The Use of Polilights in the Detection of Seminal Fluid
Contents
1. can be used successfully to locate saliva stains for DNA analysis The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight however the Polilight has particular application in instances where a bloodstain may have been concealed with paint Overall the Polilight is a relatively safe simple noninvasive and nondestructive technique suitable for use in forensic casework KEYWORDS forensic science forensic biology alternate light source Polilight fluorescence acid phosphatase luminol Phadebas Detection of seminal stains on items such as clothing and bed ding can be of significance in sexual assault cases Similarly lo cating potential saliva or bloodstains on clothing or at crime scenes can also assist investigations Polilight is a versatile light source that produces intense narrow bands of light at wavelengths between 310 and 650 nm Polilight has application in latent fin gerprint detection 1 and may offer an alternative to other more commonly used chemical based screening tests for biological flu ids such as the acid phosphatase AP test for seminal fluid Phadebas paper for saliva or luminol for blood Polilight can potentially provide a rapid less labor intensive presumptive screening test particularly for large surfaces before further tests are used to confirm the presence of spermatozoa or blood In 1991 Stoilovic 1 demonstrated that the excitation sp2. 3 samples were prepared from areas identified as Polilight positive AP negative and screened for the presence of spermatozoa No spermatozoa were detected and no male profiles were observed following DNA analysis There were 19 concordant results 47 5 16 positive for both tests and three negative These results indicate the propensity of the Polilight test to detect stains in general whether they happen to be seminal stains saliva stains or stains of some other origin On the whole the Polilight method appears to be more sensitive than specific but given the relatively low incidence of false neg atives and its intended use as a presumptive test only it remains a very useful method for screening Experience may assist an ex aminer to better differentiate stains All stains identified whether by Polilight or AP would require further examination e g cy tologically to confirm the presence of spermatozoa Comparison of Seminal Fluid Saliva and Bloodstains with Other Fluid Stains Under Polilight All of the fluids present saliva blood semen urine tea vag inal and nasal secretions were detectable under Polilight Fig 7 For the majority of fluids tested the best results were obtained VANDENBERG AND VAN OORSCHOT THE DETECTION OF STAINS USING POLILIGHT 367 ee FIG 7 Equal volumes of fluids on white nylon at 450nm excitation viewed through orange goggles Clockwise from bottom right semen ur
3. Identification of Mock Casework Saliva Stains Using Polilight Mock casework saliva stains were also examined under Poli light These stains were prepared in the following manner tan colored nylon elastane stockings were worn for 3 min as a mouth gag a cream polyester cotton pillow case was wrapped around a person s face and held firmly in place for 3 min a white polyester cotton T shirt was spat on a white nylon elastane bra was licked and fingers that had been sucked on for 3 min were pressed down firmly on a pair of white cotton elastane boxer shorts in an attempt to transfer saliva An attempt was also made to recover DNA via the tape lift technique from an area identified under Polilight on the nylon elastane stockings as a potential saliva stain and DNA analysis was performed as above Examination of the Effects of Potentially Interfering Substances or Conditions on the Identification of Bloodstains Using Polilight Bloodstains 500 uL on white cotton cloth were mixed with a variety of substrates or exposed to different environmental con ditions and examined under Polilight to assess the effects These samples were prepared in the following manner blood mixed with soil 0 25 g and left to dry blood mixed with rust 0 25 g and left to dry dried blood covered with white fingerprint powder 0 25 g dried blood covered with black fingerprint pow der 0 25 g blood mixed with semen 250 uL each and left to dry b
4. Polilight vs AP AP testing of 40 casework exhibits after examination with Polilight was performed in accordance with standard proce dures within the Biology Division at the Victoria Police Forensic Services Laboratory The results of both examinations were recorded along with case details A positive control was tested before beginning the AP testing to ensure that the reagent was working properly Effects of Laundry Detergents To examine the effect of liquid laundry detergents under Polilight 2 3 mL of each detergent was placed onto individual VANDENBERG AND VAN OORSCHOT THE DETECTION OF STAINS USING POLILIGHT 363 TABLE Effect of washing with laundry detergents Stain Visible Under Polilight Description of Stain on nee rete gr eae White Polyester Before Washing After Washing Preen Yes Yes Sard f No No White King No No Earth Choice Yes No Omo g Yes No Cold Power Yes Yes Cuddly fabric softener Yes No Seminal fluid control Yes No samples of white polyester allowed to dry for 24h and observed under Polilight A list of the laundry detergents used is given in Table 1 The samples were then washed in warm or cold water as specified by the instructions on the detergent label and allowed to dry again for 24h No further detergent was added during wash ing The samples were then observed again under Polilight Comparison of Saliva and Blood to Other Fluids under Polilight
5. 0 Seminal stain on wood under three coats of white acrylic based paint a natural light and b 450 nm excitation viewed through orange goggles VANDENBERG AND VAN OORSCHOT THE DETECTION OF STAINS USING POLILIGHT 369 Another study 10 has also investigated the detectibility of bloodstains under painted surfaces using Polilight In the study a bloodstain on a painted wooden surface was painted over with successive coats of a latex paint Blood droplets that were not visible without the light source after more than two coats of the paint had been applied were locatable using the Polilight It is well known that luminol is not specific to blood and that other substances such as iron and copper compounds can produce visible chemiluminescence when exposed to luminol An experi enced practitioner may be able to distinguish luminescence cat alyzed by hemoglobin from that of luminescence produced from other substances based on the intensity duration or spatial dis tribution of the luminescence However Quickenden and Creamer 16 have shown that there are a number of substances that impart luminescence intensities comparable with hemoglobin when sprayed with luminol including enamel paint Dulux which produces a chemiluminescent intensity identical to the naked eye to that of hemoglobin Quickenden and Creamer 16 also showed that gloss acrylic paint Taubman matte finish paint Dulux and flat oil based paint produc
6. A number of biological and nonbiological fluids were placed in different locations on a single piece of white nylon and an attempt was made to differentiate them under Polilight These fluids in cluded 500 uL of saliva blood semen urine semen free vaginal swabs resuspended in dH 0 tea and nasal secretions undeter mined amount Recovery of DNA from Saliva Stains Identified with Polilight To determine whether the Polilight could aid in the determi nation of which areas to target for DNA analysis an attempt was made to identify prepared saliva stains using the Polilight The prepared stains included a known amount of saliva 200 uL ap plied to a known corner of a nylon square an undetermined amount of saliva applied to a known corner of a second nylon square and an undetermined amount of saliva placed in a corner of a third nylon square where the corner chosen was not known to the person performing the Polilight examination recovery of DNA All undetermined amounts of saliva were deposited by an individual placing a corner of the nylon in their mouth for 3 min The same individual s saliva was used in all cases this individ ual s DNA profile was known and to avoid possible contamina tion issues this individual was not involved in the process of recovering the DNA As a negative control a sample was also taken of the opposite corner to the saliva applied to a known cor ner of the second nylon square The tape l
7. Full profile known location Undetermined amount Yes 500 Full profile known location Undetermined amount Yes 500 Full profile unknown location Negative control No 0 No profile could be readily identified DNA profiles obtained from tape lifts of these white stained areas matched the DNA profile of the source of the sample No additional alleles were observed indi cating an absence of extraneous contamination DNA could be recovered from stains comprising 200uUL of saliva and more DNA was isolated from stains on nylon that had been placed in the mouth for 3 min These results show that Polilight can be used to successfully locate saliva stains on nylon from which DNA can then be recovered using the tape lift technique Identification of Mock Casework Saliva Stains Using Polilight Mock casework saliva stains were examined under Polilight These stains could be successfully located on the pillowcase bra stocking and T shirt Only on the boxer shorts was there no ap parent staining Table 3 shows that the most useful wavelength for detecting saliva on mock casework exhibits was 450 nm orange with stained areas on garments appearing white under Polilight Fig 8 DNA was successfully recovered from the stocking via a tape lift of an area of white staining identified using Polilight The profile obtained from this tape lift matched the DNA profile of the source of the sample These results further demonstrate that Polilight
8. TECHNICAL NOTE J Forensic Sci March 2006 Vol 51 No 2 doi 10 1111 j 1556 4029 2006 00065 x Available online at www blackwell synergy com Nicholas Vandenberg B Sc Hons and Roland A H van Oorschot Ph D The Use of Polilight in the Detection of Seminal Fluid Saliva and Bloodstains and Comparison with Conventional Chemical Based Screening Tests ABSTRACT Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes The use of a versatile light source may assist in their detection The ability of Polilight to locate potential semen saliva and blood stains on a range of substrates and at different dilutions was tested We also tested the use of Polilight in comparison with conventional chemical based presumptive screening tests such as acid phosphatase AP Phadebas and luminol often used in casework for detecting potential semen saliva and blood stains respec tively The Polilight was able to locate stains that were not apparent to the naked eye The color of the material on which a stain is deposited can have an effect on the detectibility of the stain The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity In a comparative study between the AP test and Polilight on 40 casework exhibits one false negative result was observed when using the Polilight On a series of mock casework exhibits it was determined that the Polilight
9. VAN OORSCHOT THE DETECTION OF STAINS USING POLILIGHT 365 FIG 3 Seminal fluid in a condom at 450nm excitation viewed through orange goggles Top used condom Bottom unused unrolled condom The narrow absorption band for blood dictates that the most functional wavelength setting to use for bloodstains is 415 nm with yellow goggles Under Polilight the majority of blood stains on the various materials tested were easily detectable at this wavelength with the exception of a bloodstain on green polar fleece For seminal stains the variability resulting from the degree of absorbency of materials was not as apparent using Polilight as reported previously in other studies 2 For example we found that seminal stains on highly absorbent blue velour and dark green polar fleece were easily detectable with Polilight although not as apparent to the naked eye under normal light Fig 5 Seminal stains observed with Polilight on material that appeared to have very little absorbency such as nylon where the stain appeared to sit on the surface of the fabric did not appear to be significantly greater in intensity than seminal stains on more absorbent material e g cotton polyester data not shown However for saliva and bloodstains absorbency of the material appears to be a factor Saliva stains that sat on top of a surface gave better results than saliva stains for the same material that appeared to have mostly been abso
10. acrylic based paint and observed under Polilight 364 JOURNAL OF FORENSIC SCIENCES Further 0 5 mL bloodstains on brick metal and plasterboard were also painted over using up to three coats of the white low sheen water based paint and these stains were also observed un der Polilight after the addition of each new coat of paint Lastly a bloodstain sandwich was prepared and observed under Poli light The sandwich was prepared as follows a piece of wood was first painted with white acrylic based paint and allowed to dry then a 0 5 mL bloodstain was applied and left to dry before the bloodstain was painted over with another coat of the white acrylic based paint Comparison Between Polilight and the Phadebas Method for the Identification of Saliva The Phadebas method is a test for amylase activity which is generally found at high levels in human saliva 9 Amylase activity may be absent in some people s saliva and can be found in body fluids other than saliva hence the test is only presump tive In this method a dye linked to an insoluble substrate is ren dered soluble by the action of the amylase enzyme In this study the method is performed using treated blotting paper The paper is prepared as follows Phadebas tablets essentially blue starch are dissolved in dH2O 0 9g into 100 mL and the solution is sprayed onto one side of a sheet of blotting paper The treated paper has a speckled blue a
11. al information reprints not available from author Nicholas Vandenberg Victoria Police Forensic Services Center Forensic Drive Macleod Victoria 3085 Australia E mail nicholas vandenberg police vic gov au
12. been subjected to environmental insult The type of material and in particular its color is a factor in the detection of saliva and seminal stains and the degree of absorb ency also appears to be a factor in the detection of saliva and bloodstains When examining exhibits for stains using the Poli light it is important to be mindful of the background color of the garment and its potential fluorescence and to choose appropriate excitation emission conditions in order to enhance the contrast between the stain and the background It is also important to be mindful of the fact that in most instances the life history of gar ment will be unknown and as such other fluorescent staining may be present possibly caused by other fluids that are known to flu oresce e g urine To a degree these can be identified by dif ferences in appearance from at least bloodstains and also through comparison with the appearance of stains with the naked eye under natural light The Polilight was found to be relatively poor at distinguishing between different types of biological fluid stains with a relatively high incidence of false positives when examining casework exhibits for seminal stains Further it has been shown that the presence of residual laundry detergent and other fluorescent substances may mimic the appearance of poten tial seminal stains However when screening casework exhibits for seminal stains the incidence of false negatives was relativel
13. can be used to successfully locate saliva for DNA analysis this time in a mock casework type scenario Examination of the Effects of Potentially Interfering Substances or Conditions on the Identification of Bloodstains Using Polilight In all cases the bloodstain was apparent at 415 nm yellow gog gles However of all the contaminants overlayed over blood black fingerprint powder was the most poorly differentiated from the stain appearing as a dark brown powder on a dark brown stain Soil blood and rust flakes were also compared under Polilight at 415nm data not shown Soil blood and rust flakes are generally indistinguishable from each other in terms of size and TABLE 3 Detection of saliva stains on potential exhibits Comparison at Three Set Wavelengths Stain 415 nm 450 nm 505 nm Sample Description Detected Yellow Orange Red T shirt spit Yes Poor Good Dull Bra licked Yes Very poor Good Very poor Boxer shorts transfer No Pillow case head wrap Yes Dull Dull Very poor Stocking mouth gag Yes Good Good Very poor Good strong staining Dull obvious but dull staining Poor weak staining Very poor very weak staining 368 JOURNAL OF FORENSIC SCIENCES FIG 8 Spit on a white cotton T shirt at 450 nm excitation viewed through orange goggles color with perhaps the only difference being that soil may some times appear more granular Humidity appears to have more of a degradin
14. d green paint were better at blocking out the appearance of a bloodstain For bloodstains on wood when painted over with the water based paint the order of decreasing visibility was white gt yellow gt green colored paint Although 0 5mL volumes of blood were used for each substrate paint combination small spots of blood of 1 2 mm diameter were also made next to the 0 5mL stains and were painted over in the same way These small spots were also just as visible under Polilight at 415 nm yellow goggles as the larger 0 5 mL stains Only in the instance where two coats of the darker green colored paint were applied over these spots did they become difficult to detect under Polilight By comparison a 0 5 mL seminal stain dried on wood was even more prominent than the bloodstains under Polilight when paint ed over with white acrylic based paint One coat of paint was enough to completely block out the presence of the seminal stain under natural light such that its location could not be determined however the seminal stain was clearly visible under Polilight up FIG 9 Bloodstain on wood under one coat of white acrylic based paint a natural light and b 415 nm excitation viewed through yellow goggles to three coats of paint later both at 415 nm yellow goggles and at 450 nm orange goggles Fig 10 The bloodstain sandwich paint bloodstain paint was also readily detectable at 415 nm yel low goggles FIG 1
15. ectrum of semen was broad i e fluorescence could be generated using wavelengths from 300 to 480nm and that blood had a strong narrow absorption band around 415nm These properties of semen and blood can be exploited to enable their detection 1 However to date there remains an apparent dearth of literature on the application of alternate light sources such as Polilight for detecting biological fluid stains One previous study however using a PL 10 Rofin Dingley Australia identified that the most significant factor in the detec tion of semen stains using fluorescence is the nature of the ma Victoria Police Forensic Services Center Macleod VIC 3085 Australia Received 4 June 2005 and in revised form 5 Sept 2005 accepted 1 Oct 2005 published 13 Feb 2006 Copyright 2006 by American Academy of Forensic Sciences terial on which a stain may be deposited 2 This study reported a degree of difficulty in detecting seminal stains on materials that were either highly absorbent dark colored or were made of ma terial that itself demonstrated strong fluorescence 2 Further it is known that brighteners in detergents and fabric conditioners can cause cloth to fluoresce under ultraviolet UV light and that their effect is additive 3 Background fluorescence may potentially mask fluorescence from seminal stains In the case of saliva stains in particular any improvement in the ability to target an area for DNA a
16. ed no detectable chemilumines cence when these substances were treated with luminol The paints examined in our study also showed no detectable chemi luminescence when luminol reagent was applied to a neat sample of each paint Dried bloodstains on wood covered with one coat of white acrylic paint or white yellow or green water based paint did not produce detectable chemiluminescence from the blood stain when sprayed with luminol presumably because the paint had formed a physical barrier between the reagent and the blood stain itself preventing a chemical reaction from taking place A DNA result was obtained from a bloodstain on wood under two coats of the white water based paint where the bloodstain was located first with the Polilight The profile recovered from the bloodstain under the paint matched the source of the stain Comparison Between Polilight and the Phadebas Method for the Identification of Saliva When the Phadebas method was used to identify the location of saliva stains on mock casework exhibits the results were the same as when using the Polilight Table 3 with staining de tectable on all exhibits except for on the boxer shorts All saliva stains on garments produced the typical pale blue diffuse staining characteristic of a positive Phadebas reaction in a region on the paper corresponding to the location of the saliva stain on the gar ment Again positive Phadebas reactions were equally observ ab
17. g effect on the visual quality of a bloodstain under Polilight than for a similar stain stored at room temperature for a significantly longer duration of time Equal mixtures of seminal fluid blood or saliva blood generally appear light brown and much more diffuse than a blood stain of comparable volume on the same material No distinct sa liva or seminal component is observable among these mixtures under Polilight Results of experiments in which 0 5 mL dried bloodstains were successively painted over show that for all the substrate paint combinations examined bloodstains were detectable under Poli light at 415 nm yellow goggles regardless of which of the tested surfaces the bloodstain was on or what paint was used with the exception of a bloodstain on metal and even then this stain be came undetectable only when three coats of a white water based paint were applied Figure 9 shows how readily a bloodstain can be located under a painted surface using Polilight On the whole bloodstains under paint were easier to see using the Polilight when the paint was dry not wet Under natural light to the naked eye the color of a bloodstain was undetectable after as little as one coat of either the acrylic or water based paint The white acrylic based paint though was better than the white water based paint at physically concealing bloodstains under natural light Under Poli light the colored paints especially the darker colore
18. he perspective of obtaining a false positive result from residual laundry detergents through routine washing This was in fact found to be the case with two common household products Preen and Cold Power Table 1 Both these detergents left apparent residual staining after washing in cold water that appeared bright white in color under Polilight and could be confused with a suspect seminal stain This staining effect was not observable under natural light The detergent stains were powdery looking in appearance and not as solid or discrete as a neat sem inal stain typically appears The results of this experiment are also supported by observations made in analyzing casework exhibits with Polilight where numerous small white background stains that were not observed under natural light were observed on garments under Polilight and recorded as possible stains It may be that some of these background stains were caused by brighteners in detergents and fabric conditioners Given that the fluorescent effects of brighteners are accumulative it seems rea sonable to assume that background stains would be more com mon on undergarments or light colored clothing as one would generally expect these items to be washed more often In other instances stains observed on casework exhibits under Polilight corresponded to pre existing stains visible on the garment under natural light and could be ruled out as seminal fluid stain
19. her useful wavelengths combinations of goggles were 415 nm yellow goggles for stains on dark colored materials and 505 nm red to reduce background emissions for garments that demonstrate high background fluorescence i e to maximize con trast The Polilight is suitable for detecting seminal and saliva stains on a range of materials including wool cotton nylon pol yester and blends e g cotton polyester where the stain is not apparent to the naked eye under natural light Fig 1 The color of the material was found to affect the strength of the stain s ap pearance Seminal stains on pink nylon red cotton and pink cot ton polka dot are difficult to detect Fig 2 However seminal FIG 1 Seminal stain on a multicolored cotton print a natural light and b 450nm excitation viewed through orange goggles stains on the same type of material but of a different color were readily observed Seminal fluid in a condom could also be ob served Fig 3 Some difficulty was noted when attempting to observe seminal stains on checked fabric data not shown Saliva stains on pink nylon were difficult to detect data not shown Saliva staining on blue and white checked cotton weave could not be observed under Polilight compared with the same volume of saliva on a flannel alphabet print Fig 4 FIG 2 Seminal stain on pink cotton white polka dot at 450 nm excitation viewed through orange goggles VANDENBERG AND
20. hing or rain or simply through coming into contact with another surface which could then absorb part or all of the stain It should be noted that a seminal stain that becomes diluted may be different in appearance to seminal fluid that is diluted before becoming a stain We found that gentle cold water machine washing without detergent of a seminal stain on white polyester produces a negative result with Polilight sug gesting that the fluorescent properties of semen may be complete ly removed from the fabric by simple washing Further it is known that it is possible to still obtain a weak AP result from seminal stains on fabric machine washed in cold water without a detergent 12 However another study has reported weak 366 JOURNAL OF FORENSIC SCIENCES FIG 6 Diluted seminal fluid on white polyester at 450nm excitation viewed through orange goggles Clockwise from top left 1 1000 1 100 1 10 1 5 1 2 neat semen fluorescence from a seminal stain that was AP negative after washing with a detergent 2 Saliva stains of varying amounts on white nylon were examined under Polilight 450nm orange goggles At this wavelength saliva stains were detectable down to the smallest amount tested SL The serial dilution of saliva on nylon showed that saliva is detectable down to a 1 10 dilution under Polilight The edges of the stains are strongest and appear white Under natural light these same saliva stains are not visible bel
21. ift technique was chosen as the means of recovering DNA as would routinely be performed in casework in our foren sic laboratory This technique involves the repeat application of a section of tape to a surface area until the tape loses its stickiness thereby concentrating trace amounts of material into a small area Tape lifts of the saliva stains that were prepared on separate pieces of white nylon and examined under Polilight were then submit ted for DNA analysis DNA analysis was performed using the Chelex extraction method 7 Extracted samples were quantitated using the Quan tiblot method Perkin Elmer Foster City CA in order to de termine the amount of DNA present Amplification of extracted DNA was performed using the AmpFISTR Profiler Plus Am plification Kit Applied Biosystems Foster City CA on a PE 9600 under standard conditions 8 Where possible 1 ng of the total extracted DNA for a given sample was used in each ampli fication Amplified samples 1 uL were combined with an internal size standard ROX 400 HD and resolved on an ABI PRISM 310 Genetic Analyser Applied Biosystems Polymerase chain reaction PCR fragment sizes were determined using GeneScan Analysis 3 1 software Allelic assignment was made by reference to allelic ladders using Genotyper version 2 5 software A reagent blank for the extraction of DNA was also performed along with positive and negative controls for amplification
22. ine nasal secretions tea vaginal fluid saliva Center blood at 450 nm orange goggles although blood and urine were also detectable at 415 nm yellow and semen and urine at 505 nm red At 450 nm orange all stains appeared white with the exception of blood which appeared dark brown Urine and blood were solid stains and urine appeared the most intense of all stains while the seminal stain had strong thick edges Vaginal and saliva stains appeared the most similar both having thin white edges under Polilight The tea stain had slightly thicker edges Nasal secre tions appeared as a very weak but solid smear under Polilight however this staining is not apparent in Fig 7 By comparison under natural light the bloodstain appeared red brown in color urine and semen had a yellowish tinge and the tea stain appeared brownish yellow Therefore to the trained eye and at a wavelength of 450 nm orange goggles only blood urine and possibly undiluted semen are differentiable from each other whereas the tea stain saliva vaginal and nasal secretions are difficult to tell apart with per haps the tea stain distinguishable from the others by comparison under natural light While this experiment was designed to directly compare fluid stains from different sources it is clear that Polilight cannot be used to differentiate these stains or act in any way as a confirm atory test Polilight is intended for use as a screening aid su
23. it able for identifying the location of a possible stain which may then be examined further to establish what type of biological ma terial the stain is if any In this regard then it is more important that all types of biological material tested were simply shown to be readily detectable Also it is interesting to note that where the bloodstain overlaps the vaginal fluid stain the vaginal fluid stain is no longer visible Fig 7 Given the intensity of blood and urine stains it could reasonably be expected that if performing an examination in a casework scenario e g examining underwear there might be physical masking of weaker fluorescence from semen saliva vag inal stains by blood or urine staining i e the bloodstain phys ically covers the seminal stain This suggests that in cases where a mixture of possible fluids is suspected further chemical based screening tests e g AP may be more suitable Recovery of DNA from Saliva Stains Identified with Polilight Table 2 shows the results of tape lifts of saliva deposited on white nylon Each of the stained regions were easily identifiable under Polilight as an area of white staining even when the lo cation of staining was previously unknown to the examiner it TABLE 2 Recovery of DNA from saliva stains on white nylon identified with Polilight Stain Identified DNA from Profiler Sample Description With Polilight Tape Lift ng Plus Result Known amount Yes 50
24. le whether the paper was wet or left to dry and all positive controls worked For both the Phadebas method and Polilight the best result was obtained from the nylon stocking that was used as a mouth gag The negative result for the boxer shorts from both Polilight and Phadebas method suggests that no or undetect able levels of saliva were present on the shorts the most likely explanation for this being that no or too little transfer of saliva took place from the fingers onto the shorts when the experiment was set up DNA was successfully recovered via a tape lift of an area on the nylon stockings corresponding to an area of pale blue diffuse staining The profile obtained from this tape lift matched the DNA profile of the sample s originator again indicating that the Phade bas method can be used to successfully locate saliva for DNA analysis this time in a mock casework type scenario i Of the different biological fluid stains tested with Phadebas paper only saliva gave a positive result Blood semen vaginal fluid and urine were all negative which concurs with the findings of other studies in which amylase activity was measured in these stains and found to be absent 17 18 Some transfer of blood and urine was visible on the Phadebas paper with the naked eye un der natural light This indicates a possible potential loss of sample DNA in cases where Phadebas paper is used on mixed stains This effect may be a pote
25. liva and blood were collected and stored at 4 C before use Blood was collected in ethylene diamine tetraacetic acid tubes A half milliliter of each fluid was placed on a sample of each material and left for 24h to dry before being examined The seminal and saliva stains were then examined at each of the 12 wavelength settings available white light UV 415 450 470 490 505 530 555 590 620 and 650 nm with the Polilight While bloodstains were examined at 415 nm only Serial Dilution of Seminal Fluid Saliva and Blood Serial dilutions of seminal fluid saliva and blood were per formed using distilled water dH O down to a concentration of 1 100 000 in a final volume of 500 uL The dilution ratios tested were 1 2 1 5 1 10 1 100 1 1000 1 10 000 1 100 000 Undiluted samples were also prepared for comparison purposes A prelim inary study of seminal fluid saliva and blood on white wool cotton polyester nylon and paper showed that seminal fluid sa liva and blood were least diffuse on polyester nylon and cotton respectively Hence dilutions of seminal fluid saliva and blood were applied to individual samples of these fabrics left to dry for 24h or 7 days in the case of blood to destroy any viruses that may be present and then observed under Polilight Samples of var ying amounts of saliva and blood 50 5 uL were also applied to white nylon and white cloth respectively and observed under Polilight
26. lood mixed with saliva 250 uL each and left to dry dried blood stored at 37 C for 4 weeks and a bloodstain stored for 15 years at ambient room temperature A neat bloodstain 500 L on white cotton cloth was also included for reference purposes Loose soil blood and rust flakes were also prepared separately in a Petri dish for comparative purposes Additionally as part of the study on the effects of interfering substances 0 5 mL dried bloodstains prepared on wood were suc cessively painted over with either a white acrylic based paint or a white light yellow or dark green low sheen water based paint using separate rollers for each stain paint combination Three coats were applied using the white paints while only two coats were applied using the colored paints The bloodstains were ob served under Polilight after the application of each new coat of paint both when the paint was wet and dry Also although 0 5 mL volumes of blood were used for each substrate paint combination small spots of blood 1 2 mm diameter were made next to the 0 5 mL stains After observing the bloodstains under Polilight a 5 x 5mm sample of a bloodstain under two coats of the white water based paint was cut out and subjected to DNA analysis No attempt was made to scrape off or otherwise separate the bloodstain from the wood or the paint when sampling For comparative purposes a seminal stain was also painted over with three coats of the white
27. nalysis would be an advan tage in casework especially if the examination can be performed by noninvasive nondestructive means such as Polilight Luminol is an extremely sensitive method for the detection of blood but like Polilight it requires darkness and after application of the luminol reagent the chemiluminescent intensity diminishes over time while repeated application may diffuse a bloodstain pattern on a nonporous surface 4 i In this study we used a Polilight PL 500 Rofin to examine seminal stains saliva stains and bloodstains on a variety of ma terials of differing texture and color that are often observed as substrates in forensic casework We test the degree of sensitivity that the Polilight possesses for detecting these stains by applying serial dilutions of seminal fluid saliva and blood onto known fabrics and examining the resultant stains under Polilight We also examine the potential background effects of laundry deter gents on selected materials and the effects of washing these materials under Polilight The efficiency of the Polilight tech nique compared with the AP test was investigated by performing 361 362 JOURNAL OF FORENSIC SCIENCES the AP test on a number of casework samples that had firstly been examined by Polilight Further we attempted to recover DNA from saliva stains sub sequent to these stains being identified using Polilight This in cluded the identification of saliva
28. ntial drawback of the Phadebas method in cases where the amount of blood or saliva for testing is limited A mixture of blood and saliva was tested with Phadebas paper and was found to be positive The identification of saliva stains among blood where saliva would react with Phadebas paper and blood would not or a blood saliva mixture where the pres ence of saliva in the mixture is still detectable is important be cause in certain casework scenarios it may be of assistance to screen for the presence of saliva in the vicinity of blood staining One example of this is where a defendant claims that the de ceased s blood was deposited on their clothing as a result of ex pectoration A positive result for saliva with an appropriate distribution could go some way to supporting this claim with due consideration to the presumptive nature of the Phadebas method i e potential for false positives or negatives On the whole these two methods produced comparable results both are useful for screening for potential saliva stains with an obvious advantage to the Polilight method in that it is both quicker to perform and a noninvasive technique Conclusion Our results show that the Polilight is able to detect seminal fluid saliva and bloodstains on a variety of fabric types even when the stain is dilute In the case of bloodstains the stain is still detectable even when a variety of contaminants are present or the stain has
29. ow a 1 2 dilution Ray 10 also reported that using a Polilight saliva stains on cotton at dilutions of 1 10 were visible while without the Polilight even neat saliva stains were not visible Bloodstains of varying amounts on white cotton were examined under Polilight 415 nm yellow goggles At this wavelength bloodstains were detectable down to the smallest amount tested 5 uL The serial dilution of blood on cotton showed that blood is detectable down to a 1 1000 dilution under Polilight data not shown The stains are typically solid dark brown in color but fade as the stains become more dilute Below a dilution of 1 10 only the edges of the stain are visible Under natural light these same stains are visible down to 1 1000 dilution data not shown Hence Polilight is of little benefit for detecting bloodstains on a light colored background as in most cases the staining will be readily observed with the naked eye under natural light or bright white light Swander and Stities 13 observed the loss of visual detection of coloring staining of bloodstains on cotton at dilu tions of more than 1 500 The sensitivity of luminol has been re ported to be 1 1 000 000 14 which is greater than any such visual determination of the presence of blood Effects of Laundry Detergents Given that a seminal stain can be removed from fabric through simple washing the effect of using a laundry detergent was im portant more so from t
30. ppearance When dried the paper is tested against a suspect stain by placing it face down on the stained area and wetting the back of the paper with dH O The location of the paper relative to the exhibit is marked and a weight is used to ensure even contact The reaction takes 45 min A positive result appears as pale blue diffuse staining on the treated side of the paper in the region corresponding to the location of the saliva stain as opposed to negative areas which retain the speckled blue background The paper is dried again to better visualize any positive staining When using each new piece of Phadebas paper the corner of the treated side is folded over and a positive control is applied 50 uL neat saliva Phadebas paper was used to identify the location of saliva stains on mock casework exhibits As the Polilight method is noninvasive the exhibits used were those previously prepared for analysis with the Polilight The Phadebas method was also performed on a number of other biological fluid stains including 500 uL amounts of saliva blood semen free vaginal swabs resuspended in dH O semen and urine all dried on white nylon Results and Discussion Seminal Fluid Saliva and Bloodstains on Various Materials The most useful general condition for observing fluorescence of seminal or saliva stains on various materials using the Polilight was with the wavelength set to 450nm while wearing orange goggles Ot
31. rbed into the fabric A bloodstain on highly absorbent polar fleece also gave a poor result data not shown Kobus et al FIG 4 Saliva stains on cloth at 450 nm excitation viewed through orange goggles Left blue and white checked cotton weave Right multicolored flannelette alphabet print FIG 5 Seminal stain on green polyester polar fleece a natural light and b 450 nm excitation viewed through orange goggles 2 used Rhodamine 6G to investigate the effects of absorption on fluorescence and found fleecy fabrics to be very absorbent poorly fluorescent Serial Dilution of Seminal Fluid Saliva and Blood The sensitivity of the Polilight was investigated in dilution experiments It was found that seminal stains on white polyester could be readily observed with the Polilight when the seminal fluid was diluted down to as little as a 1 part in 100 Interestingly it is the edges of a seminal stain that remain visible at the lower concentrations this may be a useful visual guide when examining casework exhibits Fig 6 A previous study 10 has reported visible seminal stains on cotton under Polilight at dilutions greater than 1 25 where these same stains were not visible with out the aid of a light source By comparison the detection limit for AP in seminal stains has been reported at a dilution of 1 100 11 The possibility exists that a seminal stain on a casework exhibit might become diluted through was
32. re the deposition of such fluids is suspected The Polilight s advantage is that it is a rel atively safe simple noninvasive and nondestructive technique to use References 1 Stoilovic M Detection of semen and bloodstains using the Polilight as a light source Forensic Sci Int 1991 51 289 96 2 Kobus HJ Silenieks E Scharnberg J Improving the effectiveness of flu orescence for the detection of seminal stains on fabrics J Forensic Sci 2002 47 8 19 23 3 Lloyd JBF Forensic significance of fluorescent brighteners their qualita tive TLC characterization in small quantities of fiber and detergents J Forensic Sci Soc 1977 17 145 52 4 Bray T Stenlake N Armitage S Fluorescein vs luminol and Leuco Crys tal Violet LCV as an alternative for bloodstain detection Proceedings of the 17th International Symposium on the Forensic Sciences 2004 28 March 2 April Wellington ANZFSS 2004 118 5 Soukos NS Crowley K Bamberg MP Gillies R Doukas AG Evans R et al A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy Forensic Sci Int 2000 114 133 8 6 Auvdel MJ Comparison of laser and ultraviolet techniques used in the detection of body secretions J Forensic Sci 1987 32 326 45 7 Walsh P Metzger D Higuchi R Chelex 100 as a medium for simple ex traction of DNA for PCR based typing from forensic material Biotech nique 1991 10 506 13 8 PE Applied Biosystems AmpFISTR p
33. rofiler plus PCR amplification kit users manual Foster City CA PE Applied Biosystems 2000 9 Willott GM An Improved tests for the detection of salivary amylase in stains J Forensic Sci Soc 1974 14 341 4 10 Ray B Use of alternate light sources for detection of body fluids SWAFS J 1992 14 30 3 11 Kearsey J Louie H Poon H Validation study of the onestep ABAcard PSA test kit for RCMP casework Can Soc Forensic Sci J 2001 34 63 72 12 Crowe G Moss D Elliot D The effect of laundering on the detection of acid phosphatase and spermatozoa on cotton t shirts Can Soc Forensic Sci J 2000 33 1 5 13 Swander CJ Stities JG Evaluation of the ABAcard HemaTrace for the forensic identification of human blood Midwestern Association of For ensic Scientists 1998 1 5 14 Castello A Alvarez M Verdu F Accuracy reliability and safety of luminol in bloodstain investigation Can Soc Forensic Sci J 2002 35 113 21 15 Rofin Polilight PL500 version 2 instruction manual Australia Rofin 2002 Vol 5 28 16 Quickenden TI Creamer JI A study of common interferences with the forensic luminol test for blood Luminescence 2001 16 295 8 17 Baxter SJ Rees B The identification of saliva in stains in forensic case work Med Sci Law 1975 15 37 41 18 Tsutsumi H Higashide K Mizuno Y Tamaki K Katsumata Y Identifi cation of saliva stains by determination of the specific activity of amylase Forensic Sci Int 1991 50 37 42 Addition
34. s Ex amples of substances that fluoresce under Polilight and may appear as stains on a garment under natural light include urea grease lipstick and ink 15 Polilight vs AP Forty casework items e g underwear clothing and including 12 bed sheets or quilts were examined using the AP test subse quent to Polilight Overall the incidence of false negative results using the Poli light was relatively low one in 40 or 2 5 False negative re sults are defined as test results that were Polilight negative but AP positive where the presence of spermatozoa was confirmed cytologically Seminal material on a black polyester dress pro duced the one false negative result under Polilight The inci dence of false positives using the Polilight is relatively high 20 in 40 or 50 False positives are defined as test results that were Polilight positive but AP negative All of these 20 false posi tives however were listed as possible only which we defined as similar in appearance to a seminal stain i e they appear white under Polilight but they are not as discrete or convincing as a seminal stain The types of items that gave false positive results included among others a burgundy woollen jumper a red polyester dress a black cotton track suit top khaki cargo pants a navy blue pair of cotton underwear a light blue woollen blanket and a pink cotton bed sheet In a small number of cases n
35. screen out any reflected incident light or other competing light To achieve this filtering effect a series of different colored goggles can be used The shade of goggles worn yellow orange red becomes darker as the wavelength of incident excitation light is progressively shifted toward the longer wavelength end of the spectrum These colored goggles are long pass filters that let light through above a certain wavelength while cutting out light below that wavelength The excitation spectrum of semen has been measured from 300 to 480nm and does in fact stretch beyond 480nm 1 and as such fluorescence from semen can be generated using wavelengths anywhere from 300 to 500 nm The emission spectra for semen are also broad and cover the region from 400 to 700 nm 1 Studies on the fluorescent properties of saliva are limited One study which examined the fluorescence profile of saliva swabs from skin reports an excitation peak for saliva at 282 nm i e in the UV range although excitation scans were only performed between 200 and 320 nm 5 However another study 6 used an argon ion laser which has a continuous wave operation of 454 5 514 5 nm to successfully locate semen and saliva stains on items of clothing The excitation spectrum of saliva was not determined in this study although fluorescence of saliva stains was achievable using excitation wavelengths higher than 320 nm and although the success rate was relatively lo
36. stains set up in mock casework type scenarios The appearance of a variety of biological and nonbiological fluids under Polilight was examined as were the effects of potentially interfering substances on the identification of bloodstains Finally a comparison was also made of the efficiency of the Polilight technique and the Phadebas test for detecting saliva stains Materials and Methods Light Sources The Polilight used in this study was a PL 500 Rofin The light source for the PL 500 is a 500 W Xenon arc lamp There are 12 wavelength settings available to choose from ranging from 415 to 650nm also including white light and UV options Bandwidths range from 100 nm for the 450 nm setting blue light commonly used for general screening to 27nm for the 555 nm band green orange light The range of wavelengths allows for the fluorescence from seminal and saliva stains to be observed against a variety of backgrounds including those that may them selves be fluorescent The Polilight PL 500 has power and fine filter i e wavelength tuning options In this study the output power was set at P7 the fine filter tuning function was not utilized Observation of Fluorescence Fluorescence is defined as the property of absorbing light of short wavelength and emitting light of longer wavelength The fluorescence must be observed at a wavelength greater than the incident excitation light and therefore a filter is required to
37. w for saliva 30 it was still higher than when using UV light sources alone to detect saliva on these materials 21 The higher success rate could be attributed in part to the laser s higher intensity radiation 6 The excitation and emission spectra of blood on the other hand are well defined Blood does not show significant fluorescence but does have a strong absorption band around 395 435 nm with a maximum at 415nm 1 Thus the appearance of a bloodstain may be enhanced by the use of Polilight set to the 415nm wavelength setting Preparation of Stains Fresh seminal fluid saliva and blood were stained onto cotton swabs and a range of colored fabrics including white yellow green red and blue colored wool white black yellow pink green red blue brown and purple cotton as well as multicolored cotton and flannelette prints checked and polka dot cotton weaves white pink and blue nylon white polyester green po lar fleece blue velour pink satin polyester and black crepe Stains were also prepared on blends of fabrics such as black and white polyester spandex blue cotton elastane fashion denim white cotton elastane boxer shorts a white nylon elastane bra a black and white polyester cotton T shirt and on other materials including white and blue synthetic carpet pine wood dried leaves and for seminal fluid only a condom and for blood only glass brick metal and plasterboard Seminal fluid sa
38. y low and it should be remembered that in essence the Polilight s function is as a screening aid it should not be used to classify stains but to merely locate stains which will then require further analysis Ideally the examination of forensic exhibits with the Polilight would be best performed in conjunction with other screening tests to better target an area of interest e g screening for seminal stains with the Polilight in conjunction with spot testing using the phosphatesmo KM test or a p30 test 370 JOURNAL OF FORENSIC SCIENCES The Polilight was found to be as good as the Phadebas method when screening for potential saliva stains with the obvi ous advantage that it is a noninvasive technique It has been shown that the Polilight can be used to successfully locate saliva stains on nylon from which DNA can then be recovered Although the sensitivity of the luminol method is greater than that of Polilight in the detection of blood the ability of the Polilight to locate seminal stains and blood stains suitable for DNA analysis under painted surfaces where chemical based tests such as luminol cannot suggests that the Polilight has clear ap plications in forensic casework Hence despite some apparent limitations in relation to back ground color absorbency of the fabric a stain may be deposited on the Polilight is a useful screening tool for locating seminal fluid saliva and bloodstains in cases whe
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