Search

Basophils are the rarest circulating white blood cells (WBCs), but they play important roles in allergic disorders and other diseases. To enhance diagnostic capabilities, it would be desirable to isolate and analyze basophils efficiently from small blood samples. In 100 μL of whole blood, there are typically ≈103 basophils, outnumbered by ≈105 WBCs and ≈108 red blood cells (RBCs). Basophils' low abundance has therefore presented a significant challenge in their isolation from whole blood. Conventional in‐bulk basophil isolation methods require lengthy processing steps and cannot work with small volumes of blood. Herein, a parallelized integrated basophil isolation device (pi‐BID) is reported for the negative immunomagnetic selection of basophils directly from four samples of 100 μL of whole blood, in parallel, within 14 min including sample preparation time. The pi‐BID interfaces directly with standard sample tubes, and uses a single pressure source to drive the flow in parallel microfluidic channels. Compared with conventional in‐bulk basophil isolation, the pi‐BID is >3× faster, and has higher purity (≈93%) and similar recovery (≈67%). Compared with other microfluidic devices for the immunomagnetic isolation of WBC subtypes, the pi‐BID achieves 10× higher enrichment of target cells from whole blood, with no prior removal of RBCs necessary. [ABSTRACT FROM AUTHOR]

Background: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore‐labeled avidin, a positively‐charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. Methods: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01–10,000 ng/mL) and four control conditions (no stimulant, anti‐IgE, fMLP (N‐formylmethionine‐leucyl‐phenylalanine), and anti‐FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. Results: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+/CD63− cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. Conclusions: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy. [ABSTRACT FROM AUTHOR]

Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 μL of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9% ± 3.6% and recovery of 95.6% ± 3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment. [ABSTRACT FROM AUTHOR]

Ion concentration polarization (ICP) accomplishes preconcentration for bioanalysis by localized depletion of electrolyte ions, thereby generating a gradient in electric field strength that facilitates electrokinetic focusing of charged analytes by their electromigration against opposing fluid flow. Such ICP focusing has been shown to accomplish up to a million-fold enrichment of nucleic acids and proteins in single-stage preconcentrators. However, the rate at which the sample volume is swept is limited, requiring several hours to achieve these high enrichment factors. This limitation is caused by two factors. First, an ion depleted zone (IDZ) formed at a planar membrane or electrode may not extend across the full channel cross section under the flow rate employed for focusing, thereby allowing the analyte to "leak" past the IDZ. Second, within the IDZ, large fluid vortices lead to mixing, which decreases the efficiency of analyte enrichment and worsens with increased channel dimensions. Here, we address these challenges with faradaic ICP (fICP) at a three-dimensional (3D) electrode comprising metallic microbeads. This 3D-electrode distributes the IDZ, and therefore, the electric field gradient utilized for counter-flow focusing across the full height of the fluidic channel, and its large area, microstructured surface supports smaller vortices. An additional bed of insulating microbeads restricts flow patterns and supplies a large area for surface conduction of ions through the IDZ. Finally, the resistance of this secondary bed enhances focusing by locally strengthening sequestering forces. This easy-to-build platform lays a foundation for the integration of enrichment with user-defined packed bed and electrode materials. [ABSTRACT FROM AUTHOR]