|Year : 2015 | Volume
| Issue : 2 | Page : 159-162
Potential Application of Quantitative Prostate-specific Antigen Analysis in Forensic Examination of Seminal Stains
Zhenping Liu1, Yang Yang1, Zhikan Pan1, Xiaoxu Zhu2, Shi Feng2, Dong Zhao2
1 Criminal Investigation Detachment, Jinhua Bureau of Public Security, Jinhua 321000, China
2 Collaborative Innovation Center of Judicial Civilization, Key Laboratory of Evidence Science, University of Political Science and Law, Ministry of Education, Beijing, China
|Date of Web Publication||27-Nov-2015|
Institute of Evidence Law and Forensic Science, China University of Political Science and Law, 25 Xitucheng Road, Haidian, Beijing 100088
Source of Support: None, Conflict of Interest: None
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.
Keywords: Forensic biology, prostate-specific antigen, rapid quantitative fluorescence analyzer, semen
|How to cite this article:|
Liu Z, Yang Y, Pan Z, Zhu X, Feng S, Zhao D. Potential Application of Quantitative Prostate-specific Antigen Analysis in Forensic Examination of Seminal Stains. J Forensic Sci Med 2015;1:159-62
|How to cite this URL:|
Liu Z, Yang Y, Pan Z, Zhu X, Feng S, Zhao D. Potential Application of Quantitative Prostate-specific Antigen Analysis in Forensic Examination of Seminal Stains. J Forensic Sci Med [serial online] 2015 [cited 2019 Oct 17];1:159-62. Available from: http://www.jfsmonline.com/text.asp?2015/1/2/159/169640
| Introduction|| |
Prostate-specific antigen (PSA) is a serine protease produced by the mature male prostate gland and surrounding cells of the urethral epithelium. In addition, it is known by other names such as γ-seminoprotein, E1 protein, P30, and PA. The normal PSA level in human semen was reported to be 0.24–5.5 mg/mL, with an average of 1.92 mg/mL. PSA detection is regarded to be useful because of its high sensitivity; the PSA epitope, however, is present in other body fluids, but it is still highly recommended for the semen diagnosis workflow at the forensic laboratory. PSA detection can avoid the effects caused by the presence or absence of sperm in semen samples and interference caused by vaginal fluid and saliva. The PSA test can differentiate between semen samples from humans and animals, and it is especially applicable for semen of individuals with azoospermia or who have undergone vasectomy , but also diagnosis for normospermic semen prior/after ejaculation or from nonspermatic aliquots. In current forensic casework, PSA is primarily detected using gold-labeled test strips. The basic principle of the gold-labeled double-antibody sandwich method is to detect PSA by immunological methods and chromatography techniques. In forensic practice, false-positive and false-negative results often occur,,, because of hysteresis effects caused by overly concentrated samples or low sensitivity of reagent strips  because sensitivity varies among strips from different manufacturers. This study introduces a quantitative fluorescence assay for PSA detection in suspicious seminal stains in forensic examination.
| Materials and Methods|| |
A total of 48 vaginal swabs were collected for examination as real casework samples. Approximately half of a cotton swab tips were placed in 1.5-mL Eppendorf tubes, and 1 mL TNE (tris sodium chloride ethylenediaminetetraacetic acid buffer) was added to submerge the samples for 30 min. The samples were then sufficiently vortexed for 15 s and centrifuged for 3 min at 13,000 ×g. Blank swabs were used as negative controls. Supernatants (15 μL) were extracted and added to test tubes containing 145 μL buffer solution, followed by repeated pipetting and mixing. Then, 75 μL buffer solution with the samples were added to sample wells of the reaction plate and allowed to stand at room temperature for 15 min. A PSA fluorescence quantitative assay was performed on an i-CHROMA Readerwith ancillary PSA detection reagents (Boditech Med Inc., Chuncheon-si, Korea). The reaction plate was inserted into the PSA rapid fluorescence quantitative analyzer (i-CHROMA Reader) for automatic scanning, and the results were read from the display screen. Conventional strip PSA tests were performed according to the manufacturer's protocol (Biowin Ltd., Qingdao, China). This study introduced a PSA rapid quantitative fluorescence analyzer, the i-CHROMA Reader. The external dimensions of the portable i-CHROMA Reader used in this study are 185 mm × 80 mm × 250 mm, and underlying principles of this analyzer include the rapid quantitative immunofluorescence technique and dry chemical chromatography. During the assay process, immune complexes are formed within samples. By analyzing the value of the detection zone/quality control zone and comparing it with the calibration curve generated by standard samples of different concentrations, the concentration of PSA in the seminal sample can be determined. PSA concentrations can be quantified and read out: >50 ng/mL, 2–50 ng/mL (in this range properly quantifying specific values) and <2 ng/mL.
The end closer to the "maximum" mark on the test strip was inserted into the sample solution, and results were observed after 5 min. DNA was isolated from mixed seminal samples through a two-step differential extraction method. Using the 800 μL sample solution remaining from the quantitative PSA assay and strip PSA test for the first step of digestion, DNA was obtained from precipitated cells through the silica-bead method. Extracted DNA was amplified with AmpFLSTR ® Identifiler ® Plus PCR Amplification Kit (Life Technologies, Carlsbad, CA, USA) on the 9700 PCR System (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. PCR products underwent electrophoresis (voltage 3 kV; time 2500 s) using the 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA), and results were analyzed using GeneMapper ID v3.2 software (Life Technologies, Carlsbad, CA, USA).
| Results and Discussion|| |
The quantitative determination of PSA in seminal stains admixed with female components can help forensic inspectors to make correct decisions based on the PSA amount in the sample and thereby type the DNA profile of a single male, which will greatly improve the detection efficiency and provide valuable evidence for litigation and trial procedures. Compared with the conventional strip PSA test, major advantages of this rapid quantitative fluorescence assay are as follows: (1) Because this assay is a quantitative immunofluorescence test with numerical test results rather than positive or negative, it is more intuitive, specific, objective, and rigorous for use in semen identification. (2) The analyzer has stricter quality control, as this product has a built-in calibration curve, which waives the requirement for positive and negative controls. (3) In addition, the analyzer has a built-in chip for each pack of disposables, which records the manufacture date of the antibody batch. If expired antibodies are used, or improper storage is detected, then the error messages are displayed during the examination. (4) Detection is quicker than other methods, as each test needs only 2 s for 100 results recorded automatically. (5) This assay has a higher sensitivity (of 2 ng/mL) compared to the conventional strip PSA test.
Quantitative fluorescence PSA assay and conventional PSA strip test, together with male DNA findings are shown in [Table 1]. Thirty-nine samples out of 48 case samples [Table 1] were quantified as having PSA >2 ng/mL by the quantitative PSA assay, and the male component was detected. The male DNA component was not detectable for all nine samples with PSA <2 ng/mL; thus, quantitative PSA results were in good agreement with DNA results. However, DNA results were not so correlated with conventional PSA test [five cases, [Table 1]. Out of nine samples with PSA 20–50 ng/mL, five were found to have a single male DNA profile, and the other four had male and female mixed DNA profiles. The latter may suggest that samples within that PSA range could contain biological fluid but at minimum adequate levels to be detected as single male DNA profiles compared to female DNA. Therefore for samples with PSA >50 ng/mL, the exact PSA quantity can be determined by a serial dilution, and the amount of DNA template added for subsequent DNA typing could be adjusted to obtain a single male DNA profile. Samples with PSA 20–50 ng/mL may result in a mixed DNA profile; therefore we recommend the two-cycle digestion method  and the column-based sperm enrichment  to reduce the impact of female epithelial cells and increase the male component for a higher probability of obtaining single male DNA profiles. Furthermore, among five samples with PSA 2–20 ng/mL, conventional strip PSA test showed weak positive results for two samples and negative results for the other three samples; either Y-STR or male and female mixed autosomal DNA profiles were detected in all five samples partial DNA results are shown in [Figure 1]. Therefore, the two PSA-negative results of the conventional strip test appeared to be false-negatives. For samples containing a small amount of male semen, DNA typing tends to show mixed profiles; hence, the microscopic laser cutting method may be applied to identify suspects in the case of single male DNA profiles. Male DNA typing failed for all seven samples with negative results of the conventional strip PSA test and with PSA <2 ng/mL from the quantitative assay. Two samples were detected as positive by the strip PSA test; however, the quantitative fluorescence assay detected PSA <2 ng/mL and no male DNA was detected in the samples which may suggest that results were detected as false-positive by the strip test. Considering the latter, the PSA fluorescence quantitative assay has much higher sensitivity compared to the conventional strip PSA test.
|Figure 1: DNA typing with the ABI Identifiler-Plus kit of a sample with prostate-specific antigen 5.25 ng/mL as detected by the i-CHROMA Reader|
Click here to view
It should be noted that although no male DNA profile was detected from any of the nine samples with PSA <2 ng/mL in this study, such cases should not be disregarded as sperm nuclear DNA may still be detected. Future investigation on PSA denaturation would be useful to explain some results in the forensic laboratory, as urea, pH, and other factors could cause PSA denaturation, submergence of bodies in water could cause PSA dissolution. In addition, semen samples from males with azoospermia or those who have undergone vasectomy contain PSA but no sperm; thus, the conventional strip PSA test will indicate positive results while DNA test may indicate negative results for male DNA typing. In such cases, inspectors may consider the nonquantitative strip PSA test result as a false-positive and may further investigate, thus losing the opportunity to disclose the fact that the suspect's semen does not contain sperms. With the aid of the quantitative assay for PSA described in this study, cases with PSA >50 ng/mL as detected by quantitative fluorescence analysis and with no male DNA profile should be suspected of having azoospermia (note also aliquots of normospermic semen with no sperm but semen fluid with other cell nuclei).
| Conclusion|| |
The PSA rapid quantitative fluorescence analyzer (i-CHROMA Reader) was proved a more reliable, accurate, and easy-to-use method than the strip test. The i-CHROMA is smaller, portable, affordable, and convenient and it is, therefore, suitable for on-site evidence screening and forensic DNA laboratories. From a technical point of view, the fluorescence quantitative PSA assay can greatly improve both test sensitivity and accuracy. Based on quantitative results of the PSA test for seminal samples, individualized inspection schemes can be developed to improve procedural efficiency and quality.
Thanks for the support from the Program for Young Innovative Research Team in China University of Political Science and Law.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rao AR, Motiwala HG, Karim OM. The discovery of prostate-specific antigen. BJU Int 2008;101:5-10.
Martínez P, Santiago B, Alcalá B, Atienza I. Semen searching when sperm is absent. Sci Justice 2015;55:118-23.
Wang MC, Papsidero LD, Chu TM. Prostate-specific antigen, p30, gamma-seminoprotein, and E1. Prostate 1994;24:107-10.
Healy DA, Hayes CJ, Leonard P, McKenna L, O'Kennedy R. Biosensor developments: application to prostate-specific antigen detection. Trends Biotechnol 2007;25:125-31.
Romero-Montoya L, Martínez-Rodríguez H, Pérez MA, Argüello-García R. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases. Forensic Sci Int 2011;206:111-8.
Simich JP, Morris SL, Klick RL, Rittenhouse-Diakun K. Validation of the use of a commercially available kit for the identification of prostate specific antigen (PSA) in semen stains. J Forensic Sci 1999;44:1229-31.
Laux DL, Barnhart JP. Validation of the Seratec® SeraQuant™ for the quantitation of prostate-specific antigen levels on immunochromatographic membranes. J Forensic Sci 2011;56:1574-9.
Lunetta P, Sippel H. Positive prostate-specific antigen (PSA) reaction in post-mortem rectal swabs: a cautionary note. J Forensic Leg Med 2009;16:397-9.
Levine B, Titus JM, Moore K, Fowler D. Use of prostate specific antigen in the identification of semen in postmortem cases. Am J Forensic Med Pathol 2004;25:288-90.
Denison SJ, Lopes EM, D'Costa L, Newman JC. Positive prostate-specific antigen (PSA) results in semen-free samples. Can Soc Forensic Sci J 2004;37:197-206.
Talthip J, Chirachariyavej T, Peonim AV, Atamasirikul K, Teerakamchai S. An autopsy report case of rape victim by the application of PSA test kit as a new innovation for sexual assault investigation in Thailand. J Med Assoc Thai 2007;90:348-51.
Sato I, Sagi M, Ishiwari A, Nishijima H, Ito E, Mukai T. Use of the "SMITEST" PSA card to identify the presence of prostate-specific antigen in semen and male urine. Forensic Sci Int 2002;127:71-4.
Tian H, Hühmer AF, Landers JP. Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. Anal Biochem 2000;283:175-91.
Liu ZP, Wu WW, Pan ZK, Gao H, Chen YM. Thesis Collection of the First Nationwide Public Security DNA Database Construction and Application Seminar: Application of Two-cycle Female Epithelial Cell Digestion Method in Detection of Mixed Stain of Seminal Stain. Beijing: China People's Public Security University Press; 1999. p. 307-8.
Shao W, Jiang XH, Sun XK, Liu F, Jin P. The study of separation techniques of sperm collecting column. Chin J Forensic Med 2009;24:194-7.
Sanders CT, Sanchez N, Ballantyne J, Peterson DA. Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis. J Forensic Sci 2006;51:748-57.
Mischler TW, Reineke EP. Immunological identification of human seminal stains. J Crim Law Criminol 1996;57:107-11.