SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence.

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (ts=6.567, df=1, P=0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.

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

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.

Developmental validation of the SPERM HY-LITER™ kit for the identification of human spermatozoa in forensic samples.

With sexual assault evidence, the visualization of spermatozoa confirms that ejaculation has occurred. However, microscopic examination of spermatozoa is a laborious process and can sometimes result in sperm cells being overlooked. Here, we present the developmental validation of the SPERM HY-LITER™ kit, which contains a human sperm-specific mouse monoclonal antibody coupled to a fluorescent Alexa 488 dye. The kit was tested using samples of human semen, saliva, blood, and urine, various animal semen extracts, sexual lubricants, and a commercially available spermicidal film. Postcoital vaginal swabs, degraded semen samples, and samples prepared with sample fixation techniques that deviated from the kit-provided protocol were also tested. In each case, the SPERM HY-LITER™ kit was demonstrated to bind only to human sperm cell heads. Limitations to this fluorescent staining procedure include nonspecific staining and increased background fluorescence with extreme heat fixation in some samples.

Developmental validation of RSID-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva.

Current methods for forensic identification of saliva generally assay for the enzymatic activity of alpha-amylase, an enzyme long associated with human saliva. Here, we describe the Rapid Stain IDentification (RSID-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID-Saliva detects less than 1 microL of saliva. The sensitivity of RSID-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.

Developmental validation of a novel lateral flow strip test for rapid identification of human blood (Rapid Stain Identification--Blood).

Human blood is the body fluid most commonly encountered at crime scenes, and blood detection may aid investigators in reconstructing what occurred during a crime. In addition, blood detection can help determine which items of evidence should be processed for DNA-STR testing. Unfortunately, many common substances can cause red-brown stains that resemble blood. Furthermore, many current human blood detection methods are presumptive and prone to false positive results. Here, the developmental validation of a new blood identification test, Rapid Stain Identification--Blood (RSID--Blood), is described. RSID--Blood utilizes two anti-glycophorin A (red blood cell membrane specific protein) monoclonal antibodies in a lateral flow strip test format to detect human blood. We present evidence demonstrating that this test is accurate, reproducible, easy to use, and highly specific for human blood. Importantly, RSID--Blood does not cross-react with ferret, skunk, or primate blood and exhibits no high-dose hook effect. Also, we describe studies on the sensitivity, body fluid specificity, and species specificity of RSID--Blood. In addition, we show that the test can detect blood from a variety of forensic exhibits prior to processing for DNA-STR analysis. In conclusion, we suggest that RSID--Blood is effective and useful for the detection of human blood on forensic exhibits, and offers improved blood detection when compared to other currently used methods.

Automated forensic DNA purification optimized for FTA card punches and identifiler STR-based PCR analysis.

Forensic labs globally face the same problem-a growing need to process a greater number and wider variety of samples for DNA analysis. The same forensic lab can be tasked all at once with processing mixed casework samples from crime scenes, convicted offender samples for database entry, and tissue from tsunami victims for identification. Besides flexibility in the robotic system chosen for forensic automation, there is a need, for each sample type, to develop new methodology that is not only faster but also more reliable than past procedures. FTA is a chemical treatment of paper, unique to Whatman Bioscience, and is used for the stabilization and storage of biological samples. Here, the authors describe optimization of the Whatman FTA Purification Kit protocol for use with the AmpFlSTR Identifiler PCR Amplification Kit.

Use of applied biosystems 7900HT sequence detection system and Taqman assay for detection of quinolone-resistant Neisseria gonorrhoeae.

Mutations in quinolone resistance-determining regions (QRDRs) have been associated with quinolone-resistant Neisseria gonorrhoeae (QRNG). Since diagnostic nucleic acid amplification tests for gonococci are now in frequent use, molecular detection of QRNG could facilitate surveillance in the absence of culture. Here we describe a real-time molecular assay for detecting QRDR mutations in gonococci.