A Novel Device for the Evaluation of Hemostatic Function in Critical Care Settings.

Major surgical procedures often result in significant intra- and postoperative bleeding. The ability to identify the cause of the bleeding has the potential to reduce the transfusion of blood products and improve patient care. We present a novel device, the Quantra Hemostasis Analyzer, which has been designed for automated, rapid, near-patient monitoring of hemostasis. The Quantra is based on Sonic Estimation of Elasticity via Resonance Sonorheometry, a proprietary technology that uses ultrasound to measure clot time and clot stiffness from changes in viscoelastic properties of whole blood during coagulation. We present results of internal validation and analytical performance testing of the technology and demonstrate the ability to characterize the key functional components of hemostasis.

HDL particle number measured on the Vantera®, the first clinical NMR analyzer.

OBJECTIVES: Nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to the measurement of high-density lipoprotein (HDL) particles, providing particle concentrations for total HDL particle number (HDL-P), HDL subclasses (small, medium, large) and weighted, average HDL size for many years. Key clinical studies have demonstrated that NMR-measured HDL-P was more strongly associated with measures of coronary artery disease and a better predictor of incident cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C). Recently, an NMR-based clinical analyzer, the Vantera(®), was developed to allow lipoprotein measurements to be performed in the routine, clinical laboratory setting. The aim of this study was to evaluate and report the performance characteristics for HDL-P quantified on the Vantera(®) Clinical Analyzer. DESIGN AND METHODS: Assay performance was evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In order to ensure that quantification of HDL-P on the Vantera(®) Clinical Analyzer was similar to the well-characterized HDL-P assay on the NMR profiler, a method comparison was performed. RESULTS: The within-run and within-lab imprecision ranged from 2.0% to 3.9%. Linearity was established within the range of 10.0 to 65.0 µmol/L. The reference intervals were different between men (22.0 to 46.0 µmol/L) and women (26.7 to 52.9 µmol/L). HDL-P concentrations between two NMR platforms, Vantera(®) Clinical Analyzer and NMR Profiler, demonstrated excellent correlation (R(2) = 0.98). CONCLUSIONS: The performance characteristics, as well as the primary tube sampling procedure for specimen analysis on the Vantera(®) Clinical Analyzer, suggest that the HDL-P assay is suitable for routine clinical applications.

NMR measurement of LDL particle number using the Vantera Clinical Analyzer.

BACKGROUND: The Vantera Clinical Analyzer was developed to enable fully-automated, high-throughput nuclear magnetic resonance (NMR) spectroscopy measurements in a clinical laboratory setting. NMR-measured low-density lipoprotein particle number (LDL-P) has been shown to be more strongly associated with cardiovascular disease outcomes than LDL cholesterol (LDL-C) in individuals for whom these alternate measures of LDL are discordant. OBJECTIVE: The aim of this study was to assess the analytical performance of the LDL-P assay on the Vantera Clinical Analyzer as per Clinical Laboratory Standards Institute (CLSI) guidelines. RESULTS: Sensitivity and linearity were established within the range of 300-3500 nmol/L. For serum pools containing low, medium and high levels of LDL-P, the inter-assay, intra-assay precision and repeatability gave coefficients of variation (CVs) between 2.6 and 5.8%. The reference interval was determined to be 457-2282 nmol/L and the assay was compatible with multiple specimen collection tubes. Of 30 substances tested, only 2 exhibited the potential for assay interference. Moreover, the LDL-P results from samples run on two NMR platforms, Vantera Clinical Analyzer and NMR Profiler, showed excellent correlation (R(2)=0.96). CONCLUSIONS: The performance characteristics suggest that the LDL-P assay is suitable for routine testing in the clinical laboratory on the Vantera Clinical Analyzer, the first automated NMR platform that supports NMR-based clinical assays.

Permanganate diffusion and reaction in sedimentary rocks.

In situ chemical oxidation using permanganate has frequently been used to treat chlorinated solvents in fractured bedrock aquifers. However, in systems where matrix back-diffusion is an important process, the ability of the oxidant to migrate and treat target contaminants within the rock matrix will likely determine the overall effectiveness of this remedial approach. In this study, a series of diffusion experiments were performed to measure the permanganate diffusion and reaction in four different types of sedimentary rocks (dark gray mudstone, light gray mudstone, red sandstone, and tan sandstone). Results showed that, within the experimental time frame (~2 months), oxidant migration into the rock was limited to distances less than 500 µm. The observed diffusivities for permanganate into the rock matrices ranged from 5.3 × 10(-13) to 1.3 × 10(-11) cm(2)/s. These values were reasonably predicted by accounting for both the rock oxidant demand and the effective diffusivity of the rock. Various Mn minerals formed as surface coatings from reduction of permanganate coupled with oxidation of total organic carbon (TOC), and the nature of the formed Mn minerals was dependent upon the rock type. Post-treatment tracer testing showed that these Mn mineral coatings had a negligible impact on diffusion through the rock. Overall, our results showed that the extent of permanganate diffusion and reaction depended on rock properties, including porosity, mineralogy, and organic carbon. These results have important implications for our understanding of long-term organic contaminant remediation in sedimentary rocks using permanganate.

Coupled diffusion and abiotic reaction of trichlorethene in minimally disturbed rock matrices.

Laboratory experiments were performed using minimally disturbed sedimentary rocks to measure the coupled diffusion and abiotic reaction of trichloroethene (TCE) through rock core samples. Results showed that, for all rock types studied, TCE dechlorination occurred, as evidenced by generation of acetylene, ethene, and/or ethane daughter products. First-order bulk reaction rate constants for TCE degradation ranged from 8.3 × 10(-10) to 4.2 × 10(-8) s(-1). Observed reaction rate constants showed a general correlation to the available ferrous iron content of the rock, which was determined by evaluating the spatial distribution of ferrous iron relative to that of the rock porosity. For some rock types, exposure to TCE resulted in a decrease in the effective diffusivity. Scanning electron microscopy (SEM) indicated that the decrease in the effective diffusivity was due to a decrease in the porosity that occurred after exposure to TCE. Overall, these coupled diffusion and reaction results suggest that diffusion of TCE into rock matrices as well as the rate and extent of back-diffusion may be substantially mitigated in rocks that contain ferrous iron or other naturally occurring reactive metals, thereby lessening the impacts of matrix diffusion on sustaining dissolved contaminant plumes in bedrock aquifers.

Analytical performance of RNA isolated from BD SurePath™ cervical cytology specimens by the PreTect™ HPV-Proofer assay.

Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.

Recovery of DNA from BD SurePath cytology specimens and compatibility with the Roche AMPLICOR Human Papillomavirus (HPV) Test.

BACKGROUND: The AMPLICOR HPV Test has been validated for use with cervical cells collected in liquid-based preservative fluids, such as BD SurePath. It is currently recommended, however, that residual BD SurePath samples be stored at 4 degrees C prior to testing. OBJECTIVES: The aim of this study was to demonstrate that DNA isolated from SurePath cervical cytology specimens and stored at ambient temperature was also compatible with the AMPLICOR HPV Test. STUDY DESIGN: DNA was extracted using the AmpliLute Media Sample Preparation Kit. Amplification and detection of HPV was performed both as directed by the manufacturer and with minor protocol modifications. RESULTS: Cervical specimens collected in SurePath preservative fluid remained stable for testing with the AMPLICOR HPV Test for at least 21 days. The performance of DNA extracted from specimens stored at room temperature was equivalent to DNA extracted from specimens stored at 4 degrees C. The beta-globin internal control was detected in all of the 146 residual SurePath cervical cytology specimens tested using the AMPLICOR HPV Test, and high-risk HPV was detected in 46.2% (18/39) of ASCUS cases, in 63.3% (19/30) of LSIL cytology specimens, and 92.3% (24/26) of HSIL cases. Concordance of AMPLICOR HPV Test results with Hybrid Capture II was 83.9%. CONCLUSIONS: The AMPLICOR HPV Test can be successfully and reproducibly performed from DNA isolated from residual SurePath cervical cytology specimens stored at ambient temperature for at least 21 days. This provides clinical laboratories flexible storage conditions for residual SurePath cytology specimens.

Screening for molecular markers of cervical papillomavirus infection: overview of methods and their clinical implications.

Molecular diagnostic adjuncts could improve the specificity of cervical cancer screening. Since persistent infection with human papillomavirus (HPV) is found in virtually 100% of cervical cancer cases, testing for markers of HPV integration may have a role in identifying underlying high-grade lesions in patients with low-grade cytologic abnormalities. Several proteins associated with the cell cycle are known to be affected by HPV integration into the host's DNA. Immunocytochemical identification of these upregulated proteins can assist in the identification of small numbers of pre-neoplastic or neoplastic cells in routine cytologic sampling.

Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR.

This study was performed to demonstrate that RNA isolated from cell lines and cervical cytology specimens stored in SurePath preservative fluid would be functional in real-time RT-PCR assays. RNA was isolated from cervical cell lines or cytology samples stored in SurePath preservative at room temperature for 2-5 weeks using five commercially available RNA purification kits, three of which contain proteinases. The quality of the RNA was assessed by real time RT-PCR amplification of GAPDH, GUSB, U1A, HPV 16 and 18 E6 mRNAs. RNA was isolated successfully from cells that were stored in SurePath preservative fluid with only the three protocols that contained proteinases. GAPDH was amplified in 98-100% of the samples, GUSB in 90-98%, and the least abundant transcript, U1A, was amplified in 81-96% of the samples. HPV 16 and 18 E6 transcripts were detected in 56% of high grade, 39% of low grade and 2% of normal samples, with a concordance between DNA genotype and E6 mRNA expression of 97%. We demonstrated that RNA can be extracted from cervical cell lines and cytology specimens stored in BD SurePath preservative fluid with three different procedures that all contain proteinases. This RNA is suitable for real-time RT-PCR applications.

Isolation of RNA from residual BD SurePath liquid-based cytology specimens and detection of HPV E6/E7 mRNA using the PreTectt HPV-Proofer assay.

Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.

Evaluation of biomarker panels for early stage ovarian cancer detection and monitoring for disease recurrence.

OBJECTIVE: To determine the utility of novel combinations of biomarkers, using both a one-step and two-step assay format, to distinguish serum of early ovarian cancer patients from that of healthy controls and to discern the utility of these biomarkers in a monitoring capacity. METHODS: For ovarian cancer detection, HE4, Glycodelin, MMP7, SLPI, Plau-R, MUC1, Inhibin A, PAI-1, and CA125 were evaluated in a cohort of 200 women with ovarian cancer and 396 healthy age-matched controls. Each biomarker was assessed by serum-based immunoassays utilizing novel monoclonal antibody pairs or commercial kits. For detection of disease recurrence, HE4, Glycodelin, MMP7 and CA125 were evaluated in 260 samples from 30 patients with OC monitored longitudinally after diagnosis. RESULTS: Based upon ROC curve analysis, the sensitivity/specificity of specific biomarker combination algorithms ranged from 59.0%/99.7% to 80.5%/96.5% for detection of early stage ovarian cancer and 76.9%/99.7% to 89.2%/97.2% for detection of late stage cancer. In monitoring evaluation of 27 patients who experienced recurrence of OC, sensitivity for predicting recurrence was 100% for the biomarker panel and 96% for CA125. At least one of the panel biomarkers was elevated earlier (range 6-69 weeks) than CA125 and prior to clinical evidence of recurrence in 14/27 (52%) patients. CONCLUSIONS: We have developed and demonstrated the utility of several one- and two-step multi-marker combinations with acceptable test characteristics for possible use in an ovarian cancer screening population. A subset of this panel may also provide adjunctive information to rising CA125 levels in disease monitoring.

Management of alcohol withdrawal delirium. An evidence-based practice guideline.

BACKGROUND: Alcohol withdrawal delirium is the most serious manifestation of alcohol withdrawal. Evidence suggests that appropriate care improves mortality, but systematic reviews are unavailable. METHODS: Articles with original data on management of alcohol withdrawal delirium underwent structured review and meta-analysis. RESULTS: Meta-analysis of 9 prospective controlled trials demonstrated that sedative-hypnotic agents are more effective than neuroleptic agents in reducing duration of delirium and mortality, with a relative risk of death when using neuroleptic agents of 6.6. Statistically significant differences among various benzodiazepines and barbiturates were not found. No deaths were reported in 217 patients from trials using benzodiazepines or barbiturates. CONCLUSIONS: Control of agitation should be achieved using parenteral rapid-acting sedative-hypnotic agents that are cross-tolerant with alcohol. Adequate doses should be used to maintain light somnolence for the duration of delirium. Coupled with comprehensive supportive medical care, this approach is highly effective in preventing morbidity and mortality.

Lipoprotein-complexed C-reactive protein and the biphasic transmittance waveform in critically ill patients.

The 'biphasic transmittance waveform' (BTW) refers to a decrease in light transmittance that often occurs prior to clotting in coagulation assays of critically ill patient plasmas. It correlates with disseminated intravascular coagulation and mortality. The present work shows that the BTW is due to the rapid formation of a precipitate and a coincident change in turbidity in re-calcified plasma. The precipitate was isolated from patient plasma and contained lipids typical of very low density lipoprotein (VLDL), plus the proteins apolipoprotein B-100 and C-reactive protein (CRP). Precipitation also occurred in normal plasma supplemented with CRP. In addition, CRP precipitated with VLDL and intermediate density lipoprotein, but not low density lipoprotein or high density lipoprotein. The Kd value for the CRP/VLDL interaction is 340 nM. The IC50 value of Ca2+ for complex formation is 5.0 mM, and epsilon-aminocaproic acid inhibits the process. In 15 plasmas with the BTW from critically ill patients, CRP was highly elevated (77-398 microg/mL) and VLDL cholesterol ranged from 0.082 to 1.32 mM. The magnitude of the turbidity change on re-calcification correlated well with the calculated level of the CRP/VLDL complex. Thus, the Ca2+-dependent formation of a complex between CRP and VLDL accounts for the BTW.