Role of Pharmacy Analytics in Creating a Data-Driven Culture for Frontline Management.

Purpose: The purpose of this article is to offer key recommendations based on the authors' experiences for utilizing pharmacy analytics to support moving beyond standard-of-practice operational metrics towards high impact reporting to drive day-to-day decisions for frontline leaders. Summary: There is a continuous and vast amount of data generated through all facets of a health system's daily operations, yet many data elements go unused and fail to contribute to value creation and increased performance at an organizational level. It is critical, therefore, for departments of pharmacy to identify and implement effective strategies to leverage data through robust business analytics and reporting, ensuring managers at every level are provided the information they need to support data-driven decisions and meaningful interventions in the day-to-day operations of the organization. At the authors' institution, development and growth of a dedicated Pharmacy Analytics (PA) team has been instrumental to the pharmacy department for generating value and proactively supporting a business intelligence strategy that focuses on a data-driven management culture. Key recommendations to leverage pharmacy analytics are provided within four overarching themes: building transparency, leveraging synergy, optimizing actionability, and prioritizing partnerships. Conclusion: Through creation of a data-driven management culture, the authors provide recommendations for leveraging pharmacy analytics to reduce costs and impact outcomes across a range of hospital pharmacy operations.

The Impact of Self-Monitoring Blood Glucose Adherence On Glycemic Goal Attainment in an Indigent Population, With Pharmacy Assistance.

Self-monitoring of bood glucose alone is not a good predictor of HbA1c goal attainment. Health plans might benefit from formulary restrictions to provide more cost-effective care, without negatively impacting glycemic control. And by using targeted inteventions, healthcare providers could help maximize SMBG's clinical benefit for patients who receive test strips. Self-monitoring of blood glucose (SMBG) can be an important tool in diabetes treatment, both for patient self-management and for guiding clinicians regarding medication adjustments. Evidence supports the association of SMBG with clinical outcomes in patients with type-1 diabetes mellitus (T1DM) although it is mixed for patients with type-2 diabetes mellitus (T2DM). The cost of SMBG comprises a substantial portion of the total cost for patients with diabetes, and test strips are one of the main expenditures of the University of North Carolina Medical Center Pharmacy Assistance Program (PAP), which provides medication coverage, including test strips, to indigent patients who have no pharmacy insurance. The objective of this study is to evaluate the utility of SMBG based on the impact of test-strip adherence on glycemic goal attainment in an indigent population that is provided with low-copay test strips. This retrospective cohort study included patients with T1DM or T2DM who were enrolled in PAP in 2016 and who received a prescription for test strips during the 90 days prior to hemoglobin A1c (HbA1c) measurement. Adherence was defined as the proportion of days covered (PDC) > 0.8. Of the 498 patients encountered, 20% of the adherent group (n = 245) and 25% of the nonadherent group (n = 253) had a goal of HbA1c < 7% (P = 0.24). There were no differences in mean HbA1c between the groups, except in the multiple daily injections (MDI) of the insulin subgroup (8.9% vs. 9.6%, P = 0.009). The adherent group was 80% less likely to have a diabetes-related hospitalization (odds ratio [OR], 0.2; 95% CI, 0.04-0.92). The total test-strip cost to PAP was more than $200,000. In conclusion, in an indigent population, adherence to SMBG does not correlate with glycemic goal attainment and imposes a substantial cost burden on the healthcare system.

Factors influencing the acceptance of referrals for clinical pharmacist managed disease states in primary care.

OBJECTIVE: Clinical pharmacists use population health methods to generate chronic disease management referrals for patients with uncontrolled chronic conditions. The purpose of this study was to compare primary care providers' (PCPs) referral responses for 4 pharmacist-managed indications and to identify provider and patient characteristics that are predictive of PCP response. DESIGN: Retrospective cohort study. SETTING: This study occurred in an academic internal medicine clinic. PARTICIPANTS: Clinical pharmacy referrals generated through a population health approach between 2012 and 2016 for hypertension, chronic pain, depression, and benzodiazepine management were included. MAIN OUTCOME MEASURES: Proportion of referrals accepted, left pending, or rejected and influencing provider and patient characteristics. RESULTS: Of 1769 referrals generated, PCPs accepted 869 (49%), left pending 300 (17%), and rejected 600 (34%). Compared with referrals for hypertension, benzodiazepine management, and depression, chronic pain referrals had the lowest likelihood of rejection (odds ratio [OR] 0.31; 95% CI 0.19-0.49). Depression referrals had an equal likelihood of being accepted or rejected (OR 1.04; 95% CI 0.66-1.64). Provider characteristics were not significantly associated with referral response, but residents were more likely to accept referrals. Patient characteristics associated with lower referral rejection included black race (OR 0.39; 95% CI 0.18-0.87), higher systolic blood pressure (OR 0.98; 95% CI 0.97-0.99), and missed visits (OR 0.24; 95% CI 0.07-0.81). CONCLUSION: The majority of referrals for clinical pharmacists in primary care settings were responded to, varying mostly between acceptance and rejection. There was variability in referral acceptance across indications, and some patient characteristics were associated with increased referral acceptance.

The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Postoperative euglycaemic diabetic ketoacidosis associated with sodium-glucose cotransporter-2 inhibitors (gliflozins): a report of two cases and review of the literature.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i)-associated euglycaemic diabetic ketoacidosis (euDKA) is a serious and increasingly recognised complication of treatment with this class of oral hypoglycaemic agents and can present a diagnostic challenge, resulting in delayed recognition, inappropriate treatment and potentially life-threatening acidosis. We present two cases of patients developing SGLT2i-associated euDKA in the early postoperative period. We support ceasing SGLT2i for 72 hours preoperatively and would suggest continuing to withhold the medication until oral intake is restored, and recommend a wider awareness of SGLT2i-associated diabetic ketoacidosis (DKA) amongst patients and their healthcare providers with an emphasis on checking ketone levels irrespective of blood glucose levels in the postoperative setting.

Voriconazole and posaconazole therapeutic drug monitoring: a retrospective study.

BACKGROUND: Therapeutic drug monitoring (TDM) aims to minimize the clinical impact of posaconazole and voriconazole pharmacokinetic variability. However, its benefits on clinical outcomes are still being defined. Additionally, TDM data are limited for posaconazole IV and delayed-release tablet formulations among specific patient populations, including critically ill. The aim of this study was to determine the percentage of therapeutic posaconazole and voriconazole drug levels across all formulations in a real-world clinical setting and elucidate factors affecting attainment of target concentrations. METHODS: This study was a retrospective cohort study conducted at the University of Colorado Hospital between September 2006 and June 2015 that evaluated patients who received posaconazole or voriconazole TDM as part of routine care. RESULTS: Voriconazole (n = 250) and posaconazole (n = 100) levels were analyzed from 151 patients. Of these, 54% of voriconazole and 69% of posaconazole levels were therapeutic. For posaconazole, 14/38 (37%), 28/29 (97%) and 27/33 (82%) levels were therapeutic for the oral suspension, IV, and delayed-release tablet, respectively. Intravenous and delayed-release tablet posaconazole were 20 fold (p < 0.01) and sevenfold (p = 0.002) more likely than the oral suspension to achieve a therapeutic level. Subsequent levels were more likely to be therapeutic after dose adjustments (OR 3.31; 95% CI 1.3-8.6; p = 0.02), regardless of timing of initial non-therapeutic level. In a multivariable logistic regression analysis, no characteristics were independently predictive of therapeutic voriconazole levels and only absence of H2RA/PPI use was independently predictive of therapeutic posaconazole levels. There was no correlation between survival and therapeutic drug levels for either voriconazole (p = 0.67) or posaconazole (p = 0.50). CONCLUSIONS: A high percentage of drug levels did not achieve TDM targets for voriconazole and posaconazole oral suspension, supporting the need for routine TDM for those formulations. The utility of TDM for the IV and delayed-release tablet formulations of posaconazole is less apparent.

Away from home meals: associations with biomarkers of chronic disease and dietary intake in American adults, NHANES 2005-2010.

BACKGROUND: Away from home (AFH) meals are known to be energy-dense and of poor diet quality. Both direct and indirect exposure (for example, neighborhood restaurant density) to AFH meals have been implicated as contributors to higher body weight and adverse health outcomes. OBJECTIVE: To examine the association of frequency of eating AFH and fast-food meals with biomarkers of chronic disease and dietary intake. DESIGN: This cross-sectional study used frequency of AFH and fast-food meal and biomarker data from the NHANES 2005-2010. Information on weekly frequency of AFH and fast-food meals was collected via questionnaire during the household interview. The metabolic biomarkers examined included body mass index (BMI), serum cholesterol (total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL)), triglycerides, glycohemoglobin and fasting glucose (n=8314, age⩾20, National Health and Nutrition Examination Surveys (NHANES) 2007-2010). Biomarkers of dietary exposure included serum concentrations of vitamins A, D, E, C, B-6, B-12, folate and carotenoids (n=4162; 2005-2006). Multiple linear and logistic regression methods adjusted for complex survey methodology and covariates. RESULTS: American adults reported a mean of 3.9 (95% confidence interval 3.7, 4.0) AFH and 1.8 (1.6, 1.9) fast-food meals/week. Over 50% of adults reported ⩾3 AFH and >35% reported ⩾2 fast-food meals/week. The mean BMI of more frequent AFH or fast-food meal reporters was higher (Ptrend⩽0.0004). Serum concentrations of total, LDL and HDL-cholesterol were related inversely with frequency of AFH meals (P<0.05). Frequencies of fast-food meals and serum HDL-cholesterol were also related inversely (P=0.0001). Serum concentrations of all examined micronutrients (except vitamin A and lycopene) declined with increasing frequency of AFH meals (P<0.05); women and ⩾50-year olds were at higher risk. CONCLUSIONS: Reporters of frequent AFH and fast-food meals had higher BMI and lower concentrations of HDL-cholesterol; however, profiles of other biomarkers did not indicate higher metabolic risk. However, the serum concentrations of nutrients with mostly plant foods as sources declined with increasing AFH meal frequency.

Compartmentalization of wards to cohort symptomatic patients at the beginning and end of norovirus outbreaks.

BACKGROUND: Outbreaks of norovirus can have a significant operational and financial impact on healthcare establishments. AIM: To assess whether containment of symptomatic patients in single rooms and bays at the beginning and end of norovirus outbreaks reduced the length of bed closure. METHODS: In 2007, we introduced a new strategy to limit the operational impact of hospital outbreaks of norovirus. Early in an outbreak, symptomatic patients were cohorted in single rooms or bays in an attempt to contain the outbreak without closing the entire ward. Once a ward had been closed, and as beds became available through discharges, patients were decanted into single rooms or empty bays with doors to facilitate earlier cleaning and opening of affected areas on the same ward. The impact of these changes was assessed by comparing outbreak data for two periods before and after implementation of the new strategy. FINDINGS: Prior to June 2007, 90% of outbreaks were managed by closure of an entire ward, compared with only 54% from June 2007 onwards. The duration of closure was significantly shorter for bays compared with entire wards, both before (3.5 vs 6, P = 0.0327) and after (3 vs 5, P < 0.0001) June 2007. When considering all outbreaks, there was a significant reduction in duration of closure after the change in strategy (6 vs 5, P = 0.007). CONCLUSION: Using ward compartmentalization to cohort affected patients at the beginning and end of norovirus outbreaks improved the efficiency of outbreak management and reduced operational disruption.

A rapid and sensitive fluorometric method for the quantitative analysis of snake venom metalloproteases and their inhibitors.

Metalloproteases are responsible for the hemorrhagic effects of many snake venoms and contribute to other pathways that lead to local tissue damage. Methods that quantify snake venom metalloproteases (SVMP) are therefore valuable tools in research on the clinical, physiological, and biochemical effects of envenomation. Comparative analysis of individual, population, and species differences requires screening of large numbers of samples and treatments, and therefore require a method of quantifying SVMP activity that is simple, rapid, and sensitive. This paper demonstrates the properties of a new fluorometric assay of SVMP activity that can provide a measure of metalloprotease activity in 1 h. The assay is reliable, with variation among replicates sufficiently small to reliably detect differences in between species (F(19,60) = 2924, p < 0.001), even for those venoms with low overall activity. It is also sensitive enough to detect differences among venoms using <2 ng of whole venom protein. We provide an example use of this assay to detect the presence of natural SVMP inhibitors in minute samples of blood plasma from rock squirrels (S. variegatus), a natural prey species for North American rattlesnakes. We propose this assay is a useful addition to the set of tools used to characterize venoms, as well as high-throughput screening of natural or synthetic inhibitors, or other novel therapeutic agents against SVMP effects.

Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study.

AIMS: To monitor the association between the course of high risk human papillomavirus (HR-HPV) infection and the development of cervical neoplasia over time, from a baseline of normal cervical cytology. METHODS: This paper presents the follow up data from a previous cross sectional analysis. Women from a screening population who had normal cytology and who were HR-HPV positive were recalled after two to three years for cytology and HPV genotyping. The development of cervical neoplasia at follow up was related to the course of HPV infection (clearance, persistence, or sequential infection) and the presence of single or multiple HPV infections at baseline. A comparator control group of women who were HPV and cytologically negative at baseline were selected from the same population. RESULTS: Twelve cases of dyskaryosis were found in women who were HPV positive at baseline; four were high grade. Only three cases of low grade dyskaryosis were found in the control group. Women with type specific persistent infections were significantly more likely to develop cervical neoplasia than women who cleared the infection (p = 0.0001) or were sequentially infected with different types (p = 0.001). Women with multiple HPV infections at baseline were no more likely to develop cervical dyskaryosis than those with a single infection. CONCLUSIONS: Type specific persistent HR-HPV infection as monitored by genotyping can identify women at increased risk of cervical neoplasia more accurately than a single or repeated presence/absence HPV test. The cost effectiveness of such an approach should be investigated by an appropriate, large scale cost-benefit analysis.

Human papillomavirus type specific DNA and RNA persistence--implications for cervical disease progression and monitoring.

In 2000, we monitored the course and persistence of human papillomavirus (HPV) infection in 54 women who were HPV positive and free of any cytological disease using HPV-DNA genotyping with a linear array assay (baseline). The impact of HPV infection on development of cervical cytological abnormality (dyskaryosis) was monitored by repeat HPV genotyping and cytological assessment 2 years later. Detection of mRNA transcripts of known HPV oncogenes E6 and E7 using NASBA methodology and specific molecular beacons for five common HPV types was also performed at both time points. A total of 11/54 (20%) women developed dyskaryosis after 2 years with 31/54 and 23/54 women exhibiting transient and persistent infections respectively, as monitored by DNA genotyping. Women who maintained type-specific persistent HPV infection were significantly more likely to develop dyskaryosis compared to those who exhibited a transient infection (P = 0.001). The presence of HPV mRNA E6/E7 transcripts was less sensitive but more specific for the detection of disease at follow up. Moreover, women who were DNA positive and also positive for mRNA transcripts at baseline were significantly more likely to harbour persistent infection compared to those in whom DNA only was detected at baseline (P = 0.013). This study highlights the importance of detecting persistent type specific HPV infection to identify those women more at risk of developing cervical abnormalities, either by repeated DNA genotyping, or potentially by RNA based techniques that may be more predictive of persistent infection if performed at a single time point.

Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.

AIMS: If human papillomavirus (HPV) testing is to be included within cervical screening programmes, the importance of multiple HPV infections in cervical neoplasia needs to be determined. This study investigated the diversity of multiple HPV types in a routine cervical screening population, and assessed associations with cervical neoplasia. METHODS: Overall HPV prevalence, type specific prevalence, and extent of multiple infection were assessed in residual material from 3444 liquid based cytology samples, using real time GP5+/GP6+ polymerase chain reaction for screening and linear array assay for genotyping. HPV status was studied in relation to age and concurrent cytological evidence of dyskaryosis. RESULTS: Twenty per cent of samples were HPV positive. HPV type diversity was broad, and multiple HPV infections occurred in half of the HPV positive samples. Younger women were significantly more likely to harbour multiple high risk HPV (HR-HPV) infections. Infections with multiple HR-HPV types were found in 3.4% of samples negative for neoplasia and in 33.3%, 41.8%, and 40.4% of samples with borderline, mild, or high grade dyskaryosis, respectively. Single HR-HPV infections were found in 4.9%, 38.6%, 45.0%, and 51.1% of negative, borderline, mild, or high grade dyskaryosis samples, respectively. CONCLUSIONS: Multiple HR-HPV infections were most prevalent in young women. Multiple HR-HPV infections were not more frequent in high grade than in low grade cervical neoplasia, reflecting common sexual transmission of multiple HR-HPV. Prospective cohort studies linking sequential loss or gain of HPV types with cytological analysis are required to assess the impact of multiple HR-HPV infections on neoplastic progression.

Transcriptional profiling of human osteoblast differentiation.

Osteoblast differentiation is a key aspect of bone formation and remodeling. To further our understanding of the differentiation process, we have developed a collection of conditionally immortalized adult human osteoblast cell lines representing discrete stages of differentiation. To evaluate changes in gene expression associated with differentiation, polyA((+)) RNA from pre-osteoblasts, early and late osteoblasts, and pre-osteocytes was subjected to gene chip analysis using the Affymetrix Hu6800 chip in conjunction with an Affymetrix custom chip enriched in bone and cartilage cDNAs. Overall, the expression of 47 genes was found to change threefold or more on both chips between the pre-osteoblastic and pre-osteocytic stages of differentiation. Many of the observed differences, including down-regulation of collagen type I and collagen-processing enzymes, reflect expected patterns and support the relevance of our results. Other changes have not been reported and offer new insight into the osteoblast differentiation process. Thus, we observed regulation of factors controlling cell cycle and proliferation, reflecting decreased proliferation, and increased apoptosis in pre-osteocytic cells. Elements maintaining the cytoskeleton, extracellular matrix, and cell-cell adhesion also changed with differentiation reflecting profound alterations in cell architecture associated with the differentiation process. We also saw dramatic down-regulation of several components of complement and other immune response factors that may be involved in recruitment and differentiation of osteoclasts. The decrease in this group of genes may provide a mechanism for controlling bone remodeling of newly formed bone. Our screen also identified several signaling proteins that may control osteoblast differentiation. These include an orphan nuclear receptor DAX1 and a small ras-related GTPase associated with diabetes, both of which increased with increasing differentiation, as well as a high mobility group-box transcription factor, SOX4, that was down-regulated during differentiation. In summary, our study provides a comprehensive transcriptional profile of human osteoblast differentiation and identifies several genes of potential importance in controlling differentiation of osteoblasts.

Retroviral vectors bearing IgG-binding motifs for antibody-mediated targeting of vascular endothelial growth factor receptors.

Targeting retroviral vectors to tumor vasculature is an important goal of cancer gene therapy. In this study, we report a novel targeting approach wherein IgG-binding peptides were inserted into the Moloney murine leukemia virus (MuLV) envelope (env) protein. The modifications on the viral env included replacement of the entire receptor binding region of the viral env with protein A (or ZZ) domains. The truncated env incorporating IgG-binding motifs (known as proteins) provided the targeting function, while the co-expressed wild-type (WT) env protein enabled viral fusion and cell entry. An anti-human VEGF receptor (Flk-1/KDR) antibody served as a molecular bridge, directing the retroviral vector to the endothelial cell. Hence, the IgG-targeted vectors bound to the Flk-1/KDR antibody which in turn bound to VEGF receptors on Kaposi sarcoma, KSY1, endothelial cells. The net effect was increased viral fusion and infectivity of IgG-bound retroviral vectors when compared to non-targeted vectors bearing WT env alone. These data provide the proof of concept that IgG-binding vector/VEGF receptor antibody complexes may be used to enhance retroviral gene delivery to activated endothelial cells.

Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice.

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.

Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18.

AIMS: To assess the validity and practicality of real time polymerase chain reaction (PCR) for human papillomavirus (HPV) testing in combination with liquid based cytology samples for cervical screening. METHODS: Real time PCR using consensus (GPS+/6+) and type specific primers was developed to detect genital HPV types. This provides rapid, efficient amplification followed by denaturation of the product and computer analysis of the kinetics data that are generated. Liquid based cytology samples were obtained from patients attending routine cervical screening clinics. DNA was extracted from the residual cellular suspension after cytology using spin columns. RESULTS: Real time PCR successfully distinguished between HPV-16 and HPV-18 on the basis of amplification with consensus primers followed by DNA melting temperature (Tm) analysis. Sensitivities of one to 10 copies of HPV-16 (mean Tm = 79.4 degrees C; 2 SD, 0.8) and four to 40 copies of HPV-18 (mean Tm = 80.4 degrees C; 2 SD, 0.4) were obtained. In a mixed population of SiHa and HeLa cells containing known copy numbers of HPV-16 and HPV-18 genomes, HPV-16 and HPV-18 products were clearly separated by Tm analysis in mixtures varying from equivalence to 111000. Together with detailed melt analysis, type specific primers from the same region of the L1 gene confirmed the differential ability of this system. The method was applied to 100 liquid based cytology samples where HPV status using conventional GP5+/6+ PCR was already known. There was 95% agreement between the methods, with 55 positives detected by conventional PCR and 59 with real time PCR. The method was then tested on 200 routine liquid based cytology samples. Approximately 10% were positive by real time PCR, most of which were classified as HPV-16 by detailed melt analysis. Thirteen (6.8%) HPV positives were identified in 189 samples showing no evidence of cervical cytological abnormality. CONCLUSIONS: Real time PCR is a rapid, efficient method for the detection of HPV with the separation of HPV-16 and HPV-18 on the basis of differential Tm. Preliminary results suggest it could prove

Evaluation of normalization procedures for oligonucleotide array data based on spiked cRNA controls.

BACKGROUND: Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. RESULTS: We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. CONCLUSIONS: Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Preparation of mRNA for expression monitoring.

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) vs. total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.