A confirmatory test for sperm in sexual assault samples using a microfluidic-integrated cell phone imaging system.

Rapid and efficient processing of sexual assault evidence to accelerate forensic investigation and decrease casework backlogs is urgently needed. Therefore, the standardized protocols currently used in forensic laboratories can benefit from continued innovation to handle the increasing number and complexity of samples being submitted to forensic labs. To our knowledge, there is currently no available rapid and portable forensic screening technology based on a confirmatory test for sperm identification in a sexual assault kit. Here, we present a novel forensic sample screening tool, i.e., a microchip integrated with a portable cell phone imaging platform that records and processes images for further investigation and storage. The platform (i) precisely and rapidly screens swab samples (<15 min after sample preparation on-chip); (ii) selectively captures sperm from mock sexual assault samples using a novel and previously published SLeX-based surface chemistry treatment (iii) separates non-sperm contents (epithelial cells and debris in this case) out of the channel by flow prior to imaging; (iv) captures cell phone images on a portable cellphone-integrated imaging platform, (v) quantitatively differentiates sperm cells from epithelial cells, using a morphology detection code that leverages Laplacian of Gaussian and Hough gradient transform methods; (vi) is sensitive within a forensic cut-off (>95% accuracy) compared to the manual counts; (vii) provides a cost-effective and timely solution to a problem which in the past has taken a great deal of time; and (viii) handles small volumes of sample (20 µL). This integration of the cellphone imaging platform and cell recognition algorithms with disposable microchips can be a new direction toward a direct visual test to screen and differentiate sperm from epithelial cell types in forensic samples for a crime laboratory scenario. With further development, this integrated platform could assist a sexual assault nurse examiner (SANE) in a hospital or sexual assault treatment center facility to flag sperm-containing samples prior to further downstream testing.

Tunable diode laser absorption spectroscopy as method of choice for non-invasive and automated detection of microbial growth in media fills.

Tunable diode laser absorption spectroscopy (TDLAS) was evaluated on its potential to detect bacterial growth of contaminated media fill vials. The target was a replacement/ automation of the traditional visual media fill inspection. TDLAS was used to determine non-invasively O2 and/or CO2 changes in headspaces of such vials being induced by metabolically active microorganisms. Four different vial formats, 34 microorganisms (inoculation volume<10 cells) and two different media (TSB/FTM) were tested. Applying parallel CO2 and O2 headspace measurements all format-organism combinations were detected within <11 days reliably with reproducible results. False negatives were exclusively observed for samples that were intentionally breached with syringes of 0.3mm in diameter. Overall it was shown that TDLAS functionality for a replacement of the visual media fill inspection is given and that investing in further validation and implementation studies is valuable. Nevertheless, some small but vincible challenges remain to have this technology in practical use.

The Application of Noninvasive Headspace Analysis to Media Fill Inspection.

The results of a proof-of-principle study demonstrating a new analytical technique for detecting microbial growth directly in pharmaceutical containers are described. This analytical technique, laser-based headspace analysis, uses tunable diode laser absorption spectroscopy to nondestructively determine gas concentrations in the headspace of a media-filled pharmaceutical container. For detecting microbial growth, the levels of headspace oxygen and carbon dioxide are measured. Once aerobic microorganisms begin to divide after the lag phase and enter the exponential growth phase, there will be significant consumption of oxygen and concomitant production of carbon dioxide in the sealed container. Laser-based headspace analysis can accurately measure these changes in the headspace gas composition. The carbon dioxide and oxygen measurement data for the representative microorganisms Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus brasiliensis were modeled using the Baranyi-Roberts equation. The mathematical modeling allowed quantitative comparisons to be made between the data from the different microorganisms as well as to the known growth curves based on microbial count. Because laser-based headspace analysis is noninvasive and can be automated to analyze the headspace of pharmaceutical containers at inspection speeds of several hundred containers per minute on-line, some potential new applications are enabled. These include replacing the current manual human visual inspection with an automated analytical inspection machine to determine microbial contamination of media fill and pharmaceutical drug product vials. LAY ABSTRACT: A novel analytical technique has been demonstrated for detecting microbial growth in media-filled pharmaceutical containers. This analytical technique, laser-based headspace analysis, uses tunable diode laser absorption spectroscopy to determine gas concentrations in the headspace of a pharmaceutical container. For detecting microbial growth, the levels of headspace oxygen and carbon dioxide are measured. The study shows that once aerobic microorganisms begin to grow after the lag phase and enter the exponential growth phase there will be a significant consumption of oxygen in the sealed container as well as a corresponding production of carbon dioxide. Headspace analysis can accurately measure and monitor these changes in the headspace gas composition and could therefore be used to detect contaminated pharmaceutical containers. Because the technique can be automated to analyze hundreds of containers a minute on-line, there are opportunities for implementing a headspace inspection machine to perform automated inspection of media fills used to validate aseptic filling operations.

Container/Closure Integrity Testing and the Identification of a Suitable Vial/Stopper Combination for Low-Temperature Storage at -80 {degrees}C.

It was recently found that after storage of a live viral vaccine at -80 °C in glass vials closed with rubber stoppers, a phenomenon was revealed which had not been observed before with other viral products stored at -20 °C: overpressure in the vials. As this phenomenon poses a serious safety problem for medical personnel as well as for the product itself, an investigation was initiated to identify the root cause of the overpressure. After exclusion of possible root causes (differences in air temperature or atmospheric air pressure during filling and quality control testing, outgassing from the formulation buffer) the remaining hypothesis involved a possible container closure integrity issue at low temperature. The glass transition temperatures (T(g)) of many rubber stopper formulations are in the range -55 to -70 °C. At storage temperatures below T(g), the rubber stopper loses its elastic properties and there is a risk that the seal integrity of the vial could be compromised. Loss of seal integrity of the vials near storage temperatures of -80 °C would result in an ingress of cold dense gas into the vial headspace. After removal of the vials from storage at -80 °C, the rubber stoppers could regain their elastic properties and the vials would quickly reseal, thereby trapping the ingressed gas, which leads to overpressure in the vial headspace. Nondestructive laser-based headspace analysis was used to investigate the maintenance of container closure integrity as a function of the filling and capping/crimping process, storage and transport conditions, and vial/stopper designs. This analytical method is based on frequency modulation spectroscopy (FMS) and can be used for noninvasive headspace measurements of headspace pressure and headspace gas composition. Changes in the vial headspace composition and/or pressure are a clear marker for vials that have lost container closure integrity. LAY ABSTRACT: After storage of a live viral vaccine at -80 °C in glass vials closed with rubber stoppers, overpressure in some of the vials was observed, posing a serious safety problem for medical personnel as well as for the product. A working hypothesis to explain this phenomenon involved a possible container closure integrity issue at these low temperatures. The glass transition temperatures (T(g)) of many rubber stopper formulations are in the range -55 to -70 °C. At storage temperatures below T(g), the rubber stopper loses its elastic properties, resulting in compromised seal integrity of the vial and ingress of cold dense gas into the vial headspace. Upon thawing, the rubber stoppers regain their elastic properties and the vials quickly reseal, thereby trapping the ingressed gas, which leads to overpressure in the vial headspace. Nondestructive, laser-based headspace analysis, which is able to detect changes in headspace pressure and gas composition, was used to investigate the maintenance of container closure integrity. Changes in the vial headspace composition and/or pressure are a clear marker for vials that have lost container closure integrity.