Analytical performance specifications based on biological variation data - considerations, strengths and limitations.

Analytical performance specifications (APS) are typically established through one of three models: (i) outcome studies, (ii) biological variation (BV), or (iii) state-of-the-art. Presently, The APS can, for most measurands that have a stable concentration, be based on BV. BV based APS, defined for imprecision, bias, total allowable error and allowable measurement uncertainty, are applied to many different processes in the laboratory. When calculating APS, it is important to consider the different APS formulae, for what setting they are to be applied and if they are suitable for the intended purpose. In this opinion paper, we elucidate the background, limitations, strengths, and potential intended applications of the different BV based APS formulas. When using BV data to set APS, it is important to consider that all formulae are contingent on accurate and relevant BV estimates. During the last decade, efficient procedures have been established to obtain reliable BV estimates that are presented in the EFLM biological variation database. The database publishes detailed BV data for numerous measurands, global BV estimates derived from meta-analysis of quality-assured studies of similar study design and automatic calculation of BV based APS.

Defining allowable total error limits in the clinical laboratory.

Allowable total error (ATE) are performance specification limits predefined for a variety of laboratory analytes. These limits define the maximum amount of error that is allowed for an assay when judging acceptability of a new assay during method verification/validation, evaluating patient or instrument comparison data, or in designing a quality control strategy. There are several widely available resources and models that can serve as a guide in selecting ATE. They may be based on legal requirements or set by providers of proficiency testing (PT) and external quality assessment schemes (EQAS). ATE can be also determined by professional expert groups or be based on biological variation of an analyte. Because there are several resources to choose from, there have been several attempts in reaching consensus on which ATE resource should be given preference. This chapter reviews several of these resources in more detail and discusses the difference between allowable total error (ATE) and observed total analytical error (TAE).

Lack of observed interference by therapeutic monoclonal antibodies in select commonly utilized immunoassays.

BACKGROUND: Therapeutic monoclonal antibodies (tmabs) have been hypothesized to interfere with immunoassay measurements, although studies investigating this potential new class of interference are lacking. This study evaluated the effects of tmabs used in cancers ipilimumab (Bristol Myers Squibb), nivolumab (Bristol Myers Squibb), pembrolizumab (Merck) and autoimmune disorders adalimumab (AbbVie), infliximab (Janssen) and vedolizumab (Takeda) in common immunoassays used in the clinical laboratory. METHODS: Residual sera from 10 randomly chosen patients were split into two tubes and spiked with same volume (approximately 5 % final volume) of either saline (control) or 6 tmabs (final concentration of 100 µg/mL each). Concentrations from sixteen analytes in 19 different assays were assessed: TSH (Roche and Beckman), free thyroxine (Roche and Siemens), cortisol (Beckman), Cancer Antigens (CA): CA19-9 (Beckman), CA15-3 (Roche), CA125 (Roche), and CA27.29 (Siemens), carcinoembryonic antigen (Beckman), alpha-fetoprotein (Beckman), thyroglobulin (Beckman) and thyroglobulin antibodies (Beckman), thyroid peroxidase antibody (Beckman), beta-human chorionic gonadotropin (Roche and Beckman), total prostate-specific antigen (Roche), parathyroid hormone (Roche) and antinuclear antibodies IgG (Werfen). The tmab spiked residual sera were compared with matched saline spiked sera and percent error was assessed against allowable total error defined from biological variation or CLIA limits. RESULTS: None of the tested immunoassays were affected by the presence of the tmabs, in samples within or outside assay reference intervals. The median % error among all immunoassays ranged between -2.0% (for TSH) to 2.7% (for TPO Ab assay). CONCLUSION: These findings demonstrate no detectable tmab interference for the assessed immunoassays using spiked preparations of the tmabs in residual human sera. The findings are limited to the tmabs and immunoassays studied here.

Pitfalls in Diagnosing Hypoglycemia Due to Exogenous Insulin: Validation and Utility of an Insulin Analog Assay.

OBJECTIVE: To overcome the limitations of commercially available insulin immunoassays which have variable detection of analog insulin and can lead to clinically discordant results and misdiagnosis in the workup of factitious hypoglycemia. PATIENTS AND METHODS: We performed analytical validation of a liquid chromatography high resolution accurate mass (LC-HRAM) immunoassay to detect insulin analogs. We completed clinical assessment using a large cohort of human serum samples from 78 unique individuals, and subsequently used the assay in the evaluation of eight individuals with high diagnostic suspicion for factitious hypoglycemia. RESULTS: The performance characteristics show that the LC-HRAM immunoassay can be applied to detect five commonly used synthetic insulin analogs (lispro, glulisine, aspart, glargine metabolite, and detemir) in human serum. Our clinical cases show that this assay could be used in the diagnosis of factitious hypoglycemia by identifying the analog insulin(s) in question. CONCLUSION: The LC-HRAM immunoassay reported here overcomes a gap in our diagnostic pathway for hypoglycemia. The results obtained from our studies suggest that this method is appropriate for use in clinical laboratories when factitious hypoglycemia is considered as a differential diagnosis.

Flower lose, a cell fitness marker, predicts COVID-19 prognosis.

Risk stratification of COVID-19 patients is essential for pandemic management. Changes in the cell fitness marker, hFwe-Lose, can precede the host immune response to infection, potentially making such a biomarker an earlier triage tool. Here, we evaluate whether hFwe-Lose gene expression can outperform conventional methods in predicting outcomes (e.g., death and hospitalization) in COVID-19 patients. We performed a post-mortem examination of infected lung tissue in deceased COVID-19 patients to determine hFwe-Lose's biological role in acute lung injury. We then performed an observational study (n = 283) to evaluate whether hFwe-Lose expression (in nasopharyngeal samples) could accurately predict hospitalization or death in COVID-19 patients. In COVID-19 patients with acute lung injury, hFwe-Lose is highly expressed in the lower respiratory tract and is co-localized to areas of cell death. In patients presenting in the early phase of COVID-19 illness, hFwe-Lose expression accurately predicts subsequent hospitalization or death with positive predictive values of 87.8-100% and a negative predictive value of 64.1-93.2%. hFwe-Lose outperforms conventional inflammatory biomarkers and patient age and comorbidities, with an area under the receiver operating characteristic curve (AUROC) 0.93-0.97 in predicting hospitalization/death. Specifically, this is significantly higher than the prognostic value of combining biomarkers (serum ferritin, D-dimer, C-reactive protein, and neutrophil-lymphocyte ratio), patient age and comorbidities (AUROC of 0.67-0.92). The cell fitness marker, hFwe-Lose, accurately predicts outcomes in COVID-19 patients. This finding demonstrates how tissue fitness pathways dictate the response to infection and disease and their utility in managing the current COVID-19 pandemic.

Challenges with At-home and Mail-in Direct-to-Consumer Testing: Preanalytical Error, Reporting Results, and Result Interpretation.

Companies that offer direct-to-consumer (DTC) testing on specimens such as saliva, blood, or urine, allow consumers to order laboratory tests without the involvement of a health care provider. This approach allows individuals to have direct access to their own laboratory results, interpret them, and make decisions regarding their health care. However, as with conventional clinical laboratory testing, there are factors that will impact the accuracy of DTC test results and limitations that consumers need to be aware of. This article focuses on challenges with DTC testing specifically related to preanalytical errors, result reporting, and result interpretation.

Forces during cellular uptake of viruses and nanoparticles at the ventral side.

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.

Development of Vitamin D Toxicity from Overcorrection of Vitamin D Deficiency: A Review of Case Reports.

Over the past two decades, vitamin D level measurements have become some of the most frequently ordered tests in the laboratory. This increase is due to a growing awareness of widespread vitamin D deficiency and scientific data suggesting the beneficial effects of vitamin D in various diseases. A literature search was carried out in PubMed for cases reporting vitamin D intoxication and overdose. Thirteen articles were included in this review. Intoxication was severe in the reported cases. Patients presented with serum vitamin D concentrations ranging between 150 and 1220 ng/mL and serum calcium concentrations between 11.1 and 23.1 mg/dL. Most of the reported patients showed symptoms of vitamin D toxicity such as vomiting, dehydration, pain, and loss of appetite. The underlying causes included manufacturing errors, overdosing by patients or prescribers, and combinations of these factors. Our literature search highlights the fact that even though vitamin D intoxication is rare, it does occur and therefore patients and prescribers should be more cognizant of the potential dangers of vitamin D overdose.

10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency.

In early 2000's vitamin-D deficiency was shown to be prevalent in several countries including the United States (US). Studies exploring the role of vitamin-D metabolism in diverse disease pathways generated an increased demand for vitamin-D supplementation and an immense public interest in measurement of vitamin-D metabolite levels. In this report, we review the role of vitamin-D metabolism in disease processes, clinical utility of measuring vitamin-D metabolites including 25-hydroxyvitamin-D (25(OH)D), 1,25-dihydroxyvitamin-D and 24,25-dihydroxyvitamin-D and discuss vitamin-D assay methodologies including immunoassays and liquid chromatography mass spectrometry (LC-MS/MS) assays. We also provide examples of vitamin-D toxicity and insight into the trends in serum 25(OH)D levels in the US population based on 10 years of data from on serum 25(OH)D values from ~5,000,000 patients who were tested at the Mayo Medical Laboratories between February 2007-February 2017.

Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step toward Personalized Mechanomedicine.

Given that dysregulation of mechanics contributes to diseases ranging from cancer metastasis to lung disease, it is important to develop methods for screening the efficacy of drugs that target cellular forces. Here, nanoparticle-based tension sensors are used to quantify the mechanical response of individual cells upon drug treatment. As a proof-of-concept, the activity of bronchodilators is tested on human airway smooth muscle cells derived from seven donors, four of which are asthmatic. It is revealed that airway smooth muscle cells isolated from asthmatic donors exhibit greater traction forces compared to the control donors. Additionally, the mechanical signal is abolished using myosin inhibitors or further enhanced in the presence of inflammatory inducers, such as nicotine. Using the signal generated by the probes, single-cell dose-response measurements are performed to determine the "mechano" effective concentration (mechano-EC50 ) of albuterol, a bronchodilator, which reduces integrin forces by 50%. Mechano-EC50 values for each donor present discrete readings that are differentially enhanced as a function of nicotine treatment. Importantly, donor mechano-EC50 values varied by orders of magnitude, suggesting significant variability in their sensitivity to nicotine and albuterol treatment. To the best of the authors' knowledge, this is the first study harnessing a piconewton tension sensor platform for mechanopharmacology.

Light-Responsive Polymer Particles as Force Clamps for the Mechanical Unfolding of Target Molecules.

Single-molecule force spectroscopy techniques are powerful tools for investigating the mechanical unfolding of biomolecules. However, they are limited in throughput and require dedicated instrumentation. Here, we report a force-generating particle that can unfold target molecules on-demand. The particle consists of a plasmonic nanorod core encapsulated with a thermoresponsive polymer shell. Optical heating of the nanorod leads to rapid collapse of the polymer, thus transducing light into mechanical work to unfold target molecules. The illumination tunes the duration and degree of particle collapse, thus controlling the lifetime and magnitude of applied forces. Single-molecule fluorescence imaging showed reproducible mechanical unfolding of DNA hairpins. We also demonstrate the triggering of 50 different particles in <1 min, exceeding the speed of conventional atomic force microscopy. The polymer force clamp represents a facile and bottom-up approach to force manipulation.

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.

Molecular Tension Probes for Imaging Forces at the Cell Surface.

Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration, and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T- or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction. Upon ligation, these receptor-ligand bonds transmit piconewton (pN) mechanical forces that are generated, in part, by the cytoskeleton. Importantly, these forces expose cryptic sites within mechanosensitive proteins and modulate the binding kinetics (on/off rate) of receptor-ligand complexes to further fine-tune mechanotransduction and the corresponding cell behavior. Over the past three decades, two categories of methods have been developed to measure cell receptor forces. The first class is traction force microscopy (TFM) and micropost array detectors (mPADs). In these methods, cells are cultured on elastic polymers or microstructures that deform under mechanical forces. The second category of techniques is single molecule force spectroscopy (SMFS) including atomic force microscopy (AFM), optical or magnetic tweezers, and biomembrane force probe (BFP). In SMFS, the experimenter applies external forces to probe the mechanics of individual cells or single receptor-ligand complexes, serially, one bond at a time. Although these techniques are powerful, the limited throughput of SMFS and the nN force sensitivity of TFM have hindered further elucidation of the molecular mechanisms of mechanotransduction. In this Account, we introduce the recent advent of molecular tension fluorescence microscopy (MTFM) as an emerging tool for molecular imaging of receptor mechanics in living cells. MTFM probes are composed of an extendable linker, such as polymer, oligonucleotide, or protein, and flanked by a fluorophore and quencher. By measuring the fluorescence emission of immobilized MTFM probes, one can infer the extension of the linker and the externally applied force. Thus, MTFM combines aspects of TFM and SMFS to optically report receptor forces across the entire cell surface with pN sensitivity. Specifically, we provide an in-depth review of MTFM probe design, which includes the extendable "spring", spectroscopic ruler, surface immobilization chemistry, and ligand design strategies. We also demonstrate the strengths and weaknesses of different versions of MTFM probes by discussing case studies involving the pN forces involved in epidermal growth factor receptor, integrin, and T-cell receptor signaling pathways. Lastly, we present a brief future outlook, primarily from a chemists' perspective, on the challenges and opportunities for the design of next generation MTFM probes.

Exercise Increases and Browns Muscle Lipid in High-Fat Diet-Fed Mice.

Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks (p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks (p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise regardless of diet. Plin5 was increased with HFD and unaffected by exercise; the respiratory exchange ratio was 15% lower in the 18-week HFD group compared with CTL (p < 0.001) and 10% lower in 18 weeks HFD-E compared with CTL-E (p < 0.001). Increased Ucp1 and Pgc1a in exercised muscle of running mice suggests that a beige/brown fat phenotype develops, which differs from the fat phenotype that induces insulin resistance in high fat feeding. This suggests that increased muscle lipid may develop a "brown" phenotype in the setting of endurance exercise training, a shift that is further promoted by HFD.

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Mechanically Induced Catalytic Amplification Reaction for Readout of Receptor-Mediated Cellular Forces.

Mechanics play a fundamental role in cell biology, but detecting piconewton (pN) forces is challenging because of a lack of accessible and high throughput assays. A mechanically induced catalytic amplification reaction (MCR) for readout of receptor-mediated forces in cells is described. Mechanically labile DNA duplexes presenting ligands are surface immobilized such that specific receptor forces denature the duplex and thus expose a blocked primer. Amplification of primers is achieved using an isothermal polymerization reaction and quantified by fluorescence readout. As a proof of concept, the assay was used to test the activity of a mechanomodulatory drug and integrin adhesion receptor antibodies. To the best of our knowledge, this is the first example of a catalytic reaction triggered in response to molecular piconewton forces. The MCR may transform the field of mechanobiology by providing a new facile tool to detect receptor specific mechanics with the convenience of the polymerase chain reaction (PCR).

A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface.

Mechanical forces between cells and their extracellular matrix (ECM) are mediated by dozens of different receptors. These biophysical interactions play fundamental roles in processes ranging from cellular development to tumor progression. However, mapping the spatial and temporal dynamics of tension among various receptor-ligand pairs remains a significant challenge. To address this issue, we have developed a synthetic strategy to generate modular tension probes combining the native chemical ligation (NCL) reaction with solid phase peptide synthesis (SPPS). In principle, this approach accommodates virtually any peptide or expressed protein amenable to NCL. We generated a small library of tension probes displaying different ligands, flexible linkers, and fluorescent reporters, enabling the mapping of integrin and cadherin tension, and demonstrating the first example of long-term (∼3 days) molecular tension imaging. This approach provides a toolset to better understand mechanotransduction events fundamental to cell biology.

Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions.

Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30-40 pN. The addition of a disulfide bridge within I27 "clamps" the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S-S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S-S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported.

Exercise Regulation of Marrow Fat in the Setting of PPARγ Agonist Treatment in Female C57BL/6 Mice.

The contribution of marrow adipose tissue (MAT) to skeletal fragility is poorly understood. Peroxisome proliferator-activated receptor (PPAR)γ agonists, associated with increased fractures in diabetic patients, increase MAT. Here, we asked whether exercise could limit the MAT accrual and increase bone formation in the setting of PPARγ agonist treatment. Eight-week-old female C57BL/6 mice were treated with 20-mg/kg · d rosiglitazone (Rosi) and compared with control (CTL) animals. Exercise groups ran 12 km/d when provided access to running wheels (CTL exercise [CTL-E], Rosi-E). After 6 weeks, femoral MAT (volume of lipid binder osmium) and tibial bone morphology were assessed by microcomputer tomography. Rosi was associated with 40% higher femur MAT volume compared with CTL (P < .0001). Exercise suppressed MAT volume by half in CTL-E mice compared with CTL (P < .01) and 19% in Rosi-E compared with Rosi (P < .0001). Rosi treatment increased fat markers perilipin and fatty acid synthase mRNA by 4-fold (P < .01). Exercise was associated with increased uncoupling protein 1 mRNA expression in both CTL-E and Rosi-E groups (P < .05), suggestive of increased brown fat. Rosi increased cortical porosity (P < .0001) but did not significantly impact trabecular or cortical bone quantity. Importantly, exercise induction of trabecular bone volume was not prevented by Rosi (CTL-E 21% > CTL, P < .05; Rosi-E 26% > Rosi, P < .01). In summary, despite the Rosi induction of MAT extending well into the femoral diaphysis, exercise was able to significantly suppress MAT volume and induce bone formation. Our results suggest that the impact of PPARγ agonists on bone and marrow health can be partially mitigated by exercise.

Nanoparticle tension probes patterned at the nanoscale: impact of integrin clustering on force transmission.

Herein we aimed to understand how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We combined molecular tension fluorescence microscopy with block copolymer micelle nanolithography to fabricate substrates with arrays of precisely spaced probes that can generate a 10-fold fluorescence response to pN-forces. We found that the mechanism of sensing ligand spacing is force-mediated. This strategy is broadly applicable to investigating receptor clustering and its role in mechanotransduction pathways.