MICROFLUIDIC LEUKAPHERESIS IS SAFE AND EFFECTIVE IN A RAT MODEL

Introduction: Therapeutic leukapheresis is a lifesaving cytoreductive treatment for pediatric leukemia patients with symptomatic hyperleukocytosis. However, the current centrifugation-based approach exposes children to significant risks due to large extracorporeal volume, excessive flow rates, and significant platelet (PLT) loss. This study assesses whether high-throughput microfluidic cell separation could replace centrifugation to ultimately mitigate these drawbacks.

Materials and

Methods: We used controlled incremental filtration (CIF), a microfluidic method for separating cells by size. In vitro, we tested the CIF devices with undiluted human whole blood (WB) donated at a local blood bank. We perfused WB samples (n=5) through the CIF devices at 1.2 mL/min and measured complete blood counts (CBC) using a hematology analyzer. Additionally, we perfused WB spiked with fluorescently-labeled, cultured leukemic blasts (MV4-11; n=5) through the devices and counted the blasts using a hemocytometer. In vivo, we compared our devices (CIF) to a resistance bridge tubing (sham) in Sprague-Dawley rats (4 males and 4 females per group) using a recirculation setup driven by arteriovenous pressure differences. We collected CBC every 15 min and assessed clinical markers of inflammation and organ injury before and after procedures.

Results, Conclusions, and Discussions: The CIF device concentrated large white blood cells (WBC) in the central retentate channel, while directing smaller cells into the adjoining filtrate channels (Figure 1a). In vitro, the collection efficiency (CE = [retentate cells/total cells processed] * 100) for the largest WBC in humans (monocytes & eosinophils) was 85.0±3.0% and 86.9±5.1%, respectively. Similarly, CE for the leukemic blasts was 90.1±2.1%. In contrast, CE for red blood cells (RBC) was 5.2±0.3%, and for PLT, it was 9.9±0.9%. In vivo, CE for the largest WBC in rats (neutrophils & eosinophils) was 74.3±20.2% and 81.5±28.3%, respectively. The CE for RBC (5.5±1.3%) and PLT (4.3±1.1%) was significantly lower. After the 3-hour microfluidic leukapheresis procedure, the rats’ circulating WBC count decreased by 45.6±2.9% relative to baseline, with no difference in PLT count. A minor reduction in RBC count occurred in both CIF and sham groups likely due to serial blood sampling (Figure 1b). End-organ histology and assessment of plasma biomarkers showed no adverse effects compared to sham control.
This study is the first to demonstrate a microfluidic device capable of selectively removing WBC from undiluted WB with high throughput and efficiency, both in vitro and in vivo. The device was safe for rats and recirculated WB for hours without any loss in separation efficiency. Particularly high efficiency of large WBC separation suggests the possibility of using CIF devices for isolating monocytes for extracorporeal photopheresis, or for detecting circulating tumor cells.

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