Novel In-Line Hemolysis Detection on a Blood Gas Analyzer and Impact on Whole Blood Potassium Results.

BACKGROUND: Preanalytical error due to hemolyzed blood samples is a common challenge in laboratory and point-of-care (POC) settings. Whole blood potassium (K+) measurements routinely measured on blood gas analyzers are particularly susceptible to hemolysis, which poses a risk for incorrect K+ results. The GEM Premier 7000 with IQM3 (GEM 7000) blood gas analyzer provides novel integrated hemolysis detection within the sample measurement process. Therefore, the GEM 7000 can detect and flag hemolyzed whole blood samples at the POC, warning the operator of potentially erroneous results. METHODS: Heparinized venous or arterial whole blood samples were used for K+ interference studies and assessed for hemolysis agreement utilizing either a traditional volumetric method or chemistry analyzer serum index measurements with the Roche cobas c311 or Abbott Alinity c. RESULTS: Hemolysis interference studies performed at 2 different K+ concentrations (3.8 and 5.3 mmol/L) identified that a plasma free hemoglobin ≥116 mg/dL can impact K+ results on the GEM 7000. Hemolysis agreement studies demonstrated an excellent agreement of >99% with the volumetric method, 98.8% with cobas H index, and 96.4% with Alinity H index. GEM 7000 K+ results were correctly flagged for both native and spiked samples. CONCLUSION: GEM 7000 hemolysis detection provides a novel technology to detect hemolysis in whole blood samples. Moreover, the GEM 7000 demonstrates excellent agreement with traditional laboratory hemolysis detection methods and offers an integrated technological solution for assuring the quality of whole blood K+ results in POC settings.

Hippocampal volumes in patients with chronic combat-related posttraumatic stress disorder: a systematic review.

The authors and others have recently demonstrated that veterans with chronic combat-related PTSD (CR-PTSD) have a twofold increased risk of dementia. To understand this increased incidence, they performed a systematic review of the literature on neuroanatomical differences between veterans with chronic CR-PTSD and control subjects (22 included studies). The hippocampus was most commonly and consistently reported to differ between groups, thereby suggesting the hypothesis that PTSD is associated with smaller hippocampi, which increases the risk for dementia. However, an alternate hypothesis is that smaller hippocampal volumes are a preexisting risk factor for PTSD and dementia. Studies are clearly needed to differentiate between these important possibilities.

CodY-mediated regulation of Streptococcus pyogenes exoproteins.

BACKGROUND: The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. RESULTS: We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. CONCLUSIONS: Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

Community-based intervention to manage an outbreak of MRSA skin infections in a county jail.

This article describes a community-based intervention to manage an outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin infections in a midwestern county jail. A systematic investigation conducted by a family medicine residency program identified 64 total cases and 19 MRSA cases between January 1 and December 31, 2007. Factors contributing to MRSA transmission included inadequate surveillance, lack of antibacterial soap, and a defective laundry process. All 19 isolates were CA-MRSA and all seven tested by pulsed-field gel electrophoresis (PFGE) were USA300. Four of the seven isolates showed variation of their PFGE patterns. A primary care approach using community-based resources effectively reduced the number of cases in this heterogeneous outbreak of CA-MRSA, with the last MRSA being isolated in October 2007.

Turbidity suppression from the ballistic to the diffusive regime in biological tissues using optical phase conjugation.

We describe the amplitude and resolution trends of the signals acquired by turbidity suppression through optical phase conjugation (TSOPC) with samples that span the ballistic and diffusive scattering regimes. In these experiments, the light field scattered through a turbid material is written into a hologram, and a time-reversed copy of the light field is played back through the sample. In this manner, the wavefront originally incident on the sample is reconstructed. We examine a range of scattering samples including chicken breast tissue sections of increasing thickness and polyacrylamide tissue-mimicking phantoms with increasing scattering coefficients. Our results indicate that only a small portion of the scattered wavefront (<0.02%) must be collected to reconstruct a TSOPC signal. Provided the sample is highly scattering, all essential angular information is contained within such small portions of the scattered wavefront due to randomization by scattering. A model is fitted to our results, describing the dependence of the TSOPC signal on other measurable values within the system and shedding light on the efficiency of the phase conjugation process. Our results describe the highest level of scattering that has been phase conjugated in biological tissues to date.

An in vivo study of turbidity suppression by optical phase conjugation (TSOPC) on rabbit ear.

We present a holography-based in vivo optical phase conjugation experiment performed on a living rabbit ear. The motion of live tissues caused the phase conjugate signal to decay with a consistent decay time of less than two seconds. We monitor the signal decay time variation after the ear is excised to postulate different mechanisms that cause the signal decay. The experimental findings address the minimum speed limit of a broad range of optical time reversal experiments for in vivo applications on tissues.

Manual-scanning optical coherence tomography probe based on position tracking.

A method based on position tracking to reconstruct images for a manual-scanning optical coherence tomography (OCT) probe is proposed and implemented. The method employs several feature points on a hand-held probe and a camera to track the device's pose. The continuous device poses tracking, and the collected OCT depth scans can then be combined to render OCT images. The tracking accuracy of the system was characterized to be about 6 microm along two axes and 19 microm along the third. A phantom target was used to validate the method. In addition, we report OCT images of a 54-stage Xenopus laevis tadpole acquired by manual scanning.

Observation of polarization-gate based reconstruction quality improvement during the process of turbidity suppression by optical phase conjugation.

We present experiments that study the impact of polarization selection on the phenomenon of turbidity suppression by optical phase conjugation. Counter to intuition, we discovered that the preferential utilization of multiply scattered light field components over their sparsely scattered counterparts via appropriate polarization selection can lead to better image reconstruction quality. This effect was observed with tissue phantoms and biological tissue sections. The physical origin of this effect and its dependence on scatterer properties are discussed.

A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

Fundamental sensitivity limit imposed by dark 1/f noise in the low optical signal detection regime.

The impact of dark 1/f noise on fundamental signal sensitivity in direct low optical signal detection is an understudied issue. In this theoretical manuscript, we study the limitations of an idealized detector with a combination of white noise and 1/f noise, operating in detector dark noise limited mode. In contrast to white noise limited detection schemes, for which there is no fundamental minimum signal sensitivity limit, we find that the 1/f noise characteristics, including the noise exponent factor and the relative amplitudes of white and 1/f noise, set a fundamental limit on the minimum signal that such a detector can detect.

Inter- and intraserotypic variation in the Streptococcus pyogenes Rgg regulon.

Human isolates of Streptococcus pyogenes, a Gram-positive bacterium, are characterized by significant genetic and phenotypic variation. The rgg locus, also known as ropB, is a global transcriptional regulator of genes associated with metabolism, stress responses, and virulence in S. pyogenes strain NZ131 (serotype M49). To assess the breadth of the Rgg regulon, the rgg gene was inactivated in three additional strains representing serotypes M1 (strains SF370 and MGAS5005) and M49 (strain CS101). Changes in gene expression were identified in the postexponential phase of growth using Affymetrix NimbleExpress Arrays. The results identified an Rgg core-regulon consisting of speB and adjacent hypothetical protein gene, spy2040, and a variable and strain-specific subregulon, ranging in size from a single gene (spy1793) in strain MGAS5005 to 43 genes in strain SF370. Thus, both interserotypic and intraserotypic variation is characteristic of the Rgg regulon in S. pyogenes.

Proteomic analysis of proteins secreted by Streptococcus pyogenes.

Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4-7. Subsequently, proteins are further separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes compared with large-format approaches, while still resolving and detecting a large proportion of the exoprotein fraction.

Spectral domain phase microscopy for local measurements of cytoskeletal rheology in single cells.

We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%.

A generalized noise variance analysis model and its application to the characterization of 1/f noise.

We present a novel generalized model for the analysis of noise with a known spectral density. This model is particularly appropriate for the analysis of noise with a 1/f(a) distribution in a homodyne interferometer. The noise model reveals that, for alpha>1, 1/f(a) noise significantly impacts the homodyne signal-to-noise ratio (SNR) for integration times that near a characteristic time, beyond which the SNR will no longer significantly improve with increasing integration time. We experimentally verify our theoretical findings with a set of experiments employing a quadrature homodyne optical coherence tomography (OCT) system, finding good agreement. The characteristic integration time is measured to be approximately 2 ms for our system. Additionally, we find that the 1/f noise characteristics, including the exponent, alpha, as well as the characteristic integration time, are system and photodetector dependent.

SNR enhancement through phase dependent signal reconstruction algorithms for phase separated interferometric signals.

We report several signal reconstruction algorithms for processing phase separated homodyne interferometric signals. Methods that take advantage of the phase of the signal are experimentally shown to achieve a signal-to-noise ratio (SNR) improvement of up to 5 dB over commonly used algorithms. To begin, we present a derivation of the SNR resulting from five image reconstruction algorithms in the context of a 3x3 fiber-coupler based homodyne optical coherence tomography (OCT) system, and clearly show the improvement in SNR associated with phase-based algorithms. Finally, we experimentally verify this improvement and demonstrate the enhancement in contrast and improved image quality afforded by these algorithms through homodyne OCT imaging of a Xenopus laevis tadpole. These algorithms can be generally applied in signal extraction processing where multiple phase separated measurements are available.

The Rgg regulator of Streptococcus pyogenes influences utilization of nonglucose carbohydrates, prophage induction, and expression of the NAD-glycohydrolase virulence operon.

The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. Finally, transcripts encoding virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase, including the immunity factor IFS and the cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which correlated with the amount of NADase and SLO activities in culture supernatant fluids. The results provide further evidence that Rgg contributes to growth phase-dependent gene regulation in strain NZ131.

Proteomic analysis of a penicillin-tolerant rgg mutant strain of Streptococcus pyogenes.

OBJECTIVES: To determine whether the transcriptional regulator Rgg contributes to penicillin-induced killing of Streptococcus pyogenes by altering a regulatory response to penicillin. METHODS: Penicillin-induced killing of a wild-type and isogenic rgg mutant strain was assessed in broth and solid media and in the presence of cerulenin, which inhibits fatty acid biosynthesis (FAB). Proteins from wild-type and rgg mutant cultures, either exposed to penicillin or not, were characterized by two-dimensional gel electrophoresis. Proteins of interest were identified with tandem mass spectrometry. RESULTS: The MIC of penicillin was 0.012 mg/L for both the wild-type strain NZ131 and an isogenic rgg mutant strain. The wild-type strain lost 1.9 log(10) cfu/mL ( approximately 80-fold) after 24 h of exposure to 0.024 mg/L penicillin compared with controls; however, the mutant strain lost 0.3 log(10) cfu/mL ( approximately 2-fold) compared with controls. Changes in the proteome of wild-type and mutant cultures were assessed 1 and 4 h after exposure to penicillin. One hour exposure was associated with increased abundance (P < 0.05) of 12 proteins associated with FAB, the pentose phosphate pathway, glycolysis and stress responses in the wild-type strain. The abundance of 8 of 12 of these proteins was greater in samples obtained from the mutant strain, even prior to penicillin exposure. After 4 h of exposure, the abundance of 16 proteins was altered in one or both strains; however, a clear functional relationship was not evident. The addition of cerulenin slightly enhanced penicillin-induced killing of wild-type strain, which supported the proteomic results. CONCLUSIONS: The results suggest that penicillin-independent changes in the cytoplasmic proteome of an rgg mutant strain of NZ131 confer tolerance to penicillin-mediated killing.

Characterization of light collection through a subwavelength aperture from a point source.

We experimentally measure and theoretically model the light transmission characteristics of subwavelength apertures. The characterization consists of translating a point source at varying vertical height and lateral displacement from the aperture and measuring the resulting transmission. We define the variation of the transmission with lateral source displacement as the collection mode point spread function (CPSF). This transmission geometry is particularly relevant to subwavelength aperture based imaging devices and enables determination of their resolution. This study shows that the achieved resolutions degrade as a function of sample height and that the behavior of sensor devices based on the use of apertures for detection is different from those devices where the apertures are used as light sources. In addition, we find that the measured CPSF is dependent on the collection numerical aperture (NA). Finally, we establish that resolution beyond the diffraction limit for a nominal optical wavelength of 650 nm and nominal medium refractive index of 1.5 is achievable with subwavelength aperture based devices when the aperture size is smaller than 225 nm.

Methods and application areas of endoscopic optical coherence tomography.

We review the current state of research in endoscopic optical coherence tomography (OCT). We first survey the range of available endoscopic optical imaging techniques. We then discuss the various OCT-based endoscopic methods that have thus far been developed. We compare the different endoscopic OCT methods in terms of their scan performance. Next, we examine the application range of endoscopic OCT methods. In particular, we look at the reported utility of the methods in digestive, intravascular, respiratory, urinary and reproductive systems. We highlight two additional applications--biopsy procedures and neurosurgery--where sufficiently compact OCT-based endoscopes can have significant clinical impacts.