Seminal Stains Research Articles

Potential Application of Quantitative Prostate-specific Antigen Analysis in Forensic Examination of Seminal Stains

The aims of this study are to use quantitative analysis of the prostate-specific antigen (PSA) in the seminal stain examination and to explore the practical value of this analysis in forensic science. For a comprehensive analysis, vaginal swabs from 48 rape cases were tested both by a PSA fluorescence analyzer (i-CHROMA Reader) and by a conventional PSA strip test. To confirm the results of these PSA tests, seminal DNA was tested following differential extraction. Compared to the PSA strip test, the PSA rapid quantitative fluorescence analyzer provided the more accurate and sensitive results. More importantly, individualized schemes based on quantitative PSA results of samples can be developed to improve the quality and procedural efficiency in the forensic seminal inspection of samples prior to DNA analysis.

Evaluation of forensic examination of extremely aged seminal stains.

The results of forensic tests, such as semen identification and short tandem repeat (STR) analysis of extremely aged seminal stains from unsolved sex crimes can provide important evidence. In this study we evaluated whether current forensic methods could be applied to seminal stains that were stored at room temperature for 33-56years (n=2, 33years old; n=1, 41years old; n=1, 44years old; n=1, 56years old). The prostatic acid phosphatase (SM-test reagent), microscopic (Baecchi stain method) and semenogelin (RSID™ Semen Laboratory Kit) tests were performed as discriminative tests for semen. In addition, the mRNA levels of the semen-specific proteins semenogelin 1 (SEMG1) and protamine 2 (PRM2) were investigated. STRs were analyzed using the AmpFlSTR® Identifiler™ PCR Amplification Kit. All samples were positive in the prostatic acid phosphatase and semenogelin tests, and sperm heads were identified in all samples. The staining degree of the aged sperm heads was similar to that of fresh sperm. Although SEMG1 mRNA was not detected in any sample, PRM2 mRNA was detected in three samples. In the STR analysis, all loci were detected in the 33-years-old sample and five loci were detected in the 56-years-old sample. We confirmed that current forensic examinations - including STR analysis - could be applied to extremely aged seminal stains. These results could be useful for forensic practice.

Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material

Procedures utilizing Chelex100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extracion of DNA from semen and very small blood stains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQα locus to obtain the DQα genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQα genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.

Examination of seminal stain by HPLC assay of phenolphthalein

Quantitative high performance liquid chromatography (HPLC) to detect semen was investigated in this study. Briefly, 1 cm of a gauze thread with a seminal stain was soaked in the reaction mixture (phenolphthalein diphosphate tetrasodium dissolved in acetate buffer) for 5–10 min, and the supernatant was analyzed by HPLC with a spectrophotometric detector. Phenolphthalein was liberated from the reagent in the presence of acid phosphatase, and the liberated phenolphthalein was detected objectively and was unaffected by blood contamination. Since liberation of phenolphthalein from the reagent occurred slightly in control negative samples, the cut-off value of the examination should be set at 1.0 μg/ml.

A case of personal identification due to detection of rare DNA types from seminal stain

Following a rape incident in an apartment in Japan, we were requested to perform a DNA analysis on a body fluid stain left on a bath towel to determine whether it could be attributed to the suspect. The acid phosphatase and prostatic-specific antigen tests confirmed it to be a seminal stain. Based on the DNA analysis by autosomal and Y-chromosome short tandem repeat (STR) systems, no inconsistency was found with the profile of the suspect with African ancestry. In this case, allele 21 of DYS390 at the Y-STR locus was examined, as it is reported to have a distinctly lower frequency in the Japanese population. Furthermore, the haplotype combinations of Y-STR at the DYS389I, DYS389II and DYS390 loci are powerful for personal identification, as these have not yet been found in the Japanese population.

Preliminary Test for Demonstration of Sweat Stains by Lactic Acid

The color reaction for detection of lactic acid was investigated for application to a preliminary test for demonstration of sweat stains. The detection of lactic acid was employed with the commercially available “Determiner-LA” kit (KYOWA medics). The positive reaction was indicated by the development of a red-purple color. The positive results could be observed in sweat stains and also seminal stains. The weak positive reaction could be seen in only one of 20 salivary stains but all urinary stains gave negative results. Therefore, it is possible to discriminate sweat stains from urinary and salivary stains by using this preliminary test. However, for the distinction between sweat and seminal stains, the acid phosphatase test (SM-test reagent) should be examined. This preliminary test was detectable from sweat stains stored for 6 months at room temperature and from stains of 16-fold diluted human sweat. This preliminary test is very useful for the demonstration of sweat stains in forensic practice.

モノクローナル抗体および動物免疫血清を用いた改良ＭＣＡＲ法

Commercially available monoclonal antibodies and animal immune serums for ABO blood-typing, were evaluated in a Mixed Cell Agglutination Reaction (MCAR)test. Eighteen kinds of the monoclonal antibodies and five kinds of animal immune serums were examined in this study. Non-specific agglutinations were observed in all of the monoclonal antibodies. These non-specific agglutinations were reduced by blocking using 1% and 10% skim milk solution or by using monoclonal antibodies mixed with skim milk (1% and 10% final concentration). No antibodies and/or immune serums of an anti-A and anti-B set had strong and specific agglutination reactions in such forensic specimens as bloodstains, vaginal stains, salivary stains and seminal stains, even if using monoclonal antibodies mixed with skim milk (1% final concentration). In order to apply the MCAR test for these samples, we equally mixed two kinds of antibodies out of three kinds of antibodies which showed strong and specific agglutination in either secretor or non-secretor samples. As the result of the examinations of the six combinations, we found that the equal mixed antibody of Kokusai's and Immucor's products was applicable to ABO typing of forensic specimens. The reactivity of this mixture was similar to that of the human polyclonal antibody. The result of the blind test of ABO blood grouping of human salivary stains obtained from 100 people corresponded to the donor's type using this method. These results suggested that the mixture of antibodies is useful to detect ABO blood typing in MCAR test.

The Effect of Laundering on the Detection of Acid Phosphatase and Spermatozoa on Cotton T-Shirts

ABSTRACTIn sexual assault cases, items submitted for forensic analysis are frequently examined for seminal fluid. The contents of a medical examination kit taken from the victim, particularly the genital swabs, are often the main source of associative DNA evidence. Depending on the circumstances, however, the examination of clothing and bedding can also be important. A standard procedure used in searching clothing is to screen for acid phosphatase and to confirm the presence of seminal fluid by examination for spermatozoa. It is frequently assumed that laundering the item in question will remove acid phosphatase and therefore testing for this enzyme is not useful. We have washed and laundered seminal stains on cotton T-shirts in a variety of ways, including dry cleaning. The cleaned fabric was screened for acid phosphatase, and the presence of spermatozoa determined. The DNA content of the cleaned stain was quantified and Short Tandem Repeat (STR) profiles were obtained using the CTT triplex. The results obtained are discussed in the context of the overall examination tactics in sexual assault cases.

Wood's lamp utility in the identification of semen.

The accurate detection of semen is critical to forensic, medical, and legal personnel. The Wood's lamp (WL) emits ultraviolet light (UVL) and has been identified as useful in rape evaluations because it is purported to cause semen to fluoresce. This study was intended to determine if semen can be distinguished from other products by WL analysis. Investigators reviewed the previous training and frequency of use of the WL by emergency medicine and pediatric emergency medicine physicians at 2 medical centers. The participants were asked to use a WL to distinguish between a semen sample (<6 hours old) and 13 commonly used products. Next, 29 semen samples were collected and evaluated under high-power microscopy and under UVL. A total of 41 physicians participated in the study (68% male). The number of years practicing in an emergency setting spanned from.3 to 25 years with a mean of 7. 1 years. A total of 51% of participants trained in emergency medicine, 23% in pediatrics and pediatric emergency medicine. A total of 22% reported formal training in the collection of forensic evidence. A total of 62% of the physicians believed they have identified semen in the past; one third felt they could differentiate semen from other products under UVL. None of the 41 physicians were able to differentiate semen from other products using a WL. Moreover, the semen samples used for the study did not fluoresce under WL analysis. None of the 29 semen samples fluoresced whether wet or dry. The medicaments most commonly mistaken for semen were A&D ointment (Cardinal Health, Inc, Dublin, OH), Surgilube (Division of Atlanta, Inc, Melville, NY), Barrier cream (Carrington Laboratories, Inc, Irving, TX), and bacitracin (Division of Atlanta, Inc, Melville, NY). Participating physicians were unable to distinguish between semen and other common products, using the WL. Although the WL has been purported to be a useful tool as a screening device for the detection of seminal stains, the investigators have found it to be unreliable. Semen, previously reported to fluoresce under WL analysis, does not appear to do so. The correct identification of semen may be complicated by the presence of previously existing ointments and creams, some of which may be iatrogenically introduced (ie, Surgilube).

Effects of solvent displacement on sensitivity and specificity of monoclonal antibodies for ABO blood grouping of forensic specimens with an absorption-elution test

Using commercially-available monoclonal antibodies (Bioclone, Neo Kokusai, Monoclonal Wako, Gamma Clone and Seraclone), ABO blood grouping of forensic specimens such as bloodstains, salivary stains, seminal stains, nails, hair and cerebral dura mater was performed with an absorption-elution test. Salivary stains, seminal stains and nails were not typed correctly using the antibodies other than Bioclone reagents, while precise grouping of bloodstains was performed using most antibodies. When hair and dura mater were tested, all of the antibodies induced weak or non-specific haemagglutination, hence correct grouping was not achieved. When the antibody solvents were displaced with 5–20% bovine serum albumin in saline, human serum of the group AB donor, or serum of chicken, sheep or bovine, titers of the reagents increased 2–8 times. Hair and dura mater were able to be typed using Bioclone reagents after solvent displacement with human AB or sheep serum, whereas displacement with the other solvents enhanced non-specific reactions.

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-GTP) was developed using a combination of monoclonal antibodies, SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10 7 times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal γ-GTP was detectable even in 14-year-old stains.

The HumD21S11 system of short tandem repeat DNA polymorphisms in Japanese and Chinese

HumD21S11 is a short tandem repeat DNA polymorphic system with a complex basic structure of (TCTA) 4-6 (TCTG) 5-6 (TCTA) 3 TA (TCTA) 3 TCA (TCTA) 2 TCCA TA (TCTA) n. Using the allelic ladder prepared by us, the distribution of alleles among Japanese and Chinese was investigated, and four new alleles 28.2, 34, 35.2, and 36.2, were discovered. DNA sequencing was performed on the newly found alleles as well as on family samples and led to the discovery of different gene structures within alleles 28 and 32. Forensic materials, including hairs and seminal stains, were tested in parallel with blood samples from the same individual and were successfully typed for D21S11.

Group-specific component subtyping in semen and seminal stains by conventional isoelectric focusing.

Group-specific component (GC) subtyping in semen and seminal stains was carried out using isoelectric focusing in carrier ampholyte-generated pH gradients and immunoblotting. In serum samples the anodal bands of GC 1F and of GC 1S disappeared by neuraminidase treatment, but in semen samples these bands remained unchanged after such treatment. The GC 2 type in semen exhibited two bands: the main GC 2 band and another fast band which focused at the position of the cathodic band of GC 1F. These seminal GC bands were unaffected by enzyme digestion. Reliable subtyping was possible in seminal stains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 8 weeks, and at 37 degrees C for up to 5 weeks. The GC subtyping by conventional isoelectric focusing after neuraminidase treatment is simple, economical and useful in medicolegal examination of seminal stains.

Analysis of the D1S80 locus by amplified fragment length polymorphism technique in the Chinese, Malays and Indians in Singapore

The distributions of the D1S80 alleles and genotypes in the Chinese, Malays and Indians in Singapore were determined by amplified fragment length polymorphism (AMP-FLP) analysis. The distributions of the observed genotypes for the three races conformed to Hardy-Weinberg expectations. The system was applied to 19 families whose paternity had been established by restriction fragment length polymorphism (RFLP) analysis. In all cases, Mendelian inheritance of the alleles at the D1S80 locus could be demonstrated. D1S80 typing on DNA recovered by differential extraction of forensic specimens which included vaginal swabs, urethral swabs and seminal stains yielded consistent results.

Potential Forensic Applications of Minisatellite Variant Repeat (MVR) Mapping Using the Polymerase Chain Reaction (PCR) at D1S8

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to samples from various human tissues. All DNA samples obtained from an individual's organs at autopsy consistently gave the same digital diploid codes. Even 1 ng of genomic DNA was sufficient to obtain authentic diploid MVR coding ladders. MVR-PCR could be reliably applied to DNA isolated from bloodstains, saliva stains, seminal stains and plucked hair roots, and should become a powerful tool for individual identification in forensic investigations.

Rapid detection of human seminal plasma proteins by membrane aspiration test (MAT)

A new dot-immunobinding method, called the membrane aspiration test (MAT), was developed for the detection of human seminal plasma (HSP) antigens. Biological materials, such as macerated vaginal swabs or extracts from seminal stains, were dotted on a nitrocellulose membrane in a disposable aspiration kit. After the drying and aspiration of a quenching solution, a suspension of gold particles (GP) coated with an immune serum to HSP was aspirated through the nitrocellulose membrane. The dotted portion on the membrane turned red after several repeated aspirations, if HSP antigen was present. Results can be obtained within a few minutes. This test has broad applicability because of its rapidity and simplicity.

ABH-related antigens in human male genital tract. A histochemical examination.

The localization of ABH related antigens in human male reproductive tract was examined using monoclonal antibodies and an avidin biotin complex method. No positive reaction with blood group antibodies on spermatozoa was observed in testis and ductus epididymidis apart from erythrocytes and endothelial cells. The expression of ABH and ABH related antigens in ductuli efferents testis, ductus epididymidis, seminal vesicle and prostate was complexly coded by a combination of H, Se, Le and X genes. The results obtained in this study indicate that the ABH antigens detected on spermatozoa of seminal stains are coating antigens and not inherent to the cell membrane, and the ABO, H, Se, Le and X genes are subjected to a tissue-dependent differential expression.

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.

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.

Typing study of human semen DIA 3 by isoelectric focusing-distribution in the Wuhan population, China

Human semen DIA 3 typing was studied by isoelectric focusing on ultra-thin-layer polyacrylamide gel which resulted in a simpler and more difinite separation of the products of DIA 3 alleles than hitherto. In 198 semen samples collected from unrelated Chinese males four different phenotypes were observed. The DIA 3 allele frequencies were calculated: DIA 3 1 = 0.7727, DIA 3 2 = 0.2172, DIA 3 3 = 0.0101. The results of the stability study of 12 laboratory-prepared semen stains stored at room temperature suggested that DIA 3 in seminal stains is a relatively stable genetic marker. Our gene frequencies have been compared to those reported in other populations.