Centrifugation‐free washing reduces buildup of potassium and free hemoglobin in washed red blood cells after the procedure
Authors: Eszter Vörös, Madeleine Lu, Dalia Lezzar, Sergey S. Shevkoplyas
Journal: American Journal of Hematology (2018)

Stored red blood cells (RBCs) are washed in saline before transfusion to remove residual plasma proteins and other contaminants present in the storage medium. However, the levels of extracellular potassium and free hemoglobin (Hb) in RBC units increase precipitously after washing because of the cellular damage induced by centrifugation. Here we tested whether our recently-developed centrifugation-free washing system could reduce the accumulation of potassium and Hb in the supernatant of washed RBCs during 24-h storage. Samples from several RBC units (n = 7, storage duration 39-42 days) were washed in normal saline either using the prototype of the centrifugation-free washing system, or via centrifugation. The indicators of mechanical damage (supernatant potassium, free Hb) and key metrics of RBC in vitro quality (ATP, 2,3-DPG, deformability) were measured at 0, 3, 6 and 24 h after washing. RBC samples washed using the centrifugation-free washing system had significantly lower concentration of free Hb (0.6 ± 0.1 vs. 0.8 ± 0.2 mg/mL) and potassium (3.3 ± 0.6 vs. 5.11 ± 0.8 mmol/L) immediately after washing, and their concentrations increased significantly less after the 24-hour storage (Hb: 1.0 ± 0.2 vs. 2.1 ± 0.3 mg/mL; potassium: 4.9 ± 0.6 vs. 10.5 ± 0.9 mmol/L), than those washed by centrifugation. None of the other measured parameters changed significantly during the short-term storage. This study shows that our centrifugation-free washing system could significantly reduce the buildup of potassium and Hb in the supernatant of washed RBCs, potentially reducing the risks that may be associated with their transfusion. Stored red blood cells (RBCs) are washed before transfusion to reduce the risk of complications associated with the residual plasma proteins, excessive potassium, free hemoglobin (Hb) and other contaminants that may be present in the unit.1, 2 Washing is typically performed by diluting a standard 350 mL RBC unit with 1-2 L of normal saline, and then concentrating the diluted RBCs back to the desired hematocrit—with a centrifuge or a centrifugation-based cell processor—and discarding the supernatant.2, 3 Centrifugation, however, subjects stored RBCs to substantial mechanical damage, inducing hemolysis and increasing fragility of the cells.3 As a result, the levels of free Hb and potassium in RBC units increase progressively after washing, reducing the clinical value of the procedure and subjecting the recipients to a host of potential adverse outcomes.4 In this study, we asked whether replacing conventional centrifugation with our recently-developed centrifugation-free washing system1 could minimize the mechanical damage incurred by RBCs during washing, and therefore reduce the accumulation of plasma free potassium and Hb in the supernatant of washed RBCs during the subsequent 24-h storage. We tested this hypothesis by sampling several RBC units (n = 7, storage duration 39-42 days), washing one halve of each sample via conventional centrifugation and the other halve using the centrifugation-free system, and measuring key metrics of RBC in vitro quality (ATP, 2,3-DPG, deformability) and the indicators of mechanical damage (supernatant potassium, free Hb) before and at 0, 3, 6 and 24 h after the washing procedure. Figure 1 shows the change in concentration of potassium and of free Hb in the supernatant of washed RBCs over the 24-h storage period. The level of potassium for conventionally washed RBCs rose precipitously (by ~2-fold), from 5.11 ± 0.8 mmol/L upon washing to 10.5 ± 0.9 mmol/L after the 24-h storage (Figure 1A). In contrast, the level of potassium in paired RBC samples washed using our centrifugation-free system increased significantly less, from 3.3 ± 0.6 mmol/L immediately following the procedure to 4.9 ± 0.6 mmol/L after the 24-h storage (Figure 1A). Similarly, the concentration of free Hb in conventionally washed RBCs rose rapidly (by >2.5-fold), from 0.8 ± 0.2 mg/mL immediately following washing to 2.1 ± 0.3 mg/mL after the 24-h storage, nearly reaching the level of free Hb in the unwashed sample, 2.8 ± 0.5 mg/mL (Figure 1B). The concentration of free Hb in RBC samples washed using the centrifugation-free system increased significantly less, from 0.6 ± 0.1 mg/mL upon washing to 1.0 ± 0.2 mg/mL after the 24-h storage (Figure 1B). Interestingly, the levels of potassium and free Hb in RBC samples 24 h after centrifugation-free washing were not significantly different than those in paired RBC samples immediately after conventional, centrifugation-based washing (Figure 1A,B). The dramatic difference in the effect of the two washing methods on the concentration of free Hb in washed RBC samples was immediately obvious when looking at the color of the supernatant (Figure 1C). None of the other measured parameters (including intracellular ATP, 2,3-DPG, and deformability) showed any significant changes over the entire duration of the 24-h postwash storage for either of the two washing methods (see Supporting Information). Centrifugation-free washing reduced the concentration of free Hb and potassium in washed RBCs significantly more than the conventional procedure (Figure 1A,B). In the absence of any additional sources of these molecules, however, there should not have been any difference between the two methods, because the concentration of free Hb and potassium in the supernatant is determined entirely by the dilution ratio, which was the same. It is therefore likely that stored RBCs damaged by centrifugation during the conventional washing procedure were the source of the additional Hb and potassium released into the supernatant immediately following the procedure, creating the differences we observed in this study. Furthermore, because both washed samples were stored under the same basic conditions (matched hematocrit, storage medium mostly replaced by saline, 2-6°C in a blood bank refrigerator) after washing, the drastic difference in the rate of free Hb and potassium buildup (Figure 1A,B) was also likely due to the use of centrifugation during washing. That is, stored RBCs damaged by centrifugation during washing continued to release additional Hb and potassium into the supernatant throughout the post-wash storage (Figure 1C). Surprisingly, the rapid rise of the two indicators of mechanical damage (supernatant potassium and Hb) within the initial 24 h after the centrifugation-based washing did not translate into the corresponding changes in key metrics of RBC in vitro quality, including deformability (see Supporting Information). This finding suggests that the probability of each individual RBCs to be damaged by centrifugation is likely independent of the cell's properties, and therefore hemolysis of damaged cells during the 24-h storage does not affect the overall quality of the sample. In contrast, our earlier work has shown that selective lysis of RBCs irreversibly damaged during hypothermic storage (sphero-echinocytes and spherocytes) via hypotonic washing produced a modest, but statistically significant improvement in the overall deformability of the sample.5 Further studies are needed to gain additional insight into how centrifugation and other types of processing affect stored RBCs at the single-cell level, and how the extent of resulting mechanical damage may depend on the individual cell properties. Although centrifugation-free washing drastically reduced the rate at which potassium and free Hb increased in the supernatant, it could not completely eliminate their buildup (Figure 1). It is therefore likely that other parts of the washing process besides centrifugation may also be damaging to stored RBCs. One possible source of mechanical damage in our centrifugation-free washing system is the contact of relatively fragile stored RBCs with the surfaces of the coiled tubing and the membrane-hydrogel component of the system.1 Another potential source of damage is the difference between osmolality of the storage medium in a typical RBC unit (~360 mmol/kg) and osmolality of saline used for washing stored RBCs (~285 mmol/kg). The reduction in osmolality of the suspending medium during washing causes mild swelling of washed RBCs, which ultimately leads to selective lysis of most spherical cells (sphero-echinocytes, spherocytes).5 Finally, the use of normal saline (0.9% NaCl solution) as the washing solution may by itself be damaging to cells, causing significantly more hemolysis than balanced, buffered crystalloid solutions (such as Plasma-Lyte A).6 The vast majority of low birth weight infants receive RBC transfusions during the first few weeks of life (typically, 10-20 mL/kg transfused over 4 h), and washing of stored RBCs in normal saline is one of the strategies currently used for reducing the amount of potentially toxic contaminants contained in the storage medium being transfused to neonates. The prototype of the centrifugation-free washing system we used in this study operated at 300 mL/h, a throughput already sufficient for washing small volumes of stored RBCs in a reasonable amount of time (eg, a 50 mL aliquot washed with 250 mL of saline in less than an hour). Such a simple, inexpensive, disposable washing system could therefore be particularly useful in the neonatal and pediatric transfusion settings. Further research and development will be needed, however, to convert our latest prototype into a fully “closed system” format, and enable proper validation in the clinical settings. All authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.