Paper-Based Cytometer for the Detection and Enumeration of White Blood Cells According to Their Immunophenotype.

Total and differential white blood cell (WBC) counts are vital metrics used routinely by clinicians to aid in the identification of diseases. However, the equipment necessary to perform WBC counts restricts their operation to centralized laboratories, greatly limiting their accessibility. Established solutions for the development of point-of-care assays, namely lateral flow tests and paper-based microfluidic devices, are inherently limited in their ability to support the detection of WBCs─the pore sizes of materials used to fabricate these devices (e.g., membranes or chromatography papers) do not permit passive WBC transport via wicking. Herein, we identify a material capable of the unimpeded transport of WBCs in both lateral and vertical directions: a coffee filter. Through in situ labeling with an enzyme-labeled affinity reagent, our paper-based cytometer detects WBCs according to their immunophenotype. Using two cultured leukocyte lines (Jurkat D1.1 T cells and MAVER-1 B cells), we demonstrate the specific, colorimetric enumeration of each target cell population across the expected physiological range for total lymphocytes, 1000-4000 cells µL-1. Additionally, we highlight a potential application of this type of device as a screening tool for detecting abnormal cell counts outside the normal physiological range and in subclasses of cell types, which could aid in the identification of certain diseases (e.g., CD4+ T lymphocytes, an important biomarker for HIV disease/AIDS). These results pave the way for a new class of paper-based devices─those capable of controlled white blood cell transport, labeling, capture, and detection─thus expanding the opportunities for low-cost, point-of-care cytometers.

Carbon conundrums: Do United States' current carbon market baselines represent an undesirable ecological threshold?

Relative frequency distribution of observed annual mortality expressed in aboveground (AG) carbon (C) (Mg CO2 e ha-1 year-1 ) summarized across supersections by forest type [Hardwood (HW) vs. Softwood (SW)] and site class (Low vs. High) based on approximately 130,000 remeasured USDA Forest Service Forest Inventory and Analysis plots across the US. Top panel summarizes conditions in plots that do and do not meet the California Air Resources Board standards based on total basal area, whereas bottom panel summarizes conditions in plots falling inside and outside of optimum relative density levels. The latter represents a biophysically-informed approach accounting for changes in tree (and carbon) packing over forest development.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP-KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3-to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3-25 nM) exhibited the lowest limits of detection (ca. µg mL-1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results.

High-Yielding Separation and Collection of Plasma from Whole Blood Using Passive Filtration.

Lateral flow tests and hand-held analyzers facilitate diagnostic testing in resource limited settings and at the point-of-care. However, many of these devices require sample preparation such as plasma separation to remove cells and isolate the liquid portion of blood. Specifically, the separation of plasma from blood is necessary for routine health assessments such as comprehensive metabolic panels and chronic HIV viral load monitoring. Away from laboratories, this type of processing has been addressed by unconventional, hand-operated centrifuge devices (high volume) or plasma separation membranes (PSM) coupled with lateral flow tests (low volume). Herein, we describe a device that separates and stores plasma from undiluted blood using only passive filtration in less than 10 min. Integrating a PSM with a prefilter and absorbent material yields a 3-fold increase in separation efficiency compared to similar devices using passive filtration. We demonstrate the reproducibility of our device across the physiological range of hematocrits (20-50%) with an average recovered plasma volume of 61.7 ± 2.6 µL. Maximum separation efficiency (53.8%, 65.6 ± 3.9 µL plasma) was achieved for a sample of whole blood (30% hematocrit) in 10 min. We evaluate the purity of our plasma sample by quantitation of hemoglobin and report hemolysis as either minimal (≤5%) or undetectable (≤1%). Specific recovery of human IgG, IFN-γ, and HIV-1 RNA indicate the diagnostic utility of plasma obtained from our device is unchanged compared to plasma obtained via centrifugation. Finally, we demonstrate the use of recovered plasma, applied via "stamping", to successfully conduct a commercial lateral flow immunochromatographic assay for tetanus antibodies. This device platform is capable of producing pure plasma samples from blood to facilitate tests in resource limited settings to improve access to healthcare.

Usability as a guiding principle for the design of paper-based, point-of-care devices - A review.

Due to their portability, versatility for supporting multiple assay formats, and potential for resulting in low-cost assays, paper-based analytical devices (PADs) are an increasingly popular format as a platform for the development of point-of-care tests. However, very few PADs have been translated successfully to their intended environments outside of academic settings. Often overlooked as a factor that inhibits translation, usability is a vital characteristic of any successful point-of-care test. Recent advancements in PAD design have demonstrated improved usability by simplifying various aspects of user operation, including sample collection, sample processing, device operation, detection, and readout/interpretation. Field testing at various stages of device design can offer critical feedback about device usability, especially when it involves the proposed end-user or other stakeholders. By highlighting advances in usability, we aim to encourage thoughtful and rigorous design at the academic prototyping stage to address one outstanding hurdle that limits the number of PADs that make it from the benchtop to the point-of-care.

Global carbon dioxide removal rates from forest landscape restoration activities.

BACKGROUND: Forest landscape restoration (FLR) has been adopted by governments and practitioners across the globe to mitigate and adapt to climate change and restore ecological functions across degraded landscapes. However, the extent to which these activities capture CO2 with associated climate mitigation impacts are poorly known, especially in geographies where data on biomass growth of restored forests are limited or do not exist. To fill this gap, we developed biomass accumulation rates for a set of FLR activities (natural regeneration, planted forests and woodlots, agroforestry, and mangrove restoration) across the globe and global CO2 removal rates with corresponding confidence intervals, grouped by FLR activity and region/climate. RESULTS: Planted forests and woodlots were found to have the highest CO2 removal rates, ranging from 4.5 to 40.7 t CO2 ha-1 year-1 during the first 20 years of growth. Mangrove tree restoration was the second most efficient FLR at removing CO2, with growth rates up to 23.1 t CO2 ha-1 year-1 the first 20 years post restoration. Natural regeneration removal rates were 9.1-18.8 t CO2 ha-1 year-1 during the first 20 years of forest regeneration, followed by agroforestry, the FLR category with the lowest and regionally broad removal rates (10.8-15.6 t CO2 ha-1 year-1). Biomass growth data was most abundant and widely distributed across the world for planted forests and natural regeneration, representing 45% and 32% of all the data points assessed, respectively. Agroforestry studies, were only found in Africa, Asia, and the Latin America and Caribbean regions. CONCLUSION: This study represents the most comprehensive review of published literature on tree growth and CO2 removals to date, which we operationalized by constructing removal rates for specific FLR activities across the globe. These rates can easily be applied by practitioners and decision-makers seeking to better understand the positive climate mitigation impacts of existing or planned FLR actions, or by countries making restoration pledges under the Bonn Challenge Commitments or fulfilling Nationally Determined Contributions to the UNFCCC, thereby helping boost FLR efforts world-wide.

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings.

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/µL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

Greenhouse gas emissions from tropical forest degradation: an underestimated source.

BACKGROUND: The degradation of forests in developing countries, particularly those within tropical and subtropical latitudes, is perceived to be an important contributor to global greenhouse gas emissions. However, the impacts of forest degradation are understudied and poorly understood, largely because international emission reduction programs have focused on deforestation, which is easier to detect and thus more readily monitored. To better understand and seize opportunities for addressing climate change it will be essential to improve knowledge of greenhouse gas emissions from forest degradation. RESULTS: Here we provide a consistent estimation of forest degradation emissions between 2005 and 2010 across 74 developing countries covering 2.2 billion hectares of forests. We estimated annual emissions of 2.1 billion tons of carbon dioxide, of which 53% were derived from timber harvest, 30% from woodfuel harvest and 17% from forest fire. These percentages differed by region: timber harvest was as high as 69% in South and Central America and just 31% in Africa; woodfuel harvest was 35% in Asia, and just 10% in South and Central America; and fire ranged from 33% in Africa to only 5% in Asia. Of the total emissions from deforestation and forest degradation, forest degradation accounted for 25%. In 28 of the 74 countries, emissions from forest degradation exceeded those from deforestation. CONCLUSIONS: The results of this study clearly demonstrate the importance of accounting greenhouse gases from forest degradation by human activities. The scale of emissions presented indicates that the exclusion of forest degradation from national and international GHG accounting is distorting. This work helps identify where emissions are likely significant, but policy developments are needed to guide when and how accounting should be undertaken. Furthermore, ongoing research is needed to create and enhance cost-effective accounting approaches.