ORIGINAL ARTICLE AS-7 improved in vitro quality of red blood cells prepared from whole blood held overnight at room temperature Margaret F. Veale,1 Geraldine Healey,1 Amrita Sran,1 Katherine A. Payne,1 Majid Zia,2 and Rosemary L. Sparrow1
BACKGROUND: Extended room temperature (RT) hold of whole blood (WB) may affect the quality of red blood cell (RBC) components produced from these donations. The availability of better RBC additive solutions (ASs) may help reduce the effects. A new AS, AS-7 (SOLX, Haemonetics Corporation), was investigated for improved in vitro quality of RBCs prepared from WB held overnight at RT. STUDY DESIGN AND METHODS: Sixteen WB units were held for 21.4 hours ± 40 minutes at 22°C on cooling plates before processing. Each pair of ABOmatched WB units were pooled, divided into a WB filter pack containing saline-adenine-glucose-mannitol (control) and a LEUKOSEP WB-filter pack containing SOLX, and processed according to manufacturer’s instructions. RBCs were stored at 2 to 6°C and sampled weekly until expiry. Glycophorin A (GPA+) and annexin V–binding microparticles (MPs) were quantitated using flow cytometry. Osmotic fragility, intracellular pH (pHi), adenosine triphosphate (ATP), 2,3diphosphoglycerate (2,3-DPG), and routine quality variables were measured. Adhesion of RBCs to human endothelial cells (ECs) was evaluated by flow perfusion under low shear stress (0.5 dyne/cm2), similar to low blood flow in microvessels. RESULTS: ATP and 2,3-DPG levels were improved for SOLX-RBCs. SOLX-RBCs maintained higher pHi, increased resistance to hypotonic stress, and reduced numbers of GPA+ MPs. No significant difference was observed between annexin V binding to MPs or adhesion of RBCs to ECs under shear stress. CONCLUSION: SOLX-stored RBCs showed increased osmotic resistance, pHi, and reduced GPA+ MPs and together with higher ATP and 2,3-DPG levels demonstrated improved in vitro RBC quality measures during 42 days of storage.
T
he red blood cell (RBC) storage lesion is characterized by biochemical and biophysical changes that occur during refrigerated storage of RBC components.1-3 Alterations to the biochemical properties of RBCs, including depletion of intracellular adenosine triphosphate (ATP) and 2,3diphosphoglycerate (2,3-DPG), may play a role not only in altered RBC metabolism, but can significantly influence the biophysical properties of RBCs.4 Irreversible changes to membrane surface area via loss of membrane and the generation of microparticles (MPs) result in shape changes that may influence RBC flow properties.5 Storage lesion changes can also be influenced by blood processing and storage conditions. The Council of Europe guidelines recommend that whole blood (WB) units can be held for up to 24 hours in conditions validated to maintain a temperature between 20 to 24°C, once rapidly cooled to room temperature (RT) before processing.6 In the United States, the Food and Drug Administration (FDA) mandates a maximum 8-hour WB hold time at
ABBREVIATIONS: EC(s) = endothelial cell(s); GPA = glycophorin A; MP = microparticle; pHi = intracellular pH; PS = phosphatidylserine; RT = room temperature; SAGM = saline-adenine-glucose-mannitol; WB = whole blood. From 1Research & Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia; and 2Hemerus Medical, LLC, St Paul, Minnesota. Address reprint requests to: Rosemary Sparrow, PhD, c/o Department of Immunology, Monash University, AMREP Campus, Commercial Road, Melbourne, VIC, 3004, Australia; e-mail: [email protected]. We acknowledge the Australian Governments that fully fund the Australian Red Cross Blood Service for the provision of blood products and services to the Australian community. Received for publication January 28, 2014; revision received May 25, 2014, and accepted May 31, 2014. doi: 10.1111/trf.12779 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
TRANSFUSION
1
VEALE ET AL.
RT, after which the WB must be refrigerated and then processed within 24 hours to RBC components.7 Previous studies have reported that longer WB hold times can significantly increase the storage-related changes that occur to RBCs, including reduced ATP and 2,3-DPG, for RBCs stored in conventional additive solutions (ASs), such as saline-adenine-glucose-mannitol (SAGM).8 Newgeneration ASs that are buffered and have different tonicity compared to the conventional unbuffered, hypertonic solutions have been shown to limit the effect of overnight RT-hold of WB on RBC biochemical variables.8,9 These types of new RBC ASs, which may reduce the storage lesion effects and enhance RBC quality, could lead to improved transfusion safety and efficacy.4,10 SOLX (Haemonetics Corporation, Braintree, MA), also known as AS-7, is a new AS invented by Hess and Greenwalt (as reviewed in Ref. 4) and designed to preserve ATP levels during storage, which is critical to maintain RBC viability. SOLX is a buffered, alkaline, hypotonic, Cl−free solution. Clinical trials of RBCs stored in SOLX showed improved 24-hour RBC recoveries after transfusion regardless of the WB hold time.11,12 The SOLX WB collection system was recently approved by the FDA for storage of blood components prepared from WB processed within 8 hours. We hypothesized that for RBCs prepared from WB units held overnight at RT, the new AS SOLX would offer improved maintenance of RBC in vitro quality during 42 days of storage compared with currently used RBC AS, SAGM. We investigated whether RBCs stored in SOLX had improved in vitro RBC biochemical variables, including levels of ATP and 2,3-DPG, as well as reduced membrane changes. Osmotic fragility was used to quantitate changes to the RBC surface area-to-volume ratio, providing information about RBC susceptibility to lysis after storage. The extent of RBC membrane loss and changes to membrane asymmetry were measured by quantitating MP accumulation and annexin V–binding MPs in the supernatant. RBC adhesion to endothelial cells (ECs) under low-shearstress conditions in vitro was used to mimic slowed blood flow conditions in postcapillary venules observed after transfusion of stored transfused RBCs.13,14 Using this method we may be able to understand the potential effects of membrane changes upon RBC flow properties.
MATERIALS AND METHODS WB collection, RBC processing, and storage WB was collected from normal volunteer donors (n = 16) according to standard procedures of the Australian Red Cross Blood Service. The study was approved by the Blood Service Human Research Ethics Committee. Briefly, WB (470 mL ± 10%) was collected into collection packs containing CPD (Pall Corporation, Portsmouth, UK). A pooland-split study design was used consisting of 2-unit pools 2
TRANSFUSION Volume **, ** **
of ABO-matched WB (i.e., n = 8 pairs). WB units to be pooled were collected within 2 hours of each other and were rapidly cooled to 20°C on cooling plates (Compocool II, Fresenius HemoCare, Bad Homburg, Germany) and held at 20°C for 21.4 hours ± 40 minutes, that is, mean time from collection to processing. WB units were pooled into a 2-L transfer bag (Teruflex, Terumo, Tokyo, Japan), mixed, and equally divided into a Pall WB filter pack (containing SAGM) and a Hemerus WB filter pack (containing SOLX). The WB units were leukoreduced using the accompanying WB in-line filter, that is, WBF3 (Pall) for the Pall WB packs or LEUKOSEP HWB-600-XL (Haemonetics) for the Hemerus WB packs. Both filters operate by gravity flow. The LEUKOSEP filter is self-priming, with an autodrain feature and does not require any mechanical pressure. The leukoreduced WB units were centrifuged at 5000 × g for 10 minutes at RT and the plasma was removed using a semiautomated blood component separator (Optipress II, Fenwal, Baxter, Maurepas, France). The paired RBC units were resuspended in the corresponding AS, that is, SAGM (100 mL) for the Pall packs and SOLX (110 mL) for the Hemerus packs. SOLX is presented as a two-part solution made up of 80 mL of SOLX AS A and 30 mL of SOLX AS B, which were combined in the satellite pack immediately before adding to the RBCs. All RBC units were stored according to Blood Service procedures at 2 to 6°C and were sampled aseptically on Days 1, 7, 14, 28, 35, and 42.
In vitro RBC quality assessment Quality assessment of RBC units was performed using standard Blood Service procedures as described previously.5 Briefly, residual white blood cell counts were determined by flow cytometry using an absolute bead count assay (TruCount tubes, BD Biosciences, San Jose, CA) on Day 1 of storage. Full blood counts were performed using an automated hematology analyzer (CellDyn 3200, Abbott, Santa Clara, CA) on each sample collection. Hematocrit (Hct) was measured manually using a microHct centrifuge (Hawskley, West Sussex, England) according to the manufacturer’s instructions. Supernatant hemoglobin (Hb) was measured as described15 and the percent (%) hemolysis was calculated based on spun Hct results, according to standard Blood Service procedures. Supernatant potassium (K+) was measured using a biochemistry analyzer (Architect, Abbott). RBC ATP and 2,3DPG were measured using commercial kits (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Extracellular pH was measured at 22°C by a pH meter (Radiometer, Copenhagen, Denmark). Intracellular pH (pHi) was measured using a modification of the method described by Meryman and colleagues.16 Briefly, RBCs were pelleted by centrifugation, and the supernatant was removed. RBCs were frozen in
AS-7 IMPROVED QUALITY OF RBCs
liquid nitrogen and thawed and the pH of the lysate was measured at 22°C using a pH meter.
MediaCybernetics, Rockville, MD). The mean number of adhered RBCs per field was calculated. Results are reported as the mean number of adherent RBCs/mm2.
MP quantitation
Statistical analysis
The number of MPs in the supernatant that expressed glycophorin A (GPA) and phosphatidylserine (PS) were quantitated by flow cytometry (FACSCanto II, BD Biosciences) using an absolute bead count assay (TruCount tubes, BD Biosciences) as described previously.5 Briefly, 25 μL of supernatant from stored RBC units was incubated with either a phycoerythrin (PE)-conjugated anti-GPA or a PE-conjugated isotype control (BD Biosciences), and 10 μL of supernatant was incubated with allophycocyanin-conjugated annexin V from a commercial kit (BD Biosciences) to detect PS exposure. For each sample, 10,000 events were acquired. Sizing beads (flow cytometry size calibration kit and FluoSpheres sulfate latex beads, Molecular Probes, Invitrogen, Eugene, OR) were used to verify the appropriate setting for the MP gate to detect MPs less than 1 μm in diameter. The number of MPs/μL of supernatant was calculated according to the manufacturer’s instructions of the tubes (TruCount, BD Biosciences).
Results shown are mean ± standard deviation (SD) or standard error of the mean (SEM). Paired t tests were used to compare RBC ASs. Repeated-measures analysis of variance (ANOVA) was used to determine the influence of storage duration. Statistical analyses were performed using computer software (GraphPad PRISM, GraphPad, Inc., La Jolla, CA).
Osmotic fragility Osmotic fragility of stored RBCs was determined as described previously using a modified method of Parpart and coworkers.17 Briefly, RBCs (1 × 108/mL) from each storage time point were suspended in 1 mL of hypotonic buffered saline solutions (3.5-9.0 g/L, NaCl), or water (100% lysis control) for 30 minutes at RT. Supernatants were collected by centrifugation (1000 × g for 5 min) and hemolysis was measured at a 540 nm wavelength in a plate reader (FLUOROstar Optima, BMG, Ortenberg, Germany). The results were expressed relative to the 100% lysis control. Osmotic fragility curves were plotted at each time point and the NaCl concentration that corresponded to 50% lysis was calculated.
Adhesion to ECs Adhesion of stored RBCs to ECs under shear stress conditions was performed as described previously.18 Briefly, human umbilical vein ECs grown on collagen-coated glass coverslips were mounted into a flow perfusion chamber and perfused at 37°C with a suspension of stored RBCs at a shear stress of 0.5 dyne/cm2 to simulate low microvascular blood flow.13,14,19 Cells were visualized using an inverted microscope (Model IX71, Olympus, Tokyo, Japan), and 15 randomly selected fields on the coverslip were recorded by a CCD camera (DP71, Olympus). The recorded images were analyzed for RBC adherence to ECs using imaging software (ImagePro-Plus,
RESULTS ATP RBCs stored in SOLX maintained significantly higher levels of ATP from Day 14 of storage compared to RBCs stored in SAGM (p ≤ 0.05; Table 1). ATP was well maintained in RBCs stored in SOLX for 42 days, with ATP levels of 3.4 ± 0.8 μmol/L/g Hb compared to 1.4 ± 0.2 μmol/L/ g Hb for RBCs stored in SAGM.
2,3-DPG RBCs stored in SOLX had significantly higher levels of 2,3DPG on Days 7,14, 21, 28, and 42 of storage compared to those stored in SAGM (Table 1). RBC 2,3-DPG declined over storage in both ASs (ANOVA p < 0.0001), although SOLX maintained significantly elevated levels compared with SAGM (p < 0.05) for at least one additional week of storage.
pHi RBCs stored in SOLX maintained significantly higher pHi (p < 0.001) compared to RBCs stored in SAGM (Table 1). Both SOLX- and SAGM-stored RBCs showed a significant decline in pHi over storage (ANOVA p < 0.001).
Routine RBC variables Hemolysis, Hb, and Hct of RBCs stored in SOLX or SAGM complied with acceptance criteria recommended in the Council of Europe guidelines6 (Table 1). As expected, due to the hypotonic effect on RBC shape and cell volume, SOLX-RBCs had a higher Hct than SAGM-RBCs, as determined by the spun Hct method. SOLX-RBCs had lower concentrations of RBCs and Hb compared to SAGM-RBCs, despite having been processed from the same initial volume of WB. Supernatant K+ concentrations of RBCs stored in either AS were not significantly different and increased in both over storage.
Osmotic fragility The NaCl concentration required to induce 50% hemolysis was calculated from osmotic fragility curves and plotted Volume **, ** **
TRANSFUSION
3
VEALE ET AL.
TABLE 1. Routine variables of RBCs stored in SOLX and SAGM* 1
7
14
Storage Day 21
28
35
42
6.68 ± 0.24 6.95 ± 0.27
6.64 ± 0.23 6.95 ± 0.29
6.71 ± 0.27 7.04 ± 0.32
6.65 ± 0.19 6.96 ± 0.18
6.70 ± 0.21 6.99 ± 0.24
6.65 ± 0.20 6.98 ± 0.26
6.64 ± 0.21 6.91 ± 0.26
196 ± 7a 206 ± 7
195 ± 8 204 ± 8
196 ± 8a 206 ± 3
192 ± 8a 202 ± 3
193 ± 8a 202 ± 9
197 ± 8a 206 ± 7
196 ± 8a 206 ± 8
NT NT
0.68 ± 0.01c 0.62 ± 0.01
0.67 ± 0.01c 0.62 ± 0.01
0.67 ± 0.01c 0.62 ± 0.01
NT NT
NT NT
0.66 ± 0.02 0.64 ± 0.01
8.1 ± 3.6 7.9 ± 2.6
6.2 ± 3.7 6.0 ± 3.3
6.7 ± 1.6c 4.7 ± 1.4
7.1 ± 1.8b 3.4 ± 0.9
4.8 ± 1.8a 2.4 ± 0.8
5.3 ± 2.1c 2.1 ± 0.6
3.4 ± 0.8c 1.4 ± 0.2
6.4 ± 1.1 5.5 ± 1.5
4.6 ± 1.3c 2.4 ± 0.7
2.7 ± 1.4a 1.2 ± 0.2
1.9 ± 0.8a 1.2 ± 0.4
1.7 ± 0.4a 1.4 ± 0.6
1.6 ± 0.8 1.2 ± 0.2
1.5 ± 0.5b 1.0 ± 0.2
NT NT
0.08 ± 0.03 0.06 ± 0.02
0.10 ± 0.04 0.08 ± 0.03
0.13 ± 0.06 0.11 ± 0.03
NT NT
NT NT
0.20 ± 0.09 0.24 ± 0.05
4.3 ± 1.4 3.7 ± 1.0
18.1 ± 3.0 17.8 ± 2.7
27.2 ± 3.9 27.9 ± 3.5
36.6 ± 4.7 35.9 ± 4.1
44.9 ± 5.2 47.9 ± 4.6
49.3 ± 5.8 52.3 ± 4.7
7.0 ± 0.07 6.9 ± 0.05
6.9 ± 0.04 6.8 ± 0.04
6.8 ± 0.06 6.7 ± 0.05
6.7 ± 0.04 6.7 ± 0.02
6.6 ± 0.09 6.6 ± 0.05
6.6 ± 0.09 6.5 ± 0.05
7.2 ± 0.06c 7.1 ± 0.07
7.1 ± 0.04c 7.0 ± 0.06
7.0 ± 0.02c 6.8 ± 0.03
6.8 ± 0.09c 6.7 ± 0.06
6.8 ± 0.04b 6.7 ± 0.05
6.7 ± 0.05b 6.6 ± 0.03
6.7 ± 0.04b 6.6 ± 0.03
* The results are plotted as mean ± SD (n = 8). Significance: † n = 4, measured by spun Hct. NT = not tested.
a
p < 0.05,
relative to storage time (Fig. 1). RBCs stored in SOLX had significantly increased osmotic resistance compared to RBCs stored in SAGM (ANOVA p < 0.0004). This suggests that RBCs stored in SOLX have decreased susceptibility to hypotonic lysis. The concentration of NaCl that induced 50% RBC lysis for RBCs stored in SAGM was significantly lower in RBCs stored in SOLX from Day 7 of storage (p ≤ 0.05).
MPs in the stored supernatant RBCs stored in SOLX and SAGM showed increased numbers of GPA+ MPs and annexin V binding MPs up to Day 42 of storage (Fig. 2; p < 0.0001). Significantly fewer MPs were detected in the supernatant of RBCs stored in SOLX compared to those in SAGM on Day 42 of storage (10,475 ± 2520 and 34,797 ± 5551 MPs/μL, respectively; p < 0.001). No significant differences in the numbers of annexin V binding MPs were observed between SOLX- and SAGM-RBC units (Fig. 2B). These results suggest that RBCs stored in SOLX retain more membrane over storage than RBCs stored in SAGM. 4
39.6 ± 4.7 42.4 ± 4.2
7.1 ± 0.05 7.1 ± 0.03
TRANSFUSION Volume **, ** **
b
p < 0.01,
c
p < 0.001.
Osmotic Fragility 50% 5.0
NaCl (g/L)
RBC variable RBCs (×1012/L) SOLX SAGM Hb (g/L) SOLX SAGM Hct (L/L)† SOLX SAGM ATP (μM/g Hb) SOLX SAGM 2,3-DPG (μmol/L/g Hb) SOLX SAGM % Hemolysis† SOLX SAGM K+ (mmol/L) SOLX SAGM Extracellular pH SOLX SAGM pHi SOLX SAGM
4.5
*
**
4.0
**
***
21
28
*** ***
3.5 0
7
14
35
42
Storage Time (days) Fig. 1. Osmotic fragility of RBCs stored in SOLX (○) or SAGM (●). Osmotic fragility of RBCs stored in SOLX or SAGM was measured by incubation of the RBCs in hypotonic solutions of NaCl. The concentration of NaCl (g/L) that resulted in 50% lysis of the RBCs was calculated for each storage time point. The results are plotted as mean ± SEM (n = 8). Significance: *p < 0.05, **p < 0.01, ***p < 0.001.
AS-7 IMPROVED QUALITY OF RBCs
GPA
Flow Perfusion 0.5 dyne/cm 2
100000
A
200
RBCs adhered/mm 2
MPs/mL Log scale
*** 10000
1000
100 0
7
14
21
28
35
50 0 1
14
21
35
42
Storage Time (days)
Annexin V Binding 100000
MPs/mL Log scale
100
42
Storage Time (days)
B
150
Fig. 3. RBCs adhered to ECs under shear stress conditions. RBCs stored in SOLX (■) or SAGM (□) were measured throughout storage for their adherence to ECs under shear stress (0.5 dyne/cm2) conditions. The results are plotted as mean ± SEM (n = 8).
10000
1000
100 0
7
14
21
28
35
42
Storage Time (days)
Fig. 2. Accumulation of GPA+ and annexin V-binding RBC MPs. MPs in the supernatant of RBCs stored in SOLX (○) or SAGM (●) were quantitated by anti-GPA (A) staining and annexin V binding (B) using a flow cytometry absolute bead count assay. The results are plotted as mean ± SEM (n = 8). Significance: ***p < 0.001.
Adhesion of stored RBCs to ECs The effect of adhesion of RBCs to ECs under shear stress conditions was investigated by an in vitro flow perfusion model that mimics low microvascular blood flow. RBC adhesion to ECs increased over the storage period. However, the increase was not significant (Fig. 3). Adhesion of RBCs stored in SOLX was not significantly different to RBCs stored in SAGM.
DISCUSSION Overnight RT hold of WB before processing into RBC components is a standard procedure in many countries.8,20 In this study, RBCs prepared from WB held for 21 hours at RT and stored in the new AS, SOLX had acceptable or improved in vitro quality measures compared to RBCs stored in conventional SAGM. RBCs stored in SOLX maintained acceptable routine quality variables, including extracellular pH, hemolysis, and extracellular K+ throughout 42 days of storage. Biochemical variables including pHi, ATP, and 2,3-DPG levels were significantly better maintained in RBCs stored in SOLX compared with SAGM. The results indicate a 2-week
storage advantage for RBCs stored in SOLX compared with SAGM. These data support a previous study that showed similar results for ATP and percent hemolysis for RBC stored in SOLX that had been prepared from WB processed either immediately after collection or held for 8 or 24 hours at RT before processing.12 The formulations for SOLX and SAGM differ in tonicity, buffering, and pH. SOLX is a hypotonic, alkaline, buffered, Cl−-free AS while SAGM is a hypertonic, acidic, unbuffered saline solution.10 The higher pHi observed with SOLX RBCs may be due to a Cl− shift, which is characterized by the efflux of Cl− ions and a corresponding influx of OH− ions into the RBCs that would increase the pHi. Maintenance of RBC pHi closer to physiologic pH contributes significantly to preservation of cell hydration, synthesis of ATP and 2,3-DPG, carbohydrate metabolism, and cell morphology.1,4,21,22 The higher pHi for SOLX-RBCs and consequential higher levels of ATP and 2,3-DPG are consistent with these previous reports. Maintenance of osmotic resistance in RBCs stored in SOLX was evident, which suggested better retention of the cell surface area to cell volume ratios during storage compared to SAGM-RBCs. Lower numbers of GPA+ MPs in the supernatant of SOLX-RBCs was also consistent with improved maintenance of RBC membrane over storage.23,24 No significant differences were found in the number of PS+ MPs in the supernatant of SOLX-RBCs compared with SAGM-RBCs. This may suggest that SOLXRBCs can efficiently divest themselves of exposed PS at the cell surface and limit the loss of other membrane elements to preserve osmotic resistance. Adhesion of stored RBCs to ECs has been previously reported to increase with storage time.5,18,25,26 In this study, RBCs stored in SOLX showed a trend toward higher adhesion to ECs on Days 35 and 42 of storage compared to Volume **, ** **
TRANSFUSION
5
VEALE ET AL.
RBCs stored in SAGM, although the differences were not significant. In our previous study of the effect of extended RT hold of WB,5 RBCs were stored in an alternative alkaline, hypotonic, Cl−-free solution AS, experimental Erythrosol-4, which showed significantly increased numbers of adhered RBCs on Days 42 and 49 of storage compared to RBCs stored in SAGM. This was thought to be due to a phenomenon that resembled increased dehydration of the RBCs stored in Erythrosol-4 when the RBCs were transferred into an isotonic, physiologic flow perfusion medium, as used in this study. The increased trend in adhesion of hypotonic-stored RBCs at the latter storage time points compared to RBCs stored in SAGM may indicate differences in the effects of storage on the cell membrane properties of RBCs. It is as yet unknown whether the observed in vitro adhesiveness of stored RBCs correlates with a similar propensity in vivo, and if so, what might be the effect on vascular hemodynamics and clinical consequences, if any. It is interesting to note that similar to SOLX-RBCs, RBCs stored in hypertonic AS-1, which is a variant of SAGM, have been shown to have significantly increased adhesion to ECs and shed fewer MPs on Day 42 of storage compared to SAGM-RBCs.26 Further exploration will be needed to identify the biologic nature of the differences between RBCs stored in alternate ASs that induce changed interactions with endothelium. Our in vitro ATP and 2,3-DPG measurements of RBCs stored in SOLX suggest that there may be significant improvements to RBC quality over storage, which could be beneficial for RBC viability and survival in vivo after transfusion. Recent clinical studies in healthy individuals showed mean 24-hour posttransfusion RBC recoveries of 86% for 42-day-stored SOLX RBCs prepared from WB held for 24 hours at RT before processing.11 Shorter WB hold times of less than 2 and 6 to 8 hours at RT also showed increased in vivo RBC recoveries of 89 and 86%, respectively.11,12 These data suggest that the improvements to in vitro quality measures for RBCs stored in SOLX, with reduction of storage lesion effects, may aid significantly better clinical outcomes for recipients.11 In conclusion, RBCs prepared from WB held for 20 to 22 hours at RT and stored in SOLX had significantly higher pHi, reduced osmotic fragility, and reduced numbers of GPA+ MPs, suggesting better maintenance of membrane properties compared with the paired control RBCs stored in SAGM. These results, together with elevated biochemical markers, ATP, and 2,3-DPG, demonstrated that storage of RBCs in SOLX could yield RBCs with improved overall quality over 42 days of storage compared with RBCs stored in SAGM AS. ACKNOWLEDGMENTS We thank Australian Red Cross Blood Service Melbourne staff in Donor Services for assistance with the collection of whole blood 6
TRANSFUSION Volume **, ** **
units and processing laboratory scientists, particularly Charley Samarani, Sue Grasso, and Nicole Saleta. We thank the BMDI Cord Blood Bank, Melbourne, staff for providing umbilical cord samples for EC preparation.
CONFLICT OF INTEREST MZ was an employee and shareholder of Hemerus Medical, LLC, at the time of study and an employee of Haemonetics Corporation at the time of preparation of this manuscript. Hemerus Medical, LLC, was purchased by Haemonetics Corporation in 2013. The other authors have disclosed no conflicts of interest.
REFERENCES 1. Högman C, Meryman H. Storage parameters affecting red blood cell survival and function after transfusion. Transfus Med Rev 1999;13:275-96. 2. Tinmouth A, Chin-Yee I. The clinical consequences of the red cell storage lesion. Transfus Med Rev 2001;15:91-107. 3. Zimrin A, Hess J. Current issues relating to the transfusion of stored red blood cells. Vox Sang 2009;96:93-103. 4. Hess J. An update on solutions for red cell storage. Vox Sang 2006;91:13-9. 5. Veale MF, Healey G, Sparrow RL. Effect of additive solutions on red blood cell (RBC) membrane properties of stored RBCs prepared from whole blood held for 24 hours at room temperature. Transfusion 2011;51:25S-33S. 6. European Directorate for the Quality of Medicines & HealthCare. Guide to the preparation, use and quality assurance of blood components: recommendation. 16th ed. Strasborg: Council of Europe Publishing; 2011. 7. AABB Standard Program Committee. Standards for blood banks and transfusion services. 28th ed. Bethesda (MD): American Association of Blood Banks; 2012. 8. van der Meer P, Cancelas C, Cardigan R, et al. Evaluation of
9.
10.
11.
12.
overnight hold of whole blood at room temperature prior to component processing: effect of red cell additive solutions on in vitro red cell measures. Transfusion 2011; 51(Suppl):15S-24S. De Korte D, Kleine M, Korsten HG, et al. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage. Transfusion 2008;48:1081-9. Sparrow RL. Time to revisit red blood cell additive solutions and storage conditions: a role for “omics” analyses. Blood Transfus 2012;10(Suppl 2):s7-s11. Cancelas J, Dumont L, Maes L, et al. RBC storage in SOLX® additive solution ameliorated the storage lesion and demonstrated extended storage up to 56 days: results of a multicenter trial. Transfusion 2012;52(Suppl):47A. Maes L, Whiteley P, Sawyer S, et al. Red cells stored in SOLX for 42 days demonstrated reduced hemolysis improved morphology and exceptional in vivo recovery. Transfusion 2011;51(Suppl):69A.
AS-7 IMPROVED QUALITY OF RBCs
13. Arslan E, Sierko E, Waters JH, et al. Microcirculatory hemodynamics after acute blood loss followed by fresh and banked blood transfusion. Am J Surg 2005;190:456-62. 14. Detterich J, Alexy T, Rabai M, et al. Low-shear red blood
20. Gulliksson H, van der Meer PF. Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells. Blood Transfus 2009;7:210-15.
cell oxygen transport effectiveness is adversely affected by
21. Högman C, Knutson F, Loof H, et al. Improved mainte-
transfusion and further worsened by deoxygenation in sickle cell disease patients on chronic transfusion therapy.
nance of 2,3 DPG and ATP in RBCs stored in a modified additive solution. Transfusion 2002;42:824-9.
Transfusion 2013;53:297-305. 15. Blakney G, Dinwoodie A. A spectrophotometric scanning technique for the rapid determination of plasma hemoglobin. Clin Biochem 1975;8:96-102. 16. Meryman H, Hornblower M, Keegan T, et al. Refrigerated storage of washed red cells. Vox Sang 1991;60:88-98. 17. Parpart A, Lorenz P, Parpart E, et al. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947;26: 636-40. 18. Anniss AM, Sparrow RL. Storage duration and leukocyte content of red cell products increases adhesion of stored red blood cells to endothelium under flow conditions. Transfusion 2006;46:1561-7. 19. Walmet P, Eckman J, Wick T. Inflammatory mediators promote strong sickle cell adherance to endothelium under venular flow conditions. Am J Hematol 2003;73:215-
22. Meryman H, Hornblower M. Manipulating red cell intraand extracellular pH by washing. Vox Sang 1991;60:99-104. 23. Greenwalt T. The how and why of exocytic vesicles. Transfusion 2006;46:143-52. 24. Willekens F, Werre J, Groenen-Dopp Y, et al. Erythrocyte vesiculation: a self-protective mechanism? Br J Haematol 2008;141:549-56. 25. Relevy H, Koshkaryev A, Manny N, et al. Blood bankinginduced alteration of red blood cell flow properties. Transfusion 2007;48:136-46. 26. Sparrow RL, Sran A, Healey G, et al. In vitro measures of membrane changes reveal differences between red blood cells stored in saline-adenine-glucose-mannitol and AS-1 additive solutions: a paired study. Transfusion 2014;54:560-8.
24.
Volume **, ** **
TRANSFUSION
7
BACKGROUND: Extended room temperature (RT) hold of whole blood (WB) may affect the quality of red blood cell (RBC) components produced from these donations. The availability of better RBC additive solutions (ASs) may help reduce the effects. A new AS, AS-7 (SOLX, Haemonetics Corporation), was investigated for improved in vitro quality of RBCs prepared from WB held overnight at RT. STUDY DESIGN AND METHODS: Sixteen WB units were held for 21.4 hours ± 40 minutes at 22°C on cooling plates before processing. Each pair of ABOmatched WB units were pooled, divided into a WB filter pack containing saline-adenine-glucose-mannitol (control) and a LEUKOSEP WB-filter pack containing SOLX, and processed according to manufacturer’s instructions. RBCs were stored at 2 to 6°C and sampled weekly until expiry. Glycophorin A (GPA+) and annexin V–binding microparticles (MPs) were quantitated using flow cytometry. Osmotic fragility, intracellular pH (pHi), adenosine triphosphate (ATP), 2,3diphosphoglycerate (2,3-DPG), and routine quality variables were measured. Adhesion of RBCs to human endothelial cells (ECs) was evaluated by flow perfusion under low shear stress (0.5 dyne/cm2), similar to low blood flow in microvessels. RESULTS: ATP and 2,3-DPG levels were improved for SOLX-RBCs. SOLX-RBCs maintained higher pHi, increased resistance to hypotonic stress, and reduced numbers of GPA+ MPs. No significant difference was observed between annexin V binding to MPs or adhesion of RBCs to ECs under shear stress. CONCLUSION: SOLX-stored RBCs showed increased osmotic resistance, pHi, and reduced GPA+ MPs and together with higher ATP and 2,3-DPG levels demonstrated improved in vitro RBC quality measures during 42 days of storage.
T
he red blood cell (RBC) storage lesion is characterized by biochemical and biophysical changes that occur during refrigerated storage of RBC components.1-3 Alterations to the biochemical properties of RBCs, including depletion of intracellular adenosine triphosphate (ATP) and 2,3diphosphoglycerate (2,3-DPG), may play a role not only in altered RBC metabolism, but can significantly influence the biophysical properties of RBCs.4 Irreversible changes to membrane surface area via loss of membrane and the generation of microparticles (MPs) result in shape changes that may influence RBC flow properties.5 Storage lesion changes can also be influenced by blood processing and storage conditions. The Council of Europe guidelines recommend that whole blood (WB) units can be held for up to 24 hours in conditions validated to maintain a temperature between 20 to 24°C, once rapidly cooled to room temperature (RT) before processing.6 In the United States, the Food and Drug Administration (FDA) mandates a maximum 8-hour WB hold time at
ABBREVIATIONS: EC(s) = endothelial cell(s); GPA = glycophorin A; MP = microparticle; pHi = intracellular pH; PS = phosphatidylserine; RT = room temperature; SAGM = saline-adenine-glucose-mannitol; WB = whole blood. From 1Research & Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia; and 2Hemerus Medical, LLC, St Paul, Minnesota. Address reprint requests to: Rosemary Sparrow, PhD, c/o Department of Immunology, Monash University, AMREP Campus, Commercial Road, Melbourne, VIC, 3004, Australia; e-mail: [email protected]. We acknowledge the Australian Governments that fully fund the Australian Red Cross Blood Service for the provision of blood products and services to the Australian community. Received for publication January 28, 2014; revision received May 25, 2014, and accepted May 31, 2014. doi: 10.1111/trf.12779 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
TRANSFUSION
1
VEALE ET AL.
RT, after which the WB must be refrigerated and then processed within 24 hours to RBC components.7 Previous studies have reported that longer WB hold times can significantly increase the storage-related changes that occur to RBCs, including reduced ATP and 2,3-DPG, for RBCs stored in conventional additive solutions (ASs), such as saline-adenine-glucose-mannitol (SAGM).8 Newgeneration ASs that are buffered and have different tonicity compared to the conventional unbuffered, hypertonic solutions have been shown to limit the effect of overnight RT-hold of WB on RBC biochemical variables.8,9 These types of new RBC ASs, which may reduce the storage lesion effects and enhance RBC quality, could lead to improved transfusion safety and efficacy.4,10 SOLX (Haemonetics Corporation, Braintree, MA), also known as AS-7, is a new AS invented by Hess and Greenwalt (as reviewed in Ref. 4) and designed to preserve ATP levels during storage, which is critical to maintain RBC viability. SOLX is a buffered, alkaline, hypotonic, Cl−free solution. Clinical trials of RBCs stored in SOLX showed improved 24-hour RBC recoveries after transfusion regardless of the WB hold time.11,12 The SOLX WB collection system was recently approved by the FDA for storage of blood components prepared from WB processed within 8 hours. We hypothesized that for RBCs prepared from WB units held overnight at RT, the new AS SOLX would offer improved maintenance of RBC in vitro quality during 42 days of storage compared with currently used RBC AS, SAGM. We investigated whether RBCs stored in SOLX had improved in vitro RBC biochemical variables, including levels of ATP and 2,3-DPG, as well as reduced membrane changes. Osmotic fragility was used to quantitate changes to the RBC surface area-to-volume ratio, providing information about RBC susceptibility to lysis after storage. The extent of RBC membrane loss and changes to membrane asymmetry were measured by quantitating MP accumulation and annexin V–binding MPs in the supernatant. RBC adhesion to endothelial cells (ECs) under low-shearstress conditions in vitro was used to mimic slowed blood flow conditions in postcapillary venules observed after transfusion of stored transfused RBCs.13,14 Using this method we may be able to understand the potential effects of membrane changes upon RBC flow properties.
MATERIALS AND METHODS WB collection, RBC processing, and storage WB was collected from normal volunteer donors (n = 16) according to standard procedures of the Australian Red Cross Blood Service. The study was approved by the Blood Service Human Research Ethics Committee. Briefly, WB (470 mL ± 10%) was collected into collection packs containing CPD (Pall Corporation, Portsmouth, UK). A pooland-split study design was used consisting of 2-unit pools 2
TRANSFUSION Volume **, ** **
of ABO-matched WB (i.e., n = 8 pairs). WB units to be pooled were collected within 2 hours of each other and were rapidly cooled to 20°C on cooling plates (Compocool II, Fresenius HemoCare, Bad Homburg, Germany) and held at 20°C for 21.4 hours ± 40 minutes, that is, mean time from collection to processing. WB units were pooled into a 2-L transfer bag (Teruflex, Terumo, Tokyo, Japan), mixed, and equally divided into a Pall WB filter pack (containing SAGM) and a Hemerus WB filter pack (containing SOLX). The WB units were leukoreduced using the accompanying WB in-line filter, that is, WBF3 (Pall) for the Pall WB packs or LEUKOSEP HWB-600-XL (Haemonetics) for the Hemerus WB packs. Both filters operate by gravity flow. The LEUKOSEP filter is self-priming, with an autodrain feature and does not require any mechanical pressure. The leukoreduced WB units were centrifuged at 5000 × g for 10 minutes at RT and the plasma was removed using a semiautomated blood component separator (Optipress II, Fenwal, Baxter, Maurepas, France). The paired RBC units were resuspended in the corresponding AS, that is, SAGM (100 mL) for the Pall packs and SOLX (110 mL) for the Hemerus packs. SOLX is presented as a two-part solution made up of 80 mL of SOLX AS A and 30 mL of SOLX AS B, which were combined in the satellite pack immediately before adding to the RBCs. All RBC units were stored according to Blood Service procedures at 2 to 6°C and were sampled aseptically on Days 1, 7, 14, 28, 35, and 42.
In vitro RBC quality assessment Quality assessment of RBC units was performed using standard Blood Service procedures as described previously.5 Briefly, residual white blood cell counts were determined by flow cytometry using an absolute bead count assay (TruCount tubes, BD Biosciences, San Jose, CA) on Day 1 of storage. Full blood counts were performed using an automated hematology analyzer (CellDyn 3200, Abbott, Santa Clara, CA) on each sample collection. Hematocrit (Hct) was measured manually using a microHct centrifuge (Hawskley, West Sussex, England) according to the manufacturer’s instructions. Supernatant hemoglobin (Hb) was measured as described15 and the percent (%) hemolysis was calculated based on spun Hct results, according to standard Blood Service procedures. Supernatant potassium (K+) was measured using a biochemistry analyzer (Architect, Abbott). RBC ATP and 2,3DPG were measured using commercial kits (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Extracellular pH was measured at 22°C by a pH meter (Radiometer, Copenhagen, Denmark). Intracellular pH (pHi) was measured using a modification of the method described by Meryman and colleagues.16 Briefly, RBCs were pelleted by centrifugation, and the supernatant was removed. RBCs were frozen in
AS-7 IMPROVED QUALITY OF RBCs
liquid nitrogen and thawed and the pH of the lysate was measured at 22°C using a pH meter.
MediaCybernetics, Rockville, MD). The mean number of adhered RBCs per field was calculated. Results are reported as the mean number of adherent RBCs/mm2.
MP quantitation
Statistical analysis
The number of MPs in the supernatant that expressed glycophorin A (GPA) and phosphatidylserine (PS) were quantitated by flow cytometry (FACSCanto II, BD Biosciences) using an absolute bead count assay (TruCount tubes, BD Biosciences) as described previously.5 Briefly, 25 μL of supernatant from stored RBC units was incubated with either a phycoerythrin (PE)-conjugated anti-GPA or a PE-conjugated isotype control (BD Biosciences), and 10 μL of supernatant was incubated with allophycocyanin-conjugated annexin V from a commercial kit (BD Biosciences) to detect PS exposure. For each sample, 10,000 events were acquired. Sizing beads (flow cytometry size calibration kit and FluoSpheres sulfate latex beads, Molecular Probes, Invitrogen, Eugene, OR) were used to verify the appropriate setting for the MP gate to detect MPs less than 1 μm in diameter. The number of MPs/μL of supernatant was calculated according to the manufacturer’s instructions of the tubes (TruCount, BD Biosciences).
Results shown are mean ± standard deviation (SD) or standard error of the mean (SEM). Paired t tests were used to compare RBC ASs. Repeated-measures analysis of variance (ANOVA) was used to determine the influence of storage duration. Statistical analyses were performed using computer software (GraphPad PRISM, GraphPad, Inc., La Jolla, CA).
Osmotic fragility Osmotic fragility of stored RBCs was determined as described previously using a modified method of Parpart and coworkers.17 Briefly, RBCs (1 × 108/mL) from each storage time point were suspended in 1 mL of hypotonic buffered saline solutions (3.5-9.0 g/L, NaCl), or water (100% lysis control) for 30 minutes at RT. Supernatants were collected by centrifugation (1000 × g for 5 min) and hemolysis was measured at a 540 nm wavelength in a plate reader (FLUOROstar Optima, BMG, Ortenberg, Germany). The results were expressed relative to the 100% lysis control. Osmotic fragility curves were plotted at each time point and the NaCl concentration that corresponded to 50% lysis was calculated.
Adhesion to ECs Adhesion of stored RBCs to ECs under shear stress conditions was performed as described previously.18 Briefly, human umbilical vein ECs grown on collagen-coated glass coverslips were mounted into a flow perfusion chamber and perfused at 37°C with a suspension of stored RBCs at a shear stress of 0.5 dyne/cm2 to simulate low microvascular blood flow.13,14,19 Cells were visualized using an inverted microscope (Model IX71, Olympus, Tokyo, Japan), and 15 randomly selected fields on the coverslip were recorded by a CCD camera (DP71, Olympus). The recorded images were analyzed for RBC adherence to ECs using imaging software (ImagePro-Plus,
RESULTS ATP RBCs stored in SOLX maintained significantly higher levels of ATP from Day 14 of storage compared to RBCs stored in SAGM (p ≤ 0.05; Table 1). ATP was well maintained in RBCs stored in SOLX for 42 days, with ATP levels of 3.4 ± 0.8 μmol/L/g Hb compared to 1.4 ± 0.2 μmol/L/ g Hb for RBCs stored in SAGM.
2,3-DPG RBCs stored in SOLX had significantly higher levels of 2,3DPG on Days 7,14, 21, 28, and 42 of storage compared to those stored in SAGM (Table 1). RBC 2,3-DPG declined over storage in both ASs (ANOVA p < 0.0001), although SOLX maintained significantly elevated levels compared with SAGM (p < 0.05) for at least one additional week of storage.
pHi RBCs stored in SOLX maintained significantly higher pHi (p < 0.001) compared to RBCs stored in SAGM (Table 1). Both SOLX- and SAGM-stored RBCs showed a significant decline in pHi over storage (ANOVA p < 0.001).
Routine RBC variables Hemolysis, Hb, and Hct of RBCs stored in SOLX or SAGM complied with acceptance criteria recommended in the Council of Europe guidelines6 (Table 1). As expected, due to the hypotonic effect on RBC shape and cell volume, SOLX-RBCs had a higher Hct than SAGM-RBCs, as determined by the spun Hct method. SOLX-RBCs had lower concentrations of RBCs and Hb compared to SAGM-RBCs, despite having been processed from the same initial volume of WB. Supernatant K+ concentrations of RBCs stored in either AS were not significantly different and increased in both over storage.
Osmotic fragility The NaCl concentration required to induce 50% hemolysis was calculated from osmotic fragility curves and plotted Volume **, ** **
TRANSFUSION
3
VEALE ET AL.
TABLE 1. Routine variables of RBCs stored in SOLX and SAGM* 1
7
14
Storage Day 21
28
35
42
6.68 ± 0.24 6.95 ± 0.27
6.64 ± 0.23 6.95 ± 0.29
6.71 ± 0.27 7.04 ± 0.32
6.65 ± 0.19 6.96 ± 0.18
6.70 ± 0.21 6.99 ± 0.24
6.65 ± 0.20 6.98 ± 0.26
6.64 ± 0.21 6.91 ± 0.26
196 ± 7a 206 ± 7
195 ± 8 204 ± 8
196 ± 8a 206 ± 3
192 ± 8a 202 ± 3
193 ± 8a 202 ± 9
197 ± 8a 206 ± 7
196 ± 8a 206 ± 8
NT NT
0.68 ± 0.01c 0.62 ± 0.01
0.67 ± 0.01c 0.62 ± 0.01
0.67 ± 0.01c 0.62 ± 0.01
NT NT
NT NT
0.66 ± 0.02 0.64 ± 0.01
8.1 ± 3.6 7.9 ± 2.6
6.2 ± 3.7 6.0 ± 3.3
6.7 ± 1.6c 4.7 ± 1.4
7.1 ± 1.8b 3.4 ± 0.9
4.8 ± 1.8a 2.4 ± 0.8
5.3 ± 2.1c 2.1 ± 0.6
3.4 ± 0.8c 1.4 ± 0.2
6.4 ± 1.1 5.5 ± 1.5
4.6 ± 1.3c 2.4 ± 0.7
2.7 ± 1.4a 1.2 ± 0.2
1.9 ± 0.8a 1.2 ± 0.4
1.7 ± 0.4a 1.4 ± 0.6
1.6 ± 0.8 1.2 ± 0.2
1.5 ± 0.5b 1.0 ± 0.2
NT NT
0.08 ± 0.03 0.06 ± 0.02
0.10 ± 0.04 0.08 ± 0.03
0.13 ± 0.06 0.11 ± 0.03
NT NT
NT NT
0.20 ± 0.09 0.24 ± 0.05
4.3 ± 1.4 3.7 ± 1.0
18.1 ± 3.0 17.8 ± 2.7
27.2 ± 3.9 27.9 ± 3.5
36.6 ± 4.7 35.9 ± 4.1
44.9 ± 5.2 47.9 ± 4.6
49.3 ± 5.8 52.3 ± 4.7
7.0 ± 0.07 6.9 ± 0.05
6.9 ± 0.04 6.8 ± 0.04
6.8 ± 0.06 6.7 ± 0.05
6.7 ± 0.04 6.7 ± 0.02
6.6 ± 0.09 6.6 ± 0.05
6.6 ± 0.09 6.5 ± 0.05
7.2 ± 0.06c 7.1 ± 0.07
7.1 ± 0.04c 7.0 ± 0.06
7.0 ± 0.02c 6.8 ± 0.03
6.8 ± 0.09c 6.7 ± 0.06
6.8 ± 0.04b 6.7 ± 0.05
6.7 ± 0.05b 6.6 ± 0.03
6.7 ± 0.04b 6.6 ± 0.03
* The results are plotted as mean ± SD (n = 8). Significance: † n = 4, measured by spun Hct. NT = not tested.
a
p < 0.05,
relative to storage time (Fig. 1). RBCs stored in SOLX had significantly increased osmotic resistance compared to RBCs stored in SAGM (ANOVA p < 0.0004). This suggests that RBCs stored in SOLX have decreased susceptibility to hypotonic lysis. The concentration of NaCl that induced 50% RBC lysis for RBCs stored in SAGM was significantly lower in RBCs stored in SOLX from Day 7 of storage (p ≤ 0.05).
MPs in the stored supernatant RBCs stored in SOLX and SAGM showed increased numbers of GPA+ MPs and annexin V binding MPs up to Day 42 of storage (Fig. 2; p < 0.0001). Significantly fewer MPs were detected in the supernatant of RBCs stored in SOLX compared to those in SAGM on Day 42 of storage (10,475 ± 2520 and 34,797 ± 5551 MPs/μL, respectively; p < 0.001). No significant differences in the numbers of annexin V binding MPs were observed between SOLX- and SAGM-RBC units (Fig. 2B). These results suggest that RBCs stored in SOLX retain more membrane over storage than RBCs stored in SAGM. 4
39.6 ± 4.7 42.4 ± 4.2
7.1 ± 0.05 7.1 ± 0.03
TRANSFUSION Volume **, ** **
b
p < 0.01,
c
p < 0.001.
Osmotic Fragility 50% 5.0
NaCl (g/L)
RBC variable RBCs (×1012/L) SOLX SAGM Hb (g/L) SOLX SAGM Hct (L/L)† SOLX SAGM ATP (μM/g Hb) SOLX SAGM 2,3-DPG (μmol/L/g Hb) SOLX SAGM % Hemolysis† SOLX SAGM K+ (mmol/L) SOLX SAGM Extracellular pH SOLX SAGM pHi SOLX SAGM
4.5
*
**
4.0
**
***
21
28
*** ***
3.5 0
7
14
35
42
Storage Time (days) Fig. 1. Osmotic fragility of RBCs stored in SOLX (○) or SAGM (●). Osmotic fragility of RBCs stored in SOLX or SAGM was measured by incubation of the RBCs in hypotonic solutions of NaCl. The concentration of NaCl (g/L) that resulted in 50% lysis of the RBCs was calculated for each storage time point. The results are plotted as mean ± SEM (n = 8). Significance: *p < 0.05, **p < 0.01, ***p < 0.001.
AS-7 IMPROVED QUALITY OF RBCs
GPA
Flow Perfusion 0.5 dyne/cm 2
100000
A
200
RBCs adhered/mm 2
MPs/mL Log scale
*** 10000
1000
100 0
7
14
21
28
35
50 0 1
14
21
35
42
Storage Time (days)
Annexin V Binding 100000
MPs/mL Log scale
100
42
Storage Time (days)
B
150
Fig. 3. RBCs adhered to ECs under shear stress conditions. RBCs stored in SOLX (■) or SAGM (□) were measured throughout storage for their adherence to ECs under shear stress (0.5 dyne/cm2) conditions. The results are plotted as mean ± SEM (n = 8).
10000
1000
100 0
7
14
21
28
35
42
Storage Time (days)
Fig. 2. Accumulation of GPA+ and annexin V-binding RBC MPs. MPs in the supernatant of RBCs stored in SOLX (○) or SAGM (●) were quantitated by anti-GPA (A) staining and annexin V binding (B) using a flow cytometry absolute bead count assay. The results are plotted as mean ± SEM (n = 8). Significance: ***p < 0.001.
Adhesion of stored RBCs to ECs The effect of adhesion of RBCs to ECs under shear stress conditions was investigated by an in vitro flow perfusion model that mimics low microvascular blood flow. RBC adhesion to ECs increased over the storage period. However, the increase was not significant (Fig. 3). Adhesion of RBCs stored in SOLX was not significantly different to RBCs stored in SAGM.
DISCUSSION Overnight RT hold of WB before processing into RBC components is a standard procedure in many countries.8,20 In this study, RBCs prepared from WB held for 21 hours at RT and stored in the new AS, SOLX had acceptable or improved in vitro quality measures compared to RBCs stored in conventional SAGM. RBCs stored in SOLX maintained acceptable routine quality variables, including extracellular pH, hemolysis, and extracellular K+ throughout 42 days of storage. Biochemical variables including pHi, ATP, and 2,3-DPG levels were significantly better maintained in RBCs stored in SOLX compared with SAGM. The results indicate a 2-week
storage advantage for RBCs stored in SOLX compared with SAGM. These data support a previous study that showed similar results for ATP and percent hemolysis for RBC stored in SOLX that had been prepared from WB processed either immediately after collection or held for 8 or 24 hours at RT before processing.12 The formulations for SOLX and SAGM differ in tonicity, buffering, and pH. SOLX is a hypotonic, alkaline, buffered, Cl−-free AS while SAGM is a hypertonic, acidic, unbuffered saline solution.10 The higher pHi observed with SOLX RBCs may be due to a Cl− shift, which is characterized by the efflux of Cl− ions and a corresponding influx of OH− ions into the RBCs that would increase the pHi. Maintenance of RBC pHi closer to physiologic pH contributes significantly to preservation of cell hydration, synthesis of ATP and 2,3-DPG, carbohydrate metabolism, and cell morphology.1,4,21,22 The higher pHi for SOLX-RBCs and consequential higher levels of ATP and 2,3-DPG are consistent with these previous reports. Maintenance of osmotic resistance in RBCs stored in SOLX was evident, which suggested better retention of the cell surface area to cell volume ratios during storage compared to SAGM-RBCs. Lower numbers of GPA+ MPs in the supernatant of SOLX-RBCs was also consistent with improved maintenance of RBC membrane over storage.23,24 No significant differences were found in the number of PS+ MPs in the supernatant of SOLX-RBCs compared with SAGM-RBCs. This may suggest that SOLXRBCs can efficiently divest themselves of exposed PS at the cell surface and limit the loss of other membrane elements to preserve osmotic resistance. Adhesion of stored RBCs to ECs has been previously reported to increase with storage time.5,18,25,26 In this study, RBCs stored in SOLX showed a trend toward higher adhesion to ECs on Days 35 and 42 of storage compared to Volume **, ** **
TRANSFUSION
5
VEALE ET AL.
RBCs stored in SAGM, although the differences were not significant. In our previous study of the effect of extended RT hold of WB,5 RBCs were stored in an alternative alkaline, hypotonic, Cl−-free solution AS, experimental Erythrosol-4, which showed significantly increased numbers of adhered RBCs on Days 42 and 49 of storage compared to RBCs stored in SAGM. This was thought to be due to a phenomenon that resembled increased dehydration of the RBCs stored in Erythrosol-4 when the RBCs were transferred into an isotonic, physiologic flow perfusion medium, as used in this study. The increased trend in adhesion of hypotonic-stored RBCs at the latter storage time points compared to RBCs stored in SAGM may indicate differences in the effects of storage on the cell membrane properties of RBCs. It is as yet unknown whether the observed in vitro adhesiveness of stored RBCs correlates with a similar propensity in vivo, and if so, what might be the effect on vascular hemodynamics and clinical consequences, if any. It is interesting to note that similar to SOLX-RBCs, RBCs stored in hypertonic AS-1, which is a variant of SAGM, have been shown to have significantly increased adhesion to ECs and shed fewer MPs on Day 42 of storage compared to SAGM-RBCs.26 Further exploration will be needed to identify the biologic nature of the differences between RBCs stored in alternate ASs that induce changed interactions with endothelium. Our in vitro ATP and 2,3-DPG measurements of RBCs stored in SOLX suggest that there may be significant improvements to RBC quality over storage, which could be beneficial for RBC viability and survival in vivo after transfusion. Recent clinical studies in healthy individuals showed mean 24-hour posttransfusion RBC recoveries of 86% for 42-day-stored SOLX RBCs prepared from WB held for 24 hours at RT before processing.11 Shorter WB hold times of less than 2 and 6 to 8 hours at RT also showed increased in vivo RBC recoveries of 89 and 86%, respectively.11,12 These data suggest that the improvements to in vitro quality measures for RBCs stored in SOLX, with reduction of storage lesion effects, may aid significantly better clinical outcomes for recipients.11 In conclusion, RBCs prepared from WB held for 20 to 22 hours at RT and stored in SOLX had significantly higher pHi, reduced osmotic fragility, and reduced numbers of GPA+ MPs, suggesting better maintenance of membrane properties compared with the paired control RBCs stored in SAGM. These results, together with elevated biochemical markers, ATP, and 2,3-DPG, demonstrated that storage of RBCs in SOLX could yield RBCs with improved overall quality over 42 days of storage compared with RBCs stored in SAGM AS. ACKNOWLEDGMENTS We thank Australian Red Cross Blood Service Melbourne staff in Donor Services for assistance with the collection of whole blood 6
TRANSFUSION Volume **, ** **
units and processing laboratory scientists, particularly Charley Samarani, Sue Grasso, and Nicole Saleta. We thank the BMDI Cord Blood Bank, Melbourne, staff for providing umbilical cord samples for EC preparation.
CONFLICT OF INTEREST MZ was an employee and shareholder of Hemerus Medical, LLC, at the time of study and an employee of Haemonetics Corporation at the time of preparation of this manuscript. Hemerus Medical, LLC, was purchased by Haemonetics Corporation in 2013. The other authors have disclosed no conflicts of interest.
REFERENCES 1. Högman C, Meryman H. Storage parameters affecting red blood cell survival and function after transfusion. Transfus Med Rev 1999;13:275-96. 2. Tinmouth A, Chin-Yee I. The clinical consequences of the red cell storage lesion. Transfus Med Rev 2001;15:91-107. 3. Zimrin A, Hess J. Current issues relating to the transfusion of stored red blood cells. Vox Sang 2009;96:93-103. 4. Hess J. An update on solutions for red cell storage. Vox Sang 2006;91:13-9. 5. Veale MF, Healey G, Sparrow RL. Effect of additive solutions on red blood cell (RBC) membrane properties of stored RBCs prepared from whole blood held for 24 hours at room temperature. Transfusion 2011;51:25S-33S. 6. European Directorate for the Quality of Medicines & HealthCare. Guide to the preparation, use and quality assurance of blood components: recommendation. 16th ed. Strasborg: Council of Europe Publishing; 2011. 7. AABB Standard Program Committee. Standards for blood banks and transfusion services. 28th ed. Bethesda (MD): American Association of Blood Banks; 2012. 8. van der Meer P, Cancelas C, Cardigan R, et al. Evaluation of
9.
10.
11.
12.
overnight hold of whole blood at room temperature prior to component processing: effect of red cell additive solutions on in vitro red cell measures. Transfusion 2011; 51(Suppl):15S-24S. De Korte D, Kleine M, Korsten HG, et al. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage. Transfusion 2008;48:1081-9. Sparrow RL. Time to revisit red blood cell additive solutions and storage conditions: a role for “omics” analyses. Blood Transfus 2012;10(Suppl 2):s7-s11. Cancelas J, Dumont L, Maes L, et al. RBC storage in SOLX® additive solution ameliorated the storage lesion and demonstrated extended storage up to 56 days: results of a multicenter trial. Transfusion 2012;52(Suppl):47A. Maes L, Whiteley P, Sawyer S, et al. Red cells stored in SOLX for 42 days demonstrated reduced hemolysis improved morphology and exceptional in vivo recovery. Transfusion 2011;51(Suppl):69A.
AS-7 IMPROVED QUALITY OF RBCs
13. Arslan E, Sierko E, Waters JH, et al. Microcirculatory hemodynamics after acute blood loss followed by fresh and banked blood transfusion. Am J Surg 2005;190:456-62. 14. Detterich J, Alexy T, Rabai M, et al. Low-shear red blood
20. Gulliksson H, van der Meer PF. Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells. Blood Transfus 2009;7:210-15.
cell oxygen transport effectiveness is adversely affected by
21. Högman C, Knutson F, Loof H, et al. Improved mainte-
transfusion and further worsened by deoxygenation in sickle cell disease patients on chronic transfusion therapy.
nance of 2,3 DPG and ATP in RBCs stored in a modified additive solution. Transfusion 2002;42:824-9.
Transfusion 2013;53:297-305. 15. Blakney G, Dinwoodie A. A spectrophotometric scanning technique for the rapid determination of plasma hemoglobin. Clin Biochem 1975;8:96-102. 16. Meryman H, Hornblower M, Keegan T, et al. Refrigerated storage of washed red cells. Vox Sang 1991;60:88-98. 17. Parpart A, Lorenz P, Parpart E, et al. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947;26: 636-40. 18. Anniss AM, Sparrow RL. Storage duration and leukocyte content of red cell products increases adhesion of stored red blood cells to endothelium under flow conditions. Transfusion 2006;46:1561-7. 19. Walmet P, Eckman J, Wick T. Inflammatory mediators promote strong sickle cell adherance to endothelium under venular flow conditions. Am J Hematol 2003;73:215-
22. Meryman H, Hornblower M. Manipulating red cell intraand extracellular pH by washing. Vox Sang 1991;60:99-104. 23. Greenwalt T. The how and why of exocytic vesicles. Transfusion 2006;46:143-52. 24. Willekens F, Werre J, Groenen-Dopp Y, et al. Erythrocyte vesiculation: a self-protective mechanism? Br J Haematol 2008;141:549-56. 25. Relevy H, Koshkaryev A, Manny N, et al. Blood bankinginduced alteration of red blood cell flow properties. Transfusion 2007;48:136-46. 26. Sparrow RL, Sran A, Healey G, et al. In vitro measures of membrane changes reveal differences between red blood cells stored in saline-adenine-glucose-mannitol and AS-1 additive solutions: a paired study. Transfusion 2014;54:560-8.
24.
Volume **, ** **
TRANSFUSION
7
