DNA Recovery from Blood and Seminal Fluid Stains Exposed to Sodium Hypochlorite (Bleach)

Abstract

Physical and sexual violence remains among the most prevalent crimes in contemporary society, with women and children comprising the majority of victims. In such cases, biological evidence—such as blood, semen, and saliva—plays a critical role in the investigation and resolution of criminal incidents. However, this evidence may be compromised, either unintentionally due to environmental exposure or intentionally by the perpetrator or victim, with the aim of concealing the crime.

This study investigates the potential for DNA recovery from blood and semen stains that have been deliberately exposed to diluted sodium hypochlorite (bleach), a common household chemical used in attempts to destroy forensic evidence. Biological stains were deposited on various fabric types and subsequently subjected to washing with bleach and detergent at 90°C. The samples were then examined using ultraviolet (UV) light and luminol for preliminary detection, followed by DNA isolation and quantification procedures.

The findings demonstrate that DNA recovery is possible even after exposure to bleach, thereby affirming the viability of forensic analysis under compromised conditions. This study underscores the potential for retrieving genetic material in scenarios where biological evidence has been subjected to intentional destruction, thereby contributing valuable insights to forensic science and criminal investigations.

Introduction

Forensic genetics is a scientific discipline frequently utilized in the resolution of criminal cases, the identification of perpetrators or victims, and the establishment of relationships between crime scenes and individuals. Since the 1990s, the application of DNA analysis to biological evidence collected from crime scenes has marked a new era in forensic investigations. The identification process from collected evidence is made possible through DNA isolation techniques. By comparing DNA profiles obtained from crime scene samples with those from reference samples, identification studies are conducted.^1–6

As in other forensic cases, the presence of biological evidence is critically important in incidents of sexual assault and abuse. Depending on the victim’s ability or attempt to resist during the act, a substantial amount of biological material may be present. The seminal fluid of perpetrators can be detected in the victim’s anal, oral, or genital regions, on their body, clothing, or on items involved in the act, such as bed linens, sheets, blankets, carpets, sofa upholstery, or—if used—inside condoms. The victim’s body is among the most important sources of forensic samples.^6–9

Following the incident, the victim may experience psychological distress and attempt to cleanse themselves as a coping mechanism, or the perpetrator(s) may intentionally try to eliminate evidence. Such actions may include washing, burning, or wiping of evidence. While the goal for perpetrators is often to destroy evidence, numerous studies have shown that it is still possible to recover DNA from clothing exposed to multiple washes, including at various temperatures, and using different methods. Although limited, the literature includes some studies on DNA recovery from samples exposed to diluted sodium hypochlorite (bleach) in addition to washing. However, the concentration of diluted bleach, the duration of exposure, and the type of substrate (fabric/surface) significantly affect the quantity of DNA that can be recovered.^9–13

In this study, the aim was to recover DNA using the Spin Column Method following serological testing of two types of fabric containing blood and semen stains that had been exposed to diluted sodium hypochlorite (bleach) and subsequently washed. The primary objective was to determine whether identification is possible from evidence intentionally or unintentionally subjected to bleach—either as a means of destruction or due to psychological distress—by assessing the feasibility of DNA recovery under such conditions.

Material and Methods

In this study, semen samples were obtained from 20 healthy male volunteers aged over 18 years, and blood samples were collected from ten healthy individuals who provided informed consent to participate in the study.

The biological samples were applied as stains onto pre-sterilized denim and cotton fabric types. The stained fabrics were allowed to air-dry at room temperature and subsequently examined under ultraviolet (UV) light. Characteristic blue-white luminescence, indicative of the presence of blood and seminal fluid, was observed and documented through photography.

Following initial documentation, the stained fabric samples were exposed to diluted sodium hypochlorite (bleach) for 15 minutes. Thereafter, each participant’s samples were grouped and washed in a washing machine at 90°C with detergent.

After the washing process, the fabric samples were left to air-dry at room temperature for 24 hours. Once completely dry, the samples were re-examined under UV light and re-photographed. DNA was then extracted from the stained areas of the fabric using standard isolation procedures, and the quantity of DNA recovered from each sample was measured.

Preparation of Fabric Samples

Two types of fabric—denim and cotton—were used in this study. To prevent potential contamination, all fabric materials were new and unused. The fabrics were initially washed at 90°C using ARIEL-brand liquid detergent in a BEKO WD 854 YK washing machine, followed by a second rinse cycle without detergent. After drying at room temperature for 24 hours, the fabrics were sterilized under UV light for three hours. Sterile scissors were used to cut the fabrics into 5 × 5 cm squares, which were then prepared for the application of blood and semen samples.

Collection of Blood Samples

Blood samples were collected from 20 volunteers who provided written informed consent, using sterile syringes. All procedures were carried out under sterile conditions, with the use of appropriate personal protective equipment.

Collection of Semen Samples

Semen samples were collected from 20 male volunteers using sterile containers in their home environments. Participants were provided with detailed instructions to prevent contamination during the collection and transport process. All samples were transported to the laboratory under cold chain conditions at +4°C.

Preparation of Semen and Blood Stains

For each volunteer, four fabric samples (two cotton and two denim) were designated, with one sample of each fabric type left untreated to serve as a reference for baseline DNA quantity. Upon arrival at the laboratory, semen samples were processed immediately to prevent degradation. Using a sterile Pasteur pipette, 1 mL of semen was applied to each fabric square. Blood samples were collected from 20 volunteers using sterile syringes, and 1 mL of blood was similarly applied to the designated fabric squares. All samples were allowed to air-dry at room temperature for 24 hours and subsequently stored in paper envelopes until further analysis.

Exposure of Stained Fabrics to Diluted Sodium Hypochlorite (Bleach)

Dried and packaged fabric samples were exposed to a diluted bleach solution prepared by mixing 100 mL of ACE-brand bleach (containing 5% sodium hypochlorite) with 1 L of water. Each stained fabric piece was fully submerged in the solution for 15 minutes, then thoroughly rinsed with distilled water before being grouped for subsequent washing.

Washing of Stained Fabrics

The bleach-treated fabric samples were washed in a BEKO WD 854 YK washing machine at 90°C using Ariel liquid detergent. Blood- and semen-stained fabrics from each volunteer were processed separately to prevent cross-contamination. In total, 40 blood-stained and 40 semen-stained samples were analyzed. To ensure no carryover contamination between washes, the machine was run empty with detergent between each group.

Detection of Residual Stains Using Luminol and UV Light

After the washing process, residual biological stains were examined using both luminol testing and UV light. All visual findings were photographed and recorded using a Canon EOS 500 camera.

Detection of Blood Stains Using Luminol

Luminol reagent (Merck/Sigma-Aldrich) was freshly prepared and applied to the washed fabric samples in a dark environment. The presence of blood was confirmed by characteristic blue-white chemiluminescence, which was documented photographically.

Detection of Semen Stains Using UV Light

Semen stains were visualized using an Obelüx-brand multi-wavelength light source (350–400 nm) in combination with orange-filtered goggles. The typical blue-white fluorescence emitted by semen was observed and recorded through photographic documentation.

DNA Isolation

Following visual detection, all samples were coded based on stain type and donor identity. Under sterile conditions, 0.5 cm² sections were excised from each stained fabric and placed into 2 mL microcentrifuge tubes. DNA extraction was performed using the QIAamp® DNA Investigator Kit, according to the manufacturer’s protocol. ¹⁴

DNA Quantification

The concentration of the extracted DNA was measured using a Qubit 4.0 Fluorometer and the Qubit dsDNA HS Assay Kit. Each sample was prepared by combining 190 µL of working solution with 10 µL of extracted DNA. Quantification was performed in triplicate, and results were expressed in ng/µL. All procedures were conducted under light-sensitive conditions, with aluminum foil used to protect reagents from light exposure. ¹⁵

Statistical Analysis

The Wilcoxon signed-rank test and the Mann–Whitney U test were performed using SPSS to evaluate the impact of sodium hypochlorite treatment and fabric type on DNA recovery.

Results

Blood- and semen-stained fabric samples were treated with diluted sodium hypochlorite (bleach) and subsequently subjected to washing in an automatic washing machine at 90°C with the addition of detergent. After the washing process, the samples were air-dried at room temperature for 24 hours prior to DNA analysis. Semen stains were visualized under UV light using presumptive screening tests, whereas blood stains were identified through luminol application. Despite exposure to both chemical and thermal treatment, the biological stains remained detectable. Characteristic blue-white luminescence was observed and documented photographically (Figure 1).

Figure 1.

Note: *The images demonstrate the persistence or degradation of biological traces following chemical and thermal treatment commonly associated with attempted forensic evidence removal.

DNA was successfully recovered from untreated control samples (40 denim and 40 cotton). For blood-stained fabrics, DNA concentrations ranged from 0.109 to 5.5 ng/µL in cotton and 0.316–8.34 ng/µL in denim (Tables 1 and 2). In semen-stained fabrics, concentrations ranged from 0.076 to 14 ng/µL in cotton and 0.068–4.57 ng/µL in denim (Tables 2 and 3).

Table 1.

DNA Concentrations in Blood-stained Fabric Samples Exposed to Diluted Sodium Hypochlorite and Washed at 90°C.

Blood Samples	Untreated Cotton Samples (ng/µL)	Cotton Fabric Exposed to Diluted Sodium Hypochlorite DNA Quantification (ng/µL)	Untreated Denim Samples (ng/µL)	Denim Fabric Exposed to Diluted Sodium Hypochlorite DNA Quantification ng/µL)
1.	1.91	0.868	1.49	1.12
2.	2.99	0.358	0.98	0.947
3.	4.62	3.06	6.11	2.70
4.	2.96	1.25	0.752	0.702
5.	1.62	0.487	0.80	0.716
6.	3.013	0.456	1.32	0.929
7.	1.39	0.354	1.16	1.02
8.	4.126	1.10	0.716	0.658
9.	0.109	0.0899	0.773	0.474
10.	3.16	0.370	4.08	1.17
11.	2.5	2.09	0.982	0.828
12.	2.23	1.73	1.36	0.80
13.	3.01	2.86	0.177	0.340
14.	4.76	3.47	0.162	0.105
15.	4.68	3.29	0.484	0.430
16.	2.65	2.22	0.752	0.548
17.	5.5	3.03	0.316	0.214
18.	2.7	2.19	1.16	0.88
19.	1.96	1.96	0.716	0.606
20.	1.76	1.76	8.34	6.58
Mean	2.98	1.65	1.63	1.09
STD	1.23	1.11	2.11	1.40

Table 2.

Minimum, Maximum, and Mean Values of DNA Quantities Obtained From Blood- and Semen-stained Samples Exposed to Diluted Sodium Hypochlorite (Bleach) and Subsequently Washed at 90°C.

		Untreated Samples			Samples Exposed to Diluted Sodium Hypochlorite (Bleach)
Blood stains		Min DNA (ng/µL)	Max DNA (ng/µL)	Mean DNA (ng/µL)	Min DNA (ng/µL)	Max DNA (ng/µL)	Mean DNA (ng/µL)
	Cotton	0.109	5.5	2.98	0.0899	3.47	1.65
	Denim	0.316	8.34	1.63	0.105	6.58	1.09
Semen stains		Min DNA (ng/µL)	Max DNA (ng/µL)	Mean DNA (ng/µL)	Min DNA (ng/µL)	Max DNA (ng/µL)	Mean DNA (ng/µL)
	Cotton	0.076	14	1.9	0.056	7.9	0.9
	Denim	0.068	4.57	1.84	0.023	2.76	0.42

Following treatment with diluted sodium hypochlorite and washing at 90°C, DNA was successfully extracted from all 40 denim and 40 cotton samples. For blood-stained fabrics, DNA concentrations ranged from 0.0899 to 3.47 ng/µL in cotton and 0.105–6.58 ng/µL in denim (Table 1 and Figure 2). Similarly, in semen-stained fabrics, concentrations ranged from 0.056 to 7.9 ng/µL in cotton and 0.23–2.76 ng/µL in denim (Tables 2 and 3, and Figure 2).

Table 3.

DNA Concentrations in Semen-stained Fabric Samples Exposed to Diluted Sodium Hypochlorite and Washed at 90°C.

Semen Stains	Untreated Cotton Samples (ng/µL)	Cotton Fabric Exposed to Diluted Sodium Hypochlorite DNA Quantification (ng/µL)	Untreated Denim Samples (ng/µL)	Denim Fabric Exposed to Diluted Sodium Hypochlorite DNA Quantification (ng/µL)
1.	3.1	1.77	4.570	2.76
2.	0.0760	0.0564	3.707	0.712
3.	2.5	1.43	0.921	0.379
4.	2.1	0.822	0.471	0.234
5.	1.9	0.496	0.944	0.402
6.	1.1	0.0947	0.068	0.0380
7.	1.25	1.12	0.191	0.162
8.	1.1	0.0755	0.434	0.362
9.	1.4	0.163	1.037	0.464
10.	0.133	0.0954	0.889	0.234
11.	0606	0.319	0.889	0.520
12.	0.318	0.166	0.921	Too Low
13.	1.04	0.547	1.037	0.678
14.	0.500	0.155	0.068	0.023
15.	0.799	0.232	10.6	0.191
16.	0.680	0.432	0.471	0.084
17.	1.7	0.450	0.944	0.058
18.	0.606	0.272	3.707	0.398
19.	14	7.9	0.434	0.023
20.	4.6	1.5	4.570	0.275
Mean	1.9	0.9	1.84	0.42
STD	2.9	1.6	2.53	0.60

Figure 2.

Comparison of Minimum, Maximum, and Mean DNA Quantities Recovered From Blood- and Semen-stained Cotton and Denim Fabrics, Both Untreated and Treated with Diluted Sodium Hypochlorite Followed by Washing at 90°C.

According to the Wilcoxon Signed Ranks Test results, there was a statistically significant difference between pre- and post-exposure detection values for both blood and semen stains on cotton and denim fabrics (p < .001). These findings indicate that exposure to diluted sodium hypochlorite significantly affects the detectability of biological traces, likely reducing the visibility or detection potential of the stains. The negative Z-scores suggest that post-exposure detection levels were generally lower than those observed before treatment, implying that sodium hypochlorite has a destructive or degrading impact on biological evidence deposited on textile surfaces (Table 4).

The Mann–Whitney U test demonstrated that the reduction in DNA yield following sodium hypochlorite treatment differed significantly between cotton and denim fabrics for blood-stained samples but not for semen-stained samples (Blood: U = 287.00, p = .019; Semen: U = 200.50, p = 1.000) (Table 5).

Table 4.

Wilcoxon Test Results.

Test Statisticsa
	Blood		Semen
	Cotton Exposed to Diluted Sodium Hypochlorite—Cotton Not Exposed	Denim Exposed to Diluted Sodium Hypochlorite—Denim Not Exposed	Cotton Exposed to Diluted Sodium Hypochlorite—Cotton Not Exposed	Denim Exposed to Diluted Sodium Hypochlorite—Denim Not Exposed
Z	−3.724b	−3.509b	−3.920b	−3.920b
Asymp. sig. (2-tailed)	0.000	0.000	0.000	0.000

Notes: ^aWilcoxon signed ranks test.

^bBased on positive ranks.

Table 5.

Mann–Whitney U Test Results.

Sample	Comparison	U Value	Z Value	p (2-tailed)
Blood	Cotton & denim	113.000	–2.353	.019
Semen	Cotton & denim	200.000	0.000	1.000

Discussion

Sexual assault and abuse rank among the most prevalent crimes in contemporary society, with victims primarily comprising women and children, although male victims are also reported. In forensic investigations of such incidents, biological evidence yields the most conclusive findings. Despite its high reliability, this type of evidence is extremely vulnerable to environmental and physical factors, which may complicate its admissibility and interpretation in judicial processes. Victims, often experiencing psychological trauma, may inadvertently compromise forensic evidence by washing the clothing worn during the assault or cleaning affected surfaces such as sofas, beds, or bed linens. Similarly, perpetrators may deliberately attempt to eliminate biological traces by cleansing themselves, their clothing, or the crime scene. The use of condoms in sexual assault cases is infrequent; consequently, absorbent materials such as bedding, clothing, and upholstery often retain significant amounts of forensic biological material.^{9, 11} Studies have demonstrated that, despite attempts at intentional destruction or cleaning prompted by psychological trauma, it is still possible to recover trace amounts of DNA from laundered fabrics. ⁹

An evaluation of the findings obtained in this study, which was specifically designed to assess DNA recovery after exposure to diluted sodium hypochlorite and subsequent washing, presents comparative results for cotton and denim fabrics:

In line with the aim of this study, blood and semen samples were applied to two different fabric types—cotton and denim—and subsequently exposed to diluted sodium hypochlorite (bleach). After this chemical exposure, the fabrics were washed in a washing machine at 90°C with detergent. Despite the harsh treatment conditions, a small amount of DNA was successfully recovered from the samples.

The primary conclusion of this study is that it is possible to recover DNA from samples exposed to diluted sodium hypochlorite for 15 minutes and subsequently washed at 90°C with the addition of detergent. Variations in the amount of DNA recovered from different samples are believed to result from several factors, including the molecular structure of the fabric used, the chemical properties of the diluted sodium hypochlorite and detergent, and the duration of exposure. These variables are considered to significantly influence the amount of DNA recovered under the experimental conditions.

DNA Recovery by Fabric Type

For blood samples: A higher amount of DNA was recovered from denim fabric compared to cotton fabric (Table 1). This difference is attributed to the tightly woven and rigid structure of denim, which likely facilitates better retention of blood cells, preventing them from being easily dislodged during washing.

For semen samples: Like the results observed with blood samples, more DNA was recovered from denim fabric than from cotton fabric (Table 3). Although the biological sample type differed, the same explanation applies: the dense weave and firm structure of denim provide a surface that promotes the adherence of semen cells and reduces their loss during washing.

Despite exposure to sodium hypochlorite and subsequent washing at 90°C, DNA was still recoverable from both blood- and semen-stained fabrics. An average yield of 1.65 ng/µL was obtained from bloodstains on cotton and 1.09 ng/µL from bloodstains on denim, while semen-stained fabrics yielded 0.61 ng/µL on cotton and 0.42 ng/µL on denim. These results demonstrate that measurable quantities of DNA can persist even after combined chemical and thermal treatment.

Overall, statistical analyses demonstrated that sodium hypochlorite exposure significantly reduced DNA yields in both blood- and semen-stained fabrics, regardless of sample type (Wilcoxon signed-rank test: Z ≈ –3.80, p < .001). However, the extent of DNA reduction differed significantly between cotton and denim fabrics for blood-stained samples, but not for semen-stained samples (Mann–Whitney U test: Blood, U = 287.000, p = .019; Semen, U = 200.500, p = 1.000). These results indicate that while bleach consistently compromises DNA integrity, its impact on DNA degradation varies with fabric type in bloodstains but remains uniform across fabric types in semen stains, highlighting its destructive role in forensic DNA analysis.

Suitability of Recovered DNA for Identification

Fluorometric analysis indicated that DNA concentrations from bloodstains ranged from 0.08 to 3.47 ng/µL on cotton and from 0.105 to 6.58 ng/µL on denim. Considering that full STR profiles can be obtained with ≥0.125 ng/µL using GlobalFiler™ kits, ¹⁶ successful identifications were achievable in 95% of cotton and 90% of denim bloodstain samples, even following bleach exposure and washing at 90°C (Table 1).

For semen stains, DNA concentrations ranged from 0.05 to 7.9 ng/µL on cotton and from 0.02 to 2.76 ng/µL on denim. Full STR profiles were successfully obtained in 80% of cotton and 65% of denim samples, indicating higher identification success on denim under the same chemical and thermal treatment conditions (Table 3).

Comparison with Previous Studies

In a 2018 study, Thabet et al. examined the effects of various cleaning agents, including bleach (Clorox), disinfectant (Dettol), stain remover (Vanish), detergent (Persil), and distilled water—on DNA recovery from blood-stained cotton and silk fabrics. Their results showed significantly lower DNA yields from silk compared to cotton across all treatments, with Vanish causing the greatest reduction. Clorox-treated samples yielded the highest DNA quantities for both fabric types. Gel electrophoresis revealed that Vanish and Dettol had the most detrimental effects on DNA quality, while Clorox, Persil, and distilled water had minimal impact. ¹⁷ Unlike Thabet et al.’s study, the present research incorporated both diluted bleach exposure and washing at 90°C with detergent. DNA was successfully isolated and quantified from all cotton and denim samples containing blood and semen (Tables 1 and 3). Consistent with Thabet et al., our findings also indicate that fabrics with lower fiber density, such as cotton, show greater DNA loss than denser fabrics like denim.

In a study by Ünsal Sapan et al., semen-stained samples on two different fabric types (cotton and nylon) were washed at 40°C, 60°C, and 90°C with detergent, and subsequently analyzed for DNA presence and suitability for profiling. DNA extraction was performed using both the spin column and organic methods, with successful recovery from all samples. The organic method yielded higher DNA quantities. Notably, DNA profiles were obtainable even after washing at 90°C with detergent. ¹² In contrast to this study, the present research involved prior exposure of samples to diluted sodium hypochlorite (bleach), a chemical known for its high DNA-degrading potential. Based on the findings of Ünsal Sapan et al., which demonstrated successful profiling at 90°C, all samples in the current study were washed at this temperature following bleach treatment. Consistent with their results, DNA was successfully extracted and quantified from all samples despite the additional chemical exposure.

Similarly, de Silva Wijeyeratne and Gweon (2025) assessed the effectiveness of sodium hypochlorite for genomic DNA decontamination across different concentrations. Their results showed that low concentrations (≤0.1%) allowed partial DNA detection, whereas concentrations of 0.5% and above completely inhibited DNA amplification and fragment visualization. These findings reinforce the highly disruptive role of sodium hypochlorite on genetic material, supporting the current study’s conclusion that bleach exposure significantly compromises DNA integrity, even though measurable quantities may still persist under certain conditions. ¹⁸

Similarly, a study published in Forensic Genomics (2023) investigated the persistence of human DNA after exposure to common household cleaning agents such as soap, bleach, and hydrogen peroxide. The authors demonstrated that while bleach and hydrogen peroxide caused significant degradation, partial DNA detection and successful amplification remained possible in some cases. These findings align with the current study by emphasizing that, although sodium hypochlorite has a severe degrading effect on DNA, genetic material may still persist at detectable levels even after chemical treatment. ¹⁹

Conclusion

In this study, DNA was successfully extracted from all blood- and semen-stained fabric samples that had been treated with diluted sodium hypochlorite (bleach) for 15 minutes and subsequently laundered at 90°C using detergent. All samples were subjected to DNA isolation using the spin column method. The results indicate that DNA recovery is feasible following this protocol. Although DNA was successfully recovered from all fabric and stain types, the quantity of DNA obtained varied depending on the fabric type, suggesting a proportional relationship between fabric characteristics and DNA yield.

Although the quantity of DNA recovered was limited, likely because of diluted sodium hypochlorite, detergent, and high-temperature exposure, the results indicate that the recovered DNA was sufficient for identification purposes, even under conditions associated with significant degradation. This study provides the first evidence that DNA can be successfully recovered from blood- and semen-stained samples subjected to treatment with diluted sodium hypochlorite and subsequent laundering at 90°C with detergent.

This study demonstrates that DNA recovery is feasible even under conditions where forensic evidence has been deliberately or unintentionally compromised. Such scenarios may include attempts by perpetrators to destroy biological traces in cases of sexual assault or abuse, delays in reporting due to psychological trauma or societal pressure on the victim, or instances where victims themselves attempt to eliminate evidence due to emotional distress. Forensic evidence in these cases often consists of items such as clothing, bed linens, or other fabric-covered materials associated with the victim or offender. The findings indicate that, even after exposure to diluted sodium hypochlorite (bleach) and subsequent laundering at high temperatures with detergent, it is still possible to recover DNA from such evidence.