Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

Bioanalysis and Stability of Polymyxins.

Clinical use of the polymyxin antibiotics began approximately 10 years after their discovery in the late 1940s. Their concentrations in biological fluids were measured using microbiological methods. These methods were reasonably accurate for measuring the active polymyxin base, such as polymyxin B and colistin (polymyxin E), but were used inappropriately for measuring the concentrations of "colistin" in humans or animals following the administration of colistimethate, also known as colistin methanesulphonate (CMS). The use of polymyxins for systemic infections waned in the 1970s because of their toxicity and the preference for other antibiotics, but their value for treating infections caused by several important Gram-negative pathogens becoming resistant to other antibiotics was realized in the mid-1990s. The lack of adequate pharmacokinetic and pharmacodynamic knowledge spurred the development of methods more specific for measuring polymyxin B and colistin after their administrations as sulphate salts, and of colistin and CMS after the administration of CMS sodium. These methods have been based on high-performance liquid chromatography, detection and quantification of fluorescent derivatives of the polymyxin bases, or of the bases themselves with detection and quantification by mass spectrometry.

Measuring the Size of the Latent Human Immunodeficiency Virus Reservoir: The Present and Future of Evaluating Eradication Strategies.

One of the major barriers to the successful design and implementation of human immunodeficiency virus (HIV) curative strategies is the limited ability to sensitively, specifically, and precisely quantify and characterize the whole-body burden of replication-competent HIV in individuals on effective antiretroviral therapy. Here, we review the development and validation of assays that directly and indirectly measure the size and distribution of the reservoir in blood and tissues. We also discuss the role that treatment interruptions will have in validating these assays and ultimately as a "proof of cure."

The utility of smartphone-based quantitative analysis of SARS-CoV-2-specific antibody lateral flow assays.

OBJECTIVES: Although antibody measurements using lateral flow assay (LFA) kits are convenient, they usually require a specialized reader for quantification. However, a smartphone-based quantification application can be used as a reader for LFA kits. We investigated the quantification ability of the application for SARS-CoV-2-specific antibodies. METHODS: Eight hundred frozen serum samples from 100 healthcare professionals who received a COVID-19 vaccine were analyzed. Images of assayed LFA kits were obtained using a smartphone camera. We determined whether the ratio of color density of the test and control lines of spike protein IgG correlated with chemiluminescent immunoassay-measured titers. RESULTS: Spike protein IgG correlated well with the quantification results of the LFA kits using the application installed on a smartphone (r = 0.886). CONCLUSION: Our results suggest that smartphone-based quantitative analysis of LFA kits enables the quantification of anti-SARS-CoV-2 IgG without special devices, enabling point-of-care assessment of acquired humoral immunity in various settings.

Analytical evaluation of the cobas® 6800 system for the detection and quantification of BK Virus (BKV) and Epstein Barr Virus (EBV) at a US solid organ and hematopoietic stem cell transplant center.

The cobas® EBV and BKV assays are the first FDA-approved, quantitative assays for monitoring posttransplant reactivation of these viruses. In this study, we assessed performance of the fully-automated cobas® assays, compared with Diasorin Molecular ASR, our laboratory developed test, and demonstrated a strong interassay correlation for BK and EBV monitoring.

Optimized Method of 3D Scaffold Seeding, Cell Cultivation, and Monitoring Cell Status for Bone Tissue Engineering.

The cultivation of cells in 3D systems is commonly regarded to be more physiological than in 2D as it comes much closer to the natural situation in tissues in many different aspects. However, 3D cell culture is much more complex. Cells within the pores of a printed 3D scaffold face a special situation concerning cell-material interaction and cell adhesion, cell proliferation, and supply of medium and oxygen into the core of the scaffolds. Biological assays (for cell proliferation, viability, and activity) have been validated primarily for 2D cell cultures and need to be adapted for 3D cultures. Likewise, in imaging, a number of points need to be taken into account in order to get a clear picture of the cells in 3D scaffolds, preferably with the method of multiphoton microscopy. Here, we describe a method for pretreatment and cell seeding of porous inorganic composite scaffolds (α-TCP/HA) for bone tissue engineering and for cultivation of the cell-scaffold constructs. The analytical methods described are the cell proliferation assay and the ALP activity assay. A step-by-step protocol is provided here that safely tackles typical difficulties that arise with this 3D cell-scaffold setting. In addition, MPM imaging of cells is described both with and without labeling. The combination of biochemical assays and imaging provides valuable insights into the possibilities of analysis with this 3D cell-scaffold system.

SARS-CoV-2 Serologic Assay Needs for the Next Phase of the US COVID-19 Pandemic Response.

BACKGROUND: There is a need for validated and standardized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quantitative immunoglobulin G (IgG) and neutralization assays that can be used to understand the immunology and pathogenesis of SARS-CoV-2 infection and support the coronavirus disease 2019 (COVID-19) pandemic response. METHODS: Literature searches were conducted to identify English language publications from peer-reviewed journals and preprints from January 2020 through November 6, 2020. Relevant publications were reviewed for mention of IgG or neutralization assays for SARS-CoV-2, or both, and the methods of reporting assay results. RESULTS: Quantitative SARS-CoV-2 IgG results have been reported from a limited number of studies; most studies used in-house laboratory-developed tests in limited settings, and only two semiquantitative tests have received US Food and Drug Administration (FDA) Emergency Use Authorization (EUA). As of November 6, 2020, there is only one SARS-CoV-2 neutralization assay with FDA EUA. Relatively few studies have attempted correlation of quantitative IgG titers with neutralization results to estimate surrogates of protection. The number of individuals tested is small compared with the magnitude of the pandemic, and persons tested are not representative of disproportionately affected populations. Methods of reporting quantitative results are not standardized to enable comparisons and meta-analyses. CONCLUSIONS: Lack of standardized SARS-CoV-2 quantitative IgG and neutralization assays precludes comparison of results from published studies. Interassay and interlaboratory validation and standardization of assays will support efforts to better understand antibody kinetics and longevity of humoral immune responses postillness, surrogates of immune protection, and vaccine immunogenicity and efficacy. Public-private partnerships could facilitate realization of these advances in the United States and worldwide.

Laboratory Monitoring of Direct Oral Anticoagulants (DOACs).

The introduction of direct oral anticoagulants (DOACs), such as dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban, provides safe and effective alternative to previous anticoagulant therapies. DOACs directly, selectively, and reversibly inhibit factors IIa or Xa. The coagulation effect follows the plasma concentration-time profile of the respective anticoagulant. The short half-life of a DOAC constrains the daily oral intake. Because DOACs have predictable pharmacokinetic and pharmacodynamic responses at a fixed dose, they do not require monitoring. However in specific clinical situations and for particular patient populations, testing may be helpful for patient management. The effect of DOACs on the screening coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) is directly linked to reagent composition, and clotting time can be different from reagent to reagent, depending on the DOAC's reagent sensitivity. Liquid chromatography-mass spectrometry (LC-MS/MS) is considered the gold standard method for DOAC measurement, but it is time consuming and requires expensive equipment. The general consensus for the assessment of a DOAC is clotting or chromogenic assays using specific standard calibrators and controls. This review provides a short summary of DOAC properties and an update on laboratory methods for measuring DOACs.

Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2.

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

Novel RT-ddPCR assays for simultaneous quantification of multiple noncoding and coding regions of SARS-CoV-2 RNA.

A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.

Evaluation of analytical performance of qualitative and semi-quantitative assays in the clinical laboratory.

As is true for quantitative assays, qualitative and semi-quantitative assays, producing strict binary or ordinal results, must undergo a verification process prior to their implementation for routine clinical laboratory testing. Standard method validation parameters used for quantitative assays, however, do not apply here. Rather, contingency tables, Bayesian statistics and statistical hypothesis testing for inter-rater agreement must be used. This article provides an overview of simple, practical tools, which can be used to verify the analytical performance of such assays. Topics discussed include the verification of precision and accuracy with a single experiment approach and performing method comparison experiments for assays with binary or ordinal results. Acceptance criteria are recommended for each test to provide a standardized framework for performance assessment. The approach is appropriate for all CE/IVD-marked and CLIA-waived assays and will ensure compliance with CAP, ISO 17025 and ISO 15189 regulations.

Kidney Smartphone Diagnostics.

Here we present a method for a mobile point-of-care (POC) testing of urinary albumin concentration, a biomarker of kidney damage and cardiovascular disease. The self-testing strips are meant to be interpreted by means of a smartphone application. The limits of detection range from 0.15 to 0.30 g/L urinary albumin, though results below 0.10 g/L are presented in a quantitative manner and estimates larger than this threshold are shown as categorical variables in a qualitative manner for increasing urinary albumin concentrations. Calibrated once under standard conditions, the app enables the user to capture problem samples and calculate the corresponding concentration. Negative and positive findings must be interpreted, taking into account the inherent limitations of the method, and professional health advice must be requested for diagnostic considerations. Acknowledgment of the association between early life nutrition and long-term renal health and the adoption of preventive strategies targeting high-risk groups is key for the reduction of the burden of chronic kidney disease on a global scale.

Hormonal Smartphone Diagnostics.

Mobile point-of-care diagnostics are paramount for the provision of healthcare. Hormonal diagnostics are powerful tools to monitor timely changes in human physiology. Hormone concentrations in serum directly correlate with urine excretions with minor time delays. Therefore, rapid tests for hormones in urine have been widely used for decades as means of early diagnostics, particularly in lateral flow immunoassay formats. However, the challenge of reading and interpreting these binary tests remains. Here we present a method for utilizing mobile technologies to quantitatively read and interpret hormonal test strips. The method demonstrates the detection of a urinary by-product of progesterone, pregnanediol glucuronide (PdG), and its relation to ovulation and the fertility cycle.

Recent Advances and Trends in Microfluidic Platforms for C. elegans Biological Assays.

Microfluidics has proven to be a key tool in quantitative biological research. The C. elegans research community in particular has developed a variety of microfluidic platforms to investigate sensory systems, development, aging, and physiology of the nematode. Critical for the growth of this field, however, has been the implementation of concurrent advanced microscopy, hardware, and software technologies that enable the discovery of novel biology. In this review, we highlight recent innovations in microfluidic platforms used for assaying C. elegans and discuss the novel technological approaches and analytic strategies required for these systems. We conclude that platforms that provide analytical frameworks for assaying specific biological mechanisms and those that take full advantage of integrated technologies to extract high-value quantitative information from worm assays are most likely to move the field forward.

Multiplex Smartphone Diagnostics.

Increasing computing power in smartphones allows for their transformation into point-of-care diagnostic devices. Mobile medical diagnostic applications enable utilization of the processing capabilities of smartphones through their cameras. Hardware attachments or stand-alone versions of smartphone diagnostics have the capability to revolutionize quantitative readouts. Here, we describe a protocol for quantifying commercial colorimetric diagnostic tests with a stand-alone smartphone application. This approach can be used in the multiplexed analyses of biomarker readouts.

Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.

The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations.