Optimization of spermatozoa detection using immunofluorescent staining and laser micro-dissection.
The present study evaluated the use of an immunofluorescence-based assay for the microscopic detection of human spermatozoa, following which the fluorescence-labelled spermatozoa could be excised with a laser micro-dissection system. The Sperm Hy-Liter™ PI kit was able to detect spermatozoa from as little as 20nL of semen. No interference or non-specificity were observed when the kit was used on semen mixed with various body fluids such as blood and urine, as well as when semen was spiked onto different types of fabric. Good results could also be obtained with rectal samples which contain auto-fluorescent fecal materials through the use of dual FITC/PI filters. We also developed a method for concurrent testing of two protein biomarkers of semen (semenogelin and prostate-specific antigen) and detection of spermatozoa. This approach would maximize the evidential value from a single piece of sexual assault exhibit. The results also showed that staining by Sperm Hy-Liter™ PI does not interfere with DNA recovery, facilitating the generation of clear male DNA profiles from dissected spermatozoa, thereby making profile interpretation less complex. In summary, Sperm Hy-Liter™ PI staining was demonstrated to be sensitive, robust and specific.
DNA Profiling Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, 169078 Singapore, Singapore. Electronic address: ping_yueh_shyang@hsa.gov.sg.
Yueh Shyang
YS
Ping
DNA Profiling Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, 169078 Singapore, Singapore.
Xavier Liang Shun
XL
Chan
DNA Profiling Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, 169078 Singapore, Singapore.
Sze Kae
SK
Goh
DNA Profiling Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, 169078 Singapore, Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543 Singapore, Singapore.
Christopher Kiu Choong
CK
Syn
D015415:Biomarkers; D016172:DNA Fingerprinting; D005455:Fluorescent Antibody Technique / Q000295:instrumentation*; D049429:Forensic Pathology; D006801:Humans; D061346:Laser Capture Microdissection; D008297:Male; D017430:Prostate-Specific Antigen / Q000302:isolation & purification; D029607:Seminal Vesicle Secretory Proteins / Q000302:isolation & purification; D012742:Sex Offenses; D013094:Spermatozoa / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*
D015415:Biomarkers; D029607:Seminal Vesicle Secretory Proteins; C424482:seminal vesicle-specific antigen; D017430:Prostate-Specific Antigen
DNA; Prostate-specific antigen; Semenogelin; Sexual assault; Sperm Hy-Liter™ PI