradation [36]. In contrast, saliva contains relatively few nucleated cells, leading to lower DNA yields and rapid degradation, consistent with studies documenting pronounced molecular and microbiome changes over time [37]. Vaginal secretions typically contain variable and comparatively low DNA concentrations and are prone to rapid degradation due to endogenous enzymatic activity and microbial processes [38]. Menstrual blood, while richer in DNA than saliva or vaginal secretions, is more susceptible to degradation than peripheral blood owing to the presence of endometrial tissue; nevertheless, it demonstrates greater persistence than most other non-blood fluids [39].

3.3. Comparative Reliability of Body Fluids Under Variable Environmental and Evidence Collection Conditions

Table 5. Legend scoring system.

Score	Detectability	Persistence	Environmental Stability (Robustness)
3	Strong (high success rates; strong signals)	Long-term (weeks–months)	Highly robust (little effect)
2	Moderate (some dropout, still detectable)	Medium-term (days–weeks)	Moderately robust
1	Weak (partial signals only)	Short-term (hours–days)	Sensitive
0	Absent	Rapidly degraded	Extremely sensitive

and

Table 6. Weighed ranking of the most reliable body fluids under varying environmental and collection conditions.

Study ID	Body Fluid	Detectability (3)	Persistence (3)	Environmental Stability (3)	Substrate Type (Porous/Non-Porous)	Environmental Conditions (Controlled/Heat/Humidity/UV Exposure)	Time Since Deposition	Collection Method	Analytical Method Used	Total Score (12)
Sirker et al. 2016 [31]	Semen	3	2	2	Porous	Humidity	2	Freshly ejaculated semen was collected in a sterile 50 mL plastic tube	mRNA profiling, STR profiling, Capillary Electrophoresis	9
Sirker et al. 2016 [31]	Blood	3	3	2	Porous	Humidity	3	Venous blood was collected from a female donor by venipuncture with anticoagulation treatment.	mRNA profiling, STR profiling, Capillary Electrophoresis	11
Sirker et al. 2016 [31]	Saliva	2	1	0	Porous	Humidity	1	Saliva was collected from a female donor in a sterile 1.5-μL Eppendorf tube.	mRNA profiling, STR profiling, Capillary Electrophoresis	4
Zhang et al. 2024 [32]	Semen	2	2	1	Porous	Controlled indoor (18.4–19.4 °C), Humidity (30–40%)	2	Swabbing: Self-collected into sterile tubes, then transferred to sterile cotton swabs	16S rRNA gene high-throughput sequencing	7
Zhang et al. 2024 [32]	Saliva	2	1	0	Porous	Controlled indoor (18.4–19.4 °C), Humidity (30–40%)	0	Swabbing: Self-collected into sterile tubes, then transferred to sterile cotton swabs	16S rRNA gene high-throughput sequencing	3
Zhang et al. 2024 [32]	Vaginal Secretion	2	2	1	Porous	Controlled indoor (18.4–19.4 °C), Humidity (30–40%)	0	Swabbing: Self-collected directly onto sterile cotton swabs	16S rRNA gene high-throughput sequencing	5
Zhang et al. 2024 [32]	Menstrual blood	3	3	2	Porous	Controlled indoor (18.4–19.4 °C), Humidity (30–40%)	3	Swabbing: Self-collected directly from the vagina using sterile cotton swabs during days 2–5 of the menstrual cycle.	16S rRNA gene high-throughput sequencing	11
Johannessen et al. 2022 [9]	Vaginal Secretion	3	1	0	Porous	Controlled	1	Swabbing	mRNA profiling, STR profiling	5
Al-Kandari et al. 2016 [30]	Blood	3	2	1	Porous	Heat	3	Swabbing	Real-time PCR	6
Al-Kandari et al. 2016 [30]	Saliva	2	1	0	Porous	Heat	1	Swabbing	Real-time PCR	3
Khorwal et al. 2024 [40]	Blood	3	3	2	Porous	UV exposure, Heat	2	Blood deposited on cotton, air dried, stain cutting collected	Organic extraction + Agarose gel + RFLP	8
Khorwal et al. 2024 [40]	Saliva	2	2	1	Porous	UV exposure, Heat	1	Saliva deposited on cotton, air dried, stain cutting collected	Organic extraction + Agarose gel + RFLP	5
Mayes et al. 2019 [41]	Blood	3	3	2	Porous	Heat, Humidity, UV exposure	3	blood was collected by venipuncture into BD Vacutainer™ tubes treated with an anticoagulant (EDTA)	RT-qPCR (real-time quantitative PCR)	11
Mayes et al. 2019 [41]	Semen	3	2	2	Porous	Heat, Humidity, UV exposure	2	Semen was provided in specimen containers	RT-qPCR (real-time quantitative PCR)	9
Borde et al. 2008 [42]	Blood	2	1	0	Porous	Water Immersion: Submerged at a depth of 5 m in either a freshwater river or seawater	0	Blood deposited on fabric, air-dried, cutting collected	DNA STR profiling	3
Borde et al. 2008 [42]	Semen	3	2	2	Porous	Water Immersion: Submerged at a depth of 5 m in either a freshwater river or seawater	1	Semen deposited on fabric, air-dried, cutting collected	DNA STR profiling	8
Borde et al. 2008 [42]	Saliva	2	1	0	Porous	Dry	0	Saliva deposited on fabric, air-dried, cutting collected	DNA STR profiling	3

summarise the comparative reliability of major body fluids under varying environmental conditions and detection approaches, highlighting differences in detectability, persistence, and overall forensic performance.

Variations in reliability scoring between studies assessing the same biological fluid reflect differences in environmental exposure, substrate characteristics, analytical methodology, and time since deposition. For example, vaginal secretions evaluated under controlled laboratory conditions demonstrated greater persistence and analytical stability compared to samples exposed to elevated temperature or humidity, which accelerated molecular degradation [5,27]. Accordingly, scoring reflects contextual performance rather than inherent fluid properties alone.

Based on the weighted reliability scores summarised in Table 5 and Table 6, blood consistently achieved the highest overall reliability across studies, reflecting its strong detectability, persistence, and environmental robustness. Sirker et al. [31] reported that blood samples stored under dry conditions retained both mRNA and DNA integrity for up to 71 weeks, whereas samples exposed to humid environments exhibited moderate degradation after approximately 33 weeks. These findings indicate substantial resilience to time-related degradation, alongside clear susceptibility to moisture. In contrast, Al-Kandari et al. [30] assessed blood only under short-term, controlled indoor conditions and observed a gradual decline in DNA quantity over a ten-day period. The absence of external stressors such as heat or humidity resulted in a comparatively lower reliability score, reflecting limited environmental challenge rather than poor inherent stability. Similarly, Khorwal et al. [40] demonstrated high DNA purity and concentration in blood samples stored under stable conditions, further supporting blood’s strong reliability in mild environments. Mayes et al. [41] additionally reported that both mRNA and microRNA markers in blood exhibit high stability over extended storage periods, reinforcing the robustness of blood across multiple molecular targets and analytical platforms.

Semen displayed greater variability in reliability scores across studies, largely driven by differences in the biological targets analysed and the environmental conditions applied. In Sirker et al. [31], semen received a relatively high score due to the preservation of protamine-rich sperm cells, which maintained semen-specific mRNA markers (PRM1 and PRM2) for up to 20 weeks, particularly under dry conditions. However, increased humidity accelerated signal loss, indicating moderate environmental sensitivity. In contrast, Zhang et al. [32] reported lower reliability scores for semen when microbiome stability was used as the primary endpoint, as microbial profiles are highly sensitive to fluctuations in temperature, oxygen exposure, and moisture. In that study, substantial microbiome shifts occurred within 21–30 days, although semen remained detectable for approximately 7–14 days. Khorwal et al. [40] assessed semen reliability based on DNA concentration and purity over shorter timeframes and reported moderate degradation under typical storage conditions, resulting in intermediate reliability scores. Borde et al. [42] further demonstrated that semen deposited on non-porous substrates and protected from washing or prolonged moisture retained detectable spermatozoa and DNA for extended periods, whereas porous or water-exposed substrates showed accelerated loss, underscoring the strong influence of environmental exposure on semen reliability. Collectively, these findings suggest that semen exhibits moderate to high reliability under dry and controlled conditions, but reduced robustness when exposed to prolonged environmental stress, with RNA-based markers generally outperforming microbial indicators in terms of persistence.

The forensic reliability of vaginal secretions was consistently rated as moderate to low, with scores strongly influenced by time since deposition and environmental exposure. Johannessen et al. [9] demonstrated that vaginal mucosa-specific mRNA markers were highly detectable only within the first 12–36 h post-deposition, indicating strong short-term detectability but very limited persistence. By contrast, Zhang et al. [32] and Khorwal et al. [40] reported moderate stability over several days when evaluating vaginal samples using microbiome composition or DNA concentration. Overall, vaginal secretions appear to be most informative within a narrow temporal window and exhibit low long-term environmental robustness, resulting in moderate detectability but poor persistence under extended exposure.

Saliva consistently received the lowest reliability scores across all included studies, reflecting rapid degradation and high environmental sensitivity. Sirker et al. [31] observed that salivary mRNA markers deteriorated quickly, particularly under humid conditions. Similarly, Al-Kandari et al. [30] reported rapid loss of salivary DNA even under standard indoor storage conditions, indicating poor intrinsic stability. Zhang et al. [32] further demonstrated that salivary microbiome profiles underwent pronounced compositional shifts within 7–10 days, reinforcing the conclusion that saliva is highly susceptible to moisture, temperature variation, and substrate effects. Borde et al. [42] likewise reported markedly reduced recovery of salivary components from porous and water-exposed substrates, supporting the consistently low reliability scores assigned to saliva across differing analytical approaches. Taken together, these studies consistently indicate that saliva exhibits low detectability, limited persistence, and minimal environmental robustness.

When findings across Table 5 and Table 6 are analysed, blood and semen emerge as the most forensically reliable body fluids under variable environmental and evidence collection conditions. Blood, in particular, demonstrates superior persistence and detectability owing to its high concentration of nucleated cells, which provides a substantial reservoir of amplifiable DNA. Multiple studies have shown slower degradation of STR-targetable fragments in blood compared with saliva and vaginal secretions, even under room-temperature exposure, supporting its consistently high reliability scores [30]. The stability of haemoglobin-based signals and successful field detection further reinforce the resilience of blood in practical forensic contexts [43]. Nolan et al. [44] similarly reported prolonged persistence of blood-derived cellular material following water exposure, highlighting its strong environmental robustness.

In contrast, vaginal secretions exhibit only moderate reliability due to rapid molecular degradation following environmental exposure. Vaginal mucosal markers decline sharply beyond the initial 12–36 h post-deposition window, rendering them highly time-sensitive [9], a pattern corroborated by early microbiome instability reported by Zhang et al. [32]. Saliva consistently represents the least reliable fluid, as DNA and RNA markers are rapidly lost under both controlled and uncontrolled conditions, resulting in low persistence and poor environmental robustness across all reviewed studies [30,31,32].

Based on the comparative analysis of persistence, degradation, and methodological performance across included studies, the following best-practice recommendations are proposed (

Table 7. Recommended best practices for the forensic detection and interpretation of major biological fluids in sexual assault investigations.

Biological Fluid	Optimal Substrate for Recovery	Recommended Collection Window	Preferred Analytical Methods	Key Environmental Vulnerabilities	Major Interpretation Limitations	Practical Best-Practice Recommendation
Semen	Non-porous surfaces (plastic, metal) for surface recovery; porous fabrics may retain DNA internally	Within 72–96 h post-assault	Acid phosphatase (screening), PSA/p30, RSID-Semen, STR profiling, mRNA markers, emerging proteomics	Heat and humidity accelerate DNA degradation; UV reduces stain visibility	Azoospermic samples; mixed vaginal fluid; dilution	Prioritise early collection; combine immunological and molecular methods; interpret mixed samples cautiously
Blood (Peripheral)	Both porous and non-porous; relatively stable on dry substrates	Up to several days if protected from extreme conditions	TMB presumptive test, confirmatory haem tests, STR profiling	Prolonged humidity promotes microbial growth; UV exposure reduces DNA integrity	Cannot indicate timing of deposition without contextual data	Ensure proper drying before storage; document environmental exposure
Menstrual Blood	Porous fabrics may retain cellular material	As early as possible due to reduced haemoglobin concentration	Microscopy (cellular composition), haemoglobin quantification, ABH typing, emerging proteomics	Degradation of cellular components in high humidity	Misclassification as peripheral blood	Combine cellular, biochemical, and molecular markers for differentiation
Saliva	Non-porous surfaces yield easier surface recovery; porous substrates reduce detectability	Within 24–48 h preferred	RSID-Saliva (α-amylase), mRNA markers, microbiome profiling, proteomics	Rapid enzymatic degradation; environmental contamination	α-amylase present in other fluids (false positives)	Use confirmatory molecular markers; avoid sole reliance on amylase-based assays
Vaginal Secretions	Porous textiles may retain epithelial cells	Within 24–72 h	mRNA profiling (e.g., MYOZ1), DNA profiling, proteomic biomarkers	Heat and moisture accelerate RNA degradation	Difficult differentiation from menstrual blood or mixed semen samples	Use multi-marker approach (mRNA + proteomics where possible)
Mixed Fluids	Recovery depends on dominant fluid and substrate	Immediate collection strongly advised	STR mixture interpretation, probabilistic models, mRNA multiplex, proteomics	Differential degradation rates complicate interpretation	High risk of false exclusion or misinterpretation	Apply probabilistic interpretation frameworks; report limitations explicitly

).

The forensic and legal implications of degraded or mixed biological fluids are substantial, particularly in sexual assault investigations where evidential interpretation often occurs in the absence of eyewitness testimony. Environmental exposure, substrate characteristics, and delayed evidence collection may compromise DNA integrity and reduce the reliability of presumptive and confirmatory tests [5,27]. In mixed-fluid scenarios, overlapping biochemical or molecular markers may further complicate interpretation, increasing the risk of false-positive or false-negative conclusions if results are considered in isolation [2]. Such limitations are especially critical in judicial contexts, where biological findings may strongly influence charging decisions, trial strategy, and jury perception [4]. As highlighted by probabilistic interpretative frameworks, including Bayesian network approaches, forensic conclusions should be expressed within the context of transfer, persistence, and background probabilities rather than as absolute statements [10]. Moreover, delays in reporting and sample collection, as discussed in the context of Sexual Assault Medical Forensic Examinations [6], may substantially reduce evidential value, thereby affecting both investigative direction and prosecutorial strength. Collectively, these considerations underscore the necessity for cautious interpretation, transparent reporting of limitations, and continued development of high-specificity analytical techniques to minimise the risk of evidential misinterpretation in court.

Messenger RNA (mRNA) profiling has emerged as a molecular approach for body fluid identification based on the detection of tissue-specific gene expression markers. Analytical workflows typically involve reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) to amplify fluid-specific transcripts [8]. While mRNA provides greater tissue specificity than genomic DNA, it is generally more susceptible to degradation due to its single-stranded structure and enzymatic instability. Time-dependent degradation of mRNA has therefore been investigated as a potential indicator of time since deposition; however, transcript stability varies considerably depending on environmental exposure, substrate type, and initial biological load [5]. Consequently, although mRNA profiling enhances body fluid discrimination, its interpretative reliability in temporal estimation remains constrained by environmental and substrate-mediated degradation processes.

3.4. Emerging Proteomic Approaches for Body Fluid Identification

The field of forensic serology is undergoing a marked transition from conventional immunochromatographic assays towards advanced omic-based technologies, among which proteomics has emerged as a particularly promising approach for body fluid identification [11,45]. Traditional serological methods, although widely adopted in routine casework, may lack sufficient specificity and sensitivity when applied to complex or environmentally compromised samples. Cross-reactivity, limited discriminatory capacity, and reduced performance in low-template or mixed samples remain recognised limitations [11,46]. In response, mass spectrometry (MS)-based proteomic techniques have been developed to enable the detection of fluid-specific protein biomarkers with high analytical precision.

Developmental validation studies of multiplex liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays have demonstrated the feasibility of simultaneously identifying multiple body fluids within a single analytical workflow. These assays target characteristic peptide markers derived from proteins preferentially expressed in specific fluids, including Semenogelin I and II for semen and α-amylase for saliva [45]. Validation frameworks have assessed key forensic performance parameters such as sensitivity, specificity, reproducibility, cross-reactivity, and mixture interpretation capability. Reported findings indicate high specificity with minimal cross-reactivity between fluids, as well as robust performance in degraded and mixed samples. Importantly, protein biomarkers may persist under conditions where nucleic acids have undergone substantial degradation, thereby offering a complementary evidential avenue in samples where DNA profiling is compromised.

Comparative assessments further suggest that methodological choice significantly influences analytical reliability. Shotgun proteomic approaches, which aim to identify proteins across a broad dynamic range, have demonstrated high but imperfect specificity, with occasional low-level marker detection in non-target fluids and susceptibility to instrument carryover [11]. By contrast, targeted proteomic strategies, which focus on predefined peptide panels, have exhibited superior specificity and markedly reduced error rates in comparative evaluations, successfully differentiating blood, semen, and saliva without false-positive detection [11]. Nevertheless, targeted assays remain dependent on the inclusion of appropriate biomarker panels and may fail to detect fluids for which markers have not been incorporated, such as vaginal secretions or urine.

Efforts to streamline proteomic workflows have led to the development of protease-free or peptidomic approaches, which exploit naturally occurring endogenous proteolysis within biological fluids [46]. For example, Semenogelin proteins are physiologically cleaved by prostate-specific antigen generating characteristic peptides that can be directly targeted without the need for extensive enzymatic digestion. By reducing sample preparation to simplified extraction protocols, these approaches have demonstrated the capacity to detect seminal biomarkers in minute quantities, including sub-microlitre samples recovered from sexual assault swabs [46]. Such simplification may enhance feasibility in routine forensic laboratories.

Further innovation is evident in automated immunoaffinity mass spectrometry (IA-MS), which integrates antibody-based enrichment with MS detection [47]. Unlike traditional lateral flow immunoassays, antibodies in IA-MS serve to selectively capture and concentrate target peptides prior to MS analysis, thereby substantially increasing analytical sensitivity. Reported detection thresholds have reached sub-nanolitre levels of seminal fluid, while automation reduces manual processing time and may contribute to alleviating forensic laboratory backlogs [47].

Beyond analytical sensitivity, proteomic approaches offer contextual advantages in complex forensic reconstructions. In a recent case application, proteomic profiling enabled the identification of salivary proteins alongside gastric enzymes and dietary proteins, providing interpretative context that extended beyond conventional DNA profiling [48]. Such findings illustrate the broader evidential potential of proteomics, particularly in cases involving degraded samples, mixed fluids, or interpretative challenges not resolvable through genetic analysis alone.

Collectively, emerging proteomic methodologies demonstrate substantial promise as complementary tools to mRNA profiling and DNA methylation assays. While challenges remain regarding standardisation, instrumentation requirements, and validation across diverse forensic contexts, proteomics offers enhanced specificity, resilience in degraded samples, and expanded interpretative capability. Within sexual assault investigations—where environmental exposure, substrate effects, and delayed reporting frequently compromise nucleic acid integrity, proteomic assays represent a significant advancement in the pursuit of reliable and contextually informative body fluid identification.

4. Conclusions

This systematic review demonstrates that the forensic reliability of biological body fluids in sexual assault investigations is highly variable and strongly influenced by fluid-specific biological composition, persistence over time, and susceptibility to environmental exposure. Among the fluids examined, blood and semen consistently exhibited the highest forensic reliability, characterised by strong detectability, sustained molecular integrity, and greater resistance to degradation caused by heat, humidity, and substrate type. These properties make blood and semen particularly valuable for evidential analysis, even in cases involving delayed collection or challenging environmental conditions.

Vaginal secretions demonstrated moderate forensic reliability, providing valuable evidential information primarily within a narrow temporal window following deposition. Although highly informative in the early post-assault period, vaginal mucosal markers declined rapidly with environmental exposure, limiting their usefulness in delayed investigations. In contrast, saliva consistently displayed the lowest stability across all analytical approaches, with DNA, RNA, and microbiome signatures degrading rapidly under both controlled and uncontrolled conditions. This pronounced sensitivity to environmental stress substantially reduces the evidential value of saliva in scenarios involving delayed reporting or extended environmental exposure.

Collectively, these findings provide an evidence-based framework to support informed decision-making in sexual assault investigations. By clarifying fluid-specific degradation patterns and environmental sensitivities, this review can assist forensic practitioners in prioritising evidence collection, selecting appropriate analytical techniques, and interpreting degraded biological material more accurately. Ultimately, the adoption of fluid-informed and context-sensitive forensic strategies has the potential to enhance the reliability and evidential strength of biological evidence in sexual assault casework.