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.

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.