The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of 'whole ...blood' into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor.
In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2-3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01).
This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation-particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.
During the mitotic cycle, the rod‐shaped fission yeast cells grow only at their tips. The newly born cells grow first unipolarly at their old end, but later in the cycle, the ‘new end take‐off’ event ...occurs, resulting in bipolar growth. Photographs were taken of several steady‐state and induction synchronous cultures of different cell cycle mutants of fission yeast, generally larger than wild type. Length measurements of many individual cells were performed from birth to division. For all the measured growth patterns, three different functions (linear, bilinear and exponential) were fitted, and the most adequate one was chosen by using specific statistical criteria, considering the altering parameter numbers. Although the growth patterns were heterogeneous in all the cultures studied, we could find some tendencies. In cultures with sufficiently wide size distribution, cells large enough at birth tend to grow linearly, whereas the other cells generally tend to grow bilinearly. We have found that among bilinearly growing cells, the larger they are at birth, the rate change point during their bilinear pattern occurs earlier in the cycle. This shifting near to the beginning of the cycle might finally cause a linear pattern, if the cells are even larger. In all of the steady‐state cultures studied, a size control mechanism operates to maintain homeostasis. By contrast, strongly oversized cells of induction synchronous cultures lack any sizer, and their cycle rather behaves like an adder. We could determine the critical cell size for both the G1 and G2 size controls, where these mechanisms become cryptic.
TAKE AWAY
Most individual fission yeast cells in steady‐state cultures grow bilinearly.
In strongly oversized fission yeast cells, linear growth dominates over bilinear.
Above birth length thresholds, both the G1 and G2 size controls become cryptic.
Highlights • Flow-through device recovers more lymphocytes than density gradient centrifugation. • Cell recoveries were 85% CD3+, 89% CD19+ and 97% CD56+ with this new approach. • ...Microfluidic-isolated cells grew faster than gradient-isolated cells in culture. • Overall, the new method gives 2X higher cumulative cell yield after 7-day culture. • Devices can process 200 mL of sample in less than 1 h, with no pumping mechanism needed.
Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation‐based cell processors commonly used by hospital blood banks cannot consistently reduce IgA ...below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4‐6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4‐fold and total free protein by >10‐fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5‐fold lower than that produced by conventional centrifugation. This proof‐of‐concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation‐free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation‐based cell processors.
Millions of blood components including red blood cells, platelets, and granulocytes are transfused each year in the United States. The transfusion of these blood products may be associated with ...adverse clinical outcomes in some patients due to residual proteins and other contaminants that accumulate in blood units during processing and storage. Blood products are, therefore, often washed in normal saline or other media to remove the contaminants and improve the quality of blood cells before transfusion. While there are numerous methods for washing and volume reducing blood components, a vast majority utilize centrifugation-based processing, such as manual centrifugation, open and closed cell processing systems, and cell salvage/autotransfusion devices. Although these technologies are widely employed with a relatively low risk to the average patient, there is evidence that centrifugation-based processing may be inadequate when transfusing to immunocompromised patients, neonatal and infant patients, or patients susceptible to transfusion-related allergic reactions. Cell separation and volume reduction techniques that employ centrifugation have been shown to damage blood cells, contributing to these adverse outcomes. The limitations and disadvantages of centrifugation-based processing have spurred the development of novel centrifugation-free methods for washing and volume reducing blood components, thereby causing significantly less damage to the cells. Some of these emerging technologies are already transforming niche applications, poised to enter mainstream blood cell processing in the not too distant future.
During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged ...cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood.
Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline.
Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample.
This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.
Iodinated contrast media (Xenetix®, Ultravist®, Omnipaque®, Visipaque® and Iomeron®) used for computed tomography (CT) may decrease fibrinolysis by recombinant tissue plasminogen activator (rt-PA). ...We hypothesized that receiving iodinated contrast media before rt-PA may impair thrombolysis as measured by a new model system.
Whole blood from Wistar Kyoto rats (n = 10) was obtained and allowed to form blood clots. Thrombolysis was performed by placing individually the prepared clots into 15 mL tubes and adding 5 mL saline buffer, 100μg rt-PA and a different contrast media; adjusting the quantity of iodine to either 30 mg or 60 mg. The thrombolytic efficacy was quantified by measuring the optical density (OD415) of the supernatant at different time points, namely at 0, 30, 60, and 90 min.
There was a significant decrease in clot lysis efficiency observed in presence of iodine containing contrast media comparing to positive control group. Moreover, when the quantity of iodine was increased from 30 mg to 60 mg; the dissolution rate downturned with additional ∼50%.
In conclusion, our study suggests that high dose of iodine potentially could negatively affect the efficiency of the thrombolytic therapy performed by rt-PA.
Introduction: Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients when RBCs from IgA-deficient donors are not available. Centrifugation-based cell processors ...commonly used by hospital blood banks cannot consistently reduce IgA to levels suitable for IgA-deficient recipients in a single washing cycle, hence double washing is frequently required. Additionally, cell processors are designed to wash one RBC unit at a time, making washing several units for a multiple-unit transfusion a laborious, lengthy and logistically difficult process. Importantly, these conventional cell washers are complex, expensive machines that may not be available in smaller medical facilities, thus limiting availability of washed RBCs on demand. To address these limitations, we developed a simple, portable, disposable system capable of washing stored RBC units at practical volumetric throughput without the use of centrifugation, while reducing IgA below 0.05 mg/dL in a single run. This new washing system consists of two core modules: (i) a coil of narrow-bore plastic tubing with three sequential bifurcations for high-throughput separation of washed RBCs from washing solution, and (ii) a highly-engineered membrane-hydrogel composite for concentrating the suspension of washed RBCs to the desired level of hematocrit.
Materials and Methods: Several units of stored RBCs (n = 4, leukoreduced, 4-6 weeks storage duration) were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Samples of stored RBCs were withdrawn from the units and diluted with 0.9% saline to a hematocrit (Hct) of 10%, to match the dilution typically used in the conventional centrifugation-based washing procedure (2000 mL of saline per typical 350 mL RBC unit with a 60-65% initial Hct). The diluted RBC samples were then washed either by our new washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics.
Results and Discussion: Stored RBCs were diluted to 10% Hct with normal saline and passed through the new washing system at 5.0 mL/min to produce washed RBCs with output Hct of about 42%, and Hct of washing solution (waste) of about 2%. Overall, washing performed by either of the two methods substantially reduced hemolysis, lowered the concentration of free hemoglobin (by >4-fold) and total free protein (by >10-fold), while keeping the intracellular ATP unchanged (Table 1). Washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) from 62.9 ± 14.3% in the initial RBC units to 74.1 ± 6.9% for our experimental device, which was a more pronounced improvement than for conventional centrifugation (67.8 ± 5.1%), although the difference was not statistically significant. Importantly, our new washing system reduced the level of IgA to well below 0.05 mg/dL required by AABB in a single pass, significantly outperforming the traditional, centrifugation-based washing procedure.
Conclusions: This study demonstrated the feasibility of washing stored RBCs in normal saline for transfusion to patients with selective IgA deficiency without the use of centrifugation. Our inexpensive, portable, disposal system was significantly better than centrifugation in reducing the IgA level, and performed on par with the conventional procedure for all other key in vitro metrics of RBC quality. The ability of this new washing system to operate in line with existing infusion equipment at bedside could be particularly attractive in remote or resource-limited settings, when conventional centrifugation-based cell washers are unavailable.
Acknowledgments: This work was supported by the 2012 NIH Director's Transformative Research Award (R01HL117329).
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Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.
Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA ...below the recommended levels, hence double washing is frequently required. Here we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4–6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key
in vitro
quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hr, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4-fold and total free protein by >10-fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation-based cell processors.
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