Microfluidic cytometer for the characterization of cell lysis.

Blood cytometry and intercellular analysis typically requires lysis as a preparatory step, which can alter the results of downstream analyses. We fabricated a microfluidic cytometer to characterize erythrocyte lysis kinetics. Forward light scatter from erythrocytes was used for enumeration at specific locations on a microfluidic chip. Diffusive transport coupled with laminar flow was used to control the concentration and exposure time of the lysis reagent Zap-OGLOBIN II to erythrocytes. Standard clinical practice is to expose erythrocytes to lysis reagent for 10 min. Under optimum conditions, we achieved complete erythrocyte lysis of a blood sample in 0.7 s. A maximum lysis reaction rate of 1.55 s(-1) was extrapolated from the data. Lysis began after 0.2 s and could be initiated with a lysis reagent concentration of 1.0% (68.5 mM). An equation that related lysis reagent concentration, [A], to erythrocyte lysis, [B], was determined to be [B] = -0.77[A](0.29)t.

Evaluation of Intradermal and Subcutaneous Infusion Set Performance Under 24-Hour Basal and Bolus Conditions.

BACKGROUND: This study sought to assess the function and delivery reliability of intradermal (ID) infusion sets used with commercial insulin pumps. METHOD: Healthy subjects (n = 43) were randomized to either ID or subcutaneous (SC) arms, and received basal/bolus placebo delivery for 24 hours. Subjects received 4 of 8 infusion set combinations (ID: microneedle design A or B, with 2 pump brands [Animas or MiniMed]; SC: Teflon Quickset or steel Rapid-D, Animas pump only, with or without overtaping) and were evaluated for pump occlusion alarms, fluid leakage, pain, and tissue tolerability. A novel algorithm was developed to determine flow consistency based on fluid pressure, and the duration and occurrence rate for periods of unalarmed but interrupted flow ("silent occlusions'") were compared. RESULTS: ID delivery was successfully maintained over the 24-hour infusion period. The number of silent occlusions was lower for ID microneedle cannula design B than A (P < .01) and lower for Rapid-D SC device compared to Quick-set (P = .03). There was no significant difference in the number of occlusion alarms between the ID and SC devices with the Animas pump. However, the pumps tested with ID devices had significantly different alarm rates (MiniMed 29.5%, Animas 0%, P < .001). Leakage and tissue tolerability were comparable across devices. CONCLUSION: The ID infusion set reliably delivered diluent for an extended 24-hour period in healthy subjects and was well tolerated. Silent occlusion flow interruptions could be detected in both ID and SC infusion sets using a proprietary algorithm. This algorithm is a promising method for quantitatively evaluating infusion set flow performance.

Characterizing normal-use temperature conditions of pumped insulin.

In this study, the temperature profiles of insulin pump reservoirs during normal wear conditions across multiple seasons were characterized. Thermocouples secured in reservoirs filled with insulin diluent were loaded in infusion pumps worn by volunteers. Reservoir and ambient environmental temperature data and activity levels were logged during the course of normal daily activities in February (winter), April (spring), and August (summer). Each seasonal data set comprised 7 to 14 days of wear from 3 to 5 volunteers. Reservoir temperature profiles were generally higher than ambient temperatures, likely due to heat transfer from the wearer when the pump was placed close to the body. Temperature conditions inside pump reservoirs fluctuated between 25°C and 37°C regardless of seasonal variations. The average reservoir temperature remained close to 30°C across all seasons, notably lower than used in previously published compatibility and stability protocols (37°C). Results from this study could be utilized to develop more accurate stability and compatibility testing procedures for new insulin formulations and/or delivery devices.

Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells.

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.