Free-flow electrophoresis to clean donated blood before transfusion at the point of care: a proof-of-concept study.

Dear Sir, Blood transfusions provide life-saving oxygen-delivery capability to those with anaemia, whether due to trauma or surgery, following ablation of haematopoietic cells by cancer treatment, or as a result of other circumstances. As with any intervention, however, complications can arise. Reducing plasma and removing white blood cells from packed red blood cells decreases the immune-mediated complications which can occur with blood transfusions1. However, residual plasma in packed red blood cells can trigger transfusion side effects in some cases1. Non-immune-mediated transfusion complications may result from the accumulation, over time, of cell-turnover by-products (e.g. potassium, microparticles/vesicles, cell-free haemoglobin [CFHb] and iron)2,3. Removal of potentially harmful by-products through washing has shown clinical benefits in selected patients4. However, washing has drawbacks since it requires equipment, time, and foresight, which are not always available. Thus, a method to rinse away aforementioned haemolysate and residual donor antibodies rapidly at the point of care (POC) is needed. The aim of this research was to use principles of free-flow electrophoresis (FFE) to remove haemolysate rapidly from donor red blood cells. FFE is a method for separating objects within flowing liquid based on differences in size and charge resulting in differing electrokinetic mobilities, and can be used on relatively fragile cells, such as plasmodium-parasitized red blood cells5. To provide proof-of-concept, a FFE “rinsing” device was made from 1 μm porous stainless steel tubes (United Filtration Systems, Sterling Heights, Michigan, USA) fitted into 60 mL syringes (BD Biosciences, San Jose, California, USA). The porous tube was secured inside the syringe with rubber stoppers at each end with holes for blood inlet/outlets. Two 16-gauge needles (rinse inlet/outlet) penetrated the space between the tube and the syringe. Expired (more than 43 days old) leucocyte-reduced red blood cell units with additive solution-1 were donated by the Blood Center of Wisconsin (Milwaukee, WI, USA). The filter was angled at 20–25° so red blood cells had to climb the filter/tube to reach the outlet. Excess normal saline (>5x blood volume) was rinsed over the inner filter tube while leucocyte-reduced red blood cells were gravity-fed from 0.75–1 meter above through the filter. The filter tube was charged with a standard nine-volt battery, confirmed by a digital multimeter, as red blood cells appeared to cross the filter more readily when uncharged. For comparison, cells were washed in a three-step procedure: (i) addition of an equal volume of saline to the leuococyte-reduced red blood cell sample; (ii) centrifugation at roughly 325 g for 10 minutes; (iii) removal of the upper four-fifths of the liquid component. The three steps were repeated again and then an equal volume of saline was added and the cells resuspended. CFHb was isolated from the “rinsed” and “washed” supernatants by centrifugation, as described above, after correcting for haematocrit. Haematocrit was determined using an automated complete blood count machine (Heska, Loveland, CO, USA). CFHb concentrations were measured using UV-visible spectroscopy. The percentage CFHb removal was compared using a two-tailed Student’s t-test with an alpha of 0.05 for statistical significance. Figure 1A shows the nominal sizes of various components within red blood cell units, while Figure 1B illustrates the FFE principles used to “rinse” red blood cells from smaller by-products. Figure 1C is a diagram of the POC FFE instrument, while Figure 1D is a photograph of the prototype. Figure 1 Using free-flow electrophoresis to clean blood rapidly. Haemoglobin was measured as a surrogate marker of filtration efficacy. Figure 2A shows representative UV-visual haemoglobin spectra of supernatants from control (unfiltered/unwashed), single-washed and double-washed leucoreduced red blood cell units, and the device-filtered blood, with characteristic oxy-haemoglobin absorbance peaks at 415, 541, and 577 nm wavelength. Figure 2B shows that the device reduced CFHb by 55%, which was comparable to the 71% CFHb removal by washing twice (p>0.05). Washing by centrifugation took nearly 30 minutes, while filtration through the device consistently took less than 1 minute per 50 mL of blood. Figure 2 Comparison of the results of filtration of aged blood with the new device or gentle washing. While this is only an introductory report showing that rapid “rinsing” of blood is possible, some key limitations should be kept in mind. As a proof-of-concept study, variable amounts of saline (greater than 5x the blood volume) were used to rinse the cells, and the seemingly-scant red blood cells lost into the waste were neither collected nor examined. To rule out the possibility that results were from dilution alone, all CFHb measurements were taken after normalising to haematocrit. Anecdotally, a charge seemed to prevent red blood cells from crossing the filter; however, how much of the CFHb removal was the result of purely mechanical dialysis and how much was due to electromotive repulsion remains unknown. Further work on optimising the pore size, flow rate, temperature, geometries, pressure gradients, and electrical potential may improve filtration efficacy. Lastly, since only CFHb was examined as a measure of the haemolysate, it was not confirmed that potassium, residual antibodies, or microparticles were removed. Despite the limitations of this proof-of-concept study, the device successfully removed CFHb to a degree comparable to that achieved by washing, but in considerably less time. Further studies are warranted to bring the clinical benefits of removing haemolysate to more transfusion recipients.