Maximizing Microsampling: Measurement of Comprehensive Metabolic and Lipid Panels Using a Novel Capillary Blood Collection Device.

BACKGROUND: Demand continues to grow for patient-centric sampling solutions that enable collection of small volumes of blood outside of healthcare facilities. Various technologies have been developed to facilitate sample collection but gaps in knowledge remain, preventing these technologies from replacing standard venipuncture. METHODS: A novel blood collection device, Touch Activated Phlebotomy (TAP) II® from YourBio Health, and standard fingerstick collection using a BD Microtainer® were utilized to collect capillary serum samples. Measurements of a comprehensive metabolic and lipid panels were measured on these samples and compared to results from venous serum samples that were collected in parallel. Hemolysis was used to assess sample quality. Sample volumes obtained from self-collected TAP II samples were also determined. RESULTS: Correlation of capillary serum with respect to venous serum was demonstrated (R > 0.9) for professionally collected TAP II samples, self-collected TAP II samples, and professionally collected fingerstick samples for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, cholesterol, high-density lipoprotein, total bilirubin, and triglycerides. Results for creatinine demonstrated acceptable correlation, however, a consistent negative bias was observed. Biases (with unacceptable correlations) were also observed for measurements of carbon dioxide and potassium. Correlative results for albumin were not consistently acceptable across the collection techniques utilized while the remaining analytes tested did not demonstrate acceptable correlations under any condition. Correlation results, however, would improve with a wider distribution of analyte concentrations. CONCLUSIONS: Collections of small volumes of liquid blood continue to show potential as a patient-centric solution.

Monitoring of SARS-CoV-2 antibodies using dried blood spot for at-home collection.

The utilization of vaccines to fight the spread of SARS-CoV-2 has led to a growing need for expansive serological testing. To address this, an EUA approved immunoassay for detection of antibodies to SARS-CoV-2 in venous serum samples was investigated for use with dried blood spot (DBS) samples. Results from self-collected DBS samples demonstrated a 98.1% categorical agreement to venous serum with a correlation (R) of 0.9600 while professionally collected DBS samples demonstrated a categorical agreement of 100.0% with a correlation of 0.9888 to venous serum. Additional studies were performed to stress different aspects of at-home DBS collection, including shipping stability, effects of interferences, and other sample-specific robustness studies. These studies demonstrated a categorical agreement of at least 95.0% and a mean bias less than ± 20.0%. Furthermore, the ability to track antibody levels following vaccination with the BioNTech/Pfizer vaccine was demonstrated with serial self-collected DBS samples from pre-dose (Day 0) out to 19 weeks.

Empiricism in Microsampling: Utilizing a Novel Lateral Flow Device and Intrinsic Normalization to Provide Accurate and Precise Clinical Analysis from a Finger Stick.

BACKGROUND: Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges. We discuss herein our efforts to overcome these challenges and provide accurate and precise clinical measurements. METHODS: Microsamples of whole blood were collected via finger stick using a collection device employing laminar-flow separation of cellular blood and plasma fractions with subsequent desiccation. Samples were analyzed on modern autoanalyzers with FDA-approved reagent and calibration systems, as well as commercially available reagents with laboratory-developed assay parameters. Measured analyte concentrations from extracted dried plasma samples were normalized to a coextracted endogenous analyte, chloride. RESULTS: Chloride normalization reduced variability incurred through extraction and undefined plasma volume. Excellent correlation of normalized measurements from dried finger-stick samples (whole blood and plasma) versus matched venous samples facilitated developing mathematical transformations to provide concordance between specimen types. Independent end-to-end performance verification yielded mean biases <3% for the 5 analytes evaluated relative to venous drawn samples analyzed on FDA-approved measurement systems. CONCLUSION: Challenges inherent with this microsampling technique and alternate sample matrix were obviated through capabilities of modern autoanalyzers and implementation of chloride normalization. These results demonstrate that self-collected microsamples from a finger stick can give results concordant with those of venous samples.

Automated generation of immature dendritic cells in a single-use system.

Dendritic cells (DCs) are an indispensable part of studying human responses that are important for protective immunity against cancer and infectious diseases as well as prevention of autoimmunity and transplant rejection. These cells are also key elements of personalized vaccines for cancer and infectious diseases. Despite the vital role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remains a comparatively under-developed and inefficient process. DCs are present in very low concentrations (<1%) in blood, thus they must be generated from monocytes and the current methodology in DC generation involves a laborious process of static culture and stimulation with cytokines contained in culture medium. Herein, we describe an automated fluidic system, MicroDEN, that allows for differentiation of monocytes into immature-DCs (iDCs) utilizing continuous perfusion of differentiation media. Manual steps associated with current ex vivo monocyte differentiation are vastly reduced and an aseptic environment is ensured by the use of an enclosed cartridge and tubing network. Benchmark phenotyping was performed on the generated iDCs along with allogeneic T-cell proliferation and syngeneic antigen-specific functional assays. MicroDEN generated iDCs were phenotypically and functionally similar to well plate generated iDCs, thereby demonstrating the feasibility of utilizing MicroDEN in the broad range of applications requiring DCs.

Enzymatic Glucose Sensor Compensation for Variations in Ambient Oxygen Concentration.

Due to the increasing prevalence of diabetes, research toward painless glucose sensing continues. Oxygen sensitive phosphors with glucose oxidase (GOx) can be used to determine glucose levels indirectly by monitoring oxygen consumption. This is an attractive combination because of its speed and specificity. Packaging these molecules together in "smart materials" for implantation will enable non-invasive glucose monitoring. As glucose levels increase, oxygen levels decrease; consequently, the luminescence intensity and lifetime of the phosphor increase. Although the response of the sensor is dependent on glucose concentration, the ambient oxygen concentration also plays a key role. This could lead to inaccurate glucose readings and increase the risk of hyper- or hypoglycemia. To mitigate this risk, the dependence of hydrogel glucose sensor response on oxygen levels was investigated and compensation methods explored. Sensors were calibrated at different oxygen concentrations using a single generic logistic equation, such that trends in oxygen-dependence were determined as varying parameters in the equation. Each parameter was found to be a function of oxygen concentration, such that the correct glucose calibration equation can be calculated if the oxygen level is known. Accuracy of compensation will be determined by developing an overall calibration, using both glucose and oxygen sensors in parallel, correcting for oxygen fluctuations in real time by intentionally varying oxygen, and calculating the error in actual and predicted glucose levels. While this method was developed for compensation of enzymatic glucose sensors, in principle it can also be implemented with other kinds of sensors utilizing oxidases.

Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet.

Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.

Temperature Compensation of Oxygen Sensing Films Utilizing a Dynamic Dual Lifetime Calculation Technique.

With advances to chemical sensing, methods for compensation of errors introduced by interfering analytes are needed. In this work, a dual lifetime calculation technique was developed to enable simultaneous monitoring of two luminescence decays. Utilizing a windowed time-domain luminescence approach, the response of two luminophores is separated temporally. The ability of the dual dynamic rapid lifetime determination (DDRLD) approach to determine the response of two luminophores simultaneously was investigated through mathematical modeling and experimental testing. Modeling results indicated that lifetime predictions will be most accurate when the ratio of the lifetimes from each luminophore is at least three and the ratio of intensities is near unity. In vitro experiments were performed using a porphyrin that is sensitive to both oxygen and temperature, combined with a temperature-sensitive inorganic phosphor used for compensation of the porphyrin response. In static experiments, the dual measurements were found to be highly accurate when compared to single-luminophore measurements-statistically equivalent for the long lifetime emission and an average difference of 2% for the short lifetimes. Real-time testing with dynamic windowing was successful in demonstrating dual lifetime measurements and temperature compensation of the oxygen sensitive dye. When comparing the actual oxygen and temperature values with predictions made using a dual calibration approach, an overall difference of less than 1% was obtained. Thus, this method enables rapid, accurate extraction of multiple lifetimes without requiring computationally intense curve fitting, providing a significant advancement toward multi-analyte sensing and imaging techniques.

Dynamic windowing algorithm for the fast and accurate determination of luminescence lifetimes.

An algorithm for the accurate calculation of luminescence lifetimes in near-real-time is described. The dynamic rapid lifetime determination (DRLD) method uses a window-summing technique and dynamically selects the appropriate window width for each lifetime decay such that a large range of lifetimes can be accurately calculated. The selection of window width is based on an optimal range of window-sum ratios. The algorithm was compared to alternative approaches for rapid lifetime determination as well as nonlinear least-squares (NLLS) fitting in both simulated and real experimental conditions. A palladium porphyrin was used as a model luminophore to quantitatively evaluate the algorithm in a dynamic situation, where oxygen concentration was modulated to induce a change in lifetime. Unlike other window-summing techniques, the new algorithm calculates lifetimes that are not significantly different than the slower, traditional NLLS. In addition, the computation time required to calculate the lifetime is 4 orders of magnitude less than NLLS and 2 orders less than other iterative methods. This advance will improve the accuracy of real-time measurements that must be made on samples that are expected to exhibit widely varying lifetimes, such as sensors and biosensors.

Microparticle ratiometric oxygen sensors utilizing near-infrared emitting quantum dots.

Luminescent sensors incorporating two luminophores, an indicator and a reference, offer many advantages over intensity measurements from sensors made with one indicator dye. Quantum dots have yet to be widely employed as insensitive reference luminophores in such systems. This work describes the use of near-infrared emitting quantum dots in conjunction with a long-lifetime platinum(II) porphyrin phosphor in a microsphere-based, ratiometric oxygen sensor. The process for self-assembly of the nanocomposite system was developed, and the response and photostability of the prototypes were investigated. Results indicate the sensors possess excellent sensitivity (K(SV) = 0.00826 µM(-1)) at oxygen concentrations below 300 µM and were resistant to photobleaching. The sensor luminophores displayed minimal spectral overlap and little interference from excitation light, preventing the need for optical filters. A reversible photoenhancement of the quantum dot signal was also observed when exposed for extended periods of time. This work demonstrates the advantages of incorporating long-wavelength quantum dots into ratiometric intensity sensing schemes and highlights some key limitations that must be considered in their use.