The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events.

Clinicians frequently use influenza rapid antigen tests for diagnostic testing. We tested nasal wash samples from 1 April to 7 June 2009 from 1538 patients using the QuickVue Influenza A+B (Quidel) rapid influenza antigen test and compared the results with real-time reverse transcription polymerase chain reaction (rRT-PCR) assay (gold standard). The prevalence of 2009 pandemic influenza A (pH1N1) was 1.98%, seasonal influenza type A .87%, and seasonal influenza type B 2.07%. The sensitivity and specificity of the rapid test for pH1N1 was 20% (95% CI, 8-39) and 99% (95% CI, 98-99), for seasonal influenza type A 15% (95% CI, 2-45) and 99% (95% CI, 98-99), and for influenza type B was 31% (95% CI, 9-61) and 99% (95% CI, 98-99.7). Rapid influenza antigen tests were of limited use at a time when the prevalence of pH1N1 and seasonal influenza in the United States was low. Clinicians should instead rely on clinical impression and laboratory diagnosis by rRT-PCR.

CD8+ T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8+ T cells, as measured by IFN-γ secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN-γ-secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN-γ-secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-γ. These results indicate that measurement of IFN-γ production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN-γ ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of “preterminal differentiation” of HIV-specific CD8+ T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.

In this study population, age and elapsed time since baseline audiogram were modestly associated with decreased hearing sensitivity and increased likelihood for a non-H1 hearing profile. Aircraft type, as determined from Air Force Specialty Code, was not associated with changes in hearing sensitivity by STS metric.Greenwell BM, Tvaryanas AP, Maupin GM. Risk factors for hearing decrement among U.S. Air Force aviation-related personnel. Aerosp Med Hum Perform. 2018; 89(2):80-86.

Although the pressure injury rate of 4.9% in this cohort of patients is consistent with that reported by civilian critical care units, the rate must be interpreted with caution, because civilian study data frequently represent the entire intensive care unit length of stay. Targeted interventions for patients with increased body mass index and 2 or more critical care air transports per patient may help decrease the development of pressure injury in these patients.