Standardization of flow cytometric detection of antigen expression.

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques.

The Impact of Tumor Treating Fields on Glioblastoma Progression Patterns.

Purpose: Tumor-treating fields (TTFields) are an antimitotic treatment modality that interfere with glioblastoma (GBM) cell division and organelle assembly by delivering low-intensity, alternating electric fields to the tumor. A previous analysis from the pivotal EF-14 trial demonstrated a clear correlation between TTFields dose density at the tumor bed and survival in patients treated with TTFields. This study tests the hypothesis that the antimitotic effects of TTFields result in measurable changes in the location and patterns of progression of newly diagnosed GBM.

Development and Validation of Measurement Traceability for In Situ Immunoassays.

Background: Immunoassays for protein analytes measured in situ support a $2 billion laboratory testing industry that suffers from significant interlaboratory disparities, affecting patient treatment. The root cause is that immunohistochemical testing lacks the generally accepted tools for analytic standardization, including reference standards and traceable units of measure. Until now, the creation of these tools has represented an insoluble technical hurdle.

Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles.

The National Institute of Standards and Technology (NIST), the National Institutes of Health (NIH) and other industry stakeholders have been working together to enable fluorescence intensities of flow cytometer calibration beads to be assigned quantitative equivalent reference fluorophore (ERF) values with high accuracy and precision. The ultimate goal of this effort is to accurately quantify the number of antibodies bound to individual living cells. The expansion of this effort to assign ERF values to more than 50 fluorescence channels and particles with diameters ranging from 10 μm down to 80 nm is reported here.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic.

Stochastic Reaction-Diffusion Model of the Binding of Monoclonal Antibodies to CD4 Receptors on the Surface of T Cells.

A stochastic reaction-diffusion model was developed to describe the binding of labeled monoclonal antibodies (mAbs) to CD4 receptors on the surface of T cells. The mAbs diffused to, adsorbed on, and underwent monovalent and bivalent binding to CD4 receptors on the cell surface. The model predicted the time-dependent nature of all populations involved in the labeling process.

A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes.

The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources.

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples.

We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST calibrated pathlength cuvette.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability.

The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria.

Primary Determination of Particle Number Concentration with Light Obscuration and Dynamic Imaging Particle Counters.

Accurate number concentrations of particles in liquid media are needed to assess the quality of water, pharmaceuticals, and other liquids, yet there are limited reference materials or calibration services available with clear traceability to the International System of Units. We describe two methods, based on very simple modifications of commercial particle counter instruments, that can provide traceable number concentration measurements. One method used a light obscuration counter.

NIST Spectroscopic Measurement Standards.

Ultraviolet (UV) absorbance measurements provide a rapid and reliable method to determine protein concentrations. the National Institute of standards and technology (NIST) has developed as a pathlength standard for UV absorbance measurements for use with the new generation of microvolume spectrophotometers and short-pathlength cuvettes. short pathlengths are used with high-concentration targets to ensure that absorbance values are within the optimal range.

Development of NIST Standard Reference Material 2082, a Pathlength Standard for Measurements in the Ultraviolet Spectrum.

New spectrophotometers and cuvettes have been designed to allow the measurement of absorbance values from samples using microliter volume sizes. These measurements are done using short pathlengths to decrease the sample volumes required. The major applications for these spectrophotometers and cuvettes are samples that are difficult to obtain in large amounts, such as proteins and nucleic acids that absorb light in the ultraviolet range.

Assignment of the Number of Equivalent Reference Fluorophores to Dyed Microspheres.

A procedure will be described to assign to each dyed microsphere a number called the Equivalent number of Reference Fluorophores (ERF). The ERF unit gives the number of reference fluorophores in solution which produce the same fluorescence signal as a single dyed microsphere. In the first step, fluorescence measurements were carried out on serial dilutions of a solution of reference fluorophores.

An automated protocol for performance benchmarking a widefield fluorescence microscope.

Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to-use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications.

Optical Properties of CdSe/ZnS Nanocrystals.

Measurements are presented of the absorbance, fluorescence emission, fluorescence quantum yield, and fluorescence lifetime of CdSe/ZnS nanocrystals, also known as quantum dots (QDs). The study included three groups of nanocrystals whose surfaces were either passivated with organic molecules, modified further with carboxyl groups, or conjugated with CD14 mouse anti-human antibodies. The surface modifications had observable effects on the optical properties of the nanocrystals.

Beetroot-pigment-derived colorimetric sensor for detection of calcium dipicolinate in bacterial spores.

In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1∶1 complex with a stability constant of 1.4 × 10(5) L mol(-1).

State-of-the art comparability of corrected emission spectra. 2. Field laboratory assessment of calibration performance using spectral fluorescence standards.

In the second part of this two-part series on the state-of-the-art comparability of corrected emission spectra, we have extended this assessment to the broader community of fluorescence spectroscopists by involving 12 field laboratories that were randomly selected on the basis of their fluorescence measuring equipment. These laboratories performed a reference material (RM)-based fluorometer calibration with commercially available spectral fluorescence standards following a standard operating procedure that involved routine measurement conditions and the data evaluation software LINKCORR developed and provided by the Federal Institute for Materials Research and Testing (BAM). This instrument-specific emission correction curve was subsequently used for the determination of the corrected emission spectra of three test dyes, X, QS, and Y, revealing an average accuracy of 6.

State-of-the art comparability of corrected emission spectra. 1. Spectral correction with physical transfer standards and spectral fluorescence standards by expert laboratories.

The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods.

ACR Appropriateness Criteria® definitive external beam irradiation in stage T1 and T2 prostate cancer.

Purpose: : External beam radiation therapy is a standard of care treatment for men who present with clinically localized (T1-T2) prostate cancer. The purpose of this review was to provide clarification on the appropriateness criteria and management considerations for the treatment of prostate cancer with external beam radiation therapy.

Methods: : A panel consisting of physicians with expertise on prostate cancer was assembled and provided with a number of clinical scenarios for consensus treatment and management guidelines.

Development of bavituximab, a vascular targeting agent with immune-modulating properties, for lung cancer treatment.

Bavituximab is a chimeric monoclonal antibody directed against the membrane phospholipid phosphatidylserine. Phosphatidylserine exposure is increased on endothelial cells and apoptotic cancer cells in solid tumors, allowing tumor-specific targeting of bavituximab. Bavituximab binding results in tumor vessel occlusion and enhanced antitumor immunity.

Phase I dose-escalation study of stereotactic body radiation therapy for low- and intermediate-risk prostate cancer.

Purpose: To evaluate the tolerability of escalating doses of stereotactic body radiation therapy in the treatment of localized prostate cancer.

Patients And Methods: Eligible patients included those with Gleason score 2 to 6 with prostate-specific antigen (PSA) ≤ 20, Gleason score 7 with PSA ≤ 15, ≤ T2b, prostate size ≤ 60 cm(3), and American Urological Association (AUA) score ≤ 15. Pretreatment preparation required an enema and placement of a rectal balloon.

Recommendations for fluorescence instrument qualification: the new ASTM Standard Guide.

Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification.

Procedures for Wavelength Calibration and Spectral Response Correction of CCD Array Spectrometers.

This work describes a procedure for acquiring a spectrum of an analyte over an extended range of wavelengths and validating the wavelength and intensity assignments. To acquire a spectrum over an extended range of wavelengths with a spectrometer with a charge coupled device (CCD) array detector, it is necessary to acquire many partial spectra, each at a different angular position of the grating, and splice the partial spectra into a single extended spectrum. The splicing procedure exposes instrument dependent artifacts.

Fat-specific protein 27 regulates storage of triacylglycerol.

FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins. In this study, we investigated the function of another member of this family, FSP27 (Cidec), in apoptosis and adipocyte metabolism.