Equivalence of ELISpot assays demonstrated between major HIV network laboratories.

BACKGROUND: The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates. METHODS: We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study. FINDINGS: Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (-1.5%, 1.5%) and CEF (-0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [-15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study. INTERPRETATION: The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.

Using quantum dots to tag subsurface damage in lapped and polished glass samples.

Grinding, lapping, and polishing are finishing processes used to achieve critical surface parameters in a variety of precision optical and electronic components. As these processes remove material from the surface through mechanical and chemical interactions, they may induce a damaged layer of cracks, voids, and stressed material below the surface. This subsurface damage (SSD) can degrade the performance of a final product by creating optical aberrations due to diffraction, premature failure in oscillating components, and a reduction in the laser induced damage threshold of high energy optics. As these defects lie beneath the surface, they are difficult to detect, and while many methods are available to detect SSD, they can have notable limitations regarding sample size and type, preparation time, or can be destructive in nature. The authors tested a nondestructive method for assessing SSD that consisted of tagging the abrasive slurries used in lapping and polishing with quantum dots (nano-sized fluorescent particles). Subsequent detection of fluorescence on the processed surface is hypothesized to indicate SSD. Quantum dots that were introduced to glass surfaces during the lapping process were retained through subsequent polishing and cleaning processes. The quantum dots were successfully imaged by both wide field and confocal fluorescence microscopy techniques. The detected fluorescence highlighted features that were not observable with optical or interferometric microscopy. Atomic force microscopy and additional confocal microscope analysis indicate that the dots are firmly embedded in the surface but do not appear to travel deep into fractures beneath the surface. Etching of the samples exhibiting fluorescence confirmed that SSD existed. SSD-free samples exposed to quantum dots did not retain the dots in their surfaces, even when polished in the presence of quantum dots.