The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing.

Background: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available.

Correlation of SARS-CoV-2 RNA and nucleocapsid concentrations in samples used in INSTAND external quality assessment schemes.

Objective: In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021.

Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.

The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load.

The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018).

An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1.

Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories.

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.

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.

Microflow cytometers with integrated hydrodynamic focusing.

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel.

Quantitative assessment of cell viability based on flow cytometry and microscopy.

We compare flow cytometric and microscopic determination of cell viability by fluorescence labeling using calcein acetoxy-methyl-ester and ethidium homodimer-1 as live and dead stain, respectively. Peripheral blood monocytes served as model system and were accumulated applying density gradients. Subsequently, monocytes were further enriched by magnetic-activated or fluorescence-activated cell sorting (MACS, FACS) targeting the antigen CD14.

A microflow cytometer exploited for the immunological differentiation of leukocytes.

In this article, we demonstrate the potential of a microfluidic chip for the differentiation of immunologically stained blood cells. To this end, white blood cells stained with antibodies typically applied for the determination of the immune status were measured in the micro-device. Relative concentrations of lymphocytes and subpopulations of lymphocytes are compared to those obtained with a conventional flow cytometer.

Microfluidic structures for flow cytometric analysis of hydrodynamically focussed blood cells fabricated by ultraprecision micromachining.

We present three-dimensional microfluidic structures with integrated optical fibers, mirrors and electrodes for flow cytometric analysis of blood cells. Ultraprecision milling technique was used to fabricate different flow cells featuring single-stage and two-stage cascaded hydrodynamic focusing of particles by a sheath flow. Two dimensional focussing of the sample fluid was proven by fluorescence imaging in horizontal and vertical directions and found to agree satisfactorily with finite element calculations.

Evaluation of higher-order time-domain perturbation theory of photon diffusion on breast-equivalent phantoms and optical mammograms.

Time-domain perturbation theory of photon diffusion up to third order was evaluated for its accuracy in deducing optical properties of breast tumors using simulated and physical phantoms and by analyzing 141 projection mammograms of 87 patients with histology-validated tumors that had been recorded by scanning time-domain optical mammography. The slightly compressed breast was modeled as (partially) homogeneous diffusely scattering infinite slab containing a scattering and absorbing spherical heterogeneity representing the tumor. Photon flux densities were calculated from densities of transmitted photons, assuming extended boundary conditions.

In-vivo tissue optical properties derived by linear perturbation theory for edge-corrected time-domain mammograms.

A valuable method is described to analyze time-domain optical mammograms measured in the slab-like geometry of the slightly compressed female breast with a method based on linear perturbation theory including edge correction. Perturbations in scattering and absorption coefficients were mapped applying a computationally efficient point model.

Femtosecond mid-infrared spectroscopy of condensed phase hydrogen-bonded systems as a probe of structural dynamics.

We report the first time-resolved site-specific mid-infrared study of the photo-induced excited state hydrogen transfer reaction in 2-(2'-hydroxyphenyl)benzothiazole (HBT) with 130 fs time resolution. The transient absorption of the C=O stretching band marking the keto*-S1-state appears delayed on a time scale of 30-50 fs after electronic excitation to the enol*-S1-state. Its line center subsequently shifts up by about 3-5 cm(-1) after excitation, depending on the excitation wavelength tuned between 315 and 349 nm.

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically 'dark') S(1) state of carotenoids.

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300-1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2A(g)(-) state of rhodopin [corrected] is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2.

Spatially resolved small-angle noncollinear interferometric autocorrelation of ultrashort pulses with microaxicon arrays.

Small-angle, noncollinear, first- and second-order interferometric autocorrelation experiments with Ti:sapphire laser pulses of 9-80-fs duration have been performed with microaxicon arrays. Predictions of short-pulse spatial frequency effects were verified by comparison of interference patterns of single elements and matrices. An angular spectrum of Gaussian-shaped axicons was analyzed on the basis of linear refraction.

Acousto-optic mode locking of 3-microm Er lasers.

Er:YAG 2.94-microm lasers and Er:YLF 2.81-microm lasers were actively mode locked, with TeO(2) as the acousto-optic modulator.