The zinc test as an alternative for acid phosphatase spot tests in the primary identification of seminal traces

The value of the acid phosphatase spot test in the primary visualization and identification of seminal traces is hampered by the sensitiveness of the enzyme to biodegradation. An alternative spot test is proposed, based on the high concentration of the more stable zinc metal in seminal plasma. The proposed zinc spot test is simple and suitable for on site investigation. Although the sensitivity in fresh stains is lower than that of the acid phosphatase spot test, this is largely compensated by the lower sensitiveness to biodegradation. The specificity for semen is higher than that of the acid phosphatase spot test. In vaginal swabs it was nevertheless seen, that samples should be taken within 24 h after alleged sexual assault to give reliable results.     

Comparative study of the sensitivity and specificity of the zinc and acid phosphatase spot tests for the detection of seminal stains

The sensitivity and specificity of a zinc spot test for the detection of semen were compared with those of an acid phosphatase detection method. As screening techniques both tests were found to be very sensitive, but the zinc test was more specific and was more reliable in older and especially in deteriorated specimens. It is concluded that the zinc spot test deserves at least the same place as the acid phosphatase test in the primary investigation of suspected semen stains and might well be the test of choice in older and poorly preserved stains.     

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.     

Diagnostic Value of PSA and AP Tests for the Detection of Spermatozoa in Postmortem Swabs from the Genital and Anal Region in Males

The aim of this study was to clarify whether positive results for prostate‐specific antigen (PSA) and acid phosphatase (AP) occur in postmortem swabs from the genito‐anal region in males (n = 80; 4 regions) and females (n = 20; 3 regions) and to calculate the positive predictive value (PPV) concerning the presence of spermatozoa. In male subjects, the highest incidence of positive test results was found in urethral swabs (PSA 76%, AP 71%) and the lowest frequencies appeared in perianal and rectal swabs (15–20%). Microscopic evaluation for spermatozoa was positive between 39% in urethral swabs and 1% in rectal swabs. PPV regarding positive identification of spermatozoa was 33.3% for PSA and 31.5% for AP. The combination of both tests yielded a PPV of 38.2%. In female cases, no spermatozoa were identified, and one case was PSA‐ and AP‐positive in perianal swabs. Our findings indicate that PSA and AP tests are of limited value for the postmortem detection of spermatozoa in male subjects.     

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.     

Persistence of spermatozoa and prostatic acid phosphatase in specimens from deceased individuals during varied postmortem intervals

The survival of spermatozoa and the persistence of prostatic acid phosphatase has been an area of interest for investigators of sexual assault. However, not much documentation exists concerning the examination of a deceased individual with regard to the postmortem interval and presence of such evidence. The authors reviewed cases referred to the medical examiner's office during a 10-year period. During this time, 199 cases were both autopsied and examined for sexual assault. In particular, these examinations included procurement of swabs for Papanicolaou staining of smears and for quantitation of prostatic acid phosphatase. Most of the victims were female, although a few were male. In the majority of cases, the swabs for smears and prostatic acid phosphatase were taken from oral, vaginal, and anorectal areas in females and oral and anorectal areas in males. The smears all were stained with the routine Papanicolaou stain, and intact spermatozoa and spermatozoan heads were sought. The prostatic acid phosphatase was analyzed by the microparticle enzyme immunoassay method and reported as ng/ml. A level of greater than 100 ng/ml was considered positive. The cases were analyzed with respect to postmortem interval; presence or absence of intact spermatozoa or spermatozoan heads; presence of an elevated prostatic acid phosphatase; body location of the specimen; the time of year; location of the victim; and physical injury (anogenital) of sexual assault. The authors hope that by examining the laboratory evidence of sexual assault, a correlation can be drawn between the presence or absence of such evidence and the aforementioned variables.     

A more sensitive modification of the zinc test for seminal traces suitable for stable test paper strips.

A more sensitive modification of the zinc test for semen has been developed, which can be used either as a solution or incorporated into test papers. The latter retain their sensitivity for at least 3 months.     

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.     

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.     

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.     

Forensic identification of urine using the DMAC test: a method validation study

Forensic scientists may sometimes be asked to identify the presence of urine in cases such as harassment, rape or murder. One popular presumptive test method uses para-dimethylaminocinnamaldehyde (DMAC), favoured because it is simple, rapid and safe. This paper confirms that DMAC reacts with urea rather than creatinine, ammonia or uric acid. Sensitivity studies found that the 0.1% w/v DMAC solution currently used for urine identification detects levels of urea found in other body fluids, potentially resulting in false positives. A 0.05% w/v solution was found to be more appropriate in terms of sensitivity to urea however the test is still not specific for urine, giving positive reactions with a number of body fluids (saliva, semen, sweat and vaginal material) and other substances (foot lotion, hair removal cream and broccoli).     

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 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 examination of vaginally inserted plastic tampon applicators for genetic markers and evidence of prior sexual intercourse

Vaginally inserted plastic tampon applicators were obtained from 42 female volunteers. The applicators were examined for the presence of ABH blood group substances, phosphoglucomutase (PGM), amylase, acid phosphatase, P30, and intact spermatozoa. Each applicator was accompanied by a control blood sample, a saliva specimen, a brief sexual and menstrual history, and method of birth control of the donor. Eight of the male sexual partners of the donors submitted blood and saliva samples. One male sexual partner submitted only a saliva sample. ABH blood group substances corresponding to the donor were recovered from 36 of the 42 applicators. The remaining 6 applicators revealed a combination of the donor's and sexual partner's ABH substances. The female's PGM type was recovered from 34 of the applicators. The remaining 8 applicators failed to show PGM activity. Of the applicators, 15 indicated evidence of prior sexual intercourse by the detection of ABH substances not consistent with the applicator donor (6 samples), high levels of acid phosphatase (11 samples), or recovery of spermatozoa (8 samples) or some combination of these. All applicator samples failed to show the presence of either P30 activity or PGM factors foreign to the female.     

Acid phosphatase screening — Wetting test paper or wetting fabric and test paper?

Detecting and locating semen stains is addressed by identifying the associated acid phosphatase activity of semen. The recovery of semen stains is critical as it can, via DNA testing, address the possible source(s) of the semen and may aid in the interpretation of a case. The purpose of these experiments, carried out on behalf of the Body Fluids Forum, was to consider whether wetting the test paper alone or wetting the semen stained fabric and the test paper affected the detection and location of the semen stains on various fabric types, or the subsequent recovery of spermatozoa from these fabrics. It became evident that the preferred approach varied depending on the fabric type being tested but that more often than not, wetting both the fabric and the test paper had a detrimental effect on the recovery of spermatozoa.     

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.     

Comparative studies of the practical use of different sperm samples

Comparative Investigation of Six Different Searching Techniques for Seminal Stains The comparison of six searching methods showed, that the phosphatase test has the highest, the choline test a sufficient and the other tests a poor sensitivity. Stains of seven body secretions and excretions, 13 fruit and 17 vegetables have been investigated by damp filter paper method. The spermine-, gamma-glutamyltranspeptidase-, and the zinc test had an insufficient specificity. The phosphatase test had false positive results with kiwi fruit, cauliflower, pea, and potato; the choline test with cauliflower, pea, broccoli, leek, and tomato; the leucineaminopeptidase test with feces and strawberry.     

Use of a Y chromosome probe as an aid in the forensic proof of sexual assault

Currently, the most common procedures for the forensic identification of semen that may be present due to a sexual assault include the microscopic identification of spermatozoa, acid phosphatase activity, or the detection of PSA. However, not all cases of sexual assault result in the deposit of semen. Fluorescent In Situ Hybridization (FISH) has been found to be a very sensitive and specific method for detection of the Y chromosome from male cells. This study was undertaken to demonstrate the presence of epithelial cells of male origin in the postcoital vaginal tract using a commercially available probe. Results identified Y chromosome in intact epithelial cells on postcoital Days 1 through 4, and on Day 7. Additionally, Y chromosome positive epithelial cells were identified in vaginal swabs obtained following intercourse with no ejaculation. The method developed in this study demonstrates that FISH is a sensitive method for the identification of the presence of male epithelial cells in the postcoital vagina.     

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen

Abstract: Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate‐specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID?‐Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID?‐Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross‐react with other human or nonhuman tissues tested. RSID?‐Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA‐based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID?‐Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.     

The acid phosphatase test two minute cut-off: An insufficient time to detect some semen stains

The ability to detect semen in sexual offence cases is a crucial first step to locating stains which may be suitable for DNA profiling. Since the development of the acid phosphatase test in the late1950s by Stuart S. Kind, the process undertaken to perform the test has gone largely unchanged. The method currently accepted by operational forensic science laboratories allows 2 min for a reaction to be obtained, and until relatively recently, this has not been challenged. In this research, samples of semen were obtained from three donors and a range of dilutions for each sample were prepared. Each dilution was subjected to acid phosphatase testing using both direct testing and the ‘press test’ method. The results showed that semen could be detected in excess of 15 min in dilutions up to 1 in 400 using the press test method and in dilutions up to 1 in 1000 using the direct method. Of further significance was the observation that using the press test method, the two minute cut-off was insufficient to detect the majority of stains and in some cases, semen stains as strong as 1 in 20 dilutions. This research provides compelling evidence for protocols currently utilised in forensic practice to be reviewed in order that forensic scientists do not overlook potential evidential material that may prove suitable for body fluid identification such as DNA STR profiling.