Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

Compact devices for generation of reference trace VOC mixtures: a new concept in assuring quality at chemical and biochemical laboratories.

Volatile organic compounds (VOCs) in gas mixtures at trace level (nmol/mol) are routinely measured by chemical and biochemical laboratories as climate indicators, indoor air quality pollutants from building materials emissions, contaminants in food and beverages, and biomarkers in body fluids (blood, urine, breath) of occupational exposure or human diseases. Current analytical instruments used for measurements are gas chromatographs equipped with various injector and detector configurations. The assurance of measurement quality is done by using a huge amount of certified liquid VOC standard solutions (or gaseous VOC standard cylinders) with multiple dilutions to reach the required trace level. This causes high standard uncertainty in instrument calibrations, high cost, and high consumption of analysis and laboratory personal time. In this paper, we present the implementation of portable generators producing VOC gas standards at trace level for automatic and direct calibration of VOC detectors employed in various contexts, removing the need for preparation of matrix calibration standards in cylinders. Two compact devices in-house developed by two national metrology institutes-the Istituto Nazionale di Ricerca Metrologica (INRIM) and the Federal Institute of Metrology (METAS)-are here used to dynamically generate reference gas mixtures in an SI traceable way. The two devices are based on different technologies: diffusion and permeation, for INRIM and METAS, respectively. A metrological characterization is given and the practical implementation at chemical and biochemical laboratories is discussed. Graphical abstract Onsite calibration with transportable generation system with similar performances to primary laboratory devices.

Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR.

Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Behaviour of fluorescence emission of cyanine dyes, cyanine based fluorescent nanoparticles and CdSe/ZnS quantum dots in water solution upon specific thermal treatments.

Fluorescence techniques are widely used as detection methods in a wide range of biological imaging and analytical applications. The purpose of this work is to determine a measurement method which leads to a comparison between different classes of fluorophores in term of stability of the fluorescence signal upon thermal treatment cycles. This kind of investigation can determine whether the fluorophore performance is affected by heating/cooling cycles and to what extent. The fluorophores considered in this work were organic fluorophores belonging to the family of indocyanine dyes (IRIS3 by Cyanine Technologies S.p.A.) in their molecular form or encapsulated within silica nanoparticles, and CdSe/ZnS carboxyl quantum dots (Qdots 565 ITK by Invitrogen). The NIST Standard Reference Material® SRM 1932 fluorescein solution was used in the certified concentration as reference material in order to evaluate the repeatability of the used spectrofluorimeter. The proposed measurement protocol allows to characterize all kind of fluorophores upon thermal treatments. This allows direct comparison of their performance under temperature changes, giving useful guidelines for the selection of the most suitable fluorophore for the envisaged application. Moreover the method appears to be a promising tool for the characterisation of reference fluorescent materials. The experimental results demonstrate that each fluorophore class shows a specific behaviour. The experimental data analysis points out an important hysteresis effect for quantum dots that was not detected for cyanine molecules and was only slightly detected for cyanine doped silica nanoparticles.

In vitro dexamethasone treatment does not induce alternative ATM transcripts in cells from Ataxia-Telangiectasia patients.

Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia-Telangiectasia (A-T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A-T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A-T patients.

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

BACKGROUND: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. METHOD: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. RESULTS: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. CONCLUSIONS: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements.

The symptomatic irreproducibility of data in biomedicine and biotechnology prompts the need for higher order measurements of cells in their native and near-native environments. Such measurements may support the adoption of new technologies as well as the development of research programs across different sectors including healthcare and clinic, environmental control and national security. With an increasing demand for reliable cell-based products and services, cellular metrology is poised to help address current and emerging measurement challenges faced by end-users. However, metrological foundations in cell analysis remain sparse and significant advances are necessary to keep pace with the needs of modern medicine and industry. Herein we discuss a role of metrology in cell and cell-related R&D activities to underpin growing international measurement capabilities. Relevant measurands are outlined and the lack of reference methods and materials, particularly those based on functional cell responses in native environments, is highlighted. The status quo and current challenges in cellular measurements are discussed in the light of metrological traceability in cell analysis and applications (e.g., a functional cell count). An emphasis is made on the consistency of measurement results independent of the analytical platform used, high confidence in data quality vs. quantity, scale of measurements and issues of building infrastructure for end-users.

Synthesis, structure and non-linear optical properties of new isostructural ß-D-fructopyranose alkaline halide metal-organic frameworks: a theoretical and an experimental study.

In this work four metal-organic framework isomorphs, based on fructose and alkali-earth halogenides, were investigated to better understand the effect of the size of the cation and the different polarizability of the anion on the calculated hyperpolarizability and optical susceptibility, which are correlated to non-linear optical properties. The compounds were characterized by X-ray diffraction and the first hyperpolarizability and the second-order susceptibility were obtained from theoretical calculations. Furthermore, a new method to measure the second-harmonic (SH) efficiency on a small quantity of powder at different wavelengths of excitation was optimized and an attempt was made to assess the reduction of the SH intensity for small quantities of nano-crystals, in order to ascertain the possibility of applications in biological systems. The results of this work show that both the intrinsic nature of the anion and the induced dissociation of cations and anions by fructose play a role in the second-harmonic generating properties of such compounds.

Micro- and Macrostructured PLGA/Gelatin Scaffolds Promote Early Cardiogenic Commitment of Human Mesenchymal Stem Cells In Vitro.

The biomaterial scaffold plays a key role in most tissue engineering strategies. Its surface properties, micropatterning, degradation, and mechanical features affect not only the generation of the tissue construct in vitro, but also its in vivo functionality. The area of myocardial tissue engineering still faces significant difficulties and challenges in the design of bioactive scaffolds, which allow composition variation to accommodate divergence in the evolving myocardial structure. Here we aimed at verifying if a microstructured bioartificial scaffold alone can provoke an effect on stem cell behavior. To this purpose, we fabricated microstructured bioartificial polymeric constructs made of PLGA/gelatin mimicking anisotropic structure and mechanical properties of the myocardium. We found that PLGA/gelatin scaffolds promoted adhesion, elongation, ordered disposition, and early myocardial commitment of human mesenchymal stem cells suggesting that these constructs are able to crosstalk with stem cells in a precise and controlled manner. At the same time, the biomaterial degradation kinetics renders the PLGA/gelatin constructs very attractive for myocardial regeneration approaches.

A first approach to evaluate the cell dose in highly porous scaffolds by using a nondestructive metabolic method.

BACKGROUND: In cell-based therapies, in vitro studies on biomimetic cell-scaffold constructs can facilitate the determination of the cell dose, a key factor in guaranteeing the effectiveness of the treatment. However, highly porous scaffolds do not allow a nondestructive evaluation of the cell number. Our objective was to develop a nondestructive method for human mesenchymal stem cells dose evaluation in a highly porous scaffold for bone regeneration. MATERIALS & MEASUREMENT METHOD: Proliferation trend of human mesenchymal stem cells on Biocoral® scaffolds was measured by a resazurin-based assay here optimized for 3D cultures. The method allows to noninvasively follow the cell proliferation on biocorals over 3 weeks with very high reproducibility. CONCLUSION: This reliable method could be a powerful tool in cell-based therapies for cell dose determination.

A mechanical characterization of polymer scaffolds and films at the macroscale and nanoscale.

Biomaterials should be mechanically tested at both the nanoscale and macroscale under conditions simulating their working state, either in vitro or in vivo, to confirm their applicability in tissue engineering applications. In this article, polyester-urethane-based films and porous scaffolds produced by hot pressing and thermally induced phase separation respectively, were mechanically characterized at both the macroscale and nanoscale by tensile tests and indentation-type atomic force microscopy. All tests were conducted in wet state with the final aim of simulating scaffold real operating conditions. The films showed two distinct Young Moduli populations, which can be ascribed to polyurethane hard and soft segments. In the scaffold, the application of a thermal cooling gradient during phase separation was responsible for a nanoscale polymer chain organization in a preferred direction. At the macroscale, the porous matrices showed a Young Modulus of about 1.5 MPa in dry condition and 0.3 MPa in wet state. The combination of nanoscale and macroscale values as well as the aligned structure are in accordance with stiffness and structure required for scaffolds used for the regeneration of soft tissues such as muscles.