Comparison of enzyme assay and radioimmunoassay for the measurement of human acid phosphatase in cases of sexual assault

We investigated whether a radioimmunoassay for prostatic acid phosphatase might be used as a more specific test for the identification of semen in samples from cases of sexual assault than the measurement of total acid phosphatase enzyme activity. The results of the measurement of acid phosphatase by enzyme assay in semen and vaginal swab extracts were compared with the results of the radioimmunoassay. It was found that the radioimmunoassay is a sensitive and more specific method than the enzymic determination of acid phosphatase. Incidentally we have found that a low concentration of an immunological cross reacting acid phosphatase is present in semen free vaginal swab extracts.     

Effect of swabbing technique and duration on forensic DNA recovery

Various factors have been shown to affect performance of the conventional wet-dry double and single wet swabbing techniques to recover DNA, such as pressure and angle of application, volume and type of wetting agent, and swab type. However, casework laboratories in some jurisdictions have recently adopted different swabbing techniques that include wet-moist double swabbing and moist-dry single swabbing. Factors affecting the effectiveness of these recent techniques in maximising DNA recovery therefore need to be investigated. Here, the performance of traditional and recent swabbing techniques was compared and the impact of swabbing duration on DNA recovery was investigated. Ten µl aliquots of a known concentration of DNA extracted from human blood were deposited on pre-cleaned DNA-free cotton swatches (porous) and porcelain tiles (non-porous). Five swabbing techniques were used, of which three were double swabbing techniques: wet-moist, wet-wet and wet-dry, and two were single swabbing techniques: wet and moist-dry. For a ‘wet’ or ‘moist’ swab, 100 or 50 µL water was added, respectively. For a moist-dry swab, water was applied to one side of the swab, leaving the other side drier. Each swabbing technique was applied for two durations, 15 and 30 s per swab, with 5 reps of each combination (n = 100 plus controls). All samples were extracted and quantified, and a sub-set was profiled. The results showed that the wet-moist double swabbing technique with a swabbing duration of 30 s maximised DNA recovery from cotton. From tile, a single wet or moist-dry swab maximised DNA recovery, but increasing swabbing duration from 15 to 30 s had no impact. These data can be used to inform standardisation of DNA collection protocols across casework laboratories.     

The evaluation of an ELISA for semen-specific 19-OH prostaglandin F1 alpha/F2 alpha using ex-casework swabs and stains

The application of an enzyme-linked immunosorbent assay (ELISA) for 19-OH Prostaglandin F1 alpha/F2 alpha (19-OH PG F) to casework analysis of seminal contamination of swabs and stains is reported. The results are compared to those where the identification of semen was based on the presence of acid phosphatase and spermatozoa. Five hundred and one samples were analysed and there was good agreement between the results of ELISA and conventional methods. The determination of 19-OH PG F confirmed both the presence of semen where spermatozoa were absent and indicated semen was present when acid phosphatase and spermatozoa were both absent. The results indicate that 19-OH PG F represents a useful marker for the casework identification of semen and is particularly valuable where spermatozoa are absent.     

An evaluation of gamma-glutamyl transpeptidase (GGT) and p30 determinations for the identification of semen on postcoital vaginal swabs

The following study entails the investigation of gamma-glutamyl transpeptidase (GGT) and p30 for the identification of seminal stains in sexual assault cases. A commercial kit was used to test for GGT activity, while p30 was demonstrated with a crossed electrophoresis technique. Specificity, sensitivity, and stability of both markers were studied. Postcoital swabs from lab staff were tested for GGT and p30. In addition, 144 postcoital swabs from case material were tested for p30, spermatozoa, and acid phosphatase. Results show p30 to be a useful semen marker particularly in cases of azoospermia. However, GGT was found to be unsuitable for forensic science casework.     

The sensitivity and specificity of red-starch paper for the detection of saliva.

The detection of saliva in forensic casework is extremely important in many types of cases. This study describes a relatively sensitive method, based on a red dye bound to starch, for the detection of amylase. The sensitivity and specificity of the method has been examined by testing over 50 household products, various body fluids and five laboratory chemicals. This study demonstrated for the first time that positive results can be obtained from certain washing powders as well as other household products. As well as detecting amylase in saliva, positive Red-Starch results were obtained from faeces and urine. The method was found to be suitable for the detection of mixtures of saliva and semen in conjunction with the Brentamine test for the detection of acid phosphatase.     

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD₂₆₀/OD₂₃₀ and OD₂₆₀/OD₂₈₀) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.     

Vaginal swabs: endogenous and postcoital components

The relationship between components found on vaginal swabs was examined to determine whether the presence and quantity of a particular component could be used to predict the presence of others or to help interpret grouping results. Vaginal swabs were tested for the presence of blood, cellular material, spermatozoa, acid phosphatase, p30, ABH, Lewis, EsD, GLO I, PGM and PGM subtype. Methods included rocket electrophoresis and p-nitrophenyl phosphate assay for acid phosphatase; rocket and cross-over electrophoresis for p30. Results from 323 semen-free and postcoital vaginal swabs from known donors are presented. Comparison of methods showed that seminal acid phosphatase and p30 were detected more often by the rocket techniques. Attributing the grouping results to semen, based on the reactivity of any single component, could lead to erroneous conclusions. Activation of vaginal components in the presence of semen and endogenous vaginal levels are discussed.     

Touch DNA collection from improvised explosive devices: A comprehensive study of swabs and moistening agents

Improvised explosive devices (IEDs) are used in devastating terrorist attacks worldwide and daily in Thailand. Touch DNA deposited during IED assembly are subjected to intense heat and pressure, resulting in rare events of usable DNA profiles obtained from real casework. No study has simultaneously evaluated both swab brands and moistening agents for touch DNA collection from substrates encountered in IED evidence. In this study, we investigated the effects of swab brands and moistening agents on DNA collection from adhesive tape, a common IED substrate. A full factorial design using four cotton swab brands (two forensic and two medical cotton swabs) and six moistening agents (DNA-free water, phosphate-buffered saline, ethanol, sodium dodecyl sulfate, isopropanol, and lysis buffer) was employed (24 total combinations). Using buffy coats, we found that DNA recovery depended on both swab brands and moistening agents (p < 0.05). The optimal method recovered significantly higher DNA amount from real IED cases compared to the standard Royal Thai Police method. Percentages of high partial profiles also increased. Our results changed the standard operating protocol of the Thai police. Other commonly found substrates from IED cases are being investigated to maximize the evidential value obtained from touch DNA.     

Presumptive screening of suspected semen stain in situ using cotton swabs and bromochloroindolyl phosphate to detect prostatic acid phosphatase activity

A novel approach to the presumptive screening of questioned semen stains has been developed which enables the rapid identification of stains which are devoid of semen. Questioned semen stains can be swabbed with a moist cotton swab, and the prostatic acid phosphatase (SAP) activity transferred to the swab identified through assay with 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Controlled laboratory studies revealed that the BCIP swab procedure was as sensitive as the semiquantitative SAP test currently employed in the FBI Laboratory for the presumptive screening of semen stains. A validation study of the BCIP swab procedure in parallel with the current procedure using 4305 case evidence stains indicated that the BCIP swab procedure was as effective as the current procedure in identifying those questioned stains which lack semen. The advantage of the BCIP swab procedure is that it can be performed on questioned stains in situ and thereby avoids the requirement of removing and extracting the stain before assay of SAP activity.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

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, respectively. 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 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.     

Detection of p30 antigen in sexual assault case material.

A sandwich enzyme immunoassay for the detection of p30 prostate specific antigen was applied in 52 forensic cases. In each case, immunoassay results were compared with those of the search for spermatozoa and prostatic acid phosphatase analysis. The results indicate that the p30 assay gave no false positive and fewer false negative reactions than the acid phosphatase test. The sensitivities compared to the search for spermatozoa as a reference method were 94.8% for the p30 assay and 84.4% for the acid phosphatase test; the specificities were 95.6% and 90.0% respectively.     

Phenotyping of alpha-2-HS glycoprotein in bloodstains by isoelectric focusing and immunoblotting

A sensitive immunoblotting procedure has been applied to the detection of alpha-2-HS-glycoprotein (A2HS) phenotypes from control and casework bloodstains. A2HS phenotypes were separated by thin layer polyacrylamide gel isoelectric focusing (PAGIEF) in gels containing Pharmalyte pH 4.2-4.9. After transfer to nitrocellulose by a rapid capillary blot, the A2HS phenotypes were developed using a double antibody enzyme-immunoassay. The evaluation of A2HS phenotyping of casework material was undertaken in parallel with phosphoglucomutase (PGM) phenotyping by PAGIEF. A total of 598 water extracts from casework bloodstains have been tested. Positive results were obtained in 84% and 75% of samples for PGM and A2HS respectively. The A2HS gene frequencies A2HS*1 = 0.6420, A2HS*2 = 0.3530, and A2HS*3 = 0.0050 were determined from a survey of 1000 people in Brisbane.     

The use of acid phosphatase test papers for DNA profiling.

The acid phosphatase (AP) test is a routine assay used to screen casework items for the possible presence of semen. This colour test is carried out on filter paper which is retained after testing. Two-year-old AP test papers were found to contain sufficient DNA for short tandem repeat (STR) profiling. Prior to polymerase chain reaction (PCR) amplification, the DNA was preferentially separated into sperm depleted and sperm enriched cell fractions. The implication of these findings for past and present cases is discussed.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.     

Application of BioRobot M48 to forensic DNA extraction

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized organic/Microcon 100 based extraction procedure and magnetic extraction with BioRobot M48. The DNA concentration of DNA extracts obtained from different kinds of typical forensic material was evaluated followed by amplification with the SGM Plus or Identifiler kit and capillary electrophoresis using ABI 3100 Avant. We can conclude that BioRobot M48 is a very effective instrument for DNA extraction from most specimens and can be successfully applied in forensic laboratories.     

Development of biological standards for the quality assurance of presumptive testing reagents

Forensic scientists periodically check working test reagents with knowns or standards to verify that the presumptive testing reagents are working properly. Oftentimes, this is done with a neat body fluid such as blood or saliva that is dried onto a swab and kept in a freezer. The problem with this practice is that a degrading test reagent, for example acid phosphatase testing reagent, may test positive on a neat standard but miss a weak semen stain from a case.To ensure that presumptive testing reagents are working properly, a series of “weak” standards have been developed for the testing of acid phosphatase, amylase, creatinine and hemoglobin. The preparation and use of these biological standards will be discussed.     

A comparison of methods used in the UK and Ireland for the extraction and detection of semen on swabs and cloth samples

The recent formation of a United Kingdom and Irish working group, the Body Fluids Forum (BFF), highlighted the need to investigate different working practices prior to any inter-laboratory comparison work and identification of best practice. Various dilutions of semen were seeded onto swabs and cloth samples for each BFF member laboratory to test using their standard techniques. The results showed that the detection of acid phosphatase on swabs is best achieved using direct testing rather than on an extract from the swab. Extraction methods for spermatozoa require a balance to be achieved between using a sufficient volume of water to ensure optimal release and minimal volume to ensure a concentrated extract. PSA tests were investigated and found to be more sensitive than Choline. DNA profiles were obtained from samples in which no spermatozoa had been detected during microscopic examination.     

Immunologic sperm detection]

Proof of prostatic acid phosphatase with the enzyme-immuno-assay (EIA) is specific and as sensitive as the common phosphatase reaction. The EIA for prostatic-specific-antigen (PSA) is prostate specific as well, but less sensitive than the phosphatase assay. In a review of 30 own cases no indications for nonspecifity of the immuno assay were obtained. A positive EIA-test in absence of spermatozoa can be explained by either azoospermia or a prolonged persistence of prostatic acid phosphatase in comparison with spermatozoa.     

Validation of the use of a commercially available kit for the identification of prostate specific antigen (PSA) in semen stains

PSA is currently being used to detect and monitor quantitatively the development of prostate cancer by serum levels of PSA and has also been found to be present in high concentrations in semen. Elegantly simple, sensitive, and reproducible methods have been developed for analysis of the presence of PSA, including the Tandem-E PSA Immunoenzymetric Assay. The most common procedures for the forensic identification of semen have focused on the microscopic detection of sperm, acid phosphatase activity, and immunoelectrophoretic methods for the detection of PSA. Although these methods have been used for many years, there are problems associated with each method. The Tandem-E PSA Immunoenzymetric Assay detected PSA in 100% of the forensic casework fabric samples, 80% of the forensic casework vaginal swabs and 100% of the vasectomized individuals tested. The cut-off value was determined to be 1.77 ng/mL. These results indicate that this method can be used to identify the presence of semen in forensically significant specimens.     

An evaluation of an enzymatic choline determination for the identification of semen in casework samples

This study compares the detection of choline in seminal stains by both an enzymatic method and by the standard Florence crystal test. The tests were conducted on 293 actual casework samples. In those samples identified as containing semen, choline was detected twice as often by the enzymatic method compared to the Florence method (84.6 versus 40.3%). The choline results were correlated with spermatozoa and acid phosphatase tests. The enzymatic detection of choline in seminal stains was found to be a fast, easy, sensitive, and reliable test.