A Substitute Presumptive Test for Screening of Semen using Sodium –p– Nitrophenyl Phosphate (NaPNPP)

In forensic investigation of alleged sexual crimes, presumptive semen detection test methods are commonly used to reach preliminary identication of seminal uid in questioned samples. These methods are based on the detection of components of semen such as enzyme acid phosphatase (AP). Of these methods, the acid phosphatase identication method still remains the most reliable and widely used presumptive test due to high activity of the AP in seminal uid. In standard AP test, Bretamine Fast Blue B (FBB) reagent is used. However FBB has been explicitly classied as carcinogenic. Although FBB has been handled safely over time, there is a need at the moment to develop a simple, readily available, reliable and ecient method for screening the presence of semen in any material collected as evidence in a sexual assault crime. Given the improved sensitivity of DNA proling tests that have been introduced in to routine forensic casework over recent years, the need for improved sensitivity at this rst stage of detection has never been higher. Here we highlight a simple method using readily available reagents in standard biochemical laboratory as a substitute for the standard AP test for seminal uid identication from a crime scene. This method is based on the hydrolysis of sodium–p-nitrophenyl phosphate at pH 5.5 by the acid phosphatase to produce an intense yellow coloured complex in 15 seconds.


Abstract Background
In forensic investigation of alleged sexual crimes, presumptive semen detection test methods are commonly used to reach preliminary identi cation of seminal uid in questioned samples. These methods are based on the detection of components of semen such as enzyme acid phosphatase (AP). Of these methods, the acid phosphatase identi cation method still remains the most reliable and widely used presumptive test due to high activity of the AP in seminal uid.
In standard AP test, Bretamine Fast Blue B (FBB) reagent is used. However FBB has been explicitly classi ed as carcinogenic. Although FBB has been handled safely over time, there is a need at the moment to develop a simple, readily available, reliable and e cient method for screening the presence of semen in any material collected as evidence in a sexual assault crime.
Given the improved sensitivity of DNA pro ling tests that have been introduced in to routine forensic casework over recent years, the need for improved sensitivity at this rst stage of detection has never been higher.

Findings
Here we highlight a simple method using readily available reagents in standard biochemical laboratory as a substitute for the standard AP test for seminal uid identi cation from a crime scene. This method is based on the hydrolysis of sodium-p-nitrophenyl phosphate at pH 5.5 by the acid phosphatase to produce an intense yellow coloured complex in 15 seconds.

Conclusions
The method presented is sensitive, reliable, e cient and routinely used in standard biochemical and pathology laboratories for spectrophotometric analysis of alkaline phosphatase. It can be easily and readily applied as a preliminary test for identi cation of semen at a crime scene that involves sexual assault.

Background
Over the years several different methods have been utilized for identi cation of seminal uid at a crime scene (Laffan et al. 2011). Although new technologies are being researched and developed, traditional semen identi cation methods appear to remain the most widely applied in forensic casework (Gamblin and Morgan-Smith 2020). Of these methods acid phosphatase identi cation method still remains the most reliable and widely used presumptive test (Raju and Iyengar 1964;Rana et al. 2019).
Quantitative and qualitative AP testing is routinely used in biochemical analysis and more importantly in screening for the presence of semen in evidence found at a crime scene. AP is one of the forensically signi cant components of semen and the activity of this enzyme is usually very high in seminal uids (Gamblin and Morgan-Smith 2020). The standard screening AP test for semen involves the use of alpha-naphthyl phosphate as a substrate to produce alpha-naphthanol in the presence of AP which then has to combine with Bretamine Fast Blue B (Orthodianisidine / diazo blue dye) to form a violet / purple coloured complex within 2 minutes (Raju and Iyengar 1964).
In standard AP test, Bretamine Fast Blue B (FBB) reagent is used. However FBB has been explicitly classi ed as carcinogenic. Although FBB has been handled safely over time, there is a need to highlight sensitive, reliable, e cient and readily available methods for screening the presence of semen in any material collected as evidence in a sexual assault crime to address this gap. This is particularly important since evidence has shown that in order to get a good con rmatory DNA result, it is critical for the seminal uids to be collected within a speci c timeframe, preferably within the rst 24-48h (Gonçalves et al. 2017).
The substitute presumptive test presented here is sensitive, reliable, and e cient. The method utilizes readily available reagents routinely used in standard biochemical and pathology laboratories for spectrophotometric analysis of alkaline phosphatase (McComb et al. 1979). This method can be easily and readily applied as a substitute preliminary test for identi cation of semen at a crime scene that involves sexual assault. The method is based on the hydrolysis of sodium-p-nitrophenyl phosphate (NaPNPP) at pH 5.5 by the AP in semen to p-nitrophenol, a yellow chromophore as shown in Figure 1. Figure 1. The reaction between sodium-p-nitrophenyl phosphate (NaPNPP) and Acid Phosphatase Methods A buffered substrate consisting of 0.41g of citric acid, 1.125g of sodium citrate and 1.65mg of sodium-pnitrophenyl phosphate (NaPNPP) in 100ml distilled water is prepared at a pH of 5.5. The semen is extracted from material collected at a crime scene or swab by dampening a Whatman lter paper with distilled water and then pressed on the surface of the suspected stain on the cloth and left in position for approximately 10 minutes.
The paper is then removed and sprayed with the AP test reagents from the buffered substrate prepared using a chromatographic reagents sprayer. An intense yellow colour appears on the paper at the position where the stain has been extracted in less than 15 seconds depending on the amount of stain. As a negative control an unstained area on the known semen dilution strip doesn't exhibit any changes in colour within the 2 minutes threshold upon addition of these AP working solution.

Results
Using 0.05mL of a neat sample over a 12 hour interval for 48 hours, the results for the test are shown in Fig. 2. In the presence of sodium ions, p-nitrophenol produces the yellow coloured complex; which indicates the presence of semen. Since AP is not exclusive to human semen, it can only be used as a presumptive test for the presence of semen.

Discussion
In a standard AP test, a rapid colour change to purple is expected within 30 seconds to indicate a presumptive positive result for the presence of semen. An inconclusive result that indicates the possible presence of semen is shown by a colour change from 31-59 seconds. A presumptive negative result is indicated by no colour change at or after 60 seconds (Virkler and Lednev 2009).
However, as the method is currently implemented by operational forensic science laboratories allows 2 min for a reaction to be obtained beyond which a negative result is declared (Skalleberg and Bouzga 2016). Furthermore, recent research has shown that using the AP standard presumptive test semen could be detected in excess of 15 min depending on the dilutions using the press and test method (Lewis et al. 2012;Redhead and Brown 2013). The proposed substitute method falls within the timeframe that is indicated by standard operational forensic science laboratories.
Generally cases involving sexual assault are characterized by very low disclosure rates, reporting, prosecution, and conviction (Magalhaes et al. 2015). Due to the prevalence of sexually motivated crime, any improvements or alternatives to current practices and techniques would be welcomed.
Given the improved sensitivity of DNA pro ling tests that have been introduced in to routine forensic casework over recent years, the need for improved sensitivity at this rst stage of detection has never been higher (Laffan et al. 2011). It is therefore paramount to utilize all available robust and reliable methods for detecting semen during the forensic casework. Furthermore, AP test is one of the most common tests used by forensic laboratories for the detection of semen. Availability of this test and within the appropriate time frame as evidence indicates would be a signi cant addition to any forensic casework at hand. This substitute method presented herein is sensitive, reliable, e cient and readily available in standard laboratories. The method can additionally be used in routine forensic laboratory and experimental work owing to the health and safety concerns of the Bretamine Fast Blue B. As standard practice indicates, further con rmatory test such as DNA pro ling should be carried out adhering to the standard con rmatory procedures.

Abbreviations
NaPNPP -Sodium -p-Nitrophenyl Phosphate Figure 1 The reaction between sodium-p-nitrophenyl phosphate (NaPNPP) and Acid Phosphatase Figure 2 The results for the Acid Phosphatase test over 48 hours at a 12 hours interval