Pilot study to assess the activation status of the complement system in blood from patients with anti-neutrophil cytoplasmic antibody (ANCA-) associated vasculitis

The present research study will look at the way the complement system, an important part of the immune system, functions in people with vasculitis. Around 20 patients with vasculitis positive for the circulating antibody ANCA will take part in the study. Each patient will provide one blood sample for analysis of several complement components. The patients will be included before starting treatment, when stable in remission, or during flares of vasculitis. The aim of the research is to obtain data on the frequency of evidence of complement activation in vasculitis patients, and to evaluate the correlation of complement activation to disease related clinical and/or laboratory parameter

Population PK-PD model for tolerance evaluation to the p38 MAP kinase inhibitor BCT197

The p38 mitogen-activated protein kinase (p38) is a key signaling pathway involved in regulation of inflammatory cytokines. Unexpectedly, several clinical studies using p38 inhibitors found no convincing clinical efficacy in treatment of chronic inflammation. It was the objective of this study to characterize the population pharmacokinetics (PK) of BCT197 in heathy volunteers and to examine the relationship between BCT197 exposure and pharmacodynamics (PD) measured as inhibition of ex-vivo LPS-induced TNF, a downstream surrogate marker of p38. PK was characterized using a two-compartment model with mixed-order absorption and limited-capacity tissue binding. The PK-PD relationship revealed that suppression of TNF was counteracted, at least partly, despite continuous drug exposure. This may indicate a mechanism by which the inflammatory response acquires the ability to bypass p38. Simulations of posology dependence in drug effect revealed that an intermittent regimen may offer clinical benefit over continuous dosing, by limiting the potential impact of tolerance development

Flow cytometric method transfer: Recommendations for best practice

As with many aspects of the validation and monitoring of flow cytometric methods, the method transfer processes and acceptance criteria described for other technologies are not fully applicable. This is due to the complexity of the highly configurable instrumentation, the complexity of cellular measurands, the lack of qualified reference materials for most assays, and limited specimen stability. There are multiple reasons for initiating a method transfer, multiple regulatory settings, and multiple context of use. All of these factors influence the specific requirements for the method transfer. This recommendation paper describes the considerations and best practices for the transfer of flow cytometric methods and provides individual case studies as examples. In addition, the manuscript emphasizes the importance of appropriately conducting a method transfer on data reliability

High-sensitivity Flow Cytometric Assays: Considerations for Design Control and Sensitivity Validation for Identification of Rare Events

The current consensus recommendation papers dealing with the unique requirements for the validation of assays performed by flow cytometry address the validation of assay sensitivity only in general terms (1-3). In this paper, a detailed approach for designing and validating the sensitivity of rare event methods is described. The impact of panel design and optimization on the Lower Limit of Quantification (LLOQ) as well as suggestions for reporting data near, or below, the LLOQ will be addressed. Thus, this paper will serve to provide best practices for the development, optimization, and validation of flow cytometric assays designed to assess rare events

Implementation of highly sophisticated flow cytometry assays in multi- center clinical studies: considerations and guidance

the item has no formal formal abstract as summary of the items the table of contents is given here: Table of contents 1 Introduction: 1.1 Technical overview and applications of flow cytometry 1.2 Use of flow cytometry for biomarker analysis in human clinical studies 2 Development and Validation of flow cytometry assays for clinical use 2.1 Summary of key challenges 2.2 Considerations for the Validation of PhosFlow assays used for PK/PD assessment 3 Whole blood flow cytometry assays in multi-center clinical studies: Immunophenotyping versus PhosFlow 3.1 On-site processing of whole blood samples 3.2 Stabilization of samples: stabilizing tubes or freezing of whole blood samples 3.3 Clinical study implementation 4 Summary and Future Perspectives

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials

Background: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. Minimizing the number of intermediate technical steps of cell sample preparation will increase reproducibility of results. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. Methods: Fresh whole blood samples were obtained from consented healthy donors preserved either in PAXgene Blood RNA tubes or used for cell sorting. Target cells were enriched using magnetic microbeads and an autoMACS Pro Separator (Miltenyi). Cells were enumerated prior to magnetic cell sorting using a Siemens ADVIA® 120 Hematology System. Flow cytometric analysis for purity was performed before and after the magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). Results: Positive magnetic-activated cell separation (MACS) coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, CD45+ pan leucocytes. RNA quality from enriched cells was above eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. Conclusion: The proposed workflow generates reproducible cell-type specific transcriptome data for CD14+ -, CD3+ -, CD4+ -, CD8+ -, CD15+ -, CD19+ -, CD56+ -, and CD45+- cells, which can be translated to clinical settings and used to generate clinically relevant gene expression biomarkers from whole blood samples. This procedure facilitates the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols