Perry Haaland

A systematic approach to planning for a designed industrial experiment

Design of experiments and analysis of data from designed experiments are well-established methodologies in which statisticians are formally trained. Another critical and rarely taught skill is the planning that precedes designing an experiment. This article suggests a set of tools for presenting generic technical issues and experimental features found in industrial experiments. These tools are predesign experiment guide sheets to systematize the planning process and to produce organized written documentation. They also help experimenters discuss complex trade-offs between practical limitations and statistical preferences in the experiment. A case study involving the (computer numerical control) CNC-machining of jet engine impellers is included.

flowCore: a Bioconductor package for high throughput flow cytometry

BackgroundRecent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates.ResultsWe developed a set of flexible open source computational tools in the R package flowCore to facilitate the analysis of these complex data. A key component of which is having suitable data structures that support the application of similar operations to a collection of samples or a clinical cohort. In addition, our software constitutes a shared and extensible research platform that enables collaboration between bioinformaticians, computer scientists, statisticians, biologists and clinicians. This platform will foster the development of novel analytic methods for flow cytometry.ConclusionThe software has been applied in the analysis of various data sets and its data structures have proven to be highly efficient in capturing and organizing the analytic work flow. Finally, a number of additional Bioconductor packages successfully build on the infrastructure provided by flowCore, open new avenues for flow data analysis.

Inference for effect-saturated fractional factorials

Industrial scientists and engineers often use experimental designs in which all degrees of freedom are used to estimate effects and consequently no classical estimate of the error is possible. Robust scale estimates provide an alternative measure of the error. In this study, several such scale estimators are evaluated based on the power or related significance tests. The pseudo standard error method of Lenth provides the best overall performance. Lenths t approximation for critical values was found to be inaccurate, however, so new tables are provided. Additional recommendations are made according to the experimenters prior belief in the number of likely important factors.

The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: oxygen consumption as a potential measure of islet potency.

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding (“dO2 slope”). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized “dO2 slope” in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.

Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

Regression and calibration with nonconstant error variance

Abstract Davidian, M. and Haaland, P., 1990. Regression and calibration with nonconstant error variance. Chemometrics and Intelligent Laboratory Systems , 9: 231–248. Ordinary least squares regression analysis is generally inappropriate for calibration and regression problems when the usual assumption of constant variance across all observations does not hold. Estimators of regression parameters are of relatively poor quality and the resulting inference can be misleading. The use of standard data transformations is a common alternative but may not provide enough flexibility for some cases. The use of weighted regression with weights estimated from replicates is generally unreliable for reasonable sample sizes. However, when the error variance changes systematically with the mean response or other variables, generalized least squares (GLS) and variance function estimation (VFE) methods can be used. The GLS-VFE approach allows the experimenter to specify a model for the systematic change in variance, estimate unknown parameters, and to use this information to provide more efficient estimates of the regression parameters. In this tutorial, GLS-VFE methods are introduced and described in the context of regression and calibration. An example of calibration for a chemical assay is used to motivate discussion and illustrate the implementation of these methods using standard software packages.

A Nonparametric Regression Approach to Syringe Grading for Quality Improvement

Abstract In the biomedical products industry, measures of the quality of individual clinical specimens or manufacturing production units are often available in the form of high-dimensional data such as continuous recordings obtained from an analytical instrument. These recordings are then examined by experts in the field who extract certain features and use these to classify individuals. To formalize and quantify this procedure, an approach for extracting features from recordings based on nonparametric regression is described. These features are then used to build a classification model that incorporates the knowledge of the expert. The procedure is illustrated with the problem of grading of syringes from associated friction profile data. Features of the syringe friction profiles used in the classification are extracted via smoothing splines, and grades of the syringes are assigned by an expert tribologist. A nonlinear classification model is constructed to predict syringe grades based on the extracted fe...

IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells

BackgroundDiminished IL-2 production and lack of effector differentiation have been reported for HIV-specific T cells. In this study, we examined the prevalence of these phenomena using 8-color cytokine flow cytometry, and tested the hypothesis that these two findings were causally related. We analyzed cytokine profiles and memory/effector phenotypes of HIV-specific and CMV-specific T cells using short-term in vitro stimulation with HIV or CMV peptide pools. Nineteen HIV-positive subjects with progressive disease and twenty healthy, HIV-negative subjects were examined.ResultsAmong HIV-infected subjects, there were significantly fewer CD8+ IL-2+ T cells responding to HIV compared to CMV, with no significant difference in CD4+ IL-2+ T cells. The majority of CMV-specific T cells in both HIV-negative and HIV-positive subjects appeared to be terminally differentiated effector cells (CD8+ CD27- CD28- CD45RA+ or CD8+ CD27- CD28- CD45RA-). In HIV-positive subjects, the most common phenotype of HIV-specific T cells was intermediate in differentiation (CD8+ CD27+ CD28- CD45RA-). These differences were statistically significant, both as absolute cell frequencies and as percentages. There was a significant correlation between the absolute number of HIV-specific CD8+ IL-2+ T cells and HIV-specific CD8+ CD27- CD28- CD45RA+ terminal effector cells.ConclusionIL-2 production from antigen-specific CD8+ T cells correlates with effector cell differentiation of those cells.

Virtual kinetics: Using statistical experimental design for rapid analysis of enzyme inhibitor mechanisms

Modern automated drug-screening can generate hundreds of inhibitor leads from diverse chemical sources in a short period of time. Traditional methods of inhibitor analysis are resource intensive and limit the number of inhibitors that can be analyzed for their mechanism of inhibition. This paper presents methods we have developed for rapid estimation of both potency and mechanism of potential inhibitor leads for a biochemically complex screening target (protein kinase C) using commercially available computer programs for statistical experimental design. Our findings indicate that, with careful choice of factor levels, statistical experimental design clearly identifies the various interactions of the assay components with inhibitors. Suitably plotted, the data can be used to examine the competitive nature of the inhibitor and can provide estimates of IC50 and Michaelis constants useful for planning further kinetic work. The techniques used are amenable to automation and should be useful for identifying inhibitors that may have only marginal potency, but exhibit desirable mechanistic profiles suitable for structural analoging efforts.

Analysis of High-Throughput Flow Cytometry Data Using plateCore

Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data.