The application of autologous cancer immunotherapies in the age of memory-NK cells.

Cellular immunotherapy has revolutionized the oncology field, yielding improved results against hematological and solid malignancies. NK cells have become an attractive alternative due to their capacity to activate upon recognition of "stress" or "danger" signals independently of Major Histocompatibility Complex (MHC) engagement, thus making tumor cells a perfect target for NK cell-mediated cancer immunotherapy even as an allogeneic solution. While this allogeneic use is currently favored, the existence of a characterized memory function for NK cells ("memory-like" NK cells) advocates for an autologous approach, that would benefit from the allogeneic setting discoveries, but with added persistence and specificity. Still, both approaches struggle to exert a sustained and high anticancer effect in-vivo due to the immunosuppressive tumor micro-environment and the logistical challenges of cGMP production or clinical deployment. Novel approaches focused on the quality enhancement and the consistent large-scale production of highly activated therapeutic memory-like NK cells have yielded encouraging but still unconclusive results. This review provides an overview of NK biology as it relates to cancer immunotherapy and the challenge presented by solid tumors for therapeutic NKs. After contrasting the autologous and allogeneic NK approaches for solid cancer immunotherapy, this work will present the current scientific focus for the production of highly persistent and cytotoxic memory-like NK cells as well as the current issues with production methods as they apply to stress-sensitive immune cells. In conclusion, autologous NK cells for cancer immunotherapy appears to be a prime alternative for front line therapeutics but to be successful, it will be critical to establish comprehensives infrastructures allowing the production of extremely potent NK cells while constraining costs of production.

Analytical Performance Evaluation of Aptima Herpes Simplex Virus 1 and 2 Assay Using Hologic Panther Instrument.

OBJECTIVE: Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are responsible for a variety of human diseases. Therefore, rapid detection of these viruses in clinical laboratory is important. Recently Aptima Herpes Simplex 1 and 2 assay has been approved by the FDA. We evaluated analytical performance of this assay by comparing results obtained by analysis of cultures. MATERIALS AND METHODS: Aptima Herpes Simplex assay is a nucleic acid amplification test that can be fully automated using the Hologic Panther instrument. This is a qualitative test providing either positive or negative result. We analyzed 115 specimens collected from anogenital locations using the new Aptima Herpes Simplex assay and our current tissue culture method. RESULTS: We observed good correlation between results obtained by using Aptima assay and the tissue culture method in 101 specimens but 14 specimens showed discordant results. Further testing in a reference laboratory using PCR (polymerase chain reaction) showed results in agreement with the Aptima assay but not the tissue culture method. The cause of discordance was misidentification of HSV-2 as HSV-1 by the tissue culture method. CONCLUSIONS: Aptima Herpes simplex assay on the Hologic Panther instrument is suitable for rapid detection of herpes simplex virus in clinical laboratory.

Contamination of Some Kratom Products with Salmonella.

OBJECTIVE: Recently, the US Food and Drug Administration investigated the contamination of kratom (Mitragyna speciosa) by Salmonella, an event that caused an outbreak in 20 states after its consumption. Therefore, we investigated 16 different bags of kratom submitted for testing for potential contamination with Salmonella and other microorganisms within the Public Health Laboratory for the state of South Carolina. MATERIALS AND METHODS: All kratom powders collected were analyzed for potential contamination with bacteria by an in-house modified Food and Drug Administration's Bacteriological Analytical Manual Salmonella procedure. RESULTS: Out of 16 products analyzed, six brands have unknown manufacturers, but manufacturer information was available for the other 10 products. In total, three brands of kratom showed presence of any Salmonella species. CONCLUSIONS: Recently analyzed kratom products show a presence of Salmonella.

Comparison of SemiQuantitative Cotinine Values Obtained by the DRI Immunoassay and Values Obtained by a Liquid Chromatography-Tandem Mass Spectrometry-Based Method: The DRI Immunoassay is Suitable for Screening Purposes Only Because Semiquantitative Values May Be Unreliable.

BACKGROUND: DRI cotinine assay is suitable only for screening for cotinine in urine specimens. We studied the reliability of DRI cotinine semiquantitative values by comparing them with the cotinine concentration obtained with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. METHODS: Semiquantitative cotinine concentrations in 39 urine specimens obtained by the DRI immunoassay were compared with cotinine concentrations obtained by LC-MS/MS. RESULTS: The DRI cotinine assay consistently overestimated cotinine values obtained by the LC/MS/MS method (y = 1.1529 x + 252.24, n = 39, R2 = 0.8899) indicating that semiquantitative values obtained using the DRI assay may be unreliable. However, no false-negative results were observed using the DRI assay. CONCLUSION: DRI cotinine assay is suitable only for screening cotinine in urine specimens.

Comparison of Response of DRI Oxycodone Semiquantitative Immunoassay With True Oxycodone Values Determined by Liquid Chromatography Combined With Tandem Mass Spectrometry: Sensitivity of the DRI Assay at 100 ng/ml Cut-Off and Validity of Semiquantitative Value.

OBJECTIVE: Oxycodone is a widely used opioid for pain management and patient's compliance with therapy is often monitored by using oxycodone immunoassay. The performance of the DRI oxycodone immunoassay was compared with liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) assay. MATERIALS AND METHODS: In 48 urine specimens collected from patients taking oxycodone, urinary oxycodone concentrations were determined using LC/MS/MS and the DRI oxycodone immunoassay for application on the Cobas c 501 analyzer (Roche Diagnostics, Indianapolis, IN). RESULTS: Out of 48 specimens, 14 specimens showed oxycodone value less than 100 ng/ml, seven specimens had low positive values (between 101 and 165 ng/ml) and all other specimens had values 165 to 1789 ng/ml using the LC/MS/MS assay. The DRI oxycodone assay successfully identified all oxycodone specimens with oxycodone concentrations over the 100 ng/ml. In addition, the DRI assay also showed positive response in 11 out of 14 specimens with oxycodone values less than 100 ng/ml. However, semiquantitative values obtained by the DRI assay did not match with true oxycodone and metabolite oxymorphone concentrations combined obtained by using LC/MS/MS. CONCLUSIONS: DRI oxycodone immunoassay at 100 ng/ml is a reliable immunoassay for analysis of oxycodone in urine.

Zinc Sulfate, a Recently Introduced Urinary Adulterant Can Invalidate Urine Cotinine Test Using Immunoassay but Has Less Effect on Liquid Chromatography Combined With Tandem Mass Spectrometry-Based Test.

OBJECTIVE: Zinc sulfate is a recently introduced urinary adulterant, which causes false-negative results with immunoassays used for screening drugs of abuse in urine but whether zinc sulfate also could invalidate urine cotinine assay using immunoassay or liquid chromatography combined with mass spectrometry has never been studied. DESIGN AND METHOD: Four urine pools containing none detected to high levels of cotinine were analyzed using DRI cotinine immunoassay on the Olympus 640 analyzer as well as using liquid chromatography combined with tandem mass spectrometry. Specimens were reanalyzed after supplementing with various amounts of zinc sulfate that are known to invalidate immunoassays used for drugs of abuse testing. RESULTS: Zinc sulfate in all concentrations studied caused false-negative results using immunoassays, but zinc sulfate also reduced cotinine values by approximately 2.1%-38.4% when analyzed using liquid chromatography combined with mass spectrometry. CONCLUSIONS: Zinc sulfate caused false-negative cotinine result when DRI immunoassay was used and also had small to moderate impact on liquid chromatography combined with tandem mass spectrometry-based assay for urine cotinine.

Limitations of EMIT benzodiazepine immunoassay for monitoring compliance of patients with benzodiazepine therapy even after hydrolyzing glucuronide metabolites in urine to increase cross-reactivity: comparison of immunoassay results with LC-MS/MS values.

BACKGROUND: Benzodiazepines are widely prescribed, and compliance of patients with benzodiazepine therapy is often monitored using urine specimens. Although various commercially available benzodiazepines immunoassays are widely used for compliance monitoring, such immunoassays usually have low cross-reactivity with glucuronide metabolites. We studied the effect of hydrolyzing such glucuronide before analysis to reevaluate suitability of Enzyme multiplied immunoassay technique benzodiazepine immunoassay for monitoring compliance with benzodiazepine therapy. METHODS: In 31 urine specimens collected from patients taking benzodiazepines, the true analyte concentrations were determined (after hydrolyzing glucuronide metabolites using beta-glucuronidase) using liquid chromatography-tandem mass spectrometry. These urine specimens were reanalyzed using EMIT benzodiazepine assay (Flex Reagent Cartridge; Siemens Diagnostics) and Vista analyzer. RESULTS: We observed false negative test results with EMIT in 11 of 31 specimens analyzed where liquid chromatography-tandem mass spectrometry values were above the 200 ng/mL cutoff concentration, but EMIT benzodiazepine assay showed a negative result, indicating that despite hydrolysis of the specimen to liberate parent drug (glucuronide metabolite often has poor cross-reactivity), the false negative rate using EMIT assay was 35.5%. CONCLUSIONS: Patient compliance with benzodiazepine therapy must be monitored using a chromatographic method.

One-Year Plasma N-linked Glycome Intra-individual and Inter-individual Variability in the Chicken Model of Spontaneous Ovarian Adenocarcinoma.

Spontaneous epithelial ovarian cancer (EOC) in the chicken presents a similar pathogenesis compared with humans including CA-125 expression and genetic mutational frequencies (e.g., p53). The high prevalence of spontaneous EOC chickens also provides a unique experimental model for biomarker discovery at the genomic, proteomic, glycomic, and metabolomic level. In an effort to exploit this unique model for biomarker discovery, longitudinal plasma samples were collected from chickens at three month intervals for one year. The study described herein involved cleaving the N-glycans from these longitudinal chicken plasma samples and analyzing them via nanoLC-FTMS/MS. Glycans identified in this study were previously found in human plasma and this work provides a promising methodology to enable longitudinal studies of the N-linked plasma glycome profile during EOC progression. The structure, abundance, and intra-variability and inter-variability for 35 N-linked glycans identified in this study are reported. The full potential of the chicken model for biomarker discovery has yet to be realized, but the initial interrogation of longitudinally-procured samples provides evidence that supports the value of this strategy in the search for glycomic biomarkers.

Design, Modeling, Fabrication, and Evaluation of the Air Amplifier for Improved Detection of Biomolecules by Electrospray Ionization Mass Spectrometry.

Through a multi-disciplinary approach, the air amplifier is being evolved as a highly engineered device to improve detection limits of biomolecules when using electrospray ionization. Several key aspects have driven the modifications to the device through experimentation and simulations. We have developed a computer simulation that accurately portrays actual conditions and the results from these simulations are corroborated by the experimental data. These computer simulations can be used to predict outcomes from future designs resulting in a design process that is efficient in terms of financial cost and time. We have fabricated a new device with annular gap control over a range of 50 to 70 µm using piezoelectric actuators. This has enabled us to obtain better aerodynamic performance when compared to the previous design (2× more vacuum) and also more reproducible results. This is allowing us to study a broader experimental space than the previous design which is critical in guiding future directions. This work also presents and explains the principles behind a fractional factorial design of experiments methodology for testing a large number of experimental parameters in an orderly and efficient manner to understand and optimize the critical parameters that lead to obtain improved detection limits while minimizing the number of experiments performed. Preliminary results showed that several folds of improvements could be obtained for certain condition of operations (up to 34 folds).

Study of the ionization mechanism in hybrid laser based desorption techniques.

In order to study the ionization mechanism in MALDESI, the RASTIR source was implemented to separate in space the laser desorption event from the intersecting electrospray ionization beam. Deuterated solvents were electrosprayed from the RASTIR source which resulted in a near complete shift from [M + H(+)](1+) to [M + D(+)](1+) of the monoisotopic peak for reserpine. The purpose of these experiments is to test the hypothesis that the primary ionization pathway in MALDESI is through interaction of the laser-desorbed neutral species with the intersecting electrospray ionization beam. The combination of RASTIR coupled to MALDESI can be utilized to study small organic molecules as well as peptides and proteins. Moreover, the use of RASTIR combined with MALDESI lends itself to improve the overall efficiency of ionization.

Characterization of charge separation in the Array of Micromachined UltraSonic Electrospray (AMUSE) ion source for mass spectrometry.

An initial investigation into the effects of charge separation in the Array of Micromachined UltraSonic Electrospray (AMUSE) ion source is reported to gain understanding of ionization mechanisms and to improve analyte ionization efficiency and operation stability. In RF-only mode, AMUSE ejects, on average, an equal number of slightly positive and slightly negative charged droplets due to random charge fluctuations, providing inefficient analyte ionization. Charge separation at the nozzle orifice is achieved by the application of an external electric field. By bringing the counter electrode close to the nozzle array, strong electric fields can be applied at relatively low DC potentials. It has been demonstrated, through a number of electrode/electrical potential configurations, that increasing charge separation leads to improvement in signal abundance, signal-to-noise ratio, and signal stability.

Generation of multiply charged peptides and proteins by radio frequency acoustic desorption and ionization for mass spectrometric detection.

The design and implementation of a radio frequency acoustic desorption ionization (RADIO) source has been demonstrated for the analysis of multiply charged peptides and proteins. One muL aliquots of melittin, BNP-32, and ubiquitin ( approximately 1 mug of analyte) were deposited onto a quartz crystal microbalance (QCM) electrode before radio frequency actuation for desorption. Continuous electrospray parallel to/above the sampling surface enabled the ionization of desorbed species. Detection by a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer confirmed the intact and dissociated species observed during MS and MS/MS experiments, respectively.

Ambient aerodynamic ionization source for remote analyte sampling and mass spectrometric analysis.

The use of aerodynamic devices in ambient ionization source development has become increasingly prevalent in the field of mass spectrometry. In this study, an air ejector has been constructed from inexpensive, commercially available components to incorporate an electrospray ionization emitter within the exhaust jet of the device. This novel aerodynamic device, herein termed remote analyte sampling, transport, and ionization relay (RASTIR) was used to remotely sample neutral species in the ambient and entrain them into an electrospray plume where they were subsequently ionized and detected using a linear ion trap Fourier transform mass spectrometer. Two sets of experiments were performed in the ambient environment to demonstrate the device's utility. The first involved the remote (approximately 1 ft) vacuum collection of pure sample particulates (i.e., dry powder) from a glass slide, entrainment and ionization at the ESI emitter, and mass spectrometric detection. The second experiment involved the capture (vacuum collection) of matrix-assisted laser desorbed proteins followed by entrainment in the ESI emitter plume, multiple charging, and mass spectrometric detection. This approach is in principle a RASTIR-assisted matrix-assisted laser desorption electrospray ionization source (Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712-1716; Rapid Commun. Mass Spectrom. 2007, 21, 1150-1154.). A detailed description of the device construction, operational parameters, and preliminary small molecule and protein data are presented.

Mass measurement accuracy comparisons between a double-focusing magnetic sector and a time-of-flight mass analyzer.

We report a direct comparison of the mass measurement accuracies (MMAs) obtained on different mass spectrometry instrument types; a magnetic sector as the 'gold standard' and an electrospray ionization time-of-flight (ESI-TOF) instrument. Sixty samples, obtained from the Department of Chemistry at North Carolina State University, were analyzed on each instrument. Data are presented and compared between the different instruments. The average absolute MMAs achieved for the magnetic sector and Agilent ESI-TOF mass spectrometers were 3.0 and 1.1 ppm, respectively.

Quantitative comparison of a flared and a standard heated metal capillary inlet with a voltage-assisted air amplifier on an electrospray ionization linear ion trap mass spectrometer.

The performance characteristics (i.e., ion abundance and electrospray ion current) of a flared and blunt-ended heated metal capillary were evaluated with a voltage-assisted air amplifier on a linear ion trap mass spectrometer (LTQ-MS). The results demonstrated that a standard capillary afforded higher ion abundance than a flared capillary, thus further work is necessary to investigate conditions for which significant benefits with the flared capillary will be observed. The compatibility of a voltage-assisted air amplifier is explored for both types of capillaries and in all cases resulted in improved ion abundance and spray current.

Probing the mechanisms of an air amplifier using a LTQ-FT-ICR-MS and fluorescence spectroscopy.

We report the first quantitative assessment of electrosprayed droplet/ion focusing enabled by the use of a voltage-assisted air amplifier between an electrospray ionization emitter and a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (ESI-LTQ-FT-ICR-MS). A solution of fluorescent dye was electrosprayed with a stainless steel mesh screen placed in front of the MS inlet capillary acting as a gas-permeable imaging plate for fluorescence spectroscopy. Without use of the air amplifier, no detectable FT-ICR signal was observed, as well as no detectable fluorescence on the screen upon imaging using a fluorescence scanner. When the air amplifier was turned ON while electrospraying the fluorescent dye, FT-ICR mass spectra with high signal to noise ratio were obtained with an average ion injection time of 21 ms for an AGC target value of 5 x 10(5). Imaging of the screen using a fluorescence scanner produced a distinct spot of cross-sectional area approximately 33.5 mm(2) in front of the MS inlet capillary. These experimental results provide direct evidence of aerodynamic focusing of electrosprayed droplets/ions enabled by an air amplifier, resulting in improved electrospray droplet/ion capture efficiency and reduced ion injection time. A second set of experiments was carried out to explore whether the air amplifier assists in desolvation. By electrospraying a mix of quaternary amines, ratios of increasingly hydrophobic molecules were obtained. Observation of the solvophobic effect associated with electrospray ionization resulted in a higher abundance of the hydrophobic molecule. This bias was eliminated when the air amplifier was turned ON and a response indicative of the respective component concentrations of the molecules in the bulk solution was observed.

Remote mass spectrometric sampling of electrospray- and desorption electrospray-generated ions using an air ejector.

A commercial air ejector was coupled to an electrospray ionization linear ion trap mass spectrometer (LTQ) to transport remotely generated ions from both electrospray (ESI) and desorption electrospray ionization (DESI) sources. We demonstrate the remote analysis of a series of analyte ions that range from small molecules and polymers to polypeptides using the AE-LTQ interface. The details of the ESI-AE-LTQ and DESI-AE-LTQ experimental configurations are described and preliminary mass spectrometric data are presented.