Labeled leukocyte Imaging: Improved Leukocyte Isolation with a Novel Automated Microfluidics-Based System (2020)

541 Objectives: Autologous human leukocytes (WBCs) labeled with111In-oxine (111In) or 99mTc-exametazime (99mTc) are used to detect infection. Since these radiotracers label all blood cells, WBCs must be separated from red blood cells (RBCs) and platelets before labeling. For decades, combined hydroxyethyl starch augmented gravity sedimentation and centrifugation (conventional method) has been used to isolate WBCs, but this process is labor-intensive, time-consuming, and prone to variability. A novel microfluidic system has been developed that isolates WBCs from 40-50 mL whole blood in <30 min. The objectives of this IRB approved prospective investigation are to compare purity, labeling efficiency, label stability, and viability of WBCs isolated via conventional method to those isolated via microfluidic method for healthy and patient subjects.
 Methods: 80 mL peripheral blood was collected from up to 10 healthy donors on two separate occasions, 48 hrs apart, once for 111In labeling and once for 99mTc labeling. From up to 15 patients with suspected infections, 80 mL peripheral blood was collected on one occasion for labeling with 111In or 99mTc. From each blood draw, 40 mL was processed via conventional method, with a fixed separation time of 90 min, and 40 mL was processed via microfluidic isolation. WBC viability using trypan blue and WBC purity by Calcein-AM staining were determined, followed by labeling efficiency calculation. WBC viability (0, 1, 2, 4 h post labeling) and label stability in plasma (1, 2, 4 h post labeling) were determined. In this preliminary review, data from nine healthy subjects and two patients are reported.
 Results: Microfluidic isolation separated WBCs from 40 mL whole blood in 23.0 ± 0.9 min for healthy donors and 22.5 ± 0.7 min for patients vs. fixed 90 min for the conventional method. Total processing time (between blood draw and initiating labeling) using microfluidic isolation was 39.3 ± 5.3 min for healthy donors and 40.0 ± 0.0 min for patients vs. 123.8 ± 13.3 min for healthy donors and 115 ± 7.1 mins. for patients using the conventional method. WBC purity of microfluidic isolate, 96.6 ± 2.1% (healthy) and 94.8 ± 1.0% (patients), were significantly higher than conventional isolate, 8.7 ± 3.6% (healthy) and 11.3 ± 3.4% (patients). Most impurities in conventional isolates were platelets: 85.0 ± 6.6% (healthy) and 80.8 ± 5.6% (patients). Labeling efficiency was comparable in healthy and patient samples for the two methods. Label stability and WBC viability were superior in cells isolated with the microfluidic method compared to the conventional method.
 Conclusions: Microfluidic isolation automates WBC isolation and achieves pure WBC isolates consistently from healthy and patient blood within a much shorter time, compared to the conventional method. Such predictable turnaround time and minimal technician hands-on time are very desirable in the clinical settings. The microfluidic method achieved similar labeling efficiency with far superior WBC purity, suggesting that the radiotracer is concentrated in the WBCs, while it is likely associated with contaminating cells (RBCs and platelets) in addition to WBCs in the conventional isolate. The improved label stability and WBC viability with the microfluidic approach may contribute to improved imaging and should be evaluated in future studies.